Bispecific t-cell engager molecule combinations for use in treatment of claudin18.2 expressing solid tumors

Abstract

Provided are compositions of a multi-specific T-cell engager comprising a claudin 18.2 (CLDN18.2) binding site and a T cell binding site, in combination with a checkpoint inhibitor agent and / or one or more chemotherapy agent(s), and methods of use therefor. Methods for treating a patient having a CLDN18.2-expressing solid tumor with a bispecific T cell engager (BsTCE), comprising a first domain that binds to CLDN18.2 and a second domain that binds to a T cell, in combination with a checkpoint inhibitor agent and / or one or more chemotherapy agent(s) are disclosed. Also provided are pharmaceutical compositions comprising provided combinations, such pharmaceutical composition combinations for use in the treatment of a CLDN18.2-expressing cancer, and the use of such pharmaceutical composition combinations for the manufacture of a medicament for treating a CLDN18.2-expressing cancer.

Description

CLDN18T-200-PCT01BISPECIFIC T-CELL ENGAGER MOLECULE COMBINATIONS FOR USE IN TREATMENT OF CLAUDIN18.2 EXPRESSING SOLID TUMORSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of United States Provisional Application No. 63 / 715,985, filed November 4, 2024, which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] Incorporated by reference in its entirety herein is a computer-readable sequence listing which is submitted concurrently herewith and contained in the XML file created November 4, 2024, which is entitled "24-1601-US-PRO_Sequence-Listing.xml," and which is 34,104 bytes in size.FIELD

[0003] The disclosure generally relates to compositions of a multi-specific T-cell engager comprising a claudin 18.2 (CLDN18.2) binding site and a T cell binding site, in combination with a checkpoint inhibitor agent and / or one or more chemotherapy agent(s), and methods of use therefor. The disclosure relates to methods for treating a patient having a CLDN18.2-expressing solid tumor with a bispecific T cell engager (BsTCE), comprising a first domain that binds to CLDN18.2 and a second domain that binds to a T cell, in combination with a checkpoint inhibitor agent and / or one or more chemotherapy agent(s); as well as pharmaceutical compositions comprising combinations of a T cell engager comprising a CLDN18.2 binding site and a T cell binding site, a checkpoint inhibitor agent, and / or one or more chemotherapy agent(s), and uses in the treatment of a CLDN18.2-expressing cancer, and for the manufacture of a medicament for treating a CLDN18.2-expressing cancer.BACKGROUND

[0004] Claudins are integral transmembrane proteins located in the tight junction of epithelium and endothelium. Claudins play a critical role in maintaining the polarity of epithelial cells as well as regulating cell growth and differentiation. Among the claudins, claudin 18.2 (CLDN18.2) is a tetraspanin splice variant protein minimally expressed in healthy tissue, but highly expressed in a variety of cancers, including gastric, esophageal, pancreatic cancer, and lung tumors.CLDN18T-200-PCT01

[0005] CLDN18.2-targeted therapeutic approaches, including combinations with chemotherapy, have demonstrated beneficial effects in CLDN18.2 cancers. Despite this, CLDN18.2-targeting antibodies are characterized by short half-lives, low target affinity, unfavorable pharmacological properties, and undesirable cytokine release. Thus, there is a need in the art for safe and effective CLDN18.2 therapies, including bispecific T-cell engagers.SUMMARY

[0006] The disclosure provides methods, compositions, and combinations for treating claudin 18.2 (CLDN18.2) solid tumors using a bispecific T-cell engager (BsTCE) having high- affinity to CLDN18.2 and low-affinity to T cells. The disclosure also provides methods, compositions, and combinations for treating patients having a CLDN18.2-expressing cancer or solid tumor with a BsTCE comprising a first domain that binds to CLDN18.2 and a second domain that binds to cluster of differentiation 3 (CD3). In the methods, compositions, and combinations herein, a BsTCE is used in combination with an immune checkpoint inhibitor agent, and / or one or more chemotherapy agent(s). The disclosure further provides methods, compositions, and combinations for treating CLDN18.2-expressing cancers and solid tumors, including gastric, pancreatic, or esophageal cancer.

[0007] The disclosure provides a method of treating a Claudin 18.2 (CLDN18.2)-expressing solid tumor in a patient in need thereof, the method comprising administering to the patient: (a) a bispecific T-cell engager (BsTCE) agent; and (b) an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s); wherein the BsTCE agent comprises: (i) a first domain that binds to CLDN18.2; and (ii) a second domain that binds to cluster of differentiation 3 (CD3); and wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNis.2-VHcLDNis.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL.

[0008] The disclosure also provides a method of inducing cytotoxicity in a cell expressing Claudin 18.2 (CLDN18.2), the method comprising contacting the cell with: (a) a bispecific T-cell engager (BsTCE) agent; (b) an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s); wherein the BsTCE agent comprises: (i) a first domain that binds to CLDN18.2; and (ii) a second domain that binds to cluster of differentiation 3 (CD3); and wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptideCLDN18T-200-PCT01 chain is represented by the formula: VHcLDNi8.2-VHcLDNi8.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL.

[0009] The disclosure further provides a pharmaceutical combination comprising a bispecific T-cell engager (BsTCE) agent, an immune checkpoint inhibitor agent, and / or one or more chemotherapy agent(s); and a pharmaceutically acceptable carrier; wherein the BsTCE agent comprises: (a) a first domain that binds to Claudin 18.2 (CLDN18.2); and (b) a second domain that binds to cluster of differentiation 3 (CD3); wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNi8.2-VHcLDNi8.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL; wherein the immune checkpoint inhibitor is an immune checkpoint antibody or antigen binding fragment thereof or an immune oncologic bispecific (IO-bispecific) antibody or antigen binding fragment thereof; and wherein the one or more chemotherapy agent is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.

[0010] The disclosure further provides a combination for use in the treatment of a Claudin 18.2 (CLDN18.2)-expressing solid tumor in a patient in need thereof, wherein the combination comprises: (a) a bispecific T-cell engager (BsTCE) agent; and (b) an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s); wherein the BsTCE agent comprises: (i) a first domain that binds to CLDN18.2; and (ii) a second domain that binds to cluster of differentiation 3 (CD3); and wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNi8.2-VHcLDNi8.2-hinge- CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2- CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL.

[0011] The disclosure further provides a combination for use in inducing cytotoxicity in a cell expressing Claudin 18.2 (CLDN18.2), wherein the combination comprises: (a) a bispecific T-cell engager (BsTCE) agent; (b) an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s); wherein the BsTCE agent comprises: (i) a first domain that binds toCLDN18T-200-PCT01CLDN18.2; and (ii) a second domain that binds to cluster of differentiation 3 (CD3); and wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNi8.2-VHcLDNi8.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL.

[0012] The disclosure further provides use of a combination in the manufacture of a medicament for the treatment of a Claudin 18.2 (CLDN18.2)-expressing solid tumor in a patient in need thereof, wherein the combination comprises: (a) a bispecific T-cell engager (BsTCE) agent; and (b) an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s); wherein the BsTCE agent comprises: (i) a first domain that binds to CLDN18.2; and (ii) a second domain that binds to cluster of differentiation 3 (CD3); and wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNi8.2-VHcLDNi8.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL.

[0013] The disclosure further provides use of a combination in the manufacture of a medicament for inducing cytotoxicity in a cell expressing Claudin 18.2 (CLDN18.2), wherein the combination comprises: (a) a bispecific T-cell engager (BsTCE) agent; (b) an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s); wherein the BsTCE agent comprises: (i) a first domain that binds to CLDN18.2; and (ii) a second domain that binds to cluster of differentiation 3 (CD3); and wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHCLDNI8.2-VHCLDNIS.2- hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHCD3-CH1- hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL.

[0014] These and other features and advantages will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claimed invention is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings are included to provide a further understanding of the methods, compositions, combinations, and uses herein. The drawings illustrate one or moreCLDN18T-200-PCT01 embodiments, and together with the description serve to explain the principles and operation of the disclosure.

[0016] Figure 1: FIG. 1A depicts Total T-cells (of CD45+) from immunophenotyping of MC38-mCLDN18.2 tumors or tumors draining lymph nodes (DLn) from human CD3 (hCD3) transgenic mice (Genoway) at day 24 following tumor implantation, 72 hours after the second dose of BsTCE or control treatments. p****<0.0001; p**<0.01; p*<0.05 (One or Two-way ANOVA with multiple comparisons). FIG. IB depicts CD8+ T-cells (of TCRvB+) from immunophenotyping of MC38-mCLDN18.2 tumors or tumor draining lymph nodes (DLn) from human CD3 (hCD3) transgenic mice (Genoway) at day 24 following tumor implantation, 72 hours after the second dose of BsTCE or control treatments. p****<0.0001; p**<0.01; p*<0.05 (One or Two-way ANOVA with multiple comparisons). FIG. 1C depicts CD4+ T-cells (of TCRvB+) from immunophenotyping of MC38-mCLDN18.2 tumors or tumor draining lymph nodes (DLn) from human CD3 (hCD3) transgenic mice (Genoway) at day 24 following tumor implantation, 72 hours after the second dose of BsTCE or control treatments. p****<0.0001 ; p**<0.01; p*<0.05 (One or Two-way ANOVA with multiple comparisons). FIG. ID depicts the ratio of CD8 to CD4 T-cells from immunophenotyping of MC38-mCLDN18.2 tumors or tumor draining lymph nodes (DLn) from human CD3 (hCD3) transgenic mice (Genoway) at day 24 following tumor implantation, 72 hours after the second dose of BsTCE or control treatments. p****<0.0001; p**<0.01; p*<0.05 (One or Two-way ANOVA with multiple comparisons). FIG. IE depicts Natural Killer cells (of CD45+) from immunophenotyping of MC38- mCLDN18.2 tumors or tumor draining lymph nodes (DLn) from human CD3 (hCD3) transgenic mice (Genoway) at day 24 following tumor implantation, 72 hours after the second dose of BsTCE or control treatments. p****<0.0001; p**<0.01; p*<0.05 (One or Two-way ANOVA with multiple comparisons). FIG. IF depicts MHCII+CD86+ B-cells (of CD45+) from immunophenotyping of MC38-mCLDN18.2 tumors or tumor draining lymph nodes (DLn) from human CD3 (hCD3) transgenic mice (Genoway) at day 24 following tumor implantation, 72 hours after the second dose of BsTCE or control treatments. p****<0.0001; p**<0.01; p*<0.05 (One or Two-way ANOVA with multiple comparisons). FIG. 1G depicts CD69+ (of CD8+) intratumoral T-cells from immunophenotyping of MC38-mCLDN18.2 tumors or tumor draining lymph nodes (DLn) from human CD3 (hCD3) transgenic mice (Genoway) at day 24 following tumor implantation, 72 hours after the second dose of BsTCE or control treatments.CLDN18T-200-PCT01 p****<0.0001; p**<0.01; p*<0.05 (One or Two-way ANOVA with multiple comparisons). FIG. 1H depicts Ki67+ (of CD8+) intratumoral T-cells from immunophenotyping of MC38- mCLDN18.2 tumors or tumor draining lymph nodes (DLn) from human CD3 (hCD3) transgenic mice (Genoway) at day 24 following tumor implantation, 72 hours after the second dose of BsTCE or control treatments. p****<0.0001; p**<0.01; p*<0.05 (One or Two-way ANOVA with multiple comparisons). FIG. II depicts PD-1+ (of CD45+) intratumoral T-cells from immunophenotyping of MC38-mCLDN18.2 tumors or tumor draining lymph nodes (DLn) from human CD3 (hCD3) transgenic mice (Genoway) at day 24 following tumor implantation, 72 hours after the second dose of BsTCE or control treatments. p****<0.0001; p**<0.01; p*<0.05 (One or Two-way ANOVA with multiple comparisons). FIG. 1 J depicts GranzymeB+ (of CD8+) intratumoral T-cells from immunophenotyping of MC38-mCLDN18.2 tumors or tumor draining lymph nodes (DLn) from human CD3 (hCD3) transgenic mice (Genoway) at day 24 following tumor implantation, 72 hours after the second dose of BsTCE or control treatments. p****<0.0001; p**<0.01; p*<0.05 (One or Two-way ANOVA with multiple comparisons).

[0017] Figure 2: FIG. 2A represents serum changes of IFNg in hCD3 transgenic mice (Genoway) bearing MC38-mCLDN18.2 tumors following one dose of BsTCE (day 14 after tumor implantation). p***<0.001; p**<0.01; p*<0.05 (Two-way ANOVA with multiple comparisons). FIG. 2B represents serum changes of CXCL9 in hCD3 transgenic mice (Genoway) bearing MC38-mCLDN18.2 tumors following one dose of BsTCE (day 14 after tumor implantation). p***<0.001; p**<0.01; p*<0.05 (Two-way ANOVA with multiple comparisons).

[0018] Figure 3: FIG. 3A represents a schematic of processing gastric and pancreatic cancer samples for ex vivo treatment. FIG. 3B represents immunophenotyping of gastric cancer samples (n=9) at baseline i.e., before treatment, by flow cytometry. Each dot represents a single patient. FIG. 3C represents cytokine secretion from gastric cancer patients (n=12) processed into 300 mM thin slices and treated ex vivo with BsTCE for 2 or 3 days. Line graphs displaying production of indicated analytes as fold change of BsTCE-treated over untreated condition for individual donors. Each dot represents average from 3-4 slices per patient sample. Shaded area indicates fold change <1.5. FIG. 3D represents cytokine secretion from pancreatic cancer patients (n=12) processed into 300 mM thin slices and treated ex vivo with BsTCE for 2 or 3 days. Line graphs displaying production of indicated analytes as fold change of BsTCE-treatedCLDN18T-200-PCT01 over untreated condition for individual donors. Each dot represents average from 3-4 slices per patient sample. Shaded area indicates fold change <1.5. FIG. 3E represents correlations of log2 fold change in IFNy (comparing BsTCE to untreated condition) with CLDN18.2+ cells measured by flow cytometry (n=9) in gastric cancer patient-derived explants. Each dot represents average from 3-4 slices per patient sample; Spearman's correlation coefficient (r) and p values are indicated. FIG. 3F represents correlations of log2 fold change in IFNy (comparing BsTCE to untreated condition) with CLDN18.2+ cells measured by IHC (n=9) in gastric cancer patient- derived explants. Each dot represents average from 3-4 slices per patient sample; Spearman's correlation coefficient (r) and p values are indicated. FIG. 3G represents correlations of log2 fold change in IFNy (comparing BsTCE to untreated condition) with CLDN18.2+ cells measured by CD3+ T cell counts / mm2measured with IHC (n=8) in gastric cancer patient-derived explants. Each dot represents average from 3-4 slices per patient sample; Spearman's correlation coefficient (r) and p values are indicated.

[0019] Figure 4: FIG. 4A represents GranzymeB expression on CD8+ T cells from in vitro co-culture of PBMCs with NUGC4 gastric cancer cell line, treated with controls, BsTCE or BsTCE in combination with anti-PD-1 monoclonal antibody. PBMCs were pre-activated by coculture with NUGC4 tumor cells at 4:1 E:T ratio in the presence of BsTCE; after 7 days, activated PBMCs were collected and reseeded onto fresh NUGC4 cells for 48h in the presence of treatments. Data shown from 48h timepoint as mean + / - SEM. Each dot is an individual PBMC donor. p**<0.01; p*<0.05 (Paired T test). FIG. 4B represents IFNg expression on CD8+ T cells from in vitro co-culture of PBMCs with NUGC4 gastric cancer cell line, treated with controls, BsTCE or BsTCE in combination with anti-PD-1 monoclonal antibody. PBMCs were preactivated by co-culture with NUGC4 tumor cells at 4: 1 E:T ratio in the presence of BsTCE; after 7 days, activated PBMCs were collected and reseeded onto fresh NUGC4 cells for 48h in the presence of treatments. Data shown from 48h timepoint as mean + / - SEM. Each dot is an individual PBMC donor. p**<0.01; p*<0.05 (Paired T test). FIG. 4C represents CD25+ (of CD8+) expression on CD8+ T cells from in vitro co-culture of PBMCs with NUGC4 gastric cancer cell line, treated with controls, BsTCE or BsTCE in combination with anti-PD-1 monoclonal antibody. PBMCs were pre-activated by co-culture with NUGC4 tumor cells at 4: 1 E:T ratio in the presence of BsTCE; after 7 days, activated PBMCs were collected and reseeded onto fresh NUGC4 cells for 48h in the presence of treatments. Data shown from 48h timepointCLDN18T-200-PCT01 as mean + / - SEM. Each dot is an individual PBMC donor. p**<0.01; p*<0.05 (Paired T test). FIG. 4D represents 41BB+ (of CD8+) expression on CD8+ T cells from in vitro co-culture of PBMCs with NUGC4 gastric cancer cell line, treated with controls, BsTCE or BsTCE in combination with anti-PD-1 monoclonal antibody. PBMCs were pre-activated by co-culture with NUGC4 tumor cells at 4: 1 E:T ratio in the presence of BsTCE; after 7 days, activated PBMCs were collected and reseeded onto fresh NUGC4 cells for 48h in the presence of treatments. Data shown from 48h timepoint as mean + / - SEM. Each dot is an individual PBMC donor. p**<0.01; p*<0.05 (Paired T test).

[0020] Figure 5: FIG. 5A depicts tumor growth inhibition driven by BsTCE combined with anti-PD-1. Tumor volume vs time plots (n=10 mice per group, mean + / - SEM) in hCD3deg mice implanted with 1.5 x 106MC38-mCLDN18.2 cells. P values obtained with Two-way ANOVA (tumor volumes), p****<0.0001; p***<0.001; p*<0.05. Groups were removed from graph when <50% animals remained in the study. FIG. 5B depicts tumor growth inhibition driven by BsTCE combined with CAPOX + anti-PD-1. Tumor volume vs time plots (n=l 0 mice per group, mean + / - SEM) in hCD3deg mice implanted with 1.5 x 106MC38-mCLDN18.2 cells. Tumor growth rates estimated based on fitting each tumor's growth curve to an exponential model; p values obtained with Two-way ANOVA (tumor volumes) or with Mann- Whitney t-Test (growth rate). p****<0.0001; p***<0.001; p*<0.05. Groups were removed from graph when <50% animals remained in the study.

[0021] Figure 6: FIG. 6A depicts IFNg secretion from PBMCs co-cultured with NUGC4 tumor cells. Combinations of BsTCE with an anti-PD-1 mAb, rilvegostomig (a bispecific antibody targeting PD-1 and TIGIT), sabestomig (PD-1-TIM3 bispecific), or volrustomig (PD-1- TIGIT bispecific), with or without Oxaliplatin and 5-FU, increased IFNg and GranzymeB secretion from PBMCs in co-culture with NUGC4 tumor cells. NUGC4 cancer cell line was cocultured with healthy donor PBMCs in the presence of BsTCE. After 7 days, the PBMCs, now activated, were collected and co-cultured with fresh NUGC4 cancer cells in the presence of BsTCE, an anti-PD-1 mAb, rilvegostomig (a bispecific antibody targeting PD-1 and TIGIT), sabestomig (PD-1-TIM3 bispecific), or volrustomig (PD-1 -TIGIT bispecific), with or without Oxaliplatin and 5-FU chemotherapeutic agents. Results were generated from 6 donor pairs with a titration of BsTCE and all combination drugs. Arithmetic mean and SEM are plotted.Statistical analysis was performed by One-way ANOVA with multiple comparisons to BsTCE (*CLDN18T-200-PCT01 p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). FIG. 6B depicts the fold change of IFNg (relative to BsTCE) following treatment combinations of BsTCE with an anti-PD-1 mAb, rilvegostomig (a bispecific antibody targeting PD-1 and TIGIT), sabestomig (PD-1-TIM3 bispecific), or volrustomig (PD-1 -TIGIT bispecific), with or without Oxaliplatin and 5-FU. NUGC4 cancer cell line was co-cultured with healthy donor PBMCs in the presence of BsTCE. After 7 days, the PBMCs, now activated, were collected and co-cultured with fresh NUGC4 cancer cells in the presence of BsTCE, an anti-PD-1 mAb, rilvegostomig (a bispecific antibody targeting PD-1 and TIGIT), sabestomig (PD-1-TIM3 bispecific), or volrustomig (PD-1 -TIGIT bispecific), with or without Oxaliplatin and 5-FU chemotherapeutic agents. 48h later, the levels of the proinflammatory cytokine IFNg in supernatants were assessed by ELISA. Results were generated from 12 donor pairs with lOnM BsTCE and IO-bispecifics. Arithmetic mean and SEM are plotted. Statistical analysis was performed by One-way ANOVA with multiple comparisons to BsTCE (* p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). FIG. 6C depicts GranzymeB secretion from PBMCs co-cultured with NUGC4 tumor cells. Combinations of BsTCE with an anti-PD-1 mAb, rilvegostomig (a bispecific antibody targeting PD-1 and TIGIT), sabestomig (PD-1-TIM3 bispecific), or volrustomig (PD-1 -TIGIT bispecific), with or without Oxaliplatin and 5-FU, increased GranzymeB secretion from PBMCs in co-culture with NUGC4 tumor cells. NUGC4 cancer cell line was co-cultured with healthy donor PBMCs in the presence of BsTCE. After 7 days, the PBMCs, now activated, were collected and co-cultured with fresh NUGC4 cancer cells in the presence of BsTCE, an anti-PD-1 mAb, rilvegostomig (a bispecific antibody targeting PD-1 and TIGIT), sabestomig (PD-1-TIM3 bispecific), or volrustomig (PD-1- TIGIT bispecific), with or without Oxaliplatin and 5-FU chemotherapeutic agents. 48h later, the levels of the proinflammatory cytokine IFN-y and of GranzymeB in supernatants were assessed by ELISA. Results were generated from 6 donor pairs with a titration of BsTCE and all combination drugs (A, C) and with 12 donor pairs with lOnM BsTCE and IO-bispecifics (B, D). Arithmetic mean and SEM are plotted. Statistical analysis was performed by One-way ANOVA with multiple comparisons to BsTCE (* p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). FIG. 6D depicts the fold change of GranzymeB (relative to BsTCE) following treatment combinations of BsTCE with an anti-PD-1 mAb, rilvegostomig (a bispecific antibody targeting PD-1 and TIGIT), sabestomig (PD-1-TIM3 bispecific), or volrustomig (PD-l-TIGIT bispecific), with or without Oxaliplatin and 5-FU. NUGC4 cancer cell line was co-cultured with healthy donorCLDN18T-200-PCT01PBMCs in the presence of BsTCE. After 7 days, the PBMCs, now activated, were collected and co-cultured with fresh NUGC4 cancer cells in the presence of BsTCE, an anti-PD-1 mAh, rilvegostomig (a bispecific antibody targeting PD-1 and TIGIT), sabestomig (PD-1-TIM3 bispecific), or volrustomig (PD-1 -TIGIT bispecific), with or without Oxaliplatin and 5-FU chemotherapeutic agents. 48h later, the levels of GranzymeB in supernatants were assessed by ELISA. Results were generated from 12 donor pairs with lOnM BsTCE and IO-bispecifics. Arithmetic mean and SEM are plotted. Statistical analysis was performed by One-way ANOVA with multiple comparisons to BsTCE (* p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0022] Figure 7: FIG. 7A represents tumor cell viability following treatment combinations of BsTCE with Oxaliplatin and 5-FU (chemo) or with chemo + IO agents in NUGC4 tumor cells. NUGC4 cancer cell line was co-cultured in the presence of healthy donor PBMCs in the presence of BsTCE. After 7 days, the PBMCs were co-cultured with fresh NUGC4 cancer cells in the presence of BsTCE, an anti-PD-1 mAb, rilvegostomig (a bispecific antibody targeting PD- 1 and TIGIT), sabestomig (PD-1-TIM3 bispecific), or volrustomig (PD-1 -TIGIT bispecific), with or without Oxaliplatin and 5-FU chemotherapeutic agents. 48h later, the level of tumor cell viability was assessed using a CTG assay. Results were generated from 6 donor pairs with titration of BsTCE and all combination drugs. Arithmetic mean and SEM are plotted. In B, statistical analysis was performed by One-way ANOVA (* p<0.05, **p<0.01), showing the data with BsTCE used at 0. InM. FIG. 7B depicts a bar graph of tumor cell viability following treatment combinations of BsTCE with Oxaliplatin and 5-FU (chemo) or with chemo + IO agents in NUGC4 tumor cells. NUGC4 cancer cell line was co-cultured in the presence of healthy donor PBMCs in the presence of BsTCE. After 7 days, the PBMCs were co-cultured with fresh NUGC4 cancer cells in the presence of BsTCE, an anti-PD-1 mAb, rilvegostomig (a bispecific antibody targeting PD-1 and TIGIT), sabestomig (PD-1-TIM3 bispecific), or volrustomig (PD-1 -TIGIT bispecific), with or without Oxaliplatin and 5-FU chemotherapeutic agents. 48h later, the level of tumor cell viability was assessed using a CTG assay. Results were generated from 6 donor pairs with titration of BsTCE and all combination drugs. Arithmetic mean and SEM are plotted. In B, statistical analysis was performed by One-way ANOVA (* p<0.05, **p<0.01), showing the data with BsTCE used at O.lnM.

[0023] Figure 8: FIG. 8A depicts the fold change of IFNg from combinations of BsTCE with IO-bispecifics compared to BsTCE alone in gastric cancer patient samples treated ex vivo.CLDN18T-200-PCT01Gastric cancer samples were processed into 2um thin slices and placed in culture in the presence of BsTCE alone or IO-bispecifics alone, NIP-TCE (CD3-only binding isotype control) or combinations of BsTCE and IO-bispecifics. Bar graphs displaying production of indicated analytes as fold change over BsTCE-treated condition for individual donors. Each dot represents average from 3-4 slices per donor. Statistical analysis was performed by Two-way ANOVA (* p<0.05). FIG. 8B depicts the fold change of GranzymeB from combinations of BsTCE with IO- bispecifics compared to BsTCE alone in gastric cancer patient samples treated ex vivo. Gastric cancer samples were processed into 2um thin slices and placed in culture in the presence of BsTCE alone or IO-bispecifics alone, NIP-TCE (CD3-only binding isotype control) or combinations of BsTCE and IO-bispecifics. Bar graphs displaying production of indicated analytes as fold change over BsTCE-treated condition for individual donors. Each dot represents average from 3-4 slices per donor. Statistical analysis was performed by Two-way ANOVA (* p<0.05).

[0024] Figure 9: FIG. 9A depicts the tumor volume vs time plot (n=10 mice per group mean + / - SEM of each measurement) of hCD3deg mice (C57BL / 6 background, expressing human CD3 chains, Genoway) implanted with 1.5 x 106MC38-mCLDN18.2 cells and treated with Isotype control, BsTCE, anti-mPD-1, Rilvegostomig murine surrogate (mAZD2936), or combinations of BsTCE and each IO agent. BsTCE or Isotype were given IP at 1 mg / kg once a week for a total of two doses, starting at day 15 after tumor implantation; anti-mPD-1 or mAZD2936 given IP at 10 mg / kg and 30 mg / kg respectively, twice a week for a total of four doses, starting at day 15 after tumor implantation. FIG. 9B depicts individual animal spider plots with number of complete responses (CR) in each treatment group. An individual mouse was considered to be undergoing a complete response when the tumor volume = 0 mm3at the last day of measurement (i.e., day 32 after tumor implantation). Statistical analysis was performed by Two-way ANOVA with multiple comparisons to BsTCE, indicating p value from last measurement (* p<0.05, **p<0.01, ****p<0.0001).

[0025] Figure 10: FIG. 10A depicts a schematic of treatment regimens for combination of BsTCE with Oxaliplatin, Capecitabine and anti-PD-1, in concurrent or sequential schedules in hCD3deg mice (C57BL / 6 background, expressing human CD3 chains, Genoway) implanted with 1.5 x 106MC38-mCLDN18.2 cells. FIG. 10B depicts tumor volume vs time plots (left; n=10 mice per group, mean + / - SEM of each measurement) in concurrent (top) and sequentialCLDN18T-200-PCT01(bottom) schedules, tumor growth rates (right) estimated based on fitting each tumor's growth curve to an exponential model, p values obtained with Mann- Whitney t-Test, comparing each treatment group to BsTCE (***p<0.001, ****p<0.0001). FIG. IOC depicts individual animal spider plots with number of complete responses (CR) in the indicated treatment group. An individual mouse was undergoing a complete response when the tumor volume = 0 mm3at the last day of measurement (i.e., day 29 after tumor implantation).

[0026] Figure 11: FIG. 11 A depicts tumor volume vs time plots (left; n=10 mice per group, mean + / - SEM of each measurement). Statistical analysis was performed by Two-way ANOVA with multiple comparisons to BsTCE, indicating p value from last measurement (* p<0.05, **p<0.01). FIG. 11B shows individual animal spider plots with number of complete responses (CR) in the indicated treatment group. An individual mouse was undergoing a complete response when the tumor volume = 0 mm3at the last day of measurement (i.e., day 36 after tumor implantation).

[0027] Figure 12: FIG. 12A depicts results of cell expression assays following gemcitabine or paclitaxel administration demonstrating increases in CLDN18.2 expression on PaTu-8988s cells. Gemcitabine but not paclitaxel induced a dose dependent increase in CLDN18.2 expression. Representative example n=2. FIG. 12B depicts results of in vitro cytotoxicity assays following administration of BsTCE with gemcitabine or paclitaxel. Following treatment, PaTu- 8988 cells confluence was detected via incucyte every 2h, plotted over time, and cytotoxicity represented as AUC. BsTCE, Gemcitabine, and Paclitaxel all demonstrate monotherapy activity, with additional activity detected in combination. BsTCE potency increases in combination with either gemcitabine or paclitaxel. Increased BsTCE activity in combination with gemcitabine occurs at non-active concentrations of gemcitabine.

[0028] Figure 13: FIG. 13A depicts results of cell expression assays following administration of 5-FU and oxaliplatin, alone or in combination, demonstrating increases in CLDN18.2 expression on NUGC4 cells. FIG. 13B depicts results of in vitro cytotoxicity assays following administration of BsTCE with 5-FU / oxaliplatin. Following treatment, NUGC4 cell confluence was detected via incucyte every 2h, plotted over time, and cytotoxicity represented as AUC. BsTCE and 5-FU / oxaliplatin both demonstrate monotherapy activity, with additional activity detected in combination. BsTCE potency increases in combination with 5- FU / oxaliplatin. Increased BsTCE activity in combination with 5-FU / oxaliplatin occurs at non-CLDN18T-200-PCT01 active concentrations of 5-FU / oxaliplatin. FIG. 13C-D depicts results of repetetive stimulation cell assays following BsTCE combination with 5-FU / oxaliplatin for at least 3 weeks across 3 repetitive stimulations. NUGC4 confluence was detected via incucyte every 2h, plotted over time, and cytotoxicity represented as AUC. bsTCE and 5-FU / oxaliplatin both demonstrate monotherapy activity, with additive activity detected in combination. Inclusion of 5- FU / oxaliplatin in stimulations for 2 weeks had no impact on subsequent activity with BsTCE, 5- FU / oxaliplatin, or their additive activities.

[0029] Figure 14: FIG. 14A-L depict results of expression assays of induced T cell activation markers in PBMCx after repetitive stimulation with bsTCE or in combination with rilvegostomig. PBMCs were stimulated with 0.1 nM bsTCE in flasks for 7, 14 or 21 days alone (grey) or together with 200nM Rilvegostomig (black) at a 2: 1 E:T ratio. At baseline (before treatment) or at the end of each 7 day period, as indicated in plots, PBMCs were harvested and activation marker expression on CD8 or CD4 T cells analysed by flow cytometry. Mean values + / - SEM from n=5 shown. See FIG 14A-14L. Rilvegostomig enhances bsTCE-induced expression of T cell activation markers and leads to blockade of PD1 and TIGIT on T cells in repetitive stimulation assays.

[0030] Figure 15: FIG. 15A-D depict results of repatitive stimulation assays of cells treated with bsTCE alone or in combination with immuno-oncology agents, Rilvegostomig, Volrustomig or anti-PDl (10115). PBMCs were stimulated with 0. InM bsTCE alone or in combination with Rilvegostomig, Volrustomig or anti-PDl in flasks for 7 (Stim 1, FIG 15B), 14 (Stim 2, FIG 15C) or 21 (Stim 3, FIG 15D) days. Baseline results are depicted in FIG 15A. PBMCs were harvested after each stimulation and a bsTCE TDCC performed with NUGC4-GFP cells alone or in combination with Rilvegostomig, Volrustomig or anti-PDl. Supernatants were collected after 6 days and assessed for IFNg and GranzymeB concentrations. Mean values from n=3 shown. Enhanced secretion of IFNg and Granzyme-B was measured from T cells exposed to bsTCE combinations once a week for up to 3 weeks. FIG. 15E-G depict results of Tcell dependent cytotoxicity assays following repetetive T cell stimulation. PBMCs were stimulated with O.lnM bsTCE alone or in combination with anti-PDl (FIG 15E), Rilvegostomig (FIG 15F), or Volrustomig (FIG 15G) in flasks for 7, 14 or 21 days (number of flask stimulation indicated by Stim 1, 2, 3, respectively). PBMCs were harvested after each stimulation and a bsTCE TDCC performed with NUGC4-GFP cells alone or in combination with anti-PDl (FIG 15E),CLDN18T-200-PCT01Rilvegostomig (FIG 15F), or Volrustomig (FIG 15G) by incucyte, data presented as AUC normalised to untreated. Mean values from n=3 (FIG 15G volrustomig, FIG 15E anti-PDl combinations) or n=5 (FIG 15F rilvegostomig combination) shownRilvegostomig, Volrustomig or anti-PDl enhance bsTCE-mediated cytotoxicity of NUGC4-GFP tumour cells, partially rescuing functional decline in repetitive T cell stimulation assays.

[0031] Figure 16: FIG. 16A-B depict results of cytotoxicity of bsTCE in in vitro combinations with 5-FU, oxaliplatin and rilvegostomig. NUGC4 cells were plated in flasks, incubated overnight, PBMCs added (2: 1 E:T) and treated with BsTCE at 0.1 nM for 7 days, then pre-stimualted PBMCs were collected and cultured in a 1:4 ratio with 10,000 fresh NUGC4-GFP cells and treated with bsTCE in the presence of suboptimal bsTCE concentration (donor dependent), 200nM rilvegostomig, and 0.1 pM 5-FU + oxaliplatin. Images were acquired by incucyte every 2h for 6 days. FIG 16A depicts results of AUC, representing cytotoxicity, was quantified and shown as bar graphs with mean values + / - SEM from n=5 shown. FIG 16B depicts cytotoxicity over time as measured by NUGC4-GFP area over time normalised to Oh timepoint.

[0032] FIG. 17A-B depict results of cytotoxicity of reduced concentrations of in vitro combinations of bsTCE 5-FU, oxaliplatin and rilvegostomig. NUGC4 cells were plated in flasks, incubated overnight, PBMCs added (2: 1 E:T) and treated BsTCE used at 0.1 nM for 7 days. After that, the pre-stimulated PBMCs were collected and cultured in a 1:4 ratio with 10,000 fresh NUGC4-GFP cells and treated with bsTCE in the presence of inactive bsTCE concentration (donor dependent), 200nM rilvegostomig, and 0.01 pM 5-FU + oxaliplatin. Images were acquired by incucyte every 2h for 6 days. FIG 17A depicts results of AUC AUC, representing cytotoxicity, was quantified and shown as bar graphs with mean values + / - SEM from n=5 shown. FIG 17B depicts cytotoxicity over time as measured by NUGC4-GFP area over time normalised to Oh timepoint.

[0001] Figure 18: depicts a schematic and results of combination regimens of bsTCE administration with mAZD2936 (a murine surrogate of rilvegostomig), oxaliplatin, 5-FU in concurrent or alternative schedules and results in mouse models. FIG 18A depicts concurrent or alternating administration regimens in hCD3deg mice (C57BL / 6 background, expressing human CD3 chains, Genoway) implanted with 1.5 x 106MC38-mCLDN18.2 cells. Agents were administered on indicated days. Oxaliplatin was injected intraperitoneally (IP) at 5 mg / kg, 5FUCLDN18T-200-PCT01 was given IP at 12.5 mg / kg; BsTCE or Isotype were given IP at 1 mg / kg; mAZD2936 was given IP at 10 mg / kg.. FIG. 18B depicts tumour volume vs time plots following bsTCE concurrent or alternating combinations; mean + / - SEM of each measurement) in each treatment group. FIG. 18C depicts tumour growth rates estimated based on fitting each tumour’s growth curve to an exponential model, p values obtained with one-way ANOVA with multiple comparisons; FIG. 18D depicts Tumour growth rates for each individual mouse estimated based on fitting each tumour’s growth curve to an exponential modelDETAILED DESCRIPTION

[0033] The disclosure relates to methods, compositions, and combinations for treating a CLDN18.2-expressing cancer (e.g., solid tumor) using a bispecific claudin 18.2 (CLDN18.2) and cluster of differentiation 3 (CD3)-targeting T-cell engager (BsTCE), designed with high-affinity to CLDN18.2 and low-affinity to human CD3. The BsTCE used in the methods, compositions, and combinations herein is characterized by specific antitumor activity, bystander killing capability, and triggering of limited peripheral TNF-a and IL-6 release. In the methods, compositions, and combinations herein, the BsTCE is used in combination with a checkpoint inhibitor agent and / or one or more chemotherapy agent(s) to provide improvements over prior CLDN18.2 therapies for safe and effective treatment of CLDN18.2 solid tumors.

[0034] This disclosure demonstrates that administration of a CLDN18.2 and CD-3 targeting T-cell engager (BsTCE) presents specific antitumor activity when administered to a patient having a CLDN18.2 cancer (e.g., solid tumor). The disclosure also demonstrates that combining the CLDN18.2 and CD-3 targeting BsTCE with one or more of an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s) improves T cell activation, circulating levels of pro-inflammatory cytokines, tumor death, tumor volume, tumor control, and / or inhibits tumor growth rate.

[0035] Reference will now be made to exemplary embodiments of the claimed invention. While the claimed invention will be described in conjunction with the exemplary embodiments, it will be understood that it is not intended to limit the claimed invention to those embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents, as may be included within the spirit and scope of the claimed invention, as defined by the appended claims. Those of ordinary skill in the art may make modifications and variations to the embodimentsCLDN18T-200-PCT01 described herein without departing from the spirit or scope of the claimed invention. In addition, although certain methods and materials are described herein, other methods and materials that are similar or equivalent to those described herein can also be used to practice the claimed invention.

[0036] In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).

[0038] All publications, patent applications, patents, and other references mentioned or discussed herein are expressly incorporated by reference in their entireties to the same extent as if each individual publication, patent application, patent, or other reference was specifically and individually indicated to be incorporated by reference. This includes, but is not limited to, U.S. Patent Application Publication No. US 2023 / 0134183 and U.S. Patent Nos. 11,939,382, 11,279,759, and 10,457,732.

[0039] As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. As utilized in accordance with the disclosure, unless otherwise indicated, all technical and scientific terms shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular. However, it should be understood that other meanings known or understood by those having ordinary skill in the art are also possible, and within the scope of the claimed invention.

[0040] As used herein, the terms "a," "an," and "the," as used herein, are understood to be singular or plural unless the context clearly dictates otherwise. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components unlessCLDN18T-200-PCT01 otherwise indicated or dictated by its context. That is, as used herein, the singular forms "a," "an," and "the" also include plural referents unless the context clearly indicates otherwise.

[0041] Use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives unless otherwise indicated. Thus, unless specifically stated or apparent from context, the term "or" as used herein is understood to be inclusive. As used herein, the terms "or" and "and / or" can describe multiple components in combination or exclusive of one another. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z."

[0042] Throughout this disclosure, unless the context specifically indicates otherwise, the terms "comprise" and "include" and variations thereof (e.g., "comprises," "comprising," "includes," and "including") will be understood to indicate the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other component, feature, element, or step or group of components, features, elements, or steps.

[0043] In the disclosure, a concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Ranges provided herein are understood to be shorthand for all of the values within the range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0044] Unless specifically stated or apparent from context, the terms "about" and "approximately," as used herein, are understood as meaning within a range of normal tolerance in the art, for example within 2 standard deviations of the mean, or mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. The term "about" is understood as meaning within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value or range of values (e.g., about 5% also means 4.5% - 5.5%). Additionally, "about" can mean a range of up to ±10%.CLDN18T-200-PCT01Percentages disclosed herein can vary in amount by ±10% from values disclosed and remain within the scope of the contemplated disclosure. Furthermore, particularly with respect to biological systems or processes, "about" can mean up to an order of magnitude of a value. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of "about" should be assumed to be within an acceptable error range for that particular value or composition. Unless otherwise clear from context, all numerical values provided herein are modified by the term "about."

[0045] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects herein, which are understood by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0046] It is noted that terms, such as "typically," are not utilized herein to limit the scope of the claimed subject matter or to imply that certain features are critical, essential, or even important to the structure or function of the claimed subject matter. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment herein.Compounds

[0047] As used herein, the terms "T-cell engager" and "bispecific T-cell engager (BsTCE)" both include, but are not limited to, a bispecific antibody or antibody fragment thereof or other immunoglobulin-like formats that possess the ability to bind to an antigen of interest and a T cell. In one embodiment of the methods, compositions, and combinations, a BsTCE binds the CLDN18.2 antigen on a tumor cell and the CD3 antigen on a T cell (CLDN18.2xCD3). Upon binding of such BsTCE, T cells become activated and mount an immune response directed at the CLDN18.2-expressing tumor cell. In some embodiments of the methods, compositions, and combinations, the CLDN18.2xCD3 BsTCE comprises three polypeptides chains, wherein the first polypeptide chain is represented by the formula VHcLDNi8.2-VHcLDNis.2-hinge-CH2-CH3, the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3, and the third polypeptide chain is represented by the formula: VLCD3-CL.CLDN18T-200-PCT01

[0048] As used herein, "T cell" refers to a class of lymphocytes that mature in the thymus. T cells play an important role in cell-mediated immunity and are different from other lymphocytes(e.g., B cells) in that T cell receptors are present on the cell surface. The term "T cell," as used herein, includes all types of immune cells that express CD3, including, e.g., T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells (NKT cells), T regulatory cells (Tregs), and gamma-delta T-cells.

[0049] In methods, compositions, and combinations herein, CLDN18.2xCD3 BsTCE is comprised of the amino acid sequences disclosed in U.S. Patent Publication No. US2023 / 0134183, which is hereby incorporated in its entirety. The amino acid sequences for theCLDN18.2xCD3 BsTCE chains are set forth below:TABLE A: BsTCE SequencesCLDN18T-200-PCT01

[0050] In some embodiments, an alternative format and sequences for a CLDN18.2 BsTCE is used in the methods, compositions, and combinations provided herein. Exemplary alternative formats and amino acid sequences are disclosed in U.S. Patent Publication No. US 2023 / 0134183, which is hereby incorporated in its entirety.

[0051] In an alternate embodiment, the structure of the CLDN18.2xCD3 BsTCE is HCAb-HC-Fab having an arrangement of polypeptide chain 1 of N'-VH_B-hinge-CH2-CH3-C; an arrangement of polypeptide chain 2 of N'-VH_B-hinge-CH2-CH3-linker-VH_A-CHl-C; and an arrangement of polypeptide chain 3 of N'-VL_A-CL-C'.

[0052] In an alternate embodiment, the structure of the CLDN18.2xCD3 BsTCE is HCAb-LC-Fab, having an arrangement of polypeptide chain 1 of N'-VH_B-hinge-CH2-CH3-C; an arrangement of polypeptide chain 2 of N'-VH_B-hinge-CH2-CH3-linker-VL_A-CL-C'; and an arrangement of polypeptide chain 3 of N'-VH_A-CH1-C.

[0053] In an alternate embodiment, the structure of the CLDN18.2xCD3 BsTCEa is VH-VH_HC-Fab, having an arrangement of polypeptide chain 1 of N'-VH_B-linker-VH_B-hinge- CH2-CH3-C; an arrangement of polypeptide chain 2 of N'-VH_B-linker-VH_B-hinge-CH2- CH3-linker-VH_A-CHl-C; and an arrangement of polypeptide chain 3 of N'-VH_A-CL-C'.CLDN18T-200-PCT01

[0054] In an alternate embodiment, the structure of the CLDN18.2xCD3 BsTCEb is VH- VH_LC-Fab, having an arrangement of polypeptide chain 1 of N'-VH_B-linker-VH_B-hinge- CH2-CH3-C; an arrangement of polypeptide chain 2 of N'-VH_B-linker-VH_B-hinge-CH2- CH3-linker_VL_A_CL-C; and an arrangement of polypeptide chain 3 of N'_VH-A-CH1-C'.

[0055] In an alternate embodiment, the structure of the CLDN18.2xCD3 BsTCEc is VH_HC-Fab-Fc-Dual VH, having an arrangement of polypeptide chain 1 N'-VH_B-linker- VH_B-hinge-CH2-CH3-C'; an arrangement of polypeptide chain 2 of N'-VH_B-linker-VH_A- CHl-hinge-CH2-CH3- C'; and an arrangement of polypeptide chain 3 of N'-VL A-CL-C

[0056] In an alternate embodiment, the structure of the CLDN18.2xCD3 BsTCEd is Fab-Fc- VH-VH-VH, having an arrangement of polypeptide chain 1 N'-VH_B-linker-VH_B-linker- VH_B-hinge-CH2-CH3-C'; an arrangement of polypeptide chain 2 of N'-VH_A-CHl-hinge- CH2-CH3- C'; and an arrangement of polypeptide chain 3 of N'-VL A-CL-C'

[0057] In an alternate embodiment, the structure of the CLDN18.2xCD3 BsTCEe is Fab-Fc- scFv(VH-HL), having an arrangement of polypeptide chain 1 N'-VH_B-linker-VL_B-hinge- CH2-CH3-C; an arrangement of polypeptide chain 2 of N'-VH_A-CHl-hinge-CH2-CH3- C'; and an arrangement of polypeptide chain 3 of N'-VL A-CL-C.

[0058] The term "antibody," as used herein, refers to a protein capable of recognizing and specifically binding to an antigen. Ordinary or conventional mammalian antibodies comprise a tetramer, which is typically composed of two identical pairs of polypeptide chains, each pair consisting of one "light" chain (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). The term "antibody" includes antibodies having variable and constant regions substantially corresponding to human germline immunoglobulin sequences. In some embodiments, human antibodies are produced in non-human mammals, including, but not limited to, rodents, such as mice and rats, and lagomorphs, such as rabbits. In other embodiments, human antibodies are produced in hybridoma cells. In still other embodiments, human antibodies are produced recombinantly.

[0059] The terms "heavy chain" and "light chain," as used herein, refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids that typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant domainCLDN18T-200-PCT01 responsible for effector function. Thus, in a naturally occurring antibody, a full-length heavy chain immunoglobulin polypeptide includes a variable domain (VH) and three constant domains (CHI, CH2, and CH3), wherein the VH domain is at the amino-terminus of the polypeptide and the CH3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide includes a variable domain (VL) and a constant domain (CL), wherein the VL domain is at the amino-terminus of the polypeptide and the CL domain is at the carboxyl- terminus.

[0060] Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgMl and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgAl and IgA2. The variable regions of each light / heavy chain pair typically form an antigen binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope. From the amino-terminus to the carboxyl-terminus, both light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0061] The terms "antigen" or "target antigen," as used herein, refer to a molecule or a portion of a molecule capable of being bound by an antibody (or an antibody-like binding molecule or a binding molecule comprising immunoglobulin domains), and additionally is capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. A target antigen may have one or more epitopes. With respect to each target antigen recognized by an antibody-like binding protein, the antibody-like binding protein is capable of competing with an intact antibody that recognizes the target antigen.

[0062] The term "epitope" includes any determinant, typically a polypeptide determinant, capable of specifically binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certainCLDN18T-200-PCT01 embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope is a region of an antigen that an antibody binds. In certain embodiments, the BsTCE used in the methods, compositions, and combinations is said to specifically bind an antigen when it specifically recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0063] As used herein, the term "specific" with respect to a binding molecule (e.g., a BsTCE, an antibody, a bispecific antibody, an antibody-like binding protein) used in the methods, compositions, and combinations means that the binding molecule recognizes a particular antigen but does not substantially recognize or bind to other molecules (including, e.g., other similar molecule(s)) in a sample. For example, a binding molecule that specifically binds to an antigen of one form (e.g., one allele or species) would not bind to the antigen from one or more alternate forms (e.g., allele or species). In some cases, one form (e.g., allele) is similar in different species and thus, a binding molecule that specifically binds one allelic form in one species also specifically binds that allele (i.e., orthologue) in an alternate species. In some cases, the term "specificity" or "specific binding" is used to refer to the interaction of an antibody, a protein, or a peptide with a second molecule, meaning that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) in the molecule; for example, an antibody generally recognizes and binds to a particular protein structure rather than a protein. The terms "polypeptide," "peptide," and "protein" (if single-stranded) are used interchangeably in the disclosure.

[0064] The terms "affinity" or "binding affinity" is presented as a dissociation constant, or "Kp," which refers to the interaction between a binding molecule and an antigen target and is expressed in the units of moles / liter. A binding molecule typically has a dissociation constant (Kp) of 10'5to 10'12moles / liter or less, or 10'7to 10'12moles / liter or less, or 10'3to 10'12moles / liter, and / or with a binding affinity of at least 107M’1, or at least 108M’1, or at least 109M’ \ or at least 1012M’1. Any !< / > value greater than 10'4moles / liter is generally considered to indicate non-specific binding. Therefore, the lower the !< / > value, the greater the affinity. In some embodiments, a binding molecule will bind to a desired antigen with an affinity !< / > less than 500 nM, or less than 200 nM, or less than 150 nM, or less than 100 nM, or less than 50 nM, or less than 10 nM, or less than 1 nM, or less than 500 pM, or less than 200 pM. In some embodiments, a binding molecule binds with an affinity !< / > less than 500 nM, or less than 400CLDN18T-200-PCT01 nM, or less than 300 nM, or less than 200 nM, or less than 150 nM, or less than 100 nM. In some embodiments, a low affinity binding molecule binds an antigen with an affinity K / > less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM. In some embodiments, a low affinity binding molecule binds CD3 with an affinity K / > less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM. In some embodiments, a low affinity binding molecule binds CD3 with an affinity K / > less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM. In some embodiments, a low affinity binding molecule binds CD3 with an affinity I< / > of about 170 nM. High affinity or very strong binding is often associated with greater efficacy, but it is not always the case that the greater the affinity the greater the efficacy. In some embodiments, a binding molecule binds with an affinity I< / > less than 10 nM, or less than 1 nM, or less than 500 pM, or less than 200 pM, or less than 150 pM. In some embodiments, a high affinity binding molecule binds with an affinity !< / > less than 5 nM, or less than 2 nM, or less than 1 nM, or less than 500 pM, or less than 200 pM, or less than 150 pM. In some embodiments, a high affinity binding molecule binds CLDN18.2 with an affinity I< / > less than 5 nM, or less than 2 nM, or less than 1 nM, or less than 500 pM, or less than 200 pM, or less than 150 pM. In some embodiments, a high affinity binding molecule binds CLDN18.2 with an affinity K / ) less than 5 nM, or less than 2 nM, or less than 1 nM, or less than 500 pM, or less than 200 pM, or less than 150 pM and greater than 50 pM. In some embodiments, a high affinity binding molecule binds CLDN18.2 with an affinity K / > of about 100 pM

[0065] In one embodiment of the methods, compositions, and combinations, a heavy chain only antibody (VHH) is used. VHH are small (15kDa) antibodies that lack light chains. VHH are comprised of four framework regions and three CDRs and with a single variable domain for antigen recognition (Hoey etal., Exp. Biol. Med. (Maywood) 244(17): 1568-76 (2019)). The single VHH domain is comprised of three hypervariable loops (H1-H3) with antibody binding affinities comparable to conventional antibodies. The VHH format is characterized by amino acid substitutions in the framework 2 that are normally positioned in the VL interface of a conventional VH domain. The VHH domain is characterized by a long CDR3 loop when compared to a VH domain, allowing the CDR3 to act as a surrogate VL domain. VHH are highly soluble, display rapid tissue penetration, and are well expressed allowing for recognition of antigens that may go unnoticed by conventional antibodies.CLDN18T-200-PCT01

[0066] In some embodiments of the methods, compositions, and combinations, an antibody binding fragment is used. Such antibody binding fragments may contain only portions of an antibody molecule such as Fab, F(ab')2 , Fab', scFv, di-scFv, and sdAb fragments. Such fragments have use as therapeutic agents. In addition, specific residues in the variable domains can be altered to improve binding specificity and / or stability of antibodies and antibody fragments.

[0067] Single chain variable fragment (scFv) constructs comprise a VH and a VL domain of an antibody contained in a single polypeptide chain wherein the domains are separated by a flexible linker of sufficient length (more than 12 amino acid residues), that forces intramolecular interaction, allowing self-assembly of the two domains into a functional epitope binding site. These small proteins (MW -25,000 Da) generally retain specificity and affinity for their antigen in a single polypeptide and can provide a convenient building block for larger, antigen-specific molecules. Exemplary, non-limiting variants of scFv include but are not limited to tandem di- scFvs, tandem tri-scFvs, diabodies, and tri(a)bodies.

[0068] Antibody fragments (Fab and Fab2), resulting from proteolytic digestion of an antibody also exhibit antigen binding activity. Antibody fragments can also be produced recombinantly.

[0069] Also contemplated for use in the methods, compositions, and combinations are Fv fragments, consisting only of the variable domains of the heavy and light chains associated with each other are monovalent for antigen binding. An advantage of using antibody fragments rather than whole antibodies in therapy lies in their smaller size. They are likely to be less immunogenic than whole antibodies and more able to penetrate tissues. Combining two different scFvs results in bispecific antibody formats with minimal molecular mass, termed sc-BsAbs or Ta-scFvs.

[0070] Diabodies are small bivalent and bispecific antibody fragments. The fragments comprise a VH connected to a VL on the same polypeptide chain, by using a linker that is short (e.g., less than, e.g., 12 amino acid residues), to allow pairing between the two domains on the same chain. The domains are forced to pair intermolecularly with the complementary domains of another chain and create two antigen-binding sites. Diabodies are similar in size to a Fab fragment.CLDN18T-200-PCT01

[0071] As used herein, the terms "bispecific antibody" or "BsAb" refer to an antibody that can bind two unique antigen-binding sites, each an epitope. Typically, a BsAb is bivalent for binding to each of two epitopes; when the BsAb binds two different epitopes, the BsAb has multi-specificity of a conventional bispecific antibody. In some embodiments, a BsAb comprises two heavy-light chain pairs derived from a specific binding protein, wherein the heavy and light chains each comprise a variable region (e.g., VL and VH), which together form a first binding unit, and wherein the heavy chains each further comprises a second binding unit (e.g., an scFv domain attached to Fc or Fab). Where the first and second binding units bind different epitopes, each heavy-light chain pair is bispecific and the two pairs together are bivalent for each epitope.

[0072] In one aspect, provided are methods, compositions, and combinations for treating CLDN18.2-expressing cancers or solid tumors using a BsTCE in combination with an immune checkpoint inhibitor agent.

[0073] As used herein, the terms "immune checkpoint inhibitor" or "immune checkpoint inhibitor agent" refer to an antibody, antibody-like binding protein, or other agent that blocks immune derived checkpoint proteins. Interfering with immune checkpoint inhibitors allows for immune activation and T cell mediated targeting of cancer cells. As used herein, checkpoint proteins found on T cells or cancer cells include PD-1 / PD-L1 and CTLA-4 / B7-1 / B7-2.

[0074] As used herein, the terms "anti-PD-1 antibody" and "anti-PD-Ll antibody" refer to antibodies or antigen binding fragments thereof that block progressive disease (PD)-l and programmed death ligand (PD-L1) pathways, respectively. Increased expression of immunosuppressive cell populations and inhibitory signaling molecules, such as PD-1, have been observed in cancer. Tumor aggressiveness, disease progression, and high mortality in patients with cancer are associated with overexpression of PD-L1 thereby demonstrating the importance of PD-1 / PD-L1 pathways in cancer. Therefore, therapeutic agents that are capable of blocking PD-L1 possess the ability to improve clinical outcomes by reversing the immunosuppressive environment characteristic of cancer and by stimulating host immunity against the cancer. In some embodiments the immune checkpoint inhibitor comprises an anti- PD-1 antibody or an anti-PD-Ll antibody. In some embodiments, the anti-PD-1 antibody or anti-PD-Ll antibody is a monoclonal antibody. In some embodiments, the anti-PD-1 antibody or anti-PD-Ll antibody is a monoclonal antibody.CLDN18T-200-PCT01

[0075] Programmed Death Ligand 1 (PD-L1) is a part of a complex system of receptors and ligands that are involved in controlling T-cell activation. In normal tissue, PD-L1 is expressed on T cells, B cells, dendritic cells, macrophages, mesenchymal stem cells, bone marrow- derived mast cells, as well as various non-hematopoietic cells. Its normal function is to regulate the balance between T-cell activation and tolerance through interaction with its two receptors: programmed death 1 (PD-1) and CD50 (also known as B7-1 or B7.1). PD-L1 is also expressed by tumors and acts at multiple sites to help tumors evade detection and elimination by the host immune system. PD-L1 is expressed in a broad range of cancers with a high frequency. In some cancers, expression of PD-L1 has been associated with reduced survival and unfavorable prognosis. Antibodies that block the interaction between PD-L1 and its receptors are able to relieve PD-L1 -dependent immunosuppressive effects and enhance the cytotoxic activity of antitumor T cells in vitro. Durvalumab is a human monoclonal antibody directed against human PD-L1 and capable of blocking the binding of PD-L1 to both the PD-1 and CD50 receptors.

[0076] Durvalumab is a human monoclonal antibody of the immunoglobulin G 1 kappa subclass that blocks the interaction of PD-L1, but not PD-L2, with PD-1 on T cells and CD80 (B7.1) on immune cells. Blockade of PD-L1 / PD-1 and PDL1 / CD80 interactions releases the inhibition of immune responses, including those that result in tumor elimination. Thus, durvalumab has the technical effect of stimulating the antitumor immune response by binding to PD-L1 and shifting the balance toward an antitumor response. Durvalumab has the additional technical effect of reducing antibody-dependent cellular toxicity and complement dependent toxicity. The durvalumab antibody is disclosed in U.S. Patent No. 9,493,565 (referred therein as "2.14H9OPT"), which is incorporated by reference herein in its entirety.

[0077] The fragment crystallizable (Fc) domain of durvalumab contains a triple mutation in the constant domain of the IgGl heavy chain that reduces binding to the complement component Clq and the Fey receptors responsible for mediating antibody-dependent cell-mediated cytotoxicity (ADCC). In certain embodiments, the triple mutation refers to the IgGl Fc region comprising a L234F / L235E / P331S triple mutation (EU numbering; see also U.S. Patent No. 9,493,565). Durvalumab can relieve PD-L1 -mediated suppression of human T-cell activation in vitro and inhibits tumor growth in a xenograft model via a T-cell dependent mechanism.Table B: DurvalumabCLDN18T-200-PCT01

[0078] Additional anti-PD-Ll antibodies for use in the methods, compositions, and combinations include, but are not limited to, atezolizumab (TECENTRIQ), avelumab (BAVENCIO), BMS-936559, lodapohmab, cosibelimab, CX-072, FAZ053, KN035, and MDX- 1105.

[0079] Programmed Death- 1 (PD-1 or PD1) is an approximately 31 kD type 1 membrane protein member of the extended CD28 / CTLA-4 family of T cell regulators. PD-1 is expressed on activated T cells, B cells, and monocytes. PD-1 is a receptor responsible for down-regulation of the immune system following activation by binding of PDL-1 or PDL-2. This process is exploited in many tumors via the over-expression of PD-L1, leading to a suppressed immune response. PD-1 is a well-validated target for immune mediated therapy in oncology, with positive results from clinical trials in the treatment of melanoma and non-small cell lung cancers (NSCLC), among others. Antagonistic inhibition of the PD-l / PD-L-1 interaction increases T- cell activation, enhancing recognition and elimination of tumor cells by the host immune system.CLDN18T-200-PCT01Anti-PD-1 antibodies provide enhanced anti-tumor therapy response in combination with chemotherapy and other targeted therapies. This class of antibodies are often used in combination with anti-CTLA-4 and / or anti-PD-Ll antibodies that block a combination of immune checkpoint receptors and ligands. Anti-PD-1 antibodies for use in the methods, compositions, and combinations include, but are not limited to, nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), cemiplimab (LIBTAYO), dostarlimab (JEMPERLI), retifanlimab (ZYNYZ), toripalimab (LOQTORZI), vopratelimab, spartalizumab, pidilizumab, MEDI0680, cetrehmab, AMP-224, AMP-514, PF-06801591, tislehzumab, ABBV-181, BI 754091, and camrelizumab.

[0080] In some embodiments, the anti-PD-1 antibody or anti-PD-Ll antibody is a polyclonal antibody. In some embodiments, the anti-PD-1 antibody or anti-PD-Ll antibody is a bispecific antibody. In one embodiment, the bispecific molecule that binds two epitopes is bivalent for the first and second epitopes.

[0081] In some embodiments of the methods, compositions, and combinations, the immune checkpoint inhibitor agent comprises an immune-oncological (lO)-bispecific antibody. As used herein, the term "IO-bispecific antibody" refers to immune-oncological antibodies that are engineered to target two immune checkpoints. An IO-bispecific antibody also refers to an antibody designed to target tumor antigens and immunomodulatory receptors. IO bispecific antibodies include, but are not limited to, one or more of PD-1 in combination with T cell immunoreceptor with Ig and ITIM domain (TIGIT; e.g., rilvegostomig), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3; e.g., sabestomig) or cytotoxic T-lymphocyte- associated antigen 4 (CTLA4; e.g., volrustomig). In some embodiments of the methods, compositions, and combinations, the IO-bispecific antibody is administered with a BsTCE. In some embodiments of the methods, compositions, and combinations, a bispecific protein that binds PD-1 and CTLA-4 is used. In some embodiments, a bispecific protein that binds to PD-1 and TIM3 is used. In some embodiments, a bispecific protein that binds PD-1 and TIGIT is used. Suitable bispecific molecules for use in methods, compositions, and combinations are disclosed in U.S. Patent No. 10,457,732, which is hereby incorporated by reference in its entirety.

[0082] Rilvegostomig is a bispecific antibody directed against PD-1 and the co-inhibitory molecule and immune checkpoint inhibitor T-cell immunoreceptor with immunoglobulin (Ig)CLDN18T-200-PCT01 and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT), with potential immune checkpoint inhibitory and antineoplastic activities. Rilvegostomig simultaneously inhibits PD-1 and TIGIT and their downstream signaling pathways thereby restoring immune function via activation of T-cells and T-cells mediated immune response. Inhibition of TIGIT prevents the interaction of TIGIT with its ligands CD112 and CD155 allowing for a T-cell- mediated immune response against cancer cells. Rilvegostomig is disclosed in U.S. Patent No. 11,939,382, which is incorporated by reference herein in its entirety. The amino acid sequences of rilvegostomig are set forth below: Table C: Rilvegostomig sequencesCLDN18T-200-PCT01

[0083] Sabestomig is a monovalent, bispecific, humanized IgGl monoclonal antibody that binds PD-1 and an epitope in the TIM-3 IgV domain without blocking phosphatidylserine binding, thereby inhibiting growth of solid tumors. Sabestomig is disclosed in U.S. Patent Nos.11,939,382 and 10,457,732, both of which are incorporated by reference herein in their entireties. The amino acid sequences of sabestomig are set forth below:Table D: Sabestomig sequencesCLDN18T-200-PCT01

[0084] Volrustomig is an engineered fragment crystallizable (Fc) domain bispecific human immunoglobulin G1 (IgGl) monoclonal antibody directed against PD-1 and cytotoxic T- lymphocyte-associated antigen 4 (CTLA4). Volrustomig targets and binds to both PD-1 and CTLA4 expressed on tumor- infiltrating T lymphocytes (TILs), and inhibits the PD-1- and CTLA4-mediated downregulation of T-cell activation and proliferation. Volrustomig degrades PD-1 thereby restoring immune function and activating a sustained cytotoxic T-lymphocyte (CTL) -mediated immune response against tumor cells. Volrustomig is disclosed in U.S. Patent Nos. 11,939,382 and 10,457,732 both of which are incorporated by reference herein in their entireties. The amino acid sequences of volrustomig are set forth below: Table E: Volrustomig sequencesCLDN18T-200-PCT01

[0085] As used herein, the term "chemotherapy" refers to both platinum-based and non- platinum-based chemotherapy. In some embodiments of the methods, compositions, and combinations, chemotherapy refers to treatment comprising one or more non-platinum-based agents, including but not limited to, 5 -fluorouracil (5-FU) and derivatives thereof, anthracycline- containing agents, cyclophosphamide, doxorubicin (Adriamycin), folinic acid, epirubicin, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxanes, gemcitabine, vinblastine, vincristine, vemurafenib, bevacizumab, cetuximab, afatinib, and erlotinib.

[0086] In some embodiments of the methods, compositions, and combinations, chemotherapy comprises a fluoropyrimidine compound selected from 5 -fluorouracil (5-FU) and derivatives thereof, floxuridine, capecitabine, tegafur, and / or doxifluridine. In some embodiments, chemotherapy comprises one or more platinum-based chemotherapy including, but not limited to, one of one or more of carboplatin, cisplatin, nedaplatin, and / or oxaliplatin. In some embodiments, chemotherapy comprises an anthracycline-containing compound including, but not limited to, daunorubicin, epirubicin, and / or doxorubicin.

[0087] In some embodiments of the methods, compositions, and combinations, chemotherapy comprises a fluoropyrimidine compound selected from 5 -fluorouracil (5-FU) and derivatives thereof, floxuridine, capecitabine, tegafur, and / or doxifluridine; a platinum-based chemotherapy selected from one of one or more of carboplatin, cisplatin, nedaplatin, and / or oxaliplatin; and / or an anthracycline-containing compound selected from daunorubicin, epirubicin, and / or doxorubicin.

[0088] In some embodiments of the methods, compositions, and combinations, a single chemotherapeutic agent is administered. In some embodiments, one or more chemotherapy agents are administered concurrently. In some embodiments, chemotherapy comprises more than one chemotherapeutic agent. In some embodiments, chemotherapy comprises a standard chemotherapy regimen, for example, standard chemotherapy regimens include, but are not limited to: EOX, which is a chemotherapy regimen comprising a combination of epirubicin, oxaliplatin, and capecitabine; ECF, which is a chemotherapy regimen comprising a combinationCLDN18T-200-PCT01 of epirubicin, cisplatin, and 5 -fluorouracil; ECX, which is a chemotherapy regimen comprising a combination of epirubicin, cisplatin and capecitabine; EOF is a chemotherapy regimen comprising a combination of epirubicin, oxaliplatin, and 5 -fluorouracil; FLO, which is a chemotherapy regimen comprising a combination of 5- fluorouracil, folinic acid, and oxaliplatin; FOLFOX, which is a chemotherapy regimen comprising a combination of folinic acid (leucovorin), 5 -fluorouracil, and oxaliplatin; SOX, which is a chemotherapy regimen comprising a combination of tegafur, gimeracil, oteracil, and oxaliplatin; CAPOX, which is a chemotherapy regimen comprising a combination of capecitabine and oxaliplatin; DCF, which is a chemotherapy regimen comprising a combination of docetaxel, cisplatin, and 5-fluorouracil; and FLOT, which is a chemotherapy regimen comprising a combination of docetaxel, oxaliplatin, 5- fluorouracil, and folinic acid. In certain embodiments, methods provided herein comprise a chemotherapy regimen selected from the group consisting of EOX chemotherapy, ECF chemotherapy, ECX chemotherapy, EOF chemotherapy, FLO chemotherapy, FOLFOX chemotherapy, SOX chemotherapy, CAPOX chemotherapy, DCF chemotherapy and FLOT chemotherapy.

[0089] In certain embodiments of the methods, compositions, and combinations, the chemotherapy comprises oxaliplatin and / or 5-fluorouracill (5-FU). In certain embodiments, the chemotherapy comprises gemcitabine and / or paclitaxel. In certain embodiments, the chemotherapy comprises gemcitabine and / or nab-paclitaxel. In certain embodiments, the chemotherapy comprises a CAPOX chemotherapy regimen.

[0090] In some embodiments of the methods, compositions, and combinations, the BsTCE is administered with an immune checkpoint inhibitor or antigen binding fragment thereof; and / or one or more chemotherapy agent(s). In some embodiments, the chemotherapy agent(s) is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline- containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine. In some embodiments of the methods, compositions, and combinations, the BsTCE is administered with an immune checkpoint inhibitor selected from a PD-1 or PD-L1 antibody or antigen binding fragment thereof; and / or one or more chemotherapy agent(s) selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed,CLDN18T-200-PCT01 gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine. In some embodiments of the methods, compositions, and combinations, the BsTCE is administered with an immune checkpoint inhibitor selected from a PD-1 or PD-L1 antibody or antigen binding fragment thereof; and one or more chemotherapy agent(s) selected from a fluoropyrimidine compound selected from 5 -fluorouracil (5-FU) and derivatives thereof, floxuridine, capecitabine, tegafur, and / or doxifluridine; a platinum-based chemotherapy selected from one of one or more of carboplatin, cisplatin, nedaplatin, and / or oxaliplatin; an anthracy cline- containing compound selected from daunorubicin, epirubicin, and / or doxorubicin, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine. In certain embodiments, the BsTCE is administered with an immune checkpoint inhibitor selected from a PD-1 or PD-L1 antibody or antigen binding fragment thereof; and one or more chemotherapy agent(s) selected from oxaliplatin and / or 5-fluorouracill (5-FU). In certain embodiments, the BsTCE is administered with an immune checkpoint inhibitor selected from a PD-1 or PD-L1 antibody or antigen binding fragment thereof; and one or more chemotherapy agent(s) selected from oxaliplatin and / or capecitabine. In certain embodiments, the BsTCE is administered with an immune checkpoint inhibitor selected from a PD-1 or PD-L1 antibody or antigen binding fragment thereof; and one or more chemotherapy agent(s) selected from gemcitabine and / or paclitaxel. In certain embodiments, the BsTCE is administered with an immune checkpoint inhibitor selected from a PD-1 or PD-L1 antibody or antigen binding fragment thereof; and one or more chemotherapy agent(s) selected from gemcitabine and / or nab-paclitaxel. In certain embodiments, the BsTCE is administered with an immune checkpoint inhibitor selected from a PD-1 or PD-L1 antibody or antigen binding fragment thereof; and one or more chemotherapy agent(s) selected from a CAPOX or FOLFOX chemotherapy regimen.Cancer and Cancer Therapy

[0091] As used herein, "cancer" is intended to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of their histopathological types or stages of invasiveness. Examples include, but are not limited to, solid tumors, hematologic cancer, soft tissue tumors and metastatic lesions. Claudin 18.2 (CLDN18.2) is highly expressed in several types of cancer, including gastric, esophageal, and pancreatic cancer, and its expression is maintained in metastases. CLDN18.2 isCLDN18T-200-PCT01 present in about 60% of gastric and gastroesophageal junction cancers (G / GEJCs), and aberrant expression has also been found in pancreatic cancer (PDAC), esophageal adenosarcoma (EAC), biliary tract cancer (BTC), and with less frequency, in ovarian, colorectal, and non-small cell lung cancers; whereas normal tissue expression of CLDN18.2 is highly limited to differentiated cells of the gastric epithelium where it is localized to tight junctions (Sahin etal., Clin. Cancer Res. 14(23): 7624-34 (2008); Shinozaki etal., Virchows Archiv. 459(1): 73-80 (2011); W611 et al., Int. J. Cancer 134(3): 731-39 (2013)). CLDN18.2-expressing cancers include, but are not limited to, solid tumors expressing CLDN18.2, for example, gastrointestinal cancer, gastric cancer, esophageal cancer, lung cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, pancreaticobiliary cancer, ovarian cancer, nonsmall-cell lung cancer, head and neck sarcoma, and melanoma. CLDN18.2 cancer also refers to metastatic lesions of the foregoing. In some embodiments, the methods, compositions, and combinations are useful for the treatment of a CLDN solid tumor cancer, wherein the cancer is selected from gastrointestinal cancer, ovarian cancer, lung cancer, renal cancer, hepatocellular cancer, breast cancer, bladder cancer, head and neck sarcoma, and melanoma. In certain embodiments, the methods, compositions, and combinations are useful for the treatment of a gastrointestinal cancer, wherein the gastrointestinal cancer is gastric cancer, pancreatic cancer, gastroesophageal junction cancer, biliary tract cancer, esophageal cancer, or colorectal cancer. In some embodiments, the methods, compositions, and combinations are useful for the treatment of gastric cancer (GC) or pancreatic ductal adenocarcinoma (PDAC).

[0092] Prognosis of gastric cancer is related to tumor extent and in localized distal gastric cancer the cure rate is above 50%. However, early-stage disease accounts for only 10-20% of all cases diagnosed in the United States. The remaining patients present with metastatic disease in either regional or distant sites. Gastric cancer is defined by stage and is based on TNM (tumor, node, metastasis) classification. The current gastric cancer standard of care regimen includes capecitabine plus oxaliplatin (CAPOX + anti-PD-1 therapy). CAPOX is a standard first-line treatment for advanced, unresectable, or metastatic gastric cancer. When combined with an anti- PD-1, the combination can improve overall survival. Further, immune checkpoint inhibitors plus cytotoxic drugs or dual blockade with ipilimumab and nivolumab, can be utilized, irrespective of PD-L1 expression. Such PD-L1 immune checkpoint inhibitors plus cytotoxic drugs offer a new standard of care immunotherapy in advanced gastric cancer. Cytotoxic regimens can include oneCLDN18T-200-PCT01 or more of CAPOX (capecitabine + oxaliplatin), XP (capecitabine + cisplatin), FOLFOX (fluoropyrimidine + leucovorin + oxaliplatin), SOX (S-l + oxaliplatin), or CF (cisplatin + fluoropyrimidine).

[0093] Clinical proof of concept for CLDN18.2 targeting has been demonstrated in G / GEJC with zolbetuximab, a murine-human chimeric IgGl antibody that binds CLDN18.2 and mediates tumor cell death through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). When combined with capecitabine and oxaliplatin (CAPOX) or with folinic acid, fluouracil, and oxaliplatin (FOLFOX), zolbetuximab showed improvements in progression-free survival as compared to with placebo plus CAPOX of FOLFOX in randomized phase III trials (Shah et al., Nat. Med. 29(8): 2133-41 (2023); Shitara et al., Lancet 401(10389): 1655-68 (2023)) leading to its approval in 2024 by the FDA. Other therapeutic modalities against CLDN18.2 are in clinical development such as ADCs and CAR- Ts (Qi etal., Nat. Med. 30(8): 2224-34 (2024); Xu et al., J. Clin. Oncol. 41(4_suppl): abstract 352 (2023)).

[0094] In an effort to target Gastric, GEJ, and pancreatic cancers, the disclosure describes a BsTCE targeting CD3 and CLDN18.2 to monovalently bind CD3 with low affinity via a Fab domain and bivalently bind CLDN18.2 with high affinity via dual antibody VH domains. This BsTCE has specific binding to CLDN18.2 and CD3, induces potent CLDN18.2-dependent and T cell- dependent cellular cytotoxicity (TDCC) in vitro, as well as bystander killing of CLDN18.2- negative cells only in the presence of CLDN18.2-positive cells. In vivo, the BsTCE induces potent tumor control in both humanized and syngeneic tumor models and increases T cell levels in treated tumors. When compared to a high CD3 affinity T cell engager, the BsTCE induces lower cytokine release in vitro and in vivo, indicative of providing an improved therapeutic index in the clinic.

[0095] As used herein, the terms "treat," "treatment," or "treating" when used in the context of treating cancer refer to reducing disease pathology, reducing or eliminating disease symptoms, promoting increased survival rates, and / or reducing discomfort. For example, treating can refer to the ability of a therapy when administered to a patient, to reduce disease symptoms, signs, or causes. Treating also refers to mitigating or decreasing at least one clinical symptom and / or inhibition or delay in the progression of the condition and / or prevention or delay of the onset of a disease or illness.CLDN18T-200-PCT01

[0096] As used herein, the terms "patient" or "subject" refer to any subject, particularly a human subject, for whom diagnosis, prognosis, or therapy is desired. In one aspect, methods of treating a patient identified as having a CLDN18.2-expressing tumor are provided.

[0097] The terms "administration" or "administering," as used herein, refer to providing, contacting, and / or delivering a compound or compounds by any appropriate route to achieve the desired effect. Administration may include, but is not limited to, oral, sublingual, parenteral (e.g., intravenous, subcutaneous, intracutaneous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, intratumoral, intralymphatic, or intracranial injection), transdermal, topical, buccal, rectal, vaginal, nasal, ophthalmic, via inhalation, and via an implantable. In some embodiments, one or more agent(s) is administered by intravenous infusion. In some embodiments, one or more agent is administered by subcutaneous or intramuscular injection. In some embodiments, one or more agent is administered by intratumoral or intralymphatic injection.

[0098] The terms "pharmaceutical composition" or "pharmaceutical combination," as used herein, refer to a composition or combination capable of inducing a desired therapeutic effect when properly administered to a subject. In one aspect, a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a BsTCE combination is provided. The terms "pharmaceutically acceptable carrier" or "physiologically acceptable carrier," as used herein, refer to one or more formulation materials suitable for accomplishing or enhancing delivery of one or more BsTCE combinations. In some embodiments, the BsTCE combinations disclosed herein is formulated with a pharmaceutically acceptable carrier, excipient, or stabilizer, as pharmaceutical compositions. In certain embodiments, such pharmaceutical compositions are suitable for administration to a human or non-human animal via any one or more routes of administration using methods known in the art. A pharmaceutically acceptable carrier includes one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable preparations may also contain compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. Other contemplated carriers, excipients, and / or additives, which are utilized in the formulations described herein include, for example, flavoring agents, antimicrobial agents, sweeteners,CLDN18T-200-PCT01 antioxidants, antistatic agents, lipids, protein excipients such as serum albumin, gelatin, casein, salt-forming counterions such as sodium, and the like. These and additional known pharmaceutical carriers, excipients, and / or additives suitable for use in the formulations described herein are known in the art, for example, as listed in "Remington: The Science & Practice of Pharmacy," 21st ed., Lippincott Williams & Wilkins, (2005), and in the "Physician's Desk Reference," 60th ed., Medical Economics, Montvale, NJ (2005). Pharmaceutically acceptable carriers are selected that are suitable for the mode of administration, solubility, and / or stability desired or required.

[0099] Pharmaceutical compositions and pharmaceutical combinations are contemplated by the disclosure. In some embodiments, the pharmaceutical compositions and pharmaceutical combinations are used for treating a CLDN18.2-expressing solid tumor in a patient in need thereof or for inducing cytotoxicity in a cell expressing CLDN18.2. Such pharmaceutical compositions and pharmaceutical combinations comprise a BsTCE agent; and an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s). In the pharmaceutical compositions and pharmaceutical combinations, the BsTCE agent comprises a first domain that binds to CLDN18.2; and a second domain that binds to cluster of differentiation 3 (CD3); wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNi8.2-VHcLDNi8.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL.

[0100] In some pharmaceutical compositions and pharmaceutical combinations, the immune checkpoint inhibitor agent is an immune checkpoint inhibitor antibody or antigen binding fragment thereof. In some pharmaceutical compositions and pharmaceutical combinations, the immune checkpoint inhibitor agent is a monoclonal or polyclonal antibody or antigen binding fragment thereof that specifically binds PD-1 or PD-L1.

[0101] In other pharmaceutical compositions and pharmaceutical combinations, the immune checkpoint inhibitor agent is an IO bispecific antibody or antigen binding fragment thereof comprising a first domain that binds PD-1 and a second domain that binds a T cell immunoreceptor with Ig and ITIM domain (TIGIT), a T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3), or a cytotoxic T-lymphocyte associated protein 4 (CTLA- 4). In one embodiment, the IO bispecific antibody that binds PD-1 and TIGIT is rilvegostomig.CLDN18T-200-PCT01In one embodiment, the IO bispecific antibody that binds PD-1 and TIM3 is sabestomig. In one embodiment, the IO bispecific antibody that binds PD-1 and CTLA-4 is volrustomig.

[0102] The pharmaceutical compositions and pharmaceutical combinations contemplated herein may also include one or more chemotherapy agents sekected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine. In certain embodiments, the fluoropyrimidine compound is one or more of 5-fluorouracil (5-FU) and derivatives thereof, floxuridine, capecitabine, tegafur, and / or doxifluridine. The platinum-based chemotherapy can include one or more of carboplatin, cisplatin, nedaplatin, and / or oxaliplatin. In certain embodiments, the anthracycline-containing compound is one or more of daunorubicin, epirubicin, and / or doxorubicin. In some pharmaceutical compositions and pharmaceutical combinations, the chemotherapy agent is oxaliplatin and / or 5-FU, gemcitabine and / or paclitaxel, and / or a CAPOX or FOLFOX chemotherapy regimen.

[0103] A variety of dosing regimens are contemplated by the disclosure. In some embodiments, the BsTCE is optionally administered with immune checkpoint inhibitor, and / or an IO-bispecific, and / or one or more chemotherapy agent(s). The chemotherapy agents can be administered prior to the BsTCE or prior to the administration of the BsTCE and the immune checkpoint inhibitor agent. The BsTCE and immune checkpoint inhibitor agent can also be administered following the last dose of the chemotherapy agents. The immune checkpoint inhibitor agent can be administered on the same day or alternate days as the BsTCE agent with the BsTCE and immune checkpoint inhibitor agent administered concurrently or in separate applications.

[0104] In one embodiment, the BsTCE agent, immune checkpoint inhibitor agent, and one or more chemotherapy agent(s) are administered sequentially. In one embodiment of sequential administration, the one or more chemotherapy agent(s) are administered on consecutive days according to a recommended dosing schedule followed by administration of the immune checkpoint inhibitor agent and / or the BsTCE agent. In one embodiment, the one or more chemotherapy agent(s) are oxaliplatin and capecitabine, wherein an initial dose of oxaliplatin and capecitabine is followed by three consecutive daily doses of capecitabine. The following day, the immune checkpoint inhibitor and BsTCE are administered followed by administration ofCLDN18T-200-PCT01 only the immune point inhibitor agent four days later. Administration of the immune checkpoint inhibitor agent and BsTCE agent is then repeated one week later.

[0105] In one embodiment, the BsTCE agent, immune checkpoint inhibitor agent, and one or more chemotherapy agent(s) are administered concurrently. In one embodiment of concurrent administration, the one or more chemotherapy agent(s), the BsTCE agent, and the immune checkpoint inhibitor agent are administered on the same day. In one embodiment, the one or more chemotherapy agent(s) are oxaliplatin and capecitabine, which are administered via recommended guidelines. Following the initial administration, capecitabine is administered for three consecutive days with an immune checkpoint inhibitor agent administered with the last dose of capecitabine. Four days later, the BsTCE agent and the immune checkpoint inhibitor agent are administered followed by the immune checkpoint inhibitor agent at 3 days, 7 days, and 10 days after administration of the BsTCE agent and immune checkpoint inhibitor agent.It is to be understood that particular aspects described herein are not limited to specific embodiments presented and can vary. It also will be understood that the terminology used herein is for the purpose of describing particular aspects and embodiments and, unless specifically defined herein, is not intended to be limiting. Moreover, particular embodiments disclosed herein can be combined with other embodiments disclosed herein, as would be recognized by a skilled person, without limitation.

[0106] Without limiting the disclosure, a number of embodiments are described below.Embodiments

[0107] Embodiment 1 : A method of treating a Claudin 18.2 (CLDN18.2)-expressing solid tumor in a patient in need thereof, the method comprising administering to the patient:(a) a bispecific T-cell engager (BsTCE) agent; and(b) an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s); wherein the BsTCE agent comprises:(i) a first domain that binds to CLDN18.2; and(ii) a second domain that binds to cluster of differentiation 3 (CD3); and wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNis.2-VHcLDNis.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL.CLDN18T-200-PCT01

[0108] Embodiment 2: A method of inducing cytotoxicity in a cell expressing Claudin 18.2 (CLDN18.2), the method comprising contacting the cell with:(a) a bispecific T-cell engager (BsTCE) agent;(b) an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s); wherein the BsTCE agent comprises:(i) a first domain that binds to CLDN18.2; and(ii) a second domain that binds to cluster of differentiation 3 (CD3); and wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNi8.2-VHcLDNis.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL.

[0109] Embodiment 3 : The method of either embodiment 1 or embodiment 2, wherein the immune checkpoint inhibitor agent is an immune checkpoint inhibitor antibody or antigen binding fragment thereof or an immune-oncologic (IO) bispecific antibody or antigen binding fragment thereof.

[0110] Embodiment 4: The method of any one of embodiments 1-3, wherein the solid tumor or the cell is gastrointestinal cancer, ovarian cancer, lung cancer, renal cancer, hepatocellular cancer, breast cancer, bladder cancer, head and neck sarcoma, or melanoma.

[0111] Embodiment 5 : The method of embodiment 4, wherein the gastrointestinal cancer is gastric cancer, pancreatic cancer, gastroesophageal junction cancer, biliary tract cancer, esophageal cancer, or colorectal cancer.

[0112] Embodiment 6: The method of embodiment 5, wherein the gastrointestinal cancer is gastric cancer (GC) or pancreatic ductal adenocarcinoma (PDAC).

[0113] Embodiment 7: The method of any one of embodiments 1-6, wherein the VHCLDNIS.2 variable heavy domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 5; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 6; and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 7; and wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1).

[0114] Embodiment 8: The method of any one of embodiments 1-7, wherein the VHCD3 variable heavy domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HCDR3CLDN18T-200-PCT01 comprising the amino acid sequence of SEQ ID NO: 11; and the VLCDS variable light domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 13; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 14; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 15; and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM.

[0115] Embodiment 9: The method of any one of embodiments 1-8, wherein:(a) the first polypeptide chain comprises two VHCLDNIS.2 domains comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 8;(b) the second polypeptide chain comprises a VHCD3 domain comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 12; and(c) the third polypeptide chain comprises a VLCD3 domain comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 16; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM.

[0116] Embodiment 10: The method of any one of embodiments 1-9, wherein the immune checkpoint agent comprises a domain that specifically binds PD-1 or PD-L1.

[0117] Embodiment 11 : The method of embodiment 10, wherein the immune checkpoint inhibitor agent is a monoclonal antibody or antigen binding fragment that specifically binds PD-1 or PD-Ll.

[0118] Embodiment 12: The method of embodiment 10, wherein the immune checkpoint inhibitor agent is a polyclonal antibody or antigen binding fragment thereof that specifically binds PD-1 or PD-L1.

[0119] Embodiment 13: The method of any one of embodiments 1-12, wherein the immune checkpoint inhibitor agent is an IO-bispecific antibody comprising:(a) a first domain that specifically binds PD-1; and(b) a second domain that specifically binds:CLDN18T-200-PCT01(i) a T cell immunoreceptor with Ig and ITIM domain (TIGIT);(ii) T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3); or(iii) cytotoxic T-lymphocyte associated protein 4 (CTLA-4).

[0120] Embodiment 14: The method of embodiment 13, wherein the IO-bispecific antibody specifically binds PD-1 and TIGIT.

[0121] Embodiment 15: The method of embodiment 14, wherein the IO-bispecific antibody is rilvegostomig.

[0122] Embodiment 16: The method of embodiment 13, wherein the IO-bispecific antibody specifically binds PD-1 and TIM3.

[0123] Embodiment 17: The method of embodiment 16, wherein the IO-bispecific antibody is sabestomig.

[0124] Embodiment 18: The method of embodiment 13, wherein the IO-bispecific antibody specifically binds PD-1 and CTLA-4.

[0125] Embodiment 19: The method of embodiment 18, wherein the IO-bispecific antibody is volrustomig.

[0126] Embodiment 20: The method of any one of embodiments 1-19, wherein the one or more chemotherapy agent is a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.

[0127] Embodiment 21 : The method of embodiment 20, wherein the one or more chemotherapy agent is a fluoropyrimidine compound selected from 5 -fluorouracil (5-FU) and derivatives thereof, floxuridine, capecitabine, tegafur, and / or doxifluridine; a platinum-based chemotherapy selected from one of one or more of carboplatin, cisplatin, nedaplatin, and / or oxaliplatin; and / or an anthracycline-containing compound selected from daunorubicin, epirubicin, and / or doxorubicin.

[0128] Embodiment 22: The method of embodiment 21, wherein the one or more chemotherapy agent is selected from oxaliplatin and / or 5-FU; gemcitabine and / or paclitaxel; and / or a CAPOX or FOLFOX chemotherapy regimen.CLDN18T-200-PCT01

[0129] Embodiment 23 : The method of any one of embodiments 1 -22, wherein the method comprises administering to the patient a bispecific T-cell engager (BsTCE) agent and one or more chemotherapy agent(s); wherein:(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; and wherein the one or more chemotherapy agent is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.

[0130] Embodiment 24: The method of any one of embodiments 1-22, wherein the method comprises administering to the patient a bispecific T-cell engager (BsTCE) agent and an immune checkpoint inhibitor agent; wherein:(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; andCLDN18T-200-PCT01 wherein the immune checkpoint inhibitor agent is an immune checkpoint antibody or antigen binding fragment thereof or an immune oncologic bispecific (IO-bispecific) antibody or antigen binding fragment thereof.

[0131] Embodiment 25: The method of any one of embodiments 1-22, wherein the method comprises administering to the patient a bispecific T-cell engager (BsTCE) agent, an immune checkpoint inhibitor agent, and one or more chemotherapy agent(s); wherein:(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; wherein the immune checkpoint inhibitor agent is an immune checkpoint antibody or antigen binding fragment thereof or an immune oncologic bispecific (IO-bispecific) antibody or antigen binding fragment thereof; and wherein the one or more chemotherapy agent is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.

[0132] Embodiment 26: The method of any one of embodiments 1-25, wherein the method results in one or more of:(a) an increase in T-cell activation;(b) an increase in circulating levels of pro-inflammatory cytokines;(c) an increase in tumor cell death; and / or(d) a reduction in tumor volume.

[0133] Embodiment 27: The method of embodiment 26, wherein the increased circulating levels of pro inflammatory cytokines include interferon gamma and / or Granzyme B.CLDN18T-200-PCT01

[0134] Embodiment 28: The method of any one of embodiments 1-27, wherein one or more agent(s) is administered by a route selected from oral, sublingual, parenteral, transdermal, topical, buccal, rectal, vaginal, nasal, ophthalmic, inhalate, and implantable.

[0135] Embodiment 29: The method of embodiment 28, wherein one or more agent(s) is administered by intravenous infusion.

[0136] Embodiment 30: The method of embodiment 28, wherein one or more agent(s) is administered by subcutaneous or intramuscular injection.

[0137] Embodiment 31 : The method of embodiment 28, wherein one or more agent(s) is administered by intratumoral or intralymphatic injection.

[0138] Embodiment 32: The method of any of embodiments 1-31, wherein the BsTCE agent, and the one or more chemotherapy agent(s), and / or the immune checkpoint inhibitor agent are administered sequentially.

[0139] Embodiment 33: The method of embodiment 1-32, wherein administration of the BsTCE agent is subsequent to the administration of one or more chemotherapy agent(s) and / or concurrently with the immune checkpoint inhibitor agent.

[0140] Embodiment 34: The method of embodiment 33, wherein the one or more chemotherapy agent(s) is administered prior to administration of the BsTCE agent and the immune checkpoint inhibitor agent.

[0141] Embodiment 35: The method of embodiment 34, wherein the BsTCE agent and the immune checkpoint inhibitor agent are administered subsequent to the last dose of the one or more chemotherapy agent(s).

[0142] Embodiment 36: The method of embodiment 35, wherein the immune checkpoint inhibitor agent is administered on the same day as the BsTCE agent.

[0143] Embodiment 37: The method of embodiment 35, wherein the immune checkpoint inhibitor agent is administered concurrently with the BsTCE agent on alternate administrations, and administration of the BsTCE agent is alone on other administrations.

[0144] Embodiment 38: The method of any one of embodiments 1-31, wherein the one or more chemotherapy agent(s), the BsTCE agent, and the immune checkpoint inhibitor agent are administered concurrently.CLDN18T-200-PCT01

[0145] Embodiment 39: The method of embodiment 38, wherein the chemotherapy agent(s), the BsTCE agent, and the immune checkpoint inhibitor agent are administered in separate applications.

[0146] Embodiment 40: The method of embodiment 38, wherein the concurrent administration comprises an initial administration of the one or more chemotherapy agent(s), the BsTCE agent, and the immune checkpoint inhibitor agent, followed by subsequent administration of the one or more chemotherapy agent(s) concurrently or sequentially with the immune checkpoint inhibitor agent, which are administered concurrently, sequentially, or without the BsTCE agent.

[0147] Embodiment 41 : A pharmaceutical combination comprising a bispecific T-cell engager (BsTCE) agent and a pharmaceutically acceptable carrier, an immune checkpoint inhibitor agent and a pharmaceutically acceptable carrier, and / or one or more chemotherapy agent(s); and a pharmaceutically acceptable carrier; wherein the BsTCE agent comprises:(a) a first domain that binds to Claudin 18.2 (CLDN18.2); and(b) a second domain that binds to cluster of differentiation 3 (CD3); wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNis.2-VHcLDNis.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL; wherein the immune checkpoint inhibitor is an immune checkpoint antibody or antigen binding fragment thereof or an immune oncologic bispecific (IO-bispecific) antibody or antigen binding fragment thereof; and wherein the one or more chemotherapy agent is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.

[0148] Embodiment 42: The pharmaceutical combination of embodiment 41, wherein the pharmaceutical composition comprises a bispecific T-cell engager (BsTCE) agent and a pharmaceutically acceptable carrier, and one or more chemotherapy agent(s); and a pharmaceutically acceptable carrier;CLDN18T-200-PCT01 wherein:(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; and wherein the one or more chemotherapy agent is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.

[0149] Embodiment 43: The pharmaceutical combination of embodiment 41, wherein the pharmaceutical composition comprises a bispecific T-cell engager (BsTCE) agent and a pharmaceutically acceptable carrier, and an immune checkpoint inhibitor agent and a pharmaceutically acceptable carrier; wherein:(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; and wherein the immune checkpoint inhibitor is an immune checkpoint antibody or antigen binding fragment thereof or an immune oncologic bispecific (IO-bispecific) antibody or antigen binding fragment thereof.CLDN18T-200-PCT01

[0150] Embodiment 44: The pharmaceutical combination of embodiment 41, wherein the pharmaceutical composition comprises a bispecific T-cell engager (BsTCE) agent and a pharmaceutically acceptable carrier, an immune checkpoint inhibitor agent and a pharmaceutically acceptable carrier, and one or more chemotherapy agent(s) and a pharmaceutically acceptable carrier; wherein:(a) the first polypeptide chain of the BsTCE agent comprises a SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; wherein the immune checkpoint inhibitor is an immune checkpoint antibody or antigen binding fragment thereof or an immune oncologic bispecific (IO-bispecific) antibody or antigen binding fragment thereof; and wherein the one or more chemotherapy agent is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.

[0151] Embodiment 45: A combination for use in the treatment of a Claudin 18.2 (CLDN18.2)-expressing solid tumor in a patient in need thereof, wherein the combination comprises:(a) a bispecific T-cell engager (BsTCE) agent; and(b) an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s); wherein the BsTCE agent comprises:(i) a first domain that binds to CLDN18.2; and(ii) a second domain that binds to cluster of differentiation 3 (CD3); andCLDN18T-200-PCT01 wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNi8.2-VHcLDNis.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL.

[0152] Embodiment 46: A combination for use in inducing cytotoxicity in a cell expressing Claudin 18.2 (CLDN18.2), wherein the combination comprises:(a) a bispecific T-cell engager (BsTCE) agent;(b) an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s); wherein the BsTCE agent comprises:(i) a first domain that binds to CLDN18.2; and(ii) a second domain that binds to cluster of differentiation 3 (CD3); and wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNi8.2-VHcLDNi8.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL.

[0153] Embodiment 47: The combination for use of either embodiment 45 or embodiment 46, wherein the immune checkpoint inhibitor agent is an immune checkpoint inhibitor antibody or antigen binding fragment thereof or an immune-oncologic (IO) bispecific antibody or antigen binding fragment thereof.

[0154] Embodiment 48: The combination for use of any one of embodiments 45-47, wherein the solid tumor or the cell is gastrointestinal cancer, ovarian cancer, lung cancer, renal cancer, hepatocellular cancer, breast cancer, bladder cancer, head and neck sarcoma, or melanoma.

[0155] Embodiment 49: The combination for use of embodiment 48, wherein the gastrointestinal cancer is gastric cancer, pancreatic cancer, gastroesophageal junction cancer, biliary tract cancer, esophageal cancer, or colorectal cancer.

[0156] Embodiment 50: The combination for use of embodiment 49, wherein the gastrointestinal cancer is gastric cancer (GC) or pancreatic ductal adenocarcinoma (PDAC).

[0157] Embodiment 51 : The combination for use of any one of embodiments 45-50, wherein the VHCLDNIS.2 variable heavy domain comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 5; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 6; and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 7; andCLDN18T-200-PCT01 wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1).

[0158] Embodiment 52: The combination for use of any one of embodiments 45-51, wherein the VHCDS variable heavy domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 11; and the VLCDS variable light domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 13; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 14; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 15; and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM.

[0159] Embodiment 53: The combination for use of any one of embodiments 45-52, wherein:(a) the first polypeptide chain comprises two VHCLDNIS.2 domains comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 8;(b) the second polypeptide chain comprises a VHCD3 domain comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 12; and(c) the third polypeptide chain comprises a VLCD3 domain comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 16; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM.

[0160] Embodiment 54: The combination for use of any one of embodiments 45-53, wherein the immune checkpoint agent comprises a domain that specifically binds PD-1 or PD-L1.

[0161] Embodiment 55: The combination for use of embodiment 54, wherein the immune checkpoint inhibitor agent is a monoclonal antibody or antigen binding fragment that specifically binds PD-1 or PD-L1.CLDN18T-200-PCT01

[0162] Embodiment 56: The combination for use of embodiment 54, wherein the immune checkpoint inhibitor agent is a polyclonal antibody or antigen binding fragment thereof that specifically binds PD-1 or PD-L1.

[0163] Embodiment 57: The combination for use of any one of embodiments 45-56, wherein the immune checkpoint inhibitor agent is an IO-bispecific antibody comprising:(a) a first domain that specifically binds PD-1; and(b) a second domain that specifically binds:(i) a T cell immunoreceptor with Ig and ITIM domain (TIGIT);(ii) T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3); or(iii) cytotoxic T-lymphocyte associated protein 4 (CTLA-4).

[0164] Embodiment 58: The combination for use of embodiment 57, wherein the IO- bispecific antibody specifically binds PD-1 and TIGIT.

[0165] Embodiment 59: The combination for use of embodiment 58, wherein the IO- bispecific antibody is rilvegostomig.

[0166] Embodiment 60: The combination for use of embodiment 57, wherein the IO- bispecific antibody specifically binds PD-1 and TIM3.

[0167] Embodiment 61 : The combination for use of embodiment 60, wherein the IO- bispecific antibody is sabestomig.

[0168] Embodiment 62: The combination for use of embodiment 57, wherein the IO- bispecific antibody specifically binds PD-1 and CTLA-4.

[0169] Embodiment 63: The combination for use of embodiment 62, wherein the IO- bispecific antibody is volrustomig.

[0170] Embodiment 64: The combination for use of any one of embodiments 45-63, wherein the one or more chemotherapy agent is a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.

[0171] Embodiment 65: The combination for use of embodiment 64, wherein the one or more chemotherapy agent is a fluoropyrimidine compound selected from 5 -fluorouracil (5-FU) and derivatives thereof, floxuridine, capecitabine, tegafur, and / or doxifluridine; a platinum-based chemotherapy selected from one of one or more of carboplatin, cisplatin, nedaplatin, and / orCLDN18T-200-PCT01 oxaliplatin; and / or an anthracycline-containing compound selected from daunorubicin, epirubicin, and / or doxorubicin.

[0172] Embodiment 66: The combination for use of embodiment 65, wherein the one or more chemotherapy agent is selected from oxaliplatin and / or 5-FU; gemcitabine and / or paclitaxel; and / or a CAPOX or FOLFOX chemotherapy regimen.

[0173] Embodiment 67: The combination for use of any one of embodiments 45-66, wherein the combination comprises a bispecific T-cell engager (BsTCE) agent and one or more chemotherapy agent(s); wherein:(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; and wherein the one or more chemotherapy agent is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.

[0174] Embodiment 68: The combination for use of any one of embodiments 45-66, wherein the combination comprises a bispecific T-cell engager (BsTCE) agent and an immune checkpoint inhibitor agent; wherein:(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; andCLDN18T-200-PCT01(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; and wherein the immune checkpoint inhibitor agent is an immune checkpoint antibody or antigen binding fragment thereof or an immune oncologic bispecific (IO-bispecific) antibody or antigen binding fragment thereof.

[0175] Embodiment 69: The combination for use of any one of embodiments 45-66, wherein the combination comprises a bispecific T-cell engager (BsTCE) agent, an immune checkpoint inhibitor agent, and one or more chemotherapy agent(s); wherein:(a) the first polypeptide chain of the BsTCE agent comprises a SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; wherein the immune checkpoint inhibitor agent is an immune checkpoint antibody or antigen binding fragment thereof or an immune oncologic bispecific (IO-bispecific) antibody or antigen binding fragment thereof; and wherein the one or more chemotherapy agent is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.

[0176] Embodiment 70: The combination for use of any one of embodiments 45-69, wherein administration of the combination results in one or more of:(a) an increase in T-cell activation;CLDN18T-200-PCT01(b) an increase in circulating levels of pro-inflammatory cytokines;(c) an increase in tumor cell death; and / or(d) a reduction in tumor volume.

[0177] Embodiment 71 : The combination for use of embodiment 70, wherein the increased circulating levels of pro inflammatory cytokines include interferon gamma and / or Granzyme B.

[0178] Embodiment 72: The combination for use of any one of embodiments 45-71, wherein administration of the one or more agent(s) is by a route selected from oral, sublingual, parenteral, transdermal, topical, buccal, rectal, vaginal, nasal, ophthalmic, inhalate, and implantable.

[0179] Embodiment 73: The combination for use of embodiment 72, wherein administration of the one or more agent(s) is by intravenous infusion.

[0180] Embodiment 74: The combination for use of embodiment 72, wherein administration of the one or more agent(s) is by subcutaneous or intramuscular injection.

[0181] Embodiment 75: The combination for use of embodiment 72, wherein administration of the one or more agent(s) is by intratumoral or intralymphatic injection.

[0182] Embodiment 76: The combination for use of any of embodiments 45-75, wherein the BsTCE agent, and the one or more chemotherapy agent(s), and / or the immune checkpoint inhibitor agent are administered sequentially.

[0183] Embodiment 77: The combination for use of any of embodiments 45-75, wherein the BsTCE agent is subsequent to the administration of the one or more chemotherapy agent(s) and / or concurrently with the immune checkpoint inhibitor agent.

[0184] Embodiment 78: The combination for use of embodiment 77, wherein administration of the one or more chemotherapy agent(s) occurs prior to administration of the BsTCE agent and the immune checkpoint inhibitor agent.

[0185] Embodiment 79: The combination for use of embodiment 78, wherein administration of the BsTCE agent and the immune checkpoint inhibitor agent occurs subsequent to administration of the last dose of the one or more chemotherapy agent(s).

[0186] Embodiment 80: The combination for use of embodiment 79, wherein administration of the immune checkpoint inhibitor agent occurs concurrent with administration of the BsTCE agent.CLDN18T-200-PCT01

[0187] Embodiment 81 : The combination for use of embodiment 79, wherein administration of the immune checkpoint inhibitor agent occurs concurrent with the BsTCE agent on alternate administrations, and administration of the BsTCE agent occurs alone on other administrations.

[0188] Embodiment 82: The combination for use of any one of embodiments 45-75, wherein administration of the one or more chemotherapy agent(s), the BsTCE agent, and the immune checkpoint inhibitor agent occurs concurrently.

[0189] Embodiment 83: The combination for use of embodiment 82, wherein administration of the chemotherapy agent(s), the BsTCE agent, and the immune checkpoint inhibitor agent occurs in separate applications.

[0190] Embodiment 84: The combination for use of embodiment 82, wherein concurrent administration of the one or more agents(s) comprises an initial administration of the one or more chemotherapy agent(s), the BsTCE agent, and the immune checkpoint inhibitor agent, followed by subsequent administration of the one or more chemotherapy agent(s) concurrently or sequentially with the immune checkpoint inhibitor agent, wherein administration occurs concurrently, sequentially, or without the BsTCE agent.

[0191] Embodiment 85: Use of a combination in the manufacture of a medicament for the treatment of a Claudin 18.2 (CLDN18.2)-expressing solid tumor in a patient in need thereof, wherein the combination comprises:(a) a bispecific T-cell engager (BsTCE) agent; and(b) an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s); wherein the BsTCE agent comprises:(i) a first domain that binds to CLDN18.2; and(ii) a second domain that binds to cluster of differentiation 3 (CD3); and wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNis.2-VHcLDNis.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL.

[0192] Embodiment 86: Use of a combination in the manufacture of a medicament for inducing cytotoxicity in a cell expressing Claudin 18.2 (CLDN18.2), wherein the combination comprises:(a) a bispecific T-cell engager (BsTCE) agent;CLDN18T-200-PCT01(b) an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s); wherein the BsTCE agent comprises:(i) a first domain that binds to CLDN18.2; and(ii) a second domain that binds to cluster of differentiation 3 (CD3); and wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNi8.2-VHcLDNis.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL.

[0193] Embodiment 87: The use of either embodiment 85 or embodiment 86, wherein the immune checkpoint inhibitor agent is an immune checkpoint inhibitor antibody or antigen binding fragment thereof or an immune-oncologic (IO) bispecific antibody or antigen binding fragment thereof.

[0194] Embodiment 88: The use of any one of embodiments 85-87, wherein the or the cell is gastrointestinal cancer, ovarian cancer, lung cancer, renal cancer, hepatocellular cancer, breast cancer, bladder cancer, head and neck sarcoma, or melanoma.

[0195] Embodiment 89: The use of embodiment 88, wherein the gastrointestinal cancer is gastric cancer, pancreatic cancer, gastroesophageal junction cancer, biliary tract cancer, esophageal cancer, or colorectal cancer.

[0196] Embodiment 90: The use of embodiment 89, wherein the gastrointestinal cancer is gastric cancer (GC) or pancreatic ductal adenocarcinoma (PDAC).

[0197] Embodiment 91 : The use of any one of embodiments 85-90, wherein the VHCLDNIS.2 variable heavy domain comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 5; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 6; and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 7; and wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1).

[0198] Embodiment 92: The use of any one of embodiments 85-91, wherein the VHCD3 variable heavy domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 11; and the VLCD3 variable light domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 13; an LCDR2CLDN18T-200-PCT01 comprising the amino acid sequence of SEQ ID NO: 14; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 15; and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM.

[0199] Embodiment 93: The use of any one of embodiments 85-92, wherein:(a) the first polypeptide chain comprises two VHCLDNIS.2 domains comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 8;(b) the second polypeptide chain comprises a VHCDS domain comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 12; and(c) the third polypeptide chain comprises a VLCD3 domain comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 16; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM.

[0200] Embodiment 94: The use of any one of embodiments 85-93, wherein the immune checkpoint agent comprises a domain that specifically binds PD-1 or PD-L1.

[0201] Embodiment 95: The use of embodiment 94, wherein the immune checkpoint inhibitor agent is a monoclonal antibody or antigen binding fragment that specifically binds PD- 1 or PD-Ll.

[0202] Embodiment 96: The use of embodiment 94, wherein the immune checkpoint inhibitor agent is a polyclonal antibody or antigen binding fragment thereof that specifically binds PD-1 or PD-L1.

[0203] Embodiment 97: The use of any one of embodiments 85-96, wherein the immune checkpoint inhibitor agent is an IO-bispecific antibody comprising:(a) a first domain that specifically binds PD-1; and(b) a second domain that specifically binds:(i) a T cell immunoreceptor with Ig and ITIM domain (TIGIT);(ii) T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3); orCLDN18T-200-PCT01(iii) cytotoxic T-lymphocyte associated protein 4 (CTLA-4).

[0204] Embodiment 98: The use of embodiment 97, wherein the IO-bispecific antibody specifically binds PD-1 and TIGIT.

[0205] Embodiment 99: The use of embodiment 98, wherein the IO-bispecific antibody is rilvegostomig.

[0206] Embodiment 100: The use of embodiment 97, wherein the IO-bispecific antibody specifically binds PD-1 and HM3.

[0207] Embodiment 101: The use of embodiment 100, wherein the IO-bispecific antibody is sabestomig.

[0208] Embodiment 102: The use of embodiment 97, wherein the IO-bispecific antibody specifically binds PD-1 and CTLA-4.

[0209] Embodiment 103: The use of embodiment 102, wherein the IO-bispecific antibody is volrustomig.

[0210] Embodiment 104: The use of any one of embodiments 85-103, wherein the one or more chemotherapy agent is a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.

[0211] Embodiment 105: The use of embodiment 104, wherein the one or more chemotherapy agent is a fluoropyrimidine compound selected from 5 -fluorouracil (5-FU) and derivatives thereof, floxuridine, capecitabine, tegafur, and / or doxifluridine; a platinum-based chemotherapy selected from one of one or more of carboplatin, cisplatin, nedaplatin, and / or oxaliplatin; and / or an anthracycline-containing compound selected from daunorubicin, epirubicin, and / or doxorubicin.

[0212] Embodiment 106: The use of embodiment 105, wherein the one or more chemotherapy agent is selected from oxaliplatin and / or 5-FU; gemcitabine and / or paclitaxel; and / or a CAPOX or FOLFOX chemotherapy regimen.

[0213] Embodiment 107: The use of any one of embodiments 85-106, wherein the combination comprises a bispecific T-cell engager (BsTCE) agent and one or more chemotherapy agent(s); wherein:CLDN18T-200-PCT01(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; and wherein the one or more chemotherapy agent is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.

[0214] Embodiment 108: The use of any one of embodiments 85-106, wherein the combination comprises a bispecific T-cell engager (BsTCE) agent and an immune checkpoint inhibitor agent; wherein:(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; and wherein the immune checkpoint inhibitor agent is an immune checkpoint antibody or antigen binding fragment thereof or an immune oncologic bispecific (IO-bispecific) antibody or antigen binding fragment thereof.CLDN18T-200-PCT01

[0215] Embodiment 109: The use of any one of embodiments 85-106, wherein the combination comprises a bispecific T-cell engager (BsTCE) agent, an immune checkpoint inhibitor agent, and one or more chemotherapy agent(s); wherein:(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; wherein the immune checkpoint inhibitor agent is an immune checkpoint antibody or antigen binding fragment thereof or an immune oncologic bispecific (IO-bispecific) antibody or antigen binding fragment thereof; and wherein the one or more chemotherapy agent is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.

[0216] Embodiment 110: The use of any one of embodiments 85-109, wherein administration of the combination results in one or more of:(a) an increase in T-cell activation;(b) an increase in circulating levels of pro-inflammatory cytokines;(c) an increase in tumor cell death; and / or(d) a reduction in tumor volume.

[0217] Embodiment 111: The use of embodiment 110, wherein the increased circulating levels of pro inflammatory cytokines include interferon gamma and / or Granzyme B.

[0218] Embodiment 112: The use of any one of embodiments 85-111, wherein administration of the one or more agent(s) is by a route selected from oral, sublingual, parenteral, transdermal, topical, buccal, rectal, vaginal, nasal, ophthalmic, inhalate, and implantable.CLDN18T-200-PCT01

[0219] Embodiment 113: The use of embodiment 112, wherein administration of the one or more agent(s) is by intravenous infusion.

[0220] Embodiment 114: The use of embodiment 112, wherein administration of the one or more agent(s) is by subcutaneous or intramuscular injection.

[0221] Embodiment 115: The use of embodiment 112, wherein administration of the one or more agent(s) is by intratumoral or intralymphatic injection.

[0222] Embodiment 116: The use of any of embodiments 85-115, wherein the BsTCE agent, the one or more chemotherapy agent(s), and / or the immune checkpoint inhibitor agent are administered sequentially.

[0223] Embodiment 117: The use of any of embodiments 85-115, wherein administration of the BsTCE agent is subsequent to the administration of the one or more chemotherapy agent(s) and / or concurrently with the immune checkpoint inhibitor agent.

[0224] Embodiment 118: The use of embodiment 117, wherein administration of the one or more chemotherapy agent(s) occurs prior to administration of the BsTCE agent and the immune checkpoint inhibitor agent.

[0225] Embodiment 119: The use of embodiment 118, wherein administration of the BsTCE agent and the immune checkpoint inhibitor agent occurs subsequent to administration of the last dose of the one or more chemotherapy agent(s).

[0226] Embodiment 120: The use of embodiment 119, wherein administration of the immune checkpoint inhibitor agent occurs concurrently with administration of the BsTCE agent.

[0227] Embodiment 121: The use of embodiment 119, wherein administration of the immune checkpoint inhibitor agent occurs concurrent with the BsTCE agent on alternate administrations, and administration of the BsTCE agent occurs alone on other administrations.

[0228] Embodiment 122: The use of any one of embodiments 85-115, wherein administration of the one or more chemotherapy agent(s), the BsTCE agent, and the immune checkpoint inhibitor agent occurs concurrently.

[0229] Embodiment 123: The use of embodiment 122, wherein administration of the chemotherapy agent(s), the BsTCE agent, and the immune checkpoint inhibitor agent occurs in separate applications.

[0230] Embodiment 124: The use of embodiment 122, wherein concurrent administration of the one or more agents(s) comprises an initial administration of the one or more chemotherapyCLDN18T-200-PCT01 agent(s), the BsTCE agent, and the immune checkpoint inhibitor agent, followed by subsequent administration of the one or more chemotherapy agent(s) concurrently or sequentially with the immune checkpoint inhibitor agent, wherein administration occurs concurrently, sequentially, or without the BsTCE agent.EXAMPLES

[0231] The claimed invention is further illustrated by the following Examples, which should not be construed as limiting. bsTCE induces T cell activation, cytokine secretion, and tumor cell cytotoxicity in vitro, and in vivo, which effects are further potentiated in combination with clinically relevant chemotherapies and / or immunotherapies (e.g., rilvegostomig). Those of skill in the art will recognize that the claimed invention may be practiced with variations of the disclosed materials, formulations, and methods, and such variations are regarded as within the scope of the claimed invention.

[0232] General Methods: bsTCE cytotoxicity was assessed in vitro using gastric and PDAC cell lines. Combinatorial activity was assessed with 5-FU / oxaliplatin, gemcitabine / paclitaxel, and immunotherapies, e.g., volrustomig, sabestomig, rilvegostomig across multiple rounds of bsTCE treatment. Cytokine secretion was measured by ELISA, and T cell phenotypes were analyzed using spectral flow cytometry. In vivo efficacy was assessed in C57BL / 6-hCD3 mice bearing MC38-mCLDN18.2 tumours.

[0233]

[0234] Example 1: BsTCE Activates CD3 cells in Tumors and Tumor Draining Lymph Nodes

[0235] Human CD3 (hCD3) transgenic mice were implanted subcutaneously with B- hCLDN18.2-MC38 cells for three weeks, followed by administration of a control treatment or 0.1 mg / kg, 1 mg / kg, or 5 mg / kg of a CLDN18.2xCD3 bispecific T cell engager (BsTCE) for 72 hours. The total number of CD45+ T-cells significantly decreased in tumor DLn (draining lymph node) with greatest reduction of CD45+ T-cells at 5 mg / kg (FIG. 1A). Treatment with the CLDN18.2xCD3 BsTCE induced an increase of CD8+ T-cells within the tumor (FIG. IB), while CD4+ T-cells significantly decreased in both the DLn and tumor (FIG. 1C). Further, the ratio of CD8 to CD4 cells increased in both DLn and tumors, with the most significant increaseCLDN18T-200-PCT01 for 5 mg / kg treatment in DLn (FIG. ID). Finally, the CLDN18.2xCD3 BsTCE treatment induced significant increases of both NK cells (FIG. IE) and MHCII+CD86+ B-cells (FIG. IF) significantly increased in the tumor, with the greatest significant increase induced by the 5 mg / kg treatment.

[0236] Intratumoral T-cells see a significant increase in GranzymeB for all CLDN18.2xCD3 BsTCE treatment groups (FIG. 1G). While the percentage of CD69+ T-cells increased for all experimental treatment groups, the most significant increase occurred in the 1 mg / kg treatment (FIG. 1H) The percentage of CD8 cells with positive expression of Ki67 was consistent among all groups except 1 mg / kg treatment, which had a significantly higher population of T-cells (FIG. II). Finally, T-cells expressing PD-1+ increased with the dosage of CLDN18.2xCD3 BsTCE treatment, with the greatest significance between the control and 5 mg / kg treatment (FIG. 1J)

[0237] Example 2: Serum Cytokine and Chemokine Changes Following BsTCE Administration

[0238] Human CD3 (hCD3) transgenic mice were subcutaneously implanted with MC38- mCLDN18.2 tumors for two weeks, followed by receiving either a placebo or 0.2 mg / kg, 0.5 mg / kg, or 1 mg / kg dose of the CLDN18.2xCD3 BsTCE. IFNg, CXCL9, and CXCL10 were each measured (pg / mL) at 6-, 24-, and 48-hours following treatment. Both IFNg (FIG. 2A) and CXCL9 (FIG. 2B) induced significant increases in the first 24 hours, followed by a reduction observed at 48 hours. Conversely, CXCL10 demonstrated comparable levels for the first 6 hours, a significant increase at 24 hours and further increased levels of CXCL10 at 48 hours (FIG. 2C)

[0239] Example 3: Immnophenotyping of Ex Vivo Cancer Gastric Cancer Samples

[0240] Tumor resections were obtained from gastric and pancreatic cancer patients and 6 mm punch biopsy explants were prepared. Explants were then embedded in agarose and generated in 300 mM slices. Resulting slices were cultured ex vivo in the presence of the CLDN18.2xCD3 BsTCE for 2-3 days (FIG. 3A).

[0241] Gastric cancer patient samples were analyzed by immune cell composition or by the percentage of cell population of CD8+ and CD4+ T-cells expressing different cellular markers (FIG. 3B) The highest populations of immune cells pre-treatment were CD3+CD45+ T-cells and CD3+CD4+ T-cells (FIG. 3B). Among CD8+ T-cells, the highest population (greater thanCLDN18T-200-PCT0160%) of T-cells were CD69+, PD-1+, or TIGIT+ (FIG. 3B). In CD4+ T-cells, ICOS+ or PD-1+ expression was highest in pretreated gastric cancer cells (FIG. 3B).

[0242] After treatment with the CLDN18.2xCD3 BsTCE, there was substantial fold change of cytokine secretion compared to the untreated biopsies of both gastric cancer (FIG. 3C) and pancreatic cancer (FIG. 3D). After treatment with the CLDN18.2xCD3 BsTCE for 2 or 3 days, IFNg, GranzymeB, Perforin, and TNFa secretion increased at a 10-fold to 1000-fold increase for IFNg, a 10 fold to 100 fold increase of GranzymeB, and approximately a 10 fold increase of both Perforin and TNFa secretion in gastric cancer (FIG. 3C) and pancreatic cancer (FIG. 3D).

[0243] The log2 fold change correlations were analyzed examining IFNg with CLDN18.2+ cells by flow cytometry (FIG. 3E) or IHC (FIG. 3F), as well as CD3+ T-cell counts vs IFNg by IHC (FIG. 3G) of gastric cancer patient-derived explants. Baseline CLDN18.2 expression (FIG. 3E-3F), but not CD3+ T-cell content (FIG. 3G) is significantly correlated with CLDN18.2xCD3 BsTCE -induced secretion of IFNg.

[0244] Example 4: BsTCE Administration in Combination with Anti-PD-1 Monoclonal Antibody Administration and Chemotherapy In Vitro and In Vivo

[0245] A, In vitro NUGC4 gastric cancer cells were co-cultured in vitro with PBMCs to analyze cytokine secretion and marker expression of CD8+ T-cells (FIG 4). PBMCs were preactivated by coculturing with NUGC4 tumor cells at a 4: 1 E:T ratio in the presence of the CLDN18.2xCD3 BsTCE for 7 days. After 7 days, the activated PBMCs were collected and reseeded with fresh NUGC4 cells for 48 hours in the presence of treatments. There was a significant increase of GranzymeB (FIG. 4A), CD25+ (FIG. 4C), and 41BB- marker expression (FIG. 4D) after exposure to BsTCE in combination with an anti-PD-1 treatment versus the CLDN18.2xCD3 BsTCE alone. IFNg (FIG. 4B) also increased with the anti-PD-1 combination treatment compared to the CLDN18.2xCD3 BsTCE treatment alone.

[0246] B, In vivo The combination treatment was further studied by incorporating gastric cancer standard of care regimen (CAPOX + anti-PD-1 treatment) in hCD3deg mice. Mice were implanted with 1.5 xl 06MC38-mCLDN 18.2 cells. Mice treated with placebo CLDN 18.2xCD3 BsTCE, anti-mPD-1, and CLDN18.2xCD3 BsTCE + anti-mPD-1 treatments were evaluated for tumor volume over 4 weeks (FIG 5A). There was a highly significant reduction in tumor volume (mm3) between the placebo and the CLDN18.2xCD3 BsTCE treatment, which was further decreased when the anti-PD-1 treatment also was incorporated (FIG. 5A). WhenCLDN18T-200-PCT01 comparing the placebo group to Oxaliplatin + Capecitabine (CAPOX), CLDN18.2xCD3 BsTCE, CAPOX + BsTCE, and CAPOX + BsTCE + Anti-mPD-1, the tumor volume was flat and significantly reduced versus the placebo and other treatment groups over 4 weeks (FIG. 5B).

[0247] Finally, the growth rate was also analyzed among these treatment groups. The combination of CAPOX + the CLDN18.2xCD3 BsTCE + anti-mPD-1 treatment had a negative tumor growth rate compared to the other treatments or the placebo group (FIG. 5C).

[0248] Example 5: BsTCE Combination of BsTCE with Anti-PD-1, lO-Bispecific Antibodies and Chemotherapy In Vitro and Ex Vivo

[0249] NUGC4 gastric cancer cells were co-cultured with healthy donor PBMCs in the presence of the CLDN18.2xCD3 BsTCE. After 7 days, the activated PBMCs were collected and co-cultured with fresh NUGC4 cancer cells in the presence of the CLDN18.2xCD3 BsTCE in combination with an anti-PD-1 mAb, rilvegostomig (a bispecific antibody targeting PD-1 and TIGIT), sabestomig (bispecific for PD-1 and TIM3), or volrustomig (bispecific for PD-1 and CTLA-4). The treatment combinations were further evaluated for efficacy with the addition of Oxaliplatin and 5-FU chemotherapeutic agents. After 48 hours, IFNg and GranzymeB levels in supernatants was assessed by ELISA.

[0250] IFNg and GranzymeB secretion (pg / mL) was assessed between the CLDN18.2xCD3 BsTCE, the CLDN18.2xCD3 BsTCE + chemotherapy, BsTCE + IO, and BsTCE + chemotherapy + IO treatments with varying concentrations of the CLDN18.2xCD3 BsTCE (nM) (FIG. 6A, 6C). While chemotherapy only modestly increased IFNg and GranzymeB, the addition of bispecific antibodies greatly increases secretion (FIG. 6A, 6C).

[0002] Twelve donor pairs treated with 10 nM the CLDN18.2xCD3 BsTCE and IO- bispecific antibodies were then analyzed for the fold change of IFNg (FIG. 6B) and GranzymeB (FIG. 6D) relative to the CLDN18.2xCD3 BsTCE treatment alone. The addition of bispecific antibodies induced a positive fold change compared to the CLDN18.2xCD3 BsTCE alone (FIG.6B, 6D)

[0251] Tumor viability also was assessed between the CLDN18.2xCD3 BsTCE, chemotherapy, and the bispecific drug treatment groups (FIG. 7A). While chemotherapy decreased the overall percentage of tumor cell viability, the viability decreases most in the presence of bispecific antibodies (FIG. 7A). Tumor cell viability decreased most with the combination of the CLDN18.2xCD3 BsTCE, chemotherapy, and sabestomig (FIG. 7B).CLDN18T-200-PCT01

[0252] The effects of cytokine secretion from intratumoral gastric cancer patient samples were examined in the presence combination of the CLDN18.2xCD3 BsTCE with IO-bispecific antibodies. Gastric samples were processed into 2 mM thin slices with the CLDN18.2xCD3 BsTCE alone, IO-bispecifics alone, NTP-TCE (CD3-only binding isotype control), or combinations of the CLDN18.2xCD3 BsTCE and IO-bispecifics. The CLDN18.2xCD3 BsTCE combined with IO-bispecifics induced increased secretion of IFNg (FIG. 8A) and Granzyme B (FIG. 8B) from selected patient samples, compared to the CLDN18.2xCD3 BsTCE sole administration. Specifically, for IFNg, there was an observed increase in 2 / 5 samples treated with the CLDN18.2xCD3 BsTCE and rilvegostomig, in 4 / 4 samples treated with the CLDN18.2xCD3 BsTCE and sabestomig, and in 3 / 4 samples treated with the CLDN18.2xCD3 BsTCE and volrustomig (FIG. 8A).

[0253] Example 6: Combining BsTCE Administration with Immunotherapy and Chemotherapy in hCD3deg Mice

[0254] The CLDN18.2xCD3 BsTCE was combined with anti-mouse PD-1 and rilvegostomig to evaluate tumor control in hCD3deg mice. Mice were implanted with 1.5 x 106MC38- mCLDN18.2 cells and tumor volumes (mm3) were evaluated over 4 weeks.

[0255] The combination of the CLDN18.2xCD3 BsTCE and anti-mPD-1 administration showed significantly lower tumor volume compared to the CLDN18.2xCD3 BsTCE or anti- mPD-1 sole treatments and isotype control (FIG. 9A). Concurrently, the CLDN18.2xCD3 BsTCE and mCLDN18.2xCD3 BsTCE rilvegostomig combination administration also maintained significantly lower tumor volume compared to the CLDN18.2xCD3 BsTCE or mCLDN18.2xCD3 BsTCE sole administration and isotype controls (FIG. 9A). The combination administration further demonstrated high efficacy with six of the 10 experimental mice showing a complete response with treatment versus mCLDN18.2xCD3 BsTCE sole administration (5 / 10) or combination administration with anti-mPD-1 (2 / 10) (FIG. 9B). The anti-mPD-1 sole administration and the CLDN18.2xCD3 BsTCE sole administration had comparable efficacy to the isotype control with zero of the ten subjects receiving treatment achieved a complete response (FIG. 9B)

[0256] The CLDN18.2xCD3 BsTCE was next combined with capecitabine, oxaliplatin (CAPOX), and anti-PD-1 in vivo using hCD3deg mice implanted with 1.5 x 106MC38- mCLDN18.2 cells. Four concurrent or sequential regimens were implemented (FIG. 10A) andCLDN18T-200-PCT01 resulted in significant tumor volume compared to the CLDN18.2xCD3 BsTCE alone in both concurrent and sequential schedules (FIG. 10B). Inclusion of anti-PD-1 in these regimens results in prolonged tumor control (beyond day 25) compared to the CLDN18.2xCD3 BsTCE alone or in quadruple combination with chemotherapy. Additionally, the concurrent quadruple combo led to 3 / 10 complete responses (FIG. 10C).

[0257] Finally, the effects of combining the CLDN18.2xCD3 BsTCE with gemcitabine using hCD3deg mice implanted with 1.5 x 106MC38-mCLDN18.2 cells was evaluated. Combination administration of the CLDN18.2xCD3 BsTCE with gemcitabine resulted in 8 / 10 complete responses compared to 0 / 10 responses in the CLDN18.2xCD3 BsTCE sole administration or 7 / 10 complete responses in gemcitabine treatment (FIG. 11B). Though there is not a significant difference in tumor volumes, the CLDN18.2xCD3 BsTCE, combination with gemcitabine led to no tumors regrowing above 500 mm3 at the end of the experiment compared to two in the gemcitabine sole administration group (FIG. 11A).

[0258] Example 7: BsTCE Administration with Chemotherapy

[0259] A, Gemcitabine increases CLDN18.2 expression on pancreatic cancer cells 300,000 PaTu-8988s cells (a pancreatic adenocarcinoma cell line) were plated in each well of a 6-well plate and treated with various concentrations of Gemcitabine (Gem) or Paclitaxel (Pac) for 48h. CLDN18.2 expression was detected by flow cytometry and technical replicates were plotted as Mean Fluorescence Intensity (MFI). Gemcitabine but not paclitaxel induced a dose dependent increase in CLDN18.2 expression. Representative example n=2. See FIG. 12A. Gemcitabine, but not paclitaxel) upregulated CLDN18.2 expression in vitro.

[0260] a BsTCE effectively combines with gemcitabine and paclitaxel in in vitro cytotoxicity assays 10.000 PaTu-8988s cells were plated overnight in each well of a 96-well plate, PBMCs were added (4: 1 E:T), and treated with various concentrations of bsTCE + / - Gemcitabine (n=4) or Paclitaxel (n=3) for 7 days. PaTu-8988s confluence was detected via incucyte every 2h, plotted over time, and cytotoxicity represented as AUC. BsTCE, Gemcitabine, and Paclitaxel all demonstrate activity alone, with additive activity detected when treated in combination. BsTCE potency increases in combination with either gemcitabine or paclitaxel, with notable increases in BsTCE activity detected in combination with gemcitabine at low concentrations of gemcitabine that do not demonstrate activity alone. See FIG. 12B and FIG. 12C. Mean EC50 values are shown in the following Table 1.CLDN18T-200-PCT01Table 1: Mean EC 50IBGemcitabine 10 1 0.1 0.01 No(uM) chemoBsTCE 0.22 0.11 0.08 0.05 0.17(nM)1CPaclitaxel 1 0.01 0.0001 No(uM) chemoBsTCE 0.03 0.02 0.09 0.11(nM)

[0261] C. 5-FU / Oxaliplatin increases CLDN18.2 expression on NUGC4 cells 240.000 NUGC4 cells were plated overnight in each well of a 12- well plate and then treated with luM, 0.1 uM, O.OluM (left to right) of 5-FU / oxaliplatin (F / OX) for 48h. CLDN18.2 expression was detected by flow cytometry and technical replicates are plotted as Mean Flourescence Intensity (MFI). 5- FU and oxaliplatin alone, or in combination induced a dose dependent increase in CLDN18.2 expression. n=3. See FIG. 13A.

[0262] BsTCE effectively combines with 5-FU / oxaliplatin in in vitro cytotoxicity assays

[0263] 10,000 NUGC4 cells were plated overnight in each well of a 96-well plate, PBMCs added (4:1 E:T), and treated with indicated concentrations of BsTCE + / - 5-FU+oxaliplatin (equimolar) for 7 days (n=5). NUGC4 confluence was detected via incucyte every 2h, plotted over time, and T cell-dependent cellular cytotoxicity (TDCC) represented as AUC. BsTCE and 5-FU / oxaliplatin both demonstrate activity alone, with additive activity detected in combination. BsTCE potency increases in combination with 5-FU / oxaliplatin. Increased BsTCE activity in combination with 5-FU / oxaliplatin occurs at non-active concentrations of 5-FU / oxaliplatin. See FIG. 13B. Mean EC50 values shown in the Table 2 below. Mean values + / - SEM from n=5 shown.

[0264] Table 2: Mean EC50F / OX 10 1 0.1 0.01 No(uM) chemoCLDN18T-200-PCT01BsTCE 0.01466 0.009915 0.008794 0.01137 0 01285(nM)

[0265] : E, BsTCE combination with 5-FU / oxaliplatin is tolerable across repetitive stimulations NUGC4 cells were plated in flasks, incubated overnight, PBMCs added (2:1 E:T) and treated with various concentrations of BsTCE, + / - 0.1 uM 5-FU+oxaliplatin (equimolar) for 7 days (1 pre-stim) or 14 days (2 pre-stim). After 7 or 14 days, cells were harvested and a T celldependent cellular cytotoxicity (TDCC) assay performed as described above at E:T 4: 1 with indicated concentrations of BsTCE alone (FIG 13C) or in the presence of 0.1 uM 5- FU+oxaliplatin (equimolar; FIG 13D) for 7 days (n=3). NUGC4 confluence was detected via incucyte every 2h, plotted over time, and cytotoxicity represented as AUC. bsTCE and 5- FU / oxaliplatin both demonstrate activity alone, with additive activity detected in combination. Inclusion of 5-FU / oxaliplatin in flask stimulations for 2 weeks had no impact on subsequent activity with BsTCE, 5-FU / oxaliplatin, or their additive activities. Mean values + / - SEM from n=3 1 shown. See FIG. 13D. TDCC assay with bsTCE alone using pre-stimulated PBMCs See FIG13C and TDCC assay with bsTCE + O.luM 5-FU+oxaliplatin using pre-stimulated PBMCs See FIGI 3D.

[0266] In sum, administration of chemotherapy agents, 5FU, Oxaliplatin combination, or Gemcitabine, but not paclitaxel, upregulated CLDN18.2 expression in gastric or PDAC cell lines in vitro. See Figs 12A-13A. Combination of bsTCE with chemotherapy agents increased bsTCE potency (See Figs 12B, 12C, 13B); and in repetitive stimulation assays, bsTCE-activated T cells maintained cytotoxicity for up to three weeks even with continuous exposure to 5-FU and oxaliplatin (See Fig 13C-13D).

[0267] Example 8: BsTCE Administration with Immunotherapy

[0268] A, Rilvegostomig enhances bsTCE-induced expression of T cell activation markers and leads to blockade of PD1 and TIGIT on T cells in repetitive stimulation assay PBMCs were stimulated with O.lnM bsTCE in flasks for 7, 14 or 21 days alone (grey) or together with 200nM Rilvegostomig (black) at a 2: 1 E:T ratio. At baseline (before treatment) or at the end of each 7 day period, as indicated in plots (FIG 14A-FIG 14L), PBMCs were harvested and activation marker expression on CD8 or CD4 T cells analysed by flow cytometry. Mean values + / - SEM from n=5 shown. See FIG 14A-FIG 14L.CLDN18T-200-PCT01

[0269] B: Fig 14B: Rilvegostomig. Volrustomig or anti-PDl enhance secretion of IFNg and Granzyme-B from T cells exposed to bsTCE once a week for 3 weeks (repetitive stimulation assay) PBMCs were stimulated with 0.1 nM bsTCE alone or in combination with Rilvegostomig, Volrustomig or a monoclonal anti-PDl having the same specificity as the PD1 directed arm of Rilvegostomig and Volrustomig in flasks for 7, 14 or 21 days (number of flask stimulation indicated by Stim 1, 2, 3, respectively). PBMCs were harvested after each stimulation and a bsTCE TDCC performed with NUGC4-GFP cells alone or in combination with Rilvegostomig, Volrustomig or anti-PDl. Supernatants were collected after 6 days and assessed for IFNg and GranzymeB concentrations. Mean values from n=3 shown. See FIG 15A-15D.

[0270] C: Rilvegostomig. Volrustomig or anti-PDl enhance bsTCE-mediated cytotoxicity of NUGC4-GFP tumour cells, partially rescuing functional decline in repetitive T cell stimulation assays PBMCs were stimulated with 0.1 nM bsTCE alone or in combination with Rilvegostomig, Volrustomig or a monoclonal anti-PDl having the same specificity as the PD1 directed arm of Rilvegostomig and Volrustomig in flasks for 7, 14 or 21 days (number of flask stimulation indicated by Stim 1, 2, 3, respectively). PBMCs were harvested after each stimulation and a bsTCE TDCC performed with NUGC4-GFP cells alone or in combination with Rilvegostomig, Volrustomig or anti-PDlby incucyte, and data presented as AUC normalised to untreated. Mean values from n=3 (volrustomig (FIG 15G), anti-PDl (FIG 15E) combinations) or n=5 (rilvegostomig (FIG 15F) combination) shown. See FIG 15E-15G.

[0271] bsTCE treatment in vitro induced T cell expression of activation and co-stimulatory markers, as well as PD-1 and TIGIT expression (FiG 14A). In vitro, rilvegostomig led to PD1- TIGIT blockade, as measured by flow cytometry using competitive antibodies (Fig 14A). Combination with rilvegostomig enhanced bsTCE-induced expression of T cell activation markers in repetitive T cell stimulation assays (Fig 14A). Combination with rilvegostomig, volrustomig or anti-PDl led to increased cytokine secretion (Fig 14B) and cytotoxicity (Fig 14C), and partially rescued functional decline in repetitive T cell stimulation assays (Figs 14B,C).

[0272] Example 9: BsTCE Administration with Immunotherapy and Chemotherapy

[0273] A, bsTCE in vitro combinations with 5-FU, oxaliplatin and rilvegostomig leads to superior cytotoxicity compared to single agent administration NUGC4 cells were plated in flasks, incubated overnight, PBMCs added (2: 1 E:T) and treated BsTCE used at 0.1 nM for 7CLDN18T-200-PCT01 days. After that, the pre-stimualted PBMCs were collected and cultured in a 1:4 ratio with 10,000 fresh NUGC4-GFP cells and treated with bsTCE in the presence of suboptimal bsTCE concentration (donor dependent), 200nM rilvegostomig, and 0.1 pM 5-FU + oxaliplatin. Images were acquired by incucyte every 2h for 6 days. AUC, representing cytotoxicity, was quantified and shown as bar graphs (left) with mean values + / - SEM from n=5 shown. NUGC4-GFP area over time normalised to Oh timepoint, is shown on the right. See FIG 16A-16B. In vitro, combination of suboptimal concentration of bsTCE with suboptimal concentration of 5-FU and oxaliplatin and rilvegostomig, led to increased cytotoxicity of NUGC4 tumour cells (See Fig 16A-B).

[0274] B: Inactive concentrations of bsTCE 5-FU, oxaliplatin and rilvegostomig. in combination, lead to superior cytotoxicity compared to single agent administration, in vitro NUGC4 cells were plated in flasks, incubated overnight, PBMCs added (2:1 E:T) and treated BsTCE used at 0.1 nM for 7 days. After that, the pre- stimulated PBMCs were collected and cultured in a 1:4 ratio with 10,000 fresh NUGC4-GFP cells and treated with bsTCE in the presence of inactive bsTCE concentration (donor dependent), 200nM rilvegostomig, and 0.01 pM 5-FU + oxaliplatin. Images were acquired by incucyte every 2h for 6 days. AUC was quantified and shown as bar graphs (left) with mean values + / - SEM from n=5 shown. NUGC4-GFP area over time, normalised to Oh timepoint, is shown on the right. See FIG 17A-17B. Combination of bsTCE with 5-FU and oxaliplatin, as well as with rilvegostomig, all used at concentrations that are not active as single agent administration, led to increased cytotoxicity of NUGC4 tumour cells (See Fig 17A-B).

[0275] Example 10: BsTCE Administration with Immunotherapy and Chemotherapy

[0276] bsTCE concurrent or alternating combinations with 5-FU. oxaliplatin and mAZD2936 lead to superior tumour growth inhibition compared to single agent administration

[0277] FIG 18A depicts a schematic of treatment regimens designed for combination of bsTCE with Oxaliplatin, 5-FU and mAZD2936 (a murine surrogate rilvegostomig). See FIG 18A. Concurrent or alternative schedules were designed for administration in hCD3deg mice (C57BL / 6 background, expressing human CD3 chains, Genoway) implanted with 1.5 x 106MC38-mCLDN18.2 cells. Compounds were administered on indicated days, with oxaliplatin injected intraperitoneally (IP) at 5 mg / kg, 5FU delivered intraperitonally (IP) at 12.5 mg / kg; BsTCE or Isotype control were delivered IP at 1 mg / kg; mAZD2936 was delivered IP at 10CLDN18T-200-PCT01 mg / kg. See FIG 18A. RILFOX refers to combination of rilvegostomig (mAZD2936) + 5FU + oxaliplatin.

[0278] Tumour volume was measured over the course of treatment and FIG 18B depicts tumor volume vs time plots (n=10 mice per group, mean + / - SEM of each measurement) for each treatment group. FIG 18C depicts resulting tumour growth rates estimated based on fitting each tumour’s growth curve to an exponential model, p values obtained with one-way ANOVA with multiple comparisons, and FIG 18D depicts summary tumour growth rates for each individual mouse estimated based on fitting each tumour’s growth curve to an exponential model. In vivo, bsTCE treatment led to upregulation of PD1 and TIGIT expression on CD8+ T cels; and combination of bsTCE with 5-FU and oxaliplatin and a surrogate of rilvegostomig demonstrated superior tumor growth inhibition, compared with single agent (See FIG 18A-18D).

[0279] In conclusion, our findings demonstrate bsTCE effects to induce T cell activation, cytokine secretion, and tumor cell cytotoxicity in vitro, and in vivo are further potentiated in combination with clinically relevant chemotherapies and / or immunotherapies, including rilvegostomig and volrustomig.

Claims

CLDN18T-200-PCT01WHAT IS CLAIMED IS:Claim 1 : A method of treating a Claudin 18.2 (CLDN 18.2)-expressing solid tumor in a patient in need thereof, the method comprising administering to the patient:(a) a bispecific T-cell engager (BsTCE) agent; and(b) an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s); wherein the BsTCE agent comprises:(i) a first domain that binds to CLDN 18.2; and(ii) a second domain that binds to cluster of differentiation 3 (CD3); and wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNis.2-VHcLDNis.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL.Claim 2: A method of inducing cytotoxicity in a cell expressing Claudin 18.2 (CLDN18.2), the method comprising contacting the cell with:(a) a bispecific T-cell engager (BsTCE) agent;(b) an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s); wherein the BsTCE agent comprises:(i) a first domain that binds to CLDN 18.2; and(ii) a second domain that binds to cluster of differentiation 3 (CD3); and wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNis.2-VHcLDNis.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL.Claim 3 : The method of either claim 1 or claim 2, wherein the immune checkpoint inhibitor agent is an immune checkpoint inhibitor antibody or antigen binding fragment thereof or an immune-oncologic (IO) bispecific antibody or antigen binding fragment thereof.CLDN18T-200-PCT01Claim 4: The method of any one of claims 1-3, wherein the solid tumor or the cell is gastrointestinal cancer, ovarian cancer, lung cancer, renal cancer, hepatocellular cancer, breast cancer, bladder cancer, head and neck sarcoma, or melanoma.Claim 5: The method of claim 4, wherein the gastrointestinal cancer is gastric cancer, pancreatic cancer, gastroesophageal junction cancer, biliary tract cancer, esophageal cancer, or colorectal cancer.Claim 6: The method of claim 5, wherein the gastrointestinal cancer is gastric cancer (GC) or pancreatic ductal adenocarcinoma (PDAC).Claim 7: The method of any one of claims 1-6, wherein the VHCLDNIS.2 variable heavy domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 5; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 6; and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 7; and wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1).Claim 8: The method of any one of claims 1-7, wherein the VHCDS variable heavy domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 11 ; and the VLCDS variable light domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 13; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 14; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 15; and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM.Claim 9: The method of any one of claims 1-8, wherein:(a) the first polypeptide chain comprises two VHCLDNIS.2 domains comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 8;CLDN18T-200-PCT01(b) the second polypeptide chain comprises a VHCD3 domain comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 12; and(c) the third polypeptide chain comprises a VLCD3 domain comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 16; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM.Claim 10: The method of any one of claims 1-9, wherein the immune checkpoint agent comprises a domain that specifically binds PD-1 or PD-L1.Claim 11 : The method of claim 10, wherein the immune checkpoint inhibitor agent is a monoclonal antibody or antigen binding fragment that specifically binds PD-1 or PD-L1.Claim 12: The method of claim 10, wherein the immune checkpoint inhibitor agent is a polyclonal antibody or antigen binding fragment thereof that specifically binds PD-1 or PD-L1.Claim 13: The method of any one of claims 1-12, wherein the immune checkpoint inhibitor agent is an IO-bispecific antibody comprising:(a) a first domain that specifically binds PD-1; and(b) a second domain that specifically binds:(i) a T cell immunoreceptor with Ig and ITIM domain (TIGIT);(ii) T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3); or(iii) cytotoxic T-lymphocyte associated protein 4 (CTLA-4).Claim 14: The method of claim 13, wherein the IO-bispecific antibody specifically bindsPD-1 and TIGIT.Claim 15: The method of claim 14, wherein the IO-bispecific antibody is rilvegostomig.CLDN18T-200-PCT01Claim 16: The method of claim 13, wherein the IO-bispecific antibody specifically bindsPD-1 and TIM3.Claim 17: The method of claim 16, wherein the IO-bispecific antibody is sabestomig.Claim 18: The method of claim 13, wherein the IO-bispecific antibody specifically bindsPD-1 and CTLA-4.Claim 19: The method of claim 18, wherein the IO-bispecific antibody is volrustomig.Claim 20: The method of any one of claims 1-19, wherein the one or more chemotherapy agent is a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline- containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.Claim 21 : The method of claim 20, wherein the one or more chemotherapy agent is a fluoropyrimidine compound selected from 5 -fluorouracil (5-FU) and derivatives thereof, floxuridine, capecitabine, tegafur, and / or doxifluridine; a platinum-based chemotherapy selected from one of one or more of carboplatin, cisplatin, nedaplatin, and / or oxaliplatin; and / or an anthracycline-containing compound selected from daunorubicin, epirubicin, and / or doxorubicin.Claim 22: The method of claim 21, wherein the one or more chemotherapy agent is selected from oxaliplatin and / or 5-FU; gemcitabine and / or paclitaxel; and / or a CAPOX or FOLFOX chemotherapy regimen.Claim 23 : The method of any one of claims 1 -22, wherein the method comprises administering to the patient a bispecific T-cell engager (BsTCE) agent and one or more chemotherapy agent(s); wherein:CLDN18T-200-PCT01(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises a SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; and wherein the one or more chemotherapy agent is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.Claim 24: The method of any one of claims 1-22, wherein the method comprises administering to the patient a bispecific T-cell engager (BsTCE) agent and an immune checkpoint inhibitor agent; wherein:(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 1 ;(b) the second polypeptide chain of the BsTCE agent SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; and wherein the immune checkpoint inhibitor agent is an immune checkpoint antibody or antigen binding fragment thereof or an immune oncologic bispecific (IO-bispecific) antibody or antigen binding fragment thereof.CLDN18T-200-PCT01Claim 25: The method of any one of claims 1-22, wherein the method comprises administering to the patient a bispecific T-cell engager (BsTCE) agent, an immune checkpoint inhibitor agent, and one or more chemotherapy agent(s); wherein:(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; wherein the immune checkpoint inhibitor agent is an immune checkpoint antibody or antigen binding fragment thereof or an immune oncologic bispecific (IO-bispecific) antibody or antigen binding fragment thereof; and wherein the one or more chemotherapy agent is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.Claim 26: The method of any one of claims 1-25, wherein the method results in one or more of:(a) an increase in T-cell activation;(b) an increase in circulating levels of pro-inflammatory cytokines;(c) an increase in tumor cell death; and / or(d) a reduction in tumor volume.Claim 27: The method of claim 26, wherein the increased circulating levels of pro inflammatory cytokines include interferon gamma and / or Granzyme B.CLDN18T-200-PCT01Claim 28: The method of any one of claims 1-27, wherein one or more agent(s) is administered by a route selected from oral, sublingual, parenteral, transdermal, topical, buccal, rectal, vaginal, nasal, ophthalmic, inhalate, and implantable.Claim 29: The method of claim 28, wherein one or more agent(s) is administered by intravenous infusion.Claim 30: The method of claim 28, wherein one or more agent(s) is administered by subcutaneous or intramuscular injection.Claim 31 : The method of claim 28, wherein one or more agent(s) is administered by intratumoral or intralymphatic injection.Claim 32: The method of any of claims 1-31, wherein the BsTCE agent, and the one or more chemotherapy agent(s), and / or the immune checkpoint inhibitor agent are administered sequentially.Claim 33: The method of of claim 32, wherein administration of the BsTCE agent is subsequent to the administration of one or more chemotherapy agent(s) and / or concurrently with the immune checkpoint inhibitor agent.Claim 34: The method of claim 33, wherein the one or more chemotherapy agent(s) is administered prior to administration of the BsTCE agent and the immune checkpoint inhibitor agent.Claim 35: The method of claim 34, wherein the BsTCE agent and the immune checkpoint inhibitor agent are administered subsequent to the last dose of the one or more chemotherapy agent(s).Claim 36: The method of claim 35, wherein the immune checkpoint inhibitor agent is administered on the same day as the BsTCE agent.CLDN18T-200-PCT01Claim 37: The method of claim 35, wherein the immune checkpoint inhibitor agent is administered concurrently with the BsTCE agent on alternate administrations, and administration of the BsTCE agent is alone on other administrations.Claim 38: The method of any one of claims 1-31, wherein the one or more chemotherapy agent(s), the BsTCE agent, and the immune checkpoint inhibitor agent are administered concurrently.Claim 39: The method of claim 38, wherein the chemotherapy agent(s), the BsTCE agent, and the immune checkpoint inhibitor agent are administered in separate applications.Claim 40: The method of claim 38, wherein the concurrent administration comprises an initial administration of the one or more chemotherapy agent(s), the BsTCE agent, and the immune checkpoint inhibitor agent, followed by subsequent administration of the one or more chemotherapy agent(s) concurrently or sequentially with the immune checkpoint inhibitor agent, which are administered concurrently, sequentially, or without the BsTCE agent.Claim 41 : A pharmaceutical combination comprising a bispecific T-cell engager (BsTCE) agent and a pharmaceutically acceptable carrier, an immune checkpoint inhibitor agent and a pharmaceutically acceptable carrier, and / or one or more chemotherapy agent(s) and a pharmaceutically acceptable carrier; wherein the BsTCE agent comprises:(a) a first domain that binds to Claudin 18.2 (CLDN18.2); and(b) a second domain that binds to cluster of differentiation 3 (CD3); wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNis.2-VHcLDNis.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL;CLDN18T-200-PCT01 wherein the immune checkpoint inhibitor is an immune checkpoint antibody or antigen binding fragment thereof or an immune oncologic bispecific (IO-bispecific) antibody or antigen binding fragment thereof; and wherein the one or more chemotherapy agent is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.Claim 42: The pharmaceutical combination of claim 41, wherein the pharmaceutical composition comprises a bispecific T-cell engager (BsTCE) agent and a pharmaceutically acceptable carrier, and one or more chemotherapy agent(s) and a pharmaceutically acceptable carrier; wherein:(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; and wherein the one or more chemotherapy agent is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.Claim 43: The pharmaceutical combination of claim 41, wherein the pharmaceutical composition comprises a bispecific T-cell engager (BsTCE) agent and a pharmaceutically acceptable carrier, and an immune checkpoint inhibitor agent and a pharmaceutically acceptable carrier; wherein:CLDN18T-200-PCT01(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; and wherein the immune checkpoint inhibitor is an immune checkpoint antibody or antigen binding fragment thereof or an immune oncologic bispecific (IO-bispecific) antibody or antigen binding fragment thereof.Claim 44: The pharmaceutical combination of claim 41, wherein the pharmaceutical composition comprises a bispecific T-cell engager (BsTCE) agent and a pharmaceutically acceptable carrier, an immune checkpoint inhibitor agent and a pharmaceutically acceptable carrier, and one or more chemotherapy agent(s) and a pharmaceutically acceptable carrier; wherein:(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; wherein the immune checkpoint inhibitor is an immune checkpoint antibody or antigen binding fragment thereof or an immune oncologic bispecific (IO-bispecific) antibody or antigen binding fragment thereof; andCLDN18T-200-PCT01 wherein the one or more chemotherapy agent is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.Claim 45: A combination for use in the treatment of a Claudin 18.2 (CLDN18.2)-expressing solid tumor in a patient in need thereof, wherein the combination comprises:(a) a bispecific T-cell engager (BsTCE) agent; and(b) an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s); wherein the BsTCE agent comprises:(i) a first domain that binds to CLDN 18.2; and(ii) a second domain that binds to cluster of differentiation 3 (CD3); and wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNis.2-VHcLDNis.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL.Claim 46: A combination for use in inducing cytotoxicity in a cell expressing Claudin 18.2 (CLDN18.2), wherein the combination comprises:(a) a bispecific T-cell engager (BsTCE) agent;(b) an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s); wherein the BsTCE agent comprises:(i) a first domain that binds to CLDN 18.2; and(ii) a second domain that binds to cluster of differentiation 3 (CD3); and wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNis.2-VHcLDNis.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL.Claim 47: The combination for use of either claim 45 or claim 46, wherein the immune checkpoint inhibitor agent is an immune checkpoint inhibitor antibody or antigen bindingCLDN18T-200-PCT01 fragment thereof or an immune-oncologic (IO) bispecific antibody or antigen binding fragment thereof.Claim 48: The combination for use of any one of claims 45-47, wherein the solid tumor or the cell is gastrointestinal cancer, ovarian cancer, lung cancer, renal cancer, hepatocellular cancer, breast cancer, bladder cancer, head and neck sarcoma, or melanoma.Claim 49: The combination for use of claim 48, wherein the gastrointestinal cancer is gastric cancer, pancreatic cancer, gastroesophageal junction cancer, biliary tract cancer, esophageal cancer, or colorectal cancer.Claim 50: The combination for use of claim 49, wherein the gastrointestinal cancer is gastric cancer (GC) or pancreatic ductal adenocarcinoma (PDAC).Claim 51 : The combination for use of any one of claims 45-50, wherein the VHCLDNIS.2 variable heavy domain comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 5; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 6; and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 7; and wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1).Claim 52: The combination for use of any one of claims 45-51, wherein the VHCDS variable heavy domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 11; and the VLcosvariable light domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 13; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 14; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 15; and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM.Claim 53: The combination for use of any one of claims 45-52, wherein:CLDN18T-200-PCT01(a) the first polypeptide chain comprises two VHCLDNIS.2 domains comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 8;(b) the second polypeptide chain comprises a VHCDS domain comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 12; and(c) the third polypeptide chain comprises a VLCD3 domain comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 16; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM.Claim 54: The combination for use of any one of claims 45-53, wherein the immune checkpoint agent comprises a domain that specifically binds PD-1 or PD-L1.Claim 55: The combination for use of claim 54, wherein the immune checkpoint inhibitor agent is a monoclonal antibody or antigen binding fragment that specifically binds PD-1 or PD- Ll.Claim 56: The combination for use of claim 54, wherein the immune checkpoint inhibitor agent is a polyclonal antibody or antigen binding fragment thereof that specifically binds PD-1 or PD-L1.Claim 57: The combination for use of any one of claims 45-56, wherein the immune checkpoint inhibitor agent is an IO-bispecific antibody comprising:(a) a first domain that specifically binds PD-1; and(b) a second domain that specifically binds:(i) a T cell immunoreceptor with Ig and ITIM domain (TIGIT);(ii) T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3); or(iii) cytotoxic T-lymphocyte associated protein 4 (CTLA-4).CLDN18T-200-PCT01Claim 58: The combination for use of claim 57, wherein the IO-bispecific antibody specifically binds PD-1 and TIGIT.Claim 59: The combination for use of claim 58, wherein the IO-bispecific antibody is rilvegostomig.Claim 60: The combination for use of claim 57, wherein the IO-bispecific antibody specifically binds PD-1 and HM3.Claim 61 : The combination for use of claim 60, wherein the IO-bispecific antibody is sabestomig.Claim 62: The combination for use of claim 57, wherein the IO-bispecific antibody specifically binds PD-1 and CTLA-4.Claim 63: The combination for use of claim 62, wherein the IO-bispecific antibody is volrustomig.Claim 64: The combination for use of any one of claims 45-63, wherein the one or more chemotherapy agent is a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.Claim 65: The combination for use of claim 64, wherein the one or more chemotherapy agent is a fluoropyrimidine compound selected from 5 -fluorouracil (5-FU) and derivatives thereof, floxuridine, capecitabine, tegafur, and / or doxifluridine; a platinum-based chemotherapy selected from one of one or more of carboplatin, cisplatin, nedaplatin, and / or oxaliplatin; and / or an anthracycline-containing compound selected from daunorubicin, epirubicin, and / or doxorubicin.CLDN18T-200-PCT01Claim 66: The combination for use of claim 65, wherein the one or more chemotherapy agent is selected from oxaliplatin and / or 5-FU; gemcitabine and / or paclitaxel; and / or a CAPOX or FOLFOX chemotherapy regimen.Claim 67: The combination for use of any one of claims 45-66, wherein the combination comprises a bispecific T-cell engager (BsTCE) agent and one or more chemotherapy agent(s); wherein:(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; and wherein the one or more chemotherapy agent is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.Claim 68: The combination for use of any one of claims 45-66, wherein the combination comprises a bispecific T-cell engager (BsTCE) agent and an immune checkpoint inhibitor agent; wherein:(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3;CLDN18T-200-PCT01 wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; and wherein the immune checkpoint inhibitor agent is an immune checkpoint antibody or antigen binding fragment thereof or an immune oncologic bispecific (IO-bispecific) antibody or antigen binding fragment thereof.Claim 69: The combination for use of any one of claims 45-66, wherein the combination comprises a bispecific T-cell engager (BsTCE) agent, an immune checkpoint inhibitor agent, and one or more chemotherapy agent(s); wherein:(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; wherein the immune checkpoint inhibitor agent is an immune checkpoint antibody or antigen binding fragment thereof or an immune oncologic bispecific (IO-bispecific) antibody or antigen binding fragment thereof; and wherein the one or more chemotherapy agent is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.Claim 70: The combination for use of any one of claims 45-69, wherein administration of the combination results in one or more of:(a) an increase in T-cell activation;CLDN18T-200-PCT01(b) an increase in circulating levels of pro-inflammatory cytokines;(c) an increase in tumor cell death; and / or(d) a reduction in tumor volume.Claim 71 : The combination for use of claim 70, wherein the increased circulating levels of pro inflammatory cytokines include interferon gamma and / or Granzyme B.Claim 72: The combination for use of any one of claims 45-71, wherein administration of the one or more agent(s) is by a route selected from oral, sublingual, parenteral, transdermal, topical, buccal, rectal, vaginal, nasal, ophthalmic, inhalate, and implantable.Claim 73 : The combination for use of claim 72, wherein administration of the one or more agent(s) is by intravenous infusion.Claim 74: The combination for use of claim 72, wherein administration of the one or more agent(s) is by subcutaneous or intramuscular injection.Claim 75: The combination for use of claim 72, wherein administration of the one or more agent(s) is by intratumoral or intralymphatic injection.Claim 76: The combination for use of any of claims 45-75, wherein the BsTCE agent, and the one or more chemotherapy agent(s), and / or the immune checkpoint inhibitor agent are administered sequentially.Claim 77: The combination for use of any of claims 45-75, wherein administration of theBsTCE agent is subsequent to the administration of one or more chemotherapy agent(s) and / or concurrently with the immune checkpoint inhibitor agent.Claim 78: The combination for use of claim 77, wherein administration of the one or more chemotherapy agent(s) occurs prior to administration of the BsTCE agent and the immune checkpoint inhibitor agent.CLDN18T-200-PCT01Claim 79: The combination for use of claim 78, wherein administration of the BsTCE agent and the immune checkpoint inhibitor agent occurs subsequent to administration of the last dose of the one or more chemotherapy agent(s).Claim 80: The combination for use of claim 79, wherein administration of the immune checkpoint inhibitor agent occurs concurrently with administration of the BsTCE agent.Claim 81 : The combination for use of claim 79, wherein administration of the immune checkpoint inhibitor agent occurs concurrent with the BsTCE agent on alternate administrations, and administration of the BsTCE agent occurs alone on other administrations.Claim 82: The combination for use of any one of claims 45-75, wherein administration of the one or more chemotherapy agent(s), the BsTCE agent, and the immune checkpoint inhibitor agent occurs concurrently.Claim 83 : The combination for use of claim 82, wherein administration of the chemotherapy agent(s), the BsTCE agent, and the immune checkpoint inhibitor agent occurs in separate applications.Claim 84: The combination for use of claim 82, wherein concurrent administration of the one or more agents(s) comprises an initial administration of the one or more chemotherapy agent(s), the BsTCE agent, and the immune checkpoint inhibitor agent, followed by subsequent administration of the one or more chemotherapy agent(s) concurrently or sequentially with the immune checkpoint inhibitor agent, wherein administration occurs concurrently, sequentially, or without the BsTCE agent.Claim 85: Use of a combination in the manufacture of a medicament for the treatment of a Claudin 18.2 (CLDN18.2)-expressing solid tumor in a patient in need thereof, wherein the combination comprises:(a) a bispecific T-cell engager (BsTCE) agent; andCLDN18T-200-PCT01(b) an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s); wherein the BsTCE agent comprises:(i) a first domain that binds to CLDN 18.2; and(ii) a second domain that binds to cluster of differentiation 3 (CD3); and wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNis.2-VHcLDNis.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL.Claim 86: Use of a combination in the manufacture of a medicament for inducing cytotoxicity in a cell expressing Claudin 18.2 (CLDN18.2), wherein the combination comprises:(a) a bispecific T-cell engager (BsTCE) agent;(b) an immune checkpoint inhibitor agent and / or one or more chemotherapy agent(s); wherein the BsTCE agent comprises:(i) a first domain that binds to CLDN 18.2; and(ii) a second domain that binds to cluster of differentiation 3 (CD3); and wherein the BsTCE agent comprises three polypeptide chains, wherein the first polypeptide chain is represented by the formula: VHcLDNis.2-VHcLDNis.2-hinge-CH2-CH3; the second polypeptide chain is represented by the formula: VHcD3-CHl-hinge-CH2-CH3; and the third polypeptide chain is represented by the formula: VLCD3-CL.Claim 87: The use of either claim 85 or claim 86, wherein the immune checkpoint inhibitor agent is an immune checkpoint inhibitor antibody or antigen binding fragment thereof or an immune-oncologic (IO) bispecific antibody or antigen binding fragment thereof.Claim 88: The use of any one of claims 85-87, wherein the or the cell is gastrointestinal cancer, ovarian cancer, lung cancer, renal cancer, hepatocellular cancer, breast cancer, bladder cancer, head and neck sarcoma, or melanoma.CLDN18T-200-PCT01Claim 89: The use of claim 88, wherein the gastrointestinal cancer is gastric cancer, pancreatic cancer, gastroesophageal junction cancer, biliary tract cancer, esophageal cancer, or colorectal cancer.Claim 90: The use of claim 89, wherein the gastrointestinal cancer is gastric cancer (GC) or pancreatic ductal adenocarcinoma (PDAC).Claim 91 : The use of any one of claims 85-90, wherein the VHCLDNIS.2 variable heavy domain comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 5; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 6; and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 7; and wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1).Claim 92: The use of any one of claims 85-91, wherein the VHCDS variable heavy domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 11 ; and the VLCDS variable light domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 13; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 14; and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 15; and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM.Claim 93: The use of any one of claims 85-92, wherein:(a) the first polypeptide chain comprises two VHCLDNIS.2 domains comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 8;(b) the second polypeptide chain comprises a VHCD3 domain comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 12; andCLDN18T-200-PCT01(c) the third polypeptide chain comprises a VLCD3 domain comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 16; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM.Claim 94: The use of any one of claims 85-93, wherein the immune checkpoint agent comprises a domain that specifically binds PD-1 or PD-L1.Claim 95: The use of claim 94, wherein the immune checkpoint inhibitor agent is a monoclonal antibody or antigen binding fragment that specifically binds PD-1 or PD-L1.Claim 96: The use of claim 94, wherein the immune checkpoint inhibitor agent is a polyclonal antibody or antigen binding fragment thereof that specifically binds PD-1 or PD-L1.Claim 97: The use of any one of claims 85-96, wherein the immune checkpoint inhibitor agent is an IO-bispecific antibody comprising:(a) a first domain that specifically binds PD-1; and(b) a second domain that specifically binds:(i) a T cell immunoreceptor with Ig and ITIM domain (TIGIT);(ii) T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3); or(iii) cytotoxic T-lymphocyte associated protein 4 (CTLA-4).Claim 98: The use of claim 97, wherein the IO-bispecific antibody specifically binds PD-1 and TIGIT.Claim 99: The use of claim 98, wherein the IO-bispecific antibody is rilvegostomig.Claim 100: The use of claim 97, wherein the IO-bispecific antibody specifically binds PD-1 and TIM3.CLDN18T-200-PCT01Claim 101 : The use of claim 100, wherein the IO-bispecific antibody is sabestomig.Claim 102: The use of claim 97, wherein the IO-bispecific antibody specifically binds PD-1 and CTLA-4.Claim 103: The use of claim 102, wherein the IO-bispecific antibody is volrustomig.Claim 104: The use of any one of claims 85-103, wherein the one or more chemotherapy agent is a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline- containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.Claim 105: The use of claim 104, wherein the one or more chemotherapy agent is a fluoropyrimidine compound selected from 5 -fluorouracil (5-FU) and derivatives thereof, floxuridine, capecitabine, tegafur, and / or doxifluridine; a platinum-based chemotherapy selected from one of one or more of carboplatin, cisplatin, nedaplatin, and / or oxaliplatin; and / or an anthracycline-containing compound selected from daunorubicin, epirubicin, and / or doxorubicin.Claim 106: The use of claim 105, wherein the one or more chemotherapy agent is selected from oxaliplatin and / or 5-FU; gemcitabine and / or paclitaxel; and / or a CAPOX or FOLFOX chemotherapy regimen.Claim 107: The use of any one of claims 85-106, wherein the combination comprises a bispecific T-cell engager (BsTCE) agent and one or more chemotherapy agent(s); wherein:(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; andCLDN18T-200-PCT01(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; and wherein the one or more chemotherapy agent is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.Claim 108: The use of any one of claims 85-106, wherein the combination comprises a bispecific T-cell engager (BsTCE) agent and an immune checkpoint inhibitor agent; wherein:(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; and wherein the immune checkpoint inhibitor agent is an immune checkpoint antibody or antigen binding fragment thereof or an immune oncologic bispecific (IO-bispecific) antibody or antigen binding fragment thereof.Claim 109: The use of any one of claims 85-106, wherein the combination comprises a bispecific T-cell engager (BsTCE) agent, an immune checkpoint inhibitor agent, and one or more chemotherapy agent(s); wherein:CLDN18T-200-PCT01(a) the first polypeptide chain of the BsTCE agent comprises SEQ ID NO: 1 or an amino acid sequence at least 90% identical to SEQ ID NO: 1;(b) the second polypeptide chain of the BsTCE agent comprises SEQ ID NO: 2 or an amino acid sequence at least 90% identical to SEQ ID NO: 2; and(c) the third polypeptide chain of the BsTCE agent comprises SEQ ID NO: 3 or an amino acid sequence at least 90% identical to SEQ ID NO: 3; wherein the BsTCE agent does not specifically bind to Claudin 18.1 (CLDN18.1) and wherein the CD3 binding affinity is less than 200 nM, or less than 190 nM, or less than 180 nM, or less than 175 nM and greater than 150 nM; wherein the immune checkpoint inhibitor agent is an immune checkpoint antibody or antigen binding fragment thereof or an immune oncologic bispecific (IO-bispecific) antibody or antigen binding fragment thereof; and wherein the one or more chemotherapy agent is selected from a platinum-based chemotherapy, a fluoropyrimidine compound, an anthracycline-containing compound, cyclophosphamide, folinic acid, ifosfamide, irinotecan, methotrexate, pemetrexed, gimeracil, oteracil, docetaxel, paclitaxel, taxane, gemcitabine, vinblastine, and / or vincristine.Claim 110: The use of any one of claims 85-109, wherein administration of the combination results in one or more of:(a) an increase in T-cell activation;(b) an increase in circulating levels of pro-inflammatory cytokines;(c) an increase in tumor cell death; and / or(d) a reduction in tumor volume.Claim 111: The use of claim 110, wherein the increased circulating levels of pro inflammatory cytokines include interferon gamma and / or Granzyme B.Claim 112: The use of any one of claims 85-111, wherein administration of the one or more agent(s) is by a route selected from oral, sublingual, parenteral, transdermal, topical, buccal, rectal, vaginal, nasal, ophthalmic, inhalate, and implantable.CLDN18T-200-PCT01Claim 113: The use of claim 112, wherein administration of the one or more agent(s) is by intravenous infusion.Claim 114: The use of claim 112, wherein administration of the one or more agent(s) is by subcutaneous or intramuscular injection.Claim 115: The use of claim 112, wherein administration of the one or more agent(s) is by intratumoral or intralymphatic injection.Claim 116: The use of any of claims 85-115, wherein the BsTCE agent, the one or more chemotherapy agent(s), , and / or the immune checkpoint inhibitor agent are administered sequential.Claim 117: The use of any of claims 85-115, wherein administration of the BsTCE agent is subsequent to the administration of the one or more chemotherapy agent(s) and / or concurrently with the immune checkpoint inhibitor agent.Claim 118: The use of claim 117, wherein administration of the one or more chemotherapy agent(s) occurs prior to administration of the BsTCE agent and the immune checkpoint inhibitor agent.Claim 119: The use of claim 118, wherein administration of the BsTCE agent and the immune checkpoint inhibitor agent occurs subsequent to administration of the last dose of the one or more chemotherapy agent(s).Claim 120: The use of claim 119, wherein administration of the immune checkpoint inhibitor agent occurs concurrently with administration of the BsTCE agent.Claim 121 : The use of claim 119, wherein administration of the immune checkpoint inhibitor agent occurs concurrent with the BsTCE agent on alternate administrations, and administration of the BsTCE agent occurs alone on other administrations.CLDN18T-200-PCT01Claim 122: The use of any one of claims 85-115, wherein administration of the one or more chemotherapy agent(s), the BsTCE agent, and the immune checkpoint inhibitor agent occurs concurrently.Claim 123: The use of claim 122, wherein administration of the chemotherapy agent(s), the BsTCE agent, and the immune checkpoint inhibitor agent occurs in separate applications.Claim 124: The use of claim 122, wherein concurrent administration of the one or more agents(s) comprises an initial administration of the one or more chemotherapy agent(s), the BsTCE agent, and the immune checkpoint inhibitor agent, followed by subsequent administration of the one or more chemotherapy agent(s) concurrently or sequentially with the immune checkpoint inhibitor agent, wherein administration occurs concurrently, sequentially, or without the BsTCE agent.

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