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Purpose: Treatments are limited for metastatic melanoma and metastatic triple-negative breast cancer (mTNBC). This pilot phase I trial (NCT03060356) examined the safety and feasibility of intravenous RNA-electroporated chimeric antigen receptor (CAR) T cells targeting the cell-surface antigen cMET. Experimental Design: Metastatic melanoma or mTNBC subjects had at least 30% tumor expression of cMET, measurable disease and progression on prior therapy. Patients received up to six infusions (1 × 10e8 T cells/dose) of CAR T cells without lymphodepleting chemotherapy. Forty-eight percent of prescreened subjects met the cMET expression threshold. Seven (3 metastatic melanoma, 4 mTNBC) were treated. Results: Mean age was 50 years (35–64); median Eastern Cooperative Oncology Group 0 (0–1); median prior lines of chemotherapy/immunotherapy were 4/0 for TNBC and 1/3 for melanoma subjects. Six patients experienced grade 1 or 2 toxicity. Toxicities in at least 1 patient included anemia, fatigue, and malaise. One subject had grade 1 cytokine release syndrome. No grade 3 or higher toxicity, neurotoxicity, or treatment discontinuation occurred. Best response was stable disease in 4 and disease progression in 3 subjects. mRNA signals corresponding to CAR T cells were detected by RT-PCR in all patients’ blood including in 3 subjects on day +1 (no infusion administered on this day). Five subjects underwent postinfusion biopsy with no CAR T-cell signals seen in tumor. Three subjects had paired tumor tissue; IHC showed increases in CD8 and CD3 and decreases in pS6 and Ki67. Conclusions: Intravenous administration of RNA-electroporated cMET-directed CAR T cells is safe and feasible. Significance: Data evaluating CAR T therapy in patients with solid tumors are limited. This pilot clinical trial demonstrates that intravenous cMET-directed CAR T-cell therapy is safe and feasible in patients with metastatic melanoma and metastatic breast cancer, supporting the continued evaluation of cellular therapy for patients with these malignancies.

We conducted a phase I clinical trial of anti-BCMA chimeric antigen receptor T cells (CART-BCMA) with or without anti-CD19 CAR T cells (huCART19) in multiple myeloma (MM) patients responding to third- or later-line therapy (phase A, N = 10) or high-risk patients responding to first-line therapy (phase B, N = 20), followed by early lenalidomide or pomalidomide maintenance. We observed no high-grade cytokine release syndrome (CRS) and only one instance of low-grade neurologic toxicity. Among 15 subjects with measurable disease, 10 exhibited partial response (PR) or better; among 26 subjects responding to prior therapy, 9 improved their response category and 4 converted to minimal residual disease (MRD)–negative complete response/stringent complete response. Early maintenance therapy was safe, feasible, and coincided in some patients with CAR T-cell reexpansion and late-onset, durable clinical response. Outcomes with CART-BCMA + huCART19 were similar to CART-BCMA alone. Collectively, our results demonstrate favorable safety, pharmacokinetics, and antimyeloma activity of dual-target CAR T-cell therapy in early lines of MM treatment.Significance:CAR T cells in early lines of MM therapy could be safer and more effective than in the advanced setting, where prior studies have focused. We evaluated the safety, pharmacokinetics, and efficacy of CAR T cells in patients with low disease burden, responding to current therapy, combined with standard maintenance therapy.This article is highlighted in the In This Issue feature, p. 101

Study procedures. Participant study procedures including cell collection/manufacturing timeline as well as infusion schedule from eligibility confirmation onward.

Purpose:Treatments are limited for metastatic melanoma and metastatic triple-negative breast cancer (mTNBC). This pilot phase I trial (NCT03060356) examined the safety and feasibility of intravenous RNA-electroporated chimeric antigen receptor (CAR) T cells targeting the cell-surface antigen cMET.Experimental Design:Metastatic melanoma or mTNBC subjects had at least 30% tumor expression of cMET, measurable disease and progression on prior therapy. Patients received up to six infusions (1 × 10e8 T cells/dose) of CAR T cells without lymphodepleting chemotherapy. Forty-eight percent of prescreened subjects met the cMET expression threshold. Seven (3 metastatic melanoma, 4 mTNBC) were treated.Results:Mean age was 50 years (35–64); median Eastern Cooperative Oncology Group 0 (0–1); median prior lines of chemotherapy/immunotherapy were 4/0 for TNBC and 1/3 for melanoma subjects. Six patients experienced grade 1 or 2 toxicity. Toxicities in at least 1 patient included anemia, fatigue, and malaise. One subject had grade 1 cytokine release syndrome. No grade 3 or higher toxicity, neurotoxicity, or treatment discontinuation occurred. Best response was stable disease in 4 and disease progression in 3 subjects. mRNA signals corresponding to CAR T cells were detected by RT-PCR in all patients’ blood including in 3 subjects on day +1 (no infusion administered on this day). Five subjects underwent postinfusion biopsy with no CAR T-cell signals seen in tumor. Three subjects had paired tumor tissue; IHC showed increases in CD8 and CD3 and decreases in pS6 and Ki67.Conclusions:Intravenous administration of RNA-electroporated cMET-directed CAR T cells is safe and feasible.Significance:Data evaluating CAR T therapy in patients with solid tumors are limited. This pilot clinical trial demonstrates that intravenous cMET-directed CAR T-cell therapy is safe and feasible in patients with metastatic melanoma and metastatic breast cancer, supporting the continued evaluation of cellular therapy for patients with these malignancies.

mRNA signals corresponding to CAR T cells in peripheral blood

Supplementary Figure 1.: Representative immunohistochemical staining for Subject 74 with metastatic melanoma; lymph node biopsy. Panel A: pS6 staining prior to infusion; Panel B: pS6 staining at post-infusion biopsy; Panel C: Ki-67 staining prior to infusion; Panel D: Ki-67 staining at post-infusion biopsy.

Patient population

Response

Supplementary Figure 2: Representative immunohistochemical staining for Subject 27 with metastatic triple-negative breast cancer; lymph node biopsy. Panel A shows CD8 staining prior to infusion and Panel B shows CD8 staining at the time of post-infusion biopsy.

IHC analysis of paired tumor tissue

IHC analysis of CD8 in preinfusion and postinfusion tumor tissue. Three subjects had preinfusion and postinfusion tumor tissue available for analysis; postinfusion tumor tissue demonstrated an increase in cytoxic CD8+ cells in all 3 subjects.

Adverse events, clinically relevant (# subjects includes those with the given toxicity at least possibly related to study treatment)

Supplemental tables: (1) Subject characteristics. (2) Cytogenetic profiles and high-risk features. (3) Prior treatment exposures and refractoriness. (4) CAR T cell product characteristics. (5) Products that did not meet target dose. (6) Adverse events of grade 3-4. (7) Cytokine release syndrome and ICANS. (8). Maintenance therapy. Supplemental Figures: (1) Study schematic and subject disposition, (2) Correlates of manufacturing success, (3) Hematopoietic recovery, (4) Post-infusion T cell phenotypes, (5) Correlates of in vivo expansion and manufacturing success, (6) Late post-infusion CAR T cell re-expansion, (7) Soluble BCMA, (8) Late-onset clinical responses, (9) MM cell BCMA expression, (10) Pre- and post-treatment Sox2-specific T cell responses in CART-BCMA monotherapy patients, (11) Pre- and post-treatment Sox2-specific T cell responses in CART-BCMA + huCART19 combination therapy patients, (12) Sustained post-treatment SOX2-specific T-cell responses.

We conducted a phase I clinical trial of anti-BCMA chimeric antigen receptor T cells (CART-BCMA) with or without anti-CD19 CAR T cells (huCART19) in multiple myeloma (MM) patients responding to third- or later-line therapy (phase A, N = 10) or high-risk patients responding to first-line therapy (phase B, N = 20), followed by early lenalidomide or pomalidomide maintenance. We observed no high-grade cytokine release syndrome (CRS) and only one instance of low-grade neurologic toxicity. Among 15 subjects with measurable disease, 10 exhibited partial response (PR) or better; among 26 subjects responding to prior therapy, 9 improved their response category and 4 converted to minimal residual disease (MRD)–negative complete response/stringent complete response. Early maintenance therapy was safe, feasible, and coincided in some patients with CAR T-cell reexpansion and late-onset, durable clinical response. Outcomes with CART-BCMA + huCART19 were similar to CART-BCMA alone. Collectively, our results demonstrate favorable safety, pharmacokinetics, and antimyeloma activity of dual-target CAR T-cell therapy in early lines of MM treatment.Significance:CAR T cells in early lines of MM therapy could be safer and more effective than in the advanced setting, where prior studies have focused. We evaluated the safety, pharmacokinetics, and efficacy of CAR T cells in patients with low disease burden, responding to current therapy, combined with standard maintenance therapy.This article is highlighted in the In This Issue feature, p. 101

Fig. S1. CARTmeso cell manufacturing and treatment schedules. Fig. S2. Detection of human anti-chimeric antibody responses in patients post-CARTmeso cell infusion. Fig. S3. Serum cytokines and chemokines in PDA patient 21211-101. Fig. S4. CARTmeso cytolytic activity against allogeneic and autologous pancreatic cancer cell lines Table S1. Patient demographics. Table S2. Treatment-related adverse events. Table S3. Protoarray analysis of serum samples from mesothelioma patient 17510-105 Table S4. Protoarray analysis of serum samples from pancreatic cancer patient 21211-101

Supplementary Figure 1. : Representative immunohistochemical staining for Subject 74 with metastatic melanoma; lymph node biopsy. Panel A: pS6 staining prior to infusion; Panel B: pS6 staining at post-infusion biopsy; Panel C: Ki-67 staining prior to infusion; Panel D: Ki-67 staining at post-infusion biopsy.

Supplementary Figure 2: Representative immunohistochemical staining for Subject 27 with metastatic triple-negative breast cancer; lymph node biopsy. Panel A shows CD8 staining prior to infusion and Panel B shows CD8 staining at the time of post-infusion biopsy.

Purpose: Treatments are limited for metastatic melanoma (MM) and triple-negative breast cancer (mTNBC). This pilot phase I trial (NCT03060356) examined the safety and feasibility of intravenous RNA- electroporated chimeric antigen receptor (CAR) T cells targeting the cell-surface antigen cMET. Patients and Methods: MM or mTNBC subjects had at least 30% tumor expression of cMET, measurable disease and progression on prior therapy. Patients received up to 6 infusions (1x10e8 T cells / dose) of CAR T cells without lymphodepleting chemotherapy. Forty-eight percent of prescreened subjects met the cMET expression threshold. Seven (3 MM, 4 mTNBC) were treated. Results: Mean age was 50 years (35-64); median ECOG 0 (0-1); median prior lines of chemotherapy/immunotherapy were 4/0 for TNBC and 1/3 for melanoma subjects. Six patients experienced grade (G) 1 or 2 toxicity. Toxicities in at least 1 patient included anemia, fatigue and malaise. One subject had grade 1 cytokine release syndrome. No G 3 or higher toxicity, neurotoxicity, or treatment discontinuation occurred. Best response was stable disease in 4 and disease progression in 3 subjects. Messenger RNA signals corresponding to CAR T cells were detected by RT-PCR in all patients’ blood including in 3 subjects on Day +1 (no infusion administered on this day). Five subjects underwent post-infusion biopsy with no CAR T cell signals seen in tumor. Three subjects had paired tumor tissue; immunohistochemistry showed increases in CD8 and CD3 and decreases in pS6 and Ki67. Conclusions: Intravenous administration of RNA-electroporated cMET-directed CAR T cells is safe and feasible.

Supplemental Information and Legends

S3. Serial quantification of serum IL6 from a patient with prolonged subjectove myalgia post IT injection of mRNA c-Met CAR T cells.

S1. Clinical trial schema. S2. Representative immunohistochemistry (IHC). S3. Histology and IHC of tumor tissue pre and post intratumoral injection of RNA CAR T c- Met from one patient. S4. Histology and IHC of tumor tissue pre and post intratumoral injection with lidocaine (1 mL) and RNA CAR T c-Met (1 mL) from another patient. S5. Characterization of CD68+ tumor infiltrated immune cells using multiplex IHC analyses as described (1) in pre and post IT injection of RNA CAR T c-Met tumor tissue sections from two patients.

Chimeric antigen receptors (CAR) are synthetic molecules that provide new specificities to T cells. Although successful in treatment of hematologic malignancies, CAR T cells are ineffective for solid tumors to date. We found that the cell-surface molecule c-Met was expressed in ∼50% of breast tumors, prompting the construction of a CAR T cell specific for c-Met, which halted tumor growth in immune-incompetent mice with tumor xenografts. We then evaluated the safety and feasibility of treating metastatic breast cancer with intratumoral administration of mRNA-transfected c-Met-CAR T cells in a phase 0 clinical trial (NCT01837602). Introducing the CAR construct via mRNA ensured safety by limiting the nontumor cell effects (on-target/off-tumor) of targeting c-Met. Patients with metastatic breast cancer with accessible cutaneous or lymph node metastases received a single intratumoral injection of 3 × 107 or 3 × 108 cells. CAR T mRNA was detectable in peripheral blood and in the injected tumor tissues after intratumoral injection in 2 and 4 patients, respectively. mRNA c-Met-CAR T cell injections were well tolerated, as none of the patients had study drug–related adverse effects greater than grade 1. Tumors treated with intratumoral injected mRNA c-Met-CAR T cells were excised and analyzed by immunohistochemistry, revealing extensive tumor necrosis at the injection site, cellular debris, loss of c-Met immunoreactivity, all surrounded by macrophages at the leading edges and within necrotic zones. We conclude that intratumoral injections of mRNA c-Met-CAR T cells are well tolerated and evoke an inflammatory response within tumors. Cancer Immunol Res; 5(12); 1152–61. ©2017 AACR.

Off-target toxicity due to the expression of target antigens in normal tissue represents a major obstacle to the use of chimeric antigen receptor (CAR)-engineered T cells for treatment of solid malignancies. To circumvent this issue, we established a clinical platform for engineering T cells with transient CAR expression by using in vitro transcribed mRNA encoding a CAR that includes both the CD3-ζ and 4-1BB costimulatory domains. We present two case reports from ongoing trials indicating that adoptive transfer of mRNA CAR T cells that target mesothelin (CARTmeso cells) is feasible and safe without overt evidence of off-tumor on-target toxicity against normal tissues. CARTmeso cells persisted transiently within the peripheral blood after intravenous administration and migrated to primary and metastatic tumor sites. Clinical and laboratory evidence of antitumor activity was shown in both patients, and the CARTmeso cells elicited an antitumor immune response revealed by the development of novel antiself antibodies. These data show the potential of using mRNA-engineered T cells to evaluate, in a controlled manner, potential off-tumor on-target toxicities and show that short-lived CAR T cells can induce epitope spreading and mediate antitumor activity in patients with advanced cancer. Thus, these findings support the development of mRNA CAR-based strategies for carcinoma and other solid tumors. Cancer Immunol Res; 2(2); 112–20. ©2013 AACR.

Chimeric antigen receptor (CAR) T cells have not induced meaningful clinical responses in solid tumors. Loss of T cell stemness, poor expansion capacity, and exhaustion during prolonged tumor antigen exposure are major causes of CAR T cell therapeutic resistance. Single-cell RNA-sequencing analysis of CAR T cells from a first-in-human trial in metastatic prostate cancer identified two independently validated cell states associated with antitumor potency or lack of efficacy. Low expression of PRDM1 , encoding the BLIMP1 transcription factor, defined highly potent TCF7 [encoding T cell factor 1 (TCF1)]–expressing CD8 + CAR T cells, whereas enrichment of HAVCR2 [encoding T cell immunoglobulin and mucin-domain containing-3 (TIM-3)]–expressing CD8 + T cells with elevated PRDM1 was associated with poor outcomes. PRDM1 knockout promoted TCF7 -dependent CAR T cell stemness and proliferation, resulting in marginally enhanced leukemia control in mice. However, in the setting of PRDM1 deficiency, a negative epigenetic feedback program of nuclear factor of activated T cells (NFAT)–driven T cell dysfunction was identified. This program was characterized by compensatory up-regulation of NR4A3 and other genes encoding exhaustion-related transcription factors that hampered T cell effector function in solid tumors. Dual knockout of PRDM1 and NR4A3 skewed CAR T cell phenotypes away from TIM-3 + CD8 + and toward TCF1 + CD8 + to counter exhaustion of tumor-infiltrating CAR T cells and improve antitumor responses, effects that were not achieved with PRDM1 and NR4A3 single knockout alone. These data underscore dual targeting of PRDM1 and NR4A3 as a promising approach to advance adoptive cell immuno-oncotherapy.

MazF is an Escherichia coli-derived endoribonuclease that selectively cleaves ACA sequences of mRNA prevalent in HIV. We administered a single infusion of autologous CD4 T lymphocytes modified to express a Tat-dependent MazF transgene to 10 HIV-infected individuals (six remaining on antiretroviral therapy [ART]; four undergoing treatment interruption post-infusion) in order to provide a population of HIV-resistant immune cells. In participants who remained on ART, increases in CD4 and CD8 T cell counts of ~200 cells/mm(3) each occurred within 2 weeks of infusion and persisted for at least 6 months. Modified cells were detectable for several months in the blood and trafficked to gastrointestinal lymph tissue. HIV-1 Tat introduced ex vivo to the modified CD4(+) T cells induced MazF expression in both pre- and post-infusion samples, and MazF expression was detected in vivo post-viral-rebound during ATI. One participant experienced mild cytokine release syndrome. In sum, this study of a single infusion of MazF-modified CD4 T lymphocytes demonstrated safety of these cells, distribution to lymph tissue and maintenance of Tat-inducible MazF endoribonuclease activity, as well as sustained elevation of blood CD4 and CD8 T cell counts. Future studies to assess effects on viremia and latent proviral reservoir are warranted.

This phase I study investigated the safety and activity of lentiviral-transduced chimeric antigen receptor (CAR)-modified autologous T cells redirected against mesothelin (CART-meso) in patients with malignant pleural mesothelioma, ovarian carcinoma, and pancreatic ductal adenocarcinoma. Fifteen patients with chemotherapy-refractory cancer (n = 5 per indication) were treated with a single CART-meso cell infusion. CART-meso cells were engineered by lentiviral transduction with a construct composed of the anti-mesothelin single-chain variable fragment derived from the mouse monoclonal antibody SS1 fused to intracellular signaling domains of 4-1BB and CD3zeta. Patients received 1–3 × 10(7) or 1–3 × 10(8) CART-meso cells/m(2) with or without 1.5 g/m(2) cyclophosphamide. Lentiviral-transduced CART-meso cells were well tolerated; one dose-limiting toxicity (grade 4, sepsis) occurred at 1–3 × 10(7)/m(2) CART-meso without cyclophosphamide. The best overall response was stable disease (11/15 patients). CART-meso cells expanded in the blood and reached peak levels by days 6–14 but persisted transiently. Cyclophosphamide pre-treatment enhanced CART-meso expansion but did not improve persistence beyond 28 days. CART-meso DNA was detected in 7/10 tumor biopsies. Human anti-chimeric antibodies (HACA) were detected in the blood of 8/14 patients. CART-meso cells were well tolerated and expanded in the blood of all patients but showed limited clinical activity. Studies evaluating a fully human anti-mesothelin CAR are ongoing.

Chimeric antigen receptor (CAR) T cells have demonstrated promising efficacy, particularly in hematologic malignancies. One challenge regarding CAR T cells in solid tumors is the immunosuppressive tumor microenvironment (TME), characterized by high levels of multiple inhibitory factors, including transforming growth factor (TGF)-β. We report results from an in-human phase 1 trial of castration-resistant, prostate cancer-directed CAR T cells armored with a dominant-negative TGF-β receptor (NCT03089203). Primary endpoints were safety and feasibility, while secondary objectives included assessment of CAR T cell distribution, bioactivity and disease response. All prespecified endpoints were met. Eighteen patients enrolled, and 13 subjects received therapy across four dose levels. Five of the 13 patients developed grade ≥2 cytokine release syndrome (CRS), including one patient who experienced a marked clonal CAR T cell expansion,98% reduction in prostate-specific antigen (PSA) and death following grade 4 CRS with concurrent sepsis. Acute increases in inflammatory cytokines correlated with manageable high-grade CRS events. Three additional patients achieved a PSA reduction of ≥30%, with CAR T cell failure accompanied by upregulation of multiple TME-localized inhibitory molecules following adoptive cell transfer. CAR T cell kinetics revealed expansion in blood and tumor trafficking. Thus, clinical application of TGF-β-resistant CAR T cells is feasible and generally safe. Future studies should use superior multipronged approaches against the TME to improve outcomes.

Chimaeric antigen receptor (CAR) T cells can generate durable clinical responses in B-cell haematologic malignancies. The manufacturing of these T cells typically involves their activation, followed by viral transduction and expansion ex vivo for at least 6 days. However, the activation and expansion of CAR T cells leads to their progressive differentiation and the associated loss of anti-leukaemic activity. Here we show that functional CAR T cells can be generated within 24 hours from T cells derived from peripheral blood without the need for T-cell activation or ex vivo expansion, and that the efficiency of viral transduction in this process is substantially influenced by the formulation of the medium and the surface area-to-volume ratio of the culture vessel. In mouse xenograft models of human leukaemias, the rapidly generated non-activated CAR T cells exhibited higher anti-leukaemic in vivo activity per cell than the corresponding activated CAR T cells produced using the standard protocol. The rapid manufacturing of CAR T cells may reduce production costs and broaden their applicability.

The field of cancer immunotherapy has led to successful treatments such as monoclonal antibodies (MAb), inhibitory receptor (IR) blockade, and adoptive cell transfer (ACT). ACT modified to express chimeric antigen receptors (CARs) can redirect T cells to tumor antigens and has shown remarkable clinical efficacy in patients with relapse and refractory leukemia and lymphoma. 90% of pediatric patients with acute lymphoblastic leukemia (ALL) respond to CD19 CAR T cell therapy (CAR T19), although only 26-35% of patients with chronic lymphocytic leukemia (CLL) show complete responses (CR). It is unclear why responses are less frequent in CLL compared to ALL. The heavy pre-treatment received by CLL patients likely contributes to lower response rates with CAR T cell therapy, and disease progression is worsened due to profound T cell defects characterized by elevated expression of IRs such as PD-1 and CTLA-4. Fraietta et. al. assessed CLL patient apheresis samples and CART19 products to show that CR was associated with elevated levels of CD27+PD1-CD8+ T memory cells whereas the non-responders (NR) showed an exhausted phenotype with high levels of multiple IRs. We studied 14 patients with advanced, heavily pretreated CLL who received at least one dose of CART19. Patients with CRs exhibited high in vivo expansion and persistence of infused CAR T cells, as opposed to NR’s. Importantly, at peak levels of in vivo CAR expansion, NR’s had elevated levels of CTLA-4 expression which correlated with poor CLL patient responses to CART19 therapy. In summary, these data suggest that eliminating CTLA-4 mediated T cell inhibition can be clinically beneficial. Thus, we tested the hypothesis that disruption of CTLA4 would improve CAR T cell efficacy in CLL based on numerous observations. Our data demonstrate that knockout (KO) of CTLA-4 in T cells from healthy donors using CRISPR Cas9 technology leads to maintenance of surface CAR expression and higher tumor clearance in a chronic re-stimulation model using CART19 cells against NALM6 tumor cells. In xenograft models of ALL, KO of CTLA-4 increases the anti-tumor efficacy of CART19 cells. We then performed CTLA4 disruption in T cells banked from CLL patients that did not respond to CAR T cell therapy to determine whether dysfunctional CAR T infusion products can be invigorated by CTLA-4 KO. In both chronic re-stimulation model and xenograft models of NALM6, CTLA-4 KO CD19 CAR T cells from CLL patients maintained surface CAR expression, exhibited enhanced tumor clearance, and higher survival rates relative to subject-matched unedited CAR T cells. Thus, suggesting that CTLA4 disrupted CAR T cell products may enhance the success rate of CAR T cell therapy for CLL patients. This technology can be feasibly expanded to other tumor indications to increase the overall efficacy of CAR T cells. These IND-enabling studies will support the translation of this therapy to the clinic. Citation Format: Sangya Agarwal, Angela Aznar Gomez, Tong Da, Shunichiro Kuramitsu, Weimin Kong, Pranali Ravikumar, Mercy Gohil, Megan M. Davis, Joseph A. Fraietta, Gabriela Plesa, David L. Porter, Regina M. Young, Carl H. June. Disruption of cell-intrinsic checkpoint regulator CTLA-4 in CD19 directed CAR T cells provides clinical efficacy in CLL patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5571.

Pancreatic ductal adenocarcinoma (PDAC) is resistant to T-cell mediated immunotherapy. We engineered T cells to transiently express an mRNA encoding a chimeric antigen receptor (CAR) specific for mesothelin—a protein that is over-expressed by PDAC cells. We performed a phase 1 study to evaluate the safety and efficacy of adoptive cell therapy with autologous mesothelin-specific CAR T cells (CARTmeso cells) in 6 patients with chemotherapy-refractory metastatic PDAC. Patients were given intravenous CARTmeso cells 3 times weekly for 3 weeks. None of the patients developed cytokine release syndrome or neurologic symptoms and there were no dose limiting toxicities. Disease stabilized in 2 patients, with progression-free survival times of 3.8 and 5.4 months. We used FDG-positron emission tomography/computed tomography imaging to monitor the metabolic active volume (MAV) of individual tumor lesions. The total MAV remained stable in 3 patients and decreased by 69.2% in 1 patient with biopsy-proven mesothelin expression; in this patient, all liver lesions had a complete reduction in FDG uptake at 1 month compared to baseline, although there was no effect on the primary PDAC. Transient CAR expression was detected in patients’ blood after infusion and led to expansion of new immunoglobulin G proteins. Our results provide evidence for the potential anti-tumor activity of mRNA CARTmeso cells, as well as PDAC resistance to the immune response. Keywords: immune response heterogeneity, immune therapy, anti-tumor immunity, pancreatic cancer treatment

BackgroundMost women with epithelial ovarian cancer develop uniformly incurable disease recurrence. Chimeric antigen receptor (CAR) T cells pair the MHC-independent tumor-recognition capabilities of monoclonal antibodies with the cytotoxicity of effector T cells. The success of CAR T cell therapy in solid tumors has been hindered by (1) difficulty identifying highly expressed, tumor-specific, cell surface target antigens; (2) limited trafficking and infiltration; and (3) suboptimal cytotoxic activity. Alpha folate receptor (FRα) is a transmembrane protein involved in cellular folate transport; expression has been reported in 80% of ovarian cancer, with limited physiologic expression on epithelial cells including bronchial, renal, and intestinal tissue. We hypothesize that intraperitoneal administration of alpha folate receptor (FRα) directed CAR T cells with dual 4-1BB and TCRzeta signaling domains will circumvent the above challenges and be safe, feasible, and elicit anti-tumor responses.MethodsWe initiated a first-in-human phase I clinical trial to evaluate the feasibility, safety and preliminary efficacy of intraperitoneal administration of FRα directed CAR T cells with and without antecedent lymphodepleting chemotherapy (LDC) in women with recurrent high grade serous ovarian cancer. The lentivirally-transduced CAR is composed of a MOv19 anti-FRα-specific single chain variable fragment fused to 4-1BB and TCRzeta signaling domains. Eligible patients have persistent or recurrent high grade serous epithelial ovarian, fallopian tube, or primary peritoneal carcinoma that is not platinum refractory and expresses ≥2+ FRα staining in ≥70% of tumor cells. Subjects must have an ECOG performance status 0–1, measurable disease, adequate hematologic and organ function, and must have progressed on at least two prior chemotherapy regimens for advanced disease. Patients undergo biopsy pre-infusion and Day +14 after infusion. After same-day placement of an intraperitoneal catheter by Interventional Radiology, CAR T cells are administered via a single infusion on three dose cohorts: Cohort 1 (starting cohort), 1–3x107/m2 cells without LDC; Cohort 2, 1–3x107/m2 CAR T cells after LDC; Cohort 3, 1–3x108/m2 cells after LDC. Catheter is removed after infusion. A 3+3 dose escalation design to determine maximum tolerated dose (MTD) yields approximately 9 to 18 subjects. The primary objective is safety and feasibility, and secondary objectives are anti-tumor response (endpoints: overall response rate based on RECIST v 1.1 and irRECIST when feasible, progression-free survival and overall survival). Correlative endpoints include CAR T cell engraftment and persistence in peripheral blood and body fluids examined via quantitative PCR of CAR T DNA, and bioactivity of CAR T cells. Enrollment is ongoing.Trial RegistrationThis trial is registered at ClinicalTrials.gov (NCT03585764).Ethics ApprovalThis study was approved by the Institutional Review Board at the University of Pennsylvania (IRB 830111). All subjects provided written informed consent prior to any study-related procedures.

Chimeric antigen receptors (CAR) are synthetic molecules that provide new specificities to T cells. Although successful in treatment of hematologic malignancies, CAR T cells are ineffective for solid tumors to date. We found that the cell-surface molecule c-Met was expressed in ∼50% of breast tumors, prompting the construction of a CAR T cell specific for c-Met, which halted tumor growth in immune-incompetent mice with tumor xenografts. We then evaluated the safety and feasibility of treating metastatic breast cancer with intratumoral administration of mRNA-transfected c-Met-CAR T cells in a phase 0 clinical trial (NCT01837602). Introducing the CAR construct via mRNA ensured safety by limiting the nontumor cell effects (on-target/off-tumor) of targeting c-Met. Patients with metastatic breast cancer with accessible cutaneous or lymph node metastases received a single intratumoral injection of 3 × 107 or 3 × 108 cells. CAR T mRNA was detectable in peripheral blood and in the injected tumor tissues after intratumoral injection in 2 and 4 patients, respectively. mRNA c-Met-CAR T cell injections were well tolerated, as none of the patients had study drug–related adverse effects greater than grade 1. Tumors treated with intratumoral injected mRNA c-Met-CAR T cells were excised and analyzed by immunohistochemistry, revealing extensive tumor necrosis at the injection site, cellular debris, loss of c-Met immunoreactivity, all surrounded by macrophages at the leading edges and within necrotic zones. We conclude that intratumoral injections of mRNA c-Met-CAR T cells are well tolerated and evoke an inflammatory response within tumors. Cancer Immunol Res; 5(12); 1152–61. ©2017 AACR.

Cytokine release syndrome (CRS) is a potentially severe systemic toxicity seen after adoptive T-cell therapy and caused by T-cell activation and proliferation and is associated with elevated circulating levels of cytokines such as C-reactive protein, interleukin-6 (IL-6), and interferon-γ and has previously been described as a systemic response in hematologic malignancies. A 52-year-old woman with BRCA 1 mutation positive heavily pretreated advanced recurrent serous ovarian cancer was treated under a compassionate use protocol with autologous mesothelin-redirected chimeric antigen receptor T cells (CART-meso). Autologous T cells were transduced to express a receptor composed of an extracellular antimesothelin single-chain variable fragment fused to 4-1BB and TCR-zeta signaling domain. This patient was infused with 3×10 CART-meso T cells/m without lymphodepletion and developed compartmental CRS confined to the pleural cavities. The compartmental CRS was evidenced by an increase in IL-6 and accumulation of CART-meso T cells in pleural fluid compared with peripheral blood and was successfully treated the anti-IL6 receptor antagonist tocilizumab on D21 after the T-cell infusion. This is the first description of a compartmental CRS in a patient with solid malignancy. This response could be due to malignant pleural fluid creating an environment where T cells could interact with tumor cells and suggests localized on-target CAR-T-cell activation.

Background & Aim The Clinical Cell and Vaccine Production Facility (CVPF) at UPenn is a FACT accredited resource with a 25 year record of novel first-in-human cell and gene therapy clinical trials. CVPF is integrated with research, process development, and correlative studies laboratories, QC/QA, clinical operations and monitoring, apheresis facilities and infusion sites. CVPF has two clean room facilities in which cell immunotherapy products are currently manufactured in support of >15 investigational new drug (IND) protocols. Methods, Results & Conclusion CVPF recently developed a clean room space utilization model to increase production capacity. Previously, a product occupied a single processing suite for the length of manufacture. Improvements to the processing room layout and implementation of a “bioreactor nursery” allowed for expansion of multiple products in a single space. Other benefits included fewer equipment pieces required and built-in redundancy. Additionally, we developed a standardized production slot model to reduce the rate of unfilled manufacturing slots and maximize facility efficiency. A standardized schedule along with the application of scheduling software has improved resource management, effort tracking, production task scheduling and capacity projections. Standardized scheduling is more efficient in scheduling manufacturing staff, sample transport, environmental monitoring, facility cleaning, equipment maintenance, QC testing, batch record preparation and review and product release. We developed an accelerated training program that reduced time-to-proficiency (TTP) for new hires through resource assignment, dedicated training space and re-designed curricula. A mock processing suite was established outside of the clean room space to allow for non-GMP materials to be used during training, greatly reducing cost. Still, staff recruitment, retention and training continue to be a challenge for academic manufacturing facilities as staff renewal limits production capacity and results in fewer patients enrolled in clinical trials. CVPF joined forces with University leadership to develop a novel metrics based career advancement track for cell therapy technologists. These techniques for increased productivity coupled with improved training methods and staff retention initiatives can be adopted by academic cell therapy facilities to optimize staffing, reduce costs and increase efficiencies, thereby increasing production capacity and patient clinical trial enrollment.

CRISPR takes first steps in humans CRISPR-Cas9 is a revolutionary gene-editing technology that offers the potential to treat diseases such as cancer, but the effects of CRISPR in patients are currently unknown. Stadtmauer et al. report a phase 1 clinical trial to assess the safety and feasibility of CRISPR-Cas9 gene editing in three patients with advanced cancer (see the Perspective by Hamilton and Doudna). They removed immune cells called T lymphocytes from patients and used CRISPR-Cas9 to disrupt three genes ( TRAC, TRBC , and PDCD1 ) with the goal of improving antitumor immunity. A cancer-targeting transgene, NY-ESO-1, was also introduced to recognize tumors. The engineered cells were administered to patients and were well tolerated, with durable engraftment observed for the study duration. These encouraging observations pave the way for future trials to study CRISPR-engineered cancer immunotherapies. Science , this issue p. eaba7365 ; see also p. 976

BACKGROUND. CAR T cells are a promising therapy for hematologic malignancies. B cell maturation antigen (BCMA) is a rational target in multiple myeloma (MM). METHODS. We conducted a phase I study of autologous T cells lentivirally transduced with a fully human, BCMA-specific CAR containing CD3ζ and 4-1BB signaling domains (CART-BCMA), in subjects with relapsed/refractory MM. Twenty-five subjects were treated in 3 cohorts as follows: cohort 1, 1 × 108 to 5 × 108 CART-BCMA cells alone; cohort 2, cyclophosphamide (Cy) 1.5 g/m2 plus 1 × 107 to 5 × 107 CART-BCMA cells; cohort 3, Cy 1.5 g/m2 plus 1 × 108 to 5 × 108 CART-BCMA cells. No prespecified BCMA expression level was required. RESULTS. CART-BCMA cells were manufactured and expanded in all subjects. Toxicities included cytokine release syndrome and neurotoxicity, which were grade 3–4 in 8 (32%) and 3 (12%) subjects, respectively, and reversible. One subject died at day 24 from candidemia and progressive myeloma, following treatment for severe cytokine release syndrome and encephalopathy. Responses (based on treated subjects) were seen in 4 of 9 (44%) in cohort 1, 1 of 5 (20%) in cohort 2, and 7 of 11 (64%) in cohort 3, including 5 partial, 5 very good partial, and 2 complete responses, 3 of which were ongoing at 11, 14, and 32 months. Decreased BCMA expression on residual MM cells was noted in responders; expression increased at progression in most. Responses and CART-BCMA expansion were associated with CD4/CD8 T cell ratio and frequency of CD45RO–CD27+CD8+ T cells in the premanufacturing leukapheresis product. CONCLUSION. CART-BCMA infusions with or without lymphodepleting chemotherapy are clinically active in heavily pretreated patients with MM. TRIAL REGISTRATION. {"type":"clinical-trial","attrs":{"text":"NCT02546167","term_id":"NCT02546167"}}NCT02546167. FUNDING. University of Pennsylvania-Novartis Alliance and NIH.

The advent of engineered T cells as a form of immunotherapy marks the beginning of a new era in medicine, providing a transformative way to combat complex diseases such as cancer. Following FDA approval of CAR T cells directed against the CD19 protein for the treatment of acute lymphoblastic leukemia and diffuse large B cell lymphoma, CAR T cells are poised to enter mainstream oncology. Despite this success, a number of patients are unable to receive this therapy due to inadequate T cell numbers or rapid disease progression. Furthermore, lack of response to CAR T cell treatment is due in some cases to intrinsic autologous T cell defects and/or the inability of these cells to function optimally in a strongly immunosuppressive tumor microenvironment. We describe recent efforts to overcome these limitations using CRISPR/Cas9 technology, with the goal of enhancing potency and increasing the availability of CAR-based therapies. We further discuss issues related to the efficiency/scalability of CRISPR/Cas9-mediated genome editing in CAR T cells and safety considerations. By combining the tools of synthetic biology such as CARs and CRISPR/Cas9, we have an unprecedented opportunity to optimally program T cells and improve adoptive immunotherapy for most, if not all future patients.

This abstract was withdrawn by the authors.

125 Background: Prostate specific membrane antigen (PSMA) is a highly expressed tumor-associated antigen potentially amenable to chimeric antigen receptor-modified T (CAR-T) cell therapy for castration-resistant prostate cancer (CRPC). However, a primary challenge to the success of CAR-T therapy in CRPC is the immunosuppressive microenvironment, characterized by high levels of TGFβ. The immunosuppressive functions of TGFβ can be inhibited in T cells using a dominant negative TGFβ receptor (TGFβRdn), thereby enhancing antitumor immunity. Methods: We conducted a first-in-human phase 1 clinical trial to evaluate the feasibility, safety and preliminary efficacy of PSMA-directed/TGFβ-insensitive CAR-T cells (CART-PSMA-TGFβRdn) in patients with metastatic CRPC (NCT03089203). In a 3+3 dose-escalation design, patients received a single dose of 1-3 x 107/m2 (Cohort 1) or 1-3 x 108/m2 (Cohort 2) CART-PSMA-TGFβRdn cells without lymphodepleting (LD) chemotherapy. In Cohort 3, one patient received 1-3 x 108/m2 CART-PSMA-TGFβRdn cells following a LD chemotherapy regimen of cyclophosphamide and fludarabine (Cy/Flu). In Cohort -3, three patients received 1-3 x 107/m2 CART-PSMA-TGFβRdn cells following Cy/Flu. Patients underwent metastatic tumor biopsies at baseline and on day 10 following treatment. Quantitative PCR of CART-PSMA-TGFβRdn DNA was performed at serial timepoints to evaluate for CAR-T expansion and persistence in peripheral blood and trafficking to target tissues. Multiplex cytokine analysis assessed CART-PSMA-TGFβRdn bioactivity. Results: Ten patients received CART-PSMA-TGFβRdn therapy across dose-level cohorts. All CART-PSMA-TGFβRdn infusion products met target transduction efficiency. Evaluation of CAR-T cellular kinetics demonstrated dose-dependent peripheral blood T cell expansion, as well as tumor tissue trafficking in post-treatment tumor biopsies. At Cohort 2 and above, 5 of 7 treated patients developed grade ≥2 cytokine release syndrome (CRS). Marked increases in inflammatory cytokines (IL-6, IL-15, IL-2, IFNγ) correlated with high-grade CRS events. One grade 5 adverse event (sepsis) occurred in Cohort 3. PSA decline was observed in 6 of 10 patients (median decline -33.2%, range -11.6% to -98.3%), and PSA30 response occurred in 4 of 10 patients (including one patient achieving PSA < 0.1 ng/mL). Conclusions: Adoptive cellular therapy with CART-PSMA-TGFβRdn is safe and feasible in patients with metastatic CRPC. A dose-dependent and lymphodepletion chemotherapy-dependent relationship was observed with CART-PSMA-TGFβRdn cell expansion, cytokine expression, CRS, and anti-tumor effect. Correlative cell trafficking and paired tumor Nanostring analyses will be presented. Future clinical investigations seek to enhance anti-tumor efficacy, while optimizing the therapeutic window. Clinical trial information: NCT03089203.

10035 Background: Advanced relapsed/refractory melanoma and metastatic triple-negative breast cancer are lethal diseases for which effective therapies are limited. We conducted a pilot phase I clinical trial (NCT03060356) to establish the safety and feasibility of intravenous autologous chimeric antigen receptor (CAR) T cell immunotherapy targeting cMET, a cell-surface antigen that is highly expressed in these cancers. Methods: Subjects had metastatic or unresectable melanoma (Mel) or triple-negative breast cancer (BC) with ≥30% expression of cMET on archival tissue or screening biopsy. Eligible subjects had measurable disease and progression on at least 1 prior therapy. Patients (pts) received up to 6 doses (1x108 total T-cells per dose) of RNA electroporated anti-cMET CAR T cells over a 2-week period without antecedent lymphodepleting chemotherapy. Subjects underwent pre- and post-infusion biopsies. The primary objective was to determine feasibility and safety of treatment. Results: 77 subjects (39 mel, 38 BC) were prescreened for tumor cMET expression and 37 (17 mel, 20 BC) met the eligibility threshold. Seven pts (4 BC, 3 Mel) received cMET-directed CAR T infusions on study. Mean age was 50 years (35-64); median (M) ECOG 0 (0-1); M prior lines of chemotherapy/immunotherapy were 4/0 for BC pts and 1/3 for Mel pts. 6 of 7 pts received all planned CAR T cell infusions, and 1 received 5 infusions. 5 pts experienced grade (G) 1 or G 2 toxicity that was possibly or definitely related to study. Toxicities occurring in ≥1 pt included: anemia (n = 3), fatigue (n = 2), and malaise (n = 2). No G ≥3 toxicities or cytokine release syndrome were observed. No pts discontinued therapy due to toxicity. Best response was stable disease in 4 pts (2 BC, 2 Mel) and PD in 3 pts (2 BC, 1 Mel). Messenger RNA signals corresponding to CAR T cells were detected by RT-PCR in the peripheral blood of all pts during the infusion period and in 2 pts after the infusion period. 6 pts underwent baseline biopsy and 4 pts underwent post-infusion biopsy. Immunohistochemical stains of CD3, CD4, CD8, CD163, L26, PD1, PDL1, Foxp3, Ki67, Granzyme B and Phospho-S6 were performed on pre- and post-treatment tissue biopsies and are being evaluated. Conclusions: Intravenous administration of RNA-electroporated cMET-directed CAR T cells was safe and feasible. Future directions include examination of this target using a lentiviral construct in combination with lymphodepleting chemotherapy. Clinical trial information: NCT03060356.