Molecular Therapy

A Genetically Engineered Primary Human Natural Killer Cell Platform for Cancer Immunotherapy

Enhancing natural killer (NK) cell cytotoxicity by blocking inhibitory signaling could lead to improved NK-based cancer immunotherapy. Thus, we have developed a highly efficient method for editing the genome of human NK cells using CRISPR/Cas9 to knock out inhibitory signaling molecules. Our method efficiently edits up to 90% of primary peripheral blood NK cells. As a proof-of-principle we demonstrate highly efficient knockout of ADAM17 and PDCD1, genes that have a functional impact on NK cells, and demonstrate that these gene-edited NK cells have significantly improved activity, cytokine production, and cancer cell cytotoxicity. Furthermore, we were able to expand cells to clinically relevant numbers, without loss of activity. We also demonstrate that our CRISPR/Cas9 method can be used for efficient knockin of genes by delivering homologous recombination template DNA using recombinant adeno-associated virus serotype 6 (rAAV6). Our platform represents a feasible method for generating engineered primary NK cells as a universal therapeutic for cancer immunotherapy.

CAR T Cells Targeting MISIIR for the Treatment of Ovarian Cancer and Other Gynecologic Malignancies

The prognosis of patients diagnosed with advanced ovarian or endometrial cancer remains poor, and effective therapeutic strategies are limited. The Müllerian inhibiting substance type 2 receptor (MISIIR) is a transforming growth factor β (TGF-β) receptor family member, overexpressed by most ovarian and endometrial cancers while absent in most normal tissues. Restricted tissue expression, coupled with an understanding that MISIIR ligation transmits apoptotic signals to cancer cells, makes MISIIR an attractive target for tumor-directed therapeutics. However, the development of clinical MISIIR-targeted agents has been challenging. Prompted by the responses achieved in patients with blood malignancies using chimeric antigen receptor (CAR) T cell therapy, we hypothesized that MISIIR targeting may be achieved using a CAR T cell approach. Herein, we describe the development and evaluation of a CAR that targets MISIIR. T cells expressing the MISIIR-specific CAR demonstrated antigen-specific reactivity in vitro and eliminated MISIIR-overexpressing tumors in vivo. MISIIR CAR T cells also recognized a panel of human ovarian and endometrial cancer cell lines, and they lysed a battery of patient-derived tumor specimens in vitro, without mediating cytotoxicity of a panel of normal primary human cells. In conclusion, these results indicate that MISIIR targeting for the treatment of ovarian cancer and other gynecologic malignancies is achievable using CAR technology.

The Vascular Disrupting Agent CA4P Improves the Antitumor Efficacy of CAR-T Cells in Preclinical Models of Solid Human Tumors

Chimeric antigen receptor (CAR) T cell therapy remains relatively ineffective against solid tumors due to inadequate infiltration and in vivo expansion of CAR-T cells. Unlike hematological malignancies, solid tumors have vascular barriers that hinder CAR-T cells from reaching the tumor site. Here, we demonstrated that combretastatin A-4 phosphate (CA4P), a vascular disrupting agent (VDA), can significantly improve the infiltration ability of CAR-T cells in solid tumors as evidenced by elevated levels of IFN-γ. Moreover, combined treatment with CA4P and CAR-T cells greatly increased the therapeutic efficiency of the CAR-T cells in subcutaneous ovarian cancer mouse xenograft models and patient-derived xenograft (PDX) models of colon and ovarian carcinoma. Our findings highlight CA4P as an effective antitumor agent candidate for combination with CAR-T cells in clinical applications to treat solid tumors.

Targeted degradation of the HPV oncoprotein E6 reduces tumor burden in cervical cancer

Human papillomavirus (HPV) remains a global health burden, yet there are no targeted therapies for HPV-related cancers. The HPV E6 protein is essential for tumorigenesis and immune evasion, making it an attractive target for antiviral drug development. In this study, we developed an E6-targeting proteolysis targeting chimera (PROTAC) that inhibits the growth of HPV

An oncolytic adenovirus co-expressing a bi-specific T cell engager and IL-2 for the treatment of ovarian cancer

BiTE-secreting T cells rationally combine with PD-1 blockade and vaccine boosting to reshape antitumor immunity in ovarian cancer

Despite some clinical success, ovarian cancer (OC) patients rarely achieve durable benefit from current immunotherapies, suggesting a need for strategies that improve OC immune recognition. We previously reported that engineered T cells secreting folate receptor alpha (FRα)-targeted bispecific T cell engagers (FR-B T cells) elicit robust antitumor responses in OC, in part by engaging endogenous T cells. Here, we use clinical OC specimens and preclinical OC to evaluate FR-B T cells combined with PD-1 blockade. Assessing the tumor microenvironment during acute and prolonged FR-B T cell + anti-PD-1 responses revealed broad immune cell engagement/reorganization. Early CD8+ T cell-driven responses and myeloid cell influx were followed by accumulation of CXCL13-producing macrophages, activated B cells, and effector memory CD4+ T cells with durable response, hallmarks that were diminished with progressive disease. Resistant OC (characterized by FRα loss and metabolic reprogramming) emerged at disease relapse, suggesting a need to target additional vulnerabilities to extend responses. As FR-B T cells promoted epitope spreading beyond FRα, we employed a booster vaccine to enhance antitumor immunity, improving OC control. Our findings point to rationally combining FR-B T cells with PD-1 blockade in OC and an opportunity to apply personalized cancer vaccines to limit OC relapse.

A platform to deliver single and bi-specific Cas9/guide RNA to perturb genes in vitro and in vivo

Although CRISPR-Cas9 technology is poised to revolutionize the treatment of diseases with underlying genetic mutations, it faces some significant issues limiting clinical entry. They include low-efficiency in vivo systemic delivery and undesired off-target effects. Here, we demonstrate, by modifying Cas9 with phosphorothioate-DNA oligos (PSs), that one can efficiently deliver single and bi-specific CRISPR-Cas9/guide RNA (gRNA) dimers in vitro and in vivo with reduced off-target effects. We show that PS-Cas9/gRNA-mediated gene knockout preserves chimeric antigen receptor T cell viability and expansion in vitro and in vivo. PS-Cas9/gRNA mediates gene perturbation in patient-derived tumor organoids and mouse xenograft tumors, leading to potent tumor antitumor effects. Further, HER2 antibody-PS-Cas9/gRNA conjugate selectively perturbs targeted genes in HER2

Sustainable Tumor-Suppressive Effect of iPSC-Derived Rejuvenated T Cells Targeting Cervical Cancers

Immunotherapy utilizing induced pluripotent stem cell (iPSC) technology has great potential. Functionally rejuvenated cytotoxic T lymphocytes (CTLs) can survive long-term as young memory T cells in vivo, with continuous tumor eradication. Banking of iPSCs as an unlimited "off-the-shelf" source of therapeutic T cells may be feasible. To generate safer iPSCs, we reprogrammed human papilloma virus type 16 (HPV16) E6-specific CTLs by Sendai virus vector without cotransduction of SV40 large T antigen. The iPSCs efficiently differentiated into HPV16-specific rejuvenated CTLs that demonstrated robust cytotoxicity against cervical cancer. The tumor-suppressive effect of rejuvenated CTLs was stronger and more persistent than that of original peripheral blood CTLs. These rejuvenated HPV16-specific CTLs provide a sustained tumor-suppressive effect even for epithelial cancers and constitute promising immunotherapy for cervical cancer.

Cancer-secreted exosomal miR-1468-5p promotes tumor immune escape via the immunosuppressive reprogramming of lymphatic vessels

Cancer-associated lymphatic endothelial cells (LECs) are an active barrier to the effector arm of the anti-tumor immune response; however, it remains unclear how LECs become immunosuppressive in the tumor microenvironment (TME). Exosomal microRNAs (miRNAs) have recently been implicated in intercellular crosstalk within the TME. Here, we report a mechanistic model via which cervical cancer-secreted, exosome-encapsulated microRNA (miR)-1468-5p promotes lymphatic PD-L1 upregulation and lymphangiogenesis to impair T cell immunity. Subsequently, exosomal miR-1468-5p epigenetically activates the JAK2/STAT3 pathway in LECs by directly targeting homeobox containing 1 (HMBOX1) in the SOCS1 promoter, activating an immunosuppressive program that allows cancer cells to escape anti-cancer immunity. Furthermore, clinical data reveal that high serum exosomal miR-1468-5p levels correlate with TME immunosuppressive status and poor prognosis in cervical cancer (CCa) patients. Taken together, our results suggest that cancer-secreted exosomal miR-1468-5p instructs LECs to form an integrated immunosuppressive TME component and may be a prognostic biomarker and therapeutic target for CCa.

Overcoming effector T cell exhaustion in ovarian cancer ascites with a novel adenovirus encoding for a MUC1 bispecific antibody engager and IL-2 cytokine

T cell-focused cancer immunotherapy including checkpoint inhibitors and cell therapies has been rapidly evolving over the past decade. Nevertheless, there remains a major unmet medical need in oncology generally and immuno-oncology specifically. We have constructed an oncolytic adenovirus, Ad5/3-E2F-d24-aMUC1aCD3-IL-2 (TILT-322), which is armed with a human aMUC1aCD3 T cell engager and IL-2. TILT-322 treatment stimulated T cell cytotoxicity through the increased presence of granzyme B, perforin, and interferon-gamma. Additional immune profiling indicated TILT-322 increased gamma delta T cell activation and impacted other cell types such as natural killer cells and natural killer-like T cells that are traditionally involved in cancer immunotherapy. TILT-322 treatment also decreased the proportion of exhausted CD8

Sodium valproate drives propionylation-mediated epigenetic reprogramming to enhance mesothelin CAR-T cell therapy in solid tumors