OA20886A - Antibodies binding to plasmodium circumsporozoite protein and uses thereof. - Google Patents

Antibodies binding to plasmodium circumsporozoite protein and uses thereof. Download PDF

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OA20886A
OA20886A OA1202100486 OA20886A OA 20886 A OA20886 A OA 20886A OA 1202100486 OA1202100486 OA 1202100486 OA 20886 A OA20886 A OA 20886A
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seq
antibody
amino acid
acid sequence
chain variable
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OA1202100486
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Davide Corti
Luca PICCOLI
Katja Fink
Elisabetta CAMERONI
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Humabs Biomed Sa
Vir Biotechnology, Inc.
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Publication of OA20886A publication Critical patent/OA20886A/en

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Abstract

The present invention provides antibodies targeting Plasmodium sporozoites, in particular Plasmodium circumsporozoite protein. The invention also provides nucleic acids that encode such antibodies. In addition, the invention provides the use of the antibodies of the invention in prophylaxis and treatment malaria.

Description

ANTIBODIES BINDING TO PLASMODIUM CIRCUMSPOROZOITE PROTEIN AND USES THEREOF
The présent invention relates to the field of malaria médication, in particular to malaria vaccination and to antibodies binding to plasmodium sporozoites, in particular to plasmodium circumsporozoite protein.
Malaria is one of the most severe public health problems worldwide. Malaria is caused by parasitic protozoans of the genus Plasmodium, The genus Plasmodium includes about 200 species with P. falciparum, P. vivax, P. ovale, and P. malariae together accounting for nearly ail human infections with Plasmodium species. Among those Plasmodium species, P. falciparum accounts for the overwhelming majority of malaria deaths. Malaria symptoms typîcally include fever, feeling tired, vomiting, and headaches. In severe cases it can cause yellow skin, seizures, coma, or death.
Malaria is a mosquito-borne disease, which is most commonly transmitted b y an infected female Anophèles mosquito. For example, during Plasmodium falciparum infection, the female Anopheles mosquito injects a small number of sporozoites (-10-100) into the skin of a vertebrate host, after which they travel to the liver to invade hépatocytes (Crompton et al. (2014) Annu Rev Immunol 32, 157-187). In hépatocytes the sporozoites reproduce asexually (tîssue schizogony) and mature into schizonts, which rupture to release merozoites. Merozoîtes infect red blood cells, ring stage trophozoites mature into schizonts, which rupture releasing merozoites. Other merozoites develop into sexual erythrocytic stages (gamétocytes). When a mosquito bites an infected vertebrate host, gamétocytes are taken up with the blood and mature in the mosquito gut. The male and female gamétocytes fuse and form an ookinete — a fertilized, motile zygote. Ookinetes develop into new sporozoites that migrate to the insect’s salivary glands to infect a new vertebrate host.
Malaria symptoms are caused by blood stage parasites. In contrast, sporozoites are not associated with clinical symptoms, however, in sporozoite and liver stages of the life cycle of Plasmodium parasite numbers in the host are low and their éradication can completely abrogate infection. Accordingly, the sporozoite and liver stages of the P. falciparum parasite represent key targets of current malaria médication candidates, as a médication that successfully protects against these stages would be able to prevent both malaria infection and transmission. Therefore, subunit vaccines based on circumsporozoite protein (CSP), such as RTS,S, are at the center of the malaria vaccine effort.
The Plasmodium circumsporozoite protein (CSP) is a secreted protein of the sporozoite stage of Plasmodium. CSP forms a dense coat on the surface of the parasite and has been hypothesized to médiate many of the initial interactions between the sporozoite and its two hosts (Ménard R., 2000, Microbes Infect. 2:633-642; Sinnis P. and Nardin E., 2002, Sporozoite antigens: biology and immunology of the circumsporozoite protein and thrombospondin related anonymous protein. In Malaria Immunology. P. Perhnann and M. Troye-Blomberg, editors. S. Karger AG, Basel, Switzerland. 70-96). The structure and fonction of CSP is highly conserved across the various strains of Plasmodium that infect humans, non-human primates and rodents. The aminoacid sequence of CSP comprises an immunodominant central repeat région, that is diverse across Plasmodium species (NANP-repeat région in case of P. falciparum). Flanking the repeats are two conserved motifs at the N- and C- termini, namely région l, a 5-aa sequence at the N terminus of the repeats, and a known cell-adhesive motif C-terminal to the repeats termed the type I thrombospondin repeat (TSR). Those conserved motifs are implicated in protein Processing as the parasite travels from the mosquito to the mammalian vector.
CSP is known to play a crucial rôle in the migration of the sporozoites from the midgut walls of infected mosquitoes to the mosquito salîvary glands. Additionally, CSP is involved in hépatocyte binding in the mammalian host with the N-terminus and central repeat région of CSP initîally facilîtate parasite binding. On the hépatocyte surface proteolytic cleavage at région 1 of the Nterminus exposes the adhesive domain of the C-terminus, thereby priming the parasites for invasion of the liver (Coppî et al. (2005) JExp Med 201, 27-33).
At présent, the most advanced malaria vaccine candidate is RTS,S (RTS,S/AS01; trade name Mosquirix), which is a recombinant protein-based malaria vaccine. RTS,S is a hybrid protein particle, formulated in a multi-component adjuvant named AS01. The RTS,S vaccine antigen consists of 19 NANP amino acid repeat units followed by the complété C-terminal domain minus the GPI anchor of the CS antigen, fosed to the Hepatitis B virus S protein. Multisite clinical trials in sub-Saharan Africa hâve shown that RTS,S confers modest and short-lived protection against clinical malaria.
Another factor that has complicated the development of malaria médications is the difficulty in identifyîng robust correlates of protection. Antibodies hâve been shown to inhibit sporozoite invasion of hépatocytes in in vitro functional assays, but their rôle in the protection of malaria-vaccinated individuals remains unclear.
Recently, however, very potent anti-malaria antibodies were described, which are spécifie for Plasmodium falciparum circumsporozoite protein (CSP) (Tan J, Sack BK, Oyen D, et al. A public antibody lineage that potently inhibits malaria infection through dual binding to the circumsporozoite protein. Nat Med. 2018;24(4):401 -407. doi:10.1038/nm.4513). The authors of this study showed that the most potent antibodies - including antibodies “MGU10” and “MGH2” - simultaneously target epitopes in (i) the NANP-repeat région of CSP and (ii) an N-terminal région of CSP covering the junction between the N-terminai domain and the NANP-repeats. Moreover, this study showed that the extreme potency of those antibodies was due to their dual specificity, while antibodies targeting only one of the CSP epitopes were typically less potent.
In view of the above, it is the object of the présent invention to overcome the drawbacks of prior art outlined above. In particular, it is the object of the présent invention to provide anti-malaria antibodies, which bind to both, (i) the NANP-repeat région of CSP and (ii) an N-terminal région of CSP covering the junction between the N-terminal domain and the NANP-repeats, with high affmity.
This object is achieved by means of the subject-matter set out below and in the appended daims.
Although the présent invention is described in detail below, it is to be understood that this invention is not limited to the particular méthodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is not intended to limit the scope of the présent invention which will be limited only by the appended daims. Unless defined otherwise, ail technical and scientific ternis used herein hâve the same meanings as commonly understood by one of ordinary skill in the art.
In the following, the éléments of the présent invention will be described. These éléments are listed with spécifie embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variousiy described examples and embodiments should not be construed to limit the présent invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed éléments. Furthermore, any permutations and combinations of ail described éléments in this application should be considered disclosed by the description of the présent application unless the context îndicates otherwise.
Throughout this spécification and the daims which follow, unless the context requires otherwise, the term comprise, and variations such as comprises and comprising, will be understood to impi y the inclusion of a stated member, integer or step but not the exclusion of any other nonstated member, integer or step. The tenu consist of is a particular embodiment of the tenu comprise, wherein any other non-stated member, integer or step is excluded. In the context of the présent invention, the tenu comprise encompasses the tenu consist of. The term “comprising” thus encompasses “încluding” as well as “consisting” e.g., a composition “comprising” X may consist exclusively of X or may include something additional e.g., X + Y.
The tenus a and an and the and similar reference used in the context of describing the invention (especially in the context of the daims) are to be construed to cover both the singular and the plural, unless otherwise indîcated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indîcated herein, each individual value is incorporated into the spécification as if it were individually recited herein. No language in the spécification should be construed as indicating any non-datmed element essential to the practice of the invention.
The word “substantially” does not exclude “completel y” e.g., a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the définition of the invention.
The term “about” in relation to a numerical value x means x ± 10%, for example, x ± 5%, or x ± 7%, or x ± 10%, or x ± 12%, or x ± 15%, or x ± 20%.
The term “disease” as used herein is intended to be generally synonymous, and is used interchangeably with, the ternis “disorder” and “condition” (as in medical condition), in that ail reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to hâve a reduced duration or quality of life.
As used herein, reference to “treatment” of a subject or patient is intended to include prévention, prophylaxis, atténuation, amelioration and therapy. The terms “subject” or “patient” are used interchangeably herein to mean ail mammais including humans. Examples of subjects include humans, cows, dogs, cats, horses, goats, sheep, pigs, and rabbits. In some embodiments, the patient is a human.
Doses are often expressed in relation to the bodyweight. Thus, a dose which is expressed as [g, mg, or other unit]/kg (or g, mg etc.) usually refers to [g, mg, or other unit] “per kg (or g, mg etc.) bodyweight”, even if the tenn “bodyweight” is not explicitly mentioned.
The tenn “binding” and similar reference usually means “specifically bindîng”, which does not encompass non-speciftc sticking.
As used herein, the terni “antibody” encompasses varions fonns of antibodies including, without being limited to, whole antibodies, antibody fragments (such as antigen binding fragments), human antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies and genetically engineered antibodies (variant or mutant antibodies) as long as the characteristic properties according to the invention are retained. In some embodiments, the antibody is a human antibody. Tn some embodiments, the antibody is a mono clonal antibody. For example, the antibody is a human monoclonal antibody.
As described above, the term “antibody” generally also includes antibody fragments. Fragments of the antibodies may retain the antigen-binding activity of the antibodies. Such fragments are referred to as “antigen-binding fragments”. Antigen-binding fragments include, but are not limited to, single chain antibodies, Fab, Fab’, F(ab')2, Fv or scFv. Fragments of the antibodies can be obtained from the antibodies by methods that include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by Chemical réduction. Altematively, fragments of the antibodies can be obtained by recombinant means, for example by cloning and ex pressing a part (fragment) of the sequences of the heavy and/or light chain. The invention also encompasses sîngle-chain Fv fragments (scFv) derived from the heavy and light chains of an antibody of the invention. For example, the invention includes a scFv comprising the CDRs from an antibody of the invention. Also included are heavy or light chain monomers and dimers, single domain heavy chain antibodies, single domain light chain antibodies, as well as single chain antibodies, e.g., single chain Fv in which the heavy and light chain variable domains are joined by a peptide linker. Antibody fragments of the invention may be contained in a variety of structures known to the person skilled in the art. In addition, the sequences of the invention may be a comportent of multispecific molécules in which the sequences of the invention target the epitopes of the invention and other régions of the molécule bind to other targets. Although the spécification, including the daims, may, in some places, refer expli ci tly to antigen binding fragment(s), antibody fragment(s), varîant(s) and/or derivative(s) of antibodies, it is understood that the tenu “antibody” includes ail categories of antibodies, namely, antigen binding fragment(s), antibody fragment(s), variant(s) and derivative(s) of antibodies.
Human antibodies are wdl-known in the State of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animais (e.g., mi ce) that are capable, upon immunizatîon, of producing a full repertoîre or a sélection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human genn-line immunoglobulin gene array in such germ-line mutant mice will resuit in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc. NatL Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year Immunol. 7 (1993) 3340). Human antibodies can also be produced in phage display libraries (Hoogenboom, H. R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J. D., et al., J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole et al. and Boemer et al. are also available for the préparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boemer, P., et al., J. Immunol. 147 (1991) 86-95). In some embodiments, human monoclonal antibodies are prepared by using improved EBV-B cell immortalization as described in Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR, Murphy BR, Rappuoli R, Lanzavecchia A. (2004): An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med. 10(8):871-5. As used herein, the tenu “variable région” (variable région of a light chain (VL), variable région of a heavy chain (VH)) dénotés each of the pair of light and heavy chains which is involved directly in binding the antibody to the antigen.
Antibodies of the invention can be of any isotype (e.g., IgA, IgG, IgM i.e. an α, γ or μ heavy chain). For example, the antibody is of the IgG type. Withîn the IgG isotype, antibodies may be IgGl, IgG2, IgG3 or IgG4 subclass, for example IgGl. Antibodies of the invention may hâve a k or a λ light chain. In some embodiments, the antibody is of IgGl type and has a κ light chain.
Antibodies according to the présent invention may be provided in purified fonn. Typically, the antibody will be présent in a composition that is substantially free of other polypeptides e.g., where less than 90% (by weight), usually less than 60% and more usually less than 50% of the composition is made up of other polypeptides.
Antibodies according to the présent invention may be immunogenic in human and/or in non-human (or heterologous) hosts e.g., in mice. For example, the antibodies may hâve an idiotope that is immunogenic in non-human hosts, but not in a human host. Antibodies of the invention for human use include those that cannot be easily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, etc. and cannot generally be obtained by humanization or from xeno-mice.
As used herein, a “neutralizing antibody” is one that can neutralize, i.e., prevent, inhibit, reduce, impede or interfère with, the ability of a pathogen to initiate and/or perpetuate an infection in a host. The terms “neutralizing antibody” and “an antibody that neutralizes” or “antibodies that neutralize” are used înterchangeably herein. These antibodies can be used alone, or in combination, as prophylactic or tberapeutic agents upon appropriate formulation, in association with active vaccination, as a diagnostic tool, or as a production tool as described herein.
As used herein, the term “mutation” relates to a change in the nucleic acid sequence and/or in the amîno acid sequence in comparison to a reference sequence, e.g. a corresponding genomic sequence. A mutation, e.g. in comparison to a genomic sequence, may be, for example, a (naturally occurring) somatic mutation, a spontaneous mutation, an induced mutation, e.g. induced by enzymes, Chemicals or radiation, or a mutation obtained by site-directed mutagenesis (molecular biology methods for making spécifie and intentional changes in the nucleic acid sequence and/or in the amino acid sequence). Thus, the terms “mutation” or “mutating” shall be understood to also include physically making a mutation, e.g. in a nucleic acid sequence or in an amîno acid sequence. A mutation includes substitution, délétion and insertion of one or more nucleotîdes or amîno acids as well as inversion of several successive nucléotides or amino acids. To achieve a mutation in an amino acid sequence, a mutation may be introduced into the nucléotide sequence encoding said amino acid sequence in order to express a (recombinant) mutated polypeptide. A mutation may be achieved e.g., by altering, e.g., by site-directed mutagenesis, a codon of a nucleic acid molécule encoding one amino acid to resuit in a codon encoding a different amino acid, or by synthesizing a sequence variant, e.g., by knowing the nucléotide sequence of a nucleic acid molécule encoding a polypeptide and by designing the synthesis of a nucleic acid molécule comprising a nucléotide sequence encoding a variant of the polypeptide without the need for mutating one or more nucléotides of a nucleic acid molécule.
Several documents are cited throughout the text of this spécification. Each of the documents cited herein (including ail patents, patent applications, scientîfic publications, manufacturer's spécifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such discio sure by virtue of prior invention.
It is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the présent invention which will be limited only by the appended ciaims. Unless defined otherwise, ail technical and scientîfic terrns used herein hâve the saine meamngs as commonly understood by one of ordinary skill in the art.
Antibodies and antigen-binding fragments thereof
Recently, very potent anti-malaria antibodies were described, which are spécifie for Plasmodium falciparum circumsporozoite protein (CSP) (Tan J, Sack BK, Oyen D, et al. A public antibody lineage that potently inhibits malaria infection through dual binding to the circumsporozoite protein. Nat Med. 2018;24(4):401-407. doi:10.1038/nm.4513). The authors of this study showed that the most potent antibodies simultaneously target epitopes in (i) the NANP-repeat région of CSP and (ii) an N-terminal région of CSP covering the junction between the N-terminal domain and the NANP-repeats. Moreover, this study showed that the extreme potency of the antibodies was due to their dual specîficity, while antibodies targeting only one of the CSP epitopes were typically less potent. The most potent dual-specific antibodies described in this study include antibodies “MGU10” and “MGH2”.
Based thereon, the présent inventors designed sequence variants of antibodies MGU10 and MGH2 by artificially introducing mutations in the CDR or framework régions. Accordingiy, antibodies MGU10 and MGH2 may be referred to herein as “parental” antibodies, while the antibodies of the présent invention represent sequence variants or “variant” antibodies of said “parental” antibodies. The variant antibodies were then tested for their dual specifïcity as described in Tan et al. (Tan J, Sack BK, Oyen D, et al. A public antibody lineage that potently inhibits malaria infection through dual binding to the circumsporozoite protein. Nat Med. 2018;24(4):401-407. doi:10.1038/nm.4513). The variant antibodies of the présent invention show a surprisîngly high affinity for the target epitopes in CSP (the NANP-repeat région of CSP and the N-terminal région of CSP covering the junction between the N-terminal domain and the NANP-repeats).
In a first aspect the présent invention provides an (isolated) antibody, or an antigen-binding fragment thereof, comprising (i) the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively, and the light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; or (ii) the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively; or (iii) the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively, and the light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively; or (iv) the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively, and the light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 28, respectively; or (v) the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively, and the light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 28, respectively.
In general, the antibody, or an antigen-bindîng fragment thereof, according to the présent invention, typically comprises (at least) three complementarity determining régions (CDRs) on a heavy chain and (ai least) three CDRs on a light chain. In general, complementarity determining régions (CDRs) are the hypervariable régions présent in heavy chain variable domains and light chain variable domains. Typically, the CDRs of a heavy chain and the connected light chain of an antibody together form the antigen receptor. Usually, the three CDRs (CDR1, CDR2, and CDR3) are arranged non-consecutively in the variable domain. Since antigen receptors are typically composed of two variable domains (on two different polypeptide chains, i.e. heavy and light chain: heavy chain variable région (VH) and light chain variable région (VL)), there are typically six CDRs for each antigen receptor (heavy chain: CDRH1, CDRH2, and CDRH3; light chain: CDRL1, CDRL2, and CDRL3). A classical single antibody molécule usually has two antigen receptors and therefore contains twelve CDRs. The CDRs on the heavy and/or light chain may be separated by framework régions, whereby a framework région (FR) is a région in the variable domain which is less “variable” than the CDR. For example, a chain (or each chain, respectively) may be composed of four framework régions, separated by three CDR’s.
The sequences of the heavy chains and light chains of exemplary antibodies of the invention, comprising three different CDRs on the heavy chain and three different CDRs on the light chain were determined. The position of the CDR amino acids are defined according to the 1MGT numbering System (IMGT: http://www.imgt.org/; cf. Lefranc, M.-P. et al. (2009) Nucleic Acids Res. 37, D1006-D1012).
In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 70% or more (i.e. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 13 and a light chain variable région comprising the amino acid sequence having 70% or more (i.e. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO; 8, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO; 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are maintained.
In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an ami no acid sequence having 70% or more (i.e. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 7 and a light chain variable région comprising the amino acid sequence having 70% or more (i.e. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively) are maintained.
In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 70% or more (i.e. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 16 and a light chain variable région comprising the amino acid sequence having 70% or more (i.e. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 8, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ TD NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are maintained.
In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 70% or more (i.e. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 11 and a light chain variable région comprising the amino acid sequence having 70% or more (i.e. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and
SEQ ID NO: 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth în SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively) are maintained.
In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 70% or more (i.e. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 13 and a light chain variable région comprising the amino acid sequence having 70% or more (i.e. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively) are maintained.
In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 70% or more (i.e. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 16 and a light chain variable région comprising the amino acid sequence having 70% or more (i.e. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively) are maintained.
ïn some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 70% or more (i.e. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 104 and a light chain variable région comprising the amino acid sequence having 70% or more (i.e. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more) identity to SEQ ID NO: 8, wherein the CDR sequences as defined above (heavy chain CDRI, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectîvely; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectîvely) are maintained.
In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 70% or more (i.e. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 104 and a light chain variable région comprising the amino acid sequence having 70% or more (i.e. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectîvely; and light chain CDRI, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectîvely) are maintained.
In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 70% or more (i.e. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 24 and a light chain variable région comprising the amino acid sequence having 70% or more (i.e. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 29, wherein the CDR sequences as defined above (heavy chain CDRI, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectîvely; and light chain CDRI, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 20, SEQ ID NO: 21 or 22, and SEQ ID NO: 28, respectîvely) are maintained.
Sequence identity is usually calculated with regard to the fuli Iength of the reference sequence (i.e. the sequence recîted in the application). Percentage identity, as referred to herein, can be determined, for example, using BLAST using the default parameters specified by the NCBI (the National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/) [Blosum 62 matrix; gap open penalty=l 1 and gap extension penalty=l].
A “sequence variant” has an altered sequence in which one or more of the amino acids in the reference sequence is/are deleted or substituted, and/or one or more amino acids is/are inserted into the sequence of the reference amino acid sequence. As a resuit of the alterations, the amino acid sequence variant has an amino acid sequence which is at least 70% identical to the reference sequence. Variant sequences which are at least 70% identical hâve no more than 30 alterations, i.e. any combination of délétions, insertions or substitutions, per 100 amino acids of the reference sequence.
In general, while it is possible to hâve non-conservative amino acid substitutions, the substitutions are usually conservative amino acid substitutions, in which the substituted amino acid has similar structural or Chemical properties with the corresponding amino acid in the reference sequence. By way of example, conservative amino acid substitutions involve substitution of one alîphatic or hydrophobie amino acids, e.g. alanine, valine, leucîne and isoleucine, with another; substitution of one hydoxyl-containîng amino acid, e.g. serine and threonine, with another; substitution of one acidic residue, e.g. glutamic acid or aspartic acid, with another; replacement of one amide-containing residue, e.g. asparagine and glutamine, with another; replacement of one aromatic residue, e.g. phenylalanine and tyrosine, with another; replacement of one basic residue, e.g. lysine, arginine and histidîne, with another; and replacement of one small amino acid, e.g., alanine, serine, threonine, méthionine, and glycine, with another.
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include the fusion to the N- or C-terminus of an amino acid sequence to a reporter molécule or an enzyme.
In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 75% or more (i.e. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 13 and a light chain variable région comprising the amino acid sequence having 75% or more (i.e. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 8, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth în SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amîno acid sequence having 75% or more (i.e. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 7 and a light chain variable région comprising the amîno acid sequence having 75% or more (i.e. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively) are maintained. In some embodiments, the antibody ofthe invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 75% or more (i.e. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 16 and a light chain variable région comprising the amino acid sequence having 75% or more (i.e. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 8, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ TD NO; 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 75% or more (i.e. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 11 and a light chain variable région comprising the amino acid sequence having 75% or more (i.e. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO; 1, SEQ ID NO: 2, and SEQ ID NO; 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 75% or more (i.e. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 13 and a light chain variable région comprising the amino acid sequence having 75% or more (i.e. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 75% or more (i.e. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 16 and a light chain variable région comprising the amino acid sequence having 75% or more (i.e. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO; 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: I, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 75% or more (i.e. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 24 and a light chain variable région comprising the amino acid sequence having 75% or more (i.e. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 29, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 20, SEQ ID NO: 21 or 22, and SEQ ID NO: 28, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 75% or more (i.e. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 104 and a light chain variable région comprising the amino acid sequence having 75% or more (i.e. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 8, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set ibrth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are mainîained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 75% or more (i.e. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 104 and a light chain variable région comprising the amino acid sequence having 75% or more (i.e. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more) identity to SEQ ID NO; 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are maintained.
In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 80% or more (i.e. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 13 and a light chain variable région comprising the amino acid sequence having 80% or more (i.e. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 8, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO; 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 80% or more (i.e. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 7 and a light chain variable région comprising the amino acid sequence having 80% or more (i.e. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 80% or more (i.e. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 16 and a light chain variable région comprising the amino acid sequence having 80% or more (i.e. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 8, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 80% or more (i.e. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 11 and a light chain variable région comprising the amino acid sequence having 80% or more (i.e. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO; 2, and SEQ ID NO: 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO;
5, and SEQ ID NO: 14, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 80% or more (i.e. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 13 and a light chain variable région comprising the amino acid sequence having 80% or more (i.e. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO; 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 80% or more (i.e, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 16 and a light chain variable région comprising the amino acid sequence having 80% or more (i.e. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 80% or more (i.e. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 24 and a light chain variable région comprising the amino acid sequence having 80% or more (i.e. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 29, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 20, SEQ ID NO: 21 or 22, and SEQ ID NO: 28, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 80% or more (i.e. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 104 and a light chain variable région comprising the amino acid sequence having 80% or more (i.e. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 8, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 80% or more (i.e. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 104 and a light chain variable région comprising the amino acid sequence having 80% or more (i.e.
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are maintained.
In some embodiments, the antibody of the invention, or the antigen-bindîng fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 85% or more (i.e. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 13 and a light chain variable région comprising the amino acid sequence having 85% or more (i.e. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO; 8, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 85% or more (i.e. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 7 and a light chain variable région comprising the amino acid sequence having 85% or more (i.e. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO; 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 85% or more (i.e. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 16 and a light chain variable région comprising the amino acid sequence having 85% or more (i.e. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 8, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO; 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 85% or more (i.e. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 11 and a light chain variable région comprising the amino acid sequence having 85% or more (i.e. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDRI, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectîvely; and light chain CDRI, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectîvely) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 85% or more (i.e. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 13 and a light chain variable région comprising the amino acid sequence having 85% or more (i.e. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDRI, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectîvely; and light chain CDRI, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectîvely) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 85% or more (i.e. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 16 and a light chain variable région comprising the amino acid sequence having 85% or more (i.e. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDRI, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectîvely; and light chain CDRI, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectîvely) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 85% or more (i.e. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 24 and a light chain variable région comprising the amino acid sequence having 85% or more (i.e. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 29, wherein the CDR sequences as defined above (heavy chain CDRI, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectîvely; and light chain CDRl, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 20, SEQ ID NO: 21 or 22, and SEQ ID NO: 28, respect!vely) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 85% or more (i.e. 86%, 87%, 88%, 89%, 90%, 91%, 5 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 104 and a light chain variable région comprising the amino acid sequence having 85% or more (i.e. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 8, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO; 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and 10 light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO; 4, SEQ ID NO: 5, and SEQ ID NO; 6, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 85% or more (i.e. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 104 and a light 15 chain variable région comprising the amino acid sequence having 85% or more (i.e. 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDRl, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDRl, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, 20 SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are maintained.
In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 90% or more (i.e. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 25 13 and a light chain variable région comprising the amino acid sequence having 90% or more (i.e. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 8, wherein the CDR sequences as defined above (heavy chain CDRl, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDRl, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID 30 NO; 6, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 90% or more (i.e. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identity to SEQ ID NO: 7 and a light chain variable région comprising the amino acid sequence having 90% or more (i.e. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)îdentity to SEQ ID NO: 15, whereîn the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 90% or more (i.e. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 16 and a light chain variable région comprising the amino acid sequence having 90% or more (i.e. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identity to SEQ ID NO: 8, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 90% or more (i.e. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 11 and a light chain variable région comprising the amino acid sequence having 90% or more (i.e. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 90% or more (i.e. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 13 and a light chain variable région comprising the amino acid sequence having 90% or more (i.e. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO; 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 90% or more (i.e. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 16 and a light chain variable région comprising the amino acid sequence having 90% or more (i.e. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ TD NO: 14, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 90% or more (i.e. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 24 and a light chain variable région comprising the amino acid sequence having 90% or more (i.e. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 29, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO; 19, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 20, SEQ ID NO: 21 or 22, and SEQ ID NO: 28, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 90% or more (i.e. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO; 104 and a light chain variable région comprising the amino acid sequence having 90% or more (i.e. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 8, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth m SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 90% or more (i.e. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 104 and a light chain variable région comprising the amino acid sequence having 90% or more (i.e. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO; 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are maintained.
In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 95% or more (i.e. 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 13 and a light chain variable région comprising the amino acid sequence having 95% or more (i.e. 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 8, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 95% or more (i.e. 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 7 and a light chain variable région comprising the amino acid sequence having 95% or more (i.e. 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as deftned above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively) are maintained. In some embodiments, the antibody ofthe invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 95% or more (i.e. 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 16 and a light chain variable région comprising the amino acid sequence having 95% or more (i.e. 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 8, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ TD NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 95% or more (i.e. 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 11 and a light chain variable région comprising the amino acid sequence having 95% or more (i.e. 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO; 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 95% or more (i.e. 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 13 and a light chain variable région comprising the amino acid sequence having 95% or more (i.e. 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 95% or more (i.e. 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 16 and a light chain variable région comprising the amino acid sequence having 95% or more (i.e. 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: l, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 95% or more (i.e. 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 24 and a light chain variable région comprising the amino acid sequence having 95% or more (i.e. 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 29, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 20, SEQ ID NO: 21 or 22, and SEQ ID NO: 28, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 95% or more (i.e. 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 104 and a light chain variable région comprising the amino acid sequence having 95% or more (i.e. 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 8, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: l, SEQ ID NO: 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are maintained. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, comprises a heavy chain variable région comprising an amino acid sequence having 95% or more (i.e. 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 104 and a light chain variable région comprising the amino acid sequence having 95% or more (i.e. 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 15, wherein the CDR sequences as defined above (heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO; 2, and SEQ ID NO: 12, respectively; and light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively) are maintained.
In some embodiments, the antibody, or the antigen-binding fragment thereof, comprises (i) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 13 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 8; or (ii) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 7 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 15; or (iii) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 16 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 8; or (iv) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 11 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 15; or (v) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO; 13 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 15; or (vi) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 16 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 15; or (vii) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 24 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 29; or (viii) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 104 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 8; or (ix) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 104 and a light chain variable région comprising the amino acid sequence as set forth in SEQ IDNO: 15.
The présent invention also provides an antibody, or an antigen-binding fragment thereof, comprising (i) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 11 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 8; or (ii) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 13 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 8; or (iii) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 7 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 15; or (iv) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 16 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 8; or (v) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 11 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 15; or (vi) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 13 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 15; or (vii) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 16 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 15; or (viii) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 24 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 29; or (ix) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO; 104 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 8; or (x) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 104 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 15.
The CDR and VH/VL sequences of exemplified antibodies of the invention, namely antibodies MGUlOvariantl (MGUIOvl), MGU10variant2 (MGU10v2), MGU10variant3 (MGU10v3), MGU10variant4 (MGU10v4), MGU10variant5 (MGU10v5), MGU1 Ovariantô (MGU10v6), MGU10variant7 (MGU10v7), MGU 10variant8 (MGU10v8), MGU10variant9 (MGU10v9), and MGH2variantI (MGH2vl), and their respective wild-type référencé antibodies MGU10 and MGH2, are shown in Table 1 below.
Table 1: CDR and VH/VL sequences of exemplified antibodies of the invention and their respective référencé antibodies MGU10 and MGH2.
Antibody name Heavy chain Light chain
CDR1 CDR2 CDR3 VH CDRI CDR2 CDR3 VL
MGU 10 1 2 3 7 4 5 6 8
MGUlOvl 1 2 3 11 4 5 6 8
MGU10v2 1 2 12 13 4 5 6 8
MGU10v3 1 2 3 7 4 5 14 15
MGU10v4 1 2 12 16 4 5 6 8
MGU10v5 1 2 3 11 4 5 14 15
MGU10v6 1 2 12 13 4 5 14 15
MGU!0v7 1 2 12 16 4 5 14 15
MGU10v8 1 2 12 104 4 5 6 8
MGU10v9 1 2 12 104 4 5 14 15
MGH2 17 18 19 24 20 21/22 23 25
MGH2vl 17 18 19 24 20 21/22 28 29
In particular, the antibody of the invention, or an antigen-binding fragment thereof, binds (specifically) to Plasmodium falciparum sporozoîtes. The antibody, or the antigen-binding fragment thereof, may provide protection against Plasmodium (falciparum), in particular the antibody, or the antigen-binding fragment thereof, may inhibit or reduce (symptoms of) Plasmodium (falciparum) infection. Accordingly, the antibody of the invention, or an antigenbinding fragment thereof, may prevent, reduce, inhibit and/or neutralize infection with Plasmodium falciparum. More specifically, the antibody according to the présent invention, or the antigen-binding fragment thereof, may (specifically) bind to Plasmodium circumsporozoite protein (CSP), such as the Plasmodium falciparum circumsporozoite protein (PfCSP) according to SEQ ID NO: 33.
In other words, the antibody according to the présent invention, or the antigen-binding fragment thereof, may be able to recognize an epitope, in particular a CSP epitope. In some embodiments, the antibody of the invention, or an antigen-binding fragment thereof, binds (specifically) to the NANP-repeat région of Plasmodium falciparum circumsporozoite protein (PfCSP). In some embodiments, the antibody of the invention, or an antigen-binding fragment thereof, binds (specifically) to the N-terminal région of Plasmodium falciparum circumsporozoite protein, which covers the junction between the N-terminal domain and the NANP-repeats of circumsporozoite protein. Typically, the antibody of the invention, or an antigen-binding fragment thereof, may be monospecific regarding its paratopes (i.e., the antibody or antigenbinding fragment may contaîn only one single kind/type of antigen-binding site(s)); ail antigenbinding site(s) of the antibody or antigen-binding fragment may hâve the same CDR or VH/VL sequences) - but, at the same time, the antibody or antigen-binding fragment may be “dualspecific” regarding the target epitopes at CSP (i.e., the antibody or antigen-binding fragment can recognize two (or more) epitopes on CSP, in particular the two epitopes described herein). Accordingly, a single paratope of the antibodies of the invention, or antigen-binding fragments thereof, may be able to bind to both, the NANP-repeat région of PfCSP and the N-terminal région of PfCSP, which covers the junction between the N-terminal domain and the NANPrepeats of circumsporozoite protein. The NANP-repeat région of CSP is well-known to those skilled in the art. For example, the NANP-repeat région of CSP may hâve an amino acid 29 sequence as set forth in SEQ ID NO: 34. For example, the N-terminal région of CSP, which covers the junction between the N-terminal domain and the NANP-repeats of circumsporozoite protein may hâve an amino acid sequence as set forth in SEQ ID NO: 35 or 105. Accordingly, the antibody according to the présent invention, or the antigen-binding fragment thereof, may bind (specifically) to a peptide according to SEQ ID NO: 34 and/or to a peptide according to SEQ ID NO: 35 or 105.
Standard methods to assess binding of the antibody according to the présent invention, or the antigen-binding fragment thereof, are known to those skilled in the art and include, for ex ample, ELISA (enzyme-linked immunosorbent assay). An exemplary standard ELISA may be perfonned as follows: ELISA plates may be coated with a sufficient amount (e.g., I pg/ml) of the protein/complex/particle to which binding of the antibody is to be tested. For example, for testing binding to CSP or an epitope thereof as outlined above, a CSP protein (e.g., SEQ ID NO: 33) and/or fragments/epitopes thereof (e.g., peptides of SEQ ID NO: 34 or 35/105) may be used. ELISA plates may be coated directly or indirectiy (e.g., by coating plates first with avidin and incubaiing them later with biotinylated protein/complex/particle to which binding of the antibody is to be tested). After the first coating step (avidin or, directly, with the protein/complex/particle to which binding of the antibody is to be tested) plates may be blocked, e.g. with a 1% w/v solution of Bovine Sérum Albumin (BSA) in PBS. Before the coated plates are incubated with the antibody to be tested, they may be washed. To détermine, for example, ECzo-values, the plates are typically incubated with different concentrations of the antibody to be tested (“titration”). Antibody binding can be revealed, for example, using goat anti-human IgG, e.g. coupled to alkaline phosphatase. Plates may then be washed, the required substrate (e.g., p-NPP) may be added and plates may be read, e.g. at a wavelength of 405 nm to détermine optical density values. The relative affinities of antibody binding may be determined by measuring the concentration of the antibody required to achieve 50% maximal binding at saturation (ECsq). The ECsq values may be calculated by interpolation of binding curves fitted with a four-parameter nonlinear régression with a variable slope. A spécifie example of such an ELISA is described in Example 2, which may be performed (in essentially the same way) also with other antibodies or antigen-binding fragments.
In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, has an EC50 value below 103 ng/ml, e.g. below 200 or 100 ngdnl, for binding to CSP (e.g., SEQ ID NO: 33), such as to a fragment/epitope thereof (e.g., SEQ ID NO: 34 and/or 35/105). For example, the antibody of the invention, or the antigen-binding fragment thereof, may hâve an EC50 value below 10* ng/ml, e.g. below 200 or 100 ng/ml, for binding to a peptide of SEQ ID NO: 34 and for binding to a peptide to SEQ ID NO: 35 or 105. More specifïcally, the antibody of the invention, or the antigen-binding fragment thereof, may hâve an ECjq value below 30 ng/ml for binding to a peptide of SEQ ID NO: 34 and for binding to a peptide to SEQ ID NO: 35 or 105. For example, the antibody of the invention, or the antigen-binding fragment thereof, may hâve an EC50 value below 29 ng/ml (e.g., below 28 or 27 ng/ml) for binding to a peptide of SEQ ID NO: 34 and an EC50 value below 26 ng/mi (e.g., below 23 or 21 ng/ml) for binding to a peptide to SEQ ID NO: 35 or 105.
In some embodiments, the antibody, or an antigen-binding fragment thereof, according to according to the présent invention comprises a variable région of the heavy chain of the antibody, or of the antigen-binding fragment thereof, (VH), which is encoded by a nucleic acid comprising a gene (segment) of the VH3 gene family, such as the gene (segment) VH3-3O.
To study and quantitate virus înfectivity (or “neutralizatîon”) in the laboratory the person skilled in the art knows various standard “neutralizatîon assays”. For a neutralizatîon assay animal viruses are typically propagated in cells and/or cell lines. For example, in a neutralizatîon assay cuitured cells may be incubated with a fixed amount of Plasmodium falciparum sporozoites in the presence (or absence) of the antibody to be tested. As a reado ut for example flow cytometry may be used. Alternative!y, also other readouts are conceivable.
In some instances, the antibody of the invention, or an antigen-binding fragment thereof, may reduce gliding motility of Plasmodium sporozoites. Plasmodium sporozoites are transmitted by mosquito bites into the skin of their vertebrate host. Before sporozoites enter the blood stream, they move rapidly through the dermis, powered by an actomyosîn System, using a form of locomotion referred to as “gliding motility”. Accordingly, sporozoite motility is a key prerequisite for parasite transmission and successful infection of the vertebrate host.
Gliding motility of sporozoites can be assessed by in in vitro assays, wherein the sporozoite is allowed to glide on a fiat surface, e.g. on a giass surface. Gliding trails of sporozoites can be visualized by covering the surface with an anti-CSP antibody, which detects CSP shed by sporozoites during gliding. The anti-CSP antibody itself may be labelled (e.g. biotin) or a secondary labelled antibody against the anti-CSP antibody may be used to visualize the trails.
For testing the effect of compounds on sporozoite gliding, sporozoites may be preîncubated with test compounds before they are allowed to glide. Detailed protocols for gliding assays are known in the art and described, for example, in Example 4 or in Prinz, H. L., Sattler, J. M. & Frischknecht, F. (2017) Plasmodium Sporozoite Motility on Fiat Substrates. Bioprotocol 7, e2395. Moreover, ex vivo imaging technologies making use of human tissue may be employed to déterminé the gliding motility of sporozoites, e.g. as described in Winkel, B.M.F., de Rome, C.M., van Oosterom, M.N. et al. (2019) Quantification of wild-type and radiation attenuated Plasmodium falciparum sporozoite motility in human skin. Sci Rep 9, 13436. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, reduces Plasmodium sporozoite gliding motility to a greater extent than its parental antibody MGU10 or MGH2, respectively. This may be easily assessed by directly comparing the effects of the parental antibody (MGU10 or MGH2) and its variant antibody (or antigen-binding fragment thereof) according to the présent invention in the sanie gliding motility assay, e.g. as described in Ex ample 4.
In some instances, the antibody of the invention, or an antigen-binding fragment thereof, may reduce cell traversai of Plasmodium sporozoites. As sporozoites move towards the liver, they can enter and exit host cells within transient vacuoles, a process known as cell traversai. Traversai allows the sporozoites to cross cellular barri ers and évadé the host immune response, thereby representing a key prerequisite for successful infection of the vertebrate host.
Cell traversai of sporozoites can be assessed by in in vitro assays, wherein sporozoites are incubated with host cells in a co-culture. For visualization, varions (e.g., fluorescent) labels may be used, for example, in the co-culture or sporozoites may be pre-incubated with a label (e.g., as described in Ex ample 5). Alternative! y, genetically modified Plasmodium strains may be used, which express, e.g., fluorescent labels. For testing the effect of compounds on traversai, sporozoites may be pre-incubated with test compounds before they are co-cultured with host cells. Detailed protocols for sporozoite traversai assays are known in the art and described, for example, in Example 5; in Schleicher, T.R., Yang, J., Freudzon, M. et al. (2018) A mosquîto salivary gland protein partially inhibits Plasmodium sporozoite cell traversai and transmission. Nat Commun 9, 2908; or in Sinnis, P., De La Vega, P., Coppi, A., Krzych, U., & Mota, Μ. M. (2013). Quantification of sporozoite invasion, migration, and development by microscopy and tlow cytometry. Methods in molecular biology (Clifton, N.J.), 923, 385»00. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, reduces
Plasmodium sporozoite cell traversai to a greater extent than its parental antibody MGU10 or MGH2, respectîvely. This may be easily assessed by directly comparing the effects of the parental antibody (MGU10 or MGH2) and its variant antibody (or antigen-binding fragment thereof) according to the présent invention in the same traversai assay, e.g. as described in Example 5.
In some instances, the antibody of the invention, or an antigen-binding fragment thereof, may reduce invasion and/or maturation of Plasmodium sporozoites. Sporozoite invasion of hépatocytes and subséquent maturation into exoerythrocytic forms îs an essential step in the establishment of malaria infection.
Invasion and/or maturation of sporozoites can be assessed by in in vüro assays, wherein sporozoites are incubated with host cells (e.g., hépatocytes). For visualîzation, varîous (e.g., fluorescent) labels may be used (e.g., as described in Example 3). Alternatively, genetically modified Plasmodium strains may be used, which express, e.g., fluorescent labels. For testing the effect of compounds on invasion and/or maturation, sporozoites may be pre-incubated with test compounds before they are co-încubated with host cells. Detailed protocols for sporozoite invasion/maturation assays are known in the art and described, for example, in Example 3; in Kaushansky, A., Rezakhani, N., Mann, H. & Kappe, S. H. (2012) Development of a quantitative flow cytometry based assay to assess infection by Plasmodium falciparum sporozoites. Molecular and biochemical parasitology 183, 100-103; in Rodriguez-Galân A, Salman AM, Bowyer G, Collins KA, Longley RJ, Brod F, Ulaszewska M, Ewer KJ, Janse CJ, Khan SM, Hafalla JC, Hill AVS, Spencer AJ. (2017) An in vitro assay to measure antibody-mediated inhibition of P. berghei sporozoite invasion against P. falciparum antigens. S ci Rep 5;7(1): 17011; or in Sinnis, P., De La Vega, P., Coppi, A., Krzych, U., & Mota, Μ. M. (2013). Quantification of sporozoite invasion, migration, and development by microscopy and flow cytometry. Methods in molecular biology (Clifton, N.J.), 923, 385XJ0. In some embodiments, the antibody of the invention, or the antigen-binding fragment thereof, reduces Plasmodium sporozoite invasion/maturation to a greater extent than its parental antibody MGU10 or MGH2, respectîvely. This may be easily assessed by directly comparing the effects of the parental antibody (MGU10 or MGH2) and its variant antibody (or antigen-binding fragment thereof) according to the présent invention in the same invasion/maturation assay, e.g. as described in Example 3.
In some embodiments, the antibody of the invention, or an antigen-binding fragment thereof, may exhibit increased stabiiity as compared to its parental antibody MGU10 or MGH2, respectively. It is understood that for comparison, the variant antibody of the invention and its parental antibody are tested under the same conditions (i.e., side-by-side). For example, the antibody of the invention, or an antigen-binding fragment thereof, may exhibit increased stabiiity as compared to its parental antibody MGU10 or MGH2, respectively, at a pH below 6, such as pH 5.5 or 5.6. This may be achieved, for example, by storing the antibodies in a buffer comprising 50mM Na-Acetate and 50mM NaCl, at pH 5.5. Moreover, the antibodies may be exposed to heat stress, e.g. about 40°C, for testing their stabiiity. Stabiiity test usually continue for at least several days, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days. In some instances, stabiiity is tested over 14 or 15 days.
In some embodiments, the antibody of the invention is a human antibody. In some embodiments, the antibody of the invention is a monoclonal antibody. For example, the antibody of the invention is a human monoclonal antibody.
Antibodies of the invention can be of any isotype (e.g., IgA, IgG, IgM i.e. an α, γ or μ heavy chain). For example, the antibody is of the IgG type. Within the IgG isotype, antibodies may be IgGl, IgG2, IgG3 or IgG4 subclass, for example IgGl. Antibodies of the invention may hâve a κ or a λ light chain. In some embodiments, the antibody has a lambda or kappa light chain. In some embodiments, the antibody is of IgG 1 type and has a lambda or kappa light chain.
In some embodiments, the antibody is of the human IgGl type. The antibody may be of any allotype. The tenn “allotype” refers to the allelic variation found among the IgG subclasses. For ex ample, the antibody may be of the Glml (or Glm(a)) allotype, of the Glm2 (or Glm(x)) allotype, of the Glm3 (or Glm(f)) allotype, and/or of the Glml7 (or Gm(z)) allotype. The Glm3 and Glml7 allotypes are located at the same position in the CH1 domain (position 214 according to EU numbering). Glm3 corresponds to R214 (EU), while Glml 7 corresponds to K214 (EU). The Glml allotype is located in the CH3 domain (at positions 356 and 358 (EU)) and refers to the replacements E356D and M358L. The Glm2 allotype refers to a replacement of the alanine in position 431 (EU) by a glycine. The Glml allotype may be combined, for example, with the Glm3 or the Glml7 allotype. In some embodiments, the antibody is of the allotype Glm3 with no Glml (Glm3,-1). In some embodiments, the antibody is of the Glml7,l allotype. In some embodiments, the antibody is of the Glm3,l allotype. In some embodiments, the antibody is of the allotype Glml7 with no Glml (Glml7,-1). Optionally, these allotypes may be combined (or not combined) with the Glm2, Glm27 or Glm28 allotype. For example, the antibody may be of the G 1ml 7,1,2 allotype.
In some embodiments, the antibody according to the présent invention, or an antigen binding fragment thereof, comprises an Fc moiety. The Fc moiety may be derived from human origin, e.g. from human IgG 1, IgG2, IgG3, and/or IgG4, such as human IgGl.
As used herein, the term “Fc moiety” refers to a sequence derived from the portion of an immunoglobulin heavy chain beginning in the hinge région just upstreain of the papain cleavage site (e.g., residue 216 in native IgG, takîng the first residue of heavy chain constant région to be 114) and ending at the C-termînus of the immunoglobulin heavy chain. Accordingly, an Fc moiety may be a complété Fc moiety or a portion (e.g., a domain) thereof. A complété Fc moiety comprises at least a hinge domain, a CH2 domain, and a CH3 domain (e.g., EU amino acid positions 216-446). An additional lysine residue (K) is sometimes présent at the extreme Cterminus of the Fc moiety, but is often cleaved from a mature antibody.
Each of the amino acid positions within an Fc moiety hâve been numbered herein according to the art-recognîzed EU numbering System of Kabat, see e.g., by Kabat et al., in “Sequences of Proteins of Immunological Interest”, U.S. Dept. Health and Human Services, 1983 and 1987. The EU index or EU index as in Kabat or EU numbering refers to the numbering of the EU antibody (Edelman GM, Cunningham BA, Gall WE, Gottlieb PD, Rutishauser U, Waxdal MJ. The covalent structure of an entire gammaG immunoglobulin molécule. Proc Natl Acad Sci U S A. I969;63(l):78-85; Kabat E.A., National Institutes of Health (U.S.) Office of the Director, “Sequences of Proteins of Immunological Interest”, 5th édition, Bethesda, MD : U.S. Dept. of Health and Human Services, Public Health Service, National Institutes of Health, 1991, hereby entirely incorporated by reference).
In some embodiments, in the context of the présent invention an Fc moiety comprises at least one of: a hinge (e.g., upper, middie, and/or lower hinge région) domain, a CH2 domain, a CH3 domain, or a variant, portion, or fragment thereof. An Fc moiety may comprise at least a hinge domain, a CH2 domain or a CH3 domain. The Fc moiety may be a complété Fc moiety. The Fc moiety may also comprises one or more amino acid insertions, délétions, or substitutions relative to a naturally-occurring Fc moiety. For example, at least one of a hinge domain, CH2 domain or
CH3 domain (or portion thereof) may be deleted. For example, an Fc moiety may comprise or consist of: (i) hinge domain (or portion thereof) fused to a CH2 domain (or portion thereof), (ii) a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof), (iii) a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof), (iv) a hinge domain (or portion thereof), (v) a CH2 domain (or portion thereof), or (vi) a CH3 domain or portion thereof.
It will be understood by one of ordinary skill in the art that the Fc moiety may be modified such that it varies in amino acid sequence from the complété Fc moiety of a naturally occurring immunoglobulin molécule, while retaining at least one désirable function conferred b y the naturally-occurring Fc moiety. Such fonctions include Fc receptor (FcR) binding, antibody halflife modulation, ADCC function, proteîn A binding, protein G binding, and complément binding. The portions of naturally occurring Fc moieties, which are responsible and/or essential for such fonctions are well known by those skilled in the art.
For example, to activate the complément cascade Clq bînds to at least two molécules of IgGl or one molécule of IgM, attached to the antigénie target (Ward, E. S., and Ghetie, V., Ther. Immunol. 2 (1995) 77-94). Burton, D. R., described (Mol. Immunol. 22 (1985) 161-206) that the heavy chain région comprising amino acid residues 318 to 337 is involved in complément fixation. Duncan, A. R., and Winter, G. (Nature 332 (1988) 738-740), using site directed mutagenesis, reported that Glu318, Lys320 and Lys322 form the binding site to Clq. The rôle of Glu318, Lys320 and Lys 322 residues in the binding of Clq was confïrmed by the ability of a short synthetic peptide containing lhese residues to inhîbît complément mediated lysis.
For example, FcR binding can be mediated by the interaction of the Fc moiety (of an antibody) with Fc receptors (FcRs), which are specialized cell surface receptors on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily, and were shown to médiate both the removal of antibody-coated pathogens by phagocytosis of immune complexes, and the lysis of érythrocytes and various other cellular targets (e.g. tumor cells) coated with the corresponding antibody, via antibody dépendent cell mediated cytotoxicity (ADCC; Van de Winkel, J. G., and Anderson, C. L., J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin classes; Fc receptors for IgG antibodies are referred to as FcyR, for IgE as FcsR, for IgA as FcctR and so on and néonatal Fc receptors are referred to as FcRn. Fc receptor binding is described for example in Ravetch, J. V., and Kinet, J. P., Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P. J., et al., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J Lab. Clin. Med. 126 (1995) 330-341; and Gessner, J. E., et al., Ann. Hematol. Ί6 (1998) 231-248.
Cross-lînkîng of receptors by the Fc domain of native IgG antibodies (FcyR) triggers a wide variety of effector functions including phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory medîators, as well as immune complex clearance and régulation of antibody production. Therefore, the Fc moiety may provide cross-linking of receptors (FcyR). In humans, three classes of FcyR hâve been characterized, which are: (i) FcyRI (CD64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils; (ii) FcyRII (CD32), which binds complexed IgG with medium to low affinity, is widely expressed, in particular on leukocytes, is known to be a central player in antibody-mediated immun ity, and which can be divided into FcyRIIA, FcyRIIB and FcyRI IC, which perform different functions in the immune System, but bind with similar low affinity to the IgG-Fc, and the ectodomains of these receptors are highly homologuons; and (iii) FcyRI II (CD16), which binds IgG with medium to low affinity and exists as two types: FcyRIIIA found on NK cells, macrophages, eosinophils and some monocytes and T cells and mediating ADCC and FcyRIIIB, which is highly expressed on neutrophils. FcyRIIA is found on many cells involved in killing (e.g. macrophages, monocytes, neutrophils) and seems able to activate the killing process. FcyRIIB seems to play a rôle in inhibitory processes and is found on B-cells, macrophages and on mast cells and eosinophils. Important!y, 75% of ail FcyRIIB is found in the liver (Ganesan, L. P. et al., 2012: FcyRIIb on liver sinusoïdal endothélium clears small immune complexes. Journal of Immunology 189: 4981-4988). FcyRIIB is abundantly expressed on Liver Sinusoïdal Endothélium, called LSEC, and in Kupffer cells in the liver and LSEC are the major site of small immune complexes clearance (Ganesan, L. P. et al., 2012: FcyRIIb on liver sinusoïdal endothélium clears small immune complexes. Journal of Immunology 189: 49814988).
Accordingly, antibodies, and antigen binding fragments thereof, of the invention may be able to bind to FcyRIIb, for example antibodies comprising an Fc moiety for binding to FcyRIIb, in particular an Fc région, such as, for example IgG-type antibodies. Moreover, it is possible to engineer the Fc moiety to enhance FcyRIIB binding by introducing the mutations S267E and L328F as described by Chu, S. Y. et al., 2008: Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD 19 and FcyRIIb with Fc-engineered antibodies. Molecular Immunology 45, 3926- 3933. Thereby, the clearance of immune complexes can be enhanced (Chu, S., et ah, 2014: Accelerated Clearance of IgE In Chimpanzees Is Mediated By Xmab7195, An Fc-Engineered Antibody With Enhanced Affinity For Inhibitory Receptor FcyRIIb. Am J Respir Crit, American Thoracic Society International Conférence Abstracts). Accordingly, the antibodies, or antigen binding fragments thereof, of the invention may comprise an engineered Fc moiety with the mutations S267E and L328F, in particular as described by Chu, S. Y. et al., 2008: Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcyRIIb with Fc-engineered antibodies. Molecular Immunology 45, 3926-3933.
On B-cells it seems to fonction to suppress forther immunoglobuiin production and isotype switching to say for example the IgE class. On macrophages, FcyRI IB acts to inhibit phagocytosis as mediated through FcyRIlA. On eosinophils and mast cells the b form may help to suppress activation of these cells through IgE binding to its separate receptor.
Regarding FcyRI binding, modification in native IgG of at least one of E233-G236, P238, D265, N297, A3 27 and P329 reduces binding to FcyRI. IgG2 residues at positions 233-236, substituted into IgGl and IgG4, reduces binding to FcyRI by 103-fold and eliminated the human monocyte response to antibody-sensîtized red blood cells (Annour, K. L., et al. Eur. J. Immunol. 29 (1999) 2613-2624). Regarding FcyRII binding, reduced binding for FcyRIIA is found e.g. for IgG mutation of at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292 and K414. Regarding FcyRIII binding, reduced binding to FcyRHIA is found e.g. for mutation of at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V3O3, A327, K338 and D376. Mapping ofthe binding sites on human IgGl for Fc receptors, the above mentioned mutation sites and methods for measuring binding to FcyRI and FcyRIIA are described in Shields, R. L., et al., J, Biol. Chem. 276 (2001) 6591-6604. For example, a single (S239D or I332E), double (S239D/I332E), and triple mutations (S239D/I332E/A330L) improved the affinity against human FcyRIlla. Furthermore, the addition of the mutation G236A to S239D/I332E improved not only FcyRIIa:FcyRIIb ratio, but also enhanced binding to FcyRIlla. Accordingly, the mutations G236A/S239D/A330L/I332E were described to enhance engagement of FcyRIIa and FcyRIlla.
Regarding binding to the crucial FcyRII, two régions of native IgG Fc appear to be critical for interactions of FcyRIIs and IgGs, namely (i) the lower hinge site of IgG Fc, in particular amîno acid residues L, L, G, G (234 — 237, EU numbering), and (ii) the adjacent région of the CH2 domain of IgG Fc, in partîcular a loop and strands in the upper CH2 domain adjacent to the lower hinge région, e.g. in a région of P331 (Wines, B.D., et al., J. Immunol. 2000; 164: 5313 — 5318). Moreover, FcyRI appears to bind to the same site on IgG Fc, whereas FcRn and Protein A bind to a different site on IgG Fc, which appears to be at the CH2-CH3 interface (Wines, B.D., et al., J. Immunol. 2000; 164: 5313 - 5318).
For example, the Fc moiety may comprise or consist of at least the portion of an Fc moiety that is known in the art to be required for FcRn binding or extended half-life. Altematively or additionally, the Fc moiety of the antibody of the invention comprises at least the portion of known in the art to be required for Protein A binding and/or the Fc moiety of the antibody of the invention comprises at least the portion of an Fc molécule known in the art to be required for protein G binding. The Fc moiety may comprise at least the portion known in the art to be required for FcyR binding. As outlined above, an Fc moiety may thus at least comprise (i) the lower hinge site of native IgG Fc, in partîcular amino acid residues L, L, G, G (234 - 237, EU numbering), and (ii) the adjacent région of the CH2 domain of native IgG Fc, in partîcular a loop and strands in the upper CH2 domain adjacent to the lower hinge région, e.g. in a région of P331, for example a région of at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids in the upper CH2 domain of native IgG Fc around P331, e.g. between amino acids 320 and 340 (EU numbering) of native IgG Fc.
In some embodiments, the antibody, or antigen binding fragment thereof, according to the présent invention comprises an Fc région. As used herein, the tenn “Fc région” refers to the portion of an immunoglobulin formed by two or more Fc moîetîes of antibody heavy chains. For example, the Fc région may be monomeric or “single-chain” Fc région (i.e., a scFc région). Single chain Fc régions are comprised of Fc moieties linked wîthin a single polypeptide chain (e.g., encoded in a single contiguous nucleic acid sequence). Exemplary scFc régions are disclosed in WO 2008/143954 A2. The Fc région may be dimeric. A “dîmeric Fc région” or “dcFc” refers to the dîmer formed by the Fc moieties of two separate immunoglobulin heavy chains. The dimeric Fc région may be a homodimer of two identical Fc moieties (e.g., an Fc région of a naturally occurring immunoglobulin) or a heterodimer of two non-identical Fc moieties.
The Fc moieties of the Fc région may be of the same or different class and/or subclass. For example, the Fc moietîes may be derived from an immunoglobulin (e.g., a human immunoglobulin) of an IgGl, IgG2, IgG3 or IgG4 subclass. The Fc moietîes of the Fc région may be ofthe same class and subclass. However, the Fc région (or one or more Fc moietîes of an Fc région) may also be chimeric, whereby a chimeric Fc région may comprise Fc moietîes derived from different immunoglobulin classes and/or subclasses. For example, at least two of the Fc moietîes of a dimeric or single-chain Fc région may be from different immunoglobulin classes and/or sub classes. Additionally or altematively, the chimeric Fc régions may comprise one or more chimeric Fc moietîes. For example, the chimeric Fc région or moiety may comprise one or more portions derived from an immunoglobulin of a first subclass (e.g., an IgGl, IgG2, or IgG3 subclass) while the remainder of the Fc région or moiety is of a different subclass. For example, an Fc région or moiety of an Fc polypeptide may comprise a CH2 and/or CH3 domain derived from an immunoglobulin of a first subclass (e.g., an IgGl, IgG2 or IgG4 subclass) and a hinge région from an immunoglobulin of a second subclass (e.g., an IgG3 subclass). For example, the Fc région or moiety may comprise a hinge and/or CH2 domain derived from an immunoglobulin of a first subclass (e.g., an IgG4 subclass) and a CH3 domain from an immunoglobulin of a second subclass (e.g., an IgGl, IgG2, or IgG3 subclass). For example, the chimeric Fc région may comprise an Fc moiety (e.g., a complété Fc moiety) from an immunoglobulin for a first subclass (e.g., an lgG4 subclass) and an Fc moiety from an immunoglobulin of a second subclass (e.g., an IgGl, IgG2 or IgG3 subclass). For example, the Fc région or moiety may comprise a CH2 domain from an IgG4 immunoglobulin and a CH3 domain from an IgGl immunoglobulin. For example, the Fc région or moiety may comprise a CH1 domain and a CH2 domain from an IgG4 molécule and a CH3 domain from an IgGl molécule. For example, the Fc région or moiety may comprise a portion of a CH2 domain from a particular subclass of antibody, e.g., EU positions 292-340 of a CH2 domain. For example, an Fc région or moiety may comprise amino acids a positions 292-340 of CH2 derived from an IgG4 moiety and the remainder of CH2 derived from an IgGl moiety (altematively, 292-340 of CH2 may be derived from an IgGl moiety and the remainder of CH2 derived from an IgG4 moiety).
Moreover, an Fc région or moiety may (additionally or altematively) for example comprise a chimeric hinge région. For example, the chimeric hinge may be derived, e.g. in part, from an IgGl, IgG2, or IgG4 molécule (e.g., an upper and lower middle hinge sequence) and, in part, from an IgG3 molécule (e.g., an middle hinge sequence). In another example, an Fc région or moiety may comprise a chimeric hinge derived, in part, from an IgGl molécule and, in part, from an IgG4 molécule. In another example, the chimeric hinge may comprise upper and lower hinge domains from an IgG4 molécule and a middle hinge domain from an IgGl molécule. Such a chimeric hinge may be made, for example, by introducing a proline substitution (Ser228Pro) at EU position 228 in the middle hinge domain of an IgG4 hinge région. In other embodiments, the chimeric hinge can comprise amino acids at EU positions 233-236 are from an IgG2 antibody and/or the Ser228Pro mutation, wherein the remaining amino acids of the hinge are from an IgG4 antibody (e.g,, a chimeric hinge of the sequence ESKYGPPCPPCPAPPVAGP). Further chimeric hinges, which may be used in the Fc moiety of the antibody according to the présent invention are described in US 2005/0163783 Al.
In some embodiments, the Fc moiety, or the Fc région, comprises or consists of an amino acid sequence derived from a human immunoglobulin sequence (e.g., from an Fc région or Fc moiety from a human IgG molécule). However, polypeptides may comprise one or more amino acids from another mammalian species. For example, a primate Fc moiety or a primate binding site may be included in the subject polypeptides. Altematively, one or more murine amino acids may be présent in the Fc moiety or in the Fc région.
In some embodiments, the antibody according to the présent invention comprises, in particular in addition to an Fc moiety as described above, other parts derived from a constant région, in particular from a constant région of IgG, such as a constant région of (human) IgGl. The antibody according to the présent invention may comprise, in particular in addition to an Fc moiety as described above, ail other parts of the constant régions, in particular ail other parts of the constant régions of IgG (such as (human) IgGl).
Example sequences of constant régions are the amino acid sequences according to SEQ ID N Os: 30 - 32. For example, the amino acid sequence of IgGl CH1-CH2-CH3 is according to SEQ ID NO: 30 or a sequence variant thereof (including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity. In some embodiments, the amino acid sequence of IgGl CH1-CH2-CH3 may be according to SEQ ID NO: 103 or a sequence variant thereof (including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity, wherein the mutation(s) M428L and/or N434S may be maintained.
As outlined above, an antibody according to the présent invention may comprise a (complété) Fc région derived from human IgGl. In some embodiments, the antibody according to the présent invention comprises, in particular in addition to a (complété) Fc région derived from human IgGl also ail other parts of the constant régions of IgG, such as ail other parts of the constant régions of (human) IgGl.
In some embodiments, the antibody according to the présent invention comprises a (complété) Fc moiety/Fc région, wherein the interaction/binding with FcR is not compromised. In general, binding of the antibody to an Fc receptor may be assessed by varions methods known to the skilled person, such as ELISA (Hessell AJ, Hangartner L, Hunter M, Havenith CEG, Beurskens FJ, Bakker JM, Lanigan CMS, Landucci G, Forthal DN, Parren P WHI, et al.: Fc receptor but not complément binding is important in antibody protection against HIV. Nature 2007, 449:101— 104; Grevys A, Bem M, Foss S, Bratlie DB, Moen A, Gunnarsen KS, Aase A, Michaelsen TE, Sandlie I, Andersen JT: Fc Engineering of Human IgGl for Altered Binding to the Néonatal Fc Receptor Affects Fc Effector Functions. 2015, 194:5497-5508) or flow-cytometry (Perez LG, Costa MR, Todd CA, Haynes B F, Montefiori DC: Utilîzation of immunoglobulin G Fc receptors by human immunodeficiency virus type 1: a spécifie rôle for antibodies against the membraneproximal extemal région of gp41. J Virol 2009, 83:7397-7410; Piccoli L, Campo I, Fregni CS, Rodriguez BMF, Minola A, Sallusto F, Luisetti M, Corti D, Lanzavecchia A: Neutralization and clearance of GM-CSF by autoantibodies in puhnonary alveolar proteinosis. Nat Commun 2015, 6:1-9).
In general, the antibody according to the présent invention may be glycosylated. N-linked glycans attached to the CH2 domain of a heavy chain, for instance, can influence Clq and FcR binding, with glycosylated antibodies having lower affmity for these receptors. Accordingly, the CH2 domain of the Fc moiety of the antibody according to the présent invention may comprise one or more mutations, in which a glycosylated residue is substituted by a non-glycosylated residue. For example, the antibody's glycans do not lead to a human immunogenic response after administration.
Furthermore, the antibody according to the présent invention can be modified b y introducing (random) amino acid mutations into particular région of the CH2 or CH3 domain of the heavy chain in order to alter their binding affmity for FcR and/or their sérum half-lîfe in comparison to unmodified antibodies. Examples of such modifications include, but are not limited to, substitutions of at least one amino acid from the heavy chain constant région seiected from the group consisting of amino acid residues 250, 314, and 428. Further examples of such Fc modifications are described in Saxena A, Wu D. Advances in Therapeutic Fc Engineering Modulation of IgG-Associated Effector Functions and Sérum Half-life. Front Immunol. 2016;7:580, which is incorporated herein by référencé. In some embodiments, the antibody may comprise the “YTE” mutations (M252Y/S254T/T256E; EU numbering). In some embodiments, the antibody may comprise the mutations M428L and/or N434S in the heavy chain constant région (EU numbering). For example, the antibody may comprise a heavy chain constant région comprising an amino acid sequence as set forth in SEQ ID NO: 103; or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 103, wherein the mutations M428L and N434S are maintained.
In some embodiments, the antibody may comprise a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 100; or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 100, wherein the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively, and the mutations M428L and N434S are maintained.
In some embodiments, the antibody may comprise a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 102; or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 102, wherein the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively, and the mutations M428L and N434S are maintained.
Antibodies of the invention also include hybrid antibody molécules that comprise the six CDRs from an antibody of the invention as defined above and one or more CDRs from another antibody to an antigen. For example, the antibody may be bispecific.
Variant antibodies are also included withîn the scope of the invention. Thus, variants of the sequences recited in the application are also included within the scope of the invention. Such variants include natural variants generated by somatic mutation in vivo during the immune response or in vitro upon culture of immortalized B cell clones. Altematively, variants may arise due to the degeneracy of the genetic code or may be produced due to errors in transcription or translation.
Antibodies of the invention may be provîded in purified form. Typically, the antibody will be présent in a composition that is substantially free of other polypeptides e.g., where less than 90% (by weight), usually less than 60% and more usually less than 50% of the composition is made up of other polypeptides.
Antibodies of the invention may be immunogenic in non-human (or heterologous) hosts e.g., in mi ce. In parti cul ar, the antibodies may hâve an idiotope that is immunogenic in non-human hosts, but not in a human host. In particular, antibodies of the invention for human use include those that cannot be easily isolated from hosts such as mi ce, goats, rabbîts, rats, non-primate mammals, etc. and cannot generalïy be obtained by humanization or from xeno-mice.
Nucleic Acids
In another aspect, the invention also provides a nucleic acid molécule comprising a polynucleotide encoding the antibody according to the présent invention, or an antigen-binding fragment thereof, as described above.
In some embodiments, the polynucleotide encoding the antibody, or an antigen-binding fragment thereof, may be codon-optimîzed.
The nucleic acid molécule may comprise a nucleic acid sequence as set forth in any one of SEQ ID NOs 36 - 39 or 106; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
The présent invention also provides a nucleic acid molécule comprising a polynucleotide according to any one of SEQ ID NOs 40 - 99. Moreover, the présent invention also provides a nucleic acid molécule comprising a polynucleotide according to any one of SEQ ID NOs 40-99 or 106.
In some embodiments, the nucleic acid molécule comprises (î) a polynucleotide according to any one of SEQ ID NOs 40 — 45 and a polynucleotide according to any one of SEQ ID NOs 46 - 51 ; or (ii) a polynucleotide according to any one of SEQ ID NOs 64 - 66, a polynucleotide according to any one of SEQ ID NOs 67 - 69, a polynucleotide according to any one of SEQ ID NOs 70 - 72, a polynucleotide according to any one of SEQ ID NOs 73 - 75, a polynucleotide according to any one of SEQ ID NOs 76 - 78, and a polynucleotide according to any one ofSEQ ID NOs 79-81.
In certain embodiments, the nucleic acid molécule comprises (i) a polynucleotide according to any one of SEQ ID NOs 36, 37, 40 - 45 and 106, and a polynucleotide according to any one ofSEQ ID NOs 38, 46 - 51; or (ii) a polynucleotide according to any one of SEQ ID NOs 64 - 66, a polynucleotide according to any one of SEQ ID NOs 67 - 69, a polynucleotide according to any one of SEQ ID NOs 70 - 72, a polynucleotide according to any one of SEQ ID NOs 73 - 75, a polynucleotide according to any one of SEQ TD NOs 76 — 78, and a polynucleotide according to any one of SEQ ID NOs 79-81.
In some embodiments, the nucleic acid molécule comprises (î) a polynucleotide according to any one of SEQ ID NOs 52 — 57 and a polynucleotide according to any one of SEQ ID NOs 58 - 63; or (ii) a polynucleotide according to any one of SEQ ID NOs 82 - 84, a polynucleotide according to any one of SEQ ID NOs 85 - 87, a polynucleotide according to any one ofSEQ ID NOs 88 - 90, a polynucleotide according to any one of SEQ ID NOs 91 — 93, a polynucleotide according to any one of SEQ ID NOs 94 - 96, and a polynucleotide according to any one ofSEQ IDNOs 97-99.
In certain embodiments, the nucleic acid molécule comprises (î) a polynucleotide according to any one of SEQ ID NOs 52 - 57 and a polynucleotide according to any one of SEQ ID NOs 39, 58 - 63; or (ii) a polynucleotide according to any one of SEQ ID NOs S2 - 84, a polynucleotide according to any one of SEQ ID NOs 85 - 87, a polynucleotide according to any one of SEQ ID NOs 88 - 90, a polynucleotide according to any one of SEQ ID NOs 91 - 93, a polynucleotide according to any one of SEQ ID NOs 94 — 96, and a polynucleotide according to any one of SEQ ID NOs 97 — 99.
The one or more polynucleotide!s) may encode an antibody, in particular or the two variable régions thereof (as described in option (i)) or the six CDRs thereof (as described in option (ii)).
In certain embodiments, the one or more polynucleotide(s) may be selected such that they encode together (i) the six CDRs, (ii) the variable régions VH and VL; or the light and heavy chain of any one of the exemplified antibodies MGUlOvl, MGU10v2, MGU10v3, MGU10v4, MGU10v5, MGUlOvô, MGU10v7, MGU10v8, MGU10v9, or MGH2vl.
Examples of nucleic acid molécules and/or polynucleotides include, e.g., a recombinant polynucleotide, a vector, an oligonucleotide, an RNA molécule such as an rRNA, an mRNA, an mîRNA, an siRNA, or a tRNA, or a DNA molécule such as a cDNA. Nucleic acids may encode the light chain and/or the heavy chain of an antibody. In other words, the light chain and the heavy chain of the antibody may be encoded by the same nucleic acid molécule (e.g., in bicistronic manner). Alternative! y, the light chain and the heavy chain of the antibody may be encoded by distinct nucleic acid molécules.
Due to the redundancy of the genetic code, the présent invention also comprises sequence variants of nucleic acid sequences, which encode the same amino acid sequences. The polynucleotide encoding the antibody (or the complété nucleic acid molécule) may be optimîzed for expression of the antibody. For example, codon optimization ofthe nucléotide sequence may be used to improve the efficiency of translation in expression Systems for the production of the antibody. Moreover, the nucleic acid molécule may comprise heterologous éléments (i.e., éléments, which in nature do not occur on the same nucleic acid molécule as the coding sequence for the (heavy or light chain of) an antibody. For example, a nucleic acid molécule may comprise a heterologous promotor, a heterologous enhancer, a heterologous UTR (e.g., for optimal translation/expression), a heterologous Poly-A-tail, and the like.
A nucleic acid molécule is a molécule comprising nucleic acid components. The term nucleic acid molécule usually refers to DNA or RNA molécules. It may be used synonymous with the term “polynucleotide”, i.e. the nucleic acid molécule may consist of a polynucleotide encoding the antibody. Alternatively, the nucleic acid molécule may also comprise further éléments in addition to the polynucleotide encoding the antibody. Typically, a nucleic acid molécule is a polymer comprising or consisting of nucléotide monomers which are covalently linked to each other by phosphodi ester-bonds of a sugar/phosphate-backbone. The term “nucleic acid molécule” also encompasses modifïed nucleic acid molécules, such as base-modified, sugarmodified or backbone-modified etc. DNA or RNA molécules.
In general, the nucleic acid molécule may be manipulated to insert, delete or alter certain nucleic acid sequences. Changes from such manipulation include, but are not limited to, changes to introduce restriction sites, to amend codon usage, to add or optimize transcription and/or translation regulatory sequences, etc. It is also possible to change the nucleic acid to alter the encoded amino acids. For example, it may be useful to introduce one or more (e.g., 1,2,3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions, délétions and/or insertions into the antibody’s amino acid sequence. Such point mutations can modify effector funétions, antigen-binding affinity, post-translational modifications, immunogenicity, etc., can introduce amino acids for the attachment of covalent groups (e.g., labels) or can introduce tags (e.g., for purification purposes). Alternatively, a mutation in a nucleic acid sequence may be “silent”, i.e. not reflected in the amino acid sequence due to the redundancy of the genetic code. In general, mutations can be introduced in spécifie sites or can be introduced at random, followed by sélection (e.g., molecular évolution). For instance, one or more nucleic acids encoding any of the light or heavy chains of an (exemplary) antibody can be randomly or directionally mutated to introduce different properties in the encoded amino acids. Such changes can be the resuit of an itérative process wherein initial changes are retained and new changes at other nucléotide positions are introduced. Further, changes achîeved in independent steps maybe combined.
In some embodiments, the polynucleotide encoding the antibody, or an antigen-binding fragment thereof, (or the (complété) nucleic acid molécule) may be codon-optimized. The skilled artisan is aware of varions tools for codon optimization, such as those described in: Ju Xîn Chin, Bevan Kai-Sheng Chung, Dong-Yup Lee, Codon Optimization OnLine (COOL): a web-based multiobjective optimization platform for synthetic gene design, Bioinformatics, Volume 30, Issue 15, 1 August 2014, Pages 2210-2212; or in: Grote A, Hiller K, Scheer M, Munch R, Nortemann B,
Hempel DC, Jahn D, JCat: a noveî tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Res. 2005 Jul l;33(Web Server issue):W526-31; or, for example, Genscript’s OptimumGene™ algorithm (as described in US 2011/0081708 Al).
For example, the nucleic acid molécule of the invention may comprise a nucleic acid sequence as set forth in any one of SEQ ID NOs 36 - 39; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
The présent invention also provides a combination of a First and a second nucleic acid molécule, wherein the first nucleic acid molécule comprises a polynucleotide encoding the heavy chain of the antibody, or an antigen-binding fragment thereof, of the présent invention; and the second nucleic acid molécule comprises a polynucleotide encoding the corresponding light chain of the same antibody, or the same antigen-binding fragment thereof. The above description regarding the (general) features of the nucleic acid molécule of the invention applies accordingly to the first and second nucleic acid molécule of the combination. Accordingly, one or both of the polynucleotides encoding the heavy and/or light chain(s) of the antibody, or an antigen-binding fragment thereof, is/are codon-optimized. For example, the combination may comprise a nucleic acid sequence as set forth in any one of SEQ ID NOs 36 - 39 or 106; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
The présent invention also provides a combination of a first and a second nucleic acid molécule, wherein — the first nucleic acid molécule comprises a polynucleotide encoding the heavy chain of an antibody, or an antigen-binding fragment thereof, the polynucleotide comprising: (a) a nucléotide sequence according to any one of SEQ ID NOs 40 - 45; or (b) a nucléotide sequence according to any one of SEQ ID NOs 64 - 66, a nucléotide sequence according to any one of SEQ ID NOs 67 - 69, and a nucléotide sequence according to any one of SEQ ID NOs 70-72; and — the second nucleic acid molécule comprises a polynucleotide encoding the light chain of an antibody, or an antigen-binding fragment thereof, the polynucleotide comprising: (c) a nucléotide sequence according to any one of SEQ ID NOs 46-51; or (d) a nucléotide sequence according to any one of SEQ ID NOs 73 — 75, a nucléotide sequence according to any one of SEQ ID NOs 76 - 78, and a nucléotide sequence according to any one of SEQ ID NOs 79-81.
Again, the above description regarding the (general) features of the nucleic acid molécule of the 5 invention applies accordingly to the first and second nucleic acid molécule of the combination,
The présent invention also provides a combination of a first and a second nucleic acid molécule, wherein — the first nucleic acid molécule comprises a polynucleotide encoding the heavy chain of an 10 antibody, or an antigen-binding fragment thereof, the polynucleotide comprising: (a) a nucléotide sequence according to any one of SEQ ID NOs 36, 37, 40 - 45 and 106; or (b) a nucléotide sequence according to any one of SEQ ID NOs 64 - 66, a nucléotide sequence according to any one of SEQ ID NOs 67 - 69, and a nucléotide sequence according to any one of SEQ ID NOs 70 — 72; and —-the second nucleic acid molécule comprises a polynucleotide encoding the light chain of an antibody, or an antigen-binding fragment thereof, the polynucleotide comprising: (c) a nucleotîde sequence according to any one of SEQ ID NOs 38, 46 - 51; or (d) a nucléotide sequence according to any one of SEQ ID NOs 73 - 75, a nucléotide sequence according to any one of SEQ ID NOs 76 - 78, and a nucléotide sequence according to any one of
SEQ ID NOs 79-81.
The présent invention also provides a combination of a first and a second nucleic acid molécule, wherein
-— the first nucleic acid molécule comprises a polynucleotide encoding the heavy chain of an 25 antibody, or an antigen-binding fragment thereof, the polynucleotide comprising: (a) a nucléotide sequence according to any one of SEQ ID NOs 52 - 57; or (b) a nucléotide sequence according to any one of SEQ ID NOs 82 - 84, a nucleotîde sequence according to any one of SEQ ID NOs 85 - 87, and a nucleotîde sequence according to any one of SEQ ID NOs 88 - 90; and
-— the second nucleic acid molécule comprises a polynucleotide encoding the light chain of an antibody, or an antigen-binding fragment thereof, the polynucleotide comprising: (c) a nucleotîde sequence according to any one of SEQ ID NOs 58 - 63; or (d) a nucleotîde sequence according to any one of SEQ ID NOs 91 - 93, a nucleotîde sequence according to any one of SEQ ID NOs 94 - 96, and a nucléotide sequence according to any one of SEQ ID NOs 97 - 99.
The présent invention also provides a combination of a first and a second nucleic acid molécule, 5 wherein —- the first nucleic acid molécule comprises a polynucleotide encodîng the heavy chain of an antibody, or an antigen-binding fragment thereof, the polynucleotide comprising: (a) a nucléotide sequence according to any one of SEQ ID NOs 52 - 57; or (b) a nucléotide sequence according to any one of SEQ ID NOs S2 - 84, a nucléotide sequence according [O to any one of SEQ ID NOs 85 - 87, and a nucléotide sequence according to any one of
SEQ ID NOs 88 -90; and — the second nucleic acid molécule comprises a polynucleotide encoding the light chain of an antibody, or an antigen-binding fragment thereof, the polynucleotide comprising: (c) a nucléotide sequence according to any one of SEQ ID NOs 39, 58 - 63; or (d) a nucléotide 15 sequence according to any one of SEQ ID NOs 91 - 93, a nucléotide sequence according to any one of SEQ ID NOs 94 - 96, and a nucléotide sequence according to any one of SEQ ID NOs 97-99.
In particular, the first and the second nucleic acid molécules may be selected such that they 20 encode together (i) the six CDRs, (ii) the variable régions VH and VL; or the light and heavy chain of any one of the exemplified antibodies MGUlOvl, MGU10v2, MGU10v3, MGU10v4, MGU10v5, MGUlOvô, MGU10v7, MGU10v8, MGU10v9, or MGH2vl.
Again, the above description regarding the (general) features of the nucleic acid molécule of the 25 invention applies accordingly to the first and second nucleic acid molécule of the combination.
The nucleic acid sequences of SEQ ID NOs 40 - 99 and 106 are codon-optimized for antibody expression.
Vector
Further included within the scope of the invention are vectors, for example, expression vectors, comprising a nucleic acid molécule according to the présent invention. Usually, a vector comprises a nucleic acid molécule as described above.
The présent invention also provides a combination of a first and a second vector, wherein the first vector comprises a first nucleic acid molécule as described above (for the combination of nucleic acid molécules) and the second vector comprises a second nucleic acid molécule as described above (for the combination of nucleic acid molécules), in particular wherein the first and the second nucleic acid molécules are selected from the same (embodiment of) combination of nucleic acid molécules as described above. More specifically, the first and the second nucleic acid molécules may be selected such that they encode together (i) the six CDRs, (ii) the variable régions VH and VL; or the light and heavy chain of any one of the exemplified antibodies MGUlOvl, MGU10v2, MGU10v3, MGU10v4, MGU10v5, MGU10v6, MGU10v7, MGU10v8, MGU10v9, or MGH2vl.
A vector is usually a recombinant nucleic acid molécule, i.e. a nucleic acid molécule which does not occur in nature. Accordîngly, the vector may comprise heterologous éléments (i.e., sequence éléments of different origin in nature). For example, the vector may comprise a multi cloning site, a heterologous promotor, a heterologous enhancer, a heterologous sélection marker (to identify cells comprising said vector in comparison to cells not comprising saîd vector) and the like. A vector in the context of the présent invention is suitable for incorporating or harboring a desired nucleic acid sequence. Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors etc. A storage vector is a vector which allows the convenient storage of a nucleic acid molécule. Thus, the vector may comprise a sequence corresponding, e.g., to a (heavy and/or light chain of a) desired antibody according to the présent invention. An expression vector may be used for production of expression products such as RNA, e.g. mRNA, or peptides, polypeptides or proteins. For example, an expression vector may comprise sequences needed for transcription of a sequence stretch of the vector, such as a (heterologous) promoter sequence. A cloning vector is typically a vector that contains a cloning site, which may be used to incorporate nucleic acid sequences into the vector. A cloning vector may be, e.g., a plasmid vector or a bactériophage vector. A transfer vector may be a vector which is suitable for transferring nucleic acid molécules into cells or organisms, for example, viral vectors. A vector in the context of the présent invention may be, e.g., an RNA vector or a DNA vector. For example, a vector in the sense of the présent application comprises a cloning site, a sélection marker, such as an antibiotic résistance factor, and a sequence suitable for multiplication of the vector, such as an origin of réplication. A vector in the context of the présent application may be a plasmid vector.
Cells
In a further aspect, the présent invention also provides cell expressing the antibody according to the présent invention; and/or comprising the vector (or the combination of vectors) according the présent invention.
Examples of such cells include but are not limited to, eukaryotic cells, e.g., yeast cells, animal cells or plant cells. Other examples of such cells include but are not limited, to prokaryotic cells, e.g. E. coli. In some embodiments, the cells are mammalian cells, such as a mammalian cell line. Examples include human cells, CHO cells, HEK293T cells, PER.C6 cells, NSO cells, human liver cells, myeloma cells or hybridoma cells.
The cell may be transfected with a vector according to the présent invention, for example with an expression vector. The term “transfectîon” refers to the introduction of nucleic acid molécules, such as DNA or RNA (e.g. mRNA) molécules, into cells, e.g. into eukaryotic or prokaryotic cells. In the context of the présent invention, the tenn “transfectîon” encompasses any method known to the skilled person for introducing nucleic acid molécules into cells, such as into mammalian cells. Such methods encompass, for example, electroporation, lipofection, e.g. based on cationic lipids and/or liposomes, calcium phosphate précipitation, nanopartide based transfectîon, virus based transfectîon, or transfectîon based on cationic polymers, such as DEAEdextran or polyethylenimine etc. In some embodiments, the introduction is non-viral.
Moreover, the cells of the présent invention may be transfected stably or transiently with the vector according to the présent invention, e.g. for expressing the antibody according to the présent invention. In some embodiments, the cells are stably transfected with the vector according to the présent invention encoding the antibody according to the présent invention. In other embodiments, the cells are transiently transfected with the vector according to the présent invention encoding the antibody according to the présent invention.
Accordingly, the présent invention also provides a recombinant host cell, which heterologously expresses the antibody of the invention or the antigen-binding fragment thereof. For example, the cell may be of another specîes than the antibody (e.g., CHO cells expressing human antibodies). In some embodiments, the cell type of the cell does not express (such) antibodies in nature. Moreover, the host cell may impart a post-translational modification (PTM; e.g., glycosylation) on the antibody that is not présent in their native State. Such a PTM may resuit in a functional différence (e.g., reduced immunogenicity). Accordingly, the antibody of the invention, or the antigen-binding fragment thereof, may hâve a post-translational modification, which is distinct from the naturally produced antibody (e.g., an antibody of an immune response in a human).
Production of Antibodies
Antibodies according to the invention can be made by any method known in the art. For example, the general methodology for making monoclonal antibodies using hybridoma technology is well known (Kohler, G. and Milstein, C}. 1975; Kozbar et al. 1983). In some embodiments, the alternative EBV immortalization method described in WO2004/076677 is used.
In some embodiments, the method as described in WO 2004/076677, which is incorporated herein by reference, is used. In this method B cells producing the antibody of the invention are transformed with EBV and a polyclonal B cell activator. Additional stimulants of cellular growth and différentiation may optionally be added during the transformation step to further enhance the efficiency. These stimulants may be cytokines such as IL-2 and IL-15. In one aspect, IL-2 is added during the immortalization step to further improve the efficiency of immortalization, but its use is not essential. The immortalized B cells produced using these methods can then be cultured using methods known in the art and antibodies isolated therefrom.
Another exemplified method is described in WO 2010/046775. In this method plasma cells are cultured in limited numbers, or as single plasma cells in microwell culture plates. Antibodies can be isolated from the plasma cell cultures. Further, from the plasma cell cultures, RNA can be extracted and PCR can be performed using methods known in the art. The VH and VL régions of the antibodies can be amplified by RT-PCR (reverse transcriptase PCR), sequenced and cloned into an expression vector that is then transfected into HEK293T cells or other host cells. The cloning of nucleic acid in expression vectors, the transfection of host cells, the culture of the transfected host cells and the isolation of the produced antibody can be done using any methods known to one of skill in the art.
The antibodies may be further purified, if desired, using filtration, centrifugation and various chromatographie methods such as HPLC or affinity chromatography. Techniques for purification of antibodies, e.g., monoclonal antibodies, including techniques for producing pharmaceutical-grade antibodies, are well known in the art.
Standard techniques of molecular biology may be used to préparé DNA sequences encoding the antibodies of the présent invention. Desired DNA sequences may be synthesized completely or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain réaction (PCR) techniques may be used as appropriate.
Any suîtable host cell/vector System may be used for expression of the DNA sequences encoding the antibody molécules of the présent invention. Eukaryotîc, e.g., mammalian, host cell expression Systems may be used for production of antibody molécules, such as complété antibody molécules. Suitable mammalian host cells include, but are not limited to, CHO, HEK293T, PER.C6, NSO, myeloma or hybridoma cells. Also, prokaryotic, e.g. bacterial host cell expression Systems may be used for the production of antibody molécules, such as complété antibody molécules. Suitable bacterial host cells include, but are not limited to, E. coli cells.
The présent invention also provides a process for the production of an antibody molécule according to the présent invention comprising culturing a (heterologous) host cell comprising a vector encoding a nucleic acid of the présent invention under conditions suitable for expression of protein from DNA encoding the antibody molécule of the présent invention, and isolating the antibody molécule.
For production of the antibody comprising both heavy and light chains, a cell line may be transfected with two vectors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Altematively, a single vector may be used, the vector including sequences encoding light chain and heavy chain polypeptides.
Antibodies according to the invention may be produced by (î) expressing a nucleic acid sequence according to the invention in a host cell, e.g. by use of a vector according to the présent invention, and (ii) isolating the expressed antibody product. Additionally, the method may include (iü) purifying the isolated antibody. Transformed B cells and cultured plasma cells may be screened for those producing antibodies of the desired specificity or function.
The screening step may be carried ont by any immunoassay, e.g., ELIS A, by staining of tissues or cells (including transfected cells), by neutralization assay or by one of a number of other methods known in the art for identifying desired specificity or function. The assay may select on the basis of simple récognition of one or more antigens, or may select on the additional basis of a desired function e.g., to select neutralizing antibodies rather than just antigen-binding antibodies, to select antibodies that can change characteristics of targeted cells, such as their signaling cascades, their shape, their growth rate, their capability of influencing other cells, their response to the influence by other cells or by other reagents or by a change in conditions, their différentiation status, etc.
Individual transformed B cell clones may then be produced from the positive transformed B cell culture. The cloning step for separating individual clones from the mixture of positive cells may be carried ont using limiting dilution, micro manipulai ion, single cell déposition by cell sorti ng or another method known in the art.
Nucleic acid from the cultured plasma cells can be isolated, cloned and expressed in HEK293T cells or other known host cells using methods known in the art.
The immortalized B cell clones or the transfected host-cells of the invention can be used in varions ways e.g., as a source of monoclonal antibodies, as a source of nucleic acid (DNA or mRNA) encoding a monoclonal antibody of interest, for research, etc.
The invention also provides a composition comprising immortalized B memory cells or transfected host cells that produce antibodies according to the présent invention.
The immortalized B cell clone or the cultured plasma cells of the invention may also be used as a source of nucleic acid for the cloning of antibody genes for subséquent recombinant expression. Expression from recombinant sources may be more common for pharmaceutical purposes than expression from B cells or hybridomas e.g., for reasons of stability, reproducibility, culture ease, etc.
Thus the invention also provides a method for preparing a recombinant cell, comprising the steps of: (i) obtaining one or more nucleic acids (e.g., heavy and/or light chain mRNAs) from the B cell clone or the cultured plasma cells that encodes the antibody of interest; (ii) inserting the nucleic acid into an expression vector and (iii) transfecting the vector into a (heterologous) host cell in order to permit expression of the antibody of interest in that host cell.
Similarly, the invention also provides a method for preparing a recombinant cell, comprising the steps of: (i) sequencing nucleic acid(s) from the B cell clone or the cultured plasma cells that encodes the antibody of interest; and (ii) using the sequence information from step (i) to préparé nucleic acid(s) for insertion into a host cell in order to permit expression of the antibody of interest in that host cell. The nucleic acid may, but need not, be manipulated between steps (i) and (ii) to introduce restriction sites, to change codon usage, and/or to optimize transcription and/or translation regulatory sequences.
Furthermore, the invention also provides a method of preparing a transfected host cell, comprising the step of transfecting a host cell with one or more nucleic acids that encode an antibody of interest, wherein the nucleic acids are nucleic acids that were derived from an immortalized B cell clone or a cultured plasma cell of the invention. Thus the procedures for first preparing the nucleic acid(s) and then using it to transfect a host cell can be performed at different times b y different people in different places (e.g., in different countries).
These recombinant cells of the invention can then be used for expression and culture purposes. They are particularly useful for expression of antibodies for large-scale pharmaceutical production. They can also be used as the active ingrédient of a pharmaceutical composition. Any suitable culture technique can be used, încluding but not limited to static culture, roller bottle culture, ascites fluid, hollow-fiber type bioreactor cartridge, modular minifermenter, stirred tank, microcarrier culture, ceramic core perfusion, etc.
Methods for obtaining and sequencing immunoglobulin genes from B cells or plasma cells are well known in the art (e.g., see Chapter 4 of Kuby Immunology, 4th édition, 2000).
The transfected host cell may be a eukaryotîc cell, încluding yeast and animal cells, particularly mammalian cells (e.g., CHO cells, NS0 cells, human cells such as PER.C6 or HKB-11 cells, myeloma cells, or a human liver cell), as well as plant cells. In some embodiments, the transfected host cell is a mammalian cell, such as a human cell. In some embodiments, expression hosts can glycosylate the antibody of the invention, particularly with carbohydrate structures that are not themselves immunogenic in humans. In some embodiments the transfected host cell may be able to grow in serum-free media. In further embodiments the transfected host cell may be able to grow in culture without the presence of animal-derived products, The transfected host cell may also be cultured to give a cell line.
The invention also provides a method for preparîng one or more nucleic acid molécules (e.g., heavy and light chain genes) that encode an antibody of interest, comprising the steps of: (i) preparing an immortalized B cell clone or culturing plasma cells according to the invention; (iî) obtaining from the B cell clone or the cultured plasma cells nucleic acid that encodes the antibody of interest. Further, the invention provides a method for obtaining a nucleic acid sequence that encodes an antibody of interest, comprising the steps of: (i) preparing an immortalized B cell clone or culturing plasma cells according to the invention; (ii) sequencing nucleic acid from the B cell clone or the cultured plasma cells that encodes the antibody of interest.
The invention further provides a method of preparing nucleic acid molecule(s) that encode an antibody of interest, comprising the step of obtaining the nucleic acid that was obtained from a transformed B cell clone or cultured plasma cells of the invention. Thus the procedures for first obtaining the B cell clone or the cultured plasma cell, and then obtaining nucleic acid(s) from the B cell clone or the cultured plasma cells can be performed at different times by different people in different places (e.g., in different countries).
The invention also comprises a method for preparing an antibody (e.g., for pharmaceutical use) according to the présent invention, comprising the steps of: (i) obtaining and/or sequencing one or more nucleic acids (e.g., heavy and light chain genes) from the seiected B cell clone or the cultured plasma cells expressing the antibody of interest; (ii) inserting the nucleic acîd(s) into or using the nucleic acîd(s) sequence(s) to préparé an expression vector; (iii) transfecting a host cell that can express the antibody of interest; (iv) culturing or sub-culturing the transfected host cells under conditions where the antibody of interest is expressed; and, optionally, (v) purifying the antibody of interest.
The invention also provides a method of preparing the antibody of interest comprising the steps of: culturing or sub-culturing a transfected host cell population, e.g. a stably transfected host cell population, under conditions where the antibody of interest is expressed and, optionally, purifying the antibody of interest, wherein said transfected host cell population has been prepared by (î) providing nucleic acid(s) encoding a selected antibody of interest that is produced by a B cell clone or cultured plasma cells prepared as described above, (ii) inserting the nucleic acid(s) into an expression vector, (iii) transfecting the vector in a host cell that can express the antibody of interest, and (iv) culturing or sub-culturing the transfected host cell comprising the inserted nucleic acids to produce the antibody of interest. Thus the procedures for first preparing the recombinant host cell and then culturing it to express antibody can be perfonned at very different times by different people in different places (e.g., in different countries).
Pharmaceutical Composition
The présent invention also provides a pharmaceutical composition comprising one or more of (i) the antibody of to the présent invention, or an antigen-binding fragment thereof;
(îi) the nucleic acid or a combination of nucleic acids according to the présent invention;
(iii) the vector or a combination of vectors according to the présent invention; and/or (iv) the cell expressing the antibody according to the présent invention or comprising the vector according to the présent invention and, optionally, a pharmaceutically acceptable diluent or carrier.
In other words, the présent invention also provides a pharmaceutical composition comprising the antibody according to the présent invention, the nucleic acid according to the présent invention, the vector according to the présent invention and/or the cell according to the présent invention.
The pharmaceutical composition may optionally also contain a pharmaceutically acceptable carrier, diluent and/or excipient. Although the carrier or excipient may facilitate administration, ît should not itself induce the production of antibodies harmful to the individual receiving the composition. Nor should it be toxic. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus partiel es. In some embodiments, the pharmaceutically acceptable carrier, diluent and/or excipient in the pharmaceutical composition according to the présent invention is not an active component in respect to P. falciparum infection and/or malaria.
Pharmaceutically acceptable salts can be used, for example minerai acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organie acids, such as acétates, propionates, malonates and benzoates.
Pharmaceutically acceptable carriers in a pharmaceutical composition may additionally contain liquids such as water, saline, glycerol and éthanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be présent in such compositions, Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the subject.
Pharmaceutical compositions of the invention may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g., a lyophilized composition, similar to Synagis™ and Herceptin®, for reconstitution 15 with stérile water containing a preservative). The composition may be prepared for topical administration e.g., as an ointment, cream or powder. The composition may be prepared for oral administration e.g., as a tablet or capsule, as a spray, or as a syrup (optionally flavored). The composition may be prepared for pulmonary administration e.g., as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The 20 composition may be prepared for nasal, aurai or ocular administration e.g., as drops. The composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a subject. For example, a lyophilized antibody may be provided in kit form with stérile water or a stérile buffer.
In some embodiments, the (only) active ingrédient in the composition is the antibody according to the présent invention. As such, it may be susceptible to dégradation in the gastrointestinal tract. Thus, if the composition is to be adminîstered by a route using the gastrointestinal tract, the composition may contain agents which protect the antibody from dégradation but which release the antibody once it has been absorbed from the gastrointestinal tract.
A thorough discussion of pharmaceutically acceptable carriers is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th édition, ISBN: 0683306472.
Pharmaceutical compositions of the invention generally hâve a pH between 5.5 and 8.5, in some embodiments this may be between 6 and 8, for example about 7. The pH may be maintained by the use of a buffer. The composition may be stérile and/or pyrogen free. The composition may be isotonie with respect to humans. In some embodiments pharmaceutical compositions of the invention are supplied in hermetîcaliy-sealed containers.
Within the scope of the invention are compositions présent in several forms of administration; the forms include, but are not limited to, those forms suitable for parentéral administration, e.g., by injection or infusion, for ex ample by bolus injection or continuons infusion. Where the product is for injection or infusion, it may take the form of a suspension, solution or émulsion in an oily or aqueous vehicle and it may contain formulatory agents, such as suspending, preservative, stabilizing and/or dispersîng agents. Altematively, the antibody may be in dry form, for reconstitution before use with an appropriate stérile liquid,
A vehicle is typically understood to be a material that is suitable for storing, transporting, and/or administering a compound, such as a pharmaceutically active compound, in particular the antibodies according to the présent invention. For example, the vehicle may be a physiologically acceptable liquid, which is suitable for storing, transporting, and/or administering a pharmaceutically active compound, in particular the antibodies according to the présent invention. Once formulated, the compositions of the invention can be administered directly to the subject. In some embodiments the compositions are adapted for administration to mammalian, e.g,, human subjects.
The pharmaceutical compositions of this invention may be administered by any number of routes including, but not limited to, oral, intravenous, întramuscular, intra-arterial, intram edullary, intraperitoneal, intrathecal, intraventricular, transdermal, transcutaneous, topical, subeutaneous, intranasal, enterai, sublingual, intravaginal or rectal routes. Hyposprays may also be used to administer the pharmaceutical compositions of the invention. Optionally, the pharmaceutical composition may be prepared for oral administration, e.g. as tablets, capsules and the like, for topical administration, or as injectable, e.g. as liquid solutions or suspensions. In some embodiments, the pharmaceutical composition is an injectable. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection are also encompassed, for example the pharmaceutical composition may be in lyophilized form.
For injection, e.g. intra venons, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingrédient may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicîty and stability. Those of relevant skill in the art are well able to préparé suitable solutions using, for example, isotonie vehîcles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer’s Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required. Whether it is an antibody, a peptide, a nucleic acid molécule, or another pharmaceutically useful compound according to the présent invention that is to be given to an individual, administration is usually in a “prophylactically effective amount11 or a ”therapeutîcally effective amount” (as the case may be), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will dépend on the nature and severity of what is being treated. For injection, the pharmaceutîcal composition according to the présent invention may be provided for example in a pre-fdled syringe.
The inventive pharmaceutîcal composition as defined above may also be administered orally in any orally acceptable dosage form încluding, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnésium stéarate, are also typically added. For oral administration in a capsule fonn, useful diluents include lactose and dried comstarch. When aqueous suspensions are required for oral use, the active ingrédient, i.e. the inventive transporter cargo conjugate molécule as defined above, is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
The inventive pharmaceutîcal composition may also be administered topically, especially when the target of treatment includes areas or organs readîly accessible by topical application, e.g. încluding accessible épithélial tissue. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the inventive pharmaceutîcal composition may be formulated in a suitable ointment, containing the inventive pharmaceutîcal composition, particularly its components as defined above, suspended or dissolved in one or more carriers. Carriers for topical administration include, but are not lîmited to, minerai oil, liquîd petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Altematively, the inventive pharmaceutîcal composition can be formulated in a suitable lotion or cream. In the context of the présent invention, suitable carriers include, but are not limited to, minerai oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
Dosage treatment may be a single dose schedule or a multiple dose schedule. In particular, the pharmaceutical composition may be provided as single-dose product. In some embodiments, the amount of the antibody in the pharmaceutical composition - in particular if provided as singledose product - does not exceed 200 mg, for example it does not exceed 100 mg or 50 mg.
For a single dose, e.g. a daily, weekly or monthly dose, the amount of the antibody in the pharmaceutical composition according to the présent invention, may not exceed 1 g or 500 mg. In some embodiments, for a single dose, the amount of the antibody in the pharmaceutical composition according to the présent invention, may not exceed 200 mg, or 100 mg. For example, for a single dose, the amount of the antibody in the pharmaceutical composition according to the présent invention, may not exceed 50 mg.
Pharmaceutical compositions typically include an “effective” amount of one or more antibodies of the invention, i.e. an amount that is sufficient to treat, ameliorate, attenuate, reduce or prevent a desired disease or condition, or to exhibit a détectable therapeutic effect. Therapeutic effects also include réduction or atténuation in pathogenic potency or physical symptoms. The précisé effective amount for any particular subject will dépend upon their size, weight, and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. The effective amount for a gîven situation is detennined by routine expérimentation and is within the judgment of a clinician. For purposes of the présent invention, an effective dose may generally be from about 0.005 to about 100 mg/kg, for example from about 0.0075 to about 50 mg/kg or from about 0.01 to about 10 mg/kg. In some embodiments, the effective dose will be from about 0.02 to about 5 mg/kg, of the antibody of the présent invention (e.g. amount of the antibody in the pharmaceutical composition) in relation to the bodyweight (e.g., in kg) of the individual to which it is administered.
Moreover, the pharmaceutical composition according to the présent invention may also comprise an additional active component, which may be a further antibody or a component, which is not an antibody. Accordingly, the pharmaceutical composition according to the présent invention may comprise one or more of the additional active components.
The antibody according to the présent invention can be présent either in the same pharmaceutical composition as the additional active component or, altematively, the antibody according to the présent invention is comprised by a first pharmaceutical composition and the additional active component is comprised by a second pharmaceutical composition different from the first pharmaceutical composition. Accordingly, if more than one additional active component is envisaged, each additional active component and the antibody according to the présent invention may be comprised in a different pharmaceutical composition. Such different pharmaceutical compositions may be administered either combined/simultaneously or at separate times or at separate locations (e.g. separate parts of the body).
The antibody according to the présent invention and the additional active component may provide an additive therapeutic effect, such as a synergistic therapeutic effect. The term “synergy” is used to describe a combined effect of two or more active agents that is grenier than the sum of the individual effects of each respective active agent. Thus, where the combined effect of two or more agents results in “synergistic inhibition” of an activity or process, it is intended that the inhibition of the activity or process is greater than the sum of the inhibitory effects of each respective active agent. The term “synergistic therapeutic effect” refers to a therapeutic effect observed with a combination of two or more thérapies wherein the therapeutic effect (as measured by any of a number of parameters) is greater than the sum of the individual therapeutic effects observed with the respective individual thérapies.
In some embodiments, a composition of the invention may include antibodies of the invention, wherein the antibodies may make up at least 50% by weight (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) of the total protein in the composition. In the composition of the invention, the antibodies may be in purified form.
The présent invention also provides a method of preparing a phannaceutical composition comprising the steps of: (i) preparing an antibody of the invention; and (ii) admixing the purified antibody with one or more pharmaceutically-acceptable carriers.
In other embodiments, a method of preparing a pharmaceutical composition comprises the step of: admixing an antibody with one or more phannaceutically-acceptable carriers, wherein the antibody is a monoclonal antibody that was obtained from a transformed B cell or a cultured plasma cell of the invention.
As an alternative to delivering antibodies or B cells for therapeutic purposes, it is possible to deliver nucleic acid (typically DNA) that encodes the monoclonal antibody of interest derived from the B cell or the cultured plasma cells to a subject, such that the nucleic acid can be expressed in the subject in situ to provide a desired therapeutic effect. Suitable gene therapy and nucleic acid delivery vectors are known in the art.
Pharmaceutical compositions may include an antimicrobial, particularly if packaged in a multiple dose format. They may comprise detergent e.g., a Tween (polysorbate), such as Tween 80. Détergents are generally présent at low levels e.g., less than 0.01%. Compositions may also include sodium salts (e.g., sodium chloride) to give tonicity. For example, a concentration of 10+2mg/nil NaCl is typical.
Further, pharmaceutical compositions may comprise a sugar alcohol (e.g., mannitol) or a disaccharide (e.g., sucrose or trehalose) e.g., at around 15-30 mg/ml (e.g., 25 mg/ml), particularly if they are to be lyophilized or if they include material which has been reconstituted from lyophilized material. The pH of a composition for lyophilîzation may be adjusted to between 5 and 8, or between 5.5 and 7, or around 6.1 prior to lyophilîzation.
The compositions of the invention may also comprise one or more immunoregulatory agents. In some embodiments, one or more of the immunoregulatory agents include(s) an adjuvant.
Medical Treatments and Uses
In a further aspect, the présent invention provides the use of the antibody according to the présent invention, or an antigen-binding fragment thereof, the nucleic acid molécule (or the combination of nucleic acid molécules) according to the présent invention, the vector (or the combination of vectors) according to the présent invention, the cell according to the présent invention or the pharmaceutical composition according to the présent invention in prophylaxis and/or treatment of malaria; or in (ii) dîagnosis of malaria. Accordingly, the présent invention also provides a method of reducing malaria, or lowering the risk of P. falciparum infection, comprising: administering to a subject in need thereof, a therapeutically effective amount of the antibody, or an antigen-binding fragment thereof, according to the présent invention, the nucleic acid molécule (or the combination of nucleic acid molécules) according to the présent invention, the vector (or the combination of vectors) according to the présent invention, the cell according to the présent invention or the pharmaceutical composition according to the présent invention. Moreover, the présent invention also provides the use of the antibody according to the présent invention, or an antigen-binding fragment thereof, the nucleic acid molécule (or the combination of nucleic acid molécules) according to the présent invention, the vector (or the combination of vectors) according to the présent invention, the cell according to the présent invention or the phannaceutical composition according to the présent invention in the manufacture of a médicament for prophylaxis, treatment or atténuation of malaria.
Methods of diagnosis may include contacting an antibody with a sample. Such samples may be isolated from a subject, for example an isolated tissue sample taken from, for example, nasal passages, sinus cavities, salivary glands, lung, liver, pancréas, kidney, ear, eye, placenta, alimentary tract, heart, ovanes, pituitary, adrenals, thyroid, brain, skin or blood, such as plasma or sérum. The methods of diagnosis may also include the détection of an antigen/antibody complex, in particular following the contacting of an antibody with a sample. Such a détection step is typically performed at the bench, i.e. without any contact to the human or animal body. Examples of détection methods are well-known to the person skilled in the art and include, e.g., ELISA (enzyme-linked immunosorbent assay).
Prophylaxis of malaria refers in particular to prophylactic settings, wherein the subject was not diagnosed with malaria (either no diagnosis was performed or diagnosis results were négative) and/or the subject does not show symptoms of malaria. In therapeutic settings, in contrast, the subject is typically diagnosed with malaria and/or showing symptoms of malaria. Of note, the tenns “treatment” and “therapy”/”therapeutic” of malaria include (complété) cure as well as attenuation/reductîon of malaria and/or related symptoms.
BRÏEF DESCRIPTION OF THE FIGURES
In the following a brief description of the appended figures will be given. The figures are intended to illustrate the présent invention in more detail. However, they are not întended to limit the subject matter of the invention in any way.
Figure 1 shows for Example 3 the results of the sporozoite invasion/maturation assay for a control antibody (A) and for antibodies MGU 10 (B), MGU10v2 (C) and MGU10v2_LS (D) with fïve dilutions tested for each antibody.
5 Figure 2 shows for Example 4 the results of the sporozoite gliding assay for a control antibody (A) and for antibodies MGU 10 (B) and MGU10v2_LS (C) with fïve dilutions tested for each antibody.
Figure 3 shows for Exampïe 5 the results of the sporozoite traversai assay for a control
10 antibody (A) and for antibodies MGU10 (B) and MGU10v2_LS (C) with fïve dilutions tested for each antibody.
Figure 4 shows for Example 6 the dimerization (Dimers) and aggregation (HMWs) of antibodies MGU10v2_LS (upper panel) and MGU10_LS (lower panel) at 40°C in
15 two distinct buffers as indicated.
Figure 5 20 shows for Example 7 the results of the in vivo protection conferred by MGU 10 antibodies in a challenge study; with bioluminescence (A) and percent inhibition (B).
F igure 6 shows for Ex ample S the results of the in vivo protection conferred by MGH2 antibodies in a challenge study; with bioluminescence (A) and percent inhibition (B).
25 Figure 7 shows for Example 9 the binding of MGU10v2 and MGUlOvS to peptides NANP (A) and NPDP19 (B).
EXAMPLES
In the following, particular examples illustrating varions embodiments and aspects of the invention are presented. However, the présent invention shah not to be limited in scope by the spécifie embodiments described herein. The following préparations and examples are gîven to enable those ski lied in the art to more clearly understand and to practice the présent invention. The présent invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally équivalent are within the scope of the invention. Indeed, varions modifications of the invention in addition to those described herein will beconie readily apparent to those skîlled in the art from the foregoing description, accompanyîng figures and the examples below. Ail such modifications fall within the scope of the appended daims.
Example 1: Design of variants of antibodies MGU10 and MGH2
Recently, very potent anti-malaria antibodies were described, which are spécifie for Plasmodium falciparum circumsporozoite protein (CSP) (Tan J, Sack BK, Oyen D, et al. A public antibody lineage that potently inhibits malaria infection through dual binding to the circumsporozoite protein. Nat Med. 2018;24(4):401-407. doi:10.1038/nm.4513). The most potent dual-specific antibodies described in this study include antibodies “MGU10” and “MGH2”.
Based thereon, the présent inventors designed the following variants of MGU10 (SEQ ID NOs: 1 -8) and MGH2 (SEQ ID NOs: 17 - 27), which exhibit amino acid mutations in the heavy and/or light chain of said reference antibodies:
1. MGUIOvariantl (MGUlOvl), which differs from MGU10 in the framework régions (FR) of the heavy chain variable région (VH; SEQ ID NO: 11);
2. MGU10variant2 (MGU10v2), which differs from MGU10 in the heavy chain CDR3 (CDRH3: SEQ ID NO: 12; VH: SEQ IDNO: 13);
3. MGU10variant3 (MGU10v3), which differs from MGU10 in the light chain CDR3 (CDRL3: SEQ ID NO: 14; light chain variable région (VL): SEQ ID NO: 15);
4. MGU 10variant4 (MGU10v4), which represents a combination of MGUlOvl and
MGU10v2 and, therefore, differs from MGU 10 in the heavy chain CDR3 (CDRH3; SEQ ID NO: 12) and in the heavy chain FR (VH; SEQ ID NO: 16);
5. MGU 10vanant5 (MGU10v5), which represents a combination of MGUlOvl and
MGU10v3 (including the VH of MGUlOvl and the VL of MGU10v3) and, therefore, differs from MGU10 in the heavy chain FR (VH; SEQ ID NO: 11) and in the CDRL3 (CDRL3: SEQ ID NO: 14; VL: SEQ ID NO: 15);
6. MGU 1 Ovariantô (MGUlOvô), which represents a combination of MGU10v2 and
MGU10v3 (including the VH of MGU10v2 and the VL of MGU10v3) and, therefore, differs from MGU 10 in the CDRH3 (CDRH3: SEQ ID NO; 12; VH; SEQ ID NO: 13) and in the CDRL3 (CDRL3: SEQ ID NO: 14; VL: SEQ ID NO: 15);
7. MGU 10variant? (MGU10v7), which represents a combination of MGUlOvl, MGU10v2 and MGU10v3 (including the VH of MGU 10v4 and the VL of MGU10v3) and. therefore, differs from MGU10 in the CDRH3 (SEQ ID NO; 12) and heavy chain FR (VH; SEQ ID NO: 16) and intheCDRL3 (CDRL3: SEQ ID NO: 14; VL: SEQ ID NO: 15); and
8. MGH2variantl (MGH2vl), which differs from MGH2 in the light chain CDR3 (CDRL3: SEQ ID NO: 28; VL: SEQ ID NO: 29).
An overview over the SEQ ID NOs of CDR and VH/VL sequences of the variants in comparison with MGU 10 and MGH2, respectively, is provide in Table 2:
Antibody name Heavy chain Light chain
CDR1 CDR2 CDR3 VH CDR1 CDR2 CDR3 VL
MGU 10 1 2 3 7 4 5 6 8
MGUlOvl 1 2 3 11 4 5 6 8
MGU10v2 1 2 12 13 4 5 6 8
MGU10v3 1 2 3 7 4 5 14 15
MGU10v4 1 2 12 16 4 5 6 8
MGU10v5 1 2 3 11 4 5 14 15
MGU10v6 1 2 12 13 4 5 14 15
MGU10v7 1 2 12 16 4 5 14 15
MGH2 17 18 19 24 20 21/22 23 25
MGH2vl 17 18 19 24 20 21/22 28 29
Table 2.
Example 2: Antibodies of the invention show dual specificity
Tan et al., 201S showed that the most potent antibodies of their study - including MGU10 and MGH2 - simultaneously target epitopes in (i) the NANP-repeat région of CSP and (ii) an Nterminal région of CSP covering the jonction between the N-terminal domain and the NANPrepeats (Tan J, Sack BK, Oyen D, et al. A public antibody lineage that potently inhibits malaria infection through dual binding to the circumsporozoite protein. Nat Med. 2018;24(4):401-407. doî:10.1038/nm.4513). Moreover, this study showed that the extreme potency ofthose antibodies was due to their dual specificity, while antibodies targeting only one of the CSP epitopes were typically less potent.
Accordingly, antibodies of the présent invention were produced and tested for their capacity to bind to both of the epitopes described by Tan et al., 2018: (i) the NANP-repeat région of CSP and (ii) the N-terminal région of CSP covering the junction between the N-terminal domain and the NANP-repeats.
To this end, antibodies MGUlOvl, MGU10v2, MGU10v3 and MGH2vl were produced. Namely, the antibodies were synthetized by Genscript and subcloned in vectors for the expression of IgGl and kappa or lambda chains. The purified plasmids of heavy and light chains were combined and used to transfect Expi293F cells (ThermoFisher Scientific) using polyethylenimine (micro-scale transfection (600 μΐ) in a 96-well plate). The transfected cells were harvested on day 6 and supematants were collected by centrifugation and filtration.
Total IgGs présent in the supematants were quantified using 96-well MaxiSorp plates (Nunc) coated with 10 pg/ml goat anti-human IgG (SouthemBiotech). Plates were then blocked with PBS with 1% BSA and incubated with titrated monoclonal antibodies, using Certified Référencé Material 470 (ERMs-DA470, Sigma-Aldrich) as a standard. Plates were then washed and 69 încubated with 1/500 alkaline phosphatase (AP)-conjugated goat anti-human IgG (Southern Bîotech). Substrate (para-nitrophenyl phosphate (p-NPP), Sigma) was added and plates were read at wavelength of 405 nm to déterminé optical density (OD) values.
To test spécifie antibody bînding to (i) the NANP-repeat région of CSP and (ii) the N-terminal région of CSP covering the junction between the N-terminal domain and the NANP-repeats, ELISA plates were coated with 10 pg/ml of avidin (Sigma). Plates were blocked with PBS with 1% BSA and încubated with 1 pg/ml biotinylated NANP-peptide (SEQ ID NO; 34) or with 1 pg/ml biotinylated NPDP-peptide (SEQ ID NO; 35). The plates were washed and încubated with titrated monoclonal antibodies, followed by 1/500 AP-conjugated goat anti-human IgG (Southern Bîotech) and pNPP substrate. EC50 (ng/ml) values were calculated for every sample tested by nonlinear régression analysis using the GraphPad Prism 7 software.
The results are shown in Table 3 for MGU10 and its variants MGUlOvl, MGU10v2 and
MGU10v3 and in Table 4 for MGH2 and its variant MGH2vl ;
Antibody N AN P ECM (ng/ml) NPDP EC5U (ng/ml)
MGU10 34.3 26.1
MGUlOvl 28.51 22.54
MGUlOvl 26.79 20.45
MGU10v3 26.28 25.08
Table 3.
Antibody NANP EC5o (ng/ml) NPDP EC50 (ng/ml)
MGH2 27.88 48.8
MGH2vl 22.32 65.82
Table 4.
Surprisingly, ail variants tested for MGU10 show an increased binding affinîty for both CSP epitopes, while MGH2vl only shows an increased binding affinîty for the NANP-repeat région in comparison to MGH2.
Example 3: Sporozoite invasion/maturation assay
Variant antibody MGU10v2 was selected for further characterization in functional assays. The sporozoite invasion/maturation assay is a functional assay for testing the antibodies’ effects on the infectîvity of sporozoites. Sporozoite invasion of hépatocytes and subséquent maturation into exoerythrocytic forms is an essential step in the establishment of malaria infection.
Cryopreserved human primary hépatocytes were seeded in a microtiter plate and incubated for 2 days. Salivary gland Plasmodium falcipantm NF54 sporozoites were isolated from An. stephensi mosquitoes infected with P. falciparum. For each well, sporozoites were pre-incubated with serially diluted antibody sample for 30 minutes and thereafter transferred onto the hépatocytes. After 3 hours, non-invaded sporozoites were washed off and the cells were incubated for 4 days. Cells were fixed and stained with anti-HSP70 and DAPI. The number of hépatocyte nuclei and HSP70 positive forms were quantified by automated high content imaging.
In this assay, variant antibody MGU10v2 was compared to the parental antibody MGU10. In addition, Fc variant “MGU10v2_LS” of MGU10v2 was tested, which dîffers from variant antibody MGU10v2 only in that it comprises the mutations M428L and N434S (EU numbering) in the heavy chain constant région (amino acid sequence of MGU10v2_LS heavy chain: SEQ ID NO: 100). Accordingly, the variable régions of MGU10v2_LS are identical to those of MGU10v2. An irrelevant antibody was used as control. Five dilutions were tested per antibody sample with two replicates per dilution. 3SP2/Atovaquone treated sporozoites were used as MIN control and vehicle treated sporozoites as MAX control.
Data are expressed as total number of hépatocyte nuclei, total number of HSP70 positive forms, % infected hépatocytes. IC50 values were estimated using a four parameter non-linear régression model using least squares to find the b est fit. The resulting values (expressed in pg/ml) are presented in Table 5 below:
Antibody IC50 (pg/ml)
MGU10 0.418
MGU10v2 0.302
MGU10v2_LS 0.179
control > 10
Table 5.
Graphîcal représentations of the results are shown in Figure 1. Ail antibodies, with the exception of the control antibody, functîonally blocked the development of liver stage schizonts in human primary hépatocytes. Interestingly, variant antibody MGU10v2 was more effective than parental antibody MGU10 and the addîtîonal Fc variant MGU10v2_LS was most efficient.
Example 4: Sporozoite gliding assay
The sporozoite gliding assay is a fiinctional assay, wherein the effects of compounds on the sporozoites’ gliding motility can be assessed. Plasmodium sporozoites are transmitted into the skin of their vertebrate host through the bite of an infectious mosquito. Sporozoite motility is a key prerequisite for parasite transmission and successful infection of the vertebrate host. Motility constitutes the first parasite mechanism that can be inhibited and is, therefore, of interest for intervention strategies.
Plates were coated with anti-CSP mAb 3SP2 to capture shed CSP (circumsporozoite protein). Fresh salivary gland sporozoites were isolated from An. stephensi mosquitoes infected with P. falciparum and pre-incubated with serially diluted sample (5 dilutîons/sample) for 30 min and then transferred into the 3SP2 coated wells. After 90 minutes sporozoites were washed off and gliding trails were fixed and stained with biotinylated-anti-CSP-antibody followed by streptavidin-AF555. Gliding trails were captured by automated high content imaging and total gliding trail length was analyzed using machine leaming algorithms.
In this assay, variant antibody MGU10v2_LS was compared to the parental antibody MGU10. An irrelevant antibody was used as control. Five dilutions were tested per antibody sample with two replicates per dilution. 3SP2/Gramicidin treated sporozoites were used as MIN control and vehîcle treated sporozoites as MAX control.
Total gliding trails were quantified by image analysis and reported as relative fluorescence counts. 1C50 values were estimated using a four parameter non-linear régression model using least squares to find the best fit. The resulting values (expressed in pg/ml) are presented in Table 6 below:
Antibody IC50 (gg/ml)
MGU10 0.975
MGU10v2_LS 0.748
control > 10
Table 6.
Graphical représentations of the results are shown in Figure 2. Antibodies MGU10 and MGUI0v2_LS, but not the control antibody, functionally blocked gliding motility of P. falciparum sporozoites. Variant antibody MGU10v2_LS was more effective than parental antibody MGU10.
Example 5: Sporozoite traversai assay
Plasmodium sporozoites are deposited in the skin of the vertebrate host. As sporozoites move towards the liver, they can enter and exit host cells within transient vacuoles, a process known as cell traversai. Traversai allows the sporozoites to cross cellular barri ers and évadé the host immune response, thereby representing a key prerequisite for successful infection of the vertebrate host. The sporozoite traversai assay is a functional assay, wherein the effects of compounds on the sporozoites’ cell traversai can be assessed.
Human hepatoma (HC-04) cells were seeded in microtiter plates and grown to near confluence. Fresh P. falciparum salîvary gland sporozoites were isolated from An. stephensi mosquitoes and pre-incubated with dîluted IgG for 30 minutes before adding rhodamîn-dextran. Following incubation for 1 hour at 37°C, cell nuclei were stained with DAPL Fluorescence levels of traversed cells were quantified using a high content automated imager.
In this assay, variant antibody MGU10v2_LS was compared to the parental antibody MGU10. An irrelevant antibody was used as control. Five dilutions were tested per antibody sample with two replicates per dilution. 3SP2/Cytochalasin D treated sporozoites were used as MIN control and vehicle treated sporozoites as MAX control.
Data were expressed as % traversed cells relative to the MIN and MAX Controls of the assay plate. IC50 values were estimated using a four parameter non-linear régression model using least squares to find the best fit. The resulting values (expressed in pg/ml) are presented in Table 7 below:
Antibody IC50 (qgml)
MGU10 1.928
MGU10v2_LS 1.356
control > 10
Table 7.
Graphical représentations of the results are shown in Figure 3. Antibodies MGU10 and MGU10v2_LS, but not the control antibody, functionally blocked traversai of P. falciparum sporozoites. Variant antibody MGU10v2_LS was more effective than parental antibody MGU10.
Example 6: Stability of antibodies
To test their stability, variant antibody MGU10v2_LS was comparée! to its parental version MGU1Û_LS. MGU10_LS differs from parental antibody MGU10 only in that it comprises the mutations M428L and N434S (EU numbering) in the heavy chain constant région (ami no acid sequence of MGU10_LS heavy chain; SEQ ID NO: 101) Accordingly, the variable régions of MGU10_LS are identical to those of MGU10. Variant antibody MGU10v2_LS and its parental version MGU10_LS were exposed to heat stress under different conditions.
To this end, variant antibody MGU10v2_LS and its parental version MGU10_LS were incubated at 40°C in sodium acetate buffer at pH 5.6 for two weeks. The formation of aggregates and dîmers (high and low molecular weight species) was assessed by size exclusion chromatography. Results are shown in Table 8 below;
MGU10 LS MGU10v2_LS
%HMW S %monomer %LMW S %HMW S %monomer %LMW S
unstresse d 0.63 99 0.37 0.22 99.38 0.4
40°C 4.65 92.83 2.52 4.29 93.09 2.62
Table 8. HMWS: high molecular weight species, indicating aggregation; LMWS: low molecular weight species, representing dimer formation; %monomer indicates antibodies which do not dimerize or aggregate.
To further assess stability and aggregation of mAb MGU10, parental and developed mAbs were tested in two different buffers with different pH. To compare mAb dimerization and aggregation in different buffers, mAbs batches were buffer-exchanged to 50mM Na-Acetate, 50mM NaCl, pH 5.5 or to 20mM Na-Citrate, 50mM NaCl, pH 6.0 upon purification. Size exclusion chromatography was used to assess the molecular weight of the mAb species after 4 and 15 days at 40°C, using a BEH450 SEC Protein Standard Mix 5 component protein mixture (Thyroglobulin, IgG, BSA, Myoglobin, Uracil) for size calculation,
Results are shown in Figure 4. Variant antibody MGU10v2_LS formed less high molecular weight species (less aggregates) when kept at 4Û°C in 50mM Na-Acetate/50mM NaCl buffer, pH 5.5, for two weeks as compared to its parental version MGU10_LS, Accordingly, variant antibody MGU10v2_LS shows increased stability compared to the parental antibody.
Example 7: In vivo protection conferred by variant antibody MGU10v2 - challenge study
To assess in vivo protection against P. berghei chimeric parasite expressing full-length P. falciparum CSP, C57BL/6 mice (n = 5 per group) were injected i.v. with 54.5 pg/mouse of variant antibody MGU10v2_LS or with 100 pg/mouse of the respective parental antibody MGU10_LS. As négative control, unrelated antibody AB-1245 (100 pg/mouse) was used. Fortyeight hours after antibody injection, antibody-treated mice as well as an additional group of naive mice (n = 5) were challenged with 2x103 chimeric P. berghei sporozoites expressing full-length P. falciparum CSP injected i.v. Forty-two hours after challenge, mice were injected with 100 pl of D-Luciferin (30 mg/mL), anesthetized with isoflurane and imaged with the IVTS spectrum to measure the bioluminescence expressed by the chimeric parasites. % inhibition was calculated in comparison to the naive group (representing 100% infection).
Results are shown in Figure 5. In contrast to unrelated control antibody AB-1245, both variant antibody MGU10v2_LS and the respective parental antibody MGU10_LS significantly inhîbited infection with the parasite in vivo with 80.76 (MGU10v2_LS) and 69.60% inhibition (MGU10_LS), respectively. Accordingly, a stronger inhibition of infection was achieved with variant antibody MGUl0v2_LS as compared to its parental antibody MGU10_LS.
Example 8: In vivo protection conferred bv variant antibody MGH2vl - challenge study
To assess in vivo protection against P. berghei chimeric parasite ex pressing full-length P. falciparum CSP, C57BL/6 mîce (n = 5 per group) were injected i.v. with 100 p^mouse of variant antibody MGH2vl_LS or with the respective parental antibody MGH2_LS. Fc variants MGH2vl_LS and MGH2 LS differ from MGH2vl and MGH2, respectively, only in two mutations in the Fc région, i.e. the respective variable régions are maintained. Namely, variant antibody MGH2vl_LS dîffers from variant antibody MGH2v] only in that it comprises the mutations M428L and N434S (EU numbering) in the heavy chain constant région (amino acid sequence of MGH2vl_LS heavy chain: SEQ ID NO: 102). Accordingly, antibody MGH2_LS differs from antibody MGH2 only in that it comprises the mutations M428L and N434S (EU numbering) in the heavy chain constant région (amino acid sequence of MGH2_LS heavy chain: SEQ ID NO: 102). As variant antibody MGH2vl differs from parental antibody MGH2 only in the light chain CDR3 (VL), the heavy chain amino acid sequences of MGH2_LS and MGH2vl_LS are identical.
As négative control, unrelated antibody AB-1245 (100 pg/mouse) was used. Forty-eight hours after antibody injection, antibody-treated mice as well as an additional group of naive mice (n = 5) were challenged with 2x103 chimeric P. berghei sporozoites expressing full-length P. falciparum CSP injected i.v. as described in Example 7. Forty-two hours after challenge, mice were injected with 100 pl of D-Luciferin (30 mg/mL), anesthetized with isoflurane and imaged with the IVIS spectrum to measure the bioluminescence expressed by the chimeric parasites. % inhibition was calculated in comparison to the naîve group (representing 100% infection).
Results are shown in Figure 6. In contrast to unrelated control antibody AB-1245, both variant antibody MGH2vl_LS and the respective parental antibody MGH2 LS inhibited infection with the parasite in vivo with 38.62% (MGH2vl_LS) and 25.23% inhibition (MGH2_LS), respectively. Accordingly, a stronger inhibition of infection was achieved with variant antibody MGH2vl_LS as compared to its parental antibody MGH2vl_LS.
Example 9: Design and testing of a further variant of MGU10/MGU10v2
As shown above in Example 6, MGU10v2, which differs from MGU10 in mutation D106E, shows increased stability. Without being bound to any theory, the présent inventors believe that mutation D106E removes an isomerization motif and thereby increases the stability of antibody MGU10v2 as shown in Example 6.
In view thereof, a further variant of MGU 10 was designed, wherein - in comparison to MGU 10v2 - the additional mutation D97E was introduced in the framework région of the heavy chain (VH) of MGU10v2 in order to remove a further isomerization motif (to further increase the antibody’s stability and manufacturability). Accordingly, MGU10v8 comprises the same CDR sequences as MGU10v2; and the same VU sequence as MGU10v2 and its parental antibody MGU 10. The VH of MGU10v8 comprises an amino acid sequence as set forth in SEQ ID NO: 104. SEQ ID NO: 106 provides an exemplary nucléotide sequence encodingthe MGHv8 VH.
New variant antibody MGU10v8 was expressed essentially as described in Example 2 using ExpiCHO cells (in higher volume, e.g. 25ml or 100 ml or more), and binding to (i) the NANPrepeat région of CSP (“ΝΑΝΡ’’-peptide) and (ii) the N-terminal région of CSP covering the junction between the N-terminal domain and the NANP-repeats (uNPDP”-peptide) were tested in ELISA. To this end, Pierce™ Streptavidin coated plates (Life Technologies) were used to coat either biotinylated NANP-peptide (N-term biotinylation; SEQ ID NO: 34) or biotinylated NPDP19-peptide (N-term biotinylation; SEQ ID NO: 105), each at 5 pg/ml in blocking buffer (PBS, 1% BSA). Plates were washed (PBS, 0.05% Tween 20) before addition of titrated antibodies MGU10v2 or MGU10v8 for 90min at RT. After another wash step, secondary antibody goat anti-human IgG HRP F(ab’)2 fragment Fcg spécifie (Jackson ImmunoResearch) was added at 0.8 pg/ml. Sure Blue TMB (Bioconcept) was used for color development that was stopped with 1% HCl in water. Optical density at 450nm was detected using an ELISA Reader ELx808IU (Biotek).
Results are shown in Figure 7. As shown in Figure 7, binding to both peptides, NANP and NPDP19, is very similar in variant antibodies MGU10v2 and MGU10v8.
TABLE OF SEQUENCES AND SEQ ID NUMBERS (SEQUENCE LISTING):
SEQ ID NO Sequence Remarks
Amino acid sequences
MGU10
SEQ ID NO: 1 GFAFSNYG CDRH1
SEQ ID NO: 2 IWHDGSLK CDRH2
SEQ ID NO: 3 TVWYLETPDDGFDI CDRH3
SEQ ID NO: 4 HGHTSKA CDRL1
SEQ ID NO: 5 VNSDGSH CDRL2
SEQ ID NO: 6 QAWDSGIWV CDRL3
SEQ ID NO: 7 QVQLVESGGGVVQPGRSLRLSCAASGFAFSNYGM NWVRQAPGKGLEWVAVIWHDGSLKYYTQSVKG RFTISRDNAKNTLFLQMDSLSADDTAMYYCTVWY LETPDDGFDIWGRGTMVTVSS VH
SEQ ID NO: 8 QLVLTQPPSASASLGVSVTLTCTLSHGHTSKAIAW HQQQPGKGPRYLMKVNSDGSHTKGAAVPDRFSG STSGAERHFTISNLQSDDEADYYCQAWDSGIWVF GGGTKLTVL VE
SEQ IDNO: 9 QVQLVESGGGVVQPGRSLRLSCAASGFAFSNYGM NWVRQAPGKGLEWVAVIWHDGSLKYYTQSVKG RFTISRDNAKNTLFLQMDSLSADDTAMYYCTVWY LETPDDGFDIWGRGTMVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFP A VLQS SGLYSLSS V VTVPS SSLGTQTYICN V NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLS PGK Heavy chain
SEQIDNO: 10 QLVLTQPPSASASLGVSVTLTCTLSHGHTSKAIAW HQQQPGKGPRYLMKVNSDGSHTKGAAVPDRFSG STSGAERIIFTISNLQSDDEADYYCQAWDSGIWVF GGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATL VCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQ SNNKYAAS S YLSLTPEQWKS HRS YSCQVTHEGST VEKTVAPTECS Light chain
MGUlOvariantl (MGUlOvl)
SEQ ID NO: 11 QVQLVESGGGVVQPGRSLRLSCAASGFAFSNYG MHWVRQAPGKGLEWVAVIWHDGSLKYYTQSVK GRFTISRDNAKNTLFLQMDSLSADDTAMYYCTV WYLETPDDGFD1WGQGTMVTV SS VH
MGUlOvariantl (MGUlOvl)
SEQ IDNO: 12 TVWYLETPDEGFDI CDRH3
SEQ IDNO: 13 QVQLVESGGGVVQPGRSLRLSCAASGFAFSNYG MNWVRQAPGKGLEWVAVIWHDGSLKYYTQSVK GRFTISRDNAKNTLFLQMDSLSADDTAMYYCTV WYLETPDEGFDIWGRGTMVTVSS VH
MGU10variant3 (MGU10v3)
SEQIDNO: 14 QAWESGIWV CDRL3
SEQIDNO: 15 QLVLTQPPSASASLGVSVTLTCTLSHGHTSKAIAW HQQQPGKGPRYLMKVNSDGSHTKGAAVPDRFSG STSGAERHFTISNLQSDDEADYYCQAWESGIWVF GGGTKLTVL VL
MGU10variant4 (MGU10v4)
SEQ IDNO: 16 QVQLVESGGGVVQPGRSLRLSCAASGFAFSNYG MHWVRQAPGKGLEWVAVIWHDGSEKYYTQSVK GRFTISRDNAKNTLFLQMDSLSADDTAMYYCTV WYLETPDEGFDIWGQGTMVTVSS VH
MGHl
SEQ IDNO: 17 GFSFSSYA _______ CDRH1
SEQIDNO: 18 TRYDGSNK CDRH2
SEQ ID NO: 19 AKVGDGTVAGTIDY CDRH3
SEQ ID NO: 20 QSLVYSDGNTY CDRL1
SEQIDNO: 21 KVS CDRL2
SEQ ID NO: 22 LIYKVSNRD CDRL2 long
SEQ ID NO: 23 MQGTHWWT CDRL3
SEQ ID NO: 24 QVQLVESGGGVVQPGGSLRLSCTASGFSFSSYAMHW VRQAPGKGLEWVAYTRYDGSNKFYLDSVQGRFTIS RDNSKNTLYLEMDSLRLEDTAVYFCAKVGDGTVAG TIDYWGQGTLVTVSS VH
SEQ ID NO: 25 YIVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYL NWYQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGT DFTLKISRVEAEDVGVYYCMQGTHWWTFGQGTKV EIK VL
SEQ ID NO: 26 QVQLVESGGGVVQPGGSLRLSCTASGFSFSSYAMHW VRQAPGKGLEWVAYTRYDGSNKFYLDSVQGRFTIS RDNSKNTLYLEMDSLRLEDTAVYFCAKVGDGTVAG TIDYWGQGTLVTVS S ASTKGPS VFPLAPS SKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ S SGLYSLSSV VTVPS S SLGTQT YICNVNHKP SNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHE ALHSHYTQKSLSLSPGK Heavy chain
SEQ ID NO: 27 YIVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYL NWYQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGT DFTLKISRVEAEDVGVYYCMQGTHWWTFGQGTKV EIKRTV AAP S VFIFPP SDEQLKSGTAS VVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Light chain
MGH2variantl (MGH2vl)
SEQIDNO: 28 MQGTHFWT CDRL3
SEQ ID NO: 29 YIVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNT YLNWYQQRPGQSPRRLIYKVSNRDSGVPDRFSGS GSGTDFTLKISRVEAEDVGVYYCMQGTHFWTFG QGTKVEIK VL
Constant régions
SEQ ID NO: 30 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS S LGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPP SRDELTKNQV SLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK heavy chain constant région
SEQ ID NO: 31 GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGA VTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLS LTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS lambda light chain
SEQ ID NO: 32 RTVAAPSVFIFPPSDEQLK.SGTASVVCLLNNFYPREA KVQ WKVDN ALQSGNSQES VTEQDSKD STY SLS STLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC kappa light chain
SEQ ID NO: 33 MMRKL AILS V SSFLFVEALFQE YQC YGS S SNTRVLNE LNYDNAGTNLYNELEMNYYGKQENWYSLKKNSRSL GENDDGNNEDNEKLRKPKHKKLKQPADGNPDPNAN PNVDPNANPNVDPNANPNVDPNANPNANPNANPNA NPNANPNANPNANPNANPNANPNANPNANPNANPN ANPNANPNANPNANPNANPNVDPNANPNANPNANP NANPNANPNANPNANPNANPNANPNANPNANPNAN PNANPNANPNANPNANPNANPNANPNKNNQGNGQG HNMPNDPNRNVDENANANSAVKNNNNEEPSDKHIK EYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKP KDELDYANDIEKKICKMEKCSSVFNVVNSS1GLIMVL SFLFLN PfCSP
SEQ ID NO: 34 NANPNANPNANPNANPNA N ANP-peptide
SEQ TDNO: 35 KQPADGNPDPNANPN NPDP-peptide
Nucleic acid sequences
SEQ ID NO: 36 CAGGTGCAGCTGGTGGAGAGCGGAGGAGGAGTGGTG CAGCCAGGCCGGTCCCTGAGACTGTCTTGCGCCGCCA GCGGCTTCGCCTTITCCAACTACGGAATGCACTGGGT GCGGCAGGCACCTGGCAAGGGACTGGAGTGGGTGGC CGTGATCTGGCACGACGGATCCCTGAAGTACTATACA CAGTCTGTGAAGGGCAGGTTCACCATCTCTCGCGACA ACGCCAAGAATACACTGTTTCTGCAGATGGATTCTCT GAGCGCCGACGATACCGCCATGTACTATTGTACAGTG TGGTATCTGGAGACCCCCGACGATGGCTTCGATATCT GGGGCCAGGGCACCATGGTGACAGTGAGCTCC MGUlOvl VH
SEQ IDNO: 37 CAGGTGCAGCTGGTGGAGAGCGGAGGAGGAGTGGTG CAGCCAGGCCGGTCCCTGAGACTGTCTTGCGCCGCCA GCGGCTTCGCCTTTTCCAACTACGGCATGAATTGGGT GCGGCAGGCACCTGGCAAGGGACTGGAGTGGGTGGC CGTGATCTGGCACGACGGATCCCTGAAGTACTATACA CAGTCTGTGAAGGGCAGGTTCACCATCTCTCGCGACA ACGCCAAGAATACACTGTTTCTGCAGATGGATTCTCT GAGCGCCGACGATACCGCCATGTACTATTGTACAGTG TGGTATCTGGAGACCCCCGACGAGGGCTTCGATATCT GGGGCAGGGGCACCATGGTGACAGTGAGCTCC MGU10v2 VH
SEQ ID NO: 38 CAGCTGGTGCTGACACAGCCACCTAGCGCCTCCGCCT CTCTGGGCGTGAGCGTGACCCTGACATGCACCCTGTC CCACGGCCACACCTCTAAGGCAATCGCATGGCACCAG CAGCAGCCAGGCAAGGGACCACGGTACCTGATGAAG GTGAACAGCGACGGATCCCACACAAAGGGAGCAGCA GTGCCAGATCGGTTCAGCGGCTCCACATCTGGCGCCG AGAGACACTTTACCATCTCTAATCTGCAGAGCGACGA TGAGGCCGACTACTATTGTCAGGCCTGGGAGTCCGGA ATCTGGGTGTTCGGAGGAGGAACAAAGCTGACCGTG CTG MGU10v3 VL
SEQ ID NO: 39 TACATCGTGATGACCCAGTCCCCCCTGTCTCTGCCTGT GACACTGGGCCAGCCTGCCTCTATCAGCTGCCGGAGC TCCCAGAGCCTGGTGTACTCCGACGGCAACACCTACC TGAATTGGTATCAGCAGAGGCCAGGACAGTCCCCACG GAGACTGATCTATAAGGTGTCTAACAGGGACAGCGG AGTGCCAGATCGCTTCTCCGGATCTGGAAGCGGAACC GACTTTACACTGAAGATCTCTCGGGTGGAGGCCGAGG ATGTGGGCGTGTACTATTGTATGCAGGGCACCCACTT CTGGACATTTGGCCAGGGCACAAAGGTGGAGATCAA G MGH2vl VL
SEQ ID NO: 40 CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTG CAGCCCGGCAGAAGCCTGAGACTGAGCTGCGCCGCC AGCGGCTTCGCCTTCAGCAACTACGGCATGAACTGGG TGAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGG CCGTGATCTGGCACGACGGCAGCCTGAAGTACTACAC CCAGAGCGTGAAGGGCAGATTCACCATCAGCAGAGA CAACGCCAAGAACACCCTGTTCCTGCAGATGGACAGC CTGAGCGCCGACGACACCGCCATGTACTACTGCACCG TGTGGTACCTGGAGACCCCCGACGACGGCTTCGACAT CTGGGGCAGAGGCACCATGGTGACCGTGAGCAGC MGU10 VHvl
SEQ ID NO: 41 CAGGTTCAGTTAGTCGAGAGTGGGGGCGGGGTTGTGC AACCAGGGAGATCGTTGAGGCTCAGCTGCGCAGCCA GTGGCTTCGCCTTCAGTAATTACGGCATGAACTGGGT TAGGCAGGCTCCTGGCAAAGGTTTGGAGTGGGTAGCA GTGATTTGGCATGACGGCTCTTTGAAATATTACACAC AGAGTGTGAAAGGAAGATTCACAATCAGCAGAGACA ACGCCAAGAATACTCTGTTCCTGCAGATGGATTCCCT GTCAGCCGACGACACGGCCATGTATTACTGTACCGTG TGGTATCTCGAGACACCCGATGATGGCTTCGACATCT GGGGGAGAGGCACCATGGTTACCGTGAGCAGC MGU10 VHv2
SEQ ID NO: 42 CAGGTCCAGCTGGTCGAGTCAGGCGGGGGCGTTGTCC AACCGGGACGCTCTTTGCGATTATCTTGCGCAGCGTC CGGCTTTGCGTTCAGTAATTATGGCATGAACTGGGTC CGACAAGCTCCCGGAAAAGGGCTGGAATGGGTTGCG GTGATTTGGCATGACGGAAGCTTGAAGTACTATACGC AGTCAGTGAAAGGAAGGTTCACAATTTCACGGGATA ATGCGAAGAACACTCTATTCCTACAGATGGACTCACT TTCCGCTGACGACACCGCCATGTATTACTGCACCGTTT GGTACTTGGAAACGCCGGACGACGGGTTTGATATCTG GGGCAGAGGGACAATGGTTACCGTTTCCTCA MGU10 VHv3
SEQ IDNO: 43 CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCA GCCCGGCAGAAGCCTGAGACTGAGCTGCGCCGCCAGCG MGU10 heavy chain variant 1
GCTTCGCCTTCAGCAACTACGGCATGAACTGGGTGAGAC AGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATC TGGCACGACGGCAGCCTGAAGTACTACACCCAGAGCGT GAAGGGCAGATTCACCATCAGCAGAGACAACGCCAAGA ACACCCTGTTCCTGCAGATGGACAGCCTGAGCGCCGACG ÀCACCGCCATGTACTACTGCACCGTGTGGTACCTGGAGA CCCCCGACGACGGCTTCGACATCTGGGGCAGAGGCACC ATGGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCCAG CGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCG GCGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACT TCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCC CTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAG AGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGT GCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCA ACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAG AAGGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTG CCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCAG CGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGAT
GATCAGCAGAACCCCCGAGGTGACCTGCGTGGTGGTGG ACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGG TACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAA GCCCAGAGAGGAGCAGTACAACAGCACCTACAGAGTGG TGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAAC GGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCT
GCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGG GCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCC AGCAGAGACGAGCTGACCAAGAACCAGGTGAGCCTGAC CTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGT GGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACA AGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCT TCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGG CAGCAGGGCAACGTGTTCAGCTGCAGCGTGCTGCACGA GGCCCTGCACAGCCACTACACCCAGAAGAGCCTGAGCC TGAGCCCCGGCAAG
SEQ ID NO: 44 CAGGTTCAGTTAGTCGAGAGTGGGGGCGGGGTTGTGCA ACCAGGGAGATCGTTGAGGCTCAGCTGCGCAGCCAGTG GCTTCGCCTTCAGTAATTACGGCATGAACTGGGTTAGGC AGGCTCCTGGCAAAGGTTTGGAGTGGGTAGCAGTGATTT GGCATGACGGCTCTTTGAAATATTACACACAGAGTGTGA AAGGAAGATTCACAATCAGCAGAGACAACGCCAAGAAT ACTCTGTTCCTGCAGATGGATTCCCTGTCAGCCGACGAC ACGGCCATGTATTACTGTACCGTGTGGTATCTCGAGACA CCCGATGATGGCTTCGACATCTGGGGGAGAGGCACCAT GGTTACCGTGAGCAGCGCTTCCACCAAAGGCCCATCCGT CTTCCCACTGGCCCCCTCCTCAAAAAGTACCTCAGGAGG GACCGCCGCCTTGGGCTGCCTTGTAAAAGACTACTTCCC TGAGCCCGTTACTGTCTCTTGGAACTCCGGCGCTCTGAC CTCCGGCGTTCATACGTTTCCTGCCGTGCTTCAGTCCAGT GGCTTGTATTCCCTGAGCTCTGTAGTCACCGTGCCGAGC AGTAGCCTCGGCACACAGACGTACATATGTAACGTGAA TCACAAGCCATCTAACACTAAAGTCGATAAAAAAGTAG AGCCTAAAAGCTGTGATAAAACCCACACATGTCCGCCAT GTCCCGCGCCCGAACTGTTGGGCGGGCCCAGTGTGTTCC TATTCCCACCAAAACCGAAAGACACCTTGATGATCAGTC GCACACCTGAGGTAACCTGCGTGGTCGTCGACGTCTCCC ACGAAGACCCCGAGGTCAAGTTTAACTGGTATGTTGATG GCGTCGAGGTACACAATGCAAAAACCAAACCAAGGGAA GAACAATATAATAGTACATATCGCGTGGTGAGTGTCCTC ACCGTGCTCCACCAGGACTGGCTAAATGGG.4AGGAGTA TAAATGCAAGGTGAGCAACAAGGCACTCCCGGCCCCAA TTGAGAAAACCATTTCCAAAGCCAAGGGCCAACCACGA GAACCACAGGTCTACACCCTCCCACCTTCACGCGACGAG TTGACAAAGAATCAAGTGTCTCTCACCTGTCTTGTGAAG GGGTTTTATCCCAGTGATATCGCGGTGGAATGGGAGAGC AATGGACAACCAGAGAACAACTATAAGACCACCCCGCC TGTCCTGGATTCCGACGGATCTTTCTTCCTTTATTCAAAG TTGACCGTGGACAAGTCCCGGTGGCAGCAAGGGAATGT GTTCAGCTGCTCGGTGC'FCCATGAGGCCCTTCACAGCCA CTATACGCAGAAGAGCTTATCTCTGAGCCCTGGGAAG SI GU 10 heavy chain variant 2
SEQ ID NO: 45 CAGGTCCAGCTGGTCGAGTCAGGCGGGGGCGTTGTCCA ACCGGGACGCTCTTTGCGATTATCTTGCGCAGCGTCCGG CTTTGCGTTCAGTAATTATGGCATGAACTGGGTCCGACA AGCTCCCGGAAAAGGGCTGGAATGGGTTGCGGTGATTT GGCATGACGGAAGCTTGAAGTACTATACGCAGTCAGTG aaaggaaggttcacaatttcacgggàtaatgcgaagaa CACTCTATTCCTACAGATGGACTCACTTTCCGCTGACGA CACCGCCATGTATTACTGCACCGTTTGGTACTTGGAAAC GCCGGACGACGGGTTTGATATCTGGGGCAGAGGGACAA TGGTTACCGTTTCCTCAGCCAGTACGAAGGGGCCCTCAG TATTTCCGCTAGCGCCGAGCTCAAAGTCGACATCTGGGG GCACAGCAGCACTGGGATGTCTGGTCAAAGATTACTTCC CCGAGCCTGTAACCGTTAGTTGGAATAGTGGTGCCTTAA CGAGTGGGGTTCATACATTTCCAGCGGTACTCCAGTCCT CAGGGCTCTACTCCTTATCAAGCGTCGTTACAGTCCCAA GTTCATCGCTAGGTACTCAAACTTACATCTGCAATGTTA ACCATAAGCCCAGCAATACCAAAGTCGACAAAAAAGTC GAACCGAAGTCCTGCGACAAGACGCACACGTGTCCACC TTGTCCTGCCCCGGAGTTATTGGGCGGCCCGTCGGTGTT CTTGTTTCCTCCCAAACCGAAGGATACCCTAATGATTTC GAGGACGCCAGAAGTAACATGTGTTGTGGTCGATGTATC TCATGAAGACCCAGAGGTTAAGTTCAACTGGTATGTCGA TGGCGTCGAAGTACACAACGCAAAGACCAAACCCAGGG AAGAACAGTACAATAGTACTTATAGGGTTGTATCAGTAC TTACGGTCCTGCATCAGGACTGGCTTAACGGTAAAGAGT ACAAATGTAAGGTGTCTAATAAGGCACTGCCCGCGCCA ATTGAAAAAACCATCTCGAAAGCTAAGGGCCAGCCCAG AGAACCTCAAGTGTACACGCTTCCGCCGAGTCGCGACG AACTGACTAAGAACCAGGTTTCTCTGACTTGCCTAGTTA AAGGTTTCTACCCGTCGGACATAGCAGTCGAATGGGAA AGCAACGGCCAGCCGGAGAACAACTACAAGACCACGCC TCCGGTGCTCGATTCGGATGGGTCTTTCTTTTTATATTCG AAATTAACCGTGGATAAAAGTCGGTGGCAACAAGGTAA TGTTTTCAGTTGTTCTGTCCTTCACGAAGCCCTACATTCG CACTACACGCAAAAGAGTTTAAGTTTGTCACCGGGGAA G MGU10 heavy chain variant 3
SEQ ID NO: 46 CAGCTGGTGCTGACCCAGCCCCCCAGCGCCAGCGCCA GCCTGGGCGTGAGCGTGACCCTGACCTGCACCCTGAG CCACGGCCACACCAGCAAGGCCATCGCCTGGCACCA GCAGCAGCCCGGCAAGGGCCCCAGATACCTGATGAA GGTGAACAGCGACGGCAGCCACACCAAGGGCGCCGC CGTGCCCGACAGATTCAGCGGCAGCACCAGCGGCGC CGAGAGACACTTCACCATCAGCAACCTGCAGAGCGA CGACGAGGCCGACTACTACTGCCAGGCCTGGGACAG CGGCATCTGGGTGTTCGGCGGCGGCACCAAGCTGACC GTGCTG MGU10 VLvl
SEQ 1D NO: 47 CAGTTGGTGTTGACCCAACCACCGTCTGCCTCTGCAA GTCTGGGGGTGTCTGTGACACTTACTTGCACGCTGTCT CACGGGCACACAAGCAAGGCCATTGCTTGGCACCAA CAGCAGCCTGGCAAGGGACCTCGGTACTTGATGAAG GTCAACTCCGACGGCAGTCATACCAAAGGTGCAGCA GTGCCGGATAGATTTTCCGGCTCCACAAGTGGCGCGG AGCGCCACTTTACCATCTCCAACCTTCAGAGCGATGA CGAAGCTGATTATTATTGTCAGGCCTGGGACTCAGGC ATATGGGTATTCGGGGGGGGGACCAAGCTCACCGTGT TA MGU10 VLv2
SEQ ID NO: 48 CAACTCGTTCTCACCCAACCGCCTTCAGCCAGCGCCA GTCTCGGAGTATCGGTGACCCTTACGTGCACACTCTC ACACGGCCACACGTCGAAGGCTATAGCCTGGCATCAG CAACAACCGGGCAAAGGACCGCGTTATCTAATGAAG GTCAATTCCGACGGATCTCATACAAAAGGCGCCGCCG TACCTGACCGCTTTAGCGGGAGTACGTCCGGGGCAGA GCGTCATTTCACGATAAGTAATCTCCAGTCGGATGAC GAGGCTGACTATTATTGTCAGGCCTGGGACTCAGGTA TTTGGGTATTTGGAGGGGGGACCAAGTTGACGGTCTT A MGU10 VLv3
SEQ ID NO: 49 CAGCTGGTGCTGACCCAGCCCCCCAGCGCCAGCGCCA GCCTGGGCGTGAGCGTGACCCTGACCTGCACCCTGAG CCACGGCCACACCAGCAAGGCCATCGCCTGGCACCA GCAGCAGCCCGGCAAGGGCCCCAGATACCTGATGAA GGTGAACAGCGACGGCAGCCACACCAAGGGCGCCGC CGTGCCCGACAGATTCAGCGGCAGCACCAGCGGCGC CGAGAGACACTTCACCATCAGCAACCTGCAGAGCGA CGACGAGGCCGACTACTACTGCCAGGCCTGGGACAG CGGCATCTGGGTGTTCGGCGGCGGCACCAAGCTGACC GTGCTGGGCCAGCCCAAGGCCGCCCCCAGCGTGACCC TGTTCCCCCCCAGCAGCGAGGAGCTGCAGGCCAACAA GGCCACCCTGGTGTGCCTGATCAGCGACTTCTACCCC GGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGC CCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGC AAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTAC CTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGA AGCTACAGCTGCCAGGTGACCCACGAGGGCAGCACC GTGGAGAAGACCGTGGCCCCCACCGAGTGCAGC MGU10 light chain variant 1
SEQ ID NO: 50 CAGTTGGTGTTGACCCAACCACCGTCTGCCTCTGCAA GTCTGGGGGTGTCTGTGACACTTACTTGCACGCTGTCT CACGGGCACACAAGCAAGGCCATTGCTTGGCACCAA CAGCAGCCTGGCAAGGGACCTCGGTACTTGATGAAG GTCAACTCCGACGGCAGTCATACCAAAGGTGCAGCA GTGCCGGATAGATTTTCCGGCTCCACAAGTGGCGCGG AGCGCCACTTTACCATCTCCAACCTTCAGAGCGATGA CGAAGCTGATTATTATTGTCAGGCCTGGGACTCAGGC ATATGGGTATTCGGGGGGGGGACCAAGCTCACCGTGT TAGGGCAACCGAAAGCGGCACCCAGTGTGACCCTGTT TCCCCCCAGCAGTGAGGAACTCCAGGCAAATAAGGC CACTTTGGTCTGTCTGATTAGTGATTTTTATCCCGGGG CAGTCACCGTGGCTTGGAAAGCGGACTCTTCTCCCGT AAAAGCCGGAGTCGAGACCACTACACCGTCTAAGCA GAGTAATAACAAATATGCTGCTAGCTCTTACCTGTCC CTGACACCAGAACAGTGGAAGTCCCATAGGAGTTATA GCTGCCAGGTCACACACGAGGGGAGCACCGTGGAGA AGACAGTTGCACCCACTGAGTGCTCC MGU10 light chain variant 2
SEQ ID NO: 51 CAACTCGTTCTCACCCAACCGCCTTCAGCCAGCGCCA GTCTCGGAGTATCGGTGACCCTTACGTGCACACTCTC ACACGGCCACACGTCGAAGGCTATAGCCTGGCATCAG CAACAACCGGGCAAAGGACCGCGTTATCTAATGAAG GTCAATTCCGACGGATCTCATACAAAAGGCGCCGCCG TACCTGACCGCTTTAGCGGGAGTACGTCCGGGGCAGA GCGTCATTTCACGATAAGTAATCTCCAGTCGGATGAC GAGGCTGACTATTATTGTCAGGCCTGGGACTCAGGTA TTTGGGTATTTGGAGGGGGGACCAAGTTGACGGTCTT AGGCCAGCCTAAGGCGGCGCCGTCTGTTACTTTATTC CCTCCTTCTTCGGAGGAGCTTCAGGCCAACAAGGCCA CCCTTGTATGTCTTATATCCGACTTTTATCCTGGAGCC GTTACTGTTGCGTGGAAGGCCGACTCGTCGCCTGTCA AGGCCGGGGTCGAGACTACGACCCCTTCAAAGCAAA GTAACAATAAGTACGCTGCAAGCTCTTATCTGTCACT AACGCCTGAGCAGTGGAAGTCGCACAGATCATATAG CTGCCAGGTTACCCATGAAGGGAGCACTGTGGAAAA AACCGTTGCACCAACTGAATGCAGC MG U10 light chain variant 3
SEQ ID NO: 52 CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTG CAGCCCGGCGGCAGCCTGAGACTGAGCTGCACCGCC AGCGGCTTCAGCTTCAGCAGCTACGCCATGCACTGGG TGAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGG CCT ACACCAGATACGACGGCAGCAACAAGTTCT ACCT GGACAGCGTGCAGGGCAGATTCACCATCAGCAGAGA CAACAGCAAGAACACCCTGTACCTGGAGATGGACAG CCTGAGACTGGAGGACACCGCCGTGTACTTCTGCGCC AAGGTGGGCGACGGCACCGTGGCCGGCACCATCGAC TACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC MGH2 VHvl
SEQ LD NO: 53 CAGGTGCAGCTGGTGGAAAGCGGGGGGGGAGTCGTG CAGCCTGGCGGCTCTTTGCGGCTGTCTTGTACGGCTTC TGGATTCTCATTCTCTTCCTACGCCATGCACTGGGTCC GCCAGGCGCCCGGGAAGGGGCTGGAATGGGTTGCCT ACACAAGGTATGATGGTTCAAACAAGTTCTACTTAGA TTCAGTGCAGGGTAGATTCACTATAAGCCGGGACAAT AGTAAGAACACTCTCTACCTAGAAATGGACTCTCTCA GACTGGAAGATACCGCTGTGTACTTCTGTGCTAAGGT TGGGGACGGCACCGTTGCCGGCACAATCGACTATTGG GGACAAGGGACCCTCGTCACAGTCAGCTCG MGH2 VHv2
SEQ ID NO: 54 CAAGTCCAGCTTGTCGAGTCGGGGGGAGGGGTTGTCC AGCCTGGTGGTAGCTTACGCCTGAGTTGTACAGCATC GGGGTTTAGCTTCTCTTCCTATGCGATGCACTGGGTG AGACAGGCTCCCGGAAAGGGCTTAGAGTGGGTGGCC TACACTCGGTATGACGGTTCGAATAAGTTTTACTTAG ACAGCGTTCAGGGTAGGTTCACCATCTCACGTGATAA TAGTAAGAATACATTATATCTTGAGATGGACAGCCTT CGGTTGGAGGATACTGCCGTCTACTTTTGTGCTAAGG TAGGCGATGGTACGGTAGCAGGCACGATAGATTACTG GGGCCAAGGAACGTTGGTCACTGTCTCTTCA MGH2 VHv3
SEQ ID NO: 55 CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCA GCCCGGCGGCAGCCTGAGACTGAGCTGCACCGCCAGCG GCTTCAGCTTCAGCAGCTACGCCATGCACTGGGTGAGAC AGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCTACACC AGATACGACGGCAGCAACAAGTTCTACCTGGACAGCGT GCAGGGCAGATTCACCATCAGCAGAGACAACAGCAAGA ACACCCTGTACCTGGAGATGGACAGCCTGAGACTGGAG GACACCGCCGTGTACTTCTGCGCCAAGGTGGGCGACGG CACCGTGGCCGGCACCATCGACTACTGGGGCCAGGGCA CCCTGGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCC AGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAG CGGCGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGACT ACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGC GCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTG CAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGAC CGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTG CAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACA AGAAGGTGGAGCCCAAGAGCTGCGACAAGACCCACACC TGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCC AGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTG ATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTGGT GGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACT GGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACC AAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGAGT GGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGA ACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCC CTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAA GGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCC CCAGCAGAGACGAGCTGACCAAGAACCAGGTGAGCCTG ACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCC GTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTA CAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCT TCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGAT GGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGCTGCAC GAGGCCCTGCACAGCCACTACACCCAGAAGAGCCTGAG CCTGAGCCCCGGCAAG MGH2 heavy chain variant 1
SEQ ID NO: 56 CAGGTGCAGCTGGTGGAAAGCGGGGGGGGAGTCGTG CAGCCTGGCGGCTCTTTGCGGCTGTCTTGTACGGCTTC TGGATTCTCATTCTCTTCCTACGCCATGCACTGGGTCC GCCAGGCGCCCGGGAAGGGGCTGGAATGGGTTGCCT ACACAAGGTATGATGGTTCAAACAAGTTCTACTTAGA TTCAGTGCAGGGTAGATTCACTATAAGCCGGGACAAT AGTAAGAACACTCTCTACCTAGAAATGGACTCTCTCA GACTGGAAGATACCGCTGTGTACTTCTGTGCTAAGGT TGGGGACGGCACCGTTGCCGGCACAATCGACTATTGG GGACAAGGGACCCTCGTCACAGTCAGCTCGGCCAGTA CCAAGGGCCCCAGTGTGTTCCCTTTGGCCCCTTCTAGC AAATCAACGTCAGGGGGGACAGCCGCCCTTGGGTGTC TTGTGAAAGATTATTTTCCTGAGCCGGTGACCGTTTCC TGGAATAGTGGAGCACTGACAAGCGGCGTACATACCT TCCCAGCAGTGCTCCAATCAAGTGGGCTATACAGTCT GAGTAGCGTGGTCACCGTGCCATCTTCCTCTCTGGGA ACTCAAACCTATATTTGCAACGTTAATCACAAACCTT CTAATACGAAAGTCGATAAGAAGGTAGAACCGAAGT CCTGCGACAAAACCCACACATGCCCTCCGTGCCCAGC CCCTGAGCTACTGGGCGGCCCCTCTGTGTTTTTGITTC CCCCAAAACCTAAGGATACCTTAATGATCTCAAGAAC ACCCGAGGTGACCTGCGTCGTGGTAGATGTTTCTCAC GAGGACCCTGAGGTTAAATTTAATTGGTACGTGGACG GCGTGGAGGTCCATAACGCCAAGACTAAACCAAGAG AAGAGCAGTACAACTCCACATACAGGGTTGTGTCTGT GCTGACGGTCTTACACCAAGACTGGCTTAACGGCAAA GAGTATAAATGCAAGGTGAGTAATAAAGCGCTTCCTG CCCCAATCGAAAAGACCATCAGTAAAGCCAAAGGAC AGCCCAGGGAGCCTCAAGTCTACACGTTACCTCCCTC AAGGGATGAGCTCACCAAAAACCAGGTGTCTCTGACC TGCCTGGTTAAGGGCTTTTATCCTTCAGACATCGCTGT TGAGTGGGAATCAAATGGGCAGCCAGAAAATAACTA TAAAACTACCCCTCCTGTGCTGGACAGTGACGGTTCG TTCTTCCTCTATAGTAAGTTGACCGTGGATAAAAGCC GATGGCAGCAGGGAAATGTGTTCAGCTGCTCTGTACT ACATGAGGCCCTCCACAGTCACTATACGCAGAAGTCA CTGAGTCTAAGTCCAGGGAAG MGH2 heavy chain variant 2
SEQ ID NO: 57 CAAGTCCAGCTTGTCGAGTCGGGGGGAGGGGTTGTCC AGCCTGGTGGTAGCTTACGCCTGAGTTGTACAGCATC GGGGTTTAGCTTCTCTTCCTATGCGATGCACTGGGTG AGACAGGCTCCCGGAAAGGGCTTAGAGTGGGTGGCC TACACTCGGTATGACGGTTCGAATAAGTTTTACTTAG ACAGCGTTCAGGGTAGGTTCACCATCTCACGTGATAA TAGTAAGAATACATTATATCTTGAGATGGACAGCCTT CGGTTGGAGGATACTGCCGTCTACTTTTGTGCTAAGG TAGGCGATGGTACGGTAGCAGGCACGATAGATTACTG GGGCCAAGGAACGTTGGTCACTGTCTCTTCAGCATCT ACCAAGGGTCCATCTGTCTTCCCACTGGCCCCATCCTC CAAAAGCACGAGCGGAGGCACCGCTGCTCTAGGGTG TCTCGTCAAGGACTACTTTCCCGAGCCAGTGACAGTT AGTTGGAATTCCGGTGCACTTACGTCGGGGGTTCACA CATTCCCAGCAGTGCTGCAGTCGAGCGGCCTCTACAG CTTGTCCTCAGTCGTAACTGTTCCATCCAGTTCGCTCG GGACTCAGACTTACATCTGCAATGTAAACCACAAACC ATCCAATACGAAGGTGGATAAAAAGGTTGAGCCTAA GTCATGCGACAAGACACATACGTGCCCACCATGTCCC GCGCCAGAGTTGCTTGGCGGACCCAGCGTCTTTCTGT TCCCACCTAAACCCAAGGACACGTTGATGATTAGCAG GACCCCCGAAGTTACTTGTGTCGTGGTGGATGTAAGC CATGAAGACCCAGAGGTGAAATTTAACTGGTATGTGG ATGGAGTTGAAGTCCATAATGCGAAGACAAAACCTC GAGAGGAACAATATAACTCCACGTATCGAGTCGTGTC CGTACTCACAGTGTTACATCAAGATTGGTTAAATGGT AAAGAGTACAAATGCAAGGTTTCGAATAAAGCACTG CCAGCGCCGATCGAAAAGACTATCTCAAAGGCAAAA GGCCAGCCCCGGGAGCCTCAAGTATATACGCTGCCGC CATCGCGCGACGAGTTAACAAAAAACCAAGTATCGTT GACGTGTTTGGTGAAAGGTTTTTACCCTrCGGATATA GCCGTGGAGTGGGAATCCAACGGTCAACCAGAGAAC AACTATAAGACAACCCCACCAGTCTTAGATAGTGATG GCTCTTTCTTCCTCTATAGTAAACTAACGGTCGATAAA TCTCGCTGGCAGCAGGGCAACGTCTTTTCGTGTTCGG TTTTACATGAAGCTCTACATAGTCACTATACCCAGAA GAGTCTATCTCTAAGCCCCGGCAAG MGH 2 heavy chain variant 3
SEQ IDNO: 58 TACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCG TGACCCTGGGCCAGCCCGCCAGCATCAGCTGCAGAAG CAGCCAGAGCCTGGTGTACAGCGACGGCAACACCTA CCTGAACTGGTACCAGCAGAGACCCGGCCAGAGCCC CAGAAGACTGATCTACAAGGTGAGCAACAGAGACAG CGGCGTGCCCGACAGATTCAGCGGCAGCGGCAGCGG CACCGACTTCACCCTGAAGATCAGCAGAGTGGAGGCC GAGGACGTGGGCGTGTACTACTGCATGCAGGGCACCC ACTGGTGGACCTTCGGCCAGGGCACCAAGGTGGAGA TCAAG MGH2 VLvl
SEQ ID NO: 59 TACATTGTTATGACCCAGAGTCCTCTTTCACTGCCTGT GACCCTTGGCCAGCCTGCCTCCATCAGCTGCCGGTCC AGCCAATCTCTCGTGTACTCCGACGGCAATACCTACC TGAACTGGTATCAACAACGACCCGGCCAGTCACCCAG ACGCCTGATCTATAAGGTCAGCAATCGGGACAGCGGC GTCCCGGATAGGTTCTCAGGTTCAGGTTCAGGCACCG ATTTCACGCTGAAAATTAGTAGAGTTGAGGCAGAAGA TGTCGGCGTGTACTACTGTATGCAGGGTACCCATTGG TGGACCTTTGGGCAGGGCACAAAAGTAGAGATTAAG MGH2 VLv2
SEQ ID NO: 60 TACATAGTAATGACGCAGAGTCCTCTGTCCTTACCAG TTACACTGGGCCAACCTGCATCTATATCGTGTCGATC ATCTCAGTCCCTCGTGTACTCAGATGGAAATACGTAT TTGAACTGGTATCAACAGCGTCCGGGACAGAGCCCTC GCCGTTTAATCTACAAAGTTAGTAACCGAGACAGTGG CGTTCCTGACCGTTTCTCAGGATCAGGTTCCGGGACA GATTTCACCTTAAAAATAAGCAGGGTTGAAGCTGAGG ACGTGGGGGTTTATTATTGCATGCAGGGTACCCACTG GTGGACTTTTGGACAGGGTACGAAGGTTGAGATCAAG MGH2 VLv3
SEQ IDNO: 61 ' fACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCG MGH2 light
rGACCCTGGGCCAGCCCGCCAGCATCAGCTGCAGAAG 2AGCCAGAGCCTGGTGTACAGCGACGGCAACACCTAC 2TGAACTGGTACCAGCAGAGACCCGGCCAGAGCCCCA 3AAGACTGATCTACAAGGTGAGCAACAGAGACAGCG 3CGTGCCCGACAGATTCAGCGGCAGCGGCAGCGGCAC CGACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGAG □ACGTGGGCGTGTACTACTGCATGCAGGGCACCCACT GGTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAA GAGAACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCC CCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGC GTGGTGTGCCTGCTGAACAACTTCTACCCCAGAGAGG CCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGA GCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACA GCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGAC CCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTA CGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCC GTGACCAAGAGCTTCAACAGAGGCGAGTGC chain variant 1
SEQ ID NO: 62 TACATTGTTATGACCCAGAGTCCTCTTTCACTGCCTGT MGH2 light
GACCCTTGGCCAGCCTGCCTCCATCAGCTGCCGGTCC AGCCAATCTCTCGTGTACTCCGACGGCAATACCTACC TGAACTGGTATCAACAACGACCCGGCCAGTCACCCAG ACGCCTGATCTATAAGGTCAGCAATCGGGACAGCGGC GTCCCGGATAGGTTCTCAGGTTCAGGTTCAGGCACCG ATTTCACGCTGAAAATTAGTAGAGTTGAGGCAGAAGA TGTCGGCGTGTACTACTGTATGCAGGGTACCCATTGG TGGACCTTTGGGCAGGGCACAAAAGTAGAGATTAAG CGGACTGTGGCAGCTCCCTCAGTCTTTATATTTCCCCC ATCCGATGAGCAGTTGAAAAGCGGGACCGCATCAGTI GTGTGTCTGTTGAACAACTTTTACCCTCGGGAGGCCA AGGTGCAGTGGAAGGTTGATAACGCTTTACAGTCAGG CAATTCTCAGGAAAGTGTAACAGAACAGGATTCTAAG GACTCAACTTATAGCCTCTCCAGCACCCTCACATTGTC AAAGGCCGACTATGAGAAGCACAAAGTGTATGCGTG TGAGGTTACACATCAGGGCCTGAGCTCTCCGGTAACA AAGTCTTTTAAC AGGGGAGAGTGC chain variant 2
SEQ ID NO: 63 TACATAGTAATGACGCAGAGTCCTCTGTCCTTACCAG TTACACTGGGCCAACCTGCATCTATATCGTGTCGATC ATCTCAGTCCCTCGTGTACTCAGATGGAAATACGTAT TTGAACTGGTATCAACAGCGTCCGGGACAGAGCCCTC GCCGTTTAATCTACAAAGTTAGTAACCGAGACAGTGG CGTTCCTGACCGTTTCTCAGGATCAGGTTCCGGGACA GATTTCACCTTAAAAATAAGCAGGGTTGAAGCTGAGG acgtgggggtttattattgcatgcagggtacccactg GTGGACTTTTGGACAGGGTACGAAGGTTGAGATCAAG CGGACCGTTGCAGCCCCGTCCGTCTTTATTTTCCCCCC GTCCGATGAACAATTGAAATCCGGTACAGCGTCAGTA GTATGCCTCCTGAACAATTTTTATCCTCGGGAAGCCA AGGTTCAGTGGAAAGTGGATAACGCACTTCAGTCCGG CAACAGTCAGGAGTCTGTGACCGAGCAAGACTCTAA AGACTCAACCTACTCTCTTTCCTCGACCCTGACTCTTT CAAAGGCGGATTATGAGAAGCACAAAGTTTATGCCTG TGAAGTAACACATCAAGGCTTGTCGTCACCGGTTACC AAATCGTTCAACAGAGGGGAGTGC MGH2 light chain variant 3
SEQ ID NO: 64 GGCTTCGCCTTCAGCAACTACGGC MGU 10
SEQ ID NO: 65 GGCTTCGCCTTCAGTAATTACGGC MGU 10
SEQ ID NO: 66 GGCTTTGCGTTCAGTAATTATGGC MGU 10
SEQ ID NO: 67 ATCTGGCACGACGGCAGCCTGAAG MGU 10
SEQ ID NO: 68 ATTTGGCATGACGGCTCTTTGAAA MGU 10
SEQ ID NO: 69 ATTTGGCATGACGGAAGCTTGAAG MGU 10
SEQ ID NO: 70 ACCGTGTGGTACCTGGAGACCCCCGACGACGGCTTCG ACATC MGU 10 CDRH3vl
SEQ ID NO: 71 ACCGTGTGGTATCTCGAGACACCCGATGATGGCTTCG ACATC MGU 10 CDRH3v2
SEQ ID NO: 72 ACCGTTTGGTACTTGGAAACGCCGGACGACGGGTTTG ATATC MGU 10 CDRH3v3
SEQ ID NO: 73 CACGGCCACACCAGCAAGGCC MGU 10
SEQ ID NO: 74 CACGGGCACACAAGCAAGGCC MGU 10
SEQ ID NO: 75 CACGGCCACACGTCGAAGGCT MGU 10
SEQ ID NO: 76 GTCAATTCCGACGGATCTCAT MGU 10
SEQ ID NO: 77 GTCAACTCCGACGGCAGTCAT MGU 10
SEQ ID NO: 78 GTGAACAGCGACGGCAGCCAC MGU 10
SEQ ID NO: 79 CAGGCCTGGGACAGCGGCATCTGGGTG MGU 10
SEQ ID NO: 80 CAGGCCTGGGACTCAGGCATATGGGTA MGU 10
SEQ ID NO: 81 CAGGCCTGGGACTCAGGTATTTGGGTA MGU10
SEQ ID NO: 82 GGCTTCAGCTTCAGCAGCTACGCC MGH2
SEQ ID NO: 83 GGATTCTCATTCTCTTCCTACGCC MGH2
SEQ ID NO: 84 GGGTTTAGCTTCTCTTCCTATGCG MGH2
SEQ ID NO: 85 ACTCGGTATGACGGTTCGAATAAG MGH2
SEQ ID NO: 86 ACAAGGTATGATGGTTCAAACAAG MGH2
SEQ ID NO: 87 ACCAGATACGACGGCAGCAACAAG MGH2
SEQ ID NO: 88 GCCAAGGTGGGCGACGGCACCGTGGCCGGCACCATC GACTAC_______________________________ MGH2 CDRH3vl
SEQ ID NO: 89 GCTAAGGTTGGGGACGGCACCGTTGCCGGCACAATCG ACTAT MGH2 CDRH3v2
SEQ ID NO: 90 GCTAAGGTAGGCGATGGTACGGTAGCAGGCACGATA GATTAC MGH2 CDRH3v3
SEQ IDNO: 91 CAGAGCCTGGTGTACAGCGACGGCAACACCTAC MGH2 ΓΠΡΙ HH
SEQ ID NO: 92 CAATCTCTCGTGTACTCCGACGGCAATACCTAC MGH2 rnwi 1,,7
SEQ ID NO: 93 CAGTCCCTCGTGTACTCAGATGGAAATACGTAT MGH2 cnn T 1v3
SEQ ID NO: 94 AAAGTTAGT MGH2
SEQ ID NO: 95 AAGGTCAGC MGH2
SEQ ID NO: 96 AAGGTGAGC Π MGH2
SEQ ID NO: 97 ATGCAGGGCACCCACTGGTGGACC MGH2
SEQ ID NO: 98 ATGCAGGGTACCCATTGGTGGACC MGH2
SEQ ID NO: 99 ATGCAGGGTACCCACTGGTGGACT MGH2
Amino acid séquences __
SEQ IDNO: 100 QVQLVESGGGVVQPGRSLRLSCAASGFAFSNYGMNW VRQAPGKGLEWVAVIWHDGSLKYYTQSVKGRFTISRD NAKNTLFLQMDSLSADDTAMYYCTVWYLETPDEGFDI WGRGTMVTV S S ASTKGPS VFPLAPS SKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLS PGK MGU10v2_LS heavy chain
SEQ IDNO: 101 ^VQLVESGGGVVQPGRSLRLSCAASGFAFSNYGMNW VRQAPGKGLEWVAVIWHDGSLKYYTQSVKGRFTISRD 4AKNTLFLQMDSLSADDTAMYYCTVWYLETPDDGFDI WGRGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEP VTVS WNSGALTSGVHTFPAVLQS SGLYS L SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC dkthtcppcpapellggpsvflfppkpkdtlmisrtpev TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YN STYR WS VLTVLHQDWLNGKEYKCKV SNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSP GK MGU10_LS heavy chain
SEQ IDNO: 102 QVQLVESGGGVVQPGGSLRLSCTASGFSFSSYAMHWV RQAPGKGLEWVAYTRYDGSNKFYLDSVQGRFTISRDN SKNTLYLEMDSLRLEDTAVYFCAKVGDGTVAGTIDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSD IA VEWESNGQ PENNYKTTPPVLD S DGSFFLYSK LTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPG K MGH2/MGH2 vl_LS heavy chain
SEQ IDNO: 103 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVLHEALHSHYTQKSLSLSPGK heavy chain constant région LS
SEQ 1DNO: 104 QVQLVESGGGVVQPGRSLRLSCAASGFAFSNYGMNW VRQAPGKGLEWVAVIWHDGSLKYYTQSVKGRFTISRD NAKNTLFLQMDSLSAEDTAMYYCTVWYLETPDEGFD1 WGRGTMVTVSS MGU10v8 VH
SEQIDNO: 105 KQPADGNPDPNANPNVDPN NPDP19peptide
Nucleic acid sequences
SEQ IDNO: 106 ACCGGTGTACATTCTCAGGTGCAGCTGGTGG AGTCCGGAGGAGGAGTGGTGCAGCCAGGCAG GAGCCTGAGGCTGTCTTGCGCTGCTTCCGGAT TCGCCTTTAGCAACTACGGCATGAATTGGGTG AGGCAGGCTCCTGGCAAGGGACTGGAGTGGG TGGCTGTGATCTGGCACGACGGCAGCCTGAA GTACTATACACAGTCTGTGAAGGGCAGATTC ACCATCTCTCGCGATAACGCTAAGAATACAC TGTTTCTGCAGATGGACTCTCTGTCCGCCGAG GATACCGCTATGTACTATTGTACAGTGTGGTA TCTGGAGACCCCAGACGAGGGCTTCGATATC TGGGGCAGAGGCACCATGGTGACAGTGTCCA GCGCGTCGAC MGU10v8 VH
100

Claims (20)

1. An antibody, or an antigen-binding fragment thereof, comprising (i) the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, respectively, and the light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; or (ii) the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively; or (iii) the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO; 12, respectively, and the light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 14, respectively; or (iv) the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively, and the light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 28, respectively; or (v) the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively, and the light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 28, respectively.
2. The antibody, or an antigen-binding fragment thereof, of claim 1, wherein the antibody, or the antigen-binding fragment thereof, comprises (i) a heavy chain variable région comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO; 13 and a light chain variable région comprising the amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO: 8, wherein the CDR sequences as defined in claim 1 are maintained; or (ii) a heavy chain variable région comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO: 7 and a light chain variable région comprising the amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO: 15, wherein the CDR sequences as defined in claim 1 are maintained; or (iii) a heavy chain variable région comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO: 16 and a light chain variable région comprising the amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95%
101 identity to SEQ ID NO: 8, wherein the CDR sequences as defined in claim 1 are maintaîned; or (iv) a heavy chain variable région comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO: 11 and a light chain variable région comprising the amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO: 15, wherein the CDR sequences as defined in claim 1 are maintaîned; or (v) a heavy chain variable région comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO: 13 and a light chain variable région comprising the amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO: 15, wherein the CDR sequences as defined in claim 1 are maintaîned; or (vi) a heavy chain variable région comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO: 16 and a light chain variable région comprising the amîno acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO: 15, wherein the CDR sequences as defined in claim 1 are maintaîned; or (vii) a heavy chain variable région comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO: 24 and a light chain variable région comprising the amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO: 29, wherein the CDR sequences as defined in claim 1 are maintaîned; or (viii) a heavy chain variable région comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO: 104 and a light chain variable région comprising the amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO: 8, wherein the CDR sequences as defined in claim 1 are maintaîned; or (ix) a heavy chain variable région comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO: 104 and a light chain variable région comprising the amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO: 15, wherein the CDR sequences as defined in claim 1 are maintaîned.
3. The antibody, or the antigen-binding fragment thereof, of any one of the previous daims, wherein the antibody, or the antigen-binding fragment thereof, comprises (i) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 13 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 8; or (ii) a heavy cbain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 7 and a light chain variable région comprising the amino acid sequence as set
102 forth in SEQ ID NO: 15; or (iii) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 16 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 8; or (iv) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 11 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 15; or (v) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 13 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 15; or (vi) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 16 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 15; or (vii) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 24 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 29; or (viii) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 104 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 8; or (îx) a heavy chain variable région comprising an amino acid sequence as set forth in SEQ ID NO: 104 and a light chain variable région comprising the amino acid sequence as set forth in SEQ ID NO: 15.
4. The antibody, or an antigen-binding fragment thereof, of any one of the previous daims, wherein the antibody, or the antigen-binding fragment thereof, bînds to Plasmodium falciparum sporozoites, preferably to circumsporozoite protein of Plasmodium falciparum.
5. The antibody, or an antigen-binding fragment thereof, of any one of the previous daims, wherein the antibody, or the antigen-binding fragment thereof, neutralizes infection with Plasmodium falciparum.
6. The antibody, or an antigen-binding fragment thereof, of any one of the previous daims, wherein the antibody, or the antigen-binding fragment thereof, binds to the NANP-repeat région of circumsporozoite protein of Plasmodium falciparum.
7. The antibody, or an antigen-binding fragment thereof, of any one of the previous daims, wherein the antibody, or the antigen-binding fragment thereof, binds to the N-terminal région of Plasmodium falciparum circumsporozoite protein, which covers the jonction between the N-terminal domain and the NANP-repeats of circumsporozoite protein.
103
8. The antibody, or an antigen-binding fragment thereof, of claîm 33 or 34, wherein the antibody bînds to a peptide according to SEQ ID NO: 34 and/or a peptide according to SEQ ID NO: 35 or 105.
9. The antibody, or an antigen-binding fragment thereof, of any one of the previous daims, wherein the antibody, or the antigen-binding fragment thereof, is a human antibody and/or a monoclonal antibody.
10. The antibody of any one of the previous daims, wherein the antibody comprises an Fc moiety.
11. The antibody, or an antigen-binding fragment thereof, of any one of the previous daims, wherein the antibody, or the antigen-binding fragment thereof, comprises the mutations M428L and/or N434S in the heavy chain constant région.
12. The antibody of any one of the previous daims, wherein the antibody is of the IgG type.
13. A nucleic acid molécule comprising a polynucleotide encoding the antibody, or an antigenbinding fragment thereof, of any one of daims 1-12.
14. A combination of a first and a second nucleic acid molécule, wherein the first nucleic acid molécule comprises a polynucleotide encoding the heavy chain of the antibody, or an antigen-binding fragment thereof, of any one of daims 1-12; and the second nucleic acid molécule comprises a polynucleotide encoding the corresponding light chain of the same antibody, or the same antigen-binding fragment thereof.
15. A vector comprising the nucleic acid molécule of daim 13 or the combination of nucleic acid molécules of daim 14.
16. A combination of a first and a second vector, wherein the first vector comprises a first nucleic acid molécule as defined in daim 14 and the second vector comprises the corresponding second nucleic acid molécule as defined in daim 14.
104
17. A cell expressing the antibody, or an antigen-binding fragment thereof, of any one of daims 1 - J 2, or comprising the vector of claim 15 or the combination of vectors of claim 16.
18. A pharmaceutical composition comprising the antibody, or an antigen-binding fragment thereof, of any one of daims 1-12, the nucleic acid of daim 13, the combination of nucleic acids of—claim 14, the vector of claim 15, the combination of vectors of claim 16 or the cell of daim 17, and, optionally, a pharmaceutically acceptable diluent or carrier.
19. The antibody, or an antigen-binding fragment thereof, of any one of daims 1-12, the nucleic acid of daim 13, the combination of nucleic acids of-daim 14, the vector of daim 15, the combination of vectors of claim 16, the cell of claim 17, or the pharmaceutical composition of claim 18 for use in the prophylaxis or treatment of malaria.
20. Use of the antibody, or an antigen-binding fragment thereof, of any one of daims 1 - 12, the nucleic acid of claim 13, the combination of nucleic acids of-daim 14, the vector of daim 15, the combination of vectors of daim 16, the cell of daim 17, or the pharmaceutical composition of claim 18 in the manufacture of a médicament for prophylaxis, treatment or atténuation of malaria.
OA1202100486 2019-04-30 2020-04-30 Antibodies binding to plasmodium circumsporozoite protein and uses thereof. OA20886A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
EPPCT/EP2019/061135 2019-04-30

Publications (1)

Publication Number Publication Date
OA20886A true OA20886A (en) 2023-06-27

Family

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