MULTISPECIFIC ANTIBODIES AGAINST SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 The present invention relates to the field of multispecific antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). In particular, the present invention relates to multispecific antibodies binding to distinct epitopes of the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2. The present invention also relates to the use of such antibodies, e.g. for prophylaxis or treatment of coronavirus disease-2019 (COVID-19) or for (in-v/'tro) diagnostic purposes. Antibody therapy is largely considered as a promising prophylactic and therapeutic option for treatment of COVID-19, with various antibodies currently being in clinical development. For example, Robbiani et al. reported potently neutralizing human antibodies targeting different epitopes of the receptor-binding domain (RBD) of the spike (S) protein ofSARS-CoV- 2, which were isolated from COVID-19 patients, including antibodies C121, C135 and C144 (Robbiani, D.F., Gaebler, C., Muecksch, F. et al. Convergent antibody responses to SARS- CoV-2 in convalescent individuals. Nature 584, 437-442 (2020)). However, viral escape mutations, which can confer resistance to antibodies, and possibly vaccines, were observed (Weisblum, Yiska et al. Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants. bioRxiv: the preprint server for biology 2020.07.21.214759.22 Jul.2020, doi:10.1101/2020.07.21.214759. Preprint). Antibody cocktails including distinct antibodies have challenges for production (two molecules needed instead of one) and formulation. They also complicate novel approaches like antibody mRNA delivery. In view of the above, it is the object of the present invention to overcome the drawbacks of the prior art outlined above and to provide an antibody, or antigen-binding fragment thereof, which potently neutralizes SARS-CoV-2 and its escape mutants. In addition, it is an object of the present invention to provide an antibody, or antigen-binding fragment thereof, which fully inhibits binding of SARS-CoV-2 to hACE2, the cellular receptor for SARS-CoV-2. Furthermore, it is an object of the present invention to provide an antibody, or antigen-binding fragment thereof, which prevents the generation of viral escape mutants and/or resists to their insurgence.

This object is achieved by means of the subject-matter set out below and in the appended claims.

Although the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, 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 meanings as commonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise. Throughout this specification and the claims which follow, unless the context requires otherwise, the term "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step but not the exclusion of any other non-stated member, integer or step. The term "consist of" is a particular embodiment of the term "comprise", wherein any other non-stated member, integer or step is excluded. In the context of the present invention, the term "comprise" encompasses the term "consist of". The term "comprising" thus encompasses "including" as well as "consisting" e.g., a composition "comprising" X may consist exclusively of X or may include something additional e.g., X + Y.

The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the 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 is 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 essential to the practice of the invention.

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

The term "about" in relation to a numerical value x means x ± 10%, for example, x + 5%, or x ± 7%, or x ± 10%, or x ± 12%, or x ± 15%, or x + 20%.

The term "disease" as used herein is intended to be generally synonymous, and is used interchangeably with, the terms "disorder" and "condition" (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life. As used herein, reference to "treatment" of a subject or patient is intended to include prevention, prophylaxis, attenuation, amelioration and therapy. The terms "subject" or "patient" are used interchangeably herein to mean all mammals including humans. Examples of subjects include humans, cows, dogs, cats, horses, goats, sheep, pigs, and rabbits. In some embodiments, the patient is a human.

Doses are often expressed in relation to the bodyweight. Thus, a dose which is expressed as [g, mg, or other unit]/kg (or g, mg etc.) usually refers to [g, mg, or other unit] "per kg (or g, mg etc.) bodyweight", even if the term "bodyweight" is not explicitly mentioned.

The term "binding" and similar reference usually means "specifically binding", which does not encompass non-specific sticking.

As used herein, the term "antibody" encompasses various forms of antibodies including, without being limited to, whole antibodies, antibody fragments (such as antigen binding fragments), human antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies and genetically engineered antibodies (variant or mutant antibodies) as long as the characteristic properties according to the invention are retained. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a monoclonal antibody. For example, the antibody is a human monoclonal antibody.

As described above, the term "antibody" generally also includes antibody fragments. Fragments of the antibodies may retain the antigen-binding activity of the antibodies. Such fragments are referred to as "antigen-binding fragments". Antigen-binding fragments include, but are not limited to, single chain antibodies, Fab, Fab', F(ab')2, Fv or scFv. Fragments of the antibodies can be obtained from the antibodies by methods that include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, fragments of the antibodies can be obtained by recombinant means, for example by cloning and expressing a part (fragment) of the sequences of the heavy and/or light chain. The invention also encompasses single-chain Fv fragments (scFv) derived from the heavy and light chains of an antibody of the invention. For example, the invention includes a scFv comprising the CDRs from an antibody of the invention. Also included are heavy or light chain monomers and dimers, single domain heavy chain antibodies, single domain light chain antibodies, as well as single chain antibodies, e.g., single chain Fv in which the heavy and light chain variable domains are joined by a peptide linker. Antibody fragments of the invention may be contained in a variety of structures known to the person skilled in the art. In addition, the sequences of the invention may be a component of multispecific molecules in which the sequences of the invention target the epitopes of the invention and other regions of the molecule bind to other targets. Although the specification, including the claims, may, in some places, refer explicitly to antigen binding fragment(s), antibody fragment(s), variant(s) and/or derivative(s) of antibodies, it is understood that the term "antibody" includes all categories of antibodies, namely, antigen binding fragment(s), antibody fragment(s), variant(s) and derivative(s) of antibodies.

Human antibodies are well-known in the state of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001 ) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line 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. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al., /. Immunol. 147 (1991 ) 86-95). In some embodiments, human monoclonal antibodies are prepared by using improved EBV-B cell immortalization as described in Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR, Murphy BR, Rappuoli R, Lanzavecchia A. (2004): An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med. 10(8):871 -5. As used herein, the term "variable region" (variable region of a light chain (V _L ), variable region of a heavy chain (V _H )) denotes each of the pair of light and heavy chains which is involved directly in binding the antibody to the antigen. Antibodies of the invention can be of any isotype (e.g., IgA, IgG, IgM i.e. an a, y or p heavy chain). For example, the antibody is of the IgG type. Within the IgG isotype, antibodies may be IgGI , lgG2, lgG3 or lgG4 subclass, for example IgGI . Antibodies of the invention may have a K or a A light chain. In some embodiments, the antibody is of IgGI type and has a K light chain.

Antibodies according to the present 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% (by weight), usually less than 60% and more usually less than 50% of the composition is made up of other polypeptides.

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

As used herein, a "neutralizing antibody" is one that can neutralize, i.e., prevent, inhibit, reduce, impede or interfere with, the ability of a pathogen to initiate and/or perpetuate an infection in a host. The terms "neutralizing antibody" and "an antibody that neutralizes" or "antibodies that neutralize" are used interchangeably herein. These antibodies can be used alone, or in combination, as prophylactic or therapeutic agents upon appropriate formulation, in association with active vaccination, as a diagnostic tool, or as a production tool as described herein.

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

As used herein, the term "mutation" relates to a change in the nucleic acid sequence and/or in the amino acid sequence in comparison to a reference sequence, e.g. a corresponding genomic sequence. A mutation, e.g. in comparison to a genomic sequence, may be, for example, a (naturally occurring) somatic mutation, a spontaneous mutation, an induced mutation, e.g. induced by enzymes, chemicals or radiation, or a mutation obtained by site- directed mutagenesis (molecular biology methods for making specific and intentional changes in the nucleic acid sequence and/or in the amino acid sequence). Thus, the terms "mutation" or "mutating" shall be understood to also include physically making a mutation, e.g. in a nucleic acid sequence or in an amino acid sequence. A mutation includes substitution, deletion and insertion of one or more nucleotides or amino acids as well as inversion of several successive nucleotides or amino acids. To achieve a mutation in an amino acid sequence, a mutation may be introduced into the nucleotide sequence encoding said amino acid sequence in order to express a (recombinant) mutated polypeptide. A mutation may be achieved e.g., by altering, e.g., by site-directed mutagenesis, a codon of a nucleic acid molecule encoding one amino acid to result in a codon encoding a different amino acid, or by synthesizing a sequence variant, 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 the need for mutating one or more nucleotides of a nucleic acid molecule. Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. It is to be understood that th is 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 meanings as commonly understood by one of ordinary skill in the art. Multispecific antibodies The present invention is based, amongst other findings, on the discovery that multispecific antibodies, and antigen-binding fragments thereof, that bind specifically to distinct (neutralizing) epitopes of the receptor-binding domain (RBD) of the spike (S) protein of SARS- CoV-2, minimize or eliminate the generation of SARS-CoV-2 escape mutants. The antibodies of the present invention potently neutralize SARS-CoV-2 and its escape mutants and fully inhibit binding of SARS-CoV-2 to hACE2, the cellular receptor for SARS-CoV-2. Furthermore, the antibodies of the present invention prevent the generation of viral escape mutants. n a first aspect the present invention provides a multispecific antibody binding to distinct (neutralizing) epitopes of the receptor-binding domain (RBD) of the spike (S) protein of SARS- CoV-2. In other words, the present invention provides an isolated multispecific antibody, or an antigen binding fragment thereof, that comprises at least two epitope binding sites, which specifically bind to distinct (neutralizing) epitopes of the receptor-binding domain (RBD)ofhe spike (S) protein of SARS-CoV-2. Importantly, in contrast to conventional ("ordinary") antibodies exhibiting just one single specificity, multispecific antibodies are able to bind to at least two different epitopes. In the present case, the multispecific antibodies specifically bind to (at least two) distinct epitopes of the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2. Accordingly, as used herein, the term "multispecific" refers to the ability to bind to at least two different epitopes, e.g. in the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2. Preferably, the antibody, or antigen binding fragment thereof, according to the present invention is bispecific, trispecific, tetraspecific or pentaspecific, more preferably the antibody, or the antigen binding fragment thereof, is bispecific, trispecific or tetraspecific, even more preferably the antibody, or the antigen binding fragment thereof, is bispecific or trispecific, and most preferably the antibody, or the antigen binding fragment thereof, is bispecific. As used herein, terms like "bispecific", trispecific", "tetraspecific" etc. refer to the number of different epitopes to which the antibody, or the antigen binding fragment thereof, can bind to. For example, conventional monospecific IgG-type antibodies have two identical epitope binding sites (paratopes) and can, thus, only bind to identical epitopes (but not to different epitopes). A multispecific antibody, in contrast, has at least two different types of epitope binding sites (paratopes) and can, thus, bind to at least two different epitopes. As used herein, the term "paratope" refers to an epitope-binding site of the antibody. Accordingly, the terms "paratope" and "epitope binding site" are used herein interchangeably. Moreover, a single "specificity" may refer to one, two, three or more identical paratopes in a single antibody. The actual number of paratopes in one single antibody molecule is referred to as "valency". Preferably, the antibody, or antigen binding fragment thereof, according to the present invention is bivalent, trivalent, tetravalent, hexavalent or octavalent, more preferably, the antibody, or the antigen binding fragment thereof, is bivalent or tetravalent, most preferably, the antibody, or the antigen binding fragment thereof, is bivalent. Most preferably, the antibody, or antigen binding fragment thereof, according to the present invention is bispecific and bivalent. It is also preferred that the antibody, or antigen binding fragment thereof, according to the present invention comprises a single copy of each of the distinct epitope binding sites specifically binding to at least two distinct epitopes of the receptor-binding domain (RED) of the spike (S) protein of SARS-CoV-2.

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

In particular, the multispecific antibody, or the antigen binding fragment thereof, may comprise two or more paratopes, wherein some paratopes may be identical so that all paratopes of the antibody belong to at least two different types of paratopes and, hence, the antibody has at least two specificities. Preferably, however, the multispecific antibody or antigen binding fragment thereof according to the present invention comprises a single paratope for each specificity, e.g. two (distinct) paratopes (i.e., with distinct specificities, distinct epitope binding sites) in case of a bispecific antibody. Thus, "one specificity" refers in particular to one or more paratopes exhibiting the same specificity (which typically means that such one or more paratopes are identical) and, thus, "two specificities" may be realized by two, three, four five, six or more paratopes as long as they refer to only two specificities. For example, a multispecific antibody may comprise one single paratope for each (of the at least two) specificity, i.e. the multispecific antibody comprises in total at least two paratopes. Preferably, a bispecific antibody comprises one single paratope for each of the two specificities, i.e. the antibody comprises in total two paratopes. Alternatively, the antibody comprises exactly two (identical) paratopes for each of the two specificities, i.e. the antibody comprises in total four paratopes. Alternatively, the antibody may comprise three (identical) paratopes for each of the two specificities, i.e. the antibody comprises in total six paratopes. In general, a multispecific antibody may comprise the paratopes (specificities) of two or more conventional (monospecific) antibodies (referred to as "parental" antibodies). Accordingly, the multispecific antibody, or the antigen-binding fragment thereof, may be derived from two or more monospecific parental antibodies (e.g., naturally occurring antibodies, such as antibodies isolated from humans). In this context, the expression "derived from" means that the multispecific antibody includes the set of six CDRs (preferably the entire heavy and light chain variable regions) of each of the parental antibodies.

The multispecific antibody, or the antigen binding fragment thereof, according to the present invention may be of any multispecific antibody format. In particular, multispecific antibodies preferably encompass "whole" antibodies, such as whole IgG- or IgG-like molecules, while antigen binding fragments in the context of the present invention preferably refer to small recombinant formats, such as a format based on bispecific T-cell engagers (BiTE®s; except that in the context of the present invention both specificities target the RBD of SARS-CoV-2, accordingly, the T-cell specificity of BiTESs may be replaced by a (second) RBD specificity), tandem single chain variable fragment molecules (taFvs), diabodies (Dbs), single chain diabodies (scDbs) and various other derivatives of these (cf. bispecific antibody formats as described by Byrne H. et al. (2013) Trends Biotech, 31 (1 1 ): 621 -632 with Figure 2 showing various bispecific antibody formats; Weidle U.H. et al. (2013) Cancer Genomics and Proteomics 10: 1 -18, in particular Fig. 1 showing various bispecific antibody formats; and Chan, A.C. and Carter, P.J. (2010) Nat Rev Immu 10: 301 -316 with Fig. 3 showing various bispecific antibody formats). Examples of bispecific antibody formats include, but are not limited to, CrossMAb, knob-in-hole (KIH), DVD-lg, quadroma, chemically coupled Fab (fragment antigen binding), and BiTE® (bispecific T cell engager). In one embodiment of the present invention the antibody is preferably a CrossMAb.

Thus, the antibody, or the antigen binding fragment thereof, according to the present invention may be selected from the group comprising Fabs-in-tandem-lg (FIT-lg); DVD-lg; hybrid hybridoma (quadroma); Multispecific anticalin platform (Pieris); Diabodies; Single chain diabodies; Tandem single chain Fv fragments; TandAbs, Trispecific Abs (Affimed) (105- 1 10 kDa); Darts (dual affinity retargeting; Macrogenics); Bispecific Xmabs (Xencor); Bispecific T cell engagers (Bites; Amgen; 55kDa); Triplebodies; Tribody = Fab-scFv Fusion Protein (CreativeBiolabs) multifunctional recombinant antibody derivates (110 kDa); Duobody platform (Genmab); Dock and lock platform; Knob into hole (KIH) platform; Humanized bispecific IgG antibody (REGN1979) (Regeneron); Mab ² bispecific antibodies (F-Star); DVD- Ig = dual variable domain immunoglobulin (Abbott); kappa-lambda bodies; TBT) = tetravalent bispecific tandem Ig; and CrossMAb.

The antibody, or the antigen binding fragment thereof, according to the present invention may be selected from bi specific IgG-like antibodies (BsIgG) comprising CrossMAb; DAF (two- in-one); DAF (four-in-one); DutaMab; DT-IgG; Knobs-in-holes common EC; Knobs-in-holes assembly; Charge pair; Fab-arm exchange; SEEDbody; Triomab; LUZ-Y; Fcab; K/.-body; and Orthogonal Fab. These bispecific antibody formats are shown and described for example in Spiess C, Zhai Q. and Carter P.J. (2015) Molecular Immunology 67: 95-106, in particular Fig. 1 and corresponding description, e.g. p. 95 - 101.

In some embodiments, the antibody, or the antigen binding fragment thereof, according to the present invention may be selected from bispecific antibody fragments comprising Nanobody; Nanobody-HAS; BiTE; Diabody; DART; TandAb; scDiabody; sc-Diabody-CH3; Diabody-CH3; Triple Body; Miniantibody; Minibody; TriBi minibody; scFv-CH3 KIH; Fab- scFv; scFv-CH-CL-scFv; F(ab')2; F(ab ^, )2-scFv2; scFv-KIH; Fab-scFv-Fc; Tetravalent HCAb; scDiabody-Fc; Diabody-Fc; Tandem scFv-Fc; and Intrabody. These bispecific antibody formats are shown and described for example in Spiess C, Zhai Q. and Carter P.J. (2015) Molecular Immunology 67: 95-106, in particular Fig. 1 and corresponding description, e.g. p. 95 - 101.

In some embodiments, the antibody, or the antigen binding fragment thereof, according to the present invention may be selected from IgG-appended antibodies with an additional antigen-binding moiety comprising DVD-IgG; lgG(H)-scFv; scFv-(H)lgG; lgG(L)-scFv; scFV- (L)lgG; lgG(L,H)-Fv; lgG(H)-V; V(H)-lgG; lgG(L)-V; V(L)-lgG; KIH IgG-scFab; 2scFv-lgG; IgG- 2scFv; scFv4-lg; scFv4-lg; Zybody; and DVI-IgG (four-in-one). These bispecific antibody formats are shown and described for example in Spiess C, Zhai Q. and Carter P.J. (2015) Molecular Immunology 67: 95-106, in particular Fig. 1 and corresponding description, e.g. p. 95 - 101.

Preferably, the multispecific antibody is a CrossMAb (immunoglobulin domain crossover technology) and/or a knob-in-hole (KIH) antibody.

For example, to generate a CrossMAb, a CH1-CL crossover (as compared to the parental antibodies) between the heavy chain (HC) and the LC may be introduced to drive heterodimerization of the heavy chains. This usually results in the connection of the VL (light chain variable region) to the CH1 domain of the heavy chain constant region. The CH1 domain may be modified by addition of two Ser at the N-terminus and of the sequence EPKSC to the C-terminus resulting in the modified CH1 domain. In the heavy chain, the CH1 domain is usually replaced with the CL domain and the VH is connected to the crossover-modified constant region of the heavy chain (CL-CH2-CH3). In the CL domain, the residues A and S at the N-terminus may be substituted with residues R and T, respectively; the residues EPKSC may be removed.

The multispecific antibody of the invention may use the "knobs-in-holes" (KIH) technology. This technology refers to produce either a "knob" or a "hole" in the CH3 region of each heavy chain of the antibody to promote heterodimerization. In this method, the two heavy chains are engineered such that knob half-antibody preferentially partners with the hole half antibody, and application of disulfide reduction/reoxidation covalently binds the two to generate a multispecific antibody with different arms. For example, in one heavy chain residue Thr366 (Kabat numbering) may be substituted with Trp (to introduce a "knob") and, optionally, Ser354 (Kabat numbering) may be substituted with Cys (to introduce an additional stabilizing disulfide bond). In the other heavy chain of the same antibody, for example, the CH3 domain may be modified by substitution of residues Thr366, Leu368, and Tyr407 (Kabat numbering) with Leu, Ala, and Vai, respectively (to introduce a "hole" of the KiH technology); optionally, Tyr349 (Kabat numbering) may be additionally substituted with Cys (to introduce an additional stabilizing disulfide bond). More preferably, the antibody is a combined CrossMAb/KIH antibody, wherein the CrossMAb technology and the KIH technology are combined. Thereby, the correct association of heavy and light chain can be ensured.

In some embodiments, the multispecific antibody, or the antigen binding fragment thereof, neutralizes SARS-CoV-2. While administration of monospecific neutralizing anti-SARS-CoV- 2 antibodies may lead to the generation of a SARS-CoV-2 escape mutant (which the administered monospecific antibody cannot potently neutralize anymore), the multispecific antibody, or the antigen binding fragment thereof, of the present invention may inhibit or reduce (the occurrence of) the generation of SARS-CoV-2 escape mutants. Preferably, the multispecific antibody, or the antigen binding fragment thereof, neutralizes SARS-CoV-2 (wild-type), SARS-CoV-2 naturally occurring mutants and SARS-CoV-2 escape mutants, which may be generated in response to monospecific antibodies, such as the parental antibodies of the multispecific antibodies of the invention. For example, the multispecific antibody, or the antigen binding fragment thereof, may neutralize one or more SARS-CoV-2 (spike protein) escape mutants selected from the group consisting of N331 A, E340K, T345A, R346S, V367F, R408I, K417A, W436R, K444E, E484G, E484A/F486A, F490A, Q493R, T500A and Y505A. Preferably, the multispecific antibody, or the antigen binding fragment thereof, may neutralize one or more SARS-CoV-2 (spike protein) escape mutants selected from the group consisting of R346S, Q493R and E484G. More preferably, the multispecific antibody, or the antigen binding fragment thereof, may neutralize one or more SARS-CoV-2 (spike protein) escape mutant Q493R more potently than each of its parental monospecific antibodies. In some embodiments, the multispecific antibody, or the antigen binding fragment thereof, may neutralize SARS-CoV-2 D614G. In some embodiments, the multispecific antibody, or the antigen binding fragment thereof, may neutralize a pangolin coronavirus (which may be considered as a threat for cross-over events from animals to humans).

To study and quantitate neutralization in the laboratory, the person skilled in the art knows various standard "neutralization assays". For a neutralization assay, the viruses (to be neutralized) are typically propagated in cells and/or cell lines. For example, in a neutralization assay cultured cells may be incubated with a fixed amount of SARS-CoV-2 in the presence (or absence) of the antibody to be tested. As a readout, for example flow cytometry may be used. Alternatively, also other readouts are conceivable.

Preferably, the multispecific antibody, or the antigen binding fragment thereof, of the invention reduces or inhibits binding of SARS-CoV-2 spike protein to human angiotensinconverting enzyme 2 (hACE2). Binding of SARS-CoV-2 spike protein to ACE2 is essential for virus infectivity. Accordingly, reduced or inhibited binding of SARS-CoV-2 spike protein to ACE2 reduces or inhibits virus infectivity. In some embodiments, the multispecific antibody, or the antigen binding fragment thereof, of the invention fully inhibits binding of SARS-CoV- 2 spike protein to human angiotensin-converting enzyme 2 (hACE2). In particular, binding of the multispecific antibody, or the antigen binding fragment thereof, of the invention to SARS- CoV-2 spike protein can preferably make all ACE2 binding sites of SARS-CoV-2 spike protein unavailable for ACE2 binding; e.g., all (three) ACE2 binding sites of a SARS-CoV-2 spike protein trimer may be made unavailable (and, thereby, inhibited) by the multispecific antibody. This may be achieved, for example, by direct occupation of an ACE2 binding site in SARS-CoV-2, and/or by locking the RBD of SARS-CoV-2 Spike protein in a conformation incapable to recognize ACE2.

Standard methods to assess binding of the antibody according to the present invention, or the antigen-binding fragment thereof, are known to those skilled in the art and include, for example, SPR (surface plasmon resonance; e.g. as described in Hearty S, Leonard P, O'Kennedy R. Measuring antibody-antigen binding kinetics using surface plasmon resonance. Methods Mol Biol. 2012;907:41 1 -42. doi: 10.1007/978-1 -61779-974-7_24) or ELISA (enzyme-linked immunosorbent assay). Thereby, the relative affinities of antibody binding may be determined by measuring the concentration of the antibody (EC ₅₀ ) required to achieve 50% maximal binding at saturation.

An exemplary standard ELISA may be performed as follows: ELISA plates may be coated with a sufficient amount (e.g., 1 pg/ml) of the protein/complex/particle to which binding of the antibody is to be tested (e.g., SARS-CoV-2 spike protein). Plates may then be incubated with the antibody to be tested. After washing, antibody binding can be revealed. To this end, e.g., a labelled antibody recognizing the test antibody may be used, such as goat anti-human IgG coupled to alkaline phosphatase. Plates may then be washed, the required substrate (e.g., p- NPP) may be added and plates may be read, e.g. at 405 nm. The relative affinities of antibody binding may be determined by measuring the concentration of mAb (EC ₅₀ ) required to achieve 50% maximal binding at saturation. The ECso values may be calculated by interpolation of binding curves fitted with a four-parameter nonlinear regression with a variable slope.

To test the antibody's ability to inhibit viral binding to a target, e.g. of SARS-CoV-2 spike protein to ACE2, the ELISA may be performed as described above, while after addition (and incubation and washing) of the test antibody, the viral target (e.g., hACE-mFc) may be added (e.g., at saturating concentration), usually followed by another incubation and washing step. To reveal inhibition of binding, a labelled antibody recognizing the target may be used, such as goat anti-mouse IgG (if the target comprises murine Fc, such as hACE-mFc) coupled to alkaline phosphatase.

The receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 comprises the angiotensin-converting enzyme 2 (ACE2) binding site. The ACE2 binding site is illustrated in Figure 13. In particular, the following amino acid residues of the RBD of the SARS-CoV-2 S protein are involved in ACE2 binding (defined as being within 6A of ACE2): Arg403, Lys417, Val445, Gly446, Tyr449, Tyr453, Leu455, Phe456, Tyr473, Ala475, Gly476, Ser477, Thr478, Gly485, Phe486, Asn487, Tyr489, Phe490, Gln493, Ser494, Tyr495, Gly496, Phe497, Gln498, Pro499, Thr500, Asn501 , Gly502, Val5O3, Gly504, and Tyr505. In some embodiments, the multispecific antibody targets the ACE2 binding site in the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, e.g. one or more (as many as possible, preferably all) of the above-mentioned amino acid residues of the RBD of the SARS-CoV-2 S protein involved in ACE2 binding. In some embodiments, the multispecific antibody targets (i) the ACE2 binding site in the receptor-binding domain (RBD) of the spike protein of SARS- CoV-2, and (ii) a distinct (neutralizing) epitope in the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2.

In some embodiments, the antibody, or the antigen binding fragment thereof, according to the present invention binds specifically to the SARS-CoV-2 RBD epitope of antibody C121 . In some embodiments, the antibody, or the antigen binding fragment thereof, according to the present invention binds specifically to the SARS-CoV-2 RBD epitope of antibody C135. In some embodiments, the antibody, or the antigen binding fragment thereof, according to the present invention binds specifically to the SARS-CoV-2 RBD epitope of antibody C144. Antibodies C121 , C135 and C144 are described, for example, in Robbiani, D.F., Gaebler, C, Muecksch, F. et al. Convergent antibody responses to SARS-CoV-2 in convalescent individuals. Nature 584, 437-442 (2020). Furthermore, the RBD epitopes, to which antibodies C121 , C135 and C144 bind to are illustrated in Figure 1 showing the amino acid residues of RBD within 6A from C121 , C135 and C144, respectively.

Preferably, the antibody, or the antigen binding fragment thereof, according to the present invention binds (with one of its specificities/paratopes) specifically to the SARS-CoV-2 RBD epitope of antibody C121 and (with another of its specificities/paratopes) specifically to the SARS-CoV-2 RBD epitope of antibody C135. For example, the antibody, or the antigen binding fragment thereof, according to the present invention may also bind (with one of its specificities/paratopes) specifically to the SARS-CoV-2 RBD epitope of antibody C144 and (with another of its specificities/paratopes) specifically to the SARS-CoV-2 RBD epitope of antibody C135.

Accordingly, the antibody, or the antigen binding fragment thereof, according to the present invention, may be derived from a combination of two (or all three) of the parental antibodies C121 , C135 and C144; preferably a combination of (i) C121 and C135, or (ii) C144 and C135.

In general, a single epitope binding site (also referred to as "paratope" or "antigen receptor") of the antibody according to the present invention, or the antigen binding fragment thereof, preferably comprises three complementarity determining regions (CDRs) on a heavy chain and three CDRs on a light chain. In general, complementarity determining regions (CDRs) are the hypervariable regions present in heavy chain variable domains and light chain variable domains. Typically, the CDRs of a heavy chain and the connected light chain of an antibody together form the antigen receptor (epitope binding site/paratope). Usually, the three CDRs (CDR1 , CDR2, and CDR3) are arranged non-consecutively in the variable domain. Since epitope binding sites (paratopes) are typically composed of two variable domains (for example, on two different polypeptide chains, i.e. heavy and light chain), there are six CDRs for each epitope binding site (heavy chain: CDRH1 , CDRH2, and CDRH3; light chain: CDRL1 , CDRL2, and CDRL3). For example, a classical IgG antibody molecule usually has two antigen receptors and therefore contains twelve CDRs. The CDRs on the heavy and/or light chain may be separated by framework regions, whereby a framework region (FR) is a region in the variable domain which is less "variable" than the CDR. For example, a variable region (or each variable region, respectively) may be composed of four framework regions, separated by three CDR's.

Preferably, the antibody, or antigen binding fragment thereof, according to the present invention comprises at least one CDR of the following exemplified antibodies. More preferably, the antibody, or antigen binding fragment thereof, according to the present invention comprises all six CDRs (of an epitope binding site) of the following exemplified antibodies. 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 exemplified antibodies. Still more preferably, the antibody, or antigen binding fragment thereof, according to the present invention comprises all six CDRs (of an epitope binding site) of two of the following exemplified antibodies. Particularly 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 two of the following exemplified antibodies.

Table 1 shows the SEQ ID NO's of the amino acid sequences of the heavy chain CDR's (CDRH1 , CDRH2, and CDRH3), the light chain CDR's (CDRL1 , CDRL2, and CDRL3), the heavy chain variable region (referred to as "VH") and the light chain variable region (referred to as "VL") of parental antibodies C121 , C135 and C144 of exemplified multispecific antibodies of the invention: In some embodiments, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and light chain CDR1 , CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

In some embodiments, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences having at least 75% sequence identity with the amino acid sequences of SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and light chain CDR1 , CDR2, and CDR3 sequences having at least 75% sequence identity with the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

Preferably, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and light chain CDR1 , CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

More preferably, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences having at least 85% sequence identity with the amino acid sequences of SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and light chain CDR1 , CDR2, and CDR3 sequences having at least 85% sequence identity with the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

Even more preferably, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences having at least 90% sequence identity with the amino acid sequences of SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and light chain CDR1 , CDR2, and CDR3 sequences having at least 90% sequence identity with the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

Still more preferably, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences having at least 95% sequence identity with the amino acid sequences of SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and light chain CDR1 , CDR2, and CDR3 sequences having at least 95% sequence identity with the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

Particularly preferably, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and light chain CDR1 , CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

In some embodiments, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 1 1 , SEQ ID NO: 12, and SEQ ID NO: 13, respectively, and light chain CDR1 , CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

In some embodiments, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences having at least 75% sequence identity with the amino acid sequences of SEQ ID NO: 11 , SEQ ID NO: 12, and SEQ ID NO: 13, respectively, and light chain CDR1 , CDR2, and CDR3 sequences having at least 75% sequence identity with the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

Preferably, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 1 1 , SEQ ID NO: 12, and SEQ ID NO: 13, respectively, and light chain CDR1 , CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

More preferably, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences having at least 85% sequence identity with the amino acid sequences of SEQ ID NO: 1 1 , SEQ ID NO: 12, and SEQ ID NO: 13, respectively, and light chain CDR1 , CDR2, and CDR3 sequences having at least 85% sequence identity with the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

Even more preferably, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences having at least 90% sequence identity with the amino acid sequences of SEQ ID NO: 1 1 , SEQ ID NO: 12, and SEQ ID NO: 13, respectively, and light chain CDR1 , CDR2, and CDR3 sequences having at least 90% sequence identity with the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

Still more preferably, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences having at least 95% sequence identity with the amino acid sequences of SEQ ID NO: 11 , SEQ ID NO: 12, and SEQ ID NO: 13, respectively, and light chain CDR1 , CDR2, and CDR3 sequences having at least 95% sequence identity with the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

Particularly preferably, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences as set forth in SEQ ID NO: 11 , SEQ ID NO: 12, and SEQ ID NO: 13, respectively, and light chain CDR1 , CDR2, and CDR3 sequences as set forth in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

In some embodiments, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 21 , SEQ ID NO: 22, and SEQ ID NO: 23, respectively, and light chain CDR1 , CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively.

In some embodiments, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences having at least 75% sequence identity with the amino acid sequences of SEQ ID NO: 21 , SEQ ID NO: 22, and SEQ ID NO: 23, respectively, and light chain CDR1 , CDR2, and CDR3 sequences having at least 75% sequence identity with the amino acid sequences of SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively.

Preferably, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 21 , SEQ ID NO: 22, and SEQ ID NO: 23, respectively, and light chain CDR1 , CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively.

More preferably, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences having at least 85% sequence identity with the amino acid sequences of SEQ ID NO: 21 , SEQ ID NO: 22, and SEQ ID NO: 23, respectively, and light chain CDR1 , CDR2, and CDR3 sequences having at least 85% sequence identity with the amino acid sequences of SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively.

Even more preferably, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences having at least 90% sequence identity with the amino acid sequences of SEQ ID NO: 21 , SEQ ID NO: 22, and SEQ ID NO: 23, respectively, and light chain CDR1 , CDR2, and CDR3 sequences having at least 90% sequence identity with the amino acid sequences of SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively. Still more preferably, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences having at least 95% sequence identity with the amino acid sequences of SEQ ID NO: 21 , SEQ ID NO: 22, and SEQ ID NO: 23, respectively, and light chain CDR1 , CDR2, and CDR3 sequences having at least 95% sequence identity with the amino acid sequences of SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively.

Particularly preferably, the antibody, or an antigen-binding fragment thereof, comprises (i) heavy chain CDR1 , CDR2, and CDR3 sequences as set forth in SEQ ID NO; 21 , SEQ ID NO; 22, and SEQ ID NO: 23, respectively, and light chain CDR1 , CDR2, and CDR3 sequences as set forth in SEQ ID NO; 24, SEQ ID NO: 25, and SEQ ID NO; 26, respectively.

In some embodiments, the antibody, or the antigen binding fragment thereof, comprises (a) a first epitope binding site comprising CDRH1 , CDRH2, and CDRH3 amino acid sequences and CDRL1 , CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs: 1 1 - 16, respectively; or 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 CDRH1 , CDRH2, and CDRH3 amino acid sequences and CDRL1 , CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs: 21 - 26, respectively; or 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 the antigen binding fragment thereof, comprises (a) a first epitope binding site comprising CDRH1 , CDRH2, and CDRH3 amino acid sequences and CDRL1 , CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs: 1 - 6, respectively; or 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 CDRH1 , CDRH2, and CDRH3 amino acid sequences and CDRL1 , CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs: 11 - 16, respectively; or 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 the antigen binding fragment thereof, comprises

(a) a first epitope binding site comprising CDRH1 , CDRH2, and CDRH3 amino acid sequences and CDRL1 , CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs: 1 1 - 16, respectively; and

(b) a second epitope binding site comprising CDRH1 , CDRH2, and CDRH3 amino acid sequences and CDRL1 , CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs: 21 - 26, respectively.

Particularly preferably, the antibody, or the antigen binding fragment thereof, comprises (a) a first epitope binding site comprising CDRH1 , CDRH2, and CDRH3 amino acid sequences and CDRL1 , CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs: 1 - 6, respectively; and

(b) a second epitope binding site comprising CDRH1 , CDRH2, and CDRH3 amino acid sequences and CDRL1 , CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs: 11 - 16, respectively.

In some embodiments, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 7; and a light chain variable region (VL) amino acid sequence having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 8.

In some embodiments, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 75% sequence identity with the amino acid sequences of SEQ ID NO: 7; and a light chain variable region (VL) amino acid sequence having at least 75% sequence identity with the amino acid sequences of SEQ ID NO: 8.

Preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 7; and a light chain variable region (VL) amino acid sequence having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 8.

Preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 85% sequence identity with the amino acid sequences of SEQ ID NO: 7; and a light chain variable region (VL) amino acid sequence having at least 85% sequence identity with the amino acid sequences of SEQ ID NO: 8.

More preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 90% sequence identity with the amino acid sequences of SEQ ID NO: 7; and a light chain variable region (VL) amino acid sequence having at least 90% sequence identity with the amino acid sequences of SEQ ID NO: 8.

More preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 95% sequence identity with the amino acid sequences of SEQ ID NO: 7; and a light chain variable region (VL) amino acid sequence having at least 95% sequence identity with the amino acid sequences of SEQ ID NO: 8.

Even more preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 96% sequence identity with the amino acid sequences of SEQ ID NO: 7; and a light chain variable region (VL) amino acid sequence having at least 96% sequence identity with the amino acid sequences of SEQ ID NO: 8. Even more preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 97% sequence identity with the amino acid sequences of SEQ ID NO: 7; and a light chain variable region (VL) amino acid sequence having at least 97% sequence identity with the amino acid sequences of SEQ ID NO: 8.

Still more preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 98% sequence identity with the amino acid sequences of SEQ ID NO: 7; and a light chain variable region (VL) amino acid sequence having at least 98% sequence identity with the amino acid sequences of SEQ ID NO: 8.

Still more preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 99% sequence identity with the amino acid sequences of SEQ ID NO: 7; and a light chain variable region (VL) amino acid sequence having at least 99% sequence identity with the amino acid sequences of SEQ ID NO: 8.

Particularly preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 7; and a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 8.

In some embodiments, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 17; and a light chain variable region (VL) amino acid sequence having at least 70% sequence identity with the amino acid sequences of SEQ ID NO:1 8.

In some embodiments, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 75% sequence identity with the amino acid sequences of SEQ ID NO: 17; and a light chain variable region (VL) amino acid sequence having at least 75% sequence identity with the amino acid sequences of SEQ ID NO: 18.

Preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 17; and a light chain variable region (VL) amino acid sequence having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 18.

Preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 85% sequence identity with the amino acid sequences of SEQ ID NO: 17; and a light chain variable region (VL) amino acid sequence having at least 85% sequence identity with the amino acid sequences of SEQ ID NO: 18.

More preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 90% sequence identity with the amino acid sequences of SEQ ID NO: 17; and a light chain variable region (VL) amino acid sequence having at least 90% sequence identity with the amino acid sequences of SEQ ID NO: 18.

More preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 95% sequence identity with the amino acid sequences of SEQ ID NO: 17; and a light chain variable region (VL) amino acid sequence having at least 95% sequence identity with the amino acid sequences of SEQ ID NO: 18.

Even more preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 96% sequence identity with the amino acid sequences of SEQ ID NO: 17; and a light chain variable region (VL) amino acid sequence having at least 96% sequence identity with the amino acid sequences of SEQ ID NO: 18. Even more preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 97% sequence identity with the amino acid sequences of SEQ ID NO: 17; and a light chain variable region (VL) amino acid sequence having at least 97% sequence identity with the amino acid sequences of SEQ ID NO: 18.

Still more preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 98% sequence identity with the amino acid sequences of SEQ ID NO: 17; and a light chain variable region (VL) amino acid sequence having at least 98% sequence identity with the amino acid sequences of SEQ ID NO: 18.

Still more preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 99% sequence identity with the amino acid sequences of SEQ ID NO: 17; and a light chain variable region (VL) amino acid sequence having at least 99% sequence identity with the amino acid sequences of SEQ ID NO: 18.

Particularly preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 17; and a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 18.

In some embodiments, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 27; and a light chain variable region (VL) amino acid sequence having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 28.

In some embodiments, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 75% sequence identity with the amino acid sequences of SEQ ID NO: 27; and a light chain variable region (VL) amino acid sequence having at least 75% sequence identity with the amino acid sequences of SEQ ID NO: 28.

Preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: T7,- and a light chain variable region (VL) amino acid sequence having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 28.

Preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 85% sequence identity with the amino acid sequences of SEQ ID NO: 27; and a light chain variable region (VL) amino acid sequence having at least 85% sequence identity with the amino acid sequences of SEQ ID NO: 28.

More preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 90% sequence identity with the amino acid sequences of SEQ ID NO: 27; and a light chain variable region (VL) amino acid sequence having at least 90% sequence identity with the amino acid sequences of SEQ ID NO: 28.

More preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 95% sequence identity with the amino acid sequences of SEQ ID NO: 27; and a light chain variable region (VL) amino acid sequence having at least 95% sequence identity with the amino acid sequences of SEQ ID NO: 28.

Even more preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 96% sequence identity with the amino acid sequences of SEQ ID NO: 27; and a light chain variable region (VL) amino acid sequence having at least 96% sequence identity with the amino acid sequences of SEQ ID NO: 28. Even more preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 97% sequence identity with the amino acid sequences of SEQ ID NO: 27; and a light chain variable region (VL) amino acid sequence having at least 97% sequence identity with the amino acid sequences of SEQ ID NO: 28.

Still more preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 98% sequence identity with the amino acid sequences of SEQ ID NO: 27; and a light chain variable region (VL) amino acid sequence having at least 98% sequence identity with the amino acid sequences of SEQ ID NO: 28.

Still more preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence having at least 99% sequence identity with the amino acid sequences of SEQ ID NO: 27; and a light chain variable region (VL) amino acid sequence having at least 99% sequence identity with the amino acid sequences of SEQ ID NO: 28.

Particularly preferably, the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 27; and a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 28.

In some embodiments, the antibody, or the antigen binding fragment thereof, comprises

(a) a first epitope binding site comprising a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 17 or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 18 or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and (b) a second epitope binding site comprising a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 27 or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 28 or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

Preferably, the antibody, or the antigen binding fragment thereof, comprises

(a) a first epitope binding site comprising a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 7 or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 8 or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and

(b) a second epitope binding site comprising a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 1 7 or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 1 8 or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

More preferably, the antibody, or the antigen binding fragment thereof, comprises

(a) a first epitope binding site comprising a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 1 7 and a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 18; and (b) a second epitope binding site comprising a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 27 and a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 28.

Particularly preferably, the antibody, or the antigen binding fragment thereof, comprises

(a) a first epitope binding site comprising a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 7 and a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 8; and

(b) a second epitope binding site comprising a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 17 and a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 18.

In some embodiments, the antibody, or the antigen binding fragment thereof, comprises a polypeptide chain having the amino acid sequence according to SEQ ID NO: 40; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. Preferably, the antibody, or the antigen binding fragment thereof, comprises a polypeptide chain having the amino acid sequence according to SEQ ID NO: 40.

In some embodiments, the antibody, or the antigen binding fragment thereof, comprises a polypeptide chain having the amino acid sequence according to SEQ ID NO: 41 ; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. Preferably, the antibody, or the antigen binding fragment thereof, comprises a polypeptide chain having the amino acid sequence according to SEQ ID NO: 41.

In some embodiments, the antibody, or the antigen binding fragment thereof, comprises

(a) a first heavy chain having the amino acid sequence according to SEQ ID NO: 29; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and a first light chain having the amino acid sequence according to SEQ ID NO: 30; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and

(b) a second heavy chain having the amino acid sequence according to SEQ ID NO: 19; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and a second light chain having the amino acid sequence according to SEQ ID NO: 20; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

Preferably, the antibody, or the antigen binding fragment thereof, comprises

(a) a first heavy chain having the amino acid sequence according to SEQ ID NO: 9; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and a first light chain having the amino acid sequence according to SEQ ID NO: 10; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and

(b) a second heavy chain having the amino acid sequence according to SEQ ID NO: 1 9; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and a second light chain having the amino acid sequence according to SEQ ID NO: 20; or a sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. More preferably, the antibody, or the antigen binding fragment thereof, comprises

(a) a first heavy chain having the amino acid sequence according to SEQ ID NO: 29; and a first light chain having the amino acid sequence according to SEQ ID NO: 30; and

(b) a second heavy chain having the amino acid sequence according to SEQ ID NO: 19; and a second light chain having the amino acid sequence according to SEQ ID NO:

20.

Particularly preferably, the antibody, or the antigen binding fragment thereof, comprises

(a) a first heavy chain having the amino acid sequence according to SEQ ID NO: 9; and a first light chain having the amino acid sequence according to SEQ ID NO: 10; and

(b) a second heavy chain having the amino acid sequence according to SEQ ID NO: 19; and a second light chain having the amino acid sequence according to SEQ ID NO: 20.

Preferably, each CDR or each variable region comprised in the antibody, or the antigen binding fragment thereof, may be a human CDR or human variable region, respectively. More preferably, the antibody of the invention is a human antibody, i.e. an antibody comprising human variable and constant regions (which includes antibodies with human variable and constant regions, wherein one or more amino acids are mutated, e.g., due to Fc modifications, the construction of the multispecific antibody (e.g., CrossMAb and/or knob-in-hole). Accordingly, a "human" multispecific antibody is usually derived from (at least) two human parental antibodies (which may be antibodies isolated from humans). In particular, a "human" antibody does usually not comprise sequences from non-human antibodies or humanized sequences. In some embodiments, the antibody of the invention is a monoclonal antibody. For example, the antibody of the invention may be a human monoclonal antibody.

Antibodies of the invention can be of any isotype (e.g., IgA, IgG, IgM i.e. an a, y or μ heavy chain). Preferably, the antibody may be of the IgG type. Within the IgG isotype, antibodies may be IgGI , lgG2, lgG3 or lgG4 subclass, for example IgGI . Antibodies of the invention may have a K or a A light chain. In some embodiments, the antibody is of IgGI type and has a lambda or kappa light chain. In some embodiments, the antibody according to the present invention, or an antigen binding fragment thereof, comprises an Fc moiety. The Fc moiety may be derived from human origin, e.g. from human IgG 1 , lgG2, lgG3, and/or lgG4, such as human IgGI .

As used herein, the term "Fc moiety" refers to a sequence derived from the portion of an immunoglobulin heavy chain beginning in the hinge region just upstream of the papain cleavage site (e.g., residue 216 in native IgG, taking the first residue of heavy chain constant region to be 1 14) and ending at the C-terminus of the immunoglobulin heavy chain. Accordingly, an Fc moiety may be a complete Fc moiety or a portion (e.g., a domain) thereof. A complete Fc moiety comprises at least a hinge domain, a CH2 domain, and a CH3 domain (e.g., EU amino acid positions 216-446). An additional lysine residue (K) is sometimes present at the extreme C-terminus of the Fc moiety, but is often cleaved from a mature antibody.

Each of the amino acid positions within an Fc moiety have been numbered herein according to the art-recognized EU numbering system of Kabat, see e.g., by Kabat et al., in "Sequences of Proteins of Immunological Interest", U.S. Dept. Health and Human Services, 1983 and 1987. The EU index or EU index as in Kabat or EU numbering refers to the numbering of the EU antibody (Edelman GM, Cunningham BA, Gall WE, Gottlieb RD, Rutishauser U, Waxdal MJ. The covalent structure of an entire gammaG immunoglobulin molecule. Proc Nat! Acad Sci U S A. 1969;63(1 ):78-85; Kabat E.A., National Institutes of Health (U.S.) Office of the Director, "Sequences of Proteins of Immunological Interest", 5 ^th edition, Bethesda, MD : U.S. Dept, of Health and Human Services, Public Health Service, National Institutes of Health, 1991 , hereby entirely incorporated by reference).

In some embodiments, in the context of the present invention an Fc moiety 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. An Fc moiety may comprise at least a hinge domain, a CH2 domain or a CH3 domain. The Fc moiety may be a complete Fc moiety. The Fc moiety may also comprises one or more amino acid insertions, deletions, or substitutions relative to a naturally-occurring Fc moiety. For example, at least one of a hinge domain, CH2 domain or CH3 domain (or portion thereof) may be deleted. For example, an Fc moiety may comprise or consist of: (i) hinge domain (or portion thereof) fused to a CH2 domain (or portion thereof), (ii) a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof), (iii) a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof), (iv) a hinge domain (or portion thereof), (v) a CH2 domain (or portion thereof), or (vi) a CH3 domain or portion thereof.

In some embodiments, the antibody, or antigen binding fragment thereof, according to the present invention comprises an Fc region. As used herein, the term "Fc region" refers to the portion of an immunoglobulin formed by two or more Fc moieties of antibody heavy chains. For example, the Fc region may be monomeric or "single-chain" Fc region (i.e., a scFc region). Single chain Fc regions are comprised of Fc moieties 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. The Fc region may be dimeric. A "dimeric Fc region" or "dcFc" refers to the dimer formed by the Fc moieties of two separate immunoglobulin heavy chains. The dimeric Fc region may be a homodimer of two identical Fc moieties (e.g., an Fc region of a naturally occurring immunoglobulin) or a heterodimer of two non-identical Fc moieties.

In some embodiments, the Fc moiety, or the Fc region, comprises or consists of an amino acid sequence derived from a human immunoglobulin sequence (e.g., from an Fc region or Fc moiety from a human IgG molecule).

In some embodiments, the antibody according to the present invention comprises, in particular in addition to an Fc moiety as described above, other parts derived from a constant region, in particular from a constant region of IgG, such as a constant region of (human) IgG 1 . The antibody according to the present invention may comprise, in particular in addition to an Fc moiety as described above, all other parts of the constant regions, in particular all other parts of the constant regions of IgG (such as (human) IgGI ).

It will be understood by one of ordinary skill in the art that the Fc moiety may be modified such that it varies in amino acid sequence from the complete Fc moiety of a naturally occurring immunoglobulin molecule, while retaining at least one desirable function conferred by the naturally-occurring Fc moiety. Such functions include Fc receptor (FcR) binding, antibody half-life modulation, ADCC function, protein A binding, protein G binding, and complement binding. The portions of naturally occurring Fc moieties, which are responsible and/or essential for such functions are well known by those skilled in the art.

In some embodiments, the antibody may comprise GRLR substitutions (G236R/L328R) known to diminish binding to all FcyR classes.

Furthermore, the antibody according to the present invention can be modified by introducing (random) amino acid mutations into particular region of the CH2 or CH3 domain of the heavy chain in order to alter their binding affinity for FcR and/or their serum half-life in comparison to unmodified antibodies. Examples of such modifications include, but are not limited to, substitutions of at least one amino acid from the heavy chain constant region selected from the group consisting of amino acid residues 250, 314, and 428. Further examples of such Fc modifications are described in Saxena A, Wu D. Advances in Therapeutic Fc Engineering - Modulation of IgG-Associated Effector Functions and Serum Half-life. Front Immunol. 2016;7:580, which is incorporated herein by reference. In some embodiments, the antibody may comprise the "YTE" mutations (M252Y/S254T/T256E; EU numbering). In some embodiments, the antibody may comprise the mutations M428L and/or N434S in the heavy chain constant region (EU numbering).

Without being bound to any theory, it is believed that antibody-dependent enhancement (ADE) is brought about by the binding of the Fc moiety of the antibody, in particular, the Fc moiety of the heavy chain of an IgG molecule, to an Fc receptor, e.g., an Fey receptor on a host cell. It is thus preferred that the antibody according to the present invention, or an antigen binding fragment thereof, comprises one or more mutations in the Fc moiety, which inhibit or reduce ADE. The mutation(s) may be any mutation that reduces binding of the antibody to an Fc receptor (FcR), in particular reduces binding of the antibody to an Fey receptor (FcyR). On the other hand, it is preferred that the antibody according to the present invention comprises a (complete) Fc moiety/Fc region, wherein the interaction/binding with FcRn is not compromised. Accordingly, it is particularly preferred that the antibody according to the present invention, or an antigen binding fragment thereof, comprises one or more mutations in the Fc moiety, which (i) reduce(s) binding of the antibody to an Fey receptor, but do(es) not compromise interaction with FcRn. One example of such a mutation is the "LALA" mutation (Leu234Ala and Leu235Ala; Kabat numbering). Accordingly, it is preferred that the Fc moiety of an antibody of the invention comprises a substitution at positions CH2 4 (Leu234), CH2 5 (Leu235), or both. In general, the amino acid at positions 4 and 5 of CH2 of the wild-type IgG 1 and lgG3 is a leucine ("L"). Preferably, the antibody according to the present invention comprises an amino acid at position CH2 4, CH2 5, or both, that is not an L. More preferably, antibody according to the present invention comprises an alanine ("A") at position CH2 4, or CH2 5, or both. Most preferably, the antibody according to the present invention comprises both, a CH2 L4A and a CH2 L5A substitution (Leu234Ala and Leu235Ala; Kabat numbering). Such antibodies are referred to herein as a "LALA" variant. In addition, the Fc moiety may comprise a PG mutation (Pro329Gly; Kabat numbering). In some embodiments, the antibody comprises both, the LALA and the PG mutation in its Fc region ("LALA-PG"). The L234A, L235A, P329G (LALA-PG) variant typically eliminates complement binding and fixation as well as Fcy-dependent ADCC.

As outlined above, an antibody according to the present invention may comprise a (complete) Fc region derived from human IgGI . In some embodiments, the antibody according to the present invention comprises, in particular in addition to a (complete) Fc region derived from human IgGI also all other parts of the constant regions of IgG, such as all other parts of the constant regions of (human) IgG 1 .

In some embodiments, the Fc moiety (in particular constant domains CH2 and CH3 of the heavy chains) may comprise one or more of the following amino acid substitutions to modulate the antibody effector function and/or to improve the antibody circulation half-life: LALA (Leu234Ala and Leu235Ala; Kabat numbering), PG (Pro329Gly; Kabat numbering), GRLR (Gly236Arg and Leu328Arg; Kabat numbering), LS (Met428Leu and Asn434Ser; Kabat numbering) or combinations thereof.

Example sequences of constant regions are the amino acid sequences according to SEQ ID NOs: 31 - 35. For example, the amino acid sequence of IgGI CH1 -CH2-CH3 is according to SEQ ID NO: 32 or a sequence variant thereof (including, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity.

Variant antibodies are also included within the scope of the invention. Thus, variants of the sequences recited in the application are also included within the scope of the invention. Such variants include natural variants generated by somatic mutation in vivo during the immune response or in vitro upon culture of immortalized B cell clones. Alternatively, variants may arise due to the degeneracy of the genetic code or may be produced due to errors in transcription or translation.

Antibodies of the invention may be 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% (by weight), usually less than 60% and more usually less than 50% of the composition is made up of other polypeptides.

Antibodies of the invention may be immunogenic in non-human (or heterologous) hosts e.g., in mice. In particular, the antibodies may have an idiotope that is immunogenic in non-human hosts, but not in a human host. In particular, antibodies of the invention for human use include those that cannot be easily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, etc. and cannot generally be obtained by humanization or from xeno-mice.

Nucleic Acids

In another aspect, the invention also provides a nucleic acid molecule comprising a polynucleotide encoding the antibody according to the present invention, or an antigenbinding fragment thereof, as described above.

The antibody according to the present invention, or an antigen-binding fragment thereof, may be a single-chain antibody, or it may comprise multiple (e.g., (exactly) two) chains, such as a heavy chain and a light chain. While a single-chain antibody is usually encoded by a single nucleic acid molecule, an antibody comprising more than one (e.g., two) chains may encoded by a single nucleic acid molecule (e.g., in a bi- or multicistronic manner) or by a plurality of (separate) nucleic acid molecules, wherein each nucleic acid molecule comprises at least one polynucleotide encoding a fragment or a chain of said antibody, or of the antigen binding fragment thereof. Accordingly, the present invention also provides a plurality of nucleic acid molecules encoding the antibody, or the antigen binding fragment thereof, according to the present invention, wherein each nucleic acid molecule comprises at least one polynucleotide encoding a fragment or a chain of said antibody, or of the antigen binding fragment thereof.

In general, the nucleic acid molecule may be mono-, bi-, or multicistronic, such as tricistronic. A bicistronic or multicistronic nucleic acid molecule is typically a nucleic acid molecule that typically may have two (bicistronic) or more (multicistronic) open reading frames (ORFs). An open reading frame in this context is a sequence of codons that is translatable into a peptide or protein. Preferably, the inventive nucleic acid molecule contains at least two coding polynucleotides, for example (exactly) two coding polynucleotides, which encode distinct chains of the antibody, or antigen-binding fragment.

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

It is also preferred that the antibody, or the antigen binding fragment thereof, according to the present invention comprises (exactly) two distinct polypeptide chains, such as, for example, a heavy chain and a light chain. This may be illustrated by a classical native IgG molecule, which comprises two identical heavy chains and two identical light chains and, thus, two distinct polypeptide chains (even though the antibody finally comprises four polypeptide chains, they may be encoded by two (distinct) polynucleotides). Preferably, such two distinct polypeptide chains (e.g., a heavy chain and a light chain) of the antibody, or the antigen binding fragment thereof, according to the present invention may be encoded by two distinct polynucleotides, which may be located on the same nucleic acid molecule, e.g. in a bicistronic nucleic acid molecule, or on (exactly two) distinct nucleic acid molecules (e.g., each nucleic acid molecule may be monocistronic). Accordingly, the nucleic acid molecule according to the present invention may be bicistronic, in particular it may comprise (exactly) two polynucleotides encoding (together) the antibody, or the antigen binding fragment thereof, according to the present invention. Moreover, the antibody, or the antigen binding fragment thereof, according to the present invention may be encoded by a plurality of (e.g., monocistronic) nucleic acid molecules. For example, an antibody comprising two distinct polypeptide chains may be encoded by two polynucleotides located on two distinct nucleic acid molecules.

Examples of nucleic acid molecules and/or polynucleotides include, e.g., a recombinant polynucleotide, a vector, an oligonucleotide, an RNA molecule such as an rRNA, an mRNA, an miRNA, an siRNA, or a tRNA, or a DNA molecule such as a cDNA. Nucleic acids may encode the light chain and/or the heavy chain of an antibody. In other words, the light chain and the heavy chain of the antibody may be encoded by the same nucleic acid molecule (e.g., in bicistronic manner). Alternatively, the light chain and the heavy chain of the antibody may be encoded by distinct nucleic acid molecules.

Due to the redundancy of the genetic code, the present invention also comprises sequence variants of nucleic acid sequences, which encode the same amino acid sequences. The polynucleotide encoding the antibody (or the complete nucleic acid molecule) may be optimized for expression of the antibody. For example, codon optimization of the nucleotide sequence may be used to improve the efficiency of translation in expression systems for the production of the antibody. Moreover, the nucleic acid molecule may comprise heterologous elements (i.e., elements, which in nature do not occur on the same nucleic acid molecule as the coding sequence for the (heavy or light chain of) an antibody. For example, a nucleic acid molecule may comprise a heterologous promotor, a heterologous enhancer, a heterologous UTR (e.g., for optimal translation/expression), a heterologous Poly-A-tail, and the like. A nucleic acid molecule is a molecule comprising nucleic acid components. The term nucleic acid molecule usually refers to DNA or RNA molecules. It may be used synonymous with the term "polynucleotide", i.e. the nucleic acid molecule may consist of a polynucleotide encoding the antibody. Alternatively, the nucleic acid molecule may also comprise further elements in addition to the polynucleotide encoding the antibody. Typically, a nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers which are 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 basemodified, sugar-modified or backbone-modified etc. DNA or RNA molecules.

In general, the nucleic acid molecule may be manipulated to insert, delete or alter certain nucleic acid sequences. Changes from such manipulation include, but are not limited to, changes to introduce restriction sites, to amend codon usage, to add or optimize transcription and/or translation regulatory sequences, etc. It is also possible to change the nucleic acid to alter the encoded amino acids. For example, it may be useful to introduce one or more (e.g;, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions, deletions and/or insertions into the antibody's amino acid sequence. Such point mutations can modify effector functions, antigen-binding affinity, post-translational modifications, immunogenicity, etc., can introduce amino acids for the attachment of covalent groups (e.g., labels) or can introduce tags (e.g., for purification purposes). Alternatively, a mutation in a nucleic acid sequence may be "silent", i.e. not reflected in the amino acid sequence due to the redundancy of the genetic code. In general, mutations can be introduced in specific sites or can be introduced at random, followed by selection (e.g., molecular evolution). For instance, one or more nucleic acids encoding any of the light or heavy chains of an (exemplary) antibody can be randomly or directionally mutated to introduce different properties in the encoded amino acids. Such changes can be the result of an iterative process wherein initial changes are retained and new changes at other nucleotide positions are introduced. Further, changes achieved in independent steps may be combined.

In some embodiments, the polynucleotide encoding the antibody, or an antigen-binding fragment thereof, (or the (complete) nucleic acid molecule) may be codon-optimized. The skilled artisan is aware of various tools for codon optimization, such as those described in: Ju Xin Chin, Bevan Kai-Sheng Chung, Dong-Yup Lee, Codon Optimization Online (COOL): a web-based multi-objective optimization platform for synthetic gene design, Bioinformatics, Volume 30, Issue 15, 1 August 2014, Pages 2210-2212; or in: Grote A, Hiller K, Scheer M, Munch R, Nortemann B, Hempel DC, Jahn D, JCat: a novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Res. 2005 Jul 1 ;33(Web Server issue):W526-31 ; or, for example, Genscript's OptimumGene™ algorithm (as described in US 201 1/0081708 A1 ).

Vector

Further included within the scope of the invention are vectors, for example, expression vectors, comprising a nucleic acid molecule according to the present invention. Usually, a vector comprises a nucleic acid molecule as described above.

The present invention also provides a plurality of vectors comprising the plurality of nucleic acid molecules as described above.

A vector is usually a recombinant nucleic acid molecule, i.e. a nucleic acid molecule which does not occur in nature. Accordingly, the vector may comprise heterologous elements (i.e., sequence elements of different origin in nature). For example, the vector may comprise a multi cloning site, a heterologous promotor, a heterologous enhancer, a heterologous selection marker (to identify cells comprising said vector in comparison to cells not comprising said vector) and the like. A vector in the context of the present invention is suitable for incorporating or harboring a desired nucleic acid sequence. Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors etc. A storage vector is a vector which allows the convenient storage of a nucleic acid molecule. Thus, the vector may comprise a sequence corresponding, e.g., to a (heavy and/or light chain of a) desired antibody according to the present invention. An expression vector may be used for production of expression products such as RNA, e.g. mRNA, or peptides, polypeptides or proteins. For example, an expression vector may comprise sequences needed for transcription of a sequence stretch of the vector, such as a (heterologous) promoter sequence. A cloning vector is typically a vector that contains a cloning site, which may be used to incorporate nucleic acid sequences into the vector. A cloning vector may be, e.g., a plasmid vector or a bacteriophage vector. A transfer vector may be a vector which is suitable for transferring nucleic acid molecules into cells or organisms, for example, viral vectors. A vector in the context of the present invention may be, e.g., an RNA vector or a DNA vector. For example, a vector in the sense of the present application comprises a cloning site, a selection marker, such as an antibiotic resistance factor, and a sequence suitable for multiplication of the vector, such as an origin of replication. A vector in the context of the present application may be a plasmid vector.

Cells

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

Examples of such cells include but are not limited to, eukaryotic cells, e.g., yeast cells, animal cells or plant cells. Other examples of such cells include but are not limited, to prokaryotic cells, e.g. £. co/i. In some embodiments, the cells are mammalian cells, such as a mammalian cell line. Examples include human cells, CHO cells, HEK293T cells, PER.C6 cells, NSO cells, human liver cells, myeloma cells or hybridoma cells.

The cell may be transfected with a vector according to the present invention, for example with an expression vector. The term "transfection" refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g. mRNA) molecules, into cells, e.g. into eukaryotic or prokaryotic cells. 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, such as into mammalian cells. Such methods encompass, for example, electroporation, lipofection, e.g. based on cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle based transfection, virus based transfection, or transfection based on cationic polymers, such as DEAE-dextran or polyethylenimine etc. In some embodiments, the introduction is non-viral.

Moreover, the cells of the present invention may be transfected stably or transiently with the vector according to the present invention, e.g. for expressing the antibody according to the present invention. In some embodiments, the cells are stably transfected with the vector according to the present invention encoding the antibody according to the present invention. In other embodiments, the cells are transiently transfected with the vector according to the present invention encoding the antibody according to the present invention.

Accordingly, the present invention also provides a recombinant host cell, which heterologously expresses the antibody of the invention or the antigen-binding fragment thereof. For example, the cell may be of another species than the antibody (e.g., CHO cells expressing human antibodies). In some embodiments, the cell type of the cell does notexpress (such) antibodies in nature. Moreover, the host cell may impart a post-translational modification (PTM; e.g., glycosylation) on the antibody that is not present in their native state. Such a PTM may result in a functional difference (e.g., reduced immunogenicity). Accordingly, the antibody of the invention, or the antigen-binding fragment thereof, may have a post-translational modification, which is distinct from the naturally produced antibody (e.g., an antibody of an immune response in a human).

Pharmaceutical Composition

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

(i) the antibody of the present invention, or an antigen-binding fragment thereof;

(ii) the nucleic acid or the plurality of nucleic acids of the present invention;

(iii) the vector or the plurality of vectors of the present invention; and/or

(iv) the cell expressing the antibody according to the present invention or comprising the vector according to the present invention and, optionally, a pharmaceutically acceptable excipient, diluent or carrier. In other words, the present invention also provides a pharmaceutical composition comprising the antibody according to the present invention, the nucleic acid according to the present invention, the vector according to the present invention and/or the cell according to the present invention.

The pharmaceutical composition may optionally also contain 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. Nor should it be toxic. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. In some embodiments, the pharmaceutically acceptable carrier, diluent and/or excipient in the pharmaceutical composition according to the present invention is not an active component in respect to SARS-CoV-2 infection or COVID-19.

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

Pharmaceutically acceptable carriers in a pharmaceutical composition may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, 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 the subject.

Pharmaceutical compositions of the invention may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g., a lyophilized composition, similar to Synagis™ and Herceptin®, for reconstitution with sterile water containing a preservative). The composition may be prepared for topical administration e.g., as an ointment, cream or powder. The composition may be prepared for oral administration e.g., as a tablet or capsule, as a spray, or as a syrup (optionally flavored). The composition may be prepared for pulmonary administration e.g., as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g., as drops. The composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a subject. For example, a lyophilized antibody may be provided in kit form with sterile water or a sterile buffer.

In some embodiments, the (only) active ingredient in the composition is the antibody according to the present invention. As such, it may be susceptible to degradation in the gastrointestinal tract. Thus, if the composition is to be administered by a route using the gastrointestinal tract, the composition may contain agents which protect the antibody from degradation but which release the antibody once it has been absorbed from the gastrointestinal tract.

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

Pharmaceutical compositions of the invention generally have a pH between 5.5 and 8.5, in some embodiments this may be between 6 and 8, for example about 7. The pH may be maintained by the use of a buffer. The composition may be sterile and/or pyrogen free. The composition may be isotonic with respect to humans. In some embodiments pharmaceutical compositions of the invention are supplied in hermetically-sealed containers.

Within the scope of the invention are compositions present in several forms of administration; the forms include, but are not limited to, those forms suitable for parenteral administration, e.g., by injection or infusion, for example by bolus injection or continuous infusion. Where the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents, such as suspending, preservative, stabilizing and/or dispersing agents. Alternatively, the antibody may be in dry form, for reconstitution before use with an appropriate sterile liquid.

A vehicle is typically understood to be a material that is suitable for storing, transporting, and/or administering a compound, such as a pharmaceutically active compound, in particular the antibodies according to the present invention. For example, the vehicle may be a physiologically acceptable liquid, which is suitable for storing, transporting, and/or administering a pharmaceutically active compound, in particular the antibodies according to the present invention. Once formulated, the compositions of the invention can be administered directly to the subject. In some embodiments the compositions are adapted for administration to mammalian, e.g., human subjects.

The pharmaceutical compositions of this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal or rectal routes. Hyposprays may also be used to administer the pharmaceutical compositions of the invention. Optionally, the pharmaceutical composition may be prepared for oral administration, e.g. as tablets, capsules and the like, for topical administration, or as injectable, e.g. as liquid solutions or suspensions. In some embodiments, the pharmaceutical composition is an injectable. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection are also encompassed, for example the pharmaceutical composition may be in lyophilized form.

For injection, e.g. intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well 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, as required. Whether it is an antibody, a peptide, a nucleic acid molecule, or another pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is usually in an "effective amount", e.g. in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. For injection, the pharmaceutical composition according to the present invention may be provided for example in a pre-filled syringe.

The inventive pharmaceutical composition 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. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient, i.e. the inventive transporter cargo conjugate molecule as defined above, is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

The inventive pharmaceutical composition may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, e.g. including accessible epithelial tissue. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the inventive pharmaceutical composition may be formulated in a suitable ointment, containing the inventive pharmaceutical composition, particularly its components as defined above, suspended or dissolved in one or more carriers. Carriers for topical administration include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the inventive pharmaceutical composition can be formulated in 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, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water. Dosage treatment may be a single dose schedule or a multiple dose schedule. In particular, the pharmaceutical composition may be provided as single-dose product. In some embodiments, the amount of the antibody in the pharmaceutical composition - in particular if provided as single-dose product - does not exceed 200 mg, for example it does not exceed 100 mg or 50 mg.

For a single dose, e.g. a daily, weekly or monthly dose, the amount of the antibody in the pharmaceutical composition according to the present invention, may not exceed 1 g or 500 mg. In some embodiments, for a single dose, the amount of the antibody in the pharmaceutical composition according to the present invention, may not exceed 200 mg, or 100 mg. For example, for a single dose, the amount of the antibody in the pharmaceutical composition according to the present invention, may not exceed 50 mg.

Pharmaceutical compositions typically include an "effective" amount of one or more antibodies of the invention, i.e. an amount that is sufficient to treat, ameliorate, attenuate, reduce or prevent a desired disease or condition, or to exhibit a detectable therapeutic effect. Therapeutic effects also include reduction or attenuation in pathogenic potency or physical symptoms. The precise effective amount for any particular subject will depend upon their size, weight, and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. The effective amount for a given situation is determined by routine experimentation and is within the judgment of a clinician. For purposes of the present invention, an effective dose may generally be from about 0.005 to about 100 mg/kg, for example from about 0.0075 to about 50 mg/kg or from about 0.01 to about 10 mg/kg. In some embodiments, the effective dose will be from about 0.02 to about 5 mg/kg, of the antibody of the present invention (e.g. amount of the antibody in the pharmaceutical composition) in relation to the bodyweight (e.g., in kg) of the individual to which it is administered.

Moreover, the pharmaceutical composition according to the present invention may also comprise an additional active component, which may be a further antibody or a component, which is not an antibody. Accordingly, the pharmaceutical composition according to the present invention may comprise one or more of the additional active components. The antibody according to the present invention can be present either in the same pharmaceutical composition as the additional active component or, alternatively, the antibody according to the present invention is comprised by a first pharmaceutical composition and the additional active component is comprised by a second pharmaceutical composition different from the first pharmaceutical composition. Accordingly, if more than one additional active component is envisaged, each additional active component and the antibody according to the present invention may be comprised in a different pharmaceutical composition. Such different pharmaceutical compositions may be administered either combined/simultaneously or at separate times or at separate locations (e.g. separate parts of the body).

The antibody according to the present invention and the additional active component may provide an additive therapeutic effect, such as a synergistic therapeutic effect. The term "synergy" is used to describe a combined effect of two or more active agents that is 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 "synergistic inhibition" of an activity or process, it is intended that the inhibition of the activity or process is greater than the sum of the inhibitory effects of each respective active agent. The term "synergistic therapeutic effect" refers to a therapeutic effect observed with a combination of two or more therapies wherein the therapeutic effect (as measured by any of a number of parameters) is greater than the sum of the individual therapeutic effects observed with the respective individual therapies.

In other embodiments, the pharmaceutical composition according to the present invention may not comprise an additional active component (in addition to the antibody of the invention or respective nucleic acids, vectors or cells as described above).

In some embodiments, a composition of the invention may include antibodies of the invention, wherein the antibodies may make up at least 50% by weight (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) of the total protein in the composition. In the composition of the invention, the antibodies may be in purified form. The present invention also provides a method of preparing a pharmaceutical composition comprising the steps of: (i) preparing an antibody of the invention; and (ii) admixing the purified antibody with one or more pharmaceutically acceptable excipients, diluents or carriers.

In other embodiments, a method of preparing a pharmaceutical composition comprises the step of: admixing an antibody with one or more pharmaceutically-acceptable carriers, wherein the antibody is a monoclonal antibody that was obtained from a transformed B cell or a cultured plasma cell of the invention.

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

Pharmaceutical compositions may include an antimicrobial, particularly if packaged in a multiple dose format. They may comprise detergent e.g., a Tween (polysorbate), such as Tween 80. Detergents are generally present at low levels e.g., less than 0.01 %. Compositions may also include sodium salts (e.g., sodium chloride) to give tonicity. For example, a concentration of 10±2mg/ml NaCI is typical.

Further, pharmaceutical compositions may comprise a sugar alcohol (e.g., mannitol) or a disaccharide (e.g., sucrose or trehalose) e.g., at around 15-30 mg/ml (e.g., 25 mg/ml), particularly if they are to be lyophilized or if they include material which has been reconstituted from lyophilized material. The pH of a composition for lyophilization may be adjusted to between 5 and 8, or between 5.5 and 7, or around 6.1 prior to lyophilization.

The compositions of the invention may also comprise one or more immunoregulatory agents. In some embodiments, one or more of the immunoregulatory agents include(s) an adjuvant. Medical Treatments, Kits and Uses

Medical treatments

In a further aspect, the present invention provides the use of the antibody according to the present invention, or an antigen-binding fragment thereof, the nucleic acid molecule (or the plurality of nucleic acid molecules) according to the present invention, the vector (or the plurality of vectors) according to the present invention, the cell according to the present invention or the pharmaceutical composition according to the present invention as a medicament. In particular, the antibody according to the present invention, or an antigenbinding fragment thereof, the nucleic acid molecule (or the plurality of nucleic acid molecules) according to the present invention, the vector (or the plurality of vectors) according to the present invention, the cell according to the present invention or the pharmaceutical composition according to the present invention may be used in prophylaxis and/or treatment of SARS-CoV-2 infection or COVID-19; or in (ii) diagnosis of SARS-CoV-2 infection or COVID-19.

Accordingly, the present invention also provides a method of ameliorating or reducing SARS- CoV-2 infection or COVID-19, or lowering the risk of SARS-CoV-2 infection or COVID-19 in a subject, comprising: administering to the subject, a therapeutically effective amount of the antibody, or an antigen-binding fragment thereof, according to the present invention, the nucleic acid molecule (or the plurality of nucleic acid molecules) according to the present invention, the vector (or the plurality of vectors) according to the present invention, the cell according to the present invention or the pharmaceutical composition according to the present invention. Moreover, the present invention also provides the use of the antibody according to the present invention, or an antigen-binding fragment thereof, the nucleic acid molecule (or the plurality of nucleic acid molecules) according to the present invention, the vector (or the plurality of vectors) according to the present invention, the cell according to the present invention or the pharmaceutical composition according to the present invention in the manufacture of a medicament for prophylaxis, treatment or attenuation of SARS-CoV- 2 infection or COVID-19. Prophylaxis of SARS-CoV-2 infection or COVID-19 refers in particular to prophylactic settings, wherein the subject was not diagnosed with SARS-CoV-2 infection or COVID-19 (either no diagnosis was performed or diagnosis results were negative) and/or the subject does not show symptoms of SARS-CoV-2 infection or COVID-19. In therapeutic settings, in contrast, the subject is typically diagnosed with SARS-CoV-2 infection or COVID-19 and/or showing symptoms of SARS-CoV-2 infection or COVID-19. Of note, the terms "treatment" and "therapy'7"therapeutic" of SARS-CoV-2 infection or COVID-19 include (complete) cure as well as attenuation/reduction of SARS-CoV-2 infection or COVID-19 and/or related symptoms.

In some embodiments, prophylaxis, treatment or attenuation of SARS-CoV-2 infection or COVID-19 refers to infection with one or more variants of SARS-CoV-2. Variants of SARS- CoV-2, for which prophylaxis, treatment or attenuation of SARS-CoV-2 infection or COVID- 19 may be provided, include, but are not limited to, one or more of alpha (B.1 .1 .7; 501 Y.V1 ), beta (B.1 .351 ; 501 Y.V2), gamma (P.1 ; 501 Y.V3), delta (B.1 .617.2) and omicron (B.1 .1 .529). In some embodiments, prophylaxis, treatment or attenuation of SARS-CoV-2 infection or COVID-19 refers to infection with SARS-CoV-2 delta (B.1.617.2). In some embodiments, prophylaxis, treatment or attenuation of SARS-CoV-2 infection or COVID-19 refers to infection with SARS-CoV-2 omicron (B.1 .1 .529).

In some embodiments the subject may be a human. One way of checking efficacy of therapeutic treatment involves monitoring disease symptoms after administration of the composition of the invention. Treatment can be a single dose schedule or a multiple dose schedule. In one embodiment, an antibody, antibody fragment, nucleic acid, vector, cell or composition according to the invention is administered to a subject in need of such treatment. Such a subject includes, but is not limited to, one who is particularly at risk of or susceptible to SARS-CoV-2 infection or COVID-19, including, for example, medical personnel, individuals with ineffective vaccine responses, in particular elderly and/or immunocompromised subjects. Further Use and Kits

In a further aspect, the present invention also provides the use of the antibody, or an 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 plurality of nucleic acid molecules according to the present invention, the plurality of vectors according to the present invention, the cell according to the present invention or the pharmaceutical composition according to the present invention for monitoring the quality of an anti-SARS-CoV-2 vaccine and/or immune response in absence of a vaccine by checking that the antigen of said vaccine contains the specific epitope in the correct conformation. Preferred antigens comprised by such an anti-SARS-CoV-2 vaccine to be checked include the spike protein of SARS-CoV-2 (in particular its RBD or its ACE2 binding site), or any other molecule/complex comprising or consisting of the spike protein of SARS-CoV-2, its RBD or its ACE2-binding site.

Antibodies and fragments thereof as described in the present invention may also be used for the diagnosis of SARS-CoV-2 infection or COVID-19. They can also be used as reagents and bio-recognition elements for detection of the presence of RBD, S, SARS-CoV-2 viral particles or their mutants in a sample of interest, e.g. food, environmental samples, water and more. They can also be used as reagents to assess the ability of other antibodies, sera or samples to inhibit ACE2 binding by SARS-CoV-2 and other related viruses and mutants. Methods of diagnosis may include contacting an antibody with a sample. Such samples may be isolated from a subject, for example an isolated tissue sample taken from, for example, nasal passages, sinus cavities, salivary glands, lung, liver, pancreas, kidney, ear, eye, placenta, alimentary tract, heart, ovaries, pituitary, adrenals, thyroid, brain, skin or blood, such as plasma or serum. For example, the antibody, or an antigen-binding fragment thereof, may be contacted with an (isolated) blood sample (e.g., whole blood, plasma or serum). The methods of diagnosis may also include the detection of an antigen/antibody complex, in particular following the contacting of an antibody with a sample. Such a detection step is typically performed at the bench, i.e. without any contact to the human or animal body. Examples of detection methods are well-known to the person skilled in the art and include, e.g., ELISA (enzyme-linked immunosorbent assay), SPR (Surface Plasmon Resonance), microscopy with fluorescently labelled antibodies. Accordingly, the diagnosis may be performed in vitro, for example by using an isolated sample as described above (and an in vitro detection step of an anti gen/anti body complex). Accordingly, the antibody, or an antigen-binding fragment thereof, may be used in (in vitro) diagnosis of SARS-CoV-2 infection or COVID-19.

In a further aspect, the present invention also provides a kit of parts comprising at least one antibody, or antigen binding fragment thereof, according to the present invention, at least one nucleic acid according to the present invention, the plurality of nucleic acids according to the present invention, at least one vector according to the present invention, the plurality of vectors according to the present invention, at least one cell according to the present invention, and/or at least one pharmaceutical composition according to the present invention. In addition, the kit may comprise means for administration of the antibody, or an 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 plurality of nucleic acid molecules according to the present invention, the plurality of vectors according to the present invention, the cell according to the present invention or the pharmaceutical composition according to the present invention, such as a syringe or a vessel, a leaflet, and/or a leaflet.

Such a kit may be used as described above for its components, for example regarding the medical use. Moreover, such kits may be provided for diagnosis or as ACE2 inhibition kit, for example to prove that other antibodies or sera are capable of inhibiting ACE2.

BRIEF DESCRIPTION OF THE FIGURES

In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the invention in any way. Figure 1 shows the epitopes (residues) in the RBD of SARS-CoV-2 Spike (S) protein targeted by the parental antibodies C121 , C135, and C144. The epitope regions of each mAbs are highlighted in dark-grey on the SARS-CoV-2 RBD surface (white). Residues of RBD within 6A from the corresponding mAb are listed in tables.

Figure 2 shows for Example 1 a schematic representation of the bispecific constructs LVL-scFv-BiS1 , LVL-scFv-BiS2, LVLX1 , and LVLX2. The different domains of LVL-scFv-BiS1 and LVL-scFv-BiS2 are connected via (GGGGSh linkers.

Figure 3 shows for Example 1 the sequence of the CH2-CH3 domains (wild-type and Fc mutants). Residues substituted in each Fc mutant are highlighted in dark grey.

Figure 4 shows for Example 2 atomic structures of the binding of the parental monospecific antibodies to the SARS-CoV-2 Spike trimer. Only one IgG per trimer can be accomodated.

Figure 5 shows for Example 2 atomic structures of the binding of bispecific antibody LVLX2 to the SARS-CoV-2 Spike trimer. In contrast to the parental antibodies, multiple IgG-like bispecific antibodies can bind simultaneously and occupy all ACE2 binding sites on a spike trimer.

Figure 6 shows for Example 3 a) the association (K _a , K _on , left) and dissociation rate (Kd, K _Off , right) of the bispecific LVLX2, parental monospecific IgG antibodies C121 and C135, and C135 Fab plotted against decreasing concentrations of RBD immobilized on a SPR surface. It shows simultaneous engagement of both antigen binding sites of the bispecific to a single RBD molecule. The rates are normalized against the value at the highest RBD concentration for each molecule. Kon (left) is not affected by avidity effects and remains equal at all RBD concentrations; so does the Koff for the Fab, which has only monovalent binding and thus no avidity. Koff becomes faster with decreasing RBD concentration for parental IgG antibodies C121 and C135. This indicates loss of avidity (resulting in faster dissociation) when the monoclonal IgG cannot engage both arms simultaneously on two adjacent RBDs (1 IgG, 2RBDs). Intramolecular avidity is absent by definition in IgGs, since only one binding site per RBD is present. By contrast, the bispecific maintains avidity even at low RBD concentration due to simultaneous intra-molecular bivalent binding to one RBD (1 bispecific, 1 RBD). b) values corresponding to the charts in a), c) SPR traces used to calculate the rates.

Figure 7 shows for Example 4 a) Kinetic parameters for the binding of C121 , 035, and LVLX2 to SARS-CoV-2 full spike protein and its RBD; related SPR traces in b). c) SPR determined binding affinities, K _D , of LVLX2 and the two parental monospecific antibodies for several viral mutants and one animal sequence. The LVLX2 bispecific binds to mutants not recognized by the individual parental monospecific antibodies.

Figure 8 shows for Example 5 inhibition of hACE2 binding to SARS-CoV-2 spike protein by LVLX2 and parental monospecific antibodies C121 and C135. Only the bispecific antibody can achieve complete inhibition.

Figure 9 shows for Example 6 the results of an in vitro neutralization assay of HIV-SARS- CoV-2-pseudovirus by bispecific antibodies LVLX1 , LVLX2, LVL-scFv-BiS1 , and LVL-scFv-BiS2, all neutralizing the virus at low nanomolar concentration; sub-nanomolar for LVLX2.

Figure 10 shows for Example 6 the results of an in vitro neutralization assay of wild-type SARS-CoV-2 and of 3 viral mutants generated by exposure of the virus to the C121 and C135 monospecific parental antibodies (R346S, E484G, and Q493R). LVLX2 fully neutralizes the viruses even when the monospecific parental antibodies fail. LVLX2 achieves better or equal neutralization levels than the monospecific parental antibodies (first page) and it is always better than the C121 and C135 Fabs (second page). Figure 1 1 shows for Example 7 the Western blot showing the presence of human ACE2 (upper bands) in the lungs of humanized mice. Histone H3 expression levels are shown at the bottom as control.

Figure 12 shows for Example 7 (A) humanized mice expressing human ACE2 in the lungs treated with LVLX2, C121 IgG or an unrelated isotype control. Following infection with SARS-CoV-2, pathological signs and weight loss were evident in the groups treated with C121 IgG or an unrelated isotype control antibody, whereas animals treated with LVLX2 had normal weight and absence of any pathological signs. Remarkably, mice treated with the C121 monospecific antibody showed weight loss and pathological signs following development of the viral escape mutation E484D. This residue is at the center of the C121- RBD interface (atomic structure in (B)) and its mutation prevents C121 binding and neutralization. By preventing escape mutants and/or neutralizing the mutated virus, the bispecific antibody offers a clear advantage over the monospecific parental antibodies.

Figure 13 illustrates the angiotensin-converting enzyme 2 (ACE2) binding site in the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2.

Figure 14 shows for Example 8 the results of an in vitro neutralization assay of SARS- CoV-2 B.1.617.2 by bispecific antibody LVLX2. LVLX2 also neutralizes the delta variant of SARS-CoV-2 at low nanomolar concentration. EXAMPLES

In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.

Example 1 : c antibodies

Design:

Four different bispecific antibodies (LVL-scFv-BiS1 , LVL-scFv-BiS2, LVLX1 , and LVLX2) and Fc-variants thereof were designed by combination of neutralizing monoclonal antibodies C121 , C135, and C144, which target different epitopes of the RBD of SARS-CoV-2 Spike (S) protein (Robbiani, D.F., Gaebler, C, Muecksch, F. et al. Convergent antibody responses to SARS-CoV-2 in convalescent individuals. Nature 584, 437-442 (2020)). Accordingly, these bispecific antibodies were obtained by combination of neutralizing monoclonal antibodies targeting different epitopes of SARS-CoV-2 Spike (S) protein, and its Receptor Binding Domain (RBD) in particular. Parental monoclonal antibodies for bispecific design were selected based on their binding, neutralization and structural properties. Not every pair of parental monospecific antibodies can be made into a bispecific due to the distance and relative position of the individual epitopes. Bispecific antibodies were thus designed from aptly selected monospecific antibody pairs (parental antibodies) based on structural biology analysis. Parental monospecific antibody pairs were chosen through an iterative process utilizing atomic-level molecular dynamics simulations to verify stability and feasibility of the desired constructs.

Figure 1 shows the epitopes (residues) in the RBD of SARS-CoV-2 Spike (S) protein targeted by the parental antibodies C121 , C135, and C144. Figure 2 shows a schematic representation of the bispecific constructs of the present invention.

LVL-scFv-BiS1 was constructed by sequential connection of the C-terminus of one domain to the N-terminus of the following domain. Specifically, the variable region of the light chain (VL) of C144 (SEQ ID NO: 28) was connected to the variable region of the heavy chain (VH) of C144 (SEQ ID NO: 27), followed by the VH of C135 (SEQ ID NO: 17), and by the VL of C135 (SEQ ID NO: 18). Heavy and light domains were connected through a 15 amino-acid linker (SEQ ID NO: 38 = (GGGGS) ₃ ). The two variable fragments (VL+VH) were linked with a 25 amino-acid linker (SEQ ID NO: 39 = (GGGGS) ₅ ), resulting in the full sequence of LVL- scFv-BiS1 (SEQ ID NO: 40). Similarly, LVL-scFv-BiS2 was constructed from monoclonal antibodies C121 and C135 resulting in the sequence according to SEQ ID NO: 41 .

Bispecific antibody LVLX1 was constructed by combination of parental antibodies C144 and C135 (based on CrossMAb technology) resulting in the combination of two different light chains and two different heavy chains (Figure 1 ). The VL of C144 (SEQ ID NO: 28) was connected to the kappa isotype constant region of the light chain (CL; SEQ ID NO: 31 ; UniProt P01834) resulting in the full C144 light chain (LC ^C144 ; SEQ ID NO: 30). The VH of C144 was connected to Immunoglobulin G (IgG) 1 heavy chain constant region (CH1 -CH2-CH3; SEQ ID NO: 32; UniProt P01857). The CH3 domain was modified to introduce the "knob" of the "knobs-into-holes" (KiH) methodology; specifically, residue Thr366 (Kabat numbering; Thr249 in SEQ ID NO: 32) was modified into a Trp. Additionally, Ser354 (Kabat numbering; Ser237 in SEQ ID NO: 32) was modified into a Cys to introduce an additional stabilizing disulfide bond with the parent heavy chain resulting in the C144 LVLX1 heavy chain (HC ^c144 ; SEQ ID NO: 29). For the C135 moiety of bispecific antibody LVLX1 , a CH1 -CL crossover between the heavy chain (HC) and the LC was introduced to drive heterodimerization of the LVLX1 heavy chains. This resulted in the connection of the VL of C135 (SEQ ID NO: 18) to the CH1 domain (residues 1 -98 of sequence UniProt P01857; SEQ ID NO: 33) of the heavy chain constant region (SEQ ID NO: 32). The CH1 domain was modified by introduction of two Ser at the N-terminus and of the sequence EPKSC to the C-terminus, resulting in the modified CH1 domain (SEQ ID NO: 34) and the full C135 light chain (LC ^C135 ; SEQ ID NO: 20) of LVLX1 . The VH of C135 (SEQ ID NO: 17) was connected to the crossover-modified constant region of the heavy chain (CL-CH2-CH3; SEQ ID NO: 35). The heavy chain constant region (SEQ ID NO: 32) was modified as follow: The CH1 domain (residues 1-98 of SEQ ID NO: 32) was replaced by the CL domain (SEQ ID NO: 31 ; UniProt P01834) in which the residues R and T at the N-terminal were substituted with residues A and S; the residues EPKSC (residues 99-103 of SEQ ID NO: 32) were removed; the CH3 domain was modified to introduce the "hole" of the KiH methodology by substitution of residues Thr366, Leu368, and Tyr407 (Kabat numbering; Thr249, Leu251 , and Tyr290 in SEQ ID NO: 32) for Leu, Ala, and Vai, respectively; and Tyr349 (Kabat numbering; Tyr232 in SEQ ID NO: 32) was modified into a Cys to introduce an additional stabilizing disulfide bond with the parent heavy chain. The modification listed above resulted in the HC ^c135 of LVLX1 with sequence according to SEQ ID NO: 19.

Similarly, LVLX2 was constructed from monoclonal antibodies C121 and C135. The sequences of HC ^c135 and LC ^C135 are the same as for LVLX1 (SEQ ID NOs 19 and 20, respectively); while the HC ^C121 and LC ^C121 were constructed as outlined above for C144 (in LVLX1 ) resulting in the sequences according to SEQ ID NOs 9 and 10, respectively.

Fc-variants of L VLX1 and L VLX2:

In addition to the above described bispecific antibodies LVLX1 and LVLX2, bispecific antibodies with mutations in the Fc moiety (constant domains CH2 and CH3 of the heavy chains HC ^C121 , HC ^c135 , and HC ^C144 ) were designed and produced to modulate the antibody effector function and/or to improve the antibody circulation half-life. The introduced mutations were LALA (Leu234Ala and Leu235Ala; Kabat numbering), PG (Pro329Gly; Kabat numbering), GRLR (Gly236Arg and Leu328Arg; Kabat numbering), IS (Met428Leu and Asn434Ser; Kabat numbering), and combination thereof, as shown in Figure 3. LALA-PG variants of LVLX1 and LVLX2 bind to S protein and RBD with low nanomolar KD according to SPR experiments and they neutralize pseudo-virus in vitro at low nanomal concentration. Preparation of bispecific antibody constructs:

A N-terminal signal peptide (residues 1 -1 9; SEQ ID NO: 36; UniProt P01 743) and a C- terminal 6x HisTag (HHHHHH) preceded by Gly-Ser (GS) spacer, were added to the sequences of LVL-scFv-BiS1 (SEQ ID NO: 40) and LVL-scFv-BiS2 (SEQ ID NO: 41 ). The constructs were synthetized and subcloned into the mammalian expression vector pcDNA3.1 (+) by Genscript.

The constructs for light chains LC ^C144 , LC ^C121 , and LC ^C135 (SEQ ID NOs 30, 1 0 and 20, respectively) and heavy chains HC ^C144 , HC ^C121 , and HC ^C135 (SEQ ID NOs 29, 9 and 19, respectively) were synthetized and subcloned into the mammalian expression vector pcDNA3.1 (+) by Genscript. Signal peptides were included at the N-terminus of the variable sequences: residues 1 -1 9 (SEQ ID NO: 36; UniProt P01 743) for the heavy chains and residues 1 -20 (SEQ ID NO: 37; UniProt P06312) for the light chains.

Expression:

The bispecific antibodies were produced by transient PEI (polyethylenimine, Polysciences) transfection in Expi293F cells (ThermoFisher). The day before transfection (Day 0) the cells, maintained in Expi-293 Expression Medium (Gibco), were seeded at a density of 2x10 ⁶ ml ¹ in shake flask (Corning) and incubated at 37°C, 120 rpm, 8% CO ₂ . The day after (Day 1 ), the cells at a density of 2.5 x 10 ⁶ ml"' were transfected with a mixture of plasmid DNA and PEI.

In brief, a solution of PEI (10 pg/ ml transfection) in Opti-MEM (volume 5% of the transfection volume) was added to a solution of plasmid DNA (in Opti-MEM, Gibco; volume 5% of the transfection volume) was added. 1 pg/mL transfection was used for LVL-scFv-BiS1 and LVL- scFv-BiS2. 0.25 pg/mL transfection of plasmids encoding for LC ^C144 , LC ^C135 , HC ^C144 , and HC ^C135 was used for LVLX1 and of plasmids encoding for LC ^C121 , LC ^C135 , HC ^C121 , and HC ^c135 was used for LVLX2.

After 20 min incubation at room temperature, the DNA/PEI solution was added dropwise to the cells suspension under shacking. On day 3, the transfected cells suspension was treated with Soy Hydrolysate SOX (Sigma; volume ratio 1 /50 of transfection volume), D-glucose solution (Sigma; volume ratio 1 /1 50 of transfection volume), and Expi-293 Expression Medium (volume ratio 1/3 of transfection volume). The supernatant containing the secreted bispecific antibody was harvested after 5-7 days by centrifugation.

Antibodies purification:

The bispecific antibodies LVL-scFv-BiS1 and LVL-scFv-BiS2 were purified from the cell supernatants by HiTrap™ Chelating HP (Cytiva; elution with PBS containing 500 mM Imidazole at pH 7.2) and Hiload Superdex 75 16/60 column (Cytiva).

The bispecific antibodies LVLX1 and LVLX2 were purified with HiTrap™ Protein A HP (Cytiva; elution 100 mM Glycine at pH 2.7), and Hiload Superdex 200 16/60 column (Cytiva).

Antibodies characterization:

The purified proteins were analyzed by SDS-PAGE for purity; dynamic light scattering (DLS, on a DynaPro NanoStar by Wyatt Technology) to ensure a monomeric state and lack of aggregation; circular dichroism (CD) to assess folding and secondary structure.

Bispecitic antibodies bind SAKS-Cov-z spike contormations not ava parental antibodies and occupy all ACE2 binding site on the S trimer

Next, 3D binding structures of the parental monospecific antibodies and the bispecific constructs of Example 1 to SARS-CoV-2 spike protein was analyzed in siiico and compared to the parental monospecific antibodies. Experimental structures of the Fab portion of the monospecific parental antibodies binding to one RBD are available (PDB ID 7K8X; 7K8Z; 7K90). The structures of two identical Fabs (for monoclonals) or two different ones (for bispecifics, e.g. C121 and C135 for LVLX2) were manually connected to the constant antibody regions in siiicoto form a full IgG antibody (parental monoclonal) or aptly modified to form a CrossMab (bispecifics). The full IgGs were subjected to 400ns atomistic molecular dynamics simulations with GROMACS and the AMBER99SB-ILDN force field to obtain a realistic and stable IgG conformation. The resulting IgG was superimposed on the experimental structure of the S protein in complex with the parental monoclonal antibodies Fab. A further 400ns of atomistic molecular dynamics simulations were performed on the S- Antibody complex to verify feasibility and stability of the designed constructs and their ability to occupy the ACE2 binding sites. The process was repeated for all antibodies and for different spike conformations: 3 RBD in the Up conformation, 3 Down, 2 RBD up and 1 RBD down; 1 RBD up and 2 RBD Down.

Results are shown in Figures 4 (for parental monospecific antibodies) and 5 (for bispecific construct LVLX2). Each spike trimer is composed of three identical units with one RBD each. Structural data have shown that the RBDs can be in either an 'up' or 'down' conformation, with only the former being able to bind ACE2 and, thus, infect human cells. Any combination of up/down RBD is available to the trimer: 3 up, 3 down, 2 up 1 down and 2 down 1 up.

Figure 4 shows that the parental monospecific antibodies C121 , 035 and C144 cannot engage all RBDs in all conformations. Despite occupying the ACE2 binding site on one RBD, for instance, parental antibodies C121 and C144 cannot occupy three of them simultaneously on the spike due to steric clashes. Although the parental monospecific antibodies can bind across RBDs in selected conformations, only one of them can be present on each Spike protein at the same time (Figure 4).

The bispecific construct LVLX2, in contrast, can (i) bind to conformations not available to individual monospecific antibodies; and (ii) occupy every ACE2 binding site on the Spike trimer (Figure 5).

Surface Plasmon Resonance (SPR) assays was used to verify that bispecific antibodies LVLX1 and LVLX2 can bind their targets with their two distinct specificities simultaneously.

First of all, A C121/RBD complex was formed and the data show that LVLX2 binds to it, proving that its C135 moiety is active. Similarly, binding to a C135/RBD complex indicates that the C121 moiety is functional. Then, an avidity assay was used to show that both arms engage their target simultaneously. To this end, analysis and comparison of kinetics parameters at different RBD concentrations were performed as follows: RBD was immobilized on the surface of CM5 SPR chips at 5, 15, 75 and 150 nM. Increasing concentration of antibodies (3.12, 6.25, 12.5, 25, 50 nM) were injected using a Single-cycle kinetics setting. Analyte responses were corrected for unspecific binding and buffer responses through control channels. Curve fitting and data analysis were performed with Biacore™ Insight Evaluation Software. Results are shown in Figure 6.

Briefly, avidity effects resulting in slower dissociation arise when an antibody has bivalent binding to antigens immobilized on an SPR chip surface. Monoclonal IgG C121 and C135 show avidity at high antigen concentration due to intermolecular binding to adjacent RBDs (1 monoclonal IgG, 2 RBDs). Intermolecular binding is prevented at low concentration, when the RBDs are distant from each other and thus only monovalent binding to the single epitope on each RBD is possible, leading to loss of avidity and faster dissociation. By contrast, LVLX2 has avidity even at low RBD concentration due to simultaneous, bivalent, intramolecular binding (1 bispecific to 1 RBD). For comparison, Fab constructs with a single binding arm do not show avidity at any concentration.

Example 4: T he bispecitic antibody LVLX2 binds to 5AKS-Cov-2 5 protein, its RBD a viral mutants that are not recognized by the individual parental antibodies

The binding kinetics of the bispecific antibody LVLX2 and its parental monoclonal antibodies C121 and C135 were measured at 25 °C on a Biacore™ 8K SPR instrument (Surface Plasmon Resonance, GE Healthcare) using 10 mM HEPES pH 7.4, 150 mM NaCI, 3 mM EDI A and 0.005% Tween-20 as running buffer. SARS-CoV-2 S protein, RBD and various mutations were immobilized on the surface of a CMS chip (Cytiva) through standard amine coupling; one immobilized antigen per channel/experiment. Increasing concentrations of antibodies (3.12, 6.25, 12.5, 25, and 50 nM) were injected using a single-cycle kinetics setting; analyte responses were corrected for unspecific binding and buffer responses by use of control channels. Curve fitting and data analysis were performed with Biacore™ Insight Evaluation Software.

Results are shown in Figure 7. Bispecific antibody LVLX2 binds S protein and RBD in the low nanomolar range. LVLX2 binds several viral mutants, including the currently circulating and dominant D614G viral mutation, a pangolin coronavirus (considered a threat for cross-over events from animals to humans) and SARS-CoV-2 mutants that the individual parental monospecific antibodies C121 and C135 cannot bind to.

Example 5:

Binding of SARS-CoV2 to human angiotensin-converting enzyme 2 (hACE2) is an essential prerequisite for virus infectivity. Therefore, the ability of the bispecific antibodies to inhibit or reduce binding of the SARS-CoV2 spike protein to hACE2 was investigated.

To this end, ELISA plates were coated at 4 °C with the spike protein and then washed and blocked with PBS+10% FCS. Antibodies were added at different dilutions, and incubated 1 h at 25 °C; after washing, hACE-mFc was added at constant saturating concentration and left 1 h at 25 °C. After further washing, bound hACE was detected using goat anti-mouse IgG coupled to alkaline phosphatase (SouthernBiotech).

Results are shown in Figure 8. LVLX2 was designed to occupy the ACE2 binding site on the RBD with the C121 arm. As outlined above, ELISA assays were used to verify the ability of LVLX2 to inhibit the binding of recombinant ACE2 to SARS-CoV2 S protein. In line with the structural information, binding of C135 to immobilized S protein does not prevent subsequent binding of ACE2 (Figure 8). Surprisingly, complete inhibition is also not achieved when C121 is complexed with S, despite it occupying the ACE2 binding site. Evidently, there are ACE2 bindings sites not accessible by the monospecific parental antibody C121 in S protein conformations. By contrast, full ACE2 inhibition is synergistically obtained by bispecific LVLX2. Only simultaneous binding by the bispecific moieties, in other words, can make all ACE2 binding sites unavailable either by direct occupation or by locking the RBD in a conformation incapable to recognize ACE2 (e.g. 'down' conformation). In summary, LVLX2 can fully inhibit binding of the spike protein to ACE2 whereas the individual monospecific parental antibodies fail to do so.

Neutralization of SARS-CoV-2: Bispecifics neutralize viral sequences that cannot be neutralized by the parental monospecific antibodies

To investigate in vitro neutralization of SARS-CoV2, the four exemplified bispecific antibodies of Example 1 LVLX1 , LVLX2, LVL-scFv-BiS1 and LVL-scFv-BiS2 were tested in a neutralization assay. For LVLX1 and LVLX2 LALA-PG Fc mutants (differing from wt Fc in the LALA-PG mutation described above) were used. To this end, fourfold serially diluted monoclonal antibodies were incubated with SARS-CoV-2 pseudotyped virus, containing also a luciferase reported gene, for 1 h at 37 °C. The mixture was subsequently incubated with 293T _ACE2 cells (expressing human ACE2) for 48 h. Cells were then washed twice with PBS and lysed. Luciferase activity in lysates, indicating presence of pseudovirus in the cells, was measured. The obtained relative luminescence units were normalized to those derived from cells infected with SARS-CoV-2 pseudotyped virus in the absence of antibody.

Results are shown in Figure 9. The four exemplified bispecific antibodies of Example 1 neutralize SARS-CoV-2 pseudoviruses in vitro at low nanomolar concentration (EC50 <1 nM for LVLX2).

Next, neutralization of bispecific antibody LVLX2 was compared to its parental monospecific antibodies C121 and C135 (as IgG and Fab versions). To this end, neutralization assays were performed essentially as described above. LVLX2, C121 and C135 (as IgG and Fab versions) were tested for neutralization of wild-type SARS-CoV2 and of SARS-CoV2 viral escape mutations, which are generated by exposure of the virus to either C135 or C121 , namely, Q493R, E484G and R346S.

Results are shown in Figure 10. The bispecific antibody LVLX2 and the parental monospecific antibody C121 fully neutralize the currently circulating SARS-CoV-2 sequence; while the parental monospecific antibody C135 neutralizes it to a lesser extent. LVLX2, but not the individual parental monospecific antibodies, fully neutralizes the viral escape mutations generated by exposure of the virus to either C135 or C121. The bispecific is always significantly better than monovalent Fab versions of the parental antibodies. Due to the atomic configuration of the Spike protein, the parental antibodies, but not the bispecific, can only bind to all S conformations as Fab and not as IgG (Figure 4 and 5).

In vivo protection of mice from SARS-CoV-2

To assess the clinical potential of bispecific antibody LVLX2, its ability to protect animals from SARS-CoV-2 infection was investigated.

To mimic human COVID-19 disease in mice, an adenovirus vector expressing human ACE2 was delivered to mice by forced inhalation under general anesthesia, reaching both lower and upper respiratory tracts. This gentle delivery system is important to mimic the real environment of SARS-CoV-2 infection in humans. Expression of hACE2 protein in lungs of transduced animals was confirmed by western blot (Figure 1 1 ) at 1 , 2 and 4 weeks post - delivery of hACE2. This system mimics the human COVID-19 disease, including damage caused by lung inflammation and cytokine response rather than the virus itself.

These mice with 'humanized lungs' were intraperitoneally injected with 150 pg/mouse (corresponding to ~7.5mg/kg) of antibody (LVLX2, C121 or unrelated isotype control antibody) at day -1 , followed by intranasal inoculation with 1 x10 ⁴ pfu of SARS-CoV-2 (strain SARS-CoV-2/human/Czech Republic/951/2020) at day 0.

Results are shown in Figure 12. A control group treated with an unrelated antibody (isotype control) showed statistically significant weight loss (25-30 %) and pathological effects including lung inflammation with alveoli replaced by inflammatory infiltrates containing lymphocytes, macrophages, neutrophils and fibroblasts. Alveolar septa were thickened in areas close to infiltrates. Increased number of activated macrophages with foamy cytoplasm in regions with minimally changed morphology were detected, suggesting significant cytokine production and pro-inflammatory action, which resembles pathological findings seen in human patients with COVID-19.

In contrast thereto, administration of LVLX2 prevented any pathological signs in lungs from the SARS-CoV-2 infected animals, which also showed decreased viral titers. In 7 out of 8 mice the viral titer was below the limit of detection on day 5 post infection, as demonstrated by plaque assay. None of the mice had any significant weight loss over the whole experimental period (Figure 12). Viral RNA levels in spleen samples were significantly lower (at the detection limit) in mice that received LVLX2, indicating substantially reduced viremia.

Remarkably, animals that received the monospecific parental antibody C121 , the potent in vitro neutralizer employed in LVLX2, also showed lung pathology and weight loss (~20 %). Sequencing of the viral material recovered from these animals revealed an E484D mutation, whereas virus sequences determined from control mice had a wild-type genotype. E484 forms intermolecular H-bonds at the core of the C121/RBD interface (Figure 12) and E484D conferred resistance to C121 neutralization. Weight loss in C121 -treated mice becomes apparent at a later time point than in control animals, suggestive of loss of protection when the escaped virus takes prevalence.

Example 8: Neutralization of SARS-CoV-2 delta variant

To investigate in vitro neutralization of the delta variant (B.1.617.2) of SARS-CoV2, the exemplified bispecific antibody LVLX2 was tested in a neutralization assay.

To this end, different LVLX2 antibody dilutions were mixed in a flat-bottomed tissue culture microtitre plate with an equal volume of 100 median tissue culture infectious dose of infectious virus that was isolated from patients with COVID-19, sequenced, titrated and incubated at 33 °C in 5% CO2. After 1 h, 3 x 10 ⁴ Vero E6 cells were added to each well. After 3 days of incubation, cells were stained with Gram's crystal violet solution plus 5% formaldehyde 40% m/v for 30 min. Microtitre plates were then washed in water. Wells were analysed to evaluate the degree of cytopathic effect compared to untreated control. Results are shown in Figure 14. The bispecific antibody LVLX2 potently neutralizes also the delta variant (B.1 .617.2) of SARS-CoV-2 in vitro at nanomolar concentration.

TABLE OF SEQUENCES AND SEQ ID NUMBERS (SEQUENCE LISTING):