Antibody drug conjugates offer a targeted cancer treatment for the delivery of potent cytotoxic drugs. Derivatives of the natural product dolastatin 10 containing pyridines and other basic amines were examined with the objective of determining if a more hydrophilic auristatin derivative would be potent enough for use as part of an ADC. This may be advantageous if a less hydrophobic drug makes a better ADC. A pyridine derivative, monomethyl auristatin PYE, showed the greatest potency when tested in vivo. While only a modest tumor growth inhibition was observed when the HCC1954 human breast cancer xenografts were treated with"non-cleavable" linker ADCs, tumor regression was seen when treated with an enzymatically degradable "cleavable" linker ADC when conjugated to trastuzumab. Based on these studies, monomethyl auristatin PYE shows promise for use as an ADC payload.

Synthetic analogues of the natural occurring dolastatin 10 are of great interest in cancer due to their potent in vitro activity and their uses as payloads in antibody drug conjugates (ADCs). Modification of the dolastatin 10 core scaffold has mainly focused on modifications of the P1, N-terminus, and P5, C-terminus, with minimal attention to the P2 subunit. In this paper we discuss the introduction of heteroatoms to the P2 side chain, which results in potent activity in vitro. The most active compounds contained azides in the P2 unit and required a phenylalanine-derived P5 subunit.

Therapies targeting HER2 have transformed the treatment of patients with HER2-expressing breast and gastric cancers. Unfortunately, many patients recur following HER2-targeted treatments and new therapies are needed. Multiple antibody-drug conjugate (ADC) technologies are being explored in this setting, some of which utilize the anti-HER2 antibody trastuzumab. Here we present the preclinical characterization of a new anti-HER2 biparatopic ADC, ZW49, which is generated from the conjugation of a novel N-acyl sulfonamide auristatin payload to the inter-chain disulfide bond cysteines of the bispecific anti-HER2 IgG1 antibody ZW25, via a protease cleavable linker. A series of in vitro and in vivo experiments were performed to characterize ZW49 as a potential therapeutic candidate. In cellular binding assays, it was confirmed that the payload conjugation to ZW25 did not affect the antibody's binding to HER2-expressing cells. ZW49 displayed potent in vitro cytotoxicity in multiple cancer cell lines expressing HER2 and was efficacious in multiple patient-derived xenograft (PDX) models. In mice bearing the HBCx-13b HER2 3+ PDX, two doses of ZW49 administered two weeks apart generated tumor regressions. Furthermore, preliminary results from PDX models with lower levels of HER2 expression treated with ZW49 also generated regressions. In nonhuman primates ZW49 administered intravenously every two weeks for three doses was well tolerated. Based on these findings, we are proceeding with further development of ZW49 as a therapeutic candidate in HER2-expressing cancers. Citation Format: Kevin J. Hamblett, Phil W. Hammond, Stuart D. Barnscher, Vincent K. Fung, Rupert H. Davies, Grant R. Wickman, Andrea Hernandez, Tong Ding, Adam S. Galey, Geoffrey C. Winters, Jamie R. Rich, John S. Babcook. ZW49, a HER2-targeted biparatopic antibody-drug conjugate for the treatment of HER2-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3914.

IL-12 is a cytokine produced by antigen-presenting cells that increases T cell proliferation, IFN gamma mediated Th1 effector functions and T and NK cell cytotoxicity. While these effects generate potent anti-tumor immunity in mouse models, the high toxicity of IL-12 in cancer patients has limited its clinical utility. Potency attenuation and intratumoral cytokine localization may improve IL-12 tolerability, but these approaches can reduce efficacy or be limited by intratumoral delivery or tumor antigen expression. To improve both tolerability and efficacy of IL-12, we engineered IL-12Fc fusions with anti-IL-12 antibodies to block IL-12 potency. Using linkers designed to be cleaved by highly active intratumoral proteases, blocking antibodies are released specifically in the tumor microenvironment, thereby increasing intratumoral IL-12 activity. Single chain IL-12 was fused to one C-termini of an Azymetric™ Fc heterodimer. To the other Fc C-termini, an anti-IL-12 scFv was fused via a protease-cleavable linker in order to block IL-12 activity. In addition to antibody blockade, modifications of IL-12Fc fusions were used to further limit IL-12Fc activity. In vitro potency was determined by CD8T cell IFN gamma release. Anti-IL-12 scFv cleavage in the presence of recombinant enzyme or human tumor material was assessed by reducing CE-SDS and LC/MS, respectively. All IL-12Fc fusions were produced with favorable biophysical characteristics. Modified IL-12Fc molecules had up to 500x reduced potency, while antibody blocked IL-12Fc molecules had up to 100,000x reduced potency compared to IL-12Fc control. In vitro cleavage of the anti-IL-12 scFv from IL-12Fc recovered potency to that of the corresponding non-masked IL-12Fc molecules. Reducing CE-SDS confirmed that anti-IL-12 scFvs were cleaved from IL-12Fc in the presence of recombinant enzyme. Cleavage of anti-IL-12 scFvs also occurred when variants were incubated in pancreatic tumor cell supernatant or human tumor tissue lysate. These results indicate that antibody blockade and re-activation by protease cleavage is a promising strategy to localize the activity of IL-12 to the tumor microenvironment while potentially limiting off-tumor toxicities. Further modifying antibody blocked IL-12 has the potential to better fine tune therapeutic index. We are pursuing tumor-specific IL-12 fusions for clinical application in tumors with high intratumoral protease activity. Citation Format: Jennifer Leah Bishop, Ryan Blackler, Gesa Volkers, Maya Poffenberger, Irene Yu, Joel Smith, Akram Khodabandehloo, Sifa Arrafi, Desmond Lau, Liz Stangle, Leisa Stenberg, Patricia Zwierzchowski, Iulia Dude, David Douda, Grant Wickman, Jeff Proctor, Gerry Rowse, Laurence Madera, Genevieve Desjardins, Nicole Afacan, Stuart Barnscher, David Mills, Thomas Spreter, Surjit Dixit. Increasing the therapeutic index of IL12 by engineering for tumor specific protease activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1788.

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