Human Chimeric Antigen Receptor T-cell Research Articles

Overexpressing Bcl-2 enhances murine chimeric antigen receptor T cell therapy against solid tumor.

Chimeric antigen receptor T (CART) cell therapy has demonstrated promising potential in the treatment of hematologic malignancies. However, its application to solid tumors is limited due to the restrictive nature of the tumor microenvironment, resulting in functional failure and poor persistence of CART cells. Overexpression of Bcl-2 in human CART cells (hCART) has been found to significantly enhance their anti-apoptotic effects both in vitro and in vivo. Nevertheless, the evaluation of hCART cells in preclinical studies has predominantly relied on immunodeficient mice xenograft tumor models, making it challenging to assess the impact of hCART cells on normal tissues and the immune system. We established a murine CART (mCART) that overexpresses Bcl-2 and targets the epidermal growth factor receptor variant III (EGFRvIII), named EGFRvIII·mCART-Bcl2. It demonstrated superior proliferation, cytotoxicity, and anti-apoptotic capabilities in vitro. In an immunocompetent mouse model of abdominal metastasis of colorectal cancer, EGFRvIII·mCART-Bcl2 exhibited improved survival of CART in the abdomen, increased tumor clearance, and significantly prolonged overall mouse survival. In summary, our study provides evidence that the introduction of Bcl-2 into mCART cells can enhance their therapeutic efficacy against solid tumors while ensuring safety.

Tissue-resident memory CAR T cells with stem-like characteristics display enhanced efficacy against solid and liquid tumors

Chimeric antigen receptor (CAR) Tcells demonstrate remarkable success in treating hematological malignancies, but their effectiveness in non-hematopoietic cancers remains limited. This study proposes enhancing CAR Tcell function and localization in solid tumors by modifying the epigenome governing tissue-residency adaptation and early memory differentiation. We identify that a key factor in human tissue-resident memory CAR Tcell (CAR-TRM) formation is activation in the presence of the pleotropic cytokine, transforming growth factor β (TGF-β), which enforces a core program of both "stemness" and sustained tissue residency by mediating chromatin remodeling and concurrent transcriptional changes. This approach leads to a practical and clinically actionable invitro production method for engineering peripheral blood Tcells into a large number of "stem-like" CAR-TRM cells resistant to tumor-associated dysfunction, possessing an enhanced ability to accumulate in situ and rapidly eliminate cancer cells for more effective immunotherapy.

Abstract 1847: Engineered cell surface tag-targeted IL-2 and IL-21 selectively and safely enhance CAR-T anti-tumor activity via different mechanisms

Abstract The treatment of some patients with hematological malignancies has been transformed by chimeric antigen receptor T (CAR-T) cells. However, a large clinical need remains for more effective CAR-T therapies and to expand their use to broader patient groups including those with solid tumors. The exogenous administration of IL-2 and IL-21 were shown to enhance the engraftment, persistence, and functionality of CAR-Ts in preclinical models. However, clinical use of such cytokines is limited due to their pleiotropic nature that can result in toxicity and undesired effects on endogenous immune cells. To overcome this, we applied our cis-targeting technology to develop CAR-T specific IL-2 and IL-21 molecules that selectively activate CAR-Ts and exhibit minimal activity on non-CAR cells. Cis-targeted IL-2 and IL-21 molecules are comprised of a targeting antibody fused to an affinity-attenuated cytokine mutein, the activity of which is rescued due to the avidity provided by the targeting arm. This enables the highly selective delivery of cytokine support to human CAR-T cells. We engineered two types of fusions targeting either 1) the CAR directly without blocking CAR antigen recognition (CAR-IL2) or 2) an exogenous cell surface tag (non-signaling truncated epidermal growth factor receptor [EGFRt]) co-expressed with the CAR (EGFRt-IL2 and EGFRt-IL21). We characterized these molecules in vitro and in vivo. Both CAR- and EGFRt-targeted molecules demonstrated >100-fold preferential STAT activation of CAR-T cells over non-CAR cells. Unexpectedly, CAR-targeted fusions induced substantial antigen independent cytokine release, whereas fusions targeting the EGFRt tag did not. We found that antigen independent cytokine release was more suppressed by a LCK inhibitor rather than a JAK inhibitor, suggesting that downstream CAR signaling was the major contributor, compared to cytokine receptor signaling. In vivo studies examined the anti-tumor activity of the tag-targeted cytokine fusions given their desired safety profile. A single dose of either EGFRt-IL2 or EGFRt-IL21 on day 1 or day 7 following a sub-optimal infusion of CD19 CAR-T cells (0.1 x106) induced strong tumor regression and relapse free survival for >70 days in an aggressive NALM-6 leukemia model. EGFRt-IL21 induced a sustained 10-fold expansion of CAR-T numbers over PBS in the blood by day 7. In contrast, EGFRt-IL2 induced a delayed but greater expansion (1000-fold over PBS) by day 14 suggestive of a mechanistic difference of the two cis-targeted molecules. Analysis of EGFRt-IL2 and EGFRt-IL21 in a solid tumor model using CAR-T cells targeting endogenous tumor antigen showed similar anti-tumor effects. Cis-targeted IL-2 and IL-21 fusion molecules directed by anti-tag EGFRt antibodies confer selective enhancement of CAR-T cells with a minimal safety risk, representing a promising approach as an adjuvant CAR-T therapy. Citation Format: Nathan D. Mathewson, Wei Chen, Paul Bessette, Sara Sleiman, Meghana Sukthankar, Kelly D. Moynihan, Chris Kimberlin, Terrence Park, Audrey Hollingsworth, Saar Gill, Andy Yeung, Ivana Djuretic. Engineered cell surface tag-targeted IL-2 and IL-21 selectively and safely enhance CAR-T anti-tumor activity via different mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1847.

Metabolic reprogramming via an engineered PGC-1α improves human chimeric antigen receptor T-cell therapy against solid tumors

BackgroundCellular immunotherapies for cancer represent a means by which a patient’s immune system can be augmented with high numbers of tumor-specific T cells. Chimeric antigen receptor (CAR) therapy involves genetic...

Engineered exosome-mediated messenger RNA and single-chain variable fragment delivery for human chimeric antigen receptor T-cell engineering

Background aimsMost current chimeric antigen receptor (CAR) T cells are generated by viral transduction, which induces persistent expression of CARs and may cause serious undesirable effects. Messenger RNA (mRNA)-based approaches in manufacturing CAR T cells are being developed to overcome these challenges. However, the most common method of delivering mRNA to T cells is electroporation, which can be toxic to cells. MethodsThe authors designed and engineered an exosome delivery platform using the bacteriophage MS2 system in combination with the highly expressed protein lysosome-associated membrane protein 2 isoform B on exosomes. ResultsThe authors’ delivery platform achieved specific loading and delivery of mRNA into target cells and achieved expression of specific proteins, and anti-CD3/CD28 single-chain variable fragments (scFvs) expressed outside the exosomal membrane effectively activated primary T cells in a similar way to commercial magnetic beads. ConclusionsThe delivery of CAR mRNA and anti-CD3/CD28 scFvs via designed exosomes can be used for ex vivo production of CAR T cells with cancer cell killing capacity. The authors’ results indicate the potential applications of the engineered exosome delivery platform for direct conversion of primary T cells to CAR T cells while providing a novel strategy for producing CAR T cells in vivo.

EBAG9 silencing exerts an immune checkpoint function without aggravating adverse effects

Chimeric antigen receptor (CAR) Tcells have revolutionized treatment of B cell malignancies. However, enhancing the efficacy of engineered Tcells without compromising their safety is warranted. The estrogen receptor-binding fragment-associated antigen 9 (EBAG9) inhibits release of cytolytic enzymes from cytotoxic T lymphocytes. Here, we examined the potency of EBAG9 silencing for the improvement of adoptive Tcell therapy. MicroRNA (miRNA)-mediated EBAG9 downregulation in transplanted cytolytic CD8+ Tcells (CTLs) from immunized mice improved their cytolytic competence in a tumor model. In tolerant female recipient mice that received organ transplants, a minor histocompatibility antigen was turned into a rejection antigen by Ebag9 deletion, indicating an immune checkpoint function for EBAG9. Considerably fewer EBAG9-silenced human CAR Tcells were needed for tumor growth control in a xenotransplantation model. Transcriptome profiling did not reveal additional risks regarding genotoxicity or aberrant differentiation. A single-step retrovirus transduction process links CAR or TCR expression with miRNA-mediated EBAG9 downregulation. Despite higher cytolytic efficacy, release of cytokines associated with cytokine release syndrome remains unaffected. Collectively, EBAG9 silencing enhances effector capacity of TCR- and CAR-engineered Tcells, results in improved tumor eradication, facilitates efficient manufacturing, and decreases the therapeutic dose.

S207: EFFICACY AND SAFETY OF A THIRD GENERATION CD20 CART (MB-106) FOR TREATMENT OF RELAPSED/REFRACTORY FOLLICULAR LYMPHOMA (FL)

Background: Follicular lymphoma (FL) is incurable and novel treatments with high efficacy and low toxicity are needed. Chimeric antigen receptor T-cells (CAR-T) targeting CD19 are effective, and axicabtagene ciloleucel is currently approved by FDA for treatment of patients (pts) with relapsed FL, but toxicities like cytokine-release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) may limit its use. MB-106 is a fully human 3rd-generation CD20-targeted CAR-T product with both 4-1BB and CD28 costimulatory domains. Aims: We present the results of the FL cohort from our ongoing phase I/II clinical trial investigating MB-106 for B-cell lymphoma/CLL. Methods: Pts with R/R B-cell malignancies including FL were eligible after confirmation of CD20 expression. Prior treatment with CD19 CAR-T is permitted. Lymphodepletion (LD) consists of cyclophosphamide (Cy) ± fludarabine (Flu). CAR-T cells are administered at one of 4 dose levels (DL): DL1: 3.3x105, DL2: 1x106, DL3: 3.3x106, DL4: 1x107 CAR T cells/kg. A continual reassessment method dose escalation design was used to find the maximally tolerated dose. CAR-T was infused in the outpatient setting except for the first pt of each dose cohort (overnight observation). Initial treatment response is assessed on day 28 and best response is reported here. CRS and ICANS are graded per ASTCT. Results: Between Dec 2019 and Dec 2021, 16 pts with FL were treated and had day 28 assessment. Median age was 61.5 years (range: 45 – 81). High-risk features included pts with progression of disease within 24 months of first-line chemotherapy (POD24) (n=11; 69%), history of histologic transformation (n=3; 19%), prior treatment with a CD19 targeted CAR-T (n=1; 6%). 6 pts (37.5%) had a PI3 kinase inhibitor. Median time between leukapheresis and LD was 15 days (range: 9-21) and 2 pts received bridging therapy with lenalidomide (1) and high-dose corticosteroids (1). All DLs were reached (DL0=1, DL1=1, DL2=4, DL3=7, DL4=2), with no dose-limiting toxicities. All CRS events were grade 1 (n=4; 25%) or grade 2 (n=1; 6%), with no grade ≥ 3 CRS events. There was no occurrence of ICANS of any grade. No pts had tumor lysis syndrome or Gr 3-4 infections. In the first 28 days, thrombocytopenia (Gr 3-4: 12.5%) and neutropenia (Gr 3-4: 94%) were common but there were no bleeding complications, and the rate of febrile neutropenia was 25%. Overall response (ORR) rate was 94% (15/16) and a complete response (CR) rate was 75% (12/16). Pts who received DL3 or DL4, had an ORR 100% and CR rate of 89%. The single patient who had previously been treated with a CD19 targeted CAR-T achieved a CR. With median follow-up of 10 months, one patient died, from complications of myelodysplastic syndrome (MDS) while still in remission; the MDS was attributed to the prior treatments (7 lines of chemo-based therapy and radioimmunotherapy). Most of the remissions are ongoing and 4 pts have relapsed during the follow-up median 7 months after treatment (range 3-10.5). CAR T cells were detectable by flow cytometry at last available timepoint (up to 2 years) for 15 of 16 patients. One patient had undetectable CAR T cells by day 201. Summary/Conclusion: Treatment with MB-106, a third generation CD20 targeting CAR-T, resulted in high ORR and CR rates and CAR-T persistence in FL pts and was associated with favorable safety profile with no occurrence of Gr 3 or Gr 4 CRS and no ICANS event of any grade. A multicenter trial for treatment of B-cell malignancies including FL will start enrollment in 2022.

B7-H3 Specific CAR T Cells for the Naturally Occurring, Spontaneous Canine Sarcoma Model.

One obstacle for human solid tumor immunotherapy research is the lack of clinically relevant animal models. In this study, we sought to establish a chimeric antigen receptor (CAR) T-cell treatment model for naturally occurring canine sarcomas as a model for human CAR T-cell therapy. Canine CARs specific for B7-H3 were constructed using a single-chain variable fragment derived from the human B7-H3-specific antibody MGA271, which we confirmed to be cross-reactive with canine B7-H3. After refining activation, transduction, and expansion methods, we confirmed target killing in a tumor spheroid three-dimensional assay. We designed a B7-H3 canine CAR T-cell and achieved consistently high levels of transduction efficacy, expansion, and in vitro tumor killing. Safety of the CAR T cells were confirmed in two purposely bred healthy canine subjects following lymphodepletion by cyclophosphamide and fludarabine. Immune response, clinical parameters, and manifestation were closely monitored after treatments and were shown to resemble that of humans. No severe adverse events were observed. In summary, we demonstrated that similar to human cancers, B7-H3 can serve as a target for canine solid tumors. We successfully generated highly functional canine B7-H3-specific CAR T-cell products using a production protocol that closely models human CAR T-cell production procedure. The treatment regimen that we designed was confirmed to be safe in vivo. Our research provides a promising direction to establish in vitro and in vivo models for immunotherapy for canine and human solid tumor treatment.

An NK-like CAR T cell transition in CAR T cell dysfunction

Chimeric antigen receptor (CAR) Tcell therapy has achieved remarkable success in hematological malignancies but remains ineffective in solid tumors, due in part to CAR Tcell exhaustion in the solid tumor microenvironment. To study dysfunction of mesothelin-redirected CAR Tcells in pancreatic cancer, we establish a robust model of continuous antigen exposure that recapitulates hallmark features of Tcell exhaustion and discover, both invitro and in CAR Tcell patients, that CAR dysregulation is associated with a CD8+ T-to-NK-like Tcell transition. Furthermore, we identify a gene signature defining CAR and TCR dysregulation and transcription factors, including SOX4 and ID3 as key regulators of CAR Tcell exhaustion. Our findings shed light on the plasticity of human CAR Tcells and demonstrate that genetic downmodulation of ID3 and SOX4 expression can improve the efficacy of CAR Tcell therapy in solid tumors by preventing or delaying CAR Tcell dysfunction.

PI3Kδ/γ inhibition promotes human CART cell epigenetic and metabolic reprogramming to enhance antitumor cytotoxicity

AbstractCurrent limitations in using chimeric antigen receptor T(CART) cells to treat patients with hematological cancers include limited expansion and persistence in vivo that contribute to cancer relapse. Patients with chronic lymphocytic leukemia (CLL) have terminally differentiated T cells with an exhausted phenotype and experience low complete response rates after autologous CART therapy. Because PI3K inhibitor therapy is associated with the development of T-cell–mediated autoimmunity, we studied the effects of inhibiting the PI3Kδ and PI3Kγ isoforms during the manufacture of CART cells prepared from patients with CLL. Dual PI3Kδ/γ inhibition normalized CD4/CD8 ratios and maximized the number of CD8+ T-stem cell memory, naive, and central memory T-cells with dose-dependent decreases in expression of the TIM-3 exhaustion marker. CART cells manufactured with duvelisib (Duv-CART cells) showed significantly increased in vitro cytotoxicity against CD19+ CLL targets caused by increased frequencies of CD8+ CART cells. Duv-CART cells had increased expression of the mitochondrial fusion protein MFN2, with an associated increase in the relative content of mitochondria. Duv-CART cells exhibited increased SIRT1 and TCF1/7 expression, which correlated with epigenetic reprograming of Duv-CART cells toward stem-like properties. After transfer to NOG mice engrafted with a human CLL cell line, Duv-CART cells expressing either a CD28 or 41BB costimulatory domain demonstrated significantly increased in vivo expansion of CD8+ CART cells, faster elimination of CLL, and longer persistence. Duv-CART cells significantly enhanced survival of CLL-bearing mice compared with conventionally manufactured CART cells. In summary, exposure of CART to a PI3Kδ/γ inhibitor during manufacturing enriched the CART product for CD8+ CART cells with stem-like qualities and enhanced efficacy in eliminating CLL in vivo.

CAR T-Cells Depend on the Coupling of NADH Oxidation with ATP Production.

The metabolic milieu of solid tumors provides a barrier to chimeric antigen receptor (CAR) T-cell therapies. Excessive lactate or hypoxia suppresses T-cell growth, through mechanisms including NADH buildup and the depletion of oxidized metabolites. NADH is converted into NAD+ by the enzyme Lactobacillus brevis NADH Oxidase (LbNOX), which mimics the oxidative function of the electron transport chain without generating ATP. Here we determine if LbNOX promotes human CAR T-cell metabolic activity and antitumor efficacy. CAR T-cells expressing LbNOX have enhanced oxygen as well as lactate consumption and increased pyruvate production. LbNOX renders CAR T-cells resilient to lactate dehydrogenase inhibition. But in vivo in a model of mesothelioma, CAR T-cell’s expressing LbNOX showed no increased antitumor efficacy over control CAR T-cells. We hypothesize that T cells in hostile environments face dual metabolic stressors of excessive NADH and insufficient ATP production. Accordingly, futile T-cell NADH oxidation by LbNOX is insufficient to promote tumor clearance.

Itacitinib (INCB039110), a JAK1 Inhibitor, Reduces Cytokines Associated with Cytokine Release Syndrome Induced by CAR T-cell Therapy.

T cells engineered to express a chimeric antigen receptor (CAR) are a promising cancer immunotherapy. Such targeted therapies have shown long-term relapse-free survival in patients with B-cell leukemia and lymphoma. However, cytokine release syndrome (CRS) represents a serious, potentially life-threatening side effect often associated with CAR T-cell therapy. CRS manifests as a rapid (hyper)immune reaction driven by excessive inflammatory cytokine release, including IFNγ and IL6. Many cytokines implicated in CRS are known to signal through the JAK-STAT pathway. Here we study the effect of blocking JAK pathway signaling on CAR T-cell proliferation, antitumor activity, and cytokine levels in in vitro and in vivo models. We report that itacitinib, a potent, selective JAK1 inhibitor, was able to significantly and dose-dependently reduce levels of multiple cytokines implicated in CRS in several in vitro and in vivo models. Importantly, we also report that at clinically relevant doses that mimic human JAK1 pharmacologic inhibition, itacitinib did not significantly inhibit proliferation or antitumor killing capacity of three different human CAR T-cell constructs (GD2, EGFR, and CD19). Finally, in an in vivo model, antitumor activity of CD19-CAR T cells adoptively transferred into CD19+ tumor-bearing immunodeficient animals was unabated by oral itacitinib treatment. Together, these data suggest that itacitinib has potential as a prophylactic agent for the prevention of CAR T cell-induced CRS, and a phase II clinical trial of itacitinib for prevention of CRS induced by CAR T-cell therapy has been initiated (NCT04071366).

Third Generation CD20 Targeted CAR T-Cell Therapy (MB-106) for Treatment of Patients with Relapsed/Refractory B-Cell Non-Hodgkin Lymphoma

Third Generation CD20 Targeted CAR T-Cell Therapy (MB-106) for Treatment of Patients with Relapsed/Refractory B-Cell Non-Hodgkin Lymphoma

Demonstrating the Manufacture of Human CAR-T Cells in an Automated Stirred-Tank Bioreactor.

Chimeric antigen receptor T-cell (CAR-T) therapies have proven clinical efficacy for the treatment of hematological malignancies. However, CAR-T cell therapies are prohibitively expensive to manufacture. The authors demonstrate the manufacture of human CAR-T cells from multiple donors in an automated stirred-tank bioreactor. The authors successfully produced functional human CAR-T cells from multiple donors under dynamic conditions in a stirred-tank bioreactor, resulting in overall cell yields which were significantly better than in static T-flask culture. At agitation speeds of 200 rpm and greater (up to 500 rpm), the CAR-T cells are able to proliferate effectively, reaching viable cell densities of >5 × 106 cells ml-1 over 7 days. This is comparable with current expansion systems and significantly better than static expansion platforms (T-flasks and gas-permeable culture bags). Importantly, engineered T-cells post-expansion retained expression of the CAR gene and retained their cytolytic function even when grown at the highest agitation intensity. This proves that power inputs used in this study do not affect cell efficacy to target and kill the leukemia cells. This is the first demonstration of human CAR-T cell manufacture in stirred-tank bioreactors and the findings present significant implications and opportunities for larger-scale allogeneic CAR-T production.

Improving the anti-solid tumor efficacy of CAR-T cells by inhibiting adenosine signaling pathway

ABSTRACT Chimeric antigen receptor T (CAR-T) cell therapy has been applied successfully in treating hematologic malignancies; however, it shows very limited efficacy in treating solid tumors. Adenosine is one of the key immunosuppressive metabolites in tumor microenvironment (TME) of solid tumors. Although the effect of adenosine has been well studied using mouse CAR-T cells, its effect on human CAR-T cells has not been fully elucidated. In particular, there was no evaluation of the CAR-T cells with blocked adenosine signaling in tumor xenograft animal model, which is essential for determining the feasibility of future clinical trials. In this study, we found the expression of A2a receptor (A2AR) and A2b receptor (A2BR) both upregulated in human-derived CAR-T cells, and only A2AR was responsible for adenosine-induced impairment of CAR-T cell function. Disrupting A2AR gene in human CAR-T cells with CRISPR-Cas9 increased the anti-tumor function and prevented the exhaustion of CAR-T cells in vitro. Furthermore, CRL5826-CDX model and two patient-derived xenograft solid tumor models were applied to evaluate the efficacy of A2AR knock-out CAR-T cells, which showed superior capability of inhibiting tumor growth. Taken together, these results demonstrate that A2AR knock-out CAR-T cells have the potential of being an improved CAR-T cell therapy in treating solid tumors.

Combined Antitumor Effects of Sorafenib and GPC3-CAR T Cells in Mouse Models of Hepatocellular Carcinoma.

Our previous study indicated that GPC3-targeted chimeric antigen receptor (CAR) Tcell therapy has a high safety profile in patients with hepatocellular carcinoma (HCC). However, theresponse rate requires further improvement. Here, we analyzed the combined effect of GPC3-CAR Tcells and sorafenib in both immunocompetent and immunodeficient mouse models of hepatocellular carcinoma. In immunocompetent mouse model, mouse CAR (mCAR) Tcells induced regression of small tumors (approximately 130mm3 tumor volume) but had no effect on large, established tumors (approximately 400mm3 tumor volume). Sorafenib, at a subpharmacologic but not a pharmacologic dose, augmented the antitumor effects of mCAR Tcells, in part by promoting IL12 secretion in tumor-associated macrophages (TAMs) and cancer cell apoptosis. In an immunodeficient mouse model, both subpharmacologic and pharmacologic doses of sorafenib had limited impacts on the function of human CAR (huCAR) Tcells invitro and showed synergistic effects with huCAR Tcells invivo, which can at least partially be ascribed to the upregulated tumor cell apoptosis induced by the combined treatment. Thus, this study applied two of the most commonly used mouse models for CAR Tcell research and demonstrated the clinical potential of combining sorafenib with GPC3-targeted CAR Tcells against HCC.

Abstract PR06: Dual-specific T-cells and an indirect vaccine eradicate large solid tumors

Abstract While immunotherapy can eliminate substantial burdens of some leukemias, the ultimate challenge remains the eradication of large solid tumors and metastases for most cancers. Here we generate dual-specific T-cells expressing a chimeric antigen receptor (CAR) specific for Her2 and a TCR specific for the melanocyte protein (gp100). Injection of T-cells, together with a vaccine that contains a recombinant vaccinia virus expressing gp100, induced durable complete remission of a variety of Her2+ tumors and established metastases, some in excess of 150 mm2, in immunocompetent mice expressing Her2 in normal tissues. Tumor destruction occurred rapidly over seven days and was associated with an extensive infiltrate of T-cells. Mice that had rejected tumors were resistant to rechallenge with the same Her2+ tumor cells, indicating the formation of immune memory. Furthermore, we have established methods to transduce dual-specific T-cells from human peripheral blood with both a TCR specific for gp100 and a CAR for Her2. From as little as 1 ml of human buffy coat, we could generate sufficient numbers of cells for a course of treatment for a patient. The stimulation of gp100 through TCR enhanced the human dual-specific CAR T-cell proliferation, secretion of IFN-γ and killing of Her2+ human cancer cells in vitro. These characteristics were identified to be important for eradicating tumors in the mouse models. Taken together, our data provide valuable information for the development of CAR T-cell therapies for patients with solid cancers and evidence for pursuing a phase I clinical trial. Citation Format: Clare Y. Slaney, Bianca von Scheidt, Phillip K. Darcy, Michael H. Kershaw. Dual-specific T-cells and an indirect vaccine eradicate large solid tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr PR06.

Low Dose of Human scFv-Derived BCMA-Targeted CAR-T Cells Achieved Fast Response and High Complete Remission in Patients with Relapsed/Refractory Multiple Myeloma

▪Introduction: B Cell Mature Antigen (BCMA)-targeted chimeric antigen receptor T (CAR-T) cell therapy emerges as promising treatment for patients with relapse/refractory multiple myeloma (RRMM). Previous studies indicate patients who receive high-dose CAR-T cells may achieve better remission but have worse adverse events, like cytokine release syndrome (CRS). To solve this dilemma, we have developed novel autologous CAR-T therapeutics CT053 that are genetically modified T cells comprising an extracellular anti-BCMA human scFv and an intracellular 4-1BB costimulatory motif connected to a CD3-zeta T cell activation domain.Methods: A multi-center investigator-initiated clinical study is designed to evaluate CT053 in patients with RRMM who have failed in the prior treatment with ≥2 regimens, including a proteasome inhibitor, an immunomodulatory agent, and anti-CD38 monoclonal antibody. All patients have ≥50% BCMA expression on malignant cells. Patients are subjected to the lymphodepletion with 20-25 mg/m2 fludarabine and 300-500 mg/m2 cyclophosphamide daily for 2-4 days prior to receiving single-dose infusion of CT053 CAR-T cells. In case of progressive disease, patient may be dosed again on basis of investigators' evaluation of the disease status, BCMA expression and CAR-T persistence. Most enrolled patients received a single dose of 1.5 x 108 cells, except for 1 patient who received 0.5 x 108 cells and 1 patient who was infused with 1.8 x 108 cells. The primary outcome measure is incidence of adverse events (AEs), including dose-limiting toxicities (DLTs) and CAR T related AEs. Additional outcome measures include clinical response assessed according to the IMWG Uniform Response Criteria for Multiple Myeloma, overall and progression-free survival, pharmacokinetics and pharmacodynamic of CT053.Results: The study was performed in compliance with the declaration of Helsinki. As of the data cut-off date (July 10th, 2018), 16 patients (median 55 [39 to 67] years old) with a median of 3.9 (0.4 to 16.7) years since MM diagnosis, were infused with CT053. Patients had a median of 4 prior different regimens (range 2 to 10), and 56% (9/16) patients received prior autologous or allogeneic stem cell transplant.Among 16 patients, no neurotoxicity and no dose-limiting toxicities (DLT) were observed. The most common grade≥3 CAR-T related AEs were 3 thrombocytopenia (19%), 3 leukopenia (19%), 2 anemia (13%), 2 neutropenia (13%), 2 fever (13%) (Figure 1A). CRS was reported in 3 patients, including 1 Grade 3, 1 Grade 2 and 1 Grade 1, who had rapid recovery after Tocilizumab administration.13/16 patients were eligible for initial evaluation of early clinical response with a median observation period of 8 (4 to 36) weeks. Overall response rate (ORR) in 13/13 patients was 100% post treatment. 12/13 patients (92%) quickly achieved partial response (4 PR), very good PR (6 VGPR), and complete response (2 CR) within 4 weeks post single-dose infusion (Figure 1B). 5/12 patients (42%) who were dosed at ≥1.5 x 108 CT053 CAR-T cells obtained CR at a median of 8 weeks post treatment. Durable responses from 4 weeks towards the data cut-off date were found in 12/13 patients (92%). One relapse from VGPR by the Week 12 was reported in a patient who had aggressive RRMM at enrollment and received the reduced dose of lymphodepletion regimen at 19 mg/m2 fludarabine and 192 mg/m2 cyclophosphamide for 2 days prior to CT053 infusion. Because positive BCMA expression on malignant cells was verified at relapse, the patient was re-dosed with CT053 at the Week 16 and subjected to the further evaluation.All patients had detectable CAR-T expansion from Day 3 post CT053 infusion. Expansion peaks were found on Day 7 (5/13), Day 14 (6/13) and Day 21 (2/13). 11/13 patients had notable persistence of CT053 CAR-T cells up to 4-6 months. The only relapsed patient had the lowest CAR-T expansion peak among 13 patients, indicating the potential correlation between CAR-T expansion and response outcome.Conclusions: Data from this early-stage clinical study showed the unparalleled safety and efficacy of CT053 CAR-T cells. Major AEs were transient, manageable, and reversible. 100% ORR in 13/13 evaluable patients were reported post single-dose infusion of 0.5~1.8 x 108 cells. 5/12 patients who were dosed at ≥1.5 x 108 CAR-T cells rapidly achieved durable CR at median of 8 weeks, suggesting CT053 could be developed as competitive therapeutics to treat patients with RRMM. DisclosuresRuan:CARsgen Therapeutics: Employment. Xiao:CARsgen Therapeutics: Employment, Equity Ownership. Wang:CARsgen Therapeutics: Employment, Equity Ownership. Li:CARsgen Therapeutics: Employment, Equity Ownership.

Abstract 4979: Development of KITE-585: A fully human BCMA CAR T-cell therapy for the treatment of multiple myeloma

Abstract Background: Multiple myeloma (MM) is a usually fatal malignancy of plasma cells, with no current therapy considered curative. About 15% of patients diagnosed with MM are stratified as high risk with poor treatment outcomes and short (2-3 years) survival from diagnosis. Standard risk patients tend to live longer but undergo chronic and/or high intensity therapy and likely experience a relapsing and remitting disease pattern. Therefore, there is still a considerable unmet need for innovative therapies that improve outcomes in MM. One such approach is to use adoptive transfer of engineered autologous T cells expressing a chimeric antigen receptor (CAR) directed against malignant cells. The efficacy of CAR T cells directed against hematological malignancies, particularly CD19-expressing B cell leukemia and lymphomas, has been demonstrated in multiple clinical studies. KITE-585 was developed as a CAR T cell immunotherapy product candidate directed against B cell maturation antigen (BCMA). BCMA is nearly ubiquitously expressed on MM cells, plasma cells and subsets of mature B cells, but with limited or absent expression on other tissues. Methods: We generated >50 fully human IgGs directed against BCMA using the BCMA protein as antigen and selection criteria including affinity, cross-reactivity and poly-specificity. Following assessment of the binding of the IgGs to a MM cell line known to express BCMA, >10 IgGs were identified that met the criteria for affinity and selectivity and had a >50-fold binding over background. The 8 IgGs that demonstrated the highest specific binding were then sequence-converted to single-chain variable fragments (scFvs) and incorporated into CARs. Results: In all but one case, human T cells engineered to express these CAR constructs exhibited specific cytolytic activity against MM cell lines (NCI-H929 and MM.1s). These CAR T cells demonstrated killing efficiencies of >95% at effector:target ratios of 1:1 over a 24-hour period. Similarly antigen-specific production of inflammatory cytokines was observed in response to target cell lines in vitro. Assessment of antigen-dependent proliferation over a 5 day period revealed >80% proliferation in the 7 constructs that showed cytolytic activity in vitro. Multiple different anti-BCMA CAR constructs representing distinct epitope binding bins of BCMA were then selected for in vivo evaluation. In two disseminated tumor models of luciferase labeled NCI-H929 or MM.1s cells injected intravenously (i.v.), a single i.v. injection of anti-BCMA CAR T-cells delayed the progression of disease and significantly increased survival when compared to control treatment. Conclusions: The results of these studies highlight the potential of targeting BCMA with adoptive transfer of engineered T cells for the treatment of MM. Given these positive findings, progress towards Phase 1 clinical studies in MM patients with KITE-585 is continuing. Citation Format: Gregor B. Adams, Jun Feng, Atefah Ghogha, Armen Mardiros, Jodi Murakami, Tammy Phung, Ruben Rodriguez, Stuart Sievers, Tassja J. Spindler, Jed Wiltzius, Clare Yarka, Sean C. Yoder, Tony Polverino. Development of KITE-585: A fully human BCMA CAR T-cell therapy for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4979. doi:10.1158/1538-7445.AM2017-4979