This study investigated a dual crosslinking paradigm, consisting of (a) photocrosslinking with Rose Bengal (RB) and green light followed by (b) chemical crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) to enhance collagen gel stiffness. In group 1, 50 μL collagen constructs of 2% (w/v) type I collagen containing 10 μM RB were allowed to gel spontaneously at 37 °C. In group 2, the spontaneous gels were exposed to green light (532 nm). In group 3, the photochemically crosslinked gels were subsequently treated with a 1-h exposure to 33 mM EDC/6 mM NHS. Samples (n = 18) were subjected to 0.08% (w/v) collagenase digestion, and the storage modulus of samples was measured by rheometry. Viability of encapsulated chondrocytes was measured by live/dead assay. Chondrocytes were ≥ 95% viable in all constructs at 10 days in vitro. Resistance to collagenase digestion increased as; spontaneous gels (2 h) < photochemical gels (3-4 h) < dual crosslinked gels (>24 h). The storage modulus of dual-crosslinked constructs was increased 5-fold over both photocrosslinked and spontaneous gels. As the dual crosslinking paradigm did not reduce encapsulated chondrocyte viability, these crosslinked collagen hydrogels could be a useful tool for the practical delivery of encapsulated chondrocytes to articular defects.

Engineered, off-the-shelf, allogeneic natural killer (NK) cell therapy is an attractive approach for targeting solid tumors, given their emerging clinical safety, efficacy, and intrinsic anti-tumor recognition and activity. However, improvements to support persistence and maintain durable anti-tumor activity within the tumor microenvironment may be necessary to achieve meaningful clinical efficacy. Here we describe the preclinical activity of CAT-248, a CD70-directed CAR-NK cell therapy, engineered using the TcBuster™ Transposon System (Bio-Techne) multiplexed with CRISPR/Cas9 editing. CD70 is highly expressed in many tumor types while normal tissue expression is restricted to a subset of activated immune cells. CAT-248 is an allogeneic, healthy donor peripheral blood-derived NK cell product designed for durable efficacy against CD70 expressing tumors. CAT-248 is engineered to express CD70 CAR, interleukin 15 (IL-15), and transforming growth factor β (TGFβ) dominant negative receptor (DNR). In addition, CAT-248 includes CRISPR/Cas9 knockout of CD70 to mitigate fratricide due to endogenous CD70 expression in activated NK cells. IL-15 enhances persistence of CAT-248 to enable durable efficacy, and TGFβ DNR enables CAT-248 to maintain high activity in TGFβ-enriched and immunosuppressive solid tumor microenvironments. CAT-248 is manufactured using transposon-based engineering which enables stable integration of the three transgenes and CRISPR/Cas9 knockout of CD70 in a single electroporation step, resulting in 40-80% CAR expression and 80-90% knockout of CD70 in CAT-248 NK cells. CAT-248 activity was characterized across a panel of in vitro assays to evaluate the function of CD70 CAR, TGFβ DNR, and IL-15 transgenes. CD70-directed cytotoxicity was assessed against a panel of tumor cell lines with a broad range of CD70 expression. In vitro, CAT-248 cells demonstrated both CAR-dependent cytotoxicity and over 2-fold greater secretion of effector cytokines IFNγ and TNFα than control NK cells. TGFβ DNR effectively prevented TGFβ-induced SMAD phosphorylation and TGFβ-induced downregulation of DNAM-1, an NK cell activating receptor. IL-15 secretion enabled in vitro NK cell expansion over a 9-day time course without exogenous cytokine support. To confirm cytolytic activity in vivo, CAT-248 cells were administered therapeutically in a 786-O CD70+ renal cell carcinoma xenograft model. CAT-248 cells effectively controlled tumor, demonstrating &gt;99% reduction in tumor burden relative to control NK cells (p&lt;0.01). Further, CAT-248 cells demonstrated significant in vivo persistence beyond 4 weeks post-dosing in peripheral blood. Overall, the results demonstrate the potential for CAT-248 as a novel off-the-shelf, cryopreserved, allogeneic NK cell therapy for CD70-positive renal cell carcinoma and other solid tumor malignancies. Citation Format: Bashar Hamza, Eugene Choi, Dominic Picarella, Finola Moore, Angela Nunez, Henry Moreno, Marilyn Marques, Alexia Barandiaran, Meghan Walsh, Kisha Pradhan, Krista Daniel, Kelly Becker, Jennifer Johnson, Karl Malakian, Keith H. Wong, Mark A. Omobono, Charlotte Franco, Andres Alvarez, Jenna Nguyen, Billy Blanco, Priya Khurana, Beatriz Marques, Adriana Rivera, Michele McAuliffe, Jae-Woong Chang, Joshua Krueger, Beau R. Webber, Branden S. Moriarity, Joseph Gold, Cherry Thomas, Vipin Suri. CAT-248, an allogeneic CD70-directed CAR-NK cell therapy effectively controls CD70-positive tumor xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2898.