IMMU-48. RATIONAL DEVELOPMENT AND CHARACTERIZATION OF MULTIVALENT TARGETING TANDEM AND PARALLEL CAR T CELLS FOR GLIOMA

Abstract High grade glioma is the most common malignant primary brain tumor. Despite the best available therapy, the median survival is limited. Chimeric antigen receptor (CAR)-T cells effectively attack target positive tumor cells, demonstrating a promising possible treatment. Based on our previous clinical and pre-clinical studies, we utilized epidermal growth factor receptor variant III (EGFRvIII) and interleukin 13 receptor subunit alpha 2 (IL13Rα2) targeting single-chain variable fragments (scFvs) to generate bi-specific tandem CAR T cells and bi-specific parallel CAR T cells to decrease the potential of antigen escape and tumor recurrence. 5, 10, 15 or 20 flexible amino acids were utilized to link scFvs in tandem CARs. Ribosomal “skipping” 2A peptides were utilized to generate parallel CAR T cells. Flow-based IL13Rα2 staining on glioma stem cells (GSCs) showed 7 out 11 cases positive (64%) heterogeneous intratumoral expression. Tandem CAR T cells and parallel CAR T cells upregulated CD69 expression (P< 0.0001), generated IFNγ, interleukin 2 and TNFα (P< 0.0001), as well as exhibiting cytotoxic activity (P< 0.0001) when compared against un-transduced T cells, in co-culture with either single- or double-target positive GSCs. There was no significant difference between different lengths of linker in tandem CAR T cells. Parallel CAR T cells generated a larger cytokine response (P< 0.0001) and more effectively killed target positive cells (P< 0.05) than tandem CAR T cells. In summary, glioma associated target expression is heterogeneous in GSCs. Multivalent tandem CAR T cells and parallel CAR T cells effectively respond to target expressing tumor cells. The function of tandem CAR T cells is not relative with the length of linker. Parallel CAR T cells are a more promising strategy in generating gene modified multivalent targeting T cells.

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shRNA-Interleukin-6 Modified CD19-Specific Chimeric Antigen Receptor T Cell Significantly Improves the Safety in Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2019-132067 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2621-2621

Author(s):

Liqing Kang ◽

Xiaowen Tang ◽

Nan Xu ◽

Minghao Li ◽

Jingwen Tan ◽

...

Keyword(s):

T Cells ◽

Interleukin 6 ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Mouse Serum ◽

Single Chain ◽

Car T Cells ◽

Significant Difference ◽

Car T ◽

Short Hairpin

[Background] An urgent need exists to enhance the safety in treating hematologic malignancies with CAR-T therapy by reducing the CAR-T-related cytokine release syndrome (CRS) . Interleukin-6 (IL-6) is a central driver of CRS and neurotoxicity; hence, inhibition of the IL-6 of T cells via gene engineering may improve the safety of CAR-T therapy. [Objective] Investigation of the efficacy and safety of IL-6-targeting short hairpin (sh) RNA in the CART-19 (referred to ssCART-19) to determine whether the IL-6 shRNA in T cells can reduce the severe CRS incidence of ssCART-19 treatment. [Methods]We designed a short hairpin RNA sequence which targets the 3'UTR region of the human IL-6 transcript, and the sequence was added to a CAR construct containing the CD19 target single chain variable fragment (scFv), the EF1a promoter, the co-stimulated domain of 4-1BB and the CD3zeta domain. In vitro study, While there is no significant difference in the transduction efficiency, proliferation ability and cytotoxicity efficacy of ssCART-19 comparing to regular CART-19, there was clear inhibition of the IL-6 expression. IL-6 shRNA mediated gene silence of ssCART-19 significantly inhibited IL-6 gene expression at both the mRNA level (P<0.001) and the soluble cytokines level (P≤0.0001). IL-6 expression profile from ssCART-19 showed consistently maintained the lower level over the entire 150 hours of experiment period compared to regular CART-19 cells (P<0.001 ). And add the supernatants from regular CART-19/Raji co-culture and ssCART-19/Raji co-culture system to the primary induced monocytes, respectively, ssCART-19 could significantly reduce the monocytes derived IL-6 expression levels compared to regular CART-19. In vivo study, the preclinical study showed the consistent results that ssCART-19 significantly reduced the mouse serum IL-6 levels compared to regular CART-19, but with similar anti-tumor efficacy. In the clinical trail, 13 patients with the similar tumor burden baseline administrated with ssCART-19 (n=7) or regular CART-19 (n=6) cells with a dose of 5-10x106 CAR-T cells per kilogram over three consecutive days (10%, 30%, 60% split dose). While all patients from both groups achieved complete response and the CAR-T cells exhibit similar expansion ability, patients treated with ssCART-19 had lower CRS grade and significantly lower IL-6 level in the human serum compared to patients treated with regular CART-19 (the peak value of IL-6, P=0.0285, the IL-6 AUC(0-Tmax), P=0.0217). CRS emerged in 6/6 patients in regular CART-19 cohort and 6/7 patients in ssCART-19 cohort, severe CRS with grade 3 or higher was observed in 83.3% of the patients (5/6) treated with regular CART-19 cohort versus only 42.8% of the patients (3/7) treated with ssCART-19 cohorts. Tocilizumab was given to 66.7 % (4/6) of the patients in the regular CART-19 cohort and two patients needed more than one treatment with tocilizumab. In the regular CART-19 group one patient occurred CRES. There was no CAR T-related death. [Conclusion]Our study demonstrated that inhibition of CAR-T derived IL-6 expression by shRNA interfering technology could significantly reduce the severe CRS incidence without affecting their immune-oncotherapy efficacy in treating r/r B-ALL patients, which may provide a potential technology to improve the safety profile and promote the extended use of the CAR-T therapy without sacrificing efficacy. Disclosures No relevant conflicts of interest to declare.

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Contextual reprogramming of CAR-T cells for treatment of HER2+ cancers

Journal of Translational Medicine ◽

10.1186/s12967-021-03132-6 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Zhifen Yang ◽

Lingyu Li ◽

Ahu Turkoz ◽

Pohan Chen ◽

Rona Harari-Steinfeld ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Growth Factor Receptor ◽

Checkpoint Inhibition ◽

Single Chain ◽

Tev Protease ◽

Car T Cells ◽

Car T

Abstract Background Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells combined with checkpoint inhibition may prevent T cell exhaustion and improve clinical outcomes. However, the approach is limited by cumulative costs and toxicities. Methods To overcome this drawback, we created a CAR-T (RB-340-1) that unites in one product the two modalities: a CRISPR interference-(CRISPRi) circuit prevents programmed cell death protein 1 (PD-1) expression upon antigen-encounter. RB-340-1 is engineered to express an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3ζ co-stimulatory domains linked to the tobacco etch virus (TEV) protease and a single guide RNA (sgRNA) targeting the PD-1 transcription start site (TSS). A second constructs includes linker for activation of T cells (LAT) fused to nuclease-deactivated spCas9 (dCas9)-Kruppel-associated box (KRAB) via a TEV-cleavable sequence (TCS). Upon antigen encounter, the LAT-dCas9-KRAB (LdCK) complex is cleaved by TEV allowing targeting of dCas9-KRAB to the PD-1 gene TSS. Results Here, we show that RB-340-1 consistently demonstrated higher production of homeostatic cytokines, enhanced expansion of CAR-T cells in vitro, prolonged in vivo persistence and more efficient suppression of HER2+ FaDu oropharyngeal cancer growth compared to the respective conventional CAR-T cell product. Conclusions As the first application of CRISPRi toward a clinically relevant product, RB-340-1 with the conditional, non-gene editing and reversible suppression promotes CAR-T cells resilience to checkpoint inhibition, and their persistence and effectiveness against HER2-expressing cancer xenografts.

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TriBAFF-CAR-T cells eliminate B-cell malignancies with BAFFR-expression and CD19 antigen loss

Cancer Cell International ◽

10.1186/s12935-021-01923-x ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Guangchao Li ◽

Qing Zhang ◽

Zhi Liu ◽

Huijuan Shen ◽

Yangmin Zhu ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Tumor Cells ◽

Interleukin 2 ◽

B Cell Malignancies ◽

Car T Cells ◽

Car T ◽

Hematological Tumor ◽

Antigen Loss

Abstract Background To investigate the effect of TriBAFF-CAR-T cells on hematological tumor cells. Methods TriBAFF-CAR-T and CD19-CAR-T cells were co-cultured with BAFFR-bearing B-cell malignancies at different effector/target ratios to evaluate the anti-tumor effects. In vivo, TriBAFF-CAR-T and CD19-CAR-T cells were intravenously injected into Raji-luciferase xenograft mice. CD19 antigens losing lymphoblasts was simulated by Raji knocking out CD19 (CD19KO) to investigate the effect of TriBAFF-CAR-T cells on CD19KO Raji. Results Both TriBAFF-CAR-T and CD19-CAR-T cells significantly induced the lysis of Raji, BALL-1, and Jeko-1. Moreover, when CD19-CAR-T cells specifically caused the lysis of K562 with overexpressed CD19, the lethal effect of TriBAFF-CAR-T cells was also specific for BAFFR-bearing K562 with increasing levels of interleukin-2 and INF-γ. The TriBAFF-CAR-T have the same effect with CD19-CAR-T cells in treating Raji xenofraft mice. TriBAFF-CAR-T cells also have great effect in CD19KO Raji cells. Conclusions In this study, we successfully constructed novel TriBAFF-CAR-T cells to eliminate BAFFR-bearing and CD19 antigen loss in hematological tumor cells.

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TRAIL-R1-targeted chimeric antigen receptor T cells exhibit dual antitumor effects

10.22541/au.162187087.70131237/v1 ◽

2021 ◽

Author(s):

AiShun Jin ◽

Yaru Nai ◽

Li Du ◽

Meiying Shen ◽

Tingting Li ◽

...

Keyword(s):

T Cells ◽

Tumor Cells ◽

Human Monoclonal Antibody ◽

Death Receptor ◽

Target Antigen ◽

Single Chain ◽

Antitumor Effects ◽

Apoptotic Pathway ◽

Car T Cells ◽

Car T

Tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL-R1) has limited expression in normal tissues but highly expression in a broad range of tumors, making it an attractive target for cancer immunotherapy. We have previously prepared a fully human monoclonal antibody targeting TRAIL-R1 (TR1419), which can specifically induce apoptosis in antigen-positive tumor cells. Here, we prepared the TR1419CAR-T cells using the single chain variable fragment (scFv) from TR1419, which were evaluated for the phenotypes and function. The TR1419CAR-T cells induced cytolysis of TRAIL-R1-positive tumor cells not only via activation of the death receptor-dependent apoptotic pathway, but also via T-cell mediated cytotoxicity. Furthermore, compared to the second-generation TR1419-28ζ and TR1419-BBζ CAR-T cells, the third-generation TR1419-28BBζ CAR-T cells had greater sensitivity to target antigen, exhibited a better proliferative ability, but showed slightly higher PD-1 expression after antigen stimulation. Altogether, TR1419CAR-T cells, especially TR1419-28BBζCAR-T cells could be a promising treatment strategy for TRAIL-R1 positive tumors.

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112 Rational design of chimeric antigen receptor T cells against glypican 3 decouples toxicity from therapeutic efficacy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0112 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A124-A124

Author(s):

Letizia Giardino ◽

Ryan Gilbreth ◽

Cui Chen ◽

Erin Sult ◽

Noel Monks ◽

...

Keyword(s):

T Cells ◽

Chimeric Antigen Receptor ◽

Antigen Receptor ◽

Single Chain ◽

High Affinity ◽

Car T Cells ◽

Car T ◽

Animal Care ◽

Cytotoxic Function

BackgroundChimeric antigen receptor (CAR)-T therapy has yielded impressive clinical results in hematological malignancies and it is a promising approach for solid tumor treatment. However, toxicity, including on-target off-tumor antigen binding, is a concern hampering its broader use.MethodsIn selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we compared CAR bearing a low and high affinity single-chain variable fragment (scFv,) binding to the same epitope and cross-reactive with murine GPC3. We characterized low and high affinity CAR-T cells immunophenotype and effector function in vitro, followed by in vivo efficacy and safety studies in hepatocellular carcinoma (HCC) xenograft models.ResultsCompared to the high-affinity construct, the low-affinity CAR maintained cytotoxic function but did not show in vivo toxicity. High-affinity CAR-induced toxicity was caused by on-target off-tumor binding, based on the evidence that high-affinity but not low-affinity CAR, were toxic in non-tumor bearing mice and accumulated in organs with low expression of GPC3. To add another layer of safety, we developed a mean to target and eliminate CAR-T cells using anti-TNFα antibody therapy post-CAR-T infusion. This antibody functioned by eliminating early antigen-activated CAR-T cells, but not all CAR-T cells, allowing a margin where the toxic response could be effectively decoupled from anti-tumor efficacy.ConclusionsSelecting a domain with higher off-rate improved the quality of the CAR-T cells by maintaining cytotoxic function while reducing cytokine production and activation upon antigen engagement. By exploring additional traits of the CAR-T cells post-activation, we further identified a mechanism whereby we could use approved therapeutics and apply them as an exogenous kill switch that would eliminate early activated CAR-T following antigen engagement in vivo. By combining the reduced affinity CAR with this exogenous control mechanism, we provide evidence that we can modulate and control CAR-mediated toxicity.Ethics ApprovalAll animal experiments were conducted in a facility accredited by the Association for Assessment of Laboratory Animal Care (AALAC) under Institutional Animal Care and Use Committee (IACUC) guidelines and appropriate animal research approval.

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IMMU-10. ENGINEERED PD-L1 RECEPTORS AUGMENT ANTI-TUMOR ACTIVITY OF HER2 CAR T CELLS IN HIGH GRADE GLIOMA

Neuro-Oncology ◽

10.1093/neuonc/nox083.120 ◽

2017 ◽

Vol 19 (suppl_4) ◽

pp. iv29-iv29

Author(s):

Daniel Landi ◽

Kristen Fousek ◽

Malini Mukherjee ◽

Ankita Shree ◽

Heba Samaha ◽

...

Keyword(s):

T Cells ◽

High Grade Glioma ◽

High Grade ◽

Car T Cells ◽

Car T ◽

Anti Tumor Activity

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121 ICAM-1-specific affinity tuned CAR T cells expressing SSTR2 for real-time imaging

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.121 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A130-A130

Author(s):

Jingmei Hsu ◽

Eric von Hofe ◽

Michael Hsu ◽

Koen Van Besien ◽

Thomas Fahey ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Phase I ◽

Tumor Cells ◽

Somatostatin Receptor ◽

Laboratory Animals ◽

Therapeutic Window ◽

Car T Cells ◽

Car T

BackgroundThe use of CAR T cells for solid tumors has a number of challenges, such as lack of tumor-specific targets, CAR T cell exhaustion, and the immunosuppressive tumor microenvironment. To address these challenges, AffyImmune has developed technologies to affinity tune and track CAR T cells in patients. The targeting moiety is affinity tuned to preferentially bind to tumor cells overexpressing the target while leaving normal cells with low basal levels untouched, thereby increasing the therapeutic window and allowing for more physiological T cell killing. The CAR T cells are designed to express SSTR2 (somatostatin receptor 2), which allows for the tracking of CAR T cells in vivo via PET/CT scan using FDA-approved DOTATATE.MethodsAIC100 was generated by affinity tuning the I-domain of LFA-1, the physiological ligand to ICAM-1. Various mutants with 106-fold difference in affinity were evaluated for affinity. This allowed structure activity relationships to be conducted using CAR T cells expressing the various affinity mutants against targets with varying antigen densities. The variant with micromolar affinity was clearly the most effective in non-clinical animal models. AIC100 is currently being evaluated to assess safety, CAR T expansion, tumor localization, and preliminary activity in patients with advanced thyroid cancer in a phase I study (NCT04420754). Our study uses a modified toxicity probability interval design with three dosage groups of 10 x 106, 100 x 106, and 500 x 106 cells.ResultsPreclinical studies demonstrated greater in vivo anti-tumor activity and safety with lower affinity CAR T cells. A single dose of AIC100 resulted in tumor elimination and significantly improved survival of animals. AIC100 activity was confirmed in other high ICAM-1 tumor models including breast, gastric, and multiple myeloma. In a Phase I patient given 10-million CAR T cells, near synchronous imaging of FDG and DOTATATE revealed preliminary evidence of transient CAR T expansion and tumor reduction at multiple tumor lesions, with the peak of CAR T density coinciding with the spike in CAR T numbers in blood.ConclusionsWe have developed affinity tuned CAR T cells designed to selectively target ICAM-1 overexpressing tumor cells and to spatiotemporally image CAR T cells. Near-synchronous FDG and DOTATATE scans will enhance patient safety by early detection of off-tumor CAR T activity and validation of tumor response. We anticipate that our ‘tune and track’ technology will be widely applicable to developing potent yet safe CAR T cells against hard-to-treat solid cancers.Trial RegistrationNCT04420754Ethics ApprovalIRB number19-12021154IACUC (animal welfare): All animal experiments were performed in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals. Animal handling protocols were approved by the Institutional Laboratory Animal Use and Care Committee of Weill Cornell Medicine (Permit Number: 2012–0063).

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Bispecific c-Met/PD-L1 CAR-T Cells Have Enhanced Therapeutic Effects on Hepatocellular Carcinoma

Frontiers in Oncology ◽

10.3389/fonc.2021.546586 ◽

2021 ◽

Vol 11 ◽

Author(s):

Wei Jiang ◽

Tao Li ◽

Jiaojiao Guo ◽

Jingjing Wang ◽

Lizhou Jia ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

T Cells ◽

Carcinoma Cells ◽

Therapeutic Effects ◽

Single Chain ◽

Antitumor Activities ◽

Hepatocellular Carcinoma Cells ◽

Car T Cells ◽

Car T

T cells expressing chimeric antigen receptors, especially CD19 CAR-T cells have exhibited effective antitumor activities in B cell malignancies, but due to several factors such as antigen escape effects and tumor microenvironment, their curative potential in hepatocellular carcinoma has not been encouraging. To reduce the antigen escape risk of hepatocellular carcinoma, this study was to design and construct a bispecific CAR targeting c-Met and PD-L1. c-Met/PD-L1 CAR-T cells were obtained by lentiviral transfection, and the transfection efficiency was monitored by flow cytometry analysis. LDH release assays were used to elucidate the efficacy of c-Met/PD-L1 CAR-T cells on hepatocellular carcinoma cells. In addition, xenograft models bearing human hepatocellular carcinoma were constructed to detect the antitumor effect of c-Met/PD-L1 CAR-T cells in vivo. The results shown that this bispecific CAR was manufactured successfully, T cells modified with this bispecific CAR demonstrated improved antitumor activities against c-Met and PD-L1 positive hepatocellular carcinoma cells when compared with those of monovalent c-Met CAR-T cells or PD-L1 CAR-T cells but shown no distinct cytotoxicity on hepatocytes in vitro. In vivo experiments shown that c-Met/PD-L1 CAR-T cells significantly inhibited tumor growth and improve survival persistence compared with other groups. These results suggested that the design of single-chain, bi-specific c-Met/PD-L1 CAR-T is more effective than that of monovalent c-Met CAR-T for the treatment of hepatocellular carcinoma., and this bi-specific c-Met/PD-L1 CAR is rational and implementable with current T-cell engineering technology.

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