Enfortumab vedotin is a Nectin-4-directed antibody-drug conjugate designed to deliver the microtubule-disrupting agent monomethyl auristatin E (MMAE) to tumor cells. Using preclinical models of urothelial cancer (UC), we expand the understanding of the multifaceted mechanism of action for enfortumab vedotin that includes direct cytotoxicity on Nectin-4-positive tumor cells, indirect bystander effect on neighboring Nectin-4-negative tumor cells, and MMAE-mediated induction of immunogenic cell death (ICD) and associated increase in activated immune cells in the tumor microenvironment. Importantly, vaccination with enfortumab vedotin-treated tumor cells results in protection against tumor rechallenge in mice, consistent with antitumor immunity. MMAE-mediated ICD induction modulates the tumor microenvironment in a complementary manner to immune checkpoint inhibition. Accordingly, enfortumab vedotin plus PD-1 inhibitor shows enhanced antitumor activity in vivo. These preclinical findings provide mechanistic hypotheses that may be relevant to the improved clinical outcomes observed for enfortumab vedotin plus pembrolizumab relative to chemotherapy.

Antibody humanization, binding assays, conjugation, mass spec, additional in vivo details

Supplementary Table 1: Surface antigen density impacts in vitro potency of anti-LIV-1 ADC . MCF-7 ATCC cells from three sources with varying levels of LIV-1 expression were tested with SGN-LIV1A for cytotoxicity. Lower antigen density resulted in decreased potency.

Background Enfortumab vedotin (EV) is a first-in-class Nectin-4-directed antibody-drug conjugate (ADC) with demonstrated improved overall survival in patients with previously treated advanced-stage urothelial carcinoma.1 EV is comprised of a fully human Nectin-4-directed monoclonal antibody conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) by a protease cleavable maleimidocaproyl-valine-citrulline linker. EV has a multifaceted mechanism of action. Previously, we demonstrated that EV induces antitumor activity in vitro via direct cytotoxicity on Nectin-4-expressing malignant cells and indirect bystander activity on neighboring Nectin-4­ negative cells, both of which are mediated by MMAE release within target cells. Here, we expand upon the mechanism of action and show EV induces tumor cell killing in a manner leading to immunogenic cell death (ICD) and improves antitumor responses when combined with checkpoint inhibitors. Methods The ability of EV to induce hallmarks of ICD was evaluated in vitro in Nectin-4-expressing human urothelial carcinoma cell lines. Immune activation associated with ICD was assessed in vitro in monocytes co-cultured with EV-treated tumor cells and in vivo by immunohistochemistry, RNA-seq, flow cytometry, and immune cytokine profiling. The effects of EV plus anti-PD-1 on tumor growth inhibition, the tumor microenvironment, and immune memory were evaluated in syngeneic mouse models engineered to express human Nectin-4. Antitumor immune memory was also assessed in mice vaccinated with EV-treated cells. Results In vitro, EV induced ICD via MMAE-mediated microtubule disruption and concomitant endoplasmic reticulum (ER) stress, as evidenced by increased phosphorylation of JNK, extracellular release of inflammatory mediators ATP and HMGB1, and cell surface exposure of calreticulin. Xenograft tumors treated with EV demonstrated upregulation of MHC genes as well as genes involved in ER stress, autophagy, and type I interferon response. Additionally, there were noted increases in both macrophages and dendritic cells along with cytokines involved in chemoattraction and T-cell stimulation. Consistent with ICD induction, vaccination with EV-treated Nectin-4-expressing tumor cells promoted antitumor immunity and provided protection against tumor rechallenge. Lastly, the combination of EV with PD-1 inhibition improved antitumor activity and durable immunity in vivo, consistent with complementary modes of action of these two anticancer agents. Conclusions These data provide insight into the clinical activity observed with EV and bolster the scientific rationale to combine EV with checkpoint inhibitors, which is currently an area of active clinical investigation across multiple studies.2-6 References Powles T, Rosenberg JE, Sonpavde GP, Loriot Y, Duran I, Lee JL, et al. Enfortumab Vedotin in Previously Treated Advanced Urothelial Carcinoma. N Engl J Med. 2021;384(12):1125–35. Epub 2021/02/13. doi:10.1056/NEJMoa2035807. PubMed PMID: 33577729; PubMed Central PMCID: PMCPMC8450892. Friedlander TW, Milowsky MI, Bilen MA, Srinivas S, McKay RR, Flaig TW, et al. Study EV-103: Update on durability results and long term outcome of enfortumab vedotin + pembrolizumab in first line locally advanced or metastatic urothelial carcinoma (la/mUC). Journal of Clinical Oncology 2021;39(15_suppl):4528. doi: 10.1200/JCO.2021.39.15_suppl.4528. Galsky MD, Necchi A, Shore ND, Plimack ER, Jia C, Sbar E, et al. KEYNOTE-905/EV-303: Perioperative pembrolizumab or pembrolizumab plus enfortumab vedotin (EV) and cystectomy compared to cystectomy alone in cisplatin-ineligible patients with muscle-invasive bladder cancer (MIBC). J Clin Oncol. 2021;39(6_suppl):TPS507. doi: 10.1200/JCO.2021.39.6_suppl.TPS507. Heijden MSVD, Gupta S, Galsky MD, Derleth CL, Lee S, Kataria RS, et al. Study EV-302: A two-arm, open-label, randomized controlled phase 3 study of enfortumab vedotin in combination with pembrolizumab versus chemotherapy in previously untreated advanced urothelial carcinoma (aUC) (trial in progress). J Clin Oncol 2022;40(6_suppl):TPS589. doi: 10.1200/JCO.2022.40.6_suppl.TPS589. Hoimes CJ, Bedke J, Loriot Y, Nishiyama H, Fang X, Kataria RS, et al. KEYNOTE-B15/EV-304: Randomized phase 3 study of perioperative enfortumab vedotin plus pembrolizumab versus chemotherapy in cisplatin-eligible patients with muscle-invasive bladder cancer (MIBC). J Clin Oncol 2021;39(15_suppl):TPS4587. doi: 10.1200/JCO.2021.39.15_suppl.TPS4587. ClinicalTrials.gov [Internet] Bethesda (MD): U.S. National Library of Medicine. 2000 – . ClinicalTrials.gov Identifier: NCT04960709. Treatment Combination of Durvalumab, Tremelimumab and Enfortumab Vedotin or Durvalumab and Enfortumab Vedotin in Patients With Muscle Invasive Bladder Cancer Ineligible to Cisplatin or Who Refuse Cisplatin (VOLGA). 2021 Jul 14 [cited 2022 Jul 22]. Available from: https://clinicaltrials.gov/ct2/show/NCT04960709. Ethics Approval All animal studies were conducted in accordance with protocols reviewed and approved by the Institutional Animal Care and Use Committee at Seagen, Astellas, or the external testing facilities that conducted the studies.

Abstract The antibody-drug conjugate enfortumab vedotin (EV; AGS22C3E) targets Nectin-4 expressing tumor cells by delivering MMAE, a potent microtubule disrupting agent, to induce cell death. EV has demonstrated single agent activity and encouraging activity (71% ORR) when combined with pembrolizumab (anti-PD-1) in the 1L setting of cis-ineligible metastatic urothelial carcinoma (mUC) (EV-103, NCT03288545). Here we demonstrated that EV may promote multiple mechanisms of action including bystander cell killing and hallmarks of immunogenic cell death (ICD) including ER stress, immune cell recruitment and activation. Two urothelial carcinoma models, T-24 and UM-UC-3, were engineered to express Nectin-4, and both were sensitive to EV in vitro and in vivo. In these Nectin-4 expressing cell lines, EV internalized with Nectin-4, trafficked to lysosomal vesicles, and released intracellular MMAE as shown by intracellular MMAE accumulation. In addition, EV demonstrated a bystander effect by release of the cell permeable MMAE from Nectin-4 positive cells to kill Nectin-4 negative cancer cells in an admixed cellular assay. Furthermore, EV treated cells exhibited early markers of ICD such as induction of the ER stress response through phosphorylation of JNK in Nectin-4 expressing cells. The ER protein calreticulin and protein chaperones like HSP70 are translocated from the intracellular compartments and exposed on the cell surface to signal for immune cell recruitment upon EV treatment. Additional hallmarks of ICD were observed with EV included extracellular release of ATP and HMGB1 in both the T-24 and UM-UC-3 Nectin-4 expressing cell lines. Immune cell recruitment and activation in the T-24 Nectin-4 xenograft model that demonstrated potent anti-tumor activity were measured in vivo using IHC, RNA-seq, flow cytometry, and immune cytokine paneling. Immune profiling assessment showed an enhancement of the immune cell markers in 6 of 7 tumors compared to untreated animals or non-binding ADC control treated animals. Analysis of gene signatures using RNA-seq support our qualitative IHC analysis of immune cell recruitment. Additional gene signature analyses identified altered gene transcripts associated with microtubule disruption, mitotic arrest, and ER stress. In addition, Luminex assessment of mouse cytokines showed coincident changes in RNA expression of genes associated with macrophage activation such MIP1α and MIPβ, suggesting EV treatment can promote APC activation. This mechanistic data and ongoing research by which EV induces cell death to promote immune cell recruitment and activation provides a potential mechanism underpinning the clinical benefit observed with EV as a monotherapy or in combination with pembrolizumab in mUC. Citation Format: Bernard A. Liu, Devra Olson, Katie Snead, John Gosink, Elena-Marie Tenn, Margo Zaval, Anthony Cao, Disha Sahetya, Albina Nesterova, Kelly Hensley, Julia Cochran, Shyra Gardai, Timothy S. Lewis. Enfortumab vedotin, an anti-Nectin-4 ADC demonstrates bystander cell killing and immunogenic cell death anti-tumor activity mechanisms of action in urothelial cancers [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 5581.

Tucatinib is an investigational, oral, small molecule tyrosine kinase inhibitor that is highly selective for the kinase domain of HER2, without significant inhibition of EGFR. Recently, HER2CLIMB (NCT02614794), a pivotal, randomized, international, double-blind trial that evaluated tucatinib or placebo in combination with trastuzumab and capecitabine in patients with HER2+ metastatic breast cancer (MBC) with or without brain metastases, after progression with trastuzumab, pertuzumab, and ado-trastuzumab emtansine (T-DM1), showed superior progression-free and overall survival in patients treated on the tucatinib arm. Adverse events in the tucatinib arm were primarily low grade and occurred at rates similar to what was observed in the placebo arm. In this report, we characterize the in vitro and in vivo activity of tucatinib alone and in combination with T-DM1 in HER2-amplified preclinical models. In vitro assays demonstrate that tucatinib potently suppresses HER2-meditated signaling pathways, including phosphorylation of HER2, HER3, AKT and ERK. Ex vivo analysis of tucatinib-treated xenografts shows similar suppression of signal transduction pathways. Tucatinib inhibits the proliferation of HER2+ breast cancer (BC) cell lines in vitro with single digit nanomolar potencies, but was inactive in blocking cell proliferation of BC cell lines lacking amplified HER2. In HER2-amplified BC cell line derived- and patient-derived xenograft models, tucatinib suppresses growth of tumors as a single agent and shows improved activity when combined with T-DM1. To evaluate activity and drug penetration in HER2+ metastatic CNS tumors, we developed and characterized a red-shifted luciferase-expressing BT-474 BC cell line which was stereotaxically implanted into the brain. In vivo bioluminescence imaging was used to evaluate the efficacy of tucatinib and T-DM1 in CNS tumors as single agents and in combination. Histological assessment of treated tumors suggests that tucatinib can penetrate the blood-brain tumor barrier to suppress HER2 signaling. These data are further supported by a drug penetration study of CNS-implanted BT-474 tumors. Quantitative autoradiography analysis demonstrates that 14C-labeled tucatinib penetrates the CNS tumor mass with drug concentration levels exceeding measurements in normal brain regions. These data demonstrate that tucatinib is a uniquely selective and highly potent inhibitor of HER2 signaling, with activity against HER2+ breast cancer xenograft and brain metastasis models. Tucatinib is effective against implanted CNS tumors, suggesting that therapeutically relevant drug concentrations are achievable. These preclinical findings are consistent with clinical data showing tucatinib activity in patients with HER2+ MBC with brain metastases and support the continued development of tucatinib in patients with HER2+ BC. Citation Format: Devra J. Olson, Anita Kulukian, Janelle D. Taylor, Margo C. Zaval, Albina Nesterova, Kelly M. Hensley, Michelle L. Ulrich, Nicole S. Stevens, Scott R. Peterson. Preclinical characterization of tucatinib in HER2-amplified xenograft and CNS implanted tumors [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 1962.

Melanotransferrin (CD228/MFI2/MELTF) is a cell-surfaced glycosylphosphatidylinoitol (GPI)-anchored glycoprotein that belongs to the transferrin family of iron-binding proteins. CD228 was first described as an oncofetal protein highly expressed on malignant melanoma cells. Data from The Cancer Genome Atlas (TCGA) suggests that CD228 has broad expression across many types of carcinomas and it was recently described as a potential biomarker of invasive colorectal carcinoma. In this study, we characterize protein expression of CD228 using an immunohistochemical (IHC) assay and describe pre-clinical antitumor activity of SGN-CD228A, a potent CD228-targeting antibody-drug conjugate (ADC). We found that in addition to melanoma, CD228 is highly expressed in mesothelioma, non-small cell lung (NSCL), breast, colorectal, and pancreatic carcinomas. Monoclonal antibodies (mAbs) specific for human CD228 were evaluated and a lead antibody was selected based on binding characteristics, internalization properties, and cytotoxic activity as an ADC. SGN-CD228A is a humanized anti-CD228 mAb to which eight molecules of MMAE, a potent microtubule disrupting cytotoxic drug, have been conjugated via a β-glucuronidase-cleavable linker, which incorporates a PEG side chain and self-stabilizing maleimide to achieve homogenous conjugation with decreased plasma clearance and increased preclinical antitumor activity. Interestingly, when evaluating drug linkers, we found that changing the linker from a di-peptide to β-glucuronidase resulted in a striking improvement in the cytotoxicity of MMAE, likely due to unique trafficking and recycling of CD228. We examined 50 carcinoma cell lines and found 41 had >10,000 CD228 receptors per cell of which 60% had EC50 values <10ng/ml, and 30% had EC50 values between 10-100ng/ml when treated with SGN-CD228A in vitro. We also evaluated antitumor activity of SGN-CD228A in melanoma and NSCLC xenograft and PDX models. In the Colo-853 and Sk-Mel-5 melanoma models, a single dose of 0.33 mg/kg caused tumor delay whereas 1.0 mg/kg resulted in 5/8 and 4/8 durable complete responses (CRs), respectively. Similarly, in the squamous NSCLC model, Calu-1, a single dose of 1.0 mg/kg produced 6/6 durable responses (DRs). Similar results were obtained in NSCLC PDX models with 3 out of 4 models achieving tumor delay or DRs when dosed with 1.0 mg/kg and durable (CRs) at 3.0 mg/kg. Additionally, in a mouse TNBC PDX clinical trial (n=22), we found that SGN-CD228A achieved durable CRs even in low expressing PDX models. In summary, CD228 is a highly expressed carcinoma target and the novel glucuronide-MMAE ADC, SGN-CD228A, shows potent antitumor activity in vitro and in vivo. Citation Format: Sharsti L. Sandall, Marsha Mason, Devra Olson, Rebecca Mazahreh, Disha Sahetya, Lori Westendorf, Chris Leiske, Brian Schimpf, Liem Nguyen, Madhu Katepalli, Esther Trueblood, Christopher Hale, Albina Nesterova, Jason Wall, Timothy S. Lewis. SGN-CD228A: A novel humanized anti-CD228 antibody-drug conjugate for the treatment of solid tumors [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 2688.

Multiple myeloma (MM) is an incurable hematologic malignancy of transformed plasma cells. New targeted biological therapeutics are needed to increase the stringency and durability of remissions. In this study we describe SGN-CD48A, a potent CD48-targeting antibody-drug conjugate (ADC) utilizing a novel glucuronide-monomethylauristatin E (MMAE) linker, under development for the treatment of MM. CD48, or SLAMF2 (Signaling Lymphocyte Activation Molecule family member 2), is a GPI-anchored membrane protein in the SLAM family of immunoreceptors. CD48 is expressed on B and T lymphocytes, natural killer (NK) cells, and other immune cell types where it functions to modulate immune cell activation, proliferation, and differentiation. CD48 is also a tumor antigen broadly expressed in MM. We observed CD48 expression on the surface of malignant plasma cells in 90% (90/100) of human multiple myeloma patient samples examined by flow cytometry. Monoclonal antibodies (mAbs) specific for human CD48 were evaluated and a lead antibody was selected based on binding characteristics and cytotoxic activity against myeloma cells as an auristatin ADC. SGN-CD48A is a humanized anti-CD48 mAb to which eight molecules of MMAE, a potent microtubule disrupting cytotoxic drug, have been conjugated via a β-glucuronidase-cleavable linker. This novel glucuronide-MMAE drug-linker incorporates a PEG side chain and self-stabilizing maleimide to achieve homogenous drug-to-antibody ratio (DAR) 8 conjugates with decreased plasma clearance and increased preclinical antitumor activity. Following binding of CD48 at the myeloma cell surface, SGN-CD48A internalizes and traffics to lysosomal vesicles. Intracellular MMAE drug released from SGN-CD48A in myeloma cells induced cell cycle arrest at G2/M phase, phospho-histone H3 (Ser-10) phosphorylation, and caspase 3/7 dependent apoptotic cell death. SGN-CD48A demonstrated potent cytotoxic activity (EC50 values 1.0 - 11 ng/mL) against a panel of human MM cell lines, with CD48 expression levels of 135,000 - 480,000 receptors per cell. In contrast, SGN-CD48A had negligible cytotoxic activity against normal resting human B, NK, and T lymphocytes. We evaluated the in vivo antitumor activity of SGN-CD48A in disseminated MM cell line mouse xenograft models. In the NCI-H929 and EJM xenograft models, a single intraperitoneal dose of 0.3 mg/kg SGN-CD48A produced durable complete remissions in 8/8 and 6/8 mice, respectively. Similarly, in the U-266 xenograft model, a single dose of 1.0 mg/kg SGN-CD48A produced durable complete remissions in 7/8 mice. Neither unconjugated mAb nor a non-binding control MMAE ADC were active in these MM xenograft models, demonstrating that targeted delivery of MMAE drug through CD48 binding is required for activity. In summary, CD48 is a highly expressed new multiple myeloma target and the novel glucuronide-MMAE ADC SGN-CD48A shows potent antitumor activity against cell line models of MM.

Abstract 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 (< 2 hours) and traffics to lysosomal vesicles. PBD dimers released from SGN-CD352A in lysosomes induce a dose dependent DNA damage signaling response in MM cells, activating ATM and ATR kinases, and caspase 3/7 dependent apoptotic cell death results within 48 hours. SGN-CD352A demonstrated potent cytotoxic activity (EC50 values 6.6 - 52.6 pM) against a panel of human myeloma and lymphoma cell lines, with efficient cancer cell killing observed at CD352 expression levels as low as 3,500 receptors per cell. In contrast, SGN-CD352A had no effect on the viability of resting human T lymphocytes and minimal cytotoxic activity on B lymphocytes. We evaluated the in vivo antitumor activity of SGN-CD352A in disseminated MM cell line mouse xenograft models. In the MM.1R xenograft model, a single intraperitoneal dose of 30 μg/kg SGN-CD352A produced durable complete remissions in 10/10 mice. Similarly, in the U-266 xenograft model, a single dose of 100 μg/kg SGN-CD352A produced durable complete remissions in 9/10 mice and significant (P <0.0001) tumor delay at a single dose of 30 μg/kg. Neither unconjugated h20F3ec mAb nor a non-binding control PBD dimer ADC produced remissions in these MM xenograft models, demonstrating that targeted delivery of PBD dimer drug through CD352 binding is required for activity. In summary, CD352 is a newly validated multiple myeloma tumor antigen and the novel PBD dimer ADC SGN-CD352A shows potent antitumor activity against cell line models of MM at clinically relevant doses. 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 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 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

TPS1143 Background: First identified as an estrogen-inducible gene in a breast cancer cell line, LIV-1 is a multispan transmembrane protein of the solute-carrier family 39 with putative zinc transp...

Abstract 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 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

CD70, a transmembrane type II protein, is a member of the tumor necrosis factor (TNF) family. CD70 has been previously reported to be highly expressed in solid tumors (renal cell and nasopharyngeal carcinomas) as well as hematologic malignancies and has very limited expression in normal cells (activated lymphocytes, dendritic cells). An extensive expression profiling study for CD70 in carcinomas has been a challenge in the past due to a lack of an anti-CD70 reagent that works well in formalin-fixed paraffin embedded (FFPE) tissues. Murine monoclonal antibodies were generated and validated for specific CD70 staining and a protocol for immunohistochemical (IHC) analysis using FFPE samples was developed. Tissue microarray analysis of various types of tumors confirmed CD70 expression in kidney cancers (70% of cases). In addition, we report novel detection of significant CD70 expression in carcinomas such as pancreatic (25% of cases), larynx/pharynx (22%), ovarian (15%), skin (13%), lung (10%), and colon (9%). We also detected CD70 expression in tumor cell lines from the same indications by IHC and flow cytometric analyses. Using CD70+ ovarian and pancreatic cell lines, we show in vitro efficacy of our anti-CD70 antibody-drug conjugate, SGN-75. These results demonstrate the potential application of anti-CD70 ADCs for treatment of ovarian and pancreatic cancers. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4652.

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.

CD70 is a member of the TNF family whose interaction with its cognate receptor CD27 regulates immune effector and memory responses, and blockade of CD70-CD27 interactions inhibits the onset of EAE and cardiac allograft rejection in mice. Immunohistochemical analysis revealed the presence of CD70+ cells in inflamed tissues of lupus, rheumatoid arthritis and Crohn's disease patients. We have developed a humanized anti-human CD70 monoclonal antibody, SGN-70, that can selectively deplete CD70+ antigen-specific T cells and down-regulate expression of cytokines and chemokines including IFN-γ, TNF-α, VEGF and IP-10, as well as the co-stimulatory molecules CD40L, 4-1BB, ICOS and OX40. In a T-cell directed co-stimulation of peripheral blood lymphocyte culture system, SGN-70 decreased expansion of both T and B cells. The differentiation of CD20+CD38+ peripheral blood B cells to CD20-CD38+ plasmablasts was also inhibited by SGN-70, as were levels of secreted Ig. The in vivo activity of SGN-70 was evaluated in SCID-huPBMC mice immunized with tetanus toxoid (TT). Treatment with SGN-70 significantly reduced anti-TT antibody titers compared to untreated mice or to those that received non-binding control Ig. Altogether, our data indicate that SGN-70 can modulate immune responses mediated by both T and B lymphocytes, and provide a rationale to test SGN-70 clinically in autoimmune indications.

656 A novel oncofetal antigen Glypican-3 (GPC-3) has been reported to be highly expressed in primary hepatocellular carcinoma (HCC) and melanoma. Our data using a commercial anti-GPC-3 mAb confirmed a strong signal in 70% of HCC tumors tested by immunohistochemistry. Expression profiling of 32 normal human tissues showed that expression of GPC-3 was restricted to placenta and faint cytoplasmic staining in normal kidney. We also evaluated breast, lung, uterus and ovarian cancer tissue microarrays for GPC-3 expression. Thirty-two out of 42 ovarian tumors (76%) showed elevated GPC-3 expression. We found moderate to high levels of expression of GPC-3 in HCC, colon cancer and melanoma cell lines and undetectable levels in normal renal cells by quantitative FACS. Using an anti-GPC-3 mAb conjugated to the potent cytotoxic agent monomethylauristatin F, we showed specific growth inhibition of GPC-3+ cells in vitro, thus indicating internalization of the antibody-target complex. With the restricted normal tissue expression of GPC-3 and efficient internalization of the antibody-drug conjugate (ADC), an anti-GPC-3 ADC may be useful for targeted therapy of hepatocellular carcinomas. Possible additional indications are in ovarian cancer and melanomas.