CLDN18.2-targeting chimeric antigen receptors and methods of use

Abstract

Disclosed herein are VH-only single domain binders that target claudin 18.2 (CLDN18.2) and chimeric antigen receptors (CARs) that contain the same, engineered T cells containing the CLDN18.2-targeting CARS, and methods of treating cancer (e.g., gastric cancer or pancreatic cancer) using the CAR-T cells.

Description

DFCI IP No. 3561Attorney Docket No. 91016-431320CLDN18.2-TARGETING CHIMERIC ANTIGEN RECEPTORS AND METHODS OF USERELATED APPLICATIONS

[0001] This application claims priority to CN Provisional Application No. 202510028444.4 filed on January 8, 2025, and CN Provisional Application No. 202511224534.7, filed on August 29, 2025, both of which are incorporated by reference in their entirety herein.STATEMENT REGARDING SEQUENCE LISTING

[0002] The name of the.xml file containing the Sequence Listing is "91016-431320SequenceListing". The.xml file is 66,905 bytes, was created on January 7, 2026, and is being submitted electronically, concurrent with the filing of this specification.FIELD

[0003] The present disclosure relates to a novel nucleic acid construct for the expression of a chimeric antigen receptor (CAR) targeting claudin 18.2 (CLDN18.2) and expressing the same in an immune cell. Also described herein are methods of treating cancer using the same.BACKGROUND

[0004] Globally, gastric cancer has the fifth highest incidence and mortality of any malignancy. See, for example, Sung, H. et al. A Cancer J Clin 71, 209-249 (2021) and Ferlay, J. etal. IntJ Cancer 149, 778- 789 (2021). While biomarker selected strategies are increasingly driving frontline management the median Overall Survival (OS) for advanced disease is 14-17 months. See, for example, Rha, S. Y. et al. Lancet Oncol 24, 1181-1195 (2023), J. A. Njigian, Y. Y. etal. Ann Oncol 35, S877-S878 (2024), and J. A. Njigian, Y. Y. et al. Journal of Clinical Oncology 42, 2012-2020 (2024). Standard biomarker testing includes assessment of PD-L1 expression, HER2 expression and / or amplification, and mismatch repair (MMR) or microsatellite instability testing (MSI) and, more recently, claudin 18.2 (CLDN18.2) testing. On average these strategies improve survival by roughly 2-4DFCI IP No. 3561Attorney Docket No. 91016-431320months versus chemotherapy alone and managing toxicities, which impact quality of life (QoL), is central to optimizing patient outcomes.

[0005] The CLDN18.2 isoform has been identified as a target of interest given its high prevalence in both primary lesions and metastases of upper gastrointestinal cancers. See, for example, Choi, E. etal., Scientific Reports 2024 14:1 14, 1-9 (2024), Klempner, S. et al., ESMO Open 8, (2023), Shitara, K. et al. Gastric Cancer 27, 1058 (2024), and Kim, H. D. etal. Sci Rep 13, 20047 (2023). Furthermore, rapid expansion of the therapeutic landscape, a lack of understanding of the pathological injury in human samples and pre-clinical models represents a current clinical and scientific knowledge gap. There is a clear need for cell therapies to treat gastric cancers, pancreatic cancer, esophageal cancer, ovarian cancer, and non-small cell lung cancers with high efficacy and fewer side effects.SUMMARY

[0006] Described herein are novel chimeric antigen receptors comprising fully-human VH-only single domain binders that target claudin 18.2 (CLDN18.2), which, when engineered into a CAR-T for cell therapy, may be used to treat cancer (e.g., gastric cancer or pancreatic cancer).

[0007] Thus, one embodiment described herein is a chimeric antigen receptor (CAR) comprising an amino acid having formula I:R1 - R2 - R3 - R4 - R5 (I),wherein:R1 comprises a VH-only single domain binder for binding claudin 18.2 (CLDN18.2) comprising a heavy chain variable region comprising: a) a complementarity-determining region 1 (CDR1) having an amino acid sequence with 0 to 2 amino acid substitutions compared to SEQ ID NOs: 1, 4, 29, or 32; b) a complementarity-determining region 2 (CDR2) having an amino acid sequence with 0 to 2 amino acid substitutions compared to SEQ ID NOs: 2, 5, 30, or 33; or c) a complementarity-determining region 3 (CDR3) having an amino acid sequence with 0 to 2 amino acid substitutions compared to SEQ ID NOs: 3, 6, 31, or 34; R2 comprises a hinge region (e.g., a cluster of differentiation 28 (CD28) hingeDFCI IP No. 3561Attorney Docket No. 91016-431320region); R3 comprises a transmembrane region (e.g., a CD28 transmembrane region); R4 comprises a costimulatory region (e.g., a 4-1 BB costimulatory region); and R5 comprises an intracellular region (e.g., a cluster of differentiation 3 zeta (CD3 intracellular region).

[0008] In one aspect of the CAR, the heavy chain variable region comprises human framework regions. In another aspect of the CAR, the heavy chain variable region comprises: a) a CDR1 having an amino acid sequence selected from SEQ ID NOs: 1, 4, 29, or 32; b) a CDR2 having an amino acid sequence selected from SEQ ID NOs: 2, 5, 30, or 33; or c) a CDR3 having an amino acid sequence selected from SEQ ID NOs: 3, 6, 31, or 34. In another aspect of the CAR, the heavy chain variable region comprises: a) a CDR1 having the amino acid sequence of SEQ ID NO: 1; a CDR2 having the amino acid sequence of SEQ ID NO: 2; and a CDR3 having the amino acid sequence of SEQ ID NO: 3; b) a CDR1 having the amino acid sequence of SEQ ID NO: 4; a CDR2 having the amino acid sequence of SEQ ID NO: 5; and a CDR3 having the amino acid sequence of SEQ ID NO: 6; c) a CDR1 having the amino acid sequence of SEQ ID NO: 29; a CDR2 having the amino acid sequence of SEQ ID NO: 30; and a CDR3 having the amino acid sequence of SEQ ID NO: 31; or d) a CDR1 having the amino acid sequence of SEQ ID NO: 32; a CDR2 having the amino acid sequence of SEQ ID NO: 33; and a CDR3 having the amino acid sequence of SEQ ID NO: 34.

[0009] In another aspect of the CAR described herein, the heavy chain variable region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 7, 9, 35, or 37. In another aspect, the CAR is encoded by a nucleic acid sequence of SEQ ID Nos: 8, 10, 36, or 38. In yet another aspect of the CAR described herein, R2 comprises an amino acid sequence according to SEQ ID NO: 13; R3 comprises an amino acid sequence according to SEQ ID NO: 15; R4 comprises an amino acid sequence according to SEQ ID NO: 17; and R5 comprises an amino acid sequence according to SEQ ID NO: 19.

[0010] In another aspect of the CAR described herein, the CAR comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 21, 23, 39, or 41. In another aspect, the CAR is encoded by a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 22, 24, 40, or 42. In another aspect of the CAR, the binder has no binding affinity to CLDN18.1.DFCI IP No. 3561Attorney Docket No. 91016-431320

[0011] Another aspect of the CAR described herein is a vector comprising any of the nucleic acids described herein or a nucleic acid sequence according to SEQ ID NOs: 25-28 or 43-46. Another aspect described herein is an immune cell (e.g., a T cell) transfected with any of the vectors described herein. Another aspect described herein is a pharmaceutical composition, comprising any of the immune cells (e.g., T cells) described herein.

[0012] Another embodiment described herein is a method of treating cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of a T cell engineered with any of the nucleic acids described herein or any of the pharmaceutical compositions described herein.

[0013] Another aspect described herein is a method of limiting gastrointestinal toxicity in a patient previously identified as having cancer, the method comprising administering to the patient an effective amount of a T cell engineered with any of the nucleic acids described herein or any of the pharmaceutical compositions described herein.

[0014] Another embodiment described herein is a method of limiting stomach tissue atrophy in a patient previously identified as having cancer, the method administering to the patient an effective amount of a T cell engineered with any of the nucleic acids described herein or any of the pharmaceutical compositions described herein.

[0015] In one aspect, in any of the methods described herein, the cancer is selected from gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, and non-small cell lung cancer. In some aspect of the any of the methods described herein, the step of administering comprises intravenously administering.BRIEF DESCRIPTION OF THE FIGURES

[0016] FIGURE 1 depicts an exemplary schematic of a construct to express a CLDN18.2-targeting CAR using an anti-CLDN18.2 VH-only single domain binder.(aCLDN18.2 VH), a hinge region, a transmembrane region, a costimulatory region (e.g., a 4-1 BB costimulatory region), and an intracellular region (e.g., a CD3ζ intracellular region).

[0017] FIGURES 2A-2F show binding affinity and kinetic parameters of VH-only single domain binders and the IgG-format VHA / L binder from the control anti-CLDN 18.2DFCI IP No. 3561Attorney Docket No. 91016-431320CAR, CT041, to CLDN18.2. Figs. 2A-2E show graphs describing the affinity in nanomolarity (nM) of antibodies constructed with the VH-only single domain binder of SEQ ID NO: 37, VH-only binder 4 (Fig. 2A), the VH-only single domain binder of SEQ ID NO: 7, VH-only binder 1 (Fig. 2B), the VH-only single domain binder of SEQ ID NO: 9, VH-only binder 2 (Fig. 2C), the VH-only single domain binder of SEQ ID NO: 35, VH-only binder 3 (Fig. 2D), or the VHVL binder in IgG format from the control CLDN18.2-targeting CAR, CT041. (Fig. 2E) to Claudin 18.2 (CLDN18.2). Fig. 2F illustrates a two-dimensional isoaffinity plot comparing the affinity distribution, specifically comparing the association rate constant (Kon) and dissociation rate constant (Koff) of the various VH-only single domain binders and the CT041 binder. The diagonal lines within the plot represent equal or "iso" affinity.

[0018] FIGURES 3A-3C depict in vitro characterization of the various CARs comprising VH-only single domain binders. Fig. 3A and Fig. 3B are graphs depicting cytotoxicity in OE19s cells (Fig. 3A) and SNU620s cells (Fig. 3B) measured using percentage cell killing (%) normalized to an initial count of cells. CT041 and zolbetuximab are positive controls and an irrelevantly-targeted CAR ( / .e., a non-CLDN18.2 targeting CAR) was used as a negative control. Fig. 3C shows a graph depicting the tonic signaling, namely the non-specific, sustained activation of T cells in a ligand-independent manner, of the CAR displaying the indicated VH-only single domain binders ( / .e. single domain CARs) as a function of their transduction efficiency.

[0019] FIGURES 4A-4C depict the binding affinity of the VH Fes targeting human CLDN18.2 correlates with on -target / off -tumor toxicity. NSG-DKO animals were injected with OE19, treated with CLDN18.2 targeted VH CAR T cells (either CAR T cells having SEQ ID NO: 21, CAR T cells having SEQ ID NO: 23), CT041-scFv CAR T cells, or BCMA-scFv CAR T cells on day 16. Animals were monitored for tumor control as shown in Fig. 4A and overall survival as shown in Fig. 4B. Fig. 4C shows a graph of the survival of mice having tumors after treatment with the indicated VH-only single domain binder CAR, CT041-scFv CAR T cells or BCMA-scFv CAR T cells and the cause of death as either CAR toxicity (square) or the tumor (circle). Mice that survived are indicated with a triangle.DFCI IP No. 3561Attorney Docket No. 91016-431320

[0020] FIGURES 5A-5D show CLDN18.2-targeted VH binder-expressing CARs have therapeutic window targeting a pancreatic xenograft model. Male NSG-DKO mice were engrafted with pancreatic xenograft model PATU8998s subcutaneously and dosed by tail vein with either 1x106, 3x105CT041-scFv, VH Binder 1, VH Binder 2, or 1x106BCMA-scFv CAR-T after tumor volume had reached 100 mm3at day 1 (n=5 / arm). Fig. 5A shows tumor volume and Fig. 5B depicts a spider plot of individual body weights (thin lines), with mean overlay (heavy lines). Analyzed via Two-way ANOVA. Fig. 5C illustrates overall survival (Log-rank Mantel-Cox). Fig. 5D shows a swimmers plot of cause of death. Data representative of two independent experiments in male and female mice. **p<0.01,*** p<0.001 ****p<0.0001.

[0021] FIGURES 6A-6F depicts binding affinity correlates with body weight changes in NSG-DKO mice treated with CLDN18.2-targeted VH binder-expressing CARs. Fig.6A shows a graph of the bodyweight loss for animals treated with various VH-only single domain binders, CARS, CT041 or irrelevantly-targeted CAR-T (BCMA-scFv). Fig. 6B shows a representative IHC image of stomach of a mouse treated with irrelevantly-targeted CAR-T showing normal architecture. Fig. 6C-Fig. 6F show representative mIF images of stomach of a mouse showing absence of irrelevantly-targeted CAR-T in stomach tissue.

[0022] FIGURES 7A-7F depict erosive gastritis after zolbetuximab. Fig. 7A shows pretreatment endoscopic images in a gastric cancer patient. Fig. 7B shows after 4 doses of zolbetuximab plus chemotherapy, follow up endoscopy shows grossly visible gastric mucosal injury. Fig. 7C-Fig. 7D illustrate H& E and IHC from biopsies of tumor-uninvolved gastric epithelium, taken prior to treatment demonstrate strong CLDN18.2 (43-14A clone) expression (magnification at 4x). Fig. 7E-Fig. 7F depict biopsies from tumor-uninvolved gastric epithelium post-treatment demonstrate erosive gastritis with regenerative changes and preserved CLDN18.2 expression (magnification at 4x).

[0023] FIGURES 8A-8B illustrate sequence alignment of mouse and human CLDN18.2 exon 1b, corresponding to region of CAR binding specificity. Fig. 8A shows genomic architecture demonstrating the alternative splicing of CLDN18 with inclusion of exon 1a resulting in lung specific CLDN18.1 isoform and inclusion of exon 1b into the gastricDFCI IP No. 3561Attorney Docket No. 91016-431320specific CLDN18.2 isoform. Fig. 8B shows the amino acid sequence alignment of mouse and human CLDN18.2 exon 1b, distinct at two positions using BLASTp.

[0024] FIGURES 9A-9L illustrate additional examples of erosive gastritis after zolbetuximab including clinical resolution after treatment cessation. Fig. 9A-Fig. 9D show pre-zolbetuximab with chemotherapy treatment endoscopic images. Fig. 9E-Fig 9H show on treatment endoscopy images. Fig. 9I-Fig. 9L show resolution of gastritis noted on endoscopy performed at the time of progression, after 8 months as in Fig. 9I, after 1 month as in Fig. 9J, or after 6 months as shown in Fig. 9K-9L.

[0025] FIGURES 10A-10H demonstrate treatment with CT041-scFv CLDN18.2-targeted CAR T cells leads to intolerable on-target / off-target tumor toxicity. NSG-DKO mice bearing OE19 xenografts were left untreated (UTD) or treated on day 13 with 1x106CT041-scFv or BCMA-scFv CAR T by tail vein (n=3-4). Fig. 10A depicts tumor volume over time, comparing BCMA-scFv and CT041-scFv CAR treatment. Data are presented asmean ± SEM. Fig. 10B shows body weight, comparing BCMA-scFv and CT041-scFv.Animals reached humane endpoint if bodyweight declined by 20% or due to tumor progression. Data are presented as mean SEM. Fig. 10C illustrates overall survival. Fig.10D shows stomach from a CT041-scFv treated animal at the time of sacrifice due to toxicity. H& E shows atrophy of normal architecture and inflammation (scale bar 100 pm).Fig. 10E depicts tissue stained via immunofluorescence for CLDN18.2, Fig. 10F shows human CD3, Fig. 10G shows an epithelial marker, PANCK. DAPI identifies cell nuclei. Fig.10H shows a merge demonstrating strong gastric CLDN18.2 expression with colocalization of human CD3+ T cells. Scale bars 20 pm. Fig. 10A and Fig. 10B were statistically analyzed via an unpaired, two-sided t-test Fig. 10C was evaluated via log-rank Manel-Cox test, *p<0.05; **p<0.01.

[0026] FIGURES 11A-11L illustrate CT041-scFv CAR T treated tumor shows concurrent tumor CAR T cell infiltration and absence of gastric tissue infiltration or injury by irrelevant CAR T. Representative images from a CT041 CAR T treated tumor. Scale bar = 1 mm; with ROI detail shows tumor sample from an animal 3 weeks post CAR T injection shown in Fig. 10, stained using the same protocol. Fig. 11A depicts H& EDFCI IP No. 3561Attorney Docket No. 91016-431320staining which reveals a small area of tumor with T-cell infiltration and necrosis.Representative images from a CT041 CAR T treated tumor. The sample was also stained via mIF for CLDN18.2 as shown in Fig. 11 C, for CD3 as shown in Fig. 11 D, and pan-cytokeratin (PANCK) in Fig. 11E and merged as shown in Fig. 11 F; DAPI is staining for nuclear visualization; scale bar = 50 pm. Representative images of the stomach of an irrelevantly targeted BCMA CAR T cell control animal 2 weeks after CAR T injection. Fig.11G depicts H& E staining which shows normal tissue structure. Scale bar = 200 pm with the square indicating the corresponding region show in Fig. 11I-11L. Positive staining for CLDN18.2 as shown in Fig. 111, negative staining for CD3 as shown in Fig. 11 J, pan-cytokertain (PANCK) staining shown in Fig. 11 K, and a merged image of all markers shown in Fig. 11L; DAPI stained for nuclear visualization; scale bar - 50 pm.

[0027] FIGURES 12A-12I show toxicity from CT041-scFV CAR T is dependent on dose and not tumor burden. Fig. 12A depicts an experimental schematic of Fig. 12B-Fig. 12E indicating injection of OE19 xenografts subcutaneously, staggered such that animals were treated with CAR T cells once tumors reached ~100 mm3or ~200 mm3, to model low or high burden disease. Non-tumor bearing animals were also treated. All animals received 1x106CT041-scFv or BCMA-scFv CAR T cells by tail vein. Fig. 12B shows tumor volume and Fig. 12C shows a spider plot of individual mouse body weights (thin solid or dotted lines) with mean overlay (heavy dashed lines), significance shown relative to high tumor cohort via two-way ANOVA analysis; shown in Fig. 12D is overall survival analyzed via Log-rank Mantel-Cox test. Fig. 12E shows individual cause of death shown as swimmers plot. Fig. 12F-Fig. 121 depict NSG-DKO mice engrafted with OE19 subcutaneously and dosed by tail vein with either 1 x105, 3x105or 1x106CT041-scFv or 1x106BCMA-scFv CAR-T after tumor volume had reached 200mm3on day 13. (representative of n=2 in male and female mice). Fig. 12F shows a spider plot of body weight (thin solid or dotted line), with mean overlay (heavy dashed lines), significance shown relative to 1x106dose, and two-way ANOVA. Fig. 12G shows tumor volume, (Fig. 12F-Fig. 12G both measured weekly). Two-way ANOVA, relative to 1x106dose. Fig. 12H shows overall survival (Logrank Mantel-Cox). Fig. 121 shows individual cause of death is shown in swimmers plot.DFCI IP No. 3561Attorney Docket No. 91016-431320Arrowheads are animals still alive at the end of the experiment; squares are animals which succumbed to toxicity; circles are animals which succumbed to tumor. *p<0.05, **p<0.01, and ****p<0.0001, ns: not significant.

[0028] FIGURES 13A-13F show CAR T infiltration into normal gastric tissue occurs regardless of presence of tumor. Representative mIF images of stomachs from mice treated with CT041-scFv 1x106CAR-T cells. Shown on the left are non-tumor bearing on the right, tumor bearing for each figure. Fig. 13A-Fig. 13B show the epithelial marker PANCK, Fig. 13C-Fig. 13D show the T cell marker human CD3, and Fig. 13E-Fig. 13F show the merged images. Also included is the nuclear stain DAPI. Scale bar = 50 pm. Representative mIF images comparing, on the left of each figure, tumor PATU8998s and, on the right of each figure, murine stomach both from non-CLDN18.2 CAR-T treated mice.Fig. 13A and Fig. 13B show whole slide images with pan-cytokeratin (panCK), CLDN18.2, and nuclei (DAPI); scale bar 1 mm. Fig. 13D-Fig. 13H show high-magnification views of insets; scale bar: 20 pm. Fig. 13C and Fig. 13D show panCK; Fig. 13E-Fig. 13F show CLDN18.2; Fig. 13G-Fig. 13H show merged panCK and CLDN18.2 expression.

[0029] FIGURES 14A-14D show the affinity towards muCLDN18.2 is similar as towards huCLDN18.2 across binders. Binding affinity towards muCLDN18.2 demonstrating its similarity to huCLDN18.2 across binders, as measured by multicycle kinetics, of as in Fig. 14A for VH Binder 1, in Fig. 14B for VH Binder 2 and Fig. 14C the control CT041 in IgG format to human CLDN18.2. Fig. 14D depicts an isoaffinity plot for CLDN18.2 binders against mouse CLDN18.2. All data is shown as mean± standard deviation (n = 3).

[0030] FIGURES 15A-15B illustrate targeted epitopes are overlapping between CT041 and Experimental CARs. Fig. 15A schematic of the CLDN18.2 and the residues within the ECL that differ between CLDN18.2 and 18.1. Five chimeric constructs - ECL1-0, ECL1-1, ECL1-2, EC1-3, ECL1-4 - were generated by mutating the indicated residues, highlighted in white (with arrowheads) within the predicted Alphafold structure, to analogous CLDN18.1 residues. Fig. 15B depicts heatmaps showing the percent viable target cells ( / .e. wildtypeDFCI IP No. 3561Attorney Docket No. 91016-431320CLDN 18.2, ECL1-1, ECL1-2, ECL1-3) in cytotoxicity experiments when cocultured with the designated CAR.

[0031] FIGURES 16A-16L depict toxicity and affinity correlate with CAR-T cell infiltration into the stomach. Fig. 16A-Fig. 16L show representative images from gastric tissue of differentially treated animals. The top panels of each set within the figures show low-magnification multiplexed immunofluorescence (mIF; with PANCK, CD3, CLDN18.2, and DNA) overview of the entire stomach (scale bar = 1 mm); the middle panel of each set within the figures shows high magnification mIF view of gastric mucosa (scale bar = 50 pm); and the bottom panel of each set within the figures depict a high magnification hematoxylin and eosin (H& E) view of gastric mucosa (scale bar = 50 pm). Stomach from Fig. 16A, Fig. 16E, and Fig. 161 CT041-scFv. Fig. 16B, Fig. 16F, Fig. 16J 5797-VH i.e. VH Binder 2, Fig. 16C, Fig. 16G, Fig. 16K 5795-VH i.e. VH Binder 1, and Fig. 16D, Fig.16H, Fig. 16L BCMA-scFv CAR treated animals.

[0032] FIGURES 17A-17H show representative gating strategy to determine transduction efficiency of CAR T cells prior to normalization. Each CAR T cell preparation was analyzed for morphology as shown in Fig. 17A, singlets as shown in Fig.17B, viability as in Fig. 17C, and transduction efficiency shown in Fig. 17D-17H (TE). Data shown is pre-normalization. In all experiments, treatment groups receive the same number of viable CAR+ T-cells and total T cells. This is accomplished by normalizing each CAR T preparation to the lowest transduction efficiency in that experiment by adding mock transduced T cells from the same donor.

[0033] FIGURE 18 depicts a series of line plots showing that novel human HCAbs targeting CLDN18.2 show binding to CHOK1 cells overexpressing human CLDN18.2, and not CHOK1 cells overexpressing CLDN18.1. This is in contrast to PR002725, a mAb that binds both CLDN18.1 and 18.2.

[0034] FIGURE 19 shows a series of line plots depicting binding of the human HCAbs to HEK293 cells overexpressing non-human orthologs of CLDN18.2. Specifically, the novel human HCAbs show targeting of CLDN18.2 and good binding activity to HEK293DFCI IP No. 3561Attorney Docket No. 91016-431320cells overexpressing cynomologous CLDN18.2 and HEK293 cells overexpressing mouse CLDN18.2.

[0035] FIGURE 20 shows a series of line plots depicting the binding of the novel human HCAbs in primary human cell lines. The human HCAbs target CLDN18.2 via binding activity in primary cell lines known to overexpress CLDN18.2 including NUGC4 and SNU620. Additionally, the cell line SNU620 harbors the M149L mutation in CLDN18.2.DETAILED DESCRIPTION

[0036] The present disclosure relates to a novel chimeric antigen receptor (CAR) targeting claudin 18.2 (CLDN18.2) and expressing the CLDN18.2-targeting CAR in a T cell. Also described herein is a method of treating cancer using the same.

[0037] Claudin 18 (CLDN18) belongs to a group of transmembrane proteins participating in tight junctions. It has two isoforms, CLDN18.1, primarily restricted to the lung, and CLDN18.2, which in normal tissues, is limited to the differentiated epithelial cells of the stomach. See, for example, Niimi, T. et al Mol Cell Biol 21, 7380-7390 (2001). CLDN18.2 was detected by IHC in almost 80% of primary gastric adenocarcinomas, and the majority of cases investigated showed similar staining patterns between primary and involved lymph nodes. See, for example, Sahin, U. et al. Clinical Cancer Research 14, 7624-7634 (2008). In addition to expression in gastric adenocarcinoma, CLDN18.2 has been detected in pancreatic, esophageal, ovarian and a limited number of non-small cell lung cancers. See, for example, Tureci, O etal. Oncoimmunology 8.1: e1523096 (2019), Moentenich, V. et al. Oncol Lett 19, 3665 (2020), Wagner, P. et al. Virchows Archiv 485, 63 (2024), and Micke, P. et al. Int J Cancer 135, 2206-2214 (2014).

[0038] The limited normal tissue expression and distribution coupled with the high prevalence of tumor expression (inherent tumor specificity) makes CLDN18.2 an attractive therapeutic target. In the trials of both zolbetuximab (chimeric IgG 1 antibody, formerly IMAB-362) and CT041 (a CLDN18.2-targeted CAR T cell therapy), some of the most common, treatment emergent adverse events (not expected to be related to required coadministered chemotherapy) were nausea and vomiting. Nausea was seen in 76.0% of theDFCI IP No. 3561Attorney Docket No. 91016-431320patients who received zolbetuximab and chemotherapy, and in 56.2% of those who received chemotherapy with placebo. Vomiting occurred in 66.8% and 34.2%, respectively. Around 30% of patients in these trials had been previously treated with gastrectomy, and both gastro-intestinal (Gl)-related toxicities were more likely to occur in patients with intact stomachs. CT041 treated patients also experienced high rates of gastrointestinal toxicity, with any grade Gl disorder occurring in 92% of patients, including 20% of patients with > grade 3 toxicity. Nausea and vomiting occurred in 67% and 53% of patients, respectively. Gastric mucosa injury (which included gastritis, erosive gastritis, and mucosal lesions) were reported in 8.2% of patients. See, for example, Shitara, K. etal. The Lancet. 401.10389 (2023): 1655-1668 and Shah, M. A. etal. Nature Medicine 29:829, 2133-2141 (2023). Additional reports of Gl hemorrhage and perforation were also made. See, for example, Qi, C. et al. Nature Medicine 30:830, 2224-2234 (2024). However, the overall rate of prior gastrectomy in this trial is unknown. The mechanism of CLDN18.2 therapy related nausea and vomiting is hypothesized to involve off-tumor / on-target binding to normal CLDN18.2 expressed in gastric epithelium. The VH-only single domain binders disclosed herein and CAR-T cells encoding the same induce low-levels of on-target / off-target toxicity while having anti-tumor efficacy.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0040] As used herein, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.DFCI IP No. 3561Attorney Docket No. 91016-431320

[0041] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In some embodiments, the terms "about" or "approximately" when preceding a numerical value indicates the value plus or minus a range of 10%, 5%, or 1%.

[0042] The term “comprise”, “comprises”, and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of” is meant to include, and be limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase and be limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

[0043] The term “coding sequence” as used herein refers to a segment of a polynucleotide that encodes for protein or polypeptide. The region or sequence is bounded nearer the 5’ end by a start codon and nearer the 3’ end with a stop codon. Coding sequences may also be referred to as open reading frames.

[0044] As will be appreciated by the skilled practitioner, slight changes in nucleic acid sequence do not necessarily alter the amino acid sequence of the encoded polypeptide. This disclosure embraces the degeneracy of codon usage as would be understood by one of ordinary skill in the art. For example, as known in the art, different codons will code for the same amino acid.

[0045] As used herein, the phrase “codon degenerate nucleic acid sequence” when used with reference to a nucleic acid sequence refers to a nucleic acid sequence that differs from the referenced sequence, but that encodes a polypeptide having the same amino acid sequence as that encoded by the referenced sequence. Also used herein the term "degenerate variant" refers to a polynucleotide encoding a polypeptide (such as a CAR) that includes a sequence that is degenerate based on the genetic code (i.e., the 20 naturalDFCI IP No. 3561Attorney Docket No. 91016-431320amino acids may be specified by more than one codon). All degenerate nucleotide sequences encoding the described amino acid sequences are included.

[0046] As used herein, "an effective amount" refers to an amount that causes relief of symptoms of a disorder or disease as noted through clinical testing and evaluation, patient observation, and / or the like. An "effective amount" may further designate a dose that causes a detectable change in biological or chemical activity. The detectable changes may be detected and / or further quantified by one skilled in the art for the relevant mechanism or process. Moreover, an "effective amount" may designate an amount that maintains a desired physiological state, i.e., reduces or prevents significant decline and / or promotes improvement in the condition of interest. For example, this may be the amount necessary to prevent, treat, or ameliorate a disease, for example, inhibiting or suppressing a tumor or symptoms of a cancer (e.g., a gastric cancer). In embodiments, an effective amount is the amount necessary to significantly inhibit or reduce cancer cell proliferation or migration, invasion, or adhesion. A cellular response manifests as significantly reduced tumor size, reduced or inhibited disease progression, and improvement in survival in a subject or patient. More particularly, an effective amount provides improvement in important cancer endpoints, Overall Survival (OS), Disease-Free Survival (DFS), Objective Response Rate, Complete Response Rate or Progression Free Survival (PFS). See, for example, Department of Health and Human Services, Food and Drug Admin, Clinical Trial Endpoints for the Approval of Cancer Drugs and Biologies: Guidance for Industry (2018); E. A.Eisenhauer et al., New Response Evaluation Criteria in Solid Tumours: Revised RECIST Guideline (Version 1.1), 45 Eur. J. Cancer 228 (2009).

[0047] An “expression vector” or “vector” is any genetic element, e.g., a plasmid, a minicircle, a nanoplasmid, chromosome, virus, transposon, behaving either as an autonomous unit of polynucleotide replication within a cell (i.e. capable of replication under its own control) or being rendered capable of replication by insertion into a host cell chromosome, having attached to it another polynucleotide segment, so as to bring about the replication and / or expression of the attached segment. A vector is sometimes referred to as a construct. Recombinant DNA vectors are vectors having recombinant DNA. A vector mayDFCI IP No. 3561Attorney Docket No. 91016-431320include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements.

[0048] As used herein, the term "individual" and "subject" are often used interchangeably and refer to any human or domestic animal that may be treated with the methods disclosed herein. Suitable subjects e.g., patients) include humans and domestic animals or pets (such as a cat or dog). Non-human primates and human patients are included. In one embodiment, subjects may include human patients that have been diagnosed with cancer. As used herein, the term "patient" refers to a subject that may receive a treatment of a disease or condition.

[0049] As used herein, the term “nucleic acid construct” also known as a DNA / RNA construct, is a segment of DNA / RNA that has been genetically engineered using a combination of different DNA / RNA sequences to achieve specific goals. These constructs are often used in genetic engineering and molecular biology for research, therapeutic, and biotechnological purposes.

[0050] The term “operably linked” as used herein refers to the physical and / or functional linkage of a DNA segment to another DNA segment in such a way as to allow the segments to function in their intended manners. A DNA sequence encoding a gene product is operably linked to a regulatory sequence when it is linked to the regulatory sequence, such as, for example, promoters, enhancers and / or silencers, in a manner, which allows modulation of transcription of the DNA sequence, directly or indirectly. For example, a DNA sequence is operably linked to a promoter when it is ligated to the promoter downstream with respect to the transcription initiation site of the promoter, in the correct reading frame with respect to the transcription initiation site and allows transcription elongation to proceed through the DNA sequence. An enhancer or silencer is operably linked to a DNA sequence coding for a gene product when it is ligated to the DNA sequence in such a manner as to increase or decrease, respectively, the transcription of the DNA sequence. Enhancers and silencers may be located upstream, downstream or embedded within the coding regions of the DNA sequence. A DNA for a signal sequence is operably linked to DNA coding for aDFCI IP No. 3561Attorney Docket No. 91016-431320polypeptide if the signal sequence is expressed as a pre-protein that participates in the secretion of the polypeptide. Linkage of DNA sequences to regulatory sequences is typically accomplished by ligation at suitable restriction sites or via adapters or linkers inserted in the sequence using restriction endonucleases known to one of skill in the art. The term “promoter” refers to a region of a polynucleotide that initiates transcription of a coding sequence. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (towards the 5’ region of the sense strand). Some promoters are constitutive as they are active in all circumstances in the cell, while others are regulated and are activated in response to specific stimuli, e.g., an inducible promoter. The term “promoter activity” and its grammatical equivalents as used herein refer to the extent of expression of nucleotide sequence that is operably linked to the promoter whose activity is being measured. Promoter activity may be measured directly by determining the amount of RNA transcript produced, for example by Northern blot analysis or indirectly by determining the amount of product coded for by the linked nucleic acid sequence, such as a reporter nucleic acid sequence linked to the promoter.

[0051] “Polynucleotide” as used herein refers to a polymeric form of nucleotides or nucleic acids of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double and single stranded DNA, triplex DNA, as well as double and single stranded RNA. It also includes modified, for example, by methylation and / or by capping, and unmodified. Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5' to 3' direction.

[0052] As used herein, the phrase “variant” when used with reference to a nucleic acid or polypeptide refers to a nucleic acid or polypeptide that differs from the referenced nucleic acid or polypeptide (for example, differing by at least one amino acid substitution from a wild-type sequence) but possesses the primary function of the referenced polypeptide. For example, a functional variant of a polypeptide that serves as a transmembrane domain is a fragment of that polypeptide that also serves as a transmembrane domain. When used with reference to a nucleic acid, the phrase “variant” refers to a nucleic acid that differs from theDFCI IP No. 3561Attorney Docket No. 91016-431320referenced nucleic acid but encodes a polypeptide having the same primary function as the polypeptide encoded by the referenced nucleic acid.

[0053] Contemplated herein are conservative variants of the amino acid sequences described herein. A protein is a conservative variant where it contains conservative amino acid substitutions that do not substantially affect or decrease the affinity of a protein. For example, a CAR (e.g., expressing a heavy chain variable region) that binds its cognate antigen (e.g., CLDN18.2) may include at least 1, 2, 5, 10, or 15 conservative substitutions, for example, and bind the cognate antigen. Conservative amino acid substitution tables providing functionally similar amino acids are well-known to one of ordinary skill in the art. The following groups are examples of amino acids that are considered conservative substitutions for one another: 1) serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0054] The terms “transfection,” “transformation,” “nucleofection,” or “transduction” as used herein refer to the introduction of one or more exogenous polynucleotides into a host cell or organism by using physical, chemical, and / or electrical methods. The nucleic acid sequences and vectors disclosed herein may be introduced into a cell or organism by any such methods, including, for example, by electroporation, calcium phosphate coprecipitation, strontium phosphate DNA co- precipitation, liposome mediated-transfection, DEAE dextran mediated- transfection, polycationic mediated- transfection, tungsten particle- facilitated microparticle bombardment, viral, and / or non- viral mediated transfection. In some cases, the method of introducing nucleic acids into the cell or organism involve the use of viral, retroviral, lentiviral, or transposon, or transposable element - mediated (e.g., Sleeping Beauty) vectors.

[0055] The terms “identical” and its grammatical equivalents as used herein or “sequence identity” in the context of two nucleic acid sequences or amino acid sequences of polypeptides refer to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. A “comparison window”, as may be used herein, refers to a segment of at least about 20 contiguousDFCI IP No. 3561Attorney Docket No. 91016-431320positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981); by the alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci U. S. A., 85:2444 (1988); by computerized implementations of these algorithms (including, but not limited to CLUSTAL in the PC / Gene program by Intelligences, Mountain View Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., U. S. A.); the CLUSTAL program is well described by Higgins and Sharp, Gene, 73:237-244 (1988) and Higgins and Sharp, CABIOS, 5:151-153 (1989); Corpet et al., Nucleic Acids Res., 16:10881-10890 (1988); Huang et al., Computer Applications in the Biosciences, 8:155-165 (1992); and Pearson et al., Methods in Molecular Biology, 24:307-331 (1994). Alignment may also be performed by inspection and manual alignment. In one class of embodiments, the nucleic acids described herein are at least 80%, 85%, 90%, 98% 99% or 100% identical to a reference polypeptide, or a fragment thereof, e.g., as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids may also be described with reference to a starting nucleic acid, e.g., they may be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99% or 100% identical to a reference nucleic acid or a fragment thereof, e.g., as measured by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. When one molecule is said to have certain percentage of sequence identity with a larger molecule, it means that when the two molecules are optimally aligned, the percentage of residues in the smaller molecule finds a match residue in the larger molecule in accordance with the order by which the two molecules are optimally aligned.

[0056] “Homology” is generally inferred from sequence identity between two or more nucleic acids (or sequences thereof). The precise percentage of identity betweenDFCI IP No. 3561Attorney Docket No. 91016-431320sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence identity is routinely used to establish homology. Higher levels of sequence identity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more may also be used to establish homology. Methods for determining sequence identity percentages (e.g., BLASTP and BLASTN using default parameters) are described herein and are generally available. Nucleic acids and / or nucleic acid sequences are “homologous” when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. The homologous molecules may be termed “homologs.” For example, any naturally occurring proteins may be modified by any available mutagenesis method. When expressed, the mutagenized nucleic acid encodes a polypeptide that is homologous to the protein encoded by the original nucleic acid.

[0057] The term “pharmaceutically acceptable” refers to a molecule or composition that, when administered to a recipient, is not deleterious to the recipient thereof, or that any deleterious effect is outweighed by a benefit to the recipient thereof. With respect to a carrier, diluent, or excipient used to formulate a composition as disclosed herein, a pharmaceutically acceptable carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof, or any deleterious effect must be outweighed by a benefit to the recipient.

[0058] The term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one portion of the body to another (e.g., from one organ to another). Each carrier present in a pharmaceutical composition must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient, or any deleterious effect must be outweighed by a benefit to the recipient. The pharmaceutically acceptable carriers of use are conventional (e.g., as described in Remington, The Science and Practice of Pharmacy, 22nd Edition, Loyd V., ed., Pharmaceutical Press, 2012). In general, the nature of the carrier will depend on the mode of administration. Some examples of materials which may serve as pharmaceutically acceptable carriers comprise: sugars, suchDFCI IP No. 3561Attorney Docket No. 91016-431320as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogenfree water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations. For instance, parenteral formulations typically comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions or the like as a vehicle. Pharmaceutical compositions may additionally include minor amounts of non-toxic auxiliary substances for stability.

[0059] The term “pharmaceutical composition” refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant subject or population. In some embodiments, a pharmaceutical composition may be formulated for administration in solid or liquid form, comprising, without limitation, a form adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as aDFCI IP No. 3561Attorney Docket No. 91016-431320pessary, cream, or foam; sublingually; ocularly; transdermally, as, for example, nasally, pulmonary, and to other mucosal surfaces; or infusion.

[0060] As used herein, "treatment”, "treat", and "treating" refer to reversing, alleviating, mitigating, or slowing the progression of, or inhibiting the progress of, a disorder or disease or symptoms associated with such disorder or disease, and as described in more detail herein.

[0061] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0062] CARs have been used as an immunotherapy to treat cancer by genetically modifying an immune cell (e.g., a T cell) to target and kill cancer cells. CARs include antigen binding domains that recognize and bind to specific proteins on the cancer cell-surface. There are currently six CAR-T cell therapies approved by the Food and Drug Administration. See, for example, cancer. gov / about-cancer / treatment / research / car-t-cells. Thus, without being bound by any theory, it is believed that an T cell modified by the expression of a CAR specific for a cancer cell target may amplify the T cell’s ability to target these malignant cells. CARs may include (1) an extracellular antigen-binding motif (e.g., VH-only single domain binder), (2) linking / transmembrane motifs, and (3) an intracellular domain, including a costimulatory domain and an activity domain (e.g., CD137 (4-1 BB) and CD247 (CD3Q-derived costimulatory domain and an activity domain, respectively). As used herein, the term “chimeric antigen receptor T cell” means a T cell which is genetically modified to express a chimeric antigen receptor (CAR) with specificity for a particular antigen. The CAR may have specificity for any known antigen, including but not limited to, claudin, such as CLDN18.2. A CAR-T cell targeting CLDN18.2 may be referred to as an anti-CLDN18.2 CAR-T cell. In some aspects, the CAR T cell expresses an anti-CLDN18.2 VH-only single domain binder or functional fragment thereof.DFCI IP No. 3561Attorney Docket No. 91016-431320

[0063] Accordingly, one embodiment described herein is an amino acid comprising a CLDN18.2-targeting CAR. For example, a CLDN18.2 targeting CAR may include an antigen binding domain comprising a heavy chain variable fragment (VH) or a derivative thereof. The term “heavy chain variable region means” refers to an antibody fragment, the heavy chain variable region, that binds CLDN18.2. Heavy chain variable regions within the scope of heavy chain variable region means are the disclosed heavy chain variable regions and functional equivalents thereto. Functional equivalent heavy chain variable regions comprise different specific amino acid residues but bind CLDN18.2. Functional equivalent heavy chain variable regions would differ ^substantially to bind CLDN18.2. Methods of making these heavy chain variable regions or fragments thereof are routine (see, e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)).

[0064] Herein, “binding” (or “binds”) refers to the well understood interaction between a heavy chain variable region or fragment thereof and a target protein, peptide, or polysaccharide, such as CLDN18.2. Binding may be measured in a variety of ways (see, e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)), for example, by activity (such as cancer cell viability) or immunoassays (such as ELISA or western blotting). A particular heavy chain variable region or fragment thereof binds to a particular target protein, peptide, or polysaccharide and does not bind in a significant amount to other proteins or polysaccharides present in a sample or subject disclosed herein. Binding occurs between the disclosed heavy chain variable region and epitopes of CLDN18.2. Herein, "epitope" refers to discrete sites of an antigen recognized by the disclosed heavy chain variable region antibody fragments. Epitopes may be linear or three-dimensional. A heavy chain variable region or fragment thereof binds to a target protein when the interaction has a KD of less than 10-6M, such as less than 10-7M, less than 10-8M, less than 10-9M, or less than 10-10M.

[0065] Another embodiment described herein is a CAR comprising an amino acid having formula I: R1 - R2 - R3 - R4 - R5 (I), wherein: R1 comprises a VH-only single domain binder for binding claudin 18.2 (CLDN18.2); R2 comprises a hinge region; R3 comprises aDFCI IP No. 3561Attorney Docket No. 91016-431320transmembrane region; R4 comprises a costimulatory region; and R5 comprises an intracellular region.

[0066] Another embodiment described herein is a CAR comprising an amino acid having formula I: R1 - R2 - R3 - R4 - R5 (I), wherein: R1 comprises a VH-only single domain binder for binding claudin 18.2 (CLDN18.2); R2 comprises a cluster of differentiation 28 (CD28) hinge region; R3 comprises a CD28 transmembrane region; R4 comprises a 4-1 BB costimulatory region; and R5 comprises a cluster of differentiation 3 zeta (CD3ζ) intracellular region.

[0067] In another embodiment, a VH-only single domain binder may comprise any of the embodiments of R1 described herein, i.e., a VH-only single domain binder may be a polypeptide that is not a component of a CAR. In one aspect, a VH-only single domain binder targeting CLDN18.2 comprising any of the amino acid sequences or nucleic acid sequences of R1, as described herein. Therefore, any description of R1 herein may describe either R1 as a VH-only single domain binder or a CAR comprising a VH-only single domain binder of R1.

[0068] In one aspect, R1 comprises a VH-only single domain binder for binding claudin 18.2 (CLDN18.2) comprising a heavy chain variable region. For example, R1 comprises a VH-only single domain binder for binding claudin 18.2 (CLDN18.2) comprising a heavy chain variable region comprising complementarity-determining regions (CDRs) which are the variable parts of the binder that are directly involved in antigen binding. The variable region of a binder (e.g., a VH-only single domain binder) is specific for the target antigen, i.e. CLDN18.2 antigen, and the identity of a target antigen is determined by the interaction of CDRs and the target antigen. Canonically, each VH domain contains three CDRs: CDR1, CDR2, and CDR3. Assignment of amino acids to CDR domains within the heavy chain variable region of the present disclosure is based on the well-known IMGT numbering conventions. See, for example, Lefranc, M. P., et al., Dev. Comp. Immunol., 27, 55-77 (2003).

[0069] In another aspect, R1 comprises a VH-only single domain binder for binding claudin 18.2 (CLDN18.2) comprising a heavy chain variable region comprising: a) aDFCI IP No. 3561Attorney Docket No. 91016-431320complementarity-determining region 1 (CDR1) having an amino acid sequence with 0 to 2 amino acid substitutions compared to SEQ ID NOs: 1, 4, 29, or 32; b) a complementaritydetermining region 2 (CDR2) having an amino acid sequence with 0 to 2 amino acid substitutions compared to SEQ ID NOs: 2, 5, 30, or 33; or c) a complementaritydetermining region 3 (CDR3) having an amino acid sequence with 0 to 2 amino acid substitutions compared to SEQ ID NOs: 3, 6, 31, or 34 (See Table 2 for sequences). For example, CDR1 may have an amino acid sequence with 0, 1, or 2 amino acid substitutions compared to SEQ ID NOs: 1, 4, 29, or 32, CDR2 may have an amino acid sequence with 0, 1, or 2 amino acid substitutions compared to SEQ ID NOs: 2, 5, 30, or 33; or CDR3 may have an amino acid sequence with 0, 1, or 2 amino acid substitutions compared to SEQ ID NOs: 3, 6, 31, or 34 (See Table 2 for sequences).

[0070] In another aspect R1 comprises a VH-only single domain binder for binding claudin 18.2 (CLDN18.2) comprising a heavy chain variable region, the heavy chain variable region comprises: a) a CDR1 having an amino acid sequence selected from SEQ ID NOs: 1, 4, 29, or 32; b) a CDR2 having an amino acid sequence selected from SEQ ID NOs: 2, 5, 30, or 33; or c) a CDR3 having an amino acid sequence selected from SEQ ID NOs: 3, 6, 31, or 34 (See Table 2 for sequences).

[0071] In other aspects R1 comprises a VH-only single domain binder for binding claudin 18.2 (CLDN18.2) comprising a heavy chain variable region, the heavy chain variable region comprises: a) a CDR1 having the amino acid sequence of SEQ ID NO: 1; a CDR2 having the amino acid sequence of SEQ ID NO: 2; and a CDR3 having the amino acid sequence of SEQ ID NO: 3; b) a CDR1 having the amino acid sequence of SEQ ID NO: 4; a CDR2 having the amino acid sequence of SEQ ID NO: 5; and a CDR3 having the amino acid sequence of SEQ ID NO: 6; c) a CDR1 having the amino acid sequence of SEQ ID NO: 29; a CDR2 having the amino acid sequence of SEQ ID NO: 30; and a CDR3 having the amino acid sequence of SEQ ID NO: 31; or d) a CDR1 having the amino acid sequence of SEQ ID NO: 32; a CDR2 having the amino acid sequence of SEQ ID NO: 33; and a CDR3 having the amino acid sequence of SEQ ID NO: 34 (See Table 2 for sequences).DFCI IP No. 3561Attorney Docket No. 91016-431320

[0072] In one aspect R1 comprises a VH-only single domain binder for binding claudin 18.2 (CLDN18.2) comprising a heavy chain variable region, the heavy chain variable region may comprise a CDR1 having the amino acid sequence of SEQ ID NO: 1; a CDR2 having the amino acid sequence of SEQ ID NO: 2; and a CDR3 having the amino acid sequence of SEQ ID NO: 3. In another aspect, the heavy chain variable region may comprise a CDR1 having the amino acid sequence of SEQ ID NO: 4; a CDR2 having the amino acid sequence of SEQ ID NO: 5; and a CDR3 having the amino acid sequence of SEQ ID NO: 6 (See Table 2 for sequences). In another aspect, the heavy chain variable region may comprise a CDR1 having the amino acid sequence of SEQ ID NO: 29; a CDR2 having the amino acid sequence of SEQ ID NO: 30; and a CDR3 having the amino acid sequence of SEQ ID NO: 31. In another aspect, the heavy chain variable region may comprise a CDR1 having the amino acid sequence of SEQ ID NO: 32; a CDR2 having the amino acid sequence of SEQ ID NO: 33; and a CDR3 having the amino acid sequence of SEQ ID NO: 34 (See Table 2 for sequences).

[0073] In another aspect, R1 comprises a VH-only single domain binder for binding claudin 18.2 (CLDN18.2) comprising a heavy chain variable region, the heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity) to SEQ ID NOs: 7, 9, 35, or 37 (See Table 2 for sequences). For example, in one aspect, the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NOs: 7, 9, 35, or 37. In another aspect, the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 7, 9, 35, or 37. In another aspect, the heavy chain variable region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 7, 9, 35, or 37. In another aspect, the heavy chain variable region comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NOs: 7, 9, 35, or 37. In another aspect, the heavy chain variable region comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NOs: 7, 9, 35, or 37. In another aspect, the heavy chain variable region comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NOs:DFCI IP No. 3561Attorney Docket No. 91016-4313207, 9, 35, or 37. In another aspect, the heavy chain variable region comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 7, 9, 35, or 37. In another aspect, the heavy chain variable region comprises an amino acid sequence having at least 99.9% sequence identity to SEQ ID NOs: 7, 9, 35, or 37. In another aspect, the heavy chain variable region comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NOs: 7, 9, 35, or 37.

[0074] In another aspect, R1 comprises a VH-only single domain binder for binding claudin 18.2 (CLDN18.2) comprising a heavy chain variable region, the heavy chain variable region comprising an amino acid sequence having 0-10 amino acid substitutions (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions) compared to a sequence selected from SEQ ID NOs: 7, 9, 35, or 37 (See Table 2 for sequences). For example, in one aspect, the heavy chain variable region comprises an amino acid sequence having 0-10 amino acid substitutions compared to a sequence according to SEQ ID NO: 7. In one aspect, the heavy chain variable region comprises an amino acid sequence having 0-10 amino acid substitutions compared to a sequence according to SEQ ID NO: 9. In one aspect, the heavy chain variable region comprises an amino acid sequence having 0-10 amino acid substitutions compared to a sequence according to SEQ ID NO: 35. In one aspect, the heavy chain variable region comprises an amino acid sequence having 0-10 amino acid substitutions compared to a sequence according to SEQ ID NO: 37.

[0075] In another aspect, a VH-only single domain binder of R1 described herein is encoded by a nucleic acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity) to SEQ ID Nos: 8, 10, 36, or 38, or a codon degenerate nucleic acid sequence thereof (See Table 2 for sequences). In another aspect, the VH-only single domain binder of R1 is encoded by a nucleic acid sequence having at least 85% sequence identity to SEQ ID Nos: 8, 10, 36, or 38. In another aspect, the VH-only single domain binder of R1 is encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID Nos: 8, 10, 36, or 38. In another aspect, the VH-only single domain binder of R1 is encoded by a nucleic acid sequence having at least 95% sequence identity to SEQ ID Nos: 8, 10, 36, or 38. InDFCI IP No. 3561Attorney Docket No. 91016-431320another aspect, the VH-only single domain binder of R1 is encoded by a nucleic acid sequence having at least 96% sequence identity to SEQ ID Nos: 8, 10, 36, or 38. In another aspect, the VH-only single domain binder of R1 is encoded by a nucleic acid sequence having at least 97% sequence identity to SEQ ID Nos: 8, 10, 36, or 38. In another aspect, the VH-only single domain binder of R1 is encoded by a nucleic acid sequence having at least 99% sequence identity to SEQ ID Nos: 8, 10, 36, or 38. In another aspect, the VH-only single domain binder of R1 is encoded by a nucleic acid sequence having at least 95% sequence identity to SEQ ID Nos: 8, 10, 36, or 38. In another aspect, the VH-only single domain binder of R1 is encoded by a nucleic acid sequence having at least 99.9% sequence identity to SEQ ID Nos: 8, 10, 36, or 38. In another aspect, the VH-only single domain binder of R1 is encoded by a nucleic acid sequence selected from SEQ ID Nos: 8, 10, 36, or 38. In another aspect, the VH-only single domain binder of R1 is encoded by the nucleic acid sequence according to SEQ ID Nos: 8, 10, 36, or 38. In another aspect, the VH-only single domain binder of R1 is encoded by the nucleic acid sequence according to SEQ ID No: 8. In another aspect, the VH-only single domain binder of R1 is encoded by the nucleic acid sequence according to SEQ ID No: 10. In another aspect, the VH-only single domain binder of R1 is encoded by the nucleic acid sequence according to SEQ ID No: 36. In another aspect, the VH-only single domain binder of R1 is encoded by the nucleic acid sequence according to SEQ ID No: 38.

[0076] In another aspect, the VH-only single domain binder of R1 described herein is encoded by a nucleic acid sequence having 0-20 nucleic acid substitutions (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 nucleic acid substitutions) compared to a sequence selected from SEQ ID Nos: 8, 10, 36, or 38, or a codon degenerate nucleic acid sequence thereof (See Table 2 for sequences). For example, in one aspect, the VH-only single domain binder of R1 is encoded by a nucleic acid sequence having 0-20 nucleic acid substitutions (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 nucleic acid substitutions) compared to the sequence according to SEQ ID No: 8. In one aspect, the VH-only single domain binder of R1 is encoded by a nucleic acid sequence having 0-20 nucleic acid substitutions (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 nucleic acid substitutions) compared to the sequenceDFCI IP No. 3561Attorney Docket No. 91016-431320according to SEQ ID No: 10. In one aspect, the VH-only single domain binder of R1 is encoded by a nucleic acid sequence having 0-20 nucleic acid substitutions (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 nucleic acid substitutions) compared to the sequence according to SEQ ID No: 36. In one aspect, the VH-only single domain binder of R1 is encoded by a nucleic acid sequence having 0-20 nucleic acid substitutions (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 nucleic acid substitutions) compared to the sequence according to SEQ ID No: 38.

[0077] In some aspects, the VH-only single domain binder of R1 described herein comprises complementary determining means. The phrase “complementary determining means” as used herein describes one or more of the three CDRs that form specific interactions with the target antigen, i.e., CLDN18.2. CDRs within the scope of complementary determining means are the disclosed CDRs and functional equivalents thereto. Functional equivalent CDRs comprise different specific amino acid residues but bind CLDN18.2. Functional equivalent CDRs would differ ^substantially, would bind CLDN18.2, and have a therapeutic effect.

[0078] Surrounding the CDRs in a variable region (e.g., a heavy chain variable region) are the framework regions (FRs), which are more conserved than the CDRs and provide structural support to the variable region. These FRs help with proper folding and stability of the binder molecule and maintaining the correct positioning of the CDRs to interact effectively with the antigens. In some aspects, the heavy chain variable region comprises human or humanized framework regions. For example, in some aspects, the heavy chain variable region comprises sequences that are human framework regions. See, for example, Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018). Exemplary human framework regions include the framework regions of SEQ ID NOs: 7, 9, 35, and 37. For example, heavy chain variable regions disclosed herein may have four framework regions, termed FR1 (e.g., SEQ ID NO: 47, EVQLX1ESGGGX2VQPGX3SLRLSCAAS), FR2 (e.g., SEQ ID NO: 48, MX1WVRQAPGKGLEWVX2X3), FR3 (e g., SEQ ID NO: 49, YYADSVKGRFTISRX1NSX2X3X4LYLQMX5SLRX6EDTAX7YY), and FR4, where Xnis anyDFCI IP No. 3561Attorney Docket No. 91016-431320amino acid. In some aspects, the heavy chain variable region comprises sequences that are not mouse framework regions. In some aspects, the heavy chain variable region comprises sequences that are not camelid framework regions.

[0079] In some aspects, the VH-only single binding domain is human. For example, in some aspects, the VH-only single binding domain was produced in an animal (e.g., a mouse) in which the native variable heavy and light (VH / VL) chain loci are removed, and a human VH chain locus is inserted. Therefore, antibodies produced by these mice only contain a human VH chain. See, for example, Example 1.

[0080] Various hinge regions are contemplated for R2. In some aspects, the hinge region comprises a hinge domain derived from CD3, CD4, CD8a, CD28, lgG1, lgG2, or lgG4. In some aspects the hinge region comprises a hinge domain derived from CD28. In some aspects, R2 comprises a CD28 hinge region. In some aspects, R2 comprises a CD28 hinge region having the amino acid sequence of SEQ ID NO: 13 (See Table 2 for sequences). The hinge region of a CAR is an extracellular structure that links the antigen binding domain (e.g., the VH-only single domain binder) and the transmembrane domains.Typically, the hinge domain is a flexible amino acid sequence that allowed the antigen binding domain and the transmembrane domains to move at least partially independently of one another. Thus, the hinge region impacts the antigen binding domain’s access to the target epitope and ability of CAR recognition of the target cells. See, for example, Sterner et al., Blood cancer journal 11.4 (2021 ): 69. Thus, in one aspect the CD28 hinge region of R2 comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 13 (See Table 2 for sequences). In some aspects, the CD28 hinge region of R2 comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 13. In some aspects, the CD28 hinge region of R2 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13. In some aspects, the CD28 hinge region of R2 comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 13. In some aspects, the CD28 hinge region of R2 comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 13. In some aspects, the CD28 hingeDFCI IP No. 3561Attorney Docket No. 91016-431320region of R2 comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 13. In some aspects, the CD28 hinge region of R2 comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 13. In some aspects, the CD28 hinge region of R2 comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 13. In some aspects, the CD28 hinge region of R2 comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 13. In some aspects, the CD28 hinge region of R2 comprises an amino acid sequence having 0-10 amino acid substitutions (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions) compared to SEQ ID NO: 13.

[0081] In some aspect, R2 comprises a CD28 hinge region encoded by the nucleic acid sequence of SEQ ID NO: 14, or a codon degenerate nucleic acid sequence thereof. Thus, in one aspect, the CD28 hinge region of R2 is encoded by a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 14, or a codon degenerate nucleic acid sequence thereof. In another aspect, the CD28 hinge region of R2 is encoded by a nucleic acid sequence having 0-20 nucleic acid substitutions (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 nucleic acid substitutions) compared to SEQ ID NO: 14, ora codon degenerate nucleic acid sequence thereof.

[0082] Various transmembrane domains are contemplated as R3. In some aspects, the transmembrane domain comprises a transmembrane domain derived from CD3, CD4, CD8a, CD28, or CD137. Particularly, in some aspects, R3 comprises a transmembrane domain derived from CD28. In some aspects, R3 comprises a CD28 transmembrane region. In some aspects, R3 comprises a CD28 transmembrane region having the amino acid sequence of SEQ ID NO: 15 (See Table 2 for sequences). The transmembrane domain serves to facilitate activation of the CAR upon antigen-binding domain binding of the target, by transducing signals to the intracellular domains. Thus, in one aspect the CD28 transmembrane region of R3 comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 15 (See Table 2 for sequences). In some aspects,DFCI IP No. 3561Attorney Docket No. 91016-431320the CD28 transmembrane region of R3 comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 15. In some aspects, the CD28 transmembrane region of R3 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15. In some aspects, the CD28 transmembrane region of R3 comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 15. In some aspects, the CD28 transmembrane region of R3 comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 15. In some aspects, the CD28 transmembrane region of R3 comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 15. In some aspects, the CD28 transmembrane region of R3 comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 15. In some aspects, the CD28 transmembrane region of R3 comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 15. In some aspects, the CD28 transmembrane region of R3 comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 15. In some aspects, the CD28 transmembrane region of R3 comprises an amino acid sequence having 0-10 amino acid substitutions (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions) compared to SEQ ID NO: 15.

[0083] In some aspect, R3 comprises a CD28 transmembrane region encoded by the nucleic acid sequence of SEQ ID NO: 16, or a codon degenerate nucleic acid sequence thereof (See Table 2 for sequences). Thus, in one aspect, the CD28 transmembrane region of R3 is encoded by a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 16, or a codon degenerate nucleic acid sequence thereof. In another aspect, the CD28 transmembrane region of R3 is encoded by a nucleic acid sequence having 0-20 nucleic acid substitutions (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 nucleic acid substitutions) compared to SEQ ID NO: 16, or a codon degenerate nucleic acid sequence thereof.

[0084] Various intracellular domains or intracellular signaling means are contemplated. In embodiments, R4 comprises a signaling domain or a signaling domain and a costimulatory domain. The signaling domain may be derived from CD3ζ, CD27, CD28, CD40, KIR2DS2,DFCI IP No. 3561Attorney Docket No. 91016-431320MyD88, or 0X40. A costimulatory domain may be derived from of CD3y, CD35, CD3£, CD3, CD27, CD40, CD28, CD72, CD80, CD86, CLEC-1, 4-1BB, TYROBP (DAP12), Dectin-1, FcaRI, FcyRI, FcyRII, FcyRIII, FCERI, IL-2RB, ICOS, KIR2DS2, MyD88, 0X40, and ZAP70. In some aspects, R4 comprises a 4-1 BB costimulatory region. In some aspects, R4 comprises a 4-1 BB costimulatory region having the amino acid sequence of SEQ ID NO: 17 (See Table 2 for sequences). 4-1 BB is a widely used costimulatory domain used in CAR cells. CAR architecture using 4-1 BB exhibits long term function and persistence. See, for example, Singh et al., Experimental & Molecular Medicine 56.1 (2024): 32-39. Thus, in one aspect, the 4-1 BB costimulatory region of R4 comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 17. In some aspects, the 4-1 BB costimulatory region of R4 comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 17. In some aspects, the 4-1 BB costimulatory region of R4 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17. In some aspects, the 4-1 BB costimulatory region of R4 comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 17. In some aspects, the 4-1 BB costimulatory region of R4 comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 17. In some aspects, the 4-1 BB costimulatory region of R4 comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 17. In some aspects, the 4-1 BB costimulatory region of R4 comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 17. In some aspects, the 4-1 BB costimulatory region of R4 comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 17. In some aspects, the 4-1 BB costimulatory region of R4 comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 17. In some aspects, the 4-1 BB costimulatory region of R4 comprises an amino acid sequence having 0-10 amino acid substitutions (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions) compared to SEQ ID NO: 17.

[0085] In some aspect, R4 comprises a 4-1 BB costimulatory region encoded by the nucleic acid sequence of SEQ ID NO: 18, or a codon degenerate nucleic acid sequence thereofDFCI IP No. 3561Attorney Docket No. 91016-431320(See Table 2 for sequences). Thus, in one aspect, the 4-1 BB costimulatory region of R4 is encoded by a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 18, or a codon degenerate nucleic acid sequence thereof. In another aspect, the 4-1 BB costimulatory region of R4 is encoded by a nucleic acid sequence having 0-20 nucleic acid substitutions (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 nucleic acid substitutions) compared to SEQ ID NO: 18, or a codon degenerate nucleic acid sequence thereof.

[0086] In some aspects, R5 comprises a CD3ζ intracellular region. In some aspects, R5 comprises cluster of differentiation 3 zeta (CD3 ) intracellular region having the amino acid sequence of SEQ ID NO: 19 (See Table 2 for sequences). The CD3ζ intracellular region facilitates intracellular signaling after antigen-binding domain binding to activate the T cells. Thus in one aspect, the CD3ζ intracellular region of R5 comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 19 (See Table 2 for sequences). In some aspects, the CD3ζ intracellular region of R5 comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 19. In some aspects, the CD3ζ intracellular region of R5 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 19. In some aspects, the CD3ζ intracellular region of R5 comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 19. In some aspects, the CD3 intracellular region of R5 comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 19. In some aspects, the CD3 intracellular region of R5 comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 19. In some aspects, the CD3 intracellular region of R5 comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 19. In some aspects, the CD3 intracellular region of R5 comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 19. In some aspects, the CD3ζ intracellular region of R5 comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 19. In some aspects, the CD3ζ intracellular region of R5 comprises an amino acid sequenceDFCI IP No. 3561Attorney Docket No. 91016-431320having 0-10 amino acid substitutions (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions) compared to SEQ ID NO: 19.

[0087] In some aspect, R5 comprises a CD3ζ intracellular region encoded by the nucleic acid sequence of SEQ ID NO: 20, or a codon degenerate nucleic acid sequence thereof (See Table 2 for sequences). Thus, in one aspect, the CD3ζ intracellular region of R5 is encoded by a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 20, or a codon degenerate nucleic acid sequence thereof. In another aspect, the CD3ζ intracellular region of R5 is encoded by a nucleic acid sequence having 0-20 nucleic acid substitutions (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 nucleic acid substitutions) compared to SEQ ID NO: 20, or a codon degenerate nucleic acid sequence thereof.

[0088] In some aspects, R1 through R5 comprise the complete coding region of the CAR. For example, in some aspects, the CAR comprises an amino acid selected from SEQ ID NOs: 21, 23, 39, or 41. In some aspects, the CAR comprises the amino acid according to SEQ ID NO: 21. In some aspects, the CAR comprises the amino acid according to SEQ ID NO: 23. In some aspects, the CAR comprises the amino acid according to SEQ ID NO: 39. In some aspects, the CAR comprises the amino acid according to SEQ ID NO: 41. In another aspect, the CAR is encoded by a nucleic acid selected from SEQ ID NOs: 22, 24, 40, or 42. For example, in some aspects, the CAR is encoded by the nucleic acid according to SEQ ID NO: 22. In some aspects, the CAR is encoded by the nucleic acid according to SEQ ID NO: 24. In some aspects, the CAR is encoded by the nucleic acid according to SEQ ID NO: 40. In some aspects, the CAR is encoded by the nucleic acid according to SEQ ID NO: 42.

[0089] In some aspects, CARs provided herein are encoded with codon optimized nucleic acid sequences.

[0090] Any of the VH-only single domain binders, or CARs comprising the same, target claudin 18.2 (CLDN18.2).

[0091] Targets may be determined by measuring binding affinities of the binders or CARs comprising the same, to various cell-surface proteins. Antibodies comprising any of theDFCI IP No. 3561Attorney Docket No. 91016-431320binders or antigen-binding regions described herein may be used to determine relative binding affinities of the binders or antigen-binding regions to their specific target antigens. For example, the binding affinities of binders or CARs comprising the same may be determined as described in Example 2.

[0092] In some aspects, the VH-only single domain binder of R1 has a binding affinity from about 1 nM to about 100 nM to CLDN18.2 (e.g., about 2 nM to about 60 nM, about 2 nM to about 30 nM or about 4 nM to about 58 nM). For example, in some aspects, the VH-only single domain binder of R1 has a binding affinity from about 1 nM to about 10 nM, about 2 nM to about 7 nM, about 2 nM to about 6 nM, about 3.5 nM to about 5.5 nM, from 20 nM to about 30 nM, about 21 nM to about 26 nM, about 21 nM to about 25 nM, about 21 to about 23 nM, from about 30 nM to about 60 nM, about 35 nM to about 55 nM, about 40 nM to about 50 nM, about 45 nM to about 49 nM, about 47 to about 49 nM, about 50 nM to about 60 nM, about 56 to about 58 nM. In some aspects, the VH-only single domain binder of R1 has a binding affinity from about 2 nM to about 6 nM to CLDN18.2. In some aspects, the VH-only single domain binder has a binding affinity from about 18 nM to about 25 nM to CLDN18.2. In some aspects, the VH-only single binder of R1 has a binding affinity of about 47.8 ± 13 nM. In some aspects, the VH-only single binder of R1 has a binding affinity of about 21.8 ± 2 nM.. In some aspects, the VH-only single binder of R1 has a binding affinity of about 4.53 ± 3 nM. In other aspects, the VH-only single binder of R1 has a binding affinity of about 56.9 ± 14 nM.

[0093] Furthermore, the binders, or CARs comprising the binders, target CLDN18.2 and do not target the isoform claudin 18.1(CLDN18.1). For example, in some aspects, the VH-only single domain binder has no binding affinity to CLDN18.1. In other aspects, the VH-only single domain binder may have reduced, decreased, or lowered binding affinity to CLDN18.1 as compared to binding affinity to CLDN18.2. In other aspects, the VH-only single domain binder has a binding affinity of greater than 100 mM to CLDN18.1.

[0094] In some aspects, VH-only binders described herein and CARs comprising the same have functional advantages facilitated by the size and content of VH-only binders. For example, in some aspects, VH-only binders described herein and CARs comprising theDFCI IP No. 3561Attorney Docket No. 91016-431320same cause low levels of toxicity or no toxicity, exhibit anti-tumor efficacy (e.g., prevent tumor growth or decrease the size and / or number of existing tumors), induce low levels of host anti-binder or anti-CAR immunity, have higher efficiency transduction of primary T cells compared to a binder comprising heavy and light chains (e.g., CAR comprising an scFv), induce low levels on tonic signaling, induce low levels of T cell exhaustion, and / or induce delayed T cell exhaustion (e.g., do not induce early exhaustion). Any of these features may be measured as described in Examples 2-4.

[0095] In some aspects, the nucleic constructs described herein are engineered into a vector (e.g., a recombinant vector). To obtain expression of the nucleic acid sequences, the sequences may be incorporated in a vector having one or more control sequences operably linked to the nucleic acid to control its expression. The vectors may include other sequences such as promoters or enhancers to drive the expression of the inserted nucleic acid, nucleic acid sequences so that the polypeptide or peptide is produced as a fusion and / or nucleic acid encoding secretion signals so that the polypeptide produced in the host cell is secreted from the cell. Polypeptide may then be obtained by transforming the vectors into host T cells in which the vector is functional, culturing the host cells so that the polypeptide is produced, and recovering the polypeptide from the host cells or the surrounding medium. Prokaryotic and eukaryotic cells are used for this purpose in the art, including strains of E. coli, yeast, and eukaryotic cells such as COS or CHO cells.

[0096] Suitable vectors include, but are not limited to, plasmids, transposons, bacteriophages, cosmids or virus-based vectors. Vectors may contain polynucleotide sequences which are necessary to effect ligation or insertion of the vector into a desired host cell and to effect the expression of the attached segment. Such sequences differ depending on the host organism; they include promoter sequences to effect transcription, enhancer sequences to increase transcription, ribosomal binding site sequences and transcription and translation termination sequences. Alternatively, expression vectors may be capable of directly expressing nucleic acid sequence products encoded therein without ligation or integration of the vector into host cell DNA sequences. In some aspects, the vector is an “episomal expression vector” or “episome,” which is able to replicate in a hostDFCI IP No. 3561Attorney Docket No. 91016-431320cell, and persists as an extrachromosomal segment of DNA within the host cell in the presence of appropriate selective pressure (see, e.g., Conese et al., Gene Therapy, 11:1735-1742 (2004)). Representative commercially available episomal expression vectors include, but are not limited to, episomal plasmids that utilize Epstein Barr Nuclear Antigen 1 (EBNA1) and the Epstein Barr Virus (EBV) origin of replication (oriP).

[0097] In some aspects, chimeric antigen receptors provided herein are encoded by a vector. In some aspects, chimeric antigen receptors provided herein are encoded by a plasmid vector. In some aspects, chimeric antigen receptors provided herein are encoded by a lentiviral plasmid vector.

[0098] In one aspect, a vector comprises a nucleic acid selected from SEQ ID NOs: 25-28 or 43-46. For example, in some aspects, a vector comprises the nucleic acid according to SEQ ID NO: 25. In some aspects, a vector comprises the nucleic acid according to SEQ ID NO: 26. In some aspects, a vector comprises the nucleic acid according to SEQ ID NO: 27. In some aspects, a vector comprises the nucleic acid according to SEQ ID NO: 28. In some aspects, a vector comprises the nucleic acid according to SEQ ID NO: 43. In some aspects, a vector comprises the nucleic acid according to SEQ ID NO: 44. In some aspects, a vector comprises the nucleic acid according to SEQ ID NO: 45. In some aspects, a vector comprises the nucleic acid according to SEQ ID NO: 46.

[0099] Suitable vectors may be transfected into any suitable host cell. For example, vectors expressing CARs described herein are receptor proteins that have been engineered for expression on an immune cell and to target a specific antigen (“target antigen”) as well as activate the immune cell. CARs are used in therapies, such as immune cell therapy, including T and NK cell therapy. CARs may be engineering into allogeneic immune cells (i.e., immune cells from a donor are engineered) or autologous immune cells (i.e., immune cells from a patient or subject that are re-introduced after engineering). CARs typically include (1 ) an extracellular antigen-binding motif (e.g., antibody or fragment thereof, such as heavy chain variable region(s)), (2) linking / transmembrane motifs, and (3) an intracellular domain (e.g., a signaling, such as such as a signaling domain derived from CD137 (4-IBB), and / or costimulatory domain, such as a costimulatory domain derived fromDFCI IP No. 3561Attorney Docket No. 91016-431320CD247 (CD3 )). In embodiments, the CAR expresses CD8+ or CD4+ on the immune cell surface (such as a CAR-NK or CAR-T cell surface).

[0100] Various immune cells are contemplated for the engineered cells of the present disclosure. In embodiments, the immune cell is selected from the group consisting of T cells, natural killer (NK) cells, macrophages, and dendritic cells. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is selected from the list consisting of: a cytotoxic lymphocyte, T cell, cytotoxic T cell (CD8+ T cell), T helper cell (CD4+ T cell), a[3 T cell and / or yb T cell, Th17 T-cell, and NKT (NKT) cell. In some aspects, the immune cell is a CD8+ T cell. In some aspects, the immune cell is a CD4+ T cell.

[0101] For example, a vector comprising a nucleic acid encoding any of the CARs described herein may be transfected into a T cell to produce an engineered T cell expressing the CAR, referred to as a CAR-T cell. For example, in some aspects, a CAR-T cell expresses a CAR comprising an amino acid selected from SEQ ID NOs: 21, 23, 39, or 40. In another aspect, a CAR-T cell expresses a CAR encoded by a nucleic acid selected from SEQ ID NOs: 22, 24, 40, or 42.

[0102] Pharmaceutical compositions comprising the engineered T cells (e.g., the CAR-T cells) described herein are also contemplated, and may further comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present disclosure may be formulated for cellular infusion, parenteral administration, e.g., intravascular (intravenous or intra-arterial), intraperitoneal, intramuscular administration. Thus, another aspect described herein is a pharmaceutical composition comprising the nucleic acid constructs or vectors described herein.

[0103] In some aspects, compositions described herein comprise an effective amount of an engineered T cell composition, alone or in combination with one or more therapeutic agents. The dosages and dosage regimen to achieve the desired therapeutic resultDFCI IP No. 3561Attorney Docket No. 91016-431320depending on the means of administration and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the engineered cell to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the engineered cell of the present disclosure are outweighed by the therapeutically beneficial effects. Thus, the T cell compositions described herein may be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, surgery, or immunotherapy. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, antiinflammatories, chemotherapeutics, radiotherapeutics, therapeutic antibodies, or other active and ancillary agents.

[0104] In some aspects, the compositions described herein are administered in any method that is most appropriate for treating the disease or disorder of the patient. In some embodiments, the active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant subject or population. In some embodiments, a pharmaceutical composition may be formulated for administration in solid or liquid form, comprising, without limitation, a form adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces; or infusion. In some aspects, the compositions described herein are administered intravenously.

[0105] In one aspect, any of the pharmaceutical compositions described herein comprises a pharmaceutically acceptable carrier. In some aspects, the carrier may be sterile and / orDFCI IP No. 3561Attorney Docket No. 91016-431320suspended or otherwise contained in a unit dosage form including one or more measured doses of the composition suitable for administration to a subject of an effective amount of the engineered cells disclosed herein. Medications for use in treatment may also be included in some embodiments. In one aspect, the unit dosage form may be in a sealed vial that contains sterile contents or a syringe for injection into a subject, lyophilized for subsequent solubilization and administration, or in a solid or controlled release dosage.

[0106] Another aspect described herein is a method of treating a cancer in a subject in need thereof comprising administering an effective amount of a composition comprising the engineered T cells expressing the CAR constructs as described herein. In some aspects, the cancer may be any cancer. Exemplary cancers include, but are not limited to, is gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, and non-small cell lung cancer. In some aspects, the cancer is gastric cancer. Other cancers which may overexpress the target antigen, i.e., CLDN18.2, are also contemplated herein. In some aspects, the target antigen is expressed or overexpressed on cancer cells, but not healthy cells.

[0107] Another aspect described herein is a method of limiting gastrointestinal toxicity in a patient previously identified as having cancer comprising administering an effective amount of a composition comprising the engineered T cells expressing the CAR constructs as described herein.

[0108] Another aspect described herein is a method of limiting stomach tissue atrophy in a patient previously identified as having cancer comprising administering an effective amount of a composition comprising the engineered T cells expressing the CAR constructs as described herein.

[0109] Another aspect described herein is a method of treating treatment-resistant or chemotherapy-resistant cancer, comprising administering to a patient or subject in need thereof a pharmaceutical composition provided herein (e.g., a patient or subject with or at risk of treatment-resistant or chemotherapy-resistant cancer).

[0110] The quantity and frequency of administration for any of the methods described herein will be determined by such factors as the condition of the patient, and the type andDFCI IP No. 3561Attorney Docket No. 91016-431320severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[0111] In some aspects of any of the methods described herein, the step of administering comprises any appropriate method of administration. For example, appropriate methods of administration may include oral administration, parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection, topical administration, intravaginally or intrarectally, sublingually, ocularly, transdermally, nasally, pulmonarily, or as an infusion. In some aspects, the step of administering comprises intravenously administering.

[0112] Also described herein is the use of an immune cell, a T cell or a pharmaceutical composition as described herein for the manufacture or preparation of a medicament for the treatment of cancer in a patient. In some aspects, the cancer is selected from a gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, and non-small cell lung cancer.

[0113] The CARs and / or engineered cells (e.g., CAR-T cells) described herein may also be used in combination therapy. In embodiments, the subject is treated with engineered cells disclosed herein in combination with one or more additional therapies to treat cancer, for example, radiation, surgery, bone marrow transplantation, chemotherapy, immunotherapy, hormone therapy, targeted therapy, or other engineered cell therapies. Use of the engineered cells of the of the present disclosure in combination with chemotherapy or other immunotherapy is contemplated. In embodiments, the additional treatment is directed to targeting similar or the same antigens (e.g., one or more engineered cells of provided herein may be used in combination, such as engineered cells expressing CD8+ and engineered cells expressing CD4+ at the cell surface). As used herein, “combination” therapy or use in combination refers to the administration of the engineered cells of the present disclosure to a patient in conjunction with (i.e., before, simultaneously, or following) any number of relevant treatments.

[0114] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparentDFCI IP No. 3561Attorney Docket No. 91016-431320to one of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.EXAMPLESExample 1. Initial VH-only single domain binder discovery.

[0115] HCAb Harbour Mice® (Version 2.1) were immunized with HEK293 cells overexpressing human CLDN18.2. B cell and plasma cells from the blood, spleen, and marrow were obtained, and specific and productive VH secreting cells were identified through SBC (Single B Cell) screening on the Beacon optofluidic system, single B / plasma cell sequencing, followed by confirmation using 24-well transient transfection supernatants to generate bivalent VH-Fc format binders. (HCAb Harbour Mice® are a line of transgenic mice knocked out for murine variable heavy and light (VHA / L) chain loci and knocked in for only the human VH chain locus. Therefore, antibodies produced by these mice only contain a human VH chain). VH-Fc format binders were purified via size exclusion chromatography by HPLC and screened for target specific binding via flow cytometry against CHO-K1 cells engineered to express either human CLDN18.2 (positive binding) or human CLDN18.1 (negative for binding) (Fig. 18). The binders were further screened for target specific binding via flow cytometry against HEK293 cells engineered to overexpress cynomologous CLDN18.2 or HEK293 cells overexpressing mouse CLDN18.2 (Fig. 19). Binding was further confirmed to cell lines that endogenously express CLDN18.2, NUGC4_D8 cells and SNU620 cells (SNU620 has M149L mutation) (Fig. 20).

[0116] Four VH-only single domain binders were identified in this discovery campaign (Table 1)DFCI IP No. 3561Attorney Docket No. 91016-431320Table 1. Identified VH-only single domain bindersVH-only single domain binder Amino Acid DNASEQ ID NO: SEQ ID NO:VH-only binder 1 7 8VH-only binder 2 9 10VH-only binder 3 35 36VH-only binder 4 37 38Example 2. Assessing binding kinetics of antibodies expressing VH-only single domain binders.

[0117] Binding kinetics of CLDN 18.2-targeting human VH-only single domain binders was assessed using surface plasmon resonance (SPR). SPR experiments were done on the Nicoya AltoSPR using PBS supplemented with 1% BSA and 0.1% Tween 20 running buffer (Nicoya Lifesciences PBS-T, Kitchener, ON, Canada). To generate a capture surface, Protein A (Nicoya Lifesciences ALTO- R-PROA-KIT) diluted in a sodium acetate buffer, pH 5.0 was amine coupled to the sensor surface of a 16-channel carboxyl cartridge (Nicoya Lifesciences KC-CBX-PEG-16) via EDC and NHS.

[0118] Anti-CLDN18.2 heavy chain only antibodies expressed as bivalent, IgG-reformatted binders (HCAbs) and a custom-produced IgG-reformatted antibody using the binding domain of CT041 (WuXi Biologies, Shanghai, China) were diluted to 10 pg / mL in running buffer and individually captured on sensor surfaces previously immobilized with Protein A. Recombinant human CLDN18.2-his (Genscript Z03504-10, Piscataway, NJ, USA) was diluted to 1800 nM in running buffer and then serially diluted 3-fold for a total of five concentrations as well as a baseline control sample at 0 nM. Each dilution of rhCLDN 18.2 was flowed over the captured antibodies from low to high concentration with a regeneration of 10 mM glycine-HCI pH 1.5 (Nicoya Lifesciences ALTO-R-GLYHCI-1.5) in between each cycle. Resulting sensorgrams were double referenced and, where applicable, fit to a 1:1 Langmuir binding model. All kons, koffs and KDs were reported as mean ± standard deviation from a total of four separate experiments.DFCI IP No. 3561Attorney Docket No. 91016-431320

[0119] Multicycle kinetics for each bivalent, IgG-reformatted binder are shown (Fig. 2A-Fig.2F). Binding affinity ranged from 4.53 ± 3 nM (antibody with VH-only binder 2) to 56.9 ± 14 nM (antibody with VH-only binder 3). Specifically, the binding affinity for the VH-only binder 4 (5781 ) was determined as 47.8 ± 13 nM. The binding affinity for the VH-only binder 1 (5795) was determined as 21.8 ± 2 nM. The binding affinity for the VH-only binder 2 (5797) was determined as 4.53 ± 3 nM, and the binding affinity for the VH-only binder 3 (5800) was determined as 56.9 ± 14 nM.Example 3. In vitro characterization of VH-only single binding domain CAR T cells

[0120] Individual CAR constructs containing each the identified VH-only single domain binders were generated. Briefly, a modified version of a third-generation self-inactivating (SIN) lentiviral transfer plasmid (CMV-GFP-WPRE) was used as an acceptor vector to clone candidate CAR sequences. A CAR construct incorporating a cytomegalovirus (CMV) promoter, the VH-only single domain binder, a CD28 hinge region, a CD28 transmembrane region, a 4-1 BB co-stimulatory region, and a CD3 intracellular domain were incorporated. The construct also incorporated the gene encoding a vexGFP reporter.

[0121] As benchmarks, CAR utilizing clinically developed CAR T cells were made, Here, the scFv sequence from CT041, (see, Jiang, H. et al. Journal of the National Cancer Institute 111, 409-418 (2019)) and the binding domains of zolbetuximab were engineered into the same second-generation CAR backbone as above. CAR T cells expressing the VH-only single domain binders, specifically CARs expressing VH-only binders 1-4, along with controls, CT041-scFv CAR and, in some instances, Zolbetuximab-scFv CAR, were assessed for cytotoxicity, tonic signaling, and transduction efficiency.

[0122] To assess cytotoxicity, SNU620 and OE19 cell lines were stably transduced with luciferase. 10,000 target cells were plated in 96-well plates in triplicate with CAR+ T cells at the indicated effector-to-target (E: T) ratios; cells were then incubated for 24 h. Cell viability was determined by an luciferase-dependent assay with OneGlo substrate E6110 (Promega, Madison, Wl, USA), where % cytotoxicity = (BLIControl- BLISAMPLE) / BLIMAX; BLI Control = mean target cell alone value of that experiment (irrelevantly targeted CAR wasDFCI IP No. 3561Attorney Docket No. 91016-431320used as a control). Bioluminescence was read on an Agilent Cytation 5 (Santa Clara, CA, USA). To assess tonic signaling, or CD3ζ signaling in absence of antigen-CAR binding, CAR were transduced into Jurkat T cell lymphoma reporter cells which have been gene modified to express GFP under the endogenous control of NR4A1 (Nur77), a gene specifically transcribed downstream of CD3ζ signaling. Jurkat reporter CAR cells were plated and monitored for GFP (Nur77 surrogate) for several days after transduction in the absence of target antigen and monitored for % GFP positivity. Low GFP positivity in absence of antigen is a desirable quality as it suggests signaling through the CAR is antigen dependent. Transduction efficiency was determined by flow cytometry for vexGFP expression, a reporter placed downstream of the CAR.CAR-T cell production

[0123] Lenti-X 293T cells (Takara) were seeded at 50,000 cells / cm2the day before transfection using TranslT-293 Transfection Reagent (Mirus). The relative amounts of plasmids for a 15 cm plate (145 cm2) were as follow: Packaging psPAX2 (Gag-Pol-Rev-Tat, Addgene 12260): 3 pmol, envelop pMD2. G (VSVg, Addgene 12259): 1.6 pmol, PKR inhibitor (pAdv, Promega #E1711): 0.9 pmol, transfer: 3.7 pmol. For other types of plates, the amounts of plasmids were adjusted proportionally to the surface. The media was changed with fresh media 24 hrs. post-transfection. The viral supernatant was harvested 48 hrs. post-transfection. Viral supernatants were cleared from debris after centrifugation (500 g, 5 min, 4 °C) followed by 0.45 pm filtration. Viral supernatants were used directly for transduction or concentrated by ultracentrifugation (110,000 g, 1 hr., 4 °C), aliquoted and stored at -80 °C. T cells were activated as above. After at least 2 days, cells were transduced with lentivirus via spinoculation (2000 g, 1 hr., 30 °C), monitored daily and kept at 0.5-1 e6cells / mL. Transduction efficiency could be enhanced using 1 mg / mL Poloxamer 338 (P338) (Sigma Aldrich #P2164021). vexGFP expression was measured to determine transduction efficiency in each CAR-T cell batch 5-6 days after transduction by flow cytometry analysis using the Northern Lights Spectral Flow Cytometer and SpectroFlo software (Cytek), further processed in FlowJo.CellsDFCI IP No. 3561Attorney Docket No. 91016-431320

[0124] OE19, a human esophageal cancer derived cell line, was acquired from the DSMZ collection (Braunschweig, Germany) and were cultured according to manufacturer’s recommendation. SNU620, a human gastric adenocarcinoma cell line, was acquired through the Korean Cell Line Bank and cultured per manufacturers recommendation. They were tested monthly for mycoplasma using PCR (Bulldog Bio). Human peripheral blood mononuclear cells (PBMCs), isolated by ficoll density centrifugation or purified T cells (RosetteSep Human T Cell Enrichment Cocktail, StemCell #15061) were stimulated with TransAct (Miltenyi), 10 pL for 5e6PBMCs / mL or for 1e6 T cells / mL, in complete RPMI medium: RPMI (Gibco #118750-093) supplemented with 10% heat inactivated Fetal Bovine Serum (FBS) (Life Technologies #10438026), Penicillin-Streptomycin (P / S) (Life Technologies #15140122), Glutamax (GX) (Life Technologies #35050061). The human recombinant cytokines IL-2 (200 U / mL final), IL-7 (5 ng / mL final) and IL-15 (10 ng / mL final) were added.

[0125] Results showed differential killing, especially at lower effector to target ratios (E: T) with T cells expressing antibody with VH-only binder 2 at a level comparable to CT041-scFv CAR T cells, on both SNU620 and OE19 cells. Most CLDN18.2-VH only single domain CAR T cells had high transduction efficiency similar to that of CT041-scFv, and low tonic signaling (Fig. 3A-Fig. 3C).Luciferase based cytotoxicity

[0126] SNU1s overexpressing domain specific constructs were made GFP / luciferase positive by stably transducing with gamma retrovirus expressing the cDNA as described previously. 10,000 of these target cells were plated in 96-well plates in triplicate with CAR+ T cells at the indicated effector-to-target (E: T) ratios; cells were then incubated for 24 hours. Cell viability was determined by a luciferase dependent assay with OneGlo substrate E6110 (Promega Madison, Wl, USA), where % cytotoxicity = (BLI Control -BLISAMPLE) / BLIMAX; BLI Control = mean target cell alone value of that experiment with a non-targeting control. Bioluminescence was read on an Agilent Cytation 5. (Santa Clara, CA, USA).DFCI IP No. 3561Attorney Docket No. 91016-431320Example 4. In vivo assessment of VH-only single binding domain CAR T cells for treating cancer

[0127] VH-only single domain binder-expressing CAR T cells were tested in vivo for efficacy. NSG-MHC class l / ll double knock out mice (NSG-DKO), which protects mice from potentially confounding human T cell mediated xenogeneic graft vs host disease, were used. See, for example, Jiang, H. et al., Journal of the National Cancer Institute. 111, 409-418 (2019). Mice were engrafted with OE19 cells and treated with a 1x106engineered CAR T cell dose. Nearly all animals treated with CLDN18.2-targeted CAR T cell therapy showed anti-tumor response compared to irrelevantly targeted CAR T cell (BCMA-targeted CAR T cell) treated controls (Fig. 4A-Fig. 4C). Mice treated with CT041-scFv CAR T cells all lost weight and required sacrifice due to toxicity, substantially limiting survival extension.Animals treated with the similar affinity CAR-T cells expressing antibody with VH-only binder 2 also showed some toxicity requiring euthanasia, but remaining animals showed long term tumor control. However, animals treated with CAR T cells expressing VH-only binder 1, were the only group to completely escape early toxicity requiring sacrifice, with most animals exhibiting initial tumor clearance and long term (>100 days) tumor control, despite ultimately having tumor recurrence (Fig. 4B-Fig. 4C).

[0128] The CAR T cells expressing VH-only binders 1-3 were screened in vivo for toxicity. NSG-DKO mice were injected with 1x106CLDN18.2 VH, CT041-scFv CAR T cells, or irrelevantly targeted (BCMA-scFv) CAR T cells. Toxicity, as assessed by weight loss, was seen in the CLDN18.2-targeted arms proportionally to the binding affinity of the binder within the CAR construct. While CAR T cells expressing VH-only single domain binder 2 had similar binding kinetics to CT041, toxicity onset and severity was worse in CT041-scFv treated animals (Fig. 5A).

[0129] Animals were sacrificed when they reached end point due to either toxicity or tumor progression, and stomach tissues were collected. Sections of stomachs from these animals were stained with hematoxylin and eosin (H& E) and multiplex immunofluorescence (mIF) for presence of CLDN18.2 and human CD3 to identify T cell infiltration. Animals treated with CT041-scFv CAR T cells show atrophy and disrupted stomach architecture on H& E.DFCI IP No. 3561Attorney Docket No. 91016-431320Sections showed the presence of CLDN18.2, and dense T cell infiltration into the stomach tissue. In irrelevantly targeted CAR-T cell control treated animals, normal stomach architecture was preserved. See, Fig. 11A-Fig. 11 L. While CLDN18.2 was present in these stomach sections as well, there was no corresponding T cell infiltrate.

[0130] Taken together these results show the efficacious treatment of gastric tumors with CAR T cells expressing VH-only binders 1-2.In vivo Mouse Model Experiments

[0131] NSG-DKO mice (JAX strain #025216), were purchased from The Jackson Laboratory (Bar Harbor, ME USA). Animals were allowed to acclimate for at least 5 days before initiation of a study. Xenografts were produced as follows: 4 x 106cells were resuspended with 50% Matrigel (Corning) and were implanted subcutaneously in the flank of the NSG- DKO mice. Tumors were allowed to establish and animals grouped to normalize tumor volume prior to single IV injection of either control oBCMA CAR-T or aCLDN18.2 CAR-T cells. Tumor were measured with a caliper and volumes were determined using the formula, Tumor volume = (length x width2) / 2. Tumor volumes and body weights were measured weekly.Hematoxylin and Eosin (H& E) Staining

[0132] Tissue sections were cut from FFPE blocks, and the first slide was used for H& E staining. To begin, the slides were baked at 60 °C for 1 hour to remove paraffin. They were then deparaffinized by treating with three changes of xylene, each for 2 minutes, followed by rehydration in three changes of 100% ethanol for 2 minutes each, 95% ethanol for 1 minute, and water for 5 minutes. The slides were stained with hematoxylin for 5 seconds, followed by three water washes. A bluing reagent was applied for 30 seconds, followed by three additional water washes. The slides were then stained with eosin for 15 seconds, followed by dehydration in three changes of 100% ethanol for 2 minutes each and three changes of xylene for 2 minutes each. Finally, the slides were mounted with a permanent coverslipping medium for optimal tissue staining for histological evaluation.Multiplex Immunofluorescence (mIF)DFCI IP No. 3561Attorney Docket No. 91016-431320

[0133] To evaluate changes in T cell infiltration and CLDN18.2 expression, a customized multiplex immunofluorescence (mIF) panel was developed. The panel included monoclonal antibodies targeting CLDN18.2 (a tight junction cell surface marker), CD3 (a T cell marker), pan-cytokeratin (pan-CK, to distinguish epithelial cells), and the DNA-binding dye 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) as a nuclear marker. Staining conditions for each antibody were optimized using immunohistochemistry (IHC) and singleplex immunofluorescence. Following optimization, all antibodies were combined into a single mIF assay. Multiplex staining was performed on a Leica BOND RX Research Stainer (Leica Biosystems). The staining protocol included sequential steps: antigen retrieval, primary antibody incubation, secondary antibody detection with Opal polymer HRP, and fluorescent labeling using tyramide signal amplification. Fluorescent images were captured on the PhenoImager HT multispectral imaging system (Akoya Biosciences, Hopkinton, MA).Initially, an overview image was acquired at 10* magnification. Regions of interest (ROIs) were then selected, and multispectral images were captured at 20 magnification. After image acquisition, spectral unmixing was performed using a supervised machine learning algorithm in the inFORM 3.0.0 software (Akoya Biosciences). The unmixed component data was exported and processed using QuPath vO.5.1 (open-source software available on GitHub). Each image was carefully inspected to confirm that the staining was specific to the expected cellular compartment. Images were prepared using a representative ROI for visualization.Statistical methods

[0134] Analyses were performed with GraphPad Prism 10 (version 10.4). Test used are indicated in the corresponding figure legends. Significance was considered for two-sided P < 0.05 as the following: p = *<0.05, **<0.01, ***<0.001, ****<0.0001.Example 5. Clinically significant erosive gastritis from on target / off-tumor activity targeting CLDN18.2

[0135] To better understand potential histopathologic correlates to the high rates of nausea and vomiting in non-gastrectomy patients treated with zolbetuximab plus chemotherapy, 50DFCI IP No. 3561Attorney Docket No. 91016-431320out of 58 sequential treated patients underwent paired pre- and on-treatment upper endoscopy (EGD) during standard therapy. Among these 50 zolbetuximab-treated patients where EGD was performed, it was observed that 30 (60%) of these showed endoscopic changes to their gastric mucosa, including redness, erosion, and / or superficial ulcers, which were consistent with findings suggestive of gastritis. Amongst this cohort, 32 were male and 18 female, with 17 and 13 patients developing gastritis respectively, thus while there was a trend toward female predominance, this was not statistically significant.Examples are provided, where prior to treatment, endoscopy demonstrated normal mucosa (Fig. 7A, Fig. 9A-Fig. 9D), while EGD on-treatment showed grossly visible gastritis (Fig.7B, Fig. 9E-Fig. 9H). In one patient example, endoscopic tissue sampling of the tumor uninvolved gastric epithelium was performed before (Fig. 7C-Fig. 7D) and after (Fig. 7E-Fig. 7F) treatment with four doses of zolbetuximab and chemotherapy. Samples from this patient were stained for CLDN18.2 expression using the approved 43-1 A anti-CLDN18.2 antibody clone (Roche Ventana, Oro Valley, AZ). Follow up biopsies demonstrated erosive gastritis, denuded normal architecture and preserved CLDN18.2 expression (Fig. 7E-Fig.7F). Notably, all patients with gastritis were able to continue zolbetuximab-based therapy, patients with follow up EGD at the time of progressive disease often showed improvement in gastric injury (Fig. 9I-Fig. 9L).Endoscopic image collection

[0136] Analysis included patients treated with chemotherapy plus zolbetuximab in National Cancer Center Hospital East until September 2024. Patients were prospectively enrolled in the biomarker study; it was reviewed and approved by the Institutional Review Boards (UMIN000019129) Chemotherapy plus zolbetuximab was given similar to previous study protocols.Example 6. Pre-clinical model ofCLDN18.2 targeted CAR T cell therapy using the same scFv binder as CT041 reproduces on-target / off-tumor toxicity

[0137] To evaluate the efficacy and toxicity profile of CAR T cell therapy targeting CLDN18.2 a CAR utilizing the scFv sequence from CT041 was engineered. A secondDFCI IP No. 3561Attorney Docket No. 91016-431320generation CAR design incorporating the CT041 scFv was engineered, a 4-1 BB costimulatory domain, and a CD3ζ activation domain. To evaluate this construct in vivo, NSG-MHC class l / ll double knock out mice (NSG-DKO) were employed which protects mice from potentially confounding human T cell mediated xenogeneic graft vs host disease. Animals were subcutaneously engrafted with OE19 cell line, which endogenously expresses CLDN18.2 and was derived from a human gastric cardia / gastro-esophageal junction adenocarcinoma CT041-scFv CAR T cells and irrelevantly targeted BCMA-scFv human CAR T cells (dose: 1x106CAR+ cells) were evaluated in mice bearing tumors with average size ~100 mm3. Tumors in animals treated with CT041-scFv CAR T cells regressed (Fig.10A), however animals rapidly succumbed to weight loss and failure to thrive (Fig. 10B-Fig. 10C). In control mice, treated with either BCMA-scFv CAR T cells or no cell injection, all tumors progressed and there was no evidence of toxicity from treatment. Animals were sacrificed when they reached end point due to either toxicity or tumor progression and stomach tissues were collected. Sections of stomachs from these animals were stained with hematoxylin and eosin (H& E) and multiplex immunofluorescence (mIF) for presence of CLDN18.2 and human CD3 to identify T cell infiltration. Animals treated with CT041-scFv CAR T cells show atrophy and disrupted stomach architecture on H& E (Fig. 10D). Sections clearly show presence of CLDN18.2 (Fig. 10E), and dense T cell infiltration (Fig. 10F) into the stomach tissue. Any remaining tumor from treated animals was also collected. While scant tumor remained in CT041 -scFv treated animals at the time of sacrifice (Fig. 11 A-Fig.11 B), CDLN18.2 positive tumor cells were identified (Fig. 11C), surrounded also by a dense human T cell infiltrate (Fig. 11D-Fig. 11F) In irrelevantly targeted CAR-T cell control treated animals, normal stomach architecture is preserved (Fig. 11G-Fig. 11H). While CLDN18.2 is clearly present in these stomach sections as well (Fig. 111), there is no corresponding T cell infiltrate (Fig. 11J-Fig. 11 L).Cell lines

[0138] OE19 and PATU8988s cell lines were acquired from the DSMZ collection (Braunschweig, 440 Germany) and were cultured according to manufacturer’s recommendation. They were 441 tested monthly for mycoplasma using PCR (Bulldog Bio).DFCI IP No. 3561Attorney Docket No. 91016-431320Human peripheral blood 442 mononuclear cells (PBMCs), isolated by ficoll density centrifugation or purified T cells 443 (RosetteSep Human T Cell Enrichment Cocktail, StemCell #15061) were stimulated with 444 TransAct (Miltenyi), 10 pL for 5x106PBMCs / mL or for 1x106T cells / mL, in complete RPMI 445 medium: RPMI (Gibco #118750-093) supplemented with 10% heat inactivated Fetal Bovine 446 Serum (FBS) (Life Technologies #10438026), Penicillin-Streptomycin (P / S) (Life Technologies 447 #15140122), Glutamax (GX) (Life Technologies #35050061). The human recombinant 448 cytokines IL-2 (200 U / mL final), IL-7 (5 ng / mL final) and IL-15 (10 ng / mL final) were added.

[0139] To generate cell lines overexpressing chimeric CLDN18.1 / 18.2 constructs, HEK293S (ATCC, Manassas, VA, USA) were engineered to overexpress chimeric CLDN18 using lentiviral constructs for the CMV-driven expression of each construct (i.e. ECL1 -1, 1-2, 1 -3) as well as a puromycin marker for selection. Since the two isoforms only differ in 5 distinctly extracellular amino acids centered in three structurally distinct regions, each chimeric construct harbored 1 or 2 mutations in the ECL. ECL1-1 mutations (N45Q and Q47E) centered on the unstructured, N-terminus of the pore-lining loop, ECL1-2 mutations (N37D and A42S) near the ‘GLW signature sequence within the [3-fold of the loop, and ECL1-3 (E56Q) formed by the residues sandwiched within the disulfide bond in the loop Transduced HEK293s overexpressing chimeric constructs were selected with 1.25 pg / mL puromycin (Thermo Fisher Scientific, Waltham, MA #A1113803) for 5-7 days prior to inclusion in cytotoxicity assays.Example 7. On-target / off-tumor toxicity is independent of tumor burden, but demonstrates dose dependence, without sufficient therapeutic window

[0140] To investigate if excess antigen from large tumor burden drove toxicity, an experimental approach where tumor was established in two cohorts of mice, 1 week apart was developed. These two cohorts of mice, high burden (~200 mm3) and low burden (~100 mm3) groups and a third tumor-free cohort, were injected with 1x106CT041-scFv CAR+ T cells on the same day (Fig. 12A). While tumor bearing animals again showed almost complete clearance of tumor, regardless of tumor burden (Fig. 12B), all CT041-scFvDFCI IP No. 3561Attorney Docket No. 91016-431320CAR T cell treated animals lost substantial body weight (Fig. 12C) and reached humane endpoint within 22 days. Ultimately, irrespective of tumor burden, survival was no longer for CT041-scFv CAR T cell treated mice, limited by toxicity; than for control animals treated with irrelevantly targeted CAR T cells, where survival was limited by lack of tumor control (Fig. 12D-12E). Data representative of two models with distinct tumor types (OE19, PATU8998s). Dense T cell infiltration at the stomach is present regardless if tumor is present or not (Fig. 13A-Fig. 13F). In the normal gastric mucosa CLDN18.2 was uniformly expressed in differentiated epithelial cells and was enriched at the basolateral cell membrane. In contrast, in tumor cells CLDN18.2 expression was more heterogeneous, where it was expressed at a lower intensity than normal gastric mucosa, and exhibited membranous and cytoplasmic subcellular expression without basolateral enrichment, suggesting loss of membrane polarization more consistent with non-junctional staining (Fig.13A-Fig. 13H). Clinically, CLDN18.2 expression can be upregulated or downregulated in gastric loss of membrane polarization as compared to stomach adenocarcinomas relative to endogenous expression levels, where downregulation may even correlate with development, infiltration and proliferation; the malignant transformation also alters the localization of CLDN18.2. These clinical findings were observed here, with tumor samples showing reduced expression of CLDN18.2 and loss of membrane polarization as compared to stomach (Fig. 13A-Fig. 13H).

[0141] To test whether CAR T cell dose modulates severity of toxicity, animals were engrafted with OE19; when tumors reached 200 mm3they were treated with a narrow dose de-escalation of CT041-scFV CAR T cells (starting with the commonly used xenograft model dose 1x106and continuing with more clinically relevant weight based dose 3x105, and 1x105). In contrast to the results observed with tumor burden, CAR T cell number impacted both toxicity and efficacy in a clear dose-dependent manner. Animals treated with lower doses had less body weight loss (Fig. 12F) but also had less effective tumor clearance (Fig. 12G). Throughout this narrow dose de-escalation, while lower doses showed some benefit, the tradeoff between anti-tumor efficacy and on-target / off-tumor toxicity limited improvement in overall survival (Fig. 12H). Consistent with this, examining tumor vs non-DFCI IP No. 3561Attorney Docket No. 91016-431320tumor related mortality as visualized with a swimmers plot, shows that the majority of animals at the higher 3 x105-1 x106doses succumb to toxicity, while animals treated with the lowest dose, 1x105CAR T cells, show less toxicity, but ultimately less durable tumor control, as all but one animal ultimately succumbed to tumor progression prior to Day 100 (Fig 12H)Example 8. A VH-only binder with lower affinity mitigates on-target / off-tumor toxicity while preserving anti-tumor efficacy

[0142] In order to determine if the therapeutic window targeting CLDN18.2 could be enhanced with CAR-T cells utilizing alternative binders, a fully-human heavy chain only antibody (HCAb) discovery campaign was conducted. Briefly, transgenic mice knocked out for murine variable heavy and light (VHA / L) chain loci and knocked in for only the human VH chain locus (HCAb mice; were immunized with HEK293 cells over-expressing recombinant human CLDN18.2 and binders were obtained through Single B Cell screening on the Beacon Optofluidic system and subsequent VDJ sequencing. From this HCAb discovery campaign, two highly active and specific binders, “5795” and “5797” were identified and cloned into the same 4-1 BB / CD3 containing second-generation CAR construct, hereafter called “5795-VH” or “VH Binder 1” and “5797-VH” or “VH Binder 2”.Multicycle kinetics for each bivalent, IgG-reformatted binder against human CLDN18.2 was assessed by surface plasmon resonance (SPR), revealing a 10-fold range of affinities for the binding elements within the three CARs. CT041 IgG had highest affinity with a KDof 3.64 ± 0.2 nM; 5797-lgG i.e. VH Binder 2, had a slightly lower affinity of 4.5 ± 3 nM, while 5795-IgG i.e. VH Binder 1 had the lowest affinity of 21.8 ± 2 nM (Fig.2A-Fig.2F). Affinity of each binder to mouse claudin 18.2 is similar to human claudin 18.2 and follows this same trend (Fig 14A-Fig. 14D).

[0143] The binding epitope within human claudin 18.2 was further characterized. Taking advantage of the non-binding CLDN18.1 isoform, structurally proximate residues that differed between isoform 18.1 and 18.2 were mutated to those found in CLDN18.1, resultingDFCI IP No. 3561Attorney Docket No. 91016-431320in three chimeric 18.1 / 18.2 constructs. Cell lines bearing these chimeric constructs were then subjected to killing by 5795 (VH binder 1), 5797-VH (VH binder 2) and CT041-scFv CAR-T in vitro. While all CAR candidates require WT ECL1-2 and ECL1-3 residues, the VH binder based-CARs additionally rely more heavily on interactions with ECL1-1 residues than CT041. In contrast, CT041-scFv CAR-T were able to kill cells expressing chimeric CLDN18.2 harboring 18.1 residues in ECL1-1, indicating less activity dependent binding at this site (Fig. 15B).

[0144] These candidates were then screened in vivo for toxicity. Mice were injected with 1x106CLDN18.2 VH, CT041-scFv CAR T cells, or irrelevantly targeted (BCMA-scFv) CAR T cells. Toxicity, as assessed by weight loss, was seen in the CLDN18.2-targeted arms proportionally to the binding affinity of the binder within the CAR construct (Fig. 4A). Next binders were tested in vivo for efficacy. As before, animals were engrafted with OE19 and treated with 1x106CAR T cell dose. Nearly all animals treated with CLDN18.2-targeted CAR T cell therapy showed anti-tumor response (Fig. 4B) compared to BCMA treated controls. As expected however, animals treated with CT041-scFv CAR T cells all lost weight and required sacrifice due to toxicity, substantially limiting survival extension.Animals treated with the similar affinity 5797-VH (VH binder 2) CAR T cells also showed some toxicity requiring euthanasia, but remaining animals showed long term tumor control. Animals treated with 5795-VH (VH binder 1) CAR T cells, however, were the only group to completely escape early toxicity requiring sacrifice, with most animals exhibiting initial tumor clearance and significantly prolonged median overall survival (mOS) relative to CT041-scFv treated animals (CT041-scFv mOS 39 d vs 5795-VH (VH binder 1) mOS 124 d p<0.001 ), despite ultimately having tumor recurrence (Fig. 4B, Fig. 4C).

[0145] The therapeutic window across these CARs in a pancreatic cancer xenograft model, PATU8988s, was also investigated (Fig. 5A-Fig. 5D). Tumor were allowed to engraft and expand for 2 weeks when NSG DKO mice were treated with a single dose of either 3 x105or 1 x1065795-VH (VH binder 1), 5797-VH (VH binder 2), or CT041-scFv CAR-T and compared 1 x106BCMA-scFv irrelevantly targeted control treated animals. All animals treated with either CT041, or 5797 (VH binder 2) based CAR-T succumbed toDFCI IP No. 3561Attorney Docket No. 91016-431320toxicity, even at the lower dose, while 5795 (VH binder 1) based CAR-T had long term tumor control at both doses, with minimal toxicity in this tumor model (mOS 31-36 d for CT041 -scFv groups and mOS not reached by 60 d for 5795-VH groups (VH binder 1 ); p<0.01). Stomach was harvested from animals in this model with the CT041-scFv CAR showing the highest degree of CAR-T cell infiltration into the stomach and the 5795-VH (VH binder 1) CAR-T showing the lowest (Fig. 16A-Fig. 16H). This also correlated with degree of tissue disruption, with almost complete atrophy and erosion in the CT041-scFv treated group, only around 1% atrophy and some erosion in the 5797-VH (VH binder 2) treated animal, very little if any tissue disruption in the 5795-VH (VH binder 1) animal and normal stomach architecture in the BCMA-scFv control (Fig. 16I-16L).EQUIVALENTS

[0146] Various aspects of the present disclosure may be used alone, in combination, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and is, therefore, not limited in its application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.Table 2. Exemplary Sequences.SEQ Name Type SequenceID NO:7 VH-only binder AA EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGI 1 SGSGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCEKVPDGY MIPYYNYNGMDVWGQGTTVTVSS1 CDR1 AA GFTFSSYA (SEQ ID NO: 1)2 CDR2 ISGSGGRT (SEQ ID NO: 2)3 CDR3 EKVPDGYMIPYYNYNGMDV (SEQ ID NO: 3)9 VH-only binder AA EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAL 2 VSYDGSKKYYADSVKGRFTISRENSQKMLYLQMDSLRVEDTAMYYCTRDFPT IPGTRDAFDIRGQGTMVTVSS4 CDR1 AA GFTFSSYG (SEQ ID NO: 4)5 CDR2 VSYDGSKK (SEQ ID NO: 5)6 CDR3 TRDFPTIPGTRDAFDI (SEQ ID NO: 6)8 VH-only binder DNA GAGGTGCAACTGTTGGAATCCGGAGGTGGACTCGTCCAACCTGGGGGTA 1 GTCTGAGGCTCTCTTGCGCGGCTAGTGGATTTACGTTCAGCTCATACGCTATGAGCTGGGTCAGACAAGCTCCCGGCAAGGGACTCGAATGGGTATCTGDFCI IP No. 3561Attorney Docket No. 91016-431320GTATTTCTGGTAGTGGTGGAAGGACATACTACGCAGATTCAGTGAAAGGC AGATTTACAATAAGTCGAGATAACTCCAAAAATACTCTTTACCTGCAAATGA ATAGCCTCCGGGCAGAGGATACAGCAGTTTACTACTGTGAGAAAGTACCC GACGGATATATGATCCCATATTACAACTACAACGGGATGGATGTGTGGGG GCAGGGCACCACTGTTACTGTTTCAAGC10 VH-only binder DNA GAGGTGCAGTTGGTAGAATCAGGCGGGGGAGTCGTTCAACCTGGCCGAT 2 CTCTCCGGCTGTCTTGCGCCGCATCCGG I l l i ACATTTAGCAGTTATGGCA TGCACTGGGTCCGGCAGGCTCCGGGCAAAGGACTGGAATGGGTGGCACT TGTGTCATACGACGGTTCCAAGAAGTACTATGCGGACTCCGTGAAAGGAC GGTTTACCATCTCCAGAGAGAATTCCCAGAAAATGTTGTACCTGCAAATGG ACTCCCTGAGGGTAGAAGATACGGCGATGTACTATTGCACGCGGGA I l l i CCTACGATCCCTGGGACCAGGGATGCGTTTGATATTCGAGGCCAAGGCAC GATGGTTACTGTGTCTAGC11 hkappa signal AA MVLQTQVFISLLLWISGAYGpeptide12 hkappa signal DNA atggtgctgcagacccaggtgttcatcagcctgctgctgtggatctctggcgcctatggapeptide13 CD28 hinge AA IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPregion14 CD28 hinge DNA atcgaagtaatgtacccgcccccttatttggataacgaaaagtctaatggtaccataatccacgtcaaaggca region agcatttgtgcccgagtcccctttttcccggcccctccaaaccc15 CD28 AA FWVLVVVGGVLACYSLLVTVAFIIFWVtransmembraneregion16 CD28 DNA ttttgggtattggtagttgtgggaggtgtccttgcgtgctacagccttcttgtgacggtggcattcatcattttttgggtt transmembraneregion17 4-1 BB coAA KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL stimulatoryregion18 4-1 BB coDNA aagcggggcagaaagaagctgctctacatcttcaagcagcccttcatgcggcccgtgcagaccacacaag stimulatory aggaagatggctgctcctgcagattccccgaggaagaagaaggcggctgcgagctgregion19 CD3zeta AA RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR intracellular RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY region DALHMQALPPR20 CD3zeta DNA agagtgaagttcagcagatccgccgacgctccagcctatcagcagggccaaaaccagctgtataacgagct intracellular gaacctggggagaagagaagagtacgacgtgctggataagcggagaggcagagatcctgaaatgggcg region gcaagcccagacggaagaatcctcaagagggcctgtataatgagctgcagaaagacaagatggccgag gcctacagcgagatcggaatgaagggcgagcgcagaagaggcaagggacacgatggactgtaccagg gcctgagcaccgccaccaaggatacctatgacgcactgcacatgcaggccctgccacctaga 21 CAR expressing AA ERVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVS VH-only binder GISGSGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCEKVPD 1 GYMIPYYNYNGMDVWGQGTTVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHL CPSPLFPGPSKPFVWLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPF MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNE LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR22 CAR expressing DNA GAGGTGCAACTGTTGGAATCCGGAGGTGGACTCGTCCAACCTGGGGGTA VH-only binder GTCTGAGGCTCTCTTGCGCGGCTAGTGGATTTACGTTCAGCTCATACGCT 1 ATGAGCTGGGTCAGACAAGCTCCCGGCAAGGGACTCGAATGGGTATCTG GTATTTCTGGTAGTGGTGGAAGGACATACTACGCAGATTCAGTGAAAGGC AGATTTACAATAAGTCGAGATAACTCCAAAAATACTCTTTACCTGCAAATGA ATAGCCTCCGGGCAGAGGATACAGCAGTTTACTACTGTGAGAAAGTACCC GACGGATATATGATCCCATATTACAACTACAACGGGATGGATGTGTGGGG GCAGGGCACCACTGTTACTGTTTCAAGCatcgaagtaatgtacccgcccccttatttggata acgaaaagtctaatggtaccataatccacgtcaaaggcaagcatttgtgcccgagtcccctttttcccggcccctccaaacccttttgggtattggtagttgtgggaggtgtccttgcgtgctacagccttcttgtgacggtggcattcatcDFCI IP No. 3561Attorney Docket No. 91016-431320attttttgggttaagcggggcagaaagaagctgctctacatcttcaagcagcccttcatgcggcccgtgcagac cacacaagaggaagatggctgctcctgcagattccccgaggaagaagaaggcggctgcgagctgagagt gaagttcagcagatccgccgacgctccagcctatcagcagggccaaaaccagctgtataacgagctgaac ctggggagaagagaagagtacgacgtgctggataagcggagaggcagagatcctgaaatgggcggcaa gcccagacggaagaatcctcaagagggcctgtataatgagctgcagaaagacaagatggccgaggccta cagcgagatcggaatgaagggcgagcgcagaagaggcaagggacacgatggactgtaccagggcctg agcaccgccaccaaggatacctatgacgcactgcacatgcaggccctgccacctaga23 CAR VH-only AA EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAL binder 2 VSYDGSKKYYADSVKGRFTISRENSQKMLYLQMDSLRVEDTAMYYCTRDFPT IPGTRDAFDIRGQGTMVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLF PGPSKPFVWLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQ TTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR24 CAR VH-only DNA GAGGTGCAGTTGGTAGAATCAGGCGGGGGAGTCGTTCAACCTGGCCGAT binder 2 CTCTCCGGCTGTCTTGCGCCGCATCCGG I l l i ACATTTAGCAGTTATGGCA TGCACTGGGTCCGGCAGGCTCCGGGCAAAGGACTGGAATGGGTGGCACT TGTGTCATACGACGGTTCCAAGAAGTACTATGCGGACTCCGTGAAAGGAC GGTTTACCATCTCCAGAGAGAATTCCCAGAAAATGTTGTACCTGCAAATGG ACTCCCTGAGGGTAGAAGATACGGCGATGTACTATTGCACGCGGGA I l l i CCTACGATCCCTGGGACCAGGGATGCGTTTGATATTCGAGGCCAAGGCAC GATGGTTACTGTGTCTAGCatcgaagtaatgtacccgcccccttatttggataacgaaaagtctaa tggtaccataatccacgtcaaaggcaagcatttgtgcccgagtcccctttttcccggcccctccaaacccttttgg gtattggtagttgtgggaggtgtccttgcgtgctacagccttcttgtgacggtggcattcatcattttttgggttaagc ggggcagaaagaagctgctctacatcttcaagcagcccttcatgcggcccgtgcagaccacacaagagga agatggctgctcctgcagattccccgaggaagaagaaggcggctgcgagctgagagtgaagttcagcaga tccgccgacgctccagcctatcagcagggccaaaaccagctgtataacgagctgaacctggggagaagag aagagtacgacgtgctggataagcggagaggcagagatcctgaaatgggcggcaagcccagacggaag aatcctcaagagggcctgtataatgagctgcagaaagacaagatggccgaggcctacagcgagatcgga atgaagggcgagcgcagaagaggcaagggacacgatggactgtaccagggcctgagcaccgccacca aggatacctatgacgcactgcacatgcaggccctgccacctaga25 vector insert DNA atggtgctgcagacccaggtgttcatcagcctgctgctgtggatctctggcgcctatggaGAGGTGCAAC (including signal TGTTGGAATCCGGAGGTGGACTCGTCCAACCTGGGGGTAGTCTGAGGCT peptide) of VH- CTCTTGCGCGGCTAGTGGATTTACGTTCAGCTCATACGCTATGAGCTGGG only binder 1 TCAGACAAGCTCCCGGCAAGGGACTCGAATGGGTATCTGGTATTTCTGGT AGTGGTGGAAGGACATACTACGCAGATTCAGTGAAAGGCAGATTTACAAT AAGTCGAGATAACTCCAAAAATACTCTTTACCTGCAAATGAATAGCCTCCG GGCAGAGGATACAGCAGTTTACTACTGTGAGAAAGTACCCGACGGATATA TGATCCCATATTACAACTACAACGGGATGGATGTGTGGGGGCAGGGCACC ACTGTTACTGTTTCAAGCatcgaagtaatgtacccgcccccttatttggataacgaaaagtctaatg gtaccataatccacgtcaaaggcaagcatttgtgcccgagtcccctttttcccggcccctccaaacccttttgggt attggtagttgtgggaggtgtccttgcgtgctacagccttcttgtgacggtggcattcatcattttttgggttaagcgg ggcagaaagaagctgctctacatcttcaagcagcccttcatgcggcccgtgcagaccacacaagaggaag atggctgctcctgcagattccccgaggaagaagaaggcggctgcgagctgagagtgaagttcagcagatcc gccgacgctccagcctatcagcagggccaaaaccagctgtataacgagctgaacctggggagaagagaa gagtacgacgtgctggataagcggagaggcagagatcctgaaatgggcggcaagcccagacggaagaa tcctcaagagggcctgtataatgagctgcagaaagacaagatggccgaggcctacagcgagatcggaatg aagggcgagcgcagaagaggcaagggacacgatggactgtaccagggcctgagcaccgccaccaagg atacctatgacgcactgcacatgcaggccctgccacctaga26 vector insert DNA atggtgctgcagacccaggtgttcatcagcctgctgctgtggatctctggcgcctatggaGAGGTGCAGT (including signal TGGTAGAATCAGGCGGGGGAGTCGTTCAACCTGGCCGATCTCTCCGGCT peptide) of VH- GTCTTGCGCCGCATCCGG I I I I ACATTTAGCAGTTATGGCATGCACTGGGT only binder 2 CCGGCAGGCTCCGGGCAAAGGACTGGAATGGGTGGCACTTGTGTCATAC GACGGTTCCAAGAAGTACTATGCGGACTCCGTGAAAGGACGGTTTACCAT CTCCAGAGAGAATTCCCAGAAAATGTTGTACCTGCAAATGGACTCCCTGA GGGTAGAAGATACGGCGATGTACTATTGCACGCGGGA I l l i CCTACGATC CCTGGGACCAGGGATGCGTTTGATATTCGAGGCCAAGGCACGATGGTTACTGTGTCTAGCatcgaagtaatgtacccgcccccttatttggataacgaaaagtctaatggtaccataatcDFCI IP No. 3561Attorney Docket No. 91016-431320cacgtcaaaggcaagcatttgtgcccgagtcccctttttcccggcccctccaaacccttttgggtattggtagttgt gggaggtgtccttgcgtgctacagccttcttgtgacggtggcattcatcattttttgggttaagcggggcagaaag aagctgctctacatcttcaagcagcccttcatgcggcccgtgcagaccacacaagaggaagatggctgctcc tgcagattccccgaggaagaagaaggcggctgcgagctgagagtgaagttcagcagatccgccgacgctc cagcctatcagcagggccaaaaccagctgtataacgagctgaacctggggagaagagaagagtacgacg tgctggataagcggagaggcagagatcctgaaatgggcggcaagcccagacggaagaatcctcaagag ggcctgtataatgagctgcagaaagacaagatggccgaggcctacagcgagatcggaatgaagggcgag cgcagaagaggcaagggacacgatggactgtaccagggcctgagcaccgccaccaaggatacctatga cgcactgcacatgcaggccctgccacctaga27 vector DNA atggtgctgcagacccaggtgttcatcagcctgctgctgtggatctctggcgcctatggaGAGGTGCAAC expressing VH- TGTTGGAATCCGGAGGTGGACTCGTCCAACCTGGGGGTAGTCTGAGGCT only binder 1 CTCTTGCGCGGCTAGTGGATTTACGTTCAGCTCATACGCTATGAGCTGGG TCAGACAAGCTCCCGGCAAGGGACTCGAATGGGTATCTGGTATTTCTGGT AGTGGTGGAAGGACATACTACGCAGATTCAGTGAAAGGCAGATTTACAAT AAGTCGAGATAACTCCAAAAATACTCTTTACCTGCAAATGAATAGCCTCCG GGCAGAGGATACAGCAGTTTACTACTGTGAGAAAGTACCCGACGGATATA TGATCCCATATTACAACTACAACGGGATGGATGTGTGGGGGCAGGGCACC ACTGTTACTGTTTCAAGCatcgaagtaatgtacccgcccccttatttggataacgaaaagtctaatg gtaccataatccacgtcaaaggcaagcatttgtgcccgagtcccctttttcccggcccctccaaacccttttgggt attggtagttgtgggaggtgtccttgcgtgctacagccttcttgtgacggtggcattcatcattttttgggttaagcgg ggcagaaagaagctgctctacatcttcaagcagcccttcatgcggcccgtgcagaccacacaagaggaag atggctgctcctgcagattccccgaggaagaagaaggcggctgcgagctgagagtgaagttcagcagatcc gccgacgctccagcctatcagcagggccaaaaccagctgtataacgagctgaacctggggagaagagaa gagtacgacgtgctggataagcggagaggcagagatcctgaaatgggcggcaagcccagacggaagaa tcctcaagagggcctgtataatgagctgcagaaagacaagatggccgaggcctacagcgagatcggaatg aagggcgagcgcagaagaggcaagggacacgatggactgtaccagggcctgagcaccgccaccaagg atacctatgacgcactgcacatgcaggccctgccacctagaGGCTCCGGCGCCACGAACTTC TCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTGGATC CATGGTtAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTG GTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCG AGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTG CACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTC AGCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGC ACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACC ATCAGCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTT CGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTC AAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACA GCCACAACGTCTATATCACGGCCGACAAGCAGAAGAACGGCATCAAGGC GAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCC GACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGC CCGACAACCACTACCTGTTCATCCAGTCCGCCCTGAGCAAAGACCCCAAC GAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGA TCACTCACGGCATGGACGAGCTGTACAAGTAA28 vector DNA atggtgctgcagacccaggtgttcatcagcctgctgctgtggatctctggcgcctatggaGAGGTGCAGT expressing VH- TGGTAGAATCAGGCGGGGGAGTCGTTCAACCTGGCCGATCTCTCCGGCT only binder 2 GTCTTGCGCCGCATCCGG I I I I ACATTTAGCAGTTATGGCATGCACTGGGT CCGGCAGGCTCCGGGCAAAGGACTGGAATGGGTGGCACTTGTGTCATAC GACGGTTCCAAGAAGTACTATGCGGACTCCGTGAAAGGACGGTTTACCAT CTCCAGAGAGAATTCCCAGAAAATGTTGTACCTGCAAATGGACTCCCTGA GGGTAGAAGATACGGCGATGTACTATTGCACGCGGGA I l l i CCTACGATC CCTGGGACCAGGGATGCGTTTGATATTCGAGGCCAAGGCACGATGGTTAC TGTGTCTAGCatcgaagtaatgtacccgcccccttatttggataacgaaaagtctaatggtaccataatc cacgtcaaaggcaagcatttgtgcccgagtcccctttttcccggcccctccaaacccttttgggtattggtagttgt gggaggtgtccttgcgtgctacagccttcttgtgacggtggcattcatcattttttgggttaagcggggcagaaag aagctgctctacatcttcaagcagcccttcatgcggcccgtgcagaccacacaagaggaagatggctgctcc tgcagattccccgaggaagaagaaggcggctgcgagctgagagtgaagttcagcagatccgccgacgctc cagcctatcagcagggccaaaaccagctgtataacgagctgaacctggggagaagagaagagtacgacgtgctggataagcggagaggcagagatcctgaaatgggcggcaagcccagacggaagaatcctcaagagDFCI IP No. 3561Attorney Docket No. 91016-431320ggcctgtataatgagctgcagaaagacaagatggccgaggcctacagcgagatcggaatgaagggcgag cgcagaagaggcaagggacacgatggactgtaccagggcctgagcaccgccaccaaggatacctatga cgcactgcacatgcaggccctgccacctagaGGCTCCGGCGCCACGAACTTCTCTCTGT TAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTGGATCCATGGTt AGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGC TGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGA GGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACC GGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCAGCTACG GCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTC TTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCAGCTT CAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGC GACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAC GTCTATATCACGGCCGACAAGCAGAAGAACGGCATCAAGGCGAACTTCAA GATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTAC CAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC ACTACCTGTTCATCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGC GATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCACG GCATGGACGAGCTGTACAAGTAA35 VH-only binder AA EVQLLESGGGSVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAI 3 SGSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKVPDGY LLPYYSYYDRDVWGQGTTVTVSS29 CDR1 AA GFTFSSYA (SEQ ID NO: 29)30 CDR2 ISGSGGNT (SEQ ID NO: 30)31 CDR3 VKVPDGYLLPYYSYYDRDV (SEQ ID NO: 31)36 VH-only binder DNA GAGGTGCAACTGCTGGAATCTGGAGGCGGGTCCGTCCAGCCAGGAGGAT 3 CTTTGCGCCTCTCTTGTGCCGCCTCCGGATTCACA I l l i CAAGTTACGCTA TGTCCTGGGTCAGACAAGCTCCCGGAAAGGGGCTGGAGTGGGTCAGCGC TATTTCCGGGTCAGGCGGCAACACATATTATGCTGACTCAGTCAAGGGTA GATTCACAATCAGTAGGGATAACTCTAAAAACACGCTGTACCTGCAGATGA ACAGCCTCCGAGCAGAAGACACAGCGGTCTACTATTGTGTGAAAGTTCCC GACGGTTATCTTCTCCCCTACTATAGCTACTATGACAGAGATGTTTGGGGG CAGGGAACCACCGTCACAGTGTCAAGC37 VH-only binder AA EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYAMRWVRQAPGKGLEWVSGI 4 SGSGGRTYYADSVKGRFTISRDNSRNTLYLQMNSLRAEDTAVYYCEKVPDTP MVPYYSYDLNVWGHGTTVTVSS37 VH-only binder AA EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYAMRWVRQAPGKGLEWVSGI 4 SGSGGRTYYADSVKGRFTISRDNSRNTLYLQMNSLRAEDTAVYYCEKVPDTP'MVPYYSYDLNVWGHGTTVTVSS32 CDR1 AA GFTFSGYA (SEQ ID NO: 32)33 CDR2 ISGSGGRT (SEQ ID NO: 33)34 CDR3 EKVPDTPMVPYYSYDLNV (SEQ ID NO: 34)38 VH-only binder DNA GAGGTGCAACTGCTGGAGAGCGGAGGTGGCCTGGTACAACCTGGAGGCA 4 GCCTCCGGCTGTCTTGCGCGGCGAGCGGATTTACTTTCAGCGGGTACGC CATGAGATGGGTTCGACAAGCGCCGGGAAAGGGCCTGGAATGGGTATCA GGCATCAGCGGATCAGGCGGACGGACGTATTATGCTGATAGTGTGAAGG GCAGATTCACCATCAGTAGGGATAATAGCAGAAACACGCTTTACCTGCAAA TGAACAGCCTCCGAGCAGAAGATACTGCGGTGTATTACTGCGAGAAAGTA CCTGACACCCCGATGGTTCCGTACTATTCATACGATCTCAACGTCTGGGG ACATGGGACTACTGTGACTGTTTCCAGC39 CAR expressing AA EVQLLESGGGSVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAI VH-only binder SGSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKVPDGY 3 LLPYYSYYDRDVWGQGTTVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPS PLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRP VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLG RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRDFCI IP No. 3561Attorney Docket No. 91016-43132040 CAR expressing DNA GAGGTGCAACTGCTGGAATCTGGAGGCGGGTCCGTCCAGCCAGGAGGAT VH-only binder CTTTGCGCCTCTCTTGTGCCGCCTCCGGATTCACA I l l i CAAGTTACGCTA 3 TGTCCTGGGTCAGACAAGCTCCCGGAAAGGGGCTGGAGTGGGTCAGCGC TATTTCCGGGTCAGGCGGCAACACATATTATGCTGACTCAGTCAAGGGTA GATTCACAATCAGTAGGGATAACTCTAAAAACACGCTGTACCTGCAGATGA ACAGCCTCCGAGCAGAAGACACAGCGGTCTACTATTGTGTGAAAGTTCCC GACGGTTATCTTCTCCCCTACTATAGCTACTATGACAGAGATGTTTGGGGG CAGGGAACCACCGTCACAGTGTCAAGCatcgaagtaatgtacccgcccccttatttggataa cgaaaagtctaatggtaccataatccacgtcaaaggcaagcatttgtgcccgagtcccctttttcccggcccctc caaacccttttgggtattggtagttgtgggaggtgtccttgcgtgctacagccttcttgtgacggtggcattcatcatt ttttgggttaagcggggcagaaagaagctgctctacatcttcaagcagcccttcatgcggcccgtgcagacca cacaagaggaagatggctgctcctgcagattccccgaggaagaagaaggcggctgcgagctgagagtga agttcagcagatccgccgacgctccagcctatcagcagggccaaaaccagctgtataacgagctgaacctg gggagaagagaagagtacgacgtgctggataagcggagaggcagagatcctgaaatgggcggcaagcc cagacggaagaatcctcaagagggcctgtataatgagctgcagaaagacaagatggccgaggcctacag cgagatcggaatgaagggcgagcgcagaagaggcaagggacacgatggactgtaccagggcctgagc accgccaccaaggatacctatgacgcactgcacatgcaggccctgccacctaga41 CAR expressing AA EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYAMRWVRQAPGKGLEWVSGI VH-only binder SGSGGRTYYADSVKGRFTISRDNSRNTLYLQMNSLRAEDTAVYYCEKVPDTP 4 MVPYYSYDLNVWGHGTTVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSP LFPGPSKPFVWLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR42 CAR expressing DNA GAGGTGCAACTGCTGGAGAGCGGAGGTGGCCTGGTACAACCTGGAGGCA VH-only binder GCCTCCGGCTGTCTTGCGCGGCGAGCGGATTTACTTTCAGCGGGTACGC 4 CATGAGATGGGTTCGACAAGCGCCGGGAAAGGGCCTGGAATGGGTATCA GGCATCAGCGGATCAGGCGGACGGACGTATTATGCTGATAGTGTGAAGG GCAGATTCACCATCAGTAGGGATAATAGCAGAAACACGCTTTACCTGCAAA TGAACAGCCTCCGAGCAGAAGATACTGCGGTGTATTACTGCGAGAAAGTA CCTGACACCCCGATGGTTCCGTACTATTCATACGATCTCAACGTCTGGGG ACATGGGACTACTGTGACTGTTTCCAGCatcgaagtaatgtacccgcccccttatttggata acgaaaagtctaatggtaccataatccacgtcaaaggcaagcatttgtgcccgagtcccctttttcccggcccc tccaaacccttttgggtattggtagttgtgggaggtgtccttgcgtgctacagccttcttgtgacggtggcattcatc attttttgggttaagcggggcagaaagaagctgctctacatcttcaagcagcccttcatgcggcccgtgcagac cacacaagaggaagatggctgctcctgcagattccccgaggaagaagaaggcggctgcgagctgagagt gaagttcagcagatccgccgacgctccagcctatcagcagggccaaaaccagctgtataacgagctgaac ctggggagaagagaagagtacgacgtgctggataagcggagaggcagagatcctgaaatgggcggcaa gcccagacggaagaatcctcaagagggcctgtataatgagctgcagaaagacaagatggccgaggccta cagcgagatcggaatgaagggcgagcgcagaagaggcaagggacacgatggactgtaccagggcctg agcaccgccaccaaggatacctatgacgcactgcacatgcaggccctgccacctaga43 vector insert DNA atggtgctgcagacccaggtgttcatcagcctgctgctgtggatctctggcgcctatggaGAGGTGCAAC (including signal TGCTGGAATCTGGAGGCGGGTCCGTCCAGCCAGGAGGATCTTTGCGCCT peptide) of VH- CTCTTGTGCCGCCTCCGGATTCACA I l l i CAAGTTACGCTATGTCCTGGGT only binder 3 CAGACAAGCTCCCGGAAAGGGGCTGGAGTGGGTCAGCGCTATTTCCGGG TCAGGCGGCAACACATATTATGCTGACTCAGTCAAGGGTAGATTCACAATC AGTAGGGATAACTCTAAAAACACGCTGTACCTGCAGATGAACAGCCTCCG AGCAGAAGACACAGCGGTCTACTATTGTGTGAAAGTTCCCGACGGTTATC TTCTCCCCTACTATAGCTACTATGACAGAGATGTTTGGGGGCAGGGAACC ACCGTCACAGTGTCAAGCatcgaagtaatgtacccgcccccttatttggataacgaaaagtctaat ggtaccataatccacgtcaaaggcaagcatttgtgcccgagtcccctttttcccggcccctccaaacccttttgg gtattggtagttgtgggaggtgtccttgcgtgctacagccttcttgtgacggtggcattcatcattttttgggttaagc ggggcagaaagaagctgctctacatcttcaagcagcccttcatgcggcccgtgcagaccacacaagagga agatggctgctcctgcagattccccgaggaagaagaaggcggctgcgagctgagagtgaagttcagcaga tccgccgacgctccagcctatcagcagggccaaaaccagctgtataacgagctgaacctggggagaagag aagagtacgacgtgctggataagcggagaggcagagatcctgaaatgggcggcaagcccagacggaagaatcctcaagagggcctgtataatgagctgcagaaagacaagatggccgaggcctacagcgagatcggaDFCI IP No. 3561Attorney Docket No. 91016-431320atgaagggcgagcgcagaagaggcaagggacacgatggactgtaccagggcctgagcaccgccacca aggatacctatgacgcactgcacatgcaggccctgccacctaga44 vector insert DNA atggtgctgcagacccaggtgttcatcagcctgctgctgtggatctctggcgcctatggaGAGGTGCAAC (including signal TGCTGGAGAGCGGAGGTGGCCTGGTACAACCTGGAGGCAGCCTCCGGCT peptide) of VH- GTCTTGCGCGGCGAGCGGATTTACTTTCAGCGGGTACGCCATGAGATGG only binder 4 GTTCGACAAGCGCCGGGAAAGGGCCTGGAATGGGTATCAGGCATCAGCG GATCAGGCGGACGGACGTATTATGCTGATAGTGTGAAGGGCAGATTCACC ATCAGTAGGGATAATAGCAGAAACACGCTTTACCTGCAAATGAACAGCCTC CGAGCAGAAGATACTGCGGTGTATTACTGCGAGAAAGTACCTGACACCCC GATGGTTCCGTACTATTCATACGATCTCAACGTCTGGGGACATGGGACTA CTGTGACTGTTTCCAGCatcgaagtaatgtacccgcccccttatttggataacgaaaagtctaatgg taccataatccacgtcaaaggcaagcatttgtgcccgagtcccctttttcccggcccctccaaacccttttgggta ttggtagttgtgggaggtgtccttgcgtgctacagccttcttgtgacggtggcattcatcattttttgggttaagcggg gcagaaagaagctgctctacatcttcaagcagcccttcatgcggcccgtgcagaccacacaagaggaagat ggctgctcctgcagattccccgaggaagaagaaggcggctgcgagctgagagtgaagttcagcagatccg ccgacgctccagcctatcagcagggccaaaaccagctgtataacgagctgaacctggggagaagagaag agtacgacgtgctggataagcggagaggcagagatcctgaaatgggcggcaagcccagacggaagaat cctcaagagggcctgtataatgagctgcagaaagacaagatggccgaggcctacagcgagatcggaatg aagggcgagcgcagaagaggcaagggacacgatggactgtaccagggcctgagcaccgccaccaagg atacctatgacgcactgcacatgcaggccctgccacctaga45 vector DNA atggtgctgcagacccaggtgttcatcagcctgctgctgtggatctctggcgcctatggaGAGGTGCAAC expressing VH- TGCTGGAATCTGGAGGCGGGTCCGTCCAGCCAGGAGGATCTTTGCGCCT only binder 3 CTCTTGTGCCGCCTCCGGATTCACA I l l i CAAGTTACGCTATGTCCTGGGT CAGACAAGCTCCCGGAAAGGGGCTGGAGTGGGTCAGCGCTATTTCCGGG TCAGGCGGCAACACATATTATGCTGACTCAGTCAAGGGTAGATTCACAATC AGTAGGGATAACTCTAAAAACACGCTGTACCTGCAGATGAACAGCCTCCG AGCAGAAGACACAGCGGTCTACTATTGTGTGAAAGTTCCCGACGGTTATC TTCTCCCCTACTATAGCTACTATGACAGAGATGTTTGGGGGCAGGGAACC ACCGTCACAGTGTCAAGCatcgaagtaatgtacccgcccccttatttggataacgaaaagtctaat ggtaccataatccacgtcaaaggcaagcatttgtgcccgagtcccctttttcccggcccctccaaacccttttgg gtattggtagttgtgggaggtgtccttgcgtgctacagccttcttgtgacggtggcattcatcattttttgggttaagc ggggcagaaagaagctgctctacatcttcaagcagcccttcatgcggcccgtgcagaccacacaagagga agatggctgctcctgcagattccccgaggaagaagaaggcggctgcgagctgagagtgaagttcagcaga tccgccgacgctccagcctatcagcagggccaaaaccagctgtataacgagctgaacctggggagaagag aagagtacgacgtgctggataagcggagaggcagagatcctgaaatgggcggcaagcccagacggaag aatcctcaagagggcctgtataatgagctgcagaaagacaagatggccgaggcctacagcgagatcgga atgaagggcgagcgcagaagaggcaagggacacgatggactgtaccagggcctgagcaccgccacca aggatacctatgacgcactgcacatgcaggccctgccacctagaGGCTCCGGCGCCACGAAC TTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTGG ATCCATGGTtAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCC TGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGG CGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATC TGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCT TCAGCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCA GCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGC ACCATCAGCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGA CTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACA ACAGCCACAACGTCTATATCACGGCCGACAAGCAGAAGAACGGCATCAAG GCGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCG CCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCT GCCCGACAACCACTACCTGTTCATCCAGTCCGCCCTGAGCAAAGACCCCA ACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGG GATCACTCACGGCATGGACGAGCTGTACAAGTAA46 vector DNA atggtgctgcagacccaggtgttcatcagcctgctgctgtggatctctggcgcctatggaGAGGTGCAAC expressing VH- TGCTGGAGAGCGGAGGTGGCCTGGTACAACCTGGAGGCAGCCTCCGGCT only binder 4 GTCTTGCGCGGCGAGCGGATTTACTTTCAGCGGGTACGCCATGAGATGGGTTCGACAAGCGCCGGGAAAGGGCCTGGAATGGGTATCAGGCATCAGCGDFCI IP No. 3561Attorney Docket No. 91016-431320GATCAGGCGGACGGACGTATTATGCTGATAGTGTGAAGGGCAGATTCACC ATCAGTAGGGATAATAGCAGAAACACGCTTTACCTGCAAATGAACAGCCTC CGAGCAGAAGATACTGCGGTGTATTACTGCGAGAAAGTACCTGACACCCC GATGGTTCCGTACTATTCATACGATCTCAACGTCTGGGGACATGGGACTA CTGTGACTGTTTCCAGCatcgaagtaatgtacccgcccccttatttggataacgaaaagtctaatgg taccataatccacgtcaaaggcaagcatttgtgcccgagtcccctttttcccggcccctccaaacccttttgggta ttggtagttgtgggaggtgtccttgcgtgctacagccttcttgtgacggtggcattcatcattttttgggttaagcggg gcagaaagaagctgctctacatcttcaagcagcccttcatgcggcccgtgcagaccacacaagaggaagat ggctgctcctgcagattccccgaggaagaagaaggcggctgcgagctgagagtgaagttcagcagatccg ccgacgctccagcctatcagcagggccaaaaccagctgtataacgagctgaacctggggagaagagaag agtacgacgtgctggataagcggagaggcagagatcctgaaatgggcggcaagcccagacggaagaat cctcaagagggcctgtataatgagctgcagaaagacaagatggccgaggcctacagcgagatcggaatg aagggcgagcgcagaagaggcaagggacacgatggactgtaccagggcctgagcaccgccaccaagg atacctatgacgcactgcacatgcaggccctgccacctagaGGCTCCGGCGCCACGAACTTC TCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTGGATC CATGGTtAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTG GTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCG AGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTG CACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTC AGCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGC ACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACC ATCAGCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTT CGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTC AAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACA GCCACAACGTCTATATCACGGCCGACAAGCAGAAGAACGGCATCAAGGC GAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCC GACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGC CCGACAACCACTACCTGTTCATCCAGTCCGCCCTGAGCAAAGACCCCAAC GAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGA TCACTCACGGCATGGACGAGCTGTACAAGTAA47 Framework AA EVQLX1ESGGGX2VQPGX3SLRLSCAAS, where Xnis any amino acid region 148 Framework AA MX1WVRQAPGKGLEWVX2X3, where Xnis any amino acidregion 249 Framework AA YYADSVKGRFTISRX1NSX2X3X4LYLQMX5SLRX6EDTAX7YY, where Xnis any region 3 amino acid50 CAR expressing AA EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGI VH-only binder SGSGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCEKVPDGY 1 MIPYYNYNGMDVWGQGTTVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPS PLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRP VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLG RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR51 vector insert DNA atggtgctgcagacccaggtgttcatcagcctgctgctgtggatctctggcgcctatggaGAGGTGCAGT (including signal TGGTAGAATCAGGCGGGGGAGTCGTTCAACCTGGCCGATCTCTCCGGCT peptide) of VH- GTCTTGCGCCGCATCCGG I I I I ACATTTAGCAGTTATGGCATGCACTGGGT only binder 2 CCGGCAGGCTCCGGGCAAAGGACTGGAATGGGTGGCACTTGTGTCATAC GACGGTTCCAAGAAGTACTATGCGGACTCCGTGAAAGGACGGTTTACCAT CTCCAGAGAGAATTCCCAGAAAATGTTGTACCTGCAAATGGACTCCCTGA GGGTAGAAGATACGGCGATGTACTATTGCACGCGGGA I l l i CCTACGATC CCTGGGACCAGGGATGCGTTTGATATTCGAGGCCAAGGCACGATGGTTAC TGTGTCTAGCatcgaagtaatgtacccgcccccttatttggataacgaaaagtctaatggtaccataatc cacgtcaaaggcaagcatttgtgcccgagtcccctttttcccggcccctccaaacccttttgggtattggtagttgt gggaggtgtccttgcgtgctacagccttcttgtgacggtggcattcatcattttttgggttaagcggggcagaaag aagctgctctacatcttcaagcagcccttcatgcggcccgtgcagaccacacaagaggaagatggctgctcc tgcagattccccgaggaagaagaaggcggctgcgagctgagagtgaagttcagcagatccgccgacgctccagcctatcagcagggccaaaaccagctgtataacgagctgaacctggggagaagagaagagtacgacgDFCI IP No. 3561Attorney Docket No. 91016-431320tgctggataagcggagaggcagagatcctgaaatgggcggcaagcccagacggaagaatcctcaagag ggcctgtataatgagctgcagaaagacaagatggccgaggcctacagcgagatcggaatgaagggcgag cgcagaagaggcaagggacacgatggactgtaccagggcctgagcaccgccaccaaggatacctatgacgcactgcacatgcaggccctgccacctaga

Claims

DFCI IP No. 3561Attorney Docket No. 91016-431320What is claimed:

1. A chimeric antigen receptor (CAR) comprising an amino acid having formula I:R1 - R2 - R3 - R4 - R5 (I),wherein:R1 comprises a VH-only single domain binder specific for binding claudin 18.2 (CLDN18.2) comprising a heavy chain variable region comprising:a) a complementarity-determining region 1 (CDR1) having an amino acid sequence with 0 to 2 amino acid substitutions compared to SEQ ID NOs: 1, 4, 29, or 32;b) a complementarity-determining region 2 (CDR2) having an amino acid sequence with 0 to 2 amino acid substitutions compared to SEQ ID NOs: 2, 5, 30, or 33; orc) a complementarity-determining region 3 (CDR3) having an amino acid sequence with 0 to 2 amino acid substitutions compared to SEQ ID NOs: 3, 6, 31, or 34;R2 comprises a cluster of differentiation 28 (CD28) hinge region;R3 comprises a CD28 transmembrane region;R4 comprises a 4-1 BB costimulatory region; andR5 comprises a cluster of differentiation 3 zeta (CD3 intracellular region.

2. The CAR of claim 1, wherein heavy chain variable region comprises human framework regions.

3. The CAR of claim 1 or claim 2, wherein the heavy chain variable region comprises:DFCI IP No. 3561Attorney Docket No. 91016-431320a) a CDR1 having an amino acid sequence selected from SEQ ID NOs: 1, 4, 29, or 32;b) a CDR2 having an amino acid sequence selected from SEQ ID NOs: 2, 5, 30, or 33; orc) a CDR3 having an amino acid sequence selected from SEQ ID NOs: 3, 6, 31, or 34.

4. The CAR of any one of claims 1 -3, wherein the heavy chain variable region comprises:a) a CDR1 having the amino acid sequence of SEQ ID NO: 1; a CDR2 having the amino acid sequence of SEQ ID NO: 2; and a CDR3 having the amino acid sequence of SEQ ID NO: 3;b) a CDR1 having the amino acid sequence of SEQ ID NO: 4; a CDR2 having the amino acid sequence of SEQ ID NO: 5; and a CDR3 having the amino acid sequence of SEQ ID NO: 6;c) a CDR1 having the amino acid sequence of SEQ ID NO: 29; a CDR2 having the amino acid sequence of SEQ ID NO: 30; and a CDR3 having the amino acid sequence of SEQ ID NO: 31; ord) a CDR1 having the amino acid sequence of SEQ ID NO: 32; a CDR2 having the amino acid sequence of SEQ ID NO: 33; and a CDR3 having the amino acid sequence of SEQ ID NO: 34.

5. The CAR of any one of claims 1 -4, wherein the heavy chain variable region comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 7, 9, 35, or 37.

6. The CAR of any one of claims 1-5, encoded by a nucleic acid sequence of SEQ ID Nos: 8, 10, 36, or 38.

7. The CAR of claim 1, wherein:DFCI IP No. 3561Attorney Docket No. 91016-431320R2 comprises an amino acid sequence according to SEQ ID NO: 13;R3 comprises an amino acid sequence according to SEQ ID NO: 15;R4 comprises an amino acid sequence according to SEQ ID NO: 17; andR5 comprises an amino acid sequence according to SEQ ID NO: 19.

8. The CAR of claim 7, wherein the CAR comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 21, 23, 39, or 41.

9. The CAR of claim 7, wherein the CAR is encoded by a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 22, 24, 40, or 42.

10. The CAR of any one of claims 7-9, wherein the binder has no binding affinity to CLDN18.1.

11. A vector comprising the nucleic acid of claim 10 or a nucleic acid sequence according to SEQ ID NOs: 25-28 or 43-46.

12. A T cell transfected with the vector of claim 11.

13. A pharmaceutical composition, comprising the T cell of claim 12.

14. Use of the T cell of claim 12 or the pharmaceutical composition of claim 13 for the manufacture or preparation of a medicament for the treatment of cancer in a patient15. The use of claim 14, wherein the cancer is selected from a gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, and non-small cell lung cancer.DFCI IP No. 3561Attorney Docket No. 91016-43132016. The use of claim 14 or claim 15, wherein the cancer is gastric cancer.

17. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of the T cell of claim 12 or the pharmaceutical composition of claim 13.

18. A method of limiting gastrointestinal toxicity in a patient previously identified as having cancer, the method comprising administering to the patient an effective amount of the T cell of claim 12 or the pharmaceutical composition of claim 13.

19. A method of limiting stomach tissue atrophy in a patient previously identified as having cancer, the method comprising administering to the patient an effective amount of the T cell of claim 12 or the pharmaceutical composition of claim 13.

20. The method of any one of claims 17-19, wherein the cancer is selected from gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, and non-small cell lung cancer.

21. The method of claim 20, wherein the cancer is gastric cancer.

22. The method of any one of claims 17-19, wherein the step of administering comprises intravenously administering.

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