The use of chimeric antigen receptor (CAR)-modified T cells as a therapy for hematologic malignancies and solid tumors is becoming more widespread. However, the infusion of a T-cell product targeting a single tumor-associated antigen may lead to target antigen modulation under this selective pressure, with subsequent tumor immune escape. With the purpose of preventing this phenomenon, we have studied the impact of simultaneously targeting two distinct antigens present on tumor cells: namely mucin 1 and prostate stem cell antigen, both of which are expressed in a variety of solid tumors, including pancreatic and prostate cancer. When used individually, CAR T cells directed against either tumor antigen were able to kill target-expressing cancer cells, but tumor heterogeneity led to immune escape. As a combination therapy, we demonstrate superior antitumor effects using both CARs simultaneously, but this was nevertheless insufficient to achieve a complete response. To understand the mechanism of escape, we studied the kinetics of T-cell killing and found that the magnitude of tumor destruction depended not only on the presence of target antigens but also on the intensity of expression-a feature that could be altered by administering epigenetic modulators that upregulated target expression and enhanced CAR T-cell potency.
Broader implementation of cell-based therapies has been hindered by the logistics associated with the expansion of clinically relevant cell numbers ex vivo. To overcome this limitation, Wilson Wolf Manufacturing developed the G-Rex, a cell culture flask with a gas-permeable membrane at the base that supports large media volumes without compromising gas exchange. Although this culture platform has recently gained traction with the scientific community due to its superior performance when compared with traditional culture systems, the limits of this technology have yet to be explored. In this study, we investigated multiple variables including optimal seeding density and media volume, as well as maximum cell output per unit of surface area. Additionally, we have identified a novel means of estimating culture growth kinetics. All of these parameters were subsequently integrated into a novel G-Rex “M” series, which can accommodate these optimal conditions. A multicenter study confirmed that this fully optimized cell culture system can reliably produce a 100-fold cell expansion in only 10 days using 1L of medium. The G-Rex M series is linearly scalable and adaptable as a closed system, allowing an easy translation of preclinical protocols into the good manufacturing practice.
Background: Umbilical cord blood transplants (UCB) have greatly improved alternative donor options, but have been historically limited by higher rates of primary graft failure, delayed immune reconstitution, higher treatment-related mortality, and finite cell doses. Prior to infusion, UCB grafts require thawing and washing using manual (Rubinstein) or automated methods. Our institution has validated the use of Sepax for automated UCB graft processing with ex vivo viability of > 94% up to 24 hours after processing. For quality assurance and improvement, we are now investigating the clinical outcomes of Sepax and non-Sepax UCB transplants at our center. Objective: Compare engraftment outcomes for Sepax and non-Sepax processed umbilical cord blood transplants. Methods: We retrospectively reviewed clinical outcomes of UCB transplants at our center from 2000-2015. Data collected for initial analyses were service (pediatric or adult), diagnosis, presence of neutrophil engraftment (ANC >500 x 3 consecutive days), and number of cases of primary graft failure (Sepax or Non-Sepax). Data collected for future analyses included transplant characteristics, time to neutrophil and platelet (>20 K x 7 consecutive days), UCB infusion reactions, presence of pre/engraftment syndrome, acute and chronic GVHD, day 100, 180 and 365 mortality with causes of death, estimated cost of Sepax and non-Sepax processing, viability (Sepax and non-Sepax), and infused total nucleated cell dose/kg recipient (Sepax and non-Sepax). Fisher's exact test was used to compare groups. Results: Between 1/1/2000 and 9/3/2015, our center performed a total of 349 UCB transplants (283 pediatric, 66 adult) with cumulative incidence (CI) of primary graft failure (PGF) of 15.5% in pediatrics (n ¼ 44) and 22.7% in adults (n¼16). Between 1/1/2000 and 7/30/2014 (177.5 months), 335 UCB transplants were done using non-Sepax manual processing with CI of PGF of 17.3% (n ¼ 44 of 264 pediatric, n¼ 14 of 63 adult). From 8/1/2014-9/3/2015 (13.7 months), 14 cord blood transplants were done using Sepax processing with CI of PGF of 7.1% (n¼ 0 of 11 pediatric; n¼ 1 of 3 adult). There were no statistically significant differences between engraftment and PGF for Sepax and non-Sepax processed cords for entire group (P ¼ 0.48), pediatric recipients (P¼ 0.37), or adult recipients (P¼ 0.57). Conclusion: Sepax (automated) cord blood processing results are equivalent to standard manual methods, with the advantage of product viability of >94% at 24 hours postprocessing. This technology has been adopted as our standard cord blood processing method in our cellular therapy lab. Our clinical observations suggest shortened time to neutrophil engraftment using Sepax processing, but data is limited for analysis at the present time. Future analyses will fully evaluate Sepax and non-Sepax based engraftment kinetics and transplant-related complications.
While adoptive transfer of cytotoxic T lymphocytes (CTLs) engrafted with chimeric antigen receptors (CAR) has produced objective clinical responses in vivo, the infused cells usually fail to persist long-term, limiting benefit. We have recently demonstrated that CTL persistence can be improved by engineering cells to express the IL-7 receptor alpha chain (IL-7R) which is physiologically absent on CTLs. However, this approach requires access to clinical grade cytokine and biodistribution to T lymphocytes at tumor sites may be insufficient. To circumvent these problems we have prepared two CTL products: one expressing a tumor-specific CAR in combination with IL-7R (product #1), and the second engineered to co-express the same CAR and produce IL-7 cytokine (product #2). In this way, both products have anti-tumor activity mediated through the CAR, while cytokine produced from CTL#2 should support the survival and persistence of the IL-7R-expressing CTL#1. A binary system such as this should be intrinsically safer than incorporating a positive feedback loop of both cytokine and receptor in a single cell. As a proof of this principle, we used the SFG-CAR that targets the ≤ light chain expressed on B cell malignancies: SFG-CAR/IL-7R-GFP(#1) and SFG-CAR/IL-7cyto-mOrange(#2). EBV-CTLs from 3 donors were transduced with each vector. FACS analysis of transgene expression indicated that all were expressed at approximately equivalent levels: CTL#1 (CAR, IL-7Rα, and GFP; 58%±15, 53%±18, 57.8%±12) and CTL#2 (CAR and mOrange; 54%±18 and 52%±20). The modified CTL were functional, and cells transduced with either vector were able to kill the α+ B cell tumors Daudi as evaluated by Cr51 assay (72%±13 and 69%±25, respectively) at an R:S of 40:1. Preliminary data indicates that CTLs#1 were able to proliferate in response to exogenous IL-7 administration, and IL-7 cytokine production from CTLs#2 was directly proportional to the antigenic stimulation provided to the cells. In addition, when CTLs#1 and #2 were cultured together we observed that CTLs#2 were capable of promoting the expansion of their CTL counterpart in a more efficient way than that compared to exogenous administration of IL-7 or IL-2. These results indicate that binary control of CAR modified CTLs is possible through transgenic expression of IL-7R and IL-7 cytokine in two different CTL populations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3501. doi:1538-7445.AM2012-3501
Objective: We have previously reported that an HLA 8 of 8 allele-matched unrelated donor (8/8 MUD) is superior to a related donor with HLA-1 antigen mismatch in the graftversus-host (GVH) direction (RD/1AG-MM-GVH) in transplantation for leukemia (Kanda J, et al. Blood 2012). However, the risk of relapse during the unrelated donor coordination period biases this comparison. Therefore, we performed decision analysis of donor selection in allogeneic stem cell transplantation for acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) in first remission (CR1); this method can consider various factors including risk of relapse during the donor coordination period and the decrease in quality of life (QOL) as a result of chronic graft-versus-host disease (GVHD). Methods: The incidences of relapse during the coordination period of 8/8MUD or RD/1AG-MM-GVH were estimated using the data from published studies on chemotherapeutic treatment for AML and ALL. Transition probabilities after transplantation were estimated using the database of the Transplant Registry Unified Management Program for the Japan Society for Hematopoietic Cell Transplantation. The expected 5-year survival probabilities with or without QOL adjustments were estimated using TreeAgePro software. One-way sensitivity analysis was performed by varying each transition-probability value within its plausible range. Results: In transplantation for AML-CR1, the expected 5-year survival probability was higher on selection of 8/8MUD than RD/1AG-MM-GVH (59% vs. 47%), and this superiority remained unchanged by sensitivity analysis of various factors, including the interval between achievement of CR1 and actually receiving transplantation. In transplantation for ALL-CR1, the 5-year survival probability was higher on selection of 8/8 MUD (48% vs. 43%). In one-way sensitivity analysis, the 5-year survival probability was higher on selection of RD/1AG-MM-GVH when the interval between CR1 and 8/8 MUD transplantation was !7 months. However, 8/8 MUD was superior after QOL adjustments. If the 5-year survival rate was increased by 3% (7% after QOL adjustment) in transplantation using RD/1AG-MM-GVH, the merit of selecting RD/1AG-MM-GVH outweighs that of 8/8 MUD. Conclusions: 8/8 MUD should be prioritized in transplantation for AML-CR1. In transplantation for ALL-CR1, RD/1AG-MM-GVH should be prioritized only when the interval between CR1 and 8/8 MUD transplantation is expected to be long. However, MUD should be prioritized if QOL is considered.
348 Chimeric antigen receptors (CARs) are artificial molecules that can be used to redirect T cell immune response against antigens expressed on the surface of tumor cells. Recent encouraging clinical data from our group and others has shown that T cells engineered with these molecules can effectively traffic to distant tumor sites, penetrate even bulky disease, and eradicate disseminated tumors. Although promising, most current protocols expand engineered T cells non-specifically using IL2 and OKT3, which often results in a decrease in the frequency of transgenic populations over time. Additionally, cell expansion using conventional cultureware is complicated and labor intensive, which limits the broader application of this therapy. With the purpose of optimizing and streamlining CAR-T cell manufacture, we assessed whether cell expansion could be improved by: (i) supplementing non-specific stimuli (IL2) with an artificial antigen presenting cell (a-APC) engineered to express cognate antigen and co-stimulatory molecules, and (ii) efficiently and rapidly expanding cells in a simple and scalable gas permeable culture device (G-Rex), developed by Wilson Wolf Manufacturing for expanding suspension cells. As a proof of principle, we sought to expand T cells engineered with a CAR targeting the prostate cancer antigen, PSCA. We first generated an antigen-expressing a-APC cell line by modifying K562 cells, which already expressed a range of co-stimulatory molecules including CD80, CD86, and 41BBL, with a retroviral vector encoding the PSCA antigen. After the co-culture of CAR-PSCA T cells with the irradiated a-APC, we found that a-APCs co-expressing PSCA antigen, CD80, and 41BBL were the most effective in inducing T cell expansion, with a 1.9 fold increase in total cell numbers when compared with CAR T cells expanded in the presence of IL2 alone. We also saw an increase in the frequency of transgenic CAR-modified T cells in cultures expanded in the presence of a-APCs co-expressing PSCA antigen, CD80, and 41BBL, which increased from 36.5% CAR-modified cells to 88.1% after 10 days of culture. In contrast, the percentage of transgenic T cells was sustained when culture in the presence of IL2 (36.5% on day 0 and 37.2% on day 10). Thus, culture of CAR-T cells with antigen-expressing a-APCs not only improves total cell output, but also enriches for transgene-expressing. Next, to assess whether we could scale up cell production for clinical application we transferred the engineered a-APCs and CAR-PSCA modified T cells (at a 2:1 ratio) into a static GMP-compliant G-Rex with a surface area of 100cm2. In these G-Rex devices, O2 and CO2 are exchanged across a silicone membrane at the base, which allows for the addition of an increased depth of medium above the cells, providing more nutrients while the waste products are diluted. These culture conditions have been shown to increase cell output when compared with conventional commercial products such as bags, flasks, and 24-well tissue culture plates, without increasing the number of cell doublings. From an initial seeding density of 25E+06 CAR-modified T cells (0.25E+06 cells per cm2), we obtained a total of 2200–2500E+06 cells (22-25E+06 T cells per cm2) within 10 days of culture. Thus, without any intervention we obtained a 93 fold increase in cell numbers using only 1 liter of T cell culture media. As expected, the co-culture of antigen-expressing a-APCs with CAR-T cells also resulted in an enrichment of transgenic T cells (from 33.2% to 81.7% after 10 days of culture). Thus, we achieved a 2.4±1.2 fold increase in the frequency of transgenic T cells. Taken together the total T cell fold expansion (93) and the enrichment for the transgene (2.4±1.2), we calculate a 223.5±111.6 fold expansion of CAR T cells with 10 days of culture. Importantly we demonstrated the robustness of this manufacture process by successfully extending this approach to other CAR T cell products. Disclosures: Vera: Wilson Wolf Manufacturing: Consultancy.
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