scholarly journals Pre-clinical validation of a novel pan-cancer CAR-T cell immunotherapy against nfP2X7

2021 ◽  
Author(s):  
Veronika Bandara ◽  
Jade Foeng ◽  
Batjargal Gundsambuu ◽  
Todd Norton ◽  
Silvana Napoli ◽  
...  
Keyword(s):  
T Cells ◽  
Clinical Activity ◽  
Solid Cancer ◽  
Multiple Cancer ◽  
Car T Cells ◽  
Car T ◽  
Specific Cytotoxicity ◽  
Cancer Types ◽  
Breast And Prostate Cancer

Abstract Chimeric antigen receptor (CAR)-T immunotherapy is a novel treatment that genetically modifies the patient’s own T cells to target and kill malignant cells. CAR-T cells demonstrated robust clinical activity against certain B-cell malignancies. However, identification of tumour-specific antigens expressed on multiple cancer types, especially on solid cancers, remains a major challenge. P2X purinoceptor 7 (P2X7) is an ATP gated cation channel that forms homotrimers and heterotrimers at the cell surface. When functioning normally, it controls ion transport in response to ATP. A dysfunctional version of P2X7, named nfP2X7, has been identified on cancer cells from a range of tissues, while being undetectable on healthy cells. We generated prototype nfP2X7-targeting human CAR-T cells, which demonstrated effective antigen-specific cytotoxicity against twelve solid cancer types including breast, prostate, lung, colorectal, brain and skin in vitro. In preclinical xenograft mouse models of aggressive breast and prostate cancer, CAR-T cells targeting nfP2X7 exhibited robust anti-tumour efficacy. These data indicate CAR-T cells targeting nfP2X7 have potential as a novel broad-spectrum cancer immunotherapy for solid tumours in humans.

scholarly journals Reducing Hinge Flexibility of CAR-T Cells Prolongs Survival In Vivo With Low Cytokines Release

2021 ◽  
Vol 12 ◽  
Author(s):  
Ang Zhang ◽  
Yao Sun ◽  
Jie Du ◽  
Yansheng Dong ◽  
Honggang Pang ◽  
...  
Keyword(s):  
T Cells ◽  
Similar Efficacy ◽  
Tumor Burden ◽  
Car T Cells ◽  
High Tumor Burden ◽  
Car T ◽  
Specific Cytotoxicity ◽  
Hinge Domain

Chimeric antigen receptor (CAR)-modified T cells targeting CD19 demonstrate unparalleled responses in B cell malignancies. However, high tumor burden limits clinical efficacy and increases the risk of cytokine release syndrome and neurotoxicity, which is associated with over-activation of the CAR-T cells. The hinge domain plays an important role in the function of CAR-T cells. We hypothesized that deletion of glycine, an amino acid with good flexibility, may reduce the flexibility of the hinge region, thereby mitigating CAR-T cell over-activation. This study involved generating a novel CAR by deletion of two consecutive glycine residues in the CD8 hinge domain of second-generation (2nd) CAR, thereafter named 2nd-GG CAR. The 2nd-GG CAR-T cells showed similar efficacy of CAR expression but lower hinge flexibility, and its protein affinity to CD19 protein was lower than that of 2nd CAR-T cells. Compared to the 2nd CAR-T cells, 2nd-GG CAR-T cells reduced proinflammatory cytokine secretion without diminishing the specific cytotoxicity toward tumor cells in vitro. Furthermore, 2nd-GG CAR-T cells prolonged overall survival in an immunodeficient mouse model bearing NALM-6 when tumor burden was high. This study demonstrated that a lower-flexibility of CD8α hinge improved survival under high tumor burden and reduced proinflammatory cytokines in preclinical studies. While there is potential for improved safety and efficacy, yet this needs validation with clinical trials.

scholarly journals Fast, efficient and virus-free generation of TRAC-replaced CAR T cells

2021 ◽  
Author(s):  
Jonas Kath ◽  
Weijie Du ◽  
Bernice Thommandru ◽  
Rolf Turk ◽  
Leila Amini ◽  
...  
Keyword(s):  
T Cells ◽  
Gene Editing ◽  
Cell Receptor ◽  
Homology Directed Repair ◽  
Car T Cells ◽  
Human T Cells ◽  
Efficient Gene ◽  
Car T ◽  
Specific Cytotoxicity

AbstractChimeric Antigen Receptor (CAR) redirected T cells are a potent treatment option for certain hematological malignancies. Recently, site-specific insertion of CARs into the T cell receptor (TCR) alpha constant (TRAC) locus using gene editing and adeno-associated viruses was shown to generate CAR T cells with improved functionality over their retrovirally transduced counterparts. However, the development of viruses for gene transfer is complex and associated with extensive costs at early clinical stages. Here, we provide an economical and virus-free method for efficient CAR insertion into the TRAC locus of primary human T cells via CRISPR-Cas mediated homology-directed repair (HDR). While the toxicity induced by transfected double-stranded template (donor) DNA was not fully prevented by pharmacological means, the combination of DNA-sensor inhibitors and HDR enhancers resulted in highly efficient gene editing with TCR-to-CAR replacement rates reaching up to 68%. The resulting TCR-deficient CAR T cells show antigen-specific cytotoxicity and cytokine production in vitro. Our GMP-compatible non-viral platform technology lays the foundation for clinical trials and fast-track generation of novel CAR T cells applicable for autologous or allogeneic off-the-shelf use.

scholarly journals Generation of Multiplexed Engineered, Off-the-Shelf CAR T Cells Uniformly Carrying Multiple Anti-Tumor Modalities to Prevent Tumor Relapse

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-11
Author(s):  
Chiawei Chang ◽  
Eigen Peralta ◽  
Gloria Hsia ◽  
Bi-Huei Yang ◽  
Wen-I Yeh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fold Increase ◽  
Cell Receptor ◽  
Control Group ◽  
Antigen Specificity ◽  
Car T Cells ◽  
Car T ◽  
Specific Cytotoxicity

The development of chimeric antigen receptor (CAR) T cell therapeutics is widely recognized as a significant advancement for the treatment of cancer. However, several obstacles currently impede the broad use of CAR T cells, including the inherent process variability, cost of manufacturing, the absolute requirement for precise and uniform genetic editing in the allogeneic setting, and the challenge to keep pace with clonal heterogeneity and tumor growth. Utilizing our previously described induced pluripotent stem cell (iPSC)-derived T (iT) cell platform, we illustrate here the unique ability to address these challenges by creating a consistent CAR iT cell product that can be repeatedly manufactured in large quantities from a renewable iPSC master cell bank that has been engineered to mitigate the occurrence of graft versus host disease (GvHD), antigen escape and tumor relapse. Utilizing our proprietary cellular reprogramming and engineering platform and stage-specific T cell differentiation protocol, we demonstrate that iPSC can be engineered at the single cell level to generate a fully characterized clonal iPSC line, which can then be accessed routinely to yield CAR iT cells in a highly scalable manufacturing process (>100,000 fold expansion). Through bi-allelic targeting of a CAR into the T cell receptor alpha constant (TRAC) region, we generated CAR iT cells with uniform CAR expression (99.0 ± 0.5% CAR+) and complete elimination of T cell receptor (TCR) expression to avoid GvHD in the allogeneic setting. We elected to utilize the 1XX-CAR configuration, which has demonstrated superior anti-tumor performance relative to other CAR designs and when introduced into iT cells displayed enhanced antigen specificity (% specific cytotoxicity at E:T=10:1, antigen positive group: 86.4 ± 7.8; antigen null group: 8.9 ± 3.5). To enhance persistence without reliance on exogenous cytokine support, we engineered signaling-fusion complexes, including IL-7 receptor fusion (RF), into iPSC and studied its impact on iT phenotype, persistence, and efficacy. In vitro, IL-7RF clones demonstrated improved anti-tumor activity in a serial antigen dependent tumor challenge assay (Day 10, relative tumor counts, IL-7RF group: 1.95 ± 0.01; control group: 57.56 ± 4.55, P<0.000001). In a preclinical in vivo model of disseminated leukemia, IL-7RF clones demonstrate enhanced tumor growth inhibition (Day 34, Log [BLI], IL-7RF group: 6.68 ± 1.93; control group: 9.99 ± 0.23, P=0.0143). We next investigated a unique strategy to incorporate multi-antigen targeting potential into anti-CD19 1XX CAR iT cells with the addition of a high-affinity non-cleavable CD16 (hnCD16) Fc receptor. The combination of hnCD16 with anti-CD19 1XX CAR culminated in iT cells capable of multi-antigen specificity through combinatorial use with monoclonal antibodies to tackle antigen escape. Utilizing CD19 negative leukemia cells as targets, superior antibody-dependent cellular cytotoxicity (ADCC) was demonstrated by the combination of hnCD16 CAR iT and Rituximab (% specific cytotoxicity at E:T=1:1, hnCD16 group + Rituximab: 75.64 ± 2.12; control group + Rituximab: 16.98 ± 3.87, P<0.001). To address T cell fitness, the role of CD38 knockout (KO) in T cells was investigated, which we have previously shown to mediate NK cell resistance to oxidative stress induced apoptosis. CD38 gene was disrupted at the iPSC stage to generate 1XX-CAR T cells that lack CD38 expression (% CD38+ population, CD38WT group: 69.67 ± 24.34; CD38KO group: 0.12 ± 0.11) and upon antigen mediated stimulation, CD38KO CAR iT cells showed higher percentages of degranulation (2.3-fold increase in CD107a/b), and IFNγ (4.1-fold increase) and TNFα (2.5-fold increase) production. Antigen specific in vitro tumor killing also was enhanced in CD38KO CAR iT cells (EC50, 3.2-fold decrease). Lastly, to avoid the potential host-mediated rejection, the inclusion of allogeneic defense receptor (ADR) which has been shown to significantly reduce host-mediated rejection will be discussed. Collectively, the described studies demonstrate that iPSCs are an ideal cellular source to generate large-quantities of uniformly multi-edited off-the-shelf CAR T cell products that include a best-in-class CAR design, enhanced product modalities, and complete elimination of TCR expression to avoid the potential of GvHD while maintaining high anti-tumor efficacy in allogeneic setting. Disclosures Hsia: Fate Therapeutics Inc.: Current Employment. Clarke:Fate Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Lee:Fate Therapeutics, Inc.: Current Employment. Robbins:Fate Therapeutics, Inc.: Current Employment. Denholtz:Fate Therapeutics, Inc: Current Employment. Hanok:Fate Therapeutics, Inc.: Current Employment. Carron:Fate Therapeutics, Inc.: Current Employment. Navarrete:Fate Therapeutics, Inc.: Current Employment. ORourke:Fate Therapeutics, Inc.: Current Employment. Sung:Fate Therapeutics, Inc.: Current Employment. Gentile:Fate Therapeutics, Inc.: Current Employment. Nguyen:Fate Therapeutics, Inc.: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.

scholarly journals 549 Design and in vitro efficacy of TRP-1 CAR T cells to target melanoma

2021 ◽  
Vol 141 (5) ◽  
pp. S95
Author(s):  
R.S. Shivde ◽  
D. Jaishankar ◽  
A. Thomas ◽  
I. Le Poole
Keyword(s):  
T Cells ◽  
Car T Cells ◽  
Car T

98 ATA3271: an armored, next-generation off-the-shelf, allogeneic, mesothelin-CAR T cell therapy for solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A109-A109
Author(s):  
Jiangyue Liu ◽  
Xianhui Chen ◽  
Jason Karlen ◽  
Alfonso Brito ◽  
Tiffany Jheng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Next Generation ◽  
Phase 3 ◽  
T Cell Therapy ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T

BackgroundMesothelin (MSLN) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein with high expression levels in an array of malignancies including mesothelioma, ovaria, non-small cell lung cancer, and pancreatic cancers and is an attractive target antigen for immune-based therapies. Early clinical evaluation of autologous MSLN-targeted chimeric antigen receptor (CAR)-T cell therapies for malignant pleural mesothelioma has shown promising acceptable safety1 and have recently evolved with incorporation of next-generation CAR co-stimulatory domains and armoring with intrinsic checkpoint inhibition via expression of a PD-1 dominant negative receptor (PD1DNR).2 Despite the promise that MSLN CAR-T therapies hold, manufacturing and commercial challenges using an autologous approach may prove difficult for widespread application. EBV T cells represent a unique, non-gene edited approach toward an off-the-shelf, allogeneic T cell platform. EBV-specific T cells are currently being evaluated in phase 3 trials [NCT03394365] and, to-date, have demonstrated a favorable safety profile including limited risks for GvHD and cytokine release syndrome.3 4 Clinical proof-of-principle studies for CAR transduced allogeneic EBV T cell therapies have also been associated with acceptable safety and durable response in association with CD19 targeting.5 Here we describe the first preclinical evaluation of ATA3271, a next-generation allogeneic CAR EBV T cell therapy targeting MSLN and incorporating PD1DNR, designed for the treatment of solid tumor indications.MethodsWe generated allogeneic MSLN CAR+ EBV T cells (ATA3271) using retroviral transduction of EBV T cells. ATA3271 includes a novel 1XX CAR signaling domain, previously associated with improved signaling and decreased CAR-mediated exhaustion. It is also armored with PD1DNR to provide intrinsic checkpoint blockade and is designed to retain functional persistence.ResultsIn this study, we characterized ATA3271 both in vitro and in vivo. ATA3271 show stable and proportional CAR and PD1DNR expression. Functional studies show potent antitumor activity of ATA3271 against MSLN-expressing cell lines, including PD-L1-high expressors. In an orthotopic mouse model of pleural mesothelioma, ATA3271 demonstrates potent antitumor activity and significant survival benefit (100% survival exceeding 50 days vs. 25 day median for control), without evident toxicities. ATA3271 maintains persistence and retains central memory phenotype in vivo through end-of-study. Additionally, ATA3271 retains endogenous EBV TCR function and reduced allotoxicity in the context of HLA mismatched targets. ConclusionsOverall, ATA3271 shows potent anti-tumor activity without evidence of allotoxicity, both in vitro and in vivo, suggesting that allogeneic MSLN-CAR-engineered EBV T cells are a promising approach for the treatment of MSLN-positive cancers and warrant further clinical investigation.ReferencesAdusumilli PS, Zauderer MG, Rusch VW, et al. Abstract CT036: A phase I clinical trial of malignant pleural disease treated with regionally delivered autologous mesothelin-targeted CAR T cells: Safety and efficacy. Cancer Research 2019;79:CT036-CT036.Kiesgen S, Linot C, Quach HT, et al. Abstract LB-378: Regional delivery of clinical-grade mesothelin-targeted CAR T cells with cell-intrinsic PD-1 checkpoint blockade: Translation to a phase I trial. Cancer Research 2020;80:LB-378-LB-378.Prockop S, Doubrovina E, Suser S, et al. Off-the-shelf EBV-specific T cell immunotherapy for rituximab-refractory EBV-associated lymphoma following transplantation. J Clin Invest 2020;130:733–747.Prockop S, Hiremath M, Ye W, et al. A Multicenter, Open Label, Phase 3 Study of Tabelecleucel for Solid Organ Transplant Subjects with Epstein-Barr Virus-Driven Post-Transplant Lymphoproliferative Disease (EBV+PTLD) after Failure of Rituximab or Rituximab and Chemotherapy. Blood 2019; 134: 5326–5326.Curran KJ, Sauter CS, Kernan NA, et al. Durable remission following ‘Off-the-Shelf’ chimeric antigen receptor (CAR) T-Cells in patients with relapse/refractory (R/R) B-Cell malignancies. Biology of Blood and Marrow Transplantation 2020;26:S89.

scholarly journals Nanoparticle T-cell engagers as a modular platform for cancer immunotherapy

Leukemia ◽  
2021 ◽  
Author(s):  
Kinan Alhallak ◽  
Jennifer Sun ◽  
Katherine Wasden ◽  
Nicole Guenthner ◽  
Julie O’Neal ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Escape ◽  
Multiple Cancer ◽  
Cancer Antigen ◽  
Cancer Antigens ◽  
Car T ◽  
Modular Platform

AbstractT-cell-based immunotherapy, such as CAR-T cells and bispecific T-cell engagers (BiTEs), has shown promising clinical outcomes in many cancers; however, these therapies have significant limitations, such as poor pharmacokinetics and the ability to target only one antigen on the cancer cells. In multiclonal diseases, these therapies confer the development of antigen-less clones, causing tumor escape and relapse. In this study, we developed nanoparticle-based bispecific T-cell engagers (nanoBiTEs), which are liposomes decorated with anti-CD3 monoclonal antibodies (mAbs) targeting T cells, and mAbs targeting the cancer antigen. We also developed a nanoparticle that targets multiple cancer antigens by conjugating multiple mAbs against multiple cancer antigens for T-cell engagement (nanoMuTEs). NanoBiTEs and nanoMuTEs have a long half-life of about 60 h, which enables once-a-week administration instead of continuous infusion, while maintaining efficacy in vitro and in vivo. NanoMuTEs targeting multiple cancer antigens showed greater efficacy in myeloma cells in vitro and in vivo, compared to nanoBiTEs targeting only one cancer antigen. Unlike nanoBiTEs, treatment with nanoMuTEs did not cause downregulation (or loss) of a single antigen, and prevented the development of antigen-less tumor escape. Our nanoparticle-based immuno-engaging technology provides a solution for the major limitations of current immunotherapy technologies.

scholarly journals Immunotherapy using IgE or CAR T cells for cancers expressing the tumor antigen SLC3A2

2021 ◽  
Vol 9 (6) ◽  
pp. e002140
Author(s):  
Giulia Pellizzari ◽  
Olivier Martinez ◽  
Silvia Crescioli ◽  
Robert Page ◽  
Ashley Di Meo ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
T Cells ◽  
T Cell ◽  
Type I ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T Cell ◽  
Tumor Associated Antigen ◽  
Car T

BackgroundCancer immunotherapy with monoclonal antibodies and chimeric antigen receptor (CAR) T cell therapies can benefit from selection of new targets with high levels of tumor specificity and from early assessments of efficacy and safety to derisk potential therapies.MethodsEmploying mass spectrometry, bioinformatics, immuno-mass spectrometry and CRISPR/Cas9 we identified the target of the tumor-specific SF-25 antibody. We engineered IgE and CAR T cell immunotherapies derived from the SF-25 clone and evaluated potential for cancer therapy.ResultsWe identified the target of the SF-25 clone as the tumor-associated antigen SLC3A2, a cell surface protein with key roles in cancer metabolism. We generated IgE monoclonal antibody, and CAR T cell immunotherapies each recognizing SLC3A2. In concordance with preclinical and, more recently, clinical findings with the first-in-class IgE antibody MOv18 (recognizing the tumor-associated antigen Folate Receptor alpha), SF-25 IgE potentiated Fc-mediated effector functions against cancer cells in vitro and restricted human tumor xenograft growth in mice engrafted with human effector cells. The antibody did not trigger basophil activation in cancer patient blood ex vivo, suggesting failure to induce type I hypersensitivity, and supporting safe therapeutic administration. SLC3A2-specific CAR T cells demonstrated cytotoxicity against tumor cells, stimulated interferon-γ and interleukin-2 production in vitro. In vivo SLC3A2-specific CAR T cells significantly increased overall survival and reduced growth of subcutaneous PC3-LN3-luciferase xenografts. No weight loss, manifestations of cytokine release syndrome or graft-versus-host disease, were detected.ConclusionsThese findings identify efficacious and potentially safe tumor-targeting of SLC3A2 with novel immune-activating antibody and genetically modified cell therapies.

scholarly journals 114 Preclinical development of a novel iPSC-derived CAR-MICA/B NK cell immunotherapy to overcome solid tumor escape from NKG2D-mediated mechanisms of recognition and killing

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A126-A126
Author(s):  
John Goulding ◽  
Mochtar Pribadi ◽  
Robert Blum ◽  
Wen-I Yeh ◽  
Yijia Pan ◽  
...  
Keyword(s):  
T Cells ◽  
Cancer Immunotherapy ◽  
Tumor Immunity ◽  
Immune Cell ◽  
Nk Cell ◽  
Tumor Escape ◽  
Car T Cells ◽  
Car T

BackgroundMHC class I related proteins A (MICA) and B (MICB) are induced by cellular stress and transformation, and their expression has been reported for many cancer types. NKG2D, an activating receptor expressed on natural killer (NK) and T cells, targets the membrane-distal domains of MICA/B, activating a potent cytotoxic response. However, advanced cancer cells frequently evade immune cell recognition by proteolytic shedding of the α1 and α2 domains of MICA/B, which can significantly reduce NKG2D function and the cytolytic activity.MethodsRecent publications have shown that therapeutic antibodies targeting the membrane-proximal α3 domain inhibited MICA/B shedding, resulting in a substantial increase in the cell surface density of MICA/B and restoration of immune cell-mediated tumor immunity.1 We have developed a novel chimeric antigen receptor (CAR) targeting the conserved α3 domain of MICA/B (CAR-MICA/B). Additionally, utilizing our proprietary induced pluripotent stem cell (iPSC) product platform, we have developed multiplexed engineered, iPSC-derived CAR-MICA/B NK (iNK) cells for off-the-shelf cancer immunotherapy.ResultsA screen of CAR spacer and ScFv orientations in primary T cells delineated MICA-specific in vitro activation and cytotoxicity as well as in vivo tumor control against MICA+ cancer cells. The novel CAR-MICA/B design was used to compare efficacy against NKG2D CAR T cells, an alternative MICA/B targeting strategy. CAR-MICA/B T cells showed superior cytotoxicity against melanoma, breast cancer, renal cell carcinoma, and lung cancer lines in vitro compared to primary NKG2D CAR T cells (p<0.01). Additionally, using an in vivo xenograft metastasis model, CAR-MICA/B T cells eliminated A2058 human melanoma metastases in the majority of the mice treated. In contrast, NKG2D CAR T cells were unable to control tumor growth or metastases. To translate CAR-MICA/B functionality into an off-the-shelf cancer immunotherapy, CAR-MICA/B was introduced into a clonal master engineered iPSC line to derive a multiplexed engineered, CAR-MICA/B iNK cell product candidate. Using a panel of tumor cell lines expressing MICA/B, CAR-MICA/B iNK cells displayed MICA specificity, resulting in enhanced cytokine production, degranulation, and cytotoxicity. Furthermore, in vivo NK cell cytotoxicity was evaluated using the B16-F10 melanoma cell line, engineered to express MICA. In this model, CAR-MICA/B iNK cells significantly reduced liver and lung metastases, compared to untreated controls, by 93% and 87% respectively.ConclusionsOngoing work is focused on extending these preclinical studies to further support the clinical translation of an off-the-shelf, CAR-MICA/B iNK cell cancer immunotherapy with the potential to overcome solid tumor escape from NKG2D-mediated mechanisms of recognition and killing.ReferenceFerrari de Andrade L, Tay RE, Pan D, Luoma AM, Ito Y, Badrinath S, Tsoucas D, Franz B, May KF Jr, Harvey CJ, Kobold S, Pyrdol JW, Yoon C, Yuan GC, Hodi FS, Dranoff G, Wucherpfennig KW. Antibody-mediated inhibition of MICA and MICB shedding promotes NK cell-driven tumor immunity. Science 2018 Mar 30;359(6383):1537–1542.

105 A third-generation human GUCY2C-targeted CAR-T cell for colorectal cancer immunotherapy

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A116-A116
Author(s):  
Trevor Baybutt ◽  
Adam Snook ◽  
Scott Waldman ◽  
Jonathan Stem ◽  
Ellen Caparosa ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
T Cells ◽  
Cancer Cells ◽  
Inflammatory Cytokines ◽  
Department Of Defense ◽  
Thomas Jefferson ◽  
Third Generation ◽  
Car T Cells ◽  
Car T

BackgroundColorectal cancer (CRC) presents a significant public health burden, responsible for the second most cancer-related deaths in the United States, with an increasing incidence in young adults observed globally.1,2 While the blockade of immune checkpoints received FDA approval as a CRC therapeutic, only patients with microsatellite instability, accounting for 15% of sporadic cases, demonstrate partial or complete responses.3 We present a third-generation chimeric antigen receptor (CAR)-T cell directed towards the extracellular domain of the mucosal antigen guanylyl cyclase C (GUCY2C), which is over-expressed in 80% of CRC cases, as a therapeutic alternative for late stage disease. Here, we demonstrate that human GUCY2C CAR-T cells can selectively kill GUCY2C-expressing colorectal cancer cells in vitro and produce inflammatory cytokines in response to antigenic stimulation.MethodsPeripheral blood mononuclear (PBMCs) cells were isolated from leukoreduction filters obtained from the Thomas Jefferson University Hospital Blood Donor Center (IRB #18D.495). Magnetic Activated Cell Sorting (MACS) technology was used to negatively select pan-T cells (Miltenyi Biotec), followed by activation and expansion using anti-CD3, anti-CD28, and anti-CD2 coated microbeads (Miltenyi Biotec) and supplemented with IL-7 and IL-15 (Biological Resources Branch Preclinical Biologics Repository – NCI). T-cells were transduced with a lentiviral vector encoding the anti-GUCY2C CAR. Our CAR utilizes a single chain variable fragment of human origin directed towards the extracellular domain of GUCY2C, the CD28 hinge, transmembrane, and intracellular signaling domain (ICD), 4-1BB (CD137) ICD, and CD3ζ ICD. CAR-T cells were used for experiments between 10 to 14 days after activation in vitro using the xCELLigence real time cytotoxicity assay and intracellular cytokine staining.ResultsGUCY2C-directed CAR-T cells specifically lysed the GUCY2C-expressing metastatic CRC cell line T84, while the control CAR did not. GUCY2C-negative CRC cells were not killed by either. In addition to cell killing, GUCY2C-directed CAR-T cells of both the CD8+ and CD4+ co-receptor lineage produced the inflammatory cytokines IFN-γ and TNFα in response to GUCY2C antigen.ConclusionsWe demonstrate that human GUCY2C-directed CAR-T cells can selectively target GUCY2C-expressing cancer cells. We hypothesize that GUCY2C-directed CAR-T cells present a viable therapeutic option for metastatic CRC. In vivo animal models to examine this potential are currently on-going.AcknowledgementsThis work was supported by the Department of Defense Congressionally Directed Medical Research Programs (W81XWH-17-1-0299, W81XWH-191-0263, and W81XWH-19-1-0067) to AES and Targeted Diagnostic & Therapeutics to SAW. AES is also supported by a DeGregorio Family Foundation Award. SAW is supported by the National Institutes of Health (NIH) (R01 CA204881, R01 CA206026, and P30 CA56036), and the Department of Defense Congressionally Directed Medical Research Program W81XWH-17-PRCRP-TTSA. SAW and AES were also supported by a grant from The Courtney Ann Diacont Memorial Foundation. SAW is the Samuel M.V. Hamilton Professor of Thomas Jefferson University. JS, EC, and AZ were supported by an NIH institutional award T32 GM008562 for Postdoctoral Training in Clinical Pharmacology.Ethics ApprovalThis study was approved by the Thomas Jefferson University Institutional Review Board (IRB Control #18D.495) and the Institutional Animal Care and Use Committee (Protocol #01529).ReferencesSiegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin2020;70: 7–30. doi:10.3322/caac.21590Araghi M, Soerjomataram I, Bardot A, Ferlay J, Cabasag CJ, Morrison DS, et al. Changes in colorectal cancer incidence in seven high-income countries: a population-based study. Lancet Gastroenterol Hepatol 2019;4: 511–518. doi:10.1016/S2468-1253(19)30147-5Overman MJ, McDermott R, Leach JL, Lonardi S, Lenz H-J, Morse MA, et al. Nivolumab in patients with metastatic DNA mismatch repair-deficient or microsatellite instability-high colorectal cancer (CheckMate 142): an open-label, multicentre, phase 2 study. Lancet Oncol 2017;18: 1182–1191. doi:10.1016/S1470-2045(17)30422-9

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