BTN3a-binding protein and medical uses thereof

Abstract

The present invention relates to a BTN3A-binding protein and medical uses thereof. Specifically, the present invention relates to the BTN3A-binding protein, a method thereof for activating γδ T cells and promoting cytokine release in γδ T cells, a method for treating cancer, and medical uses.

Description

BTN3A binding protein and its medical use

[0001] This disclosure claims priority to the Chinese patent application "BTN3A binding protein and its medical use" (application number: 2023109765101) filed on August 4, 2023, and is incorporated herein by reference. Technical Field

[0002] The present disclosure relates to the field of biomedicine, and in particular to a BTN3A binding protein and methods for activating γδT cells and treating cancer, as well as related pharmaceutical uses. Background Art

[0003] The emergence of immunotherapy has transformed the landscape of traditional cancer treatment. Currently, most immunotherapy approaches focus on enhancing the anti-tumor immune response of αβT cells. However, other lymphocyte subsets with anti-tumor potential are still worthy of attention. γδT cells are a special type of T cell subset with innate immune characteristics, accounting for approximately 0.5%-5% of total T cells in the human body. They can directly recognize antigen signals transmitted by specific ligands for activation without relying on the major histocompatibility complex (MHC) and antigen-presenting cells (APCs). T cells all express TCRs and are divided into αβT and γδT based on the different TCR genes. αβT cells express Vα and VβTCR chains, while γδT cells express Vγ and VδTCR chains. γδT cells have a total of seven VγTCR chains (Vγ2, 3, 4, 5, 8, 9, and 11) and four Vδ chains (Vδ1, 2, 3, and 5). Based on the different δ chains, human γδT cells are divided into three major subpopulations: Vδ1T, Vδ2T, and Vδ3T. Vδ1T cells are primarily found in mucosal epithelial tissues, Vδ2T cells are primarily found in peripheral blood, and Vδ3T cells are primarily distributed in the liver and intestines. The Vγ9Vδ2T cell subpopulation, which accounts for approximately 1 to 5% of total T cells in peripheral blood, is the most studied subpopulation. Studies have shown that activated Vγ9Vδ2T cells can kill cancer cells by secreting cytokines such as IFN-γ and TNF-α, or releasing perforins and granzymes. Activated Vγ9Vδ2T cells also proliferate, further exerting an anti-tumor effect.

[0004] In recent years, butyrophilin subfamily 3, member A (BTN3A) has emerged as a key molecule regulating Vγ9Vδ2 T cell function. The BTN3A family comprises three isoforms: BTN3A1 (also known as CD277), BTN3A2, and BTN3A3. These proteins are expressed by three distinct genes on human chromosome 6 and are expressed in humans and nonhuman primates (NHPs). The BTN3A family is a type I transmembrane protein. The extracellular domain of each BTN3A isoform consists of an IgV domain distal to the cell membrane and an IgC domain proximal to the cell membrane. The extracellular domains of the three isoforms share up to 95% sequence identity. The cytoplasmic regions of BTN3A1 and BTN3A3 contain a B30.2 domain, which is absent in BTN3A2. Recent studies have found that under bacterial or viral stimulation and under stress conditions, phosphorylated antigens (pAg) that gradually accumulate within cells are recognized and bound by the intracellular B30.2 domain of the BTN3A protein. After binding to pAg, the extracellular domain of the BTN3A protein changes conformation, further promoting the formation of a complex with the BTN2A1 protein on the cell membrane. The protein then binds to the Vγ9 chain of Vγ9Vδ2 T cells through the IgV domain of BTN2A1, thereby activating Vγ9Vδ2 T cells to function. Because BTN3A3 has a poor affinity for pAg, only BTN3A1 can sense the increase in intracellular pAg and activate Vγ9Vδ2 T cells.

[0005] Recent studies have found that by designing antibodies that target the extracellular domain of BTN3A1, they can replace intracellular pAg and more efficiently activate Vγ9Vδ2 T cells. Furthermore, BTN3A1 antibodies have the potential to enhance the anti-tumor ability of αβ T cells.

[0006] The present disclosure provides an anti-BTN3A antibody with a new sequence structure, which has excellent activity in activating γδT cells and promoting the release of cytokines by γδT cells, thereby enhancing the killing of tumor cells by γδT cells, and has good drugability and potential to become a clinical drug.

[0007] Summary of the Invention

[0008] The present disclosure provides BTN3A binding proteins, encoding nucleic acids, vectors, host cells, pharmaceutical compositions, and methods for activating γδT cells, promoting γδT cell cytokine release, treating or preventing diseases (e.g., cancer), and related pharmaceutical uses.

[0009] BTN3A binding protein

[0010] The present disclosure provides a BTN3A binding protein comprising a domain that specifically binds to BTN3A, wherein the domain comprises a heavy chain variable region (VH) and / or a light chain variable region (VL). In some embodiments, the BTN3A binding protein comprises one or more domains that specifically bind to BTN3A.

[0011] The present disclosure provides BTN3A binding proteins comprising a heavy chain variable region (VH) and / or a light chain variable region (VL).

[0012] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 17-19, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 20-22, respectively.

[0013] HCDR1 (SEQ ID NO: 17): X1YYX2X3, wherein X1 is selected from D or N, X2 is selected from I or M, and X3 is selected from T or N;

[0014] HCDR2 (SEQ ID NO: 18): X4IYGSX5NTX6YASWAKG, wherein X4 is selected from V or I, X5 is selected from S or D, and X6 is selected from V or Y;

[0015] HCDR3(SEQ ID NO:19):X7X8X9X 10 SSAX 11 X 12 X 13 X 14 , wherein X7 is selected from G or N, X8 is selected from Y or L, X9 is selected from L or D, X 10 Selected from A or Y, X 11 Selected from D or Y, X 12 Selected from I or F, X 13 Does not exist or is H, X 14 Does not exist or is 1;

[0016] LCDR1 (SEQ ID NO: 20): QX 15 SQSVYNNNRLA, where X 15 Select from S or A;

[0017] LCDR2 (SEQ ID NO: 21): X 16 ASX 17 LAS, where X 16 Select from D or E, X 17 Select from T or K;

[0018] LCDR3 (SEQ ID NO: 22): QX 18 YYSGX 19 IX 20 X 21 , where X 18 Selected from T or G, X 19 Select from Y or F, X 20 Select from W or Y, X 21 Select A or P.

[0019] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, 31-35, and 39-44, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 2, 4, 36-38, and 45-47.

[0020] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence of any one of SEQ ID NOs: 1 and 31-35, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence of any one of SEQ ID NOs: 2 and 36-38.

[0021] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 5-7, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NOs: 8-10, respectively;

[0022] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence of any one of SEQ ID NOs: 3 and 39-44, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence of any one of SEQ ID NOs: 4 and 45-47;

[0023] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 11-13, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 14-16, respectively.

[0024] The above CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems, for example, according to the Kabat numbering system.

[0025] In some embodiments, the BTN3A binding protein comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 1, 3, 31-35, 39-44, or an amino acid sequence having at least 80% or at least 90% identity thereto, and / or a light chain variable region as shown in any one of SEQ ID NOs: 2, 4, 36-38, 45-47, or an amino acid sequence having at least 80% or at least 90% identity thereto.

[0026] In some embodiments, the BTN3A binding protein comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 1, 31-35, or an amino acid sequence having at least 80% or at least 90% identity thereto, and a light chain variable region as shown in any one of SEQ ID NOs: 2, 36-38, or an amino acid sequence having at least 80% or at least 90% identity thereto.

[0027] In some specific embodiments, the BTN3A binding protein comprises:

[0028] a heavy chain variable region as set forth in SEQ ID NO: 1, or an amino acid sequence at least 80%, at least 90% identical thereto, and a light chain variable region as set forth in SEQ ID NO: 2, or an amino acid sequence at least 80%, at least 90% identical thereto;

[0029] a heavy chain variable region as shown in any one of SEQ ID NOs: 31-35, or an amino acid sequence at least 80% or at least 90% identical thereto, and a light chain variable region as shown in SEQ ID NO: 36, or an amino acid sequence at least 80% or at least 90% identical thereto;

[0030] A heavy chain variable region as shown in any one of SEQ ID NOs: 31-35, or an amino acid sequence at least 80% or at least 90% identical thereto, and a light chain variable region as shown in SEQ ID NO: 37, or an amino acid sequence at least 80% or at least 90% identical thereto; or

[0031] A heavy chain variable region as shown in any one of SEQ ID NOs: 31-35, or an amino acid sequence at least 80%, at least 90% identical thereto, and a light chain variable region as shown in SEQ ID NO: 38, or an amino acid sequence at least 80%, at least 90% identical thereto.

[0032] In some embodiments, the BTN3A binding protein comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 3, 39-44, or an amino acid sequence having at least 80% or at least 90% identity thereto, and a light chain variable region as shown in any one of SEQ ID NOs: 4, 45-47, or an amino acid sequence having at least 80% or at least 90% identity thereto.

[0033] In some specific embodiments, the BTN3A binding protein comprises:

[0034] a heavy chain variable region as set forth in SEQ ID NO: 3, or an amino acid sequence at least 80%, at least 90% identical thereto, and a light chain variable region as set forth in SEQ ID NO: 4, or an amino acid sequence at least 80%, at least 90% identical thereto;

[0035] a heavy chain variable region as shown in any one of SEQ ID NOs: 39-44, or an amino acid sequence at least 80%, at least 90% identical thereto, and a light chain variable region as shown in SEQ ID NO: 45, or an amino acid sequence at least 80%, at least 90% identical thereto;

[0036] a heavy chain variable region as set forth in any one of SEQ ID NOs: 39-44, or an amino acid sequence at least 80%, at least 90% identical thereto, and a light chain variable region as set forth in SEQ ID NO: 46, or an amino acid sequence at least 80%, at least 90% identical thereto;

[0037] A heavy chain variable region as shown in any one of SEQ ID NOs: 39-44, or an amino acid sequence at least 80%, at least 90% identical thereto, and a light chain variable region as shown in SEQ ID NO: 47, or an amino acid sequence at least 80%, at least 90% identical thereto.

[0038] In some embodiments, the aforementioned BTN3A binding protein is an anti-BTN3A antibody or an antigen-binding fragment thereof.

[0039] In some embodiments, the aforementioned antibody is a rabbit-derived antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0040] In some embodiments, the aforementioned antibody is a recombinant antibody.

[0041] In some embodiments, the aforementioned antibody is a monospecific antibody or a multispecific antibody (eg, a bispecific antibody, a trispecific antibody, a tetraspecific antibody).

[0042] In some embodiments, the aforementioned antibodies or antigen-binding fragments thereof are single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above.

[0043] In some embodiments, the aforementioned antibodies or antigen-binding fragments thereof are humanized, backmutated, affinity matured, T cell epitope (TCE) removed / reduced, antibody deamidation reduced, and / or antibody isomerization reduced.

[0044] In some embodiments, after TCE removal / reduction engineering, there are one or more changes in one or more CDRs, which result in reduced immunogenicity of the BTN3A binding protein.

[0045] In some embodiments, the humanization process uses a human germline template whose heavy chain framework region is derived from IGHV3-66 and whose light chain framework region is derived from IGkV1-27.

[0046] In some embodiments, the BTN3A binding protein further comprises an immunoglobulin Fc region. In some specific embodiments, the Fc region is an Fc region of human IgG1, human IgG2, human IgG3, or human IgG4. In some specific embodiments, the Fc region may be an Fc region with reduced effector function, for example, the Fc region may have a mutation, an exemplary IgG with reduced effector function Fc regions include those with the following substitutions: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); H268Q / V309L / A330S / A331S (IgG2); C220S / C226S / C229S / P238S (IgG1); C226S / C229S / E233P / L234V / L235A (IgG1); L234F / L235E / P331S (IgG1); or S267E / L328F (IgG1), wherein “ / ” represents “and”. In some embodiments, the Fc region is a human IgG1 Fc region having L234F, L235E and / or P331S mutations, optionally with K446 removed. In some embodiments, the Fc region has SEQ ID NO: 23, 24, or an amino acid sequence having at least 80% or 90% identity thereto.

[0047] In some embodiments, the Fc region has increased stability or has mutations that increase the stability of the Fc region compared to the wild-type Fc region. In some embodiments, the Fc region can enable the binding protein to form a dimeric molecule. In some embodiments, the Fc region can extend the in vivo half-life of the binding protein.

[0048] In some embodiments, the BTN3A binding protein comprises a heavy chain as shown in any one of SEQ ID NOs: 25, 27, 48-52, 56-61, or an amino acid sequence having at least 80% or at least 90% identity thereto, and / or a light chain as shown in any one of SEQ ID NOs: 26, 28, 53-55, 62-64, or an amino acid sequence having at least 80% or at least 90% identity thereto.

[0049] In some specific embodiments, the BTN3A binding protein comprises:

[0050] a heavy chain as set forth in SEQ ID NO: 25, or an amino acid sequence at least 80%, at least 90% identical thereto, and a light chain as set forth in SEQ ID NO: 26, or an amino acid sequence at least 80%, at least 90% identical thereto;

[0051] a heavy chain as set forth in any one of SEQ ID NOs: 48-52, or an amino acid sequence at least 80%, at least 90% identical thereto, and a light chain as set forth in SEQ ID NO: 53, or an amino acid sequence at least 80%, at least 90% identical thereto;

[0052] A heavy chain as shown in any one of SEQ ID NOs: 48-52, or an amino acid sequence at least 80%, at least 90% identical thereto, and a light chain as shown in SEQ ID NO: 54, or an amino acid sequence at least 80%, at least 90% identical thereto; or

[0053] A heavy chain as shown in any one of SEQ ID NOs: 48-52, or an amino acid sequence at least 80%, at least 90% identical thereto, and a light chain as shown in SEQ ID NO: 55, or an amino acid sequence at least 80%, at least 90% identical thereto.

[0054] In some specific embodiments, the BTN3A binding protein comprises:

[0055] a heavy chain as set forth in SEQ ID NO: 27, or an amino acid sequence at least 80%, at least 90% identical thereto, and a light chain as set forth in SEQ ID NO: 28, or an amino acid sequence at least 80%, at least 90% identical thereto;

[0056] a heavy chain as depicted in any one of SEQ ID NOs: 56-61, or an amino acid sequence at least 80%, at least 90% identical thereto, and a light chain as depicted in SEQ ID NO: 62, or an amino acid sequence at least 80%, at least 90% identical thereto;

[0057] A heavy chain as shown in any one of SEQ ID NOs: 56-61, or an amino acid sequence at least 80%, at least 90% identical thereto, and a light chain as shown in SEQ ID NO: 63, or an amino acid sequence at least 80%, at least 90% identical thereto; or

[0058] A heavy chain as shown in any one of SEQ ID NOs: 56-61, or having an amino acid sequence at least 80%, at least 90% identical thereto, and a light chain as shown in SEQ ID NO: 64, or having an amino acid sequence at least 80%, at least 90% identical thereto.

[0059] In the present disclosure, "at least 80% (sequence) identity" encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity; "at least 90% (sequence) identity" encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity.

[0060] In some embodiments, the BTN3A binding protein specifically binds to one or more of BTN3A1, BTN3A2, and BTN3A3, for example:

[0061] It specifically binds to BTN3A1 alone, but not to BTN3A2 or BTN3A3;

[0062] It specifically binds to BTN3A2 alone, but not to BTN3A1 or BTN3A3;

[0063] It specifically binds to BTN3A3 alone, but not to BTN3A1 or BTN3A2;

[0064] Specifically binds to BTN3A1 and BTN3A2, but not BTN3A3;

[0065] Specifically binds to BTN3A2 and BTN3A3, but not BTN3A1;

[0066] Specifically binds to BTN3A1 and BTN3A3, but not BTN3A2;

[0067] It also specifically binds to BTN3A1, BTN3A2, and BTN3A3.

[0068] In some specific embodiments, the aforementioned BTN3A1, BTN3A2, and BTN3A3 are primate or human BTN3A1, BTN3A2, and BTN3A3.

[0069] In some embodiments, the BTN3A binding proteins of the present disclosure have one or more of the following characteristics:

[0070] (i) as measured by ELISA with an EC of 0.1 nM, 0.05 nM, 0.02 nM, 0.01 nM or lower 50 Binds to human BTN3A1. The ELSIA detection method is well known in the art, such as described in Part 1 of Example 3 of the present disclosure.

[0071] (ii) with an EC of 50 nM, 20 nM, 15 nM, 10 nM, 8 nM, 5 nM or less as measured by FACS 50 Binding to human BTN3A1 on cells, the FACS detection method is well known in the art, such as described in Part 2 of Example 3 of the present disclosure;

[0072] (iii) as measured by Biacore with a K of 5E-10 M, 2E-10 M, 1E-10 M or less D Binding to human BTN3A1, the Biacore detection method is well known in the art, such as described in Part 3 of Example 3 of the present disclosure;

[0073] (iv) with an EC of 10 nM, 5 nM, 2 nM, 1 nM or lower 50 Inducing the activation and proliferation of γδT cells (e.g., Vγ9Vδ2T cells), the EC 50 The detection method is well known in the art, such as described in Section 4 of Example 3 of the present disclosure; In some embodiments, the induction occurs when γδT cells are co-cultured with cancer cells (e.g., SKOV-3 cells);

[0074] (v) inducing γδT cells (eg, Vγ9Vδ2T cells) to secrete cytokines (eg, IFNγ); in some embodiments, the induction occurs when the γδT cells are co-cultured with cancer cells (eg, SKOV-3 cells).

[0075] The present disclosure also provides a BTN3A binding protein comprising any one or any combination of the following CDRs: HCDR1, HCDR2, and HCDR3 having amino acid sequences shown in SEQ ID NOs: 17-19, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences shown in SEQ ID NOs: 20-22, respectively.

[0076] In some embodiments, the present disclosure provides the aforementioned BTN3A binding protein variants, which have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations compared to the heavy chain variable region of the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, 31-35, 39-44 and / or the light chain variable region of the amino acid sequence shown in any one of SEQ ID NOs: 2, 4, 36-38, 45-47; the amino acid mutations may be conservative replacements, substitutions or modifications, and / or deletions or additions that do not affect the function; the amino acid mutations may occur in the CDR region and / or the FR region.

[0077] In some embodiments, proteins or molecules are provided that bind to or compete for binding to the same epitope as the aforementioned BTN3A binding proteins of the present disclosure.

[0078] In some embodiments, proteins or molecules are provided that block the binding of the aforementioned BTN3A binding proteins of the present disclosure to BTN3A (including BTN3A1, BTN3A2 and / or BTN3A3).

[0079] In some embodiments, a protein or molecule is provided whose binding to BTN3A (including BTN3A1, BTN3A2 and / or BTN3A3) is blocked by the aforementioned BTN3A binding protein of the present disclosure.

[0080] In some embodiments, a protein or molecule is provided, comprising a combination of any one or more heavy chain variable regions and light chain variable regions disclosed herein, wherein the heavy chain variable region is selected from SEQ ID NOs: 1, 3, 31-35, 39-44, and the light chain variable region is selected from SEQ ID NOs: 2, 4, 36-38, 45-47. For example, the protein or molecule is a conjugate, and the conjugate may comprise any detectable label.

[0081] Polynucleotides and vectors

[0082] The present disclosure provides polynucleotides encoding the BTN3A binding proteins of the present disclosure. The nucleic acids of the present disclosure may be RNA, DNA (such as cDNA). According to some embodiments of the present disclosure, the nucleic acids of the present disclosure are substantially isolated nucleic acids.

[0083] The nucleic acids of the present disclosure may also be in the form of, may be present in, and / or may be part of, a vector, such as a plasmid, cosmid, YAC, or viral vector. The vector may particularly be an expression vector, i.e., a vector that provides for expression of the BTN3A binding protein in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). The expression vector typically comprises at least one nucleic acid of the present disclosure, operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). It is common knowledge for those skilled in the art to select such elements and their sequences for expression in a particular host. Regulatory elements and other elements useful or necessary for expression of the BTN3A binding protein of the present disclosure are, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, and reporter genes.

[0084] The nucleic acids of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and / or recombinant DNA technology) based on the information of the amino acid sequence of the polypeptides of the present disclosure, and / or can be isolated from suitable natural sources.

[0085] host cells

[0086] The present disclosure provides recombinant host cells that express or are capable of expressing one or more BTN3A binding proteins of the present disclosure and / or contain polynucleotides or vectors of the present disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0087] Examples of bacterial cells include cells of gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0088] Exemplary fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0089] Examples of mammalian cells include HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0090] However, the present disclosure may also be used with amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

[0091] Preparation method

[0092] The present disclosure provides a method for preparing a BTN3A binding protein, comprising: expressing the target protein in a host cell as described above, and isolating the target protein from the host cell. Optionally, a purification step may be included, for example, purification using an A or G Sepharose FF column containing an adjusted buffer, washing away non-specifically bound components, eluting the bound antibodies using a pH gradient method, detecting with SDS-PAGE, and collecting. Optionally, filtration and concentration are performed using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The obtained product must be immediately frozen, such as at -70°C, or freeze-dried.

[0093] Methods for producing and purifying antibodies are well known in the art and can be found in, for example, the Cold Spring Harbor Manual of Antibody Laboratory Techniques (Chapters 5-8 and 15).

[0094] The binding proteins disclosed herein (such as engineered antibodies or antigen-binding fragments) can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. Mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to antigens. Positive clones are expanded and cultured in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes the antibodies can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange.

[0095] Composition

[0096] The present disclosure provides compositions comprising the aforementioned BTN3A-binding protein of the present disclosure. For example, pharmaceutical compositions are provided, comprising an effective amount of the aforementioned BTN3A-binding protein for treating, alleviating, or preventing a disease, and at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0097] In some embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of the BTN3A binding protein in a unit dose, or the amount of the BTN3A binding protein in a unit dose of the pharmaceutical composition may be 0.1-2000 mg, and in some embodiments, 1-1000 mg.

[0098] In some embodiments, an article or product (e.g., a kit) is provided that includes the aforementioned BTN3A binding protein. Optionally, the article comprises a container and a label. The container, for example, a bottle, syringe, or test tube, holds a composition effective for treating a condition. The label on or associated with the container indicates that the composition is used to treat the selected condition.

[0099] In some embodiments, the aforementioned disease is cancer.

[0100] Methods of treatment and pharmaceutical uses

[0101] The present disclosure provides methods for using the aforementioned BTN3A binding proteins, polynucleotides, and compositions (including pharmaceutical compositions) for treating, alleviating, preventing, and diagnosing diseases or disorders.

[0102] In some embodiments, a method for improving, alleviating, treating or preventing a disease is provided, comprising administering to a subject in need thereof an effective amount of the aforementioned BTN3A binding protein, polynucleotide, composition (including pharmaceutical composition).

[0103] In some embodiments, the present disclosure provides uses of the BTN3A binding proteins, polynucleotides, and compositions (including pharmaceutical compositions) for preparing drugs for improving, alleviating, treating, or preventing diseases.

[0104] In some embodiments, the aforementioned disease is a disease or disorder associated with overexpression of BTN3A.

[0105] In some embodiments, the aforementioned disease is a disease or condition mediated by BTN3A.

[0106] In some embodiments, the aforementioned disease is cancer.

[0107] In some embodiments, a method for activating γδT cells and promoting cytokine release by γδT cells is provided, comprising administering or contacting the γδT cells with a BTN3A binding protein, polynucleotide, or composition (including a pharmaceutical composition), wherein the γδT cells can be in vivo or in vitro in a subject in need thereof. In some specific embodiments, the γδT cells are Vγ9Vδ2T cells.

[0108] Detection

[0109] The present disclosure provides detection uses of BTN3A binding proteins, polynucleotides, and compositions. The present disclosure also provides methods, systems, or devices for in vivo or in vitro detection of BTN3A, comprising treating a sample with the aforementioned binding proteins, polynucleotides, and compositions of the present disclosure.

[0110] In some embodiments, an in vitro detection method, system, or device may include, for example:

[0111] (1) contacting a sample with a BTN3A binding protein, polynucleotide, or composition disclosed herein;

[0112] (2) detecting a complex formed between the aforementioned binding protein, polynucleotide, and sample; and / or

[0113] (3) contacting a reference sample (e.g., a control sample) with the binding protein and nucleic acid; and

[0114] (4) Determining the extent of complex formation by comparison with a reference sample. A change (e.g., a statistically significant change) in complex formation in the sample compared to the control sample indicates the presence of BTN3A in the sample.

[0115] In some embodiments, a kit is also provided, comprising the aforementioned BTN3A binding protein and polynucleotide, and instructions for diagnostic use. The kit may also contain at least one additional reagent, such as a marker or an additional diagnostic agent. For in vivo use, the BTN3A binding protein may be formulated as a pharmaceutical composition.

[0116] Definition of terms

[0117] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in the present disclosure, all other technical and scientific terms used in the present disclosure have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0118] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0119] "BTN3A" has its ordinary meaning in the art and refers to human BTN3A polypeptide, illustratively, BTN3A1 set forth in NP_008979.3 (Genbank Accession NO), BTN3A2 set forth in NP_008978.2 (Genbank Accession NO), or BTN3A3 set forth in NP_008925.1 (Genbank Accession NO).

[0120] "BTN3A binding protein" encompasses any protein that can specifically bind to BTN3A or any molecule comprising the protein, including but not limited to anti-BTN3A antibodies, antigen-binding fragments thereof, or conjugates thereof as defined herein. In some embodiments, the BTN3A binding protein may comprise a linker and / or a portion having effector function, such as a half-life extending portion (e.g., an immunoglobulin single variable domain that binds serum albumin) and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region.

[0121] "Antibodies" encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Antibodies may refer to immunoglobulins, which are tetrapeptide chains composed of two heavy chains and two light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant regions of immunoglobulins' heavy chains differ, resulting in different antigenicity. Based on this, immunoglobulins can be divided into five classes, or so-called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Igs of the same class can be further divided into different subclasses based on differences in the amino acid composition of their hinge regions and the number and position of heavy chain disulfide bonds, such as IgG, which can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either kappa or lambda chains. The approximately 110 amino acids near the N-terminus of antibody heavy and light chains vary greatly in sequence and constitute the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region comprises three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). These three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0122] The antibodies of the present disclosure may be polyclonal, monoclonal, xenogeneic, allogeneic, isogenic, or modified forms thereof, with monoclonal antibodies being particularly suitable for use in a number of embodiments. In general, the antibodies of the present disclosure are recombinant antibodies. As used herein, "recombinant" refers generally to products such as cells or nucleic acids, proteins, or vectors, indicating that the cells, nucleic acids, proteins, or vectors have been modified by the introduction of heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or that the cells are derived from cells so modified. For example, recombinant cells express genes that are not present in the native (non-recombinant) cell form or express native genes that are abnormally expressed, underexpressed, or not expressed at all.

[0123] "Antigen-binding fragments" encompass single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and preparing these antigen-binding fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0124] For the determination or definition of CDRs, the definitive depiction of CDRs and the identification of residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. This can be accomplished by any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions. The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8). The Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the position of certain structural loop regions (see, e.g., Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83). The AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group that model antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd.). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from primary sequence (see those described in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198). Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In the conformational definition, the positions of the CDRs can be identified as residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166).In addition, other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental results that a particular residue or residue group does not significantly affect antigen binding. As used in this disclosure, CDRs can refer to CDRs defined by any method known in the art (including combinations of methods). The correspondence between the various numbering systems is well known to those skilled in the art.

[0125] A "domain" of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and in many cases can be added, removed, or transferred to other proteins without loss of function of the rest of the protein and / or the domain.

[0126] "Antibody framework (FR)" refers to the portion of a variable domain that serves as a scaffold for the antigen binding loops (CDRs) of that variable domain.

[0127] "Humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting non-human CDR sequences into the human antibody variable region framework. It can overcome the strong immune response induced by chimeric antibodies due to carrying a large amount of non-human protein components. In order to avoid a decrease in activity while reducing immunogenicity, the fully human antibody variable region can be subjected to minimal reverse mutation to maintain activity. Examples of "humanization" include "humanizing" a VHH domain derived from Camelidae by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at the corresponding positions in the VH domain of a human conventional tetrapeptide chain structure antibody. The humanized VHH domain may contain one or more completely human framework region sequences, and in some specific embodiments, may contain the human framework region sequence of IGHV3. Humanization methods such as protein surface amino acid humanization (resurfacing) and antibody humanization universal framework transplantation (CDR grafting to a universal framework), that is, CDR "grafting" on other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Scaffolds and technologies suitable for the CDR transplantation are known in the art. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the VBase human germline sequence database, as well as in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies disclosed herein also include humanized antibodies after affinity maturation of CDRs by phage display. In addition, in order to avoid a decrease in immunogenicity and the resulting decrease in activity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.

[0128] An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody for the antigen, compared to a parent antibody that does not possess such alterations. For example, an "affinity matured" BTN3A binding protein or anti-BTN3A antibody has one or more changes in one or more CDRs that result in increased affinity for the antigen compared to its parent antibody. Affinity matured antibodies can be prepared by methods known in the art, for example, as described in Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813; Shier et al., 1995, Gene 169:147-155; Yelton et al., 1995, Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J. MoI. Biol. 226(3):889-896; KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

[0129] Typically, the BTN3A binding proteins of the present disclosure will be expressed as preferably 10 -7 to 10 -10 Mole / liter (M), more preferably 10 -8 to 10 -10 mol / L, even more preferably 10 -9 to 10 -10 or lower dissociation constant (K D ), and / or with at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, more preferably at least 10 -10 The association constant (KA) of M binds to the antigen or target protein (i.e. BTN3A1, BTN3A2, BTN3A3). Any value greater than 10 -4 M's K DValues ​​are generally considered to indicate nonspecific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competitive assays) as described herein.

[0130] "Binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen). The binding affinity between two molecules can be determined by determining the dissociation constant (K D K can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods (Biacore). D The rate constants corresponding to the association and dissociation of a monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D Through equation K D = kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods and can even be calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9: 340-362). For example, K can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). D Other standard assays for assessing the binding ability of an antibody to a target antigen are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can also be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore TM The K of each antibody / antigen complex can be compared by comparing the K D The K values ​​can be used to compare the binding affinities associated with different molecular interactions, for example, the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the K values ​​of the interactions of interest (e.g., the specific interaction between an antibody and an antigen). D The K values ​​were compared with those of non-target interactions (e.g., control antibodies known not to bind BTN3A). D The value is evaluated.

[0131] A "conservative substitution" refers to a substitution with another amino acid residue having properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have non-polar side chains. In addition, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when substituting an amino acid residue in a group that exhibits similar properties as described above, it will not exhibit specific changes in properties.

[0132] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0133] "Nucleic acid" or "polynucleotide" are used interchangeably in this disclosure to refer to any DNA or RNA molecule, whether single-stranded or double-stranded, and, in the case of single-stranded DNA, its complementary sequence, preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0134] "Host cell" includes individual cells or cell cultures that can be or have been recipients of vectors for incorporating polynucleotide inserts. Host cells include the progeny of a single host cell, and due to natural, accidental or intentional mutations, the progeny may not necessarily be identical (in morphology or genomic DNA complement) to the original parent cell. Host cells include cells transfected and / or transformed in vivo with the polynucleotides of the present disclosure. "Cell," "cell line," and "cell culture" are used interchangeably, and all such designations include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be precisely identical in terms of DNA content. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included.

[0135] "Inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking. "Inhibit growth" (eg, involving cells) is intended to include any measurable decrease in cell growth.

[0136] "Administer," "apply," and "treat" as applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid, for example, therapeutic, pharmacokinetics, diagnostic, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, as well as contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "apply," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. When applied to humans, veterinary medicine, or research subjects, it refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0137] "Treatment" means administering an internal or external therapeutic agent, such as a binding protein or a pharmaceutical composition thereof, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases or symptoms thereof, and for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the treated subject or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of the therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical test method commonly used by a physician or other health care professional to evaluate the severity or progression of the symptoms. Although an embodiment of the present disclosure (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the symptoms of the target disease in a certain subject, it should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0138] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or signs of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0139] "Optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and the description includes instances where the event or circumstance occurs or does not occur. "And / or" should be taken as specifically disclosing that each of the two specified features or components has or does not have the other. Thus, the term "and / or" as used in phrases such as "A and / or B" in the present disclosure includes "A and B," "A or B," "A" (alone), and "B" (alone). Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be understood to have an inclusive meaning, rather than an exclusive or exhaustive meaning; that is, the meaning of "including but not limited to." In the context of mutations contained in the Fc region in the present disclosure, " / " means "and," for example, "L234A / L235A" means "L234A and L235A," i.e., the Fc contains L234A and L235A mutations; the amino acid positions of the mutations in the Fc region of the present disclosure are all defined according to the EU numbering system.

[0140] The "subject" and "patient" of the present disclosure refer to mammals, especially primates, and especially humans. BRIEF DESCRIPTION OF THE DRAWINGS

[0141] FIG1 shows the ELISA test results of the binding ability of anti-BTN3A1 chimeric antibody to human BTN3A1 recombinant protein.

[0142] FIG2 shows the FACS detection results of the binding ability of anti-BTN3A1 chimeric antibodies to human BTN3A1 overexpressing cells.

[0143] Figure 3 shows the results of the anti-BTN3A1 chimeric antibody's ability to activate γδT cells. Vγ9Vδ2 T cells were co-cultured with A375 target cells, incubated with the test antibody, and the secreted human IFNγ content in the cell supernatant was measured by ELISA.

[0144] Figure 4 shows the results of the BTN3A1 chimeric antibody-mediated cytotoxicity test against A375 cells by γδT cells. Vγ9Vδ2T cells and A375 cells were co-cultured and incubated with the test antibody. The cells were lysed with the reagent and the luminescence activity was measured using a microplate reader.

[0145] Figure 5 shows the results of the 20A3 humanized antibody's ability to activate γδT cells. Vγ9Vδ2 T cells were co-cultured with A375 target cells, incubated with the test antibody, and the secreted human IFNγ content in the cell supernatant was measured by ELISA.

[0146] FIG6A shows the results of FACS detection of the binding ability of the humanized antibody 17G4 to human BTN3A1-overexpressing cells, and FIG6B shows the results of FACS detection of the binding ability of the humanized antibody 20A3 to human BTN3A1-overexpressing cells.

[0147] Figure 7A shows the FACS detection results of 20A3-H2L2's binding ability to human BTN3A1-overexpressing cells, Figure 7B shows the FACS detection results of 20A3-H2L2's binding ability to human BTN3A2-overexpressing cells, and Figure 7C shows the FACS detection results of 20A3-H2L2's binding ability to human BTN3A3-overexpressing cells.

[0148] Figure 8 shows the results of the 20A3-H2L2 activation ability test on γδT cells. Vγ9Vδ2T cells were co-cultured with SKOV-3 target cells, incubated with the test antibody, and the secreted human IFNγ content in the cell supernatant was measured by ELISA.

[0149] Figure 9 shows the results of the 20A3-H2L2-mediated cytotoxicity test against SKOV-3 cells by γδT cells. Vγ9Vδ2T cells and SKOV-3 cells were co-cultured and incubated with the test antibody. The cells were lysed with the reagent and the luminescence activity was measured using a microplate reader. DETAILED DESCRIPTION

[0150] The following examples further illustrate the present disclosure, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples herein where specific conditions are not specified generally follow conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Techniques and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents where the specific source is not specified are commercially available.

[0151] Example 1. Screening and preparation of rabbit monoclonal antibodies against human BTN3A1

[0152] 1. Immune antigens and screening antigens

[0153] Human BTN3A1 recombinant protein with mouse IgG2a Fc and his tags (h-BTN3A1-mFc-his), biotin-modified human BTN3A1 recombinant protein with his and avi tags (Bio-h-BTN3A1-his-avi), and his-tagged human BTN3A1 recombinant protein (h-BTN3A1-his) are all commercial protein reagents. The sources of their respective sequences are shown in Table 1. These protein reagents can be used in the following experiments.

[0154] Table 1. Sources of recombinant protein amino acid sequences

[0155] 2. Screening of Rabbit Monoclonal Antibodies Against Human BTN3A1

[0156] Antibodies were produced by immunizing New Zealand white rabbits.

[0157] Experimental Methods: The immunizing antigen was a recombinant human BTN3A1 protein with a mouse IgG2a Fc and his-tag (h-BTN3A1-mFc-his). The primary immunization was emulsified with an equal volume of antigen in complete Freund's adjuvant (CFA) at a dose of 300 μg / animal. For subsequent booster immunizations, the antigen was emulsified with an equal volume of antigen in incomplete Freund's adjuvant (IFA) at a dose of 150 μg / animal. Immunizations were performed on days 0, 22, 36, and 50. Blood was collected on day 42 for analysis, and antibody titers in New Zealand white rabbit serum were determined by ELISA. Spleens from rabbits with high titers detected by ELISA were isolated, and B cells binding to the biotinylated human BTN3A1 (Bio-h-BTN3A1-his-avi) recombinant protein were isolated by flow cytometry. The cells were plated in 96-well plates at a density of less than one B cell per well. After two weeks of culture, the B cell supernatant was collected.

[0158] 3. ELISA Binding Assay of Anti-BTN3A1 Rabbit Monoclonal Antibody to BTN3A1 Recombinant Protein

[0159] The binding properties of rabbit B cell supernatant to human BTN3A1 were detected by ELISA experiment.

[0160] Experimental Method: A 96-well microtiter plate was coated with His-tagged human BTN3A1 (h-BTN3A1-his) recombinant protein. After adding the supernatant of a rabbit monoclonal antibody, antibody-antigen binding activity was detected by adding a secondary antibody (HRP-conjugated anti-primary Fc antibody) and the HRP substrate TMB. A 96-well microtiter plate was coated with His-tagged human BTN3A1 (h-BTN3A1-his) recombinant protein at a concentration of 1 μg / mL and incubated overnight at 4°C. The coated plate was spin-dried and patted dry on absorbent paper. 150 μL / well of blocking solution was added and the plate was incubated at 37°C for 1 hour. Spin-dry and pat dry on absorbent paper. Anti-BTN3A1 test antibody was added to each well and incubated at 37°C for 1 hour. Discard the supernatant and pat dry. Add 100 μL of HRP-conjugated goat anti-rabbit IgG secondary antibody diluted 1:5000 in diluent to each well and incubate at 37°C for 1 hour. Wash the plate using a plate washer and rinse with PBS five times, soaking for 30 seconds each wash. Add 100 μL of substrate solution (TMB) per well and incubate for 5 minutes. Finally, add 100 μL of 1 mol / L sulfuric acid to terminate the reaction. Measure the OD450 value at 450 nm using a Tecan-infinite F50 microplate reader.

[0161] We found that two rabbit-derived monoclonal antibodies, r17G4 and r20A3, had high binding activity to human BTN3A1 recombinant protein.

[0162] Table 2. ELISA binding results of anti-BTN3A1 rabbit monoclonal antibody to human BTN3A1 recombinant protein

[0163] 4. Binding experiment of anti-BTN3A1 rabbit monoclonal antibody to BTN3A1-expressing cells

[0164] The binding properties of rabbit B cell supernatant to HEK293 cells expressing human BTN3A1 protein were detected by flow cytometry (FACS).

[0165] Experimental method: A cell line transiently expressing human BTN3A1 was constructed. After adding the antibody, the binding characteristics of the antibody and antigen were detected by adding a secondary antibody. The expression plasmid containing the human BTN3A1 gene sequence was transiently transfected into HEK293 cells. 2×10 5 Transiently transfected HEK293 cells were centrifuged at 300g for 5 minutes, the supernatant was removed, 100 μL of the test antibody was added, and the cells were incubated at 4°C for 1 hour. The supernatant was removed by centrifugation, and the cells were washed three times with 200 μL of wash buffer (PBS + 2% FBS). 100 μL of a 1:500 dilution of anti-rabbit IgG secondary antibody was added and the cells were incubated at 4°C for 1 hour. The supernatant was removed by centrifugation, and the cells were washed three times with 200 μL of wash buffer (PBS + 2% FBS). The cells were resuspended in 100 μL of PBS and analyzed by flow cytometry.

[0166] The results showed that r17G4 and r20A3 could significantly bind to HEK293 cells expressing BTN3A1.

[0167] Table 3. Binding assay results of anti-BTN3A1 rabbit monoclonal antibody to BTN3A1-expressing HEK293 cells

[0168] 5. γδ T cell activation experiment with anti-BTN3A1 rabbit monoclonal antibody

[0169] The IFNγ secretion content of Vγ9Vδ2T cells was measured by functional experiments to detect the activation ability of rabbit B cell supernatant.

[0170] Experimental method: Vγ9Vδ2 T cells were co-cultured with A375 target cells, and rabbit B cell supernatant was added and mixed. The cells were incubated in a 5% CO2 incubator at 37°C for 24 hours, and the content of human IFNγ secreted in the cell supernatant was determined using an ELISA kit.

[0171] The results in Table 4 show that r17G4 and r20A3 can significantly produce IFNγ after incubation with Vγ9Vδ2T cells, indicating that the antibodies have the function of activating Vγ9Vδ2T cells.

[0172] Table 4. Results of the activation experiment of Vγ9Vδ2 T cells by anti-BTN3A1 rabbit monoclonal antibody

[0173] Example 2. Preparation of human-rabbit chimeric antibodies against BTN3A1 rabbit monoclonal antibody

[0174] 1. Sequencing of Anti-BTN3A1 Rabbit Monoclonal Antibody

[0175] B cells corresponding to the above antibody strains in the logarithmic growth phase were taken, RNA was extracted with Trizol, reverse transcribed into cDNA, PCR amplified using rabbit Ig-Primer, subcloned into a vector, and finally the sequence of the rabbit monoclonal antibody was obtained after sequencing.

[0176] The sequences of the heavy chain variable region (HCVR) and light chain variable region (LCVR) of rabbit monoclonal antibodies r17G4 and r20A3 are as follows:

[0177] >r17G4 HCVR

[0178] >r17G4 LCVR

[0179] >r20A3 HCVR

[0180] >r20A3 LCVR

[0181] The underlined CDR sequences are determined according to the Kabat numbering system, see Table 5.

[0182] Table 5. CDR sequences of r17G4 and r20A3 (according to the Kabat numbering system)

[0183] The two antibodies have the following general CDR structures:

[0184] HCDR1 (SEQ ID NO: 17): X1YYX2X3, wherein X1 is selected from D or N, X2 is selected from I or M, and X3 is selected from T or N;

[0185] HCDR2 (SEQ ID NO: 18): X4IYGSX5NTX6YASWAKG, wherein X4 is selected from V or I, X5 is selected from S or D, and X6 is selected from V or Y;

[0186] HCDR3(SEQ ID NO:19):X7X8X9X 10 SSAX 11 X 12 X 13 X 14, wherein X7 is selected from G or N, X8 is selected from Y or L, X9 is selected from L or D, X 10 Selected from A or Y, X 11 Selected from D or Y, X 12 Selected from I or F, X 13 Does not exist or is H, X 14 Does not exist or is 1;

[0187] LCDR1 (SEQ ID NO: 20): QX 15 SQSVYNNNRLA, where X 15 Select from S or A;

[0188] LCDR2 (SEQ ID NO: 21): X 16 ASX 17 LAS, where X 16 Select from D or E, X 17 Select from T or K;

[0189] LCDR3 (SEQ ID NO: 22): QX 18 YYSGX 19 IX 20 X 21 , where X 18 Selected from T or G, X 19 Select from Y or F, X 20 Selected from W or Y, X 21 Select A or P.

[0190] 2. Construction and Preparation of Human-Rabbit Chimeric Antibodies

[0191] The obtained heavy and light chain variable regions were ligated to a human IgG1 heavy chain constant region (Fc region) with L234F / L235E / P331S mutations (numbered according to the Eu nomenclature system) and a human Cκ light chain constant region, respectively, to construct human-rabbit chimeric full-length antibodies 17G4 and 20A3. The sequences are as follows:

[0192] >IgG1 Fc region (L234F / L235E / P331S)

[0193] >IgG1 Fc region (L234F / L235E / P331S and K446 removed)

[0194] >17G4 HC

[0195] >17G4 LC

[0196] >20A3 HC

[0197] >20A3 LC

[0198] Also provided herein is the sequence of the positive anti-BTN3A1 antibody mAb1 (derived from WO2020025703, as SEQ ID NOs: 4 and 6 in the patent), whose heavy chain constant region is an IgG1 Fc region (L234F / L235E / P331S with K446 removed).

[0199] >mAb1 HC

[0200] >mAb1 LC

[0201] In the above sequence, the underlined portion of the full-length heavy chain is the human IgG1 Fc region, and the underlined portion of the full-length light chain is the human Cκ region.

[0202] The gene sequence encoding the aforementioned antibody was synthesized and subcloned into the pcDNA3.1 expression vector. The expression vector and the transfection reagent PEI were transfected into CHO cells at a ratio of 1:2 and incubated in a CO2 incubator for 4-5 days. The expressed antibody was centrifuged, the supernatant was recovered, and the antibody was purified according to conventional methods. After testing, the target antibody was obtained.

[0203] Example 3. Verification of the function and effect of anti-BTN3A1 human-rabbit chimeric antibody

[0204] 1. ELISA binding assay of chimeric antibody and BTN3A1 recombinant protein

[0205] The binding properties of anti-BTN3A1 human-rabbit chimeric antibody to human BTN3A1 were detected by ELISA experiment.

[0206] Experimental Method: A 96-well microtiter plate was coated with His-tagged human BTN3A1 (h-BTN3A1-his) recombinant protein. Anti-BTN3A1 human-rabbit chimeric antibodies were added at varying concentrations. Antibody-antigen binding activity was detected by adding a secondary antibody (HRP-conjugated anti-Fc primary antibody) and the HRP substrate TMB. A 96-well microtiter plate was coated with His-tagged human BTN3A1 (h-BTN3A1-his) recombinant protein at a concentration of 1 μg / mL and incubated overnight at 4°C. The coated plate was spin-dried and patted dry on absorbent paper. 150 μL / well of blocking buffer was added and the plate was incubated at 37°C for 1 hour. The plate was spin-dried and patted dry on absorbent paper. The anti-BTN3A1 test antibody was added to each well and incubated at 37°C for 1 hour. Discard the supernatant and pat dry. Add 100 μL of HRP-conjugated anti-human IgG secondary antibody diluted 1:5000 in diluent to each well and incubate at 37°C for 1 hour. Wash the plate using a plate washer and rinse with PBS five times, soaking for 30 seconds each wash. Add 100 μL of substrate solution (TMB) per well and incubate for 5 minutes. Finally, add 100 μL of 1 mol / L sulfuric acid to terminate the reaction. Measure the OD value at 450 nm using a microplate reader (Tecan-infinite F50).

[0207] The results are shown in Table 6 and Figure 1. IgG1 is the isotype control antibody, as is the case in other examples of the present disclosure. The results showed that 17G4 and 20A3 significantly bound to the human BTN3A1 recombinant protein, and their binding activity was comparable to that of mAb1.

[0208] Table 6. ELISA binding results of anti-BTN3A1 human-rabbit chimeric antibodies to human BTN3A1 recombinant protein

[0209] 2. FACS binding assay of chimeric antibodies to cells overexpressing BTN3A1

[0210] Flow cytometry (FACS) was used to detect the binding properties of the anti-BTN3A1 human-rabbit chimeric antibody.

[0211] Experimental method: A cell line overexpressing human BTN3A1 was constructed to knock out the background expression of BTN3A protein. After adding the antibody, the binding characteristics of the antibody and antigen were detected by adding a secondary antibody.

[0212] The cell line construction process involves infecting HEK293 cells with a lentivirus carrying a short hairpin RNA sequence that knocks out human BTN3A (BTN3A1, BTN3A2, and BTN3A3), and obtaining a stably overexpressing monoclonal cell line using antibiotic selection and the infinite dilution method. An expression plasmid carrying the human BTN3A1 gene sequence is then transfected into cells in which basal expression of BTN3A has been knocked out, and a stably overexpressing monoclonal cell line is obtained using antibiotic selection and the infinite dilution method.

[0213] 2 × 10 cells were seeded per well in a 96-well plate. 5 Overexpressing cells. Centrifuge at 300g for 5 minutes, remove the supernatant, add 100μL of the antibody to be tested, and incubate at 4°C for 1 hour. Remove the supernatant by centrifugation, wash three times with 200μL of wash buffer (PBS + 2% FBS), add 100μL of 1:500 diluted anti-human IgG secondary antibody labeled with Alexa Fluor 488 (Invitrogen, A-11013), and incubate at 4°C for 1 hour. Remove the supernatant by centrifugation, wash three times with 200μL of wash buffer (PBS + 2% FBS). Resuspend the cells in 100μL of PBS and analyze by flow cytometry.

[0214] The results are shown in Table 7 and Figure 2. The results showed that 17G4 and 20A3 significantly bound to human BTN3A1 overexpressing cells, and the binding strength was slightly stronger than that of mAb1.

[0215] Table 7. Binding assay results of anti-BTN3A1 human-rabbit chimeric antibodies to human BTN3A1 overexpressing cells

[0216] 3. Affinity Determination of Chimeric Antibodies for BTN3A1 Recombinant Protein

[0217] Experimental Methods: Detection was performed using a Biacore 8K instrument (GE Healthcare). A Protein A sensor chip was used, and the mobile phase consisted of HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20). Each test antibody was prepared in HBS-EP+ buffer as a ligand and captured using Protein A on the chip channel. Human BTN3A1 antigen protein (BTN-HM1A1) was prepared in HBS-EP+ buffer as the analyte. A two-fold serial dilution was performed starting at 100 nM, with a total of seven dilution points. The analyte was passed through the experimental and reference channels at a flow rate of 30 μL / min, with an association time of 90 s and a dissociation time of 500 s. Regeneration buffer (10 mM Glycine pH 1.5) (GE Healthcare, 29238268-AA) was used for 30 s at a flow rate of 10 μl / min. Data were processed using Biacore 8K Evaluation software. The signal value of the corresponding reference channel (Fc 1) was subtracted from the signal value of the detection channel (Fc 2) to generate a corrected signal curve. Affinity kinetics curves were fitted to a 1:1 Langmuir binding model, and the association rate, Ka, and dissociation rate, Kd, ​​as well as the dissociation constant (i.e., affinity, KD), were calculated.

[0218] The affinities of the anti-BTN3A1 antibodies to human BTN3A1 are shown in Table 8. The results showed that 17G4 and 20A3 bound to human BTN3A1 protein with high affinity, which was stronger than the affinity of mAb1 for human BTN3A1.

[0219] Table 8. Affinity of anti-BTN3A1 human-rabbit chimeric antibodies to human BTN3A1

[0220] 4. Chimeric Antibody Activation Experiment on γδT Cells

[0221] The in vitro activation function of anti-BTN3A1 human-rabbit chimeric antibody on γδT cells was detected by measuring the amount of IFNγ secreted by γδT cells.

[0222] Experimental method: Vγ9Vδ2 T cells were co-cultured with A375 target cells, and gradient concentrations of anti-BTN3A1 human-rabbit chimeric antibodies were added. The cells were incubated in a 37°C 5% CO2 incubator, and the content of human IFNγ secreted in the cell supernatant was determined using an ELISA kit.

[0223] The results are shown in Table 9 and Figure 3. 17G4 and 20A3 have obvious activation functions on Vγ9Vδ2 T cells.

[0224] Table 9. Results of the γδT cell activation experiment using anti-BTN3A1 human-rabbit chimeric antibodies

[0225] Example 4. Humanization of anti-BTN3A1 rabbit antibody

[0226] In this example, based on the typical VH / VL structures of the rabbit-derived monoclonal antibodies 17G4 and 20A3, the heavy and light chain variable region sequences were compared with an antibody germline database to obtain human germline templates with high homology. The human germline heavy chain framework region is derived from a human heavy chain, and the human germline light chain framework region is derived from a human kappa light chain. The disclosed antibodies 17G4 and 20A3 preferably use the human germline heavy chain template IGHV3-66 and the human germline light chain template IGkV1-27. The CDR regions of the rabbit-derived monoclonal antibodies were transplanted onto the selected humanized templates, replacing the existing CDR regions of the humanized templates to construct the variable regions. Then, based on the three-dimensional structure of the rabbit-derived monoclonal antibodies, back mutations were performed on buried residues, residues that directly interact with the CDR regions, and residues that have a significant impact on the conformation of the VH and VL regions. Chemically labile amino acid residues in the CDR regions were optimized to generate a series of humanized antibodies.

[0227] The heavy chain variable regions of each humanized antibody of 17G4 are as follows:

[0228] >17G4 H1CVR

[0229] >17G4 H2CVR

[0230] >17G4 H3CVR

[0231] >17G4 H4CVR

[0232] >17G4 H5CVR

[0233] The light chain variable regions of each humanized antibody of 17G4 are as follows:

[0234] >17G4 L1CVR

[0235] >17G4 L2CVR

[0236] >17G4 L3CVR

[0237] In the above sequences, the underlined CDRs are the same as SEQ ID NOs: 5-10.

[0238] The heavy chain variable regions of each humanized antibody of 20A3 are as follows:

[0239] >20A3 H1CVR

[0240] >20A3 H2CVR

[0241] >20A3 H3CVR

[0242] >20A3 H4CVR

[0243] >20A3 H5CVR

[0244] >20A3 H6CVR

[0245] The light chain variable regions of each humanized antibody of 20A3 are as follows:

[0246] >20A3 L1CVR

[0247] >20A3 L2CVR

[0248] >20A3 L3CVR

[0249] In the above sequences, the underlined CDRs are the same as SEQ ID NOs: 11-16.

[0250] The above heavy chain variable regions and light chain variable regions were ligated to the human IgG1 heavy chain constant region and human Cκ light chain constant region with L234F / L235E / P331S mutations, respectively, to obtain full-length sequences.

[0251] The full-length heavy chain sequences of each humanized antibody of 17G4 are as follows:

[0252] >17G4 H1C

[0253] >17G4 H2C

[0254] >17G4 H3C

[0255] >17G4 H4C

[0256] >17G4 H5C

[0257] The full-length light chain sequences of each humanized antibody of 17G4 are as follows:

[0258] >17G4 L1C

[0259] >17G4 L2C

[0260] >17G4 L3C

[0261] The full-length heavy chain sequences of each humanized antibody of 20A3 are as follows:

[0262] >20A3 H1C

[0263] >20A3 H2C

[0264] >20A3 H3C

[0265] >20A3 H4C

[0266] >20A3 H5C

[0267] >20A3 H6C

[0268] The full-length sequences of the light chains of the 20A3 humanized antibodies are as follows:

[0269] >20A3 L1C

[0270] >20A3 L2C

[0271] >20A3 L3C

[0272] The above-mentioned humanized full-length heavy chain sequence and full-length light chain sequence were combined to obtain the following humanized molecules:

[0273] Table 10. Anti-BTN3A1 humanized antibodies

[0274] Example 5. Verification of the function and effect of anti-BTN3A1 humanized antibody

[0275] 1. Antibody Activation of γδT Cells

[0276] The aforementioned humanized molecules of 17G4 and 20A3 were expressed and purified, and the ability of the humanized antibodies to activate γδT cells was detected according to the method in Part 4 of Example 3.

[0277] The results are shown in Table 11 and Figure 5. The results showed that the activities of all humanized molecules of 20A3 were not significantly decreased compared with the parent 20A3.

[0278] Table 11. Results of the γδT cell activation experiment with 20A3 humanized antibody

[0279] 2. Antibody Binding Experiment on Cells Overexpressing BTN3A1

[0280] According to the method in part 2 of Example 3, the ability of the anti-BTN3A1 humanized antibody to bind to cells overexpressing human BTN3A1 was detected.

[0281] The results are shown in Table 12 and Figures 6A and 6B. The cell binding abilities of the humanized molecules 17G4 and 20A3 were comparable to those of their corresponding chimeric antibodies, and were both superior to those of the control antibody.

[0282] Table 12. Binding results of 17G4 and 20A3 humanized antibodies to cells overexpressing human BTN3A1

[0283] 3. Affinity Determination of Antibodies and BTN3A1 Recombinant Protein

[0284] According to the method of Part 3 of Example 3, the affinity of 20A3-H2L2 to the human BTN3A1 recombinant protein was tested.

[0285] The results are shown in Table 13. The results showed that 20A3-H2L2 had a high binding affinity to the human BTN3A1 recombinant protein, and the affinity of 20A3-H2L2 to human BTN3A1 was stronger than that of mAb1.

[0286] Table 13. Affinity of 20A3-H2L2 for human BTN3A1 recombinant protein

[0287] 4. Antibody Binding Experiment on BTN3A-Overexpressing Cells

[0288] Flow cytometry (FACS) was used to examine the binding ability of 20A3-H2L2 to other BTN3A family proteins. Following the method in Part 2 of Example 3, HEK293 cell lines overexpressing human BTN3A2 and human BTN3A3 were constructed to knock out the background BTN3A protein. The binding properties of 20A3-H2L2 were tested against HEK293 cells overexpressing human BTN3A1, human BTN3A2, and human BTN3A3, respectively, to knock out the background BTN3A protein.

[0289] The results are shown in Table 14 and Figures 7A, 7B, and 7C. The results showed that both 20A3-H2L2 and mAb1 could bind to human BTN3A1, human BTN3A2, and human BTN3A3, and that 20A3-H2L2 had stronger binding ability than mAb1.

[0290] Table 14. Binding assay results of 20A3-H2L2 on cells overexpressing BTN3A family proteins

[0291] 5. Antibody γδ T Cell Activation Experiment

[0292] The in vitro activation of γδT cells by 20A3-H2L2 was assessed by measuring IFNγ secretion by γδT cells. Vγ9Vδ2 T cells were co-cultured with SKOV-3 cells, which overexpress the BTN3A protein. Gradual concentrations of the test antibody were added and the cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. The secreted human IFNγ in the cell supernatant was measured using an ELISA kit.

[0293] The results are shown in Table 15 and Figure 8. The results showed that 20A3-H2L2 had a stronger ability to activate Vγ9Vδ2 T cells than mAb1. 50 Compared to the experimental results in Example 5, Part 1, using A375 cells with low BTN3A expression, 20A3-H2L2 in this example showed stronger activation activity than mAb1, indicating that higher BTN3A expression on tumor cells helps further enhance the activation activity of 20A3-H2L2 on Vγ9Vδ2 T cells.

[0294] Table 15. Activation experiment of γδT cells by 20A3-H2L2

[0295] 6. Antibody-activated γδT cells against SKOV-3

[0296] The ability of 20A3-H2L2 to mediate γδT cell killing of tumor target cells was evaluated by measuring the killing of tumor target cells by γδT cells. Vγ9Vδ2T cells and SKOV-3 cells (luc-labeled) were mixed and seeded in 96-well plates. 20A3-H2L2, mAb1, or isotype control (IgG1) were added at varying concentrations. After co-culture for 24 hours, the cells were lysed with Bright-Glo™ reagent (Promega) for 5 minutes, and the luminescence activity was measured using an Envision multi-mode microplate reader (PerkinElmer).

[0297] The results are shown in Table 16 and Figure 9. The results showed that 20A3-H2L2 activated Vγ9Vδ2T cells to kill SKOV-3 more effectively than mAb1. 50 EC is the control antibody 50 About 1 / 3 of.

[0298] Table 16. Experimental results of 20A3-H2L2 activating γδT cells to kill SKOV-3 cells

Claims

1. A BTN3A binding protein comprising a domain that specifically binds to BTN3A, wherein the domain comprises a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, 31-35, and 39-44, The light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 2, 4, 36-38, and 45-47, wherein the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; Preferably, 1) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 3 and 39-44, The light chain variable region comprises LCDR1, LCDR2, LCDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 4, 45-47; or 2) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 1 and 31-35, The light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 2 and 36-38; More preferably, 1) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 11-13, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 14-16, respectively; or 2) The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 5-7, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 8-10, respectively.

2. The BTN3A binding protein according to claim 1, comprising: A heavy chain variable region as shown in any one of SEQ ID NOs: 1, 3, 31-35, 39-44, or an amino acid sequence having at least 90% identity thereto, and A light chain variable region as shown in any one of SEQ ID NOs: 2, 4, 36-38, 45-47, or an amino acid sequence having at least 90% identity thereto; Preferably, 1) comprising a heavy chain variable region as shown in any one of SEQ ID NOs: 1, 31-35, or an amino acid sequence having at least 90% identity thereto, and a light chain variable region as shown in any one of SEQ ID NOs: 2, 36-38, or an amino acid sequence having at least 90% identity thereto; or, 2) comprising a heavy chain variable region as shown in any one of SEQ ID NOs: 3, 39-44, or an amino acid sequence having at least 90% identity thereto, and a light chain variable region as shown in any one of SEQ ID NOs: 4, 45-47, or an amino acid sequence having at least 90% identity thereto; More preferably, 1) comprising a heavy chain variable region as shown in SEQ ID NO: 1, or an amino acid sequence having at least 90% identity thereto, and a light chain variable region as shown in SEQ ID NO: 2, or an amino acid sequence having at least 90% identity thereto; comprising a heavy chain variable region as shown in any one of SEQ ID NOs: 31-35, or an amino acid sequence having at least 90% identity thereto, and a light chain variable region as shown in SEQ ID NO: 36, or an amino acid sequence having at least 90% identity thereto; comprising a heavy chain variable region as shown in any one of SEQ ID NOs: 31-35, or an amino acid sequence having at least 90% identity thereto, and a light chain variable region as shown in SEQ ID NO: 37, or an amino acid sequence having at least 90% identity thereto; comprising a heavy chain variable region as shown in any one of SEQ ID NOs: 31-35, or an amino acid sequence having at least 90% identity thereto, and a light chain variable region as shown in SEQ ID NO: 38, or an amino acid sequence having at least 90% identity thereto; or, 2) comprising a heavy chain variable region as shown in SEQ ID NO: 3, or an amino acid sequence having at least 90% identity thereto, and a light chain variable region as shown in SEQ ID NO: 4, or an amino acid sequence having at least 90% identity thereto; comprising a heavy chain variable region as shown in any one of SEQ ID NOs: 39-44, or an amino acid sequence having at least 90% identity thereto, and a light chain variable region as shown in SEQ ID NO: 45, or an amino acid sequence having at least 90% identity thereto; comprising a heavy chain variable region as shown in any one of SEQ ID NOs: 39-44, or an amino acid sequence having at least 90% identity thereto, and a light chain variable region as shown in SEQ ID NO: 46, or an amino acid sequence having at least 90% identity thereto; The invention comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 39-44, or an amino acid sequence having at least 90% identity thereto, and a light chain variable region as shown in SEQ ID NO: 47, or an amino acid sequence having at least 90% identity thereto.

3. A BTN3A binding protein comprising a domain that specifically binds to BTN3A, wherein the domain comprises a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 17-19, respectively. The light chain variable region comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences shown in SEQ ID NOs: 20-22, respectively.

4. The BTN3A binding protein according to any one of claims 1 to 3, which is an anti-BTN3A antibody or an antigen-binding fragment thereof, wherein the antibody is preferably a rabbit antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; and the antigen-binding fragment is preferably a scFv, Fab or F(ab′)2 fragment.

5. The BTN3A binding protein according to any one of claims 1 to 4, which is an anti-BTN3A antibody or an antigen-binding fragment thereof that is humanized, backmutated, affinity matured, has T cell epitopes removed, has reduced antibody deamidation, and / or has reduced antibody isomerization; Preferably, the human germline template used in the humanization process has a heavy chain framework region derived from IGHV3-66 and a light chain framework region derived from IGkV1-27.

6. The BTN3A binding protein according to any one of claims 1 to 5, further comprising an Fc region of an immunoglobulin; Preferably, the Fc region is the Fc region of human IgG1, human IgG2, human IgG3 or human IgG4; More preferably, the Fc region is an Fc region of human IgG1 having L234F, L235E and / or P331S mutations, the mutated amino acid positions of the Fc region being defined according to the EU numbering system, optionally, K446 is removed.

7. The BTN3A binding protein according to any one of claims 1 to 3, 5 to 6, comprising: A heavy chain as shown in any one of SEQ ID NOs: 25, 27, 48-52, 56-61, or having an amino acid sequence at least 80%, at least 90% identical thereto, and A light chain as shown in any one of SEQ ID NOs: 26, 28, 53-55, 62-64, or an amino acid sequence having at least 80%, at least 90% identity thereto; Preferably, 1) comprising a heavy chain as shown in any one of SEQ ID NOs: 25, 48-52, or an amino acid sequence having at least 80%, at least 90% identity thereto, and a light chain as shown in any one of SEQ ID NOs: 26, 53-55, or an amino acid sequence having at least 80%, at least 90% identity thereto; or 2) a heavy chain as shown in any one of SEQ ID NOs: 27, 56-61, or having an amino acid sequence at least 80%, at least 90% identical thereto, and a light chain as shown in any one of SEQ ID NOs: 28, 62-64, or having an amino acid sequence at least 80%, at least 90% identical thereto; More preferably, 1) comprising a heavy chain as shown in SEQ ID NO: 25, or an amino acid sequence having at least 80%, at least 90% identity thereto, and a light chain as shown in SEQ ID NO: 26, or an amino acid sequence having at least 80%, at least 90% identity thereto; A heavy chain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 48-52, or having at least 80%, at least 90% identity thereto, and a light chain as shown in SEQ ID NO: 53, or having at least 80%, at least 90% identity thereto; A heavy chain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 48-52, or having at least 80%, at least 90% identity thereto, and a light chain as shown in SEQ ID NO: 54, or having at least 80%, at least 90% identity thereto; A heavy chain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 48-52, or having at least 80%, at least 90% identity thereto, and a light chain as shown in SEQ ID NO: 55, or having at least 80%, at least 90% identity thereto; or 2) comprising a heavy chain as shown in SEQ ID NO: 27, or an amino acid sequence having at least 80%, at least 90% identity thereto, and a light chain as shown in SEQ ID NO: 28, or an amino acid sequence having at least 80%, at least 90% identity thereto; A heavy chain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 56-61, or having at least 80%, at least 90% identity thereto, and a light chain as shown in SEQ ID NO: 62, or having at least 80%, at least 90% identity thereto; A heavy chain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 56-61, or having at least 80%, at least 90% identity thereto, and a light chain as shown in SEQ ID NO: 63, or having at least 80%, at least 90% identity thereto; A heavy chain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 56-61, or having at least 80%, at least 90% identity thereto, and a light chain as shown in SEQ ID NO: 64, or having at least 80%, at least 90% identity thereto.

8. The BTN3A binding protein according to any one of claims 1 to 7, which specifically binds to one or more of BTN3A1, BTN3A2, and BTN3A3, preferably, specifically binds to BTN3A1, BTN3A2, and BTN3A3.

9. A polynucleotide encoding the BTN3A binding protein according to any one of claims 1 to 8; Preferably, the polynucleotide is DNA or RNA.

10. A vector comprising the polynucleotide according to claim 9.

11. A host cell containing or expressing the polynucleotide according to claim 9 or the vector according to claim 10.

12. A method for preparing a BTN3A binding protein, comprising: Expressing the polynucleotide of claim 9 or the vector of claim 10 in the host cell of claim 11, and isolating the expressed BTN3A binding protein from the host cell; Optionally, the method further comprises the step of purifying the BTN3A binding protein.

13. A pharmaceutical composition comprising: The BTN3A binding protein according to any one of claims 1 to 8, and At least one pharmaceutically acceptable excipient, diluent or carrier.

14. A method for treating or preventing cancer comprising: A therapeutically or prophylactically effective amount of the BTN3A binding protein of any one of claims 1 to 8, the polynucleotide of claim 9, the vector of claim 10, or the pharmaceutical composition of claim 13 is administered to a subject in need thereof.

15. A method for activating γδT cells and / or promoting γδT cells to release cytokines, comprising: Administering the BTN3A binding protein of any one of claims 1 to 8, the polynucleotide of claim 9, the vector of claim 10, or the pharmaceutical composition of claim 13 to the γδT cells or contacting the γδT cells with the BTN3A binding protein of any one of claims 1 to 8, the polynucleotide of claim 9, the vector of claim 10, or the pharmaceutical composition of claim 13, wherein the γδT cells may be in vivo or in vitro; The γδT cells are preferably Vγ9Vδ2T cells.

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