Changpu Yu

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.

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.

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 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.