T he superfamily of human Class II cytokines contains interleukin-10 (IL-10), the IL-10 -related interleukins , the interferons (IFN-␣, -␤ -, -, -, and -␥) and the interferon-like molecules IL-28A, IL-28B, and IL-29 (also referred to as lambda interferons). 1 Collectively, these molecules modulate innate and adaptive immune responses to environmental pathogens and protect the host against diseases such as cancer. The best-characterized class II cytokines are the type I interferons, whose expression is tightly regulated by viral infection. 2 After binding, these proteins induce a large set of interferonstimulated genes (ISGs) that inhibit viral replication and activate numerous downstream cellular responses involving dendritic cells, lymphocytes, and macrophages. 3 In addition to the type I interferons, viral infection also stimulates the rapid production of IL-28 and IL-29, a related, but distinct subset of the class II cytokine superfamily. 4,5 These proteins also possess potent antiviral activity; however, in contrast to the type I interferons, they bind a heterodimeric receptor consisting of the IL-28R␣ 4,5 subunit and the IL-10R␤ subunit, a receptor subunit that is also shared by IL-10, Chronic viral hepatitis is the leading cause of liver disease and may play a role in the pathogenesis of lesions characteristic of cirrhosis, hepatocellular carcinoma, and end-stage liver failure. The two major causes of chronic viral hepatitis are hepatitis B virus (HBV), a DNA-containing member of the Hepadnaviridae family that infects approximately 350 million people worldwide, 7 and hepatitis C virus (HCV), an RNA virus of the Flaviviridae family that infects approximately 170 million individuals worldwide. 8 IFN-␣ is an approved treatment for both types of chronic viral hepatitis and has demonstrated considerable clinical success. [9][10][11] However, this cytokine is ineffective for a substantial percentage of infected individ-

◥ HER2 Po ten cy Tucatinib blocks MAPK and PI3K/AKT signaling through inhibition of HER2 kinase activity

Targeting angiogenesis is a promising approach to the treatment of solid tumors and age-related macular degeneration (AMD). Inhibition of vascularization has been validated by the successful marketing of monoclonal antibodies (mAbs) that target specific growth factors or their receptors, but there is considerable room for improvement in existing therapies. Combination of mAbs targeting both the VEGF and PDGF pathways has the potential to increase the efficacy of anti-angiogenic therapy without the accompanying toxicities of tyrosine kinase inhibitors and the inability to combine efficiently with traditional chemotherapeutics. However, development costs and regulatory issues have limited the use of combinatorial approaches for the generation of more efficacious treatments. The concept of mediating disease pathology by targeting two antigens with one therapeutic was proposed over two decades ago. While mAbs are particularly suitable candidates for a dual-targeting approach, engineering bispecificity into one molecule can be difficult due to issues with expression and stability, which play a significant role in manufacturability. Here, we address these issues upstream in the process of developing a bispecific antibody (bsAb). Single-chain antibody fragments (scFvs) targeting PDGFRbeta and VEGF-A were selected for superior stability. The scFvs were fused to both termini of human Fc to generate a bispecific, tetravalent molecule. The resulting molecule displays potent activity, binds both targets simultaneously, and is stable in serum. The assembly of a bsAb using stable monomeric units allowed development of an anti-PDGFRB/VEGF-A antibody capable of attenuating angiogenesis through two distinct pathways and represents an efficient method for rapid engineering of dual-targeting molecules.

Tucatinib is an investigational, orally dosed, small molecule tyrosine kinase inhibitor that is highly selective for the kinase domain of HER2 without significant inhibition of EGFR. In this report, we describe the activity of tucatinib alone and in combination with trastuzumab in multiple HER2 mutant driven PDX models. Tucatinib was tested in 6 HER2 mutant PDX tumor models derived from colorectal, non-small cell lung cancer, gall bladder and gastric cancers with L755S, V77L or S310Y mutations. MCF-10A cells were engineered to express the L755S and V777L HER2 activating point mutations and the potency of tucatinib was evaluated in each of these cell lines using HER2 autophosphorylation as an endpoint. The activity of tucatinib was also evaluated in G776V_G/C and G776_YVMA in frame exon 20 insertion mutations, including the G776V_G/C mutant non-small cell lung cancer line NCI-H1781 using both cellular signaling and cell proliferation as endpoints. Tucatinib significantly inhibited tumor growth in 5 of the 6 HER2 point mutant PDX models, whereas trastuzumab alone significantly inhibited tumor growth in 1 of 6 models and the combination of both drugs was significantly active in all HER2 mutant models. Tucatinib inhibited the phosphorylation of HER2 in a L755S mutant MCF-10A cell line and inhibited the growth of 3 L755S mutant tumor models derived from colorectal, gastric and non-small cell lung cancer. Tucatinib also potently inhibited the phosphorylation of HER2 in a V777L mutant MCF-10A cell line, and in a colorectal PDX model containing a V777L mutation, tucatinib alone or in combination with trastuzumab induced tumor regressions. In two PDX models containing the S310Y extracellular domain mutation of HER2, tucatinib alone, or in combination with trastuzumab, resulted in tumor growth delays. In exon 20 insertion mutant derived cell lines, tucatinib inhibited HER2 phosphorylation, but not EGFR phosphorylation, and inhibited the phosphorylation of HER2, HER3, Erk1/2, MEK1 and AKT in the G776V_G/C HER2 mutant NCI-H1781 cell line. These data demonstrate that tucatinib, a highly potent and selective inhibitor of HER2 signaling without significant inhibition of EGFR, is active in multiple HER2 mutant driven tumor models. Importantly, tucatinib is active in models containing the L755S HER2 mutation which has been associated with lapatinib resistance preclinically (1), and trastuzumab resistance clinically (2). Taken together, these results support the idea that HER2 selective inhibition by tucatinib can block oncogenic signaling elicited by mutant HER2, and that it could potentially provide clinical benefit in patients with cancers with activating HER2 mutations. References: 1) Kancha et al., 2011 PLoS ONE, 6;e26760 2) Zhu et al., 2016 Clin. Cancer Res. 22; 4859 Citation Format: Scott Peterson, Robert Rosler, Kevin Klucher. Tucatinib, a selective small molecule HER2 inibitor, is active in HER2 mutant driven tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4222.

<div>Abstract<p>HER2 is a transmembrane tyrosine kinase receptor that mediates cell growth, differentiation, and survival. HER2 is overexpressed in approximately 20% of breast cancers and in subsets of gastric, colorectal, and esophageal cancers. Both antibody and small-molecule drugs that target HER2 and block its tyrosine kinase activity are effective in treating HER2-driven cancers. In this article, we describe the preclinical properties of tucatinib, an orally available, reversible HER2-targeted small-molecule tyrosine kinase inhibitor. In both biochemical and cell signaling experiments, tucatinib inhibits HER2 kinase activity with single-digit nanomolar potency and provides exceptional selectivity for HER2 compared with the related receptor tyrosine kinase EGFR, with a >1,000-fold enhancement in potency for HER2 in cell signaling assays. Tucatinib potently inhibits signal transduction downstream of HER2 and HER3 through the MAPK and PI3K/AKT pathways and is selectively cytotoxic in HER2-amplified breast cancer cell lines <i>in vitro</i>. <i>In vivo</i>, tucatinib is active in multiple HER2⁺ tumor models as a single agent and shows enhanced antitumor activity in combination with trastuzumab or docetaxel, resulting in improved rates of partial and complete tumor regression. These preclinical data, taken together with the phase-I tucatinib clinical trial results demonstrating preliminary safety and activity, establish the unique pharmacologic properties of tucatinib and underscore the rationale for investigating its utility in HER2⁺ cancers.</p></div>