EA046175B1 - AN ISOLATED ANTIBODY OR ITS ANTIGEN-BINDING FRAGMENT THAT SPECIFICALLY BINDS TO AN EPITOPE OF THE ZIKA VIRUS, THEIR PREPARATION AND APPLICATIONS - Google Patents

Description

The present invention relates to antibodies and antigen-binding fragments thereof that bind specifically to epitopes of the Zika virus (ZIKV). Such antibodies (I) potentially neutralize Zika virus (ZIKV) infection or (II) target ZIKV NS1 and can be used as diagnostic tools. The invention also relates to nucleic acids that encode antibodies and immortalize B cells that produce such antibodies and antibody fragments. Additionally, the invention relates to the use of antibodies and antibody fragments according to the invention in methods of diagnosing, preventing and treating ZIKV infection.

Zika virus (ZIKV), a mosquito-borne flavivirus, is a public health emergency. ZIKV was first isolated from macaques in 1947 in the Zika forests of Uganda (G. W. A. Dick, S. F. Kitchen, A. J. Haddow, Zika virus. I. Isolations and serological specificity. Trans. R. Soc. Trop. Med. Hyg. 46 , 509-520 (1952)) and the first human infection was described in Nigeria in 1954 by F.N. Macnamara, Zika virus: a report on three cases of human infection during an epidemic of jaundice in Nigeria. Trans. R. Soc. Trop. Med. Hyg. 48, 139-145 (1954). Since that time, ZIKV infections have occurred sporadically in Africa and southeast Asia (D. Musso, Van Mai Cao-Lormeau, D.J. Gubler, Zika virus: following the path of dengue and chikungunya? The Lancet. 386, 243- 244 (2015)), but epidemics were described in Micronesia in 2007 (M.R. Duffy et al., Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med. 360, 2536-2543 (2009)) and in French Polynesia 2013-14, the virus subsequently spread to other countries in Oceania (V.-M. Cao-Lormeau, D. Musso, Emerging arboviruses in the Pacific. Lancet. 384, 1571-1572 (2014); D. Musso , E. J. Nilles, V.-M. Cao-Lormeau, Rapid spread of emerging Zika virus in the Pacific area. Clin. Microbiol. Infect. 20, 0595-6 (2014). Following its introduction in Brazil in 2015, ZIKV spread rapidly and was declared a public health emergency of international concern by the World Health Organization (WHO) in February 2016 (L. R. Baden, L. R. Petersen, D. J. Jamieson, A. M. Powers, M. A. Honein , Zika Virus. N. Engl. J. Med. 374, 1552-1563 (2016); A. S. Fauci, D. M. Morens, Zika Virus in the Americas - Yet Another Arbovirus Threat. N Engl. J. Med., 160113142101009 (2016); D. L. Heymann et al., Zika virus and microcephaly: why is this situation a PHEIC? Lancet. 387, 719-721 (2016)). The main route of ZIKV infection is through the bites of Aedes mosquitoes, but the virus can also be sexually transmitted (D. Musso et al., Potential sexual transmission of Zika virus. Emerg Infect Dis. 21, 359-361 (2015)) and transmitted vertically (J. Mlakar et al., Zika Virus Associated with Microcephaly. N Engl J Med. 374, 951-958 (2016). Although most ZIKV infections are asymptomatic or cause only minor symptoms, there is evidence that ZIKV infection can lead to neurological complications such as Guillain-Barré syndrome in adults (V.-M. Cao-Lormeau and al., Guillain-Barre Syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. Lancet. 0 (2016), doi:10.1016/S0140-6736(16)00562-6) and congenital malformations, including microcephaly in developing embryos G. Calvet, R.S. Aguiar, A. Melo, S.A. Sampaio, Detection and sequencing of Zika virus from amniotic fluid of fetuses with microcephaly in Brazil: a case study. Lancet Infect Dis (2016), doi:10.1016/s1473-3099(16)00095-5; J. Mlakar et al., Zika Virus Associated with Microcephaly. N Engl J Med. 374, 951-958 (2016); E.J. Rubin, M.F. Greene, L.R. Baden, Zika Virus and Microcephaly. N Engl J Med (2016), doi:10.1056/NEJMel601862), possibly due to its ability to infect human neural progenitor cells (N. Tang et al., Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth. Stem Cell, 1 -5 (2016)).

ZIKV belongs to the flavivirus genus, 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 envelope, with icosahedral and spherical geometry.

The diameter is approximately 50 nm. The genomes are positive sense RNA and are non-segmented, approximately 10-11 kilobases in length. The flavivirus genome encodes 3 structural proteins (Capsid, prM, and Envelope) and 8 nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5, and NS5B).

While flavivirus envelope proteins (envelope, E) mediate fusion and are the primary target of neutralizing antibodies, nonstructural protein 1 (NS1) is secreted by infected cells and is involved in immune evasion and pathogenesis (D.A. Muller, P.R. Young, The flavivirus NS1 protein: molecular and structural biology, immunology, role in pathogenesis and application as a diagnostic biomarker. Antiviral Res. 98, 192-208 (2013)). Two recent structural studies have shown a high level of structural similarity between the ZIKV E protein and those of other flaviviruses, such as dengue virus (DENV), yellow fever virus (YFV), and West Nile virus (WNV), but also revealed unique characteristic features related to ZIKV neurotropism ( L. Dai et al., Structures of the Zika Virus envelope protein and Its Complex with a Flavivirus Broadly Protective Antibody. Cell Host Microbe (2016), doi:10.1016/j.chom.2016.04.013; D. Sirohi et al., The 3.8 A resolution cryo-EM structure of Zika virus. Science, aaf5316 (2016). Similarly, structural analysis of ZIKV NS1 revealed conserved features of NS1 shared by other flaviviruses, although with different electrostatic characteristics (J. Kim et al., Zika virus NS1

- 1 046175 structure reveals diversity of electrostatic surfaces among flaviviruses, 1-6 (2016)).

A phenomenon that is characteristic of certain flaviviruses is the activity of disease-enhancing cross-reactive antibodies, the production of which is caused by previous infection with heterologous viruses. In the case of dengue virus (DENV), for which 4 serotypes are known, there is epidemiological evidence that primary infection protects against re-infection with the same serotype, but is a risk factor for severe disease if re-infected with a different serotype (SB Halstead, Dengue Antibody- Dependent Enhancement: Knowns and Unknowns. Microbiol Spectr. 2, 249-271 (2014). This aggravated disease is triggered 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 increased viral replication and activation of cross-reacting memory T cells. The resulting cytokine storm is believed to be the basis of the most severe form of the disease, known as dengue hemorrhagic fever/dengue shock syndrome (SB Halstead, Neutralization and antibody-dependent enhancement of dengue viruses. Adv Virus Res. 60, 421-467 (2003) ; G. Screaton, J. Mongkolsapaya, S. Yacoub, S. Roberts, New insights into the immunopathology and control of dengue virus infection. Nat Rev Immunol. 15, 745-759 (2015). The role of antibodies in severe forms of dengue is supported by research. , indicating that waning levels of maternal antibodies in newborns are a higher risk factor for the development of severe dengue disease (SB Halstead, Neutralization and antibody-dependent enhancement of dengue viruses. Adv Virus Res. 60, 421-467 (2003); SB Halstead et al., Dengue hemorrhagic fever in infants: research opportunities ignored. Emerging Infect Dis. 8, 1474-1479 (2002); T. N. Nguyen et al., Dengue hemorrhagic fever in infants: a study of clinical and cytokine profiles. J Infect Dis. 189, 221-232 (2004); AL Rothman, Dengue: defining protective versus pathologic immunity. J Clin Invest. 113, 946-951 (2004).

Recently, it has been shown that most antibodies that react with the DENV envelope protein also bind to ZIKV, but those that recognize the major linear fusion-loop epitope (FLE) do not neutralize ZIKV but instead promote antibody-dependent enhancement (ADE) ZIKV infections (Dejnirattisai W, Supasa P, Wongwiwat W, Rouvinski A, Barba-Spaeth G, Duangchinda T, Sakuntabhai A, Cao-Lormeau VM, Malasit P, Rey FA, Mongkolsapaya J, Screaton GR: Denge virus sero-crossreactivity drives antibody -dependent enhancement of infection with zika virus. Nat Immunol. 2016 Jun 23. doi: 10.1038/ni.3515. [advance electronic publication]).

Additionally, according to WHO, the recent increase in the incidence of microcephaly and other neurological disorders associated with Zika virus infection is increasing the need for laboratory testing to detect Zika virus infection. Therefore, high antibody specificity is required to distinguish infection caused by ZIKV from infection caused by other flaviviruses. However, known Zika virus antibodies typically cross-react with other flaviviruses and are therefore not useful for differentiating ZIKV infection from infection with other flaviviruses.

In view of the above, it is an object of the present invention to provide new antibodies that specifically bind to ZIKV epitopes. It is also an object of the present invention to provide effective neutralizing antibodies to ZIKV. Such antibodies preferably do not contribute to antibody-dependent enhancement (ADE) of Zika virus infection. It is also an object of the present invention to provide highly specific anti-ZIKV antibodies useful for diagnosing and testing ZIKV infection and diagnostic methods using such antibodies.

The problem underlying the present invention is solved using the objects disclosed in the claimed claims.

Although the present invention is described in more detail below, it is intended that it is not limited to the specific methodologies, protocols and reagents disclosed herein, as they may vary. It is also understood that the terminology used in this application is not intended to limit the scope of the present invention, which is defined only by the appended claims. Unless specifically stated otherwise, all technical and scientific terms used in this application have the same meaning as commonly understood by one skilled in the art.

Next, elements of the present invention will be described. These elements are listed in specific embodiments, however, it should be appreciated that they can be combined in any manner and in any quantity. This description is to be understood as supporting and covering embodiments that combine the specifically described embodiments with any number of described and/or preferred elements. In addition, any permutations and combinations of all described elements in this application should be considered as disclosed by the description of this application, unless the context indicates otherwise.

For the entire specification and claims attached, unless the context otherwise requires, the term contain, and such variations as contains and containing, are intended to include the specified component, integer, or step, but not to exclude any other component,

- 2 046175 integer or stage. The term consists of is a specific embodiment of the term contains, where any other unspecified component, integer or step is excluded. In the context of the present invention, the term contains includes the term consists of. Therefore, the term containing covers including as well as consisting, for example, a composition containing X may consist only of X or may include some additional elements, for example X+Y.

Terms in the singular number used in the context of the description of the invention (especially in the context of the claims) should be considered to cover both the singular and the plural unless specifically stated otherwise in this application or clearly inconsistent with the context. The listing of ranges of values in this application is only intended as a shorthand way of referring individually to each individual value falling within the specified range. Unless specifically stated otherwise herein, each individual value is included in the specification in the same manner as if it were individually cited herein. Nothing in the specification should be construed as indicating any unclaimed element important to the practice of the invention.

The expression essentially does not completely exclude, for example, a composition that is substantially free of Y may be completely free of Y. If necessary, the expression essentially may be omitted from the definition according to the invention.

The term approximately in relation to the numerical value of x means x±10%.

The term disease, as used herein, is generally synonymous with and is used interchangeably with the terms disorder and condition (as a medical condition), in the sense that all reflect an abnormal condition of the human or animal body or one of its parts in which there is a disorder. normal functioning, typically manifests with varying signs and symptoms, and causes the person or animal to have a reduced length or quality of life.

As used herein, reference to treating a subject or patient includes prevention, prophylaxis, mitigation, relief and therapy. The terms subject or patient are used interchangeably in this application to refer to all mammals, including humans. Examples of subjects include humans, cows, dogs, cats, horses, goats, sheep, pigs and rabbits. In one embodiment, the patient is a human.

As used herein, the terms antigen binding fragment, fragment and antibody fragment are used interchangeably to refer to any antibody fragment of the invention that retains the antigen binding activity of the antibody. Examples of antibody fragments include, but are not limited to, single chain antibody, 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, especially antigen binding fragments, human antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies and genetically engineered antibodies (variant antibodies). or mutant antibodies), provided that all the characteristic properties of the invention are retained. Preferred are human antibodies and monoclonal antibodies, and more preferred are human monoclonal antibodies, especially recombinant human monoclonal antibodies.

Human antibodies are well known in the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be obtained from transgenic animals (eg mice) that are capable, when immunized, of producing the full range or specific human antibodies in the absence of endogenous immunoglobulin production. Transfer of human germline immunoglobulin genetic information into such mutant mouse germlines will result in the production of human antibody upon antigen stimulation (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year Immunol. 7 (1993) 3340). Human antibodies can also be produced by the phage display library method (Hoogenboom, H.R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J.D., et al., J. Mol. Biol. 222 (1991) 581-597). The methods of Cole et al. and Boerner et al. are also applicable to the production of human monoclonal antibodies (Cole et al., Monoclonal antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al. , J. Immunol. 147 (1991) 86-95). Preferably, human monoclonal antibodies are prepared by improved immortalization of EBV-B cells, as described in Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR, Murphy BR, Rappuoli R, Lanzavecchia A. (2004): An efficient method to make human monoclonal antibodies from memory in cells: potent neutralization of SARS coronavirus. Nat Med. 10(8):871-5. The term human antibody, as used herein, also covers those antibodies that have been modified, for example in the variable region, to obtain properties in accordance with the invention, as described in this application. As used herein, the term variable region (light chain variable region (VL), heavy chain variable region (VH)) refers to each

- 3 046175 a pair of light and heavy chains that are directly involved in the binding of the antibody to the antigen.

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

Preferably, the antibody or antigen binding fragment thereof may be a purified antibody, single chain antibody, Fab, Fab', F(ab') ₂ , Fv or scFv.

Thus, the antibodies may preferably be human antibodies, monoclonal antibodies, human monoclonal antibodies, recombinant antibodies or purified antibodies. The invention also provides antibody fragments of the invention, particularly fragments that retain the antigen binding activity of the antibodies. Such fragments include, but are not limited to, single chain antibodies, Fab, Fab', F(ab')2, Fv or scFv. However, in the description, including the claims, in some places, references are made to antigen-binding fragment(s), antibody fragment(s), variant(s) and/or derivative(s) of antibodies, this means that the term antibody or antibody of the invention includes all categories of antibodies, namely, antigen binding fragment(s), antibody fragment(s), antibody variant(s), and antibody derivative(s).

Antibody fragments can be produced from antibodies using 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. Antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments, single chain Fv fragments (scFv) derived from the heavy and light chains of an antibody of the invention, for example, scFvs containing CDRs from an antibody of the invention, as well as monomers and dimers of heavy and light chains, single-domain antibody heavy chain, single-domain antibody light chain, as well as single-chain antibodies, for example, single-chain Fv, in which the heavy and light chain variable domains are connected by a peptide linker.

Antibody fragments can make monovalent or multivalent interactions and can be contained in various structures, as described above. For example, scFv molecules can be synthesized to create a trivalent tribody or a tetravalent tetrabody. scFv molecules include an Fc domain fragment, leading to the formation of divalent minibodies. Additionally, the sequences are components of multispecific molecules, in which the sequences target corresponding epitopes and other regions of the molecule that bind to other targets. Examples of molecules include, but are not limited to, bispecific Fab2, trispecific Fab3, bispecific scFv, and diabodies (Holliger and Hudson, 2005, Nature Biotechnology 9: 1126-1136).

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

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

As used herein, a neutralizing antibody is an antibody that neutralizes, that is, prevents, inhibits, reduces, impedes or interferes with, the ability of a pathogen to initiate and/or establish infection in a host. The terms neutralizing antibody and antibody that neutralizes or antibodies that neutralize are used interchangeably throughout this application. These antibodies may be used alone, or in combination, as prophylactic or therapeutic agents in suitable formulations, in combination with active vaccination, as a diagnostic agent, or as a manufacturing agent, as described herein.

Doses are often expressed in relation to body weight. Thus, a dose that is expressed as [g, mg, or other unit]/kg (or g, mg, etc.) usually refers to [g, mg, or other unit] per kg (or g, mg, etc. .) body weight, even if the term body weight is not specifically stated.

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

The term vaccine, as used herein, is typically meant as a prophylactic or therapeutic material providing at least one antigen, preferably an immunogen. The antigen or immunogen can be derived from any material that is suitable for vaccination. For example, an antigen or immunogen may be derived from a pathogen,

- 4 046175 such as bacteria or viral particles, etc., or from tumor or cancerous tissue. An antigen or immunogen stimulates the body's adaptive immune system to provide an adaptive immune response. In particular, an antigen or immunogen typically refers to a substance that can be recognized by the immune system, preferably the adaptive immune system, and that is capable of triggering an antigen-specific immune response, for example, by producing antibodies and/or antigen-specific T cells as part of the adaptive immune response. Typically, the antigen may be or may contain a peptide or protein that can be presented to MHC T cells.

As used herein, a sequence variant (also referred to as a variant) refers to any change in a sequence, such that a reference sequence is any of the sequences listed in the Table of Sequences and SEQ ID Numbers (sequence listing), that is, SEQ ID NO: 1 - SEQ ID NO: 407. Thus, the term sequence variant includes nucleotide sequence variants and amino acid sequence variants. It should be noted that the sequence variants specified in this application are, in particular, functional sequence variants, that is, sequence variants that retain biological function, for example, antibodies. In the context of the present invention, such biological function is preferably neutralization of ZIKV infection, binding of the antibody to the ZIKV E protein and/or binding of the antibody to the ZIKV NS1 protein. Thus, preferred sequence variants are functional sequence variants having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the comparative sequence. Phrase its functional sequence variant having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, as used herein, indicates (I) that the sequence variant is functional as described in this application, and (II) the higher the % sequence identity, the more preferred the sequence variant.

In other words, a phrase of its functional sequence variant having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity means, in particular, that the functional sequence variant has at least 70% identity sequence identity, preferably at least 75% sequence identity, preferably at least 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 88% sequence identity, even more preferably at least 90% sequence identity , even more preferably at least 92% sequence identity, even more preferably at least 95% sequence identity, even more preferably at least 96% sequence identity, especially preferably at least 97% sequence identity, especially preferably at least 98 % sequence identity and most preferably at least 99% sequence identity to the corresponding comparative sequence.

The term sequence variant includes in particular those variants that contain mutations and/or substitutions compared to the reference sequence. Exemplary Fc component sequence variants include, but are not limited to, those that have an L to A substitution at the CH2 4, CH 2 5, or both positions.

Sequence identity is typically calculated relative to the full length of the reference sequence (ie the sequence cited in the application). Percentage identity as referred to herein can be determined, for example, using BLAST using the default parameters specified by NCBI (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/) [Blosum 62 matrix; penalty for opening a gap=11 and penalty for extending a gap=1].

As used herein, a variant nucleotide sequence has an altered sequence in which one or more nucleotides in the comparative sequence are deleted or replaced, or one or more nucleotides are inserted into the sequence of the comparative nucleotide sequence. Nucleotides are designated herein using a standard single letter designation (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 comparative amino acid sequence, that is, in the amino acid variant

- 5 046175 sequence or not. Preferred sequence variants are those nucleotide sequence variants that do not result in amino acid sequence variants (silent mutations), but non-silent mutations are possible, as well as mutant nucleotide sequences that result in an amino acid sequence that is at least 80% preferably at least 90%, more preferably at least 95% sequence identical to the comparative sequence.

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

While non-conservative amino acid substitutions are possible, it is preferred that the substitutions be conservative amino acid substitutions in which the replaced amino acid has similar structural or chemical properties to the corresponding amino acid in the comparative sequence. By way of example, conservative amino acid substitutions include replacing one of the aliphatic or hydrophobic amino acids, for example, alanine, valine, leucine and isoleucine, with another; replacing one of the hydroxyl-containing amino acids, for example, serine and threonine, with another; replacing one acid residue, for example, glutamic acid or aspartic acid, with another; replacing one amide-containing residue, for example, asparagine and glutamine, with another; replacing one aromatic residue, for example, phenylalanine and tyrosine, with another; replacing one alkaline 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.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from a single residue in polypeptides consisting of a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include fusions at the N- or C-terminus of an amino acid sequence with a reporter molecule or enzyme.

It is important that changes in the sequence variants do not interfere with the functionality of the corresponding reference sequence, in this case, for example, the functionality of the antibody sequence or antigen binding fragment thereof, to bind to the same epitope and/or to sufficiently neutralize a ZIKV infection. Methodological principles for determining which nucleotides and amino acid residues, respectively, can be substituted, inserted or deleted without affecting the specified functionality, can be found using computer programs well known in the art.

As used herein, a nucleic acid sequence or amino acid sequence having the origin of said 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 that sequence or portion thereof from which it is derived, whereby substantially identical includes sequence variants as defined 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, 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). Therefore, the corresponding portions of the peptides, proteins and nucleic acids are readily identifiable to one skilled in the art. Likewise, sequences having their origin from another sequence are usually readily identified by one skilled in the art as having their origin from the sequence.

Preferably, the nucleic acid sequence or amino acid sequence having its origin from another nucleic acid, peptide, polypeptide or protein may be identical to the original nucleic acid, peptide, polypeptide or protein (from which it originates). 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,

- 6 046175 peptide, polypeptide or protein (from which it is derived), in particular a nucleic acid sequence or 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 original nucleic acid, peptide, polypeptide or protein (from which it originates). 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.

As used herein, the term mutation refers to a change in the nucleic acid sequence and/or amino acid sequence compared to a reference sequence, for example, a corresponding genomic sequence. The mutation, for example, compared to the genomic sequence, may be, for example, a (naturally occurring) somatic mutation, a spontaneous mutation, an induced mutation, for example, induced by enzymes, chemicals or radiation, or a mutation obtained by site-directed mutagenesis ( using molecular biological techniques to produce specific and deliberate changes in the nucleic acid sequence and/or amino acid sequence). Thus, the terms mutation or mutation should be understood to also cover the physical occurrence of a mutation, for example, in a nucleic acid sequence or in an amino acid sequence. A mutation includes the substitution, deletion and insertion of one or more nucleotides or amino acids, as well as the insertion of several consecutive nucleotides or amino acids. To produce a mutation in an amino acid sequence, preferably the mutation may be introduced into a nucleotide sequence encoding said amino acid sequence to express the (recombinant) mutated polypeptide. The mutation can be carried out, for example, by changing, for example, using site-directed mutagenesis, a codon of a nucleic acid molecule encoding one amino acid to produce a codon encoding a different amino acid, or by synthesizing a sequence variant, for example, from a known nucleotide sequence of a nucleic acid molecule an acid encoding a polypeptide and by synthesizing a nucleic acid molecule containing a nucleotide sequence encoding a variant polypeptide without the need to mutate one or more nucleotides of the nucleic acid molecule.

Some documents are cited in the text of this description. Each of the documents cited in this application (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether previously or hereinafter, is hereby incorporated by reference in its entirety. Nothing contained in this application should be construed as an admission that the invention gives the right to backdate such disclosure based on an earlier invention.

It is intended that the present invention is not limited to the specific methodology, protocols and reagents described in this application, as they may vary. It should also be understood that the terminology used in this application is intended to describe preferred embodiments only, and is not intended to limit the scope of the present invention, as it will be limited only by the claims appended. Unless specifically stated otherwise, all technical and scientific terms used in this application will have the same meanings as commonly used by one skilled in the art.

Antibodies that effectively neutralize infection caused by the Zika virus.

The present invention is based, among other findings, on the discovery and isolation of antibodies that bind specifically to epitopes of the Zika virus. Such antibodies are either (i) extremely effective in neutralizing Zika virus if targeted to the antigenic determinant of the Zika virus envelope (E) protein or the ZIKV quaternary epitope or (ii) useful for diagnosing Zika virus infection if targeted to the NS1 protein of the virus Zika. Such antibodies are desirable since only very small amounts of antibodies are needed to neutralize the Zika virus. In particular, there is currently no way to prevent or treat Zika virus infection. The antibodies of the present invention are extremely effective in preventing as well as treating or mitigating Zika virus infection. In addition, due to the specificity of antibodies to the Zika virus, they do not cause ADE, but rather block ADE. For diagnosis, Zika virus-specific antibodies provide an important means of differentiating Zika virus infection from infections caused by other flaviviruses such as dengue virus.

In a first aspect, the present invention provides an isolated antibody or antigen binding fragment thereof that specifically binds to an epitope of the Zika virus and neutralizes an infection caused by the Zika virus, which comprises the amino acid sequences CDRH1, CDRH2 and CDRH3 and the amino acid sequences CDRL1, CDRL2 and CDRL3 (i) according to with SEQ ID NO: 1-5 and 7 or (ii) in accordance with SEQ ID NO: 1-4 and 6-7. Preferably they inhibit

- 7 046175 stage of the virus life cycle after the Zika virus attaches to the cell membrane.

In other words, the antibody or antigen binding fragment thereof of the present invention reduces the viral infectivity of the Zika virus.

To study and quantify viral infectivity (or neutralization) in the laboratory, one skilled in the art is familiar with various standard neutralization assays. 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 in which cultured cells are incubated with a fixed amount of Zika virus (ZIKV) in the presence (or absence) of a test antibody. To read the data, you can use, for example, flow cytometry. Alternatively, other readouts are also possible, such as determining the amount of ZIKV non-structural proteins (such as ZIKV NS1) secreted into the culture supernatant. For example, the ZIKV nonstructural protein 1 (NS1) antigen capture test (TCID50-ELISA) based on cultured tissue infectious dose-50 (TCID50) enzyme-linked immunosorbent assay (ELISA) can be used as an alternative to the standard plaque assay for Zika virus titration - in a similar manner as described for dengue virus (DENV) by Li J, Hu D-M, Ding X-X, Chen Y, Pan Y-X, Qiu L-W, Che X-Y: Enzyme-linked immunosorbent assay - format tissue culture infectious dose-50 test for titrating virus denge. PLoS ONE 2011, 6:e22553. In such an assay, it may be advantageous to use, for example, ZIKV No. 1 binding antibodies, as described herein.

Preferably, in a ZIKV neutralization assay, cultured cells, eg Vero cells, are incubated with a fixed amount of ZIKV in the presence or absence of a test antibody, for example, for about four days. After incubation, the cells can be washed and further cultured. To measure viral infectivity, flow cytometry can be used. To do this, cells can be fixed, for example, with 2% formaldehyde, permeabilized, for example, in PBS (phosphate buffered saline) 1% FCS (fetal calf serum) 0.5% saponin, and stained, for example, with mouse 4G2 antibodies. The cells can then be incubated with goat anti-mouse IgG conjugated to a dye such as Alexa Fluor488 and analyzed by flow cytometry. Alternatively, viable cells can be detected by flow cytometry using, for example, WST-1 reagent (Roche). The preferred ZIKV strain used in such a neutralization assay is ZIKV H/PF/2013.

The antibody and antigen-binding fragment described in the application have high neutralizing efficiency. The antibody concentration required to neutralize Zika virus by 50% (IC ₅₀ ) compared to no antibody controls is, for example, up to about 3 μg/ml or up to about 1 μg/ml. Preferably, the concentration of antibody required to neutralize 50% of ZIKV (IC ₅₀ ) is up to about 500 ng/ml, more preferably the concentration of the antibody of the invention required to neutralize 50% of ZIKV (IC 50 ) is up to about 250 ng /ml, even more preferably, the concentration of the antibody according to the invention required to neutralize 50% of ZIKV (IC ₅₀ ) is up to about 150 ng/ml. Most preferably, the antibody concentration required to neutralize 50% of ZIKV (IC ₅₀ ) is about 100 ng/ml or less, for example, about 90 ng/ml or less, about 80 ng/ml or less, about 70 ng/ml or less, about 60 ng/ml or less, about 50 ng/ml or less, about 45 ng/ml or less, about 40 ng/ml or less, about 35 ng/ml or less, about 30 ng/ml or less , about 25 ng/ml or less, about 20 ng/ml or less, or, especially preferably, about 15 ng/ml or less. In particular, the antibody concentration required to neutralize 50% of ZIKV (IC50) is preferably about 50 ng/ml or less. This means that only low concentrations of antibody are needed to achieve 50% neutralization of ZIKV. The antibody concentration required to neutralize 50% of ZIKV (IC ₅₀ ) can be measured using standard neutralization assays as known to one of ordinary skill in the art, or particularly as described above.

In most cases, antibody binding can be assessed using a standard ELISA (enzyme-linked immunosorbent assay), which is well known to one skilled in the art. A typical standard ELISA can be performed as follows: ELISA plates can be coated (eg overnight at 4°C) with a sufficient amount (eg 1 μg/ml) of the protein/complex/particle to which the test antibody binds (eg for DENV binding, as described below, uses DENV E proteins and/or DENV VLPs), for example, in PBS. The plates can then be blocked, for example with 1% w/v. bovine serum albumin (BSA) solution in PBS, and incubate with the test antibody (eg, approximately 1.5 hours at room temperature). After washing, antibody binding can be determined, for example, using goat anti-human IgG coupled to alkaline phosphatase. After this, the plates can be washed, the necessary substrate can be added (for example, p

- 8 046175

NPP) and plates can be analyzed, for example, at 405 nm. The relative binding affinities of an antibody can be determined by measuring the mAb concentration (EC ₅₀ ) required to achieve 50% of maximum binding at saturation. EC ₅₀ values can be calculated by interpolating binding curves fitted using nonlinear slope-variable four-parameter regression.

Preferably, the antibody or antigen-binding fragment thereof of the present invention does not substantially bind to dengue virus-like particles and/or dengue envelope protein. Thus, substantially no binding means that for an antibody or antigen-binding fragment thereof, EC ₅₀ values up to 10 ² ng/ml, preferably up to 10 ³ ng/ml, more preferably up to 5x10 ³ ng/ml, even more preferably up to 8x10 ³ ng/ml, and most preferably up to 10 ⁴ ng/ml can be determined in a standard ELISA for dengue virus-like particles (DENV VLP) and/or dengue envelope protein (DENV E protein). In other words, the concentration of antibody or antigen binding fragment required to achieve 50% maximum saturation binding (EC50) to dengue virus-like particles (DENV VLP) and/or dengue envelope protein (DENV E protein) in a standard ELISA is typically greater more than 10 ² ng/ml, preferably greater than 10 ³ ng/ml, more preferably greater than 5 x 10 ³ ng/ml, even more preferably greater than 8 x 10 ³ ng/ml, and most preferably greater 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.

ADE can be assessed by flow cytometry-based analysis 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, permeabilized, and stained with m4G2, for example, as described above for neutralization assays. The number of infected cells was determined by flow cytometry as described above for neutralization assays.

Preferably, the antibody or antigen-binding fragment thereof of the present invention is a human antibody.

Preferably, the antibody of the present invention, or an antigen binding fragment thereof, contains an Fc component. More preferably, an Fc component.

As used herein, the term Fc component refers to a sequence originating from the immunoglobulin heavy chain region beginning in the hinge region immediately upstream of the papain cleavage site (e.g., residue 216 in native IgG, taking the first residue of the heavy chain constant region as the number 114) and ending at the C-terminus of the immunoglobulin heavy chain. Thus, the Fc component may represent the entire Fc component or a portion thereof (eg, a domain). A complete Fc component contains at least a hinge domain, a CH2 domain, and a CH3 domain (eg, EU amino acid positions 216-446). Sometimes an additional lysine residue (K) is present at the very C-terminus of the Fc component, but this is often cleaved from the mature antibody. Each of the amino acid positions within the Fc component has been numbered in accordance with the EU Cabot numbering system known in the art, see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. .Health and Human Services), 1983 and 1987.

In the context of the present invention, the Fc component contains at least one: a hinge (eg, upper, middle and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant, portion or fragment thereof. In preferred embodiments, the Fc component comprises at least a hinge domain, a CH2 domain, or a CH3 domain. More preferably, the Fc component is a complete Fc component. The Fc component may also contain one or more amino acid insertions, deletions or substitutions compared to the naturally occurring Fc component. For example, at least one of the hinge domain, CH2 domain, or CH3 domain (or a portion thereof) may be deleted. For example, an Fc component may contain or consist of: (I) a hinge domain (or part thereof) fused to a CH2 domain (or part thereof), (II) a hinge domain (or part thereof) fused to a CH3 domain (or part thereof) ), (III) CH2 domain (or part thereof) fused to a CH3 domain (or part thereof), (IV) hinge domain (or part thereof), (V) CH2 domain (or part thereof), or (VI) CH3 domain or part thereof.

One skilled in the art will appreciate that the Fc component can be modified such that it differs in amino acid sequence from the full Fc component of a naturally occurring immunoglobulin molecule while retaining at least one desired function provided by the naturally occurring Fc component. Such functions include Fc receptor (FcR) binding, antibody half-life modulation, ADCC function, protein A binding, protein G binding, and complement fixation. Parts of naturally occurring Fc components that are responsible and/or essential for such functions are well

- 9 046175 which are known to those skilled in the art.

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

For example, FcR binding can be mediated by the interaction of an Fc component (antibody) with Fc receptors (FcRs), which are specialized cell surface receptors on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and are known to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes and the lysis of red blood cells and various other cellular targets (such as tumor cells) coated with the appropriate antibody by antibody-dependent cell-mediated cytotoxicity ( ADCC; Van de Winkel, J. G., and Anderson, C. L., J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their immunoglobulin class specificity; Fc receptors for IgG antibodies are designated FcyR, for IgE as FcεR, for IgA as FcaR, and so on, and neonatal Fc receptors are designated FcRn. Fc receptor binding is described, for example, in Ravetch, J.V., and Kinet, J.P., Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P.J., et al., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J Lab. Clin. Med. 126 (1995) 330-341; and Gessner, J.E., et al., Ann. Hematol. 76 (1998) 231-248.

Cross-linking of receptors by the Fc domain of native IgG antibodies (FcyR) triggers a variety of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators, as well as immune complex clearance and regulation of antibody production. Therefore, Fc components that provide receptor cross-linking (FcyR) are preferred. In humans, three classes of FcyRs have been characterized, which are: (i) FcyRI (CD64), which bind monomeric IgG with high affinity and are expressed on macrophages, monocytes, neutrophils and eosinophils; (II) FcyRII (CD32), which bind complex IgG with medium to low affinity, are widely expressed, especially on leukocytes, and are known to be a key element in antibody-mediated immunogenicity, and they can be divided into FcyRIIA, FcyRIIB and FcyRIIC , which perform different functions in the immune system, but bind with similar low activity 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 is highly expressed on neutrophils. FcyRIIA have been found on many cells involved in killing (eg, macrophages, monocytes, neutrophils) and are believed to be capable of activating the killing process. FcyRIIBs are believed to be involved in inhibitory processes and have been found on B cells, macrophages, and on mast cells and eosinophils. Importantly, 75% of all FcyRIIB was found in the liver (Ganesan, L.P. et al., 2012: FcYRIIb on liver sinusoidal endothelium clears small immune complexes. Journal of Immunology 189: 4981-4988). FcyRIIB is abundantly expressed in liver sinusoidal endothelium, called LSEC, and in Kupffer cells in the liver, and LSEC represent the major site of clearance of small immune complexes (Ganesan, L.P. et al., 2012: FcYRIIb on liver sinusoidal endothelium clears small immune complexes. Journal of Immunology 189: 4981-4988).

Thus, preference is given to those antibodies and antigen-binding fragments thereof that are capable of binding to FcYRIIb, for example, antibodies containing an Fc component for binding to FcYRIIb, especially an Fc fragment, such as, for example, IgG-type antibodies. In addition, it appears possible to engineer the Fc component to enhance FcyRIIB binding by introducing the S267E and L328F mutations, as described by Chu, S.Y. et al., 2008: Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies. Molecular Immunology 45, 3926-3933. Thus, clearance of immune complexes may be enhanced (Chu, S., et al., 2014: Accelerated Clearance of IgE In Chimpanzees Is Mediated By Xmab7195, An FcEngineered Antibody With Enhanced Affinity For Inhibitory Receptor FcYRIIb. Am J Respir Crit, American Thoracic Society International Conference Abstracts). Thus, in the context of the present invention, those antibodies or antigen binding fragments thereof are preferred which contain an engineered Fc component with the S267E and L328F mutations, particularly as described by Chu, S.Y. et al., 2008: Inhibition of B cell receptor - mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies. Molecular Immunology 45, 3926-3933.

It is believed that on B cells their function is to suppress further production of immunoglobulins.

- 10 046175 bulins and switching the isotype, for example, to the IgE class. On macrophages, FcyRIIB acts by inhibiting phagocytosis, which is mediated by FcyRIIA. On eosinophils and mast cells, the b form may help suppress the activation of these cells by IgE binding to its distinct receptor.

When considering FcyRI binding, modification in the native IgG of at least one of E233-G236, P238, D265, N297, A327 and P329 reduces binding to FcyRI. IgG2 residues at positions 233-236, substituted in IgG1 and IgG4, reduce binding to FcyRI by 10 ³ -fold and eliminate the response of human monocytes to antibody-sensitized red blood cells (Armour, KL, et al. Eur. J. Immunol. 29 (1999) 2613-2624). When considering FcyRII binding, reduced binding for FcyRIIA was found, for example, for IgG mutations of at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292 and K414. When considering FcyRIII binding, reduced binding to FcyRIIIA was found, for example, for mutation of at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303 , A327, K338 and D376. Mapping of binding sites on human IgG1 for Fc receptors, the above mutation sites, and methods for determining binding to FcyRI and FcyRIIA are described in Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604.

When considering binding to the extremely important FcyRII, two regions of the native IgG Fc are believed to be key for FcyRII and IgG interactions, namely (I) the lower hinge site of the IgG Fc, especially the amino acid residues L, L, G, G (234-237 , EU numbering), and (ii) the adjacent CH2 region of the IgG Fc domain, especially the loop and chains in the upper CH2 domain adjacent to the lower hinge region, for example, in the P331 region (Wines, B.D., et al., J. Immunol. 2000;164:5313-5318). In addition, FcyRI is believed to bind to the same site on the IgG Fc, while FcRn and protein A bind to a different site on the IgG Fc, which appears to be at the CH2-CH3 interface (Wines, B.D. , et al., J Immunol 2000;164:5313-5318).

For example, the Fc component may contain or consist of at least a portion of the Fc component that is known in the art to be required for FcRn binding or half-life extension. Alternatively or additionally, the Fc component of the antibody may contain at least a portion that is known in the art to be necessary for protein A binding and/or the Fc component of the antibody may contain at least a portion of an Fc molecule that is known in the art , is required for G protein binding. Preferably, the retained function is the neutralization of Zika virus infection, which is believed to be mediated by FcyR binding. Thus, a preferred Fc component may contain at least a portion that is known in the art to be necessary for FcyR binding. Thus, as stated above, a preferred Fc component may at least comprise (i) the lower hinge site of the native IgG Fc, particularly amino acid residues L, L, G, G (234-237, EU numbering), and (ii) an adjacent the CH2 domain region of a native IgG Fc, especially the loop and strands in the upper CH2 domain adjacent to the lower hinge region, for example in the P331 region, for example a 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 according to the present invention contains an Fc fragment. As used herein, the term Fc fragment refers to the portion of an immunoglobulin formed by two or more Fc components of an antibody heavy chain. For example, the Fc fragment may be a monomeric or single chain Fc fragment (ie, a scFc fragment). Single-stranded Fc fragments consist of Fc components linked within a single polypeptide chain (eg, encoded in a single contiguous nucleic acid sequence). Typical scFc fragments are described in WO 2008/143954 A2. Preferably, the Fc fragment is a dimeric Fc fragment. A dimeric Fc fragment or dcFc refers to a dimer formed by the Fc components of two separate immunoglobulin heavy chains. The dimeric Fc fragment may be a homodimer of two identical Fc components (eg, an Fc fragment of a naturally occurring immunoglobulin) or a heterodimer of two non-identical Fc components.

The Fc components of an Fc fragment may be from the same or different classes and/or subclasses. For example, the Fc components may be of immunoglobulin (eg, human immunoglobulin) origin of the IgG1, IgG2, IgG3, or IgG4 subclass. Preferably, the Fc components of the Fc fragment are of the same class and subclass. However, the Fc fragment (or one or more Fc components of the Fc fragment) may also be chimeric, whereby the chimeric Fc fragment may contain Fc components originating from different classes and/or subclasses of immunoglobulins. For example, at least two of the Fc components of a dimeric or single chain Fc fragment may be from different classes and/or subclasses of immunoglobulins. Additionally or alternatively, the chimeric Fc fragments may contain one or more chimeric Fc components. For example, a chimeric Fc fragment or component may contain one or more portions originating from an immunoglobulin of the first subclass (eg, IgG1, IgG2, or IgG3 subclass), while the remaining portion of the Fc fragment or component is from a different subclass. For example, an Fc fragment or component of an Fc polypeptide may contain a CH2 and/or CH3 domain having

- 11 046175 origin from an immunoglobulin of the first subclass (for example, IgG1, IgG2 or IgG4 subclass) and a hinge region from an immunoglobulin of the second subclass (for example, IgG3 subclass). For example, the Fc fragment or component may comprise 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, a chimeric Fc fragment may comprise an Fc component (eg, a complete Fc component) from a first subclass immunoglobulin (eg, an IgG4 subclass) and an Fc component from a second subclass immunoglobulin (eg, an IgG1, IgG2, or IgG3 subclass). For example, the Fc fragment or component may comprise a CH2 domain from an IgG4 immunoglobulin and a CH3 domain from an IgG1 immunoglobulin. For example, the Fc fragment or component may comprise a CH1 domain and a CH2 domain from an IgG4 molecule and a CH3 domain from an IgG1 molecule. For example, the Fc fragment or component may comprise part of a CH2 domain from a preferred subclass of the antibody, for example EU positions 292-340 of the CH2 domain. For example, the Fc fragment or component may contain amino acids at positions 292-340 of CH2 originating from the IgG4 component and the remainder of CH2 originating from the IgG1 component (alternatively, 292-340 of CH2 may be originating from the IgG1 component and the remainder CH2 , originating from the IgG4 component).

In addition, the Fc fragment or component may further or alternatively comprise, for example, a chimeric hinge region. For example, a chimeric hinge may be derived, for example, in part from an IgG1, IgG2, or IgG4 molecule (eg, an upper and lower middle hinge sequence) and, in part, from an IgG3 molecule (eg, a middle hinge sequence). In another example, the Fc fragment or component may comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule. In another example, the chimeric hinge may comprise top and bottom hinge domains from an IgG4 molecule and a middle hinge domain from an IgG1 molecule. Such a chimeric hinge can be obtained, for example, by introducing a proline substitution (Ser228Pro) at EU position 228 in the middle hinge domain of the IgG4 hinge region. In another embodiment, the chimeric hinge may contain amino acids at EU positions 233-236 from an IgG2 antibody and/or a Ser228Pro mutation, where the remaining hinge amino acids are of origin from an IgG4 antibody (eg, chimeric hinge sequence ESKYGPPPPPCPAPPVAGP). Other chimeric hinges that may be used in the Fc component of an antibody of the present invention are described in US 2005/0163783 A1.

An Fc component or Fc fragment may contain or consist of an amino acid sequence derived from a human immunoglobulin sequence (eg, an Fc fragment or Fc component from a human IgG molecule). However, the polypeptides may contain one or more amino acids from other mammalian species. For example, a primate Fc component or a primate binding site may be included in the claimed polypeptides. Alternatively, one or more murine amino acids may be present in the Fc component or Fc fragment.

Preferably, the antibody may contain, in particular in addition to the Fc component 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 accordingly may comprise, especially in addition to the Fc component as described above, all other constant region parts, in particular all other IgG constant region parts, preferably all other IgG1 constant region parts, more preferably all other constant region parts Human IgG1.

Particularly preferred constant region sequences may be amino acid sequences according to SEQ ID NOs: 145-148 (nucleic acid sequences according to SEQ ID NOs: 149-152). Preferably, the amino acid sequence of IgG1 CH1-CH2-CH3 may be the sequence according to SEQ ID NO: 145 or a functional sequence variant thereof. Even more preferably, the amino acid sequence of IgG1 CH1-CH2-CH3 may be the sequence according to SEQ ID NO: 146 or a functional sequence variant thereof, as described herein, where the LALA mutation is supported.

As stated above, a particularly preferred antibody according to the present invention contains a (full) Fc fragment derived from human IgG1. However, the antibody may contain, in particular in addition to the (full) Fc fragment originating from human IgG1, also all other 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 wishing to be bound by theory, it is believed that antibody-dependent enhancement (ADE) of Zika virus infection is likely due to the binding of the Fc component of the antibody, particularly the Fc component of the heavy chain of the IgG molecule, to an Fc receptor, e.g., the Fcy receptor on host cell. Thus, it is preferred that the antibody or antigen binding fragment thereof may contain one or more mutations in the Fc component. The mutation(s) may be any mutation that reduces the binding of the antibody to the Fc receptor (FcR). Thus, it is preferred that the antibody or antigen binding fragment thereof may comprise one

- 12 046175 or several mutations in the Fc component, which (s) reduce(s) the binding of the antibody to the Fcy receptor, but does not disrupt the interaction with FcRn. One example of such a mutation is the LALA mutation, described below.

In general, antibody binding to the Fc receptor can be assessed using various methods known to one skilled in the art, such as ELISA (Hessell AJ, Hangartner L, Hunter M, Havenith CEG, Beurskens FJ, Bakker JM, Lanigan CMS, Landucci G, Forthal DN, Parren PWHI, et al: Fc receptor but not complement binding is important in antibody protection against HIV. Nature 2007, 449:101-104; Grevys A, Bern M, Foss S, Bratlie DB, Moen A, Gunnarsen KS, Aase A, Michaelsen TE, Sandlie I, Andersen JT: Fc Engineering of Human IgG1 for Altered Binding to the Neonatal Fc Receptor Affects Fc Effector Functions. 2015, 194:5497-5508) or flow cytometry (Perez LG, Costa MR , Todd CA, Haynes BF, Montefiori DC: Utilization of immunoglobulin G Fc receptors by human immunodeficiency virus type 1: a specific role for antibodies against the membrane-proximal external region of gp41. J Virol 2009, 83:7397-7410; Piccoli L , Campo I, Fregni CS, Rodriguez BMF, Minola A, Sallusto F, Luisetti M, Corti D, Lanzavecchia A: Neutralization and clearance of GM-CSF by autoantibodies in pulmonary alveolar proteinosis. Nat Commun 2015, 6:1-9).

The antibody 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. Thus, the CH2 domain of the Fc component of an antibody may contain 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 sugars (0, 1, or 2) at the ends of the biantennary glycan chain. Preferably, the antibody glycans do not result in immunogenic responses 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 heavy chain CH2 domain to change their FcR binding affinity and/or their serum half-life compared to unmodified antibodies.

Particularly preferably, the Fc component of the antibody may contain a substitution at the CH2 4, CH2 5, or both positions. Typically, 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 both CH2 L4A and CH2 L5A substitutions. Such antibodies are referred to herein as the LALA variant. It is extremely interesting that such a LALA mutation in the Fc component 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. A typical amino acid sequence of an IgG1 CH1-CH2-CH3 containing a LALA mutation is the sequence according to SEQ ID NO: 146. 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 preferred that the antibody or antigen binding fragment thereof binds to an epitope of the Zika virus envelope protein that includes one or more amino acid residues of the Zika virus envelope protein domain III (EDIII). ZIKV includes a nucleocapsid core that contains single-stranded RNA wrapped in nuclear proteins. The nucleocapsid core is encapsulated by a lipid bilayer membrane with membrane proteins and envelope proteins. The ZIKV envelope protein (E protein) is the dominant antigen. The coat protein ectodomain contains three distinct 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. 12 for amino acid sequence alignment of EDIII of different ZIKV strains).

Thus, 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: 401):

TAAFTFTKXPAEXXHGTVTVEXQYXGXDGPCKXPXQMAVDXQTLTPVGRLITA

NPVITEXTENSKMMLELDPPFGDSYIVIGXGXKKITHHWHRS where X can be any (naturally occurring) amino acid.

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 having the following amino acid sequence (SEQ ID NO: 407):

X ₁ GX2X3YSLCTAAFTFTKX ₄ PAEX ₅ X ₆ HGTVTVEX7QYX ₈ GX9DGPCKX ₁ oPXiiQM

AVDXuQTLTPVGRLITANPVITEXBTXMNSKMMLELDPPFGDSYIVIGXisGXie

X ₁₇ KITHHWHRSG where X1 can be any (naturally occurring) amino acid, preferably

- 13 046175 specifically K, A, or E;

X2 may be any (naturally occurring) amino acid, preferably V, F, or L;

X3 may be any (naturally occurring) amino acid, preferably S or F;

X ₄ can be any (naturally occurring) amino acid, preferably I or V;

X ₅ can be any (naturally occurring) amino acid, preferably T or V;

X ₆ can be any (naturally occurring) amino acid, preferably L or D;

X ₇ can be any (naturally occurring) amino acid, preferably V or G;

X ₈ can be any (naturally occurring) amino acid, preferably or G;

X ₉ can be any (naturally occurring) amino acid except R, preferably T or A;

X ₁₀ can be any (naturally occurring) amino acid, preferably V or I;

X ₁₁ can be any (naturally occurring) amino acid, preferably or V;

X ₁₂ can be any (naturally occurring) amino acid, preferably M or T;

X ₁₃ may be any (naturally occurring) amino acid, preferably S or G;

X ₁₄ can be any (naturally occurring) amino acid, preferably E or K;

X ₁₅ may be any (naturally occurring) amino acid, preferably V or I;

X ₁₆ can be any naturally occurring amino acid, preferably E, A, K, or D; And

X ₁₇ can be any (naturally occurring) amino acid, preferably E, A, or K, more preferably K or A.

For example, EDIII extends from amino acid 309 to amino acid 403 of the E protein of ZIKV strain ZIKV H/PF/2013 (Genbank accession number KJ776791). Thus, 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: 402):

TAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITA

NPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRS.

Surprisingly, the inventors of the present invention have discovered that antibody binding to domain III of the Zika virus envelope protein (EDIII) exhibits (i) increased neutralization of ZIKV and (ii) reduced cross-reactivity with DENV (specifically, essentially no cross-reactivity with DENV) , compared to antibody binding to domain I/II of the Zika virus envelope protein (EDI/II).

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 EDI-EDIII in the hinge region. The EDIII lateral ridge and the EDI-EDIII hinge region are known to one skilled in the art and are described, for example, in Zhao, H., Fernandez, E., Dowd, K.A., Speer, S.D., Platt, D.J., Gorman, M.J., Govero, J., Nelson, C. A., Pierson, T. C., Diamond, M. S., et al. (2016). Structural Basis of Zika Virus-Specific Antibody Protection. Cell 166(4): 1016-27 and in Kostyuchenko VA, Lim EX, Zhang S, Fibriansah G, Ng TS, Ooi JS, Shi J, Lok SM. Structure of the thermally stable Zika virus. Nature. 2016 May 19;533(7603):425-8. Without wishing to be bound by theory, it is believed that (i) binding to LR may inhibit fusion by delaying the viral fusion transition state and (ii) binding to the EDI-EDIII hinge and EDIII may interfere with the movement of EDIII to form a three-dimensional structure after fusion, stopping thus membrane synthesis.

Thus, the claimed antibody or antigen-binding fragment thereof (is capable of) inhibiting the ZIKV post-attachment step). Postattachment typically refers to any stage of ZIKV infection after ZIKV has attached to the cell membrane (the cell targeted by ZIKV). For example, they are preferably capable of preventing membrane synthesis. In addition, before

- 14 046175 respectfully described in the application, the antibody or antigen-binding fragment thereof is capable of causing aggregation of ZIKV (particles), as well as inhibiting the post-attachment step of ZIKV and (ii) causing aggregation of ZIKV (particles).

The antibody or antigen binding fragment thereof can also bind to a quaternary epitope present on the ZIKV infectious virion. Despite significant neutralizing activity, such antibodies typically exhibit undetectable binding to recombinant ZIKV E protein or ZIKV EDIII in a standard ELISA (as described above), that is, when tested in vitro, especially in purified form (i.e., E ZIKV protein outside/without virion, virus-like particle, etc.). Thus, undetectable binding typically means that EC50 up to 10,000 ng/ml is undetectable in a standard ELISA. In other words, if the EC50 detected in a standard ELISA is above 10,000 ng/ml, then this is designated as undetectable binding.

Thus, such antibodies are also referred to herein as neutralizing but non-E binding (NNB) antibodies. The quaternary epitope displayed on the infectious ZIKV virion is typically a conformational epitope. For example, a quaternary epitope exposed on an infectious ZIKV virion may be formed at the monomer interface of two envelope proteins, forming a dimer (envelope dimer epitope; EDE), or it may be formed within adjacent dimers (herringbone epitope).

In general, the antibody or antigen binding fragment thereof preferably contains (at least) three complementarity determining regions (CDRs) on the heavy chain and (at least) three CDRs on the light chain. Typically, complementarity determining regions (CDRs) are hypervariable regions present on the heavy chain variable domains and the light chain variable domains. Typically, the CDRs of the heavy chain and the associated light chain of an antibody together form an antigen receptor. Typically, three CDRs (CDR1, CDR2, and CDR3) are located non-sequentially in the variable domain. Because antigen receptors typically consist of two variable domains (on two different polypeptide chains, i.e., heavy and light chain), there are six CDRs for each antigen receptor (heavy chain: CDRH1, CDRH2, and CDRH3; light chain: CDRL1, CDRL2, and CDRL3). A single antibody molecule typically has two antigen receptors and therefore contains twelve CDRs. The CDRs on the heavy and/or light chain may be separated by framework regions, whereby a framework region (FR) is a region in a variable domain that is less variable than a CDR. For example, a chain (or each chain, respectively) may consist of four framework regions separated by three CDRs.

The heavy chain and light chain sequences of typical antibodies, containing three different CDRs on the heavy chain and three different CDRs on the light chain, have been determined. CDR amino acid positions are defined according to the IMGT numbering system (IMGT: http://www.imgt.org/; cf. Lefranc, M.-P. et al. (2009) Nucleic Acids Res. 37, D1006-D1012).

In table 1 provides SEQ ID NOs for the heavy chain CDR amino acid sequences (CDRH1, CDRH2, and CDRH3) and the heavy chain variable region (denoted VH) of exemplary antibodies in accordance with the present invention.

- 15 046175

Table 1

Antibody name 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 237 238 239 240 ZKA4 241 242 243 244 ZKA5 245 246 247 248 ZKA6 249 250 251 252 ZKA7 253 254 255 256 ZKA8 257 258 259 260 ZKA76 261 262 263 264 ZKA117 265 266 267 268 ZKB27 269 270 271 272 ZKB29 273 274 275 276 ZKB34 277 278 279 280 ZKB39 281 282 283 284 ZKB46 285 286 287 288 ZKB53 289 290 291 292 ZKC26 293 294 295 296 ZKD5 297 298 299 300

- 16 046175

ZKD7 301 302 303 304 ZKD8 305 306 307 308 ZKD15 309 310 311 312 ZKD16 313 314 315 316 ZKD17 317 318 319 320 ZKD20 321 322 323 324 ZKA134 325 326 327 328 ZKA246 329 330 331 332 ZKA256 333 334 335 336 ZKB42 337 338 339 340 ZKB85 341 342 343 344 ZKB47 345 346 347 348 ZKC6 349 350 351 352 ZKA160 353 354 355 356 ZKA172 357 358 359 360 ZKA174 361 362 363 364 ZKA189 365 366 367 368 ZKA195 369 370 371 372 ZKA215 373 374 375 376 ZKA218 377 378 379 380 ZKB75 381 382 383 384 ZKB83 385 386 387 388 ZKC3 389 390 391 392 ZKC18 393 394 395 396 ZKD1 397 398 399 400

In table 2 below sets forth SEQ ID NOs for the amino acid sequences of the light chain CDRs (CDRL1, CDRL2, and CDRL3) and the light chain variable region (referred to as VL) of exemplary antibodies in accordance with the present invention.

table 2

Name CDRL1 CDRL2 CDRL2 CDRL3 VL antibodies long 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, preferably, the antibody or antigen binding fragment thereof of the invention comprises a heavy chain variable region (VH) with the amino acid sequence of SEQ ID NO: 8 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and/or light chain variable region (VL) with the amino acid sequence according to SEQ ID NO: 9 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least at least 98% or at least 99% sequence identity.

The antibody or antigen binding fragment thereof may also comprise 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 at least one CDR, preferably at least one heavy chain CDRH3, contains or consists of an amino acid sequence as specified in any of SEQ

- 17 046175

ID NO: 3, 75, 39, 21, 57, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311 , 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363, 367, 371, 375, 379, 383, 387, 391, 395, and 399, or a variant of its functional sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

The antibody or antigen binding fragment thereof may comprise 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 at least one CDR, preferably at least one heavy chain CDRH3, contains or consists of an amino acid sequence as defined in any of SEQ ID NOs: 3, 21, 39, 57 and 75 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. The antibody or antigen binding fragment thereof may comprise 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 at least one CDR, preferably at least one heavy chain CDRH3, contains or consists of an amino acid sequence as defined in any of SEQ ID NOs: 3, 21, 39 and 75 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96% , at least 97%, at least 98%, or at least 99% sequence identity. The antibody or antigen binding fragment thereof may comprise 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 at least one CDR, preferably at least one heavy chain CDRH3, contains or consists of an amino acid sequence according to SEQ ID NO: 3 or according to SEQ ID NO: 75; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. Also, the antibody or antigen-binding fragment thereof may comprise a heavy chain containing at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain containing at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein at least one CDR, preferably at least one heavy chain CDRH3, contains or consists of an amino acid sequence according to SEQ ID NO: 21 or according to SEQ ID NO: 39; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In addition, the antibody or antigen binding fragment thereof may comprise a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain containing at least one CDRL1, at least one CDRL2 and at least one at least one CDRL3, wherein at least one CDR, preferably at least one heavy chain CDRH3, contains or consists of an amino acid sequence according to SEQ ID NO: 3 or a functional sequence variant thereof having 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% sequence identity.

The antibody or antigen binding fragment thereof may comprise 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 (I) at least one heavy chain CDRH1 contains an amino acid sequence as defined in any of SEQ ID NO: 1, 19, 37, 55, 73, 237, 241, 245, 249, 253, 257, 261, 265 , 269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 3 65 , 369, 373, 377, 381, 385, 389, 393, and 397, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, of 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

- 18 046175 number of sequences;

(II) at least one CDRH2 contains an amino acid sequence as defined in any of SEQ ID NO: 2, 20, 38, 56, 74, 238, 242, 246, 250, 254, 258, 262, 266, 270, 274 , 278, 282, 286, 290, 294, 298, 302, 306, 310, 314, 318, 322, 326, 330, 334, 338, 342, 346, 350, 354, 358, 362, 366, 370, 3 74 , 378, 382, 386, 390, 394, and 398, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88% , at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and/or (III) at least one heavy chain CDRH3 contains an amino acid sequence as defined in any of SEQ ID NO: 3, 21, 39, 57, 75, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363 , 367, 371, 375, 379, 383, 387, 391, 395, and 399, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

An antibody or an antigen-binding fragment thereof may also contain a heavy chain containing at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain containing at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein (I) at least one heavy chain CDRH1 contains an amino acid sequence as defined in any of SEQ ID NOs: 1, 19, 37, 55 and 73 or a functional sequence variant thereof having at least 70% of at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

(II) at least one CDRH2 contains an amino acid sequence as defined in any of SEQ ID NOs: 2, 20, 38, 56 and 74 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and/or (III) at least one heavy chain CDRH3 contains an amino acid sequence as defined in any of SEQ ID NOs: 3, 21, 39, 57 and 75 or a functional sequence variant thereof having at least 70% of at least at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97 %, at least 98% or at least 99% sequence identity.

The antibody or antigen binding fragment thereof may further comprise 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 (I) at least one heavy chain CDRH1 contains an amino acid sequence as defined in any of SEQ ID NOs: 1, 19, 37 and 73 or a functional sequence variant thereof having at least 70% of at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

(II) at least one CDRH2 contains an amino acid sequence as defined in any of SEQ ID NO: 2, 20, 38 and 74 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and/or (III) at least one heavy chain CDRH3 contains an amino acid sequence as defined in any of SEQ ID NOs: 3, 21, 39 and 75 or a functional sequence variant thereof having at least 70% of at least 75 %, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

Finally, the antibody or antigen binding fragment thereof may comprise a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain containing at least one CDRL1, at least one CDRL2 and at least one

- 19 046175 at least one CDRL3, wherein (I) at least one heavy chain CDRH1 contains an amino acid sequence in accordance with SEQ ID NO: 1 or in accordance with SEQ ID NO: 73; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

(II) at least one CDRH2 contains an amino acid sequence in accordance with SEQ ID NO: 2 or in accordance with SEQ ID NO: 74; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and/or (III) at least one heavy chain CDRH3 contains an amino acid sequence according to SEQ ID NO: 3 or according to SEQ ID NO: 75; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

Also, the antibody or antigen-binding fragment thereof may comprise a heavy chain containing at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain containing at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein (I) at least one heavy chain CDRH1 contains an amino acid sequence according to SEQ ID NO: 19 or according to SEQ ID NO: 37; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

(II) at least one CDRH2 contains an amino acid sequence according to SEQ ID NO: 20 or according to SEQ ID NO: 38; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and/or (III) at least one heavy chain CDRH3 contains an amino acid sequence according to SEQ ID NO: 21 or according to SEQ ID NO: 39; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

The antibody or antigen binding fragment thereof may comprise 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 (I) at least one heavy chain CDRH1 contains an amino acid sequence according to SEQ ID NO: 1 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 % sequence identity;

(ii) at least one CDRH2 contains an amino acid sequence in accordance with SEQ ID NO: 2 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and/or (III) at least one heavy chain CDRH3 contains an amino acid sequence in accordance with SEQ ID NO: 3 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, according to at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

The antibody or antigen binding fragment thereof may comprise a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain

- 20 046175 a chain containing at least one CDRL1, at least one CDRL2 and at least one CDRL3, where (I) at least one CDRL1 contains an amino acid sequence as specified in any of SEQ ID NO: 4, 22, 40, 58 and 76 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

(II) at least one CDRL2 contains an amino acid sequence as defined in any of SEQ ID NO: 5, 6, 23, 24, 41, 42, 59, 60, 77 and 78 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96% , at least 97%, at least 98%, or at least 99% sequence identity; and/or (III) at least one CDRL3 amino contains an amino acid sequence as defined in any of SEQ ID NO: 7, 25, 43, 61 and 79 or a functional sequence variant thereof having 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% sequence identity.

In addition, the antibody or antigen binding fragment thereof may comprise a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain containing at least one CDRL1, at least one CDRL2 and at least one at least one CDRL3, wherein (I) at least one CDRL1 contains an amino acid sequence as defined in any of SEQ ID NO: 4, 22, 40 and 76 or a functional sequence variant thereof having at least 70%, at least 75 %, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity;

(II) at least one CDRL2 contains an amino acid sequence as defined in any of SEQ ID NO: 5, 6, 23, 24, 41, 42, 77 and 78 or a functional sequence variant thereof having at least 70%, according to at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and/or (III) at least one CDRL3 amino contains an amino acid sequence as defined in any of SEQ ID NO: 7, 25, 43 and 79 or a functional sequence variant thereof having at least 70%, at least 75% , at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, according to at least 98% or at least 99% sequence identity.

In addition, the antibody or antigen binding fragment thereof may comprise a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain containing at least one CDRL1, at least one CDRL2 and at least one at least one CDRL3, where (I) at least one CDRL1 contains an amino acid sequence in accordance with SEQ ID NO: 4 or in accordance with SEQ ID NO: 76; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

(II) at least one CDRL2 contains an amino acid sequence as defined in any of SEQ ID NOs: 5, 6, 77 and 78 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and/or (III) at least one CDRL3 amino contains an amino acid sequence in accordance with SEQ ID NO: 7 or in accordance with SEQ ID NO: 79; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

- 21 046175

Also, the antibody or antigen-binding fragment thereof may comprise a heavy chain containing at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain containing at least one CDRL1, at least one CDRL2 and at least one CDRL3, where (I) at least one CDRL1 contains an amino acid sequence in accordance with SEQ ID NO: 22 or in accordance with SEQ ID NO: 40; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

(II) at least one CDRL2 contains an amino acid sequence as defined in any of SEQ ID NOs: 23, 24, 41 and 42 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and/or (III) at least one CDRL3 amino contains an amino acid sequence in accordance with SEQ ID NO: 25 or in accordance with SEQ ID NO: 43; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

In addition, the antibody or antigen binding fragment thereof may also comprise a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain containing at least one CDRL1, at least one CDRL2 and at least one CDRL3, where (I) at least one CDRL1 contains an amino acid sequence in accordance with SEQ ID NO: 4 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least at least 99% sequence identity;

(II) at least one CDRL2 contains an amino acid sequence according to SEQ ID NO: 5 or 6, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and/or (III) at least one CDRL3 amino contains an amino acid sequence according to SEQ ID NO: 7 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 % sequence identity.

The antibody or antigen binding fragment thereof described herein may comprise CDRH1, CDRH2, and CDRH3 amino acid sequences (I) according to SEQ ID NO: 1-3; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (II) in accordance with SEQ ID NO: 19-21; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (III) in accordance with SEQ ID NO: 37-39; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (IV) in accordance with SEQ ID NO: 55-57; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (V) in accordance with SEQ ID NO: 73-75; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least

- 22 046175 at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (VI) in accordance with SEQ ID NO: 237-239; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (VII) in accordance with SEQ ID NO: 241-243; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (VIII) in accordance with SEQ ID NO: 245-247; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (IX) in accordance with SEQ ID NO: 249-251; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (X) in accordance with SEQ ID NO: 253-255; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XI) in accordance with SEQ ID NO: 257-259; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XII) in accordance with SEQ ID NO: 261-263; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XIII) in accordance with SEQ ID NO: 265-267; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XIV) in accordance with SEQ ID NO: 269-271; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XV) in accordance with SEQ ID NO: 273-275; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XVI) in accordance with SEQ ID NO: 277-279; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XVII) in accordance with SEQ ID NO: 281-283; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XVIII) in accordance with SEQ ID NO: 285-287; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XIX) in accordance with SEQ ID NO: 289-291; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XX) in accordance with SEQ ID NO: 293-295; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to less

- 23 046175 at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (XXI) in accordance with SEQ ID NO: 297-299; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XXII) in accordance with SEQ ID NO: 301-303; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XXIII) in accordance with SEQ ID NO: 305-307; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XXIV) in accordance with SEQ ID NO: 309-311; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XXV) in accordance with SEQ ID NO: 313-315; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XXVI) in accordance with SEQ ID NO: 317-319; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XXVII) in accordance with SEQ ID NO: 321-323; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XXVIII) in accordance with SEQ ID NO: 325-327; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XXIX) in accordance with SEQ ID NO: 329-331; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XXX) in accordance with SEQ ID NO: 333-335; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XXXI) in accordance with SEQ ID NO: 337-339; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XXXII) in accordance with SEQ ID NO: 341-343; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XXXIII) in accordance with SEQ ID NO: 345-347; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XXXIV) in accordance with SEQ ID NO: 349-351; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XXXV) in accordance with SEQ ID NO: 353-355; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least

- 24 046175

85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (XXXVI) in accordance with SEQ ID NO: 357-359; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XXXVII) in accordance with SEQ ID NO: 361 - 363; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XXXVIII) in accordance with SEQ ID NO: 365-367; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XXXIX) in accordance with SEQ ID NO: 369-371; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XL) in accordance with SEQ ID NO: 373-375; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XLI) in accordance with SEQ ID NO: 377-379; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XLII) in accordance with SEQ ID NO: 381-383; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XLIII) in accordance with SEQ ID NO: 385-387; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XLIV) in accordance with SEQ ID NO: 389-391; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XLV) in accordance with SEQ ID NO: 393-395; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or (XLVI) in accordance with SEQ ID NO: 397-399; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

Thus, the antibody or antigen binding fragment thereof may comprise CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences (I) according to SEQ ID NOs: 1-5 and 7; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (II) in accordance with SEQ ID NO: 1-4 and 6-7; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (III) in accordance with SEQ ID NO: 19-23 and 25; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98% or according

- 25 046175 at least 99% sequence identity; (IV) in accordance with SEQ ID NO: 19-22 and 24-25; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (V) in accordance with SEQ ID NO: 37-41 and 43; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (VI) in accordance with SEQ ID NO: 37-40 and 42-43; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (VII) in accordance with SEQ ID NO: 55-59 and 61; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (VIII) in accordance with SEQ ID NO: 55-58 and 60-61; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (IX) in accordance with SEQ ID NO: 73-77 and 79; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or (X) in accordance with SEQ ID NO: 73-76 and 78-79; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

The antibody or antigen binding fragment thereof may comprise CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences (I) according to SEQ ID NOs: 1-5 and 7; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (II) in accordance with SEQ ID NO: 1-4 and 6-7; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (III) in accordance with SEQ ID NO: 19-23 and 25; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (IV) in accordance with SEQ ID NO: 19-22 and 24-25; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (V) in accordance with SEQ ID NO: 37-41 and 43; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (VI) in accordance with SEQ ID NO: 37-40 and 42-43; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (VII) in accordance with SEQ ID NO: 73-77 and 79; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or (VIII) in accordance with SEQ ID NO: 73-76 and 78-79; or variants of their functional sequence

- 26 046175 of activity having 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% sequence identity.

Another antibody or antigen binding fragment thereof may contain CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences (I) in accordance with SEQ ID NOs: 1-5 and 7; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (II) in accordance with SEQ ID NO: 1-4 and 6-7; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (III) in accordance with SEQ ID NO: 73-77 and 79; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or (IV) in accordance with SEQ ID NOs: 73-76 and 78-79; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

Also, the antibody or antigen binding fragment thereof may contain CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences (I) in accordance with SEQ ID NOs: 19-23 and 25; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (II) in accordance with SEQ ID NO: 19-22 and 24-25; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (III) in accordance with SEQ ID NO: 37-41 and 43; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or (VI) in accordance with SEQ ID NO: 3740 and 42-43; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

The antibody or antigen binding fragment thereof may comprise CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences (I) according to SEQ ID NOs: 1-5 and 7; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or (II) in accordance with SEQ ID NO: 1-4 and 6-7; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

Additionally, the antibody or antigen binding fragment thereof may comprise a heavy chain variable region (VH) and, optionally, a light chain variable region (VL), wherein the heavy chain variable region (VH) contains or consists of an amino acid sequence as defined in any of SEQ ID NO : 8, 26, 44, 62, 80, 240, 244, 248, 252, 256, 260, 264, 268, 272, 276, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316 , 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, and 400; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

- 27 046175

In addition, the antibody or antigen binding fragment thereof may comprise (I) a heavy chain variable region (VH) with the amino acid sequence of SEQ ID NO: 8 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least at least 98% or at least 99% sequence identity and/or light chain variable region (VL) with the amino acid sequence according to SEQ ID NO: 9 or a functional sequence variant thereof having at least 70%, at least 75% , at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, according to at least 98% or at least 99% sequence identity; (II) a heavy chain variable region (VH) with an amino acid sequence according to SEQ ID NO: 26 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85 %, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical sequences and/or a light chain variable region (VL) with an amino acid sequence according to SEQ ID NO: 27 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (III) a heavy chain variable region (VH) with an amino acid sequence according to SEQ ID NO: 44 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85 %, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical sequences and/or a light chain variable region (VL) with an amino acid sequence according to SEQ ID NO: 45 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (IV) a heavy chain variable region (VH) with an amino acid sequence according to SEQ ID NO: 62 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85 %, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical sequences and/or a light chain variable region (VL) with an amino acid sequence according to SEQ ID NO: 63 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; or (V) a heavy chain variable region (VH) with an amino acid sequence according to SEQ ID NO: 80 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and/or light chain variable region (VL) with the amino acid sequence according to SEQ ID NO: 81 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 % sequence identity.

The antibody or antigen binding fragment thereof may comprise (I) a heavy chain variable region (VH) with the amino acid sequence of SEQ ID NO: 8 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and/or a light chain variable region (VL) with the amino acid sequence according to SEQ ID NO: 9 or a functional sequence variant thereof having at least 70%, at least 75%, at least at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98 % or at least 99% sequence identity; (II) a heavy chain variable region (VH) with an amino acid sequence in accordance with SEQ ID NO: 26 or a variant of a functional sequence thereof

- 28 046175 of 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% sequence identity and/or light chain variable region (VL) with the amino acid sequence according to SEQ ID NO: 27 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (III) a heavy chain variable region (VH) with an amino acid sequence according to SEQ ID NO: 44 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85 %, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical sequences and/or a light chain variable region (VL) with an amino acid sequence according to SEQ ID NO: 45 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; or (IV) a heavy chain variable region (VH) with an amino acid sequence according to SEQ ID NO: 80 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and/or light chain variable region (VL) with the amino acid sequence according to SEQ ID NO: 81 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 % sequence identity.

Another antibody or antigen-binding fragment thereof may comprise (I) a heavy chain variable region (VH) with an amino acid sequence according to SEQ ID NO: 8 or a functional sequence variant thereof having at least 70%, at least 75%, at least at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98 % or at least 99% sequence identity and/or light chain variable region (VL) with the amino acid sequence according to SEQ ID NO: 9 or a functional sequence variant thereof having at least 70%, at least 75%, according to at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; or (II) a heavy chain variable region (VH) with an amino acid sequence according to SEQ ID NO: 80 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and/or light chain variable region (VL) with the amino acid sequence according to SEQ ID NO: 81 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 % sequence identity.

Also, the antibody or antigen binding fragment thereof may contain (I) a heavy chain variable region (VH) with the amino acid sequence in accordance with SEQ ID NO: 26 or a functional sequence variant thereof having at least 70%, at least 75%, at least at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98 % or at least 99% sequence identity and/or a light chain variable region (VL) with the amino acid sequence according to SEQ ID NO: 27 or a functional sequence variant thereof having at least 70%, at least 75%, according to at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; or (II) a heavy chain variable region (VH) with an amino acid sequence in accordance with SEQ ID NO: 44 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, according to

- 29 046175 at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and/or a light chain variable region (VL) with the amino acid sequence of SEQ ID NO: 45 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

The antibody or antigen binding fragment thereof may comprise a heavy chain variable region (VH) with the amino acid sequence of SEQ ID NO: 8 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least at least 99% sequence identity and/or light chain variable region (VL) with the amino acid sequence in accordance with SEQ ID NO: 9 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80% , at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or according at least 99% sequence identity.

Thus, the antibody or antigen binding fragment thereof may be gZKA190, gZKA64, gZKA230, gZKA185 or gZKA78.

The inventors of the present invention have isolated monoclonal antibodies (mAbs), which are referred to herein as ZKA190, ZKA64, ZKA230, ZKA185 and ZKA78 (see Tables 1 and 2, Example 1). Based on these antibodies, particularly the VH and VL genes of these antibodies, the terms gZKA190, gZKA64, gZKA230, gZKA185 and gZKA78, as used herein, refer to the corresponding generic antibodies or antigen-binding fragments thereof.

Namely, gZKA190 refers to an antibody or antigen binding fragment thereof having a CDRH1 amino acid sequence according to SEQ ID NO: 1, a CDRH2 amino acid sequence according to SEQ ID NO: 2, a CDRH3 amino acid sequence according to SEQ ID NO: 3, CDRL1 an amino acid sequence according to SEQ ID NO: 4, a CDRL2 amino acid sequence according to SEQ ID NO: 5 or 6, and a CDRL3 amino acid sequence according to SEQ ID NO: 7. The heavy chain variable region (VH) preferably has an amino acid sequence in according to SEQ ID NO: 8 and the light chain variable region (VL) preferably has the amino acid sequence according to SEQ ID NO: 9.

gZKA64 refers to an antibody or antigen binding fragment thereof having a CDRH1 amino acid sequence according to SEQ ID NO: 73, a CDRH2 amino acid sequence according to SEQ ID NO: 74, a CDRH3 amino acid sequence according to SEQ ID NO: 75, a CDRL1 amino acid sequence according to according to SEQ ID NO: 76, a CDRL2 amino acid sequence according to SEQ ID NO: 77 or 78, and a CDRL3 amino acid sequence according to SEQ ID NO: 79. The heavy chain variable region (VH) preferably has the amino acid sequence according to SEQ ID NO: 80 and the light chain variable region (VL) preferably has the amino acid sequence according to SEQ ID NO: 81.

gZKA230 refers to an antibody or antigen binding fragment thereof having a CDRH1 amino acid sequence according to SEQ ID NO: 37, a CDRH2 amino acid sequence according to SEQ ID NO: 38, a CDRH3 amino acid sequence according to SEQ ID NO: 39, a CDRL1 amino acid sequence according to according to SEQ ID NO: 40, CDRL2 amino acid sequence according to SEQ ID NO: 41 or 42, and CDRL3 amino acid sequence according to SEQ ID NO: 43. The heavy chain variable region (VH) preferably has the amino acid sequence according to SEQ ID NO: 44 and the light chain variable region (VL) preferably has the amino acid sequence according to SEQ ID NO: 45.

gZKA185 refers to an antibody or antigen binding fragment thereof having a CDRH1 amino acid sequence according to SEQ ID NO: 19, a CDRH2 amino acid sequence according to SEQ ID NO: 20, a CDRH3 amino acid sequence according to SEQ ID NO: 21, a CDRL1 amino acid sequence according to according to SEQ ID NO: 22, CDRL2 amino acid sequence according to SEQ ID NO: 23 or 24, and CDRL3 amino acid sequence according to SEQ ID NO: 25. The heavy chain variable region (VH) preferably has the amino acid sequence according to SEQ ID NO: 26 and the light chain variable region (VL) preferably has the amino acid sequence according to SEQ ID NO: 27.

- 30 046175 gZKA78 refers to an antibody or antigen binding fragment thereof having a CDRH1 amino acid sequence according to SEQ ID NO: 55, a CDRH2 amino acid sequence according to SEQ ID NO: 56, a CDRH3 amino acid sequence according to SEQ ID NO: 57, CDRL1 an amino acid sequence according to SEQ ID NO: 58, a CDRL2 amino acid sequence according to SEQ ID NO: 59 or 60, and a CDRL3 amino acid sequence according to SEQ ID NO: 61. The heavy chain variable region (VH) preferably has an amino acid sequence in according to SEQ ID NO: 62 and the light chain variable region (VL) preferably has the amino acid sequence according to SEQ ID NO: 63.

As noted, preferably the antibody of the invention is a single chain antibody, Fab, Fab', F(ab')2, Fv or scFv.

A second object of the present invention is the use of an antibody or an antigen binding fragment thereof according to the invention to prevent or treat an infection caused by the Zika virus. Preferably in subjects with a diagnosed Zika virus infection, or in subjects exhibiting symptoms of Zika infection, or in a subject not exhibiting symptoms of Zika infection. Preferably, the subject may be a pregnant woman.

A third aspect of the present invention is the use of an antibody or an antigen-binding fragment thereof according to the invention to monitor the quality of a Zika virus vaccine by verifying that the antigen of said vaccine contains a specific epitope in the correct conformation.

Nucleic acid molecule.

In another aspect, the invention also provides a nucleic acid molecule comprising a polynucleotide encoding an antibody or antigen binding fragment thereof, in accordance with the present invention, as described above.

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. Preferred are nucleic acid sequences encoding portions or all of the light and heavy chains and CDRs of the antibodies of the present invention.

Preferred is a nucleic acid molecule whose polynucleotide sequence contains or consists of a nucleic acid sequence according to any of SEQ ID NOs: 10-18, 28-36, 46-54, 64-72, and 82-90; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

Thus, preferably provided herein are nucleic acid sequences encoding parts or all of the light and heavy chains, especially the VH and VL sequences and CDRs of exemplary antibodies of the invention. In table 1 and 2 present the SEQ ID numbers for the CDR and VH and VL amino acid sequences of the antibodies described in the application.

In table 3 below are the SEQ ID numbers of the nucleic acid sequences encoding the CDRs and VH and VL of the antibodies disclosed in the application.

A nucleic acid molecule is a molecule containing, preferably consisting of, nucleic acid components. The term nucleic acid molecule preferably refers to DNA and RNA molecules. In particular, it is used synonymously with the term polynucleotide. Preferably, the nucleic acid molecule is a polymer containing or consisting of nucleotide monomers that are covalently linked to each other via phosphodiester bonds of the sugar phosphate backbone. The term nucleic acid molecule also covers modified nucleic acid molecules, such as DNA or RNA molecules with modified bases, modified sugars or modified backbones, etc.

In table 3 shows representative CDR and variable heavy chain (VH) and variable light chain (VL) nucleic acid sequences of five representative antibodies according to the present invention (ZKA190, ZKA64, ZKA230, ZKA185, ZKA78).

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Table 3

ZKA190 SEQ ID NO. Nucleic acid sequence CDRH1 10 ggattcaccttcagtaaatatggc CDRH2 eleven atatcatatgagggaagtaataaa CDRH3 12 gcgaaatcggggacccaatactatgatactactggttatgagtatagg ggtttggaatactttggctac CDRL1 13 cagagtgttagtagcagttac CDRL2 14 gatgcatcc CDRL2 long 15 ctcatctatgatgcatccagcaggggcc CDRL3 16 cagcagtatggtaggtcaaggtggaca VH 17 caggtgcagctggtggagtctgggggaggcgtggtccagcctgggagg tccctgagactctcctgtgcagcctctggattcaccttcagtaaatat ggcatgcactgggtccgccaggct ccaggcaaggggctggagtgggtg gcagttatatcatatgagggaagtaataaatattatgcagactccgtg aagggccgattcaccatct ccagagacaattccaagaacacgctgtat ctgcaaatgaacagcctgagagctgaggacacggcagtgtattactgt gcgaaatcggggacccaatactatgatactactggttatgagtatagg ggtttggaatactttggctactggggccagggaaccctggtcaccgtc tcctcag VL 18 gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggg gaaagagccaccctctcctgcagggccagtcagagtgttagtagcagt tacttagcctggtaccagcagaaa cgtggccaggctcccaggctcctc atctatgatgcatccagcaggccactggcatcccagacaggttcagt ggcagtgggtctgg gacagacttcactctcaccatcagcagactggag cctgaagattttgcagtgtattactgtcagcagtatggtaggtcaagg tggacattcggccaagggaccaaggtggaaatcaaac ZKA185 SEQ ID NO. Nucleic acid sequence CDRH1 28 ggatatagttttaccagttactgg CDRH2 29 tttgatcctagtgactctcaaacc CDRH3 thirty gcgagaagatattgtagtagtagttgttatgtggacaat CDRL1 31 gcattgccaaataaattt CDRL2 32 gaggacaac CDRL2 long 33 gtcatctatgaggacaacaaacga ccc CDRL3 34 tactcaacagacagcagttctaatcccctgggagta VH 35 gaagtgcagctggtgcagtccggagcagaggtgaaaaagcccggggag tctctgaggatctcctgtaagggttctggatatagttttaccagttac tggatcacctgggtgcgccagatgcccgggaaaggcctggagtggatg gcgaagtttgatcctagtgactctcaaaccaactacagcccgtccttc caaggcca cgtcaccatctcagttgacaagtccatcagcactgcctac ttgcagtggagcagcctgaaggcctcggacaccgccatgtattactgt gcgagaagatattgtagtagtagttgttatgtggacaattggggc

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cagggaaccctggtcaccatcttctcag VL 36 tcctatgagctgacacagccaccctcggtgtcagtgtccccaggacaa acggccaggatcacctgctctggagatgcattgccaaataaatttgct tattggtaccggcagaagtcaggccaggcccctgttctggtcatctat gaggacaacaaacgaccctccgggatccctgagagattctctggctcc agctcagggacaatggccaccttgactat cagtggggcccaggtggag gatgaagctgactaccactgttactcaacagacagcagttctaatccc ctgggagtattcggcggagggaccaagctgaccgtcctag ZKA230 SEQ ID NO. Nucleic acid sequence CDRH1 46 ggtggctccatcagtagtgactac CDRH2 47 atctattacagtgggagcacc CDRH3 48 gcgaggagggaggaagtatgattccctttgggggagttttgcttttgat ate CDRL1 49 agctccaacatcggaggtaattat CDRL2 50 attaatgat CDRL2 long 51 ctcatctgtattaatgatcaccggccc CDRL3 52 gcaacatgggatgacagcctgggtggccttgta VH 53 caggtgcagctgcaggagtcgggcccaggcctggtgaagccttcggag accctgtccctcacctgcgcagt ctctggtggctccatcagtagtgac tactggagctggatccggcagcccccagggaaggga ctggagtggatt gggtatatctattacagtgggagcaccaactacaacccctccctcaag agtcgagtcaccatatca gtagacacgtccaagaaccacttctccctg aagctgaactctgtgaccgctgcggacacggccgtgtattactgtgcg aggagggaggaagtatgattccctttgggggagttttgcttttgatatc tggggccaagggacaatggtcaccgtctcttcag VL 54 cagtctgtgctgactcagccaccctcagcgtctgggacccccggggcag agggtcaccatctcttgttctggaagcagctccaacatcggaggtaat tatgtatactggtaccagcagctcccaggaacggcccccaaactcctc atctgtattaatgatcaccggccctcaggggtccctgaccgattctct ggctccaagtctggcacctca gcctccctggccatcagtgggctccag tccgaggatgaggctgattatta ctgtgcaacatgggatgacagcctg ggtggccttgtattcggcggagggaccaagctgaccgtcctag

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ZKA7 8 SEQ ID NO. Nucleic acid sequence CDRH1 64 ggcttcacttttagtaactatgca CDRH2 65 atcggggcgcaacggggactctatc CDRH3 66 gtgaaagatctggccatccccgagtcctacagaattgaagctgattat CDRL1 67 cagtccgtgctgtaccgctctaacaacaagaattac CDRL2 68 tggggcttca CDRL2 long 69 ctgatctattgggcttcaacccgggaa CDRL3 70 cagcagtactattctagtcctcgaact VH 71 gaggtgcagctggcagaatcaggcgggggactggtccagcctggcggc agcctgacactgtcttgcagtggatcaggcttcacttttagtaactat gcaatggtgtgggcaaggcaggctcctgggaagggactggagtatgtc tctggcatcgggcgcaacggggactctatctactatactgatagtgtg aagggccggttcaccatca gcagagacaatagcaaatccatggtgtac ctgcagatgagctccctgcgaaccgaagacacagcagtgtactattgc gtgaaagatctggccatccccgagtcctacagaattgaagctgattat tggggacagggcaccctggtcatcgtgagcgccg VL 72 gacatcgtgatgacacagtctccagatagtctggcagtcagtctgggg gagagggccactattaa ctgcaagagctcccagtccgtgctgtaccgc tctaacaacaagaattacctgtcttggtatcagcagaagcccggacag ccccctaaactgctgatctattgggcttcaacccgggaaagcggcgtc ccagacagattctca ggcagcgggtccggaacagacttcaccctgaca attagccccctgcaggcagaggacgtggctgtctactattgtcagcag tactattctagtcctcgaactttcggccaggggaccaaggtggaaatc aaac ZKA6 4 SEQ ID NO. Nucleic acid sequence CDRH1 82 ggctacaccttcacagggtatcac CDRH2 83 attaaccctaattctggcgggacc CDRH3 84 gctcggatgagctcctctatttggggcttcgatcat CDRL1 85 cagtctgtgctgattaac CDRL2 86 ggagcatcc

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CDRL2 long 87 ctgatctatggagcatcctccagggct CDRL3 88 cagcagtacaatgattggccccctatcaca VH 89 caggtgcagctggtccagagcggagcagaggtgaagaaacccggcgcc tcagtgaaggtcagctgcaaagcttccggctacaccttcacaggtat cacatcgactgggtgaggcaggcaagaggacaggga ctggaatggatg ggacggattaaccctaattctggcgggaccaactacgcccagaagttt cagggccgagtgactatgaccaga gacaccagcatctccacagcttat atgcagctgtcccggctgagatctgacgatagtgccgtctactattgt gctcggatgagctcctctatttggggcttcgatcattgggggcaggga acactggtgactgtcagttcag VL 90 gagatcgtgatgactcagtctccagccaccctgtcagtcagcccagga gaacgggcaaccctgtcttgcagagcctcccagtctgtgctgattaac ctggcttggtaccagcagaagccaggccaggcaccccgactgctgatc tatggagcatcctccagggctaccggcattcctgcacgcttcagtgga tcaggaagcggaac agagtttaccctgacaatctctagtctgcagtcc gaagacttcgctgtctactattgtcagcagtacaatgattggccccct atcacatttggccaggggactagactggagatcaagc

Thus, the nucleic acid molecule sequence may comprise or consist of a nucleic acid sequence according to any of SEQ ID NOs: 10-18, 28-36, 46-54, 64-72, and 82-90; or its variant of the functional sequence.

Nucleic acid sequences may also include nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with respect to the nucleic acid encoding the CDR, VH sequence and/or VL sequence, used in a (typical) antibody, for example, in the sequences presented in table. 3.

In general, a nucleic acid molecule can be manipulated to insert, delete, or alter certain nucleic acid sequences. Changes in such manipulation include, but are not limited to, changes to introduce restriction sites, to change the frequency of the codon used, to add or optimize transcription and/or translation of regulatory 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 tails (eg, for purification). Mutations can be introduced at specific sites, or they can be introduced randomly, followed by selection (eg, molecular evolution). For example, one or more nucleic acids encoding the CDR regions, VH sequence and/or VL sequence of a (typical) antibody can be randomly or specifically altered to introduce various properties into the encoded amino acids. Such changes can lead to an iterative process in which the original changes are maintained and new changes are introduced at other nucleotide positions. Moreover, changes carried out at independent stages can be combined. Various properties introduced into the encoded amino acids may include, but are not limited to, altered affinity.

Vector.

Another object of the present invention is an expression vector containing a nucleic acid molecule in accordance with the present invention.

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 suitable for incorporating or anchoring 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 allows suitable storage

- 35 046175 nucleic acid molecule. Thus, the vector may contain a sequence corresponding to, for example, a desired antibody or antibody fragment thereof. An expression vector can be used to produce expression products such as RNA, eg mRNA, or peptides, polypeptides or proteins. For example, the expression vector may contain sequences necessary for transcription of the sequence, such as a promoter sequence. A cloning vector is typically a vector containing a cloning site that can be used to incorporate nucleic acid sequences into the vector. The cloning vector may be, for example, a plasmid vector or a bacteriophage vector. A transfer vector may be a vector that is suitable for transferring nucleic acid molecules into cells or organisms, such as viral vectors. The vector in the context of the present invention may be, for example, an RNA vector or a DNA vector. Preferably, the vector is a DNA molecule. For example, a vector in the context of the present application contains a cloning site, a selectable marker, such as an antibiotic resistance factor, and a sequence suitable for propagation of the vector, such as an origin of replication. Preferably, the vector in the context of the present application is a plasmid vector.

Cells.

Finally, the present invention also relates to a cell expressing an antibody or antigen binding fragment thereof according to the present invention; and containing a vector in accordance with 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 a mammalian cell line. Preferred examples include human cells, CHO cells, HEK293T cells, PER.C6 cells, NS0 cells, human liver cells, myeloma cells or hybridoma cells.

In particular, the cell can be transfected with an expression vector in accordance with the present invention. 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 the skilled person for introducing nucleic acid molecules into cells, preferably eukaryotic cells, for example, mammalian cells. Such methods include, for example, electroporation, lipofection, for example, based on cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or transfection based on cationic polymers, such as DEAE-dextran or polyethylenimine and etc. Preferably, the introduction is non-viral.

Cells can be stably or transiently transfected with a vector in accordance with the present invention, for example, to express an antibody or antigen binding fragment thereof, in accordance with the present invention. Preferably, the cells are stably transfected with a vector encoding an antibody or an antigen binding fragment thereof. Alternatively, it is also preferred that the cells are transiently transfected with vector B encoding an antibody or an antigen binding fragment thereof.

Optional additional characteristic features of antibodies.

Antibodies can be associated, for example, with a drug for delivery to the treatment site or associated with a detectable label to facilitate visualization of the site containing cells of interest. Methods for binding antibodies to drugs and detectable labels are well known in the art, as are methods of imaging using detectable labels. Labeled antibodies can be used in a variety of assays using a variety of labels. Detection of the formation of an antibody-antigen complex between an antibody and an epitope of interest can be facilitated by coupling the detectable substance to the antibody. Suitable detection refers to the inclusion of the use of labels such as radionuclides, enzymes, coenzymes, fluorescent substances, chemiluminescent substances, chromogens, enzyme substrates or cofators, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes and others. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-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, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include 125I, 131I, 35S, or 3H. Such labeled reagents can be used in a variety of well-known assays, such as radioimmunoassays, enzyme-linked immunosorbent assays, eg ELISA, fluorescent immunoassays, etc. Therefore, labeled antibodies can be used in such assays, for example, as described in US 3,766,162; US 3,791,932; US 3,817,837; and US 4,233,402.

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The antibody may be conjugated to a therapeutic moiety, such as a cytotoxin, a therapeutic agent, or a radioactive metal ion or radioactive isotope. Examples of radioactive isotopes 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, In-111, etc. Such antibody conjugates can be used to modify a given biological response; the drug component should not be considered as limited to classical chemical therapeutics. For example, the drug component may be a protein or polypeptide having a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, Pseudomonas aeruginosa exotoxin, or diphtheria toxin.

Technologies for conjugating such a therapeutic component to antibodies are well known. See, for example, Arnon et al. (1985) Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy, in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.), cc. 243256; edited by Hellstrom et al (1987) Antibodies for Drug Delivery, in Controlled Drug Delivery, ed. Robinson et al. (2nd ed.; Marcel Dekker, Inc.), cc. 623-653; Thorpe (1985) Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review, in Monoclonal Antibodies '84: Biological and Clinical Applications, ed. Pinchera et al. ss. 475-506 (Editrice Kurtis, Milano, Italy, 1985); Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibodies in Cancer Therapy, in Monoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin et al. (Academic Press, New York, 1985), cc. 303-316; and Thorpe et al. (1982) Immunol. Rev. 62:119-158.

Alternatively, the antibody or antibody fragment thereof can be conjugated to a second antibody or antibody fragment thereof to form an antibody heteroconjugate, as described in US Pat. No. 4,676,980. Additionally, linkers can be used between tags and antibodies according to the invention, for example, as 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 treatments using conjugated and unconjugated antibodies, administered simultaneously or sequentially, for example, as described in WO 00/52031; WO 00/52473.

Antibodies can also be attached to a solid support. Additionally, the antibodies of the invention, or functional antibody fragments thereof, can be chemically modified by covalent conjugation to a polymer, for example, increasing their circulating half-life. Examples of polymers and methods for attaching them to peptides are presented in US 4,766,106; US 4,179,337; US 4,495,285 and US 4,609,546. In some embodiments, the polymers may be selected from polyoxyethylated polyols and polyethylene glycol (PEG). PEG is soluble in water at room temperature, and has the general formula: R(O-CH ₂ -CH ₂ ) _n OR, where R can represent hydrogen or a protecting group such as an alkyl or alkanol group. Preferably, the protecting group may have between 1 and 8 carbons. For example, the protecting group is methyl. The symbol n represents a positive integer. In one embodiment, n is in the range from 1 to 1000. In another embodiment, n is in the range from 2 to 500. Preferably, PEG has an average molecular weight in the range of 1000 to 40,000, more preferably PEG has a molecular weight in the range of 2000 to 20,000, even more preferably PEG has a molecular weight in the range from 3000 to 12000. In addition, PEG may have at least one hydroxy group, for example, PEG may have a terminal hydroxy group. For example, it is a terminal hydroxy group that is activated to react with the free amino group on the inhibitor. However, it should be taken into account that the type and number of reactive groups can be varied to achieve a covalent PEG/antibody conjugation.

Water-soluble polyoxyethylated polyols are also suitable in this case. These include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), and others. In one embodiment, POG is used. Without wishing to be bound by any theory, since the glycerol backbone of polyoxyethylated glycerol is the same backbone that occurs naturally in, for example, animals and humans in mono-, di-, triglycerides, this branch should not be considered a foreign agent 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 circulating half-life is the liposome. Methods for preparing liposome delivery systems are known to one skilled in the art. Other drug delivery systems are known in the art and are described, for example, in references in Poznansky et al. (1980) and Poznansky (1984).

Preferably, the antibodies of the invention may be in purified form. Typically, antibodies will be present in the composition when they are substantially free of other polypeptides, for example, where less than 90% (by weight), typically less than 60%, and more preferably less than 50% of the composition consists of other polypeptides.

Antibodies may be immunogenic in non-human (or heterologous) hosts, such as mice. In particular, antibodies may have an idiotope that is immunogenic in

- 37 046175 hosts that differ from humans, but not from a human host. In particular, antibodies include those 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 .

Obtaining antibodies.

Antibodies can be obtained using any method known in the art. For example, the conventional methodology for producing monoclonal antibodies using hybridoma technology is well known (Kohler, G. and Milstein, S., 1975; Kozbar et al. 1983). An alternative EBV immortalization method described in WO 2004/076677 can also be used.

A preferred method is described in WO 2004/076677. In this method, antibody-producing B cells are transformed with EBV and a polyclonal B cell activator. Additional stimulators of cell growth and differentiation may optionally be added during the transformation step to enhance efficacy. These stimulators may be cytokines such as IL2 and IL-15. In one aspect, IL-2 is added during the immortalization step to further improve the immortalization efficiency, but their use is not essential. The immortalized B cells obtained using these methods can then be cultured using methods known in the art and antibodies can be isolated therefrom.

Another preferred method is described in WO 2010/046775. In this method, plasma cells are cultured in limited quantities, or as single plasma cells in microwell culture plates. Antibodies can be isolated from plasma cell cultures. In addition, RNA can be extracted from plasma cell cultures and PCR can be performed using methods known in the art. The VH and VL regions of the antibodies can be amplified by RT-PCR (reverse transcriptase PCR), sequenced, and cloned into an expression vector, which is then transfected into HEK293T cells or other host cells. Cloning nucleic acid into expression vectors, transfecting host cells, culturing transfected host cells, and isolating the resulting antibody can be accomplished using any methods known to one skilled in the art.

Antibodies can be further purified, if necessary, using filtration, centrifugation and various chromatographic techniques such as HPLC or affinity chromatography. Technologies for purifying antibodies, such as monoclonal antibodies, including technologies for producing pharmaceutical grade antibodies, are well known in the art.

Antibody fragments 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. The antibody fragments of the invention preferably include Fab, Fab', F(ab')2 and Fv fragments. Single chain Fv fragments, scFv, may contain antibody CDRs. The prior art also describes monomers and dimers of heavy and light chains, single-domain antibody heavy chain, single-domain antibody light chain, as well as single-chain antibodies, for example, single-chain Fv, in which the heavy and light chain variable domains are connected by a peptide linker.

Antibody fragments can convey monovalent or multivalent interactions and are contained in various structures, as described above. For example, scFv molecules can be synthesized to create a trivalent tribody or a tetravalent tetrabody. scFv molecules can include an Fc domain fragment, resulting in divalent minibodies. Additionally, the sequences can be a component of multispecific molecules, in which the sequences target epitopes and other regions of the molecule for binding to other targets. Typical molecules include, but are not limited to, bispecific Fab2, trispecific Fab3, bispecific scFv, and diabodies (Holliger and Hudson, 2005, Nature Biotechnology 9: 1126-1136).

Standard molecular biology techniques can be used to prepare DNA sequences encoding antibodies or antibody fragments. Desired DNA sequences can be synthesized in whole or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques can be used as suitable techniques.

Any suitable host cell/vector system can be used to express DNA sequences encoding antibody molecules or fragments thereof. Bacterial, eg E. coli, and other microbial systems can be used in particular for the expression of antibody fragments such as Fab and F(ab')2 fragments, and in particular Fv fragments and single chain antibody fragments, eg single chain Fv. Expression systems based on eukaryotic, eg mammalian, host cells can be used to produce large antibody molecules, including complete antibody molecules. Suitable mammalian host cells include, but are not limited to, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells.

- 38 046175

A method for producing an antibody molecule involves culturing a host cell containing a vector encoding a nucleic acid under conditions suitable for expressing a protein from DNA encoding the antibody molecule and isolating the antibody molecule.

An antibody molecule may contain only a heavy and light chain polypeptide, in which case only the sequence encoding the heavy chain or light chain polypeptide needs to be used to transfect host cells. To produce products containing both heavy and light chains, a cell line can be transfected with two vectors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector may be used that includes sequences encoding light chain and heavy chain polypeptides.

Alternatively, antibodies can be produced by (i) expressing a nucleic acid sequence in a host cell, for example, by use of a vector, and (ii) isolating the expressed antibody product. Additionally, the method may include (iii) purifying the isolated antibody. Transformed B cells and cultured plasma cells can be screened to find those that produce antibodies with the desired specificity or function.

The screening step can be performed by an immunoassay, such as an ELISA, by staining tissues or cells (including transfected cells), by a neutralization assay, or by one of various other methods known in the art to identify the desired specificity or function. The assay can be selected based on simple recognition of one or more antigens, or can be selected based on desired function, for example, to select neutralizing antibodies that not only bind to an antigen, to select antibodies that can alter characteristics of target cells, such as their transmission cascades signals, their shape, their growth rate, their ability to influence other cells, their response to the influence of other cells or other reagents or to changes in conditions, their differentiated states, etc.

Individual transformed B cell clones can then be obtained from the positively transformed B cell culture. The cloning step to separate individual clones from a mixture of positive cells can be accomplished using serial dilution, micromanipulation, single cell deposition by cell sorting, or other method known in the art.

Nucleic acid from cultured plasma cells can be isolated, cloned and expressed in HEK293T cells or other known host cells using methods known in the art.

Immortalized B cell clones or transfected host cells can be used in various ways, for example, as a source of monoclonal antibodies, as a source of nucleic acid (DNA or mRNA) encoding a monoclonal antibody of interest for research, etc.

It is possible to prepare a composition containing immortalized memory B cells or transfected host cells that produce antibodies in accordance with the present invention.

An immortalized B cell clone or cultured plasma cells can also be used as a source of nucleic acid to clone antibody genes for subsequent recombinant expression. Expression from recombinant sources is more common for pharmaceutical purposes than expression from B cells or hybridomas, for example, for reasons of stability, reproducibility, ease of culture, etc.

Thus, a method for producing a recombinant cell may include the steps of: (i) obtaining one or more nucleic acids (eg, heavy and/or light chain mRNA) from a clone of B cells or cultured plasma cells that encode an antibody of interest; (II) inserting a nucleic acid into an expression vector; and (III) transfecting the vector into a host cell to permit expression of the antibody of interest in that host cell.

Likewise, a method for producing a recombinant cell may include the steps of: (I) sequencing nucleic acid(s) from a clone of B cells or cultured plasma cells that encode an antibody of interest; and (II) using the sequence information from step (I) to prepare nucleic acid(s) for insertion into a host cell to permit expression of the antibody of interest in that host cell. The nucleic acid may, but not necessarily, be manipulated between steps (I) and (II) to introduce restriction sites, to change codon usage frequency, and/or to optimize transcription and/or translation of regulatory sequences.

In addition, the method of producing a transfected host cell may include the step of transfecting the host cell with one or more nucleic acids that encode an antibody of interest, where the nucleic acids are nucleic acids that are derived from a clone of immortalized B cells or cultured plasma cells according to the invention. Thus, the procedures for the first preparation of nucleic acid(s)

- 39 046175 slot(s) and its subsequent use for transfection of a host cell, can be carried out at different times by different people in different places (eg, in different countries).

These recombinant cells can then be used for expression and culture. They are particularly suitable for the expression of antibodies for pharmaceutical production on an industrial scale. They can also be used as an active component of a pharmaceutical composition. Any suitable culture technique may be used, including, but not limited to, static culture, spinner flask culture, ascites fluid, hollow fiber bioreactor cartridge, modular minifermenter, mixing tank, microcarrier culture, ceramic rod perfusion, etc.

Methods for obtaining and sequencing immunoglobulin genes from B cells or plasma cells are well known in the art (eg, see Section 4 of Kuby Immunology, 4th ed., 2000).

The transfected host cell may be a eukaryotic cell, including yeast and animal cells, especially mammalian cells (eg, CHO cells, NS0 cells, human cells such as PER.C6 or HKB-11 cells, myeloma cells, or a human liver cell ), as well as plant cells, with mammalian cells being preferred. Preferred hosts for expression may glycosylate the antibody, particularly with carbohydrate structures that are not themselves immunogenic in humans. In one embodiment, the transfected host cell is capable of growing in a serum-free medium. In a further embodiment, the transfected host cell is capable of growing without the presence of products of animal origin. The transfected host cell can also be cultured to produce a cell line.

A method of producing one or more nucleic acid molecules (eg, heavy and light chain genes) that encode an antibody of interest may comprise the steps of: (i) preparing an immortalized B cell clone or culturing plasma cells; (ii) obtaining from a clone of B cells or cultured plasma cells a nucleic acid that encodes an antibody of interest. In addition, a method of obtaining a nucleic acid sequence that encodes an antibody of interest may include the steps of: (I) preparing an immortalized B cell clone or culturing plasma cells in accordance with the invention; (ii) sequencing nucleic acid from a clone of B cells or cultured plasma cells that produce the antibody of interest.

A method of obtaining a nucleic acid molecule(s) that encodes an antibody of interest may include the step of obtaining the nucleic acid from a transformed clone of B cells or cultured plasma cells. Thus, the procedures for first obtaining a B cell clone or cultured plasma cell and then obtaining nucleic acid(s) from the B cell clone or cultured plasma cells can be performed at different times by different people in different places (for example, in different countries).

A method of producing an antibody (eg, for pharmaceutical use) may include the steps of: (i) obtaining and/or sequencing one or more nucleic acids (eg, heavy and light chain genes) from a selected clone of B cells or cultured plasma cells expressing the antibody , of interest; (II) inserting the nucleic acid(s) into or using the nucleic acid(s) sequence(s) to prepare an expression vector; (III) transfecting a host cell that can express the antibody of interest; (iv) culturing or sub-culturing the transfected host cells under conditions in which the antibody of interest is expressed; and, optionally, (V) purifying the antibody of interest.

A method for producing an antibody may include the steps of: culturing or subculturing a transfected population of host cells, e.g., a stably transfected population of host cells, under conditions in which the antibody of interest is expressed, and optionally purifying the antibody of interest, wherein said transfected population host cells was obtained by (I) providing nucleic acid(s) encoding the selected antibody of interest that is produced by a clone of B cells or cultured plasma cells prepared as described above, (II) inserting the nucleic acid(s) into an expression vector, (III) transfecting the vector into a host cell that can express the antibody of interest, and (IV) culturing or subculturing the transfected host cell containing the inserted nucleic acids to produce the antibody , of interest. Thus, the procedures for first preparing a recombinant host cell and then culturing it for antibody expression can be performed at very different times by different people in different places (eg, different countries).

Pharmaceutical composition.

The present invention also provides a pharmaceutical composition containing:

- 40 046175 (I) an antibody or an antigen-binding fragment thereof in accordance with the present invention;

(II) a nucleic acid encoding an antibody or antigen-binding agent thereof according to the present invention;

(III) an expression vector containing a nucleic acid in accordance with the present invention; or (IV) a cell expressing an antibody or antigen-binding fragment thereof in accordance with the present invention or containing a vector in accordance with the present invention;

(V) a pharmaceutically acceptable excipient, diluent or carrier.

Although the carrier or excipient may facilitate administration, it should not actually induce the production of antibodies harmful to the individual receiving the composition. It should also not be toxic. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, copolymers of amino acids and inactivated viral particles. In general, pharmaceutically acceptable carriers in a pharmaceutical composition may be active ingredients or inactive ingredients. Preferably, the pharmaceutically acceptable carrier in the pharmaceutical composition is an inactive component against Zika virus infection.

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

Pharmaceutically acceptable carriers in the pharmaceutical composition may further contain liquids such as water, saline, glycerin and ethanol. Additionally, auxiliary substances such as wetting or emulsifying agents or pH buffering agents may be present in such compositions. Such carriers enable pharmaceutical compositions to be prepared in the form of tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions for ingestion by a subject.

Pharmaceutical compositions can be prepared in various forms. For example, the compositions may be formulated as injectables, or as liquid solutions or suspensions. Solid forms suitable for solution or suspension in liquid excipients prior to injection may also be prepared (eg, a lyophilized composition like 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 a tablet or capsule, in the form of a spray, or in the form of a syrup (optionally flavored). The composition can be prepared for pulmonary administration, for example, in the form of an inhaler, using a 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 designed such that the combination composition is reconstituted only before administration to a subject. For example, the lyophilized antibody may be provided in the form of a kit with sterile water or sterile buffer.

If the active component in the composition is an antibody molecule, an antibody fragment, or variants and derivatives thereof, it is susceptible to degradation in the gastrointestinal tract. Therefore, if the composition is administered by the gastrointestinal tract, the composition may contain agents that protect the antibody from degradation, but which release the antibody after it has been absorbed from the gastrointestinal tract.

A comprehensive discussion of pharmaceutically acceptable carriers is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th ed., ISBN: 0683306472.

Pharmaceutical compositions typically have a pH in the range of 5.5 to 8.5, in some embodiments it may be from 6 to 8, and in other embodiments about 7. The pH may be maintained by the use of a buffer. The composition may be sterile and/or pyrogen-free. The composition may be isotonic in relation to humans. In one embodiment, the pharmaceutical compositions of the invention are supplied in hermetically sealed containers.

The compositions may be presented in various forms of administration; forms include, but are not limited to, those forms that are suitable for parenteral administration, for example, by injection or infusion, for example, by bolus injection or continuous infusion. If the product is intended for injection or infusion, it may be in the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and it may contain formulation auxiliaries such as suspending, preservative, stabilizing and/or dispersing agents. Alternatively, the antibody molecule may be in anhydrous form for reconstitution with a suitable sterile liquid before use. By excipient is typically meant a material that is suitable for storing, transporting and/or administering a compound such

- 41 046175 as a pharmaceutically active compound, in particular an antibody according to the present invention. For example, the excipient may be a physiologically acceptable liquid that is suitable for storing, transporting and/or administering a pharmaceutically active compound, especially an antibody. Once prepared, the compositions may be administered directly to the subject. In one embodiment, the compositions are adapted for administration to mammals, such as human subjects.

Pharmaceutical compositions may be administered by a variety of routes, including, but not limited to, oral, intravenous, intramuscular, intraarterial, bone marrow, intraperitoneal, intrathecal, intraventricular, transdermal, transdermal, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal, or rectal routes. . Needleless syringes can also be used to administer pharmaceutical compositions. Preferably, the pharmaceutical composition may be prepared for parenteral administration, for example, in the form of tablets, capsules, etc., for topical administration, or in the form of injectable preparations, for example, in the form of liquid solutions or suspensions, and it is particularly preferred when the pharmaceutical composition is an injectable drug. Also preferred are solid forms suitable for preparation of a solution or suspension in liquid carriers before injection, for example when the pharmaceutical composition is presented in lyophilized form.

For injection, for example intravenous, dermal or subcutaneous injection, or injection into a disease site, the active ingredient will preferably be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has a suitable pH, isotonicity and stability. Those skilled in the art can prepare suitable solutions using, for example, isotonic excipients such as sodium chloride injection, Ringer's solution for injection, lactated Ringer's solution for injection. If necessary, preservatives, stabilizers, buffers, antioxidants and/or or other excipients. Regardless of whether the polypeptide, peptide or nucleic acid molecule, or other pharmaceutically acceptable compound to be administered to an individual, administration is preferably in a prophylactically effective amount or a therapeutically effective amount (depending on the particular situation) sufficient to produce benefit in the individual. The actual amount administered, and the rate and time course of administration, will depend on the nature and severity of the condition being treated. For injection, the pharmaceutical composition in accordance with the present invention may be contained, for example, in a pre-filled syringe.

The pharmaceutical composition may 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, carriers commonly used include lactose and corn starch. Lubricants such as magnesium stearate are also typically added. For oral administration in capsule form, suitable diluents include lactose and anhydrous corn starch. When aqueous suspensions are required for oral administration, the transport cargo-conjugated molecule is combined with emulsifying and suspending agents. If necessary, certain sweeteners, flavors or colors may also be added.

The pharmaceutical composition may also be administered topically, particularly if the target of treatment involves areas or organs that can be reached by local administration, for example, including diseases of the skin or any other epithelial tissue that can be reached. Suitable topical preparations are readily prepared for each of these areas or organs. For topical applications, the pharmaceutical composition may be formulated as a suitable ointment containing the pharmaceutical composition, particularly its components as defined above, suspended or dissolved in one or more carriers. Carriers for topical administration include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and wax. Alternatively, the pharmaceutical composition may be formulated in a suitable lotion or cream. 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.

Dosage treatment may be a single dose regimen or a multiple dose regimen. In a preferred embodiment of the invention, the pharmaceutical composition is a single dose product. Preferably, the amount of antibody in such a pharmaceutical composition does not exceed 500 mg.

For example, the pharmaceutical composition may be administered every day, for example, once or more times a day, for example, once, twice, three or four times a day, preferably once or twice a day, more preferably once a day, for 1. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 or more days, for example, every day for 1 , 2, 3, 4, 5, 6 months. Preferably, the pharmaceutical composition may be administered every week, for example once or

- 42 046175 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 or more weeks, such as every week for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months or every week for 2, 3, 4 or 5 years. In addition, the pharmaceutical composition may preferably be administered every month, for example once a month or more preferably every two months for 1, 2, 3, 4 or 5 or more years. It is also preferred that administration continues throughout the life period. Additionally, only one single administration is also provided, especially for certain indications, for example, to prevent Zika virus infection in case of accidental exposure, for example, in non-immunized subjects. However, the most preferred treatment regimen is post-exposure prophylaxis (PEP), where one or more single doses are administered as quickly as possible after Zika virus infection. Also preferred is a prophylactic regimen where one or more single doses are administered to prevent Zika virus infection (ie, prior to Zika virus infection, particularly in subjects not immunized to Zika virus).

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

Pharmaceutical compositions typically include an effective amount of one or more antibodies, that is, an amount sufficient to treat, ameliorate, ameliorate, or prevent a desired disease or condition, or to produce a detectable therapeutic effect. Therapeutic effects also include reduction or attenuation of pathogenic strength or physical symptoms. The exact effective amount for any particular subject will depend on their size, weight and health status, the nature and extent of the condition, and the drugs or combination of drugs chosen for administration. The effective amount for a given situation is determined by routine experimentation and is within the discretion of the attending physician. An effective dose will generally be from about 0.005 to about 100 mg/kg, preferably from about 0.0075 to about 50 mg/kg, more preferably from about 0.01 to about 10 mg/kg, and even more preferably from about 0. 02 to about 5 mg/kg, of the antibody of the present invention (eg, the amount of antibody in the pharmaceutical composition) relative to the body weight (eg, in kg) of the individual to whom it is administered.

In addition, the pharmaceutical composition may also contain an additional active component, which may be an additional antibody or a component that is not an antibody. The additional active component is preferably a checkpoint inhibitor. It is also preferred that the ZIKV neutralizing antibody or antigen binding fragment thereof can be combined with the ZIKV No. 1 binding antibody or antigen binding fragment thereof as an additional active component (coagent). Thus, the pathogenic role of NS1 may be blocked, in addition to neutralizing ZIKV. The pharmaceutical composition may contain one or more additional active ingredients, for example as described below, as coagents in the context of combination therapy.

The antibody or antigen binding fragment may be present either in the same pharmaceutical composition as an additional active component or, preferably, the antibody or antigen binding fragment constitutes a first pharmaceutical composition and the additional active component constitutes a second pharmaceutical composition different from the first pharmaceutical composition. Thus, if more than one additional active ingredient is provided, each additional active ingredient and antibody or antigen binding fragment preferably constitutes a different pharmaceutical composition. Such different pharmaceutical compositions may be administered either in combination/simultaneously or at different periods of time or at different locations (eg, separate body parts).

Preferably, the antibody or antigen binding fragment and the additional active component provide an additive therapeutic effect or, preferably, a synergistic therapeutic effect. The term synergy 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, if the combined effect of two or more agents results in synergistic inhibition of an activity or process, then this means that the inhibition of the activity or process is greater than the sum of the inhibitory effects of each respective active agent. The term synergistic therapeutic effect refers to the therapeutic effect observed when two or more therapies are combined, where the therapeutic effect (as measured by any of the various parameters) is greater than the sum of the individual therapeutic effects observed in the respective individual therapies.

The pharmaceutical composition may contain an antibody according to gZKA190, gZKA64, gZKA230, gZKA185, gZKA78 or an antigen binding fragment thereof and a pharmaceutically acceptable

- 43 046175 carrier.

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

A method for preparing a pharmaceutical composition may include the steps of: (I) preparing an antibody; and (ii) mixing the purified antibody with one or more pharmaceutically acceptable carriers.

The method of preparing a pharmaceutical composition may also include the steps of: mixing the antibody with one or more pharmaceutically acceptable carriers, where the antibody is a monoclonal antibody that is obtained from a transformed B cell or cultured plasma cell according to the invention.

As an alternative to delivering antibodies or B cells for therapeutic purposes, it is possible to deliver a nucleic acid (typically DNA) that encodes a monoclonal antibody of interest (or an active fragment thereof) originating from a B cell or cultured plasma cell to a subject, thereby that the nucleic acid can be expressed in situ in subjects to provide the desired therapeutic effect. Suitable gene therapy and vectors for delivering nucleic acids are known in the art.

Pharmaceutical compositions may include an antimicrobial agent, particularly if packaged in a multiple dose format. They may contain a detergent, for example, Tween (polysorbate), such as Tween 80. Detergents are usually present in low amounts, for example less than 0.01%. The compositions may also include sodium salts (eg, sodium chloride) to impart tonicity. For example, a concentration of 10 ± 2 mg/ml NaCl is typical.

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 lyophilized or if they include material that is reconstituted from lyophilized material. The pH of the lyophilization composition can be adjusted to a range of 5 to 8, or a range of 5.5 to 7, or approximately 6.1 prior to lyophilization.

The compositions may also contain one or more immunoregulatory agents, in particular, the one or more immunoregulatory agents include(s) an adjuvant.

Medical treatments, kits and applications.

Medical treatments.

Another aspect of the present invention is a method of preventing or treating an infection caused by the Zika virus in a subject, comprising administering to the subject an antibody or antigen binding fragment thereof according to the present invention, a nucleic acid according to the present invention, a vector according to the present invention, a cell or pharmaceutical composition in accordance with the present invention.

The invention also relates to the use of a nucleic acid according to the invention, a vector according to the invention, a cell according to the invention and a pharmaceutical composition according to the invention for the prevention or treatment of infection caused by the Zika virus.

It is further an object of this invention to use an antibody or an antigen-binding fragment thereof according to the invention and a nucleic acid according to the invention for diagnosing an infection caused by the Zika virus.

Diagnostic methods may involve contacting an antibody or antibody fragment with a sample. Such samples may be isolated from a subject, for example, an isolated tissue sample taken from, for example, the nasal passages, sinus cavities, salivary glands, lungs, liver, pancreas, kidneys, ear, eye, placenta, digestive tract, heart, ovaries, pituitary gland, adrenal glands, thyroid gland, brain, skin or blood, preferably plasma or serum. Diagnostic methods may also include detection of an antigen/antibody complex, particularly after contact of the antibody or antibody fragment with a sample. This detection step is typically carried out in a laboratory, 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, ELISA (enzyme-linked immunosorbent assay).

Prevention of Zika virus infection refers specifically to preventive settings where 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 . Thus, preventing Zika virus infection involves post-exposure prophylaxis (PEP), which is preventive treatment after a possible Zika virus infection, such as a mosquito bite in an area affected by the Zika virus. Prevention of Zika virus infection is preferable for high-risk subjects such as pregnant women.

In contrast, in a therapeutic setting, the subject is typically infected with Zika virus, is diagnosed with Zika virus infection, and/or exhibits symptoms of infection,

- 44 046175 caused by the Zika virus. Of course, the terms treatment and therapy/therapeutic ZIKV infections include (complete) treatment as well as mitigation of ZIKV infection.

Preferred methods for diagnosing Zika virus infection are diagnostic methods such as, for example, those involving the use of a neutralizing antibody or antigen binding fragment thereof and/or a ZIKV #1 binding antibody or antigen binding fragment thereof.

Thus, an antibody or an antigen binding fragment thereof according to the present invention, a nucleic acid according to the present invention, a vector according to the present invention, a cell according to the present invention or a pharmaceutical composition according to the present invention is preferably used for treating an infection, caused by the Zika virus in subjects in subjects exhibiting symptoms of Zika infection.

It is also preferred that an antibody or antigen binding fragment thereof according to the present invention, a nucleic acid according to the present invention, a vector according to the present invention, a cell according to the present invention or a pharmaceutical composition according to the present invention is used to prevent and/or treatment of 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 according to the present invention, a nucleic acid according to the present invention, a vector according to the present invention, a cell according to the present invention or a pharmaceutical composition according to the present invention is used to prevent and/or treat an infection caused by the Zika virus in pregnant women, especially to prevent intrauterine infection. For example, this could be done in a similar manner to preventing intrauterine HCMV infection, as described in Nigro G, Adler SP, La Torre R, Best AM, Congenital Cytomegalovirus Collaborating Group: Passive immunization during pregnancy for congenital cytomegalovirus infection; N Engl J Med 2005, 353:1350-1362.

Without wishing to be limited by theory, it is believed that the antibody, or antigen binding fragment thereof, in accordance with the present invention can cross the placenta by interacting with FcRn when administered to a pregnant woman, for example, by (iv) injection or other route of administration. It is important that the interaction of LALA antibody variants with FcRn is not disrupted. It is believed that FcRn is already expressed in the first trimester in the placenta.

Alternatively, the antibody or antigen-binding fragment thereof can also be administered into the extra-amniotic space.

The antibody or antigen binding fragment, nucleic acid, vector, cell or pharmaceutical composition thereof can be used to prevent or treat Zika virus infection by administration up to seven days after (possible) Zika virus infection, preferably up to five days after (possible) Zika virus infection. infection with Zika virus, more preferably up to four days after (possible) infection with Zika virus, even more preferably up to three days after (possible) infection with Zika virus, and most preferably up to one day after (possible) infection with Zika virus. Such a treatment regimen may be suitable in therapeutic settings as well as in prophylactic settings, particularly for post-exposure prophylaxis (PEP).

In PEP, typically the first administration of an antibody or antigen-binding fragment, nucleic acid, vector, cell, or pharmaceutical composition should be administered as quickly as possible after a possible ZIKV infection, for example, after a mosquito bite in a ZIKV-affected area. Thus, in PEP, the first administration of an antibody or antigen binding fragment, nucleic acid, vector, cell or pharmaceutical composition thereof can typically be administered up to one or more days after (possible) ZIKV infection, as described above.

Also, an antibody or antigen-binding fragment, nucleic acid, vector, cell, or pharmaceutical composition thereof for preventing and/or treating Zika virus infection may be administered up to three months before (possible) Zika virus infection, preferably up to one month before (possible) Zika virus infection, more preferably up to two weeks before (possible) Zika virus infection, even more preferably up to one week before (possible) Zika virus infection, and most preferably up to one day before (possible) Zika virus infection. This treatment regimen applies especially to preventive settings.

In general - and especially with PEP - after the first administration of an antibody or antigen-binding fragment thereof, nucleic acid, vector, cell or pharmaceutical composition, one or more subsequent administrations can then be made, preferably a single dose per day or every other day for 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1, 15, 16, 17, 18, 19, 20, or 21 days. Also, after the first administration of an antibody or an antigen-binding fragment, nucleic acid,

- 45 046175 torus, cell or pharmaceutical composition, then one or more subsequent administrations can be made, preferably a single 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. After the first administration of an antibody or antigen binding fragment, nucleic acid, vector, cell or pharmaceutical composition, one or more subsequent administrations can then be made, preferably a single dose every 2 or 4 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. Also, after the first administration of an antibody or antigen binding fragment, nucleic acid, vector, cell or pharmaceutical composition, one or more subsequent administrations may then be made, preferably a single 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. Also, after the first administration of an antibody or antigen-binding nucleic acid fragment thereof, vector, cell or pharmaceutical composition, one or more subsequent administrations may then be made, preferably a single dose once or twice a year 1, 2, 3, 4, 5, 6. 7, 8, 9, or 10 years.

In a preferred embodiment of the invention, an antibody or antigen-binding fragment thereof of the invention, a nucleic acid of the invention, a vector of the invention, a cell of the invention or a pharmaceutical composition of the invention is administered up to seven days after (possible) infection with the Zika virus. Preferably, the antibody or antigen-binding fragment thereof , in accordance with the present invention, a nucleic acid in accordance with the present invention, a vector in accordance with the present invention, a cell in accordance with the present invention or a pharmaceutical composition in accordance with the present invention is administered in a (unit) dose of 0.005 to 100 mg/kg body weight bodies.

The antibody or antigen binding fragment, nucleic acid, vector, cell or pharmaceutical composition can be administered by any of the various possible routes, such as oral, intravenous, intramuscular, intraarterial, bone marrow, intraperitoneal, intrathecal, intraventricular, transdermal, transdermal, topical, subcutaneous, intranasal , enteral, sublingual, intravaginal or rectal routes. Preferred is intravenous administration or subcutaneous administration or intramuscular administration, and more preferable is intravenous administration or subcutaneous administration.

In pregnant women, the antibody or antigen-binding fragment thereof can also be administered intra- or extra-amniotically, for example by injection.

Combination therapy.

Administration of an antibody or antigen binding fragment, nucleic acid, vector, cell or pharmaceutical composition in the methods and uses can be performed alone or in combination with a coagent (also referred to as an additional active component in this application) that is particularly useful for preventing and/or treating infection ZIKV.

The antibody or Antigen-binding fragment, nucleic acid, vector, cell, or pharmaceutical composition thereof may be administered to a subject prior to, concurrently, or sequentially with other therapeutic regimens or coagents useful for treating and/or preventing ZIKV infection. The specified antibody, nucleic acid, vector, cell or pharmaceutical composition, which is administered simultaneously with the specified coagents, can be administered in the same or different compositions and by the same or different routes of administration.

In a preferred embodiment of the invention, the coagents may be, for example, checkpoint inhibitors.

Thus, an antibody or antigen binding fragment, nucleic acid, vector, cell or pharmaceutical composition thereof can be administered in combination with a checkpoint inhibitor for (medical) applications as described herein.

Checkpoint inhibitors aim to block PD-1/PD-L1 and/or CTLA4 and therefore include αntu-PD-1 antibodies, anti-PD-LT antibodies and anti-CTLA4 antibodies.

A ZIKV neutralizing antibody or antigen binding fragment thereof can be combined with a ZIKV #1 binding antibody or antigen binding fragment thereof as an additional active component (coagent). Thus, the pathogenic role of NS1 may be blocked, in addition to neutralizing ZIKV. Thus, the ZIKV #1 binding antibody or antigen binding fragment thereof may serve as a preferred additional active component (coagent).

The antibody or antigen binding fragment may be present either in the same pharmaceutical composition as an additional active component (coagent) or, preferably, the antibody or antigen binding fragment constitutes the first pharmaceutical composition and the additional active component (coagent) constitutes a second pharmaceutical composition different from the first. pharmaceutical composition. Thus, if more than one additional active ingredient (coagent) is provided, then each additional active ingredient (coagent) and antibody or antigen binding fragment constitutes a different pharmaceutical composition. Such pharmaceutical compositions can be administered either in combination

- 46 046175 but/at the same time or at different times or in different locations (for example, separated parts of the body).

The antibody or antigen binding fragment and the additional active component (coagent) may provide an additive therapeutic effect or, preferably, a synergistic therapeutic effect. The term synergy 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, if the combined effect of two or more agents results in synergistic inhibition of an activity or process, then this means that the inhibition of the activity or process is greater than the sum of the inhibitory effects of each respective active agent. The term synergistic therapeutic effect refers to the therapeutic effect observed when two or more therapies are combined, where the therapeutic effect (as measured by any of the various parameters) is greater than the sum of the individual therapeutic effects observed in the respective individual therapies.

Additional uses and kits.

As already noted, another aspect of the present invention is the use of an antibody or antigen binding fragment thereof in accordance with the present invention, a nucleic acid in accordance with the present invention, a vector in accordance with the present invention, a cell or a pharmaceutical composition in accordance with the present invention for quality monitoring Zika virus vaccine by verifying that the vaccine antigen contains the specific epitope in the correct conformation. The Zika virus vaccine is subject to testing whether it includes the ZIKV envelope protein or any other molecule/complex containing or consisting of (I) ZIKV E protein domain III (EDIII) as described above or (II) a ZIKV quaternary epitope as described above .

In addition, the present invention relates to the use of an antibody or an antigen-binding fragment thereof according to the present invention, a nucleic acid according to the present invention, for diagnosing an infection caused by the Zika virus.

An antibody or antigen-binding fragment, nucleic acid, vector, cell, or pharmaceutical composition thereof may also be used to determine whether an isolated 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 isolated from a subject, for example, an isolated tissue sample taken from, for example, the nasal passages, sinus cavities, salivary glands, lungs, liver, pancreas, kidneys, ear, eye, placenta, digestive tract, heart, ovaries, pituitary gland, adrenal glands, thyroid gland, brain, skin or blood, preferably plasma or serum. Diagnostic methods may also include detection of an antigen/antibody complex, particularly after contact of the antibody or antibody fragment with a sample. This detection step is typically carried out in a laboratory, 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, ELISA (enzyme-linked immunosorbent assay).

A final aspect of the present invention is a kit for preventing or treating infection caused by the Zika virus, comprising at least one antibody or antigen binding fragment thereof in accordance with the present invention, at least one nucleic acid in accordance with the present invention, at least one vector in in accordance with the present invention, at least one cell in accordance with the present invention or at least one pharmaceutical composition in accordance with the present invention and instructions for their use. Additionally, the kit may contain means for administering the antibody or antigen binding fragment thereof, nucleic acid, vector, cell or pharmaceutical composition, such as a syringe or vial, package insert and/or administration coagent as described above.

Antibodies that specifically bind to the NS1 protein of the Zika virus.

The isolated antibody or antigen-binding fragment described herein specifically binds to the NS1 protein of the ZIKV virus.

ZIKV NS1 protein (non-structural protein 1) is found intracellularly, secreted and associated on the cell surface, and in particular, the secreted ZIKV NS1 protein is typically found in body fluids, such as serum, saliva, urine, etc., of ZIKV-infected subjects. Secreted and associated on the cell surface, NS1 is extremely immunogenic and induces the formation of antibodies. NS1 is known to be an important biomarker for the early diagnosis of ZIKV infection. Thus, an antibody or antigen-binding fragment thereof that specifically binds to the NS1 protein of ZIKV virus is suitable, for example, for diagnosing ZIKV infection.

In general, binding can be assessed using a standard ELISA, as known to one skilled in the art and as described above. Thus, the relative binding affinities of an antibody can be determined by measuring the antibody concentration (EC50) required to achieve 50% of maximum binding at saturation. Preferably EC50 antibody or anti

- 47 046175 gene binding fragment to the ZIKV NS1 protein is no more than 50 ng/ml, preferably said EC ₅₀ is no more than 25 ng/ml, more preferably said EC ₅₀ is no more than 15 ng/ml, even more preferably the said EC ₅₀ is no more than 10 ng/ml, and most preferably the said EC ₅₀ is no more than 5 ng/ml, such as, for example, about 2 or 3 ng/ml.

Preferably, an antibody or antigen binding fragment thereof that specifically binds to the NS1 protein of ZIKV virus (ZIKV) does not substantially bind to the NS1 protein of dengue virus (DENV). Thus, substantially no binding means that for an antibody or antigen-binding fragment thereof, there is no EC ₅₀ value of up to 10 ² ng/ml, preferably up to 10 ³ ng/ml, more preferably up to 5x10 ³ ng/ml, yet more preferably up to 8x10 ³ ng/ml, and most preferably up to 10 ⁴ ng/ml, which can be determined in a standard ELISA for dengue virus (DENV) NS1 protein. In other words, the concentration of antibody or antigen-binding fragment required to achieve 50% maximum binding at saturation (EC ₅₀ ) to dengue virus (DENV) NS1 protein in a standard ELISA is typically greater than 10 ² ng/ml, preferably greater than 10 ³ ng/ml, more preferably greater than 5 x 10 ³ ng/ml, even more preferably greater than 8 x 10 ³ ng/ml, and most preferably greater than 10 ⁴ ng/ml.

Also, an antibody or antigen-binding fragment thereof that specifically binds to the NS1 protein of ZIKA virus (ZIKV) does not bind substantially to the NS1 protein of yellow fever virus (YFV), NS1 protein of West Nile virus (WNV), or NS1 protein of Japanese encephalitis virus (JEV). ) and/or NS1 protein of tick-borne encephalitis virus (TBEV). Thus, substantially no binding means that for an antibody or antigen-binding fragment thereof, there is no EC ₅₀ value of up to 10 ² ng/ml, preferably up to 10 ³ ng/ml, more preferably up to 5x10 ³ ng/ml, yet more preferably up to 8x10 ³ ng/ml, and most preferably up to 10 ⁴ ng/ml, which can be determined in a standard ELISA for yellow fever virus (YFV) NS1 protein, West Nile virus (WNV) NS1 protein, NS1 protein Japanese encephalitis virus (JEV) and/or NS1 protein of tick-borne encephalitis virus (TBEV). In other words, the concentration of an antibody or antigen-binding fragment required to achieve 50% of the maximum saturation binding (EC50) to yellow fever virus (YFV) NS1 protein, West Nile virus (WNV) NS1 protein, Japanese encephalitis virus (JEV) NS1 protein and/or NS1 protein of tick-borne encephalitis virus (TBEV) in a standard ELISA is typically greater than 10 ² ng/ml, preferably greater than 10 ³ ng/ml, more preferably greater than 5x10 ³ ng/ml, even more preferably greater than 8x10 ³ ng/ml, and most preferably greater than 10 ⁴ ng/ml.

Preferably, the antibody that specifically binds to the NS1 protein of ZIKV virus is a human antibody. The antibody that specifically binds to the ZIKV NS1 protein may be a monoclonal antibody, preferably a human monoclonal antibody. In addition, an antibody that specifically binds to the NS1 protein of ZIKV virus may be a recombinant antibody.

An antibody or antigen-binding fragment thereof that specifically binds to the ZIKA virus (ZIKV) NS1 protein may contain an Fc component. An antibody or antigen-binding fragment thereof that specifically binds to the ZIKV NS1 protein may further contain a CH2 L4A mutation, a CH2 L5A mutation, or both.

An antibody or antigen binding fragment thereof that specifically binds to the NS1 protein of ZIKV virus may not contain an Fc component. Preferably, the antibody of the present invention or antigen binding fragment thereof that specifically binds to the ZIKV NS1 protein is a purified antibody, single chain antibody, Fab, Fab', F(ab') ₂ , Fv or scFv. An antibody or antigen binding fragment thereof that specifically binds to the ZIKV NS1 protein can be labeled, for example, by biotinylation of Fab, Fab' or F(ab') ₂ fragments.

An antibody or antigen-binding fragment thereof that specifically binds to the ZIKA virus (ZIKV) NS1 protein can bind to the S1 antigenic determinant and/or to the S2 antigenic determinant of the Zika virus NS1 protein. The inventors of the present invention have surprisingly discovered that anti-ZIKV NS1 antibodies that bind to the S1 antigenic determinant and/or the S2 antigenic determinant of the ZIKV NS1 protein do not cross-react with the dengue virus NS1 protein (DENV NS1). Anti-ZIKV NS1 antibodies, which bind to neither the S1 antigenic determinant nor the S2 antigenic determinant of the ZIKV NS1 protein, in contrast, typically cross-react with DENV NS1. This unexpected finding indicates that the S1 and S2 antigenic determinants on ZIKV NS1 can be used to distinguish ZIKV #1-specific antibodies from antibodies that cross-react with DENV NS1.

An antibody or antigen-binding fragment thereof that specifically binds to the ZIKA virus (ZIKV) NS1 protein can bind to the S2 antigenic determinant of the Zika virus NS1 protein. The S2 antigenic determinant is highly conserved across multiple ZIKV lineages, but not

- 48 046175 is homologous in sequence and structure to the corresponding NS1 site of other flaviviruses, thus providing unique specificity for ZIKV.

The S1 and S2 antigenic determinants of the ZIKV NS1 protein were identified by the inventors of the present invention as described in Example 3, FIG. 6. Regardless of whether an antibody binds to the antigenic determinant S1 and/or S2, it can be readily identified by one skilled in the art using cross-competition studies, for example as described below or in Example 3, where the S1-specific antibody with according to gZKA15 (SEQ ID NO: 91-99) and/or S2 specific antibody according to gZKA35 (SEQ ID NO: 127-135) can be used as the second antibody. In such a competition assay, the presence of any competition (total or partial) with gZKA15 and/or gZKA35 indicates that the test antibody binds to the S1 antigenic determinant and/or to the S2 antigenic determinant, respectively.

In general, commercially available protein-protein binding characterization systems, such as, for example, the Octet® RED96 System supplied by ForteBio, can be used for competitive analysis, particularly according to the manufacturer's instructions.

In a typical competition assay, for example, using the Octet® RED96 System supplied by ForteBio, ZIKV-NS1 protein (eg, diluted to 2.5 μg/ml in PBS) can be immobilized (eg, for 7-9 min) on the surface of a coated APS sensory type. The coated biosensors can then be placed into wells containing a blocking buffer (eg, 0.1% BSA in PBS; eg, for 6 min) to block unoccupied Biosensor binding sites. The coated Biosensors can then be incubated (eg for 8 min) with the antibody/test antibodies (eg diluted in blocking buffer, eg at 10 μg/ml). After binding of test antibodies (step 1), Biosensors were moved to wells containing second antibodies, eg gZKA15 and/or gZKA35 (eg, for 8 min) (step 2). Thus, it is possible to determine competition, partial competition, or no competition in step 2, depending on whether no association (competition), low association (partial competition), or (strong) association (no competition) was found.

As described above, the antibody or antigen binding fragment thereof preferably contains (at least) three complementarity determining regions (CDRs) on the heavy chain and (at least) three CDRs on the light chain. In general, complementarity determining regions (CDRs) are hypervariable regions present in the heavy chain variable domains and the light chain variable domains. Typically, the heavy chain CDR and associated light chain of an antibody together form an antigen receptor. Typically, the three CDRs (CDR1, CDR2, and CDR3) are not sequentially located in the variable domain. Because antigen receptors typically consist of two variable domains (on two different polypeptide chains, i.e., heavy and light chain), there are six CDRs for each antigen receptor (heavy chain: CDRH1, CDRH2, and CDRH3; light chain: CDRL1, CDRL2, and CDRL3). A single antibody molecule typically has two antigen receptors and therefore contains twelve CDRs. The CDRs on the heavy and/or light chain may be separated by framework regions, whereby a framework region (FR) is a region in the variable domain that is less variable than the CDRs. For example, a chain (or each chain, respectively) may consist of four framework regions separated by three CDRs.

The Applicant has determined the heavy chain and light chain sequences of five representative antibodies, containing three different CDRs on the heavy chain and three different CDRs on the light chain. CDR amino acid positions are defined according to the IMGT numbering system (IMGT: http://www.imgt.org/; cf. Lefranc, M.-P. et al. (2009) Nucleic Acids Res. 37, D1006-D1012).

In table 4 provides SEQ ID NOs for the amino acid sequences of the heavy chain CDRs (CDRH1, CDRH2, and CDRH3) and the heavy chain variable region (denoted VH) of exemplary antibodies in accordance with the present invention.

- 49 046175

Table 4

Antibody name CDRH1 CDRH2 CDRH3 VH ZKA15 91 92 93 98 ZKA25 109 110 111 116 ZKA35 127 128 129 134 ZKA10 153 154 155 156 ZKA18 157 158 159 160 ZKA28 161 162 163 164 ZKA29 165 166 167 168 ZKA33 169 170 171 172 ZKA39 173 174 175 176 ZKA43 177 178 179 180 ZKA44 181 182 183 184 ZKA46 185 186 187 188 ZKA50 189 190 191 192 ZKA54 193 194 195 196 ZKB18 197 198 199 200 ZKB20 201 202 203 204 ZKB21 205 206 207 208 ZKB23 209 210 211 212 ZKC29 213 214 215 216 ZKC31 217 218 219 220 ZKC32 221 222 223 224 ZKC33 225 226 227 228 ZKC34 229 230 231 232 ZKD25 233 234 235 236

In table 5 below sets forth SEQ ID NOs for the amino acid sequences of the light chain CDRs (CDRL1, CDRL2, and CDRL3) and the light chain variable region (referred to as VL) of exemplary antibodies in accordance with the present invention.

Table 5

Name CDRL1 CDRL2 CDRL2 CDRL3 VL antibodies long ZKA15 94 95 96 97 99 ZKA25 112 FROM 114 115 116 ZKA35 130 131 132 133 135

Thus, an antibody or antigen binding fragment thereof may comprise amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to at least one of the CDR sequences, VH sequences and/or VL sequences presented in table. 4 and/or in table. 5.

The antibody or antigen binding fragment thereof may comprise 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 at least one CDR, preferably at least one heavy chain CDRH3, contains or consists of an amino acid sequence as defined in any of SEQ ID NO: 93, 111, 129, 155, 159, 163, 167, 171, 175 , 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, and 235; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least

- 50 046175

90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

The antibody or antigen binding fragment thereof may comprise 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 at least one CDR, preferably at least one heavy chain CDRH3, contains or consists of an amino acid sequence as defined in any of SEQ ID NOs: 93, 111 and 129, or a functional sequence variant thereof having at least 70 %, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. Thus, it is preferred that at least one CDR, preferably at least one heavy chain CDRH3, contains or consists of an amino acid sequence according to SEQ ID NO: 93 or a functional sequence variant thereof having at least 70% at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. It is also preferred that at least one CDR, preferably at least one heavy chain CDRH3, contains or consists of an amino acid sequence according to SEQ ID NO: 111 or a functional sequence variant thereof having 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% sequence identity. In addition, it is also preferred that at least one CDR, preferably at least one heavy chain CDRH3, contains or consists of an amino acid sequence according to SEQ ID NO: 129 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least at least 97%, at least 98%, or at least 99% sequence identity.

The antibody or antigen binding fragment thereof may comprise 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 at least one CDR, preferably at least one heavy chain CDRH3, contains or consists of an amino acid sequence according to SEQ ID NO: 129, or a functional sequence variant thereof having at least 70%, at least 75% , at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, according to at least 98% or at least 99% sequence identity.

Also, the antibody or antigen-binding fragment thereof may comprise a heavy chain containing at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain containing at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein (I) at least one heavy chain CDRH1 contains an amino acid sequence as defined in any of SEQ ID NO: 91, 109, 127, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, and 233; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

(II) at least one CDRH2 contains an amino acid sequence as defined in any of SEQ ID NO: 92, 110, 128, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198 , 202, 206, 210, 214, 218, 222, 226, 230, and 234; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and/or (III) at least one heavy chain CDRH3 contains an amino acid sequence as defined in any of SEQ ID NO: 93, 111, 129, 155, 159, 163, 167, 171, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, and 235; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical after

- 51 046175 evidence.

Another antibody or antigen binding fragment thereof may comprise a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain containing at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein (I) at least one heavy chain CDRH1 contains an amino acid sequence as defined in any of SEQ ID NOs: 91, 109 and 127, or a functional sequence variant thereof having at least 70%, at least 75% , at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, according to at least 98% or at least 99% sequence identity;

(II) at least one CDRH2 contains an amino acid sequence as defined in any of SEQ ID NOs: 92, 110 and 128, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80 %, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and/or (III) at least one heavy chain CDRH3 contains an amino acid sequence as defined in any of SEQ ID NOs: 93, 111 and 129, or a functional sequence variant thereof having at least 70%, at least 75% , at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, according to at least 98% or at least 99% sequence identity.

Another antibody or antigen binding fragment thereof may comprise a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain containing at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein (I) at least one heavy chain CDRH1 contains an amino acid sequence in accordance with SEQ ID NO: 91 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, according to at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity;

(ii) at least one CDRH2 contains an amino acid sequence in accordance with SEQ ID NO: 92 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and/or (III) at least one heavy chain CDRH3 contains an amino acid sequence in accordance with SEQ ID NO: 93 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, according to at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

Also, the antibody or antigen-binding fragment thereof may comprise a heavy chain containing at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain containing at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein (I) at least one heavy chain CDRH1 contains an amino acid sequence in accordance with SEQ ID NO: 109 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, according to at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity;

(ii) at least one CDRH2 contains an amino acid sequence in accordance with SEQ ID NO: 110 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and/or (III) at least one heavy chain CDRH3 contains an amino acid sequence in accordance with SEQ ID NO: 111 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, according to at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

The antibody or antigen-binding fragment thereof may comprise a heavy chain containing

- 52 046175 at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain containing at least one CDRL1, at least one CDRL2 and at least one CDRL3, where (I) at least one Heavy chain CDRH1 contains the amino acid sequence according to SEQ ID NO: 127 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88% , at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity;

(ii) at least one CDRH2 contains an amino acid sequence in accordance with SEQ ID NO: 128 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and/or (III) at least one heavy chain CDRH3 contains an amino acid sequence in accordance with SEQ ID NO: 129 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, according to at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

The antibody or antigen binding fragment thereof may comprise 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 (I) at least one CDRL1 contains an amino acid sequence as defined in any of SEQ ID NOs: 94, 112 and 130, or a functional sequence variant thereof having at least 70%, at least 75%, at least at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98 % or at least 99% sequence identity;

(II) at least one CDRL2 contains an amino acid sequence as defined in any of SEQ ID NOs: 95, 96, 113, 114, 131 and 132, or a functional sequence variant thereof having at least 70% of at least 75 %, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and/or (III) at least one CDRL3 amino contains an amino acid sequence as defined in any of SEQ ID NOs: 97, 115 and 133, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least at least 98% or at least 99% sequence identity.

Another antibody or antigen binding fragment thereof may comprise a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain containing at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein (I) at least one CDRL1 contains an amino acid sequence in accordance with SEQ ID NO: 94 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity;

(II) at least one CDRL2 contains an amino acid sequence according to SEQ ID NO: 95 or 96, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and/or (III) at least one CDRL3 amino contains an amino acid sequence according to SEQ ID NO: 97 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 % sequence identity.

Also, the antibody or antigen-binding fragment thereof may comprise a heavy chain containing at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain containing at least one CDRL1, at least one CDRL2 and at least one CDRL3, where

- 53 046175 (I) at least one CDRL1 contains an amino acid sequence in accordance with SEQ ID NO: 112 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity;

(II) at least one CDRL2 contains an amino acid sequence according to SEQ ID NO: 113 or 114, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and/or (III) at least one CDRL3 amino contains an amino acid sequence according to SEQ ID NO: 115 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 % sequence identity.

The antibody or antigen binding fragment thereof may comprise 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 (I) at least one CDRL1 contains an amino acid sequence according to SEQ ID NO: 130 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85 %, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical sequences;

(II) at least one CDRL2 contains an amino acid sequence according to SEQ ID NO: 131 or 132, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and/or (III) at least one CDRL3 amino contains an amino acid sequence according to SEQ ID NO: 133 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 % sequence identity.

The antibody or antigen binding fragment thereof may comprise CDRH1, CDRH2, and CDRH3 amino acid sequences (I) according to SEQ ID NO: 91-93; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (II) in accordance with SEQ ID NO: 109-111; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (III) in accordance with SEQ ID NO: 127-129; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (IV) in accordance with SEQ ID NO: 153-155; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (V) in accordance with SEQ ID NO: 157 - 159; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (VI) in accordance with SEQ ID NO: 161-163; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (VII) in accordance with SEQ ID NO: 165-167; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, of

- 54 046175 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 sequences; (VIII) in accordance with SEQ ID NO: 169-171; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (IX) in accordance with SEQ ID NO: 173-175; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (X) in accordance with SEQ ID NO: 177-179; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XI) in accordance with SEQ ID NO: 181-183; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XII) in accordance with SEQ ID NO: 185-187; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XIII) in accordance with SEQ ID NO: 189-191; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XIV) in accordance with SEQ ID NO: 193-195; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XV) in accordance with SEQ ID NO: 197-199; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XVI) in accordance with SEQ ID NO: 201-203; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XVII) in accordance with SEQ ID NO: 205-207; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XVIII) in accordance with SEQ ID NO: 209-211; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XIX) in accordance with SEQ ID NO: 213215; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XX) in accordance with SEQ ID NO: 217-219; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XXI) in accordance with SEQ ID NO: 221-223; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (XXII) in accordance with SEQ ID NO: 225-227; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least

- 55 046175

85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (XXIII) in accordance with SEQ ID NO: 229-231; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or (XXIV) in accordance with SEQ ID NO: 233-235; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

The antibody or antigen binding fragment thereof may comprise CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences (I) according to SEQ ID NOs: 91-95 and 97; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (II) in accordance with SEQ ID NO: 91-94 and 96-97; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (III) in accordance with SEQ ID NO: 109-113 and 115; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (IV) in accordance with SEQ ID NO: 109-112 and 114-115; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (V) in accordance with SEQ ID NO: 127-131 and 133; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or (VI) in accordance with SEQ ID NO: 127-130 and 132-133; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

The antibody or antigen binding fragment thereof may comprise CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences (I) according to SEQ ID NOs: 127-131 and 133; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or (II) in accordance with SEQ ID NO: 127-130 and 132-133; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

Also, the antibody or antigen binding fragment thereof may comprise a heavy chain variable region (VH) and, optionally, a light chain variable region (VL), wherein the heavy chain variable region (VH) contains or consists of an amino acid sequence as defined in any of SEQ ID NO : 98, 116, 134, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, and 236; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

The antibody or antigen binding fragment thereof may comprise (I) a heavy chain variable region (VH) with the amino acid sequence according to SEQ ID NO: 98 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least

- 56 046175

92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity and/or light chain variable region (VL) with the amino acid sequence in accordance with SEQ ID NO: 99 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (II) a heavy chain variable region (VH) with an amino acid sequence according to SEQ ID NO: 116 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85 %, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical sequences and/or a light chain variable region (VL) with the amino acid sequence according to SEQ ID NO: 117 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; or (III) a heavy chain variable region (VH) with an amino acid sequence according to SEQ ID NO: 134 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and/or light chain variable region (VL) with the amino acid sequence according to SEQ ID NO: 135 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 % sequence identity.

The antibody or antigen binding fragment thereof may comprise a heavy chain variable region (VH) with the amino acid sequence of SEQ ID NO: 134 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least at least 99% sequence identity and/or light chain variable region (VL) with the amino acid sequence in accordance with SEQ ID NO: 135 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80% , at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or according at least 99% sequence identity.

The antibody or antigen binding fragment thereof may be gZKA15, gZKA25, or gZKA35, more preferably the antibody or antigen binding fragment thereof may be gZKA25 or gZKA35, even more preferably the antibody or antigen binding fragment thereof may be gZKA35.

The inventors of the present invention have isolated a monoclonal antibody (mAb) in accordance with the present invention, which are referred to herein as ZKA15, ZKA25 and ZKA35 (see Tables 4 and 5, Example 1). Based on these antibodies, particularly the VH and VL genes of these antibodies, the terms gZKA15, gZKA25 and gZKA35, as used herein, refer to the corresponding generic antibodies or antigen-binding fragments thereof.

Namely, gZKA15 refers to an antibody or an antigen binding fragment thereof having a CDRH1 amino acid sequence according to SEQ ID NO: 91, a CDRH2 amino acid sequence according to SEQ ID NO: 92, a CDRH3 amino acid sequence according to SEQ ID NO: 93, CDRL1 an amino acid sequence according to SEQ ID NO: 94, a CDRL2 amino acid sequence according to SEQ ID NO: 95 or 96, and a CDRL3 amino acid sequence according to SEQ ID NO: 97. The heavy chain variable region (VH) preferably has an amino acid sequence in according to SEQ ID NO: 98 and the light chain variable region (VL) preferably has the amino acid sequence according to SEQ ID NO: 99.

gZKA25 refers to an antibody or antigen binding fragment thereof having a CDRH1 amino acid sequence according to SEQ ID NO: 109, a CDRH2 amino acid sequence according to SEQ ID NO: 110, a CDRH3 amino acid sequence according to SEQ ID NO: 111, a CDRL1 amino acid sequence according to according to SEQ ID NO: 112, CDRL2 amino acid sequence according to SEQ ID NO: 113 or 114, and CDRL3 amino acid sequence according to SEQ ID NO: 115. The heavy chain variable region (VH) preferably has the amino acid sequence according to SEQ ID NO:

- 57 046175

116 and the light chain variable region (VL) preferably has the amino acid sequence according to SEQ ID NO: 117.

gZKA35 refers to an antibody or antigen binding fragment thereof having a CDRH1 amino acid sequence according to SEQ ID NO: 127, a CDRH2 amino acid sequence according to SEQ ID NO: 128, a CDRH3 amino acid sequence according to SEQ ID NO: 129, a CDRL1 amino acid sequence according to according to SEQ ID NO: 130, CDRL2 amino acid sequence according to SEQ ID NO: 131 or 132, and CDRL3 amino acid sequence according to SEQ ID NO: 133. The heavy chain variable region (VH) preferably has the amino acid sequence according to SEQ ID NO: 134 and the light chain variable region (VL) preferably has the amino acid sequence according to SEQ ID NO: 135.

The detailed description given above entitled Preparation of Antibodies (Section above Preparation of Antibodies) and Optional Additional Features of Antibodies (section above Optional Additional Features of Antibodies) apply to all antibodies, and antigen binding fragments thereof, as described herein - that is, these sections apply not only to neutralizing antibodies and their antigen-binding fragments, but also to No. 1 protein-binding antibodies and their antigen-binding fragments.

The antibody or antigen binding fragment thereof may be labeled, for example biotinylated.

The antibody or antigen binding fragment thereof can be conjugated to an enzyme such as horseradish peroxidase (HRP). Conjugation of antibodies to HRP is described, for example, in Wisdom GB. Conjugation antibodies to horseradish peroxidase. Methods Mol Biol. 2005;295:127-30 or in Antibodies - a laboratory manual. Ed. Edward A. Greenfield, second edition 2012, Cold Spring Harbor Laboratory Press, ISBN: 9781936113811.

Antibodies, or antigen-binding fragments thereof, may be associated with a detectable label, for example, to enable measurement, such as quantitation, or to facilitate visualization. Labeled antibodies can be used in a variety of assays, especially immunoassays, using a large number of labels. Preferred labels include radionuclides, enzymes, coenzymes, fluorescent substances, chemiluminescent substances, chromogens, enzyme substrates or cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes (eg, fluorescent dyes, tandem dyes), etc. Examples of suitable ones enzymes include horseradish peroxidase (HRP), alkaline phosphatase, β-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, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include 125I, 131I, 35S, or 3H. Such labeled reagents can be used in a variety of well known assays such as radioimmunoassays, enzyme immunoassays such as ELISA, fluorescence immunoassays, etc., preferably ELISA. Therefore, labeled antibodies in accordance with the present invention can be used in such assays, for example, as described in US 3,766,162; US 3,791,932; US 3,817,837; and US 4,233,402.

Preferred labels include (I) enzymes as described above, for example horseradish peroxidase (HRP) or alkaline phosphatase, especially in binding blocking assays, Western blots, ELISAs and immunohistochemistry; (ii) prosthetic group complexes as described above, for example streptavidin/biotin and avidin/biotin, especially in ELISA and immunohistochemistry; (III) fluorescent substances as described above, such as fluorescent dyes and fluorescent proteins (eg (enhanced) green fluorescent protein (EGFP); TagBFP, Turquoise, Venus, KO2, Cherry, Apple, Kate2), especially in immunofluorescence and flow cytometry; and (IV) tandem dyes in flow cytometry.

The antibody or antigen binding fragment thereof may be biotinylated. Biotinylation is rapid, specific, and unlikely to interfere with the natural function of the molecule due to the small size of biotin (MB = 244.31 g/mol). Biotin binds to streptavidin and avidin with extremely high affinity, fast forward reaction rates and high specificity. Biotin binding to streptavidin and avidin is resistant to excessive heat, pH, and proteolysis, making it possible to trap biotinylated molecules under a variety of conditions. The antibody or antigen binding fragment thereof may be biotinylated chemically or enzymatically. Chemical biotinylation uses various conjugative chemistries to achieve non-specific biotinylation of amines (e.g. NHS coupling ensures biotinylation of any primary amines in the antibody, see below). Enzymatic biotinylation leads to biotinylation of a specific lysine within a certain sequence

- 58 046175 activity by using bacterial biotin ligase.

The antibody or its fragment can also be used as a label. In this case, the antibody or antigen-binding fragment thereof is conjugated to a second antibody or antibody fragment thereof to form an antibody heteroconjugate, as described, for example, in US 4,676,980. In this case, the second antibody may optionally be labeled.

Methods for linking antibodies to tags are well known in the art. For example, in an antibody or antigen binding fragment thereof, a lysine side chain that terminates in a primary amine (-NH2) can be used to covalently attach tags to the antibody or antigen binding fragment thereof. Various labeling procedures have been described in the literature. For example, a suitable labeling approach may be selected from the group consisting of NHS esters, heterobifunctional reagents, carbodiimides, and sodium periodate.

NHS esters can be used especially for labeling with fluorescent dyes. The fluorescent dye tag can be purchased in an activated form of the tag with an integrated NHS ester (also called 'succinimidyl ester'). The activated dye can be reacted under suitable conditions with the antibody or an antigen-binding fragment thereof (eg, via a lysine group). Excess reaction dye can be removed (eg, by column chromatography) before the labeled antibody or antigen binding fragment thereof is used in an immunoassay.

Heterobifunctional reagents can be used when the label is a protein molecule (eg, HRP, alkaline phosphatase, or phycoerythrin). In this case, the antibody or antigen binding fragment and label thereof may have multiple amines. In this situation, some of the lysines on one molecule (eg, on an antibody or on its antigen-binding fragment) can be modified to create a new reactive group (X) and the lysines on a tag to create a different reactive group (Y) (or vice versa). The 'heterobifunctional reagent' is then used to introduce Y groups, which subsequently react with X groups when the antibody and tag are mixed, thereby creating heterodimeric conjugates.

Carbodiimides, such as EDC, can especially be used to create covalent bonds between amine- and carboxyl-containing molecules. Carbodiimides activate carboxyl groups, and the activated intermediate is then attacked by an amine (eg, provided by a lysine residue on an antibody or antigen-binding fragment thereof). Carbodiimides can especially be used to conjugate antibodies to carboxylated particles (eg, latex particles, magnetic beads), and to other carboxylated surfaces such as microwell plates or chip surfaces. Carbodiimides can also be used to attach dyes or protein tags to antibodies or antigen-binding fragments thereof.

Sodium periodate can be used particularly for horseradish peroxidase (HRP) labeling. Periodate activates carbohydrate chains on the HRP molecule to create aldehyde groups that are able to react with lysines on the antibody or antigen-binding fragment. Because HRP itself has only a few lysines, it is relatively easy to create antibody-HRP conjugates without significant HRP polymerization.

Linkers can optionally be used between tags and antibodies of the invention, for example as 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.

Thus, it is possible to obtain a complex containing (I) an antibody or an antigen-binding fragment thereof, in accordance with the present invention; and (II) a label as described above.

Such a complex is preferably conjugated with a label to an antibody or an antigen-binding fragment thereof. Preferably, the label and the antibody or antigen binding fragment thereof may be covalently linked.

For example, the complex may be a fusion protein comprising (i) an antibody of the present invention and (ii) a tag that is a peptide or protein, such as a fluorescent peptide or protein, such as EGFP.

The nucleic acid molecule containing a polynucleotide encoding an antibody or an antigen binding fragment or complex thereof may be a fusion protein.

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.

In table 4 and 5 provide SEQ ID numbers for the amino acid sequences for the CDRs and VH and VL of representative antibodies.

In table 6 below provides SEQ ID numbers for exemplary nucleic acid sequences encoding the CDRs and VH and VL of exemplary antibodies.

The nucleic acid molecule may be a molecule containing, preferably

- 59 046175 consisting entirely of nucleic acid components. The term nucleic acid molecule preferably refers to DNA and RNA molecules. In particular, it is used synonymously with the term polynucleotide. A nucleic acid molecule may be a polymer containing or consisting of nucleotide monomers that are covalently linked to each other via phosphodiester bonds of the sugar phosphate backbone. The term nucleic acid molecule also covers modified nucleic acid molecules, such as DNA or RNA molecules with modified bases, modified sugars or modified backbones, etc.

In table 6 shows representative CDR and variable heavy chain (VH) and variable light chain (VL) nucleic acid sequences of three representative antibodies of the present invention (ZKA15, ZKA25, ZKA35).

Table 6

ZKA15 SEQ ID NO. Nucleic acid sequence CDRH1 100 Ggtggcttcatcaatagttactac CDRH2 101 Atctataaaagtgggagcacc CDRH3 102 Gcgagagatccctacggtgactacgttaaggcttttgatatt CDRL1 103 Cagagcctcctgcatagtaatggatacaactat CDRL2 104 Ttggggttct CDRL2 long 105 Ctgatctatttgggttctaatcgggcc

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CDRL3 106 Atgcaagctctacaaactgtcact VH 107 caggtgcagctgcaggagtcggggccaggactggtgaagccttcggaga ccctgtccctcacctgcactgtctccggtggcttcatcaatagttacta ctggagctggatccggcagcccgccgggaagggactggagtggattggg cgtatctataaaagtgggagcaccaactacaacccctccctcaagagtc gagtcaccatgtcactaga cacgtccaagtaccagttctccctgaagct gaggtctgtgaccgccgctgacacggccgtgtattactgtgcgagagat ccctacggtgactacgttaaggcttttgatatttggggccaagggacaa tggtcaccgtctcttcag VL 108 gatattgtgatgactcagtctccactctccctgcccgtcacccctggag agccggcctccatctcctgcaggtctagtcagagcctcctgcatagtaa tggatacaactatttgaattggtacctgcagaagccagggcagtctcca cagctcctgatctatttgggttctaatcgggcctccggggtccctgaca ggttcagtggcag tggatcaggcacagattttacactgaaaatcagcag agtggaggctgaggatgttggggtttattactgcatgcaagctctacaa actgtcactttcggccctgggaccaaagtggatatcaaac ZKA25 SEQ ID NO. Nucleic acid sequence CDRH1 118 Ggattcacctttagaagtcattgg CDRH2 119 Ataaaggaagatggatatgagaaa CDRH3 120 Gcgagagatttgaggtatatagtgggagaggtttcgacccc CDRL1 121 Aaattgggggataaatat CDRL2 122 Caagatagc CDRL2 long 123 Gtcatctatcaagatagcaagcggccc CDRL3 124 Caggcgtgggacagcagcactgtggta VH 125 gaggtgcagttggtggagtctgggggaggcttggtccggcctggggggt ccctgagactctcctgtgcagcctctggattcacctttagaagtcattg gatgagttgggtccgccaggctccagggaaggggctggagtgggtggcc aacataaaggaagatggatatgagaaatactatgtggactctgtgaagg gccgattcaccat ctccagagacaacgccaagaactcactgtatctgca aatgaagagcctgagagccgaggacacggccgtgtattactgtgcgaga gatttgagggtatatagtgggagaggtttcgacccctggggccagggaa ccctggtcaccgtctcctcag VL 126 tcctatgagctgactcagccaccctcactgtccgtgtccccaggacaga cagccagcatcacctgctctggagataaattggggataaatatgcttg ctggtatcagcagaagccaggccagtcccctgtgttggtcatctatcaa

- 61 046175

gatagcaagcggccctcagggatccctgcgcgattctctggctccaact ctgggaacacagccactctgaccatcagcgggacccaggctatggatga ggctgactattactgtcaggcgtgggacagcagcactgtggtattcggt ggagggaccaagctgaccgtcctag ZKA35 SEQ ID NO. Nucleic acid sequence CDRH1 136 Ggtggctccatcagcactggtggttactac CDRH2 137 Atctattacagtgggaacacc CDRH3 138 Gcgaaaggaggagggagggagcgaccctttgactac CDRL1 139 Agctccaacatcggaagaaattat CDRL2 140 Aggaataat CDRL2 long 141 Ctcatctataggaataatcagcggccc CDRL3 142 Gtagcatgggatgacagccggagtggttttgtggta VH 143 caggtgcagctgcaggagtcgggcccaggactggtgaagccttcacaga ccctgtccctcacctgcactgtctctggtggctccatcagcactggtgg ttactactggagctggatccgccagcacccagggaagggcctggagtgg attggttacatctattacagtgggaacacctactacaacccgtccctca agagtcgagttaccatatca gttgacacctctaagaagcagttctccct gaagctgagctctgtgactgccgcggacacggccgtgtattachgtgcg aaaggaggagggagggagcgaccctttgactactggggccagggaaccc tggtcaccgtctcctcag VL 144 cagtctgtgctgactcagccaccctcagcgtctgggacccccgggcaga gggtcaccatctcttgttctggaagcagctccaacatcggaagaaatta tgtagactggtaccagcaactcccaggaacggcccccaaactcctcatc tataggaataatcagcggccctcaggggtccctgagcgattctctggct ccaagtctggcacctcagcct ccctggccatcagtgggctccggtccga ggatgaggctgattattactgtgtagcatgggatgacagccggagtggt tttgtggtattcggcggagggaccaaggtgaccgtcctag

The nucleic acid molecule sequence may comprise or consist of a nucleic acid sequence according to any of SEQ ID NOs: 100-108, 118-126, and 136-144.

Nucleic acid sequences may also include nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with respect to the nucleic acid encoding the CDR, VH sequence and/or VL sequence, used in a (typical) antibody, for example, to the sequences presented in table. 6.

In general, a nucleic acid molecule can be manipulated to insert, delete, or alter certain nucleic acid sequences. Changes in such manipulation include, but are not limited to, changes to introduce restriction sites, to change the frequency of the codon used, to add or optimize transcription and/or translation of regulatory 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 tails (eg, for purification). Mutations are possible

- 62 046175 introduced into specific sites or can be introduced randomly, followed by selection (eg molecular evolution). For example, one or more nucleic acids encoding the CDR regions, VH sequence and/or VL sequence of a (typical) antibody of the invention may be randomly or targetedly mutated to introduce different properties into the encoded amino acids. Such changes may result in an iterative process in which the original substitutions are maintained and new substitutions are introduced at other nucleotide positions. Moreover, substitutions carried out at independent stages can be combined. Various properties introduced into the encoded amino acids may include, but are not limited to, altered affinity.

The invention also relates to expression vectors containing a nucleic acid molecule in accordance with the present invention.

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 suitable for incorporating or anchoring 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 enables suitable storage of a nucleic acid molecule. Thus, the vector may contain a sequence corresponding to, for example, a desired antibody or fragment thereof. An expression vector can be used to produce expression products such as RNA, eg mRNA, or peptides, polypeptides or proteins. For example, an expression vector may contain sequences necessary for transcription of a sequence stronger than the vector, such as a promoter sequence. A cloning vector is typically a vector containing a cloning site that can be used to incorporate nucleic acid sequences into the vector. The cloning vector may be, for example, a plasmid vector or a bacteriophage vector. A transfer vector may be a vector that is suitable for transferring nucleic acid molecules into cells or organisms, such as viral vectors. The vector in the context of the present invention may be, for example, an RNA vector or a DNA vector. Preferably, the vector is a DNA molecule. For example, a vector in the context of the present application contains a cloning site, a selectable marker, such as an antibiotic resistance factor, and a sequence suitable for propagation of the vector, such as an origin of replication.

Preferably, the vector in the context of the present application is a plasmid vector.

The invention also relates to a cell expressing an antibody or antigen binding fragment thereof in accordance with the present invention; and containing a vector in accordance with 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 a mammalian cell line. Preferred examples include human cells, CHO cells, HEK293T cells, PER.C6 cells, NS0 cells, human liver cells, myeloma cells or hybridoma cells.

In particular, the cell can be transfected with an expression vector. 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 the skilled person for introducing nucleic acid molecules into cells, preferably eukaryotic cells, for example, mammalian cells. Such methods include, for example, electroporation, lipofection, for example, based on cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or transfection based on cationic polymers, such as DEAE-dextran or polyethylenimine and etc. Preferably, the introduction is non-viral.

Cells can be stably or transiently transfected with a vector, for example, to express an antibody or an antigen-binding fragment thereof. Cells can be stably transfected with a vector encoding an antibody or an antigen-binding fragment thereof. Alternatively, cells can be transiently transfected with a vector encoding an antibody or an antigen-binding fragment thereof.

The invention also relates to a pharmaceutical composition as described above in the context of neutralizing antibodies, wherein the detailed description as described above thus applies to an antibody or antigen binding fragment thereof that binds the ZIKV NS1 protein. However, the composition can also be used for non-pharmaceutical purposes, such as for diagnosis (ZIKV infection) or for analytical purposes.

Preferably, the composition may be presented in liquid form, for example, to provide an antibody or antigen-binding fragment thereof in a liquid, for example, in a diagnostic assay. The liquid (carrier) can be selected according to the purpose, e.g. depending on the analysis

- 63 046175 for. Preferably, the composition may contain PBS (phosphate buffered saline) or other buffer. Such buffers are preferably biological buffers, and therefore the composition may contain any of MES, BIS-TRIS, ADA, PIPES, ACES, MOPsO, BIS-TRIS propane, BES, MOPS, TES, HEPES, DIPSO, TAPSO, Trizma, POPSO, HEPPS, TRICINE, Gly-Gly, BICINE, HEPBS, TAPS, AMPD, AMPSO, CHES, CAPSO, AMP, CAPS and CABS. The composition may also contain Ringer's solution. Additionally, the composition may also contain Tris, for example Tris-HCl.

The composition may also contain a detergent, for example, Tween (polysorbate), such as Tween 20 or Tween 80. The detergents are preferably present in low amounts, for example less than 0.01%. The compositions may also include sodium salts (eg, sodium chloride) to impart tonicity. For example, a typical NaCl concentration is 10 ± 2 mg/ml.

Additionally, the composition may optionally contain a protein stabilizer, BSA (bovine serum albumin) or HSA (human serum albumin). Other examples of protein stabilizers that may optionally be included in the composition include buffers, for example, as described above; salts such as sodium chloride; amino acids such as histidine, glycine and arginine; polyols/disaccharides/polysaccharides such as trehalose and sucrose (disaccharides), mannitol and sorbitol (sugar alcohols); surfactants such as polysorbate 20, polysorbate 80, and proteins such as HSA or BSA; polymers such as dextran and polyethylene glycol; and antioxidants.

In addition, the composition may optionally contain a preservative such as sodium azide. Preservatives are typically used to prevent contamination by microorganisms.

The invention also relates to a kit containing an antibody or an antigen-binding fragment thereof, it may optionally contain one or more of the following components:

(I) one or more solutions, for example, for use in a diagnostic assay, for example, for diluting an antibody or antigen-binding fragment thereof;

(ii) package insert, for example, with instructions for use;

(III) a label as described above and, optionally, solutions and/or other components necessary for labeling; and/or (IV) vials or devices, for example, suitable for diagnostic analysis, for example, one or more ELISA plates.

Preferably, the kit as described above may also contain a color development substrate. Examples of such a substrate include p-NPP, especially when detected by alkaline phosphatase; or an enzyme such as ABTS, TMB or OPD, especially in the case of horseradish peroxidase (HRP). Optionally, the substrate can be diluted in a suitable buffer, for example, a buffer as described above in the context of the composition in accordance with the present invention. Alternatively, the substrate and buffer may be provided as separate components in the kit.

As for the label, the kit may also contain a streptavidin-conjugated enzyme or other system to detect binding of the probe antibody. For example, the probe antibody can be formulated in murine form, in which case binding can be detected by an anti-mouse secondary antibody—without the need for biotinylation. The anti-mouse secondary antibody is typically polyclonal and/or cross-absorbable so as not to react with human antibodies.

In addition, the kit may contain one or more ELISA plates. More preferably, these ELISA plates are pre-coated with ZIKV-NS1 protein. Optionally, such pre-coated ELISA plates can be pre-blocked.

Diagnosis of infection caused by the Zika virus.

An antibody or antigen-binding fragment thereof that binds to the ZIKV NS1 protein, kit or composition can be used to diagnose Zika virus (ZIKV) infection.

Diagnosis of Zika virus (ZIKV) infection is typically performed in vitro, for example, in an isolated sample from the subject being diagnosed. Preferred isolated subject samples include body fluid samples and tissue samples. A body fluid sample is preferred. Preferred body fluids for diagnosing ZIKV infection include blood (eg, whole blood, plasma, serum), saliva, and urine. Most preferred is blood, especially plasma or serum.

Thus, the use of an antibody or antigen-binding fragment thereof that binds to the ZIKV NS1 protein, complex, composition or kit allows one to determine whether an isolated sample (body fluid), such as an isolated blood sample, is infected with Zika virus, as described above, Preferred Fluids organisms used to diagnose ZIKV infection include blood (eg, whole blood, plasma, serum), saliva, and urine. Most preferred is blood, especially plasma or serum.

Various diagnostic tests can be used to diagnose Zika virus infection. Preferred diagnostic tests are immunoassays. Preferred examples of immunoassays include ELISA, immunofluorescence, immunohistochemistry and flow cytometry. Preferably, the diagnosis includes ELISA.

- 64 046175

For example, a standard ELISA, a sandwich ELISA, or a binding blockade assay can be used.

Preferably, the diagnostic assay detects (I) the ZIKV NS1 protein itself; and/or (II) the (presence of) anti-ZIKV NS1 antibody in the (isolated) sample of the subject to be diagnosed.

Preferably, a binding blocking assay is used. In this assay, an isolated sample from the subject to be diagnosed (e.g., a sample of body fluid such as blood (e.g., whole blood, plasma, serum), saliva, and urine) is added to an ELISA plate coated with ZIKV NS1 protein and incubated ( for example, for at least about 30 minutes or at least about one hour) to allow binding. Thereafter, an antibody or antigen-binding fragment thereof is added (as a probe antibody), which is preferably labeled, for example biotinylated or conjugated with horseradish peroxidase (HRP). After additional incubation time (e.g., at least about 1 minute, preferably at least about 3 minutes, more preferably at least about 5 minutes, even more preferably at least about 10 minutes, most preferably at least about 15 minutes) , the inhibition of binding of an antibody or antigen binding fragment thereof can be determined in accordance with the present invention.

In general, inhibition of binding indicates the presence of anti-ZIKV NS1 antibodies in the subject's sample, thus indicating ZIKV infection of the subject. In contrast, binding inhibition is typically not expected in samples from uninfected subjects. Importantly, this assay using ZIKV #1-binding antibodies does not yield positive results in subjects who have already been infected with other flaviviruses. Flaviviruses typically induce large amounts of antibodies that cross-react with ZIKV. In other words, this assay is highly specific and independent of cross-reacting Abs.

Thus, a binding block assay for in-vitro diagnosis of Zika virus infection is possible, involving the following steps:

(I) adding an isolated sample from the subject to be diagnosed to a plate coated with the ZIKV NS1 protein and incubating said sample on said plate, (II) adding an antibody or antigen binding fragment or complex thereof, (III) determining whether said antibody or complex is inhibited from binding by antigen-binding fragment.

Preferably, the isolated sample from the subject to be diagnosed is selected from blood, saliva or urine; preferably the sample is a blood sample, such as whole blood, plasma or serum.

It is also preferred that the antibody, or antigen binding fragment thereof, added in step (II) is labeled, preferably biotinylated or horseradish peroxidase (HRP) conjugated.

In addition, the isolated sample from the subject to be diagnosed is preferably diluted, for example, 1:5 - 1:50, preferably 1:5-1:25, for example, 1:10.

Preferably, the incubation time in step (I) is at least 5 minutes, preferably at least 15 minutes, more preferably at least 30 minutes, even more preferably at least 45 minutes and most preferably at least 60 minutes.

In step (II), after adding the antibody or antigen binding fragment thereof, the antibody or antigen binding fragment thereof may be incubated for at least 1 minute, preferably at least 3 minutes, more preferably at least 5 minutes, even more preferably at least 10 minutes and most preferably at least 15 minutes.

Preferably, the antibody or antigen binding fragment thereof that is used as a probe antibody in the binding blocking assay is a preferred antibody or antigen binding fragment thereof. For example, the antibody or antigen binding fragment thereof may preferably be an antibody or antigen binding fragment thereof that binds to the S2 NS1 antigenic determinant of the Zika virus protein. Most preferably, the antibody or antigen binding fragment thereof that is used as a probe antibody in a binding blocking assay is an antibody or antigen binding fragment thereof comprising CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences (I) according to with SEQ ID NOs: 127-131 and 133; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or (II) in accordance with SEQ ID NO: 127-130 and 132-133; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. Particularly preferably

- 65 046175 The antibody or antigen binding fragment thereof, in accordance with the present invention, which is used as a probe antibody in a binding blocking assay, is an antibody or antigen binding fragment thereof containing a heavy chain variable region (VH) with the amino acid sequence in accordance with SEQ ID NO: 134 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity and/or light chain variable region (VL) with the amino acid sequence according to SEQ ID NO: 135 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. For example, the inhibition of binding of typical biotinylated antibodies or antigen binding fragments thereof can be assessed by determining the optimal concentration of the antibody or antigen binding fragment thereof in accordance with the present invention to achieve 70% maximum binding to the ZIKV NS1 protein. For example, the optimal concentrations of typical antibodies gZKA15, gZKA25 and gZKA35 to achieve 70% maximum binding to the ZIKV NS1 protein may be 38, 17 and 7 ng/ml, respectively. After performing the above binding inhibition assay, a substrate such as p-NPP can be added and the ELISA plate can be run at 405 nm and the percentage binding inhibition value can be calculated according to the following equation (I):

(I) % Inhibit = (1-[(OD of sample - OD of negative counter)/ (OD of positive counter - OD of negative counter)]) x 100 where % inhibition refers to the percentage inhibition of binding of an antibody or antigen-binding fragment according to this invention with ZIKV NS1 protein; The OD of a sample refers to the optical density of the sample; The OD of negative control refers to the optical density of the negative control; and OD positive control refers to the optical density of the positive control.

This assay provides certain advantages, such as the ability to detect clinical, subclinical, and asymptomatic ZIKV infections at the population level while distinguishing them from infections caused by other flaviviruses such as DENV. In particular, the diagnostic assay in accordance with the present invention provides higher accuracy than direct binding ELISA assays.

In addition, diagnosis (in vitro) of Zika infection (in an isolated sample) can be performed using an antibody or antigen binding fragment thereof that binds to the ZIKV NS1 protein, complex, composition or kit to determine whether the isolated sample (body fluid) is such as the isolated blood sample is infected with the Zika virus.

Preferred isolated (subject) samples include body fluid samples and tissue samples. A body fluid sample is preferred. Preferred body fluids for diagnosing ZIKV infection include blood (eg, whole blood, plasma, serum), saliva, and urine. Most preferred is blood, especially plasma or serum. In addition, preferred diagnostic tests are immunoassays. Preferred examples of immunoassays include ELISA, immunofluorescence, immunohistochemistry and flow cytometry. Preferably, the diagnosis includes ELISA. Most preferably, a binding blocking assay is used as described above.

Preferably, the method (in vitro) for diagnosing Zika infection (in an isolated sample) may comprise the steps of:

(I) contacting the isolated sample with an antibody or antigen-binding fragment thereof that binds to the ZIKV NS1 protein, complex or composition.

More preferably, the method (in vitro) for diagnosing Zika infection (in an isolated sample) may include the following steps:

(0) adding an isolated sample from the subject to be diagnosed (eg, a sample of a body fluid such as blood (eg, whole blood, plasma, serum), saliva and urine) onto an ELISA plate coated with the ZIKV NS1 protein;

(I') further adding the antibody or antigen binding fragment thereof to the ELISA plate, wherein the antibody or antigen binding fragment thereof is preferably labeled, for example biotinylated;

(II) optionally, washing the ELISA plate; and (III) determining inhibition of binding of the antibody or antigen-binding fragment thereof.

The present invention also provides the use of a neutralizing antibody or antigen-binding nucleic acid fragment thereof, vector, cell or pharmaceutical composition for the treatment or prevention of ZIKV infection in subjects with a diagnosed infection, you

- 66 046175 called the Zika virus.

A proposed method for preventing and/or treating Zika virus infection may include the following steps:

(I) diagnosing a Zika virus infection in a subject by administering an antibody or antigen-binding fragment thereof that binds to the ZIKV NS1 protein, complex, composition, or kit; and (II) administering to said subject a neutralizing antibody or antigen binding fragment, nucleic acid, vector, cell, or pharmaceutical composition.

Preferably, in this method of preventing and/or treating Zika virus infection, step (I) diagnosing the Zika virus infection can be performed as an in-vitro diagnosis on an isolated sample (body fluid), such as an isolated blood sample.

A composite kit (kit) containing (I) an antibody or an antigen-binding fragment thereof that binds to the ZIKV NS1 protein, complex, composition; and (II) the neutralizing antibody or antigen binding fragment, nucleic acid, vector, cell or pharmaceutical composition thereof, suitable for use in the method as described above. By using such a method and/or such a kit, ZIKV infection can be specifically diagnosed as well as prevented and/or treated.

Description of the figures

Fig. 1 shows the reactivity (ELISA) of both ZIKV and DENV1 neutralizing activity of antibodies obtained from four ZIKV immunocompetent donors (ZKA, ZKB, ZKC and ZKD) to the ZIKV E protein and DENV1-4 and to the EDIII domain of the ZIKV E protein; NNB - neutralizing, but not E protein binding, antibodies.

Fig. 2 shows the reactivity (ELISA) of antibodies obtained from four ZIKV immunocompetent donors (ZKA, ZKB, ZKC and ZKD) to the NS1 protein from ZIKV, DENV1-4 and other flaviviruses. YFV - yellow fever virus; WVN - West Nile virus; JEV - Japanese encephalitis virus; and TBEV - tick-borne encephalitis virus (but not determined).

Fig. 3 shows the binding of ZKA190, ZKA78 and ZKA64 antibodies to ZIKV and DENV1 E and to ZIKV ED III proteins as measured by ELISA.

Fig. 4 shows the binding of ZKA185 and ZKA190 antibodies to ZIKV E, DENV1 VLP and ZIKV EDIII proteins as measured by ELISA.

Fig. 5 shows the binding of ZKA15, ZKA25 and ZKA35 antibodies to ZIKV and DENV1-4 NS1 proteins as measured by ELISA.

Fig. 6 shows for Example 3 mapping of the ZIKV NS1 antigenic determinant protein using cross-competitive Octet binding assays. (A-B) Cross-competition array was performed using Octet 24 mAbs specific for ZIKV NS1 (A) or cross-reactive with DENV NS1 (B). +, absence of secondary Ab binding; +/-, partial loss of secondary mAb binding; -, secondary mAb binding. Crossed out cells were not tested. (C) Map of antigenic determinants targeted by ZIKV #1-specific mAbs as determined using BLI (Octet) cross-competition.

Fig. 7 shows for Example 4 a binding blockade assay using mAb ZKA35 as a probe to detect ZIKV NS1 in the plasma of ZIKV-immune (n=4), DENV-immune (n=5) and control donors (n=48) (1/10 breeding). Plasma samples were tested to determine their ability to bind NS1 (open circles) and inhibit the binding of biotinylated mAb ZKA35 to NS1 (solid circles).

Fig. 8 shows for Example 5 the neutralizing activity of ZKA190, ZKA64, ZKA64-LALA, ZKA230 and ZKA78 antibodies against ZIKV (H/PF/2013 strain) and DENV1 on Vero cells as determined by flow cytometry (% cells infected).

Fig. 9 shows for Example 5 the neutralizing activity of ZKA190, ZKA64, ZKA185, ZKA230 and ZKA78 antibodies against ZIKV (H/PF/2013 strain) on Vero cells as determined by cell viability assay (wst-1, Roche).

Fig. 10 shows for Example 6 the infection enhancing activity (ADE, antibody dependent enhancement) of ZKA190, ZKA64, ZKA64-LALA, ZKA185, ZKA230 and ZKA78 antibodies for ZIKV (H/PF/2013 strain) on non-permissive K562 cells as determined by flow cytometry (% infected cells).

Fig. 11 shows for Example 6 that four ZIKV-immune plasmas and one DENV-immune plasma exhibited similar activity in enhancing ZIKV infection of K562 cells (top panel). This ADE effect was completely blocked in all five immune plasmas by the EDIII-specific ZKA64-LALA antibody (lower panel).

Fig. 12 shows the amino acid alignment of the EDIII region for 39 ZIKV strains from Asian lineages since 2013 (including the prototype strain MR766 of the African lineage, isolated in 1947).

Fig. 13 shows for example 5 the neutralizing activity of ZKA190 and ZKA190-LALA anti

- 67 046175 body against three ZIKV strains (H/PF/2013, MR766 and MRS_OPY_Martinique_PaRi_2015) on Vero cells as determined by flow cytometry (% of cells infected).

Fig. 14 shows for Example 7 NS1 a binding block analysis for European residents. BOB values are presented for samples collected in Italy and Switzerland. VACV values in samples from ZIKV, primary and secondary DENV-, WNV-, and CHIKV-infected individuals and a panel of blood samples from healthy donors from Switzerland are graphically presented.

Fig. 15 shows for Example 8 the neutralization of ZKA190 and C8 mAbs 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 presented. Data are representative of at least two independent experiments.

Fig. 16 shows for Example 9 neutralization and enhancement of ZIKV infection by antibody ZKA190. (A) Neutralization of ZIKV PRVABC59 strain hNPC infection by ZKA190, ZKA190LALA, and control mAb, as determined by plaque formation assay on Vero cells (left panel) and indirect immunofluorescence of infected hNPCs using fluorophore-labeled anti-E antibody (right panel). (B) ADE of ZIKV infection of nonpermissive K562 cells with ZKA190 and ZKA190-LALA. (C) ADE was induced in K562 cells when ZIKV was preincubated with serial dilutions of plasma serum from various ZIKV-positive patients (left panel). When ZKA190 LALA was added to ZIKV-serum complexes, ADE was inhibited (right panel). (D) ADE was induced in K562 cells when ZIKV was preincubated with serial dilutions of prM cross-reacting mAb (DV62) derived from a DENV-immunocompetent donor. ZKA190-LALA inhibits ADE from ZIKV when complexed with the prM-reactive antibody DV62. (E) Effect on ADE induced by peak enhanced dilution of plasma DENV2 (left panel) or anti-prM DV62 mAb (right panel) using serial dilutions of the indicated mAbs.

Fig. 17 shows for example 10 the identification of the ZKA190 epitope and analysis of its conservation in ZIKV strains. (A) Overlay of [ ¹⁵ N/ ¹ H]-HSQC spectra of ^-labeled ZIKV EDIII in the absence (black) or presence (red) of unlabeled ZKA190 Fab. The differences identify the EDIII residues involved in antibody binding. (B) NMR epitope mapping of ZKA190 Fab in complex with ZKV EDIII. Chemical shift perturbation (CSP, y-axis) was graphically represented relative to the numbers of EDIII residues. Residues involved in antibody binding are highlighted in red. (C) Residues in the FG loop identified by NMR epitope mapping are partially buried in the E protein of mol A but are largely exposed in molecules B and C. EDIII E protein is highlighted in blue. Residues identified by NMR epitope mapping are colored magenta, except for those in the FG loop, which are colored green. Adjacent E proteins are shown as a gray surface. (D) Level of conservation of amino acid residues in the ZKA190 epitope, as calculated by sequence analysis of 217 ZIKV strains found in the ZIKV Source Database (NCBI) as of November 24, 2016. (E) Open representation showing complementarity loading between epitope and paratope as a result of mooring. The epitope and paratope boundaries are outlined in green. The boundaries between Fab heavy and light chains and their respective recognition regions on EDIII are shown as yellow dashed lines.

Fig. 18 shows for example 10 the ZKA190 epitope identified by NMR and docking. (A) Schematic representation of the 12 lowest energy NMR structures of ZIKV EDIII, with residues involved in ZKA190 binding highlighted in red. Flexibility is visible at the N-terminus of the structure. (B) Model of the ZKA190:EDIII complex obtained from mooring and molecular simulations, supported by NMR results. The epitope on EDIII (gray) identified by NMR is highlighted in red. The heavy and light chains of ZKA190 are indicated in dark and light green, respectively. EDIII residues that are or are not involved in antibody binding, if mutated, are shown as orange and blue sticks, respectively. (C) NMR identified ZKA190 epitope (red) accessible on the virus surface (white).

Fig. 19 shows for Example 10 the binding of wt or mutated EDIII to ZKA190 IgG. SPR data and binding kinetics are presented. Shown are EDIII mutants that are involved (in red) or not involved in binding as shown in the figure.

Fig. 20 shows for example 11 the results of confocal microscopy experiments. ZIKV was incubated at 10 thousand times the IC50 value of either ZKA190 Fab or full-length IgG spiked into Vero cells. The ZIKV:antibody complex was found intracellularly (green) and colocalized with endosomes (red, yellow overlap). Endosomes and acidic organelles are indicated by Lysotracker in red; Alexa-488 conjugated ZKA190 is represented in green. Nuclei were stained with DAPI (blue).

Fig. 21 shows for Example 12 the prophylactic and therapeutic efficacy of ZKA190. (A) ZKA190 is effectively protective against ZIKV infection when administered prophylactically to mice (A129 in (A) and AG129 in (B)) when exposed to a lethal dose of ZIKV strain MP17451. In ex

- 68 046175 experiments used N=4-8 mice per group. Kaplan-Meier survival curves are shown (A). Significance was determined using the Mantel-Cox log-rank test. Panel A, top left: ZKA190 at 5, 1 and 0.2 mg/kg rel. Control mAb, P = 0.0031; ZKA190 at a dose of 0.04 mg/kg rel. Control mAb, P = 0.0116; ZKA190-LALA at a dose of 5, 1, 0.2 and 0.04 mg/kg rel. Control mAb, P = 0.0031. Panel A, top right: Morbidity assessment of mice observed for 14-15 days (two different assessment methods were used; see Dowall, S.D., Graham, V.A., Rayner, E., Atkinson, B., Hall, G., Watson, R. J., Bosworth, A., Bonney, L. C., Kitchen, S., and Hewson, R. (2016). A Susceptible Mouse Model for Zika Virus Infection. PLoS Negl Trop Dis 10, e0004658-13). bottom panels: body weight of mice Panel B: ZKA190 or ZKA190-LALA administered at 15 mg/kg at various time points after ZIKV infection Panel B, top left: A Kaplan-Meier survival curve shown Experiments used N=5 mice per group. Significance was determined using the Mantel-Cox log-rank test. ZKA190 and ZKA190-LALA were administered on either day 1, 2, 3, or 4 rel. Control, P = 0.0016. Panel B, top right: Incidence estimates mice were observed for 14 days according to (Dowall et al., 2016).Mice were observed for a period of 14 days to determine body weight loss (Panel B, bottom panels). The control antibody was MPE8, specific for RSV F protein (Corti, D., et al. Cross-neutralization of four paramyxoviruses by a human monoclonal antibody. Nature 501, 439-443 (2013)).

Fig. 22 shows for Example 12 the preventive efficacy of anti-ZIKV EDIII-specific mAb ZKA190 against ZIKV strains MP1741. (A) Viremia measured as PFU/mL on day 5 in the blood of all animals is shown. (B) Viral load was measured as genomic copies/ml by qPCR on day 5 in the blood of all animals and in the blood and designated tissues if animals were discarded at the end of the study or if humane endpoints were met. (C) Mice were observed over a 14-day period to determine body weight loss (D) IgG concentrations in human serum on day 5 blood samples. Significance was determined in comparison with a control antibody using the nonparametric unpaired Mann-Whitney-Wilcoxon test. *p < 0.05; **p < 0.01; ***p<0.001.

Fig. 23 shows for Example 12 the therapeutic efficacy of the anti-ZIKV EDIII-specific mAb ZKA190. (A) Viral loads were measured as PFU on day 5 in the blood of all animals. (B) Viral loads were measured as genomic copies by qPCR on day 5 in the blood of all animals and in the blood and designated tissues if animals were discarded at the end of the study or if humane endpoints were met. Significance was determined in comparison with a control antibody using the nonparametric unpaired Mann-Whitney-Wilcoxon test. *p < 0.05; **p < 0.01. (C) IgG concentration in human serum on day 5 blood samples.

Examples

The following examples present illustrative embodiments of the present invention. The following examples are presented for the purpose of illustration only and to assist one skilled in the art in using the invention. The examples are not intended to limit the scope of the invention in any other way.

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) from 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 were then immortalized with EBV (Epstein-Barr virus) and CpG (CpG oligodeoxynucleotide 2006) in multiple parallel wells as previously described (Traggiai, E. et al., Nat. Med. 10 , 871-875, 2004) and then the culture supernatants were tested in a primary screen using a parallel 384-well microneutralization assay and binding assay (ELISA) to test their binding to the ZIKV NS1 protein or to the ZIKV E protein. The results of the binding assay are presented in FIG. 1 (binding to ZIKV E protein) and FIG. 2 (binding to ZIKV NS1 protein).

Neutralization assays were performed on Vero cells. In a 384-well plate, ZIKV H/PF/2013, which resulted in an infection (moi, multiplicity of infection) of 0.35, was incubated with supernatants for 1 hour at 37% (5% CO ₂ ) before adding pre-plated 5 thousand. Vero cells. They were incubated for an additional 5 days, after which the supernatant was removed and WST-1 reagent (Roche) was added. Positive cultures were collected and propagated. From positive cultures, VH and VL sequences were obtained by RT-PCR. Antibodies were cloned into human IgG1 and Ig kappa or Ig lambda expression vectors (provided by Michel Nussenzweig, Rockefeller University, New York, US) essentially as described (Tiller T, Meffre E, Yurasov S, Tsuiji M, Nussenzweig MC, Wardemann H (2008) Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J Immunol Methods 329: 112-124). Monoclonal antibodies were generated 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 affinity purified with

- 69 046175 using Protein A or Protein G chromatography (GE Healthcare) and desalted against PBS.

In fig. 1 provides an overview of selected ZIKV neutralizing antibodies (see Tables 1 and 2 for their CDR amino acid sequences and heavy/light chain variable regions). The last two columns in Fig. 1 describes the neutralization activities (IC ₅₀ ) for ZIKV and DENV1 (if determined). Other columns provide binding activities (EC ₅₀ ) of antibodies to ZIKV E protein (ZIKV E), DENV1 E protein (DENV1 E), DENV2 E protein (DENV2 E), DENV3 E protein (DENV3 E), DENV4 E protein (DENV4 E) , DENV1 virus-like particle (DENV1 VLP), DENV2 virus-like particle (DENV2 VLP), DENV3 virus-like particle (DENV3 VLP), DENV4 virus-like particle (DENV4 VLP), and to the EDIII domain of the ZIKV E protein (DIII ZKA).

Additional antibodies were isolated to determine their ability to bind to the ZIKV NS1 protein (see Fig. 2). Positive cultures were collected and propagated. From positive cultures, VH and VL sequences were obtained by RT-PCR. Antibodies were cloned into human IgG1 and Ig kappa or Ig lambda expression vectors (provided by Michel Nussenzweig, Rockefeller University, New York, US) essentially as described (Tiller T, Meffre E, Yurasov S, Tsuiji M, Nussenzweig MC, Wardemann H (2008) Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J Immunol Methods 329: 112-124). Monoclonal antibodies were generated 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 affinity purified by Protein A or Protein G chromatography (GE Healthcare) and desalted against PBS.

In fig. 2 provides an overview of selected antibodies that bind the ZIKV #1 protein (see Tables 4 and 5 for their CDR amino acid sequences and heavy/light chain variable regions). In particular, in FIG. Figure 2 shows the binding activity (EC ₅₀ ) of antibodies to the NS1 protein of ZIKV (ZIKV NS1), NS1 protein of DENV1 (DENV1 NS1), NS1 protein of DENV2 (DENV2 NS1), NS1 protein of DENV3 (DENV3 NS1), NS1 protein of DENV4 (DENV4 NS1), NS1 protein of yellow fever virus (YFV NS1), S1 protein of West Nile virus N (WNV NS1), NS1 protein of Japanese encephalitis virus (JEV NS1), and NS1 protein of tick-borne encephalitis virus (TBEV NS1).

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

In Example 1, a large number of ZIKV neutralizing antibodies were identified and characterized with respect to their specificity for ZIKV, particularly ZIKV E protein and ZIKV EDIII, as well as their cross-reactivity with DENV. Subsequently, antibodies ZKA190 (SEQ ID NO: 1-18), ZKA185 (SEQ ID NO: 19-36), ZKA230 (SEQ ID NO: 37-54), ZKA64 (SEQ ID NO: 73-90) and ZKA 78 ( SEQ ID NO: 55-72) described in Example 1 were selected and further tested against the E protein of ZIKV (ZIKV), ZIKV EDIII (DIIIZI) and also tested against the E protein of dengue virus (DENV, serotype number 1 ) using ELISA. For this purpose, a standard ELISA was used. Briefly, ELISA plates were coated with ZIKV E protein at 1 or 3 μg/ml, blocked with 10% FCS in PBS, incubated with human serum or antibodies, and washed. Bound antibodies were detected by incubation with AP-conjugated goat anti-human IgG (Southern Biotech). After this, the plates were washed, the substrate (p-NPP, Sigma) was added and the plates were analyzed at 405 nm. The relative binding affinities of the monoclonal antibody were determined by measuring the antibody concentration (EC ₅₀ ) required to achieve 50% 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 (DIII ZI) with low EC ₅₀ values, thus indicating that ZKA64 and ZKA190 bind to domain III of the ZIKV E protein (EDIII). ZKA78 binds to ZIKV E but not to ZIKV EDIII, indicating that ZKA78 binds to ZIKV E but is not targeted to the EDIII region. Despite their significant ZIKV neutralizing activity (see Fig. 1), antibodies ZKA185 and ZKA230 did not show any detectable binding to ZIKV E and ZIKV EDIII (Fig. 4). Thus, ZKA185 and ZKA230 are designated as neutralizing but non-E protein binding (NNB) antibodies. Those NNB antibodies presumably recognize quaternary epitopes that are displayed on ZIKV infectious virions, but not on soluble proteins.

In addition, none of ZKA190, ZKA185, ZKA230, and ZKA64 exhibited any detectable binding to DENV E proteins (Fig. 1, DENV1-4 serotypes, and Figs. 3 and 4), indicating that ZKA190, ZKA185, ZKA230, and ZKA64 are specific to ZIKV and do not cross-react with dengue virus. In contrast, ZKA78, which is predicted to bind to ZIKV EDI/II but not ZIKV E DIII (see Fig. 3), binds to DENV proteins (Figs. 1 and 3), indicating that ZKA78 is cross-reactive antibody that binds to both ZIKV and DENV.

To further confirm these results, ZIKV E protein-binding antibodies ZKA190, ZKA64, and ZKA78 were further tested against the E protein of dengue virus (DENV, serotype numbers 1-4). ZKA64 and ZKA190 do not bind to the E protein of DENV1-4, thus confirming that ZKA64 and ZKA190 are specific to ZIKV. In contrast, ZKA78 binds to DENV1-4 E, confirming that ZKA78 is a cross-reactive antibody that binds to the E protein of both ZIKV and DENV (see Fig. 1).

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Example 3. Characteristics of ZIKV NSl-specific antibodies for serological diagnostics.

In Example 1, a large number of #1-reactive antibodies were identified and then characterized with respect to their specificity for ZIKV NS1 and cross-reactivity with other flavivirus NS1 proteins (Fig. 2). Subsequently, antibodies ZKA15 (SEQ ID NO: 91-108), ZKA25 (SEQ ID NO: 109 - 126) and ZKA35 (SEQ ID NO: 127-144) were further characterized with respect to their binding to ZIKV NS1 and DENV1 NS1, DENV2 NS1, DENV3 NS1 and DENV4 NS1. For this purpose, a standard ELISA was used. Briefly, ELISA plates were coated with 1 μg/ml ZIKV NS1 protein, blocked with 10% FCS in PBS, incubated with human serum or antibodies, and washed. Bound antibodies were detected by incubation with AP-conjugated goat anti-human IgG (Southern Biotech). After this, the plates were washed, substrate (p-NPP, Sigma) was added and the plates were analyzed at 405 nm. The relative binding affinities of the monoclonal antibody were determined by measuring the antibody concentration (EC ₅₀ ) required to achieve 50% maximum binding at saturation.

The results are presented in Fig. 5. All three antibodies (ZKA15, ZKA25 and ZKA35) bind with high affinity to ZIKV NS1, but not to DENV1-4 NS1 antigens (Fig. 5).

To further investigate antibody binding to ZIKV NS1, bilayer interferometric competition assays were used. Created a cross-competitive array using bilayer interferometry (BLI; Octet) on 13 antibodies specific for ZIKV NS1 (i.e., not cross-reactive with DENV NS1), namely antibodies ZKA24, ZKA15, ZKA32, ZKA19, ZKA50, ZKA37, ZKA46, ZKA10 , ZKA48, ZKA35, ZKA25, ZKA44, and ZKA30 (see Fig. 6A). As can be seen in FIG. 2, none of these 13 antibodies exhibited detectable binding to DENV NS1.

Competition assays and determination of antigenic determinants were performed at 37°C using the Octet RED96 system, ForteBio. ZIKV-NS1 protein, diluted to 2.5 μg/ml in PBS, was immobilized for 7-9 min on the surface of an APS-coated sensor chip. Coated biosensors were placed in wells containing blocking buffer (0.1% BSA in PBS) for 6 min to block unoccupied Biosensor binding sites. The Biosensors coating was then incubated for 8 min with a set of single purified ZIKV-NS1-specific mAbs diluted in blocking buffer at 10 μg/ml. After binding the first batch of mAbs (step 1), Biosensors were moved into wells containing different mAbs for 8 min (step 2). Association of the second mAb resulted in recognition of a different antigenic determinant compared to the first mAb (eg, non-competitive). Competition or partial competition was determined at step 2, where no association or minor association was found, respectively. A cross-competition matrix was generated by multiple competition runs to predict antigenic determinant mapping on ZIKV NS1.

The results are presented in Fig. 6A and 6C. First, all of the ZIKV NS1-specific antibodies tested bound to the S1 and/or S2 antigenic determinant(s) (Fig. 6A). However, some of the antibodies did not compete with others. For example, ZKA15 did not compete for binding with ZKA25 and ZKA35 and vice versa (Fig. 6A). Thus, the ZKA15 antibody was assigned the S1 antigenic determinant, while the ZKA25 and ZKA35 antibodies were assigned the S2 antigenic determinants (Fig. 6C). Finally, based on the antibodies used, antigenic determinants (S1 and S2) on ZIKV NS1 were identified (Fig. 6C).

Additionally, the binding of 10 antibodies cross-reacting with the ZIKV NS1 protein and with the DENV NS1 protein (namely, ZKA18, ZKA29, ZKA39, ZKA53, ZKA54, ZKB19, ZKB23, ZKC29, ZKC33, and ZKC34; Fig. 6B) to antigenic determinants was examined. S1 and/or S2 on ZIKV NS1. As can be seen in FIG. 2, all of these 10 antibodies exhibit binding to DENV NS1. These 10 cross-reacting antibodies were tested in a cross-competition assay as described above (for ZIKV NS1-specific antibodies) against the ZIKV NS1 S1-specific antibody ZKA15 and against the ZIKVNS1 S2-specific antibody ZKA35.

The results are presented in Fig. 6B. Of interest, none of the ten cross-reacting antibodies tested competed with ZKA 15 and/or ZKA35 for binding to the S1 and/or S2 antigenic determinant(s) on ZIKV NS1 (Fig. 6B). These results suggest that the antigenic determinant of ZKA15 and ZKA35 is not targeted by NS1 cross-reacting antibodies. Thus, the NS1 antigenic determinants S1 and S2 are targeted by ZIKV-specific, but not cross-reacting, antibodies.

Example 4. Use of ZIKV No. 1-specific antibodies for the diagnosis of ZIKV infection.

In this example, the utility of the ZIKV No. 1-specific antibodies of the present invention for the diagnosis of ZIKV infection was examined. More specifically, the use of ZIKV No. 1-specific antibodies according to the present invention to specifically detect the presence or absence of antibodies produced to ZIKV NS1 in plasma samples of ZIKV- or DENV-infected donors was determined.

For this purpose, a binding blockade assay was used. Specifically, the ability of ZIKV #1-reactive antibodies in plasma to inhibit the binding of biotinylated ZKA35 antibody to ZIKV NS1 was determined. For this purpose, ZIKV No. 1-specific antibody ZKA35 was biotinylated,

- 71 046175 using EZ-Link NHS-PEO solid phase biotinylation kit (Pierce). Tagged ZKA35 was tested for binding to ZIKV NS1 and the optimal concentration of ZKA35 to achieve 70% maximum binding was determined. Plasma samples from ZIKV- (n=4), DENV-immune (n=5) and control (n=48) donors (1/10 dilution) were added to ZIKV NS1-coated ELISA plates. After 1 h, biotinylated anti-ZIKV NS1 antibody ZKA35 was added at a concentration providing 70% maximum binding, and the mixture was incubated at room temperature for 15 minutes. The plates were washed, substrate (p-NPP, Sigma) was added and the plates were analyzed at 405 nm. The percentage inhibition value was calculated as follows: (1-[(OD of sample - OD of negative counter)/(OD of positive counter - OD of negative counter)])x 100.

The results are presented in Fig. 7. It should be noted that binding of the ZKA35 antibody to the S2 antigenic determinant on NS1 was inhibited only by plasma samples from ZIKV-immunocompetent donors, but not by DENV-immunocompetent donors, and this binding was also not inhibited by 48 control plasma samples (Fig. 7). Therefore, this study can be used to specifically detect clinical and subclinical ZIKV infections at the population level.

Example 5 Antibodies in accordance with the present invention effectively neutralize ZIKV infection.

The isolated antibodies ZKA190, ZKA185, ZKA230, ZKA64 and ZKA78 were tested for their ability to neutralize ZIKV and DENV1 infection in vitro.

Neutralization of DENV and ZIKV infection by antibodies was measured using a flow cytometry microneutralization assay. Various antibody dilutions were mixed with ZIKV (MOI 0.35) or attenuated DENV1 (all at MOI 0.04) for 1 h at 37°C and added to 5000 Vero cells/well in 96-well flat-bottom plates. After four days for ZIKV and five days for DENV, cells were fixed with 2% formaldehyde, permeabilized in PBS 1% FCS 0.5% saponin, and stained with mouse mAb 4G2. Cells were incubated with Alexa Fluor488-conjugated goat anti-mouse IgG (Jackson Immuno-Research, 115485164) and analyzed by flow cytometry. In other cases, ZIKV neutralization data were also determined by measuring cell viability using WST-1 reagent (Roche). The neutralization titer (50% inhibitory concentration [IC ₅₀ ]) was expressed as the concentration of antibody that reduced infection by 50% compared to cell-only control wells.

The results are presented in Fig. 8, 9 and 13. EDIII-specific mAbs ZKA64 and ZKA190 and NNB mAb ZKA230 are extremely effective in neutralizing ZIKV (strain H/PF/2013), with IC ₅₀ values of 93, 9 and 10 ng/ml, respectively (Fig. 8, top panel). In contrast, the cross-reactive antibody ZKA78 only partially neutralized ZIKV infectivity and cross-neutralized DENV1 infectivity (Fig. 8, bottom panels). Similar data were obtained by modifying the ZIKV-induced cytopathic effect determined using the WST-1 reagent (Fig. 9). In this second assay, the NNB antibody ZKA185 was also included in the panel of antibodies tested and exhibited an IC ₅₀ similar to the top performing antibodies ZKA190 (EDIII-specific) and ZKA230 (NNB).

It is important to note that the highly effective ZKA64 and ZKA190 antibodies, in addition to their ability to neutralize the ZIKV H/PH/2013 strain (this example), also bind to the E protein and EDIII derived from ZIKV strains MR766 and SPH2015, respectively (Fig. 1 and Fig. 3). ZKA190 and ZKA190-LALA were also confirmed to effectively neutralize two additional ZIKV strains (MR766 and MRS_OPY_Martinique_PaRi_2015) (Fig. 13). Taken together, the results indicate that the extremely effective ZKA64 and ZKA190 antibodies cross-react with multiple ZIKV strains of different genotypes and origins (East Africa and Asia from Uganda, French Polynesia, Martinique and Brazil).

Example 6 LALA mutation inhibits antibody-dependent enhancement of ZIKV infection by serum antibodies.

Neutralizing antibodies were also tested to determine their ability to enhance ZIKV infection in nonpermissive K562 cells (antibody-dependent enhancement assay, ADE assay). ADE was measured by flow-through assay 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 was determined by flow cytometry.

The results are presented in Fig. 10. All antibodies enhanced ZIKV infection in nonpermissive K562 cells over a wide range of concentrations, including those that completely neutralized ZIKV infection on Vero cells (Fig. 10). It should be noted that while the EDIII-specific antibodies ZKA64 and ZKA190 completely neutralize ZIKV infection of K562 cells above 1 μg/ml, the NNB antibody ZKA230 fails to do so, a result that may be due to different mechanisms of neutralization of free viruses compared to Fc -gamma receptor internalized viruses. In contrast, cross-reacting ZKA78, which only partially neutralizes ZIKV infectivity, effectively enhances ZIKV infection of K562 cells. These results suggest that cross-reacting antibodies caused by either

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ZIKV or DENV infection may mediate heterologous ADE.

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

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

The results are presented in Fig. 11. In homologous settings, four ZIKV-immune plasmas collected from convalescent patients and one DENV-immune plasma showed a similar ability to enhance ZIKV infection of K562 cells (Fig. 11, top panel), and this ADE effect was completely blocked by the EDIn-specific ZKA64- LALA antibody (Fig. 11, bottom panel).

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

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

Example 7: Analysis of samples from European residents using ZIKV #1-specific antibodies to diagnose ZIKV infection.

This Example is based on the binding blockade assay described in Example 4. To further evaluate the specificity of the ZIKV NS1 VACV assay, a large number of samples obtained from patients infected with DENV, WNV, or chikungunya virus (CHIKV) were tested.

For this purpose, a binding blockade assay was used. Polystyrene plates were coated overnight with 1 μg/ml ZIKV NS1 and blocked for 1 h with PBS containing 1% BSA. Plasma or serum (1:10 dilution) was added to NSI-coated ELISA plates. Thereafter, for example after 1 hour, an equal volume of biotinylated anti-K81 ZKA35 was added and the mixture was incubated, for example, at room temperature for 15 minutes. The plates were washed and alkaline phosphatase-conjugated streptavidin was added, for example, for 30 minutes. The plates were again incubated and substrate was added. The percentage inhibition value was calculated as follows: (1-[(OD of sample-OD of negative counter)/(OD of positive counter-OD of negative counter)])x 100.

The results are presented in Fig. 14. Thirty-one of 32 samples (96.9%) from WNV patients collected more than 10 days after symptom onset were scored negative. It should be noted that only a positive assessment was obtained from a sample collected in 2016. Two of the 27 samples from DENV patients collected more than 10 days after the onset of symptoms were assessed as positive, and two positive samples were from secondary DENV infections. Additionally, none of the samples obtained from patients with chikungunya or YFV vaccines tested positive. A large number of plasma samples from Swiss blood donors (n=116) collected between 2010 and 2016 were also tested. None of these samples were rated positively. The results obtained confirm and strengthen the high sensitivity and specificity of the NS1 BOB ELISA assay.

Example 8 The antibody of the present invention neutralizes ZIKV more effectively than the prior art antibody EDE1 mAb C8.

To compare the neutralizing antibodies of the present invention with the effective neutralizing anti-ZIKV antibodies of the prior art, the neutralization performance of ZKA190 was compared with the same parameters for the effective neutralizing mAb of the prior art EDE1 C8 (Barba-Spaeth G, Dejnirattisai W, Rouvinski A, Vaney MC, Medits I, Sharma A, Simon-Loriere E, Sakuntabhai A, Cao-Lormeau VM, Haouz A, England P, Stiasny K, Mongkolsapaya J, Heinz FX, Screaton GR, Rey FA. Structural basis of potent Zika-denge virus antibody cross-neutralization. Nature. 2016 Aug 4;536(7614):48-53). Neutralization of both antibodies was tested against a panel of four different ZIKV strains (H/PF/2013; MR766, MRS-OPY and PV10552).

Briefly, neutralization of ZIKV infection by mAb was measured using an assay

- 73 046175 micro-neutralization based on flow cytometry. Various dilutions of mAb were mixed with ZIKV (MOI 0.35) for 1 h at 37°C and added to 5000 Vero cells/well in 96-well flat-bottom plates. After four days for ZIKV, cells were fixed with 2% formaldehyde, permeabilized in PBS containing 1% fetal bovine serum (Hyclone) and 0.5% saponin, and stained with mouse mAb 4G2. Cells were incubated with Alexa Fluor488-conjugated goat anti-mouse IgG (Jackson Immuno-Research, 115485164) and analyzed by flow cytometry. The neutralization titer (50% inhibitory concentration [IC ₅₀ ]) was expressed as the concentration of antibody that reduced infection by 50% compared to virus-only control wells.

The results are presented in Fig. 15. ZKA190 mAb effectively neutralizes African, Asian and African strains with IC ₅₀ ranging from 0.6 to 8 ng/ml. In comparison, the prior art C8 antibody is approximately 24 times less potent.

Example 9 Further characterization of the ZKA190 antibody.

The effectiveness of the ZKA190 antibody was further studied in vitro and in vivo. To do this, mAbs were synthesized in wild-type (wt) IgG1 and IgG1 Fc-LALA formats. Briefly, VH and VL sequences were cloned into human IgyL IgK and Ig/.. expression vectors (kindly provided by Michel Nussenzweig, Rockefeller University, New York, NY, USA) essentially as described (Tiller T, Meffre E, Yurasov S, Tsuiji M, Nussenzweig MC, Wardemann H: Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J Immunol Methods 2008, 329:112-124). Recombinant mAbs were prepared by transiently transfecting EXP1293 cells (Invitrogen) purified by Protein A chromatography (GE Healthcare) and desalted in PBS. To produce the LALA variant, each of the heavy chains was mutated at amino acids 4 and 5 of the CH2 domain by replacing it with alanine instead of the naturally occurring leucine using site-directed mutagenesis. As described above, LALA variants (human IgG1 antibodies) do not bind to Fc-gamma receptors and complement.

As shown in FIG. 15A and described in Example 8, ZKA190 was tested against a panel of four ZIKV strains. ZKA190 mAb effectively neutralized African, Asian and American strains with IC ₅₀ ranging from 0.004 to 0.05 nM (Fig. 15A; 0.6 to 8 ng/ml).

Because ZIKV has been shown to infect human neutral progenitor cells (hNPCs), resulting in increased cellular toxicity, cell cycle dysregulation, and reduced cell growth, ZKA190 and ZKA190-LALA were tested in hNPCs. For this purpose, adult male fibroblasts obtained from the Movement Disorders Bio-Bank (Neurogenetics Unit of the Neurological Institute 'Carlo Besta', Milan) were reprogrammed using the CytoTune-iPS 2.0 Sendai kit (Life Technologies). hiPSCs were maintained under food-free conditions in mTeSR1 (Stem Cell Technologies). To obtain embryoid bodies (EB), dissociated hiPSCs were 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 in Marchetto MCN, Carromeu C, Acab A, Yu D, Yeo GW, Mu Y, Chen G, Gage FH, Muotri AR: A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell 2010, 143:527-539). To obtain rosettes, EBs were plated 10 days later on Matrigel-coated plates (1:100, Matrigel with reduced growth factors, Corning) in DMEM/F12 (Sigma) with N2 (1:100), nonessential amino acids (1%, ThermoFisher Scientific) and penicillin/streptomycin. After 10 days, cells were passaged with Accutase (Sigma) and seeded into Matrigel-coated 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 (3x104) were seeded on coverslips in 24-well plates 3 days before infection with the PRVABC59 strain. Virus stock solution was incubated with mAb for 1 h before adding to hNPC to obtain an MOI of 0.5. After virus absorption for 4 h, the cell supernatant was removed and fresh medium containing mAb was re-added. The supernatant was collected 96 h postinfection to measure virus titers by plaque assay on Vero cells. Cells were fixed in 4% paraformaldehyde (PFA, Sigma) in phosphate-buffered saline (PBS, Euroclone) for 30 min for indirect immunofluorescence. Fixed cells were permeabilized for 30 min (minutes) 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 was used for detection: anti-envelope (1:200, Millipore, MAB10216). Thereafter, cells were washed with PBS and incubated for 1 h with Hoechst and anti-mouse Alexa Fluor-488 secondary antibodies (1:1,000 in blocking solution, ThermoFisher Scientific). After washing with PBS, the cells were washed and strengthened again.

The results are presented in Fig. 16A. Both antibodies, ZKA190 and ZKA190-LALA, completely abolished ZIKV infection and replication in hNPCs.

Next, the ability of ZKA190 and ZKA190-LALA to induce ADE in the K562 cell line was tested as described in Example 6. Briefly, ADE was measured by flow assay using K562 cells.

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Briefly, for ZKA190, ZKA190 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 mAb. The number of infected cells was determined by flow cytometry. For ZKA190-LALA, ZIKV (MOI 0.175) was mixed with plasma from primary ZIKV-infected donors for 30 min at 37°C. ZKA190-LALA was added at a concentration of 50 μg/ml, 5000 K562 cells/well were mixed and incubated for three days. Cells were then stained with 4G2 and analyzed by flow cytometry. The results are presented in Fig. 16V. ZKA190 supports ADE from 0.0001-1 nM; as predicted, ZKA190-LALA does not exhibit any ADE activity. The ability of ZKA190-LALA to inhibit ADE induced by plasma from four ZIKV-immunocompetent donors in K562 cells was also tested. The results are presented in Fig. 16C. It was found that ZKA190-LALA completely inhibited ADE induced by antibodies in plasma (Fig. 16C).

Anti-prM antibodies constitute part of the predominant antibodies produced during the human immune response to flaviviruses and have been shown to enhance viral infection in vitro (Dejnirattisai, W., Jumnainsong, A., Onsirisakul, N., Fitton, P., Vasanawathana, S., Limpitikul, W., Puttikhunt, C., Edwards, C., Duangchinda, T., Supasa, S., et al (2010). Cross-reacting antibodies enhance dengue virus infection in humans. Science 328, 745 -748). K562 cells were preincubated with serial dilutions of the prM cross-reactive antibody DV62 (Beltmamello, M., Williams, K.L., Simmons, S.R., Macagno, A., Simonelli, L., Quyen, N.T.H., Sukupolvi-Petty, S., Navarro -Sanchez, E., Young, P.R., de Silva, A.M., et al. (2010). The human immune response to dengue virus is dominated by highly cross-reactive antibodies endowed with neutralizing and enhancing activity. Cell Host Microbe 8, 271 -283), originating from a DENV immunocompetent donor. The results are presented in Fig. 16D. DV62 cross-reacts with the ZIKV prM protein and causes ADE over a wide range of concentrations (Fig. 16D). ZKA190LALA could completely block the anti-prM DV62 mAb-induced ADE of immature or partially immature ZIKV particles (Fig. 16D).

Finally, the ability of varying concentrations of ZKA190, ZKA190-LALA, and ZKA190 Fab to induce or block ADE for ZIKV was tested in the presence of increasing concentrations of antiDENV2 in human plasma or DV62. The results are presented in Fig. 16E. ZKA190 at low concentrations increases prM DV62 ADE-mediated 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 plasma DENV and mAb DV62. ZKA190-LALA, as well as its Fab fragment, reduces ADE at concentrations above 0.06 nM, indicating that both inhibited viral infections are at a post-attachment stage, such as fusion.

Example 10 ZKA190 binds to the conserved and highly accessible region of EDIII.

To determine the ZKA190 epitope at the residue level, resolution NMR spectroscopy was used as described in Bardelli, M., Livoti, E., Simonelli, L., Pedotti, M., Moraes, A., Valente, A.P., and Varani, L. (2015). Epitope mapping by solution NMR spectroscopy. J. Mol. Recognize 28, 393-400; Simonelli, L., Beltramello, M., Yudina, Z., Macagno, A., Calzolai, L., and Varani, L. (2010). Rapid structural characterization of human antibody-antigen complexes through experimentally validated computational docking. J Mol Biol 396, 1491-1507; and Simonelli, L., Pedotti, M., Beltramello, M., Livoti, E., Calzolai, L., Sallusto, F., Lanzavecchia, A., and Varani, L. (2013). Rational Engineering of a Human Anti-Dengue Antibody through Experimentally Validated Computational Docking. PLoS ONE 8, e55561.

Briefly, spectra were recorded on a Bruker Avance 700 MHz NMR spectrometer at 300 K. To establish framework resonances in standard triple resonance experiments (HNCO, HN(CA)CO, HN(CO)CACB, HNCACB were used, with side chains annotated using HCCHTOCSY and HBHA(CO)NH experiments. All NMR experiments were performed using Topspin 2.1 (Bruker Biospin) and analyzed using CARA. NOESY cross peaks were automatically determined using CYANA noeassign macro, based on manually determined chemical shifts. Upper distance limiters used for structural calculations on CYANA using a standard artificial annealing protocol were obtained from 70 ms ¹⁵ N- and ¹³ C-resolved NOESY spectra. Frame speakers for ZIKV EDIII were obtained from N relaxation measurements recorded on 600 and 700 MHz spectrometers. Proton detection versions were used CPMG (R2), inversion-recovery (R1) and ¹⁵ N { ¹ H}-stationary NOE. The delay parameters for T2 series were in the range from 0 to 0.25 s and for T1 series in the range from 0.02 to 2 s . In ^{the 15} N{1H}-NOE experiment, a relaxation delay of 5 s was used. R1 and R2 relaxation rates were obtained from a least squares fit of the corresponding exponential functions to the measured data using in-house written scripts. Relaxation data were analyzed in a model-free approach using DYNAMICS software. The ROTDIF program was used to calculate the total correlation time from the relaxation data (8.5 ns). NMR epitope mapping was performed as previously described (Bardelli et al., 2015; Simonelli et al., 2010; 2013). Briefly, overlap of ¹⁵ NHSQC spectra of EDIII-labeled free or bound ZKA190 Fabs provided the opportunity

- 75 046175 identification of EDIII residues whose NMR signals changed upon complex formation, indicating that they are involved in ZKA190 binding. Changes were identified by manual observation and by chemical shift perturbation (CSP), CSP=((Δδ _H ) ² +(Δδ _N /10) ² ) ^1/2 . NMR samples typically included 800 μM [ ^15S , ^13C ]-labeled EDIII in 20 mM sodium phosphate, 50 mM NaCl, pH 6.0. Perdeuterated (nominally 70%) ² H, ¹⁵ N EDIII samples were used for NMR epitope mapping with EDIII:ZKA190 Fab ratio 1:1.1; The EDIII concentration was typically 0.4 mM.

Because the NMR signal is highly dependent on the local chemical environment, changes in complex formation identify antigen residues that are involved in antibody binding, either directly or through allosteric effects. By comparing the NMR spectra of free and bound EDIII (Figure 17A), the residues involved on ZKA190 are mapped to the LR in EDIII, specifically the BC, DE and FG loops, and as part of the EDI-EDIII hinge (Figure 18A) . These residues are virtually identical among the 217 known ZIKV strains, with the exception of substitutions at V341I and E393D in the Uganda 1947 isolate (Fig. 17D). These mutations are also present in the MR766 strain, which are effectively neutralized by ZKA190 (Fig. 15A). Analysis of the ZKA190 epitope on the uncomplexed ZIKV structure showed that the epitope is readily accessible except for the FG loop at the 5-pleated apex (FIGS. 18B and 17C, molecule A).

Computational docking followed by molecular dynamics simulations, guided and informed by epitope information from NMR data as well as EDIII mutagenesis, revealed that ZKA190 binds across the interface by shape and charge complementarity (Figures 18B and 17E). The docking indicates that there are no direct contacts between ZKA190 and the FG loop on EDIII, suggesting that changes in its NMR signals upon antibody binding are due to allosteric effects. This view is supported by the fact that mutation of the FG loop residues in recombinant EDIII, but not in other epitope regions, has no effect on the binding affinity of ZKA190 for EDIII (Fig. 18B and 19).

Example 11. ZKA190 neutralization mechanisms.

The ability of ZKA190 to effectively neutralize the virus may involve inhibition of either cell attachment or membrane synthesis. A further mechanism may involve virus inactivation by cross-linking of viral particles.

ZKA190 Fab 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 rotation required for viral fusion with the host cell membrane (Bressanelli, S., Stiasny, K., Allison, S.L., Stura, E.A. , Duquerroy, S., Lescar, J., Heinz, F. X., and Rey, F. A. (2004). Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation. Embo J 23, 728-738; Modis, Y., Ogata, S., Clements, D., and Harrison, S. C. (2004). Structure of the dengue virus envelope protein after membrane fusion. Nature 427, 313-319). Alternatively, ZKA190 may prevent ZIKV from attaching to target cells.

The ability of ZKA190 to inhibit membrane synthesis was confirmed by confocal microscopy analysis. For this purpose, Vero cells were seeded at 7500 cells/well onto 12 mm diameter 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 minutes at room temperature. Acidified endosomes were identified using Lysotracker red (Invitrogen) by adding dye (50 nM) to the cells during the last 30 min of incubation before fixation. After fixation, extensive washes were carried out in PBS and 50 mM glycine, and finally coverslips were prepared for microscopic analysis using Vectashield Fluorescence Histology Medium with DAPI (Vector Laboratories). Samples were analyzed by confocal microscopy using a Leica TCS SP5 microscope with a 63x/1.4 N.A. lens. Image analysis and processing were performed using FIJI software.

The results are presented in Fig. 20. Analysis by confocal microscopy showed that ZKA190 (Fab or IgG) can enter Vero cells only when complexed with ZIKV, at neutralizing concentrations exceeding the IC ₅₀ by 10 thousand times (Fig. 20).

Example 12: In vivo characterization of EDIII-specific mAb ZKA190.

To evaluate their preventive and therapeutic properties, ZKA190 and ZKA190-LALA were tested in A129 mice treated with a lethal dose of ZIKV strain MP1751 (African strain).

To test their prophylactic efficacies, ZKA190 and ZKA190-LALA were administered one day before viral challenge.

Female A129 (IFN-alpha/beta receptor -/-) and wild-type 129Sv/Ev mice at 58 weeks of age were treated with mAbs (ZKA190, ZKA190-LALA and control antibody MPE8 (Corti, D., et al. Crossneutralization of four paramyxoviruses by a human monoclonal antibody. Nature 501, 439-443 (2013)), diluted in PBS in various tissues by intraperitoneal route (i.p.) in a volume of 500 μl. MAb was administered either

- 76 046175 1 day or 1, 2, 3 or 4 days after viral challenge. Animals were challenged subcutaneously with 102 PFU of ZIKV (strain MP1751) and then challenged for 14 days. Body weight and temperature were monitored daily and clinical observations were recorded at least twice daily. On day 5 after challenge, 50 μl of blood was collected from each animal into RNA-protected tubes (Qiagen, UK) and frozen at -80°C. At the end of the study (14 days postchallenge) or when animals reached humane endpoints, organs were sampled after necropsy, and blood and sections from the brain, spleen, liver, kidneys, and ovaries were collected for virological analyses.

Tissue samples from A129 mice were weighed and homogenized in PBS using ceramic beads and an automated homogenizer (Precellys, UK) using six 5 s cycles at 6500 rpm with a 30 s rest period. Two hundred ml of tissue homogenate or blood solution was transferred into 600 μl of RLT buffer (Qiagen, UK) for RNA extraction using the RNeasy Mini extraction kit (Qiagen, UK); samples were passed through QIAshredder (Qiagen, UK) as in the initial stage. A ZIKV specific real-time RT-PCR assay was used to detect viral RNA from animal subjects. Primer and probe sequences were adapted from Quick et al., 2017 (Quick, J, Grubaugh ND, Pullan ST, Claro IM, Smith AD, Gangavarapu K, Oliveira G, Robles-Sikisaka R, Rogers TF, Beutler NA, et al: Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nat Protoc 2017, 12:1261-1276) with internal optimization and validation carried out to ensure optimal master mix and cyclization conditions. Real-time RT-PCR was performed using a Superscript III Platinum One-step qRT-PCR kit (Life Technologies, UK). The final master mix (total reaction mixture) (15 µl) contained 10 µl of 2x reaction mixture, 1.2 µl of PCR grade water, 0.2 µl of 50 mM MgSO ₄ , 1 µl of each primer (ZIKV 1086 and ZIKV 1162c both at 18 μM working concentration), 0.8 μl of probe (ZIKV 1107-FAM at 25 μM working concentration) and 0.8 μl of SSIII enzyme mixture. Five μl of messenger RNA was added to the master mix, resulting in a final reaction volume of 20 μl. The cyclization conditions used were 50°C for 10 min, 95°C for 2 min, followed by 45 cycles of 95°C for 10 s and 60°C for 40 s, plus a final cooling step of 40°C for 30 With. Quantitative analysis using fluorescence was performed after the end of each 60°C stage. Reactions were run and analyzed on a 7500 Fast platform (Life Technologies, UK) using 7500 software version 2.0.6. Quantification of viral load in samples was performed using serial dilutions of a quantitative RNA oligonucleotide (Integrated DNA Technologies). The oligonucleotide contained the 77 bases of ZIKV RNA targeted for this assay based on GenBank accession number AY632535.2 and was synthesized at 250 nmol with HPLC purification.

The results are presented in Fig. 21, 22 and 23. ZKA190 and ZKA190-LALA showed protection against mortality and morbidity in mice at concentrations of 5, 1 or 0.2 mg/kg (Fig. 21A-B). ZKA190-LALA, and to a lesser extent ZKA190, slowed morbidity and mortality compared to the control group at a dose of 0.04 mg/kg. Viral titers in the blood and organs were significantly reduced compared to treated animals receiving control antibody, even in the presence of serum antibody levels below 1 μg/ml (Fig. 22A-D).

To evaluate the therapeutic potential of ZKA190, we administered ZKA190 and ZKA190-LALA at different time points after ZIKV infection. At a dose of 15 mg/kg, survival rates of 80-100% were achieved, and morbidity was significantly reduced even with treatment four days after infection (Fig. 21E-G). 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 notable trend toward greater reductions with early treatment (FIGS. 23A-21C). Of note, ZKA190-LALA showed a significant reduction in antiviral activity in the day 5 blood sample compared to ZKA190 when the mAb was administered four days postinfection, a result that may be related to the impaired ability of the LALA variant to facilitate rapid clearance of enveloped virions.

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Tables of sequences and SEQ ID numbers

ZKA190 SEQ ID NO. Amino acid sequence CDRH1 1 GFTFSKYG CDRH2 2 ISYEGSNK CDRH3 3 AKSGTQYYDTTGYEYRGLEYFGY CDRL1 4 QSVSSSY CDRL2 5 DAS CDRL2 long 6 LIYDASSRA CDRL3 7 QQYGRSRWT VH 8 QVQLVESGGGVVQPGRSLRLSCAASGTFFSKYGMHWVRQAPGKGLEWVAV ISYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSG TQYYDTTGYEYRGLEYFGYWGQGTLVTVSS VL 9 EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQQKRGQAPRLLIY DASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGRSRWTFG QGTKVEIK

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CDRHL 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 gcagactccgtgaagggccgattcaccatctcca gagacaattcc aagaacacgctgtatctgcaaatgaacagcctgagagctgaggac acggcagtgtattactgtgcgaaatcggggacccaatactatgat actactggttatgagtataggggtttggaatactttggctactgg ggccagggaaccctggtcaccgtctcctcag VL 18 gaaattgtgttgacgcagtctccaggcaccctgtctttgtctcca ggggaaagagccaccctctcctgcagggccagtcagagtgttagt agcagttacttagcctggtaccagcagaaacgtggccaggctccc aggctcctcatctatgatgcatccagcagggccactggcatccca gacaggttcagtggcagtgggtctgg gacagacttcactctcacc atcagcagactggagcctgaagattttgcagtgtattactgtcag cagtatggtaggtcaaggtggacattcggccaagggaccaaggtg gaaatcaaac

ZKA185 SEQ ID NO. Amino acid sequence CDRHL 19 GYSFTSYW CDRH2 20 FDPSDSQT CDRH3 21 ARRYCSSSSCYVDN CDRL1 22 ALPNKF

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CDRL2 23 EDN CDRL2 long 24 VIYEDNKRP CDRL3 25 YSTDSSSNPLGV VH 26 EVQLVQSGAEVKKPGESLRISCKGSGYSFTSYWITWVRQMPGKGLEWMAK FDPSDSQTNYSPSFQGHVTISVDKSISTAYLQWSSLKASDTAMYYCARRY CSSSSCYVDNWGQGTLVTIFS VL 27 SYELTQPPSVSVSPGQTARITCSGDALPNKFAYWYRQKSGQAPVLVIYED NKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYHCYSTDSSSNPLGVF GGGTKLTVL

ZKA185 SEQ ID NO. Nucleic acid sequence CDRH1 28 ggatatagttttaccagttactgg CDRH2 29 tttgatcctagtgactctcaaacc CDRH3 thirty gcgagaagatattgtagtagtagttgttatgtggacaat CDRL1 31 gcattgccaaataaattt CDRL2 32 gaggacaac CDRL2 length Y 33 gtcatctatgaggacaacaaacgaccc CDRL3 34 tactcaacagacagcagttctaatcccctgggagta VH 35 gaagtgcagctggtgcagtccggagcagaggtgaaaaagcccgggg agtctctgaggatctcctgtaagggttctggatatagttttaccag ttactggatcacctgggtgcgccagatgcccgggaaaggcctggag tggatggcgaagtttgatcctagtgactctctcaaaccaactacagcc cgtccttccaaggccacg tcaccatctcagttgacaagtccatcag cactgcctacttgcagtggagcagcctgaaggcctcggacaccgcc atgtattactgtgcgagaagatattgtagtagtagttgttatg tggacaattggggccagggaaccctggtcaccatcttctcag VL 36 tcctatgagctgacacagccaccctcggtgtcagtgtccccaggac aaacggccaggatcacctgctctggagatgcattgccaaataaatt tgcttattggtaccggcagaagtcaggccaggcccctgttctggtc atctatgaggacaacaaacgaccctccgggatccctgagagattct ctggctccagctcagggacaatggccaccttgact atcagtggggc ccaggtggaggatgaagctgactaccactgttactcaacagacagc agttctaatcccctgggagtattcggcggagggaccaagctgaccg

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tcctag

ZKA230 SEQ ID NO. Amino acid sequence 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 QVQLQESGPGLVKPSETLSLTCAVSGGSISSDYWSWIRQPPGKGLEWIGY IYYSGSTNYNPSLKSRVTISVDTSKNHFSLKLNSVTAADTAVYYCARRRK YDSLWGSFAFDIWGQGTMVTVS S VL 45 QSVLTQPPSASGTPGQRVTISCSGSSSNIGGNYVYWYQQLPGTAPKLLIC INDHRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCATWDDSLGGLV FGGGTKLTVL

ZKA230 SEQ ID NO. Nucleic acid sequence CDRH1 46 ggtggctccatcagtagtgactac CDRH2 47 atctattacagtgggagcacc CDRH3 48 gcgaggagggaggaagtatgattccctttgggggagttttgcttttg atatc CDRL1 49 agctccaacatcggaggtaattat CDRL2 50 attaatgat CDRL2 length Y 51 ctcatctgtattaatgatcaccggccc

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CDRL3 52 gcaacatgggatgacagcctgggtggccttgta VH 53 caggtgcagctgcaggagtcgggcccaggcctggtgaagccttcgg agaccctgtccctcacctgcgcagtctctggtggctccatcagtag tgactactggagctggatccggcagcccccagggaagggactggag tggattgggtatatctattacagtgggagcaccaactacaacccct ccctcaagagtcgagtcaccatatcagtagacac gtccaagaacca cttctccctgaagctgaactctgtgaccgctgcggacacggccgtg tattactgtgcgaggagggaagtatgattccctttgggggagtt ttgcttttgatatctggggccaagggacaatggtcaccgtctcttc ag VL 54 cagtctgtgctgactcagccaccctcagcgtctgggacccccgggc agaggtcaccatctcttgttctggaagcagctccaacatcggagg taattatgtatactggtaccagcagctcccaggaacggcccccaaa ctcctcatctgtattaatgatcaccggccctcaggggtccctgacc gattctctggctccaagtctggcacctca gcctccctggccatcag tgggctccagtccgaggatgaggctgattattactgtgcaacatgg gatgacagcctgggtggccttgtattcggcggagggaccaagctga ccgtcctag

ZKA7 8 SEQ ID NO. Amino acid sequence 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 PRTFGQGTKVEIK

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ZKA7 8 SEQ ID NO. Nucleic acid sequence 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 actgatagtgtgaagggccggttcaccatca gcagagacaatagc aaatccatggtgtacctgcagatgagctccctgcgaaccgaagac acagcagtgtactattgcgtgaaagatctggccatccccgagtcc tacagaattgaagctgattattggggacagggcaccctggtcatc gtgagcgccg VL 72 gacatcgtgatgacacagtctccagatagtctggcagtcagtctg ggggagagggccactattaactgcaagagctcccagtccgtgctg taccgctctaacaacaagaattacctgtcttggtatcagcagaag cccggacagccccctaaactgctgatctattgggcttcaacccgg gaaagcggcgtcccagacagattctcaggca gcgggtccggaaca gacttcaccctgacaattagccccctgcaggcagaggacgtggct gtctactattgtcagcagtactattctagtcctcgaactttcggc caggggaccaaggtggaaatcaaac

ZKA6 4 SEQ ID NO. Amino acid sequence CDRH1 73 GYTFTGYH CDRH2 74 INPNSGGT CDRH3 75 ARMSSIWGFDH

- 83 046175

CDRLl 76 QSVLIN CDRL2 77 G.A.S. CDRL2 long 78 LIYGASSRA CDRL3 79 QQYNDWPPIT VH 80 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYHIDWVRQARGQGLEWMGRI NPNSGGTNYAQKFQGRVTMTRDTSISTAYMQLSRLRSDDSAVYYCARMSSS IWGFDHWGQGTLVTVSS VL 81 EIVMTQSPATLSVSPGERATLLSCRASQSVLINLAWYQQKPGQAPRLLIYGA SSRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNDWPPITFGQG TRLEIK

ZKA6 4 SEQ ID NO. Nucleic acid sequence CDRHL 82 ggctacaccttcacagggtatcac CDRH2 83 attaaccctaattctggcgggacc CDRH3 84 gctcggatgagctcctctatttggggcttcgatcat CDRLl 85 cagtctgtgctgattaac CDRL2 86 ggagcatcc CDRL2 length Y 87 ctgatctatggagcatcctccagggct CDRL3 88 cagcagtacaatgattggccccctatcaca VH 89 caggtgcagctggtccagagcggagcagaggtgaagaaacccggcg cctcagtgaaggtcagctgcaaagcttccggctacaccttcacagg gtatcacatcgactgggtgaggcaggcaagaggacagggactggaa tggatgggacggattaaccctaattctctggcgggaccaactacgccc agaagtttcagggccgagtgactatgaccagaga caccagcatctc cacagcttatatgcagctgtcccggctgagatctgacgatagtgcc gtctactattgtgctcggatgagctcctctatttggggcttcgatc attgggggcagggaacactggtgactgtcagttcag VL 90 gagatcgtgatgactcagtctccagccaccctgtcagtcagcccag gagaacgggcaaccctgtcttgcagagcctcccagtctgtgctgat taacctggcttggtaccagcagaagccaggccaggcaccccgactg ctgatctatggagcatcctccagggctaccggcattcctgcacgct tcagtggatcaggaagcggaacagag tttaccctgacaatctctag tctgcagtccgaagacttcgctgtctactattgtcagcagtacaat gattggccccctatcacatttggccaggggactagactggagatca

- 84 046175

age

ZKA15 SEQ ID NO. Amino acid sequence CDRH1 91 GGFINSYY CDRH2 92 IYKSGST CDRH3 93 ARDPYGDYVKAFDI CDRL1 94 QSLLHSNGYNY CDRL2 95 LGS CDRL2 length Y 96 LIYLGSNRA CDRL3 97 MQALQTVT VH 98 QVQLQESGPGLVKPSETLSLTCTVSGGFINSYYWSWIRQPAGKGLE WIGRIYKSGSTNYNPSLKSRVTMSLDTSKYQFSLKLRSVTAADTAV YYCARDPYGDYVKAFDIWGQGTMVTVSS VL 99 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLNWYLQKPG QSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMQALQTVTFGPGTKVDIK

ZKA15 SEQ ID NO. Nucleic acid sequence CDRH1 100 ggtggcttcatcaatagttactac CDRH2 101 atctataaaagtgggagcacc CDRH3 102 gcgagagatccctacggtgactacgttaaggcttttgatatt CDRL1 103 cagagcctcctgcatagtaatggatacaactat CDRL2 104 ttggggttct CDRL2 length Y 105 etgatctatttgggttctaategggee CDRL3 106 atgcaagctctacaaactgtcact

- 85 046175

VH 107 caggtgcagctgcaggagtcggggccaggactggtgaagccttcgg agaccctgtccctcacctgcactgtctccggtggcttcatcaatag ttactactggagctggatccggcagcccgccgggaagggactggag tggattgggcgtatctataaaagtgggagcaccaactacaacccct ccctcaagagtcgagtcaccatgtcactagaca cgtccaagtacca gttctccctgaagctgaggtctgtgaccgccgctgacacggccgtg tattactgtgcgagagatccctacggtgactacgttaaggcttttg atatttggggccaagggacaatggtcaccgtctcttcag VL 108 gatattgtgatgactcagtctccactctccctgcccgtcacccctg gagagccggcctccatctcctgcaggtctagtcagagcctcctgca tagtaatggatacaactatttgaattggtacctgcagaagccaggg cagtctccacagctcctgatctatttgggttctaatcgggcctccg gggtccctgacaggttcagtggcagt ggatcaggcacagattttac actgaaaatcagcagagtggaggctgaggatgttggggtttattac tgcatgcaagctctacaaactgtcactttcggccctgggaccaaag tggatatcaaac

ZKA25 SEQ ID NO. Amino acid sequence CDRH1 109 GFTFRSHW CDRH2 110 IKEDGYEK CDRH3 111 ARDLRVYSGRGFDP CDRL1 112 KLGDKY CDRL2 113 QDS CDRL2 length Y 114 VIYQDSKRP CDRL3 115 QAWDSSTW VH 116 EVQLVESGGGLVRPGGSLRLSCAASGFTFRSHWMSWVRQAPGKGLE WVANIKEDGYEKYYVDSVKGRFTISRDNAKNSLYLQMKSLRAEDTA VYYCARDLRVYSGRGFDPWGQGTLVTVSS VL 117 SYELTQPPSLSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPVLV IYQDSKRPSGIPARESGSNSGNTATLTISGTQAMDEADYYCQAWDS STWFGGGTKLTVL

ZKA25 SEQ ID Nucleic acid sequence NO.

- 86 046175

CDRHL 118 ggattcacctttagaagtcattgg CDRH2 119 aaaaggaagatggatatgagaaa CDRH3 120 gcgagagatttgaggtatatagtgggagaggtttcgacccc CDRL1 121 aaattgggggataaatat CDRL2 122 caagatagc CDRL2 long 123 gtcatctatcaagatagcaagcggccc CDRL3 124 caggcgtgggacagcagcactgtggta VH 125 gaggtgcagttggtggagtctgggggaggcttggtccggcctgggg ggtccctgagactctcctgtgcagcctctggattcacctttagaag tcattggatgagttgggtccgccaggctccaggaaggggctggag tgggtggccaacataaaggaagatggatatgagaaatactatgtgg actctgtgaagggccgattcaccatctccag agacaacgccaagaa ctcactgtatctgcaaatgaagagcctgagagccgaggacacggcc gtgtattactgtgcgagagatttgagggtatatagtgggagaggtt tcgacccctggggccagggaaccctggtcaccgtctcctcag VL 126 tcctatgagctgactcagccaccctcactgtccgtgtccccaggac agacagccagcatcacctgctctggagataaattgggggataaata tgcttgctggtatcagcagaagccaggccagtcccctgtgttggtc atctatcaagatagcaagcggccctcagggatccctgcgcgattct ctggctccaactctgggaacacagccactctgaccat cagcgggac ccaggctatggatgaggctgactattactgtcaggcgtgggacagc agcactgtggtattcggtggagggaccaagctgaccgtcctag

ZKA35 SEQ ID NO. Amino acid sequence CDRHL 127 GGSISTGGYY CDRH2 128 IYYSGNT CDRH3 129 AKGGGRERPFDY CDRL1 130 SSNIGRNY CDRL2 131 RNN CDRL2 length Y 132 LIYRNNQRP CDRL3 133 VAWDDSRSGFW

- 87 046175

VH 134 QVQLQESGPGLVKPSQTLSLTCTVSGGSISTGGYYWSWIRQHPGKG LEWIGYIYYSGNTYYNPSLKSRVTISVDTSKKQFSLKLSSVTAADT AVYYCAKGGGRERPFDYWGQGTLVTVSS VL 135 QSVLTQPPSASGTPGQRVTISCSGSSSNIGRNYVDWYQQLPGTAPK LLIYRNNQRPSGVPERFSGSKSGTSASLAISGLRSEDEADYYCVAW DDSRSGFWFGGGTKVTVL

ZKA35 SEQ ID NO. Nucleic acid sequence CDRH1 136 ggtggctccatcagcactggtggttactac CDRH2 137 atctattacagtgggaacacc CDRH3 138 gcgaaaggaggagggagggagcgaccctttgactac CDRL1 139 agctccaacatcggaagaaattat CDRL2 140 aggaataat CDRL2 length Y 141 ctcatctataggaataatcagcggccc CDRL3 142 gtagcatgggatgacagccggagtggttttgtggta VH 143 caggtgcagctgcaggagtcgggcccaggactggtgaagccttcac agaccctgtccctcacctgcactgtctctggtggctccatcagcac tggtggttactactggagctggatccgccagcacccagggaagggc ctggagtggattggttacatctattacagtgggaacacctactaca acccgtccctcaagagtcgagttaccatatca gttgacacctctaa gaagcagttctccctgaagctgagctctgtgactgccgcggacacg gccgtgtattactgtgcgaaaggaggagggagggagcgaccctttg actactggggccagggaaccctggtcaccgtctcctcag VL 144 cagtctgtgctgactcagccaccctcagcgtctgggacccccggggc agaggtcaccatctcttgttctggaagcagctccaacatcggaag aaattatgtagactggtaccagcaactcccaggaacggcccccaaa ctcctcatetataggaataatcagcggccctcaggggtccctgage gattctctggctccaagtctggcacctcagcct ccctggccatcag tgggctccggtccgaggatgaggctgattattactgtgtagcatgg gatgacagccggagtggttttgtggtattcggcggagggaccaagg tgaccgtcctag

Constant SEQ ID Subsequence

- 88 046175

plots NO. IgGl CH1-CH2- SNZ ak 145 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgGl CH1-CH2- SNZ LALA ak. 146 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgG SC ac 147 RTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC IgG CL ac 148 GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRS YSCQVTHEGSTVEKTVAPTECS IgGl CH1-CH2- SNZ nucle 149 gcgtcgaccaagggcccatcggtcttccccctggcaccctcc tccaagagcacctctgggggcacagcggccctgggctgcctg gtcaaggactacttccccgaacctgtgacggtctcgtggaac tcaggcgccctgaccagcggcgtgcacaccttcccggctgtc ctacagtcctcaggactctactccctcagcagcgt ggtgacc gtgccctccagcagcttgggcacccagacctacatctgcaac gtgaatcacaagcccagcaacaccaaggtggacaagagagtt gagcccaaatcttgtgacaaaactcacacatgcccaccgtgc ccagcacctgaactcctggggggaccgtcagtcttcctcttc cccccaaaacccaaggacaccctcatgatctcccggacc cct gaggtcacatgcgtggtggtggacgtgagccacgaAgaCcct gaggtcaagttcaactggtacgtggacggcgtggaggtgcat aatgccaagacaaagccgcgggaggagcagtacaacagcacg taccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg ctgaatggcaagggagtacaagtgcaagg tctccaacaaagcc ctcccagcccccatcgagaaaaccatctccaaagccaaaggg cagccccgagaaccacaggtgtacaccctgcccccatcccgg

- 89 046175

gaggagatgaccaagaaccaggtcagcctgacctgcctggtc aaaggcttctatcccagcgacatcgccgtggagtggggagagc aatgggcagccggagaacaactacaagaccacgcctcccgtg ctggactccgacggctccttcttcctctatagcaagctcacc gtggacaagagcaggtggcagcaggggaacgtcttct catgc tccgtgatgcatgaggctctgcacaaccactacacgcagaag agcctctccctgtccccgggtaaa IgGl CH1-CH2- CH3 LALA nucle 150 gcgtcgaccaagggcccatcggtcttccccctggcaccctcc tccaagagcacctctgggggcacagcggccctgggctgcctg gtcaaggactacttccccgaacctgtgacggtctcgtggaac tcaggcgccctgaccagcggcgtgcacaccttcccggctgtc ctacagtcctcaggactctactccctcagcagcgt ggtgacc gtgccctccagcagcttgggcacccagacctacatctgcaac gtgaatcacaagcccagcaacaccaaggtggacaagagagtt gagcccaaatcttgtgacaaaactcacacatgcccaccgtgc ccagcacctgaaGCCGCGgggggaccgtcagtcttcctcttc cccccaaaacccaaggacaccctcatgatctcccgg acccct gaggtcacatgcgtggtggtggacgtgagccacgaagaccct gaggtcaagttcaactggtacgtggacggcgtggaggtgcat aatgccaagacaaagccgcgggaggagcagtacaacagcacg taccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg ctgaatggcaagggagtacaagtgcaaggt ctccaacaaagcc ctcccagcccccatcgagaaaaccatctccaaagccaaaggg cagccccgagaaccacaggtgtacaccctgcccccatcccgg gaggagatgaccaagaaccaggtcagcctgacctgcctggtc aaaggcttctatcccagcgacatcgccgtggagtggggagagc aatgggcagccggagaacaactacaagaccacgcct cccgtg ctggactccgacggctccttcttcctctatagcaagctcacc gtggacaagagcaggtggcagcaggggaacgtcttctcatgc tccgtgatgcatgaggctctgcacaaccactacacgcagaag agcctctccctgtccccgggtaaa IgG SC nucle 151 cgTacGgtggctgcaccatctgtcttcatcttcccgccatct gatgagcagttgaaatctggaactgcctctgttgtgtgcctg ctgaataacttctatcccagagaggccaaagtacagtggaag gtggataacgccctccaatcgggtaactcccaggagagtgtc acagagcaggacagcaaggacagcacctacagcctca gcagc accctgacgctgagcaaagcagactacgagaaacacaaagtc tacgcctgcgaagtcacccatcagggcctgagctcgcccgtc acaaagagcttcaacaggggagagtgt IgG CL nucle 152 ggtcagcccaaggctgccccctcggtcactctgttcccgccc tcctctgaggagcttcaagccaacaaggccacactggtgtgt

- 90 046175

ctcataagtgacttctacccgggagccgtgacagtggcttgg aaagcagatagcagccccgtcaaggcgggagtggagaccacc acaccctccaaacaaagcaacaacaagtacgcggccagcagc tatctgagcctgacgcctgagcagtggaagtcccacagaagc tacagctgccaggtcacgcatgaagggagcaccgtggaga ag acagtggcccctacagaatgttca

ZKA10 SEQ ID NO. Amino acid sequence CDRH1 153 GFTFSDSY CDRH2 154 ISSSSPFT CDRH3 155 ARGLVRDGYKWLYFFDY VH 156 QVQLVESGGGLVEPRGSLRLSCAASGFTFSDSYMSWIRQAPGKGLE WISYISSSSPFTNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTA VYYCARGLVRDGYKWLYFFDYWGQGTLVTVSS ZKA18 SEQ ID NO. Amino acid sequence CDRH1 157 GFTFSSYG CDRH2 158 IWYDGSNK CDRH3 159 ARDDSGYSEPFDY VH 160 QVQLVESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLE WVAVIWYDGSNKYYADSVKGRFTITRDNSKNTLYLQMNSLRPEDTA VYYCARDDSGYSEPFDYWGQGTLVTVSS ZKA2 8 SEQ ID NO. Amino acid sequence CDRH1 161 GFTVSRNY CDRH2 162 IYSGGST CDRH3 163 ARWINDAFDI VH 164 EVQLVESGGGLIQPGGSLRLSCAASGFTVSRNYMSWVRQAPGKGLE WVSVIYSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV YYCARWINDAFDIWGQGTMVTVS S ZKA2 9 SEQ ID NO. Amino acid sequence CDRH1 165 GFTFSRYS CDRH2 166 ISPRSTTI CDRH3 167 AREDCNGVCYRVDY VH 168 EVQLVESGGGLVQPGGSLRLSCVVSGFTFSRYSMNWVRQAPGKGLE WVSYISPRSTTIYYADSVEGRFTVSRDNAKNSLYLQLNSLRAEDTA VYYCAREDCTNGVCYRVDYWGQGTLVTVSS

- 91 046175

ZKA33 SEQ ID NO. Amino acid sequence CDRH1 169 GFTFSRNW CDRH2 170 IKEDGNEK CDRH3 171 ARPFHQGGYAYGLAY VH 172 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNWMTWVRQAPGKGLE WVANIKEDGNEKYYVD SVKGRFTIS RDNAKN S LYLQMN S LRAEDTA VYYCARPFHQGGYAYGLAYWGQGTLVTVSS ZKA3 9 SEQ ID NO. Amino acid sequence CDRH1 173 GFTFSTYS CDRH2 174 ISPSSSTI CDRH3 175 AREYCSGGSCYLLDY VH 176 EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYSMNWVRQAPGKGLE WVSYISPSSSTIYYPDSLKGRFTISRDNAKNSLYLQMDSLRAEDTA QYYCAREYCSGGSCYLLDYWGQGTLVTVSS ZKA43 SEQ ID NO. Amino acid sequence CDRH1 177 GGSITSYY CDRH2 178 SHYSGST CDRH3 179 ARGIYSGKNWFDP VH 180 QVQLQESGPGLVKPSETLSLTCTVYGGSITSYYWTWIRQPPGKGLE WIGYSHYSGSTNYNPSLKSRVTISIDTSKSQFSLNLNSVTAADTAV YYCARGIYSGKNWFDPWGQGTLVTVS S ZKA44 SEQ ID NO. Amino acid sequence CDRH1 181 GFTVSTSY CDRH2 182 IYSSGST CDRH3 183 ARVSLGGLDP VH 184 EVQLVESGGGLIQPGGSLRLSCVASGFTVSTSYMNWVRQAPGKGLE WVSVIYSSGSTYYADSVKGRFTISRNTSKNTLYLQMNSLRAEDTAV YYCARVSLGGLDPWGQGTPVTVSS ZKA46 SEQ ID NO. Amino acid sequence CDRH1 185 GFSLSNGRMG CDRH2 186 IFSNDEK CDRH3 187 ARVE FRAGNY LD S VH 188 QVTLKESGPVLVKPTETLTLTCTVSGFSLSNGRMGVSWIRQPPGKA LEWLAHIFSNDEKYYSTSLKNRLTISKDTSKSQVVLTMTNMDPVDT

- 92 046175

ATYYCARVEFRAGNYLDSWGQGTLVTVSS ZKA50 SEQ ID NO. Amino acid sequence CDRH1 189 GYTFTNSW CDRH2 190 IYPGDSDT CDRH3 191 ARQPFFDY VH 192 EVQLVQSGAQVKKPGESLKISCKASGYTFTNSWIGWVRQMPGKGLE WMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTA MYYCARQPFFDYWGQGTLVTVSS ZKA5 4 SEQ ID NO. Amino acid sequence CDRH1 193 GYTFTGYY CDRH2 194 INANSGGT CDRH3 195 AHSDIVWPSDDYYALDV VH 196 QVQLVQSGAEVKKPGASVKVSCKTSGYTFTGYYMHWVRQAPGQGLE WMGWINANSGGTNFAQRFQGRVTMTWDT SISTAYMELS RLRS DDTA VYYCAHSDIVWPSDDYYALDVWGQGTTVTVS S ZKB18 SEQ ID NO. Amino acid sequence CDRH1 197 GYSFTSYW CDRH2 198 IYPGDSDT CDRH3 199 ARQTPGDY VH 200 EVQLVQSGAEVKKPGESLKISCKTFGYSFTSYWIGWVRQMPGKGLE WMGMIYPGDSDTRYSPSFQGQVTISADMSISTAYLQWSSLKASDTA MYYCARQTPGDYWGQGTLVTVSS ZKB2 0 SEQ ID NO. Amino acid sequence CDRH1 201 GYFFTRYV CDRH2 202 INTDNGST CDRH3 203 ARGTGRDGYNSFFAN VH 204 QVQLVQSGAEVKKPGASVRVSCKASGYFFTRYVILWVRQAPGQRPE WMGWINTDNGSTRYSQKFQGRVTITKDTSATTAYMDLSSLKSDDTA VYYCARGTGRDGYNSFFANWGQGTLVTVSP ZKB21 SEQ ID NO. Amino acid sequence CDRH1 205 GYTFTGYS CDRH2 206 IDTNSGDT CDRH3 207 ARDRERHPFSY VH 208 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYSIHWVRQAPGQGLA WMGRIDTNSGDTNYAERFQGRVTMTRDT SISTAYMEVRRLRS DDTA

- 93 046175

VYYCARDRERHPFSYWGQGTLVTVSS ZKB2 3 SEQ ID NO. Amino acid sequence CDRHL 209 GGSISSGDYS CDRH2 210 ITHSGTT CDRH3 211 ARHFGWFDP VH 212 QLQLQESGSGLVKPSQTLSLTCAVSGGSISSGDYSWSWIRQPPGKG LEWIGYITHSGTTYFNPSLKSRVTISVDRSRNQFSLKVTSVTAADT AVYYCARHFGWFDPWGQGTLVTVSS ZKC2 9 SEQ ID NO. Amino acid sequence CDRHL 213 GGSISSGEYF CDRH2 214 IHNRGNT CDRH3 215 ARGGGDLVWPDSIWDYYGMDV VH 216 QVQLQESGPGLVRPSQTLSLTCTVSGGSISSGEYFWTWIRQHPKKG LEWIGYIHNRGNTYYNPSLKSRLSISLDTSKNHLSLRLSSVTAADT AV Y Y C ARGGGD L VWPD SIWD Y Y GMD VW GQGTTVTVSS ZKC31 SEQ ID NO. Amino acid sequence CDRHL 217 GGSISSGGYH CDRH2 218 IYYSGST CDRH3 219 ARDRSEPGEYHYYYYAMDV VH 220 QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYHWSWIRQHPGKG LEWIGYIYYSGSTYYNPSLKRRVTISVDTSKNQFSLKLSSVSAADT AVYYCARDRSEPGEYHYYYYAMDVWGQGTTVTVSS ZKC32 SEQ ID NO. Amino acid sequence CDRHL 221 GFTVSSNY CDRH2 222 IYSSGST CDRH3 223 ARGKKGNAFDI VH 224 EVQLVESGGDLIQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKGLE WVSVIYSSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAGDTAV Y Y CARGKKGNAFDIW GQ GTVVTVS S ZKC33 SEQ ID NO. Amino acid sequence CDRHL 225 GDSISSRTFS CDRH2 226 IYYSGST CDRH3 227 ARRNAE F F S FWS Y Y GMDV VH 228 QVQLQESGPGLVKPSQTLSLTCTVSGDSISSRTFSWSWIRQPPGKG

- 94 046175

LEWVGHIYYSGSTDYNPSLKSRISISIDTSKNQFSLKLSSVTAADT AVYYCARRNAEFFSFWSYYGMDVWGHGTAVIVSS ZKC34 SEQ ID NO. Amino acid sequence CDRH1 229 GGSINSGGYY CDRH2 230 ILHSGNT CDRH3 231 ARAGDYYSGYVPPEY VH 232 QVQLQESGPGLVKPSQTLSLTCAVSGGSINSGGYYWSWVRQHPGKG LEWIGYILHSGNTNYNPSLKSRVNIFVDTSENQFSLKLRSVTAADT AIYFCARAGDYYSGYVPPEYWGPGTLVTVSS ZKD2 5 SEQ ID NO. Amino acid sequence CDRH1 233 GFTVSSNY CDRH2 234 IYSGGST CDRH3 235 ARFGGNPSFDY VH 236 EVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKGLE WVSVIYSGGSTYYANSVKGRFTISRDKSKNTLYLQMNNLRAEDTAV YFCARFGGNPSFDYWGQGTLVTVSS

ZKA3 SEQ ID NO. Amino acid sequence CDRH1 237 GFIFSNYA CDRH2 238 IGGKGDSI CDRH3 239 VKDLAVLESDRLEVDQ VH 240 EVQLAESGGGLVQPGGSLRLSCSGSGFIFSNYAMVWARQAPGKGLE YVSGIGGKGDSIYHIDSVKGRFTISRDNSKRTVYLQMSRLRTEDTA VYYCVKDLAVLESDRLEVDQWGQGTLVIVSA ZKA4 SEQ ID NO. Amino acid sequence CDRH1 241 GFTFSSYV CDRH2 242 TSYDGSNK CDRH3 243 ARGPVPYWS GE S Y S GAY FD F VH 244 QVQLVESGGGVVQPGRSLRLSCAASGFFSSYVMHWVRQAPGKGLE WVTVTSYDGSNKYYADSVKGRFTISRDNAKNTLYLQMNSLRGEDTA IYYCARGPVPYWSGESYSGAYFDFWGQGILVTVS S ZKA5 SEQ ID NO. Amino acid sequence CDRH1 245 GFTFSNYY CDRH2 246 MSSSETIK

- 95 046175

CDRH3 247 ARSGIETVAGSIDYYGMDV VH 248 QVQLVESGGGLVKPGGSLRLSCAGSGTFFSNYYMTWIRQAPGKGLE LVSYMSSSETIKYYADSVKGRFTISRDNAKNSLYLQMNSLRADDTA RYYCARSGIETVAGSIDYYGMDVWGHGTPVTVSS ZKA6 SEQ ID NO. Amino acid sequence CDRH1 249 DFTVSNYA CDRH2 250 VSYDGSNK CDRH3 251 ATGVTMFQGAQTNAEYLHY VH 252 QVHLVESGGGVVQPGRSLRLSCEASDFTVSNYAMHWVRQAPGKGLE WVAWSYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA LYYCATGVTMFQGAQTNAEYLHYWGQGS LVTIS S ZKA7 SEQ ID NO. Amino acid sequence CDRH1 253 GFTFSRYG CDRH2 254 VSGDGSST CDRH3 255 VKDFWSGDQSLESDF VH 256 EVQLVESGGGLVQPGGSLRLSCSASGTFFSRYGMVWARQAPGKGLE YLSGVSGDGSSTYYANSVKGRFTISRDNSKNTLYLHMSRLRDEDTA MYYCVKDFWSGDQSLESDFWGQGALVTVS S ZKA8 SEQ ID NO. Amino acid sequence CDRH1 257 GFTFSAHA CDRH2 258 ISNEDYT CDRH3 259 VKDFGTSPQTDF VH 260 DERLVESGGGLVQPGGSLRLVCSASGFTFSAHAMHWVRQPPGKGLE YVSTISRNEDYTYYADSVKGRFTISRDNSKNSLYLQMRRLRPEDTA IYYCVKDFGTSPQTDFWGQGTLVAVSS ZKA7 6 SEQ ID NO. Amino acid sequence CDRH1 261 GFTFSTYF CDRH2 262 ISSTGSYK CDRH3 263 ARPFHSEYTYGLDAFDI VH 264 EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYFMHWVRQAPGKGLE WVASISSTGSYKFYADSVKGRFTISRDNTKNSLFLQMNSLRAEDTA VFYCARPFHSEYTYGLDAFDIWGQGTMLTVSS ZKA117 SEQ ID NO. Amino acid sequence CDRH1 265 GGSIRTNSY CDRH2 266 ISYSGST

- 96 046175

CDRH3 267 ARLNDGSTVTTSSYFDY VH 268 QLQLQESGPGLVKPSETLSLTCTVSGGSIRRTNSYWGWIRQTTGKG LQWIGSISYSGSTFYNPSLKSRVTISLDTSKDHFSLELSSVTAADT AIYYCARLNDGSTVTTSSYFDYWGQGTLVTVSS ZKB2 7 SEQ ID NO. Amino acid sequence CDRH1 269 GYSFTSSW CDRH2 270 IDPSDSYT CDRH3 271 ARHDYSVSENGMDV VH 272 EVQLVQSGAEVKKPGESLRISCKASGYSFTSSWINWVRQMPGKGLE WMGRIDPSDSYTTYNPSFQGHVTISVDKSIGTAYLQWNSLRASDTA MYYCARHDYSVSENGMDVWGQGTTVTVS S ZKB2 9 SEQ ID NO. Amino acid sequence CDRH1 273 GFTFSSYT CDRH2 274 ISYDGSHK CDRH3 275 ARRSYSISCFDY VH 276 QVQLVESGGGVVQPGRSLRLSCAASGFFSSYTMHWVRQAPGKGLE WVAVISYDGSHKFYADSVKGRFTISRDNSKDTLYLQMNSLRAEDTA LYYCARRSYSISCFDYWGQGTLVTISS ZKB3 4 SEQ ID NO. Amino acid sequence CDRH1 277 GFTFSRSG CDRH2 278 VSYDGSNK CDRH3 279 AKDLTMVRGVHYYYYVMDV VH 280 QVQLVESGGGVVQPGRSLRLSCAASGFTFSRSGMHWVRQAPGKGLE WVAWSYDGSNKYYSDSVKGRFTISRDNSKNTLYLQMNSLRVEDTA VYYCAKDLTMVRGVHYYYYVMDVWGQGTTVTVSS ZKB3 9 SEQ ID NO. Amino acid sequence CDRH1 281 GYTFDDYY CDRH2 282 INPHRGGT CDRH3 283 VRDQYCDGGNCYGIHQPHYGMDV VH 284 QVQLVQSGAEVKKPGASLKVSCKASGYTFDDYYIHWVRQAPGQGLE WLGRINPHRGGTNYAQKFQGRVIMTLDMSISTTYMELRRITSDDAA VYYCVRDQYCDGGNCYGIHQPHYGMDVWGQGTTVTVSS ZKB4 6 SEQ ID NO. Amino acid sequence CDRH1 285 GYSFTSYW CDRH2 286 IDPSDSYT

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CDRH3 287 ARRE Y S S S S GQEDWFD P VH 288 EVQLVQSGAEVKKPGESLRISCKGSGYSFTSYWISWVRQMPGKGLE WMGRIDPSDSYTNYSPSFQGHVTISADKSISTAYLQWSSLKASDTA MYYCARREY S S S S GQEDWFDPWGQ GT LVTVS S ZKB53 SEQ ID NO. Amino acid sequence CDRH1 289 GFTFSSYA CDRH2 290 ISYDGSNR CDRH3 291 ARHVEQLPSSGYFQH VH 292 QVQLVESGGGVVQPGRSLRLSCAASGFFSSYAMHWVRQTPGKGLE WVTVISYDGSNRYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTA VYYCARHVEQLPSSGYFQHWGQGTLVTVSS ZKC2 6 SEQ ID NO. Amino acid sequence CDRH1 293 GFIFSDFY CDRH2 294 IGHDGSYI CDRH3 295 ARAHGGFRH VH 296 QVQVVESGGGLVKPGGSLRLSCAASGFIFSDFYMSWMRQAPGKGLE WVAYIGHDGSYILYADSVKGRFTISRDNAKNSLFLRMNSLRVEDTA VYYCARAHGGFRHWGQGTVVAVS P ZKD 5 SEQ ID NO. Amino acid sequence CDRH1 297 GFTFTSYG CDRH2 298 ISYDGSNK CDRH3 299 ARDRDHYDLWNAYTFDY VH 300 QVQLVESGGGVVQPGRSLRLSCAASGFTFTSYGMHWVRQTPGKGLD WVAVISYDGSNKYYADSVKGRFTISRDNSKDTLYLQMNSLRAADTA LYYCARDRDHYDLWNAYTFDYWGQGTLVTVSS ZKD 7 SEQ ID NO. Amino acid sequence CDRH1 301 GFTFSNYA CDRH2 302 ISYDVSDK CDRH3 303 AGGPLGVWIKPSNAEHFHH VH 304 QVQLVESGGGVVQPGRSLRLSCAASGTFFSNYAMHWVRQAPGKGLE WVAVISYDVSDKYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTA AYYCAGGPLGVWIKPSNAEHFHHWGQGTLVTVSS ZKD 8 SEQ ID NO. Amino acid sequence CDRH1 305 GFTFINYA CDRH2 306 ISYDGSNK

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CDRH3 307 ATDADAY GD S GANFHY VH 308 QVQLVESGGGVVQPGKSLRLSCAASGFTFINYAIHWVRQAPGKGLE WVAVISYDGSNKFYTDSVKGRFTISRDNSKNTLYLQMNSLRADDTA VYYCATDADAYGDSGANFHYWGQGTLVTVSS ZKD15 SEQ ID NO. Amino acid sequence CDRH1 309 DASISSGGFS CDRH2 310 IYSSGDT CDRH3 311 ARAHTPTSKFYYYYAMDV VH 312 QLQLQESGSGLVKPSQTLSLTCTVSDASISSGGFSWSWIRQPLGKG LEWLGYIYSSGDTFYNPSLQGRVTMSVDIFRSQFSLKLTSVTAADT AMYYCARAHTPTSKFYYYYAMDVWGQGTTVTVSS ZKD16 SEQ ID NO. Amino acid sequence CDRH1 313 GFTFSDHF CDRH2 314 SRNKPNSYTT CDRH3 315 AKVGGCYGGDCHVENDY VH 316 EVQLVESGGDLVQPGGSLRLSCVASGFTFSDHFMDWVRQAPGKGLE WVGRSRNKPNSYTTEYAASVKGRFSISRDDSKKALYLQMNSLQTED TAVYYCAKVGGCYGGDCHVENDYWGQGTLVTVSS ZKD17 SEQ ID NO. Amino acid sequence CDRH1 317 GFIFSDYA CDRH2 318 ISYDGSSR CDRH3 319 ARGYCSSGTCFSTNAEYFHP VH 320 QVQMVESGGGVVQPGTSLRLSCATSGFIFSDYAMHWVRQAPGKGLE WVAVISYDGSSRLYADSVKGRFTVSRDNSKNTLYLQMHSLRAGDTA VYYCARGYCSSGTCFSTNAEYFHPWGQGTLATISS ZKD2 0 SEQ ID NO. Amino acid sequence CDRH1 321 GFTFSDHF CDRH2 322 SRNKPNSYTT CDRH3 323 ARVGGCNGGDCHVENDY VH 324 EVQLVESGGGLVQPGGSLRLSCVASGFTFSDHFMDWVRQAPGKGLE WVGRSRNKPNSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLQTED TAVYYCARVGGCNGGDCHVENDYWGQGTLVTVSS ZKA134 SEQ ID NO. Amino acid sequence CDRH1 325 GGTFSAYA CDRH2 326 IIPFFGTA

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CDRH3 327 ARSDIVSTTRGYHHYGMDV VH 328 QVHLVQSGAEVKKPGSSVNVSCKASGGTFSAYAISWVRQAPGQGLE WMGGIIPFFGTAYYAQKFKGRVTVTADKSTSTVYMEMTSLRSEDTA VYYCARSDIVSTTRGYHHYGMDVWGQGTTVTVSS ZKA246 SEQ ID NO. Amino acid sequence CDRH1 329 GYTFSDYY CDRH2 330 INPYSGGT CDRH3 331 ARGFTMISDREFDP VH 332 QVQLVQSGAEVKRPGASVKVSCKASGYTFSDYYMHWVRQAPGQGLE WMGRINPYSGGTNYAQKFHGRVTVTRDTSISTVYMELRGLRSDDTA VYYCARGFTMISDREFDPWGQGT LVTVS S ZKA256 SEQ ID NO. Amino acid sequence CDRH1 333 GFTFSTYW CDRH2 334 IKQDGSEK CDRH3 335 ARDPGYDDFWSGSYSGSFDI VH 336 EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYWMTWVRQAPGKGLE WVANIKQDGSEKYYVDSVKGRFTISRDNTKNSLYLQVNSLRAEDTA IYYCARDPGYDDFWSGSYSGSFDIWGQGTMVTVSS ZKB42 SEQ ID NO. Amino acid sequence CDRH1 337 GFTFNNYG CDRH2 338 ISYDGNKK CDRH3 339 VKYGERINGYSDPFDH VH 340 QVQVVESGGGVVQPGRSLRLFCAASGFTFNNYGMHWVRQAPGKGLE WVALISYDGNKKYYADSVKGRFSISRDNSKNTLYLQMNRLRSGDTA VYHCVKYGERINGYSDPFDHWGQGTLVTVSS ZKB8 5 SEQ ID NO. Amino acid sequence CDRH1 341 GYTFTTYA CDRH2 342 INTNTGNP CDRH3 343 ARVIVPYAFDI VH 344 QVQLVQSGSELKKPGASVKVSCKASGYTFTTYAMNWVRQAPGQGPE WVGWINTNTGNPTYAQGFTGRFVLSLDTSVSTAFLQISSLKAEDTA VYYCARVIVPYAFDIWGQGTMVTVS S ZKB47 SEQ ID NO. Amino acid sequence CDRH1 345 GYTFTNYY CDRH2 346 INPSGGPT

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CDRH3 347 ARDQYGGYARYGMDV VH 348 QVQLVQSGAEVKKPGASVKVSCQASGYTFTNYYMHWVRQAPGQGLE WMGIINPSGGPTSYAQKFQGRVTMTTDTSTSTVYMELSSLRSEDTA VYYCARDQYGGYARYGMDVWGQGTTVTVS S ZKC6 SEQ ID NO. Amino acid sequence CDRHL 349 GYTFTGYY CDRH2 350 INPNSGGT CDRH3 351 ARVSDWGFAFDI VH 352 QVQLVQSGTEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLE WMGRINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSGLRSDDTA VYYCARVSDWGFAFDIWGQGTMVTVSQ ZKA160 SEQ ID NO. Amino acid sequence CDRHL 353 GGSITSYS CDRH2 354 IFYSGST CDRH3 355 ARDQTMPVWVGGMDV VH 356 QVQLQESGPGLVKPSETLSLTCTVSGGSITSYSWSWIRQPPGKGLE WIGYIFYSGSTDYNPSLKSRVTISVDTSKDQFSLRLRSVTAADTAV YYCARDQTMPVWVGGMDVWGQGTTVTVSS ZKA172 SEQ ID NO. Amino acid sequence CDRHL 357 GYIFTRYW CDRH2 358 IDPSDSYT CDRH3 359 ARQE TARED GMAV VH 360 EVQLVQSGAEVKKPGKSLRISCKGSGYIFTRYWISWVRQMPGKGLE WMGRIDPSDSYTNYSPSFQGHVTISADKSISTAYLQWSSLKASDTA MYYCARQETAREDGMAVWGQGTTVTVSS ZKA174 SEQ ID NO. Amino acid sequence CDRHL 361 GGSMSNSYYH CDRH2 362 IYYSGST CDRH3 363 ARNPVFNPLTLTHDAFDI VH 364 QLQLQESGPGLVKPSETLSLTCTVSGGSMSNSYYHWGWIRQPPGKG LEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLNSVTAADT AVYYCARNPVFNPLTLTHDAFDIWGQGTMVTVSS ZKA189 SEQ ID NO. Amino acid sequence CDRHL 365 GFTFSSYA CDRH2 366 ISGSGDNT

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CDRH3 367 AKWPYYDFWSGSESYFDP VH 368 GVQLLESGGALVQPGKSLRLSCAASGFFSSYALTWVRQAPGKGLQ WVSAISGSGDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCAKWPYYDFWSGSESYFDPWGQGTLVTVSS ZKA195 SEQ ID NO. Amino acid sequence CDRH1 369 GYNFPSYW CDRH2 370 IDPSDSYT CDRH3 371 ARADCRSTSCYLVFE VH 372 EVQLVQSGAEVKKPGESLRISCKDSGYNFPSYWIHWVRQMPGKGLE WMGTIDPSDSYTNYSPSFQGHVTISADKSISTAYLQWSSLKASDTA MYYCARADCRSTSCYLVFEGQGTLVTVSS ZKA215 SEQ ID NO. Amino acid sequence CDRH1 373 GYTFTSYW CDRH2 374 IDPSDSHT CDRH3 375 ARHALPNYFDS VH 376 EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWISWVRQMPGKGLE WMGRIDPSDSHTDYSPSFQGHVTISADKSISAAYLQWSSLKASDTA MYYCARHALPNYFDSWGQGTLVTVSS ZKA218 SEQ ID NO. Amino acid sequence CDRH1 377 GFPFSSYW CDRH2 378 INSDGRNT CDRH3 379 ARGGYDYDSSGCFDY VH 380 EVQLVESGGGLVQPGGSLRLSCAASGFPFSSYWMHWVRQAPGKGLV WVSRINSDGRNTNYADSVKGRFTISRDNAENTVYLQMNSLRAEDTA VYYCARGGYDYDSSGCFDYWGQGTLVTVSS ZKB7 5 SEQ ID NO. Amino acid sequence CDRH1 381 GFTFSNYA CDRH2 382 ISGTGGST CDRH3 383 AKDSASRGGYCSGGVCYLNPGHHDY VH 384 EVQVLESGGGLLQPGGSLRLSCAASGTFFSNYAMSWVRQAPGKGLE WVSTISGTGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCAKDSASRGGYCSGGVCYLNPGHHDYWGQGTLVTVSS ZKB8 3 SEQ ID NO. Amino acid sequence

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CDRH1 385 GYSFTNYW CDRH2 386 IDPSDSYT CDRH3 387 ARLRGSLYCSGGRCYSVPGETPNWFDP VH 388 EVQLVQSGAEVKKPGESLRISCKGSGYSFTNYWITWVRQMPGKGLE WMGSIDPSDSYTNYSPSFQGHVTISADWSINTAYLQWSSLKASDTA KYYCARLRGSLYCSGGRCYSVPGETPNWFDPWGQGTLVTVSS ZKC3 SEQ ID NO. Amino acid sequence CDRH1 389 GGSITSYY CDRH2 390 IYYSGST CDRH3 391 ARVGGAPYYYYGMDV VH 392 QVQLQESGPGLVKPSETLSLTCTVSGGSITSYYWSWIRQPPGKGLE WIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAV YYCARVGGAPYYYYGMDVWGQGTTVTVS S ZKC18 SEQ ID NO. Amino acid sequence CDRH1 393 GFTFGDYA CDRH2 394 IRSKAYGGTT CDRH3 395 SRDHTGTTYAFDI VH 396 EVQLVESGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLE WVGFIRSKAYGGTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTED TAVYYCSRDHTGTTYAFDIWGQGTMVTVSQ ZKD1 SEQ ID NO. Amino acid sequence CDRH1 397 GFTFSSYG CDRH2 398 IWYDGSNK CDRH3 399 ARDRRGYGDYVGYYYGMDV VH 400 QVQLVESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLE WVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VY Y CARDRRGY GD YVGY Y Y GMDVW GQGTTVTVSS

Name SEQ ID NO. Amino acid sequence ZIKV EDIII generic 401 TAAFTFTKXPAEXXHGTVTVEXQYXGXDGPCKXPXQMAVDXQTLT PVGRLITANPVITEXTENSKMMLELDPPFGDSYIVIGXGXKKITH HWHRS ZIKV H/PF/2013 EDIII 402 TAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLT PVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITH HWHRS

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ZIKV-NS1 direct primer 403 TGGAGTTCAACTGACGGTCG ZIKV-NSl reverse primer 404 TACCCCGAACCCATGATCCT Gapdh direct primer 405 GGCAAGTTCAAAGGCACAGTC Gapdh reverse primer 406 CAC CAG SAT CAC C C CAT T T ZIKV EDIII generic 407 X ₁ GX2X ₃ YSLCTAAFTFTKX4PAEX ₅ X ₆ HGTVTVEX ₇ QYX ₈ GX ₉ DGPC KXioPXnQMAVDX^QTLTPVGRLITANPVITEX^TX^NSKMMLEL dppfgdsyivigx ₁₅ gx ₁₆ x ₁₇ kithhwhrsg where XI can be any (naturally occurring) amino acid, preferably K, A, or E; X2 may be any (naturally occurring) amino acid, preferably V, F, or L; CH may be any (naturally occurring) amino acid, preferably S or F; X4 can be any (naturally occurring) amino acid, preferably I or V; X5 may be any (naturally occurring) amino acid, preferably T or V; X6 may be any (naturally occurring) amino acid, preferably L or D; X7 may be any (naturally occurring) amino acid, preferably V or G; X8 may be any (naturally occurring) amino acid, preferably or G; X9 can represent any

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Claims (42)

(naturally occurring) amino acid other than R, preferably T or A; XY may be any (naturally occurring) amino acid, preferably V or I; XII can be any (naturally occurring) amino acid, preferably or V; X12 may be any (naturally occurring) amino acid, preferably M or T; X13 may be any (naturally occurring) amino acid, preferably S or G; X14 can be any (naturally occurring) amino acid, preferably E or K; X15 may be any (naturally occurring) amino acid, preferably V or I; X16 may be any naturally occurring amino acid, preferably E, A, K, or D; And X17 may be any (naturally occurring) amino acid, preferably E, A, or K, more preferably K or A * sequences in bold represent CDR regions (nucleotide or aa) and underlined residues represent mutated residues, according to compared to the germline sequence CLAIM 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of the Zika virus and neutralizes an infection caused by the Zika virus, wherein the antibody or antigen-binding fragment thereof comprises the amino acid sequences CDRH1, CDRH2 and CDRH3 and the amino acid sequences CDRL1, CDRL2 and CDRL3 (i ) in accordance with SEQ ID NO: 1-5 and 7 or (ii) in accordance with SEQ ID: 1-4 and 6-7. 2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that the antibody or antigen-binding fragment thereof does not substantially bind to dengue virus-like particles and/or to the dengue virus envelope protein. 3. An antibody or an antigen-binding fragment thereof according to claim 1 or 2, characterized in that the antibody or an antigen-binding fragment thereof does not contribute to an antibody-dependent enhancement of infection caused by the Zika virus. 4. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that the antibody or an antigen-binding fragment thereof is a human antibody. 5. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that the antibody or an antigen-binding fragment thereof is a monoclonal antibody. 6. An antibody or an antigen-binding fragment thereof according to any one of claims 1-5, characterized in that the antibody or an antigen-binding fragment thereof is an IgG type. 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 contains an Fc fragment. 8. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that the antibody or antigen-binding fragment thereof contains a mutation in the Fc fragment, said mutation reduces the binding of the antibody to the Fc receptor. 9. The antibody or antigen-binding fragment thereof according to claim 7 or 8, characterized in that the antibody or antigen-binding fragment thereof contains a CH2 L4A mutation, a CH2 L5A mutation, or both. - 105 046175 10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that the antibody or antigen-binding fragment thereof binds to domain III of the Zika virus envelope protein. 11. The antibody or antigen binding fragment thereof according to claim 10, wherein domain III of the Zika virus envelope protein contains or consists of the amino acid sequence as set forth in SEQ NO: 401 or 407. 12. The antibody or antigen binding fragment thereof according to claim 11, wherein domain III of the Zika virus envelope protein contains or consists of the amino acid sequence as set forth in SEQ NO: 402. 13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, 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 EDI-EDIII of the hinge region. 14. The antibody or antigen-binding fragment thereof according to claim 13, wherein the epitope of the Zika virus envelope protein includes one or more amino acid residues of the lateral ridge (LR) in EDIII and one or more amino acid residues of the EDI-EDIII hinge region. 15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that the antibody or antigen-binding fragment thereof binds to a quaternary epitope present on the infectious Zika virus virion. 16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, characterized in that the antibody or antigen-binding fragment thereof can inhibit the stage of the virus life cycle after the Zika virus attaches to the cell membrane. 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 contains a heavy chain variable region (VH) with an amino acid sequence in accordance with SEQ ID NO: 8 or a variant of its functional sequence, having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, according to at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and/or a light chain variable region (VL) with the amino acid sequence according to SEQ ID NO: 9 or a functional variant thereof sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. 18. The antibody or antigen-binding fragment thereof according to any of the preceding claims, characterized in that the antibody or antigen-binding fragment thereof is a purified antibody. 19. The antibody or antigen-binding fragment thereof according to any one of claims 1-6 or 10-18, characterized in that the antibody or antigen-binding fragment thereof is a single chain antibody, Fab, Fab', F(ab)2, Fv or scFv. 20. Use of an antibody or antigen-binding fragment thereof according to any of the preceding claims for the prevention or treatment of infection caused by the Zika virus. 21. Use according to claim 20 in subjects diagnosed with Zika virus infection or in subjects exhibiting symptoms of Zika virus infection. 22. Use according to claim 20, wherein the subject does not exhibit symptoms of Zika infection. 23. Use according to claim 20, wherein the subject is a pregnant woman. 24. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 19 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. 25. A nucleic acid molecule containing a polynucleotide encoding an antibody or an antigen-binding fragment according to any one of claims 1 to 19. 26. The nucleic acid molecule according to claim 25, where the polynucleotide sequence contains or consists of a nucleic acid sequence in accordance with any of SEQ ID NO: 10-18, 28-36, 46-54, 64-72 and 82-90; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. 27. An expression vector containing a nucleic acid molecule according to claim 25 or 26. 28. A cell for expressing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19, containing a vector according to claim 27. 29. A pharmaceutical composition for the prevention or treatment of infection caused by the Zika virus, containing an antibody or an antigen-binding fragment according to any one of claims 1 to 19, a nucleic acid according to claims 25, 26 or a vector according to claim 27, where the pharmaceutical composition additionally contains pharmaceutically an acceptable excipient, diluent or carrier. 30. The pharmaceutical composition according to claim 29, which is a single dose product. 31. Pharmaceutical composition according to any one of claims 29, 30, where the amount of antibody or anti - 106 046175 gene-binding fragment does not exceed 500 mg. 32. Use of a nucleic acid according to any one of claims 25, 26 for the prevention or treatment of infection caused by the Zika virus. 33. Use of the vector according to claim 27 for the prevention or treatment of infection caused by the Zika virus. 34. Use of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19 for diagnosing an infection caused by the Zika virus. 35. Use of a nucleic acid according to any one of claims 25, 26 for diagnosing an infection caused by the Zika virus. 36. A kit for preventing or treating infection caused by the Zika virus, containing at least one antibody or antigen-binding fragment according to any one of claims 1 to 19, at least one nucleic acid according to any of claims 25, 26, at least one vector according to claim 27 or at least one pharmaceutical composition according to any of claims 29-31 and instructions for their use, for the prevention or treatment of infection caused by the Zika virus. 37. A method of preventing or treating an infection caused by the Zika virus in a subject, where the method includes administering to a subject in need thereof an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19, a nucleic acid according to any one of claims 25, 26 , a vector according to claim 27 or a pharmaceutical composition according to any of claims 29-31. 38. The method of claim 37, wherein the antibody or antigen-binding fragment, nucleic acid, vector or pharmaceutical composition thereof is administered up to seven days after (possible) Zika virus infection. 39. The method of claim 37, wherein the antibody or antigen-binding fragment, nucleic acid, vector or pharmaceutical composition thereof is administered in combination with a checkpoint inhibitor. 40. The method according to claim 37, where the antibody is administered in a (single) dose of 0.04 to 15 mg/kg. 41. The method of claim 37, wherein the subject has been diagnosed with a Zika virus infection or is exhibiting symptoms of a Zika virus infection. 42. The method according to claim 37 or 41, where the subject is a pregnant woman. - 107 -