Almost 270,000 US women will be diagnosed with breast cancer (BC) this year, anddespite advances in treatment, ~40,000 women will die. ER/PR+ (endocrineresponsive), Her2- tumors occur in approximately 40% of breast cancer patients andare candidates for drugs targeting estrogen responsiveness, such as letrozole orfulvestrant. CDK4 targeting drugs (CDK4i) like palbociclib, abemaciclib, or ribociclib arenow approved in combination with letrozole or fulvestrant as a front- and second-linetherapy for metastatic ER/PR+, Her2- patients. However, while combined ER andCDK4i treatment significantly extends Progression Free Survival (PFS), those treatedinvariably develop resistance. Thus, drug resistance remains an urgent unmet need, as current treatments only marginally improve Overall Survival (OS) for these patients.Resistance to palbociclib develops because of compensation by another kinase, CDK2, suggesting that to be effective, therapies must be developed to inhibit both CDK4 andCDK2. Concarlo has taken a different approach to inhibit CDK4 and CDK2 by targetingp27Kip1 (p27). p27 interacts specifically with CDK4/6 and CDK2, and is responsible forturning these kinases ON and OFF. This transition from ON to OFF is mediated by aspecific modification to p27 itself, by the tyrosine kinase BRK (breast tumor relatedkinase). Published work has shown that a naturally occurring ALTernatively splicedform of BRK, ALT, can bind to p27, blocking BRK's association, and preventing BRK'sphosphorylation of p27. This locks CDK4 and CDK2 into the OFF conformation andsimultaneously inhibits activities of both kinases. As the naked 144 aa ALT peptidedoes not enter cells, it was formulated within a lipid nano-particle (NP), and called IpY.1.IpY.1 inhibits proliferation of HR+ and triple negative (TN) BC cells, but not non-cancerous breast cells MCF10A. IpY.1 arrests CDK4i-resistant and endocrine-resistantBC cells and it is both cytostatic and cytotoxic, demonstrating that p27 is a viabletherapeutic target to combat drug resistance. In vivo, IpY reduces tumor volumes and increase OS in cell line-derived xenografts (CDX). In order to convert manufacturing of the therapeutic peptide portion of IpY from recombinant to synthetic production, Concarlo truncated the large ALT peptide and bioengineered it to a more stable form. IpY.20 has a comparable IC50 as IpY.1 in HR+and TNBC cells and does not cause growth arrest in non-cancerous MCF10A cells. Using the fluorescent-labeled variant of IpY.20 (fluo-IpY.20) injected into tumor-bearingsyngeneic CDX model, followed by IVIS imaging, we could detect fluo-IpY.20 in tumorsas early as 15 min and up to 24h post injection. 6-10% of the total fluorescent signal isallocated to tumor at all timepoints. In addition, fluo-IpY.20 inhibits the activation of itstarget in tumors within 24h, suggesting fluo-IpY.20 not only reaches tumors but alsoengages with its target. IpY.20 induces tumor regression in CDX models and animmunocompetent genetically engineered model that overexpresses the potent Erbb2oncogene. Overall, repeated dosing of IpY.20 does not exhibit significant adverseeffects and is well tolerated in immunocompetent mice. However, in these treated mice, there was an increase in platelets and production of some cytokines compared to vehicle-treated mice, suggesting that an immune response may have been initiated. By targeting p27 instead of the conserved CDK4 or CDK2 active sites, IpY.20 will bemore selective and have fewer off-target effects than the ATP-competitive smallmolecule CDK4is currently in use. As the majority of the drug resistance seen withCDK4 inhibitory drugs is a result of compensatory CDK2 activity, IpY.20 preventsacquired drug resistance by hitting both CDK4 and CDK2 simultaneously, resulting in amore durable response to the drug. Concarlo is currently in manufacturing with IpY.20.

Citation Format: Grace Chen, Stacy W Blain, Irina Jilishitz, Allison Vanlnwegen, Lingyue Yan, Yun Wu. Developing IpY: A novel inhibitor for the treatment of ER+ CDK4i-resistant breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-16-18.