CAR-T Cell Activation Research Articles

CAR-T manufactured from frozen PBMC yield efficient function with prolonged in vitro production.

Chimeric antigen receptor (CAR)-T cells are engineered to identify and eliminate cells expressing a target antigen. Current manufacturing protocols vary between commercial CAR-T cell products warranting an assessment of these methods to determine which approach optimally balances successful manufacturing capacity and product efficacy. One difference between commercial product manufacturing methods is whether T cell engineering begins with fresh (unfrozen) patient cells or cells that have been cryopreserved prior to manufacture. Starting with frozen PBMC material allows for greater manufacturing flexibility, and the possibility of collecting and storing blood from patients prior to multiple lines of therapy. We prospectively analyzed if second generation anti-CD19 CAR-T cells with either CD28 or 4-1BB co-stimulatory domains have different phenotype or function when prepared side-by-side using fresh or cryopreserved PBMCs. We found that cryopreserved PBMC starting material is associated with slower CAR-T cell expansion during manufacture but does not affect phenotype. We also demonstrate that CAR-T cell activation, cytokine production and in vitro anti-tumor cytotoxicity were not different when CAR-T cells were manufactured from fresh or cryopreserved PBMC. As CAR-T cell therapy expands globally, the need for greater flexibility around the timing of manufacture will continue to grow. This study helps support the concept that cryopreservation of PBMCs could be the solution to these issues without compromising the quality of the final CAR-T product.

Membrane anchored IL-18 linked to constitutively active TLR4 and CD40 improves human T cell antitumor capacities for adoptive cell therapy

BackgroundAdoptive transfer of tumor-infiltrating lymphocytes (TILs) or blood T cells genetically redirected by an antitumor TCR or CAR induces a strong antitumor response in a proportion of patients with cancer;...

A living drug: application of CAR-T therapy for lymphoid malignancies and beyond

The ongoing development of novel personalized cancer therapies has resulted in the implementation of T cells enriched with synthetic chimeric antigen receptors, known as chimeric antigen receptors T cell (CAR-T) cells, into clinical practice. CAR-T cells are able to recognize and bind specific antigens present on the surface of target cells — so-called tumor-associated antigens. This innovative method has been approved for the treatment of hematological malignancies and may also serve as a bridge to hematopoietic stem cell transplantation. The production of the drug containing modified T cells consists of several steps — leukapheresis, T cell activation, transduction and expansion of the final CAR-T cells. Activation of CAR-T cells occurs through a pathway independent of the major histocompatibility complex, which is often associated with uncontrolled responses from the immune system and adverse reactions such as cytokine release syndrome. CAR-T therapy can only be performed in certified centers, and requires close cooperation between experienced specialists of different medical disciplines. This is what determines its effectiveness. Every step from collection and cryopreservation, through transport and modification, to thawing and infusion is strictly controlled because it has a critical impact on the quality and efficiency of the drug. Despite its proven benefits, CAR-T therapy remains available only to patients who meet well-defined criteria. These however are liable to change with the emergence of new indications.

Size-dependent activation of CAR-T cells.

As the targets of chimeric antigen receptor (CAR)-T cells expand to a variety of cancers, autoimmune diseases, viral infections, and fibrosis, there is an increasing demand for identifying new antigens and designing new CARs that can be effectively activated. However, the rational selection of antigens and the design of CARs are limited by a lack of knowledge regarding the molecular mechanism by which CARs are activated by antigens. Here, we present data supporting a "size exclusion" model explaining how antigen signals are transmitted across the plasma membrane to activate the intracellular domains of CARs. In this model, antigen engagement with CAR results in a narrow intermembrane space that physically excludes CD45, a bulky phosphatase, out of the CAR zone, thus favoring CAR phosphorylation by kinases, which further triggers downstream pathways leading to T cell activation. Aligned with this model, increasing the size of CAR extracellular domains diminished CAR-T activation both in vitro and in a mouse lymphoma model; membrane-proximal epitopes activated CAR-Ts better than membrane-distal epitopes. Moreover, increasing the size of CD45 by antibody conjugation enhanced the activation of CARs that recognize membrane-distal epitopes. Consistently, CAR-Ts expressing CD45RABC, the larger isoform, were activated to a higher level than those expressing a smaller isoform CD45RO. Together, our work revealed that CAR-T activation depends on the size difference between the CAR-antigen pair and CD45; the size of CAR, antigen, and CD45 can thus be targets for tuning CAR-T activation.

Abstract 693: TGFβ-mediated targeting of the extracellular matrix enhances the migration and cytotoxicity of CAR-T cells in 3D models of ovarian cancer

Abstract We have assessed the utility of 3-dimensional (3D) in vitro human cell models to understand barriers to chimeric antigen receptor (CAR)-T cell activity in high-grade serous ovarian cancer, (HGSOC) a disease with a poor response to immunotherapy. We defined mucin-1 (MUC1) as a potential target in HGSOC biopsies and the HGSOC cell lines, OvCAR3 and G164. We then generated CAR-T cells against MUC1 and tested them in spheroid and collagen gel cultures. In spheroids, although CAR-T cells killed OvCAR3 cells, G164 cells failed to induce CAR-T cell activation or cytotoxicity. However, when we added primary omental fibroblasts from ovarian cancer patients to G164 spheroids, CAR-T cells were activated and cytotoxic. Fibroblast conditioned medium also activated CAR-T cells to kill G164 cells in spheroids and this was due to their production of C-C motif chemokine ligand 2 (CCL2). Further experiments revealed that CCL2 produced by fibroblast stimulated CCR2/4 positive CAR-T cells to a higher state of activation, which enhanced the cytotoxicity of CAR-T cells against G164 cells. We then investigated CAR-T cell activity in co-cultures of OvCAR3 or G164 cells and primary fibroblasts embedded in collagen. CAR-T cells migrated into OvCAR3 gels and killed the malignant cells during a three-day period. However, CAR-T cells failed to migrate into gels with G164 cells and there was no malignant cell killing. Gels containing G164 cells had denser extracellular matrix (ECM) than OvCAR3 gels, as measured by staining for collagens and fibronectin. Previously, we showed that transforming growth factor-beta (TGFβ) secreted by HGSOC cells acted on fibroblasts to induce the production of ECM in collagen gels.1 Treating G164 gels with the TGFβ receptor inhibitor SB431542 reduced ECM density, stimulated CAR-T cell migration and restored CAR-T cell cytotoxicity against G164 cells. Using these different human 3D models we have demonstrated that malignant cell intrinsic factors can cause resistance to CAR-T cells. Sensitivity to CAR-T cell killing can be modulated both positively and negatively by fibroblasts. Targeting ECM along with CAR-T cell therapy might improve the efficiency of CAR-T cells in solid tumors. 1Delaine-Smith et al, iScience, 2021 Citation Format: Joash Dominic Joy, Beatrice Malacrida, Florian Laforêts, Panoraia Kotantaki, Eleni Maniati, Sarah Hopkins, Ianire Calleja, Sara Brett, Takis Athanasopoulos, Sadfer Ali, Peter Emery-Billcliff, Ida Ricciardelli, Charlotte Kay, Jayne Colebrook, Magda Ali, Katherine Strong, Frances Balkwill. TGFβ-mediated targeting of the extracellular matrix enhances the migration and cytotoxicity of CAR-T cells in 3D models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 693.

Abstract 2855: Antitumor activity of BAFF-R targeting CAR T-cells on chronic lymphocytic leukemia

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in adults with CD19-targeting CAR T-cell therapy showing an excellent initial curative response. However, 10-20% of patients who receive anti-C19 CAR T-cell treatment experience relapse due to CD19 antigen loss. B-cell activating factor receptor (BAFF-R) that is expressed on the surface of normal and neoplastic B cells mediates the survival of mature peripheral B cells along with the B-cell receptor. Since B-CLL is characterized by apoptotic disruption and subsequent accumulation of mature B cells, BAFF-R is a logical, novel target for CAR T-cell therapy directed against B-CLL, especially if CD19 antigen loss occurs. In this study, we first manufactured BAFF-R CAR T-cells by infecting healthy donor Tnaïve/memory (Tn/mem) cells with BAFF-R CAR lentivirus before evaluating the antitumor activity of BAFF-R CAR T-cells on wild type MEC-1 (a B-CLL cell line), modified MEC-1, and primary cell lines. Antigen-specific cytotoxicity and activation of the BAFF-R CAR T-cells or control CD19 CAR T-cells were determined upon incubation with B-CLL cell lines and patient-derived primary B-CLL by measuring degranulation and IFN-γ release, respectively. BAFF-R CAR T-cells demonstrated substantial cytotoxicity against MEC-1, CD19-deficient MEC-1, and primary B-CLL cell lines; CD19 CAR T-cells showed no effect on CD19-deficient MEC-1 cells but did show effects with primary B-CLL and MEC-1 cells. Similarly, the release of IFN-γ was greater when BAFF-R CAR T-cells were co-cultured with CD19-deficient MEC-1 or primary B-CLL compared to CD19 CAR T-cells co-cultured with CD19-deficient MEC-1. In summary, BAFF-R CAR T-cells exert significant antitumor properties not only on patient-derived primary B-CLL cells but also on a CD19-deficient B-CLL cell line. We, therefore, assert BAFF-R-targeting CAR T-cells to be a novel therapeutic avenue for patients newly diagnosed with B-CLL and patients experiencing relapsed B-CLL after anti-CD19 therapy. Citation Format: Yan Luo, Kaihin Lui, Tommy T. To, Andrew liu, Farah Yassine, Mohamed A. Kharfan Dabaja, Martha E. Gadd, Hong Qin. Antitumor activity of BAFF-R targeting CAR T-cells on chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2855.

Abstract 2843: Development of a translatable CD19 CAR-T platform for drug discovery

Abstract Chimeric Antigen Receptor (CAR)-T cell therapy has achieved great success with specificity, efficacy, safety and ingenuity. CAR cell technology is advancing rapidly, with ever increasing capabilities for cancer treatment both with regard to tumor types and the CAR cell used (NK, Macrophage, T). To date, the most successful application remains the treatment of B cell lymphoma using CD19-specific CAR-T cells. Here we describe how to efficiently generate and expand a CD19 CAR-T cell and validate their efficacy in in-vitro and in-vivo studies. HLA-A*02:01+ CD3+ T-cells were transduced to express a clinically tested CD19-specific CAR to >80% efficiency and reproducibly expanded to >68 fold over a 19-day period. After sufficient expansion, cells were validated in in-vitro studies to assess their efficacy, utilizing a HLA-A*02:01+ patient derived xenograft (PDX) CD19+ cell line, alongside human cancer cell lines RAJI (CD19+) and K562 (CD19-). Flow cytometry analysis revealed the potent ability of CD19-CAR-T cells to kill CD19+ targets in a dose-dependent fashion. Highly specific killing at all E:T ratios was observed, with negligible off target killing identified. Supportive supernatant analysis showed highly elevated pro-inflammatory cytokine concentrations, identifying enhanced activation of CAR-T cells in the presence of CD19+ target cells only. After successful in-vitro batch testing for on-target killing CAR-T cells were released for in-vivo survival studies. Mice were inoculated with the same strains of CD19+ cells used during in-vitro validation to produce a tumor burdened model. CAR-T cells or non-transduced cells from the same donor were transfused and survival rates, tumor progression and tolerability assessments were observed over several weeks. Ex-vivo tissue and sera analysis further characterized these highly efficacious human CAR-T cells. These assays represent the capability to produce, expand and study CAR-effector cells both in-vitro and in-vivo. Careful consideration of human target cells enables the efficient translation of in-vitro efficacy data to whole organism mouse models. Citation Format: Lauren Kelsey, Jezrom Self-Fordham, Eva Oswald, Ilona Aylott, Daniel Rocca, Robert Nunan, David Harris, Julia Schueler, Rhiannon Jenkinson. Development of a translatable CD19 CAR-T platform for drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2843.

Abstract 2848: Incorporation of inhibitory signaling domains into chimeric antigen receptors (iCAR) designed for self-regulation of canonical CAR-T to treat solid tumors

Abstract Chimeric antigen receptor (CAR)-T cell therapy is a groundbreaking cancer treatment that has produced remarkable clinical efficacy for hematopoietic malignancies, yet “on-target off-tumor” toxicity due to lack of target specificity limits the therapeutic potential of CAR-T cells in solid tumors. We are developing dual CAR-T cells composed of a canonical activator CAR (aCAR), intended to elicit efficacy in solid tumors, and an inhibitory CAR (iCAR) designed to efficiently inhibit the aCAR activity in normal tissue and vital organs. The iCAR and aCAR scFvs of this system bind distinct cell-surface antigens that are widely co-expressed on the normal epithelial from which solid tumors originate. Upon antigen binding, signal propagation through iCAR cytoplasmic domain (iDomain) counteracts aCAR induction of CAR T-cell activation and killing; thus protecting normal tissue. Dual CAR-T cells are activated and kill when exposed to tumor cells that have lost iCAR target expression due to chromosomal loss-of-heterozygosity (LOH) which is common in all cancers. iCAR-targeted LOH generates absolute and irreversible tumor specificity therefore tumor-specific overexpression of the iCAR or aCAR targets is not required. We have carried out a comprehensive screen using human T-cells to identify the most effective inhibitory iDomains derived from cell-surface receptors that naturally inhibit or moderate immune cell activation. The cell-based assay suite developed for this screen consisted of cell lines that co-express iCAR and aCAR target at clinically relevant levels and isogenic partners that mimic LOH through CRISPR editing of the iCAR target. The expression of dual CARs in T cells as biscistronic constructs introduced with lentiviral vectors was successful in a limited number of cases, and it was necessary to develop alternative methods of iCAR aCAR co-expression to complete the screen. Combining aCAR transduction with iCAR mRNA Electroporation (T-REP) proved to be a useful method to disentangle iDomain potency and expression level. The screen identified new iDomains that may expand the potential of iCAR technology in developing strategies to treat solid tumors without compromising efficacy for safety. Citation Format: David Bassan, Jason Yi, Neta Chaim, Nir Bujanover, Sarit Tabak, Tanya Kim, Yael Lopesco, Leehee Weinberger, Kristina Vucci, Michael Weist, Caitlin Schnair, Gregor B. Adams, Orit Foord, Frank J. Calzone, Rick Kendall, Adi Sharbi-Yunger. Incorporation of inhibitory signaling domains into chimeric antigen receptors (iCAR) designed for self-regulation of canonical CAR-T to treat solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2848.

Inducible localized delivery of an anti-PD-1 scFv enhances anti-tumor activity of ROR1 CAR-T cells in TNBC

BackgroundChimeric antigen receptor (CAR)-T cells can induce powerful immune responses in patients with hematological malignancies but have had limited success against solid tumors. This is in part due to the immunosuppressive tumor microenvironment (TME) which limits the activity of tumor-infiltrating lymphocytes (TILs) including CAR-T cells. We have developed a next-generation armored CAR (F i-CAR) targeting receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is expressed at high levels in a range of aggressive tumors including poorly prognostic triple-negative breast cancer (TNBC). The F i-CAR-T is designed to release an anti-PD-1 checkpoint inhibitor upon CAR-T cell activation within the TME, facilitating activation of CAR-T cells and TILs while limiting toxicity.MethodsTo bolster potency, we developed a F i-CAR construct capable of IL-2-mediated, NFAT-induced secretion of anti-PD-1 single-chain variable fragments (scFv) within the tumor microenvironment, following ROR1-mediated activation. Cytotoxic responses against TNBC cell lines as well as levels and binding functionality of released payload were analyzed in vitro by ELISA and flow cytometry. In vivo assessment of potency of F i-CAR-T cells was performed in a TNBC NSG mouse model.ResultsF i-CAR-T cells released measurable levels of anti-PD-1 payload with 5 h of binding to ROR1 on tumor and enhanced the cytotoxic effects at challenging 1:10 E:T ratios. Treatment of established PDL1 + TNBC xenograft model with F i-CAR-T cells resulted in significant abrogation in tumor growth and improved survival of mice (71 days), compared to non-armored CAR cells targeting ROR1 (F CAR-T) alone (49 days) or in combination with systemically administered anti-PD-1 antibody (57 days). Crucially, a threefold increase in tumor-infiltrating T cells was observed with F i-CAR-T cells and was associated with increased expression of genes related to cytotoxicity, migration and proliferation.ConclusionsOur next-generation of ROR1-targeting inducible armored CAR platform enables the release of an immune stimulating payload only in the presence of target tumor cells, enhancing the therapeutic activity of the CAR-T cells. This technology provided a significant survival advantage in TNBC xenograft models. This coupled with its potential safety attributes merits further clinical evaluation of this approach in TNBC patients.

A next generation inducible armored CAR to overcome the immunosuppressive tumor microenvironment and enhances cytotoxicity of CAR-T and TILs.

e14517 Background: The full potential of CAR-T cells in clinic is limited by the immunosuppressive tumour microenvironment (TME), which induces expression of exhaustion markers and limits the activity of tumour infiltrating lymphocytes (TILs) including CAR-T cells. We developed a next generation inducible Armored CAR platform (aCAR) that releases an anti-PD-1 checkpoint inhibitor upon CAR-T cell activation, limiting payload release exclusively to the TME, thereby reducing the risk of systemic exposure. This differentiated strategy facilitates activation of CAR-T cells and TILs within the TME and has the potential for lower toxicity. Methods: A ROR1-targeting second-generation CAR containing a 41BB-CD3ζ intracellular domain was cloned into a lentivirus transfer vector alongside a prototypic anti-PD1 antibody. The CAR was under the control of a constitutively active promoter, and the PD1 targeting payload was controlled by an inducible promoter that was activated upon engagement of CAR with ROR1 on tumour cells. CAR-T cells were subject to in vitro co-culture assays with target ROR1+ tumour cells and their cytotoxic responses evaluated by flow cytometry. Levels and binding functionality of released payload were analyzed by ELISA and flow cytometry. In vivo xenograft models were performed in NSG mice with tumor growth assessed by bioluminescent imaging (BLI) and caliper measurement of the tumour volume. Results: Non-Armored CAR cells displayed potent and specific cytotoxic responses directed towards ROR1+ TNBC and NSCLC cells lines at different effector to target (E:T) ratios. The results were equivalent or superior to CARs generated with comparator anti-ROR1 antibodies, which may be due to the ROR1 CAR targeting a membrane proximal epitope within the ROR1 frizzled domain. The aCAR cells released measurable levels of anti-PD1 payload within 5 hours of binding to ROR1 on tumors and enhanced the cytotoxic effects at challenging 1:10 E:T ratios. Established PDL1+ TNBC xenograft models using the aCAR cells and comparing with non-Armored CAR cells displayed a qualitative abrogation in tumor growth by BLI, which was confirmed and shown to be significant by caliper measurement of the tumor volume. Continuing the experiment out to 3 months showed a significant survival advantage for the animals receiving aCAR. All other cohorts were terminated by day 70, however 20% of the aCAR cohort survived at day 95. Conclusions: Our next generation inducible aCAR platform enables the release of an immune stimulating payload only in the presence of target tumor cells, enhancing the therapeutic activity of the CAR-T cells and limiting payload exposure to the site of action. This technology provided a significant survival advantage in challenging in vivo xenograft models. This coupled with its potential safety attributes merits further clinical evaluation of this approach.

Delivery of CAR-T cells in a transient injectable stimulatory hydrogel niche improves treatment of solid tumors.

Adoptive cell therapy (ACT) has proven to be highly effective in treating blood cancers, but traditional approaches to ACT are poorly effective in treating solid tumors observed clinically. Novel delivery methods for therapeutic cells have shown promise for treatment of solid tumors when compared with standard intravenous administration methods, but the few reported approaches leverage biomaterials that are complex to manufacture and have primarily demonstrated applicability following tumor resection or in immune-privileged tissues. Here, we engineer simple-to-implement injectable hydrogels for the controlled co-delivery of CAR-T cells and stimulatory cytokines that improve treatment of solid tumors. The unique architecture of this material simultaneously inhibits passive diffusion of entrapped cytokines and permits active motility of entrapped cells to enable long-term retention, viability, and activation of CAR-T cells. The generation of a transient inflammatory niche following administration affords sustained exposure of CAR-T cells, induces a tumor-reactive CAR-T phenotype, and improves efficacy of treatment.

Signaling Dynamics of TSHR-Specific CAR-T Cells Revealed by FRET-Based Biosensors.

Although most patients with thyroid cancers have good prognosis and long-term survival, some patients are refractory to traditional therapeutic approaches and face a high risk of mortality. CAR-T therapy provides an attractive strategy to treat these patients. Considering the limited expression in thyroid tissues, thyroid-stimulating hormone receptor (TSHR) has been considered as a promising candidate as CAR-T target. However, it is still a challenge to find the optimal CAR design for the treatment of thyroid cancers. Dynamic signaling cascade is initiated by CAR molecules during CAR-T cell activation. The development of FRET-based biosensors enables us to detect the signaling dynamics of key kinases during CAR-T cell activation with high spatiotemporal resolution. Here using the ZAP70 and ERK biosensors, we visualized the dynamics of ZAP70 and ERK activities in TSHR-specific CAR-T cells upon antigen stimulation. We first constructed several TSHR-targeting CARs for the treatment of advanced thyroid cancers. The TSHR CAR-T cells with CD28 or 4-1BB co-stimulatory signaling domains exhibited potent cytotoxicity in vitro. By FRET imaging, we observed rapid increase of ZAP70 and ERK activities in TSHR CAR-T cells upon target cell binding. Even though CD28-based CAR-T cells had similar ZAP70 activation dynamics as 4-1BB-based CAR-T cells, they displayed slightly enhanced ERK activation, which may contribute to their faster anti-tumor kinetics in vivo. These results demonstrated the efficacy of TSHR CAR-T cells to treat advanced thyroid cancers. Our study indicated the potential of applying FRET biosensors to optimize the design of CAR for effective CAR-T therapy.

A Reversible Chemogenetic Switch for Chimeric Antigen Receptor T Cells.

As a revolutionary cancer treatment, the chimeric antigen receptor (CAR) T cell therapy suffers from complications such as cytokine release syndromes and T cell exhaustion. Their mitigation desires controllable activation of CAR-T cells that is achievable through regulatory display of CARs. By embedding the hepatitis C virus NS3 protease (HCV-NS3) between the single-chain variable fragment (scFv) and the hinge domain, we showed that the display of anti-CD19 scFv on CAR-T cells was positively correlated to the presence of a clinical HCV-NS3 inhibitor asunaprevir (ASV). This novel CAR design that allows the display of anti-CD19 scFv in the presence of ASV and its removal in the absence of ASV creates a practically reversible chemical switch. We demonstrated that the intact CAR on T cells can be repeatedly turned on and off by controlling the presence of ASV in a dose dependent manner both in vitro and in vivo, which enables delicate modulation of CAR-T activation during cancer treatment.

Recent advances in the prevention and management of cytokine release syndrome after chimeric antigen receptor T-cell therapy

Adoptive immunotherapy has recently garnered widespread interests owing to the successful application of chimeric antigen receptor T cell therapy. CAR-T cells are “living drugs” that can live in patients for several years and act as an effective antitumor agent. Over the last few years, five types of CAR-T cells have been approved by Food and Drug Administration (FDA) for treatment of hematologic malignancies. Despite their impressive clinical efficacy, the current application of CAR-T cell therapy is restricted by the uncontrollable release of cytokines (cytokine release syndrome and cytokine release syndrome) due to serious treatment-related toxicities resulting from synchronous activation and rapid proliferation of CAR-T cells. CRS is the most common toxicity and its severity can range from low-grade physical symptoms to a high-grade syndrome linked with life-threatening multiple organ dysfunction. Treatment-related deaths from severe CRS have been reported, suggesting the importance of appropriate intervention. Gaining a better understanding of CRS and developing new treatments for CRS are active areas of laboratory and clinical research. Herein, we summarize the current studies on prevention and management of CRS to expand the safety and applicability of CAR-T cell therapy in various malignancies.

IMMU-45. COMBINATION OF EGFRVIII CAR T-CELL THERAPY AND PARACRINE GAM MODULATION FOR THE TREATMENT OF GBM

Abstract The GBM immune tumor microenvironment mainly consists of protumoral glioma-associated microglia and macrophages (GAMs). We have previously shown that blockade of CD47, a ‘don't eat me’-signal overexpressed by GBM cells, rescued GAMs' phagocytic function in mice. However, monotherapy with CD47 blockade has been ineffective in treating human solid tumors to date. Thus, we propose a combinatorial approach of local CAR T cell therapy with paracrine GAM modulation for a synergistic elimination of GBM. We generated humanized EGFRvIII CAR T-cells by lentiviral transduction of healthy donor human T-cells and engineered them to constitutively release a soluble SIRPγ-related protein (SGRP) with high affinity towards CD47. Tumor viability and CAR T-cell proliferation were assessed by timelapse imaging analysis in co-cultures with endogenous EGFRvIII-expressing BS153 cells. Tumor-induced CAR T-cell activation and degranulation were confirmed by flow cytometry. CAR T-cell secretomes were analyzed by liquid chromatography-mass spectrometry. Immunocompromised mice were orthotopically implanted with EGFRvIII+ BS153 cells and treated intratumorally with a single CAR T-cell injection. EGFRvIII and EGFRvIII-SGRP CAR T-cells killed tumor cells in a dose-dependent manner (72h-timepoint; complete cytotoxicity at effector-target ratio 1:1) compared to CD19 controls. CAR T-cells proliferated and specifically co-expressed CD25 and CD107a in the presence of tumor antigen (24h-timepoint; EGFRvIII: 59.3±3.00%, EGFRvIII-SGRP: 52.6±1.42%, CD19: 0.1±0.07%). Differential expression analysis of CAR T-cell secretomes identified SGRP from EGFRvIII-SGRP CAR T-cell supernatants (-Log10qValue/Log2fold-change= 3.84/6.15). Consistent with studies of systemic EGFRvIII CAR T-cell therapy, our data suggest that intratumoral EGFRvIII CAR T-cells were insufficient to eliminate BS153 tumors with homogeneous EGFRvIII expression in mice (Overall survival; EGFRvIII-treated: 20%, CD19-treated: 0%, n= 5 per group). Our current work focuses on the functional characterization of SGRP binding, SGRP-mediated phagocytosis, and on the development of a translational preclinical model of heterogeneous EGFRvIII expression to investigate an additive effect of CAR T-cell therapy and GAM modulation.

A Novel Cotinine-Based System for Switchable Chimeric Antigen Receptor T Cell Immunotherapy

Autologous T cells engineered to express a chimeric antigen receptor (CAR-T cells) have shown impressive outcomes in hematologic malignancies. However, safety concerns derived from the inability to control CAR-T cells in vivo remain a significant challenge to expand the application of CAR-T cell therapy. Switchable CAR-T cells can allow for turning on and off the activation and cytotoxicity of CAR-T cells in vivo, potentially increasing the safety of next-generation CART products. To this goal, CAR constructs targeting non-tumor antigens, e.g., haptens and peptide tags, can be designed and combined with tumor-targeting units that are fused to the hapten or peptide tag. In this study, we developed a novel switchable CAR-T system using cotinine as the hapten and an anti-cotinine CAR-T cell product. Of note, cotinine is physiologically absent in human and pharmacologically inert. A mouse antibody to cotinine was developed and successfully humanized. The anti-cotinine CAR confirmed to be displayed on T-cell surface. We first tested this approach to target epidermal growth factor receptor 2 (HER2) in ovarian cancer. To target HER2, we developed an affibody-based antigen binding domain characterized by high stability, broad spectrum of specificity, and feasibility of large scale chemical synthesis. In in vitro cytotoxicity and cytokine release assays, the activity of CAR-T cells was controlled by cotinine-tagged anti-HER2 affibody in a HER2-specific and dose-dependent manner. We synthesized the cotinine-tagged HER2 affibodies using single isoform cotinine and selected the optimal cotinine-tagged anti-HER2 affibody at the cotinine-based switchable CAR-T system. In an orthotopic ovarian cancer model using NSG mice engrafted with SKOV3, tumor regression was observed only when CAR-T cells were administered in combination with the cotinine-tagged HER2 affibody. In subsequent studies, we demonstrated that the cotinine-based switchable CAR-T system can be applied to other tumor targets such as mesothelin and epidermal growth factor receptor (EGFR). Additional studies are ongoing to determine the kinetics of the on and off CAR-T activation of this system and define the optimal dosing strategy to ensure anti-tumor effect or to interrupt toxicity. In conclusion, we have developed a novel switchable cotinine-based CAR-T system to control CAR-T function in vivo and that could be broadly applicable to wide variety of tumors. DisclosuresKim: AbClon Inc.: Current Employment. Lee: AbClon Inc.: Current Employment. Ruella: viTToria biotherapeutics: Research Funding; Tmunity: Patents & Royalties; AbClon: Consultancy, Research Funding; BMS, BAYER, GSK: Consultancy; Novartis: Patents & Royalties. Chung: AbClon Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Bispecific CAR-T Cells Targeting Both BCMA and CD24: A Potentially Treatment Approach for Multiple Myeloma

Introduction: Anti-myeloma BCMA-specific chimeric antigen receptor (CAR) T-cell therapies represent a promising new treatment strategy, with high response rates observed in the early stages of therapy. However, the responses are not durable. One known mechanism of relapse has been traced to the loss of BCMA expression following long-term CAR-T therapy. Another potential reason is that while BCMA CAR-T cells eliminate the bulk of BCMA-positive MM cells, a small subset of BCMA-negative, very drug-resistant MM cells, such as tumor-initiating cells (TICs) survive and seed relapses. There is a strong correlation between the presence of MM TICs with minimal residual disease, acquired drug resistance and relapse. This suggests that TIC-targeted therapies could improve outcomes. We have previously demonstrated that MM cells expressing CD24 also exhibit features of TICs, e.g. self-renewal, increased expression of embryonic stem cell genes and drug resistance. We have generated bispecific CAR-T cells which recognize both BCMA and CD24 antigens and have tested their therapeutic efficacy in MM cells in vitro and in vivo models.Methods: We constructed a bispecific BCMA-CD24 CAR vector, with 2 complete CAR units: BCMA CAR and CD24 CAR. P2A was inserted between these two CARs. The BCMA CAR contains a safety switch in the hinge region, and a CD28 co-activation domain with CD3ζ. The CD24 CAR contained a 4-1BB co-activation domain with CD3ζ. To decrease the risk of severe immunological side effects, we integrated RQR8, an immunological safety switch with epitopes for CD34 and CD20 as a suicide molecule into the hinge region. Lentivirus particles were used to transduce primary human T cells. CAR-T cells were detected on day 7 by flow cytometry using antibodies to CD34. We performed co-culture killing assays, detected the T cell activation marker CD69 and measured the cytokines in the supernatant. We determined whether BCMA-CD24 CAR-T cells targeted the TIC population by flow cytometry and microscopy. The NOD. Cg-Prkdc scidIl2rg tm1Wjl/SzJ (NSG) xenograft mouse model was used for in vivo studies. 8-week-old NSG mice were administered 2 × 10 6 MM cells by intravenous injection. On day 7 after MM cells injection, 1 × 10 6 CAR-T cells were administered. Mice were weighed and monitored for signs of distress every two days. Bioluminescence images were acquired 10 min after D-luciferin injection. Myeloma progression was monitored every 7 days until the mice develop hind limb paralysis or the bioluminescence signal (ROI) is more than 2 × 10 10.Results: CAR-T cells were detected by flow cytometry using the RQR8-specific CD34 antibody. The BCMA-CD24 CAR was found to be expressed on roughly 13% of T-cells. To determine the selective lysis by the CAR-T cells, we performed co-culture killing assays in which MM cell lines over-expressing CD24 (ARP-1 CD24OE or OCI CD24OE cells) were incubated with CAR-T cells. When the CAR-T: MM ratio was 5:1, the lysis percentage of target cells was 99% (ARP-1 CD24OE) and 89% (OCI CD24OE). CAR-T cell activation was determined by increased CD69 expression and IL-2 production. As expected, exposure to CD24 + MM cells resulted in strong activation of CAR-T cells, and CAR-T cells did target and kill the TIC population. Bioluminescence imaging showed CAR-T mediated antitumor activity, yielding near-complete tumor clearance. Additionally, mice treated with CAR-T cells exhibited increased survival compared with mice in the control groups.Conclusion and Significance: This study developed a BCMA-CD24 CAR-T, a novel MM immunotherapy. We have demonstrated strong cytotoxic activity and selectivity for MM cells in vitro and in vivo. Future studies will be aimed at determining if BCMA-CD24 CAR-T can target TIC-mediated relapses. DisclosuresNo relevant conflicts of interest to declare.

Engineered Removal of PD-1 From the Surface of CD19 CAR-T Cells Results in Increased Activation and Diminished Survival.

CAR-T cell therapy is the most advanced way to treat therapy resistant hematologic cancers, in particular B cell lymphomas and leukemias, with high efficiency. Donor T cells equipped ex vivo with chimeric receptor recognize target tumor cells and kill them using lytic granules. CAR-T cells that recognize CD19 marker of B cells (CD19 CAR-T) are considered the gold standard of CAR-T therapy and are approved by FDA. But in some cases, CD19 CAR-T cell therapy fails due to immune suppressive microenvironment. It is shown that tumor cells upregulate expression of PD-L1 surface molecule that binds and increases level and signal provided by PD-1 receptor on the surface of therapeutic CAR-T cells. Induction of this negative signaling results in functional impairment of cytotoxic program in CAR-T cells. Multiple attempts were made to block PD-1 signaling by reducing binding or surface level of PD-1 in CAR-T cells by various means. In this study we co-expressed CD19-CAR with PD-1-specific VHH domain of anti-PD-1 nanobody to block PD-1/PD-L1 signaling in CD19 CAR-T cells. Unexpectedly, despite increased activation of CAR-T cells with low level of PD-1, these T cells had reduced survival and diminished cytotoxicity. Functional impairment caused by disrupted PD-1 signaling was accompanied by faster maturation and upregulation of exhaustion marker TIGIT in CAR-T cells. We conclude that PD-1 in addition to its direct negative effect on CAR-induced signaling is required for attenuation of strong stimulation leading to cell death and functional exhaustion. These observations suggest that PD-1 downregulation should not be considered as the way to improve the quality of therapeutic CAR-T cells.

Speed and Location Both Matter: Antigen Stimulus Dynamics Controls CAR-T Cell Response.

Despite the success in B-cell malignancies, chimeric antigen receptor (CAR)-T cell therapies have not yet demonstrated consistent efficacy across all patients and tumor types, particularly against solid tumors. Higher rates of T cell exhaustion are associated with inferior clinical outcomes following CAR-T cell therapy, which is prevalent in solid tumors. T cell exhaustion may originate from persistent and chronic antigen stimulation by tumor cells that resist and/or evade T cell-mediated killing. We exploited CAR-T exhaustion with a classic negative feedback model (incoherent feedforward loop, IFFL) to investigate the balance between CAR-T cell activation and exhaustion under different antigen presentation dynamics. Built upon the experimental and clinical data, we hypothesize that the speed and anatomical location of antigenic stimulation are both crucial to CAR-T cell response. Chronic antigenic stimulation as well as the harsh tumor microenvironment present multiple barriers to CAR-T cell efficacy in solid tumors. Many therapeutic strategies are individually insufficient to improve of CAR-T responses against solid tumors, as they clear but one of the many barriers CAR-T cells face in solid tumors. A combination strategy targeting multiple barriers holds promise to improve CAR-T therapy in solid tumors.

Pembrolizumab for B-cell lymphomas relapsing after or refractory to CD19-directed CAR T-cell therapy

AbstractCD19-directed chimeric antigen receptor–modified (CAR T) T cells achieve durable remissions in about 30% to 40% of relapsed/refractory large B-cell lymphomas. T-cell exhaustion and/or an immunosuppressive tumor microenvironment may contribute to CAR T-cell failure. Pembrolizumab, an anti-PD1 immune checkpoint inhibitor, may reverse T-cell exhaustion after CAR T-cell therapy. We treated 12 patients with B-cell lymphomas who were either refractory to (n = 9) or relapsed after (n = 3) CD19-directed CAR T-cell (4-1BB–costimulated) therapy with pembrolizumab 200 mg IV every 3 weeks. Median time from CAR T-cell infusion to first pembrolizumab dose was 3.3 months (range, 0.4-42.8 months). Pembrolizumab was well tolerated, and the only grade ≥3 adverse events related to pembrolizumab were neutropenia (n = 3; 25%). Best overall response rate after pembrolizumab was 25% (3 of 12 patients; 1 complete response; 2 partial responses). One (8%) patient had stable disease; thus, 4 of 12 (33%) patients had clinical benefit. After pembrolizumab, 4 patients with clinical benefit had an increase in percentage of CAR T cells by mass cytometry by time of flight (CyTOF); 3 of 4 of these patients also had increases in CAR19 transgene levels by quantitative polymerase chain reaction. Deep immune profiling using CyTOF revealed increased CAR T-cell activation and proliferation and less T-cell exhaustion in clinical responders. Together, PD1 blockade with pembrolizumab after CD19-directed CAR T-cell therapy appears safe and may achieve clinical responses in some patients with B-cell lymphomas refractory to or relapsed after CAR T-cell therapy. This trial was registered at www.clinicaltrials.gove as #NCT02650999.