Generation and in vivo validation of an IL-12 fusion protein based on a novel anti-human FAP monoclonal antibody

Abstract

Background: In this study, we describe the generation of a fully human monoclonal antibody (named '7NP2') targeting human fibroblast activation protein (FAP), an antigen expressed in the microenvironment of different types of solid neoplasms.

Methods: 7NP2 was isolated from a synthetic antibody phage display library and was improved by one round of mutagenesis-based affinity maturation. The tumor recognition properties of the antibody were validated by immunofluorescence procedures performed on cancer biopsies from human patients. A fusion protein consisting of the 7NP2 antibody linked to interleukin (IL)-12 was generated and the anticancer activity of the murine surrogate product (named mIL12-7NP2) was evaluated in mouse models. Furthermore, the safety of the fully human product (named IL12-7NP2) was evaluated in Cynomolgus monkeys.

Results: Biodistribution analysis in tumor-bearing mice confirmed the ability of the product to selectively localize to solid tumors while sparing healthy organs. Encouraged by these results, therapy studies were conducted in vivo, showing a potent antitumor activity in immunocompetent and immunodeficient mouse models of cancer, both as single agent and in combination with immune checkpoint inhibitors. The fully human product was tolerated when administered to non-human primates.

Conclusions: The results obtained in this work provided a rationale for future clinical translation activities using IL12-7NP2.

Keywords: antibodies, neoplasm; antigens; cytokines; immunotherapy.

Figure 1

Microscopic fluorescence analysis of human fibroblast activated protein (hFAP) expression on tissue human microarray and colon healthy and malignant samples from donors with IgG1(7NP2)-fluorescein isothiocyanate (FITC). Microscopic fluorescence analysis of hFAP expression on healthy and malignant colon samples (A), pancreas specimens (B), and brain metastasis (C) from donors detected with IgG1(7NP2)-FITC and IgG1(KSF)-FITC (negative control). Cryosections were stained with anti-FITC (green, AlexaFluor 488); blood vessels were stained with anti-CD31 (red, AlexaFluor 594); cell nuclei were stained with DAPI (blue). 20× magnification, scale bars=100 µm.

Figure 2

Preclinical characterization of mIL12-7NP2. (A) Schematic representation of mIL12-7NP2; (B) sodium dodecyl-sulfate polyacrylamide gel electrophoresis, 10% gel in non-reducing (NR) and reducing (R) conditions of purified mIL12-7NP2; (C) size exclusion chromatogram of mIL12-7NP2; (D) interferon-gamma (IFN-γ) induction assay by mIL12-7NP2 in freshly isolated BALB/c splenocytes. Cells were resuspended at 5×10⁶ cells/mL and incubated for 5 days at 37°C and 5% CO₂ with or without 0.1 pM of the interleukin-12 (IL-12) derivatives. IFN-γ was measured in cultured supernatants by sandwich ELISA; (E–F) quantitative biodistribution analysis of radioiodinated mIL12-7NP2 in BALB/c nude mice bearing SKRC52 renal cell carcinoma (E) or HT1080 (F) fibrosarcoma. Results are expressed as percentage of injected dose per gram of tissue (%ID/g±SEM; n=4–5); (G) microscopic fluorescence analysis of tumor-targeting performance of mIL12-7NP2 in CT26-hFAP tumor and organs from BALB/c mice. Cryosections were stained with ProteinA-AlexaFluor 488; cell nuclei were stained with DAPI (blue). 20× magnification, scale bars=100 µm; (H) therapeutic performance of mIL12-7NP2 in BALB/c nude mice bearing SKRC52-hFAP human renal cell carcinoma. Data represent mean tumor volume±SEM, n=6 mice per group; (I) therapeutic performance of mIL12-7NP2 in BALB/c mice bearing CT26-hFAP colon carcinoma. Data represent mean tumor volume±SEM, n=3-5 mice per group. CR, complete response; hFAP, human fibroblast activated protein; IP-10; IFN-inducible protein 10; i.v., intravenous; TNF, tumor necrosis factor. *P<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Figure 3

In vitro characterization of IL12-7NP2 (A) Schematic representation of IL12-7NP2; (B) sodium dodecyl-sulfate polyacrylamide gel electrophoresis, 10% gel in non-reducing (NR) and reducing (R) conditions of purified IL12-7NP2; (C) size exclusion chromatogram of IL12-7NP2; (D–E) biological activity assay on human peripheral blood mononuclear cells (D) and on NK-92 cells (E) incubated with IL12-7NP2 and interleukin-12 (IL-12) reference standard; (F) surface plasmon resonance of IL12-7NP2 (250 nM) on streptavidin chip coated with human FAP (hFAP) (correction for change in the refraction index was applied); (G) pharmacokinetic analysis conducted in Cynomolgus monkeys injected once at the dose of 1 mg/kg, 0.2 mg/kg, or 0.04 mg/kg of IL12-7NP2. Blood samples were collected on day 1 and day 22 of the safety toxicology study, before dosing, at 5, 15, 60, 120, 240, and 360 min after end of dosing.

Figure 4

Hematology, coagulation, and serum chemistry analysis of Cynolmoulgus monkeys administered with IL12-7NP2 at different doses. Hematology parameters evaluated at predose, day 2, day 8, and day 26 of (A) white blood cells (WBC) count, lymphocytes (LYAB) count, neutrophils (LUAB) count, and monocytes (MAB) count of Cynomolgus monkeys administered with IL12-7NP2 (1 mg/kg, 0.2 mg/kg, and 0.04 mg/kg); (B) red blood cell (RBC) count, reticulocytes (RAB) count, hemoglobin (HGB), and hematocrit (HCT) of Cynomolgus monkeys administered with IL12-7NP2 (1 mg/kg, 0.2 mg/kg, and 0.04 mg/kg); (C) platelets count (PLT), prothrombin time (PT), partial prothrombin time (PPT), and fibrinogen (FIB) of Cynomolgus monkeys administered with IL12-7NP2 (1 mg/kg, 0.2 mg/kg, and 0.04 mg/kg); (D) serum chemistry parameters evaluated at predose, day 8, and day 26: aspartate aminotrasnferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), and alkaline phosphatase (AP) of Cynomolgus monkeys administered with IL12-7NP2 (1 mg/kg, 0.2 mg/kg, and 0.04 mg/kg).

Figure 5

Flow cytometry analysis of Cynomolgus monkeys peripheral blood cells. Flow cytometry analysis of Cynomolgus monkeys blood cells; monkeys were administered with 1 mg/kg (A), 0.2 mg/kg (B), or 0.04 mg/kg (C). Samples were collected at predose, day 2, day 5, day 22 (predose, 24, and 96 hours after dosing). Per cent (%) of CD20⁺, CD16⁺, CD8⁺, and CD4⁺ cells were analyzed. NK, natural killer.

Figure 6

Quantification of interleukin-6 (IL-6), IFN-inducible protein 10 (IP-10), interferon-gamma (IFN-γ), and neopterin in Cynolmogus monkey serum. Blood collection timepoints for cytokine plasma levels (IFN-γ (A), IL-6 (B), IP-10 (C), and neopterin (D)) were at day 1 (predose, 6, 12, 24, and 96 hours after dosing) and at day 22 (predose, 6, 12, 24, and 96 hours after dosing).