PSMA / γδTCR-binding protein and pharmaceutical use thereof

Abstract

The present disclosure relates to a PSMA / γδTCR-binding protein and a pharmaceutical use thereof. Specifically, the present disclosure relates to a PSMA / TRGV9-binding protein, a PSMA-binding protein, a polynucleotide, a vector, a pharmaceutical composition, a method for treating diseases (e.g. cancer), and a pharmaceutical use.

Description

PSMA / γδTCR binding protein and its pharmaceutical uses

[0001] This disclosure claims priority to Chinese patent application CN202411571777.3, filed on November 6, 2024. Technical Field

[0002] This disclosure relates to the biomedical field, specifically to PSMA / γδTCR binding protein, PSMA binding protein, and methods for activating γδT cells, treating cancer, and related pharmaceutical uses. Background Technology

[0003] γδT cells are a group of naturally occurring T cells capable of recognizing and killing tumor cells. They possess both antigen-presenting and cytotoxic functions, bridging innate and adaptive immunity. Unlike αβTCRs, the two chains that make up the γδT cell TCR are the γ chain and the δ chain, and their antigen recognition is not restricted by the MHC (Exp Mol Med. 2021 Mar; 53(3):318-327). γδT cells are relatively few in number, accounting for approximately 1%-5% of PBMCs. Based on the different δ chains, they can be divided into four categories: δ1, δ2, δ3, and δ5 (Front Immunol. 2022 Jun 16; 13:915837), with δ1 and δ2 being the most numerous. δ1 can pair with different γ chains to form different TCRs, and is mainly distributed in tissues such as skin and mucous membranes; δ2 mainly pairs with γ9 to form the γ9δ2 subtype, which is mainly distributed in peripheral blood, accounting for up to 95% of the total peripheral blood γδT (Front Immunol. 2022 Jun 16; 13:915837).

[0004] γδT cells play a crucial role in anti-tumor activity, employing various mechanisms to kill tumor cells. These include recognizing the phosphorylated BTN2A / BTN3A complex activated by the TCR on target cells, initiating a killing signal, and exerting their killing function. Additionally, they exhibit a killing pathway similar to NK cells, expressing receptors such as NKG2D on their cell surface, which bind to corresponding ligands on tumor cells to exert tumor-killing effects. γδT cells infiltrate to varying degrees in various tumors. A large-scale analysis of 5782 tumors across 25 tumor types revealed that γδT cell infiltration predicts a good prognosis and is the most favorable cell population among all immune cells. Analysis of 3238 samples from 14 non-brain cancer solid tumors also showed that γδT cell infiltration is an indicator of good prognosis (Oncoimmunology. 2017 Feb 6; 6(3):e1284723).

[0005] Early clinical studies primarily focused on in vivo activation and in vitro expansion followed by reinfusion. Although the anti-tumor effects shown in different studies were relatively limited, no serious side effects were reported, suggesting that γδT cells have good safety profiles. To improve the effectiveness of γδT cell therapy, many companies have increased anti-tumor activity by enhancing γδT cell targeting. One approach involves using TAA-γδT bispecific antibodies to increase γδT cell targeting. TAA-γδT bispecific antibodies bind to TAA at one end and γδTCR at the other, specifically activating γδT cells and inducing their killing of tumor cells. LAVA therapeutics is a representative example (Front Immunol. 2022 Jun 16; 13:915837). TAA-γδT bispecific antibodies have shown good anti-tumor activity both in vitro and in vivo.

[0006] Prostate cancer is the second most common and fifth leading cause of cancer death among men. Globally, there were 1.4 million new cases and 375,000 deaths in 2020. In China, an estimated 125,600 new cases and 56,200 deaths were projected in 2022. The mortality rate for prostate cancer in China is higher than the global average. The proportion of Chinese patients initially diagnosed with metastatic prostate cancer is significantly higher than in the United States, and the five-year survival rate for prostate cancer patients in China is much lower than in the United States. Developing safer and more effective treatments for prostate cancer is of paramount importance.

[0007] Prostate-specific membrane antigen (PSMA) is an important target for prostate cancer treatment. PSMA is a type 2 transmembrane protein composed of 750 amino acids, consisting of an extracellular domain, a transmembrane domain, and an intracellular domain. Besides prostate tissue, PSMA is expressed in small amounts in tissues such as the small intestine and salivary glands. Compared to normal tissues, PSMA is highly expressed in prostate cancer tumor tissues, and it also maintains high levels of expression in castration-resistant prostate cancer. This provides a foundation for PSMA as an important target for prostate cancer treatment. Various forms of drugs have been developed targeting PSMA. Among them, PSMA-CD3 bispecific antibodies are a highly promising drug form, utilizing activated T cells to kill tumor cells. However, excessive activation and release of cytokines lead to cytokine release syndrome, posing a significant challenge to the application of this type of drug. Due to the good safety profile of γδT cells, targeting and killing PMSA-positive cells with γδT cells holds promise for achieving antitumor activity with even better safety. Early clinical data from LAVA-1207, developed by LAVA Therapeutics, show promising safety and preliminary efficacy.

[0008] This study aims to develop a more potent PSMA / γδTCR binding protein that can recruit and activate γδT to specifically kill PSMA-positive tumor cells, exhibiting good tumor-killing activity, safety, and drug-likeness. Summary of the Invention

[0009] This disclosure provides a PSMA / TRGV9 binding protein, a PSMA binding protein encoding a nucleic acid, a vector, a host cell, a pharmaceutical composition, and methods for treating or preventing diseases (e.g., cancer) and related pharmaceutical uses.

[0010] PSMA-binding protein

[0011] This disclosure provides a PSMA-binding protein. In some embodiments, it is capable of binding PSMA. In some embodiments, it is capable of binding PSMA epitopes. In some embodiments, it is capable of binding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.) PSMA epitopes.

[0012] In some embodiments, a PSMA-binding protein is provided that includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.) immunoglobulin single variable domains. In some specific embodiments, the PSMA-binding protein includes two or more immunoglobulin single variable domains, wherein any two immunoglobulin domains bind the same or different epitopes.

[0013] In some embodiments, a PSMA-binding protein is provided comprising an immunoglobulin single variable domain 1, said immunoglobulin single variable domain 1 comprising CDR1, CDR2 and / or CDR3 of any sequence in SEQ ID NO:23, 33-37. In some embodiments, said immunoglobulin single variable domain 1 comprises CDR1, CDR2 and CDR3 of any sequence in SEQ ID NO:23, 33-37.

[0014] In some embodiments, a PSMA-binding protein is provided comprising an immunoglobulin single variable domain 2, said immunoglobulin single variable domain 2 comprising CDR1, CDR2 and / or CDR3 of any sequence in SEQ ID NO:24, 38-42. In some embodiments, said immunoglobulin single variable domain 2 comprises CDR1, CDR2 and CDR3 of any sequence in SEQ ID NO:24, 38-42.

[0015] In some implementations, the CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific implementations, the CDR is defined according to the Kabat numbering system.

[0016] In some embodiments, a PSMA-binding protein is provided, comprising an immunoglobulin single variable domain 1, said immunoglobulin single variable domain 1 comprising CDR1, CDR2 and / or CDR3 as shown in SEQ ID NO:25-27, respectively. In some embodiments, said immunoglobulin single variable domain 1 comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO:25-27, respectively.

[0017] In some embodiments, a PSMA-binding protein is provided, comprising an immunoglobulin single variable domain 2, said immunoglobulin single variable domain 2 comprising CDR1, CDR2 and / or CDR3 as shown in SEQ ID NO:28-30, respectively. In some embodiments, said immunoglobulin single variable domain 2 comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO:28-30, respectively.

[0018] In some embodiments, a PSMA-binding protein is provided that includes a single variable immunoglobulin domain, wherein the single variable immunoglobulin domain includes one, two, or three of the aforementioned CDR1, CDR2, and CDR3.

[0019] In some embodiments, the immunoglobulin single variable domain is humanized, reversed mutation, affinity maturation, T-cell epitope removal, antibody deamidation reduced, and / or antibody isomerization reduced. In some embodiments, the immunoglobulin single variable domain is humanized. In some specific embodiments, the heavy chain template used in the humanization process of the immunoglobulin single variable domain is IGHV3-30*02.

[0020] In some embodiments, a PSMA-binding protein is provided, comprising an immunoglobulin single variable domain 1, said immunoglobulin single variable domain 1 comprising an amino acid sequence shown in or having at least 80% or at least 90% identity with any of SEQ ID NO: 23, 33-37.

[0021] In some embodiments, a PSMA-binding protein is provided, comprising an immunoglobulin single variable domain 2, said immunoglobulin single variable domain 2 comprising an amino acid sequence shown or having at least 80% or at least 90% identity with any of SEQ ID NO:24, 38-42.

[0022] In the context of this 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; and "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.

[0023] In some embodiments, the aforementioned PSMA-binding protein comprises or is an anti-PSMA antibody or its antigen-binding fragment. In some embodiments, the anti-PSMA antibody or its antigen-binding fragment is a recombinant antibody or a fragment thereof. In some embodiments, the anti-PSMA antibody is a monospecific antibody or a multispecific antibody (e.g., a bispecific antibody, a trispecific antibody, etc.).

[0024] In some embodiments, the antibody or its antigen-binding fragment is a linear antibody, a single-chain antibody, a nanobody, a peptide antibody, a domain antibody and a diabody, a triabody and a tetrabody, a tandem di-scFv, or a tandem tri-scFv.

[0025] In some embodiments, when the aforementioned PSMA-binding protein contains a single variable immunoglobulin domain, it can be a camel antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof. In some embodiments, the single variable immunoglobulin domain is a single-domain antibody (sdAb) or a VHH, or the aforementioned PSMA-binding protein itself is a single-domain antibody or a VHH.

[0026] In some embodiments, the aforementioned PSMA-binding protein may contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) of the aforementioned immunoglobulin single variable domains. These immunoglobulin single variable domains may form dimer or multimer molecules.

[0027] In some embodiments, a PSMA-binding protein is provided that includes at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) immunoglobulin single variable domain 1, and / or at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) immunoglobulin single variable domain 2.

[0028] In some specific embodiments, the PSMA-binding protein comprises at least one immunoglobulin single variable domain 1 and at least one immunoglobulin single variable domain 2. The immunoglobulin single variable domain 1 and the immunoglobulin single variable domain 2 may be located on the same polypeptide chain or on different polypeptide chains.

[0029] In some embodiments, the aforementioned PSMA-binding protein further includes a human immunoglobulin Fc region; for example, the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4. As used in this disclosure, "immunoglobulin IgG" includes wild-type IgG or a variant thereof.

[0030] In some implementations, the Fc has a mutation at position C220, such as C220A.

[0031] In some embodiments, the Fc region is the Fc region of human IgG1. In some specific embodiments, the Fc region may be an Fc region with reduced effector function. For example, an Fc region with reduced antibody-dependent cytotoxicity (ADCC), antibody-dependent cytophagy (ADCP), and / or complement-dependent cytotoxicity (CDC).

[0032] In some specific implementations, the Fc region contains mutations that reduce effector function. Exemplary IgG Fc regions with reduced effector function include, but are not limited to, 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 / P 238S (IgG1); C226S / C229S / E233P / L234V / L235A (IgG1); L234F / L235E / P331S (IgG1); L234F / L235E (IgG1); L234F or L235E (IgG1); L234A or L235A (IgG1) or S267E / L328F (IgG1).

[0033] In some implementations, the Fc region is an Fc region that enhances effector function, such as an Fc region that enhances antibody-dependent cytotoxicity (ADCC), antibody-dependent cytophagy (ADCP), and / or complement-dependent cytotoxicity (CDC) with enhanced effector function.

[0034] An exemplary IgG1 Fc region includes substitutions having the following: 239D; 239E; 239K, 241A; 262A; 264D; 264L; 264A; 264S; 265A; 265S; 265V; 296A; 301A; 332E; 239D / 332E; 239D / 330S / 332E; 239D / 330L / 332E; 298A / 333A / 334A; 247I / 339D; 247I / 339Q; 280H / 290S; 280H / 290S / 298D; 280 H / 290S / 298V; 243L / 292P / 300L; 243L / 292P / 300L / 396L; 243L / 292P / 300L / 305I / 396L; 236A / 239D / 332E; 326A / 333A; 326W / 333S; 290E / 298G / 299A; 290N / 298G / 299A; 290E / 298G / 299A / 326E; or 290N / 298G / 299A / 326E; or any combination of the above positions. The mutations are defined according to the EU numbering system.

[0035] An exemplary IgG1 Fc region includes substitutions having the following: S239D; S239E; S239K; F241A; V262A; V264D; V264L; V264A; V264S; D265A; D265S; D265V; F296A; Y296A; R301A; I332E; S239D / I332E; S239D / A330S / I332E; S239D / A330L / I332E; S298A / D333A / K334A; P247I / A339D; P247I / A339Q; D280H / K290S; D280H / K290S / S298D; D 280H / K290S / S298V; F243L / R292P / Y300L; F243L / R292P / Y300L / P396L; F243L / R292P / Y300L / V305I / P396L; G236A / S239D / I332E; K326A / E333A; K326W / E333S; K290E / S298G / T299A; K290N / S298G / T299A; K290E / S298G / T299A / K326E; or K290N / S298G / T299A / K326E, or any combination of the above positions.

[0036] In some implementations, the immunoglobulin single variable domain in the aforementioned PSMA-binding protein is directly or via a linker to the Fc region. The linker can be a non-functional amino acid sequence of 1-20 or more amino acids in length, without secondary or higher structures. For example, the linker could be (G... m S n ) h or (G) m Q n ) h Or (GGNGT) h (SEQ ID NO:53) or (YGNGT) h (SEQ ID NO:54) or (EPKSS) h (SEQ ID NO:55) or (A) m S n ) h As shown, m is selected from integers 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), n is selected from integers 0-8 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from integers 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). For example, the connector is selected from G4S (SEQ ID NO: 56), GS, GAP, (G4S)2 (SEQ ID NO: 57), (G4S)3 (SEQ ID NO: 58), (G4S)4 (SEQ ID NO: 59), (G4S)5 (SEQ ID NO: 60), ASGS (SEQ ID NO: 61), A3S (SEQ ID NO: 62), etc.

[0037] In some implementations, the aforementioned PSMA-binding protein is: a protein that specifically binds to PSMA, or an anti-PSMA antibody or its antigen-binding fragment.

[0038] In some embodiments, the aforementioned PSMA-binding protein comprises an amino acid sequence as shown in any of SEQ ID NO:31-32, 43-44 or having at least 80% or at least 90% sequence identity with it.

[0039] In some implementations, the aforementioned PSMA-binding protein has a function or property selected from at least one of the following:

[0040] (a) EC with ≤10 nM 50 The EC binds to LNCaP cells. 50For example, ≤10nM, ≤8nM, ≤7nM, ≤5nM, ≤4nM, ≤3nM, ≤2nM, ≤1nM, ≤0.5nM, ≤0.2nM, ≤0.1nM. The EC 50 It is obtained through FACS detection, which is a commonly used affinity detection method in the art, such as that described in Embodiment 10 of this disclosure;

[0041] (b) Specific binding to PSMA.

[0042] In some implementations, the aforementioned protein binds to the K+ of PSMA. D The value can be ≤1×10 -7 M, for example, ≤1×10 - 8 M, or ≤1×10 -9 M, or ≤1×10 -10 M.

[0043] In some embodiments, the aforementioned PSMA-binding protein encompasses variants of a single variable domain of an immunoglobulin, wherein the variant, compared to any one of SEQ ID NO:23-24, 33-42, has one or more amino acid mutations, "a plurality of" encompassing 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid mutations may be conserved substitutions, replacements, or modifications, and / or deletions or additions that do not affect function; the amino acid mutations may occur in the CDR region and / or FR region.

[0044] In some implementations, the aforementioned PSMA-binding protein encompasses a functional portion of a single variable domain of an immunoglobulin or a variant thereof, such as CDR3, CDR3-FR4, CDR2-FR3-CDR3, CDR2-FR3-CDR3-FR4, FR2-CDR2-FR3-CDR3-FR4, CDR1-FR2-CDR2-FR3-CDR3-FR4, and FR1-CDR1-FR2-CDR2-FR3-CDR3.

[0045] In some embodiments, a protein is provided that binds to or competitively binds to the same antigenic epitope in the aforementioned PSMA-binding protein of this disclosure, which is an immunoglobulin single variable domain.

[0046] In some embodiments, a protein is provided whose binding to PSMA is blocked by a single variable immunoglobulin domain in the aforementioned PSMA-binding protein of this disclosure.

[0047] In some embodiments, a protein (or molecule) is provided comprising any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) immunoglobulin single variable domains as disclosed herein. For example, the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO:25-27, or comprises any of the sequences shown in SEQ ID NO:23, 33-37. The protein (or molecule) may be a conjugate or fusion protein formed with other compounds or other peptides, and the conjugate may, for example, contain any detectable marker.

[0048] In some embodiments, a protein (or molecule) is provided comprising any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) immunoglobulin single variable domains as disclosed herein. For example, the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO:28-30, or comprises any of the sequences shown in SEQ ID NO:24, 38-42. The protein (or molecule) may be a conjugate or fusion protein formed with other compounds or other peptides, and the conjugate may, for example, contain any detectable marker.

[0049] In some embodiments, a protein (or molecule) is provided comprising any one or more of the aforementioned immunoglobulin single variable domains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) disclosed herein; and at least one antigen-binding domain that does not bind to PSMA. In some embodiments, a protein (or molecule) is provided comprising any one or more of the aforementioned immunoglobulin single variable domains that specifically bind to PSMA; and at least one antigen-binding domain that specifically binds to a second target. In some embodiments, the second target is a TCR. In some embodiments, the second target is a γδTCR. In some embodiments, the second target is a γ9δ2TCR. In some embodiments, the second target is a γ9δ1TCR. In some embodiments, the second target is the T cell receptor γ variable region 9 (TRGV9).

[0050] T cell receptor-binding protein

[0051] This disclosure provides a T-cell receptor-binding protein.

[0052] In some embodiments, it is capable of binding to the TCR. In some embodiments, it is capable of binding to the γδTCR. In some embodiments, it is capable of binding to the γ9 chain of the TCR. In some embodiments, it is capable of binding to the γ9δ2TCR. In some embodiments, it is capable of binding to the γ9δ1TCR. In some embodiments, it is capable of binding to the T cell receptor γ variable region 9 (TRGV9).

[0053] In some embodiments, a TRGV9 binding protein is provided, comprising a single immunoglobulin variable domain comprising CDR1, CDR2, and / or CDR3 from any of the sequences in SEQ ID NO:5, 16-19. In some embodiments, the single immunoglobulin variable domain comprises CDR1, CDR2, and CDR3 from any of the sequences in SEQ ID NO:5, 16-19.

[0054] In some implementations, the CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific implementations, the CDR is defined according to the Kabat numbering system.

[0055] In some embodiments, a TRGV9 binding protein is provided, comprising a single immunoglobulin variable domain comprising CDR1, CDR2, and / or CDR3 as shown in SEQ ID NO:6-8. In some embodiments, the single immunoglobulin variable domain comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO:6-8.

[0056] In some embodiments, a TRGV9 binding protein is provided, which includes an immunoglobulin single variable domain, said immunoglobulin single variable domain including one, two or three of the aforementioned CDR1, CDR2 and CDR3.

[0057] In some implementations, the single variable domain of the immunoglobulin is humanized, reversed through mutation, affinity maturation, T-cell epitope removal, antibody deamidation reduced, and / or antibody isomerization reduced.

[0058] In some implementations, the single variable domain of the immunoglobulin is humanized. In some specific implementations, the heavy chain template used in the humanization process of the single variable domain of the immunoglobulin is IGHV3-30*02.

[0059] In some embodiments, a TRGV9 binding protein is provided, comprising an immunoglobulin single variable domain comprising an amino acid sequence shown in or having at least 80% or at least 90% identity with any of SEQ ID NO:5, 16-19.

[0060] In the context of this 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; and "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.

[0061] In some embodiments, the aforementioned TRGV9 binding protein comprises or is an anti-TRGV9 antibody or its antigen-binding fragment. In some embodiments, the anti-TRGV9 antibody or its antigen-binding fragment is a recombinant antibody or a fragment thereof. In some embodiments, the anti-TRGV9 antibody is a monospecific antibody or a multispecific antibody (e.g., a bispecific antibody, a trispecific antibody, etc.).

[0062] In some embodiments, the antibody or its antigen-binding fragment is a linear antibody, a single-chain antibody, a nanobody, a peptide antibody, a domain antibody and a diabody, a triabody and a tetrabody, a tandem di-scFv, or a tandem tri-scFv.

[0063] In some implementations, when the aforementioned TRGV9 binding protein contains a single variable immunoglobulin domain, it can be a camel antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof.

[0064] In some implementations, the single variable domain of the immunoglobulin is a single domain antibody (sdAb) or VHH, or the aforementioned TRGV9 binding protein itself is a single domain antibody or VHH.

[0065] In some embodiments, the aforementioned TRGV9 binding protein may contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) of the aforementioned immunoglobulin single variable domains. These immunoglobulin single variable domains may form dimer or multimer molecules.

[0066] In some embodiments, the aforementioned TRGV9 binding protein further includes a human immunoglobulin Fc region; for example, the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4. As used in this disclosure, "immunoglobulin IgG" includes wild-type IgG or a variant thereof. In some embodiments, the Fc region is the Fc region of human IgG1.

[0067] In some specific implementations, the Fc region may be an Fc region with reduced effector function. For example, an Fc region with reduced antibody-dependent cytotoxicity (ADCC), antibody-dependent cytophagy (ADCP), and / or complement-dependent cytotoxicity (CDC).

[0068] In some specific implementations, the Fc region may have mutations, and exemplary IgG Fc regions with reduced effector function include, but are not limited to, 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 / P 238S (IgG1); C226S / C229S / E233P / L234V / L235A (IgG1); L234F / L235E / P331S (IgG1); L234F / L235E (IgG1); L234F or L235E (IgG1); L234A or L235A (IgG1) or S267E / L328F (IgG1).

[0069] In some implementations, the immunoglobulin single variable domain of the aforementioned TRGV9 binding protein is directly or via a linker to the Fc region. The linker can be a non-functional amino acid sequence of 1-20 or more amino acids without secondary or higher structures. For example, the linker is such as (G... m S n ) h or (G) m Q n ) h Or (GGNGT) h Or (YGNGT) h Or (EPKSS) h or (A) m S n ) hAs shown, m is selected from integers 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), n is selected from integers 0-8 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from integers 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). For example, the connector is selected from G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, A3S, etc.

[0070] In some embodiments, the aforementioned TRGV9 binding protein is: a protein that specifically binds to γδTCR, or an anti-γδTCR antibody or its antigen-binding fragment; a protein that specifically binds to the γ9 chain of TCR, or an anti-TCR γ9 chain antibody or its antigen-binding fragment; a protein that specifically binds to the variable region (Vγ9) of the γ9 chain of TCR, or an anti-Vγ9TCR antibody or its antigen-binding fragment; a protein that specifically binds to γ9δ2TCR, or an anti-γ9δ2TCR antibody or its antigen-binding fragment; a protein that specifically binds to γ9δ1TCR, or an anti-γ9δ1TCR antibody or its antigen-binding fragment; or a protein that specifically binds to TRGV9, or an anti-TRGV9 antibody or its antigen-binding fragment.

[0071] In some embodiments, the aforementioned TRGV9 binding protein comprises an amino acid sequence as shown in SEQ ID NO:9 or 20, or having at least 80% or at least 90% sequence identity with it.

[0072] In some implementations, the aforementioned TRGV9 binding protein has a function or property selected from at least one of the following:

[0073] (a) EC with ≤10 nM 50 The EC binds to γ9δ2T cells. 50 For example, ≤5nM, ≤4nM, ≤3nM, ≤2nM, ≤1nM, ≤0.5nM, ≤0.2nM, ≤0.1nM. The EC 50 It is obtained through FACS detection, which is a commonly used affinity detection method in the art, such as that described in Embodiment 5 of this disclosure;

[0074] (b) Specifically binds to the γ9 chain of TCR, for example, specifically binds to γ9δ1TCR and γ9δ2TCR;

[0075] (c) γ8 chains that do not bind TCR, for example, those that do not bind γ8δ2TCR;

[0076] (d) The variable region (TRGV9) of the γ9 chain that specifically binds to TCR;

[0077] (e) Specifically binds to VγδCαβ chimeric TCRs, but does not bind to VαβCγδ chimeric TCRs;

[0078] Wherein, (b)-(e) can be obtained by conventional detection methods in the art, such as those described in Embodiment 4 of this disclosure.

[0079] In some implementations, the aforementioned protein binds to the K of TRGV9. D The value can be ≤1×10 -7 M, for example, ≤1×10 - 8 M, or ≤1×10 -9 M, or ≤1×10 -10 M.

[0080] In some embodiments, the aforementioned TRGV9 binding protein encompasses variants of a single variable domain of an immunoglobulin, wherein the variant, compared to any one of SEQ ID NO:5, 16-19, has one or more amino acid mutations, "a plurality of" covering 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid mutations may be conserved substitutions, replacements, or modifications, and / or deletions or additions that do not affect function; the amino acid mutations may occur in the CDR region and / or FR region.

[0081] In some implementations, the aforementioned TRGV9 binding protein encompasses a functional portion of a single variable domain of an immunoglobulin or a variant thereof, such as CDR3, CDR3-FR4, CDR2-FR3-CDR3, CDR2-FR3-CDR3-FR4, FR2-CDR2-FR3-CDR3-FR4, CDR1-FR2-CDR2-FR3-CDR3-FR4, and FR1-CDR1-FR2-CDR2-FR3-CDR3.

[0082] In some embodiments, a protein is provided that binds to or competitively binds to the same antigenic epitope in the aforementioned TRGV9 binding protein of this disclosure, which is an immunoglobulin single variable domain.

[0083] In some embodiments, a protein is provided whose binding to TRGV9 is blocked by a single variable immunoglobulin domain in the aforementioned TRGV9-binding protein of this disclosure.

[0084] In some embodiments, a protein (or molecule) is provided comprising any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) immunoglobulin single variable domains as disclosed herein. For example, the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 6-8, or comprises any of the sequences shown in SEQ ID NO: 5, 16-19. The protein (or molecule) may be a conjugate or fusion protein formed with other compounds or other peptides, and the conjugate may, for example, contain any detectable marker.

[0085] PSMA / T cell receptor (TCR) binding protein

[0086] This disclosure provides a binding protein that binds PSMA and TCR. In some embodiments, the binding protein binds PSMA and γδTCR. In some embodiments, the binding protein binds the γ9 chain of PSMA and TCR. In some embodiments, the binding protein binds the variable region (Vγ9) of the γ9 chain of PSMA and TCR. In some embodiments, a protein is provided that binds PSMA and γ9δ2TCR. In some embodiments, the binding protein binds PSMA and γ9δ1TCR. In some embodiments, the binding protein binds PSMA and TRGV9. In some embodiments, the "binding" is simultaneous or sequential binding.

[0087] In some embodiments, this disclosure provides a PSMA / TRGV9 binding protein comprising a first antigen-binding domain that specifically binds PSMA and a second antigen-binding domain that specifically binds TRGV9.

[0088] In some embodiments, the first antigen-binding domain includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, etc.) immunoglobulin single variable domains 1 that specifically bind PSMA.

[0089] In some implementations, the first antigen-binding domain includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, etc.) immunoglobulin single variable domains 2 that specifically bind PSMA.

[0090] In some embodiments, the immunoglobulin single variable domain 1 and immunoglobulin single variable domain 2 bind to the same antigenic epitope. In some embodiments, the immunoglobulin single variable domain 1 and immunoglobulin single variable domain 2 bind to different antigenic epitopes.

[0091] In some embodiments, immunoglobulin single variable domain 1 and immunoglobulin single variable domain 2 are located on the same polypeptide chain. In some embodiments, immunoglobulin single variable domain 1 and immunoglobulin single variable domain 2 are located on different polypeptide chains.

[0092] In some implementations, any two immunoglobulin variable domains in the aforementioned first antigen-binding domain (e.g., any two immunoglobulin variable domains 1, any two immunoglobulin variable domains 2, or any one immunoglobulin variable domain 1 and any one immunoglobulin variable domain 2) may be directly connected, indirectly connected, or not connected (e.g., located on two different polypeptide chains).

[0093] In some specific embodiments, the immunoglobulin single variable domain 1 comprises CDR1, CDR2, and / or CDR3 from any of the sequences in SEQ ID NO:23, 33-37.

[0094] In some specific embodiments, the immunoglobulin single variable domain 2 comprises CDR1, CDR2, and / or CDR3 from any of the sequences in SEQ ID NO:24, 38-42.

[0095] In some implementations, the CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific implementations, the CDR is defined according to the Kabat numbering system.

[0096] In some embodiments, the immunoglobulin single variable domain 1 comprises CDR1, CDR2, and / or CDR3 as shown in SEQ ID NO:25-27, respectively.

[0097] In some embodiments, the immunoglobulin single variable domain 2 comprises CDR1, CDR2, and / or CDR3 as shown in SEQ ID NO:28-30, respectively.

[0098] In some embodiments, the first antigen-binding domain comprises a single variable immunoglobulin domain that specifically binds to PSMA, which includes one, two, or three of the aforementioned CDR1, CDR2, and CDR3.

[0099] In some embodiments, the immunoglobulin single variable domain is humanized, reversed mutation, affinity maturation, T-cell epitope removal, antibody deamide reduction, and / or antibody isomerization reduction modification. In some embodiments, the immunoglobulin single variable domain is humanized. In some specific embodiments, the heavy chain template used in the humanization process of the immunoglobulin single variable domain is IGHV3-30*02.

[0100] In some embodiments, the immunoglobulin single variable domain 1 comprises an amino acid sequence shown in or having at least 80% or at least 90% identity with any of SEQ ID NO:23, 33-37.

[0101] In some embodiments, the immunoglobulin single variable domain 2 comprises an amino acid sequence shown in or having at least 80% or at least 90% identity with any of SEQ ID NO:24, 38-42.

[0102] In some embodiments, the second antigen-binding domain includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, etc.) specific-binding TRGV9 immunoglobulin single variable domain 3, wherein the immunoglobulin single variable domain 3 includes CDR1, CDR2, and / or CDR3 from any of the sequences in SEQ ID NO:5, 16-19. In some embodiments, the immunoglobulin single variable domain 3 includes CDR1, CDR2, and CDR3 from any of the sequences in SEQ ID NO:5, 16-19.

[0103] In some implementations, the CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific implementations, the CDR is defined according to the Kabat numbering system.

[0104] In some embodiments, the immunoglobulin single variable domain 3 comprises CDR1, CDR2, and / or CDR3 as shown in SEQ ID NO:6-8, respectively.

[0105] In some embodiments, the immunoglobulin single variable domain 3 comprises one, two, or three of the aforementioned CDR1, CDR2, and CDR3 that specifically bind PSMA.

[0106] In some implementations, the immunoglobulin single variable domain 3 is humanized, reversed by mutation, affinity maturation, T-cell epitope removal, antibody deamidation reduction, and / or antibody isomerization reduction modification.

[0107] In some embodiments, the immunoglobulin single variable domain 3 is humanized. In some specific embodiments, the heavy chain template used in the humanization process of the immunoglobulin single variable domain 3 is IGHV3-30*02.

[0108] In some embodiments, the immunoglobulin single variable domain 3 comprises an amino acid sequence shown in or having at least 80% or at least 90% identity with any of SEQ ID NO:5, 16-19.

[0109] In some embodiments, the aforementioned PSMA / TRGV9 binding protein has a valence ratio of the first antigen-binding domain to the second antigen-binding domain between 2:1 and 1:2, for example, 2:1, 1:1, or 1:2. In some specific embodiments, the valence ratio of the first antigen-binding domain to the second antigen-binding domain is 2:1 or 1:1. As used in this disclosure, "valence ratio" refers to the ratio of the number of immunoglobulin single variable domains in the antigen-binding domain that bind to the antigen epitope.

[0110] In some embodiments, the aforementioned PSMA / TRGV9 binding protein comprises or is an anti-PSMA / TRGV9 antibody or its antigen-binding fragment. In some embodiments, the anti-PSMA / TRGV9 antibody or its antigen-binding fragment is a recombinant antibody or a fragment thereof. In some embodiments, the anti-PSMA / TRGV9 antibody is a multispecific antibody (e.g., a bispecific antibody, a trispecific antibody).

[0111] In some specific implementations, the anti-PSMA / TRGV9 antibody is a bispecific antibody that binds to one epitope on the PSMA antigen and one epitope on the TRGV9 antigen.

[0112] In some specific implementations, the anti-PSMA / TRGV9 antibody is a bispecific antibody that binds to two epitopes on the PSMA antigen and one epitope on the TRGV9 antigen.

[0113] In some embodiments, the antibody or its antigen-binding fragment is a linear antibody, single-chain antibody, nanobody, peptide antibody, domain antibody and diabody, triabody and tetrabody, tandem di-scFv, or tandem tri-scFv. In some embodiments, the antibody or its antigen-binding fragment is a camel antibody, chimeric antibody, humanized antibody, fully human antibody, or its antigen-binding fragment.

[0114] In some embodiments, the immunoglobulin single variable domain in the anti-PSMA / TRGV9 antibody or its antigen-binding fragment is a single-domain antibody or VHH. In some specific embodiments, the antigen-binding fragment includes, but is not limited to: Fab, Fv, sFv, Fab', F(ab')2, linear antibody, single-chain antibody, scFv, sdAb, sdFv, nanobody, peptide antibody, domain antibody, diabody, triabody and tetrabody, tandem di-scFv, and tandem tri-scFv.

[0115] In some implementations, the aforementioned PSMA / TRGV9 binding protein also includes the Fc region of human immunoglobulins; for example, the Fc region of human IgG1, IgG2, IgG3, or IgG4.

[0116] In some embodiments, the aforementioned PSMA / TRGV9 binding protein further includes a human immunoglobulin Fc region; for example, the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4. As used in this disclosure, "immunoglobulin IgG" includes wild-type IgG or a variant thereof.

[0117] In some specific implementations, the Fc region may be an Fc region with reduced effector function, such as an Fc region with reduced antibody-dependent cytotoxicity (ADCC), antibody-dependent cytophagy (ADCP), and / or complement-dependent cytotoxicity (CDC).

[0118] In some specific embodiments, the Fc region may have mutations, and exemplary IgG Fc regions with reduced effector function include substitutions having the following: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); H268Q / V309L / A330S / A331S (IgG2); C220S / C226S / C229S / P2 38S (IgG1); C226S / C229S / E233P / L234V / L235A (IgG1); L234F / L235E / P331S (IgG1); L234F / L235E (IgG1); L234F or L235E (IgG1); L234A or L235A (IgG1) or S267E / L328F (IgG1).

[0119] In some specific embodiments, the Fc region comprises a first subunit (Fc1) and a second subunit (Fc2). In some specific embodiments, mutations are introduced that cause the two subunits (Fc1, Fc2) of the Fc region to pair and form a dimer, or mutations that reduce homodimerization are introduced. In some specific embodiments, the first and second subunits contain knock-in-hole mutations. For example, within the CH3 / CH3 interface, one, two, or more amino acid residues in the CH3 domain of Fc1 are mutated with one or more amino acid residues having a larger side chain volume, thereby creating a protrusion (or pestle, knob) on the surface of the CH3 domain of Fc1; one, two, or more amino acid residues in the CH3 domain of Fc2 that interact with the CH3 domain of Fc1 are mutated with amino acid residues having a smaller side chain volume, thereby creating a depression (or hole, hole) on the surface of the CH3 domain of Fc2 that interacts with the CH3 domain of Fc1.

[0120] In some specific embodiments, Fc1 contains one or more amino acid substitutions selected from positions 354, 356, 358, and 366, and Fc2 contains one or more amino acid substitutions selected from positions 349, 356, 358, 366, 368, and 407. In some specific embodiments, Fc1 contains a mutation at position 366, and Fc2 contains mutations selected from positions 366, 368, and 407, or any combination thereof; in some specific embodiments, Fc1 contains a mutation at position 354 or 356, and Fc2 contains a mutation at position 349; in some specific embodiments, Fc1 contains a mutation at position 354 or 356, and Fc2 contains mutations at positions 349, 366, 368, and 407.

[0121] In some specific embodiments, Fc1 contains one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W, and Fc2 contains one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V. In some specific embodiments, Fc1 contains a 366W mutation, and Fc2 contains a mutation selected from 366S, 368A, and 407V, or any combination thereof; in some specific embodiments, Fc1 contains a 354C or 356C mutation, and Fc2 contains a 349C mutation; or in some specific embodiments, Fc1 contains a 354C / 366W mutation, and Fc2 contains a 349C / 366S / 368A / 407V mutation.

[0122] In some specific embodiments, Fc1 contains the T366W mutation, and Fc2 contains a mutation selected from T366S, L368A, and Y407V, or any combination thereof; Fc1 contains the S354C or E356C mutation, and Fc2 contains the Y349C mutation; or Fc1 contains the S354C / T366W mutation, and Fc2 contains the Y349C / T366S / L368A / Y407V mutation. In some specific embodiments, the amino acid sequence of Fc1 is as shown in SEQ ID NO: 46, and the amino acid sequence of Fc2 is as shown in SEQ ID NO: 45.

[0123] In some implementations, the aforementioned PSMA / TRGV9 binding protein includes a linker.

[0124] In some embodiments, the aforementioned single immunoglobulin variable domain in the PSMA / TRGV9 binding protein is directly or via a linker connected to the Fc region. In some embodiments, two adjacent single immunoglobulin variable domains located on the same polypeptide chain in the aforementioned PSMA / TRGV9 binding protein are directly or via a linker connected.

[0125] In some specific embodiments, the linker can be a non-functional amino acid sequence of 1-20 or more amino acids without secondary or higher structures. For example, the linker is such as (G m S n ) h or (G) m Q n ) h Or (GGNGT) h Or (YGNGT) h Or (EPKSS) h or (A) m S n ) hAs shown, m is selected from integers 1-8, n is selected from integers 0-8, and h is independently selected from integers 1-20. For example, the connector is selected from G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, A3S, etc.

[0126] In some embodiments, the PSMA / TRGV9 binding protein comprises a first polypeptide chain and a second polypeptide chain; wherein the first polypeptide chain and the second polypeptide chain are selected from any one of the following groups:

[0127] (a) The first polypeptide chain, from the N-terminus to the C-terminus, includes: a second antigen-binding domain and a first Fc subunit; and, the second polypeptide chain, from the N-terminus to the C-terminus, includes: a first antigen-binding domain and a second Fc subunit; or,

[0128] (b) The first polypeptide chain from the N-terminus to the C-terminus includes: a second antigen-binding domain and a first antigen-binding domain-Fc first subunit; and the second polypeptide chain from the N-terminus to the C-terminus includes: a first antigen-binding domain and an Fc second subunit.

[0129] In some embodiments, the PSMA / TRGV9 binding protein comprises any one of the following first polypeptide chains and second polypeptide chains:

[0130] (1) The first polypeptide chain from the N-terminus to the C-terminus includes: VHH3-[linker 1]a-Fc first subunit; and the second polypeptide chain from the N-terminus to the C-terminus includes: VHH1-[linker 2]b-Fc second subunit;

[0131] (2) The first polypeptide chain from the N-terminus to the C-terminus includes: VHH3-[linker 1]a-Fc first subunit; and the second polypeptide chain from the N-terminus to the C-terminus includes: VHH2-[linker 3]c-Fc second subunit;

[0132] (3) The first polypeptide chain from the N-terminus to the C-terminus includes: VHH3-[linker 4]d-VHH1-[linker 5]e-first subunit; and the second polypeptide chain from the N-terminus to the C-terminus includes: VHH1-[linker 2]b-Fc second subunit;

[0133] (4) The first polypeptide chain from the N-terminus to the C-terminus includes: VHH3-[linker 4]d-VHH1-[linker 5]e-first subunit; and the second polypeptide chain from the N-terminus to the C-terminus includes: VHH2-[linker 3]c-Fc second subunit.

[0134] In some specific implementation schemes, VHH1 is any of the aforementioned immunoglobulin single variable domain 1, VHH2 is any of the aforementioned immunoglobulin single variable domain 2, and VHH3 is any of the aforementioned immunoglobulin single variable domain 3.

[0135] For example, VHH1 comprises amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO:25-27, respectively. For example, VHH1 comprises an amino acid sequence as shown in any one of SEQ ID NO:23, 33-37, or having at least 80% or at least 90% identity with it.

[0136] For example, VHH2 comprises amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO:28-30, respectively. For example, VHH2 comprises an amino acid sequence as shown in any one of SEQ ID NO:24, 38-42, or having at least 80% or at least 90% identity with it.

[0137] For example, VHH3 comprises amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO:6-8, respectively. For example, VHH3 comprises amino acid sequences as shown in any one of SEQ ID NO:5, 16-19, or having at least 80% or at least 90% identity with them.

[0138] In some specific embodiments, the VHH1 comprises an amino acid sequence as shown in SEQ ID NO:34 or an amino acid sequence having at least 80% or at least 90% identity with it.

[0139] In some specific embodiments, the VHH2 comprises an amino acid sequence as shown in SEQ ID NO:40 or an amino acid sequence having at least 80% or at least 90% identity with it.

[0140] In some specific embodiments, the VHH3 comprises an amino acid sequence as shown in SEQ ID NO:19 or an amino acid sequence having at least 80% or at least 90% identity with it.

[0141] In some specific implementation schemes, a, b, c, d, and e are independently selected from 0 or 1.

[0142] In some specific implementations, connector 1, connector 2, connector 3, connector 4, and connector 5 are independently selected from any of the aforementioned connectors. In some specific implementations, connector 1, connector 2, connector 3, connector 4, and connector 5 are independently selected from A3S, G4S, or (G4S)2.

[0143] In some embodiments, the PSMA / TRGV9 binding protein includes a first polypeptide chain and a second polypeptide chain; wherein:

[0144] The first polypeptide chain comprises an amino acid sequence shown in or having at least 80% or at least 90% identity with any of SEQ ID NO:47 or 49; and,

[0145] The second polypeptide chain contains an amino acid sequence shown in or having at least 80% or at least 90% identity with any of SEQ ID NO:48 or 50;

[0146] In some specific embodiments, the PSMA / TRGV9 binding protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are selected from any one of the following groups:

[0147] The first polypeptide chain contains an amino acid sequence shown in or having at least 80% or 90% identity with SEQ ID NO:47, and the second polypeptide chain contains an amino acid sequence shown in or having at least 80% or 90% identity with SEQ ID NO:48.

[0148] The first polypeptide chain contains an amino acid sequence shown in or having at least 80% or at least 90% identity with SEQ ID NO:49, and the second polypeptide chain contains an amino acid sequence shown in or having at least 80% or at least 90% identity with SEQ ID NO:48.

[0149] The first polypeptide chain comprises an amino acid sequence shown in SEQ ID NO:49 or having at least 80% or at least 90% identity with it, and the second polypeptide chain comprises an amino acid sequence shown in SEQ ID NO:50 or having at least 80% or at least 90% identity with it; or,

[0150] The first polypeptide chain contains an amino acid sequence shown in or having at least 80% or 90% identity with SEQ ID NO:47, and the second polypeptide chain contains an amino acid sequence shown in or having at least 80% or 90% identity with SEQ ID NO:50.

[0151] In some implementations, the aforementioned PSMA / TRGV9 binding protein has a function or property selected from at least one of the following:

[0152] (a) EC with ≤10 nM 50 The EC binds to γ9δ2T cells. 50 For example, ≤5nM, ≤4nM, ≤3nM, ≤2nM, ≤1nM, ≤0.5nM, ≤0.2nM, ≤0.1nM. The EC 50 It is obtained through FACS detection, which is a commonly used affinity detection method in the art, such as that described in embodiment 13 of this disclosure;

[0153] (b) Specifically binds to the γ9 chain of TCR, for example, specifically binds to γ9δ1TCR, specifically binds to γ9δ2TCR;

[0154] (c) γ8 chains that do not bind TCR, for example, those that do not bind γ8δ2TCR;

[0155] (d) The variable region (TRGV9) of the γ9 chain that specifically binds to TCR;

[0156] (e) Specifically binds to VγδCαβ chimeric TCRs, but does not bind to VαβCγδ chimeric TCRs;

[0157] Wherein, (b)-(e) can be obtained by conventional detection methods in the art, such as those described in Embodiment 4 of this disclosure;

[0158] (f) Specifically binds to PSMA protein or PSMA-positive cells, and does not bind or binds very little to PSMA-negative cells (e.g., PBMCs or DU145), for example, using the detection methods of Examples 13 or 14 of this disclosure;

[0159] (g) Combining cynomolgus monkey PSMA and human PSMA proteins; for example, using the method shown in Example 12;

[0160] (h) Mediating γδT cell killing of tumor cells, for example, using the detection method of Example 16 of this disclosure; PSMA expression level-dependent mediating of γδT cell killing of tumor cells, for example, using the detection method of Example 16 of this disclosure.

[0161] (i) Promote the proliferation activity of γδT cells in PBMCs, for example, by using the method provided in Example 17 of this disclosure, the PSMA / TRGV9 binding protein in this disclosure can significantly increase the proportion of γδT cells in PBMCs.

[0162] In some implementations, the aforementioned PSMA / TRGV9 binding protein binds to the K+ of TRGV9. D The value can be ≤1×10 -7 M, for example, ≤1×10 -8 M, or ≤1×10 -9 M, or ≤1×10 -10 M.

[0163] In some implementations, the aforementioned PSMA / TRGV9 binding protein binds to the K+ of PSMA. D The value can be ≤1×10 -7 M, for example, ≤1×10 -8 M, or ≤1×10 -9 M, or ≤1×10 -10 M.

[0164] In some embodiments, the immunoglobulin single variable domain in the aforementioned PSMA / TRGV9 binding protein encompasses variants having one or more amino acid mutations compared to any one of SEQ ID NO:5, 16-19. And / or, the variants have one or more amino acid mutations compared to any one of SEQ ID NO:23, 33-37. And / or, the variants have one or more amino acid mutations compared to any one of SEQ ID NO:24, 38-42. "A plurality of" is, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid mutations can be conserved substitutions, replacements, or modifications, and / or deletions or additions that do not affect function; the amino acid mutations can occur in the CDR region and / or FR region.

[0165] In some embodiments, a protein is provided that binds to or competitively binds to the same TRGV9 and / or PSMA antigenic epitopes as described above with respect to the PSMA / TRGV9 binding protein of this disclosure.

[0166] In some embodiments, a protein is provided whose binding to TRGV9 and / or PSMA is blocked by the aforementioned PSMA / TRGV9 binding protein of this disclosure.

[0167] In some embodiments, a protein (or molecule) is provided that comprises any of the PSMA / TRGV9 binding proteins disclosed herein. The protein (or molecule) may be a conjugate, coupling compound, or fusion protein formed with other compounds or peptides. For example, a conjugate may contain any detectable tag.

[0168] Polynucleotides and carriers

[0169] This disclosure provides polynucleotides encoding the PSMA-binding protein and PSMA / TRGV9-binding protein of this disclosure. The nucleic acid of this disclosure may be RNA, DNA, or cDNA. According to some embodiments of this disclosure, the nucleic acid of this disclosure is a substantially isolated nucleic acid.

[0170] The nucleic acids disclosed herein may also be in vector form, may be present in a vector and / or may be part of a vector, such as a plasmid, sticky-terminal plasmid, YAC, or viral vector. The vector may be, in particular, an expression vector, providing a means for expressing PSMA-binding proteins and PSMA / TRGV9-binding proteins in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). This expression vector typically contains at least one nucleic acid of this disclosure, operably linked to one or more suitable expression regulatory elements (e.g., promoters, enhancers, terminators, etc.). Selection of these elements and their sequences for expression in a particular host is common knowledge to those skilled in the art. Regulatory elements and other elements useful or necessary for the expression of the PSMA-binding proteins and PSMA / TRGV9-binding proteins of this disclosure include, for example, promoters, enhancers, terminators, integrators, selection markers, leader sequences, and reporter genes.

[0171] The nucleic acids disclosed herein can be prepared or obtained by known means (e.g., by automated DNA synthesis and / or recombinant DNA technology) based on information about the amino acid sequence of the polypeptides disclosed herein, and / or can be isolated from suitable natural sources.

[0172] host cells

[0173] This disclosure provides recombinant host cells that express or are capable of expressing one or more of the PSMA-binding proteins, PSMA / TRGV9-binding proteins, and / or containing polynucleotides or vectors of this disclosure. In some embodiments, the host cells are bacterial cells, fungal cells, or mammalian cells.

[0174] Bacterial cells include, for example, cells of Gram-negative bacterial strains (such as Escherichia coli, Proteus, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus strains).

[0175] Fungal cells include, for example, cells of species from the genera *Trichoderma*, *Neurospora*, and *Aspergillus*; or cells of species from the genera *Saccharomyces* (e.g., *Saccharomyces cerevisiae*), *Schizosaccharomyces* (e.g., *Schizosaccharomyces pombe*), *Pichia* (e.g., *Pichia pastoris* and *Pichia methanolica*), and *Hansenula*.

[0176] Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc.

[0177] However, this disclosure may also use amphibian cells, insect cells, plant cells, and any other cells in the art used for expressing heterologous proteins.

[0178] Preparation method

[0179] This disclosure provides methods for preparing PSMA-binding proteins and PSMA / TRGV9-binding proteins.

[0180] In some embodiments, the method includes: expressing the target protein in a host cell as described above, and isolating the target protein from the host cell.

[0181] In some embodiments, the method includes: expressing the aforementioned vector or polynucleotide in a host cell, the vector or polynucleotide encoding a target protein, and then isolating the target protein.

[0182] In some embodiments, the method further includes a purification step, for example, purification using an A or G Sepharose FF column containing adjusted buffer to wash away non-specifically bound components, followed by elution of bound antibodies using a pH gradient, detection by SDS-PAGE, and collection. Exemplarily, filtration and concentration are performed using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product must be immediately frozen, such as at -70°C, or lyophilized.

[0183] Methods for producing and purifying antibodies are well-known and available in existing technologies, such as Cold Spring Harbor’s Guide to Antibody Laboratory Techniques (Chapters 5-8 and 15).

[0184] The engineered antibodies or antigen-binding fragments disclosed herein 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. Recombinant immunoglobulin expression vectors can stably transfect 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 human antigens. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. Cultures secreting antibodies can be purified and collected using conventional techniques. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving and ion exchange.

[0185] Composition

[0186] This disclosure provides compositions comprising the aforementioned PSMA-binding protein, PSMA / TRGV9-binding protein, and / or a carrier encoding any of the aforementioned proteins, or a polynucleotide. In some embodiments, a pharmaceutical composition is provided comprising an effective amount of the aforementioned PSMA-binding protein, PSMA / TRGV9-binding protein, carrier, and / or polynucleotide for treating, alleviating, or preventing disease, and at least one pharmaceutically acceptable excipient, diluent, or excipient.

[0187] In some specific embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of PSMA-binding protein, PSMA / TRGV9-binding protein, carrier, and / or polynucleotide per unit dose, or the amount of PSMA-binding protein, PSMA / TRGV9-binding protein, carrier, and / or polynucleotide per unit dose of the pharmaceutical composition may be 0.1-2000 mg, and in some specific embodiments, 1-1000 mg.

[0188] In some specific embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of PSMA-binding protein and / or PSMA / TRGV9-binding protein per unit dose, or the amount of PSMA-binding protein and / or PSMA / TRGV9-binding protein per unit dose may be 0.1-2000 mg, and in some specific embodiments, 1-1000 mg.

[0189] In some embodiments, an article or product (such as a kit) is provided that contains the aforementioned PSMA-binding protein, PSMA / TRGV9-binding protein, carrier, and / or polynucleotide. In some embodiments, the article includes a container and a label. Containers include, for example, bottles, syringes, and test tubes. The container contains a composition effective for treating a disease or condition. A label on or attached to the container indicates that the composition is intended to treat the selected disease or condition.

[0190] In some embodiments, the aforementioned disease is a proliferative disorder or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the aforementioned disease or condition is prostate cancer.

[0191] Treatment methods and pharmaceutical uses

[0192] This disclosure provides methods for using the aforementioned PSMA-binding protein and PSMA / TRGV9-binding protein, which encode polynucleotides, carriers, or compositions (including pharmaceutical compositions) to treat, alleviate, prevent, or diagnose diseases or conditions.

[0193] Some implementation schemes provide methods for improving, alleviating, treating, or preventing disease, including administering to a subject an effective amount for improving, alleviating, treating, or preventing the disease:

[0194] (1) The aforementioned PSMA-binding protein or its encoded polynucleotide, carrier, or pharmaceutical composition disclosed herein;

[0195] (2) The aforementioned PSMA / TRGV9 binding protein or its encoded polynucleotide, carrier, or pharmaceutical composition disclosed herein.

[0196] In some implementations, the use of the foregoing (1)-(2) for preparing a medicament to improve, alleviate, treat or prevent disease is provided.

[0197] In some embodiments, this disclosure provides methods for recruiting and / or activating T cells, which include:

[0198] (1) The steps of contacting the PSMA binding protein or its encoded polynucleotide, carrier, or pharmaceutical composition disclosed herein with T cells;

[0199] (2) The steps of contacting the PSMA / TRGV9 binding protein or its encoded polynucleotide, carrier, or pharmaceutical composition disclosed herein with T cells.

[0200] In some implementations, the T cells are γδT cells.

[0201] In some embodiments, this disclosure provides methods for recruiting and / or activating T cells, which include:

[0202] (1) Administering the aforementioned PSMA-binding protein or its encoded polynucleotide, carrier, or pharmaceutical composition to a subject;

[0203] (2) Administer the aforementioned PSMA / TRGV9 binding protein or its encoded polynucleotide, carrier, or pharmaceutical composition to a subject.

[0204] In some specific implementation schemes, the subject suffers from a cell proliferation disorder or condition.

[0205] In some implementation schemes, the aforementioned disease or condition is cancer.

[0206] In some implementation schemes, the aforementioned disease or condition is prostate cancer.

[0207] In some specific implementation plans, the aforementioned disease or condition is PSMA-positive cancer.

[0208] In some specific implementation plans, the aforementioned disease or condition is PSMA-positive prostate cancer.

[0209] Some embodiments provide a method of treating a disease, including administering a therapeutically effective amount of any of the aforementioned TRGV9 binding protein and γδT cells of this disclosure to a subject in need. Some embodiments provide a method of treating a disease, including administering a therapeutically effective amount of any of the aforementioned PSMA / TRGV9 binding protein and γδT cells of this disclosure to a subject in need.

[0210] In some implementation schemes, the aforementioned disease or condition is a cell proliferation disease or condition.

[0211] In some implementation schemes, the aforementioned disease or condition is cancer.

[0212] In some implementation schemes, the aforementioned disease or condition is prostate cancer.

[0213] In some specific implementation plans, the aforementioned disease or condition is PSMA-positive cancer.

[0214] In some specific implementation plans, the aforementioned disease or condition is PSMA-positive prostate cancer.

[0215] In some embodiments, this disclosure provides for any of the following uses of the PSMA-binding protein or its encoded polynucleotide, carrier, or pharmaceutical composition:

[0216] (1) To prepare medicines for treating diseases or symptoms;

[0217] (2) Used to treat diseases or symptoms;

[0218] (3) Prepare drugs for recruiting and / or activating T cells;

[0219] (4) Used to recruit and / or activate T cells;

[0220] (5) Prepare drugs for recruiting and / or activating γδT cells;

[0221] (6) Used to recruit and / or activate γδT cells.

[0222] In some embodiments, this disclosure provides for any of the following uses of the PSMA / TRGV9 binding protein or its encoded polynucleotide, carrier, or pharmaceutical composition:

[0223] (7) To prepare medicines for treating diseases or symptoms;

[0224] (8) Used to treat diseases or symptoms;

[0225] (9) Preparation of drugs for recruiting and / or activating T cells;

[0226] (10) Used to recruit and / or activate T cells;

[0227] (11) Prepare drugs for recruiting and / or activating γδT cells;

[0228] (12) Used to recruit and / or activate γδT cells.

[0229] In some embodiments, for any of the foregoing uses, the disease or condition is a proliferative disease or condition. In some embodiments, the foregoing disease or condition is cancer.

[0230] In some implementation schemes, the aforementioned disease or condition is prostate cancer.

[0231] In some specific implementation plans, the aforementioned disease or condition is PSMA-positive cancer.

[0232] In some specific implementation plans, the aforementioned disease or condition is PSMA-positive prostate cancer.

[0233] Detection

[0234] This disclosure provides the detection uses of PSMA-binding proteins, PSMA / TRGV9-binding proteins encoding polynucleotides, and compositions thereof. This disclosure also provides methods, systems, or apparatus for in vivo or in vitro detection of PSMA and TRGV9, comprising treating a sample with the aforementioned binding proteins, polynucleotides, or compositions of this disclosure.

[0235] In some embodiments, a kit is also provided comprising the aforementioned PSMA-binding protein, PSMA / TRGV9-binding protein encoding a polynucleotide, a composition, and may also include diagnostic instructions for use. The kit may also contain at least one additional reagent, such as a marker or additional diagnostic agent. For in vivo use, the PSMA-binding protein, PSMA / TRGV9-binding protein encoding a polynucleotide, may be formulated as a pharmaceutical composition. Attached Figure Description

[0236] Figure 1 is a schematic diagram of the anti-PSMA / γδTCR antibody.

[0237] Figure 2 shows the binding activity of the anti-PSMA / γδTCR antibody to human γδT cells.

[0238] Figure 3 shows the binding activity of the anti-PSMA / γδTCR antibody to human PBMC cells.

[0239] Figures 4A to 4D show the binding activity of anti-PSMA / γδTCR antibodies against cells with different PSMA expression levels. Figure 4A shows the binding activity with 22Rv1 cells, Figure 4B shows the binding activity with DU145-hPSMA (medium) cells, Figure 4C shows the binding activity with LNCaP cells, and Figure 4D shows the binding activity with DU145-hPSMA (high) cells.

[0240] Figure 5 shows the binding activity of anti-PSMA / γδTCR antibody on PSMA-negative DU145 cells.

[0241] Figures 6A to 6D show the cytotoxic activity of anti-PSMA / γδTCR antibodies against cells with different PSMA expression levels. Figure 6A shows the cytotoxic activity against 22Rv1 cells, Figure 6B shows the cytotoxic activity against DU145-hPSMA (medium) cells, Figure 6C shows the cytotoxic activity against LNCaP cells, and Figure 6D shows the cytotoxic activity against DU145-hPSMA (high) cells.

[0242] Figures 7A and 7B show the killing activity of anti-PSMA / γδTCR antibody against 22Rv1 in the PBMC system. Figure 7A shows the killing results of PBMC donor #XC11053, and Figure 7B shows the killing results of PBMC donor #XW0801372W.

[0243] Figures 8A to 8C evaluate the ability of anti-PSMA / γδTCR antibody to promote the proliferation of γδT cells in PBMCs. Figure 8A shows the results for PBMC donor #XC11053, Figure 8B shows the results for PBMC donor XC11061, and Figure 8C shows the results for PBMC donor sc12369.

[0244] Figures 9A and 9B evaluate the antitumor activity of anti-PSMA / γδTCR antibodies in a mouse bone metastasis model. Figure 9A shows the tumor-inhibiting activity of different antibodies in mice, and Figure 9B shows the changes in mouse body weight. Detailed Implementation

[0245] definition

[0246] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this disclosure, all other technical and scientific terms used in this disclosure shall have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0247] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0248] "TRGV9" refers to a polypeptide that, when expressed on the surface of γδT cells, can form a T cell receptor. γδT cells expressing TRGV9 are among the earliest T cells to develop in human fetuses and are the major γδT cell subset in the peripheral blood of healthy adults. "TRGV9" includes any TRGV9 variant, isotype, or species homolog that is naturally expressed by cells (including T cells) or that can be expressed on cells transfected with a gene or cDNA encoding the polypeptide. In a specific embodiment, TRGV9 is human TRGV9. An exemplary human TRGV9 amino acid sequence is provided by GenBank accession number NG_001336.2.

[0249] "PSMA" refers to prostate-specific membrane antigen protein. For example, PSMA is also known as human prostate-specific membrane antigen protein, glutamate carboxypeptidase 2 (EC: 3.4.17.21), cell growth inhibitory gene 27 protein, folic acid hydrolase 1, folylpoly-gamma-glutamate carboxypeptidase (FGCP), glutamate carboxypeptidase II (GCPII), membrane glutamate carboxypeptidase (mGCP), N-acetylated-α-linked acidic dipeptidase I (NAALAD enzyme I), or pteroylpoly-gamma-glutamate carboxypeptidase (UniProtKB-Q04609(FOLH1_HUMAN)), etc.

[0250] The term "TRGV9 binding protein" encompasses any protein capable of specifically binding TRGV9 (or its epitopes) or any molecule containing said protein, including but not limited to antibodies against TRGV9 as defined herein, their antigen-binding fragments, or conjugates or fusion proteins thereof. In some embodiments, "TRGV9 binding protein" may comprise at least one (e.g., 1, 2, 3, 4, 5, 6, or more) single-domain antibodies that specifically bind TRGV9 as described in the embodiments of this disclosure. In some embodiments, the "TRGV9 binding protein" of this disclosure, in addition to comprising the immunoglobulin single variable domain of TRGV9, may also comprise a linker and / or a portion having effector function, such as a half-life extension portion (e.g., the immunoglobulin single variable domain binding serum albumin) and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region. In some embodiments, "TRGV9 binding protein" encompasses the anti-γδTCR antibodies described in the embodiments of this disclosure.

[0251] The term "PSMA-binding protein" encompasses any protein capable of specifically binding to PSMA (or its epitopes) or any molecule containing said protein, including but not limited to antibodies against PSMA as defined in this disclosure, their antigen-binding fragments, or conjugates or fusion proteins thereof. In some embodiments, a "PSMA-binding protein" may comprise at least one (e.g., 1, 2, 3, 4, 5, 6, or more) single-domain antibodies that specifically bind to PSMA as described in the embodiments of this disclosure. In some embodiments, the "PSMA-binding protein" of this disclosure, in addition to comprising a single immunoglobulin variable domain of PSMA, may also comprise a linker and / or a portion having effector function, such as a half-life-extending portion (e.g., a single immunoglobulin variable domain binding to serum albumin) and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region. In some embodiments, "PSMA-binding protein" encompasses the anti-PSMA antibodies described in the embodiments of this disclosure.

[0252] "PSMA / TRGV9 binding protein" encompasses any protein or molecule capable of specifically binding to PSMA (or its epitope) and TRGV9 (or its epitope), including but not limited to antibodies, peptides, fusion proteins of antibodies and peptides, or conjugates thereof. In some embodiments, "PSMA / TRGV9 binding protein" encompasses the anti-PSMA / γδTCR multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.) described in the embodiments of this disclosure.

[0253] The term "multispecific antibody" refers to an antibody that is specific to at least two different epitopes (e.g., 2, 3, 4, 5, 6, 7, etc.). Such epitopes may be on the same or different target antigens. If the epitopes are on different targets, these targets may be on the same cell or different cells or cell types. In some embodiments, the epitopes are partially overlapping or non-overlapping. In some embodiments, the epitopes are non-overlapping epitopes.

[0254] The term "antibody" encompasses 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. Classical four-chain antibodies can refer to immunoglobulins, which are tetrapeptide chains composed of two heavy chains and two light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in its heavy chain, different subclasses can be distinguished; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into κ chains or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain. The sequence of approximately 110 amino acids near the N-terminus of the antibody heavy and light chains varies considerably, forming the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, forming the constant region (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0255] The antibodies disclosed herein can be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, or modified forms thereof, with monoclonal antibodies being particularly suitable in many embodiments. Generally, the antibodies disclosed herein are recombinant antibodies. As used herein, “recombinant” refers to products such as cells or nucleic acids, proteins, or vectors, indicating that said cells, nucleic acids, proteins, or vectors have been modified by introducing heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or that said cells are derived from such modified cells. For example, recombinant cells express genes not present in native (non-recombinant) cell forms or express native genes that are abnormally expressed, poorly expressed, or not expressed at all.

[0256] The determination or definition of a CDR can be accomplished by resolving the structure of the antibody and / or the structure of the antibody-ligand complex, thereby enabling the definitive depiction of the CDR and the identification of residues containing the antibody binding site. This can be achieved using 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 definition, and conformation definition. The Kabat numbering system is the standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8). The Chothia numbering system is similar to the Kabat numbering system, but it takes into account the location of certain structural loop regions (see, for example, Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83). The AbM numbering system uses a computer program integration suite produced by the Oxford Molecular Group to model antibody structures (see, for example, Martin et al., 1989, ProcNatl Acad Sci (USA), 86: 9268-9272; "AbMTM, 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 a de novo approach to model the tertiary structure of antibodies from basic sequences (see those described in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" in PROTEINS, Structural, Function and Genetics Suppl., 3: 194-198). Contact definitions are based on the analysis of available complex crystal structures (see, for example, MacCallum et al., 1996, J. Mol. Biol., 5: 732-45). In the conformational definition, the position of CDR can be identified as a residue that contributes enthalpy to antigen binding (see, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166).Other CDR boundary definitions may not strictly follow one of the methods described above, but still overlap with at least a portion of the Kabat CDR, although they may be shortened or lengthened depending on the predicted or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used in this disclosure, a CDR may refer to a CDR defined by any method (including combinations of methods) known in the art. The correspondence between various numbering systems is well known to those skilled in the art.

[0257] A "domain" of a polypeptide or protein refers to a folded protein structure that can maintain its tertiary structure independently of the rest of the protein. Generally, a domain is responsible for a single functional property of a protein and, in many cases, can be added to, removed from, or transferred to other proteins without losing the function of the rest of the protein and / or the domain itself.

[0258] "Immunoglobulin domain" refers to a globular region of an antibody chain (such as the chain of a conventional tetrapeptide chain antibody or the chain of a heavy chain antibody), or a polypeptide that is essentially composed of such globular regions.

[0259] An immunoglobulin variable domain refers to an immunoglobulin domain that is essentially composed of a frame region and a complementarity-determining region (CDR), referred to in this art and hereinafter as four "frame regions": "Frame Region 1" or "FR1", "Frame Region 2" or "FR2", "Frame Region 3" or "FR3", and "Frame Region 4" or "FR4". These frame regions are separated by three complementarity-determining regions (CDRs): "Complementarity-determining Region 1" or "CDR1", "Complementarity-determining Region 2" or "CDR2", and "Complementarity-determining Region 3" or "CDR3". Therefore, the general structure or sequence of an immunoglobulin variable domain can be represented as: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The immunoglobulin variable domain confers antigen specificity due to the presence of antigen-binding sites.

[0260] "Antibody framework (FR)" refers to a portion of a variable domain that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain.

[0261] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which can be a heavy chain or light chain domain, including VH, VHH, or VL domains) that can form a functional antigen-binding site without interacting with other variable domains (e.g., without the VH / VL interaction required between the VH and VL domains of a conventional four-chain monoclonal antibody). Examples of "immunoglobulin single variable domain" include nanobodies (including VHH, humanized VHH, and / or camelified VH, such as camelified human VH), IgNAR, domains, and (single-domain) antibodies (such as dAbs) that are VH domains or derived from VH domains. TM ) and antibodies that are VL domains or derived from VL domains (such as dAbs) TM Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. A specific example of an immunoglobulin single variable domain is the “VHH domain” (or simply “VHH”) as defined below.

[0262] "VHH," also known as heavy chain single-domain antibody, VHH, VHH domain, VHH antibody fragment, VHH antibody, or nanobody, is a variable domain of an antigen-binding immunoglobulin called a "heavy chain antibody" (i.e., "antibody lacking a light chain"). (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH" is used to distinguish this variable domain from the heavy chain variable domain (referred to herein as the "VH domain" or VH) and the light chain variable domain (referred herein as the "VL domain" or VL) present in conventional tetrapeptide chain antibody structures. The VHH domain specifically binds to epitopes without the need for other antigen-binding domains (unlike the VH or VL domains in conventional tetrapeptide chain antibodies, where the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable, and highly efficient antigen-recognition unit formed by a single immunoglobulin domain. Terms include "heavy chain single-domain antibody," "VHH domain," "VHH," and "V..." H "H domain", "VHH antibody fragment", "VHH antibody", as well as" The term "domain" ("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) is used interchangeably. VHHs include, but are not limited to, naturally occurring antibodies produced by camelids, or antibodies produced by camelids that have been humanized, or those obtained through phage display technology. The total number of amino acid residues in a VHH will typically be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in this disclosure. Methods for obtaining VHHs that bind to specific antigens or epitopes have previously been disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240(2000)185-195; Li et al., J Biol Chem., 287(2012)13713-13721; Deffar et al., African Journal of Biotechnology Vol.8(12), pp.2645-2652, 17 June, 2009 and WO94 / 04678.

[0263] As is known in the art regarding VH and VHH domains, the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number). This means that, in general, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence. Other numbering systems or encoding rules include Chothia, IMGT, and AbM.

[0264] "Humanized antibody," also known as CDR-grafted antibody, refers to an antibody generated by grafting a non-human CDR sequence into the variable region framework of a human antibody. This can overcome the strong immune response induced by chimeric antibodies due to the presence of a large number of non-human protein components. To avoid a decrease in activity along with a decrease in immunogenicity, the variable region of the fully human antibody can be minimally reverse-mutated to maintain activity. Examples of "humanization" include the substitution of a VHH domain derived from camelids with one or more amino acid residues in the original VHH sequence by replacing one or more amino acid residues present at the corresponding position in the VH domain of a conventional human tetrapeptide chain antibody (also referred to herein as "sequence optimization"; in addition to humanization, "sequence optimization" may also encompass other modifications to the sequence by providing one or more mutations that enhance the properties of the VHH, such as removing potential post-translational modification sites). The humanized VHH domain may contain one or more fully human framework region sequences, and in some specific embodiments, may contain the human framework region sequence of IGHV3. Humanization methods include protein surface amino acid resurfacing and antibody humanization using a universal framework grafting method (CDR grafting to a universal framework), which involves "grafting" the CDR onto other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Suitable scaffolds and techniques for CDR grafting are known in the art. Germline DNA sequences of human heavy and light chain variable region genes, for example, can be found in the VBase human germline sequence database and in Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies disclosed herein also include humanized antibodies further matured by phage display with affinity for the CDR. Furthermore, to avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reversion mutations can be performed on the human antibody variable region framework sequence to maintain activity.

[0265] "Affinity-matured" antibodies are those that have one or more alterations in one or more hypervariable regions (HVRs) compared to parental antibodies that do not possess such alterations, resulting in improved affinity of the antibody for the antigen. For example, an "affinity-matured" TRGV9 binding protein or anti-TRGV9 antibody has one or more alterations in one or more CDRs that result in increased affinity for the antigen compared to its parent antibody. Affinity-matured antibodies can be prepared, for example, by methods known in the art as described below: 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): 889896; KS Johnson and RE Hawkins, “Affinity maturation of antibodies using phage display”, Oxford University Press 1996.

[0266] Typically, the PSMA / TRGV9 binding protein and PSMA binding protein disclosed herein will be measured in a preferred 10 as in Biacore, KinExA, or Fortibio assays. -7 Up to 10 -10 mol / L (M), more preferably 10 -8 Up to 10 -10 moles per liter, or even more preferably 10 -9 Up to 10 -10 or a lower dissociation constant (K) D ), and / or 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 it is intended to bind to (i.e., PSMA, TRGV9). Any protein greater than 10... -4 M of K DValues ​​are generally considered to indicate nonspecific binding. The specific binding of antigen-binding proteins to antigens or epitopes 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., radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assays) as described in this disclosure.

[0267] "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 a portion thereof with an antigen). The binding affinity between two molecules can be determined by determining the dissociation constant (K). D Quantification can be achieved by using methods such as surface plasmon resonance (SPR) (Biacore) to measure the kinetics of complex formation and dissociation. D The rate constants corresponding to the binding and dissociation of monovalent complexes are called the binding 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 even for complex mixtures, it can be calculated using methods such as those described by Caceci et al. (1984, Byte 9: 340-362). For example, K can be determined using a double-filtered nitrocellulose filter combined with determinations such as those disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). D Other standard assays for assessing the binding ability of antibodies to target antigens are known in the art, including, for example, ELISA, Western blotting, RIA, and flow cytometry, as well as other assays exemplified elsewhere in this disclosure. Antibody binding kinetics and binding affinity can also be determined using standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore. TM Evaluation can be performed using a system or KinExA. The Kelvin values ​​of individual antibody / antigen complexes can be compared. D The binding affinity is used to compare the binding affinity of different molecules associated with their interactions, for example, comparing the binding affinity of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by identifying and comparing the K-value of the target interaction (e.g., the specific interaction between an antibody and an antigen). D Values ​​of K for non-target interactions (e.g., control antibodies known not to bind IGF-1R or TRGV9) D The value is evaluated.

[0268] "Conservative substitution" refers to the substitution with another amino acid residue that has 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. Furthermore, 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 nonpolar side chains. Additionally, 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 amino acid residues in the group exhibiting similar properties as described above are substituted, it will not show a specific change in properties.

[0269] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are 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 percentage of 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 multiplied by 100%. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of homology is obtained by aligning the two sequences.

[0270] The terms "nucleic acid" and "polynucleotide" are used interchangeably in this disclosure and refer to any single-stranded or double-stranded DNA or RNA molecule, and in the case of a single-stranded molecule, its complementary sequence, preferably double-stranded DNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "effectively linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0271] "Host cell" includes individual cells or cell cultures that may be, or have been, recipients of vectors for incorporating polynucleotide inserts. Host cells include progeny of a single host cell, and progeny may not necessarily be identical to the original parent cell (in morphology or genomic DNA complementation) due to natural, accidental, or intentional mutations. Host cells include cells transfected and / or transformed in vivo with the polynucleotides of this disclosure. "Cell," "cell line," and "cell culture" are used interchangeably, and all such names include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be exactly identical in DNA content. This includes mutant progeny with the same function or biological activity as those screened from the originally transformed cells.

[0272] "Inhibit" or "block" are used interchangeably and cover both partial and complete inhibition / blockage. "Inhibit growth" (e.g., involving cells) is intended to include any measurable reduction in cell growth.

[0273] "Giving," "applying," and "treatment," when 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 the animal, human, subject, cell, tissue, organ, or biological fluid, such as in therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and a cell, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cell. "Giving," "applying," and "treatment" also mean the in vitro and ex vivo treatment of, for example, cells, by means of a reagent, diagnostic agent, composition, or another cell. When applied to humans, veterinary, or research subjects, it refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0274] "Treatment" means administering, either internally or externally, a therapeutic agent, such as a pharmaceutical composition comprising any of the binding proteins of this disclosure or thereof, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases or their symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in a treated subject or population in an amount that effectively relieves symptoms of one or more diseases, whether by inducing the regression of such symptoms or inhibiting their development to any clinically measurable degree. The amount of therapeutic agent that effectively relieves symptoms of any specific disease (also referred to as the "therapeuticly effective amount") can vary depending on a variety of factors, such as the subject's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been relieved can be evaluated using any clinical test method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating the symptoms of the target disease in a particular subject, they should reduce 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 the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0275] "Effective amount" includes an amount sufficient to improve or prevent the symptoms or condition of a medical condition. Effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount used on 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. Effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxicity. Subjects in this disclosure may be animal or human subjects.

[0276] "Optional" or "optionally" means that the event or circumstance described below may, but does not necessarily, occur, and the description includes the possibility that the event or circumstance may or may not occur. "And / or" should be interpreted as specifically disclosing that each of the two specified features or components has or does not have the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" in this 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."

[0277] In the context of mutations contained in the Fc region in this disclosure, " / " means "and", for example, "354C / 366W" means "354C and 366W", that is, the Fc region contains the 354C and 366W mutations; the amino acid positions of the mutations in the Fc region of this disclosure are all numbered according to the EU numbering system.

[0278] In this disclosure, “subject” and “patient” refer to mammals and encompass any mammal. Examples of mammals include, but are not limited to, mice, rats, rabbits, guinea pigs, monkeys, humans, or such animals as humans.

[0279] Example

[0280] The following embodiments are used to further describe this disclosure, but these embodiments are not intended to limit the scope of this disclosure.

[0281] Experimental methods not specified in the embodiments or test examples of this disclosure are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. See Sambrook et al., Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory; Methods of Modern Molecular Biology, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY.

[0282] Reagents without a specified source are routine reagents purchased from the market.

[0283] Example 1. Design and preparation of γδT antigen

[0284] The TCR (γδT cell receptor) on the surface of γδT cells is a heterodimeric membrane protein composed of γ and δ chains. Amino acids 1-242 of the γ9 chain (Protein data bank, 1HXM, chain B) of the human γδTCR extracellular region were selected, with a 3C restriction enzyme site, a leucine zipper, and a FLAG tag added sequentially to the C-terminus. Amino acids 1-229 of the δ2 ​​chain (Protein data bank, 1HXM, chain A) of the human γδTCR extracellular region were selected, with a 3C restriction enzyme site, a leucine zipper, and a His8 tag added sequentially to the C-terminus. Furthermore, Q180C and V168C mutations were introduced into the constant regions of the γ and δ chains, respectively, to form interchain disulfide bonds, further reducing homodimerization mismatch between the γ and δ chains. The recombinant monkey (Macaca mulatta) γ9δ2TCR protein was obtained by selecting amino acids 1-241 of the monkey γ9 chain (sequence published in patent number: US2019144540A1 SEQ ID NO:42), with a 3C restriction site, a leucine zipper, and a FLAG tag added sequentially to the C-terminus; amino acids 1-229 of the monkey δ2 chain (sequence published in patent number: US20190144540A1 SEQ ID NO:37) were selected, with a 3C restriction site, a leucine zipper, and a His8 tag added sequentially to the C-terminus; in addition, Q179C and V168C mutations were introduced into the constant regions of the γ and δ chains, respectively, to form interchain disulfide bonds.

[0285] Recombinant human γ9 chain (Q180C)

[0286] Recombinant human δ2 chain (V168C)

[0287] Recombinant monkey γ9 chain (Q179C)

[0288] Recombinant monkey δ2 chain (V168C)

[0289] Plasmids (pTT5 vectors) carrying the target protein encoding genes were synthesized separately. Plasmids encoding recombinant human γ9 chain (SEQ ID NO:1) and human δ2 chain (SEQ ID NO:2) were mixed in a 1:1 ratio. Plasmids encoding recombinant monkey γ9 chain (SEQ ID NO:3) and monkey δ2 chain (SEQ ID NO:4) were mixed in a 1:1 ratio and transfected into Expi293 cells (purchased from Thermo). After transient transfection and expression for 7 days, the supernatant was collected after centrifugation at 4°C and purified by His-tag purification resin to obtain recombinant human γ9δ2TCR protein and monkey γ9δ2TCR protein. The proteins were stored at -80°C for later use.

[0290] Example 2. Screening of anti-γδT single-domain antibodies

[0291] Adult healthy alpacas were immunized with in vitro expanded human γ9δ2T cells as immunogens, and specific antibodies were screened using the recombinant human γ9δ2TCR protein from Example 1 via phage display.

[0292] Specifically, the initial immunization dose was 2E7 cells per alpaca. Three weeks after the initial immunization, a booster immunization was administered at a dose of 2E7 cells per alpaca. Subsequent booster immunizations were administered at 3-week intervals. Serum samples were collected one week after each booster immunization, and antibody titers in the alpaca serum were detected using Protein ELISA and FACS.

[0293] The specific procedure for the Protein ELISA is as follows: Recombinant human γ9δ2TCR protein was diluted to 2 μg / mL with 0.05 M carbonate buffer (pH 9.6), 100 μL / well, and coated overnight at 4°C. The plate was blocked with PBST buffer containing 5% skim milk for 1 hour and washed 3 times. Alpaca serum was serially diluted 1:2000 in blocking buffer, incubated at 37°C for 45 min, and washed 5 times. 100 μL of horseradish peroxidase-labeled goat anti-Alpaca IgG secondary antibody (AlpVHHs, 053-404-005, diluted 1:10000 with PBS) was added to each well, and the plate was incubated at 37°C for 45 min and washed 5 times. Finally, 100 μL of TMB chromogenic solution was added to each well for color development, and 50 μL of stop solution was added after 5 minutes to terminate the reaction. The absorbance at 450 nm was read using a microplate reader.

[0294] The specific FACS detection procedure is as follows: Collect cells and resuspend them to 4E6 / mL. Add serially diluted alpaca serum to 50 μL of cells and incubate at 4°C for 1 hour. Wash twice with 1% BSA / PBS buffer, discard the supernatant, add 1:200 diluted Anti alpaca IgG iFluor647 (AlpVHHs, 053-404-009), and incubate at 4°C in the dark for 45 min. Wash twice with 1% BSA / PBS buffer, resuspend in 200 μL buffer, and then perform FACS detection.

[0295] Tests showed that the serum titer of the immunized alpaca was greater than 128kJ.

[0296] One week after each of the three immunizations, 50 mL of peripheral blood was collected to separate lymphocytes (PBMCs). Total RNA was extracted from the PBMCs using RNAiso Plus reagent to construct a phage library, which yielded a phage library with a capacity of 2.56E9.

[0297] Antibodies against recombinant human γ9δ2TCR protein were panned using phage display. Antibodies that cross-binded with monkey γ9δ2TCR protein were identified using ELISA, and their binding activity to in vitro expanded human γ9δ2T cells was determined using FACS. Positive monoclonal single-domain antibodies were screened using the above ELISA and FACS detection methods, and their sequences are shown below.

[0298] >SDP01346 Variable Area

[0299] Table 1. CDR sequences of anti-human γδTCR single-domain antibodies (Kabat numbering rules)

[0300] Example 3. Construction and functional validation of anti-γδTCR chimeric antibody

[0301] 1. Expression of chimeric antibodies

[0302] The nucleotide sequence encoding the single-domain antibody was cloned into the pTT5 vector and transfected into ExpiCHO cells. After 8 days, the cells were centrifuged to remove the cells, and the cell culture medium was collected and filtered. The harvested cell culture medium was purified using a nickel affinity column (HisTrap excel, GE). The bound antibody was eluted with 300mM imidazole, and the medium was desalted and replaced with PBS to obtain the target antibody.

[0303] SDP01346 full length

[0304] 2. Binding of chimeric antibodies to γ9δ2 T cells

[0305] The procedure for detecting the cellular affinity of anti-γδTCR single-domain antibodies is as follows: The serially diluted antibody molecules are placed in a 1×10⁻⁶ medium... 5 γ9δ2T cells were incubated at 4°C for 1 hour. After washing away excess antibody, DyLight 405-AffiniPure Goat Anti-Alpaca IgG and VHH domain antibody (Jackson, Cat#128-475-232) were added and incubated at 4°C for 30 minutes. After washing away excess antibody, the cells were resuspended in 200 μL of 2% FBS / PBS buffer and the fluorescence signal on the cell surface was read using a Thermo Attune NxT flow cytometer. The results are shown in Table 2.

[0306] Table 2. Binding ability of anti-γδTCR chimeric antibodies

[0307] The results showed that SDP01346 had good binding activity on γ9δ2T cells.

[0308] Example 4. Identification of the antigen-binding region of anti-γδTCR antibody

[0309] This embodiment uses a protein-based ELISA to detect the binding region between the anti-γδTCR antibody and the antigen. Using a method similar to that in Example 1, recombinant human γ9δ1TCR protein (dimeric proteins of SEQ ID NO:1 and SEQ ID NO:10), human γ8δ2TCR protein (dimeric proteins of SEQ ID NO:11 and SEQ ID NO:2), human VγδCαβ chimeric TCR protein (dimeric proteins of SEQ ID NO:12 and SEQ ID NO:13), and human VαβCγδ chimeric TCR protein (dimeric proteins of SEQ ID NO:14 and SEQ ID NO:15) were prepared.

[0310] The binding activity of anti-γδTCR antibodies to the aforementioned antigens was identified using ELISA to determine the approximate binding region. The specific detection procedure for the Protein ELISA is as follows: 1 μg / mL of recombinant TCR protein was coated onto plates and incubated overnight at 4°C. The plates were then blocked with PBST buffer containing 1% BSA for 1 hour and washed three times. The antibody was diluted to 30 nM with blocking buffer and incubated at 37°C for 1 hour. After washing three times, the plates were incubated with a 1:10000 dilution of anti-Goat Anti-Alpaca IgG, VHH domain antibody for 1 hour. The plates were washed three times with PBST, and 100 μL of TMB chromogenic solution was added to each well for color development. The reaction was terminated with stop solution after 15 minutes. The absorbance at 450 nm was read using a microplate reader.

[0311] Recombinant human δ1 chain

[0312] Recombinant human γ8 chain

[0313] Recombinant human Vγ9Cβ chain

[0314] Recombinant human Vδ2Cα chain

[0315] Recombinant human Vβ7Cγ chain

[0316] Recombinant human Vα13Cδ chain

[0317] The ELISA results are shown in Table 3. The absorbance values ​​of antibody SDP01346 for different antigens are different. If the absorbance value is more than twice the background value (about 0.1), it is determined to be bound to the same antigen. As shown in Table 3, this antibody binds to the γ9δ1TCR protein but not the γ8δ2TCR protein, indicating that the antibody binds to the γ9 chain; this antibody binds to the VγδCαβ chimeric TCR protein but not the VαβCγδ chimeric TCR, indicating that the antibody binds to the variable region (V region) of the TCR. In summary, it can be clearly concluded that antibody SDP01346 binds to TRGV9.

[0318] Table 3. Identification results of chimeric antibody-antigen binding regions

[0319] Example 5. Humanization of anti-γδTCR antibody

[0320] The variable region sequence was compared with the antibody germline database to obtain a human germline template with high homology. Among them, the human germline heavy chain template used for SDP01346 was IGHV3-30. After predicting the structure of the monoclonal antibody through homology modeling, the CDR of the single-domain antibody was embedded into a suitable human GermLine frame (Bioinformation.2014;10(4):180-186;Methods Mol Biol.2019;1904:213-230), and then a reversion mutation was introduced into the frame region.

[0321] The obtained humanized molecule sequence is as follows:

[0322] >SDP01346 VH1

[0323] >SDP01346 VH2

[0324] >SDP01346 VH3

[0325] >SDP01346 VH4

[0326] After humanization of the single-domain antibody, a full-length antibody was constructed, and GGGGSHHHHHHH (SEQ ID NO:63) was added after the corresponding VHH.

[0327] Table 4. Correspondence between antibody names and antibody structures.

[0328] The following example provides the full-length sequence of SDP01378.

[0329] SDP01378 full length

[0330] The following section describes the FACS assay for the binding of humanized monoclonal antibody to γ9δ2T cells.

[0331] Affinity assay for humanized single-domain antibody SDP01346: The serially diluted antibody molecules were analyzed at 1×10⁻⁶... 5 γ9δ2T cells were incubated at 4°C for 1 hour. Excess antibody was washed away, and DyLight 405-AffiniPure Goat Anti-Alpaca IgG, VHH domain antibody (Jackson, Cat#128-475-232) was added. The cells were incubated at 4°C for 30 minutes, and after washing away excess antibody, they were resuspended in 200 μL of 2% FBS / PBS buffer. The fluorescence signal on the cell surface was read using a Thermo Attune NxT flow cytometer. The results are shown in Table 5.

[0332] Table 5. Affinity Detection of Humanized Molecules in SDP01346

[0333] The affinity ranking was SDP01376 (0.76 nM) > SDP01377 (0.77 nM) > SDP01378 (1.06 nM) > SDP01375 (1.27 nM), all of which were in the nanomolar range, indicating that the affinity was good.

[0334] Max MFI (Maximum Mean Fluorescence Intensity) reflects the total amount of antibody binding to cell surface antigens / the saturation of binding sites; the higher the value, the greater the total amount of binding. SDP01378 has the highest Max MFI, indicating that it binds the most to the TRGV9 antigen on the surface of γ9δ2T cells, and has the best binding efficiency.

[0335] Example 6. Design and preparation of PSMA antigen

[0336] 1. PSMA antigen protein

[0337] Prostate-specific membrane antigen (PSMA) is an N-terminal intracellular single-pass transmembrane protein. The full-length human PSMA sequence is referenced from the uniprot database Q04609, with its extracellular domain consisting of amino acids 44 to 750. The histagged human PSMA extracellular domain protein was purchased from Acro (catalog number PSA-H52H3). The full-length monkey (Macaca fascicularis) PSMA sequence is referenced from the uniprot database A0A2K5VNZ0, with its extracellular domain consisting of amino acids 44 to 750. The histagged monkey PSMA extracellular domain protein was purchased from Acro (catalog number PSA-C5247). The full-length mouse (Mus musculus) PSMA sequence is referenced from the uniprot database O35409, with its extracellular domain consisting of amino acids 44 to 752. The histagged mouse PSMA extracellular domain protein was purchased from Acro (catalog number PSA-M5245).

[0338] Human PSMA sequence (including signal peptide)

[0339] 2. PSMA cell line

[0340] The human PSMA cell line used for selection was LNCaP. The monkey PSMA cell line used for selection was the CHO-K1 cell line (CHO-K1 / cyno PSMA) that stably expresses monkey PSMA. The sequences used for stable cell construction are as follows: >Monkey PSMA sequence (containing signal peptide)

[0341] Example 7. Screening of anti-PSMA single-domain antibodies

[0342] Adult healthy Bactrian camels were immunized with recombinant human PSMA protein as an immunogen, and specific antibodies were screened using the recombinant human PSMA protein from Example 6 with phage display.

[0343] Specifically, the initial immunization dose was 200 μg per camel, with CFA adjuvant added. Two weeks after the initial immunization, a booster immunization was administered, with a dose of 200 μg per camel, with IFA adjuvant added. Subsequent booster immunizations were administered at two-week intervals. Serum samples were collected one week after each booster immunization, and antibody titers in camel serum were detected using protein-based ELISA and cell-based FACS.

[0344] The specific ELISA detection procedure is as follows: Recombinant human PSMA protein was coated at 0.2 μg / well in 0.05 M carbonate buffer (pH 9.6) overnight at 4°C; the plate was blocked with PBST buffer containing 1% skim milk for 1 hour and washed 3 times; camel serum was serially diluted 1:200 in blocking buffer, incubated at 37°C for 1 hour, and washed 3 times; 100 μL of horseradish peroxidase-conjugated goat anti-Llama IgG (H+L) secondary antibody (Antai Zhiyuan) was added to each well, incubated at 37°C for 1 hour, and washed 4 times. Finally, 100 μL of TMB chromogenic solution was added to each well for color development, and 50 μL of stop solution was added after 5 minutes to terminate the reaction. The absorbance at 450 nm was read using a microplate reader.

[0345] The specific FACS detection procedure is as follows: Collect LNCaP cells and resuspend them to 4E6 / mL. Add 50 μL of serially diluted camel serum to each cell, and incubate at 4°C for 1 hour. Wash twice with 1% BSA / PBS buffer, discard the supernatant, add 1:200 diluted Anti-llama IgG 488 (Antai Zhiyuan), and incubate at 4°C in the dark for 30 minutes. Wash twice with 1% BSA / PBS buffer, resuspend in 250 μL buffer, and then perform FACS detection.

[0346] Tests showed that the serum titer of the immunized camels was greater than 10. 2 k.

[0347] One week after each of the three immunizations, 50 mL of peripheral blood was collected to isolate peripheral blood cells (PBMCs). Total RNA was extracted from the PBMCs using RNAiso Plus reagent to construct a phage library, resulting in a phage library with a capacity of 3.2E9. The phage immune library was panned using biotinylated recombinant human PSMA protein, and positive monoclonal single-domain antibodies were screened using a phage ELISA assay. The sequences are shown below.

[0348] >SDP05591 Variable Area

[0349] >SDP05586 Variable Area

[0350] Table 6. CDR sequences of anti-human PSMA single-domain antibodies (Kabat numbering rules)

[0351] Example 8. Construction and functional verification of anti-PSMA chimeric antibody

[0352] 1. Expression of chimeric antibodies

[0353] The nucleotide sequence encoding the single-domain antibody was cloned into the pTT5 vector by adding the human IgG1 hinge-CH2-CH3 sequence. The plasmid was then extracted and transfected into ExpiCHO cells. After 8 days of expression, the cells were centrifuged to remove them, and the cell culture medium was collected and filtered. The harvested cell culture medium was purified using Protein A magnetic beads, desalted, and replaced with PBS to obtain the target chimeric antibody.

[0354] SDP05591 full length (abruptly changes to hinge region C220A)

[0355] SDP05586 full length (abruptly changes to hinge region C220A) Note: Underlined text indicates CDR sequence, italic text indicates IgG1 hinge-CH2-CH3 sequence.

[0356] 2. The binding activity of chimeric antibodies to cells

[0357] The procedure for detecting the cellular affinity of anti-PSMA single-domain antibodies is as follows: Serially diluted antibody molecules are placed in a 1 x 10⁻⁶ medium... 5 LNCaP cells were incubated at 4°C for 1 hour, excess antibody was washed away, and mouse Alexa Flour 647-labeled anti-human Fc antibody was added. After incubation at 4°C for 30 minutes, excess antibody was washed away, and the cells were resuspended in 200 μL of 2% FBS / PBS buffer. Fluorescence signals on the cell surface were read using a Thermo Attune NxT flow cytometer. Table 7 summarizes the binding activities of each antibody. The results showed that SDP05591 and SDP05586 exhibited good binding activity on LNCaP cells and also showed cross-binding activity with monkey PSMA.

[0358] Table 7. Cell binding ability of anti-PSMA chimeric antibodies

[0359] Example 9. Epitope grouping among anti-PSMA antibodies

[0360] Epitope grouping was performed using a competitive ELISA against anti-PSMA antibodies. The specific steps were as follows: Antibody 1 (SDP05586, SDP05591) was diluted to 1 μg / mL and added to 96-well plates, 50 μL / well. The plates were incubated overnight at 4°C. After blocking with 1% BSA / PBST, human PSMA protein was mixed with excess antibody 2 (30 μg / mL, approximately 200 nM) and added to the 96-well plates. A positive control (antigen only) was also added. The plates were incubated at room temperature for 1 h. After washing with PBST, anti-His tag HRP antibody was added and incubated at room temperature for 1 h. After washing with PBST, ATBS was added for color development, and OD415 was detected. The inhibition rate was calculated. Inhibition rate (%) = 100 - (OD415(Ag+Ab2) / OD415(Ag)). When the inhibition rate (%) < 10%, the two antibodies have the same epitope; when 20% < inhibition rate (%) < 80%, the epitopes of the two antibodies partially overlap; when the inhibition rate (%) > 90%, the epitopes of the two antibodies are different.

[0361] The inhibition rate (%) is shown in Table 8. According to the results analysis, SDP05591 and SDP05586 target different epitopes.

[0362] Table 8. Inhibition rate of anti-PSMA antibody epitopes by group (%)

[0363] Example 10. Humanization of anti-PSMA antibody

[0364] The variable region sequence was compared with the antibody germline database to obtain human germline templates with high homology. Among them, the human germline heavy chain template used for SDP05591 was IGHV3-30*02; the human germline heavy chain template used for SDP05586 was IGHV3-30*02. After predicting the structure of the monoclonal antibody through homology modeling, the CDR of the single-domain antibody was embedded into a suitable human GermLine frame (Bioinformation.2014;10(4):180-186;Methods Mol Biol.2019;1904:213-230), and then a reversion mutation was introduced into the frame region.

[0365] The obtained humanized molecule sequence is as follows:

[0366] >SDP05591 VH1

[0367] >SDP05591 VH2

[0368] >SDP05591 VH3

[0369] >SDP05591 VH4

[0370] >SDP05591 VH5

[0371] >SDP05586 VH1

[0372] >SDP05586 VH2

[0373] >SDP05586 VH3

[0374] >SDP05586 VH4

[0375] >SDP05586 VH5

[0376] After humanization of the single-domain antibody, a full-length antibody was constructed, and a human IgG1 hinge -CH2-CH3 was added after the corresponding VH.

[0377] Table 9. Correspondence between antibody names and antibody structures.

[0378] The following examples provide the full-length sequences of SDP05617 and SDP05603.

[0379] SDP05617 full length (abruptly changes to hinge region C220A)

[0380] SDP05603 full length (abruptly changes to hinge region C220A)

[0381] The following section describes the FACS assay for the binding of humanized monoclonal antibodies to LNCaP cells.

[0382] Affinity assay for humanized single-domain antibodies SDP05617 and SDP05603: The serially diluted antibody molecules were analyzed at 1 x 10⁻⁶ ppm. 5 LNCaP cells were incubated at 4°C for 1 hour, excess antibody was washed away, and mouse Alexa Flour 647-labeled anti-human Fc antibody was added. The cells were incubated at 4°C for 30 minutes, and after washing away excess antibody, they were resuspended in 200 μL of 2% FBS / PBS buffer. The fluorescence signal on the cell surface was read using a Thermo Attune NxT flow cytometer. The results are shown in Table 10.

[0383] Table 10. Affinity Detection of Humanized Molecules in SDP05591 and SDP05586

[0384] SDP05591 humanized molecules had comparable affinity, so SDP05617 was selected based on a combination of expression level and purity. SDP05586 humanized molecules had comparable affinity, so SDP05603, with a moderate number of reversion mutations, was selected.

[0385] Further analysis was conducted to determine the binding kinetics of antibodies SDP05617 and SDP05603. Experimental protocol: Antibodies SDP05617 and SDP05603 were diluted to 1 μg / mL with HBS-EP+ buffer at a flow rate of 10 μL / min, capturing antibodies up to 200 RU. His-tagged human PSMA antigen (purchased from Acro, catalog number PSA-H52H3) was diluted with HBS-EP+ buffer at specific concentration gradients of 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM. The flow rate for sample analysis was set to 30 μL / min. The binding time was 120 s, and the dissociation time was 600 s. Regeneration was then performed using pH 1.5 Gly-HCl buffer at a flow rate of 30 μL / min for 30 s. The response signal is plotted with analysis time on the x-axis and response value on the y-axis. The obtained data is fitted using BIAcore 8K analysis software, and a 1:1 Langmuir binding model is used to determine the kinetic constants such as binding rate constant (Ka), dissociation rate constant (Kd), and dissociation equilibrium constant (KD).

[0386] Table 11 summarizes the binding activities of each antibody. In terms of affinity, the KD of SDP05617 is less than that of SDP05603 (1.12E-08M), indicating that SDP05617 has a stronger affinity for human PSMA.

[0387] Table 11. Binding activity of antibodies to human PSMA antigen protein

[0388] Example 11. Design and preparation of anti-PSMA×γδTCR antibody

[0389] Based on the anti-PSMA and anti-γδT antibodies screened in the above embodiments, anti-PSMA×γδTCR antibodies were designed, including two 1+1 asymmetric structures (SDP02604 and SDP02605), one PSMA bivalent 2+1 asymmetric structure (SDP02607), and one PSMA dual epitope 1+1+1 asymmetric structure (SDP02597). These four multispecific antibody structures are shown in Figure 1. All employ KIH (knob into hole) to avoid heavy chain mismatch, and the constant region sequences are as follows.

[0390] >IgG1 Fc2(L234F / L235E / Y349C / T366S / L368A / Y407V)

[0391] >IgG1 Fc1(L234F / L235E / S354C / T366W)

[0392] SDP02597's first polypeptide chain includes the SDP01346 VH4-linker-SDP05591 VH2-constant region from the N-terminus to the C-terminus, and the second polypeptide chain includes the SDP05586 VH3-constant region from the N-terminus to the C-terminus. SDP02604's first polypeptide chain includes the SDP01346 VH4-constant region from the N-terminus to the C-terminus, and the second polypeptide chain includes the SDP05586 VH3-constant region from the N-terminus to the C-terminus. SDP02605's first polypeptide chain includes the SDP01346 VH4-constant region from the N-terminus to the C-terminus, and the second polypeptide chain includes the SDP05591VH2-constant region from the N-terminus to the C-terminus. The first polypeptide chain of SDP02607 includes the SDP01346 VH4-linker-SDP05591 VH2-constant region from the N-terminus to the C-terminus, and the second polypeptide chain includes the SDP05591 VH2-constant region from the N-terminus to the C-terminus.

[0393] The sequences of the four bispecific antibodies are as follows:

[0394] >SDP02597 H1

[0395] >SDP02597 H2

[0396] >SDP02604 H1

[0397] SDP02604 H2 is identical to SEQ ID NO:48;

[0398] SDP02605 H1 is identical to SEQ ID NO:49;

[0399] >SDP02605 H2

[0400] SDP02607 H1 is identical to SEQ ID NO:47;

[0401] SDP02607 H2 is identical to SEQ ID NO:50;

[0402] The control antibody is SDP03779 (LAVA-1207), and its sequence is as follows.

[0403] >SDP03779 H1

[0404] >SDP03779 H2

[0405] Example 12. Detection of binding affinity between anti-PSMA / γδTCR antibody and human / monkey antigen

[0406] A Protein A biosensor chip (Cat.#29139121-AB, Cytiva) was used. Each antibody was prepared as a ligand using HBS-EP+ buffer to capture Protein A on the chip channels. Human PSMA (Acro Biosystems, Cat#PSA-H52H3) / cynomolgus PSMA (Acro Biosystems, Cat#PSA-C5247) / human γ9δ2 / cynomolgus γ9δ2 antigens were prepared as analytes using HBS-EP+ buffer and serially diluted 2-fold. The diluted antigens were flowed through the experimental and reference channels at a flow rate of 30 μL / min for 80 seconds for binding and 300 seconds for dissociation. Regeneration buffer was 10 mM Glycine pH 1.5 (GE Healthcare, BR-1003-54) at a flow rate of 10 μL / min for 30 seconds. Data were analyzed using Biacore 8K evaluation software.

[0407] The results are shown in Table 12. SDP02597 showed the strongest binding activity to human PSMA, while SDP02605 and SDP02607 exhibited nanomolar binding activities. SDP02604, with a binding KD of 28 nM, was stronger than SDP03779. SDP03779 did not bind to cynomolgus monkey PSMA. The affinity of SDP02597, SDP02604, SDP02605, and SDP02607 for cynomolgus monkey PSMA was comparable to that for human PSMA.

[0408] Table 12. Binding kinetic parameters of antibody to human / cynomolgus monkey PSMA protein

[0409] Example 13. Detection of the binding activity of anti-PSMA / γδTCR antibody with human γ9δ2T cells and PBMCs

[0410] The binding activity of anti-PSMA / γδTCR antibody to human γ9δ2T cells and human PBMCs was detected using FACS assays. Human γ9δ2T cells were obtained by induction with zoledronic acid and IL-2. The binding activity of anti-PSMA / γδTCR antibody on γδT cells was evaluated by detecting the fluorescence signal of the antibody on the cell surface, and the binding strength was assessed based on the intensity of the fluorescence signal. Specifically, serially diluted antibody molecules and control molecules were incubated at 1E5 cells per cell at 4°C for 1 hour. Excess antibody was washed away, and mouse Alexa Flour 647-labeled anti-human Fc antibody (Jackson, Cat#209-605-098) was added. The cells were incubated at 4°C for 30 minutes, and after washing away excess antibody, the cells were resuspended in 200 μL of 2% FBS / PBS buffer. The fluorescence signal on the cell surface was read using a Thermo Attune NxT flow cytometer.

[0411] The results are shown in Figure 2 and Table 13. SDP02597, SDP02604, SDP02605, SDP02607 and SDP03779 showed high affinity for γδT-terminal EC. 50 The effective concentrations were 1.101 nM, 0.9148 nM, 0.8855 nM, 1.070 nM, and 2.296 nM, respectively. None of the antibodies tested exhibited nonspecific binding on PBMCs (Figure 3).

[0412] Table 13. Detection of γδT cell binding activity Note: - indicates no binding activity.

[0413] Example 14. Detection of the binding activity of anti-PSMA / γδTCR antibody with PSMA-positive and PSMA-negative cells

[0414] The binding activity of PSMA / γδTCR antibodies to cells with different PSMA expression levels and negative cells was detected using FACS assays. 22Rv1 cells and LNCaP cells naturally expressing human PSMA were purchased from ATCC, and DU145 cells not expressing human PSMA were purchased from the Chinese Academy of Sciences Cell Bank. DU145-hPSMA (medium) and DU145-hPSMA (high) were obtained by selecting single clones after transfecting DU145 cells with human PMSA-expressing lentivirus. The PMSA binding activity of anti-PSMA / γδTCR antibodies on different cells was assessed by detecting the fluorescence signal of the antibody binding on the cell surface; the intensity of the fluorescence signal was used to evaluate the antibody binding strength. Specifically, serially diluted antibody molecules and control molecules were incubated in 1E5 cells at 4°C for 1 hour. Excess antibody was washed away, and mouse-derived Alexa Flour 647-labeled anti-human Fc antibody (Jackson, Cat#209-605-098) was added. The cells were incubated at 4°C for 30 minutes, and after washing away excess antibody, the cells were resuspended in 200 μL of 2% FBS / PBS buffer. The fluorescence signal on the cell surface was read using a Thermo Attune NxT flow cytometer.

[0415] The results are shown in Figures 4A-4D and Table 14. The binding activity trends of SDP02597, SDP02604, SDP02605, SDP02607 and SDP03779 on cells with different PSMA expression levels are consistent, and the antibodies tested did not bind nonspecifically on PSMA-negative cells (Figure 5).

[0416] Table 14. Cell binding activity assay at different PSMA expression levels Note: - indicates no binding activity.

[0417] Example 15. Detection of PSMA / γδTCR antibody-mediated γδT cell killing activity against tumor cells

[0418] In this embodiment, the lactate dehydrogenase (LDH) assay was used to evaluate the antibody-mediated killing activity of γδT cells against target cells with different PSMA expression levels.

[0419] LNCaP cells naturally express high levels of PSMA, and the cell culture medium is RPMI 1640 (Gibco, Cat#10491A-01, the same below) containing 10% inactivated fetal bovine serum. 22Rv1 cells express low to medium levels of PSMA, and the cell culture medium is RPMI 1640. DU145-hPSMA (medium) and DU145-hPSMA (high) were obtained by selecting single clones after transfecting DU145 cells with human PMSA-expressing lentivirus, and the culture medium is MEM (GIBCO, Cat#11095072) containing 10% inactivated fetal bovine serum and sodium pyruvate. After digestion, the target cells were resuspended in RPMI 1640 medium with 2% serum, and the density was adjusted to 7E4 cells / mL. Then, 50 μL / well was seeded into 96-well plates, and 50 μL of serially diluted test antibody was added. 50 μL of culture medium was added to each well. γδT cells were collected and resuspended in RPMI 1640 containing 2% fetal bovine serum, adjusting the cell density to a final E:T ratio of 1:1. 50 μL / well were seeded into the above experimental plates and incubated at 37°C in a 5% CO2 incubator for 24 hours. The cell culture plates were then removed, and the cell culture supernatant was collected by centrifugation (400 g, 5 min) using CytoTox. The Non-Radioactive Cytotoxicity Assay kit (Promega, G1780) is used to detect LDH levels. Refer to the kit's instruction manual for specific instructions.

[0420] The results, as shown in Figures 6A to 6D and Table 15, indicate that the EC50 values ​​of SDP02597, SDP02604, SDP02605, and SDP02607 on all cells were significantly lower than those of the control SDP03779, demonstrating that the cytotoxic activity of these four antibodies was significantly stronger than that of the control. With increasing PSMA expression levels on the cell surface, the EC50 values ​​of each antibody decreased (e.g., the EC50 of SDP02597 was 2.47 in DU145-hPSMA (medium) and 1.04 in DU145-hPSMA (high), indicating that higher antigen expression levels resulted in better cytotoxic effects even at low concentrations.

[0421] Table 15. Killing effect of PSMA / γδTCR antibody on cells expressing different antigens Note: - indicates no lethal activity.

[0422] Example 16. Detection of PSMA / γδTCR antibody-mediated PBMC killing activity against tumor cells

[0423] This embodiment assesses the antibody-mediated killing activity of PBMCs against PSMA-expressing 22Rv1 cells by detecting changes in tumor cell fluorescence signals. 22Rv1 cells carrying the Luc signal were trypsinized and then mixed with PBMC cells at an E:T ratio of 10:1. Serially diluted antibodies were added, and the mixture was incubated at 37°C in a 5% CO2 incubator for 120 hours. The cell culture plates were then removed, and 100 μL / well of BrightGlo assay reagent was added to detect the Luc signal.

[0424] The results, as shown in Figures 7A and 7B and Table 16, indicate that the cytotoxic activity of SDP02597 and SDP02607 was significantly stronger than that of SDP03779 on two different PBMC donors.

[0425] Table 16. PSMA / γδTCR antibody-mediated killing of tumor cells by PBMCs Note: - indicates no lethal activity.

[0426] Example 17. Detection of PSMA / γδTCR antibody-induced γδT cell proliferation activity in PBMCs

[0427] In this embodiment, flow cytometry was used to evaluate the activity of anti-PSMA / γδTCR bispecific antibody in inducing γδT cell proliferation in PBMCs.

[0428] Take 800 μL (1×10⁻⁶) of PBMC 6 DU145-hPSMA cells were added to 24-well plates; after washing twice with PBS, the cell density was adjusted to 5 × 10⁶ cells / well. 5 Cells / mL, add 1 mL to the corresponding well; prepare the antibody to be tested to the appropriate concentration in culture medium, then add 200 μL to the well. Incubate at 37℃, 5% CO2, changing the medium halfway every 3 days. After 7 days, wash the cells twice with PBS, and stain with CD3 antibody (BD, Cat#564713), Vδ2TCR antibody (BD, Cat#555739), and Vγ9TCR antibody (BD, Cat#555732) to identify changes in the proportion of γδT cells.

[0429] The results are shown in Figures 8A-8C. In PBMC cells derived from the three donors #XC11053, #XC11061, and #sc12369, after 7 days of stimulation, SDP02597 and SDP02607 significantly increased the proportion of γδT cells.

[0430] Example 18. Evaluation of the antitumor activity of PSMA / γδTCR antibody in a mouse model of prostate cancer bone metastasis.

[0431] Experimental NOG mice, female, 6-8 weeks old, were provided by Vital River. Housing environment: SPF grade. DU145-hPSMA cells expressing Luciferase were used at a rate of 2 × 10⁻⁶. 5 Five NOG mice were randomly assigned to five groups of eight mice each, with one or more mice injected into the tibia: vehicle, γδT, SDP02597 (6.0 mg / kg), and SDP02607 (6.0 mg / kg). γδT was administered intravenously (iv) on days 1, 5, and 8, while the antibody was administered intraperitoneally (ip) on days 1 and 8. Luc signals were measured twice weekly during the administration and observation periods, and the values ​​were recorded. The relative tumor proliferation rate T / C (%) was calculated as (T - T0) / (C - C0) × 100, where T and C represent the tumor volumes at the end of the experiment in the treatment and control groups, respectively; and T0 and C0 represent the tumor volumes at the beginning of the experiment. The tumor inhibition rate TGI (%) was calculated as 1 - T / C (%).

[0432] The results are shown in Figures 9A and 9B and Table 17. The bispecific antibodies disclosed herein can effectively inhibit tumors, and no significant changes in body weight were observed in mice in any of the treatment groups.

[0433] Table 17. Antitumor activity of bispecific antibodies in the DU145-hPSMA bone metastasis model Note: *** represents P<0.001.

Claims

1. PSMA / TRGV9 binding protein, which includes: Specifically binds to the first antigen-binding domain of PSMA; and It specifically binds to the second antigen-binding domain of TRGV9; in, The first antigen-binding domain includes: Immunoglobulin single variable domain 1, wherein the immunoglobulin single variable domain 1 comprises CDR1, CDR2 and CDR3 in any of the sequences of SEQ ID NO:23, 33-37; And / or, Immunoglobulin single variable domain 2, wherein the immunoglobulin single variable domain 2 comprises CDR1, CDR2 and CDR3 in any of the sequences of SEQ ID NO:24, 38-42; The CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system; Preferably, the first antigen-binding domain comprises: The immunoglobulin single variable domain 1 comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO:25-27, respectively; And / or, The immunoglobulin single variable domain 2 comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO:28-30, respectively.

2. The PSMA / TRGV9 binding protein according to claim 1, wherein: The immunoglobulin single variable domain 1 comprises an amino acid sequence shown in or having at least 80% or at least 90% identity with any of SEQ ID NO:23, 33-37; and / or, The immunoglobulin single variable domain 2 comprises an amino acid sequence shown in or having at least 80% or at least 90% identity with any of SEQ ID NO:24, 38-42.

3. The PSMA / TRGV9 binding protein according to claim 1 or 2, wherein, The second antigen-binding domain includes an immunoglobulin single variable domain 3, wherein the immunoglobulin single variable domain 3 includes CDR1, CDR2 and CDR3 in any of the sequences of SEQ ID NO:5, 16-19; wherein the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system. Preferably, the immunoglobulin single variable domain 3 comprises amino acid sequences CDR1, CDR2 and CDR3 as shown in SEQ ID NO:6-8.

4. The PSMA / TRGV9 binding protein of claim 3, wherein, The immunoglobulin single variable domain 3 comprises the amino acid sequence shown in any one of SEQ ID NO: 5, 16-19 or an amino acid sequence having at least 80% or at least 90% identity with it.

5. PSMA / TRGV9 binding protein, which includes: Specifically binds to the first antigen-binding domain of PSMA; and It specifically binds to the second antigen-binding domain of TRGV9; wherein The second antigen-binding domain includes an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain includes CDR1, CDR2 and CDR3 in any of the amino acid sequences shown in SEQ ID NO:5, 16-19; wherein the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system. Preferably, the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO:6-8, respectively; Preferably, the immunoglobulin single variable domain comprises an amino acid sequence shown in or having at least 80% or at least 90% identity with any of SEQ ID NO: 5, 16-19.

6. The PSMA / TRGV9 binding protein according to any one of claims 1-5, wherein, The immunoglobulin single variable domain is modified by humanization, reverse mutation, affinity maturation, removal of T cell epitopes, reduction of antibody deamide and / or reduction of antibody isomerization. Preferably, the humanized heavy chain template for the immunoglobulin single variable domain is selected from IGHV3-30*02 and / or IGHV3-30.

7. The PSMA / TRGV9 binding protein according to any one of claims 1-6, wherein, The valence ratio of the first antigen-binding domain to the second antigen-binding domain is between 2:1 and 1:2, preferably 2:1 or 1:

1.

8. The PSMA / TRGV9 binding protein according to any one of claims 1-7, 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.

9. The PSMA / TRGV9 binding protein according to claim 8, wherein, The Fc region comprises a first subunit and a second subunit; Preferably, the first and second subunits of the Fc region contain a knock-into-hole mutation; Preferably, The first subunit of the Fc region contains a mutation at position 366, and the second subunit contains a mutation selected from positions 366, 368, or 407, or any combination thereof. The first subunit of the Fc region contains a mutation at position 354 or 356, and the second subunit contains a mutation at position 349; or The first subunit of the Fc region contains a mutation at position 354 or 356, and the second subunit contains a mutation at position 349, 366, 368 or 407, or any combination thereof. The mutation is defined according to the EU numbering system; More preferably, The first subunit of the Fc region contains the 366W mutation, and the second subunit contains a mutation selected from 366S, 368A and 407V or any combination thereof. The first subunit of the Fc region contains a 354C or 356C mutation, and the second subunit contains a 349C mutation; or The first subunit of the Fc region contains the 354C / 366W mutation, and the second subunit contains the 349C / 366S / 368A / 407V mutation.

10. The PSMA / TRGV9 binding protein according to any one of claims 1-9, comprising a linker; Preferably, the connector is (G) m S n ) h 、(G m Q n ) h (GGNGT) h (YGNGT) h (EPKSS) h or (A) m S n ) h ,in, m is independently selected from integers 1-8, n is independently selected from integers 0-8, and h is independently selected from integers 1-20; more preferably, the connector is A3S, G4S, or (G4S)2.

11. A PSMA / TRGV9 binding protein comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are selected from any one of the following groups: The first polypeptide chain, from the N-terminus to the C-terminus, includes: It specifically binds to the TRGV9 second antigen-binding domain and the Fc first subunit; And, the second polypeptide chain from the N-terminus to the C-terminus includes: a PSMA-specific first antigen-binding domain and an Fc second subunit; or, The first polypeptide chain, from the N-terminus to the C-terminus, includes: a TRGV9 second antigen-binding domain specifically binding to the PSMA first antigen-binding domain - Fc first subunit; and the second polypeptide chain, from the N-terminus to the C-terminus, includes: a PSMA first antigen-binding domain specifically binding to the PSMA second subunit. Preferably, the first polypeptide chain and the second polypeptide chain are selected from any one of the following groups: (1) The first polypeptide chain from the N-terminus to the C-terminus includes: VHH3-[linker 1] a -Fc first subunit; and, the second polypeptide chain from the N-terminus to the C-terminus includes: VHH1-[linker 2] b -Fc second subunit; (2) The first polypeptide chain from the N-terminus to the C-terminus includes: VHH3-[linker 1] a -Fc first subunit; and, the second polypeptide chain from the N-terminus to the C-terminus includes: VHH2-[linker 3] c -Fc second subunit; (3) The first polypeptide chain from the N-terminus to the C-terminus includes: VHH3-[linker 4] d -VHH1-[Connector 5] e - First subunit; and, the second polypeptide chain from the N-terminus to the C-terminus includes: VHH1-[linker 2] b -Fc second subunit; (4) The first polypeptide chain from the N-terminus to the C-terminus includes: VHH3-[linker 4] d -VHH1-[Connector 5] e - First subunit; and, the second polypeptide chain from the N-terminus to the C-terminus includes: VHH2-[linker 3] c -Fc second subunit; Preferably, connector 1, connector 2, connector 3, connector 4, and connector 5 are independently selected from A3S, G4S, or (G4S)2; a, b, c, d, and e are independently selected from 0 or 1. Preferably, the VHH1 comprises amino acid sequences CDR1, CDR2 and CDR3 as shown in SEQ ID NO:25-27, and / or the VHH1 comprises an amino acid sequence as shown in any one of SEQ ID NO:23, 33-37 or having at least 80% or at least 90% identity with it. VHH2 contains amino acid sequences CDR1, CDR2 and CDR3 as shown in SEQ ID NO:28-30, and / or VHH2 contains an amino acid sequence as shown in any one of SEQ ID NO:24, 38-42 or having at least 80% or at least 90% identity with it; And / or, VHH3 contains amino acid sequences CDR1, CDR2 and CDR3 as shown in SEQ ID NO:6-8, and / or VHH3 contains an amino acid sequence as shown in any one of SEQ ID NO:5, 16-19 or having at least 80% or at least 90% identity with it.

12. A PSMA / TRGV9 binding protein, comprising a first polypeptide chain and a second polypeptide chain; wherein, The first polypeptide chain comprises an amino acid sequence shown in or having at least 80% or at least 90% identity with any of SEQ ID NO:47 or 49; and, The second polypeptide chain contains an amino acid sequence shown in or having at least 80% or at least 90% identity with any of SEQ ID NO:48 or 50; Preferably, the first polypeptide chain and the second polypeptide chain are selected from any one of the following groups: The first polypeptide chain contains an amino acid sequence shown in or having at least 80% or at least 90% identity with SEQ ID NO:47, and the second polypeptide chain contains an amino acid sequence shown in or having at least 80% or at least 90% identity with SEQ ID NO:48; The first polypeptide chain contains an amino acid sequence shown in or having at least 80% or at least 90% identity with SEQ ID NO:49, and the second polypeptide chain contains an amino acid sequence shown in or having at least 80% or at least 90% identity with SEQ ID NO:48; The first polypeptide chain comprises an amino acid sequence shown in or having at least 80% or 90% identity with SEQ ID NO:49, and the second polypeptide chain comprises an amino acid sequence shown in or having at least 80% or 90% identity with SEQ ID NO:50; or, The first polypeptide chain contains an amino acid sequence shown in or having at least 80% or at least 90% identity with SEQ ID NO:47, and the second polypeptide chain contains an amino acid sequence shown in or having at least 80% or at least 90% identity with SEQ ID NO:

50.

13. The PSMA / TRGV9 binding protein according to any one of claims 1-12, wherein it is an anti-PSMA / TRGV9 antibody.

14. A PSMA-binding protein comprising a single variable immunoglobulin domain that specifically binds to PSMA; wherein, The immunoglobulin single variable domain includes: CDR1, CDR2, and CDR3 from any sequence of SEQ ID NO:23, 33-37; or, CDR1, CDR2, and CDR3 in any of the sequences of SEQ ID NO:24, 38-42; The CDR is defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system; Preferably, the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO:25-27; or, CDR1, CDR2 and CDR3 as shown in SEQ ID NO:28-30.

15. The PSMA-binding protein according to claim 14, comprising an amino acid sequence shown in or having at least 80% or at least 90% identity with any of SEQ ID NO:23, 33-37, or an amino acid sequence shown in or having at least 80% or at least 90% identity with any of SEQ ID NO:24, 38-42.

16. The PSMA-binding protein according to claim 14 or 15, wherein, The immunoglobulin single variable domain is modified by humanization, reverse mutation, affinity maturation, removal of T cell epitopes, reduction of antibody deamide and / or reduction of antibody isomerization. Preferably, the humanized heavy chain template for the immunoglobulin single variable domain is IGHV3-30*02.

17. The PSMA-binding protein according to any one of claims 14-16, 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.

18. The PSMA-binding protein according to any one of claims 14-17, further comprising an antigen-binding domain that specifically binds to a T-cell receptor; wherein the T-cell receptor is preferably the T-cell receptor γ9 chain, more preferably the T-cell receptor γ variable region 9 (TRGV9).

19. A polynucleotide encoding the PSMA / TRGV9 binding protein of any one of claims 1-13 or the PSMA binding protein of any one of claims 14-16, or a combination thereof; Preferably, the polynucleotide is DNA or RNA.

20. A carrier comprising the polynucleotide of claim 19.

21. A host cell containing or expressing the polynucleotide of claim 19 or the vector of claim 20.

22. A pharmaceutical composition comprising: The PSMA / TRGV9 binding protein according to any one of claims 1-13, the PSMA binding protein according to any one of claims 14-16, the polynucleotide according to claim 19, or the vector according to claim 20; In addition, optionally one or more pharmaceutically acceptable excipients, diluents, or excipients.

23. A method for preparing the PSMA / TRGV9 binding protein according to any one of claims 1-13 or the PSMA binding protein according to any one of claims 14-16, comprising: The host cells of claim 21 express PSMA / TRGV9 binding protein, or PSMA binding protein; or, The polynucleotide of claim 19 or the vector of claim 20 is expressed in a host cell, wherein the polynucleotide or the vector encodes a PSMA / TRGV9 binding protein or a PSMA binding protein. And, the PSMA / TRGV9 binding protein or PSMA binding protein expressed in the host cells; Optionally, the process further includes a step of purifying the PSMA / TRGV9 binding protein or the PSMA binding protein.

24. Use of the PSMA / TRGV9 binding protein of any one of claims 1-13, the PSMA binding protein of any one of claims 14-16, the polynucleotide of claim 19, the carrier of claim 20, or the pharmaceutical composition of claim 22 in the preparation of a medicament for treating cancer; Preferably, the cancer is a PSMA-positive cancer; Preferably, the cancer is prostate cancer.

25. A method for recruiting and / or activating T cells, comprising: The step of contacting T cells with the PSMA / TRGV9 binding protein of any one of claims 1-13, the polynucleotide of claim 19, the carrier of claim 20, or the pharmaceutical composition of claim 22; Preferably, the T cells are γδT cells.

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