Development of cell-penetrating bispecific antibodies targeting the N-terminal domain of androgen receptor for prostate cancer therapy†

Abstract

Castration-resistant prostate cancer cells exhibit continued androgen receptor signaling in spite of low levels of ligand. Current therapies to block androgen receptor signaling act by inhibiting ligand production or binding. We developed bispecific antibodies capable of penetrating cells and binding androgen receptor outside of the ligand-binding domain. Half of the bispecific antibody molecule consists of a single-chain variable fragment of 3E10, an anti-DNA antibody that enters cells. The other half is a single-chain variable fragment version of AR441, an anti-AR antibody. The resulting 3E10-AR441 bispecific antibody enters human LNCaP prostate cells and accumulates in the nucleus. The antibody binds to wild-type, mutant and splice variant androgen receptor. Binding affinity of 3E10-AR441 to androgen receptor (284 nM) was lower than that of the parental AR441 mAb (4.6 nM), but could be improved (45 nM) through alternative placement of the affinity tags, and ordering of the VH and VK domains. The 3E10-AR441 bispecific antibody blocked genomic signaling by wild-type or splice variant androgen receptor in LNCaP cells. It also blocked non-genomic signaling by the wild-type receptor. Furthermore, bispecific antibody inhibited the growth of C4-2 prostate cancer cells under androgen-stimulated conditions. The 3E10-AR441 biAb can enter prostate cancer cells and inhibits androgen receptor function in a ligand-independent manner. It may be an attractive prototype agent for prostate cancer therapy.

GABARAPL1 Promotes AR+ Prostate Cancer Growth by Increasing FL-AR/AR-V Transcription Activity and Nuclear Translocation

“…Recently, much focus has been placed on the development of inhibitors that target AR NTD. Agents targeting the AR NTD are currently being explored (22–24). However, the AR NTD structure contains a high degree of intrinsic disorder and, therefore, has been difficult to target using structure-based drug design (25).…”

Breaking androgen receptor addiction of prostate cancer by targeting different functional domains in the treatment of advanced disease

“… QW-07 Binds unknown region of AR NTD, suppressing interactions with AREs and coactivators [ 108 ] . BiAb 3E10-AR441 Bispecific antibody complementary to aa 302-318 in NTD [ 109 ] . AR-DBD targeting: inhibits AR interaction with DNA Pyridium pamoate Interacts with AR-DBD already bound to DNA to prevent RNA pol II recruitment [ 110 , 111 ] .…”

“…AR441 (a known anti-AR antibody [129] was used as the scFv component due to its epitope being located in the NTD of AR, creating the antibody BiAb 3E10-AR441. The antibody was able to inhibit the AR transcriptional activity by binding to and inactivating the NTD of full-length AR and AR-Vs and reducing transactivation of AR target genes leading to inhibition of androgen-dependent cell growth in vitro [107] . Whether this antibody can penetrate cancer cells in vivo remains be determined.…”

Non-nuclear AR Signaling in Prostate Cancer

“…3E10-AR441 (Figure 4A) is a bispecific antibody with the uncommon ability to enter the target cell and simultaneously bind two different targets (Goicochea et al, 2017). It is composed by two parts; one is a single-chain variable fragment of 3E10, an high-affinity DNA antibody, the other part is a single-chain variable fragment that target the AR NTD around aminoacid 441.…”