scholarly journals 765 Contextual secretion of nanoscale interleukin (IL)-12 by CAR T cells for the treatment of cancer

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A813-A813
Author(s):  
Zhifen Yang ◽  
Maggie Bobbins ◽  
Hana Choi ◽  
Ofir Stefanson ◽  
Jin Yang ◽  
...  
Keyword(s):  
T Cells ◽  
Cancer Cells ◽  
Systemic Toxicity ◽  
Car T Cells ◽  
Car T ◽  
Her 2 ◽  
Ifn Γ ◽  
Fadu Cells

BackgroundInterleukin(IL)-12 activates T cells and macrophages pivoting the switch that turns chronic into acute inflammation and results in cancer rejection. However, despite formidable antitumor effects in preclinical models, its clinical utilization is limited by severe systemic toxicity. Here, we present a conditional, antigen-dependent, non-editing CRISPR-activation (CRISPRa) circuit (RB-2-12) that purposefully induces minimally effective doses of IL-12 for autocrine activation of CAR-T.MethodsRB-2-12 is a CAR T cell engineered to express the IL-12 heterodimer via conditional transcription of its two endogenous subunits p35 and p40. The circuit includes a lentiviral constructs encoding an anti-HER2 (4D5) single chain variable fragment, with CD28 and CD3ζ co-stimulatory domains linked to a tobacco etch virus (TEV) protease and two single guide RNAs (sgRNA) targeting the promoter region for IL-12A orL-12B. A second constructs encodes linker for activation of T cells, complexed to nuclease-deactivated/dead Cas9 (dCas9)-VP64-p65-Rta transcriptional activator (VPR) via a TEV-cleavable linker (LdCV). Activation of CAR brings CAR-TEV in proximity to LdCV releasing dCas9 for nuclear localization to the regulatory regions and conditionally and reversibly induce nanoscale expression of the p70 heterodimer. RB-2-12 was compared in vitro to control (cRB-2-12, lacking the IL-12 sgRNAs).ResultsRB-2-12 induced autocrine production of low concentrations of IL-12 upon exposure to HER2+ FaDu cancer cells resulting in significantly enhanced production of interferon (IFN)-γ, cytotoxic activity and proliferation (figure 1a). These effects were comparable to co-culturing conventional HER2-specific CAR-T cells with a modified FaDu cell line expressing high doses of IL-12 (figure 1b).Abstract 765 Figure 1Conditional autocrine release of nanoscale-dose p70/IL-12 byConditional autocrine release of nanoscale-dose p70/IL-12 by RB-2-12 resulting in enhanced IFN-γ production, cytotoxicity and proliferation in vitro after three days of exposure to FaDu cells (figure 1a). Constitutive high dose release of p70-IL-12 by HER2+ Fadu cells engineered to constitutively express IL-12 (FaDu/IL-12) and its effect on IFN-γ secretion, cytotoxicity and proliferation of conventional HER-2-specific CAR T-cells three days after exposure to FaDu or FaDu/IL-12 cancer cells (figure 1b). N.T. = non-transduced T cells; NOsg = cRB-2-12 CAR-T cells missing the sgRNAs for the two IL-12 subunits; IL12sg = complete product incorporating the full CRISPRa functions.ConclusionsWe have previously shown that tandem suppression of PD-1 expression upon HER-2 CAR activation using CRISPR interference enhances anti-cancer properties of CAR-T cells in vivo against HER2-FaDu xenografts by promoting their persistence and long-term tumor colonization (companion abstract submitted to SITC annual meeting). We hypothesize that addition of a Th1 polarizing component such as IL-12 will exponentially increase the efficacy of reprogrammed CAR-T cells by combining enhancement of effector functions to cellular fitness. At the same time, the autocrine effects of nanoscale IL-12 production limit the risk of off-tumor leakage and systemic toxicity. Such cumulative synthetic biology approaches are currently investigated in vitro and in vivo model systems. Current work is testing the effectiveness of RB-2-12 in vivo against FaDu xenografts.AcknowledgementsNoneTrial RegistrationN.A.Ethics ApprovalNot ApplicableConsentNot ApplicableReferencesNone

scholarly journals 153 Nanoscale, antigen-dependent, IL-12 delivery by CAR T cells plus PD-L1 blockade for cancer treatment

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A162-A163
Author(s):  
Zhifen Yang ◽  
Francesco Marincola
Keyword(s):  
T Cells ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Car T Cells ◽  
Car T ◽  
Ifn Γ ◽  
High Doses ◽  
Fadu Cells

BackgroundInterleukin(IL)-12 activates T cells pivoting the switch that turns lingering inflammation into acute inflammation and cancer rejection. However, its clinical utilization is limited by severe systemic toxicity. IL-12 is a potent inducer of PD-1 expression in T cells. Here, we present a conditional, antigen-dependent, non-editing CRISPR-activation (CRISPRa) circuit (RB-312) that delivers nanoscale doses of IL-12 for autocrine activation of CAR-T cells. RB-312 was also tested in combination with PD-L1 blocking antibody (atezolizumab).MethodsRB-312 is a CAR T cell engineered to express the IL-12 heterodimer via conditional transcription of its two endogenous subunits p35 and p40. The circuit includes two lentiviral constructs with one encoding HER2-specific chimeric antigen receptor and two sgRNAs targeting IL-12A or IL-12B and the other encoding linker for activation of T cells, complexed to dead Cas9 (dCas9)-VP64-p65-Rta transcriptional activator (VPR) (LdCV). Activation of CAR allows the release of dCas9 for nuclear localization and hence conditionally and reversibly induces the secretion of IL-12 p70 heterodimer.ResultsRB-312 induced low concentrations of IL-12 upon exposure to HER2+ FaDu cancer cells engineered to overexpress PD-L1 and this resulted in significantly enhanced production of IFN-γ, cytotoxicity and CAR-T proliferation (figure 1A). These effects were comparable to co-culturing conventional HER2 CAR with FaDu cells modified to express high doses of IL-12 (figure 1B). In vivo administration of RB-312 significantly enhanced survival of mice carrying FaDu xenografts compared to mice treated with the respective conventional HER2 CAR or cRB-312 (control lacking the IL-12 sgRNAs, figure 2A). RB-312 induced a strong upregulation of PD-1 in CAR-T cells in vivo (figure 2B). The critical role of the PD-1/PD-L1 interaction was demonstrated in vitro by comparing RB-312 proliferation when exposed to FaDu overexpressing PD-L1 or PD-L1 knock out cells (figure 3A). Indeed, combined treatment of RB-312 and atezolizumab resulted in significant reduction in tumor growth (figure 3B and C) and significantly enhanced survival (figure 3D).Abstract 153 Figure 1Conditional autocrine release of nanoscale-dose p70/IL-12 by RB-312 resulting in enhanced IFN-γ production in vitro after three days of exposure to HER2+ FaDu cells (figure 1A), and the level of IFN-γ production was comparable to co-culturing conventional HER2-specific CAR-T cells with a modified FaDu cell line engineered to constitutively express high doses of IL-12 (FaDu/IL-12, figure 1B)Abstract 153 Figure 2Intra-tumoral administration of RB-312 extended survival in mice carrying FaDu xenografts compared to NT (non-transduced T cells), HER2 CAR (conventional HER2 CAR-T cells) and cRB-312 CAR-T cells missing the sgRNAs for the two IL-12 subunits (figure 2A). Analysis of necropsy material demonstrated that PD-1 expression was dramatically increased in RB-312 compared with the respective control cRB-312 (figure 2B)Abstract 153 Figure 3RB-312 cellular function in vivo. PD-L1 expression by FaDu cell lines is a critical mechanism of repression of RB-312 function. In vitro CAR-T proliferation of RB-312 upon stimulation with FaDu tumor cells (orange solid lines) or FaDu/PD-L1 knockout tumor cells (orange dashed lines) over 6-day time course (figure 3A). In vivo efficacy of intra-tumoral RB-312 against FaDu tumor cells with (orange solid lines) or without (orange dashed lines) addition of PD-L1 blocking antibody atezolizumab (administered intravenously at 5 mg/kg twice per week), as shown by tumor growth followed till day 29 and scatter plot at day 29 (figure 3B), tumor growth spider plots (figure 3C) and Kaplan-Meier survival curve (figure 3D)ConclusionsWe concluded that addition of a Th1 polarizing component such as IL-12 exponentially increases the efficacy of reprogrammed CAR-T cells by combining enhancement of effector functions to cellular fitness. The autocrine effects of nanoscale IL-12 production limit the risk of off-tumor leakage and systemic toxicity. Here, we tested the combination of PD-1/PD-L1 blockade with IL-12-induced CAR-T cell activation demonstrated dramatic synergistic effects. We are currently evaluating the intrinsic combination of IL-12 delivery and PD-L1 resistance for the next generation of RB-312 product eliminating the need for systemic checkpoint blockade.

Preclinical evaluation of anti-ROR1 CAR T cells employing a ROR1 binding SCFV derived from the clinical stage mab cirmtuzumab.

2020 ◽  
Vol 38 (5_suppl) ◽  
pp. 41-41
Author(s):  
Charles E. Prussak ◽  
Christopher Oh ◽  
Juliana Velez Lujan ◽  
Sharon Lam ◽  
Jieyu Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
Cancer Cells ◽  
In Vivo Studies ◽  
Tumor Escape ◽  
Cancer Stemness ◽  
2Nd Generation ◽  
Car T Cells ◽  
Car T

41 Background: Chimeric antigen receptor (CAR)-modified T cells (CAR-T) were generated targeting cells expressing ROR1, which is present on many malignant cancers and has been associated with cancer stemness and chemo-resistance. The ROR1 CAR utilizes the humanized single-chain fragment variable (scFv) binding domain of UC-961 (cirmtuzumab), which exhibits high affinity and specificity for human ROR1 and has demonstrated an excellent safety profile in Phase 1 studies. Methods: CAR constructs with varying spacer regions and intracellular co-stimulatory domains, using the scFV of cirmtuzumab, were constructed and used to generate CAR-T cells from healthy donors. These ROR1 CAR-T cells were tested for cytotoxicity against lymphoid cancer cells in vitro and in vivo studies that employed immune-deficient mice engrafted with labeled human leukemia cells MEC1 or MEC1-ROR1, which had been transfected to stably express ROR1. Results: The 2nd generation and 3rd generation CAR-T-cells with analogous spacer regions were comparably potent and selectively cytotoxic for cells bearing the ROR1 target antigen. But the 2nd generation CARs demonstrated greater potency in vitro even at low effector to target ratios. For the in vivo studies, mice received a single injection of ROR1 CAR-T cells or activated T cells from the same donor as a control. The ROR1 CAR-T cells rapidly cleared the leukemic cells from the animals, whereas animals receiving control T cells or no therapy quickly succumbed to progressive disease within 3 weeks. The administered CAR-T products remained highly active following administration and could be detected for ≥ 3 months without evidence for T cell exhaustion. Conclusions: The generated CAR-T cells utilizing constructs with the Fv of cirmtuzumab, a humanized mAb highly specific for ROR1, onco-embryonic surface antigen, effectively and selectively killed neoplastic cells bearing ROR1 both in vitro and in vivo. As ROR1 expression and signaling has been associated with cancer stemness and chemo-resistance utilizing ROR1 CAR-T therapy to target cancer cells might mitigate tumor escape. These data strongly support the rationale for continued development of our ROR1 CAR-T.

scholarly journals Engineering light-controllable CAR T cells for cancer immunotherapy

2020 ◽  
Vol 6 (8) ◽  
pp. eaay9209 ◽  
Author(s):  
Ziliang Huang ◽  
Yiqian Wu ◽  
Molly E. Allen ◽  
Yijia Pan ◽  
Phillip Kyriakakis ◽  
...  
Keyword(s):  
T Cells ◽  
Cancer Cells ◽  
Cancer Immunotherapy ◽  
Tumor Antigens ◽  
Nuclear Translocation ◽  
Car T Cells ◽  
Car T ◽  
Target Cancer

T cells engineered to express chimeric antigen receptors (CARs) can recognize and engage with target cancer cells with redirected specificity for cancer immunotherapy. However, there is a lack of ideal CARs for solid tumor antigens, which may lead to severe adverse effects. Here, we developed a light-inducible nuclear translocation and dimerization (LINTAD) system for gene regulation to control CAR T activation. We first demonstrated light-controllable gene expression and functional modulation in human embryonic kidney 293T and Jurkat T cell lines. We then improved the LINTAD system to achieve optimal efficiency in primary human T cells. The results showed that pulsed light stimulations can activate LINTAD CAR T cells with strong cytotoxicity against target cancer cells, both in vitro and in vivo. Therefore, our LINTAD system can serve as an efficient tool to noninvasively control gene activation and activate inducible CAR T cells for precision cancer immunotherapy.

scholarly journals Effective and persistent antitumor activity of HER2-directed CAR-T cells against gastric cancer cells in vitro and xenotransplanted tumors in vivo

2017 ◽  
Vol 9 (10) ◽  
pp. 867-878 ◽  
Author(s):  
Yanjing Song ◽  
Chuan Tong ◽  
Yao Wang ◽  
Yunhe Gao ◽  
Hanren Dai ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
T Cells ◽  
Antitumor Activity ◽  
Cancer Cells ◽  
Gastric Cancer Cells ◽  
Car T Cells ◽  
Car T

scholarly journals CAR T Cells Equipped With a Fully Human scFv Targeting Trop2 Can Be Used to Treat Pancreatic Cancer

2021 ◽  
Author(s):  
Hong Jia Zhu ◽  
Yujie Jia ◽  
Jingwen Tan ◽  
Xiaoyan Fang ◽  
Jing Ye ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
T Cells ◽  
Cancer Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Pancreatic Cancer Cells ◽  
Car T

Abstract Purpose: Chimeric antigen receptor (CAR) T cell therapy has demonstrated clinical success in treating haematologic malignancies but has not been effective against solid tumours thus far. Trop2 is a tumour-related antigen broadly overexpressed on a variety of tumours and has been reported as a promising target for pancreatic cancers. Our study aimed to determine whether CAR T cells designed with a fully human Trop2-specific single-chain fragment variable (scFv) can be used in the treatment of Trop2-positive pancreatic tumours.Methods: We designed Trop2-targeted chimeric antigen receptor engineered T cells with a novel human anti-Trop2 scFv (2F11) and then investigated the cytotoxicity, degranulation, and cytokine secretion profiles of the anti-Trop2 CAR T cells when they were exposed to Trop2+ cancer cells in vitro. We also studied the antitumour efficacy and toxicity of Trop2-specific CAR T cells in vivo using a BxPC-3 pancreatic xenograft model.Results: Trop2-targeted CAR T cells designed with 2F11 effectively killed Trop2-positive pancreatic cancer cells and produced high levels of cytotoxic cytokines in vitro. In addition, Trop2-targeted CAR T cells, which persistently circulate in vivo and efficiently infiltrate into tumour tissues, significantly blocked and even eliminated BxPC-3 pancreatic xenograft tumour growth without obvious deleterious effects observed after intravenous injection into NSG mice. Moreover, disease-free survival was efficiently prolonged.Conclusion: These results show that Trop2-targeted CAR T cells equipped with a fully human anti-Trop2 scFv could be a potential treatment strategy for pancreatic cancer and could be useful for clinical evaluation.

scholarly journals Tri-Specific CD19xCD20xCD22 VHH CAR-T Cells (LCAR-AIO) Eradicate Antigen-Heterogeneous B Cell Tumors, Enhance Expansion, and Prolong Persistence in Preclinical In Vivo Models

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1700-1700
Author(s):  
Zhe (joy) Zhou ◽  
Yue Han ◽  
Hong-Bo Pan ◽  
Cai-Jun Sang ◽  
Dong-Lin Shi ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Treatment Efficacy ◽  
Cytolytic Activity ◽  
Publicly Traded ◽  
Car T Cells ◽  
Car T ◽  
Ifn Γ

Abstract Introduction: Anti-CD19 CAR-T therapy has achieved remarkable treatment efficacy in B cell lymphoma. However, targeting CD19 antigen alone can only benefit about half of patients with B cell malignancies. The FDA-approved CD19 CAR-T therapies all use same binder, which is murine FMC63 scFv targeting CD19 and up to 39%-88% of patients have relapsed. Possible mechanisms of relapse include mutations or downregulation of the targeted antigen, CD19, however, the targetable expression of CD20 and CD22 is preserved. In addition, immunogenicity against murine FMC63 scFv could have a negative impact on possible re-dosing regimen. To overcome these limitations, we designed and developed a novel tri-specific VHH CAR-T, targeting three antigens that include CD19, CD20 and CD22, for treating patients who relapsed from prior CAR-T therapies. Methods: We engineered mono-, bi-, or tri-specific VHH CAR constructs targeting CD19, CD20 and/or CD22 respectively in a lentiviral vector. The mono-, bi- or tri-specific CAR-T cells were tested against tumor lines expressing single, dual or triple antigens in an in vitro cytotoxicity assay. In addition, we evaluated the contribution of different CAR backbones, and possible combinations of scFv, VH or VHH to CAR design. We hypothesized that our lead tri-specific VHH CAR-T, LCAR-AIO, would potently inhibit tumors with heterogeneous Ag expression and prevent Ag escape. To validate this, we compared in vitro cytolytic activity and cytokine production of LCAR-AIO CAR-T to anti-CD19 FMC63 CAR-T against CD19 +CD20 +CD22 + Raji.Luc and CD19KOCD20 +CD22 +Raji.Luc cells . In vivo treatment efficacy and CAR-T persistence were also investigated in NCG murine model xenografted with Raji tumor line. 0.3x10 6 CAR +T cells or dose-matched untransduced T cells were given to NCG mice four days post i.v. implantation of Raji.Luc tumor cells. Tumor growth was monitored weekly by bioluminescence imaging until achieved endpoint (55 days), and CAR-T persistence was determined using genomic DNA level. Results: Tri-specific VHH CAR-T cells can mediate dose-dependent cytotoxicity against Raji tumor lines. Compared to mono- or bi-specific VHH or scFv CAR-T, tri-specific VHH CAR-T demonstrated equal or better cytolytic activity. Our lead tri-VHH CAR-T, LCAR-AIO, was able to specifically lyse K652 over-expressing single target such as CD19, CD20 or CD22, at the similar level to mono-specific CD19, CD20 or CD22 VHH CAR-T. Since no blocking effect of recognition against these three antigens was observed, our result suggested that all three VHHs in LCAR-AIO are functional. In comparison to anti-CD19 FMC63 scFv CAR-T, LCAR-AIO exhibited higher lytic activity and IFN-γ production against Raji.Luc tumor lines in vitro. In addition, LCAR-AIO retained its robust lytic activity and IFN-γ production when co-cultured with CD19KO-Raji.Luc cells while anti-CD19 FMC63 scFv CAR-T could not, suggesting LCAR-AIO may prevent tumor escape due to loss of CD19. Furthermore, comparison of LCAR-AIO to mono-scFv CAR-T (anti-CD19 FMC63-BBz, anti-CD20 Leu16-BBz or anti-CD22 m971-BBz) was performed in NCG mice xenografted with Raji cell line, LCAR-AIO exhibited better T cell expansion, longer persistence, and superior efficacy in eliminating tumors. Conclusions: Based on in vitro and in vivo preclinical data, tri-specific CD19xCD20xCD22 VHH CAR-T can be effective targeting tumors lack of CD19 expression, therefore, it has the potential of treating relapsed patients with prior CD19 CAR-T therapy. The feasibility of making tri-specific CAR-T would help to extend this technology to solid cancers where heterogeneity poses a major challenge at current stage. Figure 1 Figure 1. Disclosures Zhou: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Han: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Pan: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Sang: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Shi: Legend Biotech: Current Employment. Feng: Legend Biotech: Current Employment. Xiao: Legend Biotech: Current Employment. Zhuang: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Wang: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Fan: Legend Biotech: Current Employment, Current equity holder in publicly-traded company.

767 Interferon gamma reduces CAR-T exhaustion and toxicity without compromising therapeutic efficacy in hematologic malignancies

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A815-A815
Author(s):  
Stefanie Bailey ◽  
Sonika Vatsa ◽  
Amanda Bouffard ◽  
Rebecca Larson ◽  
Irene Scarfo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cancer Cells ◽  
Tumor Cells ◽  
Interferon Gamma ◽  
Hematologic Malignancies ◽  
Car T Cells ◽  
Car T

BackgroundChimeric antigen receptor (CAR) T cell therapy has shown remarkable efficacy in hematologic malignancies, ultimately leading to its FDA approval for relapsed/refractory acute lymphoblastic leukemia and large cell lymphomas in 2017. Despite the success of CAR T cells in the clinic, toxicities such as cytokine release syndrome (CRS) can be severe. Attempts to mitigate these effects have primarily focused on the blockade of macrophage-derived cytokines, such as IL-6 and IL-1B. Herein, we show that the pharmaceutical blockade or genetic deletion of interferon gamma (IFNg, a CAR-T-derived cytokine that strongly correlates with CRS in the clinic, appears to be a viable target for the reduction of CAR-T-associated toxicities.MethodsPharmacologic (blocking antibody) and genetic (CRISPR/Cas9) approaches were used to block IFNg signaling and/or production by CAR T cells. In vitro CAR-T function and cytotoxicity was tested using ELISA, flow cytometry and short-/long-term killing assays prior to their assessment in vivo. NSG mice were injected with Nalm6 or JeKo-1 cancer cells prior to treatment with IFNg-modified CAR-T and tumor size and IFNg production were measured. To determine how the loss of IFNg might affect innate immune cells, CAR-T, macrophages and tumor cells were co-cultured and assessed by flow cytometry, immunofluorescence, Luminex and RNA sequencing.ResultsIFNg could be blocked using an anti-IFNg antibody or CRISPR/Cas9 editing of the CAR T cells without affecting T cell activation, proliferation or cytokine production (IL-2, TNFa, GM-CSF). Successful blockade of the IFNg signaling pathway was confirmed by reduced phosphorylation of JAK1, JAK2 and STAT1, even in the presence of exogenous IFNg. Loss of IFNg did not reduce the cytotoxic potential or persistence of CAR-T against hematologic malignancies in vitro or in vivo. When cultured with macrophages and cancer cells, IFNg knockout (IFNgKO) CAR-T yielded decreased levels of IL-1B, IL-6, IL-13, MCP1 and CXCL10, indicating a reduction in macrophage activation induced by CAR-T in the absence of IFNg. Serum from tumor-bearing mice treated with IFNgKO CAR-T elicited lower activation of macrophages in vitro compared to those treated with IFNg-producing CAR-T cells. Furthermore, IFNgKO CAR T cells co-cultured with tumor cells and macrophages demonstrated less exhaustion as shown by reduced expression of PD1, Tim3 and Lag3 and increased IFNgKO CAR-T expansion.ConclusionsCollectively, these data suggest that IFNg is not required for the efficacy of CAR-T in hematologic malignancies and can potentially be targeted to reduce toxicity and enhance CAR-T efficacy and persistence in the clinic.

scholarly journals Effective antitumor activity of 5T4‐specific CAR‐T cells against ovarian cancer cells in vitro and xenotransplanted tumors in vivo

MedComm ◽  
2020 ◽  
Vol 1 (3) ◽  
pp. 338-350
Author(s):  
Cuiyu Guo ◽  
E Dong ◽  
Qinhuai Lai ◽  
Shijie Zhou ◽  
Guangbing Zhang ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
T Cells ◽  
Antitumor Activity ◽  
Cancer Cells ◽  
Ovarian Cancer 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 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.

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