HER2/HER3 dimerization resulting from overexpression of HER2 or neuregulin (NRG1) in cancer leads to HER3-mediated oncogenic activation of phosphoinositide 3-kinase (PI3K) signaling. Although ...ligand-blocking HER3 antibodies inhibit NRG1-driven tumor growth, they are ineffective against HER2-driven tumor growth because HER2 activates HER3 in a ligand-independent manner. In this study, we describe a novel HER3 monoclonal antibody (LJM716) that can neutralize multiple modes of HER3 activation, making it a superior candidate for clinical translation as a therapeutic candidate. LJM716 was a potent inhibitor of HER3/AKT phosphorylation and proliferation in HER2-amplified and NRG1-expressing cancer cells, and it displayed single-agent efficacy in tumor xenograft models. Combining LJM716 with agents that target HER2 or EGFR produced synergistic antitumor activity in vitro and in vivo. In particular, combining LJM716 with trastuzumab produced a more potent inhibition of signaling and cell proliferation than trastuzumab/pertuzumab combinations with similar activity in vivo. To elucidate its mechanism of action, we solved the structure of LJM716 bound to HER3, finding that LJM716 bound to an epitope, within domains 2 and 4, that traps HER3 in an inactive conformation. Taken together, our findings establish that LJM716 possesses a novel mechanism of action that, in combination with HER2- or EGFR-targeted agents, may leverage their clinical efficacy in ErbB-driven cancers.
Despite an improving therapeutic landscape, significant challenges remain in treating the majority of patients with advanced ovarian or renal cancer. We identified the cell-cell adhesion molecule ...cadherin-6 (
) as a lineage gene having significant differential expression in ovarian and kidney cancers. HKT288 is an optimized CDH6-targeting DM4-based antibody-drug conjugate (ADC) developed for the treatment of these diseases. Our study provides mechanistic evidence supporting the importance of linker choice for optimal antitumor activity and highlights CDH6 as an antigen for biotherapeutic development. To more robustly predict patient benefit of targeting CDH6, we incorporate a population-based patient-derived xenograft (PDX) clinical trial (PCT) to capture the heterogeneity of response across an unselected cohort of 30 models-a novel preclinical approach in ADC development. HKT288 induces durable tumor regressions of ovarian and renal cancer models
, including 40% of models on the PCT, and features a preclinical safety profile supportive of progression toward clinical evaluation.
We identify CDH6 as a target for biotherapeutics development and demonstrate how an integrated pharmacology strategy that incorporates mechanistic pharmacodynamics and toxicology studies provides a rich dataset for optimizing the therapeutic format. We highlight how a population-based PDX clinical trial and retrospective biomarker analysis can provide correlates of activity and response to guide initial patient selection for first-in-human trials of HKT288.
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Antibody-drug conjugates (ADCs) are of great interest as targeted cancer therapeutics. Preparation of ADCs for early stage screening is constrained by purification and biochemical analysis techniques ...that necessitate burdensome quantities of antibody. Here we describe a method, developed for the maytansinoid class of ADCs, enabling parallel conjugation of antibodies in 96-well format. The method utilizes ∼ 100 µg of antibody per well and requires <5 µg of ADC for characterization. We demonstrate the capabilities of this system using model antibodies. We also provide multiple examples applying this method to early-stage screening of maytansinoid ADCs. The method can greatly increase the throughput with which candidate ADCs can be screened in cell-based assays, and may be more generally applicable to high-throughput preparation and screening of different types of protein conjugates.
Src signaling plays an important role in prostate cancer (PrCa) progression. It has previously been shown that Src interacts with androgen receptor (AR) and enhances AR transactivation. Although it ...has been shown that Src promotes AR activity, the underlying pathway has not been defined. To help characterize the Src-AR pathway, the cellular localizations of Src, p-Src, AR, pAR, and Prostate Specific Antigen (PSA, an AR target gene) were analyzed in androgen-dependent (AD) LNCaP cells and in androgen-independent (AI) castration-resistant C4-2B cells. Using sub-cellular fractionation, the data showed that treatment of AD cells with synthetic androgen R1881 increased p-Src, AR, pAR, and PSA in the nucleus, while the levels of c-Src remained unchanged. Treatment of AI cells with R1881 increased pSrc and AR in the nucleus, while the levels of c-Src and PSA remained unchanged. When using immunofluorescence microscopy, R1881 did not appear to increase the nuclear levels of p-Src or c-Src, so perhaps this technique is not as sensitive or quantitative as subcellular fractionation immunoblots. The presence of PSA in the nucleus was unexpected given its well proven role as a secreted protein. Nuclear PSA was observed upon androgen stimulation in AD and AI cells, and in the nucleus of AI cells upon androgen deprivation. Given PSA's ability to induce cell division and decrease apoptosis when transfected into cells, its presence in the nucleus may imply that PSA acts there to help induce tumorigenesis. The effect of Src on AR activity was further studied by transfection of a dominant negative src (SrcK298M) in AD and AI cells. Transfection with SrcK298M did not affect PSA expression in LNCaP cells, but strongly inhibited PSA levels in AI cells. Integrin signaling through Src was investigated in PrCa by ligand binding assay in AD and AI cells. The data showed that alpha v beta 3 integrin (but not alpha v beta 6) upon attachment to fibronectin or TGF-beta-latency associated peptide (TGF- beta-LAP) increases p-Src levels in AD and AI cells, while the levels of c-Src, PSA, and AKT remain unchanged. Thus, alpha v beta 3 integrin facilitates Src signaling, but the activation does not appear to affect AR transactivation. In conclusion, these data show that Src is required for AR activity and, consequently, PSA expression in AI prostate cancer cells, but not in AD cells. These data also suggest that the nuclear co-localization of p-Src, AR and PSA might allow macromolecular interactions, which can further enhance AR transactivation and promote disease progression. With respect to the switch in tumor progression from an AD to AI state, the data indicate that the integrin-Src pathway does not include AKT or PSA (and not AR by deduction), so perhaps other non-AR pathways help facilitate tumor growth at the AI state.
Antibody-drug conjugates (ADCs) are of great interest as targeted cancer therapeutics. Preparation of ADCs for early stage screening is constrained by purification and biochemical analysis techniques ...that necessitate burdensome quantities of antibody. Here we describe a method, developed for the maytansinoid class of ADCs, enabling parallel conjugation of antibodies in 96-well format. The method utilizes ∼100 µg of antibody per well and requires <5 µg of ADC for characterization. We demonstrate the capabilities of this system using model antibodies. We also provide multiple examples applying this method to early-stage screening of maytansinoid ADCs. The method can greatly increase the throughput with which candidate ADCs can be screened in cell-based assays, and may be more generally applicable to high-throughput preparation and screening of different types of protein conjugates.
Conversations Karkala, John Alphonso; Dodd, Maya S.; Bhattacharya, Sayan ...
South Asian review (South Asian Literary Association),
01/2000, Volume:
21, Issue:
1
Journal Article
Conversations Karkala, John Alphonso; Dodd, Maya S.; Bhattacharya, Sayan ...
South Asian review (South Asian Literary Association),
20/1/1/, Volume:
21, Issue:
1
Journal Article
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