Secretion of bispecific protein of anti-PD-1 fused with TGF-ß trap enhances antitumor efficacy of CAR-T cell therapy.

Despite the remarkable success of chimeric antigen receptor-modified T (CAR-T) cell therapy for blood malignancies, the clinical efficacy of this novel therapy in solid tumor treatment is largely limited by the immunosuppressive tumor microenvironment (TME). For instance, immune checkpoints (e.g., programmed cell death protein 1 [PD-1]/programmed death ligand 1 [PD-L1]) in TME play an important role in inhibiting T cell proliferation and functions. Transforming growth factor ß (TGF)-ß secreted by cancer cells in TME induces regulatory T cells (Tregs) and inhibits cytotoxic T cells. To overcome the inhibitory effect of immune checkpoints, we have previously engineered CAR-T cells to secrete anti-PD-1 to block the PD-1/PD-L1 pathway activity, a step demonstrating superior antitumor efficacy compared with conventional CAR-T cells. In this study, we engineered CAR-T cells that secrete bispecific trap protein co-targeting PD-1 and TGF-ß, with the aim of further improving antitumor immunity. Compared with conventional CAR-T cells and anti-PD-1-secreting CAR-T cells, data from in vitro and in vivo experiments showed that CAR-T cells with trap protein secretion further attenuated inhibitory T cell signaling, enhanced T cell persistence and expansion, and improved effector function and resistance to exhaustion. In the xenograft mouse model, CAR-T cells with trap protein secretion exhibited significantly enhanced antitumor immunity and efficacy. With these observations, we demonstrate the potential of trap protein self-secreting CAR-T cells as a potent therapy for solid tumors.

Evaluating CAR-T Cell Therapy in a Hypoxic 3D Tumor Model.

Despite its revolutionary success in hematological malignancies, chimeric antigen receptor T (CAR-T) cell therapy faces disappointing clinical results in solid tumors. The poor efficacy has been partially attributed to the lack of understanding in how CAR-T cells function in a solid tumor microenvironment. Hypoxia plays a critical role in cancer progression and immune editing, which potentially results in solid tumors escaping immunosurveillance and CAR-T cell-mediated cytotoxicity. Mechanistic studies of CAR-T cell biology in a physiological environment has been limited by the complexity of tumor-immune interactions in clinical and animal models, as well as by a lack of reliable in vitro models. A microdevice platform that recapitulates a 3D tumor section with a gradient of oxygen and integrates fluidic channels surrounding the tumor for CAR-T cell delivery is engineered. The design allows for the evaluation of CAR-T cell cytotoxicity and infiltration in the heterogeneous oxygen landscape of in vivo solid tumors at a previously unachievable scale in vitro.

Adnectin-Based Design of Chimeric Antigen Receptor for T Cell Engineering.

Although chimeric antigen receptor (CAR)-engineered T cell therapy has achieved encouraging clinical trial results for treating hematological cancers, further optimization can likely expand this therapeutic success to more patients and other cancer types. Most CAR constructs used in clinical trials incorporate single chain variable fragment (scFv) as the extracellular antigen recognition domain. The immunogenicity of nonhuman scFv could cause host rejection against CAR T cells and compromise their persistence and efficacy. The limited availability of scFvs and slow discovery of new monoclonal antibodies also limit the development of novel CAR constructs. Adnectin, a class of affinity molecules derived from the tenth type III domain of human fibronectin, can be an alternative to scFv as an antigen-binding moiety in the design of CAR molecules. We constructed adnectin-based CARs targeting epithelial growth factor receptor (EGFR) and found that compared to scFv-based CAR, T cells engineered with adnectin-based CARs exhibited equivalent cell-killing activity against target H292 lung cancer cells in vitro and had comparable antitumor efficacy in xenograft tumor-bearing mice in vivo. In addition, with optimal affinity tuning, adnectin-based CAR showed higher selectivity on target cells with high EGFR expression than on those with low expression. This new design of adnectin CARs can potentially facilitate the development of T cell immunotherapy for cancer and other diseases.

Masked Chimeric Antigen Receptor for Tumor-Specific Activation.

Adoptive cellular therapy based on chimeric antigen receptor (CAR)-engineered T (CAR-T) cells is a powerful form of cancer immunotherapy. CAR-T cells can be redirected to specifically recognize tumor-associated antigens (TAAs) and induce high levels of antitumor activity. However, they may also display "on-target off-tumor" toxicities, resulting from low-level expression of TAAs in healthy tissues. These adverse effects have raised considerable safety concerns and limited the clinical application of this otherwise promising therapeutic modality. To minimize such side effects, we have designed an epidermal growth factor receptor (EGFR)-specific masked CAR (mCAR), which consists of a masking peptide that blocks the antigen-binding site and a protease-sensitive linker. Proteases commonly active in the tumor microenvironment can cleave the linker and disengage the masking peptide, thereby enabling CAR-T cells to recognize target antigens only at the tumor site. In vitro mCAR showed dramatically reduced antigen binding and antigen-specific activation in the absence of proteases, but normal levels of binding and activity upon treatment with certain proteases. Masked CAR-T cells also showed antitumor efficacy in vivo comparable to that of unmasked CAR. Our study demonstrates the feasibility of improving the safety profile of conventional CARs and may also inspire future design of CAR molecules targeting broadly expressed TAAs.

Nanogel-Integrated pH-Responsive Composite Hydrogels for Controlled Drug Delivery.

A novel pH-sensitive hydrogel system consisting of poly(methacrylic acid-g-ethylene glycol) (P(MAA-g-EG)) and acryloyl group modified-cholesterol-bearing pullulan (CHPOA) nanogels was developed for the controlled delivery of an anticonvulsant drug, pregabalin (PGB). Here, the hydrophilic hydrogel network provides the pH-sensitive swelling behavior, whereas nanogel components form separate reservoirs for the delivery of drugs with different hydrophobicities. These nanocarrier-integrated hybrid gels were synthesized through both surface-initiated and bulk photopolymerization approaches. The swelling and drug release behavior of these pH-responsive hydrogels synthesized by different photopolymerization approaches at visible and UV light wavelenghts were studied at acidic and basic pH values. Nanogel-integrated hydrogels exhibited higher swelling behavior compared to plain hydrogels in reversible swelling experiments. Similarly, the presence of nanogels in hydrogel network enhanced the loading and release percentages of PGB and the release was analyzed to describe the mode of transport through the network. In vitro cytotoxicity assay suggests that hydrogels in altered groups are nontoxic. This is the first report about the visible light-induced synthesis of a pH-responsive network incorporated CHPOA nanogels. Responsive and multifunctional properties of this system could be used for pH-triggered release of therapeutic molecules for clinical applications.