A novel fully-human potency-matched dual cytokine-antibody fusion protein targets carbonic anhydrase IX in renal cell carcinomas

Abstract

Posted on non-commercial preprint serversThe data that support the findings of this study are openly available in bioRxiv. Contribution to the fieldThere is a growing interest in the antibody-based targeted delivery of pro-inflammatory cytokines for tumor therapy, which may be complementary or alternative to immune checkpoint inhibitors for immunotherapeutic applications. In this article, we have described a novel fusion protein, featuring antibody moieties specific to carbonic anhydrase IX, as well as interleukin-2 and tumor necrosis factor as pro-inflammatory cytokines, which combines the ability to recognize a tumor-associated antigen on the surface of renal cell carcinomas with the simultaneous display of two potent therapeutic payloads. The newly developed product (termed IL2-XE114-TNF mut ) exhibited favorable biochemical characteristics, the ability to preferentially localize at the tumor site, a cancer growth inhibition activity and a suitable pharmacokinetic profile in Cynomolgus monkey. IL2-XE114-TNF mut may therefore represent an attractive candidate for the immunotherapy of renal cell carcinoma. AbstractCertain 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-TNF mut , 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 (TNF mut ). 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-TNF mut 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-TNF mut showed the expected plasma concentration of ~1500 ng/ml at early time points, indicating the absence of any in vivo trapping events, and a half-life of ~2 hours. IL2-XE114-TNF mut 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.

Antibody-based delivery of interleukin-9 to neovascular structures: Therapeutic evaluation in cancer and arthritis

“…IL2, IL12, IL15, TNF) can promote strong neoplastic mass immune cells infiltration and strong anti-tumor efficacy. 40,44,45,48,65,66…”

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

“… 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 …”

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 pro-inflammatory cytokines including interleukin-2 (IL2), intereleukin-12 (IL12) and tumor necrosis factor (TNF) [1][2][3][4][5][6] . In several cases antibody-cytokine fusion proteins (i.e.…”

“…However, its use in RCC at high dose produces durable complete response in a small portion of patients but with severe systemic toxicity 23,24 . 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 .…”