Pharmacologically active levels of anti-TGFbeta2 aptamers can be sustained in the ocular fluid and local tissue environment over a 12-h period after single administration. Daily subconjunctival administration of PEGylated anti-TGFbeta2 aptamers should allow further pharmacological evaluation of these agents in a rabbit conjunctival scarring model. Perioperative administration, via subconjunctival injection, may prove to be an effective means to deliver therapeutic quantities of TGFbeta2 aptamer conjugates in trabeculectomy procedures.

The specific down-regulation of gene expression in cells is a powerful method for elucidating a gene's function. A common method for suppressing gene expression is the elimination of mRNA by RNAi or antisense. Alternatively, oligonucleotide-derived aptamers have been used as protein-directed agents for the specific knock-down of both intracellular and extracellular protein activity. Protein-directed methods offer the advantage of more closely mimicking small molecule therapeutics' mechanism of activity. Furthermore, protein-directed methods may synergize with RNA-directed methods since the two methods attack gene expression at different levels. Here we have knocked down a well-characterized intracellular protein's activity, NFκB, by expressing either aptamers or small interfering RNAs (siRNAs). Both methods can diminish NFκB's activity to similar levels (from 29 to 64%). Interestingly, expression of both aptamers and siRNAs simultaneously, suppressed NFκB activity better than either method alone (up to 90%). These results demonstrate that the expression of intracellular aptamers is a viable alternative to siRNA knock-down. Furthermore, for the first time, we show that the use of aptamers and siRNA together can be the most effective way to achieve maximal knock-down of protein activity.

Purpose The solid tumor microenvironment (TME) drives T cell dysfunction and inhibits the effectiveness of immunotherapies such as chimeric antigen receptor-based T cell (CAR T) cells. Early data has shown that modulation of T cell metabolism can improve intratumoral T cell function in preclinical models. Experimental design We evaluated GPC3 expression in human normal and tumor tissue specimens. We developed and evaluated BOXR1030, a novel CAR T therapeutic co-expressing glypican-3 (GPC3)-targeted CAR and exogenous glutamic-oxaloacetic transaminase 2 (GOT2) in terms of CAR T cell function both in vitro and in vivo. Results Cell surface expression of tumor antigen GPC3 was observed by immunohistochemical staining in tumor biopsies from hepatocellular carcinoma, liposarcoma, squamous lung cancer, and Merkel cell carcinoma patients. Compared to control GPC3 CAR alone, BOXR1030 (GPC3-targeted CAR T cell that co-expressed GOT2) demonstrated superior in vivo efficacy in aggressive solid tumor xenograft models, and showed favorable attributes in vitro including an enhanced cytokine production profile, a less-differentiated T cell phenotype with lower expression of stress and exhaustion markers, an enhanced metabolic profile and increased proliferation in TME-like conditions. Conclusions Together, these results demonstrated that co-expression of GOT2 can substantially improve the overall antitumor activity of CAR T cells by inducing broad changes in cellular function and phenotype. These data show that BOXR1030 is an attractive approach to targeting select solid tumors. To this end, BOXR1030 will be explored in the clinic to assess safety, dose-finding, and preliminary efficacy (NCT05120271).

PurposeThe solid tumor microenvironment (TME) drives T cell dysfunction and inhibits the effectiveness of immunotherapies such as chimeric antigen receptor-based T cell (CAR T) cells. Early data has shown that modulation of T cell metabolism can improve intratumoral T cell function in preclinical models.Experimental DesignWe evaluated GPC3 expression in human normal and tumor tissue specimens. We developed and evaluated BOXR1030, a novel CAR T therapeutic co-expressing glypican-3 (GPC3)-targeted CAR and exogenous glutamic-oxaloacetic transaminase 2 (GOT2) in terms of CAR T cell function both in vitro and in vivo.ResultsExpression of tumor antigen GPC3 was observed by immunohistochemical staining in tumor biopsies from hepatocellular carcinoma, liposarcoma, squamous lung cancer, and Merkel cell carcinoma patients. Compared to control GPC3 CAR alone, BOXR1030 (GPC3-targeted CAR T cell that co-expressed GOT2) demonstrated superior in vivo efficacy in aggressive solid tumor xenograft models, and showed favorable attributes in vitro including an enhanced cytokine production profile, a less-differentiated T cell phenotype with lower expression of stress and exhaustion markers, an enhanced metabolic profile and increased proliferation in TME-like conditions.ConclusionsTogether, these results demonstrated that co-expression of GOT2 can substantially improve the overall antitumor activity of CAR T cells by inducing broad changes in cellular function and phenotype. These data show that BOXR1030 is an attractive approach to targeting select solid tumors. To this end, BOXR1030 will be explored in the clinic to assess safety, dose-finding, and preliminary efficacy (NCT05120271).Statement of Translational RelevanceChimeric antigen receptor-based T cell (CAR T) therapeutics have revolutionized the field of oncology. Despite early successes targeting hematological malignancies, substantial challenges limit application of CAR T therapy in solid tumors, in part due to the suppressive tumor microenvironment which drives T cell exhaustion and metabolic dysfunction. Glutamic-oxaloacetic transaminase 2 (GOT2) is a mitochondrial enzyme in glutamine metabolism and contributes to cellular redox balance.Glypican-3 (GPC3) is an oncofetal tumor antigen with restricted expression on normal tissues and high prevalence in several solid tumors. We describe BOXR1030, a novel CAR T therapeutic co-expressing GPC3-targeted CAR and exogenous GOT2. Compared to T cells expressing CAR alone, BOXR1030 T cells demonstrated superior in vivo efficacy and have favorable attributes including enhanced cytokine production, a less-differentiated phenotype with lower expression of exhaustion markers, and an enhanced metabolic profile. These data support BOXR1030 as a potential treatment to explore in select solid tumor indications.

GPC3 is an oncofetal tumor antigen that is an attractive target for CAR T-cell therapy due to its highly restricted expression on normal tissue and high prevalence in several adult and pediatric solid tumors, including hepatocellular carcinoma and squamous cell lung carcinoma. However, solid tumors create an unfavorable microenvironment that restricts critical nutrients, drives T-cell dysfunction, and inhibits the effectiveness of cellular therapies. BOXR1030 is a first-in-class engineered cell therapy identified from a screen of > 100 bolt-on transgenes, composed of a humanized GPC3-targeting CAR and glutamic-oxaloacetic transaminase 2 (GOT2) mitochondrial enzyme to enhance T-cell metabolic function. GOT2 plays a central metabolic role, linking multiple pathways involved in biosynthesis and cellular energy production, and addition of the GOT2 bolt-on leads to increased amino acid uptake and improved antioxidant capacity of BOXR1030 T cells. As the first product candidate from the BOXR platform, BOXR1030 represents a novel approach to modulate T-cell metabolism, specifically engineered to overcome immunosuppressive challenges that limit conventional CAR T cells. In preclinical studies across tumor microenvironment (TME) conditions including hypoxia, nutrient restriction, and exhaustion driven by chronic stimulation, BOXR1030 T cells had improved function relative to control CAR T cells lacking the GOT2 bolt-on. These functional changes result in superior in vivo anti-tumor activity without altering target specificity. BOXR1030 activity is target-dependent with no evidence of off-target cytotoxicity or cytokine release in the presence of GPC3-negative cell lines. Binding specificity of BOXR1030 T cells was evaluated against > 5000 human membrane and secreted proteins, and no off-target CAR interactions were identified. In tumor-bearing NSG mice, no abnormalities in hematology, clinical chemistry, or gross pathology were observed with BOXR1030 treatment. In addition, no evidence for target-independent proliferation of BOXR1030 T cells was observed in vivo. These preclinical data demonstrate the robust activity and specificity of BOXR1030 and support clinical investigation. BOXR1030 has potential application in GPC3-positive solid tumor indications with significant unmet medical need. IND-enabling studies are currently in progress to support a future first-in-human trial in subjects with GPC3-positive tumors. Citation Format: Eugene Choi, Kathleen Whiteman, Tapasya Pai, Taylor Hickman, Tyler Johnson, Taylor Friedman, Avani Parikh, Madaline Gilbert, Binzhang Shen, Glen J. Weiss, Seth Ettenberg, Kathleen E. McGinness, Greg Motz. BOXR1030: A first-in-class CAR T-cell therapy co-expressing GOT2 enhances T-cell metabolic function for the treatment of GPC3-positive solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2184.