Pradip Bajgain

Kinetics of Tumor Destruction by Chimeric Antigen Receptor-modified T Cells

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.

Fine-tuning the CAR spacer improves T-cell potency

ABSTRACT The adoptive transfer of genetically engineered T cells expressing chimeric antigen receptors (CARs) has emerged as a transformative cancer therapy with curative potential, precipitating a wave of preclinical and clinical studies in academic centers and the private sector. Indeed, significant effort has been devoted to improving clinical benefit by incorporating accessory genes/CAR endodomains designed to enhance cellular migration, promote in vivo expansion/persistence or enhance safety by genetic programming to enable the recognition of a tumor signature. However, our efforts centered on exploring whether CAR T-cell potency could be enhanced by modifying pre-existing CAR components. We now demonstrate how molecular refinements to the CAR spacer can impact multiple biological processes including tonic signaling, cell aging, tumor localization, and antigen recognition, culminating in superior in vivo antitumor activity.

Optimizing the production of suspension cells using the G-Rex "M" series

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.

190. Adaptive CAR T Cell Design

Chimeric antigen receptor (CAR) T cell therapy has recently emerged as an attractive approach for the treatment of CD19-expressing hematological malignancies. However extending the success of this strategy to other targets has proven to be more complicated that simply replacing the scFv. To address this issue we have implemented a form of adaptive CAR design whereby a series of sequential modifications are made to a single domain and subsequently tested in vitro and in vivo to assess activity. We illustrate the utility of such a strategy using our published CAR-PSCA (Anurathapan U. et al, Mol Ther. 2014) as a template (CAR-PSCA v1.0) with subsequent iterations coded as versions 2.0, 3.0, 4.0 and 5.0. We now demonstrate how modifications made to a single CAR structural domain can result in enhanced (i) T cell migration, (ii) antigen recognition, and (iii) cell phenotype, ultimately producing superior anti-tumor effects. First, with CAR v2.0 and v3.0 we were able to improve T cell migration, which was evident in NSG mice engrafted s.c. with Capan-1 and treated i.v. with FFluc+ T cells. Ten days post CAR administration we saw a 2 log increase in the T cell signal at the tumor site (4.5±2.3×105 p/s vs 4.8±0.5×107 p/s vs 4.0±1.1×107 p/s, CAR v1.0, v2.0 and v3.0 respectively). Subsequently to enhance in vivo T cell persistence, we next generated CAR v4.0, which resulted in a less differentiated T cell phenotype (Tnaive: 1.8±0.6% to 19.2±4.0%, TCM: 10.4±1.4% to 14.1±3.0%, TEM: 83.5±1.2% to 53.7±6.9% and TEMRA: 4.3±0.9% to 12.9±1.7% - CAR v3.0 and CAR v4.0, respectively). When administered to Capan-1-engrafted NSG mice CAR v4.0 T cells exhibited enhanced in vivo longevity as measured using bioluminescence imaging (7.3±4.6×107 p/s CAR v3.0 vs 2.8±1.7×108 p/s CAR v4.0 T cells - day 35 post-administration). Finally, antigen recognition of CAR-PSCA was further improved in v5.0 where a final modification to the same domain produced superior anti-tumor effects against a PSCA-dim target tumor cell line (DU145) in a 6hr 51Cr-release assay (20.7±5.8% vs 48.4±5.2%, CAR v4.0 vs CAR v5.0, 40:1 E:T). Overall, therefore, implementation of this adaptive design produced a CART cell product with enhanced in vivo anti-tumor activity. This was clearly illustrated when we compared the tumor volume of NSG mice treated with CAR v1.0 or CAR v5.0 T cells (1309±143 mm3 vs 510±53 mm3 on Day 66). Specific details of the modifications conducted in this adaptive CAR design will be presented.

451. Robust Manufacture of CAR-T Cells

Although adoptive transfer of chimeric antigen receptor (CAR)-modified T cells has produced promising clinical responses, the broader application of this therapy has been hindered by prolonged and complicated cell production methods. In the current work, we have overcome this limitation through the incorporation of a gas permeable culture device (G-Rex) to support T cell expansion. This culture system consists of a suite of devices, all of which contain a gas permeable silicone membrane, which allows gas exchange to occur at the base. This configuration allows for the culture of T cells with an unconventionally large volume of media per unit of surface area (10ml of media/cm2), thereby supporting uninterrupted cell growth without media exchange. Importantly, this system is simple to use and can be placed in a regular incubator as the G-Rex does not require active agitation or perfusion. To evaluate the utility of this system for the expansion of CAR T cells, we transduced healthy donor-derived primary T cells with a CAR targeting the prostate cancer antigen - PSCA (previously generated and characterized by our group). Three days after retroviral transduction, transgenic CAR T cells (transduction efficiency of 83.6±6%) from 3 donors were transferred to G-Rex100M devices (surface area of 100cm2) in 1000ml of complete T cell media (10ml/cm2) at low (total of 25E+06 CAR T cells), intermediate (50E+06 CAR T cells) and high (100E+06 CAR T cells) cell densities (250E+03 cells/cm2, 500E+03 cells/cm2 and 1000E+03 cells/cm2, respectively). Subsequently, the cell cultures were monitored by glucose and lactic acid assessment. After 10 days of culture, the average fold-expansion was similar for all conditions (24.3±10.4, 35.5±7.8, 29.8±2.1 for low, intermediate, and high cell densities, respectively). Interestingly though, the donor-to-donor variability was decreased significantly at the higher cell density (SD of 10.4, 7.8, and 2.1 for cell densities of 250E+03 cells/cm2, 500E+03 cells/cm2 and 1000E+03 cells/cm2, respectively), highlighting the importance of identifying the optimal seeding density to support robust manufacture. Notably, no media replenishment was required and the only culture manipulation performed was the addition of IL2 (50U/ml) 3 times/week. Importantly, T cells manufactured using this optimized method expressed higher levels of central memory and activation markers (CD62L and CD25) and demonstrated superior anti-tumor activity when compared to cells maintained in conventional tissue-culture plates. To further simplify the manufacturing process, we have now developed a semi-automated, closed system (GatheRex) for cell collection, which can be paired with G-Rex and allows collection of cells in a small volume (100ml) in under five minutes.

751. Improving CAR T Cell Function by Reversing the Immunosuppressive Tumor Environment of Pancreatic Cancer

Adoptive transfer of T cells redirected to tumor-associated antigens (TAAs) by expression of chimeric antigen receptors (CARs) can produce tumor responses, even in patients with resistant malignancies. To target pancreatic ductal adenocarcinoma (PDAC), we generated T cells expressing a CAR directed to the TAA prostate stem cell antigen (PSCA). T cells expressing this CAR were able to kill PSCA(+) tumor cell lines CAPAN1 and K562-PSCA but not PSCA(-)293T cells (74±4%, 73±6% and 9±3% specific lysis, respectively, 10:1 E:T, n=3). Although these CAR-T cells had potent anti-tumor activity, pancreatic tumors employ immune evasion strategies such as the production of inhibitory cytokines, which limit in vivo CAR-T cell persistence and effector function. Indeed, when we examined the serum of patients with pancreatic cancer (n=8) we found the levels of the immunosuppressive cytokine IL4 to be elevated relative to patients with benign pancreatic disorders or normal healthy controls (14.25±19.48 pg/mL vs 7.28±9.03 vs 1.13±1.42 pg/mL). Thus, to protect our CAR-PSCA T cells from the negative influences of IL-4, we generated a chimeric cytokine receptor in which the IL4 receptor exodomain was fused to the IL7 receptor endodomain (IL4/7 ChR). Transgenic expression of this molecule in CAR-PSCA T cells can invert the inhibitory effects of tumor-derived IL4 to instead promote the proliferation of the effector CAR T cells. In preliminary experiments, we successfully co-expressed both CAR-PSCA and IL4/7 ChR (47.5±12.3% double-positive cells, n=4) on primary T cells. These T cells retained their tumor-specific activity (80±8% specific lysis against CAPAN1, 10:1 E:T, n=3) and when cultured in conditions that mimic the tumor milieu (IL4 12.5 ng/ml), CAR-PSCA 4/7R ChR-modified T cells continued to expand (increase from 2×10e6 cells on day 0 to 5.53±8.46×10e10 cells on day 28), unlike unmodified CAR-PSCA T cells which plateaued at 3.84±5.43×10e8 cells (n=4). Indeed, in the presence of IL4, transgenic cells had a selective advantage (comprising 44.8±11.0% of the population on day 0 and 87.6±10.0% on day 28; n=4). However, even after prolonged cytokine exposure these T cells remained both antigen- and cytokine-dependent. In conclusion, CAR-PSCA 4/7 ChR-modified tumor-specific T cells can effectively target pancreatic cancer cells and are equipped to expand, persist, and retain their cytotoxic function even in the presence of high levels of IL4 in the tumor microenvironment.

518. Artificial Mouse Model: An Animal-Free System for Assessment of CAR-T Cell Function

Although numerous studies have sought to better understand tumor:T cell interactions, their experimental systems have primarily been restricted to the use of in vitro “two dimensional” assays or in vivo SCID mouse models. Both systems, however, have proved to be poor predictors of clinical T cell activity. In addition, SCID models are expensive, time consuming, and lack physiological relevance. Therefore, to study T cell function we have tested a novel, animal-free system called the Artificial-Mouse (Art Mouse) developed by Wilson Wolf Corporation. The Art Mouse is a gas permeable culture system that contains multiple chambers connected in series in order to allow the dynamic study of T cell migration, expansion and anti-tumor effects. These properties can be monitored by periodic bioluminescence imaging and flow cytometric analysis. We first explored the ability of the Art Mouse to generate a chemokine gradient. Seventy-two hours after adding 24μg of MCP1 in compartment 1 (C1), we detected the following concentrations: C1 (196.79ng/ml), C2 (78.52ng/ml), C3 (56.80ng/ml), C4 (9.79ng/ml), C5 (2.52ng/ml) and C6 (0.64ng/ml). To evaluate if these conditions would drive T cell migration, 24μg of MCP1 was added in C1 and 3 days later 20×106 CAR-PSCA T cells marked with firefly-luciferase GFP (FFluc-GFP) were placed in C6. After 5 days we observed an accumulation of CAR-PSCA Tcells in C1 (increase from 1.29×108 to 2.09×108 photons/second; p/s), with no accumulation observed in the control (no MCP1). Importantly, engraftment of the pancreatic tumor cell line CAPAN1 (which produces IL8 and CXCL1) in C1 also induced CAR-PSCA T cell migration (increase from 0.92×108 to 1.45×108 p/s). To assess the utility of this new tool in evaluating anti-tumor effects we established a 3D tumor model by seeding C1 with 1×106 FFLuc-GFP-marked CAPAN1 cells (PSCA+ve pancreatic tumor cell line). Subsequently, 20×106 CAR-PSCA T cells were added to C6. We then monitored the tumor signal by frequent bioluminescence imaging and observed a progressive decrease in the tumor signal (2.0±.32×108 to 1.4±.09×105 p/s, day 0 to day 30). In contrast, in the absence of T cells the tumor signal progressively increased (1.7±.59×108 to 1.3±.29×109 p/s, day 0 to day 30). Notably, this model resembles the growth kinetics and anti-tumor effects observed in SCID xenograft models but an important distinction between the two platforms is the substantially lower variability in the Art Mouse (Std Dev of 0.32×108 vs 3.31×108p/s, Art Mouse vs SCID mice). Thus, this preliminary data suggests that the Art Mouse can serve as an animal-free alternative to study dynamic T cell features including migration, expansion and anti-tumor effects.