A Novel Fully-Human Potency-Matched Dual Cytokine-Antibody Fusion Protein Targets Carbonic Anhydrase IX in Renal Cell Carcinomas

Abstract

Certain cytokines synergize in activating anti-cancer immunity at the site of disease and it may be desirable to generate biopharmaceutical agents, capable of simultaneous delivery of cytokine pairs to the tumor. In this article, we have described the cloning, expression and characterization of IL2-XE114-TNFmut, a dual-cytokine biopharmaceutical featuring the sequential fusion of interleukin-2 (IL2) with the XE114 antibody in scFv format and a tumor necrosis factor mutant (TNFmut). The fusion protein recognized the cognate antigen (carbonic anhydrase IX, a marker of hypoxia and of renal cell carcinoma) with high affinity and specificity. IL2-XE114-TNFmut formed a stable non-covalent homotrimeric structure, displayed cytokine activity in in vitro tests and preferentially localized to solid tumors in vivo. The product exhibited a partial growth inhibition of murine CT26 tumors transfected for carbonic anhydrase IX. When administered to Cynomolgus monkey as intravenous injection, IL2-XE114-TNFmut showed the expected plasma concentration of ~1,500 ng/ml at early time points, indicating the absence of any in vivo trapping events, and a half-life of ~2 h. IL2-XE114-TNFmut may thus be considered as a promising biopharmaceutical for the treatment of metastatic clear-cell renal cell carcinoma, since these tumors are known to be sensitive to IL2 and to TNF.

The present and future of immunocytokines for cancer treatment

“…However, some immunocytokines, which carry multiple different payloads have been developed. These dual-cytokine immunocytokines have currently only been tested in in vitro and pre-clinical models [ 133 – 138 ]. With the cytokine repertoire’s scale and variety, many combinations can be made which could have synergetic effects in vivo.…”

Carbonic Anhydrase IX: A Renewed Target for Cancer Immunotherapy

“…De Luca et al ( 2019) reported the characterization of fusion proteins targeting CAIX while simultaneously linked to IL2 and a low-potency TNF mutant (mut). Mice implanted with CAIX positive murine colon adenocarcinoma cells CT-26 treated with the fusion protein IL2-Anti-CAIX(XE114)-TNFmut and IL2-Anti-CAIX(F8)-TNFmut showed around 60% reduction in tumor volume compared to the control group injected with PBS after 18 days of treatment [28].…”

Sortase-Mediated Site-Specific Modification of Interleukin-2 for the Generation of a Tumor-Targeting Acetazolamide–Cytokine Conjugate

“…The active delivery of anticancer cytokines to tumor tissue has been extensively explored with the aim to enhance their therapeutic window and circumvent adverse events associated with their systemic administration. To this respect, tumor-specific antibodies have been used as selective vehicles for the targeted delivery of proinflammatory cytokines including interleukin-2 (IL2), interleukin-12 (IL12), and the tumor necrosis factor. − In several cases, antibody–cytokine fusion proteins (i.e., immunocytokines) have demonstrated selective tumor uptake and antitumor activity in murine models of cancer, and several products have been advanced into clinical trial. ,,− However, the use of antibodies for pharmacodelivery of bioactive payloads to solid tumors may be limited by their relatively large size, which can lead to slow extravasation and poor tumor tissue penetration combined with extended systemic half-life . These limitations can be partly counteracted using small antibody fragments (such as scFv or diabodies) …”

“…The IL2 cytokine is a potent inducer of cytotoxic T cells and NK cells and was one of the first immunotherapeutic agents approved by FDA for the treatment of metastatic melanoma and RCC . However, its use in RCC at high dose produces durable complete response in a small portion of patients but with severe systemic toxicity. , The antibody-based delivery to tumors has shown the potential to improve the therapeutic index of IL2 in immunocompetent mouse models of cancer. ,,, …”

“…29,30 The antibody-based delivery to tumors has shown the potential to improve the therapeutic index of IL2 in immunocompetent mouse models of cancer. 2,3,5,6 Traditional strategies for covalent bioconjugation allow limited control over the site and frequency of the modification, resulting in heterogeneous products with potential loss of biological activity of the modified protein. 31 Sortase A (SrtA) is a sequence-specific transpeptidase that catalyzes the ligation between LPXTG-containing polypeptides (Sortag) and oligoglycine-terminated moieties.…”