CLDN18.2 / 4-1BB binding protein and its pharmaceutical use

Abstract

The present disclosure relates to CLDN18.2 / 4-1BB binding proteins and pharmaceutical uses thereof. Specifically, the present disclosure relates to CLDN18.2 / 4-1BB binding proteins, 4-1BB binding proteins, CD16A binding proteins, and methods for treating cancer and related pharmaceutical uses thereof.

Description

[Technical Field]

[0001] This application claims priority from Chinese patent application CN202211513673.8 filed on November 29, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to the field of biopharmaceuticals, and in particular to CLDN18.2 / 4-1BB binding proteins, 4-1BB binding proteins, CD16A binding proteins, and methods and related pharmaceutical uses thereof for treating cancer. [Background technology]

[0003] The Claudin protein family is an important tight junction protein that generally consists of four transmembrane domains and two extracellular loops. Its main role is to maintain normal molecular exchange between cells. Claudin18, a member of the Claudin protein family, has two splice variants, Claudin18 splice variant 1 (CLDN18.1) and 2 (CLDN18.2), which differ by eight amino acids in the first extracellular loop. The two variants have distinct physiological distribution differences: CLDN18.1 is predominantly expressed in normal lung tissue, while CLDN18.2 is primarily expressed in the stomach. In healthy tissues, CLDN18.2 is normally embedded in the gastric mucosa and is barely accessible to antibodies. However, upon malignant transformation, disruption of cell-cell junctions exposes CLDN18.2's antibody-binding epitope, making it a tumor-specific target. CLDN18.2 is ectopically expressed in various epithelial tumors, including gastric, esophageal, and pancreatic cancers, providing a biological basis for tumor-targeted therapy targeting CLDN18.2. IMAB362, an IgG1 subtype anti-CLDN18.2 antibody developed by Astellas, demonstrated a response rate of 63.2% in patients with advanced metastatic gastric and gastroesophageal cancer when used in combination with chemotherapy, surpassing existing data on chemotherapy alone (2021 ASCO). This demonstrates the clinical value of CLDN18.2 as a tumor-specific antigen.

[0004] 4-1BB (CD137, TNFRSF9) is a transmembrane protein that belongs to the tumor necrosis factor receptor superfamily. + and CD4 +4-1BB is a costimulatory molecule expressed on the cell surface of T cells, regulatory T cells (Tregs), NK cells, NKT cells, B cells, and neutrophils. In T cells, 4-1BB is not constitutively expressed but is inducibly expressed after T cell receptor (TCR) activation. 4-1BB is expressed on the cell surface in a monomeric or dimeric form and forms a trimer after binding to its natural ligand, 4-1BBL, which signals via TNFR-associated factor (TRAF)-2 and TRAF-1. The initial signal transduction pathway of 4-1BB involves the polyubiquitination of K-63, activating the nuclear factor (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, resulting in T cell proliferation, maturation, and prolonged survival, as well as cytokine production. Studies have shown that antibody agonists against 4-1BB can promote the antitumor function of T cells in mice (Murillo et al., Clin Cancer Res. 2008, 14(21):6895-906). Antibodies that activate 4-1BB can enhance antitumor immune responses and trigger tumor killing by immune cells by increasing the expression of costimulatory molecules and inducing T cell survival and proliferation in many models. Existing 4-1BB-activating antibodies include Bristol Myers Squibb's (BMS) Urelumab (WO2005035584), a human IgG4 antibody; Pfizer's Utomilumab (Fisher et al., Cancer Immunol. 2012, 61:1721-1733); and Adagene's ADG106 (WO2019037711A1), a human IgG4 antibody. However, the development of anti-tumor antibodies targeting 4-1BB has not progressed well, mainly because 4-1BB not only activates T cells inside the tumor but also activates T cells in the periphery (e.g., the liver), which can cause liver inflammation and further severe liver damage (Todd Bartkowiak et al., Clin Cancer Res, 24(5) March 1, 2018). This narrows the therapeutic window of 4-1BB.

[0005] In light of the excellent tumor specificity and targeting properties of CLDN18.2 and the problems inherent in prior art agonistic antibodies targeting 4-1BB, the present disclosure provides an anti-4-1BB single domain antibody with a novel structure. We have designed and developed an anti-CLDN18.2 / 4-1BB bispecific antibody that activates 4-1BB by binding to CLDN18.2 in tumor tissue, thereby avoiding 4-1BB-induced liver toxicity and expanding the therapeutic range of the antibody. The efficacy of the anti-CLDN18.2 / 4-1BB bispecific antibody can be further improved by using an Fc with enhanced effector function (e.g., ADCC, ADCP). The present disclosure also provides an anti-CD16A single domain antibody with a novel structure that, together with an anti-CLDN18.2 antibody and an anti-4-1BB single domain antibody, forms an anti-CLDN18.2 / 4-1BB / CD16A trispecific antibody, of which the anti-CD16A single domain antibody can enhance the ADCC function of the antibody. The anti-CLDN18.2 / 4-1BB bispecific antibody and anti-CLDN18.2 / 4-1BB / CD16A trispecific antibody according to the present disclosure can prevent the antibody from killing 4-1BB-positive T cells through ADCC, contributing to improved antitumor effects and exhibiting favorable safety and efficacy. Furthermore, the antibodies according to the present disclosure have favorable pharmacological activity, drug development potential, and expression level. As described above, the antibodies according to the present disclosure have the potential to be excellent candidate drugs for clinical tumor treatment. Summary of the Invention

[0006] The present disclosure provides 4-1BB binding proteins, CD16A binding proteins, CLDN18.2 / 4-1BB binding proteins, CLDN18.2 / 4-1BB / CD16A binding proteins, their encoding nucleic acids, vectors, host cells, pharmaceutical compositions, and methods for treating or preventing cancer and related pharmaceutical uses thereof.

[0007] 4-1BB binding protein The present disclosure provides a 4-1BB binding protein comprising an immunoglobulin single variable domain, the immunoglobulin single variable domain comprising CDR1, CDR2, and CDR3 in the amino acid sequence set forth in any one of SEQ ID NOs: 10, 18-21, wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems. The immunoglobulin single variable domain specifically binds to the 4-1BB antigen or a fragment thereof.

[0008] In some embodiments, a 4-1BB binding protein is provided that comprises any one or any combination of the above CDR1, CDR2, and CDR3.

[0009] In some embodiments, a 4-1BB binding protein is provided, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of said immunoglobulin single variable domain are set forth in SEQ ID NOs: 11, 12, 13, or set forth in SEQ ID NOs: 11, 12, 22, respectively, with the CDRs defined according to the Kabat numbering system.

[0010] In some embodiments, the immunoglobulin single variable domain in the 4-1BB binding protein is modified by humanization, affinity maturation, removal / reduction of T-cell epitopes (TCEs), reduction of antibody deamidation, and / or reduction of antibody isomerization.

[0011] In some embodiments, said immunoglobulin single variable domain is obtained by TCE removal / reduction and it has one or more changes in one or more CDRs, said changes resulting in a reduction in the immunogenicity of the 4-1BB binding protein.

[0012] In some embodiments, the immunoglobulin single variable domain has been modified by humanization. The heavy chain framework regions (FR) of the human germline template used for the humanization are derived from IGHV3-64*04, IGHV3-23*03 and / or IGHV3-74*01. In some embodiments, FR1 is derived from IGHV3-64*04, FR2 is derived from IGHV3-23*03, and FR3 is derived from IGHV3-74*01.

[0013] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain in the 4-1BB binding protein is set forth in any one of SEQ ID NOs: 10, 18-21, or has at least 80%, at least 90%, sequence identity to any one of SEQ ID NOs: 10, 18-21, respectively.

[0014] In the present disclosure, "at least 80% (sequence) identity" covers 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" covers 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.

[0015] In some embodiments, the 4-1BB-binding protein comprises or is an antibody or antigen-binding fragment thereof that specifically binds to 4-1BB or a fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a camelid antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a recombinant antibody or fragment thereof.

[0016] In some specific embodiments, the antibody or antigen-binding fragment thereof is a linear antibody, a single-chain antibody, a nanobody, a peptibody, a domain antibody, or a multispecific antibody (bispecific antibody, diabody, triabody, tetrabody, tandem di-scFv, tandem tri-scFv).

[0017] In some embodiments, the immunoglobulin single variable domain in the 4-1BB binding protein is a single domain antibody or a VHH.

[0018] In some embodiments, the present disclosure provides 4-1BB binding proteins comprising one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the above immunoglobulin single variable domains, which may be the same or different and may form dimeric or multimeric molecules.

[0019] In some embodiments, the 4-1BB binding protein further comprises a human immunoglobulin Fc region, eg, the Fc region is a human IgG1, IgG2, or IgG4 Fc region.

[0020] In some embodiments, the Fc has a mutation at position C220, eg, C220A.

[0021] In some embodiments, the Fc region is an Fc region that increases effector function, for example, increases antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC).

[0022] Exemplary IgG1 Fc regions contain the following substitutions: 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 ... 90S / 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 any of the above positions. The above mutations are defined according to the EU numbering system.

[0023] Exemplary IgG1 Fc regions contain the following substitutions: 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, D28 0H / 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 any of the above positions.

[0024] In some embodiments, the Fc region is an Fc region with reduced effector function, eg, reduced ADCC, ADCP, and / or CDC. Exemplary Fc regions with reduced effector function contain the following substitutions: N297A or N297Q (IgG1), L234A / L235A (IgG1), V234A / G237A (IgG2), L235A / G237A / E318A (IgG4), H268Q / V309L / A330S / A331S (IgG2), C220S / C226S / C229S / P238S (IgG1), C226S / C229S / E233P / L234V / L235A (IgG1), L234F / L235E / P331S (IgG1), or S267E / L328F (IgG1).

[0025] In some specific embodiments, the 4-1BB binding protein comprises a human IgG1 Fc region, wherein the Fc region has the C220A / S267E / L328F, C220A / L234A / L235A / N297A, S267E / L328F, or L234A / L235A / N297A mutations. In some specific embodiments, the 4-1BB binding protein comprises a human IgG4 Fc region, wherein the human IgG4 Fc region has the S228P mutation.

[0026] In some embodiments, the 4-1BB binding protein further comprises a human immunoglobulin Fc region, wherein the Fc region is set forth in any one of SEQ ID NOs: 14 to 16, or has at least 80%, at least 90%, sequence identity to any one of SEQ ID NOs: 14 to 16.

[0027] In some embodiments, a 4-1BB binding protein is provided having the amino acid sequence set forth in SEQ ID NO: 17 or at least 80%, at least 90% sequence identity thereto.

[0028] In the present disclosure, in the context of mutations contained in the Fc region, " / " indicates "and", for example, "L234A / L235A" indicates "L234A and L235A", i.e., the Fc contains the L234A and L235A mutations, and in the present disclosure, all mutated amino acid positions in the Fc region are defined according to the EU numbering system.

[0029] In some embodiments, the Fc region contained in the 4-1BB binding protein allows the binding protein to form a dimeric molecule.

[0030] In some embodiments, the Fc region contained in the 4-1BB binding protein can extend the in vivo half-life of the binding protein.

[0031] In some embodiments, the immunoglobulin single variable domain in the 4-1BB binding protein is linked to the Fc region directly or via a linker. The linker may be a non-functional amino acid sequence having a length of 1 to 20 or more amino acids and lacking secondary or higher structure. For example, the linker may be a flexible linker, such as G4S (SEQ ID NO: 102), GS, GAP, (G4S)2 (SEQ ID NO: 103), (G4S)3 (SEQ ID NO: 104), (G4S)4 (SEQ ID NO: 105), (G4S)5 (SEQ ID NO: 106), ASGS (SEQ ID NO: 107), and further, for example, (G4S)2.

[0032] In some embodiments, the 4-1BB binding protein of the present disclosure is an anti-4-1BB antibody or an antigen-binding fragment thereof, or a conjugate or fusion protein comprising the antibody or antigen-binding fragment.

[0033] In some embodiments, the 4-1BB binding protein has at least one activity selected from the following: (a)≦10 -7 K D binding to human 4-1BB or an epitope thereof at a value (b) When not cross-linked with FcγRIIb (i.e., CD32b), it weakly activates or does not activate the 4-1BB signaling pathway, for example, at an antibody concentration of 100 nM, the degree of activation when not cross-linked with FcγRIIb is 10% or less of the activity under saturating antibody concentration conditions when cross-linked with FcγRIIb; (c) When cross-linked with FcγRIIb, it relatively strongly activates or strongly activates the 4-1BB signaling pathway, e.g., EC 50 is less than 1 nM, (d) activating T cells and / or promoting T cell proliferation; (e) inhibiting tumor growth;

[0034] Among these, for detecting activation of the 4-1BB signaling pathway in (b) and (c), reference is made to the 4-1BB / NF-κB luciferase reporter gene assay in Example 2, for example.

[0035] In some embodiments, the K of the 4-1BB binding protein of the present disclosure binds 4-1BB. D The value is ≦1×10 -7 M, e.g., ≤ 1 × 10 -8 M, or ≦1×10 -9 M, or ≦1×10 -10 It may also be M.

[0036] In some embodiments, the 4-1BB binding proteins of the present disclosure can inhibit tumor growth by at least about 10%, e.g., at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%.

[0037] In some embodiments, the 4-1BB binding proteins of the present disclosure include variants having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations compared to any one of SEQ ID NOs: 10, 18-21, which may be conservative replacements, substitutions or modifications, and / or deletions or additions that do not affect function, and which may occur in the CDR and / or FR regions.

[0038] In some embodiments, an anti-4-1BB antibody or antigen-binding fragment thereof is provided that binds to or competitively binds to the same epitope as an immunoglobulin single variable domain in a 4-1BB binding protein according to the present disclosure.

[0039] In some embodiments, an anti-4-1BB antibody or antigen-binding fragment thereof is provided that blocks binding of an immunoglobulin single variable domain in a 4-1BB-binding protein of the present disclosure to 4-1BB (e.g., human 4-1BB). In some specific embodiments, the anti-4-1BB antibody or antigen-binding fragment thereof can also activate T cells and / or promote T cell proliferation.

[0040] In some embodiments, there is provided an anti-4-1BB antibody or antigen-binding fragment thereof whose binding to 4-1BB (eg, human 4-1BB) is blocked by an immunoglobulin single variable domain in a 4-1BB binding protein according to the present disclosure.

[0041] In some embodiments, a protein or molecule is provided that comprises the immunoglobulin single variable domains of any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) 4-1BB binding proteins according to the present disclosure, wherein the immunoglobulin single variable domains are the same or different. For example, the protein or molecule is a complex, and the complex may, for example, include any detectable label.

[0042] CD16A binding protein The present disclosure provides a CD16A binding protein comprising an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain: comprising CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 23, 35 to 39, or CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 24, 40 to 43, Wherein, the above CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system.

[0043] In some embodiments, a CD16A binding protein is provided that comprises an immunoglobulin single variable domain comprising any one or any combination of the CDR1, CDR2, and CDR3 described above.

[0044] In some embodiments, a CD16A binding protein is provided that comprises an immunoglobulin single variable domain comprising CDR1, CDR2, and CDR3, wherein: The amino acid sequences of the CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 25, 26, and 27, respectively. The amino acid sequences of the CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 28, 29, and 30, respectively. Alternatively, the amino acid sequences of the CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 28, 44 and 30, respectively, wherein the CDR1, CDR2 and CDR3 are CDRs defined according to the Kabat numbering system.

[0045] In some embodiments, the immunoglobulin single variable domain in the CD16A binding protein is modified by humanization, affinity maturation, removal / reduction of T-cell epitopes (TCEs), reduction of antibody deamidation, and / or reduction of antibody isomerization.

[0046] In some embodiments, the immunoglobulin single variable domain is obtained by TCE removal / reduction and has one or more changes in one or more CDRs, which changes result in a reduction in the immunogenicity of the CD16A binding protein.

[0047] In some embodiments, the immunoglobulin single variable domain has been modified by humanization. The heavy chain framework regions (FR) of the human germline template used for the humanization are derived from IGHV3-23*04 or IGHV3-20*04. In some embodiments, when the parent immunoglobulin single variable domain is SEQ ID NO: 23, the heavy chain framework regions (FR) of the human germline template used for humanization are derived from IGHV3-23*04, and when the parent immunoglobulin single variable domain is SEQ ID NO: 24, the heavy chain framework regions (FR) of the human germline template used for humanization are derived from IGHV3-20*04.

[0048] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain in the CD16A binding protein is set forth in any one of SEQ ID NOs: 23, 35-39, respectively, or has at least 80%, at least 90%, sequence identity to any one of SEQ ID NOs: 23, 35-39, respectively, or is set forth in any one of SEQ ID NOs: 24, 40-43, respectively, or has at least 80%, at least 90%, sequence identity to any one of SEQ ID NOs: 24, 40-43, respectively.

[0049] In some embodiments, the immunoglobulin single variable domain in the CD16A binding protein specifically binds to CD16A and does not specifically bind to CD16B.

[0050] In some embodiments, the CD16A binding protein comprises or is an antibody or antigen-binding fragment thereof that specifically binds to CD16A. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a camelid antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a recombinant antibody or fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a linear antibody, a single-chain antibody, a nanobody, a peptibody, a domain antibody, or a multispecific antibody (such as a bispecific antibody, a diabody, a triabody, a tetrabody, a tandem di-scFv, or a tandem tri-scFv).

[0051] In some embodiments, the immunoglobulin single variable domain in the CD16A binding protein is a single domain antibody or a VHH.

[0052] In some embodiments, the present disclosure provides CD16A binding proteins comprising one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the above immunoglobulin single variable domains, which may be the same or different and may form dimeric or multimeric molecules.

[0053] In some embodiments, the CD16A binding protein further comprises a human immunoglobulin Fc region, e.g., the Fc region is a human IgG1, IgG2, or IgG4 Fc region. In some embodiments, the Fc region may be an Fc region with enhanced effector function, where the effector function is ADCC, ADCP, and / or CDC. For example, the Fc region may have mutations, such as an exemplary IgG1 Fc region with increased effector function. The Fc region comprises the following substitutions or any combination thereof: 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 / S29 8D, D280H / 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 any of the above positions.

[0054] In some other embodiments, the Fc region may be an Fc region with reduced effector function, e.g., the Fc region may have mutations, such as an exemplary IgG with reduced effector function. The Fc region contains the following substitutions: N297A or N297Q (IgG1), L234A / L235A (IgG1), V234A / G237A (IgG2), L235A / G237A / E318A (IgG4), H268Q / V309L / A330S / A331S (IgG2), C220S / C226S / C229S / P238S (IgG1), C226S / C229S / E233P / L234V / L235A (IgG1), L234F / L235E / P331S (IgG1), or S267E / L328F (IgG1).

[0055] The mutations capable of increasing or decreasing the effector function of the Fc region used in the present disclosure are all known in the art, and are disclosed in, for example, WO2019220369A, WO2021027850A, and WO2020180712A, all of which are incorporated herein in their entireties.

[0056] In some specific embodiments, the Fc region is represented by any one of SEQ ID NOs: 14 to 16, or has at least 80%, or at least 90%, sequence identity to any one of SEQ ID NOs: 14 to 16.

[0057] In some embodiments, the Fc region contained in the CD16A binding protein can enable the binding protein to form a dimeric molecule and increase the in vivo half-life of the binding protein.

[0058] In some embodiments, the immunoglobulin single variable domain in the CD16A binding protein is linked to the Fc region directly or via a linker. The linker may be a non-functional amino acid sequence having a length of 1 to 20 or more amino acids and lacking secondary or higher structure. For example, the linker is a flexible linker such as G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, and further for example, (G4S)2.

[0059] In some embodiments, the CD16A binding protein of the present disclosure is an anti-CD16A antibody or antigen-binding fragment thereof, or a conjugate or fusion protein comprising the antibody or antigen-binding fragment.

[0060] In some embodiments, the CD16A binding protein has at least one activity selected from the following: (a)≦10 -7 K D specifically binds to human CD16A or an epitope thereof at a value (b) does not bind or weakly binds to human CD16B or an epitope thereof, wherein the weak binding is 10% or less of the specific binding affinity to human CD16A or an epitope thereof; (c) binds to the CD16A 158V (i.e., CD16A 176V) variant and CD16A 158F (i.e., CD16A 176F) with the same or similar affinity; (d) activating or enhancing NK cell-mediated ADCC; (e) activating or enhancing macrophage-mediated ADCP; (f) inhibiting tumor growth;

[0061] In some embodiments, the K of the CD16A binding protein of the present disclosure binds CD16A. D The value is ≦1×10 -7 M, for example, ≦1×10 -8 M, or ≦1×10 -9 M, or ≦1×10 -10It's M.

[0062] In some embodiments, the CD16A binding proteins of the present disclosure can inhibit tumor growth by at least about 10%, e.g., at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%.

[0063] In some embodiments, the CD16A binding proteins of the present disclosure encompass variants having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations compared to any one of SEQ ID NOs: 23-24, 35-43, which may be conservative replacements, substitutions, or modifications, and / or deletions or additions that do not affect function, and which may occur in the CDR and / or FR regions.

[0064] In some embodiments, an anti-CD16A antibody or antigen-binding fragment thereof is provided that binds to or competitively binds to the same epitope as an immunoglobulin single variable domain in a CD16A binding protein according to the present disclosure.

[0065] In some embodiments, an anti-CD16A antibody or antigen-binding fragment thereof is provided that blocks binding of an immunoglobulin single variable domain in a CD16A binding protein of the present disclosure to CD16A (eg, human CD16A).

[0066] In some embodiments, an anti-CD16A antibody or antigen-binding fragment thereof is provided whose binding to CD16A (eg, human CD16A) is blocked by an immunoglobulin single variable domain in a CD16A binding protein according to the present disclosure.

[0067] In some embodiments, a protein or molecule is provided that comprises the immunoglobulin single variable domains of any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) CD16A binding proteins according to the present disclosure, wherein the immunoglobulin single variable domains are the same or different. For example, the protein or molecule is a complex, and the complex may, for example, include any detectable label.

[0068] CLDN18.2 / 4-1BB binding protein In a first aspect, the present disclosure provides a CLDN18.2 / 4-1BB binding protein that comprises a first antigen-binding domain that specifically binds to 4-1BB and a second antigen-binding domain that specifically binds to CLDN18.2, and that can specifically bind to 4-1BB and CLDN18.2 simultaneously or separately.

[0069] In some embodiments, when the second antigen-binding domain that specifically binds to CLDN18.2 does not bind to CLDN18.2, the first antigen-binding domain that specifically binds to 4-1BB does not activate 4-1BB signaling, or binds to the 4-1BB antigen but does not activate 4-1BB signaling.

[0070] In some embodiments, the CLDN18.2 / 4-1BB binding protein has enhanced effector function, e.g., the CLDN18.2 / 4-1BB binding protein comprises an Fc region with enhanced effector function and / or a third antigen-binding domain that specifically binds to CD16A.

[0071] In some embodiments, the Fc region with enhanced effector function has increased binding to FcγR, for example, increased binding to FcγRIIB (CD32B) and increased binding to FcγRIIIA (CD16A). In some embodiments, it has reduced, impaired, or no binding to FcγRIIIB (CD16B).

[0072] In some embodiments, the Fc region with enhanced effector function increases binding to C1q.

[0073] In some embodiments, the effector function-enhanced Fc region has a normal or higher glycosylation level compared to a wild-type Fc region.

[0074] In some embodiments, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC).

[0075] In a second aspect, the present disclosure provides a CLDN18.2 / 4-1BB binding protein comprising a first antigen-binding domain that specifically binds to 4-1BB, a second antigen-binding domain that specifically binds to CLDN18.2, and a third antigen-binding domain that specifically binds to CD16A, and which can specifically bind to 4-1BB, CLDN18.2, and CD16A simultaneously or separately.

[0076] In some embodiments, when the second antigen-binding domain that specifically binds to CLDN18.2 does not bind to CLDN18.2, the first antigen-binding domain that specifically binds to 4-1BB does not activate 4-1BB signaling, or binds to the 4-1BB antigen but does not activate 4-1BB signaling.

[0077] In the above-described embodiment, the first antigen-binding domain that specifically binds to 4-1BB, the second antigen-binding domain that specifically binds to CLDN18.2, and the third antigen-binding domain that specifically binds to CD16A may be independently selected from, for example, Fab, Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH, VL, or VHH), and scFv. For example, the first antigen-binding domain that specifically binds to 4-1BB and the third antigen-binding domain that specifically binds to CD16A are VHH, and the second antigen-binding domain that specifically binds to CLDN18.2 is Fab.

[0078] Regarding the first antigen-binding domain that specifically binds to 4-1BB: In some embodiments, the first antigen-binding domain in the CLDN18.2 / 4-1BB binding protein that specifically binds to 4-1BB comprises an immunoglobulin single variable domain, which comprises CDR1, CDR2, and CDR3 in the amino acid sequence set forth in any one of SEQ ID NOs: 10, 18 to 21, wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. The immunoglobulin single variable domain specifically binds to the 4-1BB antigen or a fragment thereof.

[0079] In some embodiments, the immunoglobulin single variable domain comprises any one or any combination of the CDR1, CDR2, and CDR3 described above.

[0080] In some embodiments, the amino acid sequences of CDR1, CDR2 and CDR3 of said immunoglobulin single variable domains are set forth in SEQ ID NOs: 11, 12, 13, or set forth in SEQ ID NOs: 11, 12, 22, respectively, which are CDRs defined according to the Kabat numbering system.

[0081] In some embodiments, the amino acid sequences of CDR1, CDR2 and CDR3 of said immunoglobulin single variable domains are set forth as GFTFSSYA (SEQ ID NO: 66), INSGGEST (SEQ ID NO: 67), AKHPLTFTIATMNDYDY (SEQ ID NO: 68), or as SEQ ID NOs: 66, 67, AKHPLTYTIATMNDYDY (SEQ ID NO: 69), respectively, which are CDRs defined according to the IMGT numbering system.

[0082] In some embodiments, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are set forth in GFTFSSY (SEQ ID NO: 70), NSGGES (SEQ ID NO: 71), SEQ ID NO: 13, or set forth in SEQ ID NOs: 70, 71, 22, respectively, which are CDRs defined by the Chothia numbering system.

[0083] In some embodiments, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domains are set forth in GFTFSSYAMS (SEQ ID NO: 72), DINSGGESTF (SEQ ID NO: 73), SEQ ID NO: 13, or set forth in SEQ ID NOs: 72, 73, 22, respectively, which are the CDRs defined by the AbM numbering system.

[0084] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domains are set forth as SSYAMS (SEQ ID NO: 74), WVSDINSGGESTF (SEQ ID NO: 75), AKHPLTFTIATMNDYD (SEQ ID NO: 76), or as set forth as SEQ ID NOs: 74, 75, AKHPLTYTIATMNDYD (SEQ ID NO: 77), respectively, which are the CDRs defined by the Contact numbering system.

[0085] In some embodiments, the immunoglobulin single variable domain is modified by humanization, affinity maturation, removal / reduction of T-cell epitopes, reduction of antibody deamidation and / or reduction of antibody isomerization.

[0086] In some embodiments, the immunoglobulin single variable domain is obtained by removing / reducing T-cell epitopes and has one or more changes in one or more CDRs, which changes result in a reduction in the immunogenicity of the binding protein.

[0087] In some embodiments, the immunoglobulin single variable domain has been modified by humanization. The heavy chain framework regions (FR) of the human germline template used for the humanization are derived from IGHV3-64*04, IGHV3-23*03 and / or IGHV3-74*01. In some embodiments, FR1 is derived from IGHV3-64*04, FR2 is derived from IGHV3-23*03, and FR3 is derived from IGHV3-74*01.

[0088] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain is set forth in any one of SEQ ID NOs: 10, 18-21, or has at least 80% sequence identity to any one of SEQ ID NOs: 10, 18-21, respectively.

[0089] In some embodiments, the first antigen-binding domain that specifically binds to 4-1BB in the CLDN18.2 / 4-1BB binding protein comprises a 4-1BB binding protein of the present disclosure, or comprises urelumab, utomilumab, ADG106, and the anti-4-1BB antibodies or antigen-binding fragments thereof described in WO2005035584A, WO2019037711A, US20190055314A, WO2019014328A3, and US20210206867A, all of which are incorporated herein by reference in their entireties.

[0090] For the second antigen-binding domain that specifically binds to CLDN18.2: In some embodiments, the second antigen-binding domain that specifically binds to CLDN18.2 in the CLDN18.2 / 4-1BB binding protein comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0091] In some embodiments, the VH of the second antigen-binding domain that specifically binds to CLDN18.2 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence set forth in SEQ ID NO: 63, and the VL comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence set forth in SEQ ID NO: 64. The CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system, for example, according to the Kabat numbering system.

[0092] In some embodiments, the VH of the second antigen-binding domain that specifically binds to CLDN18.2 comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NOs: 57, 58, and 59, and the VL comprises LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NOs: 60, 61, and 62, which are CDRs defined according to the Kabat numbering system.

[0093] In some embodiments, the VH of the second antigen-binding domain that specifically binds to CLDN18.2 comprises HCDR1, HCDR2, and HCDR3 represented by GYTFTSYW (SEQ ID NO: 78), IHPNSGST (SEQ ID NO: 79), and ARLKTGNSFDY (SEQ ID NO: 80), and the VL comprises LCDR1, LCDR2, and LCDR3 represented by QSLLNSGNQKNY (SEQ ID NO: 81), WA, and SEQ ID NO: 62, with the CDRs defined according to the IMGT numbering system.

[0094] In some embodiments, the VH of the second antigen-binding domain that specifically binds to CLDN18.2 comprises HCDR1, HCDR2, and HCDR3 set forth in GYTFTSY (SEQ ID NO: 82), HPNSGS (SEQ ID NO: 83), and SEQ ID NO: 59, and the VL comprises LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NOs: 60, 61, and 62, respectively, which are CDRs defined by the Chothia numbering system.

[0095] In some embodiments, the VH of the second antigen-binding domain that specifically binds to CLDN18.2 comprises GYTFTSYWMH (SEQ ID NO: 84), MIHPNSGSTN (SEQ ID NO: 85), and HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO: 59, and the VL comprises LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NOs: 60, 61, and 62. These are CDRs defined by the AbM numbering system.

[0096] In some embodiments, the VH of the second antigen-binding domain that specifically binds to CLDN18.2 comprises HCDR1, HCDR2, and HCDR3 represented by TSYWMH (SEQ ID NO: 86), WMGMIHPNSGSTN (SEQ ID NO: 87), and ARLKTGNSFD (SEQ ID NO: 88), and the VL comprises LCDR1, LCDR2, and LCDR3 represented by LNSGNQKNYLTWY (SEQ ID NO: 89), LLIYWASTRE (SEQ ID NO: 90), and QNAYTYPF (SEQ ID NO: 91), which are CDRs defined by the Contact numbering system.

[0097] In some embodiments, the VH of the second antigen-binding domain that specifically binds to CLDN18.2 comprises an amino acid sequence set forth in SEQ ID NO: 63 or having at least 80%, at least 90% identity thereto, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 64 or having at least 80%, at least 90% identity thereto.

[0098] In some embodiments, the second antigen-binding domain that specifically binds to CLDN18.2 further comprises a human immunoglobulin Fc region, for example, the Fc region is a human IgG1, IgG2, or IgG4 Fc region.

[0099] In some embodiments, the second antigen-binding domain that specifically binds to CLDN18.2 comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 45 or has at least 80%, at least 90% identity thereto, and the amino acid sequence of the light chain is set forth in SEQ ID NO: 46 or has at least 80%, at least 90% identity thereto.

[0100] The present disclosure incorporates all of the anti-CLDN18.2 antibodies disclosed in WO2020200196A, and any of the antibodies or antigen-binding fragments thereof may be used as the second antigen-binding domain specifically binding to CLDN18.2 disclosed herein. In some embodiments, the second antigen-binding domain specifically binding to CLDN18.2 includes the anti-CLDN18.2 antibodies or antigen-binding fragments thereof disclosed in WO2021027850A, WO2014146672A, WO2021025177A, WO2016180782A, and WO2021254481A, and the above patents are incorporated herein in their entireties.

[0101] Regarding the third antigen-binding domain that specifically binds to CD16A: In some embodiments, the third antigen-binding domain in the CLDN18.2 / 4-1BB binding protein that specifically binds to CD16A comprises an immunoglobulin single variable domain, which comprises CDR1, CDR2, and CDR3 in the amino acid sequence set forth in any one of SEQ ID NOs: 23, 35 to 39, or CDR1, CDR2, and CDR3 in the amino acid sequence set forth in any one of SEQ ID NOs: 24, 40 to 43, wherein CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system.

[0102] In some embodiments, the immunoglobulin single variable domain comprises any one or any combination of the CDR1, CDR2, and CDR3 described above.

[0103] In some embodiments, the amino acid sequences of CDR1, CDR2 and CDR3 of said immunoglobulin single variable domains are set forth in SEQ ID NOs: 25, 26 and 27, respectively, which are CDRs defined according to the Kabat numbering system.

[0104] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domains are set forth as GFAFSTYA (SEQ ID NO: 92), INSDGSST (SEQ ID NO: 93), and AKGWISSPVWGDYVPPV (SEQ ID NO: 94), respectively, which are CDRs defined according to the IMGT numbering system.

[0105] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domains are set forth in GFAFSTY (SEQ ID NO: 95), NSDGSS (SEQ ID NO: 96), and SEQ ID NO: 27, respectively, which are the CDRs defined by the Chothia numbering system.

[0106] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domains are set forth as GFTFSTYAMY (SEQ ID NO: 97), TINSDGSSTR (SEQ ID NO: 98), and SEQ ID NO: 27, respectively, which are the CDRs defined by the AbM numbering system.

[0107] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domains are set forth as STYAMY (SEQ ID NO: 99), WVSTINSDGSSTR (SEQ ID NO: 100), and AKGWISSPVWGDYVPP (SEQ ID NO: 101), respectively, which are the CDRs defined by the Contact numbering system.

[0108] In some embodiments, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domains are set forth in SEQ ID NOs: 28, 29 and 30, respectively, which are CDRs defined according to the Kabat numbering system.

[0109] In some embodiments, the immunoglobulin single variable domain is modified by humanization, affinity maturation, removal / reduction of T-cell epitopes, reduction of antibody deamidation and / or reduction of antibody isomerization.

[0110] In some embodiments, the immunoglobulin single variable domain is obtained by removing / reducing T-cell epitopes and has one or more changes in one or more CDRs, which changes result in a reduction in the immunogenicity of the binding protein.

[0111] In some embodiments, the immunoglobulin single variable domain has been modified by humanization. The heavy chain framework regions (FR) of the human germline template used for the humanization are derived from IGHV3-23*04 or IGHV3-20*04. In some embodiments, when the parent immunoglobulin single variable domain is SEQ ID NO: 23, the heavy chain framework regions (FR) of the human germline template used for humanization are derived from IGHV3-23*04, and when the parent immunoglobulin single variable domain is SEQ ID NO: 24, the heavy chain framework regions (FR) of the human germline template used for humanization are derived from IGHV3-20*04.

[0112] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain is set forth in any one of SEQ ID NOs: 23, 35-39 or has at least 80%, at least 90% sequence identity thereto, or is set forth in any one of SEQ ID NOs: 24, 40-43 or has at least 80%, at least 90% sequence identity thereto.

[0113] In some embodiments, the third antigen-binding domain that specifically binds to CD16A comprises a CD16A binding protein of the present disclosure, or an anti-CD16A antibody or antigen-binding fragment thereof described in WO2006125668A, WO2007009065A, or WO2016177846A, all of which are incorporated herein by reference in their entireties.

[0114] Exemplary CLDN18.2 / 4-1BB binding proteins according to the present disclosure are provided below: In some embodiments, in the CLDN18.2 / 4-1BB binding protein, the first antigen-binding domain that specifically binds to 4-1BB comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 in the amino acid sequence set forth in any one of SEQ ID NOs: 10, 18 to 21, and the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are set forth in SEQ ID NOs: 11, 12, and 13, or SEQ ID NOs: 11, 12, and 22, respectively, according to the Kabat numbering system. In some specific embodiments, the amino acid sequence of the immunoglobulin single variable domain is set forth in any one of SEQ ID NOs: 10, 18 to 21, respectively, or has at least 80%, at least 90%, sequence identity to any one of SEQ ID NOs: 10, 18 to 21. In some specific embodiments, the immunoglobulin single variable domain is a single domain antibody or a VHH.

[0115] In some embodiments, in the CLDN18.2 / 4-1BB binding protein, the second antigen-binding domain that specifically binds to CLDN18.2 comprises a VH and a VL. In some specific embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 63, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 64. The CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system, for example, the Kabat numbering system. In some specific embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NOs: 57, 58, and 59, and the VL comprises LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NOs: 60, 61, and 62. In some specific embodiments, the VH comprises an amino acid sequence set forth in SEQ ID NO: 63 or having at least 80%, at least 90% identity thereto, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 64 or having at least 80%, at least 90% identity thereto. In some specific embodiments, in the CLDN18.2 / 4-1BB binding protein, the second antigen-binding domain that specifically binds to CLDN18.2 comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 45 or having at least 80%, at least 90% identity thereto, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 46 or having at least 80%, at least 90% identity thereto.

[0116] Alternatively, in some embodiments, the CLDN18.2 / 4-1BB binding protein further comprises a third antigen-binding domain that specifically binds to CD16A. In some specific embodiments, the third antigen-binding domain that specifically binds to CD16A comprises an immunoglobulin single variable domain, which comprises CDR1, CDR2, and CDR3 in the amino acid sequence set forth in any one of SEQ ID NOs: 23, 35-39, or CDR1, CDR2, and CDR3 in the amino acid sequence set forth in any one of SEQ ID NOs: 24, 40-43, wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are set forth in SEQ ID NOs: 25, 26, and 27, or SEQ ID NOs: 28, 29, and 30, respectively, according to the Kabat numbering system. In some specific embodiments, the amino acid sequence of the immunoglobulin single variable domain is set forth in, or has at least 80%, at least 90% sequence identity with, any one of SEQ ID NOs: 23, 35 to 39, respectively, or is set forth in, or has at least 80%, at least 90% sequence identity with, any one of SEQ ID NOs: 24, 40 to 43, respectively. In some specific embodiments, the immunoglobulin single variable domain is a single domain antibody or a VHH.

[0117] In some embodiments, the CLDN18.2 / 4-1BB binding protein has one or more (e.g., 2, 3, 4, 5, 6) first antigen-binding domains that specifically bind to 4-1BB and / or has one or more (e.g., 2, 3, 4) second antigen-binding domains that specifically bind to CLDN18.2, and optionally, further has one or more (e.g., 2, 3, 4, 5, 6) third antigen-binding domains that specifically bind to CD16A. In some specific embodiments, the CLDN18.2 / 4-1BB binding protein has two first antigen-binding domains that specifically bind to 4-1BB and two second antigen-binding domains that specifically bind to CLDN18.2. In some specific embodiments, the CLDN18.2 / 4-1BB binding protein has two first antigen-binding domains that specifically bind to 4-1BB, two second antigen-binding domains that specifically bind to CLDN18.2, and two third antigen-binding domains that specifically bind to CD16A.

[0118] In some embodiments, in the CLDN18.2 / 4-1BB binding protein, the valency ratio of the first antigen-binding domain that specifically binds to 4-1BB to the second antigen-binding domain that specifically binds to CLDN18.2 is between 6:1 and 1:3 (e.g., 4:1 and 1:2), for example, 1:1, 1:2, 2:1, 1:3, or 3:1. In some embodiments, in the CLDN18.2 / 4-1BB binding protein, the valency ratio of the first antigen-binding domain that specifically binds to 4-1BB, the second antigen-binding domain that specifically binds to CLDN18.2, and the third antigen-binding domain that specifically binds to CD16A is between 6:3:1 and 1:3:6, for example, 1:1:1, 1:2:2, 2:2:1, 2:1:2, 1:2:1, 3:1:3, 3:1:2, 2:1:3, 3:1:1, or 3:2:1.

[0119] In some embodiments, in the above-mentioned CLDN18.2 / 4-1BB binding protein, a first antigen-binding domain that specifically binds to 4-1BB is located at the N-terminus and / or C-terminus of a second antigen-binding domain that specifically binds to CLDN18.2, and optionally, in some embodiments, a third antigen-binding domain that specifically binds to CD16A is located at the N-terminus and / or C-terminus of the second antigen-binding domain that specifically binds to CLDN18.2.

[0120] In some embodiments, the CLDN18.2 / 4-1BB binding protein further comprises a human immunoglobulin Fc region, for example, the Fc region is a human IgG1, IgG2, or IgG4 Fc region.

[0121] In some specific embodiments, the Fc region enables the binding protein to form a dimeric molecule.

[0122] In some specific embodiments, the Fc region comprises a mutation that extends the in vivo half-life, where the half-life is determined by FcRn binding affinity. Extended half-life allows for reduced dosage and / or less frequent administration of the drug to patients. For example, the Fc region comprises an M252Y, S254T, and / or T256E mutation. In some specific embodiments, the Fc region is an Fc region with enhanced effector function, e.g., an Fc region with enhanced ADCC, ADCP, and / or CDC, or an Fc region with reduced fucosylation. The Fc region may have mutations, and exemplary IgG1 with enhanced effector function are: The Fc region comprises the following substitutions or any combination thereof: 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 / S29 8D, D280H / 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 any of the above positions. In some specific embodiments, the CLDN18.2 / 4-1BB binding protein is a S239D / I332E mutated human IgG1 Fc region.

[0123] In some other specific embodiments, the Fc region may be an Fc region with reduced effector function, e.g., the Fc region may have mutations, such as an exemplary IgG with reduced effector function. The Fc region contains the following substitutions: N297A or N297Q (IgG1), L234A / L235A (IgG1), V234A / G237A (IgG2), L235A / G237A / E318A (IgG4), H268Q / V309L / A330S / A331S (IgG2), C220S / C226S / C229S / P238S (IgG1), C226S / C229S / E233P / L234V / L235A (IgG1), L234F / L235E / P331S (IgG1), or S267E / L328F (IgG1).

[0124] In the present disclosure, in a CLDN18.2 / 4-1BB binding protein (e.g., an anti-CLDN18.2 / 4-1BB bispecific antibody, an anti-CLDN18.2 / 4-1BB / CD16A trispecific antibody), selecting an Fc region with enhanced effector function is significantly superior to an Fc region with unchanged or reduced effector function.

[0125] In some specific embodiments, the Fc comprises the amino acid sequence shown in SEQ ID NO: 65. In other specific embodiments, the Fc comprises the amino acid sequence shown in any one of SEQ ID NOs: 14 to 16.

[0126] In some embodiments, the Fc region in the CLDN18.2 / 4-1BB binding protein comprises a first subunit, Fc1, and a second subunit, Fc2, which can associate with each other.

[0127] In some embodiments, Fc1 and Fc2 contain amino acid mutations such that Fc1 preferentially pairs with or forms heterodimers with Fc2 compared to Fc1. In some embodiments, the mutations are in CH3 of Fc1 and Fc2. In some embodiments, the amino acid mutations in Fc1 and Fc2 result in greater electrostatic complementarity than wild-type antibodies that do not contain the mutations. In some embodiments, the amino acid mutations in Fc1 and Fc2 result in greater spatial complementarity than wild-type antibodies that do not contain the mutations.

[0128] In some embodiments, for Fc1 and Fc2, for example, one 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 within the CH3 / CH3 interface to create a protrusion (or knob) on the surface of the CH3 domain of Fc1, and one or more, preferably two or three, 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 to create a depression (or hole) on the surface of the CH3 domain of Fc2 that interacts with the CH3 domain of Fc1. In some embodiments, the input residue with a larger side chain volume is phenylalanine (F), tyrosine (Y), arginine (R), or tryptophan (W). In some embodiments, the input residue with a smaller side chain volume is serine (S), alanine (A), valine (V), or threonine (T).

[0129] In some specific embodiments, the Fc1 comprises at least one or at least two amino acid mutations (hole mutation modifications) selected from T366S, L368A, and Y407V, and the Fc2 comprises T366W (knob mutation modification), or the Fc1 comprises T366W (knob mutation modification) and the Fc2 comprises at least one or at least two amino acid mutations (hole mutation modifications) selected from T366S, L368A, and Y407V.

[0130] In some specific embodiments, in Fc1 and Fc2, for example, CH3 may contain a mutation of a native non-cysteine ​​to a cysteine, e.g., Fc1 contains S354C and Fc2 contains Y349C, or Fc1 contains Y349C and Fc2 contains S354C.

[0131] In some specific embodiments, Fc1 and Fc2 comprise the following amino acid mutations or combinations thereof, e.g., at the Fc1 CH3 / Fc2 CH3 interface: T366Y / Y407T, T366W / Y407A, T366Y / Y407T, T394W / F405A, T366Y / F405AT394W / Y407T, T366W / F405WT394S / Y407A, F405W / T394S, D399C / K392C, T366W / T366S / L368A / Y407V, T366W / D399C / T366S / L368A / K392C / Y407V, T366W / K392C / T366S / D399C / L368A / Y407V, S354C / T366W / Y349C / T366S / L368A / Y407V, Y349C / T366W / S354C / T366S / L368A / Y407V, E356C / T366W / Y349C / T366S / L368A / Y407V, Y349C / T366W / E356C / T366S / L368A / Y407V, E357C / T366W / Y349C / T366S / L368A / Y407V, and Y349C / T366W / E357C / T366S / L368A / Y407V.

[0132] In some specific embodiments, Fc1 and Fc2 further comprise amino acid mutations that allow an electrostatic interaction interface to be formed between Fc1 and Fc2 (e.g., CH3 and CH3). Examples of amino acid mutations that form an electrostatic interaction interface include K370E / D399K / K439D / D356K / E357K / K409D, K409D / D399K, K409E / D399K, K409E / D399R, K409D / D399R, D339K / E356K, D399K / E356K / K409D / K392D, and D399E / D399R. 9K / E356K / K409D / K439D, D399K / E357K / K409D / K370D, D399K / E356K / E357K / K409D / K392D / K370D, D399K / E357K / K409D / K392D, K392D / K409D / D399K, and K409D / K360D / D399K.

[0133] In some embodiments, Fc1 and / or Fc2 comprise domains from different antibody subtypes, eg, from different antibody subtype CH3s.

[0134] Furthermore, the present disclosure incorporates methods for enhancing heterodimerization by modifying the CH3 region of the Fc region described in WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO2007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012058768, WO2013157954, and WO2013096291.

[0135] In some embodiments, in the CLDN18.2 / 4-1BB binding protein, the first antigen-binding domain that specifically binds to 4-1BB and the second antigen-binding domain that specifically binds to CLDN18.2 are linked directly or via a linker. In some embodiments, in the CLDN18.2 / 4-1BB binding protein, the first antigen-binding domain that specifically binds to 4-1BB, the second antigen-binding domain that specifically binds to CLDN18.2, and the third antigen-binding domain that specifically binds to CD16A are linked directly or via a linker. In some embodiments, the first antigen-binding domain that specifically binds to 4-1BB is linked to an Fc region directly or via a linker. In some embodiments, the third antigen-binding domain that specifically binds to CD16A is linked to the light chain constant region of the second antigen-binding domain that specifically binds to CLDN18.2 directly or via a linker.

[0136] In some specific embodiments, the linker is, for example, m S n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h wherein m and n are each independently selected from integers of 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from integers of 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some specific embodiments, the linker may be a non-functional amino acid sequence having a length of 1 to 20 or more amino acids and lacking secondary or higher structures. In some specific embodiments, the linker is a flexible linker. In some specific embodiments, the linker is selected from G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, and ASGS, for example, (G4S)2 or (G4S)3.

[0137] In some specific embodiments, the CLDN18.2 / 4-1BB binding protein comprises a first polypeptide chain and a second polypeptide chain, the first and second polypeptide chains arranged from N-terminus to C-terminus as follows: (1) First polypeptide chain: [VH of second antigen-binding domain that specifically binds to CLDN18.2]-CH1-Fc region-[linker 1]a-[first antigen-binding domain that specifically binds to 4-1BB]; Second polypeptide chain: [VL of the second antigen-binding domain that specifically binds to CLDN18.2]-CL (2) a first polypeptide chain: [a first antigen-binding domain that specifically binds to 4-1BB]-[linker 1]a-[VH of a second antigen-binding domain that specifically binds to CLDN18.2]-CH1-Fc region; Second polypeptide chain: [VL of the second antigen-binding domain that specifically binds to CLDN18.2]-CL (3) a first polypeptide chain: [VH of a second antigen-binding domain that specifically binds to CLDN18.2]-CH1-Fc region; Second polypeptide chain: [first antigen-binding domain that specifically binds to 4-1BB]-[linker 1]a-[VL of second antigen-binding domain that specifically binds to CLDN18.2]-CL (4) a first polypeptide chain: [VH of a second antigen-binding domain that specifically binds to CLDN18.2]-CH1-Fc region; Second polypeptide chain: [VL of second antigen-binding domain that specifically binds to CLDN18.2]-[Linker 1]a-CL[first antigen-binding domain that specifically binds to 4-1BB] (5) First polypeptide chain: [VH of second antigen-binding domain that specifically binds to CLDN18.2]-CH1-Fc region-[linker 2]b-[first antigen-binding domain that specifically binds to 4-1BB]; Second polypeptide chain: [VL of second antigen-binding domain that specifically binds to CLDN18.2]-[Linker 1]a-CL[first antigen-binding domain that specifically binds to 4-1BB] (6) First polypeptide chain: [VH of second antigen-binding domain that specifically binds to CLDN18.2]-CH1-Fc region-[linker 3]c-[first antigen-binding domain that specifically binds to 4-1BB]; A second polypeptide chain: [VL of a second antigen-binding domain that specifically binds to CLDN18.2]-CL-[linker 4]d-[a third antigen-binding domain that specifically binds to CD16A], and (7) First polypeptide chain: [VH of a second antigen-binding domain that specifically binds to CLDN18.2]-CH1-Fc region-[linker 3]c-[third antigen-binding domain that specifically binds to CD16A]; Second polypeptide chain: [VL of second antigen-binding domain that specifically binds to CLDN18.2]-CL-[linker 4]d-[first antigen-binding domain that specifically binds to 4-1BB] and wherein - represents a peptide bond, linker is a polypeptide capable of performing a linking function, linker 1, linker 2, linker 3, and linker 4 may be the same or different, and a, b, c, and d may independently be selected from 1 or 0, for example, a, b, c, and d are all 1. The linkers are, for example, independently selected from G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, and ASGS, for example, (G4S)2 or (G4S)3.

[0138] In some embodiments, a CLDN18.2 / 4-1BB binding protein is provided, comprising a first and a second polypeptide chain. In some specific embodiments, the amino acid sequence of the first polypeptide chain is set forth in any one of SEQ ID NOS: 47-49 or has at least 80%, at least 90%, sequence identity thereto, and the amino acid sequence of the second polypeptide chain is set forth in SEQ ID NO: 46 or has at least 80%, at least 90%, sequence identity thereto.

[0139] In some specific embodiments, the amino acid sequence of the first polypeptide chain is set forth in any one of SEQ ID NOs: 50, 52, and 53, or has at least 80%, at least 90%, sequence identity thereto, and the amino acid sequence of the second polypeptide chain is set forth in SEQ ID NO: 51 or 54, or has at least 80%, at least 90%, sequence identity thereto, e.g., the amino acid sequence of the first polypeptide chain is set forth in SEQ ID NO: 50 or 52, or has at least 80%, at least 90%, sequence identity thereto, and the amino acid sequence of the second polypeptide chain is set forth in SEQ ID NO: 51, or has at least 80%, at least 90%, sequence identity thereto, e.g., the amino acid sequence of the first polypeptide chain is set forth in SEQ ID NO: 53, or has at least 80%, at least 90%, sequence identity thereto, and the amino acid sequence of the second polypeptide chain is set forth in SEQ ID NO: 54, or has at least 80%, at least 90%, sequence identity thereto.

[0140] In some specific embodiments, the amino acid sequence of the first polypeptide chain is set forth in SEQ ID NO: 55 or 56 or has at least 80%, at least 90% sequence identity thereto, and the amino acid sequence of the second polypeptide chain is set forth in SEQ ID NO: 46 or has at least 80%, at least 90% sequence identity thereto.

[0141] In some embodiments, a CLDN18.2 / 4-1BB binding protein is provided that includes first and second polypeptide chains selected from: (1) The amino acid sequence of the first polypeptide chain is represented by any one of SEQ ID NOs: 47 to 49, and the amino acid sequence of the second polypeptide chain is represented by SEQ ID NO: 46; (2) the amino acid sequence of the first polypeptide chain is represented by SEQ ID NO: 50 or 52, and the amino acid sequence of the second polypeptide chain is represented by SEQ ID NO: 51; (3) the amino acid sequence of the first polypeptide chain is represented by SEQ ID NO: 53, and the amino acid sequence of the second polypeptide chain is represented by SEQ ID NO: 54; (4) The amino acid sequence of the first polypeptide chain is represented by SEQ ID NO: 55 or 56, and the amino acid sequence of the second polypeptide chain is represented by SEQ ID NO: 46.

[0142] In some embodiments, a CLDN18.2 / 4-1BB binding protein according to the present disclosure has at least one activity selected from the following: (a)≦10 -7 K D binding to human 4-1BB or an epitope thereof at a value (b) When not cross-linked by CLDN18.2 (or when not bound to CLDN18.2), it weakly activates or does not activate the 4-1BB signal transduction pathway, for example, at an antibody concentration of 100 nM, the degree of activation when not cross-linked by CLDN18.2 (or when not bound to CLDN18.2) is 10% or less of the activity when cross-linked by CLDN18.2 (or when bound to CLDN18.2) under saturating antibody concentration conditions; (c) When cross-linked with CLDN18.2 (or not bound to CLDN18.2), it relatively strongly activates or strongly activates the 4-1BB signaling pathway, e.g., EC 50 is less than 1 nM, (d) activating T cells and / or promoting T cell proliferation; (e) inhibiting tumor growth; (f) having enhanced ADCC and / or ADCP function.

[0143] Among these, for detecting activation of the 4-1BB signaling pathway in (b) and (c), reference is made to the 4-1BB / NF-κB luciferase reporter gene assay in Example 2, for example.

[0144] In some embodiments, a CLDN18.2 / 4-1BB binding protein of the present disclosure can inhibit tumor growth by at least about 10%, e.g., at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%.

[0145] In some embodiments, the CLDN18.2 / 4-1BB binding protein of the present disclosure includes variants, which have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid mutations compared to the first and second polypeptide chains in any one of the combinations (1) to (4) above, and the amino acid mutations may be conservative replacements, substitutions, or modifications, and / or deletions or additions that do not affect function.

[0146] In some embodiments, a CLDN18.2 / 4-1BB binding protein is provided that binds to or competitively binds to CLDN18.2 and / or 4-1BB with the CLDN18.2 / 4-1BB binding protein of the present disclosure, or that binds to or competitively binds to the same epitope on CLDN18.2 and / or 4-1BB as the CLDN18.2 / 4-1BB binding protein described above. When the CLDN18.2 / 4-1BB binding protein further binds to CD16A, a CLDN18.2 / 4-1BB binding protein is provided that binds to or competitively binds to CLDN18.2 and / or 4-1BB and / or CD16A with the CLDN18.2 / 4-1BB binding protein described above, or that binds to or competitively binds to the same epitope on CLDN18.2 and / or 4-1BB and / or CD16A as the CLDN18.2 / 4-1BB binding protein described above.

[0147] In some embodiments, a CLDN18.2 / 4-1BB binding protein is provided that blocks the binding of the CLDN18.2 / 4-1BB binding protein of the present disclosure to CLDN18.2 and / or 4-1BB, and selectively blocks the binding of the CLDN18.2 / 4-1BB binding protein of the present disclosure to CD16A.

[0148] In some embodiments, a protein or molecule is provided, comprising any of the CLDN18.2 / 4-1BB binding proteins of the present disclosure. For example, the protein or molecule is a complex, and the complex may include, for example, any detectable label.

[0149] Polynucleotides and Vectors The present disclosure provides polynucleotides encoding the 4-1BB binding proteins, CD16A binding proteins, and CLDN18.2 / 4-1BB binding proteins of the present disclosure. The nucleic acids of the present disclosure may be RNA, DNA, or cDNA. According to some embodiments of the present disclosure, the nucleic acids of the present disclosure are essentially isolated nucleic acids.

[0150] The nucleic acids of the present disclosure may be in the form of, present in, and / or part of a vector, such as a plasmid, cosmid, YAC, or viral vector. The vector may be, in particular, an expression vector, i.e., a vector that allows for the expression of a 4-1BB binding protein, a CD16A binding protein, or a CLDN18.2 / 4-1BB binding protein in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). Such expression vectors typically contain at least one nucleic acid of the present disclosure, operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). The selection of these elements and their sequences for expression in a particular host is within the skill of the art. Regulatory and other elements useful or necessary for expression of the 4-1BB binding proteins, CD16A binding proteins, and CLDN18.2 / 4-1BB binding proteins of the present disclosure are, for example, promoters, enhancers, terminators, integration factors, selectable markers, leader sequences, and reporter genes.

[0151] Nucleic acids according to the present disclosure may be prepared or obtained by known methods (e.g., automated DNA synthesis and / or recombinant DNA techniques) based on information about the amino acid sequence of a polypeptide according to the present disclosure, and / or may be isolated from a suitable natural source.

[0152] host cell The present disclosure provides recombinant host cells that express or are capable of expressing one or more 4-1BB binding proteins, CD16A binding proteins, CLDN18.2 / 4-1BB binding proteins of the present disclosure, and / or that comprise a polynucleotide or vector of the present disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0153] Bacterial cells include, for example, cells of Gram-negative strains (e.g., Escherichia coli, Proteus, and Pseudomonas) and Gram-positive strains (e.g., Bacillus, Streptomyces, Staphylococcus, and Lactococcus).

[0154] Fungal cells include, for example, cells of species of Trichoderma, Neurospora, and Aspergillus, or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0155] Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0156] However, the present disclosure may also be used with amphibian cells, insect cells, plant cells and any other cells in the art for expressing heterologous proteins.

[0157] Preparation method The present disclosure provides methods for preparing 4-1BB binding proteins, CD16A binding proteins, and CLDN18.2 / 4-1BB binding proteins, comprising expressing the target protein in the host cells described above and isolating the target protein from the host cells. Optionally, a purification step may be included, for example, by purifying on an A or G Sepharose FF column containing a conditioned buffer to wash away nonspecifically bound components, followed by elution of the bound antibody using a pH gradient, detection by SDS-PAGE, and collection. Optionally, the product may be filtered and concentrated using conventional methods. Soluble components and multimers may be removed using conventional methods such as molecular sieves or ion exchange. The resulting product should be immediately frozen, e.g., at -70°C, or lyophilized.

[0158] Methods for producing and purifying antibodies are well known in the art and can be found, for example, in Cold Spring Harbor's Antibody Laboratory Techniques Manual (Chapters 5-8 and 15).

[0159] The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. Mammalian expression systems result in 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 expanded in serum-free medium in bioreactors to produce antibodies. The culture medium from which the antibodies are secreted can be purified and collected by conventional techniques. The antibodies can be concentrated by filtration using conventional methods. Soluble admixtures and multimers can be removed using conventional methods, such as molecular sieving or ion exchange.

[0160] composition The present disclosure provides compositions comprising the 4-1BB binding protein, CD16A binding protein, or CLDN18.2 / 4-1BB binding protein of the present disclosure, for example, pharmaceutical compositions comprising an effective amount of the 4-1BB binding protein, CD16A binding protein, or CLDN18.2 / 4-1BB binding protein for treating, mitigating, or preventing cancer, and at least one medicament-acceptable excipient, diluent, or carrier.

[0161] In some specific embodiments, a unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of the 4-1BB binding protein, CD16A binding protein, or CLDN18.2 / 4-1BB binding protein, or the amount of the 4-1BB binding protein, CD16A binding protein, or CLDN18.2 / 4-1BB binding protein contained in a unit dose of the pharmaceutical composition is 0.1 to 2000 mg, and in some specific embodiments, 1 to 1000 mg.

[0162] In some embodiments, an article of manufacture or product (e.g., a reagent kit) is provided that includes at least one container that independently contains the 4-1BB binding protein, CD16A binding protein, or CLDN18.2 / 4-1BB binding protein. Optionally, the article of manufacture includes a container and a label. The container is, for example, a vial, syringe, or test tube. The container holds a composition that is effective for treating a medical condition. A label on or associated with the container indicates that the composition is used for treating a selected medical condition.

[0163] Therapeutic Methods and Pharmaceutical Uses The present disclosure provides methods for treating, mitigating, preventing, or diagnosing diseases or conditions using the above-mentioned 4-1BB binding proteins, CD16A binding proteins, CLDN18.2 / 4-1BB binding proteins, polynucleotides, and compositions (including pharmaceutical compositions).

[0164] In some embodiments, methods are provided for ameliorating, alleviating, treating, or preventing a disease, comprising administering to a subject an ameliorating, alleviating, treating, or preventing effective amount of the 4-1BB binding protein, CD16A binding protein, CLDN18.2 / 4-1BB binding protein, polynucleotide, or composition (including pharmaceutical composition).

[0165] In some embodiments, the present disclosure provides a use of a 4-1BB binding protein, CD16A binding protein, CLDN18.2 / 4-1BB binding protein polynucleotide, or composition (including a pharmaceutical composition) for the preparation of a medicament for ameliorating, alleviating, treating, or preventing a disease.

[0166] In some embodiments, the disease is a proliferative condition or any other disease or condition characterized by uncontrolled cell proliferation, such as cancer. In this disclosure, cancer and tumor may be used interchangeably.

[0167] In some embodiments, the cancer is a solid tumor or a hematological tumor.

[0168] In some embodiments, the cancer is advanced or metastatic.

[0169] In some embodiments, the disease is CLDN18.2-associated or CLDN18.2-positive, such as CLDN18.2-positive cancer. In some embodiments, a Claudin18.2-associated or CLDN18.2-positive disease can be diagnosed by detecting or measuring Claudin18.2-expressing cells using an antibody or antibody fragment according to the present disclosure. Known immunodetection methods can be used to detect cells expressing a polypeptide, preferably immunoprecipitation, fluorescent cell staining, or immunohistochemical staining. Furthermore, fluorescent antibody staining using an FMAT8100HTS system (Applied Biosystems) can also be used. In the present disclosure, the sample to be measured for detecting or measuring a target antigen (e.g., Claudin18.2) is not particularly limited, and may be any sample that may contain cells expressing the target antigen (e.g., Claudin18.2), such as histiocytes, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium.

[0170] In some embodiments, the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, blood cancer, or any other disease or condition characterized by uncontrolled cell proliferation, or a combination thereof.

[0171] detection The present disclosure provides detective uses of 4-1BB binding proteins, CD16A binding proteins, CLDN18.2 / 4-1BB binding proteins, polynucleotides, and compositions. The present disclosure further provides methods, systems, or devices for detecting 4-1BB, CLDN18.2, or CD16A in vivo or in vitro, comprising treating a sample with the binding proteins, polynucleotides, or compositions of the present disclosure.

[0172] In some embodiments, the in vitro detection method, system, or device comprises, e.g., (1) contacting a sample with a 4-1BB binding protein, CD16A binding protein, CLDN18.2 / 4-1BB binding protein, polynucleotide, or composition of the present disclosure; (2) detecting a complex formed between the binding protein, the polynucleotide, and the sample; and / or (3) contacting a reference sample (e.g., a control sample) with a binding protein, a nucleic acid, and (4) determining the extent of complex formation by comparison with a reference sample; For example, a change (e.g., a statistically significant change) in complex formation in the sample compared to a control sample indicates the presence of 4-1BB, CLDN18.2, and CD16A in the sample.

[0173] In some embodiments, a reagent kit is further provided, which includes the 4-1BB binding protein, CD16A binding protein, CLDN18.2 / 4-1BB binding protein, and polynucleotide, and may further include a diagnostic manual. The reagent kit may further include at least one additional reagent, such as a marker or an additional diagnostic agent. For in vivo use, the 4-1BB binding protein, CD16A binding protein, or CLDN18.2 / 4-1BB binding protein may be prepared as a pharmaceutical composition.

[0174] Definition of Terms In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined in this disclosure, all other technical and scientific terms used in this disclosure have the meanings commonly understood by those of ordinary skill in the art.

[0175] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0176] "CLDN18 protein" or "CLDN18" is a protein encoded by the Claudin-18 gene in humans and belongs to the tight junction protein family. Claudin-18 can regulate intercellular molecular flow. The Claudin-18 protein structure contains four transmembrane domains and two extracellular loops, with its N- and C-termini located in the cytoplasm. Claudin-18 has two splice variants, Claudin-18.1 and Claudin-18.2, respectively. The two sequences differ by eight amino acids in the first extracellular loop only. Claudin-18.1 and Claudin-18.2 have different expression profiles. Claudin-18.1 is selectively expressed in normal lung cells, while Claudin-18.2 is severely restricted in normal cells but frequently allosterically activated and overexpressed in several types of tumors (e.g., gastric, lung, and pancreatic cancers).

[0177] "CD16," also known as FcγRIII, is a low-affinity receptor for the Fc fragment of IgGs involved in antibody-dependent cellular cytotoxicity (ADCC). Human FcγRIII has two subtypes, FcγRIIIA (i.e., CD16A) and FcγRIIIB (i.e., CD16B), whose extracellular immunoglobulin binding domain sequences share 96% sequence identity (van de Winkel and Capel, 1993, Immunol Today 14(5):215-221). CD16A is a transmembrane receptor expressed on macrophages, mast cells, and NK cells. In NK cells, the α-chain of CD16A binds to immunoreceptor tyrosine-based activation motifs (ITAMs) including the FcεRI γ-chain and / or the T cell receptor (TCR) / CD3 ζ-chain (Wirthmueller et al., 1992, J. Exp. Med. 175:1381-1390), inducing signal transduction leading to cytokine production and cytotoxicity. CD16B is present on polymorphonuclear granulocytes (PMNs) as a glycosylphosphatidylinositol (GPI)-anchored receptor (FcγRIIIB subtype) that is unable to induce tumor cell killing (van de Winkel and Capel, 1993, supra). Furthermore, CD16B is present in serum as a soluble receptor, and when it binds to antibodies in vivo, it may form immune complexes that can cause adverse reactions.

[0178] "4-1BB protein" or "4-1BB" is also known as CD137, tumor necrosis factor receptor superfamily 9, a member of the TNF receptor superfamily (TNFRSF), and CD8 + and CD4 +4-1BB is a costimulatory molecule expressed on the surface of cells such as T cells, regulatory T cells (Tregs), NK cells, NKT cells, B cells, and neutrophils. 4-1BB belongs to the costimulatory molecules and is expressed after immune cell activation. Human 4-1BB protein has NCBI accession number NP_001552.2. In the present disclosure, "4-1BB" may optionally include any of the proteins or fragments or variants thereof, including (but not limited to) the known or wild-type 4-1BB described in this disclosure, and any naturally occurring splice variants, amino acid variants, or isoforms, e.g., human 4-1BB shown in SEQ ID NO: 11.

[0179] The term "4-1BB-binding protein" covers any molecule capable of specifically binding to the 4-1BB protein or an epitope thereof, including, but not limited to, antibodies, antigen-binding fragments thereof, or complexes thereof as defined herein against 4-1BB. A "4-1BB-binding protein" according to the present disclosure may comprise at least one (e.g., 1, 2, 3, 4, 5, 6, or more) immunoglobulin single variable domain (e.g., VHH) that binds to 4-1BB. A "4-1BB-binding protein" according to the present disclosure may also comprise, in addition to the 4-1BB-containing immunoglobulin single variable domain, a linker and / or a moiety with effector function, such as a half-life extending moiety (e.g., an immunoglobulin single variable domain that binds serum albumin) and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region.

[0180] "CD16A binding protein" takes a similar definition to "4-1BB binding protein" and covers any molecule capable of specifically binding to CD16A or its epitope, including, but not limited to, antibodies, antigen-binding fragments or conjugates thereof, and fusion proteins as defined in the present disclosure against CD16A.

[0181] The term "CLDN18.2 / 4-1BB binding protein" encompasses any molecule capable of specifically binding to the CLDN18.2 protein or an epitope thereof and the 4-1BB protein or an epitope thereof, including, but not limited to, antibodies, polypeptides, fusion proteins of antibodies and polypeptides, or complexes thereof. In some embodiments, the term "CLDN18.2 / 4-1BB binding protein" encompasses an anti-CLDN18.2 / 4-1BB bispecific antibody in the examples of the present disclosure. In some embodiments, the "CLDN18.2 / 4-1BB binding protein" of the present disclosure further comprises a CD16A-binding domain and can further bind to CD16A, for example, an anti-CLDN18.2 / 4-1BB / CD16A trispecific antibody in the examples of the present disclosure.

[0182] The term "antibody" encompasses various antibody structures that exhibit the desired antigen-binding activity, 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). Antibodies may also refer to immunoglobulins, which have a tetrapeptide chain structure consisting of two heavy chains and two light chains linked by interchain disulfide bonds. Immunoglobulins differ in the amino acid composition and sequence of the heavy chain constant region, resulting in different antigenicities. Therefore, immunoglobulins can be divided into five types, or immunoglobulin isotypes, IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Ig of the same type can be further divided into different subclasses based on the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as kappa chains or lambda chains depending on the difference in their constant regions. Each of the five types of Ig may have either a kappa chain or a lambda chain. In antibody heavy and light chains, the sequence of approximately 110 amino acids near the N-terminus is highly variable and forms the variable region (V region), while the remaining amino acid sequence near the C-terminus is relatively stable and forms the constant region (C region). The variable region contains three hypervariable regions (HVRs) and four framework regions (FRs) whose sequences are relatively conserved. The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). 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 carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2 and LCDR3, and the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3.

[0183] Antibodies of the present disclosure may be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, or modified forms thereof, with monoclonal antibodies being particularly applicable in some embodiments. Generally, antibodies of the present disclosure are recombinant antibodies. As used herein, "recombinant" broadly refers to a product, such as a cell or a nucleic acid, protein, or vector, that has been modified by introducing a heterologous nucleic acid or protein or by modifying a naturally occurring nucleic acid or protein, or that the cell is derived from a cell that has been so modified. For example, recombinant cells express genes that are not present in the native (non-recombinant) cell form, or express naturally occurring genes that are aberrantly expressed, under-expressed, or not expressed at all.

[0184] "Antigen-binding fragment" covers single-chain antibodies (i.e., full-length heavy and light chains), Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH or VL or VHH), scFv, bivalent, trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody and epitope-binding fragments of any one of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for producing and preparing these antigen-binding fragments are known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0185] The determination or definition of CDRs can be achieved by elucidating the structure of an antibody and / or the structure of an antibody-ligand complex, thereby enabling accurate delineation of CDRs and identification of antibody binding site residues. This can be achieved by any one of a variety of techniques known to those skilled in the art, such as X-ray crystallography. Various analytical methods can be used to identify CDRs, including, but not limited to, the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions. The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8). The Chothia numbering system is similar to the Kabat numbering system, but takes into account the location of several structural loop regions. (See, e.g., Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83.) The AbM numbering system uses an integrated suite of computer programs from the Oxford Molecular Group that model antibody structure (see, e.g., Martin et al., 1989, ProcNatl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK, Oxford Molecular, Ltd.). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from the base sequence (see "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" in Samudrala et al., 1999, PROTEINS, Structure, Function and Genetics Suppl., 3:194-198).Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In conformational definitions, CDR positions can be identified as residues that contribute enthalpicly to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166). It should be noted that other CDR boundary definitions may not strictly adhere to one of the above methods, but still overlap with at least a portion of the Kabat CDRs, albeit shortened or extended by prediction or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used in this disclosure, CDR can refer to a CDR defined by any method (including a combination of methods) known in the art. The correspondence between each numbering system is familiar to those skilled in the art and is illustratively shown in Table 1 below.

[0186] [Table 1]

[0187] The CDR amino acid residues of the VL and VH regions of the antibodies of the present disclosure conform in number and position to the known Kabat numbering system.

[0188] In the present disclosure, "effector function" refers to a biological activity that can be attributed to the antibody Fc region and varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), Fc receptor (FcR) binding, cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. For example, ADCC is an immune mechanism that results in the lysis of antibody-coated target cells by immune effector cells. Target cells are cells that are specifically bound by an antibody or derivative thereof comprising an Fc region. "Enhanced ADCC" is defined as an improvement in the number of target cells lysed in a given time period using a given concentration of antibody in the medium surrounding the target cells via the ADCC mechanism defined above, and / or a reduction in the concentration of antibody in the medium surrounding the target cells required to achieve lysis of a given number of target cells in a given time period via the ADCC mechanism. Enhanced ADCC is relative to ADCC mediated by the same antibody produced from the same type of host cell, but without further engineering, by the same standard manufacturing, purification, preparation, and storage methods known to those skilled in the art. For example, the enhancement in ADCC mediated by an antibody containing an ADCC-enhancing amino acid substitution in its Fc region is relative to the ADCC mediated by the same antibody without that amino acid substitution in the Fc region. Suitable assays for measuring ADCC are known in the art (see, e.g., WO2006 / 082515 or WO2012 / 130831).

[0189] 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, domains are responsible for specific functional properties of the protein and can often be added, removed, or transferred to other proteins without impairing the function of the other parts and / or domains of the protein.

[0190] "Immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a conventional tetrapeptide chain structure antibody or a chain of a heavy chain antibody), or a polypeptide consisting essentially of such a globular region. Immunoglobulin domains are characterized by maintaining the immunoglobulin fold characteristic of antibody molecules.

[0191] An "immunoglobulin variable domain" refers to an immunoglobulin domain that is essentially composed of four "framework regions," referred to herein and hereinafter as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively, which are spaced apart by three "complementarity-determining regions" or "CDRs," referred to herein and hereinafter as "complementarity-determining region 1" or "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3," respectively. The general structure or sequence of an immunoglobulin variable domain may therefore be depicted as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The immunoglobulin variable domain contains the antigen-binding site and thus confers specificity for the antigen.

[0192] "Antibody framework (FR)" refers to that part of a variable domain that serves as a framework for the complementarity determining regions (CDRs) of that variable domain.

[0193] The term "immunoglobulin single variable domain" is typically used to refer to an immunoglobulin variable domain (which may be a heavy or light chain domain and comprises a VH, VHH or VL domain) that is capable of forming a functional antigen-binding site when it does not interact with other variable domains (e.g., in the absence of the necessary VH / VL interactions between the VH and VL domains of a conventional four-chain monoclonal antibody). Examples of "immunoglobulin single variable domains" are nanobodies (including VHH, camelized VH such as humanized VHH and / or camelized human VH), IgNARs, domains, (single domain) antibodies (e.g., dAbs) as or derived from a VH domain.TM ) and VL domains or (single domain) antibodies (e.g. dAbs TM ) Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (e.g., VH or VHH domains) are generally preferred. One specific example of an immunoglobulin single variable domain is a "VHH domain" (or abbreviated "VHH"), defined as follows:

[0194] "VHH domain" refers to a heavy chain single domain antibody, VHH, VH H These variable domains are also called "heavy-chain antibodies" (i.e., "light-chain-depleted antibodies"), and are also called domains, VHH antibody fragments, VHH antibodies, or nanobodies. These variable domains are antigen-binding immunoglobulins called "heavy-chain antibodies" (i.e., "light-chain-depleted antibodies") (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 these variable domains from the heavy-chain variable domain (referred to in the present disclosure as a "VH domain" or VH) and the light-chain variable domain (referred to in the present disclosure as a "VL domain" or VL) present in conventional antibodies with a tetrapeptide chain structure. A VHH domain specifically binds to an epitope without the need for another antigen-binding domain (this is the opposite of the VH or VL domain in a conventional tetrapeptide chain antibody, where the epitope is recognized by both the VL and VH domains). A VHH domain is a small, stable, and efficient antigen-recognition unit formed by a single immunoglobulin domain. The terms "heavy chain single domain antibody," "VHH domain," "VHH," and "V" are used interchangeably. HThe terms "H domain," "VHH antibody fragment," "VHH antibody," "Nanobody®," and "Nanobody® domain" ("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) may be used interchangeably. "VHH" includes, but is not limited to, natural antibodies produced in camelids, and may also be antibodies produced in camelids and then humanized, or may be antibodies screened using phage display technology. The total number of amino acid residues in a VHH is usually in the range of 110-120, and often between 112-115. However, it should be noted that both relatively short and relatively long 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 publications: 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.

[0195] As is known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions based on the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the Kabat numbering allows). This generally means that the Kabat numbering may or may not correspond to the actual number of amino acid residues in the actual sequence. Other numbering systems or conventions include Chothia, IMGT, and AbM.

[0196] A "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting non-human CDR sequences onto a human antibody variable region framework. This antibody can overcome the strong immune response induced by chimeric antibodies containing a large amount of non-human protein components. To avoid a decrease in activity associated with reduced immunogenicity, activity can be maintained by performing minimal back mutations on the fully human antibody variable region. An example of "humanization" includes a VHH domain derived from Camelidae, which can be "humanized" by substituting one or more amino acid residues in the original VHH sequence with one or more amino acid residues present at the corresponding positions in the VH domain of a normal human tetrapeptide chain structure antibody (also referred to as "sequence optimization" in the present disclosure, which may include, in addition to humanization, one or more mutations added to the sequence to confer improved properties of the VHH, such as removal of potential post-translational modification sites). A humanized VHH domain may comprise one or more fully human framework region sequences, and in some specific embodiments, may comprise human framework region sequences of IGHV3. Humanization methods include, for example, protein surface amino acid resurfacing and antibody humanization universal framework grafting (CDR grafting to a universal framework), in which CDRs are "grafted" onto other "stents" (including, but not limited to, human stents or non-immunoglobulin stents). Suitable stents and techniques for CDR grafting are known in the art. For example, germline DNA sequences of human heavy and light chain variable region genes 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 of the present disclosure also include phage-displayed humanized antibodies that have undergone affinity maturation of their CDRs.Furthermore, in order to avoid a decrease in activity due to a decrease in immunogenicity, the activity can be maintained by performing minimal back mutations or reverse mutations on the framework sequences of the variable regions of the human antibody.

[0197] An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) that improve the affinity of the antibody for its antigen, compared to a parent antibody that does not possess such alterations. For example, an "affinity matured" 4-1BB binding protein or anti-4-1BB antibody may have one or more changes in one or more CDRs that increase its affinity for the antigen, compared to the parent antibody. Affinity matured antibodies can be prepared by methods known in the art, for example, Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad. Sci. USA 91:3809-3813; Shier et al., 1995, Gene 169:147-155; Yelton et al., 1995, Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J. MoI. Biol. 226(3):889-896; KS Johnson & RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

[0198] Typically, the 4-1BB binding protein, CLDN18.2 / 4-1BB binding protein, CD16A binding protein of the present disclosure has a specific activity, preferably 10 -7 ~10 -10 moles / liter (M), more preferably 10 -8 ~10 -10 moles / liter, more preferably 10 -9 ~10 -10or 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 It binds to the antigen or target protein (i.e., 4-1BB, CLDN18.2, CD16A) with an association constant (KA) of M. -4 Any K greater than M D Values ​​are also generally considered to represent non-specific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be measured by any suitable known method, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays) described in this disclosure.

[0199] "Epitope" refers to the site on an antigen that binds to an immunoglobulin or antibody. An epitope may be formed from adjacent amino acids or from juxtaposed non-adjacent amino acids formed by tertiary folding of a protein. Epitopes formed from adjacent amino acids typically persist after exposure to denaturing solvents, whereas epitopes formed by tertiary folding typically disappear after treatment with denaturing solvents. An epitope typically contains at least 3-15 amino acids in a unique spatial conformation. Methods for determining the epitope that binds to a given antibody are well known in the art and include Western blotting and immunoprecipitation detection assays. Methods for determining the spatial conformation of an epitope include techniques known in the art and described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0200] "Binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen). The binding affinity between two molecules is determined by the dissociation constant (K D) can be quantified by determining the K. For example, surface plasmon resonance (SPR) methods (Biacore) can be used to measure the kinetics of complex formation and dissociation. D The rate constants corresponding to the association and dissociation of the monovalent complex are called the association rate constant ka (or k) and the dissociation rate constant kd (or koff), respectively. D is K D The dissociation constants ka and kd are related by the equation ka = kd / ka. The value of the dissociation constant can be determined directly by well-known methods and can also be calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9:340-362). For example, the K can be calculated by double filtration nitrocellulose filter binding assays 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 an antibody to a target antigen are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays mentioned elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can be determined by standard assays known in the art, such as surface plasmon resonance (SPR), e.g., Biacore. TM The K of each antibody / antigen complex may be evaluated by the KinExA system or KinExA. D By comparing K values, it is possible to compare the binding affinities associated with interactions with different molecules, for example, to compare the binding affinities of different antibodies to a given antigen. Similarly, the specificity of an interaction can be determined by comparing the K values ​​of the interaction of interest (e.g., the specific interaction between an antibody and an antigen). D values ​​and K of non-target interactions (e.g., a known control antibody that does not bind to 4-1BB, CLDN18.2, or CD16A). D It can be evaluated by determining and comparing values.

[0201] "Conservative substitution" refers to a substitution of an amino acid residue with another amino acid residue having similar properties 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. Furthermore, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it is clear to those skilled in the art that even when amino acid residues in the above-mentioned group showing similar properties are substituted, it does not show a specific change in properties.

[0202] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When every position in two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, when each position in two DNA molecules is occupied by the same nucleotide, 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, if 6 out of 10 positions in two sequences are matched or homologous when the sequences are optimally aligned, the two sequences are 60% homologous. Typically, two sequences are compared when aligned to obtain the maximum percentage homology.

[0203] "Nucleic acid" or "polynucleotide" may be used interchangeably in this disclosure and refer to any DNA or RNA molecule, single- or double-stranded, and, if single-stranded, to a molecule of its complementary sequence, preferably double-stranded DNA. A nucleic acid is "operatively linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operatively linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.

[0204] A "host cell" includes an individual cell or cell culture that may be, or has been, a recipient of a vector for incorporating a polynucleotide insert. A host cell includes the progeny of a single host cell, and due to natural, accidental, or deliberate mutation, the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell. A host cell includes cells transfected and / or transformed in vivo with a polynucleotide according to the present disclosure. "Cell," "cell line," and "cell culture" may be used interchangeably, and all such designations include their progeny. It should also be understood that due to deliberate or unintentional mutation, all progeny may not be precisely identical in DNA content. Mutant progeny that have the same function or biological activity as screened for from the originally transformed cell are included.

[0205] "Inhibition" or "blocking" may be used interchangeably and covers both partial and complete inhibition / blocking. "Inhibition of growth" (e.g., of a cell) is intended to include any measurable decrease in cell growth.

[0206] "Inhibits the growth of" or "growth inhibition" means inhibiting the growth or proliferation of cells.

[0207] A "proliferative disorder" refers to a medical condition associated with some degree of abnormal cell proliferation. In one embodiment, the proliferative condition refers to cancer. A "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Cancer," "proliferative disorder," and "tumor" are not mutually exclusive when referred to in this disclosure.

[0208] "Cancer prevention" means delaying, inhibiting, or preventing the onset of cancer in a subject where the onset of cancer or tumor formation in the subject has not been confirmed, but a cancer susceptibility has been identified, e.g., as determined by genetic screening or other methods. It further includes treating a subject with a precancerous condition, thereby halting the progression of the precancerous condition to a malignant tumor or causing its regression.

[0209] "Giving," "administration," and "treatment," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to contact of an exogenous agent, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid, e.g., therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of a reagent with a cell and contact of a reagent with a fluid, where the fluid contacts the cell. "Giving," "administration," and "treatment" also refer to treating, e.g., cells, ex vivo and in vitro, with a reagent, diagnostic, binding composition, or through another cell. When applied to a human, veterinary, or research subject, they refer to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic uses.

[0210] "Treatment" refers to the administration of an internal or external therapeutic agent, such as one comprising any one of the binding proteins or pharmaceutical compositions thereof disclosed herein as a therapeutic agent, to a subject suffering from, at risk of, or prone to one or more proliferative diseases or symptoms thereof, where the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered to the subject or population being treated in an amount that effectively alleviates one or more disease symptoms, whether by inducing regression of such symptoms or by inhibiting such symptoms from progressing to any clinically measurable extent. The amount of therapeutic agent that effectively alleviates any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on several factors, including the disease state, age and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Reduction of disease symptoms can be assessed by any clinical detection method commonly used by physicians or other professional health care providers to assess the severity or progression of the condition. An embodiment of the present disclosure (e.g., a method of treatment or product) may be ineffective in alleviating a target disease symptom in a subject, but should alleviate the target disease symptom in a statistically significant number of subjects, as determined by any statistical testing method known in the art, such as, for example, a Student's t-test, a chi-squared test, a Mann and Whitney U test, a Kruskal-Wallis test (H test), a Jonckheere-Terpstra test, and a Wilcoxon test.

[0211] An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or pathology of a medical condition. An effective amount also refers to an amount sufficient to enable or facilitate diagnosis. The effective amount used in a subject can vary depending on factors such as the condition being treated, the subject's overall health, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or human subject.

[0212] "Optionally" or "optionally" means that the subsequently described event or circumstance may, but need not, occur; the description includes cases where the event or circumstance occurs and cases where the event or circumstance does not occur. "And / or" should be considered to specifically indicate that each of the two specified features or components may or may not have the other. Thus, the term "and / or," as used in the phrase "A and / or B" in this disclosure, includes "A and B," "A or B," "A" (alone) and "B" (alone). Unless the context clearly indicates otherwise, throughout the specification and claims, words such as "comprise," "have," "contain," and the like, should be understood to have an inclusive meaning, i.e., "including but not limited to," rather than an exclusive or exhaustive meaning.

[0213] As used herein, a "subject" or "patient" refers to mammals, particularly primates, and especially humans. [Brief explanation of the drawings]

[0214] [Figure 1] This shows the results of a FACS binding test of antibodies to human 4-1BB antigen, awaiting measurement. [Figure 2] This shows the results of 4-1BB / NF-κB luciferase reporter gene detection before and after FcγRIIb cross-linking of the antibody awaiting measurement. [Figure 3] The results of FACS detection of the binding of anti-4-1BB antibody C5 and its humanized antibodies C5_V1, C5_V2, and C5_V3 to human 4-1BB on the surface of HEK293 cells. Urelumab, an isotype IgG1 antibody, was used as a control. [Figure 4] These are the results of detecting the activation effect of the anti-4-1BB antibody C5 and its humanized antibodies C5_V1, C5_V2, and C5_V3 on the NF-κB signaling pathway. Urelumab and an isotype IgG1 antibody were used as controls. [Figure 5]This is the result of FACS detection of the binding of C5_V2 and TCE-removed C5_V2-YTI to human 4-1BB on the surface of HEK293 cells. Urelumab, an isotype IgG1 antibody, was used as a control. [Figure 6] Binding experiments of anti-CD16A antibodies to cells overexpressing human CD16A, human CD16B, and cynomolgus monkey CD16. In binding experiments with cells overexpressing human CD16B, VHH-2-LALA, which can bind to human CD16B, was used as a control (VHH-2 is from patent US20190276554). Isotype IgG was also used as a control in each experiment. [Figure 7A] and [Figure 7B] Binding of anti-CD16A antibodies 34, 501 and humanized anti-CD16A antibodies 34H3, 501-V3NQ to cells overexpressing human CD16A or CD16B. In binding experiments with cells overexpressing human CD16B, VHH-2-LALA, which can bind to human CD16B, was used as a control (VHH-2 is from patent US20190276554). Isotype IgG was also used as a control in each experiment. [Figure 8] FIG. 1 is a schematic diagram of the structure of the CLDN18.2 / 4-1BB bispecific antibody. [Figure 9] FIG. 1 is a schematic diagram of the structure of the CLDN18.2 / 4-1BB / CD16A trispecific antibody. [Figure 10] This is the result of detecting the binding of the antibody 1903×C5_V2-YTI-LALA after C5_V2-YTI modification to human 4-1BB on the surface of HEK293 cells. 1903×C5_V2-LALA, C5_V2, and isotype IgG1 were used as controls. [Figure 11] The results show that the antibody 1903×C5_V2-YTI-LALA after C5_V2-YTI modification detects the activation of the NF-κB signaling pathway. 1903×C5_V2-LALA, TJ-CD4B, and isotype IgG1 were used as controls. [Figure 12]Figure 12A shows the binding activity of the CLDN18.2 / 4-1BB multifunctional antibodies 1903×C5_V2×34H3 and 1903×C5_V2 to the cell surface CLDN18.2 / CD16A V176 / 4-1BB antigen. Figure 12A shows the binding activity of the antibodies to NUGC4-hi18.2 cells, using 1903, IMAB362, TJ-CD4B, and isotype IgG1 as controls. Figure 12B shows the binding activity of the antibodies to CHOK1-CD16A V176A cells, using IMAB362, TJ-CD4B, 34H-Fc, and isotype IgG1 as controls. Figure 12C shows the binding activity of the antibodies to HEK293-Hu4-1BB cells, using TJ-CD4B, C5-_V2, and isotype IgG1 as controls. [Figure 13] Figure 13A shows the results of NK cell killing against CLDN18.2-expressing target cells induced by the CLDN18.2 / 4-1BB multifunctional antibodies 1903×C5_V2-YTI×34H3 and 1903×C5_V2-YTI. IMAB362, isotype IgG1, was used as a control. Figure 13A shows ADCC of PBMCs against NUGC4-hi18.2 cells (donor SC190061), Figure 13B shows ADCC of PBMCs against NUGC4-hi18.2 cells (donor S2001102), Figure 13C shows ADCC of PBMCs against SNU601 cells (donor SC190061), and Figure 13D shows ADCC of PBMCs against SNU601 cells (donor S2001102). [Figure 14] These are the results of detecting the phagocytosis of tumor cells by macrophages induced by the CLDN18.2 / 4-1BB multifunctional antibodies 1903×C5_V2-YTI×34H3 and 1903×C5_V2-YTI. IMAB362, isotype IgG1, was used as a control. [Figure 15]These are the results of CLDN18.2-dependent 4-1BB / NF-κB luciferase reporter gene detection using the CLDN18.2 / 4-1BB multifunctional antibodies 1903×C5_V2×34H3 and 1903×C5_V2. The left side shows the results before CLDN18.2 cross-linking, and the right side shows the results after CLDN18.2 cross-linking. In all cases, TJ-CD4B, ADG-106, and isotype IgG1 were used as controls. [Figure 16A] and [Figure 16B] These are the results of detecting the activation of CLDN18.2 / 4-1BB multifunctional antibodies 1903×C5_V2-YTI×34H3 and 1903×C5_V2-YTI against T lymphocytes from donors 1 and 2. TJ-CD4B and isotype IgG1 were used as controls. [Figure 17] This is the result of detecting NK T cell killing induced by the CLDN18.2 / 4-1BB multifunctional antibody 1903×C5_V2-YTI×34H3 and 1903×C5_V2-YTI. Isotype IgG1 was used as a control. [Figure 18] Figure 18 shows the tumor-inhibitory effects of the CLDN18.2 / 4-1BB multifunctional antibodies 1903×C5_V2-YTI×34H3 and 1903×C5_V2-YTI on MC38-hCLDN18.2-transplanted tumors. Vehicle and TJ-CD4B were used as controls. Figure 18A shows the tumor volume, and Figure 18B shows the mouse body weight. [Figure 19] The effects of the CLDN18.2 / 4-1BB multifunctional antibodies 1903×C5_V2-YTI×34H3 and 1903×C5_V2-YTI on mouse ALT / AST were shown. Vehicle and TJ-CD4B were used as controls. [Figure 20] Pharmacokinetic curve of CLDN18.2 / 4-1BB multifunctional antibody 1903×C5_V2 in h4-1BB transgenic mice, where 1903 was used as a control. DETAILED DESCRIPTION OF THE INVENTION

[0215] The present disclosure will be further explained in the following examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods for which specific conditions are not specified in the examples of the present disclosure generally follow standard conditions, such as those in the Cold Spring Harbor Antibody Technology Laboratory Manual and the Molecular Cloning Manual, or conditions suggested by raw material or product manufacturers. Reagents for which specific sources are not specified are standard commercially available reagents.

[0216] The proteins used in this disclosure include human 4-1BB protein (Human 4-1BB / TNFSF9 Protein, His Tag, purchased from Acrobiosystems, product number 41B-H5227), human 4-1BB protein (Biotin / His Tag) (Biotinylated Human 4-1BB / TNFRSF9 Protein, Avitag 商標 , His Tag, purchased from Acrobiosystems, Product No. 41B-H82E3), and monkey 4-1BB protein (Cynomolgus / Rhesus macaque 4-1BB / TNFRSF9 Protein, His Tag, purchased from Acrobiosystems, Product No. 41B-C52H4), the amino acid sequences of which begin and end with Leu24-Gln186.

[0217] The above protein reagents can be used in the experiments of each embodiment of the present disclosure, including use as immunization antigens, antigen screening, and identification of activity and function. The amino acid sequences of the control molecules used in the present disclosure are shown in Table 2.

[0218] [Table 2-1] [Table 2-2]

[0219] Example 1. Screening and preparation of anti-4-1BB single domain antibodies 1. Alpaca Immunization, Titer Detection, and Affinity Panning of the Phage Library Alpacas were immunized with His-tagged human 4-1BB recombinant protein (Acrobiosystems, 41B-H5227) for a total of four immunizations, once every two weeks. For the first immunization, 0.5 mg of antigen was mixed with 1 mL of complete Freund's adjuvant (CFA) and injected subcutaneously. For the following three immunizations, 0.25 mg of antigen was mixed with 1 mL of incomplete Freund's adjuvant (IFA) and injected subcutaneously. Blank serum was collected before immunization, and 50 mL of peripheral blood was collected one week after the third and one week after the fourth immunization. PBMCs were isolated, total RNA was extracted, and purity was confirmed. After two rounds of nested PCR, the nanobody target fragments were ligated into phage display vectors. A phage library was generated by electroporation.

[0220] [ka]

[0221] To obtain anti-4-1BB nanobodies that simultaneously recognize both human and monkey antigens, we used a two-round cross-screening strategy of human and monkey antigens. The antigens used in the first and second rounds of screening were human 4-1BB and monkey 4-1BB, respectively. Phages specifically binding to 4-1BB were eluted using the Gly-HCl acid elution method in each round of screening. Ninety-six clones (192 clones in total) were randomly selected from the first and second rounds of titration plates, and positive clones were screened using phage ELISA, with optical density at 450 nm determined. The positive clones were sequenced. Based on the sequencing results, sequence alignment and phylogenetic tree analysis were performed, and 14 unique sequences were identified, including H27, H170, C3, C5, and C145. The sequence of C5 is as follows:

[0222] >C5 [ka] (Note: The underlined parts are the CDR regions.)

[0223] [Table 3]

[0224] 2. Expression and purification of VHH-Fc fusion proteins The C5 sequence was linked to human IgG1-Fc (SEQ ID NO: 16, where the underlined mutations are) with C220A, S267E, and L328F mutations (numbered according to the Eu system). The sequence of the linked VHH-Fc fusion protein is as follows: Furthermore, introducing mutations such as L234A, L235A, and N297A (numbered according to the Eu system) into the Fc of human IgG1 completely eliminated antibody FcγR-mediated effector function (e.g., as shown in SEQ ID NO: 15), and introducing S228P (numbered according to the Eu system) into the Fc of human IgG4 stabilized the antibody molecule and prevented half-molecule formation (e.g., as shown in SEQ ID NO: 16). Both of these are selectable IgG Fc sequences. In SEQ ID NOs: 14 to 16, the underlined mutations are Fc mutations.

[0225] >Human IgG1-Fc (including C220A, S267E, and L328F mutations) [ka] >Human IgG1-Fc (including C220A, L234A, L235A, and N297A mutations) [ka] >Human IgG4-Fc (including S228P mutation) [ka]

[0226] An exemplary antibody sequence is shown in SEQ ID NO: 17, in which C5 is linked to SEQ ID NO: 14.

[0227] >C5-Fc [ka] (Note: The italicized parts are Fc.)

[0228] The plasmid was constructed and transiently transfected into cells, and the antibody was expressed and purified. The target antibody was obtained by detection.

[0229] Example 2. Detection of antigen-binding activity of anti-4-1BB antibody and its agonist activity 1. Detection of binding ability to 4-1BB antigen The binding activity of the anti-4-1BB single domain antibody to human 4-1BB protein was detected using a flow cytometer.

[0230] HEK293-Hu4-1BB cells were obtained by transient transfection of HEK293 cells (ATCC CRL-1573) with a gene expressing human 4-1BB protein (CD137 cDNA ORF Clone, Human, C-OFPSpark tag, purchased from Sino Biological, Cat# HG10041-ACR). The cell culture medium was DMEM (Gibco, Cat# 11995065) containing 10% fetal bovine serum. The experimental medium was sterile PBS (phosphate buffer, pH 7.40) containing 2% fetal bovine serum. HEK293-Hu4-1BB cells were washed twice with the experimental medium and cultured at a concentration of 1 × 10 5HEK293-Hu4-1BB cells were seeded at 1 / well in a 96-well U-bottom plate, and various concentrations of anti-4-1BB VHH-Fc samples were added. The cells were incubated at 4°C for 1 hour, then washed twice with experimental medium. Goat anti-human IgG (H+L) Alexa Fluor 488 antibody (Thermo, Cat# A11013) was then added, washed twice, and the fluorescent signal was read using a flow cytometer. Urelumab monoclonal antibody was used as a positive control, and the MFI values ​​for each antibody are shown in Figure 1.

[0231] As a result, the anti-4-1BB antibodies have different degrees of binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, among which C5 has good cell membrane surface antigen binding activity.

[0232] 2. Detection of agonist activity against the 4-1BB signaling pathway The agonistic activity of anti-4-1BB antibodies was evaluated using a 4-1BB / NF-κB reporter gene.

[0233] HEK293 cells (ATCC CRL-1573) were transiently transfected with a gene expressing human 4-1BB (CD137 cDNA ORF Clone, Human, C-OFPSpark tag, purchased from Sino Biological, Cat. #HG10041-ACR) and an NF-κB reporter gene (pGL4.32[luc2P / NF-κB-RE / Hygro] Vector, purchased from Promega, Cat. #E849A) to obtain HEK293-Hu4-1BB / NF-κB double-transfected cells. 4-1BB activation could be characterized by the activation level of the NF-κB signaling pathway. HEK293 cells were transiently transfected with FcγRIIb plasmid (CD32B / Fcgr2b cDNA ORF Clone, Human, N-His tag, purchased from Sino Biological, Cat. #HG10259-NH) to obtain HEK293 cells with high FcγRIIb expression. The cell culture medium was DMEM (Gibco, Cat. #11995065) containing 10% fetal bovine serum. HEK293-Hu4-1BB / NF-κB cells (2 × 10 6 50 μL of 100 μg / mL of 100 μL ... 6 10 × 10 μL of gradient diluted anti-4-1BB antibody was added to each well and incubated at 37°C for 6 hours. The cells were removed, and an equal volume of Bio-Glo Luciferase Assay System reagent (Promega, Cat# G7940) was added to each well. The cells were incubated in the dark for 5 minutes, and the fluorescent signal was measured using an Envision plate reader (PerkinElmer, 2150). EC 50 The EC value and Emax value (fluorescence intensity of the control group without antibody) were calculated. 50 The in vitro cellular agonist activity of the anti-4-1BB antibody was evaluated based on the values. The results are shown in Figure 2 and Table 4.

[0234] The results showed that without FcγRIIb (i.e., CD32b) cross-linking, the anti-4-1BB antibodies showed weaker activation of the 4-1BB / NF-κB luciferase reporter gene signaling pathway than the urelumab control, suggesting that the anti-4-1BB antibodies of the present disclosure are safer. After FcγRIIb cross-linking, the anti-4-1BB antibodies showed significant activation of the 4-1BB / NF-κB luciferase reporter gene signaling pathway, with C5 having the strongest activation ability and comparable to the urelumab control. Based on the activity results, sequence C5 was screened and humanized, having low background activation before FcγRIIb cross-linking and even stronger activation activity after FcγRIIb cross-linking.

[0235] [Table 4]

[0236] Example 3. Modification of anti-4-1BB antibody 1. Humanization The amino acid numbers of the CDR and FR regions (human framework regions, framework) of the C5 sequence were labeled using the Kabat numbering system. The FR1, FR2, and FR3 sequences were aligned with the antibody germline database to obtain highly homologous human FR germline templates. FR1 is derived from IGHV3-64*04, FR2 from IGHV3-23*03, and FR3 from IGHV3-74*01. To reduce immunogenicity in humans, each of the above human germline FR regions was substituted and inserted into the original sequence. Key amino acids that affect antibody structure and function were backmutated to restore avidity and activity. The humanized sequences are as follows:

[0237] >C5_V1 [ka] >C5_V2 [ka] >C5_V3 [ka] (Note: The underlined parts are the CDR regions.)

[0238] The above three humanized sequences were each linked to human IgG1-Fc (SEQ ID NO: 14). Plasmids were constructed, transiently transfected, expressed, and purified. The specific process was as follows: 60 μL of transfection reagent was diluted with culture medium and mixed uniformly with 15 μg of plasmid. After incubation at 37°C for 15 minutes, the mixed transfection solution was added dropwise to 30 mL of cell suspension and cultured on a shaker for one week to allow expression. The supernatant was then collected and subjected to Protein A affinity purification. The cells were eluted with citrate buffer (pH 3.4), dialyzed against 1x PBS buffer, and frozen for storage.

[0239] 2. Removal of TCE sites Then, T-cell epitopes (TCEs) were predicted for the C5_V2 sequence, and the CDR3 sequence of C5_V2 was modified based on the prediction results to reduce the number of TCEs. The F mutation in the CDR3 region was mutated to Y (F99Y, according to the Kabat numbering system) to obtain a TCE-optimized version of the C5_V2 sequence, designated C5_V2-YTI, and its sequence is as follows: >C5_V2-YTI [ka]

[0240] That is, according to the Kabat numbering system, the amino acid sequence of CDR1 in C5_V2-YTI is shown in SEQ ID NO: 11, the amino acid sequence of CDR2 is shown in SEQ ID NO: 12, and the amino acid sequence of CDR3 is shown in HPLTYTIATMNDYDY (SEQ ID NO: 22).

[0241] The C5_V2-YTI sequence was linked to human IgG1-Fc (SEQ ID NO: 14). The plasmid was constructed, transiently transfected, expressed and purified.

[0242] 3. WO2023093899 was incorporated as a whole by modifying the amino acids in the VH framework (FR) of the anti-4-1BB single domain antibody to obtain C5_V2-YTI-AA (corresponding to C5_V2-YTI-53 shown in the 30th sequence in WO2023093899).

[0243] Example 4. Detection of antigen-binding activity and agonist activity of modified anti-4-1BB antibodies 1. Detection of binding ability to 4-1BB antigen The antigen-binding activity of the humanized antibodies was detected by the FACS detection method described in Example 2. The results are shown in Figure 3 and Table 5.

[0244] As a result, the humanized anti-4-1BB antibodies had different degrees of binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, and C5_V1, C5_V2, and C5_V3 all maintained good binding activity.

[0245] [Table 5]

[0246] 2. Detection of agonist activity against the 4-1BB signaling pathway The activation of the humanized antibody in the 4-1BB / NF-κB signal was detected by an NF-κB luciferase reporter gene experiment as described in Example 2, and the results are shown in FIG.

[0247] As a result, after FcγRIIb cross-linking, the humanized anti-4-1BB antibodies showed activation of the 4-1BB / NF-κB luciferase reporter gene signaling pathway, with C5, C5_V1, C5_V2, and C5_V3 exhibiting activation capabilities comparable to those of the urelumab control.

[0248] [Table 6]

[0249] 3. Detection of antigen-binding ability of TCE-removed humanized 4-1BB antibody TCE removal was optimized for humanized antibody C5_V2, and the resulting antibody was named C5_V2-YTI (see Example 3 for details). The antigen-binding activities of C5_V2 and C5_V2-YTI were compared using the FACS detection method described in Example 2, and the results are shown in Figure 5 and Table 7. As a result, C5_V2-YTI maintained good binding activity to 4-1BB on the surface of HEK293-Hu4-1BB cells, corresponding to C5_V2.

[0250] [Table 7]

[0251] Example 5. Process for obtaining anti-CD16A nanobodies 1. Screening of a Natural Library of Human CD16A Research has revealed that the human CD16A gene, FcγRIIIb (CD16B), shares a sequence highly similar to that of CD16A, with over 97% homology. CD16B is primarily expressed on neutrophils, with small amounts of soluble CD16B present in the blood. Therefore, we needed to screen for antibodies specific to CD16A that could not recognize CD16B. First, we performed a first round of panning against a camel natural library using biotin-tagged human CD16A protein (CDA-H82E8, ACROBiosystems) at a concentration of 5 μg / mL, followed by elution with Gly-HCl at pH 2.2 to obtain first-round phage. The phage were then amplified and subjected to a second round of panning. During the second round, negative screening was performed with 5 μg / mL of biotin-tagged human CD16B protein (CDB-H82E4). Phage bound to human CD16B were removed, and then further screened with 3 mg / mL of human CD16A protein. The elution product was then obtained from the second round of panning. Finally, phage ELISA was performed on the second round of panning product. Phages that recognized human CD16A but not human CD16B were selected and sequenced to obtain the target antibody sequence.

[0252] 2. Camel Immunization and Screening for Human CD16A CD16A-specific antibodies were screened and prepared by a combination of camel immunization and phage display. The immunization strategy is shown in Table 8.

[0253] [Table 8]

[0254] After isolating PBMCs from the blood sample collected at the fourth immunization, a library was constructed. The screening strategy was consistent with the screening of the camel natural library in Example 2.1. As a result of the screening, two candidate molecules, 34 and 501, were finally selected for further development, with the sequences shown below: >34 [ka] >501 [ka]

[0255] [Table 9]

[0256] 3. Cell-binding verification of candidate molecules We examined the binding ability of candidate molecules to CHO cell lines overexpressing human CD16A-V176 (human CD16A-V176 protein sequence: AAH36723.1), CD16B (human CD16B protein sequence: O75015.2), or cynomolgus monkey CD16 (monkey CD16 sequence: NP_001270121.1). A positive antibody, VHH-2, with high affinity for human CD16B was selected as a control (from patent US20190276554). We found that the candidate molecules had sub-nM to nM levels of binding to both human CD16A and monkey CD16, but very weak binding to human CD16B (see Figure 6).

[0257] The above VHH-2 was linked to a human IgG1-Fc (mutations are underlined) with C220A, L234A, and L235A mutations (numbered according to the Eu system), and VHH-2 was placed at the N-terminus of the Fc. The resulting sequence, designated VHH-2-LALA, is as follows: >Human CD16B-positive antibody VHH-2-LALA [ka] Human CD16A-V176 (AAH36723.1) amino acid sequence: [ka] >Human CD16B (O75015.2) [ka] >Cynomolgus monkey CD16 (NP_001270121.1) [ka]

[0258] 4. Humanization of candidate molecules The anti-human CD16A antibodies 34 and 501 were humanized and their binding activity was confirmed in human CD16A-overexpressing cell lines. The humanized templates for 34 were IGHV3-23*04, with several back mutations. The humanized templates for 501 were IGHV3-20*04, with several back mutations. The amino acid sequences of the humanized versions of 34 and 501 are as follows: >34H1 [ka] >34H2 [ka] >34H3 [ka] >34H4 [ka] >34H5 [ka] >501V1 [ka] >501V2 [ka] >501V3 [ka] >501-V3NQ [ka]

[0259] Of these, CDR1 of 501-V3NQ is represented by SEQ ID NO: 28, CDR2 is represented by CINWQGGRTQYGDSVKG (SEQ ID NO: 44), and CDR3 is represented by SEQ ID NO: 30.

[0260] 5. Identification of cellular binding of humanized candidate molecules The antibodies were linked to human IgG1-Fc, expressed by transient transfection, and then subjected to cell binding assays. 34H3 and 501-V3NQ were selected for further development and dual antibody combination. The cell binding results for 34H3 and 501-V3NQ are shown in Figures 7A and 7B, and are similar to those of the parent antibody before humanization.

[0261] Example 6. Preparation of CLDN18.2 / 4-1BB multifunctional antibody 1. Design, expression, and purification of CLDN18.2 / 4-1BB bispecific antibody (CLDN18.2×4-1BB) Based on the screening results of the humanized anti-4-1BB nanobody, the C5_V2 clone and the CLDN18.2 monoclonal antibody sequence (from WO2020200196A1) were selected to construct a CLDN18.2 x 4-1BB bispecific antibody. Among them, the sequence of CLDN18.2 monoclonal antibody 1903 is as follows: CLDN18.2 monoclonal antibody 1903 heavy chain (SEQ ID NO: 45) [ka] CLDN18.2 monoclonal antibody 1903 light chain (SEQ ID NO: 46) [ka]

[0262] The italicized portion in the heavy chain is the heavy chain constant region, and the italicized portion in the light chain is the light chain constant region.

[0263] The 1903 CDRs, defined according to the Kabat numbering convention, are as follows: HCDR1 is SYWMH (SEQ ID NO: 57), HCDR2 is MIHPNSGSTNYNEKFKG (SEQ ID NO: 58), and HCDR3 is LKTGNSFDY (SEQ ID NO: 59); LCDR1 is KSSQSLLNSGNQKNYLT (SEQ ID NO: 60), LCDR2 is WASTRES (SEQ ID NO: 61), and LCDR3 is QNAYTYPFT (SEQ ID NO: 62).

[0264] The sequences of the heavy and light chain variable regions and the Fc portion of the antibody are as follows: >1903 VH (SEQ ID NO: 63) [ka] EVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQRLEWMG MIHPNSGSTNYNEKFKG RVTITRDTSASTAYMELSSLRSEDTAVYYCAR LKTGNSFDY WGQGTTVTVSS >1903 VL (SEQ ID NO: 64) [ka] >IgG1 Fc (containing S239D, I332E mutations) (SEQ ID NO: 65) [ka]

[0265] The bispecific antibody molecule used the CLDN18.2 monoclonal antibody sequence described above, but with the S239D / I332E mutation in its Fc region. One C5_V2 was fused to each of the two CLDN18.2 heavy chains at their C-termini, using (G4S)2 as a linker, to obtain the bispecific antibody molecule 1903×C5_V2. In the case of 1903×C5_V2, according to the molecular naming conventions described herein, 1903 represents the variable region of the CLDN18.2 monoclonal antibody, and C5_V2 represents the 4-1BB nanobody clone number. The bispecific antibody molecule 1903×C5_V2-YTI was obtained by replacing C5_V2 with C5_V2-YTI, from which TCE had been removed. The antibody structure is shown in Figure 8. The 1903×C5_V2-YTI bispecific antibody molecule was further optimized by varying two amino acid residues at the C-terminus of C5_V2-YTI to remove pre-ADA. The optimized bispecific antibody molecule was designated 1903×C5_V2-YTI-AA.

[0266] The amino acid sequence of the above bispecific antibody molecule is as follows: >1903×C5_V2 heavy chain (SEQ ID NO: 47) [ka] >1903×C5_V2-YTI heavy chain (SEQ ID NO: 48) [ka] >1903×C5_V2-YTI-AA heavy chain (SEQ ID NO: 49) [ka]

[0267] The italicized parts in the heavy chain are the heavy chain constant regions, and the underlined parts are linkers. The light chain amino acid sequences of 1903×C5_V2, 1903×C5_V2-YTI, and 1903×C5_V2-YTI-AA are all shown in SEQ ID NO:46.

[0268] Transient transfection and expression: For example, using a 30 mL expression system, 60 μL of transfection reagent was diluted with culture medium and mixed evenly with 15 μg of plasmid. The mixture was then incubated at 37°C for 15 minutes, and the cell suspension was added dropwise to the mixed transfection solution while shaking. The mixture was then placed on a shaker and cultured for one week to allow expression. The supernatant was then collected and centrifuged at 8000 rpm for 5 minutes.

[0269] Antibody purification: First, a Protein A affinity chromatography column was equilibrated with 20 mL of 1x PBS at a flow rate of 1 mL / min. The loading flow rate was 1 mL / min, and then washed with 20 mL of 1x PBS at a flow rate of 1 mL / min. The column was then eluted with citrate buffer (pH 3.4) at 1 mL / min. The fraction was collected and the absorbance at 280 nm was measured using a NanoDrop device. Finally, the concentrated protein was transferred to a dialysis bag and dialyzed in a beaker of 1x PBS. The target antibody molecules were obtained by detection.

[0270] 2. Design, expression, and purification of CLDN18.2 / 4-1BB / CD16A trispecific antibody (CLDN18.2 x 4-1BB x CD16A) Based on the screening results of the humanized anti-4-1BB nanobody, the C5_V2 clone was selected, and based on the screening results of the humanized anti-CD16A nanobody, the 34H3 clone was selected. These were then combined with the above-mentioned CLDN18.2 monoclonal antibody 1903 to construct a CLDN18.2 x 4-1BB x CD16A trispecific antibody.

[0271] The trispecific antibody molecule was constructed using the IgG1 subtype CLDN18.2 monoclonal antibody 1903 as the scaffold, with the S239D / I332E mutation in the Fc region. One C5_V2 was fused to the C-terminus of each of the two CLDN18.2 heavy chains, with a (G4S)2 linker. One 34H3 was fused to the C-terminus of each of the two light chains, with a (G4S)3 linker. The molecular nomenclature for 1903×C5_V2×34H3 indicates that the CLDN18.2 monoclonal antibody uses the variable region of 1903, C5_V2 is the 4-1BB nanobody clone number, and 34H3 is the CD16A nanobody clone number. The trispecific antibody molecule 1903×C5_V2-YTI×34H3 was constructed by replacing C5_V2 with TCE-depleted C5_V2-YTI. The antibody structure is shown in Figure 9. The 1903×C5_V2-YTI×34H3 trispecific antibody molecule was further optimized to remove pre-ADA by varying two amino acids at the C-terminus of C5_V2-YTI and 34H3. The optimized trispecific antibody molecule was designated 1903×C5_V2-YTI×34H3-AA.

[0272] The amino acid sequence of the above trispecific antibody molecule is as follows: >1903×C5_V2×34H3 heavy chain (SEQ ID NO: 50) [ka] >1903×C5_V2×34H3 light chain (SEQ ID NO: 51) [ka] >1903×C5_V2-YTI×34H3 heavy chain (SEQ ID NO: 52) [ka] >1903×C5_V2-YTI×34H3 light chain (SEQ ID NO: 51) >1903×C5_V2-YTI×34H3-AA heavy chain (SEQ ID NO: 53) [ka] >1903×C5_V2-YTI×34H3-AA light chain (SEQ ID NO: 54) [ka]

[0273] In the above sequence, the italicized part in the heavy chain is the heavy chain constant region, the italicized part in the light chain is the light chain constant region, and the underlined part is the linker.

[0274] The target antibody molecule was obtained by transient cell transfection, expression, and antibody purification according to the method described in Part 1 of Example 6 above.

[0275] Example 7. Antigen binding activity of C5_V2-YTI The antigen-binding activity of C5_V2, 1903×C5_V2-LALA, and 1903×C5_V2-YTI-LALA against human 4-1BB was compared. C5_V2 was linked to human IgG1-Fc with C220A / S267E / L328F mutations, and its sequence is shown in SEQ ID NO: 17. 1903×C5_V2-LALA was obtained by introducing L234A / L235A mutations into the Fc of the 1903×C5_V2 heavy chain (SEQ ID NO: 47), but without the S239D / I332E mutations (SEQ ID NO: 55) in the Fc, and its light chain was still shown in SEQ ID NO: 46. 1903xC5_V2-YTI-LALA is a heavy chain obtained by introducing L234A / L235A mutations into the Fc of the 1903xC5_V2-YTI heavy chain (SEQ ID NO: 48), and does not have the S239D / I332E mutations in the Fc (SEQ ID NO: 56), while the light chain is still represented by SEQ ID NO: 46. The sequence is as follows: >1903×C5_V2-LALA heavy chain (SEQ ID NO: 55) [ka] >1903×C5_V2-YTI-LALA heavy chain (SEQ ID NO: 56) [ka]

[0276] The italicized parts in the heavy chain are the heavy chain constant regions, and the underlined parts are linkers. The light chain sequences of 1903×C5_V2-LALA and 1903×C5_V2-YTI-LALA are both shown in SEQ ID NO:46.

[0277] The binding activity of the C5_V2-YTI modified antibody to human 4-1BB protein was detected by FACS experiments. HEK293-Hu4-1BB cells are HEK293 cells overexpressing human 4-1BB protein, and the cell culture medium is DMEM (Gibco, Cat#11965092) containing 10% fetal bovine serum and 100 μg / mL hygromycin B. After resuscitation, the cells were passaged and adjusted. The experimental medium is sterile PBS (phosphate buffer, pH 7.40) containing 2% fetal bovine serum. HEK293-Hu4-1BB cells were washed twice with the experimental medium and cultured at a concentration of 1 × 10 5 Cells were seeded at 1 / well in a 96-well plate, and various concentrations of samples to be measured were added. The cells were incubated at 4°C for 1 hour, then washed twice with experimental medium. Alexa Fluor 647-mouse anti-human (IgG, Fcγ fragment specific) antibody (Jackson, Cat#209-605-098) was then added. After washing twice, the fluorescent signal was read using a flow cytometer. The results are shown in Figure 10 and Table 10.

[0278] FACS detection results showed that the 1903×C5_V2-YTI-LALA antibody had strong binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, corresponding to 1903×C5_V2-LALA and C5V2, and that C5_V2 and C5_V2-YTI had the same binding ability to 4-1BB.

[0279] [Table 10]

[0280] Example 8. 4-1BB / NF-κB luciferase reporter gene detection experiment for C5_V2-YTI The agonist activity of the antibody was assessed using a 4-1BB / NF-κB reporter gene. HEK293 cells (ATCC CRL-1573) were transiently transfected with a gene expressing human 4-1BB (CD137 cDNA ORF Clone, Human, C-OFPSpark tag, Sino Biological, Cat. #HG10041-ACR) and the NF-κB genome (pGL4.32[luc2P / NF-κB-RE / Hygro] Vector, Promega, Cat. #E849A) to obtain HEK293-Hu4-1BB / NF-κB double-transfected cells. Activation of Hu4-1BB by the NF-κB signaling pathway could be characterized. NUGC4-hi18.2 cells were obtained by stable transfection of NUGC4 cells with a gene expressing human CLDN18.2 protein. The cell culture medium was RPMI1640 (Gibco, Cat# 10491A-01) containing 10% inactivated fetal bovine serum and 10 μg / mL puromycin. HEK293-Hu4-1BB / NF-κB cells (2 × 10 6 50 μL of CLDN18.2-expressing NUGC4 cells (2.5 × 10 cells / mL) was seeded into a 96-well cell culture plate, and 40 μL of medium or 40 μL of CLDN18.2-expressing NUGC4 cells (2.5 × 10 cells / mL) was seeded into a 96-well cell culture plate. 6 10 x 10 μL of gradient diluted antibody was added to each well and incubated at 37°C for 6 hours. The cells were removed, and an equal volume of Bio-Glo Luciferase Assay System reagent (Promega, Cat# G7940) was added to each well. The cells were incubated in the dark for 5 minutes, and the fluorescent signal was measured using an Envision plate reader. EC 50 The fluorescence intensity and Emax (fluorescence intensity of the control group without antibody) were calculated, and the in vitro cellular agonist activity of the anti-4-1BB antibody was evaluated. The results are shown in Figure 11 and Table 11.

[0281] As a result, it was revealed that the 1903×C5_V2-YTI-LALA antibody had no background activation, but its agonistic activity against 4-1BB after cross-linking with CLDN18.2 was equivalent to that of 1903×C5_V2-LALA, and that C5_V2 and C5_V2-YTI had the same activation ability against 4-1BB.

[0282] [Table 11]

[0283] Example 9. Detection of antigen binding affinity of CLDN18.2 / 4-1BB multifunctional antibody The antigen affinity of the CLDN18.2 / 4-1BB multifunctional antibody was detected using surface plasmon resonance (SPR). A CM5 sensor chip was selected for the experiment, and HBS-EP+ buffer solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) was used as the mobile phase. Anti-human IgG (Fc) antibody was prepared at 30 μg / mL in 10 mM sodium acetate buffer (pH 5.0), and the immobilization program was used to automatically immobilize the anti-human IgG (Fc) antibody channel via amino coupling. Each antibody to be measured was prepared as a ligand in HBS-EP+ buffer solution and captured together with the anti-human IgG (Fc) antibody in the chip channel. Human 4-1BB protein (Acro Biosystems, 41B-H522a) and cynomolgus monkey 4-1BB protein (Acro Biosystems, 41B-C52H4) were used as antigens (i.e., analytes) in HBS-EP+ buffer. The analytes were diluted two-fold. The diluted antibodies were allowed to bind for 1 minute through the experimental and reference channels at a flow rate of 30 μL / min, followed by dissociation for 15 minutes. 10 mM Glycine pH 1.5 (GE Healthcare, BR-1003-54) was added to the regeneration buffer at a flow rate of 10 μL / min for 30 seconds. Zeba Spin Desalting Columns (Thermo, 89882) were used, and data were analyzed using Biacore 8K evaluation software.

[0284] The results are shown in Table 12, which demonstrate that the antigen-binding affinity of the CLDN18.2×4-1BB multifunctional antibody to human and cynomolgus monkey 4-1BB proteins is comparable to that of the parent 4-1BB VHH antibody C5_V2.

[0285] [Table 12]

[0286] Example 10. Detection of antigen-binding activity of CLDN18.2 / 4-1BB multifunctional antibody The binding activity of the CLDN18.2 / 4-1BB multifunctional antibody to human CLDN18.2 / CD16A / 4-1BB protein was detected by FACS experiments. NUGC4-hi18.2 cells were obtained by stable transfection of NUGC4 cells with a gene expressing human CLDN18.2 protein. The cell culture medium was RPMI1640 (Gibco, Cat#10491A-01) containing 10% inactivated fetal bovine serum and 10 μg / mL puromycin. CHO-K1-CD16A V176 cells were obtained by overexpressing human CD16A V176 protein in CHO-K1 cells. The cell culture medium was Ham's F12 + GlutaMAX containing 10% fetal bovine serum and 200 μg / mL hygromycin B. TM The experimental medium was sterile PBS (phosphate buffered saline, pH 7.40) containing 2% fetal bovine serum. NUGC4-hi18.2, CHO-K1-CD16A V158, or HEK293-Hu4-1BB cells were washed twice with the experimental medium and cultured at a concentration of 1 × 10. The HEK293-Hu4-1BB cells were cultured in DMEM (Gibco, Cat# 11965092) containing 10% fetal bovine serum and 100 μg / mL hygromycin B. After resuscitation, the cells were passaged and the cell condition was adjusted. The experimental medium was sterile PBS (phosphate buffered saline, pH 7.40) containing 2% fetal bovine serum. NUGC4-hi18.2, CHO-K1-CD16A V158, or HEK293-Hu4-1BB cells were washed twice with the experimental medium and cultured at a concentration of 1 × 10. 5 Cells were seeded at 1000p / well in a 96-well plate, and various concentrations of samples to be measured were added. The cells were incubated at 4°C for 1 hour, then washed twice with experimental medium. Alexa Fluor 647-mouse anti-human (IgG, Fcγ fragment specific) antibody (Jackson, Cat#209-605-098) was added, washed twice, and the fluorescent signal was read using a flow cytometer. The results are shown in Figures 12A-12C and Tables 13-15.

[0287] FACS analysis showed that the CLDN18.2 / 4-1BB multifunctional antibody had strong binding ability to CLDN18.2 on the surface of NUGC44-hi18.2 cells, comparable to the binding abilities of the CLDN18.2 monoclonal antibody 1903 and the control antibody TJ-CD4B, and stronger than the control antibody IMAB362. The CLDN18.2 / 4-1BB multifunctional antibody also had good binding ability to CD16A V176 on the surface of CHO-K1-CD16A V176 cells, stronger than the control antibody IMAB362. The CLDN18.2 / 4-1BB multifunctional antibody also had strong binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, comparable to the binding ability of the 4-1BB monoclonal antibody C5_V2, and slightly stronger than TJ-CD4B.

[0288] [Table 13] [Table 14] [Table 15]

[0289] Example 11. Experiment on the induction of antibody-mediated cell-mediated cell cytotoxicity (ADCC) by CLDN18.2 / 4-1BB multifunctional antibody in vitro A lactate dehydrogenase (LDH) assay was used to evaluate antibody-mediated ADCC activity of NK cells against CLDN18.2-expressing target cells. NUGC4-hi18.2 cells were obtained by stable transfection of NUGC4 cells with a gene expressing human CLDN18.2 protein, and the cell culture medium was RPMI 1640 (Gibco, Cat. #10491A-01) supplemented with 10% inactivated fetal bovine serum and 10 μg / mL puromycin. SNU601 cells were purchased from Nanjing Kebai Biosciences (CBP60507), and the cell culture medium was RPMI 1640 (Gibco, Cat. #10491A-01) supplemented with 10% fetal bovine serum. Cryopreserved PBMCs were isolated from fresh human blood and cultured after resuscitation in RPMI1640 medium (Gibco, Cat#10491A-01) containing 10% fetal bovine serum and incubated overnight at 37°C. The next day, different target cells were digested and resuspended in phenol red-free RPMI1640 medium (Gibco, Cat#11835-030) containing 2% fetal bovine serum at a density of 2 × 10 5 The cells were adjusted to 1 mL / mL, then 50 mL / well of the cells were seeded into a 96-well plate, and 10 x 10 mL of gradient diluted antibody was added. The cells were incubated at 37°C in a 5% CO2 incubator for 0.5 hours.

[0290] PBMCs were collected and resuspended in phenol red-free RPMI 1640 containing 2% fetal bovine serum. The effector / target ratio was adjusted appropriately depending on the target cells, and the cell density was adjusted accordingly. 40 μL / well of the cells were seeded into the experimental plate and incubated for 4 hours in an incubator at 37°C and 5% CO2. The cell culture plate was removed and centrifuged (400 g, 5 minutes) to collect the cell culture supernatant. LDH levels were detected using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Reagent Kit (Promega, G1780). For specific procedures, please refer to the reagent's instruction manual.

[0291] The results are shown in Figures 13A to 13D and Table 16. The CLDN18.2 / 4-1BB multifunctional antibody exhibited excellent ADCC activity against target cells with different levels of CLDN18.2 expression, and was stronger than the control antibody IMAB362. Among these, SC190061 (176F) is the PBMC donor number of the CD16A-F176 mutant, and S2001102 (176V) is the PBMC donor number of the CD16A-V176 mutant.

[0292] [Table 16]

[0293] Example 12. Experiments showing that CLDN18.2 / 4-1BB multifunctional antibody induces antibody-mediated cellular phagocytosis (ADCP) in vitro PBMCs were isolated from fresh human blood and then CD14-positive cells were isolated using human CD14 microbeads (Miltenyi Biotec, 130-050-201). + Monocytes were sorted and these CD14 + Monocytes were cultured in macrophage differentiation medium RPMI 1640 (Gibco, Cat. #10491A-01) containing 50 ng / mL recombinant human macrophage colony-stimulating factor (rhM-CSF, PeproTech, Cat. #300-25) and 10% fetal bovine serum. After 6 days of differentiation, macrophages became adherent and antennal. Macrophages were digested with trypsin for 5 minutes, gently scraped with a scraper, and resuspended in RPMI 1640 medium containing 10% fetal bovine serum at a density of 4 × 10. 5The solution was adjusted to cells / mL. Then, 100 μL / well was seeded into a 96-well plate and incubated overnight at 37°C. The next day, NUGC4-hi18.2 cells were labeled with CellTrace Far Red (Invitrogen, C34564) for 15 minutes at 37°C. After washing twice with PBS, NUGC4-hi18.2 was added to the wells containing macrophages at a ratio of 5 NUGC4-hi18.2 per macrophage at 50 μL / well, and 4 x 50 μL of gradient diluted antibody was added. Target cell phagocytosis was then allowed for 4 hours. After phagocytosis, the cells were washed three times with PBS and then stained with FITC anti-human / mouse CD11b (Tonbo, 35-0112-M100) at a fixed ratio for 30 minutes. After washing twice with PBS, the cells were analyzed by flow cytometry. CD11b-positive cells were gated and stained with Far Red. + / CD11b + Phagocytosis was measured by assessing the percentage of double-positive cells.

[0294] The results are shown in Figure 14 and Table 17, which demonstrate that the CLDN18.2 / 4-1BB multifunctional antibody has good ADCP function, which is stronger than that of the control antibody IMAB362.

[0295] [Table 17]

[0296] Example 13. CLDN18.2-dependent 4-1BB / NF-κB luciferase reporter gene detection experiment of CLDN18.2 / 4-1BB multifunctional antibody The NF-κB luciferase reporter gene experiment described in Example 8 was used to detect CLDN18.2-dependent 4-1BB / NF-κB signal activation.

[0297] The results are shown in Figure 15 and Table 18, demonstrating that the CLDN18.2 / 4-1BB multifunctional antibody has no background activation and its agonistic activity in the CLDN18.2-dependent 4-1BB / NF-κB signaling pathway corresponds to TJ-CD4B.

[0298] [Table 18]

[0299] Example 14. T lymphocyte activation experiment Freshly isolated and purified PBMCs were resuspended in RPMI1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum to a density of 1 × 10 6 NUGC4-hi18.2 cells were obtained by stable transfection of NUGC4 cells with a gene expressing human CLDN18.2 protein. The cell culture medium was RPMI1640 (Gibco, Cat# 10491A-01) containing 10% inactivated fetal bovine serum and 10 μg / mL of puromycin. After resuscitation, the cells were passaged to adjust the cell condition and maintain a density of 1.25 × 10 cells / mL. 6 The concentration of PBMC cells (1 × 10 cells / mL) was adjusted. A 96-well plate was coated with 0.25 μg / mL of anti-CD3 antibody OKT3 (Invitrogen, Cat#16-0037-85) at 100 μL / well and incubated at 37°C for 2 hours. After that, residual antibody was washed off with PBS. Then, 100 μL of PBMC cells (1 × 10 cells / mL) were added per well. 5 cells / well) and 80 μL of medium or NUGC4-hi18.2 cells (1 × 10 5 The cells (cells / well) were seeded into a 96-well plate coated with OKT3, and 20 μL / well of the samples to be measured at 10× different concentrations were added. The plates were then incubated at 37°C in a 5% CO2 incubator for 3 days. The cell culture plates were removed and centrifuged (400 g, 5 minutes) to collect the cell culture supernatant. IL-2 levels were then detected using a human IL-2 detection reagent kit (Cisbio, Cat#62HIL02PEG). For specific procedures, please refer to the instructions for the reagent.

[0300] The results are shown in Figures 16A and 16B and demonstrate that in two PBMC donors, after CLDN18.2 cross-linking, both of these CLDN18.2 / 4-1BB multifunctional antibodies were able to stimulate IL-2 secretion, comparable to the control antibody TJ-CD4B.

[0301] Example 15. Experiment on T cell killing by NK cells PBMCs were isolated from fresh human blood and then purified using EasySep 商標 T cells were selected using a Human T Cell Enrichment Kit (Stemcell, Cat. #17951). T75 culture flasks (Corning, Cat. #430641) were coated with 0.25 μg / mL of the anti-CD3 antibody OKT3 (Invitrogen, Cat. #16-0037-85) and incubated at 37°C for 2 hours, after which residual antibody was washed off with PBS. Freshly isolated T cells were resuspended in RPMI1640 (Gibco, Cat. #10491A-01) containing 10% fetal bovine serum and seeded into OKT3-coated T75 culture flasks at a density of 2 × 10 6 After 48 hours of incubation, activated T cells were labeled with CellTrace Far Red (Invitrogen, C34564) for 15 minutes at 37°C, washed twice with PBS, and then resuspended in phenol red-free RPMI1640 (Gibco, Cat#11835-030) containing 2% fetal bovine serum at a density of 2 × 10 5 After adjusting the concentration to cells / mL, 50 μL / well was seeded into a 96-well plate, and 10×10 μL of gradient diluted antibody was added. The plate was incubated at 37°C in a 5% CO incubator for 0.5 hours.

[0302] Freshly resuscitated PBMCs from the same donor were collected and resuspended in phenol red-free RPMI 1640 containing 2% fetal bovine serum. The cell density was adjusted to an effector / target ratio of 25:1 (PBMC:activated T cells). Fresh PBMCs were seeded into the experimental plates at 40 μL / well and incubated in an incubator at 37°C with 5% CO for 6 hours. After incubation, the cell culture plates were removed and stained with PI (Absin, abs9358) at a fixed ratio for 10 minutes. After washing twice with PBS, the cells were analyzed by flow cytometry. CellTrace Far Red-positive cells were gated and then stained with PI. + / Far Red + The killing effect of NK cells on activated T cells in PBMCs was measured by assessing the percentage of double-positive cells.

[0303] The results are shown in Figure 17 and show that the CLDN18.2x4-1BB bispecific antibody 1903xC5_V2-YTI was not found to induce killing of NK T cells and was comparable to the homozygous control IgG1, while the CLDN18.2x4-1BBxCD16A polyspecific antibody 1903xC5_V2-YTIx34H3 was shown to induce only slight killing of NK T cells.

[0304] Example 16. Evaluation of efficacy, pharmacokinetics and safety 1. In vivo efficacy study of the CLDN18.2 / 4-1BB multifunctional antibody in the murine colon cancer model MC38-hCLDN18.2 MC38-hCLDN18.2 cells (provided by Biocytogen) were cultured at 2 × 10 5 The cells were subcutaneously inoculated into B-h4-1BB humanized mice (provided by Biocytogen) at 100 μL / mouse, and the tumors grew to approximately 102 mm 3Once tumors reached the normal size, 24 mice were randomly assigned based on tumor volume. Six mice per group were assigned to four groups: vehicle, 1903×C5_V2-YTI (0.26 mg / kg), 1903×C5_V2-YTI×34H3 (0.3 mg / kg), and TJ-CD4B (0.3 mg / kg). Tumor volumes were measured twice weekly during the treatment and observation periods and recorded.

[0305] The tumor volume (TV) was calculated using the formula TV = 1 / 2 × a × b 2 where a and b represent the long and short diameters of the measured tumor, respectively.

[0306] Relative tumor growth rate T / C%=(T-T0) / (C-C0)×100%, tumor inhibition rate TGI%=1-T / C%.

[0307] CR% (Complete Tumor Regression Rate) = Complete tumor regression (<102mm 3 ) Number of mice / Number of mice enrolled.

[0308] The results are shown in Figure 18A and Table 19, which demonstrate that the CLDN18.2 / 4-1BB multifunctional antibody has good anti-tumor activity, which is superior to the control antibody TJ-CD4B.

[0309] [Table 19]

[0310] 2. Toxicity detection of CLDN18.2 / 4-1BB multifunctional antibody in the mouse colon cancer model MC38-hCLDN18.2 CLDN18.2 / 4-1BB multifunctional antibody cells (provided by Biocytogen) were added at 2 × 10 5 The cells were subcutaneously inoculated into B-h4-1BB humanized mice (provided by Biocytogen) at 100 μL / mouse, and the tumors were approximately 102 mm 3Once tumors reached the tumor size, 24 mice were randomly assigned to four groups (6 mice per group): vehicle, 1903×C5_V2-YTI (0.26 mg / kg), 1903×C5_V2-YTI×34H3 (0.3 mg / kg), and TJ-CD4B (0.3 mg / kg). Mice were treated every two days. During the treatment and observation periods, mice were weighed and recorded twice weekly. Blood samples were taken on day 17 for AST and ALT measurements.

[0311] The results are shown in Figures 18B and 19. The results showed that the mice's weights remained stable during administration, suggesting that the CLDN18.2 / 4-1BB multifunctional antibody had no obvious toxicity or side effects. ALT / AST detection results on day 17 showed that the CLDN18.2 / 4-1BB multifunctional antibody had no obvious hepatotoxicity at an effective antitumor dose.

[0312] 3. Single-dose pharmacokinetic study of the CLDN18.2 / 4-1BB multifunctional antibody in the murine colon cancer model MC38-hCLDN18.2 5 × 10 MC38-hCLDN18.2 cells 5 The cells were subcutaneously inoculated into B-h4-1BB humanized mice (provided by Biocytogen) at 100 μL / mouse, and the tumors were approximately 110 mm 3 Once tumors reached the tumor size, six mice were randomly assigned to two groups (three mice per group) and administered a single dose of 1903 (10 mg / kg) and 1903 × C5_V2 (12 mg / kg) intravenously.

[0313] Blood samples were collected from the experimental mice at the following time points after administration: 0.25 h, 6 h, 24 h, 72 h, 144 h, 240 h, 336 h, 408 h, and 504 h. Blood concentrations were detected by ELISA. The bottom of the plate was coated with goat anti-human IgG, Fc (Rockland, 609-101-017) and incubated at 4°C for 14–18 h. After washing three times with 300 μL of PBS, a blocking reagent containing 3% BSA was added and incubated at room temperature for 1 h. After washing three times with 300 μL of PBS, serum samples were added and incubated at room temperature for 2 h. After washing three times with 300 μL of PBS, 50 μL of goat anti-human IgG-HRP (BETHYL, A80-304P) solution was added to each well. The plate was sealed with plate sealing film and incubated at room temperature for 1 h. The detection concentration of the secondary antibody was 1:20,000, and the dilution solution was 1% BSA-PBS + 0.5% mouse serum.

[0314] The results are shown in Figure 20 and Table 20. The CLDN18.2 / 4-1BB multifunctional antibody exhibited pharmacokinetic parameters similar to those of monoclonal antibodies, primarily due to antibody distribution mediated by the CLDN18.2 terminus. PK characteristics at equimolar doses were similar to those of the CLDN18.2 parent monoclonal antibody 1903. These results suggest that the blood concentration of the CLDN18.2 / 4-1BB multifunctional antibody in mice was not affected by the 4-1BB terminus and exhibited typical pharmacokinetic characteristics of a CLDN18.2-targeting antibody.

[0315] [Table 20]

Claims

1. 1. A 4-1BB binding protein comprising an immunoglobulin single variable domain, said immunoglobulin single variable domain comprising: comprising CDR1, CDR2, and CDR3 in the amino acid sequence set forth in any one of SEQ ID NOs: 10, 18 to 21, wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system; Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 of said immunoglobulin single variable domains are set forth in SEQ ID NOs: 11, 12, 13, or set forth in SEQ ID NOs: 11, 12, 22, respectively. 4-1BB binding protein.

2. the immunoglobulin single variable domain has been modified by humanization, affinity maturation, removal of T-cell epitopes, reduction of antibody deamidation and / or reduction of antibody isomerization; Preferably, the heavy chain framework regions of the human germline template used in the humanization modification process are IGHV3-64*04, IGHV3-23*03 and / or IGHV3-74*01. The 4-1BB binding protein of claim 1.

3. the amino acid sequence of said immunoglobulin single variable domain is set forth in any one of SEQ ID NOs: 10, 18-21 or has at least 80%, at least 90% sequence identity thereto; Preferably, the 4-1BB binding protein is an anti-4-1BB nanobody or VHH. The 4-1BB binding protein according to claim 1 or 2.

4. further comprising an immunoglobulin Fc region; Preferably, the Fc region is a human IgG1, human IgG2, or human IgG4 Fc region. A 4-1BB binding protein according to any one of claims 1 to 3.

5. 5. The 4-1BB binding protein of any one of claims 1 to 4, comprising an amino acid sequence set forth in SEQ ID NO: 21 or having at least 80%, at least 90% identity thereto.

6. a first antigen-binding domain that specifically binds to 4-1BB; A second antigen-binding domain that specifically binds to CLDN18.2; A CLDN18.2 / 4-1BB binding protein comprising: The first antigen-binding domain comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises: comprising CDR1, CDR2, and CDR3 in the amino acid sequence set forth in any one of SEQ ID NOs: 10, 18 to 21, wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system; Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 of said immunoglobulin single variable domains are set forth in SEQ ID NOs: 11, 12, 13, or set forth in SEQ ID NOs: 11, 12, 22, respectively. CLDN18.2 / 4-1BB binding protein.

7. the immunoglobulin single variable domain has been modified by humanization, affinity maturation, removal of T-cell epitopes, reduction of antibody deamidation and / or reduction of antibody isomerization; Preferably, the heavy chain framework regions of the human germline template used in the humanization modification process are IGHV3-64*04, IGHV3-23*03 and / or IGHV3-74*01. The CLDN18.2 / 4-1BB binding protein of claim 6.

8. The CLDN18.2 / 4-1BB binding protein of claim 6 or 7, wherein the amino acid sequence of the immunoglobulin single variable domain is set forth in any one of SEQ ID NOs: 10, 18-21 or has at least 80%, at least 90% sequence identity thereto.

9. the second antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL); The VH comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 63, The VL comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence represented by SEQ ID NO: 64, the HCDR and LCDR are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems; Preferably, the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 57, 58, and 59, and the VL comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 60, 61, and 62; The CLDN18.2 / 4-1BB binding protein of any one of claims 6 to 8.

10. The VH comprises an amino acid sequence set forth in SEQ ID NO: 63 or having at least 80%, at least 90% identity thereto; The VL comprises an amino acid sequence set forth in SEQ ID NO: 64 or having at least 80%, at least 90% identity thereto; The CLDN18.2 / 4-1BB binding protein of claim 9.

11. a first antigen-binding domain that specifically binds to 4-1BB; A second antigen-binding domain that specifically binds to CLDN18.2; A CLDN18.2 / 4-1BB binding protein comprising: When the second antigen-binding domain does not bind to CLDN18.2, the first antigen-binding domain that specifically binds to 4-1BB does not activate 4-1BB signaling, and the CLDN18.2 / 4-1BB binding protein has an enhanced effector function; Preferably, the CLDN18.2 / 4-1BB binding protein comprises an Fc region for enhancing the effector function of the CLDN18.2 / 4-1BB binding protein, and / or the CLDN18.2 / 4-1BB binding protein comprises a third antigen-binding domain that specifically binds to CD16A, Preferably, said effector function is antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC), Preferably, the first antigen-binding domain and the second antigen-binding domain are the first antigen-binding domain and the second antigen-binding domain defined in any one of claims 6 to 10. CLDN18.2 / 4-1BB binding protein.

12. a first antigen-binding domain that specifically binds to 4-1BB; A second antigen-binding domain that specifically binds to CLDN18.2; a third antigen-binding domain that specifically binds to CD16A; and A CLDN18.2 / 4-1BB binding protein comprising: Preferably, when the second antigen-binding domain that specifically binds to CLDN18.2 does not bind to CLDN18.2, the first antigen-binding domain that specifically binds to 4-1BB cannot activate the 4-1BB signal; Preferably, the first antigen-binding domain and the second antigen-binding domain are the first antigen-binding domain and the second antigen-binding domain defined in any one of claims 6 to 10. CLDN18.2 / 4-1BB binding protein.

13. the third antigen-binding domain comprises an immunoglobulin single variable domain, the immunoglobulin single variable domain comprising: comprising CDR1, CDR2, and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 23, 24, 35 to 43; wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system; Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 of said immunoglobulin single variable domains are set forth in SEQ ID NOs: 25, 26, 27, or set forth in SEQ ID NOs: 28, 29, 30, or set forth in SEQ ID NOs: 28, 44, 30, respectively. The CLDN18.2 / 4-1BB binding protein of claim 12.

14. the immunoglobulin single variable domain in said third antigen-binding domain is modified by humanization, affinity maturation, removal of T-cell epitopes, reduction of antibody deamidation and / or reduction of antibody isomerization; Preferably, the heavy chain framework regions of the human germline template used in the humanization modification process are derived from IGHV3-23*04 or IGHV3-20*04. The CLDN18.2 / 4-1BB binding protein of claim 13.

15. The CLDN18.2 / 4-1BB binding protein of any one of claims 12 to 14, wherein the amino acid sequence of the immunoglobulin single variable domain in the third antigen-binding domain is set forth in any one of SEQ ID NOs: 23, 24, 35 to 43, or has at least 80%, at least 90% sequence identity thereto.

16. further comprising an Fc region of an immunoglobulin; Preferably, the Fc region is a human IgG1, human IgG2, or human IgG4 Fc region; More preferably, the Fc region is an effector-enhanced Fc region. The CLDN18.2 / 4-1BB binding protein of any one of claims 6 to 15.

17. When the Fc region is an Fc region of human IgG1, the Fc region can be any of 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, 280H / 290S / 298V, 243 and any one or any combination of the amino acid mutations L / 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, wherein said mutations are defined according to the EU numbering system; Preferably, the Fc region comprises 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, D280H / 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 thereof; The CLDN18.2 / 4-1BB binding protein of claim 16.

18. further comprising a linker; Preferably, the amino acid sequence of the linker is (G ｍ S ｎ ) ｈ or (GGNGT) ｈ Or (YGNGT) ｈ or (EPKSS) ｈ wherein m and n are each independently selected from integers of 1 to 8, and h is independently selected from integers of 1 to 20; More preferably, the linker is (G ４ S) ２ , (G ４ S) ３ is a linker represented by The CLDN18.2 / 4-1BB binding protein of any one of claims 6 to 17.

19. the second antigen-binding domain comprises a heavy chain and a light chain; the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 45 or has at least 80%, at least 90% sequence identity thereto; The amino acid sequence of the light chain is set forth in SEQ ID NO: 46 or has at least 80%, at least 90% sequence identity thereto; The CLDN18.2 / 4-1BB binding protein of any one of claims 6 to 18.

20. (1) The amino acid sequence of the first polypeptide chain is set forth in any one of SEQ ID NOs: 47 to 49 or has at least 80%, at least 90%, sequence identity thereto; the amino acid sequence of the second polypeptide chain is set forth in SEQ ID NO: 46 or has at least 80%, at least 90% sequence identity thereto; (2) The amino acid sequence of the first polypeptide chain is set forth in any one of SEQ ID NOs: 50, 52, and 53, or has at least 80%, at least 90%, sequence identity thereto; The amino acid sequence of the second polypeptide chain is set forth in SEQ ID NO: 51 or 54 or has at least 80%, at least 90% sequence identity thereto; The polypeptide comprises a first polypeptide chain and a second polypeptide chain selected from 20. The CLDN18.2 / 4-1BB binding protein of any one of claims 6 to 19.

21. 1. A CD16A binding protein comprising an immunoglobulin single variable domain, said immunoglobulin single variable domain comprising: comprising CDR1, CDR2, and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 23, 24, 35 to 43; wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system; Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 of said immunoglobulin single variable domains are set forth in SEQ ID NOs: 25, 26, 27, or set forth in SEQ ID NOs: 28, 29, 30, or set forth in SEQ ID NOs: 28, 44, 30, respectively. CD16A binding protein.

22. the immunoglobulin single variable domain has been modified by humanization, affinity maturation, removal of T-cell epitopes, reduction of antibody deamidation and / or reduction of antibody isomerization; Preferably, the heavy chain framework regions of the human germline template used in the humanization modification process are derived from IGHV3-23*04 or IGHV3-20*04.

22. The CD16A binding protein of claim 21.

23. the amino acid sequence of said immunoglobulin single variable domain is set forth in any one of SEQ ID NOs: 23, 24, 35-43 or has at least 80%, at least 90% sequence identity thereto; Preferably, the CD16A binding protein is an anti-CD16A nanobody or VHH. A CD16A binding protein according to claim 21 or 22.

24. further comprising a human immunoglobulin Fc region; Preferably, the Fc region is a human IgG1, human IgG2, or human IgG4 Fc region. A CD16A binding protein described in any one of claims 21 to 23.

25. A CD16A binding protein according to any one of claims 21 to 24, which does not specifically bind to CD16B.

26. A polynucleotide encoding a CLDN18.2 / 4-1BB binding protein according to any one of claims 6 to 20, a 4-1BB binding protein according to any one of claims 1 to 5, or a CD16A binding protein according to any one of claims 21 to 25, Preferably, the polynucleotide is DNA or RNA. Polynucleotide.

27. A vector comprising the polynucleotide of claim 26.

28. 28. A host cell containing or expressing a polynucleotide according to claim 26 or a vector according to claim 27.

29. A method for preparing a CLDN18.2 / 4-1BB binding protein, a 4-1BB binding protein, or a CD16A binding protein, comprising: Expressing the polynucleotide of claim 26 or the vector of claim 27 in a host cell of claim 28, and isolating the expressed CLDN18.2 / 4-1BB binding protein, 4-1BB binding protein, or CD16A binding protein from the host cell; Optionally, further comprising purifying the CLDN18.2 / 4-1BB binding protein, 4-1BB binding protein, or CD16A binding protein; method.

30. A pharmaceutical composition comprising a CLDN18.2 / 4-1BB binding protein according to any one of claims 6 to 20, a 4-1BB binding protein according to any one of claims 1 to 5, a CD16A binding protein according to any one of claims 21 to 25, and at least one medicament excipient, diluent or carrier.

31. A method for treating cancer, comprising the steps of (1) or (2) below: (1) Administering to a subject in need thereof a therapeutically effective amount of a CLDN18.2 / 4-1BB binding protein according to any one of claims 6 to 20, or a pharmaceutical composition according to claim 30, wherein the cancer is preferably CLDN18.2 positive; or (2) administering to a subject in need thereof a therapeutically effective amount of a 4-1BB binding protein according to any one of claims 1 to 5, a CD16A binding protein according to any one of claims 21 to 25, a polynucleotide according to claim 26, a vector according to claim 27, or a pharmaceutical composition according to claim 30; A method comprising:

32. 32. The method of claim 31, wherein the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, renal cancer, squamous cell carcinoma, blood cancer, or any combination thereof.

33. Use as shown in (1) or (2) below: (1) Use of the CLDN18.2 / 4-1BB binding protein according to any one of claims 6 to 20, the polynucleotide according to claim 26, or the vector according to claim 27 for the preparation of a drug for treating cancer, preferably wherein the cancer is CLDN18.2-positive; or (2) Use of a 4-1BB binding protein described in any one of claims 1 to 5 or a CD16A binding protein described in any one of claims 21 to 25, a polynucleotide described in claim 26, or a vector described in claim 27 for use in the preparation of a drug for treating cancer.

34. 34. The use of claim 33, wherein the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, renal cancer, squamous cell carcinoma, blood cancer, or any combination thereof.

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