Antibodies that bind to the gamma-delta T cell receptor

Abstract

The present invention relates to an antibody capable of binding to the human Vy9V52 T cell receptor. The present invention further relates to a pharmaceutical composition comprising the antibody of the invention and to the use of the antibody of the invention for medical treatment.

Description

[Technical field]

[0001] The present invention relates to novel antibodies capable of binding to the V52 chain of the human Vy9V52 T cell receptor. The present invention further relates to pharmaceutical compositions comprising the antibodies of the invention and to the use of the antibodies of the invention for medical treatment. [Background technology]

[0002] Gamma-delta (γδ) T cells are T cells that express a T cell receptor (TCR) consisting of gamma and delta chains. The majority of γδ T cells express TCRs that contain Vγ9 and Vδ2 regions. Vγ9Vδ2 T cells can react against a wide range of pathogens and tumor cells. It is understood that this broad reactivity is mediated by phosphoantigens that can specifically activate this T cell subset in a TCR-dependent manner. The broad antimicrobial and antitumor reactivity of Vγ9Vδ2 T cells suggests a direct involvement in the immune control of cancer and infectious diseases.

[0003] Agents capable of activating Vγ9V52 T cells may be useful in the treatment of infectious diseases or cancer, as they may promote Vγ9V52 T cell reactivity against pathogens or infected or cancer cells. WO2015156673 describes antibodies capable of binding to Vγ9V52 TCR and activating Vγ9V52 T cells. WO2020060405 describes bispecific antibodies that bind to both Vγ9V52 T cells and tumor cell targets, thus potentially recruiting Vγ9V52 T cells to tumors and thus stimulating a therapeutic effect.

[0004] Recombinant production of antibodies in host cells often results in heterogeneous products, including different forms of antibodies with various types and degrees of post-translational modifications of the polypeptide chain. Such heterogeneity is undesirable for antibody products for medical use, since post-translational modifications can alter the functional properties of the antibody, for example, with respect to affinity for the target antigen, with respect to pharmacokinetic properties, product stability, aggregation, etc.

[0005] The present invention provides improved Vy9V52 TCR binding antibody sequences which result in a more homogenous product when produced in a host cell and which maintain good functional properties in terms of target binding and functional effect on target cells, as well as good structural properties such as stability. Summary of the Invention

[0006] The inventors surprisingly found that the antibody 5C8 described in WO2015156673 is sulfated at a site in the antibody that was not predicted to be subject to this post-translational modification. Sulfation occurs partially in different host cells, resulting in a heterologous antibody product.

[0007] Surprisingly, the tyrosine residue targeted for sulfation, located in the CDR3 region, known to be a major determinant of antigen-binding specificity in the antigen-binding region of an antibody, can be mutated to phenylalanine or serine without affecting the antigen-binding properties of the antibody.

[0008] Removal of sulfation sites through mutation resulted in a more homogeneous antibody product.

[0009] Thus, in a first aspect, the present invention provides an antibody comprising a first antigen-binding region capable of binding to human V52, said first antigen-binding region comprising a CDR1 sequence as set forth in SEQ ID NO:1, a CDR2 sequence as set forth in SEQ ID NO:2, and a CDR3 sequence as set forth in SEQ ID NO:3.

[0010] In a further aspect, the invention provides a bispecific antibody comprising a first binding region capable of binding human V52 as defined herein and a second antigen binding region capable of binding a second antigen, the second antigen being preferably human EGFR. In further aspects, the invention relates to pharmaceutical compositions comprising the antibodies of the invention, to the use of the antibodies of the invention in medicine, as well as to nucleic acid constructs, expression vectors for producing the antibodies of the invention, and to host cells comprising such nucleic acid constructs or expression vectors. Furthermore, the invention relates to a process for producing the antibodies of the invention, which avoids sulfation and results in a more homogeneous product.

[0011] Further aspects and embodiments of the invention are described below. [Brief description of the drawings]

[0012] [Figure 1] Figure 1: Representative chromatogram of size-exclusion profile of Protein-A purified LAVA compound (showing VHH 5C8) using a Superdex-75 column. Fractions of the dominant monomer peak (fractions 1E11-1G2) were pooled and quantified. [Diagram 2] Figure 2: Representative example of lab-chip polyacrylamide gel electrophoresis of purified VHH 5C8. Left: non-reducing, right: reducing conditions. [Figure 3A] Figure 3: HP-SEC characterization of purified VHH 5C8 (A) and VHH 5C8var1 (B). [Figure 3B] Same as above. [Figure 4A] Figure 4: Representative HP-SEC profile of purified bispecific VHH (bsVHH) 1D12var5-5C8var1. B: bsVHH 1D12var5-5C8var1 batch expressed and purified by protein A affinity chromatography from the supernatant of Pichia pastoris. C: bsVHH 1D12var5-5C8var1 batch expressed and purified by both protein A and size exclusion chromatography from HEK-293 E cells. [Figure 4B] Same as above. [Diagram 5] Figure 5: LabChip analysis of purified VHHs 5C8var1-Y105F and 5C8var1-Y105S under non-reducing conditions. [Figure 6A] Figure 6: HP-SEC analysis of VHHs 5C8var1-Y105F (A) and 5C8var1-Y105S (B). [Figure 6B] Same as above. [Figure 7] Figure 7: Affinity measurement of VH fragment binding to recombinant Vγ9Vδ2-TCR protein using BLI. Protein mass (response, nm) is plotted as a function of time. The dotted vertical line separates the association phase (left) from the dissociation phase (right). A: VHH 5C8var1; B: VHH 5C8var1-Y105F; C: VHH 5C8var1-Y105S. The black line represents the data fitted to the actual responses measured. [Figure 8] Figure 8: Both bsVHH 7D12var8-5C8var1-Y105F and bsVHH 7D12-5C8 induce potent Vγ9Vδ2 T cell activation and lead to Vγ9Vδ2 T cell-mediated tumor cell lysis. A: 4 h degranulation assay: The percentage of CD107A (LAMP-1)+ Vγ9Vδ2 T cells is plotted as a function of the antibody concentration used. Left: 7D12-5C8 (non-humanized); Right: 7D12var8-5C8var1-Y105F. B: 24 h cytotoxicity assay showing the A431 tumor cell killing percentage as a function of the antibody concentration used. Left: 7D12-5C8 (non-humanized); Right: 7D12var8-5C8var1-Y105F. [Figure 9] Figure 9: Binding of 7D12var8-5C8var1(Y105F)-Fc to primary γδ T cells isolated from healthy human PBMCs using flow cytometry. The two panels represent two different donors. [Figure 10] Figure 10: Binding of 7D12var8-5C8var1(Y105F)-Fc to EGFR on tumor cells by cell-based ELISA. [Figure 11]FIG. 11: 7D12var8-5C8var1(Y105F)-Fc-induced γδ T cell degranulation dependent on the A431 cell line. [Figure 12] FIG. 12: Viability of A-388 cells in co-culture with γδ T cells and 7D12-5C8. [Figure 13] Figure 13: Lysed tumor cells after 4 h culture of dissociated tumor cell suspensions (primary CRC: n=10, peritoneal CRC metastases: n=5, hepatic CRC metastases: n=3, primary HNSCC: n=5, and primary NSCLC: n=4) with healthy donor-derived Vγ9Vδ2 T cells (E:T ratio of 1:1) and 7D12-5C8 (50 nM) or media control. [Figure 14] Figure 14: Lysed tumor cells after 24 h culture of dissociated tumor cell suspensions (peritoneal CRC metastases: n=4) with healthy donor-derived Vy9V52 T cells (1:1 E:T ratio) and 7D12-5C8var1(Y105S)-Fc (50 nM), gp120-5C8var1(Y105S)-Fc (50 nM) or media control. [Figure 15] Figure 15: Structure of constructs for non-human primate studies. [Figure 16] Figure 16: Binding of 7A5-7D12var8-Fc to antigen targets. [Figure 17] Figure 17: 7A5-7D12var8-Fc-mediated degranulation and cytotoxicity. [Figure 18] Figure 18: PK analysis of 7A5-7D12var8-Fc concentrations in the blood of the three treated animals. Concentration-time curves are shown indicative of a molecule with IgG-like PK. [Figure 19] Figure 19: Total number of T cells (CD3+, left graph) and number of Vγ9 positive cells (as a percentage of the CD3+ population) in the blood of treated animals. Arrows indicate compound injection. Numbers in the legend indicate the number of monkeys treated. [Figure 20] Figure 20: Levels of IL-6 cytokine in the blood of treated animals over time. Only low levels of cytokine were observed, with release being fairly limited after the first injection. Arrows indicate the moment of treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] definition As used herein, the term "human V52" refers to the rearranged 52 chain of the Vy9V52-T cell receptor (TCR). UniProtKB-A0JD36 (A0JD36_HUMAN) provides examples of variable TRDV2 sequences. V52 is part of the delta chain of the Vy9V52-TCR. Antibodies capable of binding to human V52 can bind to an epitope located entirely within the variable region, or to an epitope located within the constant region, or to an epitope that is a combination of residues of the variable and constant regions of the delta chain.

[0014] As used herein, the term "human Vy9" refers to the rearranged y9 chain of the Vy9V52-T cell receptor (TCR). UniProtKB-Q99603_HUMAN provides examples of variable TRGV9 sequences.

[0015] As used herein, the term "EGFR" refers to the human EGFR protein (UniProtKB-P00533(EGFR_HUMAN)).

[0016] The term "antibody" refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either, that has the ability to specifically bind to an antigen under typical physiological conditions having a half-life of a significant period of time, such as at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 days or more, or any other relevant functionally defined period of time (e.g., a sufficient time to induce, promote, enhance, and / or potentiate a physiological response associated with the antibody binding to the antigen and / or a sufficient time for the antibody to recruit effector activity). The antigen-binding region (or antigen-binding domain) that interacts with the antigen may include both the heavy and light chain variable regions of the immunoglobulin molecule, or may be a single domain antigen-binding region, e.g., only the heavy chain variable region. The constant region of the antibody, when present, may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells and T cells) and components of the complement system, such as C1q, the first component in the classical pathway of complement activation.

[0017] The Fc region of an immunoglobulin is defined as the fragment of an antibody that is typically generated after digestion of the antibody with papain that contains the two CH2-CH3 regions of the immunoglobulin and a binding region, e.g., a hinge region. The constant domains of the antibody heavy chain define the antibody isotype, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE. The Fc region mediates the effector functions of the antibody using cell surface receptors called Fc receptors and proteins of the complement system.

[0018] As used herein, the term "hinge region" is intended to refer to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216 to 230 according to EU numbering.

[0019] As used herein, the term "CH2 region" or "CH2 domain" is intended to refer to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to EU numbering. However, the CH2 region may be of any of the other subtypes described herein.

[0020] As used herein, the term "CH3 region" or "CH3 domain" is intended to refer to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to EU numbering. However, the CH3 region may be of any of the other subtypes described herein.

[0021] References to amino acid positions of Fc regions / Fc domains in the present invention are according to EU numbering (Edelman et al., Proc Natl Acad Sci US A. 1969 May; 63(1):78-85; Kabat et al., Sequences of proteins of immunological interest. 5th Edition-1991 NIH Publication No. 91-3242).

[0022] As indicated above, the term antibody as used herein includes fragments of antibodies that retain the ability to specifically bind to an antigen, unless otherwise stated or clearly contradicted by the context. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antibody" include: (i) Fab' or Fab fragments, i.e., monovalent fragments consisting of VL, VH, CL and CH1 domains or monovalent antibodies as described in WO2007059782; (ii) F(ab')2 fragments, i.e., bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting essentially of VH and CH1 domains; and (iv) Fv fragments consisting essentially of the VL and VH domains of a single arm of an antibody. Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but may be linked by a synthetic linker that allows the VL and VH regions to be paired using recombinant techniques to produce a single protein chain (known as a single-chain antibody or single-chain Fv (scFv), e.g., Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)) that forms a monovalent molecule. Such single-chain antibodies are encompassed within the term antibody, unless the context indicates otherwise. Although such fragments are generally included in the meaning of antibodies, they collectively and each independently are unique features of the present invention and exhibit different biological properties and usefulness. The term antibody also includes polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies and humanized antibodies, as well as antibody fragments produced by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques, unless otherwise specified.

[0023] In some embodiments of the antibody of the present invention, the first antigen-binding region or the second antigen-binding region, or both, are single-domain antibodies. Single-domain antibodies are well known to those skilled in the art, see, for example, Hamers-Casterman et al. (1993) Nature 363:446, Roovers et al. (2007) Curr Opin Mol Ther 9:327, and Krah et al. (2016) Immunopharmacol Immunotoxicol 38:21. Single-domain antibodies include a single CDR1, a single CDR2, and a single CDR3. Examples of single-domain antibodies are heavy chain-only antibodies, antibodies that do not naturally contain light chains, single-domain antibodies derived from conventional antibodies, and variable fragments of engineered antibodies. Single-domain antibodies can be derived from any species, including mouse, human, camel, llama, shark, goat, rabbit, and cow. For example, single domain antibodies can be derived from antibodies raised in Camelidae, such as camel, dromedary, llama, alpaca and guanaco. Like whole antibodies, single domain antibodies can selectively bind to a specific antigen. Single domain antibodies may contain only the variable domains of an immunoglobulin chain, i.e., CDR1, CDR2 and CDR3 and framework regions. Such antibodies are also called Nanobodies®, or VH.

[0024] As used herein, the term "immunoglobulin" is intended to refer to a class of structurally related glycoproteins consisting of two paired polypeptide chains, one paired light (L) chains and one paired heavy (H) chains, all four of which may be interconnected by disulfide bonds. As used herein, the terms "immunoglobulin heavy chain", "immunoglobulin heavy chain", and "heavy chain" are intended to refer to one of the immunoglobulin chains. The heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH), which define the immunoglobulin isotype. The heavy chain constant region typically consists of three domains, CH1, CH2, and CH3. The heavy chain constant region further includes a hinge region. Within the structure of an immunoglobulin (e.g., IgG), the two heavy chains are interconnected via disulfide bonds in the hinge region. Equivalent to the heavy chain, each light chain typically consists of several regions: a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions (or hypervariable regions that are highly variable in sequence and / or form structurally defined loops), also called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDR sequences may be determined by using various methods, for example, the methods provided by Chothia and Lesk (1987) J. Mol. Biol. 196:901 or Kabat et al. (1991) Sequence of protein of immunological interest, fifth edition. NIH publication. Various methods of CDR determination and amino acid numbering can be compared at www.abysis.org (UCL).

[0025] As used herein, the term "isotype" refers to an immunoglobulin (sub)class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotype thereof, such as IgG1m(za) and IgG1m(f), encoded by heavy chain constant region genes. Each heavy chain isotype may be combined with either a kappa (k) or lambda (λ) light chain. The antibodies of the present invention may have any isotype.

[0026] The term "parent antibody" should be understood as an antibody that is identical to the antibody according to the invention, but where the parent antibody does not have one or more specific mutations. A "variant" or "antibody variant" or "variant of a parent antibody" of the present invention is an antibody molecule that comprises one or more mutations compared to the "parent antibody". The amino acid substitution may replace a natural amino acid with another naturally occurring amino acid or a non-naturally occurring amino acid derivative. The amino acid substitution may be conservative or non-conservative. In the context of the present invention, a conservative substitution may be defined by a substitution within a class of amino acids reflected in one or more of the following three tables: [Table 1] [Table 2] [Table 3]

[0027] In the context of the present invention, substitutions in the variant are indicated as original amino acid-position-substituted amino acid.

[0028] Three-letter or one-letter codes are used, including the codes Xaa and X to denote amino acid residues. Thus, the description "T366W" means that the variant contains a substitution of tryptophan for threonine at the variant amino acid position corresponding to amino acid 366 in the parent antibody.

[0029] Additionally, the term "substitution" encompasses substitutions with any one of the other 19 naturally occurring amino acids or with other amino acids, such as unnatural amino acids. For example, substitutions of the amino acid T at position 366 include each of the following substitutions: 366A, 366C, 366D, 366G, 366H, 366F, 366I, 366K, 366L, 366M, 366N, 366P, 366Q, 366R, 366S, 366E, 366V, 366W, and 366Y.

[0030] As used herein, the term "full length antibody" refers to an antibody containing all of the heavy and light chain constant domains, as well as the variable domains that correspond to those normally present in a wild-type antibody of that isotype.

[0031] The term "chimeric antibody" refers to an antibody whose variable region is derived from a non-human species (e.g., from a rodent) and whose constant region is derived from a different species, such as human. Chimeric antibodies may be produced by genetic engineering. Chimeric monoclonal antibodies for therapeutic applications are developed to reduce antibody immunogenicity.

[0032] The term "humanized antibody" refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain that has been modified to contain a high level of sequence homology to the human variable domain. This can be achieved by grafting the six non-human antibody complementarity determining regions (CDRs) that together form the antigen binding site into a homologous human acceptor framework region (FR). Substitution of framework residues from the parent antibody (i.e., non-human antibody) into the human framework region (back mutation) may be required to fully reconstitute the binding affinity and specificity of the parent antibody. Structural homology modeling may help to identify amino acid residues in the framework region that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions, optionally including one or more amino acid back mutations to non-human amino acid sequences, and optionally a fully human constant region. Optionally, additional amino acid modifications, not necessarily back mutations, may be introduced to obtain a humanized antibody with favorable characteristics such as affinity and biochemical properties. Humanization of non-human therapeutic antibodies is performed to ensure that the humanized antibody maintains the specificity and binding affinity of the antibody of non-human origin while at the same time minimizing its immunogenicity in humans.

[0033] The term "multispecific antibody" refers to an antibody having at least two different specificities, such as at least three different, typically non-overlapping, epitopes. Such epitopes may be on the same or different target antigens. When the epitopes are on different targets, such targets may be on the same cell or different cells or cell types. In some embodiments, a multispecific antibody may comprise one or more single domain antibodies.

[0034] The term "bispecific antibody" refers to an antibody that has specificity for two different, typically non-overlapping epitopes. Such epitopes may be on the same or different targets. When the epitopes are on different targets, such targets may be on the same cell or different cells or cell types. In some embodiments, a bispecific antibody may comprise one or two single domain antibodies.

[0035] Examples of different classes of multispecific antibodies, such as bispecific antibodies, include: (i) IgG-like molecules with complementary CH3 domains that force heterodimerization; (ii) recombinant IgG-like dual targeting molecules, where each of the two sides of the molecule contains an Fab fragment or a portion of an Fab fragment of at least two different antibodies; (iii) IgG fusion molecules, where a full-length IgG antibody is fused to an extra Fab fragment or a portion of an Fab fragment; (iv) Fc fusion molecules, where a single chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region or a portion thereof; (v) IgG-like molecules, where a single chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region or a portion thereof; ) Fab fusion molecules, where different Fab fragments are fused together to a heavy chain constant domain, Fc region or portion thereof; and (vi) ScFv and bispecific antibody-based antibodies and heavy chain antibodies (e.g., domain antibodies, Nanobodies®), where different single chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, Nanobodies®) are fused to each other or to another protein or carrier molecule fused to a heavy chain constant domain, Fc region or portion thereof.

[0036] Examples of IgG-like molecules with complementary CH3 domain molecules include Triomab® (Trion Pharma / Fresenius Biotech), Knobs-into-Holes (Genentech), CrossMAb (Roche) and electrocompatible (Amgen, Chugai, Oncomed), LUZ-Y (Genentech, Wranik et al. J. Biol. Chem. 2012, 287(52):43331-9, doi:10.1074 / jbc.M112.397869. Epub 2012 Nov 1), DIG bodies and PIG bodies (Pharmabcine, WO 2010134666, WO 2014081202), strand-displacement engineered domain bodies (SEED bodies) (EMD Serono), Biclonics (Merus, WO2013157953), FcΔAdp (Regeneron), bispecific IgG1 and IgG2 (Pfizer / Rinat), asymmetric scaffolds (Zymeworks / Merck), mAb-Fv (Xencor), bivalent bispecific antibodies (Roche, WO2009080254) and DuoBody® molecules (Genmab).

[0037] Examples of recombinant IgG-like dual targeting molecules include dual targeting (DT)-Ig (GSK / Domantis, WO2009058383), Two-in-one antibodies (Genentech, Bostrom, et al 2009. Science 323,1610-1614), cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star), Zybodies™ (Zyngenia, LaFleur et al. MAbs. 2013 Mar-Apr;5(2):208-18), the common light chain approach, κλBodies (NovImmune, WO2012023053) and CovX-body® (CovX / Pfizer, Doppalapudi, VR, et al. 2007. Bioorg. Med. Chem. Lett. 17, 501-506), but are not limited to these.

[0038] Examples of IgG fusion molecules include, but are not limited to, dual variable domain (DVD)-Ig (Abbott), dual domain double-headed antibody (Unilever; Sanofi Aventis), IgG-like bispecific antibody (ImClone / Eli Lilly, Lewis et al. Nat Biotechnol. 2014 Feb;32(2):191-8), Ts2Ab (MedImmune / AZ, Dimasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92) and BsAb (Zymogenetics, WO2010111625), HERCULES (Biogen Idec), scFv fusion (Novartis), scFv fusion (Changzhou Adam Biotech Inc) and TvAb (Roche).

[0039] Examples of Fc fusion molecules include, but are not limited to, ScFv / Fc fusions (Academic Institution, Pearce et al Biochem Mol Biol Int. 1997 Sep;42(6):1179), SCORPION (Emergent BioSolutions / Trubion, Blankenship JW, et al. AACR 100th Annual meeting 2009(Abstract #5465); Zymogenetics / BMS, WO 2010111625), Dual Affinity Retargeting Technology (Fc-DART™) (MacroGenics) and Dual (ScFv)2-Fab (National Research Center for Antibody Medicine-China).

[0040] Examples of Fab fusion bispecific antibodies include, but are not limited to, F(ab)2 (Medarex / AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock® (DNL) (ImmunoMedics), bivalent bispecific antibodies (Biotecnol) and Fab-Fv (UCB-Celltech).

[0041] Examples of ScFv-based antibodies, bispecific antibody-based antibodies and domain antibodies include bispecific T cell engagers (BiTE®) (Micromet, Tandem Bispecific Antibodies (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART™) (MacroGenics), single chain bispecific antibodies (Academic, Lawrence FEBS Lett. 1998 Apr 3;425(3):479-84), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack, WO 2010059315) and COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 Aug;88(6):667-75), dual targeting nanobodies® (Ablynx, Hmila et al., FASEB J. 2010), and dual targeting heavy chain only domain antibodies.

[0042] In the context of an antibody that binds to an antigen, the term "binds" or "specifically binds" refers to the binding of an antibody to a predetermined antigen or target (e.g., human V52 or human EGFR), which binding typically occurs within about 10 -6 M or less, e.g., about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 M or less, about 10 -8 M and under, etc. -7 K below M D Alternatively, K DValues ​​can be determined, for example, using surface plasmon resonance (SPR) technology in a BIAcore T200 or biolayer interferometry (BLI) in an Octet RED96 instrument using the antigen as the ligand and a binding moiety or binding molecule as the analyte. Specific binding means that the antibody has a K that is at least 10-fold lower, e.g., at least 100-fold lower, e.g., at least 1,000-fold lower, e.g., at least 10,000-fold lower, e.g., at least 100,000-fold lower, such as at least 100,000-fold lower, than its affinity for binding to a non-specific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). D The term "binding moiety" or "binding molecule" refers to a binding moiety or molecule that binds to a given antigen with an affinity corresponding to the K D If the K is very low (i.e., the binding moiety or binding molecule is very specific), then the degree to which the affinity is lower can be determined by the K of the binding moiety or binding molecule, such that the degree to which the affinity for the antigen is lower than the affinity for a non-specific antigen can be at least 10,000-fold. D As used herein, the term "K D (M)" refers to the dissociation equilibrium constant of a particular interaction between an antigen and a binding moiety or binding molecule.

[0043] In the context of the present invention, "competition" or "capable of competing" or "competing" refers to any detectably significant decrease in the tendency for a particular binding molecule (e.g., an EGFR antibody) to bind to a particular binding partner (e.g., EGFR) in the presence of another molecule (e.g., a different EGFR antibody) that binds to the binding partner. Typically, competition means at least about a 25% reduction in binding due to the presence of another molecule, e.g., an antibody, as determined, for example, by ELISA analysis or flow cytometry using a significant amount of two or more competing molecules, e.g., antibodies, e.g., at least about a 50% reduction, e.g., at least about a 75% reduction, e.g., at least about a 90% reduction, of binding due to the presence of another molecule, e.g., an antibody. Further methods for determining binding specificity by competitive inhibition can be found, for example, in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley InterScience NY, (1992, 1993), and Muller, Meth. Enzymol. 92, 589-601 (1983)).

[0044] In one embodiment, the antibody of the invention binds to the same epitope on EGFR as antibody 7D12 and / or the same epitope on Vδ2 as antibody 5C8. There are several methods available for mapping antibody epitopes on target antigens known in the art, including but not limited to cross-linking mass spectrometry, which allows identification of peptides that are part of the epitope, and X-ray crystallography, which identifies individual residues on the antigen that form the epitope. Epitope residues can be determined to be all amino acid residues with at least one atom 5 Å or less from the antibody. 5 Å was selected as the epitope cut-off distance to allow for possible water-mediated hydrogen bonds with atoms within the van der Waals radius. Epitope residues can then be determined to be all amino acid residues with at least one atom 8 Å or less. 8 Å or less is selected as the epitope cut-off distance to allow for the extension of arginine amino acids. Cross-linking mass spectrometry begins by conjugating the antibody and antigen with a mass-tagged chemical cross-linker. The presence of the complex is then confirmed using high-mass MALDI detection. After cross-linking chemistry, the Ab / Ag complex is very stable, so many different enzymes and digestion conditions can be applied to the complex to provide many different overlapping peptides. Identification of these peptides is performed using high resolution mass spectrometry and MS / MS techniques. The identity of the cross-linked peptides is determined using mass tags linked to the cross-linking reagents. After MS / MS fragmentation and data analysis, the cross-linked, antigen-derived peptides are part of the epitope, while the antibody-derived peptides are part of the paratope. All residues between the most N-terminal and C-terminal cross-linked residues from each individual cross-linked peptide found are considered to be part of the epitope or paratope. The epitope of antibody 7D12 was determined by X-ray crystallography described in Schmitz et al. (2013) Structure 21:1214 and consists of a flat surface on domain III (residues R353, D355, F357, Q384, N420) that corresponds to the domain III ligand binding site.

[0045] As used herein, the terms "first" and "second" antigen-binding regions do not refer to their orientation / location within the antibody, i.e., they have no meaning with respect to the N-terminus or C-terminus. The terms "first" and "second" simply serve to accurately and consistently refer to two distinct antigen-binding regions in the claims and specification.

[0046] As used herein, "% sequence identity" refers to the number of identical nucleotide or amino acid positions shared by different sequences, taking into account the number of gaps and the length of each gap that must be introduced for optimal alignment (i.e., % identity = number of identical positions / total number of positions x 100). The % identity between two nucleotide or amino acid sequences may be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988), which is incorporated into the ALIGN program (version 2.0) using the PAM120 weight residue table, gap length penalty of 12, and gap penalty of 4.

[0047] Further aspects and embodiments of the invention As described above, in a first aspect, the present invention relates to an antibody comprising a first antigen-binding region capable of binding to human V52, said first antigen-binding region comprising a CDR1 sequence as set forth in SEQ ID NO:1, a CDR2 sequence as set forth in SEQ ID NO:2, and a CDR3 sequence as set forth in SEQ ID NO:3.

[0048] In one embodiment, X1 of SEQ ID NO: 1 is S (Ser). In another embodiment, X1 of SEQ ID NO: 1 is G (Gly).

[0049] In one embodiment, X2 of SEQ ID NO:3 is F (Phe). In another embodiment, X2 of SEQ ID NO:3 is S (Ser).

[0050] In one embodiment, X1 of SEQ ID NO: 1 is S (Ser) and X2 of SEQ ID NO: 3 is F (Phe).

[0051] In one embodiment, X1 of SEQ ID NO: 1 is S(Ser) and X2 of SEQ ID NO: 3 is S(Ser).

[0052] In one embodiment, X1 of SEQ ID NO: 1 is G (Gly) and X2 of SEQ ID NO: 3 is F (Phe).

[0053] In one embodiment, X1 of SEQ ID NO: 1 is G (Gly) and X2 of SEQ ID NO: 3 is S (Ser).

[0054] In a preferred embodiment, the antibody is capable of activating human Vy9V52 T cells. Activation of Vy9V52 T cells can be measured by measuring changes in gene expression and / or (surface) marker expression (e.g. activation markers such as CD25, CD69, or CD107a) and / or secreted protein (e.g. cytokine or chemokine) characteristics. In a preferred embodiment, the antibody is capable of inducing activation (e.g. upregulation of CD69 and / or CD25 expression), resulting in degranulation marked by increased CD107a expression and / or cytokine production (e.g. TNF, IFNγ) by Vy9V52 T cells.

[0055] In a further preferred embodiment, the antibody is capable of increasing the number of CD107a positive cells by at least 2-fold, such as at least 5-fold, for example at a concentration of 1 nM, preferably 100 pM, preferably 10 pM, preferably 1 pM, even more preferably 100 fM, when tested as described herein in Example 9. In another preferred embodiment, the antibody of the invention has an EC50 value for increasing the percentage of CD107a positive cells of 100 pM or less, such as 50 pM or less, such as 25 pM or less, such as 20 pM or less, for example 15 pM or less, when tested using Vy9V52 T cells and A431 target cells as described herein in Example 9.

[0056] In one embodiment, the first antigen-binding region is a single domain antibody. Thus, in one embodiment, the antibody of the invention comprises a single domain antibody capable of binding to human V52, wherein said first antigen-binding region comprises the CDR1 sequence set forth in SEQ ID NO:1, the CDR2 sequence set forth in SEQ ID NO:2 and the CDR3 sequence set forth in SEQ ID NO:3.

[0057] In another embodiment, the first antigen-binding region is humanized, and preferably the antigen-binding region comprises the sequence as set forth in SEQ ID NO:4, or · comprising or consisting of a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence set forth in SEQ ID NO:4.

[0058] In one embodiment, X1 of SEQ ID NO:4 is S(Ser). In another embodiment, X1 of SEQ ID NO:4 is G(Gly). In one embodiment, X2 of SEQ ID NO:4 is F(Phe). In another embodiment, X2 of SEQ ID NO:4 is S(Ser). In one embodiment, X1 of SEQ ID NO:4 is S(Ser) and X2 of SEQ ID NO:4 is F(Phe). In one embodiment, X1 of SEQ ID NO:4 is S(Ser) and X2 of SEQ ID NO:4 is S(Ser). In one embodiment, X1 of SEQ ID NO:4 is G(Gly) and X2 of SEQ ID NO:4 is F(Phe). In one embodiment, X1 of SEQ ID NO:4 is G(Gly) and X2 of SEQ ID NO:4 is S(Ser).

[0059] In some embodiments, the antibody of the present invention is a multispecific antibody, such as a bispecific antibody. Thus, in one embodiment, the antibody further comprises a second antigen-binding region. In one embodiment, the second antigen-binding region is a single domain antibody.

[0060] In further embodiments, the antibody is a bispecific antibody, where both the first and second antigen-binding regions are single domain antibodies. In further embodiments, the multispecific antibody is a bispecific antibody, where the first antigen-binding region is a single domain antibody and the second antigen-binding region is a single domain antibody.

[0061] In one embodiment, the antibody of the invention comprises a second antigen-binding region, the second antigen-binding region capable of binding to human EGFR. Bispecific antibodies targeting both Vγ9Vδ2-T cells and EGFR have been shown to induce potent Vγ9Vδ2 T cell activation and tumor cell lysis in both in vitro and in vivo mouse xenograft models (de Bruin et al. (2018) Oncoimmunology 1, e1375641).

[0062] In a further embodiment, the antibody comprises a second antigen-binding region, the second antigen-binding region comprising the CDR1 sequence set forth in SEQ ID NO:5, the CDR2 sequence set forth in SEQ ID NO:6, and the CDR3 sequence set forth in SEQ ID NO:7.

[0063] In one embodiment, the second antigen-binding region is humanized.

[0064] In a further embodiment, the antibody comprises a second antigen-binding region, the second antigen-binding region comprising: the sequence as set forth in SEQ ID NO:8, or comprising or consisting of a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence set forth in SEQ ID NO:8.

[0065] In a further embodiment, the antibody competes (i.e., is capable of competing) with an antibody having the sequence set forth in SEQ ID NO:8 for binding to human EGFR, and preferably the antibody binds to the same epitope on human EGFR as the antibody having the sequence set forth in SEQ ID NO:8.

[0066] In a further embodiment, an antibody of the invention comprises a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region comprises the CDR1 sequence set forth in SEQ ID NO:1, the CDR2 sequence set forth in SEQ ID NO:2 and the CDR3 sequence set forth in SEQ ID NO:3, and wherein the second antigen-binding region comprises the CDR1 sequence set forth in SEQ ID NO:5, the CDR2 sequence set forth in SEQ ID NO:6 and the CDR3 sequence set forth in SEQ ID NO:7.

[0067] In a further embodiment, the antibody of the invention comprises a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region comprises the sequence set forth in SEQ ID NO: 4 and the second antigen-binding region comprises the sequence set forth in SEQ ID NO: 8.

[0068] X1 of SEQ ID NO:4 is S (Ser) and X2 of SEQ ID NO:3 is F (Phe); or

[0069] X1 of SEQ ID NO:4 is S(Ser) and X2 of SEQ ID NO:4 is S(Ser), or

[0070] X1 of SEQ ID NO:4 is G (Gly) and X2 of SEQ ID NO:4 is F (Phe); or

[0071] X1 in SEQ ID NO:4 is G (Gly), and X2 in SEQ ID NO:4 is S (Ser).

[0072] In a further embodiment, the antibody is capable of mediating killing of human EGFR expressing cells. In a preferred embodiment, the antibody is capable of increasing Vy9V52 T cell mediated killing of EGFR expressing cells, such as A431 cells, by at least 50%, such as at least 25%, such as at least 2-fold, when tested as described in Example 9 herein.

[0073] In a further embodiment, the antibody is incapable of mediating killing of EGFR-negative cells, such as EGFR-negative human cells.

[0074] In one embodiment, the antibody comprises a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region and the second antigen-binding region are covalently linked via a peptide linker, e.g., a linker having 1-20 amino acids in length, e.g., 1-10 amino acids in length, e.g., 2, 3, 4, 5, 6, 7, 8 or 10 amino acids in length. In one embodiment, the peptide linker comprises or consists of the sequence GGGGS set forth in SEQ ID NO:9.

[0075] In another embodiment, the antibody comprises a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region capable of binding to human V52 is located C-terminal to the second antigen-binding region capable of binding to human EGFR.

[0076] In one embodiment of the present invention, the antibody further comprises a half-life prolonging domain. In one embodiment, the antibody has a terminal half-life of greater than about 168 hours when administered to a human subject. Most preferably, the terminal half-life is 336 hours or more. As used herein, the "terminal half-life" of an antibody refers to the time it takes for the serum concentration of a polypeptide to decrease by 50% in vivo during the final stage of elimination.

[0077] In one embodiment, the antibody further comprises a half-life prolonging domain, the half-life prolonging domain being an Fc region. In a further embodiment, the antibody is a multispecific antibody, such as a bispecific antibody comprising an Fc region. Various methods for making bispecific antibodies have been described in the art, for example as reviewed by Brinkmann and Kontermann (2017) MAbs 9:182. In one embodiment of the invention, the Fc region is a heterodimer comprising two Fc polypeptides, a first antigen-binding region is fused to the first Fc polypeptide, a second antigen-binding region is fused to the second Fc polypeptide, and the first and second Fc polypeptides comprise asymmetric amino acid mutations that favor the formation of heterodimers over the formation of homodimers (see, for example, Ridgway et al. (1996) 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng 9:617). In further embodiments herein, the CH3 regions of the Fc polypeptides comprise the asymmetric amino acid mutations, preferably the first Fc polypeptide comprises a T366W substitution and the second Fc polypeptide comprises a T366S, L368A and Y407V substitution, or vice versa, and the amino acid positions correspond to human IgG1 according to the EU numbering system. In further embodiments, the cysteine ​​residue at position 220 of the first and second Fc polypeptides is deleted or substituted, and the amino acid positions correspond to human IgG1 according to the EU numbering system. In further embodiments, the region comprises a hinge sequence set forth in SEQ ID NO: 10.

[0078] In some embodiments, the first and / or second Fc polypeptide contains a mutation that renders the antibody inactive, i.e., unable to mediate effector function or has reduced ability to mediate effector function. In one embodiment, the inactive Fc region is further unable to bind C1q. In one embodiment, the first and second Fc polypeptides contain a mutation at position 234 and / or 235, preferably the first and second Fc polypeptides contain L234F and L235E substitutions, the amino acid positions corresponding to human IgG1 according to the EU numbering system. In another embodiment, the antibody contains a L234A mutation, a L235A mutation, and a P329G mutation. In another embodiment, the antibody contains a L234F mutation, a L235E mutation, and a D265A mutation.

[0079] In a preferred embodiment, the first antigen-binding region comprises the sequence set forth in SEQ ID NO:4, and the second antigen-binding region comprises the sequence set forth in SEQ ID NO:8; - the first Fc polypeptide comprises the sequence set forth in SEQ ID NO: 11 and the second Fc polypeptide comprises the sequence set forth in SEQ ID NO: 12, or - the first Fc polypeptide comprises the sequence set forth in SEQ ID NO:11 and the second Fc polypeptide comprises the sequence set forth in SEQ ID NO:12.

[0080] In a further preferred embodiment, the antibody comprises or consists of the sequences set forth in SEQ ID NO:16 and SEQ ID NO:17.

[0081] In a further preferred embodiment, the antibody comprises or consists of the sequences set forth in SEQ ID NO:16 and SEQ ID NO:18.

[0082] In a further main aspect, the present invention relates to a pharmaceutical composition comprising an antibody according to the invention as described herein and a pharma- ceutically acceptable excipient.

[0083] The antibody can be formulated with pharma- ceutically acceptable excipients according to conventional techniques, such as those disclosed in Rowe et al. 2012 Handbook of Pharmaceutical Excipients, ISBN 9780857110275. The pharma-ceutically acceptable excipients and any other carriers, diluents or adjuvants should be suitable for the antibody and the selected mode of administration. The suitability of excipients and other components of the pharmaceutical composition is determined based on the lack of significant negative effect on the desired biological properties of the selected antibody or pharmaceutical composition of the present invention (e.g., less than substantial effect on antigen binding (relative inhibition of 10% or less, relative inhibition of 5% or less, etc.)).

[0084] Pharmaceutical compositions may include diluents, fillers, salts, buffers, surfactants (e.g., non-ionic surfactants such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizing agents, and / or other materials suitable for inclusion in a pharmaceutical composition. Additional pharma- ceutically acceptable excipients include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption delaying agents, etc. that are physiologically compatible with the antibodies of the invention.

[0085] In a further main aspect, the present invention relates to an antibody according to the invention as described herein for use as a medicament.

[0086] The antibody according to the invention makes it possible to generate a microenvironment favourable for the killing of tumour cells by Vy9V52 T cells.Thus, in a preferred embodiment, the antibody is for use in the treatment of cancer.

[0087] In one embodiment, the antibody is for use in treating primary or metastatic colon or colorectal cancer. In another embodiment, the antibody is for use in treating cancer of the peritoneum. In another embodiment, the antibody is for use in treating liver cancer. In another embodiment, the antibody is for use in treating head and neck squamous cell carcinoma (HNSCC). In another embodiment, the antibody is for use in treating non-small cell lung cancer (NSCLC). In another embodiment, the antibody is for use in treating squamous cell carcinoma of the skin.

[0088] Likewise, the present invention relates to a method of treating a disease comprising the administration of a multispecific antibody according to the invention as described herein to a human subject in need thereof, in one embodiment the disease is cancer.

[0089] In some embodiments, the antibody is administered as a monotherapy. However, the antibody of the invention may also be administered as a combination therapy, i.e., in combination with other therapeutic agents relevant to the disease or condition being treated.

[0090] "Treatment" or "treating" means administering an effective amount of an antibody according to the invention for the purpose of alleviating, ameliorating, halting, eradicating (curing) or preventing symptoms or conditions. An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. An effective amount of a polypeptide, such as an antibody, may vary depending on factors such as the stage of the disease, age, sex, and weight of the individual, as well as the ability of the antibody to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the antibody are outweighed by the therapeutically beneficial effects. Administration may be by any suitable route, but is typically parenteral, such as intravenous, intramuscular, or subcutaneous.

[0091] The multispecific antibodies of the present invention are typically produced recombinantly, i.e., by expressing a nucleic acid construct encoding the antibody in a suitable host cell, followed by generating the recombinant antibody produced from the cell culture. The nucleic acid construct can be produced by standard molecular biology techniques well known in the art. The construct is typically introduced into the host cell using an expression vector. Suitable nucleic acid constructs and expression vectors are known in the art. Host cells suitable for recombinant expression of antibodies are well known in the art and include CHO, HEK-293, Expi293F, PER-C6, NS / 0 and Sp2 / 0 cells.

[0092] Thus, in a further aspect, the present invention relates to a nucleic acid construct encoding an antibody according to the invention. In one embodiment, the construct is a DNA construct. In another embodiment, the construct is an RNA construct.

[0093] In a further aspect, the present invention relates to an expression vector comprising a nucleic acid construct encoding the antibody according to the invention.

[0094] In a further aspect, the present invention relates to a host cell comprising one or more nucleic acid constructs encoding an antibody according to the invention, or an expression vector comprising a nucleic acid construct encoding an antibody according to the invention.

[0095] In a further aspect, the invention relates to a process for producing an antibody of the invention comprising expressing in a host cell one or more nucleic acids encoding an antibody according to the invention.

[0096] In a further aspect, the invention relates to a process for producing a clinical batch of an antibody of the invention comprising expressing in a host cell one or more nucleic acids encoding an antibody according to the invention. As used herein, "clinical batch" refers to a product composition suitable for use in humans.

[0097] In a further aspect, the invention relates to a process for producing an antibody that has no tyrosine sulfation comprising expressing in a host cell one or more nucleic acids encoding an antibody according to the invention.

[0098] In a further aspect, the invention relates to a process for avoiding tyrosine sulfation of an antibody capable of activating human Vy9V52 T cells, said process comprising constructing a nucleic acid encoding an antibody of the invention and producing said antibody by expression of said nucleic acid in a host cell.

[0099] In a further aspect, the invention relates to a process for generating a homogenous antibody preparation of an antibody capable of activating human Vy9V52 T cells, said process comprising constructing a nucleic acid encoding an antibody of the invention and producing said antibody by expression of said nucleic acid in a host cell.

[0100] In one embodiment, the host cell in the above manufacturing process is a mammalian cell, such as a CHO cell or a HEK cell, or a yeast cell, such as a Pichia pastoris cell. [Table 4-1] [Table 4-2] [Table 4-3]

[0101] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the mention of any references, articles, publications, patents, patent publications, and patent applications herein is not and should not be construed as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country throughout the world. EXAMPLES

[0102] Example 1 | Production and purification of VHH compounds VHH compounds were primarily produced by transient transfection of the encoding plasmid in HEK293-E 253 cells and purification of the protein from conditioned medium (after 1 week of production) by protein A affinity chromatography followed by preparative gel filtration. The dominant monomeric peak (fractions 1E11-1G2) observed by preparative size exclusion using a Superdex-75 column was purified: Figure 1.

[0103] The purified protein was shown to migrate as a single band under reducing and non-reducing conditions in polyacrylamide gel electrophoresis: Figure 2 shows a representative example.

[0104] Example 2 | HP-SEC analysis of purified VHH compounds A Waters Acquity ARC-bio system was used for HP-SEC analysis of purified VHH compounds. 10ug of antibody (10ug of antibody at a concentration of 1mg / mL) was injected onto a Waters BEH200 SEC column (bead size 2.5μm, column dimensions 7.8×300mm). The mobile phase consisted of 50mM sodium phosphate, 0.2M sodium chloride buffer pH 7.0, with a buffer rate of 0.8mL / min used for the run. Proteins were detected by measuring the absorbance at a wavelength of 214nm. Total analysis time was 15min per injection. VH compounds were produced and purified as described in Example 1. Surprisingly, when VHH 5C8 (SEQ ID NO: 13) (previously described in WO2015156673) and 5C8var1 (SEQ ID NO: 14) (previously described in WO2020060405) were tested for integrity and monomericity, two peaks were observed: FIG. 3.

[0105] Example 3 | Mass analysis of 5C8 revealed an additional 80 Da mass To determine whether the two isoforms of 5C8 differed in mass, a protein preparation of 5C8 was analyzed by LC-ESI-MS mass spectrometry. The major species found in this analysis was 5C8 without post-translational modifications or signal peptide. The second species was a protein with a mass difference of +80.3 Daltons (Da), indicating possible sulfation or phosphorylation. This was further investigated by treatment with phosphatase or sulfatase and subsequent LC-ESI-MS mass spectrometry of peptides after protein digestion. Sulfatase treatment reduced the mass of a peptide containing Y105, the seventh residue of CDR3 (SEQ ID NO: 15), by 80 Da, whereas phosphatase treatment had no effect. This demonstrates that Y105 in 5C8 is post-translationally modified by sulfation. This sulfation was present in approximately 30% of the protein preparations.

[0106] Example 4 | 1D12var5-5C8var1 containing the same anti-Vy9V52 VHH show the same heterogeneity in HP-SEC and the same additional mass of 80 Da. 1D12var5-5C8var1 is a bispecific VH compound composed of an anti-CD1d VHH linked to 5C8var1 via a flexible linker (described in SEQ ID NO: 87 of WO2020060405). The protein was expressed in HEK293E cells as described above. In addition, protein preparations were obtained from different expression systems and the bispecific VHH was also expressed in Pichia pastoris and Chinese Hamster Ovary (CHO) cells. When the different protein preparations were tested in HP-SEC analysis, a pre-peak was systematically observed: Figure 4.

[0107] The observed pre-peak again indicates a significant percentage of the protein that is a different isoform. Since 5C8 VH is sulfated and 5C8var1 contains exactly the same CDR3 sequence, 1D12var5-5C8var1 batches were also analyzed by mass spectrometry for their molecular weight. Depending on the protein batch, 15%-40% were found to contain an additional 80 Da mass. This is consistent with the sulfation observed for VHH 5C8.

[0108] Example 5 | In silico analysis of VHH 5C8 and 5C8var1 Homology models of 5C8 and 5C8var1 were constructed using Maestro (Schrodinger) based on PDB ID 5M2W. CDR1 and CDR3 required refinement by de novo loop prediction using Prime (Schrodinger). The generated model showed that CDR3 residue Y105 was located at 205.1 Å in the 5C8var1 model. 2 and 122.2 Å in the model of 5C8. 2 We show that the β-amino acid sequence of ... [Table 5]

[0109] Example 6 | Design and generation of 5C8var1 VH CDR3 mutants Y105F and Y105S Using the homology model described in Example 5, mutations were introduced to prevent sulfation of Y105 in 5C8 and 5C8var1. Two different mutants were designed based on the model structure of VHH: Y105S (retaining the alcohol function) and Y105F (retaining the aromatic ring). Residue Y105 is the seventh residue of CDR3, and by introducing the mutation, the effect on binding was predicted. As described above, both mutations were designed in the humanized VH sequence 5C8var1, and both proteins were produced in HEK293E cells and purified. The CDR3 amino acid sequence of the humanized VHH remained identical to that of the non-humanized VHH.

[0110] Both 5C8var1-Y105F and 5C8var1-Y105S were successfully purified and appeared as monomeric proteins in preparative size exclusion (data not shown). Both proteins were highly pure (Figure 5) and migrated as single species in polyacrylamide gel electrophoresis.

[0111] Example 7 | HP-SEC analysis of purified VHHs containing designed CDR3 mutations HP-SEC analysis was performed as described for 5C8. Both 5C8var1-Y105F as well as 5C8var1-Y105S were analyzed (Figure 6).

[0112] As can be concluded from the HP-SEC analysis of purified VHH molecules containing the designed CDR3 mutations, no heterogeneity was observed any more for any of the mutants, indicating that the observed post-translational modification Y105 is absent and the protein is homogeneous.

[0113] Example 8 | Affinity measurements of 5C8var1, 5C8var1-Y105F and 5C8va1-Y105S using Biolayer Interferometry (BLI) show no difference in affinity Binding of 5C8var1 VH antibody fragment and variants 5C8var1-Y105F and 5C8var1-Y105S to the Vy9Vδ2 TCR was measured by biolayer interferometry using an Octet RED96 instrument (ForteBio). Recombinant human Vy9Vδ2-Fc fusion protein (20 μg / ml) was captured as ligand on an anti-human Fc capture biosensor. Sensorgrams were recorded when the ligand-captured biosensor was incubated with serially diluted VHH antibody fragments (40-0.63 nM) in 10x kinetic buffer (ForteBio). A global data fit to a 1:1 binding model was used to determine the k on (association rate constant) and koff Using these values, KD = k off / k on The equilibrium dissociation constant (KD) was calculated using the formula:

[0114] As can be concluded from Figure 7 and Table 3, the KD values ​​found for the two different Y105 VHH mutants were not substantially different from those found for 5C8var1. In particular, the Y105F mutant had an affinity similar to that found for 5C8var1. [Table 6]

[0115] Example 9 | Fully retained functionality of anti-(EGFR x Vy9V52 TCR) bispecific VHH containing the Y105F mutation To determine whether the equal affinity of VHH 5C8var1-Y105F compared to 5C8var1 translates to comparable functionality, a bispecific VHH 7D12var8-5C8var1-Y105F was designed, in which the humanized anti-EGFR VHH 7D12var8 (based on the VHH described in Gainkam et al. (2008) J Nucl Med 49(5):788) was linked to the 5C8var1-Y105F VHH via a G4S linker to form 7D12var8-5C8var1-Y105F. The two VHH molecules were separated by a flexible G4S linker sequence. This molecule was produced and purified as described above and then tested for its ability to induce Vy9V52 T cell activation and cause T cell-mediated tumor cell lysis in a dependent EGFR positive tumor cell line (A431). Briefly, Vγ9Vδ2 T cells were isolated from the blood of healthy donors using magnetic activated cell sorting (MACS) in combination with anti-Vδ2 antibodies according to standard procedures. These cells were then expanded for one week using a mixture of cytokines and irradiated feeder cells, namely a mixture of JY cell line and PBMCs from different donors. Vγ9Vδ2 T cells were always >90% pure (stained double positive for Vγ9 and Vδ2 in FACS) when used in the assay. A431 cell line (ATCC, Cat. No. CRL-1555) was cultured according to the supplier's recommendations. For activation or cytotoxicity assays, 50,000 tumor target cells were seeded in 96-well tissue culture plates the day before the assay. The next day, 50,000 expanded and purified Vγ9Vδ2 T cells were added to the medium together with a range of concentrations of bispecific VHH compounds. For activation assays, Vy9Vδ2-T cell degranulation was assessed using a mixture of labeled anti-CD3 and anti-CD107A antibodies added to the mixture. After 4 hours, cells were harvested, washed and analyzed by FACS for expression of the degranulation marker CD107A. For cytotoxicity assays, the supernatants of the co-cultures were examined the next day for the presence of proteolytic enzymes (indicative of cell death) using the CytoTox-Glo Cytotoxicity Assay Kit: (Promega G9290).Detergent-mediated cell lysis was used to set 100% killing at the end of the assay. Figure 8 shows the data.

[0116] Figure 8 shows that 7D12var8-5C8var1-Y105F, as well as non-humanized 7D12-5C8, induced potent Vγ9Vδ2 T cell activation and tumor cell lysis. These results are consistent with the potency of the non-humanized "precursor" molecule without the Y105 mutation 7D12wt-5C8. Table 4 shows the EC50 values ​​obtained after curve fitting. 7D12var8-5C8var1-Y105F had a slightly lower EC50 in the cytotoxicity assay compared to 7D12-5C8. [Table 7]

[0117] The maximal level of tumor cell killing was slightly lower for 7D12var8-5C8var1-Y105F compared to that observed for 7D12-5C8, however these were two different measurements using two different Vy9V52 T cell donors, and this maximal level of cytotoxicity may be particularly donor dependent.

[0118] Example 10 | Temperature stability of VHH 5C8var1 containing the Y105 mutation was not altered To determine whether the mutations introduced in the different variants affected the thermal stability of the VHH folding, the melting temperatures of the mutants were measured using NanoDSF (differential scanning fluorimetry). The antibody samples were diluted using PBS until they were equal to the lowest concentration sample. The antibody samples were then filled into nanoDSF grade capillaries and measured with a Prometheus NT.48. During the experiment, the temperature was increased from 20 to 95 °C. The intrinsic fluorescence of the protein was detected at 350 and 330 nm and recorded together with the amount of reflected light. From these measurements, the apparent melting temperature (Tm) and the onset of aggregation (Tagg) were determined. For all three antibody fragments, the onset melting temperature (T) at which the VHH was fully unfolded was determined. on) and melting temperatures (Tm) were reported (Table 4). The melting temperatures measured for 5C8var1-Y105F and 5C8var1-Y105S were consistent with the melting temperature of 5C8var1: Table 4. [Table 8]

[0119] Example 11 | Half-life extended (Fc-containing) bispecific constructs To obtain a molecule with a longer in vivo plasma half-life, the 7D12var8-5C8var1-Y105F bispecific VHH was reformatted into a therapeutic antibody format containing a human Fc. Both VHH domains were linked to a human IgG1 Fc (i.e., CH2 and CH3) domain with the following characteristics: the VH domain was linked to a modified hinge (AAA followed by SDKTHTCPPCP, with cysteine ​​220 deleted) and human CH2 and CH3 domains. The CH2 domain was Fc-silenced by the LFLE mutation pair (L234F, L235E) and the CH3 domain was mutagenized with a "knob-into-hole" mutant (knob: T366W and hole: T366S, L368A and Y407V) that heterodimerizes upon co-expression of the two chains in the same cell. This mutation pair has been described in the scientific literature (Ridgway et al. (1996) Protein Eng 9:617). The sequences of the constructs are set forth in SEQ ID NO:16 and SEQ ID NO:17. The resulting antibody construct 7D12var8-5C8var1(Y105F) with Fc region was designated 7D12var8-5C8var1(Y105F)-Fc. Similarly, a construct was prepared in which Y at position 105 was replaced with S (7D12var8-5C8var1(Y105S)-Fc). The sequences of the constructs are set forth in SEQ ID NO:16 and SEQ ID NO:18.

[0120] The proteins were produced via co-transfection of the two encoding expression vectors in HEK293E cells and purification from culture supernatants by protein A affinity chromatography followed by preparative size-exclusion chromatography as described in Example 1. This resulted in a highly monomeric protein preparation.

[0121] Example 12 | Binding of 7D12var8-5C8var1(Y105F)-Fc to primary Vγ9Vδ2 T cells isolated from healthy human PBMCs To demonstrate binding of 7D12var8-5C8var1(Y105F)-Fc to the Vγ9Vδ2 T cell receptor (TCR), human Vγ9Vδ2 T cells were isolated from healthy peripheral blood mononuclear cells (PBMCs) by magnetic activated cell sorting (MACS) and then expanded as described (de Bruin et al., Clin. Immunology 169(2016), 128-138; de Bruin et al., J. Immunology 198(1)(2017), 308-317). Expanded polyclonal and pure (>95%) Vy9V52 T cells were then seeded at a density of 50000 cells / well and incubated for 1 h at 4°C with either 7D12var8-5C8var1(Y105F)-Fc antibody or GP120-5C8var1(Y105F)-Fc antibody as a positive control in a half-log titration starting at 100 nM. Binding of antibodies to Vy9V52 TCR was visualized by flow cytometry using a fluorescently labeled secondary anti-IgG1 antibody. Figure 9 shows the mean fluorescence intensity (MFI) signal of anti-IgG1 antibody staining measured by flow cytometry for two different PBMC donors (D336 and D339). The sigmoidal curve highlights significant binding of 7D12var8-5C8var1(Y105F)-Fc to Vγ9Vδ2 T cells, with a half-maximal effective concentration (EC50) in the low nanomolar range (approximately 3 nM).

[0122] Example 13 | Binding of 7D12var8-5C8var1(Y105F)-Fc to EGFR-positive tumor cells by cell-based ELISA The binding of 7D12var8-5C8var1(Y105F)-Fc to epidermal growth factor receptor (EGFR) was tested in a cell-based enzyme-linked immunosorbent assay (ELISA) using the EGFR-expressing tumor cell lines A-431, HCT-116 and HT-29. For this purpose, tumor cells were first seeded at different concentrations on day -1, reaching a concentration of approximately 50000 cells / well on day 0. On day 0, half-log titrations of 7D12var8-5C8var1(Y105F)-Fc antibody or GP120-5C8var1(Y105F)-Fc antibody as a negative control, starting at 100 nM, were added for 1 h at 37°C. The bound antibodies were then labeled in a second incubation step with anti-IgG1-HRP for 1 h at 37°C. Secondary antibody binding was then disrupted by the addition of 3,3',5,5'-tetramethylbenzidine, a colorimetric change was induced by HRP, and the reaction was subsequently stopped by the addition of H2SO4. Optical density (OD) was then measured in a UV spectrometer at a wavelength of 450 nm. Figure 10 shows that 7D12var8-5C8var1(Y105F)-Fc strongly binds to A-431, HCT-116 and HT-29 tumor cells with an EC50 of approximately 7 nM, whereas the non-targeting control antibody did not measurably bind to any of the cell lines tested.

[0123] Example 14 | A-431 cell-dependent degranulation of Vγ9Vδ2 T cells induced by 7D12var8-5C8var1(Y105F)-Fc To examine the ability of 7D12var8-5C8var1(Y105F)-Fc to activate Vy9V52 T cells, Vy9V52 T cells were first isolated and expanded as described above. Vy9V52 T cells were then cultured with A-431 tumor cells at a 1:1 E:T ratio in the presence of different concentrations of 7D12var8-5C8var1(Y105F)-Fc antibody and PE-labeled anti-CD107a fluorescent antibody. After 24 hours, cells were harvested and stained with fluorescently labeled anti-Vy9 and anti-CD3 antibodies to distinguish Vy9V52 T cells from tumor cells. Flow cytometry was used to examine the extent of CD107a expression on Vy9V52 T cells, which reflects target-dependent degranulation. Figure 11 shows that increasing concentrations of 7D12var8-5C8var1(Y105F)-Fc efficiently induced Vγ9Vδ2 T cells to degranulate in response to A-431 cells. The EC50 for Vγ9Vδ2 T cell degranulation induced by 7D12var8-5C8var1(Y105F)-Fc is in the picomolar range (approximately 40-90 pM).

[0124] Example 15 | Antibody 7D12-5C8 induces T cell-mediated target cytotoxicity To investigate whether the bispecific VHH 7D12-5C8 is efficient in inducing Vγ9Vδ2 T cell-mediated cytotoxicity against target cells, the viability of A-388 epidermal tumor cell line (ATCC, CRL-7905) was evaluated in a co-culture setting with Vγ9Vδ2 T cells and bsVHH antibody fragments. The assay used Vγ9Vδ2 T cells isolated from healthy PBMCs as previously described, but subsequently frozen and stored at -150°C. Frozen Vγ9Vδ2 T cells were thawed and rested overnight in IL-2-supplemented medium. A-388 tumor cells were seeded alone or together with resting Vγ9Vδ2 T cells at a ratio of 1:1 or 1:0.1, with or without 7D12-5C8 (10 nM). As a further control, Vy9V52 T cells were seeded alone with or without antibody 7D12-5C8 (10 nM). After 72 hours, cell viability was determined by addition of ATP Lite (Perkin Elmer, 6016731) and reading the luminescence signal with a microplate reader. Figure 12 shows the ATP-derived fluorescent signal, which represents the metabolic activity of live cells and thus the number of live cells. It can be observed that at an E:T ratio of 1:1, the antibody induces a reduction of viable cells by about 50%, while untreated co-cultures of A-388 and Vy9V52 T cells are unaffected, highlighting its ability to induce T cell-mediated cytotoxicity.

[0125] Example 16 | Tumor cell killing by 7D12-5C8 and 7D12-5C8var1(Y105S)-Fc-activated Vγ9Vδ2 T cells To investigate whether bsVHH 7D12-5C8 and antibody 7D12-5C8var1(Y105S)-Fc were able to induce Vy9V52 T cell-mediated cytotoxicity against patient-derived tumor cells, the viability of such tumor cells was assessed in a co-culture setting with Vy9V52 T cells and antibodies. A variety of different types of tumor cells were tested.

[0126] Tissue samples (i.e. primary and metastatic tumor material from colon, peritoneum and liver, head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC)) were collected from cancer patients at Amsterdam UMC (location VUmc) after written informed consent. Tissues were cut into small pieces using a surgical blade (size No. 22; Swann Morton Ltd), resuspended in dissociation medium consisting of IMDM supplemented with 0.1% DNAse I, 0.14% collagen A, 5% FCS, 100 IU / ml sodium penicillin, 100 μg / ml streptomycin sulfate and 2.0 mM L-glutamine, transferred to a sterile flask containing a stir bar and incubated on a magnetic stirrer for 45 min in a 37°C water bath. After incubation, the cell suspension was passed through a 100 μM cell strainer. Tumors were dissociated three times and then cells were washed for viable cell counts using trypan blue exclusion. Dissociated patient-derived tumor cells were incubated with healthy donor-derived Vy9V52 T cells (1:1 E:T ratio) in the presence or absence of 50 nM 7D12-5C8 for 4 h or in the presence or absence of 7D12-5C8var1(Y105S)-Fc or gp120-5C8var1(Y105S)-Fc for 24 h.

[0127] Adherent cells were detached after the incubation period using trypsin-EDTA, if necessary, resuspended in FACS buffer (PBS supplemented with 0.5% bovine serum albumin and 20 μg / ml NaN3) and incubated with fluorochrome-conjugated antibodies for 30 min at 4°C, after which staining was measured by flow cytometry using an LSR Fortessa XL-20 (BD).

[0128] Live cells were identified using the live / dead marker 7AAD in combination with 123 counting eBeads™ according to the manufacturer's instructions. Flow cytometry data were analyzed using Kaluza Analysis Version 1.3 (Beckman Coulter) and FlowJo Versions 10.6.1 and 10.7.2 (Becton Dickinson).

[0129] 7D12-5C8 and 7D12-5C8var1(Y105S)-Fc-induced Vγ9Vδ2 T cell-mediated cytotoxicity of tumor cells was assessed by incubating expanded healthy donor-derived Vγ9Vδ2 T cells with single cell suspensions of various malignancies (primary CRC, peritoneal and liver, CRC metastases in head and neck squamous cell carcinoma and non-small cell lung cancer).

[0130] As shown in FIG. 13, 7D12-5C8 induced substantial lysis of patient tumor cells by Vy9V52 T cells (mean % of lysis induced by 7D12-5C8: CRC primary 52.3% and p-value 0.0003, CRC peritoneum 46.0% and p-value 0.0052, CRC liver 31.8% and p-value 0.0360, head and neck squamous cell carcinoma 46.1% and p-value 0.0187 and non-small cell lung carcinoma 64.1% and p-value 0.0153).

[0131] Furthermore, as shown in Figure 14, 7D12-5C8var1(Y105S)-Fc induced a significant amount of lysis of patient tumor cells by Vy9V52 T cells (mean % of lysis induced by 7D12-5C8var1(Y105S)-Fc: 71.2% and p<0.0001 and 0.0012). The control compound gp120-5C8var1(Y105S)-Fc also did not induce any measurable tumor cell lysis.

[0132] Example 17 | Design, generation and purification of constructs for non-human primate studies For in vivo studies in non-human primates, a construct was generated with a binding domain that cross-reacts with the cynomolgus Vγ9 TCR chain (FIG. 15). This binding domain was based on the antibody 7A5, a TCR Vγ9-specific antibody (Janssen et al., J. Immunology 146(1)(1991), 35-39). Antibodies based on 7A5 have been found to bind to cynomolgus Vγ9Vδ2 T cells (see Example 1 of WO2021052995). A bispecific Fc-containing antibody was constructed that includes 7A5 and anti-EGFR VHH 7D12var8. The molecule contained a human IgG1 Fc tail engineered for heterodimerization using knob-in-hole technology (KiH; Carter et al., 2001 Imm. Meth. 2001:248, 7; knob: T366W; hole: T366S, L368A and Y407V). A Vγ9-binding scFv of the 7A5 antibody was attached to the "knob" chain and the EGFR-binding VHH 7D12var8 was cloned in-frame with the "hole" chain of the KiH Fc pair. In addition, the upper hinge was engineered to "AAASDKTHTCPPCP" to remove the cysteine ​​(C220) that normally bridges to CL and to change "EPK" to "AAA" to provide further flexibility. The N-terminal portion of CH2 was engineered to disrupt Fc receptor (CD16, 32 and 64) interactions (silencing mutations L234F, L235E) while maintaining FcRN binding. The resulting construct was designated 7A5-7D12var8-Fc.

[0133] The molecules were produced by transient co-transfection of two plasmids encoding the two different chains in HEK293E cells and purified from culture supernatants by Protein A affinity chromatography followed by preparative size-exclusion chromatography (Example 1). The molecules were shown to bind to either target with an apparent affinity of about 3 nanomolar (nM) using ELISA and recombinant forms of both antigens (Figure 16). The functionality of the molecules was demonstrated by showing that they triggered target-dependent activation (CD107a expression) of in vitro expanded Vy9V52 T cells and subsequent T cell-mediated tumor cell lysis (Figure 17).

[0134] Example 18 | Bispecific antibody 7A5-7D12var8-Fc was well tolerated in an exploratory multiple-dose non-human primate (NHP: cynomolgus monkey) study

[0135] In a multiple-dose exploratory NHP study, three female cynomolgus monkeys were administered 7A5-7D12var8-Fc at doses of 1 mg / kg, 5 mg / kg, and 23 mg / kg, respectively. The antibody was administered as a 30-minute infusion at 5 mL / kg, with infusions every 4 weeks. The first two dose groups (one animal per dose) at 1 and 5 mg / kg were administered simultaneously, and after three doses (once per week), the third dose group (23 mg / kg) received the first dose. Blood was collected periodically from the animals for PK analysis, analysis of clinical chemistry parameters, measurement of cytokine levels, and analysis of blood cell subsets by flow cytometry. One day after the final dose, the animals were euthanized and tissues were harvested and prepared for histopathology and immunohistochemistry (IHC).

[0136] Pharmacokinetic analysis of 7A5-7D12var8-Fc concentrations in the blood of treated animals (measured by ELISA, FIG. 18) revealed that the antibody exhibited IgG-like PK with a half-life ranging from 84 to 127 hours. In animals dosed at 1 mg / kg, the antibody had a short half-life after the third injection, likely due to a possible anti-drug antibody (ADA) response in the animals.

[0137] The clearance values ​​obtained were 0.36-0.72 mL / h / kg, and the volume of distribution was 58.5-115.2 mL / kg. Systemic exposure increased dose-proportionally between 1 and 23 mg / kg. However, no accumulation was observed after repeated dosing.

[0138] The compound could be detected by IHC in different tissues (lymph node, muscle, skin and colon) and, as expected, there was a dose-proportional intensity of compound staining in these tissues (data not shown). Flow cytometry analysis of blood cells showed some transient reduction in lymphocytes (Figure 19) related to treatment, as is often observed in these types of multiple dose studies. Figure 19 shows a transient reduction in T cell counts at each time point 2 hours after dosing. However, T cell counts returned to baseline levels 2 days after injection.

[0139] In contrast, Vγ9 positive T cells were reduced in number in peripheral blood and did not regain their previous frequency. These cells were almost absent during the course of the study, indicating a specific pharmacodynamic effect of the compound. Measurement of cytokines in the blood of treated animals showed that the treatment caused little cytokine release, which was almost exclusively limited to the first injection of the compound. Figure 20 shows the measured IL-6 levels as an example.

[0140] As a general conclusion, treatment of NHPs with 7A5-7D12var8-Fc was very well tolerated and did not show clinical signs of toxicity. Furthermore, no gross or microscopic abnormalities were observed in the examined organs by histopathology (data presented here). In comparison, anti-EGFRxCD3 BiTE was lethal to NHPs at a dose of 31 μg / kg / day infusion (Lutterbuese et al., Proc Natl Acad Sci US 2010:107(28),12605).

Claims

1. An antibody comprising a first antigen-binding region capable of binding to human EGFR, wherein the antigen-binding region comprises or consists of a sequence set forth in SEQ ID NO:8 or a sequence having at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% sequence identity to the sequence set forth in SEQ ID NO:

8.

2. The antibody of claim 1 , wherein the first antigen-binding region is a single domain antibody.

3. The antibody of claim 1 or 2, wherein the antibody is a bispecific antibody.

4. A pharmaceutical composition comprising the antibody of any one of claims 1 to 3 and a pharma- ceutically acceptable excipient.

5. A nucleic acid construct encoding the antibody of any one of claims 1 to 3, an expression vector comprising said nucleic acid, or a host cell comprising one or more nucleic acid constructs encoding the antibody of any one of claims 1 to 3.

6. A method for producing an antibody described in any one of claims 1 to 3, the method comprising expressing one or more nucleic acids encoding the antibody in a host cell.

7. The antibody of claim 1, wherein the antibody is a bispecific antibody. the first antigen-binding region comprises a complementarity determining region (CDR) 1 sequence set forth in SEQ ID NO:5, a CDR2 sequence set forth in SEQ ID NO:6, and a CDR3 sequence set forth in SEQ ID NO:7; the second antigen-binding region binds to human V52, the second binding region comprising the CDR1 sequence set forth in SEQ ID NO:1, the CDR2 sequence set forth in SEQ ID NO:2 and the CDR3 sequence set forth in SEQ ID NO:3; The antibody according to any one of claims 1 to 3.

8. - X1 of SEQ ID NO: 1 is S (Ser) and X2 of SEQ ID NO: 3 is F (Phe), or - The bispecific antibody described in claim 7, in which X1 of SEQ ID NO: 1 is S (Ser) and X2 of SEQ ID NO: 3 is S (Ser).

9. The bispecific antibody according to claim 7 or 8, wherein X 1 in SEQ ID NO: 1 is S (Ser) and X 2 in SEQ ID NO: 3 is F (Phe).

10. The bispecific antibody of claim 7, wherein the second antigen-binding region is a single domain antibody.

11. A bispecific antibody according to any one of claims 7 to 10, wherein the first antigen-binding region comprises or consists of a sequence as set forth in SEQ ID NO:4 or a sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% sequence identity to the sequence as set forth in SEQ ID NO:

4.

12. The bispecific antibody of any one of claims 7 to 11, wherein the antibody is capable of mediating the killing of human EGFR-expressing cells.

13. The bispecific antibody of claim 7, wherein the antibody further comprises a half-life prolonging domain, e.g., an Fc region.

14. The bispecific antibody of claim 12 or 13, wherein the Fc region is a heterodimer comprising two Fc polypeptides, the first antigen-binding region being fused to a first Fc polypeptide and the second antigen-binding region being fused to a second Fc polypeptide, and the first and second Fc polypeptides comprising asymmetric amino acid mutations that favor heterodimer formation over homodimer formation.

15. A bispecific antibody as described in claim 13 or 14, wherein the CH3 regions of the Fc polypeptides comprise the asymmetric amino acid mutations, preferably wherein the first Fc polypeptide comprises a T366W substitution and the second Fc polypeptide comprises T366S, L368A and Y407V substitutions, or vice versa, and wherein the amino acid positions correspond to human IgG1 according to the EU numbering system.

16. A bispecific antibody according to any one of claims 12 to 15, wherein the first and second Fc polypeptides comprise a mutation at position 234 and / or 235, preferably wherein the first and second Fc polypeptides comprise L234F and L235E substitutions, said amino acid positions corresponding to human IgG1 according to the EU numbering system.

17. The antibody comprising a second antigen-binding region, the second antigen-binding region comprising the sequence set forth in SEQ ID NO:4, and the first antigen-binding region comprising the sequence set forth in SEQ ID NO:8; - said first Fc polypeptide comprises the sequence set forth in SEQ ID NO: 11 and said second Fc polypeptide comprises the sequence set forth in SEQ ID NO: 12, or - the first Fc polypeptide comprises the sequence as set forth in SEQ ID NO: 12 and the second Fc polypeptide comprises the sequence as set forth in SEQ ID NO:

11.

18. A bispecific antibody according to any one of claims 12 to 17, wherein the antibody comprises or consists of the sequences set out in SEQ ID NO: 16 and SEQ ID NO: 17, or comprises or consists of the sequences set out in SEQ ID NO: 16 and SEQ ID NO:

18.

19. A pharmaceutical composition comprising the bispecific antibody of any one of claims 7 to 18 and a pharma- ceutically acceptable excipient.

20. Use of a bispecific antibody according to any one of claims 1 to 18 for the manufacture of a medicament, preferably a medicament for use in the treatment of cancer.

21. The use of claim 20, wherein the cancer is selected from rectal cancer, peritoneal cancer, liver cancer, head and neck squamous cell carcinoma, non-small cell lung cancer and squamous cell carcinoma of the skin.

22. A nucleic acid construct encoding a bispecific antibody according to any one of claims 7 to 18, an expression vector comprising said nucleic acid, or a host cell comprising one or more nucleic acid constructs encoding a bispecific antibody according to any one of claims 7 to 18.

23. A method for producing a bispecific antibody according to any one of claims 7 to 18, comprising expressing one or more nucleic acids encoding the bispecific antibody in a host cell.

24. 20. A process for producing an antibody that is free of tyrosine sulfation, comprising expressing one or more nucleic acids encoding a bispecific antibody according to any one of claims 7 to 18 in a host cell, said host cell being preferably a Chinese hamster ovary cell, a human embryonic kidney cell or a Pichia pastoris cell.