Second-generation Chimeric Antigen Receptor Research Articles

Two-Year Follow-up of Investigator-Initiated Phase 1 Trials of the Safety and Efficacy of Fully Human Anti-Bcma CAR T Cells (CT053) in Relapsed/Refractory Multiple Myeloma

Background: B cell maturation antigen (BCMA) is a potential therapeutic target in multiple myeloma. CT053 comprises autologous T cells transduced with a second-generation chimeric antigen receptor (CAR) utilizing a fully human BCMA-specific single-chain fragment variant (25C2) with high binding affinity. CT053 was firstly studied in a single-arm, open-label Phase I, investigator-initiated program (NCT03716856, NCT03302403, and NCT03380039) in eastern China. Here we present the 24-month follow-up results of the study. Methods: The multicenter exploratory Phase 1 studies included adult subjects with relapsed/ refractory multiple myeloma (RRMM) who had received at least 2 prior lines of myeloma treatment. After preconditioning treatment with fludarabine and cyclophosphamide for 2-4 days, 21 subjects received one cycle of 1.5 × 108 T cells CT053 CAR-BCMA T cells. Three subjects respectively received 0.5 × 108, 1 × 108, or 1.8 × 108 cells. The primary objective was subject safety. The secondary objectives were pharmacokinetics and efficacy. Efficacy was assessed according to IMWG 2016 criteria. Results: A total of 24 subjects with a median age of 60.1 years (range, 38.5-69.9) were enrolled from Sept. 10, 2017 to Sept. 22, 2018. The subjects had a median of 4.5 (range, 2-11) prior lines of therapy, and 41.7% (10/24) underwent autologous stem cell transplantation. At baseline, 10 subjects (41.7%) had concomitant extramedullary involvement, 8 subjects (33.3%) had ECOG scores 2-3, and 9 subjects (37.5%) reported ISS Grade III. As of June 30, 2020, 9 subjects completed 24 months of follow-up with responses including 8 stringent complete response (sCR) and 1 complete response (CR). Also, 15 subjects discontinued prior to completing the 24-month follow-up, of whom 13 discontinued due to disease progression (PD), and 2 discontinued for other anticancer therapy. The overall response rate was 87.5% (21/24) including 79.2% (19/24) with complete responses or stringent complete responses (3 CR, 16 sCR). The median duration of response (DOR) was 21.8 months (95%CI: 9.2, not evaluable [NE]). The median progression-free survival (PFS) was 18.8 months (95%CI: 10.1, NE), with 6-month and 12-month PFS rates of 87% and 60.9%, respectively. Thirteen subjects progressed with median PFS of 10.2 months (range, 0.9-23 months): 3 progressed within 6 months, 6 progressed within 6-12 months, and 4 within 12-24 months. Compared to 9 subjects with persistent CR/sCR, the 13 progressed subjects had a higher percentage of ECOG scores 2-3 (46.2% vs 22.2%), ISS Grade III (53.9% vs 11.1%) and high-risk cytogenetics profiles (53.8% vs 33.3%). Rates of concomitant extramedullary diseases were similar, 46.2% and 44.4%, respectively. Hematological toxicities were the most common treatment-related adverse events of grade (G) 3 or higher, including leukopenia (83.3%), neutropenia (85%), lymphocytopenia (79.2%) and thrombocytopenia (20.8%). In general, cytokine release syndrome (CRS) occurred at 1-4 days and resolved in a median 6 days (range, 3-9 days). Low-grade CRS was reported in 15 of 24 (62.5%) subjects. All CRS events (4 G1, 11 G2) resolved within 2-8 days; among them, 9 patients received a low dose of tocilizumab 4-6 mg/kg. One patient experienced G3 neurotoxicity, presenting as epilepsy and accompanied by simultaneous G2 CRS. This patient fully recovered within 3 days after treatment with methylprednisolone, diazepam and sodium valproate. Six patients (25%) experienced 10 cases of treatment-related serious adverse events (SAEs), including lung infection (3), gastroenteritis (1), neutropenic infection (1), fever (1) and hematological toxicities (4). By the cutoff date, one subject died of SAE a (bone morrow failure and neutropenic infection) and PD, and seven subjects died of PD. CAR-BCMA T cell expansion was detectable as early as day 1-7 after infusion and reached peak values on day 7-21 with the highest concentration at 4.5×105 copies/µg genomic DNA. Median T cell persistence was 172 days. The longest persistence of CAR-BCMA copies was measured at 341 days and continues. No immunogenicity was detected. Conclusion: These studies demonstrated that CT053 had excellent efficacy in RRMM, showing early, deep and durable response with 21.8 months DOR. CT053 was well tolerated among the subjects. Figure Disclosures Li: CARsgen Therapeutics Co. LtD: Current Employment, Current equity holder in private company. Wang:CARsgen Therapeutics Corp.: Current Employment. Xiao:CARsgen Therapeutics Corp.: Current Employment. Yuan:CARsgen Therapeutics Corp.: Current Employment. Ma:CARsgen Therapeutics Corp.: Current Employment.

Tonic Signaling Leads to Off-Target Activation of T Cells Engineered with Chimeric Antigen Receptors That Is Not Seen in T Cells Engineered with T Cell Antigen Coupler (TAC) Receptors

Chimeric antigen receptors (CARs) are powerful tools that enable MHC-independent activation of T cells. Recent reports have indicated that constitutive, low-level (tonic) signaling by CARs can impair the utility of the engineered T cells. The single-chain antibody (scFv) binding domain was one of the features determined to promote tonic signaling. We have recently developed a novel chimeric receptor, known as the T cell antigen coupler (TAC), that is less prone to tonic signaling than second-generation CARs. The TAC consists of a scFv-based antigen binding domain, a CD3-binding domain that couples the TAC to endogenous T cell receptor (TCR), and a transmembrane and cytoplasmic coreceptor (CD4) domain. In contrast to CARs, this design enables TAC-T cells to signal through the endogenous TCR, which we propose provides a fidelity to natural T cell signal regulation. Interestingly, we have recently reported that CAR-T cells have a greater propensity for off-target activation than TAC-T cells, suggesting a safety advantage to TAC-T cells (Helsen et al., Nat. Comm., 2019). Further characterization of the differences between CAR- and TAC-T cell signal initiation and activation is required to understand how their design affects sensitivity, specificity and regulation of T cell activation. Examination of the activation requirements for BCMA-specific CAR-T cells and TAC-T cells confirmed that TAC-T cells are reliant upon the endogenous TCR for T cell activation whereas CAR-T cells are TCR-independent. TRAC knock-out CAR-T cells retained potent effector function at levels similar to CAR-T cells with intact TCR expression, whereas TRAC knock-out TAC T-cells showed significant impairment in effector function. Consistent with TCR-dependence, the immunological synapse produced by TAC-T cells displays all the hallmarks of a conventional immunological synapse, whereas CAR-T cells form unconventional synapses. Unlike TAC-T cells, immunological synapses formed by CAR-T cells display non-uniform central supramolecular activation clusters, disperse Lck distribution, a lack of an LFA-1 associated adhesion ring (Figure), as well as more disperse delivery of perforin to the cell interface. CAR-T cells also formed synapses faster than TAC-T cells. This suggests that while TAC T-cells are beholden to the requirement of organized, mature synapse formation, CAR T-cells can rapidly form less structurally organized synapses. Transcriptional profiling of CAR-T cells in the absence of antigen stimulation revealed a basal activation status associated with upregulation of Nur77, a transcription factor that is downstream of TCR activation. Transcriptional profiling of TAC-T cells failed to reveal evidence of TCR signaling in the absence of stimulation. Further evaluation of CAR- and TAC- T cells in the absence of stimulation revealed elevated levels of CD69, PD-1 and LAG-3 in CAR-T cells compared with TAC-T cells, as well as higher expression of IL-2, IFNγ, and TNF in CAR-T cells. Interestingly, the level of tonic signaling was dependent on the antigen-binding scFV, as otherwise identical BCMA-specific CAR- and TAC-T cells displayed different levels of CD69, PD-1 and LAG-3 depending on the identity of the BCMA-specific scFv. Despite different levels of basal activation, both CAR- and TAC-T cells displayed comparable activation kinetics as measured by upregulation of CD69 and Ki-67, as well as proliferation. However, the elevated level of basal activation rendered the CAR-T cells more easily activated by a cross-reactive off-target antigen that failed to stimulate TAC-T cells carrying the same binding domain. These data suggest that the TAC receptor offers a valuable alternate platform to CAR-T cells. The antigen-binding scFv domain has a direct impact on tonic signaling and basal activation in CAR-T cells. Conversely, TAC-T cells are less susceptible to basal activation and this works suggests that the TAC receptor can deploy scFv binding domains that are not suitable for CARs. This work was supported by Triumvira Immunologics and Genome Canada. Figure 1 Disclosures Bramson: McMaster University: Current Employment, Patents & Royalties; Triumvira Immunologics: Current Employment, Current equity holder in private company, Research Funding.

Single and Dual Targeting Chimeric Antigen Receptor T-Cell Therapy in Acute Myeloid Leukemia

BACKGROUND: Discovery of chimeric antigen receptors (CARs) specific for tumour-associated antigens are emerging to be an effective form of immunotherapy for cancer treatment in recent years. However, the lack of a compelling acute myeloid leukemia (AML)-specific cell surface antigen and the safety concerns for myeloid-directed CAR T therapies causing prolonged myeloablation/aplasia which necessitate bone marrow transplantation make it challenging to develop CARTs for AML. In spite of these challenges, the high relapse rate of the disease i.e. 43% and 18% patients never attaining clinical remission (CR) with front-line induction treatments highlight an unmet need for developing improved CAR T vectors with enhanced specificity towards leukemic blasts in refractory/resistant cases with poor cytogenetics. Therefore, we aim to develop improved CAR T vectors for AML that produce safe and consistent responses in patients with high-risk disease. Herein, we demonstrate the in-vitro data for (a) efficacy of a second-generation CAR expressing six single-chain variable fragments (scFv) with different affinities for CD123 [interleukin three receptor alpha (IL3RA; CD123), a molecule over expressed on AML blasts and leukemic stem cells (LSC)] and (b) evaluate the cytotoxic effects of a dual targeting CARCD123/CD33 (against CD123 and CD33; an important myeloid marker specifically expressed on bulk AML disease) to enhance specificity towards leukemic cells therefore reducing "on-target off-organ effects". METHOD: Six lentiviral vectors with CAR against CD123 were constructed i.e. two high affinity (4nM kD & 4nM kD with a point mutation resulting in amino acid change K136Q), two moderate (56nM kD & 56nM kD with mutation at A105G) and two low affinity vectors (101nM kD & 101nM kD with mutation at V24G). To improve the specificity of the single targeting CARCD123, the high affinity (4nM kD, K136Q) vector was utilized to generate two dual targeting (CARCD123/CD33) constructs containing the activation domain (CD3ζ) directed against CD33 and the costimulatory domain (either CD28 or 41BB) directed against CD123. All constructs were transduced (MOI 1:5) into peripheral blood mononuclear cells (PBMCs) from healthy donors or AML patients and their cytotoxicity was examined by flowcytometry on leukemic cell lines; Kg1, U937, K562 [Fig:1a], Ramos wild type (CD19+, CD123-), artificially engineered Ramos cells (transduced by lentiviral vectors with CD123 and/or CD33 cDNA) i.e. Ramos 123 (CD123+), Ramos 33 (CD33+), Ramos 123/33 (CD123+ and CD33+) and AML mononucelar cells (MNCs). RESULTS: Flowcytometric analysis confirmed the expansion of T cells from PBMCs and cytotoxicity of the eight CAR constructs against target cells in in-vitro co-culture assay. High affinity CARCD123 (4nM kD & 4nM kD K136Q) T cells demonstrated enhanced cytotoxicity [Fig 1a] compared to moderate (56nM kD, 56nM kD A105G) CARCD123 in all leukemic cell lines while the low affinity (101nM kD, 101nM kD V24G) vectors had no effect. Efficacy of the high affinity CARCD123 constructs was validated on Kg1 [Fig 1b] and Ramos 123+ target cells by the increasing effector: target ratios (1:2, 1:4 & 1:10). Similar cytotoxic effects were also consistently observed against autologous AML MNCs (target cells) [n=4] and allogenic (effector cells are PBMCs from healthy donors) AML MNCs [Fig 1c] [n=3]. T cell activation was confirmed by ELISA and showed increased IFN-γ (500-2000 fold) and TNF-α (150-200 fold) levels after 24hr co-culture. Furthermore, we also elucidated the exclusive cytotoxicity of the two dual targeting CARCD123/CD33 in Ramos 123+/33+ cells [Fig 1d] with no effect being observed on Ramos 123+, Ramos 33+ and Ramos wild type (123-/33-) cell lines. This confirmed the absence of non-specific targeting, validated the improved specificity of the CARs towards leukemic cells and demonstrated a potential to reduce deleterious "on-target but off organ effects". CONCLUSION: In summary, we illustrate in-vitro data establishing the importance of scFv on CAR T cell cytotoxicity and exemplify for the first time an improved specificity of CARTs by targeting two antigens simultaneously in AML. Future work will involve examining the in-vivo dynamics of CAR CD123 and CAR CD123 CD33 on the hematopoietic system and on disease pathogenesis with an aim to proceed to phase I clinical trial. Figure 1 Disclosures Mufti: BMS, Novartis: Research Funding; Abbvie, Novartis: Consultancy.

Loss of TET2 Uncouples Proliferative and Effector Functions in CAR T Cells

Chimeric antigen receptor (CAR) T cells have opened a new paradigm for the treatment of leukemia and lymphoma. Their production, however, is laborious, requiring tens of millions of CAR T cells per infusion. This constraint could be significantly alleviated if safe and more efficacious T cells could be generated. In a patient with chronic lymphocytic leukemia, treated with anti-CD19 CAR T cells, a recent report described the emergence of a single T cell clone that at its expansion peak accounted for 94% of circulating CAR T cells, coinciding with the development of cytokine release syndrome and tumor regression (Fraietta et. al. Nature 2018). Insertional mutagenesis in this T cell had disrupted an allele of TET2, an epigenetic regulator mediating the oxidation of 5-methylcytosine. The other allele appeared to bear an inherited hypomorphic variant, resulting in the near complete loss of TET2 function in this clone. To understand the mechanisms accounting for this chance clinical finding, we investigated the effect of TET2 loss in human T cells engineered to express different chimeric receptors. Using CRISPR/Cas9, we edited TET2 in T cells engineered to express a CD19-specific second-generation CAR encompassing the costimulatory domain of either CD28 or 4-1BB (Rv-1928z and Rv-19BBz). TET2 disruption enhanced the in vivo anti-tumor activity of Rv-19BBz but not Rv-1928z CAR T cells tested under stress test conditions using limiting CAR T cell doses (as previously described in a human B cell acute lymphoblastic leukemia (B-ALL) NALM6 model, Zhao et. al. Cancer Cell 2015). Since Rv-1928z induces potent effector differentiation but limited persistence compared to Rv-19BBz, we hypothesized that loss of TET2 could amplify the expansion and persistence of 4-1BB-costimulated T cells but not override the differentiation program imparted by Rv-1928z. To test this hypothesis, we utilized two orthogonal approaches known to limit exhaustion and increase persistence of CD28-costimulated CAR T cells, Rv-1928z co-expressed with 4-1BB ligand (Rv-1928z-41BBL) and 1928z driven by the TRAC promoter (TRAC-1928z). Disruption of TET2 enhanced the anti-tumor efficacy of both these CAR T cells and promoted acquisition of a central memory phenotype. However, over time (50-200 days), TET2-edited TRAC-1928z and Rv-1928z-41BBL attained a hyper-proliferative phenotype ultimately requiring euthanasia due to splenomegaly and extensive CAR T cell accumulation in various organs. Post-mortem analysis found no evidence of NALM6 in these mice. This was in contrast to stress test studies with Rv-1928z and Rv-19BBz where most mice succumbed to NALM6 progression. These observations established an essential role for CAR signaling in determining the phenotypic outcome of TET2 loss in T cells. To examine the long-term effects of TET2 disruption in the context of all 4 receptors, we treated human B-ALL bearing mice with curative doses of all 4 CAR T cells and followed them for up to 200 days. We found that all 4 CAR expressing TET2-edited T cells could eventually attain a hyper-proliferative phenotype, but with varying frequency depending on the CAR design (Rv-1928z-41BBL and TRAC-1928z > Rv-19BBz > Rv-1928z). To assess their effector function, NALM6-bearing mice were infused with adoptively transferred hyper-proliferative TET2-edited CAR T cells. Strikingly, these T cells were unable to elicit any tumor control, despite their maintaining a central memory phenotype as assessed by flow cytometry. This loss of effector function was observed for all 4 CAR T cell types, suggesting a discrepancy between function and flow cytometric phenotype. Transcriptional, methylation and genome accessibility studies revealed a unique T cell state wherein the proliferative program is uncoupled from effector response. We identified a unique transcriptional and epigenetic signature that is manifested in a loss of effector function while maintaining robust proliferation. This state stands in contrast to the classically described T cell exhaustion state where loss of effector function is preceded by loss of proliferative ability. TET2 disruption thus promotes a CAR T cell proliferative program that depends on the CAR design but does not in itself enhance anti-tumor activity. Disclosures Sadelain: Atara: Patents & Royalties, Research Funding; Mnemo: Patents & Royalties; Minerva: Other: Biotechnologies , Patents & Royalties; Fate Therapeutics: Patents & Royalties, Research Funding; Takeda: Patents & Royalties, Research Funding.

Orvacabtagene Autoleucel (orva-cel; JCARH125): A Fully Human BCMA-Targeted Second-Generation CAR T Cell Product Characterized By a Predominant Central Memory Phenotype with High in Vitro and In Vivo Proliferative Potential and Sustained In Vivo Persistence

Background: Orva-cel is an investigational B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR) T cell product genetically modified with a lentiviral vector to express a CAR construct with a unique fully human single-chain variable fragment, optimized spacer, and 4-1BB costimulatory and CD3ζ activation domains. Orva-cel is currently being evaluated for efficacy and safety in the ongoing phase 1/2 EVOLVE study (NCT03430011) in heavily pretreated patients with relapsed/refractory multiple myeloma. We characterized orva-cel drug products, manufactured using the process in place for the phase 2 portion of the study and intended for commercial manufacturing, for CAR+ T cell purity, phenotype, and function. Methods: Immunophenotyping was performed by flow cytometry of both surface and intracellular markers, including CD3, CD4, CD8, CD45, CCR7, CD45RA, CD28, CD27, and active caspase 3. Cytokine production after challenge with BCMA+ target cells was assessed by intracellular cytokine staining and Luminex multiplex assay of secreted cytokines, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and granzyme B (GrB). CAR-mediated in vitro proliferative capacity was measured after anti-idiotypic antibody stimulation using the IncuCyte Live-Cell Analysis System (Sartorius, Göttingen, Germany). In vivo CAR+ T cell proliferation and persistence were assessed by quantitative polymerase chain reaction (qPCR). Results: The orva-cel manufacturing process was designed to enable consistent production of highly pure CD3+ cell products (median frequency of CD3+ T cells, 99.96%; quartiles 1-3 interquartile range, 99.9%-100.0%; n = 81). Orva-cel drug products were characterized by high frequencies of less-differentiated CAR+ T cells, leading to a dominant central memory-like population (CCR7+CD45RA- CAR+ T cells) and substantial frequencies of naïve-like cells (CCR7+CD45RA+ CAR+ T cells) (Figure). When assayed for in vitro functional activity, orva-cel drug products showed robust antigen-specific cytokine and effector molecule production (IFN-γ, TNF-α, IL-2, and GrB) upon challenge with BCMA+ tumor cells, as well as vigorous proliferation in response to CAR stimulation. Preliminary correlative analysis suggested that the early memory phenotype may be linked to increased CAR+ T cell proliferative capacity, as determined by in vitro experiments and in vivo PK parameters (ie, maximum CAR+ T cell concentration observed in the blood [Cmax], time to Cmax, area under the curve from Day 0 to 28 [AUC0-28], and CAR+ T cell persistence at Month 3 and Month 6). Consistent with the early CAR T cell memory phenotype, qPCR analysis showed robust in vivo proliferation of CAR+ T cells after infusion, with a median Cmax of 1.54 × 105 transgene copies/µg DNA and median AUC0-28 of 1.61 × 106 transgene copies/µg DNA*day, as well as long-term in vivo persistence, with CAR+ T cells detected in 69% of patients at 6 months postinfusion. Conclusions: The orva-cel manufacturing process results in drug products characterized by highly pure T cells, with high frequencies of early memory and polyfunctional CAR+ T cells. Orva-cel drug products showed robust antigen-specific degranulation, production of multiple cytokines, sustained in vitro and in vivo proliferation, and in vivo persistence. Disclosures Colonna: Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Navarro:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Devries:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Beckett:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment. Amsberry:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Radhakrishnan:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Piasecki:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Heipel:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Li:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Kavita:Bristol Myers Squibb Company: Current Employment, Current equity holder in publicly-traded company. Works:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment. Mujacic:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment.

Endogenous TCR promotes in vivo persistence of CD19-CAR-T cells compared to a CRISPR/Cas9-mediated TCR knockout CAR

AbstractAnti-CD19 chimeric antigen receptor (CAR) T cells showed significant antileukemic activity in B-precursor acute lymphoblastic leukemia (ALL). Allogeneic, HLA-mismatched off-the-shelf third-party donors may offer ideal fitness of the effector cells, but carry the risk of graft-versus-host disease. Knockout (KO) of the endogenous T-cell receptor (TCR) in CD19-CAR-T cells may be a promising solution. Here, we induced a CRISPR/Cas9-mediated KO of the TCRβ chain in combination with a second-generation retroviral CAR transduction including a 4-1BB costimulatory domain in primary T cells. This tandem engineering led to a highly functional population of TCR-KO-CAR-T cells with strong activation (CD25, interferon γ), proliferation, and specific killing upon CD19 target recognition. TCR-KO-CAR-T cells had a balanced phenotype of central memory and effector memory T cells. KO of the endogenous TCR in T cells strongly ablated alloreactivity in comparison with TCR-expressing T cells. In a patient-derived xenograft model of childhood ALL, TCR-KO-CAR-T cells clearly controlled CD19+ leukemia burden and improved survival in vivo. However, coexpression of endogenous TCR plus CAR led to superior persistence of T cells and significantly prolonged leukemia control in vivo, confirmed by a second in vivo model using the leukemia cell line NALM6. These results point toward an essential role of the endogenous TCR for longevity of the response at the price of alloreactivity. In conclusion, anti-CD19 CAR T cells with a CRISPR/Cas9-mediated TCR-KO are promising candidates for nonmatched third-party adoptive T-cell transfer with high antileukemic functionality in the absence of alloreactivity, but long-term persistence in vivo is better in the presence of the endogenous TCR.

Abstract 2173: CRISPR-Cas9 gene editing of inhibitory receptor signaling pathways improves CAR T cell activity against glioblastoma

Abstract Early clinical trials have demonstrated the therapeutic potential of chimeric antigen receptor (CAR) T cells against glioblastoma (GBM), but also highlighted the obstacles that need to be addressed to improve their anti-tumor activity. GBM is highly immunosuppressive and thus necessitates endowing adoptively transferred CAR T cells with resistance mechanisms to achieve sustained treatment responses. This is, in part, due to the effect of immune-inhibitory receptors, including PD1, CTLA4, LAG3 and BTLA, that antagonize the tumor-directed T cells. A common mechanism of T cell inhibition involves the recruitment of Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) to the immune synapse upon engagement of multiple co-inhibitory receptors. SHP2, in turn, de-phosphorylates key signaling proteins involved in T cell activation. We hypothesized that disrupting SHP2 expression will offset multiple co-inhibitory pathways, thereby improving the anti-tumor activity of CAR T cells. Using CRISPR/Cas9 gene editing, we disrupted the SHP2-encoding gene locus, PTPN11, in primary human T cells by generating and testing a library of single-guide RNA (sgRNA). We selected the top candidate that reproducibly reduced the SHP2 protein expression to <10%. A HER2-targeting second-generation CAR (HER2-CAR) was then expressed in SHP2 knock-out (SHP2KO) T cells via gamma retroviral vector transduction. SHP2KO CAR T cell manufacturing following a single round of stimulation was optimized to minimize ex vivo manipulation of the cells. The phenotypic profiles of SHP2KO and wild-type HER2-CAR T cells were comparable at baseline. Loss of SHP2 did not alter the CAR T cell viability, expansion or proliferative capacity in comparison to the wild-type CAR T cells. Mass cytometry (CyTOF)-based analysis of SHP2KO CAR T cells after encountering GBM cells demonstrated the preservation of the central memory (CCR7+CD45RA-) T cell subset. The majority of PD1+ SHP2KO CAR T cells co-expressed functional activation markers such as ICOS and CD107a. The PD1+ T cell subset was comprised of mostly T-betHi progenitor population and only a smaller percentage of EomesHi terminally exhausted population. Functionally, HER2-specific SHP2KO CAR T cells from different donors (n=4) exhibited improved anti-tumor activity that was sustained in long-term cytotoxicity assays. In an experimental orthotopic LN229-GBM mouse model, SHP2KO HER2-CAR T cells better controlled the growth of established xenografts and improved survival in comparison to wild-type HER2-CAR T cells. In conclusion, our results show that disrupting SHP2 expression using a CRISPR/Cas9 gene editing strategy could improve the CAR T cell function against GBM and warrants further clinical testing. Citation Format: Khaled Sanber, Zeid Nawas, Vita Salsman, Ahmed Gad, Pretty Rose Matthew, Daniel Landi, Ciaran Lee, Amel Sengal, Rikhia Chakraborty, Sujith Joseph, Ahmed Nabil, Meenakshi Hegde. CRISPR-Cas9 gene editing of inhibitory receptor signaling pathways improves CAR T cell activity against glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2173.

Abstract 6530: Old target, but new approach: Targeting cancer-associated MUC1 with ADC and Car-T

Abstract Purpose: The characteristics of aberrant overexpression and distinct features of cancer-associated (CA)-MUC1 has been often considered as a promising target in most of the solid cancers. MUC1 is composed of α and β subunits to form a full length of MUC1, and α subunit is released and circulated into the blood in cancer, which is used as biomarkers such as CA 15-3 and CA 27-29. As those α subunit-targeting strategies were failed in the numerous clinical trials due to, at least in part, the diminished α subunit of MUC1 in cancer, we have developed a humanized Ab, PAb001 targeting the residual β subunit of MUC1, called as an onco-tethered (OT)-MUC1. To prove the concept of this strategy, an antibody-drug conjugate (ADC) and chimeric antigen receptor (CAR) T cells were applied to assess its efficacies. Methods: We produced a monoclonal antibody (mAb) against OT-MUC1 from mouse and sequentially generated its derivative humanized antibody, PAb001. Then, the target product profiles (TPP) were determined by SPR, ELISA, FACS, immunohistochemical analysis, pilot tissue cross-reactivity, and pharmacokinetics (PK) analysis. Then, to evaluate anti-cancer efficacies, we either conjugated PAb001 with Monomethyl auristatin E (MMAE) or generated the second-generation CAR T cells and then sequentially tested in vitro and in vivo cancer models. Results: Based on the TPP criteria, PAb001 antibody was chosen over a hundred of candidate antibodies tested. Next, it was conjugated with MMAE, treated and measured its cytotoxicity in ovarian and breast cancer cell lines, including triple-negative breast cancers (TNBC). As a result, PAb001-ADC selectively inhibited the growth of cancer cell lines, depending on the expression level of MUC1. Also, it showed 80-100 % tumor growth inhibition rates (TGI) in the cell lines- and patient-derived xenograft models. Lastly, OT-MUC1 Car-T cells showed significant and selective growth inhibition of cancer cells. Conclusion: Taken together, the preclinical studies of PAb001-ADC and Car-T indicate the high therapeutic potentials targeting OT-MUC1 in cancer, which further provided the rationale for clinical development. Citation Format: Jin G Jung, Min-Seok Kim, Hwa-Chul Jung, Hoil Choi, Kyung-Duk Moon. Old target, but new approach: Targeting cancer-associated MUC1 with ADC and Car-T [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6530.

Multiple Signaling Roles of CD3ε and Its Application in CAR-T Cell Therapy

A Tcell receptor (TCR) mediates antigen-induced signaling through its associated CD3ε, δ, γ, and ζ, but the contributions of different CD3 chains remain elusive. Using quantitative mass spectrometry, we simultaneously quantitated the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of all CD3 chains upon TCR stimulation. A subpopulation of CD3ε ITAMs was mono-phosphorylated, owing to Lck kinase selectivity, and specifically recruited the inhibitory Csk kinase to attenuate TCR signaling, suggesting that TCR is a self-restrained signaling machinery containing both activating and inhibitory motifs. Moreover, we found that incorporation of the CD3ε cytoplasmic domain into a second-generation chimeric antigen receptor (CAR) improved antitumor activity of CAR-T cells. Mechanistically, the Csk-recruiting ITAM of CD3ε reduced CAR-T cytokine production whereas the basic residue rich sequence (BRS) of CD3ε promoted CAR-T persistence via p85 recruitment. Collectively, CD3ε is a built-in multifunctional signal tuner, and increasing CD3 diversity represents a strategy to design next-generation CAR.

Efficacy and safety of second-generation CAR T-cell therapy in diffuse large B-cell lymphoma: A meta-analysis.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma (NHL), representing 30% of all lymphoma cases. Within the first 2-3 years following immunochemotherapy, 30-40% of patients will experience a relapse or a refractory disease, thereby exhibiting a poor prognosis. High-dose immunotherapy followed by autologous stem cell transplantation is the standard care for relapsed/refractory (RR) patients with DLBCL. However, >60% of patients are ineligible for a transplant, presenting a therapeutic challenge. Chimeric antigen receptor (CAR) T-cell therapy has shown promising efficacy in patients with DLBCL, including those with R/R disease. The present study conducted a meta-analysis that showed highly favorable outcomes [objective response rate (ORR): 69%; complete remission (CR): 49%] in B-cell NHL patients (n=419) who were treated with second-generation CAR T cells. The response rate varied in different types of B-cell NHL. In 306 patients with R/R DLBCL eligible for rate evaluation, the ORR and CR rate mean estimates were 68% [95% confidence interval (CI), 55-79%] and 46% (95% CI, 38-54%), respectively. Thus, the findings indicated that immunotherapy with CAR T cells has improved outcomes for patients with R/R DLBCL and other subtypes of B-cell NHL compared with standard chemotherapy regimens. The study revealed that grade ≥3 anemia (34%) and thrombocytopenia (30%) were the most common adverse effects of CAR T-cell therapy. Incidence of grade ≥3 cytokine release syndrome and neurotoxicity associated with CAR T-cell therapy was effectively managed.

Novel car design reversing the PD-1 signal boosts the efficacy of car-T cells with low cytokine release and no off-target effect

Background & Aim Purpose The immunosuppressive tumor microenvironment (TIME) and cytokine release syndrome (CRS) hinder the progression of chimeric antigen receptor (CAR) T cell therapy. Strategies that block or reverse PD-1/PD-L1 inhibitory signals through CAR structural redesign to overcome TIME are extensively studied but still need be improved. Targeting the widely expressed PD-L1 without increasing the off-tumor effect while reduce cytokines release are critical challenges. Based on the above considerations, we engineered a novel bispecific CAR T cell type targeting CD19 and PD-L1 by incorporating the PD-1 extracellular domain next to the transmembrane domain (TMD) of CD19-targeting CAR domain, which we named EMD.PD-1 CAR. Methods, Results & Conclusion Experimental design In this study, we firstly engineered a novel CD19/PD-L1 bispecific CARs by incorporating the PD-1 domain into CD19 CAR and constructed second-generation CAR as control. Second, to assess the function of the novel anti-CD19 CAR T cells via antigen-specific stimulation, the antitumor functions, cytokine release, and off-target effects were studied in vitro and in vivo. We demonstrated that PD-L1 stimulation alone is not sufficient to activate EMD.PD-1 CAR T cells for the production of a antigen-specific immune response, including specific lysis, cytokines release, and proliferation. When challenged with CD19+PDL1+ tumor cells, EMD.PD-1 CARs showed a stronger specific immune response than 2nd CAR in vitro. Moreover, EMD.PD-1 CAR T cells exhibited only moderate specific cytokine release compared with 2nd CAR T cells when challenged with target tumor cells. In xenograft NOD/SCID/γc−/−(NSG) mouse model bearing CD19+PD-L1+ tumor cells, our results demonstrated that EMD.PD-1 CAR prolonged the overall survival and decreased the tumor burden compared with 2nd CAR. In addition, EMD.PD-1 CAR T cells showed a higher fraction of stem cell memory (TSCM) and central memory (TCM) T cell phenotype and lower PD-L1, TIM-3, CTLA-4, and intrinsic PD-1 expression in flow cytometry, CAR T cells demonstrated decreased differentiation and exhaustion marker expression. In conclusion, when faced with heterogeneous tumors with high expression of immunosuppressive signals, traditional second-generation CAR T cells are insufficient. The novel CAR, called EMD.PD-1 CAR with a low-avidity affinity to PD-L1 and moderate cytokine release can address this critical challenge.

Abstract A31: Comparing IL-12 and IL-18 secreting CAR T-cell efficacy

Abstract MUC16, a subunit of CA125 that is overexpressed on epithelial ovarian cancers, can be targeted by T cells via the incorporation of the 4h11m28m chimeric antigen receptor (CAR) into the T-cell surface. However, the microenvironment of ovarian cancers consists of regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages that act to immunosuppress CAR T-cell function, thus abrogating CAR T-cell efficacy. To overcome this immunosuppression, our lab has developed IL-12 and IL-18 secreting CAR T cells, which have shown enhanced efficacy in a syngeneic ovarian cancer model when compared to second-generation CAR T cells. While IL-18 secreting CAR T cells have proven to be superior to IL-12 secreting CAR T cells in various leukemia and lymphoma mouse models, this observation does not translate to the ovarian cancer model. Mechanistic studies into the effects of IL-18 and IL-12 in a syngeneic ovarian cancer model are currently under way in order to better understand what each cytokine is doing to the tumor microenvironment as well as to the CAR T cell. With better understanding of the effects of IL-18 and IL-12, we can contribute to the immunobiology of these cytokines as well as harness their efficacy to apply to other tumor models. Citation Format: Christina E. Bebernitz, Renier J. Brentjens. Comparing IL-12 and IL-18 secreting CAR T-cell efficacy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A31.

Abstract A67: Augmented antitumor activity of NK-92 MI cells expressing a chimeric antigen receptor targeting human mesothelin

Abstract Adoptive natural killer (NK) cell immunotherapy using primary NK cells has been challenging, to date. The use of an ”off-the -shelf” NK cell line where multiple genetic changes can be introduced has advantages. In this study, we explored the use of the NK-92MI cell line expressing a chimeric antigen receptor (CAR) targeting human mesothelin, the current target in our human CAR-T cell therapy trials. The NK-92MI cell line (which makes its own IL-2) was purchased from ATCC and transduced with a lentivirus encoding GFP (vector control) or an anti-human mesothelin scFv-41BB-CD3 ζ second-generation CAR (MesoCAR). Addition of IL-21 enhanced transduction. The transduced cells (MesoCar-NK-92MI) were sorted by flow cytometry to achieve an expression level of >98%. The NK markers NKp30, NKp44, NKG2D, 2B4, and CD56 were highly expressed on the Car-NK-92MI cell line (from 75% to 99.9%). To test the antitumor effects in vitro, three types of human tumor lines transduced to express human mesothelin were tested. Lung cancer (A549Meso), mesothelioma (EMMeso), and head and neck cancer (SSC15Meso) cells were exposed to vector control NK-92MI or MesoCar-NK-92MI cells for 22 hours at varying E:T ratios. The supernatants were collected for detection of cytokines and granzyme B. We also determined the killing function of MesoCar-NK-92MI cells after irradiation of the cells at 10 Gy. To test in vivo activity, NSG mice were injected in the flank with 5 million EMMeso cells. After tumors were established, each mouse was intravenously injected with 10 million vector control NK-92MI cells or MesoCar-NK-92MI cells with a boost injection of 5 million effectors on days 4, 8, and 11. Tumor volumes were assessed over time and mice were sacrificed on day 18. In vitro killing results showed augmented antitumor effect in MesoCar-NK-92MI compared to vector control NK-92MI. Irradiated MesoCar-NK-92MI cells still possessed good antitumor activity with only 32% loss of activity on average. Increased cytokine and granzyme B secretion was also seen with the MesoCar-NK-92MI cell line (with or without irradiation) compared to NK-92MI cells. In animal studies, irradiated MesoCar-NK-92MI cells significantly inhibited tumor growth by an average of 59% with no obvious toxicity or tumor growth in other organs. The non-CAR-expressing NK-92MI cells had significantly less antitumor activity. In conclusion, use of an irradiated NK-92MI cell line expressing a CAR targeting human mesothelin could become a safe and possible “off-the-shelf” adoptive cell therapy that could be used alone or in combination with CAR-T cells or other treatments. Citation Format: Jing Sun, Patrick Woodruff, Soyeon Kim, Estela Noguera-Ortega, Evguenia Arguiri, Maria Liousia, Steven Maceko, Edmund Moon, Steven Albelda. Augmented antitumor activity of NK-92 MI cells expressing a chimeric antigen receptor targeting human mesothelin [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A67.

A Small Number of HER2 Redirected CAR T Cells Significantly Improves Immune Response of Adoptively Transferred Mouse Lymphocytes against Human Breast Cancer Xenografts.

HER2 positive JIMT-1 breast tumors are resistant to trastuzumab treatment in vitro and develop resistance to trastuzumab in vivo in SCID mice. We explored whether these resistant tumors could still be eliminated by T cells redirected by a second-generation chimeric antigen receptor (CAR) containing a CD28 costimulatory domain and targeting HER2 with a trastuzumab-derived scFv. In vitro, T cells engineered with this HER2 specific CAR recognized HER2 positive target cells as judged by cytokine production and cytolytic activity. In vivo, the administration of trastuzumab twice weekly had no effect on the growth of JIMT-1 xenografts in SCID mice. At the same time, a single dose of 2.5 million T cells from congenic mice exhibited a moderate xenoimmune response and even stable disease in some cases. In contrast, when the same dose contained 7% (175,000) CAR T cells, complete remission was achieved in 57 days. Even a reduced dose of 250,000 T cells, including only 17,500 CAR T cells, yielded complete remission, although it needed nearly twice the time. We conclude that even a small number of CAR T lymphocytes can evoke a robust anti-tumor response against an antibody resistant xenograft by focusing the activity of xenogenic T cells. This observation may have significance for optimizing the dose of CAR T cells in the therapy of solid tumors.

Third-generation anti-CD19 chimeric antigen receptor T-cells incorporating a TLR2 domain for relapsed or refractory B-cell lymphoma: a phase I clinical trial protocol (ENABLE)

IntroductionAutologous T-cells transduced to express a chimeric antigen receptor (CAR) directed against CD19 elicit high response rates in relapsed or refractory (r/r) B-cell non-Hodgkin lymphoma (B-NHL). However, r/r B-NHL remissions...

Rapid and Effective Generation of Nanobody Based CARs using PCR and Gibson Assembly.

Recent approval of chimeric antigen receptor (CAR) T cell therapy by the European Medicines Agency (EMA)/Federal and Drug Administration (FDA) and the remarkable results of CAR T clinical trials illustrate the curative potential of this therapy. While CARs against a multitude of different antigens are being developed and tested (pre)clinically, there is still a need for optimization. The use of single-chain variable fragments (scFvs) as targeting moieties hampers the quick generation of functional CARs and could potentially limit the efficacy. Instead, nanobodies may largely circumvent these difficulties. We used an available nanobody library generated after immunization of llamas against Cluster of Differentiation (CD) 20 through DNA vaccination or against the ectodomain of CD33 using soluble protein. The nanobody specific sequences were amplified by PCR and cloned by Gibson Assembly into a retroviral vector containing two different second-generation CAR constructs. After transduction in T cells, we observed high cell membrane nanoCAR expression in all cases. Following stimulation of nanoCAR-expressing T cells with antigen-positive cell lines, robust T cell activation, cytokine production and tumor cell lysis both in vitro and in vivo was observed. The use of nanobody technology in combination with PCR and Gibson Assembly allows for the rapid and effective generation of compact CARs.

Understanding the Spatial Topology of Artificial Immunological Synapses Assembled in T Cell-Redirecting Strategies: A Major Issue in Cancer Immunotherapy.

T cell-redirection strategies aim to selectively eliminate cancer cells by physically linking T lymphocytes with cancer cells using tumor-targeted cell-cell bridging (CCB) molecules, such as membrane-anchored chimeric antigen receptors (CARs) or soluble bispecific antibodies (bsAbs) that specifically recognize a cell-surface tumor-associated antigen (TAA) (Blanco et al., 2019). In the CAR approach, a TAA-specific antibody is genetically fused to intracellular T cell signaling domains. CARs have evolved greatly since their initial description, as single-chain antibody fragment (scFv)-based receptors containing the signaling domain of the CD3ζ chain (CD247) of the T cell receptor (TCR) (Eshhar et al., 1993). Subsequently, constructs incorporating signaling domains of costimulatory molecules (e.g., CD28 or 4-1BB) in tandem with the CD3ζ signaling domain were generated (Finney et al., 1998). Engrafting T-cells with such receptors, termed second-generation CARs, enables sustained proliferation and increased cytokine secretion. Third-generation CARs contain two costimulatory domains, in addition to the CD3ζ signaling domain (Carpenito et al., 2009; Milone et al., 2009). Current CAR-T cell therapy involves the isolation of autologous T cells using leukapheresis, followed by in vitro stimulation, genetic modification to express the TAA-specific CAR, and expansion to infuse back into the patient (Blanco et al., 2019). The bsAbs are designed to simultaneously bind to the TAA in the surface of tumor cells and the CD3e chain of the TCR/CD3 complex in the surface of T cells (Blanco et al., 2019). More than a 100 different bsAb formats have been reported, including small bsAbs composed only by two antigen-binding sites, IgG-like bsAbs and larger and non-IgG bsAbs formed by different antigen-binding moieties, often combined with oligomerization modules (Nunez-Prado et al., 2015; Brinkmann and Kontermann, 2017). By connecting CD3 signaling molecules with a recognition process independent of the TCR variable domains, T cells can be hot-wired to recognize a user-defined cell-surface TAA that is not associated with the major histocompatibility complex (MHC) to activate effector cell responses and kill cancer cells (Blanco et al., 2019). Nonetheless, the precise molecular mechanisms by which T cells are activated through these CCB molecules are poorly understood. The administration of bsAbs and CAR-T cells has achieved remarkable clinical outcomes in hematological tumors, and several products have been approved by regulatory agencies for clinical use. Blinatumomab, an anti-CD19xanti-CD3 bsAb designed in the BiTE (bispecific T cell-engager) format, was approved by the US Food and Drug Administration (FDA) for the treatment of relapsed or refractory B cell acute lymphoblastic leukemia (B-ALL) (Przepiorka et al., 2015). Two CD19-specific second generation CAR-T cell products, tisagenlecleucel and axicabtagene ciloleucel (axi-cel), have been approved by the US FDA for the treatment of pediatric and young adult patients with relapsed or refractory B-ALL (Maude et al., 2018) and adult patients with relapsed or refractory diffuse large B cell lymphomas (Neelapu et al., 2017), respectively. However, the utility of these approaches in the treatment of solid tumors targeting TAAs has been limited by organ toxicities related to activation of T cell effector functions by non-tumor cells expressing low levels of the TAA, as well as systemic cytokine-associated toxicities (Alonso-Camino et al., 2016).

Development and characterisation of NKp44-based chimeric antigen receptors that confer T cells with NK cell-like specificity.

ObjectivesOne of the reasons as to why chimeric antigen receptors (CAR)‐T cell therapy for malignancies other than CD19‐ or BCMA‐positive tumors has yet to produce remarkable progress is the paucity of targetable antigens. NKp44 is only expressed by activated natural killer cells and detects a variety of transformed cells, while it reportedly does not react with normal tissues. The aim of this study is to develop CAR‐T cell that can target multiple types of tumor cells.MethodsWe created a series of novel CAR constructs in first‐generation (1G) and second‐generation (2G) CAR format with the extracellular immunoglobulin‐like domain of NKp44 (NKp44‐CAR).ResultsTransduction of the best 1G construct into human primary T cells led to specific cytotoxic effects and cytokine secretion upon encountering multiple types of neoplastic cells including AML, T‐ALL and childhood solid tumors. Replacement of the extracellular hinge domain of NKp44 with that of CD8α resulted in diminished CAR function. The 1G NKp44‐CAR‐T cells exhibited significantly better tumor control in long‐term co‐culture assays compared with activated NK cells, as well as with NK cells transduced with identical NKp44‐CAR. T cells transduced with the best 2G‐CAR construct with 4‐1BB co‐stimulatory domain proliferated at significantly higher levels upon single antigen exposure and showed significantly better tumor control compared with the 1G‐CAR and 2G‐CAR with CD28 co‐stimulatory domain.ConclusionsNKp44‐based CAR endows T cells with NK cell‐like anti‐tumor specificity. The CAR gene created in this study will be useful for the development of novel gene‐modified T‐cell immunotherapy.

MUC1 as a target for CAR-T therapy in head and neck squamous cell carinoma.

The modification of chimeric antigen receptor (CAR) endowing T cells with tumor‐specific cytotoxicity induces antitumor immunity. However, the structural characteristics of solid tumors, the loss of specific antigens, and the strong immunosuppressive environment are challenges to treat solid tumors with CAR‐T therapy. The purpose of our study was to find and verify the potentials of CAR‐T therapies for patients with head and neck squamous cell carcinoma (HNSCC). First, we selected MUC1 as our research target and verified its differential expression in cancer tissues and adjacent non‐neoplastic tissues (ANNT). Next, we constructed a second‐generation CAR and validated the cytotoxic function in vitro. In our study, we found that exogenous addition human IL22 recombinant protein could increase the MUC1 expression and enhance the function of T cells. In addition, we constructed a fourth‐generation CAR that secretes IL22. Finally, we verified the antitumor function of two different CAR‐T cells in vitro and in vivo, respectively. CAR‐MUC1‐IL22 T cells were found to have a stronger and more effective cytotoxic function against MUC1 + HNSCC cells. Taken together, these results demonstrate the potential effectiveness of CAR‐T in the treatment of patients with HNSCC and provide evidence‐based of MUC1 + CAR‐T therapy.

Chimeric Antigen Receptor T Cell Therapy Pipeline at a Glance: A Retrospective and Systematic Analysis from Clinicaltrials.Gov

Background: Chimeric Antigen Receptor (CAR) T cell therapy is a promising cancer immunotherapy that is growing exponentially. The doubling time of medical knowledge in 2010 was 3.5 years, and the projection for 2020 is just 73 days. In the last five years, the number of PubMed publications on cancer applications of CAR T cells has tripled. Therefore, to remain updated in the field represents a challenge for patients, care providers and researchers. In this review we provide a focused summary of the currently ongoing clinical trials, with a comprehensive overview of advances in CAR T cell therapy, beyond CD19, emphasizing on antigenic targets, development phases, and leading sponsor pharmaceutical companies. Methods: We retrieved the available data from the national registry of clinical trials (clinicaltrials.gov) using the following keywords: "CAR T cell", "CAR T cell and cancer", "chimeric antigen receptor", "CAR T AND tumor antigen", 'CAR T cell antigens", "Tumor antigens targeted by CAR T cells", "engineered T cells", "modified T cell", "CAR T cells in Cancer", "CAR T cell therapy", "CAR T cell therapy AND Cancer" until December 31, 2018 and manually excluded the trials unrelated to CAR T-cell therapies on cancer, by reviewing the detailed information provided on the website as well as preliminary data published. Results: The analysis included 271 clinical trials posted on the clinicaltrials.gov website from the United States by the cut-off date. For efficacy analysis, we retrieved information from 52 trials, by NCT number on a PubMed search. The majority of CAR T clinical research is focused on hematological cancer (57%), followed by CNS 8%, GI 6%, Skin 5%, Genitourinary 4%, Breast 4%, Gynecologic 4%, Respiratory 3%, Sarcoma 2%, Mesothelioma 2% and others 5%. The most used target in CAR T cell therapy and the leaders in phase 3 trials are CD19 (42%) and BCMA (12%), followed by CD20, NY-ESO-1, Mesothelin, HER2, GD2, MAGE-A3 and CD30. An essential step in CAR T cell therapy development is the selection of the right antigen/target. Here, we provide an overview of the clinically relevant targets that are actively being using by clinical trials in the United States. For example, CD19 appears to be a leading target regarding CAR T cell therapy on cancer with 116 trials (42% of total CAR T cells trials) on going just in the United States with a significant increment in the previous years. Similarly, with BCMA is one of the targets with more phase 3 trials (Figure 1) with promising results on patients with Multiple Myeloma with and the objective response of 85%, CR 45%, and PFS of 11.8 months. Second-generation CARs with either CD28 or 4-1BB as costimulatory signaling domain are preferred, with 4-1BB being the most commonly chosen. Conclusions: Our findings show growing trends in the development of CAR T cell-based therapies, combination and possible retargeting therapies in the future for solid tumor and hematologic malignances; taking into account the amount of important information and the complexity of the database, we have developed this analysis to understand how to generate in the future a friendly platform for researchers and patients to have an detailed overview of the clinical trials in cellular therapies Disclosures No relevant conflicts of interest to declare.