WO2016044736A1 - Anticorps modifiés et leurs procédés de production - Google Patents

Anticorps modifiés et leurs procédés de production Download PDF

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Publication number
WO2016044736A1
WO2016044736A1 PCT/US2015/050960 US2015050960W WO2016044736A1 WO 2016044736 A1 WO2016044736 A1 WO 2016044736A1 US 2015050960 W US2015050960 W US 2015050960W WO 2016044736 A1 WO2016044736 A1 WO 2016044736A1
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antibody
variable domain
amino acid
heavy chain
human
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PCT/US2015/050960
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English (en)
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Deepangi PANDIT
Aleksandr TKACHEV
Roberto BARBERENA PELÁEZ
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Cell Signaling Technology, Inc.
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Publication of WO2016044736A1 publication Critical patent/WO2016044736A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/461—Igs containing Ig-regions, -domains or -residues form different species
    • C07K16/467—Igs with modifications in the FR-residues only
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]

Definitions

  • Humanized antibodies are commonly used as therapeutic agents for cancer and autoimmune disease. There is a need for alternative methods of humanizing antibodies, particularly for antibodies derived from non-rodents.
  • This disclosure is based, at least in part, on the development of methods for modifying antibodies, e.g., for humanization.
  • the disclosure features altered (e.g., humanized) antibodies that include cysteine residues at Kabat positions H35A and H50.
  • the altered antibodies are humanized versions of parental rabbit antibodies (e.g., rabbit antibodies derived from germline V gene IGHV1S40*01, IGHV1S43*01, IGHV1S45*01,
  • cysteine residues cysteine residues at Kabat positions H35A and H50 form a disulfide bond.
  • an antibody disclosed herein bind to an antigen.
  • the disclosure also features altered antibody heavy chain variable domains that include non-human (e.g., rabbit) Complementarity Determining Region (CDR) amino acid residues which bind an antigen incorporated into a human antibody variable domain, and further comprising cysteine residues at positions H35A and H50, utilizing the numbering system set forth in Kabat.
  • the rabbit antibody heavy chain variable region is derived from a germline V gene selected from the group consisting of
  • the rabbit antibody heavy chain variable region is derived from a germline V gene selected from the group consisting of IGHV1S43*01, IGHV1S45*01, IGHV1S47*01, IGHV1S54*01, IGHVlS7*01, and IGHV1S8*01.
  • the disclosure features humanized variants of a non-human (e.g., rabbit) parent antibodies that binds an antigen, wherein the humanized variants include Complementarity Determining Region (CDR) amino acid residues of the non-human parent antibody incorporated into a human antibody variable domain, and further comprises cysteine residues at positions H35A and H50, utilizing the numbering system set forth in Kabat.
  • CDR Complementarity Determining Region
  • the rabbit antibody heavy chain variable region is derived from a germline V gene selected from the group consisting of IGHV1S40*01, IGHV1S43*01, IGHV1S45*01, IGHV1S47*01, IGHV1S54*01, IGHV1S7*01, and IGHV1S8*01, or a subset thereof.
  • the rabbit antibody heavy chain variable region is derived from a germline V gene selected from the group consisting of IGHV 1 S43 * 01 , IGHV1S45*01, IGHV1S47*01, IGHV1S54*01, IGHV1S7*01, and IGHV1S8*01.
  • the disclosure also features nucleic acids that encode altered (e.g., humanized) heavy chain variable domains that include cysteine residues at Kabat positions H35A and H50.
  • the altered heavy chain variable domains are humanized versions of parental rabbit heavy chain variable domains.
  • the parental rabbit heavy chain variable domains are derived from germline V genes IGHV1S40*01,
  • the parental rabbit heavy chain variable domains are derived from germline V genes IGHV1S43*01, IGHV1S45*01, IGHV1S47*01, IGHV1S54*01, IGHV1S7*01, or IGHV1S8*01.
  • the disclosure also features vectors that include the nucleic acids disclosed herein and cells (e.g., recombinant cells) that contain a nucleic acid or vector disclosed herein.
  • the disclosure also features methods of producing altered antibodies that include culturing such host cells and isolating an altered antibody from the cell culture.
  • the disclosure features methods that include modifying the sequence of an antibody heavy chain variable domain by substituting or inserting one or both of Kabat positions H35A and H50 with cysteine residues, such that the resultant antibody heavy chain variable domain has cysteine residues at Kabat positions H35A and H50.
  • the disclosure features methods for making altered (e.g., humanized) antibodies that include non-human, donor Complementarity Determining Region (CDR) amino acid residues and human Framework Region (FR) amino acid residues, that include the steps of: (a) obtaining the amino acid sequences of a donor heavy chain variable domain and of a human heavy chain variable domain; (b) identifying CDR amino acid sequences in the donor and the human heavy chain variable domain sequences; (c) substituting donor CDRs for the corresponding human CDRs; (d) substituting or inserting one or both of residues at Kabat positions H35A and H50 in the human variable domain FR sequence to cysteine residues, such that both positions H35A and H50 are cysteine residues; and (e) preparing a humanized antibody which binds antigen, wherein the humanized antibody comprises a heavy chain variable domain amino acid sequence determined according to the above steps.
  • CDR Complementarity Determining Region
  • FR human Framework Region
  • the disclosure features methods for making an altered (e.g., humanized) antibody that includes non-human Complementarity Determining Region (CDR) amino acid residues and human Framework Region (FR) amino acid residues, wherein the method includes providing a donor, non-human antibody heavy chain variable domain amino acid sequence having CDR amino acid residues and FR amino acid residues; obtaining the amino acid sequence of a human antibody heavy chain variable domain having CDR amino acid residues and FR amino acid residues; substituting or inserting one or both of Kabat positions H35A and H50 in the human variable domain FR sequence to cysteine residues, such that both residues H35A and H50 are cysteine residues; and preparing a humanized antibody which binds an antigen, wherein the humanized antibody comprises an amino acid sequence determined according to the above steps.
  • CDR Complementarity Determining Region
  • FR human Framework Region
  • the donor heavy chain variable domain is a rabbit heavy chain variable domain, e.g., a rabbit heavy chain variable domain derived from a germline V gene selected from the group consisting of IGHV1S40*01,
  • IGHV1S8*01 or a subset thereof, or a rabbit heavy chain variable domain derived from a germline V gene selected from the group consisting of IGHV1S43*01, IGHV1S45*01, IGHV1S47*01, IGHV1S54*01, IGHV1S7*01, and IGHV1S8*01.
  • the antibody binds to an antigen selected from the group consisting of IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18, IFN-a, IFN- ⁇ , TNF-a, BAFF, CXCL13, IP-10, CD3, CD19, CD20, CD22, CD30 (TNFRSF8), IGF-1R, VEGF, EPO, EGF, HRG, MIF, IL-6R, VEGFR, EpCAM, alpha-V integrin, amyloid-beta, PD-1, PD-Ll, colony stimulating factors, hepatocyte growth factor, 4- 1BB, 5AC, 5T4, activin receptor-like kinase 1, ACVR2B, angiopoietin 2, angiopoietin 3, AOC3 (VAP-1), B7-H3, B7-H4, BLyS, C-X-C chemokine receptor type 4,
  • the disclosure features altered (e.g., humanized) antibodies, nucleic acids, and cells made by the methods disclosed herein.
  • the methods and compositions disclosed herein can provide altered (e.g., humanized) antibodies that retaining significant binding activity as compared to the parental antibodies and efficient means for altering (e.g., humanizing) antibodies while retaining significant binding activity.
  • the methods and compositions can also result in increased stability of altered antibodies.
  • Standard reference works setting forth the general principles of recombinant DNA technology include Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York (1989); Kaufman et al, Eds., Handbook of Molecular and Cellular Methods in Biology in Medicine, CRC Press, Boca Raton (1995); McPherson, Ed., Directed Mutagenesis: A Practical Approach, IRL Press, Oxford (1991). Standard reference works setting forth the general principles of antibody technology include Greenfield, Ed., Antibodies: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, New York (2014); Coligan et al, Eds., Current Protocols in Immunology, DOI:
  • FIG. 1 is an alignment of rabbit immunoglobulin V gene amino acid sequences (SEQ ID NO. 1).
  • FIG. 2 is a ribbon diagram representation of the tertiary structure of a rabbit heavy chain variable region showing a disulfide bond between cysteine residues at Chothia positions H35 and H50 (equivalent to Kabat positions H35A and H50). Framework regions are indicated in green, CDR regions are indicated in red, and cysteine residues H35 and H50 are indicated in yellow with space-filling representation.
  • This application describes the use of certain structural features in creation of altered antibodies, e.g., for humanization of non-human antibodies. It has been discovered that incorporating cysteine residues at conserved positions can significantly maintain antigen- binding function.
  • this application discloses altered (e.g., humanized) antibodies, methods of designing altered (e.g., humanized) antibodies, methods of altering (e.g., humanizing) antibodies, methods of producing altered (e.g., humanized) antibodies and encoding nucleic acids and cells, and antibodies, nucleic acids, and cells produced by the methods disclosed herein.
  • this application discloses altered (e.g., humanized) antibodies that include cysteine residues at Kabat positions H35A and H50. These positions are equivalent to H35 and H50 by the numbering system of Chothia and Cys40 and Cys55 by the numbering system of IMGT. It has been found that cysteines at these positions are conserved in several rabbit immunoglobulin V genes and that incorporating cysteines into altered antibodies derived in part from such V genes results in improved activity (e.g., antigen binding). Incorporation of cysteines at these positions in other altered antibodies can also result in improved activity, such as antigen binding, stability, or other properties.
  • the rabbit V gene IGHV1S54*01 includes a cysteine only at Kabat position H50, although incorporation of a corresponding cysteine at Kabat position H35A is predicted to also result in improved activity.
  • an antibody e.g., a rabbit antibody
  • a rabbit antibody may be considered to be derived from a particular germline V gene based on amino acid homology to the germline gene.
  • the amino acid sequence of the subject antibody is compared to the amino acid sequence of each of all known germline V genes of the same species from which the antibody is derived. The comparison is made using the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2: 482-489 (1981)) (e.g., as implemented in the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis.
  • the germline V gene with the highest score is considered to be the germline V gene from which the antibody was derived.
  • the antibody will be considered to be derived from each of those two or more germline V genes.
  • antibody as used herein is meant to include intact immunoglobulin molecules (e.g., IgGl, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA) for any species (e.g., human, rodent, camelid), as well as antigen binding domain fragments thereof, such as Fab, Fab', F(ab') 2 ; variants thereof such as scFv, Fv, Fd, dAb, bispecific scFvs, diabodies, linear antibodies (see U.S. Pat. No.
  • an antibody binding domain is meant any portion of an antibody that retains specific binding activity of the intact antibody (i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody's target molecule).
  • An "epitope” is smallest portion of a target molecule capable being specifically bound by the antigen binding domain of an antibody. The minimal size of an epitope may be about five or six to seven amino acids.
  • Non- limiting antigen binding domains include the heavy chain and/or light chain CDRs of an intact antibody, the heavy and/or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact antibody.
  • Antibodies disclosed herein include but are not limited to polyclonal, monoclonal, monospecific, polyspecific antibodies and fragments thereof and chimeric antibodies that include an immunoglobulin binding domain fused to another polypeptide.
  • Native antibodies are made up of two classes of polypeptide chains, light chains and heavy chains.
  • a non- limiting antibody of the disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains.
  • the heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgA or IgD or sub-isotype including IgGl, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgEl, IgE2, etc.
  • the light chain can be a kappa light chain or a lambda light chain.
  • a single native antibody comprises two identical copies of a light chain and two identical copies of a heavy chain.
  • the heavy chains which each contain one variable domain (V H ) and multiple constant domains, bind to one another via disulfide bonding within their constant domains to form the "stem" of the antibody.
  • the light chains which each contain one variable domain (V L ) and one constant domain, each bind to one heavy chain via disulfide binding.
  • the variable domain of each light chain is aligned with the variable domain of the heavy chain to which it is bound.
  • the variable regions of both the light chains and heavy chains contain three hypervariable regions sandwiched between four more conserved framework regions (FR). These hypervariable regions, known as the complementary determining regions (CDRs), form loops that comprise the principle antigen binding surface of the antibody (see Kabat, E. A.
  • the four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding domain.
  • Each antigen-binding region of an antibody is typically composed of two domains, a heavy chain variable region (VH) and a light chain variable region (VL).
  • the VH and VL regions each contain three hypervariable regions between four more conserved framework regions (FR).
  • These hypervariable regions known as the complementary determining regions (CDRs), form loops that comprise the principal antigen binding surface of the antibody (see Kabat, E. A. et a., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)).
  • the four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen- binding domain.
  • the variable domain of each light chain is aligned with the variable domain of the heavy chain to which it is bound.
  • Template selection for antibody modeling is typically performed by comparing light and heavy chain variable regions or framework regions of the target antibody to be modeled to available light and heavy chain variable regions or framework regions of template antibodies with existing structures.
  • a template having matching pair of light and heavy chains framework regions compared to the target sequence, can be selected based on alignment obtained using Smith- Waterman algorithm (Smith and Waterman, 1981, J. Mol. Biol, 147: 195-197) with BLOSUM62 (Henikoff et al, 1992, Proc. Natl. Acad. Sci. USA, 89: 10915-19) scoring matrix.
  • the templates can be ranked by the average framework sequence similarity of the heavy and the light chain, and the best template chosen as the one with the highest average similarity. If a template with a matching pair of heavy and light chain is unavailable, multiple templates can be assessed in context with the target sequence.
  • a set of loop sequences and conformations can be derived individually for each loop position (LI, L2, L3, HI, H2, and H3).
  • Each of the six resulting loop databases can be dynamically filtered based on the query sequence, loop length, and the stem residue geometry of the framework template that has been selected.
  • all remaining loop candidates can be clustered and ranked by the cluster size, and the "representative loop" of each cluster defined as the one with the highest sequence similarity (as defined by BLOSUM62) to the query loop within that cluster.
  • the representative loop with the highest sequence similarity can then be chosen.
  • the similarity cutoff for H3 loops and non-H3 loops can be set at 0.3 and 0.6, respectively.
  • the initial homology model can be constructed by first copying over the backbone coordinates and also side chains for conserved residues in the frame- work region, then mutating the non- conserved residues in the framework, and finally grafting. See, e.g., Zhu et al, 2014, Proteins, 82: 1646-55.
  • Antibodies disclosed herein can be derived from any species of animal, including mammals.
  • Non-limiting exemplary native antibodies include antibodies derived from human, camelids (e.g., camels and llamas), chickens, horses, bovines, sheep, rabbits, and rodents (e.g., rats, mice, and hamsters), including transgenic rodents genetically engineered to produce human antibodies (see, e.g., Lonberg et al, WO 93/12227; U.S. Pat. No.
  • Native antibodies are the antibodies produced by a host animal.
  • altered antibodies refer to antibodies wherein the amino acid sequence has been varied from that of a native or naturally-occurring antibody. Because of the relevance of recombinant DNA techniques to this application, one need not be confined to the sequences of amino acids found in native antibodies; antibodies can be redesigned to obtain desired characteristics. The possible variations are many and range from the changing of just one or a few amino acids to the complete redesign of, for example, the variable or constant region.
  • Changes in the constant region can be made to improve or alter characteristics, such as complement fixation, interaction with membranes, and other effector functions (e.g., complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cell phagocytosis (ADCP)). Additionally, changes in the variable region can be made to improve or alter characteristics, such as antigen binding.
  • characteristics such as complement fixation, interaction with membranes, and other effector functions
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cell phagocytosis
  • the antibody or fragment or derivative thereof can be useful in medicine, in particular in therapy, diagnosis, prognosis and/or monitoring of a disease, in particular a disease as described herein, preferably cancer.
  • Antigen-specificity can be measured with respect to any antigen.
  • the antigen can be any substance to which an antibody can bind including, but not limited to, peptides, proteins or fragments thereof; carbohydrates; organic and inorganic molecules; receptors produced by animal cells, bacterial cells, and viruses; enzymes; agonists and antagonists of biological pathways; hormones; and cytokines.
  • antigens include, but are not limited to, IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18, IFN-a, IFN- ⁇ , TNF-a, BAFF, CXCL13, IP-10, CD3, CD19, CD20, CD22, CD30 (TNFRSF8), IGF-1R, VEGF, EPO, EGF, HRG, MIF, IL-6R, VEGFR, EpCAM, alpha-V integrin, amyloid-beta, PD-1, PD-L1, colony stimulating factors, hepatocyte growth factor, hepcidin, TP As, interferons, tumor associated antigens (including tumor antigens specific to particular human cancers), HIV antigens such as env and gag and pol, influenzae antigens, bird flu antigens, et al.
  • tumor associated antigens including tumor antigens specific to particular human cancers
  • HIV antigens such as env and gag
  • Additional exemplary antigens include 4-lBB, 5AC, 5T4, activin receptor-like kinase 1, ACVR2B, angiopoietin 2, angiopoietin 3, AOC3 (VAP-1), B7-H3, B7-H4, BLyS, C-X-C chemokine receptor type 4, C242 antigen, C5, CA-125, carbonic anhydrase 9, cardiac myosin, CCL11 (eotaxin-1), CCR2, CCR4, CCR5, CD11, CDl la, CD18, CD125, CD138, CD140a, CD146, CD147 (basigin), CD 15, CD 152, CD 154 (CD40L), CD 164, CD 167a, CD 167b, CD2, CD200, CD221 , CD23 (IgE receptor), CD24, CD25 (alpha chain of IL-2 receptor), CD27, CD274, CD28, CD3 epsilon, CD33, CD37, CD38, CD4, CD40,
  • glycoprotein glycoprotein, myostatin, NARP-1, Navl .7, NCA-90 (granulocyte antigen), nectin04, neural apoptosis-regulated proteinase 1, NGF, NOGO-A, Notch receptor, NRP1, OX-40, oxLDL, PCSK9, PD-L2, PDCD 1 , PDGF-R alpha, phosphate-sodium co-transporter,
  • phsophatidylserine PDGF-R beta, PEM, PSMA, RANKL, RHD, Rhesus factor, RON, RTN4, sclerostin, scr-like kinase Fyn3, SDC1, selectin P, sialoglycotope CA6, SLAMF7, SLC44A4, SLITRK6, sodium-dependent phosphate transport protein 2b, SOST, sphingosine- 1 -phosphate, STEAP1, T-cell receptor, TAG-72, TEM1, tenascin C, tissue factor (TF), TFPI, TGF beta 1, TGF beta 2, TGF beta, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, tumor-associated calcium signal transducer 2, TWEAK receptor, TYRPl (glycoprotein 75), VEGF-A, VEGFR-1, VEGFR-2, vimentin, and VWF.
  • TF tissue factor
  • TFPI tissue factor
  • Antibodies, including altered antibodies can be made in any expression systems including both prokaryotic and eukaryotic expression systems or using phage display methods (see, e.g., PCT Publication No. WO 91/17271, PCT Publication No. WO 92/01047; U.S. Pat. No. 5,969,108; U.S. Pat. No. 6,331,415; US Pat. No. 7,498,024, and U.S. Pat. No. 7,485,291, which are herein incorporated by reference in their entirety).
  • Antibodies can be engineered in numerous ways. They can be made as single-chain antibodies (including small modular immunopharmaceuticals or SMIPs), Fab and F(ab') 2 fragments, etc. Antibodies can be humanized, chimerized, deimmunized, or fully human.
  • modified antibodies can be functionally equivalent or near equivalent to the above- mentioned native antibodies.
  • modified antibodies provide improved stability or/and therapeutic efficacy.
  • modified antibodies include those with conservative substitutions of amino acid residues, and one or more deletions or additions of amino acids that do not significantly deleteriously alter the antigen binding utility.
  • Substitutions can range from changing or modifying one or more amino acid residues to complete redesign of a region as long as the therapeutic utility is maintained.
  • Antibodies of this application can be modified post-translationally (e.g., acetylation, and/or
  • phosphorylation or can be modified synthetically (e.g., the attachment of a labeling group).
  • the altered antibodies disclosed herein include CDR grafted humanized antibodies.
  • the humanized antibody comprises heavy and/or light chain CDRs of a non-human (e.g., rabbit) donor immunoglobulin and heavy chain and light chain frameworks and constant regions of a human acceptor immunoglobulin.
  • Non-limiting methods for humanizing antibodies are disclosed in U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370 each of which is incorporated herein by reference in its entirety.
  • the antibody fragments are truncated chains (truncated at the carboxyl end). In certain embodiments, these truncated chains possess one or more immunoglobulin activities (e.g., complement fixation activity).
  • immunoglobulin activities e.g., complement fixation activity.
  • truncated chains include, but are not limited to, Fab fragments (consisting of the VL, VH, CL and CHI domains); Fd fragments (consisting of the VH and CHI domains); Fv fragments (consisting of VL and VH domains of a single chain of an antibody); dAb fragments (consisting of a VH domain); isolated CDR regions; (Fab') 2 fragments, bivalent fragments (comprising two Fab fragments linked by a disulphide bridge at the hinge region).
  • the truncated chains can be produced by conventional biochemical techniques, such as enzyme cleavage, or recombinant DNA techniques, each of which is known in the art.
  • These polypeptide fragments may be produced by proteolytic cleavage of intact antibodies by methods well known in the art, or by inserting stop codons at the desired locations in the vectors using site-directed mutagenesis, such as after CHI to produce Fab fragments or after the hinge region to produce (Fab') 2 fragments.
  • Single chain antibodies may be produced by joining VL- and VH-coding regions with a DNA that encodes a peptide linker connecting the VL and VH protein fragments
  • Fv usually refers to the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain (i.e., a VL domain and a VH domain) in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V H -V L dimer. Collectively, the CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising three CDRs specific for an antigen) has the ability to recognize and bind antigen, although likely at a lower affinity than the entire binding site.
  • Single- chain Fv or “scFv” antibody fragments comprise the V H and V L domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains that enables the scFv to form the desired structure for antigen binding.
  • Papain digestion of an intact antibody produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily.
  • the Fab fragment contains the entire light chain (i.e., the constant domain (CL) and variable domain (VL) of the light chain) together with the first constant domain (CHI) and variable region (VH) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. For example, pepsin treatment of an antibody yields an F(ab') 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen. In other words, an F(ab') 2 fragment comprises two disulfide linked Fab fragments. Other chemical couplings of antibody fragments are also known.
  • the antigen-binding fragment can be a diabody.
  • diabody refers to a small antibody fragment with two antigen-binding sites, which fragment comprises a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) in the same polypeptide chain (V H -V L ).
  • Diabodies can be prepared by constructing scFv fragments with short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a multivalent fragment, i.e., a fragment having two antigen-binding sites.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. Acad. Sci. USA, 90: 6444-48 (1993).
  • single chain antibodies, and chimeric, humanized or primatized (CDR-grafted) antibodies, as well as chimeric or CDR-grafted single chain antibodies, comprising portions derived from different species, are also encompassed by the present disclosure as antigen-binding fragments of an antibody.
  • the various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.
  • nucleic acids encoding an altered (e.g., humanized) chain can be expressed to produce a contiguous protein. See, e.g., U.S. Pat. Nos. 4,816,567 and 6,331,415; U.S. Pat. No. 4,816,397;
  • functional fragments of antibodies including fragments of chimeric, humanized, primatized or single chain antibodies, can also be produced.
  • Functional fragments of the subject antibodies retain at least one antigen binding domain function and/or modulation function of the full-length (i.e., intact) antibody from which they are derived. Since the immunoglobulin-related genes contain separate functional regions, each having one or more distinct biological activities, the genes of the antibody fragments may be fused to functional regions from other genes (e.g., enzymes, U.S. Pat. No. 5,004,692, which is incorporated by reference in its entirety) to produce fusion proteins or conjugates having novel properties.
  • % identical for two polypeptides or two polynucleotides is intended a similarity score produced by comparing the amino acid sequences of the two polypeptides or by comparing the nucleotides sequences of the two polynucleotides using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) and the default settings for determining similarity. Bestfit uses the local homology algorithm of Smith and Waterman ⁇ Advances in Applied Mathematics 2: 482-489 (1981)) to find the best segment of similarity between two sequences.
  • ELISA ELISA
  • RIA RIA
  • surface plasmon resonance Kinetic parameters, such as dissociation constant, on rate, and off rate, may be measured by surface plasmon resonance using, e.g., a BIAcore sensor.
  • an antibody disclosed herein e.g., an altered or humanized antibody
  • 500 nM or less e.g., 200 nM or less, 100 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 pM or less, or 1 pM or less
  • 500 nM or less e.g., 200 nM or less, 100 nM or less,
  • polypeptide As used herein, the terms “polypeptide”, “peptide” and “protein” are used
  • polymers of amino acids of any length may be linear or branched, and it may comprise modified amino acids. Where the amino acid sequence is provided, unless otherwise specified, the sequence is in an N-terminal to C- terminal orientation. In some embodiments, the polymer may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • the polypeptides disclosed herein are based upon antibodies, the polypeptides can occur as single chains or associated chains.
  • nucleic acid molecule refers to polymers of nucleotides of any length, and include, without limitation, DNA, R A, DNA/R A hybrids, and modifications thereof. Unless otherwise specified, where the nucleotide sequence is provided, the nucleotides are set forth in a 5 ' to 3 ' orientation. Thus, the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as un
  • any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports.
  • the 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms.
  • Other hydroxyls may also be derivatized to standard protecting groups.
  • Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0-methyl-, 2'-0-allyl, 2'-fluoro- or 2'- azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.
  • One or more phosphodiester linkages may be replaced by alternative linking groups.
  • linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S ("thioate”), P(S)S ("dithioate”), "(0)NR2 ("amidate”), P(0)R, P(0)OR * , CO or CH2
  • each R or R is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether ( ⁇ 0 ⁇ ) linkage, aryl, alkenyl, cycloalkyl,
  • Altered antibodies include those produced by nucleotide substitutions, deletions, and/or insertions to alter the encoded polypeptide sequence. Methods of creating such substitutions, deletions, and/or insertions are well known in the arts of molecular biology and antibody engineering.
  • the present disclosure also provides recombinant vectors (e.g., an expression vectors) that include a polynucleotide sequence encoding an altered antibody sequence (e.g., a heavy or light chain sequence), host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the
  • recombinant binding agent polypeptides e.g., antibodies
  • fragments thereof by recombinant techniques.
  • a "vector” is any construct capable of delivering one or more polynucleotide(s) of interest to a host cell when the vector is introduced to the host cell.
  • An "expression vector” is capable of delivering and expressing the one or more
  • polynucleotide(s) of interest as encoded polypeptide in a host cell introduced with the expression vector.
  • the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, poly-A tail, etc., either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.
  • vectors are inserted into the host cell by any means including, without limitation, electroporation, fusion with a vector-containing liposomes, chemical transfection (e.g., DEAE-dextran), transformation, transvection, and infection and/or transduction (e.g., with recombinant virus).
  • vectors include viral vectors (which can be used to generate recombinant virus), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.
  • a polynucleotide disclosed herein may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus, or may use a replication defective virus. In the latter case, viral propagation generally will occur only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al, 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al, 1989, Ann. N.Y. Acad Sci. 569:86- 103; Flexner et al, 1990, Vaccine 8: 17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, and
  • DNA may also be "naked,” as described, for example, in Ulmer et al, 1993, Science 259: 1745- 1749, and reviewed by Cohen, 1993, Science 259: 1691-1692.
  • the uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cells.
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid.
  • the vector may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • the methods disclosed herein may be practiced with vectors comprising cis- acting control regions to the polynucleotide of interest. Appropriate trans-acting factors may be supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
  • the vectors provide for specific expression, which may be inducible and/or cell type-specific (e.g., those inducible by environmental factors that are easy to manipulate, such as temperature and nutrient additives).
  • DNA insert comprising an antibody-encoding or polypeptide- encoding polynucleotide disclosed herein may be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan.
  • the expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors may include at least one selectable marker.
  • markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • bacterial cells such as E. coli, Streptomyces and Salmonella typhimurium cells
  • fungal cells such as yeast cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells
  • plant cells Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
  • Non-limiting bacterial promoters suitable for use include the E. coli lacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter.
  • Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (1986).
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • secretion signals may be incorporated into the expressed polypeptide.
  • the signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide may be expressed in a modified form, such as a fusion protein ⁇ e.g., a GST-fusion), and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
  • a binding agent or antibody of the disclosure may comprise a heterologous region from an immunoglobulin that is useful to solubilize proteins.
  • an immunoglobulin that is useful to solubilize proteins.
  • US 7,253,264 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
  • the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties.
  • binding agents and antibodies can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose
  • Polypeptides of the present disclosure include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present disclosure may be glycosylated or may be non-glycosylated. In addition, polypeptides of the disclosure may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • the disclosure provides a method for producing a recombinant binding agent or antibody by culturing a recombinant host cell (as described above) under conditions suitable for the expression of the fusion polypeptide and recovering the polypeptide.
  • Culture conditions suitable for the growth of host cells and the expression of recombinant polypeptides from such cells are well known to those of skill in the art. See, e.g., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel FM et al, eds., Volume 2, Chapter 16, Wiley Interscience.
  • the disclosure also provides immortalized cell lines that produce an antibody obtained by methods disclosed herein. For example, hybridoma clones, constructed as described above, that produce monoclonal antibodies to the target molecule disclosed herein are also provided.
  • the disclosure includes recombinant cells producing an antibody as disclosed herein, which cells may be constructed by well known techniques; for example the antigen combining site of the monoclonal antibody can be cloned by PCR and single-chain antibodies produced as phage-displayed recombinant antibodies or soluble antibodies in E. coli (see, e.g., ANTIBODY ENGINEERING PROTOCOLS, 1995, Humana Press, Sudhir Paul editor.).
  • the altered antibodies of the present disclosure may be analyzed in various methods.
  • the binding agents of the disclosure may be used in any known assay method, such competitive binding assays, direct and indirect sandwich assays, and
  • the binding agents may be detectably labeled (e.g., with a fluorophore such as FITC or phycoerythrin or with an enzyme substrate, such as a substrate for horse radish peroxidase) for easy detection.
  • a fluorophore such as FITC or phycoerythrin
  • an enzyme substrate such as a substrate for horse radish peroxidase
  • the binding agents of the disclosure may be used for in vivo diagnostic assays, such as in vivo imaging.
  • the antibody is labeled with a radionucleotide (such as 3 H, m In, 14 C, 32 P, 99 Tc, or 123 I) so that the cells or tissue of interest can be localized using immunoscintiography.
  • a radionucleotide such as 3 H, m In, 14 C, 32 P, 99 Tc, or 123 I
  • Methods of conjugating labels to a binding agent are known in the art.
  • binding agents disclosed herein need not be labeled, and the presence thereof can be detected using a labeled antibody, which binds to the binding agent.
  • a rabbit antibody derived in part from germline IGHV1S43*01 was subjected to humanization by grafting of the rabbit CDRs onto a human framework region. Substitutions/insertions of cysteines in the human framework regions at Kabat positions H35A and H50 resulted in significantly higher antigen binding as compared to antibodies without the substitutions/insertions. Substitution or insertion at just one of the positions was not sufficient to increase antigen binding.
  • Structural modeling of the parental rabbit antibody (FIG. 2) predicted that the cysteines formed a disulfide bond that constrained the structure of the antibody, particularly at CDRH2. This example demonstrates that incorporation of cysteine residues at Kabat positions H35A and H50 can maintain antigen binding for antibodies derived from germline V genes that include cysteine residues at one or both positions.

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Abstract

L'invention concerne des anticorps modifiés (par exemple, humanisés), des acides nucléiques codant pour de tels anticorps, des cellules comprenant les acides nucléiques et des procédés de conception et de production des anticorps modifiés (par exemple, humanisés).
PCT/US2015/050960 2014-09-18 2015-09-18 Anticorps modifiés et leurs procédés de production WO2016044736A1 (fr)

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US9920123B2 (en) 2008-12-09 2018-03-20 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture

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US6180370B1 (en) * 1988-12-28 2001-01-30 Protein Design Labs, Inc. Humanized immunoglobulins and methods of making the same
CA2800450A1 (fr) * 2000-02-10 2001-08-16 Abbott Laboratories Anticorps de liaison de l'interleukine 18 humaine et procedes de preparation et d'utilisation
WO2005016950A1 (fr) * 2003-08-07 2005-02-24 Epitomics, Inc. Methodes destinees a humaniser des anticorps monoclonaux de lapin

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Publication number Priority date Publication date Assignee Title
US6180370B1 (en) * 1988-12-28 2001-01-30 Protein Design Labs, Inc. Humanized immunoglobulins and methods of making the same
CA2800450A1 (fr) * 2000-02-10 2001-08-16 Abbott Laboratories Anticorps de liaison de l'interleukine 18 humaine et procedes de preparation et d'utilisation
WO2005016950A1 (fr) * 2003-08-07 2005-02-24 Epitomics, Inc. Methodes destinees a humaniser des anticorps monoclonaux de lapin

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PAN, R ET AL.: "Rabbit Anti-HIV-1 Monoclonal Antibodies Raised By Immunization Can Mimic The Antigen-Binding Modes Of Antibodies Derived From HIV-1-Infected Humans.", JOURNAL OF VIROLOGY, vol. 87, no. 18, 17 July 2013 (2013-07-17), pages 10221 - 10231 *

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Publication number Priority date Publication date Assignee Title
US9920123B2 (en) 2008-12-09 2018-03-20 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture

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