EA043940B1 - MULTI-SPECIFIC ANTIBODIES SPECIFIC TO ZIKA VIRUS EPITOPES AND THEIR APPLICATION - Google Patents

Description

The invention relates to multispecific antibodies and their antigen-binding fragments that specifically bind to various epitopes of the Zika virus (ZIKV). Such antibodies effectively neutralize Zika virus (ZIKV) infection and minimize or eliminate the production of Zika virus escape mutants. The invention also relates to nucleic acids that encode such antibodies and antibody fragments. The invention also relates to the use of antibodies and antibody fragments of the present invention for the prevention and treatment of ZIKV infection.

Zika virus (ZIKV) is a flavivirus, transmitted by mosquitoes, and poses a public health threat. ZIKV was first isolated from macaques in 1947 in the Zika forest in Uganda (Dick G.W.A. et al., Trans. R. Soc. Trop. Med. Hyg. 46, 1952, 509-520) and the first infection in humans was described in Nigeria in 1954 (Macnamara F.N., Trans. R. Soc. Trop. Med. Hyg. 48, 1954, 139-145). Since then, ZIKV infections have been periodically reported in Africa and Southeast Asia (Musso D., CaoLormeau V.-M., Gubler D. J., The Lancet. 386, 2015, 243-244), and epidemics were reported in Macronesia in 2007 (Duffy M.R. et al., N Engl J Med. 360, 2009, 2536-2543) and French Polynesia in 2013-2014, with the subsequent spread of the virus to other countries in Oceania (Cao-Lormeau V.-M., Musso D. Lancet. 384, 2014, 1571-1572); Musso D., Nilles E. J., Cao-Lormeau V.-M. Clin. Microbiol. Infect. 20, 2014, 0595-6). After its emergence in Brazil in 2015, ZIKV spread rapidly and in February 2016, the World Health Organization (WHO) declared its spread a public health emergency of international concern (Baden L.R. et al. N. Engl. J Med. 374, 2016, 1552-1563; Fauci A. S., Morens D. M. N Engl J Med, 160113142101009, 2016; Heymann D. L. et al. Lancet. 387, 2016, 719-721). The main spread of ZIKV infection is through the bites of Aedes mosquitoes, but the virus can also be spread sexually (Musso D. et al. Emerg Infect Dis. 21, 2015, 359-361) and transmitted vertically (from parents to children) (Mlakar J. et al. co-author N Engl J Med 374, 2016, 951-958). Although most ZIKV infections are asymptomatic or have mild symptoms, there is evidence that ZIKV infection can cause neurological complications, such as Guillain-Barré syndrome in adults (CaoLormeau V.-M. et al. Lancet. 0, 2016, doi:10.1016 /S0140-6736(16)00562-6) and birth defects, including microcephaly in the developing fetus (Calvet G. et al. Lancet Infect Dis 2016, doi: 10.1016/s1473-3099(16)00095-5; Mlakar J. with al N Engl J Med 374, 2016, 951-958; Rubin E. J. et al N Engl J Med 2016, doi:10.1056/NEJMe1601862), probably due to its ability to infect human neuronal progenitor cells (Tang H. et al Stem Cell, 2016, 1-5).

ZIKV belongs to the genus Flavivirus, which also includes West Nile virus, dengue virus, tick-borne encephalitis virus, yellow fever virus, and several other viruses that can cause encephalitis. Flaviviruses have an icosahedral or spherical envelope. Diameter is about 50 nm. The genomes are positively polarized linear strands of RNA and are unsegmented, about 10-11 kb. in length. The flavivirus genome encodes 3 structural proteins (capsid, prM and envelope) and 8 non-structural proteins (NS1, nS2A, NS2B, NS3, NS4A, NS4B, nS5 and NS5B).

Although flavivirus envelope proteins (E - envelope) mediate fusion and are the main target of neutralizing antibodies, nonstructural protein 1 (NS1) is secreted by infected cells and is involved in immune evasion and pathogenesis (Muller D.A., Young P.R. Antiviral Res. 98, 2013, 192 -208). Two recent structural studies have shown a high level of structural similarity between the E protein of ZIKV and the same protein of other flaviviruses, such as dengue virus (DENV), yellow fever virus (YFV) and West Nile virus (WNV), but also reveal unique properties , which may be associated with ZIKV neurotropism (Dai L. et al., Cell Host Microbe (2016), doi:10.1016/j.chom.2016.04.013; Sirohi D. et al., Science, aaf5316 (2016). Similarly Thus, structural analysis of ZIKV NS1 revealed conserved features of NS1 of other flaviviruses, albeit with different electrostatic properties (Kim J., 1-6 (2016)).

A phenomenon that is common to some flaviviruses is the disease-enhancing activity of cross-reactive antibodies induced by previous infection with heterologous viruses. In the case of dengue virus (DENV), which has 4 known serotypes, there is epidemiological evidence that primary infection protects against reinfection with the same serotype but is a risk factor for severe disease if reinfected with a different serotype (Halstead S.B., Microbiol Spectr. 2, 2014, 249-271). Acute disease is caused by E and prM-specific antibodies that fail to neutralize incoming virus but instead enhance its uptake by Fc receptor (FcR+)-expressing cells, leading to enhanced viral replication and activation of cross-reactive memory T cells. The resulting cytokine storm is thought to underlie the most severe form of the disease, known as dengue hemorrhagic fever/dengue shock syndrome (Halstead S.B., Adv Virus Res. 60, 2003, 421-467; Screaton G., Mongkolsapaya J., Yacoub S., Roberts C. Nat Rev Immunol 15, 2015, 745-759). The role of antibodies in severe dengue is supported by studies showing that decreased levels of maternal antibodies in children pose an increased risk of developing severe dengue (Halstead S.B., Adv Virus Res. 60, 2003, 421-467; Halstead S.B. et al., Emerging Infect Dis 8, 2002, 1474-1479); Nguyen

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T.H. et al., J Infect Dis. 189, 2004, 221-232; Rothman A.L., J Clin Invest. 113, 2004, 946-951).

It was previously shown that most antibodies that reacted to the DENV envelope protein also bound to ZIKV, but those that recognized the large linear fusion-loop epitope (FLE) did not neutralize ZIKV and instead promoted antibody-dependent enhancement ( ADE) ZIKV infection (Dejnirattisai W., Nat Immunol. 2016, June 23, doi: 10.1038/ni.3515. [published online ahead of print]).

In addition, organisms with high mutation rates, such as various viruses such as the Zika virus, often use so-called mutation escape as a mechanism to avoid destruction by host cells. Namely, the virus can protect itself from the host’s immune responses due to mutations in its genotype and phenotype (elimination due to mutations). Accordingly, the development of escape mutations (that is, the emergence of viruses carrying escape mutations) may reduce the effectiveness of antibody drugs.

In view of the above, the object of the present invention is to develop multispecific antibodies that can neutralize the Zika virus (ZIKV). Such antibodies do not promote preferential antibody-dependent enhancement (ADE) of Zika virus infection. It is also an object of the present invention to provide highly specific anti-ZIKV antibodies that eliminate or minimize the occurrence of ZIKV mutants protected by mutations.

The solution to the problem underlying the present invention is given in the claims.

Although the present invention is described in detail below, it should be noted that the present invention is not limited to the specific methodologies, protocols and reagents described herein, as they may vary. It should also be noted that the terminology used in the present invention is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise specified, all technical and scientific terms used in this invention have the same meanings as used by those skilled in the art.

The following is a description of the elements of the present invention. These elements are listed in the description of some specific embodiments of the present invention, however, it should be understood that they can be combined in any manner and in any quantity to create additional embodiments of the present invention. The various examples and preferred embodiments of the present invention described should not be construed as limiting the present invention to only the embodiments shown. This description should be considered as supporting and covering embodiments of the present invention that combine specific embodiments with any number of the described and/or preferred elements. In addition, any permutations and combinations of all described elements in the invention are to be considered as disclosed in the description of the present invention, unless the context indicates otherwise.

In the present specification and in the claims below, unless the context otherwise requires, the term contains, includes, and includes means the inclusion of a particular subject matter, integer, or step, but does not exclude any other subject matter, integer, or step. . The concept consists of means specifically the concept contains and excludes any unspecified element, integer or stage. In the context of the present invention, the concept includes includes the concept consists of. Thus, the concept containing covers the concept of including as well as consisting, for example, a composition containing X may consist solely of X or may include something additional, for example, X + Y.

In the context of the present invention (especially in the context of the claims), both singular and plural entities may be implied unless otherwise indicated or the context clearly contradicts them. The listing of ranges of values in the present invention is intended to serve as a concise way of individually referring to each individual value within the range. Unless otherwise indicated in the present invention, each individual value is included in the specification as if it were separately stated therein. The language in this specification should not be construed as indicating any unclaimed element essential to the practice of the invention.

The term essentially does not exclude the concept completely, for example, a composition that is substantially free of Y may be completely free of Y. If necessary, the word essentially may be omitted from the description of the present invention.

The concept roughly applied to the numerical expression χ means ±10%.

The concept of disease in the context of the present invention is synonymous and is used interchangeably with the concepts of disorder and condition (state of health), since they all reflect an abnormal condition of the human or animal body or any of its parts, leading to dysfunction, which usually manifests itself in changes in symptoms and symptoms and leads to a decrease in the duration or quality of life in a person or animal.

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In the present invention, references to treating a subject or patient include prevention, amelioration, improvement, and therapy. The terms subject or patient are used interchangeably in the present invention to refer to all mammals, including humans. Subjects include, for example, humans, cows, dogs, cats, horses, goats, sheep, pigs and rabbits. In one embodiment of the present invention, the patient is a human.

In the present invention, the terms antigen binding fragment, fragment and antibody fragment are used interchangeably to refer to any fragment of the antibody of the present invention that retains the antigen binding activity of the antibody. Examples of antibody fragments include, but are not limited to, single chain antibodies, Fab, Fab', F(ab') ₂ , Fv or scFv. In addition, the term antibody as used herein includes both antibodies and antigen-binding fragments thereof.

As used herein, the term antibody covers various forms of antibodies, including, but not limited to, whole antibodies, antibody fragments, particularly antigen binding fragments, human antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies and genetically engineered antibodies (variants or mutant antibodies). as long as the characteristic properties according to the present invention are maintained. Monoclonal antibodies are preferred, and particularly preferred are monoclonal antibodies with human CDRs or human variable regions. In particular, the antibodies or antigen-binding fragments thereof according to the present invention are preferably derived from human antibodies (ie, they contain CDRs and/or variable regions of human antibodies). More preferably, the antibodies, or antigen binding fragments thereof, of the present invention also contain human antibody constant regions. Most preferably, all constant and variable regions of antibodies or antigen binding fragments thereof according to the present invention are of human origin, that is, human constant and variable regions, such as human antibody constant and variable regions.

Human antibodies are well known to those skilled in the art (van Dijk M.A., van de Winkel J.G., Curr. Opin. Chem. Biol. 5, 2001, 368-374). Human antibodies can also be produced in transgenic animals (eg mice) that are capable of producing a full spectrum of or selecting for human antibodies after immunization in the absence of endogenous immunoglobulin production. Transfer of the human germline immunoglobulin gene array into the germline of such mutant mice results in the production of human antibodies upon challenge with the antigen (see, for example, Jakobovits A. et al., Proc. Nail. Acad. Sci. USA 90, 1993, 2551-2555 ; Jakobovits A. et al., Nature 362, 1993, 255258; Bruggemann M. et al., Year Immunol. 7, 1993, 3340). Human antibodies can also be produced in phage display libraries (Hoogenboom H.R., Winter G., J. Mol. Biol. 227, 1992, 381-388; Marks J.D. et al., J. Mol. Biol. 222, 1991, 581-597 ). Methods Cole et al. and Boerner et al. can also be used to obtain human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner P. et al., J. Immunol. 147, 1991, 86-95) . Preferably, human monoclonal antibodies are produced using improved immortalization of EBV-B cells, as described in Traggiai E. et al., Nat Med. 10(8), 2004, 871-875. In the context of the present invention, the term human antibody also includes modified antibodies, for example, in the variable region or constant region, which exhibit the properties of the present invention.

The antibodies of the present invention may be of any isotype (eg, IgA, IgG, IgM, ie α, γ or μ heavy chain), but are preferably IgG. Within the IgG isotype, antibodies can be from the IgG1, IgG2, IgG3 or IgG4 subclasses, with the IgG1 subclass being preferred. Antibodies of the present invention may have a k or λ light chain.

The antibody of the present invention, or an antigen binding fragment thereof, can be a purified antibody or a single chain antibody, for example, a bispecific single chain fragment Fv(scFv).

The invention also provides fragments of the antibodies of the present invention, in particular fragments that retain the antigen binding activity of the antibodies. Although the description of the present invention, including the claims, may in some places expressly refer to antigen-binding fragment(s), antibody fragment(s), antibody variant(s), and/or antibody derivative(s), it is intended that the term antibody or antibody as used herein The invention includes all categories of antibodies, namely antigen-binding fragment(s), antibody fragment(s), antibody variant(s), and antibody derivative(s). The antibody fragments of the present invention can be prepared from antibodies by methods that include digestion with enzymes such as pepsin or papain and/or cleavage of disulfide bonds by chemical reduction. Alternatively, antibody fragments can be produced by cloning and expressing portions of the heavy or light chain sequences. For example, the invention includes a bispecific scFv containing a CDR from an antibody of the present invention.

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The antibody fragments of the present invention may impart monovalent or multivalent interactions and may be contained in a variety of structures. For example, scFv molecules can be synthesized to create a trivalent triantibody or a tetravalent tetraantibody.

ScFv molecules can include an Fc region domain, resulting in the formation of divalent miniantibodies.

Antibodies of the present invention may be provided in purified form. Typically, the antibody will be in a composition that is substantially free of other polypeptides, such as less than 90% (by weight) of other polypeptides in the composition, typically less than 60%, and most often less than 50%.

Antibodies of the present invention may be immunogenic in hosts in humans and/or other (heterologous) species, such as mice. For example, antibodies may have an idiotope that is immunogenic in non-human hosts, but not in the human host. Antibodies of the present invention for human use include antibodies that cannot be readily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, etc., and generally cannot be obtained by humanization or from xeno-mice.

In the context of the present invention, the term neutralizing antibody means an antibody that can neutralize, that is, prevent, inhibit, reduce or interfere with the ability of a pathogen to initiate and/or maintain infection in a host. The terms neutralizing antibody and antibody that neutralizes or antibodies that neutralize are used interchangeably in the present invention. These antibodies can be used alone or in combination, as prophylactic or therapeutic agents when appropriately formulated, in combination with active vaccination, as a diagnostic tool, or as a production tool, as described in the present invention.

Doses are often designed based on body weight. For example, a dose expressed in [g, mg or other unit]/kg (or g, mg, etc.) usually refers to [g, mg, or other unit] per kg (or g, mg, etc. ) body weight, even if the concept of body weight is not precisely specified.

The term specifically binding and similar references do not include non-specific adhesion.

In the context of the present invention, the term vaccine generally means a prophylactic or therapeutic material providing at least one antigen, preferably an immunogen. The antigen or immunogen can be obtained from any material that is suitable for vaccination. For example, the antigen or immunogen may be derived from a pathogen such as bacteria or viral particles, etc., or from a tumor or cancerous tissue. An antigen or immunogen stimulates the body's adaptive immune system to produce an adaptive immune response. In particular, antigen or immunogen generally refers to a substance that can be recognized by the immune system, preferably the adaptive immune system, and that is capable of inducing an antigen-specific immune response, for example, through the production of antibodies and/or antigen-specific T cells as part of the adaptive immune response. Typically, the antigen may be, or may include, a peptide or protein that can be presented by the major histocompatibility complex (MHC) to T cells.

In the context of the present invention, the term sequence variant (also simply variant) refers to any change in a reference sequence such that the original sequence is any of the sequences listed in the Sequence Tables and SEQ ID NOs (Sequence Listing) section, i.e. .e. from the sequences of SEQ ID NO: 1 to SEQ ID NO: 273. Thus, the term sequence variant includes nucleotide sequence variants and amino acid sequence variants. It should be noted that the sequence variants referred to in the present invention are, in particular, functional sequence variants, that is, sequence variants that support a biological function, for example, an antibody. In the context of the present invention, such supported biological function is preferably neutralization of ZIKV infection and/or binding of the antibody to ZIKV E protein. Thus, preferred sequence variants are functional sequence variants that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different at least 90, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical original sequence.

Thus, preferred sequence variants are functional sequence variants that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different at least 90, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to the original sequences.

In the present invention, the expression is a functional sequence variant that

- 4 043940 is identical to the original sequence by at least 70, at least 75, at least 80, at least 85, at least 88, at least 90, by at least 92, at least 95, at least 96, at least 97, at least 98, or at least 99% means that (i) the sequence variant is functional as described in the present invention, and (ii) the higher the percentage of sequence identity, the more preferred that sequence variant is. In other words, the expression is a functional sequence variant that is at least 70 identical to the original sequence, at least 75 identical, at least 80 identical, at least 85 identical, at least 88 identical, at least 90, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% means, in specifically, that the functional sequence variant is at least 70% identical, preferably at least 75% identical, preferably at least 80% identical, more preferably at least 85% identical to the parent sequence , more preferably at least 88 identical, even more preferably at least 90 identical, even more preferably at least 92 identical, and even more preferably at least 95 identical, and even more preferably at least 96% identical, particularly preferably at least 97% identical, particularly preferably at least 98% identical, and most preferably at least 99% identical.

The term sequence variant includes, in particular, those variants that contain mutations and/or substitutions with respect to the original sequence. Examples of Fc fragment sequence variants include, but are not limited to, those variants that have an L to A substitution at the CH2 4 position, CH2 5 position, or both.

Sequence identity is usually calculated relative to the total length of the original sequence (ie the sequence that is provided in the invention). The percent identity as described in the present invention can be determined, for example, using BLAST using default parameters defined by NCBI (the National Center for Biotechnology Information; http://www.ncbi.nlm.nih .gov/) [Blosum 62 matrix; penalty for gap opening = 11 and penalty for gap extension = 1].

In the context of the present invention, the term nucleotide sequence variant has an altered sequence in which one or more nucleotides in the reference sequence are deleted or replaced, or one or more nucleotides are inserted into the sequence of the original nucleotide sequence. In the present invention, nucleotides are designated by a standard one-letter code (A, C, G or T). Due to the degeneracy of the genetic code, a variant of the nucleotide sequence can either lead to a change in the corresponding original amino acid sequence, that is, to a variant amino acid sequence, or not. Preferred sequence variants are those nucleotide sequence variants that do not result in amino acid sequence variants (silent mutations), but other non-silent mutations are also within the scope of the present invention, in particular mutant nucleotide sequences that result in an amino acid sequence that is at least 80, sequentially at least 90, more sequentially at least 95% of the sequence is identical to the control sequence.

An amino acid sequence variant has an altered sequence in which one or more amino acids are deleted or substituted from the reference sequence, or one or more amino acids are inserted from the reference sequence. As a result of the changes, the amino acid sequence variant has an amino acid sequence that is at least 80 identical to the control sequence, preferably at least 90 identical, more preferably at least 95 identical, most preferably at least 95 identical , is 99% identical to the control sequence. Sequence variants that are at least 90% identical have no more than 10 changes, that is, any combination of deletions, insertions, or substitutions of 100 amino acids of the reference sequence.

Although it is possible to have non-conservative amino acid substitutions, it is preferred that the substitutions are conservative amino acid substitutions in which the substituted amino acid has similar structural or chemical properties to the corresponding amino acid in the original sequence. By way of example, conservative amino acid substitutions include replacing one aliphatic or hydrophobic amino acid, such as alanine, valine, leucine and isoleucine, with another; substitution of one hydroxyl-containing amino acid, for example, serine and threonine, with another; replacement of one acid residue, for example glutamic acid or aspartic acid, with another; substitution of one amide-containing residue, for example, asparagine and glutamine, with another; substitution of one aromatic residue, for example, phenylalanine and tyrosine, with another; substitution of one basic residue, such as lysine, arginine and histidine, with another; and replacing one small amino acid, such as alanine, serine, threonine, methionine and glycine, with another.

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Amino acid sequence insertions include amino- and/or carboxy-terminal fusions ranging from one residue to a polypeptide containing one hundred or more residues, as well as sequence insertions of one or more amino acid residues. Examples of terminal insertions include fusion of the N- or C-terminus of an amino acid sequence with a reporter molecule or enzyme.

It is important to note that changes in sequence variants do not eliminate the corresponding functionality of the original sequence, in this case, for example, the functionality of the antibody sequence, or an antigen binding fragment thereof, to bind to the same epitope and/or to substantially neutralize ZIKV infection. Techniques for determining which nucleotides and amino acid residues, respectively, can be substituted, inserted or deleted without affecting such functionality are found using computer programs known in the art.

In the context of the present invention, the concept of a nucleic acid sequence or an amino acid sequence derived from a particular nucleic acid, peptide, polypeptide or protein refers to the origin of the nucleic acid, peptide, polypeptide or protein. Preferably, the nucleic acid sequence or amino acid sequence that is derived from a particular sequence has an amino acid sequence that is substantially identical to the sequence or portion thereof from which it is derived, whereby being substantially identical includes sequence variants as described above. Preferably, the nucleic acid sequence or amino acid sequence that is derived from a particular peptide or protein is derived from a corresponding domain in the particular peptide or protein. Thus, the concept corresponding refers in particular to the same functionality. For example, the extracellular domain corresponds to another extracellular domain (of another protein), or the transmembrane domain corresponds to another transmembrane domain (of another protein). The corresponding portions of the peptides, proteins and nucleic acids are thus easily identified by those skilled in the art. Moreover, sequences derived from another sequence are usually easily identified by those skilled in the art because they are derived from that sequence.

Preferably, the nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may be identical to the original nucleic acid, peptide, polypeptide or protein (from which it is derived). However, a nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may also have one or more mutations relative to the original nucleic acid, peptide, polypeptide or protein (from which it is derived), in particular the nucleic acid sequence or an amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may be a variant of the functional sequence, as described above, of the parent nucleic acid, peptide, polypeptide or protein (from which it is derived). For example, in a peptide/protein, one or more amino acid residues may be replaced by other amino acid residues, or insertions or deletions of one or more amino acid residues may occur.

In the context of the present invention, the term mutation refers to a change in the nucleic acid sequence and/or amino acid sequence relative to a reference sequence, for example, a corresponding genomic sequence. The mutation, for example, compared to the genomic sequence, can be, for example, a (natural) somatic mutation, a spontaneous mutation, an induced mutation, for example caused by enzymes, chemicals or radiation, or a mutation obtained by site-directed mutagenesis (molecular biology techniques for making specific and deliberate changes to the nucleic acid sequence and/or amino acid sequence). Thus, the concepts of mutation and mutation should also be understood as the physical action of creating a mutation, for example, in a nucleic acid sequence or in an amino acid sequence. Mutations include substitutions, deletions and insertions of one or more nucleotides or amino acids, as well as inversions of several consecutive nucleotides or amino acids. To produce a mutation in an amino acid sequence, it is preferred that the mutation can be introduced into a nucleotide sequence encoding said amino acid sequence to express the (recombinant) mutated polypeptide. A mutation can be achieved, for example, by changing, for example by site-directed mutagenesis, a codon of a nucleic acid molecule encoding one amino acid to produce a codon encoding another amino acid, or by synthesizing a sequence variant, for example, knowing the nucleotide sequence of a nucleic acid molecule, encoding a polypeptide, and synthesizing a designed nucleic acid molecule containing a nucleotide sequence that encodes a variant polypeptide, without having to mutate one or more nucleotides of the nucleic acid molecule.

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Several documents are cited throughout this specification. Each of the documents cited (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) above or below in the text is incorporated herein by reference. Nothing in the present invention should be construed as an admission that the present invention is not entitled to precede such disclosure by virtue of a prior invention.

It should be noted that the present invention is not limited to the specific methodology, protocols and reagents described in the present invention, as they may vary. It should also be understood that the terminology used in the present invention is intended only to describe certain embodiments of the present invention and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as commonly accepted by those skilled in the art.

Multispecific antibodies that bind to specific epitopes of the Zika virus.

The present invention is based, among other results, on the discovery that multispecific antibodies and their antigen-binding fragments specifically bind to different epitopes of the Zika virus.

Such antibodies minimize or eliminate the formation of Zika virus mutants that escape antibodies (antibody escape mutants). In particular, there are currently no preventive/treatment measures for Zika virus infection. Antibodies meeting this criterion are highly effective in preventing, treating or mitigating Zika virus infection. Moreover, due to the specificity of Zika virus antibodies, they do not cause ADE, but rather block ADE.

The first aspect of the present invention relates to individual multispecific antibodies or antigen binding fragments thereof that specifically bind to different epitopes of the Zika virus. In other words, the present invention provides an isolated multispecific antibody or antigen binding fragments thereof that contain at least two epitope binding sites that specifically bind to different epitopes of the Zika virus.

It is important that, unlike ordinary antibodies, which exhibit only one specificity, multispecific antibodies are able to bind to at least two different epitopes. In this case, multispecific antibodies specifically bind to (at least two) different epitopes of the Zika virus.

Thus, in the context of the present invention, the term multispecific refers to the ability to bind to at least two different epitopes, e.g. on different antigens, such as different Zika virus (ZIKV) proteins, or on the same antigen, e.g. on the same ZIKV protein. Preferably, the multispecific antibodies, or antigen binding fragments thereof, of the present invention bind to at least two different epitopes on the same ZIKV protein, most preferably to at least two different epitopes on the (E) envelope protein of the virus Zika.

Preferably, the multispecific antibodies, or antigen binding fragments thereof, of the present invention are bispecific, trispecific, tetraspecific or pentaspecific, more preferably, the antibody, or antigen binding fragment thereof, is bispecific or trispecific, and most preferably, the antibody, or antigen binding fragment thereof, is bispecific.

In the context of the present invention, the terms bispecific, trispecific, tetraspecific are related to the number of different epitopes to which an antibody, or an antigen-binding fragment thereof, can bind. For example, conventional monospecific IgG antibodies have two identical epitope binding sites (paratopes) and thus can only bind to identical epitopes (not to different epitopes). In contrast, a multispecific antibody has at least two different types of epitope binding sites (paratopes) and can thus bind to at least two different epitopes. In the context of the present invention, the term paratope refers to the epitope-binding site of an antibody. Accordingly, the terms paratope and epitope binding site are used interchangeably herein.

In addition, one specificity may refer to one, two, three or more of the same paratopes in a single antibody. The actual number of paratopes in one antibody molecule is called valence. Preferably, the antibody or antigen binding fragment thereof of the present invention is bivalent, trivalent, tetravalent, hexavalent or octavalent, more preferably the antibody or antigen binding fragment thereof is divalent or tetravalent, most commonly the antibody or antigen binding fragment thereof is tetravalent.

Most preferably, the antibody, or antigen binding fragment thereof, of the present invention is bispecific or tetravalent.

It is also preferred that the antibody, or antigen binding fragment thereof, of the present invention includes exactly two (identical) copies of each of the different epitope binding sites specifically binding to at least two different Zika virus epitopes.

For example, one native IgG antibody is monospecific and bivalent because it has two identical paratopes (two identical copies). However, a multispecific antibody includes at least two (different) paratopes. Thus, the term multispecific refers to antibodies and antigen-binding fragments that have more than one paratope and are capable of binding to two or more different epitopes. The term multispecific antibodies/antigen-binding fragments includes in particular bispecific antibodies as described above, but typically also a protein, for example an antibody, a scaffold molecule that binds in particular three or more different epitopes, i.e. antibodies having three or more paratopes .

In particular, a multispecific antibody, or antigen binding fragment thereof, may include two or more paratopes, some of the paratopes may be identical, such that all paratopes of the antibody belong to at least two different types of paratopes and therefore the antibody has at least two specificities.

For example, a multispecific antibody, or antigen binding fragment thereof, of the present invention may include four paratopes, with each two paratopes being identical (having equal specificity) and thus the antibody or fragment thereof being bispecific and tetravalent (two identical paratopes for each of the two specificities ). Thus, the concept of one specificity refers in particular to one or more paratopes exhibiting equal potency (which usually means that such one or more paratopes are identical) and thus two specificities can be achieved by two, three, four , five, six or more paratopes, since they belong to only two specificities. For example, a multispecific antibody may include a single paratope for each (at least two) specificities, i.e. a multispecific antibody includes a total of at least two paratopes. For example, a bispecific antibody contains a single paratope for each of the two specificities, i.e. the antibody includes only two paratopes. Most preferably, the antibody includes exactly two (identical) paratopes for each of the two specificities, i.e. the antibody includes only four paratopes. Alternatively, the antibody may include three (identical) paratopes for each of the two specificities, i.e. the antibody includes only six paratopes.

In the context of the present invention, the term antigen means any structural substance that serves as a target for receptors of the adaptive immune response, in particular as a target for antibodies, T-cell receptors and/or B-cell receptors. An epitope, also called an antigenic determinant, is a part (or fragment) of an antigen that is recognized by the immune system, in particular by antibodies, T-cell receptors and/or B-cell receptors. Thus, one antigen has at least one epitope, i.e. one antigen contains one or more epitopes. An antigen may be (i) a peptide, polypeptide or protein, (ii) a polysaccharide, (iii) a lipid, (iv) a lipoprotein or lipopeptide, (v) a glycolipid, (vi) a nucleic acid, or (vii) a small molecule drug or toxin. Thus, the antigen may be a peptide, protein, polysaccharide, lipid, a combination thereof including lipoproteins and glycolipids, a nucleic acid (e.g., DNA, siRNA, shRNA, antisense oligonucleotides, decoy DNA, plasmid), or a small molecule drug (e.g., cyclosporine A, paclitaxel, doxorubicin, methotrexate, 5-aminolevulinic acid) or any combination thereof. Preferably, the antigen is selected from (i) a peptide, polypeptide or protein, (ii) a polysaccharide, (iii) a lipid, (iv) a lipoprotein or lipopeptide and (v) a glycolipid; more preferably, the antigen is a peptide, polypeptide or protein.

The antibody, or antigen binding fragment thereof, of the present invention binds to at least two different epitopes of the Zika virus. At a minimum, different Zika virus antigens may be located at different Zika virus epitopes, such as different Zika virus (ZIKV) proteins, or the same Zika virus antigen, for example, on the same ZIKV protein. Preferred examples of ZIKV antigens/proteins are capsid, prM, envelope proteins and non-structural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5 and NS5B. Most preferably, the antibody, or antigen binding fragment thereof, of the present invention binds to (at least two) different epitopes of the Zika virus envelope (ZIKV E protein). In other words, most preferably, the antibody, or antigen binding fragment thereof, of the present invention contains at least two epitope binding sites that specifically bind to different epitopes of the Zika virus envelope protein (ZIKV E protein). ZIKV comprises a nucleocapsid core, which contains single-stranded RNA surrounded by core proteins. The nucleocapsid core is encapsulated by a lipid bilayer membrane with membrane and envelope proteins. The ZIKV envelope protein (E protein) is the dominant antigen. The structure and domains of the ZIKV E protein are described, for example, in Dai L. et al., Cell Host Microbe. 19(5), 2016, 696-704.

Preferably, the (at least two) different Zika virus epitopes to which the antibody, or antigen binding fragment thereof, binds, of the present invention are non-overlapping epitopes. In particular, the amino acids forming the first epitope of the Zika virus to which the antibody, or antigen-binding fragment thereof, binds, of the present invention are different from the amino acids forming the second epitope of the Zika virus, to which the antibody, or antigen-binding fragment thereof, binds, of the present invention. invention.

The antibody, or antigen binding fragment thereof, of the present invention may be in any format. In particular, multispecific antibodies preferably encompass whole antibodies, for example whole IgG or IgG-like molecules, while antigen binding fragments in the context of the present invention preferably refer to small recombinant formats, for example, a format based on bispecific T cell activators (BiTE®; except that in the context of the present invention, both specificities are aimed at the Zika virus, accordingly, the T-cell specificity of BiTE® can be replaced by a (second) specificity of ZIKV), tandem single chain variable fragment (taFv) molecules, diantibodies (Db ), single chain diantibodies (scDb) and various other derivatives thereof (bispecific antibody formats are described in Byrne H. et al., Trends Biotech, 31(11), 2013, 621-632 in Fig. 2, which shows various bispecific antibody formats ; Weidle U.H., et al., Cancer Genomics and Proteomics 10, 2013, 1-18, in particular, Fig. 1 shows various formats of bispecific antibodies; Chan A.C., Carter P.J. Nat Rev Immu 10, 2010, 301-316, where in FIG. 3 shows various formats of bispecific antibodies). Examples of bispecific antibody formats include, but are not limited to, Fabs-in-tandem-Ig, DVD-Ig, quadhybridoma, chemically linked Fab (fragment-binding antigen) and BiTE® (bispecific T-cell activator). In one embodiment of the present invention, preferably the antibody is a Fabs-in-tandem-Ig (FIT-Ig) or DVD-Ig antibody.

Thus, the antibody, or antigen-binding fragment thereof, of the present invention may be selected from the group consisting of Fabs-in-tandem-Ig (FIT-Ig); DVD-Ig; hybrid hybridoma (quadroma); multispecific anticalin platform (Pieris); diantibodies; single chain antibodies; tandem single-chain Fv fragments; tandem antibodies (TandAbs), trispecific antibodies (Trispecific Abs) (Affimed) (105-110 kDa); Dart (dual affinity retargeting retargeting antibody with double affinity; Macrogenics); bispecific antibodies Xmab (Xencor); bispecific T cell activators (Bispecific T cell engagers - Bites; Amgen; 55 kDa); Triplebodies; Tribody = Fab-scFv fusion protein (CreativeBiolabs) derivatives of multifunctional recombinant antibodies (110 kDa); Duobody platform (Genmab); Dock and lock platform; Knob into hole platform (KIH); humanized bispecific IgG antibody (REGN1979) (Regeneron); Mab ² bispecific antibodies (F-Star); DVD-Ig = double variable domain immunoglobulin (Abbott); kappa lambda antibodies; TBTI = tetravalent bispecific tandem Ig; and CrossMab.

The antibody, or antigen binding fragment thereof, of the present invention may be selected from bispecific IgG-like antibodies (BsIgG), including CrossMab; DAF (two-in-one); DAF (four-in-one); DutaMab; DT-IgG; Knobs-in-holes, usually LC; Knob into hole assembly (Knob into hole - KIH); charged pair (Charge pair); Fab-arm exchange (exchange of Fab fragments); SEEDbody fusion proteins; Triomab; LUZ-Y; Fcab; κλ-body; orthogonal Fab. Such bispecific antibody formats are described, for example, by Spiess C., Zhai Q., Carter P.J. Molecular Immunology 67, 2015, 95-106, in particular in FIG. 1 and in the corresponding description, for example, on pages 95-101.

Preferably, the antibody, or antigen binding fragment thereof, of the present invention may be selected from bispecific antibody fragments including nanobodies, nanobodies-HAS; BiTE; diantibodies; DART; TandAb; scDiabody; sc-Diabody-CH3; Diabody-CH3; Triple Body; mini-antibodies; minibody; TriBi minibody; scFv-CH3 KIH; Fab-scFv; scFv-CH-CL-scFv; F(ab')2; F(ab')2-scFv2; scFv-KIH; Fab-scFv-Fc; tetravelent HCAb; scDiabody-Fc; Diabody-Fc; Tandem scFv-Fc; Intrabody. Such bispecific antibody formats are described, for example, by Spiess C., Zhai Q., Carter P.J. Molecular Immunology 67, 2015, 95-106, in particular in FIG. 1 and in the corresponding description, for example, on pages 95-101.

More preferably, the antibody, or antigen binding fragment thereof, of the present invention may be selected from IgG-linked antibodies with an additional antigen binding fragment, which include DVD-IgG; IgG(H)-scFv; scFv-(H)IgG; IgG(L)-scFv; scFV-(L)IgG; IgG(L,H)Fv; IgG(H)-V; V(H)-IgG; IgG(L)-V; V(L)-IgG; KIH IgG-scFab; 2scFv-IgG; IgG-2scFv; scFv4-Ig; scFv4-Ig; Zybody; and DVI-IgG (four-in-one). Such bispecific antibody formats are described, for example, by Spiess C., Zhai Q., Carter P.J. Molecular Immunology 67, 2015, 95-106, in particular in FIG. 1 and in the corresponding description, for example, on pages 95-101. Of such antibody fragments, DVD-Ig (Double Variable Domain Immunoglobulin (Abbott)) is even more preferred. This antibody format is described in detail, for example, in the publication of Wu C. et al., Nat Biotechnol. 25(11), 2007, 12901297; or in DiGiammarino E., Ghayur T., Liu J. Methods Mol Biol. 899, 2012, 145-156.

Most preferably, the antibody, or antigen-binding fragment thereof, of the present invention is an IgG-coupled antibody of Fabs-in-tandem-Ig (FIT-Ig) format. In other words, the antibody, or antigen binding fragment thereof, of the present invention is most preferably in the Fabs-in-tandem-Ig (FIT-Ig) format. The Fabs-in-tandem-Ig (FIT-Ig) format is described in detail, for example in WO 2015/103072 A1, incorporated herein by reference, or in Gong S. et al., MAbs 2017. Similar to DVD-Ig , also FIT-Ig is a bispecific tetravalent antibody of a symmetrical format. FIT-Ig can be produced using three polypeptides: Polypeptide 1 typically comprises the light chain of an outer Fab hybridized, preferably without linkers, to the N-terminal heavy chain region of an inner Fab; polypeptide 2 typically includes the variable and CH1 heavy chain regions of the outer Fab; and polypeptide 3 typically includes the light chain of an internal Fab. Thus, a FIT-Ig format antibody typically includes an internal Fab and an external Fab.

Preferably, the antibody of the present invention, or antigen-binding agent thereof, neutralizes Zika virus infection. In other words, the antibody, or antigen binding fragment thereof, of the present invention reduces the infectivity of the Zika virus.

To study and quantify virus infectivity (or neutralization) in vitro, various standard neutralization assays are known to those skilled in the art. For neutralization assays, animal viruses are typically propagated in cells and/or cell lines. In the context of the present invention, a neutralization assay is preferred, wherein the cultured cells are incubated with a fixed amount of Zika virus (ZIKV) in the presence (or absence) of the antibody of interest. To obtain data, for example, liquid cytometry can be used. In another embodiment of the present invention, it is also possible to measure other indicators, for example, the amount of ZIKV non-structural proteins (eg, ZIKV NS1) secreted into the culture supernatant. For example, the ZIKV nonstructural protein 1 (NS1) antigen capture enzyme-linked immunosorbent assay (ELISA), tissue culture infectious dose-50 (TCID50) test (TCID50-ELISA) can be used as an alternative to the standard viral plaque count method for virus titration Zika - similar to the method described for dengue virus (DENV) in the work of Li J. et al., PLoS ONE 2011, 6:e22553. In such a study, for example, ZIKV NS1 binding antibodies described in the present invention may be useful.

In a preferred embodiment of the present invention, for a ZIKV neutralization assay, cultured cells, eg Vero cells, are incubated with a specified amount of ZIKV in the presence or absence of the antibody of interest, for example, for about four days. After incubation, cells can be washed and further cultured. Liquid cytometry can be used to measure virus infectivity. For this purpose, cells can be fixed, for example, with 2% formaldehyde, permeabilization agents, for example, 1% FCT (fetal calf serum) in PBS (phosphate-buffered saline), 0.5% saponin and stained, for example, with a mouse antibody 4G2. The cells can then be incubated with a goat anti-mouse IgG conjugated to a dye such as Alexa Fluor488 and examined by liquid cytometry. In another embodiment of the present invention, viable cells can be detected by liquid cytometry using, for example, WST-1 reagent (Roche). The preferred ZIKV strain for use in this neutralization assay is ZIKV H/PF/2013.

The antibody and antigen binding fragment of the present invention have high neutralizing activity. The antibody concentration required to neutralize 50% of the ZIKV virus (IC ₅₀ ), compared to controls without antibodies, is, for example, up to about 3 μg/ml or up to about 1 μg/ml. Preferably, the concentration of the antibody of the present invention required to neutralize 50% of the ZIKV virus (IC ₅₀ ) is about 500 ng/ml, more preferably, the concentration of the antibody of the present invention required to neutralize 50% of the ZIKV virus (IC ₅₀ ) is about 250 ng /ml, even more preferably, the concentration of the antibody of the present invention required to neutralize 50% of the ZIKV virus (IC ₅₀ ) is about 150 ng/ml. Most preferably, the concentration of the antibody of the present invention required to neutralize 50% of the ZIKV virus (IC ₅₀ ) is about 100 ng/ml or less, for example, about 90 or less, about 80 or less, about 70 or less, about 60 or less, about 50 or less, about 45 or less, about 40 or less, about 35 or less, about 30 or less, about 25 or less, about 20 or less, or, especially preferably, about 15 ng/ml or less. In particular, the concentration of the antibody of the present invention required to neutralize 50% of the ZIKV virus (IC ₅₀ ) is preferably about 50 ng/ml or less. This means that only low concentrations of antibody are required to neutralize 50% of the ZIKV virus. The concentration of the antibody of the present invention required for 50% neutralization of ZIKV (IC ₅₀ ) can be measured using standard neutralization assay methods known to those skilled in the art or, in particular, as described above.

In general, antibody binding can be assessed using a standard ELISA (enzyme-linked enzyme-linked assay) method, which is well known in the art. A variation of the standard ELISA method can be as follows: ELISA plates can be coated (e.g. overnight at 4°C) with a sufficient amount (e.g. 1 µg/ml) of white

- 10 043940 ca/complex/particles to which the test antibody binds (for example, for the DENV binding described below, DENV E proteins and/or DENV VLP are used), for example, in PBS. The plates are then blocked, for example, with a 1 wt.% solution of bovine serum albumin (BSA) in PBS and incubated with the antibody of interest (for example, about 1.5 hours at room temperature). After washing, antibody binding can be detected, for example, using goat anti-human IgG coupled to alkaline phosphatase. The plates can then be washed, the desired substrate (eg p-NPP) can be added and the plates can be read at, for example, 405 nm. The relative binding affinity of an antibody can be determined by measuring the mAb concentration (EC ₅₀ ) required to achieve 50% of the maximum binding at the visit. EC ₅₀ values can be calculated by interpolating binding curves fitted with four-parameter non-linear variable slope regression.

Preferably, the antibody, or antigen binding fragment thereof, of the present invention does not significantly bind to Dengue virus-like particles and/or the Dengue virus envelope protein. More preferably, the antibody, or antigen binding fragment thereof, of the present invention does not significantly bind to Dengue virus-like particles and/or to the envelope protein of the Dengue virus of any of the four DENV serotypes DENV1, DENV2, DENV3 and DENV4. Thus, substantially no binding means that the antibody, or antigen binding fragment thereof, has an EC ₅₀ value of less than ¹⁰² ng/ml, preferably 103, more preferably ^5x103 , even more preferably ^8x103 , and most preferably up to 104 ng/ml. ml, which can be determined by a standard ELISA method for Dengue virus-like particles (DENV VLP) and/or Dengue virus envelope protein (DENV E protein). In other words, the concentration of antibody, or antigen-binding fragment thereof, required to achieve 50% of the maximum binding at saturation (EC ₅₀ ) of the Dengue virus-like particles (DENV VLP) and/or the envelope protein of the Dengue virus (DENV E protein) in a standard ELISA method is usually is more than 10 ² , preferably more than 103, more preferably more than 5x10 ³ , even more preferably more than 8x10 ³ and most preferably more than 10 ⁴ ng/ml.

Preferably, the antibody, or antigen binding fragment thereof, of the present invention does not promote antibody-dependent enhancement (ADE) of Zika virus infection. More preferably, the antibody, or antigen-binding fragment thereof, of the present invention blocks the antibody-dependent enhancement (ADE) of Zika virus. ADE can be assessed by liquid cytometry using, for example, cultured cells or cell lines, such as K562 cells. For example, test antibodies and ZIKV can be mixed for 1 hour at 37°C and added to 5000 K562 cells/well. After four days, cells can be fixed, saturated and stained with m4G2, for example, as described above in neutralization methods. The number of infected cells is determined by liquid cytometry, as described above for the neutralization method.

Preferably, the antibody of the present invention, or an antigen binding fragment thereof, eliminates the formation of antibody escape mutants of ZIKV. Elimination of the propagation of escape mutants can be easily determined by those skilled in the art. For example, to determine the propensity of an antibody or antibody fragment to form escape mutants, ZIKV is repeatedly passaged in the presence of sub-neutralizing concentrations of the antibody or antigen-binding fragment of interest. In Examples 10 and 11 of the present invention, ZIKV escape mutants (MARMs) typically appear after the third or fourth passage of ZIKV. Accordingly, the antibody can be read to eliminate the propagation of escape mutants of the virus if still escape mutants of ZIKV are not detected after five passages of the virus (ZIKV) in the presence of subneutralizing concentrations of the antibody, or antigen binding fragment thereof, of the present invention. More preferably, ZIKV escape mutants are not detected after six passages of the virus (ZIKV) in the presence of sub-neutralizing concentrations of the antibody, or antigen binding fragment thereof, of the present invention. Even more preferably, ZIKV escape mutants are not detected after seven passages of the virus (ZIKV) in the presence of sub-neutralizing concentrations of the antibody, or antigen binding fragment thereof, of the present invention. Most preferably, ZIKV escape mutants are not detected after eight passages of the virus (ZIKV) in the presence of sub-neutralizing concentrations of the antibody, or antigen binding fragment thereof, of the present invention.

For example, to assess the propensity of an antibody or antibody fragment to form escape mutants, ZIKV, such as strain H/PF/2013, is incubated with various subneutralizing concentrations of the antibody/antigen-binding fragment of interest, e.g., for at least 30 minutes, e.g. 37°C (body temperature). (Live) cells, eg Vero cells, are then added and incubated, eg for at least one day, preferably 3-4 days, eg at about 37° C. (body temperature) to allow the virus to multiply. Supernatants are then selected from three options: the lowest concentration of the antibody/antigen-binding fragment of interest at which complete protection of the cell monolayer is observed; the concentration at which partial exposure of the CPE to the cell monolayer is observed; and the concentration at which 100% of the cell monolayer was destroyed by ZIKV CPE.

One part of the selected supernatant can be used for micro-neutralization assays and subsequent virus sequencing (to identify antibody-escape mutants), while another part of the (same) selected supernatant can be used for the next selection step (passage). Namely, for the next passage, a portion of the selected supernatant can be mixed with various sub-neutralizing concentrations of the test antibody/antigen-binding fragment and incubated as described above, followed by the addition of (live) cells and incubated as described above to finally select the supernatants again . This selection and propagation method is repeated for at least five passages, preferably at least six passages, more preferably at least seven passages, and most preferably at least eight passages.

Preferably, each CDR or each variable region of at least one epitope binding site of an antibody, or antigen binding fragment thereof, of the present invention is a human CDR or human variable region, respectively. Most preferably, all constant regions and variable regions included in the antibody, or antigen binding fragment thereof, of the present invention are human constant regions and human variable regions.

It is also preferred that the antibody, or antigen binding fragment thereof, of the present invention is a monoclonal antibody, preferably a monoclonal antibody, wherein each CDR or variable region thereof included in the antibody or antigen binding fragment thereof of the present invention is a human CDR, or human variable region , respectively.

Preferably, the antibody, or antigen binding fragment thereof, of the present invention is an IgG type, for example an IgG1 type, an IgG2 type, an IgG3 type or an IgG4 type, more preferably an IgG1 type. More preferably, the antibody, or antigen binding fragment thereof, of the present invention includes (i) a heavy chain constant region of the type IgG1 CH1-CH2-CH3, where CH1 means heavy chain constant domain 1, CH2 means heavy chain constant domain 2, CH3 means constant domain 3 heavy chains; and (ii) an IgG type light chain constant region CL, where CL means light chain constant domain. Even more preferably, the antibody, or antigen binding fragment thereof, of the present invention comprises a heavy chain constant region of the type IgG1 CH1-CH2-CH3 containing or consisting of the amino acid sequence SEQ ID NO: 91 or 92 or a functional variant thereof, and a light chain constant region an IgG CL type chain containing or consisting of the amino acid sequence of SEQ ID NO: 93 or 94, or a functional variant thereof.

Accordingly, it is preferred that the antibody of the present invention, or an antigen binding fragment thereof, includes an Fc fragment. More preferably, the Fc fragment is derived from a human, for example human IgG1, IgG2, IgG3 and/or IgG4, with human IgG1 being particularly preferred. Various formats of multispecific antibodies comprising an Fc fragment are known in the art. Preferred antibody formats comprising an Fc fragment are the IgG-attached antibody formats described above. In general, an antibody containing an Fc fragment is more effective and has a longer half-life than antibodies or antibody fragments without an Fc fragment.

In the context of the present invention, the term Fc fragment refers to the sequence derived from the portion of the immunoglobulin heavy chain starting in the hinge region immediately upstream of the papain cleavage site (for example, residue 216 in native IgG, taking the first residue of the heavy chain constant region is 114) and ending at the C-terminus of the immunoglobulin heavy chain. Accordingly, the Fc fragment may be a complete Fc fragment or a portion thereof (eg, a domain thereof). A complete Fc fragment contains at least a hinge domain, a CH2 domain, and a CH3 domain (eg, EC amino acid positions 216-446). An additional lysine residue (K) is sometimes present at the very C-terminus of the Fc fragment, but is often cleaved from the mature antibody. Each of the amino acid positions in the Fc fragment was numbered in accordance with the generally accepted Kabat numbering system in the EU (see, for example, Kabat et al., in the book: Sequences of Proteins of Immunological Interest, US Department of Health and Human Services. Services, 1983 and 1987.

Preferably, in the context of the present invention, the Fc fragment contains at least one of the following: a hinge region domain (eg, upper, middle, and/or lower hinge region), a CH2 domain, a CH3 domain, or a variant, portion, or fragment thereof. In a preferred embodiment of the present invention, the Fc fragment contains at least a hinge domain, a CH2 domain, or a CH3 domain. More preferably, the Fc fragment is a complete Fc fragment. The Fc fragment may also include one or more amino acid insertions, deletions or substitutions relative to the natural Fc fragment. For example, at least one of the following components, the hinge domain, the CH2 domain, or the CH3 domain (or a portion thereof), may be deleted. For example, an Fc fragment may include or consist of: (i) a hinge domain (or part thereof) hybridized with a CH2 domain (or part thereof), (ii) a hinge domain (or part thereof) hybridized with a CH3 domain (or part thereof) ), (iii) a CH2 domain (or part thereof) hybridized with a CH3 domain (or part thereof), (iv) a hinge domain (or part thereof), (v) a CH2 domain (or part thereof), or (vi) a domain CH3 (or parts thereof).

Those skilled in the art will appreciate that the Fc fragment can be modified such that its amino acid sequence varies relative to the complete Fc fragment of the naturally occurring immunoglobulin molecule while retaining at least one desired function provided by the naturally occurring Fc fragment. Such functions include Fc receptor (FcR) binding, antibody half-life modulation, ADCC function, protein A binding, protein G binding, and complement fixation. Parts of natural Fc fragments that are responsible and/or important for such functions are known to those skilled in the art.

For example, to activate the complement cascade, C1q binds at least two IgG1 molecules or one IgM molecule attached to an antigenic target (Ward E. S., Ghetie V., Ther. Immunol. 2, 1995, 77-94). The publication (Burton D. R., Mol. Immunol. 22, 1985, 161-206) shows that the heavy chain region, including amino acid residues 318 to 337, is involved in complement fixation. In the publication (Duncan A. R., Winter G. Nature 332, 1988, 738-740), using site-directed mutagenesis it was established that Glu318, Lys320 and Lys322 form a binding site for C1q. The role of residues Glu318, Lys320, and Lys322 in C1q binding was confirmed by the ability of a short synthetic peptide containing these residues to inhibit complement-mediated lysis.

For example, FcR binding may be mediated by the interaction of an Fc moiety (antibody) with Fc receptors (FcRs), which are specialized receptors on the surface of hematopoietic cells. Fc receptors, members of the immunoglobulin superfamily, have been shown to mediate both the clearance of antibody-coated pathogens by phagocytosis of immune complexes and the lysis of red blood cells and various other cellular targets (eg, tumor cells) coated with the appropriate antibody, antibody-mediated cell-dependent cytotoxicity. cell mediated cytotoxicity - ADCC; Van de Winkel J.G., Anderson C.L., J. Leukoc. Biol. 49, 1991, 511-524). FcRs are defined by immunoglobulin class specificity; Fc receptors for IgG antibodies are designated as FcyR, for IgE - as FcεR, for IgA - as FcaR, and so on; neonatal Fc receptors are designated FcRn. Fc receptor binding is described, for example, by Ravetch J.V., Kinet J.P., Annu. Rev. Immunol. 9, 1991, 457-492; Capel P.J. et al., Immunomethods 4, 1994, 25-34; de Haas M.C. et al., J Lab. Clin. Med. 126, 1995, 330-341; Gessner J. E. et al., Ann. Hematol. 76, 1998, 231248.

Cross-linking of receptors by the Fc domain of native IgG antibodies (FcyR) triggers a wide range of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity and release of inflammatory mediators, as well as clearance of immune complexes and regulation of antibody production. Therefore, Fc fragments that provide cross-linking of receptors (FcyR) are preferred. In humans, three classes of FcyR have been described, namely: (i) FcyRI (CD64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils; (ii) FcyRII (CD32), which binds complex IgG with medium to low affinity, is widely expressed, particularly on leukocytes, is known to be a central player in antibody-mediated immunity and which can be divided into FcyRIIA, FcyRIIB and FcyRIIC, which have different functions functions in the immune system, but binds with similar low affinity to IgG-Fc, and the ectodomains of these receptors are highly homologous; and (iii) FcyRIII (CD16), which binds IgG with moderate 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 highly expressed on neutrophils. FcyRIIA is found on many cells involved in killing (eg, macrophages, monocytes, neutrophils) and is presumably capable of activating the cell killing process. FcyRIIB is believed to be involved in inhibition processes and is found on B cells, macrophages and mast cells, as well as on eosinophils. Importantly, 75% of all FcyRIIB is found in the liver (Ganesan L.P. et al., J of Immunology 189, 2012, 4981-4988). FcyRIIB is overexpressed in liver sinusoidal endothelium, referred to as LSEC, and in Kupffer cells in liver and LSEC, major sites of clearance of small immune complexes (Ganesan L.P. et al., J of Immunology 189, 2012, 4981-4988).

Thus, the present invention prefers such bodies, and antigen binding fragments thereof, that are capable of binding to FcyRIIb, for example, antibodies comprising an Fc fragment for binding to FcyRIIb, in particular the Fc region, for example, antibodies of the IgG type. Moreover, it is possible to engineer an Fc fragment to enhance FcyRIIB binding by introducing mutations S267E and L328F, as described by Chu S.Y. et al., Molecular Immunology 45, 2008, 3926-3933. Thus, the clearance

- 13 043940 immune complexes can be enhanced (Chu S. et al, 2014, in the book: Am J Respir Crit, American Thoracic Society International Conference Abstracts). Accordingly, in the context of the present invention, such antibodies, or antigen-binding fragments thereof, are preferred which include an engineered Fc fragment with mutations S267E and L328F, in particular as described by Chu S.Y. et al., Molecular Immunology 45, 2008, 3926-3933. On B cells, it appears to act by inhibiting further immunoglobulin production and isotype switching, such as IgE class. In macrophages, FcyRIIB acts to inhibit phagocytosis mediated through FcyRIIA. On eosinophils and mast cells, the b-form may help suppress the activation of such cells through the binding of IgE to their individual receptor.

Regarding FcyRI binding, modification in native IgG of at least one residue of E233-G236, P238, D265, N297, A327 and P329 reduces FcyRI binding. IgG2 residues at positions 233-236, substituted in IgG1 and IgG4, reduce binding to FcyRI by 103 times and eliminate the response of human monocytes to antibody-sensitized erythrocytes (Armour K.L. et al., Eur. J. Immunol. 29, 1999, 2613-2624 ). Regarding FcyRII binding, it has been found that there is reduced FcyRIIA binding, for example, for IgG mutation of at least one residue of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292 and K414. Regarding FcyRIII binding, reduced binding to cyRIIIA has been found, for example, for at least one residue from E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338 and D376. Mapping of binding sites on human IgG1 for Fc receptors, the above 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.

Regarding binding to the critical FcyRII, two regions of the native IgG Fc appear to be critical for the interaction of FcyRIIs and IgG, namely (i) the lower hinge site of the IgG Fc, in particular amino acid residues L, L, G, G (234 - 237, EU numbering), and (ii) the adjacent CH2 domain region of the IgG Fc, particularly the loop and strands in the upper CH2 domain adjacent to the lower hinge region, e.g., region (Wines B.D. et al., J. Immunol. 164 , 2000, 5313-5318). Moreover, FcyRI appears to bind to the same site on the IgG Fc, whereas FcRn and protein A bind to different sites on the IgG Fc, which appears to be at the CH2-CH3 interface (Wines B.D. et al. ., J. Immunol. 164, 2000, 5313-5318).

For example, an Fc fragment may include or consist of at least a portion of an Fc fragment known to be required for FcRn binding or half-life extension. Alternatively or additionally, the Fc fragment of an antibody of the present invention includes at least a portion known in the art necessary for binding protein A and/or the Fc fragment of an antibody of the present invention, comprising at least a portion of the Fc molecule described it is known to be required for G protein binding. Preferably, the function retained is the neutralization of Zika virus infection, which is believed to be mediated by FcyR binding. Accordingly, a preferred Fc fragment includes at least a portion known in the art to be required for FcyR binding. As noted above, a preferred Fc fragment may thus at least contain (i) a lower hinge site of the native IgG Fc, in particular amino acid residues L, L, G, G (234-237, European Union (EU) numbering) ), and (ii) the adjacent region of the CH2 domain of the native IgG Fc, in particular the loops and strands in the upper CH2 domain adjacent to the lower hinge region, for example in the P331 region, for example the region of at least 3, 4, 5 , 6, 7, 8, 9 or 10 consecutive amino acids in the upper CH2 domain of the native IgG Fc around P331, for example between amino acids 320 and 340 (EU numbering) of the native IgG Fc.

Preferably, the antibody, or antigen binding fragment thereof, of the present invention contains an Fc region. In the context of the present invention, the term Fc region refers to the part of an immunoglobulin formed by two or more Fc fragments of the heavy chains of an antibody. For example, the Fc region may be a monomeric or single chain Fc region (ie, a scFc region). Single-stranded Fc regions consist of Fc fragments linked into a single polypeptide chain (eg, encoded by a single contiguous nucleic acid sequence). Examples of scFc regions are described in WO 2008/143954 A2. Preferably the Fc region is dimeric. The term dimeric Fc region or dcFc refers to a dimer formed by Fc fragments of two different immunoglobulin heavy chains. The dimeric Fc region may be a homodimer of two identical Fc fragments (eg, the Fc region of a natural immunoglobulin) or a heterodimer of two non-identical Fc fragments.

Fc fragments of the Fc region may belong to the same or different classes/subclasses. For example, Fc fragments may be derived from an immunoglobulin (eg, human immunoglobulin) of the IgG1, IgG2, IgG3, or IgG4 subclasses. Preferably, Fc fragments of the Fc region are from the same class or subclass. However, the Fc region (or one or more Fc fragments of the Fc region) may also be chimeric, whereby the chimeric Fc region may contain Fc fragments derived from different immunoglobulin classes and/or subclasses. For example, the at least two Fc fragments of a dimeric or single chain Fc region may be from different immunoglobulin classes and/or subclasses. Additionally or alternatively, chimeric Fc regions may comprise one chimeric

- 14 043940 fragment or more Fc. For example, a chimeric Fc region or fragment may include one or more portions derived from an immunoglobulin of a first subclass (eg, IgG1, IgG2, or IgG3 subclass), although the remainder of the Fc region or fragment is of a different subclass. For example, an Fc region or fragment of an Fc polypeptide may include a CH2 and/or CH3 domain derived from a first subclass immunoglobulin (eg, IgG1, IgG2, or IgG4 subclass) and a hinge region from a second subclass immunoglobulin (eg, IgG3 subclass). For example, the Fc region or fragment may include a hinge and/or CH2 domain derived from a first subclass immunoglobulin (eg, IgG4 subclass) and a CH3 domain from a second subclass immunoglobulin (eg, IgG1, IgG2, or IgG3 subclass). For example, the chimeric Fc region may include an Fc fragment (eg, a complete Fc fragment) from a first subclass immunoglobulin (eg, the IgG4 subclass) and an Fc fragment from a second subclass immunoglobulin (eg, the IgG1, IgG2, or IgG3 subclass). For example, the Fc region or fragment may include a CH2 domain from an IgG4 immunoglobulin and a CH3 domain from an IgG1 immunoglobulin. For example, the Fc region or fragment may include a CH1 domain and a CH2 domain from an IgG4 molecule and a CH3 domain from an IgG1 molecule. For example, the Fc region or fragment may contain part of the CH2 domain from a particular antibody subclass, for example, at EU positions 292-340 of the CH2 domain. For example, the Fc region or fragment may include amino acids at positions 292-340 in the CH2 domain derived from the IgG4 fragment and the CH2 residue derived from the IgG1 fragment (in another embodiment, 292-340 of the CH2 domain may be derived from the IgG1 fragment and the CH2 residue from an IgG4 fragment).

Moreover, the Fc region or Fc fragment may, additionally or otherwise, for example, include a chimeric hinge region. For example, a chimeric hinge may be derived, for example, in part, from an IgG1, IgG2 or IgG4 molecule (eg, the upper and lower hinge sequences) and, in particular, from an IgG3 molecule (eg, the middle hinge sequence). In another example, the Fc region or fragment may include a chimeric fragment derived from, in particular, an IgG1 molecule, and, in particular, an IgG4 molecule. In another example, the chimeric fragment may include the upper and lower hinge domains from an IgG4 molecule and the middle hinge domain from an IgG1 molecule. Such a chimeric fragment can be obtained, for example, by introducing a proline substitution (Ser228Pro) at position EU 228 in the middle hinge domain of the IgG4 hinge region. In another embodiment of the present invention, the chimeric hinge may include the amino acids at position EU 233-236 from the IgG2 antibody and/or the Ser228Pro mutation, with the remaining hinge amino acids being derived from the IgG4 antibody (eg, the chimeric hinge sequence ESKYGPPPPCPPCPAPPVAGP). Other chimeric hinges that can be used in the Fc fragment of the antibody of the present invention are described in US 2005/0163783 A1.

In the present invention, it is preferred that the Fc fragment, or Fc region, includes or consists of an amino acid sequence derived from a human immunoglobulin sequence (eg, an Fc region or Fc fragment from a human IgG molecule). However, the polypeptides may include one or more amino acids from another mammalian species. For example, a primate Fc fragment or primate binding site may be included in the subject's polypeptides. In another embodiment, one or more mouse amino acids may be contained in an Fc fragment or Fc region.

Preferably, the antibody of the present invention contains, in particular in addition to the Fc fragment as described above, other parts derived from the constant region, in particular from the IgG constant region, preferably from the IgG1 constant region, more preferably from the human IgG1 constant region. More preferably, the antibody of the present invention contains, in particular, in addition to the Fc fragment as described above, all other parts of the constant regions, in particular all other parts of the IgG constant regions, preferably all other parts of the IgG1 constant regions, more preferably all other parts of human IgG1 constant regions.

Particularly preferred constant region sequences are the amino acid sequences SEQ ID NO: 91-94 (encoded, for example, by the nucleic acid sequences SEQ ID NO: 95-98, respectively). Preferably, the amino acid sequence of IgG1 CH1-CH2-CH3 corresponds to SEQ ID NO: 91, or a variant of the functional sequence as described in the present invention. Even more preferably, the IgG1 amino acid sequence CH1-CH2-CH3 corresponds to SEQ ID NO: 92 or a functional variant of this sequence as described herein where the LALA mutation is retained. It was noted above that a particularly preferred antibody of the present invention contains the (complete) Fc region derived from human IgG1. More preferably, the antibody of the present invention contains, in particular, in addition to the (complete) Fc region derived from human IgG1, also all parts of the IgG constant regions, preferably all other parts of the IgG1 constant regions, more preferably all other parts of the human IgG1 constant regions .

Without being limited by theory, it is believed that antibody dependent enhancement (ADE) of Zika virus infection is caused by binding of the Fc fragment of an antibody, in particular the Fc fragment of the heavy chain of an IgG molecule. to an Fc receptor, for example the Fcy receptor on a host cell. Thus, it is preferable that the antibody of the present invention, or an

- 15 043940 tigen-binding fragment, contained one or more mutations in the Fc fragment. The mutation(s) may be any mutation that reduces the binding of the antibody to the Fc receptor (FcR), in particular reduces the binding of the antibody to the Fcy receptor (FcyR). On the other hand, it is preferable that the antibody in present amount contains the (full/complete) Fc fragment/Fc region, and the interaction/binding with FcRn is not impaired. Accordingly, it is particularly preferred that the antibody of the present invention, or an antigen binding fragment thereof, contains one or more mutations in the Fc fragment that (i) reduce the binding of the antibody to the Fcy receptor, but do not impair the interaction with FcRn. One example of such mutations is the LALA mutation, described below.

In general, the binding of an antibody to the Fc receptor can be assessed by various methods known in the art, for example, the ELISA method (Hessell A.J. et al., Nature, 449, 2007, 101-104; Grevys A. et al. Fc Engineering of Human IgG1 for Altered Binding to the Neonatal Fc Receptor Affects Fc Effector Functions. 2015, 194:5497-5508) by liquid cytometry (Perez L.G. et al., J Virol, 83, 2009, 7397-7410; Piccoli L. et al., Nat Commun 2015 , 6, 1-9).

In general, the antibody of the present invention may be glycosylated. N-linked glycans attached to the heavy chain CH2 domain, for example, can influence C1q and FcR binding, with aglycosylated antibodies having lower affinity for these receptors. Accordingly, the CH2 domain of the Fc fragment of an antibody of the present invention may include one or more mutations in which a glycosylated residue is replaced by a non-glycosylated residue. Glycan structure can also influence activity, for example, differences in complement-mediated cell death can be observed depending on the number of galactose sugar molecules (0, 1, or 2) at the end of the biantternary glycan chain. Preferably, the antibody glycans do not result in an immunogenic response in humans following administration.

In addition, the antibody of the present invention can be modified by introducing random amino acid mutations into a specific region of the CH2 or CH3 domain of the heavy chain to change their FcR binding affinity and/or their serum half-life compared 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 region selected from the group consisting of amino acid residues 250, 314, and 428.

Particularly preferably, the Fc fragment of the antibody of the present invention contains a substitution at positions CH2 4, CH2 5, or both. In general, the amino acid at positions 4 and 5 of wild-type IgG1 and IgG3 CH2 is leucine (L). Preferably, the antibody of the present invention contains an amino acid at the CH2 4, CH2 5 or both position that is not L. More preferably, the antibody of the present invention contains an alanine (A) at the CH2 4 or CH2 5 position or both. Most preferably, the antibody of the present invention contains both CH2 L4A and CH2 L5A substitutions. Such antibodies are referred to in the present invention as LALA. Interestingly, such a LALA mutation in the Fc fragment not only results in a lack of contribution of the corresponding antibody to antibody-dependent enhancement (ADE) of Zika virus infection, but also blocks antibody-dependent enhancement (ADE) of Zika virus infection. The typical amino acid sequence of IgG1 CH1-CH2-CH3 containing the LALA mutation corresponds to SEQ ID NO: 92. Accordingly, the amino acid sequence of IgG1 CH1-CH2-CH3 is found in accordance with SEQ ID NO: 92 or its variant functional sequence as described herein invention where the LALA mutation persists. Most preferably, the antibody is in the FIT-Ig format described above containing the LALA mutation in the Fc fragment as described in the present invention.

Preferably, the antibody, or antigen binding fragment thereof, of the present invention does not include an Fc receptor binding site. More preferably, the antibody, or antigen binding fragment thereof, does not include an Fc region, even more preferably, the antibody, or antigen binding fragment thereof, does not include an Fc fragment. Various formats of multispecific antibodies without an Fc fragment are known in the art and are described above.

As described above, the antibody, or antigen binding fragment thereof, of the present invention preferably specifically binds to (at least two) different epitopes of the Zika virus envelope protein (ZIKV E protein). More preferably, the antibody, or antigen binding fragment thereof, binds to domain III of the Zika virus envelope protein (EDIII, also referred to as DIII). In other words, it is preferable that the antibody, or antigen binding fragment thereof, of the present invention binds to an epitope of the Zika virus envelope protein that includes one or more amino acid residues of domain III of the Zika virus envelope protein (EDIII). ZIKV comprises a nucleocapsid core, which includes single-stranded RNA surrounded by core proteins. The nucleocapsid core is encapsulated by a lipid bilayer membrane with membrane and envelope proteins. The ZIKV envelope protein (E protein) is the dominant antigen. The coat protein ectodomain contains three different domains: E protein domain I (EDI), E protein domain II (EDII), and E protein domain III (EDIII). EDIII is highly conserved among different ZIKV strains (see FIG. 8 for an amino acid sequence alignment of EDIII among different ZIKV strains). Antibodies binding to domain III of the Zika virus envelope protein (EDIII) show (i) increased neutralization of ZIKV and (ii) reduced cross-reactivity with DENV (in particular, essentially no cross-reactivity with DENV) compared to antibodies binding to the domain Zika virus envelope protein I/II (EDI/II).

Accordingly, the antibody, or antigen binding fragment thereof, more preferably binds to domain III of the Zika virus envelope protein (EDIII) with EDIII having the following amino acid sequence (SEQ ID NO: 263):

TAAFTFTKXPAEXXHGTVTVEXQYXGXDGPCKXPXQMAVDXQTLTPVGRLITANPVIT

EXTENSKMMLELDPPFGDSYIVIGXGXKKITHHWHRS where X can be any (natural) amino acid. In other words, it is preferred that the antibody, or antigen binding fragment thereof, of the present invention binds to an epitope of the Zika virus envelope protein that includes one or more amino acid residues from SEQ ID NO: 263.

It is also preferred that the antibody, or antigen binding fragment thereof, of the present invention binds to domain III of the Zika virus envelope protein, (EDIII) with EDIII containing the following amino acid sequence (SEQ ID NO: 265):

X1GX2X3YSLCTAAFTFTKX4PAEX ₅ X6HGTVTVEX7QYX8GX9DGPCKX _1O PX11QM

AVDXuQTLTPVGRLITANPVITEXBTXMNSKMMLELDPPFGDSYIVIGXnGXieXn

KITHHWHRSG in which X1 can be any (natural) amino acid, preferably K, A or E,

X ₂ can be any (natural) amino acid, preferably V, F, or L,

X ₃ can be any (natural) amino acid, preferably S or F,

X4 may be any (natural) amino acid, preferably I or V, X ₅ may be any (natural) amino acid, preferably T or V, X ₆ may be any (natural) amino acid, preferably L or D, X ₇ may be any (natural) amino acid, preferably V or G, X ₈ may be any (natural) amino acid, preferably A or G, X ₉ may be any (natural) amino acid, preferably T or A , X ₁₀ may be any (natural) amino acid, preferably V or I, _X may be any (natural) amino acid, preferably A or V, X ₁₂ may be any (natural) amino acid, preferably M or T, X13 may be any (natural) amino acid, preferably S or G, X14 may be any (natural) amino acid, preferably E or K, X15 may be any (natural) amino acid, preferably V or I, X ₁₆ may be any (natural) amino acid, preferably E, A, K, or D, X ₁₇ may be any (natural) amino acid, preferably E, A, or K, more preferably K or A.

In other words, it is preferred that the antibody, or antigen binding fragment thereof, of the present invention binds to an epitope of the Zika virus envelope protein that includes one or more amino acid residues of the sequence SEQ ID NO: 265.

For example, the EDIII stretches from amino acid 309 to amino acid 403 of the E protein of ZIKV strain ZIKV H/PF/2013 (Genbank database number KJ776791). Accordingly, the antibody, or antigen binding fragment thereof, most preferably binds to domain III of the Zika virus envelope protein (EDIII) with EDIII having the following amino acid sequence (SEQ ID NO: 264):

TAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITE

STENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRS.

In other words, it is preferred that the antibody, or antigen binding fragment thereof, of the present invention binds to a Zika virus envelope protein that includes one or more amino acid residues of the sequence SEQ ID NO: 264.

More preferably, the antibody, or antigen binding fragment thereof, of the present invention binds to an epitope of the Zika virus envelope protein that includes one or more lateral ridge (LR) amino acid residues in EDIII and/or one or more amino acid residues in the hinge region EDI-EDIII. EDIII of the lateral ridge and EDI-EDIII of the hinge region are known to specialists and are described, for example, by Zhao H. et al., Cell 166(4), 2016, 1016-1027, and Kostyuchenko V.A. et al., Nature. 533(7603016), 2016, 425-428. Without being limited to any theory, it is concluded that (i) binding to LR can inhibit fusion by trapping the viral fusion transition state and (ii) binding to the EDI-EDIII hinge and EDIII can interfere with the movement of EDIII to form a trimeric structure after fusion , thereby stopping membrane fusion.

Accordingly, it is preferred that the antibody, or antigen-binding fragment thereof, of the present invention (be capable of) inhibiting the ZIKV post-attachment step. Concept

- 17 043940 post-attachment generally refers to any stage of ZIKV infection after ZIKV has attached to the membrane of a cell (the cell targeted by ZIKV). For example, it is preferred that the antibody, or antigen binding fragment thereof, of the present invention (be able to) interfere with membrane fusion. It is further preferred that the antibody, or antigen binding fragment thereof, of the present invention is capable of causing aggregation of ZIKV particles. Most preferably, the antibody, or antigen binding fragment thereof, of the present invention (is capable of) (i) inhibiting the post-attachment step of ZIKV and (ii) causing aggregation of ZIKV (particles). Particularly preferably, the antibody, or antigen binding fragment thereof, of the present invention includes at least one CDR, preferably all six CDRs, more preferably a variable region, even more preferably both variable regions (i.e., an epitope binding site), in in particular specificity, as an example of the human antibody ZKA190, which binds to ZIKV EDIII (see example 1, Fig. 1). The amino acid sequences of the CDRs and variable regions of ZKA190, which are preferably contained in the antibody, or antigen binding fragment thereof, of the present invention are described in table. 1 and 2 below.

It is also preferable that the antibody, or antigen-binding fragment thereof, despite significant neutralizing activity, such antibodies generally do not bind to recombinant ZIKV E protein or ZIKV EDIII in a standard ELISA method (as described above), i.e. when tested in vitro, particularly in purified form (ie with the ZIKV E protein outside/without the virion, virus-like particle or the like). Thus, undetectable binding usually means that EC ₅₀ up to 10,000 ng/ml was not detected using the standard ELISA method. In other words, if the EC ₅₀ detected by the standard ELISA method is above 10,000 ng/ml, then undetectable binding is observed.

Therefore, such antibodies are also referred to in the present invention as neutralizing-non-E-binding (NNB) antibodies. The quaternary epitope located on the infectious ZIKV virion is usually a conformational epitope. For example, a quaternary epitope located on an infectious ZIKV virion can form at the interface of two envelope protein monomers, forming a dimer (envelope dimer epitope EDE), or it can form through adjacent dimers (herringbone epitope).

It is particularly preferred that the antibody, or antigen binding fragment thereof, of the present invention comprises at least one CDR, preferably all six CDRs, more preferably a variable region, even more preferably both variable regions (i.e. an epitope binding site) in particular, the specificity of the exemplified human antibody ZKA230, which binds to a quaternary epitope located on the infectious ZIKV virion (see Example 1, FIG. 1). The amino acid sequences of the CDRs and variable regions of ZKA230, which are preferably included in the antibody, or antigen binding fragment thereof, of the present invention are described below in table. 1 and 2.

It is also preferably an antibody, or an antigen-binding fragment thereof, that binds to domain II of the Zika virus envelope protein (EDII). EDII is an elongated finger-like domain containing a conserved fusion loop that interacts with the host cell endosomal membrane, extending from approximately amino acid position 52 to amino acid position 132 and approximately from amino acid position 193 to amino acid position 280 of the ZIKV E protein (Dai L. et al., Cell Host Microbe, 19(5), 2016, 696-704).

Particularly preferably, the antibody, or antigen binding fragment thereof, of the present invention contains at least one CDR, preferably all six CDRs, more preferably a variable region, even more preferably both variable regions (i.e. an epitope binding site), in particular, the specificity of the exemplified human antibody ZKA185, which binds to ZIKV EDII. The amino acid sequences of the ZKA185 CDRs and variable regions that are preferably included in the antibody, or antigen binding fragment thereof, of the present invention are described below in Table. 1 and 2.

Preferably, the antibody, or antigen binding fragment thereof, of the present invention (i) binds to domain III of the Zika virus envelope protein (EDIII) and (ii) binds to domain II of the Zika virus envelope protein (EDII). That is, it is preferred that an antibody, or antigen binding fragment thereof, of the present invention includes (i) an epitope binding site that specifically binds to domain III of the Zika virus envelope protein (EDIII), and (ii) an epitope binding site that specifically binds to domain II of the Zika virus envelope protein (EDII). As described above, it is preferred that domain III of the Zika virus envelope protein includes or consists of the amino acid sequence of SEQ ID NO: 263 or 265, in particular SEQ ID NO: 264.

Moreover, as described above, it is also preferred that the antibody, or antigen binding fragment thereof, binds to an epitope of the Zika virus envelope protein that includes one or more lateral ridge (LR) amino acid residues of EDIII and/or one or more hinge region amino acid residues of EDI -EDIII. In other words, the EDIII-targeting epitope binding site binds to one or more lateral ridge (LR) amino acid residues of EDIII and/or

- 18 043940 one or more amino acid residues of the EDI-EDIII hinge region.

It is also preferred that the antibody, or antigen binding fragment thereof, of the present invention (i) binds to domain III of the Zika virus envelope protein (EDIII) and (ii) binds to a quaternary epitope present on the infectious ZIKV virion. That is, it is preferred that an antibody, or antigen binding fragment thereof, of the present invention includes (i) an epitope binding site that specifically binds to domain III of the Zika virus envelope protein (EDIII), and (ii) an epitope binding site that specifically binds to quaternary epitope present on the infectious ZIKV virion. As described above, it is preferable that domain III of the Zika virus envelope protein contains or consists of the amino acid sequence of SEQ ID NO: 263 or 265, especially SEQ ID NO: 264. Moreover, as described above, it is also preferable if the antibody, or its antigen-binding fragment binds to an epitope of the Zika virus envelope protein that includes one or more lateral ridge (LR) amino acid residues of EDIII and/or one or more hinge region amino acid residues of EDI-EDIII. That is, the EDIII-targeting epitope binding site preferably binds to one or more amino acid residues of the EDIII side ridge (LR) and/or one or more amino acid residues of the EDI-EDIII hinge region.

In general, the single epitope binding site (also referred to as paratope or antigen receptor) of an antibody of the present invention, or an antigen binding fragment thereof, preferably contains three complementarity determining regions (CDRs) in the heavy chain and (at least) three CDRs in the light chain. In general, complementarity determining regions (CDRs) are hypervariable regions contained in heavy chain variable domains and light chain variable domains.

Typically, the CDRs of the heavy chain and the associated light chain of an antibody together form the antigen receptor (epitope binding site). Typically, the three CDRs (CDR1, CDR2 and CDR3) are not sequentially located in the variable domain. Because epitope binding sites typically consist of two variable domains (e.g., on two different polypeptide chains, i.e., heavy and light chain), there are six CDRs for each epitope binding site (heavy chain: CDRH1, CDRH2, and CDRH3; light chain: CDRH1, CDRH2, and CDRH3; light chain: CDRL1, CDRL2 and CDRL3). The CDRs in the heavy and/or light chain may be separated by framework regions, the framework region (FR) being a region of the variable domain that is less variable than the CDRs. For example, a sequence (or each sequence, respectively) may consist of four framework regions separated by three CDRs.

The heavy chain and light chain sequences of various examples of human antibodies against the ZIKV E protein were determined, including three different CDRs in the heavy chain and three different CDRs in the light chain (see Tables 1 and 2 below). CDR amino acid positions are expressed according to the IMGT numbering system (IMGT: http://www.imgt.org/; cf. Lefranc M.-P. et al., Nucleic Acids Res. 37, 2009, D1006-D1012). Preferably, the antibody, or antigen binding fragment thereof, of the present invention comprises at least one CDR in the antibody examples below. More preferably, the antibody, or antigen binding fragment thereof, of the present invention includes all six CDRs (antibody binding sites) in the antibody examples below. Even more preferably, the antibody, or antigen binding fragment thereof, of the present invention includes a heavy chain variable region (VH) and a light chain variable region (VL) in the following antibody examples.

- 19 043940

Table 1

SEQ ID NO of the heavy chain CDR amino acid sequences (CDRH1, CDRH2 and CDRH3) and the heavy chain variable region (called VH) of typical antibodies

Antibody designation CDRH1 CDRH2 CDRH3 VH ZKA190 1 2 3 8 ZKA185 19 20 21 26 ZKA230 37 38 39 44 ZKA78 55 56 57 62 ZKA64 73 74 75 80 ZKA3 99 100 101 102 ZKA4 103 104 105 106 ZKA5 107 108 109 110 ZKA6 111 112 FROM 114 ZKA7 115 116 117 118 ZKA8 119 120 121 122 ZKA76 123 124 125 126 ZKA117 127 128 129 130 ZKB27 131 132 133 134 ZKB29 135 136 137 138 ZKB34 139 140 141 142 ZKB39 143 144 145 146 ZKB46 147 148 149 150 ZKB53 151 152 153 154 ZKC26 155 156 157 158 ZKD5 159 160 161 162 ZKD7 163 164 165 166 ZKD8 167 168 169 170 ZKD15 171 172 173 174 ZKD16 175 176 177 178 ZKD17 179 180 181 182 ZKD20 183 184 185 186 ZKA134 187 188 189 190 ZKA246 191 192 193 194 ZKA256 195 196 197 198 ZKB42 199 200 201 202 ZKB85 203 204 205 206 ZKB47 207 208 209 210 ZKC6 211 212 213 214 ZKA160 215 216 217 218 ZKA172 219 220 221 222 ZKA174 223 224 225 226 ZKA189 227 228 229 230 ZKA195 231 232 233 234 ZKA215 235 236 237 238 ZKA218 239 240 241 242 ZKB75 243 244 245 246 ZKB83 247 248 249 250 ZKC3 251 252 253 254 ZKC18 255 256 257 258 ZKD1 259 260 261 262

table 2

SEQ ID NO of the amino acid sequences of the light chain CDRs (CDRL1, CDRL2 and CDRL3) and the light chain variable region (called VL) of representative antibodies

Antibody designation CDRL1 CDRL2 CDRL2 long CDRL3 VL ZKA190 4 5 6 7 9 ZKA185 22 23 24 25 27 ZKA230 40 41 42 43 45 ZKA78 58 59 60 61 63 ZKA64 76 77 78 79 81

Thus, it is preferred that the antibody, or antigen binding fragment thereof, of the present invention contains amino acid sequences that are 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, is at least 99% identical to at least one of the CDR sequences, the VH sequence and/or the VL sequence presented in table. 1 and/or table. 2.

- 20 043940

Preferably, the antibody, or antigen binding fragment thereof, of the present invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3, and a light chain comprising at least , one CDRL1, at least one CDRL2 and at least one CDRL3, wherein the at least one heavy chain CDR, preferably at least one CDRH3, includes or consists of any of the amino acid sequences of SEQ ID NO: 3, 75, 39, 21, 57, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257 and 261, or a functional variant of these sequences, which is at least 70, at least 75, at least 80, at least 85, at least 88, at least 90, at least 92 is at least 95, at least 96, at least 97, at least 98, or at least 99% identical to this sequence.

More preferably, the antibody, or antigen binding fragment thereof, of the present invention contains a heavy chain including at least one CDRH1, at least one CDRH2 and at least one CDRH3, and a light chain including at least , one CDRL1, at least one CDRL2 and at least one CDRL3, wherein the at least one heavy chain CDR, preferably at least one CDRH3, includes or consists of any of the amino acid sequences of SEQ ID NO: 3, 21 and 39, 57 and 75 or a functional variant of these sequences that is 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 98 , is 99% identical to this sequence.

More preferably, the antibody, or antigen binding fragment thereof, of the present invention contains a heavy chain including at least one CDRH1, at least one CDRH2 and at least one CDRH3, and a light chain including at least , one CDRL1, at least one CDRL2 and at least one CDRL3, wherein the at least one heavy chain CDR, preferably at least one CDRH3, includes or consists of any of the amino acid sequences of SEQ ID NO: 3, 21 and 39 or a functional variant of these sequences that is 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% identical to this sequence.

It is also preferred that the antibody, or antigen binding fragment thereof, of the present invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3, and a light chain comprising at least at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein the at least one heavy chain CDR, preferably at least one CDRH3, comprises or consists of the amino acid sequence SEQ ID NO: 21 or SEQ ID NO: 39, or a functional variant of these sequences that is 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% identical to this sequence.

Most preferably, the antibody, or antigen binding fragment thereof, of the present invention comprises a heavy chain including at least one CDRH1, at least one CDRH2 and at least one CDRH3, and a light chain including at least , one CDRL1, at least one CDRL2 and at least one CDRL3, wherein the at least one CDR, preferably at least one heavy chain CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO: 3 or functional variant of this sequence, which is at least 70, at least 75, at least 80, at least 85, at least 88, at least 90, according to at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to this sequence.

More preferably, the antibody, or antigen binding fragment thereof, of the present invention contains a heavy chain including at least one CDRH1, at least one CDRH2 and at least one CDRH3, and a light chain including at least , one CDRL1, at least one CDRL2 and at least one CDRL3, wherein (i) at least one heavy chain CDRH1 includes any amino acid sequence of SEQ ID NO: 1,19, 37, 55 , 73, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, 1 91 , 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255 and 259, or a functional variant of these sequences that is 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

- 21 043940 is at least 96, at least 97, at least 98 or at least 99% identical to this sequence.

More preferably, the antibody, or antigen binding fragment thereof, of the present invention contains a heavy chain including at least one CDRH1, at least one CDRH2 and at least one CDRH3, and a light chain including at least , one CDRL1, at least one CDRL2 and at least one CDRL3, wherein the at least one heavy chain CDR, preferably at least one CDRH3, includes or consists of any of the amino acid sequences of SEQ ID NO: 3, 21 and 39 or a functional variant of these sequences that is 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% identical to this sequence;

(ii) at least one CDRH2 includes any amino acid sequence of SEQ ID NO: 2, 20, 38, 56, 74, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136 , 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236 , 240, 244, 248, 252, 256 and 260, or a functional variant of these sequences that is at least 70, at least 75, at least 80, at least 85, by 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% identical to this sequence; and/or (iii) at least one heavy chain CDRH3 contains any of the amino acid sequences of SEQ ID NO: 3, 21, 39, 57, 75, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 2 25, 229, 233, 237, 241, 245, 249, 253, 257 and 261, or a functional variant of these sequences that is at least 70 different, at least 75 different, at least 80 different, 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% identical to this sequence.

Even more preferably, the antibody, or antigen binding fragment thereof, of the present invention contains a heavy chain containing at least one CDRH1, at least one CDRH2 and at least one CDRH3, and a light chain containing at least at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein (i) at least one heavy chain CDRH1 includes any amino acid sequence of SEQ ID NO: 1,19, 37, 55 and 73 or a functional variant of these sequences that is 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% identical to this sequence;

(ii) at least one CDRH2 includes any amino acid sequence of SEQ ID NOs: 2, 20, 38, 56 and 74 or a functional variant of these sequences that is at least 70 different from 75, at least 80, at least 85, at least 88, at least 90, at least 92, at least 95, at least 96 is at least 97, at least 98 or at least 99% identical to this sequence; and/or (iii) at least one heavy chain CDRH3 contains any amino acid sequence of SEQ ID NOs: 3, 21, 39, 57 and 75 or a functional variant of these sequences that is 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 at least 96, at least 97, at least 98, or at least 99% identical to this sequence.

Even more preferably, the antibody, or antigen binding fragment thereof, of the present invention contains a heavy chain including at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain including at least , one CDRL1, at least one CDRL2 and at least one CDRL3, wherein (i) at least one heavy chain CDRH1 includes any amino acid sequence of SEQ ID NOs: 1,19 and 37, or a functional variant of these sequences that is at least 70, at least 75, at least 80, at least 85, at least 88, at least 90, at at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to this sequence;

(ii) at least one CDRH2 includes any amino acid sequence of SEQ ID NOs: 2, 20 and 38, or a functional variant of these sequences,

- 22 043940 which is 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% identical to this sequence; and/or (iii) at least one heavy chain CDRH3 contains any amino acid sequence of SEQ ID NOs: 3, 21 and 39, or a functional variant of these sequences that is at least 70 different from , 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% identical to this sequence.

Even more preferably, the antibody, or antigen binding fragment thereof, of the present invention contains a heavy chain including at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain including at least , one CDRL1, at least one CDRL2, and at least one CDRL3, wherein (i) the at least one heavy chain CDRH1 comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 73 or a functional variant of these sequences , which is at least 70, at least 75, at least 80, at least 85, at least 88, at least 90, at least 92 is at least 95, at least 96, at least 97, at least 98, or at least 99% identical to such sequence;

(ii) at least one CDRH2 includes the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 74 or a functional variant of these sequences that is 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 is at least 98 or at least 99% identical to this sequence; and/or (iii) at least one heavy chain CDRH3 contains an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 75 or a functional variant of these sequences that is at least 70 different from 75, at least 80, at least 85, at least 88, at least 90, at least 92, at least 95, at least 96 , is at least 97, at least 98, or at least 99% identical to this sequence.

It is also preferred that the antibody, or antigen binding fragment thereof, of the present invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3, and a light chain comprising at least at least one CDRL1, at least one CDRL2, and at least one CDRL3, wherein (i) the at least one heavy chain CDRH1 comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 37 or a functional variant thereof sequences that are 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% identical to such sequence;

(ii) at least one CDRH2 includes the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 38 or a functional variant of these sequences that is 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 are at least 98 or at least 99% identical to these sequences; and/or (iii) at least one heavy chain CDRH3 contains the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 39 or a functional variant of these sequences that is 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 at least 97, at least 98 or at least 99% identical to this sequence.

Most preferably, the antibody, or antigen binding fragment thereof, of the present invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3, and a light chain comprising at least at least one CDRL1, at least one CDRL2, and at least one CDRL3, wherein (i) the at least one heavy chain CDRH1 comprises the amino acid sequence of SEQ ID NO: 1 or a functional variant thereof that at least at least 70, at least 75, at least 80, at least 85, at least 88, at least 90, at least 92, at least , by 95, at least by 96, at least

- 23 043940 is at least 97, at least 98 or at least 99% identical to such sequence;

(ii) at least one CDRH2 comprises the amino acid sequence of SEQ ID NO: 2, or a functional variant thereof, that is at least 70%, at least 75%, at least 80% different, by 85, at least by 88, by at least 90, by at least 92, by at least 95, by at least 96, by at least 97, by at least 98 or at least 99% identical to these sequences; and/or (iii) at least one heavy chain CDRH3 contains the amino acid sequence of SEQ ID NO: 3 or a functional variant of this sequence that is 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, are at least 98 or at least 99% identical to this sequence.

It is also preferred that the antibody, or antigen binding fragment thereof, of the present invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3, and a light chain comprising at least at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein (i) at least one CDRL1 includes any of the amino acid sequences of SEQ ID NOs: 4, 22, 40, 58 and 76 or a functional variant of these sequences that is 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% identical to these sequences;

(ii) at least one CDRL2 includes any amino acid sequence of SEQ ID NO:, 6, 23, 24, 41, 42, 59, 60, 77 and 78, or a functional variant of these sequences, that at least by 70, by at least 75, by at least 80, by at least 85, by at least 88, by at least 90, by at least 92, by at least 95, at least 96, at least 97, at least 98, or at least 99% identical to these sequences; and/or (iii) at least one CDRL3 contains any amino acid sequence of SEQ ID NOs: 7, 25, 43, 61 and 79 or a functional variant of these sequences that is at least 70 , 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% identical to these sequences.

More preferably, the antibody, or antigen binding fragment thereof, of the present invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3, and a light chain comprising at least at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein (i) at least one CDRL1 includes any of the amino acid sequences of SEQ ID NOs: 4, 22, 40, 58 and 76 or a functional variant of these sequences that is 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% identical to these sequences;

(ii) at least one CDRL2 includes any amino acid sequence of SEQ ID NO: 5, 6, 23, 24, 41 and 42, or a functional variant of these sequences, which is at least 70 , 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% identical to these sequences; and/or (iii) at least one CDRL3 contains any of the amino acid sequences of SEQ ID NOs: 7, 25 and 43, or a functional variant of these sequences that is 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, are at least 97, at least 98 or at least 99% identical to these sequences.

Even more preferably, the antibody, or antigen binding fragment thereof, of the present invention contains a heavy chain including at least one CDRH1, at least one CDRH2 and at least one CDRH3, and a light chain including at least at least one CDRL1, at least one CDRL2, and at least one CDRL3, wherein (i) the at least one CDRL1 comprises the amino acid sequence of SEQ ID NO: 4

- 24 043940 or SEQ ID NO: 76; or a functional variant of these sequences that is 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% identical to these sequences;

(ii) at least one CDRL2 includes any of the amino acid sequences of SEQ ID NOs: 5, 6, 77 and 78, or a functional variant of these sequences that is 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 at least 97, at least 98 or at least 99% identical to these sequences; and/or (iii) at least one CDRL3 contains the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 79; or a functional variant of these sequences that is 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% identical to these sequences.

It is also preferred that the antibody, or antigen binding fragment thereof, of the present invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3, and a light chain comprising at least at least one CDRL1, at least one CDRL2, and at least one CDRL3, wherein (i) the at least one CDRL1 comprises the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 40; or a functional variant of these sequences that is 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% identical to these sequences;

(ii) at least one CDRL2 includes any amino acid sequence of SEQ ID NOs: 23, 24, 41 and 42, or a functional variant of these sequences that is 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 at least 97, at least 98 or at least 99% identical to these sequences; and/or (iii) at least one CDRL3 contains the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 43; or a functional variant of these sequences that is 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% identical to these sequences.

Most preferably, the antibody, or antigen binding fragment thereof, of the present invention comprises a heavy chain including at least one CDRH1, at least one CDRH2 and at least one CDRH3, and a light chain including at least , one CDRL1, at least one CDRL2, and at least one CDRL3, wherein (i) the at least one CDRL1 comprises the amino acid sequence of SEQ ID NO: 4 or a functional variant thereof that is at least 70, at least 75, at least 80, at least 85, at least 88, at least 90, at least 92, at least 95 is at least 96, at least 97, at least 98, or at least 99% identical to this sequence;

(ii) at least one CDRL2 includes any amino acid sequence of SEQ ID NO: 5 or 6, or a functional variant of these sequences, which is at least 70, at least 75, at least by 80, by at least 85, by at least 88, by at least 90, by at least 92, by at least 95, by at least 96, by at least 97 are at least 98 or at least 99% identical to these sequences; and/or (iii) at least one CDRL3 contains the amino acid sequence of SEQ ID NO: 7 or a functional variant of this sequence that is 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 are at least 98 or at least 99% identical to these sequences.

Preferably, the antibody, or antigen binding fragment thereof, of the present invention contains the amino acid sequences CDRH1, CDRH2 and CDRH3, (i) corresponding to the sequences of SEQ ID NO: 1-3; or functional variants of these

- 25 043940 consequences, which are at least 70, at least 75, at least 80, at least 85, at least 88, at least 90, according to at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(ii) corresponding to the sequences of SEQ ID NO: 19-21; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(iii) corresponding to the sequences of SEQ ID NO: 37-39; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(iv) corresponding to the sequences of SEQ ID NO: 55-57; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(v) corresponding to the sequences of SEQ ID NO: 73-75; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(vi) corresponding to the sequences of SEQ ID NO: 99-101; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(vii) corresponding to the sequences of SEQ ID NO: 103-105 or functional variants of these sequences that are at least 70, at least 75, at least 80, at least 85, at least at least 88, at least 90, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 98 at least 99% identical to these sequences;

(viii) corresponding to the sequences of SEQ ID NO: 107-109; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(ix) corresponding to the sequences of SEQ ID NO: 111-113; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(x) corresponding to the sequences of SEQ ID NO: 115-117; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xi) corresponding to the sequences of SEQ ID NO: 119-121; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xii) corresponding to the sequences of SEQ ID NO: 123-125; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xiii) corresponding to the sequences of SEQ ID NO: 127-129; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92,

- 26 043940 are at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xiv) corresponding to the sequences of SEQ ID NO: 131-133; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xv) corresponding to the sequences of SEQ ID NO: 135-137 or functional variants of these sequences that are at least 70, at least 75, at least 80, at least 85, at least at least 88, at least 90, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 98 at least 99% identical to these sequences;

(xvi) corresponding to the sequences of SEQ ID NO: 139-141; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xvii) corresponding to the sequences of SEQ ID NO: 143-145; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xviii) corresponding to the sequences of SEQ ID NO: 147-149; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xix) corresponding to the sequences of SEQ ID NO: 151-153; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xx) corresponding to the sequences of SEQ ID NO: 155-157; or functional variants of these sequences that are 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% identical to these sequences;

(xxi) corresponding to the sequences of SEQ ID NO: 159-161; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xxii) corresponding to the sequences of SEQ ID NO: 163-165; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xxiii) corresponding to the sequences of SEQ ID NO: 167-169; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xxiv) corresponding to the sequences of SEQ ID NO: 171-173; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xxv) corresponding to the sequences of SEQ ID NO: 175-177; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

- 27 043940 (xxvi) corresponding to the sequences of SEQ ID NO: 179-181; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xxvii) corresponding to the sequences of SEQ ID NO: 183-185; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xxviii) corresponding to the sequences of SEQ ID NO: 187-189; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xxix) corresponding to the sequences of SEQ ID NO: 191-193; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xxx) corresponding to the sequences of SEQ ID NO: 195-197; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xxxi) corresponding to the sequences of SEQ ID NO: 199-201; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xxxii) corresponding to the sequences of SEQ ID NO: 203-205; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xxxiii) corresponding to the sequences of SEQ ID NO: 207-209; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xxxiv) corresponding to the sequences of SEQ ID NO: 211-213; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xxxv) corresponding to the sequences of SEQ ID NO: 215-217; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xxxvi) corresponding to the sequences of SEQ ID NO: 219-221; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xxxvii) corresponding to the sequences of SEQ ID NO: 223-225; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xxxviii) corresponding to the sequences of SEQ ID NO: 227-229; or functional variants of these sequences that are at least 70, at least 75, at least

- 28 043940 at least 80, at least 85, at least 88, at least 90, at least 92, at least 95, at least 96, are at least 97, at least 98 or at least 99% identical to these sequences;

(xxxix) corresponding to the sequences of SEQ ID NO: 231-233; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xl) corresponding to the sequences of SEQ ID NO: 235-237; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xli) corresponding to the sequences of SEQ ID NO: 239-241; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xlii) corresponding to the sequences of SEQ ID NO: 243-245; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xliiii) corresponding to the sequences of SEQ ID NO: 247-249; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xliv) corresponding to the sequences of SEQ ID NO: 251-253; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(xlv) corresponding to the sequences of SEQ ID NO: 255-257; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences; or (xlvi) corresponding to the sequences of SEQ ID NO: 259-261; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences.

Accordingly, it is also preferred that the antibody, or antigen binding fragment thereof, of the present invention comprises the amino acid sequences CDRH1, CDRH2 and CDRH3, as well as the amino acid sequences CDRL1, CDRL2 and CDRL3, (i) corresponding to the sequences of SEQ ID NOs: 1-5 and 7; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(ii) corresponding to the sequences of SEQ ID NO: 1-4 and 6, 7; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(iii) corresponding to the sequences of SEQ ID NO: 19-23 and 25; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(iv) corresponding to the sequences SEQ ID NO: 19-22 and 24, 25; or functional variations

- 29 043940 ants of these sequences, which are at least 70, at least 75, at least 80, at least 85, at least 88, at least 90 are at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(v) corresponding to the sequences of SEQ ID NO: 37-41 and 43; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(vi) corresponding to the sequences of SEQ ID NO: 37-40 and 42, 43; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(vii) corresponding to the sequences of SEQ ID NO: 55-59 and 61; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(viii) corresponding to the sequences of SEQ ID NO: 55-58 and 60, 61; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(ix) corresponding to the sequences of SEQ ID NO: 73-77 and 79; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences; or (x) corresponding to the sequences of SEQ ID NO: 73-76 and 78, 79; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences.

More preferably, the antibody, or antigen binding fragment thereof, of the present invention comprises the amino acid sequences CDRH1, CDRH2 and CDRH3, as well as the amino acid sequences CDRL1, CDRL2 and CDRL3, (i) corresponding to the sequences of SEQ ID NOs: 1-5 and 7; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(ii) corresponding to the sequences of SEQ ID NO: 1-4 and 6, 7; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(iii) corresponding to the sequences of SEQ ID NO: 19-23 and 25; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(iv) corresponding to the sequences SEQ ID NO: 19-22 and 24, 25; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(v) corresponding to the sequences of SEQ ID NO: 37-41 and 43; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

- 30 043940 (vi) corresponding to the sequences SEQ ID NO: 37-40 and 42, 43; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(vii) corresponding to the sequences of SEQ ID NO: 73-77 and 79; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences; or (viii) corresponding to the sequences of SEQ ID NO: 73-76 and 78, 79; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences.

Even more preferably, the antibody, or antigen binding fragment thereof, of the present invention contains the amino acid sequences CDRH1, CDRH2 and CDRH3, as well as the amino acid sequences CDRL1, CDRL2 and CDRL3, (i) corresponding to the sequences of SEQ ID NOs: 1-5 and 7; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(ii) corresponding to the sequences of SEQ ID NO: 1-4 and 6, 7; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(iii) corresponding to the sequences of SEQ ID NO: 73-77 and 79; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences; or (iv) corresponding to the sequences of SEQ ID NO: 73-76 and 78, 79; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences.

It is also preferred that the antibody, or antigen binding fragment thereof, of the present invention comprises the amino acid sequences CDRH1, CDRH2 and CDRH3, as well as the amino acid sequences CDRL1, CDRL2 and CDRL3, (i) corresponding to the sequences of SEQ ID NOs: 19-23 and 25; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(ii) corresponding to the sequences of SEQ ID NO: 19-22 and 24, 25; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences;

(iii) corresponding to the sequences of SEQ ID NO: 37-41 and 43; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences; or (vi) corresponding to the sequences of SEQ ID NO: 37-40 and 42, 43; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences.

Even more preferably, the antibody, or antigen-binding fragment thereof, is

- 31 043940 of the invention contains the amino acid sequences CDRH1, CDRH2 and CDRH3, as well as the amino acid sequences CDRL1, CDRL2 and CDRL3, (i) corresponding to the sequences of SEQ ID NO: 1-5 and 7; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences; or (ii) corresponding to the sequences of SEQ ID NO: 1-4 and 6, 7; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences.

It is also preferred that the antibody, or antigen binding fragment thereof, of the present invention described above comprises the amino acid sequences CDRH1, CDRH2 and CDRH3, as well as the amino acid sequences CDRL1, CDRL2 and CDRL3, (i) corresponding to the sequences of SEQ ID NOs: 19-23 and 25; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences; or (ii) corresponding to the sequences of SEQ ID NO: 19-22 and 24, 25; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences.

Most preferably, the antibody, or antigen binding fragment thereof, of the present invention comprises (a) a first epitope binding site comprising the amino acid sequences CDRH1, CDRH2 and CDRH3, and the amino acid sequences CDRL1, CDRL2 and CDRL3, (i) corresponding to the sequences ID NO: 1 -5 and 7; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences; or (ii) corresponding to the sequences of SEQ ID NO: 1-4 and 6, 7; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences; and (b) a second epitope binding site comprising the amino acid sequences CDRH1, CDRH2 and CDRH3, as well as the amino acid sequences CDRL1, CDRL2 and CDRL3, (i) corresponding to the sequences of SEQ ID NOs: 19-23 and 25; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences; or (ii) corresponding to the sequences of SEQ ID NO: 19-22 and 24, 25; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences.

It is also preferred that the antibody, or antigen binding fragment thereof, of the present invention described above comprises the amino acid sequences CDRH1, CDRH2 and CDRH3, as well as the amino acid sequences CDRL1, CDRL2 and CDRL3, (i) corresponding to the sequences of SEQ ID NOs: 37-41 and 43; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences; or (ii) corresponding to the sequences of SEQ ID NO: 37-40 and 42, 43; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least,

- 32 043940 are 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences.

More preferably, the antibody, or antigen binding fragment thereof, of the present invention contains (a) a first epitope binding site comprising the amino acid sequences CDRH1, CDRH2 and CDRH3, and the amino acid sequences CDRL1, CDRL2 and CDRL3, (i) corresponding to the sequences ID NO: 1 -5 and 7; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences; or (ii) corresponding to the sequences of SEQ ID NO: 1-4 and 6, 7; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences; and (b) a second epitope binding site comprising the amino acid sequences CDRH1, CDRH2 and CDRH3, and the amino acid sequences CDRL1, CDRL2 and CDRL3, (i) corresponding to the sequences of SEQ ID NOs: 37-41 and 43; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences; or (ii) corresponding to the sequences of SEQ ID NO: 37-40 and 42, 43; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences.

In addition, it is also preferred that the antibody, or antigen binding fragment thereof, of the present invention comprises a heavy chain variable region (VH) and optionally a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises or consists of an amino acid sequences corresponding to any of SEQ ID NO: 8, 26, 44, 62, 80, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242, 246, 250, 2 54, 258 and 262; or a functional variant of these sequences that is 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% identical to these sequences.

Moreover, it is also preferred that the antibody, or antigen binding fragment thereof, of the present invention comprises (i) a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 8, or a functional variant thereof, which 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% identical to these sequences, and/or the amino acid sequence of the light chain variable region (VL) corresponding to sequence SEQ ID NO: 9, or a functional variant of this sequence, which is 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% identical to these sequences;

(ii) a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 26, or a functional variant of this sequence, which is 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 are at least 98% or at least 99% identical to these sequences, and/or a light chain variable region (VL) amino acid sequence corresponding to SEQ ID NO: 27, or a functional variant thereof, that are 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

- 33 043940 or at least 99% identical to these sequences;

(iii) a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 44, or a functional variant of this sequence, which is 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 are at least 98% or at least 99% identical to these sequences, and/or a light chain variable region (VL) amino acid sequence corresponding to SEQ ID NO: 45, or a functional variant thereof, that are 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% identical to these sequences;

(iv) a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 62, or a functional variant of this sequence, which is 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 at least 98% or at least 99% identical to these sequences, and/or a light chain variable region (VL) amino acid sequence corresponding to SEQ ID NO: 63, or a functional variant thereof, that is 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% identical to these sequences; or (v) a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 80, or a functional variant thereof, which is at least 70 different, at least 75 different, at least 80 different , 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% identical to these sequences, and/or a light chain variable region (VL) amino acid sequence corresponding to SEQ ID NO: 81, or a functional variant thereof, that is at least at least 70, at least 75, at least 80, at least 85, at least 88, at least 90, at least 92, at least , are 95, at least 96, at least 97, at least 98, or at least 99% identical to these sequences.

More preferably, the antibody, or antigen binding fragment thereof, of the present invention comprises (i) a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 8, or a functional variant thereof, that is 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 at least 96, at least 97, at least 98 or at least 99% identical to these sequences, and/or the amino acid sequence of the light chain variable region (VL) corresponding to the sequence of SEQ ID NO : 9, or a functional variant of this sequence, which is 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% identical to these sequences;

(ii) a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 26, or a functional variant of this sequence, which is 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 are at least 98% or at least 99% identical to these sequences, and/or a light chain variable region (VL) amino acid sequence corresponding to SEQ ID NO: 27, or a functional variant thereof, that are 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% identical to these sequences;

(iii) a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 44, or a functional variant of this sequence, which is at least 70, at least 75, at least 80, at least 85, at least 88, at least 90, at least 92, at least 95, according

- 34 043940 at least 96, at least 97, at least 98 or at least 99% identical to these sequences, and/or the amino acid sequence of the light chain variable region (VL) corresponding to the sequence SEQ ID NO: 45, or a functional variant thereof, which is 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% identical these sequences; or (iv) a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 80, or a functional variant thereof, which is 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% identical to these sequences, and/or a light chain variable region (VL) amino acid sequence corresponding to SEQ ID NO: 81, or a functional variant thereof, that is at least at least 70, at least 75, at least 80, at least 85, at least 88, at least 90, at least 92, at least , are 95, at least 96, at least 97, at least 98, or at least 99% identical to these sequences.

Even more preferably, the antibody, or antigen binding fragment thereof, of the present invention contains (i) a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 8, or a functional variant of this sequence, which is 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% identical to these sequences, and/or the amino acid sequence of the light chain variable region (VL) corresponding to the sequence of SEQ ID NO: 9, or a functional variant of this sequence, which is 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% identical to these sequences ; or (ii) a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 80, or a functional variant thereof, which is at least 70 different, at least 75 different, at least 80 different , 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% identical to these sequences, and/or a light chain variable region (VL) amino acid sequence corresponding to SEQ ID NO: 81, or a functional variant thereof, that is at least at least 70, at least 75, at least 80, at least 85, at least 88, at least 90, at least 92, at least , are 95, at least 96, at least 97, at least 98, or at least 99% identical to these sequences.

It is also preferred that the antibody, or antigen binding fragment thereof, of the present invention comprises (i) a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 26, or a functional variant thereof, that is 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% identical to these sequences, and/or the amino acid sequence of the light chain variable region (VL) corresponding to the sequence of SEQ ID NO: 27, or a functional variant of this sequence, which is 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% identical to these sequences ; or (ii) a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 44, or a functional variant thereof, which is at least 70 different, at least 75 different, at least 80 different , 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% identical to these sequences, and/or the amino acid sequence of the light chain variable region (VL) corresponding to the sequence of SEQ ID NO: 45, or a functional variable

- 35 043940 ants of this sequence, which are at least 70, at least 75, at least 80, at least 85, at least 88, at least 90 , at least for

92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences.

Even more preferably, the antibody, or antigen binding fragment thereof, of the present invention comprises a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 8, or a functional variant thereof, that is at least 70 times greater than 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% identical to these sequences, and/or the amino acid sequence of the light chain variable region (VL) corresponding to the sequence of SEQ ID NO: 9 , or a functional variant of this sequence, which is at least 70, at least 75, at least 80, at least 85, at least 88, at least 90 are at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences.

It is also preferred that the antibody, or antigen binding fragment thereof, of the present invention as described above comprises a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 26, or a functional variant of this sequence, which is 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% identical to these sequences, and/or the amino acid sequence of the light chain variable region (VL) corresponding to the sequence of SEQ ID NO: 27, or a functional variant of this sequence, which is 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% identical to these sequences .

Most preferably, the antibody, or antigen binding fragment thereof, of the present invention as described above comprises (a) a first epitope binding site comprising a variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 8, or a functional variant of this sequence, which, at least 70, at least 75, at least 80, at least 85, at least 88, at least 90, at least 92, at least at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences, and/or the amino acid sequence of the light chain variable region (VL ), corresponding to the sequence SEQ ID NO: 9, or a functional variant of this sequence that is 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% identical to these sequences; and (b) a second epitope binding site comprising a variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 26, or a functional variant of this sequence that is at least 70 different, at least 75 different, 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% identical to these sequences, and/or a light chain variable region (VL) amino acid sequence corresponding to the sequence of SEQ ID NO: 27, or a functional variant of this sequence, which is at least 70, at least 75, at least 80, at least 85, at least 88, at least 90, at least 92 is at least 95, at least 96, at least 97, at least 98, or at least 99% identical to these sequences.

It is also preferred that the antibody, or antigen binding fragment thereof, of the present invention as described above comprises a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 44, or a functional variant of this sequence, which is 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% identical to this sequence, and/or the amino acid sequence of the light chain variable region (VL) corresponding to the sequence of SEQ ID NO: 45, or a functional variant of this sequence that is at least 70, by

- 36 043940 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% identical to this sequence.

More preferably, the antibody, or antigen binding fragment thereof, of the present invention as described above comprises (a) a first epitope binding site comprising a variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 8, or a functional variant thereof, which, at least 70, at least 75, at least 80, at least 85, at least 88, at least 90, at least 92, at least at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences, and/or the amino acid sequence of the light chain variable region (VL ), corresponding to the sequence SEQ ID NO: 9, or a functional variant of this sequence, which is 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% identical to these sequences; and (b) a second epitope binding site comprising a variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 44, or a functional variant of this sequence that is at least 70 different, at least 75 different, 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% identical to these sequences, and/or a light chain variable region (VL) amino acid sequence corresponding to the sequence of SEQ ID NO: 45, or a functional variant of this sequence, which is at least 70, at least 75, at least 80, at least 85, at least 88, at least 90, at least 92 is at least 95, at least 96, at least 97, at least 98, or at least 99% identical to these sequences.

Particularly preferably, the antibody, or antigen binding fragment thereof, of the present invention is in Fabs-in-tandem-Ig (FIT-Ig) format and the outer Fab of the FIT-Ig format, including the epitope binding site, contains the amino acid sequences CDRH1, CDRH2 and CDRH3 and the amino acid sequences of CDRL1, CDRL2 and CDRL3, (i) corresponding to the sequences of SEQ ID NOs: 1-5 and 7; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences (ii) corresponding sequences SEQ ID NO: 1-4 and 6, 7; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences. More preferably, the outer Fab of the FIT-Ig format contains an epitope binding site comprising a light chain variable region (VL) amino acid sequence corresponding to the sequence of SEQ ID NO: 9, or functional variants of this sequence that are at least 70 different from at least 75, at least 80, at least 85, at least 88, at least 90, at least 92, at least 95, at least , are 96, at least 97, at least 98, or at least 99% identical to this sequence. Even more preferably, the internal Fab of the FIT-Ig format contains an epitope binding site comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 and the amino acid sequences CDRL1, CDRL2 and CDRL3, (i) corresponding to the sequences of SEQ ID NOs: 19-23 and 25; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences; or (ii) corresponding to the sequences of SEQ ID NO: 19-22 and 24, 25; or functional variants of these sequences that are at least 70 different, at least 75 different, at least 80 different, at least 85 different, at least 88 different, at least 90 different, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to these sequences. Even more preferably, the internal Fab of the FIT-Ig format contains an epitope binding site comprising a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID

- 37 043940

NO: 26, or a functional variant of these sequences that is 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% identical to this sequence , and/or a light chain variable region (VL) amino acid sequence corresponding to the sequence of SEQ ID NO: 27, or a functional variant of these sequences that is 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, is at least 98 or at least 99% identical to this sequence.

Preferably, the antibody, or antigen binding fragment thereof, of the present invention is for use as a medicament. In other words, the antibody, or antigen-binding fragment thereof, of the present invention can be used for the preparation of a drug. More preferably, the antibody, or antigen binding fragment thereof, of the present invention is intended for the prevention and/or treatment of infection caused by the Zika virus. In other words, the antibody, or antigen-binding fragment thereof, of the present invention can be used for the preparation of a medicament or used for the prevention and/or treatment of infection caused by the Zika virus. This object is described in detail below.

Nucleic acid molecules.

Another subject also provided by the present invention is a nucleic acid molecule containing at least one polynucleotide encoding an antibody, or an antigen binding fragment thereof, of the present invention as described above or a fragment thereof, wherein the fragment includes at least one CDR of an antibody, or antigen-binding fragment thereof.

A nucleic acid molecule is a molecule, preferably composed of nucleic acid components. The term nucleic acid molecule preferably refers to DNA or RNA molecules. In particular, it is used as a synonym for the concept of polynucleotide. Preferably, the nucleic acid molecule is a polymer containing or consisting of nucleotide monomers that are covalently linked to each other by sugar/phosphate backbone phosphodiester bonds. The term nucleic acid molecule also includes modified nucleic acid molecules, such as base-modified, sugar-modified or scaffold-modified molecules, such as DNA or RNA.

In a nucleic acid molecule of the present invention, an encoded fragment of an antibody or antigen binding fragment thereof of the present invention contains at least one CDR of the antibody or antigen binding fragment thereof. Table 1 and 2 represent the SEQ ID numbers of the amino acid sequences of the CDR regions, as well as the VH and VL of exemplary antibodies of the present invention. Accordingly, the nucleic acid molecule of the present invention preferably contains a polynucleotide encoding one or more amino acid sequences shown in table. 1 and 2. Preferably, the encoded fragment of an antibody or antigen binding fragment thereof of the present invention contains three CDRs, more preferably all three epitope binding site heavy chain CDRs (CDRH1, CDRH2, CDRH3) and/or all three epitope binding site light chain CDRs (CDRL1 , CDRL2, CDRL3). Accordingly, it is preferred that the encoded fragment of an antibody or antigen-binding fragment thereof contains (exactly) three or six CDRs. Accordingly, the nucleic acid molecule of the present invention includes a polynucleotide encoding three or six CDR amino acid sequences shown in table. 1 and 2, in particular the corresponding CDRH1, CDRH2 and CDRH3 sequences and/or the CDRL1, CDRL2 and CDRL3 sequences. More preferably, the encoded fragment of the antibody or antigen binding fragment thereof comprises a variable region, for example, a heavy chain variable region (VH) and/or a light chain variable region (VL). Accordingly, it is preferred that the encoded fragment of an antibody or antigen-binding fragment thereof contains (exactly) one or two variable regions. Accordingly, the nucleic acid molecule of the present invention preferably includes a polynucleotide encoding one or two variable region amino acid sequences shown in Table. 1 and 2. Most preferably, the encoded fragment of the antibody or antigen binding fragment thereof comprises the (complete) polypeptide chain of the antibody or antigen binding fragment thereof of the present invention. Such a (complete) polypeptide chain may be, for example, a (complete) heavy chain or a (complete) light chain. However, such a (complete) polypeptide chain may also include both heavy and light chain elements, as is known in the art for many multispecific antibody formats (e.g., polypeptide chains containing heavy chain constant regions, but also, for example, in addition to one or more heavy chain variable regions, light chain variable regions).

The nucleic acid molecule can be mono-, bi- or multicistronic, for example tricistronic. A bicistronic or multicistronic nucleic acid molecule is usually a molecule

- 38 043940 nucleic acid core, which typically may have two (bicistronic) or multiple (multicistronic) open reading frames (ORFs). An open reading frame in this context is a sequence of codons that is translated into a peptide or protein. More generally, a nucleic acid molecule of the present invention contains at least one polynucleotide encoding at least one antibody, or antigen binding fragment thereof, of the present invention. If more nucleic acid molecules of the present invention contain more than one coding polynucleotide, then the second, third and subsequent polynucleotides may encode other peptides/proteins and/or may encode antibodies, for example, of the present invention, or also fragments thereof, which may be the same or different from the first antibody coding region. In a preferred embodiment, the nucleic acid molecule of the present invention contains at least two coding polynucleotides, for example (exactly) two or three coding polynucleotides, all of which encode identical or different antibodies, or fragments thereof, or variants thereof. For example, individual fragments of an antibody, or antigen binding fragment thereof, of the present invention may be encoded by different polynucleotides on the same nucleic acid molecule. In yet another embodiment of the present invention, a nucleic acid molecule of the present invention may encode more than one antibody or antibody fragment in the same coding region. Thus, the nucleic acid molecule of the present invention may be mono-, bi- or multicistronic.

For example, in one preferred embodiment of the present invention, an antibody, or antigen binding fragment thereof, of the present invention may be a single chain antibody. In this case, it is preferable that the entire single chain of the antibody or antigen-binding fragment thereof is encoded by a single polynucleotide.

Accordingly, it is preferable that the nucleic acid molecule of the present invention be monocistronic, in particular, it may include a single polynucleotide encoding an antibody, or antigen binding fragment thereof, of the present invention.

It is also preferred that the antibody, or antigen binding fragment thereof, of the present invention comprises exactly two different polypeptide chains, for example, a heavy chain and a light chain. This can be illustrated by the classic native IgG molecule, which contains two identical heavy chains and two identical light chains and thus two different polypeptide chains (even if the antibody ultimately contains four polypeptide chains, they may be encoded by two (different) polynucleotides ). Preferably, such two different polypeptide chains (eg, a heavy chain and a light chain) of an antibody, or antigen binding fragment thereof, of the present invention may be encoded by two different polynucleotides, which may be located in the same nucleic acid molecule, for example, a bicistronic molecule nucleic acid or in (exactly two) separate nucleic acid molecules (for example, each nucleic acid molecule can be monocistronic). Thus, it is preferable that the nucleic acid molecule of the present invention be bicistronic, in particular it may contain (exactly) two polynucleotides encoding (together) an antibody, or antigen binding fragment thereof, of the present invention. It is also preferable that the nucleic acid molecule of the present invention contains (at least or exactly) one polynucleotide encoding an antibody fragment, or an antigen binding fragment thereof, of the present invention as described above (eg, a (full) polypeptide chain). Thus, it is preferable that the nucleic acid molecule of the present invention be monocistronic. In addition, an antibody, or antigen binding fragment thereof, of the present invention may be encoded by multiple (eg, monocistronic) nucleic acid molecules. For example, an antibody containing two different polypeptide chains may be encoded by two polynucleotides located in two different nucleic acid molecules.

It is also preferred that the antibody, or antigen binding fragment thereof, of the present invention comprises (exactly) three different polypeptide chains. Thus, it is preferred that the nucleic acid molecule of the present invention be tricistronic (eg, encoding all three polypeptide chains in such an antibody, or antigen binding fragment thereof, of the present invention). However, it is preferred that the nucleic acid molecule is monocistronic or bicistronic, for example encoding one or two polypeptide chains contained in such an antibody, or antigen binding fragment thereof, of the present invention. For example, a plurality of nucleic acid molecules, such as two or three nucleic acid molecules, may encode (together) an antibody, or antigen binding fragment thereof, of the present invention.

For example, the antibody, or antigen binding fragment thereof, of the present invention is particularly preferably in Fabs-in-tandem-Ig (FIT-Ig) format, which typically comprises the following three polypeptide chains:

polypeptide 1 containing or consisting of an outer Fab light chain and an inner Fab heavy chain, including the CH1-CH2-CH3 constant domains, preferably with the LALA mutation as described above, preferably the outer Fab light chain hybridized, preferably through linkers , with the N-terminus of the heavy chain of the internal Fab;

polypeptide 2 containing or consisting of the heavy chain of the outer Fab (VH and CH1 of the outer

Fab); and polypeptide 3 containing or consisting of a light chain of an internal Fab.

Accordingly, such a FIT-Ig may preferably be encoded by one (eg, tricistronic) nucleic acid molecule, three (eg, monocistronic) nucleic acid molecules, or two (eg, one monocistronic, one bicistronic) nucleic acid molecules.

Accordingly, it is preferable that such a nucleic acid molecule of the present level contains a polynucleotide encoding the FIT-Ig antibody polypeptide 1 of the present level. More preferably, the nucleic acid molecule does not contain polynucleotides encoding polypeptide 2 or polypeptide 3 of the FIT-Ig antibody as defined herein. Such a nucleic acid molecule is preferably monocistronic.

It is also preferable that the present amount of the nucleic acid molecule contains a polynucleotide encoding the polypeptide 2 of the FIT-Ig antibody of the present invention. More preferably, the nucleic acid molecule does not contain polynucleotides encoding polypeptide 1 or polypeptide 3 of the FIT-Ig antibody as defined herein. Such a nucleic acid molecule is preferably monocistronic.

It is also preferable that the present amount of the nucleic acid molecule contains a polynucleotide encoding the polypeptide 3 of the FIT-Ig antibody of the present invention. More preferably, the nucleic acid molecule does not contain polynucleotides encoding polypeptide 1 or polypeptide 2 of the FIT-Ig antibody as defined herein. Such a nucleic acid molecule is preferably monocistronic.

It is also preferable that the nucleic acid molecule of the present invention contains a polynucleotide encoding polypeptide 1 and a polynucleotide encoding polypeptide 2 of the FIT-Ig antibody of the present invention. More preferably, the nucleic acid molecule does not contain a polynucleotide encoding polypeptide 3 of the FIT-Ig antibody of the present invention. Such a nucleic acid molecule is preferably bicistronic.

It is also preferable that the present amount of the nucleic acid molecule contains a polynucleotide encoding polypeptide 1 and a polynucleotide encoding polypeptide 3 of the FIT-Ig antibodies of the present invention. More preferably, the nucleic acid molecule does not contain a polynucleotide encoding the polypeptide 2 of the FIT-Ig antibody of the present invention. Such a nucleic acid molecule is preferably bicistronic.

It is also preferable that the nucleic acid molecule of the present invention contains a polynucleotide encoding polypeptide 2 and a polynucleotide encoding polypeptide 3 of the FIT-Ig antibodies of the present invention. More preferably, the nucleic acid molecule does not contain a polynucleotide encoding polypeptide 1 of the FIT-Ig antibody of the present invention. Such a nucleic acid molecule is preferably bicistronic.

It is particularly preferred that the nucleic acid molecule of the present invention includes a polynucleotide encoding polypeptide 1, a polynucleotide encoding polypeptide 2, and a polynucleotide encoding polypeptide 3 of the FIT-Ig antibodies of the present invention. Such a nucleic acid molecule is preferably tricistronic.

In general, a nucleic acid molecule may be a DNA molecule or an RNA molecule. Examples of nucleic acid molecules and/or polynucleotides include, for example, a recombinant polynucleotide, a vector, an oligonucleotide, an RNA molecule, such as rRNA, mRNA, microRNA, siRNA or tRNA, or a DNA molecule, such as cDNA. Preferably the nucleic acid molecule is a plasmid DNA or an mRNA molecule. Plasmid DNA is a circular, preferably double-stranded DNA molecule.

In the table below. 3 provides SEQ ID numbers for exemplary nucleic acid sequences encoding the CDRs, as well as VH and VL, exemplary antibody sequences of the present invention. Due to the redundancy of the genetic code, the present invention also includes sequence variants of the corresponding nucleic acids and, in particular, sequence variants that encode the same amino acid sequences.

- 40 043940

Table 3

Nucleic acid sequences of CDR, variable heavy chain (VH) and variable light chain (VL) of five representative antibodies (ZKA190, ZKA64, ZKA230, ZKA185, ZKA78), given as examples

ZKA190 SEQ ID NO. Amino acid sequences CDRH1 10 ggattcaccttcagtaaatatggc CDRH2 eleven atatcatatgagggaagtaataaa CDRH3 12 gcgaaatcggggacccaatactatgatactactggttatgagtat aggggtttggaatactttggctac CDRL1 13 cagagtgttagtagcagttac CDRL2 14 gatgcatcc CDRL2 long 15 ctcatctatgatgcatccagcaggggcc CDRL3 16 cagcagtatggtaggtcaaggtggaca VH 17 caggtgcagctggtggagtctgggggaggcgtggtccagcctggg aggtccctgagactctcctgtgcagcctctggattcaccttcagt aaatatggcatgcactgggtccgccaggctccaggcaaggggctg gagtgggtggcagttatatcatatgagggaagtaataaatattat gcagactccgtgaagggccgattcaccat ctccagagacaattcc aagaacacgctgtatctgcaaatgaacagcctgagagctgaggac acggcagtgtattactgtgcgaaatcggggacccaatactatgat actactggttatgagtataggggtttggaatactttggctactgg ggccagggaaccctggtcaccgtctcctcag VL 18 gaaattgtgttgacgcagtctccaggcaccctgtctttgtctcca ggggaaagagccaccctctcctgcagggccagtcagagtgttagt agcagttacttagcctggtaccagcagaaacgtggccaggctccc aggctcctcatctatgatgcatccagcagggccactggcatccca gacaggttcagtggcagtggggt ctgggacagacttcactctcacc atcagcagactggagcctgaagattttgcagtgtattactgtcag cagtatggtaggtcaaggtggacattcggccaagggaccaaggtg gaaatcaaac ZKA185 SEQ ID NO. Amino acid sequences CDRHL 28 ggatatagttttaccagttactgg CDRH2 29 tttgatcctagtgactctcaaacc CDRH3 thirty gcgagaagatattgtagtagtagttgttatgtggacaat CDRLl 31 gcattgccaaataaattt CDRL2 32 gaggacaac CDRL2 long 33 gtcatctatgaggacaacaaacgaccc CDRL3 34 tactcaacagacagcagttctaatcccctgggagta VH 35 gaagtgcagctggtgcagtccggagcagaggtgaaaaagcccggg gagtctctgaggatctcctgtaagggttctggatatagttttacc agttactggatcacctgggtgcgccagatgcccgggaaaggcctg gagtggatggcgaagtttgatcctagtgactctcaaaccaactac agcccgtccttcca aggccacgtcaccatctcagttgacaagtcc atcagcactgcctacttgcagtggagcagcctgaaggcctcggac accgccatgtattactgtgcgagaagatattgtagtagtagtagt tgttatgtggacaattggggccagggaaccctggtcaccatcttc tcag

- 41 043940

VL 36 tcctatgagctgacacagccaccctcggtgtcagtgtccccagga caaacggccaggatcacctgctctggagatgcattgccaaataaa tttgcttattggtaccggcagaagtcaggccaggcccctgttctg gtcatctatgaggacaacaaacgaccctccgggatccctgagaga ttctctggctccagctcagggacaatggccacc ttgactatcagt ggggcccaggtggaggatgaagctgactaccactgttactcaaca gacagcagttctaatcccctgggagtattcggcggagggaccaag ctgaccgtcctag ZKA230 SEQ ID NO. Amino acid sequences CDRH1 46 ggtggctccatcagtagtgactac CDRH2 47 atctattacagtgggagcacc CDRH3 48 gcgaggagggaggaagtatgattccctttgggggagttttgctttt gatatc CDRL1 49 agctccaacatcggaggtaattat CDRL2 50 attaatgat CDRL2 long 51 ctcatctgtattaatgatcaccggccc CDRL3 52 gcaacatgggatgacagcctgggtggccttgta VH 53 caggtgcagctgcaggagtcgggcccaggcctggtgaagccttcg gagaccctgtccctcacctgcgcagtctctggtggctccatcagt agtgactactggagctggatccggcagcccccagggaagggactg gagtggattgggtatatctattacagtgggagcaccaactacaac ccctccctcaagagtcgagtcaccatat cagtagacacgtccaag aaccacttctccctgaagctgaactctgtgaccgctgcggacacg gccgtgtattactgtgcgaggagggaggaagtatgattccctttgg gggagttttgcttttgatatctggggccaagggacaatggtcacc gtctcttcag VL 54 cagtctgtgctgactcagccaccctcagcgtctgggacccccggg cagagggtcaccatctcttgttctggaagcagctccaacatcgga ggtaattatgtatactggtaccagcagctcccaggaacggccccc aaactcctcatctgtattaatgatcaccggccctcaggggtccct gaccgattctctggctccaagtctggcacctca gcctccctggcc atcagtgggctccagtccgaggatgaggctgattattactgtgca acatgggatgacagcctgggtggccttgtattcggcggagggacc aagctgaccgtcctag ZKA78 SEQ ID NO. Amino acid sequences CDRH1 64 ggcttcacttttagtaactatgca CDRH2 65 atcggggcgcaacggggactctatc CDRH3 66 gtgaaagatctggccatccccgagtcctacagaattgaagctgat tat CDRL1 67 cagtccgtgctgtaccgctctaacaacaagaattac CDRL2 68 tggggcttca CDRL2 long 69 ctgatctattgggcttcaacccgggaa CDRL3 70 cagcagtactattctagtcctcgaact VH 71 gaggtgcagctggcagaatcaggcgggggactggtccagcctggc ggcagcctgacactgtcttgcagtggatcaggcttcacttttagt aactatgcaatggtgtgggcaaggcaggctcctgggaagggactg gagtatgtctctggcatcgggcgcaacggggactctatctactat actgatagtgtgaagggccggttcacca tcagcagagacaatagc aaatccatggtgtacctgcagatgagctccctgcgaaccgaagac acagcagtgtactattgcgtgaaagatctggccatccccgagtcc tacagaattgaagctgattattggggacagggcaccctggtcatc gtgagcgccg VL ZKA64 CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL2 long CDRL3 VH 72 SEQ ID NO. 82 83 84 85 86 87 88 89 gacatcgtgatgacacagtctccagatagtctggcagtcagtctg ggggagagggccactattaactgcaagagctcccagtccgtgctg taccgctctaacaacaagaattacctgtcttggtatcagcagaag cccggacagccccctaaactgctgatctattgggcttcaacccgg gaaagcggcgtcccagacagattctcaggcagcgggtccggaaca gacttcaccctgacaattagccccctgcaggcagaggacgtggct gtctactattgtcagcagtactattctagtcc tcgaactttcggc caggggaccaaggtggaaatcaaac Amino acid sequences ggctacaccttcacaggtatcac attaaccctaattctggcgggacc gctcggatgagctcctctatttggggcttcgatcat cagtctgtgctgattaac ggagcatcc ctgatctatggagcatcctccagggct cagcagtacaatgattggccccctatcaca caggtgcagctggtccagagcggagcagaggtgaagaaacccggc gcctcagtgaaggtcagctgcaaagcttccggctacaccttcaca gggtatcacatcgactgggtgaggcaggcaagaggacagggactg gaatggatgggacggattaaccctaattctctggcgggaccaactac gcccagaagtttcagggccgagtgactatgaccaga gacaccagc atctccacagcttatatgcagctgtcccggctgagatctgacgat agtgccgtctactattgtgctcggatgagctcctctatttggggc ttcgatcattgggggcagggaacactggtgactgtcagttcag VL 90 gagatcgtgatgactcagtctccagccaccctgtcagtcagccca ggagaacgggcaaccctgtcttgcagagcctcccagtctgtgctg attaacctggcttggtaccagcagaagccaggccaggcaccccga ctgctgatctatggagcatcctccaggggctaccggcattcctgca cgcttcagtggatcaggaagcggaacaga gtttaccctgacaatc tctagtctgcagtccgaagacttcgctgtctactattgtcagcag tacaatgattggccccctatcacatttggccaggggactagactg gagatcaagc

Preferably, the sequence of the nucleic acid molecule of the present invention includes or consists of a nucleic acid sequence corresponding to the sequences of SEQ ID NO: 10-18, 28-36, 46-54, 64-72 and 82-90; or a functional variant of this sequence.

It is also preferred that the nucleic acid sequences of the present invention

- 42 043940 teniy were such nucleic acid sequences that are 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% identical to the nucleic acid, encoding CDR, VH sequence and/or VL sequence presented in table. 1 and 2, for example, to the sequences presented in table. 3.

In general, a nucleic acid molecule can be altered by insertion, deletion, or alteration of certain nucleic acid sequences. Changes from such manipulations include, but are not limited to, changes to introduce restriction sites, to change codon usage, to add or optimize transcriptional and/or translational control sequences, etc. It is also possible to modify the nucleic acid to change the encoded amino acids. For example, it may be useful to introduce one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions, deletions and/or insertions into the amino acid sequence of the antibody. Such point mutations may modify effector functions, antigen binding affinity, post-translational modifications, immunogenicity, etc., may introduce amino acids for attachment of covalent groups (eg, tags), or may introduce tags (eg, for purification purposes). Mutations may be introduced at specific sites or may be introduced randomly followed by selection (e.g. molecular evolution). For example, one or more nucleic acids encoding any of the CDR regions, the VH sequence and/or the VL sequence of a (typical) antibody of the present invention may be randomly or targetedly mutated to introduce different properties into the encoded amino acids. Such changes may be the result of a random process, with the original changes being maintained and new changes being introduced at other nucleotide positions. In addition, changes achieved at independent stages can be combined. Various properties introduced into the encoded amino acids may include, but are not limited to, increased affinity.

Another aspect of the present invention also relates to a plurality of nucleic acid molecules as described above, each nucleic acid molecule comprising at least one polynucleotide encoding an antibody fragment, or an antigen binding fragment thereof, of the present invention as described above. Preferably, a plurality of fragments encoded by a plurality of nucleic acid molecules form the antibody, or antigen binding fragment thereof, of the present invention. In other words, it is preferred that the plurality of nucleic acid molecules of the present invention encode a (complete) antibody, or a (complete) antigen binding fragment thereof, of the present invention. This means that, in particular, additional nucleic acid molecules (in addition to the plurality of nucleic acid molecules of the present invention) are not required to encode/produce the (complete) antibody, or (complete) antigen binding fragment thereof, of the present invention. For example, a plurality of nucleic acid molecules of the present invention encode all polypeptide chains of an antibody, or antigen binding fragment thereof, of the present invention.

A preferred example of the plurality of nucleic acid molecules of the present invention are (exactly) two nucleic acid molecules, wherein each of the nucleic acid molecules contains one or more than one (e.g., two or three) polynucleotides, and each polynucleotide encodes a (different) polypeptide chain an antibody, or an antigen-binding fragment thereof, of the present invention. Accordingly, the nucleic acid molecules of such a plurality of nucleic acid molecules are preferably mono- or bicistronic.

An example is two nucleic acid molecules encoding together a FIT-Ig antibody of the present invention as described above (e.g., one bicistronic nucleic acid molecule in combination with a corresponding monocistronic nucleic acid molecule, combined such that FIT polypeptides 1, 2, and 3 Igs are encoded by two nucleic acid molecules, as described above).

In another preferred embodiment, the present invention provides (exactly) three nucleic acid molecules, each of the nucleic acid molecules containing one polynucleotide encoding a (different) polypeptide chain of an antibody, or antigen binding fragment thereof, of the present invention. Accordingly, the nucleic acid molecules in such a plurality of nucleic acid molecules are preferably monocistronic. Examples are three nucleic acid molecules co-encoding the FIT-Ig antibody of the present invention as described above (e.g., three (e.g., monocistronic) nucleic acid molecules, one encoding polypeptide 1 of FIT-Ig, the second encoding polypeptide 2 of FIT -Ig and the third encodes polypeptide 3 from FIT-Ig, as described above).

Vector.

The present invention also describes vectors, such as expression vectors, containing a nucleic acid molecule of the present invention. Preferably, the vector contains mole

- 43 043940 nucleic acid kulu, as described above. The term vector refers to a nucleic acid molecule, preferably a recombinant nucleic acid molecule, that is, a nucleic acid molecule that does not occur in nature. A vector in the context of the present invention is useful for incorporating or storing a desired nucleic acid sequence. Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors, etc. A storage vector is a vector that provides convenient storage of a nucleic acid molecule. For example, the vector may contain a sequence corresponding to, for example, a desired antibody, or fragment of an antibody, of the present invention. An expression vector can be used to produce expression products, for example RNA, eg mRNA, or peptides, polypeptides or proteins. For example, an expression vector may contain sequences necessary for transcription of a portion of the vector sequence, such as a promoter sequence. A cloning vector is typically a vector that contains a cloning site useful for incorporating nucleic acid sequences into the vector. The cloning vector can be, for example, a plasmid vector or a bacteriophage vector. A transfer vector may be a vector that is useful for transferring nucleic acid molecules into cells or organisms, such as viral vectors. The vector in the context of the present invention can be, for example, an RNA vector or a DNA vector. Preferably the vector is a DNA molecule, for example plasmid DNA. For example, a vector in the context of the present invention contains a cloning site, a selectable marker, such as an antibiotic resistance trait, and a sequence useful for propagating the vector, such as an origin of replication. Preferably the vector in the context of the present invention is a plasmid vector.

Another aspect of the present invention also provides a plurality of vectors of the present invention, preferably encoding an antibody, or antigen binding fragment thereof, of the present invention. Accordingly, the preferred embodiments of the present invention and the examples of the plurality of nucleic acid molecules of the present invention described above are also applicable to a variety of vectors of the present invention.

Cells.

In another embodiment, the present invention also provides cells expressing an antibody, or an antigen binding fragment thereof, of the present invention; and/or comprising a vector of the present invention or a plurality of vectors of the present invention.

Examples of such cells include, but are not limited to, eukaryotic cells, such as yeast cells, animal cells or plant cells. Preferably, the cells are mammalian cells, more preferably mammalian cell lines. Preferred examples include human cells, CHO cells, HEK293T cells, PER.C6 cells, NS0 cells, human liver cells, myeloma cells or hybridoma cells.

In particular, a cell can be transfected with a vector of the present invention or a plurality of vectors of the present invention, preferably an expression vector or a plurality thereof. The term transfection refers to the introduction of nucleic acid molecules, such as DNA or RNA molecules (eg, mRNA), into cells, preferably eukaryotic cells. In the context of the present invention, the term transfection covers any method known to one skilled in the art for introducing nucleic acid molecules into cells, preferably eukaryotic cells, for example mammalian cells. Such methods include, for example, electroporation, lipofection, e.g., based on cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or transfection based on cationic polymers, e.g., DEAE-dextran or polyethylenimine, etc. .d. Preferably the introduction is not virus based.

Moreover, cells of the present invention can be stably or transiently transfected with a vector of the present invention or a plurality of vectors of the present invention, for example, to express an antibody, or an antigen binding fragment thereof, of the present invention. Preferably, cells are stably transfected with a vector of the present invention or a plurality of vectors of the present invention encoding an antibody, or an antigen binding fragment thereof, of the present invention. In another embodiment of the present invention, it is preferred that the cells are transiently transfected with a vector of the present invention or a plurality of vectors of the present invention encoding an antibody, or an antigen binding fragment thereof, of the present invention.

Optional additional properties of antibodies.

Antibodies of the present invention can be combined, for example, with a drug to be delivered to the site of treatment or associated with a detectable label to facilitate visualization of the site containing the cells of interest. Methods for binding antibodies to drugs and detectable labels are known to those skilled in the art, as are imaging techniques using detectable labels. Tagged antibodies can be used in a wide variety of assays using a wide variety of labels. Detection of antibody-antigen complex formation

- 44 043940 between the antibody of the present invention and the epitope of interest can be facilitated by attaching a detectable substance to the antibody. Suitable detection means include, for example, tracers such as radionuclides, enzymes, coenzymes, fluorophores, chemiluminescent compounds, chromogens, enzyme substrates or cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, and others. Examples of useful enzymes include horseradish peroxidase, alkaline phosphatase, P-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, fluorescein dichlorotriazinylamine, dansyl chloride or phycoerythrin; an example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, luciferin and aequorin; examples of suitable radioactive materials include ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ H. Such labeled reagents can be used in a variety of known assays, such as radioimmunoassays, enzyme-linked immunosorbent assays, eg ELISA, fluorescence immunoassays and others. Thus, the labeled antibodies of the present invention can be used in such assays, for example, as described in US patents 3,766,162; US 3791932; US 3817837; US 4233402.

The antibody of the present invention may be conjugated to a drug moiety, for example, a fragment of a therapeutic molecule, such as a cytotoxin, a therapeutic agent, or a radioactive metal or radioisotope. Examples of radioisotopes include, but are not limited to, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In111 and others. Such antibody conjugates can be used to modify a given biological response; the drug component should not be considered as limited to classical chemical therapeutic agents. For example, a fragment of a drug molecule may be a protein or polypeptide that has the desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, Pseudomonas aeruginosa exotoxin, or diphtheria toxin.

Methods for conjugating such a therapeutic moiety to antibodies are well known. See, for example, Arnon et al., in the book: Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld et al., 1985, (Alan R. Liss, Inc.), 243-256; Hellstrom et al. in the book: Controlled Drug Delivery, ed. Robinson et al., 1987, (2nd edition; Marcel Dekker, Inc.), 623-653; Thorpe, in: Monoclonal Antibodies '84: Biological and Clinical Applications, ed. Pinchera et al., 1985, 475-506 (Editrice Kurtis, Milan, Italy, 1985); in the book: Monoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin et al., 1985, (Academic Press, New York, 1985), 303-316; Thorpe et al., Immunol. Rev. 62, 1982, 119-158.

In another embodiment of the present invention, an antibody or antibody fragment can be conjugated to a second antibody, or a fragment thereof, to form an antibody heteroconjugate as described in US 4,676,980. Additionally, linkers can be used between tags and antibodies of the present invention, for example, according to described in US 4,831,175. Antibodies or antigen-binding fragments thereof can be directly labeled with radioactive iodine, indium, yttrium or other radioactive species known in the art, for example, as described in US 5,595,721. Treatment may consist of a combination of conjugated and unconjugated antibody treatments, administered simultaneously or sequentially, for example as described in WO 00/52031; WO 00/52473.

Antibodies of the present invention can also be attached to a solid support. Additionally, the antibodies of the present invention or functional fragments of these antibodies can be chemically modified by covalent conjugation to a polymer, for example, to increase their half-life in the bloodstream. Examples of polymers and methods for attaching peptides thereto are shown in US 4,766,106, US 4,179,337, US 4,495,285 and US 4,609,546. In some embodiments of the present invention, the polymers may be selected from polyoxyethylene polyols and polyethylene glycol (PEG). PEG is soluble in water at room temperature and has the general formula: R(O-CH2-CH2) _n OR, in which R can be hydrogen or a protecting group, such as an alkyl or alkanol group. Preferably, the protecting group may contain from 1 to 8 carbon atoms. For example, the protecting group is methyl. The letter n denotes a positive integer. In one embodiment of the present invention, n is a number from 1 to 1000. In another embodiment of the present invention, n is a number from 2 to 500. Preferably, the average molecular weight of PEG is from 1000 to 40,000, more preferably the molecular weight of PEG is from 2000 to 20000, even more preferably the molecular weight of PEG is from 3000 to 12000. In addition, PEG may contain at least one hydroxyl group, for example, PEG may contain one terminal hydroxyl group. For example, it is the terminal hydroxyl group that is activated to react with the free amino group in the inhibitor. However, it should be noted that the type and number of reactive groups may vary to achieve the covalently conjugated PEG/antibodies of the present invention.

Water-soluble polyoxyethylated polyols are also useful in the present invention. TO

- 45 043940 These include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG) and others. In one embodiment of the present invention, a POG is used. Without being bound by any theory, the authors suggest that since the glycerol backbone of the polyoxyethylated glycerol molecule is the same as in the natural molecule, for example, in animals and humans in mono-, di- and triglycerides, such branching would not necessarily be considered as foreign agents in the body. POG may have a molecular weight in the same range as PEG. Another drug delivery system that can be used to increase the half-life in the circulatory system is the liposome. Methods for preparing liposome delivery systems are known to those skilled in the art. Other drug delivery systems are known in the art and are described, for example, in references to Poznansky et al. (1980) and Poznansky (1984).

Antibodies of the present invention can be obtained in purified form. Typically, the antibody may be contained in a composition that is substantially free of other polypeptides, for example, less than 90% (by weight), typically less than 60%, and more often less than 50% of the composition consists of other polypeptides.

The antibodies of the present invention may be immunogenic in heterologous non-human hosts, such as mice. In particular, the antibodies may have an idiotope that is immunogenic in a non-human host but not in a human host. In particular, the antibodies of the present invention for use in humans include antibodies that are difficult to isolate from hosts, such as mice, goats, rabbits, rats, non-primate mammals, etc., and which are generally not can be obtained by humanization or from xeno-mice.

Pharmaceutical composition.

The present invention also provides a pharmaceutical composition comprising one or more of the following components:

(i) an antibody, or fragment of an antibody, of the present invention, (ii) a nucleic acid molecule or plurality of nucleic acid molecules of the present invention, (iii) a vector or plurality of vectors of the present invention, and/or (iv) cells of the present invention .

In other words, the present invention also provides a pharmaceutical composition comprising an antibody, or an antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a plurality of nucleic acid molecules of the present invention, a vector of the present invention, a plurality of vectors of the present invention and/or cells according to the present invention.

The pharmaceutical composition may preferably also contain a pharmaceutically acceptable carrier, diluent and/or excipient. Although the carrier or excipient may facilitate administration, it should not, in itself, induce the production of antibodies harmful to the individual to whom the composition is administered. Besides. They must not be toxic. Suitable carriers may be large, slowly metabolized macromolecules, for example, proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive viral particles. In general, pharmaceutically acceptable carriers in the pharmaceutical composition of the present invention may be active ingredients or inactive ingredients. Preferably, the pharmaceutically acceptable carrier in the pharmaceutical composition of the present invention is not an active component against Zika virus infection.

Pharmaceutically acceptable salts may be used, for example salts of mineral acids, for example hydrochlorides, hydrobromides, phosphates and sulfates, or salts of organic acids, for example acetates, propionates, malonates and benzoates.

Pharmaceutically acceptable carriers in the pharmaceutical composition may further comprise liquids such as water, saline, glycerin and ethanol. In addition, such compositions may contain auxiliary substances, for example wetting or emulsifying agents or pH buffering agents. Such carriers allow pharmaceutical compositions to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions and slurries for administration by the subject.

The pharmaceutical compositions of the present invention can be prepared in various forms. For example, the compositions may be formulated as injectables, either as liquid solutions or suspensions. Solid forms may also be prepared, which may be in the form of a solution or suspension, in liquid carriers prepared before injection (eg, lyophilized composition such as Synagis™ and Herceptin™, for reconstitution with sterile water containing a preservative). The composition may be prepared for topical administration, for example, in the form of an ointment, cream or powder. The composition may be prepared for oral administration, for example, in the form of tablets or capsules, in the form of a spray or in the form of a syrup (optionally flavored). The composition can be prepared for administration into the lungs, for example, in the form of an inhaler, using

- 46 043940 using fine powder or spray. The composition can be prepared in the form of a suppository or pessary. The composition can be prepared for nasal, ear or ocular administration, for example, in the form of drops. The composition may be in the form of a kit formulated such that the combination composition is reconstituted immediately prior to administration to the subject. For example, the lyophilized antibody may be presented in the form of a kit with sterile water or sterile buffer.

Preferably, the active ingredient in the composition is an antibody molecule, antibody fragment, variant or derivative thereof, particularly that the active ingredient in the composition is an antibody, antibody fragment or variants and derivatives thereof of the present invention. The active ingredient may be subject to degradation in the gastrointestinal tract. Thus, if the composition is to be administered through the gastrointestinal tract, the composition may contain agents that protect the antibody from degradation, but which release the antibody after it is absorbed in the gastrointestinal tract.

For a detailed discussion of pharmaceutically acceptable carriers, see Gennaro Remington: The Science and Practice of Pharmacy, 2000, 20th ed., ISBN: 0683306472.

The pharmaceutical compositions of the present invention typically have a pH of from 5.5 to 8.5, in some embodiments of the present invention it can be from 6 to 8, in other embodiments of the present invention about 7. The pH value can be maintained through the use of a buffer. The composition may be sterile and/or pyrogenic. The composition may be isotonic in relation to humans. In one embodiment of the present invention, the pharmaceutical compositions of the present invention are supplied in sealed containers.

Within the scope of the present invention are compositions formulated in several forms of administration; forms include, but are not limited to, forms suitable for parenteral administration, for example, by injection or infusion, such as bolus injection or continuous infusion. If the product is intended for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and may contain formulation agents, such as suspending, preservative, stabilizing and/or dispersing agents. In another embodiment of the present invention, the antibody molecule may be in dry form and reconstituted with a suitable sterile liquid before use. By solvent is generally meant a material that is suitable for storing, transporting and/or administering a compound, for example a pharmaceutically active compound, in particular an antibody of the present invention. For example, the solvent may be a physiologically acceptable liquid that is suitable for storing, transporting and/or administering a pharmaceutically active compound, in particular the antibodies of the present invention. Once the composition of the present invention has been formulated, it can be administered directly to the subject. In one embodiment of the present invention, the compositions are adapted for administration to mammals, such as humans.

The pharmaceutical compositions of the present invention can be administered by numerous routes, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transdermal, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal, or rectal. . Hyposprays (needleless injectors) can also be used to administer the pharmaceutical compositions of the present invention. Preferably, the pharmaceutical composition may be prepared for oral administration, for example, in the form of tablets, capsules and other forms for topical use, or in injectable forms, for example, in the form of liquid solutions or suspensions, and it is especially preferable if the pharmaceutical composition is injectable a drug. Solid forms suitable for dissolution or suspension in liquid injection carriers are also preferred, for example, a pharmaceutical composition in lyophilized form.

For injection, for example, intravenous, dermal or subcutaneous injection, or injection into a lesion, the active ingredient will preferably be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the art can prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactose Ringer's injection. If necessary, preservatives, stabilizers, buffers, antioxidants and/or other additives may be added. Whether a polypeptide, peptide or nucleic acid molecule, another pharmaceutically useful compound of the present invention, it is preferably administered to an individual in a prophylactically effective amount or in a therapeutically effective amount (as appropriate) sufficient to produce an effect beneficial to the individual. The actual amount administered, as well as the rate and duration of administration, will depend on the nature and severity of the condition being treated. For injection, the pharmaceutical composition of the present invention may be presented, for example, in a pre-filled syringe.

- 47 043940

The pharmaceutical composition of the present invention, as described above, can also be administered orally in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of oral tablets, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also commonly added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral administration, the active ingredient, that is, the molecule-cargo conjugate of the present invention as described above, is combined with emulsifying and suspending agents. Certain sweeteners, flavors or colors may also be added if desired.

The pharmaceutical composition of the present invention can also be administered topically, especially when the target of treatment includes areas or organs that are easily accessible for topical application, for example, including diseases of the skin or any other accessible epithelial tissue. Formulations suitable for topical application can be readily prepared for such areas or organs. For topical administration, the pharmaceutical composition of the present invention can be formulated as a suitable ointment containing the pharmaceutical composition of the present invention, especially the components described above, suspended or dissolved in one or more carriers. Carriers for topical use include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. In another embodiment of the present invention, the pharmaceutical composition of the present invention can be formulated as a suitable lotion or cream. In the context of the present invention, suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, wax cetyl esters, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

The dosage of treatment can be carried out according to a single-dose regimen or a multiple-dose regimen. In particular, the pharmaceutical composition may be presented in a single dose. Preferably, the amount of antibody in the pharmaceutical composition - particularly if provided as a single dose - does not exceed 200, more preferably does not exceed 100 and even more preferably does not exceed 50 mg.

For example, the pharmaceutical composition of the present invention may be administered daily, for example one to several times a day, for example one, two, three or four times a day, preferably once or twice a day, more preferably once a day for for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days or more, for example, daily for over 1, 2, 3, 4, 5, 6 months. Preferably, the present invention can be administered weekly, for example, once or twice a week for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20 or 21 weeks or more, for example, weekly for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or weekly for 2, 3, 4 or 5 years. Moreover, the pharmaceutical composition of the present invention may preferably be administered monthly, for example once a month or more preferably every other month for 1, 2, 3, 4 or 5 or more years. It is also preferable that administration be lifelong. In addition, only one administration is also envisaged, particularly for certain indications, for example, to prevent infection with Zika virus through accidental exposure, for example, in subjects who have not been immunized.

However, the most preferred treatment regimen is post-exposure prophylaxis (PEP), in which one or more single doses are administered as soon as possible after Zika virus infection. A prophylactic setting in which one or more single doses are administered to prevent Zika infection (ie, prior to Zika virus infection, especially in individuals not immunized against Zika virus) is also preferred.

In particular, it is preferred that for a single dose, such as a daily, weekly or monthly dose, preferably for a weekly dose, the amount of antibody, or antigen binding fragment thereof, in the pharmaceutical composition of the present invention does not exceed 1 g, preferably does not exceed 500 mg, more preferably does not exceed 200, even more preferably does not exceed 100, and especially preferably does not exceed 50 mg.

Pharmaceutical compositions typically include an effective amount of one or more antibodies of the present invention, that is, an amount sufficient to treat, improve, alleviate the symptoms of, or prevent a particular disease or condition, or to exhibit an apparent therapeutic effect. Therapeutic effects also include reducing or mitigating the severity of the disease or symptoms of the disease. The exact effective amount for any particular subject will depend on the size, weight and health status, the nature of the disease and its severity, and the therapeutic agents or combination of therapeutic agents selected for administration. The effective amount for a given situation is determined by routine experimentation and is at the discretion of the physician. For purposes of the present invention, the effective dose is typically from about 0.005 to about 100, preferably from about 0.0075 to about 50, more preferably from about 0.01 to about 10, and even more preferably from about 0.02 to about 5 mg/kg of an antibody of the present invention (eg, the amount of antibody in a pharmaceutical composition) relative to the body weight (in kg) of the individual being dosed.

Moreover, the pharmaceutical composition of the present invention may also include an additional active component, which may be an additional antibody or non-antibody component. The additional active component is preferably an immune checkpoint inhibitor. It is also preferable that a ZIKV neutralizing antibody, or an antigen binding fragment thereof, described in the present invention has been combined with a ZIKV NS1 binding antibody, or an antigen binding fragment thereof, as described in the present invention as an additional active component (co-agent). Thus, the pathogenic role of NS1 may be blocked in addition to neutralizing ZIKV. The pharmaceutical composition of the present invention may contain one or more additional active ingredients, for example those described below as co-agents in the context of combination therapy.

The antibody, or antigen-binding fragment thereof, of the present invention may be contained either in the same pharmaceutical composition as the additional active ingredient, or, preferably, the antibody, or antigen-binding fragment thereof, of the present invention consists of a first pharmaceutical composition, and the additional active ingredient is included in the composition of a second pharmaceutical composition different from the first pharmaceutical composition. Accordingly, if more than one additional active ingredient is provided, each additional active ingredient and the antibody, or antigen binding fragment thereof, of the present invention are preferably formulated in different pharmaceutical compositions. Such different pharmaceutical compositions can be administered either in combination/simultaneously, or at different times, or in different parts (eg, different parts of the body).

Preferably, the antibody, or antigen binding fragment, of the present invention and the additional active component provide an additional therapeutic effect or, preferably, a synergistic therapeutic effect. The term synergism is used to describe the combined effect of two or more active agents that is greater than the sum of the individual effects of each respective active agent. Thus, when the combined action of two or more agents results in synergistic inhibition of an activity or process, the inhibition of the activity or process is meant to be greater than the sum of the inhibitory effects of each respective active agent. The term synergistic therapeutic effect refers to the therapeutic effect observed when two or more therapies are combined, wherein the therapeutic effect (as measured by any one of a number of parameters) is greater than the sum of the individual therapeutic effects observed from the respective individual treatments.

A pharmaceutical composition comprising an antibody corresponding to gZKA190, gZKA64, gZKA230, gZKA185, gZKA78 or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier is preferred.

In one embodiment of the present invention, the composition of the present invention may include antibodies of the present invention, and the antibodies may constitute at least 50% by weight (e.g., 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 wt.% or more) by weight of the total protein in the composition. In such a composition, the antibodies are preferably in purified form.

The present invention also provides a method for producing a pharmaceutical composition, comprising the steps of: (i) producing an antibody of the present invention; and (ii) mixing the purified antibody with one or more pharmaceutically acceptable carriers.

In another embodiment of the present invention, a method for preparing a pharmaceutical composition includes the step of mixing an antibody with one or more pharmaceutically acceptable carriers, wherein the antibody is a monoclonal antibody that is obtained from a transformed B cell or cultured plasma cell of the present invention.

As an alternative to delivering antibodies or B cells for therapeutic purposes, a nucleic acid (usually DNA) that encodes the desired monoclonal antibody (or active fragment thereof) derived from B cells or cultured plasma cells can be delivered to a subject, which nucleic acid can be expressed in the subject's body in situ to provide the desired therapeutic effect. Appropriate gene therapy and nucleic acid delivery vectors are known in the art.

Pharmaceutical compositions may include an antimicrobial agent, especially if they are packaged in a multi-dose format. The pharmaceutical compositions may include a detergent, for example, Tween (polysorbate), for example, Tween 80. The concentration of detergents is usually low, for example, less than 0.01%. The compositions may also include sodium salts (eg, sodium chloride) to impart tonicity. For example, the usual concentration of NaCl is 10 ± 2 mg/ml.

- 49 043940

In addition, the pharmaceutical compositions may contain a sugar alcohol (eg, mannitol) or disaccharide (eg, sucrose or trehalose), for example, in an amount of about 15-30 mg/ml (eg, 25 mg/ml), especially if they are to be lyophilized or if they include material that has been reconstituted from lyophilized material. The pH of the lyophilization composition may be adjusted to a value of 5 to 8, or 5.5 to 7, or about 6.1 prior to lyophilization.

The compositions of the present invention may also contain one or more immunoregulatory agents. In one embodiment of the present invention, one or more of the immunoregulatory agents includes an adjuvant.

Medical procedures, kits and applications.

Medical procedures.

In another embodiment, the present invention provides the use of an antibody, or antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, cells of the present invention, or the pharmaceutical composition of the present invention for (i) preventing and/or treating Zika virus infection; or to (ii) diagnose Zika virus infection. Thus, the use of an antibody, or an antigen binding fragment thereof, in accordance with the present invention (and especially preferred embodiments thereof as described above), or (a plurality of) nucleic acid molecules of the present invention encoding an antibody, or an antigen binding fragment thereof, according to the present the invention is preferred for (i) preventing and/or treating Zika virus infection as described above; or to (ii) diagnose Zika virus infection as described above.

Diagnostic methods may involve contacting an antibody or antibody fragment with a sample. Such samples can be isolated from the subject, for example, from a tissue sample, for example, from the nasal passages, sinuses, salivary glands, lungs, liver, pancreas, kidneys, ears, eyes, placenta, gastrointestinal tract, heart, ovaries , pituitary gland, adrenal gland, thyroid gland, brain, skin or blood, preferably plasma or serum. Diagnostic methods may also involve detection of the antigen/antibody complex, particularly after contact of the antibody or antibody fragment with a sample. This detection stage is usually performed on a bench, that is, without any contact with the human or animal body. Examples of detection methods are well known to those skilled in the art and include, for example, the ELISA (enzyme-linked immunosorbent assay) method.

Prevention of Zika virus infection refers specifically to preventive measures when the subject has not been diagnosed with Zika virus infection (either not diagnosed or tested negative) and/or the subject is not exhibiting symptoms of Zika virus infection . Accordingly, prevention of Zika virus infection includes post-exposure prophylaxis (PEP), which is prophylactic treatment following a possible Zika virus infection, such as a mosquito bite in a Zika virus affected area. Prevention of Zika virus infection is particularly beneficial for high-risk subjects, such as those who are pregnant and/or subjects in areas affected by Zika virus (eg, subjects living in areas affected by Zika virus or traveling to areas affected by Zika virus) .

In contrast, in therapeutic settings, the subject is typically infected with Zika virus, has been diagnosed with Zika virus infection, and/or is exhibiting symptoms of Zika virus infection. It should be noted that the terms treatment and therapy/therapeutic ZIKV infections include (complete) cure as well as attenuation of ZIKV infection.

Accordingly, an antibody, or antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, a cell of the present invention, or a pharmaceutical composition of the present invention is preferably are used to treat Zika virus infection in subjects who have been diagnosed with Zika virus infection or in subjects with symptoms of Zika virus infection.

It is also preferred that an antibody, or antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, cells of the present invention, or a pharmaceutical composition of the present invention The invention is used to prevent and/or treat Zika virus infection in asymptomatic subjects. These subjects may or may not have been diagnosed with Zika virus infection.

Preferably, an antibody, or antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention

- 50 043940 of the invention, the cells of the present invention or the pharmaceutical composition of the present invention are used for the prevention and/or treatment of infection caused by the Zika virus in pregnant women, in particular for the prevention of congenital infection. For example, this can be performed in the same way as for the prevention of congenital HCMV infection as described in Nigro G. et al, N Engl J Med 353, 2005, 1350-1362.

Without wishing to be bound by any theory, it is believed that the antibody, or antigen binding fragment thereof, of the present invention can cross the placenta upon interaction with FcRn, for example, if administered to a pregnant woman, for example, by (intravenous) injection or some other route administration according to the description of the present invention. It is important that the interaction of LALA antibody variants as described herein with FcRn is not compromised. It is believed that FcRn is already expressed in the first trimester in the placenta.

In another embodiment, the antibody, or antigen binding fragment thereof, or (multiple) nucleic acid molecules of the present invention can also be introduced into the extra-amniotic space.

Preferably, an antibody, or antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, cells of the present invention, or a pharmaceutical composition of the present invention is applicable for the prevention and/or treatment of infection caused by the Zika virus, wherein the antibody, or antigen binding fragment thereof, nucleic acid, vector, plurality of nucleic acids, plurality of vectors, cells or pharmaceutical composition is administered within seven days after the (possible) infection with the Zika virus, up to up to five days after (possible) Zika virus infection, more preferably up to four days after (possible) Zika virus infection, even more preferably up to three days after (possible) Zika virus infection, and most preferably within one to two days after (possible) Zika virus infection. This treatment regimen may be useful for both therapeutic and prophylactic purposes, particularly post-exposure prophylaxis (PEP).

In PPC, typically the first administration of an antibody, or antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, cells of the present invention, or a pharmaceutical composition of the present invention. The present invention is carried out as soon as possible after a possible ZIKV infection, for example, after a mosquito bite in a ZIKV affected area. Accordingly, in PPC, the first administration of an antibody, or antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, cells of the present invention, or a pharmaceutical composition the present invention is usually carried out no later than one or more days after (possible) ZIKV infection, as described above.

It is also preferred that an antibody, or antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, cells of the present invention, or a pharmaceutical composition of the present invention The invention is used to prevent and/or treat Zika virus infection, wherein the antibody, or antigen binding fragment, nucleic acid, vector, plurality of nucleic acids, plurality of vectors, cells or pharmaceutical composition is administered three months before (possible) Zika virus infection, preferably before a month before (possible) Zika virus infection, more preferably two weeks before (possible) Zika virus infection, even more preferably a week before (possible) Zika virus infection, and most preferably one day before (possible) Zika virus infection. This treatment regimen, in particular, refers to preventive measures.

In general - and in particular in PPC - after the first administration of an antibody, or antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention , cells of the present invention, or a pharmaceutical composition of the present invention, may be followed by one or more subsequent administrations, preferably one dose once or twice daily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 1, 15, 16, 17, 18, 19, 20 or 21 days.

It is also preferred that after the first administration of an antibody, or antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, cells of the present invention, or a pharmaceutical composition of the present invention may be followed by one or more subsequent administrations, preferably one dose once or twice a week for 1, 2, 3, 4, 5, 6, 7, 8, 9,

10, 11, 12, 13, 1, 15, 16, 17, 18, 19, 20 or 21 weeks.

It is also preferred that after the first administration of an antibody, or antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, cells of the present invention, or a pharmaceutical composition of the present invention may be followed by one or more subsequent administrations, preferably one dose every two or four weeks for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1 , 15, 16, 17, 18, 19, 20 or 21 weeks. It is also preferred that after the first administration of an antibody, or antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, cells of the present invention, or a pharmaceutical composition of the present invention may be followed by one or more subsequent administrations, preferably one dose every two or four months for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1 , 15, 16, 17, 18, 19, 20 or 21 months. It is also preferred that after the first administration of an antibody, or antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, cells of the present invention, or a pharmaceutical composition of the present invention may be followed by one or more subsequent administrations, preferably one dose once or twice a year for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years.

Preferably, an antibody, or antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, a cell of the present invention, or a pharmaceutical composition of the present invention is administered in a (single) dose from 0.005 to 100, preferably in a (single) dose from 0.0075 to 50, more preferably in a (single) dose from 0.01 to 10, even more preferably in a (single) dose from 0.05 to 5, and especially preferably in a (single) dose of 0.1 to 1 mg/kg body weight.

An antibody, or antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, a cell of the present invention, or a pharmaceutical composition of the present invention can be administered by any route, such as oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transdermal, topical, intranasal, enteral, sublingual, intravaginal, or rectal. Intravenous administration or subcutaneous administration or intramuscular administration are preferred, and intravenous administration or subcutaneous administration are more preferred.

To pregnant women, the antibody, or antigen-binding fragment thereof, or nucleic acid molecule(s) of the present invention may also be administered by the intra- or extra-amniotic route, such as by injection.

Thus, the present invention also provides a method of preventing and/or treating infection caused by the Zika virus, which method includes administering to a subject in need thereof an antibody, or an antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, cells of the present invention, or a pharmaceutical composition of the present invention. Preferred embodiments of such a method correspond to the preferred embodiments of the present invention relating to medical use as described above (and below regarding combination therapy). For example, a preferred subject in this method is a subject who has been diagnosed with Zika virus infection. Other preferred subjects in this method are pregnant women.

Combination therapy.

Incorporating an antibody, or an antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, cells of the present invention, or a pharmaceutical composition of the present invention into methods and the uses of the present invention can be performed alone or in combination with concomitant agents (also referred to in the present invention as a co-active component) which are particularly useful for the prevention and/or treatment of ZIKV infection.

The present invention covers the administration of an antibody, or an antigen-binding fragment thereof,

- 52 043940 of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, cells of the present invention, or a pharmaceutical composition of the present invention, wherein administration is carried out to the subject before, concomitantly or following other treatment regimens or treatment with concomitant agents useful in the treatment and/or prevention of ZIKV infection. The specified antibody, nucleic acid, vector, cell or pharmaceutical composition, which is administered simultaneously with the specified concomitant agents, can be administered in the same or different compositions and by the same or different routes of administration.

These other treatment regimens or concomitant agents may be, for example, a checkpoint inhibitor.

Thus, in another embodiment of the present invention, an antibody, or antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, cells of the present invention or a pharmaceutical composition of the present invention is administered in combination with a checkpoint inhibitor for (medical) use as described in the present invention.

Preferred checkpoint inhibitors are aimed at blocking PD-1/PD-L1 and/or CTLA4 and thus include anti-PD-1 antibodies, anti-PD-L1 antibodies and anti-CTLA4 antibodies. Thus, the pharmaceutical composition of the present invention may include one or more additional active ingredients.

It is also preferred that a ZIKV neutralizing antibody, or an antigen binding fragment thereof, as described herein is combined with a ZIKV NS1 binding antibody, or an antigen binding fragment thereof, as an additional active component (co-agent). Thus, the pathogenic role of NS1 may be blocked in addition to neutralizing ZIKV. Accordingly, a ZIKV NS1 binding antibody, or an antigen binding fragment thereof, such as those described in PCT/EP2017/067581, incorporated by reference herein, is a preferred active component (co-agent).

An antibody, or an antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, or cells of the present invention may be in one pharmaceutical composition as an additional active component (co-agent), or, preferably, an antibody, or an antigen-binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, or the cells of the present invention are contained in a first pharmaceutical composition, and the additional active ingredient (co-agent) is contained in a second pharmaceutical composition different from the first pharmaceutical composition. Accordingly, if more than one additional active ingredient (co-agent) is used, each additional active ingredient (co-agent) is preferably included in a different pharmaceutical composition. Such different pharmaceutical compositions may be administered in combination/simultaneously, or separately at different times, or in different locations (eg, to different parts of the body).

Preferably, the antibody, or antigen binding fragment thereof, of the present invention and the additive active component (co-agent) provide an additive therapeutic effect, or preferably a synergistic therapeutic effect. The concept of synergy is used to describe the combined effect of two or more active agents, the combined effect of which exceeds the sum of the individual effects of each respective active agent. Thus, when the combined action of two or more agents results in synergistic inhibition of an activity or process, the inhibition of the activity or process is meant to be greater than the sum of the inhibitory effects of each respective active agent. The term synergistic therapeutic effect refers to the therapeutic effect observed when two or more therapeutic agents are combined, wherein the therapeutic effect (as measured by any number of parameters) is greater than the sum of the individual therapeutic effects observed with the respective individual therapies.

Additional applications and kits.

In another embodiment, the present invention provides the use of an antibody, or antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, cells of the present invention, or pharmaceutical composition of the present invention for monitoring the quality of a vaccine against the Zika virus by verifying that the antigen of said vaccine contains

- 53 043940 specific epitope in the correct conformation. Preferred antigens included in such Zika vaccine to be tested include the ZIKV envelope protein or some other molecule/complex containing or consisting of (i) ZIKV E protein domain III (EDIII) as described above, or (ii) ) ZIKV quaternary epitope as described above.

Moreover, the present invention also provides the use of an antibody, or an antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, cells of the present invention, or pharmaceutical composition of the present invention (in vitro) for diagnosing Zika virus infection.

In addition, the use of an antibody, or an antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, cells of the present invention, or a pharmaceutical composition of the present invention is provided. the present invention to determine whether a blood sample (eg, whole blood, serum and/or plasma) is infected with Zika virus.

As described above, diagnostic methods may involve contacting an antibody or antibody fragment with a sample. Such samples may be obtained from the subject, for example, from an isolated tissue sample taken, for example, from the nasal passages, sinuses, salivary glands, lungs, liver, pancreas, kidneys, ears, eyes, placenta, gastrointestinal tract, heart , ovaries, pituitary gland, adrenal glands, thyroid gland, brain, skin or blood, serum or plasma. Diagnostic methods may also include detection of the antigen/antibody complex, in particular after contact of the antibody or antibody fragment with the sample. This detection stage is usually performed on a bench, that is, without any contact with the human or animal body. Examples of detection methods are well known to those skilled in the art and include, for example, the ELISA (enzyme-linked immunosorbent assay) method.

In another embodiment, the present invention also provides a set of parts comprising at least one antibody, or antigen binding fragment thereof, of the present invention, at least one nucleic acid of the present invention, a plurality of nucleic acids of the present invention, at least , one vector of the present invention, a plurality of vectors of the present invention, at least one cell of the present invention, and/or at least one pharmaceutical composition of the present invention. In addition, the kit may include means of administering an antibody, or antigen binding fragment thereof, of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a plurality of nucleic acid molecules of the present invention, a plurality of vectors of the present invention, cells of the present invention, or a pharmaceutical composition of the present invention, for example, a syringe or vial, insert and/or accompanying agent, intended for administration as described above.

Description of the figures

Fig. 1. Reactivity (by ELISA) and neutralizing activity of ZIKV and DENV1 by antibodies obtained from four immune donors of ZIKV (ZKA, ZKB, ZKC and ZKD) to the E protein of ZIKV and DENV14, as well as to the EDIII domain of the E protein of ZIKV; NNB - neutralizing, non-E-protein binding antibodies.

Fig. 2. Binding of antibodies ZKA190, ZKA78 and ZKA64 to ZIKV E proteins and DENV1 to ZIKV EDIII according to ELISA analysis.

Fig. 3. Binding of antibodies ZKA185 and ZKA190 to ZIKV E, DENV1 VLP and ZIKV EDIII proteins according to ELISA analysis.

Fig. 4. Example 3 shows the neutralizing activity of antibodies ZKA190, ZKA64, ZKA64LALA, ZKA230 and ZKA78 against ZIKV (strain H/PF/2013) and DENV1 on Vero cells according to liquid cytometry (% of infected cells).

Fig. 5. Example 3 shows the neutralizing activity of antibodies ZKA190, ZKA64, ZKA185, ZKA230 and ZKA78 against ZIKV (strain H/PF/2013) on Vero cells, measured by cell viability reading (wst-1, Roche).

Fig. 6. In example 4, the enhancing infectious activity (ADE, antibody-dependent enhancement) of antibodies ZKA190, ZKA64, ZKA64-LALA, ZKA185, ZKA230 and ZKA78 to ZIKV (strain H/PF/2013) is shown on non-permissive K562 cells according to liquid cytometry (% infected cells).

Fig. 7. Example 4 shows that four ZIKV immune plasmas and one DENV immune plasma exhibit similar ability to enhance ZIKV infection of K562 cells (top panel). This effect of ADE was completely blocked in all five immune plasmas by the EDIII-specific antibody ZKA64-LALA (lower panel).

Fig. 8. Amino acid alignment of the EDIII region of 39 ZIKV strains from the Asian lineage from 2013

- 54 043940 (including the prototype strain MR766 of the African line, isolated in 1947).

Fig. 9. Example 3 shows the neutralizing activity of antibodies ZKA190 and ZKA190-LALA against three strains of ZIKV (H/PF/2013, MR766 and MRS_OPY_Martinique_PaRi_2015) on Vero cells according to liquid cytometry (% of infected cells).

Fig. 10. Example 5 shows neutralization of mAbs ZKA190 and C8 tested against a panel of four ZIKV strains as determined by the percentage of Vero cells infected in the presence of increasing amounts of mAb (A). IC ₅₀ values (B) and statistics (C) are also shown. Data are representative of at least two independent experiments.

Fig. 11. Example 6 shows the neutralization and enhancement of ZIKV infection by the ZKA190 antibody. (A) Neutralization of infection of ZIKV PRVABC59 hNPC strains by monoclonal antibodies (mAbs) ZKA190, ZKA190-LALA, and control mAb as determined by plaque assay on Vero cells (left panel) and indirect immunofluorescence of infected hNPCs using fluorophore-labeled anti-E protein antibodies (right panel). panel). (B) ADE of ZIKV infection in nonpermissive K562 ZKA190 and ZKA190-LALA cells. (B) ADE is induced in K562 cells when ZIKV is preincubated with serial dilutions of plasma serum from different ZIKV-positive patients (left panel). When ZKA190 LALA is added to ZIKV-serum complexes, ADE is inhibited (right panel). (D) ADE is induced in K562 cells when ZIKV is preincubated with serial dilutions of prM cross-reactive mAb (DV62) obtained from a DENV-immune donor. ZKA190-LALA inhibits ZIKV ADE upon formation of a complex with the prM-reactive antibody DV62. (E) Effect on ADE caused by peak enhancement by diluting plasma DENV2 (left panel) or anti-prM DV62 mAb (right panel) with serial dilutions of the indicated mAbs.

Fig. 12. Example 7 shows the identification of the ZKA190 epitope and analysis of its conservation in ZIKV strains.

(A) Overlay of [ ¹⁵ N,1H]-HSQC ¹⁵ N-labeled ZIKV EDIII spectra in the absence (black) or presence (red) of unlabeled Fab ZKA190. The differences are determined by the EDIII residues that are affected by antibody binding.

(B) NMR epitope mapping of Fab ZKA190 in complex with ZKV EDIII. Chemical shift perturbation (CSP, Y-axis) is plotted against the EDIII residue number. Residues affected by antibody binding are highlighted in red.

(B) Residues in the FG loop, identified by NMR epitope mapping, partially buried by the E protein in molecules A but largely exposed in molecules B and C. The EDIII of the E protein is colored blue. Residues identified by NMR epitope mapping are colored magenta, except those in the FG loop are colored green. Adjacent E proteins are shown as a gray surface.

(D) Level of amino acid residue conservation in the ZKA190 epitope calculated by analyzing sequences from 217 ZIKV strains found in the ZIKV Resources (NCBI) databases as of November 24, 2016.

(E) Open book view showing charge complementarity between epitope and paratope as determined by docking. The epitope and paratope boundaries are outlined in green. The boundaries between the Fab heavy and light chains and their corresponding recognition region on EDIII are shown by yellow dashed lines.

Fig. 13. Example 7 shows the ZKA190 epitope identified by NMR and docking. (A) Schematic representation of the 12 lowest energy NMR structures of ZIKV EDIII, with residues affected by ZKA190 binding in red. Flexibility at the N-terminus of the design is evident. (B) Model of the ZKA190:EDIII complex obtained by computational docking and molecular modeling and confirmed by NMR results. The NMR-identified epitope on EDIII (gray) is highlighted in red. The ZKA190 heavy and light chains are dark and light green, respectively. EDIII residues that do or do not affect antibody binding as a result of mutation are highlighted in orange and blue, respectively. (B) NMR-identified ZKA190 epitope (red) is accessible on the virus surface (white).

Fig. 14. Examples 7 and 10 show the binding of wt or mutated EDIII to ZKA190 IgG. SPR data and binding kinetics are shown. EDIII mutants that do or do not affect (in red) binding are marked.

Fig. 15. Example 8 shows the results of confocal microscopy experiments. ZIKV incubated with Fab ZKA190 or total IgG at a concentration of 10,000 times the IC50 is added to Vero cells. The ZIKV:antibody complex is found inside cells (green); the complex is colocalized with endosomes (red and yellow overlay). Endosomes and acidic organelles are marked with Lysotracker red; green dye Alexa488 conjugated to ZKA190. Nuclei are stained with DAPI (blue).

Fig. 16. Example 9 demonstrates the prophylactic and therapeutic effectiveness of ZKA190. (A) ZKA190 effectively protects against ZIKV infection when administered prophylactically to mice (A129 in (A) and AG129 in (B)) challenged with a lethal dose of ZIKV strain MP17451. In experiments

- 55 043940 there were 4-8 mice in groups. Kaplan-Meier survival curves are shown (A).

Significance was determined using the Mantel-Cox log series test. Panel A, top left: ZKA190 at 5, 1, and 0.2 mg/kg versus Ctr mAb, P=0.0031; ZKA190 at 0.04 mg/kg versus control mAb, P=0.0116; ZKA190-LALA at 5, 1, 0.2 and 0.04 mg/kg versus control mAb, P=0.0031. Panel A, top right: incidence rate of mice observed for 14-15 days (using two different assessment methods; see (Dowall S.D. et al., PLoS Negl Trop Dis 10, 2016, e0004658-13). Panel A, panels bottom: body weight of mice Panel B: ZKA190 or ZKA190LALA administered at 15 mg/kg at various time points after ZIKV infection Panel B, top left: Kaplan-Meier survival curve shown. Experiments used 5 mice per group. Significance determined using the log Mantel-Cox test ZKA190 and ZKA190-LALA given on days 1, 2, 3 or 4 versus control, P=0.0016 Panel B, top right: Morbidity rate of mice monitored for 14 days according to with Dowall et al., 2016. Mice are monitored for 14 days for weight loss (panel B, bottom panels) using the RSV E protein-specific antibody MPE8 as a control (Corti D. et al., Nature 501, 2013 , 439-443).

Fig. 17. Example 9 demonstrates the prophylactic effectiveness of the anti-ZIKV EDS-specific mAb ZKA190 against ZIKV MP1741 strains. (A) Viremia measured as PFU/mL on day 5 in the blood of all animals is shown. (B) Viral load is measured as genomic copies/ml by qPCR on day 5 in the blood of all animals and in the blood and specific tissues when animals are collected at the end of the study or when humane endpoints are reached. (B) Mice are monitored over a 14-day period to determine body weight loss. (D) Day 5 human serum IgG concentrations in blood samples. Significance was determined in comparison to control antibody treatment using the nonparametric unpaired Mann-Whitney U test. *p<0.05; **p<0.01; ***p<0.001.

Fig. 18. Example 9 demonstrates the therapeutic efficacy of the anti-ZIKV EDIII-specific mAb ZKA190. (A) Viral loads are measured as PFU on day 5 in the blood of all animals. (B) Viral loads are measured as genome copies by qPCR on day 5 in the blood of all animals and in specific tissues when animals are collected at the end of the study or when humane endpoints are reached. Significance was determined by comparison with control antibody treatment using the nonparametric unpaired Mann-Whitney U test. *p<0.05; **p<0.01. (B) Human serum IgG concentration on day 5.

Fig. 19. Examples 11 and 12 describe the incorporation of ZKA190 into the FIT-1 bispecific mAb and its in vitro characterization. (A) Monoclonal antibodies (mAbs) ZKA185 and ZKA230 are tested for neutralization of four ZIKV strains, as determined by the percentage of infected Vero cells in the presence of increasing amounts of mAb. Data are representative of at least two independent experiments. (B) Binding of ZKA185, ZKA230 IgG and Fab to recombinant ZIKV VLP, E and DIII antigens was assessed by ELISA. (B) ZKA190, ZKA185 and ZKA230 will be tested for their ability to neutralize H/PF/2013 (wt) and MARM 1-4. (D) Surface representation of two E protein dimers bound by ZKA190 (green); the ZKA190 NMR-derived epitope is red; positions mutated in MARM are indicated in yellow (E370), blue (T335), orange (D67), and magenta (K84). (E) FIT-1 model. Natural linkers between the inner and outer Fab provide flexible movement of the Fab within the FIT-1 antibody. The variable regions ZKA185 and ZKA190 are highlighted in blue and green, respectively. (E) Binding of FIT-1 IgG and Fab to recombinant ZIKV VLP, E, and DIII antigens was assessed by ELISA. (G) mAbs ZKA190, ZKA185, and FIT-1 were tested for their ability to neutralize four strains (IC50 values, G) and four MARMs (3) of ZIKV. (I) Neutralization of ZIKV strain H/PF/2013 by ZKA185, ZKA230, and FIT-1 IgG and Fab identified in (A). (K) Confocal microscopy experiments shown in Fig. 3. (L) Effect on ADE induced by peak dilution of anti-prM DV62 mAb or DENV2 plasma by serial dilutions of FIT-1 IgG and Fab.

Fig. 20. Therapeutic effectiveness of FIT-1. FIT-1 is highly effective against ZIKV infection when administered therapeutically to mice (A129) at various time points that are injected with a lethal dose of ZIKV strain MP17451. In experiments, 5-6 mice per group are used. (A) Kaplan-Meier survival curves. Significance was determined using the Mantel-Cox log-rank test. FIT-1 at doses of 15, 5 and 1 mg/kg was administered on day 1, on day 2 depending on the control mAb, P=0.0012; ZKA190 at doses of 15 and 5 mg/kg on day 3 depending on the control mAb, P=0.0012; ZKA190 1 mg/kg given on day 3 based on control mAb, P=0.0170. (B) Morbidity assessment of mice observed for 21 days (Dowall et al., 2016). (B) Viral loads are measured as PFU on day 5 in the blood of all animals. (D) Mice are monitored over a 21-day period for weight loss. The control mAb in panel A is MPE8 mAb (RSV-specific F protein (Corti D. et al., Nature 501, 2013, 439-443). (E) Virus load is measured by qPCR and expressed as genome copy number 5 day in the blood of all animals and in the blood and specific tissues when animals are collected at the end of the study or when humane endpoints are reached Significance is determined by comparison with control antibody treatment using a nonparametric unpaired Mann-Whitney U test. *p<0.05; **p<0.01.

Fig. 21. Example 14 shows that female AG129 mice treated with FIT-1 following Malaysian ZIKV infection are protected from mortality compared with placebo-treated mice.

Fig. 22. Example 14 shows intrauterine growth restriction (IUGR) in mice born to FIT-1-treated dams infected with ZIKV.

Fig. 23. Example 14 shows the average birth weight of pups born to FIT-1-treated dams infected with ZIKV.

Fig. 24. Example 14 shows the weight of placentas collected on the 11th day after infection from Smokies receiving FIT-1.

Fig. 25. Example 14 shows the quantification of viral RNA in (A) the fetus, (B) the placenta, (C) the maternal spleen, and (D) the maternal brain (***P<0.001, **P<0.01, vs. with MPE treatment8).

Fig. 26. Example 15 shows the survival of male AG129 mice infected with ZIKV and treated with FIT-1 24 or 72 hours after virus infection (*P<0.05 compared to treatment with the negative control MPE8).

Fig. 27. Example 15 shows the average percentage change in weight of FIT-1-treated AG129 mice at various times after ZIKV infection.

Fig. 28. Example 15 shows disease assessment of A) testes or B) epididymis of male AG129 mice treated with FIT-1 24 or 72 hours after ZIKV infection. Treatment with reactive Abs results in decreased testicular and epididymal morbidity.

Fig. 29. Example 16 shows the serum virus load in the three groups tested. The horizontal line shows LLOQ 860 GC/ml.

Examples

Possible embodiments of the present invention are presented below in the examples. These examples are presented by way of illustration only and to assist those skilled in the art in practicing the present invention. These examples are in no way intended to limit the scope of the present invention.

Example 1. Isolation of ZIKV-specific antibodies and production of monoclonal antibodies.

IgG+ memory B cells were isolated from cryopreserved peripheral blood mononuclear cells (PBMCs) of four ZIKV-infected donors (ZKA, ZKB, ZKC and ZKD) using CD22 microbeads (Miltenyi Biotec) followed by depletion of cells bearing IgM, IgD and IgA by cell sorting. Memory B cells from ZIKV-infected donors are then immortalized with EBV (Epstein-Barr virus) and CpG (CpG oligodeoxynucleotide 2006) in multiple replicate wells as described previously (Traggiai E. et al., Nat. Med. 10, 2004, 871-875) and then the culture supernatants are tested in a primary screen using a parallel 384-well micro-neutralization and binding assay (ELISA) to test their binding to the ZIKV NS1 protein or the ZIKV E protein. The results of the binding assay (binding to the ZIKV E protein) are shown in FIG. 1.

Memory B cells from ZIKV-infected donors are then immortalized with EBV (Epstein Barr virus) and CpG (CpG oligodeoxynucleotide 2006) in multiple replicate wells as described previously (Traggiai E. et al., Nat. Med. 10, 2004, 871- 875) and then the culture supernatants are tested in a primary screen using a parallel 384-well microneutralization and binding assay (ELISA) to test their binding to the ZIKV NS1 protein or the ZIKV E protein. The results of the binding assay (binding to the ZIKV E protein) are shown in FIG. 1.

Neutralization assays are performed on Vero cells. In a 384-well plate ZIKV H/PF/2013 at a multiplicity of infection (MOI) of 0.35, incubate with supernatants for 1 hour at 37°C (5% CO ₂ ) before adding to the pre-seeded 5000 cells Vero. They are incubated for a further 5 days, after which the supernatant is removed and WST-1 reagent (Roche) is added. Positive cultures were collected and propagated. From positive cultures, VH and VL sequences were recovered by RT-PCR. Antibodies were cloned into human IgG1 and Ig-kappa or lambda-Ig expression vectors (kindly provided by Michel Nussenzweig, The Rockefeller University, New York, USA) essentially as described (Tiller T. et al., J Immunol Methods 329, 2008 , 112124). Monoclonal antibodies are prepared from EBV immortalized B cells or by transient transfection of 293 Freestyle cells (Invitrogen). Supernatants from B cells or transfected cells were collected and IgG was affinity purified using Protein A or Protein G chromatography (GE Healthcare) and desalted against PBS.

Fig. 1 provides an overview of individual ZIKV neutralizing antibodies (see Tables 1 and 2 for their CDR amino acid sequences and heavy/light chain variable regions). In the last two columns of Fig. 1 shows the activity aimed at neutralizing (IC ₅₀ ) ZIKV and DENV1 (when tested). Other columns represent the binding activity (EC ₅₀ ) of antibodies to E

- 57 043940 ZIKV protein (ZIKV E), E protein DENV1 (DENV1 E), E protein DENV2 (DENV2), E protein DENV3 E (DENV3), E protein DENV4 (DENV4)), virus-like particle DENV1 (VLP DENV1), virus-like DENV2 particles (DENV2 VLP), DENV3 virus-like particles (DENV3 VLP), DENV4 virus-like particles (DENV4 VLP) and the ZIKV EDD domain E protein (DIII ZKA).

Example 2. Description of antibodies ZKA190, ZKA185, ZKA230, ZKA64 and ZKA78.

In Example 1, a large number of ZIKV neutralizing antibodies are identified and characterized by their specificity to ZIKV, in particular to ZIKV E protein and ZIKV EDIII, as well as their cross-reactivity to DENV. Antibodies ZKA190 (SEQ ID NO: 1-18), ZKA185 (SEQ ID NO: 1936), ZKA230 (SEQ ID NO: 37-54), ZKA64 (SEQ ID NO: 73-90) and ZKA 78 (SEQ ID NO: 55-72) described in example 1 are then selected and further tested against the E protein of ZIKV (ZIKV), EDIII of ZIKV (DIIIZI), and against the E protein of dengue virus (DENV, serotype number 1) by ELISA. For this purpose, a standard enzyme-linked immunosorbent assay (ELISA) is used. Briefly, ELISA plates are coated with 1 or 3 μg/ml ZIKV E protein, blocked with 10% FST in PBS, incubated with human sera or antibodies, and washed. Bound antibodies are detected by incubation with AP-conjugated goat anti-human IgG (goat anti-human IgG; Southern Biotech). The plates are then washed, substrate (p-NPP, Sigma) is added and the plates are read at 405 nm. The relative binding affinity of a monoclonal antibody is determined by measuring the antibody concentration (EC ₅₀ ) required to achieve 50% of maximum binding at saturation.

The results are presented in Fig. 3 and 4. It is noteworthy that ZKA64 and ZKA190 bind to ZIKV E and ZIKV EDIII (DIIIZI) with low EC ₅₀ values, indicating that ZKA64 and ZKA190 bind to domain III of the ZIKV E protein (EDIII). ZKA78 binds to the ZIKV E protein but not to the ZIKV EDIII protein, indicating that ZKA78 binds to the ZIKV E protein but does not target the EDIII region. Despite significant ZIKV neutralizing activity (see Fig. 1), antibodies ZKA185 and ZKA230 did not show any detectable binding to ZIKV E and ZIKV EDIII (Fig. 3). Accordingly, ZKA185 and ZKA230 are called neutralizing-non-E-binding NNB antibodies. These NNB antibodies are predicted to recognize quaternary epitopes that are present on infectious ZIKV virions but not on soluble proteins.

Moreover, none of ZKA190, ZKA185, ZKA230 and ZKA64 show any detectable binding to DENV E proteins (Fig. 1, DENV serotypes1-4 and Figs. 3 and 4), indicating that ZKA190, ZKA185 , ZKA230 and ZKA64 are specific for ZIKV and do not cross-react with dengue virus. In contrast, ZKA78 binds to the E proteins of DENV (Figs. 1 and 3) but not to ZIKV EDIII (see Fig. 2), indicating that ZKA78 is a cross-reactive antibody that binds to both ZIKV and DENV.

To further confirm these results, the ZIKV E protein-binding antibodies ZKA190, ZKA64, and ZKA78 were further tested against the E protein of dengue virus (DENV, serotypes no. 1-4). ZKA64 and ZKA190 do not bind to the E protein of DENV1-4, thereby confirming that ZKA64 and ZKA190 are specific for ZIKV. ZKA78, in contrast, binds to the DENV1-4 E protein, suggesting that ZKA78 is a cross-reactive antibody that binds to the E protein of both ZIKV and DENV (see Fig. 1).

Example 3: Isolated antibodies actively neutralize ZIKV infection.

The ability of antibodies ZKA190, ZKA185, ZKA230, ZKA64 and ZKA78 to neutralize ZIKV and DENV1 infections in vitro was determined.

Antibody neutralization of DENV and ZIKV infections is measured using a flow cytometry-based micro-neutralization method. Various antibody dilutions were mixed with ZIKV (MOI 0.35) or attenuated DENV1 (all with MOI 0.04) for 1 h at 37° and added to 5000 Vero cells/well in 96-well flat-bottom plates. After four days for ZIKV and five days for DENV, cells are fixed with 2% formaldehyde, saturated with saponin in 1% PBS 0.5% and stained with mouse mAb 4G2. Cells are incubated with goat anti-mouse IgG conjugated to Alexa Fluor488 (Jackson Immuno-Research, 115485164) and analyzed by liquid cytometry. In other cases, ZIKV neutralization data are also determined by measuring cell viability using WST-1 reagent (Roche).

The neutralization titer (50% inhibitory concentration [IC ₅₀ ]) is expressed as the concentration of antibody that reduces infection by 50% compared to control wells containing cells only.

The results are presented in Fig. 4, 5 and 9. EDIII-specific mAbs ZKA64 and ZKA190, as well as NNB mAb ZKA230, are highly active in neutralizing ZIKV (strain H/PF/2013), with IC50 values of 93, 9 and 10 ng/ml, respectively (Fig. 4, top panel). In contrast, cross-reactive ZKA78 antibodies only partially neutralized ZIKV infectivity and cross-neutralized DENV1 infectivity (Fig. 4, bottom panels). Similar data were obtained by measuring the ZIKV-induced cytopathic effect measured using the WST-1 reagent (Fig. 5). In this second NNB assay, ZKA185 was also included in the panel of antibodies tested and showed an IC value similar to the most potent ZKA190 antibody.

- 58 043940 (EDSh-specific) and ZKA230 (NNB).

It is important to note that the ultra-potent antibodies ZKA64 and ZKA190, in addition to their ability to neutralize ZIKV strain H/PH/2013 (present example), are also associated with protein E and EDIII derived from ZIKV strains MR766 and SPH2015, respectively (Fig. 1 and 2). ZKA190 and ZKA190-LALA were also confirmed to effectively neutralize two additional ZIKV strains (MR766 and MRS_OPY_Martinique_PaRi_2015) (Fig. 9). Taken together, the results show that the ultra-potent antibodies ZKA64 and ZKA190 cross-react with several ZIKV strains belonging to different genotypes and having different origins (East African and Asian from Uganda, French Polynesia, Martinique and Brazil).

Example 4 LALA mutation suppresses antibody-dependent enhancement of ZIKV infection by serum antibodies.

Neutralizing antibodies also test HA's ability to enhance ZIKV infection in nonpermissive K562 cells (antibody-dependent enhancement, ADE). ADE is measured by a flow-based method using K562 cells. Antibodies and ZIKV H/PF/2013 (MOI 0.175) were mixed for 1 h at 37°C and added to 5000 K562 cells/well. Four days later, cells were fixed, permeabilized, and stained with m4G2. The number of infected cells is determined by liquid cytometry.

The results are presented in Fig. 6. All antibodies enhanced ZIKV infection in nonpermissive K562 cells over a wide range of concentrations, including those that completely neutralized ZIKV infection in Vero cells (Fig. 6). It should be noted that although EDIII-specific antibodies ZKA64 and ZKA190 completely neutralize ZIKV infections of K562 cells at concentrations above 1 μg/ml, the NNB antibody ZKA230 does not cause such a result, which may be due to different mechanisms of neutralization of free viruses compared to Fc -gamma receptor of internalized viruses. In contrast, cross-reactive ZKA78, which only partially neutralizes ZIKV infectivity, effectively enhances ZIKV infection of K562 cells. These results indicate that cross-reactive antibodies induced by ZIKV or DENV infection can mediate heterologous antibody-dependent enhancement (ADE).

In view of this, it was investigated whether ADE could also be induced by immune serum and whether it could be blocked by neutralizing antibodies such as the LALA variant. To produce the LALA variant, each of the heavy chains is mutated at amino acids 4 and 5 of the CH2 domain by replacing native leucine with alanine using site-directed mutagenesis. It was described above that variants of LALA (human IgG1 antibodies) do not bind to Fc-gamma receptors and complement.

To study the effect of the ZKA64-LALA antibody on ZIKV ADE, an ADE inhibition assay was used. Because ZIKV ADE is observed with ZIKV- or DENV-immune plasma, ZIKV (MOI 0.175) is mixed with plasma from primary ZIKV- or DENV-infected donors for 30 min at 37°C. Antibody ZKA64-LALA is added at a concentration of 50 μg/ml, mixed with 5000 K562 cells/well and incubated for three days. The cells are then stained with 4G2 and analyzed by liquid cytometry.

The results are presented in Fig. 7. In a homologous environment, four ZIKV-immune plasma samples from convalescent patients and one DENV-immune plasma showed a similar ability to enhance ZIKV infection of K562 cells (Fig. 7, top panel), and this effect of ADE completely blocked EDIII-specific ZKA64-LALA antibody (Fig. 7, bottom panel).

It should be noted that the ADE effect of ZIKV and DENV immune plasma is completely blocked by the EDIII-specific antibody ZKA64-LALA. The ability of ADE to block the single EDIII-specific antibody LALA may be due not only to its ability to outperform serum enhancing antibodies but also to neutralize the virus after internalization into endosomes.

These results indicate that potent neutralizing antibodies, such as ZKA190, ZKA230, ZKA185, or ZKA64, developed in the form of LALA, have strong potential for use in prophylactic or therapeutic settings to prevent congenital ZIKV infection, such as in pregnant women and/or humans living in high-risk areas. Use of the LALA form avoids the risk of developing ZIKV ADE and, as shown above, can also block the ADE of pre-existing cross-reactive antibodies, for example in patients already immune to DENV.

Example 5 ZKA190 neutralizes ZIKV more strongly than the prior art antibody EDE1 mAb C8.

To compare the isolated neutralizing antibodies with the highly neutralizing prior art anti-ZIKV antibodies, the neutralization efficiency of ZKA190 is compared with the highly neutralizing prior art mAb EDE1 C8 (Barba-Spaeth G. et al., Nature, 536 (7614), 2016, 48-53 ). Neutralization of both antibodies was tested on a panel of four different strains (H/PF/2013; MR766, MRS-OPY and PV10552).

Briefly, neutralization of ZIKV infection by mAbs is measured using a liquid cytometry-based microneutralization assay. Various mAb dilutions are mixed with ZIKV (MOI

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0.35), incubate for 1 hour at 37° and add to 5000 Vero cells/well in 96-well flat-bottom plates. Four days later for ZIKV, cells are fixed with 2% formaldehyde, saturated in PBS containing 1% fetal calf serum (Hyclone) and 0.5% saponin, and stained with mouse mAb 4G2. Cells are incubated with goat anti-mouse IgG conjugated to Alexa Fluor488 (Jackson Immuno-Research, catalog number 115485164) and analyzed by liquid cytometry. The neutralization titer (50% inhibitory concentration [IC ₅₀ ]) is expressed as the concentration of antibody that reduces infection by 50% compared to control wells containing virus alone.

The results are presented in Fig. 10. ZKA190 mAb strongly neutralizes African, Asian and American strains with IC ₅₀ values ranging from 0.6 to 8 ng/ml. In comparison, the prior art antibody C8 is approximately 24 times less active.

Example 6 Additional Description of Antibody ZKA190.

The activity of the ZKA190 antibody was further studied in vitro and in vivo. For this purpose, mAbs were synthesized in wild-type (wt) IgG1 and Fc-LALA IgG1 formats. Briefly, VH and VL sequences were cloned into human expression vectors for Igy1, IgK and Igλ, (kindly provided by Michel Nussenzweig, The Rockefeller University, New York, NY, USA), essentially as published (Tiller T. c. al., J Immunol Methods, 329, 2008, 112-124). Recombinant mAbs were prepared by transient transfection of EXPI293 cells (Invitrogen), purified by Protein A chromatography (GE Healthcare) and desalted against PBS. To produce the LALA variant, each of the heavy chains is mutated at amino acids 4 and 5 of the CH2 domain by replacing native leucine with alanine using site-directed mutagenesis. As described above, LALA (human IgG1 antibody) variants do not bind to Fc-gamma receptors and complement.

As shown in FIG. 10A and described in Example 5, ZKA190 was tested against a panel of four ZIKV strains. ZKA190 mAb potently neutralized African, Asian and American strains with IC50 ranging from 0.004 to 0.05 nM (Fig. 10A; 0.6-8 ng/ml).

Because ZIKV has been shown to infect human neural progenitor cells (hNPCs), resulting in increased cell toxicity, cell cycle dysregulation, and decreased cell growth, ZKA190 and ZKA190-LALA are tested in hNPCs. For this purpose, adult male fibroblasts obtained from the Movement Disorders Bio-Bank (Department of Neurogenetics, Carlo Besta Neurological Institute, Milan) were reprogrammed using the CytoTune-iPS 2.0 Sendai kit (Life Technologies). hiPSCs are maintained under feeder-free conditions in mTeSR1 (Stem Cell Technologies). To generate embryoid bodies (EB), dissociated hiPSCs are plated in low adhesion plates in mTeSR1 supplemented with N2 (0.5x) (ThermoFisher Scientific), human Noggin (0.5 mg/ml, R&D System), SB431542 ( 5 µM, Sigma), Y27632 (10 µM, Miltenyi Biotec) and penicillin/streptomycin (1%, Sigma) (as described by Marchetto MCN et al., Cell, 143, 2010, 527-539). To obtain rosettes, EB is sown after 10 days on Matrigel-coated dishes (1: 100, Matrigel growth factor reduced, Corning) in DMEM/F12 (Sigma) with N2 (1: 100), non-essential amino acids (1%, Sigma) ThermoFisher Scientific) and a mixture of penicillin/streptomycin. After 10 days, the cells were passaged with Accutase (Sigma) and seeded in Matrigel flasks in NPC medium containing DMEM/F12, N2 (0.25%), B27 (0.5%, ThermoFisher Scientific), penicillin/streptomycin and FGF2 (20 ng/ml, ThermoFisher Scientific). hNPC (3x10 ⁴ ) were seeded on coverslips in 24-well plates 3 days before infection with strain PRVABC59. Virus stock was incubated with mAb for 1 h before addition to hNPC to obtain an MOI of 0.5. After 4 h of virus adsorption, the culture supernatant is removed and fresh medium containing mAb is re-added. The supernatant was collected 96 h postinfection to measure virus titers using a plaque assay on Vero cells. Cells are fixed in 4% paraformaldehyde (PFA, Sigma) in phosphate-buffered saline (PBS, Euroclone) for 30 min for indirect immunofluorescence. Fixed cells are saturated for 30 min in blocking solution containing 0.2% Triton X-100 (Sigma) and 10% donkey serum (Sigma) and incubated overnight at 4°C with primary antibodies in blocking solution. The following antibody is used to detect anti-envelope (1:200, Millipore, MAB10216). Cells are then washed with PBS and incubated for 1 hour with secondary antibodies Hoechst and anti-mouse Alexa Fluor-488 (1:1000 in blocking solution, ThermoFisher Scientific). After washing with PBS, the cells are washed again and monitored. The results are shown in Fig. 11A. Both ZKA190 and ZKA190-LALA antibodies completely eliminated infection and inhibited ZIKV replication in hNPCs.

The ability of ZKA190 and ZKA190-LALA to induce ADE was then tested in the K562 cell line as described in Example 4. Briefly, ADE was measured by a flow-based assay using K562 cells. Briefly, ZKA190, ZKA190 and ZIKV H/PF/2013 (MOI 0.175) were mixed for 1 h at 37° and added to 5000 K562 cells/well. Four days later, cells are fixed, saturated, and stained with m4G2 mAb. The number of infected cells is determined by flow cytometry. For ZKA190-LALA,

- 60 043940

ZIKV (MOI 0.175) was mixed with plasma from primary ZIKV-infected donors for 30 min at 37°C. ZKA190-LALA is added at a concentration of 50 μg/ml, mixed with 5000 K562 cells/well and incubated for three days. Cells are then stained with 4G2 and analyzed by flow cytometry. The results are shown in Fig. 11B. ZKA190 supports ADE from 0.0001 to 1 nM; as expected, ZKA190-LALA does not exhibit ADE activity. The ability of ZKA190-LALA to inhibit ADE induced by plasma from four ZIKV-immune donors in K562 cells was also tested. The results are shown in Fig. 11B. ZKA190-LALA was found to completely inhibit ADE induced by plasma antibodies (Fig. 11B).

Antibodies against prM (anti-prM) form part of the predominant antibodies raised during the human immune response against flaviviruses and have been shown to enhance viral infection in vitro (Dejnirattisai W. et al., 2010). Cross-reacting antibodies enhance dengue virus infection in humans (Science 328, 745-748). K562 cells are preincubated with serial dilutions of the prM cross-reactive antibody DV62 (Beltramello M. et al., Cell Host Microbe 8, 2010, 271-283) obtained from a DENV immune donor. The results are shown in Fig. 11D. DV62 cross-reacts with the ZIKV prM protein and causes ADE over a wide range of concentrations (Fig. 11D). ZKA190-LALA could completely block anti-prM DV62 mAb-induced ADE of immature or partially immature ZIKV particles (Fig. 11D).

In summary, the ability of different concentrations of FABs ZKA190, ZKA190-LALA, and ZKA190 to induce or block ZIKV ADE was tested in the presence of increasing concentrations of human plasma against DENV2 or DV62. The results are shown in Figure 11E. ZKA190 at low concentrations increases the prM of DV62-mediated ADE of ZIKV infection, consistent with its ability to promote entry of both immature and mature virions, while at concentrations above 1.3 nM (i.e., 200 ng/ml) ZKA190 blocks ADE induced by both DENV plasma and mAb DV62. ZKA190-LALA, as well as its Fab fragment, reduced ADE at concentrations above 0.06 nM, indicating inhibition in both cases of viral infection at the post-attachment stage, such as the fusion stage.

Example 7 ZKA190 binds to the conserved and highly active region of EDIII.

To determine the ZKA190 epitope at the residual level, resolution NMR spectroscopy is used, described in the publications of Bardelli M. et al., J. Mol. Recognize 28, 2015, 393-400; Simonelli L. et al., J Mol Biol 396, 2010, 1491-1507; Simonelli L. et al, PLoS ONE 8, 2013, e55561.

Briefly, spectra are recorded on a Bruker Avance 700 MHz NMR spectrometer at 300 K. Triple resonance experiments (HNCO, HN(CA)CO, HN(CO)CABC, HNCACB) are used to abstract backbone resonances (HNCO, HN(CA)CO, HN(CO)CABC, HNCACB, while side chains are annotated using HCCH-TOCSY and HBHA(CO)NH experiments. All NMR experiments were processed using Topspin 2.1 (Bruker Biospin) and analyzed using CARA. NOESY cross peaks were automatically assigned using the CYANA noeassign macro based on manual chemical shifts. Limitations at distances used for structure calculations in CYANA using a standard simulated annealing protocol, obtained from 70 ms resolution NOESY spectra at ¹⁵ N and ¹³ C. ZIKV ED III backbone dynamics are obtained from ¹⁵ N relaxation measurements recorded on 600 and 700 MHz spectrometers. Proton-detected versions of CPMG (R2), inversion recovery (R1) and use ¹⁵ N{1H}-stable NOE Delay settings for T2 series range from 0 to 0.25 s and for T1 series from 0.02 to 2 s. Experiment ¹⁵ N{1H}-NOE uses a relaxation delay of 5 s. The relaxation rates R1 and R2 are derived from a least squares fit of the corresponding exponential functions to the acquired data using pre-written scripts. The relaxation data are analyzed using a model-free approach using the DYNAMICS software package. The ROTDIF program is used to calculate the total correlation time from the relaxation data (8.5 ns). NMR epitope mapping was performed as described (Bardelli et al., 2015; Simonelli et al., 2010; 2013). Briefly, superposition of 15NHSQC spectra of labeled EDIII, free or bound to Fab ZKA190, identified EDIII residues whose NMR signal changed upon complex formation, indicating that they were affected by ZKA190 binding. Changes were detected by manual inspection and by chemical shift perturbation (CSP), CSP=((Δδн) ² +(Δδ _N /10) ² ) ^1/2 . NMR samples are typically 800 µM EDIII-labeled [ ¹⁵ N, ¹³ C] in 20 mM sodium phosphate, 50 mM NaCl, pH 6.0. Perdeuterated (nominally 70%) ^2H , ^15N EDIII samples are used for NMR epitope mapping with an EDIII:ZKA190 Fab ratio of 1:1.1; the EDIII concentration is typically 0.4 mM.

Because the NMR signal is highly dependent on the local chemical environment, changes during complex formation reveal antigen residues that are affected by antibody binding, either directly or through allosteric effects. By comparing the NMR spectra of free and bound EDIII (Figure 12A), residues affected by ZKA190 were mapped to the LR of EDIII, specifically the BC, DE and FG loops, as well as part of the EDI-EDIII loop (Figure 13A). These residues are nearly identical among the 217 known ZIKV strains, except for substitutions at positions V341I and E393D in isolate Uganda 1947 (Fig. 12D). These mutations are also present in strain MR766, which was effectively neutralized by ZKA190 (Fig.

- 61 043940

10A). Analysis of the ZKA190 epitope on the ZIKV simple structure shows that the epitope is highly accessible, with the exception of the FG loop at the 5-fold vertex (Fig. 13B and 12B, molecule A).

Calculated docking followed by molecular dynamics simulations targeting and confirming NMR-derived epitope information, as well as EDIII mutagenesis, show that ZKA190 binds through a contact region characterized by shape and charge complementarity (Figures 13B and 12E). Docking indicates that there are no direct contacts between ZKA190 and the FG loop on EDIII, suggesting that changes in NMR signals upon antibody binding are due to allosteric effects. This notion is supported by the fact that mutations of FG loop residues in recombinant EDIII, but not in other epitope regions, do not affect the binding affinity of ZKA190 for EDIII (Figs. 13B and 14).

Example 8. Neutralization mechanisms of ZKA190.

The ability of ZKA190 to effectively neutralize the virus may involve inhibition, cell attachment, or membrane fusion. Another mechanism may involve inactivation of the virus by cross-linking of viral particles.

Fab ZKA190 can neutralize ZIKV, although less efficiently than the corresponding IgG. By binding to the EDI-EDIII linker, ZKA190 (both Fab and IgG) can inhibit the ~70 degree DIII turn required for viral fusion with the host cell membrane (Bressanelli S. et al., Embo J 23, 2004, 728- 738; Modis Y. et al., Nature 427, 2004, 313-319). Alternatively, ZKA190 may prevent ZIKV from attaching to target cells.

The ability of ZKA190 to inhibit membrane fusion was confirmed by confocal microscopy analysis. To do this, Vero cells are seeded at 7500 cells/well onto 12 mm glass coverslips in 24-well plates and incubated overnight. Cells were infected with ZIKV H/PF/2013 (MOI 100) in the presence or absence of neutralizing concentrations of Alexa-488-conjugated mAb (0.7 μM) at 37°C for 3 h, washed with PBS, and fixed with 2% paraformaldehyde in PBS for 30 min at room temperature. The acidic endosome is identified using Lysotracker red (Invitrogen) by adding the dye (50 nM) to the cells during the last 30 min of incubation before fixation. Fixation is followed by extensive washes in PBS and 50 mM glycine, and finally the coverslips are prepared for microscopic analysis using Vectashield fluorescence mounting medium with DAPI (Vector Laboratories). Samples were analyzed by confocal microscopy using a Leica TCS SP5 microscope with a 63 x/1.4 H.A. objective. Image analysis and processing is carried out using FIJI software.

The results are presented in Fig. 15. Confocal microscopy analysis shows that ZKA190 (Fab or IgG) can enter Vero cells only in complex with ZIKV, at neutralizing concentrations exceeding IC ₅₀ by 10,000 times (Fig. 15).

Example 9: In vivo characterization of EDIII-specific mAb ZKA190.

ZKA190 and ZKA190-LALA are being tested in A129 mice infected with a lethal dose of ZIKV strain MP1751 (African origin) to evaluate their preventive and therapeutic properties.

To test their preventive activity, ZKA190 and ZKA190-LALA were administered one day before viral infection.

Female A129 (IFN-alpha/beta receptor -/-) and 129Sv/Ev wild-type mice were treated with mAbs (ZKA190, ZKA190-LALA and control antibody MPE8) at 5-8 weeks of age (Corti DC et al., Nature 501, 2013 , 439-443), diluted in PBS in various doses intraperitoneally in a volume of 500 μl. Monoclonal antibodies (mAbs) are administered 1 day before, or 1, 2, 3 or 4 days after infection with the virus. Animals are infected subcutaneously with 10 ² PFU ZIKV (strain MP1751) and observed for 14 days. Weight and temperature are monitored daily, and clinical observations are recorded at least twice a day. 5 after infection, 50 μl of blood is taken from each animal in RNAprotect tubes (Qiagen, UK) and frozen at -80° C. At the end of the study (14 days postinfection) or when animals reach humane endpoints, necropsies are performed and blood samples, brain sections, spleens, livers, kidneys and ovaries are collected for virological analysis.

Tissue samples from A129 mice were weighed and homogenized in PBS using ceramic beads and an automatic homogenizer (Precellys, UK) using six 5 second cycles at 6500 rpm with a 30 second interval. Two hundred µl of tissue homogenate or blood solution is transferred into 600 µl of RLT buffer (Qiagen, UK) for RNA extraction using the RNeasy Mini extraction kit (Qiagen, UK); samples are passed through a QIAshredder homogenizer (Qiagen, UK) as an initial step. A ZIKV-specific real-time PCR (RT-PCR) assay is used to detect viral RNA in experimental animals.

Primer and probe sequences were taken and adapted from (Quick et al., Nat Protoc, 12, 2017, 1261-1276) with optimization and validation to ensure optimal mastermix and cycling conditions. RT-PCR is performed using the Superscript One-Step qRT-PCR Kit

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III Platinum (Life Technologies, UK). The final mastermix (15 µl) consists of 10 µl 2x reaction mixture, 1.2 µl PCR water, 0.2 µl 50 mM MgSO ₄ , 1 µl each primer (ZIKV 1086 and ZIKV 1162c, both at a working concentration of 18 µM) 0 .8 µl probe (ZIKV 1107-FAM at 25 µM working concentration) and 0.8 µl SSIII enzyme mixture. Five μl of messenger RNA is added to the mastermix, resulting in a final reaction volume of 20 μl. The cycling conditions used were 50° for 10 min, 95° for 2 min, then 45 cycles of 95° for 10 s and 60° for 40 s, plus a final cooling step of 40° for 30 s. Quantitative analysis using fluorescence is carried out at the end of each 60° step. Reactions were carried out and analyzed on a 7500 Fast platform (Life Technologies, UK) using 7500 software version 2.0.6. Quantitative determination of viral load in samples is carried out using serial dilutions of an RNA oligonucleotide with a specified concentration (Integrated DNA Technologies). The oligonucleotide containing the 77 bases of ZIKV RNA targeted by the assay is based on GenBank reference AY632535.2 and was synthesized at 250 nmol with HPLC purification.

The results are presented in Fig. 16, 17 and 18. ZKA190 and ZKA190-LALA were shown to protect mice from death and morbidity at concentrations of 5, 1 or 0.2 mg/kg (Fig. 16A, B). ZKA190LALA and, to a lesser extent, ZKA190, slowed morbidity and mortality compared to the control group at 0.04 mg/kg. Viral titers in the blood and organs were significantly reduced compared to control animals treated with antibodies, even with serum antibody levels below 1 μg/ml (Fig. 17A-D).

To evaluate the therapeutic potential of ZKA190, the present study administered ZKA190 and ZKA190-LALA at different time points after ZIKV infection. At a dose of 15 mg/kg, survival reaches 80-100%, and morbidity is significantly reduced when treatment is given four days after infection (Fig. 16A, B). Treatment with ZKA190 and ZKA190-LALA at all time points postinfection resulted in significant reductions in virus titers compared to control antibody-treated animals, with a clear trend toward greater reductions earlier in treatment (FIGS. 18A-16B). Of note, ZKA190-LALA exhibited significantly reduced antiviral activity in a day 5 blood sample compared to ZKA190 when the mAb was given four days postinfection, which may be due to an impairment of the ability of the LALA variant to promote rapid clearance from coated virions .

One in vivo antibody-evading mutant (Monoclonal Antibody Resistant Mutant 1 - MARM1) containing an amino acid substitution in DIII (T335R, at the center of the epitope) was isolated from 16 treated mice, whereas viruses from other treated mice do not have any E mutations. Introduction of the T335R mutation into recombinant DIII showed that it abolished ZKA190 binding as determined by SPR (Fig. 14; see Experimental Methods Example 7).

Example 10: In vivo selection of ZIKV escape mutants.

The use of antibody therapeutics may result in the selection of antibody-evading mutants. To evaluate the ability of ZKA190 to select resistant mutants (MARMs) in vitro, ZIKV (H/PF/2013) was passaged in the presence of sub-neutralizing concentrations of ZKA190.

Briefly, two thousand TCID50 H/PF/2013 ZIKV in 500 μl are incubated with 250 μl containing different concentrations of mAb (8 different concentrations, starting with a final concentration of 200 μg/ml and performing serial dilutions of 1:4). The mixture is incubated for 45 minutes at 37°C, after which 250 µl of Vero cell suspension (3.2 x 106 cells) is added and incubated in a 24-well plate for three to four days at 37°C to allow virus multiplication. After each selection step, 500 μl of supernatants are taken from three options: the lowest concentration of mAb at which complete protection of the monolayer was observed, one concentration at which a partial effect of CPE on the cell monolayer was observed, and one concentration at which 100% of the cell monolayer was destroyed by ZIKV CPE. The tube is centrifuged for 5 minutes at 1000xg, an aliquot is removed and stored at -80°. Half the volume is again premixed with different concentrations of mAb to repeat the selection and propagation process. The remaining supernatant is used for micro-neutralization assays and subsequent virus sequencing.

To identify antibody-escape mutants of the selected MARM virus, genomic RNA extraction is performed, followed by one-step PCR to amplify and sequence the ZIKV E protein amplicon. Cell supernatant (140 μl) from MARM selection was used for RNA extraction using a QIAamp Viral RNA mini kit (Qiagen). cDNA synthesis and PCR amplification were performed together using Superscript III One-Step RT-PCR with a platinum tag (Platinium Taq, Invitrogen). For one reaction, use 25 μl of reaction mixture, 8 μl of sterile water, 2 mM of each primer, 1 μl of RNase (Life Technologies), 2 μl of Superscript III RT/Platinum TaqMix and 12 μl of RNA, resulting in a final reaction volume of 50 μl . For the N-terminal part of the E protein, the primer pair Zika-E-F1 5'-TGCAAACGCGGTCGCAAACCTGGTTG-3' (SEQ ID NO: 266) and ZIKV-E-R1 5'

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CGTGCCAAGGTAATGGAATGTCGTG-3' (SEQ ID NO: 267) and for the C-terminal part of the primer pair ZIKV-Ef1530 5'-AGCCTAGGACTTGATTGTGAACCGA-3' (SEQ ID NO: 268) and ZIKV-E-R2769 5'TTACAGATCCCACAACGACCGTCAG-3' (SEQ ID NO: 269) are used. Cycling conditions: 54° for 40 min, 94° for 2 min, then 45 cycles of 94° for 45 s, 50° for 45 s and 68° for 1.5 min with a final elongation step at 68° for 5 minutes and the final stage of cooling at 4°. PCR products are analyzed and extracted from a 1.5% agarose gel and further purified using the GFX DNA Purification Kit and Gel Band (GE Healthcare). For the sequencing reaction, 8 μl of purified PCR product is mixed with 2 μM primer in a final volume of 10 μl and submitted for sequencing (Microsynth). E protein N-terminal products were sequenced with ZIKV-E-F2 5'-ACTTGGTCATGATACTGCTGATTGC-3' (SEQ ID NO: 270) and ZIKV-E-R2 5'TCGGTTCACAATCAAGTCCTAGGCT-3' (SEQ ID NO: 271), C-terminal PCR with ZIKV-E-f2058 5'GCTAACCCCGTAATCACTGAAAGCA-3' (SEQ ID NO: 272) and ZIKV-E-r2248 5'AAGACTGCCATTCTCTTGGCACCTC-3' (SEQ ID NO: 273). Sequences were collected and analyzed using CLC Main Workbench software (CLC Bio, version 5).

A resistant mutant of MARM2 was isolated after three rounds of selection, and its E protein exhibited the E370K mutation in DHL. The mutation abolishes neutralization by ZKA190, although the antibody can bind to mutant DIII (Fig. 14). Mutations in MARMS in vivo (T335R) and in vitro (E370K) are located on the BC and DE loops of DIII, respectively, and are consistent with the epitope identified by NMR.

Example 11 Development of bispecific antibodies according to the present invention.

Viral antibody-evasion mutants can significantly reduce the effectiveness of therapeutic antibodies. To overcome this problem, the inventors of the present invention hypothesized that the likelihood of virus escape would be significantly reduced by combining two highly neutralizing antibodies. In this regard, a series of bispecific antibodies have been generated that combine ZKA190 with other potent neutralizing mAbs directed to different sites on the E protein. Thus, the challenge focused on two mAbs, ZKA185 and ZKA230, that are highly neutralizing and do not compete with ZKA190.

First, their ability to cross-neutralize four ZIKV strains was analyzed as described above. ZKA185, and to a lesser extent ZKA230, strongly neutralized African, Asian and American strains with IC50 ranging from 0.02 to 0.62 nM (Fig. 19A). ZKA185 binds with high affinity to recombinant ZIKV E protein and to Zika virus-like particles (VLPs), but not to isolated DIII (Fig. 19B). Conversely, ZKA230 binds to ZIKV VLPs but not to recombinant E or DIII, suggesting that it recognizes a quaternary epitope displayed only on the viral surface (Fig. 19B). IgKA and Fab ZKA185 were shown by ELISA to bind to E antigens and VLPs with similar high affinity.

To identify the epitopes of ZKA185 and ZKA230, as well as their propensity to generate antibody escape mutants, MARMs against ZKA185 (MARM3) and ZKA230 (MARM4) were isolated by passaging the virus in the presence of sub-neutralizing concentrations of antibodies as described above. MARM3 contains substitutions at K84E and D67H, which are both located in DII (Fig. 19D). MARM4 showed a mixture of different amino acid substitutions at position 84 (K to G, E or R), confirmed in multiplex sequencing experiments. Additionally, MARMs 1 through 4 were tested against ZKA190, ZKA185, and ZKA230. ZKA190 neutralizes MARMs ZKA185 and ZKA230, as well as the parental virus (Fig. 19B). ZKA185 neutralizes both ZKA190 and ZKA230 MARM. ZKA230 neutralizes only ZKA190 MARM2 and does not neutralize either ZKA190 MARM1 or ZKA185 MARM3.

To gain insight into the development of MARMs capable of escape from multiple antibody pressure, ZKA190 MARM2 (E370K) was serially passaged in the presence of ZKA185 or ZK230. Thus, double MARMs were found to occur after 3–4 passages. ZKA230 contains an additional K84E mutation, while ZKA185 is selected for the D76G mutation. These data indicate that ZIKV can evade neutralization by multiple antibodies targeting different sites when selection is carried out in a stepwise manner and confirm the high plasticity of the ZIKV E protein.

Therefore, ZKA185 was chosen to be used along with ZKA190 to develop a bispecific antibody because it strongly cross-neutralizes ZIKV strains, binds to an alternative site, and does not compete with ZKA190. The bispecific antibody was produced in a tetravalent symmetric format called Fabs-in-tandem-Ig (FIT-Ig). FIT-Igs are described in detail, for example, in WO 2015/103072 A1 and in Gong S. et al., MAbs 2017.

FIT-Ig can be produced using three polypeptides. Polypeptide 1 typically contains an outer Fab light chain fused, without linkers, to the N-terminal region of an inner Fab heavy chain. Polypeptide 2 typically contains the heavy chain variable regions and CH1 regions of the outer Fab, and polypeptide 3 typically contains the light chain of the inner Fab. Accordingly, a FIT-Ig format antibody typically contains an internal Fab and an external Fab. Two types of FIT-Ig were produced with Fab ZKA190 in either the outer or inner position. Briefly, three genes encoding FIT-Ig were codon optimized, synthesized by Genscript, and cloned as follows: i) VL of the outer Fab followed by the full constant region (lambda or kappa) fused to VH of the inner Fab. and cloned into an Igy1 expression vector (modified to encode the LALA mutation). The resulting polypeptide 1 is formed by the VL and CL of the outer Fab, the VH of the inner Fab is fused to the IgG1 CH1hinge-CH2-CH3 domains; ii) the VH gene of the outer Fab (encoding polypeptide 2 formed by VH and CH1 of the outer Fab) is cloned into a Fab expression vector (Igy1 expression vector into which a stop codon is introduced after the codon encoding cysteine CH1 residue 220); iii) the VL gene of the internal Fab is cloned into IgK or Igλ expression vectors (encoding polypeptide 3 formed by the VL and CL of the internal Fab). Recombinant FIT-Ig mAbs were prepared by transiently transfecting EXPI293 cells (Invitrogen) using a 1:3:3 molar ratio of the three constructs described above (as described in WO 2015/103072 A1), purified by Protein A chromatography (GE Healthcare) and unsalted against the FSB. Proteins were analyzed by SDS-PAGE under reduced and unreduced conditions, and their concentrations were determined using BCA (Pierce, Rockford, USA). Under unreduced conditions, FIT-Ig moves as one large band of approximately 250 kDa. Under reducing conditions, each of the FIT-Ig proteins produces two bands, one with a higher molecular weight band representing polypeptide 1 of approximately 75 kDa, and a second, lower mass band corresponding to both polypeptides 2 and 3, overlapping at approximately 25 kDa. To further study the physical properties of FIT-Ig in solution, size exclusion chromatography (SEC) is used to analyze each protein. Purified FIT-Ig in PBS was applied to a Superdex 200 Increase 5/150 GL column. All proteins were detected using UV detection at 280 and 214 nm. FIT-Ig proteins exhibit a single major peak, indicating physical homogeneity as monomeric proteins.

Example 12 In vitro description of the antibody of the present invention (FIT-1).

A bispecific antibody FIT-Ig (herein referred to as FIT-1) with ZKA190 in the outer and ZKA185 in the inner positions of the Fab (FIG. 19E) was selected and further characterized. ELISA shows that FIT-1 binds DIII, E and VLP (Fig. 19E). FIT-1 retains high neutralizing activity against ZIKV strains, with IC50 values largely similar to those of the parental antibodies ZKA190 and ZKA185 (Fig. 19G). FIT-1 was produced using an IgG1 primary antibody in LALA format, thus eliminating any possibility of causing ADE. Some evidence suggests that both ZKA190 and ZKA185 fragments in the FIT-1 format are active. First, FIT-1 binds to the E protein with higher affinity than the parental antibodies ZKA190 and ZKA185 (KD values: ZKA185 1.8 nM, ZKA190 9.3 nM, and FIT-1 KD <1 pM due to higher low dissociation rate, presumably due to avidity effects). Second, FIT-1 effectively neutralized all MARMs ZKA190, ZKA185, and ZKA230 (Fig. 19H), in contrast to individual mAbs. The neutralizing activity of the FIT-1 Fab fragment (each containing one ZKA190 and one ZKA185) was reduced only about 6-fold (Fig. 19I, right panel).

The ability of FIT-1 to select MARMs was then tested (described above). However, despite eight rounds of serial passage, MARMs were not isolated. In contrast, MARMs appeared after 3–4 passages when individual mAbs were used. These results suggest that the use of FIT-1 as a therapeutic agent is safer because the likelihood of simultaneous mutations in both DIII and DII is less.

Confocal microscopy studies using Vero cells also showed that FIT1, like ZKA190, likely inhibits viral infection at a post-attachment stage, probably at the fusion stage (Fig. 19K).

Finally, FIT-1 as well as its Fab fragment block human plasma ADE against DENV2 or DV62 at concentrations above 0.1 nM and 10 nM, respectively (Fig. 19K), demonstrating that FIT-1 does not cause ADE and can block ADE monocytic cells by poorly neutralizing cross-reactive antibodies.

Example 13 Therapeutic potential of FIT-1 in vivo.

To evaluate the therapeutic potential of FIT-1, three different doses (15, 5, and 1 mg/kg) were administered at three different time points to female A129 mice after ZIKV infection. Briefly, female A129 mice are assigned to 10 different groups (control and three different dosage groups with three different administration time points for each dose). All animals were infected subcutaneously with 10 ² PFU of ZIKV (strain MP1751) and observed for 14 days. Mice from nine treatment groups were injected intraperitoneally with 500 μl of FIT-1 bispecific antibody diluted in PBS at doses of 15, 5, or 1 mg/kg on day 1, day 2, or day 3 after ZIKV infection. The weight and temperature of all animals are monitored daily, and clinical observations are recorded at least twice daily. On day 5 after infection, 50 μl of blood is collected from each animal into an RNAprotect tube (Qiagen, UK) and frozen at -80°C. At the end of the study (14 days postinfection) or when animals reach human endpoints, necropsies are performed and blood, brain and ovary sections are collected for virological analysis.

The results are presented in Fig. 20. Data show that at a dose of 15 mg/kg, survival was 100% without evidence of morbidity when treatment was given three days after infection (Fig. 20A); virus titers are nullified and no escape mutants were detected, indicating high in vivo efficacy. Administration of 5 mg/kg leads to a survival rate of 70-100%, and escape mutants are also not detected on day 5 after infection. The lowest dose tested (i.e. 1 mg/kg) was protective when administered on day 1 or 2 but on day 3 post-challenge.

Example 14: Effect of FIT-1 on disease in the AG129 mouse model of congenital infection.

The effect of FIT-1 treatment of infected pregnant dams on pups born to infected and treated mothers was assessed in a mouse model of congenital ZIKV infection, which produces live offspring after congenital ZIKV exposure. This model uses AG129 mice, which are deficient in IFN receptors, resulting in viral replication in the placenta, transmission of the virus to the fetus, and potential long-term consequences such as intrauterine growth restriction and hearing impairment.

The effect of FIT-1 treatment on various parameters is tested in the context of intrauterine infection. To this end, mice are infected with ZIKV 7 days post-coital (DPPA) and treated with FIT-1 1 or 3 days after virus infection (see Example 13). Various disease outcomes, such as fetal and placental viral titers and intrauterine growth restriction, are assessed to determine the effect of FIT-1 treatment.

Materials and methods.

Animals. 42 female AG129 mice were used. Animals are randomly assigned to experimental groups, and each animal is tagged with ear tags. Hormonal treatment is used to stimulate estrus. Females were individually housed with males and examined for vaginal plug at 0.5 days post-coital (DPPA).

Virus. The Zika virus strain Malaysia (P6-740) is used. The required dose of infection is administered subcutaneously in an amount of 0.1 ml.

Test agent. FIT-1 is administered at a dose of 45 mg/kg. The nonspecific control antibody MPE8-LALA Ctr IgG1 is used as an isotype control in placebo treatment.

Virus quantification. The virus titer in various tissues is quantified by real-time polymerase chain reaction (RT-PCR). Total RNA was extracted from tissue samples using TRIzol (ThermoFisher Scientific, catalog number 15596018). A volume of 2 μl of RNA preparation is used for amplification. Serial dilutions of synthetic RNA covering the region of amplification are used to obtain positive controls; undiluted synthetic ZIKV RNA contains 10 ^8.0 copies/μl. Samples are subjected to 40 cycles of 15 s at 95° and 60 s at 60°, after one initial cycle of 30 min at 50° and 10 min at 95°. Samples of unknown quantity are quantified by extrapolating C(t) values using a curve obtained from serial dilutions of synthetic ZIKV RNA.

Construction of the experiment. Pregnant females are challenged 7 days postcoital (DPPA) and treated with FIT-1 24 or 72 h after virus challenge. Two females from each group are necropsied 11 days after infection with the virus. Placenta, fetal tissue, brain tissue, and spleen tissue are collected for viral titer determination by qRT-PCR. Two females in each group give birth. The occipito-frontal diameter of the head (OF) and the length from the head to the end of the body (crown rump length - CRL) are recorded with a digital caliper; intrauterine growth of mice is determined by the formula: CRLxOF. The weight of puppies and females is measured at different times.

Statistical analysis. Survival data were analyzed using Wilcoxon log-rank survival analysis (Prism 5, GraphPad Software, Inc).

Results and discussion.

FIT-1 treatment is being evaluated in a mouse model of congenital infection and disease associated with ZIKV infection. Treatment with FIT-1 is protective in pregnant females, with the vast majority of treated females protected from death, regardless of when treatment is administered (FIG. 21). Females treated with a nonspecific negative control antibody had mortality rates similar to those observed in previous studies.

There is a tendency to improve the results of determining the average size of mice. The average size of the pups from FIT-1-treated females was higher than that of the MPE8-treated control females and was similar to that of the mock-infected animals (Fig. 22). This difference is less pronounced in relation to fetal weight, and for this indicator, close average values were obtained in all groups (Fig. 23). There was a trend toward increased placental weight in females treated with FIT-1 (FIG. 24).

The virus titer in different tissues is shown in Fig. 25. There was a significant reduction in viral RNA in the fetus and placenta of dams treated with FIT-1 (Fig. 25A and B, respectively). The reduction was particularly evident in placental tissues, with an approximately 5-log ₁₀ reduction in ZIKV RNA levels. Females treated with FIT-1 also had significantly reduced viral RNA (several log ₁₀ ) in the maternal spleen and brain compared to females treated with MPE8 (Fig. 25B and D, respectively).

Discussion.

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Overall, these data support a protective role for FIT-1 in preventing or treating disease in fetuses congenitally exposed to ZIKV. There is a trend towards improvement in fetal and placental size parameters with a significant reduction in viral titer in various tissues of the mother and fetus.

Example 15. Effect of FIT-1 on disease in the AG129 mouse model of testicular infection.

Sexual transmission and persistent infection of the male reproductive tract have been reported in men infected with Zika virus (D'Ortenzio E et al., N Engl J Med 2016; 374, 2016, 2195-2198). In murine AG129 mice, severe disease is usually observed approximately 2 weeks after virus infection, including significant viral replication in the mouse testes (Julander J.G. et al., Antiviral Res 137, 2016, 14-22). Key sites of viral replication in the reproductive tract of male AG129 mice include the epididymis and testes, as well as various associated gonads.

In the present study, the effect of FIT-1 on ZIKV-infected male mice was assessed in a testis infection model. To this end, male AG129 mice are infected with ZIKV and pathology in the male reproductive tract is assessed after ZIKV infection and FIT-1 treatment.

Materials and methods.

Animals. Male AG129 mice were used. Animals are randomly assigned to experimental groups and each animal is tagged with ear tags.

Virus. The Zika virus is Puerto Rican strain (PRVABC-59). The dose for infection 10 ² CCID ₅₀ is administered by subcutaneous injection into the inguinal fold in an amount of 0.1 ml. This dose of challenge is usually lethal in untreated AG129 mice, with death occurring approximately 2 weeks after challenge.

Test agent. FIT-1 is administered at a dose of 15 mg/kg. The nonspecific control antibody MPE8-LALA Ctr IgG1 is used as an isotype control (placebo) in treatment.

Histopathology. Tissues are collected and incubated for 24 hours in neutral buffered formalin. After appropriate fixation, all collected tissues were trimmed and stored in 70% ethanol until routine processing, paraffin embedding, and cutting were performed. All tissues are analyzed by an independent, blind veterinary pathologist and issued with a detailed certificate. A scoring system was developed to assess the severity of inflammation in the reproductive tract.

Construction of the experiment. Mice are infected with ZIKV and monitored for 28 days after virus infection for survival and weight changes. Treatment with 15 mg/kg FIT-1 is given 24 or 72 hours after virus infection. A single treatment is carried out by intraperitoneal injection of 0.1 ml. The isotype-matched MPE8-LALA Crt IgG1 control antibody was administered as described above 24 h after virus challenge. To control for toxicity, the study included a group of mock virus-infected mice treated with FIT-1, as well as a simple control group without any exposure. The individual weights of female mice are determined at point 0 and every other day for 7-21 days after DPPA. Mice are observed daily for signs of disease, including conjunctivitis, hunched posture, limb weakness, or paralysis, and initial signs of disease are recorded. A group of 3 animals is necropsied on day 6 of DPPA and tissue samples are collected for histopathological analysis.

Statistical analysis. Survival data were analyzed using Wilcoxon log-rank survival analysis (Prism 5, GraphPad Software, Inc).

Results and discussion.

Zika virus (ZIKV) can persist in male reproductive tissues for long periods of up to six months, providing an obvious target for antiviral treatment. To determine the effectiveness of a bispecific anti-ZIKV antibody in preventing or reducing pathology of the male reproductive tract, groups of mice were treated 24 or 72 h after infection with a Puerto Rican isolate of ZIKV virus. Survival, weight change, and histopathology are determined to assess the effectiveness of mAb treatment. As placebo therapy, mAb of the control isotype MPE8-LALA Ctr IgG1 is used.

A significant (P<0.05) improvement in survival was observed compared to placebo mAb treatment (FIG. 26). Complete survival was observed in mice treated 24 h postinoculation with FIT-1, although mice treated 72 h postinoculation had one animal that succumbed to viral infection. This single animal was sacrificed 24 days after virus challenge, which was much later than placebo-treated animals (FIG. 26). Because infection of AG129 mice with ZIKV results in lethality that is much more severe than normal natural infection with the virus, the high levels of protection observed with FIT-1 are very promising.

The change in mean weight in FIT-1-treated mice was similar to that in mice with mock-infected control treatments, while the mean weight of MPE8-treated mice decreased rapidly after 7 DPPA, further demonstrating the potent protection of mice against disease. 043940 vania (Fig. 27).

Because the primary goal of the present study is to evaluate the effect of treatment on the male reproductive tract, and in previous studies the testes and epididymis were most severely affected after infection, special attention was paid to the histopathology of these tissues. No evidence of disease was observed in the testes or epididymis of FIT1-treated mice, except for one animal in the 24-hour treatment group (FIG. 28), further demonstrating the effectiveness of FIT-1 treatment. FIT-1 also protected mice from disease when treatment was started 72 hours after virus challenge, as none of the mice treated at this time developed testicular or epididymal disease (FIG. 28). The majority of mice in the placebo group had inflammation of the testes (2/3) and epididymis (3/3) (Fig. 28), although the severity of the disease in this study was quite mild.

Conclusion.

Treatment with FIT-1 was effective in reducing all disease parameters assessed. Therapeutic treatment up to 72 hours after viral infection was very effective.

Example 16: Prophylactic and therapeutic efficacy of FIT-1 in ZIKV-infected rhesus monkeys.

This study evaluates the preventive and therapeutic efficacy of a bispecific antibody targeting Zika virus (ZIKV) in Indian rhesus macaques (IRM) infected with ZIKV strain PRVABC59. Sixteen (16) IRMs were randomized into three treatment groups. One group receives FIT-1 one day before infection (5 mg/kg), and the second receives treatment one day after infection (15 mg/kg). The third group received isotype control (FIT-3, 5 mg/kg) the day before infection. On day 0, all IRMs are infected with 1 x 10 ⁵ PFU of ZIKV strain PRVABC59 given by subcutaneous injection. Serum, urine, and saliva are collected at predetermined time points and ZIKV load measured by quantitative RT-PCR is determined.

Methods.

Prior to the start of the study (Day 0), sixteen (16) IRMs are randomized into appropriate groups according to sex/weight using Provantis software. On days -1 or 1, animals receive either FIT-1 or FIT-3 isotype control by intravenous administration at the dose indicated in Table 1. 4 below. On day 0, all macaques are anesthetized and challenged with 0.5 ml of wild-type ZIKV strain PRVABC59 with a target challenge dose of 1.0 x 10 ⁵ PFU per animal by subcutaneous injection. Blood, urine, and saliva samples are collected at predetermined time points indicated in Table 1. 5, to assess viral load using RT-qPCR.

Table 4

Dividing animals into groups

Group Treatment Day of treatment Method of treatment, dose Antigen ¹ stimulation 1 (3 males/ 3 females) FIT-1 Day -1 Intravenous administration, 5 mg/kg 2 (3 males/3 females) FIT-1 Day +1 Intravenous administration, 15 mg/kg 3 (2 males/2 females) FIT-3 Day -1 Intravenous administration, 5 mg/kg

1Subcutaneous stimulation with antigen on day 0 in the form of 0.5 ml of wild-type ZIKV.

Table 5

Key activities

Day of treatment -1 0 1 5 10 15 20 25 thirty Treatment ZIKV antigen stimulation (subcutaneous administration) Number of observations per day All animals are observed twice a day Body mass Daily l/ Body temperature Daily l/ V Taking blood for analysis ¹ Daily y/ y/ Taking urine/saliva samples for analysis 1 eleven 1 V Virus load: RT-qPCR Daily y/ y/ y/ Blood volume (ml) 2 2 2 2 20 Euthanasia l/

On days 0 and 1, blood is taken for analysis before antigen stimulation and before treatment.

On study days -1 (groups 1 and 3) and 1 (group 2), anesthetized animals were administered either FIT-1 (baseline concentration: 3.67 mg/ml) or FIT-3 (baseline concentration: 3.79 mg/ml ) by intravenous injection into the right saphenous or cephalic vein.

ZIKV strain PRVABC59; Human/2015/Puerto Rico (American isolate) is used in this study. The preparation of the virus inoculum is carried out in a class II biological safety cabinet

- 68 043940 in BSL-2 conditions. The starting material of the virus is thawed in a water bath at 37±1°, shaken and diluted with VP-SFM, obtaining an inoculum with an appropriate concentration of 2x105 PFU/ml. Each syringe is filled with 0.5 ml of virus inoculum and stored on ice until transported to the animal facility for administration. On study day 0, all animals are anesthetized, an incision is made at the injection site, wiped with alcohol and an indelible mark is applied. Animals are inoculated subcutaneously on the anterior surface of the left forearm with 0.5 ml of ZIKV isolate at the dose indicated in Table. 4.

All macaques are observed twice daily throughout the quarantine period and monitored for signs of illness or death. Animals are observed twice daily (at least 8 hours apart) for sensitivity and clinical signs, including rash, erythema, conjunctivitis, ocular discharge and swelling. For all animals, blood is collected at the time indicated in the table. 5, for in vitro viral load testing using RT-qPCR. On the 30th day, during terminal bleeding, blood is taken, no more than 20 ml from one animal. Urine is collected from all animals at the time indicated in the table. 5, for in vitro testing using the RT-qPCR method for monitoring virus shedding. Animals are kept in separate rooms during urine collection periods throughout the study. Urine is collected directly from cell trays, placed on melting ice after collection, aliquoted, and stored at -70°C or below until ready for analysis. Saliva is collected from anesthetized animals directly into tubes (up to approximately 0.5 ml). Samples are stored on melted ice after collection and stored at -70°C or lower until ready for analysis.

Viral loads were measured using RT-qPCR to detect ZIKV genomes in serum, urine, and saliva samples collected at the time points indicated in Table 1. 5. Specimens isolated from virus-infected animals are tested using primers and probes designed to detect ZIKV strain PRVABC59. A description of PCR methods, including primer and probe sequences, has been published (Goebel et al., J. Virol. 2016). Viral RNA is isolated from biological fluids using the QIAmp Viral RNA mini kit (Qiagen, 52906). Viral RNA is eluted with sterile RNase- and DNase-free water and stored at -70° or below. The lower limit of quantitation (LLOQ) of this assay was defined as 10 copies per reaction.

An aliquot of the infecting virus inoculum is back-titrated using a standard Vero cell plaque assay to confirm the dose actually delivered. Tenfold serial dilutions of challenge inoculum are used to infect confluent Vero cell monolayers in 6-well plates that were seeded the day before. The plates are incubated at 37°C and 5% CO ₂ for 1 hour before adding a top layer of medium containing 0.5% agarose. The plates are incubated for 3 days until visible plaques form, after which they are fixed, stained with crystal violet, and counted.

Results.

Macaques were monitored twice daily for morbidity and mortality throughout the study. All animals lived to the stated date. Body weight and temperature in all animals were measured at the time points indicated in Table. 5. No significant reduction in body weight was observed during the study. All animals had their body temperature maintained within the normal range throughout the study. Clinical observations included only moderate redness at the site of infection in a few animals.

Viral loads were measured using RT-qPCR (real-time quantitative polymerase chain reaction) to detect ZIKV genomes in serum, urine, and saliva samples collected at the time points indicated in Table 1. 5. Serum viral load is shown in FIG. 29.

Animals in group 3 receiving isotype control had detectable viral load in serum the day after infection. Viral loads in three of four animals peaked above 1x105 genome copies/ml (kg/ml) on either day 2 or day 3. The mean serum viral load peaked in this group on day 3 at 1.15x105 kg/ml. Viral loads decreased below the LLOQ assay (860 kg/ml) by day 4 in one animal and by day 5 in the remaining three.

Animals in group 2, receiving 15 mg/kg FIT-1 the day after infection, had an average viral load of 3.99x103 GC/ml before treatment. On day 2, the mean serum viral load decreased to 96.5 kg/ml. Although low levels of viral RNA were observed sporadically after day 1, none of the post-treatment control points showed viral loads in animals in group 2 at or below the LLOQ by RT-qPCR analysis.

Pretreatment with 5 mg/kg FIT-1 reduced mean serum viral loads on day 1 by more than 50-fold compared with animals in group 3. Viral loads were not detected at or above the LLOQ at any of the postchallenge control time points , and by the second day, viral RNA was not detected in the serum of any of the animals from group 1.

Only sporadic, low levels of ZIKV RNA have been detected in the urine or saliva of any

- 69 043940 animals. At no time point were viral loads found at or above

LLOQ in the urine or saliva of any animal.

An aliquot of the virus inoculum used for infection was back-titrated using a standard plaque count method on Vero cells to confirm the actual dose administered.

The plaque method showed a titer of 1.7 x 105 plaque-forming units/mL (PFU/mL).

Conclusion and conclusions.

This study evaluates the prophylactic and therapeutic efficacy of FIT-1 in ZIKV-infected IRM macaques. There was no mortality throughout the study, and clinical observations were limited to mild redness at the site of infection in a few animals. Viral load, determined by RT-qPCR in serum collected after infection, is the primary endpoint of the study. Viral RNA was clearly detectable in the serum of all isotype control-treated animals, with viral loads peaking on days 2 or 3 and maintained at levels above the LLOQ of 860 GC/ml until days 4 or 5. The average peak load is 1.15x10 ⁵ . GC/ml on day 3. In contrast, prophylactic treatment with 5 mg/kg FIT-1 effectively reduced peak viral loads and time to viral clearance in ZIKV-infected IRMs. Low levels of viral RNA were detected in all six animals in this group on the first day, but by the second day no viral RNA was detected in any of the animals. At no point was ZIKV RNA detected above LLOQ 860 GC/ml in the serum of any animal in this group. Likewise, therapeutic treatment with 15 mg/kg FIT-1 the day after infection reduced both peak viral loads and time to viral clearance from serum compared with isotype control animals. Macaques in group 2 had a mean peak viral load of 3.99x103 GC/mL on day 1, but after treatment on day 1, the mean serum viral load in this group decreased to 96.5 GC/mL by day 2 . There were no animals in this group whose viral loads exceeded LLOQ 860 GC/ml during the remaining observation period.

Table of sequences and their SEQ ID numbers

ZKA190 SEQ ID NO Amino acid sequences CDRH1 1 GFTFSKYG CDRH2 2 ISYEGSNK CDRH3 3 AKSGTQYYDTTGYEYRGLEYFGY CDRL1 4 QSVSSSY CDRL2 5 DAS CDRL2 6 LIYDASSRA long CDRL3 7 QQYGRSRWT VH 8 QVQLVESGGGVVQPGRSLRLSCAASGTFFSKYGMHWVRQAPGKGLEWVAVISYEG SNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGTQYYDTTGYE YRGLEYFGYWGQGTLVTVSS VL 9 EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQQKRGQAPRLLIYDASSR ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGRSRWTFGQGTKVEIK ZKA190 SEQ ID NO Nucleic acid sequences CDRH1 10 ggattcaccttcagtaaatatggc CDRH2 eleven atatcatatgagggaagtaataaa CDRH3 12 gcgaaatcggggacccaatactatgatactactggttatgagtataggg gtttggaatactttggctac CDRL1 13 cagagtgttagtagcagttac CDRL2 14 gatgcatcc CDRL2 15 ctcatctatgatgcatccagcaggggcc long CDRL3 16 cagcagtatggtaggtcaaggtggaca VH 17 caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggt ccctgagactctcctgtgcagcctctggattcaccttcagtaaatatgg catgcactgggtccgccaggctccaggcaaggggctggagtgggtggca gttatatcatatgagggaagtaataaatattatgcagactccgtgaagg gccgattcaccatct ccagagacaattccaagaacacgctgtatctgca aatgaacagcctgagagctgaggacacggcagtgtattactgtgcgaaa tcggggacccaatactatgatactactggttatgagtataggggtttgg aatactttggctactggggccagggaaccctggtcaccgtctcctcag VL 18 gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccagggg aaagagccaccctctcctgcagggccagtcagagtgttagtagcagtta cttagcctggtaccagcagaaacgtggccaggctcccaggctcctcatc tatgatgcatccagcagggccactggcatcccagacaggttcagtggca gtgggtctgg gacagacttcactctcaccatcagcagactggagcctga agattttgcagtgtattactgtcagcagtatggtaggtcaaggtggaca ttcggccaagggaccaaggtggaaatcaaac

- 70 043940

ZKA185 SEQ ID NO Amino acid sequences CDRH1 19 GYSFTSYW CDRH2 20 FDPSDSQT CDRH3 21 ARRYCSSSSCYVDN CDRL1 22 ALPNKF CDRL2 23 EDN CDRL2 long CDRL3 VH VL ZKA185 CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL2 Ion CDRL3 VH 24 25 26 27 SEQ ID 28 29 thirty 31 32 g 33 34 3i VIYEDNKRP YSTDSSSNPLGV EVQLVQSGAEVKKPGESLRISCKGSGYSFTSYWITWVRQMPGKGLEWMAKFDP SDSQTNYSPSFQGHVTISVDKSISTAYLQWSSLKASDTAMYYCARRYCSSSSC YVDNWGQGTLVTIFS SYELTQPPSVSVSPGQTARITCSGDALPNKFAYWYRQKSGQAPVLVIYEDNKR PSGIPERFSGSSSGTMATLTISGAQVEDEADYHCYSTDSSSNPLGVFGGGTKL TVL NO. Nucleic acid sequences ggatatagttttaccagttactgg tttgatcctagtgactctcaaacc gcgagaagatattgtagtagtagtagttgttatgtggacaat gcattgccaaataaattt gaggacaac gtcatctatgaggacaacaaacgaccc tactcaacagacagcagttctaatcccctgggagta Gaagtgcagctggtgcagtccggagcagaggtgaaaaagc

Ccggggagtctctgaggatctctcctgtaagggttctggata Tagttttaccagttactggatcacctgggtgcgccagatg Cccgggaaaggcctggagtggatggcgaagtttgatccta Gtgactctcaaaccaactacagcccgtccttccaaggcca Cgtcaccatctcagttgacaagtccatcagcactgcctac T tgcagtggagcagcctgaaggcctcggacaccgccatgt attactgtgcgagaagatattgtagtagtagtagttgtta tgtggacaattggggccagggaaccctggtcaccatcttc tcag

VL 36 Tcctatgagctgacacagccaccctcggtgtcagtgtcc Ccaggaaaacggccaggatcacctgctctggagatgca Ttgccaaataaatttgcttattggtaccggcagaagtca Ggccaggcccctgttctggtcatctatgaggacaacaaa Cgaccctccgggatccctgagagattctctggctccagc Tcagggacaatggcc accttgactatcagtggggcccag Gtggaggatgaagctgactaccactgttactcaacagac Agcagttctaatcccctgggagtattcggcggagggacc aagctgaccgtcctag ZKA230 SEQ ID NO Amino acid sequences CDRH1 37 GGSISSDY CDRH2 38 IYYSGST CDRH3 39 ARRRKYD S LWG S FAFDI CDRL1 40 SSNIGGNY CDRL2 41 IND CDRL2 long 42 LICINDHRP CDRL3 43 ATWDDSLGGLV VH 44 QVQLQESGPGLVKPSETLSLTCAVSGGSISSDYWSWIRQPPGKG LEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNHFSLKLNSVTAA DTAVYYCARRRKYDSLWGSFAFDIWGQGTMVTVSS VL 45 QSVLTQPPSASGTPGQRVTISCSGSSSNIGGNYVYWYQQLPGTA PKLLICINDHRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYY CATWDDSLGGLVFGGGTKLTVL ZKA230 SEQ ID NO Nucleic acid sequences CDRH1 46 ggtggctccatcagtagtgactac CDRH2 47 atctattacagtgggagcacc CDRH3 48 gcgaggagggaggaagtatgattccctttgggggagttttgcttttgatat c CDRL1 49 agctccaacatcggaggtaattat

- 71 043940

CDRL2 50 attaatgat CDRL2 long 51 ctcatctgtattaatgatcaccggccc CDRL3 52 gcaacatgggatgacagcctgggtggccttgta VH 53 caggtgcagctgcaggagtcgggcccaggcctggtgaagccttcggagac cctgtccctcacctgcgcagtctctggtggctccatcagtagtgactact ggagctggatccggcagcccccagggaagggactggagtggattgggtat atctattacagtgggagcaccaactacaacccctccctcaagagtcgagt caccatatcagtag acacgtccaagaaccacttctccctgaagctgaact ctgtgaccgctgcggacacggccgtgtattactgtgcgaggagggaggaag tatgattccctttgggggagttttgcttttgatatctggggccaagggac aatggtcaccgtctcttcag VL 54 cagtctgtgctgactcagccaccctcagcgtctgggacccccgggcagag ggtcaccatctcttgttctggaagcagctccaacatcggaggtaattatg tatactggtaccagcagctcccaggaacggcccccaaactcctcatctgt attaatgatcaccggccctcaggggtccctgaccgattctctggctccaa gtctggcacctca gcctccctggccatcagtgggctccagtccgaggatg aggctgattattactgtgcaacatgggatgacagcctgggtggccttgta ttcggcggagggaccaagctgaccgtcctag ZKA78 SEQ ID NO Amino acid sequences CDRH1 55 GFTFSNYA CDRH2 56 IGRNGDSI CDRH3 57 VKDLAIPESYRIEADY CDRL1 58 QSVLYRSNNNKNY CDRL2 59 W.A.S. CDRL2 long 60 LIYWASTRE CDRL3 61 QQYYSSPRT VH 62 EVQLAESGGGLVQPGGSLTLSCSGSGTFFSNYAMVWARQAPGKGLEYVSG IGRNGDSIYYTDSVKGRFTISRDNSKSMVYLQMSSLRTEDTAVYYCVKDL AIPESYRIEADYWGQGTLVIVSA VL 63 DIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNKNYLSWYQQKPGQPP KLLIYWASTRESGVPDRFSGSGSGTDFTLTISPLQAEDVAVYYCQQYYSS PRTFGQGTKVEIК ZKA78 SEQ ID NO Nucleic acid sequences CDRH1 64 ggcttcacttttagtaactatgca CDRH2 65 atcggggcgcaacggggactctatc CDRH3 66 gtgaaagatctggccatccccgagtcctacagaattgaagctgattat CDRL1 67 cagtccgtgctgtaccgctctaacaacaagaattac CDRL2 68 tggggcttca CDRL2 long 69 ctgatctattgggcttcaacccgggaa CDRL3 70 cagcagtactattctagtcctcgaact VH 71 gaggtgcagctggcagaatcaggcgggggactggtccagcctggcggca gcctgacactgtcttgcagtggatcaggcttcacttttagtaactatgc aatggtgtgggcaaggcaggctcctgggaagggactggagtatgtctct ggcatcgggcgcaacggggactctatctactatactgatagtgtgaagg gccggttcacca tcagcagagacaatagcaaatccatggtgtacctgca gatgagctccctgcgaaccgaagacacagcagtgtactattgcgtgaaa gatetggccatccccgagtcctacagaattgaagctgattattgggga c agggcaccctggtcatcgtgagcgccg VL 72 gacatcgtgatgacacagtctccagatagtctggcagtcagtctggggg agagggccactattaaetgeaagagetcccagtccgtgctgtaccgctc taacaacaagaattacctgtcttggtatcagcagaagcccggacagccc cctaaactgctgatctattgggcttcaacccgggaaagcggcgtcccag acagattct caggcagcgggtccggaacagacttcaccctgacaattag ccccctgcaggcagaggacgtggctgtctactattgtcagcagtactat tctagtcctcgaactttcggccaggggaccaaggtggaaatcaaac

-72043940

ZKA64 SEQ ID NO Amino acid sequence CDRH1 73 GYTFTGYH CDRH2 74 INPNSGGT CDRH3 75 ARMSSIWGFDH CDRL1 76 QSVLIN CDRL2 77 G.A.S. CDRL2 long 78 LIYGASSRA CDRL3 79 QQYNDWPPIT VH 80 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYHIDWVRQARGQGLEWMGRINPNS GGTNYAQKFQGRVTMTRDTSISTAYMQLSRLRSDDSAVYYCARMSSSIWGFDHWG QGTLVTVSS VL ZKA64 CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL2 long CDRL3 VH VL Konstag region IgGl CHl-( aa IgGl CHl-( LALA IgG CI IgG CI IgGl CHl-( nuc 81 SEQ II 82 83 84 85 86 87 88 89 90 1ТННЯ:т 2H2-CH: 2H2-CH: . aa C aa 1 aa 2H2-CH 1 E T 3 3 3 IVMTQSP7 GIPARFSC YU ggct atta gctc cagt ggag ctga cage cage agte tags atta agte cccc tcct cagt gaga aege ette gcat egga etgt cage SEQ ID NO 91 92 93 94 95 iTLSVSPGERATLLSCRASQSVLINLAWYQQKPGQAPRLLIYGASSRA JSGSGTEFTLTISSLQSEDFAVYYCQQYNDWPPITFGQGTRLEIK Nucleic acid sequence ;acaccttcacagggtatcac laccctaattctggcgggacc :ggatgagcteetctatttggggcttcgateat ;ctgtgctgattaac rcatcc itctatggagcatcctccagggct :agtacaatgattggccccctatcaca [tgcagctggtccagagcggagcagaggtgaagaaacccggcgcctc [aaggtcagctgcaaagcttccggctacaccttcacaggtatcaca ictgggtgaggcaggcaagaggacagggactggaatggatgggacgg laccctaattctggcgggaccaactacgcccagaagtttcagggccg [actatgaccagagacaccagcatct ccacagcttatatgcagctgt [getgagatctgaegatagtgeegtetaetattgtgctcggatgagc ictatttggggcttcgatcattgggggcagggaacactggtgactgt :tcag itcgtgatgactcagtctccagccaccctgtcagtcagcccaggaga [gcaaccctgtcttgcagagcctcccagtctgtgctgattaacctgg [gtaccagcagaagccaggccaggcaccccgactgctgatctatgga ;cctccagggctaccggcattcctgcacgcttcagtggatcaggaag lacagagtttaccctgaca atctctagtctgcagtccgaagacttcg :ctactattgtcagcagtacaatgattggccccctatcacatttggc [ggactagactggagatcaagc Sequences ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISR TPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK RTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADS SPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE GSTVEKTVAPTECS gcgtcgaccaagggcccatcggtcttccccctggcaccctcctcca agagcacctctgggggcacagcggccctgggctgcctggtcaagga ctacttccccgaacctgtgacggtctcgtggaactcaggcgccctg accagcggcgtgcacaccttcccggctgtcctacagtcctcaggac tctactccctcagcag cgtggtgaccgtgccctccagcagcttggg cacccagacctacatctgcaacgtgaatcacaagcccagcaacacc aaggtggacaagagagttgagcccaaatcttgtgacaaaactcaca catgcccaccgtgcccagcacctgaactcctggggggaccgtcagt cttcctcttccccccaaaacccaaggacaccctcatgatct cccgg acccctgaggtcacatgcgtggtggtggacgtgagccacgaAgaCc ctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataa tgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgt gtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggca aggagtaca agtgcaaggtctccaacaaagccctcccagcccccat cgagaaaaccatctccaaagccaaagggcagccccgagaaccacag gtgtacaccctgcccccatcccgggaggagatgaccaagaaccagg tcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc

- 73 043940

cgtggagtggggagagcaatgggcagccggagaacaactacaagacc acgcctcccgtgctggactccgacggctccttcttcctctatagca agctcaccgtggacaagagcaggtggcagcaggggaacgtcttctc atgctccgtgatgcatgaggctctgcacaaccactacacgcagaag agcctctccctgtccccggggta aa IgGl CH1-CH2-CH3 LALA nucl 96 gcgtcgaccaagggcccatcggtcttccccctggcaccctcctcca agagcacctctgggggcacagcggccctgggctgcctggtcaagga ctacttccccgaacctgtgacggtctcgtggaactcaggcgccctg accagcggcgtgcacaccttcccggctgtcctacagtcctcaggac tctactccctcagcag cgtggtgaccgtgccctccagcagcttggg cacccagacctacatctgcaacgtgaatcacaagcccagcaacacc aaggtggacaagagagttgagcccaaatcttgtgacaaaactcaca catgcccaccgtgcccagcacctgaaGCCGCGgggggaccgtcagt cttcctcttccccccaaaacccaaggacaccctcatgat ctcccgg acccctgaggtcacatgcgtggtggtggacgtgagccacgaagacc ctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataa tgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgt gtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggca aggagtacaag tgcaaggtctccaacaaagccctcccagcccccat cgagaaaaccatctccaaagccaaagggcagccccgagaaccacag gtgtacaccctgcccccatcccgggaggagatgaccaagaaccagg tcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc cgtggagtgggagagcaatgggcagccggagaacaactacaag acc acgcctcccgtgctggactccgacggctccttcttcctctatagca agctcaccgtggacaagagcaggtggcagcaggggaacgtcttctc atgctccgtgatgcatgaggctctgcacaaccactacacgcagaag agcctctccctgtccccgggtaaa IgG CKnucl 97 cgTacGgtggctgcaccatctgtcttcatcttcccgccatctgatg agcagttgaaatctggaactgcctctgttgtgtgcctgctgaataa cttctatcccagagaggccaaagtacagtggaaggtggataacgcc ctccaatcgggtaactcccaggagagtgtcacagagcaggacagca aggacagcacct acagcctcagcagcaccctgacgctgagcaaagc agactacgagaaacacaaagtctacgcctgcgaagtcacccatcag ggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt IgG CL nucl 98 ggtcagcccaaggctgccccctcggtcactctgttcccgccctcct ctgaggagcttcaagccaacaaggccacactggtgtgtctcataag tgacttctacccgggagccgtgacagtggcttggaaagcagatagc agccccgtcaaggcgggagtggagaccaccacaccctccaaacaaa gcaacaacaagtacg cggccagcagctatctgagcctgacgcctga gcagtggaagtcccacagaagctacagctgccaggtcacgcatgaa gggagcaccgtggagaagacagtggcccctacagaatgttca

ZKA3 SEQ ID NO Amino acid sequence CDRH1 99 GFIFSNYA CDRH2 100 IGGKGDSI CDRH3 101 VKD LAVLE S DRLEVD Q VH 102 EVQLAESGGGLVQPGGSLRLSCSGSGFIFSNYAMVWARQAPGKGLEYVS GIGGKGDSIYHIDSVKGRFTISRDNSKRTVYLQMSRLRTEDTAVYYCVK DLAVLESDRLEVDQWGQGTLVIVSA ZKA4 SEQ ID NO Amino acid sequence CDRH1 103 GFTFSSYV CDRH2 104 TSYDGSNK CDRH3 105 ARGPVPYWS GE S Y S GAY FD F VH 106 QVQLVESGGGVVQPGRSLRLSCAASGFFSSYVMHWVRQAPGKGLEWVT VTSYDGSNKYYADSVKGRFTISRDNAKNTLYLQMNSLRGEDTAIYYCAR GPVPYWSGESYSGAYFDFWGQGILVTVSS

- 74 043940

ZKA5 SEQ ID NO Amino acid sequence CDRH1 107 GFTFSNYY CDRH2 108 MSSSETIK CDRH3 109 ARSGIETVAGSIDYYGMDV VH 110 QVQLVESGGGLVKPGGSLRLSCAGSGTFFSNYYMTWIRQAPGKGLELVS YMSSSETIKYYADSVKGRFTISRDNAKNSLYLQMNSLRADDTARYYCAR SGIETVAGSIDYYGMDVWGHGTPVTVSS ZKA6 SEQ ID NO Amino acid sequence CDRH1 111 DFTVSNYA CDRH2 112 VSYDGSNK CDRH3 113 ATGVTMFQGAQTNAEYLHY VH 114 QVHLVESGGGVVQPGRSLRLSCEASDFTVSNYAMHWVRQAPGKGLEWVA WSYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTALYYCAT GVTMFQGAQTNAEYLHYWGQGSLVTISS ZKA7 SEQ ID NO Amino acid sequence CDRH1 115 GFTFSRYG CDRH2 116 VSGDGSST CDRH3 117 VKDFWSGDQSLESDF VH 118 EVQLVESGGGLVQPGGSLRLSCSASGTFFSRYGMVWARQAPGKGLEYLS GVSGDGSSTYYANSVKGRFTISRDNSKNTLYLHMSRLRDEDTAMYYCVK DFWSGDQSLESDFWGQGALVTVS S ZKA8 SEQ ID NO Amino acid sequence CDRH1 119 GFTFSAHA CDRH2 120 ISNEDYT CDRH3 121 VKDFGTSPQTDF VH 122 DERLVESGGGLVQPGGSLRLVCSASGFTFSAHAMHWVRQPPGKGLEYVS TISRNEDYTYYADSVKGRFTISRDNSKNSLYLQMRRLRPEDTAIYYCVK DFGTSPQTDFWGQGTLVAVSS ZKA76 SEQ ID NO Amino acid sequence CDRH1 123 GFTFSTYF CDRH2 124 ISSTGSYK CDRH3 125 ARPFHSEYTYGLDAFDI VH 126 EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYFMHWVRQAPGKGLEWVA SISSTGSYKFYADSVKGRFTISRDNTKNSLFLQMNSLRAEDTAVFYCAR PFHSEYTYGLDAFDIWGQGTMLTVS S ZKA117 SEQ ID NO Amino acid sequence CDRH1 127 GGSIRTNSY CDRH2 128 ISYSGST CDRH3 129 ARLNDGSTVTTSSYFDY VH 130 QLQLQESGPGLVKPSETLSLTCTVSGGSIRRTNSYWGWIRQTTGKGLQW IGSISYSGSTFYNPSLKSRVTISLDTSKDHFSLELSSVTAADTAIYYCA RLNDGSTVTTSSYFDYWGQGTLVTVSS ZKB27 SEQ ID NO Amino acid sequence CDRH1 131 GYSFTSSW CDRH2 132 IDPSDSYT CDRH3 133 ARHDYSVSENGMDV VH 134 EVQLVQSGAEVKKPGESLRISCKASGYSFTSSWINWVRQMPGKGLEWMG RIDPSDSYTTYNPSFQGHVTISVDKSIGTAYLQWNSLRASDTAMYYCAR HDYSVSENGMDVWGQGTTVTVS S ZKB29 SEQ ID NO Amino acid sequence

- 75 043940

CDRHL 135 GFTFSSYT CDRH2 136 ISYDGSHK CDRH3 137 ARRSYSISCFDY VH 138 QVQLVESGGGVVQPGRSLRLSCAASGFFSSYTMHWVRQAPGKGLEWVA VISYDGSHKFYADSVKGRFTISRDNSKDTLYLQMNSLRAEDTALYYCAR RSYSISCFDYWGQGTLVTISS ZKB34 SEQ ID NO Amino acid sequence CDRHL 139 GFTFSRSG CDRH2 140 VSYDGSNK CDRH3 141 AKD L TMVRGVHΥ Υ Y YVMDV VH 142 QVQLVESGGGVVQPGRSLRLSCAASGFTFSRSGMHWVRQAPGKGLEWVA WSYDGSNKYYSDSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCAK DLTMVRGVHYYYYVMDVWGQGTTVTVS S ZKB39 SEQ ID NO Amino acid sequence CDRHL 143 GYTFDDYY CDRH2 144 INPHRGGT CDRH3 145 VRDQYCDGGNCYGIHQPHYGMDV VH 146 QVQLVQSGAEVKKPGASLKVSCKASGYTFDDYYIHWVRQAPGQGLEWLG RINPHRGGTNYAQKFQGRVIMTLDMSISTTYMELRRITSDDAAVYYCVR DQYCDGGNCYGIHQPHYGMDVWGQGTTVTVSS ZKB46 SEQ ID NO Amino acid sequence CDRHL 147 GYSFTSYW CDRH2 148 IDPSDSYT CDRH3 149 ARREYSSSSGQEDWFDP VH 150 EVQLVQSGAEVKKPGESLRISCKGSGYSFTSYWISWVRQMPGKGLEWMG RIDPSDSYTNYSPSFQGHVTISADKSISTAYLQWSSLKASDTAMYYCAR REYSSSSGQEDWFDPWGQGTLVTVSS ZKB53 SEQ ID NO Amino acid sequence CDRHL 151 GFTFSSYA CDRH2 152 ISYDGSNR CDRH3 153 ARHVEQLPSSGYFQH VH 154 QVQLVESGGGVVQPGRSLRLSCAASGFFSSYAMHWVRQTPGKGLEWVT VISYDGSNRYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCAR HVEQLPSSGYFQHWGQGTLVTVSS ZKC26 SEQ ID NO Amino acid sequence CDRHL 155 GFIFSDFY CDRH2 156 IGHDGSYI CDRH3 157 ARAHGGFRH VH 158 QVQVVESGGGLVKPGGSLRLSCAASGFIFSDFYMSWMRQAPGKGLEWVA YIGHDGSYILYADSVKGRFTISRDNAKNSLFLRMNSLRVEDTAVYYCAR AHGGFRHWGQGTVVAVS P ZKD5 SEQ ID NO Amino acid sequence CDRHL 159 GFTFTSYG CDRH2 160 ISYDGSNK CDRH3 161 ARDRDHYDLWNAYTFDY VH 162 QVQLVESGGGVVQPGRSLRLSCAASGFFTSYGMHWVRQTPGKGLDWVA VISYDGSNKYYADSVKGRFTISRDNSKDTLYLQMNSLRAADTALYYCAR DRDHYDLWNAYTFDYWGQGTLVTVS S ZKD7 SEQ ID NO Amino acid sequence CDRHL 163 GFTFSNYA

- 76 043940

CDRH2 164 ISYDVSDK CDRH3 165 AGGPLGVWIKPSNAEHFHH VH 166 QVQLVESGGGVVQPGRSLRLSCAASGTFFSNYAMHWVRQAPGKGLEWVA VISYDVSDKYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAAYYCAG GPLGVWIKPSNAEHFHHWGQGTLVTVSS ZKD8 SEQ ID NO Amino acid sequence CDRH1 167 GFTFINYA CDRH2 168 ISYDGSNK CDRH3 169 ATDADAY GD S GANFHY VH 170 QVQLVESGGGVVQPGKSLRLSCAASGFTFINYAIHWVRQAPGKGLEWVA VISYDGSNKFYTDSVKGRFTISRDNSKNTLYLQMNSLRADDTAVYYCAT DADAYGDSGANFHYWGQGTLVTVSS ZKD15 SEQ ID NO Amino acid sequence CDRH1 171 DASISSGGFS CDRH2 172 IYSSGDT CDRH3 173 ARAHTPTSKFYYYYAMDV VH 174 QLQLQESGSGLVKPSQTLSLTCTVSDASISSGGFSWSWIRQPLGKGLEW LGYIYSSGDTFYNPSLQGRVTMSVDIFRSQFSLKLTSVTAADTAMYYCA RAHTPTSKFYYYYAMDVWGQGTTVTVS S ZKD16 SEQ ID NO Amino acid sequence CDRH1 175 GFTFSDHF CDRH2 176 SRNKPNSYTT CDRH3 177 AKVGGCYGGDCHVENDY VH 178 EVQLVESGGDLVQPGGSLRLSCVASGFTFSDHFMDWVRQAPGKGLEWVG RSRNKPNSYTTEYAASVKGRFSISRDDSKKALYLQMNSLQTEDTAVYYC AKVGGCYGGDCHVENDYWGQGTLVTVSS ZKD17 SEQ ID NO Amino acid sequence CDRH1 179 GFIFSDYA CDRH2 180 ISYDGSSR CDRH3 181 ARGYCSSGTCFSTNAEYFHP VH 182 QVQMVE S GGGVVQ P GT S LRL S CAT S GFIFSDYAMHWVRQAP GKGLEWVA VISYDGSSRLYADSVKGRFTVSRDNSKNTLYLQMHSLRAGDTAVYYCAR GYCSSGTCFSTNAEYFHPWGQGTLATISS ZKD20 SEQ ID NO Amino acid sequence CDRH1 183 GFTFSDHF CDRH2 184 SRNKPNSYTT CDRH3 185 ARVGGCNGGDCHVENDY VH 186 EVQLVESGGGLVQPGGSLRLSCVASGFTFSDHFMDWVRQAPGKGLEWVG RSRNKPNSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLQTEDTAVYYC ARVGGCNGGDCHVENDYWGQGTLVTVSS ZKA134 SEQ ID NO Amino acid sequence CDRH1 187 GGTFSAYA CDRH2 188 IIPFFGTA CDRH3 189 ARSDIVSTTRGYHHYGMDV VH 190 QVHLVQSGAEVKKPGSSVNVSCKASGGTFSAYAISWVRQAPGQGLEWMG GIIPFFGTAYYAQKFKGRVTVTADKSTSTVYMEMTSLRSEDTAVYYCAR SDIVSTTRGYHHYGMDVWGQGTTVTVS S ZKA246 SEQ ID NO Amino acid sequence CDRH1 191 GYTFSDYY

- 77 043940

CDRH2 192 INPYSGGT CDRH3 193 ARGFTMISDREFDP VH 194 QVQLVQSGAEVKRPGASVKVSCKASGYTFSDYYMHWVRQAPGQGLEWMG RINPYSGGTNYAQKFHGRVTVTRDTSISTVYMELRGLRSDDTAVYYCAR GFTMISDREFDPWGQGTLVTVSS ZKA256 SEQ ID NO Amino acid sequence CDRH1 195 GFTFSTYW CDRH2 196 IKQDGSEK CDRH3 197 ARDPGYDDFWSGSYSGSFDI VH 198 EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYWMTWVRQAPGKGLEWVA NIKQDGSEKYYVDSVKGRFTISRDNTKNSLYLQVNSLRAEDTAIYYCAR DPGYDDFWSGSYSGSFDIWGQGTMVTVS S ZKB42 SEQ ID NO Amino acid sequence CDRH1 199 GFTFNNYG CDRH2 200 ISYDGNKK CDRH3 201 VKYGERINGYSDPFDH VH 202 QVQVVESGGGVVQPGRSLRLFCAASGFTFNNYGMHWVRQAPGKGLEWVA LISYDGNKKYYADSVKGRFSISRDNSKNTLYLQMNRLRSGDTAVYHCVK YGERINGYSDPFDHWGQGTLVTVSS ZKB85 SEQ ID NO Amino acid sequence CDRH1 203 GYTFTTYA CDRH2 204 INTNTGNP CDRH3 205 ARVIVPYAFDI VH 206 QVQLVQSGSELKKPGASVKVSCKASGYTFTTYAMNWVRQAPGQGPEWVG WINTNTGNPTYAQGFTGRFVLSLDTSVSTAFLQISSLKAEDTAVYYCAR VIVPYAFDIWGQGTMVTVS S ZKB47 SEQ ID NO Amino acid sequence CDRH1 207 GYTFTNYY CDRH2 208 INPSGGPT CDRH3 209 ARDQYGGYARYGMDV VH 210 QVQLVQSGAEVKKPGASVKVSCQASGYTFTNYYMHWVRQAPGQGLEWMG IINPSGGPTSYAQKFQGRVTMTTDTSTSTVYMELSSLRSEDTAVYYCAR DQYGGYARYGMDVWGQGTTVTVS S ZKC6 SEQ ID NO Amino acid sequence CDRH1 211 GYTFTGYY CDRH2 212 INPNSGGT CDRH3 213 ARVSDWGFAFDI VH 214 QVQLVQSGTEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMG RINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSGLRSDDTAVYYCAR VSDWGFAFDIWGQGTMVTVSQ ZKA160 SEQ ID NO Amino acid sequence CDRH1 215 GGSITSYS CDRH2 216 IFYSGST CDRH3 217 ARDQTMPVWVGGMDV VH 218 QVQLQESGPGLVKPSETLSLTCTVSGGSITSYSWSWIRQPPGKGLEWIG YIFYSGSTDYNPSLKSRVTISVDTSKDQFSLRLRSVTAADTAVYYCARD QTMPVWVGGMDVWGQGTTVTVS S ZKA172 SEQ ID NO Amino acid sequence CDRH1 219 GYIFTRYW CDRH2 220 IDPSDSYT CDRH3 221 ARQE TARED GMAV

- 78 043940

VH 222 EVQLVQSGAEVKKPGKSLRISCKGSGYIFTRYWISWVRQMPGKGLEWMG RIDPSDSYTNYSPSFQGHVTISADKSISTAYLQWSSLKASDTAMYYCAR QETAREDGMAVWGQGTTVTVSS ZKA174 SEQ ID NO Amino acid sequence CDRH1 223 GGSMSNSYYH CDRH2 224 IYYSGST CDRH3 225 ARNPVFNPLTLTHDAFDI VH 226 QLQLQESGPGLVKPSETLSLTCTVSGGSMSNSYYHWGWIRQPPGKGLEW IGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCA RNPVFNPLTLTHDAFDIWGQGTMVTVS S ZKA189 SEQ ID NO Amino acid sequence CDRH1 227 GFTFSSYA CDRH2 228 ISGSGDNT CDRH3 229 AKWPYYDFWSGSESYFDP VH 230 GVQLLESGGALVQPGKSLRLSCAASGFFSSYALTWVRQAPGKGLQWVS AISGSGDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK WPYYDFWSGSESYFDPWGQGTLVTVSS ZKA195 SEQ ID NO Amino acid sequence CDRH1 231 GYNFPSYW CDRH2 232 IDPSDSYT CDRH3 233 ARADCRSTSCYLVFE VH 234 EVQLVQSGAEVKKPGESLRISCKDSGYNFPSYWIHWVRQMPGKGLEWMG TIDPSDSYTNYSPSFQGHVTISADKSISTAYLQWSSLKASDTAMYYCAR ADCRSTSCYLVFEGQGTLVTVSS ZKA215 SEQ ID NO Amino acid sequence CDRH1 235 GYTFTSYW CDRH2 236 IDPSDSHT CDRH3 237 ARHALPNYFDS VH 238 EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWISWVRQMPGKGLEWMG RIDPSDSHTDYSPSFQGHVTISADKSISAAYLQWSSLKASDTAMYYCAR HALPNYFDSWGQGTLVTVS S ZKA218 SEQ ID NO Amino acid sequence CDRH1 239 GFPFSSYW CDRH2 240 INSDGRNT CDRH3 241 ARGGYDYDSSGCFDY VH 242 EVQLVESGGGLVQPGGSLRLSCAASGFPFSSYWMHWVRQAPGKGLVWVS RINSDGRNTNYADSVKGRFTISRDNAENTVYLQMNSLRAEDTAVYYCAR GGYDYDSSGCFDYWGQGTLVTVSS ZKB75 SEQ ID NO. Amino acid sequence CDRH1 243 GFTFSNYA CDRH2 244 ISGTGGST CDRH3 245 AKDSASRGGYCSGGVCYLNPGHHDY VH 246 EVQVLESGGGLLQPGGSLRLSCAASGTFFSNYAMSWVRQAPGKGLEWVS TISGTGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DSASRGGYCSGGVCYLNPGHHDYWGQGTLVTVSS ZKB83 SEQ ID NO Amino acid sequence CDRH1 247 GYSFTNYW CDRH2 248 IDPSDSYT CDRH3 249 ARLRGSLYCSGGRCYSVPGETPNWFDP VH 250 EVQLVQSGAEVKKPGESLRISCKGSGYSFTNYWITWVRQMPGKGLEWMG SIDPSDSYTNYSPSFQGHVTISADWSINTAYLQWSSLKASDTAKYYCAR LRGSLYCSGGRCYSVPGETPNWFDPWGQGTLVTVSS ZKC3 SEQ ID NO Amino acid sequence CDRH1 251 GGSITSYY CDRH2 252 IYYSGST CDRH3 253 ARVGGAPYYYYGMDV VH 254 QVQLQESGPGLVKPSETLSLTCTVSGGSITSYYWSWIRQPPGKGLEWIG YIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARV GGAPYYYYGMDVWGQGTTVTVS S ZKC18 SEQ ID NO Amino acid sequence CDRH1 255 GFTFGDYA CDRH2 256 IRSKAYGGTT CDRH3 257 SRDHTGTTYAFDI VH 258 EVQLVESGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVG FIRSKAYGGTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYC SRDHTGTTYAFDIWGQGTMVTVSQ ZKD1 SEQ ID NO Amino acid sequence CDRH1 259 GFTFSSYG CDRH2 260 IWYDGSNK CDRH3 261 ARDRRGY GD YVGY Y Y GMDV VH 262 S

- 79 043940

Designation SEQ ID NO Amino acid sequence ZIKV EDIII generic 263 TAAFTFTKXPAEXXHGTVTVEXQYXGXDGPCKXPXQMAVDXQTLTPV GRLITANPVITEXTENSKMMLELDPPFGDSYIVIGXGXKKITHHWHR S ZIKV H/PF/2013 EDIII 264 TAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPV GRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHR S ZIKV EDIII generic 265 x ₁ gx ₂ x ₃ yslctaaftftkx ₄ paex ₅ x ₆ hgtvtvex ₇ qyx ₈ gx ₉ dg PCKXwPXnQMAVDXnQTLTPVGRLITANPVITEXnTXnNSKM MLELDPPFGDSYIVIGX1 ₅ GX1 ₆ X1 ₇ KITHHWHRSG where XI can be any (natural no) amino acid, preferably K, A or E, X2 can be any -either a (natural) amino acid, preferably V, F or L, X3 can be any (natural) amino acid, preferably S or F, X4 can be any (natural) amino acid, preferably I or V, X5 can be any - either a (natural) amino acid, preferably T or V, X6 can be any (natural) amino acid, preferably L or D, X7 can be any (natural) amino acid, preferably V or G, X8 can be any a (natural) amino acid, preferably A or G, X9 can be any (natural) amino acid other than R, preferably T or A, XY can be any (natural) amino acid, preferably V or I, XII can be any -either a (natural) amino acid, preferably A or V, X12 can be any (natural) amino acid, preferably M or T, XI3 can be any (natural) amino acid, preferably S or G, X14 can be any (natural) amino acid, preferably E or K, XI5 can be any (natural) amino acid, preferably V or I, X16 can be any (natural) amino acid, preferably E, A, K or D, X17 may be any (natural) amino acid, preferably E, A or K, more preferably K or A Zika-E-Fl primer 266 TGCAAACGCGTCGCAAACCTGGTTG ZIKV-E-R1 primer 267 CGTGCCAAGGTAATGGAATGTCGTG ZIKV-Efl530 primer 268 AGCCTAGGACTTGATTGTGAACCGA ZIKV-E-R2769 primer 269 T TACAGAT C C CACAAC GAC C GT CAG ZIKV-E-F2 270 ACTTGGTCATGATACTGCTGATTGC ZIKV-E-R2 271 TCGGTTCACAATCAAGTCCTAGGCT ZIKV-E-f2058 272 GCTAACCCCGTAATCACTGAAAGCA ZIKV-E-r2248 273 AAGACTGCCATTCTCTTGGCACCTC

*Sequences in bold are CDR regions (nucleotide or amino acid (aa)) and underlined residues are mutated residues compared to the germline sequence.

LIST OF SEQUENCES <110> HUMABS BIOMED CA <12 0> MULTI-SPECIFIC ANTIBODIES SPECIFIC TO ZIKA VIRUS EPITOPES AND THEIR APPLICATION <130> HB01P034WO1 <160>273 <170> Patentin version 3.5 <210>1 <211>8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA190 CDRH1 <400> 1

-80043940

Gly Phe Thr Phe Ser Lys Tyr Gly <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA190 CDRH2 < 400>2

Ile Ser Tyr Glu Gly Ser Asn Lys <210>3 <211>23 <212> PRT <213> Artificial Sequence <220>

<223> ZKA190 CDRH3 <400>3

Ala Lys Ser Gly Thr Gln Tyr

Tyr Asp Thr Thr Gly Tyr Glu Tyr Arg

1015

Gly Leu Glu Tyr Phe Gly Tyr <210>4 <211>7 <212> PRT <213> Artificial Sequence <220>

<223> ZKA190 CDRL1 <400>4

Gln Ser Val Ser Ser Ser Tyr <210>5 <400>5

000 <210>6 <211>9 <212> PRT <213> Artificial Sequence <220>

<223> ZKA190 CDRL2 long <400> 6

Leu Ile Tyr Asp Ala Ser Ser Arg Ala

5

- 81 043940 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> ZKA190 CDRL3 <400>7

Gln Gln Tyr Gly Arg Ser Arg Trp Thr <210>8 <211>130 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA190 VH < 400>8

Gln 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Val Val Gln Pro Gly 15 Arg Ser Leu Arg Leu 20 Ser Cys Ala Ala Ser 25 Gly Phe Thr Phe Ser 30 Lys Tyr Gly Met His 35 Trp Val Arg Gln Ala 40 Pro Gly Lys Gly Leu 45 Glu Trp Val Ala Val 50 Ile Ser Tyr Glu Gly 55 Ser Asn Lys Tyr Tyr 60 Ala Asp Ser Val Lys 65 Gly Arg Phe Thr Ile 70 Ser Arg Asp Asn Ser 75 Lys Asn Thr Leu Tyr 80 Leu Gln Met Asn Ser 85 Leu Arg Ala Glu Asp 90 Thr Ala Val Tyr Tyr 95 Cys Ala Lys Ser Gly 100 Thr Gln Tyr Tyr Asp 105 Thr Thr Gly Tyr Glu 110 Tyr Arg Gly Leu Glu 115 Tyr Phe Gly Tyr Trp 120 Gly Gln Gly Thr Leu 125 Val Thr Val Ser Ser 130

< 210>9 < 211>108 < 212> PRT < 213> Artificial Sequence <220>

<223> ZKA190 VL <400> 9

- 82 043940

Glu Ile Val 1

Glu Arg Ala

Tyr Leu Ala 35

Leu

Thr 20

Trp

Thr 5

Leu

Tyr

Gln

Ser

Gln

Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1015

Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 2530

Gln Lys Arg Gly Gln Ala Pro Arg Leu Leu 4045

Ile Tyr Asp Ala Ser Ser Arg Ala Thr Gly Ile

55

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 85 90

Pro 60

Ile

Tyr

Asp Arg

Phe Ser

Ser Arg

Gly Arg

Leu Glu

Ser Arg 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220>

<223> ZKA190 CDRH1 <400> 10 ggattcacct tcagtaaata tggc <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220>

<223> ZKA190 CDRH2 <400> 11 atatcatatg agggaagtaa taaa <210> 12 <211> 69 <212> DNA <213> Artificial Sequence <220>

<223> ZKA190 CDRH3 <400> 12 gcgaaatcgg ggacccaata ctatgatact actggttatg agtatagggg tttggaatac tttggctac <210> 13 <211> 21 <212> DNA <213> Artificial Sequence

- 83 043940 <220>

<223> ZKA190 CDRL1 <400>13 cagagtgtta gtagcagtta c21 <210>14 <400>14

000 <210>15 <211>27 <212> DNA <213> Artificial Sequence <220>

< 223> ZKA190 CDRL2 long < 400>15 ctcatctatg atgcatccag cagggcc27 < 210>16 < 211>27 < 212> DNA < 213> Artificial Sequence <220>

< 223> ZKA190 CDRL3 < 400>16 cagcagtatg gtaggtcaag gtggaca27 < 210>17 < 211>391 < 212> DNA < 213> Artificial Sequence <220>

<223> ZKA190 VH <400> 17

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt aaatatggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atatcatatg agggaagtaa taaatattat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agctgaggac acggcagtgt attackgtgc gaaatcgggg 300 acccaatact atgatactac tggttatgag tataggggtt tggaatactt tggctactgg 360 ggccagggaa ccctggtcac cgtctcctca g 391

<210> 18 <211> 325 <212> DNA <213> Artificial Sequence <220>

<223> ZKA190 VL <400> 18

- 84 043940 gaaattgtgt ctctcctgca cgtggccagg gacaggttca cctgaagatt caagggacca tgacgcagtc gggccagtca ctcccaggct gtggcagtgg ttgcagtgta aggtggaaat tccaggcacc gagtgttagt cctcatctat gtctgggaca ttactgtcag ca aac ctgtctttgt agcagttact gatgcatcca gacttcactc cagtatggta ctccagggga tagcctggta gcagggccac tcaccatcag ggtcaaggtg aagagccacc ccagcagaaa tggcatccca cagactggag gacattcggc

120

180

240

300

325 < 210> 19 < 211> 8 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKA185 CDRH1 < 400>19

Gly Tyr Ser Phe Thr Ser Tyr Trp < 210>20 < 211>8 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKA185 CDRH2 < 400>20

Phe Asp Pro Ser Asp Ser Gln Thr < 210>21 < 211>14 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKA185 CDRH3 < 400>21

Ala Arg Arg Tyr Cys Ser Ser Ser Ser Cys Tyr Val Asp Asn

510 < 210>22 < 211>6 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKA185 CDRL1 < 400> 22

Ala Leu Pro Asn Lys Phe

5 <210> 23

- 85 043940 <400>23

000 <210>24 <211>9 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA185 CDRL2 long < 400>24

Val Ile Tyr Glu Asp Asn Lys Arg Pro < 210>25 < 211>12 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKA185 CDRL3 < 400>25

Tyr Ser Thr Asp Ser Ser Ser Asn Pro Leu Gly Val

510 <210>26 <211>121 <212> PRT <213> Artificial Sequence <220>

<223> ZKA185 VH <400> 26

Glu Val Gln Leu Val Gln Ser Gly 1 5

Ala Glu Val Lys Lys Pro Gly Glu

15

Ser Leu Arg Ile Ser Cys Lys Gly 20

Ser Gly Tyr Ser Phe Thr Ser Tyr 25 30

Trp Ile Thr Trp Val Arg Gln Met

40

Pro Gly Lys Gly Leu Glu Trp Met 45

Ala Lys Phe Asp Pro Ser Asp Ser

55

Gln Thr Asn Tyr Ser Pro Ser Phe 60

Gln Gly His Val Thr Ile Ser Val 65 70

Asp Lys Ser Ile Ser Thr Ala Tyr

80

Leu Gln Trp Ser Ser Leu Lys Ala 85

Ser Asp Thr Ala Met Tyr Tyr Cys

95

Ala Arg Arg Tyr Cys Ser Ser Ser

100

Ser Cys Tyr Val Asp Asn Trp Gly

105 110

- 86 043940

Gln Gly Thr Leu Val Thr Ile Phe 120 Ser 115 <210> 27 <211> 109 <212>PRT <213> Artificial Sequence <220> <223> ZKA185 VL <400> 27 Ser Tyr Glu Leu Thr Gln Pro 1 5 Pro Ser Val 10 Thr Ala Arg Ile Thr Cys Ser 20 Gly Asp 25 Ala Tyr Trp Tyr Arg Gln Lys Ser 35 Gly 40 Gln Ala Glu Asp Asn Lys Arg Pro Ser 50 55 Gly Ile Pro Ser Ser Gly Thr Met Ala Thr 65 70 Leu Thr Ile Asp Glu Ala Asp Tyr His Cys 85 Tyr Ser Thr 90 Leu Gly Val Phe Gly Gly Gly 100 <210> 28 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ZKA185 CDRH1 <400> 28 ggatatagtt ttaccagtta ctgg <210> 29 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ZKA185 CDRH2 <400> 29 tttgatccta gtgactctca aacc <210> 30 <211> 42 <212> DNA <213> Artificial Sequence Thr Lys 105 Leu

Val Ser Pro Gly Gln

Pro Asn Lys Phe Ala 30

Val Leu Val Ile Tyr 45

Arg Phe Ser Gly Ser 60

Gly Ala Gln Val Glu

Ser Ser Ser Asn Pro

Val Leu

- 87 043940 <220>

<223> ZKA185 CDRH3 <400>30 gcgagaagat attgtagtag tagtagttgt tatgtggaca at <210>31 <211>18 <212> DNA <213> Artificial Sequence <220>

< 223> ZKA185 CDRL1 < 400>31 gcattgccaa ataaattt < 210>32 < 400>32

000 < 210>33 < 211>27 < 212> DNA < 213> Artificial Sequence <220>

< 223> ZKA185 CDRL2 long < 400>33 gtcatctatg aggacaacaa acgaccc27 < 210>34 < 211>36 < 212> DNA < 213> Artificial Sequence <220>

< 223> ZKA185 CDRL3 < 400>34 tactcaacag acagcagttc taatcccctg ggagta36 < 210>35 < 211>364 < 212> DNA < 213> Artificial Sequence <220>

<223> ZKA185 VH

<400> 35 gaagtgcagc tggtgcagtc cggagcagag gtgaaaaagc ccggggagtc tctgaggatc 60 tcctgtaagg gttctggata tagttttacc agtactgga tcacctggggt gcgccagatg 120 cccgggaaag gcctggagtg gatggcgaag tttgatccta gtgactctca aaccaactac 180 agcccgtcct tccaaggcca cgtcaccatc tcagttgaca agtccatcag cactgcctac 240 ttgcagtgga gcagcctgaa ggcctcggac accgccatgt attackgtgc gagagatat 300 tgtagtagta gtagttgtta tgtggacaat tggggccagg gaaccctggt caccatcttc 360

- 88 043940 tcag

364 <210> 36 <211> 328 <212> DNA <213> Artificial Sequence <220>

<223> ZKA185 VL <400> 36 tcctatgagc tgacacagcc accctcggtg tcagtgtccc caggacaaac ggccaggatc acctgctctg gagatgcatt gccaaataaa tttgcttatt ggtaccggca gaagtcaggc caggcccctg ttctggtcat ctatgaggac aacaaac gac cctccgggat ccctgagaga ttctctggct ccagctcagg gacaatggcc accttgacta tcagtggggc ccaggtggag gatgaagctg actaccactg ttactcaaca gacagcagtt ctaatcccct gggagtattc ggcggaggga ccaagctgac cgtcctag

120

180

240

300

328 <210> 37 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA230 CDRH1 <400> 37

Gly Gly Ser Ile Ser Ser Asp Tyr

5 <210> 38 <211> 7 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA230 CDRH2 < 400> 38

Ile Tyr Tyr Ser Gly Ser Thr

5 <210> 39 <211> 17 <212> PRT <213> Artificial Sequence <220>

<223> ZKA230 CDRH3 <400> 39

Ala Arg Arg Arg Lys Tyr Asp Ser Leu

Trp Gly Ser Phe Ala Phe Asp

Ile

- 89 043940 <210> 40 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA230 CDRL1 <400> 40

Ser Ser Asn Ile Gly Gly Asn Tyr

5 <210>41 <400>41

000 <210>42 <211>9 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA230 CDRL2 long < 400> 42

Leu Ile Cys Ile Asn Asp His Arg Pro

5 <210> 43 <211> 11 <212> PRT <213> Artificial Sequence <220>

<223> ZKA230 CDRL3 <400> 43

Ala Thr Trp Asp Asp Ser Leu Gly Gly Leu Val

5 10 <210> 44 <211> 123 <212> PRT <213> Artificial Sequence <220>

<223> ZKA230 VH <400> 44

Gln Val Gln Leu Gln Glu Ser Gly Pro 1 5

Gly Leu Val Lys Pro Ser Glu

15

Thr Leu Ser Leu Thr Cys Ala Val Ser

25

Gly Gly Ser Ile Ser Ser Asp

Tyr Trp Ser Trp Ile Arg Gln Pro Pro

40

Gly Lys Gly Leu Glu Trp Ile 45

- 90 043940

Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 5560

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn His Phe Ser Leu 65 70 7580

Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 9095

Arg Arg Arg Lys Tyr Asp Ser Leu Trp Gly Ser Phe Ala Phe Asp Ile

100 105110

Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115120 <210>45 <211>110 <212> PRT <213> Artificial Sequence <220>

<223> ZKA230 VL <400>45

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

5 1015

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly Asn 20 2530

Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 4045

Ile Cys Ile Asn Asp His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 5560

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 7580

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Ser Leu 85 9095

Gly Gly Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105110 <210>46 <211>24 <212> DNA <213> Artificial Sequence <220>

<223> ZKA230 CDRH1 <400> 46 ggtggctcca tcagtagtga ctac

- 91 043940 <210> 47 <211> 21 <212> DNA <213> Artificial Sequence <220>

< 223> ZKA230 CDRH2 < 400>47 atctattaca gtgggagcac c21 < 210>48 < 211>51 < 212> DNA < 213> Artificial Sequence <220>

< 223> ZKA230 CDRH3 < 400>48 gcgaggagga ggaagtatga ttccctttgg gggagttttg cttttgatat c51 < 210>49 < 211>24 < 212> DNA < 213> Artificial Sequence <220>

< 223> ZKA230 CDRL1 < 400>49 agctccaaca tcggaggtaa ttat24 < 210>50 < 400>50

000 < 210>51 < 211>27 < 212> DNA < 213> Artificial Sequence <220>

< 223> ZKA230 CDRL2 long < 400>51 ctcatctgta ttaatgatca ccggccc < 210>52 < 211>33 < 212> DNA < 213> Artificial Sequence <220>

<223> ZKA230 CDRL3 <400>52 gcaacatggg atgacagcct gggtggcctt gta <210>53 <211>370 <212> DNA <213> Artificial Sequence

- 92 043940 <220>

<223> ZKA230 VH <400> 53 caggtgcagc tgcaggagtc gggcccaggc ctggtgaagc cttcggagac cctgtccctc acctgcgcag tctctggtgg ctccatcagt agtgactact ggagctggat ccggcagccc ccagggaagg gactggagtg gattgggtat atctat taca gtgggagcac caactacaac ccctccctca agagtcgagt caccatatca gtagacacgt ccaagaacca cttctccctg aagctgaact ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag gaggaggaag tatgattccc tttgggggag tttttgctttt gatatctggg gccaaggg ac aatggtcacc gtctcttcag <210> 54 <211> 331 <212> DNA <213> Artificial Sequence <220>

<223> ZKA230 VL <400> 54 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc tcttgttctg gaagcagctc caacatcgga ggtaattatg tatactggta ccagcagctc ccaggaacgg cccccaaact cctcatctgt attaatgat c accggccctc aggggtccct gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag tccgaggatg aggctgatta ttactgtgca acatgggatg acagcctggg tggccttgta ttcggcggag ggaccaagct gaccgtcctag

120

180

240

300

360

370

120

180

240

300

331 <210>55 <211>8 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA78 CDRH1 < 400>55

Gly Phe Thr Phe Ser Asn Tyr Ala < 210>56 < 211>8 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKA78 CDRH2 < 400> 56

Ile Gly Arg Asn Gly Asp Ser Ile

5 <210> 57

- 93 043940 <211> 16 <212> PRT <213> Artificial Sequence <220>

<223> ZKA78 CDRH3 <400>57

Val Lys Asp Leu Ala Ile Pro Glu

Ser Tyr Arg Ile Glu Ala Asp Tyr <210>58 <211>12 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA78 CDRL1 < 400>58

Gln Ser Val Leu Tyr Arg Ser Asn Asn Lys Asn Tyr

510 < 210>59 < 400>59

000 < 210>60 < 211>9 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKA78 CDRL2 long < 400>60

Leu Ile Tyr Trp Ala Ser Thr Arg Glu < 210>61 < 211>9 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKA78 CDRL3 < 400>61

Gln Gln Tyr Tyr Ser Ser Pro Arg Thr < 210>62 < 211>123 < 212> PRT < 213> Artificial Sequence <220>

<223> ZKA78 VH <400> 62

- 94 043940

Glu Val Gln Leu Ala Glu Ser Gly Gly Gly

5 10

Ser Leu Thr Leu Ser Cys Ser Gly Ser Gly

25

Ala Met Val Trp Ala Arg Gln Ala Pro Gly 35 40

Ser Gly Ile Gly Arg Asn Gly Asp Ser Ile

55

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 65 70

Leu Gln Met Ser Ser Leu Arg Thr Glu Asp

90

Val Lys Asp Leu Ala Ile Pro Glu Ser Tyr

100 105

Trp Gly Gln Gly Thr Leu Val Ile Val Ser

115 120 <210> 63 <211> 113 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA78 VL < 400> 63

Asp Ile Val Met Thr Gln Ser Pro Asp Ser 1 5 10

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser

25

Ser Asn Asn Lys Asn Tyr Leu Ser Trp Tyr

40

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser

55

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 65 70

Ile Ser Pro Leu Gln Ala Glu Asp Val Ala

90

Tyr Tyr Ser Ser Pro Arg Thr Phe Gly Gln

100 105

Val Gln Pro Gly Gly

Thr Phe Ser Asn Tyr 30

Gly Leu Glu Tyr Val 45

Tyr Thr Asp Ser Val 60

Lys Ser Met Val Tyr 80

Ala Val Tyr Tyr Cys 95

Ile Glu Ala Asp Tyr 110

Ala Val Ser Leu Gly

Ser Val Leu Tyr Arg 30

Gln Lys Pro Gly Gln 45

Arg Glu Ser Gly Val 60

Asp Phe Thr Leu Thr 80

Tyr Tyr Cys Gln Gln

Thr Lys Val Glu Ile 110

- 95 043940

Lys <210> 64 <211> 24 <212> DNA <213> Artificial Sequence <220>

<223> ZKA78 CDRH1 <400>64 ggcttcactt ttagtaacta tgca24 <210>65 <211>24 <212> DNA <213> Artificial Sequence <220>

<223> ZKA78 CDRH2 <400>65 atcgggcgca acggggactc tatc24 <210>66 <211>48 <212> DNA <213> Artificial Sequence <220>

<223> ZKA78 CDRH3 <400>66 gtgaaagatc tggccatccc cgagtcctac agaattgaag ctgattat48 <210>67 <211>36 <212> DNA <213> Artificial Sequence <220>

<223> ZKA78 CDRL1 <400>67 cagtccgtgc tgtaccgctc taacaacaag aattac <210>68 <400>68

000 <210>69 <211>27 <212> DNA <213> Artificial Sequence <220>

< 223> ZKA78 CDRL2 long < 400> 69 ctgatctatt gggcttcaac ccgggaa <210> 70

- 96 043940 <211> 27 <212> DNA <213> Artificial Sequence <220>

<223> ZKA78 CDRL3 <400>70 cagcagtact attctagtcc tcgaact27 <210>71 <211>370 <212> DNA <213> Artificial Sequence <220>

< 223> ZKA78 VH < 400>71 gaggtgcagc tggcagaatc aggcggggga ctggtccagc ctggcggcag cctgacactg60 tcttgcagtg gatcaggctt cacttttagt aactatgcaa tggtgtgggc aaggcaggct120 cctgggaagg gactggagta tgtctctgg c atcgggcgca acggggactc tatctactat180 actgatagtg tgaagggccg gttcaccatc agcagagaca atagcaaatc catggtgtac240 ctgcagatga gctccctgcg aaccgaagac acagcagtgt actattgcgt gaaagatctg300 gccatccccg agtcctacag aattgaagct gattatt ggg gacagggcac cctggtcatc360 gtgagcgccg370 < 210>72 < 211>340 < 212> DNA < 213> Artificial Sequence <220>

< 223> ZKA78 VL < 400>72 gacatcgtga tgacacagtc tccagatagt ctggcagtca gtctggggga gagggccact60 attaactgca agagctccca gtccgtgctg taccgctcta acaacaagaa ttacctgtct120 tggtatcagc agaagcccgg acagccccct aaactg ctga tctattgggc ttcaacccgg180 gaaagcggcg tcccagacag attctcaggc agcgggtccg gaacagactt caccctgaca240 attagccccc tgcaggcaga ggacgtggct gtctactatt gtcagcagta ctattctagt300 cctcgaactt tcggccaggg gaccaaggtg gaaatcaa ac340 <210>73 <211> 8 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA64 CDRH1 < 400> 73

Gly Tyr Thr Phe Thr Gly Tyr His

5

- 97 043940 <210> 74 <211> 8 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA64 CDRH2 < 400>74

Ile Asn Pro Asn Ser Gly Gly Thr <210>75 <211>12 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA64 CDRH3 < 400>75

Ala Arg Met Ser Ser Ser Ile Trp Gly Phe Asp His

510 < 210>76 < 211>6 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKA64 CDRL1 < 400>76

Gln Ser Val Leu Ile Asn < 210>77 < 400>77

000 < 210>78 < 211>9 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKA64 CDRL2 long < 400>78

Leu Ile Tyr Gly Ala Ser Ser Arg Ala <210>79 <211>10 <212> PRT <213> Artificial Sequence <220>

<223> ZKA64 CDRL3

- 98 043940 <400> 79

Gln Gln Tyr Asn Asp Trp Pro Pro Ile Thr

5 10 <210> 80 <211> 119 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA64 VH < 400> 80

Gln Val Gln Leu Val Gln Ser Gly Ala Glu

5 10

Ser Val Lys Val Ser Cys Lys Ala Ser Gly

25

His Ile Asp Trp Val Arg Gln Ala Arg Gly

40

Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr

55

Gln Gly Arg Val Thr Met Thr Arg Asp Thr 65 70

Met Gln Leu Ser Arg Leu Arg Ser Asp Asp 85 90

Ala Arg Met Ser Ser Ser Ile Trp Gly Phe

100 105

Thr Leu Val Thr Val Ser Ser

115 <210> 81 <211> 108 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA64 VL < 400> 81

Glu Ile Val Met Thr Gln Ser Pro Ala Thr

5 10

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser

25

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

40

Lys Lys Pro Gly Ala

Thr Phe Thr Gly Tyr 30

Gly Leu Glu Trp Met 45

Tyr Ala Gln Lys Phe 60

Ile Ser Thr Ala Tyr

Ala Val Tyr Tyr Cys 95

His Trp Gly Gln Gly

110

Ser Val Ser Pro Gly 15

Ser Val Leu Ile Asn

Pro Arg Leu Leu Ile 45

- 99 043940

Tyr Gly 50 Ala Ser Ser Arg Ala 55 Thr Gly Ile Pro Ala 60 Arg Phe Ser Gly Ser 65 Gly Ser Gly Thr Glu 70 Phe Thr Leu Thr Ile 75 Ser Ser Leu Gln Ser 80 Glu Asp Phe Ala Val 85 Tyr Tyr Cys Gln Gln 90 Tyr Asn Asp Trp Pro 95 Pro Ile Thr Phe Gly 100 Gln Gly Thr Arg Leu 105 Glu Ile Lys

<210> 82 <211> 24 <212> DNA <213> Artificial Sequence <220>

<223> ZKA64 CDRH1 <400> 82 ggctacacct tcacagggta tcac <210> 83 <211> 24 <212> DNA <213> Artificial Sequence <220>

<223> ZKA64 CDRH2 <400> 83 attaacccta attctggcgg gacc <210> 84 <211> 36 <212> DNA <213> Artificial Sequence <220>

<223> ZKA64 CDRH3 <400> 84 gctcggatga gctcctctat ttggggcttc gatcat <210> 85 <211> 18 <212> DNA <213> Artificial Sequence <220>

<223> ZKA64 CDRL1 <400> 85 cagtctgtgc tgattaac

<210> 86 <400> 86 000 <210> 87

- 100 043940 <211> 27 <212> DNA <213> Artificial Sequence <220>

<223> ZKA64 CDRL2 long <400>87 ctgatctatg gagcatcctc cagggct <210>88 <211>30 <212> DNA <213> Artificial Sequence <220>

<223> ZKA64 CDRL3 <400>88 cagcagtaca atgattggcc ccctatcaca <210>89 <211>358 <212> DNA <213> Artificial Sequence <220>

<223> ZKA64 VH <400> 89

caggtgcagc tggtccagag cggagcagag gtgaagaaac ccggcgcctc agtgaaggtc agctgcaaag cttccggcta caccttcaca gggtatcaca tcgactgggt gaggcaggca aggacagg gactggaatg gatggacgg attaaccta attctggcgg gaccaactac gccagaagt ttcaggggccg agtgactatg accagagaca ccagcatctc cacagcttat atgcagctgt cccggctgag atctgacgat agtgccgtct actattgtgc tcggatgagc tcctctattt ggggcttcga tcattgggggg cagggaacac tggtgactgt cagttcag

120

180

240

300

358 <210> 90 <211> 325 <212> DNA <213> Artificial Sequence <220>

< 223> ZKA64 VL <400> 90 gagatcgtga ctgtcttgca ggccaggcac cgcttcagtg gaagacttcg tgactcagtc gagcctccca cccgactgct gatcaggaag ctgtctacta tccagccacc gtctgtgctg gatctatgga cggaacagag ttgtcagcag ctgtcagtca attaacctgg gcatcctcca tttaccctga tacaatgatt gcccaggaga cttggtacca gggctaccgg caatctctag ggccccctat acgggcaacc gcagaagcca cattcctgca tctgcagtcc cacatttggc caggggacta gactggagat caagc

120

180

240

300

325 <210> 91 <211> 330 <212> PRT

- 101 043940

Ala Pro Ser Ser Lys <213> Artificial Sequence <220>

<223> IgGl CH1-CH2-CH3 aa <400> 91

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 1 5 10

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

25

Phe Pro Glu Pro Val Thr Val Ser Trp Asn

40

Gly Val His Thr Phe Pro Ala Val Leu Gln 50 55

Leu Ser Ser Val Val Thr Val Pro Ser Ser 65 70

Tyr Ile Cys Asn Val Asn His Lys Pro Ser 85 90

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr

100 105

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

115 120

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

130 135

Val Val Val Asp Val Ser His Glu Asp Pro

145 150

Tyr Val Asp Gly Val Glu Val His Asn Ala

165 170

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

180 185

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

195 200

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

210 215

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

225 230

Met Thr Lys Asn Gln Val Ser Leu Thr Cys

245 250

Leu Val Lys Asp Tyr 30

Gly Ala Leu Thr Ser 45

Ser Gly Leu Tyr Ser 60

Leu Gly Thr Gln Thr

Thr Lys Val Asp Lys 95

Thr Cys Pro Pro Cys 110

Phe Leu Phe Pro Pro

125

Pro Glu Val Thr Cys 140

Val Lys Phe Asn Trp

160

Thr Lys Pro Arg Glu

175

Val Leu Thr Val Leu

190

Cys Lys Val Ser Asn

205

Ser Lys Ala Lys Gly 220

Pro Ser Arg Glu Glu

240

Val Lys Gly Phe Tyr

255

- 102 043940

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

260 265

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

275 280

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

290 295

Val Phe Ser Cys Ser Val Met His Glu Ala 305 310

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330 <210> 92 <211> 330 <212> PRT <213> Artificial Sequence <220>

<223> IgG1 CH1-CH2-CH3 LALA aa <400> 92

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 1 5 10

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

25

Phe Pro Glu Pro Val Thr Val Ser Trp Asn

40

Gly Val His Thr Phe Pro Ala Val Leu Gln

55

Leu Ser Ser Val Val Thr Val Pro Ser Ser 65 70

Tyr Ile Cys Asn Val Asn His Lys Pro Ser

90

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

100 105

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

115 120

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

130 135

Val Val Val Asp Val Ser His Glu Asp Pro 145 150

Gly Gln Pro Glu Asn 270

Asp Gly Ser Phe Phe

285

Trp Gln Gln Gly Asn 300

His Asn His Tyr Thr

320

Ala Pro Ser Ser Lys 15

Leu Val Lys Asp Tyr 30

Gly Ala Leu Thr Ser 45

Ser Gly Leu Tyr Ser 60

Leu Gly Thr Gln Thr

Thr Lys Val Asp Lys 95

Thr Cys Pro Pro Cys

110

Phe Leu Phe Pro Pro

125

Pro Glu Val Thr Cys 140

Val Lys Phe Asn Trp

160

- 103 043940

Tyr Val Asp Gly Val Glu Val His Asn Ala

165 170

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

180 185

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

195 200

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

210 215

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

225 230

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

245 250

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

260 265

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

275 280

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

290 295

Val Phe Ser Cys Ser Val Met His Glu Ala

305 310

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330 <210> 93 <211> 107 <212> PRT <213> Artificial Sequence <220>

<223>IgG CK aa <400>93

Arg Thr Val Ala Ala Pro Ser Val Phe Ile 1 5 10

Gln Leu Lys Ser Gly Thr Ala Ser Val Val

25

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys

40

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu

55

Thr Lys Pro Arg Glu

175

Val Leu Thr Val Leu

190

Cys Lys Val Ser Asn

205

Ser Lys Ala Lys Gly 220

Pro Ser Arg Asp Glu

240

Val Lys Gly Phe Tyr

255

Gly Gln Pro Glu Asn 270

Asp Gly Ser Phe Phe 285

Trp Gln Gln Gly Asn 300

His Asn His Tyr Thr

320

Pro Pro Ser Asp Glu

Leu Leu Asn Asn Phe

Asp Asn Ala Leu Gln 45

Asp Ser Lys Asp Ser 60

- 104 043940

Thr Tyr Ser Leu 65

Ser Ser Thr Leu Thr

Leu Ser Lys Ala Asp 75

Tyr Glu 80

Lys His Lys Val

Tyr Ala Cys Glu Val 85

Thr His Gln Gly Leu 90

Ser Ser 95

Pro Val Thr Lys

100

Ser Phe Asn Arg Gly

105

Glu Cys <210> 94 <211> 106 <212> PRT <213> Artificial Sequence <220>

<223> IgG CL aa <400>94

Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser

5 1015

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 2530

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 4045

Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 5560

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 7580

Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 9095

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

100105 <210>95 <211>990 <212> DNA <213> Artificial Sequence <220>

<223> IgG1 CH1-CH2-CH3 nucl <400> 95 gcgtcgacca ggcacagcgg tggaactcag ggactctact tacatctgca agggcccatc ccctgggctg gcgccctgac ccctcagcag acgtgaatca ggtcttcccc cctggtcaag cagcggcgtg cgtggtgacc caagc ccagc ctggcaccct gactacttcc cacaccttcc gtgccctcca aacaccaagg cctccaagag ccgaacctgt cggctgtcct gcagcttggg tggacaagag cacctctggg gacggtctcg acagtcctca cacccagacc agttgagccc

120

180

240

300

- 105 043940

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacacc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcggggaga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacacc tgcccccatc ccggggagg 720 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt ggggagagcaa tggggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 cagaagagcc tctccctgtc cccggggtaaa 990

<210> 96 <211> 990 <212> DNA <213> Artificial Sequence <220> <223> IgG1 CH1-CH2-CH3 LALA nucl <400> 96 gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaacctgt gacggtctcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactactact ccctcagcag cgtggtgacc gtgccctcca gcagcttgggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaagc cgcgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacacc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcggggaga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacacc tgcccccatc ccggggagg 720 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt ggggagagcaa tggggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 cagaagagcc tctccctgtc cccggggtaaa 990

<210> 97

- 106 043940 <211> 321 <212> DNA <213> Artificial Sequence <220>

<223> IgG CK nucl <400>97 cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag120 tggaaggtgg ataacgccct ccaatc gggt aactcccagg agagtgtcac agagcaggac180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag300 agcttcaaca ggggagagtg t321 <210>98 <211> 318 <212> DNA <213> Artificial Sequence <220>

<223> IgG CL nucl <400> 98 ggtcagccca aggctgcccc ctcggtcact ctgttcccgc cctcctctga ggagcttcaa 60 gccaacaagg cccacactggt gtgtctcata agtgacttct acccggggagc cgtgacagtg 120 gcttggaaag cagatagcag ccccgtcaag gcggggagtgg agaccaccac accctccaaa 180 caaagcaaca acaagtacgc ggccagcagc tatctgagcc tgacgcctga gcagtggaag 240 tccccagaa gctacagctg ccaggtcacg catgaaggga gcaccgtgga gaagacagtg 300 gcccctacag aatgttca 318

< 210>99 < 211>8 < 212> PRT < 213> Artificial Sequence <220>

<223>ZKA3 CDRH1 <400>99

Gly Phe Ile Phe Ser Asn Tyr Ala <210>100 <211>8 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA3 CDRH2 < 400> 100

Ile Gly Gly Lys Gly Asp Ser Ile

5

- 107 043940 <210> 101 <211> 16 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA3 CDRH3 < 400> 101

Val Lys Asp Leu Ala Val Leu Glu Ser Asp 1 5 10 <210> 102 <211> 123 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA3 VH < 400> 102

Glu Val Gln Leu Ala Glu Ser Gly Gly Gly

5 10

Ser Leu Arg Leu Ser Cys Ser Gly Ser Gly

25

Ala Met Val Trp Ala Arg Gln Ala Pro Gly 35 40

Ser Gly Ile Gly Gly Lys Gly Asp Ser Ile

55

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 65 70

Leu Gln Met Ser Arg Leu Arg Thr Glu Asp

90

Val Lys Asp Leu Ala Val Leu Glu Ser Asp

100 105

Trp Gly Gln Gly Thr Leu Val Ile Val Ser

115 120

Leu Glu Val Asp Gln

Val Gln Pro Gly Gly

Ile Phe Ser Asn Tyr 30

Gly Leu Glu Tyr Val 45

His Ile Asp Ser Val 60

Lys Arg Thr Val Tyr 80

Ala Val Tyr Tyr Cys 95

Leu Glu Val Asp Gln

110 <210> 103 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA4 CDRH1 <400> 103

Gly Phe Thr Phe Ser Ser Tyr Val 1 5

- 108 043940 <210> 104 <211> 8 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA4 CDRH2 < 400>104

Thr Ser Tyr Asp Gly Ser Asn Lys < 210>105 < 211>20 < 212> PRT < 213> Artificial Sequence <220>

<223> ZKA4 CDRH3 <400>105

Ala Arg Gly Pro Val Pro Tyr

Trp Ser Gly Glu Ser Tyr Ser Gly Ala

1015

Tyr Phe Asp Phe <210>106 <211>127 <212> PRT <213> Artificial Sequence <220>

<223> ZKA4 VH <400> 106

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

25

Val Met His Trp Val Arg Gln Ala Pro

40

Thr Val Thr Ser Tyr Asp Gly Ser Asn

55

Lys Gly Arg Phe Thr Ile Ser Arg Asp 65 70

Gly Phe Thr Phe Ser Ser Tyr 30

Gly Lys Gly Leu Glu Trp Val 45

Lys Tyr Tyr Ala Asp Ser Val 60

Asn Ala Lys Asn Thr Leu Tyr 75 80

Leu Gln Met Asn Ser Leu Arg Gly Glu Asp

90

Thr Ala

Ile Tyr Tyr Cys 95

Ala Arg Gly Pro Val

100

Pro Tyr Trp Ser Gly

105

Glu Ser

Tyr Ser Gly Ala

110

- 109 043940

Tyr Phe Asp Phe Trp

115

Gly Gln Gly Ile Leu

120

Val Thr Val Ser Ser

125 <210> 107 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA5 CDRH1 <400>107

Gly Phe Thr Phe Ser Asn Tyr Tyr 15 <210>108 <211>8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA5 CDRH2 <400>108

Met Ser Ser Ser Glu Thr Ile Lys 15 <210>109 <211>19 <212> PRT <213> Artificial Sequence <220>

<223> ZKA5 CDRH3 <400>109

Ala Arg Ser Gly Ile Glu Thr Val Ala Gly Ser

510

Ile Asp Tyr Tyr Gly

Met Asp Val <210>110 <211>126 <212> PRT <213> Artificial Sequence <220>

<223> ZKA5 VH <400> 110

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu 20

Ser Cys Ala Gly Ser Gly Phe 25

Thr Phe Ser Asn Tyr

Tyr Met Thr Trp Ile Arg Gln Ala Pro

Gly Lys Gly Leu Glu Leu Val

- 110 043940

Lys Tyr Tyr Ala Asp Ser Val

Ser Tyr Met Ser Ser Ser Glu Thr Ile

55

Lys Gly Arg Phe Thr Ile Ser Arg Asp 65 70

Asn Ala Lys Asn Ser Leu Tyr

80

Leu Gln Met Asn Ser Leu Arg Ala Asp 85

Asp Thr Ala Arg Tyr Tyr Cys

95

Ala Arg Ser Gly Ile Glu Thr Val Ala 100 105

Met Asp Val Trp Gly His Gly Thr Pro

115 120

Gly Ser Ile Asp Tyr Tyr Gly

110

Val Thr Val Ser Ser

125 <210> 111 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA6 CDRH1 <400> 111

Asp Phe Thr Val Ser Asn Tyr Ala 1 5 <210> 112 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA6 CDRH2 <400> 112

Val Ser Tyr Asp Gly Ser Asn Lys

5 <210> 113 <211> 19 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA6 CDRH3 < 400> 113

Ala Thr Gly Val Thr Met Phe Gln Gly Ala Gln 1 5 10

Thr Asn Ala Glu Tyr

Leu His Tyr <210> 114

- 111 043940 <211> 126 <212> PRT <213> Artificial Sequence <220>

<223> ZKA6 VH <400>114

Gln Val His Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 1015

Ser Leu Arg Leu Ser Cys Glu Ala Ser Asp Phe Thr Val Ser Asn Tyr 20 2530

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4045

Ala Val Val Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 5560

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 7580

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 9095

Ala Thr Gly Val Thr Met Phe Gln Gly Ala Gln Thr Asn Ala Glu Tyr

100 105110

Leu His Tyr Trp Gly Gln Gly Ser Leu Val Thr Ile Ser Ser

115 120125 <210>115 <211>8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA7 CDRH1 <400>115

Gly Phe Thr Phe Ser Arg Tyr Gly 15 <210>116 <211>8 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA7 CDRH2 < 400> 116

Val Ser Gly Asp Gly Ser Ser Thr

5 <210> 117

- 112 043940

Ser Leu Glu Ser Asp Phe

15 <211> 15 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA7 CDRH3 < 400>117

Val Lys Asp 1

Phe Trp Ser Gly Asp Gln 5 < 210>118 < 211>122 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKA7 VH < 400>118

Glu 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Gly Ser Leu Arg Leu 20 Ser Cys Ser Ala Ser 25 Gly Phe Thr Phe Ser 30 Arg Tyr Gly Met Val 35 Trp Ala Arg Gln Ala 40 Pro Gly Lys Gly Leu 45 Glu Tyr Leu Ser Gly 50 Val Ser Gly Asp Gly 55 Ser Ser Thr Tyr Tyr 60 Ala Asn Ser Val Lys 65 Gly Arg Phe Thr Ile 70 Ser Arg Asp Asn Ser 75 Lys Asn Thr Leu Tyr 80 Leu His Met Ser Arg 85 Leu Arg Asp Glu Asp 90 Thr Ala Met Tyr Tyr 95 Cys Val Lys Asp Phe 100 Trp Ser Gly Asp Gln 105 Ser Leu Glu Ser Asp 110 Phe Trp Gly Gln Gly 115 Ala Leu Val Thr Val 120 Ser Ser

< 210>119 < 211>8 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKA8 CDRH1 < 400> 119

Gly Phe Thr Phe Ser Ala His Ala 1 5 <210> 120

- 113 043940 <211> 8 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA8 CDRH2 < 400>120

Ile Ser Arg Asn Glu Asp Tyr Thr <210>121 <211>12 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA8 CDRH3 < 400>121

Val Lys Asp Phe Gly Thr Ser Pro Gln Thr Asp Phe

510 < 210>122 < 211>119 < 212> PRT < 213> Artificial Sequence <220>

<223> ZKA8 VH <400> 122

Asp 1 Glu Arg Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Gly Ser Leu Arg Leu 20 Val Cys Ser Ala Ser 25 Gly Phe Thr Phe Ser 30 Ala His Ala Met His 35 Trp Val Arg Gln Pro 40 Pro Gly Lys Gly Leu 45 Glu Tyr Val Ser Thr 50 Ile Ser Arg Asn Glu 55 Asp Tyr Thr Tyr Tyr 60 Ala Asp Ser Val Lys 65 Gly Arg Phe Thr Ile 70 Ser Arg Asp Asn Ser 75 Lys Asn Ser Leu Tyr 80 Leu Gln Met Arg Arg 85 Leu Arg Pro Glu Asp 90 Thr Ala Ile Tyr Tyr 95 Cys Val Lys Asp Phe 100 Gly Thr Ser Pro Gln 105 Thr Asp Phe Trp Gly 110 Gln Gly Thr Leu Val 115 Ala Val Ser Ser

<210> 123

- 114 043940 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA76 CDRH1 <400>123

Gly Phe Thr Phe Ser Thr Tyr Phe 15 <210>124 <211>8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA76 CDRH2 <400>124

Ile Ser Ser Thr Gly Ser Tyr Lys 15 <210>125 <211>17 <212> PRT <213> Artificial Sequence <220>

<223> ZKA76 CDRH3 <400> 125

Ala Arg Pro Phe His Ser Glu Tyr Thr Tyr Gly Leu Asp Ala Phe Asp

5 10 15

Ile <210> <211> <212> <213> <220> <223> <400> 126 124 PRT Artificial Sequence ZKA76 VH 126 Glu Val 1 Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Lys Pro Gly 15 Gly Ser Leu Arg Leu 20 Ser Cys Ala Ala Ser 25 Gly Phe Thr Phe Ser 30 Thr Tyr PheMet His 35 Trp Val Arg Gln Ala 40 Pro Gly Lys Gly Leu 45 Glu Trp Val Ala Ser 50 Ile Ser Ser Thr Gly 55 Ser Tyr Lys Phe Tyr 60 Ala Asp Ser Val

- 115 043940

Lys 65 Gly Arg Phe Thr Ile 70 Ser Arg Asp Asn Thr 75 Lys Asn Ser Leu Phe 80 Leu Gln Met Asn Ser 85 Leu Arg Ala Glu Asp 90 Thr Ala Val Phe Tyr 95 Cys Ala Arg Pro Phe 100 His Ser Glu Tyr Thr 105 Tyr Gly Leu Asp Ala 110 Phe Asp Ile Trp Gly 115 Gln Gly Thr Met Leu 120 Thr Val Ser Ser

<210> 127 <211> 10 <212> PRT <213> Artificial Sequence <220>

<223> ZKA117 CDRH1 <400>127

Gly Gly Ser Ile Arg Arg Thr Asn Ser Tyr 1 510 <210>128 <211>7 <212> PRT <213> Artificial Sequence <220>

<223> ZKA117 CDRH2 <400>128

Ile Ser Tyr Ser Gly Ser Thr <210>129 <211>17 <212> PRT <213> Artificial Sequence <220>

<223> ZKA117 CDRH3 <400>129

Ala Arg Leu Asn Asp Gly Ser Thr Val

Thr Thr Ser Ser Tyr Phe Asp

1015

Tyr <210>130 <211>125 <212> PRT <213> Artificial Sequence <220>

<223> ZKA117 VH

- 116 043940 <400> 130

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly

5 10

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly

25

Asn Ser Tyr Trp Gly Trp Ile Arg Gln Thr 35 40

Trp Ile Gly Ser Ile Ser Tyr Ser Gly Ser

55

Leu Lys Ser Arg Val Thr Ile Ser Leu Asp 65 70

Ser Leu Glu Leu Ser Ser Val Thr Ala Ala

90

Cys Ala Arg Leu Asn Asp Gly Ser Thr Val

100 105

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

115 120

Val Lys Pro Ser Glu

Ser Ile Arg Arg Thr 30

Gly Lys Gly Leu Gln 45

Phe Tyr Asn Pro Ser 60

Ser Lys Asp His Phe 80

Thr Ala Ile Tyr Tyr 95

Thr Ser Ser Tyr Phe 110

Ser Ser

125 <210> 131 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKB27 CDRH1 <400> 131

Gly Tyr Ser Phe Thr Ser Ser Trp 1 5 <210> 132 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKB27 CDRH2 <400> 132

Ile Asp Pro Ser Asp Ser Tyr Thr 1 5 <210> 133 <211> 14 <212> PRT <213> Artificial Sequence <220>

<223> ZKB27 CDRH3

- 117 043940 <400> 133

Ala Arg His Asp Tyr Ser Val

Ser Glu Asn Gly Met Asp Val 10 <210> 134 <211> 121 <212> PRT <213> Artificial Sequence <220>

< 223> ZKB27 VH < 400> 134

Glu Val Gln Leu Val Gln Ser Gly 1 5

Ala Glu Val Lys Lys Pro Gly Glu

15

Ser Leu Arg Ile Ser Cys Lys Ala 20

Ser Gly Tyr Ser Phe Thr Ser Ser 25 30

Trp Ile Asn Trp Val Arg Gln Met

40

Pro Gly Lys Gly Leu Glu Trp Met 45

Gly Arg Ile Asp Pro Ser Asp Ser

55

Tyr Thr Thr Tyr Asn Pro Ser Phe 60

Gln Gly His Val Thr Ile Ser Val 65 70

Asp Lys Ser Ile Gly Thr Ala Tyr

80

Leu Gln Trp Asn Ser Leu Arg Ala 85

Ser Asp Thr Ala Met Tyr Tyr Cys

95

Ala Arg His Asp Tyr Ser Val Ser

100

Glu Asn Gly Met Asp Val Trp Gly

105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115120 <210>135 <211>8 <212> PRT <213> Artificial Sequence <220>

<223>ZKB29 CDRH1 <400>135

Gly Phe Thr Phe Ser Ser Tyr Thr 15 < 210>136 < 211>8 < 212> PRT < 213> Artificial Sequence <220>

<223> ZKB29 CDRH2

- 118 043940 <400>136

Ile Ser Tyr Asp Gly Ser His Lys < 210>137 < 211>12 < 212> PRT < 213> Artificial Sequence <220>

<223>ZKB29 CDRH3 <400>137

Ala Arg Arg Ser Tyr Ser Ile Ser Cys Phe Asp Tyr

510 <210>138 <211>119 <212> PRT <213> Artificial Sequence <220>

< 223> ZKB29 VH < 400> 138

Gln Val Gln Leu Val Glu Ser Gly

5

Gly Gly Val Val Gln Pro Gly Arg

15

Ser Leu Arg Leu Ser Cys Ala Ala 20

Ser Gly Phe Thr Phe Ser Ser Tyr 25 30

Thr Met His Trp Val Arg Gln Ala

40

Pro Gly Lys Gly Leu Glu Trp Val 45

Ala Val Ile Ser Tyr Asp Gly Ser

55

His Lys Phe Tyr Ala Asp Ser Val 60

Lys Gly Arg Phe Thr Ile Ser Arg 65 70

Asp Asn Ser Lys Asp Thr Leu Tyr

80

Leu Gln Met Asn Ser Leu Arg Ala 85

Glu Asp Thr Ala Leu Tyr Tyr Cys

95

Ala Arg Arg Ser Tyr Ser Ile Ser

100

Cys Phe Asp Tyr Trp Gly Gln Gly

105 110

Thr Leu Val Thr Ile Ser Ser

115 <210> 139 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKB34 CDRH1

- 119 043940 <400> 139

Gly Phe Thr Phe Ser Arg Ser Gly 1 5 <210> 140 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKB34 CDRH2 <400> 140

Val Ser Tyr Asp Gly Ser Asn Lys

5 <210> 141 <211> 19 <212> PRT <213> Artificial Sequence <220>

<223> ZKB34 CDRH3 <400> 141

Ala Lys Asp Leu Thr Met Val Arg Gly Val 1 5 10

Met Asp Val <210> 142 <211> 126 <212> PRT <213> Artificial Sequence <220>

< 223> ZKB34 VH < 400> 142

Gln Val Gln Leu Val Glu Ser Gly Gly Gly

5 10

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

25

Gly Met His Trp Val Arg Gln Ala Pro Gly

40

Ala Val Val Ser Tyr Asp Gly Ser Asn Lys

55

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 65 70

Leu Gln Met Asn Ser Leu Arg Val Glu Asp

Tyr Tyr Tyr Tyr Val

Val Gln Pro Gly Arg

Thr Phe Ser Arg Ser 30

Gly Leu Glu Trp Val 45

Tyr Ser Asp Ser Val 60

Lys Asn Thr Leu Tyr 80

Ala Val Tyr Tyr Cys

- 120 043940

His Tyr Tyr Tyr Tyr Val

110

Ala Lys Asp Leu Thr Met Val Arg Gly Val

100 105

Met Asp Val Trp Gly Gln Gly Thr Thr Val

115 120

Thr Val Ser Ser

125 <210> 143 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKB39 CDRH1 <400> 143

Gly Tyr Thr Phe Asp Asp Tyr Tyr 1 5 <210> 144 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKB39 CDRH2 <400> 144

Ile Asn Pro His Arg Gly Gly Thr 1 5 <210> 145 <211> 23 <212> PRT <213> Artificial Sequence <220>

<223> ZKB39 CDRH3 <400> 145

Val Arg Asp Gln Tyr Cys Asp Gly Gly Asn Cys 1 5 10

Tyr Gly Ile His Gln

Pro His Tyr Gly Met Asp Val <210> 146 <211> 130 <212> PRT <213> Artificial Sequence <220>

< 223> ZKB39 VH < 400> 146

Gln Val Gln Leu Val Gln Ser Gly Ala Glu 1 5 10

Val Lys

Lys Pro Gly Ala 15

- 121 043940

Ser Leu Lys Val Ser Cys Lys Ala 20

Tyr Ile His Trp Val Arg Gln Ala 3540

Gly Arg Ile Asn Pro His Arg Gly 5055

Ser Gly Tyr Thr Phe Asp Asp Tyr 2530

Pro Gly Gln Gly Leu Glu Trp Leu 45

Gly Thr Asn Tyr Ala Gln Lys Phe 60

Gln 65

Met

Val

Pro

Gly Arg Val Ile Met 70

Glu Leu Arg Arg Ile 85

Arg Asp Gln Tyr Cys 100

His Tyr Gly Met Asp

115

Thr Leu Asp Met Ser Ile Ser Thr Thr Tyr 75 80

Thr Ser Asp Asp Ala Ala Val Tyr Tyr Cys

95

Asp Gly Gly Asn Cys Tyr Gly Ile His Gln

105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val

120 125

Ser Ser

130 <210> 147 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223>ZKB46 CDRH1 <400>147

Gly Tyr Ser Phe Thr Ser Tyr Trp 15 < 210>148 < 211>8 < 212> PRT < 213> Artificial Sequence <220>

<223>ZKB46 CDRH2 <400>148

Ile Asp Pro Ser Asp Ser Tyr Thr 15 < 210>149 < 211>17 < 212> PRT < 213> Artificial Sequence <220>

<223> ZKB46 CDRH3 < 400>149

- 122 043940

Ala Arg Arg Glu Tyr Ser Ser Ser Ser Gly 1 5 10

Pro <210> 150 <211> 124 <212> PRT <213> Artificial Sequence <220>

< 223 > ZKB46 VH < 400 > 150

Glu Val Gln Leu Val Gln Ser Gly Ala Glu

5 10

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly

25

Trp Ile Ser Trp Val Arg Gln Met Pro Gly

40

Gly Arg Ile Asp Pro Ser Asp Ser Tyr Thr

55

Gln Gly His Val Thr Ile Ser Ala Asp Lys 65 70

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp

90

Ala Arg Arg Glu Tyr Ser Ser Ser Ser Gly

100 105

Pro Trp Gly Gln Gly Thr Leu Val Thr Val

115 120

Glu Asp Trp Phe Asp

Lys Lys Pro Gly Glu 15

Ser Phe Thr Ser Tyr 30

Gly Leu Glu Trp Met 45

Tyr Ser Pro Ser Phe 60

Ile Ser Thr Ala Tyr 80

Ala Met Tyr Tyr Cys

Glu Asp Trp Phe Asp

110

Ser <210> 151 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKB53 CDRH1 <400> 151

Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 <210> 152 <211> 8 <212> PRT <213> Artificial Sequence

- 123 043940 <220>

<223> ZKB53 CDRH2 <400>152

Ile Ser Tyr Asp Gly Ser Asn Arg < 210>153 < 211>15 < 212> PRT < 213> Artificial Sequence <220>

<223>ZKB53 CDRH3 <400>153

Ala Arg His Val Glu Gln Leu Pro

Ser Ser Gly Tyr Phe Gln His

15 < 210>154 < 211>122 < 212> PRT < 213> Artificial Sequence <220>

<223> ZKB53 VH <400> 154 Gln 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Val Val Gln Pro Gly 15 Arg Ser Leu Arg Leu 20 Ser Cys Ala Ala Ser 25 Gly Phe Thr Phe Ser 30 Ser Tyr Ala Met His 35 Trp Val Arg Gln Thr 40 Pro Gly Lys Gly Leu 45 Glu Trp Val Thr Val 50 Ile Ser Tyr Asp Gly 55 Ser Asn Arg Tyr Tyr 60 Ala Asp Ser Val Lys 65 Gly Arg Phe Thr Ile 70 Ser Arg Asp Asn Ser 75 Lys Asn Thr Leu Tyr 80 Leu Gln Met Asn Ser 85 Leu Arg Ser Glu Asp 90 Thr Ala Val Tyr Tyr 95 Cys Ala Arg His Val 100 Glu Gln Leu Pro Ser 105 Ser Gly Tyr Phe Gln 110 His Trp Gly Gln Gly 115 Thr Leu Val Thr Val 120 Ser Ser

< 210>155 < 211>8 < 212> PRT <213> Artificial Sequence

- 124 043940 <220>

<223> ZKC26 CDRH1 <400>155

Gly Phe Ile Phe Ser Asp Phe Tyr <210>156 <211>8 <212> PRT <213> Artificial Sequence <220>

< 223> ZKC26 CDRH2 < 400>156

Ile Gly His Asp Gly Ser Tyr Ile < 210>157 < 211>9 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKC26 CDRH3 < 400>157

Ala Arg Ala His Gly Gly Phe Arg His 15 < 210>158 < 211>116 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKC26 VH < 400> 158

Gln 1 Val Gln Val Val 5 Glu Ser Gly Gly Gly 10 Leu Val Lys Pro Gly 15 Gly Ser Leu Arg Leu 20 Ser Cys Ala Ala Ser 25 Gly Phe Ile Phe Ser 30 Asp Phe Tyr Met Ser 35 Trp Met Arg Gln Ala 40 Pro Gly Lys Gly Leu 45 Glu Trp Val Ala Tyr 50 Ile Gly His Asp Gly 55 Ser Tyr Ile Leu Tyr 60 Ala Asp Ser Val Lys 65 Gly Arg Phe Thr Ile 70 Ser Arg Asp Asn Ala 75 Lys Asn Ser Leu Phe 80 Leu Arg Met Asn Ser 85 Leu Arg Val Glu Asp 90 Thr Ala Val Tyr Tyr 95 Cys

- 125 043940

Ala Arg Ala His Gly Gly Phe Arg His

100105

Ala Val Ser Pro

115 <210>159 <211>8 <212> PRT <213> Artificial Sequence <220>

<223> ZKD5 CDRH1 <400>159

Gly Phe Thr Phe Thr Ser Tyr Gly 15 <210>160 <211>8 <212> PRT <213> Artificial Sequence <220>

<223> ZKD5 CDRH2 <400>160

Ile Ser Tyr Asp Gly Ser Asn Lys 15 <210>161 <211>17 <212> PRT <213> Artificial Sequence <220>

<223> ZKD5 CDRH3 <400>161

Ala Arg Asp Arg Asp His Tyr Asp Leu 15

Trp Gly Gln Gly Thr Val Val

110

Trp Asn Ala Tyr Thr Phe Asp

15

Tyr <210>162 <211>124 <212> PRT <213> Artificial Sequence <220>

<223> ZKD5 VH <400> 162

Gln Val Gln Leu 1

Val Glu Ser Gly 5

Gly Gly Val Val 10

Gln Pro Gly Arg 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

Gly Phe Thr Phe Thr Ser Tyr

- 126 043940

Gly Leu Asp Trp Val

25

Gly Met His Trp Val Arg Gln Thr Pro Gly

40

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys

55

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 65 70

Leu Gln Met Asn Ser Leu Arg Ala Ala Asp

90

Ala Arg Asp Arg Asp His Tyr Asp Leu Trp

100 105

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

115 120 <210> 163 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKD7 CDRH1 <400> 163

Gly Phe Thr Phe Ser Asn Tyr Ala 1 5 <210> 164 <211> 8 <212> PRT <213> Artificial Sequence <220>

< 223> ZKD7 CDRH2 < 400> 164

Ile Ser Tyr Asp Val Ser Asp Lys

5 <210> 165 <211> 20 <212> PRT <213> Artificial Sequence <220>

<223> ZKD7 CDRH3 <400> 165

Ala Gly Gly Pro Leu Gly Val Val Val Ile 1 5 10

Tyr Ala Asp Ser Val 60

Lys Asp Thr Leu Tyr 80

Ala Leu Tyr Tyr Cys 95

Ala Tyr Thr Phe Asp 110

Ser

Pro Ser Asn Ala Glu

His Phe His His

- 127 043940 <210> 166 < 211> 127 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKD7 VH < 400>166

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val 1 510

Gln Pro Gly Arg

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 2530

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4045

Ala Val Ile Ser Tyr Asp Val Ser Asp Lys Tyr Tyr Ala Asp Ser Val 50 5560

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 7580

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ala Tyr Tyr Cys 85 9095

Ala Gly Gly Pro Leu Gly Val Val Val Ile Lys Pro Ser Asn Ala Glu

100 105110

His Phe His His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120125 < 210>167 < 211>8 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKD8 CDRH1 < 400>167

Gly Phe Thr Phe Ile Asn Tyr Ala 15 < 210>168 < 211>8 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKD8 CDRH2 < 400> 168

Ile Ser Tyr Asp Gly Ser Asn Lys

- 128 043940 <210> 169 <211> 16 <212> PRT <213> Artificial Sequence <220>

< 223> ZKD8 CDRH3 < 400>169

Ala Thr Asp Ala Asp Ala Tyr Gly Asp 15

Ser Gly Ala Asn Phe His Tyr

15 < 210>170 < 211>123 < 212> PRT < 213> Artificial Sequence < 220>

< 223> ZKD8 VH < 400>170

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Lys

5 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ile Asn Tyr 20 2530

Ala Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4045

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Phe Tyr Thr Asp Ser Val 50 5560

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 7580

Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys 85 9095

Ala Thr Asp Ala Asp Ala Tyr Gly Asp Ser Gly Ala Asn Phe His Tyr

100 105110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115120 < 210>171 < 211>10 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKD15 CDRH1 < 400> 171

Asp Ala Ser Ile Ser Ser Gly Gly Phe Ser

- 129 043940

5 10 <210> 172 <211> 7 <212> PRT <213> Artificial Sequence <220>

<223> ZKD15 CDRH2 <400> 172

Ile Tyr Ser Ser Gly Asp Thr

5 <210> 173 <211> 18 <212> PRT <213> Artificial Sequence <220>

<223> ZKD15 CDRH3 <400> 173

Ala Arg Ala His Thr Pro Thr Ser Lys Phe 1 5 10

Asp Val <210> 174 <211> 126 <212> PRT <213> Artificial Sequence <220>

< 223> ZKD15 VH < 400> 174

Gln Leu Gln Leu Gln Glu Ser Gly Ser Gly

5 10

Thr Leu Ser Leu Thr Cys Thr Val Ser Asp

25

Gly Phe Ser Trp Ser Trp Ile Arg Gln Pro

40

Trp Leu Gly Tyr Ile Tyr Ser Ser Gly Asp

55

Leu Gln Gly Arg Val Thr Met Ser Val Asp 65 70

Ser Leu Lys Leu Thr Ser Val Thr Ala Ala 85 90

Cys Ala Arg Ala His Thr Pro Thr Ser Lys

Tyr Tyr Tyr Ala Met 15

Val Lys Pro Ser Gln

Ser Ile Ser Ser Gly 30

Gly Lys Gly Leu Glu 45

Phe Tyr Asn Pro Ser 60

Phe Arg Ser Gln Phe 80

Thr Ala Met Tyr Tyr 95

Tyr Tyr Tyr Tyr Ala

- 130 043940

Thr Val Ser Ser

125

100

105

110

Met Asp Val Trp Gly Gln Gly Thr Thr Val

115 120 <210> 175 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKD16 CDRH1 <400> 175

Gly Phe Thr Phe Ser Asp His Phe 1 5 <210> 176 <211> 10 <212> PRT <213> Artificial Sequence <220>

< 223> ZKD16 CDRH2 < 400> 176

Ser Arg Asn Lys Pro Asn Ser Tyr Thr Thr

5 10 <210> 177 <211> 17 <212> PRT <213> Artificial Sequence <220>

< 223> ZKD16 CDRH3 < 400> 177

Ala Lys Val Gly Gly Cys Tyr Gly Gly Asp Cys

5 10

His Val Glu Asn Asp

Tyr <210> 178 <211> 126 <212> PRT <213> Artificial Sequence <220>

<223> ZKD16 VH <400> 178

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val 1 5 10

Gln Pro Gly Gly

Ser Leu Arg Leu Ser Cys Val Ala Ser 20 25

Gly Phe Thr Phe Ser Asp His 30

- 131 043940

Phe Met Asp Trp Val Arg Gln Ala

40

Pro Gly Lys Gly

Leu Glu Trp Val 45

Gly Arg Ser Arg Asn Lys Pro Asn

55

Ser Val Lys Gly Arg Phe Ser Ile 65 70

Ser Tyr Thr Thr 60

Ser Arg Asp Asp 75

Glu Tyr Ala Ala

Ser Lys Lys Ala 80

Leu Tyr Leu Gln Met Asn Ser Leu Gln Thr

90

Glu Asp

Thr

Ala Val Tyr 95

Tyr Cys Ala Lys Val Gly Gly Cys Tyr Gly

100 105

Gly Asp

Cys

His Val Glu 110

Asn Asp Tyr Trp Gly Gln Gly Thr Leu Val

115 120

Thr Val

Ser

125

Ser <210> 179 <211> 8 <212> PRT <213> Artificial Sequence <220>

< 223> ZKD17 CDRH1 < 400>179

Gly Phe Ile Phe Ser Asp Tyr Ala 15 < 210>180 < 211>8 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKD17 CDRH2 < 400>180

Ile Ser Tyr Asp Gly Ser Ser Arg <210>181 <211>20 <212> PRT <213> Artificial Sequence <220>

<223> ZKD17 CDRH3 <400> 181

Ala Arg Gly Tyr Cys Ser Ser Gly Thr Cys 1 5 10

Phe Ser Thr Asn Ala Glu

Tyr Phe His Pro 20

- 132 043940 <210> 182 <211> 127 <212> PRT <213> Artificial Sequence <220>

< 223> ZKD17 VH < 400>182

Gln Val Gln Met Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Thr 1 5 1015

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Ile Phe Ser Asp Tyr 20 2530

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4045

Ala Val Ile Ser Tyr Asp Gly Ser Ser Arg Leu Tyr Ala Asp Ser Val 50 5560

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 7580

Leu Gln Met His Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys 85 9095

Ala Arg Gly Tyr Cys Ser Ser Gly Thr Cys Phe Ser Thr Asn Ala Glu

100 105110

Tyr Phe His Pro Trp Gly Gln Gly Thr Leu Ala Thr Ile Ser Ser

115 120125 < 210>183 < 211>8 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKD20 CDRH1 < 400>183

Gly Phe Thr Phe Ser Asp His Phe 15 < 210>184 < 211>10 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKD20 CDRH2 < 400> 184

Ser Arg Asn Lys Pro Asn Ser Tyr Thr Thr

5 10

- 133 043940 <210> 185 <211> 17 <212> PRT <213> Artificial Sequence <220>

<223> ZKD20 CDRH3 <400> 185

Ala Arg Val Gly Gly Cys Asn Gly Gly Asp 1 5 10

Tyr <210> 186 <211> 126 <212> PRT <213> Artificial Sequence <220>

< 223> ZKD20 VH < 400> 186

Glu Val Gln Leu Val Glu Ser Gly Gly Gly

5 10

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly

25

Phe Met Asp Trp Val Arg Gln Ala Pro Gly

40

Gly Arg Ser Arg Asn Lys Pro Asn Ser Tyr

55

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 65 70

Leu Tyr Leu Gln Met Asn Ser Leu Gln Thr

90

Tyr Cys Ala Arg Val Gly Gly Cys Asn Gly

100 105

Asn Asp Tyr Trp Gly Gln Gly Thr Leu Val

115 120

His Val Glu Asn Asp

Val Gln Pro Gly Gly

Thr Phe Ser Asp His 30

Gly Leu Glu Trp Val 45

Thr Glu Tyr Ala Ala 60

Asp Ser Lys Asn Ser 80

Asp Thr Ala Val Tyr 95

Asp Cys His Val Glu 110

Val Ser Ser

125 <210> 187 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA134 CDRH1 <400> 187

- 134 043940

Gly Gly Thr Phe Ser Ala Tyr Ala 1 5 <210> 188 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA134 CDRH2 <400> 188

Ile Ile Pro Phe Phe Gly Thr Ala 1 5 <210> 189 <211> 19 <212> PRT <213> Artificial Sequence <220>

<223> ZKA134 CDRH3 <400> 189

Ala Arg Ser Asp Ile Val Ser Thr Thr Arg

5 10

Met Asp Val <210> 190 <211> 126 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA134 VH < 400> 190

Gln Val His Leu Val Gln Ser Gly Ala Glu

5 10

Ser Val Asn Val Ser Cys Lys Ala Ser Gly

25

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly 35 40

Gly Gly Ile Ile Pro Phe Phe Gly Thr Ala 50 55

Lys Gly Arg Val Thr Val Thr Ala Asp Lys 65 70

Met Glu Met Thr Ser Leu Arg Ser Glu Asp 85 90

Tyr His His Tyr Gly

Lys Lys Pro Gly Ser

Thr Phe Ser Ala Tyr 30

Gly Leu Glu Trp Met 45

Tyr Ala Gln Lys Phe 60

Thr Ser Thr Val Tyr 80

Ala Val Tyr Tyr Cys 95

- 135 043940

Ala Arg Ser Asp Ile Val Ser

100

Thr Thr

105

Arg Gly Tyr His His Tyr Gly

110

Met Asp Val

115

Trp Gly Gln Gly Thr Thr Val

120

Thr Val Ser Ser

125 <210> 191 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA246 CDRH1 <400>191

Gly Tyr Thr Phe Ser Asp Tyr Tyr 15 <210>192 <211>8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA246 CDRH2 <400>192

Ile Asn Pro Tyr Ser Gly Gly Thr <210>193 <211>14 <212> PRT <213> Artificial Sequence <220>

<223> ZKA246 CDRH3 <400> 193

Ala Arg Gly Phe Thr Met Ile

Ser Asp Arg Glu Phe Asp Pro <210> 194 <211> 121 <212> PRT <213> Artificial Sequence <220>

<223> ZKA246 VH <400> 194

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ala 1 5 10 15

Ser Val Lys Val

Ser Cys

Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr 25 30

Tyr Met His Trp Val Arg Gln Ala Pro

Gly Gln Gly Leu Glu Trp Met

- 136 043940

Gly Arg Ile Asn Pro Tyr 50

Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 55 60

His Gly Arg Val Thr Val 65 70

Thr Arg Asp Thr Ser Ile Ser Thr Val Tyr

80

Met Glu Leu Arg Gly Leu 85

Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

95

Ala Arg Gly Phe Thr Met

100

Ile Ser Asp Arg Glu Phe Asp Pro Trp Gly

105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 <210> 195 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA256 CDRH1 <400> 195

Gly Phe Thr Phe Ser Thr Tyr Trp 1 5 <210> 196 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA256 CDRH2 <400> 196

Ile Lys Gln Asp Gly Ser Glu Lys

5 <210> 197 <211> 20 <212> PRT <213> Artificial Sequence <220>

<223> ZKA256 CDRH3 <400> 197

Ala Arg Asp Pro Gly Tyr Asp Asp Phe Trp Ser Gly Ser Tyr Ser Gly

5 10 15

Ser Phe Asp Ile 20 <210> 198

- 137 043940 <211> 127 <212> PRT <213> Artificial Sequence <220>

<223> ZKA256 VH <400>198

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 2530

Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4045

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 5560

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Ser Leu Tyr 65 70 7580

Leu Gln Val Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 9095

Ala Arg Asp Pro Gly Tyr Asp Asp Phe Trp Ser Gly Ser Tyr Ser Gly

100 105110

Ser Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120125 <210>199 <211>8 <212> PRT <213> Artificial Sequence <220>

<223>ZKB42 CDRH1 <400>199

Gly Phe Thr Phe Asn Asn Tyr Gly 15 < 210>200 < 211>8 < 212> PRT < 213> Artificial Sequence <220>

<223> ZKB42 CDRH2 <400> 200

Ile Ser Tyr Asp Gly Asn Lys Lys 1 5 <210> 201

- 138 043940 <211> 16 <212> PRT <213> Artificial Sequence <220>

<223> ZKB42 CDRH3 <400>201

Val Lys Tyr Gly Glu Arg Ile Asn Gly Tyr 1 510 <210>202 <211>123 <212> PRT <213> Artificial Sequence <220>

<223> ZKB42 VH <400>202

Gln Val Gln Val Val Glu Ser Gly Gly Gly

510

Ser Leu Arg Leu Phe Cys Ala Ala Ser Gly

2025

Gly Met His Trp Val Arg Gln Ala Pro Gly

3540

Ala Leu Ile Ser Tyr Asp Gly Asn Lys Lys

5055

Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn 6570

Leu Gln Met Asn Arg Leu Arg Ser Gly Asp

8590

Val Lys Tyr Gly Glu Arg Ile Asn Gly Tyr

100105

Asp Pro Phe Asp 15 His Val Gln Pro Gly 15 Arg Thr Phe Asn 30 Asn Tyr Gly Leu 45 Glu Trp Val Tyr 60 Ala Asp Ser Val Lys Asn Thr Leu Tyr 80 Ala Val Tyr His 95 Cys Asp Pro Phe 110 Asp His

Trp Gly Gln Gly Thr Leu Val Thr Val Ser

115120 <210>203 <211>8 <212> PRT <213> Artificial Sequence <220>

<223> ZKB85 CDRH1 <400> 203

Gly Tyr Thr Phe Thr Thr Tyr Ala 1 5 <210> 204

- 139 043940 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223>ZKB85 CDRH2 <400>204

Ile Asn Thr Asn Thr Gly Asn Pro <210>205 <211>11 <212> PRT <213> Artificial Sequence <220>

<223>ZKB85 CDRH3 <400>205

Ala Arg Val Ile Val Pro Tyr Ala Phe Asp Ile

510 < 210>206 < 211>118 < 212> PRT < 213> Artificial Sequence <220>

<223>ZKB85 VH <400>206

Gln 1 Val Gln Leu Val 5 Gln Ser Gly Ser Glu 10 Leu Lys Lys Pro Gly 15 Ala Ser Val Lys Val 20 Ser Cys Lys Ala Ser 25 Gly Tyr Thr Phe Thr 30 Thr Tyr Ala Met Asn 35 Trp Val Arg Gln Ala 40 Pro Gly Gln Gly Pro 45 Glu Trp Val Gly Trp 50 Ile Asn Thr Asn Thr 55 Gly Asn Pro Thr Tyr 60 Ala Gln Gly Phe Thr 65 Gly Arg Phe Val Leu 70 Ser Leu Asp Thr Ser 75 Val Ser Thr Ala Phe 80 Leu Gln Ile Ser Ser 85 Leu Lys Ala Glu Asp 90 Thr Ala Val Tyr Tyr 95 Cys Ala Arg Val Ile 100 Val Pro Tyr Ala Phe 105 Asp Ile Trp Gly Gln 110 Gly Thr Met Val Thr Val Ser Ser

115 <210> 207

- 140 043940 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKB47 CDRH1 <400> 207

Gly Tyr Thr Phe Thr Asn Tyr Tyr 1 5 <210> 208 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKB47 CDRH2 <400> 208

Ile Asn Pro Ser Gly Gly Pro Thr 1 5 <210> 209 <211> 15 <212> PRT <213> Artificial Sequence <220>

<223> ZKB47 CDRH3 <400> 209

Ala Arg Asp Gln Tyr Gly Gly Tyr Ala Arg

5 10 <210> 210 <211> 122 <212> PRT <213> Artificial Sequence <220>

< 223> ZKB47 VH < 400> 210

Gln Val Gln Leu Val Gln Ser Gly Ala Glu

5 10

Ser Val Lys Val Ser Cys Gln Ala Ser Gly

25

Tyr Met His Trp Val Arg Gln Ala Pro Gly

40

Gly Ile Ile Asn Pro Ser Gly Gly Pro Thr 50 55

Gln Gly Arg Val Thr Met Thr Thr Asp Thr 65 70

Gly Met Asp Val

Lys Lys Pro Gly Ala

Thr Phe Thr Asn Tyr 30

Gly Leu Glu Trp Met 45

Tyr Ala Gln Lys Phe 60

Thr Ser Thr Val Tyr 80

- 141 043940

Met Glu Leu Ser

Ser Leu Arg Ser Glu 85

Asp Thr Ala Val Tyr Tyr Cys

95

Ala Arg Asp Gln

100

Tyr Gly Gly Tyr Ala

105

Arg Tyr Gly Met Asp Val Trp

110

Gly Gln Gly Thr

115

Thr Val Thr Val Ser Ser

120 <210> 211 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKC6 CDRH1 <400> 211

Gly Tyr Thr Phe Thr Gly Tyr Tyr 1 5 <210> 212 <211> 8 <212> PRT <213> Artificial Sequence <220>

< 223> ZKC6 CDRH2 < 400> 212

Ile Asn Pro Asn Ser Gly Gly Thr 1 5 <210> 213 <211> 12 <212> PRT <213> Artificial Sequence <220>

<223> ZKC6 CDRH3 <400> 213

Ala Arg Val Ser Asp Trp Gly Phe Ala Phe Asp Ile

5 10 <210> 214 <211> 119 <212> PRT <213> Artificial Sequence <220>

<223> ZKC6 VH <400> 214

Gln Val Gln Leu Val Gln Ser Gly Thr Glu Val

5 10

Lys Lys Pro Gly Ala 15

Ser Val Lys Val Ser Cys Lys Ala Ser

Gly Tyr Thr Phe Thr Gly Tyr

- 142 043940

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln

40

Gly Leu Glu 45

Trp Met

Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn

55

Tyr Ala Gln 60

Lys Phe

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser 65 70 75

Ile Ser Thr

Ala Tyr 80

Met Glu Leu

Ser Gly Leu Arg Ser Asp Asp Thr Ala Val

90

Tyr Tyr Cys 95

Ala Arg Val

Ser Asp Trp Gly Phe Ala Phe Asp Ile Trp 100 105

Gly Gln Gly 110

Thr Met Val Thr Val Ser Gln

115 <210> 215 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA160 CDRH1 <400> 215

Gly Gly Ser Ile Thr Ser Tyr Ser 1 5 <210> 216 <211> 7 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA160 CDRH2 < 400> 216

Ile Phe Tyr Ser Gly Ser Thr

5 <210> 217 <211> 15 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA160 CDRH3 < 400> 217

Ala Arg Asp Gln Thr Met Pro Val

Trp Val Gly Gly Met Asp Val

15 <210> 218

- 143 043940 <211> 121 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKA160 VH < 400> 218

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly

5 10

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly

25

Ser Trp Ser Trp Ile Arg Gln Pro Pro Gly

40

Gly Tyr Ile Phe Tyr Ser Gly Ser Thr Asp

55

Ser Arg Val Thr Ile Ser Val Asp Thr Ser 65 70

Arg Leu Arg Ser Val Thr Ala Ala Asp Thr 85 90

Arg Asp Gln Thr Met Pro Val Trp Val Gly

100 105

Val Lys Pro Ser Glu

Ser Ile Thr Ser Tyr 30

Gly Leu Glu Trp Ile 45

Asn Pro Ser Leu Lys 60

Asp Gln Phe Ser Leu 80

Val Tyr Tyr Cys Ala

Met Asp Val Trp Gly 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 <210> 219 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA172 CDRH1 <400> 219

Gly Tyr Ile Phe Thr Arg Tyr Trp 1 5 <210> 220 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA172 CDRH2 <400> 220

Ile Asp Pro Ser Asp Ser Tyr Thr 1 5 <210> 221

- 144 043940 <211> 13 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKA172 CDRH3 < 400>221

Ala Arg Gln Glu Thr Ala Arg Glu Asp Gly Met Ala Val 1 510 < 210>222 < 211>120 < 212> PRT < 213> Artificial Sequence <220>

<223> ZKA172 VH <400>222

Glu 1 Val Gln Leu Val 5 Gln Ser Gly Ala Glu 10 Val Lys Lys Pro Gly 15 Lys Ser Leu Arg Ile 20 Ser Cys Lys Gly Ser 25 Gly Tyr Ile Phe Thr 30 Arg Tyr Trp Ile Ser 35 Trp Val Arg Gln Met 40 Pro Gly Lys Gly Leu 45 Glu Trp Met Gly Arg 50 Ile Asp Pro Ser Asp 55 Ser Tyr Thr Asn Tyr 60 Ser Pro Ser Phe Gln 65 Gly His Val Thr Ile 70 Ser Ala Asp Lys Ser 75 Ile Ser Thr Ala Tyr 80 Leu Gln Trp Ser Ser 85 Leu Lys Ala Ser Asp 90 Thr Ala Met Tyr Tyr 95 Cys Ala Arg Gln Glu 100 Thr Ala Arg Glu Asp 105 Gly Met Ala Val Trp 110 Gly Gln Gly Thr Thr 115 Val Thr Val Ser Ser 120

<210>223 <211>10 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA174 CDRH1 < 400> 223

Gly Gly Ser Met Ser Asn Ser Tyr Tyr His

5 10 <210> 224

- 145 043940 <211> 7 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA174 CDRH2 < 400>224

Ile Tyr Tyr Ser Gly Ser Thr < 210>225 < 211>18 < 212> PRT < 213> Artificial Sequence <220>

<223>ZKA174 CDRH3 <400>225

Ala Arg Asn Pro Val Phe Asn Pro Leu 15

Thr Leu Thr His Asp Ala Phe

15

Asp Ile <210>226 <211>126 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA174 VH < 400>226

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

5 1015

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Met Ser Asn Ser 20 2530

Tyr Tyr His Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 4045

Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 5560

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 7580

Ser Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 9095

Cys Ala Arg Asn Pro Val Phe Asn Pro Leu Thr Leu Thr His Asp Ala

100 105110

Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

- 146 043940

115 120125 < 210>227 < 211>8 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKA189 CDRH1 < 400>227

Gly Phe Thr Phe Ser Ser Tyr Ala < 210>228 < 211>8 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKA189 CDRH2 < 400>228

Ile Ser Gly Ser Gly Asp Asn Thr < 210>229 < 211>18 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKA189 CDRH3 < 400> 229

Ala Lys Trp Pro Tyr Tyr Asp Phe

Trp Ser Gly Ser Glu Ser Tyr Phe

15

Asp Pro <210> 230 <211> 125 <212> PRT <213> Artificial Sequence <220>

<223> ZKA189 VH <400>230

Gly Val Gln Leu Leu Glu Ser Gly Gly Ala Leu Val Gln Pro Gly Lys

5 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 2530

Ala Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val 35 4045

- 147 043940

Ser Ala Ile Ser

Lys Gly Arg Phe 65

Leu Gln Met Asn

Ala Lys Trp Pro

100

Asp Pro Trp Gly

115

Gly Ser Gly 55 Asp Asn Thr Tyr Tyr 60 Ala Asp Ser Val Thr Ile 70 Ser Arg Asp Asn Ser 75 Lys Asn Thr Leu Tyr 80 Ser 85 Leu Arg Ala Glu Asp 90 Thr Ala Val Tyr Tyr 95 Cys Tyr Tyr Asp Phe Trp 105 Ser Gly Ser Glu Ser 110 Tyr Phe Gln Gly Thr Leu 120 Val Thr Val Ser Ser 125

<210> 231 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA195 CDRH1 <400>231

Gly Tyr Asn Phe Pro Ser Tyr Trp 15 <210>232 <211>8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA195 CDRH2 <400>232

Ile Asp Pro Ser Asp Ser Tyr Thr 15 <210>233 <211>15 <212> PRT <213> Artificial Sequence <220>

<223> ZKA195 CDRH3 <400>233

Ala Arg Ala Asp Cys Arg Ser Thr Ser Cys 1 510

Tyr Leu Val Phe Glu <210>234 <211>121 <212> PRT <213> Artificial Sequence <220>

<223> ZKA195 VH

- 148 043940 <400>234

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

5 1015

Ser Leu Arg Ile Ser Cys Lys Asp Ser Gly Tyr Asn Phe Pro Ser Tyr 20 2530

Trp Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 4045

Gly Thr Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe 50 5560

Gln Gly His Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 7580

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 9095

Ala Arg Ala Asp Cys Arg Ser Thr Ser Cys Tyr Leu Val Phe Glu Gly

100 105110

Gln Gly Thr Leu Val Thr Val Ser Ser

115120 <210>235 <211>8 <212> PRT <213> Artificial Sequence <220>

<223>ZKA215 CDRH1 <400>235

Gly Tyr Thr Phe Thr Ser Tyr Trp 15 < 210>236 < 211>8 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKA215 CDRH2 < 400>236

Ile Asp Pro Ser Asp Ser His Thr <210>237 <211>11 <212> PRT <213> Artificial Sequence <220>

<223> ZKA215 CDRH3

- 149 043940 <400> 237

Ala Arg His Ala Leu Pro Asn Tyr Phe Asp 1 5 10 <210> 238 <211> 118 <212> PRT <213> Artificial Sequence <220>

< 223> ZKA215 VH < 400> 238

Glu Val Gln Leu Val Gln Ser Gly Ala Glu

5 10

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly

25

Trp Ile Ser Trp Val Arg Gln Met Pro Gly

40

Gly Arg Ile Asp Pro Ser Asp Ser His Thr

55

Gln Gly His Val Thr Ile Ser Ala Asp Lys 65 70

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp

90

Ala Arg His Ala Leu Pro Asn Tyr Phe Asp

100 105

Lys Lys Pro Gly Glu 15

Thr Phe Thr Ser Tyr 30

Gly Leu Glu Trp Met 45

Tyr Ser Pro Ser Phe 60

Ile Ser Ala Ala Tyr

Ala Met Tyr Tyr Cys 95

Trp Gly Gln Gly Thr 110

Leu Val Thr Val Ser Ser

115 <210> 239 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA218 CDRH1 <400> 239

Gly Phe Pro Phe Ser Ser Tyr Trp 1 5 <210> 240 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> ZKA218 CDRH2

- 150 043940 <400>240

Ile Asn Ser Asp Gly Arg Asn Thr < 210>241 < 211>15 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKA218 CDRH3 < 400> 241

Ala Arg Gly Gly Tyr Asp Tyr Asp

Ser Ser Gly Cys Phe Asp Tyr

15 < 210> 242 < 211> 122 < 212> PRT < 213> Artificial Sequence < 220>

< 223> ZKA218 VH < 400>242

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

5 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Ser Tyr 20 2530

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 4045

Ser Arg Ile Asn Ser Asp Gly Arg Asn Thr Asn Tyr Ala Asp Ser Val 50 5560

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Tyr 65 70 7580

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 9095

Ala Arg Gly Gly Tyr Asp Tyr Asp Ser Ser Gly Cys Phe Asp Tyr Trp

100 105110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115120 < 210>243 < 211>8 < 212> PRT < 213> Artificial Sequence <220>

<223>ZKB75 CDRH1

- 151 043940 <400> 243

Gly Phe 1 Thr Phe Ser Asn Tyr Ala 5 <210> <211> <212> <213> <220> <223> <400> Ile Ser 1 <210> <211> <212> <213> <220> <223> <400> Ala Lys 244 8 PRT Artificial Sequence ZKB75 CDRH2 244 Gly Thr Gly Gly Ser 5 245 25 PRT Artificial Sequence ZKB75 CDRH3 245 Asp Ser Ala Ser Arg Thr Gly Gly Tyr 1 Tyr Leu 5 Asn Pro Gly His His Asp Tyr 10 <210> <211> <212> <213> <220> <223> <400> Glu Val 20 246 132 PRT Artificial Sequence ZKB75 VH 246 Gln Val Leu Glu Ser Gly 25 Gly Gly 1 Ser Leu 5 Arg Leu Ser Cys Ala Ala Ser 10 Gly Ala Met 20 Ser Trp Val Arg Gln Ala 25 Pro Gly Ser Thr 35 Ile Ser Gly Thr Gly 40 Gly Ser Thr 50 Lys Gly 55 Arg Phe Thr Ile Ser Arg Asp Asn 65 Leu Gln 70 Met Asn Ser Leu Arg Ala Glu Asp

Ser Gly Gly Val Cys

Leu Gln Pro Gly Gly

Thr Phe Ser Asn Tyr 30

Gly Leu Glu Trp Val 45

Tyr Ala Asp Ser Val 60

Lys Asn Thr Leu Tyr 80

Ala Val Tyr Tyr Cys

- 152 043940

Ala Lys Asp Ser Ala Ser Arg Gly Gly Tyr Cys Ser Gly Gly Val Cys

100 105110

Tyr Leu Asn Pro Gly His His Asp Tyr Trp Gly Gln Gly Thr Leu Val

115 120125

Thr Val Ser Ser

130 <210>247 <211>8 <212> PRT <213> Artificial Sequence <220>

<223> ZKB83 CDRH1 <400>247

Gly Tyr Ser Phe Thr Asn Tyr Trp 15 <210>248 <211>8 <212> PRT <213> Artificial Sequence <220>

<223> ZKB83 CDRH2 <400>248

Ile Asp Pro Ser Asp Ser Tyr Thr 15 <210>249 <211>27 <212> PRT <213> Artificial Sequence <220>

<223>ZKB83 CDRH3 <400>249

Ala Arg Leu Arg Gly Ser Leu Tyr Cys 15

Ser Gly Gly Arg Cys Tyr Ser

15

Val Pro Gly Glu Thr Pro Asn Trp Phe Asp Pro

2025 < 210>250 < 211>134 < 212> PRT < 213> Artificial Sequence <220>

< 223 > ZKB83 VH < 400 > 250

- 153 043940

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 1015

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr 20 2530

Trp Ile Thr Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 4045

Gly Ser Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe 50 5560

Gln Gly His Val Thr Ile Ser Ala Asp Trp Ser Ile Asn Thr Ala Tyr 65 70 7580

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Lys Tyr Tyr Cys 85 9095

Ala Arg Leu Arg Gly Ser Leu Tyr Cys Ser Gly Gly Arg Cys Tyr Ser

100 105110

Val Pro Gly Glu Thr Pro Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr

115 120125

Leu Val Thr Val Ser Ser

130 <210>251 <211>8 <212> PRT <213> Artificial Sequence <220>

< 223> ZKC3 CDRH1 < 400>251

Gly Gly Ser Ile Thr Ser Tyr Tyr 15 < 210>252 < 211>7 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKC3 CDRH2 < 400>252

Ile Tyr Tyr Ser Gly Ser Thr <210>253 <211>15 <212> PRT <213> Artificial Sequence

- 154 043940 <220>

<223> ZKC3 CDRH3 <400> 253

Ala Arg Val 1

Gly Gly Ala Pro Tyr Tyr Tyr Tyr Gly Met

10

Asp Val <210> 254 <211> 121 <212> PRT <213> Artificial Sequence <220>

< 223> ZKC3 VH < 400> 254

Gln Val Gln Leu Gln Glu Ser Gly 1 5

Pro Gly Leu Val Lys Pro Ser Glu

15

Thr Leu Ser Leu Thr Cys Thr Val 20

Ser Gly Gly Ser Ile Thr Ser Tyr 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro

40

Pro Gly Lys Gly Leu Glu Trp Ile 45

Gly Tyr Ile Tyr Tyr Ser Gly Ser

55

Thr Asn Tyr Asn Pro Ser Leu Lys 60

Ser Arg Val Thr Ile Ser Val Asp 65 70

Thr Ser Lys Asn Gln Phe Ser Leu

80

Lys Leu Ser Ser Val Thr Ala Ala 85

Asp Thr Ala Val Tyr Tyr Cys Ala

95

Arg Val Gly Gly Ala Pro Tyr Tyr

100

Tyr Tyr Gly Met Asp Val Trp Gly

105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115120 <210>255 <211>8 <212> PRT <213> Artificial Sequence <220>

<223> ZKC18 CDRH1 <400>255

Gly Phe Thr Phe Gly Asp Tyr Ala 15 <210>256 <211>10 <212> PRT <213> Artificial Sequence

- 155 043940 <220>

<223> ZKC18 CDRH2 <400>256

Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr

510 < 210>257 < 211>13 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKC18 CDRH3 < 400>257

Ser Arg Asp His Thr Gly Thr Thr Tyr Ala Phe Asp 1 510

Ile < 210>258 < 211>122 < 212> PRT < 213> Artificial Sequence <220>

< 223> ZKC18 VH < 400> 258

Glu Val Gln Leu Val Glu Ser Gly 1 5

Gly Gly Leu Val Gln Pro Gly Arg

15

Ser Leu Arg Leu Ser Cys Thr Ala 20

Ser Gly Phe Thr Phe Gly Asp Tyr 25 30

Ala Met Ser Trp Phe Arg Gln Ala

40

Pro Gly Lys Gly Leu Glu Trp Val 45

Gly Phe Ile Arg Ser Lys Ala Tyr

55

Gly Gly Thr Thr Glu Tyr Ala Ala 60

Ser Val Lys Gly Arg Phe Thr Ile 65 70

Ser Arg Asp Asp Ser Lys Ser Ile

80

Ala Tyr Leu Gln Met Asn Ser Leu 85

Lys Thr Glu Asp Thr Ala Val Tyr

95

Tyr Cys Ser Arg Asp His Thr Gly

100

Thr Thr Tyr Ala Phe Asp Ile Trp

105 110

Gly Gln Gly Thr Met Val Thr Val Ser Gln

115120 < 210>259 < 211>8 < 212> PRT < 213> Artificial Sequence

- 156 043940 <220>

<223> ZKD1 CDRH1 <400> 259

Gly Phe Thr Phe Ser Ser Tyr Gly 1 5 <210> 260 <211> 8 <212> PRT <213> Artificial Sequence <220>

< 223> ZKD1 CDRH2 < 400> 260

Ile Trp Tyr Asp Gly Ser Asn Lys

5 <210> 261 <211> 19 <212> PRT <213> Artificial Sequence <220>

< 223> ZKD1 CDRH3 < 400> 261

Ala Arg Asp Arg Arg Gly Tyr Gly Asp 1 5

Tyr Val Gly Tyr Tyr Tyr Gly

15

Met Asp Val <210> 262 <211> 126 <212> PRT <213> Artificial Sequence <220>

< 223> ZKD1 VH < 400> 262

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys

55

Tyr Tyr Ala Asp Ser Val 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 65 70

Ser Lys Asn Thr Leu Tyr 75 80

- 157 043940

Leu Gln Met Asn Ser

Ala Arg Asp Arg Arg

100

Met Asp Val Trp Gly 115

Leu Arg Ala Glu Asp 90 Thr Ala Val Gly Tyr Gly Asp 105 Tyr Val Gly Tyr Gln Gly Thr 120 Thr Val Thr Val Ser 125

Tyr Tyr Cys 95

Tyr Tyr Gly 110

Ser

<210> 263 <211> 95 <212> PRT <213> Zika virus

<220> <221> <222> <223> naturally occurring amino acid misc_feature (9)..(9) Xaa can be any <220> <221> <222> <223> misc_feature (13) . . (14) Xaa can be any naturally occurring amino acid <220> <221> <222> <223> misc_feature (22).. (22) Xaa can be any naturally occurring amino acid <220> <221> <222> <223> misc_feature (25) . . (25) Xaa can be any naturally occurring amino acid <220> <221> <222> <223> misc_feature (27) . . (27) Xaa can be any naturally occurring amino acid <220> <221> <222> <223> misc_feature (33) .. (33) Xaa can be any naturally occurring amino acid <220> <221> <222> <223> misc_feature (35) .. (35) Xaa can be any naturally occurring amino acid <220> <221> <222> <223> misc_feature (41) .. (41) Xaa can be any naturally occurring amino acid <220> <221> <222> <223> misc_feature (60)..(60) Xaa can be any naturally occurring amino acid <220> <221> <222> <223> misc_feature (83)..(83) Xaa can be any naturally occurring amino acid

<220>

- 158 043940 < 221> misc_feature < 222> (85) .. (85) < 223> Xaa can be any naturally occurring amino acid < 400>263

Thr Ala Ala Phe Thr Phe Thr Lys Xaa Pro Ala Glu Xaa Xaa His Gly

5 1015

Thr Val Thr Val Glu Xaa Gln Tyr Xaa Gly Xaa Asp Gly Pro Cys Lys 20 2530

Xaa Pro Xaa Gln Met Ala Val Asp Xaa Gln Thr Leu Thr Pro Val Gly 35 4045

Arg Leu Ile Thr Ala Asn Pro Val Ile Thr Glu Xaa Thr Glu Asn Ser 50 5560

Lys Met Met Leu Glu Leu Asp Pro Pro Phe Gly Asp Ser Tyr Ile Val 65 70 7580

Ile Gly Xaa Gly Xaa Lys Lys Ile Thr His His Trp His Arg Ser

9095 < 210>264 < 211>95 < 212> PRT < 213> Zika virus < 400>264

Thr Ala Ala Phe Thr Phe Thr Lys Ile Pro Ala Glu Thr Leu His Gly

5 1015

Thr Val Thr Val Glu Val Gln Tyr Ala Gly Thr Asp Gly Pro Cys Lys

2530

Val Pro Ala Gln Met Ala Val Asp Met Gln Thr Leu Thr Pro Val Gly

4045

Arg Leu Ile Thr Ala Asn Pro Val Ile Thr Glu Ser Thr Glu Asn Ser 50 5560

Lys Met Met Leu Glu Leu Asp Pro Pro Phe Gly Asp Ser Tyr Ile Val 65 70 7580

Ile Gly Val Gly Glu Lys Lys Ile Thr His His Trp His Arg Ser

9095 < 210>265 < 211>104 < 212> PRT < 213> Artificial Sequence <220>

<223>ZIKV EDIII generic

- 159 043940

misc_feature (1)..(1) X may be any or E (naturally occurring) amino acid preferably K, A, misc_feature (3) . . (3) X may be any or L (naturally occurring) amino acid preferably V, F, misc_feature (4)..(4) X may be any (naturally occurring) amino acid preferably S or F misc_feature (17) .. (17) X may be any (naturally occurring) amino acid preferably I or V misc_feature (21) . . (21) X may be any (naturally occurring) amino acid preferably T or V misc_feature (22).. (22) X may be any (naturally occurring) amino acid preferably L or D misc_feature (30).. (30) X may be any (naturally occurring) amino acid preferably V or G misc_feature (33) .. (33) X may be any (naturally occurring) amino acid preferably A or G misc_feature (35) .. (35) X may be any preferably T (naturally or A occurring) amino acid except R, misc_feature (41) . . (41) X may be any (naturally occurring) amino acid preferably V or I misc_feature (43) .. (43) X may be any (naturally occurring) amino acid preferably A or V misc_feature (49).. (49) X may be any (naturally occurring) amino acid preferably M or T misc_feature (68)..(68) X may be any (naturally occurring) amino acid preferably S or G

misc_feature

- 160 043940 <222> (70).. (70) <223> X may be any (naturally occurring) amino acid, preferably E or K <220>

< 221> misc_feature < 222> (91) .. (91) < 223> X may be any (naturally occurring) amino acid, preferably V or I <220>

< 221> misc_feature < 222> (93)..(93) < 223> X may be any (naturally occurring) amino acid, preferably E, A, K, or D <220>

< 221> misc_feature < 222> (94) .. (94) <223> X may be any (naturally occurring) amino acid, preferably E, A, or K, more preferably K or A <400>265

Xaa Gly Xaa Xaa Tyr Ser Leu Cys Thr Ala Ala Phe Thr Phe Thr Lys

5 1015

Xaa Pro Ala Glu Xaa Xaa His Gly Thr Val Thr Val Glu Xaa Gln Tyr 20 2530

Xaa Gly Xaa Asp Gly Pro Cys Lys Xaa Pro Xaa Gln Met Ala Val Asp

4045

Xaa Gln Thr Leu Thr Pro Val Gly Arg Leu Ile Thr Ala Asn Pro Val 50 5560

Ile Thr Glu Xaa Thr Xaa Asn Ser Lys Met Met Leu Glu Leu Asp Pro 65 70 7580

Pro Phe Gly Asp Ser Tyr Ile Val Ile Gly Xaa Gly Xaa Xaa Lys Ile 85 9095

Thr His His Trp His Arg Ser Gly

100 <210>266 <211>26 <212> DNA <213> Artificial Sequence <220>

<223> Zika-E-F1 primer <400>266 tgcaaacgcg gtcgcaaacc tggttg <210>267 <211>25 <212> DNA <213> Artificial Sequence <220>

<223>ZIKV-E-R1 primer

- 161 043940 <400> 267 cgtgccaagg taatggaatg tcgtg <210> 268 <211> 25 <212> DNA <213> Artificial Sequence <220>

<223> ZIKV-Ef1530 primer <400> 268 agcctaggac ttgattgtga accga <210> 269 <211> 25 <212> DNA <213> Artificial Sequence <220>

<223> ZIKV-E-R2769 primer <400> 269 ttacagatcc cacaacgacc gtcag <210> 270 <211> 25 <212> DNA <213> Artificial Sequence <220>

<223> ZIKV-E-F2 <400> 270 acttggtcat gatactgctg attgc <210> 271 <211> 25 <212> DNA <213> Artificial Sequence <220>

<223> ZIKV-E-R2 <400> 271 tcggttcaca atcaagtcct aggct <210> 272 <211> 25 <212> DNA <213> Artificial Sequence <220>

<223> ZIKV-E-f2058 <400> 272 gctaaccccg taatcactga aagca <210> 273 <211> 25 <212> DNA <213> Artificial Sequence <220>

- 162 043940 <223> ZIKV-E-r2248 <400> 273 aagactgcca ttctcttggc acctc 25

Claims (53)

CLAIM 1. An isolated antibody or antigen-binding fragment thereof that specifically binds/specifically binds to Zika virus epitopes on the Zika virus envelope protein (ZIKV E protein), wherein the antibody or antigen-binding fragment thereof contains an epitope binding site containing the amino acid sequences CDRH1, CDRH2 and CDRH3, and also the amino acid sequences of CDRL1, CDRL2 and CDRL3 (i) according to SEQ ID NO: 19-23 and 25, respectively; or (ii) according to SEQ ID NO: 19-22 and 24, 25, respectively. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof neutralizes Zika virus infection. 3. The antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof eliminates the propagation of escape mutants of the Zika virus. 4. An antibody or an antigen-binding fragment thereof according to any one of claims 1-3, characterized in that the antibody or an antigen-binding fragment thereof does not contribute to an antibody-dependent enhancement of Zika virus infection. 5. An antibody or antigen binding fragment thereof according to any one of claims 1 to 4, characterized in that each CDR or each variable region included in the antibody or antigen binding fragment thereof is a human CDR or a humanized variable region, respectively. 6. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that the antibody or an antigen-binding fragment thereof is a monoclonal antibody. 7. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that the antibody or an antigen-binding fragment thereof is bispecific, trispecific, tetraspecific or pentaspecific. 8. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7, characterized in that the antibody or an antigen-binding fragment thereof is bivalent, trivalent, tetravalent, hexavalent or octavalent. 9. An antibody or an antigen-binding fragment thereof according to claims 1-8, characterized in that the antibody molecule contains two copies of sites that specifically bind to epitopes of the Zika virus. 10. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that the antibody or an antigen-binding fragment thereof is of the IgG type. 11. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 10, characterized in that the antibody or an antigen-binding fragment thereof includes an Fc fragment. 12. The antibody or antigen-binding fragment thereof according to claim 11, wherein the antibody or antigen-binding fragment thereof includes a mutation in the Fc fragment, wherein said mutation reduces the binding of the antibody to the Fc receptor. 13. The antibody or antigen-binding fragment thereof as claimed in claim 10 or 12, wherein the antibody or antigen-binding fragment thereof comprises a CH2 L4A mutation, a CH2 L5A mutation, or both. 14. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 13, characterized in that the antibody or an antigen-binding fragment thereof is in the Fabsin-tandem-Ig antibody format. 15. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 13, characterized in that the antibody or an antigen-binding fragment thereof is in the DVD-Ig antibody format. 16. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that the antibody or an antigen-binding fragment thereof does not include a binding site for the Fc receptor. 17. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 16, characterized in that the antibody or an antigen-binding fragment thereof binds to a domain of the Zika virus envelope protein (EDIII). 18. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, characterized in that the antibody or antigen-binding fragment thereof binds to an epitope of the Zika virus envelope protein that includes one or more lateral ridge (LR) amino acid residues in EDIII and /or one or more amino acid residues of the hinge region EDI-EDIII. 19. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 18, characterized in that the antibody or an antigen-binding fragment thereof can inhibit the step - 163 043940 after joining ZIKV. 20. The antibody or antigen-binding fragment thereof according to claim 19, characterized in that the antibody or antigen-binding fragment thereof can prevent membrane fusion. 21. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 20, characterized in that the antibody or an antigen-binding fragment thereof can cause aggregation of ZIKV (particles). 22. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 21, characterized in that the antibody or an antigen-binding fragment thereof further comprises the amino acid sequences CDRH1, CDRH2 and CDRH3 and the amino acid sequences CDRL1, CDRL2 and CDRL3, (i) corresponding to the sequences SEQ ID NO: 1-5 and 7; or functional variants of these sequences that are at least 85, at least 88, at least 90, at least 92, at least 95, at least 96, at least 97, according to at least 98 or at least 99% identical to these sequences; (ii) corresponding to the sequences of SEQ ID NO: 1-4 and 6, 7; or functional variants of these sequences that are at least 85, at least 88, at least 90, at least 92, at least 95, at least 96, at least 97, according to at least 98 or at least 99% identical to these sequences; (iii) corresponding to the sequences of SEQ ID NO: 37-41 and 43; or functional variants of these sequences that are at least 85, at least 88, at least 90, at least 92, at least 95, at least 96, at least 97, according to at least 98 or at least 99% identical to these sequences; (iv) corresponding to the sequences SEQ ID NO: 37-40 and 42, 43; or functional variants of these sequences that are at least 85, at least 88, at least 90, at least 92, at least 95, at least 96, at least 97, according to at least 98 or at least 99% identical to these sequences; (v) corresponding to the sequences of SEQ ID NO: 55-59 and 61; or functional variants of these sequences that are at least 85, at least 88, at least 90, at least 92, at least 95, at least 96, at least 97, according to at least 98 or at least 99% identical to these sequences; (vi) corresponding to the sequences of SEQ ID NO: 55-58 and 60, 61; or functional variants of these sequences that are at least 85, at least 88, at least 90, at least 92, at least 95, at least 96, at least 97, according to at least 98 or at least 99% identical to these sequences; (vii) corresponding to the sequences of SEQ ID NO: 7377 and 79; or functional variants of these sequences that are at least 85 different, at least 88 different, at least 90 different, at least 92 different, at least 95 different, at least 96 different, at least 97%, at least 98%, or at least 99% identical to these sequences; or (viii) corresponding to the sequences of SEQ ID NO: 73-76 and 78, 79; or functional variants of these sequences, at least 85, at least 88, at least 90, at least 92, at least 95, at least 96, at least 97, at least are at least 98 or at least 99% identical to these sequences. 23. The antibody or antigen-binding fragment thereof according to claim 22, characterized in that the antibody or antigen-binding fragment thereof includes (a) a first Zika virus epitope binding site containing the amino acid sequences CDRH1, CDRH2 and CDRH3, as well as the amino acid sequences CDRL1, CDRL2 and CDRL3, (i) corresponding to the sequences of SEQ ID NO: 1-5 and 7; or functional variants of these sequences that are at least 85, at least 88, at least 90, at least 92, at least 95, at least 96, at least 97, according to at least 98 or at least 99% identical to these sequences; or (ii) corresponding to the sequences of SEQ ID NO: 1-4 and 6, 7; or functional variants of these sequences that are at least 85, at least 88, at least 90, at least 92, at least 95, at least 96, at least 97, according to at least 98 or at least 99% identical to these sequences; and (b) a second Zika virus epitope binding site comprising the amino acid sequences CDRH1, CDRH2 and CDRH3, as well as the amino acid sequences CDRL1, CDRL2 and CDRL3, (i) corresponding to the sequences of SEQ ID NOs: 19-23 and 25; or (ii) corresponding to the sequences of SEQ ID NO: 19-22 and 24, 25. 24. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 23, characterized in that the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region ( VH) contains or consists of an amino acid sequence corresponding to the sequence of SEQ ID NO: 26; or a functional variant of this sequence, which is - 164 043940 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% identical to this sequence; and a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 27 or a functional sequence variant thereof that is at least 85, at least 88, at least 90, at least 92 is at least 95, at least 96, at least 97, at least 98, or at least 99% identical to this sequence. 25. The antibody or antigen-binding fragment thereof according to claim 24, wherein the antibody or antigen-binding fragment thereof further comprises (i) a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 8, or a functional variant thereof sequences that are 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% identical to this sequence, and/or a light chain variable region (VL) amino acid sequence corresponding to the sequence of SEQ ID NO: 9, or a functional variant of this sequence that is 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% identical to this sequence; (ii) a variable heavy chain (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 44, or a functional variant of this sequence that is 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% identical to this sequence, and/or a light chain variable region (VL) amino acid sequence corresponding to sequence SEQ ID NO: 45, or a functional variant of this sequence that is at least 85, at least 88, at least 90, at least 92, at least 95, at least 96, is at least 97, at least 98 or at least 99% identical to this sequence; (iii) a variable heavy chain (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 62, or a functional variant of this sequence that is 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% identical to this sequence, and/or a light chain variable region (VL) amino acid sequence corresponding to sequence SEQ ID NO: 63, or a functional variant of this sequence that is at least 85, at least 88, at least 90, at least 92, at least 95, at least 96, is at least 97, at least 98 or at least 99% identical to this sequence; or (iv) a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 80, or a functional variant of this sequence that is 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% identical to this sequence, and/or the amino acid sequence of the light chain variable region (VL), corresponding to the sequence SEQ ID NO: 81, or a functional variant of this sequence that is at least 85, at least 88, at least 90, at least 92, at least 95, at least 96 , is at least 97, at least 98, or at least 99% identical to this sequence. 26. The antibody or antigen-binding fragment thereof according to claim 25, wherein the antibody or antigen-binding fragment thereof comprises (a) a first epitope binding site comprising a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 8 , or a functional variant of this sequence that is at least 85, at least 88, at least 90, at least 92, at least 95, at least 96, at least 97, is at least 98 or at least 99% identical to this sequence, and/or a light chain variable region (VL) amino acid sequence corresponding to the sequence of SEQ ID NO: 9, or a functional variant of this sequence that is 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% identical to this sequences; and (b) a second epitope binding site comprising a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 26, or a functional variant thereof that is at least 85, at least 88, at least 88 90, at least 92, at least 95, at least 96, at least 97, at least 98 or at least 99% identical to this sequence, - 165 043940 and/or a light chain variable region (VL) amino acid sequence corresponding to the sequence of SEQ ID NO: 27, or a functional variant of this sequence that is at least 85, at least 88, at least 90, according to is at least 92, at least 95, at least 96, at least 97, at least 98, or at least 99% identical to this sequence. 27. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 26, characterized in that the antibody or an antigen-binding fragment thereof is in the Fabsin-tandem-Ig (FIT-Ig) format and the outer Fab of the FIT-Ig format contains a binding site an epitope comprising the amino acid sequences CDRH1, CDRH2 and CDRH3 and the amino acid sequences CDRL1, CDRL2 and CDRL3, (i) corresponding to the sequences of SEQ ID NOs: 1-5 and 7; or functional variants of these sequences that are at least 85, at least 88, at least 90, at least 92, at least 95, at least 96, at least 97, according to at least 98 or at least 99% identical to these sequences; or (ii) corresponding to the sequences of SEQ ID NO: 1-4 and 6, 7; or functional variants of these sequences that are at least 85, at least 88, at least 90, at least 92, at least 95, at least 96, at least 97, according to at least 98 or at least 99% identical to these sequences; and the internal Fab of the FIT-Ig format contains an epitope binding site containing the amino acid sequences CDRH1, CDRH2 and CDRH3, as well as the amino acid sequences CDRL1, CDRL2 and CDRL3 (i) according to SEQ ID NOs: 19-23 and 25; or (ii) as per SEQ ID NO: 19-22 and 24, 25. 28. The antibody or antigen binding fragment thereof according to claim 27, wherein the outer Fab of the FIT-Ig format includes a heavy chain variable region (VH) amino acid sequence corresponding to the sequence of SEQ ID NO: 8, or a functional variant of this sequence, which 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 is at least 99% identical to this sequence, and/or the amino acid sequence of the variable light chain (VL) region corresponding to the sequence of SEQ ID NO: 9, or a functional variant of this sequence that is at least 85, at least 88, at least is at least 90, at least 92, at least 95, at least 96, at least 97, at least 98, or at least 99% identical to this sequence. 29. The antibody or antigen-binding fragment thereof according to claim 27 or 28, characterized in that the internal Fab of the FIT-Ig format includes an epitope binding site containing the amino acid sequence of the heavy chain variable region (VH) corresponding to the sequence of SEQ ID NO: 26, or a functional variant of this sequence that is at least 85, at least 88, at least 90, at least 92, at least 95, at least 96, at least 97, according to is at least 98% or at least 99% identical to this sequence, and/or a light chain variable region (VL) amino acid sequence corresponding to the sequence of SEQ ID NO: 27, or a functional variant of this sequence that is at least 85% identical to 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% identical to this sequence . 30. An antibody or antigen binding fragment thereof as claimed in any one of the preceding claims, wherein the antibody or antigen binding fragment thereof is a purified antibody or a single chain antibody, for example a scFv. 31. Use of an antibody or an antigen-binding fragment thereof according to any of the previous paragraphs as a medicine. 32. A nucleic acid molecule containing at least one polynucleotide encoding an antibody or an antigen-binding fragment according to any one of claims 1 to 30. 33. The nucleic acid molecule according to claim 32, which is a mono-, bi- or polycistronic nucleic acid molecule. 34. The nucleic acid molecule according to any one of claims 32, 33, which is DNA or RNA. 35. The nucleic acid molecule according to claim 34, wherein the nucleic acid molecule is plasmid DNA or mRNA. 36. An expression vector containing a nucleic acid molecule according to any one of claims 32-35. 37. A set of nucleic acid molecules, wherein each nucleic acid molecule contains at least one polynucleotide encoding an antibody fragment or an antigen-binding fragment thereof according to any one of claims 1 to 30, where the set of nucleic acids encodes an antibody or an antigen-binding fragment thereof according to any one of claims 1 -thirty. 38. A set of vectors encoding an antibody or an antigen-binding fragment according to any one of claims 1 to 30. 39. A cell expressing an antibody or an antigen-binding fragment according to any one of claims 1- - 166 043940 30, containing a vector according to claim 36 or a set of vectors according to claim 38. 40. A pharmaceutical composition containing an antibody or its antigen-binding fragment according to any of claims 1-30, a nucleic acid molecule according to any of claims 32-35, a vector according to claim 36, a set of nucleic acid molecules according to claim 37, a set of vectors according to claim 38 and/or the cell according to claim 39, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, diluent or carrier. 41. Use of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 30 for the prevention or treatment of infection caused by the Zika virus. 42. Use according to claim 41 for the prevention or treatment of Zika virus infection in subjects diagnosed with Zika virus infection or in a subject exhibiting symptoms of Zika virus infection. 43. Use according to claim 41 in asymptomatic subjects. 44. Use according to any of paragraphs 41-43 in pregnant women. 45. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 30 for monitoring the quality of a Zika virus vaccine by verifying that the antigen of said vaccine contains a specific epitope in the correct conformation. 46. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 30 for (in vitro) diagnosis of Zika virus infection. 47. A kit for the prevention or treatment of Zika virus infection, containing at least one antibody or antigen-binding fragment thereof according to any one of claims 1-30, at least one nucleic acid according to any one of claims 32-35, at least one vector according to claim 36, a set of nucleic acid molecules according to claim 37, a set of vectors according to claim 38, at least one cell according to claim 39 or at least one pharmaceutical composition according to claim 40. 48. A method for preventing or treating Zika virus infection in a subject, which includes administering to a subject in need thereof an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 30, a nucleic acid molecule according to any one of claims 32 to 35, a vector according to claim .36, a set of nucleic acid molecules according to claim 37, a set of vectors according to claim 38, a cell according to claim 39 or a pharmaceutical composition according to claim 40. 49. The method of claim 48, wherein the subject has been diagnosed with Zika virus infection or exhibits symptoms of Zika virus infection. 50. The method according to claim 48 or 49, wherein the subject is a pregnant woman. 51. The method according to any one of claims 48-50, in which the antibody or antigen-binding fragment thereof is administered within seven days after (possible) infection with the Zika virus. 52. The method according to any one of claims 48 to 51, wherein the antibody or antigen binding fragment thereof is administered in combination with a checkpoint inhibitor. 53. The method according to any one of claims 48 to 52, wherein the antibody or antigen-binding fragment thereof is administered in a (single) dose of 0.005 to 100 mg.