No abstract

Bispecific IgG asymmetric (heterodimeric) antibodies offer enhanced therapeutic efficacy, but present unique challenges for drug development. These challenges are related to the proper assembly of heavy and light chains. Impurities such as symmetric (homodimeric) antibodies can arise with improper assembly. A new method to assess heterodimer purity of such bispecific antibody products is needed because traditional separation-based purity assays are unable to separate or quantify homodimer impurities. This paper presents a liquid chromatography-mass spectrometry (LC-MS)-based method for evaluating heterodimeric purity of a prototype asymmetric antibody containing two different heavy chains and two identical light chains. The heterodimer and independently expressed homodimeric standards were characterized by two complementary LC-MS techniques: Intact protein mass measurement of deglycosylated antibody and peptide map analyses. Intact protein mass analysis was used to check molecular integrity and composition. LC-MSE peptide mapping of Lys-C digests was used to verify protein sequences and characterize post-translational modifications, including C-terminal truncation species. Guided by the characterization results, a heterodimer purity assay was demonstrated by intact protein mass analysis of pure deglycosylated heterodimer spiked with each deglycosylated homodimeric standard. The assay was capable of detecting low levels (2%) of spiked homodimers in conjunction with co-eluting half antibodies and multiple mass species present in the homodimer standards and providing relative purity differences between samples. Detection of minor homodimer and half-antibody C-terminal truncation species at levels as low as 0.6% demonstrates the sensitivity of the method. This method is suitable for purity assessment of heterodimer samples during process and purification development of bispecific antibodies, e.g., clone selection.

No abstract

IL-12 is a pleiotropic cytokine that potently stimulates anti-tumor cytotoxic T and NK cell mediated immunity. Recombinant IL-12 reduces tumor growth in multiple mouse models, but its therapeutic application has been limited by severe toxicities. Protease dependent activation of therapeutics with high on-target, off-tumor toxicities may be used to localize activity to the tumor but achieving sufficient exposure of the activated therapeutic in the tumor microenvironment remains a challenge. We have previously shown our design strategy for masked, protease activated IL-12Fc that included optimization of blocking modules, fusion geometry, and linker design. Here we have further expanded our mechanistic evaluations by combining masked, protease activated IL-12Fc with attenuated potency engineering, and selected a lead candidate (ZW270) based on anti-tumor activity and non-human primate (NHP) tolerability. The relative activity of masked and unmasked IL-12Fc variants comprising wild type (wt) or attenuated (att) IL-12 potency was evaluated in CD8 T cell activity assays. The wt IL-12Fc displayed comparable potency to recombinant IL-12 and up to 100x reduced potency when masked. Introduction of attenuating mutations to the p40 subunit reduced the potency of the IL-12Fc by up to 20x and addition of the protease cleavable mask further reduced the potency to up to 5000x compared to wt IL-12Fc. In a single dose NHP study, masked att IL-12Fc was well tolerated up to 31.8 mg/kg, while wt IL-12Fc demonstrated a maximum tolerated dose of below 1.3 mg/kg. Despite the reduced potency of att IL-12Fc in vitro, in in vivo efficacy studies in a human PBMC engrafted xenograft model the masked att IL-12Fc was able to control tumor growth whereas masked wt IL-12Fc and wt IL-12Fc showed limited anti-tumor activity, suggesting the approach of masked, protease activated att IL-12Fc might be able to achieve a higher exposure of active cytokine in the tumor in comparison to the masked wt IL-12Fc and wt IL-12 comparator. To further investigate the potentially superior exposure response relationship of masked att IL-12Fc, we developed a quantitative systems pharmacology (QSP) model based on our experimental data and literature data. In this model the predicted ratio of IL-12-receptor occupancy by active IL-12Fc in the tumor vs. blood was 18x greater for masked att IL-12Fc than for wt IL-12Fc, whereas the ratio was under 10x greater for masked wt IL-12Fc vs. wt IL-12Fc. In summary, the affinity attenuated, masked IL-12Fc lead ZW270 has potent and superior anti-tumor activity to wt IL-12Fc and masked wt IL-12Fc, and is well tolerated in NHPs to >30 mg/kg. Our data suggests that the combined strategy of masked, protease activated IL-12Fc and attenuated IL-12 potency has the potential to widen the therapeutic index and to have superior activity to masked, protease cleavable wt IL-12 and unmasked wt IL-12 fusions. Citation Format: Maya C. Poffenberger, Jennifer L. Bishop, Ryan J. Blackler, Kevin G. Haworth, Steven Booth, Shalla Hanson, Jeff R. Proctor, I-Ting Shao, Nichole K. Escalante, Dayananda Siddappa, Joel Smith, Gursev Anmole, Saki Konomura, Nicholas A. Dawson, Sifa Arrafi, Desmond Lau, Gerry Rowse, Rupert H. Davies, Thomas Spreter von Kreudenstein. ZW270, a conditionally masked IL-12 cytokine fusion protein displaying potent anti-tumor activity absent systemic toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2935.