Minerva Biotechnologies (United States)

Abstract Purpose: To develop a MUC1*-ADC for the treatment of over 75% of solid tumor cancers. Methods: MUC1* (muk 1 star) is a growth factor receptor created after cleavage and release of the N-terminal portion of MUC1. The targeting antibody, huMNC2, has already demonstrated safety and cancer selectivity in a 1st-in-human CAR-T clinical trial for treatment of metastatic breast cancers. MNC2 is an IgG1 antibody that only recognizes the conformational epitope created when MUC1 is cleaved by specific tumor-associated enzymes. Confocal microscopy and pHlourin2 were employed to measure antibody internalization as a function of time. We conjugated MNC2 to MMAE, MMAF, Dxd and exatecan via several different linkers. MUC1*-ADCs with DARs ranging from 4-8 were tested in vitro and in vivo for efficacy against a panel of solid tumor cancers. Heterogenous MUC1* tumors were made by mixing different ratios of mCherry wild-type cancer cells with GFP MUC1* overexpressed cancer cells. Resulting H scores ranged from 10-280 (max 300). Results: Cancer cells internalized the MUC1* MNC2 antibody within 2 hours. In vitro, MUC1*-ADC IC50s ranged from 1.3nM to 20.0nM, depending on the linker-payload and cancer subtype, wherein the majority had an IC50 of about 10nM. In general, efficacy in animals was seen across multiple cancer sub-types treated with multiple MUC1*-ADCs. Efficacy in animals xenografted with heterogeneous MUC1* tumors showed a dependence on antigen density. Greatest efficacy was observed for breast cancer xenografts with an H Score >10 treated with MNC2-Deruxtecan. However, only pancreatic tumors with higher H Scores were completely eliminated by MNC2-MMAE, MNC2-Deruxtecan and MNC2-exatecan. MNC2-ADC and MNC2-CAR share the same antibody. No off-tumor toxicity was observed in animals treated with MNC2-ADC, which mimicked lack of off-target toxicities for MNC2-CAR T cells in humans. Conclusions: These data, combined with MNC2-CAR T data from our 1st-in-human trial for metastatic breast cancer, supports a conclusion that the MUC1* antibody MNC2 is safe and could have high therapeutic value as a MUC1*-ADC for multiple solid tumors with both high and low antigen density. Citation Format: Cynthia Carol Bamdad, Benoit S. Smagghe, Scott T. Moe, Mark G. Carter, Trevor J. Grant, Kevin R. Yi, Michael J. Nash, Jacy P. Marquez, Natalie K. Miller, Jac-Leen S. Nash, Dan S. Miller, Danica M. Walkley, Andrew K. Stewart. MUC1*-ADCs are effective against heterogeneous solid tumor cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6351.

TPS1130 Background: Metastatic breast cancer (MBC) remains incurable and novel immunotherapy for durable response remains an unmet need. Chimeric antigen receptor (CAR) T-cell therapy, an innovative form of immunotherapy wherein autologous T-cells are genetically modified to target tumor specific cell-surface markers, has been developed for treatment of solid tumors. huMNC2-CAR44 recognizes the growth factor receptor form of MUC1, which is the transmembrane cleavage product called MUC1*. MUC1* is a Class I growth factor receptor that is activated by ligand-induced dimerization of its truncated extra cellular domain, which activates the MAP kinase signaling pathway as well as survival pathways. Onco-embryonic growth factor NME7AB binds to an ectopic site on MUC1* that is only unmasked after MUC1 is cleaved and the tandem repeat domain is shed from the cell surface. The targeting head of the CAR, huMNC2, competes with NME7AB for binding to this ectopic site. huMNC2 does not bind to full-length MUC1, hence highly tumor-selective. 70% of solid tumor cancers express a huMNC2 reactive MUC1* and huMNC2-scFv bound robustly to 93% of the breast cancers with minimal staining of normal tissues. huMNC2-CAR44 T cells completely obliterated a variety of MUC1* positive solid tumors in NSG mice in vivo. IND enabling animal studies demonstrated that huMNC2-CAR44 T potently inhibited MUC1* positive tumors xenografted into female NSG mice, whether the tumor cells were MUC1 negative cells stably transduced with MUC1* or breast cancer cells such as T47D that naturally express MUC1*. In one study, huMNC2-CAR44 T treated mice survived tumor-free for over 12 weeks, whereas control group had to be sacrificed at 3 weeks due to disease progression. Methods: This is a first-in human, phase I/II trial evaluating the safety and efficacy of huMNC2-CAR44 T in patients with MBC. Key inclusion criteria include age ≥18 years, ECOGPS 0-1, available FFPE tumor sample, tumor IHC ≥30% MUC1* and preserved organ function. Dose escalation is standard 3+3 design with dosing levels ranging from 3.3x10^5 to 1.0x10^7 CAR+ cells/kg, and fludarabine/cyclophosphamide lymphodepletion pre-treatment. Phase I accepts patients with MBC that has progressed through at least 3 previous lines of therapy. The primary objective of Phase I is to determine safety and determine a recommended Phase II dose (RPIID), with the exploratory objectives of assessing CAR T cell expansion, persistence, tumor penetration and potential tumor escape. Six (6) patients have been enrolled and five (5) patients have been treated to date. Phase II will be comprised of 3 cohorts of 15 patients in each arm of luminal, HER2+ and triple negative breast cancers for a total of 45 patients in Phase II. Clinical trial information: NCT04020575.

The complexity of human tissue fluid precludes timely identification of cancer biomarkers by immunoassay or mass spectrometry. An increasingly attractive strategy is to primarily enrich extracellular vesicles (EVs) released from cancer cells in an accelerated manner compared to normal cells. The Vn96 peptide was herein employed to recover a subset of EVs released into the media from cellular models of breast cancer. Vn96 has affinity for heat shock proteins (HSPs) decorating the surface of EVs. Reflecting their cells of origin, cancer EVs displayed discrete differences from those of normal phenotype. GELFrEE LC/MS identified an extensive proteome from all three sources of EVs, the vast majority having been previously reported in the ExoCarta database. Pathway analysis of the Vn96-affinity proteome unequivocally distinguished EVs from tumorigenic cell lines (SKBR3 and MCF-7) relative to a non-tumorigenic source (MCF-10a), particularly with regard to altered metabolic enzymes, signaling, and chaperone proteins. The protein data sets provide valuable information from material shed by cultured cells. It is probable that a vast amount of biomarker identities may be collected from established and primary cell cultures using the approaches described here.

The MUC1 protein is aberrantly expressed on an estimated 75% of all human solid tumor cancers. We recently reported that a transmembrane cleavage product, MUC1*, is the predominant form of the protein on cancer cells [1]. Further, our evidence indicated that MUC1* functions as a growth factor receptor on tumor cells, while the full-length protein appeared to have no growth promoting activity. Here, we report that MUC1* acts as a growth factor receptor on undifferentiated human embryonic stem cells (hESCs). Cleavage of the full-length ectodomain to form MUC1*, a membrane receptor, appears to make binding to its ligand, NM23, possible. Unexpectedly, we found that newly differentiated cells no longer express the cleaved form, MUC1*, or its ligand, NM23. Newly differentiated stem cells exclusively present full-length MUC1. Antibody-induced dimerization of the MUC1* receptor on hESCs stimulated cell growth to a far greater degree than currently used methods that require the addition of exogenous basic fibroblast growth factor (bFGF) as well as factors secreted by fibroblast “feeder cells”. Further, MUC1* mediated growth was shown to be independent of growth stimulated by bFGF or the milieu of factors secreted by feeder cells. Stimulating the MUC1* receptor with either the cognate antibody or its ligand NM23 enabled hESC growth in a feeder cell-free system and produced pluripotent colonies that resisted spontaneous differentiation. These findings suggest that this primal growth mechanism could be utilized to propagate large numbers of pluripotent stem cells for therapeutic interventions.