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.

Abstract Background: CAR T cells for the treatment of solid tumor cancers has not yet achieved the same success as CAR T cells for treatment of blood cancers. Two of the hurdles that must be cleared for effective use of CAR T cells for solid tumor cancers are: 1) CAR T cell exhaustion; and 2) failure to recognize and kill low antigen expressing cancer cells. A promising approach to overcoming CAR T cell exhaustion, referred to as “1XX” was developed in the Sadelain Lab at MSKCC. Mutation of Tyrosines to Phenylalanine in ITAMs 2 and 3, of the CD3z signaling domain, restrict signaling to ITAM 1. This slowing down of signaling has been reported to be effective at eliminating or greatly reducing CAR T cell exhaustion in blood cancers [Feucht, et al 2019; Park et al 2022; Schoutrop, et al 2023]. Purpose: To evaluate the potential of the 1XX mutations to overcome CAR T cell exhaustion and inability to kill low antigen expressing cells in an animal model of breast cancer. Specifically, we tested the ability of three different CARs to eliminate human breast tumors that expressed variable levels of the target antigen, MUC1*, and their ability to suppress tumor recurrence over approximately 100 days. Methods: All three CARs were targeted to the tumor by the same antibody fragment, huMNC2, that recognizes MUC1*, which is the transmembrane cleavage product of MUC1 that functions as a potent growth factor receptor. The CARs are: 1) huMNC2-41BB-CD3z; 2) huMNC2-CD28-CD3z; and 3) huMNC2-CD28-1XX. Heterogeneous tumors expressing different levels of target antigen were made as follows. T47D breast cancer cells were engineered to express even more of the target, MUC1*, and were also engineered to fluoresce green. T47D wild type cells were engineered to fluoresce red. Heterogenous tumors consisting of 250,000 cells were implanted sub-cu into female NSG mice bearing 90-day estrogen pellets. The tumors comprised either 70% wild-type/30% overexpressing cells, or 85% wild-type/15% overexpressing cells or 92.5% wild-type/7.5% overexpressing cells. Tumor engraftment was verified by bioluminescence at Day 6 post implantation. There was a total of 150 animals, 5 animals per group. Animals were administered a single dose of CAR T cells into the tail vein on Day 7, wherein the Effector to Target ratio was either 10:1, 5:1 or 1:1. Tumor volume was measured weekly by Luciferase/Luciferin bioluminescence on an IVIS instrument. In addition, the red versus green fluorescence of the tumor was tracked periodically as an indicator of which cells, high or low antigen expressing, were being killed. Between Day 93 and Day 96, animals were sacrificed, cells were recovered from blood and spleen, recovered tumors were weighed, tumor cells dissociated and fluorescent images were captured to determine which cells escaped CAR T cell killing. Conclusions: The CAR with the 1XX mutations in CD3z, huMNC2-CD28-1XX, was much more effective at suppressing breast tumor recurrence than either CAR with wild-type CD3z. At sacrifice, significantly more CAR T cells were recovered from huMNC2-CD28-1XX than from huMNC2-41BB-CD3z or huMNC2-CD28-CD3z. At high dose, the CARs with wild-type CD3z effectively suppressed the high antigen expressing cells, but the recurrent tumors were essentially made up of the low antigen expressing cells that had escaped CAR T cell killing. At low CAR T cell dose, the CARs with wild-type CD3z appeared to become exhausted about 40 days post treatment, when tumors began to recur. Post sacrifice analysis of the recurrent tumors showed that they were made up of both high and low antigen expressing cells. huMNC2-CD28-1XX effectively killed both the high antigen and low antigen expressing cells as evidenced by the live fluorescent imaging and the post-sacrifice analysis of residual tumor. Citation Format: Cynthia Bamdad, Andrew Stewart, Benoit Smagghe, Mark Carter, Danica Walkley, Kevin Yi, Jac-Leen Nash, Michael Nash, Trevor Grant, Gregory Riley. Animal Study Compares CAR T cell Exhaustion & Ability to Kill Low Antigen expressing breast cancer cells among three CAR constructs including one with 1XX mutations [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-26-05.

Purpose: Develop CAR T cells that have increased persistence in vivo and kill low antigen expressing solid tumor cancer cells. Experimental: Three CARs were generated that were all targeted to the tumor with the same antibody fragment, huMNC2, that recognizes a cryptic epitope on the MUC1 cleavage product called MUC1* (muk 1 star). One construct comprises a 41BB co-stimulatory domain, while the second construct has a CD28 co-stimulatory domain. The first 2 CARs have a wild-type CD3z, whereas the 3rd bears Michel Sadelain’s “1XX” mutations in CD3z. These are 2 Tyrosine to Phenylalanine mutations in ITAM 2 and ITAM 3. The 1XX mutations were designed to slow signaling to eliminate the problem of CAR T cell exhaustion. Human T cells were separately transduced with the 3 CARs and tested in NSG mice bearing human breast tumors. The tumors were heterogeneous tumors comprising parent cells that fluoresce red and express medium to low amounts of the antigen, MUC1*, and cells that fluoresce green wherein the parent cells have been transduced to express more MUC1*. The percent of the tumor that expressed high levels of MUC1* was varied from 7.5% to 15% to 30%. 150 female NSG mice were implanted with 500,000 tumor cells. The animals received a single CAR T cell injection of one of the three CAR T cells at an Effector to Target ratio of 10:1, 5:1, or 1:1. The experiment was allowed to proceed for 96 days, with periodic IVIS measurements to track tumor growth with a focus on tumor recurrence. After sacrifice, residual tumors were excised, dissociated and analyzed to determine if tumor recurrence was being driven by the high antigen cells or the low antigen cells. CAR T cells were recovered from the blood and the spleen. They were enumerated and analyzed by flow cytometry for the presence of markers of exhaustion. Unpublished Results: Tumor recurrence occurred in mice treated with CAR T cells bearing wild-type CD3z, but essentially did not recur in the mice treated with CAR T cells bearing the 1XX mutations. Four hundredfold more CAR T cells were recovered from the mice treated with the CAR-1XX cells compared to CARs with wild-type CD3z, and the CAR-1XX cells appeared not to be exhausted even after 96 days. CAR-1XX T cells administered at a 1:1 E:T ratio, with only 7.5% of the tumor comprised of high antigen expressing cells, were effectively killed with no significant tumor recurrence. In contrast, the CARs with wild-type CD3z had no effect on tumor growth in this group. Surprisingly, analysis of excised tumors showed that tumor recurrence in mice treated with CAR wild-type CD3z T cells was driven by the low antigen density cells, whereas the CAR-1XX T cells killed high antigen and low antigen density cells at the same rate. Conclusions: 1XX mutations in CD3z greatly increase CAR T persistence in vivo and unexpectedly enable the killing of low antigen tumor cells. Citation Format: Cynthia C. Bamdad, Benoit J. Smagghe, Mark G. Carter, Danica M. Walkley, Jac-Leen S. Nash, Kevin R. Yi, Trevor J. Grant, Laura M. Reale, Michael J. Nash, Gregory L. Riley, Andrew K. Stewart. 1XX mutations slow CAR T cell signaling and increase in vivo persistence [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 1148.

Abstract Purpose: To develop therapeutics to treat metastasis of solid tumor cancers. To overcome obstacles to developing anti-metastasis treatments by: 1) developing an animal model that reproducibly mimics cancer metastasis and does so within a reasonable timeframe; and 2) figuring out the basic science that drives metastasis. Experimental: We discovered that all pluripotent human stem cells express a MUC1 cleavage product called MUC1* (muk 1 star). All MUC1 cleavage stops with the onset of differentiation and MUC1 goes back to its quiescent state. Over 75% of solid tumor cancers also express MUC1*. Overexpression of MUC1*, as well as enzymes that cleave MUC1 to MUC1* are predictors of poor prognosis. NME1 is a ligand of MUC1* that is secreted by both embryonic stem cells and cancer cells. As a dimer, NME1 dimerizes the MUC1* extra cellular domain to activate the MAP kinase growth pathway. In an embryo, the more stem cells there are, the more NME1 is secreted and it goes from the active dimer to an inactive hexamer (doesn’t bind MUC1*). The paradox was, “How do stem cells limit self-replication, but cancer cells do not?” Answer: A primitive growth factor, NME7AB, looks like a single chain dimer of NME1, so is always active, is expressed in the earliest, naïve stem cells and in metastatic cancer cells. Unpublished Results: Growing cancer cells in recombinant NME7AB for 10 days transforms them into metastatic cells: 1) become non-adherent; 2) enter dormancy; 3) upregulate metastatic markers by 200-fold; 4) form sub-cu tumors in mice from as few as 50 cells; iv injection leads to total metastasis in 10 days; 5) the percent of injected tumor cells that were first grown in NME7AB determines the growth of the entire tumor. We developed a monoclonal antibody that blocks interaction of NME7AB and MUC1*. This antibody reverses established metastasis of breast cancers in animals. NME7AB should be turned off early in embryogenesis, but is aberrantly re-activated in metastatic cancers. Yamanaka factors OCT4, SOX2 and NANOG bind to the promoters of NME7, MUC1 and MMP16, an enzyme that cleaves MUC1 to MUC1* and exposes the cryptic binding site for NME7AB. Conclusions: Primitive growth factor, NME7AB, drives metastasis of solid tumor cancers. Antibody that disrupts NME7AB-MUC1* interaction reverses metastasis in animals. Citation Format: Cynthia C. Bamdad, Benoit J. Smagghe, Mark G. Carter, Trevor J. Grant, Laura M. Reale, Michael J. Nash, Danica M. Walkley, Jac-Leen S. Nash, Kevin R. Yi, Andrew K. Stewart. Metastasis: Cancer cells that have turned on a Yamanaka-like pluripotency program [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 2487.

Purpose: To develop therapeutics to treat metastasis of solid tumor cancers. To overcome obstacles to developing anti-metastasis treatments by: 1) developing an animal model that reproducibly mimics cancer metastasis and does so within a reasonable timeframe; and 2) figuring out the basic science that drives metastasis. Experimental: We discovered that all pluripotent human stem cells express a MUC1 cleavage product called MUC1* (muk 1 star). All MUC1 cleavage stops with the onset of differentiation and MUC1 goes back to its quiescent state. Over 75% of solid tumor cancers also express MUC1*. Overexpression of MUC1*, as well as enzymes that cleave MUC1 to MUC1* are predictors of poor prognosis. NME1 is a ligand of MUC1* that is secreted by both embryonic stem cells and cancer cells. As a dimer, NME1 dimerizes the MUC1* extra cellular domain to activate the MAP kinase growth pathway. In an embryo, the more stem cells there are, the more NME1 is secreted and it goes from the active dimer to an inactive hexamer (doesn’t bind MUC1*). The paradox was, “How do stem cells limit self-replication, but cancer cells do not?” Answer: A primitive growth factor, NME7AB, looks like a single chain dimer of NME1, so is always active, is expressed in the earliest, naïve stem cells and in metastatic cancer cells. Unpublished Results: Growing cancer cells in recombinant NME7AB for 10 days transforms them into metastatic cells: 1) become non-adherent; 2) enter dormancy; 3) upregulate metastatic markers by 200-fold; 4) form sub-cu tumors in mice from as few as 50 cells; iv injection leads to total metastasis in 10 days; 5) the percent of injected tumor cells that were first grown in NME7AB determines the growth of the entire tumor. We developed a monoclonal antibody that blocks interaction of NME7AB and MUC1*. This antibody reverses established metastasis of breast cancers in animals. NME7AB should be turned off early in embryogenesis, but is aberrantly re-activated in metastatic cancers. Yamanaka factors OCT4, SOX2 and NANOG bind to the promoters of NME7, MUC1 and MMP16, an enzyme that cleaves MUC1 to MUC1* and exposes the cryptic binding site for NME7AB. Conclusions: Primitive growth factor, NME7AB, drives metastasis of solid tumor cancers. Antibody that disrupts NME7AB-MUC1* interaction reverses metastasis in animals. Citation Format: Cynthia C. Bamdad, Benoit J. Smagghe, Mark G. Carter, Trevor J. Grant, Laura M. Reale, Michael J. Nash, Danica M. Walkley, Jac-Leen S. Nash, Kevin R. Yi, Andrew K. Stewart. Metastasis: Cancer cells that have turned on a Yamanaka-like pluripotency program [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 2487.

Purpose: To develop an antibody therapeutic that disrupts interaction between onco-embryonic growth factor NME7AB and its cognate growth factor receptor, MUC1*, for the treatment of solid tumors and inhibition of their metastases. Normally, NME7 is only expressed and secreted by cells of a Day 3 to Day 5 human blastocyst. NME7 expression is then turned off and expression of self-regulating, adult forms of NME proteins is turned on. However, we found that NME7 expression is mistakenly turned on again in cancer cells. Cleavage of NME7 to NME7AB enables secretion from the cell, where it then binds to and dimerizes the MUC1* extracellular domain, which activates the MAP kinase signaling cascade. Methods: Because NME7AB shares homology to other adult forms of NME proteins, which are required for normal cellular function, we needed to develop a monoclonal antibody that binds to NME7AB, but not to other NME proteins, and disrupts its interaction with the MUC1* growth factor receptor as well. To investigate the effects of NME7AB on cancer growth and metastasis, we generated populations of cancer cells that had been cultured in a serum-free media to which was added a recombinant NME7AB. The parent cells, the NME7AB grown progeny, or mixed populations were implanted into NSG mice to test the effect of novel anti-NME7AB antibodies on tumor growth and metastasis. Parent tumor cells and the NME7AB-grown cells were engineered to emit light at different wavelengths so that growth of each population could be separately tracked in live animals. Results: Cancer cells that were cultured in a serum-free media containing recombinant NME7AB for 10 days acquired characteristics of more metastatic cells, often called cancer stem cells (referred to here as NME7AB-CSCs). They upregulated metastatic markers by up to 100-fold, became non-adherent, entered dormancy and formed tumors from injection of as few as 10,000 cells. The addition of an anti-NME7AB antibody during the 10-day culture period blocked this transition. NME7AB-CSCs that were injected into the tail vein of NSG mice induced widespread metastasis in 6-10 days, after which an anti-NME7AB antibody was i.v. administered. The anti-NME7AB antibody greatly reduced or cleared the metastasis. Animals implanted sub-cutaneously with NME7AB-CSCs, then administered anti-NME7AB antibody, showed a significant reduction in the spread of breast cancer cells to the liver. Mixed populations of parent cells plus NME7AB-CSCs, which emitted light at different wavelengths, were implanted sub-cutaneously and allowed to become established. The percent of the implanted tumor that was NME7AB-CSCs determined the growth rate of the entire tumor. An anti-NME7AB antibody inhibited growth of the parent cells as well as the NME7AB-CSCs. Conclusion: These data support further pre-clinical development to allow for a clinical candidate to be tested in a metastatic as well as an adjuvant setting. Citation Format: Cynthia Carol Bamdad, Benoit J. Smagghe, Mark G. Carter, Trevor J. Grant, Danica M. Page, Laura M. Reale, Michael J. Nash, Jac-Leen S. Nash, Andrew K. Stewart. Novel anti-NME7antibody inhibits metastasis of solid tumor cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3458.

Abstract Purpose: To develop a MUC1-targeted CAR T that recognizes the growth factor receptor form, MUC1*, does not bind full-length MUC1, hits a wide range of solid tumor cancers, binds to little or no normal tissues, and effectively kills tumor cells. Methods: Because MUC1 is expressed on normal epithelial tissues, we needed to develop an antibody that would only bind to the aberrant, cancerous form - MUC1*. MUC1* (muk1 star) is the transmembrane portion that remains after MUC1 is enzymatically cleaved and the bulky tandem repeat domain is shed from the cell surface. MUC1* is a growth factor receptor that is activated by ligand-induced dimerization of its truncated extracellular domain. Via a novel screen we identified antibodies that bind to a specific conformation within the ectopic epitope that is created when MUC1 is cleaved to MUC1* by enzymes secreted by the tumor microenvironment. This set of antibodies competitively inhibit the binding of onco-embryonic growth factor NME7AB to the cancerous form of MUC1*. We incorporated one of these cancer-specific antibodies into a CAR T. Results: huMNC2-CAR44 is in a 1st-in-human clinical trial [NCT04020575] for metastatic breast cancers, currently being performed at the Fred Hutchinson Cancer Research Center. Our IND-enabling studies showed that huMNC2-scFv bound robustly to 95% of breast cancer tissues, 83% ovarian cancers, 78% pancreatic cancers and 71% of lung cancer tissues, but showed little to no binding to normal tissues. In co-culture experiments, huMNC2-CAR44 T cells did not kill MUC1* negative cells, even if they expressed full-length MUC1, and the presence of MUC1* negative cells did not elicit a cytokine response from the CAR T cells. In vivo, huMNC2-CAR44 T cells inhibited or completely obliterated a variety of MUC1* positive solid tumors in NSG mice (n≥400). The human CD8+ huMNC2-CAR44 T cells expanded in animals as tumors shrunk, whereas the untransduced T cells did not. Clinical trial was slowed by COVID-19, as Seattle was the first hotbed of the virus. Thus far, there have been no serious adverse events attributed to the CAR T therapy. Even at the lowest dosage, patients have had robust CAR T cell expansion and have also had measurable signs of efficacy. Conclusions: MUC1* is the predominant form of MUC1 on cancerous tissues. Antibodies that bind to a specific conformation within the ectopic growth factor binding site in the MUC1* extra cellular domain are tumor selective. CAR T cells incorporating these antibodies are highly effective against solid tumors in animals. Robust staining of cancerous tissues and minimal to no staining of normal tissues predicts a large therapeutic window for huMNC2-CAR44 T cell dosing. Early patient responses appear to fulfill the predictions of the IND-enabling studies. We have now developed a cryopreservation formulation which enables shipping frozen product to additional clinical sites for bedside thaw and infusion. The trial is currently enrolling patients. Citation Format: Cynthia Bamdad, Andrew K. Stewart, Pengyu Huang, Benoit J. Smagghe, Scott T. Moe, Tyler E. Swanson, Thomas G. Jeon, Danica M. Page, Trevor J. Grant, Jennifer M. Specht. First-in-human CAR T targets MUC1 transmembrane cleavage product [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 57.

huMNC2-CAR44 is a second generation CAR that recognizes the growth factor receptor form, MUC1*, does not bind to full-length MUC1, hits a wide range of cancers and shows to little or no binding to normal tissues and is the first therapeutic tested in humans targeting the MUC1 transmembrane cleavage product called MUC1*. A 1st-in-human clinical trial of huMNC2-CAR44, NCT04020575, for metastatic breast cancers is underway at the Fred Hutchinson Cancer Research Center. MUC1 biology has historically been poorly understood. Several flawed reports are still widely cited in the literature. We will present data that de-bunks current MUC1 dogma. Namely, we will demonstrate that full-length MUC1 plays no role in tumorigenesis. The cleaved tandem repeat domain does not form a heterodimer with the remaining transmembrane portion. We demonstrate that elimination of full-length MUC1 greatly accelerates tumor growth in vitro and in vivo. 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. NME7AB looks like a single chain dimer of pseudo-identical domains that each can bind to a MUC1* extra cellular domain. Because it can dimerize MUC1* as a monomer, it renders the MUC1* growth factor receptor constitutively active. Adult forms of NME7AB limit self-replication by changing multimerization state from the active dimer to the inactive hexamer. Antibodies such as 5E5 and SM3 bind to aberrant, trapped glycans on O-linked glycosylation sites that are only in the tandem repeat domain, which is shed from the tumor after MUC1 cleavage. Unlike full-length MUC1, MUC1* has no sites for O-linked glycosylation, so MUC1* is missed by antibodies that target aberrant glycans. Importantly, therapeutics that target full-length MUC1 could increase tumorigenesis by enriching for cells expressing the tumorigenic MUC1* growth factor receptor. Minerva’s anti-MUC1* antibody, huMNC2, binds to the conformational epitope that is unmasked when MUC1 is cleaved to MUC1*. MMP9, which has been linked to poor prognosis and metastasis, cleaves MUC1 to a tumor-associated growth factor receptor form of MUC1*. huMNC2 and onco-embryonic growth factor NME7AB compete for binding to the same conformational epitope created when MUC1 is cleaved to MUC1* by MMP9. Neither huMNC2 nor NME7AB binds to full-length MUC1. IHC studies of thousands of human tissues – both normal and cancerous – show that the tumor associated antigen is MUC1* and not full-length MUC1. Patient-match primary and metastases show that as cancer stage progresses the amount of MUC1* increases. huMNC2-scFv bound robustly to 95% of the breast cancers, 83% ovarian, 78% pancreatic and 71% of lung cancer tissues (specimens n>2,800). There was minimal staining of normal tissues, primarily on apical surfaces which are expected to be less accessible to immune cells. In vivo, huMNC2-CAR44 T cells inhibited or completely obliterated a variety of MUC1* positive solid tumors in NSG mice (n>500). Minerva has developed next-gen CARs designed to increase persistence, and intends to file for additional INDs. Conclusions: MUC1* is the predominant form of MUC1 on cancerous tissues. Antibodies that target a conformational epitope in the membrane-proximal MUC1* extra cellular domain are tumor selective. CAR T cells targeting MUC1* extra cellular domain are highly effective against solid tumors in animals. Robust staining of cancerous tissues and minimal staining of normal tissues predicts a promising therapeutic window for huMNC2-CAR44 T cell dosing. Citation Format: Cynthia Bamdad, Andrew K Stewart, Pengyu Huang, Benoit J Smagghe, Scott T Moe, Tyler E Swanson, Thomas G Jeon, Danica M Page, Trevor J Grant, Jennifer M Specht. First-in-human chimeric antigen receptor t cells target muc1 transmembrane cleavage product [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-36.

Background Minerva Biotechnologies has opened a Phase I 1st-in-human CAR T clinical trial, NCT-04020575, for metastatic breast cancers at the Fred Hutchinson Cancer Research Center. huMNC2-CAR44 targets the truncated extra cellular domain of MUC1* (muk 1 star), which is the transmembrane cleavage product that remains after MUC1 is cleaved and the tandem repeat domain is shed from the cancer cells. No therapeutic that targets MUC1* has ever been tested in humans. All previous, failed attempts to therapeutically target MUC1 have targeted the tandem repeat domains, which are cleaved and shed from the surface of cancer cells. MUC1 cleavage increases as tumor stage increases. Cleavage and shedding of the tandem repeat domain unmasks an ectopic binding site for onco-embryonic growth factor NME7AB. The antibody fragment that targets the CAR to the tumor competes with NME7AB for binding to this same ectopic site. MUC1* growth factor receptor is activated when onco-embryonic growth factor NME7AB dimerizes its truncated extra cellular domain. Methods Autologous huMNC2-CAR44 T cells undergo a short 11-day manufacturing process, which includes an antigen stimulation step and preserves many of the cells in the naïve and central memory state. Patients are pre-treated with standard Cy-Flu lymphodepletion. Dose escalation phase is standard 3 × 3 with a starting dose 3.3 × 10e5 CAR T cells and going up to 1.0 × 10e7 cells. Patients are eligible if biopsy is greater than or equal to 30% reactive with MNC2 in a CLIA validated diagnostic assay. Results In vitro, huMNC2-CAR44 T cells killed cancer cells, but not non-cancer cells even if they expressed MUC1 or a normal form of cleaved MUC1. In NSG mice (n>300), huMNC2-CAR44 T cells eliminated MUC1* positive tumors from implanted breast cancer cells. A single CAR T cell injection eliminated tumors for 100 days; control animals had to be sacrificed at Day 20. Further, huMNC2-CAR44 T cell mediated killing increased as MUC1* density increased. In tissue micro array studies, huMNC2-scFv recognized 95% of breast cancers, across all subtypes, 83% ovarian, 78% pancreatic and 71% of lung cancers. huMNC2-scFv showed almost no binding to normal tissues and no staining of critical organs. Although patient recruitment has been slowed by COVID-19, preliminary results indicate CAR T cell expansion and possible efficacy. Conclusions Preliminary results show that patients experienced robust CAR T cell expansion with CAR-positive T cells persisting at Day 60 post huMNC2-CAR44 T cell treatment. Possible signs of efficacy were measured. Trial Registration NCT04020575

Abstract Purpose: To develop a MUC1-targeted CAR T that recognizes the growth factor receptor form, MUC1*, does not bind full-length MUC1, hits a wide range of cancers but binds to little or no normal tissues, and effectively kills tumor cells. We developed a novel CAR T, huMNC2-CAR44, that targets MUC1*, which is the transmembrane cleavage product of MUC1. A 1st-in-human clinical trial for metastatic breast cancers opened in Q4, 2019 at the Fred Hutchinson Cancer Research Center. MUC1* is a growth factor receptor that is activated when onco-embryonic growth factor NME7AB dimerizes its truncated extra cellular domain. The binding site for NME7AB is ectopic and is only unmasked after the tandem repeat domain of MUC1 is cleaved and shed from the cell surface. Unlike full-length MUC1, MUC1* has no sites for O-linked glycosylation, so antibodies such as 5E5 that bind to aberrant, trapped glycans cannot bind to the MUC1*. Methods: We developed a novel antibody screen that identifies monoclonal antibodies that bind to a conformational epitope of MUC1* that is created when MUC1 is cleaved by MMP9, which has been linked to poor prognosis and metastasis. The cancer selectivity of conformation-specific anti-MUC1* antibodies, anti-MUC1* antibodies that recognize linear epitopes, or antibodies that bind to the tandem repeat domain of full-length MUC1 was assessed in IHC studies of cancer vs normal tissue arrays. Conformation specific anti-MUC1* antibodies were incorporated into CARs, transduced into human T cells and tested in vitro and in vivo for their ability to kill MUC1* positive, but not MUC1* negative, tumor cells. Results: Anti-MUC1* antibody huMNC2 binds to the conformational epitope that is unmasked when MUC1 is cleaved to MUC1* by MMP9. huMNC2 competes with onco-embryonic growth factor NME7AB for this same conformational epitope on MUC1*. Neither huMNC2 nor NME7AB binds to full-length MUC1. IHC studies of tissue micro arrays showed that antibodies that bound to full-length MUC1 did not react with 29% of the breast cancer tissues in several arrays. Conversely, anti-MUC1* antibody MNC2 bound robustly to 95% of the breast cancer tissues in serial sections of the same arrays. Further, huMNC2-scFv bound to 83% ovarian, 78% pancreatic and 71% of lung cancer tissues, with little to no binding to normal tissues. In vivo, huMNC2-CAR44 T cells inhibited or completely obliterated a variety of MUC1* positive solid tumors in NSG mice (n>400). The human CD8+ huMNC2-CAR44 T cells expanded in the animals as tumors shrunk, whereas the untransduced T cells did not. Conclusions: MUC1* is the predominant form of MUC1 on cancerous tissues. Antibodies that target a conformational epitope in the MUC1* extra cellular domain are tumor selective. CAR T cells targeting MUC1* extra cellular domain are highly effective against solid tumors in animals. Robust staining of cancerous tissues and minimal to no staining of normal tissues predicts large therapeutic window for huMNC2-CAR44 T cell dosing. Citation Format: Cynthia C. Bamdad, Andrew K. Stewart, Pengyu Huang, Benoit J. Smagghe, Scott T. Moe, Tyler E. Swanson, Thomas G. Jeon, Danica M. Page, Ketan M. Mathavan, Trevor J. Grant. 1st-in-human CAR T targets MUC1 transmembrane cleavage product [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 4230.

Abstract The KRAS protooncogene is mutated in 40 to 50% of colon cancers. In an effort to identify strategies to treat KRAS mutant colon cancers, we previously implicated the TGF-β activated kinase (TAK1) as a candidate therapeutic target that promotes the survival of KRAS dependent cancers. In follow-up studies, we have explored and investigated the detailed mechanistic basis for TAK1 mediated survival signaling in KRAS dependent cancer cells. Using proteomic and transcriptomic analyses of proinflammatory signaling mediators, we have uncovered complex autocrine/paracrine signaling loops that are constitutively activated in KRAS dependent colon cancer cells. A central mediator of these signaling loops is the BMP7-BMP receptor (BMPR1A) pathway, which functions coordinately with oncogenic KRAS to drive TAK1 and NF-κB mediated transcriptional upregulation of proinflammatory cytokines such as GM-CSF, CCL5/RANTES and IL-8. Conversely, a number of cytokines and cytokine regulators are negatively regulated by KRAS-BMPR signaling interactions, including CXCL9/MIG and IL1RN. We previously showed that TAK1 inhibition with a small molecule agent, 5Z-7-oxozeaenol promotes apoptosis in colon cancer cells. We have now determined that 5Z-7-oxozeaenol, in addition to irreversibly inhibiting TAK1 kinase activity, also transiently inhibits the MEK kinase. Therefore, combined TAK1/MEK inhibition explains the potent killing effects that we have observed in KRAS dependent colon cancer cells. To test this empirically, we have used 2 selective kinase inhibitors targeting each respective kinase, AZ-TAK1 and AZD6244 to show either single agent can induce apoptotic cell selectively in KRAS dependent cells, with TAK1 inhibition resulting in stronger killing effects. Importantly, treatment of KRAS dependent colon cancer cells with combinations of AZ-TAK1 and AZD6244 results in additive killing effects, revealing a potential therapeutic strategy for KRAS dependent cancers in the clinic. Mechanistically, MEK and TAK1 converge on the control of NF-κB and canonical Wnt-dependent transcriptional activities. Surprisingly, we find that NF-κB and Wnt signaling mutually antagonize each other in terms of cytokine expression to create a finely-tuned balance between pro-death and pro-survival signals. We hypothesize that, as a consequence, these balanced signals allow for both efficient maintenance of tumor cell survival and as well as for communication with stromal components in the tumor microenvironment. Thus, KRAS, MEK or TAK1 blockade results in a tipping of the balance to favor pro-death signals. Citation Format: Kelsey L. McNew, William J. Whipple, Anita K. Mehta, Trevor Grant, Anurag Singh. MEK and TAK1 signaling interactions coordinately regulate inflammation and apoptosis in KRAS dependent colon cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3188. doi:10.1158/1538-7445.AM2015-3188

Despite improvements in treatment for T-Acute Lymphoblastic Leukemia (T-ALL), only 70-80% of children and 40% of adults achieve long-term remission. To discover novel targets for the development of molecular therapeutics, we conducted a dominant genetic modifier screen to identify genes whose inactivation delays the onset of leukemia. We have generated a Myc-driven zebrafish model of T-ALL that recapitulates its human counterpart genetically and pathologically. Utilizing this zebrafish model of T-ALL, we conducted a genetic screen that identified a gene encoding dihydrolipoamide S-succinyltransferase (DLST), whose heterozygous loss significantly delays the onset of leukemia in zebrafish. DLST is the E2 component of α-ketogluterate dehydrogenase complex (KGDHC), a key enzyme in the TCA cycle that converts α-ketogluterate (α-KG) to Succinyl CoA. Western blot analysis revealed that DLST was upregulated in zebrafish tumor cells, T-ALL cell lines and primary human T-ALL samples. Zebrafish T-ALL cells with heterozygous inactivation of dlst have decreased cell size and slowed cell cycle progression, compared to T-ALL cells without dlst inactivation. To demonstrate that DLST is a relevant target in human T-ALL, we used two structural analogs of α-KG (α-keto-n-valeric acid sodium salt (KV) and α-keto-β-methyl-n-valeric acid (KMV)) to inhibit the KGDHC activity. Inhibition of KGDHC by both KV and KMV decreased cell viability and promoted apoptotic cell death in a panel of human T-ALL cell lines. Genetic inactivation of DLST, via shRNA knockdown, also decreased cell viability in MOLT16 human T-ALL cell line, confirming the therapeutic potential of DLST in human T-ALL treatment. In vivo treatment of our zebrafish T-ALL model with KV promoted tumor regression within seven days of treatment. Taken together our findings indicate that DLST is an important contributor to T-ALL pathologenesis, and suggest that DLST is a potential target for further therapeutic development to treat human T-ALL. Disclosures: No relevant conflicts of interest to declare.