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CAR T-cell therapies have shown remarkable promise in treating patients (PTs) diagnosed with advanced B cell malignancies. CAR T-cell therapies are living drug characterized by robust proliferation and persistence. We reported previously that high T cell expansion ex vivo during clinical manufacturing (MFG) correlated with high in vivo expansion following infusion, which was also associated with favorable clinical outcomes in PTs receiving CAR T-cells to treat chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). There is no known single quantitative parameter can accurately predict high proliferative potential of the T-cells during the clinical MFG process. Therefore, we conducted multivariate analysis of historical data to investigate combinatorial signatures to predict high proliferative capacity during MFG. We pooled data from a total of 297 PTs (n=248 blood cancers, n=49 solid tumors) from 16 different clinical trials receiving autologous CAR T cell products manufactured at the University of Pennsylvania. We evaluated 70 variables including PT data, phenotype of starting material, and MFG performance to determine signatures that could predict with high accuracy when a population doubling level greater than 4 on day 9 of the clinical MFG process (PDL9>4) could be achieved. We performed logistic regression to assess 70 early variables to predict T cell expansion at least 5-7 days ahead of harvest. We successfully identified a 2-factor signature that predicts PDL9>4 with high sensitivity and specificity (S/S) above 79%. The model was validated from 48 available CAR T cell products that were not used for generating the model, resulting in S/S at least 79% and 67%, respectively. This information can be useful in implementing processes/mitigation strategies to increase proliferative capacity ex vivo and MFG success rate for the product itself, provide lead time with potential alternative therapy strategies for PT. Ongoing investigation is assessing the possibility of identifying a signature of factors prior to manufacture and predict clinical response at day 28. This work provides a methodology for further studies for assessing the impact of extrinsic/intrinsic factors on PTs' CAR T cell growth and efficacy and directing process development activities and decision algorithms in PT candidates for CAR T therapies. [ABSTRACT FROM AUTHOR]

With the goal of improving consistency and success of manufacturing (MFG), we analyzed the composition of apheresis products collected from patients (pts) enrolled in clinical trials of engineered T cell immunotherapies. We previously reported in a small cohort of NHL pts, that T cell cultures manufactured from fresh APH of challenging products comprised of 3:1 monocyte to lymphocytes, exhibited impaired proliferation. Microscopic observation of poorly expanding cell cultures revealed the phagocytosis of T cell stimulating anti-CD3/ anti-CD28 Dynabeads by large cells, possibly of monocyte/myeloid origin. Proliferative capacity of the T cells was rescued if the APH was cryopreserved/thawed prior to manufacture. Substantial reduction in MDSC frequency, following cryopreservation/thaw, was demonstrated by flow cytometry. Alternatively, lymphocyte enrichment via elutriation of APH removed the larger myeloid cells. Here we expand analysis of the critical quality attributes of APH products to a larger cohort of patients with hematological malignancies (HM) as well as solid tumors (ST). We analyzed 300 APH from pts on 20 clinical trials focused on HM or ST. Frequencies of lymphocytes and myeloid populations were determined via size/volume distribution on Multisizer, lineage and MDSC immunophenotyping by FACS (CD3, CD45, CD11b, CD33, HLA-DR, CD14, CD15). Population doubling levels (PDL) during ex vivo expansion were calculated at day 9 of manufacture. We report an overall 94% MFG success rate with 5 PDL. 12/19 MFG failures did not produce an infusible dose with -1.34 PDL and occurred in HM APH. APH of MFG fails showed the presence of myeloid cells that were not removed prior to MFG. APH from NHL, MM, and synovial sarcoma demonstrated MDSCs by flow cytometry. Cryopreservation/thaw and/or elutriation of APH enriched T cells from 81 % to 94%, reduced myeloid population from 38% to 11%, and removed MDSCs. Small scale test expansions comparing various MFG processes on the same APH confirmed that cryopreservation and or elutriation increases clinical MFG success. Myeloid subsets in HM APH increase the risk of MFG failure. Mitigation strategies to remove the myeloid cells to enrich for the lymphocytes can be employed, including cryopreservation or elutriation of fresh APH prior to MFG. To increase MFG feasibility, studies are continuing to investigate the effects of prior treatment on APH and the mechanism of inhibition by MDSCs. [ABSTRACT FROM AUTHOR]