Helen K. Tayton-Martin

An obstacle to cancer immunotherapy has been that the affinity of T-cell receptors (TCRs) for antigens expressed in tumors is generally low. We initiated clinical testing of engineered T cells expressing an affinity-enhanced TCR against HLA-A*01-restricted MAGE-A3. Open-label protocols to test the TCRs for patients with myeloma and melanoma were initiated. The first two treated patients developed cardiogenic shock and died within a few days of T-cell infusion, events not predicted by preclinical studies of the high-affinity TCRs. Gross findings at autopsy revealed severe myocardial damage, and histopathological analysis revealed T-cell infiltration. No MAGE-A3 expression was detected in heart autopsy tissues. Robust proliferation of the engineered T cells in vivo was documented in both patients. A beating cardiomyocyte culture generated from induced pluripotent stem cells triggered T-cell killing, which was due to recognition of an unrelated peptide derived from the striated muscle-specific protein titin. These patients demonstrate that TCR-engineered T cells can have serious and not readily predictable off-target and organ-specific toxicities and highlight the need for improved methods to define the specificity of engineered TCRs.

Abstract Abstract 472 Background: Despite recent therapeutic advances, multiple myeloma (MM) remains primarily an incurable cancer. Patients experiencing rapid recovery of T cells post auto-SCT may have improved outcomes, and spontaneous cellular responses to tumor can occur, suggesting immune mediated control of tumor is possible. We and others have investigated therapeutic cancer vaccines that have shown promise in pilot studies. However, vaccine efficacy may be limited by thymic selection which restricts the repertoire of T cell receptors (TCRs) to low affinity TCRs that cannot recognize the reduced level of antigen present on most tumor cells. We hypothesized that incorporation of affinity-enhanced tumor antigen specific TCRs into autologous T cells would overcome this limitation. Methods: We report interim results of a Phase II clinical trial (NCT01352286) to evaluate the safety and activity of autologous T cells genetically engineered to express an HLA-A201 restricted, affinity-enhanced TCR targeting an epitope shared by the NY-ESO-1 and LAGE-1 tumor antigens (gene engineered cells, or GEC). Inclusion criteria include: 1) eligibility for auto-SCT, 2) PS of 0–2, 3) high risk MM or relapse after prior therapy (prior allo-SCT excluded), 4) HLA-A201 positive, and 5) NY-ESO-1 and/or LAGE-1 positive tumor. GEC are manufactured from monocyte and T regulatory cell depleted apheresis, followed by lentivector gene transfer and T cell expansion by bead-based artificial antigen presenting cells. GEC are administered four days after high dose melphalan and two days following auto-SCT, at a dose range of 1–10 billion cells. Patients are evaluated for MM responses in accordance with the IMWG criteria at 6 weeks, and 3 and 6 months post infusion. At 3 months, patients start lenalidomide maintenance. The initial 6 patient phase is complete and a 20 patient extension phase is ongoing. Results: 19 patients have been enrolled, 12 have been infused, 3 are pending infusion, and 4 were taken off study prior to infusion due to disease progression. The average TCR transfer efficiency was 31.9% (range 18.2%-45%). 6, 4, and 1 patients have reached the 6 month, 3 month, and 6 week post infusion MM assessment time points, respectively. All treated patients are alive. 3/11 (27%) and 7/11 (64%) patients have a stringent CR or best response of VGPR or better, respectively; 3/11 (27%) have stable or partial responses, and 1/11 (9%) had progressive disease. In patients with VGPR, oligoclonal banding was common, marrow showed no expansion of clonal plasma cells, and all had normalized light chain ratios, suggesting at most minimal residual disease. These results are encouraging, especially considering that 3/11 patients had prior ASCT and 8/11 had adverse cytogenetics. GEC infusions have been well tolerated. The majority of adverse events are related to the high dose melphalan. Several patients had a transient skin rash with lymphocytosis and others had a diarrheal syndrome that occurred later than expected for melphalan-induced mucositis. In all cases GI toxicity has been self-limited or treatable with a short dose of corticosteroids. Possibly related SAEs were cytopenias such as neutropenia and thrombocytopenia, and GI and metabolism disorders such as diarrhea, colitis, hyponatremia and hypomagnesemia. GEC have been detected for up to 1 year at 1% in the circulation and bone marrow. Detailed correlative analysis is reported elsewhere in this meeting (Kalos, M. et al). Summary: This is the first test of GEC in the MM setting. Infusion of GEC administered post auto-SCT is safe, well tolerated, and leads to high response rate in a high risk myeloma population. Phase II accrual continues and a study evaluating GEC outside of auto-SCT is planned. Disclosures: Stadtmauer: celgene: Consultancy; millenium: Consultancy. Binder-Scholl:Adaptimmune : Employment. Brewer:Adaptimmune Ltd: Employment. Bennett:Adaptimmune Ltd: Employment. Gerry:Adaptimmune Ltd: Employment. Pumphrey:Adaptimmune Ltd: Employment. Smethurst:Adaptimmune Ltd: Employment. Tayton-Martin:Adaptimmune Ltd: Employment. Lilliam:Adaptimmune: Employment. Levine:TxCell: Consultancy, Membership on an entity's Board of Directors or advisory committees; University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight, financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties. Jakobsen:Adaptimmune Ltc: Employment. June:Novartis: Research Funding, entitled to receive royalties from patents licensed to Novartis, entitled to receive royalties from patents licensed to Novartis Patents & Royalties.

Abstract Abstract 755 Background: Adoptive transfer of T cells genetically engineered to express affinity-enhanced T cell receptors (TcRs) specific for self-antigens is one approach to generate potent anti-tumor immunity, and this approach is being evaluated in a number of clinical trials. Although persistence of engineered T cells is positively correlated with response rates, a recurring observation to-date has been poor T cell persistence beyond the initial treatment window. We have recently initiated a Phase II clinical trial (NCT01352286) in patients with recurrent multiple myeloma (MM), to evaluate the safety and activity of autologous T cells genetically engineered to express an HLA-A201 restricted, affinity-enhanced TCR that targets an epitope shared by the NY-ESO-1 and LAGE-1 cancer testis antigens commonly expressed by MM. Clinical study details and outcomes are reported elsewhere in this meeting (Rapoport, A, Stadtmauer, E. et al.). Here we report the in vivo expansion, bioactivity, trafficking, durable persistence, and anti-tumor activity of TcR modified cells in this trial. Methods: Subjects received high-dose melphalan, infusion of autologous hematopoietic stem cells on day 0, and infusion of gene engineered cells (GEC) on day +2. Persistence of GEC was assessed by quantitative ABI-Taqman based PCR and a qualified assay that detects the lentivirus backbone sequence. Surface expression of the introduced affinity-enhanced TcR α/β receptor was detected by multiparametric flow cytometry and TcR-specific dextramer™reagents. NY-ESO-1 and LAGE-1 antigen expression in marrow samples was assessed by quantitative ABI-taqman based PCR and inventoried primer/probe sets. Results: As of August 2012, we have screened marrow from 46 patients for NY-ESO- and LAGE-1 expression. Of these, 29 (63%) and 17 (37%) expressed LAGE-1 and NY-ESO-1, respectively by PCR. LAGE-1 co-expression was observed in all cases with NY-ESO-1. To date 12 patients have been infused with GEC. Robust engraftment was observed, with peak levels of gene-marked cells in peripheral blood and marrow ranging from 1 × 102-1 × 103 cells/μL and peak levels detected within the first 21 days post infusion. By day +100 the absolute number of GEC showed patient-specific and variable contraction, with a subset of patients maintaining GEC at 1 × 102-1 × 103 cells/μL, and a second subset where GEC contracted to 1 × 101-1 × 102cells/μL. Trafficking to and persistence of GEC in marrow was observed. At both early (up to day +30) and, in a subset of patients, late timepoints (post day+42), circulating CD3+ T cells could be detected with surface expression of the affinity enhanced TcR. Engineered cells are detected in all patients at their latest timepoint by Q-PCR. To date we see no evidence for TCR mispairing with endogenous TCR. Tumor-antigen and tumor-specific targeting by GEC was evaluated using Q-PCR and quantifying transcript levels of NY-ESO-1, LAGE-1, and CD138 (as a plasma cell marker) in marrow biopsies at enrollment, and days +42, +100, and +180. In the initial cohort of 5 patients, selective reappearance of CD138 transcript is observed in 4 cases (2 sCR, 1 VGPR, 1 PR), with non-detectable or very weak expression of LAGE-1/NY-ESO-1 transcripts, suggesting specific targeting of tumor cells that express LAGE-1/NY-ESO-1. In the fifth patient, who has progressive disease and low engineered cell persistence, CD138 and LAGE-1/NY-ESO-1 transcripts are both present above pre-treatment levels. A second infusion was recently given to this patient. Summary: Our studies show for the first time that adoptive transfer of affinity-enhanced TCR engineered T cells in MM patients results in prolonged T cell persistence, homing to marrow, and anti-tumor activity. Disclosures: Binder-Scholl: Adaptimmune: Employment. Smethurst:Adaptimmune Ltd: Employment. Brewer:Adaptimmune Ltd: Employment. Bennett:Adaptimmune Ltd: Employment. Gerry:Adaptimmune Ltd: Employment. Pumphrey:Adaptimmune Ltd: Employment. Tayton-Martin:Adaptimmune Ltd: Employment. Levine:TxCell: Consultancy, Membership on an entity's Board of Directors or advisory committees; University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight, financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties. Jakobsen:Adaptimmune Ltc: Employment. June:Novartis: Research Funding, entitled to receive royalties from patents licensed to Novartis, entitled to receive royalties from patents licensed to Novartis Patents & Royalties.

Abstract Background Despite recent therapeutic advances, multiple myeloma (MM) remains primarily an incurable cancer. Patients experiencing rapid recovery of T cells post autologous stem cell transplant (auto-SCT) may have improved outcomes, and spontaneous cellular responses to tumor can occur, suggesting immune mediated control of tumor is possible. We and others have investigated therapeutic cancer vaccines that have shown promise in pilot studies, in particular following post-transplant infusion of activated autologous T cells. However, efficacy of these approaches may be limited by thymic selection which restricts the repertoire of T cell receptors (TCRs) to low affinity TCRs that cannot recognize the low level of antigen present on most tumor cells. We hypothesized that incorporation of affinity-enhanced tumor antigen-specific TCRs into autologous T cells infused post-transplant would overcome this limitation and improve response rates in the post auto-SCT setting. Methods We report interim results of a Phase II clinical trial (NCT01352286) to evaluate the safety and activity of autologous T cells genetically engineered to express an affinity-enhanced TCR that recognizes the NY-ESO-1/LAGE-1 peptide complex HLA‐A*0201‐SLLMWITQC; these cells are infused in the setting of profound lymphodepletion that accompanies high dose chemotherapy administered during auto-SCT. Patients with high risk or relapsed MM, who are HLA‐A*0201 positive, and whose tumor is positive for NY-ESO-1 and/or LAGE-1 by RT-PCR are eligible. CD25 depleted CD4 and CD8 T cells are activated and expanded using anti-CD3/CD28 antibody conjugated microbeads, and genetically modified with a lentiviral vector containing the TCR construct at a multiplicity of infection of 1. Engineered T cells are administered four days after high dose melphalan and two days following auto-SCT, at a dose range of 1-10 billion total cells with a minimum gene modification requirement of 10%. Patients are evaluated for MM responses in accordance with the IMWG criteria at 6 weeks, and 3 and 6 months post infusion. At 3 months, patients start lenalidomide maintenance. The initial 6 patient phase is complete and a 20 patient extension phase is ongoing. Results Prior to enrollment on study, patients had received a median of 3 prior therapies including 6 with prior transplant. 50% of tumors contained high risk chromosomal abnormalities, and NY-ESO-1 expression is correlated with adverse prognosis. 20 patients (average age of 57) have been infused with an average of 2.3 X 109 engineered T cells (range 4.5 X 108-3.9 X 109); this reflects an average clinical scale transduction efficiency of 34% (range 18% – 49%). Infusions have been well tolerated, and the majority of adverse events were related to the high dose melphalan. Possibly related SAEs were neutropenia and thrombocytopenia, and GI and metabolism disorders including diarrhea, colitis, hyponatremia and hypomagnesemia. 10, 4, 2, and 2 patients have reached the 1 year, 9 month, 6 month and 3 month assessment timepoints, respectively, and 17/20 patients are alive. Best response by day 100 is sCR/CR in 2/15 (13%), nCR in 10/15 (67%), and PR in 3/15% (20%), which compares favorably to historic responses in patients undergoing first or second transplant. Engineered T cells expanded and persisted in blood and marrow at 180 days by Q-PCR and flow-cytometry in all but one case (Figure). 7 patients progressed after day 100, which was accompanied either by loss of engineered T cells or loss of tumor antigen. Detailed phenotyping and functional analysis of engineered T cells, and correlates with clinical responses, is underway. Summary This is the first clinical evaluation of engineered T cells in the MM setting. Infusions are safe, well tolerated, and are associated with encouraging responses in a high risk myeloma population. A study evaluating the engineered T cells in a non-transplant study is underway. Disclosures: Stadtmauer: Celgene: Consultancy. Binder-Scholl:Adaptimmune: Employment. Smethurst:Adaptimmune: Employment. Brewer:Adaptimmune: Employment. Bennett:Adaptimmune: Employment. Gerry:Adaptimmune: Employment. Pumphrey:Adaptimmune: Employment. Tayton-Martin:Adaptimmune: Employment. Ribeiro:Adaptimmune: Employment. Levine:Novartis: cell and gene therapy IP, cell and gene therapy IP Patents & Royalties. Jakobsen:Adaptimmune: Employment. Kalos:Novartis corporation: CART19 technology, CART19 technology Patents & Royalties; Adaptive biotechnologies: Member scientific advisory board , Member scientific advisory board Other. June:Novartis: Patents & Royalties, Research Funding.