Identification and Validation of Predictive Biomarkers to CD19- and BCMA-Specific CAR T-Cell Responses in CAR T-Cell Precursors
Authors: Wang Meng , Pruteanu Iulian , Cohen Adam D. , Garfall Alfred L , Milone Michael C. , Tian Lifeng , Gonzalez Vanessa E. , Gill Saar , Frey Noelle V. , Barrett David M. , Ruella Marco , Lacey Simon F , Svoboda Jakub , Chong Elise A. , Fraietta Joseph A , Davis Megan , Nasta Sunita D , Levine Bruce L , Siegel Don L. , Maude Shannon L. , Schuster Stephen J. , Stadtmauer Edward A. , Grupp Stephan , Porter David L. , June Carl H , Melenhorst J. Joseph 
Published: February 2020

CD19-specific chimeric antigen receptor (CAR) T cell therapies have been highly effective against B cell malignancies. We previously demonstrated that differential responses to anti-CD19 CAR T cell therapy in chronic lymphocytic leukemia (CLL) are associated with early memory T cell signature in apheresed, pre-manufacturing T-cells (CAR T-cell precursors). We tested the hypothesis that the composition of CAR-T precursor cells determines clinical efficacy in adult and pediatric Acute Lymphoblastic Leukemia (ALL), Non-Hodgkin's Lymphoma (NHL), Multiple Myeloma (MM), and CLL. Apheresed T cells were engineered to express 4-1BB plus CD3-zeta-signaling CARs targeting CD19, or B cell maturation antigen (BCMA). The same 9-day manufacturing process was used for all trials. CAR T cell kinetics were monitored using a CAR gene-specific quantitative PCR assay and standard clinical response assessments were performed.
Apheresed T cells from 36 CLL, 30 adult ALL, 58 pediatric ALL, 33 NHL, and 25 MM patients were immunophenotyped by flow cytometry. The CLL cohort was used to discover phenotypically distinct subpopulations associated with the two main response groups; these associations were validated in the remaining patient cohorts. Eight CD8+ T cell populations or clusters were identified using the shared-nearest-neighbor clustering method (PMID: 31178118) in the CLL cohort. T cell subsets exhibiting naive (cluster 6) or early memory (cluster 4) features were significantly enriched in responding patients, whereas an effector memory CD8 subpopulation (cluster 2) marked the non-responding patients. Mapping these clusters onto apheresed CD8+ T cells from the other four diseases showed that cluster 4 predicted response to CAR T cell therapy in NHL and myeloma but not in adult and pediatric ALL. We also examined the expression of activation-regulated molecules including HLA-DR, Ki67, and exhaustion-related molecules PD1, CTLA4, TIM3, and LAG3. A CD27+ CD8+ population expressing low level CTLA4 but none of the activation or negative regulatory molecules was significantly enriched in responding CLL patients; this cluster validated in NHL and myeloma.
A similar analysis on apheresed CD4+ T cells identified an early memory population (cluster 6) enriched in CLL responders, which expresses CCR7 and CD27 but not CD45RO, CD127, CD28, or other late memory/effector molecules. However, this population did not validate in any of the other diseases. Though not statistically significant, the CD4+ clusters with the largest effect size for enrichment in responders from NHL and myeloma trials exhibited early memory T cell features and lack of HLA-DR expression, suggesting that quiescent early memory state in CD4 may also be associated with clinical responses. A separate analysis of checkpoint inhibitory receptors and activation markers in memory CD4 T cell subsets confirmed the early memory, non-activated state of this population in CLL and was validated in myeloma but none of the other diseases. In vivo activation was a shared theme in CD4+ T cells for non-responding patients as well, though these CLL-defined CD4+ apheresed T cells clusters did not significantly validate in other diseases.
In summary, our data confirm and extend our predictive biomarker profile in CLL to mature B cell and plasma cell malignancies by showing that a non-cycling, non-activated early memory CD8+ T cell population in pre-manufacturing cells was validated as a biomarker in myeloma, and NHL. We also showed that responder-associated apheresed CD4+ T cells with early memory features identified in CLL after CD19 CAR T infusions are validated in myeloma after BCMA CAR T. Thus, differentiation state and in vivo activation, and potentially exhaustion, separate response groups. Our findings inform next-generation CAR T-cell manufacturing using the populations identified herein as a starting population.

Disclosures
Pruteanu: Novartis: Employment. Cohen:Poseida Therapeutics, Inc.: Research Funding. Garfall:Surface Oncology: Consultancy; Novartis: Research Funding; Janssen: Research Funding; Amgen: Research Funding; Tmunity: Research Funding. Milone:Novartis: Patents & Royalties: patents related to tisagenlecleucel (CTL019) and CART-BCMA; Novartis: Research Funding. Gill:Novartis: Research Funding; Tmunity: Research Funding; Carisma: Equity Ownership, Research Funding; Sensei: Consultancy; Aro: Consultancy; Fate: Consultancy. Frey:Novartis: Research Funding. Ruella:Nanostring: Consultancy, Speakers Bureau; Novartis: Patents & Royalties: CART for cancer; AbClon: Membership on an entity's Board of Directors or advisory committees. Lacey:Novartis: Patents & Royalties: Patents related to CAR T cell biomarkers; Tmunity: Research Funding; Novartis: Research Funding. Svoboda:Merck: Research Funding; BMS: Consultancy, Research Funding; Incyte: Research Funding; Pharmacyclics: Consultancy, Research Funding; Celgene: Research Funding; Kite: Consultancy; Seattle Genetics: Consultancy, Research Funding; Kyowa: Consultancy; AstraZeneca: Consultancy. Chong:Tessa: Consultancy; Novartis: Consultancy; Merck: Research Funding. Fraietta:LEK Consulting: Consultancy; Cabaletta: Research Funding; Tmunity: Research Funding. Davis:Cabaletta: Research Funding; Tmunity: Research Funding. Nasta:Rafael: Research Funding; Aileron: Research Funding; Takeda/Millennium: Research Funding; Incyte: Research Funding; Roche/Genentech: Research Funding; Merck: Consultancy; Atara: Research Funding; Debiopharm: Research Funding. Levine:CRC Oncology: Consultancy; Vycellix: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership; Novartis: Consultancy, Patents & Royalties, Research Funding; Cure Genetics: Consultancy; Avectas: Membership on an entity's Board of Directors or advisory committees; Brammer Bio: Membership on an entity's Board of Directors or advisory committees; Incysus: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy. Maude:Kite: Consultancy; Novartis: Consultancy. Schuster:Nordic Nanovector: Honoraria; Pfizer: Honoraria; AstraZeneca: Honoraria; Pharmacyclics: Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Loxo Oncology: Honoraria; Merck: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; Novartis: Honoraria, Patents & Royalties: Combination Therapies of CAR and PD-1 Inhibitors with royalties paid to Novartis, Research Funding; AbbVie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding. Stadtmauer:Celgene: Consultancy; Tmunity: Research Funding; Novartis: Consultancy, Research Funding; Takeda: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Abbvie: Research Funding. Grupp:Novartis: Consultancy, Research Funding; Roche: Consultancy; GSK: Consultancy; Cure Genetics: Consultancy; Humanigen: Consultancy; CBMG: Consultancy; Novartis: Research Funding; Kite: Research Funding; Servier: Research Funding; Jazz: Other: study steering committees or scientific advisory boards; Adaptimmune: Other: study steering committees or scientific advisory boards. Porter:Incyte: Membership on an entity's Board of Directors or advisory committees; American Board of Internal Medicine: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Glenmark Pharm: Membership on an entity's Board of Directors or advisory committees; Immunovative: Membership on an entity's Board of Directors or advisory committees; Genentech: Employment; Wiley and Sons: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. June:Novartis: Research Funding; Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties. Melenhorst:Novartis: Research Funding, Speakers Bureau; Parker Institute for Cancer Immunotherapy: Research Funding; Stand Up to Cancer: Research Funding; Incyte: Research Funding; IASO Biotherapeutics, Co: Consultancy; Simcere of America, Inc: Consultancy; Shanghai Unicar Therapy, Co: Consultancy; Colorado Clinical and Translational Sciences Institute: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; National Institutes of Health: Research Funding.