Timothy S. Lewis

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.

Orthogonal Cysteine Protection Enables Homogeneous Multi‐Drug Antibody–Drug Conjugates

Abstract A strategy for the preparation of homogeneous antibody–drug conjugates (ADCs) containing multiple payloads has been developed. This approach utilizes sequential unmasking of cysteine residues with orthogonal protection to enable site‐specific conjugation of each drug. In addition, because the approach utilizes conjugation to native antibody cysteine residues, it is widely applicable and enables high drug loading for improved ADC potency. To highlight the benefits of ADC dual drug delivery, this strategy was applied to the preparation of ADCs containing two classes of auristatin drug‐linkers that have differing physiochemical properties and exert complementary anti‐cancer activities. Dual‐auristatin ADCs imparted activity in cell line and xenograft models that are refractory to ADCs comprised of the individual auristatin components. This work presents a facile method for construction of potent dual‐drug ADCs and demonstrates how delivery of multiple cytotoxic warheads can lead to improved ADC activities. Lastly, we anticipate that the conditions utilized herein for orthogonal cysteine unmasking are not restricted to ADCs and can be broadly utilized for site‐specific protein modification.

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.

Characterization of SGN-CD123A, A Potent CD123-Directed Antibody–Drug Conjugate for Acute Myeloid Leukemia

Treatment choices for acute myelogenous leukemia (AML) patients resistant to conventional chemotherapies are limited and novel therapeutic agents are needed. IL3 receptor alpha (IL3Rα, or CD123) is expressed on the majority of AML blasts, and there is evidence that its expression is increased on leukemic relative to normal hematopoietic stem cells, which makes it an attractive target for antibody-based therapy. Here, we report the generation and preclinical characterization of SGN-CD123A, an antibody–drug conjugate using the pyrrolobenzodiazepine dimer (PBD) linker and a humanized CD123 antibody with engineered cysteines for site-specific conjugation. Mechanistically, SGN-CD123A induces activation of DNA damage response pathways, cell-cycle changes, and apoptosis in AML cells. In vitro, SGN-CD123A–mediated potent cytotoxicity of 11/12 CD123+ AML cell lines and 20/23 primary samples from AML patients, including those with unfavorable cytogenetic profiles or FLT3 mutations. In vivo, SGN-CD123A treatment led to AML eradication in a disseminated disease model, remission in a subcutaneous xenograft model, and significant growth delay in a multidrug resistance xenograft model. Moreover, SGN-CD123A also resulted in durable complete remission of a patient-derived xenograft AML model. When combined with a FLT3 inhibitor quizartinib, SGN-CD123A enhanced the activity of quizartinib against two FLT3-mutated xenograft models. Overall, these data demonstrate that SGN-CD123A is a potent antileukemic agent, supporting an ongoing trial to evaluate its safety and efficacy in AML patients (NCT02848248). Mol Cancer Ther; 17(2); 554–64. ©2017 AACR.

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 946: SGN-CD70A, a pyrrolobenzodiazepine (PBD) dimer linked ADC, mediates DNA damage pathway activation and G2 cell cycle arrest leading to cell death

Seattle Genetics is utilizing the highly potent DNA cross-linking molecule, pyrrolobenzodiazepine (PBD) dimer, as the payload across multiple antibody drug conjugate (ADC) platforms. Previous studies have demonstrated that PBD dimers bind to the minor groove of DNA causing an interstrand crosslink; however, it is poorly understood how the cells recognize and respond to this insult. The DNA damage pathway exists to ensure genomic fidelity by either repairing damaged DNA or forcing programmed cell death if the damage is too extensive. Cells do this in an orchestrated manner that involves sensing the damage and eliciting a signaling cascade to induce repair and cell cycle arrest and ultimately either cell cycle re-start, death, or senescence. Using SGN-CD70A, an ADC consisting of a CD70-directed engineered cysteine antibody linked to two PBD dimers, we have examined the DNA damage response and cell death kinetics in a panel of renal cell carcinoma (RCC) and non-Hodgkin lymphoma (NHL) cell lines after treatment with free PBD and SGN-CD70A. We examined the central DNA damage sensing protein kinases, ATM, ATR, and DNA-PK and find that all three are phosphorylated on activation residues within 24 hours at EC50 doses in both RCC and NHL cell lines. Similarly, the downstream checkpoint kinases, Chk1 and Chk2 are also activated, within 24 hours after treatment with SGN-CD70A. The kinetics of pChk2 activation mirror Caspase 3/7 activation, with both peaking 48-72 hours after treatment. To confirm the involvement of the DNA damage pathway in the PBD mechanism, we used small molecules to inhibit ATM (KU-60019), ATR (VE-821), DNA-PK (NU-7441), and Chk1/2 (AZD7762, LY2603618) and found that all were synergistic with SGN-CD70A and accelerated cell death kinetics. We also conducted a small molecule screen to identify other processes involved in SGN-CD70A9s activity. The Mps1 inhibitor (AZ 3146), which shortens mitosis and abrogates the mitotic checkpoint, was strongly synergistic. Conversely, molecules that cause mitotic arrest, such as microtubule inhibitors and Plk1 inhibitors did not show the same degree of synergy. To follow up on this finding we looked at cell cycle kinetics. We found that SGN-CD70A treatment alone causes a G2 arrest and a modest increase in polyploid cells whereas synergistic combinations with SGN-CD70A and DNA damage kinase and Mps1 inhibitors decrease the number of cells in G2 while markedly increasing polyploid cells. These data suggest induction of mitotic catastrophe may be part of the cellular response to SGN-CD70A treatment. Based on these results, we are currently testing whether sequencing SGN-CD70A and various mitotic inhibitors can increase synergy. Citation Format: Sharsti L. Sandall, Renee McCormick, Jamie Miyamoto, Travis Biechele, Che-Leung Law, Timothy S. Lewis. SGN-CD70A, a pyrrolobenzodiazepine (PBD) dimer linked ADC, mediates DNA damage pathway activation and G2 cell cycle arrest leading to cell death. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 946. doi:10.1158/1538-7445.AM2015-946

Abstract 1789: Auristatin-based antibody-drug conjugates are synergistic in combination with PI3K-AKT-mTOR pathway inhibitors in hematologic malignancies and carcinoma

Antibody-drug conjugates (ADCs) that couple the potent anti-mitotic agents monomethyl auristatin E or F (MMAE or MMAF) to tumor antigen specific monoclonal antibodies have demonstrated antitumor activity in preclinical models. We report that the combination of auristatin-based ADCs with inhibitors of the phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) signaling pathway improves cytotoxic activity in Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), and renal cell carcinoma (RCC) cell line models. Implicated in tumor cell growth and survival, the PI3K-AKT-mTOR pathway is frequently hyperactivated in carcinomas and hematologic malignancies with elevated AKT activity reported in ∼64% of Hodgkin Reed-Sternberg (HRS) cells and 25-52% of primary patient diffuse large B-cell lymphomas. We produced dose response curves for auristatin ADCs and PI3K-AKT-mTOR pathway inhibitors, alone and in combination, and then performed combination index (CI) analysis using the Chou-Talalay method. The CD30-targeting ADC brentuximab vedotin (SGN-35) and the CD19-targeting ADC (SGN-19A) were synergistic (CI 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 1789. doi:10.1158/1538-7445.AM2011-1789

Abstract 688: Characterization and circumvention of drug resistance mechanisms in SGN-35-resistant HL and ALCL clonal cell lines

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA SGN-35 (cAC10-vcMMAE, brentuximab vedotin) is an auristatin antibody-drug conjugate (ADC), which targets CD30, for the treatment of relapsed Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (ALCL). We developed three SGN-35 resistant (BVR) cell line models to identify mechanisms by which HL and ALCL cells may become resistant to SGN-35 therapy. Resistant DEL and Karpas-299 (ALCL) and L540cy (HL) cell lines were generated by continuous exposure to increasing dose levels of SGN-35 over time. BVR cell lines with >10-fold decrease in sensitivity to SGN-35 versus parental cell lines were then made clonal. Multiple parental and BVR cell clones for each cell type were then characterized by cytotoxicity assay for sensitivity to SGN-35 and other anti-CD30 ADCs conjugated to alternate auristatin or pyrrolobenzodiazepine (PBD) dimer chemotypes. CD30 expression was analyzed by flow cytometry to monitor downregulation of the ADC targeted cell surface antigen. Changes in multidrug resistance efflux activity were evaluated by rhodamine 123 efflux, flow cytometry of transporter protein expression, and cytotoxicity measurements in the presence of the ABC1 transporter inhibitor verapamil. Gene expression changes between parental and BVR clones were measured using RNA sequencing, and confirmed by qPCR and western blotting. The L540cy-BVR, DEL-BVR, and Karpas-299-BVR clonal cell lines displayed unique paths to resistance. The L540cy-BVR resistance was due to upregulated transporter activity, which was reversible with verapamil. A minor increase in P-gp level was detected, and an increase in other transporter gene mRNA levels was also observed. CD30 expression on the surface of L540cy-BVR cell lines was unaffected. In contrast, the DEL-BVR resistant lines had moderate decrease in CD30 expression levels combined with increased P-gp expression. These resistant lines also had increased expression of NNMT, an enzyme involved in the metabolism of xenobiotic compounds. Finally, resistance in Karpas-299-BVR cells was due entirely to loss of CD30 protein expression. While no longer sensitive to SGN-35, the L540cy-BVR and DEL-BVR clones were sensitive to new anti-CD30 ADCs in vitro. Anti-CD30 mAb cAC10 conjugated to mcMMAF and other related auristatins efficiently killed L540-BVR and DEL-BVR cells. In addition, CD30-directed PBD dimer conjugates were highly potent on these resistant cell lines. These new anti-CD30 auristatin and PBD dimer ADCs also showed significantly improved activity over SGN-35 in a subcutaneous DEL-BVR mouse xenograft model. Our data suggest that downregulation of CD30 cell surface expression and upregulation of transporter activity are potential mechanisms for SGN-35 acquired resistance in systemic ALCL and HL. Newly developed antibody-drug conjugates utilizing MMAF-like and DNA-damaging PBD dimer drug-linkers are highly effective at overcoming SGN-35 resistance in vitro. Citation Format: Timothy S. Lewis, Kristine Gordon, Fu Li, Allana Weimann, Rebecca Bruders, Jamie Miyamoto, Dana Chace, Che-Leung Law. Characterization and circumvention of drug resistance mechanisms in SGN-35-resistant HL and ALCL clonal cell lines. [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 688. doi:10.1158/1538-7445.AM2014-688

Abstract 2411: A combination trial of SGN-75 and everolimus in patients with CD70-positive metastatic renal cell carcinoma (RCC).

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background. SGN-75 is an antibody-drug conjugate composed of a humanized anti-CD70 mAb conjugated to the microtubule-disrupting agent MMAF via a plasma-stable maleimidocaproyl linker. Upon binding to CD70, SGN-75 internalizes and releases cys-mcMMAF, which binds tubulin and induces G2/M arrest and apoptosis. In a phase 1, dose-escalation trial of SGN-75 in patients with CD70-positive relapsed/refractory NHL or metastatic RCC, single-agent activity of SGN-75 was observed in both NHL and RCC patients (Thompson 2011). In RCC, objective responses were observed with SGN-75 administered IV every 3 weeks at doses of 2 and 3 mg/kg. In vitro synergy in target cell killing was observed when SGN-75 was combined with mTOR inhibitors (everoliumus, temsirolimus, or sirolimus), a PI3K/mTOR inhibitor (NVP-BEZ235), or a dual mTORC1/mTORC2 inhibitor (PP242), as assessed by the Chou-Talalay method (Lewis 2011). Consistent with this observation, xenograft experiments using RCC cell lines (786-O and UM-RC-3) and a patient-derived RCC model (108963P) also demonstrated significantly improved antitumor activity with the combination of SGN-75 and everolimus compared to either agent alone. Based on the clinical activity of SGN-75 observed in patients with metastatic RCC and the synergistic effects observed in combination with mTOR inhibitors in preclinical models, a phase 1b study of the combination of SGN‐75 and everolimus was initiated in patients with CD70-positive metastatic RCC (ClinicalTrials.gov #[NCT01677390][1]). Methods. The primary objective of this phase 1 study is to evaluate the safety and tolerability, and to identify the MTD, of SGN-75 in combination with everolimus. Pharmacokinetics, immunogenicity, and antitumor activity will also be evaluated. Eligible patients are ≥18 years old with pathologically confirmed CD70-positive metastatic RCC on archived or fresh tumor biopsy. Patients must have previously received treatment with 1 or 2 VEGF-receptor TKIs and must not have received prior treatment with any mTOR inhibitor. Patients must have measurable disease as defined in RECIST Version 1.1. SGN-75 will be administered IV every 3 weeks at protocol-defined doses up to 2 mg/kg in combination with everolimus 10 mg daily. After establishing the MTD, an expansion cohort of 15 efficacy-evaluable patients will be enrolled and treated with a dose at or below the MTD to further characterize the safety and pharmacokinetic profile and to obtain a preliminary estimate of antitumor activity of the combination. Patients who achieve CR, PR, or SD will be eligible to continue treatment in the study until disease progression. Patients who achieve a response of SD or better and discontinue study treatment prior to disease progression will be followed for progression-free survival and all patients will be followed for overall survival. Citation Format: Nancy C. Whiting, Che-Leung Law, Tim Lewis. A combination trial of SGN-75 and everolimus in patients with CD70-positive metastatic renal cell carcinoma (RCC). [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 2411. doi:10.1158/1538-7445.AM2013-2411 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01677390&atom=%2Fcanres%2F73%2F8_Supplement%2F2411.atom

Abstract 2590: Preclinical evaluation of ASG-5ME, a novel antibody-drug conjugate for pancreatic cancer

Antibody-drug conjugates (ADCs) have demonstrated promising activity and tolerability profiles in oncology clinical trials, including for Hodgkin lymphoma (brentuzumab vedotin) and breast cancer (trastuzumab-DM1), motivating the development of new ADCs targeting antigens in other prevalent cancers. An immunohistochemical survey of expression of the novel tumor antigen AGS-5 revealed strong staining in 90% of pancreatic cancer specimens tested. Importantly, this staining was largely uniform, with essentially all transformed cells demonstrating membranous staining. This observation along with the observation of equally abundant expression of AGS-5 in prostate and other cancers motivated the development of ASG-5ME, an ADC targeting AGS-5. The ADC is comprised of a fully human IgG2 monoclonal antibody conjugated to the potent tubulin-binding drug monomethylauristatin E, conjugated with an average of 3.7 drug molecules per antibody. Conjugation and analysis of this IgG2 ADC will be discussed. The resulting conjugate demonstrated antigen-dependent internalization and in vitro cytotoxicity at sub-saturating ADC concentrations, as well as potent in vivo antitumor activity in patient-derived xenograft models of pancreatic cancer. ASG-5ME has a long (12 day) T1/2 in mice. Taken together, these results support the clinical evaluation of ASG-5ME for treatment of pancreatic cancer 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 2590.