Reinfusion of CD19 CAR T cells for relapse prevention and treatment in children with acute lymphoblastic leukemia.

ABSTRACT: Relapse after CD19-directed chimeric antigen receptor (CAR)-modified T cells remains a substantial challenge. Short CAR T-cell persistence contributes to relapse risk, necessitating novel approaches to prolong durability. CAR T-cell reinfusion (CARTr) represents a potential strategy to reduce the risk of or treat relapsed disease after initial CAR T-cell infusion (CARTi). We conducted a retrospective review of reinfusion of murine (CTL019) or humanized (huCART19) anti-CD19/4-1BB CAR T cells across 3 clinical trials or commercial tisagenlecleucel for relapse prevention (peripheral B-cell recovery [BCR] or marrow hematogones ≤6 months after CARTi), minimal residual disease (MRD) or relapse, or nonresponse to CARTi. The primary endpoint was complete response (CR) at day 28 after CARTr, defined as complete remission with B-cell aplasia. Of 262 primary treatments, 81 were followed by ≥1 reinfusion (investigational CTL019, n = 44; huCART19, n = 26; tisagenlecleucel, n = 11), representing 79 patients. Of 63 reinfusions for relapse prevention, 52% achieved CR (BCR, 15/40 [38%]; hematogones, 18/23 [78%]). Lymphodepletion was associated with response to CARTr for BCR (odds ratio [OR], 33.57; P = .015) but not hematogones (OR, 0.30; P = .291). The cumulative incidence of relapse was 29% at 24 months for CR vs 61% for nonresponse to CARTr (P = .259). For MRD/relapse, CR rate to CARTr was 50% (5/10), but 0/8 for nonresponse to CARTi. Toxicity was generally mild, with the only grade ≥3 cytokine release syndrome (n = 6) or neurotoxicity (n = 1) observed in MRD/relapse treatment. Reinfusion of CTL019/tisagenlecleucel or huCART19 is safe, may reduce relapse risk in a subset of patients, and can reinduce remission in CD19+ relapse.

T cell lymphoma and secondary primary malignancy risk after commercial CAR T cell therapy.

We report a T cell lymphoma (TCL) occurring 3 months after anti-CD19 chimeric antigen receptor (CAR) T cell immunotherapy for non-Hodgkin B cell lymphoma. The TCL was diagnosed from a thoracic lymph node upon surgery for lung cancer. The TCL exhibited CD8+ cytotoxic phenotype and a JAK3 variant, while the CAR transgene was very low. The T cell clone was identified at low levels in the blood before CAR T infusion and in lung cancer. To assess the overall risk of secondary primary malignancy after commercial CAR T (CD19, BCMA), we analyzed 449 patients treated at the University of Pennsylvania. At a median follow-up of 10.3 months, 16 patients (3.6%) had a secondary primary malignancy. The median onset time was 26.4 and 9.7 months for solid and hematological malignancies, respectively. The projected 5-year cumulative incidence is 15.2% for solid and 2.3% for hematological malignancies. Overall, one case of TCL was observed, suggesting a low risk of TCL after CAR T.

Unedited allogeneic iNKT cells show extended persistence in MHC-mismatched canine recipients.

Allogeneic invariant natural killer T cells (allo-iNKTs) induce clinical remission in patients with otherwise incurable cancers and COVID-19-related acute respiratory failure. However, their functionality is inconsistent among individuals, and they become rapidly undetectable after infusion, raising concerns over rejection and limited therapeutic potential. We validate a strategy to promote allo-iNKT persistence in dogs, an established large-animal model for novel cellular therapies. We identify donor-specific iNKT biomarkers of survival and sustained functionality, conserved in dogs and humans and retained upon chimeric antigen receptor engineering. We reason that infusing optimal allo-iNKTs enriched in these biomarkers will prolong their persistence without requiring MHC ablation, high-intensity chemotherapy, or cytokine supplementation. Optimal allo-iNKTs transferred into MHC-mismatched dogs remain detectable for at least 78 days, exhibiting sustained immunomodulatory effects. Our canine model will accelerate biomarker discovery of optimal allo-iNKT products, furthering application of MHC-unedited allo-iNKTs as a readily accessible universal platform to treat incurable conditions worldwide.

Phase I Trial of Autologous RNA-electroporated cMET-directed CAR T Cells Administered Intravenously in Patients with Melanoma and Breast Carcinoma.

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.

Anti-BCMA/CD19 CAR T Cells with Early Immunomodulatory Maintenance for Multiple Myeloma Responding to Initial or Later-Line Therapy.

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.

PD1 Expression in EGFRvIII-Directed CAR T Cell Infusion Product for Glioblastoma Is Associated with Clinical Response.

The epidermal growth factor receptor variant III (EGFRvIII) has been investigated as a therapeutic target for chimeric antigen receptor (CAR) T cell therapy in glioblastoma. Earlier research demonstrated that phenotypic and genotypic characteristics in T cells and CAR T product predicted therapeutic success in hematologic malignancies, to date no determinants for clinical response in solid tumors have been identified. We analyzed apheresis and infusion products from the first-in-human trial of EGFRvIII-directed CAR T for recurrent glioblastoma (NCT02209376) by flow cytometry. Clinical response was quantified via engraftment in peripheral circulation and progression-free survival (PFS), as determined by the time from CAR T infusion to first radiographic evidence of progression. The CD4+CAR T cell population in patient infusion products demonstrated PD1 expression which positively correlated with AUC engraftment and PFS. On immune checkpoint inhibitor analysis, CTLA-4, TIM3, and LAG3 did not exhibit significant associations with engraftment or PFS. The frequencies of PD1+GZMB+ and PD1+HLA-DR+ CAR T cells in the CD4+ infusion products were directly proportional to AUC and PFS. No significant associations were observed within the apheresis products. In summary, PD1 in CAR T infusion products predicted peripheral engraftment and PFS in recurrent glioblastoma.

PSMA-targeting TGFß-insensitive armored CAR T cells in metastatic castration-resistant prostate cancer: a phase 1 trial.

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)-ß. 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-ß 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-ß-resistant CAR T cells is feasible and generally safe. Future studies should use superior multipronged approaches against the TME to improve outcomes.

Decade-long leukaemia remissions with persistence of CD4+ CAR T cells.

The adoptive transfer of T lymphocytes reprogrammed to target tumour cells has demonstrated potential for treatment of various cancers1-7. However, little is known about the long-term potential and clonal stability of the infused cells. Here we studied long-lasting CD19-redirected chimeric antigen receptor (CAR) T cells in two patients with chronic lymphocytic leukaemia1-4 who achieved a complete remission in 2010. CAR T cells remained detectable more than ten years after infusion, with sustained remission in both patients. Notably, a highly activated CD4+ population emerged in both patients, dominating the CAR T cell population at the later time points. This transition was reflected in the stabilization of the clonal make-up of CAR T cells with a repertoire dominated by a small number of clones. Single-cell profiling demonstrated that these long-persisting CD4+ CAR T cells exhibited cytotoxic characteristics along with ongoing functional activation and proliferation. In addition, longitudinal profiling revealed a population of gamma delta CAR T cells that prominently expanded in one patient concomitant with CD8+ CAR T cells during the initial response phase. Our identification and characterization of these unexpected CAR T cell populations provide novel insight into the CAR T cell characteristics associated with anti-cancer response and long-term remission in leukaemia.

BET bromodomain protein inhibition reverses chimeric antigen receptor extinction and reinvigorates exhausted T cells in chronic lymphocytic leukemia.

Chimeric antigen receptor (CAR) T cells have induced remarkable antitumor responses in B cell malignancies. Some patients do not respond because of T cell deficiencies that hamper the expansion, persistence, and effector function of these cells. We used longitudinal immune profiling to identify phenotypic and pharmacodynamic changes in CD19-directed CAR T cells in patients with chronic lymphocytic leukemia (CLL). CAR expression maintenance was also investigated because this can affect response durability. CAR T cell failure was accompanied by preexisting T cell-intrinsic defects or dysfunction acquired after infusion. In a small subset of patients, CAR silencing was observed coincident with leukemia relapse. Using a small molecule inhibitor, we demonstrated that the bromodomain and extra-terminal (BET) family of chromatin adapters plays a role in downregulating CAR expression. BET protein blockade also ameliorated CAR T cell exhaustion as manifested by inhibitory receptor reduction, enhanced metabolic fitness, increased proliferative capacity, and enriched transcriptomic signatures of T cell reinvigoration. BET inhibition decreased levels of the TET2 methylcytosine dioxygenase, and forced expression of the TET2 catalytic domain eliminated the potency-enhancing effects of BET protein targeting in CAR T cells, providing a mechanism linking BET proteins and T cell dysfunction. Thus, modulating BET epigenetic readers may improve the efficacy of cell-based immunotherapies.

Autologous CD4 T Lymphocytes Modified with a Tat-Dependent, Virus-Specific Endoribonuclease Gene in HIV-Infected Individuals.

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/mm3 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.

CRISPR-engineered T cells in patients with refractory cancer.

CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα (TRAC) and TCRß (TRBC), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1; PDCD1), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy.

Induction of resistance to chimeric antigen receptor T cell therapy by transduction of a single leukemic B cell.

We report a patient relapsing 9 months after CD19-targeted CAR T cell (CTL019) infusion with CD19- leukemia that aberrantly expressed the anti-CD19 CAR. The CAR gene was unintentionally introduced into a single leukemic B cell during T cell manufacturing, and its product bound in cis to the CD19 epitope on the surface of leukemic cells, masking it from recognition by and conferring resistance to CTL019.

Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells.

Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies1-3. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells4,5. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.

Nonviral RNA chimeric antigen receptor-modified T cells in patients with Hodgkin lymphoma.

Chimeric antigen receptor (CAR)-modified T cells are being investigated in many settings, including classical Hodgkin lymphoma (cHL). The unique biology of cHL, characterized by scant Hodgkin and Reed-Sternberg (HRS) cells within an immunosuppressive tumor microenvironment (TME), may pose challenges for cellular therapies directly targeting antigens expressed on HRS cells. We hypothesized that eradicating CD19+ B cells within the TME and the putative circulating CD19+ HRS clonotypic cells using anti-CD19-directed CAR-modified T cells (CART19) may indirectly affect HRS cells, which do not express CD19. Here we describe our pilot trial using CART19 in patients with relapsed or refractory cHL. To limit potential toxicities, we used nonviral RNA CART19 cells, which are expected to express CAR protein for only a few days, as opposed to CART19 generated by viral vector transduction, which expand in vivo and retain CAR expression. All 5 enrolled patients underwent successful manufacturing of nonviral RNA CART19, and 4 were infused with protocol-specified cell dose. There were no severe toxicities. Responses were seen, but these were transient. To our knowledge, this is the first CART19 clinical trial to use nonviral RNA gene delivery. This trial was registered at www.clinicaltrials.gov as #NCT02277522 (adult) and #NCT02624258 (pediatric).

Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia.

Tolerance to self-antigens prevents the elimination of cancer by the immune system1,2. We used synthetic chimeric antigen receptors (CARs) to overcome immunological tolerance and mediate tumor rejection in patients with chronic lymphocytic leukemia (CLL). Remission was induced in a subset of subjects, but most did not respond. Comprehensive assessment of patient-derived CAR T cells to identify mechanisms of therapeutic success and failure has not been explored. We performed genomic, phenotypic and functional evaluations to identify determinants of response. Transcriptomic profiling revealed that CAR T cells from complete-responding patients with CLL were enriched in memory-related genes, including IL-6/STAT3 signatures, whereas T cells from nonresponders upregulated programs involved in effector differentiation, glycolysis, exhaustion and apoptosis. Sustained remission was associated with an elevated frequency of CD27+CD45RO-CD8+ T cells before CAR T cell generation, and these lymphocytes possessed memory-like characteristics. Highly functional CAR T cells from patients produced STAT3-related cytokines, and serum IL-6 correlated with CAR T cell expansion. IL-6/STAT3 blockade diminished CAR T cell proliferation. Furthermore, a mechanistically relevant population of CD27+PD-1-CD8+ CAR T cells expressing high levels of the IL-6 receptor predicts therapeutic response and is responsible for tumor control. These findings uncover new features of CAR T cell biology and underscore the potential of using pretreatment biomarkers of response to advance immunotherapies.

Anti-CD19 CAR T cells with high-dose melphalan and autologous stem cell transplantation for refractory multiple myeloma.

BACKGROUND: Multiple myeloma is usually fatal due to serial relapses that become progressively refractory to therapy. CD19 is typically absent on the dominant multiple myeloma cell population but may be present on minor subsets with unique myeloma-propagating properties. To target myeloma-propagating cells, we clinically evaluated autologous T cells transduced with a chimeric antigen receptor (CAR) against CD19 (CTL019). METHODS: Subjects received CTL019 following salvage high-dose melphalan and autologous stem cell transplantation (ASCT). All subjects had relapsed/refractory multiple myeloma and had previously undergone ASCT with less than 1 year progression-free survival (PFS). RESULTS: ASCT + CTL019 was safe and feasible, with most toxicity attributable to ASCT and no severe cytokine release syndrome. Two of 10 subjects exhibited significantly longer PFS after ASCT + CTL019 compared with prior ASCT (479 vs. 181 days; 249 vs. 127 days). Correlates of favorable clinical outcome included peak CTL019 frequency in bone marrow and emergence of humoral and cellular immune responses against the stem-cell antigen Sox2. Ex vivo treatment of primary myeloma samples with a combination of CTL019 and CAR T cells against the plasma cell antigen BCMA reliably inhibited myeloma colony formation in vitro, whereas treatment with either CAR alone inhibited colony formation inconsistently. CONCLUSION: CTL019 may improve duration of response to standard multiple myeloma therapies by targeting and precipitating secondary immune responses against myeloma-propagating cells. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT02135406. FUNDING: Novartis, NIH, Conquer Cancer Foundation.

Activity of Mesothelin-Specific Chimeric Antigen Receptor T Cells Against Pancreatic Carcinoma Metastases in a Phase 1 Trial.

Pancreatic ductal adenocarcinoma (PDAC) is resistant to T-cell-mediated immunotherapy. We engineered T cells to transiently express a messenger RNA encoding a chimeric antigen receptor (CAR) specific for mesothelin, a protein that is overexpressed by PDAC cells. We performed a phase I 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 18F-2-fluoro-2-deoxy-D-glucose (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 with 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 antitumor activity of messenger RNA CARTmeso cells, as well as PDAC resistance to the immune response.