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

We developed a MUC1* targeting CAR T that is scheduled for a 1st-in-human clinical trial for metastatic breast cancers at the Fred Hutchinson Cancer Research Center in late 2019. huMNC2-CAR44 targets MUC1*, which is the transmembrane cleavage product of MUC1. 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 masked in full-length MUC1, as is the binding site for the huMNC2 antibody. Thus, huMNC2 does not bind to full-length MUC1, which is expressed on healthy epithelium. Previous attempts at a MUC1 targeting therapeutic all failed, presumably because they targeted the tandem repeat domain of full-length MUC1, which is shed after cleavage to MUC1*. huMNC2-CAR44 T cells are highly selective for cancerous tissues: first, because they target MUC1*, not the healthy full-length form. Secondly, the huMNC2 antibody recognizes a specific conformation of the MUC1* extra cellular domain that is created when MUC1 is cleaved by an enzyme that is overexpressed in many cancers, especially breast cancers. IHC studies of thousands of normal vs. cancerous human tissue specimens show that huMNC2-scFv almost exclusively binds to tumor tissues, hitting over 90% of breast, 83% ovarian, 78% pancreatic and 71% of lung cancers. Recognition of breast cancer specimens appears not to be limited by cancer sub-type. In vivo experiments of human tumors in NSG mice (n>300), show that huMNC2-CAR44 T cells robustly inhibited MUC1* positive solid tumors. A single injection of huMNC2-CAR44 T cells eliminated tumors in NSG mice which remained tumor free to Day 100, compared to control animals that had to be sacrificed at Day 20 due to excess tumor volume. Other animal experiments showed that huMNC2-CAR44 T cell efficacy increased with increasing antigen density. Dual tumor experiments of a high antigen density and low antigen density tumor in the same animal showed that adequate MUC1* density is required for a CAR T response, further supporting the idea that huMNC2-CAR44 T cells will selectively kill MUC1* positive tumors, while sparing normal tissues. Conclusion: MUC1* is the predominant form of MUC1 present on cancers. Antibodies that target conformational epitopes produced by specific subsets of cleavage enzymes make anti-MUC1* CAR T cells highly tumor selective. The generation of an anti-MUC1* CAR that recognizes a conformational epitope created when MUC1 is cleaved to MUC1* by a specific cleavage enzyme, argues that CARs could be made patient specific, based on which cleavage enzymes their tumors express. 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. First-in-human CAR T for solid tumors targets the MUC1 transmembrane cleavage product [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-11.

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.

Purpose: To develop a CAR T for solid tumors that hits a wide range of cancers, is effective and has little or no effect on normal tissues. We developed a MUC1* targeting CAR T that is scheduled for a 1st-in-human clinical trial for metastatic breast cancers at the Fred Hutchinson Cancer Research Center in Q1 2019. Unlike previous attempts at making an anti-MUC1 cancer therapeutic, huMNC2-CAR44 targets MUC1*, which is the transmembrane cleavage product. 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 masked in full-length MUC1; anti-MUC1* antibody huMNC2 binds to the same site. huMNC2 does not bind to full-length MUC1 nor other cleaved forms of MUC1 that are on some healthy tissues that need to rapidly divide. Monoclonal antibodies against the truncated MUC1* extra cellular domain were generated and screened by IHC for reactivity to cancerous tissue micro arrays (TMAs) and lack thereof on normal tissues. The best monoclonals were incorporated into CARs, transduced into human T cells and tested in vitro for specific killing of MUC1* positive but not MUC1* negative cells. We developed a novel cell line in which MUC1 is not cleaved. By adding specific cleavage enzymes, we identified antibodies that recognized conformational epitope that were created by specific cleavage enzymes. The final selection of an anti-MUC1* antibody for the targeting head of our CAR was based on its widespread binding to breast cancer tissues, low cross reactivity to normal tissues, and its recognition of a conformational epitope created when MUC1 is cleaved to MUC1* by a specific cleavage enzyme that is overexpressed in many cancers, especially breast cancers. In vivo we showed that injecting this cleavage enzyme near a MUC1/MUC1* breast tumor dramatically accelerated tumor growth, which was stopped by simultaneous injection of the cleavage enzyme and our CAR T cells. IHC studies of thousands of normal vs. cancerous human tissue specimens show that huMNC2-scFv almost exclusively binds to tumor tissues, hitting over 90% of breast, 83% ovarian, 78% pancreatic and 71% of lung cancers. Recognition of breast cancer specimens appears not to be limited by cancer sub-type. In vivo experiments of human tumors in NSG mice (n>300), show that huMNC2-CAR44 T cells inhibited or completely obliterated a variety of MUC1* positive solid tumors. Dual tumor experiments showed that adequate MUC1* density is required for a CAR T response, further supporting the idea that huMNC2-CAR44 T cells will selectively kill MUC1* positive tumors, while sparing normal tissues. Conclusion: MUC1* is the predominant form of MUC1 present on cancers. Antibodies that target conformational epitopes produced by specific subsets of cleavage enzymes make anti-MUC1* CAR T cells highly tumor selective. CARs could be patient specific based on which cleavage enzymes their tumors express. Citation Format: Cynthia C. Bamdad, Nelson D. Glennie, Andrew K. Stewart, Pengyu Huang, Benoit J. Smagghe, Tyler E. Swanson, Erin K. Hanahoe, Gregory L. Riley. First-in-human CAR T for solid tumors targets the MUC1 transmembrane cleavage product [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2323.

Abstract Purpose: Investigate the rationale for CAR Ts that target full-length MUC1 (MUC1-FL) rather than the cleaved, growth factor receptor form, MUC1*. Over 75% of solid tumors aberrantly express MUC1. However, attempts to create an effective therapeutic by targeting MUC1-FL have all failed. No therapeutic that targets the cleavage product, MUC1*, has ever been tested in humans. On cancer cells, most of the MUC1-FL is enzymatically cleaved, releasing the bulk of the extra cellular domain and leaving a truncated transmembrane protein - MUC1*. There are currently two CAR T therapeutics (based on antibody 5E5 or SM3) being developed that target aberrant O-linked glycans. Importantly, MUC1* has no sites for O-linked glycans and is not recognized by 5E5 or SM3. Experimental Procedures: Human ovarian cancer cells that express both MUC1-FL and MUC1* were implanted into the intraperitoneal (i.p.) space of female NSG mice. IVIS measurement verified engraftment. A cleavage enzyme that is overexpressed in most solid tumors and known to cleave MUC1-FL to MUC1* was injected into i.p. space to simulate the effect of a therapeutic that would eliminate cells expressing MUC1-FL. Human cancerous tissue specimens were probed with antibody VU4H5 that recognizes tandem repeats of MUC1-FL, 5E5 that recognizes aberrant O-linked glycans on tandem repeats of MUC1-FL, and anti-MUC1* antibody huMNC2. Summary New Data: Elimination of cells expressing MUC1-FL, by cleaving MUC1-FL to MUC1*, caused tumors to grow dramatically (average 221-times in only 9 days) and metastasize. IHC studies showed that 30% of breast cancers do not express MUC1-FL but only express MUC1*. No tumor specimens were positive for MUC1-FL but negative for MUC1*. Our studies show that as tumor stage progresses, expression of MUC1* increases as MUC1-FL decreases. Over 95% of breast, 83% ovarian, 78% pancreatic and 71% lung cancers reacted with our anti-MUC1* antibody huMNC2-scFv (n>3,000). huMNC2-CAR44 T cells inhibited growth of MUC1* positive and MUC1-FL/MUC1* positive tumors, even after metastasis. Conclusions: CAR T cells that target full-length MUC1 will enrich the tumor for MUC1*, increasing tumor growth and inducing metastasis. Citation Format: Cynthia C. Bamdad, Andrew K. Stewart, Luke T. Deary, Benoit J. Smagghe, Pengyu Huang, Nelson D. Glennie, Tyler E. Swanson. CAR T cells that target full-length MUC1 enrich for MUC1* and induce metastasis [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 LB-140.