Variant antibodies that bind gamma-delta T cell receptors

Abstract

The present invention relates to antibodies capable of binding to the human V [gamma] 9V [delta] 2T cell receptor. The invention further relates to pharmaceutical compositions comprising said antibodies of the invention and to the use of said antibodies of the invention for medical treatment.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to EP 22179260.9 filed on June 15, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] Electronic version of the sequence listing

[0004] The contents of the electronic sequence listing (LVAT_024_01WO_SeqList_ST26.xml; size: 101,824 bytes; and creation date: June 14, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present invention relates to novel antibodies capable of binding to the Vδ2 chain of the human Vγ9Vδ2 T cell receptor. The present invention further relates to pharmaceutical compositions comprising the antibodies of the present invention, and to the use of the antibodies 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 γ chain and a δ chain. Most γδ T cells express a TCR composed of the Vγ9 and Vδ2 regions. Vγ9Vδ2 T cells can fight a variety 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 Vγ9Vδ2T cells can be used to treat infection or cancer because these agents can promote the responsiveness of Vγ9Vδ2T cells to pathogens or infected cells or cancer cells. WO2015156673 describes antibodies that bind to Vγ9Vδ2TCR and are able to activate Vγ9Vδ2T cells. WO2020060405 describes bispecific antibodies that bind to Vγ9Vδ2T cells and tumor cell targets, thereby potentially recruiting Vγ9Vδ2T cells to tumors, thereby stimulating a therapeutic effect. Summary of the invention

[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 improved Vγ9Vδ2 TCR binding antibody sequences that produce more homogeneous products when produced in host cells, 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.

[0010] The production of bispecific antibodies comprising the Vγ9Vδ2-TCR binding antibody 5C8 in mammalian cells (described in WO2015 / 156673) showed that a subpopulation of antibody molecules was proteolytically cleaved within the CDR3 region of 5C8, resulting in heterogeneity of the antibody product, as determined by mass spectrometry analysis of purified proteins. The cleavage occurred in a sequence that was not expected to be susceptible to endoproteolytic degradation. In addition, surprisingly, even if the residue is located in the CDR3 region, this residue before the proteolytic cleavage site can be mutated without affecting the antigen-binding properties of the antibody. It is known that the CDR3 region is the main determinant of antigen binding specificity in the antigen-binding region of an antibody.

[0011] Furthermore, the inventors discovered that antibody 5C8 is sulfated at sites in the antibody that are not expected to be subject to such post-translational modifications. Sulfation occurs in part in various host cells, thereby also producing heterogeneous antibody products. Surprisingly, tyrosine residues that undergo sulfate can be mutated without affecting the antigen binding properties of the antibody, even though the amino acid is also located in the CDR3 region. Removal of sulfation sites by mutation produces a more homogeneous antibody product.

[0012] Therefore, in a first aspect, the present invention provides an antibody comprising a first 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: 12, a CDR2 sequence as shown in SEQ ID NO: 13, and a CDR3 sequence as shown in SEQ ID NO: 14.

[0013] In another aspect, the present invention provides a bispecific antibody comprising a first antigen binding region capable of binding to human Vδ2 as defined herein and a second antigen binding region capable of binding to a second antigen, wherein the second antigen is selected from human EGFR, CD123, PSMA, CD1d, CD40 and Nectin 4. In other aspects, the present invention relates to a pharmaceutical composition comprising an antibody of the present invention, the use of the antibody of the present invention in medical treatment, and to a nucleic acid construct, an expression vector for producing the antibody of the present invention, and to a host cell comprising such a nucleic acid construct or expression vector. In addition, the present invention relates to a method for producing an antibody of the present invention for producing a more uniform product.

[0014] Other aspects and embodiments of the invention are described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Representative chromatogram of 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.

[0016] Figure 2 : Representative example of labchip polyacrylamide gel electrophoresis of purified VHH 5C8. Left: non-reducing conditions; right: reducing conditions.

[0017] Figure 3: HP-SEC profiles of purified VHH 5C8 (A) and VHH 5C8var1 (B).

[0018] Figure 4: Representative HP-SEC profiles of purified bispecific VHH (bsVHH) 1D12var5-5C8var1. A: bsVHH 1D12var5-5C8var1 batch, expressed and purified from the supernatant of Pichia pastoris by protein A affinity chromatography. B: bsVHH 1D12var5-5C8var1 batch, expressed and purified from HEK-293E cells by protein A and size exclusion chromatography.

[0019] Figure 5 : Labchip analysis of purified VHH 5C8var1-Y105F and 5C8var1-Y105S under non-reducing conditions.

[0020] Figure 6: HP-SEC analysis of VHH 5C8var1-Y105F (A) and 5C8var1-Y105S (B).

[0021] Figure 7: Affinity measurement of VHH fragments binding to recombinant Vγ9Vδ2-TCR protein using BLI. 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). A: VHH5C8var1; B: VHH 5C8var1-Y105F; C: VHH 5C8var1-Y105S. The straight black line represents the data fit to the actual measured response.

[0022] Figure 8: Both bsVHH 7D12var8-5C8var1-Y105F and bsVHH 7D12-5C8 induce potent Vγ9Vδ2 T cell activation and lead to Vγ9Vδ2 T cell-mediated tumor cell lysis. A: 4-hour degranulation assay: The percentage of CD107A (LAMP-1) + Vγ9Vδ2 T cells is plotted as a function of the antibody concentration used. Left: 7D12-5C8 (non-humanized); Right: 7D12var8-5C8var1-Y105F. B: 24-hour cytotoxicity assay showing the percentage of A431 tumor cells killed as a function of the antibody concentration used. Left: 7D12-5C8 (non-humanized); Right: 7D12var8-5C8var1-Y105F.

[0023] Fig. 9 : Binding of 7D12var8-5C8var1(Y105F)-Fc to primary Vγ9Vδ2 T cells isolated from healthy human PBMCs using flow cytometry. The two panels represent two different donors.

[0024] FIG. 10 : Binding of 7D12var8-5C8var1(Y105F)-Fc to EGFR on tumor cells by cell-based ELISA.

[0025] Fig.11 : Degranulation of Vγ9Vδ2 T cells (denoted as γδ T cells) induced by 7D12var8-5C8var1(Y105F)-Fc is dependent on the A431 cell line.

[0026] Fig.12 : The viability of A-388 cells co-cultured with Vγ9Vδ2 T cells (denoted as GDT cells) and 7D12-5C8.

[0027] Fig.13 : Isolated tumor cell suspensions (primary CRC: n=10, peritoneal CRC metastasis: n=5, liver CRC metastasis: n=3, primary HNSCC: n=5 and primary NSCLC: n=4) were incubated with healthy donor-derived Vγ9Vδ2 T cells (1:1 E:T ratio) and 7D12-5C8 (50 nM) or medium control for 4 hours before lysing of tumor cells.

[0028] Fig.14 : Isolated tumor cell suspension (peritoneal CRC metastasis: n=4) was incubated 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 for 24 h before lysis of tumor cells.

[0029] Fig.15 : Construct structure for nonhuman primate studies.

[0030] Fig.16 : Binding of 7A5-7D12var8-Fc to antigenic targets.

[0031] Fig.17 :7A5-7D12var8-Fc-mediated degranulation and cytotoxicity.

[0032] Fig.18 : PK analysis of 7A5-7D12var8-Fc concentrations in the blood of three treated animals. The concentration-time curves indicate that the molecule has IgG-like PK.

[0033] Fig.19 : Total T cells (CD3+, left panel) and number of Vγ9-positive cells (percentage of CD3+ population) in the blood of treated animals. Arrows indicate compound injections. Numbers in the legend are the number of monkeys treated.

[0034] Fig. 20 : Changes in IL-6 cytokine levels in the blood of treated animals over time. Only low levels of the cytokine were observed, and the release was mainly limited to after the first injection. Arrows indicate the moment of treatment.

[0035] Fig.21 : SDS-PAGE analysis of purified VHH-Fc "heavy chain only" antibodies. 1 and 2 micrograms of purified protein were loaded to better identify (truncated) impurity products.

[0036] Fig. 22 : Deconvoluted mass spectrum of a VHH-Fc single domain antibody. Signal intensity is plotted as a function of molecular weight (Da). Signals between 78,000 and 79,000 Da are caused by deconvolution artifacts. There is a significant signal at 68,8 kDa.

[0037] Fig.23 |Amino acid sequence of CDR3 of VHH 5C8 (SEQ ID NO: 19) and modifications introduced to avoid sulfation and / or truncation (SEQ ID NO: 36). Y105 (sulfation) was changed to F, and positions 108 and 109 were changed to A (108 and 109) or K (109). In addition, immediately following the R109A mutation, the sequence LGIR in the CDR3 region was deleted (□LGIR, "truncated").

[0038] Figure 24 | Affinity measurement of VHH fragments binding to recombinant Vγ9Vδ2-TCR protein using BLI. 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). A: VHH5C8var1-Y105F; B: VHH 5C8var1-Y105F / G108A; C: VHH 5C8var1-Y105F / R109A; D: VHH5C8var1-Y105F / R109K; E: VHH 5C8var1-Y105F / R109A / □LGIR.

[0039] Fig.25 Binding of bispecific CD123xVδ2, Fc-containing molecules (variants containing position 108 and position 109 of 5C8) to primary expanded Vγ9Vδ2 T cells as measured by flow cytometry. The intensity of the fluorescent signal measured in FACS is plotted as a function of the antibody concentration used.

[0040] Figure 26 | Vγ9Vδ2T cell activation induced by bispecific molecules containing Fc, as measured by the appearance of the degranulation marker LAMP-1 (i.e., CD107A) on the surface of Vγ9Vδ2T cells. After Vγ9Vδ2T cells were co-cultured with tumor target cells (1:1E:T ratio) for 24 hours, the percentage of Vγ9Vδ2T cells expressing CD107A (degranulated, activated Vγ9Vδ2T cells) was measured by flow cytometry as a function of the concentration of the compound used in the assay. Two target cell lines were used: MOLM-13 and THP-1 (as shown).

[0041] Figure 27A-27B Fc-containing anti-CD123 x Vδ2 bispecific molecules-dependent, Vγ9Vδ2 T cell-mediated cytotoxicity of tumor cells is shown. After 24 hours of co-culture of Vγ9Vδ2 T cells with tumor target cells (1:1 E:T ratio), the percentage of live (7AAD negative) cells was determined by flow cytometry and plotted as a function of the concentration of the compound used. Fig.27A Results for MOLM-13 cells are shown. Fig.27B Results for THP-1 cells are shown.

[0042] Figure 28A-28B Analysis of purified proteins by CE-SDS (capillary electrophoresis) is shown. 1D2-Fc x 5C8var1-Y105F-Fc ( Fig.28A ) and 1D2-Fc x5C8var1-Y105F-R109A-Fc( Fig.28B ) electrophoretogram. DETAILED DESCRIPTION

[0043] definition

[0044] As used herein, the term "human Vδ2" refers to the rearranged δ2 chain of the Vγ9Vδ2-T cell receptor (TCR). Examples of variable TRDV2 sequences are given in UniProtKB-A0JD36 (A0JD36_HUMAN).

[0045] 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.

[0046] When used herein, the term "EGFR" refers to human EGFR protein (UniProtKB-P00533 (EGFR_HUMAN)).

[0047] When used in this article, the term "CD123" refers to human CD123 protein, also known as interleukin-3 receptor alpha chain (GenBank accession number NM_002183.4, NCBI reference sequence: NP_002174.1). The IL3 receptor is a heterodimer of CD123 and CD131, and CD131 is a common beta chain (NCBI reference sequence: NP_000386.1).

[0048] As used herein, the term "PSMA" refers to human prostate-specific membrane antigen protein (UniProtKB-Q04609 (FOLH1_HUMAN)).

[0049] As used herein, the term "CD40" refers to the CD40 protein, also known as tumor necrosis factor receptor superfamily member 5 (UniProtKB-P25942 (TNR5_HUMAN)), isotype I.

[0050] When used herein, the term "CD1d" refers to human CD1d protein (UniProtKB-P15813 (CD1D_HUMAN)).

[0051] When used herein, the term "Nectin 4" refers to human Nectin 4 protein (UniProtKB-Q96NY8 (NECT4_HUMAN)).

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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).

[0058] 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).

[0059] 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.

[0060] 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).

[0061] 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.

[0062] 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:

[0063] Amino acid residue categories for conservative substitutions

[0064] 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)

[0065] Alternative conservative amino acid residue substitution categories

[0066] 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W

[0067] Alternative physical and functional classification of amino acid residues

[0068]

[0069]

[0070] 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 the amino acid at position 366 of the parent antibody.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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).

[0079] 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 Mar-Apr; 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).

[0080] 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).

[0081] 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 bi-(ScFv)2-Fab (National Research Center for Antibody Medicine—China).

[0082] 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).

[0083] 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.

[0084] In the context of antibody binding to an antigen, the term "binding" or "specific binding" refers to the binding of an antibody to a predetermined antigen or target, the binding typically having an apparent affinity corresponding to a KD of about 10-6M or less, e.g., 10-7M or less, such as about 10-8M or less, such as about 10-9M or less, about 10-10M or less, or about 10-11M or even less, e.g., when determined using flow cytometry as described in the Examples herein. Alternatively, the KD value can be determined using, e.g., surface plasmon resonance (SPR) technology in a BIAcore T200 or biolayer interferometry (BLI) in an Octet RED96 instrument using an antigen as a ligand and a binding moiety or binding molecule as an analyte. Specific binding means that the antibody binds to a predetermined antigen with an affinity corresponding to a KD, which is at least 1 / 10, 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 of the affinity of the antibody for binding to a non-specific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). The degree to which the affinity is lower depends on the KD of the binding moiety or binding molecule, so 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 is lower than the affinity for the non-specific antigen by at least 1 / 10,000. 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.

[0085] 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 to bind to a specific binding partner in the presence of another molecule that binds to the specific binding partner. Typically, competition means that the binding is reduced by at least about 25%, such as at least about 50%, for example at least about 75%, such as at least 90%, due to the presence of another molecule such as an antibody, 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, for example, Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Colligan et al., ed., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley InterScience NY, (1992, 1993), and Muller, Meth. Enzymol. 92, 589-601 (1983)).

[0086] In one embodiment, the antibody of the present invention binds to the same epitope on EGFR as antibody 7D12, binds to the same epitope on CD123 as antibody 1D2, binds to the same epitope on CD1d as antibody 1D12, binds to the same epitope on PSMA as antibody LV1050, binds to the same epitope on CD40 as antibody v19, binds to the same epitope on Nectin4 as antibodies LV1184, LV1181, LV1185, LV1186, LV1178, LV1183 or LV1187, or binds to the same epitope on Vδ2 as antibody 5C8 or its variants. There are a variety of methods known in the art that can be used to locate antibody epitopes on target antigens, including but not limited to: cross-linking coupled 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 determined 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.

[0087] 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.

[0088] 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.

[0089] Other aspects and embodiments of the invention

[0090] As described above, in the first aspect, the present invention relates to an antibody comprising a first 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:12, a CDR2 sequence as shown in SEQ ID NO:13, and a CDR3 sequence as shown in SEQ ID NO:14.

[0091] In one embodiment, X1 in SEQ ID NO: 12 is S (Ser). In another embodiment, X1 in SEQ ID NO: 12 is G (Gly).

[0092] In one embodiment, X2 in SEQ ID NO: 14 is selected from the group consisting of A, C, D, E, G, H, F, I, K, L, M, N, P, Q, R, S, T, V, W and Y. In one embodiment, X2 in SEQ ID NO: 14 is F or S.

[0093] In one embodiment, X2 in SEQ ID NO: 14 is selected from the group consisting of A, C, D, E, G, H, F, I, K, L, M, N, Q, R, S, T, V, W and Y.

[0094] In one embodiment, X2 in SEQ ID NO: 14 is F (Phe). In another embodiment, X2 in SEQ ID NO: 14 is S (Ser).

[0095] In one embodiment, X1 in SEQ ID NO:12 is S, and X2 in SEQ ID NO:3 is F.

[0096] In one embodiment, X1 in SEQ ID NO: 12 is S, and X2 in SEQ ID NO: 3 is S.

[0097] In one embodiment, X1 in SEQ ID NO:12 is G, and X2 in SEQ ID NO:3 is F.

[0098] In one embodiment, X1 in SEQ ID NO: 12 is G, and X2 in SEQ ID NO: 3 is S.

[0099] In one embodiment, X3 in SEQ ID NO: 14 is selected from the group consisting of A, C, D, E, G, H, F, I, K, L, M, N, P, Q, S, T, V, W and Y.

[0100] In one embodiment, X3 in SEQ ID NO: 14 is selected from the group consisting of A, C, D, E, G, H, F, I, K, L, M, N, Q, S, T, V, W and Y.

[0101] In one embodiment, X3 in SEQ ID NO: 14 is A (Ala). In another embodiment, X3 in SEQ ID NO: 14 is K (Lys).

[0102] In one embodiment, X1 in SEQ ID NO:12 is S and X3 in SEQ ID NO:14 is A.

[0103] In one embodiment, X1 in SEQ ID NO:12 is S, and X3 in SEQ ID NO:14 is K.

[0104] In one embodiment, X1 in SEQ ID NO:12 is G, and X3 in SEQ ID NO:14 is A.

[0105] In one embodiment, X1 in SEQ ID NO:12 is G, and X3 in SEQ ID NO:14 is K.

[0106] In one embodiment, X2 in SEQ ID NO: 14 is F and X3 in SEQ ID NO: 14 is A.

[0107] In one embodiment, X2 in SEQ ID NO: 14 is F and X3 in SEQ ID NO: 14 is K.

[0108] In one embodiment, X2 in SEQ ID NO: 14 is S, and X3 in SEQ ID NO: 14 is A.

[0109] In one embodiment, X2 in SEQ ID NO: 14 is S, and X3 in SEQ ID NO: 14 is K.

[0110] In one embodiment:

[0111] (a) X1 in SEQ ID NO:12 is S, and X2 in SEQ ID NO:14 is F,

[0112] (b) X1 in SEQ ID NO: 12 is S, and X2 in SEQ ID NO: 14 is S,

[0113] (c) X1 in SEQ ID NO:12 is S, and X3 in SEQ ID NO:14 is A, or

[0114] (d) X1 in SEQ ID NO: 12 is S, and X3 in SEQ ID NO: 14 is K.

[0115] In one embodiment, X1 in SEQ ID NO:12 is S, X2 in SEQ ID NO:14 is F, and X3 in SEQ ID NO:14 is A.

[0116] In one embodiment, X1 in SEQ ID NO: 12 is S, X2 in SEQ ID NO: 14 is F, and X3 in SEQ ID NO: 14 is K.

[0117] In one embodiment, X1 in SEQ ID NO: 12 is S, X2 in SEQ ID NO: 14 is S, and X3 in SEQ ID NO: 14 is A.

[0118] In one embodiment, X1 in SEQ ID NO: 12 is S, X2 in SEQ ID NO: 14 is S, and X3 in SEQ ID NO: 14 is K.

[0119] In one embodiment, X1 in SEQ ID NO:12 is G, X2 in SEQ ID NO:14 is F, and X3 in SEQ ID NO:14 is A.

[0120] In one embodiment, X1 in SEQ ID NO: 12 is G, X2 in SEQ ID NO: 14 is F, and X3 in SEQ ID NO: 14 is K.

[0121] In one embodiment, X1 in SEQ ID NO:12 is G, X2 in SEQ ID NO:14 is S, and X3 in SEQ ID NO:14 is A.

[0122] In one embodiment, X1 in SEQ ID NO: 12 is G, X2 in SEQ ID NO: 14 is S, and X3 in SEQ ID NO: 14 is K.

[0123] In one embodiment:

[0124] (a) X1 in SEQ ID NO:12 is S (Ser), X2 in SEQ ID NO:14 is F (Phe), and X3 in SEQ ID NO:14 is A (Ala), or

[0125] (b) X1 in SEQ ID NO: 12 is S (Ser), X2 in SEQ ID NO: 14 is F (Phe), and X3 in SEQ ID NO: 14 is K (Lys).

[0126] In one embodiment:

[0127] (a) X1 in SEQ ID NO:12 is S (Ser), X2 in SEQ ID NO:14 is S (Ser), and X3 in SEQ ID NO:14 is A (Ala), or

[0128] (b) X1 in SEQ ID NO: 12 is S (Ser), X2 in SEQ ID NO: 14 is S (Ser), and X3 in SEQ ID NO: 14 is K (Lys).

[0129] In some embodiments, the present invention relates to an antibody comprising a first antigen binding region capable of binding to human Vδ2, wherein the first antigen binding region comprises a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence of SEQ ID NO: 17. In some embodiments, the antibody comprises a first antigen binding region capable of binding to human Vδ2 and comprising or consisting of an amino acid sequence of SEQ ID NO: 25. In some embodiments, the present invention relates to an antibody comprising a first antigen binding region capable of binding to human Vδ2, wherein the first antigen binding region comprises a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence of SEQ ID NO: 21. In some embodiments, the antibody comprises a first antigen binding region capable of binding to human Vδ2 and comprising or consisting of an amino acid sequence of SEQ ID NO: 29.

[0130] In some embodiments, the present invention relates to an antibody comprising a first antigen binding region capable of binding to human Vδ2, wherein the first antigen binding region comprises a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises a first antigen binding region capable of binding to human Vδ2 and comprising or consisting of an amino acid sequence of SEQ ID NO: 26. In some embodiments, the present invention relates to an antibody comprising a first antigen binding region capable of binding to human Vδ2, wherein the first antigen binding region comprises a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence of SEQ ID NO: 22. In some embodiments, the antibody comprises a first antigen binding region capable of binding to human Vδ2 and comprising or consisting of an amino acid sequence of SEQ ID NO: 30.

[0131] In some embodiments, the present invention relates to an antibody comprising a first antigen binding region capable of binding to human Vδ2, wherein the first antigen binding region comprises a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a first antigen binding region capable of binding to human Vδ2 and comprising or consisting of an amino acid sequence of SEQ ID NO: 27. In some embodiments, the present invention relates to an antibody comprising a first antigen binding region capable of binding to human Vδ2, wherein the first antigen binding region comprises a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence of SEQ ID NO: 23. In some embodiments, the antibody comprises a first antigen binding region capable of binding to human Vδ2 and comprising or consisting of an amino acid sequence of SEQ ID NO: 31.

[0132] In some embodiments, the present invention relates to an antibody comprising a first antigen binding region capable of binding to human Vδ2, wherein the first antigen binding region comprises a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence of SEQ ID NO: 20. In some embodiments, the antibody comprises a first antigen binding region capable of binding to human Vδ2 and comprising or consisting of an amino acid sequence of SEQ ID NO: 28. In some embodiments, the present invention relates to an antibody comprising a first antigen binding region capable of binding to human Vδ2, wherein the first antigen binding region comprises a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence of SEQ ID NO: 24. In some embodiments, the antibody comprises a first antigen binding region capable of binding to human Vδ2 and comprising or consisting of an amino acid sequence of SEQ ID NO: 32.

[0133] In a preferred embodiment, the antibody is capable of activating human Vγ9Vδ2T cells. The activation of Vγ9Vδ2T 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 Vγ9Vδ2T cells.

[0134] 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 present invention, when tested as described in Example 9 herein using Vγ9Vδ2 T cells and A431 target cells, increases the percentage of CD107a-positive cells by 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.

[0135] In one embodiment, the first antigen binding region is a single domain antibody. Therefore, in one embodiment, the antibody of the present invention comprises a single domain antibody capable of binding to human Vδ2, wherein the first antigen binding region comprises a CDR1 sequence as shown in SEQ ID NO: 12, a CDR2 sequence as shown in SEQ ID NO: 13, and a CDR3 sequence as shown in SEQ ID NO: 14.

[0136] In some embodiments, the antibody comprises a single domain antibody that can bind to human Vδ2 and comprises a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence of SEQ ID NO: 17. In some embodiments, the antibody comprises a single domain antibody that can bind to human Vδ2 and comprises an amino acid sequence of SEQ ID NO: 25 or consists thereof. In some embodiments, the antibody comprises a single domain antibody that can bind to human Vδ2 and comprises a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence of SEQ ID NO: 21. In some embodiments, the antibody comprises a single domain antibody that can bind to human Vδ2 and comprises an amino acid sequence of SEQ ID NO: 29 or consists thereof.

[0137] In some embodiments, the antibody comprises a single domain antibody that can bind to human Vδ2 and comprises a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises a single domain antibody that can bind to human Vδ2 and comprises an amino acid sequence of SEQ ID NO: 26 or consists thereof. In some embodiments, the antibody comprises a single domain antibody that can bind to human Vδ2 and comprises a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence of SEQ ID NO: 22. In some embodiments, the antibody comprises a single domain antibody that can bind to human Vδ2 and comprises an amino acid sequence of SEQ ID NO: 30 or consists thereof.

[0138] In some embodiments, the antibody comprises a single domain antibody that can bind to human Vδ2 and comprises a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a single domain antibody that can bind to human Vδ2 and comprises an amino acid sequence of SEQ ID NO: 27 or consists thereof. In some embodiments, the antibody comprises a single domain antibody that can bind to human Vδ2 and comprises a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence of SEQ ID NO: 23. In some embodiments, the antibody comprises a single domain antibody that can bind to human Vδ2 and comprises an amino acid sequence of SEQ ID NO: 31 or consists thereof.

[0139] In some embodiments, the antibody comprises a single domain antibody that can bind to human Vδ2 and comprises a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence of SEQ ID NO: 20. In some embodiments, the antibody comprises a single domain antibody that can bind to human Vδ2 and comprises or consists of an amino acid sequence of SEQ ID NO: 28. In some embodiments, the antibody comprises a single domain antibody that can bind to human Vδ2 and comprises a first antigen binding region that can bind to human Vδ2, wherein the first antigen binding region comprises a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence of SEQ ID NO: 24. In some embodiments, the antibody comprises a single domain antibody that can bind to human Vδ2 and comprises or consists of an amino acid sequence of SEQ ID NO: 32.

[0140] In another embodiment, the first antigen binding region is humanized, wherein preferably the antigen binding region comprises or consists of: (a) the sequence shown in SEQ ID NO: 16, 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: 16, wherein X1 is S (Ser), X2 is F (Phe), and X3 is A (Ala). In another embodiment, the first antigen binding region is humanized and comprises or consists of SEQ ID NO: 25, 26, 27 or 28. In another embodiment, the first antigen binding region is humanized and comprises or consists of SEQ ID NO: 29, 30, 31 or 32.

[0141] In some embodiments, the antibodies of the present invention are multispecific antibodies, such as bispecific antibodies. 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.

[0142] In another embodiment, the antibody is a bispecific antibody, wherein the first 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. In one embodiment, the second antigen binding region is humanized.

[0143] In some embodiments, the antibody comprises a second antigen binding region 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 (de Bruin et al. (2018) Oncoimmunology 1, e1375641).

[0144] In some embodiments, the antibody comprises a second antigen-binding region comprising the CDR1 sequence shown in SEQ ID NO: 33, the CDR2 sequence shown in SEQ ID NO: 34, and the CDR3 sequence shown in SEQ ID NO: 35. In some embodiments, the antibody comprises a second antigen-binding region comprising or consisting of SEQ ID NO: 36, or comprising an amino acid 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: 36.

[0145] In some embodiments, the second antigen binding region competes with (i.e., is capable of competing for) binding to human EGFR with an antibody having the sequence shown in SEQ ID NO:36, and preferably the second antigen binding region binds to the same epitope on human EGFR as the antibody having the sequence shown in SEQ ID NO:36.

[0146] In some embodiments, the antibody comprises (a) a first antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 8, a CDR2 sequence comprising SEQ ID NO: 13, and a CDR3 sequence selected from SEQ ID NOs: 17, 18, 19, and 20; and (b) a second antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 33, a CDR2 sequence comprising SEQ ID NO: 34, and a CDR3 sequence comprising SEQ ID NO: 35. In some embodiments, the antibody comprises (a) a first antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 8, a CDR2 sequence comprising SEQ ID NO: 13, and a CDR3 sequence selected from SEQ ID NOs: 21, 22, 23, and 24; and (b) a second antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 33, a CDR2 sequence comprising SEQ ID NO: 34, and a CDR3 sequence comprising SEQ ID NO: 35.

[0147] In some embodiments, the antibody comprises a first antigen binding region comprising or consisting of a sequence selected from SEQ ID NOs: 25, 26, 27, and 28 and a second antigen binding region comprising or consisting of SEQ ID NO: 36. In some embodiments, the antibody comprises a first antigen binding region comprising or consisting of a sequence selected from SEQ ID NOs: 29, 30, 31, and 32 and a second antigen binding region comprising or consisting of SEQ ID NO: 36.

[0148] In another embodiment, the antibody is capable of mediating killing of human EGFR expressing cells. In a preferred embodiment, when tested as described in Example 9 herein, the antibody is capable of increasing Vγ9Vδ2T 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.

[0149] In another embodiment, the antibody is unable to mediate killing of EGFR negative cells, such as EGFR negative human cells.

[0150] In one embodiment, the antibody of the present invention comprises a second antigen binding region capable of binding to human CD123.

[0151] In some embodiments, the antibody comprises a second antigen-binding region comprising the CDR1 sequence shown in SEQ ID NO: 37, the CDR2 sequence shown in SEQ ID NO: 38, and the CDR3 sequence shown in SEQ ID NO: 39. In some embodiments, the antibody comprises a second antigen-binding region comprising or consisting of SEQ ID NO: 40, or comprising an amino acid 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: 40.

[0152] In some embodiments, the second antigen binding region competes (i.e., is capable of competing) for binding to human CD123 with an antibody having the sequence shown in SEQ ID NO: 40, and preferably the second antigen binding region binds to the same epitope on human CD123 as an antibody having the sequence shown in SEQ ID NO: 40.

[0153] In some embodiments, the antibody comprises (a) a first antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 8, a CDR2 sequence comprising SEQ ID NO: 13, and a CDR3 sequence selected from SEQ ID NOs: 17, 18, 19, and 20; and (b) a second antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 37, a CDR2 sequence comprising SEQ ID NO: 38, and a CDR3 sequence comprising SEQ ID NO: 39. In some embodiments, the antibody comprises (a) a first antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 8, a CDR2 sequence comprising SEQ ID NO: 13, and a CDR3 sequence selected from SEQ ID NOs: 21, 22, 23, and 24; and (b) a second antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 37, a CDR2 sequence comprising SEQ ID NO: 38, and a CDR3 sequence comprising SEQ ID NO: 39.

[0154] In some embodiments, the antibody comprises a first antigen binding region comprising or consisting of a sequence selected from SEQ ID NOs: 25, 26, 27, and 28 and a second antigen binding region comprising or consisting of SEQ ID NO: 40. In some embodiments, the antibody comprises a first antigen binding region comprising or consisting of a sequence selected from SEQ ID NOs: 29, 30, 31, and 32 and a second antigen binding region comprising or consisting of SEQ ID NO: 40.

[0155] In some embodiments, the antibody comprises a second antigen binding region capable of binding to human PSMA. Multispecific antibodies having an antigen binding region capable of binding to human PSMA suitable for use in the present invention have been described, for example, in WO 2022 / 008646 (incorporated herein by reference).

[0156] In some embodiments, the antibody comprises a second antigen-binding region comprising the CDR1 sequence shown in SEQ ID NO: 41, the CDR2 sequence shown in SEQ ID NO: 42, and the CDR3 sequence shown in SEQ ID NO: 43. In some embodiments, the antibody comprises a second antigen-binding region comprising or consisting of SEQ ID NO: 44, or comprising an amino acid 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: 44.

[0157] In some embodiments, the second antigen binding region competes with (i.e., is capable of competing for) binding to human PSMA with the antibody having the sequence shown in SEQ ID NO:44, preferably the second antigen binding region binds to the same epitope on human PSMA as the antibody having the sequence shown in SEQ ID NO:44.

[0158] In some embodiments, the antibody comprises (a) a first antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 8, a CDR2 sequence comprising SEQ ID NO: 13, and a CDR3 sequence selected from SEQ ID NOs: 17, 18, 19, and 20; and (b) a second antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 41, a CDR2 sequence comprising SEQ ID NO: 42, and a CDR3 sequence comprising SEQ ID NO: 43. In some embodiments, the antibody comprises (a) a first antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 8, a CDR2 sequence comprising SEQ ID NO: 13, and a CDR3 sequence selected from SEQ ID NOs: 21, 22, 23, and 24; and (b) a second antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 41, a CDR2 sequence comprising SEQ ID NO: 42, and a CDR3 sequence comprising SEQ ID NO: 43.

[0159] In some embodiments, the antibody comprises a first antigen binding region comprising or consisting of a sequence selected from SEQ ID NOs: 25, 26, 27, and 28 and a second antigen binding region comprising or consisting of SEQ ID NO: 44. In some embodiments, the antibody comprises a first antigen binding region comprising or consisting of a sequence selected from SEQ ID NOs: 29, 30, 31, and 32 and a second antigen binding region comprising or consisting of SEQ ID NO: 44.

[0160] In some embodiments, the antibody comprises a second antigen binding region capable of binding to human CD1d. Antibodies having an antigen binding region capable of binding to human CD1d suitable for incorporation into the multispecific antibodies used in the present invention have been described, for example, in WO 2016 / 122320 and WO 2020 / 060405 (both incorporated herein by reference).

[0161] In some embodiments, the antibody comprises a second antigen-binding region comprising the CDR1 sequence shown in SEQ ID NO: 45, the CDR2 sequence shown in SEQ ID NO: 46, and the CDR3 sequence shown in SEQ ID NO: 47. In some embodiments, the antibody comprises a second antigen-binding region comprising or consisting of SEQ ID NO: 48, or comprising an amino acid 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: 48.

[0162] In some embodiments, the second antigen binding region competes (i.e., is capable of competing) with the antibody having the sequence shown in SEQ ID NO:48 for binding to human CD1d, preferably the second antigen binding region binds to the same epitope on human CD1d as the antibody having the sequence shown in SEQ ID NO:48.

[0163] In some embodiments, the antibody comprises (a) a first antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 8, a CDR2 sequence comprising SEQ ID NO: 13, and a CDR3 sequence selected from SEQ ID NOs: 17, 18, 19, and 20; and (b) a second antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 45, a CDR2 sequence comprising SEQ ID NO: 46, and a CDR3 sequence comprising SEQ ID NO: 47. In some embodiments, the antibody comprises (a) a first antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 8, a CDR2 sequence comprising SEQ ID NO: 13, and a CDR3 sequence selected from SEQ ID NOs: 21, 22, 23, and 24; and (b) a second antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 45, a CDR2 sequence comprising SEQ ID NO: 46, and a CDR3 sequence comprising SEQ ID NO: 47.

[0164] In some embodiments, the antibody comprises a first antigen binding region comprising or consisting of a sequence selected from SEQ ID NOs: 25, 26, 27, and 28 and a second antigen binding region comprising or consisting of SEQ ID NO: 48. In some embodiments, the antibody comprises a first antigen binding region comprising or consisting of a sequence selected from SEQ ID NOs: 29, 30, 31, and 32 and a second antigen binding region comprising or consisting of SEQ ID NO: 48.

[0165] In some embodiments, the antibody comprises a second antigen binding region capable of binding to human CD40. Antibodies with antigen binding regions capable of binding to human CD40 suitable for incorporation into the multispecific antibodies used in the present invention have been described, for example, in WO 2020 / 159368 (incorporated herein by reference).

[0166] In some embodiments, the antibody comprises a second antigen binding region comprising the CDR1 sequence shown in SEQ ID NO: 49, the CDR2 sequence shown in SEQ ID NO: 50, and the CDR3 sequence shown in SEQ ID NO: 51. In some embodiments, the antibody comprises a second antigen binding region comprising or consisting of SEQ ID NO: 52, or comprising an amino acid 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: 52.

[0167] In some embodiments, the second antigen binding region competes with (i.e., is capable of competing for) binding to human CD40 with the antibody having the sequence shown in SEQ ID NO:52, and preferably the second antigen binding region binds to the same epitope on human CD40 as the antibody having the sequence shown in SEQ ID NO:52.

[0168] In some embodiments, the antibody comprises (a) a first antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 8, a CDR2 sequence comprising SEQ ID NO: 13, and a CDR3 sequence selected from SEQ ID NOs: 17, 18, 19, and 20; and (b) a second antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 49, a CDR2 sequence comprising SEQ ID NO: 50, and a CDR3 sequence comprising SEQ ID NO: 51. In some embodiments, the antibody comprises (a) a first antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 8, a CDR2 sequence comprising SEQ ID NO: 13, and a CDR3 sequence selected from SEQ ID NOs: 21, 22, 23, and 24; and (b) a second antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 49, a CDR2 sequence comprising SEQ ID NO: 50, and a CDR3 sequence comprising SEQ ID NO: 51.

[0169] In some embodiments, the antibody comprises a first antigen binding region comprising or consisting of a sequence selected from SEQ ID NOs: 25, 26, 27, and 28 and a second antigen binding region comprising or consisting of SEQ ID NO: 52. In some embodiments, the antibody comprises a first antigen binding region comprising or consisting of a sequence selected from SEQ ID NOs: 29, 30, 31, and 32 and a second antigen binding region comprising or consisting of SEQ ID NO: 52.

[0170] In some embodiments, the antibody comprises a second antigen binding region capable of binding to human nectin 4.

[0171] In some embodiments, the antibody comprises a second antigen-binding region comprising a CDR1 sequence shown in SEQ ID NO: 53, a CDR2 sequence shown in SEQ ID NO: 54, and a CDR3 sequence shown in SEQ ID NO: 55. In some embodiments, the antibody comprises a second antigen-binding region comprising a CDR1 sequence shown in SEQ ID NO: 57, a CDR2 sequence shown in SEQ ID NO: 58, and a CDR3 sequence shown in SEQ ID NO: 59. In some embodiments, the antibody comprises a second antigen-binding region comprising a CDR1 sequence shown in SEQ ID NO: 61, a CDR2 sequence shown in SEQ ID NO: 62, and a CDR3 sequence shown in SEQ ID NO: 63. In some embodiments, the antibody comprises a second antigen-binding region comprising a CDR1 sequence shown in SEQ ID NO: 65, a CDR2 sequence shown in SEQ ID NO: 66, and a CDR3 sequence shown in SEQ ID NO: 67. In some embodiments, the antibody comprises a second antigen-binding region comprising a CDR1 sequence shown in SEQ ID NO: 69, a CDR2 sequence shown in SEQ ID NO: 70, and a CDR3 sequence shown in SEQ ID NO: 71. In some embodiments, the antibody comprises a second antigen-binding region comprising a CDR1 sequence shown in SEQ ID NO: 73, a CDR2 sequence shown in SEQ ID NO: 74, and a CDR3 sequence shown in SEQ ID NO: 75. In some embodiments, the antibody comprises a second antigen-binding region comprising a CDR1 sequence shown in SEQ ID NO: 77, a CDR2 sequence shown in SEQ ID NO: 78, and a CDR3 sequence shown in SEQ ID NO: 79.

[0172] In some embodiments, the antibody comprises a second antigen binding region, which comprises or consists of a sequence selected from SEQ ID NO: 56, 60, 64, 68, 72, 76 and 80, or comprises an amino acid 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: 56, 60, 64, 68, 72, 76 and 80.

[0173] In some embodiments, the second antigen binding region competes (i.e., is capable of competing) for binding to human Nectin 4 with an antibody having the sequence shown in SEQ ID NOs: 56, 60, 64, 68, 72, 76, and 80, and preferably the second antigen binding region binds to the same epitope on human Nectin 4 as an antibody having the sequence shown in SEQ ID NOs: 56, 60, 64, 68, 72, 76, and 80.

[0174] In some embodiments, the antibody comprises (a) a first antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 8, a CDR2 sequence comprising SEQ ID NO: 13, and a CDR3 sequence selected from SEQ ID NOs: 17, 18, 19, and 20; and (b) a second antigen binding region comprising a CDR1 sequence selected from SEQ ID NOs: 49, 53, 57, 61, 65, 69, 73, and 77, a CDR2 sequence comprising SEQ ID NOs: 50, 54, 58, 62, 66, 70, 74, and 78, and a CDR3 sequence comprising SEQ ID NOs: 51, 55, 59, 63, 67, 71, 75, and 79. In some embodiments, the antibody comprises (a) a first antigen binding region comprising a CDR1 sequence comprising SEQ ID NO: 8, a CDR2 sequence comprising SEQ ID NO: 13, and a CDR3 sequence selected from SEQ ID NOs: 21, 22, 23, and 24; and (b) a second antigen binding region comprising a CDR1 sequence comprising SEQ ID NOs: 49, 53, 57, 61, 65, 69, 73, and 77, a CDR2 sequence comprising SEQ ID NOs: 50, 54, 58, 62, 66, 70, 74, and 78, and a CDR3 sequence comprising SEQ ID NOs: 51, 55, 59, 63, 67, 71, 75, and 79.

[0175] In some embodiments, the antibody comprises a first antigen binding region comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 25, 26, 27, and 28 and a second antigen binding region comprising or consisting of SEQ ID NOs: 56, 60, 64, 68, 72, 76, and 80. In some embodiments, the antibody comprises a first antigen binding region comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 29, 30, 31, and 32 and a second antigen binding region comprising or consisting of SEQ ID NOs: 56, 60, 64, 68, 72, 76, and 80.

[0176] Exemplary combinations of binding domains are provided in Table A below.

[0177]

[0178]

[0179]

[0180]

[0181] 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: 81.

[0182] 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, D123, PSMA, CD1d, CD40 or Nectin 4. 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 N-terminus of the second antigen-binding region capable of binding to human EGFR, D123, PSMA, CD1d, CD40 or Nectin 4.

[0183] In one embodiment of the invention, the antibody 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.

[0184] 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: 82.

[0185] 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.

[0186] In preferred embodiments, the first antigen binding region comprises the sequence shown in SEQ ID NO:16, the second antigen binding region comprises a sequence selected from the group consisting of SEQ ID NOs:36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76 and 80, and (a) the first Fc polypeptide comprises the sequence shown in SEQ ID NO:83, and the second Fc polypeptide comprises the sequence shown in SEQ ID NO:84, or (b) the first Fc polypeptide comprises the sequence shown in SEQ ID NO:84, and the second Fc polypeptide comprises the sequence shown in SEQ ID NO:83.

[0187] In one embodiment, X7 in SEQ ID NO: 83 is absent. In another embodiment, X7 in SEQ ID NO: 83 is present and is K (lysine).

[0188] In one embodiment, X7 in SEQ ID NO: 84 is absent. In another embodiment, X7 in SEQ ID NO: 87 is present and is K (lysine).

[0189] In another preferred embodiment, the antibody comprises or consists of: (a) the sequence shown in SEQ ID NO:85, and (b) a sequence selected from the group consisting of the sequences shown in SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89 and SEQ ID NO:90.

[0190] In another preferred embodiment, the antibody comprises or consists of: (a) the sequence shown in SEQ ID NO:86, and (b) a sequence selected from the group consisting of the sequences shown in SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89 and SEQ ID NO:90.

[0191] In one embodiment, X7 in SEQ ID NO: 85 is absent. In another embodiment, X7 in SEQ ID NO: 85 is present and is K (lysine).

[0192] In one embodiment, X7 in SEQ ID NO: 86 is absent. In another embodiment, X7 in SEQ ID NO: 86 is present and is K (lysine).

[0193] In one embodiment, X7 in SEQ ID NO: 87 is absent. In another embodiment, X7 in SEQ ID NO: 22 is present and is K (lysine).

[0194] In one embodiment, X7 in SEQ ID NO: 87 is absent. In another embodiment, X7 in SEQ ID NO: 23 is present and is K (lysine).

[0195] In one embodiment, X7 in SEQ ID NO: 88 is absent. In another embodiment, X7 in SEQ ID NO: 24 is present and is K (lysine).

[0196] In one embodiment, X7 in SEQ ID NO: 88 is absent. In another embodiment, X7 in SEQ ID NO: 25 is present and is K (lysine).

[0197] In another main aspect, the invention relates to a pharmaceutical composition comprising an antibody according to the invention as described herein and a pharmaceutically acceptable excipient.

[0198] The antibody can be formulated with a pharmaceutically acceptable excipient according to conventional techniques, such as those disclosed in Rowe et al. 2012 Handbook of Pharmaceutical Excipients, ISBN 9780857110275). Pharmaceutically acceptable excipients and any other carriers, diluents or adjuvants should be suitable for the antibody and the selected mode of administration. The suitability of the excipients and other components of the pharmaceutical composition is determined based on the absence of significant negative effects on the desired biological properties of the selected antibody or pharmaceutical composition of the present invention (e.g., less than a substantial effect on antigen binding (10% or less relative inhibition, 5% or less relative inhibition, etc.)).

[0199] The pharmaceutical composition may include diluents, fillers, salts, buffers, detergents (e.g., nonionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in the 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.

[0200] In another main aspect, the invention relates to an antibody according to the invention as described herein for use as a medicament.

[0201] The antibodies 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.

[0202] In one embodiment, the antibody targets EGFR and is used to treat primary or metastatic colon or rectal cancer. In another embodiment, the antibody is used to treat peritoneal cancer. In another embodiment, the antibody is used to treat liver cancer. In another embodiment, the antibody is used to treat head and neck squamous cell carcinoma (HNSCC). In another embodiment, the antibody is used to treat non-small cell lung cancer (NSCLC). In another embodiment, the antibody is used to treat cutaneous squamous cell carcinoma.

[0203] In another embodiment, the antibody targets CD123 and is used to treat acute myeloid leukemia, B-cell acute lymphoblastic leukemia, hairy cell leukemia, Hodgkin's lymphoma, blastic plasmacytoid dendritic cell neoplasm, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell chronic lymphoproliferative disorder, or myelodysplastic syndrome.

[0204] In another embodiment, the antibody targets CD1d and is used to treat a hematological malignancy such as T-cell lymphoma, multiple myeloma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, B-cell lymphoma, smoldering myeloma, Hodgkin's lymphoma, myelomonocytic leukemia, lymphoplasmacytic lymphoma, hairy cell leukemia, or splenic marginal zone lymphoma, or a solid tumor such as renal cell carcinoma, melanoma, colorectal cancer, head Neck cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, gastroesophageal cancer, small intestine cancer, central nervous system tumors, medulloblastoma, hepatocellular carcinoma, ovarian cancer, glioma, neuroblastoma, urothelial carcinoma, bladder cancer, sarcoma, penile cancer, basal cell carcinoma, Merkel cell carcinoma, neuroendocrine cancer, neuroendocrine tumors, cancer of unknown primary (CUP), thymoma, vulvar cancer, cervical cancer, testicular cancer, bile duct cancer, appendix cancer, mesothelioma, ampullary cancer, anal cancer, or choriocarcinoma.

[0205] In another embodiment, the antibody targets PSMA and is used to treat prostate cancer, such as non-metastatic or metastatic prostate cancer, for example metastatic castration-resistant prostate cancer, such as treatment of refractory metastatic castration-resistant prostate cancer. For example, the antibody may comprise a first antigen binding region that is capable of binding to PSMA and is used to treat prostate cancer in patients who have failed at least one line of taxane-based chemotherapy and have optionally received a second generation or later androgen receptor targeted therapy / androgen biosynthesis inhibitor (e.g., abiraterone, enzalutamide and / or apotoxin).

[0206] In addition, antibodies that can bind to PSMA can be used to treat cancers in which PSMA is expressed on tumor neovasculature or tumor-associated endothelial cells of primary or metastatic tumors, including colorectal cancer, lung cancer, breast cancer, endometrial and ovarian cancer, gastric cancer, renal cell carcinoma, urothelial carcinoma, hepatocellular carcinoma, oral squamous cell carcinoma, thyroid tumors and glioblastoma. In addition, antibodies that can bind to PSMA can be used to treat adenoid cystic carcinoma of the head and neck.

[0207] Similarly, the invention relates to a method of treating a disease, said method comprising administering to a human subject in need thereof a multispecific antibody according to the invention as described herein. In one embodiment, the disease is cancer.

[0208] In some embodiments, the antibodies are administered as a monotherapy. However, the antibodies 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.

[0209] "Treatment" or "treating" refers to the administration of an effective amount of an antibody according to the invention to alleviate, ameliorate, prevent, eradicate (cure) or prevent symptoms or disease states. "Effective amount" refers to an effective amount at the necessary dosage and time period to achieve the desired therapeutic result. 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 by any suitable route, but is generally parenteral, such as intravenous, intramuscular or subcutaneous.

[0210] The multispecific antibodies of the present invention are generally 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 generally 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.

[0211] Therefore, in another aspect, the present invention relates to a nucleic acid construct encoding an antibody according to the present invention. In one embodiment, the construct is a DNA construct. In another embodiment, the construct is an RNA construct.

[0212] In another aspect, the present invention relates to an expression vector comprising a nucleic acid construct encoding an antibody according to the present invention.

[0213] In another aspect, the present invention relates to a host cell comprising one or more nucleic acid constructs encoding an antibody according to the present invention, or comprising an expression vector encoding a nucleic acid construct according to the present invention.

[0214] In another aspect, the invention relates to a method for producing an antibody of the invention, comprising expressing one or more nucleic acids encoding an antibody according to the invention in a host cell.

[0215] In another aspect, the invention relates to a method for making a clinical batch of an antibody of the invention, comprising expressing one or more nucleic acids encoding an antibody according to the invention in a host cell. "Clinical batch" as used herein refers to a product composition suitable for use in humans.

[0216] In another aspect, the invention relates to a method for producing an antibody free of proteolysis and / or tyrosine sulfation, comprising expressing one or more nucleic acids encoding an antibody according to the invention in a host cell.

[0217] In another aspect, the present invention relates to a method for avoiding proteolysis and / or tyrosine sulfation of antibodies capable of activating human Vγ9Vδ2T cells, the method comprising constructing a nucleic acid encoding the antibody of the present invention, and preparing the antibody by expressing the nucleic acid in a host cell.

[0218] In another aspect, the present invention relates to a method for producing a homogeneous antibody preparation of antibodies capable of activating human Vγ9Vδ2T cells, the method comprising constructing a nucleic acid encoding the antibody of the present invention, and preparing the antibody by expressing the nucleic acid in a host cell.

[0219] In one embodiment, the host cell in the above manufacturing process is a mammalian cell, such as a CHO cell or a HEK cell, or a yeast cell, such as a Pichia cell.

[0220] Table 1: Sequence Listing

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231] 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.

[0232] Example

[0233] Example 1 | Production and purification of VHH compounds

[0234] 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: Figure 1 .

[0235] In polyacrylamide gel electrophoresis, the purified protein migrates as a single band under reducing and non-reducing conditions: Figure 2 Representative examples are shown.

[0236] Example 2 | HP-SEC analysis of purified VHH compounds

[0237] HP-SEC analysis was performed on purified VHH compounds using the Waters Acquity ARC-bio system. 10 μg of antibody (10 μL antibody, 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. Protein was 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 WO2015156673) and 5C8var1 (SEQ ID NO: 14) (previously described in WO2020060405) were tested in HP-SEC analysis, two peaks were observed: Fig. 3.

[0238] Example 3 | Mass spectrometry analysis of 5C8 reveals 80Da additional mass

[0239] 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.

[0240] Example 4 | 1D12var5-5C8var1 containing the same anti-Vδ2 VHH exhibits the same heterogeneity in HP-SEC and has the same 80Da extra mass

[0241] 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 WO2020060405). 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: Figure 4.

[0242] The observed pre-peaks again 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.

[0243] Example 5 | Computer analysis of VHH 5C8 and 5C8var1

[0244] 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 The model was then analyzed for reactive residues, indicating those that were 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.

[0245] Table 2 | Predicted PTMs of 5C8 and 5C8var1 in Maestro and ModPred.

[0246]

[0247]

[0248] *Q13 deamidation predicted for 5C8 only. Type describes the PTM as predicted by Maestro.

[0249] Example 6 | Design and production of 5C8var1 VHH CDR3 mutants Y105F and Y105S

[0250] 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.

[0251] 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.

[0252] Example 7 | HP-SEC analysis of purified VHHs containing designed CDR3 mutations

[0253] HP-SEC analysis was performed as described for 5C8. Both 5C8var1-Y105F and 5C8var1-Y105S were analyzed (Figure 6).

[0254] 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.

[0255] Example 8 | Affinity measurements of 5C8var1, 5C8var1-Y105F, and 5C8va1-Y105S using biolayer interferometry (BLI) showed no difference in affinity

[0256] The binding of 5C8var1VHH antibody fragment and variants 5C8var1-Y105F and 5C8var1-Y105S to Vy9Vδ2TCR 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 for the anti-human Fc capture biosensor. Sensorgrams were recorded when the ligand capture biosensor was incubated with a series of diluted VHH antibody fragments (40 to 0.63 nM) in 10X kinetic buffer (ForteBio). The values ​​of kon (association rate constant) and koff (dissociation rate constant) were estimated using a global data fit of a 1:1 binding model. These values ​​were used to calculate KD (equilibrium dissociation constant) using KD=koff / kon.

[0257] From Figure 7 and Table 3, it can be concluded that the KD values ​​of the two different Y105 VHH mutants are not significantly different from that of 5C8var1. In particular, the Y105F mutant has a comparable affinity to 5C8var1.

[0258] Table 3 | Affinity values ​​of 5C8var1, 5C8var1-Y105F and 5C8var1-Y105S VHH binding to recombinant Vγ9Vδ2 TCR protein found in BLI measurements.

[0259] VHH compounds KD(nM)+ / -SD 5C8var1 0.81+ / -0.02 5C8var1-Y105F 0.78+ / -0.23 5C8var1-Y105S 1.59+ / -0.31

[0260] The values ​​shown are means + / - standard deviations of at least three independent measurements.

[0261] Example 9 | The functionality of the anti-(EGFR x Vδ2TCR) bispecific VHH containing the Y105F mutation is fully retained

[0262] 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 Vγ9Vδ2 T cell activation dependent on an EGFR-positive tumor cell line (A431) and to cause Vγ9Vδ2 T cell-mediated tumor cell lysis. In brief, Vγ9Vδ2T 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. Vγ9Vδ2T 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 Vγ9Vδ2T cells were added to the culture medium along with a range of bispecific VHH compounds. 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 assay, the next day, CytoTox-Glo cytotoxicity assay kit (Promega G9290) was used to check whether there was protease (indicating cell death) in the supernatant of co-culture. Detergent was used to carry out cell lysis for reaching 100% killing rate at the end of the assay. Fig. 8 shows data.

[0263] Figure 8 shows that 7D12var8-5C8var1-Y105F and non-humanized 7D12-5C8 induce potent Vγ9Vδ2 T cell activation and tumor cell lysis. These results are consistent with the efficacy 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 has a slightly lower EC50 in the cytotoxicity assay.

[0264] Table 4 | EC50 values ​​obtained by curve fitting the data shown in Figure 8 using GraphPad software.

[0265] Antibodies used EC50 degranulation (pM) EC50 cytotoxicity (pM) 7D12-5C8 10 9 7D12var8-5C8var1-Y105F 11 2.5

[0266] The maximum tumor cell killing level of 7D12var8-5C8var1-Y105F was slightly lower than that observed for 7D12-5C8. However, these are two different measurements using two different Vγ9Vδ2 T cell donors, and this maximum cytotoxicity level may be particularly donor-dependent.

[0267] Example 10 | The temperature stability of VHH 5C8var1 containing the Y105 mutation is not altered

[0268] 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). 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 starting temperature (Tagg) were determined. For all three antibody fragments, the starting melting temperature (Ton) and melting temperature (Tm) of VHH complete unfolding were reported (Table 5). The melting temperatures measured for 5C8var1-Y105F and 5C8var1-Y105S are consistent with the melting temperature of 5C8var1: Table 5.

[0269] Table 5 | 5C8var1-Y105F and 5C8var1-Y105S showed similar melting temperatures as 5C8var1, indicating that the introduced mutations did not compromise stability.

[0270] <![CDATA[T on (℃)]]> Tm(℃) 5C8var1 61.8 86.7 5C8var1-Y105F 64.5 85.5 5C8var1-Y105S 63.9 86.6

[0271] Example 11 | Bispecific constructs with extended half-life (Fc-containing)

[0272] 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.

[0273] 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.

[0274] Example 12 | Binding of 7D12var8-5C8var1(Y105F)-Fc to primary Vγ9Vδ2 T cells isolated from healthy human PBMC

[0275] 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 for half-log titration at 4°C for one hour. 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).

[0276] Example 13 | Binding of 7D12var8-5C8var1(Y105F)-Fc to EGFR-positive tumor cells by cell-based ELISA

[0277] 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 a 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 10 shows that 7D12var8-5C8var1(Y105F)-Fc strongly bound to A-431, HCT-116, and HT-29 tumor cells with an EC50 of approximately 7 nM, while the non-targeting control antibody did not bind measurably to any of the cell lines tested.

[0278] Example 14 | 7D12var8-5C8var1(Y105F)-Fc-induced Vγ9Vδ2 T cell degranulation dependent on A-431 cells

[0279] To investigate the potential of 7D12var8-5C8var1(Y105F)-Fc to activate Vγ9Vδ2T cells, Vγ9Vδ2T cells were first isolated and expanded as previously described. Next, Vγ9Vδ2T 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, the cells were harvested and stained with fluorescently labeled anti-Vγ9 and anti-CD3 antibodies to distinguish Vγ9Vδ2T cells from tumor cells. The extent of CD107a expression on Vγ9Vδ2T cells, reflecting target-dependent degranulation, was investigated using flow cytometry. Fig.11 It 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).

[0280] Example 15 | Antibody 7D12-5C8 induces T cell-mediated cytotoxicity of target cells

[0281] To investigate whether the bispecific VHH7D12-5C8 can effectively induce Vγ9Vδ2T cell-mediated cytotoxicity against target cells, the viability of the A-388 epidermoid tumor cell line (ATCC, CRL-7905) was evaluated in a co-culture environment with Vγ9Vδ2T cells and bsVHH antibody fragments. In this assay, Vγ9Vδ2T cells were used, which were isolated from healthy PBMCs as described previously, but then frozen and stored at -150°C. The frozen Vγ9Vδ2T cells were thawed and rested overnight in medium supplemented with IL-2. A-388 tumor cells were inoculated alone or with resting Vγ9Vδ2T cells at a ratio of 1:1 or 1:0.1 target cells to effector cells, with or without 7D12-5C8 (10nM). As an additional control, Vγ9Vδ2T cells were inoculated alone, with or without antibody 7D12-5C8 (10nM). 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 The ATP-derived fluorescence signal, representing the number of metabolically active cells as well as viable cells, is shown. At a 1:1 E:T ratio, an antibody-induced reduction of approximately 50% in viable 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.

[0282] Example 16 | Killing of tumor cells by 7D12-5C8 and 7D12-5C8var1(Y105S)-Fc activated Vγ9Vδ2 T cells

[0283] 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. A variety of different tumor cell types were tested.

[0284] After written informed consent, tissue samples (i.e., primary and metastatic colorectal cancer, head and neck squamous cell carcinoma (HNSCC), and non-small cell lung cancer (NSCLC) samples) 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.

[0285] 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.

[0286] 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).

[0287] Viable cells were identified using the life / death marker 7AAD in combination with 123counting eBeads™ according to the manufacturer's instructions. Flow cytometry data were analyzed using Kaluza Analysis version 1.3 (Beckman Coulter) and FlowJo versions 10.6.1 and 10.7.2 (Becton Dickinson).

[0288] 7D12-5C8 and 7D12-5C8var1(Y105S)-Fc-induced Vγ9Vδ2 T cell-mediated tumor cell cytotoxicity 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).

[0289] like Fig.13As shown, 7D12-5C8 induced Vγ9Vδ2 T cells to lyse patient tumor cells in large quantities (average % of lysis induced by 7D12-5C8: CRC primary 52.3% and p-value 0.0003, CRC peritoneum 46.0% and p-value 0.0052, CRC liver 31.8% and p-value 0.0360, head and neck squamous cell carcinoma 46.1% and p-value 0.0187 and non-small cell lung cancer 64.1% and p-value 0.0153).

[0290] 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.

[0291] Example 17 | Design, production and purification of construct 7A5-7D12var8-Fc for non-human primate studies

[0292] 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 V□9V□2 T cells (see Example 1 of WO2021052995). A bispecific Fc antibody consisting of 7A5 and anti-EGFR VHH 7D12var8 was constructed. The molecule contains a human IgG1 Fc tail and is designed to form heterodimerization using knob-in-hole technology (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 KiH Fc pair. In addition, the upper hinge was designed as "AAASDKTHTCPPCP" to remove the cysteine ​​(C220) that is usually bridged to CL, and more flexibility was introduced by changing "EPK" to "AAA". The N-terminal part 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.

[0293] 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) ( Fig.16 The functionality of the molecule was demonstrated by showing that it caused target-dependent activation (CD107a expression) of in vitro expanded Vγ9Vδ2 T cells and subsequent T cell-mediated lysis of tumor cells ( Fig.17 ).

[0294] Example 18 | Bispecific Antibody 7A5-7D12var8-Fc is Well Tolerated in an Exploratory Multi-Dose Non-Human Primate (NHP: Cynomolgus Monkey) Study

[0295] 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).

[0296] 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.

[0297] 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.

[0298] 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 ( Fig.19 ), a phenomenon that is frequently observed in these types of multiple-dose studies and is associated with the surgical procedure. Fig.19 A shows a temporary decrease in T cell counts at each time point 2 hours after administration. However, two days after injection, T cell numbers returned to baseline levels.

[0299] In contrast, the number of Vγ9-positive T cells in the peripheral blood decreased and did not return to their previous frequency. These cells were almost absent throughout the study, demonstrating that the compound had a specific pharmacodynamic effect. Measurement of cytokines in the blood of treated animals showed that the treatment caused very little cytokine release and was almost entirely limited to the first injection of the compound. Fig. 20 IL-6 levels measured as an example are shown.

[0300] 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).

[0301] Example 19 | VHH 5C8var1 is partially cleaved at position 109 in the product of an Fc-containing bispecific antibody format, regardless of the residue at position Y105

[0302] As described above, the Fc-carrying format includes two VHH molecules fused to a modified human IgG1 Fc, thereby forming a humanized "heavy chain only" antibody molecule. During the development of a cell line for large-scale production of therapeutic Fc-containing bispecific molecules (including Vδ2-binding VHH 5C8var1) in CHO cells, SDS-PAGE analysis revealed that a small portion of the product was truncated ( Fig.21 ).

[0303] LC-ESI-MS mass spectrometry analysis ( Fig. 22and Table 6) show that surprisingly, the truncated species seen in SDS-PAGE may be due to a small fraction of 5C8var1 VHH being cleaved after position 109 in VHH CDR3. This phenomenon was observed for the different Fc-containing bispecific molecules regardless of the (other) targeting VHH arm on the second chain and regardless of the amino acid at position 105 in VHH 5C8var1, and was not predicted based on computer analysis (Example 5).

[0304] Because truncations beyond position 109 result in the loss of a large portion of the VHH, this results in the production of non-functional proteins. The percentage of N-terminally truncated proteins was low (1-3%) and remained constant over time, suggesting that cleavage occurs during the intracellular production and processing of the protein.

[0305] Table 6 | Mass annotation of LC-ESI-MS measurements of VHH-Fc heavy chain only antibodies.

[0306]

[0307] Example 20 | Design of sequence variants of 5C8var1 to improve product homogeneity

[0308] The CDR3 sequence of 5C8 was changed at two positions to avoid proteolysis observed in different Fc-containing bispecific molecules. The G at position 108 (G108) was changed to A, and the R at position 109 (R109) was changed to K to maintain the same net charge, or to A: Fig.23 In addition, a variant (R109AΔGIR) was generated in which the R109 position was changed to "a" and four residues were deleted from the CDR3 region (ΔGIR).

[0309] These four different sequence variants (G108A, R109A, R109K and R109AΔLGIR) were then expressed and purified as single VHH and bispecific Fc-containing molecules, with CD123 binding targeting VHH as the second binding arm (SEQ ID NO: 12) (previously described in PCT / EP2022 / 054993 (incorporated by reference)). For purification purposes, the protein chain encoding (mutated) VHH was extended with a C tag, and the VHH was directed to the TCR fused to Fc. The protein was expressed by transient transfection of the encoding plasmid in HEK293E cells, and the secreted protein was purified from the conditioned cell culture supernatant by C tag affinity chromatography, followed by preparative size exclusion chromatography. The complete mass analysis (LC-ESI-MS) of the proteins showed that their purity was >98%, and no truncation was observed in any of the protein batches produced in these small-scale productions.

[0310] Example 21 | Affinity measurements by BLI show that position 109 is permissive.

[0311] To determine the possible effects of mutations at positions 108 and 109 on the binding properties of different VHH molecules, the binding of individual VHH proteins to a recombinant form of the Vγ9Vδ2T cell receptor was tested by biolayer interferometry (BLI) using the Octet system. Figure 24 and Table 7 show that the affinity of the original humanized 5C8 VHH (5C8var1) with the Y105F mutation was 0.74 nM, and VHH molecules containing R109A or R109K mutations in addition remained bound (without significant changes in affinity). However, the affinity of 5C8var1-Y105F containing an additional mutation at position 108 was reduced by 10 times. The R109A□LGIR variant no longer measurably binds to the Vγ9Vδ2TCR. These data indicate that, although all (mutated) VHH molecules are well expressed, only the R109 position allows the Vγ9Vδ2T cell receptor to bind and allows sequence changes. No binding was observed for the R109AΔLGIR variant, indicating that CDR3 is required for specific binding.

[0312] Table 7 | Affinity values ​​of 5C8var1-Y105F, 5C8var1-Y105F / G108A, 5C8var1-Y105F / R109A and 5C8var1-Y105F / R109KVHH binding to recombinant Vγ9Vδ2 TCR protein found in BLI measurements.

[0313] VHH cloning Affinity measured by BLI (nM) 5C8var1-Y105F 0.74 5C8var1-Y105F / G108A 9.2 5C8var1-Y105F / R109A 1.2 5C8var1-Y105F / R109K 0.82 5C8var1-Y105F-R109AΔLGIR ND

[0314] ND = not determined due to lack of binding.

[0315] Example 22 | Determination of the apparent affinity of VHH variants at positions 108 and 109 using flow cytometry

[0316] To test whether the mutations at positions 108 and 109 of the 5C8var1 VHH affect binding to cell-expressed Vγ9Vδ2 T cell receptors, the binding of the mutant VHH molecules in Fc-containing form to primary expanded Vγ9-Vδ2 T cells was tested in flow cytometry. Primary Vγ9Vδ2 T cells were isolated from the PBMC fraction of the blood of healthy volunteers and expanded using a mixed lymphocyte reaction with irradiated feeder cells and a cytokine cocktail. Aliquots of 50,000 Vγ9Vδ2 T cells were then incubated with a range of concentrations of the Fc-containing bispecific molecules, and binding was detected using a PE-labeled anti-human IgG detection antibody. Based on the results of BLI, the molecules containing the R109 mutation were indistinguishable from the original molecules in terms of apparent affinity (i.e., binding to Vγ9Vδ2 T cells) ( Fig.25 ); EC50 values ​​for binding were very similar. The bispecific molecule containing the R109AΔLGIR CDR3 did not bind significantly to cells (not shown), and the G108 mutant showed very weak binding.

[0317] Example 23 | Bispecific, Fc-containing R109 mutant molecules are equally effective in inducing Vγ9Vδ2 T cell activation and target-dependent Vγ9Vδ2 T cell-mediated cytotoxicity.

[0318] As described in Examples 14 and 15, the ability of bispecific, Fc-containing anti-CD123 x TCR bispecific molecules containing variants of positions 108 and 109 of 5C8var1 to induce target-dependent Vγ9Vδ2T cell activation (determined by analyzing CD107a expression) and Vγ9Vδ2T cell-mediated cytotoxicity of target cells was tested. Two target cell lines expressing CD123 molecules were used: MOLM-13 and THP-1. Representative results using Vγ9Vδ2T cells from a healthy donor are shown in the figure; two different Vγ9Vδ2T cell donors were tested, and the results of the two were comparable.

[0319] Figures 26 and 27 show that all bispecific molecules are able to induce Vγ9Vδ2 T cell activation and Vγ9Vδ2 T lymphocyte-mediated cytotoxicity of target cells. Variants containing R109 mutations (A or K) are as effective as the original molecule with only Y105F mutation. The EC50 value of the R109A variant (Table 8) is slightly lower than that of the other two variants.

[0320] Table 8 | EC50 values ​​for Vγ9Vδ2 T cell activation and tumor cell cytotoxicity induced by Fc-containing bispecific molecules.

[0321]

[0322] Example 24 | Antibody stability

[0323] To determine whether the introduction of the change at position 109 in the Vδ2 binding VHH has any effect on the thermal and colloidal stability of the antibody, the unfolding of the bispecific antibody containing Fc was tested using nanoDSF (differential scanning fluorescence). Table 9 gives the temperature at which unfolding and the onset temperature of turbidity (protein aggregation) are observed. Compared to the parent molecule, the R109 mutant has almost the same thermal stability.

[0324] Table 9 | Thermal stability parameters of different Fc-containing bispecific antibodies. IP: inflection point.

[0325]

[0326] Additionally, the antibodies were tested in accelerated stress tests: the proteins were incubated at high temperatures, under acidic or alkaline conditions, or under oxidative stress conditions.

[0327] For high temperatures, protein samples were adjusted to 1 g / L with phosphate buffer, pH 7.4, and incubated in a 40°C incubator for 1 week, after which samples were aliquoted and stored at 2-8°C or -80°C until analysis.

[0328] For low pH conditions, protein samples were re-filtered to 1 g / L by ultrafiltration (molecular weight cutoff (MWCO) 30 kDa) in 50 mM acetate buffer, pH 5.0, and incubated in an incubator at 40°C and low pH for 1 week. For high pH conditions, protein samples were re-filtered to 1 g / L by ultrafiltration (MWCO 30 kDa) into 100 mM phosphate buffer, pH 8.5, and incubated in an incubator at 40°C and high pH for 1 week.

[0329] For oxidative conditions, protein samples were re-filtered to 1 g / L by ultrafiltration (MWCO 30 kDa) in phosphate buffer, pH 7.4 and 0.05% H2O2 and incubated at RT for 24 h.

[0330] After incubation under acidic, alkaline or oxidizing conditions, samples were re-filtered by ultrafiltration (MWCO 10 kDa) in phosphate buffer, pH 7.4, aliquoted and stored at 2-8°C or -80°C until analysis.

[0331] Aggregates and fragments were analyzed by SEC-UV (Table 10), where the percentage of high molecular weight species (HMWS) and / or low molecular weight species (LMWS) did not change or changed only slightly after incubation under the specified accelerated stress conditions. Importantly, the changes observed for the R109 mutant were almost similar to those of the parental molecule.

[0332] Table 10 | Summary of HMWS, main peak and LMWS determined by SEC-UV of different Fc-containing bispecific antibodies before and after stress treatment.

[0333]

[0334] After reduction, the fragments were additionally analyzed by CGE / CE-SDS (Table 11). No or minimal changes in the percentage of LMWS were observed, and the changes observed for the R109 mutant were very similar to those for the parental molecule.

[0335] Table 11 | Summary of HMWS, main peak and LMWS determined by SEC-UV of different Fc-containing bispecific antibodies before and after stress treatment.

[0336]

[0337]

[0338] Example 24 | Humanization of anti-CD123 VHH 1D2 using CDR grafting

[0339] The 1D2 VHH antibody fragment was humanized using CDR transplantation technology (see, e.g., U.S. Pat. No. 5,225,539 and Williams, DG et al., 2010, Antibody Engineering, Vol. 1, Chapter 21). First, human germline sequences were identified using IgBLAST (Ye J. et al., 2013, Nucleic Acids Res. 41: W34-40). As the closest human germline sequence, V gene IGVH3-23*04 was identified (78.4% identity). This germline sequence was used to directly transplant camel CDR (with 91.8% identity to human germline IGVH3-23*04), thereby constructing SEQ ID NO: 32. Next, BLASTP (version 2.10.0+) was used to query the NCBI NR database (downloaded on September 27, 2020) to identify the human template sequence with the highest identity to the 1D2 sequence. Two VH sequences were identified with similarity scores of 70% or higher and showing similar CDR lengths, preferably identical to those in 1D2 CDR1, CDR2, and CDR3, respectively. The frameworks encoded by GenBank (Benson, DA et al., 2013, Nucleic Acids Res. 41(D1): D36-42) accession numbers CAD60357.1 and AKU38567.1 were selected as templates for 1D2 CDR grafting, and the following cDNA constructs were obtained, respectively: SEQ ID NOs: 29 and 33. Framework and CDR definitions were determined by Kabat et al. ("Sequences of Proteins of Immunological Interest", Kabat, E. et al., US Department of Health and Human Services, (1983)). To understand the effect of humanized framework residues on VHH structure, BioLuminate 4.2.156 was used. The homology model of 1D2VHH was established using the "Antibody Prediction" tool (default parameters) in . The homology model was established on the basis of PDB ID 6GKU. The CDRs were transplanted in silico to study the effects of human residues on features such as CDR loop conformation, surface hydrophobicity, and structural integrity (e.g., increased rigidity). These features of the resulting constructs were examined to design additional constructs: SEQ ID NOs: 26, 27, 28, 30, 31, 34, and 35. The sequences of these humanized 1D2-VHHs were then reformatted into bispecific VHHs, with the Vδ2-specific VHH (5C8var1; SEQ ID NO: 18) oriented as: N-terminal-humanized-anti-1D2-VHH-linker-anti-Vδ2VHH-C tag. The cDNAs encoding these molecules were then synthesized and cloned into expression vectors for expression in HEK293E cells. Proteins were prepared by transiently transfected cells and purified from culture supernatants using C-tag affinity chromatography followed by preparative size exclusion chromatography.

[0340] Example 25 | Determination of affinity of humanized 1D2-5C8var1(Y105F) variant binding to CD123 using biolayer interferometry (BLI)

[0341] In order to determine the kinetics of humanized 1D2-5C8var1 variant binding to CD123, recombinant purified CD123-Fc fusion protein (Bio-Techne / R&D Systems) was loaded onto the anti-human IgG Fc Capture sensor of the Octet Red96e (Sartorius) instrument at a density of 1nm (using a concentration of 5μg / ml). Different sensors were then immersed in different concentrations of humanized 1D2-5C8var1 variants, starting from 50nM and diluted twice; diluted in 10x kinetic buffer (10xKB) provided by the supplier. According to the sensorgram obtained, the kinetic association and dissociation rate constants were determined by curve fitting. When it can be fitted, the affinity of humanized 1D2-5C8var1 variants binding to CD123 is calculated using the association and dissociation rate constants. Two measurements were performed, and the affinity of the different humanized 1D2-5C8var1 variants for CD123 ranged from 2.6 nM (similar to the parental 1D2) to a non-binding variant, as shown in Table 12. As a reference, non-humanized (parental) 1D2 was included in these experiments.

[0342] Table 12 | Affinity of 1D2 humanized variants for recombinant CD123

[0343] SEQ ID No: KD(nM) SD 1 3.1 0.7 26 7.2 0.4 27 24.5* 28 14.9 4.3 29 20.0 7.5 30 7.7 1.5 31 29.1* 32 nb 33 44.1* 34 2.6 0.5 35 10.4 7.5

[0344] *Low binding (>20nM), measured only once, nb: no binding observed

[0345] Example 26 | Development of a cell pool stably expressing a bispecific antibody.

[0346] cDNAs encoding all protein chains (1D2-Fc (SEQ ID NO:21), 5C8var1-Y105F-Fc (SEQ ID NO:37), and 5C8var1-Y105F-R109A-Fc (SEQ ID NO:22)) were designed by reverse translation and then codon optimized at a contract research organization (CRO) for expression in Chinese hamster ovary (CHO)-CD390 cells. The cDNAs were generated by synthetic gene synthesis and cloned into a proprietary expression vector containing an antibiotic resistance (selection) gene (generating puromycin or methotrexate resistance). Two different plasmids encoding the two different chains (in the combination of 1D2-Fc x 5C8var1-Y105F-Fc and 1D2-Fc x 5C8var1-Y105F-R109A-Fc) were then co-transfected into CHO-CD390 cells at different plasmid ratios. After 48 hours, selection for puromycin and methotrexate was initiated, and after 13 days of culture, a pool of clones resistant to both antibiotics was obtained and cryopreserved.

[0347] Example 27 | Purification of bispecific antibodies from conditioned cell culture supernatant

[0348] In Infors HT multi-tube cell culture oscillator (bioreactor tube of 12ml scale, 14 days of cultivation), the soluble antibody production of selected cell pool was started using a general fed-batch scheme. After production, antibody was purified from conditional cell supernatant using protein A affinity chromatography. In brief, protein was captured on a protein AHP SpinTrap (protein A Sepharose) column (GE Healthcare) in 200mM sodium phosphate (pH7.0). After binding and washing, the antibody was eluted using pH shock (100mM sodium citrate pH3.6), and immediately by adding 1M Tris-HCl, pH9.0 neutralization solution. The amount of purified antibody was quantified using the Octet Red96 system (Sartorius) equipped with protein A sensor and human IgG as standard by biolayer interferometry (BLI).

[0349] Example 28 | Analysis of purified protein by CE-SDS (capillary electrophoresis)

[0350] Capillary electrophoresis is able to separate proteins by their apparent molecular weight and quantify all protein components by UV spectrophotometry. Sample preparation involves heat denaturation of the proteins in the presence of the denaturing agent sodium dodecyl sulfate (SDS). After denaturation, the sample components are separated based on differences in electrical mobility in a capillary containing a replaceable SDS polymer matrix. In FIG28 , 1D2-Fc x 5C8var1-Y105F-Fc ( Fig.28A ) and 1D2-Fc x5C8var1-Y105F-R109A-Fc( Fig.28B ). Analysis was performed using a PA800 Plus system (SCIEX) equipped with a PDA detector for detecting proteins at a wavelength of 220 nm. Since the proteins denature under non-reducing conditions, the main components of both samples were heterodimers with a retention time of 24-25 minutes. In the case of 1D2-Fc x 5C8var1-Y105F-Fc, a truncated version of 5C8var1-Y105F-Fc was detected, represented by "Peak 2", while the peak was not present in the electropherogram of 5C8var1-Y105F-R109A-Fc, indicating that 5C8var1-Y105F-R109A-Fc is not as easily fragmented as the observed 5C8var1-Y105F-Fc. Table 13 summarizes the quantification of various species. The major peak at 25 min corresponds to the intact heterodimer; "Peak 1" represents the minor half present in the 1D2-Fc x 5C8var1-Y105F-Fc preparation. "Peak 2" is the truncation observed at position 108 in 5C8var1-Y105F-Fc. "Peak 3" corresponds to the deglycosylated variant of the protein.

[0351] Table 13 | Quantification of different species in the electropherogram shown in Figure 28.

[0352]

[0353] Example 29 | Analysis of purified 1D2-Fc x 5C8var1-Y105F-R109A-Fc protein by mass spectrometry (MS)

[0354] The protein A purified 1D2-Fc x 5C8var1-Y105F-R109A-Fc protein was sent to CRO for MS analysis. At 37 ° C, N-glycosidase F (Roche) was used to N-deglycosylate the protein with an optimized enzyme: protein ratio overnight. The sample was diluted in 0.1% formic acid (FA) and separated using a reverse phase column (MAbPac RP 4 μm 2.1x 50mm, Thermo Scientific) on a UPLC system (Waters Acquity Premier H-class). The eluent was an aqueous solution of 0.1% FA and an acetonitrile solution of 0.1% FA. Mass spectrometry was performed using a Compact QTOF mass spectrometer (Bruker Daltonik). The recorded LC-ESI-MS spectra were summed, deconvoluted and smoothed using software DataAnalysis (Bruker Daltonik). For annotations, it was assumed that cysteine ​​was completely oxidized / reduced (depending on sample preparation). Four different batches of 1D2-Fc x 5C8var1-Y105F-R109A-Fc were analyzed. Although there was some heterogeneity in terms of C-terminal lysine cleavage and some heterogeneity in terms of glycosylation, no truncated products of 5C8var1-Y105F-R109A-Fc were found, again demonstrating that the R109A mutation inhibits the formation of truncations (data not shown).

Claims

1. An antibody comprising a first 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: 12, a CDR2 sequence as shown in SEQ ID NO: 13, and a CDR3 sequence as shown in SEQ ID NO:

14.

2. The antibody according to claim 1, wherein X1 in SEQ ID NO: 12 is S (Ser), and X2 in SEQ ID NO: 14 is F (Phe), X1 in SEQ ID NO: 12 is S (Ser), and X2 in SEQ ID NO: 14 is S (Ser), X1 in SEQ ID NO: 12 is S (Ser), and X3 in SEQ ID NO: 14 is A (Ala), or X1 in SEQ ID NO: 12 is S (Ser), and X3 in SEQ ID NO: 14 is K (Lys).

3. The antibody according to claim 1 or 2, wherein X1 in SEQ ID NO: 12 is S (Ser), X2 in SEQ ID NO: 14 is F (Phe), and X3 in SEQ ID NO: 14 is A (Ala), or X1 in SEQ ID NO: 12 is S (Ser), X2 in SEQ ID NO: 14 is F (Phe), and X3 in SEQ ID NO: 14 is K (Lys).

4. The antibody according to claim 1 or 2, wherein X1 in SEQ ID NO:12 is S (Ser), X2 in SEQ ID NO:14 is S (Ser), and X3 in SEQ ID NO:14 is A (Ala), or X1 in SEQ ID NO: 12 is S (Ser), X2 in SEQ ID NO: 14 is S (Ser), and X3 in SEQ ID NO: 14 is K (Lys).

5. The antibody according to any one of the preceding claims, wherein the first antigen binding region is a single domain antibody.

6. The antibody according to any one of the preceding claims, wherein the first antigen binding region comprises or consists of: The sequence shown in SEQ ID NO: 16, 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 as shown in SEQ ID NO: 16, wherein X1 is S (Ser), X2 is F (Phe), and X3 is A (Ala).

7. An antibody comprising a first antigen-binding region capable of binding to human Vδ2, wherein the first antigen-binding region comprises a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence selected from SEQ ID NOs: 17, 18, 19, and 20.

8. The antibody according to claim 7, comprising: (a) CDR1 sequence of SEQ ID NO: 8, CDR2 sequence of SEQ ID NO: 13, and CDR3 of SEQ ID NO: 17; (b) the CDR1 sequence of SEQ ID NO: 8, the CDR2 sequence of SEQ ID NO: 13, and the CDR3 sequence of SEQ ID NO: 18; (c) the CDR1 sequence of SEQ ID NO: 8, the CDR2 sequence of SEQ ID NO: 13, and the CDR3 sequence of SEQ ID NO: 19; or (d) CDR1 sequence of SEQ ID NO:8, CDR2 sequence of SEQ ID NO:13 and CDR3 of SEQ ID NO:

20.

9. The antibody of claim 7 or claim 8, wherein the first antigen binding region comprises or consists of a sequence selected from SEQ ID NO: 25, 26, 27 and 28.

10. An antibody comprising a first antigen-binding region capable of binding to human Vδ2, wherein the first antigen-binding region comprises a CDR1 sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 13, and a CDR3 sequence selected from SEQ ID NOs: 21, 22, 23, and 24.

11. The antibody according to claim 10, comprising: (a) CDR1 sequence of SEQ ID NO: 8, CDR2 sequence of SEQ ID NO: 13, and CDR3 of SEQ ID NO: 21; (b) the CDR1 sequence of SEQ ID NO: 8, the CDR2 sequence of SEQ ID NO: 13, and the CDR3 sequence of SEQ ID NO: 22; (c) the CDR1 sequence of SEQ ID NO: 8, the CDR2 sequence of SEQ ID NO: 13, and the CDR3 sequence of SEQ ID NO: 23; or (d) The CDR1 sequence of SEQ ID NO:8, the CDR2 sequence of SEQ ID NO:13, and the CDR3 of SEQ ID NO:

24.

12. The antibody of claim 10 or claim 11, wherein the first antigen binding region comprises or consists of a sequence selected from SEQ ID NO: 29, 30, 31 and 32.

13. The antibody according to any one of the preceding claims, wherein the antibody further comprises a second antigen binding region, and wherein the second antigen binding region is preferably a single domain antibody. The antibody according to claim 13 , wherein the antibody is a bispecific antibody. 15 . The antibody according to claim 13 , wherein the second antigen binding region is capable of binding to an antigen selected from the group consisting of CD123, PSMA, CD1d, CD40 and Nectin 4.

16. The antibody according to claim 15, wherein: (a) the second antigen binding region is capable of binding to CD123 and comprises the CDR1 sequence of SEQ ID NO: 37, the CDR2 sequence of SEQ ID NO: 38 and the CDR3 sequence of SEQ ID NO: 39; (b) the second antigen-binding region is capable of binding to PSMA and comprises the CDR1 sequence of SEQ ID NO:41, the CDR2 sequence of SEQ ID NO:42, and the CDR3 sequence of SEQ ID NO:43; (c) the second antigen-binding region is capable of binding to CD1d and comprises the CDR1 sequence of SEQ ID NO:45, the CDR2 sequence of SEQ ID NO:46 and the CDR3 sequence of SEQ ID NO:47; (d) the second antigen-binding region is capable of binding to CD40 and comprises the CDR1 sequence of SEQ ID NO:49, the CDR2 sequence of SEQ ID NO:50 and the CDR3 sequence of SEQ ID NO:51; or (e) the second antigen binding region is capable of binding to nectin 4 and comprises the CDR1 sequence selected from SEQ ID NO: 53, 57, 61, 65, 69, 73 and 77, the CDR2 selected from SEQ ID NO: 54, 58, 62, 66, 70, 74 and 78 and the CDR3 sequence selected from SEQ ID NO: 55, 59, 63, 67, 71, 75 and 79.

17. The antibody according to claim 15 or 16, wherein: (a) the second antigen binding region is capable of binding to CD123 and comprises or consists of SEQ ID NO: 40; (b) the second antigen binding region is capable of binding to PSMA and comprises or consists of SEQ ID NO: 44; (c) the second antigen binding region is capable of binding to CD1d and comprises or consists of SEQ ID NO: 48; (d) the second antigen binding region is capable of binding to CD40 and comprises or consists of SEQ ID NO:52; or (e) the second antigen-binding region is capable of binding to nectin 4 and comprises or consists of a sequence selected from SEQ ID NO: 56, 60, 64, 68, 72, 76 and 80.

18. The antibody of any one of claims 13-17, wherein the first antigen binding region and the second antigen binding region are covalently linked via a peptide linker.

19. The antibody according to any one of the preceding claims, wherein the antibody further comprises a half-life extending domain, such as an Fc region.

20. An antibody according to any of the preceding claims, wherein the antibody comprises an Fc region, wherein 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 that favor the formation of heterodimers rather than homodimers.

21. The antibody according to claim 20, wherein the CH3 region of the Fc polypeptide comprises the asymmetric amino acid mutation, 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.

22. The antibody according to claim 20 or 21, 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.

23. The antibody according to any one of claims 19 to 22, wherein The first Fc polypeptide comprises the sequence shown in SEQ ID NO: 83, and the second Fc polypeptide comprises the sequence shown in SEQ ID NO: 84, or The first Fc polypeptide comprises the sequence shown in SEQ ID NO:84, and the second Fc polypeptide comprises the sequence shown in SEQ ID NO:

83.

24. The antibody according to any one of claims 19 to 23, wherein the antibody comprises or consists of: The sequence shown in SEQ ID NO:86, and A sequence selected from the group of sequences shown in SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89 and SEQ ID NO:

90.

25. A pharmaceutical composition comprising the antibody according to any one of the preceding claims and a pharmaceutically acceptable excipient.

26. The antibody according to any one of claims 1 to 24 for use as a medicament, preferably for the treatment of cancer.

27. A method of treating a disease comprising administering the antibody of any one of claims 1 to 24 to a human subject in need thereof.

28. A nucleic acid construct encoding the antibody according to any one of claims 1 to 24.

29. An expression vector comprising the nucleic acid construct according to claim 28.

30. A host cell comprising one or more nucleic acid constructs encoding the antibody of any one of claims 1 to 25.

31. A method for making an antibody, comprising expressing in a host cell one or more nucleic acids encoding an antibody according to any one of claims 1 to 24, wherein the host cell is preferably a Chinese hamster ovary cell, a human embryonic kidney cell or a Pichia pastoris cell.

Images