Leia M. Smith

cJun overexpression in MCF-7 breast cancer cells produces a tumorigenic, invasive and hormone resistant phenotype

We have previously demonstrated decreased Jun/AP-1 activity in the breast cancer cell line MCF-7 when compared to normal or immortalized mammary epithelial cells. In this paper, we overexpress Jun in MCF-7 cells (MCF7Jun) and demonstrate that it results in diverse biologic and biochemical changes, which mimic those seen clinically in breast cancer. Overexpression of Jun causes significant alterations in the composition of AP-1, decreased junB and increased fra-1 expression and results in an increased biologic aggressiveness. MCF7Jun cells exhibit increased cellular motility, increased expression of a matrix degrading enzyme MMP-9, increased in vitro chemoinvasion and tumor formation in nude mice in the absence of exogenous estrogens. Furthermore, MCF7Jun cells are unresponsive to the growth stimulating effects of estrogen and growth inhibitory effects of tamoxifen. Analysis of the estrogen receptor (ER) expression and activity showed that the MCF7Jun cells have no detectable ER. MCF-7 cells overexpressing mutant forms of cJun were responsive to the growth stimulatory effects of estrogen indicating that full-length cJun is required to acquire the estrogen-independent phenotype in breast cancer cells.

Lymphocyte Activation Antigen CD70 Expressed by Renal Cell Carcinoma Is a Potential Therapeutic Target for Anti-CD70 Antibody-Drug Conjugates

Metastatic renal cell carcinoma (RCC) is an aggressive disease refractory to most existing therapeutic modalities. Identifying new markers for disease progression and drug targets for RCC will benefit this unmet medical need. We report a subset of clear cell and papillary cell RCC aberrantly expressing the lymphocyte activation marker CD70, a member of the tumor necrosis factor superfamily. Importantly, CD70 expression was found to be maintained at the metastatic sites of RCC. Anti-CD70 antibody-drug conjugates (ADC) consisting of auristatin phenylalanine phenylenediamine (AFP) or monomethyl auristatin phenylalanine (MMAF), two novel derivatives of the anti-tubulin agent auristatin, mediated potent antigen-dependent cytotoxicity in CD70-expressing RCC cells. Cytotoxic activity of these anti-CD70 ADCs was associated with their internalization and subcellular trafficking through the endosomal-lysosomal pathway, disruption of cellular microtubule network, and G2-M phase cell cycle arrest. The efficiency of drug delivery using anti-CD70 as vehicle was illustrated by the much enhanced cytotoxicity of antibody-conjugated MMAF compared with free MMAF. Hence, ADCs targeted to CD70 can selectively recognize RCC, internalize, and reach the appropriate subcellular compartment(s) for drug release and tumor cell killing. In vitro cytotoxicity of these ADCs was confirmed in xenograft models using RCC cell lines. Our findings provide evidence that CD70 is an attractive target for antibody-based therapeutics against metastatic RCC and suggest that anti-CD70 ADCs can provide a new treatment approach for advanced RCC patients who currently have no chemotherapeutic options.

Differential expression of the early lung cancer detection marker, heterogeneous nuclear ribonucleoprotein-A2/B1 (hnRNP-A2/B1) in normal breast and neoplastic breast cancer.

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP-A2/B1) is highly expressed during critical stages of lung development and carcinogenesis. To determine if the expression of hnRNP-A2/B1 is an informative biomarker in breast carcinogenesis, we analyzed hnRNP-A2/B1 overexpression by immunohistochemistry in archived specimens. Expression was detected in 48/85 (56.5%) primary invasive breast cancers and 7/72 (9.7%) specimens of normal breast tissue. Northern analysis of breast cancer cells also demonstrated higher level of hnRNP-A2/B1 expression compared to normal or transformed breast cells. Expression of hnRNP-A2/B1 in breast cancer cells was decreased by exposure to retinoids coordinately with decreased cell growth. These results warrant further evaluation of hnRNP-A2/B1 as a marker of breast carcinogenesis.

Potent cytotoxicity of an auristatin-containing antibody-drug conjugate targeting melanoma cells expressing melanotransferrin/p97

Identifying factors that determine the sensitivity or resistance of cancer cells to cytotoxicity by antibody-drug conjugates is essential in the development of such conjugates for therapy. Here the monoclonal antibody L49 is used to target melanotransferrin, a glycosylphosphatidylinositol-anchored glycoprotein first identified as p97, a cell-surface marker in melanomas. L49 was conjugated via a proteolytically cleavable valine-citrulline linker to the antimitotic drug, monomethylauristatin F (vcMMAF). Effective drug release from L49-vcMMAF likely requires cellular proteases most commonly located in endosomes and lysosomes. Melanoma cell lines with the highest surface p97 expression (80,000–280,000 sites per cell) were sensitive to L49-vcMMAF whereas most other cancer cell lines with lower p97 expression were resistant, as were normal cells with low copy numbers (≤20,000 sites per cell). Cell line sensitivity to L49-vcMMAF was found by immunofluorescence microscopy to correlate with intracellular fate of the conjugate. Specifically, L49-vcMMAF colocalized with the lysosomal marker CD107a within sensitive cell lines such as SK-MEL-5 and A2058. In contrast, in resistant cells expressing lower p97 levels (H3677; 72,000 sites per cell), L49-vcMMAF colocalized with caveolin-1, a protein prominent in caveolae, but not with CD107a. Thus, for antibody-drug conjugates targeting p97, antigen level and trafficking to the lysosomes are important factors for achieving robust in vitro cytotoxicity against cancer cells. Immunohistochemical analysis with L49 revealed that 62% of metastatic melanoma tumors had strong staining for p97. Overexpression of p97 in melanoma as compared with normal tissue, in conjunction with the greater sensitivity of tumor cells to L49-vcMMAF, supports further evaluation of antibody-drug conjugates for targeting p97-overexpressing tumors. [Mol Cancer Ther 2006;5(6):1474–82]

Lipid rafts remodeling in estrogen receptor–negative breast cancer is reversed by histone deacetylase inhibitor

Recently, we have found dramatic overexpression of ecto-5′-nucleotidase (or CD73), a glycosylphosphatidylinositol-anchored component of lipid rafts, in estrogen receptor–negative [ER(−)] breast cancer cell lines and in clinical samples. To find out whether there is a more general shift in expression profile of membrane proteins, we undertook an investigation on the expression of selected membrane and cytoskeletal proteins in aggressive and metastatic breast cancer cells. Our analysis revealed a remarkably uniform shift in expression of a broad range of membrane, cytoskeletal, and signaling proteins in ER(−) cells. A similar change was found in two in vitro models of transition to ER(−) breast cancer: drug-resistant Adr2 and c-Jun-transformed clones of MCF-7 cells. Interestingly, similar expression pattern was observed in normal fibroblasts, suggesting the commonality of membrane determinants of invasive cancer cells with normal mesenchymal phenotype. Because a number of investigated proteins are components of lipid rafts, our results suggest that there is a major remodeling of lipid rafts and underlying cytoskeleton in ER(−) breast cancer. To test whether this broadly defined ER(−) phenotype could be reversed by treatment with differentiating agent, we treated ER(−) cells with trichostatin A, an inhibitor of histone deacetylase, and observed reversal of mesenchymal and reappearance of epithelial markers. Changes in gene and protein expression also included increased capacity to generate adenosine and altered expression profile of adenosine receptors. Thus, our results suggest that during transition to invasive breast cancer there is a significant structural reorganization of lipid rafts and underlying cytoskeleton that is reversed upon histone deacetylase inhibition. [Mol Cancer Ther 2006;5(2):238–45]

SGN–LIV1A: A Novel Antibody–Drug Conjugate Targeting LIV-1 for the Treatment of Metastatic Breast Cancer

In this article, we describe a novel antibody–drug conjugate (ADC; SGN–LIV1A), targeting the zinc transporter LIV-1 (SLC39A6) for the treatment of metastatic breast cancer. LIV-1 was previously known to be expressed by estrogen receptor–positive breast cancers. In this study, we show that LIV-1 expression is maintained after hormonal therapy in primary and metastatic sites and is also upregulated in triple-negative breast cancers. In addition to breast cancer, other indications showing LIV-1 expression include melanoma, prostate, ovarian, and uterine cancer. SGN–LIV1A consists of a humanized antibody conjugated through a proteolytically cleavable linker to monomethyl auristatin E, a potent microtubule-disrupting agent. When bound to surface-expressed LIV-1 on immortalized cell lines, this ADC is internalized and traffics to the lysozome. SGN–LIV1A displays specific in vitro cytotoxic activity against LIV-1–expressing cancer cells. In vitro results are recapitulated in vivo where antitumor activity is demonstrated in tumor models of breast and cervical cancer lineages. These results support the clinical evaluation of SGN–LIV1A as a novel therapeutic agent for patients with LIV-1–expressing cancer. Mol Cancer Ther; 13(12); 2991–3000. ©2014 AACR.

Nuclear P27 expression in benign, borderline (LMP) and invasive tumors of the ovary and its association with prognosis: A gynecologic oncology group study

OBJECTIVE Nuclear p27 expression was examined in non-invasive and invasive ovarian tumors from a cross-sectional study, and clinical relevance of p27 was evaluated in the primary tumors from women participating in two randomized phase III treatment trials. METHODS An immunohistochemistry assay was used to detect p27 in formalin-fixed paraffin-embedded ovarian tumors from 3 distinct sources. RESULTS Among the initial 91 ovarian tumors tested, low p27 expression (<50% positive cells) was observed in 5.4% of non-invasive tumors versus 42.6% of invasive tumors (p<0.001). In 145 ovarian cancers with high-risk early stage disease, 16.5% exhibited low p27 expression, and categorized p27 was not associated with age, race, or performance status. Low expression of p27 was common in poorly differentiated tumors (35.7%) compared to moderately (15.0%) and well (9.5%) differentiated tumors (p=0.024) and rare in clear cell carcinomas (2.4%) compared to other histologies (p=0.014). In the 139 cancers with advanced disease, 60% displayed low p27 expression, and categorized p27 expression was not associated with age, race, performance status, tumor grade, histologic subtype, measurable disease status or survival. Exploratory analyses revealed an association of cyclin E to p27 ratio >1.0 with an increased risk of death (hazard ratio=1.53; p=0.017). CONCLUSIONS Low p27 expression could be associated with malignant transformation of the ovarian epithelium and FIGO stage. A cyclin E to p27 ratio >1.0 may be associated with shorter survival in these patients. Further study is required to confirm the trend for increased recurrences with low p27 expression in early stage disease.

Abstract 625: Preclinical characterization of an auristatin-based anti-CD19 drug conjugate, SGN-19A

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL CD19 has multiple attributes that make it an attractive target for an antibody-drug conjugate (ADC). CD19 is uniformly expressed on the cell surface of almost all malignancies of B cell origin, including non-Hodgkin lymphoma (NHL), B cell precursor acute lymphoblastic leukemia (B-ALL), and chronic lymphocytic leukemia (CLL). In addition, CD19 has very limited normal tissue expression, restricted only to B lymphocytes and their precursors, and it internalizes rapidly, which are favorable characteristic for targeted drug delivery. SGN-19A is an ADC comprised of a humanized anti-CD19 mAb and the potent cytotoxic drug-linker maleimidocaproyl monomethyl auristatin F (mcMMAF). Here we demonstrate internalization and trafficking, in vivo antitumor activity, and in vivo on-target pharmacodynamic effect of B cell depletion following SGN-19A treatment. Internalization and trafficking of SGN-19A through the endosomal-lysosomal pathway resulted in potent in vitro cytotoxic activity against CD19+ cell lines derived from B-ALL, follicular lymphoma, diffused large B cell lymphoma, Burkitts lymphoma, and plasma cell leukemia. In mouse xenografts modeling different B-lineage malignancies, SGN-19A demonstrated antitumor activity including tumor regression and prolonged survival at doses well below its maximum tolerated dose. We also show that the in vivo antitumor activity is directly related to the duration of the pharmacokinetic half-life among a panel of ADCs targeting CD19. Since SGN-19A cross-reacts with its ortholog expressed in non-human primates, we examined its pharmacodynamic effect on B cell depletion in cynomolgus monkeys. Depletion of peripheral B cells was observed following SGN-19A and correlated with results from terminal immunohistochemical (IHC) analysis demonstrating the depletion of Ki67+ germinal center B cells. Furthermore, using an antibody against mcMMAF we confirmed delivery of the cytotoxic agent to the B cell areas in peripheral lymphoid tissues. Taken together, these results support the further evaluation of SGN-19A as a therapy for B-lineage derived malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 625. doi:10.1158/1538-7445.AM2011-625

Abstract 4393: ASG-5ME is a novel antibody drug conjugate (ADC) for treating prostate cancers

AGS-5 is a novel transmembrane antigen discovered by Agensys using transcriptional profiling that is highly expressed in a variety of epithelial tumors. A fully human IgG2k monoclonal antibody that binds to AGS-5 with high affinity was conjugated with the anti-microtubulin drug, monomethyl auristatin E (MMAE), via a conditionally labile valine-citrulline (vc) maleimidocaproyl linker to yield an average drug: antibody ratio of 3.7. Following cell surface binding, ASG-5ME is internalized and trafficked through the endocytic pathway, where proteolytic cleavage releases free active drug that subsequently binds and disrupts microtubules. We evaluated the expression of AGS-5 protein in a large number of prostate patient tumors using IHC. ASG-5ME was evaluated for its cell cytoxicity in vitro, and its anti-tumor activity was investigated on different established xenograft tumors, using both patient-derived and cell line models of prostate cancer. The pharmacokinetics of ASG-5ME in mice was also evaluated. Our studies indicated AGS-5 protein was expressed in more than 95% of primary prostate cancer specimens and distant metastases, and on more than 90% and 80% of pancreatic and gastric cancer specimens, respectively. ASG-5ME, bound with high affinity (Kd = 0.5 nM) to both human and cynomolgus monkey AGS-5 and mediated potent dose dependent cell cytotoxicity in vitro. ASG-5ME showed significant long term tumor regression and eradication of multiple established prostate cancer xenografts, including those grown orthotopically. Phamacokinetic (PK) analysis of ASG-5ME in mice indicated that the ADC was stabile with long T1/2 of ∼12 days. When considered together these data indicate that ASG-5ME is a promising therapeutic ADC for the treatment of prostate and other AGS-5 expressing cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4393.

Abstract 3962: SGN-LIV1A: a development stage antibody drug-conjugate targeting LIV-1 for the treatment of metastatic breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC LIV-1, also known as SLC39A6 or ZIP6, is a member of the zinc transporter family and was first identified as an estrogen-inducible gene in breast cancer derived cell lines. LIV-1, as a downstream target of STAT3, promotes the epithelial to mesenchymal transition that is important in the malignant progression to metastasis. Consistent with its role in cancer, we determined by immunohistochemical (IHC) analysis that LIV-1 is expressed in subtypes of metastatic breast cancers (ER+/HER2-, HER2+ and triple negative). In healthy human tissues, LIV-1 expression is limited to four hormonally-regulated organs. The broad expression of LIV-1 in metastatic breast cancer in combination with the limited expression in vital organs makes LIV-1 an excellent target for an antibody-drug conjugate (ADC). SGN-LIV1A is an ADC consisting of a humanized anti-LIV-1 mAb conjugated to the microtubule-disrupting agent, monomethyl auristatin E, via a protease-cleavable linker. In vitro, SGN-LIV1A shows target specific internalization and cytotoxic activity against a breast cancer cell line. In vivo studies also demonstrate antitumor activity of SGN-LIV1A in preclinical xenograft models with significant delay of tumor growth compared to control groups. These findings support further evaluation and development of SGN-LIVA as a therapeutic for the treatment of metastatic breast cancer. Citation Format: Django Sussman, Leia M. Smith, Martha E. Anderson, Steve Duniho, Joshua H. Hunter, Heather Kostner, Jamie B. Miyamoto, Albina Nesterova, Lori Westendorf, Heather A. Van Epps, Nancy Whiting, Dennis R. Benjamin. SGN-LIV1A: a development stage antibody drug-conjugate targeting LIV-1 for the treatment of metastatic breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3962. doi:10.1158/1538-7445.AM2013-3962