COMPOSITIONS COMPRISING ANTIBODIES THAT BIND TO GAMMA-DELTA T-CELL RECEPTORS - Patent application

Abstract

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

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of European Patent Application No. 22179260, filed June 15, 2022, the disclosure of which is incorporated herein by reference in its entirety and for all purposes.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (LVAT_025_01WO_SeqList_ST26.xml, size: 35,054 bytes, created on June 12, 2023) are incorporated herein by reference in their entirety.

[0003] The present invention relates to novel pharmaceutical compositions comprising antibodies capable of binding to the V52 chain of the human Vy9V52 T cell receptor. The present invention further relates to the use of the pharmaceutical compositions of the invention for medical treatment. [Background technology]

[0004] Gamma-delta (γδ) T cells are T cells that express a T cell receptor (TCR) consisting of a gamma chain and a delta chain. The majority of circulating γδ T cells express TCRs containing Vγ9 and Vδ2 regions. Vγ9Vδ2 T cells can react against a wide range of pathogens and tumor cells. This broad reactivity is understood to be conferred by phosphoantigens that can specifically activate this subset of T cells in a TCR-dependent manner. The broad antibacterial and antitumor reactivity of Vγ9Vδ2 T cells suggests a direct involvement in the immune control of cancer and infectious diseases.

[0005] Agents capable of activating Vy9V52 T cells may be useful in the treatment of infectious diseases or cancer, as they may promote the reactivity of Vy9V52 T cells against pathogens or infected cells or cancer cells. WO2015 / 156673 describes antibodies that bind to the Vy9V52 TCR and are capable of activating Vy9V52 T cells. WO2020 / 060405 describes bispecific antibodies that bind to both Vy9V52 T cell and tumor cell targets and thus have the potential to recruit Vy9V52 T cells to tumors and thus stimulate a therapeutic effect.

[0006] Recombinant production of antibodies in host cells often results in a heterogeneous product, containing different forms of the antibody with varying types and degrees of post-translational modifications of the polypeptide chain. Such heterogeneity is undesirable for therapeutic antibody products, because post-translational modifications can alter the functional properties of the antibody, for example, in terms of affinity for the target antigen, pharmacokinetic properties, product stability, aggregation, etc.

[0007] The present invention provides pharmaceutical compositions comprising antibodies with improved Vy9V52 TCR binding antibody sequences, which upon production in host cells result in a more homogeneous product, yet retain 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

[0008] As described in WO2022 / 122973, the 5C8Vγ9Vδ2 TCR-binding antibody described in WO2015 / 156673 and WO2020 / 060405 is sulfated at a site in the antibody that was not predicted to be subject to this post-translational modification. Sulfation partially occurs in various host cells, resulting in heterogeneous antibody products. Surprisingly, the tyrosine residue (Y105) subject to sulfation can be mutated to phenylalanine or serine without affecting the antigen-binding properties of the antibody, even though the amino acid is located in the CDR3 region. Removal of the sulfation site by mutation resulted in a more homogeneous antibody product.

[0009] The present invention provides novel pharmaceutical compositions comprising the above-described mutant antibodies.

[0010] In some embodiments, the present disclosure provides a pharmaceutical composition comprising: (a) an antibody capable of binding to human EGFR, the antibody comprising a first antigen-binding region comprising the CDR1 sequence of SEQ ID NO: 5, the CDR2 sequence of SEQ ID NO: 6, and the CDR3 sequence of SEQ ID NO: 7; (b) 5 to 20 mM histidine, wherein the composition has a pH of 5.5 to 6.5, or 5 to 20 mM sodium acetate, wherein the composition has a pH of 5.0 to 6.0; (c) 250 to 350 mM sucrose; (d) 0.01% to 0.05% (w / v) polysorbate 80; and (e) 0 to 20 mM methionine.

[0011] In some embodiments, the composition comprises 5-20 mM histidine and has a pH of 5.5-6.5. In some embodiments, the composition comprises 10 mM histidine. In some embodiments, the composition comprises 250-300 mM sucrose. In some embodiments, the composition comprises 280 mM sucrose. In some embodiments, the composition comprises 0.01%-0.03% polysorbate 80. In some embodiments, the composition comprises 0.02% polysorbate 80. In some embodiments, the composition comprises 0.5-20 mM methionine. In some embodiments, the composition comprises 1 mM methionine. In some embodiments, the composition comprises 10 mM histidine, 280 mM sucrose, 0.02% polysorbate 80, pH 6.0, and 1 mM methionine.

[0012] In some embodiments, the composition comprises 0.2-20 mg / mL, hi some embodiments, the composition comprises 1 mg / mL or 10 mg / mL of antibody.

[0013] In some embodiments, the first antigen-binding region is a single domain antibody, and preferably comprises or consists of the sequence set forth in SEQ ID NO:8 or 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.

[0014] In some embodiments, the antibody further comprises a second antigen-binding region, which is preferably a single domain antibody, hi some embodiments, the antibody is a bispecific antibody and comprises a second antigen-binding region capable of binding to human V52.

[0015] In some embodiments, the second antigen-binding region comprises the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 2, and the CDR3 sequence of SEQ ID NO: 3. In some embodiments, the second antigen-binding region comprises or consists of SEQ ID NO: 4, or a sequence having at least 90%, such as at least 92%, e.g., at least 94%, at least 96%, e.g., at least 98% sequence identity to SEQ ID NO: 4.

[0016] In some embodiments, X1 of SEQ ID NO:1 is S and X2 of SEQ ID NO:3 is F or S. In some embodiments, the CDR1 sequence comprises SEQ ID NO:21, the CDR2 sequence comprises SEQ ID NO:2, and the CDR3 sequence comprises SEQ ID NO:19. In some embodiments, the second antigen-binding region comprises or consists of SEQ ID NO:23. In some embodiments, the CDR1 sequence comprises SEQ ID NO:21, the CDR2 sequence comprises SEQ ID NO:2, and the CDR3 sequence comprises SEQ ID NO:20. In some embodiments, the second antigen-binding region comprises or consists of SEQ ID NO:24.

[0017] In some embodiments, the present invention discloses a pharmaceutical composition comprising: (a) 1 mg / mL or 10 mg / mL of an antibody capable of binding to human EGFR and comprising a first 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; (b) 10 mM histidine; (c) 280 mM sucrose; (d) 0.02% polysorbate 80; (e) pH 6.0; and (f) 1 mM methionine.

[0018] In some embodiments, the first antigen-binding region is a single domain antibody, and the first antigen-binding region preferably comprises or consists of SEQ ID NO:8.

[0019] In some embodiments, the antibody is a bispecific antibody and comprises a second antigen-binding region capable of binding to human V52. In some embodiments, the second antigen-binding region comprises or consists of SEQ ID NO: 4. In some embodiments, the second antigen-binding region comprises or consists of SEQ ID NO: 23. In some embodiments, the second antigen-binding region comprises or consists of SEQ ID NO: 24.

[0020] In some embodiments, the present disclosure provides a pharmaceutical composition comprising: (a) an antibody capable of binding to human Vδ2 and comprising a first antigen-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; (b) 5 to 20 mM histidine, wherein the composition has a pH of 5.5 to 6.5, or 5 to 20 mM sodium acetate, wherein the composition has a pH of 5.0 to 6.0; (c) 250 to 350 mM sucrose; (d) 0.01% to 0.05% (w / v) polysorbate 80; and (e) 0 to 20 mM methionine.

[0021] In some embodiments, the composition comprises 5-20 mM histidine and has a pH of 5.5-6.5. In some embodiments, the composition comprises 10 mM histidine. In some embodiments, the composition comprises 250-300 mM sucrose. In some embodiments, the composition comprises 280 mM sucrose. In some embodiments, the composition comprises 0.01%-0.03% polysorbate 80. In some embodiments, the composition comprises 0.02% polysorbate 80. In some embodiments, the composition comprises 0.5-20 mM methionine. In some embodiments, the composition comprises 1 mM methionine. In some embodiments, the composition comprises 10 mM histidine, 280 mM sucrose, 0.02% polysorbate 80, pH 6.0, and 1 mM methionine. In some embodiments, the composition comprises 0.2-20 mg / mL of the antibody. In some embodiments, the composition comprises 1 mg / mL or 10 mg / mL of the antibody.

[0022] In some embodiments, the first antigen-binding region comprises or consists of SEQ ID NO:4, or 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.

[0023] In some embodiments, X1 of SEQ ID NO:1 is S and X2 of SEQ ID NO:3 is F or S. In some embodiments, the CDR1 sequence comprises SEQ ID NO:21, the CDR2 sequence comprises SEQ ID NO:2, and the CDR3 sequence comprises SEQ ID NO:19. In some embodiments, the first antigen-binding region comprises or consists of SEQ ID NO:23. In some embodiments, the CDR1 sequence comprises SEQ ID NO:21, the CDR2 sequence comprises SEQ ID NO:2, and the CDR3 sequence comprises SEQ ID NO:20. In some embodiments, the first antigen-binding region comprises or consists of SEQ ID NO:24.

[0024] In some embodiments, the antibody further comprises a second antigen-binding region capable of binding to human EGFR and 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. In some embodiments, the second antigen-binding region is a single domain antibody, and the first antigen-binding region preferably comprises or consists of SEQ ID NO: 8, or 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 SEQ ID NO: 8.

[0025] In some embodiments, the present disclosure provides a pharmaceutical composition comprising: (a) 1 mg / mL or 10 mg / mL of an antibody comprising a first antigen-binding region capable of binding to human V52 and 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; (b) 10 mM histidine; (c) 280 mM sucrose; (d) 0.02% polysorbate 80; (e) pH 6.0; and (f) 1 mM methionine.

[0026] In some embodiments, the first antigen-binding region is a single-domain antibody, and the first antigen-binding region preferably comprises or consists of SEQ ID NO:4. In some embodiments, X1 of SEQ ID NO:1 is S and X2 of SEQ ID NO:3 is F or S. In some embodiments, the CDR1 sequence comprises SEQ ID NO:21, the CDR2 sequence comprises SEQ ID NO:2, and the CDR3 sequence comprises SEQ ID NO:19. In some embodiments, the first antigen-binding region comprises or consists of SEQ ID NO:23. In some embodiments, the CDR1 sequence comprises SEQ ID NO:21, the CDR2 sequence comprises SEQ ID NO:2, and the CDR3 sequence comprises SEQ ID NO:20. In some embodiments, the first antigen-binding region comprises or consists of SEQ ID NO:24.

[0027] In some embodiments, the antibody is a bispecific antibody and comprises a second antigen-binding region capable of binding to human EGFR. In some embodiments, the second antigen-binding region comprises or consists of SEQ ID NO:8.

[0028] In some embodiments, the antibody further comprises an Fc region, which is preferably a heterodimer comprising two Fc polypeptides, wherein a first antigen-binding region is fused to a first Fc polypeptide and a second antigen-binding region is fused to a second Fc polypeptide, and the first and second Fc polypeptides comprise asymmetric amino acid mutations that favor heterodimer formation over homodimer formation, preferably the CH3 regions of the Fc polypeptides comprise said asymmetric amino acid mutations, and preferably the first Fc polypeptide comprises a T366W substitution and the second Fc polypeptide comprises T366S, L368A, and Y407V substitutions, or vice versa, and the amino acid positions correspond to those of human IgG1 according to the EU numbering system.

[0029] In some embodiments, the first and second Fc polypeptides comprise mutations at positions 234 and / or 235, preferably the first and second Fc polypeptides comprise L234F and L235E substitutions, wherein the amino acid positions correspond to those of human IgG1 according to the EU numbering system.

[0030] In some embodiments, 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:12 and the second Fc polypeptide comprises the sequence set forth in SEQ ID NO:11.

[0031] In some embodiments, the antibody comprises or consists of the sequences set forth in SEQ ID NO: 16 and SEQ ID NO: 17, or comprises or consists of the sequences set forth in SEQ ID NO: 16 and SEQ ID NO: 18.

[0032] In some embodiments, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject a pharmaceutical composition described herein.

[0033] In some embodiments, the present disclosure provides pharmaceutical compositions for use as a medicament, preferably for use in the treatment of cancer.

[0034] Further aspects and embodiments of the invention are described below. [Brief explanation of the drawings]

[0035] [Figure 1] Representative chromatogram of the size exclusion profile of Protein-A purified LAVA compounds (VHH 5C8 is shown) using a Superdex-75 column. Fractions of the major monomer peak (fractions 1E11-1G2) were pooled and quantified. [Figure 2] Representative example of labchip polyacrylamide gel electrophoresis of purified VHH 5C8. Left: non-reducing conditions, right: reducing conditions. [Figure 3A] Figure 1 shows the HP-SEC profile of purified VHH 5C8. [Figure 3B] Figure 1 shows the HP-SEC profile of purified VHH 5C8var1. [Figure 4A] Representative HP-SEC profile of purified bispecific VHH (bsVHH) 1D12var5-5C8var1. Batch of bsVHH 1D12var5-5C8var1 expressed from Pichia pastoris culture supernatant and purified by Protein-A affinity chromatography. [Figure 4B] Representative HP-SEC profile of purified bispecific VHH (bsVHH) 1D12var5-5C8var1 batch expressed from HEK-293 E cells and purified by both Protein-A and size exclusion chromatography. [Figure 5]Labchip analysis of VHHs 5C8var1-Y105F and 5C8var1-Y105S purified under non-reducing conditions. [Figure 6A] HP-SEC analysis of VHH 5C8var1-Y105F is shown. [Figure 6B] HP-SEC analysis of 5C8var1-Y105S is shown. [Figure 7A] Figure 1 shows affinity measurements of VHH fragments binding to recombinant Vy9V52-TCR protein using BLI. Protein mass (response in nm) is plotted as a function of time. The dotted vertical line separates the association phase (left) and the dissociation phase (right). VHH 5C8var1. The black line represents the fitted data to the actual response measured. [Figure 7B] Figure 1 shows affinity measurements of VHH fragment binding to recombinant Vy9V52-TCR protein using BLI. Protein mass (response in nm) is plotted as a function of time. The dotted vertical line separates the association phase (left) and the dissociation phase (right). VHH 5C8var1-Y105F. The black line represents the fitted data to the actual response measured. [Figure 7C] Figure 1 shows affinity measurements of VHH fragment binding to recombinant Vy9V52-TCR protein using BLI. Protein mass (response in nm) is plotted as a function of time. The dotted vertical line separates the association phase (left) and the dissociation phase (right). VHH 5C8var1-Y105S. The black line represents the fitted data to the actual response measured. [Figure 8A] Figure 8A shows that both bsVHH 7D12var8-5C8var1-Y105F and bsVHH 7D12-5C8 induce potent Vy9V52 T cell activation and cause Vy9V52 T cell-mediated tumor cell lysis. 4-hour degranulation assay: percentage of CD107A (LAMP-1)+ Vy9V52 T cells is plotted as a function of antibody concentration using either 7D12-5C8 (non-humanized) (Figure 8A) or 7D12var8-5C8var1-Y105F (Figure 8B). [Figure 8B]Figure 8A shows that both bsVHH 7D12var8-5C8var1-Y105F and bsVHH 7D12-5C8 induce potent Vy9V52 T cell activation and cause Vy9V52 T cell-mediated tumor cell lysis. 4-hour degranulation assay: percentage of CD107A (LAMP-1)+ Vy9V52 T cells is plotted as a function of antibody concentration using either 7D12-5C8 (non-humanized) (Figure 8A) or 7D12var8-5C8var1-Y105F (Figure 8B). [Figure 8C] Figure 8C shows that both bsVHH 7D12var8-5C8var1-Y105F and bsVHH 7D12-5C8 induce potent Vy9V52 T cell activation and Vy9V52 T cell-mediated tumor cell lysis. Figure 8D shows a 24-hour cytotoxicity assay showing the percentage of A431 tumor cells killed as a function of antibody concentration using either 7D12-5C8 (non-humanized) (Figure 8C) or 7D12var8-5C8var1-Y105F. [Figure 8D] Figure 8C shows that both bsVHH 7D12var8-5C8var1-Y105F and bsVHH 7D12-5C8 induce potent Vy9V52 T cell activation and Vy9V52 T cell-mediated tumor cell lysis. Figure 8D shows a 24-hour cytotoxicity assay showing the percentage of A431 tumor cells killed as a function of antibody concentration using either 7D12-5C8 (non-humanized) (Figure 8C) or 7D12var8-5C8var1-Y105F. [Figure 9] Figure 1 shows binding of 7D12var8-5C8var1(Y105F)-Fc to primary γδ T cells isolated from healthy human PBMCs using flow cytometry. Two panels represent two different donors. [Figure 10A] 1 shows the binding of 7D12var8-5C8var1(Y105F)-Fc to EGFR on tumor cells by cell-based ELISA in HT29 cells, respectively. [Figure 10B]1 shows the binding of 7D12var8-5C8var1(Y105F)-Fc to EGFR on tumor cells by cell-based ELISA in A-431 cells, respectively. [Figure 10C] 1 shows the binding of 7D12var8-5C8var1(Y105F)-Fc to EGFR on tumor cells by cell-based ELISA in HCT-116 cells, respectively. [Figure 11] 1 shows 7D12var8-5C8var1(Y105F)-Fc-induced degranulation of patient-derived γδ T cells dependent on the A431 cell line. [Figure 12] 1 shows the viability of A-388 cells in co-culture with patient-derived γδ T cells and 7D12-5C8. [Figure 13] Lysed tumor cells are shown after 4 h of incubation of dissociated tumor cell suspensions (primary CRC: n=10, peritoneal CRC metastases: n=5, liver CRC metastases: n=3, primary HNSCC: n=5, and primary NSCLC: n=4) with healthy donor-derived Vy9V52 T cells (1:1 E:T ratio) and 7D12-5C8 (50 nM) or media control. [Figure 14] Lysed tumor cells are shown after 24 h incubation of dissociated tumor cell suspensions (peritoneal CRC metastases: n=4) with Vy9V52 T cells from healthy donors (1:1 E:T ratio) and 7D12-5C8var1(Y105S)-Fc (50 nM), gp120-5C8var1(Y105S)-Fc (50 nM), or media control. [Figure 15] 1 shows the structure of the construct for non-human primate studies. [Figure 16] Binding of 7A5-7D12var8-Fc to EGFR antigen target (Figure 16A) and TCR antigen target (Figure 16B) are shown. [Figure 17] Figure 17A shows degranulation mediated by 7A5-7D12var8-Fc. Figure 17B shows cytotoxicity mediated by 7A5-7D12var8-Fc. [Figure 18]Figure 1 shows PK analysis of 7A5-7D12var8-Fc concentrations in the blood of three treated animals. Concentration-time curves are shown indicative of a molecule with IgG-like PK. [Figure 19] The total number of T cells (CD3+, FIG. 19A) and the number of Vγ9-positive cells (as a percentage of the CD3+ population) (FIG. 19B) in the blood of treated animals are shown. Arrows indicate compound injection. Numbers in the legend indicate the number of monkeys treated. [Figure 20] IL-6 cytokine levels over time in the blood of treated animals are shown. Only low levels of cytokine were observed, with release mainly limited to the first injection. Arrows indicate the moment of treatment. DETAILED DESCRIPTION OF THE INVENTION

[0036] definition The term "human V52", as used herein, refers to the rearranged 52 chain of the Vy9V52-T cell receptor (TCR). UniProtKB-A0JD36 (A0JD36_HUMAN) shows an example of a variable V52 sequence. V52 is part of the delta chain of the Vy9V52-TCR. Antibodies capable of binding to human V52 may bind to an epitope located entirely within the variable region, or may bind to an epitope located within the constant region, or may bind to an epitope that is a combination of residues from the variable and constant regions of the delta chain.

[0037] The term "human Vy9" as used herein refers to the rearranged y9 chain of the Vy9V52-T cell receptor (TCR). UniProtKB-Q99603_HUMAN shows an example of a variable TRGV9 sequence.

[0038] The term "human Vy9V52 T cell receptor," as used herein, refers to the Vy9V52 T cell (TCR) receptor found on human γδ T cells.

[0039] The term "EGFR," as used herein, refers to the human EGFR protein (UniProtKB-P00533(EGFR_HUMAN)).

[0040] The term "antibody" is intended to refer to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions for a substantial period of time, e.g., a half-life of at least about 30 minutes, at least 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 period of time sufficient to induce, promote, enhance, and / or modulate a physiological response associated with the antibody binding to the antigen and / or a period of time sufficient for the antibody to acquire effector activity). The antigen-binding region (or antigen-binding domain) that interacts with the antigen can include the variable regions of both the heavy and light chains of the immunoglobulin molecule, or can be a single-domain antigen-binding region, e.g., only the heavy chain variable region. The constant regions 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.

[0041] The Fc region of an immunoglobulin is typically defined as the fragment of an antibody produced after digestion of the antibody with papain, which contains the two CH2-CH3 regions of the immunoglobulin and a connecting region, e.g., the 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, along with cell surface receptors called Fc receptors and proteins of the complement system, mediates the effector functions of the antibody.

[0042] 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.

[0043] 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 also be of any of the other subtypes described herein.

[0044] 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 also be of any of the other subtypes described herein.

[0045] References to amino acid positions in the Fc region / Fc domain in the present invention are based on EU numbering (Edelman et al., Proc Natl Acad Sci USA. 1969 May; 63(1):78-85; Kabat et al., Sequences of proteins of immunological interest. 5th Edition - 1991 NIH Publication No. 91-3242).

[0046] As noted above, the term "antibody" as used herein includes antibody fragments that retain the ability to specifically bind to an antigen, unless otherwise specified 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 by the term "antibody" include: (i) Fab' or Fab fragments, i.e., monovalent fragments consisting of the VL, VH, CL, and CH1 domains, or the monovalent antibodies 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 the VH and CH1 domains; and (iv) Fv fragments consisting essentially of the VL and VH domains of a single antibody arm. Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they may be joined by a synthetic linker that allows them to be produced as a single protein chain using recombinant techniques; the VL and VH regions pair to form a monovalent molecule known as a single-chain antibody or single-chain Fv (scFv) (see, e.g., Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single-chain antibodies are encompassed within the term antibody unless the context clearly indicates otherwise. While such fragments are generally encompassed within the meaning of antibody, they collectively and each independently represent unique features of the present invention and exhibit distinct biological properties and utilities. The term antibody, unless otherwise specified, 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.

[0047] In some embodiments of the antibodies of the present invention, the first antigen-binding region, 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 comprise 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 produced in Camelidae species, such as camels, dromedaries, llamas, alpacas, and guanacos. Like whole antibodies, single domain antibodies can selectively bind to specific antigens. Single domain antibodies can contain only the variable domains of an immunoglobulin chain, i.e., CDR1, CDR2, and CDR3, as well as framework regions. Such antibodies are also called Nanobodies® or VHHs.

[0048] As used herein, the term "immunoglobulin" is intended to refer to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light chains (L) and one pair of heavy chains (H), all four of which may be interconnected by disulfide bonds. As used herein, the terms "immunoglobulin heavy chain," "immunoglobulin heavy chain," or "heavy chain" are intended to refer to one of the immunoglobulin chains. A 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 defines the immunoglobulin isotype. The heavy chain constant region typically consists of three domains, CH1, CH2, and CH3. The heavy chain constant region further comprises a hinge region. Within the structure of an immunoglobulin (e.g., IgG), the two heavy chains are interconnected via disulfide bonds within the hinge region. Like heavy chains, 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 regions of hypervariability (or hypervariable regions that may be hypervariable 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. CDR sequences can be determined using various methods, such as those provided by Chothia and Lesk (1987) J. Mol. Biol. 196:901 or Kabat et al. (1991) Sequence of proteins of immunological interest, fifth edition, NIH publication. Various methods for CDR determination and amino acid numbering can be compared on www.abysis.org (UCL).

[0049] 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 can be combined with either a kappa (κ) or lambda (λ) light chain. The antibodies of the present invention can have any isotype.

[0050] The term "parent antibody" should be understood as an antibody identical to the antibody according to the present invention, but which does not have one or more of the specified mutations. A "variant" or "antibody variant" or "variant of a parent antibody" of the present invention is an antibody molecule that contains one or more mutations compared to the "parent antibody." An amino acid substitution may replace a natural amino acid with another naturally occurring amino acid or a non-naturally occurring amino acid derivative. Amino acid substitutions may be conservative or non-conservative. In the context of the present invention, conservative substitutions may be defined by substitutions within a class of amino acids reflected in one or more of the following three tables:

[0051] [Table 1]

[0052] [Table 2]

[0053] [Table 3]

[0054] In the context of the present invention, substitutions in variants are indicated as original amino acid-position-substituted amino acid. To indicate amino acid residues, a three-letter code, including the codes Xaa and X, or a one-letter code is used. Thus, the notation "T366W" means that the variant contains a substitution of threonine with tryptophan at the variant amino acid position corresponding to amino acid 366 in the parent antibody.

[0055] Furthermore, the term "substitution" encompasses substitution with any one of the other 19 naturally occurring amino acids, or substitution with other amino acids, such as unnatural amino acids. For example, substitution of the amino acid T at position 366 includes 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.

[0056] The term "full length antibody," as used herein, refers to an antibody that contains all heavy and light chain constant and variable domains corresponding to those normally found in a wild-type antibody of that isotype.

[0057] The term "chimeric antibody" refers to an antibody in which the variable region is derived from a non-human species (e.g., from a rodent) and the constant region is derived from a different species, such as human. Chimeric antibodies can be produced by genetic engineering. Chimeric monoclonal antibodies for therapeutic use are developed to reduce antibody immunogenicity.

[0058] 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 with the human variable domain. This can be achieved by grafting the six non-human antibody complementarity-determining regions (CDRs), which 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) with human framework regions (backmutations) may be required to fully reconstitute the binding affinity and specificity of the parent antibody. Structural homology modeling can help identify amino acid residues within the framework regions 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 containing one or more amino acid backmutations relative to the non-human amino acid sequence, and optionally a fully human constant region. If desired, additional amino acid modifications, not necessarily backmutations, may be introduced to obtain a humanized antibody with desirable properties, such as affinity and biochemical properties. Humanization of non-human therapeutic antibodies is performed to minimize their immunogenicity in humans, while at the same time maintaining the specificity and binding affinity of the antibody of non-human origin.

[0059] The term "multispecific antibody" refers to an antibody having specificity for at least two different, e.g., at least three different, typically non-overlapping, epitopes. Such epitopes may be on the same target antigen or on different target antigens. When the epitopes are on different targets, such targets may be on the same cell or on different cells or cell types. In some embodiments, a multispecific antibody may comprise one or more single domain antibodies.

[0060] The term "bispecific antibody" refers to an antibody having specificity for two different, typically non-overlapping, epitopes. Such epitopes may be on the same target or on different targets. When the epitopes are on different targets, such targets may be on the same cell or on different cells or cell types. In some embodiments, a bispecific antibody may comprise one or two single domain antibodies.

[0061] Examples of different classes of multispecific antibodies, such as bispecific antibodies, include, but are not limited to, (i) IgG-like molecules with complementary CH3 domains to force heterodimerization, (ii) recombinant IgG-like dual targeting molecules (where the two sides of the molecule each contain 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), and (iv) Fc fusion molecules (where a single chain Fv molecule or stabilized diabody is fused to a heavy chain constant domain, Fc region, or a portion thereof). (v) Fab fusion molecules (different Fab fragments are fused together and fused to heavy chain constant domains, Fc regions or portions thereof); and (vi) scFv and diabody-based antibodies and heavy chain antibodies (e.g., domain antibodies, Nanobodies®) (different single chain Fv molecules or different diabody antibodies or different heavy chain antibodies (e.g., domain antibodies, Nanobodies®) are fused to each other or to another protein, or a carrier molecule is fused to heavy chain constant domains, Fc regions or portions thereof).

[0062] Examples of IgG-like molecules having complementary CH3 domain molecules include, but are not limited to, Triomab® (Trion Pharma / Fresenius Biotech), Knobs-into-Hole (Genentech), CrossMAb (Roche) and electrostatically matched (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-body and PIG-body (Pharmabcine, WO2010134666, WO2014081202), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Biclonics (Merus, WO2013157953), FcΔAdp (Regeneron), bispecific IgG1 and IgG2 (Pfizer / Rinat), Azymetric scaffold (Zymeworks / Merck), mAb-Fv (Xencor), bivalent bispecific antibodies (Roche, WO2009080254) and DuoBody® molecules (Genmab).

[0063] Examples of recombinant IgG-like dual-targeting molecules include, but are not limited to, Dual Targeting (DT)-Ig (GSK / Domantis, WO2009058383), Two-in-one Antibody (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).

[0064] Examples of IgG fusion molecules include, but are not limited to, Dual Variable Domain (DVD)-Ig (Abbott), Dual domain double head antibodies (Unilever, Sanofi Aventis), IgG-like Bispecific (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).

[0065] Examples of Fc fusion molecules include, but are not limited to, scFv / Fc fusion (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, WO2010111625), Dual Affinity Retargeting Technology (Fc-DART™) (MacroGenics), and Dual(ScFv)2-Fab (National Research Center for Antibody Medicine-China).

[0066] Examples of Fab-fused 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 (Biotecnol), and Fab-Fv (UCB-Celltech).

[0067] Examples of ScFv antibodies, diabody-based antibodies, and domain antibodies include, but are not limited to, Bispecific T Cell Engager (BiTE®) (Micromet, Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART™) (MacroGenics), Single-chain Diabodies (Academic, Lawrence FEBS Lett. 1998 Apr 3;425(3):479-84), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack, WO2010059315) 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.

[0068] In the context of an antibody that binds to an antigen, the terms "bind" or "specifically bind" refer to the binding of the antibody to a given antigen or target (e.g., human V52 or human EGFR), which binding typically occurs within about 10-15% confidence intervals as measured, for example, using flow cytometry as described in the Examples herein. -6 M or less, e.g. 10 -7 M or less, about 10 -8 M or less, about 10 -9 M or less, about 10 -10 M or less, or about 10 -11The apparent affinity corresponds to a KD of M or less. Alternatively, KD values can be determined, for example, using surface plasmon resonance (SPR) technology on a BIAcore T200 or biolayer interferometry (BLI) on an Octet RED96 instrument, using the antigen as the ligand and the binding moiety or binding molecule as the analyte. Specific binding means that an antibody binds to a given antigen with an affinity corresponding to a KD that is at least 10-fold lower, at least 100-fold lower, e.g., at least 1,000-fold lower, at least 10,000-fold lower, e.g., at least 100,000-fold lower, than the affinity for binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). The degree of lower affinity depends on the KD of the binding moiety or binding molecule; therefore, if the KD of the binding moiety or binding molecule is very low (i.e., the binding moiety or binding molecule is very specific), the degree to which the affinity for the antigen is lower than the affinity for a nonspecific antigen can be at least 10,000-fold lower. The term "KD" (M), as used herein, refers to the dissociation equilibrium constant of a particular interaction between an antigen and a binding moiety or binding molecule.

[0069] In the context of the present invention, "competition" or "competable" or "competing" refers to a detectably significant reduction in the tendency of 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 refers to a reduction in binding caused by the presence of another molecule, such as an antibody, of at least about 25%, such as at least about 50%, e.g., at least about 75%, at least 90%, etc., as determined, for example, by ELISA analysis or flow cytometry using sufficient amounts of two or more competing molecules, e.g., antibodies. Additional 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).

[0070] In one embodiment, an antibody of the present invention binds to the same epitope on EGFR as antibody 7D12 and / or the same epitope on Vδ2 as antibody 5C8. Several methods are available in the art for mapping antibody epitopes on target antigens, including, but not limited to, cross-linking mass spectrometry, which allows for 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 within 5 Å of the antibody. 5 Å was selected as the epitope cutoff distance, allowing for atoms within the van der Waals radius plus possible water-mediated hydrogen bonds. Epitope residues can then be determined to be all amino acid residues with at least one atom within 8 Å. 8 Å or less was selected as the epitope cutoff distance to account for the length of the extended arginine amino acid. Cross-linking mass spectrometry begins with coupling 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 chemical cross-linking, the Ab / Ag complex is extremely stable, allowing many different enzymes and digestion conditions to be applied to the complex, yielding many different overlapping peptides. These peptides are identified using high-resolution mass spectrometry and MS / MS techniques. The identity of the cross-linked peptides is determined using mass tags attached to the cross-linking reagents. After MS / MS fragmentation and data analysis, the cross-linked antigen-derived peptides are part of the epitope, and the antibody-derived peptides are part of the paratope. All residues between the most N- 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 as described in Schmitz et al. (2013) Structure 21:1214 and consists of a flat surface on domain III (residues R353, D355, F357, Q384, and N420) corresponding to the domain III ligand-binding site.

[0071] The terms "first" and "second" antigen-binding region, as used herein, do not refer to their orientation / location on the antibody, i.e., they have no meaning with respect to the N-terminus or C-terminus. The terms "first" and "second" serve only to accurately and consistently refer to two different antigen-binding regions in the claims and description.

[0072] "Percent sequence identity," as used herein, refers to the number of identical nucleotide or amino acid positions shared by different sequences (i.e., percent identity = number of identical positions / total number of positions × 100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment. The percent identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4, 11-17 (1988), as incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0073] Further aspects and embodiments of the present invention The present disclosure provides pharmaceutical compositions comprising antibodies comprising an antigen-binding region capable of binding to human V52. The present disclosure additionally provides pharmaceutical compositions comprising antibodies comprising a first antigen-binding region capable of binding to human EGFR and a second antigen-binding region capable of binding to human V52. The pharmaceutical compositions described herein may contain diluents, bulking agents, salts, buffers, detergents (e.g., non-ionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugar- or protein-free amino acids), preservatives, tissue fixatives, solubilizing agents, and / or other materials suitable for inclusion in pharmaceutical compositions. Additional pharmaceutically acceptable excipients include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants, and absorption delaying agents that are physiologically compatible with the antibodies of the present invention.

[0074] As described, in a first aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) an antibody comprising an antigen-binding region capable of binding to human Vδ2, 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; (b) histidine, wherein the composition has a pH of 5.5 to 6.5, or sodium acetate, wherein the composition has a pH of 5.0 to 6.0, and (c) 250-350 mM sucrose; (d) 0.01% to 0.05% (w / v) polysorbate 80; (e) 0 to 20 mM methionine.

[0075] In one embodiment, the composition comprises 5 to 20 mM histidine and has a pH of 5.5 to 6.5, hi one embodiment, the composition comprises 10 mM histidine and has a pH of 5.5 to 6.5.

[0076] In another embodiment, the composition comprises 5-20 mM sodium acetate and has a pH of 5.0-6.0.

[0077] In one embodiment, the composition comprises 250-300 mM sucrose, hi one embodiment, the composition comprises 280 mM sucrose.

[0078] In one embodiment, the composition comprises 0.01% to 0.03% polysorbate 80. In one embodiment, the composition comprises 0.02% polysorbate 80.

[0079] In one embodiment, the composition comprises 0.5 to 20 mM methionine, hi one embodiment, the composition comprises 1 mM methionine.

[0080] In one embodiment, the composition comprises 0.2 to 25 mg / mL of the antibody. In one embodiment, the composition comprises 0.2 to 20 mg / mL of the antibody. In one embodiment, the composition comprises 1 or 10 mg / mL of the antibody.

[0081] In one embodiment, the composition comprises: (a) 5 to 20 mM, for example, 10 mM, histidine, having a pH of 5.5 to 6.5; (b) 250 to 300 mM, for example, 280 mM, sucrose; (c) 0.01% to 0.03%, for example 0.02%, of polysorbate 80.

[0082] In one embodiment, the composition comprises: (a) 10 mM histidine, having a pH of 5.5 to 6.5; (b) 280 mM sucrose; (c) 0.02% polysorbate 80.

[0083] In one embodiment, the composition comprises: (a) 5 to 20 mM, for example, 10 mM, histidine, having a pH of 5.5 to 6.5; (b) 250 to 300 mM, for example, 280 mM, sucrose; (c) 0.01% to 0.03%, for example, 0.02%, of polysorbate 80; (d) 0.5 to 20 mM methionine, for example, 1 mM methionine.

[0084] In one embodiment, the composition comprises: (a) 10 mM histidine, having a pH of 5.5 to 6.5; (b) 280 mM sucrose; (c) 0.02% polysorbate 80; (d) 1 mM methionine.

[0085] In one embodiment, the composition contains 0.2 to 20 mg / mL, for example, 1 mg / mL or 10 mg / mL, of the antibody.

[0086] In one embodiment, the composition comprises: (a) 5 to 20 mM, for example, 10 mM, histidine, having a pH of 5.5 to 6.5; (b) 250 to 300 mM, for example, 280 mM, sucrose; (c) 0.01% to 0.03%, for example, 0.02%, of polysorbate 80; (d) 1 mg / mL of the antibody.

[0087] In one embodiment, the composition comprises: (a) 10 mM histidine, having a pH of 5.5 to 6.5; (b) 280 mM sucrose; (c) 0.02% polysorbate 80; (d) 1 mg / mL of the antibody.

[0088] In one embodiment, the composition comprises: (a) 5 to 20 mM, for example, 10 mM, histidine, having a pH of 5.5 to 6.5; (b) 250 to 300 mM, for example, 280 mM, sucrose; (c) 0.01% to 0.03%, for example, 0.02%, of polysorbate 80; (d) 0.5 to 20 mM methionine, for example, 1 mM methionine; (e) 1 mg / mL of the antibody.

[0089] In one embodiment, the composition comprises: (a) 10 mM histidine, having a pH of 5.5 to 6.5; (b) 280 mM sucrose; (c) 0.02% polysorbate 80; (d) 1 mM methionine; (e) 1 mg / mL of the antibody.

[0090] In one embodiment, the composition comprises: (a) 5 to 20 mM, for example, 10 mM, histidine, having a pH of 5.5 to 6.5; (b) 250 to 300 mM, for example, 280 mM, sucrose; (c) 0.01% to 0.03%, for example, 0.02%, of polysorbate 80; (d) 10 mg / mL of the antibody.

[0091] In one embodiment, the composition comprises: (a) 10 mM histidine, having a pH of 5.5 to 6.5; (b) 280 mM sucrose; (c) 0.02% polysorbate 80; (d) 10 mg / mL of the antibody.

[0092] In one embodiment, the composition comprises: (a) 5 to 20 mM, for example, 10 mM, histidine, having a pH of 5.5 to 6.5; (b) 250 to 300 mM, for example, 280 mM, sucrose; (c) 0.01% to 0.03%, for example, 0.02%, of polysorbate 80; (d) 0.5 to 20 mM methionine, for example, 1 mM methionine; (e) 10 mg / mL of the antibody.

[0093] In one embodiment, the composition comprises: (a) 10 mM histidine, having a pH of 5.5 to 6.5; (b) 280 mM sucrose; (c) 0.02% polysorbate 80; (d) 1 mM methionine; (e) 10 mg / mL of the antibody.

[0094] In one embodiment, the composition comprises: (a) 5 to 20 mM, for example, 10 mM, histidine, having a pH of 5.5 to 6.5; (b) 250 to 300 mM, for example, 280 mM, sucrose; (c) 0.01% to 0.03%, for example, 0.02%, of polysorbate 80; (d) 20 mg / mL of the antibody.

[0095] In one embodiment, the composition comprises: (a) 10 mM histidine, having a pH of 5.5 to 6.5; (b) 280 mM sucrose; (c) 0.02% polysorbate 80; (d) 20 mg / mL of the antibody.

[0096] In one embodiment, the composition comprises: (a) 5 to 20 mM, for example, 10 mM, histidine, having a pH of 5.5 to 6.5; (b) 250 to 300 mM, for example, 280 mM, sucrose; (c) 0.01% to 0.03%, for example, 0.02%, of polysorbate 80; (d) 0.5 to 20 mM methionine, for example, 1 mM methionine; (e) 20 mg / mL of the antibody.

[0097] In one embodiment, the composition comprises: (a) 10 mM histidine, having a pH of 5.5 to 6.5; (b) 280 mM sucrose; (c) 0.02% polysorbate 80; (d) 1 mM methionine; (e) 20 mg / mL of the antibody.

[0098] As described herein above, in a first aspect, the present invention relates to a pharmaceutical composition comprising an antibody comprising an antigen-binding region capable of binding to human V52, wherein the antigen-binding region comprises a CDR1 sequence set forth in SEQ ID NO: 1, a CDR2 sequence set forth in SEQ ID NO: 2, and a CDR3 sequence set forth in SEQ ID NO: 3.

[0099] In one embodiment, X1 of SEQ ID NO: 1 is S (Ser). In another embodiment, X1 of SEQ ID NO: 1 is G (Gly).

[0100] In one embodiment, X2 of SEQ ID NO: 3 is F (Phe). In another embodiment, X2 of SEQ ID NO: 3 is S (Ser).

[0101] In one embodiment, X1 of SEQ ID NO: 1 is S (Ser) and X2 of SEQ ID NO: 3 is F (Phe).

[0102] In one embodiment, X1 of SEQ ID NO: 1 is S(Ser) and X2 of SEQ ID NO: 3 is S(Ser).

[0103] In one embodiment, X1 of SEQ ID NO: 1 is G (Gly) and X2 of SEQ ID NO: 3 is F (Phe).

[0104] In one embodiment, X1 of SEQ ID NO: 1 is G (Gly) and X2 of SEQ ID NO: 3 is S (Ser).

[0105] In some embodiments, the invention relates to a pharmaceutical composition comprising an antibody comprising an antigen-binding region capable of binding to human V52, wherein the antigen-binding region comprises the CDR1 sequence set forth in SEQ ID NO: 21, the CDR2 sequence set forth in SEQ ID NO: 2, and the CDR3 sequence set forth in SEQ ID NO: 19. In some embodiments, the invention relates to a pharmaceutical composition comprising an antibody comprising an antigen-binding region capable of binding to human V52, wherein the antigen-binding region comprises the CDR1 sequence set forth in SEQ ID NO: 21, the CDR2 sequence set forth in SEQ ID NO: 2, and the CDR3 sequence set forth in SEQ ID NO: 20.

[0106] 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) profile. In a preferred embodiment, the antibody is capable of inducing activation (e.g. upregulation of CD69 and / or CD25 expression) that leads to degranulation characterized by increased CD107a expression and / or cytokine production (e.g. TNF, IFNγ) by Vy9V52 T cells.

[0107] 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, when tested at a concentration of, for example, 1 nM, preferably 100 pM, preferably 10 pM, preferably 1 pM, even more preferably 100 fM, as described herein in Example 9. In another preferred embodiment, the antibody of the invention has an EC50 value for increasing the proportion of CD107a positive cells by 100 pM or less, such as 50 pM or less, for example 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.

[0108] In one embodiment, the antigen-binding region is a single domain antibody. Thus, in one embodiment, the antibody in the pharmaceutical composition of the invention comprises a single domain antibody capable of binding to human V52, wherein the 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.

[0109] In another embodiment, the antigen-binding region is humanized, and preferably the antigen-binding region comprises or consists of (a) the sequence set forth in SEQ ID NO: 4, or (b) 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.

[0110] 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).

[0111] 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).

[0112] In some embodiments, the antigen binding region is humanized, preferably the antigen binding region comprises or consists of the sequence set forth in SEQ ID NO: 23. In some embodiments, the antigen binding region is humanized, preferably the antigen binding region comprises or consists of the sequence set forth in SEQ ID NO: 24.

[0113] In some embodiments, the antibody in the pharmaceutical composition 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.

[0114] In a further embodiment, the antibody is a bispecific antibody, and both the first antigen-antigen binding region and the second antigen-binding region are single domain antibodies. In a further embodiment, the multispecific antibody is a bispecific antibody, and the first antigen-binding region is a single domain antibody and the second antigen-binding region is a single domain antibody.

[0115] In one embodiment, the antibody comprised in the pharmaceutical composition of the invention comprises a second antigen-binding region, wherein the second antigen-binding region is capable of binding to human EGFR. Bispecific antibodies targeting both Vy9V52 T cells and EGFR have been shown to induce potent Vy9V52 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).

[0116] In some embodiments, the antibody comprises an 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.

[0117] In one embodiment, the antigen binding region is humanized.

[0118] In a further embodiment, the antibody comprises an antigen-binding region comprising or consisting of (a) the sequence set forth in SEQ ID NO:8, or (b) 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.

[0119] 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.

[0120] In a further embodiment, the antibody of the present 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 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.

[0121] In a further embodiment, the antibody of the present 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: 21, the CDR2 sequence set forth in SEQ ID NO: 2, and the CDR3 sequence set forth in SEQ ID NO: 19, and 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.

[0122] In a further embodiment, the antibody of the present 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: 21, the CDR2 sequence set forth in SEQ ID NO: 2, and the CDR3 sequence set forth in SEQ ID NO: 20, and 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.

[0123] 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. (a) X1 of SEQ ID NO: 4 is S (Ser) and X2 of SEQ ID NO: 4 is F (Phe), or (b) X1 of SEQ ID NO: 4 is S(Ser) and X2 of SEQ ID NO: 4 is S(Ser), or (c) X1 of SEQ ID NO: 4 is G (Gly) and X2 of SEQ ID NO: 4 is F (Phe), or (d) X1 of SEQ ID NO: 4 is G (Gly), and X2 of SEQ ID NO: 4 is S (Ser).

[0124] 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 sequence set forth in SEQ ID NO: 23 and the second antigen-binding region comprises the sequence set forth in SEQ ID NO: 8. 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 sequence set forth in SEQ ID NO: 24 and the second antigen-binding region comprises the sequence set forth in SEQ ID NO: 8.

[0125] 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 25%, such as at least 50%, for example by at least 2-fold, when tested as described in Example 9 herein.

[0126] In a further embodiment, the antibody is unable to mediate killing of EGFR-negative cells, such as EGFR-negative human cells.

[0127] 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 to 20 amino acids in length, e.g., 1 to 10 amino acids in length, such as 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.

[0128] 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.

[0129] In one embodiment, the antibody comprised in the pharmaceutical composition of the present invention 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 greater. As used herein, the "terminal half-life" of an antibody refers to the time it takes for the serum concentration of the polypeptide to decrease by 50% in vivo during the final stage of elimination.

[0130] In one embodiment, the antibody further comprises a half-life prolonging domain, wherein the half-life prolonging domain is 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 generating bispecific antibodies have been described in the art, and are reviewed, for example, by Brinkmann and Kontermann (2017) MAbs 9:182. In one embodiment of the present invention, the Fc region is a heterodimer comprising two Fc polypeptides, wherein a first antigen-binding region is fused to the first Fc polypeptide and 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 heterodimer formation over homodimer formation (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 described above, preferably wherein the first Fc polypeptide comprises a T366W substitution and the second Fc polypeptide comprises T366S, L368A, and Y407V substitutions, or vice versa, wherein the amino acid positions correspond to those of 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, wherein the amino acid positions correspond to those of human IgG1 according to the EU numbering system. In further embodiments, the regions comprise the hinge sequence set forth in SEQ ID NO: 10.

[0131] In some embodiments, the first and / or second Fc polypeptides contain mutations that render the antibody inactive, i.e., unable to mediate or with reduced ability to mediate effector function. In one embodiment, an inactive Fc region additionally cannot bind C1q. In one embodiment, the first and second Fc polypeptides contain mutations at positions 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 an L234A mutation, an L235A mutation, and a P329G mutation. In another embodiment, the antibody contains an L234F mutation, an L235E mutation, and a D265A mutation.

[0132] 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; (a) 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 (b) the first Fc polypeptide comprises the sequence set forth in SEQ ID NO:12, and the second Fc polypeptide comprises the sequence set forth in SEQ ID NO:11.

[0133] In one embodiment, X3 of SEQ ID NO: 11 is K. In another embodiment, X3 of SEQ ID NO: 11 is absent.

[0134] In one embodiment, X3 of SEQ ID NO: 12 is K. In another embodiment, X3 of SEQ ID NO: 12 is absent.

[0135] 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.

[0136] In one embodiment, X3 of SEQ ID NO: 16 is K. In another embodiment, X3 of SEQ ID NO: 16 is absent.

[0137] In one embodiment, X3 of SEQ ID NO: 17 is K. In another embodiment, X3 of SEQ ID NO: 17 is absent.

[0138] 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.

[0139] In one embodiment, X3 of SEQ ID NO: 18 is K. In another embodiment, X3 of SEQ ID NO: 18 is absent.

[0140] In a further main aspect, the present invention relates to a pharmaceutical composition according to the invention as described herein for use as a medicament.

[0141] The antibodies used in the pharmaceutical composition according to the invention make it possible to create a microenvironment favourable for tumor cell killing by Vy9V52 T cells, and therefore in a preferred embodiment the antibodies are for use in the treatment of cancer.

[0142] In one embodiment, the pharmaceutical composition is for use in treating primary or metastatic colon or colorectal cancer. In another embodiment, the pharmaceutical composition is for use in treating cancer of the peritoneum. In another embodiment, the pharmaceutical composition is for use in treating liver cancer. In another embodiment, the pharmaceutical composition is for use in treating head and neck squamous cell carcinoma (HNSCC). In another embodiment, the pharmaceutical composition is for use in treating non-small cell lung cancer (NSCLC). In another embodiment, the pharmaceutical composition is for use in treating squamous cell carcinoma of the skin.

[0143] Likewise, the present invention relates to a method for treating a disease comprising administering to a human subject in need thereof a pharmaceutical composition according to the invention as described herein, hi one embodiment, the disease is cancer.

[0144] In some embodiments, the pharmaceutical compositions are administered as monotherapy. However, the pharmaceutical compositions of the present invention may also be administered in combination therapy, i.e., in combination with other therapeutic agents relevant to the disease or condition being treated.

[0145] "Treat" or "treating" refers to administering an effective amount of a pharmaceutical composition of the present invention for the purpose of alleviating, ameliorating, inhibiting, eradicating (curing), or preventing symptoms or disease states. 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, the age, sex, and weight of the individual, and 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 are outweighed by the therapeutically beneficial effects. Administration can be by any suitable route, but is typically parenteral, such as intravenous, intramuscular, or subcutaneous.

[0146] The multispecific antibodies used in the present invention are typically produced recombinantly, i.e., by expression of a nucleic acid construct encoding the antibody in a suitable host cell, followed by purification of 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. Suitable host cells 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.

[0147] [Table 4-1] [Table 4-2] [Table 4-3]

[0148] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, mention of any reference, article, publication, patent, patent publication, or patent application cited herein is not, and should not be considered 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 in the world. [Example]

[0149] Example 1: Production and purification of VHH compounds VHH compounds were primarily produced by transient transfection of the encoding plasmid in HEK293-E253 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 major monomer peak (fractions 1E11-1G2) observed in preparative size exclusion using a Superdex-75 column was purified as shown in Figure 1.

[0150] The purified protein was shown to migrate as a single band under reducing and non-reducing conditions in polyacrylamide gel electrophoresis; a representative example is shown in Figure 2.

[0151] Example 2: HP-SEC analysis of purified VHH compounds For HP-SEC analysis of purified VHH compounds, a Waters Acquity ARC-bio system was used. 10 μg of antibody (10 μL of antibody with a concentration of 1 mg / mL) was injected onto a Waters BEH200 SEC column (2.5 μm bead size, 7.8 × 300 mm column dimensions). The mobile phase consisted of 50 mM sodium phosphate, 0.2 M sodium chloride buffer (pH 7.0), and the buffer flow rate for the run was 0.8 mL / min. Proteins were detected by measuring absorbance at a wavelength of 214 nm. The total analysis time was 15 min per injection. VHH compounds were prepared and purified as described in Example 1. Surprisingly, when VHH 5C8 (SEQ ID NO: 13) (previously described in WO2015 / 156673) and 5C8var1 (SEQ ID NO: 14) (previously described in WO2020 / 060405) were tested for integrity and monomericity by HP-SEC analysis, two peaks were observed (see Figures 3A-B).

[0152] Example 3: Mass spectrometry of 5C8 reveals an additional mass of 80 Da To determine whether the two isoforms of 5C8 differ in mass, a 5C8 protein preparation was analyzed by LC-ESI-MS mass spectrometry. The predominant species found in this analysis was 5C8 without post-translational modifications or a 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 treating with phosphatase or sulfatase and subsequent LC-ESI-MS mass spectrometry of peptides after proteolytic digestion. Sulfatase treatment reduced the mass of peptides containing Y105, the seventh residue of CDR3 (SEQ ID NO: 15), by 80 Da, whereas phosphatase treatment was shown to have no effect. This demonstrates that Y105 in 5C8 is post-translationally modified by sulfation. This sulfation was present in approximately 30% of the protein preparation.

[0153] Example 4: 1D12var5-5C8var1 containing the same anti-Vy9V52 VHH show the same heterogeneity and the same additional mass of 80 Da in HP-SEC 1D12var5-5C8var1 is a bispecific VHH compound composed of an anti-CD1d VHH linked to 5C8var1 via a flexible linker (described in SEQ ID NO: 87 of WO2020 / 060405). This protein was expressed in HEK293E cells as described above. Furthermore, protein preparations were obtained from different expression systems, and bispecific VHHs were also expressed in Pichia pastoris and Chinese hamster ovary (CHO) cells. When the different protein preparations were tested by HP-SEC analysis, pre-peaks were systematically observed (see Figures 4A-B).

[0154] The observed pre-peak indicates that a significant proportion of the protein is a different isoform. Because 5C8 VHH was shown to be sulfated and 5C8var1 contains the exact same CDR3 sequence, the 1D12var5-5C8var1 batch was also analyzed by mass spectrometry for their molecular weights. Depending on the protein batch, 15%–40% were found to contain an additional 80 Da mass. This is consistent with the sulfation observed in VHH 5C8.

[0155] Example 5: In silico analysis of VHH 5C8 and 5C8var1 Based on PDB ID 5M2W, a homology model of 5C8 and 5C8var1 was constructed using Maestro (Schroedinger). CDR1 and CDR3 required refinement by de novo loop prediction using Prime (Schroedinger). The generated model showed that CDR3 residue Y105 was located at 205.1 Å in the model of 5C8var1. 2 , 122.2 Å in the 5C8 model 2The model showed a high solvent-accessible surface area and was therefore expected to contribute to antigen binding. The model was then analyzed for reactive residues, indicating residues prone to post-translational modifications (PTMs). The protein sequence was then analyzed using ModPred, a sequence-based PTM prediction tool. Predicted modifications in both the structure and sequence are listed in Table 2. Individual predicted PTMs could not explain the mass differences observed in the HP-SEC analysis. Type indicates the PTM predicted by Maestro. *Q13 deamidation was predicted only for 5C8.

[0156] [Table 5]

[0157] Example 6: Design and production of 5C8var1 VHH CDR3 mutants Y105F and Y105S As described in Example 5, a homology model was used to introduce a mutation to prevent sulfation of Y105 in 5C8 and 5C8var1. Two different mutants were designed based on the model structure of VHH: Y105S (retaining an alcohol functional group) and Y105F (retaining an aromatic ring). Residue Y105 is the seventh residue in CDR3, and introducing a mutation was predicted to affect binding. Both mutations were designed in the humanized VHH sequence 5C8var1, and both proteins were produced in HEK293E cells and purified as described above. The CDR3 amino acid sequence of the humanized VHH remained identical to that of the non-humanized VHH.

[0158] Both 5C8var1-Y105F and 5C8var1-Y105S were successfully produced 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.

[0159] Example 7: HP-SEC analysis of purified VHHs containing designed CDR3 mutations HP-SEC analysis was performed as described for 5C8. Both 5C8var1-Y105F and 5C8var1-Y105S were analyzed. Figures 6A-6B.

[0160] As can be concluded from the HP-SEC analysis of purified VHH molecules containing the designed CDR3 mutations, no heterogeneity was observed for any of the variants, indicating the absence of the observed Y105 post-translational modification and the homogeneity of the proteins.

[0161] Example 8: Affinity measurements of 5C8var1, 5C8var1-Y105F, and 5C8var1-Y105S using Biolayer Interferometry (BLI) show no difference in affinity Binding of the 5C8var1 VHH antibody fragment and mutants 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 a ligand on an anti-human Fc capture biosensor. Sensorgrams were recorded when the ligand-captured biosensor was incubated with a dilution series of the VHH antibody fragment (40–0.63 nM) in 10x kinetics buffer (ForteBio). A 1:1 binding model was used to fit the overall data to determine k. on (association rate constant) and koff (dissociation rate constant). Using these values, KD = k off / k on was used to calculate the KD (equilibrium dissociation constant).

[0162] As can be concluded from Figures 7A-7C and Table 3, the KD values found for the two different Y105VHH mutants were not substantially different from those found for 5C8var1. In particular, the Y105F mutant had an affinity comparable to that found for 5C8var1. Values shown in Table 3 are the average of at least three independent measurements + / - standard deviation.

[0163] [Table 6]

[0164] Example 9: Functionality of anti-(EGFR x Vy9V52 TCR) bispecific VHH containing the Y105F mutation is fully preserved To determine whether the equal affinity of VHH 5C8var1-Y105F compared to 5C8var1 could be translated into equivalent functionality, we designed a bispecific VHH, 7D12var8-5C8var1-Y105F. 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 Vγ9Vδ2 T cell activation and trigger T cell-mediated tumor cell lysis in an EGFR-positive tumor cell line (A431). Briefly, Vy9V52 T cells were isolated from the blood of healthy donors using magnetic-activated cell sorting (MACS) in combination with an anti-V52 antibody according to standardized procedures. These cells were then expanded for one week using a mixture of cytokines and irradiated feeder cells, a mixture of JY cell lines and PBMCs from different donors. Vy9V52 T cells were always >90% pure when used in the assays (double-positive staining for Vy9 and V52 in FACS). The A431 cell line (ATCC, catalog no. CRL-1555) was cultured according to the supplier's recommendations. For activation or cytotoxicity assays, 50,000 tumor target cells were seeded into 96-well tissue culture plates the day before the assay. The following day, 50,000 expanded purified Vy9V52 T cells were added to the culture medium along with a range of bispecific VHH compound concentrations. In activation assays, degranulation of Vy9V52 T cells 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 proteases (indicating 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.

[0165] Figures 8A-8D show that 7D12var8-5C8var1-Y105F and non-humanized 7D12-5C8 induced potent Vy9V52 T cell activation (Figures 8A and 8B) and tumor cell lysis (Figures 8C and 8D). 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.

[0166] [Table 7]

[0167] The maximum level of tumor cell killing was slightly lower for 7D12var8-5C8var1-Y105F compared to that observed with 7D12-5C8, however, these were two different measurements using two different Vy9V52 T cell donors, and this maximum level of cytotoxicity may be particularly donor-dependent.

[0168] Example 10: The thermal stability of VHH 5C8var1 containing the Y105 mutation was unchanged To determine whether the mutations introduced into the different variants affect the thermal stability of the VHH fold, the melting temperatures of the variants were measured using NanoDSF (differential scanning fluorescence). The antibody samples were diluted using PBS until they were equivalent to the lowest concentration sample. The antibody samples were then loaded into nanoDSF-grade capillaries and measured using a Prometheus NT.48. During the experiment, the temperature was increased from 20°C to 95°C. The intrinsic fluorescence of the protein was detected at 350 nm and 330 nm and recorded along 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) was determined, at which the VHH was fully unfolded. on ) and melting temperatures (Tm) were reported (Table 5). The melting temperatures measured for 5C8var1-Y105F and 5C8var1-Y105S were consistent with the melting temperature of 5C8var1: Table 5.

[0169] [Table 8]

[0170] 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 VHH was linked to a modified hinge (AAA followed by SDKTHTCPPCP, with cysteine 220 removed) and human CH2 and CH3 domains. The CH2 domain was Fc-silenced by a pair of LFLE mutations (L234F, L235E), and the CH3 domain was mutated with "knobs-into-holes" mutations (knobs: T366W and holes: T366S, L368A, and Y407V) that force heterodimerization upon coexpression 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 an 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.

[0171] The protein was produced by cotransfection of the two encoding expression vectors in HEK293E cells and purification from the culture supernatant by protein A affinity chromatography followed by preparative size exclusion chromatography, as described in Example 1. This resulted in a highly monomeric protein preparation.

[0172] Example 12: Binding of 7D12var8-5C8var1(Y105F)-Fc to primary Vy9V52 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 plated at a concentration of 50,000 cells / well and incubated with either 7D12var8-5C8var1(Y105F)-Fc or GP120-5C8var1(Y105F)-Fc antibodies as a positive control in a half-log titration starting at 100 nM for 1 hour at 4°C. Binding of antibodies to the 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 Vy9V52 T cells, with a half-maximal effective concentration (EC50) in the low nanomolar range (approximately 3 nM).

[0173] 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. To this end, tumor cells were first seeded at various concentrations on day -1 to reach a concentration of approximately 50,000 cells / well on day 0. On day 0, a half-log titration of 7D12var8-5C8var1(Y105F)-Fc antibody or GP120-5C8var1(Y105F)-Fc antibody (as a negative control) was added to the tumor cells starting at 100 nM for 1 hour at 37°C. The bound antibody was then labeled with anti-IgG1-HRP for 1 hour at 37°C. The secondary antibody binding was then disrupted by the addition of 3,3',5,5'-tetramethylbenzidine, and the reaction was stopped by the addition of H2SO4, followed by a colorimetric change induced by HRP. The optical density (OD) was then measured in a UV spectrometer at a wavelength of 450 nm. Figures 10A-10C show that 7D12var8-5C8var1(Y105F)-Fc strongly bound to A-431 (Figure 10B), HCT-116 (Figure 10C), and HT-29 (Figure 10A) 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.

[0174] Example 14: A-431 cell-dependent Vy9V52 T cell degranulation induced by 7D12var8-5C8var1(Y105F)-Fc To investigate the potential of 7D12var8-5C8var1(Y105F)-Fc to activate Vγ9V52 T cells, Vγ9V52 T cells were first isolated and expanded as previously described. Next, Vγ9V52 T cells were 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-Vγ9 and anti-CD3 antibodies to distinguish Vγ9V52 T cells from tumor cells. Flow cytometry was used to examine the extent of CD107a expression on Vγ9V52 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 an A-431 cell-dependent manner. The EC50 for Vγ9Vδ2 T cell degranulation induced by 7D12var8-5C8var1(Y105F)-Fc is in the picomolar range (approximately 40-90 pM).

[0175] Example 15: Antibody 7D12-5C8 induces T cell-mediated target cell cytotoxicity To investigate whether the bispecific VHH7D12-5C8 was effective in inducing Vy9V52 T cell-mediated cytotoxicity against target cells, the viability of the A-388 epidermoid tumor cell line (ATCC, CRL-7905) was assessed in a coculture setting with Vy9V52 T cells and bsVHH antibody fragments. For this assay, Vy9V52 T cells were isolated from healthy PBMCs as previously described but then frozen and stored at -150°C. Frozen Vy9V52 T cells were thawed and incubated overnight in IL-2-supplemented medium. A-388 tumor cells were seeded alone or with incubated Vy9V52 T cells at a 1:1 or 1:0.1 ratio with or without 7D12-5C8 (10 nM). As an additional control, Vy9V52 T cells were seeded alone with or without the antibody 7D12-5C8 (10 nM). After 72 hours, cell viability was determined by adding ATP Lite (Perkin Elmer, 6016731) and reading the luminescence signal using a microplate reader. Figure 12 shows the ATP-derived fluorescent signal, which represents the metabolic activity of viable cells and, thereby, the number of viable cells. It can be observed that at a 1:1 E:T ratio, the antibody induces a reduction of viable cells by approximately 50%, while untreated co-cultures of A-388 and Vy9V52 T cells are unaffected, highlighting its potential to induce T cell-mediated cytotoxicity.

[0176] Example 16: Tumor cell killing by 7D12-5C8 and 7D12-5C8var1(Y105S)-Fc-activated Vy9V52 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 tumor cell types were tested.

[0177] 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 the Amsterdam University of Medicine (UMMC) (location VUmc) after written informed consent. The tissues were cut into small pieces with a surgical blade (size no. 22; Swann Morton Ltd) and resuspended in dissociation medium consisting of IMDM supplemented with 0.1% DNAse I, 0.14% collagenase A, 5% FCS, 100 IU / ml sodium penicillin, 100 μg / ml streptomycin sulfate, and 2.0 mM L-glutamine. The tissues were transferred to a sterile flask equipped with a stir bar and incubated on a magnetic stirrer in a 37°C water bath for 45 min. After incubation, the cell suspension was passed through a 100 μM cell strainer. After dissociating the tumor three times, the cells were washed for viable cell counts by trypan blue exclusion.

[0178] Dissociated patient-derived tumor cells were incubated with healthy donor-derived Vγ9Vδ2 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.

[0179] If necessary, adherent cells were detached using trypsin-EDTA after the culture period, 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).

[0180] Viable cells were identified using 123 Count eBeads™ in combination with the live / dead marker 7AAD 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).

[0181] 7D12-5C8 and 7D12-5C8var1(Y105S)-Fc-induced Vy9V52 T cell-mediated cytotoxicity of tumor cells was assessed by incubating expanded healthy donor-derived Vy9V52 T cells with single cell suspensions of various malignancies (primary CRC, CRC metastases in the peritoneum and liver, head and neck squamous cell carcinoma, and non-small cell lung cancer).

[0182] As shown in Figure 13, 7D12-5C8 induced substantial lysis of patient tumor cells by Vy9V52 T cells (mean % lysis induced by 7D12-5C8: primary CRC 52.3% and p-value 0.0003, peritoneal CRC 46.0% and p-value 0.0052, liver CRC 31.8% and p-value 0.0360, squamous cell carcinoma of the head and neck 46.1% and p-value 0.0187, non-small cell lung cancer 64.1% and p-value 0.0153).

[0183] 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 % lysis induced by 7D12-5C8var1(Y105S)-Fc: 71.2% and p<0.0001 and 0.0012). The control compound, gp120-5C8var1(Y105S)-Fc, did not induce measurable tumor cell lysis.

[0184] Example 17: Design, production 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 WO2021 / 052995). A bispecific Fc-containing antibody was constructed containing 7A5 and the anti-EGFR VHH 7D12var8. This 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). The Vγ9-binding scFv of the 7A5 antibody was attached to the "knob" strand, and the EGFR-binding VHH 7D12var8 was cloned in-frame with the "hole" strand of the KiH Fc pair. Additionally, the upper hinge was engineered to "AAASDKTHTCPPCP" (SEQ ID NO: 25) to remove the cysteine (C220) that normally bridges the CL and to introduce more flexibility by changing "EPK" to "AAA." The N-terminal portion of CH2 was engineered to suppress Fc receptor (CD16, -32, and -64) interaction (silencing mutations L234F, L235E) while maintaining FcRN binding. The resulting construct was designated 7A5-7D12var8-Fc.

[0185] This molecule was produced by transient cotransfection of two plasmids encoding the two different chains in HEK293E cells and purified from the culture supernatant by Protein-A affinity chromatography followed by preparative size-exclusion chromatography (Example 1). Using ELISA and recombinant forms of both antigens, the molecule was shown to bind to either target with an apparent affinity of approximately 3 nanomolar (nM) (Figures 16A-B). The functionality of the molecule was demonstrated by showing that it triggered target-dependent activation (CD107a expression) of in vitro expanded Vy9V52 T cells (Figure 17A) and subsequent T cell-mediated tumor cell lysis (Figure 17B).

[0186] Example 18: Bispecific antibody 7A5-7D12var8-Fc was well tolerated in an exploratory multi-dose non-human primate (NHP: cynomolgus monkey) study In a multiple-dose exploratory NHP study, 7A5-7D12var8-Fc was administered to three female cynomolgus monkeys at doses of 1 mg / kg, 5 mg / kg, and 23 mg / kg, respectively. The antibody was administered at 5 mL / kg via a 30-minute infusion, administered four times weekly. The first two dose groups (one animal per dose) at 1 and 5 mg / kg were administered simultaneously, and after three weekly doses, a 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 last dose, the animals were euthanized, and tissues were collected and prepared for histopathology and immunohistochemistry (IHC).

[0187] Pharmacokinetic analysis of 7A5-7D12var8-Fc concentrations in the blood of treated animals (measured by ELISA, Figure 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 exhibited a shorter half-life after the third injection, which may be due to a potential anti-drug antibody (ADA) response in those animals.

[0188] Clearance values found ranged from 0.36 to 0.72 mL / h / kg, and the volume of distribution ranged from 58.5 to 115.2 mL / kg. Systemic exposure increased dose-proportionally between 1 and 23 mg / kg. However, no accumulation was observed after repeated dosing.

[0189] 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 associated with the procedure, as is often observed in multiple-dose studies of this type. Figure 19A shows a transient reduction in T cell numbers at each time point 2 hours after administration. However, T cell numbers returned to baseline levels 2 days after injection.

[0190] In contrast, Vγ9-positive T cells decreased in number in the peripheral blood and did not recover to their previous frequency (FIG. 19B). These cells remained largely absent over the course of the study, indicating a specific pharmacodynamic effect of the compound. Measurement of cytokines in the blood of treated animals showed that treatment caused very little cytokine release, which was largely limited to the first injection of the compound. FIG. 20 shows the measured levels of IL-6 as an example.

[0191] In general, treatment of NHPs with 7A5-7D12var8-Fc was very well tolerated, with no clinical signs of toxicity. Additionally, histopathology revealed no gross or microscopic abnormalities in any of the organs examined (data presented here). In comparison, anti-EGFR x CD3 BiTE was lethal to NHPs at a dose of 31 μg / kg / day continuous infusion (Lutterbuese et al., Proc Natl Acad Sci USA 2010:107(28),12605).

[0192] Example 19: Stable formulation of 7D12var8-5C8var1(Y105F)-Fc A stable CHO cell clone expressing 7D12var8-5C8var1(Y105F)-Fc was generated and the antibody product was obtained by manufacturing in a bioreactor system. After harvesting and purification, the antibody product was formulated at concentrations of 1 mg / mL and 10 mg / mL using four formulation buffers as shown in Table 6.

[0193] [Table 9]

[0194] The formulated samples were subjected to several storage conditions (defined below) for up to six weeks. Additionally, several stress tests were applied. ●Storage at 5℃±3℃ ●Storage at -80℃±10℃ Accelerated storage conditions: 25°C ±2°C / 60% relative humidity (RH) ±5% ●Heat stress at 40℃±2℃ / 75%RH±5% Freeze / thaw cycle: The sample was completely frozen to -80±10°C. After that, the sample was completely thawed at room temperature (15-25°C), and then five freeze / thaw cycles were performed. ●Agitation stress: The sample was agitated at 240 rpm for 7 days at room temperature (EP 15°C to 25°C). Oxidation: Samples were spiked with 0.01% (v / v) hydrogen peroxide (H2O2) and incubated at room temperature for 24 hours. Light Stability: >1.2 million lux hours and >1000 watt hours / square meter of near-UV energy applied at 25°C±2°C / 60%RH±5%. The initial (=t0) 5°C ± 3°C sample was used as reference. The samples were analyzed using the following analytical methods shown in Table 7.

[0195] [Table 10]

[0196] The results of the stability test over time were as follows: clarity, color, pH, A 280The results of UV-VIS, SE-HPLC, CIEX-HPLC, CE-SDS, and PSMA binding, differential scanning, and peptide fingerprinting for storage temperatures of -80°C ± 10°C, 5°C ± 3°C, 25°C ± 2°C / 60% RH ± 5%, and 40°C ± 2°C / 75% RH ± 5% were highly comparable. The results indicate that the product was stable in each of the selected buffer conditions, with only minor effects observed over time. Furthermore, stress testing (freeze / thaw (five F / T cycles), light, agitation, and oxidative stress) indicated that the product was stable under each of these conditions.

[0197] A major effect of differentiating buffers was detected under thermal and oxidative stress conditions. CIEX-HPLC analysis of samples maintained at 40°C ± 2°C / 75% RH ± 5% shows a decrease in the main variants, coupled with an increase in the acidic variants, for all buffers and product concentrations tested. However, histidine-based formulations show a lower reduction compared to acetate-based formulations. Additionally, LC-MS analysis revealed after oxidative stress showed that the addition of methionine effectively prevented product oxidation.

[0198] A summary of the most affected analytical results for temperature stability and after oxidative stress are shown in Tables 8 and 9 for the 1 mg antibody / mL and 10 mg antibody / mL formulations, respectively.

[0199] In conclusion, all formulation buffers tested appear to be suitable for 7D12var8-5C8var1(Y105F)-Fc. However, the antibody was most stable by a small margin when formulated in Buffer 1.

[0200] [Table 11-1] [Table 11-2]

[0201] [Table 12-1]

Table 12-2

Table 12-3

Claims

1. 1. A pharmaceutical composition comprising: (a) an antibody capable of binding to human EGFR, comprising a first antigen-binding region comprising a CDR1 sequence of SEQ ID NO: 5, a CDR2 sequence of SEQ ID NO: 6, and a CDR3 sequence of SEQ ID NO: 7; (b) 5-20 mM histidine, wherein the composition has a pH of 5.5-6.5, or 5-20 mM sodium acetate, wherein the composition has a pH of 5.0-6.0; (c) 250-350 mM sucrose; (d) 0.01% to 0.05% (w / v) polysorbate 80; and (e) 0 to 20 mM methionine.

2. 10. The pharmaceutical composition of claim 1, wherein the composition comprises 5 to 20 mM histidine and the composition has a pH of 5.5 to 6.

5.

3. 3. The pharmaceutical composition of claim 1, wherein the composition comprises 10 mM histidine.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the composition comprises 250 to 300 mM sucrose.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the composition comprises 280 mM sucrose.

6. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the composition comprises 0.01% to 0.03% polysorbate 80.

7. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein the composition comprises 0.02% polysorbate 80.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the composition comprises 0.5 to 20 mM methionine.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein the composition comprises 1 mM methionine.

10. 10. The pharmaceutical composition of any one of claims 1 to 9, wherein the composition comprises 10 mM histidine, 280 mM sucrose, 0.02% polysorbate 80, pH 6.0, and 1 mM methionine.

11. The pharmaceutical composition of any one of claims 1 to 11, wherein the composition comprises 0.2 to 20 mg / mL of the antibody.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the composition comprises 1 or 10 mg / mL of the antibody.

13. The pharmaceutical composition of any one of claims 1 to 12, wherein the first antigen-binding region is a single domain antibody.

14. The first antigen-binding region is a single domain antibody, and the first antigen-binding region preferably comprises: a. the sequence set forth in SEQ ID NO: 8, or b. A pharmaceutical composition according to any one of claims 1 to 13, 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.

15. The pharmaceutical composition of any one of claims 1 to 14, wherein the antibody further comprises a second antigen-binding region, which is preferably a single domain antibody.

16. 16. The pharmaceutical composition of claim 15, wherein the antibody is a bispecific antibody and comprises a second antigen-binding region capable of binding to human V52.

17. 17. The pharmaceutical composition of claim 16, wherein the second antigen-binding region comprises a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 2, and a CDR3 sequence of SEQ ID NO:

3.

18. the second antigen-binding region comprises: a. SEQ ID NO: 4, or b. The pharmaceutical composition of claim 17, 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 SEQ ID NO:

4.

19. The pharmaceutical composition of claim 17, wherein X1 of SEQ ID NO: 1 is S and X2 of SEQ ID NO: 3 is F or S.

20. 20. The pharmaceutical composition of claim 19, wherein the CDR1 sequence comprises SEQ ID NO: 21, the CDR2 sequence comprises SEQ ID NO: 2, and the CDR3 sequence comprises SEQ ID NO:

19.

21. 21. The pharmaceutical composition of claim 20, wherein the second antigen-binding region comprises or consists of SEQ ID NO:

23.

22. 20. The pharmaceutical composition of claim 19, wherein the CDR1 sequence comprises SEQ ID NO: 21, the CDR2 sequence comprises SEQ ID NO: 2, and the CDR3 sequence comprises SEQ ID NO:

20.

23. 23. The pharmaceutical composition of claim 22, wherein the second antigen-binding region comprises or consists of SEQ ID NO:

24.

24. 1. A pharmaceutical composition comprising: (a) an antibody at 1 mg / mL or 10 mg / mL, the antibody comprising a first antigen-binding region capable of binding to human EGFR, the first 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; (b) 10 mM histidine; (c) 280 mM sucrose; (d) 0.02% polysorbate 80; (e) pH 6.0; (f) 1 mM methionine.

25. 25. The pharmaceutical composition of claim 24, wherein the first antigen-binding region is a single domain antibody, and the first antigen-binding region preferably comprises or consists of SEQ ID NO:

8.

26. 26. The pharmaceutical composition of claim 24 or 25, wherein the antibody is a bispecific antibody and comprises a second antigen-binding region capable of binding to human V52.

27. The pharmaceutical composition of any one of claims 24 to 26, wherein the second antigen-binding region comprises or consists of SEQ ID NO:

4.

28. 27. The pharmaceutical composition of any one of claims 24 to 26, wherein the second antigen-binding region comprises or consists of SEQ ID NO:

23.

29. 27. The pharmaceutical composition of any one of claims 24 to 26, wherein the second antigen-binding region comprises or consists of SEQ ID NO:

24.

30. 1. A pharmaceutical composition comprising: (a) an antibody comprising a first antigen-binding region capable of binding to human Vδ2, the first antigen-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; (b) 5-20 mM histidine, wherein the composition has a pH of 5.5-6.5, or 5-20 mM sodium acetate, wherein the composition has a pH of 5.0-6.0; (c) 250-350 mM sucrose; (d) 0.01% to 0.05% (w / v) polysorbate 80; and (e) 0 to 20 mM methionine.

31. 31. The pharmaceutical composition of claim 30, wherein the composition comprises 5 to 20 mM histidine and the composition has a pH of 5.5 to 6.

5.

32. 32. The pharmaceutical composition of claim 30 or 31, wherein the composition comprises 10 mM histidine.

33. 33. The pharmaceutical composition of any one of claims 30 to 32, wherein the composition comprises 250 to 300 mM sucrose.

34. 34. The pharmaceutical composition of any one of claims 30 to 33, wherein the composition comprises 280 mM sucrose.

35. 35. The pharmaceutical composition of any one of claims 30 to 34, wherein the composition comprises 0.01% to 0.03% polysorbate 80.

36. 36. The pharmaceutical composition of any one of claims 30 to 35, wherein the composition comprises 0.02% polysorbate 80.

37. 37. The pharmaceutical composition of any one of claims 30 to 36, wherein the composition comprises 0.5 to 20 mM methionine.

38. 38. The pharmaceutical composition of any one of claims 30 to 37, wherein the composition comprises 1 mM methionine.

39. 39. The pharmaceutical composition of any one of claims 30-38, wherein the composition comprises 10 mM histidine, 280 mM sucrose, 0.02% polysorbate 80, pH 6.0, and 1 mM methionine.

40. 40. The pharmaceutical composition of any one of claims 30 to 39, wherein the composition comprises 0.2 to 20 mg / mL of the antibody.

41. 41. The pharmaceutical composition of any one of claims 30 to 40, wherein the composition comprises 1 or 10 mg / mL of the antibody.

42. 42. The pharmaceutical composition of any one of claims 30 to 41, wherein the first antigen-binding region comprises or consists of SEQ ID NO: 4, or 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.

43. The pharmaceutical composition according to any one of claims 30 to 42, wherein X1 of SEQ ID NO: 1 is S and X2 of SEQ ID NO: 3 is F or S.

44. 44. The pharmaceutical composition of claim 43, wherein the CDR1 sequence comprises SEQ ID NO: 21, the CDR2 sequence comprises SEQ ID NO: 2, and the CDR3 sequence comprises SEQ ID NO:

19.

45. 45. The pharmaceutical composition of claim 44, wherein the first antigen-binding region comprises or consists of SEQ ID NO:

23.

46. 44. The pharmaceutical composition of claim 43, wherein the CDR1 sequence comprises SEQ ID NO: 21, the CDR2 sequence comprises SEQ ID NO: 2, and the CDR3 sequence comprises SEQ ID NO:

20.

47. 47. The pharmaceutical composition of claim 46, wherein the first antigen-binding region comprises or consists of SEQ ID NO:

24.

48. 48. The pharmaceutical composition of any one of claims 30 to 47, wherein the antibody further comprises a second antigen-binding region capable of binding to human EGFR and 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.

49. 49. The pharmaceutical composition of claim 48, wherein the second antigen-binding region is a single domain antibody and the first antigen-binding region preferably comprises or consists of SEQ ID NO: 8 or 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 SEQ ID NO:

8.

50. 1. A pharmaceutical composition comprising: (a) an antibody at 1 mg / mL or 10 mg / mL, comprising a first antigen-binding region capable of binding to human Vδ2, the first antigen-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; (b) 10 mM histidine; (c) 280 mM sucrose; (d) 0.02% polysorbate 80; (e) pH 6.0; (f) 1 mM methionine.

51. 51. The pharmaceutical composition of claim 50, wherein the first antigen-binding region is a single domain antibody, and the first antigen-binding region preferably comprises or consists of SEQ ID NO:

4.

52. 52. The pharmaceutical composition of claim 50 or 51, wherein X1 of SEQ ID NO: 1 is S and X2 of SEQ ID NO: 3 is F or S.

53. 53. The pharmaceutical composition of claim 52, wherein the CDR1 sequence comprises SEQ ID NO: 21, the CDR2 sequence comprises SEQ ID NO: 2, and the CDR3 sequence comprises SEQ ID NO:

19.

54. 54. The pharmaceutical composition of claim 53, wherein the first antigen-binding region comprises or consists of SEQ ID NO:

23.

55. 53. The pharmaceutical composition of claim 52, wherein the CDR1 sequence comprises SEQ ID NO: 21, the CDR2 sequence comprises SEQ ID NO: 2, and the CDR3 sequence comprises SEQ ID NO:

20.

56. 56. The pharmaceutical composition of claim 55, wherein the first antigen-binding region comprises or consists of SEQ ID NO:

24.

57. 57. The pharmaceutical composition of any one of claims 50 to 56, wherein the antibody is a bispecific antibody and comprises a second antigen-binding region capable of binding to human EGFR.

58. 58. The pharmaceutical composition of claim 57, wherein the second antigen-binding region comprises or consists of SEQ ID NO:

8.

59. 59. The pharmaceutical composition of any one of claims 1 to 58, wherein the antibody further comprises an Fc region, the Fc region preferably being a heterodimer comprising two Fc polypeptides, wherein the first antigen-binding region is fused to a first Fc polypeptide and the second antigen-binding region is fused to a second Fc polypeptide, the first and second Fc polypeptides comprising asymmetric amino acid mutations that favor heterodimer formation over homodimer formation, preferably the CH3 regions of the Fc polypeptides comprising the asymmetric amino acid mutations, preferably the first Fc polypeptide comprising a T366W substitution and the second Fc polypeptide comprising T366S, L368A, and Y407V substitutions, or vice versa, and wherein the amino acid positions correspond to those of human IgG1 according to the EU numbering system.

60. 60. The pharmaceutical composition of claim 59, wherein the first and second Fc polypeptides comprise mutations at positions 234 and / or 235, preferably wherein the first and second Fc polypeptides comprise L234F and L235E substitutions, and wherein the amino acid positions correspond to human IgG1 according to the EU numbering system.

61. a. 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 b. The pharmaceutical composition of claim 59 or 60, wherein said first Fc polypeptide comprises the sequence set forth in SEQ ID NO: 12 and said second Fc polypeptide comprises the sequence set forth in SEQ ID NO:

11.

62. 62. The pharmaceutical composition of any one of claims 1 to 61, wherein the antibody comprises or consists of the sequences set forth in SEQ ID NO: 16 and SEQ ID NO: 17, or comprises or consists of the sequences set forth in SEQ ID NO: 16 and SEQ ID NO:

18.

63. 63. A method of treating cancer in a subject, comprising administering to the subject a pharmaceutical composition according to any one of claims 1 to 62.

64. A pharmaceutical composition according to any one of claims 1 to 62 for use as a medicament, preferably for the treatment of cancer.

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