Abstract Purpose: Develop more effective cancer therapeutics by simultaneously targeting multiple drivers of growth, metastasis and resistance. Methods: MUC1 is aberrantly expressed on over 75% of solid tumor cancers. Although cloned 30 years ago, there is still no approved MUC1-targeted drug. Previous attempts, including those targeting trapped glycans, targeted the tandem repeat domain, which is shed after cleavage by cancer-associated enzymes. MUC1* with a 45-amino acid ecd (extracellular domain) is the only form that has been shown to function as a growth factor receptor via dimerization of ecd by embryonic growth factors. We show that where MUC1 is cleaved determines the conformation of the remaining ecd. Even within a truncated 45-amino acid ecd, there are distinct conformations that only exist on cancer cells and others that are only on progenitor cells. All these species have been mistakenly grouped together as “MUC1-C”. We developed monoclonal antibodies that specifically bind to each conformation and mapped their expression on cancer vs normal tissues vs progenitor cells. We then generated bi- and tri-specific ADCs that simultaneously bind to 2 or 3 cancer targets. Results: Data show there are 2 distinct MUC1* receptors that drive tumor growth and metastasis. Targeting one and not the other led to tumor recurrence. We developed bi- and tri-specific antibodies that simultaneously bind to both MUC1* growth factor receptors, and HER2 or alpha-v-beta-6. In antibody internalization experiments, multi-specific antibodies were internalized faster and more completely than mono-specifics. In ADC format, bi-specific antibodies that hit both MUC1* growth factor receptors inhibited tumor recurrence in animals. ADCs incorporating both a MUC1* antibody and Trastuzumab killed Trastuzumab-resistant cancer cells with an IC50 of 0.05 nM compared to 0.73 nM on Trastuzumab sensitive parent cells. Indeed, in Phase I huMNC2-CAR44 trial, patients who had acquired resistance to Trastuzumab or Fam-Trastuzumab Deruxtecan-nxki were best responders in our trial with 75% DCR, 11-month OS, 11.3 month increased survival in patients with high MUC1* expression. In animals, our novel linker payload out-performed industry gold standard Deruxtecan. Conclusions: It is critical that MUC1-targeted therapeutics hit the cancer-specific forms of MUC1*. Therapeutics that incorporate antibodies that bind to hematopoietic stem cells, for example, would be disastrous if given to cancer patients. Therapeutics that target full-length MUC1, whether aberrantly glycosylated or not, could give initial positive responses via ADC bystander killing. However, the hypothesis is that by killing cells expressing full-length MUC1, which plays no role in cell growth, the MUC1*-positive cell population would be enriched and would accelerate tumor growth and metastasis. Citation Format: Cynthia Carol Bamdad, Benoit J. Smagghe, Scott Moe, Mark G. Carter, Kevin R. Yi, Michael J. Nash, Robert McDermott, Trevor J. Grant, Salvatore Marchese, Daniel S. Miller, Natalie K. Miller, Andrew K. Stewart, . Bi- and tri-specific ADCs that target two distinct growth factor receptor forms of MUC1* and HER2 or alpha-v-beta-6 inhibit tumor recurrence and overcome acquired resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6941.

Abstract Purpose: Develop therapeutic antibodies that block the tumorigenic action of MUC1* ligands and bacterial mimics thereof. Methods: Ligand fishing using the MUC1*ecd-45 peptide as bait identified NME1 and NME7-AB as ligands of MUC1*. Dimeric NME1 and monomeric NME7-AB, which has two MUC1* binding domains, dimerize MUC1* extracellular domain (ecd) and activate MAP kinase growth pathway. IF microscopy showed NME7-AB is secreted by all Day 3 cells of a human blastocyst, then only by cells of the Inner Cell Mass by Day 5. NME7-AB is then replaced by adult form, NME1, which only binds MUC1* when a dimer. At higher concentration, NME1 switches from dimer to hexamer, which doesn’t bind MUC1* but instead triggers differentiation. NME7 is cytoplasmic until cleavage removes an N-terminal DM10 fragment, which allows secretion, then ‘NME7-AB’. Alternative splice variant NME7-X1 is transcribed without the DM10 domain, so is always secreted and active. Cancer cells that have acquired resistance to cancer drugs, increase expression of MUC1*, NME7-AB and NME7-X1. We mapped the MUC1* interaction site on NME7-AB and generated antibodies that block the interaction. We discovered a subset of antibiotic-resistant bacteria that make NME7-AB mimics that function like human NME7-AB. We developed monoclonal antibodies that specifically bind to these bacterial mimics. Results: Growing cancer cells with recombinant NME7-AB as the only growth factor, enabled anchorage independent cell growth, induced upregulation of metastasis markers and formed tumors as well as metastasis from injection of as few as 10,000 cells within 10-20 days. 50-times as many parent cells did not. A novel antibody, prevented NME7-AB from inducing upregulation of metastatic markers, and prevented anchorage independent growth. In animals, NME7-AB monoclonal antibodies reversed total metastasis in a few days. They also inhibited metastasis of established sub-cu tumors. Similarly, bacterial mimics of NME7-AB induced upregulation of metastatic markers and, in animals, accelerated tumor growth and their metastasis. We developed monoclonals antibodies that bind to the interaction region on bacterial mimics. Conclusions: MUC1 has long been an elusive target for cancer therapeutics. However, there has been little focus on its onco-embryonic ligand NME7-AB. NME7-AB is turned off early in embryogenesis but is turned on again in all MUC1-positive cancers. Levels of NME7 determine growth rate and metastatic potential of human cancers. Demonstration of NME7-AB antibodies inhibiting cancer and metastasis in animals xenografted with human cancers supports the further clinical development of NME7-AB targeted therapeutics. Discovery of a molecular link between specific bacteria and acceleration of human cancers may be an important area for future research. Citation Format: Cynthia Carol Bamdad, Benoit J. Smagghe, Mark G. Carter, Kevin R. Yi, Michael J. Nash, Trevor J. Grant, Daniel S. Miller, Natalie K. Miller, Andrew K. Stewart. Antibodies that bind to primitive human growth factor NME7-AB block interaction with MUC1* growth factor receptor to inhibit cancer growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2234.

To develop a treatment for HER2 positive patients whose tumors have stopped responding to Enhertu HER2 positive BT474 breast cancer cells were induced to become Herceptin resistant (trastuzumab resistant) in vitro via prolonged exposure to sub-lethal levels of the drug. Analysis of the trastuzumab-resistant cells showed that they had greatly increased expression of MUC1*, known to have anti-apoptotic and pro-growth properties. Recall that 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 pHrodo show that MNC2 is rapidly and robustly internalized by the cancer cells. We conjugated MNC2 to deruxtecan to yield an MNC2-deruxtecan with a DAR of 7.4. Female NSG mice were xenografted with either BT474-wt cells or the trastuzumab-resistant cells, BT474-Res2. Tumors were allowed to engraft then treated with trastuzumab or MNC2-deruxtecan. Herceptin had little to no effect on BT474-Res2 implanted tumors. However, MNC2-deruxtecan potently killed the trastuzumab resistant tumors in a matter of weeks. MNC2 conjugated to other linker-payloads similarly killed the trastuzumab resistant tumors. Treated animals showed no obvious signs of toxicities. Patients with HER2 positive breast cancers have benefitted greatly from the new ADC Enhertu (fam-trastuzumab deruxtecan). However, most patients, develop resistance to HER2-directed therapies and experience progressive disease leaving them with few treatment options. Here we’ve shown that HER2 positive breast cancer cells acquire resistance to trastuzumab by increasing expression of the powerful growth factor receptor MUC1*. These MUC1*-targeting ADCs offer an effective treatment for these patients. These data support a conclusion that a MUC1*-ADC is safe and could have high therapeutic value for patients who have stopped responding to Enhertu. Cynthia C. Bamdad, Benoit J. Smagghe, Scott T. Moe, Kevin R. Yi, Trevor J. Grant, Mark G. Carter, Daniel S. Miller, Michael J. Nash, Jacy P. Marquez, Natalie K. Miller, JacLeen S. Nash, Robert McDermot, Andrew K. Stewart. MUC1*-deruxtecan kills tumors that have acquired resistance to trastuzumab containing therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 558.

TPS2683 Background: MUC1 has been one of the most important therapeutic targets for solid tumor cancers for the past 20+ years. However, no MUC1 targeted therapeutic has yet succeeded or has been granted FDA approval. Although MUC1 is overexpressed on cancer cells, it is also widely expressed on normal epithelial cells. We previously reported that a MUC1 transmembrane cleavage product, MUC1* (muk 1 star), is a Class I growth factor receptor activated by ligand-induced dimerization of its truncated extra cellular domain. We generated monoclonal antibodies that recognize the specific conformation that is created when MUC1 is cleaved by specific enzymes expressed in the tumor microenvironment. 93% of human breast cancer tissues are recognized by one of these antibodies, huMNC2, which importantly does not bind to normal MUC1. We have incorporated this antibody into two CARs, wherein one bears the “1XX” mutations in CD3z that greatly increase CAR-T cell persistence, inhibit exhaustion and enable the killing of low antigen expressing cancer cells. Methods: Phase I for huMNC2-CAR44, an autologous frozen product, opened January, 2020, but as a 1st-in-human trial was paused during COVID. All patients receive 300 mg/m2 cyclophosphamide and 30 mg/m2 fludarabine for 3 days prior to CAR-T treatment. Dose escalation from 3.3x105 – 1x107 CAR+ cells/kg follows a standard 3 + 3 design. In addition to standard organ function inclusion criteria, the trial was first open to breast cancer patients whose cancer had progressed after at least 2 or 3 prior therapies while metastatic, yet there was no limit on the number of prior therapies. A recent patient biopsy had to be at least 30% positive for MUC1* in a CLIA validated IHC assay. Importantly, there were no limits on the vein-to-vein time. Dose levels 1 and 2 for huMNC2-CAR44 were completed without DLTs. Phase I for huMNC2-CAR22, which bore the 1XX mutations in CD3z to increase persistence and increase the killing of low antigen expressing cells opened September, 2023. Based on responses of initial huMNC2-CAR44 patients, inclusion criteria were amended to an Enrichment Trial Design. The number of prior therapies was limited to less than or equal to 10. Anticipated survival at the time of product infusion should be at least 3 months. Vein-to-vein time, which historically had been 15-16 days at one site and as high as 83 days at another site, would be targeted to 16- 22 days. The tumors of eligible patients need to have a MUC1* membrane positive H-score equal to or greater than 120 out of a possible 300, defined as high MUC1* positivity. Based on IHC analysis of human breast cancer TMAs, the H-score restriction would include roughly 40% of breast cancers. The increased ability of huMNC2-CAR22 to kill low antigen expressing cancer cells may support future studies of patients expressing low levels of MUC1*. Clinical trial information: NCT04020575 .

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 Purpose: First-in-human Phase I study for advanced MUC1* positive breast cancer with autologous T cells engineered to express either a chimeric antigen receptor, huMNC2-CAR44 or huMNC2-CAR22, which specifically bind to a cleaved form of MUC1 (MUC1*); evaluate the safety and preliminary anti-tumor activity. Methods: MUC1* (muk 1 star) is the growth factor receptor form of MUC1, created by cleavage and release of the N-terminal portion of MUC1. The targeting antibody, huMNC2, only recognizes the conformational epitope created when MUC1 is cleaved by specific tumor-associated enzymes that are correlated with poor prognosis. huMNC2 does not bind to full-length MUC1, which is expressed on all normal epithelial cells. huMNC2-scFv was incorporated into huMNC2-CAR44, comprising a CD8 hinge and transmembrane region, 4-1BB costimulatory domain and wild-type CD3z. huMNC2-scFv was also incorporated into huMNC2-CAR22 wherein the hinge, transmembrane and co-stimulatory portions were derived from CD28 and CD3z bears the 1XX mutations to increase in vivo persistence. Inclusion criteria require that the patient’s tumor is at least 30% MUC1* positive and that patient has progressed through 2 or 3 prior therapies, while in the metastatic setting. Patients receive standard Cy/Flu lymphodepletion approximately 3-days before CAR T treatment, administered at 1 of 4 dose levels ranging from 3.3X10^5 up to 1.0X10^7 CAR+ T cells. Results: To date, 8 patients have been treated with huMNC2-CAR44. No patients experienced neuro toxicities. No off-target toxicities were observed. 3 patients experienced CRS Grade 1-3. In 6 of the 8 patients, side effects were non-existent or minimal. However, one patient experienced a Grade 5 SAE that was deemed to be possibly related to the treatment. Best responses include Partial Responses and Stable Disease at a low CAR-T dose. Greatest efficacy was observed for patients whose biopsy showed H Scores >120. Patients are currently being enrolled for treatment with huMNC2-CAR22, where the 1XX mutations are expected to increase in vivo persistence, durability of response and reduced incidence of CRS. Conclusions: These data support a conclusion that the MUC1* antibody, huMNC2, is safe and could have high therapeutic value as a CAR T treatment for solid tumors with moderate to high antigen density. As the huMNC2-CAR22 (1XX) trial proceeds, we will assess if patient responses mirror our animal results that show that the 1XX mutations confer increased persistence, reduced exhaustion and the ability to kill tumors with low antigen density. Citation Format: Cynthia Carol Bamdad, Joanne E. Mortimer, Yuan Yuan, Jennifer M. Specht, Andrew K. Stewart, Benoit J. Smagghe, Stephen C. Lin, Mark G. Carter, Tim W. Synold, Mark D. Fleming, Stanley R. Hamilton, Vishwas Parekh, Danica M. Walkley, Qing Liu-Michael, Kevin R. Yi, Jac-Leen S. Nash, Michael J. Nash, Stephen J. Forman. Phase I first-in-human MUC1* targeted autologous CAR T cells for the treatment of metastatic breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT096.

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.

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.

IntroductionDirect oral anticoagulants (DOAC) are commonly prescribed and measuring drug levels may be useful in a number of contexts. However, data on DOAC level measurement and their clinical utility in real-world studies are limited. MethodsWe carried out a 2-year retrospective cohort study of DOAC levels measured at our institution. ResultsOne hundred and sixty-nine levels measured in 113 patients were included in the final analysis. Our patients had a median age of 69.9. AF was the commonest indication for anticoagulation. Median turnaround time for inpatient levels was 92minutes. Median FXa inhibitor levels within 6hours of last dose and following one half-life were similar to those described previously. However, the range of levels was wider than expected. Importantly, some levels remained in an on therapy range even after 3 half-lives. There was no correlation between dabigatran level and time from last dose. The reason for request varied with setting; 23 outpatient levels were to monitor drug efficacy, whereas 54 and 43 inpatient levels were collected in the context of bleeding and emergency surgery respectively. 60.3% of levels had an impact on clinical decision making. ConclusionOur real-world study demonstrates that DOAC levels can be performed in a timely manner to influence clinical decision making. In addition, it suggests there is a wide variation in levels such that it can be difficult to predict in the real world. Overall, this supports the wider use of DOAC levels to help guide clinicians in managing patients taking these drugs.

#### What you need to know A 78 year old woman presents with a three month history of easy bruising. She also feels tired and has lost her appetite. She has hypertension and a history of myocardial infarction, and has been taking amlodipine and aspirin for the last five years. Clinical examination reveals bruising to the arms, legs, and abdomen, and poor oral health. ### How should I assess the patient? ### History Discuss the bruises . Ask questions such as: Bruising limited to the limbs suggests trauma, and that on the trunk and other areas is more suggestive of an underlying bleeding disorder.1 Bruises are considered to represent a bleeding disorder when five or more (greater than 1 cm in size) occur simultaneously in exposed areas. Petechiae, and haematomas, when occurring without trauma, are suggestive of a bleeding disorder2(Box 1). #### Box 1: Causes of easy bruising in adults ##### Common ##### Rare

HaemophiliaVolume 22, Issue 2 p. e111-e113 Letter to the Editor A survey of platelet aggregometry tests in a tertiary centre G. Webster, G. Webster University of Manchester, Manchester, UKSearch for more papers by this authorM. J. Nash, M. J. Nash Department of Haematology, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UKSearch for more papers by this authorJ. Thachil, Corresponding Author J. Thachil Department of Haematology, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK Correspondence: Dr Jecko Thachil, Department of Haematology, Central Manchester University Hospitals NHS Foundation Trust, Oxford Road, Manchester M13 9WL, UK. Tel.: +44 161 276 4812; fax: +44 161 276 8085; e-mail: [email protected]Search for more papers by this author G. Webster, G. Webster University of Manchester, Manchester, UKSearch for more papers by this authorM. J. Nash, M. J. Nash Department of Haematology, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UKSearch for more papers by this authorJ. Thachil, Corresponding Author J. Thachil Department of Haematology, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK Correspondence: Dr Jecko Thachil, Department of Haematology, Central Manchester University Hospitals NHS Foundation Trust, Oxford Road, Manchester M13 9WL, UK. Tel.: +44 161 276 4812; fax: +44 161 276 8085; e-mail: [email protected]Search for more papers by this author First published: 08 February 2016 https://doi.org/10.1111/hae.12889Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1Harrison P, Mackie I, Mumford A et al. British Committee for Standards in Haematology. Guidelines for the laboratory investigation of heritable disorders of platelet function. Br J Haematol 2011; 155: 30–44. 2Dawood BB, Lowe GC, Lordkipanidze M et al. Evaluation of participants with suspected heritable platelet function disorders including recommendation and validation of a streamlined agonist panel. Blood 2012; 120: 5041–9. 3Langdown J, Luddington RJ, Huntington JA, Baglin TP. A hereditary bleeding disorder resulting from a premature stop codon in thrombomodulin (p.Cys537Stop). Blood 2014; 124: 1951–6. 4Zia AN, Chitlur M, Rajpurkar M et al. Thromboelastography identifies children with rare bleeding disorders and predicts bleeding phenotype. Haemophilia 2015; 21: 124–32. Volume22, Issue2March 2016Pages e111-e113 ReferencesRelatedInformation

This report provides details on strategies and tools for reducing IROPs impacts on passengers. The guidebook will assist communication and coordination as airports and airlines implement IROPS contingency plans. Included are communication checklists and a strategy for obtaining and maintaining stakeholder contacts; a list of federal flight data resources and other technologies, which allow for expedited communication regarding diversions on a national, regional, and local scale; flow diagrams to illustrate the integration of communication and collaboration processes; case studies of a variety of scenarios depicting IROPS responses; scenarios and instructions for conducting tabletop exercises; and a tool to assist in predicting the risks associated with national, regional, and local IROPS events to improve planning and response. This CD-based tool (CRP-CD-180) includes a response plan for stakeholders’ involvement in assessing the likelihood and severity of reoccurrence of IROPS impacts, data sources to help alert an airport when an IROPS event is likely to occur, and the ability to create reports on IROPS risk levels for the stakeholders. The case studies and tabletop exercises also serve as training materials and can be customized for any airport.

Acquired Haemophilia (AH) is an autoimmune bleeding disorder, which despite being rare, can be fatal. It occurs in patients with previously normal haemostasis who spontaneously develop IgG autoantibodies against factor VIII. Unlike congenital haemophilia, it manifests as spontaneous bleeding into skin and soft tissues. The presentation can be masked in patients who are receiving warfarin where the bleeding is often attributed to warfarin therapy, as in the case described in this report. Consideration of AH is important in patients taking anticoagulants, when coagulopathy and bleeding fails to correct with usual measures.

HaemophiliaVolume 21, Issue 6 p. e494-e496 Letter to the Editor Management of a myocardial infarction in a patient with classical acquired haemophilia patient in partial remission D. J. M. Routledge, Corresponding Author D. J. M. Routledge Clinical Haematology, Central Manchester NHS Foundation Trust, Manchester, UK Correspondence: David J. M. Routledge, Clinical Haematology, Central Manchester NHS Foundation Trust, Oxford Road, M13 9WL Manchester, UK. Tel.: 01612761234; fax: 01612768085; e-mail: daveroutledge@doctors.org.ukSearch for more papers by this authorD. Fraser, D. Fraser Haematology Manchester Royal Infirmary, Manchester, UKSearch for more papers by this authorJ. Thachil, J. Thachil Haematology Manchester Royal Infirmary, Manchester, UKSearch for more papers by this authorM. J. Nash, M. J. Nash Haematology Manchester Royal Infirmary, Manchester, UKSearch for more papers by this author D. J. M. Routledge, Corresponding Author D. J. M. Routledge Clinical Haematology, Central Manchester NHS Foundation Trust, Manchester, UK Correspondence: David J. M. Routledge, Clinical Haematology, Central Manchester NHS Foundation Trust, Oxford Road, M13 9WL Manchester, UK. Tel.: 01612761234; fax: 01612768085; e-mail: daveroutledge@doctors.org.ukSearch for more papers by this authorD. Fraser, D. Fraser Haematology Manchester Royal Infirmary, Manchester, UKSearch for more papers by this authorJ. Thachil, J. Thachil Haematology Manchester Royal Infirmary, Manchester, UKSearch for more papers by this authorM. J. Nash, M. J. Nash Haematology Manchester Royal Infirmary, Manchester, UKSearch for more papers by this author First published: 20 July 2015 https://doi.org/10.1111/hae.12757Citations: 2Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume21, Issue6November 2015Pages e494-e496 RelatedInformation