CLDN18.2 and GUCY2C targeted antagonist combination therapy

Abstract

The present disclosure provides a method for treating a tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of a combination of a CLDN18.2 antagonist and a GUCY2C antagonist or an antagonist of CLDN18.2 and GUCY2C. The disclosure also provides multispecific chimeric antigen receptor constructs, combinations of chimeric antigen receptors, engineered immune cells, and methods of use thereof. The disclosure further relates to the activation and expansion of cells for therapeutic use, especially for chimeric antigen receptor-based T cell immunotherapy.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to International Patent Application No. PCT / CN2023 / 108169, filed on July 19, 2023, the entire contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list that has been filed electronically and is hereby incorporated by reference in its entirety. Technical Field

[0005] This disclosure relates to a method for treating a tumor in a subject of need, the method comprising administering to the subject an effective amount of a combination of a CLDN18.2 antagonist and a GUCY2C antagonist, or an antagonist of CLDN18.2 and GUCY2C. This disclosure also relates to multispecific chimeric antigen receptor constructs, combinations of chimeric antigen receptors, engineered immune cells, and methods of using them. This disclosure further relates to the activation and expansion of cells for therapeutic purposes, particularly for chimeric antigen receptor-based T-cell immunotherapy. Background Technology

[0006] CLDN18.2 (dense protein 18.2) is an important target for the prevention and treatment of primary tumors such as gastric cancer, esophageal cancer, pancreatic ductal carcinoma, lung cancer (such as non-small cell lung cancer (NSCLC)), ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer and their metastases (especially Krukenberg's tumor, peritoneal metastases, lymph node metastases and other metastatic gastric cancers)). Ugur Sahin et al. (2008) reported that 96% of gastric cancer patients (46 / 48), 63% of pancreatic ductal carcinoma patients (7 / 11), 60% of esophageal cancer patients (6 / 10), and 41% of non-small cell lung cancer patients (30 / 73) had CLDN18.2 positive tissue samples (mRNA, RT-PCR method).

[0007] GUCY2C (guanylate cyclase 2C, GCC) belongs to the receptor guanylate cyclase family and is a membrane-bound guanylate cyclase. As a single transmembrane protein receptor, GCC expression in normal tissues is primarily limited to the apical membrane of intestinal polarized epithelial cells and occurs only in the duodenum and rectum, not in normal gastric and esophageal tissues. Activation of the GCC signaling pathway regulates various physiological processes, including intestinal epithelial cell proliferation, differentiation, and metabolism, which are crucial for epithelial barrier renewal and water and electrolyte balance.

[0008] GCC is primarily associated with intestinal disorders, including functional gastrointestinal disorders (such as irritable bowel syndrome and constipation), inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), and cancers (gastrointestinal cancers). GCC is stably expressed in primary colorectal cancer, but it is abnormally highly expressed in metastatic colorectal cancer, and is considered a specific biomarker for metastatic colorectal cancer. It can be used to identify colorectal cancer patients, stage colorectal cancer, and serve as the strongest independent prognostic marker for colorectal cancer. Stansa Biotechnology's GCC & CD19 conjugate CAR-T achieved a 50% objective response rate (ORR) in a Phase I clinical trial, with a median overall survival (mOS) of 12–17 months. Based on standard algorithms for CRS and diarrhea, this therapy was well-tolerated and had reasonable toxicity with appropriate intervention. These clinical trial results indicate that immunotherapy targeting GCC is safe, manageable, and promising.

[0009] Several CAR-T products targeting CLDN18.2 exist, but their median progression-free survival (mPFS) is relatively short. For example, CT041 from CStone Pharmaceuticals has an mPFS of only 4.2 months (Nat Med. 2022 Jun; 28(6):1189-1198). Antigen loss and tumor heterogeneity may be among the main causes of tumor recurrence and disease progression. Summary of the Invention

[0010] In one aspect, this disclosure provides a method for treating a tumor in a subject of need, the method comprising administering to the subject an effective amount of a combination of a CLDN18.2 antagonist and a GUCY2C antagonist, or an antagonist of CLDN18.2 and GUCY2C. The tumor to be treated may be a CLDN18.2-positive tumor, a GUCY2C-positive tumor, or a double-positive tumor of CLDN18.2 and GUCY2C.

[0011] CLDN18.2 antagonists, GUCY2C antagonists, and / or antagonists of CLDN18.2 and GUCY2C can be of various classes; for example, antagonists can be selected from engineered receptors, engineered immune cells, antibodies, antibody-drug conjugates (ADCs), aptamers, and small RNAs. In some embodiments, the engineered receptor is selected from the group consisting of engineered T-cell receptors (TCRs), chimeric antigen receptors (CARs), T-cell antigen conjugates (TACs), or portions thereof.

[0012] In some embodiments, a CLDN18.2 antagonist is an engineered immune cell comprising an engineered receptor specifically targeting CLDN18.2, the engineered receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain containing at least one anti-CLDN18.2 binding moiety. In some embodiments, a GUCY2C antagonist is an engineered immune cell comprising an engineered receptor specifically targeting GUCY2C, the engineered receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain containing at least one anti-GUCY2C binding moiety. In some embodiments, the engineered receptor specifically targeting CLDN18.2 and the engineered receptor specifically targeting GUCY2C are expressed in different engineered immune cells. Accordingly, the combination of the CLDN18.2 antagonist and the GUCY2C antagonist is a combination of a first group of engineered immune cells comprising the engineered receptor specifically targeting CLDN18.2 as described above and a second group of engineered immune cells comprising the engineered receptor specifically targeting GUCY2C as described above.

[0013] In some embodiments, the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising a first engineered receptor specifically targeting CLDN18.2 and a second engineered receptor specifically targeting GUCY2C, i.e., the two engineered receptors are co-expressed in the same immune cell. More specifically, the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising a first engineered receptor specifically targeting CLDN18.2 and a second engineered receptor specifically targeting GUCY2C, wherein (1) the first engineered receptor specifically targeting CLDN18.2 comprises: a first extracellular antigen-binding domain, a first transmembrane domain, and a first intracellular signal transduction domain containing at least one anti-CLDN18.2 binding portion, and (2) the second engineered receptor specifically targeting GUCY2C comprises: a second extracellular antigen-binding domain, a second transmembrane domain, and a second intracellular signal transduction domain containing at least one anti-GUCY2C binding portion.

[0014] In some embodiments, the engineered immune cells co-expressing the first engineered receptor and the second engineered receptor have been transduced by two separate vectors, the first vector containing nucleic acid encoding the first engineered receptor specifically targeting CLDN18.2, and the second vector containing nucleic acid encoding the second engineered receptor specifically targeting GUCY2C.

[0015] In some embodiments, the engineered immune cells co-expressing the first engineered receptor and the second engineered receptor have been transduced with a vector containing nucleic acid encoding a first engineered receptor specifically targeting CLDN18.2, which is operatively linked to a second engineered receptor specifically targeting GUCY2C. In some embodiments, the engineered immune cells express the first engineered receptor specifically targeting CLDN18.2, which is operatively linked to the second engineered receptor specifically targeting GUCY2C via a cleavable adapter, wherein the cleavable adapter can be readily cleaved under suitable conditions. In some embodiments, the cleavable adapter is selected from P2A, T2A, E2A, and F2A.

[0016] In some embodiments, the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising a first CAR (a single CLDN18.2-specific CAR) targeting CLDN18.2 and a second CAR (a single GUCY2C-specific CAR) targeting GUCY2C, wherein (1) the first CAR comprises a first extracellular antigen-binding domain, a first transmembrane domain and a first intracellular signal transduction domain containing at least one anti-CLDN18.2 binding portion, and (2) the second CAR comprises a second extracellular antigen-binding domain, a second transmembrane domain and a second intracellular signal transduction domain containing at least one anti-GUCY2C binding portion; and wherein the first CAR is operatively connected to the second CAR, the first CAR being located at the N-terminus or C-terminus of the second CAR targeting GUCY2C, optionally the first CAR being operatively connected to the second CAR via a cleavable adapter, or the first CAR and the second CAR being disconnected due to cleavage by the cleavable adapter.

[0017] In some embodiments, the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising an engineered receptor that co-targets CLDN18.2 and GUCY2C, the engineered receptor comprising: an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain containing at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety. The engineered receptor may be selected from the group consisting of engineered T-cell receptors (TCRs), chimeric antigen receptors (CARs), T-cell antigen conjugates (TACs), or portions thereof.

[0018] In some embodiments, the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising a CAR (“CLDN18.2×GUCY2C-specific CAR”) co-targeting CLDN18.2 and GUCY2C, wherein the CLDN18.2×GUCY2C-specific CAR comprises: (1) an extracellular antigen-binding domain containing at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety, (2) a transmembrane domain and (3) an intracellular signal transduction domain; and wherein the anti-CLDN18.2 binding moiety is located at the N-terminus or C-terminus of the anti-GUCY2C binding moiety, optionally the anti-CLDN18.2 binding moiety being operatively linked to the anti-GUCY2C binding moiety via a peptide linker such as a GS linker, for example (G4S)3.

[0019] In some embodiments, the anti-CLDN18.2 binding portion and the anti-GUCY2C binding portion are selected from Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), mini antibody, biantibody, single-domain antibody (sdAb), or VHH domain.

[0020] In some embodiments, the anti-CLDN18.2 binding portion is anti-CLDN18.2 VHH, optionally comprising CDR1, CDR2, and CDR3 as shown in the amino acid sequence of SEQ ID NO: 1, and optionally comprising (i) CDR1 containing the amino acid sequence of SEQ ID NO: 23, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 24, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO: 25. In some embodiments, the anti-CLDN18.2 VHH comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 1. For example, the anti-CLDN18.2 VHH may comprise an amino acid sequence having a CDR identical to the CDR of SEQ ID NO: 1 and having an amino acid sequence having an amino acid sequence having at least 85%, 90%, or 95% identical to the frame region of SEQ ID NO: 1.

[0021] In some embodiments, the anti-GUCY2C binding portion is anti-GUCY2C VHH, optionally comprising CDR1, CDR2, and CDR3 as shown in the amino acid sequence of SEQ ID NO: 2, and optionally comprising (i) CDR1 containing the amino acid sequence of SEQ ID NO: 26, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 27, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-GUCY2C VHH comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 2. For example, the anti-GUCY2C VHH may comprise an amino acid sequence having a CDR identical to the CDR of SEQ ID NO: 2 and having an amino acid sequence having an amino acid sequence having at least 85%, 90%, or 95% identical to the frame region of SEQ ID NO: 2.

[0022] In some embodiments, the transmembrane domain is derived from proteins selected from the group consisting of CD8α, ICOS, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

[0023] In some embodiments, the intracellular signal transduction domain comprises a primary intracellular signal transduction domain of an immune cell, optionally derived from CD3ζ. In some embodiments, the intracellular signal transduction domain comprises a co-stimulatory signal transduction domain. The co-stimulatory signal transduction domain may be derived from a co-stimulatory molecule selected from the group consisting of: CD28, 4-1BB (CD137), CD27, OX40, CD30, CD40, CD3, LFA-1, ICOS (CD278), NTBA, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 ligand, and combinations thereof.

[0024] In some embodiments, the CAR specifically targeting CLDN18.2 as disclosed herein comprises:

[0025] (1) A hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain, optionally the hinge domain being derived from CD8α or CD28; and / or

[0026] (2) The signal peptide located at the N-terminus of this extracellular antigen-binding domain,

[0027] Optionally, the signal peptide is derived from CD8α.

[0028] In some embodiments, the CAR that specifically targets GUCY2C comprises:

[0029] (1) A hinge domain located between the N-terminus of the extracellular antigen-binding domain and the transmembrane domain, optionally wherein the hinge domain is derived from CD8α or CD28; and / or

[0030] (2) A signal peptide located at the N-terminus of the extracellular antigen-binding domain, optionally wherein the signal peptide is derived from CD8α.

[0031] In some embodiments, the CAR specifically targeting CLDN18.2 comprises the amino acid sequence shown in any one of SEQ ID NO: 12 and 19. In some embodiments, the CAR specifically targeting GUCY2C comprises the amino acid sequence shown in any one of SEQ ID NO: 13 and 21-22.

[0032] In some embodiments, the CLDN18.2 antagonist is an engineered immune cell comprising a polypeptide containing the amino acid sequence shown in any one of SEQ ID NO: 12 and 19. In some embodiments, the GUCY2C antagonist is an engineered immune cell comprising a polypeptide containing the amino acid sequence shown in any one of SEQ ID NO: 13 and 21-22. In some embodiments, the combination of the CLDN18.2 antagonist and the GUCY2C antagonist comprises a first group of engineered immune cells and a second group of engineered immune cells, or comprises a first group of engineered immune cells and a second group of engineered immune cells, wherein the first group of engineered immune cells contains a polypeptide containing the amino acid sequence of SEQ ID NO: 12, and the second group of engineered immune cells contains a polypeptide containing the amino acid sequence of SEQ ID NO: 13. In some embodiments, the ratio of the first group of engineered immune cells to the second group of engineered immune cells is in the range of 10:1 to 1:10, such as 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 or 1:9.

[0033] In some embodiments, the CAR that co-targets CLDN18.2 and GUCY2C comprises:

[0034] (1) A hinge domain located between the N-terminus of the extracellular antigen-binding domain and the transmembrane domain, optionally wherein the hinge domain is derived from CD8α or CD28; and / or

[0035] (2) A signal peptide located at the N-terminus of the extracellular antigen-binding domain, optionally wherein the signal peptide is derived from CD8α.

[0036] In some embodiments, the CAR co-targeting CLDN18.2 and GUCY2C comprises the amino acid sequence shown in any one of SEQ ID NO: 14-15. In some embodiments, the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising:

[0037] (1) A polypeptide containing any one of the amino acid sequences shown in SEQ ID NO: 14-18; or

[0038] (2) A first polypeptide and a second polypeptide, wherein the first polypeptide comprises an amino acid sequence shown in any one of SEQ ID NO: 12 and 19, and the second polypeptide comprises an amino acid sequence shown in any one of SEQ ID NO: 13 and 20-22.

[0039] In some embodiments, the engineered immune cells are selected from the group consisting of T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof.

[0040] In some embodiments, the tumor is selected from gastric cancer, esophageal cancer, pancreatic ductal carcinoma, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colorectal cancer, liver cancer, head and neck cancer, gallbladder cancer and their metastases.

[0041] In some embodiments, the subject is resistant to at least one CLDN18.2 agent and / or the subject is resistant to at least one GUCY2C agent.

[0042] In one aspect, this paper provides a multispecific chimeric antigen receptor (CAR) construct (“CLDN18.2×GUCY2C specific CAR”) that binds to CLDN18.2 and GUCY2C, comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain containing at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety.

[0043] In some embodiments, the multispecific chimeric antigen receptor (CAR) construct that binds to CLDN18.2 and GUCY2C comprises:

[0044] (a) A polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain containing at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety; or

[0045] (b) A first polypeptide and a second polypeptide, the first polypeptide comprising a first extracellular antigen-binding domain, a first transmembrane domain and a first intracellular signal transduction domain containing at least one anti-CLDN18.2 binding moiety, the second polypeptide comprising a second extracellular antigen-binding domain, a second transmembrane domain and a second intracellular signal transduction domain containing at least one anti-GUCY2C binding moiety, optionally the first polypeptide and the second polypeptide are linked into a single chain via a cleavable linker, optionally the first transmembrane domain and the second transmembrane domain and the first intracellular signal transduction domain are the same or different.

[0046] In some embodiments, the anti-CLDN18.2 binding portion is located at the N-terminus or C-terminus of the anti-GUCY2C binding portion, and optionally the anti-CLDN18.2 binding portion is operatively linked to the anti-GUCY2C binding portion via a peptide linker (such as a GS linker, e.g., (G4S)3). The anti-CLDN18.2 binding portion and / or the anti-GUCY2C binding portion may be selected from Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), mini-antibody, biantibody, single-domain antibody (sdAb), or VHH domain.

[0047] In some embodiments, the anti-CLDN18.2 binding portion is anti-CLDN18.2 VHH, optionally comprising CDR1, CDR2, and CDR3 as shown in the amino acid sequence of SEQ ID NO: 1, and optionally comprising (i) CDR1 containing the amino acid sequence of SEQ ID NO: 23, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 24, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO: 25. In some embodiments, the anti-CLDN18.2 VHH comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 1.

[0048] In some embodiments, the anti-GUCY2C binding portion is anti-GUCY2C VHH, optionally comprising CDR1, CDR2, and CDR3 as shown in the amino acid sequence of SEQ ID NO: 2, and optionally comprising (i) CDR1 containing the amino acid sequence of SEQ ID NO: 26, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 27, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-GUCY2C VHH comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 2.

[0049] The transmembrane domain may be derived from proteins selected from the group consisting of CD8α, ICOS, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the transmembrane domain is derived from CD8α or ICOS, and optionally the transmembrane domain comprises an amino acid sequence shown in any one of SEQ ID NO: 5-6 or an amino acid sequence that is at least 85%, 90%, or 95% identical to an amino acid sequence shown in any one of SEQ ID NO: 5-6.

[0050] In some embodiments, the intracellular signal transduction domain comprises a primary intracellular signal transduction domain; optionally, the primary intracellular signal transduction domain is derived from CD3ζ, and optionally, the primary intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 11. The intracellular signal transduction domain may further comprise at least one co-stimulatory signal transduction domain. The co-stimulatory signal transduction domain may be derived from co-stimulatory molecules selected from: CD28, 4-1BB (CD137), CD27, OX40, CD30, CD40, CD3, LFA-1, ICOS (CD278), NTBA, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 ligands, and combinations thereof. In some embodiments, the co-stimulatory signal transduction domain is derived from 4-1BB, CD28, ICOS, or NTBA. Optionally, the co-stimulatory signal transduction domain comprises any one of the amino acid sequences of SEQ ID NO: 7-10 or an amino acid sequence that is at least 85%, 90%, or 95% identical to any one of SEQ ID NO: 7-10.

[0051] In some embodiments, the multispecific CAR construct further comprises:

[0052] (1) A hinge domain located between the N-terminus of the extracellular antigen-binding domain and the transmembrane domain, optionally wherein the hinge domain is derived from CD8α or CD28; and / or

[0053] (2) A signal peptide located at the N-terminus of the extracellular antigen-binding domain, optionally wherein the signal peptide is derived from CD8α.

[0054] In some embodiments, (a) the polypeptide of the multispecific CAR construct comprises any one of the amino acid sequences shown in SEQ ID NO: 14-15, and / or (b) the first polypeptide of the multispecific CAR construct comprises any one of the amino acid sequences shown in SEQ ID NO: 12 and 19, and (b) the second polypeptide of the multispecific CAR construct comprises any one of the amino acid sequences shown in SEQ ID NO: 13 and 20-22.

[0055] In one aspect, this article provides a combination of a CLDN18.2 antagonist and a GUCY2C antagonist, wherein the antagonist is selected from antibodies, aptamers, antibody-drug conjugates (ADCs), small RNAs, engineered receptors, and engineered immune cells.

[0056] In some embodiments, the combination of the CLDN18.2 antagonist and the GUCY2C antagonist comprises a first group of engineered immune cells and a second group of engineered immune cells, wherein:

[0057] (1) The CLDN18.2 antagonist is the first group of engineered immune cells comprising an engineered receptor that specifically targets CLDN18.2, the engineered receptor comprising: an extracellular antigen-binding domain containing at least one anti-CLDN18.2 binding moiety, a transmembrane domain, and optionally an intracellular signal transduction domain; and / or

[0058] (2) The GUCY2C antagonist is the second group of engineered immune cells containing an engineered receptor that specifically targets GUCY2C, the engineered receptor comprising: an extracellular antigen-binding domain containing at least one anti-GUCY2C binding moiety, a transmembrane domain, and optionally an intracellular signaling domain. The engineered receptor specifically targeting CLDN18.2 and the engineered receptor specifically targeting GUCY2C can be expressed in different engineered immune cells.

[0059] The engineered receptor may be selected from the group consisting of engineered T-cell receptors (TCRs), chimeric antigen receptors (CARs), T-cell antigen conjugates (TACs), or portions thereof. In some embodiments, the engineered receptor specifically targeting CLDN18.2 is a CAR specifically targeting CLDN18.2, and / or the engineered receptor specifically targeting GUCY2C is a CAR specifically targeting GUCY2C.

[0060] In some embodiments, the anti-CLDN18.2 binding portion and / or the anti-GUCY2C binding portion is selected from Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), mini antibody, biantibody, single-domain antibody (sdAb), or VHH domain.

[0061] In some embodiments, the anti-CLDN18.2 binding portion is anti-CLDN18.2 VHH, optionally comprising CDR1, CDR2, and CDR3 as shown in the amino acid sequence of SEQ ID NO: 1, and optionally comprising (i) CDR1 containing the amino acid sequence of SEQ ID NO: 23, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 24, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO: 25. In some embodiments, the anti-CLDN18.2 VHH comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 1.

[0062] In some embodiments, the anti-GUCY2C binding portion is anti-GUCY2C VHH, optionally comprising CDR1, CDR2, and CDR3 as shown in the amino acid sequence of SEQ ID NO: 2, and optionally comprising (i) CDR1 containing the amino acid sequence of SEQ ID NO: 26, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 27, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-GUCY2C VHH comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 2.

[0063] The transmembrane domains of the CAR specifically targeting CLDN18.2 and the CAR specifically targeting GUCY2C may be derived from proteins selected from the group consisting of CD8α, ICOS, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. Optionally, the transmembrane domains of the CAR specifically targeting CLDN18.2 and the CAR specifically targeting GUCY2C may be derived from CD8α or ICOS and contain an amino acid sequence that is at least 85%, 90%, or 95% identical to any of the amino acid sequences shown in SEQ ID NO: 5-6.

[0064] In some embodiments, the intracellular signal transduction domain of the CAR specifically targeting CLDN18.2 and / or the CAR specifically targeting GUCY2C includes a primary intracellular signal transduction domain, optionally derived from CD3ζ, optionally containing the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 11. In some embodiments, the intracellular signal transduction domain of the CAR specifically targeting CLDN18.2 and the CAR specifically targeting GUCY2C further includes a co-stimulatory signal transduction domain. This co-stimulatory signal transduction domain may be derived from co-stimulatory molecules selected from CD28, 4-1BB (CD137), CD27, OX40, CD30, CD40, CD3, LFA-1, ICOS (CD278), NTBA, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 ligands, and combinations thereof.

[0065] In some embodiments, the CAR specifically targeting CLDN18.2 comprises (1) a hinge domain located between the N-terminus of the extracellular antigen-binding domain and the transmembrane domain, optionally wherein the hinge domain is derived from CD8α or CD28; and / or (2) a signal peptide located at the N-terminus of the extracellular antigen-binding domain, optionally wherein the signal peptide is derived from CD8α. In some embodiments, the CAR specifically targeting GUCY2C comprises (1) a hinge domain located between the N-terminus of the extracellular antigen-binding domain and the transmembrane domain, optionally wherein the hinge domain is derived from CD8α or CD28; and / or (2) a signal peptide located at the N-terminus of the extracellular antigen-binding domain, optionally wherein the signal peptide is derived from CD8α.

[0066] In some embodiments, the CAR specifically targeting CLDN18.2 comprises the amino acid sequence shown in any one of SEQ ID NO: 12 and 19, and / or the CAR specifically targeting GUCY2C comprises the amino acid sequence shown in any one of SEQ ID NO: 13 and 20-22.

[0067] In some embodiments, the CLDN18.2 antagonist is a first group of engineered immune cells comprising a polypeptide containing the amino acid sequence shown in any one of SEQ ID NO: 12 and 19, and / or the GUCY2C antagonist is a second group of engineered immune cells comprising a polypeptide containing the amino acid sequence shown in any one of SEQ ID NO: 13 and 20-22. In some embodiments, the CLDN18.2 antagonist is a first group of engineered immune cells comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 12, and the GUCY2C antagonist is a second group of engineered immune cells comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 13.

[0068] In some embodiments, the combination comprises the CLDN18.2 antagonist and the GUCY2C antagonist in ratios ranging from 10:1 to 1:10, such as 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 or 1:9.

[0069] In some embodiments, the engineered immune cells are selected from the group consisting of T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof.

[0070] On one hand, this article provides a nucleic acid comprising:

[0071] (1) The nucleic acid sequence encoding the multispecific CAR construct as disclosed herein; or

[0072] (2) A first nucleic acid sequence encoding a first engineered receptor specifically targeting CLDN18.2, the first engineered receptor comprising: a first extracellular antigen-binding domain, a first transmembrane domain, and a first intracellular signal transduction domain containing at least one anti-CLDN18.2 binding moiety; and a second nucleic acid sequence encoding a second engineered receptor specifically targeting GUCY2C, the second engineered receptor comprising: a second extracellular antigen-binding domain, a second transmembrane domain, and a second intracellular signal transduction domain containing at least one polypeptide containing the GUCY2C binding moiety.

[0073] Optionally, the first nucleic acid sequence and the second nucleic acid sequence are linked by a nucleic acid sequence encoding a self-cleavable peptide (such as P2A, E2A, F2A or T2A).

[0074] In some embodiments, the nucleic acid comprises:

[0075] (1) A nucleic acid sequence encoding a polypeptide containing any one of the amino acid sequences shown in SEQ ID NO: 14-18; or

[0076] (2) A first nucleic acid sequence encoding a first polypeptide containing the amino acid sequences shown in any one of SEQ ID NO: 12 and 19, and a second nucleic acid sequence encoding a second polypeptide containing the amino acid sequences shown in any one of SEQ ID NO: 13 and 20-22.

[0077] In one aspect, this article provides a vector comprising nucleic acids as disclosed herein. In another aspect, this article provides an engineered immune cell comprising a multispecific CAR, nucleic acid, or vector as disclosed herein.

[0078] In some embodiments, the engineered immune cell comprises an engineered receptor specifically targeting CLDN18.2, the engineered receptor comprising: an extracellular antigen-binding domain, a transmembrane domain, and optionally an intracellular signaling domain containing at least one anti-CLDN18.2 binding moiety; and an engineered receptor specifically targeting GUCY2C, the engineered receptor comprising: an extracellular antigen-binding domain, a transmembrane domain, and optionally an intracellular signaling domain containing at least one anti-GUCY2C binding moiety. The engineered immune cell may be derived from the group consisting of T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof.

[0079] In some embodiments, the engineered immune cells comprise:

[0080] (1) A polypeptide containing any one of the amino acid sequences shown in SEQ ID NO: 14-18; or

[0081] (2) A first polypeptide and a second polypeptide, wherein the first polypeptide comprises an amino acid sequence shown in any one of SEQ ID NO: 12 and 19, and the second polypeptide comprises an amino acid sequence shown in any one of SEQ ID NO: 13 and 20-22.

[0082] In one respect, this article provides a pharmaceutical composition comprising a multispecific CAR construct, combination, nucleic acid or engineered immune cell as disclosed herein, and a pharmaceutically acceptable carrier.

[0083] In one aspect, this article provides a multispecific CAR construct, combination, nucleic acid, or engineered immune cell as disclosed herein for use in treating a subject's tumor, optionally wherein the tumor is CLDN18.2 positive and / or GUCY2C positive. The tumor may be selected from gastric cancer, esophageal cancer, pancreatic ductal carcinoma, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colorectal cancer, liver cancer, head and neck cancer, gallbladder cancer, and their metastases. The subject may be resistant to at least one CLDN18.2 agent and / or wherein the subject is resistant to at least one GUCY2C agent. Attached Figure Description

[0084] Figure 1 A to Figure 1 B shows the IHC analysis of CLDN18.2 and GUCY2C expression in primary and metastatic gastric cancer (GC) tissues.

[0085] Figure 2 A to Figure 2 C shows the CAR structure scheme for cells (e.g., T cells) expressing CLDN18.2 CAR and / or GUCY2C CAR. Figure 2 A shows two types of single antigen-specific CARs, such as a single dentin 18.2-specific CAR (Si-CLDN18.2 CAR) that recognizes and binds to dentin 18.2, or a single GUCY2C-specific CAR (Si-GCC CAR) that recognizes and binds to GUCY2C. Figure 2 B shows the structure of a tandem bispecific CAR, which contains an extracellular antigen-binding region on the same CAR and can recognize and bind two different antigens, such as dense protein 18.2 and GUCY2C. Figure 2 C shows a split-type bispecific CAR design in which cells simultaneously express two CARs that target two antigens, such as duracin 18.2 and GUCY2C, respectively.

[0086] Figure 3 A to Figure 3 C shows the effects of different CAR-T cell types on target cells (e.g., Hep3b-CLDN18.2-GUCY2C.Luc cells) at different E:T ratios of 2:1 and 0.5:1. Figure 3 A), Hep3b-CLDN18.2.Luc cells ( Figure 3 B), Hep3b-GUCY2C.Luc cells ( Figure 3 C)) in vitro cytotoxicity.

[0087] Figure 4 A to Figure 4B shows the cytokines (IFN-γ and TNF-α) released in vitro by CAR-T cells in different target cell mixtures (mixtures 1-7) in a heterogeneous model.

[0088] Figure 5 A to Figure 5 C shows the cell expansion and CAR expression of CAR-T cells in the repeated challenge assay. Detailed Implementation

[0089] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Further, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. More specifically, unless the context clearly specifies otherwise, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural indicators. Thus, for example, reference to “a protein” includes multiple proteins; reference to “a cell” includes a mixture of multiple cells, etc. In this application, unless otherwise stated, the use of “or” means “and / or.” Furthermore, the use of the term “comprising” and other forms such as “comprises” and “comprised” is non-limiting. Additionally, the scope provided in this specification and the appended claims includes both endpoints and all points between endpoints.

[0090] The term "antagonist" refers to a substance that interferes with or inhibits the physiological effects of a target antigen or the signal transduction pathway mediated by the target antigen. Various types of antagonists are known in the art, including but not limited to engineered immune cells, engineered receptors such as engineered T-cell receptors (TCRs), chimeric antigen receptors (CARs), T-cell antigen-coupled agents (TACs) or portions thereof, antibodies, antibody-drug conjugates (ADCs), aptamers, small RNAs, and inhibitors of chemical compounds.

[0091] As used herein, the term "combination of CLDN18.2 antagonist and GUCY2C antagonist" refers to a combination of a first substance that specifically antagonizes CLDN18.2 but not GUCY2C and a second substance that specifically antagonizes GUCY2C but not CLDN18.2. The first and second substances may be separate, linked, fused, or complexed. The CLDN18.2 antagonist may be a first CAR having an extracellular antigen-binding domain that binds CLDN18.2 but not GUCY2C, and the GUCY2C antagonist may be a second CAR having an extracellular antigen-binding domain that binds GUCY2C but not CLDN18.2. The first and second CARs may be separate peptides or linked together on the same chain. A CLDN18.2 antagonist can be a first group of engineered immune cells expressing a CAR that binds to CLDN18.2 but not to GUCY2C, and a GUCY2C antagonist can be a second group of engineered immune cells expressing a CAR that binds to GUCY2C but not to CLDN18.2. If the first and second substances can be linked or fused together as a new molecule that retains the antagonistic ability of both substances, then the new molecule can also be considered a "CLDN18.2 and GUCY2C antagonist".

[0092] As used herein, the term "CLDN18.2 and GUCY2C antagonist" refers to a substance that has the ability to specifically antagonize both CLDN18.2 and GUCY2C simultaneously. A CLDN18.2 and GUCY2C antagonist can be a CAR having an extracellular antigen-binding domain capable of binding to both CLDN18.2 and GUCY2C. A CLDN18.2 and GUCY2C antagonist can be an engineered immune cell expressing a CAR having an extracellular antigen-binding domain capable of binding to both CLDN18.2 and GUCY2C. A CLDN18.2 and GUCY2C antagonist can be an engineered immune cell expressing a first CAR and a second CAR, wherein the first CAR has an extracellular antigen-binding domain that binds to CLDN18.2 but not GUCY2C, and the second CAR has an extracellular antigen-binding domain that binds to GUCY2C but not CLDN18.2. Antagonists of CLDN18.2 and GUCY2C can also be anti-CLDN18.2×GUCY2C antibodies.

[0093] The term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide construct comprising an extracellular antigen-binding or ligand-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain comprises a functional signaling domain derived from a stimulatory molecule. CARs can be single-specific or multi-specific (e.g., bispecific). The domains in a CAR polypeptide construct can be on the same polypeptide chain, for example, constituting a chimeric fusion protein. The domains in a CAR construct can be discontinuous, for example, on different polypeptide chains, as provided in a split-type CAR construct as described below.

[0094] As used herein, the term "multispecific tandem CAR" refers to a recombinant polypeptide construct containing an extracellular antigen-binding region comprising more than one operatively tandemly linked antigen-binding moieties having more than one antigen-binding specificity. For example, a multispecific tandem CAR can be a multispecific or multivalent CAR containing an extracellular antigen-binding region comprising more than one copy of the same VHH, wherein these VHHs are operatively tandemly linked. A multispecific tandem CAR can be a multispecific CAR containing two or more VHHs with different antigen specificities, wherein a first VHH is tandemly linked to a second VHH in the extracellular antigen-binding region. A multispecific tandem CAR can be two CARs linked by an inelastic linker.

[0095] As used herein, the term "multispecific split CAR" refers to a CAR construct comprising two CARs (either in one chain or in separate chains), each containing an extracellular antigen-binding region with different antigen specificities. When in one chain, the two CARs are typically operatively linked via a cleavable linker.

[0096] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that binds specifically to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain or intact immunoglobulins, and can be derived from natural or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.

[0097] As used herein, the term "antigen-binding moiety" refers to an antibody fragment comprising a portion of one or more CDRs or any other antibody fragment that binds to an antigen but does not contain the complete structure of the native antibody. Examples of antigen-binding moieties include, but are not limited to, variable domains, variable regions, single variable domains (i.e., VHHs), nanobodies, domain antibodies, biantibodies, Fab, Fab', F(ab')2, Fv fragments, single-chain Fv fragments (scFv), disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized biantibodies (ds biantibodies), multispecific antibodies, camel-derived single-domain antibodies, and bivalent domain antibodies. An antigen-binding moiety is capable of binding to the same antigen as the antigen bound by the parent antibody. A more detailed description of the antigen-binding part is found in the following references: Spiess et al., (2015) Molecular Immunology 67: 95-106 and Brinkman et al., mAbs, 9(2), pp. 182-212 (2017), which are incorporated herein by reference in their entirety.

[0098] As used herein, the term "single-domain antibody" or "sdAb" refers to a single monomeric variable antibody domain that is capable of binding an antigen (e.g., a single-domain antibody binding CLDN18.2 or GUCY2C). Single-domain antibodies include VHH domains as described herein. Examples of single-domain antibodies include, but are not limited to, antibodies naturally lacking the light chain, such as those from camel species (e.g., llamas), single-domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. Single-domain antibodies (e.g., VHH domain antibodies) can be derived from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, and cattle. For example, as described herein, single-domain antibodies can be derived from camel species (e.g., antibodies produced in camels, llamas, dromedary camels, alpacas, and guanacos). Other species besides camels can produce heavy-chain antibodies naturally lacking the light chain; VHHs derived from such other species are within the scope of this disclosure. The single-domain antibody (e.g., VHH) provided herein may have a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The single-domain antibody may be genetically fused or chemically conjugated to another molecule (e.g., a pharmaceutical agent) as described herein. The single-domain antibody may be part of a larger binding molecule (e.g., a multispecific antibody or a chimeric antigen receptor).

[0099] As used herein, the term "complementarity-determining region" or "CDR" refers to the sequence of amino acids within an antibody variable domain that confers antigen specificity and binding affinity. For example, typically, three CDRs are present in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3), and three CDRs are present in each light chain variable region (LCDR1, LCDR2, and LCDR3). The extent of CDRs and frame regions can be precisely identified using methods known in the art, such as those defined by Kabat, Dr. Martin's website, Chothia, AbM, EU, and contact, all of which are well-known in the art. See, for example, Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242; Martin A. “Antibody bioinformatics website of Dr. Andrew Martin's lab at UCL,” last updated July 31, 2018; Chothia et al. (1989) Nature 342:877; Chothia, C., et al. (1987) J. Mol. Biol. 196:901-917; Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948; Edelman et al., Proc Natl Acad Sci US A. May 1969; 63(1):78-85; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs.Correspondences or comparisons between different defined numbering methods can be found, for example, at http: / / www.imgt.org / (see also Giudicelli V et al., IMGT, the international ImMunoGeneTics database. NucleicAcids Res. (1997) 25:206-11; Lefranc MP et al., Unique database numbering system for immunogenetic analysis. Immunol Today (1997) 18:509; and Lefranc MP et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol. (2003) 27:55-77). In some cases, a scheme is specified for identifying one or more specific CDRs, such as CDRs defined by IMGT, Kabat, AbM, Chothia, or Contact methods. One or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number. For exemplary numbering of the VHH domain according to Kabat, see, for example, Deschacht et al., 2010. J Immunol 184: 5696-704. In other cases, the specific amino acid sequence of the CDR is given. It should be noted that the CDR region can also be defined by a combination of various numbering systems, such as the combination of the Kabat and Chothia numbering systems, the combination of the Kabat and AbM numbering systems, or the combination of the Kabat and IMGT numbering systems. Therefore, the term "CDR as shown in a particular VH or VHH" includes, but is not limited to, any CDR as defined by the exemplary CDR numbering systems described above. Once a variable region (e.g., the VHH domain, VH, or VL domain) is given, those skilled in the art will understand that the CDR within that region can be defined by different numbering systems or combinations thereof.

[0100] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a CAR that can generate signals that promote immune effector functions in CAR-containing cells (e.g., CAR-T cells or CAR-expressing NK cells). Examples of immune effector functions, for instance, in CAR-T cells or CAR-expressing NK cells include cytolytic activity and cofactor activities, including cytokine secretion. Intracellular signaling domains can transduce effector function signals and direct cells to perform specialized functions. While the entire intracellular signaling domain can be used, in many cases, it is not necessary to use the whole chain.

[0101] The term "stimulatory molecule" refers to a molecule expressed by T cells that provides one or more primary intracellular signaling sequences that stimulately regulate primary activation of the TCR complex at least some aspects of the T cell signaling pathway. In one aspect, primary signaling is initiated, for example, by the binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, which leads to the mediation of T cell responses, including but not limited to proliferation, activation, differentiation, etc. The stimulatory primary signaling domain may contain a signaling motif known as an immune receptor tyrosine-based activation motif or ITAM. Examples of ITAM-containing primary intracellular signaling sequences specifically used in this disclosure include, but are not limited to, those derived from: TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, CD66d, DAP10, and DAP12. In the specific CARs disclosed herein, the intracellular signal transduction domain contains intracellular signal transduction sequences, such as the primary signal transduction sequence of CD3-ζ, which may be derived from human or non-human species, such as mice, rodents, monkeys, apes, etc.

[0102] The term "co-stimulatory signaling domain" refers to the intracellular portion of a co-stimulatory molecule. An intracellular signaling domain may contain the entire intracellular portion of the molecule from which it originates, the entire native intracellular signaling domain, or a functional fragment thereof. Co-stimulatory molecules are homologous binding partners on T cells that specifically bind to co-stimulatory ligands, thereby mediating T cell co-stimulatory responses (e.g., but not limited to proliferation). Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are essential for an effective immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), NK cell activating receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD137 / CD18), etc. 278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11 d. ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGΒ2, CD18, LFA-1, ITGB 7. NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM 1. CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and ligands that specifically bind to CD83.

[0103] The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers are described in this article, including but not limited to stomach cancer, pancreatic cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer. The terms "tumor" and "cancer" are used interchangeably in this article.

[0104] CLDN18.2 antagonist and GUCY2C antagonist

[0105] In one aspect, this disclosure provides a method for treating a tumor in a subject of need, the method comprising administering to the subject an effective amount of a combination of a CLDN18.2 (densin 18.2) antagonist and a GUCY2C antagonist, or an antagonist of CLDN18.2 and GUCY2C. The tumor may be CLDN18.2 positive and / or GUCY2C positive. In another aspect, this disclosure also provides a combination of a CLDN18.2 antagonist and a GUCY2C antagonist, as well as an antagonist of CLDN18.2 and GUCY2C, and methods of using them to treat a variety of diseases.

[0106] CLDN18.2 antagonists, GUCY2C antagonists, and / or antagonists of both CLDN18.2 and GUCY2C can be of various classes; for example, antagonists can be selected from engineered receptors, engineered immune cells, antibodies, antibody-drug conjugates (ADCs), aptamers, and small RNAs. CLDN18.2 antagonists may include monospecific antibodies, bispecific antibodies, ADCs, CAR-T cells redirected to target CLDN18.2, such as zotuximab (Zolbetuximab) (IMAB362), TST001, BNT141, AMG910, or LM-302. GUCY2C antagonists may include those known in the art, such as anti-GUCY2C monospecific antibodies, ADC antibodies (e.g., TAK-264 (ADC-DGN549), TAK-164), bispecific antibodies (PF-07062119 (GCCⅹCD3)), or Ad5-GUCY2C-PADRE. Antagonists for CLDN18.2 and GUCY2C may include bispecific antibodies against CLDN18.2 and GUCY2C (GCCⅹCLDN18.2).

[0107] CLDN18.2 antagonists can be engineered immune cells expressing an engineered receptor that specifically targets CLDN18.2, the engineered receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain containing at least one anti-CLDN18.2 binding moiety. GUCY2C antagonists can be engineered immune cells containing an engineered receptor that specifically targets GUCY2C, the engineered receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain containing at least one anti-GUCY2C binding moiety. In some embodiments, the engineered receptor is selected from the group consisting of engineered T-cell receptors (TCRs), chimeric antigen receptors (CARs), T-cell antigen conjugates (TACs), or portions thereof. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR). The anti-CLDN18.2 binding moiety can be anti-CLDN18.2VHH, and optionally, the anti-CLDN18.2VHH comprises CDR1, CDR2, and CDR3 as shown in the amino acid sequence of SEQ ID NO: 1. The anti-CLDN18.2 VHH CDRs (CDR1-3) can be determined according to the Kabat numbering scheme, IMGT numbering scheme, AbM numbering scheme, Chothia numbering scheme, Contact numbering scheme, or a combination thereof. In some embodiments, the anti-CLDN18.2 VHH comprises (i) CDR1 containing the amino acid sequence of SEQ ID NO: 23, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 24, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO: 25. The anti-CLDN18.2 VHH may comprise the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 1. The anti-GUCY2C binding portion is the anti-GUCY2C VHH, and optionally the anti-GUCY2C VHH comprises CDR1, CDR2, and CDR3 as shown in the amino acid sequence of SEQ ID NO: 2. The anti-GUCY2C VHH CDRs (CDR1-3) can be determined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or a combination thereof. In some embodiments, anti-GUCY2C VHH comprises (i) CDR1 containing the amino acid sequence of SEQ ID NO: 26, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 27, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO: 28.Anti-GUCY2C VHH may contain the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO: 2.

[0108] In some embodiments, the CLDN18.2 antagonist is an engineered immune cell comprising a CAR specifically targeting CLDN18.2 (referred to herein as a “single CLDN18.2-specific CAR”), the CAR comprising a polypeptide having an amino acid sequence shown in any one of SEQ ID NO: 12 and 19, and / or the GUCY2C antagonist is an engineered immune cell comprising a CAR specifically targeting GUCY2C (referred to herein as a “single GUCY2C-specific CAR”), the CAR comprising a polypeptide having an amino acid sequence shown in any one of SEQ ID NO: 13 and 21-22. The engineered immune cells may be selected from the group consisting of: T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof.

[0109] Accordingly, the combination of a CLDN18.2 antagonist and a GUCY2C antagonist can comprise two groups of engineered immune cells that target CLDN18.2 and GUCY2C, respectively. The first and second groups of engineered immune cells can be administered simultaneously or sequentially. This combination can also be considered an antagonist of CLDN18.2 and GUCY2C when the engineered immune cells simultaneously express both an engineered receptor specifically targeting CLDN18.2 and an engineered receptor specifically targeting GUCY2C.

[0110] The combination of CLDN18.2 antagonists and GUCY2C antagonists offers certain benefits. In cancer treatment, combination therapies targeting more than one antigen can have improved efficacy compared to monotherapy targeting a single antigen. Cancer cells are genetically unstable, allowing them to evade targeted therapies by mutating or losing genes encoding target antigens. By targeting two or more different epitopes or antigens on cancer cells, combination therapies can make it more difficult for cancer cells to completely evade antagonism. In one aspect, this document provides a method for treating a subject's tumor by utilizing the benefits of combining CLDN18.2 antagonists and GUCY2C antagonists. In some embodiments, the method includes administering a combination of CLDN18.2 antagonists and GUCY2C antagonists. In some other embodiments, the method includes administering antagonists of CLDN18.2 and GUCY2C as disclosed herein.

[0111] Combination of CLDN18.2 antagonist and GUCY2C antagonist

[0112] In one aspect, this paper provides a combination (referred to herein as the "combination") of CLDN18.2 antagonists and GUCY2C antagonists with different antigen specificities, which may have complementary or synergistic effects. The combination may comprise a CLDN18.2 antagonist and a GUCY2C antagonist, wherein the CLDN18.2 antagonist is an anti-CLDN18.2 monoclonal antibody or a corresponding ADC, and the GUCY2C antagonist is an anti-GUCY2C monoclonal antibody or a corresponding ADC. The CLDN18.2 antagonist may be an engineered receptor specifically targeting CLDN18.2, and the GUCY2C antagonist may be an engineered receptor specifically targeting GUCY2C. The combination may include a CLDN18.2 antagonist and a GUCY2C antagonist, wherein the CLDN18.2 antagonist is a first group of engineered immune cells expressing an engineered receptor that specifically targets CLDN18.2, and the GUCY2C antagonist is a second group of engineered immune cells expressing an engineered receptor that specifically targets GUCY2C.

[0113] The CLDN18.2 antagonist can be an engineered receptor that specifically targets CLDN18.2, comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain containing at least one anti-CLDN18.2 binding moiety. The GUCY2C antagonist can be an engineered receptor that specifically targets GUCY2C, comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain containing at least one anti-GUCY2C binding moiety. Accordingly, the combination of the CLDN18.2 antagonist and the GUCY2C antagonist can comprise two engineered receptors that respectively target CLDN18.2 and GUCY2C.

[0114] When two engineered receptors targeting CLDN18.2 and GUCY2C are linked, fused, associated, or form a complex, the combination can also be considered an antagonist of CLDN18.2 and GUCY2C. Engineered receptors specifically targeting CLDN18.2 can be operatively linked (e.g., via a cleavable linker) to engineered receptors specifically targeting GUCY2C.

[0115] In some embodiments, the combination comprises two groups of engineered immune cells, wherein a first group of engineered immune cells expresses an engineered receptor specifically targeting CLDN18.2, and a second group of engineered immune cells expresses an engineered receptor specifically targeting GUCY2C.

[0116] In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR). Accordingly, the assembly may comprise a first CAR containing an antigen-binding domain specifically targeting CLDN18.2 (i.e., a single CLDN18.2-specific CAR) and a second CAR containing an antigen-binding domain specifically targeting GUCY2C (i.e., a single GUCY2C-specific CAR). The assembly may comprise the first and second CARs presented as a multispecific resplitable CAR construct, wherein the first and second CARs are operatively linked into a single chain via cleavable linkers (e.g., P2A, T2A, E2A, or F2A), or dissociated into two chains when the linkers are cleaved.

[0117] The combination may comprise: (1) a first CAR specifically targeting CLDN18.2 containing an extracellular antigen-binding domain (also referred to herein as a “single CLDN18.2-specific CAR”), the extracellular antigen-binding domain comprising at least one anti-CLDN18.2 binding moiety targeting the CLDN18.2 antigen or an epitope on the CLDN18.2 antigen; and (2) a second CAR specifically targeting GUCY2C containing an extracellular antigen-binding domain (also referred to herein as a “single GUCY2C-specific CAR”), the extracellular antigen-binding domain comprising at least one anti-GUCY2C binding moiety targeting the GUCY2C antigen or an epitope on the GUCY2C antigen. The first and second CARs may each comprise a polypeptide comprising, from the N-terminus to the C-terminus: an extracellular antigen-binding domain, a hinge domain (e.g., a CD8α hinge domain), a transmembrane domain (e.g., CD8α, CD28, or ICOS™), optionally a co-stimulatory signaling domain, and a primary intracellular signaling domain (e.g., derived from CD3ζ). The polypeptide may further include a signal peptide (e.g., CD8α signal peptide) at its N-terminus.

[0118] In some embodiments, the combination of the CLDN18.2 antagonist and the GUCY2C antagonist comprises a first group of engineered immune cells and a second group of engineered immune cells, wherein: (1) the CLDN18.2 antagonist is a first group of engineered immune cells comprising a CAR specifically targeting CLDN18.2 (e.g., a single CLDN18.2-specific CAR), the CAR comprising: an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain containing at least one anti-CLDN18.2 binding moiety; and / or (2) the GUCY2C antagonist is a second group of engineered immune cells comprising a CAR specifically targeting GUCY2C (e.g., a single GUCY2C-specific CAR), the CAR comprising: an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain containing at least one anti-GUCY2C binding moiety.

[0119] In some embodiments of this combination, the anti-CLDN18.2 binding portion is anti-CLDN18.2 VHH, and optionally, the anti-CLDN18.2 VHH comprises CDR1, CDR2, and CDR3 as shown in the amino acid sequence of SEQ ID NO: 1. The anti-CLDN18.2 VHH CDRs (CDR1-3) can be determined according to the Kabat numbering scheme, IMGT numbering scheme, AbM numbering scheme, Chothia numbering scheme, Contact numbering scheme, or a combination thereof. In some embodiments, the anti-CLDN18.2 VHH comprises (i) CDR1 containing the amino acid sequence of SEQ ID NO: 23, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 24, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO: 25. The anti-CLDN18.2 VHH may comprise the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-GUCY2C binding portion is anti-GUCY2C VHH, and optionally, the anti-GUCY2C VHH comprises CDR1, CDR2, and CDR3 as shown in the amino acid sequence of SEQ ID NO: 2. The anti-GUCY2C VHH CDRs (CDR1-3) can be determined according to the Kabat numbering scheme, IMGT numbering scheme, AbM numbering scheme, Chothia numbering scheme, Contact numbering scheme, or a combination thereof. In some embodiments, the anti-GUCY2C VHH comprises (i) CDR1 containing the amino acid sequence of SEQ ID NO: 26, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 27, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO: 28. The anti-GUCY2C VHH may comprise the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 2.

[0120] In some embodiments, the combination of the CLDN18.2 antagonist and the GUCY2C antagonist comprises a first group of engineered immune cells and a second group of engineered immune cells, wherein: (1) the CLDN18.2 antagonist is a first group of engineered immune cells comprising a CAR specifically targeting CLDN18.2 (e.g., a single CLDN18.2-specific CAR), the CAR comprising: an extracellular antigen-binding domain containing at least one anti-CLDN18.2 VHH, optionally a hinge domain (e.g., a CD8α hinge domain), a transmembrane domain (e.g., CD8α or CD28 or ICOS™), optionally a co-stimulatory signaling domain, and a primary intracellular signaling domain (e.g., derived from CD3ζ); and / or (2) the GUCY2C antagonist is a second group of engineered immune cells comprising a CAR specifically targeting GUCY2C (e.g., a single GUCY2C-specific CAR), the CAR comprising: an anti-GUCY2C... VHH's extracellular antigen-binding domain, optionally a hinge domain (e.g., CD8α hinge domain), a transmembrane domain (e.g., CD8α, CD28, or ICOS™), optionally a co-stimulatory signaling domain, and a primary intracellular signaling domain (e.g., derived from CD3ζ). In some embodiments, the combination of the CLDN18.2 antagonist and the GUCY2C antagonist comprises a first group of engineered immune cells and a second group of engineered immune cells, the first group of engineered immune cells comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 12, and the second group of engineered immune cells comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 13.

[0121] The CLDN18.2 antagonist and GUCY2C antagonist in this combination can be administered simultaneously or sequentially. When administered simultaneously, the CLDN18.2 antagonist and GUCY2C antagonist can be mixed in a specific ratio before administration. Alternatively, the CLDN18.2 antagonist and GUCY2C antagonist can be administered individually in a specific ratio. The amounts of CLDN18.2 antagonist and GUCY2C antagonist administered to the subject can differ. For example, when the combination contains two groups of engineered immune cells, wherein the first group of engineered immune cells expresses an engineered receptor specifically targeting CLDN18.2 and the second group of engineered immune cells expresses an engineered receptor specifically targeting GUCY2C, the ratio of the number of the first group of engineered immune cells to the number of the second group of engineered immune cells can be in a wide range from 10:1 to 1:10, such as 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 or 1:9. When the combination contains two monoclonal antibodies or ADCs, wherein the first antibody or ADC specifically targets CLDN18.2 and the second antibody or ADC specifically targets GUCY2C, the molar ratio of the first antibody to the second antibody can be in a wide range from 10:1 to 1:10, such as 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 or 1:9.

[0122] As mentioned above, combinations of CLDN18.2 antagonists and GUCY2C antagonists with different antigen-binding specificities (e.g., a combination of single CLDN18.2-specific CAR-T cells and single GUCY2C-specific CAR-T cells) can have improved efficacy in cancer immunotherapy compared to single-specific CARs or immune cells alone. By targeting two or more different epitopes or antigens on cancer cells, combinations of two or more single-antigen-specific CARs can make it more difficult for cancer cells to completely evade the targeting of CAR-expressing engineered immune cells (such as T cells).

[0123] Antagonists of CLDN18.2 and GUCY2C

[0124] On the other hand, this article provides antagonists for CLDN18.2 and GUCY2C that simultaneously target both CLDN18.2 and GUCY2C. Antagonists for CLDN18.2 and GUCY2C (referred to herein as "antagonists") may include molecules that co-target CLDN18.2 and GUCY2C, such as multispecific antibodies or multispecific chimeric receptors that co-target CLDN18.2 and GUCY2C. Antagonists for CLDN18.2 and GUCY2C may be multispecific (e.g., bispecific) antibodies targeting CLDN18.2 and GUCY2C. Antagonists for CLDN18.2 and GUCY2C may be engineered receptors that simultaneously co-target CLDN18.2 and GUCY2C. Antagonists of CLDN18.2 and GUCY2C may comprise a two-part complex, wherein one part may be an engineered receptor targeting CLDN18.2 and the other part may be an engineered receptor targeting GUCY2C, and the two parts may be linked, fused, or associated. Antagonists of CLDN18.2 and GUCY2C also encompass engineered immune cells containing engineered receptors specifically targeting CLDN18.2 and specifically targeting GUCY2C.

[0125] Antagonists of CLDN18.2 and GUCY2C can be multispecific engineered receptors that simultaneously target CLDN18.2 and GUCY2C. This multispecific engineered receptor may comprise an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain containing at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety. Correspondingly, antagonists of CLDN18.2 and GUCY2C can also be engineered immune cells expressing engineered receptors that simultaneously target CLDN18.2 and GUCY2C.

[0126] Antagonists of CLDN18.2 and GUCY2C may comprise two engineered receptors with different antigen specificities, wherein a first engineered receptor specifically targets CLDN18.2 and a second engineered receptor specifically targets GUCY2C, optionally the two engineered receptors being operatively linked. The antagonists of CLDN18.2 and GUCY2C may comprise two engineered receptors, wherein the first engineered receptor comprises an extracellular antigen-binding region, a transmembrane domain, and an intracellular signal transduction domain containing at least one anti-CLDN18.2 binding moiety, and the second engineered receptor comprises an extracellular antigen-binding region, a transmembrane domain, and an intracellular signal transduction domain containing at least one anti-GUCY2C binding moiety. The two engineered receptors may be operatively linked via a cleavable adapter that can be readily cleaved under suitable conditions. The cleavable adapter may be selected from P2A, T2A, E2A, and F2A.

[0127] Accordingly, the antagonists of CLDN18.2 and GUCY2C can also be engineered immune cells that simultaneously express a first engineered receptor and a second engineered receptor. In some embodiments, the immune cells have been transduced with two separate vectors, the first vector containing nucleic acid encoding a first engineered receptor specifically targeting CLDN18.2, and the second vector containing nucleic acid encoding a second engineered receptor specifically targeting GUCY2C. In some other embodiments, the immune cells have been transduced with a vector containing nucleic acid encoding a first engineered receptor specifically targeting CLDN18.2, which is operatively linked to a second engineered receptor specifically targeting GUCY2C.

[0128] In some embodiments, the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising an engineered receptor that co-targets CLDN18.2 and GUCY2C, the engineered receptor comprising: an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain containing at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety.

[0129] Engineered receptors may be selected from the group consisting of engineered T-cell receptors (TCRs), chimeric antigen receptors (CARs), T-cell antigen conjugates (TACs), or portions thereof. In some embodiments, the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising a first CAR targeting CLDN18.2 and a second CAR targeting GUCY2C, wherein (1) the first CAR targeting CLDN18.2 (referred herein to as a single CLDN18.2-specific CAR) comprises: a first extracellular antigen-binding domain, a first transmembrane domain, and a first intracellular signaling domain containing at least one anti-CLDN18.2 binding moiety; and (2) the second CAR targeting GUCY2C (referred herein to as a single GUCY2C-specific CAR) comprises: a second extracellular antigen-binding domain, a second transmembrane domain, and a second intracellular signaling domain containing at least one anti-GUCY2C binding moiety; and wherein the first CAR targeting CLDN18.2 is operatively connected to the second CAR targeting GUCY2C via a cleavable adapter, or the first CAR and the second CAR are not connected due to cleavage of the cleavable adapter. In some embodiments, the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising a CAR (“CLDN18.2×GUCY2C-specific CAR”) co-targeting CLDN18.2 and GUCY2C, wherein the CLDN18.2×GUCY2C-specific CAR comprises: (1) an extracellular antigen-binding domain containing at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety, (2) a transmembrane domain and (3) an intracellular signal transduction domain; and wherein the anti-CLDN18.2 binding moiety is located at the N-terminus or C-terminus of the anti-GUCY2C binding moiety, optionally the anti-CLDN18.2 binding moiety being operatively linked to the anti-GUCY2C binding moiety via a peptide linker such as a GS linker, for example (G4S)3.

[0130] The anti-CLDN18.2 binding moiety and / or the anti-GUCY2C binding moiety can be selected from Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), mini-antibody, biantibody, single-domain antibody (sdAb), or VHH domain. The anti-CLDN18.2 binding moiety is anti-CLDN18.2 VHH, and optionally, the anti-CLDN18.2 VHH contains CDR1, CDR2, and CDR3 as shown in the amino acid sequence of SEQ ID NO: 1. The anti-CLDN18.2 VHH CDRs (CDR1-3) can be determined according to the IMGT numbering scheme. The anti-CLDN18.2 VHH CDRs (CDR1-3) can be determined according to the AbM numbering scheme. The anti-CLDN18.2 VHH CDRs (CDR1-3) can be determined according to the Kabat numbering scheme. In some embodiments, anti-CLDN18.2 VHH comprises (i) CDR1 containing the amino acid sequence of SEQ ID NO: 23, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 24, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO: 25. Anti-CLDN18.2 VHH may comprise the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-GUCY2C binding portion is anti-GUCY2C VHH, and optionally, the anti-GUCY2C VHH comprises CDR1, CDR2, and CDR3 as shown in the amino acid sequence of SEQ ID NO: 2. The anti-GUCY2C VHH CDRs (CDR1-3) can be determined according to the IMGT numbering scheme. The anti-GUCY2C VHH CDRs (CDR1-3) can be determined according to the AbM numbering scheme. The anti-GUCY2C VHH CDRs (CDR1-3) can be determined according to the Kabat numbering scheme. In some embodiments, the anti-GUCY2C VHH comprises (i) CDR1 containing the amino acid sequence of SEQ ID NO: 26, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 27, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO: 28. The anti-GUCY2C VHH may contain the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 2.

[0131] The single CLDN18.2 specific CAR, the single GUCY2C specific CAR, and the CLDN18.2×GUCY2C specific CAR may each further include: (1) a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain, optionally wherein the hinge domain is derived from CD8α or CD28; and / or (2) a signal peptide located at the N-terminus of the extracellular antigen-binding domain, optionally wherein the signal peptide is derived from CD8α.

[0132] In some embodiments, the CAR co-targeting CLDN18.2 and GUCY2C comprises a polypeptide containing the amino acid sequence of either SEQ ID NO: 14 (corresponding to LG23B01 CAR) or SEQ ID NO: 15 (corresponding to LG23B02 CAR). In some embodiments, the single CLDN18.2-specific CAR and the single GUCY2C-specific CAR are operably linked via a P2A linker and contain the amino acid sequence of either SEQ ID NO: 16-18 (corresponding to LG23D01 CAR, LG23D02 CAR, and LG23D03 CAR, respectively) prior to cleavage via a 2A linker.

[0133] In some embodiments, the antagonists of CLDN18.2 and GUCY2C are engineered immune cells comprising: (1) a polypeptide containing an amino acid sequence shown in any one of SEQ ID NO: 14-18; or (2) a first polypeptide and a second polypeptide, wherein the first polypeptide contains an amino acid sequence shown in any one of SEQ ID NO: 12 and 19, and the second polypeptide contains an amino acid sequence shown in any one of SEQ ID NO: 13 and 20-22.

[0134] Engineered receptors (e.g., CAR, TCR, and TAC)

[0135] One aspect of this disclosure provides engineered cells (e.g., immune cells) expressing engineered receptors. The engineered receptor may include an extracellular antigen-binding domain and optionally an intracellular signaling domain. Exemplary engineered receptors include, but are not limited to, chimeric antigen receptors (CARs), engineered T-cell receptors (TCRs), and T-cell antigen-coupled device (TAC) receptors. The engineered receptor may include an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain that specifically bind to an antigen (e.g., CLDN18.2 or GUCY2C). The intracellular signaling domain may include a primary intracellular signaling domain and / or a co-stimulatory signaling domain. The intracellular signaling domain may include an intracellular signaling domain of a TCR helper receptor. The engineered receptor may be encoded by a heteropolynucleotide operatively linked to a promoter (e.g., a constitutive or inducible promoter).

[0136] Engineered receptors may include one or more specific binding domains that target at least one tumor antigen, and one or more intracellular effector domains, such as one or more primary intracellular signaling domains and / or co-stimulatory signaling domains.

[0137] In one aspect, this disclosure provides a chimeric antigen receptor (CAR) that binds to one or more antigens, such as CLDN18.2 and / or GUCY2C. The CAR may act as an antigen antagonist or be expressed by immune cells that subsequently act as antagonists for use in a therapy. The CAR may comprise: (a) an extracellular antigen-binding domain containing one or more antigen-binding moieties (e.g., VHH) that bind to CLDN18.2 and / or GUCY2C; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0138] Each CAR's extracellular antigen-binding domain may contain one or more antigen-binding moieties, which may be present in a variety of forms, including, for example, single-domain antibody (sdAb) or VHH domain, single-chain variable fragment (scFv), Fab, Fab', F(ab)'2, F(ab)'3, Fv, bis-scFv, (scFv)2, mini-antibody, biantibody, triantibody, tetraantibody, intracellular antibody, disulfide-stabilized Fv protein (dsFv), unibody, nanobody, affinity organism, DARPin, monoclonal antibody, idenetocin, alphabet, or designed binding protein. The antigen-binding moieties may be single-domain antibodies (e.g., VHH domains), such as camel, shark, chimeric, human, or humanized single-domain antibodies (e.g., VHH domains). CARs as disclosed herein may contain an antigen-binding domain comprising one or more (e.g., any of 1, 2, 3, 4, 5, 6, or more) VHH domains. VHHs can fuse directly with each other via peptide bonds or via peptide linkers.

[0139] CARs can be monospecific or multispecific (e.g., bispecific), monovalent or multivalent (e.g., bivalent). In some embodiments, a CAR is a multispecific (e.g., bispecific) CAR containing one or more antigen-binding moieties with different antigen-binding specificities. CARs having extracellular antigen-binding regions containing one or more VHHs targeting different epitopes or antigens can be readily recombinantly constructed and generated, thus providing an efficient platform for the preparation and screening of multivalent and multispecific CARs. Furthermore, the small footprint of VHHs allows the CAR to approach hidden antigen targets and epitopes within tumor tissue.

[0140] Depending on the desired antigen to be targeted, the CAR disclosed herein can be engineered to contain appropriate VHHs that specifically target the desired antigen. The VHHs can be arranged in any suitable order. For example, the first VHH domain can be fused to the N-terminus or C-terminus of the second VHH domain. Suitable peptide linkers can be placed between different VHHs to avoid steric hindrance between them. Exemplary bispecific chimeric antigen receptors, their exemplary sequences, constructs, and vectors are shown below.

[0141] The CAR can be a monospecific CAR or a multivalent CAR. The CAR can be a mono-CLDN18.2-specific CAR containing an extracellular antigen-binding domain comprising anti-CLDN18.2 VHH targeting the CLDN18.2 antigen. A mono-CLDN18.2-specific CAR can contain more than one copy of anti-CLDN18.2 VHH operatively linked to the extracellular antigen-binding domain. A mono-CLDN18.2-specific CAR can contain a polypeptide containing the amino acid sequence of either SEQ ID NO: 12 (corresponding to LG23A01 CAR) or SEQ ID NO: 19.

[0142] The CAR can be a single GUCY2C-specific CAR containing an extracellular antigen-binding domain comprising anti-GUCY2C VHH targeting the GUCY2C antigen. The single GUCY2C-specific CAR can contain more than one copy of anti-GUCY2C VHH operatively linked to the extracellular antigen-binding domain. The single GUCY2C-specific CAR can contain a polypeptide with an amino acid sequence containing any one of SEQ ID NO: 13 (corresponding to LG23A02 CAR) and SEQ ID NO: 20-22.

[0143] Single antigen-specific CARs (e.g., single CLDN18.2-specific CARs or single GUCY2C-specific CARs) as disclosed herein can be combined to form split CAR constructs, or the antigen-binding portions of single antigen-specific CARs can be combined to construct multispecific tandem CARs.

[0144] Multispecific CAR constructs (e.g., tandem CAR and split CAR)

[0145] In one aspect, this disclosure provides a multispecific CAR construct (e.g., a tandem CAR) comprising an extracellular antigen-binding domain that binds to CLDN18.2 and GUCY2C, the extracellular antigen-binding domain comprising at least two antigen-binding moieties targeting CLDN18.2 and GUCY2C, the at least two antigen-binding moieties being operatively tandemly linked in the extracellular binding region. This multispecific CAR construct can act as an antagonist of at least two antigens or be expressed by immune cells, which in turn act as antagonists for use in a therapy.

[0146] Multispecific CAR constructs can offer improved efficacy compared to monospecific CARs in cancer immunotherapy. Cancer cells are genetically unstable, allowing them to evade targeted therapies by mutating or losing genes encoding target antigens. By targeting two or more different epitopes or antigens on cancer cells, multispecific CAR constructs, or combinations of two or more monospecific CARs, can make it more difficult for cancer cells to completely evade the targeting of engineered immune cells (such as T cells) expressing CARs. Due to their small size, the tandemly fused VHHs contained within the extracellular antigen-binding domain of multispecific CAR constructs can retain their respective structural integrity and binding affinity to the target antigen. Engineered immune cells expressing multispecific CAR constructs that bind to different tumor antigens can overcome tumor immune escape mechanisms caused by abnormal protein-antigen processing and presentation.

[0147] In some embodiments, this disclosure provides a multispecific CAR construct (e.g., a tandem CAR) (“CLDN18.2×GUCY2C-specific CAR”) that binds to CLDN18.2 and GUCY2C, comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain containing at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety. In some embodiments, the anti-CLDN18.2 binding moiety is located at the N-terminus or C-terminus of the anti-GUCY2C binding moiety, and optionally the anti-CLDN18.2 binding moiety is operatively linked to the anti-GUCY2C binding moiety via a peptide linker (such as a GS linker, e.g., (G4S)3).

[0148] In some embodiments, this disclosure provides a multispecific CAR construct (e.g., a tandem CAR) comprising an extracellular antigen-binding domain comprising one or more copies of an anti-CLDN18.2 VHH binding to the CLDN18.2 antigen and one or more copies of an anti-GUCY2C VHH binding to the GUCY2C antigen. The VHH domains are operatively tandemly linked within the extracellular antigen-binding region. In some specific embodiments, the multispecific CAR construct (e.g., a tandem CAR) comprises an extracellular antigen-binding domain containing one anti-CLDN18.2 VHH and one anti-GUCY2C VHH, wherein the anti-CLDN18.2 VHH is located at the N-terminus of the anti-GUCY2C VHH, or wherein the anti-CLDN18.2 VHH is located at the C-terminus of the anti-GUCY2C VHH. The two VHHs may be directly linked or indirectly linked via a peptide linker (such as a GS linker, e.g., (G4S)3).

[0149] The linker is typically a peptide linker, such as a peptide linker with a length of no more than about 50 amino acids (e.g., no more than about 35, 25, 20, 15, 10, or 5). In some embodiments, the peptide linker is a GS series linker, such as a (G4S)n linker, where n is an integer from 1 to 8.

[0150] In some embodiments, a multispecific CAR construct (e.g., a tandem CAR) may comprise a polypeptide comprising, from its N-terminus to its C-terminus: an extracellular antigen-binding domain containing at least one anti-CLDN18.2 VHH and at least one anti-GUCY2C VHH, a hinge domain (e.g., a CD8α hinge domain), a transmembrane domain (e.g., CD8α, CD28, or ICOSTM), optionally a co-stimulatory signaling domain, and a primary intracellular signaling domain (e.g., derived from CD3ζ). The polypeptide may further comprise a signal peptide (e.g., a CD8α signal peptide) at its N-terminus.

[0151] In some embodiments, a multispecific CAR construct (e.g., a tandem CAR) may comprise a polypeptide comprising, from its N-terminus to its C-terminus: an extracellular antigen-binding domain containing at least one anti-GUCY2C VHH and at least one anti-CLDN18.2 VHH, a hinge domain (e.g., a CD8α hinge domain), a transmembrane domain (e.g., CD8α, CD28, or ICOSTM), optionally a co-stimulatory signaling domain, and a primary intracellular signaling domain (e.g., derived from CD3ζ). The polypeptide may further comprise a signal peptide (e.g., a CD8α signal peptide) at its N-terminus.

[0152] In some embodiments, the anti-CLDN18.2 VHH comprises CDR1, CDR2, and CDR3 as shown in the amino acid sequence of SEQ ID NO: 1, and optionally the anti-CLDN18.2 VHH comprises (i) CDR1 containing the amino acid sequence of SEQ ID NO: 23, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 24, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO: 25. In some embodiments, the anti-CLDN18.2 VHH comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-GUCY2C VHH comprises CDR1, CDR2, and CDR3 as shown in the amino acid sequence of SEQ ID NO: 2, and optionally the anti-GUCY2C VHH comprises (i) CDR1 containing the amino acid sequence of SEQ ID NO: 26, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 27, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-GUCY2C VHH comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 2.

[0153] The costimulatory signal transduction domain may be derived from costimulatory molecules selected from the group consisting of: CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 ligands, and combinations thereof. In some embodiments, the costimulatory signal transduction domain is derived from 4-1BB. In some embodiments, the costimulatory signal transduction domain comprises an amino acid sequence of any one of SEQ ID NO: 7-9. In some embodiments, a multispecific CAR construct (e.g., a tandem CAR) comprises a polypeptide containing an amino acid sequence of SEQ ID NO: 14 or 15 (corresponding to LG23B01 CAR and LG23B02 CAR).

[0154] In one aspect, this disclosure provides a multispecific CAR construct (e.g., a split-type CAR construct) comprising different chimeric antigen receptors dispersed in two peptides, wherein the two peptides can be operatively linked into a single chain via a cleavable linker. The split-type CAR construct can act as an antagonist of at least two antigens or be expressed by immune cells, which in turn act as antagonists for use in a therapy. In some embodiments, the multispecific CAR construct (e.g., a split-type CAR construct) binding to CLDN18.2 and GUCY2C comprises a first peptide and a second peptide, the first peptide comprising a first extracellular antigen-binding domain, a first transmembrane domain, and a first intracellular signaling domain, and the second peptide comprising a second extracellular antigen-binding domain, a second transmembrane domain, and a second intracellular signaling domain, wherein the first extracellular antigen-binding domain comprises at least one anti-CLDN18.2 binding moiety and the second extracellular antigen-binding domain comprises at least one anti-GUCY2C binding moiety. The anti-CLDN18.2 binding moiety may be anti-CLDN18.2 VHH. The anti-GUCY2C binding site can be an anti-GUCY2C VHH.

[0155] In some embodiments, the anti-CLDN18.2 VHH comprises CDR1, CDR2, and CDR3 as shown in the amino acid sequence of SEQ ID NO: 1, and optionally the anti-CLDN18.2 VHH comprises (i) CDR1 containing the amino acid sequence of SEQ ID NO: 23, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 24, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO: 25. In some embodiments, the anti-CLDN18.2 VHH comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-GUCY2C VHH comprises CDR1, CDR2, and CDR3 as shown in the amino acid sequence of SEQ ID NO: 2, and optionally the anti-GUCY2C VHH comprises (i) CDR1 containing the amino acid sequence of SEQ ID NO: 26, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 27, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-GUCY2C VHH comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 2.

[0156] In some embodiments, a multispecific CAR construct (e.g., a split CAR construct) comprises two peptides, wherein (1) the first peptide comprises: a first extracellular antigen-binding domain, a hinge domain (e.g., a CD8α hinge domain), a transmembrane domain (e.g., CD8α, CD28, or ICOS™), optionally a co-stimulatory signaling domain, and a primary intracellular signaling domain (e.g., derived from CD3ζ) containing at least one second ..., optionally a CD8α hinge domain, and a transmembrane domain (e.g., CD8α, CD28, or ICOS™), optionally a co-stimulatory signaling domain, and a primary intracellular signaling domain (e.g., derived from CD3ζ) containing at least one second extracellular antigen-binding domain containing at least one second extracellular antigen-binding domain, a hinge domain, optionally a CD8α hinge domain, and a primary intracellular signaling domain (e.g., derived from CD3ζ). In some embodiments, the first peptide is linked to the second peptide via a cleavable linker (e.g., a 2A linker). The first and second peptides may be in two separate peptide chains. The first and second polypeptides may each further include a signal peptide (e.g., CD8α signal peptide) at their N-terminus.

[0157] Therefore, in some embodiments, the multispecific CAR construct (e.g., a split CAR construct) comprises a first intracellular signaling domain in the CLDN18.2 CAR peptide and a second intracellular signaling domain in the GUCY2C CAR peptide, such that when the first and second peptides simultaneously bind to target cells (e.g., target CLDN18.2+GUCY2C+ cells (e.g., CLDN18.2+GUCY2C+ gastric cancer cells)), complete activation of the cells (e.g., immune cell populations) occurs, compared to activation when either the first or second peptide binds to target cells expressing either CLDN18.2 or GUCY2C. The first peptide may further comprise a co-stimulatory signaling domain (e.g., a 4-1BB, CD28, ICOS, or NTBA signaling domain), and the second peptide may comprise a primary intracellular signaling domain (e.g., a CD3ζ primary intracellular signaling domain). The first polypeptide may contain a primary signal transduction domain (e.g., a CD3ζ signal transduction domain), and the second polypeptide may further contain a co-stimulatory domain (e.g., a 4-1BB, CD28, ICOS, or NTBA signal transduction domain). Both the first and second polypeptides may contain a co-stimulatory signal transduction domain (e.g., a 4-1BB, CD28, ICOS, or NTBA signal transduction domain) and a primary intracellular signal transduction domain (e.g., a CD3ζ signal transduction domain).

[0158] The first polypeptide and / or the second polypeptide may contain a co-stimulatory signal transduction domain. The first polypeptide may contain a first co-stimulatory signal transduction domain, and the second polypeptide may contain a second co-stimulatory signal transduction domain. The co-stimulatory signal transduction domain may be derived from a co-stimulatory molecule selected from the group consisting of: CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 ligands, and combinations thereof. The first and second co-stimulatory signal transduction domains may be derived from the same or different co-stimulatory molecules. The first co-stimulatory signal transduction domain may be derived from 4-1BB, and the second co-stimulatory signal transduction domain may be derived from CD28, or vice versa. The first co-stimulatory signal transduction domain may be derived from ICOS, and the second co-stimulatory signal transduction domain may be derived from NTBA, or vice versa. The first and second co-stimulatory signal transduction domains may be derived from ICOS. The co-stimulatory signal transduction domain may contain any of the amino acid sequences shown in SEQ ID NO: 7-9.

[0159] In some embodiments, the multispecific CAR construct (e.g., a resectable CAR construct) comprises a first polypeptide containing an amino acid sequence of any one of SEQ ID NO: 12 (corresponding to LG23A01 CAR) and SEQ ID NO: 19, and a second polypeptide containing an amino acid sequence of any one of SEQ ID NO: 13 (corresponding to LG23A02 CAR) and SEQ ID NO: 20-22, optionally the first polypeptide and the second polypeptide being operatively linked by a cleavable peptide linker. Prior to cleavage via the 2A linker, the multispecific CAR construct (e.g., a resectable CAR construct) may comprise a polypeptide containing an amino acid sequence of any one of SEQ ID NO: 16-18 (corresponding to LG23D01 CAR, LG23D02 CAR, and LG23D03 CAR).

[0160] Extracellular antigen-binding domain

[0161] The CAR of this application includes an extracellular antigen-binding region comprising one or more single-domain antibodies (e.g., VHH domains). Single-domain antibodies may have the same or different origins and may have the same or different sizes. Exemplary single-domain antibodies include, but are not limited to, heavy chain variable domains from heavy-chain-only antibodies (VHHs), heavy-chain-only antibodies (such as camelid or humanized heavy-chain-only antibodies), human VHs generated from transgenic mice or rats expressing human heavy chain segments, and engineered domains and single-domain scaffolds other than those derived from antibodies. Any VHH known in the art or developed de novo may be used to construct the CAR described herein. VHHs may be derived from any species, including but not limited to mice, rats, humans, camels, llamas, lampreys, fish, sharks, goats, rabbits, and cattle. VHHs considered herein also include naturally occurring VHH molecules from species other than camelids and sharks.

[0162] VHHs originate from naturally occurring single-domain antigen-binding molecules, known as heavy-chain antibodies lacking the light chain (also referred to herein as "heavy-chain-only antibodies"). For clarity, the variable domains derived from naturally occurring heavy-chain molecules lacking the light chain are referred to herein as VHHs to distinguish them from the conventional VHs of four-chain immunoglobulins. Such VHH molecules can be derived from antibodies produced in camel species, such as camels, llamas, vicuñas, dromedary camels, alpacas, and guanacos. Other species besides camels can produce naturally occurring heavy-chain molecules lacking the light chain, and such VHHs are within the scope of this application.

[0163] VHH molecules derived from camels are approximately 10 times smaller than IgG molecules. They are single polypeptides and are highly stable, tolerating extreme pH and temperature conditions. Furthermore, they can be resistant to protease activity, unlike conventional 4-chain antibodies. In vitro expression of VHHs yields high yields of well-folded, functional VHHs. Additionally, antibodies produced in camels can recognize epitopes other than those recognized by antibodies produced in vitro using antibody libraries or via immunization of mammals other than camels (see, for example, WO 9749805). Therefore, multispecific or multivalent CARs containing one or more VHH domains can interact with targets more effectively than multispecific or multivalent CARs containing antigen-binding fragments derived from conventional 4-chain antibodies such as scFv and Fab.

[0164] VHHs can be recombinant, CDR-transplanted, humanized, camelidoid, deimmunized, and / or in vitro generated (e.g., selected via phage display). The amino acid sequence of the frame region can be altered by “camelization” of specific amino acid residues within the frame region. Camelization refers to the substitution or replacement of one or more amino acid residues in the amino acid sequence of the (naturally occurring) VH domain from a conventional 4-chain antibody with one or more amino acid residues at one or more corresponding positions in the VHH domain of a heavy chain antibody. This can be done in ways known per se, which will be clear to those skilled in the art, for example, based on further description herein. Such “camelization” substitutions can be inserted at amino acid sites that form the VH-VL interface and / or are present at the VH-VL interface and / or at so-called camelidoid marker residues, as defined herein (see, for example, WO 94 / 04678, Davies and Riechmann FEBS Letters 339: 285-290, 1994; Davies and Riechmann Protein Engineering 9 (6): 531-537, 1996; Riechmann J.Mol. Biol. 259: 957-969, 1996; and Riechmann and Muyldermans J. Immunol. Meth. 231: 25-38, 1999).

[0165] VHHs can be humanized VHHs produced from transgenic mice or rats expressing human heavy chain segments. See, for example, US 20090307787 A1, US Patent No. 8,754,287, US 20150289489 A1, US 20100122358 A1, and WO2004049794. VHHs can be affinity-matured.

[0166] Naturally occurring VHH domains targeting specific antigens or targets can be obtained from libraries of VHH sequences from camels. Such methods may or may not involve screening such libraries using one or more screening techniques known per se, employing the antigen or target, or at least a portion, fragment, antigenic determinant, or epitope thereof. Such libraries and techniques are described, for example, in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020, and WO 03 / 035694. Alternatively, modified synthetic or semi-synthetic libraries derived from VHH libraries can be used, such as VHH libraries obtained from VHH libraries using techniques described, for example, in WO 00 / 43507 (e.g., random mutagenesis and / or CDR shuffling).

[0167] The anti-CLDN18.2 binding moiety can be derived from a parental antibody, such as an anti-CLDN18.2 antibody. The anti-GUCY2C binding moiety can be derived from a parental antibody, such as an anti-GUCY2C antibody. The parental antibody can be any type of antibody, including, for example, fully human antibodies, humanized antibodies, or animal antibodies (e.g., camel VHH). The parental antibody can be known in the art, commercially available, or developed de novo.

[0168] The anti-CLDN18.2 binding moiety can be derived from a VHH that specifically binds to CLDN18.2 (such as human CLDN18.2). The anti-CLDN18.2 VHH can contain one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 1. The anti-CLDN18.2 VHH can be from camels. The anti-CLDN18.2 VHH can be humanized. The anti-CLDN18.2 VHH can contain a recipient human framework, such as a human immunoglobulin framework or a human common framework. The VHH CDRs (CDR1-3) can be determined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or a combination thereof. The CDRs of the CLDN18.2 VHH can be defined according to the Kabat numbering scheme.

[0169] The anti-CLDN18.2 VHH may comprise at least one, at least two, or all three CDRs selected from the following: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 23; (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 24; and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the anti-CLDN18.2 VHH comprises: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 23; (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 24; and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 25.

[0170] The anti-CLDN18.2 VHH may contain three CDRs, comprising: (a) CDR1 containing an amino acid sequence of SEQ ID NO: 23 or different from SEQ ID NO: 23, wherein no more than two amino acids are added, deleted, or substituted; (b) CDR2 containing an amino acid sequence of SEQ ID NO: 24 or different from SEQ ID NO: 24, wherein no more than two amino acids are added, deleted, or substituted; and (c) CDR3 containing an amino acid sequence of SEQ ID NO: 25 or different from SEQ ID NO: 25, wherein no more than two amino acids are added, deleted, or substituted. These CDRs may contain substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the VHH containing these CDRs retains its ability to bind to CLDN18.2. The anti-CLDN18.2 VHH may be affinity-matured.

[0171] The anti-CLDN18.2 VHH may comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1. The VHH sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the reference sequence may contain substitutions (e.g., conservative substitutions), insertions, or deletions, but may retain the ability to bind to CLDN18.2. For example, a total of 1 to 10 amino acids may be substituted, inserted, and / or deleted within the frame region of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-CLDN18.2 VHH comprises the amino acid sequence of SEQ ID NO: 1, including post-translational modifications of that sequence.

[0172] The anti-GUCY2C binding moiety can be derived from a VHH that specifically binds to GUCY2C (such as human GUCY2C). The anti-GUCY2C VHH can contain one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 2. The anti-GUCY2C VHH can be from camels. The anti-GUCY2C VHH can be humanized. The anti-GUCY2C VHH can contain a recipient human frame, such as a human immunoglobulin frame or a human common frame. The VHH CDRs (CDR1-3) can be determined according to the Kabat numbering scheme, IMGT numbering scheme, AbM numbering scheme, Chothia numbering scheme, Contact numbering scheme, or a combination thereof. The CDRs of the GUCY2C VHH can be defined according to the Kabat numbering scheme.

[0173] The anti-GUCY2C VHH may comprise at least one, at least two, or all three CDRs selected from the following: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 26; (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 27; and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-GUCY2C VHH comprises: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 26; (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 27; and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 28.

[0174] The anti-GUCY2C VHH may contain three CDRs: (a) CDR1 containing an amino acid sequence of SEQ ID NO: 26 or different from SEQ ID NO: 26, wherein no more than two amino acids are added, deleted, or substituted; (b) CDR2 containing an amino acid sequence of SEQ ID NO: 27 or different from SEQ ID NO: 27, wherein no more than two amino acids are added, deleted, or substituted; and (c) CDR3 containing an amino acid sequence of SEQ ID NO: 28 or different from SEQ ID NO: 28, wherein no more than two amino acids are added, deleted, or substituted. These CDRs may contain substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the VHH containing these CDRs retains its ability to bind to GUCY2C. The anti-GUCY2C VHH may be affinity mature.

[0175] The anti-GUCY2C VHH may comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 2. The VHH sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the reference sequence may contain substitutions (e.g., conservative substitutions), insertions, or deletions, but may retain the ability to bind to GUCY2C. For example, a total of 1 to 10 amino acids may be substituted, inserted, and / or deleted within the frame region of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the anti-GUCY2C VHH comprises the amino acid sequence of SEQ ID NO: 2, including post-translational modifications of that sequence.

[0176] peptide linkers

[0177] In the multispecific CAR construct described in this article, multiple VHHs can fuse with each other via peptide linkers. These VHHs can also fuse directly with each other without any peptide linkers. The peptide linkers connecting different VHHs can be the same or different. Different domains of the CAR can also fuse with each other via peptide linkers.

[0178] Depending on the structural and / or functional characteristics of the single-domain antibody and / or various domains, each peptide linker in the CAR can have the same or different lengths and / or sequences. Each peptide linker can be selected and optimized independently. The length, flexibility, and / or other properties of one or more peptide linkers used in the CAR can have some influence on properties, including but not limited to affinity, specificity, or affinity for one or more specific antigens or epitopes. For example, longer peptide linkers can be selected to ensure that two adjacent domains do not spatially interfere with each other. For example, in the multivalent or multispecific CAR construct of this application containing a VHH against a multimeric antigen, the length and flexibility of the peptide linker can allow each VHH in the CAR to bind to the antigenic determinant on each subunit of that multimer. Short peptide linkers can be provided between the transmembrane domains and intracellular signaling domains of the CAR. Peptide linkers contain flexible residues (such as glycine and serine) that allow adjacent domains to move freely relative to each other. For example, a glycine-serine dinucleotide can be a suitable peptide linker.

[0179] The peptide linker can have any suitable length. In some embodiments, the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids long. The peptide linker can be no more than about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids long. The length of the peptide linker can be any of the following: about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0180] Peptide linkers can have naturally occurring or non-natural sequences. For example, sequences derived from the hinge region of heavy-chain-only antibodies can be used as linkers. See, for example, WO 1996 / 34103. In some embodiments, peptide linkers are flexible linkers. Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n, (GGGS)n, and (GGGGS)n, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Peptide linkers may contain the amino acid sequence shown in any of SEQ ID NO: 31-37.

[0181] Transmembrane domain

[0182] The CAR of this application comprises a transmembrane domain that can fuse directly or indirectly with an extracellular antigen-binding region. The transmembrane domain may be derived from natural or synthetic sources. As used herein, a “transmembrane domain” refers to any protein structure that is thermodynamically stable in a cell membrane (e.g., a eukaryotic cell membrane). Transmembrane domains suitable for use in the CAR described herein may be derived from naturally occurring proteins. Alternatively, they may be synthetic, non-naturally occurring protein segments, such as thermodynamically stable hydrophobic protein segments in a cell membrane.

[0183] Transmembrane domains are classified based on their three-dimensional structure. For example, transmembrane domains can form α-helices, complexes of more than one α-helix, beta-barrels, or any other stable structure capable of crossing the phospholipid bilayer of the cell. Furthermore, transmembrane domains can also be classified based on their topology, including the number of times the domain crosses the membrane and the orientation of the protein. For example, single-transmembrane proteins cross the cell membrane once, while multi-transmembrane proteins cross the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7, or more times). Membrane proteins can be defined as type I, type II, or type III, depending on the topology of their terminals and one or more transmembrane segments relative to the cell's interior and exterior. Type I membrane proteins have a single transmembrane region oriented such that the N-terminus of the protein is located on the extracellular side of the lipid bilayer of the cell, while the C-terminus is located on the intracellular side. Type II membrane proteins also have a single transmembrane region, but it is oriented such that the C-terminus of the protein is located on the extracellular side of the cell's lipid bilayer, while the N-terminus is located on the intracellular side. Type III membrane proteins have multiple transmembrane segments and can be further subdivided based on the number of transmembrane segments and the positions of the N-terminus and C-terminus.

[0184] The transmembrane domain of the CAR described herein may be derived from a type I single-transmembrane protein. In some embodiments, transmembrane domains derived from multiple-transmembrane proteins may also be suitable for use in the CAR described herein. Multiple-transmembrane proteins may comprise multiple complexed (at least 2, 3, 4, 5, 6, 7, or more) α-helices or a single β-sheet structure. For example, the N-terminus and C-terminus of a multiple-transmembrane protein are located on opposite sides of a lipid bilayer; for instance, the N-terminus of the protein is located on the intracellular side of the lipid bilayer, while the C-terminus is located on the extracellular side.

[0185] As disclosed herein, the transmembrane domain of a CAR may include transmembrane domains selected from the following: α, β, or ζ chains of the T cell receptor; CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154; KIRDS2, OX40, CD2, CD27; LFA-1 (CD11a, CD18); ICOS (CD278); 4-1BB (CD137); GITR; CD40; BAFFR; HVEM (LIGHTR); SLAMF7; NKp80 (KLRF1); CD160; CD19; IL-2R β; IL-2R γ; IL-7R. a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITG AX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT Transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In some embodiments, the transmembrane domains are derived from molecules selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

[0186] The transmembrane domain may be derived from CD8α. In some embodiments, the transmembrane domain is a CD8α transmembrane domain comprising the amino acid sequence of SEQ ID NO: 5. The transmembrane domain may be derived from ICOS. In some embodiments, the transmembrane domain is an ICOS transmembrane domain comprising the amino acid sequence of SEQ ID NO: 6. The transmembrane domain may comprise an amino acid sequence having at least 85%, 90%, or 95% identity with the amino acid sequences shown in any one of SEQ ID NO: 5-6.

[0187] The transmembrane domain used in the CAR described herein may also comprise at least a portion of a synthetic, non-naturally occurring protein segment. The transmembrane domain may be a synthetic, non-naturally occurring α-helix or β-sheet. In some embodiments, the protein segment is at least about 20 amino acids, for example, at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art, for example, in U.S. Patent No. 7,052,906 B1 and PCT Publication No. WO 2000 / 032776 A2 (the relevant disclosures of which are incorporated herein by reference).

[0188] Intracellular signal transduction domains

[0189] As disclosed herein, a CAR may contain one or more intracellular signaling domains. These intracellular signaling domains are responsible for activating at least one normal effector function of the CAR-expressing immune cell. The term "effector function" refers to a cell's specialized function. Effector functions of T cells may include, for example, cytolytic activity or helper activity, including cytokine secretion. Therefore, the term "intracellular signaling domain" refers to the intracellular portion of a protein that transduces effector function signals and directs the cell to perform its specialized function. While the entire intracellular signaling domain can often be used, in many cases it is not necessary to use the entire chain.

[0190] Intracellular signaling domains can contain primary intracellular signaling domains of immune cells. Intracellular signaling domains can be essentially composed of primary intracellular signaling domains of immune cells. A “primary intracellular signaling domain” refers to an intracellular signaling sequence that acts in a stimulatory manner to induce the function of immune effectors. Primary intracellular signaling domains may contain signaling motifs called immune receptor tyrosine-based activation motifs or ITAMs. As used herein, “ITAM” is a conserved protein motif that is commonly found at the tail of signaling molecules expressed in many immune cells. ITAMs within signaling molecules are important for intracellular signal transduction, which is at least partially mediated by phosphorylation of tyrosine residues in the ITAM upon activation of the signaling molecule. ITAMs can also function as docking sites for other proteins involved in signaling pathways. Exemplary primary intracellular signaling sequences containing ITAM include those derived from CD3ζ, FcRγ (FCER1G), FcRβ (Fcε Rib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0191] In some embodiments, the primary intracellular signal transduction domain is derived from CD3ζ. The intracellular signal transduction domain may consist of intracellular signal transduction domains of CD3ζ. The primary intracellular signal transduction domain may be an intracellular signal transduction domain of wild-type CD3ζ. The primary intracellular signal transduction domain is a functional mutant of the intracellular signal transduction domain of CD3ζ containing one or more mutations. The primary intracellular signal transduction domain of wild-type CD3ζ may contain the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 11.

[0192] Co-stimulatory signal transduction domain

[0193] In addition to stimulating antigen-specific signals, many immune cells require co-stimulation to promote cell proliferation, differentiation, and survival, as well as to activate effector functions. CARs containing one or more operatively linked VHHs, as disclosed herein, may include at least one co-stimulatory signaling domain. Multispecific CAR constructs containing two reconfigurable CAR peptides, as disclosed herein, may include at least one co-stimulatory signaling domain in one of these peptides. As used herein, the term "co-stimulatory signaling domain" refers to at least a portion of a protein that mediates intracellular signal transduction to induce an immune response, such as effector function. The co-stimulatory signaling domain of the chimeric receptor described herein may be an intracellular signaling domain derived from a co-stimulatory protein that transduces signals and regulates responses mediated by immune cells such as T cells, NK cells, macrophages, neutrophils, or eosinophils. A "co-stimulatory signaling domain" may be an intracellular portion of a co-stimulatory molecule. The term "co-stimulatory molecule" refers to a homologous binding partner on immune cells (such as T cells) that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response of the immune cell, such as, but not limited to, proliferation and survival.

[0194] Intracellular signal transduction domains may contain a single co-stimulatory signal transduction domain. Intracellular signal transduction domains may contain two or more (e.g., any of about 2, 3, 4, or more) co-stimulatory signal transduction domains, such as two or more identical co-stimulatory signal transduction domains, or two or more co-stimulatory signal transduction domains from different co-stimulatory proteins. Intracellular signal transduction domains may contain a primary intracellular signal transduction domain (e.g., the intracellular signal transduction domain of CD3ζ) and one or more co-stimulatory signal transduction domains. One or more co-stimulatory signal transduction domains and the primary intracellular signal transduction domain (e.g., the intracellular signal transduction domain of CD3ζ) may be fused to each other via an optional peptide linker. The primary intracellular signal transduction domain and one or more co-stimulatory signal transduction domains may be arranged in any suitable order. One or more co-stimulatory signal transduction domains are located between the transmembrane domain and the primary intracellular signal transduction domain (e.g., the intracellular signal transduction domain of CD3ζ). Multiple co-stimulatory signal transduction domains can provide additive or co-stimulatory effects.

[0195] Activation of costimulatory signaling domains in host cells (e.g., immune cells) can induce increased or decreased cellular production and secretion of cytokines, phagocytic properties, proliferation, differentiation, survival, and / or cytotoxicity. The costimulatory signaling domain of any costimulatory molecule can be used in the CAR described herein. One or more types of costimulatory signaling domains are selected based on factors such as the type of immune cell in which the effector molecule will be expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC effector).Examples of co-stimulatory signaling domains for use in CAR can be intracellular signaling domains of co-stimulatory proteins, including but not limited to members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6); and members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF). R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, Lymphotoxin-α / TNF-β, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-α and TNF RII / TNFRSF1B; members of the SLAM family (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150); and any other co-stimulatory molecules such as CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA Class I, HLA-DR, Ikaros, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function-associated antigen-1 (LFA-1), and NKG2C.

[0196] In some embodiments, the co-stimulatory signal transduction domain is selected from the group consisting of: CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), ICOS (CD278), NTBA, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 ligands, and combinations thereof.

[0197] The intracellular signal transduction domain in the CAR of this application may include a co-stimulatory signal transduction domain derived from 4-1BB (i.e., CD137). The intracellular signal transduction domain comprises a primary intracellular signal transduction domain of CD3ζ and a co-stimulatory signal transduction domain of 4-1BB. In some embodiments, the intracellular signal transduction domain includes a co-stimulatory signal transduction domain of 4-1BB, which contains the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 7.

[0198] The intracellular signal transduction domain in the CAR of this application may include a co-stimulatory signal transduction domain derived from CD28. The intracellular signal transduction domain comprises a primary intracellular signal transduction domain of CD3ζ and a co-stimulatory signal transduction domain of CD28. The intracellular signal transduction domain may include a co-stimulatory signal transduction domain of CD28, which contains the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 8.

[0199] The intracellular signal transduction domain in the CAR of this application may include a co-stimulatory signal transduction domain derived from ICOS. The intracellular signal transduction domain may include a primary intracellular signal transduction domain of CD3ζ and a co-stimulatory signal transduction domain of ICOS. The intracellular signal transduction domain may include a co-stimulatory signal transduction domain of ICOS containing the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 9.

[0200] The intracellular signal transduction domain in the CAR of this application may include a co-stimulatory signal transduction domain derived from NTB-A. The intracellular signal transduction domain comprises the primary intracellular signal transduction domain of CD3ζ and the co-stimulatory signal transduction domain of NTB-A. The intracellular signal transduction domain may include the co-stimulatory signal transduction domain of NTB-A, which contains the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 10.

[0201] Any variants of the costimulatory signaling domains described herein are also within the scope of this disclosure, enabling the costimulatory signaling domains to modulate the immune response of immune cells. Compared to the wild-type control, the costimulatory signaling domain may contain up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5, or 8). Such a costimulatory signaling domain containing one or more amino acid variations may be referred to as a variant. Mutations of amino acid residues in the costimulatory signaling domain, relative to the non-mutated costimulatory signaling domain, may result in increased signal transduction and enhanced stimulation of the immune response. Mutations of amino acid residues in the costimulatory signaling domain, relative to the non-mutated costimulatory signaling domain, may result in decreased signal transduction and reduced stimulation of the immune response.

[0202] Hinge structural domain

[0203] The CAR of this application may include a hinge domain located between an extracellular antigen-binding domain and a transmembrane domain. A hinge domain is an amino acid segment typically found between two domains of a protein and allows for the flexibility of the protein and the movement of one or both domains relative to each other. Any amino acid sequence that provides this flexibility and movement of the extracellular antigen-binding domain relative to the transmembrane domain of the effector molecule can be used.

[0204] The hinge domain may contain approximately 10-100 amino acids, for example, approximately 15-75 amino acids, 20-50 amino acids, or 30-60 amino acids. The length of the hinge domain may be at least approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids.

[0205] The hinge domain can be a hinge domain of a naturally occurring protein (e.g., an immunoglobulin). The hinge domain of any protein known in the art that contains a hinge domain is suitable for use in the chimeric receptor described herein. The hinge domain can be at least a portion of the hinge domain of a naturally occurring protein and imparts flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8α. The hinge domain can be a portion of the hinge domain of CD8α, for example, a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8α. The hinge domain of CD8α can contain the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 4.

[0206] Hinge domains of antibodies (such as IgG, IgA, IgM, IgE, or IgD antibodies) are also suitable for use in the pH-dependent chimeric receptor systems described herein. A hinge domain may be a hinge domain connecting the constant domains CH1 and CH2 of an antibody. A hinge domain may be a hinge domain of an antibody and may also include the antibody and one or more constant regions of that antibody. A hinge domain may include the antibody and its CH3 constant region. A hinge domain may include the antibody and its CH2 and CH3 constant regions. The antibody may be an IgG, IgA, IgM, IgE, or IgD antibody. The antibody may be an IgG1, IgG2, IgG3, or IgG4 antibody. A hinge region may include the hinge region of an IgG1 antibody as well as the CH2 and CH3 constant regions. A hinge region may include the hinge region of an IgG1 antibody and its CH3 constant region.

[0207] Non-naturally occurring peptides can also be used as the hinge domain of the chimeric receptor described herein. The hinge domain located between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain of the Fc receptor can be a peptide linker, such as a (GxS)n linker, where x and n can be independent integers between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or larger.

[0208] signal peptide

[0209] The CAR of this application may include a signal peptide (also known as a signal sequence) at the N-terminus of a polypeptide. Typically, a signal peptide is a peptide sequence that targets the polypeptide to a desired site in the cell. The signal peptide can target effector molecules to cellular secretory pathways and will allow effector molecules to integrate and anchor into the lipid bilayer. Suitable signal peptides for use in the CAR described herein (including signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences) will be apparent to those skilled in the art.

[0210] The signal peptide may be derived from molecules selected from the group consisting of CD8α, GM-CSF receptor α, and IgG1 heavy chain. In some embodiments, the signal peptide is derived from CD8α. The signal peptide may comprise the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 3.

[0211] Engineered T-cell receptor (TCR)

[0212] The engineered receptor can be a modified T-cell receptor or an engineered T-cell receptor. The engineered TCR can be specific for tumor antigens. The tumor antigen can be selected from dentin 18.2 and GUCY2C. The tumor antigen can be derived from an intracellular protein of tumor cells. The tumor antigen can be expressed on the surface of tumor cells. Any TCR known in the art can be used. The TCR can have enhanced affinity for the tumor antigen. Exemplary TCRs and methods for introducing TCRs into immune cells have been described, for example, in U.S. Patent No. 5,830,755 and Kessels et al., Immunotherapy through TCR gene transfer. Nat. Immunol. 2, 957-961 (2001), which are incorporated herein by reference in their entirety.

[0213] The TCR receptor complex is an octamer formed by the variable TCR receptor α and β chains (or γ and δ chains in the case of γδ T cells) with three dimeric signaling modules CD3δ / ε, CD3γ / ε, and CD247 (the CD3ζ chain of the T cell surface glycoprotein) ζ / ζ or ζ / η. Ionizable residues in the transmembrane domains of each subunit form an interacting polar network that holds the complex together. The TCR complex has the function of activating the signaling cascade in T cells.

[0214] Engineered receptors can be engineered TCRs comprising one or more T-cell receptor (TCR) fusion proteins (TFPs). Exemplary TFPs have been described, for example, in US 20170166622 A1, which is incorporated herein by reference in its entirety. A TFP may comprise an extracellular domain of a TCR subunit comprising an extracellular domain or a portion thereof of a protein selected from the group consisting of: a TCRα chain, a TCRβ chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, a functional fragment thereof, and an amino acid sequence having at least one but no more than 20 modified amino acids. A TFP may comprise a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of: a TCRα chain, a TCRβ chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, a functional fragment thereof, and an amino acid sequence having at least one but no more than 20 modified amino acids. TFP may contain a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of: TCRα chain, TCRβ chain, TCRζ chain, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, their functional fragments, and their amino acid sequences having at least one but no more than 20 modifications.

[0215] TFP may include a TCR subunit comprising at least a portion of an extracellular domain of a TCR and an intracellular domain of a TCR containing a stimulatory domain from an intracellular signaling domain of CD3ε; and an antigen-binding domain wherein the TCR subunit is operatively linked to the antigen-binding domain, and wherein the TFP is incorporated into the TCR when expressed in T cells.

[0216] T-cell antigen-coupled receptor

[0217] Engineered receptors can be T-cell antigen-coupled (TAC) receptors. For example, an exemplary TAC receptor has been described in US20160368964 A1, which is incorporated herein by reference. A TAC may comprise an antigen-binding domain, a TCR-binding domain that specifically binds to proteins associated with the TCR complex, and a T-cell receptor signaling domain. The antigen-binding domain may be an antibody fragment that specifically binds to a tumor antigen, such as scFv or VHH. The antigen-binding domain may be a designed ankyrin repeat (DARPin) polypeptide. The tumor antigen may be selected from CLDN18.2 and GUCY2C. The tumor antigen may be an intracellular protein derived from tumor cells. The tumor antigen may be expressed on the surface of tumor cells. The protein associated with the TCR complex may be CD3, such as CD3E. The TCR-binding domain may be a single-chain antibody, such as scFv or VHH. H H. The TCR-binding domain may be derived from UCHT1. The TAC receptor may include a cytoplasmic domain and a transmembrane domain. The T cell receptor signaling domain may include a cytoplasmic domain derived from a TCR helper receptor. Exemplary TCR helper receptors include, but are not limited to, CD4, CD8, CD28, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. The TAC receptor may include a transmembrane domain and a cytoplasmic domain derived from CD4. The TAC receptor may include a transmembrane domain and a cytoplasmic domain derived from CD8 (such as CD8α).

[0218] T cell helper receptors are expressed as membrane proteins on T cells. They can stabilize the TCR:peptide:MEC complex and promote signal transduction. Two subtypes of T cell helper receptors, CD4 and CD8, exhibit strong specificity for specific MEC classes. The CD4 helper receptor stabilizes only the TCR:MEC II complex, while the CD8 helper receptor stabilizes only the TCR:MEC I complex. Differential expression of CD4 and CD8 in different T cell types leads to different functional subsets of T cells. CD8+ T cells are cytotoxic T cells.

[0219] Nucleic acid and vector

[0220] This disclosure also provides nucleic acid molecules encoding engineered receptors (e.g., CARs) as described herein. The nucleic acid molecules may be provided as messenger RNA transcripts or as DNA constructs.

[0221] In one respect, this article provides a nucleic acid comprising: (1) a nucleic acid sequence encoding a multispecific CAR construct as disclosed herein; and (2) a first nucleic acid sequence encoding a first engineered receptor specifically targeting CLDN18.2, the first engineered receptor comprising: a first extracellular antigen-binding domain, a first transmembrane domain, and a first intracellular signal transduction domain containing at least one anti-CLDN18.2 binding moiety; and a second nucleic acid sequence encoding a second engineered receptor specifically targeting GUCY2C, the second engineered receptor comprising: a second extracellular antigen-binding domain, a second transmembrane domain, and a second intracellular signal transduction domain containing at least one polypeptide containing an anti-GUCY2C binding moiety; optionally, the first nucleic acid sequence and the second nucleic acid sequence are linked by a nucleic acid sequence encoding a self-cleavable peptide (such as P2A, E2A, F2A, or T2A).

[0222] Therefore, the isolated nucleic acid molecule can encode a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular antigen-binding domain containing at least one anti-CLDN18.2 binding moiety (e.g., anti-CLDN18.2 VHH), a transmembrane domain, and an intracellular signaling domain containing, for example, a co-stimulatory signaling domain and / or a primary signaling domain (e.g., a ζ chain). The anti-CLDN18.2 binding moiety is the anti-CLDN18.2 VHH described herein, for example, a VHH containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 95%-99% identity with it. The nucleic acid molecule can encode the amino acid sequence shown in SEQ ID NO: 12 or 19.

[0223] The isolated nucleic acid molecule can encode a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular antigen-binding domain containing at least one anti-GUCY2C binding moiety (e.g., anti-GUCY2C VHH), a transmembrane domain, and an intracellular signaling domain containing, for example, a co-stimulatory signaling domain and / or a primary signaling domain (e.g., a ζ chain). The anti-GUCY2C binding moiety is the anti-GUCY2C VHH described herein, for example, a VHH containing the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 95%-99% identity with it. The nucleic acid molecule can encode any of the amino acid sequences shown in SEQ ID NO: 13 and 20-22.

[0224] In some embodiments, this document provides isolated nucleic acid molecules encoding a multispecific chimeric antigen receptor (CAR) construct, wherein the multispecific CAR construct comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain containing, for example, a co-stimulatory signaling domain and / or a primary intracellular signaling domain (e.g., a ζ chain), an anti-GUCY2C binding domain operatively linked to an anti-CLDN18.2 binding portion (e.g., anti-CLDN18.2VHH). The anti-GUCY2C binding portion is the anti-GUCY2C VHH described herein, for example, a VHH containing the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 95%-99% identity with it, and the anti-CLDN18.2 binding portion is the anti-CLDN18.2 VHH described herein, for example, a VHH containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 95%-99% identity with it.

[0225] In some embodiments, this document provides isolated nucleic acid molecules encoding a first CAR specifically targeting CLDN18.2 operatively linked to a second CAR specifically targeting GUCY2C, wherein the first CAR comprises an extracellular antigen-binding domain containing at least one anti-CLDN18.2 binding moiety (e.g., anti-CLDN18.2 VHH), a first transmembrane domain, and a first intracellular signal transduction domain, and the second CAR comprises an extracellular antigen-binding domain containing at least one anti-GUCY2C binding moiety (e.g., anti-GUCY2C VHH), a second transmembrane domain, and a second intracellular signal transduction domain, wherein the first CAR and the second CAR are operatively linked via a self-cleavable peptide (e.g., P2A, E2A, F2A, or T2A). The GUCY2C binding portion is the anti-GUCY2C VHH described herein, for example, a VHH containing the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 95%-99% identity with it, and the CLDN18.2 binding portion is the anti-CLDN18.2 VHH described herein, for example, a VHH containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 95%-99% identity with it.

[0226] In some embodiments, this document provides isolated nucleic acid molecules encoding CAR peptides comprising a signal peptide of SEQ ID NO: 3 (or a sequence having 95%-99% identity with it), an anti-CLDN18.2 VHH having an amino acid sequence of SEQ ID NO: 1 or a sequence having 95%-99% identity with it, an anti-GUCY2C VHH having an amino acid sequence of SEQ ID NO: 2 or a sequence having 95%-99% identity with it, a hinge region of SEQ ID NO: 4 (or a sequence having 95%-99% identity with it), a transmembrane domain having a sequence of SEQ ID NO: 5 or 6 (or a sequence having 95%-99% identity with it), a co-stimulatory signal transduction domain having a sequence of any one of SEQ ID NO: 7-10 (or a sequence having 95%-99% identity with it), and a CD3ζ primary intracellular signal transduction domain having a sequence of SEQ ID NO: 11 (or a sequence having 95%-99% identity with it). In some specific embodiments, the nucleic acid molecule encodes the amino acid sequence of SEQ ID NO: 14 or 15.

[0227] An isolated nucleic acid molecule can contain two nucleic acid sequences. The first nucleic acid sequence can encode a first CAR, which comprises a signal peptide of SEQ ID NO: 3 (or a sequence having 95%-99% identity with it), an anti-CLDN18.2 VHH having the amino acid sequence of SEQ ID NO: 1 or a sequence having 95%-99% identity with it, a hinge region of SEQ ID NO: 4 (or a sequence having 95%-99% identity with it), a transmembrane domain having the sequence of SEQ ID NO: 5 or 6 (or a sequence having 95%-99% identity with it), a co-stimulatory signal transduction domain having any one of SEQ ID NO: 7-10 (or a sequence having 95%-99% identity with it), and a CD3ζ primary intracellular signal transduction domain having the sequence of SEQ ID NO: 11 (or a sequence having 95%-99% identity with it). The second nucleic acid sequence can encode a second CAR, which comprises a signal peptide of SEQ ID NO: 3 (or a sequence having 95%-99% identity with it), an anti-CLDN18.2 VHH having the amino acid sequence of SEQ ID NO: 1 or a sequence having 95%-99% identity with it, a hinge region of SEQ ID NO: 4 (or a sequence having 95%-99% identity with it), a transmembrane domain having the amino acid sequence of SEQ ID NO: 5 or 6 (or a sequence having 95%-99% identity with it), a co-stimulatory signal transduction domain having any one of SEQ ID NO: 7-10 (or a sequence having 95%-99% identity with it), and a CD3ζ primary intracellular signal transduction domain having the sequence of SEQ ID NO: 11 (or a sequence having 95%-99% identity with it). The first and second nucleic acid sequences are: an amino acid sequence of ID NO: 2 or a sequence having 95%-99% identity with it; an anti-GUCY2C VHH sequence of SEQ ID NO: 4 (or a sequence having 95%-99% identity with it); a transmembrane domain of a sequence of SEQ ID NO: 5 or 6 (or a sequence having 95%-99% identity with it); a co-stimulatory signal transduction domain of a sequence of any one of SEQ ID NO: 7-10 (or a sequence having 95%-99% identity with it); and a CD3ζ primary intracellular signal transduction domain of a sequence of SEQ ID NO: 11 (or a sequence having 95%-99% identity with it), wherein the first and second nucleic acid sequences are linked by a nucleic acid sequence encoding a self-cleavable peptide (such as P2A, E2A, F2A, or T2A). The nucleic acid molecule can encode an amino acid sequence of SEQ ID NO: 12 or 19 operably linked to any one of the amino acid sequences of SEQ ID NO: 13 and 20-22. The nucleic acid molecule can also encode any one of the amino acid sequences shown in SEQ ID NO: 16-18.

[0228] In some embodiments, the nucleic acid comprises a nucleic acid sequence encoding a polypeptide having an amino acid sequence shown in any one of SEQ ID NO: 14-18. In some embodiments, the nucleic acid comprises a first nucleic acid sequence encoding a first polypeptide having an amino acid sequence shown in any one of SEQ ID NO: 12 and 19, and a second nucleic acid sequence encoding a second polypeptide having an amino acid sequence shown in any one of SEQ ID NO: 13 and 20-22.

[0229] This disclosure further covers nucleic acid sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequences described above. Using recombination methods known in the art, nucleic acid sequences encoding desired molecules can be obtained using standard techniques. For example, target genes can be synthesized or cloned.

[0230] This disclosure also provides vectors in which nucleic acid sequences as disclosed herein are inserted. Vectors derived from retroviruses (such as lentiviruses) are suitable tools for long-term gene transfer because they allow for the long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have additional advantages over vectors derived from oncoretroviruses (such as murine leukemia virus) because they can transduce non-proliferating cells (such as hepatocytes). They also have the additional advantage of low immunogenicity. Retroviral vectors can also be, for example, gamma retroviral vectors.

[0231] Gamma retroviral vectors may include, for example, a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs), and a target transgene (e.g., a gene encoding a CAR). Gamma retroviral vectors may lack viral structural genes (such as gag, pol, and env). Exemplary gamma retroviral vectors include murine leukemia virus (MLV), spleen focal formation virus (SFFV), and myeloproliferative sarcoma virus (MPSV), as well as vectors derived from them. Other gamma retroviral vectors are described, for example, in Tobias Maetzig et al., “Gammaretroviral Vectors: Biology, Technology and Application” Viruses. 2011 June; 3(6): 677-713.

[0232] The vector containing the nucleic acid encoding the desired CAR disclosed herein can be an adenoviral vector (A5 / 35). Expression of the nucleic acid encoding the CAR can be accomplished using transposons such as Sleeping Beauty, Crisper, CAS9, and zinc finger nucleases. See June et al., 2009 Nature Reviews Immunology 9.10: 704-716, which is incorporated herein by reference below.

[0233] In summary, the expression of natural or synthetic nucleic acids encoding CARs is typically achieved by operatively linking a nucleic acid encoding a CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector. These vectors are suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, a start sequence, and a promoter to regulate the expression of the desired nucleic acid sequence.

[0234] Using standard gene delivery protocols, the expression constructs disclosed herein can also be used for nucleic acid immunotherapy and gene therapy. Methods for gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, this disclosure provides gene therapy vectors.

[0235] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors, including but not limited to plasmids, phage particles, phage derivatives, animal viruses, and entrapments. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0236] In addition, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and described, for example, in Sambrook et al., 2012, *MOLECULAR CLONING: A LABORATORY MANUAL*, Volumes 1–4, Cold Spring Harbor Press, NY, and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0237] Numerous virus-based systems have been developed for transferring genes into mammalian cells. Retroviruses, for example, provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the cells of a subject, either in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0238] Additional promoter elements (e.g., enhancers) regulate the frequency of transcription initiation. These are typically located 30–110 bp upstream of the start site, although many promoters have been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is generally flexible, allowing promoter function to be maintained when elements are reversed or moved relative to each other. In the thymidine kinase (TK) promoter, the spacing between promoter elements can increase to 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can act synergistically or independently to activate transcription.

[0239] The vector may also contain a selective marker gene or a reporter gene to select cells expressing CAR from a host cell population transfected via a lentiviral vector. Both the selective marker and reporter genes can be side-linked with appropriate regulatory sequences to enable expression in host cells. For example, the vector may contain transcription and translation terminators, a start sequence, and a promoter that can be used to regulate nucleic acid sequence expression.

[0240] In some embodiments, the vector comprises more than one nucleic acid encoding a CAR. In some embodiments, the vector comprises a nucleic acid containing a first nucleic acid sequence encoding a first CAR and a second nucleic acid sequence encoding a second CAR, wherein the first nucleic acid is operatively linked to the second nucleic acid via a third nucleic acid sequence encoding a self-cleaving peptide. The self-cleaving peptide is selected from the group consisting of T2A, P2A, E2A, and F2A linkers.

[0241] As used herein, a “self-cleaving peptide” or “2A linker” refers to an oligopeptide that allows multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation. The use of the term “self-cleaving” is not intended to imply a proteolytic cleavage reaction. Various self-cleaving or 2A linkers are known to those skilled in the art, including but not limited to those found in members of the Picornaviridae virus family, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV0), Thosea asigna virus (TaV), and porcine cerebrospinal viral-1 (PTV-1); and cardiogenic viruses such as Theylvirus and encephalomyocarditis virus. 2A linkers derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as “F2A,” “E2A,” “P2A,” and “T2A,” respectively. The P2A adapter may have a sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29. The T2A adapter may have a sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30.

[0242] The vector may contain two or more nucleic acid sequences encoding a first CAR (e.g., the CLDN18.2 CAR described herein) and a second CAR (e.g., the GUCY2C CAR). The two or more nucleic acid sequences encoding these CARs may be encoded by a single nucleic acid molecule within the same frame and as a single polypeptide chain. The two or more CARs may be separated, for example, by one or more peptide cleavage sites (e.g., self-cleavage sites or substrates of intracellular proteases).

[0243] Engineered immune cells

[0244] In one aspect, this article provides an engineered immune cell comprising a multispecific CAR, nucleic acid, or vector as disclosed herein. Therefore, this disclosure provides engineered immune cells (e.g., T cells or NK cells) and methods for their use in adoptive therapy.

[0245] In some embodiments, the engineered immune cell comprises an engineered receptor specifically targeting CLDN18.2, the engineered receptor comprising: an extracellular antigen-binding domain, a transmembrane domain, and optionally an intracellular signaling domain containing at least one anti-CLDN18.2 binding moiety; and an engineered immune cell comprising an engineered receptor specifically targeting GUCY2C, the engineered receptor comprising: an extracellular antigen-binding domain, a transmembrane domain, and optionally an intracellular signaling domain containing at least one anti-GUCY2C binding moiety. In some embodiments, the engineered immune cell comprises: (1) a polypeptide containing an amino acid sequence shown in any one of SEQ ID NO: 14-18; or (2) a first polypeptide and a second polypeptide, wherein the first polypeptide contains an amino acid sequence shown in any one of SEQ ID NO: 12 and 19, and the second polypeptide contains an amino acid sequence shown in any one of SEQ ID NO: 13 and 20-22.

[0246] "Immune cells" are immune cells that can perform immune effector functions. Examples of immune cells include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.

[0247] In some embodiments, the immune cells are T cells. T cells can be αβ T cells or γδ T cells. T cells can be CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, CD4- / CD8-, or combinations thereof. T cells can produce IL-2, TFN, and / or TNF after expressing a CAR and binding to target cells (such as CLDN18.2+ or GUCY2C+ tumor cells). T cells can lyse antigen-specific target cells after expressing a CAR and binding to target cells.

[0248] In some embodiments, the immune cells are NK cells. The immune cells may be established cell lines, such as NK-92 cells.

[0249] In some embodiments, immune cells are differentiated from stem cells (such as hematopoietic stem cells, pluripotent stem cells, iPS cells, or embryonic stem cells).

[0250] Engineered immune cells as disclosed herein can be prepared by introducing CARs into immune cells, such as T cells. In some embodiments, CARs are introduced into immune cells by transfecting any isolated nucleic acid or any of the vectors described herein. CARs can also be introduced into immune cells by inserting the protein into the cell membrane while passing the cell through a microfluidic system (see, for example, U.S. Patent Application Publication No. 20140287509).

[0251] Methods for introducing vectors or isolated nucleic acids into mammalian cells are known in the art. The described vectors can be transferred into immune cells by physical, chemical, or biological methods.

[0252] Physical methods for introducing vectors into immune cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York. Vectors can be introduced into cells via electroporation. Biological methods for introducing vectors into immune cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian (e.g., human) cells. Chemical means for introducing vectors into immune cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro delivery medium is a liposome (e.g., an artificial membrane vesicle).

[0253] Transduced or transfected immune cells can be proliferated in vitro after the introduction of a vector or isolated nucleic acid. In some embodiments, the transduced or transfected immune cells are cultured to proliferate for at least about 1, 2, 3, 4, 5, 6, 7, 10, 12, or 14 days. The transduced or transfected immune cells can be further evaluated or screened to select engineered mammalian cells.

[0254] Reporter genes can be used to identify potentially transfected cells and to evaluate the function of regulatory sequences. Typically, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and encodes a polypeptide whose expression is indicated by some easily detectable property (e.g., enzymatic activity). The expression of the reporter gene is determined at an appropriate time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al. FEBS Letters 479: 79-82 (2000)). Suitable expression systems are well known and can be prepared using known techniques or are commercially available.

[0255] Other methods for confirming the presence of nucleic acids encoding CARs in engineered immune cells include, for example, molecular biological assays well known to those skilled in the art, such as DNA blotting and RNA blotting, RT-PCR and PCR; and biochemical assays, such as detecting the presence or absence of a specific peptide by, for example, immunological methods (such as ELISA and Western blotting).

[0256] Before T cells are expanded and genetically modified, their source can be obtained from an individual. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. Any number of T cell lines available in the art can be used. T cells can be obtained from units of blood collected from a subject using a variety of techniques known to a skilled craftsman, such as Ficoll isolation. Cells from an individual's circulating blood can be obtained via apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. Cells collected via apheresis can be washed to remove the plasma fraction, and the cells can be placed in an appropriate buffer or culture medium for subsequent processing steps. Cells are washed with phosphate-buffered saline (PBS). The wash solution is deficient in calcium and may be deficient in magnesium or many (if not all) of divalent cations. Surprisingly, the initial activation step in the absence of calcium results in amplified activation. It will be readily understood by those skilled in the art that the washing step can be performed using methods known to those skilled in the art, such as a semi-automated “flow” centrifuge (e.g., a Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer’s instructions. After washing, the cells can be resuspended in various biocompatible buffers, such as Ca-free buffers. 2+ Mg-free 2+ PBS, PlasmaLyteA, or other saline solutions with or without buffer can be used. Alternatively, unwanted components of a single sample can be removed, and the cells can be directly resuspended in the culture medium.

[0257] T cells can be isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by PERCOLL gradient centrifugation or countercurrent centrifugation. Specific subsets of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated using positive or negative selection techniques.

[0258] Enriching a T cell population through negative selection can be achieved by conjugating antibodies against surface markers specific to the negatively selected cells. One approach involves cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry, using a mixture of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells through negative selection, the monoclonal antibody mixture typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. It may be desirable to enrich or positively select regulatory T cells that typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, regulatory T cells can be depleted using anti-C25-conjugated beads or other similar selection methods.

[0259] T cells can be obtained directly from the patient after treatment. In this regard, it has been observed that, following certain cancer treatments, particularly those with drugs that impair the immune system, the quality of the T cells obtained shortly after treatment may be optimal, or their ability to expand in vitro may be improved, during the period when patients typically recover from treatment. Similarly, after in vitro manipulation using the methods described herein, these cells can be in a preferred state for enhanced implantation and in vivo expansion. Therefore, in the context of this disclosure, it is anticipated to collect blood cells, including T cells, dendritic cells, or other hematopoietic cells, during this recovery phase. Furthermore, mobilization (e.g., mobilization with GM-CSF) and conditioning protocols can be used to create conditions in the subject that favor the reproliferation, recycling, regeneration, and / or expansion of cell types, particularly during a defined time window following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0260] Before or after genetic modification of T cells with the CAR described herein, T cells can typically be activated and genetically amplified using methods described, for example, in the following literature: U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0261] Pharmaceutical Composition

[0262] This application further provides pharmaceutical compositions comprising any engineered immune cell containing any CAR as described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition can be prepared by mixing engineered immune cells of desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A., ed. (1980)). The pharmaceutical composition may be in the form of a lyophilized formulation or an aqueous solution.

[0263] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the doses and concentrations used and include buffers; antioxidants, including ascorbic acid, methionine, vitamin E, and sodium metabisulfite; preservatives, isotonic agents, stabilizers, metal complexes (e.g., Zn-protein complexes); chelating agents, such as EDTA, and / or nonionic surfactants.

[0264] For pharmaceutical compositions to be usable for in vivo administration, they must be sterile. Pharmaceutical compositions can be sterilized by filtration through a sterile filter membrane. Typically, the pharmaceutical compositions described herein are placed in containers with sterile inlets, such as intravenous solution bags or vials with stoppers that can be punctured by a hypodermic needle.

[0265] The route of administration is determined according to known and acceptable methods, such as by single or multiple bolus injections or by appropriate means (e.g., injection or infusion via subcutaneous, intravenous, intraperitoneal, intramuscular, intra-arterial, intralesional, or intra-articular routes, local application, inhalation, or prolonged infusion via sustained or extended release).

[0266] Methods and uses

[0267] In one aspect, this disclosure provides a method for treating a tumor in a subject in need, the method comprising administering to the subject an effective amount of a combination of a CLDN18.2 antagonist and a GUCY2C antagonist, or an antagonist of CLDN18.2 and GUCY2C. The tumor to be treated may be a CLDN18.2-positive tumor, a GUCY2C-positive tumor, or a double-positive tumor of CLDN18.2 and GUCY2C. Such methods and uses include treatment methods and uses, for example, involving the administration of a molecule, cell, or composition containing the molecule or cell to a subject suffering from a disease, condition, or disorder expressing duracin 18.2 and / or GUCY2C or related to duracin 18.2 and / or GUCY2C expression and / or wherein cells or tissues express duracin 18.2 and / or GUCY2C. In some embodiments, the subject is resistant to at least one CLDN18.2 agent and / or wherein the subject is resistant to at least one GUCY2C agent.

[0268] This application further relates to methods and compositions for use in cellular immunotherapy. In some embodiments, cellular immunotherapy is used to treat cancer, including but not limited to CLDN18.2-positive cancer, GUCY2C-positive cancer, and CLDN18.2×GUCY2C double-positive cancer. Any chimeric antigen receptor, nucleic acid, and engineered immune cells described herein can be used in methods of treating cancer. The CARs described herein can be used to treat tumors with antigen loss escape mutations and to reduce resistance to existing therapies. In some embodiments, the methods and compositions described herein can be used to treat other diseases associated with CLDN18.2 and / or GUCY2C.

[0269] In some embodiments, a method of treating cancer in an individual (such as a human individual) is provided, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising: (1) an engineered immune cell (such as a T cell) containing a CAR as disclosed herein, the CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding region containing an anti-CLDN18.2 binding moiety and an anti-GUCY2C binding moiety; (b) a transmembrane domain; and (c) an intracellular signal transduction domain; and (2) a pharmaceutically acceptable carrier. The anti-CLDN18.2 binding moiety and the anti-GUCY2C binding moiety may be a VHH, such as a camel VHH, a chimeric VHH, a human VHH, or a humanized VHH. In some embodiments, the anti-CLDN18.2 VHH and the anti-GUCY2C VHH are fused to each other via a peptide bond or a peptide linker. The length of the peptide linker may be no more than about 50 amino acids (such as no more than about 35, 25, 20, 15, 10, or 5).

[0270] In some embodiments, a method of treating an individual (such as a human individual) for a tumor or cancer is provided, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising: (1) engineered immune cells (such as T cells) containing a first CAR and a second CAR, the first CAR comprising an extracellular antigen-binding region, a transmembrane domain, and an intracellular signaling domain containing an anti-CLDN18.2 binding moiety, the second CAR comprising an extracellular antigen-binding region, a transmembrane domain, and an intracellular signaling domain containing an anti-GUCY2C binding moiety, optionally the first CAR being operatively linked to the second CAR via a cleavable adapter; and (2) a pharmaceutically acceptable carrier. In some embodiments, the anti-CLDN18.2 binding moiety and the anti-GUCY2C binding moiety are VHHs, such as camel VHHs, chimeric VHHs, human VHHs, or humanized VHHs. The transmembrane domain and the intracellular signaling domain in the first CAR and the second CAR may be the same or different.

[0271] In some embodiments, a method of treating cancer in an individual (such as a human individual) is provided, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising: (1) a first group of engineered immune cells (such as T cells) containing a CAR, the CAR comprising an extracellular antigen-binding region, a transmembrane domain, and an intracellular signaling domain containing an anti-CLDN18.2 binding moiety; (2) a second group of engineered immune cells (such as T cells) containing a CAR, the CAR comprising an extracellular antigen-binding region, a transmembrane domain, and an intracellular signaling domain containing an anti-GUCY2C binding moiety; and (3) a pharmaceutically acceptable carrier. The anti-CLDN18.2 binding moiety and the anti-GUCY2C binding moiety may be VHHs, such as camel VHHs, chimeric VHHs, human VHHs, or humanized VHHs. The ratio of the number of the first group of engineered immune cells to the number of the second group of engineered immune cells may be in the range of 10:1 to 1:10.

[0272] Engineered immune cells can be autologous. Engineered immune cells can be allogeneic. In some embodiments, the cancer is a solid cancer, including but not limited to gastric cancer, esophageal cancer, pancreatic ductal carcinoma, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colorectal cancer, liver cancer, head and neck cancer, gallbladder cancer and their metastases.

[0273] These methods are applicable to all stages of cancer, including early, advanced, and metastatic cancer. The methods described herein can be used as a first-line, second-line, third-line therapy, or in combination with other types of cancer therapies known in the art, such as chemotherapy, surgery, radiation, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radiofrequency ablation, etc.

[0274] The pharmaceutical composition can be administered in any conventional manner, including by injection, ingestion, infusion, implantation, or transplantation. The composition can be administered to a patient via artery, subcutaneous, intradermal, intratumoral, intranodular, intramedullary, intramuscular, intravenous, or intraperitoneal route. In some embodiments, the pharmaceutical composition is administered systemically. The pharmaceutical composition can be administered to an individual by infusion (e.g., intravenous infusion). Infusion techniques for immunotherapy are known in the art (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676 (1988)). In some embodiments, the pharmaceutical composition is administered to an individual by intradermal or subcutaneous injection. The composition can be administered by intravenous injection. The composition can be injected directly into a tumor or lymph node. The pharmaceutical composition can be applied topically to a tumor site, such as directly to tumor cells or to tissue containing tumor cells.

[0275] The dosage and desired drug concentration of the pharmaceutical compositions disclosed herein may vary depending on the intended specific use. Determining an appropriate dosage or route of administration is entirely within the skill of a person skilled in the art. Animal studies have provided reliable guidance for determining effective dosages for human therapy. Interspecies extrapolation of effective dosages can be performed according to the principles set forth in Mordenti, J. and Chappell, W., “The Use of Interspecies Scaling in Toxicokinetics,” in Toxicokinetics and New Drug Development, Yacobi et al., editors, Pergamon Press, New York, 1989, pp. 42-46. Within the scope of this application, different formulations will be effective for different treatments and different barriers, and administration intended to treat a particular organ or tissue may require delivery in a different manner than that intended for another organ or tissue.

[0276] Combination therapy

[0277] The CAR-expressing cells described herein can be used in combination with other known agents and therapies. As used herein, “combination” administration means the delivery of two (or more) different treatments to a subject during the course of the disorder, such as after the subject has been diagnosed with the disorder and before the disorder is cured or eliminated or treatment is discontinued for other reasons. When the delivery of the second treatment begins, the delivery of the first treatment may still be in progress, so there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous delivery” or “parallel delivery.” In other embodiments, the delivery of one treatment ends before the delivery of another treatment begins. In some embodiments of either case, the treatment is more effective due to the combination administration. For example, the second treatment is more effective than the results that would be observed if the second treatment were administered in the absence of the first treatment, such as observing an equivalent effect with less second treatment, or the second treatment reducing symptoms to a greater extent; or a similar situation is observed with respect to the first treatment. In some embodiments, the delivery results in a reduction of symptoms or other parameters related to the disorder greater than the results that would be observed if one treatment were delivered in the absence of the other treatment. The effects of the two treatments may be partially additive, fully additive, or greater than additive. This delivery method allows the effects of the first treatment to remain detectable when the second treatment is delivered.

[0278] The CAR-expressing cells described herein and at least one other therapeutic agent may be administered simultaneously (in the same or separate compositions) or sequentially. For sequential administration, the CAR-expressing cells described herein may be administered first, followed by the other agent, or the order of administration may be reversed.

[0279] CAR therapy and / or other therapeutic agents, procedures, or methods may be administered during periods of activity impairment, or during periods of remission or low-activity disease. CAR therapy may be administered before, concurrently with, after, or during periods of impairment remission.

[0280] In another respect, the CAR-expressing cells described herein can be used in combination with surgery, cytokines, radiation, or chemotherapy (such as cyclophosphamide, fludarabine, histone deacetylase inhibitors, demethylating agents, or peptide vaccines) in treatment regimens, as described by Izumoto et al. 2008 J Neurosurg 108:963-971.

[0281] Sequence Summary

[0282] This application is accompanied by a sequence listing containing multiple amino acid sequences. Tables A-C below provide a summary of the included sequences. The seven illustrative CARs disclosed herein are designated as LG23A01, LG23A02, LG23B01, LG23B02, LG23D01, LG23D02, and LG23D03.

[0283] Table A: Amino acid sequences of the CDR and variable domains of VHH

[0284]

[0285] Table B: Amino acid sequence of CAR

[0286]

[0287]

[0288]

[0289] Table C: Other relevant amino acid sequences

[0290]

[0291] Example

[0292] Example 1. IHC Analysis

[0293] To determine the expression of CLDN18.2 and GUCY2C in primary and metastatic gastric cancer (GC), IHC analysis was performed on tissue sections (ST810C) from 40 primary gastric cancer cases and 39 metastatic gastric cancer cases. The detection procedure is as follows.

[0294] FFPE tissue slides (4 μm) were melted and dehydrated at 60°C for 1 h, then deparaffinized and rehydrated using xylene and alcohol, respectively. The paraffin-coated slides were placed in citrate buffer (pH 6.0), and the entire reaction system was microwaved for heat-induced antigen retrieval. 3% hydrogen peroxide was used to block endogenous peroxidase. Blocking buffer (5% BSA) was used to block the sections overnight. The tissue slides were then stained with 2 μg / mL rabbit monoclonal anti-CLDN18.2 antibody (Abcam, 222513) and 2 μg / mL rabbit anti-GUCY2C antibody, respectively, and incubated at 37°C for 2 h. The tissue slides were then washed three times thoroughly with PBS. Goat anti-rabbit IgG H&L HRP (maxim, kit-5005) was used as the secondary antibody and dripped to cover the entire tissue slide. After incubating tissue slides in an incubator for 15 min at room temperature, 100 μL of DAB substrate solution (VECTOR LABORATORIES, SK-4105) was applied to the slides for antibody staining and development for 3 min. The slides were then scanned, cell nuclei were stained with hematoxylin, and the expression of CLDN18.2 and GUCY2C in primary and metastatic gastric cancer tissues was analyzed.

[0295] like Figure 1 A- Figure 1 As shown in B and Table 1, the IHC analysis results indicated that CLDN18.2 and GUCY2C were highly expressed in both primary and metastatic gastric cancer tissues, with 70% (28 / 40) and 66.67% (26 / 39) of samples co-expressing CLDN18.2 and GUCY2C, respectively. This IHC study expanded the coverage of tumor cells co-expressing CLDN18.2 and GUCY2C in the same individual subjects, which can be used to guide or improve treatment methods to improve drug efficacy, prolong progression-free survival, address tumor heterogeneity, and potentially expand therapeutic indications.

[0296] Table 1. Expression of CLDN18.2 and GUCY2C in human primary and metastatic gastric cancer tissues

[0297]

[0298] Example 2. CAR Component Design

[0299] This disclosure proposes an immunocellular therapy strategy co-targeting CLDN18.2 and GUCY2C to address the challenges of tumor heterogeneity and target downregulation during gastric cancer treatment. This disclosure provides CAR-T cells that simultaneously target CLDN18.2 and GUCY2C, including tandem CLDN18.2×GCC bispecific CAR-T cells, split CLDN18.2×GCC bispecific CAR-T cells, and combinations of single CLDN18.2-specific CAR-T cells and single GCC-specific CAR-T cells, exhibiting strong killing effects against CLDN18.2 single-positive target cells, GUCY2C single-positive target cells, or CLDN18.2 and GUCY2C double-positive target cells. Figure 2 As shown in B, a tandem bispecific CAR can include an extracellular antigen-binding region containing at least one CLDN18.2 binding moiety and at least one GUCY2C binding moiety, which can recognize and bind to two different antigens (CLDN18.2 and GUCY2C). Figure 2 As shown in C, the split-type bispecific CAR has two CARs that target two antigens expressed in one cell, wherein each of the two CARs has an extracellular antigen-binding region containing at least one duracin 18.2 binding moiety or at least one GUCY2C binding moiety, respectively.

[0300] Alone single CLDN18.2-specific CAR-T cells (Si-CLDN18.2 CAR-T) and alone single GUCY2C-specific CAR-T cells (Si-GCC CAR-T) were used as controls. The single CLDN18.2-specific CAR may contain an extracellular antigen domain with one or more CLDN18.2 binding moieties, and the single GUCY2C-specific CAR may contain an extracellular antigen domain with one or more GUCY2C binding moieties. In this disclosure, single CLDN18.2-specific CAR-T cells exhibit very low cytotoxicity against GUCY2C single-positive target cells, and alone single GCC-specific CAR-T cells exhibit very low cytotoxicity against CLDN18.2 single-positive target cells. This combination therapy of CAR-T cells co-targeting CLDN18.2 and GUCY2C will reach more individual subjects with gastric cancer, overcoming tumor heterogeneity and improving drug efficacy.

[0301] In order to leverage the advantages of immunocellular therapies that co-target CLDN18.2 and GUCY2C in addressing tumor heterogeneity, this disclosure also provides CAR structures with different co-stimulatory signaling domains (such as CD278 (ICOS) and NTBA) that can promote CAR-T cell proliferation or improve drug efficacy.

[0302] To confirm the feasibility of immunocellular therapy co-targeting CLDN18.2 and GUCY2C, seven CAR constructs, including LG23A01, LG23A02, LG23B01, LG23B02, LG23D01, LG23D02, and LG23D03, and CAR-T cells, as well as a combined CAR-T cell mix of equal proportions of Si-CLDN18.2 CAR-T cells (LG23A01) and Si-GUCY2C CAR-T cells (LG23A02), denoted as LG23A01+LG23A02. The CAR structures of exemplary CAR-T cells provided in this paper are shown in Table 2 and described in detail below.

[0303] Table 2. Structures of CAR constructs targeting CLDN18.2 and / or GUCY2C

[0304]

[0305] LG23A01 CAR contains a CAR backbone polypeptide that includes, from the N-terminus to the C-terminus: a CD8α signal peptide (SEQ ID NO: 3), an antigen-binding domain with anti-CLDN18.2 VHH (SEQ ID NO: 1), a CD8α hinge domain (SEQ ID NO: 4), a CD8α transmembrane domain (SEQ ID NO: 5), a 4-1BB co-stimulatory signal transduction domain (SEQ ID NO: 7), and a CD3ζ primary intracellular signal transduction domain (SEQ ID NO: 11). LG23A02 CAR contains a CAR backbone polypeptide that includes, from the N-terminus to the C-terminus: a CD8α signal peptide (SEQ ID NO: 3), an antigen-binding domain with anti-GUCY2C VHH (SEQ ID NO: 2), a CD8α hinge domain (SEQ ID NO: 4), a CD8α transmembrane domain (SEQ ID NO: 5), a 4-1BB co-stimulatory signal transduction domain (SEQ ID NO: 7), and a CD3ζ primary intracellular signal transduction domain (SEQ ID NO: 11). The LG23B01 CAR contains a CAR backbone polypeptide that comprises, from the N-terminus to the C-terminus: a CD8α signal peptide (SEQ ID NO: 3), an antigen-binding domain having anti-GUCY2CVHH (SEQ ID NO: 2) and anti-CLDN18.2 VHH (SEQ ID NO: 1) linked via a (G4S)3 linker (SEQ ID NO: 31), a CD8α hinge domain (SEQ ID NO: 4), a CD8α transmembrane domain (SEQ ID NO: 5), a 4-1BB co-stimulatory signal transduction domain (SEQ ID NO: 7), and a CD3ζ primary intracellular signal transduction domain (SEQ ID NO: 11). LG23B02 CAR contains a CAR backbone polypeptide that comprises, from the N-terminus to the C-terminus: a CD8α signal peptide (SEQ ID NO: 3), an antigen-binding domain having anti-CLDN18.2 VHH (SEQ ID NO: 1) and anti-GUCY2C VHH (SEQ ID NO: 2) linked via a (G4S)3 linker (SEQ ID NO: 21), a CD8α hinge domain (SEQ ID NO: 4), a CD8α transmembrane domain (SEQ ID NO: 5), a 4-1BB co-stimulatory signal transduction domain (SEQ ID NO: 7), and a CD3ζ primary intracellular signal transduction domain (SEQ ID NO: 11).The nucleotide sequence of each CAR backbone was chemically synthesized and cloned downstream of the constitutive hEF1α promoter in a pre-modified lentiviral vector (pLSINK-BBzBB) and operably ligated to the constitutive hEF1α promoter for in vitro transcription.

[0306] The LG23D01 CAR construct comprises two CAR backbone peptides. The first CAR backbone peptide, from the N-terminus to the C-terminus, comprises: a CD8α signal peptide (SEQ ID NO: 3), an antigen-binding domain with anti-CLDN18.2 VHH (SEQ ID NO: 1), a CD8α hinge domain (SEQ ID NO: 4), a CD8α transmembrane domain (SEQ ID NO: 5), a 4-1BB co-stimulatory signal transduction domain (SEQ ID NO: 7), and a CD3ζ primary intracellular signal transduction domain (SEQ ID NO: 11). The second CAR backbone polypeptide comprises a CD8α signal peptide (SEQ ID NO: 3), an antigen-binding domain with anti-GUCY2C VHH (SEQ ID NO: 2), a CD8α hinge domain (SEQ ID NO: 4), a CD8α transmembrane domain (SEQ ID NO: 5), a CD28 co-stimulatory signal transduction domain (SEQ ID NO: 8), and a CD3ζ primary intracellular signal transduction domain (SEQ ID NO: 11). The nucleic acid comprises nucleotide sequences encoding the backbones of the first and second CARs linked by a self-cleaving 2A (e.g., P2A, SEQ ID NO: 29) linker. The nucleotide sequences encoding both CARs are chemically synthesized and cloned downstream of a constitutive hEF1α promoter in a pre-modified lentiviral vector (pLSINK-BBzBB) and operatively ligated to the constitutive hEF1α promoter for in vitro transcription.

[0307] The LG23D02 CAR construct comprises two CAR backbone peptides. The first CAR backbone peptide, from the N-terminus to the C-terminus, comprises: a CD8α signal peptide (SEQ ID NO: 3), an antigen-binding domain with anti-CLDN18.2 VHH (SEQ ID NO: 1), a CD8α hinge domain (SEQ ID NO: 4), an ICOS transmembrane domain (SEQ ID NO: 6), an ICOS co-stimulatory signal transduction domain (SEQ ID NO: 9), and a CD3ζ primary intracellular signal transduction domain (SEQ ID NO: 11). The second CAR backbone peptide comprises: a CD8α signal peptide (SEQ ID NO: 3), an antigen-binding domain with anti-GUCY2C VHH (SEQ ID NO: 2), a CD8α hinge domain (SEQ ID NO: 4), a CD8α transmembrane domain (SEQ ID NO: 5), an NTB-A co-stimulatory signal transduction domain (SEQ ID NO: 10), and a CD3ζ primary intracellular signal transduction domain (SEQ ID NO: 11). The nucleic acid contains nucleotide sequences encoding the backbones of a first CAR and a second CAR linked by a self-cleaving 2A (e.g., P2A, SEQ ID NO: 29) linker, chemically synthesized nucleotide sequences encoding both CARs and cloned downstream of a constitutive hEF1α promoter in a pre-modified lentiviral vector (pLSINK-BBzBB) and operably linked to the constitutive hEF1α promoter for in vitro transcription.

[0308] The LG23D03 CAR construct comprises two CAR backbone peptides. The first CAR backbone peptide, from the N-terminus to the C-terminus, comprises: a CD8α signal peptide (SEQ ID NO: 3), an antigen-binding domain with anti-CLDN18.2 VHH (SEQ ID NO: 1), a CD8α hinge domain (SEQ ID NO: 4), an ICOS transmembrane domain (SEQ ID NO: 6), an ICOS co-stimulatory signal transduction domain (SEQ ID NO: 9), and a CD3ζ primary intracellular signal transduction domain (SEQ ID NO: 11). The second CAR backbone peptide comprises: a CD8α signal peptide (SEQ ID NO: 3), an antigen-binding domain with anti-GUCY2C VHH (SEQ ID NO: 2), a CD8α hinge domain (SEQ ID NO: 4), an ICOS transmembrane domain (SEQ ID NO: 6), an ICOS co-stimulatory signal transduction domain (SEQ ID NO: 9), and a CD3ζ primary intracellular signal transduction domain (SEQ ID NO: 11). The nucleic acid contains nucleotide sequences encoding the backbones of a first CAR and a second CAR linked by a self-cleaving 2A (e.g., P2A, SEQ ID NO: 29) linker, chemically synthesized nucleotide sequences encoding both CARs and cloned downstream of a constitutive hEF1α promoter in a pre-modified lentiviral vector (pLSINK-BBzBB) and operably linked to the constitutive hEF1α promoter for in vitro transcription.

[0309] Example 3. Preparation of lentiviral vectors and engineered T cells expressing CAR

[0310] A mixture of lentiviral packaging plasmids containing pMDLg.pRRE (Addgene#12251), pRSV-REV (Addgene#12253), and pMD2.G (Addgene#12259) was premixed with the CAR-expressing lentiviral vector constructed in Example 2 at a pre-optimized ratio in the presence of polyetherimide (PEI) and incubated at 25°C for 5 min. The transfection mixture was then added to HEK293 cells. The cells were then incubated overnight at 37°C in a cell culture incubator with 5% CO2. The supernatant was collected after centrifugation at 3000 g for 15 min at 4°C and subsequently ultracentrifuged through a 0.45 μm PES filter for lentiviral concentration. The supernatant was then carefully discarded, and the viral particles were carefully washed with pre-chilled DPBS. The virus was appropriately resuspended and stored at -80°C.

[0311] Human T cells were purified from commercially available PBMCs using the Miltenyi Pan T Cell Isolation Kit (catalog number 130-096-535) according to the manufacturer's protocol as described below. Cell counts were determined, and the cell suspension was centrifuged at 300 g for 10 min. The supernatant was then completely discarded, and the cell particles were resuspended at 10 μL / mL. 7 40 μL of MACS buffer (replenished with 8 μM EDTA + 0.5% FBS in DPBS) per 10 cells. 7 Add 10 μL of Pan T cell biotin-antibody mixture to each total cell, mix thoroughly, and incubate in a refrigerator (2°C–8°C) for approximately 5 min. Then add every 10 cells... 7 Add 30 μL MACS buffer to each cell. Add 10 cells per cell. 7 Add 20 μL of the Pan T cell microbead mixture to each cell. Thoroughly mix the cell suspension mixture and incubate for another 10 min in a refrigerator (2°C–8°C). Magnetic separation requires at least 500 μL. For magnetic separation, place the LS column in the magnetic field of a suitable MACS separator. Prepare the column by rinsing with 3 mL of buffer. Then apply the cell suspension to the column and collect the effluent containing unlabeled cells, representing the enriched T cell fraction. Collect additional T cells by washing the column with 3 mL of buffer and collecting the passing unlabeled cells. These unlabeled cells again represent enriched T cells and are combined with the effluent from the previous step. The combined enriched T cells are then centrifuged and resuspended in 1 L of TexMACS GMP Medium (Miltenyi #170-076-309) with 300 IU / mL IL-2.

[0312] Subsequently, according to the manufacturer's protocol, the prepared T cells were pre-activated for 48-96 h using a human T cell activation / expansion kit (Miltenyi #130-091-441), in which anti-CD3 / CD28MACSiBead particles were added at a bead to cell ratio of 1:2.

[0313] Preactivated T cells were transduced with lentiviral primary strains by adding them directly to the culture medium (TexMACS GMP Medium supplemented with 300 IU / mL IL-2). The transduced cells were then transferred to a cell culture incubator at 37°C with 5% CO2 for transgene expression.

[0314] On day 5, CAR expression levels were assessed by flow cytometry (NovoCyte). In short, 3 × 10⁻⁶ cells were collected from each group. 5T cells were collected and incubated with anti-VHH antibody (Genscript, catalog number A02172) at 4°C for 30 min. After incubation, the cells were collected and washed with DPBS, then centrifuged at 300 g for 5 min at 20°C. As shown in Table 3, the CAR expression levels of LG23A01, LG23A02, LG23B01, LG23B01, LG23D01, LG23D02, and LG23D03 CAR-T cells were 50.16%, 46.31%, 48.38%, 60.26%, 51.89%, 66.54%, and 73.63%, respectively. UnT refers to T cells that have not been transduced with CAR. Detailed test results are shown in Table 3.

[0315] Table 3. CAR expression in CAR-T cells

[0316]

[0317] Example 4. Preparation of target cells

[0318] Lentiviral cells were packaged by transiently transfecting Lenti-X 293T host cells with a mixture of plasmids including psPAX2, pMD.2G, and PLLV-CLDN18.2.Luc.PuroR or PLVX-CQBL-LUC-puro. PLLV-CLDN182.Luc.PuroR was constructed internally to express CLDN18.2 and luciferase, and PLVX-CQBL-LUC-puro was constructed internally to express GUCY2C and luciferase. Hep3b2.1-7 (ATCC, HB-8064) is a human CLDN18.2 and GUCY2C negative cell line. The Hep3b2.1-7 cell line was used as a tool mother cell to construct three target cell lines, namely CLDN18.2 single-positive target cells, GUCY2C single-positive target cells only, and CLDN18.2 and GUCY2C double-positive target cells, for evaluating CAR-T cell function. In short, 0.5 × 10⁻⁶ lentiviruses were transduced using 10 μL of PLLV-CLDN182.Luc.PuroR or PLVX-CQBL-LUC-puro. 6 Hep3b2.1-7 cells were used. Two target cell lines were selected using puromycin and EMEM (containing 10% FBS and 2 μg / mL puromycin) with the medium changed every 2-3 days. These lines were named Hep3b-CLDN18.2.Luc cells and Hep3b-GUCY2C.Luc cells. 0.5 × 10⁶ cells were transduced with 10 μL of the obtained PLVX-CQBL-LUC-puro lentivirus. 6Hep3b2.1-7-CLDN18.2.Luc cells were selected using puromycin, with the selection medium (EMEM containing 10% FBS and 2 μg / mL puromycin) being replaced every 2-3 days, to obtain Hep3b2.1-7-CLDN18.2-GUCY2C.Luc cells. After three rounds of selection, the obtained cell clones were harvested by trypsin digestion. The obtained cells were properly stored and prepared for further use. Three target cell lines (Hep3b-CLDN18.2.Luc cells overexpressing only CLDN18.2, Hep3b-GUCY2C.Luc cells overexpressing only GUCY2C, and Hep3b-CLDN18.2-GUCY2C.Luc cells expressing both CLDN18.2 and GUCY2C) were used for subsequent functional evaluation of CAR-T cells.

[0319] Example 5. Cytotoxicity of CAR-T cells

[0320] Single CLDN18.2-specific CAR-T cells (LG23A01) and single GUCY2C-specific CAR-T cells (LG23A02), tandem or split bispecific CLDN18.2×GUCY2C CAR-T cells (LG23B01, LG23B02, LG23D01, LG23D02 or LG23D03), and combined CAR-T cells (LG23A01+LG23A02) were co-incubated with Hep3b-CLDN18.2.Luc cells, Hep3b-GUCY2C.Luc cells, or Hep3b-CLDN18.2-GUCY2C.Luc cells at effector cell (CAR-positive T cell) to target cell ratios (E:T) of 4:1, 2:1, 1:1 or 0.5:1 for 20-24 h. To determine the cytotoxicity of CAR-T cells against tumor cells, the One-glo luminescent luciferase assay reagent (Promega#E6120) was prepared according to the manufacturer's protocol and added to co-cultured cells to detect residual luciferase activity in the wells. Residual luciferase activity was directly correlated with the number of surviving target cells in the wells. Specific cytotoxicity was calculated using the following formula: Specific cytotoxicity % = 100% × (1-(RLU)) 样品 -RLU min ) / (RLU UnT -RLU min RLU 样品 Represents luciferase activity measured in wells containing CAR-T cells transduced with the GCC CAR disclosed herein. RLU minThis refers to the luciferase activity determined at the start of a cytotoxicity assay in wells containing a final concentration of 1% Triton X-100, while RLU... UnT This refers to luciferase activity determined using T cells that have not been transduced with CAR.

[0321] like Figure 3 A- Figure 3 As shown in Figure C, Si-CLDN18.2 CAR-T cells (LG23A01) exhibited weak cytotoxicity against Hep3b-GUCY2C.Luc cells. Si-GUCY2C CAR-T cells (LG23A02) also exhibited weak cytotoxicity against Hep3b-CLDN18.2.Luc cells. Compared to UnT cells, at an E:T ratio of 2:1, 21.2% of Hep3b-GUCY2C.Luc cells were lysed by LG23A01 CAR-T cells. Figure 3 C) and 37.1% of Hep3b-CLDN18.2.Luc cells were lysed by LG23A02 CAR-T cells ( Figure 3 B).

[0322] Bispecific CLDN18.2×GCC CAR-T cells (LG23B01, LG23B02, LG23D01, LG23D02, LG23D03) and combined CAR-T cells (LG23A01+LG23A02) induced effective specific cytotoxicity against Hep3b-CLDN18.2-GUCY2C.Luc cells, Hep3b-GUCY2C.Luc cells, and Hep3b-CLDN18.2.Luc cells. Figure 3 As shown in Figure A, at an E / T ratio of 2:1, the lysis percentage of Hep3b-CLDN18.2-GUCY2C.Luc cells by LG23B01 CAR-T cells was 82.20%, the lysis percentage by LG23B02 CAR-T cells was 79.11%, the lysis percentage by LG23D01 CAR-T cells was 86.08%, the lysis percentage by LG23D02 CAR-T cells was 88.07%, the lysis percentage by LG23D03 CAR-T cells was 90.41%, and the lysis percentage by LG23A01+LG23A02 CAR-T cells was 88.91%. Figure 3As shown in B, at an E / T ratio of 2:1, the lysis percentage of Hep3b-CLDN18.2.Luc cells by LG23B01 CAR-T cells was 77.97%, by LG23B02 CAR-T cells 80.99%, by LG23D01 CAR-T cells 88.90%, by LG23D02 CAR-T cells 86.36%, by LG23D03 CAR-T cells 94.36%, and by LG23A01+LG23A02 CAR-T cells 80.71%. And as... Figure 3 As shown in C, at an E / T ratio of 2:1, the lysis percentage of Hep3b-GUCY2C.Luc cells by LG23B01 CAR-T cells was 74.80%, the lysis percentage of Hep3b-GUCY2C.Luc cells by LG23B02 CAR-T cells was 74.13%, the lysis percentage of Hep3b-GUCY2C.Luc cells by LG23B02 CAR-T cells was 84.58%, the lysis percentage of Hep3b-GUCY2C.Luc cells by LG23D01 CAR-T cells was 90.37%, the lysis percentage of Hep3b-GUCY2C.Luc cells by LG23D03 CAR-T cells was 87.96%, and the lysis percentage of Hep3b-GUCY2C.Luc cells by LG23A01+LG23A02 CAR-T cells was 75.94%. Bispecific CLDN18.2×GUCY2C CAR-T and combined CAR-T cells (LG23A01+LG23A02) showed effective killing effects on Hep3b-CLDN18.2.Luc cells, Hep3b-GUCY2C.Luc cells, and Hep3b-CLDN18.2-GUCY2C.Luc cells.

[0323] Example 6. IFN-γ released by CAR-T cells in vitro in a heterogeneous model

[0324] Tumor heterogeneity models were used to evaluate the in vitro ability of bispecific CLDN18.2×GCC CAR-T cells (LG23B01, LG23B02, LG23D01, LG23D02, LG23D03) and combined CAR-T cells (LG23A01+LG23A02) to cope with tumor heterogeneity. Tumor heterogeneity models were implemented by mixing Hep3b-CLDN18.2.Luc cells and Hep3b-GUCY2C.Luc cells at different ratios, as shown in Table 4.

[0325] Table 4. In vitro tumor heterogeneity models

[0326]

[0327] Effector cells and target cells were cultured at a 1:1 E / T ratio in a microcell culture incubator at 37°C for 20–24 h with 5% CO2. The volume ratio of target cell complete culture medium to effector cell complete culture medium was 1:1. The concentration of IFN-γ produced in the culture supernatant was then measured using an HTRF kit (Cisbio, catalog number 62HIFNGPEG), and the concentration of TNF-α was measured using an HTRF kit (Cisbio, catalog number 62HTNFAPEG). Briefly, the HTRF reagents were warmed to room temperature for at least 30 min before assays. 16 μL / well of supernatant from the co-culture assay was transferred to a 384-well assay plate (Greiner Bio-One, #784075), followed by the addition of 4 μL / well of premixed HTRF reagent prepared according to the kit manual. The plate was then sealed with sealing film and incubated overnight at room temperature for IFN-γ and TNF-α assays. The plate was read using an HTRF-compatible Tecan Spark 10M reader. The concentrations of IFN-γ and TNF-α were calculated using signals obtained from the standard curve provided with the kit.

[0328] like Figure 4As shown, when Hep3b-CLDN18.2 cells accounted for 100%, the concentration of IFN-γ released by LG23A01 CAR-T cells was 16157.37 pg / mL. When the proportion of Hep3b-CLDN18.2.Luc was 0%, the concentration of IFN-γ released by LG23A01 CAR-T cells decreased to 0 pg / mL. Similarly, when the proportion of Hep3b-GUCY2C.Luc cells was 100%, the concentration of IFN-γ released by LG23A02 CAR-T cells was 10046.24 pg / mL, and when the proportion of Hep3b-GUCY2C.Luc cells was 0%, the concentration of IFN-γ released by LG23A02 CAR-T cells was 18.44 pg / mL. This indicates that single CLDN18.2 specific CAR-T cells (LG23A01) and single GUCY2C specific CAR-T cells (LG23A02) cannot lyse target-negative cells in the tumor heterogeneity model. In contrast, bispecific CLDN18.2×GUCY2C CAR-T cells (LG23B01, LG23B02, LG23D01, LG23D02, and LG23D03) and combined CAR-T cells (LG23A01+LG23A02) maintained high cytokine (IFN-γ and TNF-α) release levels regardless of the ratio of Hep3b-CLDN18.2 cells to Hep3b-GUCY2C.Luc cells. This indicates that bispecific CLDN18.2×GCC CAR-T cells and combined CAR-T cells (LG23A01+LG23A02) can always maintain the ability to kill Hep3b-CLDN18.2.Luc cells or Hep3b-GUCY2C.Luc cells, which is particularly important for addressing tumor heterogeneity.

[0329] In particular, such as Figure 4As shown in Figure A, the range of IFN-γ released by LG23B01 CAR-T cells was from 10631.86 pg / mL to 6548.16 pg / mL, that of LG23B02 CAR-T cells was from 18660.56 pg / mL to 7848.62 pg / mL, that of LG23D01 CAR-T cells was from 20229.48 pg / mL to 10848.12 pg / mL, that of LG23D02 CAR-T cells was from 4269.91 pg / mL to 2450.15 pg / mL, and that of LG23D03 CAR-T cells was from 3933.10 pg / mL to 4269.91 pg / mL to 2450.15 pg / mL. The range of IFN-γ released by the combined CAR-T cells (LG23A01+LG23A02) was from 12687.05 pg / mL to 2709.00 pg / mL, and from 12687.05 pg / mL to 8895.46 pg / mL.

[0330] Similarly, such as Figure 4 As shown in B, bispecific CLDN18.2×GCC CAR-T cells (LG23B01, LG23B02, LG23D01, LG23D02 and LG23D03) and combined CAR-T cells (LG23A01+LG23A02) can also maintain a high level of TNF-α release. Specifically, the range of TNF-α released by LG23B01 CAR-T cells was from 1575.50 pg / mL to 1280.42 pg / mL, that of LG23B02 CAR-T cells was from 1750.50 pg / mL to 1198.47 pg / mL, that of LG23D01 CAR-T cells was from 3294.49 pg / mL to 2378.32 pg / mL, that of LG23D02 CAR-T cells was from 844.85 pg / mL to 437.11 pg / mL, and that of LG23D03 CAR-T cells was from 1043.96 pg / mL to 789.51 pg / mL. The range of TNF-α released by the combined CAR-T cells (LG23A01+LG23A02) was from 1685.32 pg / mL to 1139.06 pg / mL.

[0331] It should be noted that the lower concentrations of cytokines (IFN-γ and TNF-α) released by the LG23D02 and LG23D03 structures are due to the use of special co-stimulatory domain structures, which can maintain their effect on target cells even at low levels of released cytokines.

[0332] Example 7. In vitro CAR-T cell repeated challenge assay

[0333] To evaluate the persistence and exhaustion of CAR-T cells in vitro, a CAR-T cell repeated challenge assay model was established. CAR-T cells were continuously stimulated with Hep3b-CLDN18.2-GUCY2C.Luc cells for several rounds. In the first round (round 1) of repeated challenge, CAR-T cells were co-cultured with tumor cells at an E / T ratio of 2:1 in 6-well plates. After 3-4 days of co-culture, CAR-T cells were counted and the CAR percentage was detected by flow cytometry (NovoCyte). The CAR-T cells were then replaced with new plates seeded with tumor cells and cultured for another 3-4 days (round 2). The number of tumor cells added was determined by the CAR positivity percentage at the end of the previous round, and the E / T ratio was maintained at 2:1.

[0334] like Figure 5 As shown in Figure A, after five rounds of stimulation with Hep3b-CLDN18.2-GUCY2C.Luc cells, the fold increase in total T cells was 207.03 times for LG23A01 CAR-T cells, 413.51 times for LG23A02 CAR-T cells, 93.28 times for LG23B01 CAR-T cells, 100.24 times for LG23B02 CAR-T cells, 188.94 times for LG23D01 CAR-T cells, 482.41 times for LG23D02 CAR-T cells, 309.91 times for LG23D03 CAR-T cells, 2.30 times for UnT cells, and 76.96 times for LG23A01+LG23A02. Data indicates that these structures have similar amplification capabilities to LG23A01 and LG23A02 during repeated attacks.

[0335] like Figure 5As shown in Figure B, after five rounds of stimulation with Hep3b-CLDN18.2-GUCY2C.Luc cells, the fold increase in CAR-T cell count was 434.36 times for LG23A01 CAR-T cells, 1126.29 times for LG23A02 CAR-T cells, 225.68 times for LG23B01 CAR-T cells, 214.23 times for LG23B02 CAR-T cells, 428.34 times for LG23D01 CAR-T cells, 1354.47 times for LG23D02 CAR-T cells, 844.19 times for LG23D03 CAR-T cells, and 151.16 times for LG23A01+LG23A02.

[0336] like Figure 5 As shown in Figure C, after five rounds of stimulation with Hep3b-CLDN18.2-GUCY2C.Luc cells, CAR expression was 73.43% for LG23A01 CAR-T cells, 95.33% for LG23A02 CAR-T cells, 84.68% for LG23B01 CAR-T cells, 74.80% for LG23B02 CAR-T cells, 79.35% for LG23D01 CAR-T cells, 98.27% for LG23D02 CAR-T cells, 95.34% for LG23D03 CAR-T cells, and 68.74% for LG23A01+LG23A02.

Claims

1. A method for treating a tumor in a subject in need, the method comprising administering to the subject an effective amount of a combination of a CLDN18.2 antagonist and a GUCY2C antagonist, or an antagonist of CLDN18.2 and GUCY2C.

2. The method of claim 1, wherein the tumor is CLDN18.2 positive and / or GUCY2C positive.

3. The method of claim 1 or 2, wherein the CLDN18.2 antagonist, the GUCY2C antagonist, and / or the CLDN18.2 and GUCY2C antagonists are selected from engineered immune cells, engineered receptors, antibodies, antibody-drug conjugates (ADCs), aptamers, and small RNAs.

4. The method of any one of claims 1-3, wherein the combination of the CLDN18.2 antagonist and the GUCY2C antagonist comprises a first group of engineered immune cells and a second group of engineered immune cells, wherein: (1) The CLDN18.2 antagonist is a first group of engineered immune cells comprising an engineered receptor that specifically targets CLDN18.2, wherein the engineered receptor comprises: an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain containing at least one anti-CLDN18.2 binding moiety; and / or (2) The GUCY2C antagonist is a second group of engineered immune cells containing an engineered receptor that specifically targets GUCY2C, wherein the engineered receptor comprises: an extracellular antigen-binding domain, a transmembrane domain and an intracellular signal transduction domain containing at least one anti-GUCY2C binding moiety.

5. The method of any one of claims 1-3, wherein the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising a first engineered receptor specifically targeting CLDN18.2 and a second engineered receptor specifically targeting GUCY2C, wherein (1) the first engineered receptor specifically targeting CLDN18.2 comprises: a first extracellular antigen-binding domain, a first transmembrane domain, and a first intracellular signal transduction domain containing at least one anti-CLDN18.2 binding portion, and (2) the second engineered receptor specifically targeting GUCY2C comprises: a second extracellular antigen-binding domain, a second transmembrane domain, and a second intracellular signal transduction domain containing at least one anti-GUCY2C binding portion.

6. The method of claim 5, wherein the engineered immune cells have been transduced by (1) two separate vectors, the first vector containing nucleic acid encoding the first engineered receptor specifically targeting CLDN18.2, and the second vector containing nucleic acid encoding the second engineered receptor specifically targeting GUCY2C; or (2) transduced by a vector containing nucleic acid encoding the first engineered receptor specifically targeting CLDN18.2, the first engineered receptor being operatively linked to the second engineered receptor specifically targeting GUCY2C.

7. The method of any one of claims 1-3, wherein the CLDN18.2 and GUCY2C antagonist is an engineered immune cell comprising an engineered receptor co-targeting CLDN18.2 and GUCY2C, wherein the engineered receptor comprises: an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain containing at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety.

8. The method of any one of claims 3-7, wherein the engineered receptor is selected from the group consisting of engineered T-cell receptors (TCRs), chimeric antigen receptors (CARs), T-cell antigen conjugates (TACs), or portions thereof.

9. The method of any one of claims 5 or 8, wherein the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising a first CAR targeting CLDN18.2 and a second CAR targeting GUCY2C, wherein (1) the first CAR targeting CLDN18.2 comprises: a first extracellular antigen-binding domain, a first transmembrane domain, and a first intracellular signal transduction domain containing at least one anti-CLDN18.2 binding portion; (2) the second CAR targeting GUCY2C comprises: a second extracellular antigen-binding domain, a second transmembrane domain, and a second intracellular signal transduction domain containing at least one anti-GUCY2C binding portion; and wherein the first CAR targeting CLDN18.2 is operatively connected to the second CAR targeting GUCY2C via a cleavable connector, or the first CAR and the second CAR are not connected due to cleavage of the cleavable connector.

10. The method of any one of claims 7-8, wherein the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising a CAR ("CLDN18.2×GUCY2C-specific CAR") co-targeting CLDN18.2 and GUCY2C, wherein the CLDN18.2×GUCY2C-specific CAR comprises: (1) an extracellular antigen-binding domain containing at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety, (2) a transmembrane domain and (3) an intracellular signal transduction domain; and wherein the anti-CLDN18.2 binding moiety is located at the N-terminus or C-terminus of the anti-GUCY2C binding moiety, optionally the anti-CLDN18.2 binding moiety being operatively linked to the anti-GUCY2C binding moiety via a peptide linker such as a GS linker, for example (G4S)3.

11. The method according to any one of claims 4-10, wherein the anti-CLDN18.2 binding portion and / or the anti-GUCY2C binding portion is selected from Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), mini antibody, biantibody, single-domain antibody (sdAb) or VHH domain.

12. The method of any one of claims 4-11, wherein the anti-CLDN18.2 binding portion is anti-CLDN18.2 VHH, optionally the anti-CLDN18.2 VHH comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequence of SEQ ID NO: 1, and optionally the anti-CLDN18.2 VHH comprises (i) CDR1 containing the amino acid sequence of SEQ ID NO: 23, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 24, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO:

25.

13. The method of claim 12, wherein the anti-CLDN18.2 VHH comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO:

1.

14. The method of any one of claims 4-13, wherein the anti-GUCY2C binding moiety is anti-GUCY2C VHH, optionally the anti-GUCY2C VHH comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequence of SEQ ID NO: 2, and optionally the anti-GUCY2C VHH comprises (i) CDR1 containing the amino acid sequence of SEQ ID NO: 26, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 27, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO:

28.

15. The method of claim 14, wherein the anti-GUCY2C VHH comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO:

2.

16. The method of any one of claims 4-15, wherein the transmembrane domain is derived from a protein selected from the group consisting of: CD8α, ICOS, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

17. The method of any one of claims 4-16, wherein the intracellular signal transduction domain comprises a primary intracellular signal transduction domain of an immune cell, optionally said primary intracellular signal transduction domain being derived from CD3ζ.

18. The method of any one of claims 4-17, wherein the intracellular signal transduction domain comprises a co-stimulatory signal transduction domain.

19. The method of claim 18, wherein the co-stimulatory signal transduction domain is derived from a co-stimulatory molecule selected from the group consisting of: CD28, 4-1BB (CD137), CD27, OX40, CD30, CD40, CD3, LFA-1, ICOS (CD278), NTBA, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 ligands, and combinations thereof.

20. The method of any one of claims 8-9 and 11-19, wherein the CAR specifically targeting CLDN18.2 comprises: (1) A hinged domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain, optionally wherein the hinged domain is derived from CD8α or CD28; and / or (2) The signal peptide located at the N-terminus of the extracellular antigen-binding domain. Optionally, the signal peptide is derived from CD8α.

21. The method of any one of claims 8-9 and 11-20, wherein the CAR specifically targeting GUCY2C comprises: (1) A hinged domain located between the N-terminus of the extracellular antigen-binding domain and the transmembrane domain, optionally wherein the hinged domain is derived from CD8α or CD28; and / or (2) A signal peptide located at the N-terminus of the extracellular antigen-binding domain, wherein the signal peptide is optionally derived from CD8α.

22. The method of any one of claims 8-9 and 11-21, wherein the CAR specifically targeting CLDN18.2 comprises the amino acid sequence shown in any one of SEQ ID NO: 12 and 19; and / or wherein the CAR specifically targeting GUCY2C comprises the amino acid sequence shown in any one of SEQ ID NO: 13 and 21-22.

23. The method according to any one of claims 1-22, wherein: (1) The CLDN18.2 antagonist is an engineered immune cell comprising a polypeptide containing an amino acid sequence shown in either SEQ ID NO: 12 or 19, and / or (2) The GUCY2C antagonist is an engineered immune cell containing a polypeptide containing any one of the amino acid sequences shown in SEQ ID NO:13 and 21-22.

24. The method of any one of claims 1-23, wherein the combination of the CLDN18.2 antagonist and the GUCY2C antagonist comprises a first group of engineered immune cells and a second group of engineered immune cells, the first group of engineered immune cells comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 12, and the second group of engineered immune cells comprising a polypeptide containing the amino acid sequence of SEQ ID NO:

13.

25. The method of any one of claims 1-24, wherein the ratio of the CLDN18.2 antagonist and the GUCY2C antagonist in the combination is in the range of 10:1 to 1:10, such as 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 or 1:

9.

26. The method of any one of claims 10-19, wherein the CAR co-targeting CLDN18.2 and GUCY2C comprises: (1) A hinged domain located between the N-terminus of the extracellular antigen-binding domain and the transmembrane domain, optionally wherein the hinged domain is derived from CD8α or CD28; and / or (2) A signal peptide located at the N-terminus of the extracellular antigen-binding domain, wherein the signal peptide is optionally derived from CD8α.

27. The method of claim 26, wherein the CAR co-targeting CLDN18.2 and GUCY2C comprises the amino acid sequence shown in any one of SEQ ID NO: 14-15.

28. The method of any one of claims 1-27, wherein the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell, the engineered immune cell comprising: (1) A polypeptide containing any one of the amino acid sequences shown in SEQ ID NO: 14-18; or (2) A first polypeptide and a second polypeptide, wherein the first polypeptide comprises an amino acid sequence shown in any one of SEQ ID NO: 12 and 19, and the second polypeptide comprises an amino acid sequence shown in any one of SEQ ID NO: 13 and 20-22.

29. The method of any one of claims 2-28, wherein the engineered immune cells are selected from the group consisting of: T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof.

30. The method according to any one of claims 1-29, wherein the tumor is selected from gastric cancer, esophageal cancer, pancreatic ductal carcinoma, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colorectal cancer, liver cancer, head and neck cancer, gallbladder cancer and their metastases.

31. The method of any one of claims 1-30, wherein the subject is resistant to at least one CLDN18.2 agent; and / or wherein the subject is resistant to at least one GUCY2C agent.

32. A multispecific CAR construct in combination with CLDN18.2 and GUCY2C, the multispecific CAR construct comprising: (a) A polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain containing at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety; or (b) A first polypeptide and a second polypeptide, the first polypeptide comprising a first extracellular antigen-binding domain, a first transmembrane domain and a first intracellular signal transduction domain containing at least one anti-CLDN18.2 binding moiety, the second polypeptide comprising a second extracellular antigen-binding domain, a second transmembrane domain and a second intracellular signal transduction domain containing at least one anti-GUCY2C binding moiety, optionally the first polypeptide and the second polypeptide are linked into a chain via a cleavable linker, optionally the first transmembrane domain and the second transmembrane domain and the first intracellular signal transduction domain and the second intracellular signal transduction domain are the same or different.

33. The multispecific CAR construct of claim 32, wherein the anti-CLDN18.2 binding portion is located at the N-terminus or C-terminus of the anti-GUCY2C binding portion, optionally the anti-CLDN18.2 binding portion is operatively linked to the anti-GUCY2C binding portion via a peptide linker, such as a GS linker, for example (G4S)3.

34. The multispecific CAR construct according to any one of claims 32-33, wherein the anti-CLDN18.2 binding portion and / or the anti-GUCY2C binding portion is selected from Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), mini antibody, biantibody, single-domain antibody (sdAb) or VHH domain.

35. The multispecific CAR construct according to any one of claims 32-34, wherein the anti-CLDN18.2 binding moiety is anti-CLDN18.2 VHH, optionally the anti-CLDN18.2 VHH comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequence of SEQ ID NO: 1, and optionally the anti-CLDN18.2 VHH comprises (i) CDR1 containing the amino acid sequence of SEQ ID NO: 23, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 24, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO:

25.

36. The multispecific CAR construct of claim 35, wherein the anti-CLDN18.2 VHH comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO:

1.

37. The multispecific CAR construct according to any one of claims 32-36, wherein the anti-GUCY2C binding moiety is anti-GUCY2C VHH, optionally the anti-GUCY2C VHH comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequence of SEQ ID NO: 2, and optionally the anti-GUCY2C VHH comprises (i) CDR1 containing the amino acid sequence of SEQ ID NO: 26, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 27, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO:

28.

38. The multispecific CAR construct of claim 37, wherein the anti-GUCY2C VHH comprises the amino acid sequence of SEQ ID NO:2 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO:

2.

39. The multispecific CAR construct according to any one of claims 32-38, wherein the transmembrane domain is derived from a protein selected from the group consisting of: CD8α, ICOS, CD4, CD28, CD137, CD80, CD86, CD152 and PD1.

40. The multispecific CAR construct of claim 39, wherein the transmembrane domain is derived from CD8α or ICOS, and optionally the transmembrane domain comprises an amino acid sequence shown in any one of SEQ ID NO: 5-6 or an amino acid sequence that is at least 85%, 90%, or 95% identical to an amino acid sequence shown in any one of SEQ ID NO: 5-6.

41. The multispecific CAR construct according to any one of claims 32-40, wherein the intracellular signal transduction domain comprises a primary intracellular signal transduction domain; optionally, the primary intracellular signal transduction domain is derived from CD3ζ, and optionally, the primary intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO:

11.

42. The multispecific CAR construct according to any one of claims 32-41, wherein the intracellular signal transduction domain further comprises at least one co-stimulatory signal transduction domain.

43. The multispecific CAR construct of claim 42, wherein the co-stimulatory signal transduction domain is derived from a co-stimulatory molecule selected from CD28, 4-1BB (CD137), CD27, OX40, CD30, CD40, CD3, LFA-1, ICOS (CD278), NTBA, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 ligand, and combinations thereof.

44. The multispecific CAR construct of claim 42 or 43, wherein the co-stimulatory signal transduction domain is derived from 4-1BB, CD28, ICOS, or NTBA. Optionally, the co-stimulatory signal transduction domain comprises any one of the amino acid sequences of SEQ ID NO: 7-10 or an amino acid sequence that is at least 85%, 90%, or 95% identical to any one of SEQ ID NO: 7-10.

45. The multispecific CAR construct of any one of claims 32-44, wherein the multispecific CAR construct further comprises: (1) A hinged domain located between the N-terminus of the extracellular antigen-binding domain and the transmembrane domain, optionally wherein the hinged domain is derived from CD8α or CD28; and / or (2) A signal peptide located at the N-terminus of the extracellular antigen-binding domain, wherein the signal peptide is optionally derived from CD8α.

46. ​​The multispecific CAR construct according to any one of claims 32-45, wherein the polypeptide of the multispecific CAR construct of (a) comprises an amino acid sequence shown in any one of SEQ ID NO: 14-15, and / or The first polypeptide of the multispecific CAR construct of (b) comprises the amino acid sequence shown in any one of SEQ ID NO: 12 and 19, and the second polypeptide of the multispecific CAR construct of (b) comprises the amino acid sequence shown in any one of SEQ ID NO: 13 and 20-22.

47. A combination of a CLDN18.2 antagonist and a GUCY2C antagonist, wherein the antagonist is selected from antibodies, aptamers, antibody-drug conjugates (ADCs), small RNAs, engineered receptors, and engineered immune cells.

48. The combination of claim 47, wherein the combination of the CLDN18.2 antagonist and the GUCY2C antagonist comprises a first group of engineered immune cells and a second group of engineered immune cells, wherein: (1) The CLDN18.2 antagonist is a first group of engineered immune cells comprising an engineered receptor that specifically targets CLDN18.2, wherein the engineered receptor comprises: an extracellular antigen-binding domain containing at least one anti-CLDN18.2 binding moiety, a transmembrane domain, and optionally an intracellular signal transduction domain; and / or (2) The GUCY2C antagonist is a second group of engineered immune cells containing an engineered receptor that specifically targets GUCY2C, wherein the engineered receptor comprises: an extracellular antigen-binding domain, a transmembrane domain, and optionally an intracellular signal transduction domain containing at least one anti-GUCY2C binding moiety.

49. The combination of claim 47 or 48, wherein the engineered receptor specifically targeting CLDN18.2 and the engineered receptor specifically targeting GUCY2C are expressed in the different engineered immune cells.

50. The combination of any one of claims 47-49, wherein the engineered receptor is selected from the group consisting of engineered T-cell receptors (TCRs), chimeric antigen receptors (CARs), T-cell antigen conjugates (TACs), or portions thereof.

51. The combination of claim 50, wherein the engineered receptor specifically targeting CLDN18.2 is a CAR specifically targeting CLDN18.2, and / or the engineered receptor specifically targeting GUCY2C is a CAR specifically targeting GUCY2C.

52. The combination of any one of claims 48-51, wherein the anti-CLDN18.2 binding portion and / or the anti-GUCY2C binding portion is selected from Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), mini antibody, biantibody, single-domain antibody (sdAb) or VHH domain.

53. The combination of claim 52, wherein the anti-CLDN18.2 binding portion is anti-CLDN18.2 VHH, optionally the anti-CLDN18.2 VHH comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequence of SEQ ID NO: 1, and optionally the anti-CLDN18.2 VHH comprises (i) CDR1 containing the amino acid sequence of SEQ ID NO: 23, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 24, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO:

25.

54. The combination of claim 53, wherein the anti-CLDN18.2 VHH comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO:

1.

55. The combination of any one of claims 52-54, wherein the anti-GUCY2C binding portion is anti-GUCY2C VHH, optionally the anti-GUCY2C VHH comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequence of SEQ ID NO: 2, and optionally the anti-GUCY2C VHH comprises (i) CDR1 containing the amino acid sequence of SEQ ID NO: 26, (ii) CDR2 containing the amino acid sequence of SEQ ID NO: 27, and (iii) CDR3 containing the amino acid sequence of SEQ ID NO:

28.

56. The combination of claim 55, wherein the anti-GUCY2C VHH comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO:

2.

57. The combination of any one of claims 50-56, wherein the transmembrane domain of the CAR specifically targeting CLDN18.2 and the transmembrane domain of the CAR specifically targeting GUCY2C are derived from proteins selected from the group consisting of: CD8α, ICOS, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. Optionally, the transmembrane domain of the CAR specifically targeting CLDN18.2 and the transmembrane domain of the CAR specifically targeting GUCY2C are derived from CD8α or ICOS and contain an amino acid sequence shown in any one of SEQ ID NO: 5-6 or an amino acid sequence that is at least 85%, 90%, or 95% identical to an amino acid sequence shown in any one of SEQ ID NO: 5-6.

58. The combination of any one of claims 50-57, wherein the intracellular signal transduction domain of the CAR specifically targeting CLDN18.2 and / or the intracellular signal transduction domain of the CAR specifically targeting GUCY2C comprises a primary intracellular signal transduction domain. Optionally, the primary intracellular signal transduction domain is derived from CD3ζ. Optionally, the primary intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO:

11.

59. The combination of any one of claims 50-58, wherein the intracellular signal transduction domain of the CAR specifically targeting CLDN18.2 and the intracellular signal transduction domain of the CAR specifically targeting GUCY2C further comprise a co-stimulatory signal transduction domain.

60. The combination of claim 59, wherein the co-stimulatory signal transduction domain is derived from a co-stimulatory molecule selected from CD28, 4-1BB (CD137), CD27, OX40, CD30, CD40, CD3, LFA-1, ICOS (CD278), NTBA, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 ligand, and combinations thereof.

61. The combination of any one of claims 50-60, wherein the CAR specifically targeting CLDN18.2 comprises (1) a hinge domain located between the N-terminus of the extracellular antigen-binding domain and the transmembrane domain, optionally wherein the hinge domain is derived from CD8α or CD28; and / or (2) a signal peptide located at the N-terminus of the extracellular antigen-binding domain, optionally wherein the signal peptide is derived from CD8α.

62. The combination of any one of claims 50-61, wherein the CAR specifically targeting GUCY2C comprises (1) a hinge domain located between the N-terminus of the extracellular antigen-binding domain and the transmembrane domain, optionally wherein the hinge domain is derived from CD8α or CD28; and / or (2) a signal peptide located at the N-terminus of the extracellular antigen-binding domain, optionally wherein the signal peptide is derived from CD8α.

63. The combination of any one of claims 50-62, wherein the CAR specifically targeting CLDN18.2 comprises the amino acid sequence shown in any one of SEQ ID NO: 12 and 19, and / or the CAR specifically targeting GUCY2C comprises the amino acid sequence shown in any one of SEQ ID NO: 13 and 20-22.

64. The combination of any one of claims 47-63, wherein the CLDN18.2 antagonist is a first group of engineered immune cells comprising a polypeptide containing an amino acid sequence shown in any one of SEQ ID NO: 12 and 19, and / or the GUCY2C antagonist is a second group of engineered immune cells comprising a polypeptide containing an amino acid sequence shown in any one of SEQ ID NO: 13 and 20-22.

65. The combination of claim 64, wherein the CLDN18.2 antagonist is a first group of engineered immune cells comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 12, and the GUCY2C antagonist is a second group of engineered immune cells comprising a polypeptide containing the amino acid sequence of SEQ ID NO:

13.

66. The combination of any one of claims 47-65, wherein the combination comprises the CLDN18.2 antagonist and the GUCY2C antagonist in a ratio ranging from 10:1 to 1:10, such as 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 or 1:

9.

67. The combination of any one of claims 47-66, wherein the engineered immune cells are selected from the group consisting of: T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof.

68. A nucleic acid, said nucleic acid comprising: (1) Encoding the nucleic acid sequence of the multispecific CAR construct as described in any one of claims 32-46; or (2) A first nucleic acid sequence encoding a first engineered receptor specifically targeting CLDN18.2, the first engineered receptor comprising: a first extracellular antigen-binding domain, a first transmembrane domain, and a first intracellular signal transduction domain containing at least one anti-CLDN18.2 binding moiety; and a second nucleic acid sequence encoding a second engineered receptor specifically targeting GUCY2C, the second engineered receptor comprising: a second extracellular antigen-binding domain, a second transmembrane domain, and a second intracellular signal transduction domain containing at least one polypeptide containing the GUCY2C binding moiety. Optionally, the first nucleic acid sequence and the second nucleic acid sequence are linked by a nucleic acid sequence encoding a self-cleavable peptide (such as P2A, E2A, F2A or T2A).

69. The nucleic acid of claim 68, comprising: (1) A nucleic acid sequence encoding a polypeptide containing any one of the amino acid sequences shown in SEQ ID NO: 14-18; or (2) A first nucleic acid sequence encoding a first polypeptide containing an amino acid sequence shown in any one of SEQ ID NO: 12 and 19, and a second nucleic acid sequence encoding a second polypeptide containing an amino acid sequence shown in any one of SEQ ID NO: 13 and 20-22.

70. A vector comprising the nucleic acid as described in claim 68 or 69.

71. An engineered immune cell comprising a multispecific CAR construct as described in any one of claims 32-46, a nucleic acid as described in any one of claims 68-69, or a vector as described in claim 70.

72. The engineered immune cell of claim 71, wherein the engineered immune cell comprises an engineered receptor specifically targeting CLDN18.2, the engineered receptor comprising: an extracellular antigen-binding domain, a transmembrane domain, and optionally an intracellular signal transduction domain containing at least one anti-CLDN18.2 binding moiety; and an engineered receptor specifically targeting GUCY2C, the engineered receptor comprising: an extracellular antigen-binding domain, a transmembrane domain, and optionally an intracellular signal transduction domain containing at least one anti-GUCY2C binding moiety.

73. The engineered immune cell of claim 71 or 72, wherein the engineered immune cell comprises: (1) A polypeptide containing any one of the amino acid sequences shown in SEQ ID NO: 14-18; or (2) A first polypeptide and a second polypeptide, wherein the first polypeptide comprises an amino acid sequence shown in any one of SEQ ID NO: 12 and 19, and the second polypeptide comprises an amino acid sequence shown in any one of SEQ ID NO: 13 and 20-22.

74. The engineered immune cell of claim 73, wherein the engineered immune cell is derived from the group consisting of: T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof.

75. A pharmaceutical composition comprising a multispecific CAR construct as described in any one of claims 32-46, a combination as described in any one of claims 47-67, a nucleic acid as described in any one of claims 68-69, or an engineered immune cell as described in any one of claims 72-74, and a pharmaceutically acceptable carrier.

76. The multispecific CAR construct of any one of claims 32-46, the combination of any one of claims 47-67, the nucleic acid of any one of claims 68-69, or the engineered immune cell of any one of claims 72-74, for use in treating a subject’s tumor, optionally wherein the tumor is CLDN18.2 positive and / or GUCY2C positive.

77. The multispecific CAR construct, combination, nucleic acid, or engineered immune cell for use as described in claim 76, wherein the tumor is selected from gastric cancer, esophageal cancer, pancreatic ductal carcinoma, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colorectal cancer, liver cancer, head and neck cancer, gallbladder cancer, and their metastases.

78. The multispecific CAR construct, combination, nucleic acid, or engineered immune cell for use as described in claim 76, wherein the subject is resistant to at least one CLDN18.2 agent; and / or wherein the subject is resistant to at least one GUCY2C agent.

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