Rayna Venook

Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index

Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display–based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.

Therapeutic antibody targeting of individual Notch receptors.

The four receptors of the Notch family are widely expressed transmembrane proteins that function as key conduits through which mammalian cells communicate to regulate cell fate and growth. Ligand binding triggers a conformational change in the receptor negative regulatory region (NRR) that enables ADAM protease cleavage at a juxtamembrane site that otherwise lies buried within the quiescent NRR. Subsequent intramembrane proteolysis catalysed by the γ-secretase complex liberates the intracellular domain (ICD) to initiate the downstream Notch transcriptional program. Aberrant signalling through each receptor has been linked to numerous diseases, particularly cancer, making the Notch pathway a compelling target for new drugs. Although γ-secretase inhibitors (GSIs) have progressed into the clinic, GSIs fail to distinguish individual Notch receptors, inhibit other signalling pathways and cause intestinal toxicity, attributed to dual inhibition of Notch1 and 2 (ref. 11). To elucidate the discrete functions of Notch1 and Notch2 and develop clinically relevant inhibitors that reduce intestinal toxicity, we used phage display technology to generate highly specialized antibodies that specifically antagonize each receptor paralogue and yet cross-react with the human and mouse sequences, enabling the discrimination of Notch1 versus Notch2 function in human patients and rodent models. Our co-crystal structure shows that the inhibitory mechanism relies on stabilizing NRR quiescence. Selective blocking of Notch1 inhibits tumour growth in pre-clinical models through two mechanisms: inhibition of cancer cell growth and deregulation of angiogenesis. Whereas inhibition of Notch1 plus Notch2 causes severe intestinal toxicity, inhibition of either receptor alone reduces or avoids this effect, demonstrating a clear advantage over pan-Notch inhibitors. Our studies emphasize the value of paralogue-specific antagonists in dissecting the contributions of distinct Notch receptors to differentiation and disease and reveal the therapeutic promise in targeting Notch1 and Notch2 independently.

Targeting FGFR4 Inhibits Hepatocellular Carcinoma in Preclinical Mouse Models

The fibroblast growth factor (FGF)-FGF receptor (FGFR) signaling system plays critical roles in a variety of normal developmental and physiological processes. It is also well documented that dysregulation of FGF-FGFR signaling may have important roles in tumor development and progression. The FGFR4–FGF19 signaling axis has been implicated in the development of hepatocellular carcinomas (HCCs) in mice, and potentially in humans. In this study, we demonstrate that FGFR4 is required for hepatocarcinogenesis; the progeny of FGF19 transgenic mice, which have previously been shown to develop HCCs, bred with FGFR4 knockout mice fail to develop liver tumors. To further test the importance of FGFR4 in HCC, we developed a blocking anti-FGFR4 monoclonal antibody (LD1). LD1 inhibited: 1) FGF1 and FGF19 binding to FGFR4, 2) FGFR4–mediated signaling, colony formation, and proliferation in vitro, and 3) tumor growth in a preclinical model of liver cancer in vivo. Finally, we show that FGFR4 expression is elevated in several types of cancer, including liver cancer, as compared to normal tissues. These findings suggest a modulatory role for FGFR4 in the development and progression of hepatocellular carcinoma and that FGFR4 may be an important and novel therapeutic target in treating this disease.

Constitutive NOTCH3 Signaling Promotes the Growth of Basal Breast Cancers

Notch ligands signal through one of four receptors on neighboring cells to mediate cell-cell communication and control cell fate, proliferation, and survival. Although aberrant Notch activation has been implicated in numerous malignancies, including breast cancer, the importance of individual receptors in distinct breast cancer subtypes and the mechanisms of receptor activation remain unclear. Using a novel antibody to detect active NOTCH3, we report here that NOTCH3 signals constitutively in a panel of basal breast cancer cell lines and in more than one third of basal tumors. Selective inhibition of individual ligands revealed that this signal does not require canonical ligand induction. A NOTCH3 antagonist antibody inhibited growth of basal lines, whereas a NOTCH3 agonist antibody enhanced the transformed phenotype in vitro and in tumor xenografts. Transcriptomic analyses generated a Notch gene signature that included Notch pathway components, the oncogene c-Myc, and the mammary stem cell regulator Id4 This signature drove clustering of breast cancer cell lines and tumors into the common subtypes and correlated with the basal classification. Our results highlight an unexpected ligand-independent induction mechanism and suggest that constitutive NOTCH3 signaling can drive an oncogenic program in a subset of basal breast cancers. Cancer Res; 77(6); 1439-52. ©2017 AACR.

The melanosomal protein PMEL17 as a target for antibody drug conjugate therapy in melanoma

Background: A search for cell surface proteins amenable to antibody drug conjugate (ADC) therapy was performed. Results: Expression of PMEL17 was highly restricted to melanoma cells, and an ADC directed against it was efficacious. Conclusion: PMEL17 is an attractive target for ADC therapy in melanoma. Significance: Intracellular transmembrane proteins that transit the cell surface represent a new class of targets for ADCs. Melanocytes uniquely express specialized genes required for pigment formation, some of which are maintained following their transformation to melanoma. Here we exploit this property to selectively target melanoma with an antibody drug conjugate (ADC) specific to PMEL17, the product of the SILV pigment-forming gene. We describe new PMEL17 antibodies that detect the endogenous protein. These antibodies help define the secretory fate of PMEL17 and demonstrate its utility as an ADC target. Although newly synthesized PMEL17 is ultimately routed to the melanosome, we find substantial amounts accessible to our antibodies at the cell surface that undergo internalization and routing to a LAMP1-enriched, lysosome-related organelle. Accordingly, an ADC reactive with PMEL17 exhibits target-dependent tumor cell killing in vitro and in vivo.

Abstract 5619: Preclinical development of anti-SLC34A2 antibody drug conjugate as a therapeutic for non-small cell lung and ovarian cancers.

SLC34A2, also known as type II Na/Pi co-transporters NaPi2b or NaPi3b, is a member of solute carrier protein family SLC34. SLC34A2 is highly expressed on cancers of the lung, ovary and thyroid as well as on normal lung pneumocytes, and is a promising target for antigen-directed therapy. Antibody drug conjugates (ADCs) specify the delivery of a cytotoxic drug to cells expressing an accessible antigenic target. Here we have appended monomethyl auristatin E (MMAE) to an antibody recognizing SLC34A2. Anti-SLC34A2-vc-MMAE inhibits tumor growth in multiple ovarian and non-small cell lung cancer xenograft models that express SLC34A2. Anti-SLC34A2 antibody and its conjugate cross-react with both human and monkey, and toxicity studies in monkeys are conducted to identify both target- and MMAE-related effects. Anti-SLC34A2-vc-MMAE was well tolerated in monkeys at efficacious doses and produced dose-related bone marrow suppression and increased apoptosis and mitotic figures consistent with MMAE-related pharmacology. Despite high levels of expression in normal lung of non-human primate, the cross-reactive ADC exhibited an acceptable safety profile with a dose-limiting toxicity unrelated to normal tissue expression. Overall, our preclinical results suggest that the antibody drug conjugate targeting the SLC34A2 may provide an effective new therapy for the treatment of NSCLC and ovarian cancer. Citation Format: Kedan Lin, Crystal Zhang, Eric Harstad, Hajime Hiraragi, Willy Solis, Siao Ping Tsai, Keyang Xu, Maria Hristopoulos, Rayna Venook, Bonnee Rubinfeld, Paul Polakis. Preclinical development of anti-SLC34A2 antibody drug conjugate as a therapeutic for non-small cell lung and ovarian cancers. [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 5619. doi:10.1158/1538-7445.AM2013-5619

Abstract B189: Impact of site of conjugation on efficacy and pharmacokinetics of anti-NaPi3b antibody drug conjugate variants.

Introduction: Optimization of antibody-drug conjugates (ADCs) with therapeutic potential involves several critical parameters, including selection of the antibody, linker, cytotoxic drug, and drug-to-antibody ratio (DAR). To minimize the heterogeneity from conjugation, the antibody can be engineered at different conjugation sites to achieve a defined DAR at specific sites. Anti-NaPi3b-vc-MMAE (anti-NaPi3b-vc-E) is an ADC targeting sodium dependent phosphate transporter expressed in lung and ovarian cancers. Construction and production of the THIOMAB variant of antibody was reported previously. Briefly, a cysteine residue was engineered at Ala118 position of heavy chain (HC) or Val205 position of light chain (LC) to produce its THIOMAB HC and LC variants, respectively. The MMAE conjugates were produced with a drug to antibody ratio (DAR) of two. The purpose of this study was to evaluate the site of conjugation on ADC efficacy and antibody pharmacokinetics. Methods: The anti-tumor activity of anti-Napi3b ADC variants was evaluated in a mouse xenograft model of human ovarian cancer OVCAR3-X2.1 after single dose administration in a range of doses. The unconjugated antibodies including anti-NaPi3b (control) and anti-NaPi3b (HC and LC) and their respective ADC variants were administered intravenously to mice and rats; and the pharmacokinetic (PK) parameters measured by a total antibody assay were estimated by a two-compartmental model. Results: Based on comparisons of tumor growth inhibition, both LC and HC anti-Napi3b-vc-E showed clear dose-dependent inhibitory activity when given at 3 to 12 mg/kg versus the vehicle group. Non-NaPi3b-binding ADCs did not significantly affect tumor growth, indicating targeted anti-tumor activity. The activities of LC and HC variants were similar. Dose proportional pharmacokinetics of total antibody following administration of anti-NaPi3b-vc-E (HC and LC) were observed in mice and rats over the dose range tested. Total antibody clearance of the three unconjugated antibodies (control, thio HC, and thio LC) was comparable. Conjugation with MMAE only modestly increased the total antibody clearance in both LC and HC conjugates when compared to unconjugated antibodies. Overall, PK behavior of the HC and LC variants were similar. Conclusion: The results demonstrated that ADC variants with different sites of conjugation exhibited similar anti-tumor activities, which provided additional choice of conjugation sites for individual antibody. Cysteine mutation in either HC or LC of the unconjugated antibodies did not affect the PK when compared to the control. In addition, conjugation did not significantly change the overall clearance of the antibody, and the site of MMAE conjugations did not have a significant impact on the PK. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B189.