Effective anti-tumor immunity in humans has been associated with the presence of T cells directed at cancer neoantigens1, which are T cell epitopes with tumor-specific expression arising from non-silent somatic mutations. They are highly immunogenic because they are not expressed in normal tissues and hence bypass central thymic tolerance. Although neoantigens were long-envisioned as optimal targets for an anti-tumor immune response2, their systematic discovery and evaluation only became feasible with the recent availability of massively-parallel sequencing for detection of all coding mutations within tumors, and of machine learning approaches to reliably predict those mutated peptides with high-affinity binding of autologous HLA molecules. We hypothesized that vaccination with neoantigens can both expand pre-existing neoantigen-specific T cell populations and induce a broader repertoire of new T cell specificities in cancer patients, tipping the intra-tumoral balance in favor of enhanced tumor control. Here we demonstrate the feasibility, safety and immunogenicity of a vaccine that targets up to 20 predicted personal tumor neoantigens. Vaccine-induced polyfunctional CD4+ and CD8+ T cells targeted 58 (60%) and 15 (16%), respectively, of the 97 unique neoantigens used across patients. These T cells discriminated mutated from wildtype antigens, and in some cases, directly recognized autologous tumor. Of 6 vaccinated patients, 4 had no recurrence at 25 months post-vaccination, while 2 with progressive disease were subsequently treated with anti-PD-1 therapy and experienced complete tumor regression, with expansion of the repertoire of neoantigen-specific T cells. These data provide a strong rationale for further development of this approach, alone and in combination with checkpoint therapies.

Neoantigens, which are derived from tumour-specific protein-coding mutations, are exempt from central tolerance, can generate robust immune responses1,2 and can function as bona fide antigens that facilitate tumour rejection3. Here we demonstrate that a strategy that uses multi-epitope, personalized neoantigen vaccination, which has previously been tested in patients with high-risk melanoma4–6, is feasible for tumours such as glioblastoma, which typically have a relatively low mutation load1,7 and an immunologically ‘cold’ tumour microenvironment8. We used personalized neoantigen-targeting vaccines to immunize patients newly diagnosed with glioblastoma following surgical resection and conventional radiotherapy in a phase I/Ib study. Patients who did not receive dexamethasone—a highly potent corticosteroid that is frequently prescribed to treat cerebral oedema in patients with glioblastoma—generated circulating polyfunctional neoantigen-specific CD4+ and CD8+ T cell responses that were enriched in a memory phenotype and showed an increase in the number of tumour-infiltrating T cells. Using single-cell T cell receptor analysis, we provide evidence that neoantigen-specific T cells from the peripheral blood can migrate into an intracranial glioblastoma tumour. Neoantigen-targeting vaccines thus have the potential to favourably alter the immune milieu of glioblastoma.

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NKG2D ligands are widely expressed in solid and hematologic malignancies but absent or poorly expressed on healthy tissues. We conducted a phase I dose-escalation study to evaluate the safety and feasibility of a single infusion of NKG2D-chimeric antigen receptor (CAR) T cells, without lymphodepleting conditioning in subjects with acute myeloid leukemia/myelodysplastic syndrome or relapsed/refractory multiple myeloma. Autologous T cells were transfected with a g-retroviral vector encoding a CAR fusing human NKG2D with the CD3z signaling domain. Four dose levels (1 Â 10 6-3 Â 10 7 total viable T cells) were evaluated. Twelve subjects were infused [7 acute myeloid leukemia (AML) and 5 multiple myeloma]. NKG2D-CAR products demonstrated a median 75% vector-driven NKG2D expression on CD3 þ T cells. No dose-limiting toxicities, cytokine release syndrome, or CAR T cell-related neurotox-icity was observed. No significant autoimmune reactions were noted, and none of the ! grade 3 adverse events were attributable to NKG2D-CAR T cells. At the single injection of low cell doses used in this trial, no objective tumor responses were observed. However, hematologic parameters transiently improved in one subject with AML at the highest dose, and cases of disease stability without further therapy or on subsequent treatments were noted. At 24 hours, the cytokine RANTES increased a median of 1.9-fold among all subjects and 5.8-fold among six AML patients. Consistent with preclinical studies, NKG2D-CAR T cell-expansion and persistence were limited. Manufactured NKG2D-CAR T cells exhibited functional activity against autologous tumor cells in vitro, but modifications to enhance CAR T-cell expansion and target density may be needed to boost clinical activity.

Purpose Invariant NKT cells (iNKT) are innate-like CD1d-restricted T cells with immunoregulatory activity in diseases including cancer. iNKT from advanced cancer patients can have reversible defects including IFN-gamma production, and iNKT IFN-gamma production may stratify for survival. Previous clinical trials using iNKT cell activating ligand alpha-galactosylceramide have shown responses. Therefore, a phase 1 clinical trial was performed of autologous in vitro expanded iNKT cells in stage IIIB-IV melanoma. Experimental Design Residual iNKT cells (<0.05% of patient PBMC) were purified from autologous leukapheresis product using an antibody against the iNKT cell receptor linked to magnetic microbeads. iNKT cells were then expanded with CD3 mAb and IL-2 in vitro to obtain up to ~109 cells. Results Expanded iNKT cells produced IFN-gamma, but limited or undetectable IL-4 or IL-10. Three iNKT infusions each were completed on 9 patients, and produced only grade 1–2 toxicities. The 4th patient onward received systemic GM-CSF with their second and third infusions. Increased numbers of iNKT cells were seen in PBMC after some infusions, particularly when GM-CSF was also given. IFN-gamma responses to alpha-galactosylceramide were increased in PBMC from some patients after infusions, and DTH responses to Candida increased in 5/8 evaluated patients. Three patients have died, three were progression-free at 53, 60 and 65 months, three received further treatment and were alive at 61, 81, and 85 months. There was no clear correlation between outcome and immune parameters. Conclusions Autologous in vitro expanded iNKT cells are a feasible and safe therapy, producing Th1-like responses with anti-tumor potential.

Despite advances in the primary treatment of non-Hodgkin's lymphoma, relapse is common and treatment after relapse is unsatisfactory. Autologous bone marrow transplantation, although sometimes successful, has generally had disappointing results. We conducted a trial of such transplantation in patients with relapsed non-Hodgkin's lymphoma, using strict criteria in selecting patients; we included only those in whom disease was minimal after conventional treatment (nodal disease less than 2 cm and bone marrow involvement less than or equal to 5 percent on histologic examination) and whose tumor cells expressed the B1 antigen. Forty-nine patients meeting these criteria received cyclophosphamide and whole-body irradiation supported by transplantation of autologous bone marrow that had been treated in vitro with anti-B1 monoclonal antibody and complement. All patients had features of a poor prognosis, including relapse from primary chemotherapy, histologic conversion to more aggressive disease, and extra-nodal dissemination. Thirty-three patients had a history of bone marrow involvement--16 at the time that marrow was obtained. Hematologic and immunologic engraftment was achieved in all patients. Only two treatment-related deaths occurred, from venoocclusive disease of the liver and intracerebral hemorrhage, respectively. Disease-free remission without maintenance therapy has lasted from greater than 2 to greater than 52 months in 34 patients (median follow-up, greater than 11 months). These results are similar to those obtained in patients with advanced, high-grade non-Hodgkin's lymphoma treated with primary combination chemotherapy. This study demonstrates that autologous bone marrow transplantation has tolerable toxicity and high efficacy in a subset of patients who are otherwise incurable but still responsive to cytoreductive therapy. The results suggest a role for such transplantation in the treatment of selected patients with newly diagnosed non-Hodgkin's lymphoma.

Novel therapies for sickle cell disease (SCD) based on genetically engineered autologous hematopoietic stem and progenitor cells (HSPCs) are critically dependent on a safe and effective strategy for cell procurement. We sought to assess the safety and efficacy of plerixafor when used in transfused patients with SCD for HSC mobilization. Six adult patients with SCD were recruited to receive a single dose of plerixafor, tested at lower than standard (180 µg/kg) and standard (240 µg/kg) doses, followed by CD34+ cell monitoring in peripheral blood and apheresis collection. The procedures were safe and well-tolerated. Mobilization was successful, with higher peripheral CD34+ cell counts in the standard vs the low-dose group. Among our 6 donors, we improved apheresis cell collection results by using a deep collection interface and starting apheresis within 4 hours after plerixafor administration. In the subjects who received a single standard dose of plerixafor and followed the optimized collection protocol, yields of up to 24.5 × 106 CD34+ cells/kg were achieved. Interestingly, the collected CD34+ cells were enriched in immunophenotypically defined long-term HSCs and early progenitors. Thus, we demonstrate that plerixafor can be employed safely in patients with SCD to obtain sufficient HSCs for potential use in gene therapy.

BACKGROUND: Early-stage and intermediate-stage nasopharyngeal cancer (NPC) generally carry a good prognosis, but for patients with recurrent, metastatic disease, options are limited. In the current study, the authors present a phase 1/2 study to evaluate the efficacy of Epstein-Barr virus (EBV)-stimulated cytotoxic T-lymphocyte (EBV-CTL) immunotherapy in this patient population. METHODS: Screening for patients with active, recurrent, metastatic EBV-associated NPC began in February 2007, and the study was closed to accrual in January 2012. After informed consent was obtained, patients had their blood drawn to initiate manufacturing of the EBV-CTL product. During product manufacturing, patients were placed on interim standard-of-care chemotherapy, and only after disease progression on the interim chemotherapy did patients receive investigational immunotherapy. Patients were restaged every 2 months until disease progression and then followed for survival. RESULTS: A total of 28 patients were enrolled, and 21 patients were treated. There was 1 complete response achieved, and at the time of last follow-up, the patient had been in remission for >8 years since treatment. The median progression-free survival was 2.2 months, and the median overall survival was 16.7 months. Two other patients, after failing EBV-CTL immunotherapy, unexpectedly demonstrated strong responses to the chemotherapy regimens they had previously failed. Patient EBV viral load and EBV-CTL specificity for tumor-associated viral antigens did not appear to correlate with clinical response. CONCLUSIONS: A durable response was observed with EBV-CTL immunotherapy, but the overall response rate for patients with recurrent, metastatic NPC was low. Further research is necessary to increase the efficacy of EBV-specific immunotherapy in patients with incurable NPC, and to characterize mechanisms for refacilitation to chemotherapy.