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Selective enrichment process of CAR- and CAR+ transgenic sub-populations

Consolidated supplementary tables for nanostring data

AIM To characterize incidence and risk factors for delayed gastric emptying (DGE) following pancreaticoduodenectomy and examine its implications on healthcare utilization. METHODS A prospectively-maintained database was reviewed. DGE was classified using International Study Group of Pancreatic Surgery criteria. Patients who developed DGE and those who did not were compared. RESULTS Two hundred and seventy-six patients underwent pancreaticoduodenectomy (PD) (> 80% pylorus-preserving, antecolic-reconstruction). DGE developed in 49 patients (17.8%): 5.1% grade B, 3.6% grade C. Demographic, clinical, and operative variables were similar between patients with DGE and those without. DGE patients were more likely to present multiple complications (32.6% vs 4.4%, ≥ 3 complications, P < 0.001), including postoperative pancreatic fistula (POPF) (42.9% vs 18.9%, P = 0.001) and intra-abdominal abscess (IAA) (16.3% vs 4.0%, P = 0.012). Patients with DGE had longer hospital stay (median, 12 d vs 7 d, P < 0.001) and were more likely to require transitional care upon discharge (24.5% vs 6.6%, P < 0.001). On multivariate analysis, predictors for DGE included POPF [OR = 3.39 (1.35-8.52), P = 0.009] and IAA [OR = 1.51 (1.03-2.22), P = 0.035]. CONCLUSION Although DGE occurred in < 20% of patients after PD, it was associated with increased healthcare utilization. Patients with POPF and IAA were at risk for DGE. Anticipating DGE can help individualize care and allocate resources to high-risk patients.

The adoptive transfer of T cells redirected to tumor-associated antigens via transgenic expression of chimeric antigen receptors (CARs) has produced tumor responses, even in patients with refractory diseases. To target pancreatic cancer, we generated CAR T cells directed against prostate stem cell antigen (PSCA) and demonstrated specific tumor lysis. However, pancreatic tumors employ immune evasion strategies such as the production of inhibitory cytokines, which limit CAR T cell persistence and function. Thus, to protect our cells from the immunosuppressive cytokine IL-4, we generated an inverted cytokine receptor in which the IL-4 receptor exodomain was fused to the IL-7 receptor endodomain (4/7 ICR). Transgenic expression of this molecule in CAR-PSCA T cells should invert the inhibitory effects of tumor-derived IL-4 and instead promote T cell proliferation. We now demonstrate the suppressed activity of CAR T cells in tumor-milieu conditions and the ability of CAR/ICR T cells to thrive in an IL-4-rich microenvironment, resulting in enhanced antitumor activity. Importantly, CAR/ICR T cells remained both antigen and cytokine dependent. These findings support the benefit of combining the 4/7 ICR with CAR-PSCA to treat pancreatic cancer, a PSCA-expressing tumor characterized by a dense immunosuppressive environment rich in IL-4.

The adoptive transfer of chimeric antigen receptor (CAR)–modified T cells has produced tumor responses even in patients with refractory diseases. However, the paucity of antigens that are tumor selective has resulted, on occasion, in “on-target, off-tumor” toxicities. To address this issue, we developed an approach to render T cells responsive to an expression pattern present exclusively at the tumor by using a trio of novel chimeric receptors. Using pancreatic cancer as a model, we demonstrate how T cells engineered with receptors that recognize prostate stem cell antigen, TGFβ, and IL4, and whose endodomains recapitulate physiologic T-cell signaling by providing signals for activation, costimulation, and cytokine support, produce potent antitumor effects selectively at the tumor site. In addition, this strategy has the benefit of rendering our cells resistant to otherwise immunosuppressive cytokines (TGFβ and IL4) and can be readily extended to other inhibitory molecules present at the tumor site (e.g., PD-L1, IL10, and IL13). Significance: This proof-of-concept study demonstrates how sophisticated engineering approaches can be utilized to both enhance the antitumor efficacy and increase the safety profile of transgenic T cells by incorporating a combination of receptors that ensure that cells are active exclusively at the tumor site. Cancer Discov; 8(8); 972–87. ©2018 AACR. See related commentary by Achkova and Pule, p. 918. This article is highlighted in the In This Issue feature, p. 899

BackgroundAlthough mortality following pancreaticoduodenectomy is decreasing, postoperative morbidity remains high. It was hypothesized that culture-directed treatment of bacteriobilia would decrease the incidence of infectious complications following pancreaticoduodenectomy.MethodsIn a retrospective study of 197 pancreaticoduodenectomy patients, those in the control group (n = 128, 2005–2009) were given perioperative prophylactic antibiotics, whereas those in the treatment group (n = 69, 2009–2011) were continued on antibiotics until intraoperative bile culture results became available. Patients with bacteriobilia received 10 days of antibiotic treatment, which was otherwise discontinued in patients without bacteriobilia. Various complication rates were compared using Fisher's exact test for categorical variables, Wilcoxon rank sum test for ordinal variables, and a two-sample t-test for continuous variables.ResultsDemographics, comorbidities, baseline clinical characteristics, and intraoperative and postoperative variables were similar between the two groups. There were higher incidences of elevated creatinine (19% versus 4%; P = 0.004) and preoperative hyperglycaemia (18% versus 7%; P = 0.053) in the control group. Fewer patients in the control group underwent preoperative biliary stenting (48% versus 67%; P = 0.017) and intraperitoneal drains were placed at the time of resection more frequently in the control group (85% versus 38%; P < 0.001). Bacteriobilia was found in 59% of patients. Treatment of bacteriobilia was associated with a decrease in the rate of postoperative wound infections (12% in the control group versus 3% in the treatment group; P = 0.036) and overall complication severity score (1 in the control group versus 0 in the treatment group; P = 0.027).ConclusionsProlonged antibiotic therapy for bacteriobilia may decrease postoperative wound infection rates after pancreaticoduodenectomy. A randomized prospective trial is warranted to provide evidence to further support this practice.

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.

OBJECTIVE: For patients with genetically triggered thoracic aortic disease, the morbidity and mortality associated with reoperation are high, making endovascular treatment an appealing option. We evaluated the short- and mid-term outcomes of different applications of endovascular intervention in such patients. METHODS: Between January 2003 and April 2013, 60 patients received endovascular or hybrid treatment for genetically triggered thoracic aortic disease. The inclusion criteria were based on those devised by the National Registry of Genetically Triggered Thoracic Aortic Aneurysms and Cardiovascular Conditions. We included patients with thoracic aneurysm or dissection not due to trauma in a patient aged ≤50 years (n= 30), bicuspid aortic valve (BAV) and coarctation (n= 11), Marfan syndrome (n= 10), BAV with thoracic aneurysm (n= 4), Loeys–Dietz syndrome (n= 3), familial thoracic aneurysm or dissection (n= 3) and genetic mutations (n= 2). Some patients met more than one inclusion criterion. Forty-one (68.3%) patients were treated with only endovascular stent grafting. Nineteen (31.7%) patients underwent a hybrid procedure with open proximal or total arch replacement and concomitant endovascular stenting of the aortic arch or the descending thoracic aorta. Twenty-nine (48.3%) had previous cardiovascular operations (mean ± SD, 1.9 ± 1.4) before undergoing hybrid or endovascular therapy. The median follow-up was 2.3 years (interquartile interval 25–75%, 1.4–4.6 years). RESULTS: The technical success rate was 100%. In-hospital mortality was 3.3% (n= 2) and neurological events occurred in 2 patients; 1 (1.6%) had a stroke and 1 (1.6%) suffered paraparesis with partial recovery. Fifteen repeat open or endovascular interventions were required in 10 surviving patients (17.2%). Overall survival during follow-up was 94.8% (55/58). CONCLUSIONS: Endovascular technology can be helpful in treating selected young patients with genetically triggered thoracic aortic disease. Long-term studies and further evolution of endovascular technology will be necessary for it to be incorporated into the armamentarium of surgical options for this challenging patient population.

Pancreatic cancer is a leading cause of cancer mortality and the incidence of this disease is expected to continue increasing. While patients with pancreatic cancer have traditionally faced a dismal prognosis, over the past several years various advances in diagnosis and treatment have begun to positively impact this disease. Identification of effective combinations of existing chemotherapeutic agents, such as the FOLFIRINOX and the gemcitabine + nab-paclitaxel regimen, has improved survival for selected patients although concerns regarding their toxicity profiles remain. A better understanding of pancreatic carcinogenesis has identified several pre-malignant precursor lesions, such as pancreatic intraepithelial neoplasias, intraductal papillary mucinous neoplasms, and cystic neoplasms. Imaging technology has also evolved dramatically so as to allow early detection of these lesions and thereby facilitate earlier management. Surgery remains a cornerstone of treatment for patients with resectable pancreatic tumors, and advances in surgical technique have allowed patients to undergo resection with decreasing perioperative morbidity and mortality. Surgery has also become feasible in selected patients with borderline resectable tumors as a result of neoadjuvant therapy. Furthermore, pancreatectomy involving vascular reconstruction and pancreatectomy with minimally invasive techniques have demonstrated safety without significantly compromising oncologic outcomes. Lastly, a deeper understanding of molecular aberrations contributing to the development of pancreatic cancer shows promise for future development of more targeted and safe therapeutic agents.

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.

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 should invert the inhibitory effects of tumor-derived IL4 and 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 should be equipped to expand, persist, and retain their cytotoxic function even in the presence of high levels of IL4 in the tumor microenvironment. Citation Format: Somala Mohammed, Sujita Sukumaran, Pradip Bajgain, Usanarat Anurathapan, Helen E. Heslop, Cliona M. Rooney, Malcolm K. Brenner, Ann M. Leen, Juan F. Vera. Improving CAR T cell function by reversing the immunosuppressive tumor microenvironment of pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4703. doi:10.1158/1538-7445.AM2015-4703

Background: 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. Although these CAR-T cells have potent anti-tumor activity in vitro and in vivo, pancreatic tumors employ immune evasion mechanisms, such as the production of inhibitory cytokines, which limit in vivo CAR-T cell persistence and effector function. Methods: To target pancreatic ductal adenocarcinoma (PDAC), we generated T cells expressing a CAR directed to the TAA prostate stem cell antigen (PSCA). We also engineered a chimeric cytokine receptor in which the IL4 receptor exodomain was fused to the IL7 receptor endodomain (IL4/7 ChR). Expansion and selection profiles and short- and long-term anti-tumor activity of these transgenic T cells were assessed. Results: T cells expressing CAR-PSCA kill PSCA(+) tumor cell lines CAPAN1 and K562-PSCA but not PSCA(-) targets, such as 293T (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 in vitro and in vivo, pancreatic tumors employ immune evasion mechanisms, such as the production of inhibitory cytokines, which limit in vivo CAR-T cell persistence and effector function. Indeed, when the serum of patients with pancreatic cancer (n=8) was examined, 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 the 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 should invert the inhibitory effects of tumor-derived IL4 and 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 unlike unmodified CAR-PSCA T cells (from 2x106 cells on day 0 to 5.53x1010±8.46x1010 cells (CAR-PSCA 4/7R ChR) on day 28, in comparison to CAR-PSCA T cells that reached only 3.84x108±5.43x108 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), but even after prolonged cytokine exposure these T cells remained both antigen- and cytokine-dependent. Conclusions: CAR-PSCA 4/7 ChR-modified tumor-specific T cells can effectively target pancreatic cancer cells and should be equipped to expand, persist, and retain their cytotoxic function even in the presence of high levels of IL4 in the tumor microenvironment. Citation Format: Somala Mohammed, Sujita Sukumaran, Usanarat Anurathapan, Pradip Bajgain, Helen E. Heslop, Cliona M. Rooney, Malcolm K. Brenner, Ann M. Leen, Juan F. Vera. Improving CAR T cell function by reversing the immunosuppressive tumor environment of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B63.

Objective Local vancomycin treatment has been shown to decrease sternal wound complication rates. Whether a similar effect can be achieved at other surgical sites is unknown. This study investigates the effect of local vancomycin on inguinal wound complication rates after vascular procedures. Methods Retrospective analysis was performed on 454 patients who underwent open aortofemoral or infrainguinal vascular procedures between 2006 and 2011. Patients received preoperative systemic antibiotics either alone (group A) or in conjunction with intraoperative wound application of vancomycin powder and irrigation (group B). Inguinal wound infection and dehiscence over a 30-day period were recorded. Fisher exact test and multivariate regression analyses were performed. Results There were 211 patients in group A and 243 patients in group B. Both groups had similar demographics and operative characteristics. There was a small but statistically significant decrease in the 30-day incidence of overall wound infections (25.1% vs 17.2%; P = .049) for group B patients. This was primarily due to a decreased rate in superficial infections (18.9% vs 11.5%; P = .033). No significant difference in the incidence of deep wound infections (6.1% vs 5.7%; P = .692) or overall dehiscence rates (22.2% vs 17.7%; P = .239) was detected. On multivariate analysis, history of chronic obstructive pulmonary disease and increased body mass index significantly increased risk of both infection and dehiscence. Medically optimized coronary artery disease was associated with less risk for dehiscence. Conclusions Addition of intraoperative local vancomycin did not improve the rates of inguinal wound dehiscence or deep infections but had a positive impact on superficial wound infections.