1Following the success of immune checkpoint blockade (ICB) therapy against cancer, agonistic 2 antibodies targeting T cell co-stimulatory pathways, are in clinical trials. The tumor necrosis factor 3 superfamily of receptors (TNFRSF) members CD137 and OX40 are co-stimulatory receptors that 4 stimulate T cell proliferation and activation upon interaction with their cognate ligands. Activating 5 CD137 and OX40 with agonistic monoclonal antibodies stimulates the immune system due to their 6 broad expression on CD4 + and CD8 + T cells and NK cells and has antitumor effects in pre-clinical 7 models. Most TNFRSF agonist antibodies require crosslinking via Fc receptors (FcRs), which can 8 limit their clinical activity. FS120 mAb 2 ™, a dual agonist bispecific antibody targeting CD137 and 9 OX40, activated both CD4 + and CD8 + T cells in a FcR-independent mechanism, dependent on 10 concurrent binding. A mouse surrogate version of the bispecific antibody displayed antitumor 11 activity in syngeneic tumor models, independent of T regulatory cell (Treg) depletion and of FcR-12interaction, but associated with peripheral T cell activation and proliferation. When compared to a 13 crosslink-independent CD137 agonist monoclonal antibody, the FS120 surrogate induced lower liver 14 T cell infiltration. These data support initiation of clinical development of FS120, a first-in-class dual 15 agonist bispecific antibody for the treatment of human cancer. 16 17

Realizing the translational impacts of three-dimensional (3D) bioprinting for cancer research necessitates innovation in bioprinting workflows which integrate affordability, user-friendliness, and biological relevance. Herein, we demonstrate ‘BioArm’, a simple, yet highly effective extrusion bioprinting platform, which can be folded into a carry-on pack, and rapidly deployed between bio-facilities. BioArm enabled the reconstruction of compartmental tumoroids with cancer-associated fibroblasts (CAFs), forming the shell of each tumoroid. The 3D printed core-shell tumoroids showed de novo synthesized extracellular matrices, and enhanced cellular proliferation compared to the tumor alone 3D printed spheroid culture. Further, the in vivo phenotypes of CAFs normally lost after conventional 2D co-culture re-emerged in the bioprinted model. Embedding the 3D printed tumoroids in an immune cell-laden collagen matrix permitted tracking of the interaction between immune cells and tumoroids, and subsequent simulated immunotherapy treatments. Our deployable extrusion bioprinting workflow could significantly widen the accessibility of 3D bioprinting for replicating multi-compartmental architectures of tumor microenvironment, and for developing strategies in cancer drug testing in the future.

Three-dimensional (3D) bioprinting has emerged as a promising tool for constructing tumor microenvironments (TME) for cancer modelling in vitro. Realizing the translational impacts of 3D bioprinting for cancer research necessitates innovation in bioprinting workflows which integrate affordability, user-friendliness, and biological relevance. Herein, we demonstrate ‘bioArm’, a simple, yet highly effective extrusion bioprinting platform, which can be folded into a carry-on pack, and rapidly deployed between bio-facilities. BioArm enabled TME reconstruction in the form of 3D core-shell tumoroids with cancer-associated fibroblasts (CAFs). Tumoroids showed the presence of a heterogenous population of CAFs with de novo synthesized extracellular matrices, demonstrating more in vivo-like characteristics compared to conventional 2D co-culture models. Embedding the 3D printed tumoroids in an immune cell laden collagen matrix permitted tracking of the interaction between immune cells and tumoroids, and subsequent immunotherapy treatments. Our deployable extrusion bioprinting workflow could significantly widen the accessibility of 3D bioprinting for gaining mechanistic understanding in TME, and for developing strategies in cancer drug testing.