Minnal Gupta

Identification of a Titin-Derived HLA-A1–Presented Peptide as a Cross-Reactive Target for Engineered MAGE A3–Directed T Cells

T cells engineered to express affinity-enhanced TCRs directed to a MAGE A3 peptide cross-react with a similar, but unrelated, self-peptide. Cross-Reactive Adoptive Therapy Engineering T cells with enhanced affinity to cancer targets is a promising therapy. However, one key bottleneck in this strategy is the identification of targets that are expressed on cancer cells but not on normal healthy tissue. One way to identify these antigens is by looking at the family of cancer-testis antigens, which have restricted expression in normal tissue but are frequently up-regulated in tumors. Cameron et al. now report that a T cell engineered to target one such antigen—MAGE A3—cross-reacts with a peptide from a muscle protein, Titin. The authors developed a T cell that targeted a MAGE A3 antigen for use in adoptive immunotherapy. Although extensive preclinical investigations demonstrated no off-target antigen recognition, patients who received these T cells had serious adverse events, including fatal cardiac toxicity. The authors then used amino acid scanning to search for potential cross-reactivity of these T cells with an off-target peptide and identified a peptide derived from the muscle protein Titin. Because affinity-enhanced T cells are highly potent, this cross-reactivity was likely the cause of the off-target toxicity. This study highlights methods that may be used to prevent cross-reactivity in future trials of adoptive immunotherapy. MAGE A3, which belongs to the family of cancer-testis antigens, is an attractive target for adoptive therapy given its reactivation in various tumors and limited expression in normal tissues. We developed an affinity-enhanced T cell receptor (TCR) directed to a human leukocyte antigen (HLA)–A*01–restricted MAGE A3 antigen (EVDPIGHLY) for use in adoptive therapy. Extensive preclinical investigations revealed no off-target antigen recognition concerns; nonetheless, administration to patients of T cells expressing the affinity-enhanced MAGE A3 TCR resulted in a serious adverse event (SAE) and fatal toxicity against cardiac tissue. We present a description of the preclinical in vitro functional analysis of the MAGE A3 TCR, which failed to reveal any evidence of off-target activity, and a full analysis of the post-SAE in vitro investigations, which reveal cross-recognition of an off-target peptide. Using an amino acid scanning approach, a peptide from the muscle protein Titin (ESDPIVAQY) was identified as an alternative target for the MAGE A3 TCR and the most likely cause of in vivo toxicity. These results demonstrate that affinity-enhanced TCRs have considerable effector functions in vivo and highlight the potential safety concerns for TCR-engineered T cells. Strategies such as peptide scanning and the use of more complex cell cultures are recommended in preclinical studies to mitigate the risk of off-target toxicity in future clinical investigations.

NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma

Despite recent therapeutic advances, multiple myeloma (MM) remains largely incurable. Here we report results of a phase I/II trial to evaluate the safety and activity of autologous T cells engineered to express an affinity-enhanced T cell receptor (TCR) recognizing a naturally processed peptide shared by the cancer-testis antigens NY-ESO-1 and LAGE-1. Twenty patients with antigen-positive MM received an average 2.4 × 109 engineered T cells 2 d after autologous stem cell transplant. Infusions were well tolerated without clinically apparent cytokine-release syndrome, despite high IL-6 levels. Engineered T cells expanded, persisted, trafficked to marrow and exhibited a cytotoxic phenotype. Persistence of engineered T cells in blood was inversely associated with NY-ESO-1 levels in the marrow. Disease progression was associated with loss of T cell persistence or antigen escape, in accordance with the expected mechanism of action of the transferred T cells. Encouraging clinical responses were observed in 16 of 20 patients (80%) with advanced disease, with a median progression-free survival of 19.1 months. NY-ESO-1–LAGE-1 TCR–engineered T cells were safe, trafficked to marrow and showed extended persistence that correlated with clinical activity against antigen-positive myeloma.

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.An IL-6/STAT3 signature and memory CD8 T cell subset in preinfusion chimeric antigen receptor–expressing T cells associate with response in patients with high-risk chronic lymphocytic leukemia.

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.Genetically engineered T cells that induced remission in a patient with chronic lymphocytic leukaemia were found to have disruption of the TET2 gene, which caused T cell changes that potentiated their anti-tumour effects.

Adoptive immunotherapy with engineered T cells expressing and HLA-A2 restricted affinity-enhanced TCR for LAGE-1 and NY-ESO-1 in patients with multiple myeloma following auto-SCT

Meeting abstracts We report on a 26 patient phase I/II clinical trial ([NCT01352286][1]) to investigate the safety, feasibility and anti-tumor activity of T cells engineered to express an affinity-enhanced TCR that recognizes the NY-ESO-1/LAGE-1 peptide complex HLA-A*0201-SLLMWITQC. Patients with

Abstract 4575: Correlates of clinical response following autologous stem cell transplant and adoptive immunotherapy with engineered T cells expressing an affinity-enhanced T cell receptor in patients with mutliple myeloma.

Background: Adoptive immunotherapy for cancer has been limited by lack of antigen specificity, low levels of target expression, and failure to break self-tolerance. We are conducting an early phase clinical trial (NCT01352286) attempting to overcome these barriers using T cells engineered with an HLA-A0201 restricted, affinity-enhanced TCR that recognizes an epitope expressed by the NY-ESO-1 and LAGE-1 cancer testis antigens; these cells are infused in the setting of profound lymphodepletion that accompanies high-dose chemotherapy with autologous stem cell transplant (aSCT) for patients with high risk or relapsed multiple myeloma (MM). Methods: Inclusion criteria include: 1) eligibility for aSCT, 2) PS of 0-2, 3) high risk MM or relapse after prior therapy, 4) HLA-A0201 positive, and 5) NY-ESO-1 and/or LAGE-1 positive tumor by PCR. CD25 depleted T cells are activated and expanded using anti-CD3/28 antibody conjugated microbeads, and genetically modified with a lentiviral vector. T cells are administered four days after high dose melphalan and two days following auto-SCT. Patients are evaluated for MM responses in accordance with the IMWG criteria at 6 weeks, and 3 and 6 months. At 3 months, patients with adequate marrow function start lenalidomide maintenance. Blood and marrow are monitored for persistence of engineered cells by qPCR and by surface expression of the NY-ESO-1 / LAGE-1 TCR using dextramerTM reagents. NY-ESO-1 and LAGE-1 antigen expression in marrow was assessed by qRT-PCR at baseline and post infusion. Results: As of November 2012, 21 patients have been enrolled, 15 have been infused; 4 were taken off study prior to infusion due to disease progression. An average of 2.7 x 109 engineered T cells were administered per patient (range 8.3 x 108-4.2 x 109), and the average transduction efficiency was 33% (range 30%-45%). More than 50% (8/15) of patients have high risk chromosomal abnormalities, and 3 (20%) have received prior aSCT. At 3 months post aSCT, 73% of patients were in a very good partial response (VGPR) or better. Gastrointestinal toxicity resulting from autologous GVHD (aGVHD) occurred in a subset of patients at a higher rate than reported following aSCT alone or aSCT and T cell infusion, and was resolved in all cases. Infused T cells typically showed peak expansion in blood at day 14, followed by durable persistence in blood and marrow at 6-12 months in all but one patient. Disease progression is typically accompanied by very low levels or loss of engineered T cell persistence or loss of target antigen on tumor. Conclusions: We report for the first time that possible correlates of clinical response in this study include persistence of engineered T cells and loss of antigen, suggesting specific activity of the infused cells. Infusions are well tolerated with a possible risk of manageable aGVHD. Citation Format: Aaron P. Rapoport, Edward A. Stadtmauer, Dan T. Vogl, Brendan Weiss, Gwendolyn K. Binder-Scholl, Dominic P. Smethurst, Jeffrey Finkelstein, Irina Kulikovskaya, Minnal Gupta, Erica Suppa, Tatiana Mikheeva, Joanna E. Brewer, Alan D. Bennett, Andrew B. Gerry, Nick J. Pumphrey, Helen K. Tayton-Martin, Lilliam Ribeiro, Elizabeth Veloso, Zhaohui Zheng, Ashraf Z. Bados, Saul Yanovich, Gorgun Akpek, Karen Dengel, Naseem Kerr, Sunita Philip, Kelly-Marie Betts, Sandra Westphal, Bruce L. Levine, Bent K. Jakobsen, Carl H. June, Michael Kalos. Correlates of clinical response following autologous stem cell transplant and adoptive immunotherapy with engineered T cells expressing an affinity-enhanced T cell receptor in patients with mutliple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4575. doi:10.1158/1538-7445.AM2013-4575