WO2025016450A1

WO2025016450A1 - Combination of cldn18.2 and gucy2c targeted antagonist therapy

Info

Publication number: WO2025016450A1
Application number: PCT/CN2024/106351
Authority: WO
Inventors: Qiuchuan ZHUANG; Jie Mao; Hong Tang; Langyu XU; Hefei HOU; Ruixue WANG; Lian Ma; Dan Zhao
Original assignee: Nanjing Legend Biotechnology Co Ltd; Legend Biotech Ireland Ltd
Current assignee: Nanjing Legend Biotechnology Co Ltd; Legend Biotech Ireland Ltd
Priority date: 2023-07-19
Filing date: 2024-07-19
Publication date: 2025-01-23
Anticipated expiration: 2026-01-19

Abstract

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

Description

Combination of CLDN18.2 and GUCY2C Targeted Antagonist Therapy

CROSS REFERENCES

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

SEQUENCE LISTING

The instant application contains a sequence listing which has been submitted electronically and is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

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

BACKGROUND

CLDN18.2 (Claudin18.2) is an important target for the prevention and treatment of primary tumors such as gastric cancer, esophageal cancer, pancreatic ductal cancer, lung cancer such as non-small cell lung cancer (NSCLC) , ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer and its metastasis, especially Krukenberg's Tumour, peritoneal metastasis, lymph node metastasis and other metastatic gastric cancer. Ugur Sahin et al. (2008) reported that 96%of gastric cancer patients (46/48 cases) , 63%of pancreatic ductal cancer patients (7/11 cases) , 60%of esophageal cancer (6/10 cases) , and 41%of non-small cell lung cancer (30/73 cases) had CLDN18.2 positive tissue samples (mRNA, RT-PCR method) .

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

GCC is mainly associated with intestinal diseases, including functional gastrointestinal diseases (such as irritable bowel syndrome, constipation) , inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis) , and cancer (gastrointestinal cancer) . GCC is stably expressed in primary colorectal cancer, while it is abnormally highly expressed in metastatic colorectal cancer, which is considered to be a specific biomarker for metastatic colorectal cancer and can be used for identification, staging, and as the strongest independent prognostic marker for colon cancer patients. Stansa Biotechnology's GCC&CD19 coupled CAR-T has achieved an ORR of 50%in Phase I clinical trials, with a mOS of 12-17 months. According to the standard algorithm for CRS and diarrhea, this therapy has good tolerability and toxicity with appropriate intervention. The results of these human clinical trials indicate that immune therapy targeting GCC is safe, controllable, and promising.

There are currently several CAR-T products that target CLDN18.2, but their mPFS is relatively short. For example, the mPFS of CT041 from CStone Pharmaceuticals is only 4.2 months (Nat Med. 2022 Jun; 28 (6) : 1189-1198) . Antigen loss and tumor heterogeneity may be one of the main reasons for tumor recurrence and disease progression.

SUMMARY

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

The CLDN18.2 antagonist, the GUCY2C antagonist, and/or the antagonist of CLDN18.2 and GUCY2C may have a variety of categories, e.g., the antagonist may be selected from an engineered receptor, an engineered immune cell, an antibody, an antibody-drug conjugate (ADC) , an aptamer and small RNAs. In some embodiments, the engineered receptor is selected from the group consisting of an engineered T cell receptor (TCR) , a chimeric antigen receptor (CAR) , a T cell antigen coupler (TAC) or a portion thereof.

In some embodiments, the CLDN18.2 antagonist is an engineered immune cell comprising an engineered receptor specifically targeting CLDN18.2 comprising: an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the GUCY2C antagonist is an engineered immune cell comprising an engineered receptor specifically targeting GUGY2C comprising: an extracellular antigen binding domain comprising at least one anti-GUCY2C binding moiety, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the engineered receptor specifically targeting CLDN18.2 and the engineered receptor specifically targeting GUGY2C are expressed in different engineered immune cells. Correspondingly, the combination of the CLDN18.2 antagonist and the GUCY2C antagonist is a combination of a first group of engineered immune cell comprising an engineered receptor specifically targeting CLDN18.2 as described above, and a second group of engineered immune cell comprising an engineered receptor specifically targeting GUGY2C as described above.

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 GUGY2C, i.e. the two engineered receptors are co-expressed in the same immune cells. 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 GUGY2C, wherein (1) the first engineered receptor specifically targeting CLDN18.2 comprising: an first extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a first transmembrane domain, and a first intracellular signaling domain, and (2) the second engineered receptor specifically targeting GUGY2C comprising: a second extracellular antigen binding domain comprising at least one anti-GUGY2C binding moiety, a second transmembrane domain, and a second intracellular signaling domain.

In some embodiments, the engineered immune cells co-expressing the first and the second engineered receptors have been transduced by two separate vectors, the first vector comprises a nucleic acid encoding the first engineered receptor specifically targeting CLDN18.2, and the second vector comprises a nucleic acid encoding the second engineered receptor specifically targeting GUGY2C.

In some embodiments, the engineered immune cells co-expressing the first and the second engineered receptors have been transduced by a vector that comprises a nucleic acid encoding the first engineered receptor specifically targeting CLDN18.2 operably linked to the second engineered receptor specifically targeting GUGY2C. In some embodiments, the engineered immune cells express the first engineered receptor specifically targeting CLDN18.2 operably linked to the second engineered receptor specifically targeting GUGY2C via a cleavable linker, where the cleavable linker can be easily cleaved under suitable conditions. In some embodiments, the cleavable linker is selected from P2A, T2A, E2A and F2A.

In some embodiments, the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising a first CAR targeting CLDN18.2 (single-CLDN18.2 specific CAR) and a second CAR targeting GUGY2C (single-GUCY2C specific CAR) , wherein (1) the first CAR comprises a first extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a first transmembrane domain and an first intracellular signaling domain, (2) the second CAR comprises a second extracellular antigen binding domain comprising at least one anti-GUGY2C binding moiety, a second transmembrane domain and a second intracellular signaling domain; and wherein the first CAR is operably linked to the second CAR, the first CAR is located at the N-terminus or C-terminus of the second CAR targeting GUGY2C, optionally the first CAR is operably linked to the second CAR via a cleavable linker or the first CAR and the second CAR are not linked due to the cleavage of the cleavable linker.

In some embodiments, the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising an engineered receptor co-targeting CLDN18.2 and GUGY2C comprising: an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety, a transmembrane domain, and an intracellular signaling domain. The engineered receptor may be selected from the group consisting of an engineered T cell receptor (TCR) , a chimeric antigen receptor (CAR) , a T cell antigen coupler (TAC) or a portion thereof.

In some embodiments, the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising a CAR co-targeting CLDN18.2 and GUGY2C ( “CLDN18.2×GUCY2C specific CAR” ) , wherein the CLDN18.2×GUCY2C specific CAR comprises: (1) an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety, (2) a transmembrane domain, and (3) an intracellular signaling domain; and wherein the anti-CLDN18.2 binding moiety is located at the N-terminus or C-terminus of the anti-GUGY2C binding moiety, optionally the anti-CLDN18.2 binding moiety is operably linked to the anti-GUCY2C binding moiety via a peptide linker, such as a GS linker e.g. (G₄S) ₃.

In some embodiments, the anti-CLDN18.2 binding moiety and anti-GUCY2C binding moiety are selected from a Fab, a Fab’ , a F (ab’ ) 2, an Fv, a single-chain Fv (scFv) , minibody, a diabody, a single-domain antibody (sdAb) or VHH domain.

In some embodiments, the anti-CLDN18.2 binding moiety is an anti-CLDN18.2 VHH, optionally the anti-CLDN18.2 VHH comprises a CDR1, CDR2, and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 1, and optionally the anti-CLDN18.2 VHH comprises (i) a CDR1 comprises the amino acid sequence of SEQ ID NO: 23, (ii) a CDR2 comprises the amino acid sequence of SEQ ID NO: 24, and (iii) a CDR3 comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the anti-CLDN18.2 VHH comprises an amino acid sequence of SEQ ID NO: 1 or 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 the same CDRs as those of SEQ ID NO: 1 and having framework regions at least 85%, 90%or 95%identical to those of SEQ ID NO: 1.

In some embodiments, the anti-GUCY2C binding moiety is an anti-GUCY2C VHH, optionally the anti-GUCY2C VHH comprises a CDR1, CDR2, and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 2, and optionally the anti-GUCY2C VHH comprises (i) a CDR1 comprises the amino acid sequence of SEQ ID NO: 26, (ii) a CDR2 comprises the amino acid sequence of SEQ ID NO: 27, and (iii) a CDR3 comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-GUCY2C VHH comprises an amino acid sequence of SEQ ID NO: 2 or 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 the same CDRs as those of SEQ ID NO: 2 and having framework regions at least 85%, 90%or 95%identical to those of SEQ ID NO: 2.

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

In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune cell, optionally the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. The co-stimulatory signaling 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, ligands of CD83 and combinations thereof.

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

(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 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 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

In some embodiments, the CAR specifically targeting CLDN18.2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 12 and 19. In some embodiments, the CAR specifically targeting GUCY2C comprises an amino acid sequence set forth in any one of SEQ ID NOs: 13 and 21-22.

In some embodiments, the CLDN18.2 antagonist is an engineered immune cell comprising a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 12 and 19. In some embodiments, the GUCY2C antagonist is an engineered immune cell comprising a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 13 and 21-22. In some embodiments, the combination of a CLDN18.2 antagonist and a GUCY2C antagonist comprises or consists of a first group of engineered immune cells comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 12, and a second group of engineered immune cells comprising a polypeptide comprising 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 are in the 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.

In some embodiments, the CAR co-targeting CLDN18.2 and GUCY2C comprises:

In some embodiments, the CAR co-targeting CLDN18.2 and GUCY2C comprises an amino acid sequence set forth in any one of SEQ ID NOs: 14-15. In some embodiments, the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising:

(1) a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 14-18; or

(2) a first polypeptide and a second polypeptide, wherein the first polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 12 and 19, and the second polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 13 and 20-22.

In some embodiments, the engineered immune cell is selected from the group consisting of T cell, NK cell, peripheral blood mononuclear cell (PBMC) , hematopoietic stem cell, pluripotent stem cell, an embryonic stem cell, and a combination thereof.

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

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.

In one aspect, provided herein is a multi-specific chimeric antigen receptor (CAR) construct that binds to CLDN18.2 and GUCY2C ( “CLDN18.2×GUCY2C specific CAR” ) , comprising an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety, a transmembrane domain and an intracellular signaling domain.

In some embodiments, the multi-specific chimeric antigen receptor (CAR) construct that binds to CLDN18.2 and GUCY2C, comprising:

(a) a polypeptide comprising an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety, a transmembrane domain and an intracellular signaling domain; or

(b) a first polypeptide comprising a first extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a first transmembrane domain and a first intracellular signaling domain, and a second polypeptide comprising a second extracellular antigen binding domain comprising at least one anti-GUCY2C binding moiety, a second transmembrane domain and a second intracellular signaling domain, optionally the first polypeptide and the second polypeptide are linked in one chain via a cleavable linker, optionally the first and second transmembrane domains as well as the first and second intracellular signaling domains are same or different, respectively.

In some embodiments, the anti-CLDN18.2 binding moiety is located at the N-terminus or C-terminus of the anti-GUGY2C binding moiety, optionally the anti-CLDN18.2 binding moiety is operably linked to the anti-GUCY2C binding moiety via a peptide linker, such as a GS linker e.g. (G₄S) ₃. The anti-CLDN18.2 binding moiety and/or the anti-GUCY2C binding moiety may be selected from a Fab, a Fab’ , a F (ab’ ) 2, an Fv, a single-chain Fv (scFv) , minibody, a diabody, a single-domain antibody (sdAb) or VHH domain.

In some embodiments, the anti-CLDN18.2 binding moiety is an anti-CLDN18.2 VHH, optionally the anti-CLDN18.2 VHH comprises a CDR1, CDR2, and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 1, and optionally the anti-CLDN18.2 VHH comprises (i) a CDR1 comprises the amino acid sequence of SEQ ID NO: 23, (ii) a CDR2 comprises the amino acid sequence of SEQ ID NO: 24, and (iii) a CDR3 comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the anti-CLDN18.2 VHH comprises an amino acid sequence of SEQ ID NO: 1 or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the anti-GUCY2C binding moiety is an anti-GUCY2C VHH, optionally the anti-GUCY2C VHH comprises a CDR1, CDR2, and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 2, and optionally the anti-GUCY2C VHH comprises (i) a CDR1 comprises the amino acid sequence of SEQ ID NO: 26, (ii) a CDR2 comprises the amino acid sequence of SEQ ID NO: 27, and (iii) a CDR3 comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-GUCY2C VHH comprises an amino acid sequence of SEQ ID NO: 2 or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 2.

The transmembrane domain may be derived from a protein 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, optionally the transmembrane domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 5-6 or at least 85%, 90%or 95%identical to the amino acid sequence set forth in any one of SEQ ID NOs: 5-6.

In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain; optionally the primary intracellular signaling domain is derived from CD3ζ, optionally the primary intracellular signaling domain comprises an amino acid sequence of SEQ ID NO: 11 or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 11. The intracellular signaling domain may further comprise at least one co-stimulatory signaling domain. The co-stimulatory signaling domain may be 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, ligands of CD83 and combinations thereof. In some embodiments, the co-stimulatory signaling domain is derived from 4-1BB, CD28, ICOS or NTBA, optionally, the co-stimulatory signaling domain comprises any of the amino acid sequences of SEQ ID NOs: 7-10 or an amino acid sequence at least 85%, 90%or 95%identical to any of SEQ ID NOs: 7-10.

In some embodiments, the multi-specific CAR construct further comprises:

In some embodiments, the polypeptide of the multi-specific CAR construct of (a) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 14-15, and/or the first polypeptide of the multi-specific CAR construct of (b) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 12 and 19, and the second polypeptide of the multi-specific CAR construct of (b) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 13 and 20-22.

In one aspect, provided herein is a combination of a CLDN18.2 antagonist and a GUCY2C antagonist, wherein the antagonist is selected from an antibody, an aptamer, an antibody-drug conjugate (ADC) , a small RNA, an engineered receptor and an engineered immune cell.

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

(1) the CLDN18.2 antagonist is the first group of engineered immune cell comprising an engineered receptor specifically targeting CLDN18.2 comprising: an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a transmembrane domain, and optionally an intracellular signaling domain; and/or

(2) the GUCY2C antagonist is the second group of engineered immune cell comprising an engineered receptor specifically targeting GUGY2C comprising: an extracellular antigen binding domain comprising 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 GUGY2C may be expressed in different engineered immune cells.

The engineered receptor may be selected from the group consisting of an engineered T cell receptor (TCR) , a chimeric antigen receptor (CAR) , a T cell antigen coupler (TAC) or a portion 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.

In some embodiments, the anti-CLDN18.2 binding moiety and/or the anti-GUCY2C binding moiety are selected from a Fab, a Fab’ , a F (ab’ ) 2, an Fv, a single-chain Fv (scFv) , minibody, a diabody, a single-domain antibody (sdAb) or VHH domain.

The transmembrane domain of the CAR specifically targeting CLDN18.2 and the CAR specifically targeting GUCY2C may be derived from a protein 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 CAR specifically targeting GUCY2C are derived from CD8α or ICOS, and comprise an amino acid sequence set forth in any one of SEQ ID NOs: 5-6 or at least 85%, 90%or 95%identical to the amino acid sequence set forth in any one of SEQ ID NOs: 5-6.

In some embodiments, the intracellular signaling domain of the CAR specifically targeting CLDN18.2 and/or the CAR specifically targeting GUCY2C comprise a primary intracellular signaling domain, optionally, the primary intracellular signaling domain is derived from CD3ζ, optionally, the primary intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence at least 85%, 90%or 95%identical to SEQ ID NO: 11. In some embodiments, the intracellular signaling domain of the CAR specifically targeting CLDN18.2 and the CAR specifically targeting GUCY2C further comprises a co-stimulatory signaling domain. The co-stimulatory signaling domain may be 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, ligands of CD83 and combinations thereof.

In some embodiments, the CAR specifically targeting CLDN18.2 comprise (1) a hinge domain located between 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. In some embodiments, the CAR specifically targeting GUCY2C comprises (1) a hinge domain located between 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.

In some embodiments, the CAR specifically targeting CLDN18.2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 12 and 19, and/or the CAR specifically targeting GUCY2C comprises an amino acid sequence set forth in any one of SEQ ID NOs: 13 and 19-22.

In some embodiments, the CLDN18.2 antagonist is a first group of engineered immune cell comprising a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 12 and 19, and/or the GUCY2C antagonist is a second group of engineered immune cell comprising a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 13 and 19-22. In some embodiments, the CLDN18.2 antagonist is a first group of engineered immune cell comprising a polypeptide comprising an amino acid sequence of SEQ ID NO: 12, and the GUCY2C antagonist is a second group of engineered immune cell comprising a polypeptide comprising an amino acid sequence of SEQ ID NO: 13.

In some embodiments, the combination comprises a ratio of the CLDN18.2 antagonist and the GUCY2C antagonist 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.

In one aspect, provided herein is a nucleic acid comprising:

(1) a nucleic acid sequence encoding the multi-specific CAR construct as disclosed herein; or

(2) a first nucleic acid sequence encoding a first engineered receptor specifically targeting CLDN18.2 comprising: a first extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a first transmembrane domain and a first intracellular signaling domain, and a second nucleic acid sequence encoding a second engineered receptor specifically targeting GUCY2C comprising: a second extracellular antigen binding domain comprising at least one polypeptide comprising the GUCY2C binding moiety, a second transmembrane domain and a second intracellular signaling domain,

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) .

In some embodiments, the nucleic acid comprises:

(1) a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 14-18; or

(2) a first nucleic acid sequence encoding a first polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 12 and 19, and a second nucleic acid sequence encoding a second polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 13 and 20-22.

In one aspect, provided herein is a vector comprising the nucleic acid as disclosed herein. In one aspect, provided herein is an engineered immune cell comprising the multi-specific CAR, the nucleic acid or the vector as disclosed herein.

In some embodiments, the engineered immune cell comprises an engineered receptor specifically targeting CLDN18.2 comprising: an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a transmembrane domain, and optionally an intracellular signaling domain; and an engineered immune cell comprising an engineered receptor specifically targeting GUGY2C comprising: an extracellular antigen binding domain comprising at least one anti-GUCY2C binding moiety, a transmembrane domain, and optionally an intracellular signaling domain. The engineered immune cell may be derived from the group consisting of T cell, NK cell, peripheral blood mononuclear cell (PBMC) , hematopoietic stem cell, pluripotent stem cell, an embryonic stem cell, and a combination thereof.

In some embodiments, the engineered immune cell comprises:

In one aspect, provided herein is a pharmaceutical composition comprising the multi-specific CAR construct, the combination, the nucleic acid or the engineered immune cell as disclosed herein, and a pharmaceutically acceptable carrier.

In one aspect, provided herein is the multi-specific CAR construct, the combination, the nucleic acid or the engineered immune cell as disclosed herein for use in treating a tumor in a subject, optionally wherein the tumor is CLDN18.2 positive and/or GUCY2C positive. The tumor may be selected from gastric cancer, esophageal cancer, pancreatic ductal cancer, lung cancer such as non-small cell lung cancer (NSCLC) , ovarian cancer, colorectal cancer, liver cancer, head and neck cancer, gallbladder cancer and its metastasis. 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.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A-1B show the IHC analysis of CLDN18.2 and GUCY2C expression on primary and metastatic GC gastric cancer tissues.

FIGs. 2A-2C show the CAR structure scheme of CLDN18.2 CAR and/or GUCY2C CAR expressed cells (e.g., T cells) . FIG. 2A shows two types of single-antigen specific CAR, such as single-Claudin18.2 specific CAR (Si-CLDN18.2 CAR) which recognizes and binds Claudin18.2, or single-GUCY2C specific CAR (Si-GCC CAR) which recognizes and binds GUCY2C, respectively. FIG. 2B shows the structure of tandem bispecific CARs, which contain an extracellular antigen binding region on one same CAR and can recognizes and binds two different antigens, such as Claudin18.2 and GUCY2C. FIG. 2C shows the split bispecific CAR design, where a cell simultaneously expresses two CARs targeting two antigens, such as Claudin18.2 and GUCY2C, respectively.

FIGs. 3A-3C show the in vitro cytotoxicity of different CAR-T cells on target cells, e.g., Hep3b-CLDN18.2-GUCY2C. Luc cells (FIG. 3A) , Hep3b-CLDN18.2. Luc cells (FIG. 3B) , Hep3b-GUCY2C. Luc cells (FIG. 3C) , at different E: T ratios of 2: 1 and 0.5: 1, respectively.

FIGs. 4A-4B show the in vitro cytokine (IFN-γ and TNF-α) released by CAR-T cells in a heterogeneity model under different target cell mixtures (Mix 1-7) .

FIGs. 5A-5C show the cell expansion and CAR expression of CAR-T cells in re-challenge assay.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms “a” , “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “aprotein” includes a plurality of proteins; reference to “acell” includes mixtures of cells, and the like. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “comprising” as well as other forms, such as “comprises" and “comprised” is not limiting. In addition, ranges provided in the specification and appended claims include both end points and all points between the end points.

The term “antagonist” refers to a substance that interferes with or inhibits the physiological action of the target antigen or the signaling pathway mediated by the target antigen. There are a variety of types of antagonists known in the art, including but not limited to, an engineered immune cell, an engineered receptor such as an engineered T cell receptor (TCR) , a chimeric antigen receptor (CAR) , a T cell antigen coupler (TAC) or a portion thereof, an antibody, an antibody-drug conjugate (ADC) , an aptamer, small RNAs and chemical compound inhibitors.

The term “a combination of CLDN18.2 antagonist and GUCY2C antagonist” , as used herein, 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 substance and the second substance may be separated, or linked, fused or complexed. The CLDN18.2 antagonist may be a first CAR having an extracellular antigen binding domain that binds to CLDN18.2 but not GUCY2C, and the GUCY2C antagonist may be a second CAR having an extracellular antigen binding domain that binds to GUCY2C but not CLDN18.2, the first CAR and the second CAR may be separate polypeptides or linked together in the same chain. The CLDN18.2 antagonist may be a first group of engineered immune cells expressing a CAR that binds CLDN18.2 but not GUCY2C, and the GUCY2C antagonist may be a second group of engineered immune cells expressing a CAR that binds GUCY2C but not CLDN18.2. If the first substance and the second substance can be linked or fused together as a new molecule that retains the antagonizing abilities of both substances, the new molecule may also be deemed as “an antagonist of CLDN18.2 and GUCY2C” .

The term “an antagonist of CLDN18.2 and GUCY2C” , as used herein, refers to a substance that have the capability to specifically antagonize both CLDN18.2 and GUCY2C. The antagonist of CLDN18.2 and GUCY2C may be a CAR having an extracellular antigen binding domain that could bind to CLDN18.2 and GUCY2C. The antagonist of CLDN18.2 and GUCY2C may be an engineered immune cell expressing a CAR having an extracellular antigen binding domain that could bind to CLDN18.2 and GUCY2C. The antagonist of CLDN18.2 and GUCY2C may be an engineered immune cell expressing a first CAR having an extracellular antigen binding domain that binds to CLDN18.2 but not GUCY2C, and a second CAR having an extracellular antigen binding domain that binds to GUCY2C but not CLDN18.2. The antagonist of CLDN18.2 and GUCY2C may also be an anti-CLDN18.2 × GUCY2C antibody.

The term “chimeric antigen receptor” or “CAR” refers to a recombinant polypeptide construct comprising an extracellular antigen binding domain or ligand binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. The CAR may be monospecific or multi-specific (e.g., bispecific) . The domains in the CAR polypeptide construct may be in the same polypeptide chain, e.g., comprise a chimeric fusion protein. The domains in the CAR construct may be not contiguous with each other, e.g., are in different polypeptide chains, e.g., as provided in a split CAR construct as described below.

The term “multi-specific tandem CAR” , as used herein, refers to a recombinant polypeptide construct comprising an extracellular antigen binding region comprising more than one antigen-binding moieties that are operably linked in tandem, wherein the antigen-binding moieties having more than one antigen-binding specificities. For example, the multi-specific tandem CAR may be a multi-specific or multi-valent CAR comprising an extracellular antigen binding region that comprises more than one copies of a same VHH, wherein the VHHs are operably linked in tandem. The multi-specific tandem CAR may be a multi-specific CAR comprising two or more VHHs having different antigen specificities, the first VHH is linked to the second VHH in tandem in the extracellular antigen binding region. The multi-specific tandem CAR may be two CARs linked by a non-cleavable linker.

The term “multi-specific split CAR” , as used herein, refers to a CAR construct comprising two CARs, either in one chain or in separate chains, the two CARs each comprising an extracellular antigen binding region having different antigen specificities. When in one chain, the two CARs are usually operably linked via a cleavable linker.

The term "antibody" as used herein refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.

The term “antigen-binding moiety” as used herein refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure. Examples of antigen-binding moiety include, without limitation, a variable domain, a variable region, a single variable domain (i.e. VHH) , a nanobody, a domain antibody, a diabody, a Fab, a Fab' , a F (ab') 2, an Fv fragment, a single chain Fv fragment (scFv) , a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2, a bispecific dsFv (dsFv-dsFv') , a disulfide stabilized diabody (ds diabody) , a multispecific antibody, a camelized single domain antibody, and a bivalent domain antibody. An antigen-binding moiety is capable of binding to the same antigen to which the parent antibody binds. More detailed formats of antigen-binding moiety are described in 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 their entirety.

The term “single domain antibody” or “sdAb” as used herein refers to a single monomeric variable antibody domain and which is capable of antigen binding (e.g., single domain antibodies that bind to CLDN18.2 or GUGY2C) . Single domain antibodies include VHH domains as described herein. Examples of single domain antibodies include, but are not limited to, antibodies naturally devoid of light chains such as those from Camelidae species (e.g., llama) , 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) may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, and bovine. For example, a single domain antibody can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco, as described herein. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; VHHs derived from such other species are within the scope of the disclosure. The single domain antibody (e.g., VHH) provided herein may have a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single domain antibodies may be genetically fused or chemically conjugated to another molecule (e.g., an agent) as described herein. Single domain antibodies may be part of a bigger binding molecule (e.g., a multispecific antibody or a chimeric antigen receptor) .

The term "complementarity determining region" or "CDR, " as used herein, refers to the sequences of amino acids within antibody variable domains which confer antigen specificity and binding affinity. For example, in general, there are three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3) . The extent of CDRs and the framework region can be precisely identified using methodology known in the art, for example, by the Kabat definition, the definitions at Dr. Martin’s website, the Chothia definition, the AbM definition, the EU definition, and the contact definition, all of which are well known in the art. See, e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. 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 on 31 July 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 U S A. 1969 May; 63 (1) : 78-85; and Almagro, J. Mol. Recognit. 17: 132-143 (2004) . See also hgmp. mrc. ac. uk and bioinf. org. uk/abs. Correspondence or alignments between numberings according to different definitions can for example be found at http: //www. imgt. org/ (see also Giudicelli V et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids 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 instances, the scheme for identification of a particular CDR or CDRs is specified, such as the CDR as defined by the IMGT, Kabat, AbM, Chothia, or Contact method. One or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering. See, e.g., Deschacht et al., 2010. J Immunol 184: 5696-704 for an exemplary numbering for VHH domains according to Kabat. In other cases, the particular amino acid sequence of a CDR is given. It should be noted CDR regions may also be defined by a combination of various numbering systems, e.g., a combination of Kabat and Chothia numbering systems, a combination of Kabat and AbM numbering systems, or a combination of Kabat and IMGT numbering systems. Therefore, the term such as “aCDR as set forth in a specific VH or VHH” includes any CDRs as defined by the exemplary CDR numbering systems described above, but is not limited thereby. Once a variable region (e.g., a VHH domain, a VH or VL domain) is given, those skilled in the art would understand that CDRs within the region can be defined by different numbering systems or combinations thereof.

The term "intracellular signaling domain" , as used herein, refers to an intracellular portion of a CAR that can generate a signal that promotes an immune effector function of the CAR containing cell, e.g., a CAR-T cell or CAR-expressing NK cell. Examples of immune effector function, e.g., in a CAR-T cell or CAR-expressing NK cell, include cytolytic activity and helper activity, including the secretion of cytokines. The intracellular signal domain may transduce the effector function signal and directs the cell to perform a specialized function. While the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain.

The term "stimulatory molecule" refers to a molecule expressed by a T cell that provides the primary cytoplasmic signaling sequence (s) that regulate primary activation of the TCR complex in a stimulatory way for at least some aspect of the T cell signaling pathway. In one aspect, the primary signal is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A primary signaling domain that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or ITAM. Examples of an ITAM containing primary cytoplasmic signaling sequence that is of particular use in the disclosure includes, but is not limited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS" ) , FcsRI, CD66d, DAP10 and DAP12. In a specific CAR as disclosed herein, the intracellular signaling domain comprises an intracellular signaling sequence, e.g., a primary signaling sequence of CD3-zeta, which may be derived from human or a non-human species, e.g., mouse, rodent, monkey, ape and the like.

The term “co-stimulatory signaling domain” refers to the intracellular portion of a costimulatory molecule. The intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof. Costimulatory molecules are the cognate binding partner on a T cell that specifically bind with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response. Costimulatory molecules include, but are not limited to an a MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins) , activating NK cell receptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CDl la/CD18) , 4-1BB (CD137) , B7-H3, CDS, ICAM-1, ICOS (CD278) , GITR, BAFFR, LIGHT, HVEM (LIGHTR) , KIRDS2, SLAMF7, NKp80 (KLRF1) , NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226) , SLAMF4 (CD244, 2B4) , CD84, CD96 (Tactile) , CEACAM1, 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, CD 19a, and a ligand that specifically binds with CD83.

The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, stomach cancer, pancreatic cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like. The terms "tumor" and "cancer" are used interchangeably herein.

CLDN18.2 Antagonist and GUGY2C Antagonist

In one aspect, the present disclosure provides a method for treating a tumor in a subject in need thereof, comprising administering an effective amount of a combination of a CLDN18.2 (Claudin18.2) antagonist and a GUCY2C antagonist, or an antagonist of CLDN18.2 and GUCY2C to the subject. The tumor may be CLDN18.2 positive and/or GUCY2C positive. In one aspect, the present disclosure also provides a combination of a CLDN18.2 antagonist and a GUCY2C antagonist, and an antagonist of CLDN18.2 and GUCY2C, and a method of using the same for the treatment of various diseases.

The CLDN18.2 antagonist, the GUCY2C antagonist, and/or the antagonist of CLDN18.2 and GUCY2C may have a variety of categories, e.g., the antagonist may be selected from an engineered receptor, an engineered immune cell, an antibody, an antibody-drug conjugate (ADC) , an aptamer and small RNAs. The CLDN18.2 antagonist may include monospecific antibodies, bispecific antibodies, ADCs, CAR-T cells redirected to target CLDN18.2, such as Zolbetuximab (IMAB362) , TST001, BNT141, AMG910, or LM-302. The GUCY2C antagonist may include those already known in the art, such as anti-GUCY2C monospecific antibodies, ADC antibodies (such as TAK-264 (ADC-DGN549) , TAK-164) , bispecific antibodies (PF-07062119 (GCCxCD3) ) , or Ad5-GUCY2C-PADRE. The antagonist of CLDN18.2 and GUCY2C may include bispecific antibodies against CLDN18.2 and GUCY2C (GCCxCLDN18.2) .

The CLDN18.2 antagonist may be an engineered immune cell expressing an engineered receptor specifically targeting CLDN18.2 comprising: an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a transmembrane domain, and an intracellular signaling domain. The GUCY2C antagonist may be an engineered immune cell comprising an engineered receptor specifically targeting GUGY2C comprising: an extracellular antigen binding domain comprising at least one anti-GUCY2C binding moiety, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the engineered receptor is selected from the group consisting of an engineered T cell receptor (TCR) , a chimeric antigen receptor (CAR) , a T cell antigen coupler (TAC) or a portion thereof. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR) . The anti-CLDN18.2 binding moiety may be an anti-CLDN18.2 VHH, and optionally the anti-CLDN18.2 VHH comprises a CDR1, CDR2, and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 1. The anti-CLDN18.2 VHH CDRs (CDR1-3) may 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, the anti-CLDN18.2 VHH comprises (i) a CDR1 comprises the amino acid sequence of SEQ ID NO: 23, (ii) a CDR2 comprises the amino acid sequence of SEQ ID NO: 24, and (iii) a CDR3 comprises the amino acid sequence of SEQ ID NO: 25. The anti-CLDN18.2 VHH may comprise an amino acid sequence of SEQ ID NO: 1 or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 1. The anti-GUGY2C binding moiety is an anti-GUGY2C VHH, and optionally the anti-GUGY2C VHH comprises a CDR1, CDR2, and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 2. The anti-GUGY2C VHH CDRs (CDR1-3) may 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, the anti-GUGY2C VHH comprises (i) a CDR1 comprises the amino acid sequence of SEQ ID NO: 26, (ii) a CDR2 comprises the amino acid sequence of SEQ ID NO: 27, and (iii) a CDR3 comprises the amino acid sequence of SEQ ID NO: 28. The anti-GUCY2C VHH may comprise an amino acid sequence of SEQ ID NO: 2 or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the CLDN18.2 antagonist is an engineered immune cell comprising a CAR specifically targeting CLDN18.2 (referred herein as “single-CLDN18.2 specific CAR” ) comprising: a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 12 and 19, and/or the GUCY2C antagonist is an engineered immune cell comprising a CAR specifically targeting GUGY2C (referred herein as “single-GUGY2C specific CAR” ) comprising: a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 13 and 21-22. The engineered immune cell may be selected from the group consisting of T cell, NK cell, peripheral blood mononuclear cell (PBMC) , hematopoietic stem cell, pluripotent stem cell, an embryonic stem cell, and a combination thereof.

Correspondingly, a combination of the CLDN18.2 antagonist and the GUCY2C antagonist may comprise two groups of engineered immune cells targeting CLDN18.2 and GUCY2C, respectively. The first group and the second group of engineered immune cells may be administered simultaneously or sequentially. The combination may also be deemed as the antagonist of CLDN18.2 and GUCY2C when an engineered immune cell expresses both the engineered receptor specifically targeting CLDN18.2 and the engineered receptor specifically targeting GUGY2C.

The combination of CLDN18.2 antagonist and GUCY2C antagonist provides certain benefits. A combined therapy that targets more than one antigen may have improved efficacy over a single therapy that targets one antigen in cancer treatment. Cancer cells are unstable genetically, which allows them to escape from targeted therapies by mutating or losing genes encoding the target antigens. By targeting two or more different epitopes or antigens on cancer cells, combined therapies can make it more difficult for cancer cells to completely escape from antagonization. In one aspect, provided herein is a method for treating tumor in a subject by utilizing the benefits of combining CLDN18.2 antagonization and GUCY2C antagonization. In some embodiments, the method comprises administering the combination of a CLDN18.2 antagonist and a GUGY2C antagonist. In some other embodiments, the method comprises administering the antagonist of CLDN18.2 and GUCY2C as disclosed herein.

A combination of a CLDN18.2 antagonist and a GUGY2C antagonist

In one aspect, provided herein is a combination of a CLDN18.2 antagonist and a GUCY2C antagonist (referred herein as “the combination” ) with different antigen specificities that may have a complementary or synergistic effect. The combination may comprise a CLDN18.2 antagonist and a GUGY2C antagonist, wherein the CLDN18.2 antagonist is an anti-CLDN18.2 monoclonal antibody or corresponding ADC, and the GUGY2C antagonist is an anti-GUGY2C monoclonal antibody or corresponding ADC. The CLDN18.2 antagonist may be an engineered receptor specifically targeting CLDN18.2, and the GUGY2C antagonist may be an engineered receptor specifically targeting GUGY2C. The combination may comprise a CLDN18.2 antagonist and a GUGY2C antagonist, wherein the CLDN18.2 antagonist is a first group of engineered immune cells expressing engineered receptor specifically targeting CLDN18.2, and the GUGY2C antagonist is a second group of engineered immune cells expressing engineered receptor specifically targeting GUGY2C.

The CLDN18.2 antagonist may be an engineered receptor specifically targeting CLDN18.2 comprising: an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a transmembrane domain, and an intracellular signaling domain. The GUCY2C antagonist may be an engineered receptor specifically targeting GUGY2C comprising: an extracellular antigen binding domain comprising at least one anti-GUCY2C binding moiety, a transmembrane domain, and an intracellular signaling domain. Correspondingly, a combination of the CLDN18.2 antagonist and the GUCY2C antagonist may comprise two engineered receptors targeting CLDN18.2 and GUCY2C, respectively.

The combination may also be deemed as the antagonist of CLDN18.2 and GUCY2C when the two engineered receptors targeting CLDN18.2 and GUCY2C are linked, fused, associated together or forming a complex. The engineered receptor specifically targeting CLDN18.2 may be operably linked to the engineered receptor specifically targeting GUCY2C, for example, linked via a cleavable linker.

In some embodiments, the combination comprises two groups of engineered immune cells, wherein the first group of engineered immune cells express the engineered receptor specifically targeting CLDN18.2 and the second group of engineered immune cells express the engineered receptor specifically targeting GUCY2C.

In some embodiments, the engineered receptors are chimeric antigen receptors (CARs) . Correspondingly, the combination may comprise a first CAR (i.e. single-CLDN18.2 specific CAR) that comprises an antigen binding domain specifically targeting CLDN18.2 and a second CAR (i.e. single-GUCY2C specific CAR) that comprises an antigen binding domain specifically targeting GUCY2C. The combination may comprise a first CAR and a second CAR that is presented as a multi-specific split CAR construct, wherein the first CAR and the second CAR are operably linked in one chain via a cleavable linker (e.g. P2A, T2A, E2A or F2A) , or separated in two chains when the linker is cleaved.

The combination may comprise: (1) a first CAR specifically targeting CLDN18.2 (also referred herein as “single-CLDN18.2 specific CAR” ) comprising an extracellular antigen binding domain that comprises 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 GUGY2C (also referred herein as “single-GUGY2C specific CAR” ) comprising an extracellular antigen binding domain that comprises 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., a CD8α or CD28 or ICOS TM) , 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 the N-terminal.

In some embodiments, the combination of the CLDN18.2 antagonist and the GUCY2C antagonist comprises a first and a second group of engineered immune cells, wherein: (1) the CLDN18.2 antagonist is the first group of engineered immune cells comprising a CAR specifically targeting CLDN18.2 (e.g., single-CLDN18.2 specific CAR) comprising: an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a transmembrane domain, and an intracellular signaling domain; and/or (2) the GUCY2C antagonist is the second group of engineered immune cells comprising a CAR specifically targeting GUGY2C (e.g., single-GUGY2C specific CAR) comprising: an extracellular antigen binding domain comprising at least one anti-GUCY2C binding moiety, a transmembrane domain, and an intracellular signaling domain.

In some embodiments of the combination, the anti-CLDN18.2 binding moiety is an anti-CLDN18.2 VHH, and optionally the anti-CLDN18.2 VHH comprises a CDR1, CDR2, and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 1. The anti-CLDN18.2 VHH CDRs (CDR1-3) may 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, the anti-CLDN18.2 VHH comprises (i) a CDR1 comprises the amino acid sequence of SEQ ID NO: 23, (ii) a CDR2 comprises the amino acid sequence of SEQ ID NO: 24, and (iii) a CDR3 comprises the amino acid sequence of SEQ ID NO: 25. The anti-CLDN18.2 VHH may comprise an amino acid sequence of SEQ ID NO: 1 or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-GUGY2C binding moiety is an anti-GUGY2C VHH, and optionally the anti-GUGY2C VHH comprises a CDR1, CDR2, and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 2. The anti-GUGY2C VHH CDRs (CDR1-3) may 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, the anti-GUGY2C VHH comprises (i) a CDR1 comprises the amino acid sequence of SEQ ID NO: 26, (ii) a CDR2 comprises the amino acid sequence of SEQ ID NO: 27, and (iii) a CDR3 comprises the amino acid sequence of SEQ ID NO: 28. The anti-GUCY2C VHH may comprise an amino acid sequence of SEQ ID NO: 2 or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 2.

(1) the CLDN18.2 antagonist is the first group of engineered immune cells comprising a CAR specifically targeting CLDN18.2 (e.g., single-CLDN18.2 specific CAR) comprising: an extracellular antigen binding domain comprising at least one anti-CLDN18.2 VHH, optionally a hinge domain (e.g., a CD8α hinge domain) , a transmembrane domain (e.g., a CD8α or CD28 or ICOS TM) , optionally a co-stimulatory signaling domain, and a primary intracellular signaling domain (e.g., derived from CD3ζ) ; and/or (2) the GUCY2C antagonist is the second group of engineered immune cells comprising a CAR specifically targeting GUGY2C (e.g., single-GUGY2C specific CAR) comprising: an extracellular antigen binding domain comprising at least one anti-GUCY2C VHH, optionally a hinge domain (e.g., a CD8α hinge domain) , a transmembrane domain (e.g., a CD8α or CD28 or ICOS TM) , optionally a co-stimulatory signaling domain, and a primary intracellular signaling domain (e.g., derived from CD3ζ) . In some embodiments, the combination of a CLDN18.2 antagonist and a GUCY2C antagonist comprises a first group of engineered immune cell comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 12, and a second group of engineered immune cell comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 13.

The CLDN18.2 antagonist and the GUCY2C antagonist in combination may be administered simultaneously or sequentially. When the CLDN18.2 antagonist and the GUCY2C antagonist are administered simultaneously, they may be mixed at certain ratios before administration. Alternatively, the CLDN18.2 antagonist and the GUCY2C antagonist may be administered separately at certain ratios. The amounts administrated to the subject for the CLDN18.2 antagonist and the GUCY2C antagonist may be different. For example, when the combination comprises two groups of engineered immune cells with the first group of engineered immune cells expressing the engineered receptor specifically targeting CLDN18.2 and the second group of engineered immune cells expressing the engineered receptor specifically targeting GUCY2C, the ratio of the cell numbers of the first group to the second group of engineered immune cells may 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 comprises two monoclonal antibodies or ADCs with the first antibody or ADC specifically targeting CLDN18.2 and the second antibody or ADC specifically targeting GUCY2C, the molar ratio of the first antibody to the second antibody may 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.

As described above, the combination of the CLDN18.2 antagonist and the GUCY2C antagonist (e.g., a combination of single-CLDN18.2 specific CAR-T cells and single-GUGY2C specific CAR-T cells) with different antigen binding specificities may have improved efficacy over monospecific CARs or immune cells alone for cancer immunotherapy. By targeting two or more different epitopes or antigens on cancer cells, a combination of two or more single-antigen specific CARs can make it more difficult for cancer cells to completely escape from targeting by engineered immune cells (such as T cells) expressing the CARs.

Antagonist of CLDN18.2 and GUCY2C

In another aspect, provided herein is an antagonist of CLDN18.2 and GUCY2C that targets both CLDN18.2 and GUCY2C. The antagonist of CLDN18.2 and GUCY2C (referred herein as “the antagonist” ) may include a molecule co-targeting CLDN18.2 and GUCY2C, such as a multi-specific antibody or a multi-specific chimeric receptor co-targeting CLDN18.2 and GUCY2C. The antagonist of CLDN18.2 and GUCY2C may be a multi-specific (e.g., bispecific) antibody that targets CLDN18.2 and GUCY2C. The antagonist of CLDN18.2 and GUCY2C may be an engineered receptor co-targeting both CLDN18.2 and GUGY2C. The antagonist of CLDN18.2 and GUCY2C may include a complex comprising two portions, one portion can be an engineered receptor targeting CLDN18.2, and another portion can be an engineered receptor targeting GUCY2C, respectively, and the two portions may be linked, fused or associated. The antagonist of CLDN18.2 and GUCY2C also encompasses an engineered immune cell comprising an engineered receptor specifically targeting CLDN18.2 and an engineered receptor specifically targeting GUGY2C.

The antagonist of CLDN18.2 and GUCY2C may be a multi-specific engineered receptor targeting both CLDN18.2 and GUCY2C. The multi-specific engineered receptor may comprise: an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety, a transmembrane domain, and an intracellular signaling domain. Correspondingly, the antagonist of CLDN18.2 and GUCY2C may also be an engineered immune cell expressing the engineered receptor targeting both CLDN18.2 and GUCY2C.

The antagonist of CLDN18.2 and GUCY2C may comprise two engineered receptors with different antigen specificities, wherein the first engineered receptor specifically targets CLDN18.2, and a second engineered receptor specifically targets GUCY2C, optionally the two engineered receptors are operably linked. The antagonist of CLDN18.2 and GUCY2C may comprise two engineered receptors, wherein the first engineered receptor comprises an extracellular antigen binding region comprising at least one anti-CLDN18.2 binding moiety, a transmembrane domain, and an intracellular signaling domain, and the second engineered receptor comprises an extracellular antigen binding region comprising at least one anti-GUCY2C binding moiety, a transmembrane domain, and an intracellular signaling domain. The two engineered receptors may be operably linked via a cleavable linker which can be easily cleaved under suitable conditions. The cleavable linker can be selected from P2A, T2A, E2A and F2A.

Correspondingly, the antagonist of CLDN18.2 and GUCY2C may also be an engineered immune cell expressing both the first engineered receptor and the second engineered receptor. In some embodiments, the immune cell has been transduced by two separate vectors, the first vector comprises a nucleic acid encoding the first engineered receptor specifically targeting CLDN18.2, and the second vector comprises a nucleic acid encoding the second engineered receptor specifically targeting GUGY2C. In some other embodiments, the immune cell has been transduced by a vector that comprises a nucleic acid encoding the first engineered receptor specifically targeting CLDN18.2 operably linked to the second engineered receptor specifically targeting GUGY2C.

In some embodiments, the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising an engineered receptor co-targeting CLDN18.2 and GUGY2C comprising: an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety, a transmembrane domain, and an intracellular signaling domain.

The engineered receptor may be selected from the group consisting of an engineered T cell receptor (TCR) , a chimeric antigen receptor (CAR) , a T cell antigen coupler (TAC) or a portion 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 GUGY2C, wherein (1) the first CAR targeting CLDN18.2 (referred herein as single-CLDN18.2 specific CAR) comprising: a first extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a first transmembrane domain and an first intracellular signaling domain, (2) the second CAR targeting GUGY2C (referred herein as single-GUGY2C specific CAR) comprising: a second extracellular antigen binding domain comprising at least one anti-GUGY2C binding moiety, a second transmembrane domain and a second intracellular signaling domain; and wherein the first CAR targeting CLDN18.2 is operably linked to the second CAR targeting GUGY2C via a cleavable linker or the first CAR and the second CAR are not linked due to the cleavage of the cleavable linker. In some embodiments, the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising a CAR co-targeting CLDN18.2 and GUGY2C ( “CLDN18.2×GUCY2C specific CAR” ) , wherein the CLDN18.2×GUCY2C specific CAR comprises: (1) an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety, (2) a transmembrane domain, and (3) an intracellular signaling domain; and wherein the anti-CLDN18.2 binding moiety is located at the N-terminus or C- terminus of the anti-GUGY2C binding moiety, optionally the anti-CLDN18.2 binding moiety is operably linked to the anti-GUCY2C binding moiety via a peptide linker, such as a GS linker e.g. (G₄S) ₃.

The anti-CLDN18.2 binding moiety and/or anti-GUCY2C binding moiety may be selected from a Fab, a Fab’ , a F (ab’ ) 2, an Fv, a single-chain Fv (scFv) , minibody, a diabody, a single-domain antibody (sdAb) or VHH domain. the anti-CLDN18.2 binding moiety is an anti-CLDN18.2 VHH, and optionally the anti-CLDN18.2 VHH comprises a CDR1, CDR2, and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 1. The anti-CLDN18.2 VHH CDRs (CDR1-3) may be determined according to the IMGT numbering scheme. The anti-CLDN18.2 VHH CDRs (CDR1-3) may be determined according to the AbM numbering scheme. The anti-CLDN18.2 VHH CDRs (CDR1-3) may be determined according to the Kabat numbering scheme. In some embodiments, the anti-CLDN18.2 VHH comprises (i) a CDR1 comprises the amino acid sequence of SEQ ID NO: 23, (ii) a CDR2 comprises the amino acid sequence of SEQ ID NO: 24, and (iii) a CDR3 comprises the amino acid sequence of SEQ ID NO: 25. The anti-CLDN18.2 VHH may comprise an amino acid sequence of SEQ ID NO: 1 or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-GUGY2C binding moiety is an anti-GUGY2C VHH, and optionally the anti-GUGY2C VHH comprises a CDR1, CDR2, and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 2. The anti-GUGY2C VHH CDRs (CDR1-3) may be determined according to the IMGT numbering scheme. The anti-GUGY2C VHH CDRs (CDR1-3) may be determined according to the AbM numbering scheme. The anti-GUGY2C VHH CDRs (CDR1-3) may be determined according to the Kabat numbering scheme. In some embodiments, the anti-GUGY2C VHH comprises (i) a CDR1 comprises the amino acid sequence of SEQ ID NO: 26, (ii) a CDR2 comprises the amino acid sequence of SEQ ID NO: 27, and (iii) a CDR3 comprises the amino acid sequence of SEQ ID NO: 28. The anti-GUCY2C VHH may comprise an amino acid sequence of SEQ ID NO: 2 or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 2.

The single-CLDN18.2 specific CAR, single-GUGY2C specific CAR and CLDN18.2×GUCY2C specific CAR may each further comprise: (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.

In some embodiments, the CAR co-targeting CLDN18.2 and GUCY2C comprises a polypeptide comprising an amino acid sequence of any of SEQ ID NO: 14 (corresponding to LG23B01 CAR) and SEQ ID NO: 15 (corresponding to LG23B02 CAR) . In some embodiments, the single-CLDN18.2 specific CAR and the single-GUGY2C specific CAR are operably linked by a P2A linker, comprises an amino acid sequence as set forth in of any of SEQ ID NO: 16-18 (corresponding to LG23D01, LG23D02, LG23D03 CARs, respectively) prior to the cleavage by a 2A linker.

In some embodiments, the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising: (1) a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 14-18; or (2) a first polypeptide and a second polypeptide, wherein the first polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 12 and 19, and the second polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 13 and 20-22.

Engineered Receptors (e.g., CAR, TCR and TAC)

One aspect of the present disclosure provides engineered cells (e.g., immune cells) that express an engineered receptor. The engineered receptor can comprise an extracellular antigen binding domain, and optionally an intracellular signaling domain. Exemplary engineered receptors include, but are not limited to, chimeric antigen receptor (CAR) , engineered T-cell receptor (TCR) , and T-cell antigen coupler (TAC) receptor. The engineered receptor can comprise an extracellular antigen binding domain that specifically binds to an antigen (e.g., CLDN18.2 or GUGY2C) , a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain can comprise a primary intracellular signaling domain and/or a co-stimulatory signaling domain. The intracellular signaling domain can comprise an intracellular signaling domain of a TCR co-receptor. The engineered receptor can be encoded by a heterologous polynucleotide operably linked to a promoter (such as a constitutive promoter or an inducible promoter) .

The engineered receptor can comprise 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.

In one aspect, the present disclosure provides chimeric antigen receptors (CARs) bind to one or more antigen specificities (e.g., CLDN18.2 and/or GUGY2C) . A CAR may act as an antagonist of the antigen or expressed by an immune cell which in turn acts as an antagonist for use in therapies. The CAR may comprise: (a) an extracellular antigen binding domain comprising one or more antigen-binding moieties (e.g., VHH) that binds to CLDN18.2 and/or GUGY2C; (b) a transmembrane domain; and (c) an intracellular signaling domain.

The extracellular antigen binding domain of each CAR may comprise one or more antigen-binding moieties, which may exist in a variety of forms, including for example, a single-domain antibody (sdAb) or VHH domain, a single chain variable fragment (scFv) , a Fab, a Fab', a F (ab) '2, a F (ab) '3, an Fv, a bis-scFv, a (scFv) 2, a minibody, a diabody, a triabody, a tetrabody, an intrabody, a disulfide stabilized Fv protein (dsFv) , a unibody, a nanobody, an affibody, a DARPin, a monobody, an adnectin, an alphabody, or a designed binder. The antigen-binding moieties can be single domain antibodies (e.g., VHH domains) , such as a camelid, shark, chimeric, human, or humanized single domain antibodies (e.g., VHH domains) . The CARs as disclosed herein may comprise an antigen binding domain comprising one or more VHH domains (such as any one of 1, 2, 3, 4, 5, 6 or more) . The VHHs can be fused to each other directly via peptide bonds, or via peptide linkers.

The CAR may be monospecific or multi-specific (such as bispecific) , monovalent or multi-valent (such as bivalent) . In some embodiments, the CARs are multi-specific (such as bispecific) CARs comprising one or more antigen-binding moieties that have different antigen binding specificities. CARs having an extracellular antigen binding region comprising one or more VHHs targeting different epitopes or antigens can be conveniently constructed and produced recombinantly, thereby providing an efficient platform for preparation and screening of multivalent and multi-specific CARs. Additionally, the small footprint of VHHs may allow access of the CARs to hidden antigen targets and epitopes in tumor tissues.

Depending on the desired antigen to be targeted, the CARs as disclosed herein can be engineered to include the appropriate VHHs that specifically target the desired antigens. The VHHs can be arranged in any suitable order. For example, the first VHH domain is fused at the N-terminus or the C-terminus of the second VHH domain. A suitable peptide linker may be placed between different VHHs to avoid steric hindrance between the VHHs. Exemplary bispecific chimeric antigen receptors, exemplary sequences, constructs and vectors thereof are shown below.

The CAR may be a monospecific CAR or multivalent CAR. The CAR may be a single-CLDN18.2 specific CAR comprising an extracellular antigen binding domain that comprises an anti-CLDN18.2 VHH targeting the CLDN18.2 antigen. The single-CLDN18.2 specific CAR may comprise more than one copies of the anti-CLDN18.2 VHH which are operably linked in the extracellular antigen binding domain. The single-CLDN18.2 specific CAR may comprise a polypeptide comprising the amino acid sequence of any of SEQ ID NO: 12 (corresponding to LG23A01 CAR) and SEQ ID NO: 19.

The CAR may be a single-GUGY2C specific CAR comprising an extracellular antigen binding domain that comprises an anti-GUGY2C VHH targeting the GUCY2C antigen. The single-GUGY2C specific CAR may comprise more than one copies of the anti-GUCY2C VHH which are operably linked in the extracellular antigen binding domain. The single-GUGY2C specific CAR may comprise a polypeptide comprising the amino acid sequence of any of SEQ ID NO: 13 (corresponding to LG23A02 CAR) and SEQ ID NOs: 20-22.

The single-antigen specific CARs (e.g., single-CLDN18.2 specific CAR or single-GUGY2C specific CAR) as disclosed herein may be combined into a split CAR construct, or the antigen-binding moieties of the single-antigen specific CARs may be combined for the construction of multi-specific tandem CARs.

Multi-specific CAR constructs (e.g., tandem CAR and spit CAR)

In one aspect, the present disclosure provides a multi-specific CAR construct (e.g., tandem CAR) that binds to CLDN18.2 and GUGY2C comprising an extracellular antigen binding domain comprising at least two antigen-binding moieties with CLDN18.2 and GUGY2C, the at least two antigen-binding moieties are operably linked in tandem in the extracellular binding region. The multi-specific CAR construct may act as an antagonist of at least two antigens or expressed by an immune cell which in turn acts as an antagonist for use in therapies.

Multi-specific CAR construct may have improved efficacy over monospecific CARs for cancer immunotherapy. Cancer cells are unstable genetically, which allows them to escape from targeted therapies by mutating or losing genes encoding the target antigens. By targeting two or more different epitopes or antigens on cancer cells, multi-specific CAR constructs or combination of two or more monospecific CARs can make it more difficult for cancer cells to completely escape from targeting by engineered immune cells (such as T cells) expressing the CARs. Owing to their small size, tandemly fused VHHs, which are comprised in extracellular antigen binding domain of the multi-specific CAR construct, can preserve their individual structural integrity and binding affinity to target antigens. Engineered immune cells expressing the multi-specific CAR construct that binds to different tumor antigens may overcome tumor immune escape mechanisms that are due to abnormalities in protein-antigen processing and presentation.

In some embodiments, the present disclosure provides a multi-specific CAR construct (e.g., tandem CAR) that binds to CLDN18.2 and GUCY2C ( “CLDN18.2×GUCY2C specific CAR” ) , comprising an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety, a transmembrane domain and an intracellular signaling domain. In some embodiments, the anti-CLDN18.2 binding moiety is located at the N-terminus or C-terminus of the anti-GUGY2C binding moiety, optionally the anti-CLDN18.2 binding moiety is operably linked to the anti-GUCY2C binding moiety via a peptide linker, such as a GS linker e.g. (G₄S) ₃.

In some embodiments, the present disclosure provides a multi-specific CAR construct (e.g., tandem CAR) comprising an extracellular antigen binding domain that comprises one or more copies of the anti-CLDN18.2 VHH that binds to the CLDN18.2 antigen, and one or more copies of the anti-GUCY2C VHH that binds to the GUCY2C antigen. The VHH domains are operably linked in the extracellular antigen binding region in tandem. In some specific embodiments, the multi-specific CAR construct (e.g., tandem CAR) comprises an extracellular antigen binding domain comprising one anti-CLDN18.2 VHH and one anti-GUCY2C VHH, wherein the anti-CLDN18.2 VHH is located at the N terminal of the anti- GUCY2C VHH, or wherein the anti-CLDN18.2 VHH is located at the C terminal 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.

The linker is often a peptide linker, such as a peptide linker no more than about 50 (such as no more than about any one of 35, 25, 20, 15, 10, or 5) amino acids long. In some embodiments, the peptide linker is a GS series linker, such as (G4S) n linker with n being an integer of 1-8.

In some embodiments, the multi-specific CAR construct (e.g., tandem CAR) may comprise a polypeptide comprising, from the N-terminus to the C-terminus: an extracellular antigen binding domain comprising 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. a CD8α or CD28 or ICOS TM) , 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 the N terminal.

In some embodiments, the multi-specific CAR construct (e.g., tandem CAR) may comprise a polypeptide comprising, from the N-terminus to the C-terminus: an extracellular antigen binding domain comprising 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., a CD8α or CD28 or ICOS TM) , 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 the N terminal.

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

The co-stimulatory signaling 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, Ligands of CD83 and combinations thereof. In some embodiments, the co-stimulatory signaling domain is derived from 4-1BB. In some embodiments, the co-stimulatory signaling domain comprises the amino acid sequence of any of SEQ ID NOs: 7-9. In some embodiments, the multi-specific CAR construct (e.g., tandem CAR) comprise a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 or 15 (corresponding to LG23B01 CAR and LG23B02 CAR) .

In one aspect, the present disclosure provides a multi-specific CAR construct (e.g., split CAR construct) comprising different chimeric antigen receptors that are split in two polypeptides, wherein the two polypeptides may be operably are linked in one chain via a cleavable linker. The split CAR construct may act as an antagonist of at least two antigens or expressed by an immune cell which in turn acts as an antagonist for use in therapies. In some embodiments, the multi-specific CAR construct (e.g., split CAR construct) that binds to CLDN18.2 and GUCY2C comprises a first polypeptide comprising a first extracellular antigen binding domain, a first transmembrane domain and a first intracellular signaling domain, and a second polypeptide comprises 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 an anti-CLDN18.2 VHH. The anti-GUCY2C binding moiety may be an anti-GUCY2C VHH.

In some embodiments, the multi-specific CAR construct (e.g., split CAR construct) comprises two polypeptides, wherein (1) the first polypeptide comprises: a first extracellular antigen binding domain comprising at least one anti-CLDN18.2 VHH, a hinge domain (e.g., a CD8α hinge domain) , a transmembrane domain (e.g., a CD8α or CD28 or ICOS TM) , optionally a co-stimulatory signaling domain, and a primary intracellular signaling domain (e.g., derived from CD3ζ) ; and (2) the second polypeptide comprises: a second extracellular antigen binding domain comprising at least one anti-GUCY2C VHH, a hinge domain (e.g., a CD8αhinge domain) , a transmembrane domain (e.g., a CD8α or CD28 or ICOS TM) , optionally a co-stimulatory signaling domain, and a primary intracellular signaling domain (e.g. derived from CD3ζ) . In some embodiments, the first polypeptide is linked to the second polypeptide via a cleavable linker, such as 2A linker. The first polypeptide and the second polypeptide may be in two separate polypeptide chains. The first polypeptide and the second polypeptide each may further comprise a signal peptide (e.g., a CD8α signal peptide) at the N terminal.

Accordingly, in some embodiments, the multi-specific CAR construct (e.g., split CAR construct) comprises a first intracellular signaling domain in the CLDN18.2 CAR polypeptide and a second intracellular signaling domain in the GUCY2C CAR polypeptide, such that full activation of the cell, e.g., the population of immune cells, occurs when both the first polypeptide and the second polypeptide bind to a target cell, e.g., a target CLDN18.2+GUCY2C+ cell (e.g., a CLDN18.2+GUCY2C+ gastric cancer cell) , compared to activation when the first polypeptide or the second polypeptide bind to a target cell that expresses one of CLDN18.2 or GUCY2C. The first polypeptide may further comprise a co- stimulatory signaling domain, e.g., a 4-1BB, CD28, ICOS or NTBA signaling domain, and the second polypeptide may comprise a primary intracellular signaling domain, e.g., a CD3 zeta primary intracellular signaling domain. The first polypeptide may comprise a primary signaling domain, e.g., a CD3 zeta signaling domain, and the second polypeptide further comprises a costimulatory domain, e.g., a 4-1BB, CD28, ICOS or NTBA signaling domain. The first and second polypeptides may both comprise a co-stimulatory signaling domain, e.g., a 4-1BB, CD28, ICOS or NTBA signaling domain, and a primary intracellular signaling domain, e.g., a CD3 zeta signaling domain.

The first polypeptide and/or the second polypeptide may comprise a co-stimulatory signaling domain. The first polypeptide may comprise a first co-stimulatory signaling domain and the second polypeptide may comprise a second co-stimulatory signaling domain. The co-stimulatory signaling 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, Ligands of CD83 and combinations thereof. The first and the second co-stimulatory signaling domains may be derived from a same co-stimulatory molecule or different co-stimulatory molecules. The first co-stimulatory signaling domain may be derived from 4-1BB and the second co-stimulatory signaling domain may be derived from CD28, or vice versa. The first co-stimulatory signaling domain may be derived from ICOS and the second co-stimulatory signaling domain may be derived from NTBA, or vice versa. The first and the second co-stimulatory signaling domains may be derived from ICOS. The co-stimulatory signaling domain may comprise an amino acid sequence set forth in of any one of SEQ ID NOs: 7-9.

In some embodiments, the multi-specific CAR construct (e.g., split CAR construct) comprises a first polypeptide comprising the amino acid sequence of any of SEQ ID NO: 12 (corresponding to LG23A01 CAR) and SEQ ID NO: 19, and a second polypeptide comprising the amino acid sequence of any of SEQ ID NO: 13 (corresponding to LG23A02 CAR) and SEQ ID NOs: 20-22, optionally the first and the second polypeptides are operably linked by a cleavable peptide linker. The multi-specific CAR construct (e.g., split CAR construct) may comprise a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 16-18 (corresponding to LG23D01 CAR, LG23D02 CAR and LG23D03 CAR) prior to the cleavage via a 2A linker.

Extracellular Antigen Binding Domain

The CARs of the present application comprise an extracellular antigen binding region comprising one or more single domain antibodies, e.g., VHH domains. The single domain antibodies may be of the same or different origins, and of the same or different sizes. Exemplary single domain antibodies include, but are not limited to, heavy chain variable domains from heavy-chain only antibodies (VHH) , heavy-chain only antibodies such as camelid or humanized heavy-chain only antibodies, human VH produced by transgenic mice or rats expressing human heavy chain segments, and engineered domains and single domain scaffolds other than those derived from antibodies. Any VHHs known in the art or developed de novo may be used to construct the CARs described herein. The VHHs may be derived from any species including, but not limited to mouse, rat, human, camel, llama, lamprey, fish, shark, goat, rabbit, and bovine. VHHs contemplated herein also include naturally occurring VHH molecules from species other than Camelidae and sharks.

The VHH is derived from a naturally occurring single-domain antigen binding molecule known as heavy chain antibody devoid of light chains (also referred herein as “heavy chain only antibodies” ) . For clarity reasons, the variable domain derived from a heavy chain molecule naturally devoid of light chain is known herein as a VHH to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example, camel, llama, vicuna, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain molecules naturally devoid of light chain, and such VHHs are within the scope of the present application.

VHH molecules from Camelids are about 10 times smaller than IgG molecules. They are single polypeptides and can be very stable, resisting extreme pH and temperature conditions. Moreover, they can be resistant to the action of proteases which is not the case for conventional 4-chain antibodies. Furthermore, in vitro expression of VHH s produces high yield, properly folded functional VHHs. In addition, antibodies generated in Camelids can recognize epitopes other than those recognized by antibodies generated in vitro through the use of antibody libraries or via immunization of mammals other than Camelids (see, for example, WO9749805) . As such, multi-specific or multivalent CARs comprising one or more VHH domains may interact more efficiently with targets than multispecific or multivalent CARs comprising antigen binding fragments derived from conventional 4-chain antibodies such as scFvs and Fabs.

the VHH may be recombinant, CDR-grafted, humanized, camelid, de-immunized and/or in vitro generated (e.g., selected by phage display) . The amino acid sequence of the framework regions may be altered by “camelization” of specific amino acid residues in the framework regions. Camelization refers to the replacing or substitution of one or more amino acid residues in the amino acid sequence of a (naturally occurring) VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position (s) in a VHH domain of a heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein. Such “camelizing” substitutions may be inserted at amino acid positions that form and/or are present at the VH-VL interface, and/or at the so-called Camelidae hallmark 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) .

The VHH may be a humanized VHH produced by transgenic mice or rats expressing human heavy chain segments. See, e.g., US20090307787A1, U.S. Pat. No. 8,754, 287, US20150289489A1, US20100122358A1, and WO2004049794. The VHH may be affinity matured.

Naturally occurring VHH domains against a particular antigen or target, can be obtained from libraries of Camelid VHH sequences. Such methods may or may not involve screening such a library using said antigen or target, or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known per se. Such libraries and techniques are for example described in WO 99/37681, WO 01/90190, WO 03/025020 and WO 03/035694. Alternatively, improved synthetic or semi-synthetic libraries derived from VHH libraries may be used, such as VHH libraries obtained from VHH libraries by techniques such as random mutagenesis and/or CDR shuffling, as for example described in WO 00/43507.

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. A parental antibody can be any type of antibody, including for example, a fully human antibody, a humanized antibody, or an animal antibody (e.g., a camelid VHH) . The parental antibody may be already known in the art, in the market or developed de novo.

The anti-CLDN18.2 binding moiety may be derived from VHH that specifically binds to CLDN18.2, such as human CLDN18.2. The anti-CLDN18.2 VHH may comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 1. The anti-CLDN18.2 VHH may be camelid. The anti-CLDN18.2 VHH may be humanized. The anti-CLDN18.2 VHH may comprise an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. The VHH CDRs (CDR1-3) may 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 CLDN18.2 VHH may be defined according to the Kabat numbering scheme.

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

The anti-CLDN18.2 VHH may comprise three CDRs comprising: (a) a CDR1 comprising SEQ ID NO: 23 or an amino acid sequence that differs from SEQ ID NO: 23 by an amino acid addition, deletion or substitution of not more than 2 amino acids; (b) a CDR2 comprising SEQ ID NO: 24 or an amino acid sequence that differs from SEQ ID NO: 24 by an amino acid addition, deletion or substitution of not more than 2 amino acids; and (c) a CDR3 comprising SEQ ID NO: 25 or an amino acid sequence that differs from SEQ ID NO: 25 by an amino acid addition, deletion or substitution of not more than 2 amino acids. The CDRs may contain substitutions (e.g., conservative substitutions) , insertions, or deletions relative to the reference sequence, but the VHH comprising the CDRs can retain the ability to bind to CLDN18.2. The anti-CLDN18.2 VHH may be affinity matured.

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 to the amino acid sequence of SEQ ID NO: 1. A VHH sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity may contain substitutions (e.g., conservative substitutions) , insertions, or deletions relative to the reference sequence, but can retain the ability to bind to CLDN18.2. For example, a total of 1 to 10 amino acids can be substituted, inserted and/or deleted in the framework regions 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.

The anti-GUCY2C binding moiety may be derived from VHHs that specifically bind to GUCY2C, such as human GUCY2C. The anti-GUCY2C VHH may comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 2. The anti-GUCY2C VHH may be camelid. The anti-GUCY2C VHH may be humanized. The anti-GUCY2C VHH may comprise an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. The VHH CDRs (CDR1-3) may 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 GUCY2C VHH may be defined according to the Kabat numbering scheme.

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

The anti-GUCY2C VHH may comprise three CDRs comprising: (a) a CDR1 comprising SEQ ID NO: 26 or an amino acid sequence that differs from SEQ ID NO: 26 by an amino acid addition, deletion or substitution of not more than 2 amino acids; (b) a CDR2 comprising SEQ ID NO: 27 or an amino acid sequence that differs from SEQ ID NO: 27 by an amino acid addition, deletion or substitution of not more than 2 amino acids; and (c) a CDR3 comprising SEQ ID NO: 28 or an amino acid sequence that differs from SEQ ID NO: 28 by an amino acid addition, deletion or substitution of not more than 2 amino acids. The CDRs may contain substitutions (e.g., conservative substitutions) , insertions, or deletions relative to the reference sequence, but the VHH comprising the CDRs can retain the ability to bind to GUCY2C. The anti-GUCY2C VHH may be affinity matured.

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 to the amino acid sequence of SEQ ID NO: 2. A VHH sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity may contain substitutions (e.g., conservative substitutions) , insertions, or deletions relative to the reference sequence, but can retain the ability to bind to GUCY2C. For example, a total of 1 to 10 amino acids can be substituted, inserted and/or deleted in the framework regions 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.

Peptide linkers

The various VHHs in the multi-specific CAR constructs described herein may be fused to each other via peptide linkers. The VHHs may be directly fused to each other without any peptide linkers. The peptide linkers connecting different VHHs may be the same or different. Different domains of the CARs may also be fused to each other via peptide linkers.

Each peptide linker in a CAR may have the same or different length and/or sequence depending on the structural and/or functional features of the single-domain antibodies and/or the various domains. Each peptide linker may be selected and optimized independently. The length, the degree of flexibility and/or other properties of the peptide linker (s) used in the CARs may have some influence on properties, including but not limited to the affinity, specificity or avidity for one or more particular antigens or epitopes. For example, longer peptide linkers may be selected to ensure that two adjacent domains do not sterically interfere with one another. For example, in a multivalent or multi-specific CAR construct of the present application that comprise VHHs directed against a multimeric antigen, the length and flexibility of the peptide linkers may be such that it allows each VHH in the CAR to bind to the antigenic determinant on each of the subunits of the multimer. A short peptide linker may be disposed between the transmembrane domain and the intracellular signaling domain of a CAR. A peptide linker comprises flexible residues (such as glycine and serine) so that the adjacent domains are free to move relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker.

The peptide linker can be of 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 may 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 may be any of about 1 amino acid to about 10 amino acids, about 1 amino acids 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 long, 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.

The peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of heavy chain only antibodies may be used as the linker. See, for example, WO1996/34103. In some embodiments, the peptide linker is a flexible linker. 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. The peptide linker may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 31-37.

Transmembrane domain

The CARs of the present application comprise a transmembrane domain that can be directly or indirectly fused to the extracellular antigen binding region. The transmembrane domain may be derived either from a natural or from a synthetic source. As used herein, a “transmembrane domain” refers to any protein structure that is thermodynamically stable in a cell membrane, for example, a eukaryotic cell membrane. Transmembrane domains compatible for use in the CARs described herein may be obtained from a naturally occurring protein. Alternatively, it can be a synthetic, non-naturally occurring protein segment, e.g., a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

Transmembrane domains are classified based on the three dimensional structure of the transmembrane domain. For example, transmembrane domains may form an alpha helix, a complex of more than one alpha helix, a beta-barrel, or any other stable structure capable of spanning the phospholipid bilayer of a cell. Furthermore, transmembrane domains may also or alternatively be classified based on the transmembrane domain topology, including the number of passes that the transmembrane domain makes across the membrane and the orientation of the protein. For example, single-pass membrane proteins cross the cell membrane once, and multi-pass membrane proteins cross the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7 or more times) . Membrane proteins may be defined as Type I, Type II or Type III depending upon the topology of their termini and membrane-passing segment (s) relative to the inside and outside of the cell. Type I membrane proteins have a single membrane-spanning region and are oriented such that the N-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the C-terminus of the protein is present on the cytoplasmic side. Type II membrane proteins also have a single membrane-spanning region but are oriented such that the C-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the N-terminus of the protein is present on the cytoplasmic side. Type III membrane proteins have multiple membrane-spanning segments and may be further sub-classified based on the number of transmembrane segments and the location of N-and C-termini.

The transmembrane domain of the CAR described herein may be derived from a Type I single-pass membrane protein. In some embodiments, transmembrane domains from multi-pass membrane proteins may also be compatible for use in the CARs described herein. Multi-pass membrane proteins may comprise a complex (at least 2, 3, 4, 5, 6, 7 or more) alpha helices or a beta sheet structure. For example, the N-terminus and the C-terminus of a multi-pass membrane protein are present on opposing sides of the lipid bilayer, e.g., the N-terminus of the protein is present on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is present on the extracellular side.

The transmembrane domain of the CAR as disclosed herein may comprise a transmembrane domain chosen from the transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDl la, CD18) , ICOS (CD278) , 4-1BB (CD137) , GITR, CD40, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRFl) , CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226) , SLAMF4 (CD244, 2B4) , CD84, CD96 (Tactile) , CEACAM1, CRT 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 domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1.

The transmembrane domain may be derived from CD8α. In some embodiments, the transmembrane domain is a transmembrane domain of CD8α 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 a transmembrane domain of ICOS 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%identical to the amino acid sequence set forth in any one of SEQ ID NOs: 5-6.

Transmembrane domains for use in the CARs described herein can also comprise at least a portion of a synthetic, non-naturally occurring protein segment. The transmembrane domain may be a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment is at least approximately 20 amino acids, e.g., 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 by reference herein.

Intracellular signaling domain

The CARs as disclosed herein may comprise one or more intracellular signaling domains. The intracellular signaling domain is responsible for activation of at least one of the normal effector functions of the immune cell expressing the CARs. The term “effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus, the term “intracellular signaling domain” refers to the intracellular portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire cytoplasmic signaling domain can be employed, in many cases it is not necessary to use the entire chain.

The intracellular signaling domain may comprise a primary intracellular signaling domain of an immune cell. The intracellular signaling domain may consist essentially of a primary intracellular signaling domain of an immune cell. “Primary intracellular signaling domain” refers to cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector functions. The primary intracellular signaling domain may contain a signaling motif known as immunoreceptor tyrosine-based activation motif, or ITAM. An “ITAM” as used herein, is a conserved protein motif that is generally present in the tail portion of signaling molecules expressed in many immune cells. ITAMs within signaling molecules are important for signal transduction within the cell, which is mediated at least in part by phosphorylation of tyrosine residues in the ITAM following activation of the signaling molecule. ITAMs may also function as docking sites for other proteins involved in signaling pathways. Exemplary ITAM-containing primary cytoplasmic signaling sequences include those derived from CD3ζ, FcR gamma (FCER1G) , FcR beta (Fc Epsilon Rib) , CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. The intracellular signaling domain may consist of the cytoplasmic signaling domain of CD3ζ. The primary intracellular signaling domain may be a cytoplasmic signaling domain of wildtype CD3ζ. The primary intracellular signaling domain is a functional mutant of the cytoplasmic signaling domain of CD3ζ containing one or more mutations. The primary intracellular signaling domain of wildtype CD3ζ may comprise the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence at least 85%, 90%or 95%identical to SEQ ID NO: 11.

Co-stimulatory signaling domain

Many immune cells require co-stimulation, in addition to stimulation of an antigen-specific signal, to promote cell proliferation, differentiation and survival, as well as to activate effector functions of the cell. The CARs as disclosed herein comprising one or more operably linked VHHs may comprise at least one co-stimulatory signaling domain. The multi-specific CAR construct as disclosed herein comprising two spit CAR polypeptides may comprise at least one co-stimulatory signaling domain in one of the polypeptides. The term “co-stimulatory signaling domain” as used herein, refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response such as an effector function. The co-stimulatory signaling domain of the chimeric receptor described herein can be a cytoplasmic signaling domain from a co-stimulatory protein, which transduces a signal and modulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils. “Co-stimulatory signaling domain” can be the cytoplasmic portion of a co-stimulatory molecule. The term "co-stimulatory molecule" refers to a cognate binding partner on an immune cell (such as T cell) that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the immune cell, such as, but not limited to, proliferation and survival.

The intracellular signaling domain may comprise a single co-stimulatory signaling domain. The intracellular signaling domain may comprise two or more (such as about any of 2, 3, 4, or more) co-stimulatory signaling domains, e.g., two or more of the same co-stimulatory signaling domains, or two or more co-stimulatory signaling domains from different co-stimulatory proteins. The intracellular signaling domain may comprise a primary intracellular signaling domain (such as cytoplasmic signaling domain of CD3ζ) and one or more co-stimulatory signaling domains. The one or more co-stimulatory signaling domains and the primary intracellular signaling domain (such as cytoplasmic signaling domain of CD3ζ) may be fused to each other via optional peptide linkers. The primary intracellular signaling domain, and the one or more co-stimulatory signaling domains may be arranged in any suitable order. One or more co-stimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (such as cytoplasmic signaling domain of CD3ζ) . Multiple co-stimulatory signaling domains may provide additive or synergistic stimulatory effects.

Activation of a co-stimulatory signaling domain in a host cell (e.g., an immune cell) may induce the cell to increase or decrease the production and secretion of cytokines, phagocytic properties, proliferation, differentiation, survival, and/or cytotoxicity. The co-stimulatory signaling domain of any co-stimulatory molecule may be compatible for use in the CARs described herein. The type (s) of co-stimulatory signaling domain is selected based on factors such as the type of the immune cells in which the effector molecules would be expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC effect) . Examples of co-stimulatory signaling domains for use in the CARs can be the cytoplasmic signaling domain of co-stimulatory proteins, including, without limitation, 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) ; 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-alpha/TNF-beta, OX40/TNFRSF4, OX40 Ligand/TNFSF4, RELT/TNFRSF19L, TACI/TNFRSF13B, TL1A/TNFSF15, TNF-alpha, 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 alpha 4/CD49d, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/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.

In some embodiments, the co-stimulatory signaling domains are 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, ligands of CD83 and combinations thereof.

The intracellular signaling domain in the CAR of the present application may comprise a co-stimulatory signaling domain derived from 4-1BB (i.e. CD137) . The intracellular signaling domain comprises a primary intracellular signaling domain of CD3ζ and a co-stimulatory signaling domain of 4-1BB. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain of 4-1BB comprising an amino acid sequence of SEQ ID NO: 7, or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 7.

The intracellular signaling domain in the CAR of the present application may comprise a co-stimulatory signaling domain derived from CD28. The intracellular signaling domain comprises a primary intracellular signaling domain of CD3ζ and a co-stimulatory signaling domain of CD28. The intracellular signaling domain may comprise a co-stimulatory signaling domain of CD28 comprising an amino acid sequence of SEQ ID NO: 8, or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 8.

The intracellular signaling domain in the CAR of the present application may comprise a co-stimulatory signaling domain derived from ICOS. The intracellular signaling domain may comprise a primary intracellular signaling domain of CD3ζ and a co-stimulatory signaling domain of ICOS. The intracellular signaling domain may comprise a co-stimulatory signaling domain of ICOS comprising an amino acid sequence of SEQ ID NO: 9, or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 9.

The intracellular signaling domain in the CAR of the present application may comprise a co-stimulatory signaling domain derived from NTB-A. The intracellular signaling domain comprises a primary intracellular signaling domain of CD3ζ and a co-stimulatory signaling domain of NTB-A. The intracellular signaling domain may comprise a co-stimulatory signaling domain of NTB-A comprising an amino acid sequence of SEQ ID NO: 10, or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 10.

Also within the scope of the present disclosure are variants of any of the co-stimulatory signaling domains described herein, such that the co-stimulatory signaling domain is capable of modulating the immune response of the immune cell. The co-stimulatory signaling domains may comprise up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5, or 8) as compared to a wild-type counterpart. Such co-stimulatory signaling domains comprising one or more amino acid variations may be referred to as variants. Mutation of amino acid residues of the co-stimulatory signaling domain may result in an increase in signaling transduction and enhanced stimulation of immune responses relative to co-stimulatory signaling domains that do not comprise the mutation. Mutation of amino acid residues of the co-stimulatory signaling domain may result in a decrease in signaling transduction and reduced stimulation of immune responses relative to co-stimulatory signaling domains that do not comprise the mutation.

Hinge domain

The CARs of the present application may comprise a hinge domain that is located between the extracellular antigen binding domain and the transmembrane domain. A hinge domain is an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen binding domain relative to the transmembrane domain of the effector molecule can be used.

The hinge domain may contain about 10-100 amino acids, e.g., about any one of 15-75 amino acids, 20-50 amino acids, or 30-60 amino acids. IThe hinge domain may be at least about any one of 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 in length.

The hinge domain may be a hinge domain of a naturally occurring protein (e.g. an immunoglobulin) . Hinge domains of any protein known in the art to comprise a hinge domain are compatible for use in the chimeric receptors described herein. The hinge domain may be at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8α. The hinge domain may be a portion of the hinge domain of CD8α, e.g., 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 may comprise an amino acid sequence of SEQ ID NO: 4, or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 4.

Hinge domains of antibodies, such as an IgG, IgA, IgM, IgE, or IgD antibodies, are also compatible for use in the pH-dependent chimeric receptor systems described herein. The hinge domain may be the hinge domain that joins the constant domains CH1 and CH2 of an antibody. The hinge domain may be of an antibody and comprises the hinge domain of the antibody and one or more constant regions of the antibody. The hinge domain may comprise the hinge domain of an antibody and the CH3 constant region of the antibody. The hinge domain may comprise the hinge domain of an antibody and the CH2 and CH3 constant regions of the antibody. The antibody may be an IgG, IgA, IgM, IgE, or IgD antibody. The antibody may be an IgG1, IgG2, IgG3, or IgG4 antibody. The hinge region may comprise the hinge region and the CH2 and CH3 constant regions of an IgG1 antibody. The hinge region may comprise the hinge region and the CH3 constant region of an IgG1 antibody.

Non-naturally occurring peptides may also be used as hinge domains for the chimeric receptors described herein. The hinge domain located between the C-terminus of the extracellular ligand-binding domain of an Fc receptor and the N-terminus of the transmembrane domain may be a peptide linker, such as a (GxS) n linker, wherein x and n, independently can be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

Signal peptide

The CARs of the present application may comprise a signal peptide (also known as a signal sequence) at the N-terminus of the polypeptide. In general, signal peptides are peptide sequences that target a polypeptide to the desired site in a cell. The signal peptide may target the effector molecule to the secretory pathway of the cell and will allow for integration and anchoring of the effector molecule into the lipid bilayer. Signal peptides including signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences, which are compatible for use in the CARs described herein will be evident to one of skill in the art.

The signal peptide may be derived from a molecule 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 an amino acid sequence of SEQ ID NO: 3, or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 3.

Engineered T-cell receptor (TCR)

The engineered receptor can be a modified T-cell receptor or engineered T-cell receptor. The engineered TCR can be specific for a tumor antigen. The tumor antigen can be selected from Claudin 18.2 and GUGY2C. 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 of the TCRs known in the art can be used. The TCR can have an enhanced affinity to the tumor antigen. Exemplary TCRs and methods for introducing the TCRs to immune cells have been described, for example, in U.S. Pat. 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 the entirety.

The TCR receptor complex is an octomeric complex formed by variable TCR receptor α and β chains (or γ and δ chains on case of γδ T cells) with three dimeric signaling modules CD3δ/ε, CD3γ/ε and CD247 (T-cell surface glycoprotein CD3 zeta chain) ζ/ζ or ζ/η. Ionizable residues in the transmembrane domain of each subunit form a polar network of interactions that hold the complex together. TCR complex has the function of activating signaling cascades in T cells.

The engineered receptor can be an engineered TCR comprising one or more T-cell receptor (TCR) fusion proteins (TFPs) . Exemplary TFPs have been described, for example, in US20170166622A1, which is incorporated herein by reference in its entirety. The TFP can comprise an extracellular domain of a TCR subunit that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. The TFP can comprise a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. The TFP can comprise a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.

The TFP can comprise a TCR subunit comprising at least a portion of a TCR extracellular domain, and a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon; and an antigen binding domain, wherein the TCR subunit and the antigen binding domain are operatively linked, and wherein the TFP incorporates into a TCR when expressed in a T cell.

T-cell antigen coupler (TAC) receptor

The engineered receptor can be a T-cell antigen coupler (TAC) receptor. Exemplary TAC receptors have been described, for example, in US20160368964A1, which is incorporated herein by reference. The TAC can comprise an antigen binding domain, a TCR-binding domain that specifically binds a protein associated with the TCR complex, and a T-cell receptor signaling domain. The antigen binding domain can be an antibody fragment, such as scFv or VHH, which specifically binds to a tumor antigen. The antigen binding domain can be a designed Ankyrin repeat (DARPin) polypeptide. The tumor antigen can be selected from CLDN18.2 and GUGY2C. 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. The protein associated with the TCR complex can be CD3, such as CD3E. The TCR-binding domain can be a single chain antibody, such as scFv, or a V_HH. The TCR-binding domain can be derived from UCHT1. The TAC receptor can comprise a cytosolic domain and a transmembrane domain. The T-cell receptor signaling domain can comprise a cytosolic domain derived from a TCR co-receptor. Exemplary TCR co-receptors include, but are not limited to, CD4, CD8, CD28, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD 154. The TAC receptor can comprise a transmembrane domain and a cytosolic domain derived from CD4. The TAC receptor can comprise a transmembrane domain and a cytosolic domain derived from CD8 (such as CD8α) .

T cell co-receptors are expressed as membrane proteins on T cells. They can provide stabilization of the TCR: peptide: MEC complex and facilitate signal transduction. The two subtypes of T cell co-receptor, CD4 and CD8, display strong specificity for particular MEC classes. The CD4 co-receptor can only stabilize TCR: MEC II complexes while the CD8 co-receptor can only stabilize the TCR: MEC I complex. The differential expression of CD4 and CD8 on different T cell types results in distinct T cell functional subpopulations. CD8+ T cells are cytotoxic T cells.

Nucleic Acids and Vectors

The present disclosure also provides nucleic acid molecules encoding the engineered receptors (e.g., CARs) described herein. The nucleic acid molecule may be provided as a messenger RNA transcript or as a DNA construct.

In one aspect, provided herein is a nucleic acid comprising: (1) a nucleic acid sequence encoding the multi-specific CAR construct as disclosed herein; (2) a first nucleic acid sequence encoding a first engineered receptor specifically targeting CLDN18.2 comprising: a first extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a first transmembrane domain and a first intracellular signaling domain, and a second nucleic acid sequence encoding a second engineered receptor specifically targeting GUCY2C comprising: a second extracellular antigen binding domain comprising at least one polypeptide comprising the anti-GUCY2C binding moiety, a second transmembrane domain and a second intracellular signaling domain, 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) .

Accordingly, an isolated nucleic acid molecule may encode a chimeric antigen receptor (CAR) , wherein the CAR comprises extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety (e.g., anti-CLDN18.2 VHH) , a transmembrane domain, and an intracellular signaling domain comprising e.g., a costimulatory signaling domain and/or a primary signaling domain, e.g., zeta chain. The anti-CLDN18.2 binding moiety is an anti-CLDN18.2 VHH described herein, e.g., a VHH comprising an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence with 95-99%identity thereof. The nucleic acid molecule may encode an amino acid sequence set forth in SEQ ID NO: 12 or 19.

An isolated nucleic acid molecule may encode a chimeric antigen receptor (CAR) , wherein the CAR comprises extracellular antigen binding domain comprising at least one anti-GUCY2C binding moiety (e.g., anti-GUCY2C VHH) , a transmembrane domain, and an intracellular signaling domain comprising e.g., a costimulatory signaling domain and/or a primary signaling domain, e.g., zeta chain. The anti-GUCY2C binding moiety is an anti-GUCY2C VHH described herein, e.g., a VHH comprising an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence with 95-99%identity thereof. The nucleic acid molecule may encode an amino acid sequence set forth in any one of SEQ ID NO: 13 and 20-22.

In some embodiments, provided herein is an isolated nucleic acid molecule encoding a multi-specific chimeric antigen receptor (CAR) construct, wherein the multi-specific CAR construct comprises an extracellular antigen binding domain comprising an anti-GUCY2C binding moiety (e.g., anti-GUCY2C VHH) operably linked to an anti-CLDN18.2 binding moiety (e.g., anti-CLDN18.2 VHH) , a transmembrane domain, and an intracellular signaling domain comprising e.g., a co-stimulatory signaling domain and/or a primary intracellular signaling domain, e.g., zeta chain. The anti-GUCY2C binding moiety is an anti-GUCY2C VHH described herein, e.g., a VHH comprising an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence with 95-99%identity thereof, and the anti-CLDN18.2 binding moiety is an anti-CLDN18.2 VHH described herein, e.g., a VHH comprising an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence with 95-99%identity thereof.

In some embodiments, provided herein is an isolated nucleic acid molecule encoding a first CAR specifically targeting CLDN18.2 operably linked to a second CAR specifically targeting GUCY2C, wherein the first CAR comprises an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety (e.g., anti-CLDN18.2 VHH) , a first transmembrane domain and a first intracellular signaling domain, and the second CAR comprises an extracellular antigen binding domain comprising at least one anti-GUCY2C binding moiety (e.g., anti-GUCY2C VHH) , a second transmembrane domain and a second intracellular signaling domain, wherein the first CAR and the second CAR is operably linked via a self-cleavable peptide (such as P2A, E2A, F2A or T2A) . The GUCY2C binding moiety is an anti-GUCY2C VHH described herein, e.g., a VHH comprising an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence with 95-99%identity thereof, and the CLDN18.2 binding moiety is an anti-CLDN18.2 VHH described herein, e.g., a VHH comprising an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence with 95-99%identity thereof.

In some embodiments, provided herein is an isolated nucleic acid molecule encoding a CAR polypeptide comprising a signal peptide of SEQ ID NO: 3 (or a sequence with 95-99%identity thereof) , an anti-CLDN18.2 VHH having an amino acid sequence of SEQ ID NO: 1 or a sequence with 95-99%identity thereof, an anti-GUCY2C VHH having an amino acid sequence of SEQ ID NO: 2 or a sequence with 95-99%identity thereof, a hinge region of SEQ ID NO: 4 (or a sequence with 95-99%identity thereof) , a transmembrane domain having a sequence of SEQ ID NO: 5 or 6 (or a sequence with 95-99%identity thereof) , a co-stimulatory signaling domain having a sequence of any of SEQ ID NOs: 7-10 (or a sequence with 95-99%identity thereof) , and a CD3 zeta primary intracellular signaling domain having a sequence of SEQ ID NO: 11 (or a sequence with 95-99%identity thereof) . In some specific embodiments, the nucleic acid molecule encodes an amino acid sequence of SEQ ID NO: 14 or 15.

The isolated nucleic acid molecule may comprise two nucleic acid sequences. The first nucleic acid sequence may encode a first CAR comprising a signal peptide of SEQ ID NO: 3 (or a sequence with 95-99%identity thereof) , an anti-CLDN18.2 VHH having an amino acid sequence of SEQ ID NO: 1 or a sequence with 95-99%identity thereof, a hinge region of SEQ ID NO: 4 (or a sequence with 95-99%identity thereof) , a transmembrane domain having a sequence of SEQ ID NO: 5 or 6 (or a sequence with 95-99%identity thereof) , a co-stimulatory signaling domain having a sequence of any of SEQ ID NOs: 7-10 (or a sequence with 95-99%identity thereof) , and a CD3 zeta primary intracellular signaling domain having a sequence of SEQ ID NO: 11 (or a sequence with 95-99%identity thereof) , and the second nucleic acid sequence may encode a second CAR comprising a signal peptide of SEQ ID NO: 3 (or a sequence with 95-99%identity thereof) , an anti-GUCY2C VHH having an amino acid sequence of SEQ ID NO: 2 or a sequence with 95-99%identity thereof, a hinge region of SEQ ID NO: 4 (or a sequence with 95-99%identity thereof) , a transmembrane domain having a sequence of SEQ ID NO: 5 or 6 (or a sequence with 95-99%identity thereof) , a co-stimulatory signaling domain having a sequence of any of SEQ ID NOs: 7-10 (or a sequence with 95-99%identity thereof) , and a CD3 zeta primary intracellular signaling domain having a sequence of SEQ ID NO: 11 (or a sequence with 95-99%identity thereof) , wherein 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) . The nucleic acid molecule may encode the amino acid sequence of SEQ ID NO: 12 or 19 operably linked to the amino acid sequence of any of SEQ ID NOs: 13 and 20-22. The nucleic acid molecule may encode an amino acid sequence set forth in any one of SEQ ID NOs: 16-18.

In some embodiments, the nucleic acid comprises a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 14-18. In some embodiments, the nucleic acid comprises a first nucleic acid sequence encoding a first polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 12 and 19, and a second nucleic acid sequence encoding a second polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 13 and 20-22.

The present disclosure further encompasses nucleic acid molecules that have a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to the nucleic acid sequences described above. The nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, using standard techniques. For example, the gene of interest can be produced synthetically or cloned.

The present disclosure also provides vectors in which a nucleic acid sequence as disclosed herein is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. A retroviral vector may also be, e.g., a gamma retroviral vector.

A gamma retroviral vector may include, e.g., a promoter, a packaging signal (ψ) , a primer binding site (PBS) , one or more (e.g., two) long terminal repeats (LTR) , and a transgene of interest, e.g., a gene encoding a CAR. A gamma retroviral vector may lack viral structural gens such as gag, pol, and env. Exemplary gammaretroviral vectors include Murine Leukemia Virus (MLV) , Spleen-Focus Forming Virus (SFFV) , and Myeloproliferative Sarcoma Virus (MPSV) , and vectors derived therefrom. Other gamma retroviral vectors are described, e.g., in Tobias Maetzig et al., "Gammaretroviral Vectors: Biology, Technology and Application" Viruses. 2011 Jun; 3 (6) : 677-713.

The vector comprising the nucleic acid encoding the desired CAR of the disclosure may be an adenoviral vector (A5/35) . The expression of nucleic acids encoding CARs can be accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See below June et al. 2009Nature Reviews Immunology 9.10: 704-716, is incorporated herein by reference.

In brief summary, the expression of natural or synthetic nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

The expression constructs of the present disclosure may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5, 399, 346, 5, 580, 859, 5, 589, 466, incorporated by reference herein in their entireties. In another embodiment, the disclosure provides a gene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY) , and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6, 326, 193) .

A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

The vector may also contain a selectable marker gene or a reporter gene to select cells expressing the CAR from the population of host cells transfected through lentiviral vectors. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences to enable expression in the host cells. For example, the vector may contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid sequences.

In some embodiments, the vector comprises more than one nucleic acid encoding CARs. In some embodiments, the vector comprises a nucleic acid comprising 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 operably 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 linker.

As used herein, a “self-cleaving peptide” or “2A linker” refers to an oligopeptide that allow multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation. 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 of skill in the art, including, without limitation, those found in members of the Picornaviridae virus family, e.g., foot-and-mouth disease virus (FMDV) , equine rhinitis A virus (ERAV0, Thosea asigna virus (TaV) , and porcine tescho virus-1 (PTV-1) ; and carioviruses such as Theilovirus and encephalomyocarditis viruses. 2A linkers derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as “F2A, ” “E2A, ” “P2A, ” and “T2A, ” respectively. The P2A linker 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 linker 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.

The vector may comprise two or more nucleic acid sequences encoding a first CAR, e.g., a CLDN18.2 CAR described herein and a second CAR, e.g., a GUCY2C CAR. The two or more nucleic acid sequences encoding the CARs may be encoded by a single nucleic molecule in the same frame and as a single polypeptide chain. The two or more CARs, can, e.g., be separated by one or more peptide cleavage sites, (e.g., an auto-cleavage site or a substrate for an intracellular protease) .

Engineered Immune Cells

In one aspect, provided herein is an engineered immune cell comprising the multi-specific CAR, the nucleic acid or the vector as disclosed herein. Accordingly, the disclosure provides an engineered immune cell, e.g., a T cell or a NK cell, and methods of their use for adoptive therapy.

In some embodiments, the engineered immune cell comprises an engineered receptor specifically targeting CLDN18.2 comprising: an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a transmembrane domain, and optionally an intracellular signaling domain; and an engineered immune cell comprising an engineered receptor specifically targeting GUGY2C comprising: an extracellular antigen binding domain comprising at least one anti-GUCY2C binding moiety, a transmembrane domain, and optionally an intracellular signaling domain. In some embodiments, the engineered immune cell comprises: (1) a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 14-18; or (2) a first polypeptide and a second polypeptide, wherein the first polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 12 and 19, and the second polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 13 and 20-22.

“Immune cells” are immune cells that can perform immune effector functions. Examples of immune cells include peripheral blood mononuclear cells (PBMC) , natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.

In some embodiments, the immune cells are T cells. The T cells may be αβ T cells, or γδ T cells. The T cells may be CD4+/CD8-, CD4-/CD8+, CD4+/CD8+, CD4-/CD8-, or combinations thereof. The T cells may produce IL-2, TFN, and/or TNF upon expressing the CAR and binding to the target cells, such as CLDN18.2+ or GUCY2C+ tumor cells. The T cells may lyse antigen-specific target cells upon expressing the CAR and binding to the target cells.

In some embodiments, the immune cells are NK cells. The immune cells can be established cell lines, for example, NK-92 cells.

In some embodiments, the immune cells are differentiated from a stem cell, such as a hematopoietic stem cell, a pluripotent stem cell, an iPS, or an embryonic stem cell.

The engineered immune cells as disclosed herein may be prepared by introducing the CARs into the immune cells, such as T cells. In some embodiments, the CAR is introduced to the immune cells by transfecting any one of the isolated nucleic acids or any one of the vectors described herein. The CAR may also be introduced to the immune cells by inserting proteins into the cell membrane while passing cells through a microfluidic system (see, for example, U.S. Patent Application Publication No. 20140287509) .

Methods of introducing vectors or isolated nucleic acids into a mammalian cell are known in the art. The vectors described can be transferred into an immune cell by physical, chemical, or biological methods.

Physical methods for introducing the vector into an immune cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising 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 Spring Harbor Laboratory, New York. The vector may be introduced into the cell by electroporation. Biological methods for introducing the vector into an immune cell 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 the vector into an immune cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro is a liposome (e.g., an artificial membrane vesicle) .

The transduced or transfected immune cell may be propagated ex vivo after introduction of the vector or isolated nucleic acid. In some embodiments, the transduced or transfected immune cell is cultured to propagate for at least about any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days. The transduced or transfected immune cell may be further evaluated or screened to select the engineered mammalian cell.

Reporter genes may be used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al. FEBS Letters 479: 79-82 (2000) ) . Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.

Other methods to confirm the presence of the nucleic acid encoding the CARs in the engineered immune cell, include, for example, molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological methods (such as ELISAs and Western blots) .

Prior to expansion and genetic modification of the T cells, a source of T cells can be obtained from an individual. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Any number of T cell lines available in the art, may be used. T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll separation. Cells from the circulating blood of an individual may be obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. The cells are washed with phosphate buffered saline (PBS) . The wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Again, surprisingly, initial activation steps in the absence of calcium lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca²⁺-free, Mg²⁺-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

T cells may be isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+T cells, can be further isolated by positive or negative selection techniques.

Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. It may be desirable to enrich for or positively select for regulatory T cells which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.

T cells may be obtained from a patient directly following treatment. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present disclosure to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase. Further, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

Whether prior to or after genetic modification of the T cells with the CARs described herein, the T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. 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.

Pharmaceutical compositions

Further provided by the present application are pharmaceutical compositions comprising any one of the engineered immune cells comprising any one of the CARs as described herein, and a pharmaceutically acceptable carrier. Pharmaceutical compositions can be prepared by mixing the engineered immune cells having the desired degree of 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 lyophilized formulations or aqueous solutions.

Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, Vitamin E, sodium metabisulfite; preservatives, isotonicifiers, stabilizers, metal complexes (e.g. Zn-protein complexes) ; chelating agents such as EDTA and/or non-ionic surfactants.

In order for the pharmaceutical compositions to be used for in vivo administration, they must be sterile. The pharmaceutical composition may be rendered sterile by filtration through sterile filtration membranes. The pharmaceutical compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical administration, inhalation or by sustained release or extended-release means.

Methods and Uses

In one aspect, the present disclosure provides a method for treating a tumor in a subject in need thereof, comprising administering an effective amount of a combination of a CLDN18.2 antagonist and a GUCY2C antagonist, or an antagonist of CLDN18.2 and GUCY2C to the subject. The tumor to be treated may be a CLDN18.2 positive tumor, a GUCY2C positive tumor or a CLDN18.2 and GUCY2C double positive tumor. Such methods and uses include therapeutic methods and uses, for example involving administration of the molecules, cells, or compositions containing the same, to a subject having a disease, condition, or disorder expressing or associated with Claudin18.2 and/or GUCY2C expression, and/or in which cells or tissues express Claudin18.2 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.

The present application further relates to methods and compositions for use in cell immunotherapy. In some embodiments, the cell immunotherapy is for treating cancer, including but not limited to CLDN18.2 positive cancer, GUCY2C positive cancer and CLDN18.2×GUCY2C double positive cancer. Any of the chimeric antigen receptors, nucleic acids and engineered immune cells described herein may be used in the method of treating cancer. The CARs described herein may be useful for treating tumors having antigen loss escape mutations, and for reducing resistance to existing therapies. In some embodiments, the methods and compositions described herein may be used for treating other diseases that are associated with the CLDN18.2 and/or GUCY2C.

In some embodiments, there is provided a method of treating a cancer in an individual (such as a human individual) , comprising administering to the individual an effective amount of a pharmaceutical composition comprising: (1) an engineered immune cell (such as T cell) comprising a CAR as disclosed herein comprising a polypeptide comprising: (a) an extracellular antigen binding region comprising an anti-CLDN18.2 binding moiety and an anti-GUCY2C binding moiety; (b) a transmembrane domain; and (c) an intracellular signaling domain; and (2) a pharmaceutically acceptable carrier. The anti-CLDN18.2 binding moiety and an anti-GUCY2C binding moiety may be VHHs, such as camelid, chimeric, human, or humanized VHHs. 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 peptide linker may be no more than about 50 (such as no more than about any one of 35, 25, 20, 15, 10, or 5) amino acids long.

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

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

The engineered immune cell may be autologous. The engineered immune cell may be allogenic. In some embodiments, the cancer is a solid cancer, including but not limited to, gastric cancer, esophageal cancer, pancreatic ductal cancer, lung cancer such as non-small cell lung cancer (NSCLC) , ovarian cancer, colorectal cancer, liver cancer, head and neck cancer, gallbladder cancer and its metastasis.

The methods are applicable to cancers of all stages, including early stage, advanced stage and metastatic cancer. The methods described herein may be used as a first therapy, second therapy, third therapy, or combination therapy 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, radio-frequency ablation or the like, in an adjuvant setting or a neoadjuvant setting.

Administration of the pharmaceutical compositions may be carried out in any convenient manner, including by injection, ingestion, transfusion, implantation or transplantation. The compositions may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously, or intraperitoneally. In some embodiments, the pharmaceutical composition is administered systemically. The pharmaceutical composition may be administered to an individual by infusion, such as intravenous infusion. Infusion techniques for immunotherapy are known in the art (see, e.g., 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 compositions may be administered by intravenous injection. The compositions may be injected directly into a tumor, or a lymph node. The pharmaceutical composition may be administered locally to a site of tumor, such as directly into tumor cells, or to a tissue having tumor cells.

Dosages and desired drug concentration of pharmaceutical compositions of the present disclosure may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. “The Use of Interspecies Scaling in Toxicokinetics” In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp. 42-46. It is within the scope of the present application that different formulations will be effective for different treatments and different disorders, and that administration intended to treat a specific organ or tissue may necessitate delivery in a manner different from that to another organ or tissue.

Combination Therapy

A CAR-expressing cell described herein may be used in combination with other known agents and therapies. Administered "in combination" , as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. The delivery of one treatment may be still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery" . In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

A CAR-expressing cell described herein and the at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the CAR-expressing cell described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.

The CAR therapy and/or other therapeutic agents, procedures or modalities can be administered during periods of active disorder, or during a period of remission or less active disease. The CAR therapy can be administered before the other treatment, concurrently with the treatment, post-treatment, or during remission of the disorder.

In further aspects, a CAR-expressing cell described herein may be used in a treatment regimen in combination with surgery, cytokines, radiation, or chemotherapy such as Cytoxan, fludarabine, histone deacetylase inhibitors, demethylating agents, or peptide vaccine, such as that described in Izumoto et al. 2008 J Neurosurg 108: 963-971.

Summary of the sequences

Appended to the instant application is a sequence listing comprising a number of amino acid sequences. The following Table A-C provides a summary of the included sequences. Seven illustrative CARs as disclosed herein are designated as LG23A01, LG23A02, LG23B01, LG23B02, LG23D01, LG23D02 and LG23D03.

Table A: Amino acid sequences of the CDRs and variable domain of the VHHs

Table B: Amino acid sequences of the CARs

Table C: Other related amino acid sequences

EXAMPLES

Example 1. IHC analysis

To determine the expression of CLDN18.2 and GUCY2C in primary and metastatic gastric cancer (GC) , IHC analysis was conducted on tissue sections of 40 cases of primary gastric cancer (Tissuearray, ST810C) and 39 cases of metastatic gastric cancer (Tissuearray, ST810C) . The detection steps are as follows.

FFPE tissue slides (4 μm) were melted and dehydrated at 60 ℃ for 1h, then they were deparaffinized and rehydrated using xylene and alcohol, respectively. The paraffin slides were placed in citrate buffer (pH 6.0) , and the whole reactive system was placed in a microwave oven for heat-induced antigen retrieval. 3%hydrogen peroxide was used to block endogenous peroxidases. The blocking buffer (5%BSA) was used to block the sections for overnight. The tissue slides were then stained by using rabbit monoclonal anti-CLDN18.2 antibody (Abcam, 222513) at 2 μg/mL and rabbit anti-GUCY2C antibody at 2 μg/mL, and incubated at 37 ℃ for 2h, respectively. The tissue slides were then sufficiently washed with PBS for three times. Goat anti-rabbit IgG H&L HRP (maxim, kit-5005) were used as secondary antibody, and were dropped to cover the whole tissue slides. After incubating the tissue slides in an incubator at room temperature for 15 min, 100 μL DAB substrate solution (VECTOR LABORATORIES, SK-4105) was applied to the sections on the slides to reveal the color of antibody staining for 3 min. Then the sections were scanned, nuclear were stained with hematoxylin, and the expressions of CLDN18.2 and GUCY2C in primary and metastatic gastric cancer tissues were analyzed.

As shown in FIGs. 1A-1B and Table 1, IHC analysis result shows that CLDN18.2 and GUCY2C are highly expressed in both primary and metastatic gastric cancer tissues, and the samples expressing CLDN18.2 and GUCY2C simultaneously are 70% (28/40) and 66.67%(26/39) , respectively. This IHC study expands the coverage of tumor cells co-expressing both CLDN18.2 and GUCY2C in the same individual subject, which may be useful in guiding or improving therapeutic approaches to improve drug efficacy, prolong progression-free survival in patients, address tumor heterogeneity, and potentially expand indications for treatment.

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

Example 2. CAR Constructs Design

The present disclosure proposes the immune cell therapy strategy of co-targeting CLDN18.2 and GUCY2C to address the problems of tumor heterogeneity and target downregulation during the treatment of gastric cancer. This present disclosure provides CAR-T cells targeting both CLDN18.2 and GUCY2C, including tandem CLDN18.2×GCC bispecific CAR-T cells, split CLDN18.2×GCC bispecific CAR-T cells, and combination of single-CLDN18.2 specific CAR-T cells and single-GCC specific CAR-T cells, have strong killing effect on CLDN18.2 single-positive target cells, GUCY2C single-positive target cells, or CLDN18.2 and GUCY2C double-positive target cells. As shown in FIG. 2B, tandem bispecific CARs may comprise an extracellular antigen binding region comprising at least one CLDN18.2 binding moiety and at least one GUCY2C binding moiety, which can recognize and bind two different antigens Claudin18.2 and GUCY2C. As shown in FIG. 2C, the split bispecific CAR having two CARs targeting two antigens expressed in one cell, wherein each of two CARs has an extracellular antigen binding region, comprising at least one Claudin18.2 binding moiety or at least one GUCY2C binding moiety, respectively.

Single-CLDN18.2 specific CAR-T cells (Si-CLDN18.2 CAR-T) alone, and Single-GUCY2C specific CAR-T cells (Si-GCC CAR-T) alone were set as controls, respectively. Single-CLDN18.2 specific CAR may comprise an extracellular antigen domain comprising one or more CLDN18.2 binding moieties, and single-GUCY2C specific CAR may comprise an extracellular antigen domain comprising one or more GUCY2C binding moieties. In the present disclosure, the single-CLDN18.2 specific CAR-T cells have very low cytotoxicity on GUCY2C single-positive target cells, and single GCC-specific CAR-T cells alone have very low cytotoxicity on CLDN18.2 single-positive target cells. The combination of CLDN18.2 and GUCY2C co-targeted CAR-T cell therapy would cover more individual subjects having gastric cancer, so as to overcome tumor heterogeneity and improve drug efficacy.

To take the advantages of the immune cell therapy of co-targeting CLDN18.2 and GUCY2C in solving tumor heterogeneity, the present disclosure also provides some CAR structures with different co-stimulation signaling domains, such as CD278 (ICOS) and NTBA, which may promote the expansion of CAR-T cells or improve drug efficacy.

In order to confirm the feasibility of the immune cell therapy co-targeting CLDN18.2 and GUCY2C, 7 CAR constructs including LG23A01, LG23A02, LG23B01, LG23B01, LG23D01, LG23D02, LG23D03 and CAR-T cells were evaluated in subsequent experiments, as well as the combination CAR-T cells of Si-CLDN18.2 CAR-T cells (LG23A01) and Si-GUCY2C CAR-T cells (LG23A02) in equal proportions, which is represented as LG23A01+LG23A02. The CAR structures of the exemplary CAR-T cells provided here are shown in Table 2, and are described in detail as following.

Table 2. Structures of CAR Constructs targeting CLDN18.2 and/or GUCY2C

LG23A01 CAR comprises a CAR backbone polypeptide comprising from the N-terminus to the C-terminus: a CD8α signal peptide (SEQ ID NO: 3) , an antigen binding domain having an 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 signaling domain (SEQ ID NO: 7) , and a CD3ζ primary intracellular signaling domain (SEQ ID NO: 11) . LG23A02 CAR comprises a CAR backbone polypeptide comprising from the N-terminus to the C-terminus: a CD8α signal peptide (SEQ ID NO: 3) , an antigen binding domain having an anti-GUCY2C VHH (SEQ ID NO: 2) , a CD8α hinge domain (SEQ ID NO: 4) , a CD8αtransmembrane domain (SEQ ID NO: 5) , a 4-1BB co-stimulatory signaling domain (SEQ ID NO: 7) , and a CD3ζ primary intracellular signaling domain (SEQ ID NO: 11) . LG23B01 CAR comprises a CAR backbone polypeptide comprising from the N-terminus to the C-terminus: a CD8α signal peptide (SEQ ID NO: 3) , an antigen binding domain having an anti-GUCY2C VHH (SEQ ID NO: 2) and an anti-CLDN18.2 VHH (SEQ ID NO: 1) linked by 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 signaling domain (SEQ ID NO: 7) and a CD3ζprimary intracellular signaling domain (SEQ ID NO: 11) . LG23B02 CAR comprises a CAR backbone polypeptide comprising from the N-terminus to the C-terminus: a CD8α signal peptide (SEQ ID NO: 3) , an antigen binding domain having an anti-CLDN18.2 VHH (SEQ ID NO: 1) and an anti-GUCY2C VHH (SEQ ID NO: 2) linked by 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 signaling domain (SEQ ID NO: 7) , and a CD3ζ primary intracellular signaling domain (SEQ ID NO: 11) . The nucleotide sequence of each CAR backbone was chemically synthesized and cloned into a pre-modified lentiviral vector (pLSINK-BBzBB) downstream and operably linked to a constitutive hEF1α promoter for in vitro transcription, respectively.

LG23D01 CAR construct comprises two CAR backbone polypeptides. The first CAR backbone polypeptide comprises from the N-terminus to the C-terminus: a CD8α signal peptide (SEQ ID NO: 3) , an antigen binding domain having an 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 signaling domain (SEQ ID NO: 7) , and a CD3ζ primary intracellular signaling domain (SEQ ID NO: 11) . The second CAR backbone polypeptide comprises a CD8αsignal peptide (SEQ ID NO: 3) , an antigen binding domain having an 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 signaling domain (SEQ ID NO: 8) and a CD3ζ primary intracellular signaling domain (SEQ ID NO: 11) . A nucleic acid comprises a nucleotide sequence encoding the first CAR and the second CAR backbone linked by a self-cleaving 2A (e.g., P2A, SEQ ID NO: 29) linker, the nucleotide sequence encoding two CARs was chemically synthesized and cloned into a pre-modified lentiviral vector (pLSINK-BBzBB) downstream and operably linked to a constitutive hEF1α promoter for in vitro transcription.

LG23D02 CAR construct comprises two CAR backbone polypeptides. The first CAR backbone polypeptide comprises from the N-terminus to the C-terminus: a CD8α signal peptide (SEQ ID NO: 3) , an antigen binding domain having an 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 signaling domain (SEQ ID NO: 9) , and a CD3ζ primary intracellular signaling domain (SEQ ID NO: 11) . The second CAR backbone polypeptide comprises a CD8αsignal peptide (SEQ ID NO: 3) , an antigen binding domain having an anti-GUCY2C VHH (SEQ ID NO: 2) , a CD8α hinge domain (SEQ ID NO: 4) , a CD8α transmembrane domain (SEQ ID NO: 5) , a NTB-Aco-stimulatory signaling domain (SEQ ID NO: 10) and a CD3ζ primary intracellular signaling domain (SEQ ID NO: 11) . A nucleic acid comprises a nucleotide sequence encoding the first CAR and the second CAR backbone linked by a self-cleaving 2A (e.g., P2A, SEQ ID NO: 29) linker, the nucleotide sequence encoding two CARs was chemically synthesized and cloned into a pre-modified lentiviral vector (pLSINK-BBzBB) downstream and operably linked to a constitutive hEF1α promoter for in vitro transcription.

LG23D03 CAR construct comprises two CAR backbone polypeptides. The first CAR backbone polypeptide comprises from the N-terminus to the C-terminus: a CD8α signal peptide (SEQ ID NO: 3) , an antigen binding domain having an 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 signaling domain (SEQ ID NO: 9) , and a CD3ζ primary intracellular signaling domain (SEQ ID NO: 11) . The second CAR backbone polypeptide comprises a CD8αsignal peptide (SEQ ID NO: 3) , an antigen binding domain having an 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 signaling domain (SEQ ID NO: 9) and a CD3ζprimary intracellular signaling domain (SEQ ID NO: 11) . A nucleic acid comprises a nucleotide sequence encoding the first CAR and the second CAR backbone linked by a self-cleaving 2A (e.g., P2A, SEQ ID NO: 29) linker, the nucleotide sequence encoding two CARs was chemically synthesized and cloned into a pre-modified lentiviral vector (pLSINK-BBzBB) downstream and operably linked to a constitutive hEF1α promoter for in vitro transcription.

Example 3. Preparation of Lentiviral vectors and Engineered T Cells Expressing CARs

The lentivirus packaging plasmid mixture containing pMDLg. pRRE (Addgene#12251) , pRSV-REV (Addgene#12253) and pMD2. G (Addgene#12259) were pre-mixed with the lentiviral vectors expressing CARs constructed in Example 2 at a pre-optimized ratio with polyetherimide (PEI) , then incubated at 25 ℃ for 5 min. The transfection mixtures were then added into HEK293 cells. Afterwards, cells were incubated overnight in a cell incubator with 5%CO₂ at 37℃. The supernatants were collected after centrifuged at 4℃, 3000 g for 15 min, and followed by ultra-centrifugation through a 0.45 μm PES filter for lentivirus concentration. Then the supernatants were carefully discarded and the virus pellets were rinsed cautiously with pre-chilled DPBS. The viruses were resuspended properly, and stored at -80 ℃.

Human T cells were purified from commercialized PBMCs using Miltenyi Pan T cell isolation kit (Cat#130-096-535) , following manufacturer’s protocols as described below. Cell number was determined and the cell suspension was centrifuged at 300 g for 10 min. The supernatant was then discarded completely, and the cell pellets were re-suspended in 40 μL MACS buffer (DPBS supplemented with 8 μM EDTA + 0.5%FBS) per 10⁷ total cells. 10 μL of Pan T Cell Biotin-Antibody Cocktail was added per 10⁷ total cells, mixed thoroughly and incubated for about 5 min in the refrigerator (2～8 ℃) . 30μL of MACS buffer was then added per 10⁷ cells. 20 μL of Pan T Cell MicroBead Cocktail was added per 10⁷ cells. The cell suspension mixture was mixed well and incubated for additional 10 min in the refrigerator (2～8℃) . A minimum of 500 μL was required for magnetic separation. For magnetic separation, an LS column was placed in the magnetic field of a suitable MACS Separator. The column was prepared by rinsing with 3mL of buffer. The cell suspension was then applied onto the column, and flow-through containing the unlabeled cells was collected, which represented the enriched T cell fractions. Additional T cells were collected by washing the column with 3 mL of buffer and collecting unlabeled cells that passed through. These unlabeled cells were represented again with the enriched T cells, and were combined with the flow-through from previous step. The pooled enriched T cells were then centrifuged and re-suspended in TexMACS GMP Medium&1L (Miltenyi #170-076-309) with 300 IU/mL IL-2.

The prepared T cells were subsequently pre-activated for 48-96 h with human T cell activation/expansion kit (Miltenyi #130-091-441) according to manufacturer’s protocol in which anti-CD3/CD28 MACSiBead particles were added at a bead-to-cell ratio of 1: 2.

The pre-activated T cells were transduced with lentivirus stocks, by adding lentivirus stock directly into the culture medium (TexMACS GMP Medium supplemented with 300 IU/mL IL-2) . The transduced cells were then transferred to a cell culture incubator with 5%CO₂ at 37 ℃ for transgene expression.

On day 5, CAR expression levels were assessed by flow cytometry (NovoCyte) . Briefly, 3×10⁵ T cells were collected from each group, then incubated with anti-VHH antibody (Genscript, Cat. No. A02172) for 30 min at 4 ℃. Upon completion of incubation, cells were harvested and washed with DPBS, then centrifuged at 300 g, 20 ℃ for 5 min. As shown in Table 3, the CAR expression levels of LG23A01, LG23A02, LG23B01, LG23B01, LG23D01, LG23D02, LG23D03 CAR-T cells were 50.16%, 46.31%, 48.38%, 60.26%, 51.89%, 66.54%, 73.63%, respectively. UnT refers to the T cell un-transduced CAR. Detailed test results are shown in Table 3.

Table 3. CAR expression of CAR-T cells

Example 4. Preparation of target cells

Lentivirus were packaged by transient transfection of Lenti-X 293T host cells with a mix of plasmids including psPAX2, pMD. 2G and PLLV-CLDN18.2. Luc. PuroR or PLVX-CQBL-LUC-puro. PLLV-CLDN182. Luc. PuroR was constructed in house to expressing CLDN18.2 and Luciferase, and PLVX-CQBL-LUC-puro was constructed in house to expressing GUCY2C and Luciferase. Hep3b2.1-7 (ATCC, HB-8064) are human CLDN18.2 and GUCY2C negative cell lines. Hep3b2.1-7 cell lines were used as tool mother cells to construct three kinds of target cells, e.g., CLDN18.2 single-positive target cells, only GUCY2C single-positive target cells, and CLDN18.2 and GUCY2C double-positive target cells, for evaluating functions of CAR-T cells. Briefly, 0.5×10⁶ Hep3b2.1-7 cells were transduced with 10 μL PLLV-CLDN182. Luc. PuroR or PLVX-CQBL-LUC-puro lentivirus. Two target cell lines, named Hep3b-CLDN18.2. Luc cells and Hep3b-GUCY2C. Luc cells, were selected by puromycin and refreshing selection culture medium (EMEM with 10%FBS and 2 μg/mL puromycin) every 2-3 days. 0.5×10⁶ Hep3b2.1-7-CLDN18.2. Luc cells were transduced with 10 μL obtained PLVX-CQBL-LUC-puro lentivirus. The transduced cells were selected with puromycin to obtain the Hep3b2.1-7-CLDN18.2-GUCY2C. Luc cells by refreshing selection culture medium (EMEM with 10%FBS and 2 μg/mL puromycin) every 2-3 days. After 3 rounds of selection, the obtained cell clones were harvested by trypsinization. The obtained cells were well preserved and ready for further use. Three target cell lines, Hep3b-CLDN18.2. Luc cells only overexpress CLDN18.2, Hep3b-GUCY2C. Luc cells only express GUCY2C, and Hep3b-CLDN18.2-GUCY2C. Luc cells simultaneously express CLDN18.2 and GUCY2C, which were used for subsequent functional evaluation of CAR-T cells.

Example 5. Cytotoxicity of CAR-T cells

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 combination 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 the effector (CAR positive T cell) to target cell ratio (E: T) of 4: 1, 2: 1, 1: 1 or 0.5: 1 for 20-24 h, respectively. To assay the cytotoxicity of CAR-T cells on tumor cells, One-glo luminescent Luciferase assay reagents (Promega#E6120) were prepared according to manufacturer’s protocol, and were added to the co-cultured cells to detect the remaining luciferase activity in the wells. The remaining luciferase activity directly correlated to the number of viable target cells in the well. The specific cytotoxicity was calculated by the formula: Specific Cytotoxicity %=100%× (1- (RLU_sample-RLU_min) / (RLU_UnT-RLU_min) ) . RLU_sample represented for the Luciferase activity as measured in the well with CAR-T cells transduced with GCC CAR of the disclosure. RLU_min referred to the luciferase activity as determined in the well added with Triton X-100 at a final concentration of 1%when the cytotoxicity assay was initiated, and RLU_UnT referred to the luciferase activity as determined with T cells un-transduced with CAR.

As illustrated in FIGs. 3A-3C, Si-CLDN18.2 CAR-T cells (LG23A01) showed weakly cytotoxicity on Hep3b-GUCY2C. Luc cells. Si-GUCY2C CAR-T cells (LG23A02) showed weakly cytotoxicity on Hep3b-CLDN18.2. Luc cells. At the E: T ratio of 2: 1, 21.2%Hep3b-GUCY2C. Luc cells were lysed by LG23A01 CAR-T cells (FIG. 3C) , and 37.1%Hep3b-CLDN18.2. Luc cells were lysed by LG23A02 CAR-T cells (FIG. 3B) , as compared with UnT cells.

The bispecific CLDN18.2×GCC CAR-T cells (LG23B01, LG23B02, LG23D01, LG23D02, LG23D03) , and combination CAR-T cells (LG23A01+LG23A02) elicited potent specific cytotoxicity on Hep3b-CLDN18.2-GUCY2C. Luc cells, Hep3b-GUCY2C. Luc cells and Hep3b-CLDN18.2. Luc cells. As shown in FIG. 3A, the lysis percentage of Hep3b-CLDN18.2-GUCY2C. Luc cells were 82.20%under LG23B01 CAR-T cells, 79.11%under LG23B02 CAR-T cells, 86.08%under LG23D01 CAR-T cells, 88.07%under LG23D02 CAR-T cells, 90.41%under LG23D03 CAR-T cells, and 88.91%under LG23A01+LG23A02 CAR-T cells at the E/T ratio of 2: 1. As shown in FIG. 3B, the lysis percentage of Hep3b-CLDN18.2. Luc cells were 77.97%under LG23B01 CAR-T cells, 80.99%under LG23B02 CAR-T cells, 88.90%under LG23D01 CAR-T cells, 86.36%under LG23D02 CAR-T cells, 94.36%under LG23D03 CAR-T cells, and 80.71%under LG23A01+LG23A02 CAR-T cells at the E/T ratio of 2: 1. And as shown in FIG. 3C, the lysis percentage of Hep3b-GUCY2C. Luc cells was 74.80%under LG23B01 CAR-T cells, 74.13%under LG23B02 CAR-T cells, 84.58%under LG23D01 CAR-T cells, 90.37%under LG23D02 CAR-T cells, 87.96%under LG23D03 CAR-T cells, and 75.94%under LG23A01+LG23A02 CAR-T cells at the E/T ratio of 2: 1. The bispecific CLDN18.2×GUCY2C CAR-T cells and combination CAR-T cells (LG23A01+LG23A02) show potent killing effects to Hep3b-CLDN18.2. Luc cells, Hep3b-GUCY2C. Luc cells, and Hep3b-CLDN18.2-GUCY2C. Luc cells.

Example 6. IFN-γ released in vitro by CAR-T cells in a heterogeneity model

A tumor heterogeneity model is used to evaluate the in vitro ability of bispecific CLDN18.2×GCC CAR-T cells (LG23B01, LG23B02, LG23D01, LG23D02, LG23D03) , and combination CAR-T cells (LG23A01+LG23A02) to address tumor heterogeneity. The tumor heterogeneity model was achieved by mixing Hep3b-CLDN18.2. Luc cells and Hep3b-GUCY2C. Luc cells in different ratios as shown in Table 4.

Table 4. An in vitro tumor heterogeneity model

The effector cells and the target cells were cultured at the E/T ratio of 1: 1 in a microcellular incubator with 5%CO₂ at 37 ℃ for 20-24 h. The volume ratio of target cell complete medium and effector cell complete medium was 1: 1. Then the concentration of IFN-γ produced in the culture supernatant were measured by HTRF kit (Cisbio, Cat#62HIFNGPEG) and TNF-α were measured by HTRF kit (Cisbio, Cat#62HTNFAPEG) . Briefly, HTRF reagents were allowed to warm up to room temperature for at least 30 min before the assay. 16 μL/well supernatants from co-culture assay were transferred to 384 well assay plate (Greiner Bio-One, #784075) , followed by adding with 4 μL/well pre-mixed HTRF reagents prepared according to the kit manual. The plate was then sealed with parafilm and incubated overnight at room temperature for IFN-γ and TNF-α test. The plate was read on an HTRF compatible reader Tecan Spark 10M. IFN-γ and TNF-α concentration was calculated by referring to the signal obtained by standard curves provided by the kit.

As shown in FIG. 4, 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 IFN-γ released by LG23A01 CAR-T 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 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, which indicated that single-CLDN18.2 specific CAR-T cells (LG23A01) and single-GUCY2C specific CAR-T cells (LG23A02) against could not lyse target-negative cells in the tumor heterogeneity model. In contrast, the bispecific CLDN18.2×GUCY2C CAR-T cells (LG23B01, LG23B02, LG23D01, LG23D02, and LG23D03) , and combination CAR-T cells (LG23A01+LG23A02) can maintain a high cytokine (IFN-γ and TNF-α) release level regardless of the proportion of Hep3b-CLDN18.2 cells and Hep3b-GUCY2C. Luc cells. It indicates that bispecific CLDN18.2×GCC CAR-T cells and combination CAR-T cells (LG23A01+LG23A02) can always maintain the ability to kill Hep3b-CLDN18.2. Luc cells or Hep3b-GUCY2C. Luc cells, which is especially important for solving the problem of tumor heterogeneity.

Specifically, as shown in FIG. 4A, the IFN-γ released by LG23B01 CAR-T cells ranged from 10631.86 pg/mL to 6548.16 pg/mL, the IFN-γ released by LG23B02 CAR-T cells ranged from 18660.56 pg/mL to 7848.62 pg/mL, and the IFN-γ released by LG23D01 CAR-T cells ranged from 20229.48 pg/mL to 10848.12 pg/mL, the IFN-γ released by LG23D02 CAR-T cells ranged from 4269.91 pg/mL to 2450.15 pg/mL, the IFN-γ released by LG23D03 CAR-T cells ranged from 3933.10 pg/mL to 2709.00 pg/mL, the IFN-γ released by combination CAR-T cells (LG23A01+LG23A02) ranged from 12687.05 pg/mL to 8895.46 pg/mL.

Similarly, as shown in FIG. 4B, the bispecific CLDN18.2×GCC CAR-T cells (LG23B01, LG23B02, LG23D01, LG23D02, and LG23D03) , and combination CAR-T cells (LG23A01+LG23A02) can also maintain a higher concentration of TNF-α release levels. Specifically, the TNF-α released by LG23B01 CAR-T cells ranged from 1575.50 pg/mL to 1280.42 pg/mL, the TNF-α released by LG23B02 CAR-T cells ranged from 1750.50 pg/mL to 1198.47 pg/mL, the TNF-α released by LG23D01 CAR-T cells ranged from 3294.49 pg/mL to 2378.32 pg/mL, and the TNF-α released by LG23D02 CAR-T cells ranged from 844.85 pg/mL to 437.11 pg /mL, the TNF-α released by LG23D03 CAR-T cells ranged from 1043.96 pg/mL to 789.51 pg/mL, the TNF-α released by combination CAR-T cells (LG23A01+LG23A02) ranged from 1685.32 pg/mL to 1139.06 pg/mL.

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 the effect on target cells even though the released cytokine levels were low.

Example 7. In vitro CAR-T Cells re-challenge assay

To evaluate the persistence and exhaustion of CAR-T cells in vitro, CAR-T cells re-challenge assay model was set up. CAR-T cells were constantly stimulated by Hep3b-CLDN18.2-GUCY2C. Luc cells for several rounds. In the first round (round 1) of re-challenge, CAR-T cells were co-cultured with tumor cells at an E/T ratio of 2: 1 in 6-well plate. After 3-4 days’ co-culture, CAR-T cells were counted and detected CAR percentage by flow cytometry (NovoCyte) . Then CAR-T cells were replaced to a new plate seeded with tumor cells for another 3-4 days (round 2) . The number of tumor cells added was determined by the CAR positive percentage at the end of the former round, and the E/T ratio was maintained at 2: 1.

As illustrated in FIG. 5A, the expansion folds of total T were 207.03 folds for LG23A01 CAR-T cells after 5 rounds stimulation by Hep3b-CLDN18.2-GUCY2C. Luc cells, 413.51 folds for LG23A02 CAR-T cells, 93.28 folds for LG23B01 CAR-T cells, 100.24 folds for LG23B02 CAR-T cells, 188.94 folds for LG23D01 CAR-T cells, 482.41 folds for LG23D02 CAR-T cells, 309.91 folds for LG23D03 CAR-T cells, 2.30 folds for UnT cells, and 76.96 fold for LG23A01+ LG23A02. The data indicates that the amplification ability of these structures in re-challenge are similar with LG23A01 and LG23A02.

As illustrated in FIG. 5B, the expansion folds of CAR-T cells were 434.36 folds for LG23A01 CAR-T cells after 5 rounds stimulation by Hep3b-CLDN18.2-GUCY2C. Luc cells, 1126.29 folds for LG23A02 CAR-T cells, 225.68 folds for LG23B01 CAR-T cells, 214.23 folds for LG23B02 CAR-T cells, 428.34 folds for LG23D01 CAR-T cells, 1354.47 folds for LG23D02 CAR-T cells, 844.19 folds for LG23D03 CAR-T cells, and 151.16 fold for LG23A01+ LG23A02.

As illustrated in FIG. 5C, the CAR expression was 73.43%for LG23A01 CAR-T cells after 5 rounds stimulation by Hep3b-CLDN18.2-GUCY2C. Luc 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

A method for treating a tumor in a subject in need thereof, comprising administering an effective amount of a combination of a CLDN18.2 antagonist and a GUCY2C antagonist, or an antagonist of CLDN18.2 and GUCY2C to the subject.
The method of claim 1, wherein the tumor is CLDN18.2 positive and/or GUCY2C positive.
The method of claim 1 or 2, wherein the CLDN18.2 antagonist, the GUCY2C antagonist, and/or the antagonist of CLDN18.2 and GUCY2C are selected from engineered immune cells, an engineered receptor, an antibody, an antibody-drug conjugate (ADC) , an aptamer and small RNAs.
The method of any one of claims 1-3, wherein the combination of the CLDN18.2 antagonist and the GUCY2C antagonist comprises a first and a second group of engineered immune cells, wherein:

(1) the CLDN18.2 antagonist is the first group of engineered immune cells comprising an engineered receptor specifically targeting CLDN18.2 comprising: an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a transmembrane domain, and an intracellular signaling domain; and/or

(2) the GUCY2C antagonist is the second group of engineered immune cells comprising an engineered receptor specifically targeting GUGY2C comprising: an extracellular antigen binding domain comprising at least one anti-GUCY2C binding moiety, a transmembrane domain, and an intracellular signaling domain.
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 GUGY2C, wherein (1) the first engineered receptor specifically targeting CLDN18.2 comprising: an first extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a first transmembrane domain, and a first intracellular signaling domain, and (2) the second engineered receptor specifically targeting GUGY2C comprising: a second extracellular antigen binding domain comprising at least one anti-GUGY2C binding moiety, a second transmembrane domain, and a second intracellular signaling domain.
The method of claim 5, wherein the engineered immune cell has been transduced by (1) two separate vectors, the first vector comprising a nucleic acid encoding the first engineered receptor specifically targeting CLDN18.2, and the second vector comprising a nucleic acid encoding the second engineered receptor specifically targeting GUGY2C; or (2) a vector that comprises a nucleic acid encoding the first engineered receptor specifically targeting CLDN18.2 operably linked to the second engineered receptor specifically targeting GUGY2C.
The method of any one of claims 1-3, wherein the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising an engineered receptor co-targeting CLDN18.2 and GUGY2C comprising: an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety, a transmembrane domain, and an intracellular signaling domain.
The method of any one of claims 3-7, wherein the engineered receptor is selected from the group consisting of an engineered T cell receptor (TCR) , a chimeric antigen receptor (CAR) , a T cell antigen coupler (TAC) or a portion thereof.
The method of any one of claim 5 or claim 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 GUGY2C, wherein (1) the first CAR targeting CLDN18.2 comprising: a first extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a first transmembrane domain and an first intracellular signaling domain, (2) the second CAR targeting GUGY2C comprising: a second extracellular antigen binding domain comprising at least one anti-GUGY2C binding moiety, a second transmembrane domain and a second intracellular signaling domain; and wherein the first CAR targeting CLDN18.2 is operably linked to the second CAR targeting GUGY2C via a cleavable linker or the first CAR and the second CAR are not linked due to the cleavage of the cleavable linker.
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 co-targeting CLDN18.2 and GUGY2C ( “CLDN18.2×GUCY2C specific CAR” ) , wherein the CLDN18.2×GUCY2C specific CAR comprises: (1) an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety, (2) a transmembrane domain, and (3) an intracellular signaling domain; and wherein the anti-CLDN18.2 binding moiety is located at the N-terminus or C-terminus of the anti-GUGY2C binding moiety, optionally the anti-CLDN18.2 binding moiety is operably linked to the anti-GUCY2C binding moiety via a peptide linker, such as a GS linker e.g. (G₄S) ₃.
The method of any one of claims 4-10, the anti-CLDN18.2 binding moiety and/or anti-GUCY2C binding moiety are selected from a Fab, a Fab’ , a F (ab’) 2, an Fv, a single-chain Fv (scFv) , minibody, a diabody, a single-domain antibody (sdAb) or VHH domain.
The method of any one of claims 4-11, wherein the anti-CLDN18.2 binding moiety is an anti-CLDN18.2 VHH, optionally the anti-CLDN18.2 VHH comprises a CDR1, CDR2, and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 1, and optionally the anti-CLDN18.2 VHH comprises (i) a CDR1 comprises the amino acid sequence of SEQ ID NO: 23, (ii) a CDR2 comprises the amino acid sequence of SEQ ID NO: 24, and (iii) a CDR3 comprises the amino acid sequence of SEQ ID NO: 25.
The method of claim 12, wherein the anti-CLDN18.2 VHH comprises an amino acid sequence of SEQ ID NO: 1 or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 1.
The method of any one of claims 4-13, wherein the anti-GUCY2C binding moiety is an anti-GUCY2C VHH, optionally the anti-GUCY2C VHH comprises a CDR1, CDR2, and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 2, and optionally the anti-GUCY2C VHH comprises (i) a CDR1 comprises the amino acid sequence of SEQ ID NO: 26, (ii) a CDR2 comprises the amino acid sequence of SEQ ID NO: 27, and (iii) a CDR3 comprises the amino acid sequence of SEQ ID NO: 28.
The method of claim 14, wherein the anti-GUCY2C VHH comprises an amino acid sequence of SEQ ID NO: 2 or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 2.
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.
The method of any of claims 4-16, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune cell, optionally the primary intracellular signaling domain is derived from CD3ζ.
The method of any one of claims 4-17, wherein the intracellular signaling domain comprises a co-stimulatory signaling domain.
The method of claim 18, wherein the co-stimulatory signaling 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, ligands of CD83 and combinations thereof.
The method of any of claims 8-9 and 11-19, wherein the CAR specifically targeting CLDN18.2 comprises:

(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.
The method of any one of claims 8-9 and 11-20, wherein the CAR specifically targeting GUCY2C comprises:

(1) a hinge domain located between 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.
The method of any of claims 8-9 and 11-21, wherein the CAR specifically targeting CLDN18.2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 12 and 19; and/or wherein the CAR specifically targeting GUCY2C comprises an amino acid sequence set forth in any one of SEQ ID NOs: 13 and 21-22.
The method of any one of claims 1-22, wherein:

(1) the CLDN18.2 antagonist is an engineered immune cell comprising a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 12 and 19, and/or

(2) the GUCY2C antagonist is an engineered immune cell comprising a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 13 and 21-22.
The method of any one of claims 1-23, wherein the combination of a CLDN18.2 antagonist and a GUCY2C antagonist comprises a first group of engineered immune cell comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 12, and a second group of engineered immune cell comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 13.
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 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.
The method of any one of claims 10-19, wherein the CAR co-targeting CLDN18.2 and GUCY2C comprises:

(1) a hinge domain located between 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.
The method of claim 26, wherein the CAR co-targeting CLDN18.2 and GUCY2C comprises an amino acid sequence set forth in any one of SEQ ID NOs: 14-15.
The method of any one of claims 1-27, wherein the antagonist of CLDN18.2 and GUCY2C is an engineered immune cell comprising:

(1) a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 14-18; or

(2) a first polypeptide and a second polypeptide, wherein the first polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 12 and 19, and the second polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 13 and 20-22.
The method of any of claims 2-28, wherein the engineered immune cell is selected from the group consisting of T cell, NK cell, peripheral blood mononuclear cell (PBMC) , hematopoietic stem cell, pluripotent stem cell, an embryonic stem cell, and a combination thereof.
The method of any of claims 1-29, wherein the tumor is selected from gastric cancer, esophageal cancer, pancreatic ductal cancer, lung cancer such as non-small cell lung cancer (NSCLC) , ovarian cancer, colorectal cancer, liver cancer, head and neck cancer, gallbladder cancer and its metastasis.
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.
A multi-specific CAR construct that binds to CLDN18.2 and GUCY2C, comprising:

(a) a polypeptide comprising an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety and at least one anti-GUCY2C binding moiety, a transmembrane domain and an intracellular signaling domain; or

(b) a first polypeptide comprising a first extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a first transmembrane domain and a first intracellular signaling domain, and a second polypeptide comprising a second extracellular antigen binding domain comprising at least one anti-GUCY2C binding moiety, a second transmembrane domain and a second intracellular signaling domain, optionally the first polypeptide and the second polypeptide are linked in one chain via a cleavable linker, optionally the first and second transmembrane domains as well as the first and second intracellular signaling domains are same or different, respectively.
The multi-specific CAR construct of claim 32, wherein the anti-CLDN18.2 binding moiety is located at the N-terminus or C-terminus of the anti-GUGY2C binding moiety, optionally the anti-CLDN18.2 binding moiety is operably linked to the anti-GUCY2C binding moiety via a peptide linker, such as a GS linker e.g. (G₄S) ₃.
The multi-specific CAR construct of any one of claims 32-33, wherein the anti-CLDN18.2 binding moiety and/or the anti-GUCY2C binding moiety are selected from a Fab, a Fab’ , a F (ab’) 2, an Fv, a single-chain Fv (scFv) , minibody, a diabody, a single-domain antibody (sdAb) or VHH domain.
The multi-specific CAR construct of any one of claims 32-34, wherein the anti-CLDN18.2 binding moiety is an anti-CLDN18.2 VHH, optionally the anti-CLDN18.2 VHH comprises a CDR1, CDR2, and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 1, and optionally the anti-CLDN18.2 VHH comprises (i) a CDR1 comprises the amino acid sequence of SEQ ID NO: 23, (ii) a CDR2 comprises the amino acid sequence of SEQ ID NO: 24, and (iii) a CDR3 comprises the amino acid sequence of SEQ ID NO: 25.
The multi-specific CAR construct of claim 35, wherein the anti-CLDN18.2 VHH comprises an amino acid sequence of SEQ ID NO: 1 or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 1.
The multi-specific CAR construct of any one of claims 32-36, wherein the anti-GUCY2C binding moiety is an anti-GUCY2C VHH, optionally the anti-GUCY2C VHH comprises a CDR1, CDR2, and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 2, and optionally the anti-GUCY2C VHH comprises (i) a CDR1 comprises the amino acid sequence of SEQ ID NO: 26, (ii) a CDR2 comprises the amino acid sequence of SEQ ID NO: 27, and (iii) a CDR3 comprises the amino acid sequence of SEQ ID NO: 28.
The multi-specific CAR construct of claim 37, wherein the anti-GUCY2C VHH comprises an amino acid sequence of SEQ ID NO: 2 or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 2.
The multi-specific CAR construct of 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.
The multi-specific CAR construct of claim 39, wherein the transmembrane domain is derived from CD8α or ICOS, optionally the transmembrane domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 5-6 or at least 85%, 90%or 95%identical to the amino acid sequence set forth in any one of SEQ ID NOs: 5-6.
The multi-specific CAR construct of any of claims 32-40, wherein the intracellular signaling domain comprises a primary intracellular signaling domain; optionally the primary intracellular signaling domain is derived from CD3ζ, optionally the primary intracellular signaling domain comprises an amino acid sequence of SEQ ID NO: 11 or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 11.
The multi-specific CAR construct of any one of claims 32-41, wherein the intracellular signaling domain further comprises at least one co-stimulatory signaling domain.
The multi-specific CAR construct of claim 42, wherein the co-stimulatory signaling 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, ligands of CD83 and combinations thereof.
The multi-specific CAR construct of claim 42 or 43, wherein the co-stimulatory signaling domain is derived from 4-1BB, CD28, ICOS or NTBA,

optionally, the co-stimulatory signaling domain comprises any of the amino acid sequences of SEQ ID NOs: 7-10 or an amino acid sequence at least 85%, 90%or 95%identical to any of SEQ ID NOs: 7-10.
The multi-specific CAR construct of any one of claims 32-44, wherein the multi-specific CAR construct further comprises:

(1) a hinge domain located between 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.
The multi-specific CAR construct of any one of claims 32-45, wherein the polypeptide of the multi-specific CAR construct of (a) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 14-15, and/or

the first polypeptide of the multi-specific CAR construct of (b) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 12 and 19, and the second polypeptide of the multi-specific CAR construct of (b) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 13 and 20-22.
A combination of a CLDN18.2 antagonist and a GUCY2C antagonist, wherein the antagonist is selected from an antibody, an aptamer, an antibody-drug conjugate (ADC) , a small RNA, an engineered receptor and an engineered immune cell.
The combination of claim 47, wherein the combination of the CLDN18.2 antagonist and the GUCY2C antagonist comprises a first and a second group of engineered immune cells, wherein:

(1) the CLDN18.2 antagonist is the first group of engineered immune cell comprising an engineered receptor specifically targeting CLDN18.2 comprising: an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a transmembrane domain, and optionally an intracellular signaling domain; and/or

(2) the GUCY2C antagonist is the second group of engineered immune cell comprising an engineered receptor specifically targeting GUGY2C comprising: an extracellular antigen binding domain comprising at least one anti-GUCY2C binding moiety, a transmembrane domain, and optionally an intracellular signaling domain.
The combination of claim 47 or 48, wherein the engineered receptor specifically targeting CLDN18.2 and the engineered receptor specifically targeting GUGY2C are expressed in the different engineered immune cells.
The combination of claim 47-49, wherein the engineered receptor is selected from the group consisting of an engineered T cell receptor (TCR) , a chimeric antigen receptor (CAR) , a T cell antigen coupler (TAC) or a portion thereof.
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.
The combination of claim 48-51, wherein the anti-CLDN18.2 binding moiety and/or the anti-GUCY2C binding moiety are selected from a Fab, a Fab’ , a F (ab’) 2, an Fv, a single-chain Fv (scFv) , minibody, a diabody, a single-domain antibody (sdAb) or VHH domain.
The combination of claim 52, wherein the anti-CLDN18.2 binding moiety is an anti-CLDN18.2 VHH, optionally the anti-CLDN18.2 VHH comprises a CDR1, CDR2, and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 1, and optionally the anti-CLDN18.2 VHH comprises (i) a CDR1 comprises the amino acid sequence of SEQ ID NO: 23, (ii) a CDR2 comprises the amino acid sequence of SEQ ID NO: 24, and (iii) a CDR3 comprises the amino acid sequence of SEQ ID NO: 25.
The combination of claim 53, wherein the anti-CLDN18.2 VHH comprises an amino acid sequence of SEQ ID NO: 1 or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 1.
The combination of any of claims 52-54, wherein the anti-GUCY2C binding moiety is an anti-GUCY2C VHH, optionally the anti-GUCY2C VHH comprises a CDR1, CDR2, and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 2, and optionally the anti-GUCY2C VHH comprises (i) a CDR1 comprises the amino acid sequence of SEQ ID NO: 26, (ii) a CDR2 comprises the amino acid sequence of SEQ ID NO: 27, and (iii) a CDR3 comprises the amino acid sequence of SEQ ID NO: 28.
The combination of any of claim 55, wherein the anti-GUCY2C VHH comprises an amino acid sequence of SEQ ID NO: 2 or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID NO: 2.
The combination of any of claims 50-56, wherein the transmembrane domain of the CAR specifically targeting CLDN18.2 and the CAR specifically targeting GUCY2C are derived from a protein selected from the group consisting of CD8α, ICOS, CD4, CD28, CD137, CD80, CD86, CD152 and PD1,

optionally the transmembrane domain the transmembrane domain of the CAR specifically targeting CLDN18.2 and the CAR specifically targeting GUCY2C are derived from CD8αor ICOS, and comprise an amino acid sequence set forth in any one of SEQ ID NOs: 5-6 or at least 85%, 90%or 95%identical to the amino acid sequence set forth in any one of SEQ ID NOs: 5-6.
The combination of any of claims 50-57, wherein the intracellular signaling domain of the CAR specifically targeting CLDN18.2 and/or the CAR specifically targeting GUCY2C comprise a primary intracellular signaling domain,

optionally, the primary intracellular signaling domain is derived from CD3ζ,

optionally, the primary intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence at least 85%, 90%or 95%identical to SEQ ID NO: 11.
The combination of any of claims 50-58, wherein the intracellular signaling domain of the CAR specifically targeting CLDN18.2 and the CAR specifically targeting GUCY2C further comprises a co-stimulatory signaling domain.
The combination of claim 59, wherein the co-stimulatory signaling 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, ligands of CD83 and combinations thereof.
The combination of any of claims 50-60, wherein the CAR specifically targeting CLDN18.2 comprise (1) a hinge domain located between 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.
The combination of any of claims 50-61, wherein the CAR specifically targeting GUCY2C comprises (1) a hinge domain located between 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.
The combination of any one of claims 50-62, wherein the CAR specifically targeting CLDN18.2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 12 and 19, and/or the CAR specifically targeting GUCY2C comprises an amino acid sequence set forth in any one of SEQ ID NOs: 13 and 19-22.
The combination of any one of claims 47-63, wherein the CLDN18.2 antagonist is a first group of engineered immune cell comprising a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 12 and 19, and/or the GUCY2C antagonist is a second group of engineered immune cell comprising a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 13 and 19-22.
The combination of claim 64, wherein the CLDN18.2 antagonist is a first group of engineered immune cell comprising a polypeptide comprising an amino acid sequence of SEQ ID NO: 12, and the GUCY2C antagonist is a second group of engineered immune cell comprising a polypeptide comprising an amino acid sequence of SEQ ID NO: 13.
The combination of any of claims 47-65, comprising a ratio of the CLDN18.2 antagonist and the GUCY2C antagonist 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.
The combination of any of claims 47-66, wherein the engineered immune cell is selected from the group consisting of T cell, NK cell, peripheral blood mononuclear cell (PBMC) , hematopoietic stem cell, pluripotent stem cell, an embryonic stem cell, and a combination thereof.
A nucleic acid comprising:

(1) a nucleic acid sequence encoding the multi-specific CAR construct of any of claims 32-46; or

(2) a first nucleic acid sequence encoding a first engineered receptor specifically targeting CLDN18.2 comprising: a first extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a first transmembrane domain and a first intracellular signaling domain, and a second nucleic acid sequence encoding a second engineered receptor specifically targeting GUCY2C comprising: a second extracellular antigen binding domain comprising at least one polypeptide comprising the GUCY2C binding moiety, a second transmembrane domain and a second intracellular signaling domain,

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) .
The nucleic acid of claim 68, wherein comprising:

(1) a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 14-18; or

(2) a first nucleic acid sequence encoding a first polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 12 and 19, and a second nucleic acid sequence encoding a second polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 13 and 20-22.
A vector comprising the nucleic acid of claim 68 or 69.
An engineered immune cell comprises the multi-specific CAR construct of any one of claims 32-46, the nucleic acid of any of claims 68-69, or the vector of claim 70.
The engineered immune cell of claim 71, comprising an engineered receptor specifically targeting CLDN18.2 comprising: an extracellular antigen binding domain comprising at least one anti-CLDN18.2 binding moiety, a transmembrane domain, and optionally an intracellular signaling domain; and an engineered immune cell comprising an engineered receptor specifically targeting GUGY2C comprising: an extracellular antigen binding domain comprising at least one anti-GUCY2C binding moiety, a transmembrane domain, and optionally an intracellular signaling domain.
The engineered immune cell of claim 70 or 72, comprising:

(1) a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 14-18; or

(2) a first polypeptide and a second polypeptide, wherein the first polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 12 and 19, and the second polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 13 and 20-22.
The engineered immune cell of claim 73, which is derived from the group consisting of T cell, NK cell, peripheral blood mononuclear cell (PBMC) , hematopoietic stem cell, pluripotent stem cell, an embryonic stem cell, and a combination thereof.
A pharmaceutical composition comprising the multi-specific 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, and a pharmaceutically acceptable carrier.
The multi-specific 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 tumor in a subject, optionally wherein the tumor is CLDN18.2 positive and/or GUCY2C positive.
The multi-specific CAR construct, the combination, the nucleic acid or the engineered immune cell for use according to claim 76, wherein the tumor is selected from gastric cancer, esophageal cancer, pancreatic ductal cancer, lung cancer such as non-small cell lung cancer (NSCLC) , ovarian cancer, colorectal cancer, liver cancer, head and neck cancer, gallbladder cancer and its metastasis.
The multi-specific CAR construct, the combination, the nucleic acid or the engineered immune cell for use according to 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.