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Metastasis is the leading cause of cancer-related deaths, and metastatic cancers remain largely incurable due to chemoresistance. Biomarkers of metastatic cells are lacking, and probes that could be used to detect and target metastases would be highly valuable. Here we hypothesize that metastatic cancer cells express cell-surface receptors that can be harnessed for identification of molecules homing to metastases. Screening a combinatorial library in a mouse mammary tumor model of spontaneous metastasis identified cyclic peptides with tropism for cancer cells disseminated to the lungs. Two lead peptides, CLRHSSKIC and CRAGVGRGC, bound murine and human cells derived from breast carcinoma and melanoma in culture and were selective for metastatic cells in vivo. In mice, peptide CRAGVGRGC radiolabeled with 67Ga for biodistribution analysis demonstrated selective probe homing to lung metastases. Moreover, systemic administration of 68Ga-labeled CRAGVGRGC enabled noninvasive imaging of lung metastases in mice by PET. A CRAGVGRGC-derived peptide induced apoptosis upon cell internalization in vitro and suppressed metastatic burden in vivo. Colocalization of CLRHSSKIC and CRAGVGRGC with N-cadherin+/E-cadherin− cells indicated that both peptides are selective for cancer cells that have undergone the epithelial-to-mesenchymal transition. We conclude that CRAGVGRGC is useful as a probe to facilitate the development of imaging modalities and therapies targeting metastases. Significance: This study identifies new molecules that bind metastatic cells and demonstrates their application as noninvasive imaging probes and vehicles for cytotoxic therapy delivery in preclinical cancer models.

Background: Although advances in neoadjuvant and adjuvant treatments have led to enhanced survival in patients with HER2 positive breast cancer, intraoperative detection of residual disease and tumor margins (including HER2 positive DCIS) remain challenging. Real-time fluorescence imaging is being used during surgery to improve tumor visualization and identify margins. However, the lack of an approved intraoperative imaging agent that is specific for HER2 expressing cancers limits the utility of fluorescence-guided surgery (FGS) in this patient population. We examined the potential of using clinically approved anti-HER2 monoclonal antibodies (mAbs), trastuzumab and pertuzumab, for the development of fluorescent immunoconjugates dual-labeled with radioactivity to enable quantitative assessment. Methods: Trastuzumab, and pertuzumab were conjugated to the near-infrared (NIR) fluorophore, IR800CW, and radiometal chelator, deferoxamine (DFO), to permit dual labeling with 89Zr (positron emitting radionuclide). To confirm retention of HER2 specificity and functionality, flow cytometry analysis of the dual-labeled immunoconjugates was performed in HER2+ (BT474, SKBR3), HER2 moderate (MCF7), and HER2− (MDA231) cell lines. Using the same cell lines, radioactive uptake studies were employed to quantitatively measure cellular uptake study using 89Zr with and without blocking doses of unlabeled mAb. In vivo optical imaging of each immunoconjugate and IgG controls was performed in BT474 and MCF7 xenografts in BALB/c (nu/nu) mice 48 hours post injection using the BrukerXtreme system. Results: In vitro results confirmed retention of receptor mediated binding on both flow cytometry and radioactive analyses. As shown in Table 1, uptake of each immunoconjugate correlated with the degree of HER2 expression. Importantly, the addition of unlabeled mAb led to a 79-93% reduction in binding to cells with the highest HER2 expression (BT474 and SKBR3). In MDA231 cells binding of the immunoconjugates was similar to the IgG control and indicates low non-specific binding. In vivo and ex vivo analysis of HER2+ and HER2 moderate xenografts demonstrated prominent tumor uptake with minimal signal in non-clearance organs. As a result, tumor-to-muscle, tumor-to-fat pad, and tumor-to-lung ratios were >5, indicating excellent contrast in key regions where surgical excisions are performed clinically. Table 1: Uptake of 89Zr-mAb-IR800 in breast cancer cell linesCell Lines% Uptake of 89Zr-PertuzumabPertuzumab Block (Unlabeled)% Uptake of 89Zr-TrastuzumabTrastuzumab Block (Unlabeled)% Uptake of 89Zr-IgGBT47449.2±14.83.7±0.036.9±5.84.6±2.61.3±0SKBR337.9±1.77.9±0.942.8±3.52.9±1.73.5±0.4MCF721.7±2.63.2±0.44.0±0.12.5±0.11.3±0.7MDA2311.9±0.40.8±0.31.0±0.31.9±0.51.3±0.7 Conclusion: This study demonstrates the potential utility of HER2-targeted FGS to improve tumor visualization, increase the likelihood of obtaining clean surgical margins, and ultimately reduce morbidity in patients with HER2 breast cancer. Citation Format: Jo Simien, Julie Voss, A.M. Thompson, S.C. Ghosh, S. AghaAmiri, A. Azhdarinia, H.S. Tran-Cao. HER2/neu antibody-fluorophore conjugate for intraoperative detection of HER2-positive breast cancer [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 P1-02-01.

Introductory statement: To overcome enzyme-mediated resistance mechanisms associated with the DNA alkylating agent, temozolomide (TMZ), we seek to develop a peptide-drug conjugate (PDC) for selective drug delivery in cells that express the somatostatin receptor subtype-2 (SSTR2). Material and methods: The clinically used somatostatin analog, 68Ga-DOTA-TOC, was used as a model for development of the SSTR2-targeted PDC. Synthesis was performed by (i) replacing DOTA with a multimodality chelator (MMC), (ii) attaching a modified TMZ analog to MMC, and (iii) conjugating the payload moiety to TOC on solid-phase. The resulting product, MMC(TMZ)-TOC, was radiolabeled with 67Ga using cation exchange chromatography methods optimized for 68Ga. Retention of SSTR2 binding was examined in cell lines with different expression level of SSTR2 with and without blocking doses of octreotide. A membrane acid wash was performed to evaluate internalization efficiency. The cytotoxicity of MMC(TMZ)-TOC was tested in IMR-32 cells that endogenously express SSTR2, and potency was compared to free-TMZ. An alkaline comet assay was then performed to assess the DNA-damaging properties of the PDC in the presence and absence of SSTR2 blocking. Unpublished results: MMC(TMZ)-TOC was efficiently produced with chemical purity >90% as shown by high-performance liquid chromatography (HPLC). Radiochemical purity following 67Ga labeling was >95% and indicates the feasibility of using the MMC to directly label the drug conjugates. Cell-based experiments showed that specific binding of the 67Ga-labeled PDC was similar to 67Ga-DOTA-TOC and correlated with SSTR2 expression. In HCT116-SSTR2 cells that overexpress SSTR2, 14.8±4.8% of 67Ga-MMC(TMZ)-TOC and 17.0±4.2% of 67Ga-DOTA-TOC were taken up by cells. Blocking the excess octreotide reduced binding to near background levels, illustrating receptor-mediated uptake of the PDC. Acid-washing demonstrated internalization of 67Ga-MMC(TMZ)-TOC after receptor-binding, indicating retention of the agonist properties of TOC after payload conjugation. Results from the cell cytotoxicity study demonstrated that the PDC inhibited cell growth in a dose-dependent manner that was similar to free-TMZ, with the IC50 values of 81.6 and 75.6 µM for free-TMZ and MMC(TMZ)-TOC, respectively. The comet assay revealed that MMC(TMZ)-TOC was effective in causing DNA damage in cells that express SSTR2, as shown by reduced cytotoxic effects when co-incubated with octreotide. Conclusions: We showed that a clinically used SSTR2-targeted radiopharmaceutical can be converted into a PDC that retains receptor-binding and internalization properties. We also showed that the cytotoxic effects of TMZ are maintained by the PDC. Based on these results, in vivo evaluation of MMC(TMZ)-TOC is warranted to assess its potential as a targeted therapy for NETs. Citation Format: Solmaz AghaAmiri, Sukhen C. Ghosh, Lea Stitzlein, Servando Hernandez Vargas, Bruno Perlatti, Daniel M. Halperin, Ali Azhdarinia. A novel peptide-drug conjugate induces DNA damage in SSTR2-expressing cells [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 LB120.

Objectives: Positron Emission Tomography (PET) plays a pivotal role in the surgical care of cancer patients pre- and post-operatively, however, no functional imaging approaches are currently available for real-time surgical guidance. Recently, antibodies dual-labeled with radioactive and near-infrared fluorescent (NIRF) labels have emerged as potential agents to extend whole-body nuclear imaging capabilities into the operating room, but structural limitations in conventional labeling reagents necessitate a random, two-step labeling process which can reduce bioactivity and batch reproducibility. Here, we evaluate the utility of a site-specific multimodal chelator (MMC) that combines PET/NIRF labeling into a single moiety for enhanced production of immunoconjugates for hybrid imaging. Methods: An anti-epithelial cell adhesion molecule (EpCAM)-specific antibody, mAb7, was treated with varying amounts of the reducing agent dithiothreitol (DTT) to partially reduce the interchain disulfides and permit conjugation with MMC-maleimide. The number of MMC moieties per mAb were quantified by isotopic dilution using non-radioactive Cu and immunoreactivity was assessed by ELISA. 64Cu labeling was performed and samples were purified by size exclusion prior to HPLC analysis. Stability of the immunoconjugates was evaluated by HPLC. Multimodal PET/NIRF imaging was performed in an orthotopic prostate tumor model to assess in vivo targeting. Results: mAbs treated with increasing amounts of DTT had higher conjugation ratios, with values ranging from 1-8 MMC moieties per mAb. ELISAs showed no differences in binding potency for the MMC-immunoconjugates. Radiochemical yields were high for all samples (>80%) and increased as a function of conjugation ratio. Interestingly, initial 64Cu labeling of the immunoconjugate with a MMC/mAb ratio of 4 had a corresponding HPLC trace showing >95% radiochemical purity, however, labeling 8 weeks later revealed formation of a second, prominent radioactive peak. Conversely, the HPLC profile of the immunoconjugate with a MMC/mAb ratio of 2 was consistent throughout the study, suggesting a possible correlation between the extent of partial reduction and protein stability. This observation was supported by in vivo imaging findings as lesions were better visualized in mice receiving 64Cu-MMC-mAb7 with a MMC/mAb ratio of 2. Conclusions: The results indicate that site-specific conjugation of MMC is effective for dual labeling antibodies. However, partial reduction conditions must be optimized in order to obtain a multimodal immunoconjugate with suitable imaging properties and stability. Citation Format: Sukhen C. Ghosh, Barrett R. Harvey, Holly Robinson, Kenneth L. Pinkston, Nathaniel Wilganowski, Banghe Zhu, Eva M. Sevick-Muraca, Ali Azhdarinia. Comparison of dual labeling strategies for NIRF/PET hybrid imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4298. doi:10.1158/1538-7445.AM2014-4298