123 P-MUC1C-ALLO1: A fully allogeneic stem cell memory T cell (TSCM) CAR-T therapy with broad potential in solid tumor

BackgroundWhile CAR-T have demonstrated potent activity against hematologic tumors, less success has been seen with solid tumors. Here we report generation of TSCM-enriched allogeneic MUC1-C-specific CAR T cells, P-MUC1C-ALLO1, with potential for a broad range of solid tumors. The proliferative capacity and metabolic profile of TSCM CAR-T are well-suited to activity in the solid tumor setting. MUC1 is comprised of an N-terminal subunit (MUC1-N) tethered to a C-terminal subunit (MUC1-C), forming a stable complex on the cell surface. During tumorigenesis, MUC1 becomes both overexpressed and hypo-glycosylated on many carcinomas. Furthermore, MUC1 undergoes proteolytic cleavage in the tumor microenvironment, leaving behind a proteolytic ‘stump’ of MUC1-C that is over-represented in cancer, making it an attractive therapeutic target.MethodsP-MUC1C-ALLO1 is manufactured using the piggyBac® DNA Delivery System for CAR insertion and the Cas-CLOVER™ Gene Editing System to knockout both the TCR and MHC class I proteins. The addition of a selectable marker within the transposon allows for selection of a fully CAR-positive population while any residual TCR-positive cells are removed at the end of production to prevent TCR-mediated GvHD. Lastly, inclusion of a proprietary ‘booster molecule’ in our allogeneic process further improves cell expansion, along with phenotype and function, and enables the production of up to hundreds of patient doses from a single manufacturing run.ResultsSignificant doses of P-MUC1C-ALLO1 products made from multiple healthy donors were achieved and comprised of an exceptionally high-percentage of desirable TSCM cells. Preclinical evaluation of these products showed potent tumor killing and cytokine secretion against MUC1-C-positive breast and ovarian tumor cell lines. P-MUC1C-ALLO1 demonstrates potent cytotoxicity against tumor cells, and minimal killing of normal MUC1-C-positive human primary cells. In a triple negative breast cancer xenograft model, MUC1C CAR-T eliminated established MDA-MB-468 tumor cells, mounted robust T cell expansion in peripheral blood and maintained a favorable TSCM percentage over time. Likewise, in an orthotopic ovarian cancer xenograft model, intraperitoneally administered MUC1C CAR-T eliminated established OVCAR3 cells to levels below the limit of detection. All together, these data demonstrated the efficacy of the MUC1C CAR-T cells and the robustness of the allogeneic platform.ConclusionsP-MUC1C-ALLO1 is an allogeneic TSCM CAR-T therapy that has a potential to treat multiple MUC1-expressing indications. P-MUC1C-ALLO1 displayed specificity for tumor vs. normal cells as well as in vivo efficacy against xenograft models of breast and ovarian cancer. This allogeneic cell therapy is advancing rapidly towards the clinic.

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SynNotch CAR circuits enhance solid tumor recognition and promote persistent antitumor activity in mouse models

Science Translational Medicine ◽

10.1126/scitranslmed.abd8836 ◽

2021 ◽

Vol 13 (591) ◽

pp. eabd8836

Author(s):

Axel Hyrenius-Wittsten ◽

Yang Su ◽

Minhee Park ◽

Julie M. Garcia ◽

Josef Alavi ◽

...

Keyword(s):

Ovarian Cancer ◽

T Cells ◽

T Cell ◽

Antitumor Activity ◽

Solid Tumors ◽

Mouse Models ◽

Solid Tumor ◽

Car T Cells ◽

Therapeutic Benefits ◽

Car T

The first clinically approved engineered chimeric antigen receptor (CAR) T cell therapies are remarkably effective in a subset of hematological malignancies with few therapeutic options. Although these clinical successes have been exciting, CAR T cells have hit roadblocks in solid tumors that include the lack of highly tumor-specific antigens to target, opening up the possibility of life-threatening “on-target/off-tumor” toxicities, and problems with T cell entry into solid tumor and persistent activity in suppressive tumor microenvironments. Here, we improve the specificity and persistent antitumor activity of therapeutic T cells with synthetic Notch (synNotch) CAR circuits. We identify alkaline phosphatase placental-like 2 (ALPPL2) as a tumor-specific antigen expressed in a spectrum of solid tumors, including mesothelioma and ovarian cancer. ALPPL2 can act as a sole target for CAR therapy or be combined with tumor-associated antigens such as melanoma cell adhesion molecule (MCAM), mesothelin, or human epidermal growth factor receptor 2 (HER2) in synNotch CAR combinatorial antigen circuits. SynNotch CAR T cells display superior control of tumor burden when compared to T cells constitutively expressing a CAR targeting the same antigens in mouse models of human mesothelioma and ovarian cancer. This was achieved by preventing CAR-mediated tonic signaling through synNotch-controlled expression, allowing T cells to maintain a long-lived memory and non-exhausted phenotype. Collectively, we establish ALPPL2 as a clinically viable cell therapy target for multiple solid tumors and demonstrate the multifaceted therapeutic benefits of synNotch CAR T cells.

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131 Coupled CAR® technology strengthens adoptive T cell therapy by promoting rapid expansion

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0131 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A144-A144

Author(s):

Zhiyuan Cao ◽

Chengfei Pu ◽

Xianyang Jiang ◽

Xiaogang Shen ◽

Ruihong Zhu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

Solid Tumors ◽

Solid Tumor ◽

Cell Expansion ◽

T Cell Therapy ◽

Car T Cells ◽

Car T

BackgroundCAR 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.MethodsWe 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.ResultsWe 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.ConclusionsWe 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.

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Low CD19 Antigen Density Diminishes Efficacy of CD19 CAR T Cells and Can be Overcome By Rational Redesign of CAR Signaling Domains

Blood ◽

10.1182/blood-2018-99-115558 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 963-963 ◽

Cited By ~ 6

Author(s):

Robbie G. Majzner ◽

Skyler P. Rietberg ◽

Louai Labanieh ◽

Elena Sotillo ◽

Evan W. Weber ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Cytokine Production ◽

Xenograft Model ◽

Target Antigen ◽

Tumor Escape ◽

Car T Cells ◽

Car T Cell ◽

Car T

Abstract Target antigen density has emerged as a major factor influencing the potency of CAR T cells. Our laboratory has demonstrated that the activity of numerous CARs is highly dependent on target antigen density (Walker et al., Mol Ther, 2017), and high complete response rates in a recent trial of CD22 CAR T cells for B-ALL were tempered by frequent relapses due to decreased CD22 antigen density on lymphoblasts (Fry et al., Nat Med, 2018). To assess if antigen density is also a determinant of CD19 CAR T cell therapeutic success, we analyzed CD19 antigen density from fifty pediatric B-ALL patients treated on a clinical trial of CD19-CD28ζ CAR T cells. We found that patients whose CD19 expression was below a threshold density (2000 molecules/lymphoblast) were significantly less likely to achieve a clinical response than those whose leukemia expressed higher levels of CD19. In order to further understand this limitation and how it may be overcome, we developed a model of variable CD19 antigen density B-ALL. After establishing a CD19 knockout of the B-ALL cell line NALM6, we used a lentivirus to reintroduce CD19 and then FACS sorted and single cell cloned to achieve a library of NALM6 clones with varying CD19 surface densities. CD19-CD28ζ CAR T cell activity was highly dependent on CD19 antigen density. We observed decreases in cytotoxicity, proliferation, and cytokine production by CD19 CAR T cells when encountering CD19-low cells, with an approximate threshold of 2,000 molecules of CD19 per lymphoblast, below which, cytokine production in response to tumor cells was nearly ablated. Given that a CD19-4-1BBζ CAR is FDA approved for children with B-ALL and adults with DLBCL, we wondered whether CARs incorporating this alternative costimulatory domain would have similar antigen density thresholds for activation. Surprisingly, CD19-4-1BBζ CAR T cells made even less cytokine, proliferated less, and had further diminished cytolytic capacity against CD19-low cells compared to CD19-CD28ζ CAR T cells. Analysis by western blot of protein lysates from CAR T cells stimulated with varying amounts of antigen demonstrated that CD19-CD28ζ CAR T cells had higher levels of downstream signals such as pERK than CD19-4-1BBζ CAR T cells at lower antigen densities. Accordingly, calcium flux after stimulation was also significantly higher in CD19-CD28ζ than CD19-4-1BBζ CAR T cells. In a xenograft model of CD19-low B-ALL, CD19-4-1BBζ CAR T cells demonstrated no anti-tumor activity, while CD19-CD28ζ CAR T cells eradicated CD19-low leukemia cells. Therefore, the choice of costimulatory domain in CAR T cells plays a major role in modulating activity against low antigen density tumors. CD28 costimulation endows high reactivity towards low antigen density tumors. We confirmed the generalizability of this finding using Her2 CAR T cells; Her2-CD28ζ CAR T cells cleared tumors in an orthotopic xenograft model of Her2-low osteosarcoma, while Her2-4-1BBζ CAR T cells had no effect. This finding has implications for CAR design for lymphoma and solid tumors, where antigen expression is more heterogeneous than B-ALL. To enhance the activity of CD19-4-1BBζ CAR T cells against CD19-low leukemia, we designed a CAR with two copies of intracellular zeta in the signaling domain (CD19-4-1BBζζ). T cells expressing this double-zeta CAR demonstrated enhanced cytotoxicity, proliferation, cytokine production, and pERK signaling in response to CD19-low cells compared to single-zeta CARs. Additionally, in a xenograft model, CD19-4-1BBζζ CAR T cells demonstrated enhanced activity against CD19-low leukemia compared to CD19-4-1BBζ CAR T cells, significantly extending survival. The addition of a third zeta domain (CD19-4-1BBζζζ) further enhanced the activity of CAR T cells. However, inclusion of multiple copies of the costimulatory domains did not improve function. In conclusion, CD19 antigen density is an important determinant of CAR T cell function and therapeutic response. CD19-CD28ζ CARs are more efficient at targeting CD19-low tumor cells than CD19-4-1BBζ CARs. The addition of multiple zeta domains to the CAR enhances its ability to target low antigen density tumors. This serves as proof of concept that rational redesign of CAR signaling endodomains can result in enhanced function against low antigen density tumors, an important step for extending the reach of these powerful therapeutics and overcoming a significant mechanism of tumor escape. Disclosures Lee: Juno: Consultancy.

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New Generation Chimeric Antigen Receptor T-Cell Therapy (CoupledCAR) Induces High Rate Remissions in Solid Tumor

Blood ◽

10.1182/blood-2019-130703 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4631-4631

Author(s):

Lei Xiao

Keyword(s):

Clinical Trials ◽

T Cells ◽

Thyroid Cancer ◽

T Cell ◽

Cell Therapy ◽

Solid Tumors ◽

Tumor Cells ◽

T Cell Therapy ◽

Car T Cells ◽

Car T

New Generation Chimeric Antigen Receptor T-Cell Therapy ( CoupledCAR ) Induces High Rate Remissions in Solid Tumor Yu Liu1,Song Li2,Youli Luo3,Haixia Song4,Chengfei Pu5, Zhiyuan Cao 5, Cheng Lu5,Yang Hang5,Xi Huang5,Xiaogang Shen5 ,Xiaojun Hu3 , Renbin Liu1,Xiuwen Wang2,Junjie Mao3,Shihong Wei4 ,Zhao Wu5and Lei Xiao5* 1.The Third Affiliated Hospital, SUN YAT-SEN University 2.Qilu Hospital of Shandong University 3.The Fifth Affiliated Hospital, SUN YAT-SEN University 4.Gansu Procincial Cancer Hospital 5.Innovative Cellular Therapeutics *Corresponding to: Lei Xiao, [email protected] Chimeric antigen receptor (CAR) T cell therapy made significant progress for treating blood cancer such as leukemia, lymphoma, and myeloma. However, the therapy faces many challenges, such as physical barrier, tumor microenvironment immunosuppression, tumor heterogeneity, target specificity, and cell expansion in vivo for treatment of solid tumors Conventional CAR T cell therapy showed weak CAR T expansion in patients and thus achieved no or little response for treating solid tumors. Here, we generated "CoupledCAR" T cells including an anti-TSHR CAR molecule. Compared with conventional CART cells,these "CoupledCAR" T cells successfully improved the expansion of CART cells more than 100 times and enhanced CAR T cells' migration ability, allowing the CAR T cells to resist and infiltrate the tumor microenvironment and killed tumor cells. To verify the effect of "CoupledCAR" T cells on solid tumors, we have completed several clinical trials for different solid tumors, including two patients with thyroid cancer. Immunohistochemistry (IHC) results showed that thyroid stimulating hormone receptors (TSHR) were highly expressed in thyroid cancer cells. In vitro co-culture experiments showed that TSHR CAR T cells specifically recognized and killed TSHR-positive tumor cells. Animal experiments showed that TSHR CAR T cells inhibited the proliferation of TSHR-positive tumor cells. Therefore, we designed "CoupledCAR" T cells expressing a binding domain against TSHR. Further,we did clinical trials of two group patients that were successfully treated using conventional TSHR CAR T cells and the "CoupledCAR" T cells, respectively. In the first group using conventional TSHR CAR T cells, patients showed weak cell expansion and less migration ability. In the group using TSHR "CoupledCAR" T cells, patients showed rapid expansion of CAR T cells and killing of tumor cells. One month after infusion (M1), the patient was evaluated as PR(Partial Response): the lymph node metastasis disappeared, and thoracic paratracheal tumors decreased significantly. Three months after infusion (M3), the patient was evaluated as a durable response, and the tumor tissue was substantially smaller than M1. Further, two patients with colonrectal cancer were enrolled in this trial and infused "CoupledCAR" T cells. One patient achieved PR and the other one achieved SD (Stable Disease). Therefore, "CoupledCAR" T cells can effectively promote expansion, migration and killing ability of CAR T cells in patients with thyroid cancer. "CoupledCAR" T cell technology is a technological platform, which may be used to treat other cancer types. Next, we are recruiting more patients with solid tumors in clinical trials using "CoupledCAR" T cells. Disclosures No relevant conflicts of interest to declare.

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The tumor vasculature an attractive CAR T cell target in solid tumors

Angiogenesis ◽

10.1007/s10456-019-09687-9 ◽

2019 ◽

Vol 22 (4) ◽

pp. 473-475 ◽

Cited By ~ 4

Author(s):

Parvin Akbari ◽

Elisabeth J. M. Huijbers ◽

Maria Themeli ◽

Arjan W. Griffioen ◽

Judy R. van Beijnum

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Microenvironment ◽

Solid Tumors ◽

Solid Tumor ◽

Hematological Malignancies ◽

Tumor Vasculature ◽

Car T Cells ◽

Car T Cell ◽

Car T

Abstract T cells armed with a chimeric antigen receptor, CAR T cells, have shown extraordinary activity against certain B lymphocyte malignancies, when targeted towards the CD19 B cell surface marker. These results have led to the regulatory approval of two CAR T cell approaches. Translation of this result to the solid tumor setting has been problematic until now. A number of differences between liquid and solid tumors are likely to cause this discrepancy. The main ones of these are undoubtedly the uncomplicated availability of the target cell within the blood compartment and the abundant expression of the target molecule on the cancerous cells in the case of hematological malignancies. Targets expressed by solid tumor cells are hard to engage due to the non-adhesive and abnormal vasculature, while conditions in the tumor microenvironment can be extremely immunosuppressive. Targets in the tumor vasculature are readily reachable by CAR T cells and reside outside the immunosuppressive tumor microenvironment. It is therefore hypothesized that targeting CAR T cells towards the tumor vasculature of solid tumors may share the excellent effects of CAR T cell therapy with that against hematological malignancies. A few reports have shown promising results. Suggestions are provided for further improvement.

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213 AB-X integrated circuit T cells demonstrate improved potency, expansion, and specificity compared to MSLN CAR T cells

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.213 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A226-A226

Author(s):

Stephen Santoro ◽

Aaron Cooper ◽

Natalie Bezman ◽

Jun Feng ◽

Kanika Chawla ◽

...

Keyword(s):

Ovarian Cancer ◽

T Cells ◽

T Cell ◽

Integrated Circuit ◽

Logic Gate ◽

Xenograft Model ◽

Car T Cells ◽

Car T

BackgroundIn solid tumors, CAR T cell efficacy is limited by off-tumor toxicity and suppression by the tumor microenvironment (TME). AB-X is an integrated circuit T cell (ICT cell) intended for the treatment of ovarian cancer. AB-X includes a transgene cassette with two functional modules: 1) an ”AND” logic gate designed to limit off-tumor toxicity through dual tumor antigen recognition; 2) a dual shRNA-miR to resist TME suppression and improve ICT cell function. The AB-X logic gate consists of a priming receptor that induces expression of an anti-mesothelin (MSLN) CAR upon engagement of a ALPG/P (alkaline phosphatase germ-line/placental). The dual shRNA-miR mediates downregulation of FAS and PTPN2. The AB-X DNA cassette is inserted into the T cell genome at a defined novel genomic site via CRISPR-based gene editing.MethodsDual-antigen specificity of the logic gate was assessed in mice harboring MSLN+ and ALPG/P+MSLN+ K562 tumors established on contralateral flanks. Potency was measured in a subcutaneous MSTO xenograft model. Logic-gated ICT cells were compared with MSLN CAR T cells in both models. In vitro, expansion of ICT cells with the FAS/PTPN2 shRNA-miR was evaluated in a 14 day repetitive stimulation assay (RSA). In vivo, expansion and potency were measured in the MSTO xenograft model. An in vitro FAS cross-linking assay was conducted to assess the impact of FAS knockdown on FAS-mediated apoptosis.ResultsLogic-gated ICT cells demonstrated specific activity against ALPG/P+MSLN+ tumors, but had no effect against MSLN+ tumors in the K562 in vivo specificity model. In addition, logic-gated ICT cells demonstrated greater in vivo potency than MSLN CAR T cells in the MSTO xenograft model. In our RSA, ICT cells containing the FAS/PTPN2 shRNA-miR had 8-fold greater expansion than the MSLN CAR T cells. Enhanced expansion was confirmed in vivo with ICT cells demonstrating >10-fold expansion in tumors and peripheral blood, enabling comparable growth inhibition in MSTO xenografts at less than one quarter the dose of the MSLN CAR T cells. Importantly, PTPN2 knockdown resulted in balanced expansion of all T cell subsets, including CD45RA+, CCR7+ memory cells. Lastly, ICT cells containing the FAS/PTPN2 shRNA-miR were resistant to FAS-mediated apoptosis.ConclusionsAB-X ICT cells specifically recognize ALPG/P+MSLN+ tumors, demonstrate superior potency, expansion, and persistence compared with MSLN CAR T cells, and are resistant to ovarian TME suppression. AB-X will be evaluated in clinical trials for treatment of platinum resistant/refractory ovarian cancer.AcknowledgementsWe would like to acknowledge all of our colleagues at Arsenal Biosciences, without whom this work would not have been possible.

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Combining De-Glycosylating Agents with CAR-T Cells for Targeting Solid Tumors and Reducing Toxicity

Blood ◽

10.1182/blood-2018-99-116019 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4544-4544

Author(s):

Beatrice Greco ◽

Katia Paolella ◽

Barbara Camisa ◽

Valeria Malacarne ◽

Laura Falcone ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cell ◽

Solid Tumors ◽

Tumor Cells ◽

Combined Approach ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Glycosylated Proteins

Abstract Background: The adoptive transfer of CAR-T cells have shown impressive results against B-cell malignancies, but still limited efficacy against solid tumors. The discovery of the key factors regulating the activity of CAR-T cells is required to improve their antitumor potency and modulate toxicities. Since solid tumors display a wide range of glycosylation alterations, including increased N-glycan branching, we hypothesized that peptidic epitopes may be masked by glycans from CAR-T cell targeting, especially in richly glycosylated proteins. Results: To investigate if sugar chains may be sterically hulking for CAR-T cell targeting, we generated N-glycosylation-defective pancreatic tumor cell lines. This aim has been achieved by knocking-out the expression of the glycosyltransferase Mgat5, a key enzyme involved in the process of N-glycan branching, using the CRISPR-Cas9 technology. As model antigens for CAR targeting, we focused on CD44v6 and CEACAM-5 (CEA) since they are both heavily glycosylated proteins over-expressed on a wide variety of solid tumors, including pancreatic adenocarcinoma. Strikingly, the impairment of N-glycosylation resulted in a dramatic increase of tumor targeting by both CD44v6 (4-fold, p<0,001) and CEA CAR-T cells (10-fold, p<0,001). This effect associated with improved CAR-T cell activation, suggesting more proficient antigen engagement. To exploit this mechanism in order to increase the efficacy of CAR-T cells against solid tumors, we sought to block tumor N-glycosylation with the clinical-grade glucose/mannose analogue 2-Deoxy-D-glucose (2DG). Similarly to genetically induced glycosylation blockade, treatment with 2DG also sensitized tumor cells to recognition by CAR-T cells, significantly increasing their elimination (CD44v6: 3-fold, p<0,01; CEA: 13-fold, p<0,001). Notably, 2DG alone proved to be ineffective as mono-therapy, suggesting a synergistic effect with CAR-T cells. To get more insights on this mechanism, we took advantage of previous studies reporting that 2DG interference with N-glycosylation can be reverted by the addition of exogenous mannose. Of notice, mannose did revert the synergy between 2DG and CAR-T cells (p<0,05), implying that blockade of N-glycosylation rather than glycolysis is the crucial mechanism involved. These findings were further confirmed by using the N-glycosylation inhibitor tunicamycin (CD44v6: 2,5-fold; CEA: 5-fold, p<0,01) and by Western blot, looking at the presence of de-glycosylated proteins on tumor cell surface after 2DG treatment. Next, we challenged the combined approach in a pancreatic adenocarcinoma xenograft mouse model. Accordingly with in vitro data, mice receiving CAR-T cells highly benefited from 2DG administration (5-fold less tumor at 7d, p<0,05), which conversely was unable to mediate any antitumor effect alone. Interestingly, improved antitumor activity was accompanied by a decrease in the frequency of CAR-T cells expressing one or more exhaustion and senescence markers, such as TIM-3, LAG-3, PD-1 and CD57 (SPICE software analysis, p=0,0105). Finally, thanks to metabolic deregulation (Warburg effect), 2DG is expected to selectively accumulate in cancer cells compared to healthy tissues, supporting the safety of the combined approach. Accordingly, we observed that the same doses of 2DG able to enhance tumor cell recognition by CAR-T cells failed to increase the elimination of healthy cells, such as keratinocytes. Conclusions: Our results indicate that i) the glycosylation status of tumor cells regulates the efficacy of CAR-T cells, especially when targeting highly glycosylated antigens, and ii) combining CAR-T cells with the de-glycosylation agent 2DG, which preferentially accumulates in tumor masses, may pave the way for a successful immunotherapy against solid tumors. Disclosures Bonini: Intellia Therapeutics: Research Funding. Bondanza:Novartis: Employment.

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CAR-T cells to deliver engineered peptide antigens and reprogram antigen specific T cell responses against solid tumors.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.2530 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 2530-2530

Author(s):

Daniel Lee ◽

Andy J Minn ◽

Lexus R Johnson

Keyword(s):

T Cells ◽

T Cell ◽

Solid Tumors ◽

Tumor Cells ◽

In Vivo Studies ◽

Peptide Antigens ◽

Car T Cells ◽

Car T

2530 Background: Neoantigen depleted malignancies such as colorectal cancer demonstrate primary resistance to immune checkpoint blockade, and solid tumors in general have shown resistance to chimeric antigen receptor (CAR) T cell therapy. However, CAR-T cells have been shown to be capable of delivering various therapeutic molecules in a targeted fashion to the tumor microenvironment, in some cases through extracellular vesicles (EVs). In vivo studies have shown that the presentation of foreign viral peptides by solid tumors can reprogram bystander virus-specific cytotoxic T cells (CTLs) against tumor cells. In this study, we demonstrate that CAR-T cells can deliver engineered peptide antigens to solid tumors, leading to presentation on tumor cells and anti-tumor response. Methods: Second generation CAR-T cells (41BB endodomain) targeting human CD19 (19BBz) or human mesothelin (M5BBz) were generated via retroviral and lentiviral transduction respectively. CAR-T cells were engineered to co-express peptides such as SIINFEKL of ovalbumin and NLVPMVATV of CMV pp65 among others. Peptides were isolated from EVs via ultracentrifugation. For in vivo studies, C57BL/6 or NSG mice were injected on the flank with relevant tumors and treated with peptide-CAR-T cells. In vitro studies utilized flow cytometry and xCELLigence killing assays. Results: Murine 19BBz CAR-T cells expressing the SIINFEKL peptide of ovalbumin (ova-19BBz) were found to transfer SIINFEKL peptide to tumor cells via EVs in vitro and in vivo, leading to peptide presentation on MHC-I of tumor cells. This resulted in significantly delayed tumor growth in tumor bearing mice transfused with OT-I T cells to mimic an existing antigen specific T cell pool. We expanded on these findings by isolating EVs from human M5BBz CAR-T cells expressing CMV viral peptides. Peptide-CAR-T EVs were co-cultured with human ovarian cancer cells to assess presentation to Jurkat T cells. Finally, we utilized primary human T cells from CMV+ healthy donors to assess the clinical feasibility of our peptide delivery approach. Conclusions: CAR-T cells can be engineered to deliver peptides to tumor cells for presentation and subsequent targeting by antigen specific CTLs. This represents a novel strategy for the treatment of non-immunogenic tumors.

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139 Establishment of canine CAR T cells treatment model for solid tumor immunotherapy development

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0139 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A152-A153

Author(s):

Shihong Zhang ◽

Karan Kohli ◽

R Graeme Black ◽

Brian Hayes ◽

Cassandra Miller ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Solid Tumors ◽

Solid Tumor ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Signaling Domain

BackgroundChimeric antigen receptor (CAR) T cell therapy has transformed therapy for hematological malignancies but has not yet been established as standard of care for any solid tumors. One obstacle for human solid tumor immunotherapy research is the lack of clinically relevant, immunocompetent animal models. In this study, we sought to establish CAR T cells for naturally occurring canine sarcomas in client owned animals as a model for human CAR T cell therapy.MethodsArchived FFPE, freshly isolated canine solid tumor samples as well as tumor lines were tested for B7H3 expression by immunohistochemistry (IHC) and flow cytometry analysis. We designed CARs using the scFv from the human B7H3-specific antibody MGA271 and confirmed the cross-reactivity to canine B7H3 (construct information see figure 1A). A truncated EGFR (tEGFR) was included in the construct to allow for IHC and flow cytometry testing for the presence of CAR T cells. Killing efficiency was evaluated using 3D tumor spheroid killing assays to monitor dynamics. Safety of the CAR products following lymphodepletion was confirmed in two healthy dogs (figure 1B).ResultsCanine solid tumors were confirmed to be B7H3 positive in almost all cases. Using the GALV-pseudotyped retrovirus system, transduction was efficient with up to 70% CAR+ cells. Post-transduction expansion was over 100 folds. B7H3 CAR transduced canine T cells were able to eliminate B7H3+ canine tumor spheroids effectively (figure 2). Safety of the CAR T cells (dose: 1 × 109/m2) were confirmed in both healthy animals following cyclophosphamide lymphodepletion. After week 6, cetuximab was given to the subjects to deplete EGFR+ cells. Subject 2 experienced fever after CAR T cell administration. Both dogs showed elevated serum ALP and ALT levels and returned to normal (figure 3). No other treatment-related adverse events were observed. Information of the CAR T cell products can be found in table 1.Abstract 139 Figure 1Construct information and safety trial design(A) Four 2nd generation CAR constructs were generated. Two B7H3 CARs were candidates for the treatment, and two HER2 CARs served as controls, as they have been shown to kill canine cancer cells. The CARs are consisted of a single chain variable fragment (scFv, either B7H3-specific MGA271 or HER2-specific FRP5), a short hinge, a transmembrane domain (tm), a canine costimulatory signaling domain (either canine CD28 or 4-1BB) and canine CD3? signaling domain. Truncated EGFR is added in the construct for CAR+ T cell detection and facilitate the depletion of CAR T cells in vivo as a safety measure. (B) Blood from the subjects were drawn 3 weeks prior to the treatment for CAR T cell production. Cyclophosphamide (Cy, 400 mg/m2) and Fludarabine (Flu, 10 mg/m2) were given to the subjects for 2 days for lymphodepletion. CAR T cells (1 × 109/m2) and cetuximab (200 mg/m2) were given to the subjects as indicated. Blood, lymph node (LN) and bone marrow (BM) aspirates were collected for CAR T cell homing and persistence analysisAbstract 139 Figure 2Killing of canine OSA spheroids by canine CAR T ce(A) Scheme of tumor cell spheroid forming and killing. The loss of GFP can be measured for cytotoxicity readout (B) FRP5 and MGA271 CAR T cells can effectively kill canine cancer spheroids. Experiments were done in triplicates and error bars indicate SDAbstract 139 Figure 3Dynamics of peripheral lymphocytes, serum ALP and Current treatment regimen effectively decreased peripheral lymphocytes number after cyclophosphamide and fludarabine administration (D-4 and D-3) and increased serum ALP and ALT level after CAR T cell infusion (D0). Dashed line in both graphs show the upper limit of ALP and ALT levels, which are both 68U/LAbstract 139 Table 1Infused CAR T cell product informationBoth subjects are adult male beagle mixConclusionsWe demonstrated that, similar to human cancers, B7H3 is a target in canine solid tumors. We successfully generated canine B7H3 specific CAR T cell products that are highly efficient at killing canine 3D tumor spheroids using a production protocol that closely models human CAR T cell production procedure and confirmed the safety in vivo. We plan to test and optimize various approaches to enhance CAR T cell efficacy for solid tumor treatment both in vitro and in canine sarcoma patients.Ethics ApprovalThe study was approved by Fred Hutchinson Cancer Research Center‘s Institutional Animal Care and Use Committee (IACUC), approval number PROTO201900860

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131 Genetic disruption of negative immune regulators to enhance CAR-T efficacy against solid tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.131 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A140-A141

Author(s):

David Mai ◽

Omar Johnson ◽

Carl June

Keyword(s):

T Cells ◽

T Cell ◽

Solid Tumors ◽

Solid Tumor ◽

Transduction Efficiency ◽

Car T Cells ◽

Car T Cell ◽

Car T

BackgroundCAR-T cell therapy has demonstrated remarkable success in hematological malignancies but displays limited efficacy in solid tumors, which comprise most cancer cases. Recent studies suggest that CAR-T cell failure via T cell exhaustion is characterized by decreased surface CAR expression, cytotoxicity, and Th1 cytokine production, leading to reduced antitumor functionality.1 2 3 To address these issues, studies have turned to genetically knocking out or overexpressing targets associated with an exhaustion or effector phenotype, such as PD-1 knockout (KO) and c-Jun overexpression, among other candidates that are typically receptors or transcription factors.4 5 6 However, there are other underexplored factors that mediate various aspects of immune regulation. While genome-wide CRISPR screens may discover such factors, they are unlikely to reveal phenotypes for genes whose function is partially redundant, therefore promising candidates may be missed. Such candidates include post-transcriptional regulators (PTRs) that coordinate immune responses, which are less well-studied in the context of CAR-T cell function. We hypothesized that KO of these PTRs may increase CAR-T cell cytokine activity, phenotype, and persistence, potentially under long-term or exhaustion-inducing conditions, leading to increased tumor control. Ultimately, disruption of negative immune regulators could produce CAR-T cells with enhanced activity and persistence, narrowing the gap between efficacy in hematological and solid tumors.MethodsTo explore whether the disruption of two target PTRs improves solid tumor efficacy, we used CRISPR-Cas9 to genetically delete one or both PTRs in mesothelin-targeting human CAR-T cells and assayed their function in vitro and in vivo in NSG mice.ResultsWe show successful genetic deletion of these genes in post-thymic human T cells and that their disruption does not affect primary expansion (figure 1) or transduction efficiency (figure 2). These KO CAR-T cells display increased expression of co-stimulatory receptors and various cytokines (figure 3). While KO CAR-T cells are functionally similar to WT CAR-T cells in in vitro assays (figure 4), KO CAR-T cells demonstrate superior activity in vivo and can clear large, established tumors compared to WT CAR-T cells at low dose (figure 5).Abstract 131 Figure 1Expansion kinetics of KO CAR-T cellsAbstract 131 Figure 2Transduction efficiency and baseline phenotype of KO CAR-T cellsAbstract 131 Figure 3Costimulatory receptor and cytokine expression of KO CAR-T cellsAbstract 131 Figure 4In vitro cytotoxicity of KO CAR-T cellsAbstract 131 Figure 5In vivo activity of KO CAR-T cellsConclusionsThese results indicate that KO of our target PTRs may improve the potency of CAR-T cells in solid tumors and may have important implications on the development of effective solid-tumor cell therapies.ReferencesJE Wherry and M Kurachi, Molecular and cellular insights into T cell exhaustion, Nature Reviews Immunology 2015;15:486–499.EW Weber, et al. Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling. Science 2021;372:6537.S Kuramitsu et al. Induction of T cell dysfunction and NK-like T cell differentiation in vitro and in patients after CAR T cell treatment. Cell, in revision.BD Choi et al, CRISPR-Cas9 disruption of PD-1 enhances activity of university EGFRvIII CAR T cells in a preclinical model of human glioblastoma. Journal for ImmunoTherapy of Cancer 2019;7:304.RC Lynn et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature 2019;576:293–300.LJ Rupp et al. CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Scientific Reports 2017;7:737.

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