Methods of treating solid tumors

Abstract

Provided herein is a method of treating a solid tumor. In particular, provided herein is a method of treating solid tumors, such as advanced and / or metastatic solid tumors, by using bispecific antibodies.

Description

Technical Field

[0001] The present application relates to a method for treating solid tumors. In particular, the present application provides a method for treating solid tumors (such as advanced and / or metastatic solid tumors) by using a bispecific antibody. Background Art

[0002] Claudin is a family of proteins that form important components of tight cell junctions. Claudin-18 splice variant 2 (CLDN18.2) is a gastric-specific membrane protein. In healthy tissue, CLDN18.2, as a component of tight junctions, is restrictedly expressed in short-lived differentiated cells of the gastric mucosa, with limited access to antibody therapy. However, it is ectopically expressed at significant levels in primary lesions and metastases of various epithelial tumor entities, including gastric, pancreatic, esophageal, and lung adenocarcinoma cells.

[0003] CLDN 18.2 is considered a therapeutic target with great potential for gastric and other types of solid tumors, providing a new option for cancer treatment. Summary of the invention

[0004] The present application addresses clinical needs through bispecific antibodies targeting CLDN 18.2.

[0005] In one aspect, provided herein is a method for treating a solid tumor, comprising administering to a subject in need thereof a bispecific antibody, the bispecific antibody comprising: (1) an anti-claudin 18.2 (CLDN18.2) unit having binding specificity to a CLDN18.2 protein; and (2) a second antibody unit, wherein the bispecific antibody is administered to the subject at a dose of about 0.1 to about 30 mg / kg body weight.

[0006] In some embodiments, the second antibody unit has a binding specificity to a target selected from 4-1BB, PD-1, PD-L1, and CD3. In some embodiments, the second antibody comprises an anti-4-1BB unit having a binding specificity to a 4-1BB protein.

[0007] In some embodiments, the bispecific antibody is administered to a subject at a dose of about 0.1-about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to a subject at a dose of about 0.3-about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to a subject at a dose of about 1-about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to a subject at a dose of about 3-about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to a subject at a dose of about 5-about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to a subject at a dose of about 8-about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to a subject at a dose of about 12-about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to a subject at a dose of about 8-about 12 mg / kg body weight. In some embodiments, the bispecific antibody is administered to a subject at a dose of about 0.1, about 0.3, about 1, about 5, about 8, about 12, about 15 or about 30 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 5, about 8, about 12, or about 15 mg / kg body weight.

[0008] In some embodiments, the bispecific antibody is administered to the subject weekly, every two weeks, every three weeks, or monthly. In some embodiments, the bispecific antibody is administered to the subject every two weeks.

[0009] In some embodiments, the bispecific antibody is administered intravenously to the subject.

[0010] In some embodiments, the anti-CLDN18.2 unit is selected from the group consisting of a full-length antibody, Fab, Fab', F(ab')2, scFv, and sdAb. In some embodiments, the anti-CLDN18.2 unit comprises a Fab.

[0011] In some embodiments, the anti-CLDN18.2 unit comprises HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region (VH) shown in SEQ ID NO.1. In some embodiments, the anti-CLDN18.2 unit comprises: (1) HC-CDR1, which comprises the amino acid sequence shown in SEQ ID No.3 or an amino acid sequence having one or more substitutions compared to SEQ ID No.3, (2) HC-CDR2, which comprises the amino acid sequence shown in SEQ ID No.4 or an amino acid sequence having one or more substitutions compared to SEQ ID No.4, and (3) HC-CDR3, which comprises the amino acid sequence shown in SEQ ID No.5 or an amino acid sequence having one or more substitutions compared to SEQ ID No.5. In some embodiments, the anti-CLDN18.2 unit comprises: a heavy chain variable region (VH), which comprises the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 90% identity to SEQ ID NO.1.

[0012] In some embodiments, the anti-CLDN18.2 unit comprises LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region (VL) shown in SEQ ID NO.2. In some embodiments, the anti-CLDN18.2 unit comprises: (1) LC-CDR1, which comprises the amino acid sequence shown in SEQ ID No.6 or an amino acid sequence having one or more substitutions compared to SEQ ID No.6, (2) LC-CDR2, which comprises the amino acid sequence shown in SEQ ID No.7 or an amino acid sequence having one or more substitutions compared to SEQ ID No.7, and (3) LC-CDR3, which comprises the amino acid sequence shown in SEQ ID No.8 or an amino acid sequence having one or more substitutions compared to SEQ ID No.8. In some embodiments, the anti-CLDN18.2 unit comprises a light chain variable region (VL), which comprises the amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence having at least 90% identity to SEQ ID NO.2.

[0013] In some embodiments, the anti-CLDN18.2 unit comprises: (1) HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region (VH) shown in SEQ ID NO.1; and (2) LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region (VL) shown in SEQ ID NO.2. In some embodiments, the anti-CLDN18.2 unit comprises: (1) HC-CDR1, which comprises the amino acid sequence shown in SEQ ID No.3 or an amino acid sequence having one or more substitutions compared to SEQ ID No.3, (2) HC-CDR2, which comprises the amino acid sequence shown in SEQ ID No.4 or an amino acid sequence having one or more substitutions compared to SEQ ID No.4, (3) HC-CDR3, which comprises the amino acid sequence shown in SEQ ID No.5 or an amino acid sequence having one or more substitutions compared to SEQ ID No.5, (4) LC-CDR1, which comprises the amino acid sequence shown in SEQ ID No.6 or an amino acid sequence having one or more substitutions compared to SEQ ID No.6, (5) LC-CDR2, which comprises the amino acid sequence shown in SEQ ID No.7 or an amino acid sequence having one or more substitutions compared to SEQ ID No.7, and (6) LC-CDR3, which comprises the amino acid sequence shown in SEQ ID No.8 or an amino acid sequence having one or more substitutions compared to SEQ ID No.8.

[0014] In some embodiments, the anti-CLDN18.2 unit comprises: (1) a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 90% identity with SEQ ID NO.1, and (2) a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence having at least 90% identity with SEQ ID NO.2.

[0015] In some embodiments, the anti-4-1BB unit is selected from a full-length antibody, Fab, Fab', F(ab')2, scFv, and sdAb. In some embodiments, the anti-4-1BB unit comprises a scFv.

[0016] In some embodiments, the anti-4-1BB unit comprises HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region (VH) shown in SEQ ID NO.9. In some embodiments, the anti-4-1BB unit comprises: (1) HC-CDR1, which comprises the amino acid sequence shown in SEQ ID No.11 or an amino acid sequence having one or more substitutions compared to SEQ ID No.11, (2) HC-CDR2, which comprises the amino acid sequence shown in SEQ ID No.12 or an amino acid sequence having one or more substitutions compared to SEQ ID No.12, and (3) HC-CDR3, which comprises the amino acid sequence shown in SEQ ID No.13 or an amino acid sequence having one or more substitutions compared to SEQ ID No.13. In some embodiments, the anti-4-1BB unit comprises a heavy chain variable region (VH), which comprises the amino acid sequence shown in SEQ ID NO.9 or an amino acid sequence having at least 90% identity to SEQ ID NO.9.

[0017] In some embodiments, the anti-4-1BB unit comprises LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region (VL) shown in SEQ ID NO. 10. In some embodiments, the anti-4-1BB unit comprises: (1) LC-CDR1, which comprises the amino acid sequence shown in SEQ ID No. 14 or an amino acid sequence having one or more substitutions compared to SEQ ID No. 14, (2) LC-CDR2, which comprises the amino acid sequence shown in SEQ ID No. 15 or an amino acid sequence having one or more substitutions compared to SEQ ID No. 15, and (3) LC-CDR3, which comprises the amino acid sequence shown in SEQ ID No. 16 or an amino acid sequence having one or more substitutions compared to SEQ ID No. 16. In some embodiments, the anti-4-1BB unit comprises a light chain variable region (VL), which comprises the amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence having at least 90% identity to SEQ ID NO. 10.

[0018] In some embodiments, the anti-4-1BB unit comprises: (1) HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region (VH) shown in SEQ ID NO.9, and (2) LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region (VL) shown in SEQ ID NO.10. In some embodiments, the anti-4-1BB unit comprises: (1) HC-CDR1, which comprises the amino acid sequence shown in SEQ ID No.11 or an amino acid sequence having one or more substitutions compared to SEQ ID No.11, (2) HC-CDR2, which comprises the amino acid sequence shown in SEQ ID No.12 or an amino acid sequence having one or more substitutions compared to SEQ ID No.12, (3) HC-CDR3, which comprises the amino acid sequence shown in SEQ ID No.13 or an amino acid sequence having one or more substitutions compared to SEQ ID No.13, (4) LC-CDR1, which comprises the amino acid sequence shown in SEQ ID No.14 or an amino acid sequence having one or more substitutions compared to SEQ ID No.14, (5) LC-CDR2, which comprises the amino acid sequence shown in SEQ ID No.15 or an amino acid sequence having one or more substitutions compared to SEQ ID No.15, and (6) LC-CDR3, which comprises the amino acid sequence shown in SEQ ID No.16 or an amino acid sequence having one or more substitutions compared to SEQ ID No.16.

[0019] In some embodiments, the anti-4-1BB unit comprises: (1) a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO.9 or an amino acid sequence that is at least 90% identical to SEQ ID NO.9, and (2) a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO.10 or an amino acid sequence that is at least 90% identical to SEQ ID NO.10.

[0020] In some embodiments, the bispecific antibody comprises a heavy chain component comprising an amino acid sequence as set forth in SEQ ID NO. 17 or an amino acid sequence having at least 90% identity to SEQ ID NO. 17. In some embodiments, the bispecific antibody comprises a light chain component comprising an amino acid sequence as set forth in SEQ ID NO. 18 or an amino acid sequence having at least 90% identity to SEQ ID NO. 18. In some embodiments, the bispecific antibody comprises: (1) a heavy chain component comprising an amino acid sequence as set forth in SEQ ID NO. 17 or an amino acid sequence having at least 90% identity to SEQ ID NO. 17, and (2) a light chain component comprising an amino acid sequence as set forth in SEQ ID NO. 18 or an amino acid sequence having at least 90% identity to SEQ ID NO. 18.

[0021] In some embodiments, the bispecific antibody is selected from TJ001, IBI389 (Innovent), Q-1802 (QureBio), AMG-910 (Amgen), QLS31905 (Qilu Pharma), PM1032 (Biotheus), and HBM7022 (Harbour, AZ).

[0022] In some embodiments, the solid tumor overexpresses CLDN 18.2. In some embodiments, the solid tumor is selected from colorectal cancer, urogenital tract cancer, sarcoma, melanoma, hepatocellular carcinoma, gastric cancer, esophageal cancer, gastroesophageal cancer, esophageal cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, lung cancer, non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, Krukenberg tumor and lymphoma. In some embodiments, the solid tumor is an advanced solid tumor. In some embodiments, the solid tumor is a metastatic solid tumor. In some embodiments, the solid tumor is an advanced and metastatic solid tumor.

[0023] Use of a bispecific antibody in the preparation of a medicament for treating a solid tumor in a subject in need thereof, wherein the bispecific antibody comprises: (1) an anti-claudin 1 8.2 (CLDN1 8.2) unit having binding specificity to CLDN1 8.2 protein; and (2) an anti-4-1BB unit having binding specificity to 4-1BB protein, wherein the bispecific antibody is administered to the subject at a dose of about 0.1 to about 30 mg / kg body weight.

[0024] A product comprising: (1) a bispecific antibody comprising an anti-claudin 18.2 (CLDN18.2) unit having binding specificity to a CLDN18.2 protein; and an anti-4-1BB unit having binding specificity to a 4-1BB protein, and (2) a package insert indicating administration of the bispecific antibody to a subject in need thereof at a dose of about 0.1 to about 30 mg / kg body weight.

[0025] It should be understood that one, some or all of the features in the various embodiments described herein can be combined to form other embodiments of the present invention. For those skilled in the art, these and other aspects of the present invention will become apparent. These and other embodiments of the present invention are further described by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A -C is a microscopic image of three MKN45 gastric cancer cell lines expressing CLDN18.2 (respectively, "MKN45 parental", "MKN45#18" and "MKN45#14") stained with IHC membrane.

[0027] Figure 2A -C displays in Figure 1A - IHC scores of three CLDN18.2 immunohistochemical profiles of three MKN45 cell lines shown in C.

[0028] Figure 3A -B shows the IFN-γ levels at different TJ001 antibody concentrations of E:T=1:10 and E:T=1:1, respectively.

[0029] Figure 4 The pharmacokinetic profiles of mean serum concentrations of TJ001 antibody at different doses every two weeks are shown.

[0030] Figure 5A Peripheral soluble 4-1BB pharmacodynamic data for each visit at different doses are shown.

[0031] Figure 5B -C shows the peripheral soluble 4-1BB levels of C1D8 and C1D15, respectively. DETAILED DESCRIPTION

[0032] definition

[0033] Before describing the embodiments in detail, it is to be understood that this disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0034] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and so forth.

[0035] The term "about" as used herein refers to the typical error range for each numerical value that is readily known to those skilled in the art. References herein to "about" a value or parameter include (and describe) embodiments directed to that value or parameter per se.

[0036] It should be understood that various aspects and embodiments of the present disclosure include "comprising," "consisting of," and "consisting essentially of," various aspects and embodiments.

[0037] As used herein, the term "antibody" is used in the broadest sense and specifically covers intact antibodies (e.g., full-length antibodies), antibody fragments (including but not limited to Fab, F(ab')2, scFv, scFv-Fc, single domain antibodies, single heavy chain antibodies, and single light chain antibodies), monoclonal antibodies, and polyclonal antibodies, so long as they exhibit the desired biological activity (e.g., epitope binding).

[0038] As used herein, the term "isolated" antibody may refer to an antibody that is substantially free of other cellular material. In one embodiment, the isolated antibody is substantially free of other proteins from the same species. In another embodiment, the isolated antibody is expressed by cells from different species and is substantially free of other proteins from different species. In some embodiments, an "isolated" antibody is an antibody that has been identified and separated and / or recovered from components of its natural environment. The contaminating components of its natural environment are substances that interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteins or non-protein solutes. By separating using protein purification techniques known in the art, the antibody can be substantially free of naturally associated components (or components associated with the cell expression system used to produce the antibody). In some embodiments, the antibody is purified (1) to greater than 75% by weight of the antibody as determined by the Lowry method, most preferably greater than 80%, 90%, 95% or 99% by weight, or (2) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0039] As used herein, the terms "natural antibodies and immunoglobulins" are typically heterotetrameric glycoproteins of about 150,000 daltons consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain (also referred to as a "VH / VL pair") by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain. See, e.g., Chothia et al., J. Mol. Biol., 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA, 82:4592 (1985).

[0040] As used herein, the term "variable" refers to the fact that the sequence of certain parts of the variable domain varies greatly between antibodies, and is used for the binding and specificity of each specific antibody to its specific antigen. However, variability is unevenly distributed throughout the variable domain of the antibody. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the light chain variable domain and the heavy chain variable domain. The more highly conserved parts of the variable domain are called frameworks (FRs). The variable domains of natural heavy and light chains each contain four FR regions, mostly using a β-folded configuration connected by three CDRs, which form a loop connection and form a part of the β-folded structure in some cases. The CDRs in each chain are kept together very closely by the FR region, and together with the CDRs from the other chain, contribute to the formation of the antigen binding site of the antibody. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991). The constant domains are not involved directly in binding the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity. Variable region sequences of interest include humanized variable region sequences of the CD47 antibody described in detail elsewhere herein.

[0041] The term "hypervariable region (HVR)" or "complementarity determining region (CDR)" may refer to subregions of the VH and VL domains that are characterized by enhanced sequence variability and / or the formation of defined loops. These include three CDRs (H1, H2, and H3) in the VH domain and three CDRs (L1, L2, and L3) in the VL domain. H3 is believed to be crucial in conferring fine binding specificity, with L3 and H3 exhibiting the highest levels of diversity. See Johnson and Wu, in Methods in Molecular Biology 248: 1-25 (Lo, ed., Human Press, Totowa, NJ, 2003).

[0042] The delimitation of several CDR / HVR is known. Kabat complementarity determining region (CDR) is based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers to the position of the structural loop (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). AbMHVR represents a compromise between Kabat HVR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software. "Contact" HVR is based on the analysis of available complex crystal structures. The residues from each of these HVR / CDRs are indicated below. "Framework" or "FR" residues are those variable domain residues other than HVR / CDR residues.

[0043]

[0044] "Extended" HVRs are also known: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH (Kabat numbering).

[0045] "Numbering according to Kabat" may refer to the numbering system of the heavy chain variable domain or light chain variable domain compiled by Kabat et al. above for antibodies. The actual linear amino acid sequence may contain a shortening of the FR or HVR corresponding to the variable domain or fewer or additional amino acids inserted therein. For a given antibody, the Kabat numbering of the residue can be determined by comparing the homology region of the antibody sequence with the "standard" Kabat numbering sequence. Typically, Kabat numbering is used when referring to the residues of the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), and the EU numbering system or index (e.g., as the EU index in Kabat, according to the numbering of EU IgG1) is generally used when referring to the residues in the heavy chain constant region.

[0046] As used herein, the term "antibody fragment" and all grammatical variations thereof are defined as a portion of an intact antibody that contains the antigen binding site or variable region of the intact antibody, in some cases, the portion does not contain the constant heavy chain domains (i.e., CH2, CH3 and / or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of an uninterrupted sequence of consecutive amino acid residues (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide"), including but not limited to (1) single-chain Fv (scFv) molecules, (2) single-chain polypeptides containing only one light chain variable domain or fragments thereof containing the three CDRs of the light chain variable domain without the associated heavy chain portion, and (3) single-chain polypeptides containing only one heavy chain variable region or fragments thereof containing the three CDRs of the heavy chain variable region without the associated light chain portion; and multispecific or multivalent structures formed by antibody fragments. In antibody fragments comprising one or more heavy chains, the heavy chain may contain any constant domain sequence found in the non-Fc region of an intact antibody (e.g., CH1 in IgG isotypes), and / or may contain any hinge region sequence found in an intact antibody, and / or may contain a leucine zipper sequence fused to or located within the hinge region sequence or constant domain sequence of a heavy chain.

[0047] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is herein the designation for Fab' in which the cysteine ​​residues of the constant domains carry free sulfhydryl groups. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0048] As used herein, the term "monoclonal antibody" (mAb) refers to an antibody obtained from a population of substantially homogeneous antibodies (i.e., the individual antibodies constituting the population are identical except for a small amount of mutations that may occur naturally). Monoclonal antibodies are highly specific and are directed against a single antigenic site. Each mAb is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without being contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the characteristic of the antibody is obtained from a population of substantially homogeneous antibodies, and should not be construed as requiring the production of antibodies by any ad hoc method. For example, the monoclonal antibodies used according to the present invention can be prepared in immortalized B cells or their hybridomas, or can be prepared by recombinant DNA methods.

[0049] The monoclonal antibodies herein include hybrid and recombinant antibodies produced by splicing the variable (including hypervariable) domains of CD47 antibodies with constant domains (e.g., "humanized" antibodies), or light chains with heavy chains, or chains from one species with chains from another species, or fusions with heterologous proteins, regardless of the species of origin or immunoglobulin class or subclass designation, as well as antibody fragments (e.g., Fab, F(ab')2 and Fv), so long as they exhibit the desired biological activity.

[0050] The monoclonal antibodies herein specifically include chimeric antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass; and fragments of such antibodies, as long as they exhibit the desired biological activity.

[0051] As used herein, the term "treatment" refers to a clinical intervention intended to change the natural course of an individual or cell being treated during clinical pathology. The desired effects of treatment include reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. For example, if one or more symptoms associated with cancer are alleviated or eliminated, including but not limited to reducing (or destroying) the proliferation of cancer cells, reducing the symptoms caused by the disease, improving the quality of life of patients with the disease, reducing the dosage of other drugs required for treating the disease, and / or extending the survival period of the individual, the individual is successfully "treated". In some embodiments, "treating" a disease such as cancer refers to delaying the progression of the disease, that is, postponing, hindering, slowing down, blocking, stabilizing and / or delaying the development of a disease (such as cancer). This delay can be different lengths of time, depending on the history of the disease and / or the individual being treated. It is obvious to those skilled in the art that sufficient or significant delays can actually cover prevention, that is, the individual does not develop the disease. For example, advanced cancer, such as the development of metastasis, can be delayed.

[0052] As used herein, for the purposes of treatment, the term "subject" refers to any animal classified as a mammal, including humans, livestock and farm animals, and zoo animals, sport animals, or pet animals, such as dogs.

[0053] As used herein, the term "advanced solid tumor" refers to a solid tumor that is incurable or has grown beyond the initial site of origin, is locally advanced or is metastatic. In some embodiments, advanced solid tumors include but are not limited to solid tumors in stage III or stage IV.

[0054] As used herein, the term "metastatic" or "metastasis" refers to the spread of a tumor from an initial or primary site to a different or secondary site in a subject's body. It is generally distinguished from cancer invasion, which is the direct extension and penetration of cancer cells into adjacent tissues.

[0055] Anti-CLDN 18.2 Unit

[0056] Claudin-18 has two isoforms, isoform 1 and isoform 2. Isoform 2 (Claudin 18.2 or CLDN18.2) is a highly selective cell lineage marker. In normal tissues, CLDN 18.2 is strictly expressed in differentiated epithelial cells of the gastric mucosa. However, CLDN 18.2 was found to be significantly expressed in primary and metastatic gastric cancer tissues, as well as pancreatic cancer, esophageal cancer, ovarian cancer, and lung cancer tissues, indicating that CLDN18.2 is an attractive therapeutic target with great potential in gastric and other types of solid tumors.

[0057] Any anti-18.2 known in the art can be used for the bispecific antibodies of the present application. In some embodiments, the anti-CLDN18.2 unit is selected from a full-length antibody, Fab, Fab', F(ab')2, scFv, and sdAb. In some embodiments, the anti-CLDN18.2 unit comprises Fab.

[0058] In some embodiments, the anti-CLDN18.2 unit comprises HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region (VH) shown in SEQ ID NO. 1. In some embodiments, the anti-CLDN18.2 unit comprises: (1) HC-CDR1, which comprises the amino acid sequence shown in SEQ ID No. 3 or an amino acid sequence having one or more substitutions compared to SEQ ID No. 3, (2) HC-CDR2, which comprises the amino acid sequence shown in SEQ ID No. 4 or an amino acid sequence having one or more substitutions compared to SEQ ID No. 4, and (3) HC-CDR3, which comprises the amino acid sequence shown in SEQ ID No. 5 or an amino acid sequence having one or more substitutions compared to SEQ ID No. 5.

[0059] In some embodiments, the anti-CLDN18.2 unit comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO.1.

[0060] In some embodiments, the anti-CLDN18.2 unit comprises LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region (VL) shown in SEQ ID NO. 2. In some embodiments, the anti-CLDN18.2 unit comprises: (1) LC-CDR1, which comprises the amino acid sequence shown in SEQ ID No. 6 or an amino acid sequence having one or more substitutions compared to SEQ ID No. 6, (2) LC-CDR2, which comprises the amino acid sequence shown in SEQ ID No. 7 or an amino acid sequence having one or more substitutions compared to SEQ ID No. 7, and (3) LC-CDR3, which comprises the amino acid sequence shown in SEQ ID No. 8 or an amino acid sequence having one or more substitutions compared to SEQ ID No. 8.

[0061] In some embodiments, the anti-CLDN18.2 unit comprises a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence that is at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO.2.

[0062] In some embodiments, the anti-CLDN18.2 unit comprises: (1) HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region (VH) shown in SEQ ID NO.1; and (2) LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region (VL) shown in SEQ ID NO.2. In some embodiments, the anti-CLDN18.2 unit comprises: (1) HC-CDR1, which comprises the amino acid sequence shown in SEQ ID No.3 or an amino acid sequence having one or more substitutions compared to SEQ ID No.3, (2) HC-CDR2, which comprises the amino acid sequence shown in SEQ ID No.4 or an amino acid sequence having one or more substitutions compared to SEQ ID No.4, (3) HC-CDR3, which comprises the amino acid sequence shown in SEQ ID No.5 or an amino acid sequence having one or more substitutions compared to SEQ ID No.5, (4) LC-CDR1, which comprises the amino acid sequence shown in SEQ ID No.6 or an amino acid sequence having one or more substitutions compared to SEQ ID No.6, (5) LC-CDR2, which comprises the amino acid sequence shown in SEQ ID No.7 or an amino acid sequence having one or more substitutions compared to SEQ ID No.7, and (6) LC-CDR3, which comprises the amino acid sequence shown in SEQ ID No.8 or an amino acid sequence having one or more substitutions compared to SEQ ID No.8.

[0063] In some embodiments, the anti-CLDN18.2 unit comprises: (1) a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO.1, and (2) a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO.2.

[0064] Anti-4-1BB Unit

[0065] 4-1BB is an inducible co-stimulatory receptor expressed on activated T cells and natural killer (NK) cells. 4-1BB trimers are clustered by 4-1BB ligand (41BBL) trimers on T cells, triggering a signaling cascade that leads to upregulation of anti-apoptotic molecules, cytokine secretion, and enhanced effector function. On NK cells, 4-1BB signaling can increase antibody-dependent cell-mediated cytotoxicity. Agonistic monoclonal antibodies targeting 4-1BB have been developed to use 4-1BB signaling for cancer immunotherapy. Preclinical results from various induced and spontaneous tumor models have shown that targeting 4-1BB with agonist antibodies can lead to tumor clearance and durable anti-tumor immunity.

[0066] Any 4-1BB antibody known in the art can be used for the bispecific antibody of the present application. In some embodiments, the anti-4-1BB unit is selected from a full-length antibody, Fab, Fab', F(ab')2, scFv and sdAb. In some embodiments, the anti-4-1BB unit comprises scFv.

[0067] In some embodiments, the anti-4-1BB unit comprises HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region (VH) shown in SEQ ID NO. 9. In some embodiments, the anti-4-1BB unit comprises: (1) HC-CDR1, which comprises the amino acid sequence shown in SEQ ID No. 11 or an amino acid sequence having one or more substitutions compared to SEQ ID No. 11, (2) HC-CDR2, which comprises the amino acid sequence shown in SEQ ID No. 12 or an amino acid sequence having one or more substitutions compared to SEQ ID No. 12, and (3) HC-CDR3, which comprises the amino acid sequence shown in SEQ ID No. 13 or an amino acid sequence having one or more substitutions compared to SEQ ID No. 13.

[0068] In some embodiments, the anti-4-1BB unit comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO.9 or an amino acid sequence that is at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO.9.

[0069] In some embodiments, the anti-4-1BB unit comprises LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region (VL) shown in SEQ ID NO. 10. In some embodiments, the anti-4-1BB unit comprises: (1) LC-CDR1, which comprises the amino acid sequence shown in SEQ ID No. 14 or an amino acid sequence having one or more substitutions compared to SEQ ID No. 14, (2) LC-CDR2, which comprises the amino acid sequence shown in SEQ ID No. 15 or an amino acid sequence having one or more substitutions compared to SEQ ID No. 15, and (3) LC-CDR3, which comprises the amino acid sequence shown in SEQ ID No. 16 or an amino acid sequence having one or more substitutions compared to SEQ ID No. 16.

[0070] In some embodiments, the anti-4-1BB unit comprises a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO.10 or an amino acid sequence that is at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO.10.

[0071] In some embodiments, the anti-4-1BB unit comprises: (1) HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region (VH) shown in SEQ ID NO.9, and (2) LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region (VL) shown in SEQ ID NO.10. In some embodiments, the anti-4-1BB unit comprises: (1) HC-CDR1, which comprises the amino acid sequence shown in SEQ ID No.11 or an amino acid sequence having one or more substitutions compared to SEQ ID No.11, (2) HC-CDR2, which comprises the amino acid sequence shown in SEQ ID No.12 or an amino acid sequence having one or more substitutions compared to SEQ ID No.12, (3) HC-CDR3, which comprises the amino acid sequence shown in SEQ ID No.13 or an amino acid sequence having one or more substitutions compared to SEQ ID No.13, (4) LC-CDR1, which comprises the amino acid sequence shown in SEQ ID No.14 or an amino acid sequence having one or more substitutions compared to SEQ ID No.14, (5) LC-CDR2, which comprises the amino acid sequence shown in SEQ ID No.15 or an amino acid sequence having one or more substitutions compared to SEQ ID No.15, and (6) LC-CDR3, which comprises the amino acid sequence shown in SEQ ID No.16 or an amino acid sequence having one or more substitutions compared to SEQ ID No.16.

[0072] In some embodiments, the anti-4-1BB unit comprises: (1) a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO.9 or an amino acid sequence that is at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO.9, and (2) a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO.10 or an amino acid sequence that is at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO.10.

[0073] Bispecific Antibodies

[0074] As used herein, the term "bispecific antibody" refers to an antibody having two antigen-binding regions or antibody units, which have binding specificities to two different antigens or two epitopes.

[0075] In some embodiments, the bispecific antibody of the present application comprises: (1) an anti-tight junction protein 18.2 (CLDN18.2) unit having binding specificity to the CLDN18.2 protein; and (2) a second antibody unit. In some embodiments, the anti-CLDN 18.2 unit can be any anti-CLDN 18.2 unit as described herein. In some embodiments, the second antibody unit has binding specificity to a target selected from 4-1BB, PD-1, PD-L1, and CD3. In some embodiments, the second antibody comprises an anti-4-1BB unit, which has binding specificity to the 4-1BB protein. In some embodiments, the second antibody comprises an anti-4-1BB unit as described herein.

[0076] In some embodiments, the bispecific antibody of the present application comprises: (1) an anti-tight junction protein 18.2 (CLDN18.2) unit having binding specificity to the CLDN18.2 protein; and (2) an anti-4-1BB unit having binding specificity to the 4-1BB protein. In some embodiments, the bispecific antibody of the present application comprises: (1) an anti-tight junction protein 18.2 (CLDN18.2) unit as described herein having binding specificity to the CLDN18.2 protein; and (2) an anti-4-1BB unit as described herein having binding specificity to the 4-1BB protein.

[0077] In some embodiments, anti-4-1BB is a scFv and is fused to the C-terminus of the heavy chain of the anti-CLDN 18.2 unit. In some embodiments, anti-4-1BB is a scFv and is fused to the N-terminus of the heavy chain of the anti-CLDN 18.2 unit. In some embodiments, anti-4-1BB is a scFv and is fused to the C-terminus of the light chain of the anti-CLDN 18.2 unit. In some embodiments, anti-4-1BB is a scFv and is fused to the N-terminus of the light chain of the anti-CLDN 18.2 unit.

[0078] In some embodiments, the bispecific antibody comprises a heavy chain component comprising an amino acid sequence as set forth in SEQ ID NO. 17, or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 17. In some embodiments, the bispecific antibody comprises a light chain component comprising an amino acid sequence as set forth in SEQ ID NO. 18, or an amino acid sequence having at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 18. In some embodiments, the bispecific antibody comprises: (1) a heavy chain component comprising an amino acid sequence as shown in SEQ ID NO.17 or an amino acid sequence that is at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO.17, and (2) a light chain component comprising an amino acid sequence as shown in SEQ ID NO.18 or an amino acid sequence that is at least 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO.18.

[0079] In some embodiments, the bispecific antibody is selected from TJ001, IBI389 (Innovent), Q-1802 (QureBio), AMG-910 (Amgen), QLS31905 (Qilu Pharma), PM1032 (Biotheus), and HBM7022 (Harbour, AZ). In some embodiments, the bispecific antibody is TJ001.

[0080] method

[0081] In one aspect, a method for treating a solid tumor is provided herein, comprising administering a bispecific antibody as described herein to a subject in need thereof. In some embodiments, the bispecific antibody is administered intravenously to the subject. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 0.1 to about 30 mg / kg body weight. In some embodiments, the bispecific antibody is administered intravenously to the subject at a dose of about 0.1 to about 30 mg / kg body weight.

[0082] In some embodiments of the present application methods, the bispecific antibody is administered to the subject at a dose of about 0.1-about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 0.1-about 12 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 0.1-about 8 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 0.1-about 5 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 0.1-about 5 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 0.1-about 3 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 0.1-about 1 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 0.1-about 0.3 mg / kg body weight.

[0083] In some embodiments of the present method, the bispecific antibody is administered to the subject at a dose of about 1 to about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 1 to about 12 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 1 to about 8 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 1 to about 5 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 1 to about 5 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 1 to about 3 mg / kg body weight.

[0084] In some embodiments of the methods of the present application, the bispecific antibody is administered to the subject at a dose of about 3 to about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 3 to about 12 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 3 to about 8 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 3 to about 5 mg / kg body weight.

[0085] In some embodiments of the methods of the present application, the bispecific antibody is administered to the subject at a dose of about 4 to about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 4 to about 12 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 4 to about 8 mg / kg body weight.

[0086] In some embodiments of the methods of the present application, the bispecific antibody is administered to the subject at a dose of about 5 to about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 5 to about 12 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 5 to about 8 mg / kg body weight.

[0087] In some embodiments of the methods of the present application, the bispecific antibody is administered to the subject at a dose of about 6 to about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 6 to about 12 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 6 to about 8 mg / kg body weight.

[0088] In some embodiments of the present method, the bispecific antibody is administered to the subject at a dose of about 7-about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 7-about 14 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 7-about 13 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 7-about 12 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 7-about 11 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 7-about 10 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 7-about 9 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 7-about 8 mg / kg body weight.

[0089] In some embodiments of the present method, the bispecific antibody is administered to the subject at a dose of about 8-about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 8-about 14 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 8-about 13 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 8-about 12 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 8-about 11 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 8-about 10 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 8-about 9 mg / kg body weight.

[0090] In some embodiments of the present method, the bispecific antibody is administered to the subject at a dose of about 9-about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 9-about 14 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 9-about 13 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 9-about 12 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 9-about 11 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 9-about 10 mg / kg body weight.

[0091] In some embodiments of the present method, the bispecific antibody is administered to the subject at a dose of about 10-about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 10-about 14 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 10-about 13 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 10-about 12 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 10-about 11 mg / kg body weight.

[0092] In some embodiments of the methods of the present application, the bispecific antibody is administered to the subject at a dose of about 11 to about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 11 to about 14 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 11 to about 13 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 11 to about 12 mg / kg body weight.

[0093] In some embodiments of the methods of the present application, the bispecific antibody is administered to the subject at a dose of about 12 to about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 12 to about 14 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 12 to about 13 mg / kg body weight.

[0094] In some embodiments of the methods of the present application, the bispecific antibody is administered to the subject at a dose of about 13 to about 15 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 13 to about 14 mg / kg body weight.

[0095] In some embodiments of the methods of the present application, the bispecific antibody is administered to the subject at a dose of about 14 to about 15 mg / kg body weight.

[0096] In some embodiments, the bispecific antibody is administered to a subject at a dose of about 0.1, about 0.3, about 1, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 mg / kg body weight. In some embodiments, the bispecific antibody is administered to a subject at a dose of about 0.1, about 0.3, about 1, about 5, about 8, about 12, about 15, or about 30 mg / kg body weight. In some embodiments, the bispecific antibody is administered to the subject at a dose of about 5, about 8, about 12, or about 15 mg / kg body weight.

[0097] In some embodiments, bispecific antibodies are administered to subjects at dosages described herein weekly, every two weeks, every three weeks, or monthly. In some embodiments, bispecific antibodies are administered to subjects at dosages described every two weeks. In some embodiments, bispecific antibodies are administered to subjects at a dosage of about 0.1, about 0.3, about 1, about 5, about 8, about 12, about 15, or about 30 mg / kg body weight weekly, every two weeks, every three weeks, or monthly. In some embodiments, bispecific antibodies are administered to subjects at a dosage of about 0.1, about 0.3, about 1, about 5, about 8, about 12, about 15, or about 30 mg / kg body weight every two weeks. In some embodiments, bispecific antibodies are administered to subjects at a dosage of about 5, about 8, about 12, or about 15 mg / kg body weight weekly, every two weeks, every three weeks, or monthly. In some embodiments, bispecific antibodies are administered to subjects at a dosage of about 5, about 8, about 12, or about 15 mg / kg body weight every two weeks. In some embodiments, bispecific antibodies are administered to subjects at a dosage of about 5 mg / kg body weight every two weeks. In some embodiments, the bispecific antibody is administered to a subject at a dose of about 6 mg / kg body weight every two weeks. In some embodiments, the bispecific antibody is administered to a subject at a dose of about 7 mg / kg body weight every two weeks. In some embodiments, the bispecific antibody is administered to a subject at a dose of about 8 mg / kg body weight every two weeks. In some embodiments, the bispecific antibody is administered to a subject at a dose of about 9 mg / kg body weight every two weeks. In some embodiments, the bispecific antibody is administered to a subject at a dose of about 10 mg / kg body weight every two weeks. In some embodiments, the bispecific antibody is administered to a subject at a dose of about 11 mg / kg body weight every two weeks. In some embodiments, the bispecific antibody is administered to a subject at a dose of about 12 mg / kg body weight every two weeks. In some embodiments, the bispecific antibody is administered to a subject at a dose of about 13 mg / kg body weight every two weeks. In some embodiments, the bispecific antibody is administered to a subject at a dose of about 14 mg / kg body weight every two weeks. In some embodiments, the bispecific antibody is administered to a subject at a dose of about 15 mg / kg body weight every two weeks.

[0098] In some embodiments, the solid tumor overexpresses CLDN 18.2. In some embodiments, the solid tumor is selected from colorectal cancer, urogenital tract cancer, sarcoma, melanoma, hepatocellular carcinoma, gastric cancer, esophageal cancer, gastroesophageal cancer, esophageal cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, lung cancer, non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, Krukenberg tumor and lymphoma. In some embodiments, the solid tumor is an advanced solid tumor. In some embodiments, the solid tumor is a metastatic solid tumor. In some embodiments, the solid tumor is an advanced and metastatic tumor. In some embodiments, the solid tumor is in stage III or stage IV.

[0099] In another aspect, provided herein is a use of a bispecific antibody as described herein in the preparation of a medicament for treating a solid tumor in a subject in need thereof, wherein the bispecific antibody comprises: (1) an anti-claudin 18.2 (CLDN18.2) unit having binding specificity to a CLDN18.2 protein; and (2) an anti-4-1BB unit having binding specificity to a 4-1BB protein, wherein the medicament is administered to the subject at a dose of about 0.1 to about 30 mg / kg body weight.

[0100] Products

[0101] In another aspect, provided herein is an article of manufacture comprising: (1) a bispecific antibody as described herein, comprising an anti-claudin 18.2 (CLDN18.2) unit having binding specificity to a CLDN18.2 protein; and an anti-4-1BB unit having binding specificity to a 4-1BB protein, and (2) a package insert indicating administration of the bispecific antibody to a subject in need thereof at a dose of about 0.1 to about 30 mg / kg body weight.

[0102] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0103] Example

[0104] The following examples are intended to provide a complete disclosure and description of how to make and use the present invention to those of ordinary skill in the art, but are not intended to limit the scope of the invention that the inventors believe, nor are they intended to represent that the following experiments are all or the only experiments performed. Every effort has been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise stated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0105] Example 1. Cytotoxicity of TJ001 on tumor cells expressing CLDN18.2

[0106] Three MKN45 gastric cancer cell lines expressing CLDN18.2 are provided: "MKN45 Parental", "MKN45#18" and "MKN45#14". Microscopic images of each tumor cell stained with IHC membranes are shown in Figure 1A -C. The CLDN18.2 positivity of each tumor cell was characterized by immunohistochemistry (IHC). The detailed IHC scores of CLDN18.2 expression of the three MKN45 cell lines are shown in Figure 2A-C. Figure 2A As shown in -C, MKN45#14 mostly expressed CLDN18.2 + , more than 60% of CLDN18.2 ++ , showing the highest CLDN18.2 expression level (CLDN 高 MKN45#18 showed more than 50% CLDN18.2 + , but very few CLDN18.2 ++ , showing moderate CLDN18.2 expression levels (CLDN 中 MKN45 parent showed the lowest CLDN18.2 expression level (CLDN 低 ).

[0107] TJ001 is a CLDN18.2×4-1BB bispecific antibody having two heavy chain components and two light chain components, each heavy chain component having the sequence of SEQ ID NO.17, and each light chain component having the sequence of SEQ ID NO.18. Such CLDN18.2×4-1BB bispecific antibodies, including TJ001, are also described in WO 2021 / 027850, which is incorporated herein by reference. In order to test the ability of the bispecific antibody TJ001, human PBMCs were used as effector cells, and three groups of MKN45 cells were used as target cells. Effector cells were co-cultured with target cells at two ratios (E:T=1:10; 1:1). Co-culture with a lower E:T ratio (E:T=1:10) was designed to simulate the tumor microenvironment of normal tumors, while co-culture with a higher E:T ratio (E:T=1:1) was designed to simulate the tumor microenvironment of hot tumors / spots.

[0108] Serial dilutions of TJ001 were added to the mixed cultures at a final concentration starting at 100 nM. After 48 hours of co-culture, the levels of IL-2 and IFN-γ in the culture medium were measured using the IL-2 (human) LANCE Ultra TR-FRET Detection Kit and the IFN-γ (human) LANCE Ultra TR-FRET Detection Kit (PerkinElmer). Figure 3A -B, for an E:T ratio of 1:1 (hot tumor / spot), at concentrations of 1.2-33 nM, for CLDN18.2 高 Tumor cells, more consistent cytotoxicity was observed for tumor cells. For a normal E:T ratio of 1:10 (normal tumor), at concentrations of 0.4-33 nM, for CLDN18.2 高 Tumor cells and CLDN18.2 中Consistent cytotoxicity was observed for all tumor cells, indicating that tumor cells with higher CLDN18.2 expression showed a more consistent response to the TJ001 bispecific antibody.

[0109] Example 2. Phase 1 Study Protocol of TJ033721 in Subjects with Advanced or Metastatic Solid Tumors

[0110] This is an open-label, multicenter, multi-dose Phase 1 study designed to evaluate the safety, tolerability, MTD, PK, and PD of TJ033721 in subjects with advanced or metastatic solid tumors. TJ001 is a CLDN18.2×4-1BB bispecific antibody with two heavy chain components and two light chain components, each heavy chain component having a sequence of SEQ ID NO.17 and each light chain component having a sequence of SEQ ID NO.18. TJ033721 recognizes cells with high, medium, and low expression of CLDN18.2 and activates 4-1BB signaling to enhance T cell activation.

[0111] Purpose

[0112] The main objectives of the study are to:

[0113] (1) Evaluate the safety and tolerability of TJ001 in subjects with advanced or metastatic solid tumors;

[0114] (2) determine the maximum tolerated dose (MTD) or maximum administered dose (MAD) of TJ001; and

[0115] (3) Determine the recommended phase 2 dose (RP2D) of TJ001.

[0116] The secondary aims of the study are to:

[0117] (1) Characterize the pharmacokinetic (PK) profile of TJ001;

[0118] (2) evaluate the pharmacodynamic (PD) profile of TJ001;

[0119] (3) evaluate the immunogenicity of TJ001; and

[0120] (4) The preliminary antitumor activity of TJ001 was evaluated by evaluating the response rate using RECIST 1.1 and iRECIST.

[0121] The exploratory aims of the study are to:

[0122] (1) Explore the association between PD activity and efficacy and safety outcomes in subjects treated with TJ001;

[0123] (2) explore the exposure-response relationship of PD activity in subjects treated with TJ001; and

[0124] (3) Explore the association of exploratory biomarkers with PD activity in subjects treated with TJ001.

[0125] method

[0126] For the dose escalation study, a total of 40 to 48 subjects (N=40-48) were administered TJ001 at 0.1 mg / kg (Q2W), 0.3 mg / kg (Q2W), 1 mg / kg (Q2W), 3 mg / kg (Q2W), 5 mg / kg (Q2W), 8 mg / kg (Q2W), 12 mg / kg (Q2W) and 15 mg / kg (Q2W), with each dose being administered by intravenous infusion over 1-2 hours.

[0127] PK and / or PD data, efficacy in GI cancers, and safety are considered by the Scientific Review Committee (SRC) and determine the dose for the dose-expansion study.

[0128] For the determined dose level, 30 subjects (N=30) were dosed with TJ001 in each cohort. The first cohort included subjects with pathologically confirmed gastric cancer (GC), gastroesophageal junction adenocarcinoma (GEJ), or esophageal adenocarcinoma (EAC). The second cohort included subjects with pathologically confirmed pancreatic ductal adenocarcinoma (PDAC). Subjects may include subjects enrolled in the dose expansion study.

[0129] Example 3. Updated Phase 1 Study of TJ001 Based on Incremental Data

[0130] This is a multicenter, dose-escalation Phase 1 study designed to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and efficacy of TJ001 using a Bayesian Optimal Interval (BOIN) design.

[0131] Dose escalation

[0132] The dose escalation study of TJ001 was conducted in subjects with pathologically confirmed advanced or metastatic solid tumors. TJ001 is a CLDN18.2×4-1BB antibody, which contains two heavy chain components and two light chain components, each heavy chain component has the sequence of SEQ ID NO.17, and each light chain component has the sequence of SEQ ID NO.18. TJ001 recognizes cells with high, medium, and low expression of CLDN18.2, and activates 4-1BB signaling to enhance T cell activation.

[0133] One subject per cohort (N=1) was given TJ001 at 0.1 mg / kg (Q2W), 0.3 mg / kg (Q2W) for accelerated titration. In addition, 4 or more subjects per cohort (N=4+) were given TJ001 at the following doses: 1 mg / kg (Q2W), 3 mg / kg (Q2W), 5 mg / kg (Q2W), 8 mg / kg (Q2W), 12 mg / kg (Q2W), and 15 mg / kg (Q2W).

[0134] As of April 18, 2023, 50 patients were enrolled (32 in dose escalation and 18 in parallel expansion), with a median age of 66 years, ECOG 0 / 1 (34% / 66%), and a median number of previous treatment lines of three. No dose-limiting toxicity was observed below 15 mg / kg, and the maximum tolerated dose was not reached. Therefore, it can be confirmed that TJ001 is well tolerated below 15 mg / kg Q2W. The most common treatment-related adverse events (TRAEs) were grade 1 / 2 nausea (22%), fatigue (14%), and vomiting (12%), with 7 occasional grade 3 TRAEs and no TRAEs ≥ grade 4.

[0135] Figure 4 The pharmacokinetic (PK) profiles of mean serum concentrations of TJ001 at each dose level are shown. Figure 4 As shown, TJ001 exhibited linear PK at dose levels of 5 mg / kg or higher, indicating target saturation.

[0136] Among the pharmacodynamic biomarkers, peripheral soluble 4-1BB levels were measured in each cohort before initiation, C1D1 (day 1 of cycle 1), C1D2, C1D8, C1D15, C2D1, C2D15, C3D1, and C4D1. Figure 5A - shown in C. Figure 5A Peripheral soluble 4-1BB levels at these time points are shown (fold change relative to baseline) at different dose levels. Figure 5B -C shows the peripheral soluble 4-1BB levels (fold change relative to baseline) of each subject at different dose levels in C1D8 and C1D15, respectively.

[0137] like Figure 5A As shown in Figure 2-C, the induction of soluble 4-1BB was dose-dependent, with an induction plateau observed at 8-15 mg / kg and a peak at 12-15 mg / kg. It was also observed that CLDN18.2-dependent s4-1BB induction was more significant at 12 mg / kg, reflecting local T cell activation in the tumor.

[0138] CLDN18.2+ parallel dose expansion

[0139] The CLDN18.2+ parallel dose expansion of TJ001 was conducted in subjects with pathologically confirmed CLDN18.2-positive (CLDN18.2+), gastric cancer (GC), gastroesophageal junction adenocarcinoma (GEJ), esophageal adenocarcinoma (EAC), pancreatic ductal adenocarcinoma (PDAC), or cholangiocarcinoma. CLDN18.2 positivity was defined as ≥1% of tumor cells with a membrane intensity score ≥1+.

[0140] Six subjects in each cohort (N=6) were administered TJ001 at the following doses: 5 mg / kg (Q2W), 8 mg / kg (Q2W), 12 mg / kg (Q2W), and 15 mg / kg (Q2W). Among 18 patients with CLDN18.2+ gastroesophageal cancer (GEC) whose efficacy was evaluable, partial responses (PR) were observed in 1 patient at 5 and 8 mg / kg, unconfirmed PR in 2 patients at 12 mg / kg, and stable disease in 2 patients. At 12 mg / kg, an additional PR was observed in 1 patient with head and neck cancer. The expression range of CLDN18.2 in responders was 11%-100%.

[0141] Dose expansion

[0142] The dose expansion study of TJ001 was conducted in subjects with pathologically confirmed CLDN18.2-positive (CLDN18.2+), gastric cancer (GC), gastroesophageal junction adenocarcinoma (GEJ), and esophageal adenocarcinoma (EAC) as a third-line or further immunotherapy. CLDN18.2 positivity was defined as ≥1% of tumor cells with a membrane intensity score ≥1+. Based on safety, clinical PK / PD, and efficacy, 12 mg / kg was selected as the RP2D.

[0143] Thirty subjects (N=30) were administered TJ001 at 12 mg / kg (Q2W).

[0144] ***

[0145] The scope of the present disclosure is not limited by the specific embodiments described, which are intended to be a single illustration of individual aspects of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present disclosure. It is apparent to those skilled in the art that various modifications and changes can be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is intended to cover modifications and changes disclosed herein as long as they are within the scope of the appended claims and their equivalents.

[0146] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0147] The present invention has been described according to the specific embodiments found or proposed by the inventors to include preferred practice modes of the present invention. It will be appreciated by those skilled in the art that, according to the present disclosure, many modifications and variations may be made to the specific embodiments of the examples without departing from the intended scope of the present invention. For example, due to codon redundancy, the underlying DNA sequence may be changed without affecting the protein sequence. In addition, due to considerations of biological function equivalence, the protein structure may be changed without affecting the type or amount of the biological action. All such modifications are intended to be included within the scope of the appended claims.

[0148]

[0149]

Claims

1. A method for treating a solid tumor, comprising administering to a subject in need thereof a bispecific antibody, wherein the bispecific antibody comprises: (1) an anti-claudin 18.2 (CLDN18.2) unit having binding specificity to the CLDN18.2 protein; and (2) a second antibody unit, wherein the bispecific antibody is administered to the subject at a dose of about 0.1 to about 30 mg / kg body weight.

2. The method of claim 1, wherein the second antibody unit has a binding specificity to a target selected from 4-1BB, PD-1, PD-L1 and CD3.

3. The method of claim 1 or 2, wherein the second antibody unit has binding specificity to 4-1BB.

4. The method of any one of claims 1-3, wherein the bispecific antibody is administered to the subject at a dose of about 0.1 to about 15 mg / kg body weight.

5. The method of any one of claims 1-3, wherein the bispecific antibody is administered to the subject at a dose of about 0.3 to about 15 mg / kg body weight.

6. The method of any one of claims 1-3, wherein the bispecific antibody is administered to the subject at a dose of about 1 to about 15 mg / kg body weight.

7. The method of any one of claims 1-3, wherein the bispecific antibody is administered to the subject at a dose of about 3 to about 15 mg / kg body weight.

8. The method of any one of claims 1-3, wherein the bispecific antibody is administered to the subject at a dose of about 5 to about 15 mg / kg body weight.

9. The method of any one of claims 1-3, wherein the bispecific antibody is administered to the subject at a dose of about 8 to about 15 mg / kg body weight.

10. The method of any one of claims 1-3, wherein the bispecific antibody is administered to the subject at a dose of about 12 to about 15 mg / kg body weight.

11. The method of any one of claims 1-3, wherein the bispecific antibody is administered to the subject at a dose of about 8 to about 12 mg / kg body weight.

12. The method of any one of claims 1-3, wherein the bispecific antibody is administered to the subject at a dose of about 0.1, about 0.3, about 1, about 5, about 8, about 12, about 15, or about 30 mg / kg body weight.

13. The method of any one of claims 1-3, wherein the bispecific antibody is administered to the subject at a dose of about 5, about 8, about 12, or about 15 mg / kg body weight.

14. The method of any one of claims 1-13, wherein the bispecific antibody is administered to the subject weekly, every two weeks, every three weeks, or monthly.

15. The method of claim 14, wherein the bispecific antibody is administered to the subject every two weeks.

16. The method of any one of claims 1-15, wherein the bispecific antibody is administered intravenously to the subject.

17. The method of any one of claims 1 to 16, wherein the anti-CLDN18.2 unit is selected from the group consisting of a full-length antibody, Fab, Fab', F(ab')2, scFv and sdAb.

18. The method of claim 17, wherein the anti-CLDN18.2 unit comprises a Fab.

19. The method of any one of claims 1 to 18, wherein the anti-CLDN18.2 unit comprises: (1) HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region (VH) shown in SEQ ID NO. 1, and (2) LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region (VL) shown in SEQ ID NO.

2.

20. The method of any one of claims 1 to 19, wherein the anti-CLDN18.2 unit comprises: (1) HC-CDR1, which comprises the amino acid sequence shown in SEQ ID No. 3, (2) HC-CDR2, which comprises the amino acid sequence shown in SEQ ID No. 4, (3) HC-CDR3, which comprises the amino acid sequence shown in SEQ ID No. 5, (4) LC-CDR1, which comprises the amino acid sequence shown in SEQ ID No. 6, (5) LC-CDR2, which comprises the amino acid sequence shown in SEQ ID No. 7, and (6) LC-CDR3, which comprises the amino acid sequence shown in SEQ ID No.

8.

21. The method of any one of claims 1 to 20, wherein the anti-CLDN18.2 unit comprises: (1) a heavy chain variable region (VH) comprising an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence having at least 90% identity with SEQ ID NO.1, and (2) A light chain variable region (VL), which comprises the amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence having at least 90% identity with SEQ ID NO.

2.

22. The method of any one of claims 1-21, wherein the anti-4-1BB unit is selected from the group consisting of a full length antibody, Fab, Fab', F(ab')2, scFv, and sdAb.

23. The method of any one of claims 1-22, wherein the anti-4-1BB unit comprises a scFv.

24. The method of any one of claims 3-23, wherein the anti-4-1BB unit comprises: (1) HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region (VH) shown in SEQ ID NO. 9, and (2) LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region (VL) shown in SEQ ID NO.

10.

25. The method of any one of claims 3-24, wherein the anti-4-1BB unit comprises: (1) HC-CDR1, which comprises the amino acid sequence shown in SEQ ID No. 11, (2) HC-CDR2, which comprises the amino acid sequence shown in SEQ ID No. 12, (3) HC-CDR3, which comprises the amino acid sequence shown in SEQ ID No. 13, (4) LC-CDR1, which comprises the amino acid sequence shown in SEQ ID No. 14, (5) LC-CDR2, which comprises the amino acid sequence shown in SEQ ID No. 15, and (6) LC-CDR3, which comprises the amino acid sequence shown in SEQ ID No.

16.

26. The method of any one of claims 3-25, wherein the anti-4-1BB unit comprises: (1) a heavy chain variable region (VH) comprising an amino acid sequence as shown in SEQ ID NO.9 or an amino acid sequence having at least 90% identity with SEQ ID NO.9, and (2) A light chain variable region (VL), which comprises the amino acid sequence shown in SEQ ID NO.10 or an amino acid sequence having at least 90% identity with SEQ ID NO.

10.

27. The method of any one of claims 1-26, wherein the bispecific antibody comprises: (1) a heavy chain component comprising an amino acid sequence as shown in SEQ ID NO. 17 or an amino acid sequence having at least 90% identity with SEQ ID NO. 17, and (2) A light chain component comprising the amino acid sequence shown in SEQ ID NO.18 or an amino acid sequence having at least 90% identity with SEQ ID NO.

18.

28. The method of any one of claims 1-27, wherein the bispecific antibody is selected from TJ001, IBI389 (Innovent), Q-1802 (QureBio), AMG-910 (Amgen), QLS31905 (Qilu Pharma), PM1032 (Biotheus), and HBM7022 (Harbour, AZ).

29. The method of any one of claims 1-16, wherein the solid tumor overexpresses CLDN 18.

2.

30. The method of any one of claims 1-29, wherein the solid tumor is selected from the group consisting of colorectal cancer, genitourinary tract cancer, sarcoma, melanoma, hepatocellular carcinoma, gastric cancer (GC), esophageal cancer (including esophageal adenocarcinoma (EAC)), gastroesophageal cancer (including gastroesophageal junction adenocarcinoma (GEJ)), esophageal cancer, pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC), lung cancer, non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, Krukenberg tumor, and lymphoma.

31. The method of any one of claims 1-30, wherein the solid tumor is an advanced solid tumor.

32. The method of any one of claims 1-31, wherein the solid tumor is a metastatic solid tumor.

33. The method of any one of claims 1-32, wherein the solid tumor is an advanced and metastatic solid tumor.

34. Use of a bispecific antibody in the preparation of a medicament for treating a solid tumor in a subject in need thereof, wherein the bispecific antibody comprises: (1) an anti-claudin 18.2 (CLDN18.2) unit having binding specificity to the CLDN18.2 protein; and (2) an anti-4-1BB unit having binding specificity to the 4-1BB protein, wherein the drug is administered to the subject at a dose of about 0.1 to about 30 mg / kg body weight.

35. An article comprising: (1) A bispecific antibody, the bispecific antibody comprising An anti-claudin 18.2 (CLDN18.2) unit having binding specificity to the CLDN18.2 protein; and an anti-4-1BB unit having binding specificity to the 4-1BB protein, as well as (2) a package insert indicating that the bispecific antibody is to be administered to a subject in need thereof at a dose of about 0.1 to about 30 mg / kg body weight.

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