May Kung Sutherland

Osteocyte control of bone formation via sclerostin, a novel BMP antagonist

There is an unmet medical need for anabolic treatments to restore lost bone. Human genetic bone disorders provide insight into bone regulatory processes. Sclerosteosis is a disease typified by high bone mass due to the loss of SOST expression. Sclerostin, the SOST gene protein product, competed with the type I and type II bone morphogenetic protein (BMP) receptors for binding to BMPs, decreased BMP signaling and suppressed mineralization of osteoblastic cells. SOST expression was detected in cultured osteoblasts and in mineralizing areas of the skeleton, but not in osteoclasts. Strong expression in osteocytes suggested that sclerostin expressed by these central regulatory cells mediates bone homeostasis. Transgenic mice overexpressing SOST exhibited low bone mass and decreased bone strength as the result of a significant reduction in osteoblast activity and subsequently, bone formation. Modulation of this osteocyte‐derived negative signal is therapeutically relevant for disorders associated with bone loss.

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.

Lysosomal Trafficking and Cysteine Protease Metabolism Confer Target-specific Cytotoxicity by Peptide-linked Anti-CD30-Auristatin Conjugates *

The chimeric anti-CD30 monoclonal antibody cAC10, linked to the antimitotic agents monomethyl auristatin E (MMAE) or F (MMAF), produces potent and highly CD30-selective anti-tumor activity in vitro and in vivo. These drugs are appended via a valine-citrulline (vc) dipeptide linkage designed for high stability in serum and conditional cleavage and putative release of fully active drugs by lysosomal cathepsins. To characterize the biochemical processes leading to effective drug delivery, we examined the intracellular trafficking, internalization, and metabolism of the parent antibody and two antibody-drug conjugates, cAC10vc-MMAE and cAC10vc-MMAF, following CD30 surface antigen interaction with target cells. Both cAC10 and its conjugates bound to target cells and internalized in a similar manner. Subcellular fractionation and immunofluorescence studies demonstrated that the antibody and antibody-drug conjugates entering target cells migrated to the lysosomes. Trafficking of both species was blocked by inhibitors of clathrin-mediated endocytosis, suggesting that drug conjugation does not alter the fate of antibody-antigen complexes. Incubation of cAC10vc-MMAE or cAC10vc-MMAF with purified cathepsin B or with enriched lysosomal fractions prepared by subcellular fractionation resulted in the release of active, free drug. Cysteine protease inhibitors, but not aspartic or serine protease inhibitors, blocked antibody-drug conjugate metabolism and the ensuing cytotoxicity of target cells and yielded enhanced intracellular levels of the intact conjugates. These findings suggest that in addition to trafficking to the lysosomes, cathepsin B and perhaps other lysosomal cysteine proteases are requisite for drug release and provide a mechanistic basis for developing antibody-drug conjugates cleavable by intracellular proteases for the targeted delivery of anti-cancer therapeutics.

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.

5-azacytidine enhances the anti-leukemic activity of lintuzumab (SGN-33) in preclinical models of acute myeloid leukemia

Despite therapeutic advances, the poor prognoses for acute myeloid leukemia (AML) and intermediate and high-risk myelodysplastic syndromes (MDS) point to the need for better treatment options. AML and MDS cells express the myeloid marker CD33, making it amenable to CD33-targeted therapy. Lintuzumab (SGN-33), a humanized monoclonal anti-CD33 antibody undergoing clinical evaluation, induced meaningful responses in a Phase 1 clinical trial and demonstrated anti-leukemic activity in preclinical models. Recently, it was reported that 5-azacytidine (Vidaza™) prolonged the overall survival of a group of high risk MDS and AML patients. To determine whether the combination of lintuzumab and 5-azacytidine would be beneficial, a mouse xenograft model of disseminated AML was used to evaluate the combination. There was a significant reduction in tumor burden and an increase in overall survival in mice treated with lintuzumab and 5-azacytidine. The effects were greater than that obtained with either agent alone. As the in vivo anti-leukemic activity of lintuzumab was dependent upon the presence of mouse effector cells including macrophages and neutrophils, in vitro effector function assays were used to assess the impact of 5-azacytidine on lintuzumab activity. The results show that 5-azacytidine significantly enhanced the ability of lintuzumab to promote tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC) and phagocytic (ADCP) activities. These results suggest that lintuzumab and 5-azacytidine act in concert to promote tumor cell killing. Additionally, these findings provide the rationale to evaluate this combination in the clinic.

Antibody-Dependent Cell-Mediated Cytotoxicity Overcomes NK Cell Resistance in MLL-Rearranged Leukemia Expressing Inhibitory KIR Ligands but Not Activating Ligands

Purpose: Leukemias with MLL gene rearrangement are associated with a poor prognosis. Natural killer (NK) cell therapy is a potential treatment, but leukemia cells may be resistant. Here, we sought to determine the susceptibility of MLL-rearranged leukemia cells to NK cell lysis and to develop a novel immunotherapeutic approach to optimize NK cell therapy, including the use of an antibody against leukemia-associated antigen and the elimination of killer-cell immunoglobulin-like receptor (KIR)–mediated inhibition. Experimental Design: Three MLL-rearranged leukemia cell lines (RS4;11, SEM, and MV4-11) and primary leukemia blasts were assessed for surface phenotype and susceptibility to NK cell lysis with or without antibodies against CD19 (XmAb5574), CD33 (lintuzumab), or KIR ligands. Results: All three cell lines were resistant to NK cell lysis, had some inhibitory KIR ligands and protease inhibitor-9, and expressed low levels of NKG2D activating ligands and adhesion molecules. After treatment with XmAb5574 or lintuzumab, MLL-rearranged leukemia cells were efficiently killed by NK cells. The addition of pan–major histocompatibility complex class I antibody, which blocked inhibitory KIR-HLA interaction, further augmented degranulation in all three KIR2DL1, KIR2DL2/3, and KIR3DL1 subsets of NK cells based on the rule of missing-self recognition. A mouse model showed a decreased rate of leukemia progression in vivo as monitored by bioluminescence imaging and longer survival after antibody treatment. Conclusion: Our data support the use of a triple immunotherapy approach, including an antibody directed against tumor-associated antigen, KIR-mismatched NK cell transplantation, and inhibitory KIR blockade, for the treatment of NK cell–resistant MLL-rearranged leukemias. Clin Cancer Res; 18(22); 6296–305. ©2012 AACR.

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

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

Abstract 2472: SEA-CD40, a sugar engineered non-fucosylated anti-CD40 antibody with improved immune activating capabilities

SEA-CD40 is a non-fucosylated, humanized IgG1 monoclonal antibody directed against human CD40, a co-stimulatory receptor of the TNF receptor superfamily. SEA-CD40 is derived from dacetuzumab, a humanized IgG1 previously developed and studied for B-lineage malignancies. Glycosylation of the antibody Fc is essential for Fc receptor-mediated activity and non-fucosylated antibodies show improved efficacy, particularly via increased binding to low affinity FcγRIIIa. Enhanced functionality of SEA-CD40 was determined through FcγRIIIa binding affinity, antibody-dependent cellular cytotoxicity (ADCC) activity, activation of the immune response, and induction of antigen-specific T-cells. While SEA-CD40 and the parent antibody dacetuzumab bind to CD40 with similar affinity, the non-fucosylated SEA-CD40 binds equally well to the low (158F) and high (158V) affinity versions of FcγRIIIa with higher affinity than dacetuzumab. The consequence of enhanced SEA-CD40/FcγRIIIa binding is potent ADCC activity against a CD40+ lymphoma B cell line and improved agonistic signaling to antigen presenting cells (APCs). SEA-CD40 treatment of human PBMCs elicits a robust immune response as measured by increased cytokine production and up-regulation of maturation markers on APCs with maintained activity at antibody concentrations as low as 10 ng/ml. The immune stimulatory properties of SEA-CD40 were observed in vivo as increased activity in xenograft and syngenic tumor models as well as induction of cytokine production in cynomolgus monkeys. Both in-vitro and in-vivo activity of SEA-CD40 was significantly greater than with dacetuzumab. The increased functionality of SEA-CD40 occurs through the non-fucosylated Fc domain as a F(ab’)2 version loses the ability to induce ADCC, stimulate cytokines, or up-regulate APC activation markers. SEA-CD40 induction of antigen specific T-cells was assessed using human peripheral blood mononuclear cells (PBMCs) exposed to influenza antigen. In the presence of SEA-CD40 influenza specific T-cells mount a robust antigen-specific response characterized by tetramer staining and elevated production of IFNγ. SEA-CD40 activity on PBMCs from donors with melanoma, pancreatic, or breast cancer was assessed and tumor antigen specific T-cell proliferation and IFNγ production was observed. SEA-CD40 is a non-fucosylated agonistic anti-CD40 antibody that shows enhanced binding to FcγRIIIa resulting in amplified cytokine production, co-stimulatory molecule up regulation, and ultimately stimulation of antigen specific T-cell responses to viral and tumor antigens. Citation Format: Shyra J. Gardai, Angela Epp, Germein Linares, Lori Westendorf, May Sutherland, Haley Neff-LaFord, Stanford L. Peng, Che-Leung Law. SEA-CD40, a sugar engineered non-fucosylated anti-CD40 antibody with improved immune activating capabilities. [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 2472. doi:10.1158/1538-7445.AM2015-2472

Abstract 4321: Development of pyrrolobenzodiazepine-based antibody-drug conjugates for cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Pyrrolobenzodiazepine (PBD) dimers are highly cytotoxic minor groove binding DNA crosslinking agents derived from the anthramycin class of natural products. In our efforts to develop highly effective and well-tolerated treatment options for cancer patients, we have investigated several antibody-drug conjugates (ADCs) based on a PBD dimer. The fully synthetic PBD-linker, comprised of a protease-cleavable val-ala sequence and a reactive maleimide group, was conjugated via engineered cysteine residues to humanized monoclonal antibodies specific for their binding to the well-characterized cancer antigen targets, CD33 and CD70. The resulting ADCs were well-defined, monomeric and showed pronounced in vitro and in vivo activity against both hematologic and solid tumor cancer models. These included models of acute myelogenous leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma and renal cell carcinoma. The ADCs were immunologically specific, and showed potent activity in MDR-positive models, including models resistant to other ADCs. These results underscore the importance of the drug component in developing highly efficacious ADCs for cancer therapy. Citation Format: Scott C. Jeffrey, Patrick J. Burke, May K. Sutherland, David W. Meyer, Robert P. Lyon, Julie A. McEarchern, Che-Leung Law, Peter D. Senter. Development of pyrrolobenzodiazepine-based antibody-drug conjugates for 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 4321. doi:10.1158/1538-7445.AM2013-4321