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IN THE SPOTLIGHT

Wishing on a CAR: Understanding the Scope of Intrinsic T-cell Defi cits in Patients with Cancer Mark Leick and Marcela V. Maus

Cellular Immunotherapy Program, Massachusetts General Hospital Cancer Center, Boston, Massachusetts . Corresponding Author: Marcela V. Maus, Massachusetts General Hospital, Harvard Medical School, 13th Street, Building 149, Room 7.219, Charles-town, MA 02129. Phone: 617-726-5619; Fax: 617-726-5637; E-mail: mvmaus@mgh.harvard.edu doi: 10.1158/2159-8290.CD-19-0073 ©2019 American Association for Cancer Research.

Summary: Treatment with chimeric antigen receptor T cells has led to impressive and durable responses in adult and pediatric malignancies refractory to conventional therapy; however, only patients with a handful of cancers have responded thus far and signifi cant disparities exist between the response rates of pediatric and adult patients. A new extensive analysis of pediatric patient T-cell subsets at diagnosis and throughout the patients’ chemotherapy courses in a variety of solid and hematologic malignancies sheds new light on the intrinsic T-cell defi cits that may be partly to blame.

See related article by Das et al., p. 492 (9).

INTRODUCTION Two different formulations of chimeric antigen receptor

(CAR) T cells targeting the B-cell antigen CD19 have received FDA approvals in the last 2 years. Tisagenlecleucel (Kymriah) has approvals for both pediatric/young adult relapsed/refrac-tory acute lymphoblastic leukemia (ALL) as well as adult relapsed/refractory diffuse large B-cell lymphoma (DLBCL). Axicabtagene ciloleucel (Yescarta) has a single approval for relapsed/refractory DLBCL. Remarkably, despite insertion of the same transgene to patients’ T cells during the manufactur-ing of tisagenlecleucel, adult patients with DLBCL and pedi-atric patients with ALL have dramatically different durable complete response rates after at least 3 months of follow-up (29% vs. 82%, respectively; refs. 1, 2 ). Disease-specifi c response rates in DLBCL between tisagenlecleucel and axicabtagene ciloleucel are comparable among adult patients ( 3 ).

Impaired therapeutic effi cacy has been attributed to anti-gen escape, a tumor characteristic, as well as patient char-acteristics such as T-cell defi cits. Tumor antigen escape as a means of evading CAR-T therapy has been well characterized in patients with ALL treated with anti-CD19 CAR T cells. Leukemic blasts in up to 25% of pediatric patients may lose (or decrease) CD19 expression, rendering them resistant to CD19-targeted CAR-T–mediated killing ( 4 ).

The presence of early-lineage T cells (naïve and early mem-ory) prior to genetic engineering has been found to cor-relate with better expansion and to be enriched in patients with ALL compared with patients with non-Hodgkin lym-phoma (NHL; ref. 5 ). A similar population of naïve T cells (CD27 + CD45RO − CD8 + ) in the starting lymphocyte material of adult patients with chronic lymphocytic leukemia (CLL)

correlated with a favorable subsequent clinical response to tisagenlecleucel ( 6 ). Importantly, naïve T-cell numbers are known to decline with age, which could partially explain the decreased response rates in adults compared with children with regard to CAR-T therapy ( 7 ). Furthermore, intensive cytotoxic chemotherapy disproportionately depletes this key subset of naïve lymphocytes, which may ultimately become an important consideration for patients with cancer consid-ering adoptive T-cell therapy ( 8 ).

In this issue of Cancer Discovery , Das and colleagues perform an extensive analysis of T-cell fi tness and phenotyping in a large cohort of pediatric patients with cancer over time as they received progressive cycles of chemotherapy (9). The authors undertook the herculean effort of prospectively consenting and enrolling 195 newly diagnosed pediatric patients with cancer to the study, which involved serial blood draws at baseline and then after every cycle of chemotherapy (see Fig. 1 ). Each sam-ple was then analyzed at the single-cell level by fl ow cytom-etry for key T-cell subsets based on the expression of surface markers, and cultured individually in a functional expansion assay that measures T-cell growth in response to stimulation with beads coated with anti-CD3 and anti-CD28 antibodies. Importantly, this is essentially the same culture process that is used in the manufacturing of CAR T cells. A previously defi ned threshold of in vitro T-cell expansion that correlated with successful manufacturing of CAR T cells was used to split patients into three groups: those whose T-cell expansion would be a “pass,” “indeterminant,” or “fail.”

Das and colleagues found that patients with standard risk (SR) ALL had the highest initial “pass” rate at diagnosis (69%), which trended down with subsequent cycles of chemotherapy to less than 25% by cycle seven. Patients with lymphoma had lower pass rates at diagnosis (20%–30%), which also worsened over time. Patients with Ewing sarcoma had the worst pass rate (15%) at diagnosis before falling even further. Impor-tantly, expansion potential could not be inferred from the readily available clinical metric of absolute lymphocyte count.

Prior work from the authors had shown that coculture with IL7 and IL15 enriched early-lineage T cells and rescued T-cell expansion capacity from the chemotherapy-induced

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depletion among patients with NHL (5). Upon addition of these two cytokines to the bead expansion assay, the authors saw a universal increase in the percentage of stem central memory T cells in the final postexpansion samples (primarily correlating with a loss of terminal effector T cells). Reassur-ingly, no samples that initially “passed” without the addition of the cytokines “failed” when the cytokines were added. On immunophenotyping analysis, patients with SR ALL had the most naïve cells at diagnosis and this population never fell below 25% of the total T-cell population, whereas most other malignancies saw declines in this subset. Importantly, sur-face phenotyping was not sufficient to explain the variance in the functional expansion assay, suggesting some latent functional defects.

This work has important implications. With the increas-ing interest in developing and applying the CAR-T cell therapies to solid tumors, it is critical to consider the fit-ness of the T cells being used for the manufacture of CAR-T products, especially given the mounting evidence of a cor-relation with clinical outcomes. Studies like this may also provide insight into CAR-T therapy for adult malignancies like CLL, which are known to harbor inherent T-cell deficits. The authors highlight the important and unexpected find-ing of very different naïve T-cell populations between solid tumors and leukemias, remarking that, “With the available

evidence, it is not clear whether children who have these intrinsic T-cell differences are more at risk for solid tumors, or whether the tumors themselves are altering the T-cell compartment” (9).

One consideration for solid tumor and adult CAR-T applications is that many of the pediatric CAR-T patients have previously undergone allogeneic bone marrow trans-plantation as part of their treatment and thus had CAR-T products manufactured with a largely chemotherapy-naïve hematopoietic system. Ongoing efforts are under way to improve T-cell fitness via several strategies. One such option includes use of a PI3K inhibitor during manufacturing, which has been shown to preserve a less differentiated state (10). Alternatively, it may be possible to simultaneously give patients a drug that augments CAR-T cell function while receiving therapy. The Bruton tyrosine kinase inhibitor ibrutinib has been shown to enhance CAR-T engraftment, tumor clearance, proliferation, and survival as well as clini-cal response in patients with CLL (11). The use of allogeneic CAR-T cells manufactured from the blood of healthy donors may be another strategy to circumvent the “defective” T cells in patients with cancer. Some allogeneic CAR-T cells are cur-rently in clinical trials and have shown efficacy, but rejection in both the host and graft directions remains a concern. One final strategy involves simply shifting CAR-T cell therapy

Blood draws

Chemotherapycycles

A

B

SR-ALL, n = 40

SR-ALL, n = 40

Ewing sarcoman = 13

Ewing sarcoman = 13

Diagnosis Cycle 3 Cycle 5 Cycle 7/9∗

End of treatment

PassIndeterminate

Fail

NaïveStem memoryCentral memoryEffector memoryTerminal effector

Phenotype

Function

Figure 1.  Schematic of the study conducted by Das and colleagues (9). One hundred ninety-five patients with ten solid and hematologic malignancies underwent serial blood draws at diagnosis and prior to each cycle of chemotherapy. Selected results from patients with SR-ALL and Ewing sarcoma are shown. A, Results of T-cell subset phenotyping analysis via flow cytometry. B, Predicted manufacturing result based on the authors’ functional prolif-eration assay. *, Sample used for last column taken from the last cycle of chemotherapy, respectively (cycle seven for SR ALL and cycle nine for Ewing sarcoma).

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(or at least cell collection) to the first line, prior to receiving cytotoxic chemotherapy, to preserve lymphocyte integrity. This paradigm is commonly employed in patients with mul-tiple myeloma in which stem cells are collected and stored for a future autologous transplant prior to the receipt of (or early in the course of) immunomodulatory imides, which are known to impair stem-cell function. To this end, a clini-cal trial utilizing tisagenlecleucel in the up-front setting is planned (but not yet recruited) for pediatric and young adult patients with very high-risk B-cell acute lymphoblastic leukemia (NCT03792633). This trial will allow the collec-tion of T cells untouched by chemotherapy and hopefully obviate the need for successive rounds of chemotherapy and bone marrow transplantation in this high-risk population. The use of up-front T-cell therapy holds the promise of sparing these pediatric patients the long-term sequalae from intensive chemotherapy and bone marrow transplantation.

In summary, this study by Das and colleagues provides the largest and most comprehensive analysis to date of T-cell phenotype and functional fitness over time in the course of multiple malignancies in pediatric patients. This data will help inform the design and testing of CAR-T cells in both pediatric and adult populations across all malig-nancies.

Disclosure of Potential Conflicts of InterestM.V. Maus has received commercial research grants from Agen-

tus, Crispr Therapeutics, Kite Pharma, and TCR2; has ownership interest (including stock, patents, etc.) in Agentus; and is a consult-ant/advisory board for Adaptimmune, Agentus, Cellectis, Crispr Therapeutics, Kite Pharma, Novartis, Takeda, TCR2, Windmill, and Bluebird Bio. No potential conflicts of interest were disclosed by the other author.

Published online April 1, 2019.

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3. Locke FL, Ghobadi A, Jacobson CA, Miklos DB, Lekakis LJ, Oluwole OO, et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicen-tre, phase 1-2 trial. Lancet Oncol 2019;20:31–42.

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7. Shearer WT, Rosenblatt HM, Gelman RS, Oyomopito R, Plaeger S, Stiehm ER, et al. Lymphocyte subsets in healthy children from birth through 18 years of age: the pediatric AIDS clinical trials group P1009 study. J Aller Clin Immunol 2003;112:973–80.

8. Mackall C, Fleisher T, Brown M, Magrath I, Shad A, Horowitz M, et al. Lymphocyte depletion during treatment with intensive chemo-therapy for cancer. Blood 1994;84:2221–8.

9. Das RK, Vernau L, Grupp SA, Barrett DM. Naïve T-cell deficits at diagnosis and after chemotherapy impair cell therapy potential in pediatric cancers. Cancer Discov 2019;9:492–9.

10. Zheng W, O’Hear CE, Alli R, Basham JH, Abdelsamed HA, Palmer LE, et al. PI3K orchestration of the in vivo persistence of chimeric antigen receptor-modified T cells. Leukemia 2018;32:1157–67.

11. Fraietta JA, Beckwith KA, Patel PR, Ruella M, Zheng Z, Barrett DM, et al. Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia. Blood 2016;127:1117–27.

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