Vicki Fellowes

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 patient's 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.

Long-lived, self-renewing, multipotent T memory stem cells (TSCM) can trigger profound and sustained tumor regression but their rareness poses a major hurdle to their clinical application. Presently, clinically compliant procedures to generate relevant numbers of this T-cell population are undefined. Here, we provide a strategy for deriving large numbers of clinical-grade tumor-redirected TSCM starting from naive precursors. CD8(+)CD62L(+)CD45RA(+) naive T cells enriched by streptamer-based serial-positive selection were activated by CD3/CD28 engagement in the presence of interleukin-7 (IL-7), IL-21, and the glycogen synthase-3β inhibitor TWS119, and genetically engineered to express a CD19-specific chimeric antigen receptor (CD19-CAR). These conditions enabled the generation of CD19-CAR-modified CD8(+) TSCM that were phenotypically, functionally, and transcriptomically equivalent to their naturally occurring counterpart. Compared with CD8(+) T cells generated with clinical protocols currently under investigation, CD19-CAR-modified CD8(+) TSCM exhibited enhanced metabolic fitness and mediated robust, long-lasting antitumor responses against systemic acute lymphoblastic leukemia xenografts. This clinical-grade platform provides the basis for a phase 1 trial evaluating the activity of CD19-CAR-modified CD8(+) TSCM in patients with B-cell malignancies refractory to prior allogeneic hematopoietic stem cell transplantation.

Two FDA-approved agents, ferumoxides (Feridex), a suspension of superparamagnetic iron oxide (SPIO) nanoparticles and protamine sulfate, a drug used to reverse heparin anticoagulation, can be complexed and used to label cells magnetically ex vivo. Labeling stem cells with ferumoxides-protamine sulfate (FePro) complexes allows for non-invasive monitoring by MRI. However, in order for stem cell trials or therapies to be effective, this labeling technique must not inhibit the ability of cells to differentiate. In this study, we examined the effect of FePro labeling on stem cell differentiation. Viability, phenotypic expression and differential capacity of FePro labeled CD34 + hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC) were compared with unlabeled control cells. Colony-forming unit (CFU) assays showed that the capacity to differentiate was equivalent for labeled and unlabeled HSC. Furthermore, labeling did not alter expression of surface phenotypic markers (CD34, CD31, CXCR4, CD20, CD3 and CD14) on HSC, as measured by flow cytometry. SDF-1-induced HSC migration and HSC differentiation to dendritic cells were also unaffected by FePro labeling. Both FePro-labeled and unlabeled MSC were cultured in chondrogenesis-inducing conditions. Alcian blue staining for proteoglycans revealed similar chondrogenic differentiation for both FePro-labeled and unlabeled cells. Furthermore, collagen X proteins, indicators of cartilage formation, were detected at similar levels in both labeled and unlabeled cell pellets. Prussian blue staining confirmed that cells in labeled pellets contained iron oxide, whereas cells in unlabeled pellets did not. It is concluded that FePro labeling does not alter the function or differentiation capacity of HSC and MSC. These data increase confidence that MRI studies of FePro-labeled HSC or MSC will provide an accurate representation of in vivo trafficking of unlabeled cells.

Selective allodepletion is a strategy to eliminate host-reactive donor T cells from hematopoietic stem cell allografts to prevent graft-versus-host disease while conserving useful donor immune functions. To overcome fluctuations in activation-based surface marker expression and achieve a more consistent and effective allodepletion, we investigated a photodepletion process targeting activation-based changes in p-glycoprotein that result in an altered efflux of the photosensitizer TH9402. Expanded lymphocytes, generated using anti-CD3 and IL-2, were cocultured with responder cells from HLA-matched or -mismatched donors. Optimal results were achieved when cocultured cells were incubated with 7.5 muM TH9402, followed by dye extrusion and exposure to 5 Joule/cm(2) light energy at 5 x 10(6) cells/mL. In mismatched stimulator-responder pairs, the median reduction of alloreactivity was 474-fold (range, 43-fold to 864-fold) compared with the unmanipulated responder. Third-party responses were maintained with a median 1.4-fold (range, 0.9-fold to 3.3-fold) reduction. In matched pairs, alloreactive helper T-lymphocyte precursors were reduced to lower than 1:100 000, while third-party responses remained higher than 1:10 000. This establishes a clinical-scale process capable of highly efficient, reproducible, selective removal of alloreactive lymphocytes from lymphocyte transplant products performed under current Good Manufacturing Practice. This procedure is currently being investigated in a clinical trial of allotransplantation.

The present study examined the safety and relative in vivo survival of genetically engineered CD4+ T lymphocytes in human immunodeficiency virus (HIV)-infected individuals. Ten pairs of identical twins discordant for HIV infection were recruited, with the uninfected twin serving as the lymphocyte donor. Ten subjects were treated with a total of 19 separate infusions of retroviral vector-transduced CD4+ enriched T cells. Control (neo gene) or anti-HIV gene (antisense trans-activation response [TAR] element and/or trans-dominant Rev)-engineered lymphocytes were monitored in peripheral blood for 3 years, using a vector-specific PCR assay. Data from 9 of the 10 patients (15 of the 19 infusions) demonstrated preferential survival of CD4+ lymphocytes containing the anti-HIV gene(s) in the immediate weeks after infusion. In six of six patients studied long term (>100 weeks), only T cells containing the anti-HIV genes were consistently detected. In addition, a marked survival advantage of anti-HIV gene-containing T cells was observed in a patient treated during a period of high viral load. Thus, these data strongly support the hypothesis that anti-HIV genes afford a survival advantage to T cells and potential benefit to HIV-1+ individuals.