Search

Purpose: Previously, we showed that adoptive transfer of in vivo vaccine-primed and ex vivo (anti-CD3/anti-CD28) costimulated autologous T cells (ex-T) at day +12 after transplant increased CD4 and CD8 T-cell counts at day +42 and augmented vaccine-specific immune responses in patients with myeloma. Here, we investigated the safety and kinetics of T-cell recovery after infusing ex-T at day +2 after transplant.Experimental Design: In this phase I/II two-arm clinical trial, 50 patients with myeloma received autografts after high-dose melphalan followed by infusions of ex-T at day +2 after transplant. Patients also received pretransplant and posttransplant immunizations using a pneumococcal conjugate vaccine only (arm B; n = 24) or the pneumococcal conjugate vaccine plus an HLA-A2–restricted multipeptide vaccine for HLA-A2+ patients (arm A; n = 26).Results: The mean number of T cells infused was 4.26 × 1010 (range, 1.59-5.0). At day 14 after transplant, the median CD3, CD4, and CD8 counts were 4,198, 1,545, and 2,858 cells/μL, respectively. Interleukin (IL)-6 and IL-15 levels increased early after transplant and IL-15 levels correlated significantly to day 14 T-cell counts. Robust vaccine-specific B- and T-cell responses were generated. T-cell infusions were well tolerated with no effect on hematopoietic recovery. Eight patients (16%) developed a T-cell “engraftment syndrome” characterized by diarrhea and fever that was clinically and histopathologically indistinguishable from grade 1 to 3 acute graft-versus-host disease (GVHD) of the gastrointestinal tract (seven patients) and/or grade 1 to 2 cutaneous GVHD (four patients).Conclusions: Adoptive T-cell transfers achieve robust T-cell recovery early after transplant and induce moderate-to-severe autologous GVHD in a subset of patients.

Supplementary Data from Rapid Immune Recovery and Graft-versus-Host Disease–like Engraftment Syndrome following Adoptive Transfer of Costimulated Autologous T Cells

Supplementary Table 3 from Telomerase-Specific T-Cell Immunity in Breast Cancer: Effect of Vaccination on Tumor Immunosurveillance

Supplementary Table 2 from Telomerase-Specific T-Cell Immunity in Breast Cancer: Effect of Vaccination on Tumor Immunosurveillance

The human telomerase reverse transcriptase (hTERT) is nearly universally overexpressed in human cancer, contributes critically to oncogenesis, and is recognized by cytotoxic T cells that lyse tumors. CD8+ T cells specific for hTERT naturally occur in certain populations of cancer patients in remission, but it remains poorly understood whether such T cells could contribute to tumor immunosurveillance. To address this issue, we induced hTERT-specific T cells in vivo via peptide vaccination in 19 patients with metastatic breast cancer who otherwise had no measurable T-cell responses to hTERT at baseline. Tumor-infiltrating lymphocytes (TIL) were evident after, but not before vaccination, with 4% to 13% of postvaccine CD8+ TIL specific for the immunizing hTERT peptide. Induction of TIL manifested clinically with tumor site pain and pruritus and pathologically with alterations in the tumor microenvironment, featuring histiocytic accumulation and widespread tumor necrosis. hTERT-specific CD8+ T cells were also evident after vaccination in the peripheral blood of patients and exhibited effector functions in vitro including proliferation, IFN-γ production, and tumor lysis. An exploratory landmark analysis revealed that median overall survival was significantly longer in those patients who achieved an immune response to hTERT peptide compared with patients who did not. Immune response to a control cytomegalovirus peptide in the vaccine did not correlate with survival. These results suggest that hTERT-specific T cells could contribute to the immunosurveillance of breast cancer and suggest novel opportunities for both therapeutic and prophylactic vaccine strategies for cancer. [Cancer Res 2007;67(21):1–9]

Supplementary Table 1 from Telomerase-Specific T-Cell Immunity in Breast Cancer: Effect of Vaccination on Tumor Immunosurveillance

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

PURPOSE To describe long-term outcomes of anti-CD19 chimeric antigen receptor T (CART) cells in patients with relapsed or refractory chronic lymphocytic leukemia (CLL). METHODS Between January 2013 and June 2016, 42 patients with relapsed or refractory CLL were enrolled in this study and 38 were infused with anti-CD19 CART cells (CART-19). Of these, 28 patients were initially randomly assigned to receive a low (5 × 107) or high (5 × 108) dose of CART-19, and 24 were evaluable for response assessment. After an interim analysis, 10 additional patients received the selected (high) dose and of these, eight were evaluable for response. Patients were followed for a median 31.5 months (range, 2 to 75 months). RESULTS At 4 weeks, the complete and overall responses for the 32 evaluable patients were 28% (90% CI, 16% to 44%) and 44% (90% CI, 29% to 60%), respectively. The median overall survival (OS) for all patients was 64 months; there was no statistically significant difference between low- and high-dose groups ( P = .84). Regardless of dose, prolonged survival was observed in patients who achieved a CR versus those who did not ( P = .035), with median OS not reached in patients with CR versus 64 months in those without CR. The median progression-free survival was 40.2 months in patients with CR and 1 month in those without a CR ( P < .0001). Toxicity was comparable in both dose groups. CONCLUSION In patients with advanced CLL, a 5 × 108 dose of CART-19 may be more effective than 5 × 107 CART-19 at inducing CR without excessive toxicity. Attainment of a CR after CART-19 infusion, regardless of cell dose, is associated with longer OS and progression-free survival in patients with relapsed CLL.

Coronavirus Disease 2019 (COVID-19) remains a global health emergency with limited treatment options, lagging vaccine rates and inadequate healthcare resources in the face of an ongoing calamity. The disease is characterized by immune dysregulation and cytokine storm. Cyclosporine A (CSA) is a calcineurin inhibitor that modulates cytokine production and may have direct antiviral properties against coronaviruses. To test whether a short course of treatment was safe in COVID-19 patients, we treated 10 hospitalized, oxygen requiring, non-critically ill patients with CSA at a starting dose of 9mg/kg/day. Five patients experienced adverse events, none were serious, and transaminitis was most common. No subject enrolled in this trial required intensive care unit (ICU)-level care and all patients were discharged alive from the hospital. Further, CSA treatment was associated with significant reductions in serum cytokines and chemokines important in COVID-19 hyper-inflammation, including CXCL10. In conclusion, short courses of CSA appear safe and feasible in COVID-19 patients requiring oxygen and therefore, may be a useful adjunct in resource-poor or resource-limited health care settings.

Donor lymphocyte infusions (DLIs) induce potent graft versus tumor (GVT) effects for relapsed chronic myelogenous leukemia (CML) after allogeneic stem cell transplantation (SCT) but are disappointing for other diseases. Disease resistance can occur if donor T cells are not appropriately activated in vivo. Ex vivo T-cell activation might overcome disease-induced anergy and augment GVT activity. We performed a phase 1 trial of ex vivo–activated DLI (aDLI) for 18 patients with relapse after SCT. Activated donor T cells are produced through costimulation with anti-CD3– and anti-CD28–coated beads. Patients with aggressive malignancies received induction chemotherapy, and all patients received conventional DLI (median, 1.5 × 108 mononuclear cells/kg) followed 12 days later by aDLI. Activated DLI was dose escalated from 1 × 106 to 1 × 108 CD3+ cells per kilogram in 5 levels. Seven patients developed acute graft versus host disease (GVHD) (5 grade I-II, 2 grade III), and 4 developed chronic GVHD. Eight patients achieved complete remission, including 4 of 7 with acute lymphocytic leukemia (ALL), 2 of 4 with acute myelogenous leukemia (AML), 1 with chronic lymphocytic leukemia (CLL), and 1 of 2 with non-Hodgkin lymphoma (NHL). Four complete responders relapsed while 4 remain alive in remission a median 23 months after aDLI. Overall, 10 of 18 remain alive 11 to 53 months after aDLI. Adoptive transfer of costimulated activated allogeneic T cells is feasible, does not result in excessive GVHD, and may contribute to durable remissions in diseases where conventional DLI has been disappointing.

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.

In a phase 1/2 two-arm trial, 54 patients with myeloma received autografts followed by ex vivo anti-CD3/anti-CD28 costimulated autologous T cells at day 2 after transplantation. Study patients positive for human leukocyte antigen A2 (arm A, n = 28) also received pneumococcal conjugate vaccine immunizations before and after transplantation and a multipeptide tumor antigen vaccine derived from the human telomerase reverse transcriptase and the antiapoptotic protein survivin. Patients negative for human leukocyte antigen A2 (arm B, n = 26) received the pneumococcal conjugate vaccine only. Patients exhibited robust T-cell recoveries by day 14 with supraphysiologic T-cell counts accompanied by a sustained reduction in regulatory T cells. The median event-free survival (EFS) for all patients is 20 months (95% confidence interval, 14.6-24.7 months); the projected 3-year overall survival is 83%. A subset of patients in arm A (36%) developed immune responses to the tumor antigen vaccine by tetramer assays, but this cohort did not exhibit better EFS. Higher posttransplantation CD4+ T-cell counts and a lower percentage of FOXP3+ T cells were associated with improved EFS. Patients exhibited accelerated polyclonal immunoglobulin recovery compared with patients without T-cell transfers. Adoptive transfer of tumor antigen vaccine-primed and costimulated T cells leads to augmented and accelerated cellular and humoral immune reconstitution, including antitumor immunity, after autologous stem cell transplantation for myeloma. This study was registered at www.clinicaltrials.gov as NCT00499577.

Severe immune deficiency follows autologous stem cell transplantation for multiple myeloma and is associated with significant infectious morbidity. This study was designed to evaluate the utility of a pretransplantation vaccine and infusion of a primed autologous T-cell product in stimulating specific immunity to influenza. Twenty-one patients with multiple myeloma were enrolled from 2007 to 2009. Patients were randomly assigned to receive an influenza-primed autologous T-cell product or a nonspecifically primed autologous T-cell product. The study endpoint was the development of hemagglutination inhibition titers to the strain-specific serotypes in the influenza vaccine. Enzyme-linked immunospot assays were performed to confirm the development of influenza-specific B-cell and T-cell immunity. Patients who received the influenza-primed autologous T-cell product were significantly more likely to seroconvert in response to the influenza vaccine (P = .001). Seroconversion was accompanied by a significant B-cell response. No differences were observed in the global quantitative recovery of T-cell and B-cell subsets or in global T-cell and B-cell function. The provision of a primed autologous T-cell product significantly improved subsequent influenza vaccine responses. This trial was registered at www.clinicaltrials.gov as #NCT00499577.

Purpose: Previously, we showed that adoptive transfer of in vivo vaccine-primed and ex vivo (anti-CD3/anti-CD28) costimulated autologous T cells (ex-T) at day +12 after transplant increased CD4 and CD8 T-cell counts at day +42 and augmented vaccine-specific immune responses in patients with myeloma. Here, we investigated the safety and kinetics of T-cell recovery after infusing ex-T at day +2 after transplant.Experimental Design: In this phase I/II two-arm clinical trial, 50 patients with myeloma received autografts after high-dose melphalan followed by infusions of ex-T at day +2 after transplant. Patients also received pretransplant and posttransplant immunizations using a pneumococcal conjugate vaccine only (arm B; n = 24) or the pneumococcal conjugate vaccine plus an HLA-A2–restricted multipeptide vaccine for HLA-A2+ patients (arm A; n = 26).Results: The mean number of T cells infused was 4.26 × 1010 (range, 1.59-5.0). At day 14 after transplant, the median CD3, CD4, and CD8 counts were 4,198, 1,545, and 2,858 cells/μL, respectively. Interleukin (IL)-6 and IL-15 levels increased early after transplant and IL-15 levels correlated significantly to day 14 T-cell counts. Robust vaccine-specific B- and T-cell responses were generated. T-cell infusions were well tolerated with no effect on hematopoietic recovery. Eight patients (16%) developed a T-cell “engraftment syndrome” characterized by diarrhea and fever that was clinically and histopathologically indistinguishable from grade 1 to 3 acute graft-versus-host disease (GVHD) of the gastrointestinal tract (seven patients) and/or grade 1 to 2 cutaneous GVHD (four patients).Conclusions: Adoptive T-cell transfers achieve robust T-cell recovery early after transplant and induce moderate-to-severe autologous GVHD in a subset of patients.

The human telomerase reverse transcriptase (hTERT) is nearly universally overexpressed in human cancer, contributes critically to oncogenesis, and is recognized by cytotoxic T cells that lyse tumors. CD8+ T cells specific for hTERT naturally occur in certain populations of cancer patients in remission, but it remains poorly understood whether such T cells could contribute to tumor immunosurveillance. To address this issue, we induced hTERT-specific T cells in vivo via peptide vaccination in 19 patients with metastatic breast cancer who otherwise had no measurable T-cell responses to hTERT at baseline. Tumor-infiltrating lymphocytes (TIL) were evident after, but not before vaccination, with 4% to 13% of postvaccine CD8+ TIL specific for the immunizing hTERT peptide. Induction of TIL manifested clinically with tumor site pain and pruritus and pathologically with alterations in the tumor microenvironment, featuring histiocytic accumulation and widespread tumor necrosis. hTERT-specific CD8+ T cells were also evident after vaccination in the peripheral blood of patients and exhibited effector functions in vitro including proliferation, IFN-γ production, and tumor lysis. An exploratory landmark analysis revealed that median overall survival was significantly longer in those patients who achieved an immune response to hTERT peptide compared with patients who did not. Immune response to a control cytomegalovirus peptide in the vaccine did not correlate with survival. These results suggest that hTERT-specific T cells could contribute to the immunosurveillance of breast cancer and suggest novel opportunities for both therapeutic and prophylactic vaccine strategies for cancer. [Cancer Res 2007;67(21):1–9]

Background Fludarabine-based therapies prolong survival of patients (pts) with chronic lymphocytic leukemia (CLL), but deplete T-cells causing immunosuppression which increases the risk of infection and may limit control of minimal residual disease by the immune system. Adoptive immunotherapy using autologous CD3/CD28-costimulated T-cells expanded ex vivo (ACTC) enhances immune reconstitution after fludarabine-based therapy of lymphoma (Schuster Blood abstract 2007). Methods We conducted a multicenter phase I/II trial of ACTC following fludarabine- or alemtuzumab-based therapy of CLL. Eligibility required IWCLL 2008 indications for treatment of CLL. After leukapheresis, pts received fludarabine- or alemtuzumab-based therapy. Eight - 12 weeks after last immuno-chemotherapy, responding pts (CR, PR) received a single dose of ACTC prepared from autologous T-cells collected before therapy. Primary study endpoints were feasibility, defined as the ability to generate a T-cell dose of 1.0 E+10+/- 20%, and safety, defined as Results Thirty-four pts with CLL were enrolled (median age: 62). Eighteen patients were previously untreated and 16 patients had relapsed or refractory CLL. Four pts (12%) had del(17p), 11 pts (33%) del(11q), and 3 (9%) both del(17p) and del(11q). For previously untreated patients, 17 received fludarabine-cyclophosphamide-rituximab (FCR) and 1 pt received fludarabine-rituximab (FR); for relapsed patients, 10 received FCR, 3 FCR-bevacizumab, 2 alemtuzumab, and 1 oxaliplatin-fludarabine-cytarabine-rituximab (OFAR). Ten patients did not receive ACTC infusion for the following reasons: 6 pts (including 1 receiving alemtuzumab) had progressive disease prior to ACTC, 1 pt developed autoimmune hemolyic anemia during FCR, 1 pt developed ITP during FCR, 1 pt developed pure red cell aplasia during FCR, and 1 pt receiving alemtuzumab was removed from study per physician preference. Median ACTC dose was 1.0 E+10 CD3 cells (range: 3.94 E+08 -1.03 E+10). Two pts had progressive disease less than 90 days after ACTC and 1 pt was lost to follow-up. Twenty-one pts (14 CR; 2 CRi; and 5 PR at the time of ACTC infusion) were evaluable for studies of immune reconstitution at ≥90 days after ACTC. After chemotherapy prior to ACTC (D0), median CD4 count was 120 (range: 19-573); 90 days after ACTC (D+90), median CD4 count was 399 (range 62-1818) (p = 0.0003; median fold CD4 increase 2.3, range -0.5–20.9) [figure 1]. Median CD8 count also increased significantly (p = 0.004; median D0 = 86, range: 4-682; D+90 = 267, range: 18-2876; median fold CD8 increase 1.1, range: -0.8-18.4) [figure 2]. The increases in CD4 and CD8 counts between D0 and D+90 were not dependent on ACTC dose. There were no SAEs or grade 3 or higher non-hematologic toxicities related to T-cell infusion. For all 21 patients evaluable for immune reconstitution at D+90, median follow-up is 33 months with median PFS 30 months (not shown). PFS for previously untreated patients is 42 months, whereas PFS for relapsed patients is 12 months (figure 3). There was no correlation between magnitude of increase in post-infusion CD4 and CD8 counts and PFS at 1 year. Conclusions For CLL patients, ACTC production is feasible. ACTC infusion is well tolerated and results in a dose-independent acceleration of CD4+ and CD8+ cell recovery after fludarabine-based chemotherapy compared to historical controls. The application of adoptive immunotherapy with ACTC to accelerate immune reconstitution after immunochemotherapy could potentially provide an optimal immunologic milieu for early application of additional immunotherapeutic interventions. Disclosures: Levine: Novartis: cell and gene therapy IP, cell and gene therapy IP Patents & Royalties. June:Novartis: Patents & Royalties, Research Funding.

Advanced atherosclerotic lesions are characterized by lipid accumulation, chronic inflammation, and defective efferocytosis; an ideal therapy should address all aspects of this multi-factorial disease. Ixmyelocel-T, an expanded autologous multicellular therapy showing clinical promise in the treatment of severe diseases associated with atherosclerosis, includes a novel population of M2-like macrophages. We examined the properties of these macrophages to determine their potential mechanisms of action in atherosclerotic states. Flow cytometry revealed that ixmyelocel-T macrophages express surface receptors of alternative activation (CD206 and CD163) and scavenger receptors involved in lipid and apoptotic cell uptake (CD36, LDLR, CD91, MSR1, and MerTK). ELISA analysis revealed ixmyelocel-T macrophages secrete substantial levels of IL-10 (1060 278 vs. 1978 313 pg/ml, p < 0.05) and IL-1ra (12673 1242 vs. 37576 3650 pg/ml, p < 0.001) before and after overnight LPS stimulation. Baseline secretion of the pro-inflammatory cytokines IL-1b, TNFa, and IL-12 is minimal and remains low after LPS stimulation. Fluorescent microscopy and flow cytometry revealed that ixmyelocel-T readily phagocytosed apoptotic cells. Apoptosis analyzed by a caspase 3/7 assay demonstrated that ixmyelocel-T isn’t sensitive to the lipotoxic effects of modified cholesterol (87 10.78 vs. 100 14.58% of control). Real time PCR revealed that the cholesterol transporter genes ABAC1 (1.49 0.32 vs. 0.42 0.11 fold change, p < 0.01) and ABCG1 (0.41 0.13 vs. 0.07 0.03 fold change, p < 0.05) are both significantly upregulated when ixmyelocel-T macrophages are loaded with cholesterol. Ixmyelocel-T also exhibits enhanced ApoAI mediated cholesterol efflux (13.91 1.90 vs. 3.42 0.31%, p < 0.001 compared to THP-1). In addition, ixmyelocel-T effluxed cholesterol in vivo in using a mouse model for reverse cholesterol transport. These biological properties of ixmyelocel-T macrophages suggest that this cellular therapy may exert beneficial effects on atherosclerotic disease by providing macrophages that potentially limit atheroma development through secretion of anti-inflammatory cytokines, removal of apoptotic cells, and promotion of cholesterol efflux.

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 1631 Introduction: Fludarabine-based chemotherapy combinations are highly effective in patients with low-grade follicular lymphoma but cause severe and long-lasting immunosuppression related to depletion of normal T cells, especially CD4 T cells. Aside from increasing the risk of serious infections, this toxicity may limit the ability of the immune system to eliminate minimal residual disease. To determine whether adoptive transfer of autologous CD25-depleted, CD3/CD28-costimulated T cells expanded ex vivo (ACTC) improves immune reconstitution following cyclophosphamide - fludarabine chemotherapy, we conducted a phase I study in patients with relapsed/refractory follicular lymphoma. Patients and Methods: Eligible patients had previously treated, purine analog naïve, relapsed or refractory follicular lymphoma (grade 1 or 2). After leukapheresis, patients received 4 cycles of fludarabine (25 mg/m2) and cyclophosphamide (250 mg/m2) on days 1 – 3 of a 28 – 42 day cycle. Four weeks after last chemotherapy, responding patients [complete response (CR), complete response unconfirmed (CRu), and partial response (PR)] received escalating doses of ACTC prepared from autologous T cells collected prior to chemotherapy. Results: Fifteen patients were enrolled. Median age was 49 years (range: 32 – 68), median number of prior therapies was 2 (range: 1 – 3), and 14 patients (93%) were rituximab-refractory. Five patients were withdrawn from the study, 2 due to chemotherapy-related hematologic toxicity and 3 for progressive disease during chemotherapy. For 10 evaluable patients completing chemotherapy and ACTC infusion, 8 patients were in CR and 2 patients in PR at the time of ACTC infusion; 5 patients received 1 – 5 × 109 ACTC and 5 patients received 5 – 10 × 109 ACTC. There were no adverse events related to T cell infusions. Following chemotherapy and before ACTC infusion, median CD4 and CD8 counts were 91/μL (range: 15 –169) and 64/μL (range: 19 – 273), respectively; four weeks after ACTC, median CD4 and CD8 counts were 502/μL (range: 156 – 1053) and 509/μL (range: 158 – 1860). Median %CD4+FOXP3+ blood cells before chemotherapy was 15.8% (n = 8; range: 5.07 – 37.1); after chemotherapy / before ACTC, 21.4% (n = 7; range: 6.15 – 56.3); on day 60 after ACTC, 3.3% (n = 8; range: 1.5 – 20.4) (p = 0.01 for both pre ACTC and post ACTC comparisons). Before receiving chemotherapy and ACTC, all 10 patients were anergic to candida antigen by DTH skin testing; 60 days after ACTC, 5 patients developed a positive DTH response to candida antigen (p = 0.03). From the day of ACTC infusion, median progression-free survival (PFS) is 14.1 months. Most significantly, no patient relapsed or had progression of lymphoma after 14.1 months with follow-up from 16.5 – 101.5 months. In addition, PFS is significantly longer than time to progression from prior therapy (p = 0.01; see figure below). Overall survival after ACTC was 60% with median follow-up of 68 months. Conclusions: Adoptive transfer of ACTC after fludarabine - cyclophosphamide chemotherapy enhances recovery of CD4 and CD8 lymphocyte counts compared to historical observations, results in a relative reduction in peripheral blood FOXP3+ regulatory T cells, enhances recovery of lymphocyte function, and can result in prolonged remission. Our findings suggest that reversal of immunosuppression by adoptive transfer of autologous co-stimulated T-cells may significantly improve therapeutic outcome after cytotoxic therapy for patients with follicular lymphoma. Disclosures: Levine: US Patent: 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. June:Life Technologies / Invitrogen: cell culture technology patents owned by U.S. Government and licensed to Life Technology / Invitrogen, cell culture technology patents owned by U.S. Government and licensed to Life Technology / Invitrogen Patents & Royalties.

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.

Abstract 352 Background: Autologous stem cell transplantation (ASCT) for MM leads to complete responses in ∼20–40% of patients but rare cures. Patients with rapid recovery of T cells post ASCT may have improved outcomes suggesting possible immune mediated tumor control. We have shown that adoptive T-cell transfers after ASCT for MM using vaccine-primed and ex-vivo costimulated autologous T-cells in combination with pre- and post-transplant immunizations using a tumor antigen vaccine (+ GM-CSF and montanide) led to vaccine-directed T-cell responses in about 1/3 of patients and enhanced recovery of polyclonal T and B cell counts and function. We hypothesized that addition of Poly-ICLC (Hiltonol®) – a TLR-3 agonistic vaccine adjuvant could increase tumor antigen vaccine responses through better priming and boosting. Methods: We report interim results of a Phase II clinical trial (NCT01245673) evaluating safety and activity of autologous T cells primed in-vivo with a MAGE-A3 multipeptide vaccine (Orphan Drug Designation: GL-0817) mixed with GM-CSF and Poly-ICLC (Hiltonol®) +/− montanide. Inclusion criteria included measurable disease or high-risk cytogenetics. MAGE-A3 is expressed in ∼50% MM cases and more frequently in relapsed/extramedullary/proliferative disease. The MAGE-A3 vaccine has 2 HLA-A2-restricted class I peptides and 1 promiscuous class II peptide linked to an HIV-1-TAT membrane translocation sequence (Trojan peptide) to enhance peptide presentation. Vaccine-primed T cells were collected by leukapheresis, costimulated and expanded ex-vivo using anti-CD3/anti-CD28 mAb conjugated beads. T-cells were infused at day +2 after ASCT followed by booster immunizations at days 14, 42, 90, 120 and 150 post-transplant. Lenalidomide maintenance was started at day +100. Patients were evaluated for MM responses in accordance with IMWG criteria at days 60, 100, 180 post-transplant and Q3 months thereafter. T-cell and B-cell responses to the vaccine were evaluated by IFN-g or IL-2 cytokine production (all patients), dextramer binding to CD8 cells (HLA-A2 positive patients) and ELISA antibody assays at days 14, 60, 100 and 180 post-transplant. Results: 25 patients were transplanted on study. At a median followup of 6 months (range 2–18 months), 24 patients are surviving while 1 patient relapsed at about day +60 and died. Four additional patients have relapsed at 7,9,18 and 18 months post-transplant, yielding a 1-year Kaplan-Meier EFS of 77%. Of the 16 patients evaluable for response at day 100, 7/16 (44%) had CR/nCR using the study enrollment (post-induction) myeloma markers as a baseline while at day 180, 7/13 (53%) had CR/nCR. T-cell infusions were well-tolerated with no probable/definite grade 3 or higher toxicities. Vaccinations were associated with > 50 mm injection site reactions (redness, induration or both) after 1 or more immunizations in the majority of patients. Two patients developed large and prolonged inflammatory reactions which evolved into sterile abscesses. These resolved over 2–3 months with conservative management but as a result montanide was eliminated from the vaccine formulation for patients 11–25. Thereafter vaccine reactogenicity was decreased with no additional sterile abscesses. Of 16 patients tested for immune responses to date, 2 patients were unevaluable due to poor sample viability. Dextramer staining demonstrated MAGE-A3-specific CD8 T-cells in 4/4 (100%) of evaluable HLA-A2+ patients. Cytokine production in response to MAGE-A3 stimulation was seen in 11/14 (79%) patients; responses usually peaked at day 100 or day 180. Robust MAGE-A3 antibody responses were detected in 7/9 patients who received montanide in the vaccine formulation but in 0/7 patients who did not. Conclusions: Combination immunotherapy using a MAGE-A3 multipeptide tumor antigen vaccine plus vaccine-primed and costimulated autologous T-cells after ASCT for MM is well-tolerated and associated with encouraging early clinical responses. The addition of Poly-ICLC (Hiltonol®) to the vaccine formulation was associated with a high frequency of post-ASCT T-cell functional responses. The combination of Poly-ICLC +GM-CSF + Montanide led to robust injection site reactions that were occasionally severe and prolonged. Elimination of the montanide reduced injection site reactogenicity but may also have compromised the B-cell responses to the tumor antigen vaccine. Disclosures: Rapoport: Gliknik, Inc: Research Funding. Stadtmauer:Celgene: Consultancy, Speakers Bureau; Millenium: Consultancy, Speakers Bureau. Vogl:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium/Takeda: Consultancy, Research Funding; Otsuka: Consultancy; Acetylon: Research Funding. Strome:Gliknik, Inc: Equity Ownership, Patents & Royalties, Research Funding. Salazar:Oncovir, Inc: 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. June:Novartis: Research Funding, entitled to receive royalties from patents licensed to Novartis, entitled to receive royalties from patents licensed to Novartis Patents & Royalties.

TPS160 Background: IMCgp100 is a novel biologic comprising a soluble T cell receptor (TCR) fused to an antibody fragment (scFv) being developed for the treatment of metastatic melanoma. The TCR component binds to the melanoma associated antigen (Ag) gp100 which is overexpressed and presented by HLA A2 on melanoma cells. The scFv component activates T cells in physical contact with the target via CD3. Thus, IMCgp100 is expected to bind melanoma cells and stimulate the immune system to attack the target tissue via cytotoxic lymphocyte killing and via stimulation of accessory immune mechanisms.The purpose of this trial is to assess the pharmacodynamic effects of a single intratumoral dose of IMCgp100 on the tumour microenvironment and to establish what local concentration of drug is required for optimal effect. Such data will aid in future trial design and facilitate clinical development. Methods: This 2 stage exploratory study will assess the effects of 2 different doses of IMCgp100 in subjects with advance...

Background Fludarabine-based therapies prolong survival of patients (pts) with chronic lymphocytic leukemia (CLL), but deplete T-cells causing immunosuppression which increases the risk of infection and may limit control of minimal residual disease by the immune system. Adoptive immunotherapy using autologous CD3/CD28-costimulated T-cells expanded ex vivo (ACTC) enhances immune reconstitution after fludarabine-based therapy of lymphoma (Schuster Blood abstract 2007). Methods We conducted a multicenter phase I/II trial of ACTC following fludarabine- or alemtuzumab-based therapy of CLL. Eligibility required IWCLL 2008 indications for treatment of CLL. After leukapheresis, pts received fludarabine- or alemtuzumab-based therapy. Eight - 12 weeks after last immuno-chemotherapy, responding pts (CR, PR) received a single dose of ACTC prepared from autologous T-cells collected before therapy. Primary study endpoints were feasibility, defined as the ability to generate a T-cell dose of 1.0 E+10+/- 20%, and safety, defined as Results Thirty-four pts with CLL were enrolled (median age: 62). Eighteen patients were previously untreated and 16 patients had relapsed or refractory CLL. Four pts (12%) had del(17p), 11 pts (33%) del(11q), and 3 (9%) both del(17p) and del(11q). For previously untreated patients, 17 received fludarabine-cyclophosphamide-rituximab (FCR) and 1 pt received fludarabine-rituximab (FR); for relapsed patients, 10 received FCR, 3 FCR-bevacizumab, 2 alemtuzumab, and 1 oxaliplatin-fludarabine-cytarabine-rituximab (OFAR). Ten patients did not receive ACTC infusion for the following reasons: 6 pts (including 1 receiving alemtuzumab) had progressive disease prior to ACTC, 1 pt developed autoimmune hemolyic anemia during FCR, 1 pt developed ITP during FCR, 1 pt developed pure red cell aplasia during FCR, and 1 pt receiving alemtuzumab was removed from study per physician preference. Median ACTC dose was 1.0 E+10 CD3 cells (range: 3.94 E+08 -1.03 E+10). Two pts had progressive disease less than 90 days after ACTC and 1 pt was lost to follow-up. Twenty-one pts (14 CR; 2 CRi; and 5 PR at the time of ACTC infusion) were evaluable for studies of immune reconstitution at ≥90 days after ACTC. After chemotherapy prior to ACTC (D0), median CD4 count was 120 (range: 19-573); 90 days after ACTC (D+90), median CD4 count was 399 (range 62-1818) (p = 0.0003; median fold CD4 increase 2.3, range -0.5–20.9) [figure 1]. Median CD8 count also increased significantly (p = 0.004; median D0 = 86, range: 4-682; D+90 = 267, range: 18-2876; median fold CD8 increase 1.1, range: -0.8-18.4) [figure 2]. The increases in CD4 and CD8 counts between D0 and D+90 were not dependent on ACTC dose. There were no SAEs or grade 3 or higher non-hematologic toxicities related to T-cell infusion. For all 21 patients evaluable for immune reconstitution at D+90, median follow-up is 33 months with median PFS 30 months (not shown). PFS for previously untreated patients is 42 months, whereas PFS for relapsed patients is 12 months (figure 3). There was no correlation between magnitude of increase in post-infusion CD4 and CD8 counts and PFS at 1 year. Conclusions For CLL patients, ACTC production is feasible. ACTC infusion is well tolerated and results in a dose-independent acceleration of CD4+ and CD8+ cell recovery after fludarabine-based chemotherapy compared to historical controls. The application of adoptive immunotherapy with ACTC to accelerate immune reconstitution after immunochemotherapy could potentially provide an optimal immunologic milieu for early application of additional immunotherapeutic interventions. Disclosures: Levine: Novartis: cell and gene therapy IP, cell and gene therapy IP Patents & Royalties. June: Novartis: Patents & Royalties, Research Funding.

Abstract 784 BACKGROUND: Previously, we showed that adoptive transfer of in vivo vaccine-primed and ex vivo (anti-CD3/anti-CD28) costimulated autologous T cells (ex-T) at day +12 after transplant increased CD4 & CD8 levels at day +42 and augmented vaccine-specific immune responses in pts with myeloma (MM)[Nat Med 2005; 11: 1230-7]. DESIGN: We conducted a phase I/II 2-arm clinical trial in which 54 pts with advanced MM received autografts after high-dose melphalan followed by infusions of ex-T at day +2 after transplant. Pts also received pretransplant and posttransplant immunizations using a pneumococcal conjugate vaccine (PCV) only (ARM B; n = 26) or the PCV plus an HLA-A2 restricted multipeptide tumor antigen vaccine (3 hTERT peptides + 1 survivin peptide + 1 CMV peptide) for HLA-A2+ patients (ARM A; n = 28). After adoptive transfer of 4.26 × 10e10 costimulated T cells (range 1.59-5.0), the median CD3, CD4 and CD8 counts were 4198, 1545 and 2858 cells per microliter at day +14 post-transplant and 16% of pts developed significant autologous GVHD [Clin Cancer Res 2009;15: 4499 – 507]. Here we first report clinical outcomes from this trial and describe several novel observations about immune reconstitution after day +2 T-cell transfers. CLINICAL RESULTS: For the 54 total transplant pts, the median age was 55 (range 37-68), 28 (52%) were male and 21 (39%) were AA while 32 (59%) were Caucasian and 1 (2%) was Asian. 66% had IgG and 28% had IgA paraproteins while 6% excreted light chains only; 41% had abnormal cytogenetics. With a median followup of 1 year (0.25 - 2.5 years) 50 pts (93%) are alive and 31 (57%) remain event-free (EFS). The projected median EFS is 15 mos with no difference between ARMS A and B. Based on the Cox regression analysis, EFS did not correlate significantly to gender, race nor absolute lymphocyte counts (ALC). However, age correlated inversely to EFS with older pts exhibiting a lower EFS (coefficient = -0.0667, p-value = 0.0398). MM responses at day +60 and day +100 did not correlate to EFS, but the response at day +180 did correlate to EFS (p IMMUNOLOGICAL RESULTS: Preliminary results indicated that 6 of 15 pts (40%) in ARM A had positive tetramer responses to hTERT and/or survivin peptides at one or more timepoints after immunization, defined as tetramer staining by flow cytometry > 0.1% (and > 3-fold increase). Based on analysis of the first 36 patients, the mean sum of the antibody responses to 4 of the 7 serotypes of the pneumococcal conjugate vaccine (PCV; serotypes 6B, 14, 19F, 23F) were 83.55 mcg/ml at day +100 and 74.13 mcg/ml at day +180, highly protective levels, compared to 5.17 mcg/ml at enrollment. We also observed several novel features of immune system recovery after early T-cell transfers. Compared to an historical cohort of 103 MM pts who were autografted without ex-T, post-transplant quantitative IgG, IgA and IgM levels at day +180 (and days +60/+100 for IgG and IgA) were significantly higher in the pts who received day +2 T-cell transfers (IgG: 1525 mg/dl vs 1137 mg/dl, p= 0.014; IgA: 264 mg/dl vs 76 mg/dl, p = 0.0029; IgM: 58 mg/dl vs 39 mg/dl, p = 0.029). Furthermore, when IgG recovery was examined for the IgA MM pts only, the IgG level at day +180 was significantly higher in the ex-T trial (mean IgG = 810 mg/dl vs 611 mg/dl, p = 0.0478). Conversely, when IgA recovery was examined for the IgG MM pts only, the IgA level was significantly higher in the ex-T trial at days +60 (mean IgA = 129.82 mg/dl vs 71.70 mg/dl, p = 0.0036), day +100 (mean IgA = 114.43 mg/dl vs 59.44 mg/dl, p = 0.0075) and day +180 (mean IgA = 98.38 mg/dl vs 48.74 mg/dl, p = 0.0037). Remarkably, a pattern of CD8 T-cell “reprogramming” was also observed after day +2 T-cell transfers such that the % of CD8+/CD45RA+ T-cells at day +60 post-transplant was 75% vs 36% in a cohort of MM transplant pts who did not receive ex-T (p< 0.0001). In addition, the log ratio of Teffector/Tregulatory cells was significantly higher among the patients who received early T-cell transfers (2.57 vs. 1.93, p = 0.0066). CONCLUSIONS: Early adoptive transfers of anti-CD3/anti-CD28 costimulated autologous T-cells significantly enhanced post-transplant humoral and cellular immune reconstitution, a pattern which may be associated with better myeloma control and protection from infection. These data may have important implications for efforts to induce clinically significant immune responses to cancer vaccines in the transplant setting. Disclosures: Vonderheide: University of Pennsylvania and Dana Farber Cancer Institute: Patents & Royalties. June:University of Pennsylvania: Patents & Royalties.

Abstract 797 Introduction: Epidemics of influenza A virus strains have been associated with a mortality rate of 0.1% and hospitalization of approximately 200,000 people per year in the United States. The recent pandemic of swine-origin H1N1 has further emphasized the need to protect the population. Patients with multiple myeloma (MM) are particularly vulnerable to flu infection from altered humoral and cellular immunity from the disease as well as from the immunosuppressive chemotherapies, corticosteroids and radiation frequently used for treatment. In particular, high dose melphalan and ASCT, a common and efficacious treatment as part of first-line or salvage therapy for MM, is associated with post-ASCT immune suppression by virtue of low T-cell numbers, poor T-cell function and immune paresis. Despite anti-infection vaccination after ASCT, pneumococcal pneumonia and influenza are common. In an attempt to improve this outcome we previously showed that adoptive transfer of in-vivo PCV vaccine-primed and ex-vivo (anti-CD3/anti-CD28) co-stimulated autologous T cells (aT-cells) early (within 14 days) post-ASCT increased CD4 and CD8 T cell counts and induced pneumococcal conjugate vaccine-directed T and B-cell responses [Rapoport et al, Nature Medicine, 2005]. Patients who did not receive pre-collection vaccination did not show this response despite infusion of co-stimulated T-cells and post-ASCT vaccination. In the current clinical trial we investigated whether pre-harvest flu vaccination would result in a similar improvement in immunity. Methods: We performed a randomized open-label single center study of two influenza vaccine schedules for patients with high-risk MM responding after conventional induction therapy. Of 21 patients enrolled, 11 were randomly assigned to Group X and received pre- and d+14 post-transplant flu vaccine with the commercially available influenza vaccine (Fluzone) produced by Aventis Pasteur. The 10 patients in Group Y received only post-transplant vaccination. All patients received T-cell harvest, high dose melphalan and ASCT and aT-cell infusion. Immunological assessment (T-cell and B-cell subsets, T-cell repertoire, T-ELISPOT, CFSE-CBC, antibody HAI, B-ELISPOT) was conducted d+60, 100 and 180. The primary study endpoint is the immune response to influenza as measured by the serotype-specific influenza antibody response on day 100 using a standard hemagglutination inhibition assay (HAI) optimized for the vaccine administered each year. Results: The median age was 55 (range 37-68), 13 M, 8 F, 18 Caucasian, 2 African American, 1 Hispanic, IgG 57%, IgA 33%, light chain 10%. With a median follow-up of 1 year, 86% (18/21) patients are alive with 48% (10/21) alive in remission. No difference in these parameters was seen between both Groups. HAI titers, however were higher at all three time points (d+60, 100, 180) for Group X when compared to Group Y both for H3N2 (p= 0.04) and for H1N1 (p=0.07). HAI titers for Group Y remained near baseline throughout all time points while Group X peaked day 60 and remained elevated day 180. Analysis of other immune assessment is ongoing and will be presented. Conclusion: Preliminary analysis of this randomized trial of flu vaccine schedule for MM patients undergoing ASCT suggests superior flu immune reconstitution when vaccine is administered to prime autologous T-cells prior ASCT and aT-cell infusion. This result adds to our observation that an approach of vaccine priming and co-stimulated autologous T-cell infusion after high dose melphalan and ASCT for MM augments an otherwise poor response in this patient population to pneumococcal and influenza vaccination. Investigation of this approach as a platform for anti-myeloma immune therapy is ongoing. Disclosures: June: University of Pennsylvania: Patents & Royalties.

Fludarabine-based chemotherapy combinations are highly effective in patients (pts) with low-grade follicular lymphoma (FL), but cause severe and long-lasting immunosuppression due to depletion of normal CD4 T-cells. Aside from increasing the risk of serious infections, this toxicity may limit the ability of the immune system to eliminate minimal residual disease. Adoptive immunotherapy using autologous CD25-depleted, CD3/CD28-costimulated T-cells expanded ex vivo (ACTC) may enhance immune reconstitution and improve disease control. We initiated a phase I study in pts with purine analog-naive relapsed/refractory FL (grades 1 and 2). After leukapheresis, pts receive 4 cycles of fludarabine (25 mg/m2) days 1 – 3 and cyclophosphamide (250 mg/m2) days 1 – 3. Four weeks after last chemotherapy, responding patients (CR, CRu, PR) receive escalating doses of ACTC prepared from autologous T-cells collected prior to chemotherapy. Thirteen pts have been enrolled to date. Median age is 49 y (range: 32 – 68). Median number of prior therapies is 2 (range: 1 – 3). Two pts were withdrawn from the study due to hematologic toxicity related to chemotherapy, one patient was withdrawn for progressive disease during chemotherapy, and one patient has not completed first response assessment after ACTC infusion. For 9 assessable pts completing chemotherapy and ACTC infusion, 7 pts achieved a CR and 2 pts achieved a PR; 5 pts received 1 – 5 x 109 and 4 patients received 5 – 10 x 109 CD3+ ACTC. There have been no adverse events related to T-cell infusions. Median follow-up after ACTC infusion is 20 months (range: 2 – 42 months). CD4 counts increased in all patients by 1 month after T-cell infusion, with a median increase of 3.8 fold (n = 8; range: 1.5 – 71). For patients at dose level 1, the median increase was 2.2 fold (n = 4; range: 1.5 – 21); at dose level 2, it was 4.2 fold (n = 4; range: 3.8 – 71). CD8 counts also increased, with a median increase of 8.1 fold (range: 1.0 – 30). All 9 pts receiving ACTC were anergic to candida antigen by delayed type hypersensitivity (DTH) skin testing before chemotherapy; 5 pts developed a positive DTH response to candida antigen 60 days after ACTC infusion. From the start of therapy for patients receiving T-cells, median follow-up is 24 months (range: 6 – 47) with median progression-free survival of 18 months, which is significantly longer than the time to progression from last therapy (median 11 months) (p = 0.01, log-rank test). Thus, ACTC results in more rapid CD4+ lymphocyte recovery in pts after cyclophosphamide-fludarabine chemotherapy compared to historical controls and results in reconstitution of recall immunity. These encouraging clinical responses provide the rationale for clinical trials to test ACTC as a way to enhance cellular immunity to tumor-specific antigens after chemotherapy.

Retrospective studies suggest that rapid lymphocyte recovery following autologous stem cell transplants (SCT) may be associated with better outcomes. Previously we showed that adoptive transfer of in-vivo vaccine-primed and ex-vivo (anti-CD3/anti-CD28) costimulated autologous T cells (ex-T) at about day 14 post-transplant increased CD4 and CD8 T cell counts at day 42 post-transplant and induced pneumococcal conjugate vaccine-directed T and B-cell responses [Rapoport et al, Nature Medicine, 2005]. In 2 current studies, we are further investigating the impact of ex-vivo costimulated autologous T cells on vaccine responses after SCT. In the first study, we are investigating whether a similar strategy of pre- and post-transplant immunizations along with an early infusion of vaccine-primed ex-T can induce responses to a putative tumor vaccine composed of 4 HLA-A2-restricted peptides derived from survivin and hTERT in pts undergoing SCT for myeloma. In the second (randomized) trial, the impact of early ex-T on immune recovery and vaccine reponses is being tested in pediatric neuroblastoma pts. Compared to the previous study, two methodologic changes were made: The target number of T cells infused was raised 5-fold to 5 x 1010 (109/kg) T cells were infused on day + 2 to take greater advantage of homeostatic expansion mechanisms. Patients were monitored for delayed hematopoietic recovery because of this switch to early ex-T and the fact that survivin and hTERT are also expressed in hematopoietic stem cells. At the time of submission, 16 adult and 30 pediatric patients have been enrolled on these trials of whom 11 and 21, respectively, are evaluable for post-transplant hematopoietic and T-cell recovery. On the myeloma trial, the mean # of T cells infused was 3.95 x 1010 with 96% viability and a CD4/CD8 ratio of 1.8:1. At day 14 post-transplant, the median CD4 count was 1951/mcl (range 651–7668) and the median CD8 count was 4117/mcl (range 1499–39,354). The median # days to achieve an absolute neutrophil count (ANC) > 500 was 12 (range 11–14) and the median # days to achieve a PLT count >20,000/mcl was 13 days (range 0–28). Similarly, in the pediatric cohort, median CD4 and CD8 counts at day 30 were 1500 and 2100/mcl, respectively, compared to 22 and 14 in a group of pts who did not receive d+2 ex-T, with no impact on engraftment. 1 adult and 3 pediatric pts also developed an “engraftment syndrome” characterized by GHVD-like features with or without fever. The adult pt with day 14 CD4 and CD8 counts of 2,724 and 11,571 cells/mcl had clinical and histologic features of (autologous) gut GVHD. 3 pediatric pts developed pruritic rashes clinically and pathologically indistiguishable from GVHD within 14 d of ex-T infusion, with fever seen in 1. In the adult and 1 pediatric pt, steroid treatment led to complete resolution of symptoms. These combined data sets demonstrate that robust CD4 and CD8 T cells counts can be achieved as early as day 14 post-SCT when adults or children receive ex-T at day +2 post-SCT without exogenous IL-2 or other cytokine support. It appears that a subset of patients develop a T cell “engraftment syndrome” similar to autologous GVHD. The mechanisms responsible for this rapid immune cell recovery are currently under investigation.

Ex-vivo co-stimulation of autologous T-cells with anti-CD3/anti-CD28-conjugated magnetic beads followed by adoptive transfer may augment T-cell responses toward tumor antigens or infectious agents. 54 patients (pts) were treated with ex-vivo co-stimulated autologous T-cells after autotransplantation for myeloma. The median age was 56 (range 38–71), 67% were male, 20% were African-American, 22% had IgA paraproteins, 11% had del 13 or complex karyotypes and the median β2m level at diagnosis was 3.31 mg/L (range 1.09–73.7). After lymphocyte collections, pts received cyclophosphamide (4.5 g/m2) + G-CSF for stem cell mobilization, and high dose melphalan (200 mg/m2 or 140 mg/m2 for pts ≥ 70) for conditioning. T-cells were cultured for ~12 days with anti-CD3/anti-CD28-immobilized immunomagnetic beads + IL-2 supplementation (100 units/ml). During a run-in phase, 12 pts received co-stimulated T-cells post-transplant ( day +12) alone. In a second phase, 42 pts who participated in a 2 x 2 randomization, received T-cells either early (day +12) or late (day + 100) after transplant. These pts also received either 2 immunizations with the pneumococcal conjugate vaccine (PCV, Prevnar) at days +30, +90 or 3 immunizations (prior to T-cell collection, at days + 30, + 90) to test immune responses to a well-defined antigen. 42 pts received PCV immunizations with no grade 3/4 adverse events. Anti-pneumococcal antibody responses developed in 51% of 31 pts tested thus far. Details of these studies will be presented separately. 52 pts received a mean dose of 8.04 x 109 costimulated T-cells (range 1.6–11). Infusion-related adverse effects included grade I–II rigors/chills (40%), grade I–II facial/upper body rashes (12%) at a median of 13 days after T-cells, grade I cardiovascular events (10%), grade I–III hypoxia (5%), grade II fever (5%), and 1 episode of DVT. At T-cell harvesting, the mean % of CD3 + cells in culture was 94.8%, the mean T-cell doubling level was 4.81 (28-fold expansion). Among the 42 randomized pts, at day + 42 post-transplant ( 30 days after T-cell infusion for the early groups), the median CD4/CD3 count was 462/mcl (range 202–1439) for the early T-cell recipients vs 230/mcl (range 50–915) for the late T-cell recipients (T-cells not yet infused) [P=0.004]. The median CD8/CD3 counts were 1399/mcl (range 465–2810) vs 1084/mcl (range 103–3422) for the early and late T-cell recipients respectively at day +42 [P=0.04]. For clinical responses there were 11 CRs, 22 VGPRs ( 90% reduction in paraprotein levels), 18 PRs (50–90% reductions), 2 pts had no response and 1 pt was unevaluable. 2 pts had delayed reductions in M-protein levels of ~ 50% between day 42 and day 180 or 270. For the entire cohort, the probability of overall survival at 1.5 years was 78% [95%CI 64%–92%]. Infusions of ex-vivo expanded autologous T-cells are well-tolerated and associated with accelerated T-cell recovery early after autotransplantation. This study may provide a platform for combining costimulated T-cells and tumor vaccines in the autograft setting.