BT474 Xenografts Research Articles

HER2-Overexpressing Breast Cancers Amplify FGFR Signaling upon Acquisition of Resistance to Dual Therapeutic Blockade of HER2.

Purpose: Dual blockade of HER2 with trastuzumab and lapatinib or pertuzumab has been shown to be superior to single-agent trastuzumab. However, a significant fraction of HER2-overexpressing (HER2+) breast cancers escape from these drug combinations. In this study, we sought to discover the mechanisms of acquired resistance to the combination of lapatinib + trastuzumab.Experimental Design: HER2+ BT474 xenografts were treated with lapatinib + trastuzumab long-term until resistance developed. Potential mechanisms of acquired resistance were evaluated in lapatinib + trastuzumab-resistant (LTR) tumors by targeted capture next-generation sequencing. In vitro experiments were performed to corroborate these findings, and a novel drug combination was tested against LTR xenografts. Gene expression and copy-number analyses were performed to corroborate our findings in clinical samples.Results: LTR tumors exhibited an increase in FGF3/4/19 copy number, together with an increase in FGFR phosphorylation, marked stromal changes in the tumor microenvironment, and reduced tumor uptake of lapatinib. Stimulation of BT474 cells with FGF4 promoted resistance to lapatinib + trastuzumab in vitro Treatment with FGFR tyrosine kinase inhibitors reversed these changes and overcame resistance to lapatinib + trastuzumab. High expression of FGFR1 correlated with a statistically shorter progression-free survival in patients with HER2+ early breast cancer treated with adjuvant trastuzumab. Finally, FGFR1 and/or FGF3 gene amplification correlated with a lower pathologic complete response in patients with HER2+ early breast cancer treated with neoadjuvant anti-HER2 therapy.Conclusions: Amplification of FGFR signaling promotes resistance to HER2 inhibition, which can be diminished by the combination of HER2 and FGFR inhibitors. Clin Cancer Res; 23(15); 4323-34. ©2017 AACR.

Abstract 2860: 89Zr-trastuzumab immunoPET imaging to monitor src status after treatment in HER2 breast cancer

Abstract Around 30% of patients with early stage breast cancer have recurrent disease due to resistance. One of the putative causes of acquired resistance can be attributed to the hyperactivation of Src kinases, which are particularly stabilized by aberrant HER2 signaling, which itself is overexpressed in nearly 30% of early stage breast cancers. HER2 downstream effectors include Src, thus, we aim to investigate the use of a HER2-specific monoclonal antibody positron emission tomography (PET) probe, 89Zr-trastuzumab, to monitor Src therapy via surrogate HER2 expression. We hypothesize that 89Zr-trastuzumab can be a surrogate imaging tool for monitoring Src treatment. 89Zr-trastuzumab was previously developed and has demonstrated excellent specificity for breast cancer. Dasatinib-treated HER2+ BT474 (IC50 ~ 1.3 μM), SK-BR-3 (IC50 ~ 5.5 µM), and JIMT-1 (IC50 ~ 8 μM) breast cancer cells lines have shown a negative correlation between treatment and HER2. We next conducted tumorigenic studies using athymic nu/nu mice bearing JIMT-1 or BT474 xenografts, which were treated for 7 or 14 days with dasatinib (75 mg/kg/day). At the end of each treatment, the tumors were subsequently imaged with 18F-FDG, and then followed by 89Zr-trastuzumab, 24 h later. Images were analyzed using AsiPro (show version here and decay-corrected to the time of injection. Tumor uptake values were measured by drawing volumes-of-interest, expressed as % injected dose per gram (%ID/g) of tissue on regions showing the most binding. 18F-FDG did not demonstrate a predictive response in both treated and placebo tumors, whereas, HER2 PET was able to show differential response in treated vs. control cohorts. In JIMT-1 mice imaged with 89Zr-trastuzumab, a two-fold difference (p < 0.01) was observed between untreated and both treated groups (8.0 ± 1.5 %ID/g, n= 7 vs. 7-day treated: 3.8 ± 1.5 %ID/g, n=4, and 14-day-treated: 4.5 ± 1.3 %ID/g, n= 4) mice imaged with 89Zr-trastuzumab. Confirmatory ex vivo western blots of JIMT-1 tumors have shown a decrease in HER2 as treatment time increases, with a decrease in p-Src (Y-416). In vivo studies with BT-474 xenografts are currently underway. Immunohistochemistry on excised tumors, as well as treatment on a metastatic patient-derived xenograft model is currently in progress. We have shown that HER2 PET can potentially be a surrogate marker of Src treatment. The findings from this study potentially afford a powerful tool to non-invasively detect and monitor changes in Src-targeted therapy in man at the early phases of treatment. Citation Format: Brooke N. McKnight, Nerissa T. Viola-Villegas. 89Zr-trastuzumab immunoPET imaging to monitor src status after treatment in HER2 breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2860. doi:10.1158/1538-7445.AM2017-2860

Abstract 1089: Anti-tumor activity of selinexor is enhanced by palbociclib in preclinical models of HER2+ breast cancer

Abstract Introduction: XPO1 (exportin-1/ CRM1) inhibitor selinexor (KPT-330) is the first-in-class, orally bioavailable, clinical stage SINE (Selective Inhibitor of Nuclear Export) compound with marked anti-tumor activity towards solid and hematological malignancies. This activity can be further enhanced by other therapeutic agents. We have previously shown strong synergistic preclinical activity of selinexor plus trastuzumab against HER2+ breast cancer. In cancer models of acquired resistance to HER2-targeted therapies, G1/S phase cell cycle regulators Cyclin D1 and CDK4/6 are inappropriately activated. We therefore investigated the combinatorial effect of selinexor plus palbociclib, a CDK4/6 inhibitor, in HER2+ breast cancer models as a treatment option for recurrent and relapsed HER2+ breast cancers. Methods: The effects of selinexor or palbociclib single agents or in combination were tested in vitro with BT474 HER2+ breast cancer cell line. Total RNA and protein was extracted from cell lysates and analyzed by qPCR and immunoblots. In vivo, a subcutaneous BT474 xenograft mouse model was treated with selinexor (5 mg/kg or 15 mg/kg; qodx3) or palbociclib (50 mg/kg or 150 mg/kg; qd) single agents or in combination. Tumor growth and body weights were monitored for 60 days. Tumors were harvested and analyzed by immunohistochemistry (IHC). Results: Selinexor plus palbociclib was highly effective in vitro and in vivo in BT474 breast cancer cells. In in vitro assays, selinexor or palbociclib single agents demonstrated inhibitory effects on cell proliferation and showed strong synergy when combined. In vivo, the combination resulted in significant survival benefit and enhanced tumor growth inhibition compared to vehicle or either single agent. IHC analysis of xenograft tumors showed synergistic inhibition of cell proliferation by selinexor plus palbociclib. The Ki67 proliferation index determined by IHC was 25% for vehicle control, 20% for selinexor, 7% for palbociclib and 2% for the combination. Based on IHC analysis, the synergistic anti-tumor activity of selinexor plus palbociclib was achieved at multiple levels of the CDK4/6 pathway. Selinexor treatment increased p21, p27 and Rb nuclear staining. Both p21 and p27 are inhibitors of CDK4/6 while Rb is a negative regulator of cell cycle progression. CDK4/6 phosphorylates and inactivates Rb, which allows cell cycle progression. In selinexor as well as palbociclib treated samples, phosphorylated Rb in the nucleus decreased, indicating a down-regulation of the CDK4/6 pathway. Conclusion: Selinexor plus palbociclib shows synergistic inhibition of cell proliferation in vivo and in vitro in HER2+ breast cancer cell line BT474 by down-regulation of CDK4/6 pathway. This combination therapy warrants further investigation as an effective treatment option for recurrent and relapsed HER2+ breast cancer. Citation Format: Hua Chang, Sharon Friedlander, Trinayan Kashyap, Boris Klebanov, Christian Argueta, Oscar A. Gonzalez, Erkan Baloglu, Yosef Landesman, Sharon Shacham, Margaret Lee, William Senapedis. Anti-tumor activity of selinexor is enhanced by palbociclib in preclinical models of HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1089. doi:10.1158/1538-7445.AM2017-1089

An ERBB1-3 Neutralizing Antibody Mixture With High Activity Against Drug-Resistant HER2+ Breast Cancers With ERBB Ligand Overexpression.

Plasticity of the ERBB receptor network has been suggested to cause acquired resistance to anti-human epidermal growth factor receptor 2 (HER2) therapies. Thus, we studied whether a novel approach using an ERBB1-3-neutralizing antibody mixture can block these compensatory mechanisms of resistance. HER2+ cell lines and xenografts (n ≥ 6 mice per group) were treated with the ERBB1-3 antibody mixture Pan-HER, trastuzumab/lapatinib (TL), trastuzumab/pertuzumab (TP), or T-DM1. Downregulation of ERBB receptors was assessed by immunoblot analysis and immunohistochemistry. Paired pre- and post-T-DM1 tumor biopsies from patients (n = 11) with HER2-amplified breast cancer were evaluated for HER2 and P-HER3 expression by immunohistochemistry and/or fluorescence in situ hybridization. ERBB ligands were measured by quantitative reverse transcription polymerase chain reaction. Drug-resistant cells were generated by chronic treatment with T-DM1. All statistical tests were two-sided. Treatment with Pan-HER inhibited growth and promoted degradation of ERBB1-3 receptors in a panel of HER2+ breast cancer cells. Compared with TL, TP, and T-DM1, Pan-HER induced a similar antitumor effect against established BT474 and HCC1954 tumors, but was superior to TL against MDA-361 xenografts (TL mean = 2026 mm 3 , SD = 924 mm 3 , vs Pan-HER mean = 565 mm 3 , SD = 499 mm 3 , P = .04). Pan-HER-treated BT474 xenografts did not recur after treatment discontinuation, whereas tumors treated with TL, TP, and T-DM1 did. Post-TP and post-T-DM1 recurrent tumors expressed higher levels of neuregulin-1 (NRG1), HER3 and P-HER3 (all P < .05). Higher levels of P-HER3 protein and NRG1 mRNA were also observed in HER2+ breast cancers progressing after T-DM1 and trastuzumab (NRG1 transcript fold change ± SD; pretreatment = 2, SD = 1.9, vs post-treatment = 11.4, SD = 10.3, P = .04). The HER3-neutralizing antibody LJM716 resensitized the drug-resistant cells to T-DM1, suggesting a causal association between the NRG1-HER3 axis and drug resistance. Finally, Pan-HER treatment inhibited growth of HR6 trastuzumab- and T-DM1-resistant xenografts. These data suggest that upregulation of a NRG1-HER3 axis can mediate escape from anti-HER2 therapies. Further, multitargeted antibody mixtures, such as Pan-HER, can simultaneously remove and/or block targeted ERBB receptor and ligands, thus representing an effective approach against drug-sensitive and -resistant HER2+ cancers.

Abstract P6-12-09: Pan-HER, an antibody mixture with antitumor activity against drug-resistant HER2-overexpressing breast cancers with high ERBB ligand expression

Abstract Background: Amplification/overexpression of ERBB receptors and/or ligands has been associated with resistance to anti-HER2 therapies. Pan-HER is a mixture of six antibodies targeting each of the ERBB receptors, EGFR, HER2 and HER3, with synergistic pairs of antibodies. Each pair of antibodies simultaneously blocks ligand binding and/or induces target degradation, thus preventing compensatory mechanisms to anti-ERBB therapies. We examined the antitumor activity of Pan-HER against drug-sensitive and -resistant HER2+ breast cancer cells and xenografts. Results: Pan-HER exhibited potent growth inhibitory activity against a panel of HER2+ breast cancer cells (BT474, MDA-453, MDA-361, SUM190, HCC1954, UACC893 and SKBR3). Growth inhibition was associated with internalization and degradation of EGFR, HER2 and HER3. Pan-HER was superior to the combination of trastuzumab/pertuzumab (TP) against HER2+/PIK3CA mutant MDA-361, HCC1954, UACC893 and MDA-453 cells. We next compared the effect of Pan-HER against BT474, HCC1954 and MDA-361 xenografts established in nude mice to that of trastuzumab/lapatinib (TL), TP and T-DM1. All treatments were effective across the panel of xenografts. In mice with MDA-361 tumors, Pan-HER and TP were superior to TL. Immunoblot analysis showed significant downregulation of EGFR, HER2 and HER3 only in tumors treated with Pan-HER. After a complete response, treatment was discontinued. Among mice with BT474 xenografts treated with TP, TL and T-DM1, 25-50% of mice exhibited a tumor recurrence within 50 weeks of follow-up, while no recurrences were registered in mice treated with Pan-HER. Tumors recurring after TP and T-DM1 expressed significantly higher HER3 and P-HER3 protein levels and NRG1 mRNA levels. HCC1954 xenografts recurring after T-DM1 also overexpressed NRG1 mRNA compared to tumors before therapy. We next examined the effect of Pan-HER against trastuzumab-resistant HR6 (BT474) cells (Ritter et al. CCR 2007) and HCC1954 and UACC893 cells with acquired resistance to T-DM1 (TDR; IC50 &amp;gt;5-, &amp;gt;6- and 600-fold in HR6, UACC893-TDR and HCC1954-TDR cells, respectively, vs. parental cells). All T-DM1-resistant cells expressed significantly higher HER3 and P-HER3 protein levels and NRG1 mRNA and protein levels. Treatment with the HER3 neutralizing antibody LJM716 resensitized HR6 and HCC1954-TDR cells to T-DM1, suggesting a causal association between the NRG1-HER3 axis and drug resistance. Mice with HR6 tumors were treated with Pan-HER, TL, TP and T-DM1. Only Pan-HER arrested HR6 tumor growth and downregulated EGFR, HER2, HER3, P-HER3 and P-AKT. Finally, HCC1954-TDR tumors rapidly grew in vivo despite treatment with T-DM1. Administration of Pan-HER to mice bearing HCC1954-TDR xenografts growing in the presence of T-DM1, induced rapid tumor regressions. Conclusions: These data suggest that multitarget therapeutic interventions, such as Pan-HER, which simultaneously remove and/or block all ERBB receptors and ligands, are a feasible and effective approach against HER2-overexpressing cancers both sensitive and resistant to anti-HER2 therapies. Citation Format: Schwarz LJ, Hutchinson KE, Estrada MV, Sanders ME, Dugger TC, Formisano L, Guerrero AL, Red-Brewer M, Young CD, Lantto J, Pedersen MW, Kragh M, Horak ID, Arteaga CL. Pan-HER, an antibody mixture with antitumor activity against drug-resistant HER2-overexpressing breast cancers with high ERBB ligand expression [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-12-09.

Abstract P4-06-11: Differential regulation of microRNAs and integrins influences metastatic potential: Comparison between locally invasive BT-474 and metastatic MDA-MB-231 xenografts

Abstract Background: Generation of BT-474 and MDA-MB-231 xenografts in immunocompromised mice provides one means of dissecting the molecular events involved in local invasion versus distant metastasis. Recently, Glunde et al have shown an interdependence of genes involved in cell-cell and cell-matrix adhesion (E-cadherin and integrin β1) and extra-cellular matrix degradation (MMP-2 and 9) in allowing cells to metastasize. Further, Fite el al have identified a set of microRNAs (miRs) up-regulated by E-cadherin (a pre-EMT marker) and down-regulated by Vimentin (post-EMT marker) in acquiring an invasive phenotype. We have performed a detailed analysis of integrins, matrix metallo-proteases and key miRs to better understand the molecular events underlying these disparate behaviours. Methods: We injected BT-474 (N=5) and MDA-MB-231 (N=5) cells orthotopically into SCID mice. Xenografts were assessed for local growth rate and monitored for distant metastasis. The implanted tumors as well as the distant metastatic foci were harvested. Markers involved in local invasion, distant metastasis and tumor-stroma interactions including miRs were compared between BT-474 and MDA-MB-231 cell lines and their xenografts by q-RT-PCR, immunofluorescence and immunohistochemistry. Results: As expected BT-474 xenografts showed a higher rate of tumor growth when compared to MDA-MB-231. Histological examination of BT-474 tumors confirmed only locally invasive tumor growth with infiltrated blood capillaries and vessels; no macro and microscopic metastases were observed in the organs collected. On the contrary, MDA-MB-231 xenografts showed highly undifferentiated tumor growth and frank lung metastasis and extra-pulmonary tumor growth in one of the five mice injected despite slow rate of local growth. Expression of matrix metalloproteases – MMP-2 &amp; 9 was more than 30 fold upregulated in MDA-MB-231 xenografts as compared to BT-474. Elevated level of E-cadherin was observed in BT-474 but was absent in MDA-MB-231. The most interesting differences were seen in the levels of miRs and cell-surface integrins. High levels of miR-18a, miR-93 and miR-182 were observed in BT-474 implants when compared to MDA-MB-231 which had a much lower level of these miRs. On the contrary, higher levels of integrin β3, and β1 were observed in MDA-MB-231 tumors when compared to BT-474. Integrin β6 was absent in both. The reciprocal relationship between these markers is being examined and compared between locally invasive tumors and metastatic triple negative breast cancers from our case series of human specimens (N=250). Conclusion: miRs and integrins known to be involved in invasion are differentially regulated in tumors that are locally invasive compared to ones with distant metastasis. The level of the key targets of these miRs as well as additional integrins is being examined. Understanding the epigenetic regulations leading to metastasis via tumor-stroma interaction might help in discerning differential tumor behaviour. Citation Format: Lawrence PV, Desai K, Prabhu JS, Korlimarla A, Nair MG, Sridhar TS. Differential regulation of microRNAs and integrins influences metastatic potential: Comparison between locally invasive BT-474 and metastatic MDA-MB-231 xenografts [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-06-11.

PH and redox dual-responsive biodegradable polymeric micelles with high drug loading for effective anticancer drug delivery

Diblock copolymers of poly(ethylene glycol) (PEG) and biodegradable polycarbonate functionalized with GSH-sensitive disulfide bonds and pH-responsive carboxylic acid groups were synthesized via organocatalytic ring-opening polymerization of functional cyclic carbonates with PEG having different molecular weights as macroinitiators. These narrowly-dispersed polymers had predictable molecular weights, and were used to load doxorubicin (DOX) into micelles primarily through ionic interactions. The DOX-loaded micelles exhibited the requisite small particle size (<100 nm), narrow size distribution and high drug loading capacity. When exposed to endolysosomal pH of 5.0, drug release was accelerated by at least two-fold. The introduction of GSH further expedited DOX release. Effective DOX release enhanced cytotoxicity against cancer cells. More importantly, the DOX-loaded micelles with the optimized composition showed excellent antitumor efficacy in nude mice bearing BT-474 xenografts without inducing toxicity. These pH and redox dual-responsive micelles have the potential as delivery carriers to maximize the therapeutic effect of anticancer drugs.

Gene therapy using plasmid DNA-encoded anti-HER2 antibody for cancers that overexpress HER2.

Plasmid DNA-encoded antibodies, or DNA-based monoclonal antibodies (dMAbs), are delivered by intramuscular injection and in vivo electroporation (EP) and are effective in virus neutralization, although they have not been evaluated for tumor gene therapy. Here we investigated whether a dMAb was appropriate for tumor gene therapy. We constructed the expression plasmids coding for the heavy or light chain of a parental murine antibody of Herceptin with the antibody genes codon- and RNA-optimized and fused to the Kozak-IgE leader sequence in pVax1. Transfection of the plasmids into human muscle RD cells resulted in functional expression of the antibody, and this exhibited the same in vitro antiproliferative activity as Herceptin. A single intramuscular injection and in vivo EP of the plasmids (100 μg per head) resulted in high and sustained antibody expression in the sera of normal mice and in effective inhibition of tumor growth in nude mice bearing HER2-positive human breast carcinoma BT474 xenografts. The antitumor efficacy of the anti-HER2 dMAb was similar to that of four doses of intravenously injected 10 mg kg−1 Herceptin. The results demonstrate that the dMAb is effective in the treatment of HER2-positive breast cancer, suggesting that this dMAb may be applicable for tumor gene therapy.

Abstract 1242: Combination of p21-activated kinase 1 (PAK1) inhibitor FRAX1036 and Aurora-A inhibitor alisertib is effective in hormone receptor-positive breast cancer

Abstract Estrogen receptor alpha (ERα) signaling plays a critical role in the development and progression of hormone receptor-positive (HR+) breast cancer. One mechanism of tumor progression of HR+ breast cancer is phosphorylation of ERα by non-receptor protein kinases, leading to ligand-independent activation of ERα. Mitotic kinases Aurora A (AURKA) and p21-activated kinase 1 (PAK1) are frequently overexpressed in breast cancer and can phosphorylate ERα on serine 305, increasing the ability of ERα to induce transcription of target genes implicated in tumorigenesis. In addition, PAK1 phosphorylates and activates AURKA and its functional partners, suggesting possible synergistic activity in tumors overexpressing both kinases. We tested the hypothesis that combined inhibition of AURKA and PAK1 would synergistically suppress tumor growth of HR+ breast cancer cells in vitro and in vivo. Treatment of T47D HR+ breast cancer cell line with the PAK1 inhibitor FRAX1036, combined with AURKA inhibitor alisertib, improved cell killing in comparison to single agents. FRAX1036 and alisertib were then evaluated in an orthotopic xenograft model using BT474 HR+ breast cancer cell line. Ten million BT474 cells were implanted into the mammary fat pads of 6-week old NSG mice simultaneously with implantation of estrogen pellets to facilitate growth of xenografts. Mice received 21 days of treatment with FRAX1036 20mg/kg daily and alisertib 10 or 15 mg/kg BID, alone or in combination. We observed exceptional inhibition of tumor growth in the combination treatment groups. Combination of FRAX1036 20mg/kg daily with alisertib 15mg/kg BID was the most effective, with partial or near-complete responses that continued through 21 days of treatment. This combination resulted in significantly better inhibition of tumor growth than alisertib 15mg/kg BID alone or FRAX1036 20mg/kg daily alone (p = 0.003 and p = 1.29e-6, respectively). Similarly, combination treatment of FRAX1036 20mg/kg daily with alisertib 10mg/kg BID was significantly better then alisertib alone or FRAX1036 alone in the corresponding doses (p = 0.002 and p = 3.52e-7, respectively). Analysis of H&amp;E stained sections and Ki-67 proliferation index of BT474 xenograft tumors indicated a highly significant decrease in viable tumor cells and replacement by stroma after combination treatment compared to treatment with single agents or with control. Caspase-3 IHC analysis showed significantly greater proportion of apoptotic cells per tumor area in the group of mice treated with FRAX1036 and Alisertib 15mg/kg BID combination comparing to control (p = 0.008). We conclude that combining FRAX1036 and alisertib is a novel and promising approach in HR+ breast cancer. Citation Format: Elena Shagisultanova, Vladislav Korobeynikov, Anna Nikonova, Jonathan Chernoff, Virginia Borges, Erica Golemis. Combination of p21-activated kinase 1 (PAK1) inhibitor FRAX1036 and Aurora-A inhibitor alisertib is effective in hormone receptor-positive breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1242.

Abstract 2961: deBouganin conjugated to trastuzumab overcomes multiple mechanisms of T-DM1 drug resistance

Abstract DeBouganin is a T-cell epitope-depleted variant of the type I Ribosome Inactivating Protein (RIP) plant toxin Bouganin. DeBouganin binds and deadenylates the sarcin/ricin loop of the 28S subunit of the ribosomal RNA leading to protein synthesis inhibition and apoptosis. To demonstrate the potential advantages of deBouganin over current small molecule payloads, deBouganin was randomly chemically conjugated to trastuzumab with a DAR of approximately 2 and compared to T-DM1 both in vitro and in vivo. The trastuzumab-deBouganin conjugate (T-deB) demonstrated a tighter IC50 range of killing and an overall greater potency in vitro against most cells lines with high levels of Her2 expression as compared to T-DM1. In addition, unlike T-DM1, T-deB was unaffected by inhibitors of multidrug resistance (MDR) and overexpression of Bcl-2 family members. Moreover, T-deB potency was unchanged by Her2-Her3 dimerization in the presence of heregulin. Contrary to T-DM1 which showed only minimal cytotoxicity, T-deB was highly potent in vitro against tumor cells with cancer stem cell (CSC) properties by preventing the formation of tumorspheres. Furthermore, in a BT-474 xenograft study, T-deB was more efficacious than T-DM1 resulting in increased survival of the T-deB treated mice. Overall, the results demonstrate the potency and efficacy of deBouganin and emphasize the importance of using payloads with different MOAs. The data suggest that deBouganin used alone or in combination with other payloads could be a highly effective cancer therapeutic that would provide prolonged clinical benefit. Citation Format: Rachelle L. Dillon, Shilpa Chooniedass, Arjune Premsukh, Gregory P. Adams, Joycelyn Entwistle, Glen C. MacDonald, Jeannick Cizeau. deBouganin conjugated to trastuzumab overcomes multiple mechanisms of T-DM1 drug resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2961.

X66, a novel N-terminal heat shock protein 90 inhibitor, exerts antitumor effects without induction of heat shock response

Heat shock protein 90 (HSP90) is essential for cancer cells to assist the function of various oncoproteins, and it has been recognized as a promising target in cancer therapy. Although the HSP90 inhibitors in clinical trials have shown encouraging clinical efficacy, these agents induce heat shock response (HSR), which undermines their therapeutic effects. In this report, we detailed the pharmacologic properties of 4-(2-((1H-indol-3-yl)methylene)hydrazinyl)-N-(4-bromophenyl)-6-(3,5- dimethyl-1H -pyrazol-1-yl)-1,3,5-triazin-2-amine (X66), a novel and potent HSP90 inhibitor. X66 binds to the N-terminal domain in a different manner from the classic HSP90 inhibitors. Cellular study showed that X66 depleted HSP90 client proteins, resulted in cell cycle arrest and apoptosis, and inhibition of proliferation in cancer cell lines. X66 did not activate heat shock factor-1 (HSF-1) or stimulate transcription of HSPs. Moreover, the combination of X66 with HSP90 and proteasome inhibitors yielded synergistic cytotoxicity which was involved in X66-mediated abrogation of HSR through inhibition of HSF-1 activity. The intraperitoneal administration of X66 alone depleted client protein and inhibited tumor growth, and led to enhanced activity when combined with celastrol as compared to either agent alone in BT-474 xenograft model. Collectively, the HSP90 inhibitory action and the potent antitumor activity, with the anti-HSR action, promise X66 a novel HSP90-targeted agent, which merits further research and development.

Bioorthogonal Fluorophore Linked DFO-Technology Enabling Facile Chelator Quantification and Multimodal Imaging of Antibodies.

Herein we describe the development and application of a bioorthogonal fluorogenic chelate linker that can be used for facile creation of labeled imaging agents. The chelate linker is based on the trans-cyclooctene(TCO)-tetrazine(Tz) chemistry platform and incorporates deferoxamine (DFO) as a (89)Zr PET tracer and a BODIPY fluorophore for multimodal imaging. The rapid (<3 min) ligation between mAb-TCO and Tz-BODIPY-DFO chelator is monitored using fluorescence and allows for determination of labeling completion. Utilizing BODIPY as the linker between mAb and DFO facilitates in chelator quantification using spectrophotometry, allowing for an alternative to traditional methods (mass and isotope dilution assay). Radiolabeling with (89)Zr to form (89)Zr-DFO-BODIPY-trastuzumab was found to be quantitative after incubation at room temperature for 1 h (1.5 mCi/mg specific activity). The cell binding assay using HER2+ (BT474) and HER2- (BT20) cell lines showed significant binding to (89)Zr-DFO-BODIPY-trastuzumab (6.45 ± 1.87% in BT474 versus 1.47 ± 0.39% in BT20). In vivo PET imaging of mice bearing BT20 or BT474 xenografts with (89)Zr-DFO-BODIPY-trastuzumab showed high tumor conspicuity, and biodistribution confirmed excellent, specific probe uptake of 237.3 ± 14.5% ID/g in BT474 xenografts compared to low, nonspecific probe uptake in BT20 xenografts (16.4 ± 5.6% ID/g) 96 h p.i. . Ex vivo fluorescence (465ex/520em) of selected tissues confirmed superb target localization and persistence of the fluorescence of (89)Zr-DFO-BODIPY-trastuzumab. The described platform is universally adaptable for simple antibody labeling.

Abstract LB-B11: Assessment of HER3 status during lapatinib treatment in HER3-positive breast cancer using 89Zr-anti-HER3 mAb

Abstract Treatment of human epidermal growth factor receptor 2 (HER2)-driven breast cancer with the HER-targeting tyrosine kinase inhibitor lapatinib can lead to a rapid compensatory increase in expression, signaling activity and relocalization of HER3 to the plasma membrane, which may attenuate the response to lapatinib. This might imply a potential role for a more dynamic assessment of HER3 tumor status using molecular imaging techniques, such as positron emission tomography (PET), instead of immunohistochemical HER3 staining on tumor biopsies. Here, we explored the feasibility of a dynamic assessment of HER3 status during lapatinib treatment in human breast cancer xenografts using zirconium-89 labeled anti-human HER3 monoclonal antibody (mAb) as a potential tracer for animal PET imaging. The anti-human HER3 mouse mAb MAB3481 was used for all experiments. The effect of lapatinib treatment on HER3 expression and HER3 mAb internalization in human breast cancer cell lines SKBR3 and BT474 was determined using flow cytometry. Biodistribution was performed using 89Zr-anti-HER3 mAb in mice bearing BT474 or SKBR3 tumors. Mice received daily vehicle or a lapatinib dose of 25, 50 or 100 mg/kg orally. A tracer dose of 89Zr-anti-HER3 mAb combined with the aspecific tracer 111In-IgG was injected 3 days after treatment. Ex vivo organ distribution assessment of 89Zr-anti-HER3 mAb was performed 6 days after tracer injection. Ex vivo tumor analysis using western blotting, ELISA and immunohistochemistry were performed to measure HER3 levels. In vitro, lapatinib treatment resulted in a ∼2-fold increase in membranous HER3 expression and HER3 internalization in SKBR3 and BT474 tumor cells. 89Zr-anti-HER3 mAb tumor uptake was significantly higher compared to 111In-IgG uptake in BT474 (P &amp;lt; 0.01), demonstrating HER3 specific tumor uptake. SKBR3 xenografts did not show HER3 specific uptake, which was likely caused by the poor viability of the tumors. HER3 upregulation was observed in BT474 xenografts after lapatinib treatment for 9 days. The enhanced HER3 expression was related to a 76 ± 9% increase in 89Zr-anti-HER3 mAb tumor uptake at 25 mg/kg and 92 ± 27% at 50 mg/kg lapatinib. Lapatinib at the highest concentration (100 mg/kg) strongly inhibited tumor growth and did not increase tumor uptake of the tracer. In conclusion, HER3-specific uptake of 89Zr-anti-HER3 mAb was shown in breast cancer xenografts. HER3 upregulation after lapatinib treatment was related to an enhanced 89Zr-anti-HER3 mAb uptake in these xenografts. These promising data warrant future dynamic assessment of HER3 status with 89Zr-anti-HER3 mAb PET imaging. Citation Format: Arjan Kol, Martin Pool, Steven de Jong, Elisabeth GE de Vries, Marjolijn N. Lub-de Hooge, Anton GT Terwisscha van Scheltinga. Assessment of HER3 status during lapatinib treatment in HER3-positive breast cancer using 89Zr-anti-HER3 mAb. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B11.

Abstract A147: MI130004, a new ADC with a payload of marine origin shows outstanding activity against HER2-expressing tumors

Abstract PM050489 is a tubulin-binding agent originally isolated from the marine sponge Lithoplocamia lithistoides. This compound binds with very high affinity to β-tubulin at a new site, disrupting the microtubule network and impairing its function during divison, which leads to mitotic aberrations. MI130004 (a novel antibody-drug conjugate formed by PM050489, a non-hydrolysable linker and trastuzumab) was tested for its in vitro and in vivo activity against selected tumor cell lines with different levels of HER2 expression. In vitro MI130004 showed very high potency and good selectivity for tumor cells that overexpressed HER2, namely HCC-1954 (IC50, 0.036 μg/mL), SK-BR-3 (IC50, 0.017 μg/mL) and BT-474 (IC50, 0.156 μg/mL). At the cellular level, MI130004 impaired tubulin polymerization, causing disorganization and disintegration of the microtubule network which ultimately led to mitotic failure. Expressing HER2 cells of breast (BT-474 and JIMT-1), gastric (Gastric-008 and N87) and ovarian (SK-OV-3 and A2780cis) as well as negative HER2 breast (MDA-MB-231) and gastric (Hs748t) were subcutaneously implanted into immunosuppressed (SCID or athymic) mice. Tumor (ca. 115 mm3) bearing animals (N = 10/group) were randomly allocated to receive the ADC treatments (at different doses) or the appropriate control (placebo included). Treatments were administered weekly for 5 consecutive weeks. Tumor growth (as median/group) was calculated 2-3 times per week. Twenty-four hours after the first dose, representative tumors were dissected free and processed for HER2 expression (erb2) and chromatin organization (Hoescht 33258). The treatment with MI130004 induced a long lasting antitumor effect with statistically significant increases (P&amp;lt;0.05) in median survival time compared to placebo. The highest dose of MI130004 induced complete tumor remissions in mice bearing BT-474, JIMT-1, Gastric-008, N87, SK-OV-3 and A2780cis xenografts lasting up to 120, 60, 165, 63, 237 and 231 days, respectively. Also 24-h post-dosing, MI130004 induced a dose-dependent disappearance of HER2-expressing cells as well as mitotic aberrations, this last being consistent with the mechanism of action of PM050489. These results demonstrate that PM050489, a marine derived compound, is a novel and remarkable payload for the design of new ADCs with potential therapeutic anti-cancer properties. Citation Format: Pablo Aviles, Maria Jose Guillen, Juan Manuel Dominguez, Carlos M. Galmarini, Carmen Cuevas. MI130004, a new ADC with a payload of marine origin shows outstanding activity against HER2-expressing tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A147.

Abstract 1813: Tumor and normal tissue localization of trastuzumab-modified with a metal chelating polymer for auger electron radioimmunotherapy in HER2-positive xenografts

Abstract Purpose We have been investigating a potential radioimmunotherapeutic (RIT) agent using the anti-HER2 monoclonal antibody trastuzumab to address the issue of primary and acquired resistances in HER2-positive Breast cancer (BC). Trastuzumab was conjugated to a metal chelating polymer (MCP) with multiple DTPA units for labeling with the Auger electron emitter Indium-111 (111In) [trastuzumab-MCP-111In]. We aim to increase the amount of radioactivity delivered and enhance the therapeutic effect towards trastuzumab sensitive and resistant tumors. High specific activity (SA) (5.5 MBq/μg) trastuzumab-Hy-MCP-111In was more cytotoxic at killing HER2-positive BC cells sensitive and resistant than low SA (0.37MBq/μg) radioimmunoconjugates (RICs) in vitro. The current study aimed to investigate the tumor localization properties of this new RIT in mice with HER-2 positive BC xenografts. Methods Hydrazide MCP with polyglutamide backbone, 20 or 30 DTPA units, and without or with PEG chains [Hy-(PEG)-MCP-111In] were site-specifically linked to periodate-oxidized carbohydrates on the Fc domain of trastuzumab. Tumor and normal tissue distribution were assessed in CD-1 athymic mice bearing BT-474 (1×106 HER2 receptors/cell) or trastuzumab resistant TrR1 (5×105 HER2 receptors/cell) xenografts. Mice were injected intravenously with trastuzumab-Hy-(PEG)-MCP-111In (10μg; 3-4 MBq/μg), or trastuzumab-Hy-(PEG)-MCP-111In (1μg; 3-5 MBq/μg) without or with non-labeled non specific anti-CD20 rituximab (9μg). Controls consisted of mice injected with MCPs only and trastuzumab-NH-Bn-DTPA-111In. Results Trastuzumab-Hy-(PEG)-MCP-111In exhibited a HER2-mediated uptake in BT-474 tumors. Both RICs (10μg) had a higher tumor uptake (0.8±0.4 and 1.3±0.3%ID/g) than the non-targeted Hy-(PEG)-MCP-111In (0.2±0.04 and 0.3±0.1%ID/g) (p = 0.0104; p&amp;lt;0.0001). Tumor uptake was also HER2-density dependent with about a 3-fold decrease (0.4±0.1%ID/g) in TrR1-bearing mice for trastuzumab-Hy-PEG-MCP-111In. Kidneys were found to be the major normal organ accumulation in both formulations. Nevertheless, pegylation of the MCP caused a 4-fold decrease in kidneys uptake (6.9±2.5 to 1.6±0.1% ID/g; p = 0.0018). At low mass (1μg) and high SA, addition of non-labeled rituximab improved the tumor uptake of trastumab-Hy-MCP-111In by 2 fold (0.3±0.1 to 0.6±0.1%ID/g), but no change in tumor uptake was seen for trastuzumab-Hy-PEG-MCP-111In. Nonetheless, BT-474 tumor uptake of trastuzumab-Hy-(PEG)-MCP-111In was lower than the one obtained with trastuzumab-NH-Bn-DTPA-111In (13.3±3.4%ID/g). Conclusion Our results suggest that trastuzumab-Hy-(PEG)-MCP-111In could target HER2-positive xenografts sensitive and resistant to trastuzumab. Further modifications on the MCP are needed to improve the tumor uptake for Auger electron radioimmunotherapy. Citation Format: Ghislaine Ngo Ndjock Mbong, Yijie Lu, Conrad Chan, Zhongli Cai, Mitchell A. Winnik, Raymond Reilly. Tumor and normal tissue localization of trastuzumab-modified with a metal chelating polymer for auger electron radioimmunotherapy in HER2-positive xenografts. [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 1813. doi:10.1158/1538-7445.AM2015-1813

Abstract 2665: High dose intermittent scheduling of AZD8835, a novel potent and selective inhibitor of PI3Kα and PI3Kδ, identifies potential treatment strategies for PIK3CA-dependent cancers

Abstract The PIK3CA gene, encoding the p110 catalytic unit of PI3Kα, is one of the most frequently mutated oncogenes described in human cancer. Hence PI3Kα is a target subject to intensive efforts in identifying inhibitors and evaluating their therapeutic potential. To date most studies with PI3K inhibitors have used a continuous (daily) dosing schedule and although clinical responses have been reported the overall activity observed has been moderate. This may in part be due to suboptimal pathway inhibition which is capped by normal tissue toxicities such as rash, diarrhoea and hyperglycaemia. Furthermore, additional dose reduction may be required when such agents are used in combination with other therapies. Therefore in our efforts to optimise inhibition of PI3K pathway signalling, we have explored high dose intermittent scheduling as an alternative to continuous dosing. Here we describe pre-clinical studies that exemplify such concepts, centred around use of AZD8835, a PI3K inhibitor currently in Phase 1 clinical evaluation. AZD8835 is a novel and potent inhibitor of PI3Kα and PI3Kδ, with selectivity vs. PI3Kβ, PI3Kγ(IC50s of 6nM, 6nM, 431nM and 90nM respectively in enzyme assays) and other kinases. AZD8835 preferentially displays activity in tumour models with a mutant PIK3CA background, such as ER+ve breast cancer models. Such models were used in our investigations, in both cell culture and in mouse xenograft contexts. We demonstrate that a high dose intermittent schedule of single agent AZD8835 achieves greater pathway inhibition yielding significant anti-tumour responses. In the sensitive BT474 xenograft model, a dose of 100mg/kg AZD8835 BID on days 1 and 4 in a weekly schedule delivered -36% tumour regression, accompanied by a strongly elevated rapid onset apoptosis signal with 4-16% cells staining positively for cleaved-caspase3. We also evaluated AZD8835 in combination with other targeted therapeutic agents, in MCF7, BT474 and T47D breast models, observing increased sensitivity relative to single agent AZD8835; firstly with agents that target other nodes in the PI3K pathway; secondly with agents targeting parallel but interconnected driver pathways in breast disease (ER, CDK4/6). Overall the data indicate that high dose intermittent scheduling can deliver strong anti-tumour efficacy in a range of combination settings and provides a promising alternative to continuous dosing. Such schedules merit clinical evaluation. Citation Format: Kevin Hudson, Urs Hancox, Cath Trigwell, Phillippa Dudley, Lyndsey Hanson, Robert McEwen, Alys Jones, Marie Cumberbatch, Urszula Polanska, Rebecca Ellston, Oona Delpuech, Pablo Morentin Gutierrez, Lara Ward, Francisco Cruzalegui, Stephen Green. High dose intermittent scheduling of AZD8835, a novel potent and selective inhibitor of PI3Kα and PI3Kδ, identifies potential treatment strategies for PIK3CA-dependent cancers. [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 2665. doi:10.1158/1538-7445.AM2015-2665

A study of the relationship of metabolic MR parameters to estrogen dependence in breast cancer xenografts.

(1)H MRS, (31)P MRS and diffusion-weighted MRI (DW-MRI) were applied to study the metabolic changes associated with estrogen dependence in estrogen receptor (ER)-positive BT-474 and triple-negative HCC1806 breast cancer xenografts supplemented with or without 17β-estradiol (E2) at a dose of 0.18 or 0.72 mg/pellet. Furthermore, the effect of estrogen withdrawal on the metabolism of BT-474 and HCC1806 breast cancer xenografts was studied on day 0, day 2 and day 10. Increasing the dose of E2 resulted in a rapid growth and increases in the lactate level and phosphomonoester/β-nucleoside triphosphate (PME/βNTP), phosphocreatine/inorganic phosphate (PCr/Pi) and βNTP/Pi ratios in BT-474 breast cancer xenografts; however, no significant changes were found in HCC1806 breast cancer xenografts. Estrogen withdrawal resulted in a marked decrease in lactate level and PME/βNTP ratio and an observed increase in βNTP/Pi, PCr/Pi and apparent diffusion coefficient (ADC) values of BT-474 breast cancer xenografts on day 10. These data suggest that the lactate level and PME/βNTP, PCr/Pi and βNTP/Pi ratios of ER-positive tumors are closely related to ER dependence.

Mango polyphenolics suppressed tumor growth in breast cancer xenografts in mice: role of the PI3K/AKT pathway and associated microRNAs

The cytotoxic and anti-inflammatory properties of mango polyphenolics including gallic acid and gallotannins have been demonstrated in numerous types of cancers. We hypothesized that the phosphoinositide 3-kinase (PI3K)/AKT pathway and the expression of related miRNAs are involved in the chemotherapeutic activities of mango polyphenolics in a mouse xenograft model for breast cancer. The objectives of this research were to determine the tumor-cytotoxic activities of mango polyphenolics and the underlying molecular mechanisms involving posttranscriptional targets in BT474 breast cancer cells and xenografts in mice. In vitro findings showed cytotoxic effects of mango polyphenolics in BT474 breast cancer cells within a concentration range of 2.5 to 20 mg/L gallic acid equivalents. Mango polyphenolics suppressed the expression of PI3K, AKT, hypoxia inducible factor–1α, and vascular endothelial growth factor (VEGF) mRNA, and pAKT, AKT, pPI3K (p85), VEGF and nuclear factor–kappa B protein levels. The involvement of miR-126 was verified by using antagomiR for miR-126, where mango reversed the effect of the antagomiR of miR-126. In vivo, the intake of mango polyphenolics decreased the tumor volume by 73% in BT474 xenograft-bearing mice compared with the control group. In addition, mango reduced the expression of nuclear factor–kappa B (p65), pAKT, pPI3K, mammalian target of rapamycin, hypoxia inducible factor–1α, and VEGF protein in athymic nude mice. A screening for miRNA expression changes confirmed that mango polyphenolics modulated the expression of cancer-associated miRNAs including miR-126 in the xenografted tumors. In summary, mango polyphenolics have a chemotherapeutic potential against breast cancer that at least in part is mediated through the PI3K/AKT pathway and miR-126.

Abstract P3-05-13: Overexpression of insulin receptor substrate 4 can mediate acquired resistance to lapatinib-containing regimens in HER2+ breast cancer cells

Abstract Background: The HER2 pathway can be inhibited by potent targeting agents such as lapatinib (L), trastuzumab (T), or their combination (LT), but acquired and de novo resistance still occur. Resistance to these drugs remains a major hurdle in the management of HER2+ breast cancer. Consequently, elucidation of mechanisms of acquired therapeutic resistance to HER2-directed therapies is of critical importance. Methods: To obtain clues to the mechanisms for resistance we developed a panel of HER2+ breast cancer cell lines resistant to L, T, or LT. Parental cells and resistant derivatives of the HER2+ BT474 cell line were characterized by RNA-seq. Genes that were overexpressed in resistant compared to parental cells were confirmed by RT-PCR, Western blotting, and immunohistochemistry (IHC). Cell growth and cell signaling were assessed in parental and resistant cell lines after down-regulation (by siRNA) or overexpression (via an inducible cDNA) of IRS4 in the presence or absence of treatment. The effect of IRS4 overexpression on L resistance was assessed in a BT474 xenograft model. The proteins that interact with IRS4 were identified by co-immunoprecipitation with IRS4 followed by separation of the associated proteins by SDS-PAGE and microsequencing by mass spectrometry. Results: RNA-seq analysis revealed that IRS4 was the most up-regulated gene in BT474 L or LT resistant derivatives in which HER2 signaling is effectively inhibited, but not T alone, where HER2 signaling is reactivated. Western blotting and IHC validated this result and identified membrane localization of IRS4. Knockdown of IRS4 in L- or LT-resistant cells reversed resistance and restored growth inhibition. IRS4 knockdown also inhibited downstream signaling, with a reduction in pAKT but not in pMAPK. Induction of the cell cycle regulator p27 and down-regulation of survivin were observed after IRS4 knockdown. Overexpression of IRS4 cDNA in parental BT474 and SKBR3 cells led to resistance to L/LT, increased pAkt, and decreased the apoptotic marker cleaved PARP in the presence of L or the LT combination. The BT474 xenograft model showed that IRS4 overexpression in the absence of treatment had no effect on tumor growth but it significantly reduced the inhibitory effect of lapatinib (p=0.002). A group of proteins that interact with IRS4 in BT474 L-resistant cells were identified by mass spectrometry. The roles of these proteins in IRS4-mediated resistance to lapatinb-containing regimens are under investigation. Conclusion: IRS4 overexpression is a critical factor in causing resistance to lapatinib-containing regimens in BT474 cells. Investigation of IRS4 and its signaling partners in HER2+ human tumors resistant to lapatinib will be important to determine if this mechanism is also operative in patients. Citation Format: Lanfang Qin, Maria B Hahn, Xiaoyong Fu, Martin J Shea, Mario Giuliano, Sarmistha Nanda, Xiaowei Xu, Huizhong Hu, Sung Yun Jung, Laura M Heiser, Nicholas Wang, Joe W Gray, Susan G Hilsenbeck, Chad Creighton, Chad A Shaw, Gary C Chamness, Dean P Edwards, Sabrina Herrera, Carolina Gutierrez, C Kent Osborne, Rachel Schiff. Overexpression of insulin receptor substrate 4 can mediate acquired resistance to lapatinib-containing regimens in HER2+ breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-13.

Design, Synthesis, and Evaluation of Linker-Duocarmycin Payloads: Toward Selection of HER2-Targeting Antibody-Drug Conjugate SYD985.

Antibody-drug conjugates (ADCs) that are currently on the market or in clinical trials are predominantly based on two drug classes: auristatins and maytansinoids. Both are tubulin binders and block the cell in its progression through mitosis. We set out to develop a new class of linker-drugs based on duocarmycins, potent DNA-alkylating agents that are composed of a DNA-alkylating and a DNA-binding moiety and that bind into the minor groove of DNA. Linker-drugs were evaluated as ADCs by conjugation to the anti-HER2 antibody trastuzumab via reduced interchain disulfides. Duocarmycin 3b, bearing an imidazo[1,2-a]pyridine-based DNA-binding unit, was selected as the drug moiety, notably because of its rapid degradation in plasma. The drug was incorporated into the linker-drugs in its inactive prodrug form, seco-duocarmycin 3a. Linker attachment to the hydroxyl group in the DNA-alkylating moiety was favored over linking to the DNA-binding moiety, as the first approach gave more consistent results for in vitro cytotoxicity and generated ADCs with excellent human plasma stability. Linker-drug 2 was eventually selected based on the properties of the corresponding trastuzumab conjugate, SYD983, which had an average drug-to-antibody ratio (DAR) of about 2. SYD983 showed subnanomolar potencies against multiple human cancer cell lines, was highly efficacious in a BT-474 xenograft model, and had a long half-life in cynomolgus monkeys, in line with high stability in monkey and human plasma. Studies comparing ADCs with a different average DAR showed that a higher average DAR leads to increased efficacy but also to somewhat less favorable physicochemical and toxicological properties. Fractionation of SYD983 with hydrophobic interaction chromatography resulted in SYD985, consisting of about 95% DAR2 and DAR4 species in an approximate 2:1 ratio and having an average DAR of about 2.8. SYD985 combines several favorable properties from the unfractionated ADCs with an improved homogeneity. It was selected for further development and recently entered clinical Phase I evaluation.