CA2704119C - Humanized anti-venezuelan equine encephalitis virus recombinant antibodies - Google Patents

Abstract

A CDR grafted humanized recombinant antibody against infection from Venezuelan equine encephalitis virus (VEEV) comprises a human Ig framework having CDRs from murine mAb 1A4A1 VH and VL. DNA sequences, expression vectors incorporating such sequences and transformed host cells are also provided. Also provided are pharmaceutical compositions and methods of prophylaxis and treatment against VEEV infection using the humanized recombinant antibodies of the invention.

Description

CA 2,704,119 Blakes Ref: 67853/00022 4 [0001] The present invention relates to a humanized antibodies (Abs) and, more specifically, to humanized recombinant antibodies (rAbs) against infection by the Venezuelan equine encephalitis 6 virus (VEEV). The invention provides methods of prophylaxis and treatment against VEEV using 7 such antibodies.

9 [0002] Venezuelan equine encephalitis virus (VEEV), a member of the alphavirus genus of the family Togaviridae, is an important mosquito-borne pathogen in humans and equides [1]. VEEV
11 infections mainly target the central nervous system and lymphoid tissues causing severe 12 encephalitis in equines and a spectrum of human diseases ranging from unapparent or sub-clinical 13 infection to acute encephalitis. Neurological disease appears in 4-14%
of cases. The incidence of 14 human infection during equine epizootics could be up to 30%. Mortality associated with the encephalitis in children is as high as 35%. Recent outbreaks in Venezuela and Colombia in 1995 16 resulted in around 100,000 human cases with more than 300 fatal encephalitis cases [2].
17 Furthermore, VEEV is highly infectious by aerosol inhalation in humans and other animals.
18 However, there are no antiviral drugs available that are effective against VEEV although currently 19 there are two forms of IND (investigational new drug) VEEV vaccines available for human and veterinary use: TC-83, a live-attenuated Trinidad donkey strain and C-84, a formalin-inactivated TC-21 83 [3,4]. However, for various reasons, these vaccines are far from satisfactory. For example, 22 approximately 20% of recipients that receive the TC-83 vaccine fail to develop neutralizing Abs, 23 while another 20% exhibit reactogenicity. In addition, the TC-83 vaccine could revert to wild-type 24 form. The vaccine C-84 is well tolerated, but requires multiple immunizations, periodic boosts, and fails to provide protection against aerosol challenge in some rodent models.
26 [0003] Like the other alphaviruses, VEEV is an enveloped virus, consisting of three structural 27 proteins: a capsid encapsidating the viral RNA genome, and two envelope glycoproteins, El and 28 E2. El and E2 form heterodimers, which project from the virus envelope as trimer spikes. Epitopes 29 on the spikes are the targets of neutralizing Abs. Studies have shown that the viral neutralizing epitopes are mainly located on the E2 protein, and that the E2C epitope appears to be the hub of the 31 neutralization epitopes [5,6]. The murine monoclonal Ab (mAb) 1A4A1 [14]
is specific for E2C. This 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 mAb has been shown to be efficient in protecting animals from a lethal peripheral challenge with 2 virulent VEEV [7].
3 [0004] Murine mAbs, however, have serious disadvantages as therapeutic agents in humans 4 [8]. For example, one of the problems associated with using murine mAbs in humans is that they may induce an anti-mouse Ab response. Further, repeat administration of murine mAbs may result 6 in rapid clearance of the murine mAbs and anaphylaxis, which can sometimes be fatal. To 7 overcome this hurdle, the humanization of murine mAbs has been proposed, by which process 8 murine Ab frameworks are replaced by human Ab ones in order to reduce immunogenicity of Abs in 9 humans [9,10].

[0005] An effective means of immunization against VEEV is needed. In particular, a means of 11 prophylaxis against VEEV and/or a therapy for VEEV infection is desired.

13 [0006] In one aspect, the present invention provides prophylaxis and post-exposure therapy 14 against VEEV infection.
[0007] In one aspect, the invention provides a humanized rAb comprising a human 16 immunoglobulin (Ig) framework and having grafted thereon complementarity determining regions 17 (CDRs) from the murine mAb 1A4A1. In a preferred embodiment, the human Ig framework is 18 obtained from IgG1.
19 [0008] In another aspect, the invention provides a humanized rAb having specificity to the E2 envelope protein of VEEV. More specifically, the rAb has specificity to the E2c epitope of the E2 21 protein.
22 [0009] In another aspect, the invention provides a humanized rAb wherein the complementarity 23 determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) have the 24 following amino acid sequences:
CDR1: SEQ ID NO: 1 26 CDR2: SEQ ID NO: 2 27 CDR3: SEQ ID NO: 3.
28 [0010] In another aspect, the invention provides a humanized rAb wherein the complementarity 29 determining regions CDR1, CDR2 and CDR3 of the light chain variable region (VL) have the following amino acid sequences:

21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 CDR1: SEQ ID NO: 4 2 CDR2: SEQ ID NO: 5 3 CDR3: SEQ ID NO: 6.
4 [0011] In a further aspect, the invention provides a humanized rAb having a VH comprising the amino acid sequence of SEQ ID NO: 7.

6 [0012] In a further aspect, the invention provides a humanized rAb having a VL comprising the 7 amino acid sequence of SEQ ID NO: 8.

8 [0013] In another aspect, the invention provides a DNA sequence which encodes a polypeptide 9 corresponding to a CDR grafted VH having the amino acid sequence according to SEQ ID NO: 7.
[0014] In another aspect, the invention provides a DNA sequence which encodes a polypeptide 11 corresponding to a CDR grafted VL having the amino acid sequence according to SEQ ID NO: 8.
12 [0015] In a further aspect, the invention provides a DNA construct having a nucleic acid 13 sequence according to SEQ ID NO:11 or SEQ ID NO:13.
14 [0016] In another aspect, the invention provides an expressed protein comprising a humanized rAb having an amino acid sequence according to SEQ ID NO: 12 or SEQ ID NO: 14.
16 [0017] The invention provides vectors containing such DNA
sequences and host cells 17 transformed thereby.
18 [0018] In other aspects, the invention provides methods and uses for treatment and/or 19 prophylaxis against VEEV infection comprising the antibodies described herein. The invention also provides pharmaceutical preparations for such treatment or prophylaxis.

22 [0019] These and other features of the invention will become more apparent in the following 23 detailed description in which reference is made to the appended drawings wherein:
24 [0020] Figure 1 is a representation of the external structure of the VEEV.
[0021] Figures 2a to 2d schematically illustrate murine, human, chimeric and humanized Abs, 26 respectively.

21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [0022] Figures 3a to 3c schematically illustrate the humanization of the murine Ab variable 2 region.
3 [0023] Figure 4 schematically illustrates the cloning of the murine Ab VH and VL.
4 [0024] Figure 5 schematically illustrates the humanization of the Ab VH and shows its amino acid sequence (SEQ ID NO: 7).
6 [0025] Figure 6 schematically illustrates the humanization of the Ab VL and shows its amino 7 acid sequence (SEQ ID NO: 8).
8 [0026] Figure 7 schematically illustrates the design of a full Hu1A4A1IgG1 rAb gene in a single 9 open reading frame with two versions, Hu1A4A1IgG1-furin (SEQ ID NO: 11) and Hu1A4A1IgG1-2A
(SEQ ID NO: 13).
11 [0027] Figure 8 schematically illustrates the cloning of the Hu1A4A1IgG1-furin and 12 Hu1A4A1IgG1-2A genes into an adenoviral vector respectively.
13 [0028] Figure 9 schematically illustrates expression and purification of the Hu1A4A1IgG1-furin 14 and Hu1A4A1IgG1-2A rAbs.
[0029] Figures 10 and 11 illustrate the results from the SDS-PAGE
separation of the produced 16 Hu1A4A1IgG1-furin rAb.
17 [0030] Figure 12 illustrates the results from the sodium dodecyl sulfate-polyacrylamide gel 18 electrophoresis (SDS-PAGE) separation of the produced Hu1A4A1IgG1-2A
rAb.
19 [0031] Figure 13 illustrates the results of the enzyme-linked immunosorbent assays (ELISA) for the reactivity of the Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A rAbs.
21 [0032] Figure 14 schematically illustrates Hu1A4A1IgG1-2A was cleaved between the heavy 22 and light chains as expected, whereas Hu1A4A1IgG1-furin was not cleaved.
23 [0033] Figure 15 schematically illustrates the neutralization assay used in assessing the 24 neutralizing activity of the Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A rAbs against VEEV.

21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 2 [0034] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein 3 refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody 4 composition displays a single binding specificity and affinity for a particular epitope.
[0035] The term "recombinant antibody", as used herein, refers to antibodies that are prepared, 6 expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an 7 animal (e.g., a mouse) that is transgenic or transchromosomal for immunoglobulin genes or a 8 hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the 9 antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means 11 that involve splicing of immunoglobulin gene sequences to other DNA
sequences.
12 [0036] As used herein the terms "expression vector" or "cloning vector" include vectors which 13 are designed to provide transcription of the nucleic acid sequence. The transcribed nucleic acid may 14 be translated into a polypeptide or protein product. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One 16 type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which 17 additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional 18 DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous 19 replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication). Other vectors are integrated into the genome of a host cell upon introduction 21 into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors 22 are capable of directing the expression of genes to which they are operatively-linked. Such vectors 23 are referred to herein as "expression vectors" or "cloning vectors". In general, expression vectors of 24 utility in recombinant DNA techniques are often in the form of plasmids.
In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form 26 of vector. However, the invention is intended to include such other forms of expression vectors, 27 such as viral vectors or plant transformation vectors, binary or otherwise, which serve equivalent 28 functions.
29 [0037] The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the 31 recombinant expression vectors include one or more regulatory sequences, selected on the basis of 32 the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 expressed. Within a recombinant expression vector, "operatively-linked"
or "operably-linked" is 2 intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a 3 manner that allows for expression of the nucleotide sequence (e.g., in an in vitro 4 transcription/translation system or in a host cell when the vector is introduced into the host cell).
[0038] The term "regulatory sequence" is intended to include promoters, enhancers and other 6 expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well 7 known in the art such as, for example, in Goeddel, Gene Expression Technology: Methods in 8 Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those 9 that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific 11 regulatory sequences) or inducible promoters (e.g., induced in response to abiotic factors such as 12 environmental conditions, heat, drought, nutrient status or physiological status of the cell or biotic 13 such as pathogen responsive). Examples of suitable promoters include for example constitutive 14 promoters, ABA inducible promoters, tissue specific promoters and abiotic or biotic inducible promoters. It will be appreciated by those skilled in the art that the design of the expression vector 16 can depend on such factors as the choice of the host cell to be transformed, the level of expression 17 of protein desired as well as timing and location of expression, etc.
The expression vectors of the 18 invention can be introduced into host cells to thereby produce proteins or peptides, including fusion 19 proteins or peptides, encoded by nucleic acids as described herein.
[0039] The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is 21 understood that such terms refer not only to the particular subject cell but also to the progeny or 22 potential progeny of such a cell. Because certain modifications may occur in succeeding 23 generations due to either mutation or environmental influences, such progeny may not, in fact, be 24 identical to the parent cell, but are still included within the scope of the term as used herein.
[0040] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional 26 transformation or transfection techniques. As used herein, the terms "transformation" and 27 "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign 28 nucleic acid (e.g., DNA) into a host cell.
29 [0041] A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a polypeptide of the invention encoded in an open reading frame 31 of a polynucleotide of the invention. Accordingly, the invention further provides methods for 32 producing a polypeptide using the host cells of the invention. In one embodiment, the method 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 comprises culturing the host cell of invention (into which a recombinant expression vector encoding 2 a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is 3 produced. In another embodiment, the method further comprises isolating the polypeptide from the 4 medium or the host cell.
[0042] Figure 1 illustrates the external structure of the VEEV. As shown, the virus 10 includes a 6 nucleocapsid 12 enveloping the viral RNA genome. The envelope comprises glycoproteins El and 7 E2, arranged in the form of heterodimers 14. Protein E2, which is responsible for viral attachment to 8 the host cell, contains neutralizing epitopes.
9 [0043] As has been described in the prior art, the murine mAb 1A4A1 has been found to be specific to the VEEV E2 envelope protein and, further, has been found to have a strong neutralizing 11 function against VEEV. The murine mAb, however, causes a sometimes fatal allergenic reaction in 12 humans, resulting in the formation of human anti-mouse Abs (HAMA). It is for this reason that the 13 present inventors have sought to humanize the 1A4A1 mAb so as to provide an effective agent to 14 counter VEEV infection in humans. In the course of this research, humanized recombinant anti-VEEV monoclonal antibodies have recently been designed and developed [18].
Such recombinant 16 antibodies are described further herein and are the subject of Canadian patent application number 17 2,607,771 and U.S. patent application number 11/933,948, both filed on November 1, 2007. The 18 present invention provides methods and uses involving such antibodies for the prevention 19 (prophylaxis) and treatment against VEEV infection in mammals.
[0044] In vivo efficacy studies in mice have demonstrated that treatment with murine mAb 21 1A4A1 leads to protection of animals from a lethal peripheral challenge with virulent VEEV. Thus, 22 the present invention builds upon these findings by providing a humanized mAb 1A4A1 to reduce the 23 foreignness of murine mAb in humans. For doing this, the majority of the non-human protein 24 sequence (in one embodiment, more than 90%) of mAb 1A4A1 is replaced with a human Ab sequence and the resultant whole humanized mAb gene is then synthesized and cloned to an 26 expression vector such as an adenoviral vector. The recombinant adenoviral vector can be 27 delivered as a therapeutic agent for prophylaxis or treatment of VEEV
infection in humans. One 28 advantage of this method is that the vector can express the humanized Ab in the human body for a 29 long period of time. The humanized Ab can also be produced in cell culture and delivered directly as a therapeutic.
31 [0045] The humanization of the present anti-VEEV mAb 1A4A1 has not been done previously 32 and particularly not for the prophylaxis or treatment of VEEV infection.
The present invention 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 provides in one embodiment a humanized Ab, referred to herein as Hu1A4A1IgG1, that retains the 2 VEEV-binding specificity and neutralizing activity of murine 1A4A1 while not eliciting a HAMA
3 response. As described further below, the humanized Ab comprises an Ig framework of human 4 IgG1 and CDRs obtained from murine mAb 1A4A1. The rAb of the present invention is specific to an epitope of the E2 envelope glycoprotein of VEEV and, more specifically, to the E2c epitope 6 thereon.
7 [0046] The construction of the humanized Ab of the invention is schematically illustrated in 8 Figures 2a to 2d. Figure 2a illustrates schematically the structure of a murine Ab 16 containing 9 murine CDRs 18 on the respective variable regions. Figure 2b shows a human Ab 20 containing human CDRs 22. As shown in Figure 2c, a chimeric Ab 26 would comprise the murine variable 11 regions 24, containing the murine CDRs 18, joined to the constant regions of the human Ab. On the 12 other hand, Figure 2d illustrates a humanized Ab 28 according to an embodiment of the invention, 13 wherein only the murine CDRs 18 are grafted to the variable regions of the human Ab 20.
14 [0047] The substitution of the murine CDRs into the human Ig framework is illustrated also in Figures 3a to 3c. As shown, the humanized Ab variable region comprises the grafted CDRs, 18, 16 from the murine Ab.
17 [0048] The protein sequences of the rAbs of the invention include linker sequences. The 18 expressed rAbs of the invention have amino acid sequences as shown in SEQ ID NO:12 and SEQ
19 ID NO:14. The nucleic acid constructs used in transforming cells to express the above rAbs are shown in SEQ ID NO:11 and SEQ ID NO:13.
21 [0049] As illustrated further below, the humanized recombinant antibodies of the present 22 invention have been found to be effective as both a prophylaxis and a treatment against VEEV
23 infection.
24 [0050] Examples [0051] The following examples are provided to illustrate embodiments of the present invention.
26 The examples are not intended to limit the scope of the invention in any way.
27 [0052] Example 1: Construction of HulA4A11pG1 and in vitro studies 28 [0053] In the study described below, murine mAb 1A4A1 CDRs of VH, VL were grafted onto the 29 frameworks of germline variable and joining (V, J) gene segments of human Ig heavy and light chains, respectively, which were chosen based on the CDR similarities between human Igs and 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 murine mAb 1A4A1. Furthermore, the humanized VH and VL were, respectively, grafted onto 2 human gamma 1 heavy chain constant regions (CHs) and kappa 1 light chain constant region (CL) 3 to assemble the whole humanized Ab gene. The resultant whole humanized mAb gene was 4 synthesized and cloned to an adenoviral vector. After the humanized Ab was expressed in HEK 293 cells and purified with protein L column, the Ab was demonstrated to retain antigen-binding 6 specificity and neutralizing activity.
7 [0054] Materials and Methods 8 [0055] Humanization of murine mAb 1A4A1 9 [0056] Murine mAb 1A4A1 was provided by Dr. J.T. Roehrig (Division of Vector-borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Colins, CO, USA).
The VH and VL of 11 mAb 1A4A1 were cloned in a single chain variable fragment (ScFv) format, mA116 previously [7], 12 which showed to retain the same binding specificity as mAb 1A4A1 [11].
The humanization of VH
13 and VL of murine mAb 1A4A1 was done by Absalus Inc. (Mountain View, CA, USA). Briefly, in order 14 to select human VH and VL frameworks 1-3, the VH and VL amino acid sequences of murine 1A4A1 were separately subjected to IgBlast and IMGT searches against the entire human Ig germline V
16 gene segments and then human heavy and light chain germline V gene segments were selected 17 based on their highest CDR 1 and 2 similarities with those of murine 1A4A1 VH and VL without 18 consideration of framework similarity. Both human VH and VL framework 4 were selected, 19 respectively, from human heavy and light chain J gene segments based on the highest similarities between human J gene segments and murine 1A4A1 VH and VL CDR3. Finally, CDRs of murine 21 1A4A1 VH and VL were, respectively, grafted onto the frameworks of selected germline V and J
22 gene segments of human Ab heavy and light chains, resulting in humanized 1A4A1 (Hu1A4A1).
23 Furthermore, the Hu1A4A1 VH and VL were, respectively, grafted onto human gamma 1 heavy 24 chain CHs and kappa 1 light chain CL to assemble the whole humanized Ab gene, resulting in humanized 1A4A1IgG1 (Hu1A4A1IgG1). This process is illustrated in Figures 3 to 6.
26 [0057] Construction, expression and purification of Hu1A4A1IpG1 (Hu1A4A1loG1-furin and 27 Hu1A4A1IoG1-2A) 28 [0058] The Hu1A4A1IgG1 DNA sequence (-2 kb) is schematically illustrated in Figure 7. The 29 nucleic acid sequence of the Hu1A4A1IgG1-furin rAb is provided in SEQ ID
NO:11 and the nucleic acid sequence of the Hu1A4A1IgG1-2A rAb is provided in SEQ ID NO:13.

21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [0059] The Hu1A4A1IgG1 DNA sequences were synthesized as follows.
As shown in Figure 7, 2 a light chain leader sequence was provided upstream from the light chain, followed by a furin or 2A
3 linker (discussed further below) before the heavy chain. The whole DNA
sequence flanked by Kpn I
4 and Hind III was synthesized by GenScript Corporation (Scotch Plaines, NJ, USA) and cloned into pUC57 vector, resulting in pUC57-Hu1A4A1IgG1-furin or pUC57-Hu1A4A1IgG1-2A.
6 [0060] Recombinant adenovirus vectors expressing either Hu1A4A1IgG1-furin or 7 Hu1A4A1IgG1-2A were constructed using AdEasy TM system (Qbiogene, Carlsbad, CA, USA) 8 according to the manufacturer's protocol. Briefly, the Kpn I¨Hind III
fragment of Hu1A4A1IgG1-furin 9 or Hu1A4A1IgG1-2A was ligated to a Kpn I¨Hind III-digested pShuttle-CMV
vector. The resulting pShuttle construct was co-transformed with the pAdEasy-1 vector into Escherichia coli BJ5183 cells 11 to produce recombinant adenoviral genomic constructs for Hu1A4A1IgG1-furin or Hu1A4AlIgG1-2A
12 proteins. The recombinant adenoviral constructs, pAd-Hu1A4A1IgG1-furin and pAd-Hu1A4A1IgG1-13 2A were linearized with Pac I and transfected into HEK 293 cells (American Type Culture Collection, 14 Manassas, VA, USA) cultured in Dulbecco's Modified Eagle's Medium supplemented with 5% fetal bovine serum (FBS) for amplification and then the amplified adenovirus was purified by a 16 chromatographic method. This procedure is illustrated in Figure 8.
17 [0061] As illustrated in Figure 9, the expression of Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A was 18 achieved by first infecting HEK 293 cells with the recombinant adenovirus pAd-Hu1A4A1IgG1-furin 19 or pAd-Hu1A4A1IgG1-2A at a multiplicity of infection (M01) of 1. The infected cells were cultured for one week and the culture supernatant was harvested. The expressed Hu1A4A1IgG1-furin or 21 Hu1A4A1IgG1-2A was purified using protein L agarose gel from Pierce (Brockville, Ont., Canada).
22 Briefly, culture supernatant was dialyzed against phosphate buffer saline (PBS) (Sigma¨Aldrich, 23 Oakville, Ont., Canada) for 12h and then concentrated using PEG
(Sigma¨Aldrich) to less than 50 24 ml. The concentrated sample was incubated with 2m1 protein L agarose gel at 4 C for 1 h. The gel and supernatant mixture was then loaded to an empty column, which was subsequently washed with 26 binding buffer. Bound Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A was eluted with elution buffer. The 27 eluted Ab was further desalted using an excellulose column (Pierce) and then concentrated by a 28 Centracon TM YM-30 (Millipore Corp., Bedford, MA, USA).
29 [0062] The amino acid sequence of the expressed Hu1A4A1IgG1-furin is shown in SEQ ID
NO:12 and the amino acid sequence of the expressed Hu1A4A1IgG1-2A is shown in SEQ ID NO:14.
31 [0063] Cells that were transformed to express the Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A
32 humanized antibodies have been deposited at the International Depositary Authority of Canada 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 (IDAC) (National Microbiology Laboratory, Winnipeg, Manitoba, Canada) under accession numbers 2 141107-01 and 141107-02, respectively.
3 [0064] SDS-PAGE
4 [0065] Abs were separated by 10% SDS-PAGE gels using a Mini-PROTEANTm II apparatus (Bio-Rad Laboratories, Mississauga, Ont., Canada). The bands were visualized by SimplyBlue TM
6 safestain staining (Invitrogen, Burlington, Ont., Canada). The molecular weights of the samples 7 were estimated by comparison to the relative mobility values of standards of known molecular 8 weights. The SDS-PAGE analyses of the purified Hu1A4A1IgG1-furin are illustrated in Figures 10 9 and 11. Figure 12 illustrates the SDS-PAGE analysis of the purified Hu1A4A1IgG1-2A. As shown, lanes 1 and 3 correspond to purified Hu1A4A1IgG1 and control human IgG1 in a non-reducing 11 condition and lanes 2 and 4 correspond to purified Hu1A4A1IgG1 and control human IgG1 in a 12 reducing condition.
13 [0066] ELISA
14 [0067] The reactivity of purified Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A to VEEV E2 antigen was determined by ELISA. Nunc Maxisorp7m flat bottomed 96-well plates (Canadian Life 16 Technologies, Burlington, Ont., Canada) were coated overnight at 4 C
with recombinant VEEV E2 17 antigen at a concentration of 10pg/m1 in carbonate bicarbonate buffer, pH 9.6. The plates were 18 washed five times with PBS containing 0.1% TweenTm-20 (PBST) and then blocked in 2% bovine 19 serum albumin for 2h at room temperature. After five washes with PBST, the plates were incubated for 2h at room temperature with various concentrations of Hu1A4A1IgG1-furin, Hu1A4A1IgG1-2A or 21 1A4A1 Abs diluted in PBST. Following five washes with PBST, the plates were incubated for 2h at 22 room temperature with horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG fragment 23 crystallizable portion or HRP-conjugated rabbit anti-mouse IgG (Jackson ImmunoResearch 24 Laboratories Inc., West Grove, PA, USA) diluted 1:5000 in PBST. Finally, the plates were washed five times with PBST and developed for 10 min at room temperature with a 3,3',5,5'-26 tetramethylbenzidine substrate (Kirkegaard and Perry Laboratories). The reactions were read at an 27 absorbance of 650 nm by a microplate autoreader (Molecular Devices, Sunnyvale, CA, USA). The 28 results of the ELISA Hu1A4A1IgG1-antigen binding assay are illustrated in Figure 13.
29 [0068] Neutralization assay in vitro [0069] Neutralizing activity of each of Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A against VEEV
31 (strain TC-83) was analyzed by a plaque reduction assay. Briefly, each Ab was serially two-fold 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 diluted (1:32, 1:64, 1:128, etc.) and mixed with an equal volume containing 50 plaque-forming units 2 of virus per 100 pl. Afterwards, the mixtures were incubated for 1 h at room temperature, 200 pl of 3 the mixture was inoculated in duplicate into wells of six-well plates containing confluent Vero cell 4 monolayers and incubated at 37 C for 1 h. At the end of the incubation, the virus/Ab mixtures were removed from the wells before the wells were overlaid by tragacanth gum and then incubated for 2 6 days. The wells were stained with 0.3% crystal violet and plaques were counted. Neutralization titre 7 was expressed as the highest Ab dilution that inhibited 50% of virus plaques. This procedure is 8 illustrated in Figure 15.
9 [0070] Results and Discussion [0071] Different approaches have been developed to humanize murine Abs in order to reduce 11 the antigenicity of murine Abs in humans [9,10]. One widely used approach is CDR-grafting, which 12 involves the grafting of all murine CDRs onto a human Ab frameworks. The human Ab frameworks 13 are chosen based on their similarities to the frameworks of the murine Ab to be humanized. The 14 CDR-grafting approach has been proven successful in some cases. However, in many more instances, this humanization process could result in CDR conformation changes, which affect the 16 antigen-binding affinity. To restore the affinity, additional work for back-mutation of several murine 17 framework amino acids, which are deemed to be critical for CDR loop conformation, have to be 18 done.
19 [0072] Recently, Hwang et al. [12] employed an approach which consisted of grafting CDRs onto human germline Ab frameworks based on the CDR sequence similarities between the murine 21 and human Abs while basically ignoring the frameworks. Because the selection of the human 22 frameworks is driven by the sequence of the CDRs, this strategy minimizes the differences between 23 the murine and human CDRs. This approach has the potential to generate humanized Abs that 24 retain their binding affinity to their cognate antigen. Further, since all residues in frameworks are from human Ab germline sequences, the potential immunogenicity of non-human Abs is highly 26 reduced.
27 [0073] Using the above approach, and as disclosed herein, the present inventors humanized an 28 anti-VEEV murine mAb 1A4A1. The amino acid sequences of VH and VL from murine 1A4A1 were 29 first aligned with human Ig germline V and J genes. As shown in Figure 5, the human heavy chain V
gene segment H5-51 and J gene segment JH4 were selected to provide the frameworks for the 31 murine 1A4A1 VH. Similarly, as shown in Figure 6, for the murine 1A4A1 VL, the human light chain 32 V gene segment L15 and J gene segment Jk3 were selected.

21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [0074] The identities of the CDR1 and CDR2 amino acid sequences between murine 1A4A1 VH
2 and the human H5-51 gene segment were 20% and 47%, respectively, while the identity of the 3 CDR3 between murine 1A4A1 VH and the JH4 gene segment was 33%. For the light chain, the 4 identities of the CDR1 and CDR2 between murine 1A4A1 VL and the human L15 gene segment were 27% and 14%, respectively, while the identity of the CDR3 between murine 1A4A1 VL and 6 human Jk3 gene segment was 22%. The CDRs of murine 1A4A1 VH were then grafted onto the 7 frameworks of selected human Ig germline H5-51 and JH4 gene segments, while the CDRs of 8 murine 1A4A1 VL were grafted onto human L15 and Jk3 gene segments. The hu1A4A1 VH was 9 further grafted onto the human gamma 1 heavy chain CHs to form a complete heavy chain, while the VL was grafted onto the human kappa 1 light chain CL to form a whole humanized light chain. This 11 procedure is schematically illustrated in Figures 5 and 6 with the end structure being illustrated in 12 Figure 7.
13 [0075] As shown in Figure 5, the murine 1A4A1 VH CDRs grafted onto the human framework 14 comprised the following amino acid sequences:
VH CDR1: DYHVH (SEQ ID NO: 1) 16 VH CDR2: MTYPGFDNTNYSETFKG (SEQ ID NO: 2) 17 VH CDR3: GVGLDY (SEQ ID NO: 3) 18 [0076] As shown in Figure 6, the murine 1A4A1 VL CDRs grafted onto the human framework 19 comprised the following amino acid sequences:
VL CDR1: KASQDVDTAVG (SEQ ID NO: 4) 21 VL CDR2: WSSTRHT (SEQ ID NO: 5) 22 VL CDR3: HQYSSYPFT (SEQ ID NO: 6) 23 [0077] As shown in Figure 5, the VH of the humanized Ab according to the present invention 24 comprises the following amino acid sequence:
Hu-VH:

27 KGQVTISADKSISTAYLQWSSLKASDTAMYYCARGVGLDYWGQGTLVTVSS (SEQ ID NO: 7).

21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [0078] Thus, as shown in Figure 6, the VL of the humanized Ab according to the present 2 invention comprises the following amino acid sequence:
3 Hu-VL:

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYSSYPFTFGPGTKVDIKR (SEQ ID NO: 8).
6 [0079] In order to express heavy and light chains in a monocistronic construct, a six-residue 7 peptide, RGRKRR (SEQ ID NO: 9) containing the recognition site for the protease furin, designated 8 as "furin linker", or a twenty-four-residue peptide of the foot-and-mouth-disease virus (FMDV)-9 derived 2A self-processing sequence, APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:

10), designated as "2A linker", was incorporated between the two chains. The location of the furin or 2A

11 linker within the nucleic acid constructs of the Abs is illustrated in Figure 7. Furin is a ubiquitous 12 subtilisin-like proprotein convertase, which is the major processing enzyme of the secretory pathway 13 [13]. The furin minimal cleavage site is R¨X¨X¨R; however, the enzyme prefers the site R¨X-14 (K/R)¨R. An additional R at the P6 position appears to enhance cleavage.
The FMDV-derived 2A
linker is able to cleave at its own C terminus between the last two residues through an enzyme-16 independent but undefined mechanism, probably by ribosomal skip, during protein translation. To 17 get the expressed Ab to be secreted to culture media, a leader sequence was added upstream to 18 the Ab gene. Figure 7 illustrates the synthesized DNA sequence, of approximately 2 kb, including 19 the human Ab kappa light chain L15 leader sequence, the humanized light chain (VL + CL), the furin or 2A linker, and the humanized heavy chain (VH + CHI + CH2 + CH3). This sequence was then 21 cloned into an adenoviral vector. The unique restriction sites, as also shown in Figure 7, flanking the 22 V regions, which allow for efficient V region replacement and at the heavy chain V¨C region junction 23 for generation of fragment antigen-binding portion of Ab (Fab), were also designed.
24 [0080] Protein G and A columns are widely used for a quick purification for Abs because of protein G and A binding to the Fc portion of lg. However, protein G and A
cannot only bind to 26 human lg, but also bind to bovine lg, therefore they cannot be used for purification of Hu1A4A1IgG1-27 furin or Hu1A4A1IgG1-2A in our study since pAd-Hu1A4A1IgG1-furin or pAd-Hu1A4A1IgG1-2A-28 infected HEK 293 cells were cultured in the medium with 5% FBS
containing a high percentage of 29 bovine lg. Unlike protein G and A, protein L binds lg through interactions with the light chains.
Protein L only binds to lg containing light chains of type kappa 1, 3 and 4 in human and kappa 1 in 31 mouse. Most importantly, protein L does not bind to bovine lg. Since our humanized Ab has human 32 kappa 1 chain, we chose a protein L column to purify Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A to 21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 eliminate co-purification of bovine lg. In this way, the purity of Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2 2A was relatively high in SDS-PAGE as shown in Figures 10, 11 and 12.
3 [0081] When the purified product was subjected to 10% SDS-PAGE, Hu1A4A1IgG1-furin and 4 Hu1A4A1IgG1-2A showed up in a different way. As illustrated in Figure 12, Hu1A4A1IgG1-2A
showed the same patterns as a control human IgG1, one band of ¨150kDa in non-reducing condition 6 (intact disulfide bridges) and two bands, 50kDa for heavy chains and 25kDa for light chains (broken 7 disulfide bridges) in reducing condition, indicating that the 2A linker underwent self-processing 8 perfectly. On the other hand, Hu1A4A1IgG1-furin showed only one clear band of ¨75kDa in 9 reducing condition observed as illustrated in Figures 10 and 11, indicating that the furin linker was not cleaved. However, in another study (data not shown), the same furin linker sequence was 11 cleaved in another Fab construct expressed in a mammalian system. This indicated the 12 conformation of expressed Hu1A4A1IgG1-furin probably rendered the furin linker inaccessible to 13 furin or that the sequence surrounding the furin linker influenced furin cleavage.
14 [0082] The specific binding reactivities of purified Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A to VEEV E2 antigen were examined by ELISA. As illustrated in Figure 13, both versions of the 16 Hu1A4A1IgG1 were found to bind to VEEV E2 in a dose-dependent manner, similar to the binding to 17 VEEV E2 of its parental murine 1A4A1, indicating this non-cleaved Ab was still reactive to VEEV E2 18 antigen in ELISA. Furthermore, both versions were evaluated for their ability to block VEEV infection 19 in Vero cells using a standard plaque-reduction assay. The Hu1A4A1IgG1-fruin showed a neutralizing activity with 50% plaque reduction neutralization titre at 0.78 pg/ml, whereas 21 Hu1A4A1IgG1-2A showed a much higher neutralization titre at 0.1 pg/ml.
22 [0083] From the above results, it is concluded that the murine 1A4A1 Ab was successfully 23 humanized. As illustrated in Figure 14, the expressed and purified Ab of Hu1A4A1IgG1-2A was 24 cleaved between the heavy and light chains as expected; however, Hu1A4A1IgG1-furin was not cleaved. Nevertheless, the present inventors have exhibited that both versions of the Hu1A4A1IgG1 26 retained the antigen binding specificity and virus neutralizing activity. Thus, the Hu1A4A1IgG1-furin 27 or Hu1A4A1IgG1-2A discussed and characterized herein would serve as an effective prophylactic 28 and therapeutic agent against VEEV infection.
21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [0084] Example 2: In vivo study - Protection or pre-exposure prophylaxis of mice from 2 VEEV challenge by passive immunization with Hu1A4A110G1-furin or Hu1A4A1IgG1-2A
3 [0085] Materials and methods 4 [0086] Passive immunization (Pre-exposure prophylaxis) [0087] Balb/c mice aged 6-8 weeks were injected intraperitoneally (i.p) with 50 pg of 6 Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A in 100 pl PBS, human IgG in 100 pl PBS (positive control) 7 or 100 pl PBS alone (negative control) 24 h prior to VEEV challenge.
8 [0088] VEEV challenge 9 [0089] Each mouse was challenged subcutaneously (s.c.) with 30-50 plaque forming units (pfu) of virulent VEEV (Trinidad donkey, TRD) in 50 pl of Leibovitz L15 maintenance medium (L15MM) 24 11 h after passive immunization. The challenge dose approximated to 100 x 50% lethal dose (LD50).
12 Mice were examined frequently for signs of illness for 14 days, and humane endpoints were used.
13 [0090] Results 14 [0091] Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A clearance in mice [0092] To determine the half-life of Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A in mouse serum, 16 groups of 4 mice, were injected i.p. with 50 pg, each mouse, of either Hu1A4A1IgG1-furin or 17 Hu1A4A1IgG1-2A, or human anti-VEEV IgG and bled from the vein at increasing time intervals after 18 injection. The quantity of Ab present in serum samples was estimated by immunoassay.
19 Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A had a similar half-life as human anti-VEEV IgG, around 10 days.
21 [0093] Protection of mice from VEEV challenge bv passive immunization with Hu1A4A1IgG1-22 furin or HulA4AllaG1 -2A
23 [0094] Groups of 8 mice were injected i.p. with the Hu1A4A1IgG1-furin, Hu1A4A1IgG1-2A, 24 human IgG (positive control) or PBS alone (negative control) and 24h later challenged s.c. with 100 x LD50 of VEEV. None of the mice treated only with human IgG (positive control) or PBS alone 26 (negative control) survived. All the Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A
treated mice survived 27 the VEEV challenge without any clinical signs at 14 days post-challenge.

21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [0095] Discussion 2 [0096] Passive immunization of the Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A in mice (50 3 pg/mouse) 24 h before virulent VEEV challenge provided 100% protection against 100xLD50 4 challenge of VEEV when mice were treated with 50 pg/each mouse of Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A. The mice were also found to be asymptomatic throughout the 14 day 6 observation period. These results indicate that the humanized anti-VEEV
rAbs of the present 7 invention have pre-exposure prophylactic capacity against VEEV
infections. The half-lives of the 8 humanized anti-VEEV rAbs in mice was around 10 days suggesting that the humanized anti-VEEV
9 rAbs of the invention would be an effective prophylactic against VEEV for at least several weeks.
Thus, the rAbs of the invention have been demonstrated to have functionality as an immunization 11 agent against VEEV infection.
12 [0097] Example 3: In vivo study - Treatment or post-exposure therapy of mice after VEEV
13 challenae by passive immunization with Hu1A4A1IaG1-furin or Hu1A4A1laG1-14 [0098] Materials and methods [0099] Post-exposure Therapy 16 [00100] Balb/c mice aged 6-8 weeks were challenged s.c. with 100 x LD50 of virulent VEEV in 17 50 pl of L15MM per mouse. At 24 h post-challenge, mice were injected i.p with 50 pg of 18 Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A in 100 pl PBS, or 100 pl PBS alone.
Mice were examined 19 frequently for signs of illness for 20 days, and humane endpoints were used.
[00101] Results 21 [00102] The half-lives of Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A was determined above in 22 Example 2.
23 [00103] Treatment of mice after VEEV challenge using Hu1A4A1loG1-furin or Hu1A4A1loG1-2A
24 [00104] Groups of 8 mice were challenged s.c. with 100 x LD50 of VEEV.
Twenty-four hours later, the infected mice were administered i.p. with Hu1A4A1IgG1.-furin, Hu1A4A1IgG1-2A or PBS
26 alone (50 ug/mouse). All the Hu1A4A1IgG1-2A-treated mice survived throughout the observation 27 period (20 days post-challenge) with minor clinical signs. All Hu1A4A1IgG1-furin or PBS-treated 28 mice died.

21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [00105] Discussion 2 [00106] Passive immunization of the Hu1A4A1IgG1-2A in mice (50 pg/mouse) 24 h after virulent 3 VEEV challenge provided 100% protection against 100xLD50 challenge of VEEV with only minor 4 clinical signs, indicating the Hu1A4A1IgG1-2A has post-exposure therapeutic capacity against VEEV
infections. Unfortunately, Hu1A4A1IgG1-furin did not show any post-exposure therapy capacity.
6 One possible reason for this finding may be that the antigen binding capacity of uncut 7 Hu1A4A1IgG1-furin is inferior to the cleaved Hu1A4A1IgG1-2A.

9 [00107] Bibliography [00108] One or more of the following documents have been referred to in the present disclosure.
11 [00109] [1] Weaver SC, Ferro C, Barrera R, Boshell J, Navarro JC.
Venezuelan equine 12 encephalitis. Annu Rev Entomol, 2004;49:141-74.
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[00111] [3] Pittman PR, Makuch RS, Mangiafico JA, Cannon TL, Gibbs PH, Peters CJ. Long-term 16 duration of detectable neutralizing antibodies after administration of live-attenuated VEE vaccine and 17 following booster vaccination with inactivated VEE vaccine. Vaccine, 1996; 14:337-43.
18 [00112] [4] Jahrling PB, Stephenson EH. Protective efficacies of live attenuated and 19 formaldehyde-inactivated Venezuelan equine encephalitis virus vaccines against aerosol challenge in hamsters. J Clin Microbiol, 1984; 19:429-31.
21 [00113] [5] France JK, Wyrick BC, Trent DW. Biochemical and antigenic comparison of the 22 envelope glycoproteins of Venezuelan equine encephalomyelitis virus strains. J Gen Virol, 1979;
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21989275.2 CA 2,704,119 Blakes Ref: 67853100022 1 [00115] [7] Roehrig JT, Mathews JH. The neutralization site on the E2 glycoprotein of 2 Venezuelan equine encephalomyelitis (TC-83) virus is composed of multiple conformationally stable 3 epitopes. Virology, 1985; 142:347-56.
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16 [00121] [13] van den Ouweland AM, van Duijnhoven HL, Keizer GD, Dorssers LC, Van de Ven 17 WJ. Structural homology between the human fur gene product and the subtilisin-like protease 18 encoded by yeast KEX2. Nucleic Acids Res, 1990; 18:664.
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21989275.2 CA 2,704,119 Blakes Ref: 67853/00022 1 [00126] [18] Hu WG, Chau D, Wu J, Jager S, Nagata L, Humanization and mammalian 2 expression of murine monoclonal antibody against Venezuelan equine encephalitis virus. Vaccine, 3 2007; 25:3210-3214.

[00127] Although the invention has been described with reference to certain specific 6 embodiments, various modifications thereof will be apparent to those skilled in the art without 7 departing from the scope of the invention as outlined in the claims appended hereto. Any examples 8 provided herein are included solely for the purpose of illustrating the invention and are not intended 9 to limit the invention in any way. Any drawings provided herein are solely for the purpose of illustrating various aspects of the invention and are not intended to be drawn to scale or to limit the 11 invention in any way.

21989275.2

Claims (12)

1. A humanized recombinant antibody for use in the prophylaxis or treatment against infection from Venezuelan equine encephalitis virus, VEEV, in a mammal, - wherein said antibody comprises a human Ig framework having grafted thereon complementarity determining regions, CDRs, from the murine monoclonal antibody 1A4A1;
- and wherein said antibody is produced by expressing a protein encoded by a nucleic acid construct having a nucleic acid sequence according to SEQ ID NO: 13.

2. The humanized recombinant antibody for use according to claim 1, wherein said antibody has an amino acid sequence according to SEQ ID NO: 14.

3. The humanized recombinant antibody for use according to claim 1 or claim 2, wherein said antibody is encoded by an expression vector.

4. The humanized recombinant antibody for use according to claim 1 or claim 2, wherein said antibody is expressed by an isolated transformed host cell.

5. A use of a humanized recombinant antibody in inhibiting Venezuelan equine encephalitis virus (VEEV) infection in a human, the humanized recombinant antibody comprising a human Ig framework and having grafted thereon complementarity determining regions, CDRs, from the murine monoclonal antibody 1A4A1, wherein:
- the antibody is capable of inhibiting VEEV infection when administered prior to or after VEEV exposure; and, - the antibody is encoded by the nucleic acid sequence according to SEQ ID NO:
13.

6. The use according to claim 5, wherein said antibody has a VH region having the amino acid sequence according to SEQ ID NO: 7.

7. The use according to claim 5, wherein said antibody has a VL region having the amino acid sequence according to SEQ ID NO: 8.

8. The use according to claim 5, wherein said antibody has the amino acid sequence according to SEQ ID NO: 14.

9. The use according to any one of claims 5 to 8, wherein said antibody is encoded by an expression vector.

10. The use according to any one of claims 5 to 9, wherein said antibody is expressed by an isolated transformed host cell.

11. A use of a humanized recombinant antibody in the passive transfer of immunity, to a human, against Venezuelan equine encephalitis virus (VEEV) infection, after exposure to VEEV, the humanized recombinant antibody comprising a human Ig framework and having grafted thereon complementarity determining regions, CDRs, from the murine monoclonal antibody 1A4A1, wherein the antibody is encoded by the nucleic acid sequence according to SEQ ID NO:
13.

12. The use according to claim 11 wherein the antibody has the amino acid sequence according to SEQ ID NO: 14.