Malgorzata Olszewska

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.

Monitoring the Efficacy of Adoptively Transferred Prostate Cancer–Targeted Human T Lymphocytes with PET and Bioluminescence Imaging

Noninvasive imaging technologies have the potential to enhance the monitoring and improvement of adoptive therapy with tumor-targeted T lymphocytes. We established an imaging methodology for the assessment of spatial and temporal distributions of adoptively transferred genetically modified human T cells in vivo for treatment monitoring and prediction of tumor response in a systemic prostate cancer model. Methods: RM1 murine prostate carcinoma tumors transduced with human prostate-specific membrane antigen (hPSMA) and a Renilla luciferase reporter gene were established in SCID/beige mice. Human T lymphocytes were transduced with chimeric antigen receptors (CAR) specific for either hPSMA or human carcinoembryonic antigen (hCEA) and with a fusion reporter gene for herpes simplex virus type 1 thymidine kinase (HSV1tk) and green fluorescent protein, with or without click beetle red luciferase. The localization of adoptively transferred T cells in tumor-bearing mice was monitored with 2′-18F-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-ethyluracil (18F-FEAU) small-animal PET and bioluminescence imaging (BLI). Results: Cotransduction of CAR-expressing T cells with the reporter gene did not affect CAR-mediated cytotoxicity. BLI of Renilla and click beetle red luciferase expression enabled concurrent imaging of adoptively transferred T cells and systemic tumors in the same animal. hPSMA-specific T lymphocytes persisted longer than control hCEA-targeted T cells in lung hPSMA-positive tumors, as indicated by both PET and BLI. Precise quantification of T-cell distributions at tumor sites by PET revealed that delayed tumor progression was positively correlated with the levels of 18F-FEAU accumulation in tumor foci in treated animals. Conclusion: Quantitative noninvasive monitoring of genetically engineered human T lymphocytes by PET provides spatial and temporal information on T-cell trafficking and persistence. PET may be useful for predicting tumor response and for guiding adoptive T-cell therapy.

Large-Scale Clinical-Grade Retroviral Vector Production in a Fixed-Bed Bioreactor

The successful genetic engineering of patient T cells with &ggr;-retroviral vectors expressing chimeric antigen receptors or T-cell receptors for phase II clinical trials and beyond requires the large-scale manufacture of high-titer vector stocks. The production of retroviral vectors from stable packaging cell lines using roller bottles or 10- to 40-layer cell factories is limited by a narrow harvest window, labor intensity, open-system operations, and the requirement for significant incubator space. To circumvent these shortcomings, we optimized the production of vector stocks in a disposable fixed-bed bioreactor using good manufacturing practice–grade packaging cell lines. High-titer vector stocks were harvested over 10 days, representing a much broader harvest window than the 3-day harvest afforded by cell factories. For PG13 and 293Vec packaging cells, the average vector titer and the vector stocks’ yield in the bioreactor were higher by 3.2- to 7.3-fold, and 5.6- to 13.1-fold, respectively, than those obtained in cell factories. The vector production was 10.4 and 18.6 times more efficient than in cell factories for PG13 and 293Vec cells, respectively. Furthermore, the vectors produced from the fixed-bed bioreactors passed the release test assays for clinical applications. Therefore, a single vector lot derived from 293Vec is suitable to transduce up to 500 patients cell doses in the context of large clinical trials using chimeric antigen receptors or T-cell receptors. These findings demonstrate for the first time that a robust fixed-bed bioreactor process can be used to produce &ggr;-retroviral vector stocks scalable up to the commercialization phase.

Efficient human hematopoietic cell transduction using RD114- and GALV-pseudotyped retroviral vectors produced in suspension and serum-free media.

Retroviral vectors derived from the Moloney murine leukemia virus have been used in successful and promising gene therapy clinical trials. However, platforms for their large-scale production must be further developed. As a proof of principle, we reported the generation of a packaging cell line that produces amphotropic retroviral vectors in suspension and serum-free medium (SFM). In the present study, we have constructed and characterized two retroviral packaging cell lines designed for gene transfer in hematopoietic cells. These cell lines grow in suspension and SFM, and produce high-titer RD114- and gibbon ape leukemia virus (GALV)-pseudotyped vectors for a 3-month culture period. Viral particles released are as robust during repeated freeze-thaw cycles and on thermal inactivation at 37 degrees C as their counterparts produced in cells cultured adherently with serum. We also show that RD114- and GALV-pseudotyped vectors produced in suspension and SFM efficiently transduce human lymphocytes and hematopoietic stem cells. As these retroviral packaging cell lines distinctively maintain high vector titers while growing in suspension and SFM, we conclude that these cell lines are uniquely suitable for large-scale clinical-grade vector production for late-phase clinical trials involving gene transfer into hematopoietic cells.

A New Acycloguanosine-Specific Supermutant of Herpes Simplex Virus Type 1 Thymidine Kinase Suitable for PET Imaging and Suicide Gene Therapy for Potential Use in Patients Treated with Pyrimidine-Based Cytotoxic Drugs

The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene is widely used as a suicide gene in combination with ganciclovir (GCV) and as a nuclear imaging reporter gene with an appropriate reporter probe. Wild-type HSV1-tk recognizes a variety of pyrimidine and acycloguanosine nucleoside analogs, including clinically used antiviral drugs. PET of HSV1-tk reporter gene expression will be compromised in patients receiving nucleoside-based antiviral treatment. With the use of an acycloguanosine-specific mutant of the enzyme, PET of HSV1-tk reporter gene expression can be successfully performed with acycloguanosine-based radiotracers without interference from pyrimidine-based antiviral drugs. Methods: The levels of expression of wild-type HSV1-tk and HSV1-A167Ytk, HSV1-sr39tk, and HSV1-A167Ysr39tk mutants fused with green fluorescent protein (GFP) and transduced into U87 cells were normalized to the mean fluorescence of GFP measured by fluorescence-activated cell sorting. The levels of enzymatic activities of wild-type HSV1-tk and its mutants were compared by 2-h in vitro radiotracer uptake assays with 3H-2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-ethyluracil (3H-FEAU), 3H-pencyclovir (3H-PCV), and 3H-GCV and by drug sensitivity assays. PET with 18F-FEAU and 18F-9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (18F-FHBG) was performed in mice with established subcutaneous tumors, expressing wild-type HSV1-tk and its mutants, followed by tissue sampling. Results: FEAU accumulation was not detected in HSV1-A167Ysr39tk–expressing cells and xenografts. Lack of conversion of pyrimidine derivatives by the HSV1-A167Ysr39tk supermutant was also confirmed by a drug sensitivity assay, in which the 50% inhibitory concentrations for thymine 1-β-d-arabinofuranoside and bromovinyldeoxyuridine were found to be similar to those in nontransduced cells. In contrast, we found that HSV1-A167Ysr39tk could readily phosphorylate 3H-GCV at levels similar to those of wild-type HSV1-tk and HSV1-A167Ytk but showed enhanced activity with 3H-PCV in vitro and with 18F-FHBG in vivo. Conclusion: We developed a new reporter gene, HSV1-A167Ysr39tk, which exhibits specificity and high phosphorylation activity for acycloguanosine derivatives. The resulting supermutant can be used for PET with 18F-FHBG and suicidal gene therapy protocols with GCV in patients treated with pyrimidine-based cytotoxic drugs.