Abstract 2630: MEN1309, a novel antibody drug conjugate (ADC) targeting Ly75 antigen, induces complete responses in several xenografts of solid tumors

Introduction: We report on the development and preclinical validation of NBE-002, a next-generation antibody drug conjugate (ADC) for the treatment of triple-negative breast cancer (TNBC). NBE-002 consists of a humanized monoclonal antibody directed against the receptor tyrosine kinase ROR1, expressed on the surface of numerous solid cancers including TNBC, and is site-specifically conjugated to a derivative of the highly potent anthracycline PNU-159682. Results: Therapeutic efficacy of NBE-002 was evaluated in ROR1-low/-intermediate/-high patient-derived xenograft (PDX) models of TNBC, lung adenocarcinoma, ovarian carcinoma, and a variety of sarcomas. NBE-002 was found to display significant anti-tumor activity in each indication. The most pronounced effect was achieved in TNBC, where complete tumor regression was observed already at the lowest ADC dose tested (0.33 mg/kg), even in models expressing low levels of ROR1 (H-score ≤70). Since these PDX studies were performed in immune-compromised hosts, and anthracyclines are known inducers of immunogenic cell death, anti-tumor activity was also investigated in syngeneic tumor models in immune-competent hosts. Anti-tumor activity of PNU-ADCs involved activation of the immune system, as shown by evaluation of NBE-002 or a Trastuzumab-PNU conjugate (T-PNU) in ROR1- or HER2-positive syngeneic breast cancer models, respectively. Depletion of CD8 T cells severely reduced anti-tumor activity, demonstrating an important role for T cells in driving tumor regression. Furthermore, when tumor free animals were re-challenged with the same tumor, tumor growth was inhibited in the absence of any further ADC administration, indicating the development of an immunological memory. Notably, combination of ADC and checkpoint inhibition, such as α-PD1 or α-CTLA4, significantly enhanced tumor eradication following the treatment. Conclusion: Our results demonstrate that NBE-002 is a highly effective and promising targeted therapeutic for the treatment of ROR1 positive TNBC and potentially other solid tumor indications that warrants clinical development. Considering the pronounced immune-modulatory functions of the PNU payload, NBE-002 may be particularly well suited for combination therapy with immune checkpoint inhibitors. NBE-002 is currently undergoing GMP manufacturing and initiation of clinical studies is expected in mid-2020. Citation Format: Roger R. Beerli, Lorenz Waldmeier, Rémy Gébleux, Francesca Pretto, Ulf Grawunder. NBE-002, an anthracycline-based immune-stimulatory antibody drug conjugate (iADC) targeting ROR1 for the treatment of 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-197.

CD205 is a type I transmembrane glycoprotein and is a member of the C-type lectin receptor family. Analysis by mass spectrometry revealed that CD205 was robustly expressed and highly prevalent in a variety of solid malignancies from different histotypes. IHC confirmed the increased expression of CD205 in pancreatic, bladder, and triple-negative breast cancer (TNBC) compared with that in the corresponding normal tissues. Using immunofluorescence microscopy, rapid internalization of the CD205 antigen was observed. These results supported the development of MEN1309/OBT076, a fully humanized CD205-targeting mAb conjugated to DM4, a potent maytansinoid derivate, via a cleavable N-succinimidyl-4-(2-pyridyldithio) butanoate linker. MEN1309/OBT076 was characterized in vitro for target binding affinity, mechanism of action, and cytotoxic activity against a panel of cancer cell lines. MEN1309/OBT076 displayed selective and potent cytotoxic effects against tumor cells exhibiting strong and low to moderate CD205 expression. In vivo, MEN1309/OBT076 showed potent antitumor activity resulting in durable responses and complete tumor regressions in many TNBC, pancreatic, and bladder cancer cell line-derived and patient-derived xenograft models, independent of antigen expression levels. Finally, the pharmacokinetics and pharmacodynamic profile of MEN1309/OBT076 was characterized in pancreatic tumor-bearing mice, demonstrating that the serum level of antibody-drug conjugate (ADC) achieved through dosing was consistent with the kinetics of its antitumor activity. Overall, our data demonstrate that MEN1309/OBT076 is a novel and selective ADC with potent activity against CD205-positive tumors. These data supported the clinical development of MEN1309/OBT076, and further evaluation of this ADC is currently ongoing in the first-in-human SHUTTLE clinical trial.

Matriptase, is a serine protease type II transmembrane protein. Matriptase forms a cognate pair with its Kunitz type endogenous inhibitor HAI-1 and is commonly deregulated and ubiquitously expressed in a variety of human carcinomas and in particular, breast cancer. This enzyme activity is regulated by binding to its endogenous inhibitors (HAI-1 or 2). Previously, we reported the synthesis and anti-tumor studies of a novel antibody drug conjugate (ADC) targeting activated matriptase of tumor cells (Oncotarget, 2018,9(40):25983). The activated matriptase-specific monoclonal antibody (M69) was conjugated with a potent anti-mitotic toxin, monomethyl auristatin-E (MMAE) via a lysosomal protease (cathepsin B) -cleavable dipeptide linker. The ADC was found to be potent and selective against multiple activated matriptase-positive epithelial carcinoma cell lines including triple negative breast cancer (TNBC), as well as alone and in combination with bortezomib in Mantle cell lymphoma (Frontiers in Oncology, 2019,9:258). In-vivo studies showed that the ADC has potent anti-tumor effects alone or in combination with cisplatin or carboplatin against TNBC xenografts and a primary human TNBC (PDX), respectively. The chimeric ADC (mouse-human chimera) also evoked an immune response against TNBC xenografts in humanized mouse models. Here, we report the sensitivity of TNBC tumors with and without a BRCA mutation to ADC alone or in combination with a PARP inhibitor, Olaparib. We analyzed the mutational status of BRCA1, BRAC2, p53 and activated matriptase expression of four TNBC cell lines (MDA-MB-468, MDA-MB-231, HCC1806 and MDA-MB-436). All of the cell lines had p53 mutations. The MDA-MB-468 cell line with a mutation in BRCA 2, and expression of activated matriptase was the most sensitive cell line to both olaparib and the chimeric MMAE ADC, and the combination of them resulted in marked synergistic cell kill. The MDA-MB-436 cell line with a splice site mutation in BCRA1 was less sensitive to both treatments, but there was modest synergy when the two drugs were combined. In contrast the MDA-MB-231 cell line without a BRCA mutation was much less sensitive to the ADC or olaparib, and the combination did not show synergy. The HCC1806 cell line with wt BRCA was sensitive to the ADC and olaparib, but the combination resulted in antagonistic effects. Triple negative breast cancers with a BRAC2 mutation and overexpression of activated matriptase may be a subset of breast cancers that may benefit from the combination of our novel ADC and a PARP inhibitor. Citation Format: Gulam M. Rather, Zoltan Szekely, Joseph R. Bertino. An antibody drug conjugate targeting activated matriptase, exhibits synergistic cytotoxicity with a PARP inhibitor, olaparib in triple negative breast cancer [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 2212.

Introduction: A constant challenge in the field of cancer therapeutics is the presence of intrinsic resistance or the development of acquired resistance to chemotherapeutic and targeted agents. One of the approaches to overcome drug resistance is the utilization of combination therapies that simultaneously target several molecules involved in cancer development, progression, and metastasis. Numerous methods are utilized to guide the selection of the appropriate combination therapy. We propose a molecularly guided approach for therapy selection to overcome PARP inhibitor resistance. Methods: We used a multipronged strategy to characterize triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) models. We identified copy number alterations and pathogenic germline and somatic variants obtained from genomic sequencing. Proteomic and transcriptomic data were analyzed to evaluate gene expression affected by PARP inhibitor therapy. ATM expression status was also determined by immunohistochemistry. Additionally, a comprehensive literature review was performed focused on PARP inhibitor resistance mechanisms to support our rationale for combination therapy selections. We tested selected combination therapies using a cell viability screening assay in PARP inhibitor-resistant TNBC cell lines. Synergistic combinations were tested in vivo in PARP inhibitor-resistant TNBC PDX models. Results: Model specific pathogenic genomic alterations in PARP inhibitor-resistant TNBC PDX models included genes in the PI3K/AKT/mTOR, DNA damage response, MAPK, and apoptosis signaling pathways. Reverse phase protein array analysis demonstrated differential expression and upregulation of proteins involved in the PI3K/AKT/mTOR and MAPK signaling pathways. Differential gene expression data obtained by RNA sequencing analysis was aggregated to further enhance the individual molecular characterization of PDX models. Subsequently, the antitumor efficacy of selected rational combination therapies was tested in vitro and in vivo. Conclusions: Our study underscores the importance of a detailed analysis of key molecular biomarkers for therapy selection and proposal of rational combinations to overcome resistance to PARP inhibitors. The translational relevance of this method relies on the potential to recapitulate this approach in patient tumor samples and the opportunity to generate and expand PDX resistant models to test the antitumor efficacy of several rational combinations. Our ultimate goal is to identify the right personalized therapy for the individual cancer patient. Citation Format: Christian X. Cruz Pico, Kurt W. Evans, Ken Chen, Xiaofeng Zheng, Argun Akcakanat, Ming Zhao, Coya Tapia, Stephen M. Scott, Erkan Yuca, Funda Meric-Bernstam. Overcoming PARP inhibitor resistance with molecularly guided rational combinations in triple-negative breast cancer patient-derived xenograft models [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 676A.

Sacituzumab Govitecan (SG) is an antibody drug conjugate that targets the epithelial glycoprotein Trop-2. SG has been approved for the treatment of patients with triple negative breast cancer (TNBC) or estrogen receptor positive (ER+) breast cancer after they have received at least two previous systemic treatments. The objectives of this study were to identify biomarkers of SG response using breast cancer patient-derived xenografts (PDXs) and determine if SG efficacy changes when cells become carboplatin resistant (CR). Analysis of transcriptomic and proteomic levels of Trop2 using bulk RNAseq, scRNA-seq, and tandem mass tag spectrometry found that overall ER+ PDXs had significantly lower RNA (p-value less than 0.001) and protein (p-value less than 0.001) expression of Trop-2 than TNBC PDXs. Immunohistochemical assessment of 42 TNBC and 27 ER+ patient tumor samples identified variation in Trop-2 expression; within each cohort, expression varied from highly positive to completely negative. To test the association of Trop-2 with drug responsiveness, cells from 19 different PDXs were cultured in vitro and administered increasing doses of SG. TNBC cells were more responsive to SG treatments compared to ER+ cells (p-value equaling 0.005). Correlation analysis identified a positive relationship of drug response with protein abundance which was stronger in TNBC samples. In vivo studies with 7 TNBC PDXs found that over 70% were highly susceptible to SG treatments, with some tumors being completely eradicated or exhibiting a total inhibition of tumor growth which resulted in a long-term durable response after 10 weeks of treatment. In vivo across all the PDXs, Trop-2 expression alone did not strongly correlate with SG treatment success. Interestingly, CR sublines derived from 2 carboplatin sensitive PDXs were significantly less responsive to SG than their parental PDXs (p-value less than 0.001). These two CR sublines were also significantly less sensitive to the majority of 555 other drugs identified by the NCI compared to their parental PDXs. Ongoing studies are defining mechanisms of reduced SG efficacy in CR models. Overall, these studies find that SG is highly effective in many TNBC PDX models and should be utilized earlier in treatment protocols, especially in metastatic patients. Citation Format: Carson J. Walker, Julia E. Altman, Emily K. Zboril, Rachel K. Myrick, Nicole S. Hairr, David C. Boyd, Bin Hu, Mikhail G. Dozmorov, J. Chuck Harrell. Acquisition of carboplatin resistance corresponds with reduced sacituzumab govitecan efficacy in triple negative breast cancer patient derived xenografts [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 1986.

Claudin18.2 (CLDN18.2) is an isoform of CLDN18 with very limited expression in gastric epithelial cells in normal tissues, and aberrant overexpression in gastric, pancreatic, esophageal, ovarian, lung, and other solid tumors. This expression pattern presents a significant hurdle in developing an ADC targeting CLDN18.2 due to potential on-target toxicity to the gastrointestinal (GI) tract. LCB02A is an antibody-drug conjugate (ADC) that targets CLDN18.2, composed of a topoisomerase 1 inhibitor (TOPO1i) as payload (DAR4) linked to the CLDN18.2 specific binding PR301839 hClaudin18.2 (CLDN18.2) is an isoform of CLDN18 with very limited expression in gastric epithelial cells in normal tissues, and aberrant overexpression in gastric, pancreatic, esophageal, ovarian, lung, and other solid tumors. This expression pattern presents a significant hurdle in developing an ADC targeting CLDN18.2 due to potential on-target toxicity to the gastrointestinal (GI) tract. LCB02A is an antibody-drug conjugate (ADC) that targets CLDN18.2, composed of a topoisomerase 1 inhibitor (TOPO1i) as payload (DAR4) linked to the CLDN18.2 specific binding PR301839 humanized IgG1 antibody utilizing LegoChem Biosciences’ ConjuAllTM technology. LCB02A showed potent in vitro cytotoxicity in CLDN18.2 expressing gastric and pancreatic cancer cell lines. LCB02A also showed excellent anti-tumor efficacy in various gastric and pancreatic cell line-derived xenograft (CDX) models and patient-derived xenograft (PDX) models. Preliminary toxicity assessment in cynomolgus monkeys (CLDN18.2 cross-reactive species) demonstrated that LCB02A was well tolerated, with repeat doses of up to 60 mg/kg. In conclusion, LCB02A is a promising ADC for the treatment of CLDN18.2 expressing solid tumors including gastric cancer (GC) and pancreatic ductal adenocarcinoma (PDAC). umanized IgG1 antibody utilizing LegoChem Biosciences’ ConjuAllTM technology. LCB02A showed potent in vitro cytotoxicity in CLDN18.2 expressing gastric and pancreatic cancer cell lines. LCB02A also showed excellent anti-tumor efficacy in various gastric and pancreatic cell line-derived xenograft (CDX) models and patient-derived xenograft (PDX) models. Preliminary toxicity assessment in cynomolgus monkeys (CLDN18.2 cross-reactive species) demonstrated that LCB02A was well tolerated, with repeat doses of up to 60 mg/kg. In conclusion, LCB02A is a promising ADC for the treatment of CLDN18.2 expressing solid tumors including gastric cancer (GC) and pancreatic ductal adenocarcinoma (PDAC). Citation Format: Eun ji Baek, Chul-Woong Chung, Changsik Park, Taeik Jang, Yeojin Jeong, Sang-Ah Lee. LCB02A an antibody drug conjugate (ADC) targeting CLDN18.2 expressing solid tumors [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 3141.

<div>Abstract<p>CD205 is a type I transmembrane glycoprotein and is a member of the C-type lectin receptor family. Analysis by mass spectrometry revealed that CD205 was robustly expressed and highly prevalent in a variety of solid malignancies from different histotypes. IHC confirmed the increased expression of CD205 in pancreatic, bladder, and triple-negative breast cancer (TNBC) compared with that in the corresponding normal tissues. Using immunofluorescence microscopy, rapid internalization of the CD205 antigen was observed. These results supported the development of MEN1309/OBT076, a fully humanized CD205-targeting mAb conjugated to DM4, a potent maytansinoid derivate, via a cleavable N-succinimidyl-4-(2-pyridyldithio) butanoate linker. MEN1309/OBT076 was characterized <i>in vitro</i> for target binding affinity, mechanism of action, and cytotoxic activity against a panel of cancer cell lines. MEN1309/OBT076 displayed selective and potent cytotoxic effects against tumor cells exhibiting strong and low to moderate CD205 expression. <i>In vivo</i>, MEN1309/OBT076 showed potent antitumor activity resulting in durable responses and complete tumor regressions in many TNBC, pancreatic, and bladder cancer cell line–derived and patient-derived xenograft models, independent of antigen expression levels. Finally, the pharmacokinetics and pharmacodynamic profile of MEN1309/OBT076 was characterized in pancreatic tumor–bearing mice, demonstrating that the serum level of antibody–drug conjugate (ADC) achieved through dosing was consistent with the kinetics of its antitumor activity. Overall, our data demonstrate that MEN1309/OBT076 is a novel and selective ADC with potent activity against CD205-positive tumors. These data supported the clinical development of MEN1309/OBT076, and further evaluation of this ADC is currently ongoing in the first-in-human SHUTTLE clinical trial.</p></div>

<div>Abstract<p>CD205 is a type I transmembrane glycoprotein and is a member of the C-type lectin receptor family. Analysis by mass spectrometry revealed that CD205 was robustly expressed and highly prevalent in a variety of solid malignancies from different histotypes. IHC confirmed the increased expression of CD205 in pancreatic, bladder, and triple-negative breast cancer (TNBC) compared with that in the corresponding normal tissues. Using immunofluorescence microscopy, rapid internalization of the CD205 antigen was observed. These results supported the development of MEN1309/OBT076, a fully humanized CD205-targeting mAb conjugated to DM4, a potent maytansinoid derivate, via a cleavable N-succinimidyl-4-(2-pyridyldithio) butanoate linker. MEN1309/OBT076 was characterized <i>in vitro</i> for target binding affinity, mechanism of action, and cytotoxic activity against a panel of cancer cell lines. MEN1309/OBT076 displayed selective and potent cytotoxic effects against tumor cells exhibiting strong and low to moderate CD205 expression. <i>In vivo</i>, MEN1309/OBT076 showed potent antitumor activity resulting in durable responses and complete tumor regressions in many TNBC, pancreatic, and bladder cancer cell line–derived and patient-derived xenograft models, independent of antigen expression levels. Finally, the pharmacokinetics and pharmacodynamic profile of MEN1309/OBT076 was characterized in pancreatic tumor–bearing mice, demonstrating that the serum level of antibody–drug conjugate (ADC) achieved through dosing was consistent with the kinetics of its antitumor activity. Overall, our data demonstrate that MEN1309/OBT076 is a novel and selective ADC with potent activity against CD205-positive tumors. These data supported the clinical development of MEN1309/OBT076, and further evaluation of this ADC is currently ongoing in the first-in-human SHUTTLE clinical trial.</p></div>

Meeting abstracts Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype associated with high risk of early relapse and metastasis [[1][1]]. At the moment chemotherapy remains the main option for systemic therapy of TNBC patients but complete remission occurs only in 20% of the

Background: Triple negative breast cancer (TNBC) is negative for estrogen receptor (ER), progesterone receptor (PR), and HER2 (human epidermal growth factor receptor 2) and known to be highly aggressive. TNBC carries poor prognosis in all ethnicities but it is especially prevalent in women of African descent, next most prevalent in African American women, and then to a lesser degree in Caucasian women. Cancer cell lines from various ethnicities have been used to study the nature of these tumors, however, PDX models more accurately reflect the characteristics of the original human tumors. To study this aggressive form of breast cancer amongst ethnicities, including up-regulated and down-regulated genes that play major roles across these tumors, we created a multi-ethnic cohort of TNBC PDXs which allows us to model TNBC disease disparities among these populations. Methods: Several trips to Ghana, Africa (Komfo Anoyke Teaching Hospital (KATH)) were made to procure tumors from African women presenting with breast cancer. We created a protocol to successfully freeze and transport tumors back to the University of Michigan, with very limited research supplies available at KATH. Once back at the University of Michigan, tumor pieces were quickly thawed, minced into small fragments and implanted orthotopically into the fat pads of NOD/SCID IL2R gamma null (NSG) mice. For African American and Caucasian PDX development, we collected tumor specimens from lumpectomies performed at the University of Michigan hospital, Baylor College of Medicine, and Van Andel Research Institute and implanted freshly minced tumor pieces into the fat pads of NSG mice. Once the tumors reached at least one centimeter in size, mice were humanely euthanized and their tumors and organs were removed. Samples of each PDX and all organs were formalin-fixed and paraffin-embedded so expression of ER, PR and HER2 receptor status could be confirmed and cancer stem cell populations assessed, and organ metastasis analyzed, by immunohistochemistry. Results: To date we have successfully established (defined by three or more passages) six African, five African American, and five Caucasian TNBC PDXs. We collected TNBC tumors from fifty-six women, of which forty-two tumors were implanted into mice and twenty four PDXs were generated (this total also includes other ethnicities in addition to the three we are looking at in this particular study). Furthermore, we created an inclusive database which indicates those PDXs that grow as tumorspheres to not only study the characteristics and cancer stem cell properties of each of these tumors and compare them based on ethnicity, but also to determine which models are best for investigational drug studies. Conclusions: We developed a method of orthotopic implantation amenable to PDX generation, even for tumor samples collected abroad, which allow us to accurately model TNBC in different ethnicities. These TNBC PDXs afford a more thorough examination of cancer stem cell properties driving aggressiveness of TNBC among different ethnicities and can inform potential drug therapies to treat TNBC and eliminate metastases. This abstract was also presented as Poster A69. Citation Format: Tahra Kaur Luther, Evelyn Jiagge, Michael T. Lewis, David Monsma, Craig Webb, Suling Liu, Hasan Korkaya, Sean McDermott, Shawn G. Clouthier, Lisa Newman, Dafydd Thomas, Max S. Wicha. Creating a comprehensive patient-derived xenograft (PDX) bank to represent racial disparities in triple-negative breast cancer (TNBC). [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr PR09.

Background: Triple negative breast cancer (TNBC) is the most difficult to treat subtype of breast cancer with limited therapeutic options. At least 50% of TNBC patients have low epidermal growth factor receptor 2 (HER2; ERBB2) expression with the majority harboring hemizygous loss of POLR2A/chromosome 17p. For these patients the treatment with antibody-targeted amanitin conjugates (ATACs) targeting HER2 is a new promising approach. ATACs comprise a new class of antibody-drug conjugates (ADCs) using amanitin as toxic payload and are able to kill antigen low expressing cells. Amanitin binds to the eukaryotic RNA polymerase II and thereby efficiently inhibits the cellular transcription process. In the current study, in vitro and in vivo data of ATACs targeting human HER2low as well as tolerability studies are presented. HER2low TNBC is considered an interesting target for amanitin-based ADCs. Material and methods: Different HER2 expressing cell lines were treated with anti-HER2 ATACs. Cysteine reactive amanitin-linkers were conjugated site-specifically to engineered cysteine residues of an anti-HER2 antibody yielding ATACs with a DAR of 2.0. Quantitative determination of cell viability was analyzed by BrdU ELISA assay. Subcutaneous mouse xenograft models with HER2-positive cell lines were performed with single-dose treatments. In addition, ATAC efficacy was tested in HER2low heterogeneous TNBC patient derived xenograft (PDX) models with and without POLR2A deletion. Tolerability of ATACs was assessed in mice and non-human primates (NHP). Results: Anti-HER2 ATACs showed in vitro cytotoxicity on HER2 + high and low cell lines in low nanomolar to picomolar range. In mouse xenograft models, the anti-HER2 ATACs caused dose-dependent tumor regression independent of Her2 expression level. In HER2low heterogeneous TNBC PDX models anti-HER2 ATACs caused dose-dependent tumor regression. The efficacy of anti-Her2 ATACs was more pronounced in PDX models with hemizygous loss of TP53 and POLR2A reflecting a 17p deletion. Safety profiling of an optimized anti-Her2 ATAC in cynomolgus monkeys revealed a good tolerability indicating a good therapeutic window for 17p deleted TNBC. Conclusions: Targeted cytotoxic drug delivery to HER2 positive cell lines was achieved by using anti-HER2 ATACs. The mode of action of the payload amanitin led to an efficient anti-tumor potential in vitro and in vivo with good tolerability in NHP studies. TNBC PDX models with HER2low expression were sensitive to ATAC treatment. Loss of POLR2A/chromosome 17p increased susceptibility to anti-HER2 ATAC making 17p del TNBC a suitable indication for optimized anti Her2 ATACs. Citation Format: Christian Breunig, Aniko Palfi, Michael Kulke, Christian Lutz, Christoph Muller, Torsten Hechler, Andreas Pahl. Preclinical evaluation of anti-HER2 Antibody Targeted Amanitin Conjugates (ATACs) on HER2low breast cancer with chromosome 17p deletion [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 237.

Tumor cells depend on both glycolysis and oxidative phosphorylation (OXPHOS) for energy and biomass production to support cell proliferation. Recent data has demonstrated a dependence of various tumor types on mitochondrial OXPHOS, which represents an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign, IACS-010759 was identified as a potent, selective inhibitor of complex I of the electron transport chain, which is orally bioavailable and has excellent PK and physicochemical properties in preclinical species. Our group and others have demonstrated that AML, plus subsets of glioblastoma, neuroblastoma, lymphoma, melanoma, triple negative breast cancer (TNBC) and pancreatic cancer (PDAC) are highly dependent on OXPHOS to meet energy and biomass demands. Treatment of multiple cell lines and patient derived xenograft (PDX) models in several cancer types with IACS-010759 led to a robust decrease in cell viability and often an increase in apoptosis with EC50 values between 1 nM - 50 nM across multiple lines. Through a series of mechanistic studies we established that IACS-10759 blocks complex I of the electron transport at the quinone binding site. Mechanistically, response to IACS-010759 was associated with induction of a metabolic imbalances that negatively impacted energy homeostasis, aspartate biosynthesis, and NTP production due to reduced conversion of NADH to NAD+ by complex I, decreased ATP production, TCA cycle flux and nucleotide biosynthesis. Tumor growth inhibition and regression have been observed in molecularly defined subsets of TNBC and PDAC PDX xenograft models treated with IACS-010759, indicating that subsets of these indications are dependent on OXPHOS. Furthermore, treating TNBC or PDAC PDX models post-chemotherapy with IACS-010759 extends progression free survival, consistent with IACS-010759 targeting recently described metabolically adapted residual tumor cells. In orthotopic xenograft models of primary AML cells, daily oral treatment with 1-7.5 mg/kg IACS-010759 extended the median survival. Efficacy was paralleled by robust modulation of OCR, aspartate, and a gene signature levels. Therefore, these readouts (OCR, aspartate and a nanostring geneset) have been validated for use as exploratory clinical biology of response endpoints. In parallel, completion of preclinical chemistry, manufacturing and control (CMC) as well as GLP safety and tolerability studies with IACS-010759 in multiple species have enabled the selection of a clinical entry dose. As a result of the robust response in multiple cell lines, primary patient samples, and efficacy in PDX models, a Phase I clinical trial in relapsed, refractory AML was initiated in October 2016, with a parallel trial in solid tumors expected to initiate in early 2017. Initial results from the on-going AML trial will be disclosed. Citation Format: Jennifer Molina, Madhavi Bandi, Jennifer Bardenhagen, Christopher Bristow, Christopher Carroll, Edward Chang, Jason Cross, Naval Daver, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Jing Han, Judy Hirst, Sha Huang, Yongying Jiang, Zhijun Kang, Marina Konopleva, Gang Liu, Helen Ma, Polina Matre, Timothy McAfoos, Funda Meric-Bernstam, Pietro Morlacchi, Florian Muller, Marina Protopopova, Melinda Smith, Sonal Sonal, Yuting Sun, Jay Theroff, Andrea Viale, Quanyun Xu, Carlo Toniatti, Giulio Draetta, Philip Jones, M. Emilia Di Francesco, Joseph R. Marszalek. IACS-010759, a novel inhibitor of complex I in Phase I clinical development to target OXPHOS dependent tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4971. doi:10.1158/1538-7445.AM2017-4971

Tumor-initiating cells with reprogramming plasticity or stem-progenitor cell properties (stemness) are thought to be essential for cancer development and metastatic regeneration in many cancers; however, elucidation of the underlying molecular network and pathways remains demanding. Combining machine learning and experimental investigation, here we report CD81, a tetraspanin transmembrane protein known to be enriched in extracellular vesicles (EVs), as a newly identified driver of breast cancer stemness and metastasis. Using protein structure modeling and interface prediction-guided mutagenesis, we demonstrate that membrane CD81 interacts with CD44 through their extracellular regions in promoting tumor cell cluster formation and lung metastasis of triple negative breast cancer (TNBC) in human and mouse models. In-depth global and phosphoproteomic analyses of tumor cells deficient with CD81 or CD44 unveils endocytosis-related pathway alterations, leading to further identification of a quality-keeping role of CD44 and CD81 in EV secretion as well as in EV-associated stemness-promoting function. CD81 is coexpressed along with CD44 in human circulating tumor cells (CTCs) and enriched in clustered CTCs that promote cancer stemness and metastasis, supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights machine learning as a powerful tool in facilitating the molecular understanding of new molecular targets in regulating stemness and metastasis of TNBC.

Background: Biparatopic antibodies and antibody-drug conjugates (ADCs) can provide many functional benefits over their traditional monoparatopic counterparts including increased binding, greater cell surface decoration, superior receptor blockade, and enhanced internalization and payload delivery; however, not all antigens are amenable to biparatopic targeting. We sought to leverage our prior knowledge of biparatopic antibody development and our AzymetricTM technology to develop a novel biparatopic ADC against protein tyrosine kinase 7 (PTK7) that could provide a functional enhancement over monoparatopic ADCs. PTK7 is an attractive ADC target due to preliminary clinical activity demonstrated with cofetuzumab peledotin in non-small cell lung cancer, ovarian cancer, and metastatic breast cancer and evidence that PTK7 is overexpressed in other solid tumor indications including esophageal cancer, colorectal cancer, head and neck cancer, and cervical cancer. Materials and Methods: Twenty-nine PTK7-targeting paratopes were isolated from a mouse antibody discovery campaign and paratope pairs were combined into a panel of biparatopic antibodies using AzymetricTM Het_Fc mutations. A high-throughput screen was used to assess the functional properties of these antibodies and to identify the top paratope combinations. ADCs were produced by conjugating the top biparatopic and monoparatopic antibodies to ZD06519, a topoisomerase 1 inhibitor payload used in ZW191, an ADC targeting FRα under evaluation in a phase 1 clinical trial (NCT06555744). ADC activity was investigated in a series of in vitro and in vivo experiments using ovarian, lung, and breast cancer models. Results: Across multiple cancer cell lines with varying PTK7 expression we compared a lead monoparatopic antibody and cofetuzumab with the top biparatopic antibody which demonstrated greater cell surface decoration and better internalization. At clinically relevant doses, the biparatopic ZD06519 ADC was more efficacious than cofetuzumab pelidotin in triple negative breast cancer and lung cancer cell line derived xenograft models. Additional studies of the PTK7 biparatopic ADC in patient-derived xenograft models and a non-human primate tolerability study are planned. Citation Format: Luying Yang, Vincent Fung, Alex Wu, Dunja Urosev, Saki Konomura, Tik Nga Tong, Katina Mak, Elizabeth M. Porter, Diego A. Alonzo, Catrina M. Kim, Janice P. Tsui, Linglan Fu, Lemlem Degefie, Kaylee Wu, Matthew Bonderud, Jamie R. Rich, Stuart D. Barnscher. Design and development of biparatopic antibody-drug conjugates against protein tyrosine kinase 7 [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 1565.

Background: Women with triple-negative breast cancer (TNBC) have a more aggressive clinical course, with a higher propensity to metastasize and a worse outcome due to a lack of effective therapies and significant intratumoral and intertumoral heterogeneity. Development of novel therapeutic strategies represents a clear unmet need. We have developed a first-in-class compound, an oligobenzamide, ERX-41, that has anti-proliferative activity against all six molecular subtypes of TNBC. Methods: In vitro activity of ERX-41 on TNBC cell lines was tested using CellTiter glo, MTT, and apoptosis assays. Efficacy of ERX-41 was tested using TNBC patient derived explants (PDEs) ex vivo, cell line-derived xenografts (CDXs), and patient derived xenografts (PDX) in vivo. To examine the mechanism, we conducted mass spec analyses using total lysates of TNBC cells treated with vehicle or ERX-41. Results: ERX-41 demonstrated potent activity in both blocking proliferation and inducing apoptosis in 30 distinct cell line models of TNBC, (representing all six molecular subtypes of TNBC), with an IC50 that ranges from 50-250nM. Incubation of ERX-41 with PDEs from primary TNBC patient tumors ex vivo caused a significant reduction in proliferation indices, as measured by Ki67 staining. ERX-41 also decreased proliferation and increased apoptosis in explants from TNBC CDX and PDX tumors cultured ex vivo. Oral administration of ERX-41 (10 mg/kg/daily) was shown to be non-toxic and dramatically limited the growth of CDX tumors derived from MDA-MB-231, SUM-159 or D2A1 syngeneic tumors. Importantly, ERX-41 treatment also significantly reduced tumor progression in four TNBC PDX (PDX-1, PDX-89, PDX-96 and PDX-98) models compared to the vehicle treated control group. Our ultrastructural and molecular studies indicate that ERX-41 induces significant endoplasmic reticulum (ER) stress within TNBC cells but not in primary epithelial cells. Global mass spectrometry studies indicated that ERX-41 treatment resulted in the alteration [down regulation (265 proteins) or upregulation (218 proteins)] of 483 proteins out of ~4000 proteins quantified with two or more peptides. Reactome pathway analysis indicated that the top pathways modulated by ERX-41 included Intra-Golgi and retrograde Golgi-to-ER traffic, membrane trafficking and TP53 mediated apoptosis. ER stress induced by ERX-41 blocks de novo protein synthesis, and triggers ER-assisted degradation (ERAD) pathways, causing TNBC apoptotic cell death. Conclusions: ERX-41 is orally bioavailable, non-toxic, and demonstrated activity in primary PDEs, CDXs and PDXs. The ability of ERX-41 to induce ER stress and apoptotic cell death in multiple types of TNBC suggests that this drug targets a fundamental weakness in TNBC cells (the high basal level of ER stress) and can effectively overcome the heterogeneity of TNBC. These studies strongly support the further clinical translation of ERX-41. Citation Format: Suryavathi Viswanadhapalli, Xihui Liu, Shi-Hong Ma, Tae-Kyung Lee, Mengxing Li, Weiwei Tang, Junhao Liu, Xiaonan Li, Gangadhara R. Sareddy, Rajeshwar Rao Tekmal, Jung-Mo Ahn, Ratna K. Vadlamudi, Ganesh V. Raj. Preclinical evaluation of ERX-41 in triple negative breast cancer [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 1237.