Victoria Shi

Allogeneic T Cells That Express an Anti-CD19 Chimeric Antigen Receptor Induce Remissions of B-Cell Malignancies That Progress After Allogeneic Hematopoietic Stem-Cell Transplantation Without Causing Graft-Versus-Host Disease

PURPOSE Progressive malignancy is the leading cause of death after allogeneic hematopoietic stem-cell transplantation (alloHSCT). After alloHSCT, B-cell malignancies often are treated with unmanipulated donor lymphocyte infusions (DLIs) from the transplant donor. DLIs frequently are not effective at eradicating malignancy and often cause graft-versus-host disease, a potentially lethal immune response against normal recipient tissues. METHODS We conducted a clinical trial of allogeneic T cells genetically engineered to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19. Patients with B-cell malignancies that had progressed after alloHSCT received a single infusion of CAR T cells. No chemotherapy or other therapies were administered. The T cells were obtained from each recipients alloHSCT donor. RESULTS Eight of 20 treated patients obtained remission, which included six complete remissions (CRs) and two partial remissions. The response rate was highest for acute lymphoblastic leukemia, with four of five patients obtaining minimal residual disease-negative CR. Responses also occurred in chronic lymphocytic leukemia and lymphoma. The longest ongoing CR was more than 30 months in a patient with chronic lymphocytic leukemia. New-onset acute graft-versus-host disease after CAR T-cell infusion developed in none of the patients. Toxicities included fever, tachycardia, and hypotension. Peak blood CAR T-cell levels were higher in patients who obtained remissions than in those who did not. Programmed cell death protein-1 expression was significantly elevated on CAR T cells after infusion. Presence of blood B cells before CAR T-cell infusion was associated with higher postinfusion CAR T-cell levels. CONCLUSION Allogeneic anti-CD19 CAR T cells can effectively treat B-cell malignancies that progress after alloHSCT. The findings point toward a future when antigen-specific T-cell therapies will play a central role in alloHSCT.

T cells expressing an anti-B-cell maturation antigen chimeric antigen receptor cause remissions of multiple myeloma

Therapies with novel mechanisms of action are needed for multiple myeloma (MM). B-cell maturation antigen (BCMA) is expressed in most cases of MM. We conducted the first-in-humans clinical trial of chimeric antigen receptor (CAR) T cells targeting BCMA. T cells expressing the CAR used in this work (CAR-BCMA) specifically recognized BCMA-expressing cells. Twelve patients received CAR-BCMA T cells in this dose-escalation trial. Among the 6 patients treated on the lowest 2 dose levels, limited antimyeloma activity and mild toxicity occurred. On the third dose level, 1 patient obtained a very good partial remission. Two patients were treated on the fourth dose level of 9 × 10(6) CAR(+) T cells/kg body weight. Before treatment, the first patient on the fourth dose level had chemotherapy-resistant MM, making up 90% of bone marrow cells. After treatment, bone marrow plasma cells became undetectable by flow cytometry, and the patients MM entered a stringent complete remission that lasted for 17 weeks before relapse. The second patient on the fourth dose level had chemotherapy-resistant MM making up 80% of bone marrow cells before treatment. Twenty-eight weeks after this patient received CAR-BCMA T cells, bone marrow plasma cells were undetectable by flow cytometry, and the serum monoclonal protein had decreased by >95%. This patient is in an ongoing very good partial remission. Both patients treated on the fourth dose level had toxicity consistent with cytokine-release syndrome including fever, hypotension, and dyspnea. Both patients had prolonged cytopenias. Our findings demonstrate antimyeloma activity of CAR-BCMA T cells. This trial was registered to www.clinicaltrials.gov as #NCT02215967.

Lymphoma Remissions Caused by Anti-CD19 Chimeric Antigen Receptor T Cells Are Associated With High Serum Interleukin-15 Levels

Purpose T cells genetically modified to express chimeric antigen receptors (CARs) targeting CD19 (CAR-19) have potent activity against acute lymphoblastic leukemia, but fewer results supporting treatment of lymphoma with CAR-19 T cells have been published. Patients with lymphoma that is chemotherapy refractory or relapsed after autologous stem-cell transplantation have a grim prognosis, and new treatments for these patients are clearly needed. Chemotherapy administered before adoptive T-cell transfer has been shown to enhance the antimalignancy activity of adoptively transferred T cells. Patients and Methods We treated 22 patients with advanced-stage lymphoma in a clinical trial of CAR-19 T cells preceded by low-dose chemotherapy. Nineteen patients had diffuse large B-cell lymphoma, two patients had follicular lymphoma, and one patient had mantle cell lymphoma. Patients received a single dose of CAR-19 T cells 2 days after a low-dose chemotherapy conditioning regimen of cyclophosphamide plus fludarabine. Results The overall remission rate was 73% with 55% complete remissions and 18% partial remissions. Eleven of 12 complete remissions are ongoing. Fifty-five percent of patients had grade 3 or 4 neurologic toxicities that completely resolved. The low-dose chemotherapy conditioning regimen depleted blood lymphocytes and increased serum interleukin-15 (IL-15). Patients who achieved a remission had a median peak blood CAR+ cell level of 98/μL and those who did not achieve a remission had a median peak blood CAR+ cell level of 15/μL ( P = .027). High serum IL-15 levels were associated with high peak blood CAR+ cell levels ( P = .001) and remissions of lymphoma ( P < .001). Conclusion CAR-19 T cells preceded by low-dose chemotherapy induced remission of advanced-stage lymphoma, and high serum IL-15 levels were associated with the effectiveness of this treatment regimen. CAR-19 T cells will likely become an important treatment for patients with relapsed lymphoma.

A randomized controlled safety/efficacy trial of therapeutic vaccination in HIV-infected individuals who initiated antiretroviral therapy early in infection

Despite lack of vaccine efficacy, the kinetics and magnitude of HIV rebound in early-treated patients affect future clinical trial design. Single-arm trials can leave you hanging Depending on the study restraints and goals, not all clinical trials include a randomized placebo group. This is often done to minimize risk to patients but can also impair interpretation of the results. When Sneller et al. embarked on their therapeutic HIV vaccine trial, they chose to include a placebo group to get a better understanding of how their vaccine affected viral rebound upon therapy interruption. The vaccine itself generated minimal T cell activation and did not induce protective responses. Somewhat surprisingly, a proportion of individuals in the placebo arm demonstrated sustained viral suppression, although they were no longer being treated. These results suggest that any future HIV intervention trials would benefit from the inclusion of a placebo arm. Despite substantial clinical benefits, complete eradication of HIV has not been possible using antiretroviral therapy (ART) alone. Strategies that can either eliminate persistent viral reservoirs or boost host immunity to prevent rebound of virus from these reservoirs after discontinuation of ART are needed; one possibility is therapeutic vaccination. We report the results of a randomized, placebo-controlled trial of a therapeutic vaccine regimen in patients in whom ART was initiated during the early stage of HIV infection and whose immune system was anticipated to be relatively intact. The objectives of our study were to determine whether the vaccine was safe and could induce an immune response that would maintain suppression of plasma viremia after discontinuation of ART. Vaccinations were well tolerated with no serious adverse events but produced only modest augmentation of existing HIV-specific CD4+ T cell responses, with little augmentation of CD8+ T cell responses. Compared with placebo, the vaccination regimen had no significant effect on the kinetics or magnitude of viral rebound after interruption of ART and no impact on the size of the HIV reservoir in the CD4+ T cell compartment. Notably, 26% of subjects in the placebo arm exhibited sustained suppression of viremia (<400 copies/ml) after treatment interruption, a rate of spontaneous suppression higher than previously reported. Our findings regarding the degree and kinetics of plasma viral rebound after ART interruption have potentially important implications for the design of future trials testing interventions aimed at achieving ART-free control of HIV infection.

Effect of analytical treatment interruption and reinitiation of antiretroviral therapy on HIV reservoirs and immunologic parameters in infected individuals

Therapeutic strategies aimed at achieving antiretroviral therapy (ART)-free HIV remission in infected individuals are under active investigation. Considering the vast majority of HIV-infected individuals experience plasma viral rebound upon cessation of therapy, clinical trials evaluating the efficacy of curative strategies would likely require inclusion of ART interruption. However, it is unclear what impact short-term analytical treatment interruption (ATI) and subsequent reinitiation of ART have on immunologic and virologic parameters of HIV-infected individuals. Here, we show a significant increase of HIV burden in the CD4+ T cells of infected individuals during ATI that was correlated with the level of plasma viral rebound. However, the size of the HIV reservoirs as well as immune parameters, including markers of exhaustion and activation, returned to pre-ATI levels 6–12 months after the study participants resumed ART. Of note, the proportions of near full-length, genome-intact and structurally defective HIV proviral DNA sequences were similar prior to ATI and following reinitiation of ART. In addition, there was no evidence of emergence of antiretroviral drug resistance mutations within intact HIV proviral DNA sequences following reinitiation of ART. These data demonstrate that short-term ATI does not necessarily lead to expansion of the persistent HIV reservoir nor irreparable damages to the immune system in the peripheral blood, warranting the inclusion of ATI in future clinical trials evaluating curative strategies.

74. The Impact of Different Hinge and Transmembrane Components on the Function of a Novel Fully-Human Anti-CD19 Chimeric Antigen Receptor

T cells expressing anti-CD19 chimeric antigen receptors (CARs) have significant activity against B-cell malignancies in humans, but increased efficacy and decreased toxicity of anti-CD19 CAR T cells are needed. Anti-CD19 CARs that have been clinically tested have single-chain variable fragments (scFvs) derived from murine antibodies or in 1 case a humanized murine antibody. Evidence of human anti-mouse immune responses against CARs with murine scFvs has been reported (Jensen et al. Biology of Blood and Marrow Transplantation 2010; Lee et al. Lancet, 2015), providing a rationale for development of less immunogenic CARs. A CAR with a scfv derived from a fully-human antibody would eliminate these human anti-mouse immune responses and perhaps increase the persistence of anti-CD19 CAR T cells. A non-immunogenic CAR might be particularly important if multiple, temporally-separated doses of CAR T cells are administered to the same patient. We have designed and constructed fully-human anti-CD19 CARs encoded by a lentiviral vector. These CARs specifically recognize CD19, and T cells expressing the CARs exhibit a full range of functions including degranulation, cytotoxicity, proliferation, and release of a variety of cytokines. The extracellular scFv component of a CAR is connected to intracellular domains by an extracellular hinge region and a transmembrane (TM) region. In comparing anti-CD19 CARs, we noticed a difference in cytokine production by CARs with hinge and TM regions from the human CD8α molecule compared to CARs with hinge and TM regions from the human CD28 molecule. We designated a fully-human CAR with CD8α hinge and TM domains hu19-CD828Z, and we designated a CAR that was identical to hu19-CD828Z except that it had CD28 hinge and TM domains hu19-28Z. Compared to T cells expressing hu19-CD828Z, T cells expressing hu19-28Z produced much higher levels of interferon gamma (IFNγ), tumor necrosis factor (TNF), and interleukin-2. For example, after an overnight culture with CD19+ NALM6 cells, T cells expressing hu19-CD828Z yielded a mean of 5688 pg/mL IFNγ in the culture supernatant while T cells expressing hu19-28Z yielded 19396 pg/mL IFNγ (P=0.03). Similarly, after an overnight culture with CD19+ NALM6 cells, T cells expressing hu19-CD828Z yielded a mean of 2033 pg/mL TNF in the culture supernatant while T cells expressing hu19-28Z yielded 5008 pg/mL TNF (P=0.007). Cytokine levels were normalized for the percentage of T cells expressing each CAR in each experiment. This same pattern of hinge and TM domains affecting cytokine production was observed for CARs incorporating a different anti-CD19 scFv. In contrast to cytokine production, significant differences in other T-cell functions including degranulation, cytotoxicity, and proliferation were not found when hu19-CD828Z and hu19-28Z were compared. When T cells expressing hu19-CD828Z and hu19-28Z were assessed in a murine lymphoma model, there was not a statistically-significant difference in tumor eradication or survival. We are currently assessing other measures of T-cell activation to obtain a more mechanistic understanding of the differences in T-cell activation with different hinge and TM domains. Most of the clinical toxicity of anti-CD19 CAR T cells is caused by cytokine release. Compared to anti-CD19 CARs with CD28 hinge and TM domains, anti-CD19 CARs with hinge and TM regions from CD8α cause lower levels of cytokine release from T cells in vitro. This decreased cytokine release might reduce cytokine-mediated toxicity in patients. We have initiated a clinical trial of T cells expressing hu19-CD828Z. We will assess cytokine release, immunogenicity, peak blood levels, and long-term persistence of hu19-CD828Z-expressing T cells in humans.