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Impressive clinical efficacy of chimeric antigen receptor (CAR)-engineered T cell therapy for hematological malignancies have prompted significant efforts in achieving similar responses in solid tumors. The lack of truly restricted and uniform expression of tumor-associated antigens, as well as limited T cell persistence and/or tumor trafficking pose major challenges for successful translation of CAR T cell therapy in solid tumors. Recent studies have demonstrated that aberrantly glycosylated cell surface proteins on tumor cells are amenable CAR targets. Tumor-associated glycoprotein 72 (TAG72) antigen is the sialyl-Tn found on multiple O-glycoproteins expressed at high levels on the surface of several cancer types, including ovarian cancer. Here, we developed a humanized TAG72-specific CAR containing a 4-1BB intracellular co-stimulatory signaling domain (TAG72-BBζ). TAG72-BBζ CAR T cells showed potent antigen-dependent cytotoxicity and cytokine production against multiple TAG72+ ovarian cancer cell lines and patient-derived ovarian cancer ascites. Using in vivo xenograft models of peritoneal ovarian tumors, regional intraperitoneal delivery of TAG72-BBζ CAR T cells significantly reduced tumor growth, extended overall survival of mice, and was further improved with repeat infusions of CAR T cells. However, reduced TAG72 expression was observed in early recurring tumors, which coincided with a lack of T cell persistence. Taken together, we demonstrate efficacy with TAG72-CAR T cells in ovarian cancer, warranting further investigations as a CAR T cell therapeutic strategy for this disease. [ABSTRACT FROM AUTHOR]

Abstract: In efforts to define new targets for antithrombotic purposes, there is interest in utilizing antibodies targeting ligand binding domains of platelet receptors. To this end, we have recently shown that an antibody (designated C-EL2Ab), which targets the C-terminus of the 2nd extracellular loop (C-EL2) of the thromboxane A2 receptor (TPR), selectively blocks TPR-mediated platelet aggregation, under both in vitro and ex vivo experimental conditions. In the current studies we sought to determine whether C-EL2Ab exhibits in vivo antithrombotic activity, by employing a carotid artery injury thrombosis model. It was found that mice treated with C-EL2Ab, exhibited a significant increase in time for occlusion, when compared to controls such as normal rabbit IgG, or an antibody which targets a region separate from the ligand binding site (i.e., EL1). We next examined the effect of C-EL2Ab on hemostasis, and found no increase in tail bleeding times in C-EL2Ab treated mice, compared to the aforementioned controls. Collectively, these results clearly demonstrate that C-EL2Ab has anti-platelet/anti-thrombotic effects, and is devoid of increased bleeding risk. Moreover, the identification of a functionally active TPR sequence should significantly aid molecular modeling study predictions for organic derivatives which possess in vivo activity. [Copyright &y& Elsevier]

Antiplatelet therapy has been documented to reduce risks of cardiovascular disease after acute myocardial infarction, coronary artery bypass graft, and in chronic atrial fibrillation patients, amongst other risk factors. Conventional management of thrombosisbased disorders includes the use of heparin, oral anticoagulants, and the preferred antiplatelet agent aspirin. Interestingly, aspirin was not intended to be used as an antiplatelet agent; rather, after being repurposed, it has become one of themost widely prescribed antithrombotic drugs. To this end, there have been several milestones in the development of antiplatelet agents in the last few decades, such as adenosine diphosphate receptor inhibitors, phosphodiesterase inhibitors, and GPIIb/IIIa inhibitors. However, given some of the limitations of these therapies, aspirin continues to play a major role in the management of thrombotic and cardiovascular disorders and is expected to do so for years to come. [ABSTRACT FROM AUTHOR]

Abstract: There is considerable interest in discovering novel antiplatelet approaches with an enhanced safety profile. To this end, in our efforts to define new targets for antithrombotic activity, we investigated the utility of antibodies which recognize the ligand binding domains of the platelet thromboxane A2 receptor (TPR). We hypothesized that an antibody (abbreviated as C-EL2Ab), which interacts with the C-terminus of the second extracellular loop (C-EL2; i.e., ligand binding domain) of TPR exhibits antagonistic activity. Our findings demonstrate that C-EL2Ab did indeed inhibit TPR-mediated platelet aggregation. However, it was devoid of any apparent effects on aggregation triggered by ADP or the thrombin receptor activating peptides 1 or 4. Furthermore, results from radiolabeled ligand binding studies indicate that C-EL2Ab competitively displaced the classical TPR antagonist [3H]SQ29,548 from its binding sites. On the other hand, control experiments indicated that normal rabbit IgG and an antibody which targets a TPR domain separate from those involved in ligand recognition, failed to inhibit aggregation in response to TPR activation. Collectively, these findings demonstrate that C-EL2 of TPR plays a critical role in platelet activation, and establish C-EL2Ab as a function blocking antibody. Furthermore, our data suggest a potential for the therapeutic application of C-EL2Ab, which may serve either as an alternative to, or a complement for current treatments. Finally, the identification of a functionally active TPR sequence should aid molecular modeling study predictions for organic derivatives which possess in vivo activity. [Copyright &y& Elsevier]

Advancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy, given impressive recent clinical responses in hematological malignancies. Prostate cancer may be amenable to T cell-based immunotherapy since several tumor antigens, including prostate stem-cell antigen (PSCA), are widely over-expressed in metastatic disease. While antigen selectivity of CARs for solid cancers is crucial, it is problematic due to the absence of truly restricted tumor antigen expression and potential safety concerns with “on-target off-tumor” activity. Here, we show that the intracellular co-stimulatory signaling domain can determine a CAR's sensitivity for tumor antigen expression. A 4-1BB intracellular co-stimulatory signaling domain in PSCA-CARs confers improved selectivity for higher tumor antigen density, reduced T cell exhaustion phenotype, and equivalent tumor killing ability compared to PSCA-CARs containing the CD28 co-stimulatory signaling domain. PSCA-CARs exhibit robustin vivoanti-tumor activity in patient-derived bone-metastatic prostate cancer xenograft models, and 4-1BB-containing CARs show superior T cell persistence and control of disease compared with CD28-containing CARs. Our study demonstrates the importance of co-stimulation in defining an optimal CAR T cell, and also highlights the significance of clinically relevant models in developing solid cancer CAR T cell therapies. [ABSTRACT FROM AUTHOR]

Impressive clinical efficacy of chimeric antigen receptor (CAR)-engineered T cell therapy for hematological malignancies have prompted significant efforts in achieving similar responses in solid tumors. The lack of truly restricted and uniform expression of tumor-associated antigens, as well as limited T cell persistence and/or tumor trafficking pose major challenges for successful translation of CAR T cell therapy in solid tumors. Recent studies have demonstrated that aberrantly glycosylated cell surface proteins on tumor cells are amenable CAR targets. Tumor-associated glycoprotein 72 (TAG72) antigen is the sialyl-Tn found on multiple O-glycoproteins expressed at high levels on the surface of several cancer types, including ovarian cancer. Here, we developed a humanized TAG72-specific CAR containing a 4-1BB intracellular co-stimulatory signaling domain (TAG72-BBζ). TAG72-BBζ CAR T cells showed potent antigen-dependent cytotoxicity and cytokine production against multiple TAG72 + ovarian cancer cell lines and patient-derived ovarian cancer ascites. Using in vivo xenograft models of peritoneal ovarian tumors, regional intraperitoneal delivery of TAG72-BBζ CAR T cells significantly reduced tumor growth, extended overall survival of mice, and was further improved with repeat infusions of CAR T cells. However, reduced TAG72 expression was observed in early recurring tumors, which coincided with a lack of T cell persistence. Taken together, we demonstrate efficacy with TAG72-CAR T cells in ovarian cancer, warranting further investigations as a CAR T cell therapeutic strategy for this disease.

Purpose: Metastasis to the brain from breast cancer remains a significant clinical challenge, and may be targeted with CAR-based immunotherapy. CAR design optimization for solid tumors is crucial due to the absence of truly restricted antigen expression and potential safety concerns with "on-target off-tumor" activity. Here, we have optimized HER2-CAR T cells for the treatment of breast to brain metastases, and determined optimal second-generation CAR design and route of administration for xenograft mouse models of breast metastatic brain tumors, including multifocal and leptomeningeal disease. Experimental Design: HER2-CAR constructs containing either CD28 or 4-1BB intracellular costimulatory signaling domains were compared for functional activity in vitro by measuring cytokine production, T-cell proliferation, and tumor killing capacity. We also evaluated HER2-CAR T cells delivered by intravenous, local intratumoral, or regional intraventricular routes of administration using in vivo human xenograft models of breast cancer that have metastasized to the brain. Results: Here, we have shown that HER2-CARs containing the 4-1BB costimulatory domain confer improved tumor targeting with reduced T-cell exhaustion phenotype and enhanced proliferative capacity compared with HER2-CARs containing the CD28 costimulatory domain. Local intracranial delivery of HER2-CARs showed potent in vivo antitumor activity in orthotopic xenograft models. Importantly, we demonstrated robust antitumor efficacy following regional intraventricular delivery of HER2-CAR T cells for the treatment of multifocal brain metastases and leptomeningeal disease. Conclusions: Our study shows the importance of CAR design in defining an optimized CAR T cell, and highlights intraventricular delivery of HER2-CAR T cells for treating multifocal brain metastases. Clin Cancer Res; 24(1); 95-105. ©2017 AACR ., (©2017 American Association for Cancer Research.)