CN116997567A - Anti-influenza antibodies and combinations thereof - Google Patents

Anti-influenza antibodies and combinations thereof Download PDF

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CN116997567A
CN116997567A CN202180091587.XA CN202180091587A CN116997567A CN 116997567 A CN116997567 A CN 116997567A CN 202180091587 A CN202180091587 A CN 202180091587A CN 116997567 A CN116997567 A CN 116997567A
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seq
nos
antibody
binding fragment
antigen
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D·科蒂
M·S·皮祖托
A·米诺拉
E·卡梅罗尼
F·萨塔
G·斯内尔
E·费里
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Humabs Biomed SA
Vir Biotechnology Inc
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Humabs Biomed SA
Vir Biotechnology Inc
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Priority claimed from PCT/US2021/060166 external-priority patent/WO2022109317A1/en
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Abstract

The present disclosure relates in part to anti-influenza antibodies (and antigen-binding fragments thereof) and combinations thereof for use in the prevention and treatment of influenza infection. The presently disclosed combinations provide a surprising synergistic effect and may be effective in preventing, inhibiting, or neutralizing influenza infection, such as Influenza A Virus (IAV) infection, influenza B Virus (IBV) infection, or both.

Description

Anti-influenza antibodies and combinations thereof
Statement regarding sequence listing
The sequence listing relevant to the present application is provided in text format in place of paper copies and is incorporated herein by reference. The text file containing the sequence listing is named 930585_415wo_sequence_list. The text file was 174KB, created at 2021, 11, 16 days, and submitted electronically via EFS-Web.
Background
Influenza is an infectious disease that outbreaks worldwide, causing about 300 to about 500 tens of thousands of serious diseases and about 290,000 to 650,000 respiratory disease deaths each year (WHO, influenza (seasonal) condition instruction (Influenza (Seasonal) face sheet), 2018, 11, 6). The most common symptoms include: sudden fever, cough (usually dry), headache, muscle and joint pain, severe discomfort (sensory discomfort), sore throat and runny nose. Latency varies from one day to four days, even though symptoms typically begin about 2 days after exposure to the virus. Complications of influenza may include pneumonia, sinus infections, and exacerbation of past health problems (e.g., asthma or heart failure), sepsis, or exacerbation of chronic underlying disease.
Influenza is caused by an influenza virus, a group of viruses of the orthomyxoviridae (family Orthomyxoviridae) that have antigenic and genetic diversity containing the RNA genome of the negative-sense single-stranded segment. Of the four types of influenza viruses (A, B, C and D), three types (A, B and C) are known to affect humans. Influenza viruses can be classified based on the different subtypes of major surface proteins present: hemagglutinin (HA) and Neuraminidase (NA). There are at least 18 influenza a subtypes defined by the hemagglutinin ("HA") protein. The HAs can be divided into two groups. Group 1 contains H1, H2, H5, H6, H8, H9, H11, H12, H13, H16 and H17 subtypes, and group 2 includes H3, H4, H7, H10, H14 and H15 subtypes. Although all subtypes are present in birds, the H1, H2 and H3 subtypes mostly cause disease in humans. The H5, H7 and H9 subtypes cause sporadic severe infections in humans and may create new pandemics. Influenza a viruses continue to evolve, producing new variants, a phenomenon known as antigen drift. Thus, antibodies raised in response to past viruses may be insufficiently or unprotected against the new drift virus. As a result, new vaccines against the H1 and H3 viruses expected to occur must be produced annually, a process which is very expensive and not always effective. The same applies to the production of H5 influenza vaccines.
HA is the major surface protein of influenza a virus and is the primary target of neutralizing antibodies induced by infection or vaccination. Without wishing to be bound by theory, HA is responsible for binding the virus to cells with sialic acid on the membrane (such as cells in the upper respiratory tract or erythrocytes). Furthermore, HA mediates fusion of the viral envelope with the endosomal membrane after the pH HAs been lowered. HA is a homotrimeric integral membrane glycoprotein. HA trimer is composed of three identical monomers, each consisting of an intact HA0 monopeptid chain, where the HA1 and HA2 regions are linked by 2 disulfide bridges. Each HA2 region adopts an alpha helical coiled-coil structure and forms primarily the "stem" or "stalk" region of HA, while the HA1 region is a small globular domain (the "head" region of HA) containing a mixture of alpha/beta structures. The globular HA head region mediates binding to sialic acid receptors, while the HA stem mediates fusion between viral and cell membranes that are subsequently triggered at low pH in endosomes. Although immunodominant HA globular head domains have high plasticity, where different antigenic sites experience constant antigenic drift, the HA stem regions are relatively conserved among subtypes. Current influenza vaccines mostly induce immune responses against immunodominant and variable HA head regions that evolve faster than the stem regions of HA (Kirkpatrick E, qiu X, wilson PC, bahl J, krammer f. Influenza virus hemagglutinin heads evolve faster than the stalk domain (The influenza virus hemagglutinin head evolves faster than the stalk domain.) "scientific report (Sci rep.)" 2018, 7 months 11; 8 (1): 10432). Thus, the protective effect of a particular influenza vaccine is typically no more than a few years, and the influenza vaccine needs to be redeveloped each year.
There are at least 11 different neuraminidase subtypes (N1 to N11 (cdc.gov/flu/about/viruses/types.htm), respectively). Neuraminidases play a role in viral movement and spread by catalyzing the hydrolysis of sialic acid residues on proviral particles and on target cell surface glycoproteins released from infected host cells. Drugs aimed at inhibiting Neuraminidase (NAI) have been developed (e.g., oseltamivir, zanamivir, peramivir, laninamivir), although naturally acquired mutations of the IAV subtype reduce susceptibility to current NAI (Hussain et al, infection and resistance (Infection and Drug Resistance), 10:121-134 (2017)).
There is a need for novel methods for treating or preventing influenza virus infection.
Drawings
Figures 1A-1F show in vitro neutralization of influenza virus by a combination of anti-NA (neuraminidase) monoclonal antibodies and anti-HA (hemagglutinin) monoclonal antibodies. anti-NA monoclonal antibodies "FNI3" (VH: SEQ ID NO:72;VL:SEQ ID NO:78) and "FNI" (VH: SEQ ID NO:132;VL:SEQ ID NO:138) and anti-HA monoclonal antibody "FM08" (VH: SEQ ID NO:43;VL:SEQ ID NO:44; see also Kallewaard et al, cell (Cell) 166 (3): 596-608 (2016), FIG. 1A) and "FHF11" (VH: SEQ ID NO:2; VL: SEQ ID NO: 8) were evaluated for sialidase inhibition, which measures cleavage of 2' - (4-methyl umbrella) - α -D-N-acetylneuraminic acid (MUNANA). Inhibition of H1N1 Cal/09 sialidase activity by FM08+ FNI3 (FIG. 1A), FM08+ FNI9 (FIG. 1B), FHF11+ FNI9 (FIG. 1C) is shown; and inhibition of H3N2 HK/68 sialidase activity by FM08 and FNI3 (FIG. 1D), FM08 and FNI9 (FIG. 1E), FHF11 and FNI9 (FIG. 1F). The heat figures depict the neutralization (%) (upper panel; antibody concentration is shown on x-and y-axes) and synergy/antagonism scores (lower panel, reflecting the combined effects of neutralization of the antibody combination at indicated concentrations relative to the antibody alone (e.g., the effect of FM08+ FNI9 relative to the effect of FM08 alone + the effect of FNI9 alone) in μg/ml. The effect of the individual antibodies is shown in the leftmost column and bottom row of the upper graph in each of figures 1A-1F.
Figures 2A-2C show in vitro neutralization of influenza virus by a combination of anti-NA monoclonal antibodies and anti-HA monoclonal antibodies. anti-NA monoclonal antibodies "FNI" (VH: SEQ ID NO:132;VL:SEQ ID NO:138), "FNI17" (VH: SEQ ID NO:192;VL:SEQ ID NO:198) and "FNI" (VH: SEQ ID NO:204;VL:SEQ ID NO:210) and anti-HA monoclonal antibody "FM08" (VH: SEQ ID NO:43;VL:SEQ ID NO:44; see also Kallewaard et al, cell 166 (3): 596-608 (2016), FIG. 1A) were evaluated by nuclear protein staining. Inhibition of H3N 2A/hong Kong/1/1968 sialidase activity by FM08+ FNI9 (FIG. 2A), FM08+ FNI17 (FIG. 2B) and FM08+ FNI19 (FIG. 2C) is shown. The heat figures depict the neutralization (%) (upper panel; antibody concentration is shown on x-and y-axes) and synergy/antagonism scores (lower panel, reflecting the combined effects of neutralization of the antibody combination at indicated concentrations relative to the antibody alone (e.g., the effect of FM08+ FNI9 relative to the effect of FM08 alone + the effect of FNI9 alone) in μg/ml. A synergy matrix and score are generated using macsynergy ii. "1:1" indicates the ratio of anti-NA monoclonal antibody to anti-HA monoclonal antibody. The effect of the individual antibodies is shown in the leftmost column and bottom row of the upper graph in each of figures 2A-2C.
FIGS. 3A and 3B show activation of FγRIIIa (FIG. 3A; F158 allele) and FγRIIa (FIG. 3B; H131 allele) by anti-NA FNI3 and FNI9, engineered anti-HA monoclonal antibody "FHF11_v9" (VH: SEQ ID NO:37;VL:SEQ ID NO:8), and combinations thereof. Activation was measured using the NFAT-mediated luciferase reporter in engineered Jurkat cells after contact with a549 cells prior to infection with H1N 1A/PR/8/34. Activation of the comparative antibody "FM08_LS" (FM 08 carrying the M428L/N434S mutation) and the negative control antibody "K-" against the unrelated antigen was also measured.
FIGS. 4A-4B show the anti-NA monoclonal antibody "1G01-LS" (Stadlbauer et al science 366 (6464): 499-504 (2019) and 1G 01) described in these experiments carrying M428L and N434S Fc mutations); see fig. 1B; activation of fcγriiia by Stadlbauer et al, fig. 1B, wherein the VH and VL amino acid sequences of antibody 1G01 and the amino acid sequences of 1E01 and 1G04 are incorporated herein by reference), anti-HA FM08-LS, and combinations of both. Activation was measured using NFAT-mediated luciferase reporter in engineered Jurkat cells after contact with a549 cells prior to infection with H1N 1A/PR/8/34 (fig. 4A; multiplicity of infection (MOI) =6) and H3N 2A/aizhi/2/68 (fig. 4b; moi=18). Activation by the negative control antibody (FY 1-LALA) was also measured.
FIGS. 5A-5B show activation of FcgammaRIIA by anti-NA 1G01-LS, anti-HA FM08-LS, and combinations of both. Activation was measured using the NFAT-mediated luciferase reporter in engineered Jurkat cells after contact with a549 cells prior to infection with H1N 1A/PR/8/34 (fig. 5a; moi=6) and H3N 2A/aizhi/2/68 (fig. 5b; moi=6). Activation by the negative control antibody (FY 1-LALA) was also measured.
FIGS. 6A-6B show the design of an in vivo study to evaluate the control activity of a combination of an anti-NA antibody and an anti-HA antibody in BALB/c mice infected with IAV A/Podoconcha/8/34. 1G01 was used as anti-NA antibody and FM08 was used as anti-HA antibody. Figure 6A shows the dose and strain used in the study. Fig. 6B shows the time line and endpoint of the study.
FIGS. 7A-7L show the results of measurements of body weight of BALB/c mice infected with H1N 1A/Polaroid/8/34 over fifteen days after pretreatment with anti-NA 1G01, anti-HA FM08 or a combination of 1G01 and FM 08. Antibodies were administered at 1mg/kg, 0.5mg/kg, 0.25mg/kg or 0.125mg/kg the day prior to infection with LD90 (90% lethal dose) A/Paris/8/34. Body weight of mice administered with vehicle control was also measured (left panel in each figure). The data are shown below: 1mg/kg 1G01 (FIG. 7A), 1mg/kg FM08 (FIG. 7B), 1mg/kg 1G01+1mg/kg FM08 (FIG. 7C); 0.5mg/kg 1G01 (FIG. 7D), 0.5mg/kg FM08 (FIG. 7E), 0.5mg/kg 1G01+0.5mg/kg FM08 (FIG. 7F); 0.25mg/kg 1G01 (FIG. 7G), 0.25mg/kg FM08 (FIG. 7H), 0.25mg/kg 1G01+0.25mg/kg FM08 (FIG. 7I); 0.125mg/kg 1G01 (FIG. 7J), 0.125mg/kg FM08-rIgG (FIG. 7K), 0.125mg/kg 1G01+0.125mg/kg FM08 (FIG. 7L).
Figures 8A-8B show a comparison of the area peak under the negative curve in the area under the curve analysis of weight loss in BALB/c mice infected with a/polis/8/34 with IgG in serum after treatment with 1G01, FM08 or a combination of 1G01 and FM08. The area peak under the negative curve is graphically represented by the amount of each mAb administered in mg/kg (fig. 8A) or the amount of total antibody (fig. 8B).
Figures 9A-9C show the Compusyn software readings of area under the curve for weight loss for BALB/C mice infected with a/polis/8/34 after treatment with the combination 1G01 and FM08. The quantitatively defined combination index of dose-response curves, isobolograms (i.e., equivalent effective curves, fig. 9B) and (fig. 9C) synergy, additive effects and antagonism are shown (fig. 9A).
FIGS. 10A and 10B show the quantification of human IgG in the serum of BALB/c mice 24 hours after antibody injection and immediately prior to infection with LD90 (90% lethal dose) A/Podoc/8/34. BALB/c mice were injected intravenously with 1G01, FM08 or 1G01 and FM08 at 1mg/kg, 0.5mg/kg, 0.25mg/kg or 0.125mg/kg for each antibody. FIG. 10A shows human IgG reported in μg/ml in serum 24 hours after antibody injection. FIG. 10B shows a comparison of the area peak under the H1N1 negative curve from the area under the curve analysis of weight loss in BALB/c mice infected with A/Polish/8/34 (FIGS. 8A-8B) with the IgG and EC50 (half maximal effective concentration) values in serum.
FIG. 11 shows survival of BALB/c mice infected with A/Paris each/8/34 for fifteen days after treatment with 1G01, FM08, or a combination of 1G01 and FM 08. Survival of mice pretreated with vehicle control was also measured.
FIGS. 12A-12B show 0.25mg/kg 1G01, 0.25mg/kg FM08, or 0.25mg/kg 1G01+0.25mg/kg FM08 in use (FIG. 12A); or (FIG. 12B) survival of BALB/c mice infected with A/Paris/8/34 for fifteen days after treatment with 0.25mg/kg 1G01, 0.25mg/kg FM08 and 0.125mg/kg 1G01+0.125mg/kg FM 08. Survival of mice pretreated with vehicle control was also measured.
FIG. 13 shows the design of a DVD (double variable domain) bispecific antibody "FNI-L-FM 08-DVDIg1-LS" containing anti-NA (FNI 17) and anti-HA (FM 08) antigen binding domains.
FIGS. 14A-14B show in vitro inhibition of sialidase activity as determined by FNI-FM 08-DVDIg 1-LS. The comparative test preparations were FNI mAb alone, FNI17+FM08 mAb or FM08 mAb alone for H1N1 Cal/09 (FIG. 14A) and H3N2 HK/68 (FIG. 14B). The calculated IC50 values (nM) are shown below the graph in each graph.
FIGS. 15A-15B show in vitro neutralization of H5 and H7 pseudotyped viruses as determined by FM08-FNI9-DVDIg1-LS, FNI9-FM08-DVDIg1-LS, FM 08-FNI-DVDIg 1-LS and FNI-FM 08-DVDIg 1-LS. Data for the comparative antibody FM08 are also shown. FIG. 15A shows neutralization of H5/VN1194 pp. FIG. 15B shows the neutralization of H7/IT/99 pp. The calculated IC50 values (nM) are shown below the graph in each graph.
FIGS. 16A-16B show antibody activation by FcgammaRIIIa (FIG. 16A; F158 allele) and FcgammaRIIa (FIG. 16B; H131 allele). NFAT-mediated luciferase reporter gene activation in engineered Jurkat cells was used to measure activation. FM08-FNI17-DVDIg1-LS and FNI-FM 08-DVDIg1-LS were tested as well as the comparative antibodies FM 08-LS, FHF12-LS, FHF11-v9-LS and negative control antibody (FY 1-LALA).
FIG. 17 shows the dosing and treatment groups of an in vivo study used to evaluate the control activity of FNI-FM 08-DVDIg1_LS ("DVD format") in BALB/c mice infected with H1N1/PR 8/8/34. Four treatments p (test articles, "TA 1-TA-4"), FM08_LS (TA 1, "mAb-08"), FNI17_LS (TA-2, "mAb-17"), FM08_LS+ FNI17_LS (TA 3, "mAb-08+mAb-17"), and FNI17-FM08-DVDIg1-LS (TA 4, "DVD format") were evaluated.
FIGS. 18A-18D show measurements of body weight of BALB/c mice infected with influenza virus over fifteen days after pretreatment with FM08_LS (TA 1, "mAb-08"), FNI17_LS (TA 2, "mAb-17"), FM08_LS+ FNI17_LS (TA 3, "mAb-08+mAb-17"), and FNI-FM 08-DVD-LS (TA 4, "DVD format"). Antibodies were administered at 1mg/kg (FIG. 18A), 0.5mg/kg (FIG. 18B), 0.25mg/kg (FIG. 18C), or 0.125mg/kg (FIG. 18D) the day prior to infection with LD90 (90% lethal dose) H1N1/PR 8/8/34. Mice in the FNI-FM 08-DVD-LS (TA 4, "DVD version") treatment group received equivalent amounts of molecules corresponding to the body weight dose (mg/kg) of TA 1-TA 3.
FIGS. 19A-19B show infection with H1N1 PR8/8/34 and with different doses of FM08_LS or FNI17_LS (FIG. 19A); survival rate of either FM08_LS+ FNI17_LS or FNI17-FM08-DVD-LS (FIG. 19B) pretreated BALB/c mice over fifteen days.
Fig. 20 shows weight loss (reported as area peak under negative curve) from day 0 to day 14 post infection in mice infected with IAV after pretreatment with FNI17_ls, FM08_ls, fn17_ls and FM08_ls or FNI/FM 08_ls double variable domain antibody (DVD). Weight loss was also measured in mice pre-treated with vehicle control.
In the left chart, for a 1mg/kg dose (leftmost set of five bars), the order of the bars from left to right corresponds to a top-to-bottom orientation in the legend (i.e., the vehicle is the leftmost bar in the 1mg/kg quadrant; FNI/FM 08-LSDVD is the rightmost bar). At other doses, the left-to-right order of the bar graphs corresponds to the top-to-bottom orientation starting at FNI17 in the legend (i.e., FNI is the leftmost bar graph in the 0.5mg/kg quadrant; FNI/FM 08_LSDVD is the rightmost bar graph). In the smaller right chart, the bar graph (left to right) is: a vehicle; FNI17+Fm08_LS; FNI17; FM08_LS.
Detailed Description
The present disclosure relates in part to anti-influenza antibodies (and antigen-binding fragments thereof), polynucleotides encoding anti-influenza antibodies and antigen-binding fragments thereof, and combinations thereof, for use in the prevention and treatment of influenza infection.
The presently disclosed combinations provide a surprising synergistic effect and may be effective in preventing, inhibiting, or neutralizing influenza infection, such as Influenza A Virus (IAV) infection, influenza B Virus (IBV) infection, or both. The presently disclosed combinations may have improved breadth and efficacy of circulating IAV strains in humans and animals, may provide improved function to Monoclonal Antibody Resistant Mutants (MARMs) and/or viral isolates, may reduce the risk of escape mutants, may promote endogenous immune responses to influenza, have low to no nonspecific activity (e.g., against healthy subject tissue), are effective for seasonal IAVs and/or IBVs, are effective for animal IAVs, and/or possess favorable pharmacokinetic properties.
In certain aspects, provided herein are combinations and compositions comprising an anti-Hemagglutinin (HA) antibody or antigen-binding fragment thereof and an anti-Neuraminidase (NA) antibody or antigen-binding fragment thereof, or one or more polynucleotides encoding the anti-HA and anti-NA antibodies or antigen-binding fragments thereof, and their use in preventing or treating influenza infection and in the manufacture of a medicament for preventing or treating influenza infection. Also provided are methods for treating or preventing influenza infection, wherein the methods comprise administering to a subject an effective amount of an anti-HA antibody (or antigen-binding fragment thereof) and an anti-NA antibody (or antigen-binding fragment thereof), or administering to a subject who HAs received, will receive, or is receiving an anti-NA antibody (or antigen-binding fragment thereof) or one or more polynucleotides encoding the same, or administering to a subject who HAs received, will receive, or is receiving an anti-HA antibody (or antigen-binding fragment thereof) or one or more polynucleotides encoding the same.
Multispecific antibodies or antigen-binding fragments thereof comprising an anti-HA binding domain and an anti-NA binding domain are also provided, as well as related compositions and uses.
Before explaining the present disclosure in more detail, an understanding of the present invention may be helpful in providing definitions of certain terms used herein. Additional definitions are set forth throughout this disclosure.
In this specification, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the range, as well as fractions thereof (e.g., one tenth and one hundredth of an integer) as appropriate, unless otherwise indicated. Moreover, any number of ranges set forth herein in connection with any physical feature, such as a polymer subunit, size, or thickness, should be understood to include any integer within the stated range, unless otherwise indicated. As used herein, the term "about" refers to ±20% of the specified range, value or structure, unless otherwise indicated. It should be understood that the term "a/an" as used herein refers to "one or more/one or more" of the listed components. The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination thereof. As used herein, the terms "include," "have," "include," and "include" are used synonymously, and these terms and variations thereof are intended to be interpreted as non-limiting.
"optional" or "optionally" means that the subsequently described element, component, event or circumstance may or may not occur, and that the description includes instances where the element, component, event or circumstance occurs and instances where it does not.
In addition, it is to be understood that individual constructs or groups of constructs derived from various combinations of structures and subunits described herein are disclosed by the present application to the same extent as if each construct or group of constructs was set forth separately. Accordingly, the selection of a particular structure or a particular subunit is within the scope of the present disclosure.
The term "consisting essentially of … …" is not equivalent to "comprising" and refers to a specified material or step of the claims, or to a material or step that does not materially affect the basic characteristics of the claimed subject matter. For example, when the amino acid sequence of a domain, region, module, or protein comprises an extension, deletion, mutation, or combination thereof (e.g., an amino acid between amino or carboxy termini or domains), the protein domain, region, or module (e.g., binding domain) or protein "consists essentially of" a particular amino acid sequence, the extension, deletion, mutation, or combination thereof together comprise up to 20% (e.g., up to 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%) of the length of the domain, region, module, or protein and do not substantially affect (i.e., do not reduce activity by more than 50%, such as not more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain, region, module, or protein (e.g., binding protein's target binding affinity).
As used herein, "amino acid" refers to naturally occurring or synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, for example, hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α -carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.
As used herein, "mutation" refers to an alteration in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. Mutations may cause several different types of changes in the sequence, including nucleotide or amino acid substitutions, insertions or deletions.
"conservative substitutions" refer to amino acid substitutions that do not significantly affect or alter the binding characteristics of a particular protein. Typically, conservative substitutions are substitutions in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include those found in one of the following groups: group 1: alanine (Ala or A), glycine (Gly or G), serine (Ser or S), threonine (Thr or T); group 2: aspartic acid (Asp or D), glutamic acid (Glu or Z); group 3: asparagine (Asn or N), glutamine (Gln or Q); group 4: arginine (Arg or R), lysine (Lys or K), histidine (His or H); group 5: isoleucine (Ile or I), leucine (Leu or L), methionine (Met or M), valine (Val or V); group 6: phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W). Additionally or alternatively, amino acids may be divided into conservative substitution groups based on similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, for substitution, the aliphatic groupings may include Gly, ala, val, leu and Ile. Other conservative substitution sets include: sulfur-containing: met and cysteine (Cys or C); acidic: asp, glu, asn and Gln; small aliphatic, non-polar or slightly polar residues: ala, ser, thr, pro and Gly; polar negatively charged residues and amides thereof: asp, asn, glu and Gln; polar positively charged residues: his, arg and Lys; large aliphatic, nonpolar residues: met, leu, ile, val and Cys; large aromatic residues: phe, tyr and Trp. Additional information can be found in Cright on (1984) Proteins, W.H. Frieman Press and Company.
As used herein, "protein" or "polypeptide" refers to a polymer of amino acid residues. Proteins are suitable for use with naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid, and non-naturally occurring amino acid polymers. Variants of the proteins, peptides and polypeptides of the present disclosure are also contemplated. In certain embodiments, variant proteins, peptides and polypeptides comprise or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identical to an amino acid sequence of a defined or reference amino acid sequence as described herein.
"nucleic acid molecule (nucleic acid molecule)" or "polynucleotide" or "polynucleic acid" refers to a polymeric compound comprising covalently linked nucleotides, which may be composed of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine loops). Purine bases include adenine, guanine, hypoxanthine, and xanthine, and pyrimidine bases include uracil, thymine, and cytosine. The nucleic acid molecule comprises: polyribonucleic acid (RNA), including mRNA, microrna, siRNA, viral genomic RNA, and synthetic RNA; and polydeoxyribonucleic acid (DNA, also referred to as deoxyribonucleic acid), including cDNA, genomic DNA, and synthetic DNA, any of which may be single-stranded or double-stranded. If single-stranded, the nucleic acid molecule may be the coding strand or the non-coding (antisense) strand. Nucleic acid molecules encoding an amino acid sequence include all nucleotide sequences encoding the same amino acid sequence. Some forms of nucleotide sequences may also include introns to the extent that the introns will be removed by co-transcription or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence due to redundancy or degeneracy of the genetic code, or by splicing.
In some embodiments, the polynucleotide comprises a modified nucleoside, a cap-1 structure, a cap-2 structure, or any combination thereof. In certain embodiments, the polynucleotide comprises pseudouridine, N6-methyladenosine, 5-methylcytidine, 2-thiouridine, or any combination thereof. In some embodiments, the pseudouridine comprises N1-methyl pseudouridine. These features are known in the art and are described, for example, in Zhang et al, front of immunology (front. Immunol.), doi= 10.3389/fimmu.2019.00594 (2019); eyler et al, proc. Natl. Acad. Sci. USA (PNAS) 116 (46): 23068-23071; DOI 10.1073/pnas.1821754116 (2019); nance and Meier, ACS center science (ACS cent. Sci.) 2021,7,5,748-756; ori/10.1021/acscentsci.1c00197 (2021); and van Hoecke and Roose, journal of transformation medicine (j. Transitional Med) 17:54 (2019); modified nucleoside and mRNA features are discussed in https:// doi.org/10.1186/s12967-019-1804-8, incorporated herein by reference. Variants of the nucleic acid molecules of the present disclosure are also contemplated. The variant nucleic acid molecule is at least 70%, 75%, 80%, 85%, 90%, preferably 95%, 96%, 97%, 98%, 99% or 99.9% identical to a nucleic acid molecule of a defined or reference polynucleotide as described herein, or hybridizes to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate, or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 65-68 ℃. Nucleic acid molecule variants retain the ability to encode their binding domains that have the functions described herein, such as binding to a target molecule.
"percent sequence identity" refers to a relationship between two or more sequences as determined by comparing the sequences. The preferred method of determining sequence identity is designed to give the best match between the compared sequences. For example, sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first amino acid or nucleic acid sequence and the second amino acid or nucleic acid sequence for optimal alignment). Furthermore, non-homologous sequences may be ignored for comparison purposes. Unless indicated otherwise, the percent sequence identity referred to herein is calculated over the length of the reference sequence. Methods for determining sequence identity and similarity can be found in publicly available computer programs. Sequence alignment and percent identity calculations can be performed using the BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX). The mathematical algorithms used in the BLAST program can be found in Altschul et al, nucleic Acids Res 25:3389-3402,1997. In the context of the present disclosure, it should be understood that where the analysis is performed using sequence analysis software, the results of the analysis are based on the "default values" of the referenced program. "default value" means any set of values or parameters that are initially loaded when the software is first initialized.
The term "isolated" means that material is removed from its original environment (e.g., the natural environment if the material is naturally occurring). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, which is separate from some or all of the coexisting materials in the natural system, is isolated. Such nucleic acids may be part of a vector and/or such nucleic acids or polypeptides may be part of a composition (e.g., cell lysate) and still be isolated, as such vector or composition is not part of the natural environment of the nucleic acid or polypeptide. In some embodiments, "isolated" also describes an antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition that is in vitro in a human.
The term "gene" means a DNA or RNA segment involved in the production of a polypeptide chain; in some cases, the gene includes intervening sequences (introns) between regions preceding and following the coding region (e.g., 5 'untranslated regions (UTRs) and 3' UTRs) as well as individual coding segments (exons).
"functional variant (functional variant)" refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of the present disclosure but slightly different in composition (e.g., one base, atom, or functional group is different, added or removed) such that the polypeptide or encoded polypeptide is capable of performing at least one function of the parent polypeptide at an efficiency of at least 50%, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100%. In other words, when a polypeptide of the present disclosure or a functional variant of an encoded polypeptide exhibits no more than 50% performance reduction in a selected assay compared to a parent or reference polypeptide, such as an assay for measuring binding affinity (e.g., measuring association (Ka) or dissociation (K) D ) Constant ofOr tetramer staining), said functional variants having "similar binding", "similar affinity", or "similar activity".
As used herein, a "functional moiety" or "functional fragment" refers to a polypeptide or polynucleotide that includes only a domain, portion, or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% of the activity associated with the domain, portion, or fragment of the parent or reference compound, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% of the activity level of the parent polypeptide, or provides a biological benefit (e.g., effector function). A functional moiety or "functional fragment" of a polypeptide or encoded polypeptide of the present disclosure has "similar binding" or "similar activity" when said functional moiety or fragment exhibits no more than 50% performance reduction (preferably no more than 20% or 10% or no more than a logarithmic difference in affinity compared to the parent or reference) in a selected assay compared to the parent or reference polypeptide.
As used herein, the term "engineered", "recombinant" or "non-natural" is meant to include at least one genetic alteration or organism, microorganism, cell, nucleic acid molecule or vector that has been modified by the introduction of an exogenous or heterologous nucleic acid molecule, wherein such alteration or modification is introduced by genetic engineering (i.e., human intervention). Genetic alterations include, for example, modifications that introduce expressible nucleic acid molecules encoding functional RNAs, proteins, fusion proteins, or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruptions of the genetic material of the cell. Additional modifications include, for example, non-coding regulatory regions, wherein the modification alters expression of a polynucleotide, gene, or operon.
As used herein, "heterologous" or "non-endogenous" or "exogenous" refers to any gene, protein, compound, nucleic acid molecule or activity that is not native to the host cell or subject, or any gene, protein, compound, nucleic acid molecule or activity that has been altered that is native to the host cell or subject. Heterologous, non-endogenous, or exogenous includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise altered such that the structure, activity, or both, differs between native and altered genes, proteins, compounds, or nucleic acid molecules. In certain embodiments, a heterologous, non-endogenous or exogenous gene, protein, or nucleic acid molecule (e.g., receptor, ligand, etc.) may not be endogenous to the host cell or subject, but rather nucleic acid encoding such gene, protein, or nucleic acid molecule is added to the host cell by conjugation, transformation, transfection, electroporation, etc., wherein the added nucleic acid molecule may be integrated into the host cell genome or may be present as extrachromosomal genetic material (e.g., as a plasmid or other self-replicating vector). The term "homologue" or "homologue" refers to a gene, protein, compound, nucleic acid molecule or activity found in or derived from a host cell, species or strain. For example, a heterologous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to the native polynucleotide or gene and encode a homologous polypeptide or activity, but the polynucleotide or polypeptide may have an altered structure, sequence, expression level, or any combination thereof. The non-endogenous polynucleotides or genes and encoded polypeptides or activities may be from the same species, different species, or a combination thereof.
In certain embodiments, a nucleic acid molecule native to a host cell, or a portion thereof, will be considered heterologous to the host cell if it has been altered or mutated, or may be considered heterologous if the nucleic acid molecule native to the host cell has been altered with heterologous expression control sequences or has been altered with endogenous expression control sequences not normally associated with the nucleic acid molecule native to the host cell. In addition, the term "heterologous" may refer to biological activity that is different, altered, or not endogenous to the host cell. As described herein, more than one heterologous nucleic acid molecule can be introduced into a host cell as an independent nucleic acid molecule, as a plurality of separately controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof.
As used herein, the term "endogenous" or "native" refers to a polynucleotide, gene, protein, compound, molecule, or activity that is typically present in a host cell or subject.
As used herein, the term "expression" refers to the process of producing a polypeptide based on the coding sequence of a nucleic acid molecule, such as a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. The expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).
The term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when the promoter is capable of affecting the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "unlinked" means that the related genetic elements are not closely related to each other and that the function of one does not affect the other.
As described herein, more than one heterologous nucleic acid molecule can be introduced into a host cell as an independent nucleic acid molecule, as a plurality of separately controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a protein (e.g., the heavy chain of an antibody), or any combination thereof. When two or more heterologous nucleic acid molecules are introduced into a host cell, it is to be understood that the two or more heterologous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof. The reference to the amount of heterologous nucleic acid molecule or protein activity refers to the amount of encoding nucleic acid molecule or protein activity, rather than the amount of an independent nucleic acid molecule introduced into the host cell.
The term "construct" refers to any polynucleotide containing a recombinant nucleic acid molecule (or fusion protein of the present disclosure when the context clearly indicates). The (polynucleotide) construct may be present in a vector (e.g., bacterial vector, viral vector) or may be integrated in the genome. A "vector" is a nucleic acid molecule capable of transporting another nucleic acid molecule. The vector may be, for example, a plasmid, cosmid, virus, RNA vector or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semisynthetic or synthetic nucleic acid molecules. Vectors of the present disclosure also include transposon subsystems (e.g., sleeping Beauty), see, e.g., geurns et al, molecular therapy (mol. Ther.)) 8:108,2003; ma tes et al, nature genet.) (41:753, 2009). Exemplary vectors are vectors capable of autonomous replication (episomal vectors), vectors capable of delivering a polynucleotide to the genome of a cell (e.g., viral vectors), or vectors capable of expressing a nucleic acid molecule to which they are linked (expression vectors).
As used herein, an "expression vector" or "vector" refers to a DNA construct containing a nucleic acid molecule operably linked to suitable control sequences capable of effecting the expression of the nucleic acid molecule in a suitable host. Such control sequences include promoters to effect transcription, optional operator sequences to control such transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences to control transcription and translation termination. The vector may be a plasmid, phage particle, virus or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases, may be integrated into the genome itself or the polynucleotides contained in the vector may be delivered into the genome without the vector sequence. In this specification, "plasmid," "expression plasmid," "virus," and "vector" are often used interchangeably.
In the context of inserting a nucleic acid molecule into a cell, the term "introduced" means "transfection", "transformation" or "transduction" and includes reference to incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell, where the nucleic acid molecule may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
In certain embodiments, polynucleotides of the present disclosure may be operably linked to certain elements of a vector. For example, the polynucleotide sequences required to effect expression and processing of the coding sequences to which the polynucleotide sequences are linked may be operably linked. Expression control sequences may include appropriate transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation signals; stabilizing the sequence of cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., kozak consensus sequences); a sequence that enhances protein stability; and sequences that may enhance protein secretion. Expression control sequences may be operably linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
In certain embodiments, the vector comprises a plasmid vector or a viral vector (e.g., a lentiviral vector or a gamma-retroviral vector). Viral vectors include retroviruses; adenoviruses; parvovirus (e.g., adeno-associated virus); coronavirus; negative strand RNA viruses, such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai virus (Sendai)); positive strand RNA viruses, such as picornaviruses and alphaviruses; and double stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, epstein-Barr virus, cytomegalovirus) and poxviruses (e.g., vaccinia, chicken pox, and canary pox). Other viruses include, for example, norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus and hepatitis virus. Examples of retroviruses include avian leukemia-sarcoma, mammalian type C viruses, type B viruses, type D viruses, HTLV-BLV groups, lentiviruses, foamy viruses (Coffin, J.M., retrovirus family: viruses and their replication (retroviradae: the viruses and their replication), basic virology (Fundamental Virology), third edition, B.N. fields et al, editions by Philadelphia-Raven Publishers, philadelphia, 1996).
A "retrovirus" is a virus having an RNA genome that is reverse transcribed into DNA using a reverse transcriptase, and the reverse transcribed DNA is then incorporated into the host cell genome. "Gamma retrovirus" refers to a genus of the family retrovirus. Examples of gamma retroviruses include mouse stem cell virus, murine leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus.
"lentiviral vectors" include HIV-based lentiviral vectors for gene delivery, which may be integrated or non-integrated, have a relatively large packaging capacity, and can transduce a range of different cell types. Lentiviral vectors are typically produced after transient transfection of three (packaging, envelope and transfer) or more plasmids into a producer cell. Similar to HIV, lentiviral vectors enter target cells through the interaction of viral surface glycoproteins with receptors on the cell surface. Upon entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of the reverse transcription is a double stranded linear viral DNA, which is the matrix for viral integration into the DNA of the infected cell.
In certain embodiments, the viral vector may be a gamma retrovirus, e.g., a moloney murine leukemia virus (Moloney murine leukemia virus, MLV) derived vector. In other embodiments, the viral vector may be a more complex retroviral-derived vector, e.g., a lentiviral-derived vector. HIV-1 derived vectors fall into this category. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Medi-Weissna (Maedi-Visna) virus (sheep lentivirus). Methods for transducing mammalian host cells with viral particles including transgenes using retroviral and lentiviral vectors and packaging cells are known in the art and have been described, for example, in the following documents: us patent 8,119,772; walchli et al, public science library, complex (PLoS One) 6:327930,2011; zhao et al, J.Immunol.) (174:4415, 2005; engels et al, human gene therapy (hum. Gene Ther.) 14:1155,2003; frecha et al, molecular therapy 18:1748,2010; verhoeyen et al, methods of molecular biology (Methods mol. Biol.) 506:97,2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available. Other viral vectors may also be used for polynucleotide delivery, including DNA viral vectors, including, for example, adenovirus-based vectors and adeno-associated virus (AAV) -based vectors; vectors derived from Herpes Simplex Virus (HSV) include amplicon vectors, replication defective HSV and attenuated HSV (Krisky et al, gene therapy (Gene Ther.)) 5:1517, 1998.
Other vectors that may be used with the compositions and methods of the present disclosure include those derived from baculovirus and alpha-viruses (Jolly, D j.1999. Emerging Viral vectors) Friedmann t. Edit, development of human gene therapy (The Development of Human Gene therapeutic.), pages 209-40, new York: cold spring harbor laboratory (New York: cold Spring Harbor Lab) or plasmid vectors (such as sleeping americans or other transposon vectors).
When the viral vector genome comprises multiple polynucleotides to be expressed as independent transcripts in a host cell, the viral vector may also comprise additional sequences between the two (or more) transcripts, allowing for bicistronic or polycistronic expression. Examples of such sequences for viral vectors include an Internal Ribosome Entry Site (IRES), a furin cleavage site, a viral 2A peptide, or any combination thereof.
Further described herein are plasmid vectors, including DNA-based antibodies or antigen-binding fragments encoding plasmid vectors for direct administration to a subject.
As used herein, the term "host" refers to a cell or microorganism genetically modified with a heterologous nucleic acid molecule to produce a target of a polypeptide of interest (e.g., an antibody of the present disclosure).
Host cells may include any individual cell or cell culture that can receive a vector or nucleic acid for incorporation or expression of a protein. The term also encompasses progeny of a host cell, whether genetically or phenotypically identical or different. Suitable host cells may depend on the vector and may include mammalian cells, animal cells, human cells, monkey cells, insect cells, yeast cells, and bacterial cells. These cells may be induced by use of viral vectors, transformation by calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection or other methods, incorporated into vectors or other materials. See, e.g., sambrook et al, molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), 2 nd edition (Cold spring harbor laboratory, 1989).
In the case of influenza infection, a "host" refers to a cell or subject infected with influenza.
As used herein, "antigen" or "Ag" refers to an immunogenic molecule that elicits an immune response. This immune response may involve antibody production, activation of specific immune competent cells, activation of complement, antibody dependent cellular cytotoxicity, or any combination thereof. The antigen (immunogenic molecule) may be, for example, a peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid, or the like. It will be apparent that the antigen may be synthesized, recombinantly produced or derived from a biological sample. Exemplary biological samples that may contain one or more antigens include tissue samples, fecal samples, cells, biological fluids, or combinations thereof. The antigen may be produced by a cell that has been modified or genetically engineered to express the antigen. Antigens may also be present as influenza NA antigens, such as in viral particles, or expressed or presented on the surface of influenza-infected cells.
The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin or other binding molecule, domain, or protein. Epitope determinants generally contain chemically active surface groupings of molecules (such as amino acids or sugar side chains) and may have specific three dimensional structural characteristics as well as specific charge characteristics. Where the antigen is or comprises a peptide or protein, the epitope may comprise contiguous amino acids (e.g., a linear epitope), or may comprise amino acids from different portions or regions of the protein that are accessed by protein folding (e.g., discontinuous or conformational epitopes), or discontinuous amino acids that are in close proximity independent of protein folding.
Antibodies, antigen binding fragments, combinations and compositions
Disclosed herein are anti-HA antibodies and anti-NA antibodies, and the antibodies are used in the presently disclosed combinations, compositions, uses, and methods. Embodiments provided include antibodies or antigen-binding fragments thereof capable of binding to Influenza A Virus (IAV) Hemagglutinin (HA) and neutralizing infection by IAV, and/or antibodies or antigen-binding fragments thereof capable of binding to Neuraminidase (NA) from: (i) IAVs, wherein the IAV comprises a group 1IAV, a group 2IAV, or both; and (ii) Influenza B Virus (IBV) and is capable of neutralising infection by and/or inhibiting sialidase activity of said IAV and/or said IBV.
In some embodiments, the composition, combination, or therapy comprises an anti-NA antibody or antigen-binding fragment and an anti-HA antibody or antigen-binding fragment in a ratio of 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:10, 10:1, 5:1, 4:1, 3:1, 2.5:1, or 2:1.
In certain embodiments, the antibodies or antigen binding fragments of the present disclosure associate or associate with HA or NA, but not with any other molecules or components in the sample.
In certain embodiments, the antibodies or antigen binding fragments of the disclosure specifically bind to IAV HA or NA. As used herein, "specific binding" (specifically binds) "refers to the association or association of an antibody or antigen binding fragment with an antigen, wherein affinity or K a (i.e., equilibrium association constant of specific binding interactions, in units of 1/M) of equal to or greater than 10 5 M -1 (which is equal to the association rate [ K ] of the association reaction on ]And dissociation rate [ K off ]Is not significantly associated or associated with other molecules or components in the sample. Alternatively, affinity may be defined as the equilibrium dissociation constant (K d ) In M (e.g., 10 -5 M to 10 -13 M). Antibodies can be classified as "high affinity" antibodies or "low affinity" antibodies. "high affinity" antibodies refer to those K a At least 10 7 M -1 At least 10 8 M -1 At least 10 9 M -1 At least 10 10 M -1 At least 10 11 M -1 At least 10 12 M -1 Or at least 10 13 M -1 Is an anti-cancer agent of (a)A body. "Low affinity" antibodies refer to those K a At most 10 7 M -1 At most 10 6 M -1 At most 10 5 M -1 Is a human antibody. Alternatively, affinity may be defined as the equilibrium dissociation constant (K d ) In M (e.g., 10 -5 M to 10 -13 M)。
A variety of assays are known for identifying antibodies of the present disclosure that bind to a particular target, as well as determining binding domain or binding protein affinity, such as western blot, ELISA (e.g., direct, indirect, or sandwich), analytical ultracentrifugation, spectroscopy, biolayer interferometry, and surface plasmon resonance) Analysis (see, e.g., scatchard et al, annual report of the national academy of sciences of New York, ann.N.Y. Acad.Sci.) (51:660, 1949; wilson, science 295:2103,2002; wolff et al, cancer research (Cancer Res.) 53:2560,1993; U.S. patent nos. 5,283,173, 5,468,614 or equivalent). Assays for assessing affinity or apparent affinity or relative affinity are also known.
In certain examples, influenza HA and/or NA antigens can be expressed recombinantly in host cells (e.g., by transfection) and the host cells (e.g., fixed or fixed and permeabilized) can be immunostained with antibodies and purified by flow cytometry (e.g., using a ZE5 cell analyzer @ ) And FlowJo software (TreeStar) to analyze the binding to determine binding. In some embodiments, positive binding may be defined by differential antibody staining of cells expressing influenza HA and/or NA versus control (e.g., mimetic) cells.
In some embodiments, the antibodies or antigen binding fragments of the present disclosure bind to influenza HA or NA proteins as measured using biolayer interferometry or by surface plasmon resonance.
Certain properties of the presently disclosed antibodies or antigen binding fragments can be described using IC50 or EC50 values. In certain embodiments, the IC50 is the concentration of a composition (e.g., an antibody) that causes half-maximal inhibition of the indicated biological or biochemical function, activity, or response. In certain embodiments, the EC50 is the concentration of the composition that provides the half maximal response in the assay. In some embodiments, for example, to describe the ability of the presently disclosed antibodies or antigen binding fragments to neutralize influenza-caused infection, the IC50 and EC50 are used interchangeably.
Unless explicitly defined differently herein, terms understood by those skilled in the art of antibodies are each given the meaning obtained in the art. For example, the term "antibody" refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as any antigen-binding portion or fragment of an intact antibody that has or retains the ability to bind to an antigen target molecule recognized by the intact antibody, such as an scFv, fab, or Fab'2 fragment. Thus, the term "antibody" is used herein in its broadest sense and includes polyclonal and monoclonal antibodies, including whole antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen-binding (Fab) fragments, F (ab ') 2 fragments, fab' fragments, fv fragments, recombinant IgG (IgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv) and single domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or otherwise modified immunoglobulin forms such as intracellular antibodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugate antibodies, multispecific antibodies, e.g., DVD-Ig (e.g., U.S. patent No. 8,258,268, the forms of which are incorporated herein by reference in their entirety), bispecific antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, tandem diabodies, and tandem trivalent scFv. Unless otherwise indicated, the term "antibody" is to be understood as encompassing functional antibody fragments thereof. The term also encompasses whole or full length antibodies, including antibodies of any class or subclass, including IgG and subclasses thereof (IgG 1, igG2, igG3, igG 4), igM, igE, igA, and IgD.
The term "V L "or" VL "and" V H "or" VH "refers to the variable binding regions from the antibody light chain and antibody heavy chain, respectively. In certain embodiments, VL is of the kappa (kappa) class (also referred to herein as "VK"). In certain embodiments, VL is a lambda (lambda) class. Variable binding regions include discrete, well-defined sub-regions known as "complementarity determining regions" (CDRs) and "framework regions" (FR). The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and refer to amino acid sequences within the variable region of an antibody, which together typically confer antigen specificity and/or binding affinity to the antibody, wherein consecutive CDRs (i.e., CDR1 and CDR2, CDR2 and CDR 3) are separated from each other by a framework region in a primary structure. Each variable region has three CDRs (HCDR 1, HCDR2, HCDR3; LCDR1, LCDR2, LCDR3; also referred to as CDRH and CDRL, respectively). In certain embodiments, the antibody VH comprises four FRs and three CDRs as follows: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; and the antibody VL includes four FRs and three CDRs as follows: FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4. Typically, VH and VL together form an antigen binding site through their respective CDRs. In certain embodiments, one or more CDRs are not contacted with antigen and/or do not contribute energy to antigen binding.
As used herein, a "variant" of a CDR refers to a functional variant of a CDR sequence having up to 1-3 amino acid substitutions (e.g., conservative or non-conservative substitutions), deletions, or combinations thereof.
The numbering of the CDRs and framework regions can be according to any known method or protocol, such as Kabat, chothia, EU, IMGT, contact, north, martin and the AHo numbering scheme (see, e.g., kabat et al, protein sequence of interest in immunology (Sequences of Proteins of Immunological Interest), U.S. department of health and human services (US Dept. Health and Human Services), U.S. national institutes of health public health service (Public Health Service National Institutes of Health), 1991, 5 th edition, chothia and Lesk, journal of molecular biology (J.mol. Biol.) 196:901-917 (1987), lefranc et al, development and comparison immunology (dev. Comp. Immunol.). 27:55,2003, honeygger and Plu ckthun, 309:657-670 (2001), north et al, journal of molecular biology (2011) 406:228-56, doi:10.1016/j.jmb.10.2010.84, and immunology (U.S. 022.84, ml.) (2008) molecular biological journal of molecular biology (2008) 309:657-670, and molecular weight of other materials). The antibody and CDR numbering systems of these references are incorporated herein by reference. Equivalent residue positions can be noted using the antigen receptor numbering and receptor classification (Antigen receptor Numbering And Receptor Classification, ANARCI) software tools (2016, bioinformatics (Bioinformatics) 15:298-300) and used to compare different molecules. Thus, identifying CDRs of an exemplary variable domain (VH or VL) sequence as provided herein according to one numbering scheme does not exclude antibodies comprising CDRs of the same variable domain as determined using a different numbering scheme. In certain embodiments, the antibodies of the disclosure are capable of neutralizing an infection caused by influenza. As used herein, a "neutralizing antibody" is an antibody that can neutralize, i.e., prevent, inhibit, reduce, hinder, or interfere with the ability of a pathogen to initiate and/or perpetuate an infection in a host. The terms "neutralizing antibody (neutralizing antibody)" and "neutralizing antibody (an antibody that neutralizes/antibodies that neutralize)" are used interchangeably herein. In any of the presently disclosed embodiments, the antibody or antigen binding fragment is capable of preventing and/or neutralizing influenza infection in an in vitro infection model and/or an in vivo infection animal model and/or in a human. In certain embodiments, the antibody or antigen binding fragment thereof is human, humanized or chimeric.
In some embodiments, the CDRs are according to the IMGT numbering system.
In certain embodiments, (1) an anti-HA antibody or antigen-binding fragment comprises a heavy chain variable domain (VH) comprising or consisting of Complementarity Determining Regions (CDRs) H1, CDRH2, and CDRH3, and a light chain variable domain (VL) comprising or consisting of CDRL1, CDRL2, and CDRL3, (1) (i) the CDRH1 comprises or consists of an amino acid sequence as set forth in any one of: 3, 32 or 15, or a functional variant thereof, or consisting of one, two or three acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (1) (ii) the CDRH2 comprises or consists of an amino acid sequence as set forth in any one of: 4, 29, 35, 16 or 42, or a functional variant thereof, or consisting of one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (1) (iii) the CDRH3 comprises or consists of an amino acid sequence as set forth in any one of: 5 or 17, or a functional variant thereof, comprising or consisting of one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (1) (iv) the CDRL1 comprises or consists of an amino acid sequence as set forth in any one of: 9 or 21, or a functional variant thereof, comprising or consisting of one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (1) (v) the CDRL2 optionally comprises or consists of an amino acid sequence as set forth in any one of: 10 or 22, or a functional variant thereof, or consisting of one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (1) (vi) the CDRL3 comprises or consists of an amino acid sequence as set forth in any one of: 11 or 23, or a functional variant thereof, comprising or consisting of a substitution of one, two or three amino acids, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (2) the anti-NA antibody or antigen-binding fragment comprises a VH comprising CDRH1, CDRH2 and CDRH3 and a VL comprising CDRL1, CDRL2 and CDRL3, wherein CDRs are determined according to the IMGT numbering system, and wherein: (2) (i) optionally the CDRH1 comprises or consists of an amino acid sequence as set forth in any one of: 49, 61, 73, 85, 97, 109, 121, 133, 145, 157, 169, 181, 193 or 205, or a functional variant thereof comprising or consisting of one, two or three acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; (2) (ii) optionally the CDRH2 comprises or consists of an amino acid sequence as set forth in any one of: 50, 62, 74, 86, 98, 110, 122, 134, 146, 158, 170, 182, 194 or 206, or a functional variant thereof comprising or consisting of one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; (2) (iii) the CDRH3 comprises or consists of an amino acid sequence as set forth in any one of: 51, 63, 75, 218, 87, 99, 111, 123, 135, 230, 147, 159, 171, 183, 195 or 207, or a functional variant thereof comprising or consisting of one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; (2) (iv) optionally the CDRL1 comprises or consists of an amino acid sequence as set forth in any one of the following: 55, 67, 79, 91, 103, 115, 127, 139, 151, 163, 175, 187, 199, or 211, or a functional variant thereof comprising or consisting of one, two, or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; (2) (v) optionally the CDRL2 comprises or consists of an amino acid sequence as set forth in any one of: 56, 68, 80, 92, 104, 116, 128, 140, 152, 164, 176, 188, 200 or 212, or a functional variant thereof comprising or consisting of one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (2) (vi) optionally the CDRL3 comprises or consists of an amino acid sequence as shown in any one of: 57, 69, 81, 221, 224, 227, 93, 105, 117, 129, 141, 233, 239, 153, 165, 177, 189, 201, 236 or 213, or a functional variant thereof or consisting of it, said functional variant comprising a substitution with one, two or three amino acids, one or more of said substitutions optionally being a conservative substitution and/or a substitution of a germline encoded amino acid.
In further embodiments, the anti-HA antibody or antigen-binding fragment of (1) comprises the following CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences: (1) (i) SEQ ID NOS 3-5 and 9-11, respectively; (1) (ii) SEQ ID NOs 3, 29, 5 and 9-11, respectively; (1) (iii) SEQ ID NOs 32, 4, 5 and 9-11, respectively; (1) (iv) SEQ ID NOs 3, 35, 5 and 9-11, respectively; (1) (v) SEQ ID NOs 32, 35, 5 and 9-11, respectively; (1) (vi) SEQ ID NOS 15-17 and 21-23, respectively; or (1) (vii) SEQ ID NOs 15, 42, 17 and 21-23, respectively; and/or (2) the anti-NA antibody or antigen-binding fragment comprises the following CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences: (2) (i) SEQ ID NOS.49-51 and 55-57, respectively; (2) (ii) SEQ ID NOS: 61-63 and 67-69, respectively; (2) (iii) SEQ ID NOS: 73-75 and 79-81, respectively; (2) (iv) SEQ ID NOS 73, 74, 218 and 79-81, respectively; (2) (v) SEQ ID NOS 73-75, 79, 80 and 221, respectively; (2) (vi) SEQ ID NOS 73-75, 79, 80 and 224, respectively; (2) (vii) SEQ ID NOS 73-75, 79, 80 and 227, respectively; (2) (viii) SEQ ID NOs 73, 74, 218, 79, 80 and 221, respectively; (2) (ix) SEQ ID NOs 73, 74, 218, 79, 80 and 224, respectively; (2) (x) SEQ ID NOs 73, 74, 218, 79, 80 and 227, respectively; (2) (xi) SEQ ID NOS 85-87 and 91-93, respectively; (2) (xii) SEQ ID NOS 97-99 and 103-105, respectively; (2) (xiii) SEQ ID NOS 109-111 and 115-117, respectively; (2) (xiv) SEQ ID NOS 121-123 and 127-129, respectively; (2) (xv) SEQ ID NOS 133-135 and 139-141, respectively; (2) (xvi) SEQ ID NOS 133, 134, 230 and 139-141, respectively; (2) (xvii) SEQ ID NOS 133-135, 139, 141 and 233, respectively; (2) (xviii) SEQ ID NOS 133-135, 139, 141 and 236, respectively; (2) (xix) SEQ ID NOS 133-135, 139, 141 and 239, respectively; (2) (xx) SEQ ID NOS: 133, 134, 184, 139, 141 and 233, respectively; (2) (xxi) SEQ ID NOs 133, 134, 184, 139, 141 and 236, respectively; (2) (xxii) SEQ ID NOs 133, 134, 184, 139, 141 and 239, respectively; (2) (xxiii) SEQ ID NOS 145-147 and 151-153, respectively; (2) (xxiv) SEQ ID NOS 157-159 and 163-165, respectively; (2) (xxv) SEQ ID NOS 169-171 and 175-177, respectively; (2) (xxvi) SEQ ID NOS 181-183 and 187-189, respectively; (2) (xxvii) SEQ ID NOS 193-195 and 199-201, respectively; or (2) (xxviii) SEQ ID NOS: 205-207 and 211-213, respectively.
In some embodiments, the anti-HA antibody or antigen-binding fragment of (1) comprises the following CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences: (1) (i) SEQ ID NOS 3-5 and 9-11, respectively; (1) (ii) SEQ ID NOs 3, 29, 5 and 9-11, respectively; (1) (iii) SEQ ID NOs 32, 4, 5 and 9-11, respectively; (1) (iv) SEQ ID NOs 3, 35, 5 and 9-11, respectively; (1) (v) SEQ ID NOs 32, 35, 5 and 9-11, respectively; (1) (vi) SEQ ID NOS 15-17 and 21-23, respectively; or (1) (vii) SEQ ID NOs 15, 42, 17 and 21-23, respectively; and/or (2) the anti-NA antibody or antigen-binding fragment comprises the following CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences: (2) (i) SEQ ID NOS: 73-75 and 79-81, respectively; (2) (ii) SEQ ID NOS 73, 74, 218 and 79-81, respectively; (2) (iii) SEQ ID NOS 73-75, 79, 80 and 221, respectively; (2) (iv) SEQ ID NOS 73-75, 79, 80 and 224, respectively; (2) (v) SEQ ID NOS 73-75, 79, 80 and 227, respectively; (2) (vi) SEQ ID NOs 73, 74, 218, 79, 80 and 221, respectively; (2) (vii) SEQ ID NOs 73, 74, 218, 79, 80 and 224, respectively; (2) (viii) SEQ ID NOs 73, 74, 218, 79, 80 and 227, respectively; (2) (ix) SEQ ID NOS 133-135 and 139-141, respectively; (2) (x) SEQ ID NOs 133, 134, 230 and 139-141, respectively; (2) (xi) SEQ ID NOS 133-135, 139, 141 and 233, respectively; (2) (xii) SEQ ID NOS 133-135, 139, 141 and 236, respectively; (2) (xiii) SEQ ID NOS 133-135, 139, 141 and 239, respectively; (2) (xiv) SEQ ID NOS 133, 134, 184, 139, 141 and 233, respectively; (2) (xv) SEQ ID NOS 133, 134, 184, 139, 141 and 236, respectively; or (2) (xvi) SEQ ID NOS: 133, 134, 184, 139, 141 and 239, respectively.
In certain embodiments, an antibody or antigen binding fragment is provided that includes a VH sequence according to any of SEQ ID NOs 2, 14, 26, 171, 38, 50, 62, 74, 86, 183, 98, 110, 122, 134, 146, and 158 and a CDR in a VL sequence according to any of SEQ ID NOs 26, 36, 46, 56, 66, 76, 86, 96, 8, 20, 32, 44, 56, 68, 80, 92, 104, 116, 128, 140, 152, 174, 177, 180, 186, 189, 192, and 164 as determined using any known CDR numbering method (including Kabat, chothia, EU, IMGT, martin (enhanced Chothia), contact, and AHo numbering methods). In certain embodiments, the CDRs are according to IMGT numbering methods. In certain embodiments, the CDRs are according to an antibody numbering method developed by the stoichiometric group (CCG); for example, molecular Operating Environment (MOE) software (www.chemcomp.com) is used.
In some embodiments, the anti-HA antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3 of the VH amino sequence shown in SEQ ID NO. 43, and CDRL1, CDRL2 and CDRL3 of the VL amino acid sequence shown in SEQ ID NO. 44. In further embodiments, the anti-HA antibody or antigen-binding fragment comprises a VH shown in SEQ ID NO. 43 and a VL shown in SEQ ID NO. 44.
In some embodiments, the anti-NA antibody or antigen-binding fragment includes CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of antibody 1G01, as shown in FIG. 1B of Stadlebaeur et al science 366 (6464): 466-504 (2019), the amino acid sequences of which are incorporated herein by reference. In further embodiments, the anti-NA antibody or antigen-binding fragment includes the VH and VL of antibody 1G01, as shown in FIG. 1B of Stadlebaeur et al science 366 (6464): 466-504 (2019), the amino acid sequences of which are incorporated herein by reference.
In certain embodiments, (1) the anti-HA antibody or antigen-binding fragment comprises: (1) (i) a VH comprising or consisting of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to an amino acid sequence set forth in any one of: 2, 26, 28, 31, 34, 37, 14, 39, 41, and 43, wherein the sequence variations referring to SEQ ID NOs 2, 26, 28, 31, 34, 37, 14, 39, 41, or 43, respectively, are optionally included in one or more framework regions and/or the sequence variations include substitution of one or more germline encoded amino acids; and/or (1) (ii) VL comprises or consists of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to an amino acid sequence of any one of: 8, 20 or 44, wherein the sequence variation relative to SEQ ID NO 8, 20 or 44, respectively, is optionally comprised in one or more framework regions and/or the sequence variation comprises a substitution of one or more germline encoded amino acids; and/or (2) the anti-NA antibody or antigen-binding fragment comprises: (2) (i) a VH comprising or consisting of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to an amino acid sequence set forth in any one of: 48, 60, 72, 171, 84, 96, 108, 120, 132, 229, 144, 156, 168, 180, 192, 204, 241, 245 and 249, wherein the sequence variations of SEQ ID NOs 48, 60, 72, 171, 84, 96, 108, 120, 132, 229, 144, 156, 168, 180, 192, 204, 241, 245 and 249 are optionally included in one or more framework regions, and/or the sequence variations include substitutions of one or more germline encoded amino acids; and/or (2) (ii) the VL comprises or consists of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to an amino acid sequence of any one of: the sequence variations of SEQ ID NOs 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 220, 223, 226, 232, 235, 238, 210, 243, 247 and 251, respectively, wherein the sequence variations of SEQ ID NOs 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 220, 223, 226, 232, 235, 238, 210, 243, 247 and 251 are optionally included in one or more framework regions and/or the sequence variations comprise substitutions of one or more germline encoded amino acids.
In particular embodiments, the VH is encoded by or derived from VH6-1, DH3-3 and/or JH 6.
In some embodiments, (1) the VH and the VL of the anti-HA antibody or antigen-binding fragment comprise or consist of amino acid sequences according to seq id no: (1) (i) SEQ ID NOs 2 and 8, respectively; (1) (ii) SEQ ID NOS 26 and 8, respectively; (1) (iii) SEQ ID NOS 28 and 8, respectively; (1) (iv) SEQ ID NOs 31 and 8, respectively; (1) (v) SEQ ID NOS 34 and 8, respectively; (1) (vi) SEQ ID NOS 37 and 8, respectively; (1) (vii) SEQ ID NOs 14 and 20, respectively; (1) (viii) SEQ ID NOs 39 and 20, respectively; (1) (ix) SEQ ID NOs 41 and 20, respectively; or (1) (x) SEQ ID NOs 43 and 44, respectively; and/or (2) the VH and the VL of the anti-NA antibody or antigen-binding fragment comprise or consist of amino acid sequences according to seq id no: (2) (i) SEQ ID NOs 48 and 54, respectively; (2) (ii) SEQ ID NOs 60 and 66, respectively; (2) (iii) SEQ ID NOs 72 and 78 or 220 or 223, respectively; (2) (vi) SEQ ID NOS 72 and 226, respectively; (2) (vii) SEQ ID NOS 217 and 78, respectively; (2) (viii) SEQ ID NOS 217 and 220, respectively; (2) (ix) SEQ ID NOS 217 and 223, respectively; (2) (x) SEQ ID NOS 217 and 226, respectively; (2) (xi) SEQ ID NOs 84 and 90, respectively; (2) (xii) SEQ ID NOs 96 and 102, respectively; (2) (xiii) SEQ ID NOs 108 and 114, respectively; (2) (xiv) SEQ ID NOS 120 and 126, respectively; (2) (xv) SEQ ID NOS 132 and 138, respectively; (2) (xvi) SEQ ID NOS 132 and 232, respectively; (2) (xvii) SEQ ID NOS 132 and 235, respectively; (2) (xviii) SEQ ID NOS 132 and 238, respectively; (2) (xix) SEQ ID NOs 229 and 138, respectively; (2) (xx) SEQ ID NOs 229 and 232, respectively; (2) (xxi) SEQ ID NOs 229 and 235, respectively; (2) (xxii) SEQ ID NOs 229 and 238, respectively; (2) (xxiii) SEQ ID NOS 144 and 150, respectively; (2) (xxiv) SEQ ID NOS 156 and 162, respectively; (2) (xxv) SEQ ID NOS 168 and 174, respectively; (2) (xxvi) SEQ ID NOS 180 and 186, respectively; (2) (xxvii) SEQ ID NOS 192 and 198, respectively; (2) (xxviii) SEQ ID NOS 204 and 210, respectively; (2) (xxix) SEQ ID NOs 241 and 243, respectively; (2) (xxx) SEQ ID NOs 245 and 247, respectively; or (2) (xxxi) SEQ ID NOS 249 and 251, respectively.
In certain embodiments, (1) the VH and the VL of the anti-HA antibody or antigen-binding fragment comprise or consist of amino acid sequences according to seq id no: (1) (i) SEQ ID NOs 2 and 8, respectively; (1) (ii) SEQ ID NOS 26 and 8, respectively; (1) (iii) SEQ ID NOS 28 and 8, respectively; (1) (iv) SEQ ID NOs 31 and 8, respectively; (1) (v) SEQ ID NOS 34 and 8, respectively; (1) (vi) SEQ ID NOS 37 and 8, respectively; (1) (vii) SEQ ID NOs 14 and 20, respectively; (1) (viii) SEQ ID NOs 39 and 20, respectively; (1) (ix) SEQ ID NOs 41 and 20, respectively; or (1) (x) SEQ ID NOs 43 and 44, respectively; and/or (2) the VH and the VL of the anti-NA antibody or antigen-binding fragment comprise or consist of amino acid sequences according to seq id no: (2) (i) SEQ ID NOs 72 and 78 or 220 or 223, respectively; (2) (ii) SEQ ID NOs 72 and 226, respectively; (2) (iii) SEQ ID NOS 217 and 78, respectively; (2) (iv) SEQ ID NOS 217 and 220, respectively; (2) (v) SEQ ID NOS 132 and 138, respectively; (2) (vi) SEQ ID NOS 132 and 232, respectively; (2) (vii) SEQ ID NOs 132 and 235, respectively; (2) (viii) SEQ ID NOS 132 and 238, respectively; (2) (ix) SEQ ID NOs 229 and 138, respectively; (2) (x) SEQ ID NOs 229 and 232, respectively; (2) (xi) SEQ ID NOs 229 and 235, respectively; (2) (xii) SEQ ID NOs 229 and 238, respectively; (2) (xiii) SEQ ID NOS 217 and 223, respectively; (2) (xiv) SEQ ID NOS 217 and 226, respectively; (2) (xv) SEQ ID NOs 241 and 243, respectively; (2) (xvi) SEQ ID NOS 245 and 247, respectively; or (2) (xvii) SEQ ID NOS 249 and 251, respectively.
In certain embodiments, the NA is N1, N2, and/or N9.
In certain embodiments, the antibody or antigen binding fragment is capable of binding to: (1) An NA epitope comprising any one or more of the following amino acids (N1 NA numbering): r368, R293, E228, E344, S247, D198, D151, R118; and/or (2) an NA epitope comprising any one or more of the following amino acids (N2 NA numbering): r371, R292, E227, E344, S247, D198, D151, R118. It will be appreciated that the antibodies and antigen binding fragments may also bind to influenza neuraminidase which may not follow the numbering convention of N1 or N2 amino acids; the amino acids of these epitopes may correspond to the N1 or N2 amino acid residues indicated herein, as by being identical at positions that are equivalent in NA (e.g., by arrangement, 3-D structure, conservation, or a combination of these) but numbered differently. Thus, references to N1 or N2 numbering will be understood as amino acids corresponding to the recited amino acids.
Examples showing the N1 and N2 position numbers are provided in table 2 (using h1n1_california. 07.2009 and h3n2_new york. 392.2004).
In certain embodiments, the antibody or antigen binding fragment is capable of binding to: (1) NA epitopes including amino acids R368, R293, E228, D151 and R118 (N1 NA numbering); and/or (2) an NA epitope comprising amino acids R371, R292, E227, D151 and R118 (N2 NA numbering).
In certain embodiments, the antibody or antigen binding fragment is capable of binding to an epitope included in or included in an NA active site (as described herein, the NA active site includes functional amino acids that form the catalytic core and directly contact sialic acid, and structural amino acids that form the active site framework), wherein optionally the NA active site includes the following amino acids (N2 numbering): r118, D151, R152, R224, E276, R292, R371, Y406, E119, R156, W178, S179, D/N198, I222, E227, H274, E277, D293, E425. In certain embodiments, R118, D151, R152, R224, E276, R292, R371, and Y406 form the catalytic core and directly contact sialic acid. In certain embodiments, E119, R156, W178, S179, D/N198, I222, E227, H274, E277, D293, and E425 form an active site framework.
In certain embodiments, the epitope comprises or further comprises any one or more of the following NA amino acids (N2 numbering): e344, E227, S247, and D198.
In certain embodiments, the antibody or antigen binding fragment is capable of binding to NA comprising an S245N amino acid mutation and/or an E221D amino acid mutation (N2 numbering).
In certain embodiments, the NA comprises IBV NA. In certain embodiments, the antibody or antigen binding fragment is capable of binding to an IBV NA epitope comprising any one or more of the following amino acids (IBV numbering; e.g., for influenza b virus Victoria (FluB Victoria) and influenza b virus yamadata)): r116, D149, E226, R292, and R374. In some embodiments, the epitope comprises amino acids R116, D149, E226, R292, and R374.
In certain embodiments, (i) the group 1IAV NA comprises H1N1 and/or H5N1; (ii) the group 2IAV NA comprises H3N2 and/or H7N9; and/or
(iii) The IBV NA includes one or more of the following: B/Li/10/1940 (ancestor);
B/Taiwan/2/1962 (ancestor); b/brisbane/33/2008 (victoria); b/brisbane/60/2008 (victoria); B/Malaysia/2506/2004 (Victoria); B/New York/1056/2003 (Victoria); B/Florida/4/2006 (mountain county); and B/Jiangsu/10/2003 (mountain county).
In certain embodiments, the anti-HA antibody or antigen-binding fragment is capable of binding to any two or more of the following IAV subtypes: h1, H2, H3, H4, H5, H8, H9, H10, H11, H12, H13, H14, H15, H17 and H18.
In certain embodiments, the anti-HA antibody or antigen-binding fragment is capable of neutralizing an infection caused by: (i) An H1N 1IAV, wherein optionally the H1N 1IAV comprises any one or more of: a/california/07/2009, a/PR/8/34 and a/solomon islands/3/06; and (ii) an H3N2IAV, wherein optionally the H3N2IAV comprises any one or more of: A/Aizhi/2/68, A/Brisban/10/07 and A/China hong Kong/68; (i) A group 1IAV, wherein optionally said group 1IAV comprises or is an H5 IAV, wherein further optionally said H5 IAV comprises or is an H5/VN/11/94pp; and (ii) a group 2IAV, wherein optionally the group 2IAV comprises or is an H7 IAV, wherein further optionally the H7 IAV comprises or is an H7/IT/99pp, wherein optionally neutralization of infection is determined using a virus pseudotyped with the IAV.
In certain embodiments, the HA comprises: (i) H1 HA, the H1 HA optionally comprising any one or more of: a/england/195/2009; A/Brisban/59/2007; A/Solomon islands/3/2006; A/New karilidoria/20/99; A/Texas state/36/1991; A/Taiwan/01/1986; A/New Jersey state/8/1976; A/Albani/12/1951; A/Mono Sturg/1/1947; A/New York/1/1918; A/Paris/8/34; a/california/07/2009; (ii) H2 HA, said H2 HA optionally comprising a/japan/305/1957; (iii) H5 HA, optionally comprising a/vietnam/1194/2004; and (iv) H9 HA, optionally including A/China hong Kong/1073/99.
In any of the presently disclosed combinations, compositions, methods and uses, (i) the group 1IAV NA may include N1, N4, N5 and/or N8; and/or (ii) the group 2IAV NA may comprise N2, N3, N6, N7, and/or N9. In certain embodiments, (i) the N1 is N1 from any one or more of: a/california/07/2009, a/california/07/2009I 23R/H275Y, A/pig/Jiangsu/J004/2018, a/stong mole/18/2007, a/brisban/02/2018, a/michigan/45/2015, a/michibizhou/3/2001, a/netherlands/603/2009, and a/new jersey/8/1976; (ii) the N4 is from a/green duck/netherlands/30/2011; (iii) the N5 is from a/waterfowl/korea/CN 5/2009; (iv) The N8 is from a/plaque seal/new hampshire state/179629/2011; (v) The N2 is N2 from any one or more of the following: A/Washington/01/2007, A/China hong Kong/68, A/south Australia/34/2019, A/Switzerland/8060/2017, A/Singapore/INFIMH-16-0019/2016, A/Switzerland/9715293/2013, A/Ninggeler/134/17/57, A/Florida/4/2006, A/Netherlands/823/1992, A/Norway/466/2014, A/Switzerland/8060/2017, A/Texas/50/2012, and A/Victoria/361/2011; (vi) said N3 is from a/canada/rv 504/2004; (v) said N6 is from a/pig/ampere/01911/1/99; (vi) said N7 is from a/netherlands/078/03; and/or (vii) the N9 is from a/security badge/2013.
In any of the presently disclosed combinations, compositions, methods and uses, the IBV NA is NA from any one or more of the following: B/Li/10/1940 (ancestor); b/brisbane/60/2008 (victoria); B/Malaysia/2506/2004 (Victoria); B/Malaysia/3120318925/2013 (mountain county); b/wisconsin/1/2010 (mountain county); B/Sorby county/166/1998 (mountain county); B/Brisbane/33/2008; b/state of cororado/06/2017; B/Hubei-Wujiang/158/2009; b/massachusetts/02/2012; B/Netherlands/234/2011; B/Perss/211/2001; and B/pray island/3073/2013.
In any of the presently disclosed combinations, compositions, methods and uses, the NA is N1, N2 and/or N9.
The term "CL" refers to either an "immunoglobulin light chain constant region (immunoglobulin light chain constant region)" or a "light chain constant region (light chain constant region)", i.e., a constant region from an antibody light chain. The term "CH" refers to an "immunoglobulin heavy chain constant region" or "heavy chain constant region" which can be further divided into CH1, CH2 and CH3 (IgA, igD, igG) or CH1, CH2, CH3 and CH4 domains (IgE, igM) depending on the antibody isotype. The Fc region of the antibody heavy chain is further described herein. In any of the presently disclosed embodiments, the antibody or antigen binding fragment of the disclosure includes any one or more of CL, CH1, CH2, and CH 3.
In any of the presently disclosed embodiments, the antibody or antigen binding fragment of the present disclosure is directed against a subject comprising any one or more of CL, CH1, CH2, and CH 3. In certain embodiments, CL includes an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO. 254. In certain embodiments, CH1-CH2-CH3 comprises an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:280 or SEQ ID NO: 281. It will be appreciated that the production in mammalian cell lines, for example, may remove one or more C-terminal lysines of the antibody heavy chain (see, e.g., liu et al, mAb 6 (5): 1145-1154 (2014)). Thus, an antibody or antigen binding fragment of the present disclosure may comprise a heavy chain, CH1-CH3, or Fc polypeptide, with or without the presence of a C-terminal lysine residue; in other words, embodiments are contemplated in which the C-terminal residue of the heavy chain, CH1-CH3, or Fc polypeptide is not lysine, as well as embodiments in which lysine is the C-terminal residue. In certain embodiments, the compositions comprise a plurality of antibodies and/or antigen-binding fragments of the present disclosure, wherein one or more antibodies or antigen-binding fragments do not comprise a lysine residue at the C-terminus of the heavy chain, CH1-CH3, or Fc polypeptide, and wherein one or more antibodies or antigen-binding fragments comprise a lysine residue at the C-terminus of the heavy chain, CH1-CH3, or Fc polypeptide.
"Fab" (antigen binding fragment) is a portion of an antibody that binds to an antigen and includes the variable region of the heavy chain and CH1 linked to the light chain by an interchain disulfide bond. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen binding site. Pepsin treatment of the antibodies produced a single large F (ab') 2 fragment that approximately corresponded to two disulfide-linked Fab fragments with bivalent antigen binding activity and was still able to crosslink the antigen. Fab and F (ab') 2 are examples of "antigen binding fragments". Fab' fragments differ from Fab fragments by having an additional small number of residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab '-SH is herein the name for Fab' in which the cysteine residue of the constant domain bears a free thiol group. F (ab ') 2 antibody fragments were originally generated as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
Fab fragments may be linked, for example, by a peptide linker to form a single chain Fab, also referred to herein as "scFab". In these embodiments, the interchain disulfide bonds present in the native Fab may not be present and the linker is used, in whole or in part, to join (link/connect) Fab fragments in a single polypeptide chain. Heavy chain-derived Fab fragments (e.g., comprising, consisting of, or consisting essentially of vh+ch1 or "Fd") and light chain-derived Fab fragments (e.g., comprising, consisting of, or consisting essentially of vl+cl) may be linked in any arrangement to form scFab. For example, scFab may be arranged in an N-to C-terminal orientation according to (heavy chain Fab fragment-linker-light chain Fab fragment) or (light chain Fab fragment-linker-heavy chain Fab fragment). Peptide linkers and exemplary linker sequences for scFab are discussed in further detail herein.
"Fv" is a small antibody fragment containing complete antigen recognition and antigen binding sites. This fragment generally consists of a dimer of one heavy chain and one light chain variable region domain in close non-covalent association. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, but typically has less affinity than the complete binding site.
"Single chain Fv" also abbreviated "sFv" or "scFv" is a polypeptide comprising V linked into a single polypeptide chain H Antibody domains and V L Antibody fragments of antibody domains. In some embodiments, the scFv polypeptide comprises a polypeptide disposed at V H And V is equal to L Polypeptide linkers between and linking the domains that enable the scFv to retain or form the desired structure for antigen binding. Such peptide linkers can be incorporated into fusion polypeptides using standard techniques well known in the art. For reviews of scFv, see Pluckaphun, volume 113, rosenburg and Moore editions, springer-Verlag, new York, pages 269-315 (1994); borrebaeck 1995, see below. In certain embodiments, the antibody or antigen-binding fragment comprises an scFv comprising a VH domain, a VL domain, and a peptide linker connecting the VH domain to the VL domain. In particular embodiments, the scFv comprises a VH domain linked to a VL domain by a peptide linker, which may be in a VH-linker-VL orientation or in a VL-linker-VH orientation. Any scFv of the disclosure can be engineered such that the C-terminus of the VL domain is linked to the N-terminus of the VH domain by a short peptide sequence, and vice versa (i.e., (N) VL (C) -linker- (N) VH (C) or (N) VH (C) -linker- (N) VL (C). Alternatively, in some embodiments, the linker can be linked to the N-terminal portion or end of the VH domain, the VL domain, or both.
The peptide linker sequence may be selected, for example, based on: (1) it can adopt a flexible extended conformation; (2) They cannot or lack the ability to employ secondary structures that can interact with functional epitopes on the first polypeptide and the second polypeptide and/or on the target molecule; and/or (3) lack or relative lack of hydrophobic or charged residues that may react with the polypeptide and/or target molecule. Other considerations regarding linker design (e.g., length) may include the conformation or range of conformations in which VH and VL may form functional antigen-binding sites. In certain embodiments, the peptide linker sequence contains, for example, gly, asn, and Ser residues. Other near neutral amino acids, such as Thr and Ala, may also be included in the linker sequence. Other amino acid sequences that may be usefully employed as linkers include those disclosed in the following: maratea et al, gene 40:3946,1985; murphy et al, proc.83:8258 8262 (1986) of the national academy of sciences USA; us patent 4,935,233 and us patent 4,751,180. Other illustrative and non-limiting examples of linkers may include, for example, glu-Gly-Lys-Ser-Ser-Gly-Ser-Ser-Glu-Ser-Lys-Val-Asp (Chaudhary et al, proc. Natl. Acad. Sci. USA 87:1066-1070 (1990)) and Lys-Glu-Ser-Gly-Ser-Glu-Gln-Leu-Ala-Gln-Phe-Arg-Ser-Leu-Asp (Bird et al, science 242:423-426 (1988)) and pentamer Gly-Gly-Gly-Gly-Ser when present in a single iteration or repeated 1 to 5 or more times. Any suitable linker may be used and will generally be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 15 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100 amino acids in length, or less than about 200 amino acids in length, and will preferably comprise a flexible structure (which may provide flexibility and space for conformational movement between two regions, domains, motifs, fragments or modules connected by a linker), and will preferably be biologically inert and/or have a low risk of immunogenicity in humans.
scFv can be constructed using any combination of VH and VL sequences disclosed herein or any combination of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences.
In some embodiments, for example, when the first polypeptide and the second polypeptide have nonessential N-terminal amino acid regions that can be used to separate functional domains and prevent steric interference, no linker sequence is required.
During antibody development, DNA in germline variable (V), junction (J) and diversity (D) loci may rearrange and insertion and/or deletion of nucleotides in the coding sequence may occur. Somatic mutations can be encoded by the resulting sequences and can be identified by reference to corresponding known germline sequences. In some cases, somatic mutations that are not important for the desired property of the antibody (e.g., binding to influenza NA antigen) or that confer an undesired property to the antibody (e.g., increased risk of immunogenicity in the subject to whom the antibody is administered), or both, may be replaced with a corresponding germline encoded amino acid or a different amino acid such that the desired property of the antibody is improved or maintained and the undesired property of the antibody is reduced or eliminated. Thus, in some embodiments, an antibody or antigen-binding fragment of the disclosure comprises at least one or more germline-encoding amino acid in the variable region as compared to the parent antibody or antigen-binding fragment, provided that the parent antibody or antigen-binding fragment comprises one or more somatic mutations.
In certain embodiments, the antibodies or antigen binding fragments of the present disclosure are monospecific (e.g., bind to a single epitope) or multispecific (e.g., bind to multiple epitopes and/or target molecules). In some embodiments, the multispecific antibody or antigen-binding fragment comprises a binding domain specific for an HA antigen and a binding domain specific for an NA antigen. For example, binding domains comprising CDRs and/or VH and VL from any of the anti-HA antibodies disclosed herein can be used in multispecific antibodies. Antibodies and antigen binding fragments can be constructed in a variety of forms. Exemplary antibody formats are disclosed in Spiess et al, molecular immunology 67 (2): 95 (2015) and Brinkmann and Kontermann, mAb 9 (2): 182-212 (2017), which formats and methods for their preparation are incorporated herein by reference and include, for example, dual specificity T cell adaptors (BiTE), DART, pestle-cup (Knobs-in-Holes, KIH) assemblies, scFv-CH3-KIH assemblies, KIH plain light chain antibodies, tandAb, triple antibody (Triple Body), triBi miniantibodies, fab-scFv, scFv-CL-scFv, F (ab') 2-scFv2, tetravalent HCab, intracellular antibodies, crossMab, dual Action Fab (DAF) (two-in-one or four-in-one), dutaMab, DT-IgG, charge Pair (large Pair), fab arm Exchange (SEFab-m Exchange), triomp-Z-Y, igG, scFv-Fv, scFv, ig-L (see-L), scFv-L, scFv (see-L), scFv-L (see, H), scFv-L (see, F) and so-called four-called (see-called) formats, e.g., see, 6 (see-4, F, 35) and (see-about (see-4) and (see-about) by reference, PCT publication No. WO 2015/103072, which is incorporated herein by reference In its entirety), the so-called WuxiBody form (e.g., PCT publication No. WO 2019/057122, which is incorporated herein by reference In its entirety), and the so-called Elbow Insert Ig form (In-Elbow-Insert Ig format, IEI-Ig; PCT publications No. WO 2019/024979 and No. WO 2019/025391, which are incorporated herein by reference in their entirety).
In certain embodiments, an antibody or antigen-binding fragment comprises two or more VH domains, two or more VL domains, or both (i.e., two or more VH domains and two or more VL domains). In particular embodiments, the antigen-binding fragment comprises the form (N-terminal to C-terminal direction) VH-linker-VL-linker-VH-linker-VL, wherein the two VH sequences may be the same or different and the two VL sequences may be the same or different. Such linked scFv may comprise any combination of VH and VL domains arranged to bind to a given target, and may bind to one, two or more different epitopes or antigens in a format comprising two or more VH and/or two or more VL. It will be appreciated that the form incorporating a plurality of antigen binding domains may comprise VH and/or VL sequences in any combination or orientation. For example, an antigen-binding fragment may include the form VL-linker-VH-linker-VL-linker-VH, VH-linker-VL-linker-VH or VL-linker-VH-linker-VL.
The monospecific or multispecific antibodies or antigen-binding fragments of the present disclosure that are constructed include any combination of VH and VL sequences disclosed herein and/or any combination of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences. In some embodiments, a bispecific or multispecific antibody or antigen-binding fragment may comprise one, two, or more antigen-binding domains of the present disclosure (e.g., VH and VL). There may be two or more binding domains that bind to the same or different NA epitopes, and in some embodiments, a bispecific or multispecific antibody or antigen-binding fragment as provided herein comprises an additional NA-specific binding domain and/or may comprise a binding domain that binds entirely to a different antigen or pathogen.
In any of the presently disclosed embodiments, the antibody or antigen-binding fragment may be multispecific; for example, bispecific, trispecific, and the like.
In a particular embodiment, the bispecific antibody is provided in the DVD-Ig format. In further embodiments, the DVD-Ig format bispecific antibody comprises a binding domain capable of specific binding to HA antigen and a binding domain capable of specific binding to NA antigen. In still further embodiments, the binding domain capable of specifically binding to the HA antigen comprises CDRs from the variable region amino acid sequences set forth in SEQ ID NOs 43 and 44, respectively, and/or comprises VH and VL according to said variable region amino acid sequences. In certain embodiments, binding domains capable of specifically binding to NA antigen include CDRs from and/or VHs and VL according to the variable region amino acid sequences set forth in SEQ ID NOS: 72 and 78, respectively, or SEQ ID NOS: 132 and 138, respectively, or SEQ ID NOS: 192 and 198, respectively, or SEQ ID NOS: 204 and 210, respectively, or SEQ ID NOS: 241 and 243. It should be appreciated that the anti-HA binding domain and the anti-NA binding domain may be present in any orientation or arrangement in a DVD-Ig bispecific antibody; for example, the anti-HA binding domain may be disposed N-terminal to the anti-NA binding domain, or the anti-NA binding domain may be disposed N-terminal to the anti-HA binding domain.
In other embodiments, the bispecific antibody is provided in the IEI-Ig format. In further embodiments, the IEI-Ig format bispecific antibody comprises a binding domain capable of specific binding to HA antigen and a binding domain capable of specific binding to NA antigen. In still other embodiments, the binding domain capable of specifically binding to the HA antigen comprises CDRs from and/or VH and VL according to the variable region amino acid sequences set forth in SEQ ID NOs 43 and 44, respectively. In certain embodiments, the binding domains capable of specifically binding to NA antigen comprise CDRs from the variable region amino acid sequences shown as SEQ ID NOS: 72 and 78, respectively, or SEQ ID NOS: 132 and 138, respectively, or SEQ ID NOS: 192 and 198, respectively, or SEQ ID NOS: 204 and 210, respectively, or SEQ ID NOS: 241 and 243, respectively, and/or comprise VH and VL according to the variable region amino acid sequences. It is understood that the anti-HA binding domain and the anti-NA binding domain may be present in any orientation or arrangement in the IEI-Ig bispecific antibody; for example, the anti-HA binding domain may be disposed in the VH-CH1 (or VL-CL 1) elbow region of an anti-NA Fab, or the anti-NA binding domain may be disposed in the VH-CH1 (or VL-CL 1) elbow region of an anti-HA Fab.
In certain embodiments, the antibody or antigen binding fragment comprises an Fc polypeptide or fragment thereof. An "Fc" fragment or Fc polypeptide includes the carboxy-terminal portions of two antibody H chains held together by disulfide bonds (i.e., the CH2 domain and CH3 domain of IgG). Fc may include dimers comprising two Fc polypeptides (i.e., two CH2-CH3 polypeptides). An antibody "effector function" refers to biological activity attributable to an antibody Fc region (native sequence Fc region or amino acid sequence variant Fc region) and varies with antibody isotype. Examples of antibody effector functions include: c1q binding and complement dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); b cell activation. As discussed herein, the Fc domain may be modified (e.g., amino acid substitutions) to modify (e.g., improve, reduce, or eliminate) one or more functions of an Fc-containing polypeptide (e.g., an antibody of the disclosure). Such functions include, for example, fc receptor (FcR) binding, antibody half-life modulation (e.g., by binding FcRn), ADCC function, protein a binding, protein G binding, and complement binding. Amino acid modifications that modify (e.g., improve, reduce or eliminate) Fc function include, for example, T250Q/M428L, M252Y/S254T/T256E, H K/N434F, M428L/N434S, E233P/L234V/L235A/G236+A327G/A330S/P331S, E333A, S D/A330L/I332E, P I/Q311, K326W/E333S, S D/I332E/G236A, N297Q, K A, S, 228P, L E+E318A/K320A/K322A, L234A/L235A (also referred to herein as "LALA") and L234A/L235A/P329G mutations, these mutations are summarized and annotated in the Engineered Fc region (Engineered Fc Regions) published by invitogen (2011) and can be obtained on-line at InvivoGen com/PDF/review/review-Engineered-Fc-Regions-InvivoGen.
For example, to activate the complement cascade, when immunoglobulin molecules are attached to an antigen target, the C1q protein complex may bind to at least two IgG1 molecules or one IgM molecule (Ward, e.s. and Ghetie, v., "immunology (ter.immunol.))" 2 (1995) 77-94). Burton, D.R. (molecular immunology) 22 (1985) 161-206) describes the involvement of the heavy chain region, comprising amino acid residues 318 to 337, in complement fixation. Duncan, A.R. and Winter, G. ("Nature") 332 (1988) 738-740) reported that Glu318, lys320 and Lys322 form binding sites for C1q using site-directed mutagenesis. The role of Glu318, lys320 and Lys322 residues in C1q binding was demonstrated by the ability of short synthetic peptides containing these residues to inhibit complement mediated cleavage.
For example, fcR binding may be mediated through the interaction of the Fc portion (of an antibody) with an Fc receptor (FcR), which is a specialized cell surface receptor on cells including hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and are shown to mediate clearance of antibody-coated pathogens via phagocytosis of immune complexes as well as lysis of both erythrocytes coated with the corresponding antibodies and various other cellular targets (e.g., tumor cells) by antibody-dependent cell-mediated cytotoxicity (ADCC; van de Winkel, j.g. and Anderson, c.l., journal of white blood cell biology (j.leukoc.biol.) 49 (1991) 511-524). FcR is defined by its specificity for immunoglobulin classes; the Fc receptor of IgG antibodies is called fcγr, the Fc receptor of IgE antibodies is called fcεr, the Fc receptor of IgA antibodies is called fcαr, etc., and the neonatal Fc receptor is called FcRn. Such as Ravetch, j.v. and Kinet, j.p. "immunological annual review (annu. Rev. Immunol.)" 9 (1991) 457-492; capel, P.J. et al, immunization methods (Immunomethods) 4 (1994) 25-34; de Haas, M.et al, (J Lab. Clin. Med.) 126 (1995) 330-341; and Gessner, J.E. et al, annual book of hematology (Ann. Hematol.) 76 (1998) 231-248.
Crosslinking of the Fc domain of natural IgG antibodies (fcγr) to the receptor triggers a variety of effector functions including phagocytosis, antibody dependent cellular cytotoxicity and inflammatory mediator release, as well as immune complex clearance and modulation of antibody production. The Fc portion that provides crosslinking of the receptor (e.g., fcγr) is contemplated herein. In humans, three classes of fcγr have been characterized to date: (i) Fcyri (CD 64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils; (ii) Fcγrii (CD 32), which binds complex IgG with medium to low affinity, is widely expressed (particularly on leukocytes), is considered a core participant in antibody-mediated immunity, and can be divided into fcγriia, fcγriib and fcγriic, functions differently in the immune system, but binds IgG-Fc with similar low affinity, and the extracellular domains of these receptors are highly homologous; and (iii) fcγriii (CD 16), which binds IgG with medium to low affinity, and has been found to take two forms: fcγriiia, which has been found on NK cells, macrophages, eosinophils, and some monocytes and T cells, and is thought to mediate ADCC; and fcγriiib highly expressed on neutrophils.
Fcγriia is found on many cells involved in killing (e.g., macrophages, monocytes, neutrophils) and appears to be able to activate the killing process. Fcyriib appears to play a role in the inhibition process and is found on B cells, macrophages, mast cells and eosinophils. Importantly, 75% of all fcyriib has been shown to be found in the liver (Ganesan, l.p. et al 2012: "fcyriib clearance of small immune complexes on the liver sinus endothelium (fcγ RIIb on liver sinusoidal endothelium clears small immune complexes)", journal of immunology 189:4981-4988). Fcyriib is abundantly expressed on the liver sinus endothelium (called LSEC) and in Kupffer cells in the liver, and LSEC is the major site of clearance of small immune complexes (Ganesan, l.p. et al 2012: fcyriib clearance of small immune complexes on the liver sinus endothelium, journal of immunology 189:4981-4988).
In some embodiments, the antibodies and antigen binding fragments thereof disclosed herein include an Fc polypeptide or fragment thereof for binding to fcyriib, particularly an Fc region, e.g., an IgG-type antibody. In addition, the Fc portion can be engineered to enhance FcgammaRIIB binding by introducing mutations S267E and L328F, as described in Chu, S.Y. et al, 2008, inhibiting B cell receptor-mediated primary human B cell activation by co-ligation of CD19 and FcgammaRIIB with Fc engineered antibodies (Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies), molecular immunology 45, 3926-3933. Thus, clearance of immune complexes can be enhanced (Chu, S.et al 2014: accelerated clearance of IgE in chimpanzees is mediated by Xmab7195 (i.e., an Fc engineered antibody), with enhanced affinity for the inhibitory receptor FcgammaRIIB (Accelerated Clearance of IgE In Chimpanzees Is Mediated By Xmab7195, an Fc-Engineered Antibody With Enhanced Affinity For Inhibitory Receptor Fc. Gamma. RIIB) [ J. Respiratory and critical care medicine (Am J respiratory Crit) ], american society of thoracic International conference abstract (American Thoracic Society International Conference Abstracts) ]. In some embodiments, the antibodies of the present disclosure, or antigen binding fragments thereof, include an engineered Fc portion having mutations S267E and L328F, particularly as described in Chu, S.Y. et al, 2008, inhibit B cell receptor-mediated primary human B cell activation by co-ligation of CD19 and FcgammaRIIB with the Fc engineered antibody, molecular immunology 45, 3926-3933.
On B cells fcyriib may act to inhibit further immunoglobulin production and isotype switching to e.g. IgE class. On macrophages, fcyriib is thought to inhibit phagocytosis mediated by fcyriia. On eosinophils and mast cells, form b can help inhibit activation of these cells by IgE binding to its independent receptor.
With respect to fcγri binding, native IgG of at least one of E233-G236, P238, D265, N297, a327 and P329Modification reduces binding to fcyri. The substitution of the IgG2 residues at positions 233-236 to the corresponding positions IgG1 and IgG4 reduces the binding of IgG1 and IgG4 to Fcgamma by 10 3 Doubling and eliminating the response of human monocytes to antibody-sensitized erythrocytes (Armour, K.L. Et al, J. Immunol. Eur., J. Immunol.), 29 (1999) 2613-2624).
Regarding fcyrii binding, reduced binding to fcyriia is found, for example, for IgG mutations of at least one of E233-G236, P238, D265, N297, a327, P329, D270, Q295, a327, R292, and K414.
Two allelic forms of human fcyriia are the "H131" variant that binds IgG1 Fc with higher affinity and the "R131" variant that binds IgG1 Fc with lower affinity. See, e.g., bruhns et al, blood (Blood) 113:3716-3725 (2009).
Regarding fcyriii binding, a decrease in binding to fcyriiia is found, for example, for a mutation in at least one of E233-G236, P238, D265, N297, a327, P329, D270, Q295, a327, S239, E269, E293, Y296, V303, a327, K338 and D376. Mapping of the binding sites for Fc receptors on human IgG1, the above-described mutation sites and methods for measuring binding to FcgammaRI and FcgammaRIIA are described in Shields, R.L. et al, J.Biol.chem.) (276 (2001) 6591-6604).
Two allelic forms of human fcyriiia are the "F158" variant that binds IgG1 Fc with lower affinity and the "V158" variant that binds IgG1 Fc with higher affinity. See, for example, bruhns et al, blood 113:3716-3725 (2009).
Regarding binding to FcgammaRII, the two regions of native IgG Fc appear to be involved in the interaction between FcgammaRII and IgG, i.e., (i) the lower hinge site of IgG Fc, particularly amino acid residues L, L, G, G (234-237, EU numbering), and (ii) the adjacent region of the CH2 domain of IgG Fc, particularly the loop and chain in the upper CH2 domain adjacent to the lower hinge region, e.g., in the region of P331 (Wines, B.D. et al, J.Immunol.2000; 164:5313-5318). Furthermore, fcγRI appears to bind to the same site on IgG Fc, while FcRn and protein A bind to different sites on IgG Fc, which appear to be located at the CH2-CH3 interface (Wines, B.D. et al, J.Immunol.2000; 164:5313-5318).
Mutations that increase the binding affinity of an Fc polypeptide of the present disclosure or fragment thereof to an fcγ receptor (i.e., one or more) are also contemplated (e.g., as compared to a reference Fc polypeptide or fragment thereof or a reference Fc polypeptide or fragment thereof containing no mutations). See, e.g., delilo and Ravetch, cell 161 (5): 1035-1045 (2015) and Ahmed et al, journal of structural biology (J. Structure. Biol.) 194 (1): 78 (2016), the Fc mutations and techniques of which are incorporated herein by reference.
In any of the embodiments disclosed herein, the antibody or antigen binding fragment may comprise an Fc polypeptide or fragment thereof comprising a mutation selected from the group consisting of: G236A; S239D; a330L and I332E; or a combination comprising any two or more thereof; such as S239D/I332E; S239D/A330L/I332E; G236A/S239D/I332E; G236A/A330L/I332E (also referred to herein as "GAALIE"); or G236A/S239D/A330L/I332E. In some embodiments, the Fc polypeptide or fragment thereof does not comprise S239D. In some embodiments, the Fc polypeptide or fragment thereof comprises S at position 239 (EU numbering).
In certain embodiments, the Fc polypeptide or fragment thereof may comprise or consist of at least a portion of an Fc polypeptide or fragment thereof that is involved in binding to FcRn. In certain embodiments, the Fc polypeptide or fragment thereof comprises one or more amino acid modifications that improve binding affinity to FcRn (e.g., enhance binding to FcRn) (e.g., at a pH of about 6.0), and in some embodiments, thereby extending the in vivo half-life of a molecule comprising the Fc polypeptide or fragment thereof (e.g., as compared to an otherwise identical reference Fc polypeptide or fragment or antibody thereof that does not comprise a modification). In certain embodiments, the Fc polypeptide or fragment thereof comprises or is derived from an IgG Fc and the half-life extending mutations comprise any one or more of the following: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I; Q311I; D376V; T307A; E380A (EU numbering). In certain embodiments, half-life extending mutations include M428L/N434S (also referred to herein as "MLNS", "_ls" and "-LS"). In certain embodiments, the half-life extending mutation comprises M252Y/S254T/T256E. In certain embodiments, the half-life extending mutation comprises T250Q/M428L. In certain embodiments, the half-life extending mutation comprises P257I/Q311I. In certain embodiments, the half-life extending mutation comprises P257I/N434H. In certain embodiments, the half-life extending mutation comprises D376V/N434H. In certain embodiments, the half-life extending mutation comprises T307A/E380A/N434A.
In some embodiments, the antibody or antigen binding fragment comprises an Fc portion comprising substitution mutation M428L/N434S. In some embodiments, the antibody or antigen binding fragment comprises an Fc polypeptide or fragment thereof comprising the substitution mutation G236A/a330L/I332E. In certain embodiments, the antibody or antigen binding fragment comprises (e.g., igG) an Fc portion that comprises a G236A mutation, an a330L mutation, and an I332E mutation (GAALIE), and does not comprise an S239D mutation (e.g., comprises native S at position 239). In certain embodiments, the antibody or antigen binding fragment comprises an Fc polypeptide or fragment thereof comprising a substitution mutation: M428L/N434S and G236A/A330L/I332E, and optionally does not include S239D (e.g., includes S at 239). In certain embodiments, the antibody or antigen binding fragment comprises an Fc polypeptide or fragment thereof comprising a substitution mutation: M428L/N434S and G236A/S239D/A330L/I332E.
In certain embodiments, the antibody or antigen binding fragment comprises a mutation that alters glycosylation, wherein the mutation that alters glycosylation comprises N297A, N297Q or N297G, and/or the antibody or antigen binding fragment is partially or fully deglycosylated and/or partially or fully deglycosylated. Host cell lines and methods for preparing antibodies and antigen binding fragments that are partially or fully deglycosylated or partially or fully deglycosylated are known (see, e.g., PCT publication No. WO 2016/181357; suzuki et al, clinical cancer research (Clin. Cancer Res.)) 13 (6): 1875-82 (2007); huang et al, MAb 6:1-12 (2018)).
In certain embodiments, the antibody or antigen-binding fragment is capable of eliciting sustained protection in a subject, even after a detectable level of antibody or antigen-binding fragment is not found in the subject (i.e., when the antibody or antigen-binding fragment has been cleared from the subject after administration). Such protection is referred to herein as vaccine effect. Without wishing to be bound by theory, it is believed that dendritic cells can internalize complexes of antibodies with antigens and thereafter induce or contribute to an endogenous immune response against the antigen. In certain embodiments, the antibody or antigen binding fragment comprises one or more modifications, e.g., mutations in the Fc comprising G236A, A330L and I332E, which are capable of activating dendritic cells that can induce T cell immunity to an antigen, for example.
In any of the presently disclosed embodiments, the antibody or antigen binding fragment includes an Fc polypeptide or fragment thereof, including CH2 (or fragment thereof), CH3 (or fragment thereof), or CH2 and CH3, wherein CH2, CH3, or both may be of any isotype and may contain amino acid substitutions or other modifications as compared to the corresponding wild-type CH2 or CH3, respectively. In certain embodiments, the Fc polypeptides of the present disclosure include two CH2-CH3 polypeptides that associate to form a dimer.
It will be appreciated that the production in mammalian cell lines, for example, may remove one or more C-terminal lysines of the antibody heavy chain (see, e.g., liu et al, mAb 6 (5): 1145-1154 (2014)). Thus, an antibody or antigen binding fragment of the present disclosure may comprise a heavy chain, CH1-CH3, or Fc polypeptide, with or without the presence of a C-terminal lysine residue; in other words, embodiments are contemplated in which the C-terminal residue of the heavy chain, CH1-CH3, or Fc polypeptide is not lysine, as well as embodiments in which lysine is the C-terminal residue.
In any of the presently disclosed embodiments, the antibody or antigen binding fragment may be monoclonal. As used herein, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts in some cases. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single epitope of an antigen as compared to a polyclonal antibody preparation comprising different antibodies directed against different epitopes. In addition to specificity, monoclonal antibodies are advantageous in that these antibodies can be synthesized without contamination by other antibodies. The term "monoclonal" is not to be construed as requiring antibody production by any particular method. For example, monoclonal antibodies suitable for use in the present invention may be prepared by the hybridoma method described for the first time by Kohler et al, nature 256:495 (1975), or may be prepared in bacterial, eukaryotic or plant cells using recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). For example, monoclonal antibodies can also be isolated from phage antibody libraries using techniques described in Clackson et al, nature 352:624-628 (1991) and Marks et al, journal of molecular biology 222:581-597 (1991). Monoclonal antibodies may also be obtained using the methods disclosed in PCT publication No. WO 2004/076677A 2.
Antibodies and antigen binding fragments of the present disclosure include "chimeric antibodies" in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass, and the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. Nos. 4,816,567, 5,530,101 and 7,498,415; and Morrison et al, proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). For example, chimeric antibodies may include human and non-human residues. In addition, chimeric antibodies may include residues not found in the recipient antibody or the donor antibody. These modifications were made to further optimize antibody performance. For additional details, see Jones et al, nature 321:522-525 (1986); reichmann et al, nature 332:323-329 (1988); and Presta, contemporary structural biology reviews (Curr.Op. Struct. Biol.), 2:593-596 (1992). Chimeric antibodies also include primatized and humanized antibodies.
"humanized antibodies" are generally considered to have a primer from a non-human source A human antibody having one or more amino acid residues incorporated therein. These non-human amino acid residues are typically taken from variable domains. Humanization may be performed by substituting the corresponding sequences of human antibodies with non-human variable sequences according to the methods of Winter and co-workers (Jones et al, nature 321:522-525 (1986); reichmann et al, nature 332:323-327 (1988); verhoeyen et al, science 239:1534-1536 (1988)). Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567; U.S. Pat. No. 5,530,101 and 7,498,415) in which substantially less than the complete human variable domain has been replaced with a corresponding sequence from a non-human species. In some cases, a "humanized" antibody is produced by a non-human cell or animal and includes human sequences (e.g., H C Domain).
A "human antibody" is an antibody that includes only sequences present in antibodies produced by humans (i.e., sequences encoded by genes encoding human antibodies). However, as used herein, a human antibody may include residues or modifications not found in naturally occurring human antibodies (e.g., antibodies isolated from humans), including those modifications and variant sequences described herein. These are typically done to further optimize or enhance antibody performance. In some cases, the human antibody is produced by a transgenic animal. See, for example, U.S. patent No. 5,770,429; no. 6,596,541; and 7,049,426.
In certain embodiments, the antibodies or antigen binding fragments of the disclosure are chimeric, humanized or human.
In some embodiments, various pharmacokinetic ("PK") parameters are used to describe and characterize the antibodies or antigen binding fragments provided herein. Details regarding the collection of antibody serum concentrations to evaluate PK parameters are described in connection with the examples herein. The term "t 1/2 "or" half-life "refers to the elimination of the half-life of an antibody or antigen binding fragment included in a pharmaceutical composition administered to a subject. The term "C last "generally means that the final measurable plasma concentration (i.e., after which the substance is not present in the plasma at a measurable concentration).
In any of the presently disclosed embodiments, the antibody or antigen binding fragment may comprise a CH1-CH3 amino acid sequence as set forth in SEQ ID NO:252 and/or a CH1-CH3 amino acid sequence as set forth in SEQ ID NO:253 and/or a CH1-CH3 amino acid sequence as set forth in SEQ ID NO:280 and/or a CH1-CH3 amino acid sequence as set forth in SEQ ID NO: 251.
In any of the presently disclosed embodiments, the antibody or antigen-binding fragment may include the CL amino acid sequence shown as SEQ ID NO. 254.
In some embodiments, the antibody comprises the heavy chain amino acid sequence set forth in SEQ ID NO. 255. In certain embodiments, the antibody further comprises the light chain amino acid sequence set forth in SEQ ID NO. 257.
In some embodiments, the antibody comprises the heavy chain amino acid sequence set forth in SEQ ID NO. 256. In certain embodiments, the antibody further comprises the light chain amino acid sequence set forth in SEQ ID NO. 257.
In some embodiments, the antibody comprises the heavy chain amino acid sequence set forth in SEQ ID NO 270 or 272. In certain embodiments, the antibody further comprises the light chain amino acid sequence set forth in SEQ ID NO 271 or 273.
In certain embodiments, the anti-NA antibody or antigen-binding fragment comprises the heavy chain amino acid sequence set forth in SEQ ID NO:255 and the light chain amino acid sequence set forth in SEQ ID NO:257, and the anti-HA antibody or antigen-binding fragment comprises the heavy chain amino acid sequence set forth in SEQ ID NO:270 or 272 and the light chain amino acid sequence set forth in SEQ ID NO:271 or 273.
In some embodiments, the anti-NA antibody or antigen-binding fragment comprises the heavy chain amino acid sequence set forth in SEQ ID NO:256 and the light chain amino acid sequence set forth in SEQ ID NO:257, and the anti-HA antibody or antigen-binding fragment comprises the heavy chain amino acid sequence set forth in SEQ ID NO:270 or 272 and the light chain amino acid sequence set forth in SEQ ID NO:271 or 273.
Polynucleotides, vectors and host cells
In another aspect, the present disclosure provides a method of encoding any one of the presently disclosed antibodies, or antigen-binding fragments thereof, or a portion thereof (e.gSuch as CDR, VH, VL, heavy chain or light chain). In certain embodiments, the polynucleotide is codon optimized for expression in a host cell (e.g., a human cell or CHO cell). Once the coding sequence is known or identified, codon optimization can be performed using known techniques and tools, e.g., usingOptimiumGene TM Tools, etc. Codon optimized sequences include partially codon optimized sequences (i.e., one or more codons are optimized for expression in a host cell) and fully codon optimized sequences.
It is also understood that polynucleotides encoding antibodies and antigen binding fragments of the present disclosure may have different nucleotide sequences while still encoding the same antibody or antigen binding fragment due to, for example, degeneracy of the genetic code, splicing, and the like.
In any of the presently disclosed embodiments, the polynucleotide may include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some embodiments, the RNA comprises messenger RNA (mRNA).
In some embodiments, the polynucleotide comprises a modified nucleoside, a cap-1 structure, a cap-2 structure, or any combination thereof. In certain embodiments, the polynucleotide comprises pseudouridine, N6-methyladenosine, 5-methylcytidine, 2-thiouridine, or any combination thereof. In some embodiments, the pseudouridine comprises N1-methyl pseudouridine.
Also provided are vectors, wherein the vectors comprise or contain a polynucleotide as disclosed herein (e.g., a polynucleotide encoding an antibody or antigen binding fragment that binds IAV HA or IAV NA and/or IBV NA). The carrier may include any one or more of the carriers disclosed herein. In a particular embodiment, vectors comprising DNA plasmid constructs encoding antibodies or antigen binding fragments or portions thereof (e.g., so-called "DMAb"; see, e.g., muthumb et al J.infectious disease (J. Infect Dis.)) 214 (3): 369-378 (2016); muthumb et al, human vaccine and immunotherapy (Hum vaccine Immu)not) 9:2253-2262 (2013); flingai et al, science report 5:12616 (2015); and Elliott et al, NPJ vaccine (NPJ Vaccines) 18 (2017), which antibody encodes the DNA construct and related methods of use, including administration thereof, are incorporated herein by reference. In certain embodiments, the DNA plasmid construct comprises a single open reading frame encoding the heavy and light chains (or VH and VL) of an antibody or antigen binding fragment, wherein the sequence encoding the heavy chain and the sequence encoding the light chain are optionally separated by a polynucleotide encoding a protease cleavage site and/or a polynucleotide encoding a self-cleaving peptide. In some embodiments, the substitution component of the antibody or antigen binding fragment is encoded by a polynucleotide included in a single plasmid. In other embodiments, the substitution component of the antibody or antigen-binding fragment is encoded by a polynucleotide included in two or more plasmids (e.g., a first plasmid includes a polynucleotide encoding a heavy chain, VH, or vh+ch1, and a second plasmid includes a polynucleotide encoding a homologous light chain, VL, or vl+cl). In certain embodiments, a single plasmid comprises a polynucleotide encoding the heavy and/or light chain from two or more antibodies or antigen binding fragments of the disclosure. Exemplary expression vectors are available from The pVax1 obtained. The DNA plasmids of the present disclosure can be delivered to a subject by, for example, electroporation (e.g., intramuscular electroporation) or with an appropriate formulation (e.g., hyaluronidase).
In some embodiments, a method is provided that includes administering to a subject a first polynucleotide (e.g., mRNA) encoding an antibody heavy chain, VH, or Fd (vh+ch1), and administering to the subject a second polynucleotide (e.g., mRNA) encoding a cognate antibody light chain, VL, or vl+cl.
In some embodiments, therapies according to the present disclosure include delivering to a subject a single nucleic acid molecule encoding: (1) an anti-HA antibody or antigen-binding fragment thereof; and (2) an anti-NA antibody or antigen-binding fragment thereof.
In some embodiments, a therapy according to the present disclosure includes delivering to a subject a first polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof and a second polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof.
In some embodiments, a therapy according to the present disclosure includes delivering to a subject: (1) A first polynucleotide encoding a VH, vh+ch1 or heavy chain of an anti-HA antibody or antigen-binding fragment thereof; (2) A second polynucleotide encoding a cognate VL, vl+cl or light chain of an anti-HA antibody or antigen-binding fragment thereof; (3) A third polynucleotide encoding a VH, vh+ch1 or heavy chain of an anti-NA antibody or antigen-binding fragment thereof; and (4) a fourth polynucleotide encoding a cognate VL, vl+cl or light chain of an anti-NA antibody or antigen-binding fragment thereof. In some embodiments, a polynucleotide (e.g., mRNA) encoding the heavy and light chains of an antibody or antigen-binding fragment thereof is provided. In some embodiments, a polynucleotide (e.g., an mRNA) encoding two heavy chains and two light chains of an antibody or antigen-binding fragment thereof is provided. See, e.g., li, JQ., zhang, ZR., zhang, HQ. et al, intranasal delivery of replication-competent mRNA encoding neutralizing antibodies against SARS-CoV-2infection in mice (Intranasal delivery of replicating mRNAencoding neutralizing antibody against SARS-CoV-2infection in mice.) (Signal transduction and targeting therapy (Sig Transduct Target Ther), (6, 369 (2021),. Https:// doi.org/10.1038/s41392-021-00783-1, mRNA constructs encoding antibodies, vectors and related techniques thereof are incorporated herein by reference. In some embodiments, the polynucleotide is delivered to the subject by an alphavirus replicon particle (VRP) delivery system. In some embodiments, the replicon comprises a modified VEEV replicon comprising two subgenomic promoters. In some embodiments, the polynucleotide or replicon may simultaneously translate the heavy chain (or VH or vh+1) and the light chain (or VL or vl+cl) of the antibody or antigen binding fragment thereof. In some embodiments, a method is provided that includes delivering such polynucleotides or replicons to a subject.
In a further aspect, the present disclosure also provides a host cell expressing an antibody or antigen-binding fragment according to the present disclosure; or comprise or contain a vector or polynucleotide according to the present disclosure.
Embodiments of such cells include, but are not limited to, eukaryotic cells (e.g., yeast cells), animal cells, insect cells, plant cells, and prokaryotic cells, including E.coli (E.coli). In some embodiments, the cell is a mammalian cell, such as a human B cell. In certain such embodiments, the cells are mammalian cell lines, such as CHO cells (e.g., DHFR-CHO cells (Urlaub et al, proc. Natl. Acad. Sci. USA 77:4216 (1980)), human embryonic kidney cells (e.g., HEK293T cells), PER.C6 cells, Y0 cells, sp2/0 cells, NS0 cells, human hepatocytes, e.g., hepa RG cells, myeloma cells, or hybridoma cells; SV40 (COS-7) transformed monkey kidney CV1 line, hamster kidney cells (BHK), african green monkey kidney cells (VERO-76), monkey kidney cells (CV 1), human cervical cancer cells (HELA), human lung cells (W138), human liver cells (Hep G2), canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A), mouse mammary tumor (MMT 060562), TRI cells, MRC5 cells, and FS4 cells mammalian host cell lines suitable for antibody production also include mammalian host cell lines described in, for example, yazaki and Wu, methods of molecular biology, volume 248 (B.K.C.Lo. Edited, hu Mana publishers of Totowa, N.J.), pages 255-268 (2003) of Totova, N.J.).
In certain embodiments, the host cell is a prokaryotic cell, such as E.coli. Expression of peptides in prokaryotic cells such as E.coli is well established (see e.g.Pluckthun, A. (Bio/Technology) 9:545-551 (1991)). For example, antibodies can be produced in bacteria, specifically when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. nos. 5,648,237; 5,789,199; and 5,840,523.
In certain embodiments, cells may be transfected with a vector according to the present disclosure with an expression vector. The term "transfection" refers to the introduction of a nucleic acid molecule, such as a DNA or RNA (e.g., mRNA) molecule, into a cell, such as a eukaryotic cell. In the context of the present specification, the term "transfection" encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, such as eukaryotic cells, including mammalian cells. Such methods encompass, for example, electroporation, lipofection (e.g., based on cationic lipids and/or liposomes), calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or transfection based on cationic polymers (such as DEAE-dextran or polyethylenimine, etc.). In certain embodiments, the introduction is non-viral.
In addition, host cells of the present disclosure may be stably or transiently transfected with vectors according to the present disclosure, e.g., to express antibodies or antigen-binding fragments thereof according to the present disclosure. In such embodiments, the cells may be stably transfected with a vector as described herein. Alternatively, cells may be transiently transfected with vectors according to the present disclosure encoding antibodies or antigen binding fragments as disclosed herein. In any of the presently disclosed embodiments, the polynucleotide may be heterologous to the host cell.
Thus, the present disclosure also provides recombinant host cells that heterologously express the antibodies or antigen binding fragments of the disclosure. For example, the cell may be a species different from the species from which the antibody was fully or partially obtained (e.g., CHO cells expressing a human antibody or an engineered human antibody). In some embodiments, the cell type of the host cell does not express the antibody or antigen binding fragment in nature. Furthermore, the host cell may confer post-translational modification (PTM; e.g., glycosylation or fucosylation) or lack thereof to an antibody or antigen-binding fragment that is not present in its native state (or in the native state of the parent antibody from which the antibody or antigen-binding fragment was engineered or derived). Such PTMs or lack thereof may cause functional differences (e.g., reduced immunogenicity). Thus, an antibody or antigen-binding fragment of the present disclosure produced by a host cell as disclosed herein may include one or more post-translational modifications that are different from the antibody (or parent antibody) in its native state (e.g., a human antibody produced by a host cell may include one or more post-translational modifications, or may include fewer post-translational modifications such that the post-translational modifications are different from antibodies isolated from a human and/or produced by a native human B cell or plasma cell).
Insect cells useful for expressing the binding proteins of the present disclosure are known in the art and include, for example, spodoptera frugiperda Sf9 cells (Spodoptera frugipera Sf cells), spodoptera frugiperda BTI-TN5B1-4 cells (Trichoplusia ni BTI-TN5B1-4 cells), and Spodoptera frugiperda SfSWT01"Mimic TM "cell (Spodoptera frugipera SfSWT01" Mimic) TM "cell"). See, for example, palmberger et al, J.Biotechnology journal (J.Biotechnol.) 153 (3-4): 160-166 (2011). A number of baculovirus strains have been identified which can be used in combination with insect cells, in particular for transfection of Spodoptera frugiperda cells.
Eukaryotic microorganisms (such as filamentous fungi or yeasts) are also suitable hosts for cloning or expressing the protein-encoding vectors and include fungal and yeast strains having a "humanized" glycosylation pathway, thereby producing antibodies having a partially or fully human glycosylation pattern. See Gerngross, nature Biotech 22:1409-1414 (2004); li et al, nature Biotechnology 24:210-215 (2006).
Plant cells may also be used as hosts for expression of the antibodies or antigen-binding fragments of the present disclosure. For example, PLANTIBODIES TM Techniques (described, for example, in U.S. Pat. nos. 5,959,177; 6,040,498; 6,420,548; 7,125,978; and 6,417,429) employ transgenic plants to produce antibodies.
In certain embodiments, the host cell comprises a mammalian cell. In particular embodiments, the host cell is a CHO cell, HEK293 cell, per.c6 cell, Y0 cell, sp2/0 cell, NS0 cell, human liver cell, myeloma cell or hybridoma cell.
In a related aspect, the present disclosure provides a method for producing an antibody or antigen-binding fragment, wherein the method comprises culturing a host cell of the present disclosure under conditions sufficient to produce the antibody or antigen-binding fragment for a time sufficient to produce the antibody or antigen-binding fragment. For example, a method suitable for isolating and purifying recombinantly produced antibodies may include obtaining supernatant from a suitable host cell/vector system that secretes the recombinant antibodies into the culture medium, and then concentrating the culture medium using commercially available filters. After concentration, the concentrate may be applied to a single suitable purification substrate or a series of suitable substrates, such as an affinity substrate or ion exchange resin. One or more reverse phase HPLC steps can be employed to further purify the recombinant polypeptide. These purification methods can also be employed when the immunogen is isolated from its natural environment. Methods for large-scale production of one or more isolated/recombinant antibodies described herein include batch cell culture that is monitored and controlled to maintain appropriate culture conditions. Purification of the soluble antibodies can be performed according to methods described herein and known in the art and conforming to the laws and guidelines of regulatory bodies at home and abroad.
Composition and method for producing the same
Also provided herein are compositions comprising any combination of the presently disclosed anti-HA antibodies or antigen-binding fragments and the presently disclosed anti-NA antibodies or antigen-binding fragments or polynucleotides encoding the same (e.g., antibodies or antigen-binding fragments or components of these), and may further comprise a pharmaceutically acceptable carrier, excipient, or diluent. Such compositions, carriers, excipients, and diluents are discussed in further detail herein.
In certain embodiments, the composition comprises one or more vectors or polynucleotides encoding an anti-HA antibody or antigen-binding fragment, an anti-NA antibody or antigen-binding fragment, or both. In some embodiments, the composition comprises a first vector comprising a first plasmid and a second vector comprising a second plasmid, wherein the first plasmid comprises a polynucleotide encoding a heavy chain, VH, or vh+ch1, and the second plasmid comprises a polynucleotide encoding a homologous light chain, VL, or vl+cl of the antibody or antigen-binding fragment thereof.
In certain embodiments, the composition comprises a polynucleotide (e.g., mRNA) coupled to a suitable delivery vehicle or carrier. Exemplary vehicles or carriers for administration to human subjects include lipid or lipid-derived delivery vehicles, such as liposomes, solid lipid nanoparticles, oily suspensions, submicron lipid emulsions, lipid microbubbles, reverse lipid micelles, cochlear liposomes, lipid microtubules, lipid microcylinders, or Lipid Nanoparticles (LNP) or nanoscale platforms (see, e.g., li et al, wilinson theory: nanomedicine and nanobiotechnology (Wilery Interdiscip rev. Nanomedia nanobiotechnology.)) 11 (2): e1530 (2019). Principles, reagents and techniques for designing and formulating mRNA-LNP and delivering it are described, for example, in Pardi et al (journal of controlled release (J Control Release), 217445-351 (2015)); thess et al (molecular therapy) 23:1456-1464 (2015)); thran et al (EMBO molecular medicine (EMBO Mol Med) 9 (10): 1434-1448 (2017), kose et al (scientific immunology) 4eaaw6647 (2019), and Sabnis et al (molecular therapy 26:1509-1519 (2018)), which techniques include capping, codon optimization, nucleoside modification, mRNA purification, incorporation of mRNA into stable lipid nanoparticles (e.g., ionizable cationic lipid/phosphatidylcholine/cholesterol/PEG-lipid; ionizable lipid: distearoyl PC: cholesterol: polyethylene glycol lipid) and subcutaneous, intramuscular, intradermal, intravenous, intraperitoneal and intratracheal administration thereof, which are incorporated herein by reference.
Method and use
Also provided herein are methods for treating a subject having, considered to have, or at risk of having an infection caused by influenza using the antibodies or antigen binding fragments, polynucleotides, compositions, or combinations of the present disclosure. "treatment," "treatment," or "amelioration" refers to the medical management of a disease, disorder, or condition in a subject (e.g., a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat). Generally, an appropriate dose or treatment regimen comprising an antibody or composition of the present disclosure is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic/preventative benefits include improving clinical outcome; alleviating or alleviating symptoms associated with the disease; reducing the occurrence of symptoms; improving the quality of life; longer disease-free state; reducing the disease degree and stabilizing the disease state; delay or prevent disease progression; relief; survival; prolonging the survival period; or any combination thereof. In certain embodiments, the therapeutic or prophylactic/preventative benefit includes reducing or preventing hospitalization (i.e., in a statistically significant manner) for the treatment of influenza infection. In certain embodiments, the therapeutic or prophylactic/preventative benefit includes shortening the duration of hospitalization (i.e., in a statistically significant manner) for treating influenza infection. In certain embodiments, therapeutic or prophylactic/preventative benefits include reducing or eliminating the need for respiratory intervention, such as intubation and/or use of a respirator device. In certain embodiments, the therapeutic or prophylactic/preventative benefit includes reversing the pathology of end disease and/or reducing mortality.
The "therapeutically effective amount" or "effective amount" of an antibody, antigen binding fragment, polynucleotide, vector, host cell, combination, or composition of the present disclosure refers to an amount of a composition or molecule sufficient to produce a therapeutic effect comprising: improved clinical outcome; alleviating or alleviating symptoms associated with the disease; reducing the occurrence of symptoms; improving the quality of life; longer disease-free state; reducing the extent of the disease; stabilizing the disease state; delay disease progression; relief; survival; or to extend survival in a statistically significant manner. When referring to individual active ingredients administered alone, a therapeutically effective amount refers to the effect of the ingredients alone.
When referring to an antibody combination, an antibody composition, a polynucleotide combination, or a polynucleotide composition, a therapeutically effective amount refers to a combined amount of the active ingredients that produces a therapeutic effect, whether administered sequentially, or simultaneously.
Thus, in certain embodiments, methods for treating influenza infection in a subject are provided, wherein the methods comprise administering to the subject an effective amount of an antibody, antigen binding fragment, polynucleotide, vector, host cell, combination, or composition disclosed herein.
Typically, subjects that can be treated by the present disclosure are human and other primate subjects, such as monkeys and apes for veterinary purposes. Other model organisms, such as mice and rats, may also be treated according to the present disclosure. In any of the preceding embodiments, the subject may be a human subject. The subject may be male or female, and may be of any suitable age, including infant, juvenile, adolescent, adult and geriatric subjects.
Many criteria are believed to contribute to the severe symptoms associated with influenza infection or the high risk of death. These include, but are not limited to, age, occupation, general health, pre-existing health, location, and lifestyle. In some embodiments, a subject treated according to the present disclosure includes one or more risk factors.
In certain embodiments, the human subject treated according to the present disclosure is an infant, a child, a young adult, a middle-aged adult, or an elderly person. In certain embodiments, a human subject treated according to the present disclosure is less than 1 year old, either 1 year old to 5 years old, or between 5 years old and 125 years old (e.g., 5 years old, 10 years old, 15 years old, 20 years old, 25 years old, 30 years old, 35 years old, 40 years old, 45 years old, 50 years old, 55 years old, 60 years old, 65 years old, 70 years old, 75 years old, 80 years old, 85 years old, 90 years old, 95 years old, 100 years old, 105 years old, 110 years old, 115 years old, or 125 years old, including any and all ages therein or therebetween). In certain embodiments, a human subject treated according to the present disclosure is 0-19 years old, 20-44 years old, 45-54 years old, 55-64 years old, 65-74 years old, 75-84 years old, or 85 years old or older. Middle-aged and especially elderly people may be at particular risk. In particular embodiments, the human subject is 45-54 years old, 55-64 years old, 65-74 years old, 75-84 years old, or 85 years old or older. In some embodiments, the human subject is a male. In some embodiments, the human subject is a female.
In certain embodiments, a subject treated according to the present disclosure has received a vaccine against influenza, and the vaccine is determined to be ineffective, e.g., by post-vaccine infection or symptoms of the subject, by clinical diagnosis or scientific or regulatory consensus.
Prevention of infection with influenza virus refers in particular to a prevention environment in which a subject is not diagnosed with an infection caused by influenza virus (is not diagnosed or the diagnosis is negative) and/or the subject does not show or experience symptoms of an infection caused by influenza virus. The control of infection by influenza virus is particularly useful in subjects at greater risk of suffering from serious diseases or complications when infected, such as pregnant women, children (e.g., children under 59 months), elderly people, individuals suffering from chronic medical conditions (e.g., chronic heart disease, lung disease, kidney disease, metabolic disease, neurological disease, liver disease or hematological disease), and individuals suffering from immunosuppressive conditions (e.g., HIV/AIDS, receiving chemotherapy or steroids, malignancy). Furthermore, the control of infections caused by influenza viruses is particularly also useful for subjects at greater risk of acquiring influenza virus infections, e.g. subjects working or staying in public areas, particularly health care workers, e.g. due to increased exposure.
In certain embodiments, the treatment is applied in the form of control during or prior to exposure. In certain embodiments, the treatment is administered in a post-exposure prophylactic form.
In a therapeutic setting, in contrast, a subject is typically infected with influenza virus, diagnosed with influenza virus infection and/or exhibits symptoms of influenza virus. Notably, the terms "treatment" and "therapy" of an influenza virus infection/therapeutic "may refer to (complete) cure as well as attenuation/alleviation of an influenza virus infection and/or associated symptoms (e.g., attenuation/alleviation of the severity of the infection and/or symptoms, number of symptoms, duration of the infection and/or symptoms, or any combination thereof).
It is to be understood that references herein to reduced number of symptoms and/or reduced severity describe a comparison to a reference subject that did not receive the presently disclosed therapy, the reduction/reduction resulting from administration of the disclosed therapy. The reference subject may be, for example, (i) the same subject during an earlier period of time (e.g., a previous influenza a virus season), (ii) the same or similar subject in: age or age group; sex; a gestational state; chronic medical conditions (such as chronic heart disease, lung disease, kidney disease, metabolic disease, neurodevelopmental disease, liver disease or hematological disease) or lack thereof; and/or immunosuppression conditions or lack thereof, or (iii) typical subjects within a population (e.g., local, regional, or national, including the same or similar ages or age ranges and/or general disease states) during influenza virus seasons. Control may be determined, for example, by failing to diagnose an influenza infection and/or lack of symptoms associated with an influenza infection during a portion of the entire influenza season or throughout the influenza season.
In certain embodiments, the methods provided herein comprise administering a therapy according to the present disclosure to a subject at risk of immediate influenza infection. Immediate influenza infection risk typically occurs during influenza epidemics. Influenza viruses are known to circulate and cause seasonal epidemics (WHO, influenza (seasonal) condition instructions, 2018, 11, 6). In temperate climates, seasonal epidemics occur mainly during winter, whereas in tropical areas influenza can occur throughout the year, making outbreaks more irregular. For example, in the northern hemisphere, influenza epidemics are at high risk during 11, 12, 1, 2 and 3 months, while in the southern hemisphere, influenza epidemics are at high risk during 5, 6, 7, 8 and 9 months.
In some embodiments, the subject has received, is receiving, or will receive an antiviral agent. In some embodiments, the antiviral agent comprises a neuraminidase inhibitor, an influenza polymerase inhibitor, or both. In certain embodiments, the antiviral agent comprises oseltamivir, lanamivir, peramivir, zanami Weiba lo Sha Wei, or any combination thereof.
Thus, typical routes of administration of the presently disclosed compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. As used herein, the term "parenteral" includes subcutaneous injections, intravenous injection, intramuscular injection, intrasternal injection or infusion techniques. In certain embodiments, administering comprises administering by a route selected from the group consisting of: oral, intravenous, parenteral, intragastric, intrapleural, intrapulmonary, intrarectal, intradermal, intraperitoneal, intratumoral, subcutaneous, topical, transdermal, intracisternal, intrathecal, intranasal, and intramuscular. In particular embodiments, the method comprises orally administering an antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition to a subject.
The compositions according to certain embodiments of the present invention are formulated such that the active ingredient contained therein is bioavailable upon administration of the composition to a patient. The composition to be administered to a subject or patient may take one or more dosage unit forms, where, for example, a tablet may be a single dosage unit and a container of an antibody or antigen binding or polynucleotide described herein in aerosol form may hold a plurality of dosage units. Practical methods of preparing such dosage forms are known or obvious to those skilled in the art; see, for example, ramington: pharmaceutical science and practice (Remington: the Science and Practice of Pharmacy), 20 th edition (philadelphia medical science institute (Philadelphia College of Pharmacy and Science), 2000). In any event, the composition to be administered will include an effective amount of an antibody or antigen binding fragment, polynucleotide, vector, host cell, or composition of the present disclosure, in order to treat a disease or condition of interest in accordance with the teachings herein.
The composition may be in solid or liquid form. In some embodiments, the carrier is particulate, such that the composition is in the form of a tablet or powder, for example. The carrier may be a liquid, while the composition is, for example, an oral oil, an injectable liquid, or an aerosol suitable for, for example, inhalation administration. When intended for oral administration, the pharmaceutical compositions are preferably in solid or liquid form, wherein semi-solid, semi-liquid, suspension and gel forms are included herein as solid or liquid forms.
As solid compositions for oral administration, pharmaceutical compositions may be formulated as powders, granules, compressed tablets, pills, capsules, chewable tablets, caplets, and the like. Such solid compositions will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binding agents such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, tragacanth or gelatin; excipients such as starch, lactose or dextrin, disintegrants such as alginic acid, sodium alginate, sodium carboxymethyl starch, corn starch, etc.; lubricants such as magnesium stearate or hydrogenated castor oil; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate or orange flavoring; a colorant. When the composition is in the form of a capsule (e.g., a gelatin capsule), it may contain a liquid carrier such as polyethylene glycol or oil, in addition to the above types of materials.
The compositions may be in liquid form, such as elixirs, syrups, solutions, emulsions or suspensions. As two example embodiments, the liquid may be delivered orally or by injection. When intended for oral administration, preferred compositions contain, in addition to the compounds of the present invention, one or more of a sweetener, preservative, dye/colorant and flavoring agent. In compositions intended for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers and isotonic agents may be included.
Liquid pharmaceutical compositions, whether in solution, suspension or other similar forms, may include one or more of the following adjuvants: sterile diluents, such as water for injection, saline solution, preferably physiological saline, ringer's solution, isotonic sodium chloride; fixed oils such as synthetic mono-or diglycerides, polyethylene glycols, glycerol, propylene glycol or other solvents that can be used as solvents or suspending media; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine tetraacetic acid; buffers such as acetate, citrate, or phosphate; and agents for tonicity adjustment, such as sodium chloride or dextrose. Parenteral formulations may be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic. Saline is a preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.
Liquid compositions intended for parenteral or oral administration should contain an amount of an antibody or antigen binding fragment as disclosed herein such that a suitable dose will be obtained. Typically, this amount is at least 0.01% of the antibodies or antigen binding fragments in the composition. When intended for oral administration, this amount may vary from 0.1 to about 70% by weight of the composition. Certain oral pharmaceutical compositions contain from about 4% to about 75% of the antibody or antigen-binding fragment. In certain embodiments, the pharmaceutical compositions and formulations according to the present invention are prepared such that, prior to dilution, the parenteral dosage unit contains 0.01 to 10% by weight of the antibody or antigen-binding fragment.
The composition may be intended for topical application, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. For example, the matrix may include one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohols, emulsifying agents and stabilizers. The thickener may be present in the composition for topical application. If intended for transdermal administration, the composition may include a transdermal patch or an iontophoresis device. The pharmaceutical composition may be intended for rectal administration, for example, in the form of suppositories which will melt in the rectum and release the drug. Compositions for rectal administration may contain an oily base as a suitable non-irritating excipient. Such matrices include, but are not limited to, lanolin, cocoa butter, and polyethylene glycols.
The composition may include a variety of materials that modify the physical form of the solid or liquid dosage unit. For example, the composition may include a material that forms a coating shell around the active ingredient. The material forming the coating shell is generally inert and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient may be enclosed in a gelatin capsule. Compositions in solid or liquid form may include agents that bind to antibodies or antigen binding fragments of the present disclosure and thereby aid in the delivery of the compounds. Suitable agents that may serve this purpose include monoclonal or polyclonal antibodies, one or more proteins or liposomes. The composition may consist essentially of dosage units that may be administered in aerosol form. The term aerosol is used to denote a variety of systems, from systems of a colloidal nature to systems consisting of pressurized packages. Delivery may be by liquefying or compressing the gas or by a suitable pump system for dispensing the active ingredient. Aerosols may be delivered in single phase, biphasic or triphasic systems in order to deliver the active ingredient. The delivery of the aerosol includes the necessary containers, activators, valves, sub-containers, etc., which may together form a kit. The preferred aerosols may be determined by one of ordinary skill in the art without undue experimentation.
It is to be understood that the compositions of the present disclosure also encompass vector molecules (e.g., lipid nanoparticles, nanoscale delivery platforms, etc.) of polynucleotides as described herein.
The pharmaceutical compositions may be prepared by methods well known in the pharmaceutical arts. For example, a composition intended for administration by injection may be prepared by combining a composition comprising an antibody, antigen-binding fragment thereof or antibody conjugate as described herein and optionally one or more of a salt, buffer and/or stabilizer with sterile distilled water to form a solution. Surfactants may be added to promote the formation of a homogeneous solution or suspension. Surfactants are compounds that interact non-covalently with the peptide composition in order to promote the dissolution or uniform suspension of the antibody or antigen binding fragment thereof in an aqueous delivery system.
In general, the appropriate dosages and treatment regimens provide compositions in amounts sufficient to provide therapeutic and/or prophylactic benefit (as described herein, including improved clinical outcome (e.g., reduced frequency, reduced duration, or severity of diarrhea or associated dehydration or inflammation, or no disease survival and/or longer overall survival, or reduced severity of symptoms)), for prophylactic use, the dosages should be sufficient to prevent, delay onset of, or reduce severity of a disease or disorder.
Administering the composition in an effective amount (e.g., to treat influenza virus infection) that will vary depending on a variety of factors, including the activity of the particular compound used; metabolic stability and duration of action of the compound; age, weight, general health, sex, and diet of the subject; mode and time of administration; excretion rate; a pharmaceutical combination; the severity of a particular disorder or condition; and subjects undergoing therapy. In certain embodiments, after administration of a therapy according to the formulations and methods of the present disclosure, the test subject will exhibit a reduction in one or more symptoms associated with the treated disease or disorder of about 10% to about 99% as compared to a placebo-treated or other suitable control subject.
Generally, a therapeutically effective dose of the antibody or antigen binding fragment (for a 70kg mammal) is from about 0.001mg/kg (i.e., 0.07 mg) to about 100mg/kg (i.e., 7.0 g); preferably, the therapeutically effective dose (for a 70kg mammal) is from about 0.01mg/kg (i.e., 0.7 mg) to about 50mg/kg (i.e., 3.5 g); more preferably, the therapeutically effective dose (for a 70kg mammal) is from about 1mg/kg (i.e., 70 mg) to about 25mg/kg (i.e., 1.75 g). For the polynucleotides, vectors, host cells and related compositions of the present disclosure, the therapeutically effective dose may be different from the therapeutically effective dose of the antibody or antigen-binding fragment.
In certain embodiments, the method comprises administering 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times an anti-HA antibody or antigen-binding fragment, polynucleotide, vector, host cell or composition and/or an anti-NA antibody or antigen-binding fragment, polynucleotide, vector, host cell or composition to the subject.
In certain embodiments, the method comprises administering to the subject a plurality of times an anti-HA antibody or antigen-binding fragment, or polynucleotide, or vector, or host cell or composition and/or an anti-NA antibody or antigen-binding fragment, or polynucleotide, or vector, or host cell or composition, wherein the second administration or subsequent administration occurs about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours, about 48 hours, about 74 hours, about 96 hours, or more, respectively, after the first administration or prior administration.
In certain embodiments, the method comprises administering at least one time an anti-HA antibody or antigen-binding fragment, or polynucleotide, or vector, or host cell or composition and/or an anti-NA antibody or antigen-binding fragment, or polynucleotide, or vector, or host cell or composition prior to infection of the subject with influenza.
Combinations or compositions comprising an antibody, antigen-binding fragment of the disclosure (e.g., comprising an anti-HA antibody or antigen-binding fragment and an anti-NA antibody or antigen-binding fragment), polynucleotide, vector, or host cell may also be administered simultaneously, before, or after administration of one or more other therapeutic agents, such as a neuraminidase inhibitor (e.g., oseltamivir, zanamivir, peramivir, or laninamivir). Such combination therapies may include administration of a single pharmaceutical dosage formulation containing a compound of the invention and one or more additional active agents, as well as administration of a composition comprising an antibody or antigen-binding fragment of the present disclosure and each active agent in its own separate dosage formulation. For example, the anti-HA antibody or antigen-binding fragment and the anti-NA antibody or antigen-binding fragment may be administered together to the patient in a single oral dosage composition (e.g., a tablet or capsule), or each agent may be administered in a separate oral dosage formulation. Similarly, the anti-HA antibody or antigen-binding fragment and the anti-NA antibody or antigen-binding fragment may be administered together to the subject in a single parenteral dosage composition (e.g., in a physiological saline solution or other physiologically acceptable solution), or each agent may be administered in a separate parenteral dosage formulation. When separate dosage formulations are used, the composition comprising the antibody or antigen-binding fragment and one or more additional active agents may be administered at substantially the same time (i.e., simultaneously) or separately at staggered times (i.e., sequentially and in any order); combination therapy should be understood to include all such regimens.
In some embodiments, the anti-HA antibody or antigen-binding fragment or polynucleotide is administered concurrently with or within 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 36 hours, 2 to 5 days, 1-2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months of administration of the anti-NA antibody or antigen-binding fragment or polynucleotide.
In some embodiments, the anti-NA antibody or antigen-binding fragment or polynucleotide is administered concurrently with or within 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 36 hours, 2 to 5 days, 1-2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months of administration of the anti-NA antibody or antigen-binding fragment or polynucleotide.
In certain embodiments, the anti-HA antibodies or antigen-binding fragments or polynucleotides are formulated together with the anti-NA antibodies or antigen-binding fragments or polynucleotides. In certain embodiments, the anti-HA antibody or antigen-binding fragment or polynucleotide and the anti-NA antibody or antigen-binding fragment are formulated or polynucleotides separately.
In certain embodiments, the anti-HA antibody or antigen-binding fragment or polynucleotide and the anti-NA antibody or antigen-binding fragment or polynucleotide are administered sequentially. In some embodiments, the antibody (or one or more nucleic acids, host cells, vectors, or compositions, or combinations) is administered to a subject who has previously received one or more anti-inflammatory agents and/or one or more antiviral agents. In some embodiments, the antiviral agent is a ceramidase inhibitor (NAI), e.g., oseltamivir, zanamivir, peramivir, or laninamivir. In some embodiments, one or more anti-inflammatory agents and/or one or more antiviral agents are administered to a subject who has previously received an antibody (or one or more nucleic acids, host cells, vectors, or compositions). In some embodiments, the antiviral agent is a ceramidase inhibitor (NAI), e.g., oseltamivir, zanamivir, peramivir, or laninamivir.
In related aspects, the use of the presently disclosed antibodies, antigen binding fragments, vectors, host cells, and compositions (e.g., in the diagnosis, prevention, and/or treatment of influenza infection, in the manufacture of a medicament for the prevention or treatment of influenza infection) is provided.
The present disclosure also provides the following non-limiting embodiments:
embodiment 1. A combination comprising: (1) (a) an antibody or antigen-binding fragment thereof that is capable of binding to Influenza A Virus (IAV) Hemagglutinin (HA) and neutralizing an infection caused by the IAV, or (b) a polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof; and
(2) (a) an antibody or antigen binding fragment thereof capable of binding to Neuraminidase (NA) from the group consisting of: 2 (i) IAV, wherein the IAV comprises a group 1IAV, a group 2IAV, or both; and 2 (ii) an Influenza B Virus (IBV) and is capable of neutralising infection by and/or inhibiting sialidase activity of said IAV and/or said IBV, or (b) a polynucleotide encoding an anti-NA antibody or antigen binding fragment thereof.
Embodiment 2. A composition, the combination comprising: (1) (a) an antibody or antigen-binding fragment thereof that is capable of binding to Influenza A Virus (IAV) Hemagglutinin (HA) and neutralizing an infection caused by the IAV, or (b) a polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof; and
(2) (a) an antibody or antigen binding fragment thereof capable of binding to Neuraminidase (NA) from the group consisting of: 2 (i) IAV, wherein the IAV comprises a group 1IAV, a group 2IAV, or both; and 2 (ii) an Influenza B Virus (IBV) and is capable of neutralising infection by and/or inhibiting sialidase activity of said IAV and/or said IBV, or (b) a polynucleotide encoding an anti-NA antibody or antigen binding fragment thereof, and optionally a pharmaceutically acceptable carrier, excipient or diluent.
Embodiment 3 the combination according to embodiment 1 or the composition according to embodiment 2 for use in a method of treating or preventing influenza (IAV, IBV or both) infection in a subject, wherein the method comprises administering to the subject an effective amount of the composition or the combination, respectively.
Embodiment 4. The combination according to embodiment 1 or the composition according to embodiment 2 is used for the preparation of a medicament for treating or preventing influenza (IAV, IBV or both) infection in a subject.
Embodiment 5 an antibody or antigen binding fragment thereof that is capable of binding to Influenza A Virus (IAV) Hemagglutinin (HA) and neutralizing an infection by IAV, or a polynucleotide encoding an anti-HA antibody or antigen binding fragment thereof, for use in a method of treating or preventing an influenza infection in a subject, wherein the method comprises administering to a subject that HAs received, is receiving, or is about to receive an effective amount of the anti-HA antibody or antigen binding fragment thereof: (1) An effective amount of an antibody or antigen-binding fragment thereof capable of binding to Neuraminidase (NA) from the group consisting of: (i) IAVs, wherein the IAV comprises a group 1IAV, a group 2IAV, or both; and (ii) an Influenza B Virus (IBV) and is capable of neutralising infection by and/or inhibiting sialidase activity of said IAV and/or said IBV, or (2) a polynucleotide encoding an anti-NA antibody or antigen binding fragment thereof.
Embodiment 6. An antibody or antigen binding fragment thereof that is capable of binding to Neuraminidase (NA) from the group consisting of: (i) Influenza A Virus (IAV), wherein the IAV comprises a group 1IAV, a group 2IAV, or both; and (ii) Influenza B Virus (IBV), and is capable of neutralizing infection by and/or inhibiting sialidase activity of said IAV and/or said IBV, or a polynucleotide encoding an anti-NA antibody or antigen binding fragment thereof, for use in a method of treating or preventing influenza (IAV, IBV or both) infection in a subject, wherein the method comprises administering an effective amount of said anti-NA antibody or antigen binding fragment thereof to a subject that has received, is receiving or will receive an effective amount of: (a) An antibody or antigen-binding fragment thereof that is capable of binding to IAV Hemagglutinin (HA) and neutralizing an infection caused by the IAV, or (b) a polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof.
Embodiment 7. A method of treating or preventing an influenza (IAV, IBV, or both) infection in a subject, the method comprising administering to the subject an effective amount of: (1) (a) an antibody or antigen-binding fragment thereof that is capable of binding to Influenza A Virus (IAV) Hemagglutinin (HA) and neutralizing an infection caused by the IAV, or (b) a polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof; and (2) (a) an antibody or antigen-binding fragment thereof capable of binding to Neuraminidase (NA) from the group consisting of: 2 (i) IAV, wherein the IAV comprises a group 1IAV, a group 2IAV, or both; and 2 (ii) an Influenza B Virus (IBV) and is capable of neutralising infection by and/or inhibiting sialidase activity of said IAV and/or said IBV, or (b) a polynucleotide encoding an anti-NA antibody or antigen binding fragment thereof.
Embodiment 8. A method of treating or preventing an influenza (IAV, IBV, or both) infection in a subject, the method comprising administering to the subject an effective amount of: (1) An antibody or antigen-binding fragment thereof that is capable of binding to Influenza A Virus (IAV) Hemagglutinin (HA) and neutralizing an infection caused by the IAV, or (2) a polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof, wherein the subject HAs received, is receiving, or will receive the following: (a) An antibody or antigen-binding fragment thereof capable of binding to Neuraminidase (NA) from the group consisting of: (i) IAVs, wherein the IAV comprises a group 1IAV, a group 2IAV, or both; and (ii) an Influenza B Virus (IBV) and is capable of neutralising infection by and/or inhibiting sialidase activity of said IAV and/or said IBV, or (b) a polynucleotide encoding an anti-NA antibody or antigen binding fragment thereof.
Embodiment 9. A method of treating or preventing influenza infection in a subject, the method comprising administering to the subject an effective amount of: (1) An antibody or antigen-binding fragment thereof capable of binding to Neuraminidase (NA) from the group consisting of: (i) Influenza A Virus (IAV), wherein the IAV comprises a group 1IAV, a group 2IAV, or both; and (ii) an Influenza B Virus (IBV) and is capable of neutralizing an infection caused by and/or inhibiting sialidase activity of the IAV and/or IBV, or (2) a polynucleotide encoding an anti-NA antibody or antigen binding fragment thereof, wherein the subject has received, is receiving, or will receive the following: (a) An antibody or antigen-binding fragment thereof that is capable of binding to IAV Hemagglutinin (HA) and neutralizing an infection caused by the IAV, or (b) a polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof.
Embodiment 10. The combination according to embodiment 1, the composition according to embodiment 2, the combination or composition for use according to any one of embodiments 3 and 4, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5 and 6 or the method according to any one of embodiments 7 to 9, wherein: the anti-HA antibody or antigen-binding fragment of (1) comprises: (1) (i) a VH comprising or consisting of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to an amino acid sequence set forth in any one of: 43, 2, 26, 28, 31, 34, 37, 14, 39 and 41, wherein the sequence variants referring to SEQ ID NOs 43, 2, 26, 28, 31, 34, 37, 14, 39 and 41, respectively, are optionally included in one or more framework regions and/or the sequence variants include substitution of one or more germline encoded amino acids; and/or (1) (ii) VL comprises or consists of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to an amino acid sequence of any one of: SEQ ID NOS.44, 8 and 20, wherein sequence variations relative to SEQ ID NOS.44, 8 and 20, respectively, are optionally included in one or more framework regions, and/or sequence variations include substitution of one or more germline encoded amino acids; and/or (2) the anti-NA antibody or antigen-binding fragment comprises: (2) (i) a VH comprising or consisting of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to an amino acid sequence set forth in any one of: 241, 48, 60, 72, 171, 84, 96, 108, 120, 132, 229, 144, 156, 168, 180, 192, 204, 245, 249, 258 and 261, wherein the sequence variations of SEQ ID NOs 241, 48, 60, 72, 171, 84, 96, 108, 120, 132, 229, 144, 156, 168, 180, 192, 204, 245, 249, 258, 261 are optionally included in one or more framework regions, and/or the sequence variations comprise substitutions of one or more germline encoded amino acids; and/or (2) (ii) the VL comprises or consists of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to an amino acid sequence of any one of: 243, 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 220, 223, 226, 232, 235, 238, 210, 247, 251, 259 and 263, respectively, wherein the sequence variation of SEQ ID NOs 243, 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 220, 223, 226, 232, 235, 238, 210, 247, 251, 259 and 263 is optionally comprised in one or more framework regions, and/or the sequence variation comprises a substitution of one or more germline encoded amino acids, wherein preferably the anti-HA antibody or antigen binding fragment comprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NOs 43 and 44, respectively, and the anti-NA antibody or antigen binding fragment comprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NOs 241 and 243; and/or (3) the anti-HA antibody or antigen-binding fragment comprises a VH and a VL comprising the HCDR and LCDR of the VH amino acid sequence and VL amino acid sequence, respectively, shown below: (3) (i) SEQ ID NOS 43 and 44, respectively; (3) (ii) SEQ ID NOS 26 and 8, respectively; (3) (iii) SEQ ID NOs 2 and 8, respectively; (3) (iv) SEQ ID NOs 31 and 8, respectively; (3) (v) SEQ ID NOS: 34 and 8, respectively; (3) (vi) SEQ ID NOS 37 and 8, respectively; (3) (vii) SEQ ID NOs 14 and 20, respectively; (3) (viii) SEQ ID NOs 39 and 20, respectively; (3) (ix) SEQ ID NOs 41 and 20, respectively; or (1) (x) is SEQ ID NO 28 and 8, respectively, wherein optionally the HCDR and the LCDR are numbered according to IMGT; and/or (4) the anti-NA antibody or antigen-binding fragment comprises a VH and a VL comprising the HCDR and LCDR of the VH amino acid sequence and VL amino acid sequence, respectively, shown below: (4) (i) SEQ ID NOs 241 and 243, respectively; (4) (ii) SEQ ID NOs 60 and 66, respectively; (4) (iii) SEQ ID NOs 72 and 78 or 220 or 223, respectively; (4) (vi) SEQ ID NOS 72 and 226, respectively; (4) (vii) SEQ ID NOS 217 and 78, respectively; (4) (viii) SEQ ID NOS 217 and 220, respectively; (4) (ix) SEQ ID NOS 217 and 223, respectively; (4) (x) is SEQ ID NO 217 and 226, respectively; (4) (xi) SEQ ID NOs 84 and 90, respectively; (4) (xii) SEQ ID NOS 96 and 102, respectively; (4) (xiii) SEQ ID NOs 108 and 114, respectively; (4) (xiv) SEQ ID NOS 120 and 126, respectively; (4) (xv) SEQ ID NOS 132 and 138, respectively; (4) (xvi) SEQ ID NOS 132 and 232, respectively; (4) (xvii) SEQ ID NOS 132 and 235, respectively; (4) (xviii) SEQ ID NOS 132 and 238, respectively; (4) (xix) SEQ ID NOs 229 and 138, respectively; (4) (xx) SEQ ID NOs 229 and 232, respectively; (4) (xxi) SEQ ID NOs 229 and 235, respectively; (4) (xxii) SEQ ID NOs 229 and 238, respectively; (4) (xxiii) SEQ ID NOS 144 and 150, respectively; (4) (xxiv) SEQ ID NOS 156 and 162, respectively; (4) (xxv) SEQ ID NOS 168 and 174, respectively; (4) (xxvi) SEQ ID NOS 180 and 186, respectively; (4) (xxvii) SEQ ID NOS 192 and 198, respectively; (4) (xxviii) SEQ ID NOS 204 and 210, respectively; (4) (xxix) SEQ ID NOs 48 and 54, respectively; (4) (xxx) SEQ ID NOs 245 and 247, respectively; (4) (xxxi) SEQ ID NOS 249 and 251, respectively; (4) (xxxii) SEQ ID NOs 258 and 259, respectively; or (4) (xxxi) SEQ ID NO:261 and 263, respectively, wherein optionally said HCDR and said LCDR are numbered according to IMGT, wherein preferably said anti-HA antibody or antigen-binding fragment comprises a VH and a VL comprising the HCDR and LCDR of the VH amino acid sequence and VL amino acid sequence shown below, respectively: 43 and 44, and the anti-NA antibody or antigen-binding fragment comprises VH and VL comprising HCDR and LCDR of VH and VL amino acid sequences shown below, respectively: SEQ ID NOs (4) (i) 241 and 243.
Embodiment 11. The combination according to embodiment 1 or 10, the composition according to embodiment 2 or 10, the combination or composition for use according to any of embodiments 3, 4 and 10, the antibody or antigen binding fragment or polynucleotide for use according to any of embodiments 5, 6 and 10 or the method according to any of embodiments 7 to 10, wherein: (1) The VH and the VL of the anti-HA antibody or antigen-binding fragment comprise or consist of amino acid sequences according to seq id no: (1) (i) SEQ ID NOS 43 and 44, respectively; (1) (ii) SEQ ID NOS 26 and 8, respectively; (1) (iii) SEQ ID NOs 2 and 8, respectively; (1) (iv) SEQ ID NOs 31 and 8, respectively; (1) (v) SEQ ID NOS 34 and 8, respectively; (1) (vi) SEQ ID NOS 37 and 8, respectively; (1) (vii) SEQ ID NOs 14 and 20, respectively; (1) (viii) SEQ ID NOs 39 and 20, respectively; (1) (ix) SEQ ID NOs 41 and 20, respectively; or (1) (x) is SEQ ID NO 28 and 8, respectively; and/or (2) the VH and the VL of the anti-NA antibody or antigen-binding fragment comprise or consist of amino acid sequences according to seq id no: (2) (i) SEQ ID NOs 241 and 243, respectively; (2) (ii) SEQ ID NOs 60 and 66, respectively; (2) (iii) SEQ ID NOs 72 and 78 or 220 or 223, respectively; (2) (vi) SEQ ID NOS 72 and 226, respectively; (2) (vii) SEQ ID NOS 217 and 78, respectively; (2) (viii) SEQ ID NOS 217 and 220, respectively; (2) (ix) SEQ ID NOS 217 and 223, respectively; (2) (x) SEQ ID NOS 217 and 226, respectively; (2) (xi) SEQ ID NOs 84 and 90, respectively; (2) (xii) SEQ ID NOs 96 and 102, respectively; (2) (xiii) SEQ ID NOs 108 and 114, respectively; (2) (xiv) SEQ ID NOS 120 and 126, respectively; (2) (xv) SEQ ID NOS 132 and 138, respectively; (2) (xvi) SEQ ID NOS 132 and 232, respectively; (2) (xvii) SEQ ID NOS 132 and 235, respectively; (2) (xviii) SEQ ID NOS 132 and 238, respectively; (2) (xix) SEQ ID NOs 229 and 138, respectively; (2) (xx) SEQ ID NOs 229 and 232, respectively; (2) (xxi) SEQ ID NOs 229 and 235, respectively; (2) (xxii) SEQ ID NOs 229 and 238, respectively; (2) (xxiii) SEQ ID NOS 144 and 150, respectively; (2) (xxiv) SEQ ID NOS 156 and 162, respectively; (2) (xxv) SEQ ID NOS 168 and 174, respectively; (2) (xxvi) SEQ ID NOS 180 and 186, respectively; (2) (xxvii) SEQ ID NOS 192 and 198, respectively; (2) (xxviii) SEQ ID NOS 204 and 210, respectively; (2) (xxix) SEQ ID NOs 48 and 54, respectively; (2) (xxx) SEQ ID NOs 245 and 247, respectively; (2) (xxxi) SEQ ID NOS 249 and 251, respectively; (2) (xxxii) SEQ ID NOs 258 and 259, respectively; or (2) (xxxiii) SEQ ID NOs 261 and 263, respectively, wherein preferably said VH and said VL of said anti-HA antibody or antigen-binding fragment comprise or consist of amino acid sequences according to SEQ ID NOs: 43 and 44, respectively, said VH and said VL of said anti-NA antibody or antigen-binding fragment comprising or consisting of amino acid sequences according to SEQ ID NOs: SEQ ID NOS 241 and 243, respectively.
Embodiment 12 the combination according to embodiment 1, 10 or 11, the composition according to embodiment 2, 10 or 11, the combination or composition for use according to any of embodiments 3, 4, 10 and 11, the antibody or antigen binding fragment or polynucleotide for use according to any of embodiments 5, 6, 10 and 11 or the method according to any of embodiments 7 to 11, wherein: (1) The VH and the VL of the anti-HA antibody or antigen-binding fragment comprise or consist of amino acid sequences according to seq id no: (1) (i) SEQ ID NOs 43 and 44, respectively; (1) (ii) SEQ ID NOS 26 and 8, respectively; (1) (iii) SEQ ID NOS 28 and 8, respectively; (1) (iv) SEQ ID NOs 31 and 8, respectively; (1) (v) SEQ ID NOS 34 and 8, respectively; (1) (vi) SEQ ID NOS 37 and 8, respectively; (1) (vii) SEQ ID NOs 14 and 20, respectively; (1) (viii) SEQ ID NOs 39 and 20, respectively; (1) (ix) SEQ ID NOs 41 and 20, respectively; or (1) (x) is SEQ ID NO 2 and 8, respectively; and/or (2) the VH and the VL of the anti-NA antibody or antigen-binding fragment comprise or consist of amino acid sequences according to seq id no: (2) (i) SEQ ID NOs 241 and 243, respectively; (2) (ii) SEQ ID NOs 72 and 226, respectively; (2) (iii) SEQ ID NOS 217 and 78, respectively; (2) (iv) SEQ ID NOS 217 and 220, respectively; (2) (v) SEQ ID NOS 132 and 138, respectively; (2) (vi) SEQ ID NOS 132 and 232, respectively; (2) (vii) SEQ ID NOs 132 and 235, respectively; (2) (viii) SEQ ID NOS 132 and 238, respectively; (2) (ix) SEQ ID NOs 229 and 138, respectively; (2) (x) SEQ ID NOs 229 and 232, respectively; (2) (xi) SEQ ID NOs 229 and 235, respectively; (2) (xii) SEQ ID NOs 229 and 238, respectively; (2) (xiii) SEQ ID NOS 217 and 223, respectively; (2) (xiv) SEQ ID NOS 217 and 226, respectively; (2) (xv) SEQ ID NOS 72 and 78 or 220 or 223, respectively; (2) (xvi) SEQ ID NOS 245 and 247, respectively; or (2) (xvii) SEQ ID NOS 249 and 251, respectively, wherein preferably said VH and said VL of said anti-HA antibody or antigen-binding fragment comprise or consist of an amino acid sequence according to SEQ ID NO:43 and 44, respectively, said VH and said VL of said anti-NA antibody or antigen-binding fragment comprising or consisting of amino acid sequences according to SEQ ID NOs: SEQ ID NOS 241 and 243, respectively.
Embodiment 13 the combination according to any one of embodiments 1 and 10 to 12, the composition according to any one of embodiments 2 and 10 to 12, the combination or composition for use according to embodiments 3, 4, 10, 11 or 12, the antibody or antigen binding fragment or polynucleotide for use according to embodiments 5, 6, 10, 11 or 12 or the method according to any one of embodiments 7 to 12, wherein: (1) The anti-HA antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) comprising Complementarity Determining Regions (CDRs) H1, CDRH2 and CDRH3 and a light chain variable domain (VL) comprising CDRL1, CDRL2 and CDRL3, wherein the CDRs are optionally according to the IMGT numbering system, and wherein: (1) (i) the CDRH1 comprises or consists of an amino acid sequence as set forth in any one of: 274, 3, 32 and 15, or a functional variant thereof, or consisting of one, two or three acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (1) (ii) the CDRH2 comprises or consists of an amino acid sequence as set forth in any one of: 275, 4, 29, 35, 16 and 42, or a functional variant thereof or consisting of one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (1) (iii) the CDRH3 comprises or consists of an amino acid sequence as set forth in any one of: 276, 5 and 17, or functional variants thereof, or consisting of functional variants thereof, comprising one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (1) (iv) the CDRL1 comprises or consists of an amino acid sequence as set forth in any one of: 277, 9 and 21, or a functional variant thereof comprising or consisting of one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (1) (v) the CDRL2 optionally comprises or consists of an amino acid sequence as set forth in any one of: 278, 10 and 22, or a functional variant thereof, or consisting of, comprising one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (1) (vi) the CDRL3 comprises or consists of an amino acid sequence as set forth in any one of: 279, 11 and 23, or functional variants thereof, or consisting of functional variants thereof, comprising substitutions with one, two or three amino acids, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (2) the anti-NA antibody or antigen-binding fragment comprises a VH comprising CDRH1, CDRH2, and CDRH3, and a VL comprising CDRL1, CDRL2, and CDRL3, wherein: (2) (i) optionally the CDRH1 comprises or consists of an amino acid sequence as set forth in any one of: 193, 49, 61, 73, 85, 97, 109, 121, 133, 145, 157, 169, 181, 205 and 264, or a functional variant thereof comprising or consisting of one, two or three acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; (2) (ii) optionally the CDRH2 comprises or consists of an amino acid sequence as set forth in any one of: 194, 50, 62, 74, 86, 98, 110, 122, 134, 146, 158, 170, 182, 206 and 265, or functional variants thereof, or consisting of functional variants thereof, comprising one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; (2) (iii) the CDRH3 comprises or consists of an amino acid sequence as set forth in any one of: 195, 51, 63, 75, 218, 87, 99, 111, 123, 135, 230, 147, 159, 171, 183, 207 and 266, or a functional variant thereof comprising or consisting of one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; (2) (iv) optionally the CDRL1 comprises or consists of an amino acid sequence as set forth in any one of the following: 199, 55, 67, 79, 91, 103, 115, 127, 139, 151, 163, 175, 187, 211 and 267, or a functional variant thereof comprising or consisting of one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; (2) (v) optionally the CDRL2 comprises or consists of an amino acid sequence as set forth in any one of: 200, 56, 68, 80, 92, 104, 116, 128, 140, 152, 164, 176, 188, 212 and 268, or a functional variant thereof comprising or consisting of one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (2) (vi) optionally the CDRL3 comprises or consists of an amino acid sequence as shown in any one of: 201, 57, 69, 81, 221, 224, 227, 93, 105, 117, 129, 141, 233, 239, 153, 165, 177, 189, 236, 213 and 269, or a functional variant thereof comprising or consisting of a substitution with one, two or three amino acids, one or more of said substitutions optionally being a conservative substitution and/or a substitution of a germline encoded amino acid.
Embodiment 14 the combination according to any one of embodiments 1 and 10 to 13, the composition according to any one of embodiments 2 or 10 to 13, the combination or composition for use according to any one of embodiments 3, 4, 10, 11, 12 or 13, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6, 10, 11, 12 or 13 or the method according to any one of embodiments 7 to 13, wherein: (1) The anti-HA antibody or antigen binding fragment comprises the following CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences: (1) SEQ ID NOS 274-279, respectively; (1) (ii) SEQ ID NOs 3, 29, 5 and 9-11, respectively; (1) (iii) SEQ ID NOs 32, 4, 5 and 9-11, respectively; (1) (iv) SEQ ID NOs 3, 35, 5 and 9-11, respectively; (1) (v) SEQ ID NOs 32, 35, 5 and 9-11, respectively; (1) (vi) SEQ ID NOS 15-17 and 21-23, respectively; (1) (vii) SEQ ID NOs 15, 42, 17 and 21-23, respectively; or (1) (viii) SEQ ID NOs 3-5 and 9-11, respectively; and/or (2) the anti-NA antibody or antigen-binding fragment comprises the following CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences: (2) (i) SEQ ID NOS 193-195 and 199-201, respectively; (2) (ii) SEQ ID NOS: 61-63 and 67-69, respectively; (2) (iii) SEQ ID NOS: 73-75 and 79-81, respectively; (2) (iv) SEQ ID NOS 73, 74, 218 and 79-81, respectively; (2) (v) SEQ ID NOS 73-75, 79, 80 and 221, respectively; (2) (vi) SEQ ID NOS 73-75, 79, 80 and 224, respectively; (2) (vii) SEQ ID NOS 73-75, 79, 80 and 227, respectively; (2) (viii) SEQ ID NOs 73, 74, 218, 79, 80 and 221, respectively; (2) (ix) SEQ ID NOs 73, 74, 218, 79, 80 and 224, respectively; (2) (x) SEQ ID NOs 73, 74, 218, 79, 80 and 227, respectively; (2) (xi) SEQ ID NOS 85-87 and 91-93, respectively; (2) (xii) SEQ ID NOS 97-99 and 103-105, respectively; (2) (xiii) SEQ ID NOS 109-111 and 115-117, respectively; (2) (xiv) SEQ ID NOS 121-123 and 127-129, respectively; (2) (xv) SEQ ID NOS 133-135 and 139-141, respectively; (2) (xvi) SEQ ID NOS 133, 134, 230 and 139-141, respectively; (2) (xvii) SEQ ID NOS 133-135, 139, 141 and 233, respectively; (2) (xviii) SEQ ID NOS 133-135, 139, 141 and 236, respectively; (2) (xix) SEQ ID NOS 133-135, 139, 141 and 239, respectively; (2) (xx) SEQ ID NOS: 133, 134, 184, 139, 141 and 233, respectively; (2) (xxi) SEQ ID NOs 133, 134, 230, 139, 141 and 236, respectively; (2) (xxii) SEQ ID NOs 133, 134, 230, 139, 141 and 239, respectively; (2) (xxiii) SEQ ID NOS 145-147 and 151-153, respectively; (2) (xxiv) SEQ ID NOS 157-159 and 163-165, respectively; (2) (xxv) SEQ ID NOS 169-171 and 175-177, respectively; (2) (xxvi) SEQ ID NOS 181-183 and 187-189, respectively; (2) (xxvii) SEQ ID NOS 49-51 and 55-57, respectively; (2) (xxviii) SEQ ID NOS 205-207 and 211-213, respectively; or (2) (xxix) SEQ ID NOS 264-266 and 267-269, respectively.
Embodiment 15. The combination according to embodiment 13 or 14, the composition according to embodiment 13 or 14, the combination or composition for use according to embodiment 13 or 14, the antibody or antigen binding fragment or polynucleotide for use according to embodiment 13 or 14, or the method according to embodiment 13 or 14, wherein: (1) The anti-HA antibody or antigen binding fragment comprises the following CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences: (1) SEQ ID NOS 274-279, respectively; (1) (ii) SEQ ID NOs 3, 29, 5 and 9-11, respectively; (1) (iii) SEQ ID NOs 32, 4, 5 and 9-11, respectively; (1) (iv) SEQ ID NOs 3, 35, 5 and 9-11, respectively; (1) (v) SEQ ID NOs 32, 35, 5 and 9-11, respectively; (1) (vi) SEQ ID NOS 15-17 and 21-23, respectively; (1) (vii) SEQ ID NOs 15, 42, 17 and 21-23, respectively; or (1) (viii) SEQ ID NOs 3-5 and 9-11, respectively; and/or (2) the anti-NA antibody or antigen-binding fragment comprises the following CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences: (2) (i) SEQ ID NOS 193-195 and 199-201, respectively; (2) (ii) SEQ ID NOS 73, 74, 218 and 79-81, respectively; (2) (iii) SEQ ID NOS 73-75, 79, 80 and 221, respectively; (2) (iv) SEQ ID NOS 73-75, 79, 80 and 224, respectively; (2) (v) SEQ ID NOS 73-75, 79, 80 and 227, respectively; (2) (vi) SEQ ID NOs 73, 74, 218, 79, 80 and 221, respectively; (2) (vii) SEQ ID NOs 73, 74, 218, 79, 80 and 224, respectively; (2) (viii) SEQ ID NOs 73, 74, 218, 79, 80 and 227, respectively; (2) (ix) SEQ ID NOS 133-135 and 139-141, respectively; (2) (x) SEQ ID NOs 133, 134, 230 and 139-141, respectively; (2) (xi) SEQ ID NOS 133-135, 139, 141 and 233, respectively; (2) (xii) SEQ ID NOS 133-135, 139, 141 and 236, respectively; (2) (xiii) SEQ ID NOS 133-135, 139, 141 and 239, respectively; (2) (xiv) SEQ ID NOS 133, 134, 184, 139, 141 and 233, respectively; (2) (xv) SEQ ID NOS 133, 134, 184, 139, 141 and 236, respectively; (2) (xvi) SEQ ID NOs 133, 134, 184, 139, 141 and 239, respectively; (2) (xvii) SEQ ID NOS 264-266 and 267-296, respectively; or (2) (xviii) SEQ ID NOS: 73-75 and 79-81, respectively.
Embodiment 16 the combination according to any one of embodiments 1 and 10 to 15, the composition according to any one of embodiments 2 and 10 to 15, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 15, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 15 or the method according to any one of embodiments 7 to 15, wherein: (i) group 1IAV NA comprises N1, N4, N5 and/or N8; and/or (ii) the group 2IAV NA comprises N2, N3, N6, N7 and/or N9.
Embodiment 17. The combination according to embodiment 16, the composition according to embodiment 16, the combination or composition for use according to embodiment 16, the antibody or antigen binding fragment or polynucleotide for use according to embodiment 16, or the method according to embodiment 16, wherein: (i) The N1 is N1 from any one or more of the following: a/california/07/2009, a/california/07/2009I 23R/H275Y, A/pig/Jiangsu/J004/2018, a/stong holly mole/18/2007, a/brisban/02/2018, a/michigan/45/2015, a/misibbean/3/2001, a/netherlands/603/2009, a/netherlands/602/2009, a/vietnam/1203/2004, a/G4/SW/shandong/1207/2016, a/G4/SW/henna/SN 13/2018, and a/new jersey/8/1976; (ii) the N4 is from a/green duck/netherlands/30/2011; (iii) the N5 is from a/waterfowl/korea/CN 5/2009; (iv) The N8 is from A/plaque seal/new hampshire/179629/2011; (v) The N2 is N2 from any one or more of the following: A/Washington/01/2007, A/China hong Kong/68, A/south Australia/34/2019, A/Switzerland/8060/2017, A/Singapore/INFIMH-16-0019/2016, A/Switzerland/9715293/2013, A/Ninggeler/134/17/57, A/Florida/4/2006, A/Netherlands/823/1992, A/Norway/466/2014, A/Switzerland/8060/2017, A/Texas/50/2012, A/Victoria/361/2011; A/China hong Kong/2671/2019, A/SW/Mexico/SG 1444/2011, A/tank Sania/205/2010, A/Aizhi/2/1968, A/Biterhufen/21793/1972, A/Netherlands/233/1982, A/Shanghai/11/1987, A/Nanchang/933/1995, A/Fujing/45/2004 and A/Brisban/10/2007; (vi) said N3 is from a/canada/rv 504/2004; (v) said N6 is from a/pig/ampere/01911/1/99; (vi) said N7 is from a/netherlands/078/03; and/or (vii) the N9 is N9 from any one or more of: a/Anhui/2013 and a/hong Kong/56/2015.
Embodiment 18 the combination according to any one of embodiments 1 and 10 to 17, the composition according to any one of embodiments 2 and 10 to 17, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 17, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 17 or the method according to any one of embodiments 7 to 17, wherein IBV NA is NA from any one or more of the following: B/Li/10/1940 (ancestor); b/brisbane/60/2008 (victoria); B/Malaysia/2506/2004 (Victoria); B/Malaysia/3120318925/2013 (mountain county); b/wisconsin/1/2010 (mountain county); B/Sorby county/166/1998 (mountain county); B/Brisbane/33/2008; b/state of cororado/06/2017; B/Hubei-Wujiang/158/2009; b/massachusetts/02/2012; B/Netherlands/234/2011; B/Perss/211/2001; B/Texas/06/2011 (mountain county); B/Perss/211/2011; b/hong Kong/05/1972; b/pray island/3073/2013; B/Harbin/7/1994 (Victoria); and B/Washington/02/2019 (Victoria).
Embodiment 19 the combination according to any one of embodiments 1 and 10 to 18, the composition according to any one of embodiments 2 and 10 to 18, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 18, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 18 or the method according to any one of embodiments 7 to 18, wherein the NA is N1, N2 and/or N9.
Embodiment 20 the combination according to any one of embodiments 1 and 10 to 19, the composition according to any one of embodiments 2 and 10 to 19, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 19, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 19, or the method according to any one of embodiments 7 to 19, wherein the anti-NA antibody or antigen binding fragment is capable of binding to: (1) (i) an NA epitope comprising any one or more of the following amino acids (N1 NA numbering): r368, R293, E228, E344, S247, D198, D151, R118; and/or (ii) an NA epitope comprising any one or more of the following amino acids (N2 NA numbering): r371, R292, E227, E344, S247, D198, D151, R118; and/or (2) (i) NA epitopes comprising the amino acids R368, R293, E228, D151 and R118 (N1 NA numbering); and/or (ii) an NA epitope comprising amino acids R371, R292, E227, D151 and R118 (N2 NA numbering); and/or (3) an epitope comprised in or comprising an NA active site, wherein optionally the NA active epitope comprises the following amino acids (N2 numbering): r118, D151, R152, R224, E276, R292, R371, Y406, E119, R156, W178, S179, D/N198, I222, E227, H274, E277, D293, E425; and/or (4) an IBV NA epitope comprising: (i) any one or more of the following amino acids: r116, D149, E226, R292, and R374; or (ii) amino acids R116, D149, E226, R292, and R374.
Embodiment 21. The combination according to embodiment 20, the composition according to embodiment 20, the combination or composition for use according to embodiment 20, the antibody or antigen-binding fragment or polynucleotide for use according to embodiment 20, or the method according to embodiment 20, wherein the anti-NA antibody or antigen-binding fragment is capable of binding to: (1) Further comprising epitopes of any one or more of the following NA amino acids (N2 numbering): e344, E227, S247, and D198; and/or
(2) NA comprising an S245N amino acid mutation and/or an E221D amino acid mutation.
Embodiment 22 the combination according to any one of embodiments 1 and 10 to 21, the composition according to any one of embodiments 2 and 10 to 21, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 21, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 21 or the method according to any one of embodiments 7 to 21, wherein the anti-NA antibody or antigen binding fragment is capable of binding to NA comprising an S245N amino acid mutation and/or an E221D amino acid mutation.
Embodiment 23 the combination according to any one of embodiments 1 and 10 to 22, the composition according to any one of embodiments 2 and 10 to 22, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 22, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 22 or the method according to any one of embodiments 7 to 22, wherein: (i) the group 1IAV NA comprises H1N1 and/or H5N1; (ii) the group 2IAV NA comprises H3N2 and/or H7N9; and/or (iii) the IBV NA comprises one or more of: B/Li/10/1940 (ancestor); b/hong Kong/05/1972; B/Taiwan/2/1962 (ancestor); b/brisbane/33/2008 (victoria); b/brisbane/60/2008 (victoria); B/Malaysia/2506/2004 (Victoria); B/New York/1056/2003 (Victoria); B/Florida/4/2006 (mountain county); B/Jiangsu/10/2003 (mountain county); B/Texas/06/2011 (mountain county); B/Perss/211/2011; B/Harbin/7/1994 (Victoria); b/state of cororado/06/2017 (victoria); b/washington/02/2019 (victoria); B/Perss/211/2001 (mountain county); B/Hubei-wujia sentry/158/2009 (mountain county); b/wisconsin/01/2010 (mountain county); b/massachusetts/02/2012 (mountain county); and B/pray island/3073/2013 (mountain county).
Embodiment 24 the combination according to any one of embodiments 1 and 10 to 23, the composition according to any one of embodiments 2 and 10 to 23, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 23, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 23 or the method according to any one of embodiments 7 to 23, wherein the anti-HA antibody or antigen binding fragment is capable of binding to any one or more of the following IAV subtypes: h1, H2, H3, H4, H5, H8, H9, H10, H11, H12, H13, H14, H15, H17 and H18.
Embodiment 25 the combination according to any one of embodiments 1 and 10 to 24, the composition according to any one of embodiments 2 and 10 to 24, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 24, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 24, or the method according to any one of embodiments 7 to 24, wherein the anti-HA antibody or antigen binding fragment is capable of neutralizing an infection by: (i) An H1N 1IAV, wherein optionally the H1N 1IAV comprises any one or more of: a/california/07/2009, a/PR/8/34 and a/solomon islands/3/06; and (ii) an H3N 2IAV, wherein optionally the H3N 2IAV comprises any one or more of: A/Aizhi/2/68, A/Brisban/10/07 and A/China hong Kong/68; (i) A group 1IAV, wherein optionally said group 1IAV comprises or is an H5 IAV, wherein further optionally said H5 IAV comprises or is an H5/VN/11/94pp; and (ii) a group 2IAV, wherein optionally the group 2IAV comprises or is an H7 IAV, wherein further optionally the H7 IAV comprises or is an H7/IT/99pp, wherein optionally neutralization of infection is determined using a virus pseudotyped with the IAV.
Embodiment 26 the combination according to any one of embodiments 1 and 10 to 25, the composition according to any one of embodiments 2 and 10 to 25, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 25, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 25 or the method according to any one of embodiments 7 to 25, wherein the anti-HA antibody or antigen binding fragment is capable of binding to one or more of the following (i) - (iv): (i) H1 HA, the H1 HA optionally comprising any one or more of: a/england/195/2009; A/Brisban/59/2007; A/Solomon islands/3/2006; A/New karilidoria/20/99; A/Texas state/36/1991; A/Taiwan/01/1986; A/New Jersey state/8/1976; A/Albani/12/1951; A/Mono Sturg/1/1947; A/New York/1/1918; A/Paris/8/34; a/california/07/2009; (ii) H2 HA, said H2 HA optionally comprising a/japan/305/1957; (iii) H5 HA, optionally comprising a/vietnam/1194/2004; and (iv) H9 HA, optionally including A/China hong Kong/1073/99.
Embodiment 27 the combination according to any one of embodiments 1 and 10 to 26, the composition according to any one of embodiments 2 and 10 to 26, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 26, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 26 or the method according to any one of embodiments 7 to 26, wherein the anti-HA antibody or antigen binding fragment, the anti-NA antibody or antigen binding fragment or both are capable of activating human fcγriiia (optionally F158 allele).
Embodiment 28. The combination of embodiment 27, the composition of embodiment 27, the combination or composition for use of embodiment 27, the antibody or antigen-binding fragment or polynucleotide for use of embodiment 27, or the method of embodiment 27, wherein activation is determined using a host cell (optionally Jurkat cells) comprising, after incubating (e.g., 23 hours) the antibody or antigen-binding fragment with a target cell (e.g., an a549 cell) infected with IAV and/or IBV: (i) the human fcyriiia (optionally the F158 allele); and (ii) an NFAT expression control sequence operably linked to a sequence encoding a reporter gene, such as a luciferase reporter gene.
Embodiment 29. The combination of embodiments 27 or 28, the composition of embodiments 27 or 28, the combination or composition for use of embodiments 27 or 28, the antibody or antigen binding fragment or polynucleotide for use of embodiments 27 or 28, or the method of embodiments 27 or 28, wherein activation is determined after incubating the antibody or antigen binding fragment with a target cell infected with H1N1 and/or H3N2 IAV (optionally for about 23 hours), wherein optionally the H1N1 IAV is a/PR8/34, and further optionally the multiplicity of infection (MOI) is 6, and/or wherein the H3N2 IAV is a/aiknow/68, and further optionally the MOI is 18.
Embodiment 30 the combination according to any one of embodiments 1 and 10 to 29, the composition according to any one of embodiments 2 and 10 to 29, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 29, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 29 or the method according to any one of embodiments 7 to 29, wherein the IAV and/or the IBV is antiviral drug resistant, wherein optionally the antiviral drug is oseltamivir.
Embodiment 31 the combination according to any one of embodiments 1 and 10 to 30, the composition according to any one of embodiments 2 and 10 to 30, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 30, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 30 or the method according to any one of embodiment 7, wherein the IAV comprises an N1 NA comprising the following amino acid mutations: H275Y; e119d+h275Y; s247n+h275Y; I222V; and/or N294S, wherein optionally the IAV comprises CA09 or a/aiknow.
Embodiment 32 the combination according to any one of embodiments 1 and 10 to 31, the composition according to any one of embodiments 2 and 10 to 31, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 31, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 32 or the method according to any one of embodiments 7 to 31, wherein the IAV comprises an N2 NA comprising the following amino acid mutations: E119V, Q136K and/or R292K.
Embodiment 33 the combination according to any one of embodiments 1 and 10 to 32, the composition according to any one of embodiments 2 and 10 to 32, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 32, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 32 or the method according to any one of embodiments 7 to 32, wherein the anti-HA antibody or antigen binding fragment, the anti-NA antibody or antigen binding fragment or both are capable of preventing weight loss in a subject infected with the IAV and/or the IBV, optionally for (i) up to 15 days, or (ii) more than 15 days after administration of an effective amount of the antibody or antigen binding fragment.
Embodiment 34 the combination according to any one of embodiments 1 and 10 to 33, the composition according to any one of embodiments 2 and 10 to 33, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 33, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 33 or the method according to any one of embodiments 7 to 33, wherein the anti-HA antibody or antigen binding fragment, the anti-NA antibody or antigen binding fragment or both is capable of preventing weight loss of a subject suffering from an IAV infection and/or an IBV infection by more than 25%, 20%, 15%, 10% or 5% as determined by reference to the weight of the subject immediately prior to the IAV infection and/or the IBV infection.
Embodiment 35 the combination according to any one of embodiments 1 and 10 to 34, the composition according to any one of embodiments 2 and 10 to 34, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 34, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 34 or the method according to any one of embodiments 7 to 34, wherein the anti-HA antibody or antigen binding fragment, the anti-NA antibody or antigen binding fragment or both are capable of extending the survival of a subject suffering from an IAV infection and/or an IBV infection.
Embodiment 36 the combination according to any one of embodiments 1 and 10 to 35, the composition according to any one of embodiments 2 and 10 to 35, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 35, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 35 or the method according to any one of embodiments 7 to 35, wherein the anti-HA antibody or antigen binding fragment, the anti-NA antibody or antigen binding fragment or both are IgG, igA, igM, igE or IgD isoforms.
Embodiment 37 the combination according to any one of embodiments 1 and 10 to 36, the composition according to any one of embodiments 2 and 10 to 36, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 36, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 36, or the method according to any one of embodiments 7 to 36, wherein the anti-HA antibody or antigen binding fragment, the anti-NA antibody or antigen binding fragment, or both are IgG isotypes selected from: igG1, igG2, igG3 and IgG4.
Embodiment 38 the combination according to any one of embodiments 1 and 10 to 37, the composition according to any one of embodiments 2 and 10 to 37, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 37, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 37, or the method according to any one of embodiments 7 to 37, wherein the anti-HA antibody or antigen binding fragment, the anti-NA antibody or antigen binding fragment, or both, comprises a human antibody, a monoclonal antibody, a purified antibody, a single chain antibody, fab ', F (ab') 2, or Fv.
Embodiment 39 the combination according to any one of embodiments 1 and 10 to 38, the composition according to any one of embodiments 2 and 10 to 38, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 38, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 38 or the method according to any one of embodiments 7 to 38, wherein the anti-HA antibody or antigen binding fragment, the anti-NA antibody or antigen binding fragment or both are multispecific antibodies or antigen binding fragments, wherein optionally the multispecific antibodies or antigen binding fragments comprise bispecific antibodies or antigen binding fragments.
Embodiment 40 the combination according to any one of embodiments 1 and 10 to 39, the composition according to any one of embodiments 2 and 10 to 39, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 39, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 39 or the method according to any one of embodiments 7 to 39, wherein the anti-HA antibody or antigen binding fragment, the anti-NA antibody or antigen binding fragment or both comprise an Fc polypeptide or fragment thereof.
Embodiment 41. The combination according to embodiment 40, the composition according to embodiment 40, the combination or composition for use according to embodiment 40, the antibody or antigen binding fragment or polynucleotide for use according to embodiment 40, or the method according to embodiment 40, wherein the Fc polypeptide or fragment thereof comprises: (i) Mutations that enhance binding affinity (e.g., measured using Surface Plasmon Resonance (SPR) (e.g., biacore, e.g., T200 instrument, using manufacturer's protocol)) to human FcRn as compared to a reference Fc polypeptide that does not include the mutations; and/or (ii) a mutation that enhances binding affinity (e.g., measured using Surface Plasmon Resonance (SPR) (e.g., biacore, e.g., T200 instrument, using manufacturer's protocol)) to human fcγr as compared to a reference Fc polypeptide that does not include the mutation.
Embodiment 42. The combination of embodiment 41, the composition of embodiment 41, the combination or composition for use of embodiment 41, the antibody or antigen binding fragment or polynucleotide for use of embodiment 41, or the method of embodiment 41, wherein the mutation that enhances binding affinity to human FcRn comprises: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I; Q311I; D376V; T307A; E380A; or any combination thereof.
Embodiment 43 the combination according to embodiment 41 or 42, the composition according to embodiment 41 or 42, the combination or composition for use according to embodiment 41 or 42, the antibody or antigen binding fragment or polynucleotide for use according to embodiment 41 or 42 or the method according to embodiment 41 or 42, wherein the mutation that enhances binding affinity to human FcRn comprises: (i) M428L/N434S; (ii) M252Y/S254T/T256E; (iii) T250Q/M428L; (iv) P257I/Q311I; (v) P257I/N434H;
(vi) D376V/N434H; (vii) T307A/E380A/N434A; or (viii) any combination of (i) - (vii).
Embodiment 44 the combination according to any one of embodiments 41 to 43, the composition according to any one of embodiments 41 to 43, the combination or composition for use according to any one of embodiments 41 to 43, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 41 to 43 or the method according to any one of embodiments 41 to 43, wherein the mutation that enhances binding affinity to human FcRn comprises M428L/N434S.
Embodiment 45. The combination according to any one of embodiments 41 to 44, the composition according to any one of embodiments 41 to 44, the combination or composition for use according to any one of embodiments 41 to 44, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 41 to 44, or the method according to any one of embodiments 41 to 44, wherein the mutation that enhances binding to fcγr comprises: S239D; I332E; a330L; G236A; or any combination thereof.
Embodiment 46. The combination according to any one of embodiments 41 to 45, the composition according to any one of embodiments 41 to 45, the combination or composition for use according to any one of embodiments 41 to 45, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 41 to 45, or the method according to any one of embodiments 41 to 45, wherein the mutation that enhances binding to fcγr comprises: (I) S239D/I332E; (ii) S239D/A330L/I332E; (iii) G236A/S239D/I332E; or (iv) G236A/A330L/I332E, wherein the Fc polypeptide or fragment thereof optionally comprises Ser at position 239.
Embodiment 47 the combination according to any one of embodiments 1 and 10 to 46, the composition according to any one of embodiments 2 and 10 to 46, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 46, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 46 or the method according to any one of embodiments 7 to 46, wherein the anti-HA antibody or antigen binding fragment, the anti-NA antibody or antigen binding fragment or both comprises a glycosylation altering mutation, wherein the glycosylation altering mutation comprises N297A, N Q or N297G, and/or deglycosylated.
Embodiment 48 the combination according to any one of embodiments 1 and 10 to 47, the composition according to any one of embodiments 2 and 10 to 47, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 47, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 47 or the method according to any one of embodiments 7 to 47, wherein the treatment and/or the prevention comprises post-exposure control.
Embodiment 49 the combination according to any one of embodiments 1 and 10 to 48, the composition according to any one of embodiments 2 and 10 to 48, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 48, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 48, or the method according to any one of embodiments 7 to 48, wherein the subject has received, is receiving, or will receive an antiviral agent, wherein optionally the antiviral agent comprises a neuraminidase inhibitor, an influenza polymerase inhibitor, or both.
Embodiment 50 the combination according to any one of embodiments 1 and 10 to 49, the composition according to any one of embodiments 2 and 10 to 49, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 49, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 49 or the method according to any one of embodiments 7 to 49, wherein the antibody or antigen binding fragment comprises: (i) CH1-CH3, wherein the CH1-CH3 comprises or consists of an amino acid sequence shown as SEQ ID NO. 252; (ii) CH1-CH3, wherein the CH1-CH3 comprises or consists of an amino acid sequence shown as SEQ ID NO 253; (iii) CL comprising or consisting of the amino acid sequence shown as SEQ ID NO. 254; or (iv) any combination of (i) - (iii).
Embodiment 51 the combination according to any one of embodiments 1 and 10 to 50, the composition according to any one of embodiments 2 and 10 to 50, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 50, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 50 or the method according to any one of embodiments 7 to 50, wherein the anti-NA antibody or antigen binding fragment comprises: (1) A heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 255; and (2) a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 257; and/or wherein the anti-HA antibody or antigen-binding fragment comprises: (1) A heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO 270 or 272; and (2) a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:271 or 273.
Embodiment 52 the combination according to any one of embodiments 1 and 10 to 50, the composition according to any one of embodiments 2 and 10 to 51, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 51, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 51 or the method according to any one of embodiments 7 to 51, wherein the anti-NA antibody or antigen binding fragment comprises: (1) A heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 256; and (2) a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 257; and/or wherein the anti-HA antibody or antigen-binding fragment comprises:
(1) A heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO 270 or 272; and (2) a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:271 or 273.
Embodiment 53 the combination according to any one of embodiments 1 and 10 to 51, the composition according to any one of embodiments 2 and 10 to 51, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 51, the antibody or antigen-binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 51 or the method according to any one of embodiments 7 to 51, wherein the anti-NA antibody or antigen-binding fragment comprises: (1) Two heavy chains, each heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 255; and (2) two light chains, each light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 257; and/or wherein the anti-HA antibody or antigen-binding fragment comprises:
(1) Two heavy chains, each heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO 270 or 272; and (2) two light chains, each comprising or consisting of the amino acid sequence shown in SEQ ID NO:271 or 273.
Embodiment 54 the combination according to any one of embodiments 1 and 10 to 50, the composition according to any one of embodiments 2 and 10 and 52, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 50 and 52, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 50 and 52 or the method according to any one of embodiments 7 to 50 and 52, wherein the anti-NA antibody or antigen binding fragment comprises: (1) Two heavy chains, each heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 256; and (2) two light chains, each light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 257; and/or wherein the anti-HA antibody or antigen-binding fragment comprises: (1) Two heavy chains, each heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO 270 or 272; and (2) two light chains, each comprising or consisting of the amino acid sequence shown in SEQ ID NO:271 or 273.
Embodiment 55 the combination according to any one of embodiments 50 to 54, the composition according to any one of embodiments 50 to 54, the combination or composition for use according to any one of embodiments 50 to 54, the antibody or antigen-binding fragment or polynucleotide for use according to any one of embodiments 50 to 54, or the method according to any one of embodiments 50 to 54, wherein the antiviral agent comprises oseltamivir, zanamivir Weila, peramivir, balo Sha Wei, or any combination thereof.
Embodiment 56 the combination according to any one of embodiments 1 and 10 to 55, the composition according to any one of embodiments 2 and 10 to 55, the combination or composition for use according to any one of embodiments 3, 4 and 10 to 55, the antibody or antigen binding fragment or polynucleotide for use according to any one of embodiments 5, 6 and 10 to 55 or the method according to any one of embodiments 7 to 55, wherein: (i) The IAV comprises a group 1IAV, a group 2IAV, or both, wherein optionally the group 1IAV NA comprises N1, N4, N5, and/or N8; and/or the group 2IAV NA comprises N2, N3, N6, N7 and/or N9, wherein further optionally the N1 is from a/california/07/2009, from a/california/07/2009I 23R/H275Y, from a/pig/Jiangsu/J004/2018, from a/ston mole/18/2007, from a/brisban/02/2018, from a/michigan state/45/2015, from a/michigan state/3/2001, from a/netherlands/603/2009, from a/netherlands/602/2009, from a/vietnam/1203/2004, from a/G4/SW/shandong/1207/2016, from a/G4/SW/henna/SN 13/2018 and/or from a/new jersey state/8/1976; the N4 is from A/green duck/Netherlands/30/2011; the N5 is from a/waterfowl/korea/CN 5/2009; the N8 is from A/plaque seal/new hampshire/179629/2011; said N2 is from a/washington/01/2007, from a/chinese hong kong/68, from a/chinese hong kong/2671/2019, from a/south australia/34/2019, from a/swiss/8060/2017, from a/singapore/INFIMH-16-0019/2016, from a/switzerland/9715293/2013, from a/lanugreek/134/17/57, from a/florida/4/2006, from a/netherlands/823/1992, from a/norway/466/2014, from a/texas/50/2012, from a/victoria/361/2011, from a/SW/mexico/SG 1444/2011, from a/known/2/1968, from a/bitterman/21793/1972, from a/netherlands/233/1982, from a/11/1987, a/vannean/1983, a/45/florida/1995, from a/9310/1995, from a/45/samsuntan/2007; the N3 is from A/Canadian/rv 504/2004; the N6 is from A/pig/Annua/01911/1/99; said N7 is from A/Netherlands/078/03; and/or the N9 is from a/security badge/2013, from a/chinese hong kong/56/2015; and/or (ii) the IBV NA is from: B/Li/10/1940 (ancestor); b/brisbane/60/2008 (victoria); B/Malaysia/2506/2004 (Victoria); B/Malaysia/3120318925/2013 (mountain county); b/wisconsin/1/2010 (mountain county); B/Sorby county/166/1998 (mountain county); b/brisbane/33/2008 (victoria); b/state of cororado/06/2017 (victoria); B/Hubei-wujiang/158/2009 (mountain county); b/massachusetts/02/2012 (mountain county); B/Netherlands/234/2011; B/Perss/211/2001 (mountain county); b/praji island/3073/2013 (mountain county); B/Texas/06/2011 (mountain county); b/hong Kong/05/1972; B/Harbin/7/1994 (Victoria); b/washington/02/2019 (victoria); B/Perss/211/2011; or any combination thereof.
Embodiment 57. A multispecific antibody, or antigen-binding fragment thereof, comprising: (i) An antigen binding domain capable of binding to Influenza A Virus (IAV) Hemagglutinin (HA); and (ii) an antigen binding domain capable of binding to Neuraminidase (NA) from: 2 (i) IAV, wherein the IAV comprises a group 1IAV, a group 2IAV, or both; and 2 (ii) Influenza B Virus (IBV).
Embodiment 58. The multispecific antibody or antigen-binding fragment of embodiment 57, comprising a dual variable domain immunoglobulin (DVD-Ig) form.
Embodiment 59. The multispecific antibody or antigen-binding fragment of embodiment 57 or 58, comprising an intra-elbow insertion Ig (IEI-Ig) form.
Embodiment 60. The multispecific antibody of any one of embodiments 57-59, wherein: (1) The anti-HA antigen binding domain comprises the following CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences: (1) SEQ ID NOS 274-279, respectively; (1) (ii) SEQ ID NOs 3, 29, 5 and 9-11, respectively; (1) (iii) SEQ ID NOs 32, 4, 5 and 9-11, respectively; (1) (iv) SEQ ID NOs 3, 35, 5 and 9-11, respectively; (1) (v) SEQ ID NOs 32, 35, 5 and 9-11, respectively; (1) (vi) SEQ ID NOS 15-17 and 21-23, respectively; (1) (vii) SEQ ID NOs 15, 42, 17 and 21-23, respectively; (1) (vii) either SEQ ID NOS 3-5 and 9-11, respectively, or as shown in the variable domain amino acid sequences of SEQ ID NOS 43 and 44, respectively; and/or (2) the anti-NA antigen binding domain comprises the following CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences: (2) (i) SEQ ID NOS 193-195 and 199-201, respectively; (2) (ii) SEQ ID NOS: 61-63 and 67-69, respectively; (2) (iii) SEQ ID NOS: 73-75 and 79-81, respectively; (2) (iv) SEQ ID NOS 73, 74, 218 and 79-81, respectively; (2) (v) SEQ ID NOS 73-75, 79, 80 and 221, respectively; (2) (vi) SEQ ID NOS 73-75, 79, 80 and 224, respectively; (2) (vii) SEQ ID NOS 73-75, 79, 80 and 227, respectively; (2) (viii) SEQ ID NOs 73, 74, 218, 79, 80 and 221, respectively; (2) (ix) SEQ ID NOs 73, 74, 218, 79, 80 and 224, respectively; (2) (x) SEQ ID NOs 73, 74, 218, 79, 80 and 227, respectively; (2) (xi) SEQ ID NOS 85-87 and 91-93, respectively; (2) (xii) SEQ ID NOS 97-99 and 103-105, respectively; (2) (xiii) SEQ ID NOS 109-111 and 115-117, respectively; (2) (xiv) SEQ ID NOS 121-123 and 127-129, respectively; (2) (xv) SEQ ID NOS 133-135 and 139-141, respectively; (2) (xvi) SEQ ID NOS 133, 134, 230 and 139-141, respectively; (2) (xvii) SEQ ID NOS 133-135, 139, 141 and 233, respectively; (2) (xviii) SEQ ID NOS 133-135, 139, 141 and 236, respectively; (2) (xix) SEQ ID NOS 133-135, 139, 141 and 239, respectively; (2) (xx) SEQ ID NOS: 133, 134, 184, 139, 141 and 233, respectively; (2) (xxi) SEQ ID NOs 133, 134, 184, 139, 141 and 236, respectively; (2) (xxii) SEQ ID NOs 133, 134, 184, 139, 141 and 239, respectively; (2) (xxiii) SEQ ID NOS 145-147 and 151-153, respectively; (2) (xxiv) SEQ ID NOS 157-159 and 163-165, respectively; (2) (xxv) SEQ ID NOS 169-171 and 175-177, respectively; (2) (xxvi) SEQ ID NOS 181-183 and 187-189, respectively; (2) (xxvii) SEQ ID NOS 49-51 and 55-57, respectively; (2) (xxviii) SEQ ID NOS 205-207 and 211-213, respectively; or (2) (xxvix) SEQ ID NOS 264-266 and 267-296, respectively.
Embodiment 61 the multispecific antibody or antigen-binding fragment of any one of embodiments 57 to 60, wherein: the anti-HA antigen binding domain of (1) comprises: (1) (i) a VH comprising or consisting of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to an amino acid sequence of any one of the following: 43, 2, 26, 28, 31, 34, 37, 14, 39 and 41, wherein the sequence variations referring to SEQ ID NOs 43, 2, 26, 28, 31, 34, 37, 14, 39 or 41, respectively, are optionally included in one or more framework regions and/or the sequence variations include substitution of one or more germline encoded amino acids; and/or (1) (ii) VL comprises or consists of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to an amino acid sequence of any one of: 44, 8 and 20 or 44, wherein the sequence variation relative to SEQ ID NO 44, 8 or 20, respectively, is optionally comprised in one or more framework regions and/or the sequence variation comprises a substitution of one or more germline encoded amino acids; and/or (2) the anti-NA antigen binding domain comprises: (2) (i) a VH comprising or consisting of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to an amino acid sequence set forth in any one of: 241, 48, 60, 72, 171, 84, 96, 108, 120, 132, 229, 144, 156, 168, 180, 192, 204, 245, 249, 258 and 261, wherein the sequence variations of SEQ ID NOs 241, 48, 60, 72, 171, 84, 96, 108, 120, 132, 229, 144, 156, 168, 180, 192, 204, 245 and 249, 258 and 261 are optionally included in one or more framework regions, and/or the sequence variations comprise substitutions of one or more germline encoded amino acids; and/or (2) (ii) the VL comprises or consists of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to an amino acid sequence of any one of: 243, 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 220, 223, 226, 232, 235, 238, 210, 247, 251, 259 and 263, respectively, wherein the sequence variations of 243, 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 220, 223, 226, 232, 235, 238, 210, 247, 251, 259 and 263 relative to SEQ ID NOs 243, 54, 66, 78, 251, 259 and 263, respectively, optionally comprise a substitution in one or more framework regions, and/or the sequence variations comprise one or more germline encoded amino acids.
Embodiment 62. The multispecific antibody or antigen-binding fragment of any one of embodiments 57 to 61, wherein: (1) The VH and the VL of the anti-HA antigen binding domain comprise or consist of amino acid sequences according to seq id no: (1) (i) SEQ ID NOs 2 and 8, respectively; (1) (ii) SEQ ID NOS 43 and 44, respectively; (1) (iii) SEQ ID NOS 28 and 8, respectively; (1) (iv) SEQ ID NOs 31 and 8, respectively; (1) (v) SEQ ID NOS 34 and 8, respectively; (1) (vi) SEQ ID NOS 37 and 8, respectively; (1) (vii) SEQ ID NOs 14 and 20, respectively; (1) (viii) SEQ ID NOs 39 and 20, respectively; (1) (ix) SEQ ID NOs 41 and 20, respectively; or (1) (x) is SEQ ID NO 26 and 8, respectively; and/or (2) the VH and the VL of the anti-NA antigen binding domain comprise or consist of amino acid sequences according to seq id no: (2) (i) SEQ ID NOs 243 and 243, respectively; (2) (ii) SEQ ID NOs 60 and 66, respectively; (2) (iii) SEQ ID NOs 72 and 78 or 220 or 223, respectively; (2) (vi) SEQ ID NOS 72 and 226, respectively; (2) (vii) SEQ ID NOS 217 and 78, respectively; (2) (viii) SEQ ID NOS 217 and 220, respectively; (2) (ix) SEQ ID NOS 217 and 223, respectively; (2) (x) SEQ ID NOS 217 and 226, respectively; (2) (xi) SEQ ID NOs 84 and 90, respectively; (2) (xii) SEQ ID NOs 96 and 102, respectively; (2) (xiii) SEQ ID NOs 108 and 114, respectively; (2) (xiv) SEQ ID NOS 120 and 126, respectively; (2) (xv) SEQ ID NOS 132 and 138, respectively; (2) (xvi) SEQ ID NOS 132 and 232, respectively; (2) (xvii) SEQ ID NOS 132 and 235, respectively; (2) (xviii) SEQ ID NOS 132 and 238, respectively; (2) (xix) SEQ ID NOs 229 and 138, respectively; (2) (xx) SEQ ID NOs 229 and 232, respectively; (2) (xxi) SEQ ID NOs 229 and 235, respectively; (2) (xxii) SEQ ID NOs 229 and 238, respectively; (2) (xxiii) SEQ ID NOS 144 and 150, respectively; (2) (xxiv) SEQ ID NOS 156 and 162, respectively; (2) (xxv) SEQ ID NOS 168 and 174, respectively; (2) (xxvi) SEQ ID NOS 180 and 186, respectively; (2) (xxvii) SEQ ID NOS 192 and 198, respectively; (2) (xxviii) SEQ ID NOS 204 and 210, respectively; (2) (xxix) SEQ ID NOs 48 and 54, respectively; (2) (xxx) SEQ ID NOs 245 and 247, respectively; (2) (xxxi) SEQ ID NOS 249 and 251, respectively; (2) (xxxii) SEQ ID NOs 258 and 259, respectively; or (2) (xxxiii) SEQ ID NOS 261 and 263, respectively.
Embodiment 63 the multispecific antibody or antigen-binding fragment of any one of embodiments 57 to 62, comprising: (i) CH1-CH3, wherein the CH1-CH3 comprises or consists of an amino acid sequence shown as SEQ ID NO. 252; (ii) CH1-CH3, wherein the CH1-CH3 comprises or consists of an amino acid sequence shown as SEQ ID NO 253;
(iii) CL comprising or consisting of the amino acid sequence shown as SEQ ID NO. 254; or (iv) any combination of (i) - (iii).
Embodiment 64 the multispecific antibody or antigen-binding fragment of any one of embodiments 57 to 63, comprising: (1) A heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 255; and (2) a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 257.
Embodiment 65 the multispecific antibody or antigen-binding fragment of any one of embodiments 57 to 64, comprising: (1) A heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 256; and (2) a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 257.
Embodiment 66. An isolated polynucleotide encoding a multispecific antibody or antigen-binding fragment according to any one of embodiments 57 to 65.
Embodiment 67. A vector comprising the polynucleotide of embodiment 66.
Embodiment 68. A recombinant host cell comprising the isolated polynucleotide according to embodiment 66 and/or the vector according to embodiment 67, and/or expressing a multispecific antibody or antigen-binding fragment according to any one of embodiments 57 to 65.
Embodiment 69. A composition comprising a multispecific antibody or antigen-binding fragment according to any one of embodiments 57 to 65, a polynucleotide according to embodiment 66, a vector according to embodiment 67 and/or a host cell according to embodiment 68, and a pharmaceutically acceptable carrier, excipient or diluent.
Embodiment 70. A method of preventing or treating an influenza a infection, an influenza b infection, or both in a subject, the method comprising administering to the subject an effective amount of a multispecific antibody or antigen-binding fragment of any one of embodiments 57 to 65, the polynucleotide of embodiment 66, the vector of claim 67, the host cell of embodiment 68, and/or the composition of embodiment 69.
Embodiment 71 the multispecific antibody or antigen-binding fragment of any one of embodiments 57 to 65, the polynucleotide of embodiment 66, the vector of embodiment 67, the host cell of embodiment 68, and/or the composition of embodiment 69 for use in a method of treating or preventing influenza a infection, influenza b infection, or both in a subject.
Embodiment 72 the multispecific antibody or antigen-binding fragment of any one of embodiments 57 to 65, the polynucleotide of embodiment 66, the vector of embodiment 67, the host cell of embodiment 68, and/or the composition of embodiment 69 for use in preparing a method for treating or preventing an influenza a infection and/or an influenza b infection.
Embodiment 73 the multispecific antibody or antigen-binding fragment of any one of embodiments 57 to 65, the polynucleotide of embodiment 66, the vector of embodiment 67, the host cell of embodiment 68, the composition of embodiment 69, the method of embodiment 70, or the antibody or antigen-binding fragment for use of any one of embodiments 71 and 72, wherein the anti-HA antibody or antigen-binding fragment, the anti-NA antibody or antigen-binding fragment, or both, is capable of preventing weight loss of a subject having an IAV infection and/or IBV infection by greater than 25%, 20%, 15%, 10%, or 5% as determined by reference to the weight of the subject just prior to the IAV infection and/or IBV infection.
Embodiment 74 the multispecific antibody or antigen-binding fragment of any one of embodiments 57 to 65, the polynucleotide of embodiment 66, the vector of embodiment 67, the host cell of embodiment 68, the composition of embodiment 69, the method of embodiment 70, the antibody or antigen-binding fragment for use of any one of embodiments 71 to 73, wherein the anti-HA antibody or antigen-binding fragment, the anti-NA antibody or antigen-binding fragment, or both, are capable of extending the survival of a subject having an IAV infection and/or IBV infection.
Embodiment 75. A method for treating or preventing influenza infection in a subject, the method comprising administering to the subject: (1) An anti-HA antibody or antigen-binding fragment thereof comprising a VH amino acid sequence shown in SEQ ID No. 43 and a VL amino acid sequence shown in SEQ ID No. 44; and (2) an anti-NA antibody or antigen-binding fragment thereof, comprising the VH amino acid sequence shown in SEQ ID NO. 241 and the VL amino acid sequence shown in SEQ ID NO. 243.
Embodiment 76 a method for treating or preventing influenza infection in a subject, the method comprising administering to the subject a polynucleotide encoding: (1) An anti-HA antibody or antigen-binding fragment thereof comprising a VH amino acid sequence shown in SEQ ID No. 43 and a VL amino acid sequence shown in SEQ ID No. 44; and (2) an anti-NA antibody or antigen-binding fragment thereof, comprising the VH amino acid sequence shown in SEQ ID NO. 241 and the VL amino acid sequence shown in SEQ ID NO. 243.
Embodiment 77. A method for treating or preventing influenza infection in a subject, the method comprising administering to the subject: (1) A polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof comprising the VH amino acid sequence shown in SEQ ID No. 43 and the VL amino acid sequence shown in SEQ ID No. 44; and (2) a polynucleotide encoding an anti-NA antibody or antigen-binding fragment thereof, the anti-NA antibody or antigen-binding fragment thereof comprising the VH amino acid sequence shown in SEQ ID NO. 241 and the VL amino acid sequence shown in SEQ ID NO. 243.
Embodiment 78. A method for treating or preventing influenza infection in a subject, the method comprising administering to the subject: (1) An anti-HA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs 274-276, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs 277-279, respectively; and (2) an anti-NA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOS 193-195, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOS 199-201, respectively.
Embodiment 79. A method for treating or preventing influenza infection in a subject, the method comprising administering to the subject a polynucleotide encoding: (1) An anti-HA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs 274-276, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs 277-279, respectively; and (2) an anti-NA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOS 193-195, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOS 199-201, respectively.
Embodiment 80. A method for treating or preventing influenza infection in a subject, the method comprising administering to the subject: (1) A polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs 274-276, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs 277-279, respectively; and
(2) A polynucleotide encoding an anti-NA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs 193-195, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs 199-201, respectively.
Embodiment 81 the method of any one of embodiments 75 to 80 wherein the antibody or antigen binding fragment of (1) comprises the heavy chain amino acid sequence of SEQ ID NO:270 or SEQ ID NO:272 and the light chain amino acid sequence of SEQ ID NO: 271.
Embodiment 82 the method of any one of embodiments 75 to 81, wherein said antibody or antigen binding fragment of (2) comprises the heavy chain amino acid sequence of SEQ ID NO:255 or SEQ ID NO:256 and the light chain amino acid sequence of SEQ ID NO: 257.
Embodiment 83 the method of any one of embodiments 76 to 82, wherein the polynucleotide of (1) and/or the polynucleotide of (2) comprises mRNA, respectively.
Embodiment 84 the method of any one of embodiments 76 to 83, wherein the polynucleotide of (1) and/or the polynucleotide of (2) comprises a modified nucleoside, cap-1 structure, cap-2 structure, or any combination thereof, respectively.
Embodiment 85 the method of embodiment 84, wherein the polynucleotide of (1) and/or the polynucleotide of (2) comprises, respectively, pseudouridine, N6-methyladenosine, 5-methylcytidine, 2-thiouridine, or any combination thereof.
Embodiment 86. The method of embodiment 85, wherein the pseudouridine comprises N1-methyl pseudouridine.
Embodiment 87. A polynucleotide encoding: (1) An anti-HA antibody or antigen-binding fragment thereof comprising a VH amino acid sequence shown in SEQ ID No. 43 and a VL amino acid sequence shown in SEQ ID No. 44; and (2) an anti-NA antibody or antigen-binding fragment thereof, comprising the VH amino acid sequence shown in SEQ ID NO. 241 and the VL amino acid sequence shown in SEQ ID NO. 243.
Embodiment 88. A polynucleotide encoding: (1) An anti-HA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs 274-276, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs 277-279, respectively; and (2) an anti-NA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOS 193-195, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOS 199-201, respectively.
Embodiment 89. A composition comprising: (1) A polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof comprising the VH amino acid sequence shown in SEQ ID No. 43 and the VL amino acid sequence shown in SEQ ID No. 44; and (2) a polynucleotide encoding an anti-NA antibody or antigen-binding fragment thereof, the anti-NA antibody or antigen-binding fragment thereof comprising the VH amino acid sequence shown in SEQ ID NO. 241 and the VL amino acid sequence shown in SEQ ID NO. 243.
Embodiment 90. A composition comprising: (1) A polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs 274-276, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs 277-279, respectively; and (2) a polynucleotide encoding an anti-NA antibody or antigen-binding fragment thereof, the anti-NA antibody or antigen-binding fragment thereof comprising a VH comprising the CDRH1, CDRH2 and CDRH3 amino acid sequences shown in SEQ ID NOS 193-195, respectively, and a VL comprising the CDRL1, CDRL2 and CDRL3 amino acid sequences shown in SEQ ID NOS 199-201, respectively.
Embodiment 91. A combination, the combination having: (1) A polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof comprising the VH amino acid sequence shown in SEQ ID No. 43 and the VL amino acid sequence shown in SEQ ID No. 44; and (2) a polynucleotide encoding an anti-NA antibody or antigen-binding fragment thereof, the anti-NA antibody or antigen-binding fragment thereof comprising the VH amino acid sequence shown in SEQ ID NO. 241 and the VL amino acid sequence shown in SEQ ID NO. 243.
Embodiment 92. A combination, the combination having: (1) A polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs 274-276, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs 277-279, respectively; and (2) a polynucleotide encoding an anti-NA antibody or antigen-binding fragment thereof, the anti-NA antibody or antigen-binding fragment thereof comprising a VH comprising the CDRH1, CDRH2 and CDRH3 amino acid sequences shown in SEQ ID NOS 193-195, respectively, and a VL comprising the CDRL1, CDRL2 and CDRL3 amino acid sequences shown in SEQ ID NOS 199-201, respectively.
Embodiment 93 the polynucleotide of embodiment 87 or 88, the composition of embodiment 89 or 90, or the combination of embodiments 91 or 92, wherein said antibody or antigen binding fragment of (1) comprises the heavy chain amino acid sequence of SEQ ID NO:270 or 272 and the light chain amino acid sequence of SEQ ID NO: 271.
Embodiment 94 the polynucleotide of embodiment 87, 88 or 93, the composition of embodiment 89, 90 or 93, or the combination of embodiments 91, 92 or 93, wherein the antibody or antigen binding fragment of (2) comprises the heavy chain amino acid sequence of SEQ ID NO:255 or SEQ ID NO:256 and the light chain amino acid sequence of SEQ ID NO: 257.
Embodiment 95 the polynucleotide of embodiment 87, 88, 93, or 94, the composition of embodiment 89, 90, 93, or 94, or the combination of any of embodiments 91-94, wherein the polynucleotide of (1) and/or the polynucleotide of (2), respectively, comprises mRNA.
Embodiment 96 the polynucleotide of embodiment 87, 88, 93, 94, or 95, the composition of embodiment 89, 90, 93, 94, or 95, or the combination of any of embodiments 91-94, wherein the polynucleotide of (1) and/or the polynucleotide of (2) comprises a modified nucleoside, cap-1 structure, cap-2 structure, or any combination thereof, respectively.
Embodiment 97. The polynucleotide, composition, or combination of embodiment 96, wherein the polynucleotide of (1) and/or the polynucleotide of (2) comprises, respectively, pseudouridine, 2-thiouridine, N6-methyladenosine, 5-methylcytidine, or any combination thereof.
Embodiment 98. The polynucleotide, composition, or combination of embodiment 97, wherein the pseudouridine comprises N1-methyl pseudouridine.
Table 1. Certain sequences and SEQ ID no:
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TABLE 2 alignment of neuraminidase amino acid positions (H1N 1 California 07.2009 versus H3N2 New York 392.2004)
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Examples
Example 1
NA and HA resistance
Functional testing of monoclonal antibodies and combinations of these
anti-Hemagglutinin (HA) antibodies and anti-Neuraminidase (NA) antibodies were isolated from donor tonsils and PBMC samples.
For HA antibodies, peripheral Blood Mononuclear Cells (PBMCs) from anonymous donors were selected based on neutralization by corresponding sera against H5 (group 1) and H7 (group 2) influenza pseudoviruses. Donors were selected by screening serum from tonsil donor samples (n=50) for reactivity against hemagglutinin subtype H5 and H7 antigens and serum from PBMC donor samples (n=124) for reactivity against H5 and H7 subtype pseudoviruses. Binding was assessed by FACS. B memory cells from five donors were sorted by flow cytometry for input into the discovery workflow. The individually sorted B cells (n= 6,700) were co-cultured with Mesenchymal Stem Cells (MSCs) in 50 μl of culture to stimulate antibody secretion. The secreted antibodies were evaluated using binding and pseudovirus neutralization assays. Binding to HA from group I Influenza A Virus (IAV), group II IAV and influenza b virus was assessed by enzyme-linked immunosorbent assay (ELISA) to determine breadth. After infection of target cells with pseudoviral particles expressing H5 or H7 luciferase (Luc), neutralization was assessed by monitoring luciferase expression-measured in a manner that blocked viral entry and uncoating. Antibody sequences from selected B cells were cloned into cDNA and sequenced.
The clone-associated anti-HA antibodies "FHF11" and "FHF12" were selected for further investigation and sequence variants of these antibodies with one or more variable domain mutations were generated (see Table 1; SEQ ID NO: 1-42). FHF11 and FHF12 each bind to several HA circulating in animal reservoirs by FACS and to group 1 (H1, H2, H5, H9) and group 2 (H3) HA by ELISA. These antibodies also bind to H1A/pig/Jiangsu/J004/2018 by FACS, but do not exhibit multiple reactivity against healthy human epithelial type 2 (HEP-2) cells. FHF11 activates FcgammaRIIIa (F158) in the presence of H1N1 and H3N2, similar to FM08_LS or to a greater extent than FM 08_LS. FHF11 also activates FcgammaRIIa (H131) in the presence of H1N1 and H3N2, similar to FM08_LS or to a greater extent than FM 08_LS.
Various other experiments were performed to characterize FHF11 and FHF12 (parent and variant) antibodies. For example, FHF11v3, FHF11v6 and FHF11v9 bind to a panel of H3N2, H1N1, H2N2, H3N1 and H9N2 subtypes by ELISA. FHF11, FHF11v3, FHF11v6, FHF11v9, FY1 and FM08 bind to HA-5 through BLI with a Kd value of less than 1.0E-12 and similar or lower affinity than HA-7. FHF11, FHF11v3, FHF11v6 and FHF11v9 neutralize H5 pp with IC50 values between about 0.7 and 0.2 ng/mL. Antibodies were examined for neutralization of various H1N1 and H3N2 viruses and activation of fcγr in the presence of H1N1 and H3N 2. The in vivo pharmacokinetics of FHF11v9-LS, FHF12-LS and FM08-LS in tg32 mice were evaluated. The control effect and pharmacokinetics of FHF11v9 in BALB/c mice pre-treated with antibodies and then infected with H1N 1A/Podoc/8/34 or H3N 2A/hong Kong/68 in China and in SCID tg32 mice were also studied.
For NA antibodies, peripheral Blood Mononuclear Cells (PBMCs) from anonymous donors were selected based on binding of corresponding serum to N1 and N4 (G1) and N2, N3 and N9 (G2) influenza pseudoviruses. Donors were selected by screening serum from tonsil donor samples (n=50) for reactivity to neuraminidase subtypes N1 and N2 antigens, and serum from PBMC (peripheral blood mononuclear cells) donor samples (n=124) for reactivity to neuraminidase subtypes N4, N3 and N9. The neuraminidase antigen used for screening was expressed in mammalian cells, and binding was assessed by FACS flow cytometry.
B memory cells from five donors were sorted by flow cytometry for input into the discovery workflow. Individual sorted B cells (n= 39,350) were co-cultured with Mesenchymal Stromal Cells (MSCs) in 50 μl culture to stimulate antibody secretion. Secreted antibodies were evaluated by binding and NA inhibition assays. Inhibition of N1 sialidase activity was assessed using ELLA (enzyme linked lectin assay), an absorbance-based assay that utilizes the macroglycoprotein substrate fetuin as a substrate for NA cleavage of sialic acid (Lambre et al J immunomethods 1990). Inhibition of N1, N2 and N9 sialidase activity was measured using a fluorescence-based assay that measures cleavage of 2' - (4-methyl umbrella) - α -D-N-acetylneuraminic acid (MUNANA) by NA enzyme (Potier et al, analytical biochemistry 1979.
Binding to NA from group 1IAV N1A/Vietnam/1203/2004 and group 2IAV N2A/tank Sania/205/2010 and N9A/hong Kong/56/2015 was evaluated by ELISA to determine breadth. Antibody sequences from selected B cells were cloned into cDNA and sequenced.
Fourteen clone-related monoclonal antibodies ("FNI" -prefix) generated the discovery procedure. These antibodies, and antibodies containing Unmutated Common Ancestor (UCA) VH and VL, exhibited binding to the breadth of IAV and IBV NA. Antibodies FNI3 and FNI demonstrate comparable or even stronger binding to NA (N1, N2, N9) by ELISA and BLI relative to reference antibody 1G01 (Stadlbauer et al science 366 (6464): 499-504 (2019)). FNI3 and FNI show comparable or stronger binding to the set of group 1 and group 2IAV NA and stronger binding to the set of IBV NA compared to 1G 01. FNI3 and FNI inhibit sialidase activity of H3N 2IAV NA, which H3N 2IAV NA comprises glycosylation motifs at positions 245 (245 Gly+) and 247 (247 Gly+) (Wan et al, nat microbiology.) (2019): a/south Australia/34/2019, A/Switzerland/8060/2017, A/Singapore/INFIMH-16-0019/2016 and A/Switzerland/9715293/2013. FNI3 and FNI bind to N2A/south Australia/34/2019 and N1A/pig/Jiangsu/J004/2018, but do not exhibit multiple reactivity against healthy Hep2 cells. FNI3 and FNI demonstrate strong NAI activity against the IAV G1 NA, IAV G2 NA and IBV NA kits. Other experiments demonstrated neutralization of H1N1, H3N2, B/MAL (Victoria lineage), B/JIA (Shandong county lineage) and inhibitory activity of FNI3 on H1N1 and H3N2 by the FNI antibody, which FNI3 was engineered to include oseltamivir resistance mutations with greater potency than oseltamivir, FM08, and 1G 01.
Structural studies showed that CDRH3 of FNI interacted with NA active site occupied by oseltamivir. The FNI epitope is conserved in the N2 NA sequence from H3N2 (n= 60,597) and in the N1 NA sequence from H1N1 (n= 57,597), the H3N2 and H1N1 being separated between year 2000 and 2020. FNI3 and FNI9 were also tested in a mouse control model against LD 90H 1N1PR8 or H3N2 HK/68 influenza; pretreated mice showed a lack of overall dose-dependent weight loss. Survival and weight loss of mice treated with FNI3 or FNI improved over vehicle in 15 day studies infected with H3N 2A/hong Kong/8/1968. Half-lives of FNI3 and FNI9 carrying MLNS Fc mutations were improved in tg32 mice (average 12.034 days (SD 1.781 days) and 14.198 days (SD 2.014 days), respectively) compared to FM08_mlns (8.072 days; SD 1.567 days). The calculated half-life of 1g01_mlns was 12.636 days (SD 2.23 days).
Antibodies FNI and FNI19 were also studied. These antibodies have a considerable breadth of binding to FNI3 and FNI9 NA and their binding is improved compared to these antibodies against certain NA. FNI17 and FNI effectively neutralize the set of H3N2 and H1N1 IAVs and the set of IBVs. Improved neutralization was demonstrated compared to FM08 and FHF11, FNI, FNI, FNI17 and FNI19 for the H1N1 and H3N2 virus kits, as measured by nucleoprotein staining. FNI3, FNI, FNI, and FNI19 neutralize the same set of viruses with greater potency than oseltamivir and activate Jurkat cells infected with H1N1PR8 (moi=6) and fcγriiia on a549 and to a lesser extent fcγriia. These antibodies also activated fcyriiia in the presence of N1 and N2 IAV NA and IBV NA, while FNI and FNI but not FNI17 and FNI19 activated fcyriiia in the presence of N9 NA. FNI3-LS, FNI9-LS, FNI17-LS and FNI-LS showed improved half-life in SCID tg32 mice over FM08-LS, FNI9-LS and FNI-LS within 30 days after administration. FNI3-LS, FNI9-LS, FNI17-LS and FNI-LS are not multi-reactive to Hep-2 cells. Structural studies showed that FNI, FNI, 17 and FNI19 have similar docking orientations for NA. CDRH3 of these antibodies are very similar in their amino acid sequence. Sequence variants of FNI and FNI were generated. The in vivo half-life of FNI17v19 and FNI v3 was improved in tg32 mice (5 mg/kg antibody injected intravenously) compared to the corresponding parent antibodies (FNI v 19-ls=14.88±3.27 days; FNI v 17-ls=8.86±0.57 days; FNI v 3-ls=14.40±2.13 days; FNI v 9-ls=11.57±0.63 days). FNI17v19 and FNI v19 inhibit sialidase activity of a panel of influenza viruses of the H1N1, H3N2, B/Victoria lineage and B/Shandong county lineage. The survival of FNI-LS was improved over FM08-LS in twelve days in BALB/c mice infected with H1N 1A/Bodoc/8/34. Additional characterization was performed using FNI antibodies including FNI17v19 and FNI19v 3.
anti-HA FHF11 (VH: SEQ ID NO:2; VL: SEQ ID NO: 8) and anti-NA FNI3 (VH: SEQ ID NO:72;VL:SEQ ID NO:78), FNI (VH: SEQ ID NO:132;VL:SEQ ID NO:138), FNI17 (VH: SEQ ID NO:192;VL:SEQ ID NO:198), FNI-v 19 (VH: SEQ ID NO:241;VL:SEQ ID NO:243), FNI (VH: SEQ ID NO:204;VL:SEQ ID NO:210) and FNI-v 3 (VH: SEQ ID NO:245;VL:SEQ ID NO:247) are non-limiting examples of the aforementioned antibodies that bind with high affinity to antigens from various influenza viruses and have robust neutralizing activity against said influenza viruses.
The ability of the anti-na+ anti-HA antibody combination to inhibit sialidase activity was evaluated in an in vitro assay. anti-NA antibodies FNI and FNI9 and anti-HA monoclonal antibodies FHF11 and "FM08" (VH: SEQ ID NO:43;VL:SEQ ID NO:44; see also Kallewaard et al, cell 166 (3): 596-608 (2016)) were tested for sialidase inhibition using a fluorescence-based assay that measures cleavage of 2' - (4-methyl umbrella) -alpha-D-N-acetylneuraminic acid (MUNANA) (Potier et al, analytical biochemistry 1979). Inhibition of H1N1 Cal/09 sialidase activity by FM08+ FNI3 (FIG. 1A), FM08+ FNI9 (FIG. 1B) and FHF11+ FNI9 (FIG. 1C) was measured. In addition, inhibition of H3N2 HK/68 sialidase activity by FM08+ FNI3 (FIG. 1D), FM08+ FNI9 (FIG. 1E), FHF11+ FNI9 (FIG. 1F) was also tested. A heat map was generated to visualize neutralization (%) between each antibody pair tested (fig. 1A-1F, upper panel) and synergy/antagonism scores (fig. 1A-1F, lower panel) at μg/ml. These data show the synergistic neutralizing effect of the anti-ha+anti-NA antibody combination.
The ability of the anti-na+ anti-HA antibody combination to inhibit sialidase activity was also assessed using nucleoprotein staining. anti-NA antibody FNI (VH: SEQ ID NO:132;VL:SEQ ID NO:138), FNI17 (VH: SEQ ID NO:192;VL:SEQ ID NO:198) and FNI19 (VH; SEQ ID NO:204;VL:SEQ ID NO:210) were tested as anti-HA monoclonal antibody FM08. Inhibition of H3N 2A/hong Kong/1/1968 sialidase activity by FM08+ FNI9 (FIG. 2A), FM08+ FNI17 (FIG. 2B) and FM08+ FNI19 (FIG. 2C) was measured. A heat map was generated to visualize neutralization (%) between each antibody pair tested (fig. 2A-2C, upper panel) and synergy/antagonism scores (fig. 2A-2C, lower panel) at μg/ml. A synergy matrix and score are generated using macsynergy ii. These data show the synergistic neutralizing effect of the anti-ha+anti-NA antibody combination. The ability of anti-NA antibodies, anti-HA antibodies, and combinations thereof to activate FcgammaRIIIa (FIG. 3A; F158 allele) and FcgammaRIIa (FIG. 3B; H131 allele) was tested using an NFAT-driven luciferase reporter assay. After contact with a549 cells prior to infection with H1N 1A/PR/8/34, the anti-NA (FNI, FNI 9) and anti-HA variant antibody fhf11_v9 (VH: SEQ ID NO:37;VL:SEQ ID NO:8) antibodies, alone, and in combination, were tested using the NFAT-mediated luciferase reporter gene in engineered Jurkat cells. Activation of the comparative FM08_ls comprising the M428L and N434S (EU numbering) Fc mutations and the negative control antibody "K-" against the unrelated antigen was also measured. These data show that anti-ha+anti-NA antibody combinations can improve fcγr activation in the context of H1N1 influenza infection.
The anti-NA 1G01-LS, anti-HA FM08-LS and combinations of both were also tested for FcgammaRIIIa and FcgammaRIIa activation using a luciferase assay. Fcγriiia activation was measured after contact with a549 cells prior to infection with H1N 1A/PR/8/34 (fig. 4A) or H3N 2A/aizhi/2/68 (fig. 4B). Fcγriia activation was measured after contact with a549 cells prior to infection with H1N 1A/PR/8/34 (fig. 5A) or H3N 2A/aizhi/2/68 (fig. 5B). Activation by the negative control antibody (FY 1-LALA) was also measured.
Example 2
Control activity of anti-NA/anti-HA antibody combinations
The control activity of the anti-NA antibody (IG 01), the anti-HA antibody (FM 08) and combinations of both were evaluated in the IAV infected murine BALB/c model. Briefly, 1G01, FM08, 1g01+fm08 or vehicle control (fig. 6A and 6B) was administered (intravenously) to 7-8 week old BALB/c mice one day prior to intranasal infection with LD90 (90% lethal dose) with subtype H1N1 a/polo each/8/34. Each antibody was administered at 1.0, 0.5, 0.25 or 0.125mg/kg (i.v.). Baseline serum was collected at the beginning of infection and both body weight and mortality were assessed on each of 2-14 days post infection (fig. 6B). Weight measurements over fifteen days are shown in FIGS. 7A-7C (1.0 mg/kg test group), FIGS. 7D-7F (0.5 mg/kg test group), FIGS. 7G-7I (0.25 mg/kg test group), and FIGS. 7J-7L (0.125 mg/kg test group). Specifically, improvements were observed with the combination at 0.25mg/kg of each antibody.
Also shown are the comparison of the area peak under the negative curve from the area under the curve analysis of weight loss with IgG in serum for BALB/c mice infected with a/polis/8/34 after treatment with 1G01, FM08 or 1g01+fm08 (fig. 8A-8B). The area peak under the negative curve is graphically represented by the amount of each mAb administered in mg/kg (fig. 8A) or the amount of total antibody (fig. 8B). Area under the curve analysis of weight loss in BALB/c mice infected with a/polis/8/34 after treatment with 1g01+fm08 was analyzed using Compusyn software (combosyn.com).
Dose-response curves (fig. 9A) and isobolograms (i.e., equal effective curves, fig. 9B) for 50%, 75% and 90% inhibition are shown. Quantitatively defined combination indices of synergy, additive effects and antagonism were also determined (fig. 9C).
Serum human IgG was measured 24 hours after antibody injection and immediately prior to infection with LD90 (90% lethal dose) a/polo each/8/34 from mice according to the timeline shown in fig. 6B. FIG. 10A shows human IgG reported in μg/ml in serum 24 hours after antibody injection. Figure 10B shows a comparison of the area peak under the H1N1 negative curve in the area analysis under the curve of weight loss (figures 8A-8B) with IgG and EC50 (half maximal effective concentration) values in serum. Overall mortality was also measured (fig. 11, fig. 12A-12B). The survival of animals receiving even the lowest dose of antibody combination (0.125 mg/kg per mAb; FIG. 11) was improved compared to some higher doses of antibody alone.
Example 3
Additional studies
Neutralization of additional H1N1 and H3N2 viruses, and anti-HA or anti-NA Monoclonal Antibody Resistance Mutants (MARMs) influenza by anti-HA and anti-NA FNI antibody combinations (including fhf11+fni, fhf11+1g01, and FM08+ FNI) were tested in vitro. In vitro resistance selection assays were performed. Bispecific anti-HA x anti-NA antibodies were generated in the DVD-Ig and IEI-Ig formats. In vivo studies were performed using Balb/c mice to test the control activity of anti-HA alone, anti-NA alone, anti-HA x anti-NA combinations (e.g., fhf11+fni, fhf11+1g01, and FM08+ FNI) and bispecific antibodies against H1N1 PR8 and H3N2 HK/68 (LD 90). Four doses were used for each test article. The endpoint of the study was weight loss and survival up to 14 days post infection.
Example 4
Design and in vitro testing of Dual Variable Domain (DVD) bispecific antibodies
Double Variable Domain (DVD) bispecific format antibodies containing anti-NA and anti-HA antibodies were designed and generated. Representative DVD bispecific antibody FNI-L-FM 08-DVDIg1-LS containing anti-NA (FNI) and anti-HA (FM 08) antigen binding domains are shown in FIG. 13.
In vitro inhibition of sialidase activity by FNI-FM 08-DVD was evaluated. The comparator test set included FNI mAb alone, FNI17+FM08 mAb or FM08 mAb alone for H1N1Cal/09 (FIG. 14A) and H3N2 HK/68 (FIG. 14B). The IC50 value (nM) was calculated for each test group.
In vitro neutralization of H5 and H7 pseudotyped viruses by FM08-FNI9-DVD, FNI9-FM08-DVD, FM 08-FNI-DVD and FNI-FM 08-DVD was also evaluated. The comparative antibody FM08 was also tested. FIG. 15A shows neutralization of H5/VN1194 pp. FIG. 15B shows the neutralization of H7/IT/99 pp. The calculated IC50 values (nM) are shown below the graph in each graph.
Antibody activation of FcgammaRIIIa (FIG. 16A; F158 allele) and FcgammaRIIa (FIG. 16B; H131 allele) on FM08-FNI17-DVD and FNI17-FM08-DVD was tested. NFAT-mediated luciferase reporter gene activation in engineered Jurkat cells was used to measure activation. The comparative antibodies FM08_LS, FHF12-LS, FHF11-v9-LS and negative control antibody (FY 1-LALA) were also evaluated.
Example 5
In vivo testing of Double Variable Domain (DVD) bispecific antibodies
The control activity of FNI-FM 08-DVD was evaluated in the IAV infected murine BALB/c model. Briefly, FM08_LS (TA 1, "mAb-08" in FIG. 17), FNI17_LS (TA-2, "mAb-17" in FIG. 17), FM08_LS+ FNI17_LS (TA 3, "mAb-08+mAb-17" in FIG. 17) and FNI17-FM08-DVD-LS (TA 4, "DVD format" in FIG. 17) were administered (intravenously) to 7-8 week old BALB/c mice.
Antibodies were administered the day prior to intranasal infection with LD90 (90% lethal dose) of H1N1 subtype. Antibodies were administered at 0.125, 0.25, 0.5 or 1mg/kg (intravenous). Baseline serum was collected at the beginning of infection and both body weight and mortality were assessed on each of 2-14 days post infection. Body weight was measured fifteen days prior to infection with LD90 (90% lethal dose) H1N1, after pretreatment with 1mg/kg (FIG. 18A), 0.5mg/kg (FIG. 18B), 0.25mg/kg (FIG. 18C), or 0.125mg/kg (FIG. 18D) of the indicated monoclonal antibody. Mice in the FNI-FM 08-DVD-LS treatment group received equivalent amounts of molecules corresponding to the body weight dose (mg/kg) of the other three treatment groups. The overall mortality of BALB/c mice infected with H1N1 and pre-treated with either FM08_ls or FNI17_ls (fig. 19A), or FM08_ls+ FNI17_ls or FNI17-FM08-DVD-LS (fig. 19B) was also measured over fifteen days. Body weight data are summarized in figure 20.
The various embodiments described above may be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patent applications, and non-patent publications cited in this specification and/or listed in the application data sheet are incorporated herein by reference in their entirety, including U.S. provisional application No. 63/117,454, filed 11/23/2020; U.S. provisional application No. 63/125,892, provided at 12/15 of 2020; U.S. provisional application No. 63/197,254, filed on 4 th month 6 of 2021; and U.S. provisional application No. 63/261,464 filed on month 21 of 2021. Aspects of the embodiments can be modified, if necessary, to employ concepts of the various patents, applications and publications to provide yet further embodiments.
These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present disclosure.
Sequence listing
<110> Weir Biotech Co., ltd (Vir Biotechnology, inc.)
Hu Mo Boss biomedical Co., ltd (Humabs BioMed SA)
<120> anti-influenza antibodies and combinations thereof
<130> 930585.415WO
<150> US 63/117,454
<151> 2020-11-23
<150> US 63/125,892
<151> 2020-12-15
<150> US 63/197,254
<151> 2021-06-04
<150> US 63/261,464
<151> 2021-09-21
<160> 281
<170> patent In version 3.5
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cagtccccat cgagaggcct tgagtggctg ggaaggacat attacaggtc caagtggtat 180
aatgattatg cagtctctgt gaaaagtcga ataaccatca atccagacac atccaagaac 240
cagttctccc tacagttgat ctctgtgact cccgaggaca cggctgtcta ttactgtgca 300
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cctggcaagc ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180
gacaggttca gtggcagtgg gtctgggaca gacttcagtc tcaccatcag cagtctggag 240
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Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Gln Ser Leu Ser Pro Gly
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Asp Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Ser Arg Ser
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Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Ser Leu Glu
65 70 75 80
Pro Glu Asp Ser Ala Met Tyr Phe Cys Gln Tyr Tyr Gly Asp Ser Pro
85 90 95
Leu Phe Ser Phe Gly Pro Gly Thr Lys Val Asp Ile Lys
100 105
<210> 21
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> FHF12 CDR-L1 (aa)
<400> 21
Gln Ser Leu Ser Arg Ser Tyr
1 5
<210> 22
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> FHF12 CDR-L2 (aa)
<400> 22
Gly Ala Ser
1
<210> 23
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FHF12 CDR-L3 (aa)
<400> 23
Gln Tyr Tyr Gly Asp Ser Pro Leu Phe Ser
1 5 10
<210> 24
<211> 327
<212> DNA
<213> artificial sequence
<220>
<223> FHF12 Vk (co-nt)
<400> 24
gagatcgtgc tgacccagtc tcctggcaca cagagcctgt ctccaggcga cagggccacc 60
ctgtcctgca gggcttccca gagcctgtct aggtcctacc tggcctggta tcagcagaga 120
ccaggcaagc cacctaggct gctgatctac ggagcttcca gcagggctac aggcatccct 180
gacagattca gcggctctgg ctccggcacc gatttttccc tgacaatctc ttccctggag 240
ccagaggact ccgccatgta tttctgtcag tactatggcg atagcccact gttctctttt 300
ggccccggca ccaaggtgga tatcaag 327
<210> 25
<211> 381
<212> DNA
<213> artificial sequence
<220>
<223> FHF11-VH W36F (nt)
<400> 25
caggtgcagc tgcagcagtc tggaccagga ctggtgaagc ctagccagac cctgtctgtg 60
acatgcggaa tctccggcga cagcgtgtcc agccactccg ccgctttcaa ctggatcaga 120
cagagcccat ctaggggact ggagtggctg ggaaggacct actatcggag caagtggtac 180
aatgactatg ccgtgtctgt gaagtccagg atcaccatca acccagatac atccaagaat 240
cagttcagcc tgcagctgat ctctgtgacc cccgaggaca cagccgtgta ctattgtgcc 300
agagtgggcg ctatgacctt tggcctgctg acaggcggaa tggacgtgtg gggacaggga 360
accacagtga cagtgtcttc c 381
<210> 26
<211> 127
<212> PRT
<213> artificial sequence
<220>
<223> FHF11-VH W36F (aa)
<400> 26
Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Val Thr Cys Gly Ile Ser Gly Asp Ser Val Ser Ser His
20 25 30
Ser Ala Ala Phe Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu
35 40 45
Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala
50 55 60
Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn
65 70 75 80
Gln Phe Ser Leu Gln Leu Ile Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Arg Val Gly Ala Met Thr Phe Gly Leu Leu Thr Gly
100 105 110
Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 27
<211> 381
<212> DNA
<213> artificial sequence
<220>
<223> FHF11-VH W59F (nt)
<400> 27
caggtgcagc tgcagcagtc tggaccagga ctggtgaagc ctagccagac cctgtctgtg 60
acatgcggaa tctccggcga cagcgtgtcc agccactccg ccgcttggaa ctggatcaga 120
cagagcccat ctaggggact ggagtggctg ggaaggacct actatcggag caagttctac 180
aatgactatg ccgtgtctgt gaagtccagg atcaccatca acccagatac atccaagaat 240
cagtttagcc tgcagctgat ctctgtgacc cccgaggaca cagccgtgta ctattgtgcc 300
agagtgggcg ctatgacctt cggcctgctg acaggcggaa tggacgtgtg gggacaggga 360
accacagtga cagtgtcttc c 381
<210> 28
<211> 127
<212> PRT
<213> artificial sequence
<220>
<223> FHF11-VH W59F (aa)
<400> 28
Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Val Thr Cys Gly Ile Ser Gly Asp Ser Val Ser Ser His
20 25 30
Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu
35 40 45
Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Phe Tyr Asn Asp Tyr Ala
50 55 60
Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn
65 70 75 80
Gln Phe Ser Leu Gln Leu Ile Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Arg Val Gly Ala Met Thr Phe Gly Leu Leu Thr Gly
100 105 110
Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 29
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> FHF11-VH W59F CDRH2 (aa)
<400> 29
Thr Tyr Tyr Arg Ser Lys Phe Tyr Asn
1 5
<210> 30
<211> 381
<212> DNA
<213> artificial sequence
<220>
<223> FHF11v3 VH (nt)
<400> 30
caggtgcagc tgcagcagtc tggaccagga ctggtgaagc ctagccagac cctgtctgtg 60
acatgcggca tctccggcga cagcgtgtcc agctactccg ccgcttggaa ctggatcaga 120
cagagcccat ctaggggact ggagtggctg ggaaggacct actatcggag caagtggtac 180
aatgactatg ccgtgtctgt gaagtccagg atcaccatca acccagatac atccaagaat 240
cagttcagcc tgcagctgat ctctgtgacc cccgaggaca cagccgtgta ctattgtgcc 300
agagtgggcg ctatgacctt tggcctgctg acaggcggaa tggacgtgtg gggacaggga 360
accacagtga cagtgtcttc c 381
<210> 31
<211> 127
<212> PRT
<213> artificial sequence
<220>
<223> FHF11v3 VH (aa)
<400> 31
Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Val Thr Cys Gly Ile Ser Gly Asp Ser Val Ser Ser Tyr
20 25 30
Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu
35 40 45
Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala
50 55 60
Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn
65 70 75 80
Gln Phe Ser Leu Gln Leu Ile Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Arg Val Gly Ala Met Thr Phe Gly Leu Leu Thr Gly
100 105 110
Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 32
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FHF11v3 CDRH1 (aa)
<400> 32
Gly Asp Ser Val Ser Ser Tyr Ser Ala Ala
1 5 10
<210> 33
<211> 381
<212> DNA
<213> artificial sequence
<220>
<223> FHF11v6 VH (nt)
<400> 33
caggtgcagc tgcagcagtc tggaccagga ctggtgaagc ctagccagac cctgtctgtg 60
acatgcggaa tctccggcga cagcgtgtcc agccactccg ccgcttggaa ctggatcaga 120
cagagcccat ctaggggact ggagtggctg ggaaggacct actatcggag cggctggtac 180
aatgactatg ccgtgtctgt gaagtccagg atcaccatca acccagatac atccaagaat 240
cagttcagcc tgcagctgat ctctgtgacc cccgaggaca cagccgtgta ctattgtgcc 300
agagtgggcg ctatgacctt tggcctgctg acaggcggaa tggacgtgtg gggacaggga 360
accacagtga cagtgtcttc c 381
<210> 34
<211> 127
<212> PRT
<213> artificial sequence
<220>
<223> FHF11v6 VH (aa)
<400> 34
Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Val Thr Cys Gly Ile Ser Gly Asp Ser Val Ser Ser His
20 25 30
Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu
35 40 45
Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn Asp Tyr Ala
50 55 60
Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn
65 70 75 80
Gln Phe Ser Leu Gln Leu Ile Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Arg Val Gly Ala Met Thr Phe Gly Leu Leu Thr Gly
100 105 110
Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 35
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> FHF11v6 CDRH2 (aa)
<400> 35
Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn
1 5
<210> 36
<211> 381
<212> DNA
<213> artificial sequence
<220>
<223> FHF11v9 VH (nt)
<400> 36
caggtgcagc tgcagcagtc tggaccagga ctggtgaagc ctagccagac cctgtctgtg 60
acatgcggca tctccggcga cagcgtgtcc agctactccg ccgcttggaa ctggatcaga 120
cagagcccat ctaggggact ggagtggctg ggaaggacct actatcggag cggctggtac 180
aatgactatg ccgtgtctgt gaagtccagg atcaccatca acccagatac atccaagaat 240
cagttcagcc tgcagctgat ctctgtgacc cccgaggaca cagccgtgta ctattgtgcc 300
agagtgggcg ctatgacctt tggcctgctg acaggcggaa tggacgtgtg gggacaggga 360
accacagtga cagtgtcttc c 381
<210> 37
<211> 127
<212> PRT
<213> artificial sequence
<220>
<223> FHF11v9 VH (aa)
<400> 37
Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Val Thr Cys Gly Ile Ser Gly Asp Ser Val Ser Ser Tyr
20 25 30
Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu
35 40 45
Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn Asp Tyr Ala
50 55 60
Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn
65 70 75 80
Gln Phe Ser Leu Gln Leu Ile Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Arg Val Gly Ala Met Thr Phe Gly Leu Leu Thr Gly
100 105 110
Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 38
<211> 381
<212> DNA
<213> artificial sequence
<220>
<223> FHF12-VH-W36F (nt)
<400> 38
caggtgcagc tgcagcagtc tggaccagga ctggtgaagc ctagccagac cctgtctgtg 60
acatgcgcta tctccggcga cagcgtgtcc agccactccg ccgctttcaa ctggatcaga 120
cagagcccat ctaggggact ggagtggctg ggaaggacct actatcggag caagtggtac 180
aatgactatg ccgtgtccgt gaagtccagg atcaccatca acccagatac atccaagaat 240
cagttcagcc tgcagctggt gtctgtgacc cccgaggaca cagccgtgta ctattgtgct 300
agagtgggcg ccgctacctt tggcatcctg acaggcggaa tggacgtgtg gggacaggga 360
accacagtga cagtgtcttc c 381
<210> 39
<211> 127
<212> PRT
<213> artificial sequence
<220>
<223> FHF12-VH-W36F (aa)
<400> 39
Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Val Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser His
20 25 30
Ser Ala Ala Phe Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu
35 40 45
Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala
50 55 60
Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn
65 70 75 80
Gln Phe Ser Leu Gln Leu Val Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Arg Val Gly Ala Ala Thr Phe Gly Ile Leu Thr Gly
100 105 110
Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 40
<211> 381
<212> DNA
<213> artificial sequence
<220>
<223> FHF12-VH-W59F (nt)
<400> 40
caggtgcagc tgcagcagtc tggaccagga ctggtgaagc ctagccagac cctgtctgtg 60
acatgcgcta tctccggcga cagcgtgtcc agccactccg ccgcttggaa ctggatcaga 120
cagagcccat ctaggggact ggagtggctg ggaaggacct actatcggag caagttctac 180
aatgactatg ccgtgtccgt gaagtccagg atcaccatca acccagatac atccaagaat 240
cagttcagcc tgcagctggt gtctgtgacc cccgaggaca cagccgtgta ctattgtgct 300
agagtgggcg ccgctacctt tggcatcctg acaggcggaa tggacgtgtg gggacaggga 360
accacagtga cagtgtcttc c 381
<210> 41
<211> 127
<212> PRT
<213> artificial sequence
<220>
<223> FHF12-VH-W59F (aa)
<400> 41
Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Val Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser His
20 25 30
Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu
35 40 45
Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Phe Tyr Asn Asp Tyr Ala
50 55 60
Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn
65 70 75 80
Gln Phe Ser Leu Gln Leu Val Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Arg Val Gly Ala Ala Thr Phe Gly Ile Leu Thr Gly
100 105 110
Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 42
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> FHF12-CDRH2-W59F (aa)
<400> 42
Thr Tyr Tyr Arg Ser Lys Phe Tyr Asn
1 5
<210> 43
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FM08 VH
<400> 43
Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Tyr
20 25 30
Asn Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu
35 40 45
Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn Asp Tyr Ala
50 55 60
Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn
65 70 75 80
Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val
100 105 110
Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 44
<211> 103
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FM08 VL
<400> 44
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr
20 25 30
Thr His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Arg Gly Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln
85 90 95
Gly Thr Lys Val Glu Ile Lys
100
<210> 45
<211> 217
<212> PRT
<213> artificial sequence
<220>
<223> WT hIgG1 Fc
<400> 45
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
115 120 125
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215
<210> 46
<211> 19
<212> PRT
<213> artificial sequence
<220>
<223> chimeric hinge sequences
<400> 46
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val
1 5 10 15
Ala Gly Pro
<210> 47
<211> 385
<212> DNA
<213> artificial sequence
<220>
<223> FNI1 VH (wt-nt)
<400> 47
caagttcagc tggtgcagtc tggggctgag gtgaagaggc ctgggtcctc ggtgaggatc 60
tcctgcaagg cctctggtga caccttcaac aactatgttc tcagctgggt gcgacaggcc 120
cctggacaag ggcttgagtg gatgggggga atcatcccta tctctggtat cccacattac 180
gcacagaagt tccagggcag agtcgcaatt atcgcggacg aatccgcgag cacagtctac 240
atggagttga gcagcctacg atctgaggac tcggccgtat attactgtgc gagagcggtt 300
tccgattatt ttaatcgaga cctcggctgg gatgattact actttccttt gtggggccag 360
ggcaccctgg tcaccgtctc ctcag 385
<210> 48
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FNI1 VH (aa)
<400> 48
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ser
1 5 10 15
Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Asp Thr Phe Asn Asn Tyr
20 25 30
Val Leu Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Ile Ser Gly Ile Pro His Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Ala Ile Ile Ala Asp Glu Ser Ala Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ala Val Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Asp Asp
100 105 110
Tyr Tyr Phe Pro Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 49
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI1 CDRH1 (aa)
<400> 49
Gly Asp Thr Phe Asn Asn Tyr Val
1 5
<210> 50
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI1 CDRH2 (aa)
<400> 50
Ile Ile Pro Ile Ser Gly Ile Pro
1 5
<210> 51
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> FNI1 CDRH3 (aa)
<400> 51
Ala Arg Ala Val Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Asp Asp
1 5 10 15
Tyr Tyr Phe Pro Leu
20
<210> 52
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> FNI1 VH (co-nt)
<400> 52
caggtgcagc tggtgcagtc tggagctgag gtgaagaggc caggatccag cgtgcggatc 60
agctgcaagg cttctggcga caccttcaac aattacgtgc tgtcctgggt gaggcaggct 120
ccaggacagg gactggagtg gatgggcggc atcatcccca tcagcggcat ccctcactac 180
gcccagaagt ttcagggcag ggtggccatc atcgctgacg agtccgctag cacagtgtat 240
atggagctgt cttccctgag atctgaggat tccgccgtgt actattgtgc cagagccgtg 300
tccgactatt tcaaccgcga tctgggctgg gacgattact attttccact gtggggacag 360
ggcaccctgg tgacagtgag ctct 384
<210> 53
<211> 325
<212> DNA
<213> artificial sequence
<220>
<223> FNI1 Vk (wt-nt)
<400> 53
gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60
ctcttctgca gggccagtcg gagtgttagt gacaacttag cctggtacca gcagaaacct 120
ggccaggctc ccaggctcct catctttggt gcctccacca gggccactgg tgtcccagcc 180
aggttcggtg gcagtgggtc tgggacacag ttcactctca ccatcagcag cctgcagtct 240
gaagattttg cagtttatta ctgtcagcat tataatacct ggcctccgtg gaccttcggc 300
caagggacca aggtggaaat caaac 325
<210> 54
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI1 VK (aa)
<400> 54
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Phe Cys Arg Ala Ser Arg Ser Val Ser Asp Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Phe Gly Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Gly Gly
50 55 60
Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Asn Thr Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 55
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> FNI1 CDRL1(aa)
<400> 55
Arg Ser Val Ser Asp Asn
1 5
<210> 56
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> FNI1 CDRL2(aa)
<400> 56
Gly Ala Ser
1
<210> 57
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI1 CDRL3(aa)
<400> 57
Gln His Tyr Asn Thr Trp Pro Pro Trp Thr
1 5 10
<210> 58
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI1 Vk (co-nt)
<400> 58
gagatcgtga tgacccagtc tcctgccaca ctgtccgtgt ccccaggcga gagggccaca 60
ctgttctgca gggctagcag gtccgtgtcc gacaacctgg cctggtacca gcagaagcca 120
ggccaggctc ccagactgct gatctttgga gcttccacca gagctacagg cgtgccagct 180
aggttcggag gaagcggatc tggcacccag tttaccctga caatctccag cctgcagagc 240
gaggatttcg ccgtgtacta ttgtcagcac tataatacct ggcccccttg gacatttggc 300
cagggcacca aggtggagat caag 324
<210> 59
<211> 385
<212> DNA
<213> artificial sequence
<220>
<223> FNI2 VH (wt-nt)
<400> 59
caggttcagc tggtgcagtc tggggctgag gtgaagaggc ctgggtcctc ggtgagggtc 60
tcctgcaagg cttctggagc caccttcaat aaccatgttc tcacctgggt gcgacaggcc 120
cctggacaag ggcttgagtg gatgggaggg atcatccctg tctctggaaa aacaacctac 180
gcacagaagt tccagggcag agtcgcgata agcacggacg aatccgcgag cacagcctat 240
atggagttga gcagcctgag atctgaggac tcggccatat attactgtgc gagagcggtt 300
tccgattact ttaatcgaga cctcggctgg gaagattatt actttccgat ctggggccag 360
ggcaccctgg tcaccgtctc ttcag 385
<210> 60
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FNI2 VH (aa)
<400> 60
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ser
1 5 10 15
Ser Val Arg Val Ser Cys Lys Ala Ser Gly Ala Thr Phe Asn Asn His
20 25 30
Val Leu Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Val Ser Gly Lys Thr Thr Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Ala Ile Ser Thr Asp Glu Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Ala Val Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asp
100 105 110
Tyr Tyr Phe Pro Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 61
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI2 CDRH1 (aa)
<400> 61
Gly Ala Thr Phe Asn Asn His Val
1 5
<210> 62
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI2 CDRH2 (aa)
<400> 62
Ile Ile Pro Val Ser Gly Lys Thr
1 5
<210> 63
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> FNI2 CDRH3 (aa)
<400> 63
Ala Arg Ala Val Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asp
1 5 10 15
Tyr Tyr Phe Pro Ile
20
<210> 64
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> FNI2 VH (co-nt)
<400> 64
caggtgcagc tggtgcagtc tggagctgag gtgaagaggc caggatccag cgtgcgggtg 60
agctgcaagg cttctggagc taccttcaac aatcacgtgc tgacatgggt gaggcaggct 120
ccaggacagg gactggagtg gatgggcggc atcatccccg tgtccggcaa gaccacatac 180
gcccagaagt ttcagggcag ggtggctatc agcaccgatg agtccgccag cacagcttat 240
atggagctgt cttccctgag atctgaggac tccgccatct actattgtgc cagagccgtg 300
tccgactact tcaaccgcga tctgggctgg gaggactact attttcccat ctggggccag 360
ggcaccctgg tgacagtgag ctct 384
<210> 65
<211> 325
<212> DNA
<213> artificial sequence
<220>
<223> FNI2 Vk (wt-nt)
<400> 65
gacgtagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60
ctctcctgca gggccagtca gagtgttagt agcaacttgg cctggtacca gcagaaacct 120
ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tgtcccagcc 180
aggttcagtg gcagtgggtc tgggacacag ttcactctca ccatcagcag cctgcagtct 240
gaagattttg cagtttatta ctgtcagcac tataataact ggcctccgtg gacgttcggc 300
caagggacca agttggaaat caaac 325
<210> 66
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI2 VK (aa)
<400> 66
Asp Val Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Asn Asn Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 67
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> FNI2 CDRL1(aa)
<400> 67
Gln Ser Val Ser Ser Asn
1 5
<210> 68
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> FNI2 CDRL2(aa)
<400> 68
Gly Ala Ser
1
<210> 69
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI2 CDRL3(aa)
<400> 69
Gln His Tyr Asn Asn Trp Pro Pro Trp Thr
1 5 10
<210> 70
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI2 Vk (co-nt)
<400> 70
gacgtggtca tgacccagtc tcctgccaca ctgagcgtgt ctccaggaga gagggccacc 60
ctgtcctgca gggcttccca gagcgtgtcc agcaacctgg cctggtacca gcagaagcca 120
ggccaggctc ccaggctgct gatctatgga gctagcacca gagctacagg cgtgccagct 180
cgcttctctg gatccggaag cggcacacag tttaccctga caatctcttc cctgcagtct 240
gaggatttcg ccgtgtacta ttgtcagcac tacaacaatt ggcccccttg gacctttggc 300
cagggcacaa agctggagat caag 324
<210> 71
<211> 385
<212> DNA
<213> artificial sequence
<220>
<223> FNI3 VH (wt-nt)
<400> 71
caggttcagc tggtgcagtc gggggctgag gtgaagaggc ctgggtcctc ggtgaaggtc 60
tcctgcaagg cttctggagc caccttcagc aacaatgtta tagcctgggt gcgacaggcc 120
cctggacaag ggcttgagtg gatggggggg atccacccta tctctgctac agcaacctac 180
gcacagaagt tccagggcag agtcgcgatt gccgcggacg aattaacgag cacagcctac 240
atggagttga atggcctgag atctgaggac tcggccgtgt attactgtgc gagagcgggg 300
tccgattact ttaatagaga cctcggctgg gaaaattact actttgactc ctggggccag 360
ggaaccctgg tcaccgtctc gtcag 385
<210> 72
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FNI3 VH (aa)
<400> 72
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Ala Thr Phe Ser Asn Asn
20 25 30
Val Ile Ala Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile His Pro Ile Ser Ala Thr Ala Thr Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Ala Ile Ala Ala Asp Glu Leu Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Asn Gly Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ala Gly Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asn
100 105 110
Tyr Tyr Phe Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 73
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI3 CDRH1 (aa)
<400> 73
Gly Ala Thr Phe Ser Asn Asn Val
1 5
<210> 74
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI3 CDRH2 (aa)
<400> 74
Ile His Pro Ile Ser Ala Thr Ala
1 5
<210> 75
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> FNI3 CDRH3 (aa)
<400> 75
Ala Arg Ala Gly Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asn
1 5 10 15
Tyr Tyr Phe Asp Ser
20
<210> 76
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> FNI3 VH (co-nt)
<400> 76
caggtgcagc tggtgcagtc cggagctgag gtgaagaggc caggatccag cgtgaaggtg 60
tcctgcaagg ccagcggcgc taccttcagc aacaatgtga tcgcttgggt gagacaggct 120
ccaggacagg gactggagtg gatgggagga atccacccta tcagcgccac cgctacatac 180
gcccagaagt ttcagggcag agtggctatc gccgctgacg agctgacctc tacagcctat 240
atggagctga acggcctgcg cagcgaggat tccgccgtgt actattgtgc cagggctggc 300
tctgactact tcaaccggga tctgggctgg gagaattact attttgactc ctggggccag 360
ggcaccctgg tgacagtgtc ttcc 384
<210> 77
<211> 325
<212> DNA
<213> artificial sequence
<220>
<223> FNI3 Vk (wt-nt)
<400> 77
gaaatattga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60
ctctcctgca gggccagtca ggatgttagc ggcaacttag cctggtacca gcagagacct 120
ggccaggctc ccaggctcct tatctatggt gcatccacga gggccactgg tgtcccagcc 180
aggttcactg gcgctgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240
gaggattttg cactttatta ctgtcagcac tataataact ggcctccgtg gaccttcggc 300
caagggacca aggtggaaat caaac 325
<210> 78
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI3 Vk (aa)
<400> 78
Glu Ile Leu Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Val Ser Gly Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Thr Gly
50 55 60
Ala Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Leu Tyr Tyr Cys Gln His Tyr Asn Asn Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 79
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> FNI3 CDRL1(aa)
<400> 79
Gln Asp Val Ser Gly Asn
1 5
<210> 80
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> FNI3 CDRL2(aa)
<400> 80
Gly Ala Ser
1
<210> 81
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI3 CDRL3(aa)
<400> 81
Gln His Tyr Asn Asn Trp Pro Pro Trp Thr
1 5 10
<210> 82
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI3 Vk (co-nt)
<400> 82
gagatcctga tgacccagtc ccctgccaca ctgtccgtgt ccccaggaga gagggccacc 60
ctgagctgca gggcttctca ggacgtgtcc ggcaacctgg cctggtacca gcagagacca 120
ggacaggctc caaggctgct gatctatgga gcttccacca gggctacagg cgtgccagct 180
agattcaccg gcgctggaag cggcacagag tttaccctga caatctccag cctgcagtct 240
gaggatttcg ctctgtacta ttgtcagcac tacaacaatt ggcccccttg gacctttggc 300
cagggcacaa aggtggagat caag 324
<210> 83
<211> 385
<212> DNA
<213> artificial sequence
<220>
<223> FNI4 VH (wt-nt)
<400> 83
caggagcagc tggtacagtc tggggctgag gtgaagaagc cggggtcctc ggtgagggtc 60
tcctgcaagg cctctggaga caccttcagc agatatacta tcagctgggt tcgacaggcc 120
cccggacaag gacttgagtg gatgggaggg atcatcgctc tctctcgaag agcgacatac 180
gcacagaagt tccagggcag agttaccatt accgcggacg aatccgcgac cacagcctac 240
atacaactga gcggcctgac atctgacgac acggccgtat attactgtgc gagagcacac 300
tccgattact ttaatagaga cctcggctgg gaagattact actttgacta ctggggccag 360
ggaaccctgg tcaccgtctc ctcag 385
<210> 84
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FNI4 VH (aa)
<400> 84
Gln Glu Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Arg Val Ser Cys Lys Ala Ser Gly Asp Thr Phe Ser Arg Tyr
20 25 30
Thr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Ala Leu Ser Arg Arg Ala Thr Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Ala Thr Thr Ala Tyr
65 70 75 80
Ile Gln Leu Ser Gly Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ala His Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asp
100 105 110
Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 85
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI4 CDRH1 (aa)
<400> 85
Gly Asp Thr Phe Ser Arg Tyr Thr
1 5
<210> 86
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI4 CDRH2 (aa)
<400> 86
Ile Ile Ala Leu Ser Arg Arg Ala
1 5
<210> 87
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> FNI4 CDRH3 (aa)
<400> 87
Ala Arg Ala His Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asp
1 5 10 15
Tyr Tyr Phe Asp Tyr
20
<210> 88
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> FNI4 VH (co-nt)
<400> 88
caggagcagc tggtgcagtc cggagctgag gtgaagaagc caggatccag cgtgagagtg 60
agctgcaagg cttctggcga caccttctct agatacacaa tctcctgggt gcgccaggct 120
cctggacagg gactggagtg gatgggagga atcatcgctc tgagcaggcg ggccacctac 180
gctcagaagt ttcagggccg cgtgaccatc acagccgatg agtctgccac cacagcttat 240
atccagctgt ccggcctgac cagcgacgat acagccgtgt actattgtgc cagggctcac 300
agcgactact tcaaccggga tctgggctgg gaggactact attttgatta ttggggccag 360
ggcaccctgg tgacagtgtc ttcc 384
<210> 89
<211> 325
<212> DNA
<213> artificial sequence
<220>
<223> FNI4 Vk (wt-nt)
<400> 89
gaagtagtgc tgacgcagtc tccagccacc ctgtctgtgt ctctagggga aagagccatc 60
ctctcctgca gggccagtca gagtgttagc accaacttag cctggtacca gcagagacct 120
ggccaggctc ccaggctcct catctctggt gcatccacca gggccacggg tatcccagcc 180
aggttcagtg gcagtgggtc tgggacagag ttcacgctca ccatcagcag cctgcagtct 240
gaagattttg cagtttatta ctgtcagcag tataataact ggcctccgtg gacgttcggc 300
caagggacca aggtggaaat cagac 325
<210> 90
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI4 VK (aa)
<400> 90
Glu Val Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Ile Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Ser Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Arg
100 105
<210> 91
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> FNI4 CDRL1 (aa)
<400> 91
Gln Ser Val Ser Thr Asn
1 5
<210> 92
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> FNI4 CDRL2 (aa)
<400> 92
Gly Ala Ser
1
<210> 93
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI4 CDRL3 (aa)
<400> 93
Gln Gln Tyr Asn Asn Trp Pro Pro Trp Thr
1 5 10
<210> 94
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI4 Vk (co-nt)
<400> 94
gaggtggtgc tgacccagtc ccctgccaca ctgtccgtgt ccctgggaga gagggctatc 60
ctgagctgca gggctagcca gtccgtgtcc accaacctgg cctggtacca gcagagacca 120
ggacaggctc caaggctgct gatcagcgga gcttctacca gggctacagg catcccagcc 180
agattcagcg gctctggctc cggcacagag tttaccctga caatctccag cctgcagtct 240
gaggacttcg ccgtgtacta ttgtcagcag tataacaatt ggcccccttg gacctttggc 300
cagggcacaa aggtggagat cagg 324
<210> 95
<211> 385
<212> DNA
<213> artificial sequence
<220>
<223> FNI5 VH (wt-nt)
<400> 95
caggtgcagc tgatacaatc tgaggctgag gtgaagaagc ctgggtcctc ggtgagggtc 60
tcctgcaagg cttctggaga caccttcagc aaatatacta tcggctgggt gcgacaggcc 120
cccggacaag ggcttgagtg gatgggaggg atcatccctc tctctcgaac agcgacctac 180
gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgac cacagtttac 240
atgcaactga gcggcctgag atctgacgac acggccgcat attactgtgc gagagcacgc 300
tcggattact ttaatagaga cctcggctgg gacgattact actttgatta ctggggccag 360
ggaaccctgg tcaccgtctc ctcag 385
<210> 96
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FNI5 VH (aa)
<400> 96
Gln Val Gln Leu Ile Gln Ser Glu Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Arg Val Ser Cys Lys Ala Ser Gly Asp Thr Phe Ser Lys Tyr
20 25 30
Thr Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Leu Ser Arg Thr Ala Thr Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Thr Thr Val Tyr
65 70 75 80
Met Gln Leu Ser Gly Leu Arg Ser Asp Asp Thr Ala Ala Tyr Tyr Cys
85 90 95
Ala Arg Ala Arg Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Asp Asp
100 105 110
Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 97
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI5 CDRH1 (aa)
<400> 97
Gly Asp Thr Phe Ser Lys Tyr Thr
1 5
<210> 98
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI5 CDRH2 (aa)
<400> 98
Ile Ile Pro Leu Ser Arg Thr Ala
1 5
<210> 99
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> FNI5 CDRH3 (aa)
<400> 99
Ala Arg Ala Arg Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Asp Asp
1 5 10 15
Tyr Tyr Phe Asp Tyr
20
<210> 100
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> FNI5 VH (co-nt)
<400> 100
caggtgcagc tgatccagag cgaggccgag gtgaagaagc caggctccag cgtgagggtg 60
agctgcaagg cttctggcga cacattctct aagtacacca tcggatgggt gcggcaggct 120
ccaggacagg gcctggagtg gatgggcggc atcatccctc tgtctagaac agccacctac 180
gctcagaagt ttcagggccg cgtgacaatc accgctgacg agtccaccac aaccgtgtat 240
atgcagctgt ccggcctgag aagcgacgat acagccgctt actattgtgc cagggctcgg 300
tccgactact tcaaccgcga tctgggctgg gacgattact attttgatta ttggggccag 360
ggcacactgg tgaccgtgtc ttcc 384
<210> 101
<211> 325
<212> DNA
<213> artificial sequence
<220>
<223> FNI5 Vk (wt-nt)
<400> 101
gaaatagtga tgacgcagtc tccagccaac ctgtctgtgt ctccagggga aagagccacc 60
ctctcctgca gggccagtca gactgttagc accaacttag cctggtacca gcagaagcct 120
ggccaggctc ccaggctcct catctctggt gcatccacca gggccactgg tatcccagcc 180
aggttcagtg gcagtgggtc tgggacagag ttcacgctca ccatcagcag cctgcagtct 240
gaagattttg cagtttatta ctgtcagcag tataataatt ggcctccgtg gacgttcggc 300
caagggacca aggtggaaat cagac 325
<210> 102
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI5 VK (aa)
<400> 102
Glu Ile Val Met Thr Gln Ser Pro Ala Asn Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Val Ser Thr Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Ser Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Arg
100 105
<210> 103
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> FNI5 CDRL1(aa)
<400> 103
Gln Thr Val Ser Thr Asn
1 5
<210> 104
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> FNI5 CDRL2(aa)
<400> 104
Gly Ala Ser
1
<210> 105
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI5 CDRL3(aa)
<400> 105
Gln Gln Tyr Asn Asn Trp Pro Pro Trp Thr
1 5 10
<210> 106
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI5 Vk (co-nt)
<400> 106
gagatcgtga tgacccagtc ccctgctaac ctgtccgtgt ccccaggaga gagggccaca 60
ctgtcctgcc gggctagcca gaccgtgtct acaaatctgg cctggtacca gcagaagcca 120
ggacaggctc caaggctgct gatcagcgga gcttctacca gagctacagg catcccagct 180
cgcttcagcg gatctggatc cggcaccgag tttaccctga caatctccag cctgcagagc 240
gaggacttcg ccgtgtacta ttgtcagcag tataacaatt ggcccccttg gacctttggc 300
cagggcacaa aggtggagat caga 324
<210> 107
<211> 385
<212> DNA
<213> artificial sequence
<220>
<223> FNI6 VH (wt-nt)
<400> 107
caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60
tcctgcaagg cctctggagg caccttcagt agtcaagtta tcagctgggt gcgagaggcc 120
ccaggacaag ggcttgagtg gatgggaggg atcattccta tcactggaat agcgaacaac 180
gcacagaagt tccagggcag agtcacgatt accgcggacg gatccacggg cacagtctac 240
atggagttga gcagcctgag atctggggac acggccgtct attactgtgc gagagcgggt 300
tcggattatt ttaatagaga cctcggctgg gaaaattact actttgaata ttggggccag 360
ggaaccctgg tcaccgtctc ctcag 385
<210> 108
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FNI6 VH (aa)
<400> 108
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Gln
20 25 30
Val Ile Ser Trp Val Arg Glu Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Ile Thr Gly Ile Ala Asn Asn Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Gly Ser Thr Gly Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Gly Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ala Gly Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asn
100 105 110
Tyr Tyr Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 109
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI6 CDRH1 (aa)
<400> 109
Gly Gly Thr Phe Ser Ser Gln Val
1 5
<210> 110
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI6 CDRH2 (aa)
<400> 110
Ile Ile Pro Ile Thr Gly Ile Ala
1 5
<210> 111
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> FNI6 CDRH3 (aa)
<400> 111
Ala Arg Ala Gly Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asn
1 5 10 15
Tyr Tyr Phe Glu Tyr
20
<210> 112
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> FNI6 VH (co-nt)
<400> 112
caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggctccag cgtgaaggtg 60
tcttgcaagg cttccggcgg caccttctct tcccaggtca tctcttgggt gagggaggct 120
ccaggacagg gactggagtg gatgggcggc atcatcccta tcacaggcat cgccaacaat 180
gctcagaagt ttcagggcag agtgaccatc acagccgacg gcagcaccgg cacagtgtac 240
atggagctga gctctctgcg ctctggcgat accgccgtgt actattgtgc cagggctggc 300
tccgactact tcaaccggga tctgggctgg gagaattact attttgagta ttggggccag 360
ggcaccctgg tgacagtgtc cagc 384
<210> 113
<211> 325
<212> DNA
<213> artificial sequence
<220>
<223> FNI6 Vk (wt-nt)
<400> 113
gaaatcgtga tgacacagtc tccagccacc ctgtctgtat ctccagggga aagagccatc 60
ctctcctgca gggccagtca gagtgttagc acccacttag cctggtacca gcagaaacct 120
ggccaggctc ccagactcct cgtttttgat gcatccacca gggccactgg tgtcccagcc 180
agattcggtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240
gaagattctg ctgtttatta ctgtcaacac tataataact ggcctccgtg gacgttcggc 300
caagggacca acgtggaaat cagac 325
<210> 114
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI6 VK (aa)
<400> 114
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Ile Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Thr His
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Val
35 40 45
Phe Asp Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Gly Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Ser Ala Val Tyr Tyr Cys Gln His Tyr Asn Asn Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Asn Val Glu Ile Arg
100 105
<210> 115
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> FNI6 CDRL1(aa)
<400> 115
Gln Ser Val Ser Thr His
1 5
<210> 116
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> FNI6 CDRL2(aa)
<400> 116
Asp Ala Ser
1
<210> 117
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI6 CDRL3(aa)
<400> 117
Gln His Tyr Asn Asn Trp Pro Pro Trp Thr
1 5 10
<210> 118
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI6 Vk (co-nt)
<400> 118
gagatcgtga tgacccagtc tcctgccaca ctgtccgtgt ccccaggaga gagggctatc 60
ctgtcctgca gggctagcca gtccgtgtcc acccacctgg cctggtacca gcagaagcca 120
ggccaggctc ccaggctgct ggtgttcgac gctagcacca gagctacagg cgtgccagct 180
aggttcggag gaagcggatc tggcacagag tttaccctga caatctccag cctgcagtcc 240
gaggattccg ccgtgtacta ttgtcagcat tataacaatt ggcccccttg gacctttggc 300
cagggcacaa acgtggagat caga 324
<210> 119
<211> 385
<212> DNA
<213> artificial sequence
<220>
<223> FNI7 VH (wt-nt)
<400> 119
caagtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaagtc 60
tcctgtaaga cttctggagg caccttcaat aggcaagtta tcagctgggt gcgacaggcc 120
ccaggacaag gacttgagtg gatgggaggg atcctccctc ttactggtag aggggacgag 180
gcagagaggt ttcagggcag agtcaccatt accgcggacg aatctgagag tacagtctac 240
atggacttga gcagcctgag atctggggac acggccgtct attactgtgc gagagcgcgt 300
tcggattact ttaatagaga cctcggctgg gaaaattact actttgaatc ttggggccag 360
ggaaccctgg tcaccgtctc ctcag 385
<210> 120
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FNI7 VH (aa)
<400> 120
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Asn Arg Gln
20 25 30
Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Leu Pro Leu Thr Gly Arg Gly Asp Glu Ala Glu Arg Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Glu Ser Thr Val Tyr
65 70 75 80
Met Asp Leu Ser Ser Leu Arg Ser Gly Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ala Arg Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asn
100 105 110
Tyr Tyr Phe Glu Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 121
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI7 CDRH1 (aa)
<400> 121
Gly Gly Thr Phe Asn Arg Gln Val
1 5
<210> 122
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI7 CDRH2 (aa)
<400> 122
Ile Leu Pro Leu Thr Gly Arg Gly
1 5
<210> 123
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> FNI7 CDRH3 (aa)
<400> 123
Ala Arg Ala Arg Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asn
1 5 10 15
Tyr Tyr Phe Glu Ser
20
<210> 124
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> FNI7 VH (co-nt)
<400> 124
caggtgcagc tggtgcagtc cggagctgag gtgaagaagc caggctccag cgtgaaggtg 60
tcttgcaaga cctccggcgg cacattcaac aggcaggtca tcagctgggt gcggcaggct 120
ccaggacagg gactggagtg gatgggagga atcctgcctc tgaccggcag gggcgacgag 180
gccgagagat ttcagggccg cgtgaccatc acagctgatg agtccgagag caccgtgtac 240
atggacctgt cttccctgag aagcggcgat acagccgtgt actattgtgc cagggctcgg 300
tctgactatt tcaaccgcga tctgggctgg gagaattact attttgagtc ttggggccag 360
ggcaccctgg tgacagtgag ctct 384
<210> 125
<211> 325
<212> DNA
<213> artificial sequence
<220>
<223> FNI7 Vk (wt-nt)
<400> 125
gaaatcgtga tgacgcagtc tccagccacc ctgtctgtat ctccagggga aagagccacc 60
ctctcctgca gggccagtca gagtgttagt accgacttag tctggtacca gcagaaacct 120
ggccaggctc cccggctcct catttatgat gcatccacta gggccactgg tatcccagcc 180
aggttcggtg gcagggggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240
gaagattctg ctgtttatta ctgtcagcac tattcttact ggcctccgtg gacattcggc 300
caagggacca aagtggaaat caatc 325
<210> 126
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI7 VK (aa)
<400> 126
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Asp
20 25 30
Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Gly Gly
50 55 60
Arg Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Ser Ala Val Tyr Tyr Cys Gln His Tyr Ser Tyr Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Asn
100 105
<210> 127
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> FNI7 CDRL1(aa)
<400> 127
Gln Ser Val Ser Thr Asp
1 5
<210> 128
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> FNI7 CDRL2(aa)
<400> 128
Asp Ala Ser
1
<210> 129
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI7 CDRL3(aa)
<400> 129
Gln His Tyr Ser Tyr Trp Pro Pro Trp Thr
1 5 10
<210> 130
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI7 Vk (co-nt)
<400> 130
gagatcgtga tgacccagtc ccctgccaca ctgtccgtgt ccccaggaga gagagccacc 60
ctgagctgca gggctagcca gtccgtgtcc acagacctgg tgtggtacca gcagaagcca 120
ggacaggctc caaggctgct gatctatgat gcctctacca gagctacagg catcccagct 180
aggttcggag gaaggggatc cggcaccgag tttaccctga caatctccag cctgcagagc 240
gaggactccg ccgtgtacta ttgtcagcac tacagctatt ggcccccttg gaccttcggc 300
cagggcacaa aggtggagat caac 324
<210> 131
<211> 385
<212> DNA
<213> artificial sequence
<220>
<223> FNI9 VH (wt-nt)
<400> 131
caggtccacc tggtgcagtc tggggctgag gtgaaggagc ctgggtcctc ggtgacggtc 60
tcctgcaagg catctggagg cagcttcaac aaccaggcta ttagctgggt gcgacaggcc 120
ccaggacaag gccttgagtg gatgggaggg atcttcccta tctctggcac accgaccagc 180
gcacagaggt tccagggcag agtcacattt accgcggacg agtccacgac cacagtctac 240
atggatctga gcagcctgag atctgacgac acggccgtct actactgtgc gagagcgggt 300
tcggattact ttaatagaga cctcggctgg gaaaactact actttgcgtc ctggggccag 360
ggaaccctgg tcaccgtctc ctcag 385
<210> 132
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FNI9 VH (aa)
<400> 132
Gln Val His Leu Val Gln Ser Gly Ala Glu Val Lys Glu Pro Gly Ser
1 5 10 15
Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Ser Phe Asn Asn Gln
20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Phe Pro Ile Ser Gly Thr Pro Thr Ser Ala Gln Arg Phe
50 55 60
Gln Gly Arg Val Thr Phe Thr Ala Asp Glu Ser Thr Thr Thr Val Tyr
65 70 75 80
Met Asp Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ala Gly Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asn
100 105 110
Tyr Tyr Phe Ala Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 133
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI9 CDRH1 (aa)
<400> 133
Gly Gly Ser Phe Asn Asn Gln Ala
1 5
<210> 134
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI9 CDRH2 (aa)
<400> 134
Ile Phe Pro Ile Ser Gly Thr Pro
1 5
<210> 135
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> FNI9 CDRH3 (aa)
<400> 135
Ala Arg Ala Gly Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asn
1 5 10 15
Tyr Tyr Phe Ala Ser
20
<210> 136
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> FNI9 VH (co-nt)
<400> 136
caggtgcacc tggtgcagag cggagctgag gtgaaggagc caggatccag cgtgacagtg 60
tcttgcaagg cttccggcgg cagcttcaac aatcaggcta tctcctgggt gaggcaggct 120
ccaggacagg gactggagtg gatgggcggc atctttccca tctctggcac acctacctcc 180
gcccagaggt tccagggaag ggtgaccttc accgctgacg agagcaccac aaccgtgtac 240
atggatctgt cttccctgag atctgacgat accgccgtgt actattgtgc cagagctggc 300
tccgactatt tcaaccgcga tctgggctgg gagaattact attttgcttc ctggggccag 360
ggcacactgg tgaccgtgag ctct 384
<210> 137
<211> 325
<212> DNA
<213> artificial sequence
<220>
<223> FNI9 Vk (wt-nt)
<400> 137
gaaatcgtga tgacgcagtc tccagccacc ctgtctctat cttcagggga aagagccacc 60
ctctcctgca gggccagtcg gagtgttagt agcaacttag cctggtacca gcagaaacct 120
ggccaggctc ccaggctcct catttatgat gcatccacca gggccactgg tttttcagcc 180
aggttcgctg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240
gaagattctg ctatttatta ctgtcagcag tataataact ggcctccgtg gacgttcggc 300
caagggacca aggtggaaat caaac 325
<210> 138
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI9 VK (aa)
<400> 138
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Ser Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Arg Ser Val Ser Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Thr Arg Ala Thr Gly Phe Ser Ala Arg Phe Ala Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Ser Ala Ile Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 139
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> FNI9 CDRL1(aa)
<400> 139
Arg Ser Val Ser Ser Asn
1 5
<210> 140
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> FNI9 CDRL2(aa)
<400> 140
Asp Ala Ser
1
<210> 141
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI9 CDRL3(aa)
<400> 141
Gln Gln Tyr Asn Asn Trp Pro Pro Trp Thr
1 5 10
<210> 142
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI9 Vk (co-nt)
<400> 142
gagatcgtga tgacccagtc cccagccaca ctgagcctgt ccagcggaga gagggccacc 60
ctgtcctgca gggcttcccg gagcgtgtct tccaacctgg cctggtacca gcagaagcca 120
ggccaggctc ccagactgct gatctatgac gcctctacca gagctacagg cttctccgcc 180
aggtttgctg gatctggatc cggcacagag ttcaccctga caatcagctc tctgcagagc 240
gaggattctg ctatctacta ttgtcagcag tacaacaatt ggcccccttg gacctttggc 300
cagggcacaa aggtggagat caag 324
<210> 143
<211> 385
<212> DNA
<213> artificial sequence
<220>
<223> FNI10 VH (wt-nt)
<400> 143
caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaagtc 60
tcctgcaagg cttctggagg caccttgagt agtcaagtta ttagctgggt gcgacaggcc 120
ccaggacaag gactggagtg gatcggaggg atcatcccca ccactggtac agggggcgcg 180
gcagaggggt tccagggcag agtctccatt tccgcggacg aatccaggag cacagtctac 240
atggaactga ccagcctgac ttctggggac acggccgtct attattgtgc gagagcggtt 300
tcggattact ttaatagaga cctcggctgg gaaaattact actttgaatc ttggggccag 360
ggaaccctgg tcaccgtctc ctcag 385
<210> 144
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FNI10 VH (aa)
<400> 144
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Leu Ser Ser Gln
20 25 30
Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Gly Ile Ile Pro Thr Thr Gly Thr Gly Gly Ala Ala Glu Gly Phe
50 55 60
Gln Gly Arg Val Ser Ile Ser Ala Asp Glu Ser Arg Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Thr Ser Leu Thr Ser Gly Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ala Val Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asn
100 105 110
Tyr Tyr Phe Glu Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 145
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI10 CDRH1 (aa)
<400> 145
Gly Gly Thr Leu Ser Ser Gln Val
1 5
<210> 146
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI10 CDRH2 (aa)
<400> 146
Ile Ile Pro Thr Thr Gly Thr Gly
1 5
<210> 147
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> FNI10 CDRH3 (aa)
<400> 147
Ala Arg Ala Val Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asn
1 5 10 15
Tyr Tyr Phe Glu Ser
20
<210> 148
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> FNI10 VH (co-nt)
<400> 148
caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggctccag cgtgaaggtg 60
tcctgcaagg ctagcggcgg caccctgtct tcccaggtca tctcttgggt gaggcaggct 120
ccaggacagg gactggagtg gatcggcggc atcatcccta ccacaggcac aggcggagct 180
gctgagggat tccagggcag agtgtccatc agcgccgacg agtctcgctc caccgtgtac 240
atggagctga ccagcctgac atctggcgat acagccgtgt actattgtgc cagggccgtg 300
tccgactatt tcaaccggga tctgggctgg gagaattact attttgagtc ctggggccag 360
ggcaccctgg tgacagtgag ctct 384
<210> 149
<211> 325
<212> DNA
<213> artificial sequence
<220>
<223> FNI10 Vk (wt-nt)
<400> 149
gaaatcgtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60
ctctcttgca gggccagtcg gagtgttagt atcaacttag cctggtacca acagaaacct 120
ggccaggctc cccggctcct catttatgat gcatctacga gggccactgg catcccagcc 180
aggttcggtg gcagggggtc tggaacagag ttcactctca ccatcagcag cctgcagtct 240
gaagattctg ctgtttatta ctgtcagcac tataataact ggcctccgtg gacattcggc 300
caagggacca gagtggaaat caaac 325
<210> 150
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI10 VK (aa)
<400> 150
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Arg Ser Val Ser Ile Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Gly Gly
50 55 60
Arg Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Ser Ala Val Tyr Tyr Cys Gln His Tyr Asn Asn Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Arg Val Glu Ile Lys
100 105
<210> 151
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> FNI10 CDRL1(aa)
<400> 151
Arg Ser Val Ser Ile Asn
1 5
<210> 152
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> FNI10 CDRL2(aa)
<400> 152
Asp Ala Ser
1
<210> 153
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI10 CDRL3(aa)
<400> 153
Gln His Tyr Asn Asn Trp Pro Pro Trp Thr
1 5 10
<210> 154
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI10 Vk (co-nt)
<400> 154
gagatcgtga tgacccagtc ccctgccaca ctgtccgtgt ccccaggaga gagagccacc 60
ctgagctgca gggctagcag gtccgtgtcc atcaacctgg cctggtacca gcagaagcca 120
ggccaggctc ccaggctgct gatctatgac gcttctacca gggctacagg catcccagct 180
agattcggag gaaggggatc cggaacagag tttaccctga caatctccag cctgcagagc 240
gaggattccg ccgtgtacta ttgtcagcac tacaacaatt ggccaccttg gaccttcggc 300
cagggaacac gcgtggagat caag 324
<210> 155
<211> 385
<212> DNA
<213> artificial sequence
<220>
<223> FNI12 VH (wt-nt)
<400> 155
caggtgcacc tggtacagtc tggggctgag gtgaagaagc ctgggtcctc ggtgagggtc 60
tcctgcaagg cttctggaga ctccttcaac aaatatgaag tcagctgggt gcgacaggcc 120
cccggacatg gacttgagtg gatgggaggg atcatccctc tctctcctat agcgaggtac 180
gcagagaaat ttcagggcag agtcacgatt accgcggacg aattcacgag cacggtctat 240
atacaactga ccagcctgag atctgacgac acggccgtat actactgtgc gacaacacgt 300
tcggattact ttaatagaga cctcggctgg gaagattact tctttgacca ctggggccag 360
ggaaccctgg tcaccgtctc ctcag 385
<210> 156
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FNI12 VH (aa)
<400> 156
Gln Val His Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Arg Val Ser Cys Lys Ala Ser Gly Asp Ser Phe Asn Lys Tyr
20 25 30
Glu Val Ser Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Leu Ser Pro Ile Ala Arg Tyr Ala Glu Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Val Tyr
65 70 75 80
Ile Gln Leu Thr Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Thr Arg Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asp
100 105 110
Tyr Phe Phe Asp His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 157
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI12 CDRH1 (aa)
<400> 157
Gly Asp Ser Phe Asn Lys Tyr Glu
1 5
<210> 158
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI12 CDRH2 (aa)
<400> 158
Ile Ile Pro Leu Ser Pro Ile Ala
1 5
<210> 159
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> FNI12 CDRH3 (aa)
<400> 159
Ala Thr Thr Arg Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asp
1 5 10 15
Tyr Phe Phe Asp His
20
<210> 160
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> FNI12 VH (co-nt)
<400> 160
caggtgcacc tggtgcagtc tggcgccgag gtgaagaagc caggctccag cgtgagggtg 60
tcctgcaagg ctagcggcga ctctttcaac aagtacgagg tgagctgggt gagacaggct 120
ccaggacatg gactggagtg gatgggcggc atcatccccc tgtctcctat cgccagatac 180
gctgagaagt tccagggccg cgtgaccatc acagctgatg agtttacctc cacagtgtat 240
atccagctga cctccctgag gagcgacgat acagccgtgt actattgtgc taccacaagg 300
agcgactact ttaatcggga tctgggctgg gaggactatt tctttgatca ctggggccag 360
ggcaccctgg tgacagtgtc ttcc 384
<210> 161
<211> 325
<212> DNA
<213> artificial sequence
<220>
<223> FNI12 Vk (wt-nt)
<400> 161
gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60
ctctcctgca gggccagtca gagtattagc accaacttag cctggtacca gcagaaacct 120
ggccaggctc ccaggctcct catctctggt gcatccacca gggccactgg tatcccagcc 180
aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240
gaagattttg gagtttatta ctgtcagcac tataataact ggcctccgtg gacgttcggc 300
caagggacca aggtggaaat caaac 325
<210> 162
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI12 VK (aa)
<400> 162
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Thr Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Ser Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Gly Val Tyr Tyr Cys Gln His Tyr Asn Asn Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 163
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> FNI12 CDRL1(aa)
<400> 163
Gln Ser Ile Ser Thr Asn
1 5
<210> 164
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> FNI12 CDRL2(aa)
<400> 164
Gly Ala Ser
1
<210> 165
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI12 CDRL3(aa)
<400> 165
Gln His Tyr Asn Asn Trp Pro Pro Trp Thr
1 5 10
<210> 166
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI12 Vk (co-nt)
<400> 166
gagatcgtga tgacccagtc ccctgccaca ctgtccgtgt ccccaggaga gagggccacc 60
ctgagctgcc gggctagcca gtctatctcc acaaacctgg cctggtacca gcagaagcca 120
ggacaggctc caaggctgct gatcagcgga gcttctacca gagctacagg catcccagct 180
cgcttcagcg gatctggatc cggaaccgag tttaccctga caatctccag cctgcagtct 240
gaggacttcg gcgtgtacta ttgtcagcac tataacaatt ggcccccttg gacctttggc 300
cagggcacaa aggtggagat caag 324
<210> 167
<211> 385
<212> DNA
<213> artificial sequence
<220>
<223> FNI13 VH (wt-nt)
<400> 167
caggttcagc tggtgcaatc tggggctgag gtgaagaggc ctgggtcctc ggtgagggtc 60
tcctgcaagg gttctggaga caccttcaac aactatgtta tcagttgggt gcgacaggcc 120
cctggccaag ggcttgagtg gatggggggg atcatcccta tctttcaaac accaaactac 180
gcagagaagt tccagggcag agtcgcgatt accgcggacg aatccacgag cacggcctac 240
atggagttga gcagcctgag atctgaggac tcggccattt attactgtgc gagagcgaat 300
tccgattact ttaatagaga cctcggctgg gaaaattact actttgaaga ctggggccag 360
ggaaccctgg tcaccgtctc ctcag 385
<210> 168
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FNI13 VH (aa)
<400> 168
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ser
1 5 10 15
Ser Val Arg Val Ser Cys Lys Gly Ser Gly Asp Thr Phe Asn Asn Tyr
20 25 30
Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Ile Phe Gln Thr Pro Asn Tyr Ala Glu Lys Phe
50 55 60
Gln Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Ala Asn Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asn
100 105 110
Tyr Tyr Phe Glu Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 169
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI13 CDRH1 (aa)
<400> 169
Gly Asp Thr Phe Asn Asn Tyr Val
1 5
<210> 170
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI13 CDRH2 (aa)
<400> 170
Ile Ile Pro Ile Phe Gln Thr Pro
1 5
<210> 171
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> FNI13 CDRH3 (aa)
<400> 171
Ala Arg Ala Asn Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asn
1 5 10 15
Tyr Tyr Phe Glu Asp
20
<210> 172
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> FNI13 VH (co-nt)
<400> 172
caggtgcagc tggtgcagtc cggagctgag gtgaagaggc caggatccag cgtgcgggtg 60
agctgcaagg gatctggcga caccttcaac aattacgtga tcagctgggt gaggcaggct 120
ccaggacagg gactggagtg gatgggcggc atcatcccca tcttccagac ccctaactac 180
gctgagaagt ttcagggcag ggtggccatc acagctgacg agtccaccag cacagcctat 240
atggagctgt cttccctgag atctgaggat tccgctatct actattgtgc cagagctaac 300
tctgactatt tcaatcgcga tctgggctgg gagaattact attttgagga ttggggccag 360
ggcaccctgg tgacagtgag ctct 384
<210> 173
<211> 325
<212> DNA
<213> artificial sequence
<220>
<223> FNI13 Vk (wt-nt)
<400> 173
gaaagagtga tgacgcagtc tccagccacc ctttctgtgt ctccaggggg aagagccacc 60
ctctcctgca gggccagtca gagtgttggt agcaacttag cctggtacca gcagaaacct 120
ggccaggctc ccaggctcct catctatgat gcttctgcca gggccactgg tgtcccagcc 180
aggttcagtg gcagtgggtc tgggacagag ttctctctct ccatcaacag cctgcagtct 240
gaagattctg cagtttatta ctgtcagcac tataatatct ggccgccgtg gacgttcggc 300
caagggacca aggtggaaat caaac 325
<210> 174
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI13 VK (aa)
<400> 174
Glu Arg Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Gly Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Ala Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Ser Leu Ser Ile Asn Ser Leu Gln Ser
65 70 75 80
Glu Asp Ser Ala Val Tyr Tyr Cys Gln His Tyr Asn Ile Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 175
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> FNI13 CDRL1(aa)
<400> 175
Gln Ser Val Gly Ser Asn
1 5
<210> 176
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> FNI13 CDRL2(aa)
<400> 176
Asp Ala Ser
1
<210> 177
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI13 CDRL3(aa)
<400> 177
Gln His Tyr Asn Ile Trp Pro Pro Trp Thr
1 5 10
<210> 178
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI13 Vk (co-nt)
<400> 178
gagagagtga tgacccagtc tcctgctaca ctgtccgtga gcccaggagg aagggctacc 60
ctgtcctgca gggcttctca gtccgtggga agcaacctgg cttggtacca gcagaagcca 120
ggccaggccc ccagactgct gatctatgac gcttccgcta gagctaccgg cgtgccagct 180
cgcttcagcg gatctggctc cggcacagag tttagcctgt ctatcaactc cctgcagagc 240
gaggattctg ccgtgtacta ttgtcagcac tacaatatct ggccaccttg gaccttcggc 300
cagggaacaa aggtggagat caag 324
<210> 179
<211> 385
<212> DNA
<213> artificial sequence
<220>
<223> FNI14 VH (wt-nt)
<400> 179
caagttcagt tggtgcagtc tggggctgag ctgaagcggc ctgggtcctc ggtgaggatc 60
tcctgcaagg cctctggtgt caccttcaac aagtatgttc tcagctgggt gcgactggcc 120
cctggacaag ggcttgagtg gatgggagga atcatcccta tttctggtat accacattac 180
gcagagaagt tccagggcag agtcgcgatt accgcggacg aatccacgag cacagtctac 240
atggagttga gcagcctacg atctgaggac tcggccctat attactgtgc gagagcggtc 300
tccgattatt ttaatcggga cctcggctgg gatgattact actttccttt gtggggccac 360
ggcaccctgg tcaccgtctc ctcag 385
<210> 180
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FNI14 VH (aa)
<400> 180
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Arg Pro Gly Ser
1 5 10 15
Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Val Thr Phe Asn Lys Tyr
20 25 30
Val Leu Ser Trp Val Arg Leu Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Ile Ser Gly Ile Pro His Tyr Ala Glu Lys Phe
50 55 60
Gln Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Leu Tyr Tyr Cys
85 90 95
Ala Arg Ala Val Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Asp Asp
100 105 110
Tyr Tyr Phe Pro Leu Trp Gly His Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 181
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI14 CDRH1 (aa)
<400> 181
Gly Val Thr Phe Asn Lys Tyr Val
1 5
<210> 182
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI14 CDRH2 (aa)
<400> 182
Ile Ile Pro Ile Ser Gly Ile Pro
1 5
<210> 183
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> FNI14 CDRH3 (aa)
<400> 183
Ala Arg Ala Val Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Asp Asp
1 5 10 15
Tyr Tyr Phe Pro Leu
20
<210> 184
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> FNI14 VH (co-nt)
<400> 184
caggtgcagc tggtgcagtc tggagctgag ctgaagaggc caggatccag cgtgcggatc 60
agctgcaagg cttctggcgt gaccttcaac aagtacgtgc tgtcctgggt gaggctggct 120
ccaggacagg gactggagtg gatgggcggc atcatcccca tcagcggcat ccctcactac 180
gctgagaagt ttcagggcag ggtggccatc acagctgacg agtccaccag cacagtgtat 240
atggagctgt cttccctgag atctgaggat tccgccctgt actattgtgc cagagccgtg 300
tccgactatt tcaatcgcga tctgggctgg gacgattact attttcccct gtggggccat 360
ggcaccctgg tgacagtgag ctct 384
<210> 185
<211> 325
<212> DNA
<213> artificial sequence
<220>
<223> FNI14 Vk (wt-nt)
<400> 185
gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagcgccacc 60
ctcttctgca gggccagtcg gagtgttagt gacaacttag cctggtacca gcagaaacct 120
ggccaggctc ccaggctcct catctttggt gcttccacca gggccactgg tgtcccagcc 180
aggttcggtg gcagtgggtc tgggacacag ttcactctca ccatcagcag cctgcagtct 240
gaagattttg cagtttatta ctgtcagcat tataataact ggcctccgtg gacgttcggc 300
caagggacca aggtggagat caaac 325
<210> 186
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI14 VK (aa)
<400> 186
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Ser Ala Thr Leu Phe Cys Arg Ala Ser Arg Ser Val Ser Asp Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Phe Gly Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Gly Gly
50 55 60
Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Asn Asn Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 187
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> FNI14 CDRL1(aa)
<400> 187
Arg Ser Val Ser Asp Asn
1 5
<210> 188
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> FNI14 CDRL2(aa)
<400> 188
Gly Ala Ser
1
<210> 189
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI14 CDRL3(aa)
<400> 189
Gln His Tyr Asn Asn Trp Pro Pro Trp Thr
1 5 10
<210> 190
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI14 Vk (co-nt)
<400> 190
gagatcgtga tgacccagtc ccctgccaca ctgtccgtgt ccccaggaga gagcgccacc 60
ctgttctgca gggctagcag gtccgtgtcc gacaacctgg cctggtacca gcagaagcca 120
ggccaggctc ccaggctgct gatctttggc gcctctacca gagctacagg cgtgccagct 180
aggttcggag gaagcggatc tggcacacag tttaccctga caatctccag cctgcagtcc 240
gaggatttcg ccgtgtacta ttgtcagcac tataacaatt ggcccccttg gacctttggc 300
cagggcacaa aggtggagat caag 324
<210> 191
<211> 385
<212> DNA
<213> artificial sequence
<220>
<223> FNI17 VH (wt-nt)
<400> 191
caggttcaac tggtgcagtc tggggctgag gtgaagaggc ctgggtcctc ggtgaaggtc 60
tcctgcaagc cttccggagg caccttcagc aacaatgtta tcagctgggt gcgacaggcc 120
cctggacaag ggcttgagtg gatgggaggg atcatcccca cctctggtat agcaaactac 180
gcgcagaagt tccagggcag agtcgcgatt attgcggaca aatctacgag cacagtctac 240
atggcgttga gcagcctgag atctgaggac tcggccgtgt atttctgtgc cagagcgcgg 300
tccgactact tcaatagaga cctcggctgg gaagattact actttgagaa ctggggccag 360
ggaaccctgg tcaccgtctc ctcag 385
<210> 192
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FNI17 VH (aa)
<400> 192
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Pro Ser Gly Gly Thr Phe Ser Asn Asn
20 25 30
Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Thr Ser Gly Ile Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Ala Ile Ile Ala Asp Lys Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Ala Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Ala Arg Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asp
100 105 110
Tyr Tyr Phe Glu Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 193
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI17 CDRH1 (aa)
<400> 193
Gly Gly Thr Phe Ser Asn Asn Val
1 5
<210> 194
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI17 CDRH2 (aa)
<400> 194
Ile Ile Pro Thr Ser Gly Ile Ala
1 5
<210> 195
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> FNI17 CDRH3 (aa)
<400> 195
Ala Arg Ala Arg Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asp
1 5 10 15
Tyr Tyr Phe Glu Asn
20
<210> 196
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> FNI17 VH (co-nt)
<400> 196
caggtgcagc tggtgcagtc cggagctgag gtgaagaggc caggctccag cgtgaaggtg 60
agctgcaagc cttctggcgg caccttctcc aacaatgtga tcagctgggt gagacaggct 120
ccaggacagg gactggagtg gatgggagga atcatcccca catctggcat cgccaactac 180
gctcagaagt ttcagggcag ggtggccatc atcgctgata agtccaccag cacagtgtat 240
atggccctgt cttccctgag atctgaggac tccgccgtgt acttctgtgc cagggctcgg 300
tccgactact tcaaccgcga tctgggctgg gaggactact atttcgagaa ttggggccag 360
ggcaccctgg tgacagtgag ctct 384
<210> 197
<211> 325
<212> DNA
<213> artificial sequence
<220>
<223> FNI17 Vk (wt-nt)
<400> 197
gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60
ctctcctgca gggccagtca gagtgttggc agcagcttag tctggtacca gcagaaacct 120
ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tgtcccagcc 180
aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240
gaagattttg cagtttatta ctgtcagcac tataataact ggcctccgtg gacgttcggc 300
caagggacca aggtggaaat caaac 325
<210> 198
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI17 VK (aa)
<400> 198
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser
20 25 30
Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Asn Asn Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 199
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> FNI17 CDRL1(aa)
<400> 199
Gln Ser Val Gly Ser Ser
1 5
<210> 200
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> FNI17 CDRL2(aa)
<400> 200
Gly Ala Ser
1
<210> 201
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI17 CDRL3(aa)
<400> 201
Gln His Tyr Asn Asn Trp Pro Pro Trp Thr
1 5 10
<210> 202
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI17 Vk (co-nt)
<400> 202
gagatcgtga tgacccagtc tcctgccaca ctgagcgtgt ctccaggaga gagggccacc 60
ctgtcctgca gggcttccca gagcgtggga tccagcctgg tgtggtacca gcagaagcca 120
ggacaggctc caaggctgct gatctatgga gctagcacca gagctacagg cgtgccagct 180
cgcttctctg gatccggaag cggcacagag tttaccctga caatctcttc cctgcagtct 240
gaggacttcg ccgtgtacta ttgtcagcac tacaacaatt ggcccccttg gacctttggc 300
cagggcacaa aggtggagat caag 324
<210> 203
<211> 385
<212> DNA
<213> artificial sequence
<220>
<223> FNI19 VH (wt-nt)
<400> 203
caagttcagc tggtgcagtc tggggctgag gtgaagaggc ctgggtcctc ggtgagggtc 60
tcctgcaagg cttctgaagg caccttcaac aagtatactc tcacctgggt gcgacaggcc 120
cctggacagg gacttgagtg gatgggagga atcatcccta tctccggtat agcaaactac 180
gcacagaagt tccagggcag agtcgcgatt accgcggacg aatccacgac cacagcctac 240
atggaattga gcagcctaag atctgaagac tcggccgtat attactgtgc gacagcggtc 300
tccgattatt ttaatcgaga cctcggctgg gaagattact actttccgtt ctggggccag 360
ggcaccctgg tcaccgtcgc ctcag 385
<210> 204
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FNI19 VH (aa)
<400> 204
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ser
1 5 10 15
Ser Val Arg Val Ser Cys Lys Ala Ser Glu Gly Thr Phe Asn Lys Tyr
20 25 30
Thr Leu Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Ile Ser Gly Ile Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Ala Val Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asp
100 105 110
Tyr Tyr Phe Pro Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ala Ser
115 120 125
<210> 205
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI19 CDRH1 (aa)
<400> 205
Glu Gly Thr Phe Asn Lys Tyr Thr
1 5
<210> 206
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> FNI19 CDRH2 (aa)
<400> 206
Ile Ile Pro Ile Ser Gly Ile Ala
1 5
<210> 207
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> FNI19 CDRH3 (aa)
<400> 207
Ala Thr Ala Val Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asp
1 5 10 15
Tyr Tyr Phe Pro Phe
20
<210> 208
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> FNI19 VH (co-nt)
<400> 208
caggtgcagc tggtgcagtc cggagctgag gtgaagaggc caggatccag cgtgcgggtg 60
tcctgcaagg ctagcgaggg cacattcaac aagtacacac tgacctgggt gaggcaggct 120
ccaggacagg gactggagtg gatgggcggc atcatcccta tctctggcat cgccaattac 180
gctcagaagt ttcagggcag agtggccatc acagctgatg agtccaccac aaccgcctat 240
atggagctgt cttccctgag aagcgaggac tccgccgtgt actattgtgc caccgctgtg 300
agcgactatt tcaaccgcga tctgggctgg gaggactact atttcccctt ttggggccag 360
ggcacactgg tgaccgtggc ttct 384
<210> 209
<211> 325
<212> DNA
<213> artificial sequence
<220>
<223> FNI19 Vk (wt-nt)
<400> 209
gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccgggggc cagagccacc 60
ctcttctgca gggccagtcg gagtgttagt gacaacttag cctggtacca gcagaaacct 120
ggccaggctc ccaggctcct catctttggt gcatccacca gggccactgg tgtcccagcc 180
aggttcagtg gaagtgggtc tgggacacag ttcactctca ccatcagcag cctgcagtcc 240
gaagattttg cagtttatta ctgtcagcat tataatattt ggcctccgtg gacgttcggc 300
caagggacca aggtggagat caaac 325
<210> 210
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI19 VK (aa)
<400> 210
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Ala Arg Ala Thr Leu Phe Cys Arg Ala Ser Arg Ser Val Ser Asp Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Phe Gly Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Asn Ile Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 211
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> FNI19 CDRL1(aa)
<400> 211
Arg Ser Val Ser Asp Asn
1 5
<210> 212
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> FNI19 CDRL2(aa)
<400> 212
Gly Ala Ser
1
<210> 213
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI19 CDRL3(aa)
<400> 213
Gln His Tyr Asn Ile Trp Pro Pro Trp Thr
1 5 10
<210> 214
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI19 Vk (co-nt)
<400> 214
gagatcgtga tgacccagtc ccctgctaca ctgtccgtgt ccccaggagc tagggctacc 60
ctgttctgca gggctagcag gtccgtgtcc gacaacctgg cttggtacca gcagaagcca 120
ggccaggccc ccagactgct gatctttgga gctagcacca gagctacagg cgtgccagct 180
cgcttcagcg gatctggatc cggcacacag tttaccctga caatctccag cctgcagtct 240
gaggatttcg ccgtgtacta ttgtcagcac tataatatct ggcccccttg gacctttggc 300
cagggcacaa aggtggagat caag 324
<210> 215
<400> 215
000
<210> 216
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> FNI3-VH-W110F (nt)
<400> 216
caggtgcagc tggtgcagtc cggagctgag gtgaagaggc caggatccag cgtgaaggtg 60
tcctgcaagg ccagcggcgc taccttcagc aacaatgtga tcgcttgggt gagacaggct 120
ccaggacagg gactggagtg gatgggagga atccacccta tcagcgccac cgctacatac 180
gcccagaagt ttcagggcag agtggctatc gccgctgacg agctgacctc tacagcctat 240
atggagctga acggcctgcg cagcgaggat tccgccgtgt actattgtgc cagggctggc 300
tctgactact tcaaccggga tctgggcttc gagaattact attttgactc ctggggccag 360
ggcaccctgg tgacagtgtc ttcc 384
<210> 217
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FNI3-VH-W110F (aa)
<400> 217
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Ala Thr Phe Ser Asn Asn
20 25 30
Val Ile Ala Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile His Pro Ile Ser Ala Thr Ala Thr Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Ala Ile Ala Ala Asp Glu Leu Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Asn Gly Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ala Gly Ser Asp Tyr Phe Asn Arg Asp Leu Gly Phe Glu Asn
100 105 110
Tyr Tyr Phe Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 218
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> FNI3-VH-W110F CDRH3 (aa)
<400> 218
Ala Arg Ala Gly Ser Asp Tyr Phe Asn Arg Asp Leu Gly Phe Glu Asn
1 5 10 15
Tyr Tyr Phe Asp Ser
20
<210> 219
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI3-VK-W94F (nt)
<400> 219
gagatcctga tgacccagtc ccctgccaca ctgtccgtgt ccccaggaga gagggccacc 60
ctgagctgca gggcttctca ggacgtgtcc ggcaacctgg cctggtacca gcagagacca 120
ggacaggctc caaggctgct gatctatgga gcttccacca gggctacagg cgtgccagct 180
agattcaccg gcgctggaag cggcacagag tttaccctga caatctccag cctgcagtct 240
gaggatttcg ctctgtacta ttgtcagcac tacaacaatt ttcccccttg gacctttggc 300
cagggcacaa aggtggagat caag 324
<210> 220
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI3-VK-W94F (aa)
<400> 220
Glu Ile Leu Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Val Ser Gly Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Thr Gly
50 55 60
Ala Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Leu Tyr Tyr Cys Gln His Tyr Asn Asn Phe Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 221
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI3-VK-W94F CDRL3 (aa)
<400> 221
Gln His Tyr Asn Asn Phe Pro Pro Trp Thr
1 5 10
<210> 222
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI3-VK-W97F (nt)
<400> 222
gagatcctga tgacccagtc ccctgccaca ctgtccgtgt ccccaggaga gagggccacc 60
ctgagctgca gggcttctca ggacgtgtcc ggcaacctgg cctggtacca gcagagacca 120
ggacaggctc caaggctgct gatctatgga gcttccacca gggctacagg cgtgccagct 180
agattcaccg gcgctggaag cggcacagag tttaccctga caatctccag cctgcagtct 240
gaggatttcg ctctgtacta ttgtcagcac tacaacaatt ggcccccttt cacctttggc 300
cagggcacaa aggtggagat caag 324
<210> 223
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI3-VK-W97F (aa)
<400> 223
Glu Ile Leu Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Val Ser Gly Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Thr Gly
50 55 60
Ala Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Leu Tyr Tyr Cys Gln His Tyr Asn Asn Trp Pro Pro
85 90 95
Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 224
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI3-VK-W97F CDRL3 (aa)
<400> 224
Gln His Tyr Asn Asn Trp Pro Pro Phe Thr
1 5 10
<210> 225
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI3-VK-W94F-W97F (nt)
<400> 225
gagatcctga tgacccagtc ccctgccaca ctgtccgtgt ccccaggaga gagggccacc 60
ctgagctgca gggcttctca ggacgtgtcc ggcaacctgg cctggtacca gcagagacca 120
ggacaggctc caaggctgct gatctatgga gcttccacca gggctacagg cgtgccagct 180
agattcaccg gcgctggaag cggcacagag tttaccctga caatctccag cctgcagtct 240
gaggatttcg ctctgtacta ttgtcagcac tacaacaatt ttcccccttt cacctttggc 300
cagggcacaa aggtggagat caag 324
<210> 226
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI3-VK-W94F-W97F (aa)
<400> 226
Glu Ile Leu Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Val Ser Gly Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Thr Gly
50 55 60
Ala Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Leu Tyr Tyr Cys Gln His Tyr Asn Asn Phe Pro Pro
85 90 95
Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 227
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI3-VK-W94F-W97F CDRL3 (aa)
<400> 227
Gln His Tyr Asn Asn Phe Pro Pro Phe Thr
1 5 10
<210> 228
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> FNI9-VH-W110F (nt)
<400> 228
caggtgcacc tggtgcagag cggagctgag gtgaaggagc caggatccag cgtgacagtg 60
tcttgcaagg cttccggcgg cagcttcaac aatcaggcta tctcctgggt gaggcaggct 120
ccaggacagg gactggagtg gatgggcggc atctttccca tctctggcac acctacctcc 180
gcccagaggt tccagggaag ggtgaccttc accgctgacg agagcaccac aaccgtgtac 240
atggatctgt cttccctgag atctgacgat accgccgtgt actattgtgc cagagctggc 300
tccgactatt tcaaccgcga tctgggcttc gagaattact attttgcttc ctggggccag 360
ggcacactgg tgaccgtgag ctct 384
<210> 229
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FNI9-VH-W110F (aa)
<400> 229
Gln Val His Leu Val Gln Ser Gly Ala Glu Val Lys Glu Pro Gly Ser
1 5 10 15
Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Ser Phe Asn Asn Gln
20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Phe Pro Ile Ser Gly Thr Pro Thr Ser Ala Gln Arg Phe
50 55 60
Gln Gly Arg Val Thr Phe Thr Ala Asp Glu Ser Thr Thr Thr Val Tyr
65 70 75 80
Met Asp Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ala Gly Ser Asp Tyr Phe Asn Arg Asp Leu Gly Phe Glu Asn
100 105 110
Tyr Tyr Phe Ala Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 230
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> FNI9-VH-W110F CDRH3 (aa)
<400> 230
Ala Arg Ala Gly Ser Asp Tyr Phe Asn Arg Asp Leu Gly Phe Glu Asn
1 5 10 15
Tyr Tyr Phe Ala Ser
20
<210> 231
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI9-VK-W94F (nt)
<400> 231
gagatcgtga tgacccagtc cccagccaca ctgagcctgt ccagcggaga gagggccacc 60
ctgtcctgca gggcttcccg gagcgtgtct tccaacctgg cctggtacca gcagaagcca 120
ggccaggctc ccagactgct gatctatgac gcctctacca gagctacagg cttctccgcc 180
aggtttgctg gatctggatc cggcacagag ttcaccctga caatcagctc tctgcagagc 240
gaggattctg ctatctacta ttgtcagcag tacaacaatt tccccccttg gacctttggc 300
cagggcacaa aggtggagat caag 324
<210> 232
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI9-VK-W94F (aa)
<400> 232
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Ser Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Arg Ser Val Ser Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Thr Arg Ala Thr Gly Phe Ser Ala Arg Phe Ala Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Ser Ala Ile Tyr Tyr Cys Gln Gln Tyr Asn Asn Phe Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 233
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI9-VK-W94F CDRL3 (aa)
<400> 233
Gln Gln Tyr Asn Asn Phe Pro Pro Trp Thr
1 5 10
<210> 234
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI9-VK-W97F (nt)
<400> 234
gagatcgtga tgacccagtc cccagccaca ctgagcctgt ccagcggaga gagggccacc 60
ctgtcctgca gggcttcccg gagcgtgtct tccaacctgg cctggtacca gcagaagcca 120
ggccaggctc ccagactgct gatctatgac gcctctacca gagctacagg cttctccgcc 180
aggtttgctg gatctggatc cggcacagag ttcaccctga caatcagctc tctgcagagc 240
gaggattctg ctatctacta ttgtcagcag tacaacaatt ggcccccttt cacctttggc 300
cagggcacaa aggtggagat caag 324
<210> 235
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI9-VK-W97F (aa)
<400> 235
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Ser Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Arg Ser Val Ser Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Thr Arg Ala Thr Gly Phe Ser Ala Arg Phe Ala Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Ser Ala Ile Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro
85 90 95
Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 236
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI9-VK-W97F CDRL3 (aa)
<400> 236
Gln Gln Tyr Asn Asn Trp Pro Pro Phe Thr
1 5 10
<210> 237
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI9-VK-W94F-W97F (nt)
<400> 237
gagatcgtga tgacccagtc cccagccaca ctgagcctgt ccagcggaga gagggccacc 60
ctgtcctgca gggcttcccg gagcgtgtct tccaacctgg cctggtacca gcagaagcca 120
ggccaggctc ccagactgct gatctatgac gcctctacca gagctacagg cttctccgcc 180
aggtttgctg gatctggatc cggcacagag ttcaccctga caatcagctc tctgcagagc 240
gaggattctg ctatctacta ttgtcagcag tacaacaatt ttcccccttt cacctttggc 300
cagggcacaa aggtggagat caag 324
<210> 238
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI9-VK-W94F-W97F (aa)
<400> 238
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Ser Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Arg Ser Val Ser Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Thr Arg Ala Thr Gly Phe Ser Ala Arg Phe Ala Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Ser Ala Ile Tyr Tyr Cys Gln Gln Tyr Asn Asn Phe Pro Pro
85 90 95
Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 239
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> FNI9-VK-W94F-W97F CDRL3 (aa)
<400> 239
Gln Gln Tyr Asn Asn Phe Pro Pro Phe Thr
1 5 10
<210> 240
<211> 386
<212> DNA
<213> artificial sequence
<220>
<223> FNI17-v19-VH (co-nt)
<400> 240
caggtccagc tggtccagag tggggcagag gtcaaagagc cagggtcttc agtcacagtc 60
tcatgcaaag caagcggagg aacattttcc aacaatgtga tcagctgggt gaggcaggct 120
ccaggacagg gactggagtg gatgggcggc atcatcccta cctctggcat cgccaactac 180
gctcagaagt tccagggcag agtggccatc atcgctgaca agtctacctc cacagtgtat 240
atggccctgt ccagcctgag aagcgaggat tccgccgtgt acttctgcgc cagggctcgg 300
tccgactact tcaaccgcga tctgggttgg gaggactatt actttgaaaa ctgggggcag 360
ggcacactgg tcactgtctc atcagc 386
<210> 241
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FNI17-v19-VH (aa)
<400> 241
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Glu Pro Gly Ser
1 5 10 15
Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Asn
20 25 30
Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Thr Ser Gly Ile Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Ala Ile Ile Ala Asp Lys Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Ala Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Ala Arg Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asp
100 105 110
Tyr Tyr Phe Glu Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 242
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI17-v19-VK (co-nt)
<400> 242
gaaattgtga tgacccagtc tccagccact ctgtcagtct ctccagggga acgagccact 60
ctgtcatgtc gggcctctca gtccgtcggc tccagcctgg cttggtacca gcagaagcca 120
ggacaggctc ctaggctgct gatctatgga gctagcacca gggctacagg cgtgccagct 180
cggttcagcg gatctggatc cggcaccgag tttaccctga caatctcttc cctgcagtct 240
gaggacttcg ccgtgtacta ttgccagcac tacaataact ggcctccttg gacattcggg 300
caggggacaa aagtcgagat taag 324
<210> 243
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI17-v19-VK (aa)
<400> 243
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Asn Asn Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 244
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> FNI19-v3-VH (co-nt)
<400> 244
caggtccagc tggtgcagag tggtgccgag gtcaaaaagc cagggtcaag tgtcaaagtc 60
agttgtaaag catcagaggg aacattcaac aagtacacaa tcagctgggt gagacaggct 120
ccaggacagg gactggagtg gatgggcggc atcatcccta tctctggcat cgccaattac 180
gctcagaagt tccagggccg cgtggccatc acagctgacg agtccaccac aaccgcctat 240
atggagctgt ccagcctgag gtctgaggat tccgccgtgt actattgcgc caccgctgtg 300
agcgactact tcaaccggga tctgggctgg gaggactatt attttccatt ctggggtcag 360
gggacactgg tcaccgtctc ttcc 384
<210> 245
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FNI19-v3-VH (aa)
<400> 245
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Glu Gly Thr Phe Asn Lys Tyr
20 25 30
Thr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Ile Ser Gly Ile Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Ala Val Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asp
100 105 110
Tyr Tyr Phe Pro Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 246
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI19-v3-VK (co-nt)
<400> 246
gagatcgtga tgacccagtc ccctgctaca ctgtccgtgt ccccaggagc tagggctacc 60
ctgttctgca gggctagcag gtccgtgtcc gacaacctgg cttggtacca gcagaagcca 120
ggccaggccc ccagactgct gatctttgga gctagcacca gagctacagg cgtgccagct 180
cgcttcagcg gatctggatc cggcacacag tttaccctga caatctccag cctgcagtct 240
gaggatttcg ccgtgtacta ttgtcagcac tataatatct ggcccccttg gacctttggc 300
cagggcacaa aggtggagat caag 324
<210> 247
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI19-v3-VK (aa)
<400> 247
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Ala Arg Ala Thr Leu Phe Cys Arg Ala Ser Arg Ser Val Ser Asp Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Phe Gly Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Asn Ile Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 248
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> FNI9-v5-VH (co-nt)
<400> 248
caggtgcacc tggtgcagag cggagctgag gtgaaggagc caggatccag cgtgacagtg 60
tcttgcaagg cttccggcgg cagcttcaac aatcaggcta tctcctgggt gaggcaggct 120
ccaggacagg gactggagtg gatgggcggc atctttccca tctctggcac acctacctcc 180
gcccagaggt tccagggaag ggtgaccttc accgctgacg agagcaccac aaccgtgtac 240
atggatctgt cttccctgag atctgacgat accgccgtgt actattgtgc cagagctggc 300
tccgactatt tcaaccgcga tctgggctgg gagaattact attttgcttc ctggggccag 360
ggcacactgg tgaccgtgag ctct 384
<210> 249
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> FNI9-v5-VH (aa)
<400> 249
Gln Val His Leu Val Gln Ser Gly Ala Glu Val Lys Glu Pro Gly Ser
1 5 10 15
Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Ser Phe Asn Asn Gln
20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Phe Pro Ile Ser Gly Thr Pro Thr Ser Ala Gln Arg Phe
50 55 60
Gln Gly Arg Val Thr Phe Thr Ala Asp Glu Ser Thr Thr Thr Val Tyr
65 70 75 80
Met Asp Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ala Gly Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asn
100 105 110
Tyr Tyr Phe Ala Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 250
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> FNI9-v5-VK (co-nt)
<400> 250
gagattgtga tgacccagtc ccctgctacc ctgagcgtgt cccccggaga gagagctacc 60
ctgagttgcc gcgccagccg cagtgtctct gacaacctgg cttggtacca gcagaagcca 120
ggacaggctc ctaggctgct gatctatggc gcctccacca gggctacagg catcccagct 180
cggttctctg gatccggaag cggcaccgag tttaccctga caatctccag cctgcagagc 240
gaggatttcg ccgtgtacta ttgccagcat tacaacatct ggcctccttg gacattcggt 300
cagggaacta aagtggaaat taag 324
<210> 251
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> FNI9-v5-VK (aa)
<400> 251
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Arg Ser Val Ser Asp Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Asn Ile Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 252
<211> 330
<212> PRT
<213> artificial sequence
<220>
<223> IgHG1 x 01, having M428L and N434S mutations and C-terminal lysine
G1m3 CH1-CH3
<400> 252
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 253
<211> 329
<212> PRT
<213> artificial sequence
<220>
<223> IgHG1 x 01, with M428L and N434S mutations but without C-terminal lysine
G1m3 CH1-CH3
<400> 253
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly
325
<210> 254
<211> 107
<212> PRT
<213> artificial sequence
<220>
<223> kappa light chain CL
<400> 254
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 255
<211> 458
<212> PRT
<213> artificial sequence
<220>
<223> FNI17-v19 heavy chain with M428L and N434S mutations in CH3 and with C-terminal lysine
<400> 255
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Glu Pro Gly Ser
1 5 10 15
Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Asn
20 25 30
Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Thr Ser Gly Ile Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Ala Ile Ile Ala Asp Lys Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Ala Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Ala Arg Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asp
100 105 110
Tyr Tyr Phe Glu Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
130 135 140
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
145 150 155 160
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
165 170 175
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
180 185 190
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
195 200 205
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
210 215 220
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
225 230 235 240
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
245 250 255
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
260 265 270
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
275 280 285
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
290 295 300
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
305 310 315 320
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
325 330 335
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
340 345 350
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
355 360 365
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
370 375 380
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
385 390 395 400
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
405 410 415
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
420 425 430
Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr
435 440 445
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 256
<211> 457
<212> PRT
<213> artificial sequence
<220>
<223> FNI17-v19 heavy chain with M428L and N434S mutations in CH3 but without C-terminal lysine
<400> 256
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Glu Pro Gly Ser
1 5 10 15
Ser Val Thr Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Asn
20 25 30
Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Thr Ser Gly Ile Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Ala Ile Ile Ala Asp Lys Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Ala Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Ala Arg Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asp
100 105 110
Tyr Tyr Phe Glu Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
130 135 140
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
145 150 155 160
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
165 170 175
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
180 185 190
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
195 200 205
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
210 215 220
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
225 230 235 240
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
245 250 255
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
260 265 270
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
275 280 285
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
290 295 300
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
305 310 315 320
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
325 330 335
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
340 345 350
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
355 360 365
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
370 375 380
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
385 390 395 400
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
405 410 415
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
420 425 430
Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr
435 440 445
Gln Lys Ser Leu Ser Leu Ser Pro Gly
450 455
<210> 257
<211> 215
<212> PRT
<213> artificial sequence
<220>
<223> FNI17-v19 light chain
<400> 257
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Asn Asn Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
100 105 110
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
130 135 140
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
145 150 155 160
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205
Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 258
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FNI17-v13 VH
<400> 258
Gln Val Gln Leu Val Gln Ser Gly Ala Arg Val Lys Glu Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Asn
20 25 30
Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Thr Ser Gly Ile Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Ala Ile Ile Ala Asp Lys Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Ala Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Ala Arg Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asp
100 105 110
Tyr Tyr Phe Glu Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 259
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FNI17-v13 VK
<400> 259
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Asn Asn Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 260
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence FNI-UCA-IGH
<400> 260
caggtgcagc tggtgcagtc tggcgccgag gtgaagaagc caggctccag cgtgaaggtg 60
agctgcaagg cttctggcgg caccttctct tcctacgcta tctcctgggt gaggcaggct 120
ccaggacagg gactggagtg gatgggcggc atcatcccta tcttcggcac agccaactac 180
gctcagaagt ttcagggcag agtgaccatc acagccgacg agtctacctc cacagcttat 240
atggagctga gctctctgcg ctccgaggat accgccgtgt actattgtgc cagggctggc 300
agcgactact tcaaccggga tctgggctgg gagaattact attttgacta ttggggccag 360
ggcaccctgg tgacagtgtc cagc 384
<210> 261
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FNI-UCA VH
<400> 261
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ala Gly Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asn
100 105 110
Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 262
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence FNI-UCA-IGK
<400> 262
gagatcgtga tgacccagtc tcctgccaca ctgagcgtgt ctccaggaga gagggccacc 60
ctgtcctgca gggcttccca gagcgtgtcc agcaacctgg cctggtacca gcagaagcca 120
ggccaggctc ccaggctgct gatctatggc gccagcacca gagctacagg catcccagct 180
cgcttctctg gatccggaag cggcacagag tttaccctga caatctcttc cctgcagtct 240
gaggacttcg ccgtgtacta ttgtcagcag tacaacaatt ggcccccttg gacctttggc 300
cagggcacaa aggtggagat caag 324
<210> 263
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FNI-UCA VK
<400> 263
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 264
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FNI-UCA CDRH1
<400> 264
Gly Gly Thr Phe Ser Ser Tyr Ala
1 5
<210> 265
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FNI-UCA CDRH2
<400> 265
Ile Ile Pro Ile Phe Gly Thr Ala
1 5
<210> 266
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FNI-UCA CDRH3
<400> 266
Ala Arg Ala Gly Ser Asp Tyr Phe Asn Arg Asp Leu Gly Trp Glu Asn
1 5 10 15
Tyr Tyr Phe Asp Tyr
20
<210> 267
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FNI-UCA CDRL1
<400> 267
Gln Ser Val Ser Ser Asn
1 5
<210> 268
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FNI-UCA CDRL2
<400> 268
Gly Ala Ser
1
<210> 269
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FNI-UCA CDRL3
<400> 269
Gln Gln Tyr Asn Asn Trp Pro Pro Trp Thr
1 5 10
<210> 270
<211> 458
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FM08_LS heavy chain
<400> 270
Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Tyr
20 25 30
Asn Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu
35 40 45
Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn Asp Tyr Ala
50 55 60
Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn
65 70 75 80
Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val
100 105 110
Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
130 135 140
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
145 150 155 160
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
165 170 175
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
180 185 190
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
195 200 205
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
210 215 220
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
225 230 235 240
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
245 250 255
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
260 265 270
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
275 280 285
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
290 295 300
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
305 310 315 320
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
325 330 335
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
340 345 350
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
355 360 365
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
370 375 380
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
385 390 395 400
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
405 410 415
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
420 425 430
Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr
435 440 445
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 271
<211> 210
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FM08_LS light chain
<400> 271
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr
20 25 30
Thr His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Arg Gly Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln
85 90 95
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe
100 105 110
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
115 120 125
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
130 135 140
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
145 150 155 160
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr
165 170 175
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val
180 185 190
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
195 200 205
Glu Cys
210
<210> 272
<211> 458
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FM08_GAALIE_LS heavy chain
<400> 272
Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Tyr
20 25 30
Asn Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu
35 40 45
Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn Asp Tyr Ala
50 55 60
Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn
65 70 75 80
Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val
100 105 110
Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
130 135 140
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
145 150 155 160
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
165 170 175
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
180 185 190
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
195 200 205
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
210 215 220
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
225 230 235 240
Pro Ala Pro Glu Leu Leu Ala Gly Pro Ser Val Phe Leu Phe Pro Pro
245 250 255
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
260 265 270
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
275 280 285
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
290 295 300
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
305 310 315 320
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
325 330 335
Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly
340 345 350
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
355 360 365
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
370 375 380
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
385 390 395 400
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
405 410 415
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
420 425 430
Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr
435 440 445
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 273
<211> 210
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FM08 GAALIE LS light chain
<400> 273
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr
20 25 30
Thr His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Arg Gly Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln
85 90 95
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe
100 105 110
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
115 120 125
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
130 135 140
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
145 150 155 160
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr
165 170 175
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val
180 185 190
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
195 200 205
Glu Cys
210
<210> 274
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FM08 GAALIE LS light chain
<400> 274
Ser Tyr Asn Ala Val Trp Asn
1 5
<210> 275
<211> 18
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FM08 CDRH2
<400> 275
Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn Asp Tyr Ala Glu Ser Val
1 5 10 15
Lys Ser
<210> 276
<211> 16
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FM08 CDRH3
<400> 276
Ser Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met
1 5 10 15
<210> 277
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FM08 CDRL1
<400> 277
Arg Thr Ser Gln Ser Leu Ser Ser Tyr Thr His
1 5 10
<210> 278
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FM08 CDRL2
<400> 278
Ala Ala Ser Ser Arg Gly Ser
1 5
<210> 279
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FM08 CDRL3
<400> 279
Gln Gln Ser Arg Thr
1 5
<210> 280
<211> 330
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence FM08 CDRL3
<400> 280
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Ala Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 281
<211> 330
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence IgG1 GAALIE_MLNS CH1-CH3
<400> 281
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Ala Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Leu Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330

Claims (98)

1. A combination, the combination comprising:
(1) (a) an antibody or antigen-binding fragment thereof that is capable of binding to Influenza A Virus (IAV) Hemagglutinin (HA) and neutralizing an infection caused by the IAV, or (b) a polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof; and
(2) (a) an antibody or antigen binding fragment thereof capable of binding to Neuraminidase (NA) from the group consisting of: 2 (i) IAV, wherein the IAV comprises a group 1 IAV, a group 2 IAV, or both; and 2 (ii) an Influenza B Virus (IBV) and is capable of neutralising infection by and/or inhibiting sialidase activity of said IAV and/or said IBV, or (b) a polynucleotide encoding an anti-NA antibody or antigen binding fragment thereof.
2. A composition, the combination comprising:
(1) (a) an antibody or antigen-binding fragment thereof that is capable of binding to Influenza A Virus (IAV) Hemagglutinin (HA) and neutralizing an infection caused by the IAV, or (b) a polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof; and
(2) (a) an antibody or antigen binding fragment thereof capable of binding to Neuraminidase (NA) from the group consisting of: 2 (i) IAV, wherein the IAV comprises a group 1 IAV, a group 2 IAV, or both; and 2 (ii) an Influenza B Virus (IBV) and is capable of neutralising infection by and/or inhibiting sialidase activity of said IAV and/or said IBV, or (b) a polynucleotide encoding an anti-NA antibody or antigen-binding fragment thereof,
and optionally a pharmaceutically acceptable carrier, excipient or diluent.
3. The combination according to claim 1 or the composition according to claim 2 for use in a method of treating or preventing influenza (IAV, IBV or both) infection in a subject, wherein the method comprises administering to the subject an effective amount of the composition or the combination, respectively.
4. The combination according to claim 1 or the composition according to claim 2 for use in the manufacture of a medicament for treating or preventing influenza (IAV, IBV or both) infection in a subject.
5. An antibody or antigen-binding fragment thereof capable of binding to Influenza A Virus (IAV) Hemagglutinin (HA) and neutralizing infection by IAV, or a polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof,
the antibody or antigen-binding fragment thereof or the polynucleotide is used in a method of treating or preventing influenza infection in a subject,
wherein the method comprises administering an effective amount of the anti-HA antibody or antigen-binding fragment thereof to a subject who HAs received, is receiving, or will receive: (1) An effective amount of an antibody or antigen-binding fragment thereof capable of binding to Neuraminidase (NA) from the group consisting of: (i) IAVs, wherein the IAV comprises a group 1 IAV, a group 2 IAV, or both; and (ii) an Influenza B Virus (IBV) and is capable of neutralising infection by and/or inhibiting sialidase activity of said IAV and/or said IBV, or (2) a polynucleotide encoding an anti-NA antibody or antigen binding fragment thereof.
6. An antibody or antigen-binding fragment thereof capable of binding to Neuraminidase (NA) from the group consisting of: (i) Influenza A Virus (IAV), wherein the IAV comprises a group 1 IAV, a group 2 IAV, or both; and (ii) an Influenza B Virus (IBV) and is capable of neutralising infection by and/or inhibiting sialidase activity of said IAV and/or said IBV, or a polynucleotide encoding an anti-NA antibody or antigen-binding fragment thereof,
the antibody or antigen binding fragment thereof or the polynucleotide is used in a method of treating or preventing influenza (IAV, IBV or both) infection in a subject,
wherein the method comprises administering to a subject who has received, is receiving, or is about to receive an effective amount of the anti-NA antibody or antigen-binding fragment thereof: (a) An antibody or antigen-binding fragment thereof that is capable of binding to IAV Hemagglutinin (HA) and neutralizing an infection caused by the IAV, or (b) a polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof.
7. A method of treating or preventing an influenza (IAV, IBV, or both) infection in a subject, the method comprising administering to the subject an effective amount of:
(1) (a) an antibody or antigen-binding fragment thereof that is capable of binding to Influenza A Virus (IAV) Hemagglutinin (HA) and neutralizing an infection caused by the IAV, or (b) a polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof; and
(2) (a) an antibody or antigen binding fragment thereof capable of binding to Neuraminidase (NA) from the group consisting of: 2 (i) IAV, wherein the IAV comprises a group 1 IAV, a group 2 IAV, or both; and 2 (ii) an Influenza B Virus (IBV) and is capable of neutralising infection by and/or inhibiting sialidase activity of said IAV and/or said IBV, or (b) a polynucleotide encoding an anti-NA antibody or antigen binding fragment thereof.
8. A method of treating or preventing an influenza (IAV, IBV, or both) infection in a subject, the method comprising administering to the subject an effective amount of: (1) An antibody or antigen-binding fragment thereof that is capable of binding to Influenza A Virus (IAV) Hemagglutinin (HA) and neutralizing an infection caused by the IAV, or (2) a polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof,
wherein the subject has received, is receiving, or will receive the following: (a) An antibody or antigen-binding fragment thereof capable of binding to Neuraminidase (NA) from the group consisting of: (i) IAVs, wherein the IAV comprises a group 1 IAV, a group 2 IAV, or both; and (ii) an Influenza B Virus (IBV) and is capable of neutralising infection by and/or inhibiting sialidase activity of said IAV and/or said IBV, or (b) a polynucleotide encoding an anti-NA antibody or antigen binding fragment thereof.
9. A method of treating or preventing influenza infection in a subject, the method comprising administering to the subject an effective amount of: (1) An antibody or antigen-binding fragment thereof capable of binding to Neuraminidase (NA) from the group consisting of: (i) Influenza A Virus (IAV), wherein the IAV comprises a group 1 IAV, a group 2 IAV, or both; and (ii) an Influenza B Virus (IBV) and is capable of neutralising infection by and/or inhibiting sialidase activity of said IAV and/or said IBV, or (2) a polynucleotide encoding an anti-NA antibody or antigen-binding fragment thereof,
wherein the subject has received, is receiving, or will receive the following: (a) An antibody or antigen-binding fragment thereof that is capable of binding to IAV Hemagglutinin (HA) and neutralizing an infection caused by the IAV, or (b) a polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof.
10. The combination according to claim 1, the composition according to claim 2, the combination or composition for use according to any one of claims 3 and 4, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5 and 6 or the method according to any one of claims 7 to 9, wherein:
(1) The anti-HA antibody or antigen-binding fragment comprises: (1) (i) a VH comprising or consisting of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to an amino acid sequence set forth in any one of seq id nos: SEQ ID NOs 43, 2, 26, 28, 31, 34, 37, 14, 39 and 41, wherein the sequence variants referring to SEQ ID NOs 43, 2, 26, 28, 31, 34, 37, 14, 39 and 41, respectively, are optionally included in one or more framework regions and/or the sequence variants include substitution of one or more germline encoded amino acids; and/or (1) (ii) VL comprises or consists of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to any one of the following amino acid sequences: SEQ ID NOS.44, 8 and 20, wherein sequence variations relative to SEQ ID NOS.44, 8 and 20, respectively, are optionally included in one or more framework regions, and/or sequence variations include substitution of one or more germline encoded amino acids;
and/or
(2) The anti-NA antibody or antigen-binding fragment comprises: (2) (i) a VH comprising or consisting of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to an amino acid sequence set forth in any one of seq id nos: 241, 48, 60, 72, 171, 84, 96, 108, 120, 132, 229, 144, 156, 168, 180, 192, 204, 245, 249, 258 and 261, wherein the sequence variations of SEQ ID NOs 241, 48, 60, 72, 171, 84, 96, 108, 120, 132, 229, 144, 156, 168, 180, 192, 204, 245, 249, 258, 261 are optionally included in one or more framework regions, and/or the sequence variations comprise substitutions of one or more germline encoded amino acids; and/or (2) (ii) the VL comprises or consists of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to any one of the amino acid sequences: 243, 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 220, 223, 226, 232, 235, 238, 210, 247, 251, 259 and 263, respectively, wherein the sequence variations of 243, 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 220, 223, 226, 232, 235, 238, 210, 247, 251, 259 and 263 relative to SEQ ID NOs 243, 54, 66, 247, 251, 259 and 263, respectively, optionally comprise a substitution in one or more framework regions, and/or the sequence variations comprise one or more germline-encoded amino acids,
Wherein preferably the anti-HA antibody or antigen-binding fragment comprises or consists of a VH and a VL comprising or consisting of an amino acid sequence having at least 80% identity to the amino acid sequences set forth in SEQ ID NOs 43 and 44, respectively, and the anti-NA antibody or antigen-binding fragment comprises or consists of a VH and a VL comprising or consisting of an amino acid sequence having at least 80% identity to the amino acid sequences set forth in SEQ ID NOs (4) (i) 241 and 243;
and/or
(3) The anti-HA antibody or antigen-binding fragment comprises a VH and a VL comprising the HCDR and LCDR of the VH amino acid sequence and VL amino acid sequence, respectively, shown below: (3) (i) SEQ ID NOS 43 and 44, respectively; (3) (ii) SEQ ID NOS 26 and 8, respectively; (3) (iii) SEQ ID NOs 2 and 8, respectively; (3) (iv) SEQ ID NOs 31 and 8, respectively; (3) (v) SEQ ID NOS: 34 and 8, respectively; (3) (vi) SEQ ID NOS 37 and 8, respectively; (3) (vii) SEQ ID NOs 14 and 20, respectively; (3) (viii) SEQ ID NOs 39 and 20, respectively; (3) (ix) SEQ ID NOs 41 and 20, respectively; or (1) (x) is SEQ ID NO 28 and 8, respectively, wherein optionally the HCDR and the LCDR are numbered according to IMGT;
and/or
(4) The anti-NA antibody or antigen-binding fragment comprises a VH and a VL comprising the HCDR and LCDR of the VH amino acid sequence and VL amino acid sequence, respectively, shown below: (4) (i) SEQ ID NOs 241 and 243, respectively; (4) (ii) SEQ ID NOs 60 and 66, respectively; (4) (iii) SEQ ID NOs 72 and 78 or 220 or 223, respectively; (4) (vi) SEQ ID NOS 72 and 226, respectively; (4) (vii) SEQ ID NOS 217 and 78, respectively; (4) (viii) SEQ ID NOS 217 and 220, respectively; (4) (ix) SEQ ID NOS 217 and 223, respectively; (4) (x) is SEQ ID NO 217 and 226, respectively; (4) (xi) SEQ ID NOs 84 and 90, respectively; (4) (xii) SEQ ID NOS 96 and 102, respectively; (4) (xiii) SEQ ID NOs 108 and 114, respectively; (4) (xiv) SEQ ID NOS 120 and 126, respectively; (4) (xv) SEQ ID NOS 132 and 138, respectively; (4) (xvi) SEQ ID NOS 132 and 232, respectively; (4) (xvii) SEQ ID NOS 132 and 235, respectively; (4) (xviii) SEQ ID NOS 132 and 238, respectively; (4) (xix) SEQ ID NOs 229 and 138, respectively; (4) (xx) SEQ ID NOs 229 and 232, respectively; (4) (xxi) SEQ ID NOs 229 and 235, respectively; (4) (xxii) SEQ ID NOs 229 and 238, respectively; (4) (xxiii) SEQ ID NOS 144 and 150, respectively; (4) (xxiv) SEQ ID NOS 156 and 162, respectively; (4) (xxv) SEQ ID NOS 168 and 174, respectively; (4) (xxvi) SEQ ID NOS 180 and 186, respectively; (4) (xxvii) SEQ ID NOS 192 and 198, respectively; (4) (xxviii) SEQ ID NOS 204 and 210, respectively; (4) (xxix) SEQ ID NOs 48 and 54, respectively; (4) (xxx) SEQ ID NOs 245 and 247, respectively; (4) (xxxi) SEQ ID NOS 249 and 251, respectively; (4) (xxxii) SEQ ID NOs 258 and 259, respectively; or (4) (xxxi) SEQ ID NO 261 and 263, respectively, wherein optionally said HCDR and said LCDR are numbered according to IMGT,
Wherein preferably the anti-HA antibody or antigen-binding fragment comprises a VH and a VL comprising the HCDR and LCDR of the VH amino acid sequence and VL amino acid sequence shown below, respectively: 43 and 44, respectively, and the anti-NA antibody or antigen-binding fragment comprises a VH and a VL comprising the HCDR and LCDR of the VH amino acid sequence and VL amino acid sequence shown below, respectively: SEQ ID NOs (4) (i) 241 and 243.
11. The combination according to claim 1 or 10, the composition according to claim 2 or 10, the combination or composition for use according to any one of claims 3, 4 and 10, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 or the method according to any one of claims 7 to 10, wherein:
(1) The VH and the VL of the anti-HA antibody or antigen-binding fragment comprise or consist of amino acid sequences according to seq id no: (1) (i) SEQ ID NOS 43 and 44, respectively; (1) (ii) SEQ ID NOS 26 and 8, respectively; (1) (iii) SEQ ID NOs 2 and 8, respectively; (1) (iv) SEQ ID NOs 31 and 8, respectively; (1) (v) SEQ ID NOS 34 and 8, respectively; (1) (vi) SEQ ID NOS 37 and 8, respectively; (1) (vii) SEQ ID NOs 14 and 20, respectively; (1) (viii) SEQ ID NOs 39 and 20, respectively; (1) (ix) SEQ ID NOs 41 and 20, respectively; or (1) (x) is SEQ ID NO 28 and 8, respectively; and/or
(2) The VH and the VL of the anti-NA antibody or antigen-binding fragment comprise or consist of amino acid sequences according to seq id no: (2) (i) SEQ ID NOs 241 and 243, respectively; (2) (ii) SEQ ID NOs 60 and 66, respectively; (2) (iii) SEQ ID NOs 72 and 78 or 220 or 223, respectively; (2) (vi) SEQ ID NOS 72 and 226, respectively; (2) (vii) SEQ ID NOS 217 and 78, respectively; (2) (viii) SEQ ID NOS 217 and 220, respectively; (2) (ix) SEQ ID NOS 217 and 223, respectively; (2) (x) SEQ ID NOS 217 and 226, respectively; (2) (xi) SEQ ID NOs 84 and 90, respectively; (2) (xii) SEQ ID NOs 96 and 102, respectively; (2) (xiii) SEQ ID NOs 108 and 114, respectively; (2) (xiv) SEQ ID NOS 120 and 126, respectively; (2) (xv) SEQ ID NOS 132 and 138, respectively; (2) (xvi) SEQ ID NOS 132 and 232, respectively; (2) (xvii) SEQ ID NOS 132 and 235, respectively; (2) (xviii) SEQ ID NOS 132 and 238, respectively; (2) (xix) SEQ ID NOs 229 and 138, respectively; (2) (xx) SEQ ID NOs 229 and 232, respectively; (2) (xxi) SEQ ID NOs 229 and 235, respectively; (2) (xxii) SEQ ID NOs 229 and 238, respectively; (2) (xxiii) SEQ ID NOS 144 and 150, respectively; (2) (xxiv) SEQ ID NOS 156 and 162, respectively; (2) (xxv) SEQ ID NOS 168 and 174, respectively; (2) (xxvi) SEQ ID NOS 180 and 186, respectively; (2) (xxvii) SEQ ID NOS 192 and 198, respectively; (2) (xxviii) SEQ ID NOS 204 and 210, respectively; (2) (xxix) SEQ ID NOs 48 and 54, respectively; (2) (xxx) SEQ ID NOs 245 and 247, respectively; (2) (xxxi) SEQ ID NOS 249 and 251, respectively; (2) (xxxii) SEQ ID NOs 258 and 259, respectively; or (2) (xxxiii) SEQ ID NOS 261 and 263, respectively,
Wherein preferably said VH and said VL of said anti-HA antibody or antigen-binding fragment comprise or consist of amino acid sequences according to seq id no:43 and 44, respectively, said VH and said VL of said anti-NA antibody or antigen-binding fragment comprising or consisting of amino acid sequences according to SEQ ID NOs: SEQ ID NOS 241 and 243, respectively.
12. The combination according to claim 1, 10 or 11, the composition according to claim 2, 10 or 11, the combination or composition for use according to any one of claims 3, 4, 10 and 11, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6, 10 and 11 or the method according to any one of claims 7 to 11, wherein:
(1) The VH and the VL of the anti-HA antibody or antigen-binding fragment comprise or consist of amino acid sequences according to seq id no: (1) (i) SEQ ID NOs 43 and 44, respectively; (1) (ii) SEQ ID NOS 26 and 8, respectively; (1) (iii) SEQ ID NOS 28 and 8, respectively; (1) (iv) SEQ ID NOs 31 and 8, respectively; (1) (v) SEQ ID NOS 34 and 8, respectively; (1) (vi) SEQ ID NOS 37 and 8, respectively; (1) (vii) SEQ ID NOs 14 and 20, respectively; (1) (viii) SEQ ID NOs 39 and 20, respectively; (1) (ix) SEQ ID NOs 41 and 20, respectively; or (1) (x) is SEQ ID NO 2 and 8, respectively; and/or
(2) The VH and the VL of the anti-NA antibody or antigen-binding fragment comprise or consist of amino acid sequences according to seq id no: (2) (i) SEQ ID NOs 241 and 243, respectively; (2) (ii) SEQ ID NOs 72 and 226, respectively; (2) (iii) SEQ ID NOS 217 and 78, respectively; (2) (iv) SEQ ID NOS 217 and 220, respectively; (2) (v) SEQ ID NOS 132 and 138, respectively; (2) (vi) SEQ ID NOS 132 and 232, respectively; (2) (vii) SEQ ID NOs 132 and 235, respectively; (2) (viii) SEQ ID NOS 132 and 238, respectively; (2) (ix) SEQ ID NOs 229 and 138, respectively; (2) (x) SEQ ID NOs 229 and 232, respectively; (2) (xi) SEQ ID NOs 229 and 235, respectively; (2) (xii) SEQ ID NOs 229 and 238, respectively; (2) (xiii) SEQ ID NOS 217 and 223, respectively; (2) (xiv) SEQ ID NOS 217 and 226, respectively; (2) (xv) SEQ ID NOS 72 and 78 or 220 or 223, respectively; (2) (xvi) SEQ ID NOS 245 and 247, respectively; or (2) (xvii) SEQ ID NOS 249 and 251, respectively,
wherein preferably said VH and said VL of said anti-HA antibody or antigen-binding fragment comprise or consist of amino acid sequences according to seq id no:43 and 44, respectively, said VH and said VL of said anti-NA antibody or antigen-binding fragment comprising or consisting of amino acid sequences according to SEQ ID NOs: SEQ ID NOS 241 and 243, respectively.
13. The combination according to any one of claims 1 and 10 to 12, the composition according to any one of claims 2 or 10 to 12, the combination or composition for use according to any one of claims 3, 4, 10, 11 or 12, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6, 10, 11 or 12 or the method according to any one of claims 7 to 12, wherein:
(1) The anti-HA antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) comprising Complementarity Determining Regions (CDRs) H1, CDRH2 and CDRH3 and a light chain variable domain (VL) comprising CDRL1, CDRL2 and CDRL3, wherein the CDRs are optionally according to the IMGT numbering system, and wherein: (1) (i) the CDRH1 comprises or consists of an amino acid sequence as set forth in any one of: 274, 3, 32 and 15, or a functional variant thereof, or consisting of one, two or three acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (1) (ii) the CDRH2 comprises or consists of an amino acid sequence as set forth in any one of: 275, 4, 29, 35, 16 and 42, or a functional variant thereof or consisting of one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (1) (iii) the CDRH3 comprises or consists of an amino acid sequence as set forth in any one of: 276, 5 and 17, or functional variants thereof, or consisting of functional variants thereof, comprising one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (1) (iv) the CDRL1 comprises or consists of an amino acid sequence as set forth in any one of: 277, 9 and 21, or a functional variant thereof comprising or consisting of one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (1) (v) the CDRL2 optionally comprises or consists of an amino acid sequence as set forth in any one of: 278, 10 and 22, or a functional variant thereof, or consisting of, comprising one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (1) (vi) the CDRL3 comprises or consists of an amino acid sequence as set forth in any one of: 279, 11 and 23, or functional variants thereof, or consisting of functional variants thereof, comprising substitutions with one, two or three amino acids, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids;
And/or
(2) The anti-NA antibody or antigen-binding fragment comprises a VH comprising CDRH1, CDRH2 and CDRH3 and a VL comprising CDRL1, CDRL2 and CDRL3, wherein: (2) (i) optionally the CDRH1 comprises or consists of an amino acid sequence as set forth in any one of: 193, 49, 61, 73, 85, 97, 109, 121, 133, 145, 157, 169, 181, 205 and 264, or a functional variant thereof comprising or consisting of one, two or three acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; (2) (ii) optionally the CDRH2 comprises or consists of an amino acid sequence as set forth in any one of: 194, 50, 62, 74, 86, 98, 110, 122, 134, 146, 158, 170, 182, 206 and 265, or functional variants thereof, or consisting of functional variants thereof, comprising one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; (2) (iii) the CDRH3 comprises or consists of an amino acid sequence as set forth in any one of: 195, 51, 63, 75, 218, 87, 99, 111, 123, 135, 230, 147, 159, 171, 183, 207 and 266, or a functional variant thereof comprising or consisting of one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; (2) (iv) optionally the CDRL1 comprises or consists of an amino acid sequence as set forth in any one of the following: 199, 55, 67, 79, 91, 103, 115, 127, 139, 151, 163, 175, 187, 211 and 267, or a functional variant thereof comprising or consisting of one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; (2) (v) optionally the CDRL2 comprises or consists of an amino acid sequence as set forth in any one of: 200, 56, 68, 80, 92, 104, 116, 128, 140, 152, 164, 176, 188, 212 and 268, or a functional variant thereof comprising or consisting of one, two or three amino acid substitutions, one or more of which are optionally conservative substitutions and/or substitutions of germline encoded amino acids; and/or (2) (vi) optionally the CDRL3 comprises or consists of an amino acid sequence as shown in any one of: 201, 57, 69, 81, 221, 224, 227, 93, 105, 117, 129, 141, 233, 239, 153, 165, 177, 189, 236, 213 and 269, or a functional variant thereof comprising or consisting of a substitution with one, two or three amino acids, one or more of said substitutions optionally being a conservative substitution and/or a substitution of a germline encoded amino acid.
14. The combination according to any one of claims 1 and 10 to 13, the composition according to any one of claims 2 or 10 to 13, the combination or composition for use according to any one of claims 3, 4, 10, 11, 12 or 13, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6, 10, 11, 12 or 13 or the method according to any one of claims 7 to 13, wherein:
(1) The anti-HA antibody or antigen binding fragment comprises the following CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences: (1) SEQ ID NOS 274-279, respectively; (1) (ii) SEQ ID NOs 3, 29, 5 and 9-11, respectively; (1) (iii) SEQ ID NOs 32, 4, 5 and 9-11, respectively; (1) (iv) SEQ ID NOs 3, 35, 5 and 9-11, respectively; (1) (v) SEQ ID NOs 32, 35, 5 and 9-11, respectively; (1) (vi) SEQ ID NOS 15-17 and 21-23, respectively; (1) (vii) SEQ ID NOs 15, 42, 17 and 21-23, respectively; or (1) (viii) SEQ ID NOs 3-5 and 9-11, respectively;
and/or
(2) The anti-NA antibodies or antigen binding fragments include the following CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences: (2) (i) SEQ ID NOS 193-195 and 199-201, respectively; (2) (ii) SEQ ID NOS: 61-63 and 67-69, respectively; (2) (iii) SEQ ID NOS: 73-75 and 79-81, respectively; (2) (iv) SEQ ID NOS 73, 74, 218 and 79-81, respectively; (2) (v) SEQ ID NOS 73-75, 79, 80 and 221, respectively; (2) (vi) SEQ ID NOS 73-75, 79, 80 and 224, respectively; (2) (vii) SEQ ID NOS 73-75, 79, 80 and 227, respectively; (2) (viii) SEQ ID NOs 73, 74, 218, 79, 80 and 221, respectively; (2) (ix) SEQ ID NOs 73, 74, 218, 79, 80 and 224, respectively; (2) (x) SEQ ID NOs 73, 74, 218, 79, 80 and 227, respectively; (2) (xi) SEQ ID NOS 85-87 and 91-93, respectively; (2) (xii) SEQ ID NOS 97-99 and 103-105, respectively; (2) (xiii) SEQ ID NOS 109-111 and 115-117, respectively; (2) (xiv) SEQ ID NOS 121-123 and 127-129, respectively; (2) (xv) SEQ ID NOS 133-135 and 139-141, respectively; (2) (xvi) SEQ ID NOS 133, 134, 230 and 139-141, respectively; (2) (xvii) SEQ ID NOS 133-135, 139, 141 and 233, respectively; (2) (xviii) SEQ ID NOS 133-135, 139, 141 and 236, respectively; (2) (xix) SEQ ID NOS 133-135, 139, 141 and 239, respectively; (2) (xx) SEQ ID NOS: 133, 134, 184, 139, 141 and 233, respectively; (2) (xxi) SEQ ID NOs 133, 134, 230, 139, 141 and 236, respectively; (2) (xxii) SEQ ID NOs 133, 134, 230, 139, 141 and 239, respectively; (2) (xxiii) SEQ ID NOS 145-147 and 151-153, respectively; (2) (xxiv) SEQ ID NOS 157-159 and 163-165, respectively; (2) (xxv) SEQ ID NOS 169-171 and 175-177, respectively; (2) (xxvi) SEQ ID NOS 181-183 and 187-189, respectively; (2) (xxvii) SEQ ID NOS 49-51 and 55-57, respectively; (2) (xxviii) SEQ ID NOS 205-207 and 211-213, respectively; or (2) (xxix) SEQ ID NOS 264-266 and 267-269, respectively.
15. The combination according to claim 13 or 14, the composition according to claim 13 or 14, the combination or composition for use according to claim 13 or 14, the antibody or antigen binding fragment or polynucleotide for use according to claim 13 or 14 or the method according to claim 13 or 14, wherein:
(1) The anti-HA antibody or antigen binding fragment comprises the following CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences: (1) SEQ ID NOS 274-279, respectively; (1) (ii) SEQ ID NOs 3, 29, 5 and 9-11, respectively; (1) (iii) SEQ ID NOs 32, 4, 5 and 9-11, respectively; (1) (iv) SEQ ID NOs 3, 35, 5 and 9-11, respectively; (1) (v) SEQ ID NOs 32, 35, 5 and 9-11, respectively; (1) (vi) SEQ ID NOS 15-17 and 21-23, respectively; (1) (vii) SEQ ID NOs 15, 42, 17 and 21-23, respectively; or (1) (viii) SEQ ID NOs 3-5 and 9-11, respectively;
and/or
(2) The anti-NA antibodies or antigen binding fragments include the following CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences: (2) (i) SEQ ID NOS 193-195 and 199-201, respectively; (2) (ii) SEQ ID NOS 73, 74, 218 and 79-81, respectively; (2) (iii) SEQ ID NOS 73-75, 79, 80 and 221, respectively; (2) (iv) SEQ ID NOS 73-75, 79, 80 and 224, respectively; (2) (v) SEQ ID NOS 73-75, 79, 80 and 227, respectively; (2) (vi) SEQ ID NOs 73, 74, 218, 79, 80 and 221, respectively; (2) (vii) SEQ ID NOs 73, 74, 218, 79, 80 and 224, respectively; (2) (viii) SEQ ID NOs 73, 74, 218, 79, 80 and 227, respectively; (2) (ix) SEQ ID NOS 133-135 and 139-141, respectively; (2) (x) SEQ ID NOs 133, 134, 230 and 139-141, respectively; (2) (xi) SEQ ID NOS 133-135, 139, 141 and 233, respectively; (2) (xii) SEQ ID NOS 133-135, 139, 141 and 236, respectively; (2) (xiii) SEQ ID NOS 133-135, 139, 141 and 239, respectively; (2) (xiv) SEQ ID NOS 133, 134, 184, 139, 141 and 233, respectively; (2) (xv) SEQ ID NOS 133, 134, 184, 139, 141 and 236, respectively; (2) (xvi) SEQ ID NOs 133, 134, 184, 139, 141 and 239, respectively; (2) (xvii) SEQ ID NOS 264-266 and 267-296, respectively; or (2) (xviii) SEQ ID NOS: 73-75 and 79-81, respectively.
16. The combination according to any one of claims 1 and 10 to 15, the composition according to any one of claims 2 and 10 to 15, the combination or composition for use according to any one of claims 3, 4 and 10 to 15, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 15 or the method according to any one of claims 7 to 15, wherein: (i) group 1 IAV NA comprises N1, N4, N5 and/or N8; and/or (ii) the group 2 IAV NA comprises N2, N3, N6, N7 and/or N9.
17. The combination according to claim 16, the composition according to claim 16, the combination or composition for use according to claim 16, the antibody or antigen-binding fragment or polynucleotide for use according to claim 16 or the method according to claim 16, wherein:
(i) The N1 is N1 from any one or more of the following: a/california/07/2009, a/california/07/2009I 23R/H275Y, A/pig/Jiangsu/J004/2018, a/stong holly mole/18/2007, a/brisban/02/2018, a/michigan/45/2015, a/misibbean/3/2001, a/netherlands/603/2009, a/netherlands/602/2009, a/vietnam/1203/2004, a/G4/SW/shandong/1207/2016, a/G4/SW/henna/SN 13/2018, and a/new jersey/8/1976;
(ii) The N4 is from A/green duck/Netherlands/30/2011;
(iii) The N5 is from a/waterfowl/korea/CN 5/2009;
(iv) The N8 is from A/plaque seal/new hampshire/179629/2011;
(v) The N2 is N2 from any one or more of the following: A/Washington/01/2007, A/China hong Kong/68, A/south Australia/34/2019, A/Switzerland/8060/2017, A/Singapore/INFIMH-16-0019/2016, A/Switzerland/9715293/2013, A/Ninggeler/134/17/57, A/Florida/4/2006, A/Netherlands/823/1992, A/Norway/466/2014, A/Switzerland/8060/2017, A/Texas/50/2012, A/Victoria/361/2011; A/China hong Kong/2671/2019, A/SW/Mexico/SG 1444/2011, A/tank Sania/205/2010, A/Aizhi/2/1968, A/Biterhufen/21793/1972, A/Netherlands/233/1982, A/Shanghai/11/1987, A/Nanchang/933/1995, A/Fujing/45/2004 and A/Brisban/10/2007;
(vi) The N3 is from A/Canadian/rv 504/2004;
(v) The N6 is from A/pig/Annua/01911/1/99;
(vi) Said N7 is from A/Netherlands/078/03; and/or
(vii) The N9 is N9 from any one or more of the following: a/Anhui/2013 and a/hong Kong/56/2015.
18. The combination according to any one of claims 1 and 10 to 17, the composition according to any one of claims 2 and 10 to 17, the combination or composition for use according to any one of claims 3, 4 and 10 to 17, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 17 or the method according to any one of claims 7 to 17, wherein IBV NA is NA from any one or more of the following: B/Li/10/1940 (ancestor); b/brisbane/60/2008 (victoria); B/Malaysia/2506/2004 (Victoria); B/Malaysia/3120318925/2013 (mountain county); b/wisconsin/1/2010 (mountain county); B/Sorby county/166/1998 (mountain county); B/Brisbane/33/2008; b/state of cororado/06/2017; B/Hubei-Wujiang/158/2009; b/massachusetts/02/2012; B/Netherlands/234/2011; B/Perss/211/2001; B/Texas/06/2011 (mountain county); B/Perss/211/2011; b/hong Kong/05/1972; b/pray island/3073/2013; B/Harbin/7/1994 (Victoria); and B/Washington/02/2019 (Victoria).
19. The combination according to any one of claims 1 and 10 to 18, the composition according to any one of claims 2 and 10 to 18, the combination or composition for use according to any one of claims 3, 4 and 10 to 18, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 18 or the method according to any one of claims 7 to 18, wherein the NA is N1, N2 and/or N9.
20. The combination according to any one of claims 1 and 10 to 19, the composition according to any one of claims 2 and 10 to 19, the combination or composition for use according to any one of claims 3, 4 and 10 to 19, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 19 or the method according to any one of claims 7 to 19, wherein the anti-NA antibody or antigen binding fragment is capable of binding to:
(1) (i) an NA epitope comprising any one or more of the following amino acids (N1 NA numbering): r368, R293, E228, E344, S247, D198, D151, R118; and/or (ii) an NA epitope comprising any one or more of the following amino acids (N2 NA numbering): r371, R292, E227, E344, S247, D198, D151, R118;
And/or
(2) (i) an NA epitope comprising amino acids R368, R293, E228, D151 and R118 (N1 NA numbering); and/or (ii) an NA epitope comprising amino acids R371, R292, E227, D151 and R118 (N2 NA numbering);
and/or
(3) An epitope comprised in or comprising an NA active site, wherein optionally the NA active epitope comprises the following amino acids (N2 numbering): r118, D151, R152, R224, E276, R292, R371, Y406, E119, R156, W178, S179, D/N198, I222, E227, H274, E277, D293, E425;
and/or
(4) An IBV NA epitope comprising: (i) any one or more of the following amino acids:
r116, D149, E226, R292, and R374; or (ii) amino acids R116, D149, E226, R292, and R374.
21. The combination according to claim 20, the composition according to claim 20, the combination or composition for use according to claim 20, the antibody or antigen-binding fragment or polynucleotide for use according to claim 20 or the method according to claim 20, wherein the anti-NA antibody or antigen-binding fragment is capable of binding to:
(1) Further comprising epitopes of any one or more of the following NA amino acids (N2 numbering): e344, E227, S247, and D198; and/or
(2) NA comprising an S245N amino acid mutation and/or an E221D amino acid mutation.
22. The combination according to any one of claims 1 and 10 to 21, the composition according to any one of claims 2 and 10 to 21, the combination or composition for use according to any one of claims 3, 4 and 10 to 21, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 21 or the method according to any one of claims 7 to 21, wherein the anti-NA antibody or antigen binding fragment is capable of binding to NA comprising an S245N amino acid mutation and/or an E221D amino acid mutation.
23. The combination according to any one of claims 1 and 10 to 22, the composition according to any one of claims 2 and 10 to 22, the combination or composition for use according to any one of claims 3, 4 and 10 to 22, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 22 or the method according to any one of claims 7 to 22, wherein:
(i) The group 1 IAV NA comprises H1N1 and/or H5N1;
(ii) The group 2 IAV NA comprises H3N2 and/or H7N9; and/or
(iii) The IBV NA includes one or more of the following: B/Li/10/1940 (ancestor);
b/hong Kong/05/1972; B/Taiwan/2/1962 (ancestor); b/brisbane/33/2008 (victoria); b/brisbane/60/2008 (victoria); B/Malaysia/2506/2004 (Victoria); B/New York/1056/2003 (Victoria); B/Florida/4/2006 (mountain county); B/Jiangsu/10/2003 (mountain county); B/Texas/06/2011 (mountain county); B/Perss/211/2011; B/Harbin/7/1994 (Victoria); b/state of cororado/06/2017 (victoria); b/washington/02/2019 (victoria); B/Perss/211/2001 (mountain county); B/Hubei-wujia sentry/158/2009 (mountain county); b/wisconsin/01/2010 (mountain county); b/massachusetts/02/2012 (mountain county); and B/pray island/3073/2013 (mountain county).
24. The combination according to any one of claims 1 and 10 to 23, the composition according to any one of claims 2 and 10 to 23, the combination or composition for use according to any one of claims 3, 4 and 10 to 23, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 23 or the method according to any one of claims 7 to 23, wherein the anti-HA antibody or antigen binding fragment is capable of binding to any one or more of the following IAV subtypes: h1, H2, H3, H4, H5, H8, H9, H10, H11, H12, H13, H14, H15, H17 and H18.
25. The combination according to any one of claims 1 and 10 to 24, the composition according to any one of claims 2 and 10 to 24, the combination or composition for use according to any one of claims 3, 4 and 10 to 24, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 24 or the method according to any one of claims 7 to 24, wherein the anti-HA antibody or antigen binding fragment is capable of neutralizing infection by:
(i) An H1N1 IAV, wherein optionally the H1N1 IAV comprises any one or more of: a/california/07/2009, a/PR/8/34 and a/solomon islands/3/06; and
(ii) An H3N2 IAV, wherein optionally the H3N2 IAV comprises any one or more of: A/Aizhi/2/68, A/Brisban/10/07 and A/China hong Kong/68;
(i) A group 1 IAV, wherein optionally said group 1 IAV comprises or is an H5 IAV, wherein further optionally said H5 IAV comprises or is an H5/VN/11/94pp; and
(ii) A group 2 IAV, wherein optionally said group 2 IAV comprises or is an H7 IAV, wherein further optionally said H7 IAV comprises or is an H7/IT/99pp,
wherein neutralization of the optional infection is determined using the IAV pseudotyped virus.
26. The combination according to any one of claims 1 and 10 to 25, the composition according to any one of claims 2 and 10 to 25, the combination or composition for use according to any one of claims 3, 4 and 10 to 25, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 25 or the method according to any one of claims 7 to 25, wherein the anti-HA antibody or antigen binding fragment is capable of binding to one or more of the following (i) - (iv):
(i) H1 HA, the H1 HA optionally comprising any one or more of: a/england/195/2009; A/Brisban/59/2007; A/Solomon islands/3/2006; A/New karilidoria/20/99; A/Texas state/36/1991; A/Taiwan/01/1986; A/New Jersey state/8/1976; A/Albani/12/1951; A/Mono Sturg/1/1947; A/New York/1/1918; A/Paris/8/34; a/california/07/2009;
(ii) H2 HA, said H2 HA optionally comprising a/japan/305/1957;
(iii) H5 HA, optionally comprising a/vietnam/1194/2004; and
(iv) H9 HA, optionally including A/China hong Kong/1073/99.
27. The combination according to any one of claims 1 and 10 to 26, the composition according to any one of claims 2 and 10 to 26, the combination or composition for use according to any one of claims 3, 4 and 10 to 26, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 26 or the method according to any one of claims 7 to 26, wherein the anti-HA antibody or antigen binding fragment, the anti-NA antibody or antigen binding fragment or both are capable of activating human fcγriiia (optionally the F158 allele).
28. The combination according to claim 27, the composition according to claim 27, the combination or composition for use according to claim 27, the antibody or antigen-binding fragment or polynucleotide for use according to claim 27 or the method according to claim 27, wherein activation is determined using host cells (optionally Jurkat cells) comprising after incubating (e.g. 23 hours) the antibody or antigen-binding fragment with target cells (e.g. a549 cells) infected with IAV and/or IBV: (i) the human fcyriiia (optionally the F158 allele); and (ii) an NFAT expression control sequence operably linked to a sequence encoding a reporter gene, such as a luciferase reporter gene.
29. The combination according to claim 27 or 28, the composition according to claim 27 or 28, the combination or composition for use according to claim 27 or 28, the antibody or antigen binding fragment or polynucleotide for use according to claim 27 or 28 or the method according to claim 27 or 28, wherein activation is determined after incubating the antibody or antigen binding fragment with target cells infected with H1N1 and/or H3N2 IAV (optionally for about 23 hours), wherein optionally the H1N1 IAV is a/PR8/34 and further optionally the multiplicity of infection (MOI) is 6, and/or wherein the H3N2 IAV is a/aizhi/68 and further optionally the MOI is 18.
30. The combination according to any one of claims 1 and 10 to 29, the composition according to any one of claims 2 and 10 to 29, the combination or composition for use according to any one of claims 3, 4 and 10 to 29, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 29 or the method according to any one of claims 7 to 29, wherein the IAV and/or the IBV are antiviral resistant, wherein optionally the antiviral is oseltamivir.
31. The combination according to any one of claims 1 and 10 to 30, the composition according to any one of claims 2 and 10 to 30, the combination or composition for use according to any one of claims 3, 4 and 10 to 30, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 30 or the method according to any one of claims 7, wherein the IAV comprises N1 NA comprising the following amino acid mutations: H275Y; e119d+h275Y; s247n+h275Y; I222V; and/or N294S, wherein optionally the IAV comprises CA09 or a/aiknow.
32. The combination according to any one of claims 1 and 10 to 31, the composition according to any one of claims 2 and 10 to 31, the combination or composition for use according to any one of claims 3, 4 and 10 to 31, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 32 or the method according to any one of claims 7 to 31, wherein the IAV comprises N2 NA comprising the following amino acid mutations: E119V, Q136K and/or R292K.
33. The combination according to any one of claims 1 and 10 to 32, the composition according to any one of claims 2 and 10 to 32, the combination or composition for use according to any one of claims 3, 4 and 10 to 32, the antibody or antigen-binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 32 or the method according to any one of claims 7 to 32, wherein the anti-HA antibody or antigen-binding fragment, the anti-NA antibody or antigen-binding fragment or both are capable of preventing weight loss in a subject infected with the IAV and/or the IBV, optionally for (i) up to 15 days, or (ii) more than 15 days after administration of an effective amount of the antibody or antigen-binding fragment.
34. The combination according to any one of claims 1 and 10 to 33, the composition according to any one of claims 2 and 10 to 33, the combination or composition for use according to any one of claims 3, 4 and 10 to 33, the antibody or antigen-binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 33 or the method according to any one of claims 7 to 33, wherein the anti-HA antibody or antigen-binding fragment, the anti-NA antibody or antigen-binding fragment or both are capable of preventing weight loss of a subject suffering from an IAV infection and/or an IBV infection by more than 25%, 20%, 15%, 10% or 5% as determined by reference to the weight of the subject just prior to the IAV infection and/or the IBV infection.
35. The combination according to any one of claims 1 and 10 to 34, the composition according to any one of claims 2 and 10 to 34, the combination or composition for use according to any one of claims 3, 4 and 10 to 34, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 34 or the method according to any one of claims 7 to 34, wherein the anti-HA antibody or antigen binding fragment, the anti-NA antibody or antigen binding fragment or both are capable of extending the survival of a subject suffering from IAV infection and/or IBV infection.
36. The combination according to any one of claims 1 and 10 to 35, the composition according to any one of claims 2 and 10 to 35, the combination or composition for use according to any one of claims 3, 4 and 10 to 35, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 35 or the method according to any one of claims 7 to 35, wherein the anti-HA antibody or antigen binding fragment, the anti-NA antibody or antigen binding fragment or both are IgG, igA, igM, igE or IgD isotypes.
37. The combination according to any one of claims 1 and 10 to 36, the composition according to any one of claims 2 and 10 to 36, the combination or composition for use according to any one of claims 3, 4 and 10 to 36, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 36 or the method according to any one of claims 7 to 36, wherein the anti-HA antibody or antigen binding fragment, the anti-NA antibody or antigen binding fragment or both are IgG isotypes selected from: igG1, igG2, igG3 and IgG4.
38. The combination according to any one of claims 1 and 10 to 37, the composition according to any one of claims 2 and 10 to 37, the combination or composition for use according to any one of claims 3, 4 and 10 to 37, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 37 or the method according to any one of claims 7 to 37, wherein the anti-HA antibody or antigen binding fragment, the anti-NA antibody or antigen binding fragment or both comprise a human antibody, a monoclonal antibody, a purified antibody, a single chain antibody, fab ', F (ab') 2 or Fv.
39. The combination according to any one of claims 1 and 10 to 38, the composition according to any one of claims 2 and 10 to 38, the combination or composition for use according to any one of claims 3, 4 and 10 to 38, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 38 or the method according to any one of claims 7 to 38, wherein the anti-HA antibody or antigen binding fragment, the anti-NA antibody or antigen binding fragment or both are multispecific antibodies or antigen binding fragments, wherein optionally the multispecific antibodies or antigen binding fragments comprise bispecific antibodies or antigen binding fragments.
40. The combination according to any one of claims 1 and 10 to 39, the composition according to any one of claims 2 and 10 to 39, the combination or composition for use according to any one of claims 3, 4 and 10 to 39, the antibody or antigen-binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 39 or the method according to any one of claims 7 to 39, wherein the anti-HA antibody or antigen-binding fragment, the anti-NA antibody or antigen-binding fragment or both comprise an Fc polypeptide or fragment thereof.
41. The combination according to claim 40, the composition according to claim 40, the combination or composition for use according to claim 40, the antibody or antigen binding fragment or polynucleotide for use according to claim 40 or the method according to claim 40, wherein the Fc polypeptide or fragment thereof comprises:
(i) Mutations that enhance binding affinity (e.g., measured using Surface Plasmon Resonance (SPR) (e.g., biacore, e.g., T200 instrument, using manufacturer's protocol)) to human FcRn as compared to a reference Fc polypeptide that does not include the mutations; and/or
(ii) Mutations that enhance binding affinity (e.g., measured using Surface Plasmon Resonance (SPR) (e.g., biacore, e.g., T200 instrument, using manufacturer's protocol)) to human fcγr compared to a reference Fc polypeptide that does not include the mutations.
42. The combination according to claim 41, the composition according to claim 41, the combination or composition for use according to claim 41, the antibody or antigen-binding fragment or polynucleotide for use according to claim 41 or the method according to claim 41, wherein the mutation that enhances binding affinity to human FcRn comprises: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I; Q311I; D376V; T307A; E380A; or any combination thereof.
43. The combination according to claim 41 or 42, the composition according to claim 41 or 42, the combination or composition for use according to claim 41 or 42, the antibody or antigen binding fragment or polynucleotide for use according to claim 41 or 42 or the method according to claim 41 or 42, wherein the mutation that enhances binding affinity to human FcRn comprises:
(i)M428L/N434S；
(ii)M252Y/S254T/T256E；
(iii)T250Q/M428L；
(iv)P257I/Q311I；
(v)P257I/N434H；
(vi)D376V/N434H；
(vii) T307A/E380A/N434A; or (b)
(viii) Any combination of (i) - (vii).
44. The combination according to any one of claims 41 to 43, the composition according to any one of claims 41 to 43, the combination or composition for use according to any one of claims 41 to 43, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 41 to 43 or the method according to any one of claims 41 to 43, wherein the mutation that enhances binding affinity to human FcRn comprises M428L/N434S.
45. The combination according to any one of claims 41 to 44, the composition according to any one of claims 41 to 44, the combination or composition for use according to any one of claims 41 to 44, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 41 to 44 or the method according to any one of claims 41 to 44, wherein the mutation that enhances binding to fcγr comprises: S239D; I332E; a330L; G236A; or any combination thereof.
46. The combination according to any one of claims 41 to 45, the composition according to any one of claims 41 to 45, the combination or composition for use according to any one of claims 41 to 45, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 41 to 45 or the method according to any one of claims 41 to 45, wherein the mutation that enhances binding to fcγr comprises:
(i)S239D/I332E；
(ii)S239D/A330L/I332E；
(iii) G236A/S239D/I332E; or (b)
(iv) G236A/A330L/I332E, wherein the Fc polypeptide or fragment thereof optionally comprises Ser at position 239.
47. The combination according to any one of claims 1 and 10 to 46, the composition according to any one of claims 2 and 10 to 46, the combination or composition for use according to any one of claims 3, 4 and 10 to 46, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 46 or the method according to any one of claims 7 to 46, wherein the anti-HA antibody or antigen binding fragment, the anti-NA antibody or antigen binding fragment or both comprise a mutation that alters glycosylation, wherein the mutation that alters glycosylation comprises N297A, N Q or N297G, and/or deglycosylated.
48. The combination according to any one of claims 1 and 10 to 47, the composition according to any one of claims 2 and 10 to 47, the combination or composition for use according to any one of claims 3, 4 and 10 to 47, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 47 or the method according to any one of claims 7 to 47, wherein the treatment and/or the prophylaxis comprises post-exposure control.
49. The combination according to any one of claims 1 and 10 to 48, the composition according to any one of claims 2 and 10 to 48, the combination or composition for use according to any one of claims 3, 4 and 10 to 48, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 48 or the method according to any one of claims 7 to 48, wherein the subject has received, is receiving or will receive an antiviral agent, wherein optionally the antiviral agent comprises a neuraminidase inhibitor, an influenza polymerase inhibitor or both.
50. The combination according to any one of claims 1 and 10 to 49, the composition according to any one of claims 2 and 10 to 49, the combination or composition for use according to any one of claims 3, 4 and 10 to 49, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 49 or the method according to any one of claims 7 to 49, wherein the antibody or antigen binding fragment comprises:
(i) CH1-CH3, wherein the CH1-CH3 comprises or consists of an amino acid sequence shown as SEQ ID NO. 252;
(ii) CH1-CH3, wherein the CH1-CH3 comprises or consists of an amino acid sequence shown as SEQ ID NO 253;
(iii) CL comprising or consisting of the amino acid sequence shown as SEQ ID NO. 254; or (b)
(iv) Any combination of (i) - (iii).
51. The combination according to any one of claims 1 and 10 to 50, the composition according to any one of claims 2 and 10 to 50, the combination or composition for use according to any one of claims 3, 4 and 10 to 50, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 50 or the method according to any one of claims 7 to 50,
wherein the anti-NA antibody or antigen-binding fragment comprises:
(1) A heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 255; and
(2) A light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 257; and/or
Wherein the anti-HA antibody or antigen-binding fragment comprises:
(1) A heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO 270 or 272; and
(2) A light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO 271 or 273.
52. The combination according to any one of claims 1 and 10 to 50, the composition according to any one of claims 2 and 10 to 51, the combination or composition for use according to any one of claims 3, 4 and 10 to 51, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 51 or the method according to any one of claims 7 to 51,
wherein the anti-NA antibody or antigen-binding fragment comprises:
(1) A heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 256; and
(2) A light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 257; and/or
Wherein the anti-HA antibody or antigen-binding fragment comprises:
(1) A heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO 270 or 272; and
(2) A light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO 271 or 273.
53. The combination according to any one of claims 1 and 10 to 51, the composition according to any one of claims 2 and 10 to 51, the combination or composition for use according to any one of claims 3, 4 and 10 to 51, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 51 or the method according to any one of claims 7 to 51,
Wherein the anti-NA antibody or antigen-binding fragment comprises:
(1) Two heavy chains, each heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 255; and
(2) Two light chains, each comprising or consisting of the amino acid sequence shown in SEQ ID NO 257; and/or
Wherein the anti-HA antibody or antigen-binding fragment comprises:
(1) Two heavy chains, each heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO 270 or 272; and
(2) Two light chains, each comprising or consisting of the amino acid sequence shown in SEQ ID NO 271 or 273.
54. The combination according to any one of claims 1 and 10 to 50, the composition according to any one of claims 2 and 10 and 52, the combination or composition for use according to any one of claims 3, 4 and 10 to 50 and 52, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 50 and 52 or the method according to any one of claims 7 to 50 and 52,
wherein the anti-NA antibody or antigen-binding fragment comprises:
(1) Two heavy chains, each heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 256; and
(2) Two light chains, each comprising or consisting of the amino acid sequence shown in SEQ ID NO 257; and/or
Wherein the anti-HA antibody or antigen-binding fragment comprises:
(1) Two heavy chains, each heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO 270 or 272; and
(2) Two light chains, each comprising or consisting of the amino acid sequence shown in SEQ ID NO 271 or 273.
55. The combination of any one of claims 50 to 54, the composition of any one of claims 50 to 54, the combination or composition for use of any one of claims 50 to 54, the antibody or antigen-binding fragment or polynucleotide for use of any one of claims 50 to 54, or the method of any one of claims 50 to 54, wherein the antiviral agent comprises oseltamivir, zanamivir Weila nimevir, peramivir, balo Sha Wei, or any combination thereof.
56. The combination according to any one of claims 1 and 10 to 55, the composition according to any one of claims 2 and 10 to 55, the combination or composition for use according to any one of claims 3, 4 and 10 to 55, the antibody or antigen binding fragment or polynucleotide for use according to any one of claims 5, 6 and 10 to 55 or the method according to any one of claims 7 to 55, wherein:
(i) The IAV comprises a group 1 IAV, a group 2 IAV, or both, wherein optionally the group 1 IAV NA comprises N1, N4, N5, and/or N8; and/or the group 2 IAV NA comprises N2, N3, N6, N7 and/or N9, wherein further optionally the N1 is from a/california/07/2009, from a/california/07/2009I 23R/H275Y, from a/pig/Jiangsu/J004/2018, from a/ston mole/18/2007, from a/brisban/02/2018, from a/michigan state/45/2015, from a/michigan state/3/2001, from a/netherlands/603/2009, from a/netherlands/602/2009, from a/vietnam/1203/2004, from a/G4/SW/shandong/1207/2016, from a/G4/SW/henna/SN 13/2018 and/or from a/new jersey state/8/1976; the N4 is from A/green duck/Netherlands/30/2011; the N5 is from a/waterfowl/korea/CN 5/2009; the N8 is from A/plaque seal/new hampshire/179629/2011; said N2 is from a/washington/01/2007, from a/chinese hong kong/68, from a/chinese hong kong/2671/2019, from a/south australia/34/2019, from a/swiss/8060/2017, from a/singapore/INFIMH-16-0019/2016, from a/switzerland/9715293/2013, from a/lanugreek/134/17/57, from a/florida/4/2006, from a/netherlands/823/1992, from a/norway/466/2014, from a/texas/50/2012, from a/victoria/361/2011, from a/SW/mexico/SG 1444/2011, from a/known/2/1968, from a/bitterman/21793/1972, from a/netherlands/233/1982, from a/11/1987, a/vannean/1983, a/45/florida/1995, from a/9310/1995, from a/45/samsuntan/2007; the N3 is from A/Canadian/rv 504/2004; the N6 is from A/pig/Annua/01911/1/99; said N7 is from A/Netherlands/078/03; and/or the N9 is from a/security badge/2013, from a/chinese hong kong/56/2015; and/or
(ii) The IBV NA is from: B/Li/10/1940 (ancestor); b/brisbane/60/2008 (victoria); B/Malaysia/2506/2004 (Victoria); B/Malaysia/3120318925/2013 (mountain county); b/wisconsin/1/2010 (mountain county); B/Sorby county/166/1998 (mountain county); b/brisbane/33/2008 (victoria); b/state of cororado/06/2017 (victoria); B/Hubei-wujiang/158/2009 (mountain county); b/massachusetts/02/2012 (mountain county); B/Netherlands/234/2011; B/Perss/211/2001 (mountain county); b/praji island/3073/2013 (mountain county); B/Texas/06/2011 (mountain county); b/hong Kong/05/1972; B/Harbin/7/1994 (Victoria); b/washington/02/2019 (victoria); B/Perss/211/2011; or (b)
Any combination thereof.
57. A multispecific antibody, or antigen-binding fragment thereof, comprising:
(i) An antigen binding domain capable of binding to Influenza A Virus (IAV) Hemagglutinin (HA); and
(ii) An antigen binding domain capable of binding to Neuraminidase (NA) from: 2 (i) IAV, wherein the IAV comprises a group 1 IAV, a group 2 IAV, or both; and 2 (ii) Influenza B Virus (IBV).
58. The multispecific antibody or antigen-binding fragment of claim 57, which comprises a dual variable domain immunoglobulin (DVD-Ig) form.
59. The multispecific antibody or antigen-binding fragment of claim 57 or 58, which comprises an in-bent tube insertion Ig (IEI-Ig) format.
60. The multispecific antibody of any one of claims 57 to 59, wherein:
(1) The anti-HA antigen binding domain comprises the following CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences: (1) SEQ ID NOS 274-279, respectively; (1) (ii) SEQ ID NOs 3, 29, 5 and 9-11, respectively; (1) (iii) SEQ ID NOs 32, 4, 5 and 9-11, respectively; (1) (iv) SEQ ID NOs 3, 35, 5 and 9-11, respectively; (1) (v) SEQ ID NOs 32, 35, 5 and 9-11, respectively; (1) (vi) SEQ ID NOS 15-17 and 21-23, respectively; (1) (vii) SEQ ID NOs 15, 42, 17 and 21-23, respectively; (1) (vii) either SEQ ID NOS 3-5 and 9-11, respectively, or as shown in the variable domain amino acid sequences of SEQ ID NOS 43 and 44, respectively;
and/or
(2) The anti-NA antigen binding domain comprises the following CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences: (2) (i) SEQ ID NOS 193-195 and 199-201, respectively; (2) (ii) SEQ ID NOS: 61-63 and 67-69, respectively; (2) (iii) SEQ ID NOS: 73-75 and 79-81, respectively; (2) (iv) SEQ ID NOS 73, 74, 218 and 79-81, respectively; (2) (v) SEQ ID NOS 73-75, 79, 80 and 221, respectively; (2) (vi) SEQ ID NOS 73-75, 79, 80 and 224, respectively; (2) (vii) SEQ ID NOS 73-75, 79, 80 and 227, respectively; (2) (viii) SEQ ID NOs 73, 74, 218, 79, 80 and 221, respectively; (2) (ix) SEQ ID NOs 73, 74, 218, 79, 80 and 224, respectively; (2) (x) SEQ ID NOs 73, 74, 218, 79, 80 and 227, respectively; (2) (xi) SEQ ID NOS 85-87 and 91-93, respectively; (2) (xii) SEQ ID NOS 97-99 and 103-105, respectively; (2) (xiii) SEQ ID NOS 109-111 and 115-117, respectively; (2) (xiv) SEQ ID NOS 121-123 and 127-129, respectively; (2) (xv) SEQ ID NOS 133-135 and 139-141, respectively; (2) (xvi) SEQ ID NOS 133, 134, 230 and 139-141, respectively; (2) (xvii) SEQ ID NOS 133-135, 139, 141 and 233, respectively; (2) (xviii) SEQ ID NOS 133-135, 139, 141 and 236, respectively; (2) (xix) SEQ ID NOS 133-135, 139, 141 and 239, respectively; (2) (xx) SEQ ID NOS: 133, 134, 184, 139, 141 and 233, respectively;
(2) (xxi) SEQ ID NOs 133, 134, 184, 139, 141 and 236, respectively; (2) (xxii) SEQ ID NOs 133, 134, 184, 139, 141 and 239, respectively; (2) (xxiii) SEQ ID NOS 145-147 and 151-153, respectively; (2) (xxiv) SEQ ID NOS 157-159 and 163-165, respectively; (2) (xxv) SEQ ID NOS 169-171 and 175-177, respectively; (2) (xxvi) SEQ ID NOS 181-183 and 187-189, respectively;
(2) (xxvii) SEQ ID NOS.49-51 and 55-57, respectively; (2) (xxviii) SEQ ID NOS 205-207 and 211-213, respectively; or (2) (xxvix) SEQ ID NOS 264-266 and 267-296, respectively.
61. The multispecific antibody or antigen-binding fragment of any one of claims 57 to 60, wherein:
(1) The anti-HA antigen binding domain comprises: (1) (i) a VH comprising or consisting of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to an amino acid sequence set forth in any one of seq id nos: 43, 2, 26, 28, 31, 34, 37, 14, 39 and 41, wherein the sequence variations referring to SEQ ID NOs 43, 2, 26, 28, 31, 34, 37, 14, 39 or 41, respectively, are optionally included in one or more framework regions and/or the sequence variations include substitution of one or more germline encoded amino acids; and/or (1) (ii) VL comprises or consists of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to any one of the following amino acid sequences: 44, 8 and 20 or 44, wherein the sequence variation relative to SEQ ID NO 44, 8 or 20, respectively, is optionally comprised in one or more framework regions and/or the sequence variation comprises a substitution of one or more germline encoded amino acids; and/or
(2) The anti-NA antigen binding domain comprises: (2) (i) a VH comprising or consisting of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to an amino acid sequence set forth in any one of seq id nos: 241, 48, 60, 72, 171, 84, 96, 108, 120, 132, 229, 144, 156, 168, 180, 192, 204, 245, 249, 258 and 261, wherein the sequence variations of SEQ ID NOs 241, 48, 60, 72, 171, 84, 96, 108, 120, 132, 229, 144, 156, 168, 180, 192, 204, 245 and 249, 258 and 261 are optionally included in one or more framework regions, and/or the sequence variations comprise substitutions of one or more germline encoded amino acids; and/or (2) (ii) the VL comprises or consists of an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to an amino acid sequence of any one of: 243, 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 220, 223, 226, 232, 235, 238, 210, 247, 251, 259 and 263, respectively, wherein the sequence variations of 243, 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 220, 223, 226, 232, 235, 238, 210, 247, 251, 259 and 263 relative to SEQ ID NOs 243, 54, 66, 78, 251, 259 and 263, respectively, optionally comprise a substitution in one or more framework regions, and/or the sequence variations comprise one or more germline encoded amino acids.
62. The multispecific antibody or antigen-binding fragment of any one of claims 57 to 61, wherein:
(1) The VH and the VL of the anti-HA antigen binding domain comprise or consist of amino acid sequences according to seq id no: (1) (i) SEQ ID NOs 2 and 8, respectively; (1) (ii) SEQ ID NOS 43 and 44, respectively; (1) (iii) SEQ ID NOS 28 and 8, respectively; (1) (iv) SEQ ID NOs 31 and 8, respectively;
(1) (v) SEQ ID NOs 34 and 8, respectively; (1) (vi) SEQ ID NOS 37 and 8, respectively; (1) (vii) SEQ ID NOs 14 and 20, respectively; (1) (viii) SEQ ID NOs 39 and 20, respectively; (1) (ix) SEQ ID NOs 41 and 20, respectively; or (1) (x) is SEQ ID NO 26 and 8, respectively; and/or
(2) The VH and the VL of the anti-NA antigen binding domain comprise or consist of amino acid sequences according to seq id no: (2) (i) SEQ ID NOs 243 and 243, respectively; (2) (ii) SEQ ID NOs 60 and 66, respectively; (2) (iii) SEQ ID NOs 72 and 78 or 220 or 223, respectively; (2) (vi) SEQ ID NOs 72 and 226, respectively; (2) (vii) SEQ ID NOS 217 and 78, respectively; (2) (viii) SEQ ID NOS 217 and 220, respectively; (2) (ix) SEQ ID NOS 217 and 223, respectively; (2) (x) SEQ ID NOS 217 and 226, respectively; (2) (xi) SEQ ID NOs 84 and 90, respectively; (2) (xii) SEQ ID NOs 96 and 102, respectively; (2) (xiii) SEQ ID NOs 108 and 114, respectively; (2) (xiv) SEQ ID NOS 120 and 126, respectively; (2) (xv) SEQ ID NOS 132 and 138, respectively; (2) (xvi) SEQ ID NOS 132 and 232, respectively; (2) (xvii) SEQ ID NOS 132 and 235, respectively; (2) (xviii) SEQ ID NOS 132 and 238, respectively; (2) (xix) SEQ ID NOs 229 and 138, respectively;
(2) (xx) SEQ ID NOs 229 and 232, respectively; (2) (xxi) SEQ ID NOs 229 and 235, respectively; (2) (xxii) SEQ ID NOs 229 and 238, respectively; (2) (xxiii) SEQ ID NOS 144 and 150, respectively; (2) (xxiv) SEQ ID NOS 156 and 162, respectively; (2) (xxv) SEQ ID NOS 168 and 174, respectively; (2) (xxvi) SEQ ID NOS 180 and 186, respectively; (2) (xxvii) SEQ ID NOS 192 and 198, respectively; (2) (xxviii) SEQ ID NOS 204 and 210, respectively; (2) (xxix) SEQ ID NOs 48 and 54, respectively; (2) (xxx) SEQ ID NOs 245 and 247, respectively; (2) (xxxi) SEQ ID NOS 249 and 251, respectively; (2) (xxxii) SEQ ID NOs 258 and 259, respectively; or (2) (xxxiii) SEQ ID NOS 261 and 263, respectively.
63. The multispecific antibody or antigen-binding fragment of any one of claims 57 to 62, comprising:
(i) CH1-CH3, wherein the CH1-CH3 comprises or consists of an amino acid sequence shown as SEQ ID NO. 252;
(ii) CH1-CH3, wherein the CH1-CH3 comprises or consists of an amino acid sequence shown as SEQ ID NO 253;
(iii) CL comprising or consisting of the amino acid sequence shown as SEQ ID NO. 254; or (b)
(iv) Any combination of (i) - (iii).
64. The multispecific antibody or antigen-binding fragment of any one of claims 57 to 63, comprising:
(1) A heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 255; and
(2) A light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 257.
65. The multispecific antibody or antigen-binding fragment of any one of claims 57 to 64, comprising:
(1) A heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 256; and
(2) A light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 257.
66. An isolated polynucleotide encoding the multispecific antibody or antigen-binding fragment of any one of claims 57 to 65.
67. A vector comprising the polynucleotide of claim 66.
68. A recombinant host cell comprising an isolated polynucleotide according to claim 66 and/or a vector according to claim 67, and/or expressing a multispecific antibody or antigen-binding fragment according to any one of claims 57 to 65.
69. A composition comprising a multispecific antibody or antigen-binding fragment of any one of claims 57 to 65, a polynucleotide of claim 66, a vector of claim 67 and/or a host cell of claim 68, and a pharmaceutically acceptable carrier, excipient or diluent.
70. A method of preventing or treating influenza a infection, influenza b infection, or both in a subject, the method comprising administering to the subject an effective amount of a multispecific antibody or antigen-binding fragment of any one of claims 57 to 65, a polynucleotide of claim 66, a vector of claim 67, a host cell of claim 68, and/or a composition of claim 69.
71. The multispecific antibody or antigen-binding fragment of any one of claims 57 to 65, the polynucleotide of claim 66, the vector of claim 67, the host cell of claim 68 and/or the composition of claim 69 for use in a method of treating or preventing influenza a infection, influenza b infection or both in a subject.
72. A multispecific antibody or antigen-binding fragment according to any one of claims 57 to 65, a polynucleotide according to claim 66, a vector according to claim 67, a host cell according to claim 68 and/or a composition according to claim 69 for use in the preparation of a method for treating or preventing influenza a infection and/or influenza b infection.
73. The multispecific antibody or antigen-binding fragment of any one of claims 57 to 65, the polynucleotide of claim 66, the vector of claim 67, the host cell of claim 68, the composition of claim 69, the method of claim 70 or the antibody or antigen-binding fragment for use of any one of claims 71 and 72, wherein the anti-HA antibody or antigen-binding fragment, the anti-NA antibody or antigen-binding fragment, or both, is capable of preventing a weight loss of greater than 25%, 20%, 15%, 10% or 5% in a subject having an IAV infection and/or IBV infection, as determined by reference to the weight of the subject immediately prior to the IAV infection and/or IBV infection.
74. The multispecific antibody or antigen-binding fragment of any one of claims 57 to 65, the polynucleotide of claim 66, the vector of claim 67, the host cell of claim 68, the composition of claim 69, the method of claim 70, the antibody or antigen-binding fragment for use of any one of claims 71 to 73, wherein the anti-HA antibody or antigen-binding fragment, the anti-NA antibody or antigen-binding fragment, or both are capable of prolonging the survival of a subject suffering from an IAV infection and/or IBV infection.
75. A method for treating or preventing influenza infection in a subject, the method comprising administering to the subject:
(1) An anti-HA antibody or antigen-binding fragment thereof comprising a VH amino acid sequence shown in SEQ ID No. 43 and a VL amino acid sequence shown in SEQ ID No. 44; and
(2) An anti-NA antibody or antigen-binding fragment thereof comprising the VH amino acid sequence shown in SEQ ID No. 241 and the VL amino acid sequence shown in SEQ ID No. 243.
76. A method for treating or preventing influenza infection in a subject, the method comprising administering to the subject a polynucleotide encoding:
(1) An anti-HA antibody or antigen-binding fragment thereof comprising a VH amino acid sequence shown in SEQ ID No. 43 and a VL amino acid sequence shown in SEQ ID No. 44; and
(2) An anti-NA antibody or antigen-binding fragment thereof comprising the VH amino acid sequence shown in SEQ ID No. 241 and the VL amino acid sequence shown in SEQ ID No. 243.
77. A method for treating or preventing influenza infection in a subject, the method comprising administering to the subject:
(1) A polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof comprising the VH amino acid sequence shown in SEQ ID No. 43 and the VL amino acid sequence shown in SEQ ID No. 44; and
(2) A polynucleotide encoding an anti-NA antibody or antigen-binding fragment thereof comprising the VH amino acid sequence shown in SEQ ID No. 241 and the VL amino acid sequence shown in SEQ ID No. 243.
78. A method for treating or preventing influenza infection in a subject, the method comprising administering to the subject:
(1) An anti-HA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs 274-276, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs 277-279, respectively; and
(2) An anti-NA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs 193-195, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs 199-201, respectively.
79. A method for treating or preventing influenza infection in a subject, the method comprising administering to the subject a polynucleotide encoding:
(1) An anti-HA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs 274-276, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs 277-279, respectively; and
(2) An anti-NA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs 193-195, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs 199-201, respectively.
80. A method for treating or preventing influenza infection in a subject, the method comprising administering to the subject:
(1) A polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs 274-276, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs 277-279, respectively; and
(2) A polynucleotide encoding an anti-NA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs 193-195, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs 199-201, respectively.
81. The method of any one of claims 75 to 80, wherein the antibody or antigen-binding fragment of (1) comprises the heavy chain amino acid sequence of SEQ ID No. 270 or SEQ ID No. 272 and the light chain amino acid sequence of SEQ ID No. 271.
82. The method of any one of claims 75-81, wherein the antibody or antigen-binding fragment of (2) comprises the heavy chain amino acid sequence of SEQ ID No. 255 or SEQ ID No. 256 and the light chain amino acid sequence of SEQ ID No. 257.
83. The method of any one of claims 76 to 82, wherein the polynucleotide of (1) and/or the polynucleotide of (2) comprises mRNA, respectively.
84. The method of any one of claims 76-83, wherein the polynucleotide of (1) and/or the polynucleotide of (2) comprises a modified nucleoside, cap-1 structure, cap-2 structure, or any combination thereof, respectively.
85. The method of claim 84, wherein the polynucleotide of (1) and/or the polynucleotide of (2) comprises, respectively, pseudouridine, N6-methyladenosine, 5-methylcytidine, 2-thiouridine, or any combination thereof.
86. The method of claim 85, wherein the pseudouridine comprises N1-methyl pseudouridine.
87. A polynucleotide encoding:
(1) An anti-HA antibody or antigen-binding fragment thereof comprising a VH amino acid sequence shown in SEQ ID No. 43 and a VL amino acid sequence shown in SEQ ID No. 44; and
(2) An anti-NA antibody or antigen-binding fragment thereof comprising the VH amino acid sequence shown in SEQ ID No. 241 and the VL amino acid sequence shown in SEQ ID No. 243.
88. A polynucleotide encoding:
(1) An anti-HA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs 274-276, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs 277-279, respectively; and
(2) An anti-NA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs 193-195, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs 199-201, respectively.
89. A composition, the composition comprising:
(1) A polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof comprising the VH amino acid sequence shown in SEQ ID No. 43 and the VL amino acid sequence shown in SEQ ID No. 44; and
(2) A polynucleotide encoding an anti-NA antibody or antigen-binding fragment thereof comprising the VH amino acid sequence shown in SEQ ID No. 241 and the VL amino acid sequence shown in SEQ ID No. 243.
90. A composition, the composition comprising:
(1) A polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs 274-276, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs 277-279, respectively; and
(2) A polynucleotide encoding an anti-NA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs 193-195, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs 199-201, respectively.
91. A combination, the combination having:
(1) A polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof comprising the VH amino acid sequence shown in SEQ ID No. 43 and the VL amino acid sequence shown in SEQ ID No. 44; and
(2) A polynucleotide encoding an anti-NA antibody or antigen-binding fragment thereof comprising the VH amino acid sequence shown in SEQ ID No. 241 and the VL amino acid sequence shown in SEQ ID No. 243.
92. A combination, the combination having:
(1) A polynucleotide encoding an anti-HA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs 274-276, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs 277-279, respectively; and
(2) A polynucleotide encoding an anti-NA antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs 193-195, respectively, and a VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs 199-201, respectively.
93. The polynucleotide of claim 87 or 88, the composition of claim 89 or 90, or the combination of claim 91 or 92, wherein said antibody or antigen-binding fragment of (1) comprises the heavy chain amino acid sequence of SEQ ID No. 270 or SEQ ID No. 272 and the light chain amino acid sequence of SEQ ID No. 271.
94. The polynucleotide of claim 87, 88, or 93, the composition of claim 89, 90, or 93, or the combination of claims 91, 92, or 93, wherein the antibody or antigen-binding fragment of (2) comprises the heavy chain amino acid sequence of SEQ ID No. 255 or SEQ ID No. 256 and the light chain amino acid sequence of SEQ ID No. 257.
95. The polynucleotide of claim 87, 88, 93, or 94, the composition of claim 89, 90, 93, or 94, or the combination of any one of claims 91-94, wherein the polynucleotide of (1) and/or the polynucleotide of (2) comprises mRNA, respectively.
96. The polynucleotide of claim 87, 88, 93, 94, or 95, the composition of claim 89, 90, 93, 94, or 95, or the combination of any one of claims 91-94, wherein the polynucleotide of (1) and/or the polynucleotide of (2) comprises a modified nucleoside, cap-1 structure, cap-2 structure, or any combination thereof, respectively.
97. The polynucleotide, composition, or combination of claim 96, wherein said polynucleotide of (1) said polynucleotide and/or (2) said polynucleotide comprises, respectively, pseudouridine, 2-thiouridine, N6-methyladenosine, 5-methylcytidine, or any combination thereof.
98. The polynucleotide, composition, or combination of claim 97, wherein said pseudouridine comprises N1-methyl pseudouridine.
CN202180091587.XA 2020-11-23 2021-11-19 Anti-influenza antibodies and combinations thereof Pending CN116997567A (en)

Applications Claiming Priority (6)

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US63/117,454 2020-11-23
US63/125,892 2020-12-15
US63/197,254 2021-06-04
US202163261464P 2021-09-21 2021-09-21
US63/261,464 2021-09-21
PCT/US2021/060166 WO2022109317A1 (en) 2020-11-23 2021-11-19 Anti-influenza antibodies and combinations thereof

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