Usanarat Anurathapan

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.

Reversal of Tumor Immune Inhibition Using a Chimeric Cytokine Receptor

The success of adoptively transferred tumor-directed T cells requires them to survive and expand in vivo. Most tumors, however, employ immune evasion mechanisms, including the production of inhibitory cytokines that limit in vivo T-cell persistence and effector function. To protect tumor-directed T cells from such negative influences, we generated a chimeric cytokine receptor in which the interleukin (IL) 4 receptor exodomain was fused to the IL7 receptor endodomain. We thereby inverted the effects of tumor-derived IL4 so that the proliferation and activation of tumor directed cytotoxic T cells was enhanced rather than inhibited in the tumor microenvironment, resulting in superior antitumor activity. These transgenic T cells were only activated in the tumor environment since triggering required exposure to both tumor antigen (signal 1) and tumor-derived IL4 (signal 2). This selectivity supports future clinical adaptation.

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.

Clinical practice guidelines for children with cancer presenting with fever to the emergency room.

Background:  Patients with febrile neutropenia (FN) may develop severe infection, septic shock, and death. To improve the outcome of pediatric oncology patients with suspected FN, clinical practice guidelines were developed for these patients at the emergency room (ER). The objective of the present study was to evaluate compliance of the clinical practice guidelines for children with cancer presenting with fever to the ER and adverse outcomes after using the guidelines.

Comparative outcome of Thai pediatric osteosarcoma treated with two protocols: the role of high-dose methotrexate (HDMTX) in a single institute experience.

BACKGROUND High-dose methotrexate (HD-MTX) is recognized as an efficient component of therapy against pediatric osteosarcoma in combination with other drugs such as cisplatin (CDP), carboplatin (CBDCA), doxorubicin (ADM), etoposide (VP-16) and ifosfamide (IFO). OBJECTIVES To demonstrate the feasibility and effectiveness of the HD-MTX/CDP/DOX/VP-16/IFO [MTX(+)] protocol comparable to CDP/ADM/CBDCA/IFO [MTX(-)] for treating childhood osteosarcoma at Ramathibodi Hospital (1999-2014). MATERIALS AND METHODS A retrospective analysis was conducted of osteosarcoma patients aged less than 18 years treated with two chemotherapeutic regimens between 1999 and 2014. A total of 45 patients received the MTX(-) and 21 the MTX(+) protocol. RESULTS Overall limb-salvage and amputation rate were 12.9% and 77.7%, respectively. Kaplan- Meier analysis results for 3-year disease free survival (DFS) and overall survival (OS) regardless of treatment regimens were 43.4±6.0% and 53.2±6.1% respectively. The 3-year DFS and OS were improved significantly with the MTX(+) protocol compared to MTX(-) protocol (p=0.010 and p=0.009, log rank test) [69.8±10.5%, 79.8±9.1% for MTX(+) and 31.1±6.9%, 42.2±7.4% for MTX(-) protocol, respectively]. Patients with metastatic osteosarcoma treated with the MTX(+) protocol had statistically significant higher 3-year DFS and OS than those treated with the MTX(-) protocol (66.7±13.6% and 15.0±8.0% for 3-year DFS, p=0.010, 73.3±13.2% and 20±8.9% for 3-year OS, p=0.006, respectively). The independent risk factors for having inferior 3-year DFS and OS were poor histological response (tumor necrosis <90%) and treatment with the MTX(-) protocol. The multivariate analysis identified only the treatment with the MTX(-) protocol as an independent predictor of inferior OS with a hazard ratio (HR) of 3.53 (95% confidence interval of 1.2-10.41, p=0.022). CONCLUSIONS Our study demonstrated the tolerability, feasibility and efficacy of the HDMTX-based regimen improving the survival rate in pediatric osteosarcoma cases, in line with reports from developed countries.

Low plasma FVII:C and activated FVII as predictive markers for overt disseminated intravascular coagulation

In sepsis, binding of factor VII (FVII:C) and activated factor VII (FVIIa) with tissue factor is the key step of coagulation resulting in disseminated intravascular coagulation (DIC). We conducted a prospective cohort study among 47 septic patients, aged 8 months to 18.8 years. They were initially divided into three groups of no DIC (n=27), non-overt DIC (n=14) and overt DIC (n=6). Blood samples were collected at 0, 24 and 48 hours (h) after the onset of sepsis. At the onset of sepsis, FVII:C tended to be lower in the non-overt DIC [median 57 % (interquartile range [IQR] 41-80)] and overt DIC groups [33 % (23-52)] than that in the no DIC group [65 % (44-87)]. Whereas FVIIa tended to be lower in the overt DIC group [1.29 % (0.50-4.19)] than those in the non-overt DIC [3.01 % (1.01-5.24)] and no DIC groups [2.49 % (1.14-3.13)]. At 24 h, FVII:C was significantly lower in the non-overt DIC [57 % (41-101)] and overt DIC groups [31 % (28-49)] than that in the no DIC group [83 % (70-102)]. While FVIIa was significantly lower in the overt DIC group [2.15 % (0.86-3.96)] than that in the no DIC group [3.83 % (2.90-5.46)]. Using FVII:C <65 % or FVIIa <3 % at 24 h among patients without hepatic dysfunction to determine overt DIC at 24 h, the sensitivity was 83.9 % and 77.4 %, respectively, and the specificity was both 83.3 %. Patients with low FVII:C and low FVIIa at 24 h after the onset of sepsis had a 20.8-fold (95 % confidence interval [CI], 2.0-213.0, p=0.010) and 14.4-fold (95 %CI, 1.5-142.4, p=0.023) chance of overt DIC.

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.

Depletion of alloreactive T cells for tolerance induction in a recipient of kidney and hematopoietic stem cell transplantations.

Tangnararatchakit K, Tirapanich W, Anurathapan U, Tapaneya‐Olarn W, Pakakasama S, Jootar S, Slavin S, Hongeng S. Depletion of alloreactive T cells for tolerance induction in a recipient of kidney and hematopoietic stem cell transplantations.