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Exposure to replication-competent lentivirus (RCL) is a theoretical safety concern for individuals treated with lentiviral gene therapy. For certain ex vivo gene therapy applications, including cancer immunotherapy trials, RCL detection assays are used to screen the vector product as well as the vector-transduced cells. In this study, we reviewed T cell products screened for RCL using methodology developed in the National Gene Vector Biorepository. All trials utilized third-generation lentiviral vectors produced by transient transfection. Samples from 26 clinical trials totaling 460 transduced cell products from 375 subjects were evaluated. All cell products were negative for RCL. A total of 296 of the clinical trial participants were screened for RCL at least 1 month after infusion of the cell product. No research subject has shown evidence of RCL infection. These findings provide further evidence attesting to the safety of third-generation lentiviral vectors and that testing T cell products for RCL does not provide added value to screening the lentiviral vector product.

Since HIV requires CD4 and a co-receptor, most commonly C-C chemokine receptor 5 (CCR5), for cellular entry, targeting CCR5 expression is an attractive approach for therapy of HIV infection. Treatment of CD4(+) T cells with zinc-finger protein nucleases (ZFNs) specifically disrupting chemokine receptor CCR5 coding sequences induces resistance to HIV infection in vitro and in vivo. A chimeric Ad5/F35 adenoviral vector encoding CCR5-ZFNs permitted efficient delivery and transient expression following anti-CD3/anti-CD28 costimulation of T lymphocytes. We present data showing CD3/CD28 costimulation substantially improved transduction efficiency over reported methods for Ad5/F35 transduction of T lymphocytes. Modifications to the laboratory scale process, incorporating clinically compatible reagents and methods, resulted in a robust ex vivo manufacturing process capable of generating10(10) CCR5 gene-edited CD4+ T cells from healthy and HIV+ donors. CD4+ T-cell phenotype, cytokine production, and repertoire were comparable between ZFN-modified and control cells. Following consultation with regulatory authorities, we conducted in vivo toxicity studies that showed no detectable ZFN-specific toxicity or T-cell transformation. Based on these findings, we initiated a clinical trial testing the safety and feasibility of CCR5 gene-edited CD4+ T-cell transfer in study subjects with HIV-1 infection.

Melanoma-associated antigens-A (MAGE-A) are members of an extensive family of cancer/testis (CT) antigens. Although emerging studies highlight the potential role of MAGE-A family members in promoting cancer growth, the biological function of MAGE-A4 remains poorly understood. MAGE-A4 is an attractive target because it has a high frequency of expression in multiple solid tumors according to The Cancer Genome Atlas (https://cancergenome.nih.gov/). In an attempt to further characterize its expression in solid tumors, immunohistochemistry analysis was performed in 534 resected non-small cell lung carcinoma (NSCLC) cases, stage I to IV with clinicopathological information including overall survival and recurrence. MAGE-A4 expression was observed in 24% of all NSCLC cases, with higher frequency observed in squamous cell carcinoma (SCC) (51%) versus adenocarcinoma (8%). MAGE-A4 expression was observed in 23% of the primary tumors and 48% of the recurrent tumor samples. Log-rank (Mantel-Cox) analysis showed that histology diagnosis, pathological stage, and MAGE-A4 expression are independent prognostic factors in NSCLC. However, no MAGE-A4 prognostic value was observed when analyzing the SCC subtype. An HLA-A*02-restricted MAGE-A4 TCR specific for MAGE-A4230-239 peptide GVYDGREHTV was identified from a pool of parental TCRs and engineered to optimize specificity and activity (MAGE-A4c1032). TCR fine specificity was assessed initially by mapping the response of the MAGE-A4c1032T-cells to panels of synthetic peptides with combinatorial substitutions at each amino acid with every other possible amino acid (X-Scan), to identify potentially cross-reactive peptides in the human and common pathogen proteomes. Secondly, MAGE-A4c1032T-cells were screened against a wide panel of normal primary cells from multiple organ systems, induced pluripotent stem cell-derived cells (iCells), and autologous whole blood to test for off-target reactivity, and against a panel of EBV-derived B-lymphoblastic cell lines expressing a wide range of HLA molecules to assess the risk of alloreactivity. Alloreactivity was observed in antigen negative cells expressing HLA-A*02:05, and therefore this allele is exclusionary. MAGE-A4c1032T-cell potency was assessed by a variety of in vitro assays, including proliferation, IFN-γ release and cytotoxicity in response to antigen-positive tumor lines in 2D and 3D culture, and cytokine release in response to freshly prepared antigen-positive primary tumor material. In conclusion, MAGE-A4 is a promising target for SPEAR T-cell therapy, and clinical trial opened this year to treat patients with inoperable or metastatic (advanced) NSCLC (SCC, adenosquamous, or large cell carcinoma), ovarian, head and neck SCC, gastric, esophageal (SCC or adenocarcinoma), urothelial tumors and melanoma (NCT03132922). Citation Format: Joana Senra, Pamela Villalobos, Barbara Mino, Luisa Solis, Carmen Behrens, Joseph P. Sanderson, Manoj Saini, Andrew B. Gerry, Nicholas J. Pumphrey, Miguel Maroto, Boris Sepesi, Patrick Hwu, David S. Hong, Elizabeht A. Mittendorf, George S. Blumenschein, Ignacio I. Wistuba, Gwendolyn Binder-Scholl, Rafael Amado. Affinity-enhanced T-cell receptor (TCR) for adoptive T-cell therapy targeting MAGE-A4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2562.

Background: NY-ESO-1 and LAGE-1a are cancer-testis antigens that are overexpressed in patients with multiple myeloma (MM), and the incidence of these antigens correlates with tumor proliferation and other high-risk features. Genetically engineered NY-ESO-1 SPEAR (specific peptide enhanced affinity receptor) T-cells (NY-ESO-1c259T cells) recognize the peptide sequence SLLMWITQC expressed by NY-ESO-1 or LAGE-1a in the context of HLA-A*02 presentation. This study evaluated treatment with NY-ESO-1SPEAR T-cells post-autologous stem cell transplant (ASCT) in patients with advanced MM. Methods: Eligible patients were HLA-A*02:01, 02:05 or 02:06 positive, with refractory, relapsed or high risk MM associated with one or more adverse cytogenetic abnormalities. Eligible patients' tumors also expressed NY-ESO-1 and/or LAGE-1a by qPCR. The primary study endpoint was safety. Secondary objectives included overall response rate (ORR) (sCR+CR+VGPR+PR) evaluated with the International Myeloma Working Group Criteria (Rajkumar S.V. et al., Blood 2011), progression-free survival (PFS), overall survival (OS), best overall response (BOR), duration of response (DOR), as well as gene-marked cell persistence. Lymphocytes were obtained by leukapheresis, isolated, activated, transduced to express NY-ESO-1c259T cell receptor, and expanded using anti-CD3/anti-CD28 immunomagnetic beads. While the SPEAR T-cells were being manufactured, stem cell mobilization was conducted using 1.5 g/m2 of cyclophosphamide plus G-CSF, and stem cells were collected (minimum: 2 × 106 CD34+ progenitors/kg). Once the manufactured product was ready, high-dose melphalan (140-200 mg/m2) was given 2 days before stem cell infusion. Two days after the stem cell infusion, the SPEAR T-cells were infused (median dose 3.1 × 109 of transduced T-cells, range 0.5-5.1 × 109). Disease was assessed at days 42, 100, 180, 270 and 360 post-T-cell infusion, and then every 3 months. Patients meeting the criteria for lenalidomide maintenance therapy received 10 mg/day starting around day 100 post-ASCT. Results: Twenty-five patients were enrolled, and all have been treated. Median age at enrollment was 61 yr (range 45 - 72); 60% were male. Based on analyses through July 2017, ORR at day 100 was 76% (1 sCR; 12 VGPR; 6PR), and at year 1, 13 patients were progression free (52%) of which 11 were responders (1 sCR; 1 CR; 8 VGPR; 1 PR). Three patients remain disease progression-free at 39, 56 and 61 months post T-cell infusion. Median PFS was ~13 months (range 3-61 months). Eleven of 25 patients (44%) are alive, and median survival was ~35 months (range 6-68 months). The most common adverse events (experienced by >70%) were diarrhea (100%), nausea (100%), anemia (96%), decreased appetite (92%), thrombocytopenia (92%), fatigue (88%), pyrexia (84%), rash (84%), hypokalemia (76%), febrile neutropenia (72%) and vomiting (72%). Autologous GVHD (24%) was reported in 6 patients (3 G3, 3 ≤G2); all resolved with corticosteroids and supportive therapy. No fatal adverse events have been reported. Conclusions: NY-ESO-1 SPEAR T-cell therapy in the setting of autologous stem cell transplant has promising efficacy and acceptable safety. GVHD, which manifests in a similar way as reported in prior transplant studies and is more frequent with adoptive T cell transfer (engineered or not engineered), appears manageable with appropriate supportive care. Analyses of transduced cell persistence, T-cell clonality, and minimal residual disease (MRD) genetic studies are ongoing and will be presented along with the efficacy and safety data for all 25 patients. Disclosures Chagin: Adaptimmune: Employment. Faitg: Adaptimmune: Employment. Iyengar: Adaptimmune: Employment. Trivedi: Adaptimmune: Employment. Norry: Adaptimmune: Employment. Holdich: Adaptimmune: Employment. Binder-Scholl: Adaptimmune: Employment. Amado: Adaptimmune: Employment. Fang: Adaptimmune: Employment.

Despite recent therapeutic advances, multiple myeloma (MM) remains largely incurable. Herein 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 two days after autologous stem cell transplant (ASCT). 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, consistent 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.

CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene ("gene editing")--in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN)--is safe.We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance.One serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (-1.81 cells per day) was significantly less than the decline in unmodified cells (-7.25 cells per day) (P=0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients.CCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00842634.).

It is now well established that human tumors express unique antigens, however immunosuppressive mechanisms prevent natural immune responses. We hypothesized that infusion of T cells that have been genetically modified to express affinity-optimized tumor antigen-specific TCRs may overcome these barriers. We initiated several ongoing clinical studies to evaluate T cells engineered with an affinity-enhanced TCR specific for the NY-ESO-1 and LAGE-1 cancer testis antigens (NY-ESOc259-T), in synovial sarcoma (SS) and multiple myeloma (MM) patients with antigen-positive tumors.

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.

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.

We report the safety and tolerability of 87 infusions of lentiviral vector–modified autologous CD4 T cells (VRX496-T; trade name, Lexgenleucel-T) in 17 HIV patients with well-controlled viremia. Antiviral effects were studied during analytic treatment interruption in a subset of 13 patients. VRX496-T was associated with a decrease in viral load set points in 6 of 8 subjects (P = .08). In addition, A → G transitions were enriched in HIV sequences after infusion, which is consistent with a model in which transduced CD4 T cells exert antisense-mediated genetic pressure on HIV during infection. Engraftment of vector-modified CD4 T cells was measured in gut-associated lymphoid tissue and was correlated with engraftment in blood. The engraftment half-life in the blood was approximately 5 weeks, with stable persistence in some patients for up to 5 years. Conditional replication of VRX496 was detected periodically through 1 year after infusion. No evidence of clonal selection of lentiviral vector–transduced T cells or integration enrichment near oncogenes was detected. This is the first demonstration that gene-modified cells can exert genetic pressure on HIV. We conclude that gene-modified T cells have the potential to decrease the fitness of HIV-1 and conditionally replicative lentiviral vectors have a promising safety profile in T cells.

The DNA methyl transferase inhibitors 5-aza-2’-deoxycytidine (decitabine or DECI) and 5-azacytidine (azacitidine or AZA) have recently been approved for the treatment of a variety of hematologic malignancies. These cytidine analogues remove repressive epigenetic DNA methyl marks and induce the transcription of numerous genes normally silenced during the processes of cellular differentiation or transformation. DNMTis have a net anti-neoplastic activity through the up-regulation of tumor-suppressor genes involved in the induction of apoptosis, cellular differentiation and cell cycle arrest. In addition, DNMTis promote the expression of several cancer testis antigens (CTAs) by tumor cells, including NY-ESO and MAGE-A1/A3. These observations suggest that the combination of DNMTis with immunotherapeutic modalities may provide some clinical benefit. Adoptive immunotherapies using autologous CD4+ and CD8+ T cells expressing an affinity optimized NY-ESO TCR are in clinical trials and show robust clinical responses in patients across several indications including synovial sarcoma, multiple myeloma or melanoma. DNMTis were previously shown to induce the expression of NY-ESO in antigen-low or -negative tumor cells and consequently enable the activation of NY-ESO-specific T-cells. While there is compelling evidence for the combination of DNMTis with T cells containing engineered affinity-enhanced TCRs recognizing NY-ESO-1, safety data supporting this approach are lacking. Initial tests into the safety of combining these treatments investigated the effects of DECI and AZA on NY-ESO and LAGE-1 expression in a panel of primary adult human cells of different tissue origins. Our data demonstrate that treatment with either compound resulted in a significant up-regulation of NY-ESO transcription in a subset of primary cells tested. The magnitude of increase in transcript expression varied between cell types and, in some cases, was sufficient to activate T cells expressing an affinity-optimized NY-ESO-specific TCR. These findings raise important safety concerns with regards to the potential combination of DNMTi compounds with NY-ESO T-cell therapy and suggest that such co-therapies may result in on-target off-tumor toxicities. Citation Format: Bruno Laugel, Alexandra Sevko, Karen Howe, Zoltan Ferjentsik, Tiago Ferronha, Miguel Maroto, Joanna Brewer, Bent Jakobsen, Gwendolyn Binder-Scholl. DNA methyl transferase inhibitors (DNMTis) upregulate NY-ESO expression in several human primary cells: implications for co-therapy with engineered adoptive T-cell therapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2281.

3040Background: Cytokine release syndrome (CRS) is observed with adoptive T-cell therapies (ACT) with varying frequency. CRS can be life threatening and may require steroids, which hinder the effectiveness of ACT. CRS prevalence and severity within the context NY-ESO-1C259T across 5 studies in 4 tumor types were examined: synovial sarcoma (SS), ovarian cancer (OC), myeloma and melanoma. Methods: A review of CRS including evaluation of concurrent AEs and reported symptoms, cytokine levels and CRP was performed in pts treated with NY-ESO-1C259T. AEs that are known manifestations of CRS were summarized for each pt. diagnosed with CRS. CRP was measured by the hospital laboratory. Serum cytokines were measured by Luminex. Results: Of 53 pts treated with NY-ESO-1C259T, 7 were diagnosed with CRS (Table). 2/7 pts received immunomodulatory treatment for mitigation of CRS: methylprednisolone 2 mg/kg x 2 doses with prednisone taper (n=1, G3) and Tocilizumab 4 mg/kg x 1 dose (n=1 G4). Diagnosis of CRS was made within...

TPS3101Background: The immunogenic cancer testis antigen NY-ESO-1 is expressed in several cancers. While NY-ESO-1 is not expressed in vital tissues, it is expressed in approximately 60% in advance ...

It is well established that human tumors express unique antigens; however, immunosuppressive mechanisms prevent natural immune responses. We hypothesized that infusion of T cells that have been genetically modified to express affinity-optimized tumor antigen-specific TCRs may overcome these barriers. We initiated several ongoing clinical studies to evaluate T cells engineered with an affinity-enhanced TCR specific for the NY-ESO-1 and LAGE-1 cancer testis antigens (NY-ESOc259-T), in synovial sarcoma (SS) and multiple myeloma (MM) patients with antigen-positive tumors. Here we report updated phenotyping results from two MM and SS cohorts of patients. Manufactured product (MP) and baseline samples were analyzed by flow cytometry evaluating memory and activation markers (e.g. CD45RA, CCR7, ICOS, OX40, etc) and polyfunctionality markers (e.g. IFN-γ and IL-2). NY-ESOc259-T was detected by pentamer staining and its phenotype was correlated with clinical response. Upon culture NY-ESOc259-T generated different memory phenotypes. The phenotype at baseline (day -50) did not predict the phenotype acquired at the end of culture. Interestingly, in MM a positive trend with clinical response was observed in MPs bearing a higher percentage of Central Memory (CM) cells secreting IL-2 and IFN-γ, while a negative trend was found in Effector Memory (EM) and Effectors (EMRA)-dominated MPs secreting high levels of TNF-α. This trend was confirmed by analysing the absolute number of CM cells infused in a small number of patients, hinting at the existence of an “effective CM cells dose” of around 3×108 CM cells necessary to achieve clinical response. Of note, this trend was observed also in the SS cohort. Upon stimulation T cells proliferate and differentiate in vitro, but at the same time cells need time in culture to reach an effective dose. To understand these dynamics we retrospectively analysed the evolution of CM cells in relation to the length of culture. We observed a negative correlation between percentage of CM cells and culture time, while EM and EMRA cells increased with the duration of culture. This suggests the importance of reconciling time of culture with phenotype and number of cells in order to achieve that “sweet spot” critical for clinical response. Activation/costimulation markers in the SS cohort were also analysed in relation to culture conditions. MPs consistently displayed up-regulation of ICOS, CD40L and OX40, suggesting that an activated phenotype is achieved even after several days of culture. Of note, markers of immunological memory like CD27 and CD28 were also maintained up to the end of culture highlighting the fitness of the product despite the duration of the culture. These data suggest that NY-ESOc259-T may acquire a CM phenotype that positively correlates with clinical response in the cancer indications observed. Additionally, we show how the manufacturing process successfully produces a population of activated cells that express important activation markers without losing markers of fitness and memory - properties that are key to driving the anti-tumour response observed in cancer patients.

The success of adoptive T cell gene transfer for treatment of cancer and HIV is predicated on generating a response that is both durable and safe. We report long-term results from three clinical trials to evaluate gammaretroviral vector–engineered T cells for HIV. The vector encoded a chimeric antigen receptor (CAR) composed of CD4 linked to the CD3ζ signaling chain (CD4ζ). CAR T cells were detected in 98% of samples tested for at least 11 years after infusion at frequencies that exceeded average T cell levels after most vaccine approaches. The CD4ζ transgene retained expression and function. There was no evidence of vector-induced immortalization of cells; integration site distributions showed no evidence of persistent clonal expansion or enrichment for integration sites near genes implicated in growth control or transformation. The CD4ζ T cells had stable levels of engraftment, with decay half-lives that exceeded 16 years, in marked contrast to previous trials testing engineered T cells. These findings indicate that host immunosuppression before T cell transfer is not required to achieve long-term persistence of gene-modified T cells. Further, our results emphasize the safety of T cells modified by retroviral gene transfer in clinical application, as measured in >500 patient-years of follow-up. Thus, previous safety issues with integrating viral vectors are hematopoietic stem cell or transgene intrinsic, and not a general feature of retroviral vectors. Engineered T cells are a promising form of synthetic biology for long-term delivery of protein-based therapeutics. These results provide a framework to guide the therapy of a wide spectrum of human diseases.

Adoptive T-cell transfer is recognized as an innovative treatment strategy for various malignant diseases.1,2 To improve the efficacy and sometimes the safety of this approach, T cells can be genetically manipulated to modify their antigen specificity, to enhance their in vivo survival and trafficking to specific tissues, or to be eliminated in the event of undesired toxic effects.3 γ-retroviral vectors are frequently used to obtain robust and stable genetic modification of human T lymphocytes because these vectors can efficiently integrate within the genome and ensure that the inserted transgene is passed to the progeny of infected cells.

Meeting abstracts Preclinical safety testing for adoptive immunotherapies utilising T cell receptor-(TCR) engineered T cells relies on stringent target validation and a robust pre-clinical specificity testing strategy. Specific safety concerns include on-target off-tumour toxicity and off-target

The cancer-testis antigen NY-ESO-1 is expressed by many solid tumors and has limited expression by mature somatic tissues, making it a highly attractive target for tumor immunotherapy. Targeting NY-ESO-1 using engineered T cells has demonstrated clinical efficacy in the treatment of some adult tumors. Neuroblastoma is a significant cause of cancer mortality in children, and is a tumor type shown to be responsive to immunotherapies. We evaluated a large panel of primarily resected neuroblastoma samples and demonstrated that 23% express NY-ESO-1. After confirming antigen-specific activity of T cells genetically engineered to express an NY-ESO-1 directed high-affinity transgenic T cell receptor in vitro, we performed xenograft mouse studies assessing the efficacy of NY-ESO-1-targeted T cells in both localized and disseminated models of neuroblastoma. Disease responses were monitored by tumor volume measurement and in vivo bioluminescence. After delivery of NY-ESO-1 transgenic TCR T cells, we observed significant delay of tumor progression in mice bearing localized and disseminated neuroblastoma, as well as enhanced animal survival. These data demonstrate that NY-ESO-1 is an antigen target in neuroblastoma and that targeted T cells represent a potential therapeutic option for patients with neuroblastoma.

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Adoptive immunotherapy for cancer has been limited by a lack of antigen specificity, low levels of target expression, and failure to break self-tolerance. We hypothesized that infusion of genetically modified tumor-specific T cells following autologous stem cell transplant (ASCT) may overcome these barriers for multiple myeloma (MM). To test this, we conducted a phase I/II clinical trial ([NCT01352286][1]) in which T cells engineered with an HLA-A*0201 restricted, affinity-enhanced TCR recognizing NY-ESO-1 / LAGE-1 peptides (NY-ESOc259-T), were infused in the setting of profound lymphodepletion that accompanies high-dose chemotherapy given with ASCT. HLA-A*0201 MM patients eligible for ASCT, with antigen positive tumor were enrolled. NY-ESOc259-T was manufactured in a 10 day process using anti-CD3/CD28 microbeads and lentiviral vector, and was administered two days following ASCT. IMWG criteria were used to assess response at day 100 with the addition of a near complete response category (nCR) due to the common occurrence of oligoclonal banding observed following rapid post-ASCT immune reconstitution. Blood and marrow samples were taken at multiple timepoints for serum cytokine analysis, NY-ESOc259-T persistence and trafficking, multiparameter flow analysis to examine the phenotype and function of NY-ESOc259-T, and tumor biomarker analysis. 25 of 29 enrolled patients were infused. A mean of 2.8 × 109 engineered cells were administered (range 8.3 × 108-4.2 × 109), and the average transduction efficiency was 33% (range 30%-45%). Patients tended to have advanced disease (64% chromosomal abnormalities, and 24% prior ASCT). At 3 months, 67% (16/24) and 58% (14/24) of patients were in VGPR and nCR or better, respectively. Infusions were well-tolerated and no cytokine release syndrome was reported. NY-ESOc259-T persisted at 6 months in all but one patient, and in a subset of patients at 2 years; marrow infiltration was consistently observed from day 7 through day 180. NY-ESOc259-T initially displayed a dominant activated effector phenotype which converted towards a dominant effector memory phenotype by 1 year post infusion, in a pattern that mirrored clinical responses. Persisting cells demonstrated a polyfunctional response (IFN-γ and TNF-α) with a cytotoxic (CD107a and granzyme B) signature without overexpression of exhaustion markers (PD-1, LAG-3, and TIM-3). Tumor biomarker analysis is ongoing. MM relapse occurred in 13/25 patients. This data show that NY-ESOc259-T cells exhibit robust trafficking and expansion, durable persistence without exhaustion, and follow a natural immune expansion and contraction pattern consistent with an antigen-driven mechanism of action. Relapse correlated with a loss of persistence or tumor antigen escape, suggesting that targeting multiple antigens and maintenance infusions may increase durable remissions. Citation Format: Aaron Rapoport, Edward Stadtmauer, Luca Melchiori, Ryan Wong, Eduardo Davila, Gwendolyn Binder-Scholl, Tom Holdich, Dan Vogl, Brendan Weiss, Jeffrey Finkelstein, Simon Lacey, Sarah Bond, Marylene Fortin, Yoav Peretz, Joanna Brewer, Alan Bennett, Andrew Gerry, Nick Pumphrey, Helen Tayton-Martin, Lilliam Ribeiro, Ashraf Badros, Saul Yanovich, Nancy Hardy, Jean Yared, Naseem Kerr, Sunita Philip, Sandra Wesphal, Bruce L. Levine, Carl June, Michael Kalos, Bent Jakobsen. NY-ESO T cells administered post ASCT for MM exhibit extended functionality without exhaustion in a natural pattern of effector and memory programming. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4701. doi:10.1158/1538-7445.AM2015-4701 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01352286&atom=%2Fcanres%2F75%2F15_Supplement%2F4701.atom

TPS3102 Background: NY-ESO-1 (CTAG-1B) is a cancer testis antigen associated with spontaneous and vaccine-induced immunity that can lead to improved clinical outcomes. NY-ESO-1 is not expressed in vital tissues, and is expressed in approximately 40% of ovarian, 60% of advanced myeloma, and 70% of synovial sarcoma tumors. Expression is correlated with tumor proliferation and high risk features. A human-derived affinity-optimized TCR that recognizes the NY-ESO-1 derived SLLMWITQC peptide in complex with HLA‐A*0201 (NY-ESOc259) has been generated, and clinically tested as previously described using adoptive T cell transfer with supportive IL-21 Methods: Multiple clinical studies were initiated in HLA-A201+ patients with NY-ESO-1 expressing tumors. NY-ESO-1c259-engineered autologous T-cells are manufactured centrally, using a 12 day commercial-grade closed system, and are administered without supportive IL-2. 40 patients have been infused: 25/26 on a phase I/II study in adults with systemic or multifocal myel...

Meeting abstracts Engineered T cell therapy (ECT) for oncology has met significant clinical proof of success for chemotherapy resistant B cell malignancies in multiple studies. These data have underscored the transformative potential of ECT in advanced oncology. Adaptimmune specializes in the

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.

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

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 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.

Meeting abstracts Relapsed or metastatic synovial sarcoma remains a significant unmet medical need. NY-ESO-1 is an attractive target for sarcoma, since it is expressed in approximately 70% of synovial sarcomas but not on vital tissues. We generated NY-ESOc259, a human-derived affinity-enhanced T

Meeting abstracts It is now well established that human tumors express unique antigens, however tumor immune evasion mechanisms often prevent effective naturally occurring anti-tumor immune responses. We hypothesized that infusion of T cells that have been genetically modified to express affinity

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