Anti-BTN3A Antibodies and Their Use in Treating Cancer or Infectious Disorders

Abstract

To provide anti-BTN3A activating antibodies that are useful in treating cancer disorder, specifically bind to BTN3A, and activate the cytolytic function of Vγ9 / Vδ2 T cells.SOLUTION: The present invention provides a pharmaceutical composition for use in a method for treating solid tumors in a subject in need thereof, comprising administering a therapeutically effective amount of an anti-BTN3A antibody, wherein the anti-BTN3A antibody is an activating antibody able to induce the activation of Vγ9 Vδ2 T cells in co-culture with BTN3A expressing cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Herein, we disclose anti-BTN3A activating antibodies that specifically bind to BTN3A and activate the cytolytic function of Vy9 / V52 T cells. Such antibodies are particularly useful for treating cancer disorders, such as hematological cancers or solid tumors. The disclosure more specifically relates to certain humanized anti-BTN3A activating antibodies with equivalent or improved properties compared to the corresponding parent murine antibody 7.2, or chimeric versions thereof with Fc-silenced human IgG1 or IgG4 constant regions. [Background technology]

[0002] Leukocytes are cells of the immune system involved in defending the body against pathogens. These cells include lymphocytes, monocytes, and dendritic cells. Monocytes migrate from the bloodstream to other tissues and differentiate into tissue-resident macrophages or dendritic cells. Dendritic cells act as antigen-presenting cells (APCs) that activate lymphocytes. Among lymphocytes, T cells can be divided into αβ T cells and γδ T cells. The Vγ9-Vδ2 subset of γδ T cells is a key effector of the immune defense system. Vγ9-Vδ2 directly lyses pathogen-infected or abnormal cells. Furthermore, Vγ9-Vδ2 regulates immune responses by inducing dendritic cell (DC) maturation, isotype switching, and immunoglobulin production. This important cell subset of the immune system is tightly regulated by surface receptors, chemokines, and cytokines.

[0003] T cell priming is regulated by the engagement of specialized cells and the secretion of chemotactic cytokines. The two-signal hypothesis postulates that T cell activation is the result of two synergistic events. The first is the interaction between the T cell receptor (TCR) and the major histocompatibility complex (MHC) complexed with processed antigen on the surface of an antigen-presenting cell (APC). The second event is a costimulatory, antigen-independent signal involving CD28 and B7 molecules. The absence of costimulatory signals induces anergy and unresponsiveness, resulting in the absence of T cell proliferation, cytokine secretion, and cytotoxic activity. Study of these pathways provides insight into the initiation of pathological events such as autoimmune or lymphoproliferative disorders. The B7 family is an extended group of costimulatory molecules (Coyle and Gutierrez-Ramos, 2001; Sharpe and Freeman, 2002). The B7 family includes the ligands B7-1 (CD80) and B7-2 (CD86); their receptors are CD28, which mediates T cell activation, and CTLA-4 (CD152), which competes with CD28 to transmit inhibitory signals (reviewed in Alegre et al., 2001). The crucial role of CD152 as a negative regulator of T cell activation has been demonstrated by the development of lymphoproliferative disorders in CTLA-4-deficient mice. Important insights into the inhibitory function exerted by CD152 have come from studies of proliferation or cytokine production by naive T lymphocytes during T cell priming. In particular, CD152 is expressed following T lymphocyte activation and inhibits the cytolytic function of CTL clones obtained following PHA stimulation or Ag selection. B7-H1 (PD-L1, CD274) and B7-DC (PD-L2, CD273), whose receptor is PD-1 (CD279), have been found to inhibit T cell proliferation and cytokine secretion (reviewed in Sharpe and Pauken, 2018). Otherwise, different studies have shown that PD-L1 and PD-L2 engagement increases T cell proliferation and IL-10 or IFN-γ production.Other molecules related to the B7 family expressed on the surface of T cells, including B7-H2 (ICOS-L), and the more recently identified B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7, have also been implicated as checkpoint regulators of immune function (reviewed in Ni and Dong, 2017).

[0004] Henry et al. (1999) discovered that the butyrophilin (BT) coding region is located telomeric to the MHC class I region on human chromosome 6. In particular, they described two genes, Bt2 and Bt3, that encode a new group of costimulatory molecules (BT2.1, BT2.2, BT2.3, BT3.1, BT3.2, and BT3.3) belonging to the Ig superfamily (IgSF) (Linsley et al., 1992; Williams and Barclay, 1988) and that sequence similarity analysis links to the B7 family; in particular, they revealed similarity to the IgV-like extracellular domains of CD80 and CD86.

[0005] BT3 family members appear in the literature under different names: BT3.1 is also called BTF5 (Ruddy et al., 1997), or BTN3A1 (Rhodes et al., 2001), or more recently CD277 (Bensussan and Olive, 2005); BT3.2 is also called BTF4 (Ruddy et al., 1997), or BTN3A2; and finally, BT3.3 also appears as BTF3 (Ruddy et al., 1997), or BTN3A3 (Rhodes et al., 2001). BT3 has two Ig-like extracellular domains, characteristic of IgSFs.

[0006] It has been proposed that the B7 gene and MHC class I and II genes share a common ancestral gene and encode proteins involved in similar functions, such as T cell activation (Rhodes et al., 2001). The BT3 molecule has been found in immune cells such as T, B, and NK cells, monocytes, and dendritic cells, as well as hematopoietic precursors and some neoplastic cell lines. As for other costimulatory molecules, their structure is characterized by three domains: an extracellular domain that binds to ligands, a transmembrane domain, and an intracellular domain called B30.2, which is likely involved in regulating intracellular superoxide levels. To date, the ligand(s) for CD277 are still unknown (reviewed in Gu et al., 2015).

[0007] To date, various therapeutic and vaccine strategies have been proposed that rely on the modulation of T cells; several immunomodulatory antibodies against CTLA-4, PD-1, and PD-L1 have already been approved for clinical use by multiple regulatory agencies worldwide. While these drugs represent a major advance in cancer therapy, there remains unmet medical need for a large proportion of the cancer patient population who do not respond to currently available treatments.

[0008] Patent Publications WO 2012 / 080351, EP 2651441, EP 2946791, US 2014 / 0322235, and WO 2012 / 080769 refer to various antibodies against BTN3A that can activate or inhibit the cytolytic function, cytokine production, and proliferation of Vγ9 Vδ2 T cells. However, these murine antibodies were not suitable for therapeutic applications. Indeed, for administration to human patients, humanization of antibodies is now mandatory to avoid immunogenic reactions.

[0009] Humanization often requires modifying amino acids in framework regions without the certainty of maintaining the same level of potency as the original murine antibody. This is especially true when modifying amino acids immediately adjacent to the CDR regions (see, e.g., Queen patent US5,585,089).

[0010] Despite these challenges, the inventors have now selected a particular humanized antibody of activating mAb 7.2 that not only combines the preserved functional properties of the mAb 7.2 parent antibody with the predicted reduced immunogenicity in humans, but also, surprisingly, exhibits superior developability, such as improved yields in cell line production and higher thermostability, as well as strong resistance to acid and heat stress, when compared to the parent murine antibody. Furthermore, the humanized antibody mAb1 of the present disclosure advantageously binds to cynomolgus BTN3A and is well tolerated in cynomolgus primates at doses up to 100 mg / kg / week, thereby providing an excellent candidate for use as a drug in human therapy. Summary of the Invention

[0011] Accordingly, the present disclosure relates to isolated anti-BTN3A antibodies comprising a variable heavy chain polypeptide VH of SEQ ID NO: 1 and a variable light chain polypeptide VL of SEQ ID NO: 2 or SEQ ID NO: 3. Such antibodies are humanized antibodies, particularly with predicted reduced immunogenicity relative to their parent murine antibodies. Such isolated anti-BTN3A antibodies bind to human BTN3A. In particular, such isolated anti-BTN3A antibodies have a K of 10 nM or less for human BTN3A as measured by surface plasmon resonance. D and preferably a K of 5 nM or less D Combine with.

[0012] In certain embodiments, the antibodies according to the present disclosure inhibit the activation of γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with BTN3-expressing cells, as measured in a degranulation assay, at an EC of less than 5 μg / ml, preferably 1 μg / ml or less. 50 As a result, the antibody induces killing of tumor target cells regardless of their tissue origin.

[0013] In certain embodiments, the isolated anti-BTN3A antibody comprises a mutated or chemically modified IgG1 constant region that lacks or exhibits reduced Fcγ receptor binding compared to a corresponding antibody having a wild-type IgG1 isotype constant region. Typically, the mutated IgG1 constant region is the IgG1 triple mutant L247F L248E and P350S. An example of the isolated anti-BTN3A antibody is mAb1, which comprises a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:6, or mAb2, which comprises a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:7.

[0014] In other embodiments, the isolated anti-BTN3A antibodies of the present disclosure are monovalent format antibodies, preferably selected from Fab or scFv antibodies.

[0015] The isolated anti-BTN3A antibodies of the present disclosure may be used (i) as therapeutic agents or (ii) as diagnostic agents. For example, these antibodies are useful in the treatment of cancer, e.g., hematological cancers, more particularly lymphoma or leukemia. In other embodiments, these antibodies may also be used to treat solid tumors, more particularly prostate, ovarian, or endometrial cancer. Alternatively, these antibodies may be used to treat infectious disorders.

[0016] The present disclosure further relates to pharmaceutical compositions comprising the anti-BTN3A antibodies described above in combination with one or more pharmaceutically acceptable excipients, diluents or carriers, and optionally other active ingredients.

[0017] Another aspect of the present disclosure relates to a lyophilized formulation, pre-filled syringe, or vial comprising the anti-BTN3A antibody described above.

[0018] The present disclosure relates to expression vectors for the recombinant production of the above-described anti-BTN3A antibodies in host cells, typically mammalian host cells such as CHO host cells, comprising at least one nucleic acid encoding the anti-BTN3A antibodies. Embodiments of such expression vectors include nucleic acids encoding at least the heavy and light chains of mAb1 disclosed herein. Host cells comprising such expression vectors are also described herein.

[0019] Also disclosed herein is a process for producing an anti-BTN3A antibody of the present disclosure, comprising the steps of: (i) culturing a host cell as defined above for expression of said antibody by the host cell; optionally (ii) purifying said antibody; and (iii) recovering the antibody.

[0020] The present disclosure further relates to multispecific antibodies, such as bispecific antibodies, comprising at least one arm comprising a Fab or scFv comprising the VH and VL of an anti-BTN3A antibody as defined above. [Brief explanation of the drawings]

[0021] [Figure 1A] Human Vy9V52 T cells expanded from PBMCs were co-cultured with the Daudi cell line (Burkitt's lymphoma) at a 1:1 E:T ratio with the indicated concentrations of mAb1 (or corresponding isotype control) for 4 hours. Cells were stained with antibodies against CD107a and CD107b, and gating for positive expression was based on unstimulated controls. Experiments were performed using three healthy donors. [Figure 1B] Daudi cells were preincubated with the indicated concentrations of mAb1 (or the corresponding isotype control) for 1 h at 37°C. After extensive washing, mAb-pulsed Daudi cells were co-cultured with expanded human Vy9V52 T cells for 4 h at 37°C before measuring caspase 3 / 7 activity in Daudi cells. In A and B, curve fitting was obtained using the sigmoidal 4PL equation from GraphPad Prism software. [Figure 1C]Using the same protocol as previously described in (A) and (B), we evaluated the efficacy of mAb1 (and the corresponding isotype control) used at 10 μg / mL against other neoplastic cell lines (L-IPC: pancreatic ductal adenocarcinoma, HT29: colorectal adenocarcinoma, A549: lung carcinoma) compared to the Daudi cell line. [Figure 2A] mAb1 mediates BTN3A-expressing target cell killing by Vy9V52 T cells. 10,000 HL60-WT or BTN3AKO cells (acute myeloid leukemia) were co-cultured with in vitro expanded Vy9V52 T cells for 24 hours (1:1 E:T ratio) in the presence of increasing concentrations of mAb1 (or the relevant isotype control hIgG1) + / - rHuIL-2 (20 IU / ml). Cell viability was measured using a bioluminescence assay detecting ATP levels. [Figure 2B] mAb1 mediates BTN3A-expressing target cell killing by Vy9V52 T cells. 10,000 HL60-WT cells were co-cultured with in vitro expanded Vy9V52 T cells for 4 days (1:1 E to T ratio) in the presence of increasing concentrations of mAb1 (or the relevant isotype control hIgG1) + / - rHuIL-2 (20 IU / ml). Cell viability was measured daily. [Figure 2C] mAb1 mediates BTN3A-expressing target cell killing by Vy9V52 T cells. 10,000 HL60-WT cells were co-cultured with freshly isolated Vy9V52 T cells from human PBMCs for 4 days (E:T ratios of 1:1 and 1:5) in the presence of increasing concentrations of mAb1 + rHuIL-2 (20 IU / ml). Cell viability was measured daily. * marks above signals. [Figure 3] 10,000 tumor cells of different tissue origins were co-cultured with in vitro-expanded Vy9V52 T cells in the presence of different concentrations of mAb1 for 24 hours (1:1 E:T ratio). Cell viability was measured using a bioluminescence assay to detect ATP levels. Bioluminescence values ​​are shown. The four bars, from left to right, represent: (1) no mAb, (2) mAb1 0.1 μg / ml, (3) mAb1 1 μg / ml, and (4) mAb1 10 μg / ml. [Figure 4A]mAb1 promotes the proliferation and activation of cynomolgus Vγ9Vδ2 T cells. Cynomolgus whole blood from three animals was treated with red blood cell lysis buffer. After extensive washing, cells were plated at 1.5 μg / mL in medium containing 200 IU / mL rHuIL-2 and mAb1 (10 μg / mL). The percentage of Vγ9+ T cells was assessed by flow cytometry on days 0, 3, 6, 8, and 10 using specific antibodies. The graph shows the dynamics of the percentage of Vγ9+ T cells among live cells. Each curve represents an individual animal. [Figure 4B] mAb1 promotes proliferation and activation of cynomolgus Vγ9Vδ2 T cells. After 10 days of expansion, cells from each animal were co-cultured for 4 hours with Daudi, K562, or Raji target cells (1:1 E:T ratio) in the presence of culture medium, mAb1, or isotype control (10 μg / mL) and analyzed for degranulation (CD107a / b) by flow cytometry. [Figure 5] Cynomolgus blood samples collected at the indicated times after ICT01 administration were stained with a specific cocktail of antibodies to quantify T cell subsets (CD4, CD8, Vy9 T cells, regulatory T cells), B cells, monocytes, NK cells, mDCs, pDCs, and granulocytes, and analyzed by flow cytometry. The top panel shows the percentage of Vy952 T cells among CD3+ T cells for single-dose animals. The bottom panel shows the percentage of Vy952 T cells among CD3+ T cells for repeated-dose animals. Data are presented as mean ± SD for each sample collection time and group. The vertical dotted line indicates the time of ICT01 administration. DETAILED DESCRIPTION OF THE INVENTION

[0022] definition In order that the present disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0023] As used herein, the term "BTN3A" has its general meaning in the art. In certain embodiments, the term refers to a human BTN3A polypeptide, including any of BTN3A1 of SEQ ID NO: 18, BTN3A2 of SEQ ID NO: 19, or BTN3A3 of SEQ ID NO: 20.

[0024] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulins, i.e., molecules that contain an antigen-binding site that immunospecifically binds to an antigen. Thus, the term antibody encompasses not only whole antibody molecules, but also antibody fragments and antibody variants (including derivatives).

[0025] In natural rodent and primate antibodies, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. In a typical IgG antibody, the light chain contains two domains: a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement fixation, and binding to Fc receptors (FcR).

[0026] An Fv fragment is the N-terminal portion of an immunoglobulin Fab fragment and consists of the variable portions of one light chain and one heavy chain. Antibody specificity resides in the structural complementarity between the antibody-binding site and an antigenic determinant. An antibody-binding site is composed of residues primarily from hypervariable or complementarity-determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) can participate in the antibody-binding site or influence the overall domain structure and thus the binding site. Complementarity-determining regions (CDRs) are amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, an antigen-binding site typically contains six CDRs, including a set of CDRs from each of the heavy and light chain V regions. Framework regions (FR) are amino acid sequences inserted between the CDRs. Thus, the variable regions of the light and heavy chains typically contain four framework regions and three CDRs of the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0027] Residues in antibody variable domains are conventionally numbered according to the system devised by Kabat et al. This system is set forth in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (Kabat et al., 1992, hereafter "Kabat et al."). This numbering system is used herein. The Kabat residue designations do not necessarily correspond directly to the linear numbering of the amino acid residues in the SEQ ID sequence. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering, corresponding to truncations of or insertions into components, whether framework or complementarity-determining regions (CDRs) of the basic variable domain structure. The exact Kabat numbering of residues can be determined for a given antibody by aligning the homologous residues in that antibody's sequence with the "standard" Kabat numbering sequence. The CDRs of the heavy chain variable domain are located at residues 31-35 (H-CDR1), residues 50-65 (H-CDR2), and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2), and residues 89-97 (L-CDR3) according to the Kabat numbering system.

[0028] In certain embodiments, the antibodies provided herein are antibody fragments, more particularly, any protein comprising the antigen-binding domain of an antibody disclosed herein. Antibody fragments include, but are not limited to, Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabodies.

[0029] As used herein, the term "specificity" refers to the ability of an antibody to detectably bind to an epitope displayed on an antigen, such as BTN3A. In some embodiments, this term refers to an antibody that detectably binds to human BTN3A expressed on peripheral blood myeloid cells (PBMCs), preferably with an EC of less than 50 μg / ml, more preferably below 10 μg / ml, as determined in the Examples. 50 (See Table 4.) In other embodiments, the antibody binds to the antigenic recombinant polypeptide with a K of 100 nM or less, 10 nM or less, 1 nM or less, 100 pM or less, or 10 pM or less, as measured by SPR measurements as determined in the Examples. D (See Table 4)

[0030] An antibody that "cross-reacts with an antigen other than BTN3A" has a K of 10 nM or less, 1 nM or less, or 100 pM or less for that antigen other than BTN3A. D An antibody that "does not cross-react with a particular antigen" is intended to refer to an antibody that binds to that antigen with a K of 100 nM or greater. D , or a K of 1 μM or greater D , or a K of 10 μM or greater D "Antibody" is intended to refer to an antibody that binds to an antigen. In certain embodiments, such antibodies that do not cross-react with the antigen exhibit essentially undetectable binding to these proteins in standard binding assays. In certain embodiments, a humanized antibody of the present disclosure, e.g., mAb1, cross-reacts with cynomolgus monkey BTN3A1, BTN3A2, and BTN3A3, of SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, respectively, as measured, e.g., in a Biacore assay (see Table 21).

[0031] An "isolated antibody," as used herein, refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to BTN3A is substantially free of antibodies that specifically bind to antigens other than BTN3A). However, an isolated antibody that specifically binds to BTN3A may have cross-reactivity to other antigens, such as related BTN3A molecules from other species. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0032] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition that displays a single binding specificity and affinity for a particular epitope.

[0033] The phrases "antibody that recognizes an antigen" and "antibody having specificity for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen."

[0034] The term “K associ " or "K a " as used herein is intended to refer to the association rate of a particular antibody-antigen interaction, and "K dis " or "K d ", as used herein, is intended to refer to the off-rate of a particular antibody-antigen interaction.

[0035] The term “K D " as used herein means K a K d The ratio of (i.e., K d / K a ) and is expressed as a molar concentration (M). D The value can be determined using methods well established in the art.

[0036] Antibody K DMethods for determining β-glucan are by using surface plasmon resonance or by using a biosensor system such as a Biacore® system.

[0037] Specificity can be further demonstrated, for example, by an affinity / avidity ratio of about 10 to 1, about 20 to 1, about 50 to 1, about 100 to 1, 10,000 to 1, or greater for binding to a specific antigen versus nonspecific binding to other, unrelated molecules (in this case, the specific antigen is a BTN3A polypeptide). The term "affinity," as used herein, refers to the strength of binding of an antibody to an epitope.

[0038] As used herein, the term "avidity" refers to an informative measure of the overall stability or strength of an antibody-antigen complex. Avidity is controlled by three major factors: antibody epitope affinity; valency of both the antigen and antibody; and the structural arrangement of the interacting moieties. Ultimately, these factors define antibody specificity, i.e., the likelihood that a particular antibody will bind to a precise antigen epitope.

[0039] As used herein, the term "activating antibody" refers to an antibody that can directly or indirectly induce immune function of effector cells. In particular, as used herein, an activating anti-BTN3A antibody has an EC of less than 5 μg / ml, preferably 1 μg / ml or less, when co-cultured with BTN3A-expressing cells, as measured in the degranulation assay described in the Examples below. 50 It has at least the ability to induce activation of γδ T cells, typically Vγ9Vδ2 T cells, in the presence of IgG.

[0040] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

[0041] As used herein, the term "optimized" means that a nucleotide sequence has been altered to encode an amino acid sequence using codons preferred in the producing cell or organism, generally a eukaryotic cell, such as a Chinese hamster ovary cell (CHO) or a human cell. An optimized nucleotide sequence is engineered to retain entirely, or as much as possible, the amino acid sequence originally encoded by the starting nucleotide sequence. An amino acid sequence encoded by an optimized nucleotide sequence is also referred to as optimized.

[0042] As used herein, the percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.

[0043] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17, 1988) incorporated into the ALIGN program (version 2.0) using a PAM120 residue weighting table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453, 1970) incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com) using a Blossom62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0044] The percent identity between two nucleotide amino acid sequences may be determined using an algorithm such as, for example, the BLASTN program for nucleic acid sequences, which uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0045] Recombinant humanized anti-BTN3A activating antibody The antibodies of the present disclosure include selected humanized recombinant antibodies mAb1, mAb2, mAb4, and mAb5, which are structurally characterized by their variable heavy and light chain amino acid sequences and human constant regions (isotypes) as set forth in Table 1 below. [Table 1]

[0046] mAb3 and mAb6 are humanized versions of another parent murine anti-BTN3A antibody, designated mAb20.1, described in WO 2012 / 080351, and used as comparative examples.

[0047] The corresponding amino acid and nucleotide coding sequences of the constant isotype regions of IgG1, IgG4 and their mutated versions IgG1 L247F / L248E / P350S and IgG4 S241P / L248E used to generate mAb1 to mAb6 are well known in the art (Oganesyan et al., 2008; Reddy et al., 2000). The C-terminal lysine found in IgG may be naturally cleaved, and this modification does not affect the properties of the antibody; therefore, this residue may be further deleted in the constructs of mAb1 to mAb6.

[0048] The full-length light and heavy chains and corresponding coding sequences of mAb1, mAb2, mAb4 and mAb5 are shown in Table 2 below. [Table 2]

[0049] Exemplary amino acid sequences of VH CDR1 (also called HCDR1), VH CDR2 (also called HCDR2), VH CDR3 (also called HCDR3), VL CDR1 (also called LCDR1), VL CDR2 (also called LCDR2), and VL CDR3 (also called LCDR3) of some antibodies according to the present disclosure are shown in Table 3.

[0050] In Table 3, the CDR regions of the antibodies of the disclosure are depicted using Kabat numbering (Kabat et al., 1992, hereafter "Kabat et al.").

[0051] For ease of reading, the CDR regions will hereafter be referred to as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively. [Table 3]

[0052] In certain embodiments, said recombinant anti-BTN3A antibody as defined above has the following characteristics: (i) the antibody has a K of 10 nM or less, as measured, for example, by SPR as described in the Examples below; D and preferably a K of 1 nM or less D binds to BTN3A at ; (ii) the antibody has a K of 100 nM or less, as measured, for example, by SPR as described in the Examples below. D and preferably a K of 10 nM or less D cross-reacts with cynomolgus monkey BTN3A; (iii) the antibody has an EC of 50 μg / ml or less, preferably 10 μg / ml or less, as measured in the flow cytometry assay described in the Examples below. 50 binds to human PBMCs; (iv) the antibody has an EC50 of less than 5 μg / ml, preferably 1 μg / ml or less, when co-cultured with BTN3-expressing cells, as measured in the degranulation assay described in the Examples below. 50induces activation of gamma delta T cells, typically Vg9Vdeg2 T cells, The present invention has one or more of the following:

[0053] In certain embodiments that may be combined with the previous embodiments, the antibody provided herein is an antibody fragment of an antibody defined above.

[0054] Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, unibody, and scFv fragments, diabodies, single domains or nanobodies and other fragments.

[0055] Preferably, the antibody is a monovalent antibody, such as the Fab of an scFv fragment.

[0056] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which comprises a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain to create two antigen-binding sites.

[0057] Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516).

[0058] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells as described herein.

[0059] The antibody of the present disclosure is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining at least the same affinity (or better affinity) as the parent non-human antibody. In a preferred embodiment, the antibody of the present disclosure is a humanized version of the parent antibody mAb7.2 disclosed in WO2012 / 080351. A comparative example includes a humanized version of the parent antibody mAb20.1 disclosed in WO2012 / 080351.

[0060] Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody, e.g., murine mAb 7.2, and the FRs (or portions thereof) are derived from murine antibody sequences with mutations that reduce immunogenicity. Optionally, the humanized antibody also comprises at least a portion of a human constant region.

[0061] Preferably, the recombinant antibody according to the present disclosure is a humanized silenced antibody, typically a humanized silenced IgG1 or IgG4 antibody.

[0062] As used herein, the term "silent" antibody refers to an antibody that exhibits no or low FcγR binding and / or C1q binding when measured in a binding assay such as the assays described in the Examples.

[0063] In one embodiment, the term "no or low FcγR binding and / or C1q binding" means that the silenced antibody exhibits FcγR and / or C1q binding that is at least 50% less, for example, less than 80%, of the FcγR and / or C1q binding observed with a corresponding antibody having a wild-type human IgG1 or IgG4 isotype.

[0064] Framework or Fc manipulation The antibodies of the present disclosure contain modifications made to framework residues within VH and VL to reduce the immunogenicity of the antibodies compared to the corresponding murine antibody, mAb7.2.

[0065] In one particular embodiment, the antibody of the present disclosure is a humanized monoclonal antibody of the parent murine antibody mAb7.2 and comprises at least the following amino acid mutations in the VH framework region: V5Q; V11L; K12V; R66K; S74F; I75S; E81Q; S82AR; R82BS; R83T; D85E; T87S; L108S; and at least the following amino acid mutations in the Vκ framework region: T5N; V15L; R18T; V19I; K42N; A43I; D70G; F73L; Q100G.

[0066] In another specific embodiment, the antibody of the present disclosure is a humanized monoclonal antibody of the parent murine antibody mAb7.2 and contains, relative to mAb7.2, at least the following amino acid mutations in the VH framework region: V5Q; V11L; K12V; R66K; S74F; I75S; E81Q; S82AR; R82BS; R83T; D85E; T87S; L108S; and at least the following amino acid mutations in the Vκ framework region: T5N; V15L; R18T; V19I; K42N; A43I; S63T; D70G; F73L; Q100G.

[0067] In addition to modifications made within the framework regions, antibodies of the disclosure may also be engineered to contain modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity.

[0068] Additionally, the antibodies of the present disclosure may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or engineered to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below.

[0069] As used herein, the terms "isotype constant region" or "Fc region" are used interchangeably to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. The human IgG heavy chain Fc region is generally defined to include amino acid residues from position C226 or P230 to the carboxyl terminus of an IgG antibody, where numbering is according to the EU numbering system. The C-terminal lysine of the Fc region (residue K447) may be removed, for example, during antibody production or purification, or its corresponding codon may be deleted in a recombinant construct. Thus, antibody compositions of the present disclosure may include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

[0070] In one specific embodiment, the hinge region of CH1 is modified such that the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. This approach is further described by Bodmer et al. in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[0071] In another embodiment, the Fc-hinge region of the antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcus protein A (SpA) binding compared to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.

[0072] In yet another embodiment, the Fc region is altered by substituting a different amino acid residue for at least one amino acid residue to alter the effector function of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand for which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent No. 5,624,821 and U.S. Patent No. 5,648,260, both by Winter et al.

[0073] In another embodiment, one or more selected amino acid residues can be replaced with a different amino acid residue such that the antibody has altered C1q binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 by Idusogie et al.

[0074] In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is further described in WO 94 / 29351 by Bodmer et al.

[0075] In other embodiments, the Fc region is modified to reduce the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to reduce the affinity of the antibody for Fcγ receptors by modifying one or more amino acids. Such antibodies with reduced effector function, particularly reduced ADCC, include silenced antibodies.

[0076] In certain embodiments, an Fc domain of the IgG1 isotype is used, while in some embodiments, a mutated variant of an IgG1 Fc fragment is used, e.g., a silenced IgG1 Fc that reduces or eliminates the ability of the fusion polypeptide to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or bind to Fcγ receptors.

[0077] In certain embodiments, an Fc domain of the IgG4 isotype is used, while in some embodiments, a mutated variant of an IgG4 Fc fragment is used, e.g., a silenced IgG4 Fc that reduces or eliminates the ability of the fusion polypeptide to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or bind to Fcγ receptors.

[0078] Silenced effector functions can be obtained by mutation of the Fc constant portion of the antibody and have been described in the art (Baudino et al., 2008; Strohl, 2009). An example of a silent IgG1 antibody includes the triple mutation variant IgG1 L247F L248E P350S. An example of a silent IgG4 antibody includes the double mutation variant IgG4 S241P L248E.

[0079] In certain embodiments, the Fc domain is a silent Fc mutant that prevents glycosylation of the Fc domain at position 314. For example, the Fc domain contains an amino acid substitution of asparagine at position 314. Examples of such amino acid substitutions are replacement of N314 with glycine or alanine.

[0080] In yet another embodiment, the glycosylation of an antibody is altered. For example, an aglycoslated antibody can be generated (i.e., the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more variable region framework glycosylation sites can be removed, thereby making one or more amino acid substitutions that eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for an antigen. Such approaches are described in further detail in U.S. Patent No. 5,714,350 and U.S. Patent No. 6,350,861 by Co et al.

[0081] Another modification of the antibodies herein contemplated by the present disclosure is pegylation or hesylation or related techniques. Antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups are attached to the antibody or antibody fragment. Pegylation can be carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono(C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the present disclosure. See, for example, European Patent No. 0 154 316 to Nishimura et al. and European Patent No. 0 401 384 to Ishikawa et al.

[0082] Another possibility is the fusion of at least the antigen-binding region of an antibody of the present disclosure to a protein capable of binding to a serum protein, such as human serum albumin, which would increase the half-life of the resulting molecule. Such an approach is described, for example, in Nygren et al., European Patent No. 0 486 525.

[0083] In certain embodiments, the C-terminal lysine, which is commonly present on human IgG heavy chain constant domains, is engineered to reduce heterogeneity due to the commonly observed cleavage of this residue during manufacturing or storage. Such modifications do not appreciably alter the desired function of these antibodies while providing stability benefits to these molecules.

[0084] Nucleic acid molecules encoding antibodies of the present disclosure Nucleic acid molecules encoding the anti-BTN3A antibodies of the present disclosure are also disclosed herein. Exemplary variable light and heavy chain nucleotide sequences are those encoding the variable light and heavy chain amino acid sequences of any one of mAb1, mAb2, mAb4, and mAb5, which amino acid sequences can be readily derived from Tables 1 and 2, using the genetic code and, optionally, taking into account codon bias depending on the host cell type.

[0085] The present disclosure also relates to nucleic acid molecules derived from amino acid sequences that have been optimized for protein expression in mammalian cells, such as CHO cell lines.

[0086] Nucleic acids may be present in whole cells, in cell lysates, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when it has been purified from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art (Ausubel et al., 1988). Nucleic acids of the present disclosure can be, for example, DNA or RNA, and may or may not contain intron sequences. In embodiments, the nucleic acid may be present in a vector, such as a phage display vector, or in a recombinant plasmid vector.

[0087] The nucleic acids of the present disclosure can be obtained using standard molecular biology techniques. For example, after DNA fragments encoding VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, a VL- or VH-encoding DNA fragment (e.g., a VL and VH as defined in Table 1) is operably linked to another DNA molecule or to a fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked," as used in this context, is intended to mean that two DNA fragments are joined in a functional manner, for example, so that the amino acid sequences encoded by the two DNA fragments remain in frame or so that a protein is expressed under the control of a desired promoter.

[0088] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (Kabat et al., 1992), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. In some embodiments, the heavy chain constant region is selected from an IgG1 isotype, e.g., a human IgG1 isotype. In other embodiments, the heavy chain constant region is selected from an IgG4 isotype, e.g., a human IgG4 isotype. In other embodiments, the heavy chain constant region is selected from an IgG4 isotype, e.g., a human IgG4 isotype. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0089] The isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as into a Fab light chain gene) by operably linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (Kabat et al., 1992), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0090] To generate scFv genes, the VH- and VL-encoding DNA fragments are operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions connected by the flexible linker (Bird et al., 1988; Huston et al., 1988; McCafferty et al., 1990).

[0091] Monoclonal antibody-producing transfectomas (transfectomas) Preparation of Antibodies of the present disclosure can be produced, for example, in host cell transfectomas using a combination of recombinant DNA technology and gene transfection techniques that are well known in the art (Morrison, 1985).

[0092] For example, to express an antibody, or antibody fragment thereof, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology or biochemistry techniques (e.g., DNA chemical synthesis, PCR amplification, or cDNA cloning using a hybridoma expressing the antibody of interest), and the DNA can be inserted into an expression vector such that the gene is operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or, more typically, both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt-end ligation if no restriction sites are present). Using the light and heavy chain variable regions of the antibodies described herein, full-length antibody genes of any antibody isotype can be generated by inserting these variable regions into an expression vector already encoding the heavy and light chain constant regions of the desired isotype, such that the VH segment is operably linked to a CH segment(s) in the vector and the VL segment is operably linked to a CL segment in the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0093] In addition to the antibody chain genes, the recombinant expression vectors disclosed herein carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel's publication (Goeddel, 1990). Those of ordinary skill in the art will recognize that the design of the expression vector, including the selection of regulatory sequences, can depend on factors such as the choice of the host cell to be transformed, the level of expression of the desired protein, and the like. Regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and polyoma. Alternatively, non-viral regulatory sequences, such as the ubiquitin promoter or P-globin promoter, may be used. Furthermore, regulatory elements are composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe et al., 1988).

[0094] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the disclosure may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication), and a selectable marker gene. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on the host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0095] For expression of the light and heavy chains, expression vector(s) encoding the heavy and light chains are transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for introducing exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. It is theoretically possible to express the antibodies of the present disclosure in prokaryotic or eukaryotic host cells. Expression of antibodies in eukaryotic cells, such as mammalian host cells, yeast, or filamentous fungi, is contemplated because such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete correctly folded and immunologically active antibodies.

[0096] In one specific embodiment, a cloning or expression vector according to the present disclosure comprises one of the coding sequences for the heavy and light chains of any one of mAb1, mAb2, mAb4, and mAb5 operably linked to a suitable promoter sequence.

[0097] Mammalian host cells for expressing recombinant antibodies of the present disclosure include Chinese hamster ovary (CHO) cells, including dhfr-CHO cells (described in Urlaub and Chasin, 1980), the CHOK1 dhfr+ cell line, NSO myeloma cells, COS cells, and SP2 cells, e.g., GS CHO cell lines in conjunction with the GS Xceed™ Gene Expression System (Lonza), used with a DHFR selectable marker (described in Kaufman and Sharp, 1982). Once a recombinant expression vector encoding an antibody gene is introduced into the mammalian host cells, the antibody is produced by culturing the host cells for a period of time sufficient for expression of the antibody in the host cells and, optionally, secretion of the antibody into the culture medium in which the host cells are grown. The antibody can be recovered, for example, from the culture medium after secretion of the antibody, and purified using standard protein purification methods (Shukla et al., 2007).

[0098] In one particular embodiment, the host cells of the present disclosure are host cells transfected with an expression vector having coding sequences suitable for expression of mAb1, mAb2, mAb4, and mAb5, respectively, operably linked to a suitable promoter sequence.

[0099] For example, the present disclosure relates to a host cell comprising at least the nucleic acids of SEQ ID NOs: 8 and 10, which encode the heavy and light chains of mAb1, respectively.

[0100] The latter host cells may then be further cultured under conditions suitable for the expression and production of an antibody of the present disclosure selected from the group consisting of mAb1, mAb2, mAb4, and mAb5, respectively.

[0101] Alternatively, a cell-free expression system may be used for the production of any of mAb1, mAb2, mAb4, and mAb5. Typically, methods for cell-free expression of proteins or antibodies have been previously described (Stech et al., 2017).

[0102] Immunoconjugates In another aspect, the disclosure features an anti-BTN3A antibody disclosed herein, or a fragment thereof, conjugated to a therapeutic moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant), or a radiotoxin. Such conjugates are referred to herein as "immunoconjugates." Immunoconjugates that include one or more cytotoxins are referred to as "immunotoxins." A cytotoxin or cytotoxic drug includes any agent that is detrimental to (e.g., kills) cells. Examples include taxon, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, t. colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. Therapeutic agents include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine), ablating agents (e.g., mechlorethamine, thiotepa, chlorambucil), and the like. Also included are chloraxnbucil, melphalan, carmustine (BSNU), and lomustine (CCNU), cyclosporine, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), monomethyl auristatin E, and antimitotic drugs (e.g., vincristine and vinblastine).

[0103] Cytotoxins can be conjugated to the antibodies of the present disclosure using linker technology available in the art. Examples of linker types that have been used to conjugate cytotoxins to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers such as valine-citrulline linkers. For example, it is possible to select linkers that are susceptible to cleavage by low pH in the lysosomal compartment or by proteases, such as proteases preferentially expressed in tumor tissues, such as cathepsins (e.g., cathepsins B, C, and D).

[0104] For further discussion of types of cytotoxins, linkers and methods for conjugating therapeutic agents to antibodies, see also Panowski et al., 2013 for a review on antibody drug conjugates.

[0105] The antibodies of the present disclosure can also be conjugated to radioisotopes to create cytotoxic radiopharmaceuticals, also called radioimmunoconjugates. Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include iodine. 131 ,indium 111 ,yttrium 90 , and lutetium 177 Methods for preparing radioimmunoconjugates are well established in the art, including but not limited to:

[0106] Bispecific or multispecific molecules In another aspect, further disclosed herein are bispecific or multispecific molecules comprising the anti-BTN3A antibodies of the present disclosure. The antibodies can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or a ligand for a receptor), to create a bispecific molecule that binds to at least two different binding sites or target molecules. An antibody may, in fact, be derivatized or linked to more than one other functional molecule to create a multispecific molecule that binds to more than two different binding sites and / or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule, an antibody of the present invention can be operatively linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or other methods) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding moiety, to result in a bispecific molecule.

[0107] Thus, the present disclosure includes bispecific molecules comprising at least one first binding specificity for BTN3A, e.g., one antigen-binding portion of any one of mAb1, mAb2, mAb4, and mAb5, and a second binding specificity for a second target epitope.

[0108] Furthermore, in embodiments in which the bispecific molecule is multispecific, the molecule can further comprise a third binding specificity in addition to the first and second target epitopes.

[0109] In one embodiment, the bispecific molecules disclosed herein comprise as binding specificities at least one antibody or antibody fragment thereof, including Fab, Fab', F(ab'), Fv, unibody, or single-chain Fv. The antibody may be a light or heavy chain dimer, or any minimal fragment thereof, such as an Fv or single-chain construct as described in U.S. Patent No. 4,946,778 to Ladner et al.

[0110] Other antibodies that can be used in the bispecific molecules disclosed herein are murine, chimeric and humanized monoclonal antibodies.

[0111] Bispecific molecules of the present disclosure can be prepared by conjugating the component binding specificities using methods known in the art. For example, each binding specificity of a bispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, various coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (Karpovsky et al., 1984; Liu et al., 1985). Other methods include those described in Brennan et al., 1985; Glennie et al., 1987; Paulus, 1985.

[0112] Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2, or ligand x Fab fusion protein. Bispecific molecules of the disclosure can be single-chain molecules containing one single-chain antibody and a binding determinant, or single-chain bispecific molecules containing two binding determinants.

[0113] Binding of a bispecific molecule to its specific target can be determined, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioassays (e.g., growth inhibition and apoptosis), or Western blot assays. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

[0114] The antibodies of the present disclosure may also be used to prepare artificial T cell receptors (also known as chimeric T cell receptors or chimeric antigen receptors (CARs)). For example, the variable regions of the antibodies may be used to form Fabs or scFvs linked via a spacer to a transmembrane domain (typically that of CD8 alpha) and a signaling endodomain of a TCR (e.g., CD3 zeta), and optionally a costimulatory signaling domain (e.g., from 4-1BB or CD28), and produced on the surface of T cells. Such CARs may be used, for example, in adoptive transfer therapy to treat proliferative disorders.

[0115] Pharmaceutical Composition In another aspect, the disclosure provides compositions, e.g., pharmaceutical compositions, containing one or a combination of the antibodies disclosed herein, e.g., one antibody or antigen-binding portion thereof selected from the group consisting of mAb1, mAb2, mAb4, and mAb5, formulated together with a pharmaceutically acceptable carrier. Such compositions may include one or a combination of the above (e.g., two or more different) antibodies, or immunoconjugates or bispecific molecules.

[0116] The pharmaceutical compositions disclosed herein can also be administered in combination therapy, i.e., in combination with other agents. For example, the combination therapy can include an anti-BTN3A antibody of the present disclosure, e.g., an antibody or antigen-binding portion thereof selected from the group consisting of mAb1, mAb2, mAb4, and mAb5, in combination with at least one antiviral, anti-inflammatory, or another antiproliferative agent. Examples of therapeutic agents that can be used in combination therapy are described in greater detail in the section below on uses of the antibodies of the present disclosure.

[0117] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). In one embodiment, the carrier should be suitable for subcutaneous routes or intratumoral injection. Depending on the route of administration, the active compound, i.e., antibody, immunoconjugate, or bispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0118] Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are known to those skilled in the art (Remington and Gennaro, 1995). The formulation may further include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, etc.

[0119] The form of the pharmaceutical composition, the route of administration, the dosage and the regimen will, of course, depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, etc.

[0120] The pharmaceutical compositions of the present disclosure may be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.

[0121] Preferably, the pharmaceutical compositions contain a pharmaceutically acceptable vehicle for injectable formulations, and these compositions may in particular be isotonic, sterile, saline (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts) or a dried, in particular lyophilized, composition that, optionally, upon addition of sterile water or saline, allows the constitution of an injectable solution.

[0122] The dose used for administration can be adapted as a function of various parameters, in particular as a function of the mode of administration used, of the pathology involved, or alternatively of the desired duration of treatment.

[0123] To prepare pharmaceutical compositions, an effective amount of the antibody may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0124] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders or lyophilisates for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid enough that easy syringability exists. The form must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0125] Solutions of the active compounds as free bases or pharmaceutically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils.Under ordinary conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

[0126] The antibodies of the present disclosure can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, as well as organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0127] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use in the composition of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0128] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in a suitable solvent, along with various other ingredients as listed above, as needed, followed by sterile filtration. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying techniques, which yield powders of the active ingredient plus any additional desired ingredients from its previously sterile-filtered solution.

[0129] The preparation of more or highly concentrated solutions for direct injection is also being considered, in which case the use of DMSO as a solvent is envisaged to provide extremely rapid penetration and delivery of high concentrations of the active ingredient to small tumor areas.

[0130] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, although drug release capsules and the like can also be used.

[0131] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous vehicles that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dose could be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected at the proposed infusion site (see, e.g., Remington's Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0132] The antibodies of the disclosure may be formulated into a therapeutic mixture to contain about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0, or even 1.0 to about 10 milligrams per dose. Multiple doses can also be administered.

[0133] Suitable formulations for infusion or subcutaneous injection solutions of antibodies have been described in the art and are reviewed, for example, in Cui et al. (Drug Dev Ind Pharm 2017, 43(4):519-530).

[0134] In addition to compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solid forms for oral administration; sustained release capsules; and any other form currently in use.

[0135] In certain embodiments, the use of liposomes and / or nanoparticles for the introduction of antibodies into host cells is contemplated. The formation and use of liposomes and / or nanoparticles is known to those of skill in the art.

[0136] Nanocapsules can generally encapsulate compounds in a stable and reproducible manner. To avoid side effects caused by intracellular polymer overload, such ultrafine particles (size of about 0.1 μm) are generally designed using polymers that can be degraded in vivo. For use in the present disclosure, biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are considered, and such particles can be easily produced.

[0137] Liposomes are formed from phospholipids that disperse in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)). MLVs generally have diameters between 25 nm and 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs), which have diameters in the range of 200 to 500 Å and contain aqueous solutions at their cores. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0138] Uses and Methods of the Antibodies of the Disclosure The antibodies of the disclosure have in vitro and in vivo diagnostic and therapeutic utilities. For example, these molecules can be administered, e.g., to cells in culture in vitro, ex vivo or in vivo, or in a subject, e.g., in vivo, to treat, prevent, or diagnose various disorders.

[0139] As used herein, the terms "treat," "treating," or "treatment" refer to one or more of: (1) inhibiting a disease; e.g., inhibiting a disease, condition, or disorder (i.e., halting further progression of the pathology and / or symptomology) in an individual experiencing or exhibiting the pathology or symptomology of the disease, condition, or disorder; and (2) ameliorating a disease; e.g., ameliorating a disease, condition, or disorder (i.e., reversing the pathology and / or symptomology) in an individual experiencing or exhibiting the pathology or symptomology of the disease, condition, or disorder, such as reducing the severity of the disease or reducing or alleviating one or more symptoms of the disease. In particular, with respect to the treatment of tumors, the term "treatment" can also refer to inhibiting tumor growth or reducing the size of a tumor.

[0140] The antibodies of the disclosure are anti-BTN3A activating antibodies and can activate the cytolytic function, cytokine production and / or proliferation of Vy9 V52 T cells, and thereby may be used to overcome immunosuppressive mechanisms observed in cancer patients and during chronic infections.

[0141] As used herein, the terms "cancer," "hyperproliferative," and "neoplastic" refer to cells capable of autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell proliferation. Hyperproliferative and neoplastic disease states may be classified as pathological, i.e., characterizing or constituting a disease state, or as non-pathological, i.e., deviations from normal but not associated with a disease state. The terms are intended to include all types of carcinomas or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathologic type or stage of invasiveness.

[0142] The term "cancer" or "neoplasm" includes malignant tumors of various organ systems, such as those affecting the lung, breast, thyroid, lymphoid, gastrointestinal, and genitourinary tract, as well as adenocarcinomas, including most colon cancers, renal cell carcinoma, prostate cancer, and / or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine, and cancer of the esophagus.

[0143] Examples of cancer include, but are not limited to, hematological malignancies such as myeloid lineage neoplasms, including B-cell lymphoid neoplasms, T-cell lymphoid neoplasms, non-Hodgkin's lymphoma (NHL), B-NHL, T-NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), NK-cell lymphoid neoplasms, and acute myeloid leukemia.

[0144] Examples of non-hematological cancers include, but are not limited to, colon cancer, breast cancer, lung cancer, brain cancer, prostate cancer, head and neck cancer, pancreatic cancer, bladder cancer, colorectal cancer, bone cancer, cervical cancer, ovarian cancer, liver cancer, oral cancer, esophageal cancer, thyroid cancer, kidney cancer, stomach cancer, ovarian cancer, and skin cancer.

[0145] Examples of chronic infections include, but are not limited to, viral, bacterial, parasitic or fungal infections such as chronic hepatitis, lung infections, lower respiratory tract infections, bronchitis, influenza, pneumoniae and sexually transmitted diseases.

[0146] Examples of viral infections include, but are not limited to, hepatitis (HAV, HBV, HCV), herpes simplex (HSV), shingles, HPV, influenza (Flu), AIDS and AIDS-related syndromes, chickenpox (varicella), the common cold, cytomegalovirus (CMV) infection, smallpox (variola), Colorado tick fever, dengue fever, Ebola hemorrhagic fever, foot and mouth disease, Lassa fever, measles, Marburg hemorrhagic fever, infectious mononucleosis, mumps, norovirus, poliomyelitis, progressive multifocal leukoencephalopathy (PML), rabies, rubella, SARS, viral encephalitis, viral gastroenteritis, viral meningitis, viral pneumonia, West Nile disease, and yellow fever. Examples of bacterial infections include, but are not limited to, pneumonia, bacterial meningitis, cholera, diphtheria, tuberculosis, anthrax, botulism, brucellosis, campylobacteriosis, typhoid, gonorrhea, listeriosis, Lyme disease, rheumatic fever, whooping cough (pertussis), plague, salmonellosis, scarlet fever, shigellosis, syphilis, tetanus, trachoma, tularemia, typhoid fever, and urinary tract infections. Examples also include bacterial infections caused by Coxiella burnetii, Brucella abortus, Tropheryma whipplei, Mycobacterium tuberculosis, and Mycobacterium canettii.

[0147] Examples of parasitic infections include, but are not limited to, malaria, leishmaniasis, trypanosomiasis, Chagas' disease, cryptosporidiosis, fascioliasis, filariasis, amebiasis, giardiasis, pinworm infection, schistosomiasis, taeniasis, toxoplasmosis, trichinosis, and trypanosomiasis. Examples of fungal infections include, but are not limited to, candidiasis, aspergillosis, coccidioidomycosis, cryptococcosis, histoplasmosis, and tinea pedis.

[0148] Accordingly, the present disclosure relates to a method for treating one of the above-disclosed disorders in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an anti-BTN3A antibody disclosed above, typically one of mAb1, mAb2, mAb4, or mAb5.

[0149] In certain embodiments, the subject is selected among patients with a BTN3A-expressing tumor.

[0150] The antibodies for the uses disclosed above may be administered as the sole active ingredient or in conjunction with, e.g., as an adjuvant or in combination with, other drugs, such as cytokines, antiviral, anti-inflammatory drugs or cytotoxic, antiproliferative, chemotherapeutic or anti-tumor drugs, cell therapy products (e.g., γδ T cell compositions), for example, for the treatment or prevention of the above-mentioned diseases.

[0151] For example, the antibodies for use disclosed above may be used in combination with cell therapy, in particular γδ T cell therapy, chemotherapy, antineoplastic agents, or immunotherapeutic agents.

[0152] As used herein, "cell therapy" refers to a therapy comprising the in vivo administration of at least a therapeutically effective amount of a cell composition to a subject in need thereof. The cells administered to a patient may be allogeneic or autologous. The term "γδ T cell therapy" refers to a cell therapy in which the cell composition comprises γδ T cells, particularly Vγ9 / Vδ2 T cells, as an active ingredient.

[0153] A cell therapy product is a cell composition administered to the patient for therapeutic purposes, comprising a therapeutically effective dose of cells and, optionally, additional excipients, adjuvants, or other pharmaceutically acceptable carriers.

[0154] Suitable antineoplastic agents include, but are not limited to, alkylating agents (e.g., cyclophosphamide, mechlorethamine, chlorambucil, melphalan, nitrosoureas, temozolomide), anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin), taxanes (e.g., paclitaxel, docetaxel), epothilones, inhibitors of topoisorase I (e.g., irinotecan or topotecan), inhibitors of topoisorase II (e.g., etoposide, teniposide, or tafluposide), nucleotide analogs and precursor analogs (e.g., azacitidine, azathioprine, capecitabine, cytarabine, fluorouracil, anti-cancer drugs may include cyclosporine, cyclosporine, cyclosporine-3, cyclosporine-4, cyclosporine-5, cyclosporine-6, cyclosporine-7, cyclosporine-8, cyclosporine-9, cyclosporine-10, cyclosporine-11, cyclosporine-12, cyclosporine-13, cyclosporine-14, cyclosporine-15, cyclosporine-16, cyclosporine-17, cyclosporine-18, cyclosporine-19, cyclosporine-20, cyclosporine-21), cyclosporine-3, cyclosporine-4, cyclosporine-5, cyclosporine-19, cyclosporine-22), cyclosporine-4, cyclosporine-5, cyclosporine-19, cyclosporine-23), cyclosporine-4, cyclosporine-5, cyclosporine-14, cyclosporine-15, cyclosporine-16, cyclosporine-17, cyclosporine-18, cyclosporine-19, cyclosporine-23, cyclosporine-19, cyclosporine-24), cyclosporine-4, cyclosporine-5, cyclosporine-19, cyclosporine-25, cyclosporine-26), cyclosporine-4, cyclosporine-5, cyclosporine-19, cyclosporine-27), cyclosporine-4, cyclosporine-19, cyclosporine-23, cyclosporine-19, cyclosporine-24, cyclosporine-25, cyclosporine-26, cyclosporine-27, cyclosporine-2

[0155] Examples of immunotherapeutic agents include, but are not limited to, phosphoantigens (e.g., zoledronic acid or other bisphosphonates), anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-BTLA antibodies, anti-CTLA-4 antibodies, and cytokines (e.g., interleukin-2 (IL-2) (Choudhry H et al., 2018, Biomed Res Int. 2018 May 6), interleukin-15 (IL-15) (Patidar M et al., Cytokine Growth Factor Rev. 2016 October;31:49-59), interleukin-21 (IL-21) (Caccamo N. et al., PLoS One. 2012;7(7):e41940), or interleukin-33 (IL-33) (Duault C et al., J Immunol. 2016 Jan. 1;196(1):493-502), or recombinant forms and derivatives thereof, or any cytokine capable of inducing lymphocyte activity (e.g., proliferation or cytokine production or metabolic changes). The term derivative is used for any cytokine modification that can rely on pegylation (e.g., conjugation to polyethylene glycol (PEG) chains), mutations such as amino acid deletions, substitutions, or insertions, or association with enhancers (e.g., an IL15 / IL15Ra complex fused to IgG1 Fc, where IL-15 is further mutated (asn72asp) to further increase the biological activity of this complex, making it an IL-2 and IL-15Rβγ superagonist (Rhode PR et al., Cancer Immunol Res. 2016;4(1):49-60)) (Barroso-Sousa R et al., Curr Oncol Rep. 2018 Nov. 15;21(1):1).

[0156] The term "IL-2" has its general meaning, which is human interleukin-2. IL-2 is part of the body's natural immune response. IL-2 primarily regulates lymphocyte activity by binding to the IL-2 receptor.

[0157] The term "IL-15" has its general meaning and refers to human interleukin-15. Like IL-2, IL-15 binds to and signals through a complex composed of the IL-2 / IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 regulates the activation and proliferation of T and natural killer (NK) cells.

[0158] The term "IL-21" has its general meaning and refers to human interleukin-21. IL-21 has been reported to have pleiotropic properties, including, but not limited to, enhancing NK cell and CD8+ T cell cytotoxicity, regulating plasma cell differentiation, and inhibiting Treg cells.

[0159] The term "IL-33" has its general meaning and refers to human interleukin-33. IL-33 is considered an alarmin that is released upon tissue stress or injury, is a member of the IL-1 family, and binds to the ST2 receptor. IL-33 binds to T H It is known to be an effective stimulator of immune cells, natural killer (NK) cells, iNKT cells, and CD8 T lymphocytes.

[0160] The term "PD-1" has its common meaning in the art and refers to the programmed death-1 receptor. The term "PD-1" also refers to a type I transmembrane protein that belongs to the CD28-B7 signaling family of receptors that includes CD28, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), inducible costimulatory molecule (ICOS), and B- and T-lymphocyte attenuator (BTLA) (Greenwald RJ et al., 2005; Riley JL et al., 2005).

[0161] The term "BTLA" has its common meaning in the art and refers to B and T lymphocyte attenuator. The term "BTLA" also refers to CD272, which is a member of the CD28-B7 signaling family of receptors that includes CD28, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), inducible costimulatory molecule (ICOS), and programmed death-1 receptor (PD-1) (Greenwald RJ et al., 2005; Riley JL et al., 2005).

[0162] The terms "anti-PD-1 antibody" or "anti-PD-L1" have their common meaning in the art and refer to an antibody that has binding affinity for PD-1 or PD-L1, respectively, and antagonist activity to PD-1, i.e., the antibody inhibits the signaling cascade involving PD-1 and blocks PD-1 ligand binding (PD-L1; PD-L2). Such anti-PD-1 or anti-PD-L1 antibodies preferentially inactivate PD-1 with greater affinity and potency, respectively, than its interaction with other subtypes or isoforms of the CD28-B7 signaling family of receptors (CD28; CTLA-4; ICOS; BTLA). Tests and assays for determining whether a compound is a PD-1 antagonist are well known to those skilled in the art and are described, for example, in Greenwald et al., 2005; Riley et al., 2005.

[0163] Examples of such anti-PD-1 or anti-PD-L1 antibodies include, without limitation, nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, or atezolizumab.

[0164] Examples of such CTLA4 antibodies include, but are not limited to, ipilimumab.

[0165] The term "anti-BTLA antibody" has its general meaning in the art, that is, an antibody that has binding affinity for BTLA and antagonist activity, i.e., the antibody is capable of inhibiting a signal transduction cascade involving BTLA. Tests and assays for determining whether a compound is a BTLA antagonist are well known to those of skill in the art and are described, for example, in (Greenwald et al., 2005; Riley et al., 2005).

[0166] In some embodiments, the anti-BTLA antibody is selected from the antibodies described in WO 2010 / 106051, WO 2011 / 014438, and WO 2017 / 144668.

[0167] In some embodiments, the anti-BTLA antibody is the BTLA antibody (BTLA8.2) available from the hybridoma available under CNCM deposit number I-4123, such as disclosed in WO 2010 / 106051.

[0168] In some embodiments, the anti-BTLA antibody is the 4C7 mAb disclosed in WO 2011 / 014438.

[0169] In some embodiments, the anti-BTLA antibody is the 629.3 mAb disclosed in WO 2017 / 144668, or a humanized version or variant thereof.

[0170] In accordance with the foregoing, the present disclosure provides, in a further aspect: a method as defined above, comprising co-administering, e.g., simultaneously or sequentially, a therapeutically effective amount of an anti-BTN3A antibody of the disclosure and at least one second pharmaceutical agent, wherein said second pharmaceutical agent is an antiviral or antiproliferative drug or an immunotherapeutic agent (e.g., an anti-PD-1, anti-PD-L1 antibody), or a cytokine, e.g., IL-2 or IL-15, or a cell therapy product (e.g., γδ T cells), e.g., as indicated above.

[0171] In one embodiment, the antibodies of the present disclosure can be used to detect levels of soluble BTN3A or levels of cells expressing BTN3A. This can be accomplished, for example, by contacting a sample (e.g., an in vitro sample) and a control sample with an anti-BTN3A antibody under conditions that allow for the formation of a complex between the antibody and BTN3A (e.g., expressed on the surface of cells or soluble BTN3A in a blood sample). Any complexes formed between the antibody and BTN3A are detected and compared in the sample and the control. Standard detection methods well known in the art, such as ELISA and flow cytometry assays, can be performed using the compositions of the present disclosure.

[0172] Thus, in one aspect, the present disclosure further provides a method for detecting the presence or measuring the amount of BTN3A (e.g., human BTN3A antigen) in a sample, comprising contacting the sample and a control sample with an antibody or protein of the present disclosure, or an antigen-binding region thereof, that specifically binds to BTN3A under conditions that allow for the formation of a complex between the antibody or portion thereof and BTN3A. Complex formation is then detected, and a difference in complex formation between the sample compared to the control sample indicates the presence of BTN3A in the sample.

[0173] Also within the scope of this disclosure are kits consisting of the compositions disclosed herein (e.g., humanized antibodies, conjugated antibodies, and multispecific molecules) and instructions for use. The kits can further contain at least one additional reagent, or one or more additional antibodies or proteins. The kits typically include a label indicating the intended use of the contents of the kit. The term label includes any written or recorded material provided on or with the kit, or which otherwise accompanies the kit. The kit may further include a tool for diagnosing whether a patient belongs to the group that will respond to anti-BTN3A antibody treatment, as defined above.

[0174] Another therapeutic strategy is based on the use of the humanized antibodies disclosed herein as agents to selectively expand and / or activate Vy9 V52 T cells isolated from a sample from a human subject.

[0175] Accordingly, the present disclosure provides a method for administering treatment to a subject in need thereof, comprising: (a) isolating blood cells, e.g., PBMCs, from a blood sample of a subject, comprising Vy9 V52 T cells; (b) expanding Vy9 V52 T cells in vitro in the presence of any one of mAbs 1, 2, 4 and 5; (c) harvesting the expanded Vy9 V52 T cells; (d) optionally formulating the expanded Vy9 V52 T cells and administering a therapeutically effective amount of said Vy9 V52 T cells to the subject; The present invention relates to a method comprising:

[0176] The present disclosure further relates to the use of a humanized antibody (e.g., mAb1, mAb2, mAb4, or mAb5) disclosed herein as an agent to selectively expand chimeric antigen receptor (CAR) Vy9 V52 T cells. CAR γδ T cells and their use in adoptive T cell cancer immunotherapy are described, for example, in Mirzaei et al. (Cancer Lett 2016, 380(2):413-423).

[0177] The present disclosure also relates to anti-BTN3A antibodies for in vivo use as enhancers of tumor cells in γδ T cell therapy, typically in a subject suffering from cancer, in need thereof.

[0178] As used herein, the term γδ T cell therapy refers to a therapy comprising administering at least an effective amount of γδ T cells to a subject in need thereof. Such γδ T cells may be allogeneic or autologous. In certain embodiments, γδ T cells can be genetically engineered by deletion or knockout or insertion or knockin of specific genes. In certain embodiments, the γδ T cells include γδ T cells expressing a chimeric antigen receptor. γδ T cells may be ex vivo expanded and / or purified. Alternatively, γδ T cells may be included in a cell composition containing other blood cells, e.g., other cells of the immune system. For references regarding γδ T cell therapy, see Pauza CD. et al., Front Immunol. 2018 Jun. 8;9:1305. doi:10.3389; Saudemont A. et al., Front Immunol. 2018 Feb. 5;9:153. doi:10.3389.

[0179] Indeed, without being bound by any particular theory, the proposed mechanism of action of the anti-BTN3A antibodies of the present disclosure is that upon binding of the anti-BTN3A antibodies to BTN3A expressed on the surface of tumor cells, it triggers a conformational change that allows its signaling to counterreceptors on Vy9 V52 T cells.

[0180] Accordingly, the present disclosure provides a method of treating a subject suffering from cancer, e.g., a hematological malignancy, particularly a leukemia such as acute myeloid leukemia, and having tumor cells, e.g., hematological tumor cells, comprising: (i) administering to the subject an effective amount of an anti-BTN3A antibody disclosed herein, typically mAb1, mAb2, mAb4, or mAb5; (ii) administering in said subject an effective amount of a γδ T cell composition; wherein the effective amount of anti-BTN3A antibody is capable of enhancing anti-tumor cytolysis mediated by the γδ T cell composition against the tumor cells.

[0181] The present disclosure provides a method for treating a subject suffering from cancer with solid tumor cells, e.g., ovarian cancer cells, comprising: (i) administering to the subject an effective amount of an anti-BTN3A antibody disclosed herein, typically mAb1, mAb2, mAb4, or mAb5; (ii) administering in said subject an effective amount of a γδ T cell composition; wherein the effective amount of anti-BTN3A antibody is capable of enhancing anti-tumor cytolysis mediated by the γδ T cell composition against the tumor cells.

[0182] The present disclosure also relates to methods for administering treatment to a subject in need thereof, comprising the combined (simultaneous or sequential) administration of CAR T cells, e.g., CAR γδ T cells, and a humanized antibody disclosed herein (e.g., mAb1, mAb2, mAb4, or mAb5).

[0183] The invention having now been fully described, will now be further illustrated by the following examples, which are merely illustrative and are not intended to be limiting. [Example]

[0184] Selection of humanized variants 1. Description of the humanization strategy a. Design of composite human antibody™ variable region sequences Structural models of the murine 7.2 and 20.1 antibody V regions were generated using the Swiss PDB and analyzed to identify key "constraining" amino acids in the V regions that may be essential for the binding properties of the antibodies. Most residues contained within the CDRs (using both the Kabat and Chothia definitions) were considered important, along with several framework residues. From the above analysis, composite human sequences of the 7.2 and 20.1 antibodies were generated.

[0185] b. CD4+ T cell epitope avoidance Based on the structural analysis, a large preliminary set of sequence segments that could be used to create 7.2 and 20.1 humanized variants was selected and analyzed using iTope™ technology for in silico analysis of peptides binding to human MHC class II alleles (Perry et al., 2008) and TCED™ for known antibody sequence-associated T cell epitopes (Bryson et al., 2010). Sequence segments identified as significant non-human germline binders to human MHC class II or showing significant hit scores against TCED™ were discarded. This reduced the set of segments, and combinations of these segments were reanalyzed as described above to ensure that intersegment junctions did not contain potential T cell epitopes. The selected sequence segments were assembled into complete V-region sequences predicted to lack significant T cell epitopes. Next, several heavy and light chain sequences were selected for gene synthesis and expression in mammalian cells for mAbs 7.2 and 20.1.

[0186] 2. Generation and preliminary characterization of humanized variants a. Construction of humanized variant plasmids The 7.2 and 20.1 humanized variants were synthesized with flanking restriction enzyme sites for cloning into expression vector systems for human IgG4 (S241P, L248E) heavy and kappa light chains. All constructs were verified by sequencing.

[0187] b. Antibody expression Chimeric 7.2 and 20.1 (VH0 / Vκ0), two control combinations (VH0 / Vκ1, VH1 / Vκ0), and humanized heavy and light chain combinations were transiently transfected into FreeStyle™ CHO-S cells (ThermoFisher, Loughborough, UK) from the corresponding endotoxin-free DNA using the MaxCyte STX® electroporation system (MaxCyte Inc., Gaithersburg, USA). Transfections were undertaken for each antibody using OC-400 processing assemblies. Following cell harvesting, cells were cultured at 3 x 10 in CD Opti-CHO medium (ThermoFisher, Loughborough, UK) containing 8 mM L-glutamine (ThermoFisher, Loughborough, UK) and 1 x hypoxanthine-thymidine (ThermoFisher, Loughborough, UK). 6 The cells were diluted to 1000 cells / mL. 24 hours after transfection, the culture temperature was reduced to 32°C, and 1 mM sodium butyrate (Sigma, Dorset, UK) was added. Cultures were fed daily with the addition of 3.6% (of the starting volume) of feed (2.5% CHO CD Efficient Feed A (ThermoFisher, Loughborough, UK), 0.5% Yeastolate (BD Biosciences, Oxford, UK), 0.25 mM Glutamax (ThermoFisher, Loughborough, UK), and 2 g / L glucose (Sigma, Dorset, UK)). IgG supernatant titers were monitored by IgG ELISA, and transfectants were cultured for up to 14 days before harvesting the supernatant.

[0188] c. Preliminary affinity measurements: Single-cycle kinetic analysis of humanized variants binding to BTN3A To evaluate the binding of all 7.2 and 20.1 composite Human Antibody™ variants and select the antibody with the highest affinity for BTN3A, single-cycle kinetic analysis was performed on supernatants from transfected cell cultures using a Biacore T200 (serial number 1909913) running Biacore T200 Evaluation Software V2.0.1 (Uppsala, Sweden).

[0189] The antibody was diluted to a final concentration of 2 μg / ml in 2% BSA / PBS based on the concentration obtained from the supernatant titered by ELISA. At the start of each cycle, the antibody was loaded onto Fc2, Fc3, and Fc4 of a Protein A chip (GE Healthcare, Little Chalfont, UK). IgG was captured at a flow rate of 10 μl / min, giving an immobilization level (RL) of approximately 146.5 RU, the theoretical value for an RMax of approximately 50 RU. The surface was then allowed to stabilize. Single-cycle kinetic data were obtained using BTN3A1-His (Sino Biological catalog no. 15973-H08H) as the analyte and HBS-P+ (GE Healthcare, Little Chalfont, UK) as the running buffer at a flow rate of 60 μl / min to minimize any potential mass transfer effects. Multiple replicates using chimeric antibodies were performed to examine the stability of the surface and analyte in the kinetic analysis. The signal from the reference channel, Fc1 (no antibody), was subtracted from the signals from Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface. A three-point, four-fold dilution range of BTN3A1 from 1.56 nM to 25 nM was used, with no regeneration between each concentration. The association phase for three injections of increasing concentrations of BTN3A1 was monitored for 240 s each, and a single dissociation phase was monitored for 2000 s following the last injection of BTN3A1. Regeneration of the Protein A surface was performed using two injections of 10 mM glycine-HCl, pH 1.5, followed by a 240 s stabilization period.

[0190] Signals from each antibody blank (without CD277) were subtracted to correct for differences in surface stability. Single-cycle kinetics showed that all humanized variants bound to BTN3A.

[0191] d. Antibody purification Based on the Biacore, as well as the iTope™ score and percentage of humanness of each humanized variant, the 7.2 and 20.1 humanized variants with the best affinity and best iTope™ score were selected for further analysis.

[0192] The selected humanized variants, along with their chimeric versions and the most conservatively humanized variant (VH1 / Vκ1), underwent purification for further assay testing. Antibodies were purified from cell culture supernatants on a Protein A Sepharose column followed by size exclusion chromatography (SEC) (GE Healthcare, Little Chalfont, UK) using 10 mM sodium acetate, 100 mM NaCl, pH 5.5, and final formulation buffer as the mobile phase. Samples were analyzed by OD analysis using the extinction coefficient (Ec (0.1%)) based on the predicted amino acid sequence. 280nm Quantification was performed by.

[0193] The antibodies were analyzed using SDS-PAGE by loading 2 μg of each antibody onto the gel, and bands corresponding to the typical antibody profile were observed.

[0194] e. Validation of binding properties: Competitive ELISA analysis between humanized and chimeric 7.2 and 20.1 mAbs The purified variants were tested for their binding to recombinant BTN3A1-His (Sino Biological catalog number 15973-H08H) in competition with the corresponding mouse antibody. Chimeric (VH0 / Vκ0) and irrelevant human IgG4 (S241P, L248E) were tested on each plate for comparison.

[0195] BTN3A1 was diluted to 0.5 μg / ml in 1x PBS, and 100 μl / well was coated onto a 96-well ELISA plate at 4°C. The following day, plates were washed three times with 1x PBS / 0.05% Tween (PBS-T) and blocked with 200 μl of 2% milk / PBS for 1 hour at room temperature. For the diluted 96-well plates, a fixed concentration of 7.2 or 20.1 (0.15 μg / ml final concentration) was added in equal volumes to a 4-fold titration series of test antibodies, starting with 80 μg / ml diluted in blocking buffer (40 μg / ml final concentration). After washing the Nunc ELISA plate three times with PBS-T, 100 μl of the mouse / test antibody mixture was added to the ELISA plate. After a 1-hour incubation at room temperature, plates were washed three times with PBS-T, and bound mouse antibodies were detected by applying 100 μl of anti-mouse Fc HRP-conjugated secondary antibody (Sigma, Dorset, UK) diluted 1:1000 in blocking buffer for 1 hour at room temperature. For color development, plates were washed three times with PBS-T, followed by the addition of 100 μl of TMB substrate and incubation for 5 minutes at room temperature. The reaction was stopped with 100 μl of 3.0 M hydrochloric acid, and absorbance was immediately read at 450 nm using a Dynex plate reader.

[0196] I C 50 The values ​​were calculated for each variant and the relative IC 50 Values ​​are IC of humanized variants 50 IC of chimeric antibodies assayed on the same plate 50 It was calculated by dividing by

[0197] 3. Selection of humanization candidates a. Multi-cycle kinetic analysis Based on the data generated from the competitive ELISA and thermostability assessment, multi-cycle kinetic analysis was performed on most of the humanized 7.2 and 20.1 variants along with the VH0 / Vκ0 chimeric antibody using a Biacore T200 (serial number 1909913) instrument running Biacore T200 evaluation software V2.0.1 (Uppsala, Sweden).

[0198] Purified antibodies were diluted to a concentration of 2 μg / ml in 2% BSA / PBS. At the start of each cycle, each antibody was captured on Protein A at a density (RL) of approximately 146.5 RU (theoretical value for an RMax of approximately 50 RU). Following capture, the surface was allowed to stabilize before injection of BTN3A1 antigen (Sino Biological catalog number 15973-H08H). BTN3A1 was titrated over a two-fold dilution range from 25 to 0.78 nM in 0.1% BSA / HBS-P+ (running buffer). The association phase was monitored for approximately 400 s, and the dissociation phase for 35 min (2100 s). Kinetic data were obtained using a flow rate of 50 μl / min to minimize any potential mass transfer effects. Regeneration of the Protein A surface was performed using two injections of 10 mM glycine-HCl pH 1.5 at the end of each cycle. Two blank (no BTN3A) and single-concentration analyte replicates were performed for each antibody tested to check the stability of the surface and analyte during kinetic cycling. The signal from the reference channel, Fc1, was subtracted from the signals for Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface. Additionally, a blank was subtracted for each Fc to correct for any antigen-independent signal fluctuations, such as drift. Sensorgrams were fitted using a one-to-one binding mathematical model with a global RMax parameter and no bulk signal (steady-state RI = 0 RU).

[0199] b. Flow cytometric binding assay on human PBMCs The 7.2 and 20.1 humanized variants were characterized for their binding to human PBMCs isolated from the blood of healthy donors. PBMCs were isolated from buffy coats using Lymphoprep (Axis-shield, Dundee, UK) density centrifugation. PBMCs were then frozen and stored at -80°C or in liquid nitrogen until needed.

[0200] 1×10 6100 μl of cells / ml were transferred to each well of a new U-bottom 96-well plate, and the plate was then centrifuged and the supernatant discarded.

[0201] Serial dilutions of antibody, 0.001 μg / ml to 150 μg / ml, were prepared in PBS 2 mM EDTA. Human PBMCs were resuspended in 50 μl of the prepared diluted test antibody titration series.

[0202] After 30 min of incubation at 4°C in the dark, the plates were centrifuged and washed twice with 150 μl / well of PBS 2 mM EDTA, after which the wells were resuspended in 50 μl of a mixture consisting of goat anti-human antibody (PE-labeled) diluted 1 / 100 in PBS 2 mM EDTA and Live / dead neat IR diluted 1 / 500.

[0203] After 15 minutes of incubation at 4°C in the dark, the plates were centrifuged and washed once with 150 μl / well of PBS 2 mM EDTA. The wells were then resuspended in 200 μl of PBS 2 mM EDTA. Cells were analyzed on a BD LSR Fortessa hemocytometer. Data were analyzed using FlowJo software (version 10, FlowJo, LLC, Ashland, USA) (data not shown).

[0204] The same protocol was followed for cynomolgus monkey PBMCs and Daudi-Burkitt lymphoma cell lines.

[0205] c. In vitro functional efficacy: γδ-T cell degranulation assay The assay consisted of measuring the activating or inhibitory effects of 7.2 and 20.1 humanized variants and their chimeric versions on γδ T cell degranulation in the Daudi Burkitt lymphoma cell line (Harly et al., 2012). γδ T cells were expanded from PBMCs of healthy donors by culturing for 11–13 days with zoledronic acid (1 μM) and IL2 (200 Ui / ml). IL2 was added on days 5, 8, and every two days thereafter. The percentage of γδ T cells was determined at the initiation of culture and assessed by flow cytometry throughout the culture period until it reached at least 80%. Frozen or fresh γδ T cells were then used in the degranulation assay against the Daudi cell line (1:1 E:T ratio). The cells were co-cultured for 4 hours at 37°C in the presence of 10 μg / ml of 7.2 and 20.1 humanized variants and their chimeric versions. Activation with PMA (20 ng / ml) plus ionomycin (1 μg / ml) served as a positive control for γδ T cell degranulation, and medium alone served as a negative control. At the end of the 4-hour co-incubation, cells were analyzed by flow cytometry to assess the percentage of γδ T cells positive for CD107a (LAMP-1, lysosomal membrane protein-1) and CD107b (LAMP-2). Following activation-induced granule exocytosis, CD107 aggregates on the cell surface; therefore, measurement of surface CD107 is a sensitive marker for identifying recently degranulated cytolytic T cells. Results did not show any significant variation among the candidates tested, indicating similar activation effects with the chimeric 7.2 or 20.1 antibodies in the degranulation assay.

[0206] The same protocol was performed using AML blasts isolated from patients as target cells instead of Daudi cells.

[0207] d. Thermal stability analysis To assess the thermal stability of selected 7.2 and 20.1 composite Human Antibody™ variants, the melting temperatures (temperature at which 50% of the protein domains are unfolded) were determined using a fluorescence-based thermal shift assay.

[0208] All purified humanized and chimeric (VH0 / Vκ0) antibodies were diluted to a final concentration of 0.1 mg / ml in formulation buffer (10 mM sodium acetate, 100 mM NaCl, pH 5.5) containing SYPRO® Orange (ThermoFisher, Loughborough, UK) at a 1 in 1000 dilution and subjected to a temperature gradient from 25°C to 99°C over a 56-minute period on a StepOnePlus Real-Time PCR System (ThermoFisher, Loughborough, UK). 10 mM sodium acetate, 100 mM NaCl, pH 5.5 was used as a negative control. Melting curves were analyzed using Protein Thermal Stability Software (version 1.2).

[0209] e. Selection of humanization candidates Based on all the results obtained for the above experiments, three variants were selected from 35 (15 humanized variants generated for 7.2; 20 humanized variants generated for 20.1) for further characterization: 7.2 (VH2 / Vκ1), 7.2 (VH2 / Vκ2), and 20.1 (VH3 / Vκ1).

[0210] The results of the different experiments described above for mAb 7.2 and 20.1 are reported in Tables 4 and 5 for the three variants and their chimeric versions. [Table 4] [Table 5] The humanization process generates multiple 7.2 and 20.1 variants that are predicted to have reduced immunogenicity.

[0211] A selected set of three variants (7.2 VH2 / Vκ1, 7.2 VH2 / Vκ2, and 20.1 VH3 / Vκ1) showed equivalent properties to their chimeric versions in terms of affinity, binding, and potency in functional assays. Modifications made to the variant sequences to reduce immunogenicity did not alter antibody function.

[0212] Surprisingly, the thermal stability of the selected humanized variants 7.2 VH2 / Vκ1, 7.2 VH2 / Vκ2 was improved compared to the chimeric antibody, and such improved thermal stability was unexpected in this humanization process.

[0213] Antibody constant regions: A comparison of silent Fc fragments Several Fc portions were tested to silence or reduce antibody effector functions. The binding of these Fc fragments to different Fcγ receptors was assessed using Biacore, and their binding to the C1q complex was assessed by ELISA assay.

[0214] 1. Binding of Engineered Fc Moieties to Different Fcγ Receptors Using Biacore The binding ability of different isotypes of chimeric antibody 20.1 (IgG1, IgG1 [N314A], IgG1 [L247F, L248E P350S], IgG2, IgG4 [S241P], and IgG4 [S241P L248E]) to different Fcγ receptors was determined using purified antibodies and single-cycle Biacore analysis. Human Fc receptors, FcγRI, FcγRIIA (Arg167 polymorphism) and IIB, and FcγRIIIA (Phe176 polymorphism) and IIIB, were obtained from Sino Biological.

[0215] Fcγ receptors were diluted in HBS-P+ (GE Healthcare, Little Chalfont, UK) to a final concentration of 0.5 or 1.0 μg / ml. At the start of each cycle, Fcγ receptors were loaded onto Fc2, Fc3, and Fc4 (GE Healthcare, Little Chalfont, UK) of an anti-His CM5 chip. Fcγ receptors were captured at a flow rate of 5 μl / min to obtain immobilization levels of 30–180 RU, depending on the molecular weight of the Fcγ receptor. The surface was then allowed to stabilize. Single-cycle kinetic data were obtained using a chimeric antibody as the analyte at a flow rate of 30 μl / min to minimize any potential mass transfer effects. The signal from the reference channel, Fc1 (no antibody), was subtracted from the signals for Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface. A five-point, 3-fold dilution range was used for each chimeric antibody; this concentration range varied for each individual Fcγ receptor due to expected differences in affinity. The signal from each blank (no antibody) was subtracted to correct for differences in surface stability. The association phase for each of five injections of increasing concentrations of chimeric antibody was monitored for 25–180 s (depending on the Fcγ receptor ligand), and a single dissociation phase was measured for 25–300 s following the last injection of antibody. Regeneration of the anti-His surface was performed using two injections of 10 mM glycine-HCl pH 1.5 at 30 μl / min for 15 s each, followed by a 180-s stabilization period.

[0216] Single-cycle kinetic constants were determined using a standard one-to-one analytical model when possible. For strong interactions, determining affinity via kinetic experiments was generally more appropriate. However, for some Fcγ receptors, the interaction was very weak; in this scenario, data were analyzed using steady-state affinity analysis, which is particularly suitable for measuring weak to moderate interactions. Sensorgrams for the interaction of Fcγ receptors with chimeric antibodies were obtained (data not shown).

[0217] As expected, the high-affinity FcγRI receptor bound unmodified IgG1 and IgG4(S241P) with good affinity. The modified IgG1 isotype, along with IgG2 and IgG4(S241P, L248E), failed to bind FcγRI. The remaining Fcγ receptors exhibited much lower affinity interactions with the different chimeric antibodies compared to FcγRI. As expected, unmodified IgG1 exhibited the strongest binding to all four of the lower affinity receptors, and the modified versions of IgG1 exhibited significantly reduced binding to these receptors. IgG2 and IgG4(S241P) exhibited some binding to FcγRIIA and B but only weak binding to FcγRIIIA and B.

[0218] 2. Binding of the Engineered Fc Moiety to the C1q Complex by ELISA Assay Chimeric antibody 20.1 was tested as different IgG isotypes for binding to the C1q complex to determine its ability to activate the complement system.

[0219] In a U-bottom 96-well plate, a 2.5-fold dilution series (10 μg / ml to 0.04 μg / ml) of purified chimera 20.1 of different isotypes was prepared in 2% BSA / DPBS. Nunc Immuno MaxiSorp 96-well flat-bottom microtiter plates (ThermoFisher Scientific, Loughborough, UK) were precoated with this titration series at 100 μl / well and incubated overnight at 4°C. The following day, the plates were washed twice with PBST, blocked with 2% BSA / DPBS for 1 hour at room temperature, and then washed five times with PBST. Purified complement protein C1q (Pathway Diagnostics Ltd, Dorking, UK), diluted to 5 μg / ml in 2% BSA / PBS, was added to the plate (100 μl / well) and incubated for 1 hour at room temperature. After washing five times with PBST, binding of C1q complexes was detected with anti-C1q-HRP (Abcam, Cambridge, UK) (100 μl / well, diluted 1 in 100 in 2% BSA / DPBS) for 1 hour at room temperature. After washing five times with PBST, binding was detected with TMB substrate (ThermoFisher Scientific, Loughborough, UK), following which the reaction was stopped with 3 M HCl, absorbance was read at 450 nm on a Dynex Technologies MRX TC II plate reader, and binding curves were plotted.

[0220] As expected, only the unmodified IgG1 isotype showed good binding to C1q, while the other isotypes showed minimal to no binding (data not shown).

[0221] 3. Selection of Engineered Fc Fragments The relative binding of all tested isotypes on the Fcγ receptor and C1q complex is given in Table 6. [Table 6] The two engineered IgG1 L247F, L248E, P350S and IgG4 S241P, L248E Fc fragments were the only fragments that showed a total loss of binding on the FcγI receptor and C1q complex.

[0222] Based on the results obtained, two engineered IgG1 L247F, L248E, P350S and IgG4 S241P, L248E isotypes were selected for further characterization.

[0223] Creation of six humanized antibodies The three selected humanized variants were cloned and fused to two selected engineered Fc fragments to generate six different candidates: mAb1 to mAb6.

[0224] The example mAb1 to mAb6 listed in Table 1 can be produced using conventional antibody recombinant production and purification processes.

[0225] For example, the coding sequence is cloned into a production vector for recombinant expression in a mammalian production cell line.

[0226] Tables 7 and 8 below provide detailed amino acid and nucleotide sequences useful for practicing the present invention, particularly for producing nucleic acids, expression vectors and humanized antibodies derived from murine 7.2 of the present disclosure. [Table 7] [Table 8] JPEG0007825605000009.jpg201149 JPEG0007825605000010.jpg170149 JPEG0007825605000011.jpg202149 JPEG0007825605000012.jpg208149 JPEG0007825605000013.jpg66149

[0227] Developability characteristics of six humanized variants 1. Cell Line Generation a. Expression vector construction The gene sequences encoding the heavy and light chains were cloned into a vector. The gene system was used to express the antibody in CHO cells. Antibody expression was under the control of the EF1 alpha promoter. The expression vector carries a unique genetic element that shields the transgene from the silencing effects of the surrounding chromatin (Girod et al., 2007). Transcription is maintained at the highest level, independent of the transgene integration site, resulting in stable, high-level protein expression.

[0228] b. Cell line development The CHO host cell line was derived from American Type Culture Collection (ATCC) CHO-K1 CCL-61 cells and adapted to grow in suspension in preformulated BalanCD Growth A culture medium (Irvine Scientific). Cells were transfected by electroporation using the Neon transfection system (Invitrogen).

[0229] c. Single-cell cloning using the ClonePix FL instrument To keep the cellular environment unchanged throughout the entire procedure, the same medium (BalanCD Growth A, Irvine Scientific) was used as the base medium for transfection, single-cell cloning, and production. Following transfection of each vector, puromycin selection pressure was applied to generate stable pools. Diluted cells were plated in semi-solid medium (CloneMedia©; Molecular Devices), and plates were incubated at 37°C and 5% CO2 in a humidified incubator. Expanded colonies were picked using a ClonePix™ FL Imager from Molecular Devices and transferred to 96-well plates, then expanded in initial 24-well and then 6-well TC plates.

[0230] d. Fed-batch performance evaluation The growth and productivity of individual clones were evaluated in 125 ml shake flasks to select the best clones based on cell growth potential and productivity in a 10-day fed-batch process using Cell Boost 7 A+7B feed (GE Healthcare, USA). 6 Starting cell concentrations were 100 cells / ml.

[0231] The results obtained for cell growth and productivity are summarized in Table 9.

[0232] 2. Sample Preparation for Manufacturability Assessment Each candidate antibody was purified by Protein A capture from a clarified CHO cell supernatant pool; two pools of each variant were required to ensure sufficient material for testing. Protein concentration was determined for all post-capture samples by UV analysis. Sample recovery and yield were greater than 89% for the majority of samples, with all variants exhibiting similar yields. Each antibody was then buffer-exchanged into 25 mM histidine, 125 mM NaCl, pH 6.0 using a 30 kDa MWCO centrifugal filter unit until the flow-through material reached the target pH for formulation. None of the variants showed signs of protein precipitation or slowed flow during the exchange step. After buffer exchange was complete, the concentration of each variant sample was adjusted to 1.0 mg / ml using formulation buffer, and 10% PS-80 was added to a final concentration of 0.02% PS-80.

[0233] 3. Thermal stability evaluation Differential scanning fluorimetry analysis was performed to assess and compare the thermal stability of the antibody variants tested. Each variant was analyzed in triplicate, and the mean T onset , T agg and T m was determined (data not shown).

[0234] The T obtained for all tested variants onset and T m No significant differences were observed between the values. m1 The value is 61°C, and T onset The determined values ​​for T were 54-55°C for all antibodies. The T determined based on the colloid stability plots agg The values ​​ranged from 71 to 78°C.

[0235] Overall, all four selected variants show comparable thermal stability, and the variations observed do not result in significant changes in thermal stability between the antibodies tested.

[0236] 4. Forced decomposition research a. Stirring Samples of each variant were subjected to agitation stress at room temperature on an orbital shaker set at 500 rpm. One sample of each variant was agitated for 24 hours, and another sample was agitated for 48 hours. One vial of each variant was stored at room temperature as a control for up to 48 hours. No changes in appearance were observed in the agitated samples compared to the control; all samples were observed to be clear, colorless, and free of visible particulates (data not shown). Furthermore, there was no significant change in total protein content as determined by UV method (data not shown).

[0237] The effect of agitation stress on the stability of the panel of variants was assessed by SEC, reduced CGE, non-reduced CGE, and icIEF (data not shown). No significant changes in the stability of the tested antibodies were observed between agitated control and agitated stressed samples stored at room temperature, nor were any discernible trends in the accumulation of degradants over time. The % main peak determined in SEC analysis was ≥99.2% for all control and agitated samples. R-CGE analysis was ≥98.5% for all control and agitated samples. NR-CGE analysis revealed no significant trends or changes in % main peak between control and stressed samples. In conclusion, no significant differences in stability were observed between all candidate variants.

[0238] b. Freeze-thaw stress Three samples of each candidate were aliquoted into Eppendorf tubes and subjected to freeze-thaw stress. The samples were stored at -75±10°C and then thawed at room temperature. One sample from each candidate underwent three freeze-thaw cycles, another underwent six freeze-thaw cycles, and a third underwent ten cycles. All stressed samples were observed to be clear, colorless, and free of visible particulates (data not shown), and there was no significant change in total protein content as determined by UV method (data not shown).

[0239] The effect of freeze-thaw stress on the stability of the panel of variants was assessed by SEC, reduced CGE, non-reduced CGE, and icIEF (data not shown). Freeze-thaw stress had no effect on antibody stability based on SEC, R-CGE, and NR-CGE analyses.

[0240] icIEF analysis revealed significant changes in the charge heterogeneity of the tested antibodies. The concentration of basic variants decreased with successive F / T cycles, with the lowest concentration of basic variants observed at 10×F / T cycles compared to the control. The only exception was variant IgG1 7.2 VH2 / Vκ1, for which the decrease in basic variant concentration was at the same level for all tested F / T cycles (see Table 9). For variants mAb1, mAb2, and mAb3, the basic species decreased by 1.0, 1.3, and 3.4%, respectively, after 10×F / T cycles. The decrease in basic species concentration correlated with an increase in the percentage of the main peak. For variants mAb4, mAb5, and mAb6, the decrease in basic species was 4.8, 2.8, and 4.7%, respectively. The changes in variant mAb4 are mostly associated with an increase in % main peak, while the changes in variants mAb5 and mAb6 are associated with an increase in both % main peak and % acidic variant.

[0241] Overall, it was determined that the highest resistance to changes from freeze-thaw stress was observed for variants mAb1 (IgG1 7.2 VH2 / Vκ1) and mAb2 (IgG1 7.2 VH2 / Vκ2).

[0242] c. Acidic pH stress Samples from each candidate were subjected to acidic pH stress at room temperature, with each sample adjusted to pH 3.5 with HCl and maintained at room temperature for 2, 4, and 24 hours, after which the samples were neutralized with 1 M Tris, pH 7.0. All samples were observed to be clear, colorless, and free of visible particulates (data not shown), and there was no significant change in protein concentration as determined by UV method (data not shown).

[0243] The effect of acidic pH stress on the panel of variants was assessed by SEC, reducing CGE, non-reducing CGE, and icIEF (data not shown). R-CGE or NR-CGE analysis did not detect significant changes in degradation product accumulation over time for samples exposed to low pH when compared to the 48-h RT control. Charge heterogeneity analysis showed that the concentration of basic variants decreased in all samples subjected to acidic pH stress, but no clear trend was observed over time.

[0244] The overall lowest effect of acid stress on the reduction of basic variants was observed for variants mAb1 (IgG1 7.2 VH2 / Vκ1) and mAb2 (IgG1 7.2 VH2 / Vκ2). This result is in good agreement with that obtained by icIEF in freeze-thaw studies. In SEC analysis, all variants were observed to accumulate some aggregates upon exposure to acid stress, which was associated with a decrease in the % main peak, but no clear trend was observed over time. In general, aggregate accumulation was approximately 9-fold higher for IgG4 variants compared to IgG1 variants. Aggregate accumulation for IgG1 variants was 0.3-1.0% for all acid-stressed samples, while for IgG4 variants, accumulation was 4.3-7.8% for stressed samples, which is significantly higher (see Table 9).

[0245] In conclusion, it was determined that the highest resistance to acid stress was observed with variants mAb1 (IgG1 7.2 VH2 / Vκ1) and mAb2 (IgG1 7.2 VH2 / Vκ2).

[0246] d. Heat stress Samples of each variant were heat-stressed in a heat block at 50°C for 3 days, 1 week, and 2 weeks and then compared to a set of generic control samples stored at 2-8°C. Visual inspection of the samples was performed periodically, and no evidence of phase separation, changes in opacity, or precipitation was observed. All samples were observed to be clear, colorless, and free of visible particulates at all time points (data not shown). Furthermore, there was no significant change in protein concentration as determined by UV (data not shown).

[0247] The effect of heat stress on the panel of variants was assessed by SEC, R-CGE, NR-CGE, and icIEF methods, and the results revealed that all variants were sensitive to heat stress.

[0248] In SEC analysis, a clear trend of increasing aggregate concentration was observed as a function of time for all tested variants. Significantly higher accumulation of aggregates was observed for IgG4 variants compared to IgG1. After 2 weeks, the increase in % total aggregates ranged from 33.7 to 44.7% for IgG4 variants, but only 13.0 to 18.2% for IgG1 variants (see Table 9). Furthermore, the mAb1 (IgG1 7.2 VH2 / Vκ1) and mAb2 (IgG1 7.2 VH2 / Vκ2) variants accumulated less aggregates than the control mAb3 (IgG1 20.1 VH3 / Vκ1).

[0249] In R-CGE analysis, a clear trend of decreasing purity (defined as a decrease in %LC+HC) was observed as a function of time for all tested variants, but no significant differences in stability were observed among all tested variants. The level of sample degradation was comparable for all tested antibodies (data not shown). Similarly, NR-CGE data revealed a clear trend of decreasing purity (defined as a decrease in the main peak) as a function of time for all tested variants. Significantly lower purity was observed for IgG4 variants compared to IgG1 (data not shown). After 2 weeks, the % purity decreased by 34.8–41.7% for IgG4 variants, but only 20.0–30.0% for IgG1 variants (data not shown). Furthermore, the mAb1 (IgG1 7.2 VH2 / Vκ1) and mAb2 (IgG1 7.2 VH2 / Vκ2) variants showed less degradation than the mAb3 IgG1 20.1 VH3 / Vκ1. For IgG1 antibodies, the decrease in the main peak was primarily accompanied by an increase in the front main peak impurities (fragments). However, for IgG4 antibodies, accumulation of both the front main peak impurities (fragments) and the back main peak impurities (aggregates) was observed as a function of the decreasing main peak (data not shown). This result is in good agreement with the SEC data, in which significantly higher concentrations of aggregates were observed for IgG4 antibodies compared to IgG1.

[0250] The week 1 samples for the IgG4 variant were too degraded for analysis by icLEF, and the week 2 samples for all variants were too degraded for analysis. Therefore, only the 3-day data were used to assess the change in charge heterogeneity between the antibodies tested. The difference in purity, as determined by % main peak, was 12.4-14.4% for the IgG1 samples and 16.0-16.6% for the IgG4 samples (data not shown).

[0251] Overall, it was determined that heat stress-induced degradation was greater for IgG4 variants compared to IgG1 variants, and this was observed at all time points. Analysis showed that the mAb1 (IgG1 7.2 VH2 / Vκ1) and mAb2 (IgG1 7.2 VH2 / Vκ2) variants were the least susceptible to heat stress, a result consistent with data obtained for freeze-thaw and acidic pH stress. Selection of the best candidate in terms of developability characteristics In terms of productivity in cell lines, the best results were obtained with the humanized variant mAb1 (7.2 VH2 / Vκ1), with a viable cell density of 54 × 10 for mAb1 at day 10. 6 Viable cells / ml increased.

[0252] For thermal stability studies, samples were evaluated in standard matrices by DSF analysis, and T onset , T m and T agg It was decided that. onset and T m No fluctuations were observed at 1, and T agg was greater than 70° C. for all variants. The results show that mAb1, mAb2, mAb4, and mAb5 demonstrate comparable thermal stability.

[0253] In forced degradation studies, samples were exposed to agitation, freeze-thaw, acidic pH, and heat stress. Results show that under appropriate stress conditions, the panel of candidate variants exhibited different degrees of susceptibility to degradation: No significant response to agitation stress was observed by any of the analytical methods.

[0254] Although no significant response to freeze-thaw stress was observed by SEC or CGE methods, icIEF analysis revealed differences in charge heterogeneity among the tested variants, with the mAb1 (IgG1 7.2 VH2 / Vκ1) and mAb2 (IgG1 7.2 VH2 / Vκ2) variants showing the highest resistance to changes induced by freeze-thaw stress.

[0255] The response to acidic pH stress was monitored by icIEF and SEC, and all candidates accumulated several impurities upon exposure. The mAb1 (IgG1 7.2 VH2 / Vκ1) and mAb2 (IgG1 7.2 VH2 / Vκ2) variants showed the highest resistance to acidic pH stress.

[0256] The most significant stress response observed was to sample storage at 50°C. SEC, NR-CGE, and icIEF analyses revealed a significant trend of decreasing sample purity over time. The observed decrease in purity was consistently greater for IgG4 variants compared to IgG1 variants. Within the IgG1 variant group, mAb1 (7.2 VH2 / Vκ1) and mAb2 (IgG1 7.2 VH2 / Vκ2) showed the highest resistance to acidic heat stress.

[0257] The results of the developability characteristics of the different humanized candidates are summarized in Table 9 below. [Table 9]

[0258] In conclusion, the mAb1 variant showed the best results in terms of developability and was selected as the lead candidate. Functional characterization of humanized mAb7.2 variants as monovalent and bivalent molecules 1. Preparation of Fab and Fab2 fragments Pepsin digestion for aF(ab')2 generation Immobilized pepsin (Thermo Scientific kit, catalog number N°44988) in a 50% slurry was buffer exchanged into digestion buffer (20 mM sodium acetate pH 4.4) by spinning the slurry at 5000 g for 2 minutes. The supernatant was discarded, and the slurry was resuspended in 1 mL of digestion buffer, followed by an additional spin at 5000 g for 2 minutes. This step was repeated four more times (for a total of five resin washes). The resin was then resuspended in digestion buffer to the original slurry volume.

[0259] mAb4, mAb5, and mAb6 (7.2 VH2 / Vκ1, 7.2 VH2 / Vκ2, or 20.1 VH3 / Vκ1 IgG4 variants) were buffer exchanged into digestion buffer using 5 or 10 mL Zeba Spin columns (scale dependent) and concentrated to 3 mg / mL in Vivaspin concentrators (10,000 MWCO) according to the manufacturer's protocol.

[0260] 3 mg / mL of mAbs 4, 5, and 6 (previously buffer-exchanged into digestion buffer) were mixed with pepsin immobilized on the resin and incubated at 37°C with rotation for 1.5 to 2 hours.

[0261] The digestion mixture was spun down at 5000g for 2 minutes. The supernatant was removed and filtered into a new tube using an appropriate syringe and a 0.22 μm mini-filter (Merck Millipore, Millex Cat. No. SLGV004SL). The resin was washed with 1 mL of PBS and spun at 5000g for 1 minute. The supernatant was removed, filtered, and pooled with the previous supernatant. The wash step was repeated.

[0262] The digested and filtered supernatant was purified using a HiLoad 16 / 600 200 pg size-exclusion column using 10 mM sodium acetate, 100 mM NaCl, pH 5.5 as the mobile phase. Fractions corresponding to the eluted F(ab')2 fragments were pooled, concentrated, and sterile filtered.

[0263] F(ab')2 fragments were analyzed by SDS-PAGE, analytical SEC and OD 280nm reading The analysis was carried out by

[0264] b. Papain digestion for Fab production Immobilized pepsin (Thermo Scientific kit, catalog number N°20341) in a 50% slurry was buffer exchanged into digestion buffer (20 mM sodium phosphate, 10 mM EDTA, 150 mM cysteine ​​pH 7.0) by spinning the slurry at 5000 g for 2 minutes. The supernatant was discarded, and the slurry was resuspended in a larger volume of digestion buffer, followed by an additional spin at 5000 g for 2 minutes. This step was repeated four more times (for a total of five resin washes). The resin was then resuspended in digestion buffer to the original slurry volume.

[0265] mAb4, mAb5, and mAb6 (7.2 VH2 / Vκ1, 7.2 VH2 / Vκ2, or 20.1 VH3 / Vκ1 IgG4 variants) were buffer exchanged into digestion buffer using 5 or 10 mL Zeba Spin columns (scale dependent) and concentrated to 3 mg / mL in Vivaspin concentrators (10,000 MWCO) according to the manufacturer's protocol.

[0266] 3 mg / mL of mAbs 4, 5, and 6 (previously buffer-exchanged into digestion buffer) were mixed with papain immobilized on the resin and incubated at 37°C with rotation for 42 hours.

[0267] The digestion mixture was spun down at 5000 g for 2 minutes. The supernatant was removed and filtered into a new tube using an appropriate syringe and a 0.22 μm mini-filter (Merck Millipore, Millex catalog no. SLGV004SL). The resin was washed three times with PBS and spun at 5000 g for 1 minute, collecting and pooling the supernatant after each cycle. The pooled fractions were then filtered into a new tube using an appropriate syringe and a 0.22 μm mini-filter.

[0268] The digested and filtered supernatant was first buffer exchanged into 1x DPBS, pH 7.4, and then purified first using a Protein A column to remove Fc and undigested mAb, followed by a polishing step using a size exclusion (SEC) column with 10 mM sodium acetate, 100 mM NaCl, pH 5.5 as the mobile phase. Fractions corresponding to the eluted Fab fragments were pooled, concentrated, and sterile filtered.

[0269] Fab fragments were analyzed by SDS-PAGE, SEC and OD 280nm Analyzed by reading.

[0270] 2. Affinity Measurements of Humanized 7.2 and 20.1 Fab, F(ab')2 Fragment, and IgG Formats Using Biacore To evaluate the affinity of the lead humanized 7.2 and 20.1 antibodies for BTN3A in their different formats (Fab, F(ab')2, and IgG), single-cycle kinetic analysis was performed using a Biacore T200 (serial number 1909913) instrument running Biacore T200 Control software V2.0.1 and Evaluation software V3.0 (GE Healthcare, Uppsala, Sweden). All single-cycle kinetic experiments were performed at 25°C using HBS-P+ running buffer (pH 7.4) containing 0.1% BSA (GE Healthcare, Little Chalfont, UK).

[0271] BNT3A His-tagged antigen (Sino Biological, Beijing, China) was diluted in running buffer to a final concentration of 0.4 μg / ml. At the start of each cycle, BNT3A-His was captured at a flow rate of 10 μl / min onto Fc2 of a CM5 sensor chip (GE Healthcare, Little Chalfont, UK) precoupled using standard amine chemistry and a His capture kit. Different theoretical immobilization levels (RL) of approximately 34 RU, 16 RU, or 11 RU were used for the analytes Fab, F(ab')2, and IgG, respectively, to achieve an RMax of approximately 50 RU. The surface was then allowed to stabilize. Single-cycle kinetic data were obtained using purified samples (Fab, F(ab')2, and IgG) at a flow rate of 40 μl / min to minimize any potential mass transfer effects. The signal from the reference channel Fc1 (no antigen capture) was subtracted from the signal from Fc2 to correct for differences in nonspecific binding to the reference surface. The signal for a BTN3A-His blank (no analyte) was subtracted to correct for differences in surface stability. The association phase for five injections of increasing concentrations of BTN3A1 was monitored for 240 seconds each time, a single dissociation phase was monitored for 2000 seconds following the last injection of BTN3A1, and a single dissociation phase was measured for 1400 seconds following the last injection of analyte. Regeneration of the chip surface was performed using two injections of 10 mM glycine-HCl pH 1.5, followed by stabilization for 240 seconds. Raw sensorgrams were fitted with a one-to-one model for the Fab samples and a bivalent analyte model for the F(ab')2 and IgG samples, consistent with the different valencies of the analytes. Kinetic constants were calculated for each variant (see Tables 10, 11, and 12). [Table 10] [Table 11] [Table 12] 3. Selection of the best candidate in terms of affinity properties

[0272] While these differences can be observed using the Fab and F(ab')2 formats, the gap is much higher using Fab fragments, and indeed the average K of 20.1 is 20.30. D 7.2 indicates an average K of 3.22 (VH2 / Vκ1) or 3.01 (VH2 / Vκ2). D showed.

[0273] 4. mAb1 Binding Avidity to Primary T Cells and Other Cell Lines Next, mAb1 binding avidity was assessed by flow cytometry in human primary T cells and in various cell lines, including WT and BTN3A knockouts individually reconstituted with BTN3A1, BTN3A2, or BTN3A3 isoforms (data not shown). EC values ​​obtained in each tested cell type were 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.2 50 The values ​​are summarized in Table 13.

[0274] Data obtained in BTN3A KO cells clearly demonstrated that mAb1 specifically binds to its target. Furthermore, reconstitution with individual isoforms confirmed that mAb1 recognizes BTN3A1, BTN3A2, and BTN3A3 with comparable avidity. All other tested cells showed EC values ​​ranging from 9.6 nM for the Burkitt's lymphoma Daudi cell line to 112.3 nM for the colorectal adenocarcinoma cell line HT29. 50 A range of values ​​appeared positive for mAb1 binding. [Table 13] 5. mAb1 has no off-target binding The potential for off-target binding of mAb1 with other non-BTN3A molecules was assessed using Retrogenix technology. This study aimed to demonstrate the absence of off-target binding by screening cell arrays expressing >5000 human membrane receptors or secreted proteins expressed on the surface of HEK293 cells (Retrogenix platform).

[0275] Examination of the binding level of mAb1 to non-transfected HEK293 cells and to cells overexpressing BTN3A before and after cell fixation indicated that 5 μg / ml on fixed cells was an appropriate screening condition. Under these conditions, mAb1 was screened for binding to human HEK293 cells expressing 5528 individual human proteins, including cell surface membrane proteins and secreted proteins anchored to the cell surface. This revealed 10 primary hits.

[0276] Each primary hit was coexpressed with two control receptors (CD20 and EGFR) and retested with 5 μg / ml mAb1, 5 μg / ml isotype control antibody, and other positive and negative control treatments. After removing five nonspecific hits, five specific interactions remained for the test antibody. All five specific hits were BTN3A-related: two isoforms of BTN3A1, two isoforms of BTN3A2, and one isoform of BTN3A3.

[0277] The study conclusions were that no off-target interactions were identified for mAb1, indicating high specificity of mAb1 for its BTN3A epitope.

[0278] 6. mAb1 mediates Vγ9Vδ2 T cell activation and tumor cell killing Originally, a murine anti-BTN3A mAb was shown to trigger BTN3A recognition by Vγ9Vδ2 T cells, mediating (i) the expansion of this specific subset in human PBMCs, (ii) cytokine (IFNγ and TNFα) production, and (iii) activation leading to cytolysis (e.g., via perforin, granzymes, and TRAIL) of infected or transformed target cells (Harly et al., Blood, 2012 & Benyamine, A. et al., 2016, Oncoimmunology 5, e1146843).

[0279] Without being bound by any particular theory, the proposed mechanism of action of mAb1 is that binding to BTN3A expressed on the surface of tumor target cells triggers a conformational change, enabling its signaling to counterreceptors on Vγ9Vδ2 T cells. Anti-BTN3A antibody activity is routinely assessed using an in vitro assay based on coculture of tumor cell lines (targets) with primary human Vγ9Vδ2 T cells (effectors) pre-expanded from PBMCs of healthy donors for 10–14 days in the presence of rHuIL-2 (200 UI / mL) and an aminobisphosphonate (Zometa, 1 μM). At the end of the expansion phase, the purity of Vγ9Vδ2 T cells is assessed by flow cytometry, and these cells are then frozen for future use. The day before the experiment, expanded Vγ9Vδ2 T cells are thawed and cultured overnight in 200 UI / mL of rHuIL-2 to maintain in vitro viability. After co-culture, Vy9V52 T cell activation is monitored by flow cytometric detection of CD107a / b expression on γδ T cells or by quantitating caspase 3 / 7 activation as a measure of target cell killing.

[0280] First, human Vγ9Vδ2 T cells expanded from PBMCs of three different healthy donors were co-cultured with a Daudi cell line (ATCC-CCL213; Burkitt's lymphoma) using increasing concentrations of mAb1. After 4 hours, cells were analyzed for Vγ9Vδ2 T cell expression of CD107a / b by flow cytometry. Results showed a concentration-related increase in the percentage of Vγ9Vδ2 T cells expressing CD107a / b, with a mean EC 50 The cytolytic activity of Vγ9Vδ2 T cells against mAb1-pulsed Daudi cells was 0.89 nM (±0.39) (Figure 1A). In parallel, we evaluated the cytolytic activity of Vγ9Vδ2 T cells against mAb1-pulsed Daudi cells. As shown in Figure 1B, mAb1 induced target cell apoptosis in a concentration-dependent manner, resulting in an EC 50 The binding of mAb1 to L-IPC (pancreatic ductal adenocarcinoma), A549 (lung cancer epithelial cells), and HT29 (colorectal adenocarcinoma) cell lines was induced by Vγ9Vδ2 T cell activation (CD107a / b; Figure 1C, upper panel) and tumor cell lysis (caspase 3 / 7; Figure 1C, lower panel). Furthermore, Vγ9Vδ2 T cell activation (CD107a / b; Figure 1C, upper panel) and tumor cell lysis (caspase 3 / 7; Figure 1C, lower panel) were examined using tumor cell lines from different tissue origins and compared with Daudi cells. The results revealed that Vγ9Vδ2 T cell activation and subsequent tumor cell lysis were induced by binding of mAb1 to L-IPC (pancreatic ductal adenocarcinoma), A549 (lung cancer epithelial cells), and HT29 (colorectal adenocarcinoma) cell lines.

[0281] 7. mAb1 enhances Vγ9Vδ2 T cell killing of a wide range of BTN3A-expressing human cell lines, regardless of the tissue origin of the cells. a. Materials and Methods Tumor cell culture HL-60 is a human promyeloblast cell line derived from acute promyelocytic leukemia. Daudi is a human B-lymphoblast cell line derived from Burkitt's lymphoma. Jurkat is an acute T-cell leukemia cell line.

[0282] HT-29 and HCT116 are human epithelial cells derived from colorectal adenocarcinoma.

[0283] PC3 and DU145 were derived from prostate cancer (metastases, bone and brain, respectively).

[0284] SUM159 and MDA-MB-231 are triple-negative breast cancer (TNBC) cell lines.

[0285] HL60, Daudi, Jurkat, DU145, and MDA-MB-231 cells were cultured in RPMI Glutamax, 10% FBS, and 1 mM sodium pyruvate at 37°C / 5% CO2. PC3 and HT-29 cells were cultured in DMEM 10% FBS and 1 mM sodium pyruvate at 37°C / 5% CO2. HCT116 cells were cultured in McCoy's 5a medium with 10% SVF. SUM159 cells were cultured in medium F12 Nut Mix 1x plus Glutamax, 5% SVF, hydrocortisone 2 mg / ml, Humalog insulin 2 mg / ml, and nonessential amino acids.

[0286] In vitro Vγ9Vδ2 T cell expansion Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation from peripheral blood obtained from the Etablissement Français du Sang, Provence Alpes Cote d'Azur (France). To expand Vγ9Vδ2 T cells, 50,106 PBMCs were cultured at 75 cm in the presence of rHuIL-2 (200 UI / mL) and aminobisphosphonate (Zoledronate, 1 μM) for 10–14 days. 2 1.5 x 10 cells in RPMI 1640 supplemented with 10% FBS and 1% sodium pyruvate in a flask 6 From day 5, rHuIL-2 was renewed every 2 or 3 days, and the cells were resuspended at 1 × 10 6 At the end of the expansion phase, the purity of Vy9V52 T cells was assessed by flow cytometry, and when the number of Vy9V52 T cells reached 80% viable cells, these cells were frozen in FBS 20% DMSO for future use.

[0287] Purification of Vγ9Vδ2 T cells from human PBMCs Fresh human PBMCs were resuspended at 50.10 cells / ml in PBS + 2% FBS + 1 mM EDTA. Vγ9Vδ2 were isolated using the EasySep™ Human Gamma / Delta T Cell Isolation Kit (Stemcell #19255) according to the manufacturer's instructions. At the end of the procedure, cell purity was determined by flow cytometry. Live CD3+Vγ9+ cells were greater than 80%.

[0288] Vγ9Vδ2 T cell killing assay 10,000 expanded or fresh Vy9V52 T cells were co-cultured with tumor cell lines in 96-well plates at the indicated ratios (E:T 1:1 or 1:5) in RPMI 1640 + glutamax, 10% FBS + 1 mM NaPy in the presence of mAb1 or the appropriate isotype control. rHuIL-2 was used at 20 IU / ml when added to the co-cultures. After the indicated times, ATP was measured using Glo reagent (Promega #G7572), which generates a luminescent signal proportional to the number of viable cells.

[0289] b. result In addition to methods based on caspase 3 / 7 staining of target cells to monitor killing by co-culture with Vγ9Vδ2 T cells, we developed a complementary approach based on assessing the number of viable cells in culture, based on quantification of the ATP present, an indicator of metabolically active cells.

[0290] Using this assay, we monitored the survival of the acute myeloid leukemia cell lines HL60-WT or -BTN3A-KO after 24 hours of co-culture (E:T ratio 1:1) with Vy9V52 T cells grown in the presence of mAb1 or the appropriate isotype control + / - rHuIL-2 (20 IU / ml) (Figure A). Results showed a concentration-dependent decrease in HL60-WT survival in the presence of mAb1, indicating efficient BTN3A-dependent killing of target tumor cells by activated Vy9V52 T cells. Similar experiments were performed with in vitro-expanded or freshly isolated Vy9V52 T cells (Figures B and C, respectively), and cell viability was measured over a 4-day period.

[0291] Panel B shows that in vitro expanded Vy9V52 T cells control HL60-WT proliferation in a concentration-dependent manner. The addition of rHu-IL2 improves effector cell killing capacity over time, likely by providing a survival signal necessary for in vitro culture of primary T cells. Similar results were obtained using freshly isolated Vy9V52 T cells from human PBMCs cocultured with HL60-WT for 4 days at E:T ratios of 1:1 and 1:5. These results demonstrate the ability of individual Vy9V52 T cells to repeatedly bind and kill multiple tumor targets upon mAb1-mediated activation (Figure 2C).

[0292] Next, we used this assay to monitor mAb1-mediated Vy9V52 T cell killing activity against BTN3A-expressing cell lines from different tissue origins (colon, breast, prostate, T lymphoma, and Burkitt's lymphoma). HT29, PC3, DU145, MDA-MB-231, HCT116, SUM159, Jurkat, and Daudi cells were co-cultured overnight with in vitro-expanded Vy9V52 T cells in the presence of increasing concentrations of mAb1, and cell viability was measured. The results confirmed that mAb1 enhances Vy9V52 T cell killing of a wide range of BTN3A-expressing human cell lines, regardless of their tissue origin (Erreur! Source du renvoi introuvable. and Figure). [Table 14] 8. mAb1 improves Vγ9Vδ2 T cell therapy in a mouse model transplanted with a human AML cell line Because mAb1 targets BTN3A, which is not expressed in rodents, and the Vγ9Vδ2 T cell subpopulation is unique to primates, experimental proof-of-concept Vγ9Vδ2 T cell antitumor activity is typically tested in immunocompromised NSG mice transplanted with BTN3A-expressing human tumor cell lines and adoptively transferred with Vγ9Vδ2 T cells from healthy human donors (the Vγ9Vδ2 T cells in such reconstitutions are allogeneic to the tumor cell line). These models have been widely used to validate the antitumor therapeutic potential of Vγ9Vδ2 T cells in a wide range of solid and hematologic tumors (Pauza, CD et al., 2018, Immunol. 9).

[0293] Furthermore, the murine anti-human BTN3A antibody, m20.1, administered to mice receiving human Vy9V52 T cells was described to enhance animal survival and reduce leukemic burden in the blood and bone marrow of AML-bearing NSG mice (Benyamine et al., 2016, supra). In this model, m20.1 was injected in combination with RLI, a rHuIL-15 / IL-15Rα fusion protein that has been shown to expand anti-tumor lymphocyte subsets, improve their survival in mice, and enhance the anti-tumor activity of transferred human Vy9V52 T cells.

[0294] To further characterize the efficacy of mAb1 in an in vivo mouse model, we investigated the possibility that human Vy9V52 T cell transfer in combination with mAb1 could slow tumor growth and improve animal survival in NSG mice bearing AML.

[0295] a. U937 model Healthy 6- to 8-week-old female mice (n=30) were injected with 0.2 × 10 IgG on day 0 as described in Gertner-Dardenne, J. et al., 2012. J. Immunol. 188, 4701-4708. 6 On day 1, tumor burden was assessed using bioluminescence imaging, and mice received in vitro-expanded human Vγ9Vδ2 T cells (3 × 10 6 The mice were randomly assigned to six groups to receive an intravenous injection of rHuIL-15 / rHuIL-15Rα (cells) alone or in combination with anti-BTN3A mAb1 or the relevant isotype control (10 mg / kg, 200 μg / mouse). Treatment was repeated on day 7. In groups 2 and 4, the antibody was also administered on days 4 and 10. Because rHuIL-15 / rHuIL-15Rα complexes enable the expansion of human Vγ9Vδ2 T cells ( J. Immunol. (2006) 17, 6072-608), these complexes were administered together with Vγ9Vδ2 T cells.

[0296] All groups are listed in Table 16.

[0297] hIL-15 / IL-15R-Fc complexes were pre-complexed 30 min at room temperature (RT) before injection (0.2 μg hIL-15 + 1.2 μg IL-15R-Fc per mouse) and mixed with Vy9V52 T cells prior to injection. The final volume for each injection was 100 μl. Treatment mAbs were injected 4 h before Vy9V52 T cell transfer.

[0298] Our results confirmed that Vy9V52 T cell plus rHuIL-15 / rHuIL-15Rα infusion reduced tumor burden. This effect was even more impressive and was associated with a significant increase in survival when anti-BTN3A mAb1 was added together with Vy9V52 T cells (Tables 16 and 17). Importantly, cytarabine (Ara-C) (10 mg / kg in NSG mice bearing U937 tumors), one of the most effective drugs for treating acute myeloid leukemia used in this highly aggressive mouse model, improved mouse survival by 2-3 days (approximately 10%) when mAb1 was combined with human Vy9V52 T cell transfer.

[0299] These data highlight the potent anti-leukemic effect exerted by anti-BTN3A mAb in combination with Vγ9Vδ2 T cell immunotherapy in vivo. [Table 15] [Table 16] JPEG0007825605000022.jpg41149 [Table 17]

[0300] b. MOLM14 model The in vivo efficacy of mAb1 against MOLM14, an AraC-resistant human AML-derived cell line, was evaluated in a xenograft model using NSG mice engrafted with human tumor cell lines and human Vy9V52 T cells. The goal of this study was to determine the effect of repeated intravenous injections of human Vy9V52 T cells in combination with mAb1 on tumor growth and on mouse survival.

[0301] Female NSG mice, 6–8 weeks old, were infected with 0.2 × 10 per mouse in a volume of 100 μl. 6MOLM14 (CVCL_7916) cells expressing luciferase (luc2) were injected intravenously (i.v.) via the tail vein on day 0. Bioluminescence assays were performed on day 0 using a PhotonIMAGER (Biospace Lab) following the addition of endotoxin-free luciferin (30 mg / kg). Mice were randomized into uniform groups of 7 mice based on the intensity of the bioluminescence signal. 6 Human in vitro expanded Vy9V52 T cells and hIL-15 / IL-15R complexes were injected intravenously on days 1, 8, 15, 22 and 29. mAb1 or hlgG1 were injected intravenously on days 1, 5, 8, 12, 15, 19, 22, 26 and 29.

[0302] The different experimental groups are summarized in Table 19.

[0303] hIL-15 / IL-15R-Fc complexes were pre-complexed for 30 min at room temperature before injection (0.2 μg hIL-15 + 1.2 μg IL-15R-Fc per mouse) and mixed with Vy9V52 T cells prior to injection. The final volume for each injection was 100 μl. Treatment mAbs were injected 4 h before Vy9V52 T cell transfer.

[0304] Bioluminescence signals from MOLM14 cells were measured on days 0, 7, 14, 21, and 28 after cell injection to track tumor growth. Blood samples were collected on day 19 to assess the number of circulating MOLM14 cells by flow cytometry. Erythrocyte lysis was performed before staining. mCD45+ mouse cells were excluded from the analysis, and MOLM14 tumor cells were detected by their GFP expression. Acquisition was performed on an LSRII SORP hemocytometer (Becton Dickinson), and analysis was performed using FlowJo software. Daily monitoring of mice for disease symptoms (significant weight loss, wrinkled fur, hunched posture, weakness, and reduced mobility) determined the time to sacrifice for injected animals showing signs of distress.

[0305] As shown in Table 20, Vy9V52 T cells injected with an irrelevant control isotype (hlgG1) do not significantly control tumor growth. In contrast, tumor growth was strongly reduced when anti-BTN3A mAb1 was administered sequentially with human Vy9V52 T cells, as indicated by a lower bioluminescence signal. Results also showed a significant reduction in the number of circulating blast cells (assessed by flow cytometry in peripheral blood) on day 19 of the protocol (Table 20). Importantly, the anti-BTN3A mAb-dependent reduction in tumor growth resulted in a significant 45% improvement in mouse survival (compared to the respective isotype control) (Table 22). [Table 18] [Table 19] [Table 20] [Table 21]

[0306] 9. mAb1 improves Vγ9Vδ2 T cell therapy in a murine solid tumor model implanted with the human ovarian cancer cell line SKOV-3 a. Materials and Methods In vivo Vγ9Vδ2 T cell expansion Allogeneic human Vγ9Vδ2 T lymphocytes were expanded from peripheral blood mononuclear cells (PBMCs) obtained from healthy donor blood samples after Ficoll density centrifugation (Eurobio, Les Ulis, France) by the Etablissment Français du Sang (EFS, Nantes, France). For specific expansion of peripheral allogeneic human Vγ9Vδ2 T lymphocytes, PBMCs were incubated with 3 μM bromohydrin pyrophosphate (BrHPP), a gift from Innate Pharma (Marseille, France), in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 10 mg / mL streptomycin, 100 IU / mL penicillin (all from Gibco), and 100 IU / mL recombinant human IL-2 (PROLEUKIN, Novartis, Bale, Switzerland). After 4 days, the cultures were supplemented with 300 IU / mL IL-2. On day 21, purity was determined by flow cytometry (purity >90%). Pure human Vy9V52 T lymphocytes were further expanded using feeder cells (mixed and 35 Gy-irradiated Epstein-Barr virus-transformed B lymphocytes and PBMCs) and PHA-L in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 10 mg / mL streptomycin, 100 IU / mL penicillin (all from Gibco), and 100 IU / mL recombinant human IL-2 (Novartis). After 3 weeks, resting ex vivo expanded Vy9V52 T lymphocytes were used for in vivo experiments.

[0307] Mouse model On day 0, 6- to 8-week-old NSG mice were inoculated with 1 x 10 cells per mouse in a volume of 100 µL of sterile PBS. 6SKOV-3 cells (ovarian cancer cell line, SKOV-3-luc-D3, Perkin Elmer, Waltham, MA) expressing luciferase were injected intraperitoneally (i.p.). Seven days later, mice were randomized into uniform groups of 5–6 mice. On days 7 and 14, mAb treatments were performed with 200 μg of mAb1 or the appropriate isotype control (hlgG1) per mouse injected i.p. in 100 μL of sterile PBS. Four hours later, 5 × 10 cells were injected in 100 μL of sterile PBS per mouse. 6 of human in vitro expanded Vγ9Vδ2 T cells were also injected i.p.

[0308] Bioluminescence signals from SKOV-3 cells were measured 16, 23, and 30 days after tumor cell implantation to track tumor growth. Bioluminescence imaging was performed 8 minutes after intraperitoneal injection of 1.5 mg of D-luciferin (Interchim, San Diego, CA) on a Biospace imager (Biospace Lab, Nesles-la-Vallee, France) after anesthetizing mice with 2% isoflurane. The experimental endpoint was reached when mice lost 10% of their initial body weight.

[0309] b. result The in vivo efficacy of mAb1 against ovarian cancer was evaluated in a xenograft model using the human ovarian cancer cell line SKOV-3 and NSG mice transplanted with human Vy9V52 T cells. The goal of this study was to evaluate the effect of two i.p. injections of human Vy9V52 T cells in combination with mAb1 on tumor growth and on mouse survival.

[0310] NSG mice were injected intraperitoneally (ip) with SKOV-3 on day 0. Seven days later, mice were randomized into uniform groups according to treatment. Groups are listed in Table 23. [Table 22]

[0311] The results showed that human Vy9V52 T cells transferred with mAb1 significantly delayed tumor growth (Table 24) and significantly improved animal survival (Table 25). Notably, this effect was observed in the absence of pro-Vy9V52 T survival cytokines such as IL-2 or IL-15. [Table 23] [Table 24]

[0312] 10. In vivo effects on cynomolgus Vγ9Vδ2 T cells Due to the absence of BTN3A and Vγ9Vδ2 T subsets in rodents, and based on previous data documenting in vitro and in vivo PAg-mediated Vγ9Vδ2 T cell activation in cynomolgus macaques, the cynomolgus macaque (Macaca fascicularis) was selected as the only relevant species for the non-clinical safety evaluation of mAb1.

[0313] a. Materials and Methods Biacore mAb1 was evaluated for binding to recombinant human or cynomolgus monkey BTN3A1, BTN3A2, and BTN3A3 proteins (SEQ ID NOs: 21, 22, and 23, respectively) via Biacore multicycle kinetic analysis using a Biacore T200 (serial number 1909913) instrument. mAb1 was diluted to a concentration of 2 μg / ml in 2% BSA / PBS. At the start of each cycle, the antibody was captured on a Protein A surface at a density of approximately 150 RU (RL) (theoretical value to obtain an RMax of approximately 50 RU). Following capture, the surface was allowed to stabilize before injection of BTN3A antigen. BTN3A was titrated in 0.1% BSA / HBS-P+ (running buffer) over a two-fold dilution range from 25 to 0.78 nM. The association phase was monitored for 420 s, and the dissociation phase was monitored for 2000 s. Kinetic data were obtained using a flow rate of 50 μl / min to minimize any potential mass transfer effects, and the data obtained were fitted using a one-to-one binding model.

[0314] ELISA The apparent affinity of mAb1 for human and cynomolgus monkey BTN3A1 was tested by ELISA. Briefly, target binding of mAb1 was assessed using plate-immobilized recombinant BTN3A1 at 1 μg / ml in phosphate buffer (1x PBS), followed by a saturation step with block buffer (2% milk / PBS). mAb1 was titrated in block buffer over a 4-fold dilution range from 0.00122 to 20 μg / ml. Secondary antibody (goat anti-human lgk chain, HRP-conjugated antibody, Millipore AP502P, diluted 1:4000 in block buffer) and TMB solution were used for detection. Apparent affinity was expressed as EC50% (antibody concentration required to obtain 50% of the signal plateau).

[0315] Binding avidity of mAb1 to human and cynomolgus monkey CD3+ cells After red blood cell lysis, human or cynomolgus monkey PBLs were incubated with increasing concentrations of mAb1 or isotype control for 30 minutes at 4°C, washed twice, and stained with goat anti-human IgG-PE conjugated secondary antibody (eBioscience™ #12-4998-82). After two washes, cells were stained with anti-CD3-PC3 mAb (BD Bioscience #557749) and live / dead reagent (Life Technology #L10119). After washing, cells were resuspended in 200 μL of Flow buffer. Cells were then analyzed on a Cytoflex cell counter (Beckman Coulter). Data were analyzed using FlowJo software (version 10, FlowJo, LLC, Ashland, USA) gating on the viable CD3+ population. MFI values ​​from the PE channel were then calculated and plotted against concentration. Curve fitting was obtained using the sigmoidal 4PL equation from GraphPad Prism software.

[0316] BTN3A surface expression on human and cynomolgus monkey circulating cells For BTN3A surface expression on leukocytes, 100 μl of cynomolgus monkey and human whole blood was plated in a 96-well plate with a cocktail of specific antibodies (Aqua Live Dead Reagent, CD20-V450, CD8-BV605, CD4-BV650, Vg9 TCR-FITC, anti-BTN3A-PE (clone 20.1) (or mIgG1-PE for isotype control), CD3-PeCy7, CD45-AF700, and CD14-APC-H7) and incubated for 15 min at RT, protected from light. Red blood cells were then lysed using 900 μl of lysis reagent (BD Bioscience #349202) according to the manufacturer's instructions. Cells were washed and analyzed using multiparameter flow cytometry. For BTN3A surface expression on red blood cells and platelets, 100 μl of cynomolgus or human whole blood was diluted with 100 μl of PBS and incubated with a specific cocktail of antibodies (Aqua Live Dead, CD41-APC, CD45-AF700, and anti-BTN3A (clone 20.1) (or mIgG1-PE for isotype control)) for 15 min at RT, protected from light. After washing, cells were analyzed using multiparameter flow cytometry. To obtain relative quantification of BTN3A surface expression, calibration beads (CellQuant Calibrator Biocytex #7208) and goat anti-mouse IgG(H+L)-PE were used in parallel according to the manufacturer's instructions. For all cell subsets, the MFI of the PE channel was reported for anti-BTN3A and the isotype control. Analysis was performed by subtracting the MFI of the isotype control from the MFI of anti-BTN3A staining, and relative surface expression was calculated based on a standard curve obtained using calibration beads.

[0317] Cynomolgus Vγ9Vδ2 T cell in vitro expansion and activation Cynomolgus whole blood from three animals was treated with red blood cell lysis buffer. After extensive washing, leukocytes were plated in 6-well plates in 1.5 μM / ml RPMI 10% SVF in the presence of rHuIL-2 (200 IU / ml) and mAb1 (10 μg / ml). To obtain sufficient numbers of cells for functional assays, rHuIL-2 was added on days 6 and 8 to mimic the normal cell expansion protocol used with human PBMCs and improve long-term in vitro survival of Vγ9Vδ2 T cells. The percentage of Vγ9+ T cells was assessed on days 0, 6, 8, and 10 using a cocktail of specific antibodies and flow cytometry analysis (CD3-PC7 BD Bioscience #557749, Vg9 TCR-FITC clone 7A5 Invitrogen #TCR2720, Live Dead near-IR ThermoFisher #L10119).

[0318] On day 10, proliferating Vy9V52 T cells were counted and co-cultured with human tumor cell lines at a 1:1 E:T ratio. 100,000 target tumor cell lines (Raji, Daudi, and K562) were mixed with 100,000 cynomolgus Vy9V52 T cells in a 96-well plate in the presence of mAb1 or hIgG1 isotype control (10 μg / ml), or PMA (20 ng / ml) / ionomycin (1 μg / ml) used as a positive control. Vy9V52 T cell degranulation was monitored after 4 hours using CD107a / b (BD Bioscience #555800) staining and flow cytometry analysis.

[0319] Cynomolgus monkey in vivo studies in life study Cynomolgus monkeys (Macaca fascicularis), 4-6 years old and weighing 3-5 kg, of Vietnamese origin were used in this study. All animals were maintained and used in accordance with the guidelines of the Institutional Animal Care and Use Committee of the GLP animal facility. As part of breeding animal health procedures, all animals were tested for tuberculosis, and preventive treatments were documented in the breeding animal records. Upon arrival, animals were acclimated to study procedures for a period of at least 2 weeks. Clinical examinations and tests for ill health were performed. To ensure the suitability of all animals for the study, a veterinarian performed an animal health assessment before the start of the pre-treatment phase.

[0320] mAb1 was administered intravenously (infusion over 15 minutes while restrained in a chair) after disinfecting the skin of non-fasted animals. Animals in groups 1, 4, and 5 were dosed on days 1, 8, 15, and 22. Animals in groups 2 and 3 were dosed once on day 1.

[0321] Pharmacokinetics A conditional pharmacokinetic assay was developed for the quantification of mAb1 in cynomolgus monkey serum. Briefly, mAb1 was quantified using an ELISA by spectrophotometry. A streptavidin-precoated plate was used to capture a human IgG-Fc PK-biotin conjugate. mAb1 was then captured on the plate surface, and the bound analyte was detected using a peroxidase-labeled anti-species antibody, goat anti-human IgG-HRP (Fc-specific) antibody. The target range for quantification was 90 ng / mL to 10,000 ng / mL in raw serum.

[0322] Immunophenotyping Blood samples (1.0 mL) were drawn from all animals into Li heparin tubes from the median cephalic or small saphenous vein. Immunophenotyping was performed using a cocktail of specific monoclonal antibodies. Analysis of relative cell counts (lymphocyte / leukocyte percentages) was performed. Total granulocyte / lymphocyte / leukocyte counts were determined on the same day by hemoanalyzer and used to calculate absolute counts. Absolute counts of lymphocyte subpopulations were computed from the relative counts.

[0323] Receptor occupancy Blood samples (400 μL) were drawn from all animals into Li heparin tubes from the median cephalic or small saphenous vein. A labeled antibody that binds noncompetitively to BTN3A (clone 103.2) on cells was preincubated with excess ICT01 and used to determine total surface BTN3A expression on CD3+ T cells and CD19+ B cells. Free BTN3A binding sites were detected using competitive binding of unlabeled mAb1 to BTN3A, which inhibited the binding of fluorochrome-conjugated anti-BTN3A antibody (mAb1) on CD3+ T cells and CD19+ B cells. The mean fluorescence intensity (MFI) of the conjugated mAb1 was measured as a geometric mean decrease depending on the amount of BTN3A blocked by mAb1. The staining intensity of Ab7.2, which was close to that of the isotype antibody, showed complete saturation.

[0324] b.Results mAb1 binds to cynomolgus monkey BTN3A Because differential identification of the three BTN3A isoform genes was not possible from public databases, ImCheck performed targeted PCR on cDNA isolated from cynomolgus monkey PBMCs using highly conserved cDNA sequences flanking the transmembrane domains to design appropriate primers. Sequencing of the PCR products allowed the identification of the ectodomain sequences for cynomolgus monkey BTN3A1, BTN3A2, and BTN3A3. Recombinant ectodomains of cynomolgus monkey BTN3A1, BTN3A2, and BTN3A3 isoforms fused to a 6xHis tag were produced in CHO cells based on these sequences (SEQ ID NOs: 21, 22, and 23, respectively).

[0325] The recombinant proteins were tested for mAb1 binding by BIAcore and ELISA (Table 26). The BIAcore results showed that mAb1 advantageously bound to the three cynomolgus recombinant BTN3A1, BTN3A2, and BTN3A3, but with relatively low affinity for the BTN3A1 isoforms. ELISA was performed with the BTN3A1 isoforms. Interestingly, Table 26 shows comparable EC50s for mAb1 binding to recombinant human or cynomolgus BTN3A1. [Table 25]

[0326] In parallel, mAb1 binding on cynomolgus macaque PBMCs was assessed by flow cytometry. The mean EC50 for mAb1 binding to cynomolgus macaque CD3+ T cells was comparable to the EC50 for mAb1 binding to human CD3+ T cells (Table 27). Target expression on different immune cell subpopulations from cynomolgus versus human healthy donor whole blood was addressed by multiparameter flow cytometry using a PE-conjugated anti-BTN3A mAb (clone 20.1) together with a panel of phenotypic antibodies with known cross-reactivity to human and cynomolgus macaque cell surface markers (data not shown). These results indicate that BTN3A is expressed in a broad panel of peripheral blood cell populations in both species, although an apparent generally lower expression was observed in cynomolgus macaques. [Table 26] mAb1 promotes cynomolgus Vγ9Vδ2 T cell proliferation and activation in vitro Next, the functional activity of mAb1 was evaluated in vitro on cynomolgus Vy9V52 T cells. First, we evaluated whether mAb1 promoted cynomolgus Vy9V52 T cell proliferation when incubated with cynomolgus PBMCs for 10 days. As shown in Figure 4A, mAb1 promoted Vy9V52 T cell proliferation in all three animals tested, with this population reaching 60% after 10 days, a level comparable to that observed with human cells. After 10 days of proliferation, cells were co-cultured with Daudi, K562, or Raji cell lines used as target cells in the presence of mAb1 for 4 hours and analyzed for CD107a / b by flow cytometry. This experiment revealed that mAb1 induced significant Vy9V52 T cell proliferation when co-cultured with all three tumor cell lines (Figure 4B).

[0327] In conclusion, the results demonstrate that (i) mAb1 binds to cynomolgus monkey cells with a binding avidity similar to that for human cells, (ii) BTN3A is expressed on the same blood cells in humans and cynomolgus monkeys, although lower expression levels were observed in the latter species, and (iii) mAb1 promotes expansion of a Vγ9Vδ2 T cell subset in cynomolgus monkey PBMCs, and the expanded cells are reactive to mAb1-pulsed tumor target cells.

[0328] mAb1 affects the cynomolgus Vγ9Vδ2 T cell compartment in vivo In vivo studies were performed in healthy female cynomolgus monkeys aged 4-6 years, which received single or repeated intravenous infusions of mAb1 (Table 28).

[0329] The intravenous route of administration was chosen because it is the intended route of human therapy. Animals were treated with mAb1 according to a staggered escalating dose design. [Table 27] The following endpoints were assessed: clinical signs, body weight, clinical pathology (hematology, clinical chemistry and coagulation), immunophenotyping of peripheral blood leukocyte populations, activation / proliferation / differentiation markers, pharmacokinetics and pharmacodynamics (BTN3A receptor occupancy on circulating T and B cells).

[0330] After receiving a single or four repeated 15-minute infusions of mAb1, all animals survived until scheduled necropsy on study day 29. No test article-related effects on body weight or food consumption were observed. Clinical signs were consistent with findings seen in cynomolgus monkeys under laboratory housing conditions and therefore were not attributable to the test article.

[0331] Pharmacokinetics mAb1 exhibited approximately dose-proportional pharmacokinetics following intravenous (IV) administration over the dose range of 0.1 to 100 mg / kg (data not shown) and a long elimination half-life typical of an IgG mAb in the absence of target-mediated clearance. Following repeated dosing at 10 or 100 mg / kg / week for four doses (data not shown), exposure was maintained throughout the treatment period in all animals, with only minimal accumulation throughout the 4-week treatment period. Pharmacokinetic profiles for sentinel animals following administration of 1 and 10 mg / kg IV in an escalating weekly dosing regimen showed some evidence of increased clearance toward the end of the weekly dosing interval, which may be the result of the formation of ADAs to mAb1, although there was no evidence of increased clearance after the final IV dose of 100 mg / kg.

[0332] Receptor Occupancy (RO) According to the BTN3A expression profile and cell population representation in blood, mAb1 RO was measured on CD3+ T cells and CD20+ B cells.

[0333] The results show that BTN3A is rapidly occupied on both CD3+ T cells and CD20+ B cells after mAb1 injection (data not shown). Repeated dosing with 100 mg / kg mAb1 appeared to be required for full receptor occupancy throughout the weekly dosing interval.

[0334] Immunophenotyping At selected time points after each dose, blood from animals receiving mAb1 was stained with a cocktail of specific mAbs and analyzed by flow cytometry to quantify T cell subsets (CD4, CD8, Vγ9 T cells, regulatory T cells), B cells, monocytes, NK cells, mDCs, pDCs, and granulocytes and associated activation markers (CD69, CD86, CD95, granzyme B, Ki67). The analysis included the relative cell numbers (lymphocytes / leukocytes) in each population. percentage ) was extrapolated from total lymphocyte / leukocyte counts determined from simultaneously harvested blood samples and analyzed using a hematology cell counter. Absolute cell count It was included along with.

[0335] Key findings from this extensive analysis include: Vδ9+ T cells (% of CD3+) significantly declined after dosing in all animals receiving mAb1, gradually rebounding in single-dose animals. This effect appears specific, as it is not observed in CD4 and CD8 αβ T cells, suggesting γδ T cell activation and margination in tissues, as observed with CD3 engager bispecific antibodies in monkeys and humans (Smith et al., 2015 Sci Rep. 2015 Dec 11;5:17943. doi:10.1038 / srep17943.). Results are shown in Figure 5.

[0336] This study shows that mAb1 appears to be well tolerated at doses up to 100 mg / kg / week when administered by the IV route. Furthermore, among T cell subsets, Vy9V52 T cells are specifically and significantly affected by mAb1.

[0337] Reference books Alegre, M.-L., Frauwirth, K.A., andThompson, C.B. (2001). T-cellregulation by CD28 and CTLA-4. Nat. Rev. Immunol.1, 220-228. Ausubel, F.M., Brent, R., Kingston, R.E.,Moore, D.D., Seidman, J.G.,and Struhl, K. (1988). Current Protocols inMolecular Biology (John Wiley &Sons). Baudino, L., Shinohara, Y., Nimmerjahn, F.,Furukawa, J.-I., Nakata,M., Martinez-Soria, E., Petry, F., Ravetch, J.V.,Nishimura, S.-I., and Izui, S.(2008). Crucial Role of Aspartic Acid atPosition 265 in the CH2 Domain forMurine IgG2a and IgG2b Fc-AssociatedEffector Functions. J. Immunol. 181,6664-6669. Bensussan, A., and Olive, D. (2005). T-cell:Section report. Cell.Immunol. 236, 3-5. Bird, R.E., Hardman, K.D., Jacobson, J.W., Johnson,S., Kaufman,B.M., Lee, S.M., Lee, T., Pope, S.H., Riordan, G.S., and Whitlow,M. (1988).Single-chain antigen-binding proteins. Science 242, 423-426. Brennan, M., Davison, P.F., and Paulus, H.(1985). Preparation ofbispecific antibodies by chemical recombination ofmonoclonal immunoglobulin G1fragments. Science 229, 81-83. Bryson, C.J., Jones, T.D., and Baker, M.P.(2010). Prediction ofImmunogenicity of Therapeutic Proteins. BioDrugs 24, 1-8. Chapoval, A.I., Ni, J., Lau, J.S., Wilcox,R.A., Flies, D.B., Liu,D., Dong, H., Sica, G.L., Zhu, G., Tamada, K., et al.(2001). B7-H3: Acostimulatory molecule for T cell activation and IFN-γproduction.Nat. Immunol. 2, 269-274. Collins, M., Ling, V., and Carreno, B.M.(2005). The B7 family ofimmune-regulatory ligands. Genome Biol. 6, 223. Coyle, A.J., and Gutierrez-Ramos, J.C.(2001). The expanding B7superfamily: increasing complexity in costimulatorysignals regulating T cellfunction. Nat. Immunol. 2, 203-209. Dong, H., Zhu, G., Tamada, K., and Chen, L.(1999). B7-H1, a thirdmember of the B7 family, co-stimulates T-cellproliferation and interleukin-10secretion. Nat. Med. 5, 1365-1369. Freeman, G.J., Long, A.J., Iwai, Y.,Bourque, K., Chernova, T.,Nishimura, H., Fitz, L.J., Malenkovich, N., Okazaki,T., Byrne, M.C., et al.(2000). Engagement of the Pd-1 ImmunoinhibitoryReceptor by a Novel B7 FamilyMember Leads to Negative Regulation of LymphocyteActivation. J. Exp. Med. 192,1027-1034. Glennie, M.J., McBride, H.M., Worth, A.T.,and Stevenson, G.T.(1987). Preparation and performance of bispecific F(ab’gamma)2antibody containing thioether-linked Fab’ gammafragments. J. Immunol. 139,2367-2375. Goeddel, D.V. (1990). [1] Systems forheterologous gene expression.In Methods in Enzymology, (Academic Press), pp. 3-7. Gu, S., Nawrocka, W., and Adams, E.J.(2015). Sensing ofPyrophosphate Metabolites by Vγ9Vδ2 T Cells. Front. Immunol.5. Harly, C., Guillaume, Y., Nedellec, S.,Peigne, C.-M., Monkkonen, H.,Monkkonen, J., Li, J., Kuball, J., Adams, E.J., Netzer,S., et al. (2012). Keyimplication of CD277 / butyrophilin-3 (BTN3A) in cellularstress sensing by amajor human γδ T-cell subset. Blood 120, 2269-2279. Huston, J.S., Levinson, D., Mudgett-Hunter,M., Tai, M.S., Novotny,J., Margolies, M.N., Ridge, R.J., Bruccoleri, R.E.,Haber, E., and Crea, R.(1988). Protein engineering of antibody binding sites:recovery of specificactivity in an anti-digoxin single-chain Fv analogueproduced in Escherichiacoli. Proc. Natl. Acad. Sci. 85, 5879-5883. Kabat, E.A., Wu, T.T., Foeller, C., Perry,H.M., and Gottesman, K.S.(1992). Sequences of Proteins of ImmunologicalInterest (DIANE Publishing). Karpovsky, B., Titus, J.A., Stephany, D.A.,and Segal, D.M. (1984).Production of target-specific effector cells usinghetero-cross-linkedaggregates containing anti-target cell and anti-Fc gammareceptor antibodies.J. Exp. Med. 160, 1686-1701. Kaufman, R.J., and Sharp, P.A. (1982).Construction of a modulardihydrofolate reductase cDNA gene: analysis ofsignals utilized for efficientexpression. Mol. Cell. Biol. 2, 1304-1319. Khattri, R., Auger, J.A., Griffin, M.D.,Sharpe, A.H., and Bluestone,J.A. (1999). Lymphoproliferative Disorder in CTLA-4Knockout Mice IsCharacterized by CD28-Regulated Activation of Th2 Responses.J. Immunol. 162,5784-5791. Klocke, K., Sakaguchi, S., Holmdahl, R.,and Wing, K. (2016).Induction of autoimmune disease by deletion of CTLA-4 inmice in adulthood.Proc. Natl. Acad. Sci. 113, E2383-E2392. Latchman, Y., Wood, C.R., Chernova, T.,Chaudhary, D., Borde, M.,Chernova, I., Iwai, Y., Long, A.J., Brown, J.A.,Nunes, R., et al. (2001). PD-L2is a second ligand for PD-1 and inhibits T cellactivation. Nat. Immunol. 2,261-268. Linsley, P.S., Greene, J.L., Tan, P.,Bradshaw, J., Ledbetter, J.A.,Anasetti, C., and Damle, N.K. (1992).Coexpression and functional cooperation ofCTLA-4 and CD28 on activated Tlymphocytes. J. Exp. Med. 176, 1595-1604. Liu, M.A., Kranz, D.M., Kurnick, J.T.,Boyle, L.A., Levy, R., andEisen, H.N. (1985). Heteroantibody duplexes targetcells for lysis by cytotoxicT lymphocytes. Proc. Natl. Acad. Sci. 82, 8648-8652. McCafferty, J., Griffiths, A.D., Winter,G., and Chiswell, D.J.(1990). Phage antibodies: filamentous phage displayingantibody variabledomains. Nature 348, 552-554. Morrison, S.L. (1985). Transfectomasprovide novel chimericantibodies. Science 229, 1202-1207. Ni, L., and Dong, C. (2017). New B7 FamilyCheckpoints in HumanCancers. Mol. Cancer Ther. 16, 1203-1211. Oganesyan, V., Gao, C., Shirinian, L., Wu,H., and Dall’Acqua, W.F. (2008). Structural characterization of a humanFcfragment engineered for lack of effector functions. Acta Crystallogr. DBiol.Crystallogr. 64, 700-704. Panowski, S., Bhakta, S., Raab, H.,Polakis, P., and Junutula, J.R.(2013). Site-specific antibody drug conjugatesfor cancer therapy. MAbs 6,34-45. Paulus, H. (1985). Preparation andbiomedical applications ofbispecific antibodies. Behring Inst. Mitt. 118-132. Perry, L.C.A., Jones, T.D., and Baker, M.P.(2008). New approaches toprediction of immune responses to therapeuticproteins during preclinicaldevelopment. Drugs RD 9, 385-396. Reddy, M.P., Kinney, C.A.S., Chaikin, M.A.,Payne, A.,Fishman-Lobell, J., Tsui, P., Monte, P.R.D., Doyle, M.L., Brigham-Burke,M.R.,Anderson, D., et al. (2000). Elimination of Fc Receptor-DependentEffectorFunctions of a Modified IgG4 Monoclonal Antibody to Human CD4. J.Immunol. 164,1925-1933. Remington, J.P., and Gennaro, A.R. (1995).Remington: the science andpractice of pharmacy (Easton, PA: Mack Publishing). Rhodes, D.A., Stammers, M., Malcherek, G.,Beck, S., and Trowsdale,J. (2001). The Cluster of BTN Genes in the ExtendedMajor HistocompatibilityComplex. Genomics 71, 351-362. Ruddy, D.A., Kronmal, G.S., Lee, V.K.,Mintier, G.A., Quintana, L., Domingo,R., Meyer, N.C., Irrinki, A., McClelland,E.E., Fullan, A., et al. (1997). A1.1-Mb Transcript Map of the HereditaryHemochromatosis Locus. Genome Res. 7,441-456. Saverino, D., Tenca, C., Zarcone, D., Merlo,A., Megiovanni, A.M.,Valle, M.T., Manca, F., Grossi, C.E., and Ciccone, E.(1999). CTLA-4 (CD152)Inhibits the Specific LysisMediated by Human Cytolytic T Lymphocytes in aClonally Distributed Fashion. J.Immunol. 162, 651-658. Sharpe, A.H., and Freeman, G.J. (2002). TheB7-CD28 superfamily. Nat.Rev. Immunol. 2, 116-126. Sharpe, A.H., and Pauken, K.E. (2018). Thediverse functions of thePD1 inhibitory pathway. Nat. Rev. Immunol. 18, 153-167. Shukla, A.A., Hubbard, B., Tressel, T.,Guhan, S., and Low, D.(2007). Downstream processing of monoclonal antibodies-Applicationof platformapproaches. J. Chromatogr. B 848, 28-39. Stech, M., Nikolaeva, O., Thoring, L., Stocklein,W.F.M.,Wustenhagen, D.A., Hust, M., Dubel, S., and Kubick, S. (2017).Cell-freesynthesis of functional antibodies using a coupled in vitrotranscription-translationsystem based on CHO cell lysates. Sci. Rep. 7, 12030. Strohl, W.R. (2009). Optimization of Fc-mediatedeffector functionsof monoclonal antibodies. Curr. Opin. Biotechnol. 20, 685-691. Takebe, Y., Seiki, M., Fujisawa, J., Hoy,P., Yokota, K., Arai, K.,Yoshida, M., and Arai, N. (1988). SR alpha promoter: an efficient and versatile mammalian cDNAexpression system composedof the simian virus 40 early promoter and the R-U5segment of human T-cellleukemia virus type 1 long terminal repeat. Mol. Cell.Biol. 8, 466-472. Tseng, S.-Y., Otsuji, M., Gorski, K.,Huang, X., Slansky, J.E., Pai,S.I., Shalabi, A., Shin, T., Pardoll, D.M., andTsuchiya, H. (2001). B7-Dc, aNew Dendritic Cell Molecule with PotentCostimulatory Properties for T Cells. J.Exp. Med. 193, 839-846. Urlaub, G., and Chasin, L.A. (1980).Isolation of Chinese hamstercell mutants deficient in dihydrofolate reductaseactivity. Proc. Natl. Acad.Sci. U. S. A. 77, 4216-4220. Williams, A.F., and Barclay, A.N. (1988). The immunoglobulinsuperfamily-domains for cell surface recognition. Annu. Rev. Immunol. 6,381-405.

Claims

1. 1. A pharmaceutical composition for use in a method for treating a solid tumor in a subject in need thereof comprising administering a therapeutically effective amount of an anti-BTN3A antibody, wherein the anti-BTN3A antibody is an activating antibody capable of inducing activation of Vγ9Vδ2 T cells in co-culture with BTN3A-expressing cells, the anti-BTN3A antibody comprising a mutant IgG1 constant region, the mutant IgG1 constant region having no or reduced binding to an Fcγ receptor compared to a corresponding antibody having a wild-type IgG1 isotype constant region, and the mutant IgG1 constant region is the IgG1 triple mutant L247F, L248E, and P350S.

2. The anti-BTN3A antibody has a K of 5 nM or less for human BTN3A polypeptide as measured by surface plasmon resonance. Ｄ 2. The pharmaceutical composition for use according to claim 1, wherein the compound binds to the hydroxyl group of the formula (I).

3. The anti-BTN3A antibody inhibits activation of Vγ9Vδ2 T cells in co-culture with BTN3-expressing cells at an EC of 5 μg / ml or less, as measured in a degranulation assay. ５０ 3. The pharmaceutical composition for use according to claim 1 or 2, which induces

4. The pharmaceutical composition for use according to any one of claims 1 to 3, wherein the anti-BTN3A antibody has the following characteristics: i. A K of 5 nM or less for human BTN3A polypeptide as measured by surface plasmon resonance Ｄ To combine with ii. An EC of 50 μg / ml or less as measured in a flow cytometry assay ５０ binding to human PBMCs at iii. Activation of Vγ9Vδ2 T cells in co-culture with BTN3-expressing cells at an EC of 5 μg / ml or less, as measured in a degranulation assay. ５０ To guide with

5. 5. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the anti-BTN3A antibody is formulated as an infusion solution and a therapeutically effective amount of the infusion solution is administered by intravenous infusion to the subject in need thereof.

6. The pharmaceutical composition for use according to any one of claims 1 to 5, wherein the anti-BTN3A antibody is administered in combination with an anti-PD1 or anti-PD-L1 antibody.

7. The pharmaceutical composition for use according to any one of claims 1 to 6, wherein the anti-BTN3A antibody is administered in combination with IL-2 or IL-15 or derivatives thereof.

8. 8. The pharmaceutical composition for use according to any one of claims 1 to 7, wherein the anti-BTN3A antibody is administered in combination with an anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, and atezolizumab.

9. 9. The pharmaceutical composition for use according to any one of claims 1 to 8, wherein the anti-BTN3A antibody is administered in combination with pembrolizumab.

10. 10. The pharmaceutical composition for use according to any one of claims 1 to 9, wherein the anti-BTN3A antibody has the ability to induce activation of Vγ9Vδ2 T cells in co-culture with BTN3A-expressing cells, with an EC50 of 1 μg / ml or less as measured in a degranulation assay.

11. 11. The pharmaceutical composition for use according to any one of claims 1 to 10, wherein the method comprises administering a therapeutically effective amount of an anti-BTN3A activating antibody in combination with an anti-PD1 or anti-PD-L1 antibody.

Images