Koichi Sasaki

Osteosarcoma (OS) is the most prevalent primary bone malignancy in children and adolescents; however, therapeutic outcomes remain suboptimal due to tumor heterogeneity, chemoresistance, and inadequate immune activation. Doxorubicin (Dox), the standard therapy that induces immunogenic cell death, has its efficacy compromised by the immunosuppressive tumor microenvironment (TME). While interleukin-12 (IL-12) can activate and recruit various immune cells, making it an attractive combination partner, its systemic delivery is severely limited by dose-limiting toxicity. We have previously reported that intravenous injection of A3 collagen binding domain (CBD) of von Willebrand Factor preferentially accumulates into the TME of various tumor models enriched in collagen I and III. Furthermore, CBD-fused IL-12 (CBD-IL-12) demonstrated superior therapeutic effects against various cancer models compared to unmodified IL-12 due to its collagen-targeted delivery and the resulting tumor-localized inflammation. Given that the OS TME also exhibits higher collagen I and III expression compared to normal bone, we hypothesized that a CBD-IL-12 fusion protein could showcase potent anti-tumor efficacy in OS via tumor-specific accumulation. Here, we demonstrated that CBD-IL-12 exhibited 4-fold enhanced tumor accumulation compared to unmodified IL-12 and increased cytotoxic T cell infiltration by 2.2-fold within the immune-cold microenvironment in a mouse model of OS. The combination of CBD-IL-12 with Dox significantly prolonged median survival in two independent murine OS models. This coordinated approach utilizing Dox coupled with precision-targeted IL-12 immunotherapy represents a clinically translatable strategy that overcomes the inherent limitations of single-agent treatments for OS. Highlight: Collagen-targeted IL-12 increases tumor accumulation in osteosarcoma. The collagen-targeted IL-12 synergizes with doxorubicin in osteosarcoma models. Combination therapy enhances T cell differentiation and activates innate immunity.

Immunosuppressive microenvironments, the lack of immune infiltration, and antigen heterogeneity pose challenges for chimaeric antigen receptor (CAR)-T cell therapies applied to solid tumours. Previously, CAR-T cells were armoured with immunostimulatory molecules, such as interleukin 12 (IL-12), to overcome this issue, but faced high toxicity. Here we show that collagen-binding domain-fused IL-12 (CBD-IL-12) secreted from CAR-T cells to target human six transmembrane epithelial antigen of prostate 1 (STEAP1) is retained within murine prostate tumours. This leads to high intratumoural interferon-γ levels, without hepatotoxicity and infiltration of T cells into non-target organs compared with unmodified IL-12. Both innate and adaptive immune compartments are activated and recognize diverse tumour antigens after CBD-IL-12-armoured CAR-T cell treatment. A combination of CBD-IL-12-armoured CAR-T cells and immune checkpoint inhibitors eradicated large tumours in an established prostate cancer mouse model. In addition, human CBD-IL-12-armoured CAR-T cells showed potent anti-tumour efficacy in a 22Rv1 xenograft while reducing circulating IL-12 levels compared with unmodified IL-12-armoured CAR-T cells. CBD fusion to potent payloads for CAR-T therapy may remove obstacles to their clinical translation towards elimination of solid tumours.

Immunosuppressive microenvironments, the lack of immune infiltration, and antigen heterogeneity pose significant challenges for chimeric antigen receptor (CAR)-T cell therapies to tackle solid tumors. CAR-T cells were armed with immunostimulatory payloads, such as interleukin-12 (IL-12), to overcome this issue, but faced intolerable toxicity during clinical development. Here, we show that collagen-binding domain-fused IL-12 (CBD-IL-12) was retained within syngeneic murine prostate tumors, after secretion from CAR-T cells targeting human six transmembrane epithelial antigen of the prostate 1 (STEAP1). This led to equivalently high intratumoral interferon-γ levels without hepatotoxicity and infiltration of T cells into non-target organs, compared with unmodified IL-12. Both innate and adaptive immune compartments were dramatically activated and recognized diverse tumor antigens after CBD-IL-12 CAR-T cell treatment. Combination immunotherapy of CBD-IL-12 CAR-T cells and immune checkpoint inhibitors eradicated large tumors in an established prostate cancer model, without pre-conditioning chemotherapy. The therapy generated anti-tumor immunological memory. CBD-fusion to potent yet toxic payloads of CAR-T therapy may remove obstacles to their clinical translation towards elimination of solid tumors.

Six transmembrane epithelial antigen of the prostate 1 (STEAP1) is a cell surface antigen for therapeutic targeting in prostate cancer. Here, we report broad expression of STEAP1 relative to prostate-specific membrane antigen (PSMA) in lethal metastatic prostate cancers and the development of a STEAP1-directed chimeric antigen receptor (CAR) T cell therapy. STEAP1 CAR T cells demonstrate reactivity in low antigen density, antitumor activity across metastatic prostate cancer models, and safety in a human STEAP1 knock-in mouse model. STEAP1 antigen escape is a recurrent mechanism of treatment resistance and is associated with diminished tumor antigen processing and presentation. The application of tumor-localized interleukin-12 (IL-12) therapy in the form of a collagen binding domain (CBD)-IL-12 fusion protein combined with STEAP1 CAR T cell therapy enhances antitumor efficacy by remodeling the immunologically cold tumor microenvironment of prostate cancer and combating STEAP1 antigen escape through the engagement of host immunity and epitope spreading.

Strategies for reducing CRS in CAR-T cells capitalize on the specificity between CAR-T cells, cytokines, and their receptors, the role of macrophages in cytokine release, and genetic constructs to ablate CAR-T function reversibly and irreversibly.

PH-Sensitive branched β-glucan-modified liposomes promote antigen uptake and activation of dendritic cells in vitro , inducing anti-tumor immune responses in vivo .

Antibody-recruiting molecules (ARMs) are bispecific molecules composed of an antibody-binding motif and a target-binding motif that redirect endogenous antibodies to target cells to elicit immune responses. To enhance the translational potential of ARMs, it is crucial to design antibody/target-binding motifs that have strong affinity and are easy to synthesize. Here, we synthesized a novel Fc-binding ARM (Fc-ARM) that targets folate receptor (FR)-positive cancer cells, Reo-3, using a recently developed monocyclic peptide 15-Lys8Leu, which binds strongly to the Fc region of an antibody. Reo-3 bound to the Fc region of the antibody with a K_d of 5.8 nM, and recruited a clinically used antibody mixture to attack FR-positive IGROV-1 cells as efficiently as Fc-ARM2, in which a bicyclic Fc-binding peptide was used. These results indicate that 15-Lys8Leu, which can be synthesized readily, is suitable for various applications including the development of Fc-ARMs.

Synthetic small molecules that redirect endogenous antibodies to target cells are promising drug candidates because they overcome the potential shortcomings of therapeutic antibodies, such as immunogenicity and the need for intravenous delivery. Previously, we reported a novel class of bispecific molecules targeting the antibody Fc region and folate receptor, named Fc‐binding antibody‐recruiting molecules (Fc‐ARMs). Fc‐ARMs can theoretically recruit most endogenous antibodies, inducing antibody‐dependent cell‐mediated cytotoxicity (ADCC) to eliminate cancer cells. Herein, we describe new Fc‐ARMs that target prostate cancer (Fc‐ARM‐Ps). Fc‐ARM‐Ps recruited antibodies to cancer cells expressing prostate‐specific membrane antigen but did so with lower efficiency compared with Fc‐ARMs targeting the folate receptor. Upon recruitment by Fc‐ARM‐P, defucosylated antibodies efficiently activated natural killer cells and induced ADCC, whereas antibodies with intact N‐glycans did not. The results suggest that the affinity between recruited antibodies and CD16a, a type of Fc receptor expressed on immune cells, could be a key factor controlling immune activation in the Fc‐ARM strategy.

Synthetic small molecules that redirect endogenous antibodies to target cells are promising drug candidates because they overcome the potential shortcomings of therapeutic antibodies, such as immunogenicity. Previously, we reported a novel class of bispecific molecules targeting the antibody Fc region and folate receptor, named Fc-binding antibody-recruiting molecules (Fc-ARMs). Fc-ARMs can theoretically recruit most endogenous antibodies, inducing cancer cell elimination via antibody-dependent cell-mediated cytotoxicity (ADCC). Here, we describe new Fc-ARMs that target prostate cancer (Fc-ARM-Ps). Fc-ARM-Ps recruited antibodies to cancer cells expressing prostate membrane-specific antigen but did so with lower efficiency compared with Fc-ARMs targeting folate receptor. Upon recruitment by Fc-ARM-P, defucosylated antibodies efficiently activated natural killer cells and induced ADCC, whereas antibodies with intact N-glycans did not. The results suggest that the affinity between recruited antibodies and CD16a, a type of Fc receptor expressed on immune cells, could be a key factor controlling immune activation in the Fc-ARM strategy.