In the first experiment, a CEA+ and FAP+ colon adenocarcinoma tumor sample (patient 8) was incubated in a perfused system enabling three-dimensional tissue culture (bioreactor) (33). 1. Table S1. X-ray data collection and refinement statistics for human 4-1BBLCCH1/CL. Table S2. Surface plasmon resonance affinity constants of TAC4-1BBLs. Table S3. FAPC4-1BBL tumor targeting and tissue biodistribution in a CRC-bearing rhesus monkey. Table S4. Monomer content of FAPC4-1BBL in production batches. References (51C62) NIHMS1578534-supplement-supp.pdf (3.2M) GUID:?3A935900-E71F-47CB-8FB9-4CCA8C4B8C00 data: Data file S1. Primary data. NIHMS1578534-supplement-data.xlsx (3.3M) GUID:?F6D83725-A23C-4740-BFEE-7B31629408C8 Abstract Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic antiC4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fc receptorCmediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAPC4-1BBL (RG7826) and CD19C4-1BBL. In the presence of a T cell receptor transmission, they provide potent T cell costimulation purely dependent on tumor antigenCmediated hyperclustering without systemic activation by FcR binding. We could display focusing on of FAPC4-1BBL to FAP-expressing tumor stroma and lymph nodes inside a colorectal cancerCbearing rhesus monkey. Combination of FAPC4-1BBL with tumor antigenCtargeted T cell bispecific (TCB) molecules in human being tumor samples led to improved IFN- and granzyme B secretion. Further, combination of FAPC or CD19C4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral build up of triggered Rabbit Polyclonal to OR1A1 effector CD8+ T cells. FAPC Ritanserin and CD19C4-1BBL therefore represent an off-the-shelf combination immunotherapy without requiring genetic changes of effector cells for the treatment of solid and hematological malignancies. Intro Cancer immunotherapy offers changed the malignancy treatment paradigm, but there remains a substantial need for improvement, particularly for individuals with tumors lacking cytotoxic T cell infiltrates at baseline (1C3). Costimulation of triggered T cells (4), natural killer (NK) cells, and additional immune cells (5) via the tumor necrosis element receptor superfamily (TNFRSF) member 4-1BB (CD137) has emerged as a encouraging approach for malignancy immunotherapy (3, 6, 7). 4-1BB costimulation of T cells enhances proliferation, cytotoxicity, T helper cell (TH1) polarization and cytokine secretion (4, 8C10), metabolic fitness (7), changes of DNA methylation (11), and T cell memory space formation (7), and counteracts exhaustion (9) and activation-induced cell death (10). Today, two methods relying on 4-1BB agonism have entered clinical tests: (we) agonistic anti-human 4-1BB antibodies (3, 6, 12, 13) and (ii) second-/third-generation 4-1BB/CD3 chimeric antigen receptor (CAR) T cells (7, 14, 15). The agonistic anti-human 4-1BB human being immunoglobulin G4 (IgG4) antibody (antiChu4-1BB huIgG4) urelumab (BMS-663513) caused dose-dependent hepatitis in individuals (3, 6, 12, 13), likely due to 4-1BB cross-linking via FcRIIb-expressing liver-resident cells such as hepatic myeloid and sinusoidal endothelial cells (16, 17). Subsequent studies exposed that, when urelumab was given securely at a reduced dose of 0.1 mg/kg, it only mediated limited efficacy (3, 13). The antiChu4-1BB huIgG2 utomilumab (PF-05082566) displays a better security profile but lower agonistic potency (3). Therefore, despite a decade-long effort, agonistic antiChu4-1BB antibodies have not progressed beyond early stage medical trials. Second-/third-generation CAR T cell treatments successfully use 4-1BB costimulation, and although authorized and very effective in relapsed/refractory individuals with B cell malignancies, they require patient-individualized and expert-dependent infrastructure and logistics. In addition, CAR T cells have not yet been successful for treatment of solid tumors (14). Recent publications have explained bispecific tumor-targeted 4-1BB agonists (18, 19), which conquer liver toxicity but display a fast clearance from blood circulation (18). To conquer these limitations, we developed tumor antigen (TA)Ctargeted 4-1BB ligand fusion proteins (TAC4-1BBL) for systemic administration. TAC4-1BBL efficiently costimulates T cells for improved tumor cell killing in both solid and hematological cancers while avoiding liver toxicity. Here, we describe two versions of such an agonist: one focusing on CD19, indicated on normal and malignant B cells, and another focusing on fibroblast activation protein (FAP), indicated on tumor stroma and, to a lower degree, on lymphoid fibroreticular cells (20, 21). As the Fc region of the designed agonists consists of mutations abrogating cross-linked by Fc receptors (FcRs) (22), TAC4-1BBL molecules can only induce 4-1BB activation when cross-linked via TA-expressing cells, representing a replacement of Ritanserin FcR-mediated cross-linking, while Ritanserin keeping Ritanserin favorable pharmacokinetics. RESULTS Design of a tumor-targeted Ritanserin 4-1BB agonist, which is definitely self-employed of Fc-mediated hyperclustering for activity As agonistic antiChu4-1BB antibodies have failed so far in the medical center, our goal was to accomplish potent 4-1BB activation in the tumor site without systemic FcR cross-linking for a broad range of cancers. Initial experiments using human being FcR-expressing Chinese hamster ovary (CHO) cells or protein A beads confirmed that antiChu4-1BB antibodies rely on Fc-mediated cross-linking to induce 4-1BB activation (fig. S1, A to C). To conquer this dependency, we.