Compositions comprising antibodies that bind to gamma-delta T cell receptors

Abstract

The present invention relates to a pharmaceutical composition comprising an antibody capable of binding to the human V [gamma] 9V [delta] 2T cell receptor. The invention further relates to the use of the pharmaceutical composition of the invention for medical treatment.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of European application No. 22179260, filed on June 15, 2022, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0003] Reference to an electronic sequence listing

[0004] The contents of the electronic sequence listing (LVAT_025_01WO_SeqList_ST26.xml; size: 35,054 bytes; and creation date: June 12, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present invention relates to novel pharmaceutical compositions comprising antibodies capable of binding to the Vδ2 chain of the human Vγ9Vδ2 T cell receptor. The present invention further relates to the use of the pharmaceutical compositions of the present invention for medical treatment. Background Art

[0006] Gamma-delta (γδ) T cells are T cells that express a T cell receptor (TCR) composed of a gamma chain and a delta chain. Most circulating γδ T cells express a TCR composed of the Vγ9 and Vδ2 regions. Vγ9Vδ2 T cells can fight a wide range of pathogens and tumor cells. This broad reactivity is thought to be conferred by phosphoantigens that can specifically activate this T cell subset in a TCR-dependent manner. The broad antimicrobial and antitumor reactivity of Vγ9Vδ2 T cells suggests their direct involvement in the immune control of cancer and infection.

[0007] Agents that can activate Vγ9Vδ2 T cells can be used to treat infection or cancer because these agents can promote the responsiveness of Vγ9Vδ2 T cells to pathogens or infected cells or cancer cells. WO 2015 / 156673 describes antibodies that bind to Vγ9Vδ2 TCR and are able to activate Vγ9Vδ2 T cells. WO 2020 / 060405 describes bispecific antibodies that bind to Vγ9Vδ2 T cell and tumor cell targets, thereby potentially recruiting Vγ9Vδ2 T cells to tumors, thereby stimulating a therapeutic effect.

[0008] Recombinant production of antibodies in host cells often results in heterogeneous products that contain different forms of antibodies with different types and degrees of post-translational modifications of the polypeptide chains. Such heterogeneity is undesirable for antibody products for medical use, as post-translational modifications may alter the functional properties of the antibody, such as affinity for the target antigen, pharmacokinetic properties, product stability, aggregation, etc.

[0009] The present invention provides pharmaceutical compositions comprising antibodies with improved Vγ9Vδ2 TCR binding antibody sequences, which when produced in host cells will produce more homogeneous products, but still retain good functional properties in terms of target binding and functional effects on target cells, as well as good structural properties such as stability. Summary of the invention

[0010] As described in WO 2022 / 122973, the 5C8 Vγ9Vδ2 TCR binding antibodies described in WO 2015 / 156673 and WO 2020 / 060405 are sulfated at sites in the antibody that are not predicted to be subject to such post-translational modifications. Sulfation occurs in part in various host cells, resulting in heterogeneous antibody products. Surprisingly, the tyrosine residue (Y105) where sulfate is present 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 sulfation sites by mutation produces a more homogeneous antibody product.

[0011] The present invention provides a novel pharmaceutical composition comprising the mutant antibody.

[0012] In some embodiments, the present disclosure provides a pharmaceutical composition comprising: (a) an antibody comprising a first antigen binding region capable of binding to human EGFR and 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 pH of the composition is between 5.5 and 6.5, or 5-20 mM sodium acetate, wherein the pH of the composition is between 5.0 and 6.0; (c) 250-350 mM sucrose; (d) 0.01%-0.05% (weight / volume) polysorbate 80; and (e) 0-20 mM methionine.

[0013] In some embodiments, the composition comprises 5-20mM histidine, wherein the pH of the composition is between 5.5 and 6.5. In some embodiments, the composition comprises 10mM histidine. In some embodiments, the composition comprises sucrose between 250 and 300mM. In some embodiments, the composition comprises 280mM sucrose. In some embodiments, the composition comprises polysorbate 80 between 0.01% and 0.03%. In some embodiments, the composition comprises 0.02% polysorbate 80. In some embodiments, the composition comprises methionine between 0.5 and 20mM. In some embodiments, the composition comprises 1mM methionine. In some embodiments, the composition comprises 10mM histidine, 280mM sucrose, 0.02% polysorbate 80, pH 6.0 and 1mM methionine.

[0014] In some embodiments, the composition comprises between 0.2 and 20 mg / mL of the antibody. In some embodiments, the composition comprises 1 or 10 mg / mL of the antibody.

[0015] In some embodiments, the first antigen binding region is a single domain antibody. In some embodiments, the first antigen binding region is a single domain antibody, and wherein the first antigen binding region preferably comprises or consists of the sequence shown 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 with the sequence shown in SEQ ID NO: 8.

[0016] In some embodiments, the antibody further comprises a second antigen binding region, and wherein the second antigen binding region is preferably a single domain antibody. In some embodiments, the antibody is a bispecific antibody and comprises a second antigen binding region capable of binding to human Vδ2.

[0017] In some embodiments, 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. 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%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to SEQ ID NO: 4.

[0018] In some embodiments, X1 of SEQ ID NO: 1 is S, and wherein 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.

[0019] In some embodiments, the present invention discloses 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 EGFR and comprising the CDR1 sequence shown in SEQ ID NO:5, the CDR2 sequence shown in SEQ ID NO:6, and the CDR3 sequence shown 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.

[0020] In some embodiments, the first antigen binding region is a single domain antibody, and wherein the first antigen binding region preferably comprises or consists of SEQ ID NO:8.

[0021] In some embodiments, the antibody is a bispecific antibody and comprises a second antigen binding region capable of binding to human Vδ2. In some embodiments, the second antigen binding region comprises SEQ ID NO: 4 or consists thereof. In some embodiments, the second antigen binding region comprises SEQ ID NO: 23 or consists thereof. In some embodiments, the second antigen binding region comprises SEQ ID NO: 24 or consists thereof.

[0022] In some embodiments, the present disclosure provides a pharmaceutical composition comprising: (a) an antibody comprising a first antigen binding region capable of binding to human Vδ2 and comprising the CDR1 sequence shown in SEQ ID NO:1, the CDR2 sequence shown in SEQ ID NO:2, and the CDR3 sequence shown in SEQ ID NO:3; (b) 5-20 mM histidine, wherein the pH of the composition is between 5.5 and 6.5, or 5-20 mM sodium acetate, wherein the pH of the composition is between 5.0 and 6.0; (c) 250-350 mM sucrose; (d) 0.01%-0.05% (weight / volume) polysorbate 80; and (e) 0-20 mM methionine.

[0023] In some embodiments, the composition comprises 5-20mM histidine, wherein the pH of the composition is between 5.5 and 6.5. In some embodiments, the composition comprises 10mM histidine. In some embodiments, the composition comprises sucrose between 250 and 300mM. In some embodiments, the composition comprises 280mM sucrose. In some embodiments, the composition comprises polysorbate 80 between 0.01% and 0.03%. In some embodiments, the composition comprises 0.02% polysorbate 80. In some embodiments, the composition comprises methionine between 0.5 and 20mM. In some embodiments, the composition comprises 1mM methionine. In some embodiments, the composition comprises 10mM histidine, 280mM sucrose, 0.02% polysorbate 80, pH 6.0 and 1mM methionine. In some embodiments, the composition comprises the antibody between 0.2 and 20mg / mL. In some embodiments, the composition comprises 1 or 10mg / mL antibody.

[0024] 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 shown in SEQ ID NO:4.

[0025] In some embodiments, X1 of SEQ ID NO: 1 is S, and wherein 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.

[0026] In some embodiments, the antibody further comprises a second antigen binding region that is capable of binding to human EGFR and comprises the CDR1 sequence shown in SEQ ID NO: 5, the CDR2 sequence shown in SEQ ID NO: 6, and the CDR3 sequence shown in SEQ ID NO: 7. In some embodiments, the second antigen binding region is a single domain antibody, and wherein 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 with SEQ ID NO: 8.

[0027] 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 Vδ2 and comprising the CDR1 sequence shown in SEQ ID NO:1, the CDR2 sequence shown in SEQ ID NO:2, and the CDR3 sequence shown 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.

[0028] In some embodiments, the first antigen binding region is a single domain antibody, and wherein 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 wherein 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.

[0029] 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 SEQ ID NO: 8 or consists of it.

[0030] In some embodiments, the antibody further comprises an Fc region, wherein the Fc region is preferably a heterodimer comprising two Fc polypeptides, wherein the first antigen binding region is fused to the first Fc polypeptide and the second antigen binding region is fused to the second Fc polypeptide, and wherein the first Fc polypeptide and the second Fc polypeptide comprise asymmetric amino acid mutations that favor the formation of heterodimers rather than homodimers, wherein preferably the CH3 region of the Fc polypeptide comprises said asymmetric amino acid mutations, and wherein preferably 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 human IgG1 according to the EU numbering system.

[0031] In some embodiments, the first and second Fc polypeptides comprise a mutation at position 234 and / or 235, preferably the first and second Fc polypeptides comprise L234F and L235E substitutions, wherein the amino acid positions correspond to human IgG1 according to the EU numbering system.

[0032] In some embodiments, the first Fc polypeptide comprises the sequence shown in SEQ ID NO:11 and the second Fc polypeptide comprises the sequence shown in SEQ ID NO:12, or the first Fc polypeptide comprises the sequence shown in SEQ ID NO:12 and the second Fc polypeptide comprises the sequence shown in SEQ ID NO:11.

[0033] In some embodiments, the antibody comprises or consists of the sequence shown in SEQ ID NO:16 and SEQ ID NO:17, or comprises or consists of the sequence shown in SEQ ID NO:16 and SEQ ID NO:18.

[0034] In some embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject a pharmaceutical composition described herein.

[0035] In some embodiments, the present disclosure provides a pharmaceutical composition for use as a medicament, preferably for treating cancer.

[0036] Other aspects and embodiments of the invention are described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Representative chromatograms showing the size exclusion profile of Protein A purified LAVA compounds (VHH 5C8 shown) using a Superdex-75 column. Fractions of the major monomer peak (fractions 1E11-1G2) were pooled and quantified.

[0038] Figure 2A representative example of labchip polyacrylamide gel electrophoresis of purified VHH 5C8 is shown. Left: non-reducing conditions; right: reducing conditions.

[0039] Figure 3A-3B Purified VHH 5C8 ( Figure 3A ) and VHH 5C8var1( Figure 3B )’s HP-SEC profile.

[0040] Figure 4A-4B A representative HP-SEC profile of purified bispecific VHH (bsVHH) 1D12var5-5C8var1 is shown. Figure 4A : bsVHH 1D12var5-5C8var1 batch, expressed and purified from the supernatant of Pichia pastoris culture by protein A affinity chromatography. Figure 4B : bsVHH 1D12var5-5C8var1 batch, expressed and purified from HEK-293E cells by protein A and size exclusion chromatography.

[0041] Figure 5 Shown is Labchip analysis of purified VHH 5C8var1-Y105F and 5C8var1-Y105S under non-reducing conditions.

[0042] Figure 6A-6B The results for VHH 5C8var1-Y105F ( Fig. 6A ) and 5C8var1-Y105S( Figure 6B ) by HP-SEC analysis.

[0043] Figure 7A-7C Affinity measurements of VHH fragments binding to recombinant Vγ9Vδ2-TCR protein using BLI are shown. Protein mass (response, nm) is plotted as a function of time. The dashed vertical line separates the association phase (left) from the dissociation phase (right). VHH 5C8var1 ( Fig. 7A ); VHH 5C8var1-Y105F( Figure 7B ); VHH 5C8var1-Y105S( Figure 7C ). The straight black line represents the data fit to the actual measured response.

[0044] Figure 8A-8D It was shown that both bsVHH 7D12var8-5C8var1-Y105F and bsVHH 7D12-5C8 induced potent Vγ9Vδ2 T cell activation and led to Vγ9Vδ2 T cell-mediated tumor cell lysis. Figure 8A-BA 4-hour degranulation assay is shown: the percentage of CD107A (LAMP-1) + Vγ9Vδ2 T cells is plotted as a function of the expression of 7D12-5C8 (non-humanized) ( Fig. 8A ) or 7D12var8-5C8var1-Y105F( Figure 8B ) as a function of the antibody concentration. Figure 8C-Figure 8D A 24 hour cytotoxicity assay is shown showing the percentage of A431 tumor cells killed as a function of the number of cells killed using 7D12-5C8 (non-humanized) ( Figure 8C ) or 7D12var8-5C8var1-Y105F( Fig.8D ) as a function of the antibody concentration.

[0045] Fig. 9 Binding of 7D12var8-5C8var1(Y105F)-Fc to primary γδ T cells isolated from healthy human PBMCs using flow cytometry is shown. The two panels represent two different donors.

[0046] Figure 10A-10C HT29 ( Fig. 10A )、A-431( Fig. 10B ) and HCT-116( Fig. 10C Binding of 7D12var8-5C8var1(Y105F)-Fc to EGFR on tumor cells in ) cells

[0047] Fig.11 Degranulation of patient-derived γδ T cells induced by 7D12var8-5C8var1(Y105F)-Fc is shown to be dependent on the A431 cell line.

[0048] Fig.12 Viability of A-388 cells co-cultured with patient-derived γδ T cells and 7D12-5C8 is shown.

[0049] Fig.13 Shown are lysed tumor cells after 4 h of incubation of isolated 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 Vγ9Vδ2 T cells (1:1 E:T ratio) and 7D12-5C8 (50 nM) or medium control.

[0050] Fig.14Shown are tumor cell lysis after 24 h of incubation of isolated tumor cell suspensions (peritoneal CRC metastases: n=4) with healthy donor-derived Vγ9Vδ2 T cells (1:1 E:T ratio) and 7D12-5C8var1(Y105S)-Fc (50 nM), gp120-5C8var1(Y105S)-Fc (50 nM), or medium control.

[0051] Fig.15 Construct structures for nonhuman primate studies are shown.

[0052] Figure 16A-16B The binding of 7A5-7D12var8-Fc to EGFR ( Fig.16A ) and TCR( Fig. 16B ) binding of antigen targets.

[0053] Figure 17A-17B 7A5-7D12var8-Fc mediated degranulation ( Fig.17A ) and cytotoxicity ( Fig. 17B ).

[0054] Fig.18 PK analysis of 7A5-7D12var8-Fc concentrations in the blood of three treated animals is shown. The concentration-time curves indicate that the molecule has IgG-like PK.

[0055] Figure 19A-19B The total number of T cells (CD3+, Fig.19A ) and the number of Vγ9-positive cells (percentage of CD3+ population) ( Fig.19B ). Arrows indicate compound injections. Numbers in the legends are the number of monkeys that received treatment.

[0056] Fig. 20 The IL-6 cytokine levels in the blood of treated animals are shown over time. Only low levels of the cytokine were observed, and the release was mainly limited to after the first injection. The arrows indicate the time of treatment. DETAILED DESCRIPTION

[0057] definition

[0058] When used in this article, the term "human Vδ2" refers to the rearranged δ2 chain of Vγ9Vδ2-T cell receptor (TCR). UniProtKB-A0JD36 (A0JD36_HUMAN) gives an example of a variable Vδ2 sequence. Vδ2 is a part of the δ chain of Vγ9Vδ2-TCR. Antibodies that can bind to human Vδ2 can bind to an epitope that is completely located in the variable region, or to an epitope that is located in the constant region, or to an epitope that is a combination of variable region and constant region residues of the δ chain.

[0059] As used herein, the term "human Vγ9" refers to the rearranged y9 chain of the Vγ9Vδ2-T cell receptor (TCR). Examples of variable TRGV9 sequences are given in UniProtKB-Q99603_HUMAN.

[0060] When used herein, the term "human Vγ9Vδ2-T cell receptor" refers to the Vγ9Vδ2-T cell receptor (TCR) found on human γδ T cells.

[0061] When used herein, the term "EGFR" refers to human EGFR protein (UniProtKB-P00533 (EGFR_HUMAN)).

[0062] The term "antibody" is intended to refer to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of any of them, which has the ability to specifically bind to an antigen under typical physiological conditions, with a half-life of a significant period of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four 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, etc., or any other relevant functionally defined period of time (such as a time sufficient to induce, promote, enhance and / or modulate a physiological response associated with antibody binding to an antigen and / or a time sufficient for the antibody to recruit effector activity). The antigen binding region (or antigen binding domain) that interacts with an antigen may comprise the variable regions of both the heavy and light chains of an immunoglobulin molecule, or may be a single domain antigen binding region, such as only a heavy chain variable region. The constant regions of the antibodies, if 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 CIq, the first component in the classical pathway of complement activation.

[0063] The Fc region of an immunoglobulin is defined as an antibody fragment typically produced after digestion of an antibody with papain, which includes two CH2-CH3 regions of an immunoglobulin and a connecting region, such as a hinge region. The constant domain of the antibody heavy chain defines the antibody isotype, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD or IgE. The Fc region mediates the effector functions of the antibody together with cell surface receptors known as Fc receptors and complement system proteins.

[0064] 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 216-230 according to EU numbering.

[0065] 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 any other subtype described herein.

[0066] 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 any other subtype described herein.

[0067] References in the present invention to amino acid positions in the Fc region / Fc domain are according to EU numbering (Edelman et al., Proc Natl Acad Sci US A. 1969 May; 63(1):78-85; Kabat et al., Sequences of proteins of immunological interest. 5th edition - 1991 NIH Publication No. 91-3242).

[0068] As described above, unless otherwise specified or clearly contradictory to the context, as used herein, the term antibody includes antibody fragments that retain the ability to specifically bind to an antigen. It has been demonstrated that the antigen-binding function of an antibody can be performed by a fragment 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 VL, VH, CL, and CH1 domains, or monovalent antibodies as described in WO2007059782; (ii) F(ab')2 fragments, i.e., bivalent fragments comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragments consisting essentially of VH and CH1 domains; and (iv) Fv fragments consisting essentially of the VL and VH domains of a single arm of an antibody. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, the two domains can be connected using a recombinant method by a synthetic linker that enables the two domains to become a single protein chain, in which the VL region and the VH region are paired to form a monovalent molecule (called a single-chain antibody or single-chain Fv (scFv); see, for example, Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Unless otherwise indicated in the context, such single-chain antibodies are encompassed in the term antibody. Although such fragments are generally included in the meaning of antibodies, they are collectively and independently unique features of the present invention, showing different biological properties and utilities. Unless otherwise indicated, the term antibody also includes polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies and humanized antibodies, as well as antibody fragments provided by any known technology (such as enzyme cleavage, peptide synthesis and recombinant technology).

[0069] In some embodiments of the antibodies of the present invention, the first antigen binding region or the second antigen binding region or both are single domain antibodies. Single domain antibodies are well known to those skilled in the art, see, for example, Hamers-Casterman et al. (1993) Nature 363:446, Roovers et al. (2007) Curr Opin Mol Ther 9:327 and Krah et al. (2016) Immunopharmacol Immunotoxicol 38:21. Single domain antibodies include a single CDR1, a single CDR2 and a single CDR3. Examples of single domain antibodies are variable fragments of heavy chain antibodies only, antibodies that naturally do not include light chains, single domain antibodies derived from conventional antibodies and engineered antibodies. Single domain antibodies can be derived from any species, including mice, humans, camels, llamas, sharks, goats, rabbits and cattle. For example, single domain antibodies can be derived from antibodies produced in camelid species (such as camels, dromedaries, llamas, alpacas and guanacos). Like whole antibodies, single-domain antibodies are able to selectively bind to specific antigens. Single-domain antibodies may contain only the variable domains of an immunoglobulin chain, namely CDR1, CDR2, and CDR3, and the framework regions. Such antibodies are also called or VHH.

[0070] As used herein, the term "immunoglobulin" is intended to refer to a class of structurally related glycoproteins, which are composed of two pairs of polypeptide chains, a pair of light (L) chains and a pair of heavy (H) chains, all four chains may be interconnected by disulfide bonds. As used herein, the term "immunoglobulin heavy chain", "heavy chain of immunoglobulin" or "heavy chain" is intended to refer to a chain of immunoglobulin. The heavy chain generally comprises 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 generally comprises three domains CH1, CH2 and CH3. The heavy chain constant region further comprises a hinge region. In the structure of an immunoglobulin (e.g., IgG), two heavy chains are interconnected by disulfide bonds in the hinge region. Like the heavy chain, each light chain generally comprises several regions; a light chain variable region (VL) and a light chain constant region (CL). In addition, the VH and VL regions can be subdivided into regions of high variability (or hypervariable regions, which 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 is typically composed of three CDRs and four FRs arranged from amino terminus to carboxyl 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 protein of immunological interest, fifth edition NIH publication. Various methods for CDR determination and amino acid numbering can be compared at www.abysis.org (UCL).

[0071] 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 a heavy chain constant region gene. Each heavy chain isotype can be combined with a kappa (κ) or lambda (λ) light chain. The antibodies of the present invention can have any isotype.

[0072] The term "parent antibody" is to be understood as an antibody identical to the antibody according to the invention, but wherein the parent antibody does not have one or more specified mutations. A "variant" or "antibody variant" or "variant of a parent antibody" of the invention is an antibody molecule comprising one or more mutations compared to a "parent antibody". Amino acid substitutions may replace a natural amino acid with another naturally occurring amino acid, or with 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 the amino acid classes reflected in one or more of the following three tables:

[0073] Amino acid residue categories for conservative substitutions

[0074] Acidic residue Asp(D) and Glu(E) Basic residues Lys(K), Arg(R) and His(H) Hydrophilic uncharged residues Ser(S), Thr(T), Asn(N) and Gln(Q) Aliphatic uncharged residue Gly(G), Ala(A), Val(V), Leu(L) and Ile(I) Nonpolar uncharged residues Cys(C), Met(M) and Pro(P) Aromatic residues Phe(F), Tyr(Y) and Trp(W)

[0075] Alternative conservative amino acid residue substitution categories

[0076] 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W

[0077] Alternative physical and functional classification of amino acid residues

[0078]

[0079] In the context of the present invention, substitutions in variants are represented as: original amino acid - position - substituted amino acid. Three letter codes or one letter codes, including the codes Xaa and X, are used to indicate amino acid residues. Thus, the notation "T366W" means that the variant comprises a substitution of threonine with tryptophan at the variant amino acid position corresponding to amino acid position 366 of the parent antibody.

[0080] In addition, the term "substitution" includes substitutions in any of the other nineteen natural amino acids, or substitutions in other amino acids such as non-natural amino acids. For example, the 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.

[0081] The term "full length antibody" when used herein refers to an antibody that contains all heavy and light chain constant and variable domains that correspond to those domains normally found in a wild-type antibody of that isotype.

[0082] The term "chimeric antibody" refers to an antibody in which the variable region is derived from a non-human species (e.g., derived from a rodent) and the constant region is derived from a different species (such as a human). Chimeric antibodies can be produced by genetic engineering. Chimeric monoclonal antibodies for therapeutic applications are developed to reduce antibody immunogenicity.

[0083] The term "humanized antibody" refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain modified to contain a high level of sequence homology with a human variable domain. This can be achieved by transplanting the six non-human antibody complementary determining regions (CDRs) that form an antigen binding site together to a homologous human receptor framework region (FR). In order to fully rebuild the binding affinity and specificity of the parent antibody, it may be necessary to replace the framework residues from the parent antibody (i.e., non-human antibody) with human framework regions (reverse mutations). Structural homology modeling can help identify amino acid residues that are critical to the binding properties of antibodies in the framework region. Therefore, a humanized antibody may include a non-human CDR sequence, mainly a human framework region, which optionally includes one or more amino acid reverse mutations to a non-human amino acid sequence, and optionally a completely human constant region. Optionally, additional amino acid modifications that are not necessarily reverse mutations may be introduced to obtain humanized antibodies with preferred properties (such as affinity and biochemical properties). Humanization of non-human therapeutic antibodies is performed to minimize their immunogenicity in humans, while such humanized antibodies retain the specificity and binding affinity of the non-human derived antibody.

[0084] The term "multispecific antibody" refers to an antibody that is specific for at least two different, such as at least three, usually non-overlapping epitopes. Such epitopes may be on the same target antigen, or on different target antigens. If the epitopes are on different targets, such targets may be on the same cell or on different cells or cell types. In some embodiments, the multispecific antibody may include one or more single domain antibodies.

[0085] The term "bispecific antibody" refers to an antibody that has specificity for two different, usually non-overlapping epitopes. Such epitopes can be on the same or different targets. If the epitopes are on different targets, such targets can be on the same cell or on different cells or cell types. In some embodiments, a bispecific antibody may include one or two single domain antibodies.

[0086] Examples of different classes of multispecifics 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, wherein each side of the molecule contains at least two Fab fragments of different antibodies or portions of the Fab fragments; (iii) IgG fusion molecules, wherein a full-length IgG antibody is fused to an additional Fab fragment or a portion of a Fab fragment; (iv) Fc fusion molecules, wherein a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or a portion thereof; (v) Fab fusion molecules, wherein different Fab fragments are fused together, fused to a heavy chain constant domain, an Fc region, or a portion thereof; and (vi) antibodies and heavy chain antibodies based on scFv and diabodies (e.g., domain antibodies, ), wherein different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, ) are fused to each other or to another protein or carrier molecule fused to the heavy chain constant domain, Fc region or a portion thereof.

[0087] Examples of IgG-like molecules with complementary CH3 domain molecules include, but are not limited to (TrionPharma / Fresenius Biotech), Knobs-into-Holes (Genentech), CrossMAbs (Roche) and electrostatic matching (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), chain 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 molecule (Genmab).

[0088] Examples of recombinant IgG-like dual targeting molecules include, but are not limited to, dual targeting (DT)-Ig (GSK / Domantis, WO2009058383), two-in-one antibodies (Genentech, Bostrom et al. 2009. Science 323, 1610-1614), cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star), ZybodiesTM (Zyngenia, LaFleur et al. MAbs. 2013 March-April; 5(2):208-18), methods using common light chains, κλ bodies (NovImmune, WO2012023053) and (CovX / Pfizer, Doppalapudi, VR et al. 2007. Bioorg. Med. Chem. Lett. 17, 501-506).

[0089] Examples of IgG fusion molecules include, but are not limited to, dual variable domain (DVD)-Ig (Abbott), dual domain diabodies (Unilever; Sanofi Aventis), IgG-like bispecifics (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 fusions (Novartis), scFv fusions (Changzhou Adam Biotech Inc), and TvAb (Roche).

[0090] Examples of Fc fusion molecules include, but are not limited to, scFv / Fc fusions (Academic Institution, Pearce et al. Biochem Mol Biol Int. 1997 Sep; 42(6): 1179), SCORPION (Emergent BioSolutions / Trubion, Blankenship JW et al. AACR 100th Annual meeting 2009 (Abstract #5465); Zymogenetics / BMS, WO2010111625), dual affinity redirection technology (Fc-DARTTM) (MacroGenics) and bis(ScFv)2-Fab (National Research Center for Antibody Medicine—China).

[0091] Examples of Fab fusion bispecific antibodies include, but are not limited to, F(ab)2 (Medarex / AMGEN), bi- or bi-Fab (Genentech), (DNL) (ImmunoMedics), bivalent bispecific (Biotecnol), and Fab-Fv (UCB-Celltech).

[0092] Examples of scFv-based, diabodies, and domain antibodies include, but are not limited to, bispecific T cell engagers (Micromet, tandem diabodies (Tandab) (Affimed), dual affinity redirection technology (DARTTM) (MacroGenics), single-chain diabodies (Academic, Lawrence FEBS Lett. 1998 Apr 3; 425(3):479-84), TCR-like antibodies (AIT, Receptor Logics), human serum albumin ScFv fusions (Merrimack, WO2010059315) and COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 Aug; 88(6):667-75), dual targeting (Ablynx, Hmila et al., FASEB J. 2010), dual targeting heavy chain domain only antibodies.

[0093] In the context of antibody and antigen binding, the term "binding" or "specific binding" refers to the binding of an antibody to a predetermined antigen or target (e.g., human Vδ2 or human EGFR), which typically has a specific binding affinity corresponding to about 10 -6M or smaller, e.g. 10 -7 M or less, such as about 10 -8 M or less, such as about 10 -9 M or smaller, about 10 -10 M or less, or about 10 -11 The KD value can be determined by using, for example, surface plasmon resonance (SPR) technology in BIAcore T200 or biolayer interferometry (BLI) in OctetRED96 instruments using antigen as ligand and using binding moiety or binding molecule as analyte. Specific binding means that the antibody binds to a predetermined antigen with an affinity corresponding to the KD, and the affinity is at least 1 / 10 of the affinity of the antibody for binding to a non-specific antigen (e.g., BSA, casein) other than a predetermined antigen or a closely related antigen, such as at least 1 / 100, for example at least 1 / 1,000, such as at least 1 / 10,000, for example at least 1 / 100,000, for example at least 1 / 100,000. The degree to which the affinity is lower depends on the KD of the binding moiety or binding molecule, such that when the KD of the binding moiety or binding molecule is very low (i.e., the binding moiety or binding molecule is highly specific), then the affinity for the antigen can be at least 1 / 10,000 lower than the affinity for the non-specific antigen. As used herein, the term "KD" (M) refers to the dissociation equilibrium constant for a specific interaction between an antigen and a binding moiety or binding molecule.

[0094] In the context of the present invention, "competition" or "capable of competing" or "competes" refers to any detectable significant reduction in the tendency of a specific binding molecule (e.g., EGFR antibody) to bind to a specific binding partner in the presence of another molecule (e.g., different EGFR antibodies) that binds to a specific binding partner. Typically, competition means that the presence of another molecule, such as an antibody, causes a reduction in binding by at least about 25%, such as at least about 50%, for example at least about 75%, such as at least 90%, as determined by, for example, ELISA analysis or flow cytometry using a sufficient amount of two or more competing molecules (e.g., antibodies). Other methods for determining binding specificity by competitive inhibition can be found in, e.g., 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)).

[0095] In one embodiment, the antibodies of the invention bind to the same epitope on EGFR as antibody 7D12 and / or to the same epitope on Vδ2 as antibody 5C8. There are a variety of methods known in the art for localizing antibody epitopes on target antigens, including but not limited to: cross-linked mass spectrometry, which can identify peptides belonging to the epitope, and X-ray crystallography, which can identify individual residues on the antigen that form the epitope. Epitope residues can be defined as at least one atom that is less than or equal to the distance from the antibody All amino acid residues in As the epitope cutoff distance, to allow atoms within the van der Waals radius plus possible water-mediated hydrogen bonds. Next, epitope residues can be defined as those with at least one atom less than or equal to All amino acid residues less than or equal to As the epitope cutoff distance, to allow for the length of the extended arginine amino acid. Cross-linking coupled mass spectrometry first combines the antibody and antigen with a mass-tagged chemical cross-linker. High-quality MALDI detection is then used to confirm the presence of the complex. Since the Ab / Ag complex is extremely stable after cross-linking chemistry, a variety of different enzymes and digestion conditions can be applied to the complex to provide many different overlapping peptides. These peptides are identified using high-resolution mass spectrometry and MS / MS techniques. The identification of the cross-linked peptides is determined using mass tags associated with the cross-linking reagent. After MS / MS fragmentation and data analysis, the cross-linked, antigen-derived peptides are part of the epitope, while the antibody-derived peptides are part of the paratope. All residues between the most N-terminal cross-linked residue and the most C-terminal cross-linked residue found in a single cross-linked peptide are considered to be part of the epitope or paratope. The epitope of antibody 7D12 has been 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, N420) that corresponds to the ligand binding site of domain III.

[0096] When used herein, the terms "first" and "second" antigen binding regions do not refer to their orientation / position in the antibody, i.e., they have no meaning for N-terminus or C-terminus. The terms "first" and "second" are only used to correctly and consistently refer to two different antigen binding regions in the claims and the specification.

[0097] When used in this article, "% sequence identity" refers to the number of identical nucleotide or amino acid positions shared by different sequences (i.e., % identity = number of identical positions / total number of positions × 100), taking into account the number of gaps that need to be introduced for optimal alignment and the length of each gap. 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), which has been 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.

[0098] Other aspects and embodiments of the invention

[0099] The present disclosure provides a pharmaceutical composition comprising an antibody comprising an antigen binding region capable of binding to human Vδ2. The present disclosure also provides a pharmaceutical composition comprising an antibody comprising a first antigen binding region capable of binding to human EGFR and a second antigen binding region capable of binding to human Vδ2. The pharmaceutical composition described herein may include a diluent, a filler, a salt, a buffer, a detergent (e.g., a nonionic detergent, such as Tween-20 or Tween-80), a stabilizer (e.g., a sugar or protein-free amino acid), a preservative, a tissue fixative, a solubilizer, and / or other materials suitable for inclusion in a pharmaceutical composition. Other pharmaceutically acceptable excipients include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants, and absorption delaying agents that are physiologically compatible with the antibodies of the present invention.

[0100] As mentioned, in a first aspect, the present invention relates to a pharmaceutical composition comprising:

[0101] (a) an antibody comprising an antigen binding region capable of binding to human Vδ2, wherein the first antigen binding region comprises a CDR1 sequence as shown in SEQ ID NO: 1, a CDR2 sequence as shown in SEQ ID NO: 2, and a CDR3 sequence as shown in SEQ ID NO: 3;

[0102] (b) 5-20 mM histidine, wherein the pH of the composition is between 5.5 and 6.5, or 5-20 mM sodium acetate, wherein the pH of the composition is between 5.0 and 6.0;

[0103] (c) 250-350 mM sucrose;

[0104] (d) 0.01% to 0.05% (weight / volume) polysorbate 80; and

[0105] (e) 0-20 mM methionine.

[0106] In one embodiment, the composition comprises 5-20 mM histidine and the pH is between 5.5 and 6.5. In one embodiment, the composition comprises 10 mM histidine and the pH is between 5.5 and 6.5.

[0107] In another embodiment, the composition comprises 5-20 mM sodium acetate and the pH is between 5.0 and 6.0.

[0108] In one embodiment, the composition comprises between 250 and 300 mM sucrose. In one embodiment, the composition comprises 280 mM sucrose.

[0109] In one embodiment, the composition comprises between 0.01% and 0.03% polysorbate 80. In one embodiment, the composition comprises 0.02% polysorbate 80.

[0110] In one embodiment, the composition comprises between 0.5 and 20 mM methionine. In one embodiment, the composition comprises 1 mM methionine.

[0111] In one embodiment, the composition comprises between 0.2 and 25 mg / mL of the antibody. In one embodiment, the composition comprises between 0.2 and 20 mg / mL of the antibody. In one embodiment, the composition comprises 1 or 10 mg / mL of the antibody.

[0112] In one embodiment, the composition comprises:

[0113] (a) 5-20 mM, such as 10 mM histidine, and a pH between 5.5 and 6.5, and

[0114] (b) between 250 and 300 mM, such as 280 mM sucrose, and

[0115] (c) between 0.01% and 0.03%, such as 0.02% polysorbate 80.

[0116] In one embodiment, the composition comprises:

[0117] (a) 10 mM histidine, and a pH between 5.5 and 6.5, and

[0118] (b) 280 mM sucrose, and

[0119] (c) 0.02% Polysorbate 80.

[0120] In one embodiment, the composition comprises:

[0121] (a) 5-20 mM, such as 10 mM histidine, and a pH between 5.5 and 6.5, and

[0122] (b) between 250 and 300 mM, such as 280 mM sucrose, and

[0123] (c) between 0.01% and 0.03%, such as 0.02% polysorbate 80, and

[0124] (d) Between 0.5 and 20 mM methionine, such as 1 mM methionine.

[0125] In one embodiment, the composition comprises:

[0126] (a) 10 mM histidine, and a pH between 5.5 and 6.5, and

[0127] (b) 280 mM sucrose, and

[0128] (c) 0.02% polysorbate 80, and

[0129] (d) 1 mM methionine.

[0130] In one embodiment, the composition comprises between 0.2 and 20 mg / mL, such as 1 or 10 mg / mL of the antibody.

[0131] In one embodiment, the composition comprises:

[0132] (a) 5-20 mM, such as 10 mM histidine, and a pH between 5.5 and 6.5, and

[0133] (b) between 250 and 300 mM, such as 280 mM sucrose, and

[0134] (c) between 0.01% and 0.03%, such as 0.02% polysorbate 80, and

[0135] (d) 1 mg / mL of the antibody.

[0136] In one embodiment, the composition comprises:

[0137] (a) 10 mM histidine, and a pH between 5.5 and 6.5, and

[0138] (b) 280 mM sucrose, and

[0139] (c) 0.02% polysorbate 80, and

[0140] (d) 1 mg / mL of the antibody.

[0141] In one embodiment, the composition comprises:

[0142] (a) 5-20 mM, such as 10 mM histidine, and a pH between 5.5 and 6.5, and

[0143] (b) between 250 and 300 mM, such as 280 mM sucrose, and

[0144] (c) between 0.01% and 0.03%, such as 0.02% polysorbate 80, and

[0145] (d) between 0.5 and 20 mM methionine, such as 1 mM methionine, and

[0146] (e) 1 mg / mL of the antibody.

[0147] In one embodiment, the composition comprises:

[0148] (a) 10 mM histidine, and a pH between 5.5 and 6.5, and

[0149] (b) 280 mM sucrose, and

[0150] (c) 0.02% polysorbate 80, and

[0151] (d) 1 mM methionine, and

[0152] (e) 1 mg / mL of the antibody.

[0153] In one embodiment, the composition comprises:

[0154] (a) 5-20 mM, such as 10 mM histidine, and a pH between 5.5 and 6.5, and

[0155] (b) between 250 and 300 mM, such as 280 mM sucrose, and

[0156] (c) between 0.01% and 0.03%, such as 0.02% polysorbate 80, and

[0157] (d) 10 mg / mL of the antibody.

[0158] In one embodiment, the composition comprises:

[0159] (a) 10 mM histidine, and a pH between 5.5 and 6.5, and

[0160] (b) 280 mM sucrose, and

[0161] (c) 0.02% polysorbate 80, and

[0162] (d) 10 mg / mL of the antibody.

[0163] In one embodiment, the composition comprises:

[0164] (a) 5-20 mM, such as 10 mM histidine, and a pH between 5.5 and 6.5, and

[0165] (b) between 250 and 300 mM, such as 280 mM sucrose, and

[0166] (c) between 0.01% and 0.03%, such as 0.02% polysorbate 80, and

[0167] (d) between 0.5 and 20 mM methionine, such as 1 mM methionine, and

[0168] (e) 10 mg / mL of the antibody.

[0169] In one embodiment, the composition comprises:

[0170] (a) 10 mM histidine, and a pH between 5.5 and 6.5, and

[0171] (b) 280 mM sucrose, and

[0172] (c) 0.02% polysorbate 80, and

[0173] (d) 1 mM methionine, and

[0174] (e) 10 mg / mL of the antibody.

[0175] In one embodiment, the composition comprises:

[0176] (a) 5-20 mM, such as 10 mM histidine, and a pH between 5.5 and 6.5, and

[0177] (b) between 250 and 300 mM, such as 280 mM sucrose, and

[0178] (c) between 0.01% and 0.03%, such as 0.02% polysorbate 80, and

[0179] (d) 20 mg / mL of the antibody.

[0180] In one embodiment, the composition comprises:

[0181] (a) 10 mM histidine, and a pH between 5.5 and 6.5, and

[0182] (b) 280 mM sucrose, and

[0183] (c) 0.02% polysorbate 80, and

[0184] (d) 20 mg / mL of the antibody.

[0185] In one embodiment, the composition comprises:

[0186] (a) 5-20 mM, such as 10 mM histidine, and a pH between 5.5 and 6.5, and

[0187] (b) between 250 and 300 mM, such as 280 mM sucrose, and

[0188] (c) between 0.01% and 0.03%, such as 0.02% polysorbate 80, and

[0189] (d) between 0.5 and 20 mM methionine, such as 1 mM methionine, and

[0190] (e) 20 mg / mL of the antibody.

[0191] In one embodiment, the composition comprises:

[0192] (a) 10 mM histidine, and a pH between 5.5 and 6.5, and

[0193] (b) 280 mM sucrose, and

[0194] (c) 0.02% polysorbate 80, and

[0195] (d) 1 mM methionine, and

[0196] (e) 20 mg / mL of the antibody.

[0197] As described above, in the first aspect, the present invention relates to a pharmaceutical composition comprising an antibody comprising an antigen binding region capable of binding to human Vδ2, wherein the antigen binding region comprises a CDR1 sequence as shown in SEQ ID NO: 1, a CDR2 sequence as shown in SEQ ID NO: 2, and a CDR3 sequence as shown in SEQ ID NO: 3.

[0198] In one embodiment, X1 in SEQ ID NO: 1 is S (Ser). In another embodiment, X1 in SEQ ID NO: 1 is G (Gly).

[0199] In one embodiment, X2 in SEQ ID NO: 3 is F (Phe). In another embodiment, X2 in SEQ ID NO: 3 is S (Ser).

[0200] In one embodiment, X1 in SEQ ID NO: 1 is S (Ser), and X2 in SEQ ID NO: 3 is F (Phe).

[0201] In one embodiment, X1 in SEQ ID NO: 1 is S (Ser), and X2 in SEQ ID NO: 3 is S (Ser).

[0202] In one embodiment, X1 in SEQ ID NO: 1 is G (Gly), and X2 in SEQ ID NO: 3 is F (Phe).

[0203] In one embodiment, X1 in SEQ ID NO: 1 is G (Gly), and X2 in SEQ ID NO: 3 is S (Ser).

[0204] In some embodiments, the present invention relates to a pharmaceutical composition comprising an antibody comprising an antigen binding region capable of binding to human Vδ2, wherein the antigen binding region comprises a CDR1 sequence as shown in SEQ ID NO: 21, a CDR2 sequence as shown in SEQ ID NO: 2, and a CDR3 sequence as shown in SEQ ID NO: 19. In some embodiments, the present invention relates to a pharmaceutical composition comprising an antibody comprising an antigen binding region capable of binding to human Vδ2, wherein the antigen binding region comprises a CDR1 sequence as shown in SEQ ID NO: 21, a CDR2 sequence as shown in SEQ ID NO: 2, and a CDR3 sequence as shown in SEQ ID NO: 20.

[0205] In a preferred embodiment, the antibody is capable of activating human Vγ9Vδ2 T cells. The activation of Vγ9Vδ2 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 proteins (e.g., cytokines or chemokines) profiles. In a preferred embodiment, the antibody is capable of inducing activation (e.g., upregulating CD69 and / or CD25 expression), resulting in degranulation, characterized by increased CD107a expression and / or cytokine production (e.g., TNF, IFNγ) of Vγ9Vδ2 T cells.

[0206] In another preferred embodiment, when tested as described in Example 9 herein, the antibody is capable of increasing the number of CD107a-positive cells by at least 2-fold, such as at least 5-fold, for example at a concentration of 1 nM, preferably 100 pM, preferably 10 pM, preferably 1 pM, even more preferably 100 fM. In another preferred embodiment, the antibody of the invention, when tested as described in Example 9 herein using Vγ9Vδ2 T cells and A431 target cells, increases the percentage of CD107a-positive cells with an EC50 value of 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.

[0207] In one embodiment, the antigen binding region is a single domain antibody. Therefore, in one embodiment, the antibody in the pharmaceutical composition of the present invention comprises a single domain antibody capable of binding to human Vδ2, wherein the antigen binding region comprises a CDR1 sequence as shown in SEQ ID NO: 1, a CDR2 sequence as shown in SEQ ID NO: 2, and a CDR3 sequence as shown in SEQ ID NO: 3.

[0208] In another embodiment, the antigen binding region is humanized, wherein preferably the antigen binding region comprises or consists of: (a) the sequence shown 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 with the sequence shown in SEQ ID NO:4.

[0209] In one embodiment, X1 in SEQ ID NO:4 is S (Ser). In another embodiment, X1 in SEQ ID NO:4 is G (Gly). In one embodiment, X2 in SEQ ID NO:4 is F (Phe). In another embodiment, X2 in SEQ ID NO:4 is S (Ser). In one embodiment, X1 in SEQ ID NO:4 is S (Ser), and X2 in SEQ ID NO:4 is F (Phe).

[0210] In one embodiment, X1 in SEQ ID NO:4 is S (Ser), and X2 in SEQ ID NO:4 is S (Ser). In one embodiment, X1 in SEQ ID NO:4 is G (Gly), and X2 in SEQ ID NO:4 is F (Phe). In one embodiment, X1 in SEQ ID NO:4 is G (Gly), and X2 in SEQ ID NO:4 is S (Ser).

[0211] In some embodiments, the antigen binding region is humanized, wherein preferably the antigen binding region comprises or consists of the sequence shown in SEQ ID NO: 23. In some embodiments, the antigen binding region is humanized, wherein preferably the antigen binding region comprises or consists of the sequence shown in SEQ ID NO: 24.

[0212] In some embodiments, the antibody in the pharmaceutical composition of the present invention is a multispecific antibody, such as a bispecific antibody. Therefore, 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.

[0213] In another embodiment, the antibody is a bispecific antibody, wherein the first antigen-antigen binding region and the second antigen binding region are both single domain antibodies. In another embodiment, the multispecific antibody is a bispecific antibody, wherein the first antigen binding region is a single domain antibody, and the second antigen binding region is a single domain antibody.

[0214] In one embodiment, the antibody contained in the pharmaceutical composition of the present invention comprises a second antigen binding region, and the second antigen binding region is capable of binding to human EGFR. Bispecific antibodies against Vγ9Vδ2-T cells and EGFR have been shown to induce potent Vγ9Vδ2 T cell activation and tumor cell lysis in vitro and in vivo mouse xenograft models (deBruin et al. (2018) Oncoimmunology 1, e1375641).

[0215] In some embodiments, the antibody comprises an antigen binding region comprising the CDR1 sequence shown in SEQ ID NO:5, the CDR2 sequence shown in SEQ ID NO:6, and the CDR3 sequence shown in SEQ ID NO:7.

[0216] In one embodiment, the antigen binding region is humanized.

[0217] In another embodiment, the antibody comprises an antigen binding region comprising or consisting of: (a) the sequence shown 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 shown in SEQ ID NO:8.

[0218] In another embodiment, the antibody competes (i.e., is capable of competing) for binding to human EGFR with an antibody having the sequence shown in SEQ ID NO:8, and preferably the antibody binds to the same epitope on human EGFR as the antibody having the sequence shown in SEQ ID NO:8.

[0219] In another embodiment, an 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 shown in SEQ ID NO:1, the CDR2 sequence shown in SEQ ID NO:2, and the CDR3 sequence shown in SEQ ID NO:3, and wherein the second antigen binding region comprises the CDR1 sequence shown in SEQ ID NO:5, the CDR2 sequence shown in SEQ ID NO:6, and the CDR3 sequence shown in SEQ ID NO:7.

[0220] In another embodiment, an 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 shown in SEQ ID NO:21, the CDR2 sequence shown in SEQ ID NO:2, and the CDR3 sequence shown in SEQ ID NO:19, and wherein the second antigen binding region comprises the CDR1 sequence shown in SEQ ID NO:5, the CDR2 sequence shown in SEQ ID NO:6, and the CDR3 sequence shown in SEQ ID NO:7.

[0221] In another embodiment, an 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 shown in SEQ ID NO:21, the CDR2 sequence shown in SEQ ID NO:2, and the CDR3 sequence shown in SEQ ID NO:20, and wherein the second antigen binding region comprises the CDR1 sequence shown in SEQ ID NO:5, the CDR2 sequence shown in SEQ ID NO:6, and the CDR3 sequence shown in SEQ ID NO:7.

[0222] In another 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 sequence shown in SEQ ID NO: 4, and the second antigen binding region comprises the sequence shown in SEQ ID NO: 8. In a further embodiment of the present invention:

[0223] (a) X1 in SEQ ID NO:4 is S (Ser), and X2 in SEQ ID NO:4 is F (Phe), or

[0224] (b) X1 in SEQ ID NO:4 is S (Ser), and X2 in SEQ ID NO:4 is S (Ser), or

[0225] (c) X1 in SEQ ID NO:4 is G (Gly), and X2 in SEQ ID NO:4 is F (Phe), or

[0226] (d) X1 in SEQ ID NO: 4 is G (Gly), and X2 in SEQ ID NO: 4 is S (Ser).

[0227] In another embodiment, an 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 sequence shown in SEQ ID NO: 23, and the second antigen binding region comprises the sequence shown in SEQ ID NO: 8. In a further embodiment, an 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 sequence shown in SEQ ID NO: 24, and the second antigen binding region comprises the sequence shown in SEQ ID NO: 8.

[0228] In another embodiment, the antibody is capable of mediating killing of human EGFR expressing cells. In a preferred embodiment, the antibody is capable of increasing Vγ9Vδ2 T cell-mediated killing of EGFR expressing cells such as A431 cells by at least 25%, such as at least 50%, for example at least 2-fold, when tested as described in Example 9 herein.

[0229] In another embodiment, the antibody is unable to mediate killing of EGFR negative cells, such as EGFR negative human cells.

[0230] 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 by a peptide linker, for example, the length of the linker is 1 to 20 amino acids, for example 1 to 10 amino acids, such as 2, 3, 4, 5, 6, 7, 8 or 10 amino acids. In one embodiment, the peptide linker comprises or consists of the sequence GGGGS shown in SEQ ID NO:9.

[0231] 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 Vδ2 is located at the C-terminus of the second antigen binding region capable of binding to human EGFR.

[0232] In one embodiment, the antibody contained in the pharmaceutical composition of the present invention further comprises a half-life extension domain. In one embodiment, when administered to a human subject, the terminal half-life of the antibody is longer than about 168 hours. Most preferably, the terminal half-life is 336 hours or longer. The "terminal half-life" of an antibody, when used herein, refers to the time required for the serum concentration of the polypeptide to decrease by 50% in the final stage of elimination in the body.

[0233] In one embodiment, the antibody further comprises a half-life extension domain, and the half-life extension domain is an Fc region. In another embodiment, the antibody is a multispecific antibody, such as a bispecific antibody comprising an Fc region. Various methods for preparing bispecific antibodies have been described in the art, for example, by Brinkmann and Kontermann (2017) MAbs9: 182 for review. In one embodiment of the present invention, the Fc region is a heterodimer comprising two Fc polypeptides, wherein the first antigen binding region is fused to the first Fc polypeptide, and the second antigen binding region is fused to the second Fc polypeptide, and wherein the first Fc polypeptide and the second Fc polypeptide comprise asymmetric amino acid mutations, which are conducive to the formation of heterodimers rather than homodimers. (See, for example, Ridgway et al. (1996) 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng 9: 617). In another embodiment thereof, the CH3 region of the Fc polypeptide comprises said asymmetric amino acid mutation, preferably the first Fc polypeptide comprises a T366W substitution, and the second Fc polypeptide comprises a T366S, L368A and Y407V substitution, or vice versa, wherein the amino acid position corresponds to human IgG1 according to the EU numbering system. In another embodiment, the cysteine ​​residue at position 220 in the first Fc polypeptide and the second Fc polypeptide has been deleted or substituted, wherein the amino acid position corresponds to human IgG1 according to the EU numbering system. In another embodiment, the region comprises the hinge sequence shown in SEQ ID NO: 10.

[0234] In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide contain mutations that render the antibody inert, i.e., unable to mediate effector functions or reduced ability to mediate effector functions. In one embodiment, the inert Fc region is additionally unable to bind C1q. In one embodiment, the first Fc polypeptide and the second Fc polypeptide comprise mutations at positions 234 and / or 235, preferably the first Fc polypeptide and the second Fc polypeptide comprise L234F and L235E substitutions, wherein the amino acid positions correspond to human IgG1 according to the EU numbering system. In another embodiment, the antibody contains L234A mutations, L235A mutations, and P329G mutations. In another embodiment, the antibody contains L234F mutations, L235E mutations, and D265A mutations.

[0235] In a preferred embodiment, the first antigen binding region comprises the sequence shown in SEQ ID NO:4, the second antigen binding region comprises the sequence shown in SEQ ID NO:8, and

[0236] (a) the first Fc polypeptide comprises the sequence shown in SEQ ID NO: 11, and the second Fc polypeptide comprises the sequence shown in SEQ ID NO: 12, or

[0237] (b) the first Fc polypeptide comprises the sequence shown in SEQ ID NO:12, and the second Fc polypeptide comprises the sequence shown in SEQ ID NO:11.

[0238] In one embodiment, X3 in SEQ ID NO: 11 is K. In another embodiment, X3 in SEQ ID NO: 11 is absent.

[0239] In one embodiment, X3 in SEQ ID NO: 12 is K. In another embodiment, X3 in SEQ ID NO: 12 is absent.

[0240] In another preferred embodiment, the antibody comprises or consists of the sequences shown in SEQ ID NO:16 and SEQ ID NO:17.

[0241] In one embodiment, X3 in SEQ ID NO: 16 is K. In another embodiment, X3 in SEQ ID NO: 16 is absent.

[0242] In one embodiment, X3 in SEQ ID NO: 17 is K. In another embodiment, X3 in SEQ ID NO: 17 is absent.

[0243] In another preferred embodiment, the antibody comprises or consists of the sequences shown in SEQ ID NO:16 and SEQ ID NO:18.

[0244] In one embodiment, X3 in SEQ ID NO: 18 is K. In another embodiment, X3 in SEQ ID NO: 18 is absent.

[0245] In another main aspect, the invention relates to a pharmaceutical composition according to the invention as described herein for use as a medicament.

[0246] The antibodies used in the pharmaceutical composition according to the present invention are capable of creating a microenvironment that is favorable for Vγ9Vδ2 T cells to kill tumor cells. Therefore, in a preferred embodiment, the antibodies are used to treat cancer.

[0247] In one embodiment, the pharmaceutical composition is used to treat primary or metastatic colon or colorectal cancer. In another embodiment, the pharmaceutical composition is used to treat peritoneal cancer. In another embodiment, the pharmaceutical composition is used to treat liver cancer. In another embodiment, the pharmaceutical composition is used to treat head and neck squamous cell carcinoma (HNSCC). In another embodiment, the pharmaceutical composition is used to treat non-small cell lung cancer (NSCLC). In another embodiment, the pharmaceutical composition is used to treat cutaneous squamous cell carcinoma.

[0248] Similarly, the present invention relates to a method of treating a disease, said method comprising administering to a human subject in need thereof a pharmaceutical composition according to the present invention as described herein. In one embodiment, the disease is cancer.

[0249] In some embodiments, the pharmaceutical composition is administered as a monotherapy. However, the pharmaceutical compositions of the invention may also be administered in combination therapy, ie, in combination with other therapeutic agents relevant to the disease or condition to be treated.

[0250] "Treatment" or "treating" refers to the administration of an effective amount of a pharmaceutical composition according to the present invention to alleviate, improve, prevent, eradicate (cure) or prevent symptoms or disease states. "Effective amount" refers to an effective amount to achieve the desired therapeutic result at the necessary dosage and time period. The effective amount of a polypeptide such as an antibody may vary according to factors such as the disease state, 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 an amount in which any toxic or deleterious effects of the antibody outweigh the therapeutically beneficial effects. Administration can be performed by any suitable route, but is typically parenteral, such as intravenous, intramuscular or subcutaneous.

[0251] The multispecific antibodies used in the present invention are usually produced in a recombinant manner, i.e., by expressing a nucleic acid construct encoding the antibody in a suitable host cell, and then purifying the recombinant antibody produced from the cell culture. The nucleic acid construct can be prepared by standard molecular biology techniques well known in the art. The construct is usually introduced into the host cell using an expression vector. Suitable nucleic acid constructs and expression vectors are known in the art. Host cells suitable for recombinantly expressed antibodies are known in the art and include CHO, HEK-293, Expi293F, PER-C6, NS / 0 and Sp2 / 0 cells.

[0252] Table 1: Sequence Listing

[0253]

[0254]

[0255]

[0256] All references, articles, publications, patents, patent publications and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the mention of any reference, article, publication, patent, patent publication and patent application herein is not and should not be taken as an admission or any form of suggestion that it constitutes valid prior art or forms part of the common general knowledge in any country in the world.

[0257] Example

[0258] Example 1: Production and purification of VHH compounds

[0259] VHH compounds were primarily produced by transient transfection of the encoding plasmid in HEK293-E253 cells and purification of the protein from the conditioned medium (one week after production) by protein A affinity chromatography followed by preparative gel filtration. The major monomer peak (fractions 1E11-1G2) observed during preparative size exclusion using a Superdex-75 column was purified as Figure 1 shown.

[0260] In polyacrylamide gel electrophoresis, the purified protein migrated as a single band under reducing and non-reducing conditions. Figure 2 Representative examples are shown.

[0261] Example 2: HP-SEC analysis of purified VHH compounds

[0262] The purified VHH compounds were subjected to HP-SEC analysis using the Waters Acquity ARC-bio system. 10 μg of antibody (10 μL antibody at a concentration of 1 mg / mL) was injected into a Waters BEH200 SEC column (bead size 2.5 μm, column size 7.8x300 mm). The mobile phase consisted of 50 mM sodium phosphate, 0.2 M sodium chloride buffer pH 7.0, and the buffer speed used in the run was 0.8 mL / min. Proteins were detected by measuring the absorbance at a wavelength of 214 nm. The total analysis time for each injection was 15 minutes. The preparation and purification of VHH compounds are as described in Example 1. Surprisingly, when the integrity and monomericity of VHH 5C8 (SEQ ID NO: 13) (previously described in WO 2015 / 156673) and 5C8var1 (SEQ ID NO: 14) (previously described in WO 2020 / 060405) were tested in HP-SEC analysis, two peaks were observed. (See Figure 3A-3B ).

[0263] Example 3: Mass spectrometry analysis of 5C8 revealed an additional mass of 80 Da

[0264] To determine if the two isoforms of 5C8 differ in mass, a protein preparation of 5C8 was analyzed by LC-ESI-MS mass spectrometry. The major species found in this analysis was 5C8 without post-translational modifications or a signal peptide. The second species was a protein with a mass difference of +80.3 Daltons (Da), suggesting that sulfation or phosphorylation may have occurred. This was further investigated by treating with phosphatases or sulfatases and then analyzing the peptides by LC-ESI-MS mass spectrometry after proteolytic digestion. The results showed that sulfatase treatment reduced the mass of the peptide containing Y105 (the 7th residue of CDR3 (SEQ ID NO:15)) by 80Da, while phosphatase treatment had no effect. This demonstrates that Y105 in 5C8 has undergone a post-translational modification by sulfation. This sulfation is present in approximately 30% of the protein preparation.

[0265] Example 4: 1D12var5-5C8var1 containing the same anti-Vγ9Vδ2 VHH exhibits the same heterogeneity in HP-SEC and has the same 80Da extra mass

[0266] 1D12var5-5C8var1 is a bispecific VHH compound consisting of an anti-CD1d VHH coupled to 5C8var1 via a flexible linker (described in SEQ ID NO:87 in WO 2020 / 060405). This protein was expressed in HEK 293E cells as described above. In addition, protein preparations were obtained from different expression systems: the bispecific VHH was also expressed in Pichia pastoris and Chinese hamster ovary (CHO) cells. When different protein preparations were tested in HP-SEC analysis, pre-peaks were systematically observed. (See Figure 4A-4B ).

[0267] The observed pre-peaks indicated that a significant proportion of the proteins were different isoforms. Since the 5C8 VHH was shown to be sulfated and 5C8var1 contained exactly the same CDR3 sequence, the 1D12var5-5C8var1 batches were also analyzed for their molecular weight by mass spectrometry. Depending on the protein batch, between 15% and 40% of the proteins were found to contain an 80Da extra mass. This is consistent with the sulfation observed for VHH 5C8.

[0268] Example 5: In silico analysis of VHH 5C8 and 5C8var1

[0269] Based on PDB ID 5M2W, using Maestro Build homology models for 5C8 and 5C8var1. CDR1 and CDR3 require Prime De novo loop prediction was performed for refinement. The generated model showed that CDR3 residue Y105 showed a 205.1 The high solvent accessible surface area of ​​5C8 model showed 122.2 of high solvent accessible surface area and is therefore expected to aid in antigen binding. Subsequently, the reactive residues in the model were analyzed to indicate residues prone to post-translational modifications (PTMs). Next, the protein sequence was analyzed using the sequence-based PTM prediction tool ModPred. Table 2 lists the modifications predicted in the structure and sequence. A single predicted PTM could not explain the mass differences observed in the HP-SEC analysis. The type describes the PTM predicted by Maestro. *Q13 deamidation was only predicted for 5C8.

[0270] Table 2: Predicted PTMs of 5C8 and 5C8var1 in Maestro and ModPred.

[0271]

[0272]

[0273] Example 6: Design and production of 5C8var1 VHH CDR3 mutants Y105F and Y105S

[0274] Homology modeling as described in Example 5 was used to introduce mutations that prevent sulfation of Y105 in 5C8 and 5C8var1. Two different mutants were designed based on the model structure of the VHH: Y105S (retaining the alcohol function) and Y105F (retaining the aromatic ring). Residue Y105 is the 7th residue of CDR3; by introducing mutations, an effect on binding is expected. 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 remains identical to that of the non-humanized VHH.

[0275] Both 5C8var1-Y105F and 5C8var1-Y105S were produced well and appeared as monomeric proteins in preparative size exclusion (data not shown). Both proteins were of high purity ( Figure 5 ), and migrates as a single species in polyacrylamide gel electrophoresis.

[0276] Example 7: HP-SEC analysis of purified VHHs containing designed CDR3 mutations

[0277] HP-SEC analysis was performed as described for 5C8. Both 5C8var1-Y105F and 5C8var1-Y105S were analyzed. Figure 6A-6B .

[0278] From the HP-SEC analysis of purified VHH molecules containing the designed CDR3 mutations, it can be concluded that heterogeneity is no longer observed for any of the mutants. This indicates that the observed Y105 post-translational modification is absent and that the protein is homogeneous.

[0279] Example 8: Affinity measurements of 5C8var1, 5C8var1-Y105F and 5C8va1-Y105S using biolayer interferometry (BLI) showed no difference in affinity

[0280] Binding of the 5C8var1 VHH antibody fragment and variants 5C8var1-Y105F and 5C8var1-Y105S to the Vy9Vδ2 TCR was measured by biolayer interferometry using an Octet RED96 instrument (ForteBio). Recombinant human Vy9Vδ2-Fc fusion protein (20 μg / ml) was captured as a ligand to an anti-human Fc capture biosensor. Sensorgrams were recorded when the ligand capture biosensor was incubated with a series of dilutions of the VHH antibody fragment (40 to 0.63 nM) in 10X kinetic buffer (ForteBio). Global data fit of a 1:1 binding model was used to estimate k on (association rate constant) and koff These values ​​are used to calculate the dissociation rate constant using KD = k off / k on Calculate KD (equilibrium dissociation constant).

[0281] from Figure 7A-7C As well as Table 3 it can be concluded that the KD values ​​of the two different Y105 VHH mutants are not significantly different from those of 5C8var1. In particular the Y105F mutant has comparable affinity to 5C8var1. The values ​​shown in Table 3 are mean values ​​+ / - standard deviations of at least three independent measurements.

[0282] Table 3: Affinity values ​​of VHHs found in BLI measurements for binding to recombinant Vγ9Vδ2 TCR protein.

[0283] VHH compounds KD(nM)+ / -SD 5C8var1 0.81+ / -0.02 5C8var1-Y105F 0.78+ / -0.23 5C8var1-Y105S 1.59+ / -0.31

[0284] Example 9: The functionality of the anti-(EGFR x Vγ9Vδ2 TCR) bispecific VHH containing the Y105F mutation is fully retained

[0285] To determine whether the equal affinity of VHH 5C8var1-Y105F and 5C8var1 can be translated into comparable functionality, a bispecific VHH 7D12var8-5C8var1-Y105F was designed: the humanized anti-EGFR VHH 7D12var8 (based on the VHH described in Gainkam et al. (2008) J Nucl Med 49(5):788) was coupled 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 activation of Vγ9Vδ2 T cells dependent on an EGFR-positive tumor cell line (A431) and to cause T cell-mediated tumor cell lysis. In brief, Vγ9Vδ2 T cells were isolated from the blood of healthy donors using a combination of magnetic activated cell sorting (MACS) and anti-Vδ2 antibodies 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 line and PBMCs from different donors. Vγ9Vδ2 T cells were always >90% pure when used in the assay (double positive staining for Vγ9 and Vδ2 in FACS). The A431 cell line (ATCC, catalog number CRL-1555) was cultured as recommended by the supplier. For activation or cytotoxicity assays, 50,000 tumor target cells were placed in 96-well tissue culture plates the day before the assay. The next day, 50,000 amplified, purified Vγ9Vδ2 T cells were added to the culture medium along with a range of bispecific VHH compounds at a certain concentration. In the activation assay, a mixture of labeled anti-CD3 and anti-CD107A antibodies added to the mixture was used to assess Vy9Vδ2-T cell degranulation. After 4 hours, cells were harvested, washed and analyzed by FACS for the expression of degranulation marker CD107A. For cytotoxicity assays, the next day, CytoTox-Glo cytotoxicity assay kit (Promega G9290) was used to check the presence of proteases (indicating cell death) in the supernatant of co-culture. Detergents were used to perform cell lysis for reaching 100% killing rate at the end of the assay.

[0286] Figure 8A-8D 7D12var8-5C8var1-Y105F and non-humanized 7D12-5C8 induced potent Vγ9Vδ2 T cell activation ( Fig. 8A and Figure 8B ) and tumor cell lysis ( Figure 8C and Fig.8D). These results are consistent with the potency of the non-humanized "precursor" molecule 7D12wt-5C8 without the Y105 mutation. Table 4 shows the EC50 values ​​obtained after curve fitting. Compared with 7D12-5C8, 7D12var8-5C8var1-Y105F had a slightly lower EC50 in the cytotoxicity assay.

[0287] Table 4: GraphPad software Figure 8A-8D The EC50 values ​​were obtained after curve fitting of the data shown.

[0288]

[0289]

[0290] The maximum tumor cell killing level for 7D12var8-5C8var1-Y105F was slightly lower than that observed for 7D12-5C8. However, these are two different measurements made using two different Vγ9Vδ2 T cell donors, and this maximum cytotoxicity level may be particularly donor-dependent.

[0291] Example 10: The temperature stability of VHH 5C8var1 containing the Y105 mutation is not altered

[0292] In order to determine whether the mutations introduced in different variants affect the thermal stability of VHH folding, the melting temperature of the mutants was measured using NanoDSF (differential scanning fluorescence). The antibody samples were diluted with PBS until they were equal to the sample with the lowest concentration. The antibody samples were then filled into nanoDSF-grade capillaries and measured with PrometheusNT.48. During the experiment, the temperature rose from 20°C to 95°C. The intrinsic fluorescence of the protein was detected at 350 and 330nm and recorded together with the amount of reflected light. Through these measurements, the apparent melting temperature (Tm) and the aggregation initiation temperature (Tagg) were determined. For all three antibody fragments, the starting melting temperature (T on ) and melting temperatures (Tm) (Table 5). The melting temperatures measured for 5C8var1-Y105F and 5C8var1-Y105S are consistent with that of 5C8var1: Table 5.

[0293] Table 5: 5C8var1-Y105F and 5C8var1-Y105S showed similar melting temperatures as 5C8var1, indicating that the introduced mutations did not impair stability.

[0294] <![CDATA[T on (℃)]]> Tm(℃) 5C8var1 61.8 86.7 5C8var1-Y105F 64.5 85.5 5C8var1-Y105S 63.9 86.6

[0295] Example 11: Bispecific constructs with extended half-life (containing Fc)

[0296] In order to obtain a molecule with a longer plasma half-life in vivo, the 7D12var8-5C8var1-Y105F bispecific VHH was reformatted into a therapeutic antibody format containing human Fc. Both VHH domains are coupled to human IgG1 Fc (i.e., CH2 and CH3) domains, which have the following characteristics: VHH is coupled to a modified hinge (AAA, followed by SDKTHTCPPCP, cysteine ​​220 missing) and human CH2 and CH3 domains. The CH2 domain is Fc-silenced by the LFLE mutation pair (L234F, L235E), and the CH3 domain is mutated by the "knob into hole" mutation (knob: T366W and hole: T366S, L368A and Y407V), which results in heterodimerization when the two chains are co-expressed in the same cell. This pair of mutations has been described in the scientific literature (Ridgway et al. (1996) Protein Eng 9: 617). The sequence of the construct is shown in SEQ ID NO: 16 and SEQ ID NO: 17. The resulting antibody construct 7D12var8-5C8var1 (Y105F) with an Fc region was called 7D12var8-5C8var1 (Y105F)-Fc. Similarly, a construct in which Y at position 105 was replaced by S (7D12var8-5C8var1 (Y105S)-Fc) was prepared. The sequence of the construct is shown in SEQ ID NO: 16 and SEQ ID NO: 18.

[0297] The protein was prepared by co-transfecting the two expression vectors encoding the protein in HEK293E cells and purified 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.

[0298] Example 12: Binding of 7D12var8-5C8var1(Y105F)-Fc to primary Vγ9Vδ2 T cells isolated from healthy human PBMC

[0299] 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 subsequently expanded as described (de Bruin et al., Clin. Immunology 169 (2016), 128-138; de Bruin et al., J. Immunology 198(1) (2017), 308-317). The expanded polyclonal and pure (>95%) Vγ9Vδ2 T cells were then seeded at a concentration of 50,000 cells / well and incubated with 7D12var8-5C8var1(Y105F)-Fc antibody or GP120-5C8var1(Y105F)-Fc antibody as a positive control, starting at 100 nM in a half-log titration for one hour at 4°C. Binding of antibodies to Vγ9Vδ2 TCR was visualized by flow cytometry using a fluorescently labeled secondary anti-IgG1 antibody. Fig. 9 The mean fluorescence intensity (MFI) signal of anti-IgG1 antibody staining of two different PBMC donors (D336 and D339) measured by flow cytometry is shown. The sigmoidal curve emphasizes the significant binding of 7D12var8-5C8var1(Y105F)-Fc to Vγ9Vδ2 T cells, with a half-maximal effective concentration (EC50) in the low nanomolar range (approximately 3 nM).

[0300] Example 13: Binding of 7D12var8-5C8var1(Y105F)-Fc to EGFR-positive tumor cells by cell-based ELISA

[0301] The binding of 7D12var8-5C8var1(Y105F)-Fc to the 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 different 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 one hour at 37°C. The bound antibodies were then labeled in a secondary incubation step with anti-IgG1-HRP for one hour at 37°C. Secondary antibody binding was then resolved by adding 3,3′,5,5′-tetramethylbenzidine and the colorimetric change caused by HRP followed by the addition of H2SO4 to stop the reaction. The optical density (OD) was then measured at a wavelength of 450 nm in a UV spectrometer. Figure 10A-10C 7D12var8-5C8var1(Y105F)-Fc and A-431( Fig. 10B )、HCT-116( Fig. 10C ) and HT-29( Fig. 10A ) tumor cells with an EC50 of approximately 7 nM, whereas a non-targeting control antibody showed no measurable binding to any of the cell lines tested.

[0302] Example 14: 7D12var8-5C8var1(Y105F)-Fc-induced Vγ9Vδ2 T cell degranulation dependent on A-431 cells

[0303] To investigate the potential of 7D12var8-5C8var1(Y105F)-Fc to activate Vγ9Vδ2 T cells, Vγ9Vδ2 T cells were first isolated and expanded as previously described. Next, Vγ9Vδ2 T cells were then cultured with A-431 tumor cells at a 1:1E:T ratio in the presence of different concentrations of 7D12var8-5C8var1(Y105F)-Fc antibody and PE-labeled anti-CD107a fluorescent antibody. After 24 h, cells were harvested and stained with fluorescently labeled anti-Vγ9 and anti-CD3 antibodies to distinguish Vγ9Vδ2 T cells from tumor cells. The extent of CD107a expression on Vγ9Vδ2 T cells, reflecting target-dependent degranulation, was investigated using flow cytometry. Fig.11It was shown that with increasing concentrations of 7D12var8-5C8var1(Y105F)-Fc, Vγ9Vδ2 T cells were effectively induced to degranulate in a manner dependent on A-431 cells. The EC50 of 7D12var8-5C8var1(Y105F)-Fc-induced Vγ9Vδ2 T cell degranulation was in the picomolar range (approximately 40-90 pM).

[0304] Example 15: Antibody 7D12-5C8 induces T cell-mediated cytotoxicity of target cells

[0305] To investigate whether the bispecific VHH 7D12-5C8 is able to effectively induce Vγ9Vδ2 T cell-mediated cytotoxicity against target cells, the viability of the A-388 epidermoid tumor cell line (ATCC, CRL-7905) was assessed in the context of co-culture with Vγ9Vδ2 T cells and bsVHH antibody fragments. In this assay, Vγ9Vδ2 T cells were isolated from healthy PBMCs as described previously, but were subsequently frozen and stored at -150°C. Frozen Vγ9Vδ2 T cells were thawed and rested overnight in medium supplemented with IL-2. A-388 tumor cells were inoculated alone or with resting Vγ9Vδ2 T cells at a 1:1 or 1:0.1 ratio, with or without 7D12-5C8 (10 nM). As an additional control, Vγ9Vδ2 T cells were inoculated alone, with or without the antibody 7D12-5C8 (10 nM). After 72 h, cell viability was determined by adding ATP Lite (Perkin Elmer, 6016731) and reading the luminescent signal using a microplate reader. Fig.12 ATP-derived fluorescence signals, representing the metabolic activity of live cells as well as the number of live cells, are shown. At a 1:1 E:T ratio, an antibody-induced reduction of approximately 50% in live cells could be observed, whereas untreated A-388 and Vγ9Vδ2 T cell co-cultures were unaffected, highlighting its potential to induce T cell-mediated cytotoxicity.

[0306] Example 16: Killing of tumor cells by 7D12-5C8 and 7D12-5C8var1(Y105S)-Fc activated Vγ9Vδ2 T cells

[0307] To investigate whether bsVHH 7D12-5C8 and antibody 7D12-5C8var1(Y105S)-Fc are able to induce Vγ9Vδ2 T cell-mediated cytotoxicity against patient-derived tumor cells, the viability of such tumor cells was assessed in a Vγ9Vδ2 T cell and antibody co-culture setting. Several different tumor cell types were tested.

[0308] After written informed consent, tissue samples (i.e., primary and metastatic tumor material from colon, peritoneum, and liver, head and neck squamous cell carcinoma (HNSCC), and non-small cell lung cancer (NSCLC)) were collected from cancer patients at Amsterdam UMC (site VUmc). The tissue was minced into small pieces with a surgical blade (No. 22; Swann Morton Ltd), resuspended in dissociation medium consisting of IMDM supplemented with 0.1% DNase I, 0.14% collagenase A, 5% FCS, 100 IU / ml penicillin sodium, 100 μg / ml streptomycin sulfate, and 2.0 mM L-glutamine, transferred to a sterile flask with a stir bar, and incubated for 45 min in a 37°C water bath with a magnetic stirrer. After incubation, the cell suspension was passed through a 100 μM cell strainer. The tumor was dissociated three times, and then the cells were washed and viable cells were counted by trypan blue exclusion.

[0309] Isolated 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 with 7D12-5C8var1(Y105S)-Fc or gp120-5C8var1(Y105S)-Fc for 24 h.

[0310] If necessary, after the incubation period, adherent cells were detached using trypsin-EDTA and resuspended in FACS buffer (PBS supplemented with 0.5% bovine serum albumin and 20 μg / ml NaN3), incubated with fluorescent dye-labeled antibodies for 30 minutes at 4 degrees, and then the staining was measured by flow cytometry using LSR Fortessa XL-20 (BD).

[0311] The life / death marker 7AAD was used with 123counting eBeads according to the manufacturer's instructions. TM 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).

[0312] 7D12-5C8 and 7D12-5C8var1(Y105S)-Fc-induced Vγ9Vδ2 T cell-mediated cytotoxicity of tumor cells was evaluated by incubating expanded healthy donor-derived Vγ9Vδ2 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).

[0313] like Fig.13 As shown, 7D12-5C8 induced Vγ9Vδ2 T cells to lyse patient tumor cells in large quantities (average % lysis induced by 7D12-5C8: CRC primary 52.3% and p-value 0.0003, CRC peritoneum 46.0% and p-value 0.0052, CRC liver 31.8% and p-value 0.0360, head and neck squamous cell carcinoma 46.1% and p-value 0.0187 and non-small cell lung cancer 64.1% and p-value 0.0153).

[0314] In addition, if Fig.14 As shown, 7D12-5C8var1(Y105S)-Fc induced Vγ9Vδ2 T cells to lyse patient tumor cells in large amounts (average % 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 any measurable tumor cell lysis.

[0315] Example 17: Design, production and purification of constructs for non-human primate studies

[0316] For in vivo studies in nonhuman primates, a construct with a binding domain that cross-reacts with the cynomolgus monkey Vγ9 TCR chain was generated ( Fig.15). This binding domain is based on 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 monkey Vγ9Vδ2 T cells (see Example 1 of WO 2021 / 052995). A bispecific Fc antibody consisting of 7A5 and anti-EGFR VHH7D12var8 was constructed. The molecule contains a human IgG1 Fc tail and is designed to form heterodimerization using the knob-in-hole technique (KiH; Carter et al., 2001Imm.Meth.2001:248,7; knob: T366W; hole: T366S, L368A and Y407V). The Vγ9 binding scFv of the 7A5 antibody was coupled to the "knob" chain; the EGFR binding VHH 7D12var8 was cloned in frame with the "hole" chain of the KiHFc pair. In addition, the upper hinge was designed as "AAASDKTHTCPPCP" (SEQ ID NO: 25) to remove the cysteine ​​(C220) that is usually bridged to CL, and more flexibility was introduced by changing "EPK" to "AAA". The N-terminal portion of CH2 was designed to eliminate Fc receptor (CD16, -32 and -64) interactions (silent mutations L234F, L235E) while maintaining FcRN binding. The resulting construct was called 7A5-7D12var8-Fc.

[0317] The molecule was produced by transient co-transfection of two plasmids encoding the two different chains in HEK293E cells and purification 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) ( Figure 16A-16B The functionality of the molecule was demonstrated by showing that it causes target-dependent activation (CD107a expression) of in vitro expanded Vγ9Vδ2 T cells ( Fig.17A ) and subsequent T cell-mediated tumor cell lysis ( Fig. 17B ) to prove it.

[0318] Example 18: Bispecific Antibody 7A5-7D12var8-Fc is Well Tolerated in an Exploratory Multi-Dose Non-Human Primate (NHP: Cynomolgus Monkey) Study

[0319] 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 infused every half hour at 5 mL / kg; 4 infusions were administered per week. The first two dose groups (1 animal per dose) were 1 and 5 mg / kg, respectively, administered simultaneously, and after three (weekly) doses, the third dose group (23 mg / kg) received the first dose. Blood was drawn from the animals regularly for PK analysis, analysis of clinical chemistry parameters, measurement of cytokine levels, and analysis of blood cell subsets by flow cytometry. The day after the last dose, the animals were euthanized and tissues were harvested for histopathology and immunohistochemistry (IHC).

[0320] Pharmacokinetic analysis of 7A5-7D12var8-Fc concentrations in the blood of treated animals (measured by ELISA, Fig.18 ) showed that the antibody exhibited IgG-like PK with a half-life between 84 and 127 hours. In animals dosed at 1 mg / kg, the antibody showed a shorter half-life after the third injection, which may be due to the possible development of anti-drug antibody (ADA) responses in the animals.

[0321] The values ​​of clearance were found to be between 0.36 and 0.72 mL / h / kg, and the volume of distribution was between 58.5 and 115.2 mL / kg. Systemic exposure increased dose-proportionally between 1 and 23 mg / kg. However, accumulation was not observed after repeated dosing.

[0322] The compound was detectable by IHC in different tissues (lymph nodes, muscle, skin, and colon); as expected, the compound staining intensity in these tissues was proportional to the dose (data not shown). Flow cytometric analysis of blood cells showed several transient decreases in lymphocytes, a phenomenon often observed in these types of multiple dose studies and related to the surgical procedure. Fig.19A A temporary decrease in T cell counts at each time point was shown 2 hours after administration. However, two days after injection, T cell numbers returned to baseline levels.

[0323] In contrast, the number of Vγ9-positive T cells in the peripheral blood decreased and did not return to their previous frequency ( Fig.19B These cells were virtually absent throughout the study, demonstrating the specific pharmacodynamic effects of the compound. Measurement of cytokines in the blood of treated animals showed that the treatment induced very little cytokine release, which was almost entirely confined to the first injection of the compound. Fig. 20 IL-6 levels measured as an example are shown.

[0324] The overall conclusion is that treatment of NHPs with 7A5-7D12var8-Fc was very well tolerated and did not show any clinical signs of toxicity. In addition, no macroscopic or microscopic abnormalities were found in any of the organs examined in histopathology (data now shown). In contrast: anti-EGFR x CD3 BiTE is lethal to NHPs at a dose of 31 μg / kg / day of continuous infusion (Lutterbuese et al., Proc Natl Acad Sci US A 2010: 107(28), 12605).

[0325] Example 19: Stable Formulation of 7D12var8-5C8var1(Y105F)-Fc

[0326] Stable CHO cell clones expressing 7D12var8-5C8var1(Y105F)-Fc were generated and the antibody product was produced by a bioreactor system. After harvesting and purification, the antibody product was formulated into concentrations of 1 mg / mL and 10 mg / mL using the four formulation buffers shown in Table 6.

[0327] Table 6: Overview of tested formulations

[0328]

[0329] The formulated samples were subjected to various storage conditions (defined below) for up to 6 weeks. In addition, several stress tests were performed:

[0330] Store at 5℃±3℃

[0331] Store at -80℃±10℃

[0332] Accelerated storage conditions: 25℃±2℃ / 60% relative humidity (RH)±5%

[0333] Thermal stress at 40℃±2℃ / 75%RH±5%

[0334] Freeze-thaw cycles: The samples were completely frozen to -80 ± 10° C. Subsequently, the samples were completely thawed at room temperature (15-25° C.) and then subjected to five freeze-thaw cycles.

[0335] Stirring stress: The sample was stirred at 240 rpm at room temperature (EP 15°C-25°C) for 7 days.

[0336] Oxidation: Add 0.01% (vol / vol) hydrogen peroxide (H2O2) to the samples and incubate at room temperature for 24 hours.

[0337] 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%.

[0338] The initial (= t0) 5°C ± 3°C sample was used as a reference. The samples were analyzed using the analytical method shown in Table 7.

[0339] Table 7: Analytical procedures for drug product evaluation

[0340] method Evaluate Clarity Appearance color Appearance pH pH <![CDATA[A 280 (UV / VIS)]]> content SE-HPLC Completeness Cation Exchange (CIEX)-HPLC Completeness Capillary electrophoresis sodium dodecyl sulfate (CE-SDS non-reducing) Completeness CE-SDS (reduction) Completeness Activity Assay – PSMA Binding ELISA efficacy Peptide mapping Identity Differential Scanning Completeness Liquid chromatography-mass spectrometry (LC-MS) (oxidation) Completeness

[0341] The results of the stability study over time at storage temperatures of -80℃±10℃, 5℃±3℃, 25℃±2℃ / 60%RH±5% and 40℃±2℃ / 75%RH±5% showed no significant difference in clarity, color, pH, A 280 The results of the experiments showed that the product was highly comparable in terms of (UV-VIS), SE-HPLC, CIEX-HPLC, CE-SDS and PSMA binding, differential scanning and peptide fingerprinting. The measurements showed that the product was stable in each of the selected buffer conditions and only slight effects were observed over time. In addition, the results of stress tests (freeze-thaw (five F / T cycles), light, stirring and oxidative stress) showed that the product was stable in each of these conditions.

[0342] The main effect of differentiating buffers was examined under thermal and oxidative stress conditions. CIEX-HPLC analysis of samples stored at 40°C ± 2°C / 75% RH ± 5% showed a decrease in the main variant, while an increase in the acidic variant for all buffers and product concentrations tested. However, histidine-based formulations showed a lower decrease compared to acetate-based formulations. Furthermore, LC-MS analysis after oxidative stress showed that the addition of methionine could effectively prevent oxidation of the product.

[0343] Tables 8 and 9 show the analytical results for the 1 mg antibody / mL and 10 mg antibody / mL formulations most affected after temperature stability and oxidative stress, respectively.

[0344] In summary, all formulation buffers tested were suitable for 7D12var8-5C8var1(Y105F)-Fc. However, the antibody formulated in buffer 1 was the most stable, but the advantage was not significant.

[0345]

[0346]

[0347]

[0348]

Claims

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

2. The pharmaceutical composition of claim 1, wherein the composition comprises 5-20 mM histidine, wherein the pH of the composition is between 5.5 and 6.

5.

3. The pharmaceutical composition of claim 1 or 2, wherein the composition comprises 10 mM histidine.

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

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

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

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

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

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

10. The pharmaceutical composition of any one of claims 1-9, 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 1-11, wherein the composition comprises between 0.2 and 20 mg / mL of the antibody.

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

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

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the first antigen binding region is a single domain antibody, and wherein the first antigen binding region preferably comprises or consists of: a. the sequence shown 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 with the sequence shown in said 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, and wherein the second antigen binding region is preferably a single domain antibody.

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 Vδ2.

17. The pharmaceutical composition of 16, wherein 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.

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

4.

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

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

23.

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

24.

24. 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 EGFR and comprising the CDR1 sequence shown in SEQ ID NO:5, the CDR2 sequence shown in SEQ ID NO:6, and the CDR3 sequence shown 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.

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

8.

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 Vδ2.

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

4.

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

23.

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

24.

30. A pharmaceutical composition comprising: (a) an antibody comprising a first antigen-binding region capable of binding to human Vδ2 and comprising the CDR1 sequence shown in SEQ ID NO: 1, the CDR2 sequence shown in SEQ ID NO: 2, and the CDR3 sequence shown in SEQ ID NO: 3; (b) 5-20 mM histidine, wherein the pH of the composition is between 5.5 and 6.5, or 5-20 mM sodium acetate, wherein the pH of the composition is between 5.0 and 6.0; (c) 250-350 mM sucrose; (d) 0.01% to 0.05% (weight / volume) polysorbate 80; and (e) 0-20 mM methionine.

31. The pharmaceutical composition of claim 30, wherein the composition comprises 5-20 mM histidine, wherein the pH of the composition is between 5.5 and 6.

5.

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

33. A pharmaceutical composition as described in any one of claims 30-32, wherein the composition comprises between 250 and 300 mM sucrose.

34. A pharmaceutical composition as described in any one of claims 30-33, wherein the composition comprises 280 mM sucrose.

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

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

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

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

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

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

42. A pharmaceutical composition as described in any one of claims 30-41, 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 shown in SEQ ID NO:

4.

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

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

23.

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

24.

48. A pharmaceutical composition as described in any one of claims 30-47, wherein the antibody further comprises a second antigen binding region that can bind to human EGFR and comprises the CDR1 sequence shown in SEQ ID NO:5, the CDR2 sequence shown in SEQ ID NO:6, and the CDR3 sequence shown in SEQ ID NO:

7.

49. The pharmaceutical composition of claim 48, wherein the second antigen binding region is a single domain antibody, and wherein 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. 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 Vδ2 and comprising the CDR1 sequence shown in SEQ ID NO: 1, the CDR2 sequence shown in SEQ ID NO: 2, and the CDR3 sequence shown 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.

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

4.

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

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

23.

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

24.

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

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

8.

59. A pharmaceutical composition as described in any one of claims 1 to 58, wherein the antibody further comprises an Fc region, wherein the Fc region is preferably a heterodimer comprising two Fc polypeptides, wherein the first antigen binding region is fused to the first Fc polypeptide and the second antigen binding region is fused to the second Fc polypeptide, and wherein the first Fc polypeptide and the second Fc polypeptide comprise asymmetric amino acid mutations that favor the formation of heterodimers rather than homodimers, wherein preferably the CH3 region of the Fc polypeptide comprises the asymmetric amino acid mutations, and wherein preferably 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 human IgG1 according to the EU numbering system.

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

61. The pharmaceutical composition of claim 59 or 60, wherein a. the first Fc polypeptide comprises the sequence shown in SEQ ID NO: 11, and the second Fc polypeptide comprises the sequence shown in SEQ ID NO: 12, or b. The first Fc polypeptide comprises the sequence shown in SEQ ID NO:12, and the second Fc polypeptide comprises the sequence shown in SEQ ID NO:

11.

62. The pharmaceutical composition of any one of claims 1-61, wherein the antibody comprises or consists of the sequence shown in SEQ ID NO: 16 and SEQ ID NO: 17, or comprises or consists of the sequence shown in SEQ ID NO: 16 and SEQ ID NO:

18.

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

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

Images