Recent Advances and Evolving Strategies of Photothermal Immunotherapy in Pancreatic Cancer Treatment

Immune response to three doses of mRNA SARS-CoV-2 vaccines in CD19-targeted chimeric antigen receptor Tcell immunotherapy recipients.

2558 Background: Chimeric antigen receptor (CAR) T therapies for pancreatic ductal adenocarcinoma (PDAC) still face significant immunosuppressive obstacles in the tumor microenvironment (TME). We hypothesize that an optimal CAR T cell for PDAC combines targeting an ideal tumor-associated antigen (TAA) and overcomes unique immunosuppression in the PDAC TME. The retained ectodomain of Muc16 (Muc16CD) is a known TAA in ovarian cancer but has yet to be explored in pancreatic cancer. Vasoactive intestinal peptide (VIP) is an emerging checkpoint pathway for T cell function, which expresses VIP receptors (VIPR) and is abundantly expressed by PDAC. In this work, we present a novel armored CAR T cell that targets Muc16CD and antagonizes VIPRs (CAR/VIPRa) to overcome the immunosuppressive PDAC TME. Methods: Patient expression data was assessed based on data generated by the TCGA Research Network. Primary human T cells from healthy donors or PDAC patients were retrovirally transduced to express Muc16CD-directed CARs with or without secretion of novel, potent VIPR antagonist peptides. In vivo, PDAC PDX tumors were engrafted into SCID/Beige mice and treated with CAR T cells. Results: PDAC tumors have significantly increased expression of Muc16 compared to normal pancreas tissue and patients with high Muc16 expression have a significantly decreased overall survival. PDAC patient-derived tumors show robust expression of both Muc16CD and VIP. CAR/VIPRa T cells reveal that VIPR antagonism metabolically reprograms CAR T cells and drives a memory-rich product. CAR/VIPRa T cells are less activated and less exhausted by the manufacturing process, which lends to better viability and a metabolically quiescent phenotype at baseline. These distinct features allow CAR/VIPRa T cells, when antigen-stimulated, to have enhanced activation and expansion with repeated stimulation. To investigate clinical relevance, CAR/VIPRa T cells manufactured from PDAC patient blood are also significantly enriched for memory phenotypes. In vivo, CAR/VIPRa T cells have enhanced expansion, phenotype, infiltration, and persistence, which ultimately reduces PDAC tumor burden. In a patient-derived PDAC preclinical mouse model where CAR T is typically ineffective, CAR/VIPRa T cells significantly reduce tumor burden. Conclusions: This work demonstrates Muc16CD as a clinically relevant TAA target for CAR T therapy in PDAC. Furthermore, antagonizing the previously undescribed VIP checkpoint pathway in CAR T cells produces enhanced phenotypic and functional profiles. Collectively, this data demonstrates that novel CAR/VIPRa T cells create an advantageous cellular therapy product capable of treating PDAC. The long-term goal of this work is translating CAR/VIPRa T cells for the treatment of PDAC and expanding these preclinical findings of cellular therapies for other VIP-abundant tumors.

Rationale and Background: Immunotherapy of cancer has gained much attention in the past decade with the development of immune checkpoint inhibitors and chimeric antigen receptor (CAR) technology that can activate and redirect patient T cells to kill tumors that over-express a specific antigen. CARs are fusion receptors that are comprised of an antibody-derived single-chain variable fragment (scFv) coupled via hinge and transmembrane elements to a T cell signaling and co-stimulatory domain. This technology is in its early stages of development and has not been fully exploited for the treatment of metastatic epithelial cancers. We focus our studies on Pancreatic Ductal Adenocarcinomas (PDAC) and Triple Negative Breast Cancer (TNBC). However, if successful, the studies will be applicable to other epithelial tumors. For CAR-T cells to work, and to avoid off target toxicities, both the target antigen and the antibody recognizing the target have to be highly specific. The challenge is that there are few such antigen-antibody combinations for solid tumors. We have recently developed a novel patented antibody (designated TAB 004) that specifically recognizes ONLY the tumor-associated form of MUC1 (tMUC1) but not the normal form of MUC1 (nMUC1) in several subtypes of breast cancers including TNBC and in PDAC. We show compelling data that TAB004 recognizes tMUC1 in >90% of human TNBCs and 85% of PDAC but spares all normal epithelial tissues. The antigenic isoform that TAB004 recognizes is completely hidden in normal epithelia making it extremely safe for development of CAR-T cells. Hypothesis: TNBC and PDAC can be specifically targeted with the tMUC1-CAR-engineered T cells, whilst sparing normal organs. Methodology and Results: We have engineered several TAB-specific CAR constructs using the scFv fragment of TAB 004. Six constructs are developed, 3 for human T cells and 3 for mouse T cells: 1. TAB- CD28-CD3zeta (2nd generation CAR), 2. TAB-CD28-41BB-CD3zeta (3rd generation CAR), and 3. TAB-CD28-OX40-CD3zeta (3rd generation CAR). Data shows that we can successfully engineer human T cells to express the TAB-CAR on their surface and that these engineered T cells can bind specifically to tumor cells expressing the unique tMUC1 epitope, become activated, and effectively kill the tumor cells. We show that these engineered T cells only minimally bind and kill normal epithelial cell lines. However, some of the cell lines are more resistant than others. We are therefore conducting combination therapy with various drugs that are known to enhance immune based therapies including checkpoint inhibitors, COX-2 inhibitors, cyclophosphamide and others. We may also have data to show that the engineered TAB-CAR T cells kill tMUC1-expressing TNBC and PDAC cells in vivo. We will show data that TAB-CAR-T cell kill human PDAC and TNBC cells in vitro and propose to conduct the same in vivo xenograft model of human PDAC and metastatic TNBC. In the following months, we will test if TAB-CAR-T cell retards tumor growth in an orthotopic syngeneic mouse model of PDAC and metastatic BC in human MUC1.Tg immune competent mice. We further propose to demonstrate that the TAB-CAR-T cell can mediate apoptosis in the immune competent KCM mice (KC X human MUC1.Tg mice) that develop spontaneous PDAC and the MMT mice (PyVMT X human MUC1.Tg mice) that develop spontaneous mammary gland tumors. Both models mimic the human disease progression and express human MUC1 in a tissue specific manner. This is important since all normal epithelia in these mice express the nMUC1 except the tumors that expresses the target, tMUC1. Impact: If successful, this project will have a major impact and accelerate progress toward a clinical trial for PDAC and metastatic TNBC. This will be the first attempt to test the efficacy of a CAR-T cell in an immune competent, human MUC1.Tg mouse model that develops spontaneous tumors within the appropriate stromal and hormonal microenvironment. Citation Format: Pinku Mukherjee, Ru Zhou, Mahboubeh Yazdanifar, Das Roy Lopamudra. Development and future of CAR T cell therapy for pancreatic ductal adenocarcinoma and triple negative breast cancer [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A37.

Desmoplasia of Pancreatic Ductal Adenocarcinoma

Background: Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis. Despite negative margin resections of the cancer and systemic therapy, PDAC often recurs. Pancreatic cancer-associated fibroblasts (CAFs) contribute to a highly desmoplastic tumor microenvironment (TME), preventing T cell infiltration. Moreover, CAF-secreted factors suppress the anti-tumor activity of T cells and shift tumor cells toward invasive proliferative and mesenchymal phenotypes. Therefore, targeting CAFs can enhance T-cell infiltration and improve treatment outcomes. In this study, we investigate using Chimeric Antigen Receptor (CAR) T cells targeting B7-H3 to eliminate PDAC metastatic disease in a preclinical mouse model. B7-H3, an immune checkpoint molecule, is highly expressed on PDAC cells and CAFs. Our CART-B7-H3 construct includes an inducible caspase 9 (iC9) suicide gene, which allows for the controlled elimination of the CAR T cells in the event of overwhelming cytokine release syndrome. Methods: The efficacy of iC9.B7-H3 CAR T cells was tested in vitro against human PDAC and fibroblast cell lines at various ratios. The antitumor activity of iC9.B7-H3 CAR T cells was assessed in PDAC cells after exposure to CAF-conditioned media, simulating TME with immunosuppressive cytokines. To validate the therapeutic potential of iC9.B7-H3 CAR T cells in vivo, NSG mice were orthotopically engrafted with patient-derived PDAC6 cells and hCAF1 cells at a ratio of 1:9, reflecting a clinically relevant model. After three weeks, the mice underwent surgical resection of the primary tumor (distal pancreatectomy and splenectomy) and were subsequently treated systemically with iC9.B7-H3 CAR T cells. Results: In vitro, at a high effector-to-target (E:T) ratio, iC9.B7-H3 CAR T cells effectively eliminated PDAC cells and CAFs. However, their anti-tumor activity was diminished when the E:T ratio was lowered (PDAC6 tumor cell killing (TCK): 92±5% at 1:1, hCAF1 TCK: 81.67±5% at 1:1, compared to PDAC6 TCK: 26±5% at 1:10, hCAF1 TCK: 0% at 1:10, P<0.0001). PDAC cells co-cultured with CAF cells demonstrated reduced susceptibility to elimination by iC9.B7-H3 CAR T cells compared to PDAC cells alone (PDAC3 TCK: 92±5% at 1:1, PDAC3+hCAF1 (1:9 ratio) TCK: 81±5% at 1:1, P=0.002). However, incubation with CAF-conditioned media did not significantly affect the elimination of PDAC cells by iC9.B7-H3 CAR T cells, which may suggest that CAFs may require direct cell-cell contact to exert their effects. In vivo, iC9.B7-H3 CAR T cells effectively eradicated both local and distant PDAC metastases for the length of the experiment (160 days), indicating long-term efficacy in the treatment of PDAC metastases with no adverse side effects. Conclusion: These data suggest that iC9.B7-H3 CAR T cells may be an effective adjuvant treatment for the eradication of micro-metastases and the prevention of disease recurrence in PDAC patients. Citation Format: Shahrzad Arya, Seyed Amir Sanatkar, Cristina Martin, Marco Ventin, Jingyu Jia, Giulia Cattaneo, Gabriella Lionetto, David T. Ting, Xinhui Wang, Sandra Ryeom, Cristina R. Ferrone, Soldano Ferrone. Adjuvant iC9.B7-H3 CAR T cell-based immunotherapy effectively eradicates local and distant metastases in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A021.

Pancreatic cancer (PC) is one of the most malignant tumors in digestive system due to its highly invasive and metastatic properties. At present, conventional treatment strategies for PC show the limited clinical efficacy. Therefore, novel effective therapeutic strategies are urgently needed. Here, we report a case of complete remission of advanced PC induced by claudin18.2-targeted CAR-T cell therapy. The patient was a 72-year-old man who was diagnosed with pancreatic ductal adenocarcinoma 2 years ago, and he experienced tumor recurrence and multiple metastases after pancreaticoduodenectomy and multi-line chemotherapies, including liver, peritoneum, and cervical lymph node metastases. Then, the patient was referred to our department for further treatment of metastatic PC, and he was enrolled in a clinical trial of claudin18.2-targeted CAR-T cell therapy. After lymphodepleting chemotherapy, the patient received claudin18.2-targeted CAR-T cell infusion at a dose of 1.2 × 106 cells/kg on November 21, 2022. During CAR-T cell therapy, the patient experienced grade 2 cytokine release syndrome (CRS) and gastric mucosa injury, which were controlled by tocilizumab and conventional symptomatic and supportive treatment. The patient achieved a complete response (CR) 1 month after claudin18.2-targeted CAR-T cell therapy, and remained in clinical remission for 8 months. Unfortunately, the patient experienced claudin18.2-negative relapse in July, 2023. Despite antigen-negative relapse after claudin18.2-targeted CAR-T cell infusion, the patient achieved sustained remission for 8 months, which indicates that claudin18.2-targeted CAR-T cell therapy is an extremely effective therapeutic strategy for the treatment of advanced PC.

Pancreatic ductal adenocarcinoma (PDAC) remains refractory to chimeric antigen receptor (CAR) T-cell therapies because of its immunosuppressive microenvironment and a dense extracellular matrix deposited by cancer-associated fibroblasts (CAF), which impair CAR T-cell infiltration. To address these barriers, we previously developed a dual-targeting CAR-TEAM platform in which mesothelin-specific CAR T cells secrete a fibroblast activation protein (FAP)-targeting T-cell engager antibody molecule (TEAM) to simultaneously kill tumor cells and CAF. In this study, we leveraged mesothelin-targeting CAR T cells and tested rational drug combinations and optimal delivery strategies to enhance therapeutic efficacy and guide potential combinations that could be incorporated into a clinical study. Tumor mesothelin shedding by proteases and CAR T-cell dysfunction remain key obstacles to CAR T-cell efficacy. Using preclinical PDAC models, we tested mesothelin-targeting CAR T cells in combination with agents that increase tumor mesothelin expression, promote T-cell polarization and persistence, and support T-cell function. Furthermore, we compared intravenous versus intraperitoneal delivery routes to treat peritoneal metastases. We demonstrated that ibrutinib enhanced CAR T-cell expansion, Th1 skewing, and antitumor activity in PDAC. PD-1 blockade synergistically improved CAR T-cell antitumor function in a patient-derived PDAC xenograft and intraperitoneal delivery proved superior against peritoneal disease. Conversely, although an ADAM-10/-17 inhibitor prevented mesothelin shedding and improved tumor killing in vitro, it did not enhance efficacy in vivo. These findings identify clinically actionable strategies to optimize CAR T-cell therapy against PDAC. A phase I clinical trial testing meso-FAP CAR-TEAM T cells, alone or in combination with ibrutinib or PD-1 blockade, is in development.

<div><p>The success of chimeric antigen receptor (CAR) T-cell therapy against hematologic malignancies has altered the treatment paradigm for patients with these diseases. Nevertheless, the occurrence of relapse due to antigen escape or heterogeneous antigen expression on tumors remains a challenge for first-generation CAR T-cell therapies as only a single tumor antigen can be targeted. To address this limitation and to add a further level of tunability and control to CAR T-cell therapies, adapter or universal CAR T-cell approaches use a soluble mediator to bridge CAR T cells with tumor cells. Adapter CARs allow simultaneous or sequential targeting of multiple tumor antigens, control of immune synapse geometry, dose control, and the potential for improved safety. Herein, we described a novel CAR T-cell adapter platform that relies on a bispecific antibody (BsAb) targeting both a tumor antigen and the GGGGS (G<sub>4</sub>S) linker commonly used in single-chain Fv (ScFv) domains expressed on CAR T-cell surfaces. We demonstrated that the BsAb can bridge CAR T cells to tumor cells and potentiate CAR T-cell activation, proliferation, and tumor cell cytolysis. The cytolytic activity of CAR T-cells was redirected to different tumor antigens by changing the BsAb in a dose-dependent manner. This study highlights the potential of G<sub>4</sub>S-displaying CAR T cells to be redirected to engage alternative tumor-associated antigens (TAA).</p>Significance:<p>New approaches are needed to address relapsed/refractory disease and manage potential toxicities associated with CAR T-cell therapy. We describe an adapter CAR approach to redirect CAR T cells to engage novel TAA-expressing cells via a BsAb targeting a linker present on many clinical CAR T-cell therapeutics. We anticipate the use of such adapters could increase CAR T-cell efficacy and reduce potential CAR-associated toxicities.</p></div>

<div><p>The success of chimeric antigen receptor (CAR) T-cell therapy against hematologic malignancies has altered the treatment paradigm for patients with these diseases. Nevertheless, the occurrence of relapse due to antigen escape or heterogeneous antigen expression on tumors remains a challenge for first-generation CAR T-cell therapies as only a single tumor antigen can be targeted. To address this limitation and to add a further level of tunability and control to CAR T-cell therapies, adapter or universal CAR T-cell approaches use a soluble mediator to bridge CAR T cells with tumor cells. Adapter CARs allow simultaneous or sequential targeting of multiple tumor antigens, control of immune synapse geometry, dose control, and the potential for improved safety. Herein, we described a novel CAR T-cell adapter platform that relies on a bispecific antibody (BsAb) targeting both a tumor antigen and the GGGGS (G<sub>4</sub>S) linker commonly used in single-chain Fv (ScFv) domains expressed on CAR T-cell surfaces. We demonstrated that the BsAb can bridge CAR T cells to tumor cells and potentiate CAR T-cell activation, proliferation, and tumor cell cytolysis. The cytolytic activity of CAR T-cells was redirected to different tumor antigens by changing the BsAb in a dose-dependent manner. This study highlights the potential of G<sub>4</sub>S-displaying CAR T cells to be redirected to engage alternative tumor-associated antigens (TAA).</p>Significance:<p>New approaches are needed to address relapsed/refractory disease and manage potential toxicities associated with CAR T-cell therapy. We describe an adapter CAR approach to redirect CAR T cells to engage novel TAA-expressing cells via a BsAb targeting a linker present on many clinical CAR T-cell therapeutics. We anticipate the use of such adapters could increase CAR T-cell efficacy and reduce potential CAR-associated toxicities.</p></div>

A Tumor Sorting Protocol that Enables Enrichment of Pancreatic Adenocarcinoma Cells and Facilitation of Genetic Analyses

Background: Pancreatic cancer is the 3rd leading cause of cancer mortality in the USA recently surpassing breast cancer. Treatment options are limited to surgery and chemo/radiation, which is normally associated with high toxicity. Targeted immune-based therapies have shown promising results but needs further exploration. Mucin 1 (MUC1), a glycoprotein expressed on the apical surface of epithelial cells, undergoes hypoglycozylation in tumors. This tumor-form of MUC1 (tMUC1) is over-expressed in 80% of pancreatic ductal adenocarcinomas (PDA). We have developed an antibody (TAB004) which specifically detects tMUC1 and not the normal MUC1. Functional fragments of TAB004 antibody (scFv) were incorporated into the chimeric antigen receptor (CAR) construct and used to genetically modify primary human T cells. tMUC1 recognizing domain (TAB004 scFv) is linked to the co-stimulatory molecules of T cells (CD28 and CD3ζ). These CAR T cells are highly activated and reactive to MUC1 expressing tumor cells. Pancreatic cancer is highly resistant to many treatments including immunotherapy. We suggest combining our CAR T cell treatment with low dose chemotherapy drug to improve the treatment efficacy. Methods: CAR gene was transduced into human T cells using retroviral based technique. Cytotoxicity was evaluated using co-culture method with varying T cell to target cell ratios followed by MTT and LDH assay. Released IFNy, granzyme B and Perforin were measured by flow cytometry and ELISA. To investigate the mechanism involved in resistance of some PDA cells, cancer cells and T cells marker were evaluated by flow cytometry and gene expression profile of sensitive vs resistant cancer cells was assessed by Real-time PCR. Results: tMUC1-CAR-T cells show increased activation and proliferation compared to normal T cells. Engineered tMUC1-CAR T cells exhibit binding and robust cytotoxicity against a panel of PDA cell lines, associated with high IFNγ, granzyme B and Perforin release. Fortunately, the same CAR T cells display minimum binding and toxicity against normal epithelial cells. CAR T cell function is evaluated in the preclinical mouse model of PDA, as single treatment and in combination with chemotherapy drugs. Several genes expression altered in cancer cells before and after treating with CAR T cells. Combining the CAR T cells with low dose chemotherapy drug (Gemcitabine and 5-FU) improved efficacy of the treatment for resistant PDA cells. Conclusion: Our results validate the idea of using anti MUC1 CAR T cells to treat pancreatic ductal adenocarcinoma. This therapy has shown to be working synergistically with low dose chemotherapy drug with improved function in treating resistant PDA cells. Hence, tMUC1 CAR T cells have the potential to be further developed for clinical use on resistant PDA. Citation Format: Mahboubeh Yazdanifar, Ru Zhou, Shu-ta Wu, Priyanka Grover, Pinku Mukherjee. Developing a novel engineered T cell to target resistant pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 584.

ABC transporters are the active transport systems of the cell involved in the export or import of a wide variety of molecules. We discovered that a member of the ABC transporter family called ABCC3 has a critical role in pancreatic cancer. ABCC3 blockade using genetic knockdown inhibits pancreatic cancer growth in vitro and in vivo. In addition, we demonstrate that knockdown of ABCC3 reduces cell proliferation by inhibition of STAT3 and HIF1α signalling pathways, which are key regulators of pancreatic cancer progression. A focused chemical library of indenes was screened for ABCC3 inhibition using ABCC3 expressing pancreatic tumour cells. A drug development candidate, designated as S3, emerged following extensive chemical modification to optimize target selectivity and oral bioavailability. Oral administration of S3 significantly inhibited tumour growth and increased survival in several mouse models of pancreatic cancer without discernible toxicity. Interestingly, using the KPC transgenic mouse model that closely mimics human pancreatic cancer, we identified a dual activity of S3 to inhibit the growth of the primary tumour and impact the surrounding stroma. Strikingly, a significant increase in survival was achieved with S3 treatment compared to vehicle treated KPC mice. A two-fold increase in lifespan was observed from 72.5 days (median survival) in the control group to 146.5 days in the treatment group. Importantly, we observed no overt toxicity from S3 treatment at a dosage of 50 mg/kg, which generated plasma levels exceeding growth inhibitory IC50 values. Furthermore, we show that stromal cells in pancreatic tumours, which actively participate in cancer progression, are enriched for ABCC3, and that its inhibition may contribute to stroma reprogramming. In other studies, we found that S3 inhibits the closely related transporter, ABCC1, and that pharmacological inhibition of ABCC1 reduced prostate cancer cell growth in vitro and potentiated the effects of Docetaxel in vitro and in mouse models of prostate cancer in vivo. Mechanistically, we have shown that ABCC3, is overexpressed in pancreatic cancer cells and can efflux the bioactive lipid lysophosphatidylinositol (LPI) which, in turn, activates its receptor G protein-coupled receptor 55 in an autocrine mitogenic loop. Similarly, ABCC1 mediates LPI efflux in prostate cancer cells. The fact that both ABCC1 and ABCC3 transport LPI and are inhibited by S3 is not surprising considering that they share a high primary sequence identity and are known to have overlapping substrate specificity. Interestingly, unlike known ABC inhibitors, S3 has anticancer activity as a single agent. Our goal is to further study the antitumor activity of S3 alone and in combination with conventional chemotherapy or molecular targeted drugs used for the treatment of pancreatic and prostate cancer. Citation Format: Marco Falasca, Xi Chen, Gary Piazza. Novel ABC transport inhibitor as a treatment for pancreatic and prostate cancers [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 1814.

Pancreatic cancer (PC) remains one of the most challenging cancers and has the worst prognosis. Tumor-associated MUC1 (tMUC1) is overexpressed and aberrantly glycosylated in over 80% of human pancreatic ductal adenocarcinoma (PDA). Chimeric antigen receptor (CAR) engineered T cells are an emerging cancer immunotherapy strategy and recently, we successfully engineered tMUC1-specific human and mouse CAR T cells and demonstrated their effectiveness as monotherapy against PDA in vitro and in vivo. In this study, we observed varying sensitivity among human PDA cell lines in response to tMUC1-targeted CAR T cell cytolysis. Notably, highly resistant HPAFII cells released greater amounts of interferon (IFN)-regulated ICAM-1, CXCL10, and CXCL11 compared to the more sensitive MiaPaCa-2 cells following CAR T cell challenge. Blocking IFN signaling using Ruxolitinib, a JAK1/2 inhibitor (JAKi), significantly reduced the upregulation of ICAM-1 and CXCL10. Western blot analysis revealed that both type I and type II IFN signaling pathways were elevated in PDA cells upon CAR T cell treatment. JAKi effectively suppressed this signaling response, with a more pronounced impact on the type I IFN pathway. Importantly, both IFN blockade and transient knockdown of IFN receptors significantly enhanced the sensitivity of PDA cells to CAR T cell-mediated cytolysis in vitro. Further mechanistic study revealed that CAR T cells partially lose their cytolytic potential after engaging with PDA cells. Treatment with CAR T cells triggered the up-regulation of immune checkpoint PD-L1 expression on PDA cells via tumor cell’ own IFN signaling. Thus, blocking PD-L1 in HPAFII enhanced its response to CAR T cells. Similarly, neutralizing CXCL10 enhanced CAR T cell killing of HPAFII cells suggested CXCL10’s involvement in resistance to CAR T cell cytolysis. RNA-seq data indicated higher expression of multiple genes along the IFN signaling pathway which were associated with poor prognosis in PDA patients. Taken together, tumor intrinsic IFN signaling may drive immune evasion in PDA cells against tMUC1-targeted T cell-mediated immunotherapy. This identifies tumor IFN signaling as a potential therapeutic target to improve CAR T cell efficacy in PDA treatment.

Background: Pancreatic cancer is the 4th leading cause of cancer deaths in the US with very poor prognosis. Treatment options are limited to surgery and chemo/radiation that often times do not increase survival and are associated with high toxicity. Targeted immune-based therapies have shown some promise but needs further exploration. Mucin 1 (MUC1), a glycoprotein expressed on the apical surface of epithelial cells of most epithelial organs, undergoes hypoglycozylation in tumors. This tumor-form of MUC1 (tMUC1) is over-expressed in 80% of pancreatic ductal adenocarcinomas (PDAC). tMUC1 therefore remains a promising target for therapeutic intervention. We have developed a patented antibody (TAB004) which specifically detects tMUC1 and spares normal MUC1. Using a novel technology, functional fragments of TAB004 antibody (scFv) were incorporated into the chimeric antigen receptor (CAR) construct and used to genetically modify primary human T cells. ScFv domain which recognizes tMUC1 is linked to the co-stimulatory molecules of T cells (CD28 and CD3ζ). When the engineered T cells come in contact with tMUC1 expressing tumor cells, multiple T cell signaling pathways are initiated leading to fully activated cytotoxic T cells that lyse the tumor cells. Methods: Retroviral based technique was used to deliver the CAR gene into human PBMC derived primary T cells. A fluorescent tag (mKate) was fused to the C-terminus of CAR molecules, in order to visualize CAR expression on T cell membrane by fluorescent microscopy and potentially for in vivo tracking. Cytotoxicity was evaluated using co-culture method with varying T cell to target cell ratios followed by MTT assay. Intracellular IFNγ was measured by flow cytometry. Results: tMUC1-CAR-T cells show increased activation and proliferation compared to normal T cells. These cells bind strongly to tMUC1 expressing human pancreatic cancer cells forming immunologic synapse. Minimal binding of the tMUC1-CAR T cells was observed to normal or low MUC1 expressing tumors cells suggesting high specificity of these CAR T cells to tMUC1. CAR expression was distributed evenly on the cell surface of the T cells. Engineered tMUC1-CAR T cells exhibit robust cytotoxicity against a panel of PDA cell lines, associated with high IFNγ release. Fortunately, the same CAR T cells display minimum toxicity against normal epithelial cells. CAR T cell function will be evaluated in the preclinical mouse model of PDA, as single treatment and also in combination with checkpoint inhibitors and chemotherapy drugs. Conclusion: Despite the remarkable successes reported using CAR T cells in clinic, particularly CD19 CAR T for leukemia; some adverse effects have been attributed to this treatment. This highlights the urgent need for developing tumor-specific CAR T cells. This study demonstrates the specificity and effectiveness of tMUC1-CAR T cells against pancreatic cancer cells. Thus, tMUC1 CAR T cells have the potential to be further developed for future clinical use. Citation Format: Mahboubeh Yazdanifar, Ru Zhou, Shu-ta Wu, Priyanka Grover, Pinku Mukherjee. A novel anti-MUC1 CAR T cell drives immunity to pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4708. doi:10.1158/1538-7445.AM2017-4708

T cells genetically engineered to express a chimeric antigen receptor (CAR) specific for the molecule CD19 have achieved significant gains in the treatment of chemotherapy-resistant leukemia and lymphoma. Despite evidence of long-lived cures in patients with metastatic solid cancer after receiving tumor-specific T cells, no CAR-T cell treatment is currently approved for use against any solid cancer. A large majority of solid cancers express one of many tumor-associated antigens (TAAs) that are vulnerable to CAR T cell recognition. However, in nearly all cases those same TAAs are present in healthy tissues. Since CAR T cells cannot differentiate between cancer and normal cells, CAR T cell recognition of TAAs on healthy tissues provokes dose-limiting ‘on-target off-tumor’ toxicities at sites of endogenous expression (lung, intestine). Thus, there remains a pressing need to develop strategies to mitigate off-tumor toxicity of CAR T cells. To this end, we develop two immunocompetent models of CAR T cell transfer targeting TAAs abundant in metastatic colorectal cancer and pancreatic cancer. TAA-specific CAR T cells affected tumor shrinkage along with infiltrating and damaging sites of endogenous expression (primarily lung, pancreas, & colon). In both of our models, off-tumor toxicities were antigen-dependent and resulted in tissue damage, systemic inflammation, and host death that was titratable to CAR-T cell dose. TAA-specific CAR T cells infiltrated sites of endogenous expression such as the intestine, pancreas, and lungs. Notably, the respiratory system was the site of greatest pathology in both of our models, with massive CAR T cell accumulation and tissue destruction that resulted in systemic wasting and rapid weight loss. Remarkably, we found that the presence of an antigen-bearing tumor in the liver, but not other sites, greatly exacerbated CAR-T cell pulmonary infiltration, tissue destruction, and host mortality.The liver is the most common site of metastasis in patients with colorectal and pancreatic adenocarcinoma and the only site of metastasis in one-third of colorectal cancers. Our models indicated that TAA-specific CAR-T cells given for the treatment of liver metastasis result in dose-limiting pulmonary toxicity due to the presence of the TAA in the healthy lung. To address this translational barrier, we designed and validated a multiparameter genetic reprogramming targeting T cell trafficking and tissue residency introduced with the CAR to imbue CAR-T cells with liver tropism. In multiple models, CAR T cells programmed with liver-specific tropism augmented CAR T cell liver trafficking, decreased lung infiltration and toxicity, and effectively treated TAA+ liver metastasis. We aim to initiate a pilot clinical trial using tissue resident-programmed CAR T cells to treat colorectal liver metastasis. Citation Format: Alex Bartlett, Matthew Mcwhorter, Camille Collier, Chelsea Jenkins, Robert Eil. A novel approach to limit off-tumor toxicity of CAR-T cells targeting solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB344.