Importance:Chimeric antigen receptor (CAR) T-cell therapy (CART) has transformed the treatment landscape of hematologic cancer, but has negligible effects for adult solid cancers. In this trial, an autologous CAR T-cell product demonstrated antitumor activity in heavily pretreated patients with metastatic colorectal cancer (mCRC). Objective:To evaluate the safety and efficacy of guanylate cyclase-C (GCC19) CART in participants with metastatic colorectal cancer (mCRC). Design, Setting, and Participants:This single-arm, nonrandomized, phase 1 trial was conducted at the First Hospital of Jilin University from December 3, 2020, to April 13, 2022. Data analysis was conducted from May 2022 to April 2024. Adults with relapsed and refractory mCRC expressing GCC were treated with GCC19CART, a mixture of autologous CAR T cells transduced with lentiviral vectors expressing genes that encode either CD-19 CAR or GCC CAR. Main Outcomes and Measures:Safety and tolerability of CAR T-cell therapy targeting GCC in patients with mCRC without therapeutic options is capable of conferring a reasonable likeliness of clinical benefit. Other outcomes included objective response rate, progression-free survival, overall survival, and immune activation. Results:Of 15 patients 9 (60%) were women, and the median (range) age was 44 (33-61) years. Treatment with GCC19CART was associated with the development of cytokine release syndrome and diarrhea in most patients, all of which were self-limited and manageable. The objective response rate was 40%, with a partial response in 2 of 8 and 4 of 7 patients treated with either 1 × 106 cells/kg or 2 × 106 cells/kg. Median overall survival was 22.8 months (95% CI, 13.4-26.1) at data cutoff; the median progress-free survival was 6.0 months in the high dose level group (95% CI, 3.0 to not available). Conclusions and Relevance:The results of this nonrandomized clinical trial suggest that GCC19CART was safe and tolerable in heavily pretreated patients with mCRC and is the first CAR T-cell therapy known to produce objective clinical activity in refractory cancer. Given the paucity of effective therapeutics developed for colorectal cancer in recent decades, the observation that CD-19 CART target engagement can robustly induce GCC19CART target engagement sufficient to produce objective activity may serve as a foundation to develop effective cellular therapy in mCRC and other solid cancers. Trial Registration:Chinese Clinical Trial Registry: ChiCTR2000040645.

Patient 13 with extramedullary leukemia relapse compromising testis obtained CR after CART19 infusion. A, Ultrasound showed complete elimination of extramedullary relapsed masses involved in testis (red arrows). Images are from before treatment and 90 days after CART19 infusion. B, Pathology showed complete elimination of extramedullary relapsed masses involved in testis. Images are from before treatment and 30 days after CART19 infusion. Left: 10×10, right: 10×40.

Supplementary Tables including Table 1, Table 2 and Table 3

CRS complication after CART19 therapy. A, the peak serum concentration of IL-6, IFN-γ, IL-10, CRP, D-dimer and ferritin of a representative patient (Patient 15) during CRS at the indicated time-points after CART19 infusion are showed. B, D, Peak serum levels of CRP, ferritin and D-dimer in patients who developed grade 3 CRS (n=6) compared with those without CRS or with 1 or 2 CRS (n=10). Data represented the mean{plus minus}SEM. The Mann-Whitney U test was used for statistical analysis. C, Paired measurements of peak serum IL-6, CRP, ferritin and D-dimer showed strong correlations (IL-6 VS CRP, Spearman r=0.617, p=0.014; IL-6 VS ferritin, Spearman r=0.574, p=0.028; IL-6 VS D-dimer, Spearman r=0.789, p<0.01).

A, A representative diagram of the transduction efficiency evaluation of CART19s 5 days after lentivirus infection. B, Specific target cell lysis by CART19s. T cells or CART19s were co-cultured with K562-RFP cells or CD19-RFP-K562 cells at the indicated E:T ratio for overnight co-culture (24 hours). C, IFN-γ secretion measurements in supernatants in T cells or CART19 cells co-cultured with K562-RFP cells or CD19-RFP-K562 cells at the indicated E:T ratios for 24 hours.

Comparison of CART19 levels in different groups. A, Levels of CART19 cells in PB were shown as assessed by means of qPCR assay at serial time points before and after infusion of CART19 respectively. B, CAR DNA copies in BM and PB on day 7 to 9 after CART19 infusion were compared. CAR DNA copies in BM (Patient 2, 6, 11, 13) were higher than in PB. C, Paired measurements of CAR DNA copies in BM and PB on day 7 to 9 after CART19 infusion, percentage of leukemia cells after FC chemotherapy shows strong correlations (CAR DNA copies in BM VS in PB, Spearman r=0.956, p<0.001; CAR DNA copies in BM VS percentage of leukemia cells after FC chemotherapy, Spearman r=0.699, p=0.011; CAR DNA copies in BM VS percentage of leukemia cells after FC chemotherapy, Spearman r=0.755, p=0.005). Data for patients 4 and 13 are not included. C, Paired measurements of peak CAR DNA copies in BM or peak CART count after CART19 infusion and infused CART dose shows no correlations (peak CAR DNA copies in BM VS infused CART dose, Spearman r=0.248, p=0.438; peak CART count in PB VS infused CART dose, Spearman r=0.172, p=0.594).

Relapsed leukemia cells analysis by FACS.A, leukemia cells with bright CD19 expression obtained CR with MRD negative after CART19 therapy and relapsed with CD19 + leukemia cells (Patient 1). B, leukemia cells with dim CD19 expression obtained CR with MRD negative after CART19 therapy but relapsed with 2 subsets of CD19 + and - leukemia cells (Patient 2 and 3).

CART19 identification and quantification

The cumulative incidence of non-relapse mortality are shown.

Background: Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable clinical efficacy in hematologic malignancies but limited success in solid tumors. GCC19CART, the first clinical candidate from the CoupledCAR solid tumor platform, is designed to overcome the limitations of conventional CAR T-cells in solid tumor malignancies by pairing solid tumor CAR T-cells with CD19 targeting CAR T-cells to amplify proliferation and activation of the solid tumor CAR T component. GCC19CART targets guanylate cyclase-C (GCC) which is expressed in the metastatic lesions of 70%-80% of subjects with colorectal cancers. A Phase 1 investigator-initiated clinical trial is underway in China for patients with relapsed or refractory metastatic colorectal cancer who have received at least 2 prior lines of therapy. Based on a data cutoff on October 20, 2022 21 subjects have been enrolled in 2 dose escalation groups at 5 hospitals in China. Methods: Subjects are screened for GCC expression by immunohistochemistry. Eligible subjects undergo leukapheresis, a single dose of lymphodepleting chemotherapy (fludarabine 30mg/m2 and cyclophosphamide 300mg/m2) 3 days prior to infusion, and then administration of a single infusion of GCC19CART at one of two preassigned doses: 1 × 106 or 2 × 106 CAR T-cells/kg. Endpoints are safety and preliminary evidence of efficacy as determined by CT or PET/CT per RECIST 1.1 or PERCIST 1.0. All responses were confirmed by an independent third-party imaging contract research organization (CRO). Results: 13 subjects have been enrolled to dose level 1 (1 × 106 cells/kg) and 8 subjects have been enrolled to dose level 2 (2 × 106 cells/kg). The most common adverse events were cytokine release syndrome (CRS) in 21/21 subjects (Grade 1 19/21 (90.48%) or Grade 2 2/21 (9.52%)) and diarrhea in 21/21 subjects (Grade 1 6/21 (28.57%) Grade 2 5/21 (23.81%) Grade 3 9/21 (42.86%) or Grade 4 1/21 (4.76%)). Neurotoxicity was observed in 2/21 (9.52%) subjects at Grade 3 or 4 and resolved with corticosteroids. The combined overall response rate (ORR) for both dose levels was 28.6% (6/21). For dose level 1, the overall response rate (ORR) per RECIST 1.1 was 15.4% (2/13). Two subjects demonstrated a partial response (PR) while 3 additional subjects had partial metabolic response (PMR) on PET/CT with stable disease (SD) or progressive disease (PD) per RECIST 1.1. For dose level 2, The ORR per RECIST 1.1 was 50% (4/8). 4 subjects demonstrated a PR (3 at month 1, 1 at month 3 after being SD at month 1) and 2 additional subjects had PMR on PET/CT with SD per RECIST 1.1. Conclusions: preliminary data show that GCC19CART has meaningful dose dependent clinical activity and an acceptable safety profile in relapsed or refractory metastatic colorectal cancer. This trial is ongoing and updated data will be presented. A Phase 1 trial of GCC19CART in the US under a cleared IND is expected to enroll patients from mid-2022. Citation Format: Naifei Chen, Chengfei Pu, Lingling Zhao, Ning Li, Chang Wang, Yusheng Huang, Suxia Luo, Xun Li, Zhenzhou Yang, Jun Bie, Ruihong Zhu, Xi Huang, Haiyang Tang, Tingting Liang, Yizhuo Wang, Beibei Jia, Dongqi Chen, Zhao Wu, Yongping Song, Victor Lu, Lei Xiao, Jiuwei Cui. A phase 1 dose escalation study of GCC19CART - a novel CoupledCAR therapy for subjects with metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1130.

3547 Background: GCC19CART, the first clinical candidate from the CoupledCAR solid tumor platform, is designed to overcome the limitations of conventional CAR T-cells in solid tumor malignancies by pairing solid tumor CAR T-cells with CD19 targeting CAR T-cells to amplify the proliferation and activation of the solid tumor CAR T component. GCC19CART targets guanylate cyclase-C (GCC), which is expressed in the metastatic lesions of 70%-80% of subjects with colorectal cancers and largely restricted to the intestinal tract. A Phase 1 investigator-initiated clinical trial is underway in China for patients with relapsed or refractory metastatic colorectal cancer who have received at least 2 prior lines of therapy. As of a data cutoff on Jan 28, 2023 21 subjects have been enrolled in 2 dose escalation groups at 5 hospitals in China. Methods: Subjects are screened for GCC expression by immunohistochemistry. Eligible subjects undergo leukapheresis, a single dose of lymphodepleting chemotherapy (fludarabine 30mg/m 2 and cyclophosphamide 300mg/m 2 ) 3 days prior to infusion, and then administration of a single infusion of GCC19CART at one of two preassigned doses: 1x10 6 or 2x10 6 CAR T-cells/kg. All responses were confirmed by an independent third-party imaging contract research organization. Results: 13 subjects have been enrolled to dose level 1 (1x10 6 cells/kg) and 8 subjects have been enrolled to dose level 2 (2x10 6 cells/kg). The most common adverse events were cytokine release syndrome (CRS) in 21/21 subjects (Grade 1 19/21 (90.48%) or Grade 2 2/21 (9.52%)) and diarrhea in 21/21 subjects (Grade 1 6/21 (28.57%) Grade 2 5/21 (23.81%) Grade 3 9/21 (42.86%) or Grade 4 1/21 (4.76%)). All patients with grade 3 and higher side effects were well managed. Immune effector cell-associated neurotoxicity syndrome (ICANS) was observed in 2/21 (9.52%) subjects at Grade 3 or 4 and resolved with corticosteroids. The combined overall response rate (ORR) for both dose levels was 28.6% (6/21). For dose level 1, overall response rate (ORR) was 15.4% (2/13). Two subjects demonstrated a partial response (PR) while 3 additional subjects had partial metabolic response (PMR) on PET/CT with stable disease (SD) or progressive disease (PD) per RECIST 1.1. For dose level 2, The ORR was 50% (4/8). 4 subjects demonstrated a PR and 2 additional subjects had PMR on PET/CT with SD per RECIST 1.1.The median PFS was 1.9 months in the dose 1 group and 6.3 months in the dose 2 group. The median overall survival was 13.3 months in the dose 1 group and 18.3 months in the dose 2 group. Conclusions: Preliminary results demonstrate that GCC19CART has meaningful dose-dependent clinical activity and an acceptable safety profile in relapsed or refractory metastatic colorectal cancer. This trial is ongoing and updated data will be presented. A Phase 1 trial of GCC19CART in the US has opened for accrual and is expected to enroll patients in mid-2022. Clinical trial information: ChiCTR2000040645 .

Background: Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable clinical efficacy in hematologic malignancies but limited success in solid tumors. GCC19CART, the first clinical candidate from the CoupledCAR® solid tumor platform, targets guanylate cyclase-C (GCC) which is expressed in colorectal cancers. A Phase 1 investigator-initiated dose escalation trial is underway in China for patients with relapsed or refractory metastatic colorectal cancer. Based on a data cutoff on September 10, 2021, 15 patients enrolled and 12 patients completed ≥1 evaluation of response and were evaluable. Methods: Subjects with relapsed or refractory metastatic colorectal cancer are screened for GCC expression, with 70% to 80% of subjects expected to demonstrate GCC per historical data. Subjects undergo leukapheresis, a single dose of lymphodepleting chemotherapy (fludarabine 30mg/m2 and cyclophosphamide 300mg/m2) 3 days prior to infusion, and then administration of a single infusion of GCC19CART at one of three doses from 1x106, or 2x106 cells/kg. Endpoints are safety and preliminary evidence of efficacy as determined by CT or PET/CT per RECIST1.1 or PERCIST 1.0. Results: 7 subjects have been enrolled to dose level 1 (1x106 cells/kg) and 5 subjects have been enrolled to dose level 2 (2x106 cells/kg) and have a 1 month post-infusion imaging study available for review. The most common adverse events were cytokine release syndrome (CRS) in 11/12 subjects (Grade 1 10/12 (83.33%) or Grade 2 1/12 (8.33%)) and diarrhea in 12/12 subjects (Grade 1 2/12 (16.67%) Grade 2 2/12 (16.67%) Grade 3 8/12 (66.67%)). Neurotoxicity was observed in 1/12 (8.33%) subjects at Grade 4 and resolved with corticosteroids. The combined overall response rate (ORR) for both dose levels was 41.67% (5/12). For dose level 1, the overall response rate (ORR) per RECIST 1.1 was 28.57% (2/7). Two subjects demonstrated a partial response (PR) while 2 additional subjects had partial metabolic response (PMR) on PET/CT with stable disease (SD) or progressive disease (PD) per RECIST 1.1. For dose level 2, The ORR per RECIST 1.1 was 60% (3/5). 3 subjects demonstrated a PR (2 at month 1, 1 at month 3 after being SD at month 1) and an additional subject had PMR on PET/CT with SD per RECIST 1.1. Conclusions: GCC19CART demonstrated meaningful clinical activity and an acceptable safety profile in relapsed or refractory metastatic colorectal cancer. This trial is ongoing and updated data will be presented. A United States based Phase 1 trial of GCC19CART is anticipated for early 2022. Citation Format: Naifei Chen, Chengfei Pu, Lingling Zhao, Chang Wang, Ruihong Zhu, Tingting Liang, Xi Huang, Haiyang Tang, Yizhuo Wang, Beibei Jia, Dongqi Chen, Eugene Kennedy, Zhao Wu, Lei Xiao, Jiuwei Cui. Novel coupledCAR࣪technology for treating colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2747.

Abstract One key hurdle for the CAR-T cell treatment of solid tumors is the limited accessibility of solid tumor antigens outside the tumor microenvironment, which prohibits the expansion of solid tumor-targeting CAR-T cells in patients. Here, we report the characterization of prostatic acid phosphatase (PAP) as a feasible CAR-T target for prostate cancer and a novel approach, named CoupledCAR, to expand solid tumor-targeting CAR-T cells lacking solid tumor antigens based on the observation of non-transduced T cells proliferating together with CD19 CAR-T cells during the treatment of acute lymphocyte leukemia. We demonstrated that CoupledCAR can significantly enhance the expansion and antitumor efficacy of PAP CAR-T cells both in vitro and in vivo. Furthermore, we showed that the expansion of solid tumor-targeting CAR-T cells does not depend on CAR/CD3ζ stimulation through direct antigen binding with CAR but enhances the memory status of CAR-T cells and causes little exhaustion. Since the CoupledCAR system does not rely on solid tumor antigens, we propose that it can be utilized in all CAR-T and T cell therapies for the treatment of solid tumors. Citation Format: Zhiyuan Cao, Chengfei Pu, Xianyang Jiang, Guiting Han, Yuzhe Peng, Wensheng Wang, Wei Ding, Xiaogang Shen, Dongqi chen, Beibei Jia, Xiaoqiang Xu, Zhipeng Huang, Xi Huang, Wenbi Liu, Ruihong Zhu, Lee Tian, Christopher Ballas, Victor.X Lu, Zhao Wu, Lei Xiao. Antigen-independent expansion enhances efficacy of CAR-T cells against solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2838.

GCC19CART, the first clinical candidate from the CoupledCAR® solid tumor platform, targets guanylate cyclase-C (GCC) which is expressed in colorectal cancers. CoupledCAR utilizes multiple vectors to make both solid tumor targeting CAR-T and CD19 CAR-T in a single manufacturing step. An investigator-initiated dose escalation trial in China for patients with relapsed or refractory metastatic colorectal cancer (R/R mCRC) is reported here. Based on a data cutoff of April 5, 2022, 15 subjects have been enrolled and treated, with all subjects completing ≥1 evaluation of response and being evaluable. Eligible subjects undergo leukapheresis, a single dose of lymphodepleting chemotherapy (fludarabine 30mg/m2 and cyclophosphamide 300mg/m2) 3 days prior to infusion, and then administration of a single infusion of GCC19CART at one of two preassigned doses: Dose 1 (1x106) or Dose 2 (2x106) CAR T-cells/kg. Endpoints are safety and preliminary evidence of efficacy. 8 subjects have been enrolled to dose 1 and 7 subjects have been enrolled to dose 2. The most common adverse events were cytokine release syndrome (CRS) in 14/15 subjects (Grade 1 and 2) and diarrhea in 14/15 subjects (Grade 1, 2, and 3). Neurotoxicity was observed in 1 patient (Grade 4) and resolved with corticosteroids. The combined overall response rate (ORR) for both dose levels was 40% (6/15). For dose level 1, the ORR per RECIST 1.1 was 25% (2/8). For dose level 2, the ORR per RECIST 1.1 was 57% (4/7). GCC19CART demonstrated meaningful dose dependent clinical activity and an acceptable safety profile in R/R mCRC. At a dose of 2x106 CAR T-cells/kg, the ORR is 57% (4/7). It was well tolerated and toxicity was manageable. A United States based phase I trial of GCC19CART is anticipated to begin enrolling subjects in mid-2022.

Abstract Chimeric antigen receptor (CAR) T cell therapy has made significant progress in the treatment of blood cancers such as leukemia, lymphoma, and myeloma. However, the therapy faces many challenges in treating solid tumors. These challenges include physical barriers, tumor microenvironment immunosuppression, tumor heterogeneity, target specificity, and limited reactive cell expansion in vivo.Conventional CAR T cell therapy has thus far shown weak cell expansion in solid tumor patients and achieved little or no therapeutic responses. Here, we developed CAR T cells based on a novel CoupledCAR® technology to treat solid tumors. In contrast to conventional CAR T cells, CoupledCAR T cells significantly improved the expansion of the CAR T cells in vivo and enhanced the CAR T cells' migration ability and resistance to immunosuppression by the tumor microenvironment. The enhanced migration ability and resistance allow the CAR T cells to infiltrate to tumor tissue sites and increase anti-tumor activities.Specifically, we engineered CoupledCAR T cells with lentiviral vectors encoding an anti-GCC (guanylate cyclase 2C) CAR molecule. Furthermore, anti-GCC CAR T cells showed anti-tumor activities in vitro and in vivo experiments.To verify the safety and efficacy of CoupledCAR T cells for treating solid tumors, we conducted several clinical trials for different solid tumors, including seven patients with colorectal cancer. These seven patients failed multiple rounds of chemotherapy and radiotherapy. In the clinical trial, the patients were infused with autologous anti-GCC CoupledCAR T cells range from 4.9×10^5/kg to 2.9×10^6/kg. All patients using anti-GCC CoupledCAR T cells showed rapid expansion of CoupledCAR T cells and killing of tumor cells. Specifically, we observed that CoupledCAR T cells expanded significantly in the patients and infiltrated tumor tissue sites, demonstrating enhanced anti-tumor activities. PET/CT showed significant tumor shrinkage and SUV max declined, and the ongoing responses were monitored. Patient 3 achieved complete response and the best overall response rate (ORR, include complete remission, complete metabolic response, partial response, and partial metabolic response.) was 57.1% (4/7), complete remission (CR) rate was 14.3% (1/7).The clinical data demonstrated that CoupledCAR T cells effectively expanded, infiltrated tumor tissue sites, and kill tumor cells in patients with colorectal cancer. We used immunotherapy to achieve complete remission in patients with advanced colorectal cancer for the first time. We are recruiting more colorectal cancer patients to further test the safety and efficacy of anti-GCC CoupledCAR T cells. Since our CoupledCAR® technology is a platform technology, we are expanding it to treat other solid tumors using different target tumor markers. Citation Format: Lei Xiao, Song Li, Chengfei Pu, Zhiyuan Cao, Xinyi Yang, Ning Li, Youli Luo, Haiyan Zhao, Hang Yang, Xi Huang, Xiaogang Shen, Xiuwen Wang, Yongping Song, Junjie Mao, Pengfei Pang, Qun Hu, Zhao Wu, Victor Lu. Novel coupledCARTMtechnology for treating colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB146.

Abstract Chimeric antigen receptor modified T cells (CAR T) have demonstrated remarkable clinical efficacy in the treatment of B cell malignancies and multiple myeloma. Significant challenges restrict their application across solid tumors due to multiple obstacles, including the lack of robust in vivo CAR-T cell expansion and persistence, the immunosuppressive tumor microenvironment.To address these difficulties, we generated CAR T cells using a novel CoupledCAR® technology. Specifically, we engineered CoupledCAR T cells with lentiviral vectors encoding an anti-thyroid stimulating hormone receptor (TSHR) CAR molecule. Immunohistochemistry (IHC) results showed that TSHR was highly expressed in thyroid cancer cells making it an ideal tumor-specific target antigen. In vitro co-culture experiments showed that TSHR CAR T cells specifically recognized and subsequently killed TSHR-positive tumor cells. Animal model experiments showed that TSHR CAR T cells inhibited the proliferation of TSHR-positive tumor cells.To evaluate the clinical safety and efficacy of anti-TSHR CoupledCAR T cells on refractory or relapsed thyroid cancer, we treated refractory/relapsed post-thyroidectomy thyroid cancer patients according to an IRB approved protocol. We treated two patients using anti-TSHR CoupledCAR T cells and observed the rapid expansion of CAR T cells and enhanced the killing of tumor cells. One patient's best response was complete remission, and the other was near complete remission.Patient 1 Male, 64Y, Papillary Thyroid Carcinoma. In May 2017, Thyroid cancer was diagnosed, bilateral total thyroidectomy, and right cervical lymph node functional dissection were performed in Jun 2018, followed by iodine 131 isotope therapy. In December 2018, bilateral multiple cervical lymph nodes were enlarged, especially on the right side. In February 2019, right neck lymphadenectomy was performed.Patient 2 Female, 60Y, Thyroid Carcinoma. In Aug 2013, a "double lobectomy of the thyroid gland” was performed. From Oct 2013 to Jan 2014, she received iodine 131 isotope therapy. In Sep 2014, she was diagnosed with iodine - resistant thyroid cancer. From Sep to Jan 2016, 5 cycles of chemotherapy were performed. In Jun 2016, she enrolled in the Anlotinib experimental group. In Mar 2019, multiple metastases in both lungs and multiple enlarged lymph nodes in the mediastinum were observed.Patient 1: One month after infusion (M1), the patient was evaluated as PR. Three months after infusion (M3), the patient was evaluated as CR, and the patient's CR lasted from M3 to M12 after infused anti-TSHR CoupledCAR T cells , and we are still following up.Patient 2: M1, the patient was evaluated as PR (Partial Response): the tumor volume in the right lower lobe of the lung was reduced by approximately 67.51% (decreased from 65*55mm to 42*39mm). Three months after infusion (M3), compared with that before, the tumor volume was reduced by approximately 73.54% and SUV max value decreased from 14.9 to 2.8, therefore, the patient was evaluated as nCR (near complete remission).We show that TSHR is a good target for treating thyroid cancer, and our anti-TSHR CoupledCAR T cells are safe and effective for treating thyroid cancer. Recruitment is ongoing to evaluate the safety and efficacy of our CoupledCAR T cells. Further, since our CoupledCAR® technology is a platform technology, we are developing it to treat other solid tumors using different target tumor markers. Citation Format: Lei Xiao, Xingchen Liu, Keshu Zhou, Yu Liu, Yong Huang, Chengfei Pu, Zhiyuan Cao, Ruihong Zhu, Haiyang Tang, Zhipeng Huang, Hang Yang, Xi Huang, Yongping Song, Renbin Liu, Zhao Wu, Victor Lu. CoupledCARTMtechnology for treating thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB145.

Background: Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable clinical efficacy in hematologic malignancies but limited success in solid tumors. GCC19CART, the first clinical candidate from the CoupledCAR ® solid tumor platform, targets guanylate cyclase-C (GCC) which is expressed in colorectal cancers. A Phase 1 investigator-initiated dose escalation trial is underway in China for patients with relapsed or refractory metastatic colorectal cancer. Data presented here are from a single participating institution.

2528 Background: Chimeric antigen receptor (CAR) T cell therapy has made significant progress in the treatment of blood cancers such as leukemia, lymphoma, and myeloma. However, the therapy faces many challenges in treating solid tumors. These challenges include physical barriers, tumor microenvironment immunosuppression, tumor heterogeneity, target specificity, and limited expansion in vivo. Methods: We designed a CAR lentivirus vector that consisted of a humanized CD19-specific single-chain variable fragment (scFv), a 4-1BB costimulatory domain, and a CD3ζ signaling domain.The lentivirus was produced by transfecting HEK-293T cells with CAR lentiviral vectors and viral packaging plasmids. Patient’s CD3 T cells was cultured in X-VIVO medium containing 125U/mL 1interleukin-2 (IL-2), and transduced with CAR lentivirus at certain MOI 24h after stimulated by anti-CD3/CD28 magnetic beads. Transduction efficiency was evaluated at 7 to 9 days after CAR lentivirus transduction, and quality controls for fungi, bacteria, mycoplasma, chlamydia, and endotoxin were performed. After infusion, serial peripheral blood samples were collected, and the expansion and the cytokine release of CART cells were detected by FACS and QPCR,respectively. The evaluation of response level for patients were performed at month 1,month 3,and month 6 by PET/CT. Results: We engineered CoupledCAR T cells with lentiviral vectors encoding an anti-GCC (guanylate cyclase 2C) CAR molecule. To verify the safety and efficacy of CoupledCAR-T cells for treating solid tumors, we conducted several clinical trials for different solid tumors, including seven patients with colorectal cancer. These seven patients failed multiple rounds of chemotherapy and radiotherapy. In the clinical trial, the metastatic colorectal cancer patients were infused with autologous anti-GCC CoupledCAR-T cells range from 4.9×105/kg to 2.9×106/kg. We observed that CoupledCAR-T cells expanded significantly in the patients and infiltrated tumor tissue sites, demonstrating enhanced anti-tumor activities. PET/CT showed significant tumor shrinkage and SUV max declined, and the ongoing responses were monitored. Patient 3 achieved complete response and the best overall response rate (ORR, include complete remission, complete metabolic response, and partial response.) was 57.1% (4/7), complete remission (CR) rate was 14.3% (1/7). Conclusions: In conclusion, the clinical data demonstrated that CoupledCAR-T cells effectively expanded, infiltrated tumor tissue sites, and kill tumor cells in patients with colorectal cancer. We used immunotherapy to achieve complete remission in patients with advanced colorectal cancer for the first time. We are recruiting more colorectal cancer patients to further test the safety and efficacy of anti-GCC CoupledCAR T cells. Since our CoupledCAR technology is a platform technology, we are expanding it to treat other solid tumors using different target tumor markers.

Background CAR T therapy has achieved remarkable results in the treatment of hematological tumors such as leukemia, lymphoma, and multiple myeloma. However, there remains challenges in treating solid tumors. These challenges include physical barriers, tumor microenvironment immunosuppression, tumor heterogeneity and target specificity. Especially, due to tumor microenvironmental barriers, CAR T cells are not effectively exposed to tumor antigens and cannot activate co-stimulation signals on CAR molecules, thus conventional CAR T cell therapy has thus far shown weak cell expansion in solid tumor patients, achieved little or no therapeutic responses. Here, we developed CAR T cells based on a novel CoupledCAR® technology to overcome the lack of persistence of solid tumor CAR T cells in vivo. Methods We designed a 'CoupledCAR' lentivirus vector containing a single-chain variable fragment (scFv) targeting human TSHR. The lentivirus was produced by transfecting HEK-293T cells with 'CoupledCAR' lentiviral vectors and viral packaging plasmids. Patient's CD3 T cells were cultured in X-VIVO medium containing 125U/mL 1interleukin-2 (IL-2), and transduced with 'CoupledCAR' lentivirus at certain MOI. Transduction efficiency and was evaluated at 7 to 9 days after 'CoupledCAR' lentivirus transduction, and quality controls for fungi, bacteria, mycoplasma, chlamydia, and endotoxin were performed. After infusion, serial peripheral blood samples were collected, and the expansion and the cytokine release of CART cells were detected by FACS and QPCR. The evaluation of response level for patients were performed at month 1,month 3,and month 6 by PET/CT. Results We used prostatic acid phosphatase (PAP) as an exemplary CAR target for prostate cancer and demonstrated that our CoupledCAR® significantly enhanced the expansion of PAP CAR T cells in vitro and in vivo. Further, we observed that this expansion showed more memory-like phenotypes, and caused little exhaustion of PAP CAR T cells. Also, we find coupled solid tumor CAR T cells have stronger tumor killing ability. We demonstrated this simple expansion to enable the persistence of solid tumor CAR T cells and can be further applied to other kinds of T cell therapy like TCR T and TILs. Conclusions We developed a novel platform technology (CoupledCAR®) that allows solid tumor CAR T cells to rapidly expand. This initial CAR T cell expansion enabled enhanced trafficking and infiltration of the tumor tissue whereby further cell expansion occurred and thereby achieved tumor clearance. We have carried clinical trials and obtained early promising clinical data. We will further verify the safety and efficacy of this technology in the treatment of different kinds of solid tumors in the clinic research.

Dysregulated B-cell activation plays pivotal roles in systemic lupus erythematosus (SLE), which makes B-cell depletion a potential strategy for SLE treatment. The clinical success of anti-CD19 CAR-T cells in treating B-cell malignancies has attracted the attention of researchers. In this study, we aimed to investigate the feasibility of applying anti-CD19 CAR-T cell therapy to SLE treatment in a mouse disease model. We constructed murine anti-CD19 CARs with either CD28 or 4-1BB as the intracellular costimulatory motif and evaluated the therapeutic function of the corresponding CAR-T cells by infusing them into MRL-lpr mice. Furthermore, anti-CD19 CAR-T cells were transferred to MRL-lpr mice before the onset of disease to determine their role in SLE prevention. According to our observations, compared with antibody treatment, the adoptive transfer of our anti-CD19 CAR-T cells showed a more sustained B-cell-depletion effect in MRL-lpr mice. The transfer of syngeneic anti-CD19 CAR-T cells not only prevented disease pathogenesis before the onset of disease symptoms but also displayed therapeutic benefits at a later stage after disease progression. We also tried to optimize the treatment strategy and found that compared with CAR-T cells with the CD28 costimulatory motif, CAR-T cells with the 4-1BB costimulatory motif showed better therapeutic efficiency without cell enrichment. Taken together, these results show that anti-CD19 CAR-T cell therapy was effective in the prevention and treatment of a murine model of SLE, indicating its potential for clinical use in patients.