Chimeric antigen receptor-T cell (CAR-T) therapy has been effective in the treatment of hematologic malignancies, but it has shown limited efficacy against solid tumors. Here we demonstrate an approach to enhancing CAR-T function in solid tumors by directly vaccine-boosting donor cells through their chimeric receptor in vivo. We designed amphiphile CAR-T ligands (amph-ligands) that, upon injection, trafficked to lymph nodes and decorated the surfaces of antigen-presenting cells, thereby priming CAR-Ts in the native lymph node microenvironment. Amph-ligand boosting triggered massive CAR-T expansion, increased donor cell polyfunctionality, and enhanced antitumor efficacy in multiple immunocompetent mouse tumor models. We demonstrate two approaches to generalizing this strategy to any chimeric antigen receptor, enabling this simple non-human leukocyte antigen-restricted approach to enhanced CAR-T functionality to be applied to existing CAR-T designs.

We sought to develop a nanoparticle vehicle that could efficiently deliver small molecule drugs to target lymphocyte populations. The synthesized amphiphilic organic ligand-protected gold nanoparticles (amph-NPs) were capable of sequestering large payloads of small molecule drugs within hydrophobic pockets of their ligand shells. These particles exhibit membrane-penetrating activity in mammalian cells, and thus enhanced uptake of a small molecule TGF-β inhibitor in T cells in cell culture. By conjugating amph-NPs with targeting antibodies or camelid-derived nanobodies, the particles’ cell-penetrating properties could be temporarily suppressed, allowing targeted uptake in specific lymphocyte subpopulations. Degradation of the protein targeting moieties following particle endocytosis allowed the NPs to recover their cell-penetrating activity in situ to enter the cytoplasm of T cells. In vivo, targeted amph-NPs showed 40-fold enhanced uptake in CD8+ T cells relative to untargeted particles, and delivery of TGF-β inhibitor-loaded particles to T cells enhanced their cytokine polyfunctionality in a cancer vaccine model. Thus, this system provides a facile approach to concentrate small molecule compounds in target lymphocyte populations of interest for immunotherapy in cancer and other diseases.

Upper Gastrointestinal Cancers (UGCs) are a leading cause of cancer-related deaths worldwide. Paclitaxel (PTX) is frequently used for the treatment of UGCs; however, low bioavailability, reduced solubility, and dose-dependent toxicity impede its therapeutic use. PAMAMG -NH -DHA is synthesized by linking amine-terminated fourth-generation poly(amidoamine) (PAMAMG -NH ) dendrimers with omega-3 fatty acid docosahexaenoic acid (DHA). Next, PAMAMG -NH -DHA-PTX (DHATX) and PAMAMG -NH -PTX (PAX) conjugates are synthesized by subsequent covalent binding of PTX with PAMAMG -NH -DHA and PAMAMG -NH , respectively. H-NMR and MALDI-TOF analyses are performed to confirm conjugation of DHA to PAMAMG -NH and PTX to PAMAMG -NH -DHA. The cell viability, clonogenic cell survival, and flow cytometry analyses are used to determine the anticancer activity of PTX, PAX, and DHATX in UGC cell lines. The in vitro data indicate that treatment with DHATX is significantly more potent than PTX or PAX at inhibiting cellular proliferation, suppressing long-term survival, and inducing cell death in UGC cells.

Background: Paclitaxel (PTX) is frequently used for the treatment of advanced gastrointestinal (GI) cancers. However, development of drug resistance, dose-dependent toxicity, and lack of oral bioavailability are some of the major limiting factors that impede the therapeutic use of PTX for treatment of GI cancers. Drug conjugates of dendrimers, such as poly (amidoamine) (PAMAM), have been reported to effectively overcome bioavailability and dose-dependent toxicity related limitations. Therefore, in this study we synthesized and investigated the anticancer potential of omega-3 fatty acid [docosahexanoic acid (DHA)]-poly (amido)amine (PAMAM)-paclitaxel conjugates in various in vitro models of gastrointestinal cancers. Methods: DHA was conjugated to amine-terminated PAMAMG4 dendrimers using coupling agents HOBt and HBTU. PAMAMG4-DHA was then conjugated to PTX using PTX-NHS-ester. The conjugates were purified by size exclusion chromatography and characterized using 1H-NMR, MALDI-TOF-MS and high-resolution ESI-MS. The percentage payload of PTX conjugated to PAMAMG4-DHA and in vitro stability of PAMAMG4-DHA-PTX were evaluated using HPLC. Cell viability, clonogenic cell survival, and western blot analyses were done to evaluate the cell viability, survival, and induction of apoptotic proteins (P53 and P21), respectively. The in vitro cytotoxicity and induction of apoptosis was investigated in AGS, FLO-1, and MIA PaCa-2 GI cancer cell lines after treatment with PTX or PAMAMG4-DHA-PTX. Results: The 1H-NMR, MALDI-TOF and high-resolution ESI mass spectra confirmed the conjugation of DHA to PAMAM and PTX to PAMAM-DHA. The in vitro stability data showed that PAMAM-DHA-PTX conjugate was stable in human plasma for 24 hours. The cell viability data indicated that treatment with PAMAMG4-DHA-PTX is significantly more potent than PTX at inhibiting proliferation of esophageal (FLO-1: PTX IC50: 3.8±0.68 nM, PAMAMG4-DHA-PTX IC50: 1.1±0.17 nM), gastric (AGS: PTX IC50: 5.1±0.58 nM, PAMAMG4-DHA-PTX IC50: 1.4±0.23 nM), and pancreatic (MIA PaCa: PTX IC50: 5.1±0.75 nM, PAMAMG4-DHA-PTX IC50: 1.8±0.35 nM) cancer cell lines. The clonogenic cell survival data shows a similar activity where treatment with PAMAMG4-DHA-PTX exhibited enhanced inhibition of long term survival of FLO-1, AGS, and MIA PaCa cells when compared to PTX. The protein expression data further showed that compared to PTX treatment with PAMAMG4-DHA-PTX induced higher expression of P53 and P21 pro-apoptotic protein in AGS gastric cancer cell line. Conclusions: Overall our findings indicate that paclitaxel was successfully conjugated with PAMAMG4-DHA resulting in the synthesis of a novel PAMAMG4-DHA-PTX conjugate that exhibits enhanced anticancer activity than PTX alone in various in vitro models of GI cancers. Citation Format: Tanmay Dichwalkar, Samhita Bapat, Priya Pancholi, V. K. Yellepeddi, Vikas Sehdev. Omega-3 fatty acid conjugated paclitaxel dendrimers exhibit enhanced anticancer activity in various preclinical models of gastrointestinal cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2200.