Maureen Ryan

SGN-CD33A: a novel CD33-targeting antibody–drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML

Outcomes in acute myeloid leukemia (AML) remain unsatisfactory, and novel treatments are urgently needed. One strategy explores antibodies and their drug conjugates, particularly those targeting CD33. Emerging data with gemtuzumab ozogamicin (GO) demonstrate target validity and activity in some patients with AML, but efficacy is limited by heterogeneous drug conjugation, linker instability, and a high incidence of multidrug resistance. We describe here the development of SGN-CD33A, a humanized anti-CD33 antibody with engineered cysteines conjugated to a highly potent, synthetic DNA cross-linking pyrrolobenzodiazepine dimer via a protease-cleavable linker. The use of engineered cysteine residues at the sites of drug linker attachment results in a drug loading of approximately 2 pyrrolobenzodiazepine dimers per antibody. In preclinical testing, SGN-CD33A is more potent than GO against a panel of AML cell lines and primary AML cells in vitro and in xenotransplantation studies in mice. Unlike GO, antileukemic activity is observed with SGN-CD33A in AML models with the multidrug-resistant phenotype. Mechanistic studies indicate that the cytotoxic effects of SGN-CD33A involve DNA damage with ensuing cell cycle arrest and apoptotic cell death. Together, these data suggest that SGN-CD33A has CD33-directed antitumor activity and support clinical testing of this novel therapeutic in patients with AML.

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.

Antibody targeting of B-cell maturation antigen on malignant plasma cells

B-cell maturation antigen (BCMA) is expressed on normal and malignant plasma cells and represents a potential target for therapeutic intervention. BCMA binds to two ligands that promote tumor cell survival, a proliferation inducing ligand (APRIL) and B-cell activating factor. To selectively target BCMA for plasma cell malignancies, we developed antibodies with ligand blocking activity that could promote cytotoxicity of multiple myeloma (MM) cell lines as naked antibodies or as antibody-drug conjugates. We show that SG1, an inhibitory BCMA antibody, blocks APRIL–dependent activation of nuclear factor-κB in a dose-dependent manner in vitro. Cytotoxicity of SG1 was assessed as a naked antibody after chimerization with and without Fc mutations that enhance FcγRIIIA binding. The Fc mutations increased the antibody-dependent cell-mediated cytotoxicity potency of BCMA antibodies against MM lines by ∼100-fold with a ≥2-fold increase in maximal lysis. As an alternative therapeutic strategy, anti-BCMA antibodies were endowed with direct cytotoxic activity by conjugation to the cytotoxic drug, monomethyl auristatin F. The most potent BCMA antibody-drug conjugate displayed IC50 values of ≤130 pmol/L for three different MM lines. Hence, BCMA antibodies show cytotoxic activity both as naked IgG and as drug conjugates and warrant further evaluation as therapeutic candidates for plasma cell malignancies. [Mol Cancer Ther 2007;6(11):3009–18]

Preclinical Characterization of SGN-70, a Humanized Antibody Directed against CD70

Purpose: CD70 (CD27L) is a member of the tumor necrosis factor family aberrantly expressed on a number of hematologic malignancies and some carcinomas. CD70 expression on malignant cells coupled with its highly restricted expression on normal cells makes CD70 an attractive target for monoclonal antibody (mAb)–based therapies. We developed a humanized anti-CD70 antibody, SGN-70, and herein describe the antitumor activities of this mAb. Experimental Design: CD70 expression on primary tumors was evaluated by immunohistochemical staining of Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, and renal cell carcinoma tissue microarrays. The CD70-binding and cytotoxic activities of SGN-70 were tested in vitro using a number of cell-based assays. The in vivo antitumor properties of SGN-70 were tested in severe combined immunodeficient mice bearing disseminated lymphoma and multiple myeloma xenografts. Mechanism-of-action studies were conducted using SGN-70v, a variant mAb with equivalent target-binding activity but impaired Fcγ receptor binding compared with SGN-70. Results: Immunohistochemical analysis identified CD70 expression on ∼40% of multiple myeloma isolates and confirmed CD70 expression on a high percentage of Hodgkin lymphoma Reed-Sternberg cells, non-Hodgkin lymphoma, and renal cell carcinoma tumors. SGN-70 lysed CD70+ tumor cells via Fc-dependent functions, including antibody-dependent cellular cytotoxicity and phagocytosis and complement fixation. In vivo, SGN-70 treatment significantly decreased tumor burden and prolonged survival of tumor-bearing mice. Conclusions: SGN-70 is a novel humanized IgG1 mAb undergoing clinical development for the treatment of CD70+ cancers. SGN-70 possesses Fc-dependent antibody effector functions and mediates antitumor activity in vivo.

Targeting of BAFF and APRIL for autoimmunity and oncology.

BLyS and APRIL are tumor necrosis factor superfamily members shown to be important for B-cell development, maturation and survival. Recent data also indicate that these cytokines regulate the survival and maintenance of malignant B-cells in cancer patients. The key role of BlyS/APRIL in the immune system and their potential role in cancers have attracted the attention of basic scientistis and biotechnology companies alike. As a result, the pathways regulated by BLYS and APRIL have been quickly elevated as attractive targets for antibody-based and non-antibody based therapeutics. Exploitation of these pathways has not only given us enormous insights into the basic biology of the APRIL/BLyS system but it has also identified potential clinical candidates for cancer and autoimmunity. As such, multiple biotechnology companies are currently testing various therapeutic candidates in the clinic. This chapter will review BLyS and APRIL functions and discuss alternative therapeutic approaches to target BLyS and APRIL pathways for human malignancies and autoimmunity.

Therapeutic potential of SGN-CD19B, a PBD-based anti-CD19 drug conjugate, for treatment of B-cell malignancies

Patients with relapsed/refractory B-cell malignancies such as non-Hodgkin lymphoma (B-NHL) or acute lymphoblastic leukemia have a poor prognosis. Despite measurable clinical activity with new targeted therapies, many patients do not achieve a complete or durable response suggesting an opportunity to improve upon existing therapies. Here we describe SGN-CD19B, a pyrrolobenzodiazepine (PBD)-based anti-CD19 antibody drug conjugate (ADC) being investigated for treatment of B-cell malignancies, which has improved potency compared with other ADCs. CD19-expressing tumor cells rapidly internalize SGN-CD19B, and the released PBD drug induces DNA damage, resulting in G2/M cell cycle arrest and cell death. SGN-CD19B demonstrated activity against a broad panel of malignant B-cell lines and induced durable regressions in mice bearing xenografts derived from these B-cell malignancies. A single dose of SGN-CD19B induced durable regressions at 300 μg/kg (3 μg/kg drug equivalents); combination with rituximab decreased the curative dose to 100 μg/kg (1 μg/kg drug equivalents). These doses are significantly lower than the level of drug required with other ADC payloads. In cynomolgus monkeys, SGN-CD19B effectively depleted CD20+ B lymphocytes in peripheral blood and lymphoid tissues confirming that SGN-CD19B is pharmacodynamically active at well-tolerated doses. In summary, preclinical studies show SGN-CD19B is a highly active ADC, which releases a DNA cross-linking agent rather than a microtubule inhibitor. The distinct mechanism of action, broad potency, and potential to combine with rituximab suggest that SGN-CD19B may offer unique clinical opportunities in B-cell malignancies. A phase 1 clinical trial is in progress to investigate the therapeutic potential of SGN-CD19B in relapsed/refractory B-NHL. This trial was registered at www.clinicaltrials.gov as #NCT02702141.

Abstract 2541: Preclinical results of SGN-CD19A in combination with R-ICE or R-CHOP in non-Hodgkin lymphoma models

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA SGN-CD19A is an antibody drug conjugate (ADC) that recognizes CD19, a B-cell specific marker expressed in B-cell malignancies including non-Hodgkin lymphoma (NHL). SGN-CD19A is currently in a phase I clinical trial for adult patients with relapsed refractory B-cell NHL (CT.gov [NCT01786135][1]) in which it has shown evidence of single-agent activity (1). This ADC binds CD19, internalizes, and releases cys-mcMMAF, which ultimately induces apoptosis in targeted cells (2). Lymphoma tumor xenografts treated with SGN-CD19A show growth delay in a dose dependent manner. Here we show that SGN-CD19A in combination with standard of care [rituximab/ifosfamide/carboplatin/etoposide (R-ICE), rituximab/cyclophosphamide/doxorubicin/vincristine/prednisone (R-CHOP), or components] exceeds the activity of SGN-CD19A alone and results in impressive anti-tumor activity in multiple xenograft models. It was notable that in some xenograft models, SGN-CD19A + R-CHOP or SGN-CD19A + R-ICE yielded activity that was similar to SGN-CD19A plus rituximab alone. This strong interaction between rituximab and SGN-CD19A was modeled in vitro. SGN-CD19A synergized with rituximab, in the absence of cross-linking, to cause decreased cell viability in NHL cell lines. These data suggest that cell autonomous signaling contributes to the unique interaction between rituximab and SGN-CD19A. Our in vivo and in vitro data support R-CHOP, R-ICE, or rituximab as viable combination therapies for NHL patients with SGN-CD19A. 1) Uma Borate et al. A first-in-human phase 1 study of the antibody-drug conjugate SGN-CD19A in relapsed or refractory B-lineage acute leukemia and highly aggressive lymphoma. Blood 2013 122:1437 2) Che-Leung Law et al. Preclinical characterization of an auristatin-based anti-CD19 drug conjugate, SGN-19A. Abstract number 625. In: Proceedings of the 102th Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Citation Format: Heather A. Van Epps, Kerry Klussman, Martha Anderson, Weiping Zeng, Devra Olson, Maureen Ryan, Tina Albertson, Che-Leung Law. Preclinical results of SGN-CD19A in combination with R-ICE or R-CHOP in non-Hodgkin lymphoma models. [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 2541. doi:10.1158/1538-7445.AM2015-2541 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01786135&atom=%2Fcanres%2F75%2F15_Supplement%2F2541.atom

Abstract 1686: CD70 as a target in non-Hodgkin lymphoma (NHL) and renal cell carcinoma (RCC)

Background CD70 is a member of the TNF superfamily expressed primarily on activated lymphocytes. CD70 interacts with CD27 to regulate B and T cell functions. Among normal, nonlymphoid tissues, CD70 is only expressed on stromal cells of the thymic medulla and mature dendritic cells. CD70 expression in RCC and NHL highlights its potential as a target for cancer therapy. Methods An indirect immunohistochemistry (IHC) assay was developed using an anti-CD70 mouse monoclonal antibody (Ryan 2010), and validated in a CLIA-compliant laboratory. Control cell lines had receptor numbers and IHC staining intensities of ∼190,000 (3+), ∼21000 (3+), ∼4000 (1+) and 0 (0). Archived tumor tissue from consenting patients with relapsed/refractory (RR) NHL or metastatic RCC were stained and read by a board-certified pathologist at Quest Diagnostics, reporting the percentage of cells staining for CD70, intensity, and subcellular localization of the stain. Results The rates and homogeneity of combined membrane and non-membrane CD70 staining intensity ≥1+ and 3+ for each tumor type are shown in the table below. The majority of NHL (88%) and RCC (80%) samples exhibited membranous CD70 staining ≥1+; >75% exhibited 3+ staining. Among the NHL and RCC subtypes sampled, high levels of 3+ intensity were observed in diffuse large B cell lymphoma (DLBCL; N = 44, 84%), follicular lymphoma (FL; N = 26, 77%), mantle cell lymphoma (MCL; N = 6, 67%), clear cell RCC (ccRCC; N = 138, 93%), and papillary RCC (pRCC; N = 15, 67%). No chromophobe RCC samples showed membranous staining (N = 5). Conclusions CD70 is expressed in the majority of DLBCL, FL, MCL, ccRCC, and pRCC samples tested. Based on the unmet medical needs of these diseases, limited expression of CD70 on normal tissues, predominant membrane expression of CD70, and observed preclinical antitumor activity (Jeffrey 2013), a CD70-targeted antibody-drug conjugate, SGN-CD70A, has been advanced into clinical development in CD70-positive RR NHL and metastatic RCC (NCT# NCT02216890). Citation Format: Kenneth W. Wood, Elaina M. Gartner, Mechthild Jonas, Maureen Ryan, Dana A. Kennedy. CD70 as a target in non-Hodgkin lymphoma (NHL) and renal cell carcinoma (RCC). [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 1686. doi:10.1158/1538-7445.AM2015-1686

Abstract 4630: Integrin αVα6 is expressed on multiple solid tumors and is a potential therapeutic target for auristatin-based antibody-drug conjugates

Antibody-drug conjugates (ADCs) are designed to deliver a targeted, potent payload directly to the tumor cell. This targeted cytoxicity can greatly augment the antitumor activity of a naked antibody and reduce the off target toxicity of free drug. Cancers of the head and neck, pancreas, bladder, lung, ovary and breast show frequent expression of integrin α6, suggesting they may be amenable to therapeutic targeting with an anti-integrin β6 ADC. We identified a therapeutic antibody that is optimal for drug delivery by screening a large panel of anti-integrin β6 ADCs on antigen-positive tumor lines. Monoclonal antibody 15H3 showed consistent potency as an ADC on solid tumor cell lines representative of the target indications. The ADC activity of 15H3 was detectable using either monomethyl auristatin E with a protease-cleavable linker (vcMMAE) or monomethyl auristatin F (mcMMAF), which requires antibody degradation. 15H3 was selected as the lead and humanized for in vivo testing. Humanized 15H3 (h15H3) retained all the properties of the mouse parental antibody, including the ability to cross-bind multiple species (human, monkey, rat, and mouse integrin α6). In vitro analysis showed that h15H3-vcMMAE and h15H3-mcMMAF internalize within a few hours and result in antigen-specific cytotoxicity of carcinoma lines. In vivo studies of integrin-α6 positive xenografts demonstrated antitumor activity. Our data suggest that integrin β6 is a promising potential ADC target for antigen-positive solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4630. doi:1538-7445.AM2012-4630

Abstract 4394: Development of parallel conjugation and assay methodologies to screen for antibodies with optimal properties for use as antibody-drug conjugates

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The activity of antibody-drug conjugates (ADCs) on cancer cells can be affected by a multitude of factors, such as binding affinity, rate of internalization, subcellular trafficking, and efficient drug release within the target cell population. Consequently, the properties of an ideal antibody for drug delivery are not necessarily the same as those for a therapeutic naked antibody. Furthermore, the use of indirect assays involving the use of secondary antibodies to screen for optimal ADCs can be misleading, since crosslinking on the cell surface can lead to altered downstream events, and the affinity of the secondary antibody constrains the dynamic range of the assay. When seeking candidate antibodies directed against a novel antigen for ADC therapy, it is therefore most desirable to screen a large panel in the form of ADCs and evaluate their cytotoxic activities, since these results provide a direct measurement of parameters that can affect cytotoxic activity. However, when dealing with microgram quantities of a large number of antibodies as is typical of an antibody discovery campaign, the yields from conventional conjugation methodologies are limiting. We developed a novel approach that addresses this issue and have successfully applied it to the discovery of optimal ADCs out of a large panel of candidate antibodies. The technology is currently being applied to the discovery of ADCs against antigens of interest for targeted drug delivery. 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 4394.