Selective targeting of the IL23 pathway: Generation and characterization of a novel high-affinity humanized anti-IL23A antibody

Abstract

Herein, we describe the generation and characterization of BI 655066, a novel, highly potent neutralizing anti-interleukin-23 (IL23) monoclonal antibody in clinical development for autoimmune conditions, including psoriasis and Crohn's disease. IL23 is a key driver of the differentiation, maintenance, and activity of a number of immune cell subsets, including T helper 17 (Th17) cells, which are believed to mediate the pathogenesis of several immune-mediated disorders. Thus, IL23 neutralization is an attractive therapeutic approach. Designing an antibody for clinical activity and convenience for the patient requires certain properties, such as high affinity, specificity, and solubility. These properties were achieved by directed design of the immunization, lead identification, and humanization procedures. Favorable substance and pharmacokinetic properties were established by biophysical assessments and studies in cynomolgus monkeys.

Keywords: ADCC, antibody-dependent cell-mediated cytotoxicity; AUC, analytical ultracentrifugation; BI 655066; CCG, Chemical Computing Group; CDRs, complementarity-determining regions; CH, constant region; Cκ, constant kappa; DMF, dimethylformamide; EOF, electro-osmotic flow; ESI, electrospray ionization; F, phenylalanine; G, glycine; GAHA, goat anti-human IgG gamma antibody; HCLF, high concentration liquid formulation; IL12, Interleukin 12; IL12RB1, IL12 receptor subunit beta 1; IL23, Interleukin-23; IL23R, IL23 receptor; JAK2, Janus kinase 2; PBS, phosphate-buffered saline; PK, pharmacokinetic; RU, resonance units; SEC, size-exclusion chromatography; SPR, surface plasmon resonance; Th17, T helper 17 cells; UV, ultraviolet; V, variable; VH, variable heavy; Vκ, variable kappa; Y, tyrosine; biophysical assessment; humanization; immunogen design; pharmacokinetic profile; tyk2, tyrosine kinase 2.

Figure 1.

6B8 Optimization design (a, light chain; b, heavy chain). CDRs as per the CCG definition are underlined. Amino acids different from the 6B8 hit are colored red if divergent or orange if similar. ∼: Sites where binary mutations (mouse and human) were included in the library. ≈: The site mutated to G, F, or Y to eliminate a potential deamidation motif, N27T28 (colored aqua).

Figure 2.

Top-selected optimized Vκ sequences. Mouse germ-line residues are shown in black, and human germ-line residues are in blue. The Vκ sequences 65, 66, and 78 were 96, 93, and 94 percent identical to the human germ-line genes IGKV1–27*01,KJ2), respectively, and had corresponding EpiVax scores of −40, −43, and −38.

Figure 3.

Top selected optimized V_H sequences. Mouse germ-line residues are shown in black, and human germ-line residues are in blue. Amino acids changed to remove a deamidation site are shown in green. V_H sequences 02, 05, 36 and 65 were 95, 96, 96, and 98 percent identical to the human germ-line genes (IGHV1–69*08, HJ4), respectively, and had corresponding EpiVax scores of −17, −38, −17, and −34.

Figure 4.

IL23 was injected into the skin of the mouse ear every 24 h for 4 consecutive days. Mice were given a single intraperitoneal dose of vehicle or 1 mg/kg antibody 1 h before the first IL23 injection. Twenty-four hours after the last IL23 injection, the ear thickness was measured and ear tissue was harvested for cIL17 and IL22 measurement by ELISA. Values are the mean +/− SEM. Statistical analysis by ANOVA.

Figure 5.

Hydrogen/Deuterium Exchange Mass Spectrometry. P19 peptides identified and their extents of exchange with BI 655066, as compared to control. All peptides showing exchange differences are found within the p19 subunit. The y-axis shows the calculated exchange normalized per amide.

Figure 6.

Pharmacokinetics of BI 655066 in cynomolgus monkeys following a single 1 mg/kg intravenous (IV) or subcutaneous (SC) dose. Serum concentrations were measured using an IL23-capture ELISA. Data are the mean ± SD of three monkeys per dose route. Bioavailability was estimated to be >70%, and the half-life (9–12 days) was similar after intravenous or subcutaneous administration.