Safety and efficacy of targeting CD138 with a chimeric antigen receptor for the treatment of multiple myeloma.

After unprecedented successes in B-cell malignancies, chimeric antigen receptor T cells have recently been investigated for the treatment of multiple myeloma. Chimeric antigen receptor targeting T cells B-cell maturation antigen (BCMA) on malignant plasma cells have led to impressive clinical responses in recent trials. However, BCMA-negative relapses have been observed, supporting the need for complementary treatment strategies. Here, we explored the feasibility of targeting CD138 (syndecan-1), a surface marker expressed on both normal and malignant plasma cells. We showed that T cells from both healthy donors and from multiple myeloma patients, when transduced with a CD138-specific chimeric antigen receptor, can eliminate tumor cell lines and primary myeloma cells both in vitro and in vivo. CD138 is also expressed by putative myeloma stem cells identified by Hoechst staining, and these cells can be eliminated by CD138-specific chimeric antigen receptor T cells. Preclinical analyses did not identify any on target off tumor cytotoxicity against normal epithelial or endothelial cells, further supporting the rationale for the use of adoptively transferred CD138-specific chimeric antigen receptor T cells for the treatment of patients with relapsed/refractory multiple myeloma.

Clinical and immunological responses after CD30-specific chimeric antigen receptor-redirected lymphocytes.

BACKGROUND: Targeting CD30 with monoclonal antibodies in Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) has had profound clinical success. However, adverse events, mainly mediated by the toxin component of the conjugated antibodies, cause treatment discontinuation in many patients. Targeting CD30 with T cells expressing a CD30-specific chimeric antigen receptor (CAR) may reduce the side effects and augment antitumor activity. METHODS: We conducted a phase I dose escalation study in which 9 patients with relapsed/refractory HL or ALCL were infused with autologous T cells that were gene-modified with a retroviral vector to express the CD30-specific CAR (CD30.CAR-Ts) encoding the CD28 costimulatory endodomain. Three dose levels, from 0.2 × 108 to 2 × 108 CD30.CAR-Ts/m2, were infused without a conditioning regimen. All other therapy for malignancy was discontinued at least 4 weeks before CD30.CAR-T infusion. Seven patients had previously experienced disease progression while being treated with brentuximab. RESULTS: No toxicities attributable to CD30.CAR-Ts were observed. Of 7 patients with relapsed HL, 1 entered complete response (CR) lasting more than 2.5 years after the second infusion of CD30.CAR-Ts, 1 remained in continued CR for almost 2 years, and 3 had transient stable disease. Of 2 patients with ALCL, 1 had a CR that persisted 9 months after the fourth infusion of CD30.CAR-Ts. CD30.CAR-T expansion in peripheral blood peaked 1 week after infusion, and CD30.CAR-Ts remained detectable for over 6 weeks. Although CD30 may also be expressed by normal activated T cells, no patients developed impaired virus-specific immunity. CONCLUSION: CD30.CAR-Ts are safe and can lead to clinical responses in patients with HL and ALCL, indicating that further assessment of this therapy is warranted. TRIAL REGISTRATION: ClinicalTrials.gov NCT01316146. FUNDING: National Cancer Institute (3P50CA126752, R01CA131027 and P30CA125123), National Heart, Lung, and Blood Institute (R01HL114564), and Leukemia and Lymphoma Society (LLSTR 6227-08).

Inducible Caspase-9 Selectively Modulates the Toxicities of CD19-Specific Chimeric Antigen Receptor-Modified T Cells.

Immunotherapy with T cells expressing the chimeric antigen receptor (CAR) specific for the CD19 antigen (CD19.CAR-Ts) is a very effective treatment in B cell lymphoid malignancies. However, B cell aplasia and cytokine release syndrome (CRS) secondary to the infusion of CD19.CAR-Ts remain significant drawbacks. The inclusion of safety switches into the vector encoding the CAR is seen as the safest method to terminate the effects of CD19.CAR-Ts in case of severe toxicities or after achieving long-term sustained remissions. By contrast, the complete elimination of CD19.CAR-Ts when CRS occurs may jeopardize clinical responses as CRS and antitumor activity seem to concur. We have demonstrated, in a humanized mouse model, that the inducible caspase-9 (iC9) safety switch can eliminate CD19.CAR-Ts in a dose-dependent manner, allowing either a selective containment of CD19.CAR-T expansion in case of CRS or complete deletion on demand granting normal B cell reconstitution.

Clinical responses with T lymphocytes targeting malignancy-associated κ light chains.

BACKGROUND: Treatment of B cell malignancies with adoptive transfer of T cells with a CD19-specific chimeric antigen receptor (CAR) shows remarkable clinical efficacy. However, long-term persistence of T cells targeting CD19, a pan-B cell marker, also depletes normal B cells and causes severe hypogammaglobulinemia. Here, we developed a strategy to target B cell malignancies more selectively by taking advantage of B cell light Ig chain restriction. We generated a CAR that is specific for the κ light chain (κ.CAR) and therefore recognizes κ-restricted cells and spares the normal B cells expressing the nontargeted λ light chain, thus potentially minimizing humoral immunity impairment. METHODS: We conducted a phase 1 clinical trial and treated 16 patients with relapsed or refractory κ+ non-Hodgkin lymphoma/chronic lymphocytic leukemia (NHL/CLL) or multiple myeloma (MM) with autologous T cells genetically modified to express κ.CAR (κ.CARTs). Other treatments were discontinued in 11 of the 16 patients at least 4 weeks prior to T cell infusion. Six patients without lymphopenia received 12.5 mg/kg cyclophosphamide 4 days before κ.CART infusion (0.2 × 108 to 2 × 108 κ.CARTs/m2). No other lymphodepletion was used. RESULTS: κ.CART expansion peaked 1-2 weeks after infusion, and cells remained detectable for more than 6 weeks. Of 9 patients with relapsed NHL or CLL, 2 entered complete remission after 2 and 3 infusions of κ.CARTs, and 1 had a partial response. Of 7 patients with MM, 4 had stable disease lasting 2-17 months. No toxicities attributable to κ.CARTs were observed. CONCLUSION: κ.CART infusion is feasible and safe and can lead to complete clinical responses. TRIAL REGISTRATION: ClinicalTrials.gov NCT00881920. FUNDING: National Cancer Institute (NCI) grants 3P50CA126752 and 5P30CA125123 and Leukemia and Lymphoma Society (LLS) Specialized Centers of Research (SCOR) grant 7018.

Early transduction produces highly functional chimeric antigen receptor-modified virus-specific T-cells with central memory markers: a Production Assistant for Cell Therapy (PACT) translational application.

BACKGROUND: Virus-specific T-cells (VSTs) proliferate exponentially after adoptive transfer into hematopoietic stem cell transplant (HSCT) recipients, eliminate virus infections, then persist and provide long-term protection from viral disease. If VSTs behaved similarly when modified with tumor-specific chimeric antigen receptors (CARs), they should have potent anti-tumor activity. This theory was evaluated by Cruz et al. in a previous clinical trial with CD19.CAR-modified VSTs, but there was little apparent expansion of these cells in patients. In that study, VSTs were gene-modified on day 19 of culture and we hypothesized that by this time, sufficient T-cell differentiation may have occurred to limit the subsequent proliferative capacity of the transduced T-cells. To facilitate the clinical testing of this hypothesis in a project supported by the NHLBI-PACT mechanism, we developed and optimized a good manufacturing practices (GMP) compliant method for the early transduction of VSTs directed to Epstein-Barr virus (EBV), Adenovirus (AdV) and cytomegalovirus (CMV) using a CAR directed to the tumor-associated antigen disialoganglioside (GD2). RESULTS: Ad-CMVpp65-transduced EBV-LCLs effectively stimulated VSTs directed to all three viruses (triVSTs). Transduction efficiency on day three was increased in the presence of cytokines and high-speed centrifugation of retroviral supernatant onto retronectin-coated plates, so that under optimal conditions up to 88% of tetramer-positive VSTs expressed the GD2.CAR. The average transduction efficiency of early-and late transduced VSTs was 55 ± 4% and 22 ± 5% respectively, and early-transduced VSTs maintained higher frequencies of T cells with central memory or intermediate memory phenotypes. Early-transduced VSTs also had higher proliferative capacity and produced higher levels of TH1 cytokines IL-2, TNF-α, IFN-γ, MIP-1α, MIP-1ß and other cytokines in vitro. CONCLUSIONS: We developed a rapid and GMP compliant method for the early transduction of multivirus-specific T-cells that allowed stable expression of high levels of a tumor directed CAR. Since a proportion of early-transduced CAR-VSTs had a central memory phenotype, they should expand and persist in vivo, simultaneously protecting against infection and targeting residual malignancy. This manufacturing strategy is currently under clinical investigation in patients receiving allogeneic HSCT for relapsed neuroblastoma and B-cell malignancies (NCT01460901 using a GD2.CAR and NCT00840853 using a CD19.CAR).

K562-Derived Whole-Cell Vaccine Enhances Antitumor Responses of CAR-Redirected Virus-Specific Cytotoxic T Lymphocytes In Vivo.

PURPOSE: Adoptive transfer of Epstein-Barr virus (EBV)-specific and cytomegalovirus (CMV)-specific cytotoxic T cells (CTL) genetically modified to express a chimeric antigen receptor (CAR) induces objective tumor responses in clinical trials. In vivo expansion and persistence of these cells are crucial to achieve sustained clinical responses. We aimed to develop an off-the-shelf whole-cell vaccine to boost CAR-redirected virus-specific CTLs in vivo after adoptive transfer. As proof of principle, we validated our vaccine approach by boosting CMV-specific CTLs (CMV-CTLs) engineered with a CAR that targets the GD2 antigen. EXPERIMENTAL DESIGN: We generated the whole-cell vaccine by engineering the K562 cell line to express the CMV-pp65 protein and the immune stimulatory molecules CD40L and OX40L. Single-cell-derived clones were used to stimulate CMV-CTLs in vitro and in vivo in a xenograft model. We also assessed whether the in vivo boosting of CAR-redirected CMV-CTLs with the whole-cell vaccine enhances the antitumor responses. Finally, we addressed potential safety concerns by including the inducible safety switch caspase9 (iC9) gene in the whole-cell vaccine. RESULTS: We found that K562-expressing CMV-pp65, CD40L, and OX40L effectively stimulate CMV-specific responses in vitro by promoting antigen cross-presentation to professional antigen-presenting cells (APCs). Vaccination also enhances antitumor effects of CAR-redirected CMV-CTLs in xenograft tumor models. Activation of the iC9 gene successfully induces growth arrest of engineered K562 implanted in mice. CONCLUSIONS: Vaccination with a whole-cell vaccine obtained from K562 engineered to express CMV-pp65, CD40L, OX40L and iC9 can safely enhance the antitumor effects of CAR-redirected CMV-CTLs.

Effect of thyroid hormone on Mg(2+) homeostasis and extrusion in cardiac cells.

The present study investigated the effect of alteration in thyroid hormone level on Mg(2+) homeostasis in cardiac ventricular myocytes. Hyperthyroid conditions increased cardiac myocytes total Mg(2+) content by ~14% as compared to cells from eu-thyroid animals. The excess Mg(2+) was localized predominantly within cytoplasm and mitochondria, and was mobilized into the extracellular compartment by addition of isoproterenol (ISO) or cAMP but not phenylephrine (PHE). Hypothyroid conditions, instead, decreased cardiac myocytes total Mg(2+) content by ~10% as compared to cells from eu-thyroid animals. Also in this case, cytoplasm and mitochondria were the two cellular pools predominantly affected. Under hypothyroid conditions, administration of ISO or cAMP resulted in a decreased Mg(2+) extrusion as compared to that observed in cardiac cells from eu-thyroid animals. Similar changes in cellular Mg(2+) content and transport were observed in cardiac ventricular myocytes isolated from hyper- and hypo-thyroid animals, as well as in cultures of H9C2 cells rendered hyper- or hypo-thyroid under in vitro conditions. Supplementation of thyroid hormone to hypothyroid animals restored Mg(2+) level and transport to levels comparable to those observed in eu-thyroid animals. Taken together, these results indicate that changes in thyroid hormone level have a major effect on Mg(2+) homeostasis and compartmentation in cardiac cells. The enlarged Mg(2+) mobilization via beta- but not alpha(1)-adrenergic receptor stimulation further suggests that beta- and alpha(1)-adrenergic receptors target selectively different Mg(2+) compartments within the cardiac myocyte. These results provide a new rationale to interpret changes in cardiac function under hyper- or hypo-thyroid conditions.