Depleting T regulatory cells by targeting intracellular Foxp3 with a TCR mimic antibody.

Depletion of T regulatory cells (Tregs) in the tumor microenvironment is a promising cancer immunotherapy strategy. Current approaches for depleting Tregs are limited by lack of specificity and concurrent depletion of anti-tumor effector T cells. The transcription factor forkhead box p3 (Foxp3) plays a central role in the development and function of Tregs and is an ideal target in Tregs, but Foxp3 is an intracellular, undruggable protein to date. We have generated a T cell receptor mimic antibody, "Foxp3-#32," recognizing a Foxp3-derived epitope in the context of HLA-A*02:01. The mAb Foxp3-#32 selectively recognizes CD4 + CD25 + CD127low and Foxp3 + Tregs also expressing HLA-A*02:01 and depletes these cells via antibody-mediated cellular cytotoxicity. Foxp3-#32 mAb depleted Tregs in xenografts of PBMCs from a healthy donor and ascites fluid from a cancer patient. A TCRm mAb targeting intracellular Foxp3 epitope represents an approach to deplete Tregs.

A novel antibody-TCR (AbTCR) platform combines Fab-based antigen recognition with gamma/delta-TCR signaling to facilitate T-cell cytotoxicity with low cytokine release.

The clinical use of genetically modified T-cell therapies has led to unprecedented response rates in leukemia and lymphoma patients treated with anti-CD19 chimeric antigen receptor (CAR)-T. Despite this clinical success, FDA-approved T-cell therapies are currently limited to B-cell malignancies, and challenges remain with managing cytokine-related toxicities. We have designed a novel antibody-T-cell receptor (AbTCR) platform where we combined the Fab domain of an antibody with the γ and δ chains of the TCR as the effector domain. We demonstrate the ability of anti-CD19-AbTCR-T cells to trigger antigen-specific cytokine production, degranulation, and killing of CD19-positive cancer cells in vitro and in xenograft mouse models. By using the same anti-CD19 binding moiety on an AbTCR compared to a CAR platform, we demonstrate that AbTCR activates cytotoxic T-cell responses with a similar dose-response as CD28/CD3ζ CAR, yet does so with less cytokine release and results in T cells with a less exhausted phenotype. Moreover, in comparative studies with the clinically validated CD137 (4-1BB)-based CAR, CTL019, our anti-CD19-AbTCR shows less cytokine release and comparable tumor inhibition in a patient-derived xenograft leukemia model.

Targeting Alpha-Fetoprotein (AFP)-MHC Complex with CAR T-Cell Therapy for Liver Cancer.

PURPOSE: The majority of tumor-specific antigens are intracellular and/or secreted and therefore inaccessible by conventional chimeric antigen receptor (CAR) T-cell therapy. Given that all intracellular/secreted proteins are processed into peptides and presented by class I MHC on the surface of tumor cells, we used alpha-fetoprotein (AFP), a specific liver cancer marker, as an example to determine whether peptide-MHC complexes can be targets for CAR T-cell therapy against solid tumors. EXPERIMENTAL DESIGN: We generated a fully human chimeric antigen receptor, ET1402L1-CAR (AFP-CAR), with exquisite selectivity and specificity for the AFP158-166 peptide complexed with human leukocyte antigen (HLA)-A*02:01. RESULTS: We report that T cells expressing AFP-CAR selectively degranulated, released cytokines, and lysed liver cancer cells that were HLA-A*02:01+/AFP+ while sparing cells from multiple tissue types that were negative for either expressed proteins. In vivo, intratumoral injection of AFP-CAR T cells significantly regressed both Hep G2 and AFP158-expressing SK-HEP-1 tumors in SCID-Beige mice (n = 8 for each). Moreover, intravenous administration of AFP-CAR T cells in Hep G2 tumor-bearing NSG mice lead to rapid and profound tumor growth inhibition (n = 6). Finally, in an established intraperitoneal liver cancer xenograft model, AFP-CAR T cells showed robust antitumor activity (n = 6). CONCLUSIONS: This study demonstrates that CAR T-cell immunotherapy targeting intracellular/secreted solid tumor antigens can elicit a potent antitumor response. Our approach expands the spectrum of antigens available for redirected T-cell therapy against solid malignancies and offers a promising new avenue for liver cancer immunotherapy. Clin Cancer Res; 23(2); 478-88. ©2016 AACR.

Truncation of cytoplasmic tail of EIAV Env increases the pathogenic necrosis.

Equine Infectious Anemia Virus (EIAV), like other lentiviruses, has a transmembrane glycoprotein with an unusually long cytoplasmic tail (CT). Viral envelope (Env) proteins having CT truncations just downstream the putative membrane-spanning domain (PMSD) are assumed to exist among all wild-type budded virions, and also in some cell-adapted strains. To determine whether CT-truncated Env proteins can cause particularly deleterious effects on the Env expressing cells and/or their neighboring cells, plasmids encoding codon-optimized env gene including full-length (pE863) or CT-truncated (pE686* and pE676*) were transiently transfected into 293T cells, respectively. Data from intracellular protein expression and cell death assays revealed that CT-truncated Env, compared to full-length Env, not only induced comparable apoptosis, but also caused much more intensive mitochondria-mediated necrosis that could simultaneously induce significant decrease of intracellular protein expression in the Env expressing cells. Moreover, results from flow cytometric analysis showed that mitochondrial depolarization preceded the caspase activation in cells no matter which env construct was delivered, and indicated that both full-length and CT-truncated Env proteins share a common intrinsic mitochondrial pathway to induce apoptosis. Our results partially elucidate the mechanisms underlying cell death resulting from EIAV pathogenesis.