Jolanta Stefanski

CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.

Five adults with chemotherapy-refractory B-ALL were induced into molecular remissions after treatment with CD19 CAR-targeted T cells. CARving a Niche for Cancer Immunotherapy Acute lymphoblastic leukemia (ALL) is a cancer of the white blood cells that fend off infection. It’s most common in children but—as with many diseases that primarily affect children—has a much worse prognosis when it affects adults. Adults with relapsed disease have a very low chance of survival, and new therapies are desperately needed. Now, Brentjens et al. test T cells engineered to target CD19, which is expressed on both healthy B lymphocytes and B-ALL cells, in five chemotherapy-refractory adult B-ALL patients. Here, the authors treat patients with the patients’ own T cells altered to express not only CD19 but also a fusion of the costimulatory molecule CD28 with CD3ζ chain—so-called “second-generation chimeric antigen receptor (CAR) T cells.” All patients treated with these cells achieved tumor eradication and complete remission. These CAR T cells were well tolerated, although there was substantial cytokine release in some patients that correlated to tumor burden. These patients were treated with steroid therapy. Long-term follow-up in four of these patients was not possible because the CAR T cell therapy allowed these patients to be eligible for subsequent hematopoietic stem cell transplant (HSCT), which resulted in restored hematopoiesis. The remaining patient experienced a relapse of CD19+ cells that coincided with the lack of persistence of the CAR T cells from circulation. These data suggest that subsequent transfusions should be considered for patients unable to undergo HSCT. Adults with relapsed B cell acute lymphoblastic leukemia (B-ALL) have a dismal prognosis. Only those patients able to achieve a second remission with no minimal residual disease (MRD) have a hope for long-term survival in the context of a subsequent allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have treated five relapsed B-ALL subjects with autologous T cells expressing a CD19-specific CD28/CD3ζ second-generation dual-signaling chimeric antigen receptor (CAR) termed 19-28z. All patients with persistent morphological disease or MRD+ disease upon T cell infusion demonstrated rapid tumor eradication and achieved MRD− complete remissions as assessed by deep sequencing polymerase chain reaction. Therapy was well tolerated, although significant cytokine elevations, specifically observed in those patients with morphologic evidence of disease at the time of treatment, required lymphotoxic steroid therapy to ameliorate cytokine-mediated toxicities. Indeed, cytokine elevations directly correlated to tumor burden at the time of CAR-modified T cell infusions. Tumor cells from one patient with relapsed disease after CAR-modified T cell therapy, who was ineligible for additional allo-HSCT or T cell therapy, exhibited persistent expression of CD19 and sensitivity to autologous 19-28z T cell–mediated cytotoxicity, which suggests potential clinical benefit of additional CAR-modified T cell infusions. These results demonstrate the marked antitumor efficacy of 19-28z CAR-modified T cells in patients with relapsed/refractory B-ALL and the reliability of this therapy to induce profound molecular remissions, forming a highly effective bridge to potentially curative therapy with subsequent allo-HSCT.

Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia.

CD19 CAR T cell therapy induces complete remissions in 88% of 16 adult patients with relapsed or refractory acute lymphoblastic leukemia. CARving Out a Niche for CAR T Cell Immunotherapy Relapsed or refractory B acute lymphoblastic leukemia (B-ALL) in adults has a poor prognosis, with an expected median survival of less than 6 months. An emerging therapy for adult B-ALL is through T cells that target tumor cells with chimeric antigen receptors (CARs). Davila et al. now report the results of a phase 1 clinical trial of CAR T cells in 16 relapsed or refractory adult patients. The CD19-targeting CAR T cell therapy resulted in an 88% complete response rate, which allowed most of the patients to transition to allogeneic hematopoietic stem cell transplantation—the current standard of care. Moreover, the authors carefully characterized cytokine release syndrome (CRS), which is a series of toxicities associated with CAR T cell therapy. They found that serum C-reactive protein (CRP) associated with the severity of CRS, which should allow for identification of the subset of patients who will likely require therapeutic intervention with corticosteroids or interleukin-6 receptor blockade to curb the CRS. This is especially important because treatment for CRS may limit the efficacy of the CAR T cell therapy. These data support the need for further multicenter trials for CAR T cell therapy. We report on 16 patients with relapsed or refractory B cell acute lymphoblastic leukemia (B-ALL) that we treated with autologous T cells expressing the 19-28z chimeric antigen receptor (CAR) specific to the CD19 antigen. The overall complete response rate was 88%, which allowed us to transition most of these patients to a standard-of-care allogeneic hematopoietic stem cell transplant (allo-SCT). This therapy was as effective in high-risk patients with Philadelphia chromosome–positive (Ph+) disease as in those with relapsed disease after previous allo-SCT. Through systematic analysis of clinical data and serum cytokine levels over the first 21 days after T cell infusion, we have defined diagnostic criteria for a severe cytokine release syndrome (sCRS), with the goal of better identifying the subset of patients who will likely require therapeutic intervention with corticosteroids or interleukin-6 receptor blockade to curb the sCRS. Additionally, we found that serum C-reactive protein, a readily available laboratory study, can serve as a reliable indicator for the severity of the CRS. Together, our data provide strong support for conducting a multicenter phase 2 study to further evaluate 19-28z CAR T cells in B-ALL and a road map for patient management at centers now contemplating the use of CAR T cell therapy.

Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias

We report the findings from the first 10 patients with chemotherapy-refractory chronic lymphocytic leukemia (CLL) or relapsed B-cell acute lymphoblastic leukemia (ALL) we have enrolled for treatment with autologous T cells modified to express 19-28z, a second-generation chimeric antigen (Ag) receptor specific to the B-cell lineage Ag CD19. Eight of the 9 treated patients tolerated 19-28z(+) T-cell infusions well. Three of 4 evaluable patients with bulky CLL who received prior conditioning with cyclophosphamide exhibited either a significant reduction or a mixed response in lymphadenopathy without concomitant development of B-cell aplasia. In contrast, one patient with relapsed ALL who was treated in remission with a similar T-cell dose developed a predicted B-cell aplasia. The short-term persistence of infused T cells was enhanced by prior cyclophosphamide administration and inversely proportional to the peripheral blood tumor burden. Further analyses showed rapid trafficking of modified T cells to tumor and retained ex vivo cytotoxic potential of CD19-targeted T cells retrieved 8 days after infusion. We conclude that this adoptive T-cell approach is promising and more likely to show clinical benefit in the setting of prior conditioning chemotherapy and low tumor burden or minimal residual disease. These studies are registered at www.clinicaltrials.org as #NCT00466531 (CLL protocol) and #NCT01044069 (B-ALL protocol).

Manufacturing Validation of Biologically Functional T Cells Targeted to Cd19 Antigen for Autologous Adoptive Cell Therapy

On the basis of promising preclinical data demonstrating the eradication of systemic B-cell malignancies by CD19-targeted T lymphocytes in vivo in severe combined immunodeficient-beige mouse models, we are launching phase I clinical trials in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia. We present here the validation of the bioprocess which we developed for the production and expansion of clinical grade autologous T cells derived from patients with CLL. We demonstrate that T cells genetically modified with a replication-defective gammaretroviral vector derived from the Moloney murine leukemia virus encoding a chimeric antigen receptor (CAR) targeted to CD19 (1928z) can be expanded with Dynabeads CD3/CD28. This bioprocess allows us to generate clinical doses of 1928z+ T cells in approximately 2 to 3 weeks in a large-scale semiclosed culture system using the Wave Bioreactor. These 1928z+ T cells remain biologically functional not only in vitro but also in severe combined immunodeficient-beige mice bearing disseminated tumors. The validation requirements in terms of T-cell expansion, T-cell transduction with the 1928z CAR, biologic activity, quality control testing, and release criteria were met for all 4 validation runs using apheresis products from patients with CLL. Additionally, after expansion of the T cells, the diversity of the skewed Vβ T-cell receptor repertoire was significantly restored. This validated process will be used in phase I clinical trials in patients with chemorefractory CLL and in patients with relapsed acute lymphoblastic leukemia. It can also be adapted for other clinical trials involving the expansion and transduction of patient or donor T cells using any CAR or T-cell receptor.

Targeted Elimination of Prostate Cancer by Genetically Directed Human T Lymphocytes

The genetic transfer of antigen receptors is a powerful approach to rapidly generate tumor-specific T lymphocytes. Unlike the physiologic T-cell receptor, chimeric antigen receptors (CARs) encompass immunoglobulin variable regions or receptor ligands as their antigen recognition moiety, thus permitting T cells to recognize tumor antigens in the absence of human leukocyte antigen expression. CARs encompassing the CD3zeta chain as their activating domain induce T-cell proliferation in vitro, but limited survival. The requirements for genetically targeted T cells to function in vivo are less well understood. We have, therefore, established animal models to assess the therapeutic efficacy of human peripheral blood T lymphocytes targeted to prostate-specific membrane antigen (PSMA), an antigen expressed in prostate cancer cells and the neovasculature of various solid tumors. In vivo specificity and antitumor activity were assessed in mice bearing established prostate adenocarcinomas, using serum prostate-secreted antigen, magnetic resonance, computed tomography, and bioluminescence imaging to investigate the response to therapy. In three tumor models, orthotopic, s.c., and pulmonary, we show that PSMA-targeted T cells effectively eliminate prostate cancer. Tumor eradication was directly proportional to the in vivo effector-to-tumor cell ratio. Serial imaging further reveals that the T cells must survive for at least 1 week to induce durable remissions. The eradication of xenogeneic tumors in a murine environment shows that the adoptively transferred T cells do not absolutely require in vivo costimulation to function. These results thus provide a strong rationale for undertaking phase I clinical studies to assess PSMA-targeted T cells in patients with metastatic prostate cancer.

459. Evaluation of Miltenyi ExpAct and TransAct CD3/28 Beads for CAR-T Cell Manufacturing

Adoptive transfer of chimeric antigen receptor (CAR) engineered T cells is a promising emerging strategy to treat cancer patients. Large-scale manufacturing of cGMP-grade CAR T cells using patient T cells selected and activated by CTS™ Dynabeads® CD3/CD28 (Dynabeads) followed by transduction with retroviral vectors is being used in the context of many clinical trials by our laboratory and others. Although we have established a robust protocol using Dynabeads, it is important to explore alternative sources to pre-empt supply chain limitations of this critical reagent. To this end, we evaluated T cell activation with either Miltenyi TransAct CD3/28 (TransAct) beads or Miltenyi ExpAct Treg (ExpAct) beads. In small-scale experiments, PBMCs were directly activated with TransAct or ExpAct beads and compared with our standard T cell selection and activation using Dynabeads. Overall, the transduction efficiency and expansion of T cells were comparable upon activation with all three reagents. The TransAct bead-stimulated cells exhibited comparable effector memory (EM)/central memory (CM) phenotype to that of the Dynabeads stimulated cells. In line with the EM/CM phenotype, CAR T cells stimulated with either TransAct or Dynabeads and tranduced with CD19-targeted CAR demonstrated robust and comparable antitumor activity in a systemic NSG/CD19+ NALM6 tumor mouse model. We further tested the efficacy of TransAct beads using positively or negatively selected T cells in a large-scale cGMP grade CAR-T cell manufacturing setting. Both the transduction efficiency and expansion of selected CD3+ cells activated with TransAct beads and Dynabeads were comparable. CD19-targeted CAR T cells activated by either TransAct or Dynabead were subjected to an in vivo stress test by using decreasing amount of CAR-T cells to treat systemic CD19+NALM6 tumors in NSG mice. In this experimental setting, T cells stimulated with TransAct beads demonstrated equivalent if not better anti-tumor activity than T cells stimulated with Dynabeads. In conclusion, our pre-clinical results suggest that TransAct beads support efficient transduction and expansion of CAR T cells. TransAct activated T cells exhibit antitumor activity equivalent to Dynabeads activated T cells in our NSG/CD19+NAML6 stress test. Therefore, Miltenyi TransAct beads can be used as an alternative to Dynabeads to stimulate T cells in clinical trials aiming at evaluating CAR T cell safety and antitumor activity.

80. Depletion of High-Content CD14+ Cells from Apheresis Products is Critical for the Successful Transduction and Expansion of CAR T Cells During Large-Scale cGMP Manufacturing

Adoptive transfer of chimeric antigen receptor (CAR) engineered T cells holds great promise as a novel strategy to treat patients with cancer. Apheresis products are a major source of starting material for large-scale T cell selection using CD3/28 magnetic beads. At the time of collection, the composition in various cell lineages is highly variable. We have successfully manufactured CAR T cell products starting from apheresis products containing as few as 0.5% to 3% of CD3+ cells. However, we have experienced manufacturing challenges with apheresis products containing a high fraction of CD14+ cells. We conducted a study with an apheresis product derived from a patient with acute lymphoblastic leukemia containing 70% CD14+ cells. 350 × 106 CD3+ cells were selected from the cryopreserved apheresis product using CD3/28 magnetic beads either with or without a prior 1.5 hr monocyte/granulocyte adherence step. This short adherence procedure decreased the CD14+ cell composition from 70% to 45%. A second overnight depletion by adherence in T-flasks and re-stimulation with CD3/28 magnetic beads on day 1 were also performed in the depleted group to ensure the proper activation of CD3+ cells. On day 3, 350×106 vs 20 x106 total cells were recovered from each group without and with the depletion step, respectively. Selected cells from both groups were transduced using the same dilution of vector stocks at a cell concentration of either 0.4×106/mL or 0.2×106/mL in tissue culture bags. On day 7, we observed a drastic difference in transduction efficiency between the group without depletion (4.5%) and the group in which the CD14+ cells were depleted (38%). Moreover, starting with 108 × 106 selected cells on day 3, a mere total of 12.3 × 106 (14.8% CD3+) were recovered on day 11 in the group without depletion. By contrast, cells from the depleted group expanded 350 fold from day 3 to day 10 (100% CD3+). Our results suggest that high CD14+ cell content poses a manufacturing challenge when the selection of the CD3+ cells in the apheresis product is performed with CD3/CD28 magnetic beads. The CD14+ cell threshold for successful manufacturing without depletion remains to be determined. However, monitoring the CD14 cell content and depleting these cells from starting apheresis products with high CD14 content prior to CD3/CD28 magnetic bead selection are critical to ensure success of the CAR T cell manufacturing process.

Abstract CT102: Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Chimeric antigen receptors (CARs) are artificial receptors for antigen that redirect antigen specificity, activate T cells and further enhance T cell function through their costimulatory component. Three groups, including our own, have reported objective tumor responses when infusing autologous T cells genetically modified with CD19-targeted CARs into patients with chronic lymphocytic leukemia (CLL), other indolent non-Hodgkin lymphomas (NHL) and, most dramatically in patients with relapsed or refractory B cell acute lymphoblastic leukemia (B-ALL). Here we report on 16 patients with relapsed or refractory B-ALL that we treated with autologous T cells expressing the 19-28z CAR specific to the CD19 antigen. The overall complete response rate was 88%, as assessed by morphological criteria and IgH deep sequencing. This allowed us to transition a majority of these patients to a standard of care allogeneic hematopoietic stem cell transplant (allo-SCT). This therapy was as effective in high-risk patients with Ph+ disease as in those with relapsed disease following prior allo-SCT. Through systematic analysis of clinical data and serum cytokine levels over the first 21 days following T cell infusion, we have defined diagnostic criteria for a severe cytokine release syndrome (sCRS), with the goal of better identifying the subset of patients who will likely require therapeutic intervention with corticosteroids or interleukin-6 receptor blockade to curb the CRS. Additionally, we found that serum C-reactive protein (CRP), a readily available laboratory study, can serve as a reliable indicator for the severity of the CRS and a road map for patient management at centers now contemplating the use of CAR T cell therapy. Based on these remarkably robust clinical results and the toxicity management algorithm we present here, we will soon open a multi-center Phase II clinical trial to further evaluate the efficacy of 19-28z CAR T cells and prospectively validate our proposed CRS monitoring and intervention guidelines in patients who are treated with CAR therapy. Citation Format: Michel Sadelain, Renier Brentjens, Marco Davila, Isabelle Riviere, Xiuyan Wang, Shirley Bartido, Jae Park, Diana Bouhassira, Kevin Curran, Stephen Chung, Jolanta Stefanski, Oriana Borquez-Ojeda, Sergio Giralt. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT102. doi:10.1158/1538-7445.AM2014-CT102