Variant antibodies that bind to gamma-delta T cell receptors

Abstract

The present invention relates to an antibody capable of binding to the human Vγ9Vδ2 T cell receptor. The present invention further relates to a pharmaceutical composition comprising the antibody of the present invention and the use of the antibody of the present invention for medical treatment.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the priority of EP22179260.9 filed on June 15, 2022, the content of which is incorporated herein by reference.

[0002] Electronic version of the sequence listing The content of the electronic sequence listing (LVAT_024_01WO_seqList_ST26.XML; size: 101,2824 bytes; creation date: June 14, 2023) is incorporated herein by reference.

[0003] 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 the use of the antibodies of the present invention for medical treatment.

Background Art

[0004] Gamma - delta (γδ) T cells are T cells that express a T - cell receptor (TCR) containing a gamma chain and a delta chain. Most γδ T cells express a TCR containing the Vγ9 and Vδ2 regions. Vγ9Vδ2 T cells can react with a wide range of pathogens and tumor cells. It can be seen that this broad reactivity is conferred by phosphoantigens that can specifically activate this T - cell subset in a TCR - dependent manner. The broad antibacterial and antitumor reactivity of Vγ9Vδ2 T cells suggests a direct involvement in the immune control of cancer and infections.

[0005] Agents that can activate Vγ9Vδ2 T cells are useful for the treatment of infectious diseases or cancer because they promote Vγ9Vδ2 T cell reactivity towards pathogens, infected cells or cancer cells. WO2015156673 describes antibodies that bind to Vγ9Vδ2 TCRS and can activate Vγ9Vδ2 T cells. WO2020060405 describes bispecific antibodies that bind to both Vγ9Vδ2 T cells and tumor cell targets and thus have the potential to redirect Vγ9Vδ2 T cells to tumors and stimulate a therapeutic effect. SUMMARY OF THE INVENTION

[0006] Recombinant production of antibodies in host cells invariably results in products of heterogeneous origin that contain different forms of the antibody with various types and degrees of post-translational modification of the polypeptide chain. Such heterogeneity is undesirable in medical antibody products because post-translational modifications can alter the functionality of the antibody, for example, in terms of affinity for the target antigen, pharmacological properties, product stability, aggregation, etc.

[0007] The present invention provides improved Vγ9Vδ2 TCR-binding antibody sequences that, when produced in host cells, result in a more homogeneous product and retain good functionality for target binding and action on target cells, as well as good structural properties such as stability.

[0008] Production of a bispecific antibody comprising the Vγ9Vδ2-TCR-binding antibody 5C8 (described in WO2015 / 156673) in mammalian cells has been demonstrated to result in heterogeneity of the antibody product such that a diverse population of antibody molecules is proteolytically cleaved within the CDR3 region of 5C8, as determined by mass spectrometric analysis of the purified protein. This cleavage occurs at a sequence where proteolytic cleavage was not predicted to occur readily. Furthermore, surprisingly, the residue is immediately prior to the proteolytic cleavage site without adversely affecting the antigen-binding properties of the antibody even though this residue is located in the CDR3 region. The CDR3 region is known to be the major determinant of antigen-binding specificity in the antigen-binding region of an antibody.

[0009] Furthermore, the inventors have found that antibody 5C8 undergoes sulfation at a site of the antibody that was not predicted to occur for this post-translational modification. Also, by partially sulfating in various host cells, heterogeneous antibody products are obtained. Surprisingly, tyrosine residues that undergo sulfation can be mutated without adversely affecting the antigen-binding properties of the antibody, even if the amino acids are also located in the CDR3 region. Removal of the sulfation site via mutation resulted in a more homogeneous antibody product.

[0010] Thus, 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 the CDR1 sequence set forth in SEQ ID NO: 12, the CDR2 sequence set forth in SEQ ID NO: 13, and the CDR3 sequence set forth in SEQ ID NO: 14.

[0011] In a further aspect, the present invention provides a bispecific antibody comprising a first 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 a further aspect, the present invention relates to a pharmaceutical composition comprising the antibody of the present invention, the use of the antibody of the present invention in medical treatment, and nucleic acid constructs, expression vectors for producing the antibody of the present invention, and host cells comprising such nucleic acids or expression vectors. The present invention also relates to a process for producing the antibody of the present invention to obtain a more homogeneous product.

[0012] Hereinafter, further aspects and embodiments of the present invention will be described.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0022] Definitions The term "human Vδ2", as used herein, refers to the rearranged δ2 chain of the Vγ9Vδ2-T cell receptor (TCR). UniProtKB-A0JD36 (A0JD36_HUMAN) shows an example of a variable TRDV2 sequence.

[0023] The term "human Vγ9", as used herein, refers to the rearranged γ9 chain of the Vγ9Vδ2-T cell receptor (TCR). UniProtKB-Q99603_HUMAN shows an example of a variable TRGV9 sequence.

[0024] The term "EGFR", as used herein, refers to the human EGFR protein (UniProtKB-P00533 (EGFR_HUMAN)).

[0025] The term "CD123", as used herein, refers to the human CD123 protein, which is also called the 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 having the common beta chain CD131 (NCBI reference sequence: NP_000386.1).

[0026] The term "PSMA", as used herein, refers to the human prostate-specific membrane antigen protein (UniProtKB-Q04609 (FOLH1_HUMAN)).

[0027] As used herein, the term "CD40" refers to tumor necrosis factor receptor superfamily member 5 (UniProtKB - P25942 (TNR5_HUMAN)), the CD40 protein also known as isoform I.

[0028] As used herein, the term "CD1d" refers to the human CD1d protein (UniProtKB - P15813 (CD1d_HUMAN)).

[0029] As used herein, the term "nectin 4" refers to the human nectin 4 protein (UniProtKB - Q96NY8 (NECT4_HUMAN)).

[0030] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof, which, under typical physiological conditions, has the ability to specifically bind to an antigen for a significant period of time, such as at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, more than about 24 hours, more than about 48 hours, about 3, 4, 5, 6, 7 days, or more, etc., a half - life, or any other relevant functionally defined period (e.g., a time sufficient to induce, facilitate, enhance, and / or modulate a physiological response related to an antibody that binds to an antigen, and / or a time sufficient for the antibody to acquire effector activity). The antigen - binding region (or antigen - binding domain) that interacts with the antigen can include the variable regions of both the heavy and light chains of the immunoglobulin molecule, or can include only a single - domain antigen - binding region, e.g., only the heavy - chain variable region. The constant region of the antibody, when present, can 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), as well as components of the complement system such as C1q, which is the first component in the classical pathway of complement activation.

[0031] The Fc region of an immunoglobulin is typically defined as the fragment of an antibody generated after digestion of the antibody with papain, which includes two CH2-CH3 regions and a connecting region (e.g., the hinge region) of the immunoglobulin. The constant domain of the antibody heavy chain defines the antibody isotype, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE. The Fc region mediates the effector functions of the antibody, together with Fc receptors of the complement system and cell surface receptors called proteins.

[0032] 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 the EU numbering.

[0033] 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 the EU numbering. However, the CH2 region may also be of any of the other subtypes described herein.

[0034] 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 the EU numbering. However, the CH3 region may also be of any of the other subtypes described herein.

[0035] References to amino acid positions in the Fc region / Fc domain in the present invention are by EU numbering (Edelman et al., Proc Natl Acad Sci USA. 1969 May; 63(1): 78-85, Kabat et al., Sequences of proteins of immunological interest. 5th Edition - 1991 NIH Publication No. 91-3242).

[0036] As indicated above, the term antibody as used herein includes antibody fragments that retain the ability to specifically bind to an antigen, unless otherwise specified or clearly inconsistent with the context. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the term antibody are: (i) Fab’ or Fab fragments, i.e., monovalent fragments consisting of the VL, VH, CL, and CH1 domains, or monovalent antibodies described in WO2007059782; (ii) F(ab’)2 fragments, i.e., divalent fragments comprising two Fab fragments linked by disulfide bridges in the hinge region; (iii) Fd fragments consisting essentially of the VH and CH1 domains; and (iv) Fv fragments consisting essentially of the VL and VH domains of a single arm of an antibody. Furthermore, the two domains of an Fv fragment, VL and VH, are encoded by separate genes, but they can be joined by a synthetic linker that enables them to be made as a single protein chain using recombinant methods, and the VL and VH regions pair to form a monovalent molecule (known as 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)). Such single-chain antibodies are included within the term antibody unless clearly indicated by the context. Such fragments are generally included within the meaning of an antibody, but they are, collectively and each independently, unique features of the present invention and exhibit different biological properties and utilities. The term antibody includes polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, and humanized antibodies, as well as antibody fragments provided by any known technique such as enzymatic cleavage, peptide synthesis, and recombinant techniques, unless otherwise specified.

[0037] 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 contain a single CDR1, a single CDR2, and a single CDR3. Examples of single-domain antibodies are antibodies consisting of only heavy chains, antibodies that do not naturally contain a light chain, single-domain antibodies derived from conventional antibodies, and engineered variable fragments of antibodies. Single-domain antibodies can be derived from any species, including mouse, human, camel, llama, shark, goat, rabbit, and cow. For example, single-domain antibodies can be derived from antibodies produced in camelid species, such as camel, dromedary, llama, alpaca, and guanaco. Similar to full antibodies, single-domain antibodies can selectively bind to specific antigens. Single-domain antibodies can contain only the variable domain of the immunoglobulin chain, i.e., CDR1, CDR2, and CDR3, as well as the framework regions. Such antibodies are also referred to as Nanobody® or VHH.

[0038] As used herein, the term "immunoglobulin" is intended to refer to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) chains, and one pair of heavy (H) chains, all four of which are potentially interconnected by disulfide bonds. As used herein, the terms "immunoglobulin heavy chain", "heavy chain of an immunoglobulin", or "heavy chain" are intended to refer to one of the chains of an immunoglobulin. A heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH) that defines the isotype of the immunoglobulin. The heavy chain constant region typically consists of three domains, CH1, CH2, and CH3. The heavy chain constant region further includes a hinge region. Within the structure of an immunoglobulin (e.g., IgG), the two heavy chains are interconnected via disulfide bonds within the hinge region. Similar to the heavy chain, each light chain typically consists of several regions, a light chain variable region (VL) and a light chain constant region (CL). Furthermore, the VH and VL regions are also referred to as hypervariable regions (or regions that can be hypervariable in sequence and / or form structurally defined loops), or complementarity determining regions (CDRs), and can be subdivided into hypervariable regions interspersed with more conserved regions, referred to as framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, and are arranged in the following order, from amino terminus to carboxy terminus: 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).

[0039] As used herein, the term "isotype" refers to immunoglobulin (sub)classes (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM), or any allotype thereof such as IgG1m(za) and IgG1m(f) encoded by heavy chain constant region genes. Each heavy chain isotype can be combined with either a kappa (κ) or lambda (λ) light chain. The antibodies of the present invention can have any isotype.

[0040] The term "parent antibody" should be understood as an antibody that is identical to the antibody according to the present invention, but the parent antibody does not have one or more of the specific mutations. A "variant" or "antibody variant" or "variant of the parent antibody" of the present invention is an antibody molecule that contains one or more mutations as compared to the "parent antibody". Amino acid substitutions can exchange a natural amino acid for another naturally occurring amino acid, or a non-naturally occurring amino acid derivative. The amino acid substitutions can be either conservative or non-conservative. In the context of the present invention, conservative substitutions can be defined by substitutions within a class of amino acids that are reflected in one or more of the following three tables. [Table 1] [Table 2] [Table 3]

[0041] In the context of the present invention, substitutions in variants are indicated as the original amino acid-position-substituted amino acid. To indicate amino acid residues, the three-letter code containing Xaa and X, or the one-letter code is used. Thus, the notation "T366W" means that the variant contains a substitution of threonine by tryptophan at the variant amino acid position corresponding to the amino acid at position 366 in the parent antibody.

[0042] Furthermore, the term "substitution" encompasses substitution with any one of the other 19 natural amino acids or substitution with other amino acids such as non-natural amino acids. For example, substitution of the amino acid T at position 366 includes each of the substitutions 366A, 366C, 366D, 366G, 366H, 366F, 366I, 366K, 366L, 366M, 366N, 366P, 366Q, 366R, 366S, 366E, 366V, 366W, and 366Y.

[0043] As used herein, the term "full-length antibody" refers to an antibody that includes all heavy and light chain constant and variable domains corresponding to those normally found in the wild-type antibody of its isotype.

[0044] 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 use are developed to reduce antibody immunogenicity.

[0045] The term "humanized antibody" refers to a genetically engineered non-human antibody that contains human antibody constant domains and a non-human variable domain that has been modified to contain a high level of sequence homology with the human variable domain. This can be achieved by transplanting the six non-human antibody complementarity determining regions (CDRs) that together form the antigen binding site into a homologous human acceptor framework region (FR). Substitution (reverse mutation) of framework residues from the parent antibody (i.e., the non-human antibody) into the human framework region may be required to fully reconstruct the binding affinity and specificity of the parent antibody. Structural homology modeling can be useful for identifying amino acid residues within the framework region that are important for the binding properties of the antibody. Thus, a humanized antibody may include non-human CDR sequences, a human framework region optionally containing one or more amino acid reverse mutations to a non-human amino acid sequence, and optionally a fully human constant region. Optionally, additional amino acid modifications that are not necessarily reverse mutations can be introduced to obtain a humanized antibody having desirable properties such as affinity and biochemical characteristics. Humanization of non-human therapeutic antibodies is done to minimize their immunogenicity in humans, and such humanized antibodies simultaneously maintain the specificity and binding affinity of the non-human-derived antibody.

[0046] The term "multispecific antibody" refers to an antibody that has specificity for at least two different, e.g., at least three, typically non-overlapping epitopes. Such epitopes may be on the same target antigen or on different target antigens. When the epitopes are on different targets, such targets may be on the same cell or on different cells or cell types. In some embodiments, a multispecific antibody may include one or more single domain antibodies.

[0047] The term "bispecific antibody" refers to an antibody having specificity for two different, typically non-overlapping epitopes. Such epitopes may be on the same target or on different targets. When the epitopes are on different targets, such targets may be on the same cell or on different cells or cell types. In some embodiments, the bispecific antibody may comprise one or two single domain antibodies.

[0048] Examples of different classes of multispecific antibodies, such as bispecific antibodies, antibodies, etc., include: (i) IgG-like molecules having complementary CH3 domains for forcing heterodimerization; (ii) recombinant IgG-like dual-target molecules, wherein two sides of the molecule each contain a Fab fragment or a part of a Fab fragment of at least two different antibodies; (iii) IgG fusion molecules, wherein a full-length IgG antibody is fused to an extra Fab fragment or a part 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 part thereof; (v) Fab fusion molecules, wherein different Fab fragments are fused together and fused to a heavy chain constant domain, an Fc region or a part thereof; and (vi) scFv and bispecific antibody-based antibodies and heavy chain antibodies (e.g., domain antibodies, Nanobodies®), wherein different single-chain Fv molecules or different bispecific antibodies or different heavy chain antibodies (e.g., domain antibodies, Nanobodies®) are fused to each other or fused to another protein or carrier molecule fused to a heavy chain constant domain, an Fc region or a part thereof, but are not limited thereto.

[0049] Examples of IgG-like molecules having a complementary CH3 domain molecule include, but are not limited to, Triomab® (Trion Pharma / Fresenius Biotech), Knobs-into-Hole (Genentech), CrossMAb (Roche) and electrostatically matched (Amgen, Chugai, Oncomed), LUZ-Y (Genentech, Wranik et al. J. Biol. Chem. 2012, 287(52):43331-9, doi:10.1074 / jbc.M112.397869. Epub 2012 Nov 1), DIG-body and PIG-body (Pharmabcine, WO2010134666, WO2014081202), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Biclonics (Merus, WO2013157953), FcΔAdp (Regeneron), bispecific IgG1 and IgG2 (Pfizer / Rinat), Azymetric scaffold (Zymeworks / Merck), mAb-Fv (Xencor), bivalent bispecific antibodies (Roche, WO2009080254) and DuoBody® molecules (Genmab).

[0050] Examples of recombinant IgG-like bispecific molecules include, but are not limited to, Dual Targeting (DT)-Ig (GSK / Domantis, WO2009058383), Two-in-one Antibody (Genentech, Bostrom, et al 2009. Science 323, 1610-1614), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star), Zybodies (trademark) (Zyngenia, LaFleur et al. MAbs. 2013 Mar-Apr;5(2):208-18), the approach using a common light chain, κλBodies (NovImmune, WO2012023053), and CovX-body (registered trademark) (CovX / Pfizer, Doppalapudi, V.R., et al 2007. Bioorg. Med. Chem. Lett. 17, 501-506).

[0051] Examples of IgG fusion molecules include, but are not limited to, Dual Variable Domain (DVD)-Ig (Abbott), Dual domain double head antibodies (Unilever, Sanofi Aventis), IgG-like Bispecific (ImClone / Eli Lilly, Lewis et al. Nat Biotechnol. 2014 Feb;32(2):191-8), Ts2Ab (MedImmune / AZ, Dimasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92), and BsAb (Zymogenetics, WO2010111625), HERCULES (Biogen Idec), scFv fusion (Novartis), scFv fusion (Changzhou Adam Biotech Inc), and TvAb (Roche).

[0052] Examples of Fc fusion molecules include, but are not limited to, ScFv / Fc fusion (Academic Institution, Pearce et al Biochem Mol Biol Int. 1997 Sep;42(6):1179), SCORPION (Emergent BioSolutions / Trubion, Blankenship JW, et al. AACR 100th Annual meeting 2009 (Abstract #5465), Zymogenetics / BMS, WO2010111625), Dual Affinity Retargeting Technology (Fc-DARTTM) (MacroGenics), and Dual(ScFv)2-Fab (National Research Center for Antibody Medicine-China).

[0053] Examples of Fab fusion bispecific antibodies include, but are not limited to, F(ab)2 (Medarex / AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (registered trademark) (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol), and Fab-Fv (UCB-Celltech).

[0054] Examples of ScFv antibodies, diabody-based antibodies, and domain antibodies include, but are not limited to, Bispecific T Cell Engager (BiTE®) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART™) (MacroGenics), Single-chain Diabody (Academic, Lawrence FEBS Lett. 1998 Apr 3;425(3):479-84), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack, WO2010059315), and COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 Aug;88(6):667-75), dual targeting nanobodies® (Ablynx, Hmila et al., FASEB J. 2010), dual targeting heavy chain only domain antibodies.

[0055] In the context of an antibody that binds to an antigen, the terms "binds" or "specifically binds" refer to the binding of the antibody to a given antigen or target, and that binding is typically, for example, about 10-6 M or less, such as 10-7 M or less, such as about 10-8 M or less, such as about 10-9 M or less, about 10-10 M or less, or about 10-11 M or less when measured using, for example, flow cytometry as described in the examples herein, corresponding to an apparent affinity with a KD. Alternatively, the KD value can be determined using, for example, the surface plasmon resonance (SPR) technique of a BIAcore T200, or the biolayer interferometry (BLI) of an Octet RED96 instrument, using the antigen as a ligand and the binding moiety or binding molecule as an analyte. Specific binding means that the antibody binds to a given antigen with an affinity that is at least 10-fold lower, such as at least 100-fold lower, such as at least 1,000-fold lower, such as at least 10,000-fold lower, such as at least 100,000-fold lower, than the affinity for binding to non-specific antigens (e.g., BSA, casein) other than the given antigen or antigens closely related thereto. The degree of low affinity depends on the KD of the binding moiety or binding molecule, so when the KD of the binding moiety or binding molecule is very low (i.e., when the binding moiety or binding molecule is very specific), the degree to which the affinity for the antigen is lower than the affinity for non-specific antigens can be at least 10,000-fold. The term "KD" (M) as used herein refers to the dissociation equilibrium constant of a particular interaction between an antigen and a binding moiety or binding molecule.

[0056] In the context of the present invention, "compete" or "competitive" or "competing" refers to a detectable and significant decrease in the tendency of a particular binding molecule to bind to a particular binding partner in the presence of another molecule that binds to the binding partner. Typically, competition refers to, for example, at least about a 25% decrease, such as at least about 50%, such as at least about 75%, such as at least 90% decrease in binding caused by the presence of another molecule, such as an antibody, determined by, for example, ELISA assays or flow cytometry using two or more competing molecules, such as antibodies, in sufficient amounts. Additional methods for determining binding specificity by competitive inhibition can be found, for example, in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley InterScience N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92, 589-601 (1983)).

[0057] In one embodiment, the antibody of the present invention binds to the same epitope on EGFR as antibody 7D12, the same epitope on CD123 as antibody 1D2, the same epitope on CD1d as antibody 1D12, the same epitope on PSMA as antibody LV1050, the same epitope on CD40 as antibody v9, the same epitope on nectin 4 as antibody LV1184, LV1181, LV1185, LV1186, LV1178, LV1183 or LV1187, or the same epitope on Vδ2 as antibody 5C8, or binds to those antibodies 5C8, or variants thereof. There are several methods available in the art for mapping antibody epitopes on target antigens, including but not limited to cross-linking mass spectrometry that enables identification of peptides that are part of the epitope, and X-ray crystallography that identifies individual residues on the antigen that form the epitope. Epitope residues can be determined to be all amino acid residues having at least 1 atom within 5 Å from the antibody. A 5 Å was selected as the epitope cut-off distance to allow the atoms to be within the van der Waals radius + possible hydrogen bonds. Next, epitope residues can be determined to be all amino acid residues having at least 1 atom within 8 Å. Selecting an epitope cut-off distance of 8 Å or less allows for the length of the extended arginine amino acid. Cross-linking mass spectrometry begins by binding the antibody and antigen with a mass-labeled chemical cross-linker. Next, the presence of the complex is confirmed using high mass MALDI detection. After chemically cross-linking, the Ab / Ag complex is very stable, so many different and overlapping peptides can be provided by applying many different enzyme and digestion conditions to the complex. Identification of these peptides is performed using high-resolution mass spectrometry and MS / MS techniques. Identification of the cross-linked peptides is determined using mass tags linked to the cross-linking reagent. After MS / MS fragmentation and data analysis, the cross-linked peptides derived from the antigen are part of the epitope and the peptides derived from the antibody are part of the paratope. All residues between the most N-terminal and C-terminal cross-linked residues from the individual cross-linked peptides discovered are considered to be part of the epitope or paratope.The epitope of antibody 7D12 is that determined by X-ray crystallography as described in Schmitz et al. (2013) Structure 21:1214 and consists of a plane on domain III (residues R353, D355, F357, Q384, N420) corresponding to the domain III ligand binding site.

[0058] The terms "first" and "second" antigen-binding regions, as used herein, do not refer to their orientation / position in the antibody, i.e., they have no meaning with respect to the N-terminus or C-terminus. The terms "first" and "second" function only to accurately and consistently refer to two different antigen-binding regions in the claims and description.

[0059] "Percent sequence identity", as used herein, refers to the number of identical nucleotide or amino acid positions shared by different sequences (i.e., % identity = # of identical positions / total # 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 may be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988) incorporated in the ALIGN program (version 2.0) using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Further aspects and embodiments of the invention

[0060] As described above, in a first aspect, the invention relates to an antibody comprising a first antigen-binding region capable of binding to human Vδ2, wherein the first antigen-binding region comprises the CDR1 sequence set forth in SEQ ID NO: 12, the CDR2 sequence set forth in SEQ ID NO: 13, and the CDR3 sequence set forth in SEQ ID NO: 14.

[0061] In one embodiment, X1 in SEQ ID NO: 12 is S (Ser). In another embodiment, X1 in SEQ ID NO: 12 is G (Gly).

[0062] In one embodiment, X2 at 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 at SEQ ID NO: 14 is F or S.

[0063] In one embodiment, X2 at 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.

[0064] In one embodiment, X2 at SEQ ID NO: 14 is F (Phe). In another embodiment, X2 at SEQ ID NO: 14 is S (Ser).

[0065] In one embodiment, X1 at SEQ ID NO: 12 is S and X2 at SEQ ID NO: 3 is F.

[0066] In one embodiment, X1 at SEQ ID NO: 12 is S and X2 at SEQ ID NO: 3 is S.

[0067] In one embodiment, X1 at SEQ ID NO: 12 is G and X2 at SEQ ID NO: 3 is F.

[0068] In one embodiment, X1 at SEQ ID NO: 12 is G and X2 at SEQ ID NO: 3 is S.

[0069] In one embodiment, X3 at 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.

[0070] In one embodiment, X3 at 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.

[0071] In one embodiment, X3 at SEQ ID NO: 14 is A (Ala). In another embodiment, X3 at SEQ ID NO: 14 is K (Lys).

[0072] In one embodiment, X1 at SEQ ID NO: 12 is S, and X3 at SEQ ID NO: 14 is A.

[0073] In one embodiment, X1 at SEQ ID NO: 12 is S, and X3 at SEQ ID NO: 14 is K.

[0074] In one embodiment, X1 at SEQ ID NO: 12 is G, and X3 at SEQ ID NO: 14 is A.

[0075] In one embodiment, X1 at SEQ ID NO: 12 is G, and X3 at SEQ ID NO: 14 is K.

[0076] In one embodiment, X2 at SEQ ID NO: 14 is F, and X3 at SEQ ID NO: 14 is A.

[0077] In one embodiment, X2 at SEQ ID NO: 14 is F, and X3 at SEQ ID NO: 14 is K.

[0078] In one embodiment, X2 at SEQ ID NO: 14 is S, and X3 at SEQ ID NO: 14 is A.

[0079] In one embodiment, X2 at SEQ ID NO: 14 is S, and X3 at SEQ ID NO: 14 is K.

[0080] In one embodiment, (a) X1 at SEQ ID NO: 12 is S, and X2 at SEQ ID NO: 14 is F, (b) X1 at SEQ ID NO: 12 is S, and X2 at SEQ ID NO: 14 is S, (c) X1 at SEQ ID NO: 12 is S, and X3 at SEQ ID NO: 14 is A, or (d) X1 at SEQ ID NO: 12 is S, and X3 at SEQ ID NO: 14 is K.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] In one embodiment, (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 (b) At SEQ ID NO: 12, X1 is S (Ser); at SEQ ID NO: 14, X2 is F (Phe); at SEQ ID NO: 14, X3 is K (Lys).

[0090] In one embodiment, (a) At SEQ ID NO: 12, X1 is S (Ser); at SEQ ID NO: 14, X2 is S (Ser); at SEQ ID NO: 14, X3 is A (Ala); or (b) At SEQ ID NO: 12, X1 is S (Ser); at SEQ ID NO: 14, X2 is S (Ser); at SEQ ID NO: 14, X3 is K (Lys).

[0091] 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 the CDR1 sequence of SEQ ID NO: 8, the CDR2 sequence of SEQ ID NO: 13, and the CDR3 sequence of SEQ ID NO: 17. In some embodiments, the antibody comprises, or consists of, a first antigen-binding region having the amino acid sequence of SEQ ID NO: 25 that is capable of binding to human Vδ2. 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 the CDR1 sequence of SEQ ID NO: 8, the CDR2 sequence of SEQ ID NO: 13, and the CDR3 sequence of SEQ ID NO: 21. In some embodiments, the antibody comprises, or consists of, a first antigen-binding region having the amino acid sequence of SEQ ID NO: 29 that is capable of binding to human Vδ2.

[0092] 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, or consists of, a first antigen-binding region having an amino acid sequence of SEQ ID NO: 26 that is capable of binding to human Vδ2. 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, or consists of, a first antigen-binding region having an amino acid sequence of SEQ ID NO: 30 that is capable of binding to human Vδ2.

[0093] 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, or consists of, a first antigen-binding region having an amino acid sequence of SEQ ID NO: 27 that is capable of binding to human Vδ2. 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, or consists of, a first antigen-binding region having an amino acid sequence of SEQ ID NO: 31 that is capable of binding to human Vδ2.

[0094] 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, or consists of, a first antigen-binding region having an amino acid sequence of SEQ ID NO: 28 that is capable of binding to human Vδ2. 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, or consists of, a first antigen-binding region having an amino acid sequence of SEQ ID NO: 32 that is capable of binding to human Vδ2.

[0095] In preferred embodiments, the antibody is capable of activating human Vγ9Vδ2 T cells. Activation of Vγ9Vδ2 T cells can be measured by measuring profiles in gene expression and / or (surface) marker expression (e.g., activation markers such as CD25, CD69, or CD107a) and / or secreted protein (e.g., cytokine or chemokine) profiles. In preferred embodiments, the antibody is capable of inducing activation (e.g., upregulation of CD69 and / or CD25 expression) resulting in degranulation characterized by an increase in CD107a expression and / or cytokine production (e.g., TNF, IFNγ) by Vγ9Vδ2 T cells.

[0096] In a further preferred embodiment, when the antibody is tested at a concentration of, for example, 1 nM, preferably 100 pM, preferably 10 pM, preferably 1 pM, and more preferably 100 fM as described in Example 9, the number of CD107a-positive cells can be increased by at least 2-fold, for example, at least 5-fold. In another preferred embodiment, the antibody of the present invention has an EC50 value for increasing the proportion of CD107a-positive cells to 100 pM or less, for example, 50 pM or less, for example, 25 pM or less, for example, 20 pM or less, for example, 15 pM or less when tested using Vγ9Vδ2 T cells and A431 target cells as described in Example 9 herein.

[0097] In one embodiment, the first antigen-binding region is a single-domain antibody. Thus, in one embodiment, the antibody of the present invention includes a single-domain antibody capable of binding to human Vδ2, and the first antigen-binding region includes the CDR1 sequence set forth in SEQ ID NO: 12, the CDR2 sequence set forth in SEQ ID NO: 13, and the CDR3 sequence set forth in SEQ ID NO: 14.

[0098] In some embodiments, the antibody includes a single-domain antibody capable of binding to human Vδ2 and including the CDR1 sequence of SEQ ID NO: 8, the CDR2 sequence of SEQ ID NO: 13, and the CDR3 sequence of SEQ ID NO: 17. In some embodiments, the antibody includes a single-domain antibody capable of binding to human Vδ2 and including or consisting of the amino acid sequence of SEQ ID NO: 25. In some embodiments, the antibody includes a single-domain antibody capable of binding to human Vδ2 and including the CDR1 sequence of SEQ ID NO: 8, the CDR2 sequence of SEQ ID NO: 13, and the CDR3 sequence of SEQ ID NO: 21. In some embodiments, the antibody includes a single-domain antibody capable of binding to human Vδ2 and including or consisting of the amino acid sequence of SEQ ID NO: 29.

[0099] In some embodiments, the antibody comprises a single-domain antibody comprising 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, which is capable of binding to human Vδ2. In some embodiments, the antibody comprises a single-domain antibody comprising or consisting of the amino acid sequence of SEQ ID NO: 26, which is capable of binding to human Vδ2. In some embodiments, the antibody comprises a single-domain antibody comprising 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, which is capable of binding to human Vδ2. In some embodiments, the antibody comprises a single-domain antibody comprising or consisting of the amino acid sequence of SEQ ID NO: 30, which is capable of binding to human Vδ2.

[0100] In some embodiments, the antibody comprises a single-domain antibody comprising 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, which is capable of binding to human Vδ2. In some embodiments, the antibody comprises a single-domain antibody comprising or consisting of the amino acid sequence of SEQ ID NO: 27, which is capable of binding to human Vδ2. In some embodiments, the antibody comprises a single-domain antibody comprising 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, which is capable of binding to human Vδ2. In some embodiments, the antibody comprises a single-domain antibody comprising or consisting of the amino acid sequence of SEQ ID NO: 31, which is capable of binding to human Vδ2.

[0101] In some embodiments, the antibody comprises a single-domain antibody capable of binding to human Vδ2 and comprising 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 capable of binding to human Vδ2 and comprising or consisting of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the antibody comprises a single-domain antibody capable of binding to human Vδ2 and 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 single-domain antibody capable of binding to human Vδ2 and comprising or consisting of the amino acid sequence of SEQ ID NO: 32.

[0102] In another embodiment, the first antigen-binding region is humanized, and preferably, the antigen-binding region comprises or consists of (a) the sequence set forth in SEQ ID NO: 16, or (b) a sequence having at least 90%, such as at least 92%, such as at least 94%, such as at least 96%, such as at least 98% sequence identity to the sequence set forth 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.

[0103] In some embodiments, the antibodies of the invention are multispecific antibodies such as bispecific antibodies. Thus, in one embodiment, the antibody further comprises a second antigen-binding region. In one embodiment, the second antigen-binding region is a single-domain antibody.

[0104] In further embodiments, the antibody is a bispecific antibody, and both the first antigen-binding region and the second antigen-binding region are single-domain antibodies. In further embodiments, the multispecific antibody is a bispecific antibody, 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.

[0105] In some embodiments, the antibody comprises a second antigen-binding region capable of binding to human EGFR. Bispecific antibodies targeting both Vγ9Vδ2 T cells and EGFR have been shown to induce potent Vγ9Vδ2 T cell activation and tumor cell lysis in both in vitro and in vivo mouse xenograft models (de Bruin et al. (2018) Oncoimmunology 1,e1375641).

[0106] In some embodiments, the antibody comprises a second antigen-binding region comprising the CDR1 sequence set forth in SEQ ID NO: 33, the CDR2 sequence set forth in SEQ ID NO: 34, and the CDR3 sequence set forth in SEQ ID NO: 35. In some embodiments, the antibody comprises or consists of SEQ ID NO: 36, or a second antigen-binding region comprising an amino acid sequence having at least 90%, such as at least 92%, such as at least 94%, such as at least 96%, such as at least 98% sequence identity to SEQ ID NO: 36.

[0107] In some embodiments, the second antigen-binding region competes with (i.e., is capable of competing with) an antibody having the sequence set forth in SEQ ID NO: 36 for binding to human EGFR, and preferably, the second antigen-binding region binds to the same epitope on human EGFR as the antibody having the sequence set forth in SEQ ID NO: 36.

[0108] 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 NO: 17, 18, 19, 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 NO: 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.

[0109] In some embodiments, the antibody comprises a first antigen-binding region comprising or consisting of a sequence selected from SEQ ID NO: 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 NO: 29, 30, 31, and 32, and a second antigen-binding region comprising or consisting of SEQ ID NO: 36.

[0110] In a further embodiment, the antibody is capable of killing human EGF-expressing cells. In a preferred embodiment, the antibody can increase the Vγ9Vδ2T cell B-mediated killing of EGF-expressing cells, such as A431 cells, by at least 25%, such as at least 50%, such as at least 2-fold, when tested as described in Example 9 herein.

[0111] In a further embodiment, the antibody is unable to mediate the killing of EGFR-negative cells, such as EGFR-negative human cells.

[0112] In one embodiment, the antibody of the present invention comprises a second antigen-binding region capable of binding to human CD123.

[0113] In some embodiments, the antibody comprises a second antigen-binding region comprising the CDR1 sequence set forth in SEQ ID NO: 37, the CDR2 sequence set forth in SEQ ID NO: 38, and the CDR3 sequence set forth in SEQ ID NO: 39. In some embodiments, the antibody comprises, consists of, or has an amino acid sequence having at least 90%, such as at least 92%, such as at least 94%, such as at least 96%, such as at least 98% sequence identity to SEQ ID NO: 40 and comprises a second antigen-binding region.

[0114] In some embodiments, the second antigen-binding region competes (i.e., is competitive) with an antibody having the sequence set forth in SEQ ID NO: 40 for binding to human CD123, and preferably, the second antigen-binding region binds to the same epitope on human CD123 as the antibody having the sequence set forth in SEQ ID NO: 40.

[0115] 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.

[0116] In some embodiments, the antibody comprises or consists of a first antigen-binding region comprising 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 or consists of a first antigen-binding region comprising 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.

[0117] 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 are described, for example, in WO2022 / 008646, which is incorporated herein by reference.

[0118] In some embodiments, the antibody comprises a second antigen-binding region comprising the CDR1 sequence set forth in SEQ ID NO: 41, the CDR2 sequence set forth in SEQ ID NO: 42, and the CDR3 sequence set forth in SEQ ID NO: 43. In some embodiments, the antibody comprises or consists of SEQ ID NO: 44, or an amino acid sequence having at least 90%, such as at least 92%, such as at least 94%, such as at least 96%, such as at least 98% sequence identity to SEQ ID NO: 44.

[0119] In some embodiments, the second antigen-binding region competes (i.e., is capable of competing) with an antibody having the sequence set forth in SEQ ID NO: 44 for binding to human PSMA, and preferably, the second antigen-binding region binds to the same epitope on human PSMA as the antibody having the sequence set forth in SEQ ID NO: 44.

[0120] 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 NO: 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 NO: 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.

[0121] In some embodiments, the antibody comprises a first antigen-binding region comprising or consisting of a sequence selected from SEQ ID NO: 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 NO: 29, 30, 31, and 32, and a second antigen-binding region comprising or consisting of SEQ ID NO: 44.

[0122] 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 and suitable for incorporation into the multispecific antibodies used in the present invention are described, for example, in WO2016 / 122320, WO2020 / 060405 (both of which are incorporated herein by reference).

[0123] In some embodiments, the antibody comprises a second antigen-binding region comprising the CDR1 sequence set forth in SEQ ID NO: 45, the CDR2 sequence set forth in SEQ ID NO: 46, and the CDR3 sequence set forth in SEQ ID NO: 47. In some embodiments, the antibody comprises or consists of SEQ ID NO: 48, or a second antigen-binding region comprising an amino acid sequence having at least 90%, such as at least 92%, such as at least 94%, such as at least 96%, such as at least 98% sequence identity to SEQ ID NO: 48.

[0124] In some embodiments, the second antigen-binding region competes (i.e., is capable of competing) with an antibody having the sequence set forth in SEQ ID NO: 48 for binding to human CD1d, and preferably, the second antigen-binding region binds to the same epitope on human CD1d as the antibody having the sequence set forth in SEQ ID NO: 48.

[0125] 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.

[0126] In some embodiments, the antibody comprises or consists of a first antigen-binding region comprising a sequence selected from SEQ ID NOs: 25, 26, 27, and 28, and a second antigen-binding region comprising or consisting of SEQ ID NOs: 48. In some embodiments, the antibody comprises or consists of a first antigen-binding region comprising a sequence selected from SEQ ID NOs: 29, 30, 31, and 32, and a second antigen-binding region comprising or consisting of SEQ ID NOs: 48.

[0127] In some embodiments, the antibody comprises a second antigen-binding region capable of binding to human CD40. Antibodies having an antigen-binding region capable of binding to human CD40 suitable for incorporation into the multispecific antibodies used in the present invention are described, for example, in WO2020 / 159368 (incorporated herein by reference).

[0128] In some embodiments, the antibody comprises a second antigen-binding region comprising the CDR1 sequence set forth in SEQ ID NO: 49, the CDR2 sequence set forth in SEQ ID NO: 50, and the CDR3 sequence set forth in SEQ ID NO: 51. In some embodiments, the antibody comprises or consists of SEQ ID NO: 52, or an amino acid sequence having at least 90%, such as at least 92%, such as at least 94%, such as at least 96%, such as at least 98% sequence identity to SEQ ID NO: 52, as the second antigen-binding region.

[0129] In some embodiments, the second antigen-binding region competes (i.e., is capable of competing) with an antibody having the sequence set forth in SEQ ID NO: 52 for binding to human CD40, and preferably, the second antigen-binding region binds to the same epitope on human CD40 as the antibody having the sequence set forth in SEQ ID NO: 52.

[0130] 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 NO: 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 NO: 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.

[0131] In some embodiments, the antibody comprises a first antigen-binding region comprising or consisting of a sequence selected from SEQ ID NO: 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 NO: 29, 30, 31, and 32, and a second antigen-binding region comprising or consisting of SEQ ID NO: 52.

[0132] In some embodiments, the antibody comprises a second antigen-binding region capable of binding to human nectin-4.

[0133] In some embodiments, the antibody comprises a second antigen-binding region comprising the CDR1 sequence set forth in SEQ ID NO: 53, the CDR2 sequence set forth in SEQ ID NO: 54, and the CDR3 sequence set forth in SEQ ID NO: 55. In some embodiments, the antibody comprises a second antigen-binding region comprising the CDR1 sequence set forth in SEQ ID NO: 57, the CDR2 sequence set forth in SEQ ID NO: 58, and the CDR3 sequence set forth in SEQ ID NO: 59. In some embodiments, the antibody comprises a second antigen-binding region comprising the CDR1 sequence set forth in SEQ ID NO: 61, the CDR2 sequence set forth in SEQ ID NO: 62, and the CDR3 sequence set forth in SEQ ID NO: 63. In some embodiments, the antibody comprises a second antigen-binding region comprising the CDR1 sequence set forth in SEQ ID NO: 65, the CDR2 sequence set forth in SEQ ID NO: 66, and the CDR3 sequence set forth in SEQ ID NO: 67. In some embodiments, the antibody comprises a second antigen-binding region comprising the CDR1 sequence set forth in SEQ ID NO: 69, the CDR2 sequence set forth in SEQ ID NO: 70, and the CDR3 sequence set forth in SEQ ID NO: 71. In some embodiments, the antibody comprises a second antigen-binding region comprising the CDR1 sequence set forth in SEQ ID NO: 73, the CDR2 sequence set forth in SEQ ID NO: 74, and the CDR3 sequence set forth in SEQ ID NO: 75. In some embodiments, the antibody comprises a second antigen-binding region comprising the CDR1 sequence set forth in SEQ ID NO: 77, the CDR2 sequence set forth in SEQ ID NO: 78, and the CDR3 sequence set forth in SEQ ID NO: 79.

[0134] In some embodiments, the antibody comprises or consists of a sequence selected from SEQ ID NOs: 56, 60, 64, 68, 72, 76, and 80, or comprises an amino acid sequence having at least 90%, such as at least 92%, such as at least 94%, such as at least 96%, such as at least 98% sequence identity with SEQ ID NOs: 56, 60, 64, 68, 72, 76, and 80, and comprises a second antigen-binding region.

[0135] In some embodiments, the second antigen-binding region competes (i.e., is competitive) with an antibody having the sequences set forth in SEQ ID NOs: 56, 60, 64, 68, 72, 76, and 80 for binding to human nectin-4, and preferably, the second antigen-binding region binds to the same epitope on human nectin-4 as the antibody having the sequences set forth in SEQ ID NOs: 56, 60, 64, 68, 72, 76, and 80.

[0136] In some embodiments, the antibody comprises a first antigen-binding region comprising (a) 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 first antigen-binding region comprising (a) 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 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.

[0137] 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 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 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.

[0138] Exemplary combinations of binding domains are shown in Table A below. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0139] In one embodiment, the antibody comprises a first antigen-binding region and a second antigen-binding region, and the first antigen-binding region and the second antigen-binding region are covalently linked via a peptide linker, for example, a linker having a length of 1 to 20 amino acids, for example, 1 to 10 amino acids, for example, 2, 3, 4, 5, 6, 7, 8 or 10 amino acids. In one embodiment, the peptide linker comprises or consists of the sequence GGGGS set forth in SEQ ID NO: 81.

[0140] In another embodiment, the antibody comprises a first antigen-binding region and a second antigen-binding region, and the first antigen-binding region capable of binding to human Vδ2 is disposed at the C-terminus of the second antigen-binding region capable of binding to human EGFR, CD123, PSMA, CD1d, CD40, or nectin 4. In another embodiment, the antibody comprises a first antigen-binding region and a second antigen-binding region, and the first antigen-binding region capable of binding to human Vδ2 is disposed at the N-terminus of the second antigen-binding region capable of binding to human EGFR, CD123, PSMA, CD1d, CD40, or nectin 4.

[0141] In one embodiment of the present invention, the antibody further comprises a half-life extension domain. In one embodiment, the antibody has a terminal half-life longer than about 168 hours when administered to a human subject. Most preferably, the terminal half-life is 336 hours or more. As used herein, the "terminal half-life" of an antibody refers to the time in vivo at the final stage of elimination for the serum concentration of the polypeptide to decrease by 50%.

[0142] In one embodiment, the antibody further comprises a half-life extension domain, and the half-life extension domain is the Fc region. In a further embodiment, the antibody is a multispecific antibody such as a bispecific antibody comprising an Fc region. Various methods for producing bispecific antibodies are described in the art and are reviewed, for example, in Brinkmann and Kontermann (2017) MAbs 9:182. In one embodiment of the present invention, the Fc region is a heterodimer comprising two Fc polypeptides, the first antigen-binding region is fused to the first Fc polypeptide, the second antigen-binding region is fused to the second Fc polypeptide, and the first and second Fc polypeptides contain asymmetric amino acid mutations that promote heterodimer formation over homodimer formation (see, for example, Ridgway et al. (1996) ‘Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng 9:617). In a further embodiment herein, the CH3 region of the Fc polypeptide contains the above asymmetric amino acid mutations, preferably the first Fc polypeptide contains a T366W substitution and the second Fc polypeptide contains T366S, L368A and Y407V substitutions, or vice versa, and the amino acid positions correspond to human IgG1 according to the EU numbering system. In a further embodiment, the cysteine residues at position 220 of the first and second Fc polypeptides are deleted or substituted, and the amino acid positions correspond to human IgG1 according to the EU numbering system. In a further embodiment, the region comprises the hinge sequence set forth in SEQ ID NO: 82.

[0143] In some embodiments, the first and / or second Fc polypeptide comprises a mutation that inactivates the antibody, i.e., cannot mediate effector function or has reduced ability. In one embodiment, the inactivated Fc region is additionally unable to bind C1q. In one embodiment, the first and second Fc polypeptides comprise a mutation at position 234 and / or 235, preferably, the first and second Fc polypeptides comprise L234F and L235E substitutions, and the amino acid positions correspond to human IgG1 according to the EU numbering system. In another embodiment, the antibody comprises L234A mutation, L235A mutation and P329G mutation. In another embodiment, the antibody comprises L234F mutation, L235E mutation and D265A mutation.

[0144] In a preferred embodiment, the first antigen-binding region comprises the sequence set forth in SEQ ID NO: 16, the second antigen-binding region comprises a sequence selected from the sequences of SEQ ID NO: 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, and 80, and (a) the first Fc polypeptide comprises the sequence set forth in SEQ ID NO: 83, the second Fc polypeptide comprises the sequence set forth in SEQ ID NO: 84, or (b) the first Fc polypeptide comprises the sequence set forth in SEQ ID NO: 84, the second Fc polypeptide comprises the sequence set forth in SEQ ID NO: 83.

[0145] 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).

[0146] 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).

[0147] In a more preferred embodiment, the antibody comprises (a) the sequence set forth in SEQ ID NO: 85, and (b) a sequence selected from the group consisting of the sequences set forth in SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, or consists of them.

[0148] In a further preferred embodiment, the antibody comprises, or consists of, (a) the sequence set forth in SEQ ID NO: 86, and (b) a sequence selected from the group consisting of the sequences set forth in SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90.

[0149] 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).

[0150] 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).

[0151] 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).

[0152] 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).

[0153] 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).

[0154] 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).

[0155] In a further main aspect, the present invention relates to a pharmaceutical composition comprising an antibody according to the present invention described herein and a pharmaceutically acceptable excipient.

[0156] Antibodies can be formulated using pharmaceutically acceptable excipients according to the prior art as disclosed in Rowe et al., 2012 Handbook of Pharmaceutical Excipients, ISBN 9780857110275). Pharmaceutically acceptable excipients, as well as any other carriers, diluents or adjuvants, should be suitable for the antibody and the selected mode of administration. Compatibility of the excipients and other components of the pharmaceutical composition is determined based on the absence of any significant adverse effects on the desired biological properties of the selected antibody or pharmaceutical composition of the invention (e.g., less than a substantial effect on antigen binding (relative inhibition of 10% or less, relative inhibition of 5% or less, etc.)).

[0157] The pharmaceutical composition may contain diluents, fillers, salts, buffers, detergents (e.g., nonionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., amino acids without sugars or proteins), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in the pharmaceutical composition. Further 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 antibody of the invention.

[0158] In a further main aspect, the invention relates to an antibody according to the invention described herein for use as a medicament.

[0159] The antibody according to the invention enables the creation of a microenvironment beneficial for the killing of tumor cells by Vγ9Vδ2 T cells. Thus, in a preferred embodiment, the antibody is used for the treatment of cancer.

[0160] In one embodiment, the antibody targets EGFR and is used for the treatment of primary or metastatic colon or rectal cancer. In another embodiment, the antibody is used for the treatment of peritoneal cancer. In another embodiment, the antibody is used for the treatment of liver cancer. In another embodiment, the antibody is used for the treatment of head and neck squamous cell carcinoma (HNSCC). In another embodiment, the antibody is used for the treatment of non-small cell lung cancer (NSCLC). In another embodiment, the antibody is used for the treatment of cutaneous squamous cell carcinoma.

[0161] In another embodiment, the antibody targets CD123 and is used for the treatment of acute myeloid leukemia, B-cell acute lymphoblastic leukemia, hairy cell leukemia, Hodgkin lymphoma, blastic plasmacytoid dendritic cell neoplasm, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell chronic lymphoproliferative disorder or myelodysplastic syndrome.

[0162] In another embodiment, the antibody targets CD1d and is used for the treatment of hematological malignancies 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 lymphoma, myelomonocytic leukemia, lymphoplasmacytic lymphoma, hairy cell leukemia, and splenic marginal zone lymphoma, or solid tumors such as renal cell carcinoma, melanoma, colorectal cancer, head and 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 cancer, bladder cancer, sarcoma, penile cancer, basal cell carcinoma, Merkel cell carcinoma, neuroendocrine cancer, neuroendocrine tumor, cancer of unknown primary (CUP), thymoma, vulvar cancer, cervical cancer, testicular cancer, cholangiocarcinoma, appendiceal cancer, mesothelioma, ampullary cancer, anal cancer, choriocarcinoma.

[0163] In another embodiment, the antibody targets PSMA and is used for the treatment of prostate cancer, such as non-metastatic or metastatic prostate cancer, such as metastatic castration-resistant prostate cancer, such as treatment-resistant metastatic castration-resistant prostate cancer. For example, the antibody may comprise a first antigen-binding region capable of binding to PSMA and is used for the treatment of prostate cancer in patients who have not responded to at least one taxane-based chemotherapy and have optionally received second-generation and later androgen receptor-targeted therapies / androgen biosynthesis inhibitors (e.g., abiraterone, enzalutamide, and / or apalutamide).

[0164] Furthermore, an antibody capable of binding to PSMA can be used for the treatment of cancers that express PSMA on tumor neovessels or tumor-associated endothelial cells of primary or metastatic tumors, including colorectal cancer, lung cancer, breast cancer, endometrial cancer and ovarian cancer, gastric cancer, renal cell carcinoma, urothelial carcinoma, hepatocellular carcinoma, oral squamous cell carcinoma, thyroid tumors, and gliotrama. Also, an antibody capable of binding to PSMA can be used for the treatment of adenoid cystic carcinoma of the head and neck.

[0165] Similarly, the present invention relates to a method of treating a disease, comprising administering to a human subject in need thereof a multispecific antibody according to the present invention described herein. In one embodiment, the disease is cancer.

[0166] In some embodiments, the antibody is administered as a monotherapy. However, the antibodies of the present invention may also be administered in combination therapy, i.e., in combination with other therapeutic agents related to the disease or condition being treated.

[0167] "To treat" or "treatment" refers to administering an effective amount of an antibody according to the present invention for the purpose of alleviating, ameliorating, arresting, eradicating (hardening), or preventing a symptom or disease state. "Effective amount" refers to an amount effective for the dosage and period required to achieve the desired therapeutic result. The effective amount of a polypeptide such as an antibody can vary depending on factors such as the disease stage, the age, sex, and weight of the individual, and the ability of the antibody to induce the desired response in the individual. The effective amount is also one in which the therapeutically beneficial effect exceeds any toxic or detrimental effects of the antibody. Administration can be effected by any suitable route, typically parenterally, such as intravenously, intramuscularly, or subcutaneously.

[0168] The multispecific antibodies of the present invention are typically produced recombinantly, i.e., by expression of a nucleic acid construct encoding the antibody in a suitable host cell, followed by purification of the recombinant antibody produced from the cell culture. The nucleic acid construct can be produced by standard molecular biological techniques known in the art. The construct is typically introduced into the host cell using an expression vector. Suitable nucleic acid constructs and expression vectors are known in the art. Host cells suitable for the recombinant expression of antibodies are well known in the art and include CHO, HEK-293, Expi293F, PER-C6, NS / 0, and Sp2 / 0 cells.

[0169] Accordingly, in a further 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.

[0170] In a further aspect, the present invention relates to an expression vector comprising a nucleic acid construct encoding an antibody according to the present invention.

[0171] In a further aspect, the present invention relates to a host cell comprising one or more nucleic acid constructs encoding an antibody according to the present invention or an expression vector comprising a nucleic acid construct encoding an antibody according to the present invention.

[0172] In a further aspect, the invention relates to a process for producing an antibody of the invention, which comprises expressing in a host cell one or more nucleic acids encoding the antibody of the invention.

[0173] In a further aspect, the invention relates to a process for producing a clinical batch of an antibody of the invention, which comprises expressing in a host cell one or more nucleic acids encoding the antibody of the invention. As used herein, a "clinical batch" refers to a product composition suitable for use in humans.

[0174] In a further aspect, the invention relates to a process for producing an antibody without proteolysis and / or tyrosine sulfation, which comprises expressing in a host cell one or more nucleic acids encoding the antibody of the invention.

[0175] In a further aspect, the invention relates to a process for avoiding proteolysis and / or tyrosine sulfation of an antibody capable of activating human Vγ9Vδ2 T cells, the process comprising constructing a nucleic acid encoding the antibody of the invention and producing the antibody by expressing the nucleic acid in a host cell.

[0176] In a further aspect, the invention relates to a process for producing a homogeneous antibody formulation of an antibody capable of activating human Vγ9Vδ2 T cells, the process comprising constructing a nucleic acid encoding the antibody of the invention and producing the antibody by expressing the nucleic acid in a host cell.

[0177] In one embodiment, the host cell in the production process is a mammalian cell, such as a CHO cell or a HEK cell, or a yeast cell, such as a Pichia cell.

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

[0178] All references, articles, publications, patents, patent gazettes, and patent applications cited in this specification are hereby incorporated by reference in their entirety for all purposes. However, any reference to any reference, article, publication, patent, patent gazette, and patent application cited in this specification is not an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world and should not be so regarded.

Examples

[0179] Example 1 | Production and Purification of VHH Compounds Most of the VHH compounds are produced by transient transfection of the encoding plasmid in HEK293-E253 cells, purification of the protein from the conditioned medium (one week after production) by protein-A affinity chromatography, and preparative gel filtration. The dominant monomer peak (fractions 1E11 to 1G2) observed by preparative size exclusion using a Superdex-75 column was purified. Refer to Figure 1.

[0180] The purified protein was shown to migrate as a single band under reducing and non-reducing conditions in polyacrylamide gel electrophoresis. Figure 2 shows a representative example.

[0181] Example 2 | HP-SEC Analysis of Purified VHH Compounds The Waters Acquity ARC-biosystem was used for HP-SEC analysis of the purified VHH compounds. 10 μg of antibody (10 μL of antibody at a concentration of 1 mg / mL) was injected onto a Waters BEH200 SEC column (bead size 2.5 μm, column dimensions 7.8 x 300 mm). The mobile phase consisting of 50 mM sodium phosphate, 0.2 M sodium chloride buffer pH 7.0, and a buffer rate of 0.8 mL / min was used for the run. The protein was detected by measuring the absorption at a wavelength of 214 nm. The total analysis time was 15 minutes per run. As described in Example 1, the VHH compounds were produced and purified. Surprisingly, VHH 5C8 (SEQ ID NO: 13) (previously described in WO2015156673) and 5C8var1 (SEQ ID NO: 14) (previously described in WO202020060405) were tested for integrity and monomericity in HP-SEC analysis, and two peaks were observed. See Figure 3.

[0182] Example 3 | Mass Spectrometry of 5C8 Reveals an Additional Mass of 80 Da To determine whether the masses of the two isoforms of 5C8 differ, the protein formulation of 5C8 was analyzed by LC-ESI-MS mass spectrometry. The major species found in this analysis was 5C8 without post-translational modifications or signal peptides. The second species was a protein with a mass difference of +80.3 Daltons (Da) indicating possible sulfation or phosphorylation. This was further investigated by treatment with phosphatase or sulfatase, followed by LC-ESI-MS mass spectrometry analysis after proteolytic digestion. It was shown that treatment with sulfatase reduced the mass of the peptide containing Y105, the 7th residue of CDR3 (SEQ ID NO: 15), by 80 Da, while treatment with phosphatase had no effect. This indicates that Y105 in 5C8 is post-translationally modified by sulfation. This sulfation was present in approximately 30% of the protein formulation.

[0183] Example 4 | 1D12var5-5C8var1, which contains the same anti-Vδ2 VHH, shows the same heterogeneity in HP-SEC and the same excess mass of 80 Da. 1D12var5-5C8var1 is a bispecific VHH compound composed of an anti-CD1d VHH, linked to 5C8var1 (described in SEQ ID NO: 87 in WO2020060405) via a flexible linker. This protein was expressed in HEK293E cells as described above. Protein formulations were also obtained from different expression systems. The bispecific VHH was also expressed in Pichia pastoris and Chinese hamster ovary (CHO) cells. When different protein formulations were tested by HP-SEC analysis, a pre-peak was observed: Figure 4.

[0184] The observed pre-peak indicates that the apparent proportion of the protein is again different isoforms. Since it has been shown that 5C8 VHH is sulfated and 5C8var1 contains exactly the same CDR3 sequence, the 1D12var5-5C8var1 batch was also analyzed for their molecular weights by mass spectrometry. Depending on the protein batch, it was found that between 15% and 40% contained an additional mass of 80 Da. This is consistent with sulfation, as observed with VHH 5C8.

[0185] Example 5 | In silico analysis of VHH 5C8 and 5C8var1 Homology models of 5C8 and 5C8var1 were constructed using Maestro (Schrodinger) based on PDB ID 5M2W. CDR1 and CDR3 required improvement by de novo loop prediction using Prime (Schrodinger). The generated models demonstrated that the CDR3 residue Y105 exhibited a high solvent-accessible surface area of 205.1 Å2 in the 5C8var1 model and 122.2 Å2 in the 5C8 model, and thus was predicted to contribute to antigen binding. Subsequently, the models were analyzed for reactive residues, showing residues prone to post-translational modification (PTM). Next, the protein sequences were analyzed using Modpred, a sequence-based PTM prediction tool. The predicted variants in both structure and sequence are shown in Table 2. The PTMs of individual predictions could not explain the mass differences observed in HP-SEC analysis. [Table 6] *Q13 deamidation was predicted only for 5C8. The type explains the PTM as predicted by Mastro.

[0186] Example 6 | Design and production of 5C8var1 VHH CDR3 mutants Y105F and Y105S The homology model was used to introduce mutations that prevent sulfation of Y105 in 5C8 and 5C8var1 as described in Example 5. Two different mutants, Y105S (retaining the alcohol function) and Y105F (retaining the aromatic ring), were designed based on the model structure of the VHH. Residue Y105 is the 7th residue of CDR3, and introducing mutations was expected to affect binding. Both mutations were designed in the humanized VHH sequence 5C8var1, and both proteins were produced in HEK293E cells and purified as described above. The CDR3 amino acid sequence of the humanized VHH was kept identical to one of the non-humanized ones.

[0187] Both 5C8var1-Y105F and 5C8var1-Y105S were well-produced and appeared as monomeric proteins in size exclusion fractionation (data not shown). Both proteins were of high purity (Figure 5) and migrated as a single species in polyacrylamide gel electrophoresis.

[0188] Example 7 | HP-SEC Analysis of Purified VHH Containing Engineered CDR3 Mutations HP-SEC analysis was performed as described for 5C8. Both 5C8var1-Y105F and 5C8var1-Y105S were analyzed (Figure 6).

[0189] As is clear from the HP-SEC analysis of the purified VHH molecules containing the engineered CDR3 mutations, no heterogeneity was observed for any of the mutants. This indicated that there was no post-translational modification of the observed Y105 and that the protein was homogeneous.

[0190] Example 8 | Affinity Measurements of 5C8var1, 5C8var1-Y105F, and 5C8va1-Y105S Using Biolayer Interferometry (BLI) Show No Difference in Affinity. The binding of the 5C8var1 VhH antibody fragment and variants 5C8var1-Y105F and 5C8var1-Y105S to Vy9Vδ2 TCR was measured by biolayer interferometry using an Octet RED96 instrument (ForteBio). Recombinant human VY9Vδ2-Fc fusion protein (20 μg / mL) was captured as a ligand on an anti-human Fc capture biosensor. Sensorgrams were recorded when the ligand capture biosensor was incubated with a dilution series (40 - 0.63 nM) of VHH antibody fragments in 10X kinetics buffer (ForteBio). Global data fitted to a 1:1 binding model was used to estimate the values of Kon (association rate constant) and Koff (dissociation rate constant). These values were used to calculate Kd (equilibrium dissociation constant) using KD = Koff / Kon.

[0191] As is apparent from Figure 7 and Table 3, the KD values found for the two different Y105 VHH mutants are nearly identical to the values determined for 5C8var1. In particular, the Y105F mutant had an affinity equivalent to the affinity found for 5C8var1.

Table 7

[0192] Example 9 | The anti-(EGFR × Vδ2 TCR) bispecific VHH containing the Y105F mutation fully retains its function To determine whether the equivalent affinity of VHH 5C8var1-Y105F can be converted into equivalent functionality, a bispecific VHH 7D12var8-5C8var1-Y105F was designed, and a humanized anti-EGFR VHH 7D12var8 (based on the VHH described in Gainkam et al. (2008) J Nucl Med 49(5):788) was conjugated 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. After producing and purifying this molecule as described above, it was tested for its ability to induce Vγ9Vδ2 T cell activation dependent on the EGFR-positive tumor cell line (A431) and to cause Vγ9Vδ2 T cell-mediated tumor cell lysis. Briefly, Vγ9Vδ2 T cells were isolated from the blood of healthy donors using magnetic-activated cell sorting (MACS) in combination with an anti-Vδ2 antibody according to a standardized protocol. These cells were then grown for one week from another donor using a mixture of cytokines and irradiated feeder cells, which was a mixture of the JY cell line and PBMC. The Vγ9Vδ2 T cells were always >90% pure (double-positive staining for Vγ9 and Vδ2 by FACS) when used in the assay. The A431 cell line (ATCC, cat nr. CRL-1555) was cultured according to the provider's recommendations. For the activation or cytotoxicity assay, 50,000 tumor target cells were seeded into 96-well tissue culture plates prior to the assay. The next day, 50,000 expanded and purified Vγ9Vδ2 T cells were added to the medium together with a range of concentrations of the bispecific VHH compound. In the activation assay, degranulation of VY9Vδ2-T cells was evaluated using a mixture of labeled anti-CD3 and an anti-CD107A antibody added to the mixture. After 4 hours, the cells were harvested, washed, and analyzed by FACS for the expression of the degranulation marker CD107A. For the cytotoxicity assay, the next day, the supernatant of the co-culture was examined for the presence of protease (an indicator of cell death) using the CytoTox-Glo cytotoxicity assay kit (Promega G9290). Cell lysis using the wash buffer was used to achieve 100% cell death at the end of the assay. Figure 8 shows the data.

[0193] Figure 8 shows the strong Vγ9Vδ2 T cell activation and tumor cell lysis of 7D12var8-5C8var1-Y105F and non-humanized 7D12-5C8 induction. These results are consistent with the potency of the non-humanized "precursor" molecule without the Y105 mutant 7D12wt-5C8. Table 4 shows the EC50 values obtained after curve fitting. Also, 7D12var8-5C8var1-Y105F had a slightly lower EC50 in the cytotoxicity assay compared to 7D12-5C8.

Table 8

[0194] For 7D12var8-5C8var1-Y105F, the maximum level of tumor cell death was slightly lower compared to the level of tumor cell death observed with 7D12-5C8. However, these are different measurements using two different Vγ9Vδ2 T cell donors, and the maximum level of this cytotoxicity may be particularly donor-dependent.

[0195] Example 10 | The thermal stability of VHH 5C8var1 containing the Y105 mutation did not change To determine whether the mutations introduced into the different variants affect the thermal stability of the VHH folding, the melting temperatures of the mutants were measured using nanoDSF (differential scanning fluorimetry). The antibody samples were diluted with PBS until the lowest concentration sample was obtained. Then, the antibody samples were filled into nanoDSF-grade capillaries and measured with Prometheus NT.48. During the experiment, the temperature was 20 - 95°C. The intrinsic fluorescence of the protein was detected at 350 and 330 nm and recorded together with the amount of reflected light. From these measurements, the apparent melting temperature (Tm) and the onset of aggregation (Tagg) were determined. For all three antibody fragments, the onset lysis temperature (Ton) and melting temperature (Tm) at which the VHH was fully unfolded were reported (Table 5). The melting temperatures measured for 5C8var1-Y105F and 5C8var1-Y105S were consistent with the melting temperature of 5C8var1. See Table 5.

Table 9

[0196] Example 11|Half-life extended (Fc-containing) bispecific construct To obtain a molecule with a long in vivo plasma half-life, the 7D12var8-5C8var1-Y105F bispecific VHH was reshaped into a therapeutic antibody format containing human Fc. Both VHH domains were coupled to a human IgG1 Fc (i.e., CH2 and CH3) domain with the following characteristics: the VHH was modified with a hinge (AAA, followed by SDKTHTCPPCP, with cysteine 220 deleted), and coupled to the human CH2 and CH3 domains. When the two chains within the same cell were co-expressed, the CH2 domain was Fc-silenced by the LFLE mutation pair (L234F, L235E), and the CH3 domain was mutated by the "knob-into-hole" mutations (knob: T366W, and hole: T366S, L368A, and Y407V) that force heterodimerization. This mutation pair has been described in the scientific literature (Ridgway et al. (1996) Protein Eng 9:617). The sequences of the constructs are described in SEQ ID NO: 16 and SEQ ID NO: 17. The antibody construct 7D12var8-5C8var1(Y105F) with the obtained Fc region was named 7D12var8-5C8var1(Y105F)-Fc. Similarly, a construct with Y replaced by S (7D12var8-5C8var1(Y105S)-Fc) at position 105 was produced. The sequences of that construct are described in SEQ ID NO: 16 and SEQ ID NO: 18.

[0197] As described in Example 1, the protein was prepared via co-transfection of two expression vectors encoding in HEK293E cells and purification from the culture supernatant by C-tag affinity chromatography followed by preparative size exclusion chromatography. Thereby, a highly monomeric protein formulation was obtained.

[0198] Example 12|Binding of 7D12var8-5C8var1(Y105F)-Fc to primary Vγ9Vδ2 T cells isolated from healthy human PBMCs To demonstrate the binding of 7D12var8-5C8var1(Y105F)-Fc to the Vγ9Vδ2 T cell receptor (TCR), human Vγ9Vδ2 T cells were isolated from healthy peripheral blood mononuclear cells (PBMCs) by magnetic-activated cell sorting (MACS) and then expanded as described above (de Bruin et al., Clin. Immunology 169 (2016), 128-138; de Bruin et al., J. Immunology 198(1) (2017), 308-317). Next, the expanded polyclonal and pure (>95%) Vγ9Vδ2 T cells were seeded at a concentration of 50,000 cells / well and incubated at 4 °C for 1 hour in a semi-logarithmic titration starting at 100 nM with either the 7D12var8-5C8var1(Y105F)-Fc antibody or the GP120-5C8var1(Y105F)-Fc antibody as a positive control. Binding of the antibody to the Vγ9Vδ2 TCR was visualized by flow cytometry using a fluorescently labeled secondary anti-IgG1 antibody. Figure 9 shows the mean fluorescence intensity (MFI) signal of anti-IgG1 antibody staining as measured by flow cytometry for two different PBMC donors (D336 and D339). The sigmoid curve emphasizes the significant binding of 7D12var8-5C8var1(Y105F)-Fc to Vγ9Vδ2 T cells with an EC50 in the low nanomolar range (about 3 nM).

[0199] Example 13|Binding of 7D12var8-5C8var1(Y105F)-Fc to EGFR-positive tumor cells by cell-based ELISA The binding of 7D12var8-5C8var1(Y105F)-Fc to the epidermal growth factor receptor (EGFR) was tested by cell-based enzyme-linked immunosorbent assay (ELISA) using EGF-expressing tumor cell lines A-431, Hct-116, and HT-29. For this, tumor cells were first seeded at different concentrations on day -1 and reached a concentration of approximately 50,000 cells / well on day 0. On day 0, a semi-logarithmic titration of the 7D12var8-5C8var1(Y105F)-Fc antibody or the GP120-5C8var1(Y105F)-Fc antibody as a negative control was started at 100 nM and added to the tumor cells at 37 °C for 1 hour. Next, the bound antibody was labeled with anti-IgG1-HRP in a secondary incubation step at 37 °C for 1 hour. Next, 3,3’,5,5’-tetramethylbenzidine was added to separate the binding of the secondary antibody, the colorimetric change induced by HRP was detected, and subsequently H2SO4 was added to stop the reaction. Next, the optical density (OD) was measured in a UV spectrophotometer at a wavelength of 450 nm. Figure 10 shows that 7D12var8-5C8var1(Y105F)-Fc binds strongly to A-431, HCT-116, and HT-29 tumor cells with an EC50 of approximately 7 nM, whereas the non-targeted control antibody did not bind measurably to any of the tested cell lines.

[0200] Example 14|Degranulation of A-431 cell-dependent Vγ9Vδ2 T cells induced by 7D12var8-5C8var1(Y105F)-Fc To examine the potential of 7D12var8-5C8var1(Y105F)-Fc to activate Vγ9Vδ2 T cells, Vγ9Vδ2 T cells were first isolated and expanded as described above. Next, Vγ9Vδ2 T cells were cultured at a 1:1 E:T ratio with A-431 tumor cells in the presence of different concentrations of 7D12var8-5C8var1(Y105F)-Fc antibody and PE-labeled anti-CD107a fluorescent antibody. After 24 hours, the cells were harvested, stained with fluorescent-labeled anti-Vγ9 and anti-CD3 antibodies, and differentiated from tumor cells into Vγ9Vδ2 T cells. Flow cytometry was used to examine the degree of CD107a expression on Vγ9Vδ2 T cells, which reflects target-dependent degranulation. Figure 11 shows that as the concentration of 7D12var8-5C8var1(Y105F)-Fc increased, Vγ9Vδ2 T cells were induced to efficiently degranulate in an A-431 cell-dependent manner. The EC50 of Vγ9Vδ2 T cell degranulation induced by 7D12var8-5C8var1(Y105F)-Fc was in the picomolar range (about 40 - 90 pM).

[0201] Example 15 | Antibody 7D12-5C8 Induces T Cell-Mediated Cytotoxicity of Target Cells To investigate whether the bispecific VHH 7D12-5C8 is efficient in inducing Vγ9Vδ2T cell-mediated cytotoxicity against target cells, the viability of the A-388 epidermoid carcinoma cell line (ATCC, CRL-7905) was evaluated in co-culture settings with Vγ9Vδ2T cells and bsVHH antibody fragments. In this assay, Vγ9Vδ2T cells isolated from healthy PBMCs as described above, then frozen and stored at -150 °C were used. The frozen Vγ9Vδ2T cells were thawed and left overnight in IL-2-supplemented medium. A-388 tumor cells were seeded at a target to effector ratio of 1:1 or 1:0.1 alone or together with resting Vγ9Vδ2T cells, with or without 7D12-5C8 (10 nM). As an additional control, Vγ9Vδ2T cells were seeded alone, with or without the antibody 7D12-5C8 (10 nM). After 72 h, cell viability was determined by addition of ATP Lite (Perkin Elmer, 6016731) and reading of the luminescence signal by a microplate reader. Figure 12 shows the fluorescence signal derived from ATP, which represents metabolically active cells and thus the number of live cells. At a 1:1 E:T ratio, it was observed that the antibody caused a ca. 50% decrease in live cells, whereas the untreated co-culture of A-388 and Vγ9Vδ2T cells was unaffected, emphasizing the potential to induce T cell-mediated cytotoxicity.

[0202] Example 16| Tumor cell killing by Vγ9Vδ2T cells activated by 7D12-5C8 and 7D12-5C8var1(Y105S)-Fc To investigate whether bsVHH 7D12-5C8 and the antibody 7D12-5C8var1(Y105S)-Fc were able to induce Vγ9Vδ2T cell-mediated cytotoxicity against patient-derived tumor cells, the viability of such tumor cells was evaluated in co-culture settings with Vγ9Vδ2T cells and the antibody. Various types of tumor cells were tested.

[0203] Tissue samples (i.e., primary and metastatic colorectal cancer, head and neck squamous cell carcinoma (HHSCC), and non-small cell lung cancer (NSCLC) samples) were collected from cancer patients at the Amsterdam UMC (location VUmc) after written informed consent. The tissue was excised into small pieces with a surgical blade (size number 22; manufactured by SWANN), resuspended in a dissociation medium consisting of IMDM supplemented with 0.1% DNase I, 0.14% collagenase A, 5% FcS, 100 IU / mL sodium penicillin, 100 μg / mL streptomycin sulfate, and 2.0 mM L-glutamine, transferred to a sterile flask with a magnetic stir bar, and incubated for 45 minutes with a magnetic stirrer in a 37 °C water bath. After incubation, the cell suspension was passed through a 100 μM cell strainer. After dissociating the tumor three times, the cells were washed to count the viable cell number by the trypan blue exclusion method.

[0204] Dissociated patient-derived tumor cells were incubated for 4 hours with healthy donor-derived Vγ9Vδ2 T cells (1:1 E:T ratio) with or without 50 nM 7D12-5C8, or for 24 hours with 7D12-5C8var1 (Y105S)-Fc or gp120-5C8var1 (Y105S)-Fc.

[0205] Adherent cells were removed, if necessary, after a culture period using trypsin-EDTA, 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 °C, and then the staining was measured by flow cytometry using an LSR Fortessa XL-20 (BD).

[0206] Viable cells were identified using the vital marker 7AAD in combination with 123 Count eBeads*(trademark) according to the manufacturer's instructions. Flow cytometry data were analyzed using Kaluza Analysis Version 1.3 (Beckman Coulter) as well as FlowJo Version 10.6.1 and 10.7.2 (Becton Dickinson).

[0207] The cytotoxicity of 7D12-5C8 and 7D12-5C8var1(Y105S)-Fc-induced Vγ9Vδ2T cells against tumor cells was evaluated by incubating expanded Vγ9Vδ2T cells from healthy donors with single-cell suspensions of various malignant tumors (primary CRC, CRC metastases to the peritoneum and liver, head and neck squamous cell carcinoma, and non-small cell lung cancer).

[0208] As shown in Figure 13, 7D12-5C8 induced significant lysis of patient tumor cells by Vγ9Vδ2T cells (mean % lysis induced by 7D12-5C8: 52.3% for primary CRC and p-value 0.0003, 46.0% for CRC peritoneum and p-value 0.0052, 31.8% for CRC liver and p-value 0.0360, 46.1% for head and neck squamous cell carcinoma and p-value 0.0187, and 64.1% for non-small cell lung cancer and p-value 0.0153).

[0209] Furthermore, as shown in Figure 14, 7D12-5C8var1(Y105S)-Fc induced a significant amount of lysis of patient tumor cells by Vγ9Vδ2T cells (mean % lysis induced by 7D12-5C8var1(Y105S)-Fc: 71.2% and p < 0.0001 and 0.0012). The control compound gp120-5C8var1(Y105S)-Fc did not induce any measurable tumor cell lysis.

[0210] Example 17 | Design, production, and purification of the construct 7A5-7D12var8-Fc used in non-human primate studies In in vivo studies in non-human primates, constructs were generated that have a binding domain that cross-reacts with cynomolgus Vγ9 TCR chains (Figure 15). This binding domain was based on antibody 7A5, a TCR Vγ9-specific antibody (Janssen et al., J. Immunology 146(1) (1991), 35-39). Antibodies based on 7A5 were found to bind to cynomolgus Vδ9Vδ2 T cells (see Example 1 of WO2021052995). A bispecific Fc-containing antibody containing 7A5 and anti-EGFR VHH 7D12var8 was constructed. The molecule contained a human IgG1 Fc tail engineered to heterodimerize using the knob-in-hole technology (KiH; Carter et al., 2001 Imm. Meth. 2001: 248, 7; Knob: T366W; Hole: T366S, L368A and Y407V). The Vγ9-binding scFv of the 7A5 antibody was conjugated to the "knob" chain, and the EGFR-binding VHH 7D12var8 was cloned with the "hole" chain of the KIH Fc pair. Also, the upper hinge was engineered to "AAASDKTHTCPPCP" to remove the cysteine (C220) that normally bridges to the CL and improve flexibility by changing "EPK" to "AAA". The N-terminal portion of CH2 was engineered to abrogate interactions with Fc receptors (CD16, -32, and -64) while maintaining FcRn binding (silencing mutations L234F, L235E). The resulting construct was named 7A5-7D12var8-Fc.

[0211] The molecule was produced by transient co-transfection of two plasmids encoding the two different chains in HEK293E cells and purified from the culture supernatant by protein A affinity chromatography and then by preparative size exclusion chromatography (Example 1). The molecule was shown to bind to either target with an apparent affinity of approximately 3 nanomolar (nM) using ELISA and recombinant forms of both antigens (Figure 16). The functionality of the molecule was demonstrated by causing target-dependent activation (CD107a expression) of Vγ9Vδ2 T cells grown in vitro and subsequent T cell-mediated tumor cell lysis (Figure 17).

[0212] Example 18 | Bispecific antibody 7A5-7D12var8-Fc was well tolerated in a multi-dose exploratory non-human primate (NHP: cynomolgus monkey) study In the multi-dose exploratory NHP study, 7A5-7D12var8-Fc was administered to three female cynomolgus monkeys at doses of 1 mg / kg, 5 mg / kg, and 23 mg / kg, respectively. The antibody was administered by injection at 5 mL / kg over half an hour and by injection every four weeks. The first two dose groups (one animal per dose) were co-administered at 1 and 5 mg / kg, and after three (weekly) administrations, the third dose group (23 mg / kg) received the first dose. Blood was collected regularly from the animals for PK analysis, analysis of clinical chemistry parameters, measurement of cytokine levels, and analysis of blood cell subsets by flow cytometry. One day after the last administration, the animals were euthanized and tissues were collected and prepared for histopathological and immunohistochemical (IHC) examinations.

[0213] Pharmacokinetic analysis of the 7A5-7D12var8-Fc concentration in the blood of the treated animals (measured by ELISA, Figure 18) revealed that the antibody exhibited IgG-like PK with a half-life between 84 and 127 hours. In the animals administered at 1 mg / kg, the antibody showed a short half-life after the third injection, which could be caused by the anti-drug antibody (ADA) response in those animals.

[0214] The clearance values found were between 0.36 and 0.72 mL / h / kg, and the volume of distribution was between 58.5 and 115.2 mL / kg. Total body exposure increased proportionally with dose between 1 and 23 mg / kg. However, no accumulation was seen after repeated dosing.

[0215] The compound can be detected by IHC in different tissues (lymph nodes, muscle, skin, and large intestine), and as expected, there is a dose-proportional intensity of compound staining in these tissues (not shown). Flow cytometry analysis of blood cells, which is always observed in these types of multi-dose studies, showed several transient decreases in lymphocytes (Figure 19) associated with the procedure. Figure 19 shows the transient decrease in T cell counts every 2 hours after administration. However, the number of T cells returned to baseline levels 2 days after injection.

[0216] On the other hand, the number of Vγ9-positive T cells decreased in peripheral blood and did not re-amplify the frequency of the former. These cells were rarely present during the course of the study and demonstrated a specific pharmacological effect of the compound. Measurement of cytokines in the blood of treated animals showed that the treatment caused a very small cytokine release, and the release was almost limited to the first injection of the compound. Figure 20 shows the levels of IL-6 measured as an example.

[0217] As a general conclusion, treatment of NHP with 7A5-7D12var8-Fc was very well tolerated and no clinical signs of toxicity were seen. Also, in histopathology, no macroscopic or microscopic abnormalities were found in any of the organs examined (data not shown at present). In comparison, anti-EGFR x CD3 BiTE was lethal to NHP with continuous injection at 31 μg / kg / day (Lutterbuese et al., Proc Natl Acad Sci U S A 2010: 107(28), 12605).

[0218] Example 19 | VHH 5C8var1 is partially cleaved at position 109 in the production of an Fc-containing bispecific antibody format regardless of the residue at position Y105 As described above, the Fc bearing format comprises two VHH molecules that fuse to a modified human IgG1 Fc to form a humanized "heavy chain only" antibody molecule. During the development of a cell line for the large-scale production of a therapeutic Fc-containing bispecific molecule containing Vδ2-binding VHH 5C8var1 in CHO cells, SDS-PAGE analysis found that a small percentage of the product was truncated (Figure 21).

[0219] LC-ESI-MS mass spectrometry (Figures 22 and 6) surprisingly showed that the truncated species seen by SDS-PAGE could be due to a small percentage of 5C8var1 VHH cleaved after position 109, which is located in the CDR3 of the VHH. This phenomenon, not predicted based on in silico analysis (Example 5), was observed in different Fc-containing bispecific molecules, regardless of the (other) target VHH arm on the second chain and regardless of the amino acid at position 105 of VHH 5C8var1.

[0220] Truncation beyond position 109 causes the majority of the VHH to be absent, which leads to the production of non-functional proteins. The percentage of N-terminal truncated proteins was low (1-3%) and constant over time, suggesting that cleavage occurred during intracellular production and processing of the protein.

Table 10

[0221] Example 20 | Design of sequence variants of 5C8var1 to enhance product homogeneity The sequence of the CDR3 of 5C8 was changed at two positions to avoid proteolysis observed in different Fc-containing bispecific molecules. G (G108) at position 108 was changed to A, and R (R109) at position 109 was changed to K to maintain the same net charge, or changed to A, see Figure 23. Also, a variant (R109AΔGIR) was generated by changing position R109 to "A" and deleting four residues from the CDR3 region (ΔGIR).

[0222] These four different array variants (G108A, R109A, R109K, and R109AΔlGIR) were then expressed and purified as a single VHH and as a bispecific Fc-containing molecule (SEQ ID NO: 12) having the CD123-binding target VHH as the second binding arm (previously described in PCT / EP2022 / 054993, incorporated by reference). The protein chain encoding the (mutated) VHH directed to the TCR fused to Fc was grown with a purification C-tag. 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. Complete mass spectrometry analysis of the proteins (LC-ESI-MS) showed that they were all of purity greater than 98% and that no truncations were observed in any of the protein batches produced in these small-scale productions.

[0223] Example 21 | Affinity measurement by BLI shows that position 109 is acceptable To determine the possible impact of mutations at positions 108 and 109 on the binding properties of different VHH molecules, a single VHH protein was tested for binding to a recombinant form of the Vγ9Vδ2 T cell receptor by Biolayer Interferometry (BLI) using an Octet system. Figures 24 and Table 7 show that the original humanized 5C8 VHH with the Y105F mutation (5C8var1) had an affinity of 0.74 nM and that VHH molecules additionally containing the R109A or R109K mutation retained binding (the affinity did not change significantly). However, 5C8var1-Y105F containing additional mutations at position 108 had a 10-fold lower affinity. The R109A□LGIR variant did not measurably bind to the Vγ9Vδ2 TCR. These data demonstrate that all (mutant) VHH molecules were well-expressed, but only position 109 was permissive for Vγ9Vδ2 T cell receptor binding and sequence changes were observed. The lack of binding observed for the R109AΔLGIR variant indicated that the CDR3 is required for specific binding.

Table 11

[0224] Example 22 | Determination of the apparent affinity of VHH variants at positions 108 and 109 using flow cytometry To test whether the mutations at positions 108 and 109 of the 5C8var1 VHH affect the binding to the cell-expressed Vγ9Vδ2 T cell receptor, the Fc-containing form of the mutant VHH molecules was tested for binding to primary, proliferating Vγ9Vδ2 T cells by flow cytometry. Primary Vγ9Vδ2 T cells were isolated from the PBMC fraction of blood from healthy volunteers and proliferated by mixed lymphocyte reaction using a mixture of irradiated feeder cells and cytokines. Next, aliquots of 50,000 Vγ9Vδ2 T cells were incubated in the concentration range of the Fc-containing bispecific molecule, and binding was detected with a PE-labeled anti-human IgG detection antibody. Consistent with the results obtained by BLI, the molecule containing the R109 mutation was indistinguishable from the original molecule in apparent affinity (i.e., binding to Vγ9Vδ2 T cells) (Figure 25), and the EC50 value of binding was very similar. The bispecific molecule containing the R109AΔLGIR CDR3 did not bind significantly to the cells (not shown), and the G108 variant showed very weak binding.

[0225] Example 23 | Bispecific, Fc-containing R109 mutant molecules are equivalent in their ability to induce Vγ9Vδ2 T cell activation and target-dependent Vγ9Vδ2 T cell-mediated cytotoxicity. Bispecific, Fc-containing anti-CD123 x TCR bispecific molecules containing variants at positions 108 and 109 of 5C8var1 were tested for their ability to induce target-dependent Vγ9Vδ2 T cell activation (determined by analysis of CD107a expression) and Vγ9Vδ2 T cell-mediated target cell cytotoxicity as described in Examples 14 and 15. Two target cell lines expressing the CD123 molecule, MOLM-13 and THP-1, were used. The figure shows representative results using Vγ9Vδ2 T cells from one healthy donor, and two different Vγ9Vδ2 T cell donors were tested, both giving equivalent results.

[0226] Figures 26 and 27 show that all bispecific molecules were able to induce Vγ9Vδ2 T cell activation and Vγ9Vδ2 T cell-mediated target cell cytotoxicity. The variants containing the R109 mutation (either A or K) were as potent as the original molecule with only the Y105F mutation. The EC50 value of the R109A variant (Table 8) was slightly lower than those of the other two variants. [Table 12]

[0227] Example 24 | Antibody Stability To determine whether the introduction of changes at position 109 in the Vδ2-binding VHH affects the thermal and colloidal stability of the antibody, the development of an Fc-containing bispecific antibody was tested using nanoDSF (differential scanning fluorimetry). Table 9 shows the temperature at which unfolding was observed and the onset temperature of turbidity (protein aggregation). The R109 mutants had approximately the same thermal stability as the parental molecule. [Table 13]

[0228] Also, the antibody was tested in an accelerated stress test: the protein was incubated under high temperature and acidic or basic conditions, or oxidative stress conditions.

[0229] At high temperature, the protein sample was adjusted to 1 g / L in phosphate buffer, pH 7.4, and incubated in an incubator at 40 °C for 1 week. Then, the sample was aliquoted and stored at 2 - 8 °C or -80 °C until analysis.

[0230] At low pH, the protein sample was re-buffered to 1 g / L by ultrafiltration (molecular weight cut-off (MWCO) 30 kDa) against 50 mM acetate buffer, pH 5.0, and incubated in an incubator at 40 °C and low pH for 1 week. At high pH, the protein sample was re-buffered to 1 g / L by ultrafiltration (MWCO 30 kDa) against 100 mM phosphate buffer, pH 8.5, and incubated in an incubator at 40 °C and high pH for 1 week.

[0231] Under oxidative conditions, the protein sample was re-buffered to 1 g / L by ultrafiltration (MWCO 30 kDa) against phosphate buffer, pH 7.4 and 0.05% H2O2, and incubated at RT for 24 hours.

[0232] After incubation under acidic, basic or oxidative conditions, the sample was re-buffered by ultrafiltration (MWCO 10 kDa) against phosphate buffer, pH 7.4, and fractionated and stored at 2 - 8 °C or at -80 °C.

[0233] Analysis of aggregates and fragments was performed by SEC-UV (Table 10), in which the proportions of high molecular weight species (HMWS) and / or low molecular weight species (LMWS) did not change or changed only slightly after culturing under the indicated accelerated stress conditions. Importantly, the changes observed for the R109 mutant were almost the same as those of the parent molecule.

Table 14

[0234] Additional analysis of fragments was performed by CGE / CE-SDS after reduction (Table 11). The proportion of LMWS did not change or changed only slightly, and the changes observed for the R109 mutant were very similar to those found for the parent molecule.

Table 15

[0235] Example 24: Humanization of anti-CD123 VHH 1D2 using CDR grafting The 1D2 VHH antibody fragment was humanized using CDR grafting technology (see, e.g., U.S. Patent No. 5,225,539 and Williams, D.G. et al., 2010, Antibody Engineering, volume 1, Chapter 21). First, human germline sequences were identified using IgBLAST (Ye J. et al., 2013, Nucleic Acids Res. 41:W34-40). The V gene IGVH3-23*04 was identified as the closest human germline sequence (78.4% identity). Using this germline sequence, the llama CDRs (91.8% identity with the human germline IGVH3-23*04) were directly grafted to obtain the construct of SEQ ID NO: 32. Next, the NCBI NR database (downloaded on September 27, 2020) was queried using BLASTP (version 2.10.0+) to identify human template sequences demonstrating the highest identity with the 1D2 sequence. Two VH sequences were identified that demonstrated a similarity score of 70% or greater and preferably displayed similar CDR lengths that were identical to those of 1D2 CDR1, CDR2, and CDR3, respectively. The frameworks encoded by GenBank (Benson, D.A. et al., 2013, Nucleic Acids. 41(D1):D36-42) accession #CAD60357.1 and AKU38567.1 were selected as templates for grafting of the 1D2 CDRs, yielding cDNA constructs of SEQ ID NOs: 29 and 33, respectively. The definitions of the framework and CDRs were those 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 the structure of the VHH, a homology model of 1D2 VHH was generated using the “Antibody Prediction” tool (default parameters) within BioLuminate 4.2.156 (Schrodinger).The homology model was constructed based on PDB ID 6GKU. The CDRs were grafted in silico to study the effect of human residues on the loop conformation of the CDRs, surface hydrophobicity, and features such as structural integrity (e.g., increased rigidity). The resulting constructs were checked for these features and led to the design of additional constructs with SEQ ID NOs: 26, 27, 28, 30, 31, 34, and 35. Then, the sequences of these humanized 1D2-VHHs were reformatted into bispecific VHHs with Vδ2-specific VHH (5C8var1, SEQ ID NO: 18) in the direction of N-terminus-humanized-anti-1D2 VHH-linker-anti-Vδ2VHH-C-tag. Subsequently, the cDNAs encoding these molecules were synthesized and cloned into expression vectors for expression in HEK293E cells. The proteins were produced by transient transfection of cells and purified from the culture supernatant using C-tag affinity chromatography, followed by size-exclusion chromatography.

[0236] Example 25 | Affinity determination of humanized 1D2-5C8var1 (Y105F) variant for binding CD123 using Biolayer Interferometry (BLI) To determine the kinetics of binding of the humanized 1D2-5C8var1 variant to CD123, recombinant purified CD123-Fc fusion protein (Bio-Techne / R&D Systems) was loaded to an anti-human IgG Fc Capture sensor (using a concentration of 5 μg / ml) for the Octet Red96e (Sartorius) instrument to a density of 1 nm. Subsequently, different sensors were immersed in different concentrations of the humanized 1D2-5C8var1 variant, starting from 50 nM and its two-fold dilutions. Dilutions were performed in a 10-fold kinetic buffer (10×KB) provided by the supplier. From the resulting sensorgrams, the kinetic association rate constant and dissociation rate constant were determined by curve fitting. When fitting was possible, the association rate constant and dissociation rate constant were used to calculate the affinity of the humanized 1D2-5C8var1 variant for binding to CD123. The measurements were performed twice, and the affinities of different humanized 1D2-5C8var1 variants for CD123 were in the range from similar 2.6 nM to non-binding variants as compared to the parental 1D2, as shown in Table 2. For reference, non-humanized (parental) 1D2 was included in these experiments.

Table 16

[0237] Example 26|Development of a cell pool stably expressing a bispecific antibody. The 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 for expression in Chinese hamster ovary (CHO)-CD390 cells by a contract research organization (CRO). The cDNAs were generated by synthetic gene synthesis and cloned into a proprietary expression vector carrying an antibiotic resistance (selection) gene (conferring either puromycin or methotrexate resistance). Then, two different plasmids encoding two different chains (combinations of 1D2-Fc x 5C8var1-Y105F-Fc and 1D2-Fc x 5C8var1-Y105F-R109A-Fc) were co-transfected into CHO-CD390 cells at different plasmid ratios. After 48 hours, selection with both puromycin and methotrexate was initiated, and after 13 days of culture, a clone pool resistant to both antibiotics was obtained and cryopreserved.

[0238] Example 27 | Purification of Bispecific Antibodies from Conditioned Cell Culture Supernatants Production of soluble antibodies by the selected cell pool was initiated using a general fed-batch protocol in an Infors HT Multitron cell culture shaker (12 ml scale bioreactor tubes, 14 days of culture). After production, the antibodies were purified from the conditioned cell supernatant using protein-A affinity chromatography. Briefly, the protein was captured on an HP SpinTrap (protein A Sepharose) column (GE Healthcare) in 200 mM sodium phosphate (pH 7.0). After binding and washing, the antibody was eluted using a pH shock (100 mM sodium citrate pH 3.6), and the solution was immediately neutralized by the addition of 1 M Tris-HCl, pH 9.0. The amount of purified antibody was quantified by biolayer interferometry (BLI) using an Octet Red96 system (Sartorius) equipped with a protein A sensor and human IgG as a standard.

[0239] Example 28 | Analysis of Purified Protein by CE - SDS (Capillary Electrophoresis) Capillary electrophoresis can separate proteins by apparent molecular weight and quantify all proteinaceous components by UV spectroscopy. Sample preparation included heat denaturation of the protein in the presence of the denaturing agent sodium dodecyl sulfate (SDS). During denaturation, sample components were separated based on differences in electrophoretic mobility within a capillary containing an exchangeable SDS polymer matrix. In Figure 28, electropherograms of non - reducing analyses of 1D2 - Fc x 5C8var1 - Y105F - Fc (Figure 28A) and 1D2 - Fc x 5C8var1 - Y105F - R109A - Fc (Figure 28B) are shown. The analysis was performed using a PA800 Plus system (SCIEX) equipped with a PDA detector for detecting proteins at a wavelength of 220 nm. Due to protein denaturation under non - reducing conditions, the major component of both samples is a heterodimer 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 shown as "Peak 2" is detected, whereas that peak is absent from the electropherogram of 5C8var1 - Y105F - R109A - Fc, demonstrating that 5C8var1 - Y105F - R109A - Fc is less prone to fragmentation as observed for 5C8var1 - Y105F - Fc. Quantification of various species is summarized in Table 13. The major peak at 25 min corresponds to the complete heterodimer, and "Peak 1" represents a small amount of the half - body present in the preparation of 1D2 - Fc x 5C8var1 - Y105F - Fc. "Peak 2" is the truncation observed at position 108 in 5C8var1 - Y105F - Fc. "Peak 3" corresponds to the deglycosylated variant of the protein.

Table 17

[0240] Example 29 | Analysis of purified 1D2-Fc x 5C8var1-Y105F-R109A-Fc protein by mass spectrometry (MS) Protein A-purified protein 1D2-Fc x 5C8var1-Y105F-R109A-Fc was sent to a CRO for MS analysis. The protein was N-deglycosylated overnight at 37 °C using N-glycosidase F (Roche) at an optimized enzyme:protein ratio. Samples were diluted with 0.1% formic acid (FA) and separated on a UPLC-system (Waters Acquity Premier H class) using a reverse-phase column (MAbPac RP 4μm 2.1 x 50mm, Thermo Scientific). The eluents were 0.1% FA in water and 0.1% FA in acetonitrile. Mass spectrometry analysis was performed on a compact QTOF mass spectrometer (Bruker Daltonik). The reported LC-ESI-MS spectra were summed, deconvoluted, and smoothed using the software DataAnalysis (Bruker Daltonik). In the annotation, fully oxidized / reduced cysteine was assumed (depending on sample preparation). Four different batches of 1D2-Fc x 5C8var1-Y105F-R109A-Fc were analyzed. Despite some heterogeneity regarding C-terminal lysine clipping and some heterogeneity in glycosylation, the truncation products of 5C8var1-Y105F-R109A-Fc were not identified, which again demonstrated that the R109A mutation prohibits the formation of truncations (data not shown).

Claims

[Claim 1] An antibody comprising a first antigen-binding region capable of binding to human Vδ2, wherein the first antigen-binding region comprises the CDR1 sequence described in SEQ ID NO: 12, the CDR2 sequence described in SEQ ID NO: 13, and the CDR3 sequence described in SEQ ID NO: 14.

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