Abstract 642: Cell-penetrating bispecific antibodies for targeting androgen receptor signaling in advanced prostate cancer
| Autor: | Grace Chan, Michael B. Lilly, Maria N. Garnovskaya, Mary G. Blanton, Richard H. Weisbart, Nancy L. Goicochea |
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| Rok vydání: | 2015 |
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| Zdroj: | Cancer Research. 75:642-642 |
| ISSN: | 1538-7445 0008-5472 |
| Popis: | Androgen receptor (AR) plays a critical role in the development and progression of prostate cancer (PCa). Current therapies target the ligand-binding domain (LBD) of AR by inhibiting production or binding of ligand. In castration-resistant PCa (CRPC), AR isoforms lacking the LBD are highly expressed, resulting in constitutive, ligand-independent AR signaling. Therapies to block ligand-independent AR signaling have not been introduced into clinical use. We have developed bispecific antibodies (bsAbs) that penetrate PCa cells and bind to the N-terminal domain of the AR, inhibiting AR signaling. The 3E10-AR441 bsAb was engineered by connecting two single-chain variable fragments (scFv) with a linker. One half of the bsAb molecule is scFv 3E10, derived from a lupus anti-DNA antibody. This module enters cells through the ENT2 nucleoside salvage receptor and locates in the nucleus. The other half is an scFv based on the anti-AR monoclonal antibody AR441. The 3E10-AR441 bsAb is expressed in yeast as a single recombinant protein. Treatment of LNCaP with bsAb resulted in nuclear accumulation of the antibody, visualized by confocal microscopy. The 3E10-AR441 bsAb also engaged its target under denaturing and non-denaturing conditions. The bsAb could detect AR when used to probe an AR immunoblot. It could also immunoprecipitate WT AR, as well as mutant/variant AR lacking the LBD (AR(Q640X), Arv7). The scFv 3E10 alone did not bind to AR. BsAb binding affinity to AR was assessed using a competitive sandwich-type ELISA. An anti-AR antibody-coated microtiter plate was used to capture WT AR or mutant\variant AR from cell lysates. AR441-HRP antibody conjugate was mixed with increasing concentrations of 3E10-AR441 or AR441 as competitors. Binding affinity of 3E10-AR441 to WT AR (180nM) was higher than that of the parental AR441 MoAb (7nM). Affinity of bsAb was 3 fold higher to WT AR than to an AR(Q640X) mutant. The bsAb blocked genomic AR signaling in LNCaP cells, as measured by dihydrotestosterone (DHT) activation of both artificial (ARE-luciferase) and endogenous (PSA) reporters. The magnitude of inhibition was similar to that seen with enzalutamide at 5 μM. The 3E10 scFv alone had minimal effect on reporter gene expression. Non-genomic AR signaling was measured by calcium release upon DHT treatment. Calcium5 dye was added to LNCap treated with variable doses of bsAb or 3E10. The fluorescence intensity is proportional to the amount of calcium released. 3E10-AR441 blocked DHT-induced release of calcium while 3E10 did not. Current work is focused on the design of 3E10-AR441 derivatives with enhanced binding affinity towards AR mutants lacking the LBD. 3E10-AR441 bsAb is an attractive therapeutic agent due to its ability to inhibit AR function in a ligand-independent manner. Citation Format: Nancy L. Goicochea, Maria Garnovskaya, Mary Blanton, Grace Chan, Richard Weisbart, Michael Lilly. Cell-penetrating bispecific antibodies for targeting androgen receptor signaling in advanced prostate cancer. [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 642. doi:10.1158/1538-7445.AM2015-642 |
| Databáze: | OpenAIRE |
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