HK40025147B - Multispecific antibodies specifically binding to zika virus epitopes and uses thereof - Google Patents

Description

Multispecific antibodies that specifically bind to epitopes of Zika virus and uses thereof

The present invention relates to multispecific antibodies and antigen-binding fragments thereof that specifically bind to different Zika virus (ZIKV) epitopes. Such antibodies efficiently neutralize zika virus (ZIKV) infection and minimize or eliminate the generation of zika virus escape mutants. The invention also relates to nucleic acids encoding such antibodies and antibody fragments. In addition, the invention relates to the use of the antibodies and antibody fragments of the invention in the prevention and treatment of ZIKV infections.

Zika virus (ZIKV) is a mosquito-transmitted flavivirus that causes public health emergencies. ZIKV was first isolated in 1947 from macaques in Zika forest, wuganda (g.w.a.dick, s.f.kitchen, a.j.hadow, zika virus.i.isolations and clinical speciality.trans.r.soc.trop.med.hyg.46, 509-520 (1952)), and the first human infection was reported in nigeria in 1954 f.n.macnamara, zika virus: a report on thread cases of human infection and epidemic of mountain in Nigeria. Trans. R. Soc. Trop. Med. Hyg.48, 139-145 (1954)). Thereafter, ZIKV infection was reported sporadically in Africa and southeast Asia (D.Musso, van Mai Cao-Lormeau, D.J. Gubler, zika virus: folowing the path of dengue and chikungunya Lancet.386, 243-244 (2015)), but Micronisa reported an epidemic in 2007 (M.R.Duffy et al, ka virus outbox on Yap Island, federated States of Micronesia.N Engl J Med.360, 2536-2543 (2009)), farmland in 2013 to 14 years, which virus was subsequently transmitted to other countries of the continent (V.Cao-Lormeau, D.Musso, eming gift virus in Clough. 2014-2014.1572.2014.1572. Valley et al, and Mikungunyu et al in 1572. Pacific et al.1. Pacific et al.10). Since the introduction of brazil in 2015, ZIKV spread rapidly, 2016, month 2, the World Health Organization (WHO) announced it as an emergent public health event of international concern (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 american-yeet animal Virus thread.n Engl J Med, 160113101142 (2016); d.l.heymann et al, zika Virus and yeast: diagnosis of disease said Virus, phe.387, 721-2016 (2016)). The primary route of ZIKV infection is by mosquito bites of the aedes, but the Virus may also be sexually transmitted (d. Musso et al, potential sexual transmission of Zika Virus. Emerg Infect dis.21, 359-361 (2015)) and vertically transmitted (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 mild symptoms, there is evidence that ZIKV infections can lead to neurological complications, such as Guilan-Barre Syndrome in adults (V. -M.Cao-Lormeau et al, guillain-Barre Syndrome with free Virus infection in free Polynesia: a case-controlled study.Lancet.0 (2016), doi:10.1016/S0140-6736 (16) and congenital defects, including small head deformities of developing fetuses (G.Calvet, R.S.Aguiar, A.lo Me000, S.A.Sampaio, detection and diagnosis of Virus free Virus, A.M. 000, S.A.Sampaction, detection and diagnosis of free Virus infection, see [ 10.10.10: 10: J.); rubin, M.F.Greene, L.R.Baden, zika Virus and Microcephary.N Engl J Med (2016), doi:10.1056/NEJMe 1601862), probably due to Their ability to Infect Human Neural progenitor cells (H.Tang et al, zika Virus infections Human Neural progenerators and attentuates Their growth. Stem Cell,1-5 (2016)).

ZIKV belongs to the genus flavivirus and also includes west nile virus, dengue virus, tick-borne encephalitis virus, yellow fever virus and several other viruses that may cause encephalitis. Flaviviruses are encapsulated by geometric shapes having an icosahedral and a spherical shape. The diameter is about 50nm. The genome is a linear, positive sense RNA and is non-segmented, about 10kb to 11kb in length. The flavivirus genome encodes 3 structural proteins (capsid, prM and envelope) and 8 non-structural proteins (NS 1, NS2A, NS2B, NS3, NS4A, NS4B, NS5 and NS 5B).

The flavivirus envelope (E) protein mediates fusion and becomes The primary target for neutralizing antibodies, but The nonstructural protein 1 (NS 1) is secreted by infected cells and is associated with immune escape and pathogenesis (D.A. Muller, P.R. Young, the flavivirus NS1 protein: molecular and structural biology, immunology, role in pathological and application as a diagnostic biological marker. Antibiotic Res.98, 192-208 (2013)). Two recent structural studies have shown that The E Protein of ZIKV shares high structural similarity with The E proteins of other flaviviruses such as dengue Virus (DENV), yellow Fever Virus (YFV) and West Nile Virus (WNV), and also revealed unique features that may be associated with ZIKV neurotropic properties (L.Dai et al, structures of The Zika Virus Envelope Protein and Its Complex with a Flavivirus Broadly Protective antibody.cell Host Micro (2016), doi: 10.1016/j.com.2016.04.013; D.Sirohi et al, the resolution cryo-EM structure of Zika virus science, aaf5316 (2016)). Also, despite their different electrostatic properties, structural analysis of ZIKV NSl revealed conserved features with other flaviviruses NSl (j. Kim et al, zika virus NS1 structural recovery of electronic su)rfaces among flaviviruses，1-6(2016))。

A phenomenon characteristic of certain flaviviruses is the enhanced disease activity of cross-reactive antibodies caused by prior infection with a heterologous virus. For Dengue viruses (DENV) of known 4 serotypes, epidemiological evidence suggests that primary infection prevents reinfection with the same serotype, but presents a risk factor for developing severe disease after reinfection with a different serotype (S.B. Halstead, where variant Antibody-Dependent Enhancement: knowns and Unknowns. Microbiol Spectr.2, 249-271 (2014)). Exacerbated disease is caused by E and prM specific antibodies that fail to neutralize the afferent virus, but do so by expression of Fc receptors (FcRs) ⁺ ) Enhanced capture of the virus, leading to enhanced viral replication and activation of cross-reactive memory T cells. The cytokine storm produced is believed to underlie the most severe form of the disease, known as Dengue hemorrhagic fever/Dengue shock syndrome (s.b. halstead, neutral and anti-severity enhancement of Dengue viruses. Adv viruses res.60, 421-467 (2003); screaton, J.Mongkolslaya, S.Yaclub, C.Roberts, new impedances in the immunopathology and control of Dengue Virus infection. Nat Rev Immunol.15, 745-759 (2015). The effect of antibodies in severe Dengue is supported by studies showing that a decrease in maternal antibody levels in infants is indicative of a higher risk of developing severe Dengue disease (S.B.Halstead, neutralization and antibody-dependent enhancement of Dengue Virus. Adv Virus Res.60, 421-467 (2003); halstead et al, dengue ferroelectric servers in the inputs: research opportunities aligned. Operating intellectual Dis.8, 1474-1479 (2002); T.H.Nguyen et al, dengue ferroelectric servers in the inputs: a student of clinical and cytokine profiles. J. Infect Dis.189, 221-232 (2004); A.L.Rothman, dengue: refining protective substrates technical immunity. J. Clin invest.113, 946-951 (2004)).

It was recently found that most antibodies reactive with DENV envelope proteins also bind to ZIKV, but those that recognize the major linear Fusion Loop Epitope (FLE) do not neutralize ZIKV, but rather promote antibody-dependent enhancement of ZIKV infection (ADE) (Dejnitiantisai W, suspasa P, wongwat W, rouvinski A, barba-Spaeth G, duangchinda T, sakuntanbhai A, cao-Lormeau VM, malasit P, rey FA, mongkoapaya J, screaton GR: dengue virus o-cross-reactive drive anti-mutation enhancement processing of introduction with vitamin H. Im. 2016.23.23.10.1038/3515).

Furthermore, organisms with high mutation rates, such as various viruses like Zika virus, often rely on so-called "mutation escape" as a mechanism to avoid destruction by the host cell. That is, viruses can protect themselves from host immune responses by mutating their genotype and phenotype (referred to as "escape mutations"). Thus, the generation of escape mutants (i.e., viruses carrying escape mutations) can reduce the efficacy of antibody drugs.

In view of the above circumstances, an object of the present invention is to provide a multispecific antibody that effectively neutralizes ZIKV. Such antibodies preferably do not promote antibody-dependent enhancement (ADE) of zika virus infection. It is another object of the present invention to provide highly specific anti-ZIKV antibodies that eliminate or minimize the generation of ZIKV escape mutants.

The objects of the invention are achieved by the claimed subject matter.

Although the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein as these may vary. It is also to be understood that the terminology used herein is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Hereinafter, elements of the present invention will be described. These elements are listed with particular embodiments, but it should be understood that they may be combined in any manner and in any number to produce other embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and/or preferred elements. Further, any permutation and combination of all described elements in this application should be considered disclosed in the description of this application, unless the context indicates otherwise.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element, integer or step, but not the exclusion of any other element, integer or step not stated. The term "consisting of 823070, \8230compositional" is a specific embodiment of the term "comprising," excluding any other unrecited elements, integers or steps. In the context of the present invention, the term "comprising" encompasses the term "consisting of 8230; \8230;. Thus, the term "comprising" encompasses "including" as well as "consisting of 8230; \8230; e.g., a composition" comprising "X may consist of X alone, or may comprise other moieties, e.g., X + Y.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The word "substantially" does not exclude "completely", e.g., a composition that is "substantially free" of Y may be completely free of Y. The word "substantially" may be omitted from the definition of the invention, if necessary.

The term "about" with respect to the numerical value x means x ± 10%.

The term "disease" as used herein is intended to be generally synonymous with the terms "disorder" and "symptom" (as in a medical condition) and used interchangeably, as all reflect abnormal conditions of the human or animal body or a part thereof that impair normal function, and are generally manifested by discernible disorders and symptoms and result in reduced life span or reduced quality of life of the human or animal.

As used herein, "treatment" of a subject or patient is meant to include prophylaxis, prevention, attenuation, amelioration, and treatment. The terms "subject" or "patient" are used interchangeably herein 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 fragment of an antibody of the invention that retains the antigen-binding activity of the antibody. Examples of antibody fragments include, but are not limited to, single chain antibodies, fab ', F (ab') ₂ Fv or scFv. Furthermore, the term "antibody" as used herein includes antibodies and antigen binding fragments thereof.

As used herein, the term "antibody" encompasses various forms of antibodies, including but not limited to whole antibodies, antibody fragments, particularly antigen binding fragments, human antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies, and genetically engineered antibodies (variant or mutant antibodies), so long as the features of the invention are retained. Monoclonal antibodies are preferred, with monoclonal antibodies having human CDRs or human variable regions being particularly preferred. In particular, the antibodies or antigen-binding fragments thereof according to the invention are preferably derived from human antibodies (i.e. they comprise the CDRs and/or variable regions of a human antibody). More preferably, the antibody or antigen-binding fragment thereof according to the invention further comprises a constant region of a human antibody. Most preferably, all constant and variable regions of the antibody or antigen binding fragment thereof according to the invention are of human origin, i.e. human constant and variable regions, e.g. of a human antibody.

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 produced in transgenic animals (e.g., mice) that, upon immunization, are capable of producing a complete human antibody repertoire or human antibody selection without the production of endogenous immunoglobulins. Transfer of human germline immunoglobulin gene arrays in such germline mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., jakobovits, a., et al, proc.natl.acad.sci.usa 90 (1993) 2551-2555 Jakobovits, a., et al, nature 362 (1993) 255-258 bruggemann, m., et al, year immunol.7 (1993) 3340. Human antibodies can also be produced in phage display libraries (Hoogenboom, H.R., and Winter, G., J.mol.biol.227 (1992) 381-388 marks, J.D., et al, J.mol.biol.222 (1991) 581-597). The techniques of Cole et al and Boerner et al can also be used to prepare human Monoclonal Antibodies (Cole et al, monoclonal Antibodies and Cancer Therapy, alan R.Liss, p.77 (1985); and Boerner, P.147 (1991) 86-95). Preferably, e.g. Traggiai E, becker S, subbarao K, kolesnikova L, uematsu Y, gismondo MR, murphy BR, rappuoli R, lanzavecchia A. (2004): anefficient method to make human monoclonal antibodies from memory B cells: potential neutralization of SARS coronavirus. Nat med.10 (8): 871-5, human monoclonal antibodies were prepared by using modified EBV-B cell immortalization. As used herein, the term "human antibody" also includes antibodies that are modified, e.g., in the variable or constant regions, to produce the properties according to the invention described herein.

The antibody of the invention may be of any isotype (e.g., igA, igG, igM, i.e., alpha, gamma or mu heavy chain), but is preferably an IgG. In the IgG isotype, the antibody can be of the IgG1, igG2, igG3, or IgG4 subclass, with IgG1 being preferred. The antibodies of the invention may have a kappa or lambda light chain.

The antibody or antigen-binding fragment thereof according to the invention may be a purified antibody or a single chain antibody, such as a bispecific single chain Fv fragment (scFv).

The invention also provides fragments of the antibodies of the invention, particularly fragments that retain the antigen binding activity of the antibodies. Although the term "antibody" or "antibody of the invention" may, at some point in the specification including the claims, explicitly refer to binding fragments of an antigen, antibody fragments, variants and/or derivatives of an antibody, it is to be understood that the term "antibody" or "antibody of the invention" includes all classes of antibodies, i.e. antigen binding fragments, antibody variants and derivatives. Fragments of the antibodies of the invention can be obtained from the antibodies by: by methods involving digestion with enzymes such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, fragments of the antibody may be obtained by cloning and expressing a partial sequence of the heavy or light chain. For example, the invention includes bispecific scfvs comprising CDRs from an antibody of the invention. The antibody fragments of the invention may confer monovalent or multivalent interactions and are comprised in a variety of structures. For example, scFv molecules can be synthesized to produce trivalent "triabodies" or tetravalent "tetrabodies". The scFv molecule can comprise a domain of an Fc region, thereby producing a bivalent minibody.

The antibodies of the invention may be provided in purified form. Typically, the antibody will be present in a composition that is substantially free of other polypeptides, e.g., where less than 90%, typically less than 60%, more typically less than 50% by weight of the composition is made up of other polypeptides.

The antibodies according to the invention may be immunogenic in human and/or non-human (or heterologous) hosts such as mice. For example, an antibody may have an idiotype that is immunogenic in a non-human host but not immunogenic in a human host. Antibodies of the invention for use in humans include antibodies that cannot be readily isolated from a host such as a mouse, goat, rabbit, rat, non-primate mammal, and the like, and generally cannot be obtained by humanization or from xenogeneic mice.

As used herein, a "neutralizing antibody" is an antibody that can neutralize, i.e., prevent, inhibit, reduce, hinder, or interfere with the ability of a pathogen to initiate and/or perpetuate an infection in a host. The terms "neutralizing antibody" and "neutralized antibody" are used interchangeably herein. These antibodies can be used as prophylactic or therapeutic agents, alone or in combination, according to appropriate formulations, and as diagnostic tools or manufacturing tools as described herein, in conjunction with active vaccination.

The dosage is usually related to body weight. Thus, a dose expressed in [ g, mg, or other units ]/kg (or g, mg, etc.) generally refers to [ g, mg, or other units ] "per kg (or g, mg, etc.) of body weight", even if the term "body weight" is not explicitly mentioned.

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

As used herein, the term "vaccine" is generally understood to mean a prophylactic or therapeutic substance that provides at least one antigen, preferably an immunogen. The antigen or immunogen may be derived from any material suitable for vaccination. For example, the antigen or immunogen may be derived from a pathogen, e.g. from a bacterium or viral particle or the like, or from a tumor or cancerous tissue. The antigen or immunogen stimulates the adaptive immune system of the human body to provide an adaptive immune response. In particular, an "antigen" or "immunogen" generally refers to a substance that can be recognized by the immune system, preferably an adaptive immune system, and is capable of triggering an antigen-specific immune response, for example by forming antibodies and/or antigen-specific T cells as part of the adaptive immune response. Typically, an antigen may be or may comprise a peptide or protein that can be presented by MHC to a T cell.

As used herein, "sequence variant" (also referred to as "variant") refers to any change in a reference sequence, wherein the reference sequence is any sequence listed in "sequence and SEQ ID No. (sequence listing), i.e. SEQ ID NO:1 to SEQ ID NO:273. thus, the term "sequence variant" includes both nucleotide sequence variants and amino acid sequence variants. It is noted that sequence variants as referred to herein are in particular functional sequence variants, i.e. sequence variants which retain the biological function of e.g. an antibody. In the context of the present invention, such maintained biological function is preferably neutralization of ZIKV infection and/or binding of antibodies to ZIKV E proteins. 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 a reference sequence. The phrase "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" as used herein refers to (i) a sequence variant that functions as described herein, and (ii) a sequence variant that is more preferred the higher the% sequence identity. In other words, the phrase "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" especially means that the functional sequence variant has at least 70% 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, still more preferably at least 95% sequence identity, still more preferably at least 96% sequence identity, especially preferably at least 97% sequence identity, especially preferably at least 98% sequence identity, most preferably at least 99% sequence identity to the respective reference sequence.

The term "sequence variant" especially includes variants which comprise mutations and/or substitutions compared to the reference sequence. Exemplary variants of the Fc partial sequence include, but are not limited to, those having L substituted with a at position CH24, CH25, or both.

Sequence identity is typically calculated over the full length of the reference sequence (i.e., the sequence described in this application). As referred to herein, percent identity may be determined, for example, using BLAST using default parameters specified by NCBI (national center for Biotechnology information; http:// www.ncbi.nlm.nih.gov.) [ Blosum 62 matrix; gap opening penalty =11, gap extension penalty =1].

As used herein, a "nucleotide sequence variant" has an altered sequence in which one or more nucleotides in a reference sequence are deleted or substituted, or one or more nucleotides are inserted into the sequence of a reference nucleotide sequence. Nucleotides are referred to herein by the standard one letter (A, C, G or T). Due to the degeneracy of the genetic code, a "nucleotide sequence variant" may result in a change of the corresponding reference amino acid sequence, i.e. a change or no change of the "amino acid sequence variant". Preferred sequence variants are nucleotide sequence variants which do not produce amino acid sequence variants (silent mutations), but other non-silent mutations are also within the scope, in particular mutated nucleotide sequences which produce amino acid sequences having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity with a reference sequence.

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

Although non-conservative amino acid substitutions are possible, preferably, the substitutions are conservative amino acid substitutions, wherein the substituted amino acid has similar structural or chemical properties as the corresponding amino acid in the reference sequence. For example, a conservative amino acid substitution involves the substitution of one aliphatic or hydrophobic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic or hydrophobic amino acid; one hydroxyl-containing amino acid such as serine and threonine is substituted with another hydroxyl-containing amino acid; substitution of one acidic residue, such as glutamic acid or aspartic acid, with another acidic residue; one amide-containing residue, such as asparagine and glutamine, is substituted with another amide-containing residue; one aromatic residue such as phenylalanine and tyrosine is substituted with another aromatic residue; substitution of one basic residue, such as lysine, arginine and histidine, with another; one small amino acid, such as alanine, serine, threonine, methionine, and glycine, is substituted with another small amino acid.

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

Importantly, changes in sequence variants do not abrogate the function of the respective reference sequence, in which case, for example, the antibody sequence or antigen-binding fragment thereof binds to the same epitope and/or substantially neutralizes the function of ZIKV infection. By using computer programs well known in the art, guidance can be found for determining which nucleotides and amino acid residues, respectively, can be substituted, inserted or deleted without abrogating this function.

As used herein, a nucleic acid sequence or amino acid sequence "derived from" a given nucleic acid, peptide, polypeptide, or protein refers to the source of the nucleic acid, peptide, polypeptide, or protein. Preferably, the nucleic acid sequence or amino acid sequence derived from a particular sequence has substantially the same amino acid sequence as the amino acid sequence from which the sequence or part thereof is derived, whereby "substantially identical" includes sequence variants as defined above. Preferably, the nucleic acid sequence or amino acid sequence derived from a particular peptide or protein is derived from the corresponding domain in the particular peptide or protein. Thus, "corresponding" specifically refers to the same function. For example, an "extracellular domain" corresponds to another "extracellular domain" (of another protein), or a "transmembrane domain" corresponds to another "transmembrane domain" (of another protein). "corresponding" portions of peptides, proteins and nucleic acids are thus readily identifiable to those of ordinary skill in the art. Likewise, a sequence "derived from" another sequence is generally readily recognized by one of ordinary skill in the art as being derived from that sequence.

Preferably, the nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may be identical to the starting nucleic acid, peptide, polypeptide or protein (from which it was derived). However, a nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may also have one or more than one mutation relative to the starting nucleic acid, peptide, polypeptide or protein (from which it is derived), in particular the nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may be a functional sequence variant of the starting nucleic acid, peptide, polypeptide or protein (from which it is derived) as described above. For example, in a peptide/protein, one or more than one amino acid residue may be substituted with other amino acid residues, or insertion or deletion of one or more than one amino acid residue may occur.

As used herein, the term "mutation" refers to a change in the nucleic acid sequence and/or amino acid sequence as compared to a reference sequence, e.g., the corresponding genomic sequence. Mutations, for example in comparison with genomic sequences, can be, for example, (naturally occurring) somatic mutations, spontaneous mutations, induced mutations, for example induced mutations caused by enzymes, chemicals or radiation, or mutations obtained by site-directed mutagenesis (molecular biological methods for making specific and deliberate changes in nucleic acid sequences and/or amino acid sequences). Thus, the term "mutation" is to be understood as also including physically performing the mutation, for example in a nucleic acid sequence or an amino acid sequence. Mutations include substitutions, deletions and insertions of one or more nucleotides or amino acids, as well as inversions of several consecutive nucleotides or amino acids. To achieve mutation of the amino acid sequence, preferably a mutation can be introduced into the nucleotide sequence encoding the amino acid sequence to express the (recombinant) mutated polypeptide. Mutation may be achieved, for example, by altering codons of a nucleic acid molecule encoding one amino acid, e.g., by site-directed mutagenesis, to produce codons encoding a different amino acid, or by synthesizing sequence variants, e.g., by knowing the nucleotide sequence of a nucleic acid molecule encoding a polypeptide, and by designing the synthesis of a nucleic acid molecule comprising a nucleotide sequence encoding a variant of the polypeptide without mutating one or more nucleotides of the nucleic acid molecule.

Several documents are cited throughout the present specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Multispecific antibodies binding to different Zika virus epitopes

Among other findings, the present invention is based on the discovery of multispecific antibodies and antigen-binding fragments thereof that specifically bind to different epitopes of Zika virus. Such antibodies minimize or eliminate the production of escape mutants of Zika virus. In particular, there is currently no method for preventing/treating Zika virus infection. The antibodies according to the invention are very effective in preventing and treating or reducing Zika virus infection. Furthermore, due to the specificity of the antibodies to Zika virus, they did not cause ADE, but blocked ADE.

In a first aspect, the invention provides an isolated multispecific antibody or antigen-binding fragment thereof that specifically binds to different epitopes of zika virus. In other words, the invention provides an isolated multispecific antibody or antigen-binding fragment thereof comprising at least two epitope binding sites that specifically bind different epitopes of zika virus.

Importantly, in contrast to conventional ("normal") antibodies that exhibit only one monospecificity, multispecific antibodies are capable of binding at least two different epitopes. In the present case, the multispecific antibody specifically binds to (at least two) different epitopes of zika virus.

Thus, as used herein, the term "multispecific" refers to the ability to bind to at least two different epitopes, e.g., on different antigens, such as different ZIKV proteins, or on the same antigen, such as the same ZIKV protein. Preferably, the multispecific antibody or antigen-binding fragment thereof according to the present invention binds to at least two different epitopes on the same ZIKV protein, most preferably to at least two different epitopes on the zika virus envelope (E) protein.

Preferably, the antibody or antigen-binding fragment thereof according to the invention is bispecific, trispecific, tetraspecific or pentaspecific, more preferably the antibody or antigen-binding fragment thereof is bispecific, trispecific or tetraspecific, even more preferably the antibody or antigen-binding fragment thereof is bispecific or trispecific, most preferably the antibody or antigen-binding fragment thereof is bispecific.

As used herein, terms such as "bispecific," "trispecific," "tetraspecific," and the like refer to the number of different epitopes to which an antibody or antigen-binding fragment thereof can bind. For example, conventional monospecific IgG-type antibodies have two identical epitope binding sites (paratopes) and therefore can only bind to the same epitope (but not to different epitopes). In contrast, multispecific antibodies have at least two different types of epitope binding sites (paratopes), and thus can bind at least two different epitopes. As used herein, the term "paratope" refers to an epitope binding site of an antibody. Thus, the terms "paratope" and "epitope binding site" are used interchangeably herein. Furthermore, a single "specificity" may refer to one, two, three, or more than three of the same paratopes in a single antibody. The actual number of paratopes in a single antibody molecule is called the "valency". Preferably, the antibody or antigen-binding fragment thereof according to the invention is bivalent, trivalent, tetravalent, hexavalent or octavalent, more preferably the antibody or antigen-binding fragment thereof is bivalent or tetravalent, most preferably the antibody or antigen-binding fragment thereof is tetravalent.

Most preferably, the antibody or antigen binding fragment thereof according to the invention is bispecific and tetravalent.

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

For example, a single native IgG antibody is monospecific and bivalent in that it has two identical paratopes (two identical copies). However, multispecific antibodies comprise at least two (different) paratopes. Thus, the term "multispecific" refers to antibodies and antigen-binding fragments that have more than one paratope and the ability to bind two or more different epitopes. The term "multispecific antibody/antigen-binding fragment" specifically includes a bispecific antibody as defined above, but typically also includes a protein, e.g. an antibody specifically binding to three or more different epitopes, a scaffold, i.e. an antibody having three or more paratopes.

In particular, a multispecific antibody or antigen-binding fragment thereof may comprise two or more paratopes, some of which may be the same, such that all paratopes of the antibody belong to at least two different paratope types, and thus the antibody has at least two specificities. For example, a multispecific antibody or antigen-binding fragment thereof according to the present invention may comprise four paratopes, wherein each two paratopes are the same (i.e., have the same specificity), and thus, the antibody or fragment thereof is bispecific and tetravalent (two identical paratopes for each of the two specificities). Thus, "a specificity" particularly refers to one or more paratopes that exhibit the same specificity (which typically means that such one or more paratopes are the same), and thus "two specificities" can be achieved by two, three, four, five, six or more than six paratopes, as long as they refer to only two specificities. For example, a multispecific antibody may comprise one single paratope for each (at least two) specificities, i.e., the multispecific antibody comprises at least two paratopes in total. For example, a bispecific antibody comprises one single paratope for each of the two specificities, i.e., the antibody comprises a total of two paratopes. Most preferably, the antibody comprises exactly two (identical) paratopes for each of the two specificities, i.e. the antibody comprises a total of four paratopes. Alternatively, the antibody may comprise three (identical) paratopes for each of the two specificities, i.e. the antibody comprises six paratopes in total.

As used herein, the term "antigen" refers to any structural substance that serves as a target for a receptor of an adaptive immune response, in particular, an antibody, a T cell receptor, and/or a B cell receptor. An "epitope", also referred to as an "antigenic determinant", is a portion (or fragment) of an antigen that is recognized by the immune system, particularly by antibodies, T cell receptors, and/or B cell receptors. Thus, an antigen has at least one epitope, i.e., an antigen has one or more than one epitope. The antigen may be (i) a peptide, polypeptide, or protein, (ii) a polysaccharide, (iii) a lipid, (iv) a lipoprotein or lipopeptide, (v) a glycolipid, (vi) a nucleic acid, or (vii) a small molecule drug or toxin. Thus, the antigen may be a peptide, protein, polysaccharide, lipid, combination including lipoproteins and glycolipids, nucleic acid (e.g., DNA, siRNA, shRNA, antisense oligonucleotide, decoy DNA, plasmid), or small molecule drug (e.g., cyclosporine a, paclitaxel, doxorubicin, methotrexate, 5-aminolevulinic acid), or any combination thereof. Preferably, the antigen is selected from (i) a peptide, polypeptide or protein, (ii) a polysaccharide, (iii) a lipid, (iv) a lipoprotein or lipopeptide, and (v) a glycolipid; more preferably, the antigen is a peptide, polypeptide or protein.

An antibody or antigen-binding fragment thereof according to the invention binds to at least two different epitopes of Zika virus. The at least two different Zika virus epitopes may be located on different Zika virus antigens, such as different Zika virus (ZIKV) proteins, or on the same Zika virus antigen, such as on the same ZIKV protein. Preferred examples of ZIKV antigens/proteins include capsid, prM, envelope and nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5 and NS5B. Most preferably, the antibody or antigen-binding fragment thereof according to the present invention binds to (at least two) different epitopes on the envelope protein of ZIKV virus (ZIKV E protein). In other words, most preferably, the antibody or antigen-binding fragment thereof according to the present invention comprises at least two epitope binding sites that specifically bind to different epitopes on the envelope protein of ZIKV virus (ZIKV E protein). ZIKV comprises a nucleocapsid core comprising single-stranded RNA encapsulated by a core protein. The nucleocapsid core is surrounded by a lipid bilayer membrane with "membrane proteins" and "envelope proteins". The ZIKV envelope protein (E protein) is the dominant antigen. The structures and domains of ZIKV E proteins are described, for example, in Dai L, song J, lu X, deng YQ, musyoki AM, cheng H, zhang Y, yuan Y, song H, haywood J, xiao H, yah J, shi Y, qin CF, qi J, gao GF.Structure of the Zika viruses Envelope Protein and files Complex with a vitamin broad reactive antibody. Cell Host Microbe.2016 May 11;19 (5): 696-704.

Preferably, the (at least two) different epitopes of zika virus bound by the antibody or antigen binding fragment thereof according to the invention are non-overlapping epitopes. In particular, the antibodies or antigen binding fragments thereof according to the invention bind amino acids forming a first Zika virus epitope that are different from the amino acids forming a second Zika virus epitope that are bound by the antibodies or antigen binding fragments thereof according to the invention.

The antibody or antigen binding fragment thereof according to the invention may be in any antibody form. In particular, multispecific antibodies preferably encompass "whole" antibodies, e.g., intact IgG-or IgG-like molecules, whereas in the context of the present invention, antigen-binding fragments preferably refer to small recombinant formats, e.g., formats based on bispecific T-cell engagers: (Except in the context of the present invention, both specificities are directed against Zika virus, and therefore,can be specifically replaced by (second) ZIKV), tandem single chain variable fragment molecules (taFv), diabodies (Db), single chain diabodies (scDb), and various other derivatives thereof (see Byrne h. Et al (2013) Trends Biotech,31 (11):621-632, wherein figure 2 shows a plurality of bispecific antibody formats; weidle UH et al (2013) Cancer Genomics and Proteomics 10:1-18, particularly figure 1, shows a variety of bispecific antibody formats; and Chan, AC and Carter, p.j. (2010) Nat Rev Immu 10:301-316, wherein figure 3 shows various bispecific antibody formats). Examples of bispecific antibody formats include, but are not limited to, tandem Fab-Ig, DVD-Ig, quadroma cells, chemically bound Fab (fragment antigen binding) and (bispecific T cell engagers). In one embodiment of the invention, the antibody is preferably a tandem Fab-Ig (FIT-Ig) or DVD-Ig.

Thus, the antibody or antigen-binding fragment thereof according to the invention may be selected from the group consisting of tandem Fab-Ig (FIT-Ig); DVD-Ig; hybrid hybridomas (quadroma cells); the multispecific Anticalin platform (Pieries); a diabody; a single chain diabody; tandem single chain Fv fragments; tandAb, trispecific antibody (Affimed) (105 kDa to 10 kDa); dart (dual affinity retargeting; macrogenetics); bispecific Xmab (Xencor); bispecific T cell engagers (Bites; amgen;55 kDa); a triabody; trisomy = Fab-scFv fusion protein (creativolabs) multifunctional recombinant antibody derivative (110 kDa); duobody platform (Genmab); dock and lock platforms; a pestle and mortar structure (KIH) platform; humanized bispecific IgG antibody (REGNl 979) (Regeneron); mab ² Bispecific antibodies (F-Star); DVD-Ig = dual variable domain immunoglobulin (Abbott); the kappa-lambda body; TBTI = tetravalent bispecific tandem Ig; and CrossMab.

The antibody or antigen binding fragment thereof according to the invention may be selected from bispecific IgG-like antibodies (BsIgG) comprising CrossMab; DAF (two in one); DAF (four in one); dutamab; DT-IgG; common LC with a pestle and mortar structure; a pestle and mortar assembly; fab arm exchange; SEEDbody; triomab; LUZ-Y; fcab; the κ λ body; and an orthogonal Fab. These bispecific antibody formats are described, for example, in Spiess c., zhai q., and Carter p.j. (2015) Molecular Immunology 67:95-106, and in particular fig. 1 and the corresponding description, e.g. pages 95 to 101.

Preferably, the antibody or antigen binding fragment thereof according to the present invention may be selected from the group consisting of nanobodies; a nanobody-HAS; biTE; a diabody; DART; tandAb; sc diabody; sc-diabody-CH 3; diabody-CH 3; a triabody; a minibody; a minibody; a TriBi miniantibody; scFv-CH3KIH; fab-scFv; scFv-CH-CL-scFv; f (ab') 2; f (ab') 2-scFv2; scFv-KIH; fab-scFv-Fc; a tetravalent HCAb; sc diabody-Fc; a diabody-Fc; tandem scFv-Fc; and bispecific antibody fragments of intrabodies. These bispecific antibody formats are described, for example, in Spiess c., zhai q., and Carter p.j. (2015) Molecular Immunology 67:95-106, in particular fig. 1 and the corresponding description, e.g. pages 95 to 101.

More preferably, the antibody or antigen-binding fragment thereof according to the invention may be selected from the group consisting of antibodies with an additional binding domain comprising DVD-IgG; igG (H) -scFv; scFv- (H) IgG; igG (L) -scFv; scFV- (L) IgG; igG (L, H) -Fv; igG (H) -V; v (H) -IgG; igG (L) -V; v (L) -IgG; KIH IgG-scFab;2scFv-IgG; igG-2scFv; scFv4-Ig; scFv4-Ig; zybody; and DVI-IgG (four in one) antigen binding portion of IgG additional antibody. These bispecific antibody formats are described, for example, in spiess c., zhai q., and Carter p.j. (2015) Molecular Immunology 67:95-106, and in particular fig. 1 and the corresponding description, e.g. pages 95 to 101. Among those antibody formats, DVD-Ig (double variable domain immunoglobulin (Abbott)) is even more preferred. Such antibody formats are described in detail in, for example, wu C, ying H, grinnell C, bryant S, miller R, clabbers A, bose S, mcCarthy D, zhu RR, santora L, davis-Taber R, kunes Y, long E, schwartz A, sakorafas P, gu J, tarcsa E, murtaza A, ghayur T.Simulanous targeting of multiple discrete reagents by a dual-variable-domain immunoglobulin Nat Biotechnology.2007 Nov;25 (11): 1290-7; or DiGiammarinano E, ghayur T, liu J ^TM molecules for dual-specific targeting.Methods Mol Biol.2012；899：145-56。

Most preferably, the antibody or antigen-binding fragment thereof according to the invention is an IgG-additional antibody in the form of a tandem Fab-Ig (FIT-Ig). In other words, the antibody or antigen-binding fragment thereof according to the invention is most preferably in the form of a tandem Fab-Ig (FIT-Ig). Tandem Fab-Ig (FIT-Ig) formats are described in detail in, for example, WO 2015/103072 A1, which is incorporated herein by reference in its entirety, or Gong S, ren F, wu D, wu X, wu C: fab-in-tandem immunoglobulin is a novel and versatic design for engaging multiple therapeutic targets MAbs 2017. Like DVD-Ig, FIT-Ig is also a bispecific tetravalent symmetric form. FIT-Ig can be prepared using three polypeptides: polypeptide 1 typically comprises the light chain of the outer Fab, preferably without a linker, fused to the N-terminal region of the heavy chain of the inner Fab. Polypeptide 2 typically comprises the heavy chain variable region and the CH1 region of the outer Fab, and polypeptide 3 typically comprises the light chain of the inner Fab. Thus, antibodies in the form of FIT-Ig usually comprise an "internal Fab" and an "external Fab".

Preferably, the antibody or antigen-binding fragment thereof according to the present invention neutralizes zika virus infection. In other words, the antibody or antigen-binding fragment thereof according to the present invention preferably reduces the viral infectivity of zika virus.

In order to study and quantify viral infectivity (or "neutralization") in the laboratory, the skilled person is aware of 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, wherein cultured cells are incubated with a fixed amount of zika virus (ZIKV) in the presence (or absence) of the antibody to be tested. For example, a flow cytometer may be used as the reading device. Alternatively, other reading means are also conceivable, such as determining the amount of ZIKV nonstructural protein (e.g., ZIKV NS 1) secreted into the culture supernatant. For example, a tissue culture infectious dose 50 (TCID 50) test (TCID 50-ELISA) based on the ZIKV nonstructural protein 1 (NS 1) antigen capture enzyme-linked immunosorbent assay (ELISA) can be used as an alternative to the standard plaque assay for titrating Zika virus, in combination with the standard plaque assay for dengue virus (DENV) of Li J, hu D-M, ding X-X, chen Y, pan Y-X, qiu L-W, chen X-Y: enzyme-linked immunological assay-format tissue culture gene-50 test for transforming virus, plos ONE 2011,6: e22553 a similar method is described. In such assays, for example, ZIKV NS1 binding antibodies described herein may be advantageously used.

In a preferred embodiment of the ZIKV neutralization assay, cultured cells, e.g., vero cells, are incubated with a fixed amount of ZIKV, e.g., for about 4 days, in the presence or absence of the antibody to be tested. After incubation, the cells can be washed and further cultured. To measure the infectivity of the virus, a flow cytometer may be used. For this purpose, the cells can be fixed, for example, with 2% formaldehyde, permeabilized, for example, with 1% fcs (fetal calf serum) 0.5% saponin in PBS (phosphate buffered saline), and stained, for example, with the mouse antibody 4G 2. The cells can then be incubated with goat anti-mouse IgG conjugated to, for example, alexa Fluor488 dye and analyzed by flow cytometry. Alternatively, viable cells can be detected by flow cytometry using, for example, WST-1 reagent (Roche). The preferred strain of ZIKV used in this neutralization assay is ZIKV H/PF/2013.

The antibodies and antigen binding fragments of the invention have high neutralizing potency. Antibody concentration required to neutralize 50% of Zika virus (IC) compared to no-antibody control ₅₀ ) For example, up to about 3. Mu.g/ml or up to about 1. Mu.g/ml. Preferably, the concentration (IC) of the antibody of the present invention required to neutralize 50% of ZIKV ₅₀ ) Concentration of the antibody of the Invention (IC) required to neutralize up to about 500ng/ml, more preferably 50% of ZIKV ⁵⁰ ) Concentration of the antibody of the Invention (IC) required to neutralize up to about 250ng/ml, even more preferably 50% of ZIKV ₅₀ ) Up to about 150ng/ml. Most preferably, the concentration (IC) of the antibody of the invention required to neutralize 50% of ZIKV ₅₀ ) Is about 100ng/ml or less than 100ng/ml, for example about 90ng/ml or less than 90ng/ml, about 80ng/ml or less than 80ng/ml, about 70ng/ml or less than 70ng/ml, about 60ng/ml or less than 60ng/ml, about 50ng/ml or less than 50ng/ml, about 45ng/ml or less than 45ng/ml, about 40ng/ml or less than 40ng/ml, about 35ng/ml or less than 35ng/ml, about 30ng/ml or less than 30ng/ml, about 25ng/ml or less than 25ng/ml, about 20ng/ml or less than 20ng/ml, particularly preferably about 15ng/ml or less than 15ng/ml. In particular, of neutralising ZIKV50% desired concentration (IC) of the antibody of the invention ₅₀ ) Preferably about 50ng/ml or less than 50ng/ml. This means that 50% neutralization of ZIKV requires only low concentrations of antibody. Concentration (IC) of the antibody of the present invention required to neutralize 50% of ZIKV ₅₀ ) Can be determined using standard neutralization assays known to those skilled in the art, particularly as described above.

In general, the binding of the antibody can be assessed by using a standard ELISA (enzyme linked immunosorbent assay) well known to the skilled person. An exemplary standard ELISA may be performed as follows: the ELISA plate can be coated with a sufficient amount (e.g., 1 μ g/ml) of protein/complex/particle to which binding of the antibody is to be detected, e.g., in PBS, to perform the test (e.g., overnight at 4 ℃) (e.g., test DENV binding using DENV E protein and/or DENV VLPs, as described below). The plate may then be blocked, e.g., with 1% w/v solution of Bovine Serum Albumin (BSA) in PBS, and incubated with the antibody to be tested (e.g., at room temperature for about 1.5 hours). After washing, antibody binding can be demonstrated, for example, using goat anti-human IgG-bound alkaline phosphatase. The plate can then be washed, the desired substrate (e.g., p-NPP) can be added, and the plate can be read, for example, at 405 nm. Can be determined by measuring the concentration of mAb (EC) required to achieve 50% maximal binding at saturation ₅₀ ) To determine the relative affinity of antibody binding. EC can be calculated by interpolation of binding curves fitted with four-parameter non-linear regression with variable slope ₅₀ The value is obtained.

Preferably, the antibody or antigen-binding fragment thereof according to the present invention does not substantially bind to dengue virus-like particles and/or dengue envelope proteins. More preferably, the antibody or antigen-binding fragment thereof according to the invention does not substantially bind dengue virus-like particles and/or does not bind dengue envelope proteins of any of the four DENV serotypes DENV1, DENV2, DENV3 and DENV 4. Thus, "substantially non-binding" means that the EC can be determined for the antibody or antigen-binding fragment thereof in a standard ELISA for dengue virus-like particles (DENV VLPs) and/or dengue envelope proteins (DENV E proteins) ₅₀ Value not exceeding at most 10 ² ng/ml, preferably up to 10 ³ ng/ml, more preferably at most5×10 ³ ng/ml, more preferably at most 8X 10 ³ ng/ml, most preferably at most 10 ⁴ ng/ml. In other words, saturation (EC) with dengue virus-like particles (DENV VLP) and/or dengue virus envelope protein (DENV E protein) is achieved in standard ELISA ₅₀ ) The concentration of antibody or antigen-binding fragment thereof required for maximum binding of 50% under conditions is generally greater than 10 ² ng/ml, preferably greater than 10 ³ ng/ml, more preferably greater than 5X 10 ³ ng/ml, even more preferably greater than 8X 10 ³ ng/ml, most preferably greater than 10 ⁴ ng/ml。

Preferably, the antibody or antigen-binding fragment thereof according to the present invention does not promote antibody-dependent enhancement (ADE) of zika virus infection. More preferably, the antibody or antigen-binding fragment thereof according to the invention blocks antibody-dependent enhancement (ADE) of zika virus infection.

ADE can be assessed by flow cytometry-based assays using, for example, cultured cells or cell lines such as K562 cells. For example, the test antibody and ZIKV may be mixed at 37 ℃ for 1 hour and then added to 5000K 562 cells/well. After four days, the cells can be fixed, permeabilized and stained with m4G2, e.g., as described above and assays. The number of infected cells was determined by flow cytometry as described above and in the assay.

Preferably, the antibody or antigen binding fragment thereof according to the present invention eliminates the production of ZIKV escape mutants. The elimination of escape mutant production can be readily assessed by one skilled in the art. For example, to identify the propensity of an antibody or antibody fragment to generate escape mutants, ZIKV is repeatedly passaged in the presence of a sub-neutralizing concentration of the antibody or antigen-binding fragment thereof of interest. As shown in examples 10 and 11 of the present application, ZIKV escape mutants ("MARMs") typically occur after the third or fourth generation of ZIKV. Thus, if a ZIKV escape mutant is not identified even after five passages of the virus (ZIKV) in the presence of a sub-neutralizing concentration of the antibody or antigen binding fragment thereof, it is considered that the antibody eliminates the production of the ZIKV escape mutant. More preferably, according to the present invention, no ZIKV escape mutants are identified after six passages of the virus (ZIKV) in the presence of a sub-neutralizing concentration of the antibody or antigen-binding fragment thereof. Even more preferably, according to the present invention, no ZIKV escape mutants were identified after seven passages of the virus (ZIKV) in the presence of a sub-neutralizing concentration of the antibody or antigen-binding fragment thereof. Most preferably, according to the present invention, no ZIKV escape mutants were identified after eight virus (ZIKV) passages in the presence of a sub-neutralizing concentration of the antibody or antigen-binding fragment thereof.

For example, to assess the propensity of an antibody or antibody fragment to generate escape mutants, ZIKV, e.g., H/PF/2013 strain, is incubated, e.g., at about 37 ℃ (body temperature), with various sub-neutralizing concentrations of the antibody/antigen-binding fragment of interest, e.g., for at least 30 minutes. Thereafter, (live) cells such as Vero cells are added and then incubated, for example at about 37 ℃ (body temperature), for at least one day, preferably 3 to 4 days, to propagate the virus. Then, three conditions of supernatants can be selected: the lowest concentration of fully protected target antibody/antigen-binding fragment of the monolayer can be observed; a concentration at which the effect of partial CPE on the cell monolayer can be observed; it was observed that ZIKV CPE destroyed one concentration of 100% cell monolayer. One part of the selected supernatant can be used for microneutralization assays and subsequent sequencing of the virus (to identify escape mutants), while another part of the (same) selected supernatant can be used for the next selection step (passage). That is, for the next passage, (a portion of) the selected supernatant may be mixed with various sub-neutralizing concentrations of the antibody/antigen-binding fragment of interest and incubated as described above, followed by addition of (viable) cells and incubation as described above to finally select the supernatant again. The selection and propagation process is repeated for at least five generations, preferably for at least six generations, more preferably for at least seven generations, and most preferably for at least eight generations.

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

Also preferably, the antibody or antigen-binding fragment thereof according to the present invention is a monoclonal antibody, preferably a monoclonal antibody in which each CDR or each variable region contained in the antibody or antigen-binding fragment thereof according to the present invention is a human CDR or a human variable region, respectively.

Preferably, the antibody or antigen binding fragment thereof according to the invention is of the IgG type, such as the IgG1, igG2, igG3 or IgG4 type, more preferably of the IgG1 type. More preferably, the antibody or antigen-binding fragment thereof according to the invention comprises (i) a heavy chain constant region of the IgG1 CH1-CH2-CH3 type, wherein "CH1" refers to constant domain 1 of the heavy chain, "CH2" refers to constant domain 2 of the heavy chain, and "CH3" refers to constant domain 3 of the heavy chain; (ii) Light chain constant region of IgG CL type, wherein "CL" refers to the constant domain of the light chain. Even more preferably, the antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain constant region of the IgG1 CH1-CH2-CH3 type comprising a heavy chain constant region according to SEQ ID NO:91 or 92 or a functional sequence variant thereof, the light chain constant region comprising or consisting of an amino acid sequence according to SEQ ID NO:93 or 94 or a functional sequence variant thereof.

Accordingly, it is preferred that the antibody or antigen binding fragment thereof according to the present invention comprises an Fc portion. More preferably, the Fc part is derived from human origin, e.g. from human IgG1, human IgG2, human IgG3 and/or human IgG4, with human IgG1 being particularly preferred. A variety of multispecific antibody formats comprising an Fc portion are known in the art. Preferred antibody formats comprising an Fc portion are those described above for IgG addition. In general, antibodies comprising an Fc portion are more effective and exhibit a longer half-life than antibodies or antibody fragments that do not have an Fc portion.

As used herein, the term "Fc portion" refers to a sequence derived from a portion of an immunoglobulin heavy chain that begins at the hinge region immediately upstream of the papain cleavage site (e.g., residue 216 in native IgG, first residue 114 of the heavy chain constant region), and ends at the C-terminus of the immunoglobulin heavy chain. Thus, the Fc portion can be an entire Fc portion or a portion (e.g., a domain) thereof. The complete Fc portion comprises at least a hinge domain, a CH2 domain, and a CH3 domain (e.g., EU amino acids 216 to 446). Additional lysine residues (K) are sometimes present at the terminal C-terminus of the Fc portion, but are usually cleaved from the mature antibody. Each amino acid position within the Fc portion has been numbered according to the state of the art recognized Kabat EU numbering system, see, e.g., kabat et al, "Sequences of Proteins of Immunological Interest", u.s.dept.health and Human Services,1983 and 1987.

Preferably, in the context of the present invention, the Fc part comprises at least one of: a hinge (e.g., 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 portion comprises at least a hinge domain, a CH2 domain, or a CH3 domain. More preferably, the Fc portion is a complete Fc portion. The Fc portion may also comprise one or more amino acid insertions, deletions, or substitutions relative to a naturally occurring Fc portion. For example, at least one of the hinge domain, CH2 domain, or CH3 domain (or portion thereof) may be deleted. For example, the Fc portion may comprise or consist of: (ii) a hinge domain (or a portion thereof) fused to a CH2 domain (or a portion thereof), (iii) a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), (iv) a hinge domain (or a portion thereof), (v) a CH2 domain (or a portion thereof), or (vi) a CH3 domain or a portion thereof.

One of ordinary skill in the art will appreciate that the Fc portion can be modified such that its amino acid sequence differs from the entire Fc portion of a naturally occurring immunoglobulin molecule, while retaining at least one desired function conferred by the naturally occurring Fc portion. Such functions include Fc receptor (FcR) binding, antibody half-life modulation, ADCC function, protein a binding, protein G binding, and complement binding. A portion of a naturally occurring Fc portion responsible for and/or necessary for such function is well 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 and is associated with an antigen target (Ward, e.s., and Ghetie, v., ther, immunol.2 (1995) 77-94). Burton, d.r. describes the involvement of the heavy chain region in complement fixation, comprising amino acid residues 318 to 337 (mol. Immunol.22 (1985) 161-206). Duncan, A.R. and Winter, G. (Nature 332 (1988) 738-740) use site-directed mutagenesis to report that Glu318, lys320 and Lys322 form the binding site for C1 q. The role of the Glu318, lys320 and Lys322 residues in C1q binding is demonstrated by the ability of short synthetic peptides containing these residues to inhibit complement-mediated cleavage.

For example, fcR binding may be mediated through the interaction of the Fc portion (of the antibody) with Fc receptors (fcrs), which are specialized cell surface receptors on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and are shown to mediate the removal of antibody-coated pathogens by phagocytosis of immune complexes, as well as the lysis of erythrocytes and various other cellular targets (e.g., tumor cells) coated by the corresponding antibodies by antibody-dependent cell-mediated cytotoxicity (ADCC; van de Winkel, j.g., and Anderson, c.l., j.leukc.biol.49 (1991) 511-524). FcR is defined by its specificity for an immunoglobulin class; fc receptors for IgG antibodies are called fcyr, igE called fcyr, igA called fcyr, and so on, neonatal Fc receptors are called FcRn. Fc receptor binding is described, for example, in ravatch, 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 the receptor by the Fc domain of natural IgG antibodies (fcyr) triggers a variety of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, release of inflammatory mediators, and modulation of immune complex clearance and antibody production. Thus, it is preferred to provide an Fc portion (fcyr) that is receptor crosslinked. In humans, three classes of Fc γ rs have been identified, which are: (i) Fc γ RI (CD 64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils; (ii) Fc γ RII (CD 32), which binds complex IgG with moderate to low affinity and is widely expressed, in particular on leukocytes, and is known to be the central role in antibody-mediated immunity, which can be divided into Fc γ RIIA, fc γ RIIB and Fc γ RIIC and play different functions in the immune system, but with low affinity similar to that of IgG-Fc, the extracellular domains of these receptors being highly homologous; (iii) Fc γ RIII (CD 16), which binds IgG with medium to low affinity, and is classified into two types: fc γ RIIIA, which is present on NK cells, macrophages, eosinophils, and some monocytes and T cells, and mediates ADCC, and Fc γ RIIIB, which is highly expressed on neutrophils. Fc γ RIIA is found in many cells involved in killing (e.g., macrophages, monocytes, neutrophils) and appears to be able to activate the killing process. Fc γ RIIB appears to play a role in the inhibition process and is found in B cells, macrophages, and mast cells and eosinophils. Importantly, 75% of all Fc γ RIIB was found in the liver (Ganesan, l.p. et al, 2012. Fc γ RIIB is abundantly expressed on sinusoidal endothelial cells called LSEC, and is the major site of small immune complex clearance in Kupffer (Kupffer) cells and LSEC of the liver (Ganesan, l.p. et al, 2012.

Thus, in the present invention, antibodies and antigen binding fragments thereof are preferred which are capable of binding Fc γ RIIb, e.g. antibodies comprising an Fc portion, particularly an Fc region, for binding Fc γ RIIb, e.g. IgG type antibodies. Furthermore, the Fc portion can be engineered to enhance Fc γ RIIB binding by introducing mutations S267E and L328F, as described in Chu, s.y. et al, 2008: molecular Immunology 45, 3926-3933, of the Inhibition of B cell receptor-mediated activation of primary human B cells by genetic coding of CD19 and FcgammaIIb with Fc-engineered antibodies. Thus, clearance of immune complexes can be Enhanced (Chu, S., et al 2014. Thus, in the context of the present invention, preferred are antibodies or antigen-binding fragments thereof comprising an engineered Fc portion having mutations S267E and L328F, in particular as described in s.y. et al, 2008: molecular Immunology 45, 3926-3933, in the description of the Inhibition of B cell receptor-mediated activation of primary human B cells by genetic coding of CD19 and FcgammaRIIb with Fc-engineered antibodies.

On B cells, it appears to act to inhibit other immunoglobulin production and isotype switching, e.g., igE class. On macrophages, fcyriib inhibits phagocytosis mediated by fcyriia. In eosinophils and mast cells, form b can help inhibit activation of these cells by binding of IgE to its independent receptor.

Regarding Fc γ RI binding, modifications of at least one of E233-G236, P238, D265, N297, a327 and P329 in native IgG reduce binding to Fc γ RI. Substitution of IgG2 residues at positions 233 to 236 with IgG1 and IgG4, which reduced binding to Fc γ RI by 10 ³ Double, and abrogated the response of human monocytes to antibody-sensitized erythrocytes (Armour, k.l., et al eur.j. Immunol.29 (1999) 2613-2624). With respect to Fc γ RII binding, igG mutations such as at least one of E233 to G236, P238, D265, N297, a327, P329, D270, Q295, a327, R292 and K414 were found to reduce binding to Fc γ RIIA. With respect to Fc γ RIII binding, it was found that at least one mutation, for example, in E233 to G236, P238, D265, N297, a327, P329, D270, Q295, a327, S239, E269, E293, Y296, V303, a327, K338 and D376 reduces binding to Fc γ RIIIA. Maps of binding sites to Fc receptors on human IgG1, the above mentioned mutation sites and methods for measuring binding to Fc γ RI and Fc γ RIIA are described in Shields, r.l., et al, j.biol.chem.276 (2001) 6591-6604.

With respect to binding to the critical Fc γ RII, two regions of native IgG Fc appear to be critical for the interaction of Fc γ RII and IgG, namely (I) the lower hinge site of IgG Fc, in particular amino acid residues L, G (234 to 237, eu numbering), and (ii) the adjacent regions of the CH2 domain of IgG Fc, in particular the loops and chains in the upper CH2 domain adjacent to the lower hinge region, e.g. in the region of P331 (Wines, b.d., et al, j.immunol.2000; 164. Furthermore, fc γ RI appears to bind to the same site on IgG Fc, whereas FcRn and protein A bind to different sites on IgG Fc, appearing to be located at the CH2-CH3 interface (Wines, B.D., et al, J.Immunol.2000; 164.

For example, the Fc portion may comprise or consist of at least a portion of an Fc portion that is required for FcRn binding or half-life extension as known in the art. Alternatively or additionally, the Fc portion of an antibody of the invention comprises at least a portion required for protein a binding as known in the art and/or the Fc portion of an antibody of the invention comprises at least a portion of an Fc molecule required for protein G binding as known in the art. Preferably, the function retained is neutralization of Zika virus infection, assuming it is mediated by Fc γ R binding. Thus, preferred Fc portions comprise at least a portion required for Fc γ R binding as known in the art. As mentioned above, a preferred Fc part may thus comprise at least (i) the lower hinge position of a native IgG Fc, in particular amino acid residues L, G (234-237, EU numbering), and (ii) a contiguous region of the CH2 domain of a native IgG Fc, in particular the loops and chains in the upper CH2 domain adjacent to the lower hinge region, e.g. in the region of P331, e.g. a region of at least 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids in the upper CH2 domain of a native IgG Fc surrounding P331, e.g. between amino acid positions 320 and 340 (EU numbering) of a native IgG Fc.

Preferably, the antibody or antigen binding fragment thereof according to the invention comprises an Fc region. The term "Fc region" as used herein refers to the portion of an immunoglobulin formed from two or more Fc portions of an antibody heavy chain. For example, the Fc region may be a monomeric or "single chain" Fc region (i.e., scFc region). A single-chain Fc region consists of Fc portions linked within a single polypeptide chain (e.g., encoded in a single contiguous nucleic acid sequence). Exemplary scFc regions are disclosed in WO 2008/143954 A2. Preferably, the Fc region is a dimeric Fc region. "dimeric Fc region" or "dcFc" refers to a dimer formed from the Fc portions of two separate immunoglobulin heavy chains. The dimeric Fc region can be a homodimer of two identical Fc portions (e.g., the Fc region of a naturally occurring immunoglobulin) or a heterodimer of two different Fc portions.

The Fc portion of the Fc region may be of the same or different classes and/or subclasses. For example, the Fc portion may be derived from an immunoglobulin of the IgG1, igG2, igG3, or IgG4 subclass (e.g., human immunoglobulin). Preferably, the Fc portion of the Fc region has the same class and subclass. However, the Fc region (or one or more than one Fc portion of the Fc region) may also be chimeric, whereby the chimeric Fc region may comprise Fc portions derived from different immunoglobulin classes and/or subclasses. For example, at least two Fc portions of a dimeric or single chain Fc region may be from different immunoglobulin classes and/or subclasses. Additionally or alternatively, the chimeric Fc region may comprise one or more than one chimeric Fc portion. For example, a chimeric Fc region or portion can comprise one or more portions of an immunoglobulin derived from a first subclass (e.g., igG1, igG2, or IgG3 subclass), while the remainder of the Fc region or portion belongs to a different subclass. For example, an Fc region or portion of an Fc polypeptide can comprise a CH2 and/or CH3 domain derived from an immunoglobulin of a first subclass (e.g., igG1, igG2, or IgG4 subclass) and a hinge region derived from an immunoglobulin of a second subclass (e.g., igG3 subclass). For example, the Fc region or portion may comprise a hinge and/or CH2 domain derived from an immunoglobulin of a first subclass (e.g., igG4 subclass) and a CH3 domain derived from an immunoglobulin of a second subclass (e.g., igG1, igG2, or IgG3 subclass). For example, a chimeric Fc region can comprise an Fc portion (e.g., a complete Fc portion) from an immunoglobulin of a first subclass (e.g., an IgG4 subclass) and an Fc portion from an immunoglobulin of a second subclass (e.g., an IgG1, igG2, or IgG3 subclass). For example, the Fc region or portion may comprise a CH2 domain from an IgG4 immunoglobulin and a CH3 domain from an IgG1 immunoglobulin. For example, the Fc region or portion 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 region or portion may comprise a portion of a CH2 domain from a particular subclass of antibody, e.g., EU positions 292-340 of the CH2 domain. For example, the Fc region or Fc portion may comprise amino acids wherein positions 292 to 340 of CH2 are derived from an IgG4 moiety and the remainder of CH2 is derived from an IgG1 moiety (alternatively, positions 292 to 340 of CH2 may be derived from an IgG1 moiety and the remainder of CH2 is derived from an IgG4 moiety).

Furthermore, the Fc region or Fc portion may (additionally or alternatively) comprise a chimeric hinge region, for example. For example, a chimeric hinge can be derived, for example, in part, from an IgG1, igG2, or IgG4 molecule (e.g., upper, lower, middle hinge sequence) and in part from an IgG3 molecule (e.g., middle hinge sequence). In another example, the Fc region or portion may comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule. In another example, a chimeric hinge can comprise upper and lower hinge domains from an IgG4 molecule and a middle hinge domain from an IgG1 molecule. Such a chimeric hinge can be prepared, for example, by introducing a proline substitution (Ser 228 Pro) at EU 228 of the middle hinge domain of the IgG4 hinge region. In another embodiment, the chimeric hinge may comprise amino acids at EU positions 233 to 236 from an IgG2 antibody and/or a Ser228Pro mutation, wherein the remaining amino acids of the hinge are from an IgG4 antibody (e.g., the chimeric hinge of the sequence eskygppcppcpappvapp). Other chimeric hinges that can be used in the Fc portion of antibodies according to the invention are described in US 2005/0163783 A1.

In the present invention, it is preferred that the Fc portion or Fc region comprises or consists of an amino acid sequence derived from a human immunoglobulin sequence (e.g., an Fc region or Fc portion derived from a human IgG molecule). However, the polypeptide may comprise one or more than one amino acid from other mammalian species. For example, a primate Fc portion or primate binding site can be included in a subject polypeptide. Alternatively, one or more than one murine amino acid may be present in the Fc portion or Fc region.

Preferably, the antibody according to the invention comprises, in particular in addition to the Fc part as described above, further parts derived from a constant region, in particular from an IgG, preferably from an IgG1, more preferably from a human IgG1 constant region. More preferably, the antibody according to the invention comprises, in particular in addition to the Fc part as described above, all other parts of the constant region, in particular all other parts of an IgG constant region, preferably all other parts of an IgG1 constant region, more preferably all other parts of a human IgG1 constant region.

Particularly preferred sequences of the constant region are according to SEQ ID NO:91 to 94 (e.g., encoded by nucleic acid sequences according to SEQ ID NOS: 95 to 98, respectively). Preferably, the amino acid sequence of IgG1 CH1-CH2-CH3 is according to SEQ ID NO:91 or a functional sequence variant thereof. Even more preferably, the amino acid sequence of IgG1 CH1-CH2-CH3 is according to SEQ ID NO:92 or a functional sequence variant thereof, wherein the "LALA" mutation is maintained.

As mentioned above, particularly preferred antibodies according to the invention comprise a (intact) Fc region derived from human IgG 1. More preferably, the antibody according to the invention comprises, in particular in addition to the (intact) Fc region derived from human IgG1, all other parts of an IgG constant region, preferably all other parts of an IgG1 constant region, more preferably all other parts of a human IgG1 constant region.

Without being bound by any theory, it is believed that antibody-dependent enhancement (ADE) of zika virus infection is achieved by binding of the Fc portion of the antibody, particularly the Fc portion of the heavy chain of the IgG molecule, to an Fc receptor, such as an Fc γ receptor on a host cell. Thus, it is preferred that the antibody or antigen binding fragment thereof of the invention comprises one or more than one mutation in the Fc portion. The mutation may be any mutation that reduces binding of the antibody to an Fc receptor (FcR), in particular to an Fc γ receptor (Fc γ R). On the other hand, preferably, the antibody according to the invention comprises a (intact) Fc part/Fc region, wherein the interaction/binding with FcRn is not impaired. Thus, it is particularly preferred that the antibody or antigen binding fragment thereof according to the invention comprises one or more than one mutation in the Fc part which (i) reduces the binding of the antibody to the fey receptor but does not impair the interaction with FcRn. An example of such a mutation is the "LALA" mutation described below.

In general, binding of the antibody to cells of interest such as cells of the ELISA (Hessell AJ, handarter L, hunter M, havenith CEG, berkskens FJ, bakker JM, lanigan CMS, landuci G, forthal DN, parren PWHI, et al, fc Receptor fragment not Binding antigens in antigen detection HIV. Nature 2007, 449.

Generally, antibodies according to the invention may be glycosylated. For example, N-linked glycans attached to the CH2 domain of the heavy chain can affect the binding of C1q to FcR, and glycosylated antibodies have a lower affinity for these receptors. Thus, the CH2 domain of the Fc portion of the antibody according to the present invention may comprise one or more than one mutation, wherein a glycosylated residue is substituted with a non-glycosylated residue. Glycan structures may also affect activity, for example differences in complement-mediated cell death may depend on the number of galactose residues (0, 1 or 2) at the end of the glycan biantennary chain. Preferably, the glycans of the antibody do not result in a human immunogenic response after administration.

Furthermore, antibodies according to the invention may be modified by introducing random amino acid mutations into specific regions of the CH2 or CH3 domain of the heavy chain, compared to unmodified antibodies, to alter their binding affinity to FcR and/or their serum half-life. Examples of such modifications include, but are not limited to, substitution of at least one amino acid of the heavy chain constant region selected from amino acid residues 250, 314, and 428.

Particularly preferably, the Fc portion of the antibodies of the invention comprises a substitution at position CH2, CH2 or both. Typically, the amino acids at positions 4 and 5 of CH2 of wild-type IgG1 and IgG3 are leucine ("L"). Preferably, the antibody according to the invention comprises an amino acid other than L at position CH2, CH2 or both. More preferably, the antibody according to the invention comprises an alanine ("a") at position CH2, CH2 or both. Most preferably, the antibody according to the invention comprises both CH2L4A and CH2L5A substitutions. Such antibodies are referred to herein as "LALA" variants. It is noteworthy that such "LALA" mutations in the Fc portion not only resulted in the lack of help of the corresponding antibody in antibody-dependent enhancement (ADE) of zika virus infection, but also blocked the antibody-dependent enhancement (ADE) of zika virus infection. An exemplary amino acid sequence of IgG1 CH1-CH2-CH3 comprising a "LALA" mutation is according to SEQ ID NO:92 (f). Thus, as described herein, the amino acid sequence of IgG1 CH1-CH2-CH3 is preferably according to SEQ ID NO:92 or a functional sequence variant thereof, wherein the "LALA" mutation is maintained. Most preferably, the antibody is a form of FIT-Ig as described above, which contains a "LALA" mutation in the Fc portion described herein.

Preferably, the antibody or antigen binding fragment thereof according to the invention does not comprise a binding site for an Fc receptor. More preferably, the antibody or antigen-binding fragment thereof does not comprise an Fc region, even more preferably, the antibody or antigen-binding fragment thereof does not comprise an Fc portion. Various multispecific antibody formats without an Fc portion are known in the art and described above.

As mentioned above, the antibody or antigen-binding fragment thereof according to the present invention preferably specifically binds to (at least two) different epitopes on the envelope protein of ZIKV E protein. More preferably, the antibody or antigen-binding fragment thereof binds to domain III (EDIII, also referred to as "DIII") of the envelope protein of zika virus. In other words, preferably, the antibody or antigen-binding fragment thereof according to the present invention binds to an epitope of the envelope protein of zika virus comprising one or more than one amino acid residue of domain III (EDIII) of the envelope protein of zika virus. ZIKV comprises a nucleocapsid core comprising single-stranded RNA encapsulated by a core protein. The nucleocapsid core is surrounded by a lipid bilayer membrane with "membrane proteins" and "envelope proteins". The ZIKV envelope protein (E protein) is the dominant antigen. The extracellular domain of the envelope protein comprises three distinct domains: e protein domain I (EDI), E protein domain II (EDII) and E protein domain III (EDIII). EDIII is highly conserved between different ZIKV virus strains (alignment of EDIII amino acid sequences of different ZIKV virus strains is shown in fig. 8). Compared to antibodies that bind to domain I/II (EDI/II) of the envelope protein of zika virus, antibodies that bind to domain III (EDIII) of ZIKV neutralization showed (I) an increase, and (II) a decrease in cross-reactivity with DENV (particularly, essentially no cross-reactivity with DENV).

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

wherein X may be any (naturally occurring) amino acid. In other words, preferably, the antibody or antigen-binding fragment thereof according to the present invention binds to an epitope of the envelope protein of zika virus comprising one or more than one of SEQ ID NOs: 263, or a pharmaceutically acceptable salt thereof.

Also preferably, the antibody or antigen-binding fragment thereof according to the present invention more preferably binds to domain III (EDIII) of the envelope protein of Zika virus, the EDIII having the following amino acid sequence (SEQ ID NO: 265):

wherein X1 may be any (naturally occurring) amino acid, preferably 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;

x4 may 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 a or G;

X9 may be any (naturally occurring) amino acid other than R, preferably T or a;

x10 may be any (naturally occurring) amino acid, preferably V or I;

x11 may be any (naturally occurring) amino acid, preferably a 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 may 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.

In other words, preferably, the antibody or antigen-binding fragment thereof according to the present invention binds to an epitope of the envelope protein of zika virus comprising one or more than one of SEQ ID NOs: 265, or a pharmaceutically acceptable salt thereof.

For example, EDIII extends from amino acid 309 to amino acid 403 of the ZIKV E protein of the ZIKV H/PF/2013 strain (Genbank accession number KJ 776791). Thus, the antibody or antigen-binding fragment thereof most preferably binds to domain III (EDIII) of Zika virus envelope protein, which has the following amino acid sequence (SEQ ID NO: 264):

In other words, preferably, the antibody or antigen-binding fragment thereof according to the present invention binds to an epitope of the envelope protein of zika virus comprising one or more than one of SEQ ID NOs: 264, or a pharmaceutically acceptable salt thereof.

More preferably, the antibody or antigen-binding fragment thereof according to the present invention binds to an epitope of the envelope protein of zika virus comprising one or more amino acid residues of the Lateral Ridge (LR) of EDIII and/or one or more amino acid residues of the hinge region of EDI-EDIII. EDIII lateral ridges and EDI-EDIII hinge regions are known to the skilled person and are described, for example, in Zhao, h, fernandez, e, dow, k.a., sper, s.d., platt, d.j., gorman, m.j., govero, j, nelson, c.a., pierson, t.c., diamond, m.s., et al (2016). 1016-27 and Kostyuchenko VA, lim EX, zhang S, fibriansah G, ng TS, ooi JS, shi J, lok SM. Structure of the thermal stable Zika virus. Nature.2016 May 19;533 (7603): 425-8. Without being bound by any theory, it is hypothesized that (i) binding to LR may inhibit fusion by capturing the fusion transition state of the virus, and (ii) binding to the EDI-EDIII hinge and EDIII may hinder movement of EDIII to form a trimeric post-fusion structure, thereby halting membrane fusion.

Therefore, preferably, the antibody or antigen-binding fragment thereof according to the present invention (is capable of) inhibiting the post-attachment step of ZIKV. "post-attachment" generally refers to any step of ZIKV infection following ZIKV attachment to the cell membrane (of the cell targeted by the ZIKV). For example, the antibody or antigen-binding fragment thereof according to the present invention is preferably (capable of) preventing membrane fusion. Furthermore, it is also preferred that the antibody or antigen-binding fragment thereof according to the present invention is (capable of) causing aggregation of ZIKV (particles). Most preferably, the antibody or antigen-binding fragment thereof according to the present invention is (capable of) (i) inhibiting post-attachment steps of ZIKV and (ii) causing aggregation of ZIKV (particles).

Particularly preferably, the antibody or antigen binding fragment thereof according to the invention comprises at least one CDR, preferably all six CDRs, more preferably a variable region, even more preferably two variable regions (i.e. epitope binding sites), in particular the specificity of the exemplary human antibody ZKA190 binding ZIKV EDIII (see example 1, fig. 1). According to the present invention, the amino acid sequences of the CDRs and variable regions of ZKA190 preferably comprised in an antibody or antigen binding fragment thereof are described in tables 1 and 2 below.

It is also preferred that the antibody or antigen binding fragment thereof binds to a quaternary epitope displayed on the ZIKV infectious virion. Despite having a rather high neutralizing activity, such antibodies typically do not have detectable binding to recombinant ZIKV E proteins or ZIKV EDIII in standard ELISA (as described above), i.e., when tested in vitro, especially in purified form (i.e., ZIKV E proteins are "outside of, or do not have, virions, virus-like particles, etc.). Thus, "no detectable binding" typically means that no EC up to 10000ng/ml is detected in a standard ELISA ₅₀ . In other words, EC if detectable in a standard ELISA ₅₀ Above 10000ng/ml, this is termed "no detectable binding".

Accordingly, such antibodies are also referred to herein as "neutralizing non-E binding" (NNB) antibodies. The quaternary epitopes displayed on ZIKV infectious virions are typically conformational epitopes. For example, quaternary epitopes displayed on ZIKV infectious virions can be formed at the interface of two envelope protein monomers that make up a dimer ("envelope dimer epitopes"; EDEs), and can also be formed between adjacent dimers ("herringbone epitopes").

Particularly preferably, the antibody or antigen binding fragment thereof according to the invention comprises at least one CDR, preferably all six CDRs, more preferably a variable region, even more preferably two variable regions (i.e. epitope binding sites), in particular a specificity of binding to the exemplary human antibody ZKA230 on a quaternary epitope displayed on ZIKV infectious virions (see example 1, fig. 1). According to the present invention, the amino acid sequences of the CDRs and variable regions of ZKA230 preferably comprised in an antibody or antigen binding fragment thereof are described in tables 1 and 2 below.

Also preferred is an antibody or antigen-binding fragment thereof that binds to domain II (EDII) of the envelope protein of zika virus. EDII is an elongated finger domain containing a conserved fusion loop that interacts with the endosomal membrane of the Host cell, extending from about amino acid 52 to amino acid 132 of ZIKV E Protein, and from about amino acid 193 to amino acid 280 of ZIKV E Protein (Dai L, song J, lu X, deng YQ, musyoki AM, cheng H, zhang Y, yuan Y, song H, haywood J, xiao H, yan J, shi Y, qin CF, qi J, gao GF. Structure of the Zika Virus Envelope Protein and Its Complex with a vitamin broad reactive antibody. Cell Host be 11 (5): 696-704).

Particularly preferably, the antibody or antigen binding fragment thereof according to the invention comprises at least one CDR, preferably all six CDRs, more preferably variable regions, even more preferably two variable regions (i.e. epitope binding sites), in particular the specificity of the exemplary human antibody ZKA185 that binds ZIKV EDII. The amino acid sequences of the CDRs and variable regions of ZKA185 preferably comprised in an antibody or antigen binding fragment thereof according to the present invention are described in tables 1 and 2 below.

Preferably, the antibody or antigen-binding fragment thereof according to the present invention (i) binds to domain III (EDIII) of the envelope protein of zika virus and (II) binds to domain II (EDII) of the envelope protein of zika virus. In other words, preferably, the antibody or antigen-binding fragment thereof according to the present invention comprises (i) an epitope binding site that specifically binds to domain III (EDIII) of the envelope protein of zika virus, and (II) an epitope binding site that specifically binds to domain II (EDII) of the envelope protein of zika virus. As described above, it is preferred that domain III of the envelope protein of zika virus comprises SEQ ID NO:263 or 265, in particular SEQ ID NO:264 or consists thereof. Furthermore, as described above, it is also preferred that the antibody or antigen-binding fragment thereof binds to an epitope of the envelope protein of Zika virus that comprises one or more amino acid residues of the EDIII Lateral Ridge (LR) and/or one or more amino acid residues of the EDI-EDIII hinge region. In other words, the epitope binding site targeting EDIII preferably binds to one or more amino acid residues of the Lateral Ridge (LR) of EDIII and/or one or more amino acid residues of the EDI-EDIII hinge region.

Also preferably, the antibody or antigen-binding fragment thereof according to the present invention (i) binds to domain III (EDIII) of the envelope protein of zika virus and (ii) binds to a quaternary epitope displayed on ZIKV infectious virions. In other words, preferably, the antibody or antigen binding fragment thereof according to the present invention comprises (i) an epitope binding site that specifically binds to domain III (EDIII) of the Zika virus envelope protein, and (ii) an epitope binding site that specifically binds to a quaternary epitope displayed on a ZIKV infectious virion. As described above, it is preferred that domain III of the envelope protein of zika virus comprises SEQ ID NO:263 or 265, in particular SEQ ID NO:264 or consists thereof. Furthermore, as described above, it is also preferred that the antibody or antigen-binding fragment thereof binds to an epitope of the envelope protein of Zika virus that comprises one or more amino acid residues of the EDIII Lateral Ridge (LR) and/or one or more amino acid residues of the EDI-EDIII hinge region. In other words, the epitope binding site targeting EDIII preferably binds to one or more amino acid residues of the Lateral Ridge (LR) of EDIII and/or one or more amino acid residues of the EDI-EDIII hinge region.

Generally, a single epitope binding site (also referred to as a "paratope" or "antigen receptor") of an antibody or antigen binding fragment thereof according to the invention preferably comprises 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 in the heavy chain variable domain and the light chain variable domain. Typically, the CDRs of the heavy chain of an antibody and the linked light chain together form an antigen receptor (epitope binding site). Typically, the three CDRs (CDR 1, CDR2 and CDR 3) are arranged non-contiguously in the variable domain. Since epitope binding sites are typically composed of two variable domains (e.g., on two different polypeptide chains, i.e., a heavy chain and a light chain), there are six CDRs per epitope binding site (heavy chain: CDRH1, CDRH2 and CDRH3; light chain: CDRL1, CDRL2 and CDRL 3). The CDRs on the heavy and/or light chain can be separated by framework regions, where a Framework Region (FR) is a region of the variable domain that is less "variable" than the CDRs. For example, a chain (or each chain separately) may consist of four framework regions separated by three CDRs.

Sequences of heavy and light chains of various exemplary human anti-ZIKV E protein antibodies were determined, which contained three different CDRs on the heavy chain and three different CDRs on the light chain (see tables 1 and 2 below). The positions of the CDR amino Acids were determined according to the IMGT numbering system (IMGT: http:// www.imgt.org/; see Lefranc, M. -P. Et al (2009) Nucleic Acids Res.37, D1006-D1012). Preferably, the antibody or antigen-binding fragment thereof according to the invention comprises at least one CDR of the following exemplary antibodies. More preferably, the antibody or antigen-binding fragment thereof according to the invention comprises all six CDRs (of the epitope binding site) of the following exemplary antibody. Even more preferably, the antibody or antigen-binding fragment thereof according to the present invention comprises the heavy chain variable region (VH) and the light chain variable region (VL) of the following exemplary antibodies.

Table 1 shows the amino acid sequences of the heavy chain CDRs (CDRH 1, CDRH2 and CDRH 3) and heavy chain variable regions (referred to as "VH") of the exemplary antibodies in SEQ ID NOs:

table 2 below shows the amino acid sequences of the light chain CDRs (CDRL 1, CDRL2 and CDRL 3) and light chain variable regions (referred to as "VL") of the exemplary antibodies in SEQ ID NOs:

thus, preferably, an antibody or antigen-binding fragment thereof according to the invention comprises an amino acid 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% identity to at least one CDR sequence, VH sequence and/or VL sequence as set forth in table 1 and/or table 2.

Preferably, the antibody or antigen binding fragment thereof according to the invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein at least one CDR, preferably at least one heavy chain CDRH3 comprises an amino acid sequence according to SEQ ID NO: 3. 75, 39, 21, 57, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, and 261, or a functional 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 thereto.

More preferably, the antibody or antigen binding fragment thereof according to the invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein at least one CDR, preferably at least one heavy chain CDRH3 comprises a sequence according to SEQ ID NO: 3. 21, and any one of 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. More preferably, the antibody or antigen binding fragment thereof according to the invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein at least one CDR, preferably at least one heavy chain CDRH3 comprises a sequence according to SEQ ID NO: 3. 21, and 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%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

Also preferably, the antibody or antigen binding fragment thereof according to the present invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein at least one CDR, preferably at least one heavy chain CDRH3 comprises 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%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, or consists thereof.

Most preferably, the antibody or antigen binding fragment thereof according to the invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein at least one CDR, preferably at least one heavy chain CDRH3 comprises an amino acid sequence according to SEQ ID NO:3, 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 thereto.

More preferably, the antibody or antigen binding fragment thereof according to the invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein

(i) At least one heavy chain CDRH1 comprises a sequence according to SEQ ID NO: 1. 19, 37, 55, 73, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, and 259, 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 comprises a sequence according to SEQ ID NO: 2. 20, 38, 56, 74, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, and 260, or a functional 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 comprises a sequence according to SEQ ID NO: 3. 21, 39, 57, 75, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, and 261, 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.

Still more preferably, the antibody or antigen binding fragment thereof according to the invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein

(i) At least one heavy chain CDRH1 comprises a sequence according to SEQ ID NO: 1. 19, 37, 55 and 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity;

(ii) At least one CDRH2 comprises a sequence according to SEQ ID NO: 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 comprises a sequence according to SEQ ID NO: 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.

Even more preferably, the antibody or antigen binding fragment thereof according to the invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein

(i) At least one heavy chain CDRH1 comprises a sequence according to SEQ ID NO: 1. 19, and 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%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

(ii) At least one CDRH2 comprises a sequence according to SEQ ID NO: 2. 20, and 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%, 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 comprises a sequence according to SEQ ID NO: 3. 21, and 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%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

Still more preferably, the antibody or antigen binding fragment thereof according to the invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein

(i) At least one heavy chain CDRH1 comprises a sequence according to SEQ ID NO:1 or 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%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

(ii) At least one CDRH2 comprises a sequence according to SEQ ID NO:2 or 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%, 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 comprises a sequence according to SEQ ID NO:3 or 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%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

Also preferably, an antibody or antigen binding fragment thereof according to the invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein

(i) At least one heavy chain CDRH1 comprises a sequence according to SEQ ID NO:19 or 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%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

(ii) At least one CDRH2 comprises a sequence according to SEQ ID NO:20 or 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%, 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 comprises a sequence according to SEQ ID NO:21 or 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%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

Most preferably, the antibody or antigen binding fragment thereof according to the invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein

(i) At least one heavy chain CDRH1 comprises a 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 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 comprises a sequence according to 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 comprises a sequence according to SEQ ID NO:3, 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.

Also preferably, an antibody or antigen binding fragment thereof according to the invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein

(i) At least one CDRL1 comprises the amino acid sequence according to 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 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity;

(ii) At least one CDRL2 comprises a sequence according to 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 group comprises a sequence according to SEQ ID NO: 7. 25, 43, 61 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%, at least 98% or at least 99% sequence identity.

More preferably, the antibody or antigen binding fragment thereof according to the invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein

(i) At least one CDRL1 comprises a sequence according to SEQ ID NO: 4. 22, and 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%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

(ii) At least one CDRL2 comprises a sequence according to SEQ ID NO: 5. 6, 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 comprises a sequence according to SEQ ID NO: 7. 25, and 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%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

Even more preferably, the antibody or antigen binding fragment thereof according to the invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein

(i) At least one CDRL1 comprises a sequence according to SEQ ID NO:4 or 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%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

(ii) At least one CDRL2 comprises a sequence according to SEQ ID NO: 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 comprises a sequence according to SEQ ID NO:7 or 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%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

Also preferably, an antibody or antigen binding fragment thereof according to the invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein

(i) At least one CDRL1 comprises the amino acid sequence according to SEQ ID NO:22 or 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%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

(ii) At least one CDRL2 comprises the amino acid sequence according to SEQ ID NO: 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 comprises a sequence according to SEQ ID NO:25 or 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%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

Most preferably, the antibody or antigen binding fragment thereof according to the invention comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein

(i) At least one CDRL1 comprises a sequence according to 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 99% sequence identity;

(ii) At least one CDRL2 comprises the 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 comprises 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 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.

Preferably, the antibody or antigen-binding fragment thereof according to the invention comprises (i) an amino acid sequence according to SEQ ID NO:1 to 3 CDRH1, CDRH2 and CDRH3 amino acid sequences; 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) according to SEQ ID NO:19 to 21, 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) according to SEQ ID NO:37 to 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%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (iv) according to SEQ ID NO:55 to 57, 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; (v) according to SEQ ID NO:73 to 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; (vi) according to SEQ ID NO:99 to 101, 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; (vii) according to SEQ ID NO:103 to 105, 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; (viii) according to SEQ ID NO:107 to 109, 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; (ix) according to SEQ ID NO:111 to 113, 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; (x) according to SEQ ID NO:115 to 117 of CDRH1, CDRH2 and CDRH3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (xi) according to SEQ ID NO:119 to 121, 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; (xii) according to SEQ ID NO:123 to 125, 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; (xiii) according to SEQ ID NO:127 to 129 of CDRH1, CDRH2 and CDRH3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (xiv) according to SEQ ID NO:131 to 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 98%, or at least 99% sequence identity; (xv) the nucleotide sequence according to SEQ ID NO:135 to 137, 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; (xvi) the nucleotide sequence according to SEQ ID NO:139 to 141, 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; (xvii) according to SEQ ID NO:143 to 145, 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; (xviii) according to SEQ ID NO:147 to 149, or a functional sequence variant thereof having a sequence identity of 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%; (xix) according to SEQ ID NO:151 to 153, 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; (xx) according to SEQ ID NO:155 to 157 CDRH1, CDRH2 and CDRH3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (xxi) according to SEQ ID NO:159 to 161, 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; (xxii) according to SEQ ID NO:163 to 165, 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; (xxiii) the sequence according to SEQ ID NO:167 to 169, 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 to a CDRH1, CDRH2, and CDRH3 amino acid sequences; (xxiv) according to SEQ ID NO:171 to 173, 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; (xxv) according to SEQ ID NO:175 to 177, 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; (xxvi) according to SEQ ID NO:179 to 181, 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; (xxvii) according to SEQ ID NO:183 to 185 of CDRH1, CDRH2 and CDRH3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (xxviii) according to SEQ ID NO:187 to 189 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 to CDRH1, CDRH2 and CDRH3 amino acid sequences; (xxix) according to SEQ ID NO:191 to 193, 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; (xxx) the sequence according to SEQ ID NO:195 to 197, 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; (xxxi) according to SEQ ID NO:199 to 201, 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 to a CDRH1, CDRH2, and CDRH3 amino acid sequence; (xxxii) according to SEQ ID NO:203 to 205, 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; (xxxiii) according to SEQ ID NO:207 to 209, 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; (xxxiv) according to SEQ ID NO:211 to 213, 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; (xxxv) according to SEQ ID NO:215 to 217, 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; (xxxvi) according to SEQ ID NO: a CDRH1, CDRH2 and CDRH3 amino acid sequence of 219 to 221, or a functional sequence variant thereof having a sequence identity of 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%; (xxxvii) the sequence according to SEQ ID NO:223 to 225, 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; (xxxviii) the sequence according to SEQ ID NO:227 to 229, 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; (xxxix) according to SEQ ID NO:231 to 233 of CDRH1, CDRH2 and CDRH3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (xl) according to SEQ ID NO: a CDRH1, CDRH2 and CDRH3 amino acid sequence of 235 to 237, 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; (xli) according to SEQ ID NO:239 to 241, 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; (xlii) according to SEQ ID NO:243 to 245, 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; (xliii) according to SEQ ID NO:247 to 249, 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; (xliv) according to SEQ ID NO:251 to 253, 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; (xlv) according to SEQ ID NO:255 to 257 CDRH1, CDRH2 and CDRH3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; or (xlvi) according to SEQ ID NO:259 to 261, 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, preferably, the antibody or antigen-binding fragment thereof according to the invention comprises (i) an amino acid sequence according to SEQ ID NO:1 to 5 and 7 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences; 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) according to SEQ ID NO:1 to 4 and 6 to 7, and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (iii) according to SEQ ID NO:19 to 23 and 25 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (iv) according to SEQ ID NO:19 to 22 and 24 to 25 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (v) according to SEQ ID NO:37 to 41 and 43 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (vi) according to SEQ ID NO: the CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences of 37 to 40 and 42 to 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (vii) according to SEQ ID NO:55 to 59 and 61 and CDRL1, CDRH2 and CDRH3 amino acid sequences or functional sequence variants thereof having a sequence identity of 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%; (viii) according to SEQ ID NO:55 to 58 and 60 to 61, and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; (ix) according to SEQ ID NO:73 to 77 and 79 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; or (x) a sequence according to SEQ ID NO:73 to 76 and 78 to 79 and CDRL1, CDRH2 and CDRH3 amino acid sequences or CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

More preferably, the antibody or antigen binding fragment thereof according to the invention comprises (i) an amino acid sequence according to SEQ ID NO:1 to 5 and 7 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences; 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) according to SEQ ID NO:1 to 4 and 6 to 7, and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (iii) a sequence according to SEQ ID NO:19 to 23 and 25 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (iv) according to SEQ ID NO:19 to 22 and 24 to 25 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (v) according to SEQ ID NO:37 to 41 and 43 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (vi) the sequence according to SEQ ID NO:37 to 40 and 42 to 43, and CDRL1, CDRH2 and CDRH3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (vii) according to SEQ ID NO:73 to 77 and 79 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; or (viii) a sequence according to SEQ ID NO: the CDRH1, CDRH2 and CDRH3 amino acid sequences and the CDRL1, CDRL2 and CDRL3 amino acid sequences of 73 to 76 and 78 to 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

Even more preferably, the antibody or antigen-binding fragment thereof according to the invention comprises (i) a sequence according to SEQ ID NO:1 to 5 and 7 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences; 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) according to SEQ ID NO:1 to 4 and 6 to 7, and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (iii) according to SEQ ID NO:73 to 77 and 79 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; or (iv) a sequence according to SEQ ID NO:73 to 76 and 78 to 79 and CDRL1, CDRH2 and CDRH3 amino acid sequences or CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

Preferably, the antibody or antigen binding fragment thereof according to the invention comprises (i) a sequence according to SEQ ID NO: the CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences of 19 to 23 and 25; 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) according to SEQ ID NO:19 to 22 and 24 to 25 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (iii) a sequence according to SEQ ID NO:37 to 41 and 43 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; or (iv) a sequence according to SEQ ID NO: the CDRH1, CDRH2 and CDRH3 amino acid sequences and the CDRL1, CDRL2 and CDRL3 amino acid sequences of 37 to 40 and 42 to 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

Still more preferably, the antibody or antigen-binding fragment thereof according to the invention comprises (i) a heavy chain variable region according to SEQ ID NO:1 to 5 and 7 and the amino acid sequences of CDRH1, CDRH2 and CDRH3 and CDRL1, CDRL2 and CDRL 3; 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 (ii) a sequence according to SEQ ID NO:1 to 4 and 6 to 7 and CDRL1, CDRH2 and CDRH3 amino acid sequences or CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

Preferably, the antibody or antigen-binding fragment thereof according to the invention as described above comprises (i) an amino acid sequence according to SEQ ID NO:19 to 23 and 25 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences; 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 (ii) a sequence according to SEQ ID NO:19 to 22 and 24 to 25 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

Most preferably, the antibody or antigen-binding fragment thereof according to the invention comprises

(a) A first epitope binding site comprising (i) a sequence according to SEQ ID NO:1 to 5 and 7 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences; 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 (ii) a sequence according to SEQ ID NO:1 to 4 and 6 to 7, and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and

(b) A second epitope binding site comprising (i) a sequence according to SEQ ID NO:19 to 23 and 25 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences; 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 (ii) a sequence according to SEQ ID NO:19 to 22 and 24 to 25 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

Also preferably, the antibody or antigen binding fragment thereof according to the invention as described above comprises (i) an amino acid sequence according to SEQ ID NO: the CDRH1, CDRH2 and CDRH3 amino acid sequences and the CDRL1, CDRL2 and CDRL3 amino acid sequences of 37 to 41 and 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%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or (ii) a sequence according to SEQ ID NO: the CDRH1, CDRH2 and CDRH3 amino acid sequences and the CDRL1, CDRL2 and CDRL3 amino acid sequences of 37 to 40 and 42 to 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

More preferably, the antibody or antigen-binding fragment thereof according to the invention comprises

(a) A first epitope binding site comprising (i) a sequence according to SEQ ID NO:1 to 5 and 7 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences; 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 (ii) a sequence according to SEQ ID NO:1 to 4 and 6 to 7, and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and

(b) A second epitope binding site comprising (i) a sequence according to SEQ ID NO: the CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences of 37 to 41 and 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%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or (ii) a sequence according to SEQ ID NO: the CDRH1, CDRH2 and CDRH3 amino acid sequences and the CDRL1, CDRL2 and CDRL3 amino acid sequences of 37 to 40 and 42 to 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

Further, it is also preferred that the antibody or antigen binding fragment thereof according to the invention comprises a heavy chain variable region (VH) and optionally a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises an amino acid sequence according to SEQ ID NO: 8. 26, 44, 62, 80, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242, 246, 250, 254, 258, and 262, 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 thereto.

Furthermore, it is also preferred that the antibody or antigen-binding fragment thereof according to the invention comprises (i) 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 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 heavy chain variable region (VH) amino acid sequence of 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 at least 99% sequence identity; (ii) a 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% sequence identity and/or a heavy chain variable region (VH) 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) 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% sequence identity and/or a heavy chain variable region (VH) 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) 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% sequence identity and/or a heavy chain variable region (VH) 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 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 to the heavy chain variable region (VH) amino acid sequence of SEQ ID NO:81, 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 thereto.

More preferably, the antibody or antigen binding fragment thereof according to the invention comprises (i) 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 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 heavy chain variable region (VH) 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 98%, or at least 99% sequence identity; (ii) 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% sequence identity and/or a heavy chain variable region (VH) 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) 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% sequence identity and/or a heavy chain variable region (VH) 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 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 a heavy chain variable region (VH) 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 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 thereto.

Even more preferably, the antibody or antigen-binding fragment thereof according to the invention comprises (i) a sequence according to 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 to the heavy chain variable region (VH) amino acid sequence of 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 at least 99% sequence identity; or (ii) a 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 a heavy chain variable region (VH) 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 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 preferably, the antibody or antigen-binding fragment thereof according to the present invention comprises (i) 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% sequence identity to the heavy chain variable region (VH) amino acid sequence of 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; or (ii) a 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% sequence identity and/or a heavy chain variable region (VH) 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.

Still more preferably, the antibody or antigen-binding fragment thereof according to the invention comprises 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 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 heavy chain variable region (VH) 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 98%, or at least 99% sequence identity.

Also preferably, the antibody or antigen binding fragment thereof according to the invention as described above comprises 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% sequence identity and/or a heavy chain variable region (VH) 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.

Most preferably, the antibody or antigen binding fragment thereof according to the invention as described above comprises

(a) A first epitope binding site comprising 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 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 heavy chain variable region (VH) amino acid sequence of 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 at least 99% sequence identity; and

(b) A second epitope binding site comprising a 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% sequence identity and/or a heavy chain variable region (VH) 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 thereto.

Also preferably, the antibody or antigen binding fragment thereof according to the invention as described above comprises 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% sequence identity to the heavy chain variable region (VH) 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.

More preferably, the antibody or antigen binding fragment thereof according to the invention as described above comprises

(a) A first epitope binding site comprising 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 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 heavy chain variable region (VH) 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 98%, or at least 99% sequence identity; and

(b) A second epitope binding site comprising 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% sequence identity and/or a heavy chain variable region (VH) 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.

Particularly preferably, the antibody or antigen-binding fragment thereof according to the invention is in the form of a tandem Fab-Ig (FIT-Ig), and the outer Fab of the FIT-Ig format comprises an epitope binding site comprising (i) the amino acid sequence according to SEQ ID NO:1 to 5 and 7 and the amino acid sequences of CDRH1, CDRH2 and CDRH3 and CDRL1, CDRL2 and CDRL 3; 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 (ii) a sequence according to SEQ ID NO:1 to 4 and 6 to 7 and CDRL1, CDRH2 and CDRH3 amino acid sequences or CDRL1, CDRL2 and CDRL3 amino acid sequences, 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. More preferably, the external Fab in the form of FIT-Ig comprises an epitope binding site comprising 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 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 heavy chain variable region (VH) amino acid sequence of 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 at least 99% sequence identity. Even more preferably, the internal Fab of the FIT-Ig format comprises an epitope binding site comprising (i) an amino acid sequence according to SEQ ID NO: the CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences of 19 to 23 and 25; 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 (ii) a sequence according to SEQ ID NO:19 to 22 and 24 to 25 and CDRL1, CDRH2 and CDRH3 amino acid sequences 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%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. Still more preferably, the internal Fab of the FIT-Ig format comprises an epitope binding site comprising 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% sequence identity to the heavy chain variable region (VH) amino acid sequence of 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.

Preferably, the antibody or antigen binding fragment thereof according to the invention is for use as a medicament. In other words, the antibody or antigen-binding fragment thereof according to the present invention may be used in the preparation of a medicament. More preferably, the antibody or antigen-binding fragment thereof according to the invention is used for the prevention and/or treatment of Zika virus infection. In other words, the antibody or antigen-binding fragment thereof according to the invention may be used for the preparation of a medicament or for use in the prevention and/or treatment of Zika virus infection. This aspect is described in more detail below.

Nucleic acid molecules

In another aspect, the invention also provides a nucleic acid molecule comprising at least one polynucleotide encoding an antibody or antigen-binding fragment thereof according to the invention as described above, or a fragment thereof, wherein the fragment comprises at least one CDR of the antibody or antigen-binding fragment thereof.

A nucleic acid molecule is a molecule comprising, preferably consisting of, a nucleic acid component. The term nucleic acid molecule preferably refers to a DNA or RNA molecule. In particular, it is used synonymously with the term "polynucleotide". Preferably, the nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers covalently linked to each other by phosphodiester bonds of a sugar/phosphate backbone. The term "nucleic acid molecule" also encompasses modified nucleic acid molecules, such as DNA or RNA molecules that are base modified, sugar modified, or backbone modified, among others.

In the nucleic acid molecule according to the invention, the coding fragment of the antibody or antigen-binding fragment thereof according to the invention comprises at least one CDR of the antibody or antigen-binding fragment thereof. Tables 1 and 2 provide the SEQ ID numbers of the amino acid sequences of the CDRs and VH and VL of an exemplary antibody according to the present invention. Thus, the nucleic acid molecule according to the invention preferably comprises a polynucleotide encoding one or more than one of the amino acid sequences shown in table 1 and table 2. Preferably, the coding fragment of an antibody or antigen-binding fragment thereof according to the invention comprises three CDRs, more preferably all three heavy chain CDRs (CDRH 1, CDRH2, CDRH 3) of the epitope binding site and/or all three light chain CDRs (CDRL 1, CDRL2, CDRL 3) of the epitope binding site. Thus, it is preferred that the coding fragment of the antibody or antigen-binding fragment thereof comprises (exactly) three or six CDRs. Thus, the nucleic acid molecule according to the invention preferably comprises a polynucleotide encoding the amino acid sequences of three or six CDRs as shown in tables 1 and 2, in particular the corresponding CDRH1, CDRH2 and CDRH3 sequences and/or CDRL1, CDRL2 and CDRL3 sequences. More preferably, the coding fragment of the antibody or antigen-binding fragment thereof comprises a variable region, such as a heavy chain variable region (VH) and/or a light chain variable region (VL). Thus, it is preferred that the coding fragment of the antibody or antigen-binding fragment thereof comprises (exactly) one or two variable regions. Thus, the nucleic acid molecule according to the invention preferably comprises a polynucleotide encoding one or two variable region amino acid sequences as shown in tables 1 and 2. Most preferably, the coding fragment of the antibody or antigen-binding fragment thereof comprises the (intact) polypeptide chain of the antibody or antigen-binding fragment thereof according to the invention. Such a (intact) polypeptide chain may be, for example, a (intact) heavy chain or a (intact) light chain. However, such (intact) polypeptide chains may also comprise both heavy and light chain elements, as is known in the art for many multispecific antibody formats (e.g., polypeptide chains comprise a heavy chain constant region, but also, for example, a light chain variable region in addition to one or more heavy chain variable regions).

The nucleic acid molecule may be monocistronic, bicistronic, or polycistronic, such as a tricistronic. A bicistronic or polycistronic nucleic acid molecule is generally a nucleic acid molecule that may typically have two (bicistronic) or more than two (polycistronic) Open Reading Frames (ORFs). An "open reading frame" herein is a codon sequence that can be translated into a peptide or protein. More generally, according to the invention, the nucleic acid molecule of the invention comprises at least one polynucleotide encoding at least one antibody or antigen-binding fragment thereof. If the nucleic acid molecule according to the invention comprises more than one encoding polynucleotide, the second, third etc. encoding polynucleotides may encode other peptides/proteins and/or may also encode an antibody, e.g. an antibody according to the invention or a fragment thereof, which may be the same or different from the first antibody coding region. In a preferred embodiment, the nucleic acid molecule of the invention comprises at least two encoding polynucleotides, such as (exactly) two or three encoding polynucleotides, which all encode the same or different antibodies or fragments or variants thereof. For example, different fragments of an antibody according to the invention or antigen-binding fragments thereof may be encoded by different polynucleotides on the same nucleic acid molecule. In another embodiment of the invention, the nucleic acid molecule of the invention may encode more than one antibody or antibody fragment within the same coding region. In general, the nucleic acid molecules of the invention may be monocistronic, bicistronic, or polycistronic.

For example, in a preferred embodiment, the antibody or antigen-binding fragment thereof according to the invention may be a single chain antibody. In this case, it is preferred that the entire single chain of the antibody or an antigen-binding fragment thereof is encoded by one single polynucleotide. Thus, preferably, the nucleic acid molecule according to the invention is a monocistron, in particular it may comprise one (single) polynucleotide according to the invention encoding an antibody or an antigen binding fragment thereof.

Also preferably, the antibody or antigen-binding fragment thereof according to the invention comprises (exactly) two different polypeptide chains, e.g. a heavy chain and a light chain. This can be illustrated by a classical natural IgG molecule comprising two identical heavy chains and two identical light chains, and thus two different polypeptide chains (even though the antibody finally comprises four polypeptide chains, they may be encoded by two (different) polynucleotides). Preferably, such two different polypeptide chains (e.g. heavy and light chains) of an antibody or antigen-binding fragment thereof according to the invention may be encoded by two different polynucleotides which may be located on the same nucleic acid molecule, e.g. in a dicistronic nucleic acid molecule or on (exactly two) different nucleic acid molecules (e.g. each nucleic acid molecule may be monocistronic). Thus, preferably, the nucleic acid molecule according to the invention is a dicistronic molecule, in particular it may (exactly) comprise two polynucleotides (together) encoding an antibody or antigen-binding fragment thereof according to the invention. Also preferably, as mentioned above, the nucleic acid molecule according to the invention comprises (at least or exactly) one polynucleotide (e.g. a (complete) polypeptide chain) encoding a fragment of an antibody or antigen-binding fragment thereof according to the invention. Thus, preferably, the nucleic acid molecule according to the invention is a monocistron. Furthermore, an antibody or antigen-binding fragment thereof according to the invention may be encoded by a plurality (e.g. monocistronic) of nucleic acid molecules. For example, an antibody comprising two different polypeptide chains can be encoded by two polynucleotides located on two different nucleic acid molecules.

Also preferably, the antibody or antigen-binding fragment thereof according to the invention comprises (exactly) three different polypeptide chains. Thus, it is preferred that the nucleic acid molecule is a tricocistron (e.g., encoding all three polypeptide chains comprised in the antibody or antigen-binding fragment thereof according to the invention). However, the nucleic acid molecule is also preferably a monocistron or a bicistron, e.g. encoding one or both polypeptide chains comprised in such an antibody or antigen binding fragment thereof according to the present invention. For example, a plurality of nucleic acid molecules, e.g. two or three nucleic acid molecules, may (together) encode an antibody or antigen-binding fragment thereof according to the invention.

For example, the antibody or antigen-binding fragment thereof according to the invention is particularly preferably in the form of a tandem Fab-Ig (FIT-Ig) which typically comprises the following three polypeptide chains:

polypeptide 1 comprising or consisting of an outer Fab light chain and an inner Fab heavy chain comprising the constant domains CH1-CH2-CH3, preferably with a "LALA" mutation as described above, wherein preferably the outer Fab light chain is fused to the N-terminus of the inner Fab heavy chain, preferably without a linker.

-polypeptide 2 comprising or consisting of the heavy chain of the external Fab (VH and CH1 of the external Fab); and

-polypeptide 3 comprising or consisting of the light chain of an internal Fab.

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

Thus, preferably, the nucleic acid molecule according to the invention comprises a polynucleotide encoding polypeptide 1 of a FIT-Ig antibody according to the invention. More preferably, the nucleic acid molecule does not comprise a polynucleotide encoding polypeptide 2 or polypeptide 3 of a FIT-Ig antibody according to the invention. Such nucleic acid molecules are preferably monocistronic.

Also preferably, the nucleic acid molecule according to the invention comprises a polynucleotide encoding polypeptide 2 of the FIT-Ig antibody according to the invention. More preferably, the nucleic acid molecule does not comprise a polynucleotide encoding polypeptide 1 or polypeptide 3 of a FIT-Ig antibody according to the invention. Such nucleic acid molecules are preferably monocistronic.

Also preferably, the nucleic acid molecule according to the invention comprises a polynucleotide encoding polypeptide 3 of the FIT-Ig antibody according to the invention. More preferably, the nucleic acid molecule does not comprise a polynucleotide encoding polypeptide 1 or polypeptide 2 of a FIT-Ig antibody according to the invention. Such nucleic acid molecules are preferably monocistronic.

Also preferably, the nucleic acid molecule according to the invention comprises a polynucleotide encoding polypeptide 1 and a polynucleotide encoding polypeptide 2 of a FIT-Ig antibody according to the invention. More preferably, the nucleic acid molecule does not comprise a polynucleotide encoding polypeptide 3 of a FIT-Ig antibody according to the invention. Such nucleic acid molecules are preferably bicistronic.

Also preferably, the nucleic acid molecule according to the invention comprises a polynucleotide encoding polypeptide 1 and a polynucleotide encoding polypeptide 3 of a FIT-Ig antibody according to the invention. More preferably, the nucleic acid molecule does not comprise a polynucleotide encoding polypeptide 2 of the FIT-Ig antibody according to the invention. Such nucleic acid molecules are preferably bicistronic.

Also preferably, the nucleic acid molecule according to the invention comprises a polynucleotide encoding polypeptide 2 and a polynucleotide encoding polypeptide 3 of a FIT-Ig antibody according to the invention. More preferably, the nucleic acid molecule does not comprise a polynucleotide encoding polypeptide 1 of a FIT-Ig antibody according to the invention. Such nucleic acid molecules are preferably bicistronic.

Particularly preferably, the nucleic acid molecule according to the invention comprises a polynucleotide encoding polypeptide 1, a polynucleotide encoding polypeptide 2 and a polynucleotide encoding polypeptide 3 of a FIT-Ig antibody according to the invention. Such a nucleic acid molecule is preferably a tricaistron.

Typically, the nucleic acid molecule may be a DNA molecule or an RNA molecule. Examples of nucleic acid molecules and/or polynucleotides include, for example, recombinant polynucleotides, vectors, oligonucleotides, RNA molecules such as rRNA, mRNA, miRNA, siRNA or tRNA, or DNA molecules such as cDNA. Preferably, the nucleic acid molecule is a DNA plasmid or an mRNA molecule. The DNA plasmid is circular, preferably a double stranded DNA molecule.

Table 3 below provides the SEQ ID numbers of exemplary nucleic acid sequences encoding the CDRs and VH and VL of an exemplary antibody according to the present invention. Due to the redundancy of the genetic code, the present invention also encompasses sequence variants of these nucleic acid sequences, in particular such sequence variants encoding the same amino acid sequence.

Table 3 shows exemplary nucleic acid sequences for the CDRs and the heavy chain variable region (VH) and light chain variable region (VL) of five exemplary antibodies ("ZKA 190", "ZKA64", "ZKA230", "ZKA185", "ZKA 78").

Preferably, the sequence of the nucleic acid molecule according to the invention comprises the sequence according to SEQ ID NO:10 to 18, 28 to 36, 46 to 54, 64 to 72, and 82 to 90; or a functional sequence variant thereof or consists thereof.

Also preferably, the nucleic acid sequences according to the invention comprise nucleic acid sequences which are at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleic acids encoding the CDR, VH and/or VL sequences shown in table 1 and table 2, e.g. to the nucleic acids shown in table 3.

In general, nucleic acid molecules can be manipulated to insert, delete, or alter certain nucleic acid sequences. Such manipulation changes include, but are not limited to, changes to introduce restriction sites, to modify codon usage, to add or optimize transcriptional and/or translational regulatory sequences, and the like. Nucleic acids may also be altered to alter the encoded amino acids. For example, it may be useful to introduce one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions, deletions, and/or insertions into the amino acid sequence of the antibody. Such point mutations may modify effector function, antigen binding affinity, post-translational modification, immunogenicity, etc., may introduce amino acids to attach covalent groups (e.g., labels), or may introduce tags (e.g., for purification purposes). Mutations can be introduced at specific sites or can be introduced randomly and then selected (e.g., molecular evolution). For example, nucleic acids encoding any one or more than one of any CDR region, VH sequence and/or VL sequence of the (exemplary) antibodies of the invention can be randomly or directionally mutated to introduce different properties in the encoded amino acids. Such changes may be the result of an iterative process in which the initial changes are retained and new changes are introduced at other nucleotide positions. Furthermore, variations realized in separate steps may be combined. Different properties introduced into the encoded amino acids may include, but are not limited to, enhanced affinity.

In another aspect, the invention also provides a plurality of nucleic acid molecules as described above, wherein each nucleic acid molecule comprises at least one polynucleotide encoding an antibody fragment of the invention or an antigen-binding fragment thereof as described above. Preferably, according to the present invention, the plurality of fragments encoded by the plurality of nucleic acid molecules form an antibody or antigen-binding fragment thereof. In other words, preferably, a plurality of nucleic acid molecules according to the invention encode a (intact) antibody or a (intact) antigen-binding fragment thereof according to the invention. This means that, in particular, no further nucleic acid molecules (other than the plurality of nucleic acid molecules according to the invention) are required for encoding/generating the (intact) antibody according to the invention or the (intact) antigen-binding fragment thereof. For example, a plurality of nucleic acid molecules according to the invention encode all polypeptide chains of an antibody or antigen-binding fragment thereof according to the invention.

Preferred examples of a plurality of nucleic acid molecules according to the invention are (exactly) two nucleic acid molecules, wherein each nucleic acid molecule comprises one or more than one (e.g. two or three) polynucleotides, each polynucleotide encoding a (different) polypeptide chain of an antibody or antigen-binding fragment thereof according to the invention. Thus, the nucleic acid molecules of such a plurality of nucleic acid molecules are preferably monocistronic or bicistronic. Examples thereof are two nucleic acid molecules encoding together a FIT-Ig antibody according to the present invention as described above (e.g., one dicistronic nucleic acid molecule is conjugated to a corresponding monocistronic nucleic acid molecule such that FIT-Ig polypeptide 1, polypeptide 2 and polypeptide 3 are encoded by two nucleic acid molecules; as described above).

In another preferred example, the invention provides (exactly) three nucleic acid molecules, wherein each nucleic acid molecule comprises one (single) polynucleotide encoding a (different) polypeptide chain of an antibody or antigen-binding fragment thereof according to the invention. Therefore, the nucleic acid molecule in such a plurality of nucleic acid molecules is preferably a monocistron. Examples are three nucleic acid molecules (e.g., three (e.g., monocistronic) nucleic acid molecules, one encoding FIT-Ig polypeptide 1, another encoding FIT-Ig polypeptide 2, and a third encoding FIT-Ig polypeptide 3, as described above, that together encode a FIT-Ig antibody according to the present invention.

Carrier

Included within the scope of the present invention are vectors, e.g. expression vectors, comprising a nucleic acid molecule according to the invention. Preferably, the vector comprises a nucleic acid molecule as described above.

The term "vector" refers to a nucleic acid molecule, preferably to a recombinant nucleic acid molecule, i.e., a nucleic acid molecule not occurring in nature. In the context of the present invention, the vector is suitable for incorporating or comprising a desired nucleic acid sequence. Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors and the like. Storage vectors are vectors that allow for convenient storage of nucleic acid molecules. Thus, the vector may comprise, for example, a sequence corresponding to a desired antibody or antibody fragment thereof according to the invention. The expression vector may be used to produce an expression product such as RNA, e.g., mRNA, or a peptide, polypeptide or protein. For example, the expression vector may comprise sequences required for transcription of a sequence segment of the vector, such as a promoter sequence. A cloning vector is generally a vector that contains a cloning site, which can be used to incorporate a nucleic acid sequence into the vector. The cloning vector may be, for example, a plasmid vector or a phage vector. The transfer vector may be a vector suitable for transferring the nucleic acid molecule into a cell or organism, such as a viral vector. In the context of the present invention, the vector may be, for example, an RNA vector or a DNA vector. Preferably, the vector is a DNA molecule, such as a DNA plasmid. For example, a vector in the sense of the present application includes a cloning site, a selectable marker, e.g., an antibiotic resistance factor, and sequences suitable for replication of the vector, e.g., an origin of replication. Preferably, in the context of the present application, the vector is a plasmid vector.

In another aspect, the invention also provides a plurality of vectors according to the invention, preferably encoding an antibody or antigen-binding fragment thereof according to the invention. Thus, the preferred embodiments and examples of a plurality of nucleic acid molecules according to the invention as described above also apply to a plurality of vectors according to the invention.

Cells

In another aspect, the invention also provides methods of expressing an antibody or antigen-binding fragment thereof of the invention; and/or a cell comprising a vector according to the invention or a plurality of vectors according to the invention.

Examples of such cells include, but are not limited to, eukaryotic cells such as yeast cells, animal cells, or plant cells. Preferably, the cell is a mammalian cell, more preferably a mammalian cell line. Preferred examples include human cells, CHO cells, HEK293T cells, per.c6 cells, NS0 cells, human hepatocytes, myeloma cells or hybridoma cells.

In particular, cells can be transfected with a vector according to the invention or a plurality of vectors according to the invention, preferably with an expression vector or expression vectors. The term "transfection" refers to the introduction of a nucleic acid molecule, such as a DNA or RNA (e.g. mRNA) molecule, into a cell, preferably a eukaryotic cell. In the context of the present invention, the term "transfection" encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, preferably into eukaryotic cells, such as mammalian cells. Such methods include, for example, electroporation, lipofection, e.g., cationic lipid and/or liposome-based lipofection, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or cationic polymer-based transfection, e.g., DEAE-dextran or polyethyleneimine, among others. Preferably, the introduction is non-viral.

Furthermore, the cell of the invention may be stably or transiently transfected with a vector according to the invention or a plurality of vectors according to the invention, for example for expression of an antibody or antigen-binding fragment thereof according to the invention. Preferably, the cells are stably transfected with a vector according to the invention or a plurality of vectors according to the invention encoding an antibody or antigen-binding fragment thereof according to the invention. Alternatively, it is preferred to transiently transfect cells with a vector according to the present invention or a plurality of vectors according to the present invention encoding an antibody or antigen-binding fragment thereof according to the present invention.

Optional additional features of the antibody

The antibodies of the invention may, for example, be conjugated to a drug for delivery to a treatment site, or to a detectable label to facilitate imaging of a site containing cells of interest. Methods of binding antibodies to drugs and detectable labels, and methods of imaging using detectable labels, are well known in the art. Labeled antibodies can be used in a variety of assays employing a variety of labels. Detection of the formation of an antibody-antigen complex between an antibody of the invention and an epitope of interest may be facilitated by the attachment of a detectable substance to the antibody. Suitable detection means include the use of labels such as radionuclides, enzymes, coenzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, enzyme substrates or cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, and the like. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, 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; examples of suitable radioactive materials include 125I, 131I, 35S, or 3H. Such labeling agents may be used in a variety of well-known assays, such as radioimmunoassays, enzyme immunoassays such as ELISA, fluorescent immunoassays, and the like. Thus, the labeled antibodies according to the invention may be used, for example, in US 3766162; US 3791932; US 3817837; and in the assay described in US 4233402.

The antibodies of the invention may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent, or a radioactive metal ion or radioisotope. Examples of radioisotopes include, but are not limited to, I-131, I-123, I-125, Y-90, re-188, re-186, at-211, cu-67, bi-212, bi-213, pd-109, tc-99, in-111, and the like. Such antibody conjugates can be used to modify a given biological response; the drug moiety should not be construed as limited to classical chemotherapeutic agents. For example, the drug moiety may be a protein or polypeptide having a desired biological activity. Such proteins may include, for example, toxins such as abrin, ricin a, pseudomonas exotoxin, or diphtheria toxin.

The techniques for conjugating such therapeutic moieties to antibodies are well known. See, e.g., arnon et al (1985) "Monoclonal Antibodies for immunology of Drugs in Cancer Therapy," in Monoclonal Antibodies and Cancer Therapy, eds Reisfeld et al (Alan R.Liss, inc.), pp.243 to 256; edit Hellstrom et al (1987) "Antibodies for Drug Delivery," in Controlled Drug Delivery, robinson et al (second edition; marcel Dekker, inc.), pp.623 to 653; thorpe (1985) "Antibody Cariers of cytoxic Agents in Cancer Therapy: a Review, "in Monoclonal Antibodies'84: in Biological and Clinical Applications, edit Pinchera et al, pp.475 to 506 (edit Kurtis, milano, italy, 1985); "Analysis, results, and Future specificity of the Therapeutic Use of radioactive Antibodies in Cancer Therapy," in Monoclonal Antibodies for Cancer Detection and Therapy, edit Baldwin et al (Academic Press, new York, 1985), pages 303 to 316; and Thorpe et al (1982) immunol. Rev.62:119-158.

Alternatively, the antibody or antibody fragment thereof may be conjugated to a second antibody or antibody fragment thereof to form an antibody heteroconjugate, as described in US 4676980. Additionally, a linker may be used between the label and the antibody of the invention, e.g. as described in US 4831175. The antibody or antigen-binding fragment thereof may be directly labelled with radioiodine, indium, yttrium or other radioactive particles known in the art, for example as described in US 5595721. Treatment may consist of a combination therapy in which the conjugated and unconjugated antibodies are administered simultaneously or sequentially, for example according to WO00/52031; as described in WO 00/52473.

The antibodies of the invention may also be attached to a solid support. In addition, the antibodies of the invention or functional antibody fragments thereof may be chemically modified by covalent conjugation to a polymer, for example to increase their circulating half-life. Examples of polymers and methods of attaching them to peptides are described in US 4766106; US 4179337; shown in US 4495285 and US 4609546. In some embodiments, the polymer may be selected from polyoxyethylated polyols and polyethylene glycols (PEGs). PEG is soluble in water at room temperature and has the general formula: r (O-CH) ₂ -CH ₂ ) _n O-R, where R may be hydrogen, or a protecting group such as an alkyl or alkanol group. Preferably, the protecting group may have 1 to 8 carbons. For example, the protecting group is methyl. The symbol n is a positive integer. In one embodiment, n is 1 to 1000. In another embodiment, n is from 2 to 500. Preferably, the average molecular weight of PEG is from 1000 to 40000, more preferably, the molecular weight of PEG is from 2000 to 20000, even more preferably, The molecular weight of PEG is 3000 to 12000. In addition, PEG may have at least one hydroxyl group, for example PEG may have a terminal hydroxyl group. For example, the terminal hydroxyl group is activated to react with a free amino group on the inhibitor. However, it is understood that the type and number of reactive groups can be varied to achieve the covalently conjugated PEG/antibodies of the invention.

Water soluble polyoxyethylated polyols may also be used in the present invention. They include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), and the like. In one embodiment, POG is used. Without being bound by any theory, because the glycerol backbone of polyoxyethylated glycerol is the same backbone naturally found in, for example, mono-, di-, and triglycerides in animals and humans, the branch may not necessarily be considered a foreign substance in the body. The molecular weight of the POG may be the same as PEG. Another drug delivery system that can be used to increase the circulation half-life is liposomes. Methods of preparing liposome delivery systems are known to those skilled in the art. Other drug delivery systems are known in the art and are described, for example, in the literature of Poznansky et al (1980) and Poznansky (1984).

The antibodies of the invention may be provided in purified form. Typically, the antibody will be present in a composition that is substantially free of other polypeptides, e.g., where less than 90%, typically less than 60%, more typically less than 50% by weight of the composition is made up of other polypeptides.

The antibodies of the invention may be immunogenic in a non-human (or heterologous) host such as a mouse. In particular, antibodies may have unique positions that are immunogenic in a non-human host, but not in a human host. In particular, the antibody of the present invention for use in humans includes antibodies that cannot be easily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, and the like, and generally cannot be obtained by humanization or from xenogeneic mice.

Pharmaceutical composition

The present invention also provides pharmaceutical compositions comprising one or more than one of the following:

(i) An antibody or antibody fragment thereof according to the invention;

(ii) A nucleic acid molecule or a plurality of nucleic acid molecules according to the invention;

(iii) A vector or vectors according to the invention; and/or

(iv) A cell according to the invention.

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

The pharmaceutical composition may preferably further comprise a pharmaceutically acceptable carrier, diluent and/or excipient. Although the carrier or excipient may facilitate administration, it should not itself induce the production of antibodies harmful to the individual receiving the composition. It should also not be toxic. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive viral particles. Generally, the pharmaceutically acceptable carrier in the pharmaceutical composition according to the invention may be an active or inactive ingredient. Preferably, the pharmaceutically acceptable carrier in the pharmaceutical composition according to the invention is not an active ingredient with respect to Zika virus infection.

Pharmaceutically acceptable salts, such as inorganic acid salts, for example, hydrochloride, hydrobromide, phosphate and sulfate, or organic acid salts, for example, acetate, propionate, malonate and benzoate, can be used.

The pharmaceutically acceptable carrier in the pharmaceutical composition may additionally comprise liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by a subject.

The pharmaceutical composition of the present invention can be prepared in various forms. For example, the compositions may be prepared as injectables, or as liquid solutions or suspensions. Solid forms suitable for dissolution or suspension in a liquid carrier prior to injection (e.g., similar to Synagis) may also be prepared ^TM And Herceptin ^TM For reconstitution with sterile water containing a preservative). The compositions may be prepared for topical administration, for example as an ointment, cream or powder. The compositions may be prepared for oral administration, for example as tablets or capsules, as aerosols, or as syrups (optionally flavoured). The compositions may be prepared for pulmonary administration, for example as inhalants, using fine powders or sprays. The composition can be prepared into suppository or vaginal suppository. The compositions may be prepared for nasal, aural or ocular administration, for example in the form of drops. The composition may be in the form of a kit designed such that the combined composition is reconstituted immediately prior to administration to a subject. For example, lyophilized antibodies can be provided in kit form with sterile water or sterile buffer.

Preferably, according to the invention, the active ingredient in the composition is an antibody molecule, an antibody fragment or variants and derivatives thereof, in particular the active ingredient in the composition is an antibody, an antibody fragment or variants and derivatives thereof. As such, it may be susceptible to degradation in the gastrointestinal tract. Thus, if the composition is to be administered by the route of the gastrointestinal tract, the composition may comprise an agent that protects the antibody from degradation and is released once the antibody is absorbed from the gastrointestinal tract.

Gennaro (2000) Remington: the Science and Practice of Pharmacy,2 th edition, ISBN: a thorough discussion of pharmaceutically acceptable carriers is provided in 0683306472.

The pH of the pharmaceutical compositions of the present invention is typically from 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 using a buffer. The compositions may be sterile and/or pyrogen-free. The composition may be isotonic with respect to humans. In one embodiment, the pharmaceutical composition of the present invention is provided in an airtight container.

Within the scope of the present invention are compositions that exist in a variety of administration forms; such forms include, but are not limited to, those suitable for parenteral administration, for example by injection or infusion, for example by bolus injection or continuous infusion. When the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous medium, and may contain formulatory agents such as suspending, preservative, stabilising and/or dispersing agents. Alternatively, the antibody molecule may be in a dry form for reconstitution with a suitable sterile liquid prior to use. A carrier is generally understood to be a material suitable for storing, transporting and/or administering a compound, such as a pharmaceutically active compound, in particular an antibody according to the invention. For example, the carrier may be a physiologically acceptable liquid suitable for storage, transport and/or administration of a pharmaceutically active compound, in particular an antibody according to the invention. Once formulated, the compositions of the present invention can be administered directly to a subject. In one embodiment, the composition is suitable for administration to a mammal, such as a human subject.

The pharmaceutical compositions of the present invention may be administered by a number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal, or rectal routes. Painless subcutaneous jets may also be used to administer the pharmaceutical compositions of the present invention. Preferably, the pharmaceutical composition may be prepared for oral administration, e.g. as a tablet, capsule, etc., for topical administration, or as an injection, e.g. as a liquid solution or suspension, with particular preference the pharmaceutical composition is injectable. Solid forms suitable for dissolution or suspension in a liquid carrier prior to injection are also preferred, e.g. the pharmaceutical composition is in lyophilized form.

For injection, e.g. intravenous, cutaneous or subcutaneous injection, or injection at the site of disease, the active ingredient is preferably in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. One skilled in the art would be able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, ringer's injection, lactated ringer's injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included if desired. Whether a polypeptide, peptide or nucleic acid molecule, or other pharmaceutically useful compound according to the invention, to be administered to an individual, administration is preferably a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be), which is sufficient to benefit the individual. The actual amount administered, the rate of administration and the time course will depend on the nature and severity of the treatment. For injection, the pharmaceutical composition according to the invention may be provided, for example, in a pre-filled syringe.

The pharmaceutical compositions of the present invention as defined above may also be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions or solutions. For oral tablets, commonly used carriers include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When an oral aqueous suspension is required, the active ingredient, i.e. the transporter cargo conjugate molecule of the invention as defined above, is mixed with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

The pharmaceutical compositions of the present invention may also be administered topically, particularly when the target of treatment includes diseases that include areas or organs readily accessible by topical administration, such as, for example, including skin or any other accessible epithelial tissue. Suitable topical formulations are readily prepared for each of these areas or organs. For topical administration, the pharmaceutical compositions of the invention may be formulated as a suitable ointment comprising the pharmaceutical composition of the invention, in particular its components as defined above, suspended or dissolved in one or more than one carrier. Carriers for topical application include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition of the present invention may be formulated into a suitable lotion or cream. In the context of the present invention, suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, synthetic spermaceti, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

The dosage treatment may be a single dose regimen or a multiple dose regimen. In particular, the pharmaceutical composition may be provided in the form of a single dose product. Preferably, the amount of antibody in the pharmaceutical composition, particularly if provided in a single dose product form, is no more than 200mg, more preferably no more than 100mg, even more preferably no more than 50mg.

For example, the pharmaceutical composition according to the invention may be administered daily, e.g. once or several times daily, e.g. once, twice, three or four times daily, preferably once or twice daily, more preferably once daily 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 than 21 days, e.g. daily for 1, 2, 3, 4, 5, 6 months. Preferably, the pharmaceutical composition according to the invention may be administered weekly, e.g. once or twice weekly, for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks or 21 weeks or more than 21 weeks, e.g. weekly for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months, or weekly for 2 years, 3 years, 4 years or 5 years. Furthermore, the pharmaceutical composition according to the invention may preferably be administered monthly, e.g. once a month, or more preferably every other month for 1, 2, 3, 4 or 5 or more years. It is also preferred to administer continuously throughout the life cycle. Furthermore, only one administration is also envisaged, in particular for certain indications, such as prevention of 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), wherein one or more than one single dose is administered as soon as possible after zika virus infection. Also preferred is a prophylactic setting in which one or more than one single dose is administered to prevent Zika infection (i.e., prior to Zika infection, particularly in subjects that are not Zika immunized).

In particular, the amount of antibody or antigen-binding fragment thereof in the pharmaceutical composition according to the invention does not exceed 1g, preferably 500mg, more preferably 200mg, even more preferably 100mg, particularly preferably 50mg, for a single dose, e.g. a daily, weekly or monthly dose, preferably a weekly dose.

Pharmaceutical compositions typically comprise an "effective" amount of one or more than one antibody of the invention, i.e., an amount sufficient to treat, ameliorate, alleviate or prevent a target disease or disorder, or to exhibit a detectable therapeutic effect. Therapeutic effects also include reduction or alleviation of pathogenic effects or physical symptoms. The exact effective amount for any particular subject will depend upon their size, weight and health, the nature and extent of the disease, and the treatment or combination of treatments selected for administration. An effective amount for a given situation is determined by routine experimentation and is within the judgment of the clinician. For the purposes of the present invention, an effective dose is generally from about 0.005mg/kg to about 100mg/kg, preferably from about 0.0075mg/kg to about 50mg/kg, more preferably from about 0.01mg/kg to about 10mg/kg, even more preferably from about 0.02mg/kg to about 5mg/kg of an antibody of the invention (e.g., the amount of antibody in a pharmaceutical composition), relative to the body weight (e.g., in kg) of the individual to which it is administered.

Furthermore, the pharmaceutical composition according to the invention may also comprise additional active ingredients, which may or may not be other antibodies. The additional active ingredient is preferably a checkpoint inhibitor. It is also preferred that the ZIKV neutralizing antibodies or antigen-binding fragments thereof as described herein are combined with the ZIKV NS1 binding antibodies or antigen-binding fragments thereof as described herein as additional active ingredients (adjuvants). Thus, in addition to neutralizing ZIKV, the pathogenic effects of NS1 can be blocked. The pharmaceutical composition according to the invention may comprise one or more than one additional active ingredient, such as the adjuvants described below in the context of the combination therapy.

The antibody or antigen-binding fragment according to the invention may be present in the same pharmaceutical composition as the additional active ingredient or, preferably, the antibody or antigen-binding fragment according to the invention is comprised in a first pharmaceutical composition and the additional active ingredient is comprised in a second pharmaceutical composition different from the first pharmaceutical composition. Thus, if more than one additional active ingredient is envisaged, each additional active ingredient and the antibody or antigen-binding fragment according to the invention preferably consists of a different pharmaceutical composition. These different pharmaceutical compositions may be administered in combination/simultaneously or at separate times or at separate locations (e.g., separate parts of the body).

Preferably, the antibody or antigen-binding fragment according to the invention and the additional active ingredient provide an additive therapeutic effect or, preferably, a synergistic therapeutic effect. The term "synergistic" is used to describe a combined effect of two or more active agents that is greater than the sum of the individual effects of each respective active agent. Thus, where the combined effect of two or more agents results in a "synergistic inhibition" of an activity or process, the inhibition of that activity or process is greater than the sum of the inhibitory effects of each respective agent. The term "synergistic therapeutic effect" refers to a therapeutic effect observed with a combination of two or more therapeutic approaches, wherein the therapeutic effect (as measured by any one of a number of parameters) is greater than the sum of the individual therapeutic effects observed with the respective individual therapeutic approaches.

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

In one embodiment, a composition of the invention can comprise an antibody of the invention, wherein the antibody can comprise at least 50 wt% (e.g., 60 wt%, 70 wt%, 75 wt%, 80 wt%, 85 wt%, 90 wt%, 95 wt%, 96 wt%, 97 wt%, 98 wt%, 99 wt%, or greater than 99 wt%) of the total protein in the composition. In such compositions, the antibody is preferably in purified form.

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

In another embodiment, a method of preparing a pharmaceutical composition comprises the steps of: mixing an antibody with one or more than one pharmaceutically acceptable carrier, wherein the antibody is a monoclonal antibody obtained from a transformed B cell or a cultured plasma cell of the invention.

As an alternative to delivering antibodies or B cells for therapeutic purposes, nucleic acid (typically DNA) encoding a monoclonal antibody of interest (or active fragment thereof) derived from B cells or cultured plasma cells can be delivered to a subject such that the nucleic acid can be expressed in situ in the subject to provide a desired therapeutic effect. Suitable gene therapy and nucleic acid delivery vectors are known in the art.

In particular, if packaged in a multi-dose form, the pharmaceutical composition may comprise an antimicrobial agent. They may comprise a surfactant, for example a tween (polysorbate), for example tween 80. Surfactants are typically present at low levels, for example less than 0.01%. The composition may also include a sodium salt (e.g., sodium chloride) to generate tonicity. For example, typical NaCl concentrations are 10. + -.2 mg/ml.

Furthermore, in particular if the pharmaceutical composition is to be lyophilized or if the pharmaceutical composition comprises a material that has been reconstituted from a lyophilized material, the pharmaceutical composition may comprise, for example, a sugar alcohol (e.g., mannitol) or a disaccharide (e.g., sucrose or trehalose) in a range of about 15mg/ml to 30mg/ml (e.g., 25 mg/ml). Prior to lyophilization, the pH of the composition for lyophilization may be adjusted to 5 to 8, or 5.5 to 7, or about 6.1.

The compositions of the invention may also comprise one or more than one immunomodulator. In one embodiment, the one or more immunomodulatory agents comprise an adjuvant.

Drug therapy, kit and use

Medical treatment

In another aspect, the invention provides the use of an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid molecule according to the invention, a vector according to the invention, a plurality of nucleic acid molecules according to the invention, a plurality of vectors according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention for (i) preventing and/or treating zika virus infection or (ii) diagnosing zika virus infection. Thus, the use of an antibody or antigen-binding fragment thereof according to the invention (in particular a preferred embodiment thereof as described above) or a nucleic acid molecule(s) according to the invention encoding an antibody or antigen-binding fragment thereof according to the invention is preferably for (i) the prevention and/or treatment of zika virus infection as described herein; or (ii) use as described herein to diagnose Zika virus infection.

Diagnostic methods may include contacting the antibody or antibody fragment with a sample. Such a sample may be isolated from a subject, for example by extracting an isolated tissue sample from, for example, the nasal cavity, sinus cavity, salivary gland, lung, liver, pancreas, kidney, ear, eye, placenta, digestive tract, heart, ovary, pituitary, adrenal gland, thyroid, brain, skin, or blood, preferably plasma or serum. The diagnostic method may also comprise the detection of the antigen/antibody complex, in particular after the antibody or antibody fragment has been contacted with the sample. Such detection steps are usually carried out on a bench, i.e. 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 is particularly intended to mean a prophylactic environment in which the subject has not been diagnosed with Zika virus infection (has not been diagnosed or has a negative diagnosis) and/or the subject does not show symptoms of Zika virus infection. Thus, preventing Zika virus infection includes "post-exposure prevention" (PEP), i.e., prophylactic treatment after a possible Zika virus infection, e.g., after a mosquito bite in a Zika virus infected area. Preventing Zika virus infection is particularly useful in high risk subjects, such as pregnant women and/or subjects in areas affected by Zika virus (e.g., subjects who live in or travel to Zika virus-infected areas).

In contrast, in a therapeutic setting, a subject is typically infected with Zika virus, diagnosed with Zika virus infection, and/or exhibits symptoms of Zika virus infection. It is noted that the terms "treatment" and "treating" of ZIKV infection include (complete) cure as well as mitigation of ZIKV infection.

Accordingly, the antibody or antigen-binding fragment thereof according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the plurality of nucleic acid molecules according to the invention, the plurality of vectors according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention are preferably used for the treatment of a zika virus infection in a subject diagnosed with or showing symptoms of a zika virus infection.

Also preferred is an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid molecule according to the invention, a vector according to the invention, a plurality of nucleic acid molecules according to the invention, a plurality of vectors according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention for use in the prevention and/or treatment of Zika virus infection in an asymptomatic subject. Those subjects may or may not be diagnosed as infected with the Zika virus.

Preferably, the antibody or antigen-binding fragment thereof according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the plurality of nucleic acid molecules according to the invention, the plurality of vectors according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention is used for preventing and/or treating zika virus infection in a pregnant female subject, in particular for preventing congenital infection. For example, this can be done in a manner similar to the prevention of Congenital infection with HCMV, such as Nigro G, adler SP, la Torre R, best AM, genetic cytology cloning Group: a Passive ionization suppression for genetic cytokine introduction; n Engl J Med 2005, 353: 1350-1362.

Without being bound by any theory, it is hypothesized that the antibody or antigen binding fragment thereof according to the invention may cross the placenta by interacting with FcRn, e.g., where administered to a pregnant subject, e.g., by intravenous (i.v.) injection or any other route of administration described herein. Importantly, the interaction of the "LALA" variant of the antibodies described herein with FcRn is not impaired. FcRn is believed to have been expressed in the placenta of early pregnancy.

Alternatively, the antibodies or antigen-binding fragments thereof or nucleic acid molecule(s) of the invention may also be administered to the extraamniotic space.

Preferably, the antibody or antigen-binding fragment thereof according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention is for use in the prevention and/or treatment of zika virus infection, wherein the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the cell according to the invention is administered up to seven days after (possible) zika virus infection, preferably up to five days after (possible) zika virus infection, more preferably four days after (possible) zika virus infection, even more preferably three days after (possible) zika virus infection, most preferably one or two days after (possible) zika virus infection. Such treatment regimens may be useful in a therapeutic setting as well as a prophylactic setting, particularly for Post Exposure Prophylaxis (PEP).

In a PEP, typically, the first administration of the antibody or antigen-binding fragment thereof according to the present invention, the nucleic acid molecule according to the present invention, the vector according to the present invention, the nucleic acid molecules according to the present invention, the vectors according to the present invention, the cell according to the present invention or the pharmaceutical composition according to the present invention is performed as soon as possible after a possible ZIKV infection, e.g. after a mosquito bite in a ZIKV infected area. Thus, as mentioned above, in a PEP, the first administration of an antibody or antigen-binding fragment thereof according to the present invention, a nucleic acid molecule according to the present invention, a vector according to the present invention, a plurality of nucleic acid molecules according to the present invention, a plurality of vectors according to the present invention, a cell according to the present invention or a pharmaceutical composition according to the present invention is typically performed one or more days after the (possible) ZIKV infection.

Also preferred is an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid molecule according to the invention, a vector according to the invention, a plurality of nucleic acid molecules according to the invention, a plurality of vectors according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention for use in the prevention and/or treatment of zika virus infection, wherein the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the plurality of nucleic acids, the plurality of vectors, the cell according to the invention or the pharmaceutical composition according to the invention is administered up to three months prior to (possible) zika virus infection, preferably up to one month prior to (possible) zika virus infection, more preferably up to two weeks prior to (possible) zika virus infection, even more preferably up to one week prior to (possible) zika virus infection, most preferably up to one day prior to (possible) zika virus infection. Such a treatment regimen is particularly directed to a prophylactic environment.

In general, in particular in PEP, after the first administration of the antibody or antigen-binding fragment thereof according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the plurality of nucleic acid molecules according to the invention, the plurality of vectors according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention, one or more than one administration may follow, preferably a single dose is administered daily or every other day for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days, preferably for each day. It is also preferred that after the first administration of the antibody or antigen-binding fragment thereof according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the plurality of nucleic acid molecules according to the invention, the plurality of vectors according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention, one or more than one, preferably one or two, administration of a single dose per week may be followed for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks or 21 weeks, preferably for one or more than one administration per week. It is also preferred that after the first administration of the antibody or antigen-binding fragment thereof according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the plurality of nucleic acid molecules according to the invention, the plurality of vectors according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention, one or more than one, preferably a single dose every 2 weeks or 4 weeks, may be followed for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks or 21 weeks. It is also preferred that after the first administration of the antibody or antigen-binding fragment thereof according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the plurality of nucleic acid molecules according to the invention, the plurality of vectors according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention, one or more than one administration may follow, preferably a single dose is administered every 2 months or 4 months for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months or 21 months. It is also preferred that after the first administration of an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid molecule according to the invention, a vector according to the invention, a plurality of nucleic acid molecules according to the invention, a plurality of vectors according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention, one or more than one administration, preferably one or two administrations per year of a single dose for 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years or 10 years may follow.

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

The antibody or antigen-binding fragment thereof according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the plurality of nucleic acid molecules according to the invention, the plurality of vectors according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention may be administered by a number of routes, such as oral, intravenous, intramuscular, intra-arterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal or rectal routes. Intravenous or subcutaneous or intramuscular administration is preferred, intravenous or subcutaneous administration being more preferred.

In a pregnant subject, the antibody or antigen-binding fragment thereof or nucleic acid molecule(s) of the invention may also be administered, e.g., by injection, intra-or extra-amniotic membrane.

Accordingly, the present invention also provides a method of preventing and/or treating zika virus infection in a subject, wherein the method comprises administering to a subject in need thereof an antibody or antigen-binding fragment thereof according to the present invention, a nucleic acid molecule according to the present invention, a vector according to the present invention, a plurality of nucleic acid molecules according to the present invention, a plurality of vectors according to the present invention, a cell according to the present invention, or a pharmaceutical composition according to the present invention. Preferred embodiments of the method correspond to preferred embodiments of the medical use as described above (and also the embodiments below relating to combination therapy). For example, a preferred subject for this method is a subject diagnosed with or showing symptoms of Zika virus infection. Another preferred subject in the method is a pregnant subject.

Combination therapy

The administration of the antibody or antigen-binding fragment thereof according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the nucleic acid molecules according to the invention, the vectors according to the invention, the cells according to the invention or the pharmaceutical composition according to the invention may be carried out alone or in combination with adjuvants (also referred to herein as "additional active ingredients") in the methods and uses according to the invention, which are particularly useful for the prevention and/or treatment of ZIKV infections.

The invention includes the administration of an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid molecule according to the invention, a vector according to the invention, a plurality of nucleic acid molecules according to the invention, a plurality of vectors according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention, wherein it is administered to a subject prior to, simultaneously with or sequentially with other therapeutic regimens or adjuncts for the treatment and/or prevention of ZIKV infection. The antibody, nucleic acid, vector, cell or pharmaceutical composition administered concurrently with the adjuvant may be administered in the same or different composition and by the same or different route of administration.

The other treatment regimen or adjuvant may be, for example, a checkpoint inhibitor.

Thus, in a further aspect of the invention, an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid molecule according to the invention, a vector according to the invention, a plurality of nucleic acid molecules according to the invention, a plurality of vectors according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention is administered in combination with a checkpoint inhibitor for the (medical) use described herein.

Preferred checkpoint inhibitors are directed against blockade of PD-1/PD-L1 and/or CTLA4 and thus include anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA 4 antibodies. Thus, the pharmaceutical composition according to the invention may comprise one or more than one additional active ingredient.

It is also preferred that the ZIKV neutralizing antibodies or antigen-binding fragments thereof as described herein are combined with ZIKV NS 1-binding antibodies or antigen-binding fragments thereof as additional active ingredients (adjuvants). Thus, in addition to neutralizing ZIKV, the pathogenic effects of NS1 can be blocked. Thus, for example, as described in PCT/EP2017/067581, which is incorporated herein in its entirety, ZIKV NS1 binding antibodies or antigen binding fragments thereof are preferred additional active ingredients (adjuvants).

The antibody or antigen-binding fragment thereof according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the plurality of nucleic acid molecules according to the invention, the plurality of vectors according to the invention or the cell according to the invention may be present in the same pharmaceutical composition as the additional active ingredient (adjuvant) or, preferably, the antibody or antigen-binding fragment thereof according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the plurality of nucleic acid molecules according to the invention, the plurality of vectors according to the invention or the cell according to the invention is comprised in a first pharmaceutical composition and the additional active ingredient (adjuvant) is comprised in a second pharmaceutical composition different from the first pharmaceutical composition. Thus, if more than one additional active ingredient (adjuvant) is envisaged, each additional active ingredient (adjuvant) preferably constitutes a different pharmaceutical composition. These different pharmaceutical compositions may be administered in combination/simultaneously or at separate times or at separate locations (e.g., separate parts of the body).

Preferably, the antibody or antigen-binding fragment according to the invention and the additional active ingredient (adjuvant) provide an additive therapeutic effect or, preferably, a synergistic therapeutic effect. The term "synergistic" is used to describe a combined effect of two or more active agents that is greater than the sum of the individual effects of each respective active agent. Thus, where the combined effect of two or more agents results in a "synergistic inhibition" of an activity or process, the inhibition of that activity or process is greater than the sum of the inhibitory effects of each respective agent. The term "synergistic therapeutic effect" refers to a therapeutic effect with a combination of two or more therapeutic approaches observed, wherein the therapeutic effect (as measured by any one of a number of parameters) is greater than the sum of the individual therapeutic effects observed with the corresponding individual therapeutic approaches.

Other uses and kits

In another aspect, the invention also provides an antibody or antigen binding fragment thereof according to the invention, a nucleic acid molecule according to the invention, a vector according to the invention, a plurality of nucleic acid molecules according to the invention, a plurality of vectors according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention for use in monitoring the quality of an anti-zika vaccine by checking that the antigen of said vaccine comprises a specific epitope in the correct conformation. Preferred antigens comprised by such anti-zika vaccines to be tested include or consist of the ZIKV envelope protein or any other molecule/complex comprising (i) domain III (EDIII) of the ZIKV E protein as described above or (ii) a quaternary ZIKV epitope as described above.

Furthermore, the invention provides the use of an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid molecule according to the invention, a vector according to the invention, a plurality of nucleic acid molecules according to the invention, a plurality of vectors according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention for (in vitro) diagnosing Zika virus infection.

Furthermore, there is provided the use of an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid molecule according to the invention, a vector according to the invention, a plurality of nucleic acid molecules according to the invention, a plurality of vectors according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention for determining whether an isolated blood sample (e.g. whole blood, serum and/or plasma) is infected with zika virus.

As described above, the diagnostic method may comprise contacting the antibody or antibody fragment with the sample. Such a sample may be isolated from a subject, for example by extracting an isolated tissue sample from, for example, the nasal cavity, sinus cavity, salivary gland, lung, liver, pancreas, kidney, ear, eye, placenta, digestive tract, heart, ovary, pituitary, adrenal gland, thyroid, brain, skin, or blood, preferably serum or plasma. Diagnostic methods may also include detection of antigen/antibody complexes, particularly after the antibody or antibody fragment is contacted with the sample. Such detection steps are usually carried out on a bench, i.e. 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).

In another aspect, the invention also provides a kit of parts comprising at least one antibody or antigen-binding fragment thereof according to the invention, at least one nucleic acid according to the invention, a plurality of nucleic acids according to the invention, at least one vector according to the invention, a plurality of vectors according to the invention, at least one cell according to the invention and/or at least one pharmaceutical composition according to the invention. In addition, the kit may comprise means, such as a syringe or vessel, instructions and/or adjuvants to be administered as described above, for administering an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid molecule according to the invention, a vector according to the invention, a plurality of nucleic acids according to the invention, a plurality of vectors according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention.

Drawings

FIG. 1 shows reactivity (ELISA) and ZIKV and DENV1 neutralizing activity of antibodies derived from four ZIKV immune donors (ZKA, ZKB, ZKC, and ZKD) against the E proteins of ZIKV and DENV1-4 and the EDIII domain of the ZIKV E protein; NNB-neutralizing non-E protein binding antibody.

Fig. 2 shows the binding of ZKA190, ZKA78 and ZKA64 antibodies to ZIKV and DENV1E and ZIKV EDIII proteins measured by ELISA.

Figure 3 shows the binding of ZKA185 and ZKA190 antibodies to ZIKV E, DENV1 VLP and ZIKV EDIII proteins measured by ELISA.

Fig. 4 shows the neutralizing activity of ZKA190, ZKA64-LALA, ZKA230 and ZKA78 antibodies in example 3 against ZIKV (H/PF/2013 strain) and DENV1 on Vero cells as measured by flow cytometry (% of cells infected).

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

Fig. 6 shows infection-enhancing activity (ADE, antibody-dependent enhancement) of ZKA190, ZKA64-LALA, ZKA185, ZKA230, and ZKA78 antibodies of example 4 against ZIKV (H/PF/2013 strain) on unlicensed K562 cells as measured by flow cytometry (% cells infected).

Figure 7 shows that four ZIKV immune plasmas and one DENV immune plasma in example 4 show similar ability to enhance ZIKV infection of K562 cells (upper panel). EDIII-specific ZKA64-LALA antibodies completely blocked the ADE effect in all five immune plasmas (lower panel).

Figure 8 shows an amino acid alignment of the EDIII regions of 39 ZIKV strains from asian lineages since 2013 (including the prototype strain of african lineages MR766 isolated in 1947).

Fig. 9 shows the neutralizing activity of ZKA190 and ZKA190-LALA antibodies of example 3 against three ZIKV strains on Vero cells (H/PF/2013, mr766 and MRS _ OPY _ martini island _ pasi _ 2015), as measured by flow cytometry (% cells infected).

Figure 10 shows the neutralization of ZKA190 and C8 mabs of example 5 tested against a panel of four ZIKV strains, as determined by the percentage of infected Vero cells in the presence of increasing amounts of mAb (a). IC50 values (B) and statistics (C) are also shown. Data are representative of at least two independent experiments.

Fig. 11 shows neutralization and enhancement of antibody ZKA190 against ZIKV infection in example 6. (A) Neutralization of ZKA190, ZKA190-LALA and control mAb against infection with ZIKV PRVABC59 strain of hNPC was determined on Vero cells by plaque assay (left panel) and indirect immunofluorescence assay of infected hNPC using fluorophore-labeled anti-E antibody (right panel). (B) ZIKV-infected ADE of non-permissive K562 cells by ZKA190 and ZKA 190-LALA. (C) ADE induced in K562 cells when ZIKV was preincubated with serial dilutions of plasma sera from different ZIKV positive patients (left panel). When ZKA190 LALA was added to ZIKV-serum complexes, ADE was inhibited (right panel). (D) ADE induced in K562 cells when ZIKV was preincubated with serial dilutions of prM cross-reactive mAb (DV 62) derived from DENV immune donors. ZKA190-LALA inhibits ADE of ZIKV when complexed with the prM-reactive antibody DV 62. (E) The effect on ADE induced by dilution was enhanced by peaks of DENV2 plasma (left panel) or anti-prM DV62 mAb (right panel) by serial dilution of indicated mabs.

FIG. 12 shows the identification of the ZKA190 epitope in example 7 and its conservation analysis in ZIKV virus strains. (A) In the absence (black) or presence (red) of unlabeled ZKA190 Fab, ¹⁵ n-labeled ZIKV EDIII ¹⁵ N， ¹ H]Superposition of HSQC spectra. The differences identify EDIII residues affected by antibody binding. (B) NMR epitope mapping of ZKA190 Fab complexed with ZKV EDIII. Chemical shift perturbations (CSP, y-axis) are plotted against the number of EDIII residues. Residues affected by antibody binding are red in color. (C) Residues in the FG loop identified by NMR epitope mapping are partially hidden in model a of the E protein, but mostly exposed in models B and C. EDIII of the E protein was stained blue. By NThe residues identified by MR epitope mapping were stained with red, but the residues in the FG loop were stained with green. The adjacent E protein is shown as a grey surface. (D) By 24 days 11 months 2016, the level of amino acid residue conservation of the ZKA190 epitope was calculated by analyzing the sequences of 217 ZIKV strains found in the ZIKV resources (NCBI) database. (E) The open book representation shows charge complementarity between the epitope and the sublabel of the docking result. The boundaries of the epitope and the sublope are circled in green. The border between the heavy and light chain of the Fab and its corresponding blot on EDIII are shown as yellow dashed lines.

Fig. 13 shows the ZKA190 epitope identified by NMR and docking in example 7. (A) Cartoon representation of the 12 lowest energy NMR structures of ZIKV EDIII, red indicates residues affected by ZKA190 binding. The flexibility of the N-terminus of the structure is apparent. (B) Checking by NMR results calculating ZKA190 from docking and molecular modeling: EDIII complex model. The epitope on EDIII (grey) identified by NMR was red. The ZKA190 heavy and light chains are colored dark green and light green, respectively. EDIII residues that either affected or did not affect antibody binding when mutated are shown as orange and blue bars, respectively. (C) The NMR identified ZKA190 epitope (red) is accessible from the virus surface (white).

FIG. 14 shows the binding of wt or mutated EDIII to ZKA190 IgG in example 7 and example 10. SPR data and binding kinetics are shown. As shown, EDIII mutants that either affected (highlighted in red) or did not affect binding are shown.

FIG. 15 shows the results of the confocal microscopy experiment in example 8. ZIKV at concentrations over 10000 times greater than ZKA190 Fab or intact IgG IC50 values were added to Vero cells. ZIKV: the antibody complex was detected inside the cell (green) and co-localized with endosomes (red, yellow overlay). The endosome and acidic organelles are labeled with a lysosome red fluorescent probe; alexa-488 conjugated ZKA190 was green. Nuclei were stained with DAPI (blue).

Fig. 16 shows the prophylactic and therapeutic efficacy of ZKA190 of example 9. (A) ZKA190 has a strong protective effect against ZIKV infection when a lethal dose of mice challenged with the ZIKV strain MP17451 (a 129 in (a) and AG129 in (B)) was administered prophylactically. Experimental N =4 to 8 mice were used per group. A Kaplan-Meier survival curve (A) is shown. Significance was determined by using the Mantel-Cox log rank test. Panel a, upper left: ZKA190 at concentrations of 5mg/kg, 1mg/kg and 0.2mg/kg versus control mAb, P =0.0031; ZKA190 at 0.04mg/kg and control mAb were, P =0.0116; ZKA190-LALA at concentrations of 5mg/kg, 1mg/kg, 0.2mg/kg and 0.04mg/kg with control mAb, P =0.0031. Panel a, upper right: the mice were monitored for incidence score from day 14 to day 15 (using two different scoring methods; 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 summary Mouse Model for Zika Virus infection. Plos Negl tradis 10, e 0004658-13. Figure a, lower panel: mice ' body weight. Figure B: mice were administered ZKA190 or ZKA190-LALA. Figure B at different time points after ZIKV infection at a dose of 15mg/kg, upper left panel: mice's body weight-Meier survival curve =5 mice were monitored for incidence score by using either a cza map 2 or a map B with a log-t h r-t h r.p.p.p.r. (r.: the mice ' s.p.p.p.p.p.p.p.p.p.p.p.p.p.p.p.p.p.p.p.p.p.p.p.p.p.p. 14, koch. (r.p.p.p.p.p.p.p.p.p.p.p.p.: a. 1, p.p.p.p.p.p.p.p.p.p.p.p. 5. A).

Fig. 17 shows the prophylactic efficacy of the anti-ZIKV EDIII specific mAb ZKA190 of example 9 against ZIKV strain MP 1741. (A) Viremia measured at PFU/ml on day 5 in the blood of all animals is shown. (B) Viral load was measured by qPCR as genomic copies/mL in the blood of all animals on day 5 and at the end of the study slaughtering the animals or at the time of reaching the humanistic endpoint in the blood and indicated tissues. (C) weight loss in mice was monitored over a 14 day period. (D) human serum IgG concentration in blood samples on day 5. Significance compared to control antibody treatment was determined by nonparametric unpaired Mann-Whitney U test. * p is less than 0.05; * P < 0.01; * P < 0.001.

Fig. 18 shows the therapeutic efficacy of the anti-ZIKV EDIII specific mAb ZKA190 of example 9. (A) On day 5, the viral load in the blood of all animals was measured as PFU. (B) Viral load was measured as genomic copies by qPCR in the blood of all animals on day 5 and at the end of the study when the animals were slaughtered or at the humane endpoint was reached in the blood and in the indicated tissues. Significance compared to control antibody treatment was determined by nonparametric unpaired Mann-Whitney U test. * p is less than 0.05; * P < 0.01. (C) human serum IgG concentration in blood samples on day 5.

FIG. 19 shows the engineering of ZKA190 in bispecific FIT-1mAb and its in vitro characterization in examples 11 and 12. (A) The neutralizing effects of ZKA185 and ZKA230 mabs on four ZIKV strains were tested, as determined by the percentage of Vero cells infected in the presence of increasing amounts of mAb. Data are representative of at least two independent experiments. (B) ZKA185 and ZKA230 IgG and Fab binding to recombinant ZIKV VLP, E and DIII antigens was assessed by ELISA. (C) The ability of ZKA190, ZKA185 and ZKA230 to neutralize H/PF/2013 (wt) and MARM 1-4 was tested. (D) Surface representation of two E protein dimers bound by ZKA190 (green); epitopes derived from ZKA190 NMR are red; the positions of mutations in MARMs are indicated in yellow (E370), blue (T335), orange (D67) and magenta (K84). (E) FIT-1 model. The natural linker between the internal and external Fab allows the Fab to move flexibly in the FIT-1 antibody. The variable regions of ZKA185 and ZKA190 are highlighted in blue and green, respectively. (F) Binding of FIT-1IgG and Fab to recombinant ZIKV VLP, E and DIII antigens assessed by ELISA. (G) The neutralization of four strains of ZIKV (IC 50 values, G) and four MARMs (H) by ZKA190, ZKA185 and FIT-1mAb was tested. (I) Neutralization of the ZIKV H/PF/2013 strain by ZKA185, ZKA230 and FIT-1IgG and Fab as determined in (a). (J) confocal microscopy experiments, as shown in FIG. 3G. (K) The effect on ADE induced by dilution was enhanced by peaks of anti-prM DV62 mAb or DENV2 plasma by serial dilution of FIT-1IgG and Fab.

FIG. 20 shows the therapeutic efficacy of FIT-1. FIT-1 was very effective against ZIKV infection when mice challenged with the lethal dose of the ZIKV strain MP17451 (a 129) were administered therapeutically at different time points. Experimental N =5 to 6 mice were used per group. (A) shows the Kaplan-Meier survival curve. Significance was determined by using the Mantel-Cox log rank test. 15mg/kg, 5mg/kg and 1mg/kg of FIT-1 and control mAb administered on days 1, 2, P =0.0012; 15mg/kg and 5mg/kg of ZKA190 and control mAb, P =0.0012, administered on day 3; 1mg/kg ZKA190 and control mAb, P =0.0170 given on day 3. (B) Mice were monitored for an incidence score over a 21 day period (Dowall et al, 2016). (C) On day 5, the viral load in the blood of all animals was measured as PFU. (D) the mice were monitored for weight loss over 21 days. The control mAb in panel a was MPE8 mAb (specific for the RSV F protein (Corti, d., et al Cross-conjugation of four paramyxviruses by a human monoclonal antibody nature 501, 439-443 (2013)) (E) was measured in the blood of all animals on day 5 and viral load as genomic copies in blood and indicated tissues by qPCR at the end of the study slaughter or at the end of the human race.

FIG. 21 shows that female AG129 mice treated with FIT-1 after ZIKV challenge in Malaysia are protected from death compared to placebo treated mice in example 14.

FIG. 22 shows the intrauterine growth restriction (IUGR) of pups from females treated with FIT-1 and challenged with ZIKV in example 14.

FIG. 23 shows the average body weight of pups of females treated with FIT-1 and infected with ZIKV of example 14 on the day of birth.

FIG. 24 shows placental weights of 11dpi collected from females treated with FIT-1 in example 14.

Figure 25 shows quantification of viral RNA in (a) foetus, (B) placenta, (C) maternal spleen and (D) maternal brain in example 14 (P < 0.001, P < 0.01 compared to MPE8 treatment).

FIG. 26 shows the survival rate of male AG129 mice treated with FIT-1 and infected with ZIKV 24 hours or 72 hours post virus challenge (. P < 0.05 compared to MPE8 negative control treatment) in example 15.

FIG. 27 shows the average percent change in body weight of AG129 mice treated with FIT-1 at various times after challenge with ZIKV as in example 15.

FIG. 28 shows the disease score of A) testis or B) epididymis of male AG129 mice treated with FIT-1 24 hours or 72 hours after challenge with ZIKV in example 15. Treatment with active antibodies reduces testicular and epididymal disease.

Figure 29 shows the viral load in group 3 sera tested in example 16. The horizontal line indicates that LLOQ was 860GC/mL.

Examples

Exemplary embodiments of the invention are provided in the following examples. The following examples are given by way of illustration only to assist those of ordinary skill in the art in utilizing the present invention. The examples are not intended to limit the scope of the invention in any way.

Example 1: separation of ZIKV specific antibodies and preparation of monoclonal antibodies

IgG + memory B cells were isolated from cryopreserved Peripheral Blood Mononuclear Cells (PBMCs) of four ZIKV-infected donors (ZKA, ZKB, ZKC and ZKD) using CD22 microbeads (Miltenyi Biotec), and then cells carrying IgM, igD and IgA were cleared by cell sorting. Memory B cells from ZIKV infected donors were then immortalized with EBV (Epstein Barr virus) and CpG (CpG oligodeoxynucleotide, 2006) in a number of replicate wells and the culture supernatants were then tested in a primary screen, using a 384 well based microneutralization assay and binding assay (ELISA) to test their binding to ZIKV NS1 or ZIKV E proteins, as described previously (Traggiai, e.et al., nat. Med.10, 871-875, 2004). The results of the binding assay (binding to ZIKV E protein) are shown in figure 1.

Neutralization assays were performed on Vero cells. ZIKV H/PF/2013, which resulted in an infection rate (m.o.i., multiplicity of infection) of 0.35, was incubated with the supernatant at 37% (5% co 2) for 1 hour in 384-well plates, and then added to the pre-seeded 5000 Vero cells. They were incubated for an additional 5 days, then the supernatant was removed and WST-1 reagent (Roche) was added. Positive cultures were collected and expanded. VH and VL sequences were recovered from positive cultures by RT-PCR. Antibodies were cloned into human IgG1 and Ig kappa or Ig lamda expression vectors (supplied by Michel Nussenzweig, rockfeller University, new York, US) essentially as Tiller T, meffer E, yurasov S, tsuiji M, nussenzweig MC, wardemann H (2008) efficiency generation of monoclonal antibodies from single human cells B cells by single cell RT-PCR and expression vector cloning. J Immunol Methods 329: 112-124. Monoclonal antibodies were prepared either from EBV immortalized B cells or by transient transfection of 293 Freestyle cells (Invitrogen). Supernatants from B cells or transfected cells were collected and IgG was affinity purified by protein a or protein G chromatography (GE Healthcare) and desalted with PBS.

Figure 1 provides a summary of selected ZIKV neutralizing antibodies (see table 1 and table 2 for their CDRs and amino acid sequences of the heavy/light chain variable regions). The last two columns of fig. 1 provide neutralization activity (IC) of ZIKV and DENV1 (if tested) ₅₀ ). Other columns provide binding activity (EC) of antibodies to ZIKV E protein (ZIKV E), DENV 1E protein (DENV 1E), DENV 2E protein (DENV 2E), DENV 3E protein (DENV 3E), DENV 4E protein (DENV 4E), DENV1 virus-like particle (DENV 1 VLP), DENV2 virus-like particle (DENV 2 VLP), DENV3 virus-like particle (DENV 3 VLP), DENV4 virus-like particle (DENV 4 VLP) ₅₀ ) And binding activity (EC) to EDIII domain of ZIKV E protein (DIII ZKA) ₅₀ )。

Example 2: characterization of ZKA190, ZKA185, ZKA230, ZKA64 and ZKA78 antibodies

In example 1, a number of ZIKV neutralizing antibodies were identified and characterized for their specificity for ZIKV, particularly for ZIKV E protein and ZIKV EDIII, and for cross-reactivity to DENV. The antibodies ZKA190 (SEQ ID NOS: 1 to 18), ZKA185 (SEQ ID NOS: 19 to 36), ZKA230 (SEQ ID NOS: 37 to 54), ZKA64 (SEQ ID NOS: 73 to 90) and ZKA78 (SEQ ID NOS: 55 to 72) described in example 1 were then selected and further tested against ZIKV E protein ("ZIKV"), ZIKV EDIII ("DIIIZI") and DENV E protein (DENV, serotype 1) was also tested by ELISA. For this, standard ELISA was used. Briefly, ELISA plates were coated with 1. Mu.g/ml or 3. Mu.g/ml ZIKV E protein, blocked with 10% FCS in PBS, incubated with serum or human antibodies and washed. Bound antibodies were detected by incubation with goat anti-human IgG conjugated with AP (Southern Biotech). Plates were then washed, substrate (p-NPP, sigma) was added, and plates were read at 405 nm. The relative affinity of monoclonal antibody binding was determined by measuring the concentration of antibody required to achieve 50% of maximum binding at saturation (EC 50).

The results are shown in FIGS. 3 and 4. Notably, ZKA64 and ZKA190 bind to ZIKV E and ZIKV EDIII ("DIII ZI") at low EC50 values, suggesting that ZKA64 and ZKA190 bind to domain III of ZIKV E protein (EDIII). ZKA78 binds to ZIKV E but not ZIKV EDIII, indicating that ZKA78 binds to ZIKV E but not to EDIII regions. Despite their considerable ZIKV neutralizing activity (see fig. 1), antibodies ZKA185 and ZKA230 did not show any detectable binding to ZIKV E and ZIKV EDIII (fig. 3). Thus, ZKA185 and ZKA230 are referred to as "neutralizing non-E binding" (NNB) antibodies. It is assumed that those NNB antibodies recognize quaternary epitopes displayed on ZIKV infectious virions, but do not show up on soluble proteins.

Furthermore, none of ZKA190, ZKA185, ZKA230, and ZKA64 showed any detectable binding to DENV E protein (fig. 1, DENV1-4 serotypes, and fig. 3 and 4), suggesting that ZKA190, ZKA185, ZKA230, and ZKA64 are specific for ZIKV and do not cross-react to dengue virus. In contrast, ZKA78 (see fig. 2), which is assumed to bind to ZIKV EDI/II, but not to ZIKV EDII, binds to DENV E protein (fig. 1 and 3), indicating that ZKA78 is a cross-reactive antibody that binds to both ZIKV and DENV.

To further confirm these results, tests were also performed against dengue virus E proteins (DENV, serotypes 1 to 4) on ZIKV E protein binding antibodies ZKA190, ZKA64, and ZKA 78. ZKA64 and ZKA190 did not bind to DENV 1-4E protein, confirming that ZKA64 and ZKA190 are specific for ZIKV. In contrast, ZKA78 bound DENV1 to 4E, confirming that ZKA78 is a cross-reactive antibody that binds to ZIKV and the E protein of DENV (see fig. 1).

Example 3: isolated antibodies efficiently neutralize ZIKV infection

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 microneutralized flow cytometry assay. Different dilutions of the antibody were mixed with ZIKV (MOI of 0.35) or attenuated DENV1 (both MOI of 0.04) at 37 ℃ for 1 hour and added to 5000 Vero cells/well in 96-well flat-bottom plates. Four days for ZIKV treatment and five days for DENV treatment, cells were fixed with 2% formaldehyde, permeabilized in PBS 1% fcs 0.5% saponin and stained with mouse mAb 4G 2. Cells were incubated with goat anti-mouse IgG conjugated to Alexa Fluor488 (Jackson Immuno-Research, 115485164) and analyzed by flow cytometry. In other cases, ZIKV neutralization data can also be determined using WST-1 reagent (Roche) to measure cell viability. The neutralization titer (50% inhibitory concentration [ IC50 ]) is expressed as the concentration of antibody that can reduce infection by 50% compared to the cell-only control wells.

The results are shown in fig. 4, 5 and 9. The EDIII-specific mabs ZKA64 and ZKA190 and the NNB mAb ZKA230 were very effective in ZIKV neutralization (strain H/PF/2013) with IC50 values of 93ng/ml, 9ng/ml and 10ng/ml, respectively (fig. 4, upper panel). In contrast, the cross-reactive antibody ZKA78 only partially neutralizes ZIKV infectivity and cross-neutralizes DENV1 infectivity (fig. 4, bottom panel). Similar data were obtained by measuring ZIKV-induced cytopathic effects measured with WST-1 agents (fig. 5). In this second assay, the NNB antibody ZKA185 was also included in the test antibody panel and showed an IC50 similar to that of the most potent antibodies ZKA190 (EDIII specific) and ZKA230 (NNB).

It is important to note that the ultra potent ZKA64 and ZKA190 antibodies, in addition to having the ability to neutralize the ZIKV H/PH/2013 strains (this example), also bind to E protein and EDIII derived from the ZIKV strains MR766 and SPH2015, respectively (fig. 1 and 2). ZKA190 and ZKA190-LALA were also demonstrated to be effective in neutralizing two other ZIKV viral strains (MR 766 and MRs _ OPY _ martini island _ PaRi _ 2015) (fig. 9). Taken together, the results indicate that the ultra potent ZKA64 and ZKA190 antibodies cross-react with multiple ZIKV virus strains belonging to different genotypes and origins (eastern non-human and asian from urodara, farinacia, martini, and brazil).

Example 4: antibody-dependent enhancement of "LALA" mutant suppressor serum antibodies to ZIKV infection

Neutralizing antibodies were also tested for their ability to enhance ZIKV infection in non-permissive K562 cells (antibody-dependent enhancement assay, ADE assay). ADE was measured by flow cytometry-based assay using K562 cells. The antibody and ZIKV H/PF/2013 (MOI 0.175) were mixed at 37 ℃ for 1 hour and added to 5000K 562 cells/well. Four days later, cells were fixed, permeabilized and stained with m4G 2. The number of infected cells was determined by flow cytometry.

The results are shown in FIG. 6. All antibodies enhanced ZIKV infection in non-permissive K562 cells over a wide range of concentrations, including those that completely neutralized ZIKV infection on Vero cells (fig. 6). It is noteworthy that while the EDIII-specific antibodies ZKA64 and ZKA190 completely neutralized ZIKV infection of K562 cells above 1 μ g/ml, the NNB antibody ZKA230 failed to do so, a result that may be due to a different mechanism of free virus neutralization from Fc-gamma receptor internalizing virus. In contrast, cross-reacting ZKA78, which only partially neutralizes ZIKV infectivity, effectively enhanced ZIKV infection of K562 cells. These results indicate that cross-reactive antibodies caused by ZIKV or DENV infection can mediate heterologous ADE.

In view of this, it was investigated whether ADE can also be induced by immune sera and whether it can be blocked by neutralizing antibodies delivered as "LALA variants". To obtain the LALA variants, each heavy chain was mutated at amino acids 4 and 5 of the CH2 domain by using site-directed mutagenesis to replace the native leucine with alanine. As described above, LALA variants (of human IgG1 antibodies) do not bind to Fc-gamma receptors and complement.

To investigate the effect of the ZKA64-LALA antibody in ZIKV ADE, the inhibitory effect of ADE assay was used. Since ADE of ZIKV was observed using ZIKV or DENV immunized plasma, ZIKV (MOI 0.175) was mixed with plasma from the original ZIKV or DENV infected donor at 37 ℃ for 30 minutes. ZKA64-LALA antibody was added at a concentration of 50. Mu.g/ml, mixed with 5000K 562 cells/well and incubated for three days. The cells were then stained with 4G2 and analyzed by flow cytometry.

The results are shown in FIG. 7. Under homologous conditions, four ZIKV immune plasmas and one DENV immune plasma collected from convalescent patients showed similar ability to enhance ZIKV infection of K562 cells (fig. 7, top panel), and EDIII-specific ZKA64-LALA antibodies completely blocked the ADE effect (fig. 7, bottom panel).

Notably, the EDIII-specific ZKA64-LALA antibody completely blocked the ADE effect of ZIKV and DENV immune plasma. The ADE blocking ability of a single EDIII-specific LALA antibody is not only related to its ability to compete for serum-enhanced antibodies, but also to the neutralizing ability of the virus once internalized into endosomes.

These results indicate that potent neutralizing antibodies developed in the form of "LALA", such as ZKA190, ZKA230, ZKA185 or ZKA64, have great potential for use in the prevention or treatment of the environment to prevent congenital ZIKV infections, such as pregnant women and/or people living in high risk areas. The use of LALA format avoids the risk of ZIKV ADE and, as noted above, may also block ADE of pre-existing cross-reactive antibodies, e.g. for patients already immunized against DENV.

Example 5: ZKA190 neutralizes ZIKV more efficiently than the prior art antibody EDE1 mAb C8

To compare the neutralizing performance of the isolated neutralizing antibody to that of the highly neutralizing anti-ZIKV antibody of the prior art, the neutralizing performance of ZKA190 was compared to that of the highly neutralizing mAb EDE 1C 8 of the prior art (Barba-Spaeth G, dejnitrattisai W, rouvinski A, vaney MC, medits I, sharma A, simon-Lori E, sakuntabhai A, cao-Lormeau VM, haouz A, england P, stiasny K, mongkolaya J, heinz FX, screaton GR, rey FA.structural basis of post Zika-dent virus antibody cross-neutral viscosity 536.2016.761g 4 (Au48-53). The neutralizing effect of two antibodies was tested against a panel of four different ZIKV strains (H/PF/2013, MRS-OPY and PV 10552).

Briefly, neutralization of ZIKV infection by mabs was measured using a microneutralization flow cytometry-based assay. Different dilutions of mAb were mixed with ZIKV (MOI 0.35) for 1 hour at 37 ℃ and then added to 5000 Vero cells/well in 96-well flat-bottom plates. Four days after ZIKV treatment, cells were fixed with 2% formaldehyde, permeabilized in PBS containing 1% fetal bovine serum (Hyclone) and 0.5% saponin, and stained with mouse mAb 4G 2. Cells were incubated with goat anti-mouse IgG conjugated to Alexa Fluor488 (Jackson Immuno-Research, 115485164) and analyzed by flow cytometry. The neutralizing titer (50% inhibitory concentration [ IC50 ]) is expressed as the concentration of antibody that can reduce infection by 50% compared to the virus control well alone.

The results are shown in FIG. 10. The ZKA190 mAb was effective in neutralizing African, asian and American strains of virus with an IC50 ranging from 0.6ng/ml to 8ng/ml. In contrast, the prior art antibody C8 is as low as about one-third 24 in potency.

Example 6: additional characterization of antibody ZKA190

The potency of antibody ZKA190 was further investigated in vitro and in vivo. For this purpose, mAbs were synthesized in IgG1 wild type (wt) format and IgG1 Fc-LALA format. Briefly, VH and VL sequences were cloned into human Ig γ 1, ig κ and Ig λ expression vectors (supplied by Michel Nussenzweig, rockviller University, new York, NY, USA) essentially as described (Tiller T, meffere E, yurasov S, tsuiji M, nussenzweig MC, wardemann H: efficient generation of monoclonal antibodies from simple human cells B by simple single cell RT-PCR and expression vector cloning Methods 2008, 329. Recombinant mabs were produced by transient transfection of EXPI293 cells (Invitrogen), purified by protein a chromatography (GE Healthcare) and desalted with PBS. To obtain the LALA variants, each heavy chain was mutated at amino acids 4 and 5 of the CH2 domain by using site-directed mutagenesis to replace the native leucine with alanine. As described above, the LALA variant (of a human IgG1 antibody) does not bind to Fc-gamma receptors and complement.

ZKA190 was tested against a panel of four ZIKV strains as shown in fig. 10A and described in example 5. ZKA190 mAb strongly neutralized african, asian and us strains with IC50 between 0.004nM and 0.05nM (fig. 10a, 0.6ng/ml to 8 ng/ml).

Since ZIKV has been shown to infect human neural progenitor cells (hNPC), resulting in increased cytotoxicity, cell cycle dysregulation and reduced cell growth, ZKA190 and ZKA190-LALA were tested in hNPC. For this purpose, adult male fibroblasts obtained from the Movement disorder organism Bank (Movement Disorders Bio-Bank), the department of Neurogenetics of the institute for neurology, "Carlo Besta", milan, were reprogrammed using the CytoTune-iPS 2.0 Sendai kit (Life Technologies). hipscs were kept under non-feeding conditions in mTeSR1 (Stem Cell Technologies). To generate Embryoid Bodies (EBs), dissociated hipSCs were seeded in mTeSR1 (e.g., marchetto MCN, carromeu C, acab A, yu D, yeo GW, mu Y, chen G, gage FH, muoti AR: A model for neural reduction and treatment of Rett syndrome consuming human induced plodotent 2010. Cells, 143-539) supplemented with N2 (0.5X) (ThermoFisher Scientific), human noggin (0.5 mg/ml, R & D System), SB431542 (5. Mu.M, sigma), Y27632 (10. Mu.M, miltenyi Biotec) and penicillin/or streptomycin (1%, sigma). To obtain rosettes, EBs were coated after 10 days on matrigel-coated plates (1: 100, matrigel growth factor reduced, corning) in DMEM/F12 (Sigma) containing N2 (1: 100), non-essential amino acids (1%, thermoFisher Scientific) and penicillin/streptomycin. After 10 days, cells were passaged with Accutase (Sigma) and seeded onto 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 (3 × 104) was seeded on 24-well plate coverslips 3 days prior to infection with PRVABC59 virus strain. The virus stock was incubated with mAb for 1h and then added to hNPC to give an MOI of 0.5. After 4 hours of virus adsorption, the culture supernatant was removed and fresh medium containing mAb was added again. Supernatants were collected 96 hours post infection and virus titers were determined by plaque assay on Vero cells. Cells were fixed in 4% paraformaldehyde (PFA, sigma) in phosphate buffered saline (PBS, euroclone) for 30 minutes for indirect immunofluorescence. Fixed cells were permeabilized in blocking solution containing 0.2% Triton X-100 (Sigma) and 10% donkey serum (Sigma) for 30 minutes (min) and incubated overnight at 4 ℃ with primary antibody in blocking solution. The following antibodies were used for detection: anti-envelope (1: 200, millipore, MAB10216). The cells were then washed with PBS and incubated for 1h with Hoechst and an anti-mouse Alexa Fluor-488 secondary antibody (1: 1000 in blocking solution, thermoFisher Scientific). After PBS washing, cells were washed again and fixed. The results are shown in FIG. 11A. Both ZKA190 and ZKA190-LALA completely eliminated ZIKV infection and replication in hNPC.

Next, ZKA190 and ZKA190-LALA were tested for the ability to cause ADE in the K562 cell line as described in example 4. Briefly, ADE was measured by flow cytometry-based assay using K562 cells. Briefly, for ZKA190, ZKA190 and ZIKV H/PF/2013 (MOI 0.175) were mixed at 37 ℃ for 1 hour and added to 5000K 562 cells/well. Four days later, cells were fixed, permeabilized and stained with mAb m4G 2. 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 minutes at 37 ℃. ZKA190-LALA was added at 50. Mu.g/ml, mixed with 5000K 562 cells/well and incubated for three days. The cells were then stained with 4G2 and analyzed by flow cytometry. The results are shown in FIG. 11B. ZKA190 supports ADE from 0.0001nM to 1 nM; as expected, ZKA190-LALA did not show any ADE activity. ZKA190-LALA was also tested for its ability to inhibit plasma-induced ADE from four ZIKV immune donors in K562 cells. The results are shown in fig. 11C. ZKA190-LALA was found to completely inhibit plasma antibody-induced ADE (FIG. 11C).

anti-prM antibodies constitute a part of the main antibodies elicited during the course of a human immune response to flavivirus and have been shown to enhance the ability of viral infection in vitro (dejinratisai, w., jumnainsong, a., onsiriakul, n., fit, p., vasana, s., limpitikul, w., puttikhunt, c., edwards, c., durangchanda, t., supra, s., et al (2010), cross-reactive antibodies derived virus infection in human science 328, 745-748). K562 cells (Beltramello, m., williams, k.l., simmonons, c.p., macagno, a., simonelli, l., queen, n.t.h., sukuplolvi-Petty, s., navaro-Sanchez, e., young, p.r., de silvera, a.m., et al (2010) The human animal response to detection virus is doped by blue cross-reactive antibodies cross-hatching and engineering virus. Cell Host Microbe 8, 271-283) were pre-incubated with serial dilutions of prM cross-reactive antibody DV62 from DENV immune donors. The results are shown in FIG. 11D. DV62 cross-reacted with ZIKV prM protein and caused ADE over a wide range of concentrations (fig. 11D). ZKA190-LALA can completely block ADE against immature or partially immature ZIKV particles induced by PRM DV62 mAb (fig. 11D).

Finally, the ability of ZKA190, ZKA190-LALA and ZKA190 Fab at different concentrations to cause or block ZIKV ADE in the presence of enhanced concentrations of human anti-DENV 2 plasma or DV62 was tested. The results are shown in FIG. 11E. Low concentrations of ZKA190 increased the ADE of prM DV 62-mediated ZIKV infection, consistent with its ability to promote entry of immature and mature virions, while concentrations of ZKA190 above 1.3nM (i.e., 200 ng/ml) blocked ADE induced by both DENV plasma and mAb DV 62. ZKA190-LALA and its Fab fragments decrease ADE at concentrations above 0.06nM, indicating that both inhibit viral infection in post-attachment steps such as fusion.

Example 7: ZKA190 binds to a conserved and highly accessible region of EDIII

To determine the ZKA190 epitopes at the residue level, such as 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.recognit.28, 393-400; simonelli, L., beltramello, M., yudina, Z., macagno, A., calzolai, L., and Varani, L. (2010). Rapid structural characterization of human anti-organic compounds through experimental recording. 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-deep Anti-body through experimental valuable comprehensive decoding on plos ONE 8, e55561, using a solution nuclear magnetic resonance spectroscopy.

Briefly, spectra were recorded on a Bruker Avance 700MHz NMR spectrometer at 300K. For assignment of backbone resonances, standard triple resonance experiments ((HNCO, HN (CA) CO, HN (CO) CACB, HNCACB), NH experiments with side chains using HCCH-TOCSY and HBHA (CO) NH were used for annotation. All NMR experiments were treated with Topspin 2.1 (Bruker Biospin) and analyzed with CARA. According to the chemical shifts assigned manually, CYANA "Noeasign" macro-auto-attribution NOESY overlapping peaks are used ¹⁵ N and ¹³ c-resolved NOESY spectra. The framework dynamics of ZIKV EDII were recorded from 600MHz and 700MHz spectrometers ¹⁵ N relaxation is measured. CPMG (R2), inversion recovery (R1) and ¹⁵ N{ ¹ h } -steady state NOE. The delay of the T2 series is set at 0 second to 0.25 second, and the delay of the T1 series is set at 0.02 second to 2 seconds. ¹⁵ N{ ¹ The H } -NOE experiment used a relaxation delay of 5 s. The R1 and R2 relaxivity was derived from a least squares fit of the corresponding exponential function to the measured data using a homemade script. The relaxation data was analyzed in a model-free manner using the software package DYNAMICS. The program ROTDIF was used to calculate the overall correlation time from the relaxation data (8.5 ns). NMR epitope mapping was performed as previously described (Bardelli et al 2015. Briefly, of marker EDIII free from or bound to ZKA190 Fab ¹⁵ Superposition of NHSQC spectra allowed identification of EDIII residues whose NMR signals changed after complex formation, suggesting that they are affected by ZKA190 binding. Identification of changes by manual inspection and Chemical Shift Perturbation (CSP), CSP = ((Delta H) ² +(Δδ _N /10) ² ) ^1/2 . The NMR sample is typically 800. Mu.M in 20mM sodium phosphate, 50mM NaCl, pH 6.0 ¹⁵ N， ¹³ C]Tagged EDIII. Deuterated (nominally 70%) is ² H， ¹⁵ N EDIII samples were used for NMR epitope mapping, where EDIII: the ZKA190 Fab ratio is 1: 1.1; the EDIII concentration is typically 0.4mM.

Since the NMR signal is strongly dependent on the local chemical environment, changes in complex formation can recognize the antigen residues affected by antibody binding either directly or through allosteric effects. By comparing the NMR spectra of free and bound EDIII (fig. 12A), residues affected by ZKA190 were mapped to the LR of EDIII, in particular the BC, DE and FG loops and part of the EDI-EDIII hinge (fig. 13A). These residues were almost identical in 217 known ZIKV strains, except for substitutions at V341I and E393D in the udhura 1947 isolate (fig. 12D). These mutations were also present in the MR766 virus strain that was effectively neutralized by ZKA190 (fig. 10A). Analysis of the ZKA190 epitope on uncomplexed ZIKV structures showed that this epitope was highly accessible except for the FG loop at the vertex of the 5-fold (fig. 13B and 12C, molecule a).

Computational docking followed by molecular dynamics simulations and NMR-derived epitope information, as well as EDIII mutagenesis guidance and validation, indicated that ZKA190 binds through an interface characterized by shape and charge complementarity (fig. 13B and 12E). Docking showed no direct contact between ZKA190 and the FG loop on EDIII, indicating that the change in NMR signal of the antibody upon binding was from allosteric action. This view is supported by the fact that mutations of FG loop residues in recombinant EDIII but not in other epitope regions do not affect the binding affinity of ZKA190 for EDIII (fig. 13B and fig. 14).

Example 8: mechanism for ZKA190 neutralization

The ability of ZKA190 to effectively neutralize viruses may be involved in the inhibition of cell attachment or membrane fusion. A further mechanism may involve inactivation of the virus by cross-linking of the virus particles.

ZKA190 Fab can neutralize ZIKV, although less efficiently than the corresponding IgG. ZKA190 (both Fab and IgG) may inhibit the approximately 70 degree rotation of DIII required for viral fusion to the host cell membrane by binding to EDI-EDIII linkers (Bressanelli, s., stiansny, k., allison, s.l., stura, e.a., duquerroy, s., lescar, J., heinz, f.x., and Rey, f.a. (2004) Structure of a flavivirus enveloppe glycoprostrate in low-pH-induced membrane fusion. Endoscope J23, 728-738 modification, y., ogagagene, s., clements, d., harrison, s.c. (2004) Structure of the virus vector fusion, nature 319.319). Alternatively, ZKA190 may prevent ZIKV from attaching to target cells.

The ability of ZKA190 to inhibit membrane fusion is supported by confocal microscopy analysis. For this purpose, vero cells were seeded at 7500 cells/well on 12mm diameter coverslips in 24-well plates and incubated overnight. Cells were infected with ZIKV H/PF/2013 (MOI of 100) at 37 ℃ for 3 hours, washed with PBS and fixed with 2% paraformaldehyde in PBS at room temperature for 30 minutes in the presence or absence of a neutralizing concentration of Alexa-488 conjugated mAb (0.7 μ M). The acidified endosomes were identified with a lysosomal red fluorescent probe (Invitrogen) by adding a dye (50 nM) to the cells during the last 30 min before fixation. After fixation, extensive washes were performed in PBS and 50mM glycine, and finally cover slips were prepared for microscopic analysis using DAPI-containing Vectashield mounting reagent for fluorescence (Vector Laboratories). Samples were analyzed by confocal microscopy using a Leica TCS SP5 microscope with 63 x/1.4 n.a. objective. Image analysis and processing was performed with FIJI software.

The results are shown in FIG. 15. Confocal microscopy analysis showed that ZKA190 (Fab or IgG) only entered Vero cells after complexing with ZIKV, with neutralization concentrations exceeding IC50 10000 fold (fig. 15).

Example 9: in vivo characterization of EDIII-specific mAb ZKA190

To evaluate their prophylactic and therapeutic properties, ZKA190 and ZKA190-LALA were tested in a129 mice challenged with a lethal dose of the ZIKV strain MP1751 (african pedigree). To test their prophylactic efficacy, ZKA190 and ZKA190-LALA were administered the day before the virus challenge.

Female a129 mice (IFN- α/β receptor-/-) and wild-type 129Sv/Ev mice, 5 to 8 weeks old, were administered mabs (ZKA 190, ZKA190-LALA and control antibody MPE8 (Corti, d., et al Cross-immunization of four paramyxoviruses by a human monoclonal antibody, nature 501, 439-443 (2013)) diluted in PBS by intraperitoneal (i.p.) route at different doses in a volume of 500 μ Ι, administered mabs 1 day before or 1 day, 2 day, 3 day or 4 day after virus challenge animals were subcutaneously challenged with 102pfu ZIKV (strain MP 1751), body weight and temperature were monitored daily for 14 days thereafter, clinical observations were recorded at least twice daily, 50 μ l of blood was collected from each animal on day 5 post challenge into RNA protection tubes (Qiagen, UK) and frozen at-80 ℃, autopsy was performed at the end of the study (14 days post challenge) or when the animals reached the humane endpoint, and blood and sections of brain, spleen, liver, kidney and ovary were collected for virological analysis.

Tissue samples from a129 mice were weighed and homogenized in PBS using ceramic beads and an automatic homogenizer (Precellys, UK) using six 5 second cycles of 6500rpm, spaced 30 seconds apart. Two hundred μ l of the tissue homogenate or blood solution was transferred to 600 μ LRLT buffer (Qiagen, UK) and RNA extraction was performed using RNeasy Mini extraction kit (Qiagen, UK); as an initial step, the samples were passed through qiathreder (Qiagen, UK). The ZIKV specific real-time RT-PCR assay is used to detect viral RNA in subject animals. Primer and probe sequences were taken from Quick et al, 2017 (Quick, J, grubaugh ND, pullan ST, claro IM, smith AD, gangarapu K, oliveira G, robles-Sikisaka R, rogers TF, beutler NA, et al, multiplex PCR method for MinION and Illumina sequencing of Zika and other viruses direct from clinical samples Nat Protic 2017, 12 1261-1276) and were internally optimized and validated to provide optimal PCR premixes (mastermix) and cycling conditions. Real-time RT-PCR was performed using SuperScript III Platinum single-step qRT-PCR kit (Life Technologies, UK). The final PCR premix (15. Mu.l) was prepared from 10. Mu.l of the 2 × reaction mixture, 1.2. Mu.l of PCR grade water, 0.2. Mu.l of 50mM MgSO ₄ 1 mul of each primer (the working concentration of ZIKV 1086 and ZIKV 1162c is 18 mul), 0.8 mul of probe (the working concentration of ZIKV 1107-FAM is 25 mul) and 0.8 mul of SSIII enzyme mixture. Mu.l template RNA was added to the PCR premix, and the final reaction volume was 20. Mu.l. The cycling conditions used were 50 ℃ continuous10 minutes, 95 ℃ for 2 minutes, then 95 ℃ for 10 seconds and 60 ℃ for 40 seconds, plus a final cooling step of 40 ℃ for 30 seconds. Quantitative analysis was performed using fluorescence at the end of each 60 ℃ step. Reactions were run and analyzed on a 7500 Fast platform (Life Technologies, UK) using 7500 software version 2.0.6. The viral load in the sample was quantified using a series of dilutions of quantitative RNA oligonucleotides (Integrated DNA Technologies). Based on GenBank accession No. AY632535.2, the oligonucleotide contains 77 bases of the ZIKV RNA targeted by the assay and was synthesized by HPLC purification to a 250nmol scale.

The results are shown in fig. 16, 17 and 18. ZKA190 and ZKA190-LALA have been shown to protect mice from mortality and morbidity at concentrations of 5mg/kg, 1mg/kg, or 0.2mg/kg (fig. 16A-16B). ZKA190-LALA delayed morbidity and mortality compared to the control group at 0.04mg/kg, and ZKA190 delayed morbidity and mortality to a lesser extent. The virus titers in blood and organs were significantly reduced compared to control antibody treated animals, even at serum antibody levels below 1 μ g/ml (fig. 17A to 17D).

To assess the therapeutic potential of ZKA190, we administered ZKA190 and ZKA190-LALA at different time points after ZIKV infection. At a dose of 15mg/kg, the survival rate reached 80% to 100%, and the incidence was greatly reduced even after four days post-infection treatment (fig. 16E to fig. 16G). ZKA190 and ZKA190-LALA treatment at all post-infection time points resulted in a significant reduction in viral titers compared to animals treated with control antibodies, with a clear trend of greater reduction in earlier treatments (fig. 18A-16C). Notably, ZKA190-LALA had significantly reduced antiviral activity in blood at day 5 compared to ZKA190 when mAb was administered four days post-infection, a result which may be associated with an impaired ability of the LALA variant to promote rapid clearance of the coated virions.

Of these 16 treated mice, an in vivo escape mutant (monoclonal antibody resistance mutant 1, marm 1) was isolated that had amino acid substitutions in DIII (T335R in the center of the epitope), while the virus from the other treated mice did not contain any E mutation. The introduction of the T335R mutation into recombinant DIII, as determined by SPR, suggests that it abolished ZKA190 binding (fig. 14; see experimental methods of example 7).

Example 10: in vitro selection of ZIKV escape mutants

The use of antibody therapeutics may lead to the selection of escape mutants. To evaluate the ability of ZKA190 to select for resistance mutants in vitro (MARMs), ZIKV (H/PF/2013) was passaged in the presence of sub-neutralizing concentrations of ZKA 190.

Briefly, 500. Mu.l of two thousand TCID 50H/PF/2013 ZIKV were incubated with 250. Mu.l of mAb containing different concentrations (8 different concentrations, starting at a final concentration of 200. Mu.g/ml, serially diluted 1: 4). The mixture was incubated at 37 ℃ for 45 minutes and then 250. Mu.l of Vero cell suspension (3.2X 10) was added ⁶ Individual cells) and incubated in 24-well plates at 37 ℃ for three to four days to propagate the virus. After each selection step, 500 μ Ι of supernatant was selected from three conditions: the lowest mAb concentration at which the monolayer was fully protected was observed, one concentration at which the effect of partial CPE on the cell monolayer was observed, and one concentration at which ZIKV CPE destroyed 100% of the cell monolayer was observed. The tubes were centrifuged at 1000 Xg for 5 minutes, aliquoted and stored at-80 ℃. Half of the volume was again premixed with different concentrations of mAb to repeat the selection and propagation process. The remaining supernatant was used for the microneutralization assay and subsequent virus sequencing.

To identify escape mutations of selected MARM viruses, genomic RNA extraction was performed first, followed by one-step PCR to amplify ZIKV E protein amplicons and sequencing. Cell supernatants (140. Mu.l) from MARM selection were used for RNA extraction using QIAamp Viral RNA mini kit (Qiagen). cDNA synthesis and PCR amplification were performed using SuperScript III one-step RT-PCR together with platinum Taq (Invitrogen). For one reaction, 25. Mu.l of reaction mixture, 8. Mu.l of sterile water, 2. Mu.M of each primer, 1. Mu.l of RNAse inhibitor (RNAse out) (Life Technologies), 2. Mu.l of Superscript III RT/Platinum TaqMix and 12. Mu.l of RNA were required, with a final reaction volume of 50. Mu.l. For the N-terminal portion of the E protein, the primer pairs Zika-E-F1 5-. The circulation conditions are as follows: at 54 ℃ for 40 minutes and at 94 ℃ for 2 minutes; then 45 cycles of the following steps were performed: 94 ℃ for 45 seconds, 50 ℃ for 45 seconds and 68 ℃ for 1.5 minutes; the final extension step was at 68 ℃ for 5 minutes and the final cooling step was at 4 ℃. The PCR products were analyzed and extracted from 1.5% agarose gel and further purified using GFX PCR DNA and gel strip purification kit (GE Healthcare). For the sequencing reaction, 8. Mu.l of the purified PCR product was mixed with 2. Mu.M primers, the final volume was 10. Mu.l, and sent for sequencing (Microsynth). The N-terminal product of the E protein was sequenced using ZIKV-E-F2 5-. Sequences were assembled and analyzed using CLC Main Workbench software (CLC Bio, version 5).

After three rounds of selection, the resistance mutant MARM2 was isolated, the E protein of which showed the E370K mutation in DIII. Although the antibody can bind to the mutated DIII, the mutation abrogated the neutralizing effect of ZKA190 (fig. 14). Mutations in vivo (T335R) and in vitro (E370K) MARMS are located on the BC and DE loops, respectively, of DIII and are consistent with the NMR-identified epitope.

Example 11: development of bispecific antibodies according to the invention

Viral escape mutants can greatly hinder the efficacy of therapeutic antibodies. To overcome this problem, the inventors speculate that when combining two highly neutralizing antibodies, the probability of viral escape will be greatly reduced. In view of this, a series of bispecific antibodies were generated that bind ZKA190 to other potent neutralizing mabs directed to different sites on the E protein. Therefore, it focuses on two mabs, ZKA185 and ZKA230, that are highly neutralizing and do not compete with ZKA 190.

First, they were analyzed for their ability to cross-neutralize four ZIKV strains, as described above. ZKA185 and to a lesser extent ZKA230 effectively neutralized african, asian, and us strains with IC50 ranging from 0.02nM to 0.62nM (fig. 19A). ZKA185 binds with high affinity to recombinant ZIKV E protein and zika virus-like particles (VLPs), but not to isolated DIII (fig. 19B). In contrast, ZKA230 bound ZIKV VLPs, but not recombinant E or DIII, suggesting that it recognizes quaternary epitopes only shown on the surface of the virus (fig. 19B). ZKA185 IgG and Fab were shown to bind E and VLP antigens with similar high affinity by ELISA.

To identify ZKA185 and ZKA230 epitopes and their propensity to generate escape mutants, MARMs for ZKA185 (MARM 3) and ZKA230 (MARM 4) were isolated by passaging the virus in the presence of sub-neutralizing antibodies, as described above. MARM3 contains substitutions at both K84E and D67H, both of which are located on DII (FIG. 19D). MARM4 shows a mixture of different amino acid substitutions (from K to G, E or R) at position 84, which was confirmed in multiple sequencing experiments. Finally, MARMs 1 to 4 were tested for ZKA190, ZKA185, and ZKA 230. ZKA190 neutralized ZKA185 and ZKA230 MARMs as well as the parental viruses (fig. 19C). ZKA185 neutralizes ZKA190 and ZKA230 MARMs. ZKA230 neutralized only ZKA190 MARM2, but not ZKA190 MARM1 or ZKA185 MARM3.

To go deep into the development of MARMs that are able to escape multiple antibody stresses, serial passages of ZKA190 MARM2 (E370K) were performed in the presence of ZKA185 or ZK 230. Thus, it was found that double MARMs appeared after 3 to 4 passages. ZKA230 introduced an additional K84E mutation, while ZKA185 introduced a D76G mutation. These findings indicate that ZIKV is able to escape neutralization of multiple antibodies against different sites when selected in a stepwise manner and confirm the high plasticity of ZIKV E proteins.

In summary, ZKA185 was selected for use with ZKA190 in developing bispecific antibodies because it can effectively cross-neutralize ZIKV strains, bind to alternate sites, and does not compete with ZKA 190. Bispecific antibodies are prepared in a tetravalent symmetric format called tandem Fab-Ig (FIT-Ig). FIT-Ig is described, for example, in WO 2015/103072 A1 and Gong S, ren F, wu D, wu X, wu C: MAbs 2017, fab-in-tandem immunoglobulin is a novel and versatic design for engaging multiple thermal targets.

FIT-Ig may be prepared using three polypeptides. Polypeptide 1 typically comprises the light chain of the outer Fab fused to the N-terminal region of the inner Fab heavy chain, preferably without a linker. Polypeptide 2 typically comprises the heavy chain variable region and the CH1 region of the outer Fab, and polypeptide 3 typically comprises the light chain of the inner Fab. Thus, antibodies in the form of FIT-Ig usually comprise an "internal Fab" and an "external Fab". The ZKA190 Fab generated two types of FIT-Ig at either external or internal locations. Briefly, three genes encoding FIT-Ig were codon optimized, synthesized from Genscript and cloned as follows: i) The VL of the outer Fab, followed by the entire constant region (λ or κ), was fused to the VH of the inner Fab and cloned into an Ig γ 1 expression vector modified to encode the LALA mutation. The resulting polypeptide 1 is formed by fusion of the VL and CL of the external Fab, the VH of the internal Fab with the IgG1 CH 1-hinge-CH 2-CH3 domain; ii) cloning the VH gene of the external Fab (encoding polypeptide 2 formed by VH and CH1 of the external Fab) into a Fab expression vector (Ig γ 1 expression vector in which a stop codon is introduced after the codon encoding the CH1 cysteine residue 220); iii) The VL gene of the internal Fab was cloned into an Ig κ or Ig λ expression vector (encoding polypeptide 3 formed by VL and CL of the internal Fab). Recombinant FIT-Ig mAbs (as described in WO 2015/103072 A1) were prepared by transient transfection using EXPI293 cells (Invitrogen) of the above three constructs in a molar ratio of 1: 3 and purified by protein A chromatography (GE Healthcare) and desalted against PBS. Proteins were analyzed by SDS-PAGE under reducing and non-reducing conditions and their concentration was determined by BCA (Pierce, rockford, IL). Under non-reducing conditions, FIT-Ig migrated as a single major band at about 250 kDa. Under reducing conditions, two bands were produced for each FIT-Ig protein, one higher MW band being polypeptide 1 at about 75kDa and one lower MW band corresponding to polypeptides 2 and 3 overlapping at about 25 kDa. To further investigate the physical properties of FIT-Ig in solution, each protein was analyzed using Size Exclusion Chromatography (SEC). Purified FIT-Ig in PBS was applied to Superdex 200 Incase 5/150 GL. All proteins were determined using UV detection at 280nm and 214 nm. The FIT-Ig protein showed a single major peak indicating its physical homogeneity with monomeric proteins.

Example 12: in vitro characterization of the antibodies according to the invention (FIT-1)

An FIT-Ig bispecific antibody (referred to herein as FIT-1) was selected with ZKA190 at the outer Fab position and ZKA185 at the inner Fab position (FIG. 19E) and was further characterized. ELISA showed FIT-1 binding to DIII, E and VLP (FIG. 19F). FIT-1 retained high neutralizing potency against ZIKV virus strains, with IC50 values substantially similar to those of parental ZKA190 and ZKA185 antibodies (fig. 19G). FIT-1 was prepared using a LALA format of a backbone IgG1 antibody, thereby eliminating any possibility of causing ADE. Several lines of evidence indicate that both the ZKA190 and ZKA185 portions of the FIT-1 form are in an activated state. First, FIT-1 has a higher affinity for the E protein than either of the parent ZKA190 and ZKA185 antibodies (KD value: 1.8nM for ZKA185, 9.3nM for ZKA190, and FIT-1 KD < 1pM, possibly due to a slower off-rate, possibly by affinity effects). Second, FIT-1 effectively neutralized all ZKA190, ZKA185, and ZKA230 MARMs compared to a single mAb (fig. 19H). The neutralizing activity of Fab fragments of FIT-1 (each comprising one ZKA190 and one ZKA 185) was only reduced by a factor of about 6 (fig. 19I, right panel).

Next, FIT-1 was tested for its ability to select MARMs (as described above). However, despite eight successive passages, no MARM could be isolated. In contrast, MARM appeared after 3 to 4 passages with a single mAb. These results indicate that FIT-1 is safer to use as a therapeutic agent because simultaneous mutations of both DIII and DII are less likely to occur.

Confocal microscopy studies using Vero cells also showed that FIT-1 as ZKA190 may inhibit viral infection, and possibly fusion, in the post-attachment step (fig. 19J).

Finally, FIT-1 and its Fab fragments blocked ADE against DENV2 plasma or DV62 in human (fig. 19K) at concentrations above 0.1nM and 10nM, respectively, indicating that FIT-1 did not cause ADE and blocked the ADE of monocytes by poorly neutralizing cross-reactive antibodies.

Example 13: in vivo therapeutic potential of FIT-1

To assess the therapeutic potential of FIT-1, three different doses (15 mg/kg, 5mg/kg and 1 mg/kg) were administered to female A129 mice at three different time points following ZIKV infection. Briefly, female a129 mice were divided into 10 different groups (control group and three different doses, with three different administration time points for each dose). All animals were challenged subcutaneously with 102pfu of ZIKV (strain MP 1751) for 14 days. On day 1, day 2 or day 3 after the ZIKV virus challenge, 9 treatment groups of mice were administered with 15mg/kg, 5mg/kg or 1mg/kg of bispecific antibody FIT-1 diluted in PBS by intraperitoneal (i.p.) route in a volume of 500. Mu.l. Body weight and temperature of all animals were monitored daily and clinical observations were recorded at least twice daily. On day 5 post challenge, 50 μ l of blood was collected from each animal into an RNA protection tube (Qiagen, UK) and frozen at-80 ℃. At the end of the study (14 days post challenge) or when the animals reached the humanistic endpoint, necropsies were performed and blood was collected, as well as sections of brain and ovaries for virological analysis.

The results are shown in FIG. 20. The data show that at a dose of 15mg/kg, survival was 100% with no signs of disease even with treatment three days post infection (fig. 20A); elimination of viral titers and no escape mutants were detected, indicating high potency in vivo. Administration of 5mg/kg resulted in a survival rate of 70% to 100%, with no escape mutants detected at day 5 post-infection. The lowest dose tested (i.e., 1 mg/kg) served as protection when administered on day 1 or day 2 (but not day 3) post-infection.

Example 14: effect of FIT-1 on congenital infection model diseases in AG129 mice

The effect of treatment of infected mothers with FIT-1 on innate infected pups and treated mothers was evaluated in a ZIKV congenital infected mouse model that yielded live offspring after innate exposure to ZIKV. In this model, AG129 mice lacking IFN receptors were used, which resulted in viral replication in the placenta, transmission of the virus to the fetus, and potential long-term effects such as intrauterine growth restriction and hearing impairment.

The effect of FIT-1 treatment on various parameters of intrauterine infection was tested. For this purpose, mice were infected with ZIKV 7 days post-coital (dpc) and treated with FIT-1 day or 3 days post-viral challenge (see example 13). Various disease outcomes, such as viral titers in the fetus and placenta and intrauterine growth restriction, were evaluated to determine the efficacy of FIT-1 treatment.

Materials and methods:

animals: 42 female AG129 mice were used. Animal groups were randomly assigned to experimental groups and individually labeled with ear tags. Hormonal therapy is used to induce oestrus in females. Female and male individuals were placed together and checked for vaginal tampons at 0.5 days post-coital (dpc).

Virus: zika virus Malaysia (P6-740) was used. The appropriate challenge dose was administered by subcutaneous injection of 0.1 ml.

And (3) testing reagents: the dose of FIT-1 was 45mg/kg. The non-specific control antibody MPE8-LALA Ctr IgG1 was used as isotype control placebo treatment.

Quantification of the virus: viral titers were quantified for various tissues using a quantitative RT-PCR assay. Total RNA was extracted from tissue samples using TRIzol (ThermoFisher Scientific, cat # 15596018). Amplification was performed with 2. Mu.l of RNA preparation. Serial dilutions of synthetic RNA across the amplified region were used as positive controls; undiluted synthetic ZIKV RNA having a 10 ^8.0 Copies/. Mu.l. After an initial single cycle of 30 minutes at 50 ℃ and 10 minutes at 95 ℃, the samples were subjected to the following steps for 40 cycles: at 95 ℃ for 15 seconds and 60 ℃ for 60 seconds. Unknown amounts of sample were quantified by extrapolating the C (t) values using the curve generated by serial dilution of the synthetic ZIKV RNA.

Experiment design: the dams were challenged on day 7 post-coital (dpc) and treated with FIT-1 24 or 72 hours post virus challenge. At 11 days post virus challenge, 2 mice per group were necropsied. Placenta, fetal tissue, brain tissue and spleen tissue were collected to determine viral titers by QRT-PCR. Each group was then born by 2 females. Digital calipers were used to record the occipital frontal muscle diameter (OF) OF the head OF the pup and the coronal hip length (CRL) OF the pup, and the size OF the pup was determined by determining the intrauterine growth restriction by the following formula: CRL X OF. The weights of pups and dams were measured at different times.

Statistical analysis: survival data was analyzed using Wilcoxon log rank survival analysis (Prism 5, graphpad software, inc).

Results and discussion:

treatment with FIT-1 was evaluated in a mouse model of disease associated with congenital infection and ZIKV infection. Treatment with FIT-1 protected pregnant females and most of the treated females did not die whenever treated (fig. 21). The mortality rate of females treated with the non-specific negative control antibody was close to that observed in previous studies.

The trend in average cub size improved. The mean size of pups from dams treated with FIT-1 was higher than that of control MPE8 and similar to sham infected animals (fig. 22). This difference was less obvious in terms of fetal body weight, as the mean values were similar for all groups (fig. 23). The maternal mice treated with FIT-1 had a tendency to increase placental weight (FIG. 24).

Viral titers for various tissues are shown in figure 25. Significant reduction in viral RNA was observed in fetuses and placentas of maternal mice treated with FIT-1 (fig. 25A and 25B, respectively). This reduction was particularly evident in placental tissue with about a 5-log reduction in ZIKV RNA levels ₁₀ . Viral RNA was also significantly reduced in the maternal spleen and brain of FIT-1 treated animals compared to MPE8, with levels reduced by several logs ₁₀ (FIG. 25C and FIG. 25D, respectively).

And (4) conclusion:

collectively, these data support the protective role of FIT-1 in preventing or treating fetal diseases that are inherently exposed to ZIKV. A trend was observed to improve fetal and placental size parameters, while greatly reducing viral titers in various maternal and fetal tissues.

Example 15: effect of FIT-1 on AG129 mouse testis infection model diseases

Sexually transmitted and persistent infections of the male reproductive tract have been documented in men infected with ZIKV (D' Ortenzio E, matheron S, yazdanpanah Y, et al. Evaluation of Sexual Transmission of Zika virus.N Engl J Med 2016. In the AG129 mouse strain, severe disease is typically observed about 2 weeks after virus challenge, including massive replication of the virus in the mouse testis (Julander JG, siddhartan V, evans J, et al, efficacy of the broad-specific antiviral compound BCX4430 against Zika virus in cell culture and in a mouse model. Antiviral Res 2016. Key sites for viral replication in the reproductive tract of male AG129 mice include epididymis and testis, as well as various accessory gonads.

In this study, the effect of FIT-1 on ZIKV-infected male mice was evaluated in a testis infection model. For this, male AG129 mice were infected with ZIKV and the pathology of the male reproductive tract was assessed after ZIKV infection and FIT-1 treatment.

Materials and methods:

animals: male AG129 mice were used. Animal groups were randomly divided into experimental groups and labeled with ear tags, respectively.

Virus: zika virus (Puerto Rican virus strain, PRVABC-59). Administration 10 by subcutaneous injection of a 0.1ml volume in the inguinal fold ² CCID ₅₀ Challenge dose. This challenge dose was usually lethal in untreated AG129 mice, with death occurring around 2 weeks post challenge.

Testing reagent: the dose of FIT-1 was 15mg/kg. The non-specific control antibody MPE8-LALA Ctr IgG1 was used as isotype control placebo treatment.

Histopathology: tissues were collected and incubated in neutral formalin buffer for 24 hours. After appropriate fixation, all collected tissues were trimmed and fixed in 70% ethanol until routine processing, paraffin embedding and sectioning. All tissues were analyzed blindly and independently by veterinary anatomical pathology inpatients and committee certified veterinary anatomical pathologists. A scoring system was developed to grade the severity of inflammation in the reproductive tract.

Experiment design: mice were infected with ZIKV and monitored for survival and weight change 28 days after virus challenge. 24 hours or 72 hours after virus challenge, treatment was performed with 15mg/kg FIT-1. A single treatment was administered as an intraperitoneal injection in a volume of 0.1 ml. Isotype matched control antibody MPE8-LALA control IgG1 was administered as described above and treated 24 hours after virus challenge. A group of mock-infected, FIT-1 treated mice was used as toxicity control, and a group of normal controls was included. Weight calculations were performed every other day on day 0 and in 7-21dpi, respectively. Mice were observed daily for signs of disease including conjunctivitis, kyphosis, and weakness or paralysis of the limbs, and initial episodes of signs of disease were recorded. A cohort of 3 animals was necropsied at 6dpi and tissue samples were collected for histopathological analysis.

Statistical analysis: survival data was analyzed using Wilcoxon log rank survival analysis (Prism 5, graphpad software, inc).

Results and discussion:

zika virus (ZIKV) can last up to six months in male reproductive tissues, a clear target for antiviral therapy. To determine the efficacy of bispecific anti-ZIKV abs in preventing or reducing male reproductive tract disorders, groups of mice were treated 24 or 72h after challenge with ZIKV isolate virus from puerto rico. Survival, body weight change and histopathology were used to determine the efficacy of mAb treatment. Isotype control mAb, MPE8-LALA Ctr IgG1 was used as placebo treatment.

A significant (P < 0.05) increase in survival was observed compared to placebo mAb treatment (figure 26). Complete survival was observed in mice treated 24 hours after challenge with FIT-1, whereas one animal died of viral infection as a result of treatment of mice 72 hours after viral inoculation. This animal was euthanized 24 days after virus challenge, which was much later than the placebo treated animals (fig. 26). Since infection of AG129 mice with ZIKV resulted in lethality, which is much more severe than the typical natural infection with this virus, the high level of protection observed with FIT-1 is very promising.

The mean body weight change of mice treated with FIT-1 was similar to that of sham infected treated controls, while the mean body weight of mice treated with MPE8 decreased rapidly after 7dpi, further demonstrating effective protection of the mice from disease (figure 27).

Since the main objective of this study was to assess the effect of the treatment on the male reproductive tract, and in previous studies, the testicles and epididymis were most severely affected after infection, special attention was paid to the histopathology of these tissues. In the 24-hour treatment group, no disease was observed in the testis or epididymis of mice treated with FIT-1 except one animal (FIG. 28), further demonstrating the efficacy of FIT-1 treatment. FIT-1 also protected mice from disease when treatment was initiated 72 hours after virus challenge, when no disease was observed in the testes or epididymis in treated mice (fig. 28). Most of the mice in the placebo treated group had inflammation in testis (2/3) and epididymis (3/3) (FIG. 28), although the disease was less severe in this study.

And (4) conclusion:

treatment with FIT-1 was effective in reducing all disease parameters evaluated. Therapeutic treatment up to 72 hours after virus challenge was very effective.

Example 16: preventive and therapeutic efficacy of FIT-1 in ZIKV challenged rhesus monkeys

This study evaluated the prophylactic and therapeutic effects of bispecific antibodies against ZIKV virus (ZIKV) in Indian Rhesus Monkeys (IRMs) challenged with the ZIKV virus strain PRVABC 59. Sixteen (16) IRMs were randomized into three treatment groups. One group received FIT-1 (5 mg/kg) the day before challenge and the other group received treatment (15 mg/kg) the day after challenge. The third group was treated with an isotype control (FIT-3,5mg/kg) the day before challenge. 1X 10 delivered by Subcutaneous (SC) injection on day 0 ⁵ The ZIKV strain of PFU, PRVABC59, attacks all IRMs. Serum, urine and saliva were collected at predetermined time points and the ZIKV load was measured by quantitative RT-PCR.

The method comprises the following steps:

sixteen (16) IRMs were randomized into the corresponding groups by gender/weight using the proventis software prior to study day 0. On day-1 or day 1, animals received either FIT-1 or isotype control FIT-3 delivered intravenously at the doses listed in Table 4 below. On day 0, all rhesus monkeys were anesthetized and challenged with 0.5mL of wild-type ZIKV strain PRVABC59 by subcutaneous injection at a target challenge dose of 1.0 x 10 per animal ⁵ PFU. As shown in table 5, blood, urine and saliva samples were collected at predetermined time points to assess viral load by RT-qPCR.

Table 4: grouping of animals

¹ Subcutaneous challenge with 0.5mL wild type ZIKV on day 0

Table 5: key behaviors

¹ Day 0 and day 1 blood collection prior to challenge and treatment

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

ZIKV viral strain PRVABC59; human/2015/Puerto Rico (us isolate) was the challenge material used in this study. Preparation of virus inocula was carried out under BSL-2 conditions in a class II biosafety cabinet. Virus stocks were thawed in a 37 + -1 deg.C water bath, vortexed, and diluted with VP-SFM to yield 2X 10 ⁵ PFU/mL inoculum of appropriate concentration. Each syringe was filled with 0.5mL of viral inoculum and kept on ice until transferred to the animal facility for administration. On study day 0, instituteAnimals were anesthetized, clamped at the injection site, wiped with alcohol, and marked with a non-erasable marker. Animals were SC inoculated with 0.5mL of ZIKV isolates on the anterior surface of the left forearm of the animal, at doses as shown in table 4.

All rhesus monkeys were observed twice daily for morbidity and mortality throughout the quarantine and study period. Animals were observed twice daily (at least 8 hour intervals) for reactivity and clinical signs including rash, erythema, conjunctivitis, ocular secretions and swelling. For all animals, blood was collected at the time points shown in table 5 for in vitro testing by RT-qPCR viral load. The collection volume per animal did not exceed 20mL during the final blood draw on day 30. For all animals, urine was collected at the time points shown in table 5 for in vitro testing by RT-qPCR to monitor viral shedding. Animals were housed individually during urine collection for the study. Urine was collected directly from the cages, collected on wet ice, aliquoted and stored at-70 ℃ or below-70 ℃ until ready for assay testing. Saliva (saliva) from anesthetized animals was collected directly into test tubes (up to about 0.5 mL). After collection, the samples were stored on wet ice and at a temperature of-70 ℃ or below-70 ℃ until ready for assay testing.

Viral loads were measured using RT-qPCR methods to detect ZIKV genomes in serum, urine and saliva samples collected at the time points shown in table 5. Samples recovered from virus infected animals were tested using primers and probes designed to detect the ZIKV virus strain PRVABC 59. A description of PCR methods, including primer and probe sequences, has been published (Goebel et al, 2016, A Sensitive virus yield assay for evaluation of antivirals agaiset Zika virus.J.Virol. Methods). Viral RNA was isolated from the organism fluid using the QIAmp Viral RNA mini kit (Qiagen, 52906). With sterile RNase and DNase-free H ₂ O eluting the viral RNA and storing at-70 ℃ or below-70 ℃. The lower limit of quantitation (LLOQ) of this assay was determined as 10 copies per reaction.

Challenge of viral inoculum by back titration on Vero cells by standard plaque assayAliquot to confirm the actual delivered dose. Ten-fold serial dilutions of challenge inoculum were used to infect confluent monolayers of Vero cells in 6-well plates seeded the day before. Plates were CO-evaporated at 37 ℃ and 5% before addition of overlay medium containing 0.5% agarose ₂ Incubate for 1 hour. Plates were incubated for 3 days until recognizable plaques were formed, then fixed, stained with crystal violet and counted.

As a result:

rhesus monkeys were monitored twice daily for mortality and morbidity during the study. All animals survived to a predetermined termination time. The body weights and temperatures of all animals were measured at the time points shown in table 5. There was no significant weight loss during the study. Body temperatures of all animals were maintained within normal ranges throughout the study. Clinical observations were limited to mild redness at the challenge site in a few animals.

Viral loads were measured using RT-qPCR method to detect ZIKV genomes in serum, urine and saliva samples collected at the time points shown in table 5. The viral load in serum is shown in FIG. 29.

The day after challenge, the viral load in the serum of animals of group 3 treated with isotype control was detectable. Three of the four animals reached a peak viral load of 1X 10 on day 2 or day 3 ⁵ Genome copy/mL (GC/mL) or more. The mean serum viral load of this group reached 1.15X 10 on day 3 ⁵ Peak in GC/mL. By day four, the viral load of one animal dropped below the determined LLOQ (860 GC/mL), while the viral load of the remaining three animals dropped to that amount on day five.

The animals of group 2 treated with 15mg/kg FIT-1 the day after challenge had an average viral load of 3.99X 10 before treatment ³ GC/mL. On day 2, the mean serum viral load was reduced to only 96.5GC/mL. Although low levels of viral RNA were occasionally detected after day 1, the viral load detected in group 2 animals did not reach or exceed the LLOQ determined by RT-qPCR at any time point after treatment.

Pretreatment with FIT-1 at 5mg/kg reduced the average serum viral load by more than 50-fold on day 1 compared to group 3. At any time point after challenge, no viral load at or above the LLOQ determined was detected, and by day 2 no viral RNA was detected in the serum of any group 1 animals.

Only low levels of ZIKV RNA were detected occasionally in the urine or saliva of any animal. At any time point, no viral load of LLOQ or above was detected in the urine or saliva of any animal.

Aliquots of the challenge virus inoculum were back-titrated by standard plaque assay on Vero cells to confirm the actual delivered dose. The titer produced by plaque assay was 1.7X 10 ⁵ PFU/mL。

Summary and conclusion:

this study evaluated the prophylactic and therapeutic efficacy of FIT-1 in IRM challenged with ZIKV. No mortality occurred throughout the study and clinical observations were limited to mild redness at the challenge site in a few animals. Viral load detected by RT-qPCR in sera collected after challenge was the primary endpoint of the study. Viral RNA was readily detected in the sera of all animals treated with isotype control, with viral load peaking at day 2 or day 3 and maintained at levels above 860GC/mL of LLOQ until day 4 or day 5. The average peak load on day 3 was 1.15X 10 ⁵ GC/mL. In contrast, in ZIKV-infected IRM, prophylactic treatment with 5mg/kg FIT-1 was effective in reducing the peak viral load and the time to clearance of the virus. Low levels of viral RNA were detected from all six animals in the group by day 1, but no viral RNA was detected in any animal by day 2. In the sera of any animal in this group, no ZIKV RNA was detected for LLOQ above 860GC/mL at any time point. Also, treatment with FIT-1 at 15mg/kg the day after challenge reduced the peak viral load and time to clear virus from serum compared to isotype control treated animals. The rhesus monkeys of group 2 had an average peak viral load of 3.99X 10 at day 1 ³ GC/mL, but after day 1 treatment, the mean viral load in this group of sera at day 2 dropped to only 96.5 GC-And (mL). None of the animals in this group had a viral load above the LLOQ of 860GC/mL for the remainder of the observation period.

Sequence List and SEQ ID numbering

* The sequences highlighted in bold are the CDR regions (nucleotides or aa), the underlined residues are residues mutated compared to the "germline" sequence.

Claims (51)

1. An isolated multispecific antibody or antigen-binding fragment thereof that specifically binds different Zika virus epitopes, wherein the antibody or antigen-binding fragment thereof comprises an epitope binding site comprising (i) the amino acid sequences according to SEQ ID NOs: the CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences of 19 to 23 and 25; or (ii) the amino acid sequences of CDRH1, CDRH2 and CDRH3 and the amino acid sequences of CDRL1, CDRL2 and CDRL3 according to SEQ ID NOS: 19 to 22 and 24 to 25 respectively.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof neutralizes Zika virus infection.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof abrogates the production of Zika virus escape mutants.

4. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein said antibody or antigen-binding fragment thereof does not promote antibody-dependent enhancement of Zika virus infection.

5. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.

6. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is bispecific, trispecific, tetraspecific, or pentaspecific.

7. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is bivalent, trivalent, tetravalent, hexavalent, or octavalent.

8. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody molecule comprises exactly two copies of each distinct epitope binding site that specifically bind at least two distinct Zika virus epitopes.

9. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is of the IgG class.

10. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises an Fc portion.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein the antibody or antigen-binding fragment thereof comprises a mutation in the Fc portion that reduces binding of the antibody to an Fc receptor.

12. The antibody or antigen-binding fragment thereof of claim 11, wherein the antibody or antigen-binding fragment thereof comprises a CH2L4A mutation, a CH2L5A mutation, or both.

13. The antibody or antigen-binding fragment thereof of claim 1 or 2, having a tandem Fab-Ig antibody format.

14. The antibody or antigen-binding fragment thereof of claim 1 or 2, having the form of a DVD-Ig antibody.

15. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof does not comprise a binding site for an Fc receptor.

16. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof specifically binds to a different epitope on the envelope protein of Zika virus.

17. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof binds to domain III (EDIII) of the envelope protein of zika virus.

18. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof binds to an epitope of Zika virus envelope protein that comprises one or more amino acid residues of the EDIII Lateral Ridge (LR) and/or one or more amino acid residues of the EDI-EDIII hinge region.

19. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is capable of inhibiting post-attachment steps of ZIKV.

20. The antibody or antigen-binding fragment thereof of claim 19, wherein the antibody or antigen-binding fragment thereof is capable of preventing membrane fusion.

21. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is capable of causing aggregation of ZIKV particles.

22. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises (i) an amino acid sequence according to SEQ ID NO:1 to 5 and 7 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences; (ii) a polypeptide according to SEQ ID NO:1 to 4 and 6 to 7 and the amino acid sequences of CDRH1, CDRH2 and CDRH3 and CDRL1, CDRL2 and CDRL 3; (iii) a sequence according to SEQ ID NO: the CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences of 37 to 41 and 43; (iv) according to SEQ id no: the CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences of 37 to 40 and 42 to 43; (v) according to SEQ ID NO: the CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences of 55 to 59 and 61; (vi) the sequence according to SEQ ID NO: the CDRH1, CDRH2 and CDRH3 amino acid sequences and the CDRL1, CDRL2 and CDRL3 amino acid sequences of 55 to 58 and 60 to 61; (vii) according to SEQ ID NO: the CDRH1, CDRH2 and CDRH3 amino acid sequences and the CDRL1, CDRL2 and CDRL3 amino acid sequences of 73 to 77 and 79; or (viii) a sequence according to SEQ ID NO: amino acid sequences of CDRH1, CDRH2 and CDRH3 and amino acid sequences of CDRL1, CDRL2 and CDRL3 from 73 to 76 and from 78 to 79.

23. The antibody or antigen-binding fragment thereof of claim 22, wherein the antibody or antigen-binding fragment thereof comprises

(a) A first epitope binding site comprising (i) a sequence according to SEQ ID NO:1 to 5 and 7 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences; or (ii) a sequence according to SEQ ID NO:1 to 4 and 6 to 7 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences; and

(b) A second epitope binding site comprising (i) a sequence according to SEQ ID NO: the CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences of 19 to 23 and 25; or (ii) a sequence according to SEQ ID NO:19 to 22 and 24 to 25 and CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences.

24. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises 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% sequence identity; or consist thereof; and the light chain variable region (VL) comprises a 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; or consist thereof; wherein (i) is according to SEQ ID NO: the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of 19 to 23 and 25; or (ii) according to SEQ ID NO: the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 19 to 22 and 24 to 25 remain unchanged.

25. The antibody or antigen-binding fragment thereof of claim 1 or 2, further comprising (i) 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 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 heavy chain variable region (VH) 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 98%, or at least 99% sequence identity to a light chain variable region (VL) amino acid sequence of SEQ ID NO:1 to 5 and 7, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences; or (ii) according to SEQ ID NO: the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 1 to 4 and 6 to 7 remain unchanged; (ii) a 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% sequence identity to a heavy chain variable region (VH) 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 thereto, wherein (i) the amino acid sequence of SEQ ID NO: amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of 37 to 41 and 43; or (ii) according to SEQ ID NO: the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 37 to 40 and 42 to 43 remain unchanged; (iii) 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% sequence identity to a heavy chain variable region (VH) 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 to a light chain variable region (VL) amino acid sequence of SEQ id no: the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of 55 to 59 and 61; or (ii) according to SEQ ID NO: the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 55 to 58 and 60 to 61 remain unchanged; or (iv) a polypeptide 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 to a heavy chain variable region (VH) 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 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 thereto, wherein (i) the amino acid sequence of SEQ ID NO: CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of 73 to 77 and 79; or (ii) according to SEQ ID NO: the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 73 to 76 and 78 to 79 remain unchanged.

26. The antibody or antigen-binding fragment thereof of claim 25, wherein the antibody or antigen-binding fragment thereof comprises

(a) A first epitope binding site comprising a sequence according to 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 to a heavy chain variable region (VH) 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 98%, or at least 99% sequence identity; wherein (i) is according to SEQ ID NO:1 to 5 and 7 or (ii) the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 according to SEQ ID NO: the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 1 to 4 and 6 to 7 remain unchanged; and

(b) A second epitope binding site comprising 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% sequence identity to a heavy chain variable region (VH) 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; wherein (i) is according to SEQ ID NO:19 to 23 and 25 or (ii) the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 according to SEQ ID NOs: the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 19 to 22 and 24 to 25 remain unchanged.

27. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is in the form of a tandem Fab-Ig (FIT-Ig), and the outer Fab of the FIT-Ig format comprises an epitope binding site comprising (i) the amino acid sequence according to SEQ ID NO:1 to 5 and 7 and the amino acid sequences of CDRH1, CDRH2 and CDRH3 and CDRL1, CDRL2 and CDRL 3; or (ii) a sequence according to SEQ ID NO:1 to 4 and 6 to 7 and the amino acid sequences of CDRH1, CDRH2 and CDRH3 and CDRL1, CDRL2 and CDRL 3; and the internal Fab of the FIT-Ig format comprises an epitope binding site comprising (i) an amino acid sequence according to SEQ ID NO: the CDRH1, CDRH2 and CDRH3 amino acid sequences and CDRL1, CDRL2 and CDRL3 amino acid sequences of 19 to 23 and 25; or (ii) a sequence according to SEQ ID NO: the amino acid sequences of CDRH1, CDRH2 and CDRH3 and the amino acid sequences of CDRL1, CDRL2 and CDRL3 of 19 to 22 and 24 to 25.

28. The antibody or antigen-binding fragment thereof of claim 27, wherein the external Fab of the FIT-Ig format comprises an epitope binding site comprising 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 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 a heavy chain variable region (VH) 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 98%, or at least 99% sequence identity; wherein (i) is according to SEQ ID NO:1 to 5 and 7 or (ii) the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 according to SEQ ID NO: the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 1 to 4 and 6 to 7 remain unchanged.

29. The antibody or antigen-binding fragment thereof of claim 28, wherein the internal Fab of the FIT-Ig format comprises an epitope binding site comprising 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% sequence identity to a heavy chain variable region (VH) 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; wherein (i) is according to SEQ ID NO:19 to 23 and 25 or (ii) the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 according to SEQ ID NOs: the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 19 to 22 and 24 to 25 remain unchanged.

30. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is a purified antibody or a single chain antibody.

31. A nucleic acid molecule comprising at least one polynucleotide encoding the antibody or antigen-binding fragment thereof of claim 1 or 2.

32. The nucleic acid molecule of claim 31, wherein the nucleic acid molecule is a monocistron or a polycistron.

33. The nucleic acid molecule of claim 32, wherein the nucleic acid molecule is a dicistronic.

34. The nucleic acid molecule of claim 31 or 32, wherein the nucleic acid molecule is DNA or RNA.

35. The nucleic acid molecule of claim 34, wherein the nucleic acid molecule is a DNA plasmid or mRNA.

36. A vector comprising the nucleic acid molecule of claim 31 or 32.

37. A plurality of nucleic acid molecules, each nucleic acid molecule comprising at least one polynucleotide encoding a polypeptide chain of the antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the plurality of nucleic acids encode the antibody or antigen-binding fragment thereof of claim 1 or 2.

38. A plurality of vectors encoding the antibody or antigen-binding fragment thereof of claim 1 or 2.

39. A cell expressing the antibody or antigen-binding fragment thereof of claim 1 or 2; or comprising a vector according to claim 36 or a plurality of vectors according to claim 38.

40. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of claim 1 or 2, the nucleic acid molecule of claim 31 or 32, the vector of claim 36, the plurality of nucleic acid molecules of claim 37, the plurality of vectors of claim 38, and/or the cell of claim 39.

41. The pharmaceutical composition of claim 40, further comprising a pharmaceutically acceptable excipient, diluent or carrier.

42. The antibody or antigen-binding fragment thereof according to claim 1 or 2, the nucleic acid molecule according to claim 31 or 32, the vector according to claim 36, the plurality of nucleic acid molecules according to claim 37, the plurality of vectors according to claim 38, the cell according to claim 39, or the pharmaceutical composition according to claim 40 for use in preventing or treating Zika virus infection.

43. The antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, nucleic acid molecules, vectors, cells, or pharmaceutical composition for use according to claim 42, for preventing or treating Zika virus infection in a subject diagnosed with or exhibiting symptoms of Zika virus infection.

44. An antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, plurality of nucleic acid molecules, plurality of vectors, cell, or pharmaceutical composition for use according to claim 42, for use in an asymptomatic subject.

45. The antibody or antigen binding fragment thereof, nucleic acid molecule, vector, plurality of nucleic acid molecules, plurality of vectors, cell or pharmaceutical composition for use according to claim 42 or 43, for use in a pregnant subject.

46. The antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, plurality of nucleic acid molecules, plurality of vectors, cell, or pharmaceutical composition for use of claim 42 or 43, wherein the antibody or antigen-binding fragment thereof, nucleic acid, vector, plurality of nucleic acid molecules, plurality of vectors, cell, or pharmaceutical composition infection is administered up to seven days after Zika virus infection.

47. The antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, plurality of nucleic acid molecules, plurality of vectors, cell, or pharmaceutical composition for use according to claim 42 or 43, wherein the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the plurality of nucleic acid molecules, the plurality of vectors, the cell, or the pharmaceutical composition is administered in combination with a checkpoint inhibitor.

48. The antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, plurality of nucleic acid molecules, plurality of vectors, cell or pharmaceutical composition for use according to claim 42 or 43, wherein the antibody or antigen-binding fragment thereof, the nucleic acid, the plurality of nucleic acid molecules, the plurality of vectors, the vector, the cell or the pharmaceutical composition is administered at a dose of 0.005mg/kg to 100 mg/kg.

49. Use of the antibody or antigen binding fragment thereof according to claim 1 or 2 for monitoring the quality of an anti-zika vaccine by checking that the antigen comprises a specific epitope in the correct conformation.

50. Use of the antibody or antigen-binding fragment thereof according to claim 1 or 2 in the preparation of a diagnostic reagent for diagnosing Zika virus infection.

51. A kit of parts comprising at least one antibody or antigen-binding fragment thereof according to claim 1 or 2, at least one nucleic acid according to claim 31 or 32, at least one vector according to claim 36, a plurality of nucleic acid molecules according to claim 37, a plurality of vectors according to claim 38, at least one cell according to claim 39, or at least one pharmaceutical composition according to claim 40.