Here, we report on a novel bispecific antibody-derivative, designated RTX-CD47, with unique capacity for CD20-directed inhibition of CD47-SIRPα “don't eat me” signaling. RTX-CD47 comprises a CD20-targeting scFv antibody fragment derived from rituximab fused in tandem to a CD47-blocking scFv. Single agent treatment with RTX-CD47 triggered significant phagocytic removal of CD20pos/CD47pos malignant B-cells, but not of CD20neg/CD47pos cells, and required no pro-phagocytic FcR-mediated signaling. Importantly, treatment with RTX-CD47 synergistically enhanced the phagocytic elimination of primary malignant B cells by autologous phagocytic effector cells as induced by therapeutic anticancer antibodies daratumumab (anti-CD38), alemtuzumab (anti-CD52) and obinutuzumab (anti-CD20). In conclusion, RTX-CD47 blocks CD47 “don't eat me” signaling by cancer cells in a CD20-directed manner with essentially no activity towards CD20neg/CD47pos cells and enhances the activity of therapeutic anticancer antibodies directed to B-cell malignancies.

Chondroitin-sulfate proteoglycan 4 (CSPG4) is a transmembrane glycoprotein overexpressed on malignant cells in several cancer types with only limited expression on normal cells. CSPG4 is implicated in several signaling pathways believed to drive cancer progression, particularly proliferation, motility and metastatic spread. Expression may serve as a prognostic marker for survival and risk of relapse in treatment-resistant malignancies including melanoma, triple negative breast cancer, rhabdomyosarcoma and acute lymphoblastic leukemia. This tumor-associated overexpression of CSPG4 points towards a highly promising therapeutic target for antibody-guided cancer therapy. Monoclonal αCSPG4 antibodies have been shown to inhibit cancer progression by blocking ligand access to the CSPG4 extracellular binding sites. Moreover, CSPG4-directed antibody conjugates have been shown to be selectively internalized by CSPG4-expressing cancer cells via endocytosis. CSPG4-directed immunotherapy may be approached in several ways, including: (1) antibody-based fusion proteins for the selective delivery of a pro-apoptotic factors such as tumor necrosis factor-related apoptosis-inducing ligand to agonistic death receptors 4 and 5 on the cell surface; and (2) CSPG4-specific immunotoxins which bind selectively to diseased cells expressing CSPG4, are internalized by them and induce arrest of biosynthesis, closely followed by initiation of apoptotic signaling. Here we review various methods of exploiting tumor-associated CSPG4 expression to improve targeted cancer therapy.

TNFR superfamily (TNFRSF) members have important immunoregulatory functions and are of clear interest for cancer immunotherapy. Various TNFRSF agonists have been clinically evaluated, but have met with limited efficacy and/or toxicity. Recent insights indicate that ‘first-generation’ TNFRSF agonists lack efficacy as they do not effectively cross-link their corresponding receptor. Reversely, ubiquitous TNFRSF receptor(s) cross-linking by CD40 and Fas agonistic antibodies resulted in dose-limiting liver toxicity. To overcome these issues, we developed a novel pretargeting strategy exploiting recombinant fusion proteins in which a soluble form of TRAIL, FasL or CD40L is genetically fused to a high-affinity anti-fluorescein scFv antibody fragment (scFvFITC). Fusion proteins scFvFITC:sTRAIL and scFvFITC:sFasL induced potent target antigen-restricted apoptosis in a panel of cancer lines and in primary patient-derived cancer cells, but only when pretargeted with a relevant FITC-labelled antitumour antibody. In a similar pretargeting setting, fusion protein scFvFITC:sCD40L promoted tumour-directed maturation of immature monocyte-derived dendritic cells (iDCs). This novel tumour-selective pretargeting approach may be used to improve efficacy and/or reduce possible off-target toxicity of TNFSF ligands for cancer immunotherapy.