Albina Nesterova

CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers.

CD133/prominin-1 is a pentaspan transmembrane glycoprotein overexpressed in various solid tumours including colorectal and glioblastomas. CD133 was found here to be highly expressed in ⩾50% of pancreatic, gastric and intrahepatic cholangiocarcinomas. Quantitative flow cytometric analysis showed that a panel of established hepatocellular, pancreatic and gastric cancer cell lines expressed CD133 at levels higher than normal epithelial cells or bone marrow progenitor cells. A murine anti-human CD133 antibody (AC133) conjugated to a potent cytotoxic drug, monomethyl auristatin F (MMAF), effectively inhibited the growth of Hep3B hepatocellular and KATO III gastric cancer cells in vitro with IC50 values of 2–7 ng ml−1. MMAF induced apoptosis in the cancer cells as measured by caspase activation. The anti-CD133-drug conjugate (AC133-vcMMAF) was shown to internalise and colocalised with the lysosomal marker CD107a in the sensitive cell lines. In contrast, in the resistant cell line Su.86.86, the conjugate internalised and colocalised with the caveolae marker, Cav-1. Addition of ammonium chloride, an inhibitor of lysosomal trafficking and processing, suppressed the cytotoxic effect of AC133-vcMMAF in both Hep3B and KATO III. Anti-CD133-drug conjugate treatment resulted in significant delay of Hep3B tumour growth in SCID mice. Anti-CD133 antibody-drug conjugates warrant further evaluation as a therapeutic strategy to eradicate CD133+ tumours.

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]

Targeting pancreatic and ovarian carcinomas using the auristatin-based anti-CD70 antibody–drug conjugate SGN-75

Background:CD70 is an ideal target for antibody-based therapies because of its aberrant high expression in renal carcinomas and non-Hodgkin lymphomas and its highly restricted expression in normal tissues. The expression profiling of CD70 in carcinomas has been limited because of the lack of a CD70-specific reagent that works in formalin-fixed paraffin-embedded (FFPE) tissues.Methods:We generated murine monoclonal antibodies (mAbs) specific for CD70 and validated their specificity by western blot analysis and developed a protocol for immunohistochemistry on FFPE tissues. CD70+ tumour cell lines were used for testing the anti-tumour activity of the anti-CD70 antibody–drug conjugate, SGN-75.Results:We report novel detection of CD70 expression in multiple cancers including pancreatic (25%), larynx/pharynx (22%), melanoma (16%), ovarian (15%), lung (10%), and colon (9%). Our results show that pancreatic and ovarian tumour cell lines, which express high levels of endogenous or transfected CD70, are sensitive to the anti-tumour activity of SGN-75 in vitro and in vivo.Conclusion:Development of murine mAbs for robust and extensive screening of FFPE samples coupled with the detection of anti-tumour activity in novel indications provide rationale for expanding the application of SGN-75 for the treatment of multiple CD70 expressing cancers.

Anti-leukemic activity of lintuzumab (SGN-33) in preclinical models of acute myeloid leukemia.

Despite therapeutic advances, the long-term survival rates for acute myeloid leukemia (AML) are estimated to be 10% or less, pointing to the need for better treatment options. AML cells express the myeloid marker CD33, making it amenable to CD33-targeted therapy. Thus, the in vitro and in vivo anti-tumor activities of lintuzumab (SGN-33), a humanized monoclonal anti-CD33 antibody undergoing clinical evaluation, were investigated. In vitro assays were used to assess the ability of lintuzumab to mediate effector functions and to decrease the production of growth factors from AML cells. SCID mice models of disseminated AML with the multi-drug resistance (MDR)-negative HL60 and the MDR+, HEL9217 and TF1-α, cell lines were developed and applied to examine the in vivo antitumor activity. In vitro, lintuzumab significantly reduced the production of TNF-α-induced pro-inflammatory cytokines and chemokines by AML cells. Lintuzumab promoted tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) activities against MDR- and MDR+ AML cell lines and primary AML patient samples. At doses from 3 to 30 mg/kg, lintuzumab significantly enhanced survival and reduced tumor burden in vivo, regardless of MDR status. Survival of the mice was dependent upon the activity of resident macrophages and neutrophils. The results suggest that lintuzumab may exert its therapeutic effects by modulating the cytokine milieu in the tumor microenvironment and through effector mediated cell killing. Given that lintuzumab induced meaningful responses in a phase 1 clinical trial, the preclinical antitumor activities defined in this study may underlie its observed therapeutic efficacy in AML patients.

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.

Abstract 1195: SGN-CD352A: A novel humanized anti-CD352 antibody-drug conjugate for the treatment of multiple myeloma

Multiple myeloma (MM) is a hematologic malignancy of transformed plasma cells. In spite of recent advances, MM remains an incurable disease, underscoring the need to develop new targeted biological therapeutics to augment existing treatments. In this study we describe SGN-CD352A, a potent new CD352-targeting antibody-drug conjugate (ADC) under development for the treatment of MM. CD352, or SLAMF6 (Signaling Lymphocyte Activation Molecule family member 6), is a type 1 membrane protein in the SLAM family of immunoreceptors. Like other SLAM family members, CD352 is a positive regulator of natural killer (NK) cell functions. CD352 is also a tumor antigen expressed on B cell malignancies such as MM, chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). We observed CD352 expression on the surface of malignant plasma cells in 87% (13/15) of human multiple myeloma patient samples examined by flow cytometry. Monoclonal antibodies (mAbs) specific for human CD352 were produced and a lead antibody was selected based on affinity, endocytic internalization rate, and tumor cell cytotoxic activity as an ADC. SGN-CD352A is a humanized anti-CD352 engineered cysteine (ec) mAb (h20F3ec) to which two molecules of pyrrolobenzodiazepine (PBD) dimer, a potent DNA damaging cytotoxic drug, have been conjugated. Upon binding CD352 at the MM cell surface, SGN-CD352A undergoes rapid clathrin-dependent endocytosis ( Citation Format: Tim Lewis, Devra J. Olson, Kristine A. Gordon, Sharsti L. Sandall, Jamie Miyamoto, Lori Westendorf, Germein Linares, Chris Leiske, Heather Kostner, Ivan Stone, Martha Anderson, Albina Nesterova, Mechthild Jonas, Che-Leung Law. SGN-CD352A: A novel humanized anti-CD352 antibody-drug conjugate for the treatment of multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1195.

Abstract 2647: SGN-CD70A, a novel and highly potent anti-CD70 ADC, induces double-strand DNA breaks and is active in models of MDR+ renal cell carcinoma (RCC) and non-Hodgkin lymphoma (NHL)

CD70 is a member of the tumor necrosis factor superfamily that is aberrantly expressed in several solid tumors and hematologic malignancies, including clear cell and papillary renal cell carcinoma (RCC) and non-Hodgkin lymphoma (NHL). Normal expression of CD70 is limited to stromal cells of the thymic medulla, mature dendritic cells, and activated B and T lymphocytes. Thus, CD70 is an attractive target for antibody-drug conjugate (ADC) based therapy. Using a panel of CD70 positive RCC and lymphoma cell lines and xenograft models, we have previously demonstrated the antitumor activity of SGN-CD70A, a novel ADC that combines a CD70-directed engineered cysteine monoclonal antibody (h1F6ec) with a highly potent, synthetic DNA cross-linking molecule, pyrrolobenzodiazepine (PBD) dimer. The strength of these results led us to develop SGN-CD70A for clinical evaluation in RCC and lymphoma. In this report, we examine the mechanism of action for SGN-CD70A and demonstrate that the formation of double strand breaks (DSB) is an early event that precedes onset of cytotoxicity in RCC and NHL cell lines. SGN-CD70A is more potent than auristatin-based CD70 ADCs in vitro and in xenograft models, including those that are MDR positive, suggesting that the PBD chemotype may overcome common resistance mechanisms. To define the mechanism(s) of targeted cytotoxicity, we examined DNA damage pathways in Caki-1, 786-0 and UM-RC-3 (RCC, MDR+) and Raji and MHH-PREB-1 (NHL) cell lines. We utilized an immunofluorescence assay to monitor DNA damage foci using antibodies specific to the tumor suppressor p53-binding protein 1 (53BP1), Meiotic recombination 11 homolog (Mre11), and Rad50. Increased amounts of foci were observed within 6 hours of treatment with 2nM PBD to levels observed in cells exposed to 10Gy of ionizing radiation. Similarly, foci were found in SGN-CD70A-treated cells. Further evidence of damage was the co-localization of phosphorylated histone H2A.X (Ser139) to the damage foci and an increase in levels of both phosphorylated Chk1 (Ser317/345) and Chk2 (Thr68) within 4 hours of treatment. The levels of both pChk1 and pChk2 continue to increase after treatment, with peaks at 24-48 hours for pChk1 and 48-72 hours for pChk2. Concomitant, we also observed an increase in both phosphorylated ATM and phosphorylated BRCA1, confirming that SGN-CD70A in vitro activates double strand break response pathways. Ongoing research is examining DNA damage pathway activation in the corresponding xenograft models to confirm our in vitro findings. Furthermore, we are developing assays to examine pH2A.X, pChk1, and pChk2 as potential biomarkers for clinical studies with SGN-CD70A. Citation Format: Sharsti Sandall, Martha Anderson, Mechthild Jonas, Albina Nesterova, Jamie Miyamoto, Ivan J. Stone, Weiping Zeng, Che-Leung Law, Timothy S. Lewis. SGN-CD70A, a novel and highly potent anti-CD70 ADC, induces double-strand DNA breaks and is active in models of MDR+ renal cell carcinoma (RCC) and non-Hodgkin lymphoma (NHL). [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 2647. doi:10.1158/1538-7445.AM2014-2647

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

Abstract 3620: LIV-1 antibody-drug conjugate: A novel therapeutic agent for breast and prostate cancer

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. 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 by estrogen receptor-positive (ER+), hormone-treated tumors (both primary and metastatic sites) and ER-/PR-/Her2- (triple-negative) breast cancers. Hormone refractory metastatic prostate tumor samples express Liv-1, with expression confirmed in both bone and soft tissue metastatic sites by IHC. In healthy human tissues, LIV-1 expression is limited to hormonally-regulated organs (prostate, uterus, and breast). The broad expression of LIV-1 in prostate and breast cancer tumors in combination with the limited expression in vital organs makes LIV-1 an excellent target for an antibody-drug conjugate (ADC). We generated an ADC consisting of a humanized anti-LIV-1 mAb conjugated to the antitubulin agent monomethyl auristatin E (MMAE), via a plasma stable, enzyme-cleavable linker (vc). The humanized LIV-1 mAb bound with high affinity to both human and cynomolgous LIV-1. In vitro, anti-LIV-1-vcMMAE ADCs showed specific cytotoxic activity against a breast cancer cell line. In vivo studies also demonstrated antitumor activity of anti-LIV-1-vcMMAE ADCs in preclinical xenograft models with significant delay of tumor growth compared to control groups. These findings demonstrate that further evaluation and development of anti-LIV-1-vcMMAE is warranted as a promising and potential therapeutic agent for the treatment of prostate and ER+ and triple-negative breast cancers. 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 3620. doi:10.1158/1538-7445.AM2011-3620

Abstract P6-15-16: LIV-1 Antibody-Drug Conjugate: A Novel Therapeutic Agent for Breast Cancer

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. 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 86% (n=22) of estrogen receptor-positive (ER+), hormone-treated tumors (both primary and metastatic sites), and in 65% (n=20) of ER-/PR-/Her2- (triple-negative) breast cancers. In healthy human tissues, LIV-1 expression is limited to hormonally-regulated organs (prostate, uterus, and breast). We generated an antibody-drug conjugate (ADC) consisting of a humanized anti-LIV-1 mAb conjugated to the antitubulin agent monomethyl auristatin E (MMAE), via a plasma stable, enzyme-cleavable linker (vc). The humanized LIV-1 mAb bound with high affinity to both human and cynomolgous LIV-1 (2-4 nM). In vitro, anti-LIV-1-vcMMAE showed specific cytotoxic activity against a MCF-7 cell line with an IC 50 = 78 ng/ml. In vivo studies also demonstrated antitumor activity of anti-LIV-1-vcMMAE in preclinical xenograft models, including MCF-7 breast cancer cell line, with significant delay of tumor growth compared to control groups. The broad expression of LIV-1 in both hormonally-treated and triple-negative tumors and the limited expression in vital organs make LIV-1 an excellent target for an ADC. These findings support the further development of anti-LIV-1-vcMMAE as a promising therapeutic agent for the treatment of ER+ and triple-negative breast cancers. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-16.