Tumor-specific activation of folate receptor beta enables reprogramming of immune cells in the tumor microenvironment.

Folate receptors can perform folate transport, cell adhesion, and/or transcription factor functions. The beta isoform of the folate receptor (FRß) has attracted considerable attention as a biomarker for immunosuppressive macrophages and myeloid-derived suppressor cells, however, its role in immunosuppression remains uncharacterized. We demonstrate here that FRß cannot bind folate on healthy tissue macrophages, but does bind folate after macrophage incubation in anti-inflammatory cytokines or cancer cell-conditioned media. We further show that FRß becomes functionally active following macrophage infiltration into solid tumors, and we exploit this tumor-induced activation to target a toll-like receptor 7 agonist specifically to immunosuppressive myeloid cells in solid tumors without altering myeloid cells in healthy tissues. We then use single-cell RNA-seq to characterize the changes in gene expression induced by the targeted repolarization of tumor-associated macrophages and finally show that their repolarization not only changes their own phenotype, but also induces a proinflammatory shift in all other immune cells of the same tumor mass, leading to potent suppression of tumor growth. Because this selective reprogramming of tumor myeloid cells is accompanied by no systemic toxicity, we propose that it should constitute a safe method to reprogram the tumor microenvironment.

Antitumor activity of AZD0754, a dnTGFßRII-armored, STEAP2-targeted CAR-T cell therapy, in prostate cancer.

Prostate cancer is generally considered an immunologically "cold" tumor type that is insensitive to immunotherapy. Targeting surface antigens on tumors through cellular therapy can induce a potent antitumor immune response to "heat up" the tumor microenvironment. However, many antigens expressed on prostate tumor cells are also found on normal tissues, potentially causing on-target, off-tumor toxicities and a suboptimal therapeutic index. Our studies revealed that six-transmembrane epithelial antigen of prostate-2 (STEAP2) was a prevalent prostate cancer antigen that displayed high, homogeneous cell surface expression across all stages of disease with limited distal normal tissue expression, making it ideal for therapeutic targeting. A multifaceted lead generation approach enabled development of an armored STEAP2 chimeric antigen receptor T cell (CAR-T) therapeutic candidate, AZD0754. This CAR-T product was armored with a dominant-negative TGF-ß type II receptor, bolstering its activity in the TGF-ß-rich immunosuppressive environment of prostate cancer. AZD0754 demonstrated potent and specific cytotoxicity against antigen-expressing cells in vitro despite TGF-ß-rich conditions. Further, AZD0754 enforced robust, dose-dependent in vivo efficacy in STEAP2-expressing cancer cell line-derived and patient-derived xenograft mouse models, and exhibited encouraging preclinical safety. Together, these data underscore the therapeutic tractability of STEAP2 in prostate cancer as well as build confidence in the specificity, potency, and tolerability of this potentially first-in-class CAR-T therapy.

Engineered human pluripotent stem cell-derived natural killer cells with PD-L1 responsive immunological memory for enhanced immunotherapeutic efficacy.

Adoptive chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have shown promise in treating various cancers. However, limited immunological memory and access to sufficient numbers of allogenic donor cells have hindered their broader preclinical and clinical applications. Here, we first assess eight different CAR constructs that use an anti-PD-L1 nanobody and/or universal anti-fluorescein (FITC) single-chain variable fragment (scFv) to enhance antigen-specific proliferation and anti-tumor cytotoxicity of NK-92 cells against heterogenous solid tumors. We next genetically engineer human pluripotent stem cells (hPSCs) with optimized CARs and differentiate them into functional dual CAR-NK cells. The tumor microenvironment responsive anti-PD-L1 CAR effectively promoted hPSC-NK cell proliferation and cytotoxicity through antigen-dependent activation of phosphorylated STAT3 (pSTAT3) and pSTAT5 signaling pathways via an intracellular truncated IL-2 receptor ß-chain (ΔIL-2Rß) and STAT3-binding tyrosine-X-X-glutamine (YXXQ) motif. Anti-tumor activities of PD-L1-induced memory-like hPSC-NK cells were further boosted by administering a FITC-folate bi-specific adapter that bridges between a programmable anti-FITC CAR and folate receptor alpha-expressing breast tumor cells. Collectively, our hPSC CAR-NK engineering platform is modular and could constitute a realistic strategy to manufacture off-the-shelf CAR-NK cells with immunological memory-like phenotype for targeted immunotherapy.

Advances and challenges of CAR T therapy and suitability of animal models (Review).

Chimeric antigen receptors (CARs) recently gained momentum in cancer treatment due to their ability to promote T-cell mediated responses to a specific tumor-associated antigen. CARs are part of the adoptive cell transfer (ACT) strategies that utilize patients' T lymphocytes, genetically engineered to kill cancer cells. However, despite the therapy's success against blood-related malignancies, treating solid tumors has not reached its fullest potential yet. The reasons include the complex suppressive tumor microenvironment, mutations on cancer cells' target receptors, lethal side-effects, restricted trafficking into the tumor, suboptimal persistence in vivo and the lack of animal models that faithfully resemble human tumor's immunological responses. Currently, rodent models are used to investigate the safety and efficacy of CAR therapies. However, these models are limited in representing the human disease faithfully, fail to predict the adverse treatment events and overestimate the efficacy of the therapy. On the other hand, spontaneously developed tumors in dogs are more suited in CAR research and their efficacy has been demonstrated in a number of diseases, including lymphoma, osteosarcoma and mammary tumors. The present review discusses the design and evolution of CARs, challenges of CAR in solid tumors, human and canine clinical trials and advantages of the canine model.

Repolarization of Tumor-Infiltrating Myeloid Cells for Augmentation of CAR T Cell Therapies.

Although CAR T cell therapies have proven to be effective in treating hematopoietic cancers, their abilities to regress solid tumors have been less encouraging. Mechanisms to explain these disparities have focused primarily on differences in cancer cell heterogeneity, barriers to CAR T cell penetration of solid tumors, and immunosuppressive microenvironments. To evaluate the contributions of immunosuppressive tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) on CAR T cell efficacies, we have exploited the ability of a folate-targeted Toll-like receptor 7 agonist (FA-TLR7-1A) to specifically reactivate TAMs and MDSCs from an immunosuppressive to pro-inflammatory phenotype without altering the properties of other immune cells. We report here that FA-TLR7-1A significantly augments standard CAR T cell therapies of 4T1 solid tumors in immune competent mice. We further show that co-administration of the FA-TLR7-1A with the CAR T cell therapy not only repolarizes TAMs and MDSCs from an M2-like anti-inflammatory to M1-like pro-inflammatory phenotype, but also enhances both CAR T cell and endogenous T cell accumulation in solid tumors while concurrently increasing their states of activation. Because analogous myeloid cells in healthy tissues ar not altered by administration of FA-TLR7-1A, no systemic activation of the immune system nor accompanying weight loss is observed. These data argue that immunosuppressive myeloid cells contribute prominently to the failure of CAR T cells to eradicate solid tumors and suggest that methods to reprogram tumor associated myeloid cells to a more inflammatory phenotype could significantly augment the potencies of CAR T cell therapies.

Design of Neuraminidase-Targeted Imaging and Therapeutic Agents for the Diagnosis and Treatment of Influenza Virus Infections.

The last step in influenza virus replication involves the assembly of viral components on the infected cell's plasma membrane followed by budding of intact virus from the host cell surface. Because viral neuraminidase and hemagglutinin are both inserted into the host cell's membrane during this process, influenza virus-infected cells are distinguished from uninfected cells by the presence of viral neuraminidase and hemagglutinin on their cell surfaces. In an effort to exploit this difference in cell surface markers for development of diagnostic and therapeutic agents, we have modified an influenza neuraminidase inhibitor, zanamivir, for targeting of attached imaging and therapeutic agents selectively to influenza viruses and virus-infected cells. We have designed here a zanamivir-conjugated rhodamine dye that allows visual monitoring of binding, internalization, and intracellular trafficking of the fluorescence-labeled neuraminidase in virus-infected cells. We also synthesize a zanamivir-99mTc radioimaging conjugate that permits whole body imaging of the virus's biodistribution and abundance in infected mice. Finally, we create both a zanamivir-targeted cytotoxic drug (i.e., zanamivir-tubulysin B) and a viral neuraminidase-targeted CAR T cell and demonstrate that they are both able to kill viral neuraminidase-expressing cells without damaging healthy cells. Taken together, these data suggest that the influenza virus neuraminidase inhibitor, zanamivir, can be exploited to improve the diagnosis, imaging, and treatment of influenza virus infections.