Rationale: Transformed MUC1 (tMUC1) is a cancer-associated antigen that is overexpressed in >90% of triple-negative breast cancers (TNBC), a highly metastatic and aggressive subtype of breast cancer.TAB004, a murine antibody targeting tMUC1, has shown efficacy for the targeted delivery of therapeutics to cancer cells.Our aim was to evaluate humanized TAB004 (hTAB004) as a potential theranostic for TNBC.Methods: The internalization of hTAB004 in tMUC1 expressing HCC70 cells was assessed via fluorescent microscopy.hTAB004 was DOTA-conjugated and radiolabeled with Indium-111 or Actinium-225 and tested for stability and tMUC1 binding (ELISA, flow cytometry).Lastly, in vivo biodistribution (SPECT-CT), dosimetry, and efficacy of hTAB004 were evaluated using a TNBC orthotopic mouse model.Results: hTAB004 was shown to bind and internalize into tMUC1-expressing cells.A production method of 225 Ac-DOTA-hTAB004 (yield>97%, RCP>97% SA=5 kBq/µg) and 111 In-DOTA-hTAB004 (yield>70%, RCP>99%, SA=884 kBq/µg) was developed.The labeled molecules retained their affinity to tMUC1 and were stable in formulation and mouse serum.In NSG female mice bearing orthotopic HCC70 xenografts, the in vivo tumor concentration of 111 In-DOTA-hTAB004 was 65 ± 15 %ID/g (120 h post injection).A single 225 Ac-DOTA-hTAB004 dose (18.5 kBq) caused a significant reduction in tumor volume (P<0.001,day 22) and increased survival compared to controls (P<0.007).The human dosimetry results were comparable to other clinically used agents. Conclusion:The results obtained with hTAB004 suggest that the 111 In/ 225 Ac-DOTA-hTAB004 combination has significant potential as a theranostic strategy in TNBC and merits further development toward clinical translation.

Purpose: The purpose of this study was to develop an Indium-111/Actinium-225 based theragnostic utilizing the tMUC1 targeting humanized antibody, TAB004, and its evaluation in a tumor bearing rodent model.Triple negative breast cancer (TNBC) is an aggressive phenotype that comprises only 15-20% of all new breast cancer cases but is responsible for a majority of related deaths. Chemotherapy is the current mainstay treatment for TNBC but side effects and drug-resistance remain an issue, so development of targeted therapies for TNBC is critical. Tumor associated Mucin 1 (tMUC1), an aberrantly glycosylated transmembrane glycoprotein, is overexpressed in >90% of TNBCs and as such is an attractive target. The humanized antibody hTAB004 shows high affinity and selectivity to tMUC1 and was thus used to develop a theranostic strategy for TNBC.Method: hTAB004 was conjugated to DOTA-NHS, purified, formulated in a metal free buffer and radiolabeled with Indium-111 or Actinium-225 to produce 111In-DOTA-hTAB004 and 225Ac-DOTA-hTAB004, respectively. The radiolabeled molecule was evaluated for stability. Retention of affinity of cold-labeled molecules was validated.In vivo and ex vivo biodistribution studies of 111In-DOTA-hTAB004 were performed in HCC70 orthotopic xenograft tumor-bearing female NSG mice (n=3) using SPECT/CT imaging over 120h. Dosimetry analysis was performed utilizing the in vivo data. Therapeutic efficacy of 225Ac-DOTA-hTAB004 was determined in HCC70 orthotopic xenograft tumor-bearing female nude mice (n=5/group) by monitoring bodyweights and tumor sizes over time.Results: DOTA-hTAB004 was labeled successfully with both Indium-111 and Actinium-225. The radiolabeled molecules were found stable in both formulation and mouse serum and the non-radioactively labeled surrogates 115In-DOTA-hTAB004 and 139La-DOTA-hTAB004 retained their affinity to tMUC1.In vivo biodistribution data revealed increased tumor accumulation of 111In-DOTA-hTAB004 over 120 h, reaching 65±15 percent of injected dose per gram (%ID/g). The next organs of significant uptake (Spleen 8.9±0.6 %ID/g and Liver 8.3±1 %ID/g) had a 7-fold lower uptake at 120 h. All mice treated with a single injection of 225Ac-DOTA-hTAB004 (500 nCi, 18 kBq) showed a complete response with tumor volumes shrinking by > 89% within 48 days. No therapy associated toxicities were seen in any of the mice outside of tail necrosis in one animal which may have been due to dose extravasation. All the control mice, which received the DOTA-hTAB004, showed continued tumor growth and had to be sacrificed by day 35.Conclusions111In-DOTA-hTAB004 biodistribution data indicates the excellent tumor targeting capabilities of hTAB004. 225Ac-DOTA-hTAB004 therapy data shows exceptional clinical benefit in mice bearing human tumors. Given this data, radiolabeled hTAB004 is a promising targeted treatment and imaging option for TNBC.Citation Format: ohad ilovich, Vanessa Kelly, Shu-ta Wu, Vijay Gottumukkala, Richard Coelho, Rahul Puri, Pinku Mukherjee. Development and non-clinical evaluation of an111In/225Ac theranostic for triple negative breast cancer [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 LB-281.

Immunotherapy regimens have shown success in subsets of cancer patients; however, their efficacy against pancreatic ductal adenocarcinoma (PDA) remain unclear. Previously, we demonstrated the potential of TAB004, a monoclonal antibody targeting the unique tumor-associated form of MUC1 (tMUC1) in the early detection of PDA. In this study, we evaluated the therapeutic benefit of combining the TAB004 antibody with Liposomal-MSA-IL-2 in immune competent and human MUC1 transgenic (MUC1.Tg) mouse models of PDA and investigated the associated immune responses. Treatment with TAB004 + Lip-MSA-IL-2 resulted in significantly improved survival and slower tumor growth compared to controls in MUC1.Tg mice bearing an orthotopic PDA.MUC1 tumor. Similarly, in the spontaneous model of PDA that expresses human MUC1, the combination treatment stalled the progression of pancreatic intraepithelial pre-neoplastic (PanIN) lesion to adenocarcinoma. Treatment with the combination elicited a robust systemic and tumor-specific immune response with (a) increased percentages of systemic and tumor infiltrated CD45+CD11b+ cells, (b) increased levels of myeloperoxidase (MPO), (c) increased antibody-dependent cellular cytotoxicity/phagocytosis (ADCC/ADCP), (d) decreased percentage of immune regulatory cells (CD8+CD69+ cells), and (e) reduced circulating levels of immunosuppressive tMUC1. We report that treatment with a novel antibody against tMUC1 in combination with a unique formulation of IL-2 can improve survival and lead to stable disease in appropriate models of PDA by reducing tumor-induced immune regulation and promoting recruitment of CD45+CD11b+ cells, thereby enhancing ADCC/ADCP.

The purpose of this study was to develop an Indium-111/Actinium-225 based theragnostic utilizing the tMUC1 targeting humanized antibody, TAB004, and its evaluation in a tumor bearing rodent model. Triple negative breast cancer (TNBC) is an aggressive phenotype that comprises only 15-20% of all new breast cancer cases but is responsible for a majority of related deaths. Chemotherapy is the current mainstay treatment for TNBC but side effects and drugresistance remain an issue, so development of targeted therapies for TNBC is critical. Tumor associated Mucin 1 (tMUC1), an aberrantly glycosylated transmembrane glycoprotein, is overexpressed in >90% of TNBCs and as such is an attractive target. The humanized antibody hTAB004 shows high affinity and selectivity to tMUC1 and was thus used to develop a theranostic strategy for TNBC. hTAB004 was conjugated to DOTA-NHS, purified, formulated in a metal free buffer and radiolabeled with Indium-111 or Actinium-225 to produce 111In-DOTA-hTAB004 and 225Ac-DOTA-hTAB004, respectively. The radiolabeled molecule was evaluated for stability. Retention of affinity of cold-labeled molecules was validated. In vivo and ex vivo biodistribution studies of 111In-DOTA-hTAB004 were performed in HCC70 orthotopic xenograft tumor-bearing female NSG mice (n=3) using SPECT/CT imaging over 120h. Dosimetry analysis was performed utilizing the in vivo data. Therapeutic efficacy of 225Ac-DOTA-hTAB004 was determined in HCC70 orthotopic xenograft tumorbearing female nude mice (n=5/group) by monitoring bodyweights and tumor sizes over time. DOTA-hTAB004 was labeled successfully with both Indium-111 and Actinium-225. The radiolabeled molecules were found stable in both formulation and mouse serum and the non-radioactively labeled surrogates 115In-DOTA-hTAB004 and 139La-DOTA-hTAB004 retained their affinity to tMUC1.In vivo biodistribution data revealed increased tumor accumulation of 111In-DOTA-hTAB004 over 120 h, reaching 65±15 percent of injected dose per gram (%ID/g). The next organs of significant uptake (Spleen 8.9±0.6 %ID/g and Liver 8.3±1 %ID/g) had a 7-fold lower uptake at 120 h. All mice treated with a single injection of 225Ac-DOTA-hTAB004 (500 nCi, 18 kBq) showed a complete response with tumor volumes shrinking by > 89% within 48 days. No therapy associated toxicities were seen in any of the mice outside of tail necrosis in one animal which may have been due to dose extravasation. All the control mice, which received the DOTA-hTAB004, showed continued tumor growth and had to be sacrificed by day 35. 111In-DOTA-hTAB004 biodistribution data indicates the excellent tumor targeting capabilities of hTAB004. 225Ac-DOTA-hTAB004 therapy data shows exceptional clinical benefit in mice bearing human tumors. Given this data, radiolabeled hTAB004 is a promising targeted treatment and imaging option for TNBC.

e13112Background: Breast cancer (BC) remains a leading cause of cancer-related deaths worldwide. Triple negative breast cancer (TNBC) is an aggressive phenotype that comprises only 15-20% of all new BC cases but is responsible for a majority of related deaths. Chemotherapy is the current mainstay treatment for TNBC but side effects and drug-resistance remain an issue, so development of targeted therapies for TNBC is critical. Tumor associated mucin 1 (tMUC1), an aberrantly glycosylated transmembrane glycoprotein, is overexpressed in > 90% of TNBCs. We’ve developed a humanized, tMUC1-specific antibody (hTAB004) and radiolabeled it to allow for targeted TNBC treatment. Methods: hTAB004 was conjugated to DOTA-NHS, purified, formulated in a metal free buffer and radiolabeled with Indium-111 or Actinium-225 to produce 111In-DOTA-hTAB004 and 225Ac-DOTA-hTAB004, respectively. In vivo and ex vivo biodistribution studies of 111In-DOTA-hTAB004 were performed in HCC70 xenograft tumor-bearing female NSG mice (n = 3) ...

Screening for breast cancer has predominantly been done using mammography. Unfortunately, mammograms miss 50% cancers in women with dense breast tissue. Multi-modal screenings offer the best chance of enhancing breast cancer screening effectiveness. We evaluated the use of TAB004, an antibody that recognizes the tumor form of the glycoprotein MUC1 (tMUC1), to aid early detection of breast cancer. Our experimental approach was to follow tMUC1 from the tissue into circulation. We found that 95% of human breast cancer tissues across all subtypes stained positive for TAB004. In breast cancer cell lines, we showed that the amount of tMUC1 released from tumor cells is proportional to the cell's tMUC1 expression level. Finally, we showed that TAB004 can be used to assess circulating tMUC1 levels, which when monitored in the context of cancer immunoediting, can aid earlier diagnosis of breast cancer regardless of breast tissue density. In a blinded pilot study with banked serial samples, tMUC1 levels increased significantly up to 2 years before diagnosis. Inclusion of tMUC1 monitoring as part of a multi-modal screening strategy may lead to earlier stage diagnosis of women whose cancers are missed by mammography.

Screening for breast cancer has predominantly been done using mammography. Unfortunately, mammograms miss 50% cancers in women with dense breast tissue. Multi-modal screenings offer the best chance of enhancing breast cancer screening effectiveness. We evaluated the use of TAB004, an antibody that recognizes the tumor form of the glycoprotein MUC1 (tMUC1), to aid early detection of breast cancer. Our experimental approach was to follow tMUC1 from the tissue into circulation. We found that 95% of human breast cancer tissues across all subtypes stained positive for TAB004. In breast cancer cell lines, we showed that the amount of tMUC1 released from tumor cells is proportional to the cell’s tMUC1 expression level. Finally, we showed that TAB004 can be used to assess circulating tMUC1 levels, which when monitored in the context of cancer immunoediting, can aid earlier diagnosis of breast cancer regardless of breast tissue density. In a blinded pilot study with banked serial samples, tMUC1 levels increased significantly up to 2 years before diagnosis. Inclusion of tMUC1 monitoring as part of a multi-modal screening strategy may lead to earlier stage diagnosis of women whose cancers are missed by mammography.

BACKGROUND: Earlier detection of transformed cells using target-specific imaging techniques holds great promise. We have developed TAB 004, a monoclonal antibody highly specific to a protein sequence accessible in the tumor form of MUC1 (tMUC1). We present data assessing both the specificity and sensitivity of TAB 004 in vitro and in genetically engineered mice in vivo. METHODS: Polyoma Middle T Antigen mice were crossed to the human MUC1.Tg mice to generate MMT mice. In MMT mice, mammary gland hyperplasia is observed between 6 and 10 weeks of age that progresses to ductal carcinoma in situ by 12 to 14 weeks and adenocarcinoma by 18 to 24 weeks. Approximately 40% of these mice develop metastasis to the lung and other organs with a tumor evolution that closely mimics human breast cancer progression. Tumor progression was monitored in MMT mice (from ages 8 to 22 weeks) by in vivo imaging following retro-orbital injections of the TAB 004 conjugated to indocyanine green (TAB-ICG). At euthanasia, mammary gland tumors and normal epithelial tissues were collected for further analyses. RESULTS: In vivo imaging following TAB-ICG injection permitted significantly earlier detection of tumors compared with physical examination. Furthermore, TAB-ICG administration in MMT mice enabled the detection of lung metastases while sparing recognition of normal epithelia. CONCLUSIONS: The data highlight the specificity and the sensitivity of the TAB 004 antibody in differentiating normal versus tumor form of MUC1 and its utility as a targeted imaging agent for early detection, tumor monitoring response, as well as potential clinical use for targeted drug delivery.

e22153 Background: Breast cancer (BC) remains the second leading cause of cancer-related deaths for women in the US. Mammography has improved early detection, but can miss up to 25 percent of BC since mammogram sensitivity is less than 50% in women with dense breast tissue. BC is often detected in these women when a lump is discovered and the cancer is late stage. There is a critical need for an accurate diagnostic for these women. The tumor form of MUC1 (designated TMUC1),a transmembrane glycoprotein, is aberrantly glycosylated and overexpressed in ~95% of BC. We have developed an antibody (TAB004), which specifically recognizes TMUC1. Methods: 1) Using TAB004, a GMP-grade ELISA kit was used to test circulating levels of TMUC1 in sera of patients. We have tested ~250 patient sera (includes normal, benign, and malignant) collected from Duke University Cancer Center, National Cancer Institute (NCI), Early detection research network (EDRN) reference set, and Fox Chase Biorepositories. The samples represented the major BC subtypes (Luminal A, Luminal B, Triple Negative, and HER2 type). 2) Expression of bound TMUC1 on BC tissue was determined with Immunohistochemistry (IHC) using TAB004. 3) We quantified surface expression of TMUC1 using Cy5-conjugated TAB004 and flow cytometry on a panel of over 30 human BC cell lines that represented the major subtypes. 4) Finally, TAB004 conjugated to a fluorophore was used to image human BC tumors in vivo in mice. Results: 1) TMUC1 ELISA results show statistically significant differentiation between normal/benign and malignant sera distributions (p = .000 to .056 from the various institutions). 2) IHC results show strong TMUC1 expression in malignant tissue sparing normal tissue. 3) Flow cytometry data shows that all BC subtypes expressed TMUC1. 4) In vivo imaging shows localization of TAB004-fluorophore to the tumor. Conclusions: A unique tumor specific antibody based serum ELISA can be used for the early detection of breast cancer in women with dense breast tissue. Clinical adoption of the TAB004 based serum ELISA in conjunction with mammography will lead to early detection of BC in these women.

14 Background: Earlier detection of abnormal cells using target-specific techniques holds great promise. One of those targets Mucin-1 (MUC1) is expressed as a hypo-glycosylated form (i.e., tMUC1) in 95% of breast tumors and plays a crucial role in cancer progression. We have developed Tab004, a monoclonal antibody highly specific to a protein sequence accessible in tMUC1. Here we present data assessing the specificity and sensitivity of Tab004 in vitro and in genetically engineered mice in vivo overtime. Methods: We have developed human MUC1 Tg mice that were bred to mice that carry the oncogene, polyoma middle T antigen driven by the MMTV promoter. These mice designated MMT develop mammary gland tumors spontaneously and expresses the human form of tMUC1. PyVMT and C57Bl/6 served as controls. Mice develop mammary gland hyperplasia between 8-10 weeks of age that progresses to ductal carcinoma in situ by 12-14 weeks and adenocarcinoma by 18-24 weeks. Approximately 40% of the mice develop metastasis to the lung and other organs. The tumor progression appropriately mimics the human disease. MMT mice (n = 20) were injected twice monthly retro-orbitally with 12.5ug (100uL) of Tab004-conjugated to indocyanine green (ICG) and imaged thereafter from 8 to 22 weeks of age. Fluorescence was assessed 4 and 24 hrs post injection using the IVIS system. At euthanasia, tissue was collected for further analyses. Further, human breast tumor and normal mammary epithelial tissues were evaluated by immunohistochemical staining. Results: Tab004 specifically recognizes tMUC1 and not normal MUC1. In MMT mice, ICG-conjugated Tab004 allowed early detection of tumors in vivo sparing recognition of normal mammary epithelia in the C57BL/6 mice or in the PyV MT tumors. Detection with ICG-conjugated Tab004 allowed monitoring of tumor progression overtime. Importantly, ICG-conjugated Tab004 permitted significantly earlier detection than physical examination. Conclusions: The data highlight the specificity and the sensitivity of Tab004 in detecting tMUC1 in vitro, in situ and in relevant murine models in vivo. Thus, Tab004 will have significant clinical relevance for development as a targeted imaging agent and in the future for targeted drug delivery.