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ABBV-321 (serclutamab talirine), a next-generation EGFR-targeted antibody–drug conjugate (ADC) incorporates a potent pyrrolobenzodiazepine (PBD) dimer toxin conjugated to the EGFR-targeting ABT-806 affinity-matured AM1 antibody. ABBV-321 follows the development of related EGFR-targeted ADCs including depatuxizumab mafodotin (depatux-m, ABT-414), ABT-806 conjugated to monomethyl auristatin F (MMAF), and ABBV-221 (losatuxizumab vedotin), AM1 antibody conjugated to monomethyl auristatin E (MMAE). The distinct tumor selectivity of ABBV-321 differentiates it from many previous highly active antibody PBD conjugates that lack a therapeutic window. Potency of the PBD dimer, combined with increased binding of AM1 to EGFR-positive tumor cells, opens the possibility to target a wide array of tumors beyond those with high levels of EGFR overexpression or amplification, including those insensitive to auristatin-based ADCs. ABBV-321 exhibits potent antitumor activity in cellular and in vivo studies including xenograft cell line and patient-derived xenograft glioblastoma, colorectal, lung, head and neck, and malignant mesothelioma tumor models that are less sensitive to depatux-m or ABBV-221. Combination studies with ABBV-321 and depatux-m suggest a promising treatment option permitting suboptimal, and potentially better tolerated, doses of both ADCs while providing improved potency. Collectively, these data suggest that ABBV-321 may offer an extended breadth of efficacy relative to other EGFR ADCs while extending utility to multiple EGFR-expressing tumor indications. Despite its highly potent PBD dimer payload, the tumor selectivity of ABBV-321, coupled with its pharmacology, toxicology, and pharmacokinetic profiles, support continuation of ongoing phase I clinical trials in patients with advanced EGFR-expressing malignancies.

BCL2, BCL2L1 and MCL1 mRNA in CD138 primary MM patient samples and sensitivity to veneteclax ex vivo

Legends for Tables S1 to S3 and Figures S1 to S5

Caspase-dependent degradation of MCL-1 in NCI-H929 HMCL following treatment with bortezomib treatment

BCL-2 and BCL-XL IHC correlates with qPCR and immunobot analysis of HMCLs

BCL-2 family proteins dictate survival of human multiple myeloma cells, making them attractive drug targets. Indeed, multiple myeloma cells are sensitive to antagonists that selectively target prosurvival proteins such as BCL-2/BCL-XL (ABT-737 and ABT-263/navitoclax) or BCL-2 only (ABT-199/GDC-0199/venetoclax). Resistance to these three drugs is mediated by expression of MCL-1. However, given the selectivity profile of venetoclax it is unclear whether coexpression of BCL-XL also affects antitumor responses to venetoclax in multiple myeloma. In multiple myeloma cell lines (n = 21), BCL-2 is expressed but sensitivity to venetoclax correlated with high BCL-2 and low BCL-XL or MCL-1 expression. Multiple myeloma cells that coexpress BCL-2 and BCL-XL were resistant to venetoclax but sensitive to a BCL-XL–selective inhibitor (A-1155463). Multiple myeloma xenograft models that coexpressed BCL-XL or MCL-1 with BCL-2 were also resistant to venetoclax. Resistance to venetoclax was mitigated by cotreatment with bortezomib in xenografts that coexpressed BCL-2 and MCL-1 due to upregulation of NOXA, a proapoptotic factor that neutralizes MCL-1. In contrast, xenografts that expressed BCL-XL, MCL-1, and BCL-2 were more sensitive to the combination of bortezomib with a BCL-XL selective inhibitor (A-1331852) but not with venetoclax cotreatment when compared with monotherapies. IHC of multiple myeloma patient bone marrow biopsies and aspirates (n = 95) revealed high levels of BCL-2 and BCL-XL in 62% and 43% of evaluable samples, respectively, while 34% were characterized as BCL-2High/BCL-XLLow. In addition to MCL-1, our data suggest that BCL-XL may also be a potential resistance factor to venetoclax monotherapy and in combination with bortezomib. Mol Cancer Ther; 15(5); 1132–44. ©2016 AACR.

In vivo Efficacy of ABBV-321 tumor targeting.

Correlation of mRNA expression of BCL2 family members and sensitivity to venetoclax or navitoclax in HMCLs

Conceptual workflow for establishing BCL-2 family IHC cut-offs for analysis of MM patient tumor samples

qPCR primer sequences for Apopanel of BCL2 family members

Purpose: Despite the importance of the MET oncogene in many malignancies, clinical strategies targeting c-Met have benefitted only small subsets of patients with tumors driven by signaling through the c-Met pathway, thereby necessitating selection of patients with MET amplification and/or c-Met activation most likely to respond. An ADC targeting c-Met could overcome these limitations with potential as a broad-acting therapeutic.Experimental Design: ADC ABBV-399 was generated with the c-Met–targeting antibody, ABT-700. Antitumor activity was evaluated in cancer cells with overexpressed c-Met or amplified MET and in xenografts including patient-derived xenograft (PDX) models and those refractory to other c-Met inhibitors. The correlation between c-Met expression and sensitivity to ABBV-399 in tumor and normal cell lines was assessed to evaluate the risk of on-target toxicity.Results: A threshold level of c-Met expressed by sensitive tumor but not normal cells is required for significant ABBV-399–mediated killing of tumor cells. Activity extends to c-Met or amplified MET cell line and PDX models where significant tumor growth inhibition and regressions are observed. ABBV-399 inhibits growth of xenograft tumors refractory to other c-Met inhibitors and provides significant therapeutic benefit in combination with standard-of-care chemotherapy.Conclusions: ABBV-399 represents a novel therapeutic strategy to deliver a potent cytotoxin to c-Met–overexpressing tumor cells enabling cell killing regardless of reliance on MET signaling. ABBV-399 has progressed to a phase I study where it has been well tolerated and has produced objective responses in c-Met–expressing non–small cell lung cancer (NSCLC) patients. Clin Cancer Res; 23(4); 992–1000. ©2016 AACR.

ABBV-399, a c-Met Antibody Drug Conjugate that Targets Both MET Amplified and c-Met Overexpressing Tumors, Irrespective of MET Pathway Dependence. Figure S1 shows ABBV-399 is internalized upon binding to tumor cells. Figure S2 shows the down-regulation of phospho- and total c-Met and downstream signaling molecules upon treatment with ABBV-399

Supplementary Fig. S1 from ABT-263 and rapamycin act cooperatively to kill lymphoma cells in vitro and in vivo

ABT-263 is a potent, orally bioavailable inhibitor of the antiapoptotic Bcl-2 family members Bcl-2, Bcl-xL, and Bcl-w, which is currently in phase I clinical trials. Previous work has shown that this compound has low nanomolar cell-killing activity in a variety of lymphoma and leukemia cell lines, many of which overexpress Bcl-2 through a variety of mechanisms. Rapamycin is a macrolide antibiotic that inhibits the mammalian target of rapamycin complex, leading to cell cycle arrest and inhibition of protein translation. Rapamycin (and its analogues) has shown activity in a variety of tumor cell lines primarily through induction of cell cycle arrest. Activity has also been shown clinically in mantle cell lymphoma and advanced renal cell carcinoma. Here, we show that treatment of the follicular lymphoma lines DoHH-2 and SuDHL-4 with 100 nmol/L rapamycin induces substantial G0-G1 arrest. Addition of as little as 39 nmol/L ABT-263 to the rapamycin regimen induced a 3-fold increase in sub-G0 cells. Combination of these agents also led to a significant increase in Annexin V staining over ABT-263 alone. In xenograft models of these tumors, rapamycin induced a largely cytostatic response in the DoHH-2 and SuDHL-4 models. Coadministration with ABT-263 induced significant tumor regression, with DoHH-2 and SuDHL-4 tumors showing 100% overall response rates. Apoptosis in these tumors was significantly enhanced by combination therapy as measured by staining with an antibody specific for cleaved caspase-3. These data suggest that combination of ABT-263 and rapamycin or its analogues represents a promising therapeutic strategy for the treatment of lymphoma. [Mol Cancer Ther 2008;7(10):3265–74]

Supplementary Table 1 from A Small-Molecule Inhibitor of Bcl-XL Potentiates the Activity of Cytotoxic Drugs In vitro and In vivo

Inhibition of the prosurvival members of the Bcl-2 family of proteins represents an attractive strategy for the treatment of cancer. We have previously reported the activity of ABT-737, a potent inhibitor of Bcl-2, Bcl-XL, and Bcl-w, which exhibits monotherapy efficacy in xenograft models of small-cell lung cancer and lymphoma and potentiates the activity of numerous cytotoxic agents. Here we describe the biological activity of A-385358, a small molecule with relative selectivity for binding to Bcl-XL versus Bcl-2 (Ki's of 0.80 and 67 nmol/L for Bcl-XL and Bcl-2, respectively). This compound efficiently enters cells and co-localizes with the mitochondrial membrane. Although A-385358 shows relatively modest single-agent cytotoxic activity against most tumor cell lines, it has an EC50 of L for survival. In addition, A-385358 enhances the in vitro cytotoxic activity of numerous chemotherapeutic agents (paclitaxel, etoposide, cisplatin, and doxorubicin) in several tumor cell lines. In A549 non–small-cell lung cancer cells, A-385358 potentiates the activity of paclitaxel by as much as 25-fold. Importantly, A-385358 also potentiated the activity of paclitaxel in vivo. Significant inhibition of tumor growth was observed when A-385358 was added to maximally tolerated or half maximally tolerated doses of paclitaxel in the A549 xenograft model. In tumors, the combination therapy also resulted in a significant increase in mitotic arrest followed by apoptosis relative to paclitaxel monotherapy. (Cancer Res 2006; 66(17): 8731-9)

ABBV-321 (serclutamab talirine), a next-generation EGFR-targeted antibody–drug conjugate (ADC) incorporates a potent pyrrolobenzodiazepine (PBD) dimer toxin conjugated to the EGFR-targeting ABT-806 affinity-matured AM1 antibody. ABBV-321 follows the development of related EGFR-targeted ADCs including depatuxizumab mafodotin (depatux-m, ABT-414), ABT-806 conjugated to monomethyl auristatin F (MMAF), and ABBV-221 (losatuxizumab vedotin), AM1 antibody conjugated to monomethyl auristatin E (MMAE). The distinct tumor selectivity of ABBV-321 differentiates it from many previous highly active antibody PBD conjugates that lack a therapeutic window. Potency of the PBD dimer, combined with increased binding of AM1 to EGFR-positive tumor cells, opens the possibility to target a wide array of tumors beyond those with high levels of EGFR overexpression or amplification, including those insensitive to auristatin-based ADCs. ABBV-321 exhibits potent antitumor activity in cellular and in vivo studies including xenograft cell line and patient-derived xenograft glioblastoma, colorectal, lung, head and neck, and malignant mesothelioma tumor models that are less sensitive to depatux-m or ABBV-221. Combination studies with ABBV-321 and depatux-m suggest a promising treatment option permitting suboptimal, and potentially better tolerated, doses of both ADCs while providing improved potency. Collectively, these data suggest that ABBV-321 may offer an extended breadth of efficacy relative to other EGFR ADCs while extending utility to multiple EGFR-expressing tumor indications. Despite its highly potent PBD dimer payload, the tumor selectivity of ABBV-321, coupled with its pharmacology, toxicology, and pharmacokinetic profiles, support continuation of ongoing phase I clinical trials in patients with advanced EGFR-expressing malignancies.

Purpose: Despite the importance of the MET oncogene in many malignancies, clinical strategies targeting c-Met have benefitted only small subsets of patients with tumors driven by signaling through the c-Met pathway, thereby necessitating selection of patients with MET amplification and/or c-Met activation most likely to respond. An ADC targeting c-Met could overcome these limitations with potential as a broad-acting therapeutic. Experimental Design: ADC ABBV-399 was generated with the c-Met–targeting antibody, ABT-700. Antitumor activity was evaluated in cancer cells with overexpressed c-Met or amplified MET and in xenografts including patient-derived xenograft (PDX) models and those refractory to other c-Met inhibitors. The correlation between c-Met expression and sensitivity to ABBV-399 in tumor and normal cell lines was assessed to evaluate the risk of on-target toxicity. Results: A threshold level of c-Met expressed by sensitive tumor but not normal cells is required for significant ABBV-399–mediated killing of tumor cells. Activity extends to c-Met or amplified MET cell line and PDX models where significant tumor growth inhibition and regressions are observed. ABBV-399 inhibits growth of xenograft tumors refractory to other c-Met inhibitors and provides significant therapeutic benefit in combination with standard-of-care chemotherapy. Conclusions: ABBV-399 represents a novel therapeutic strategy to deliver a potent cytotoxin to c-Met–overexpressing tumor cells enabling cell killing regardless of reliance on MET signaling. ABBV-399 has progressed to a phase I study where it has been well tolerated and has produced objective responses in c-Met–expressing non–small cell lung cancer (NSCLC) patients. Clin Cancer Res; 23(4); 992–1000. ©2016 AACR.

ABT-263 is a potent, orally bioavailable inhibitor of the antiapoptotic Bcl-2 family members Bcl-2, Bcl-xL, and Bcl-w, which is currently in phase I clinical trials. Previous work has shown that this compound has low nanomolar cell-killing activity in a variety of lymphoma and leukemia cell lines, many of which overexpress Bcl-2 through a variety of mechanisms. Rapamycin is a macrolide antibiotic that inhibits the mammalian target of rapamycin complex, leading to cell cycle arrest and inhibition of protein translation. Rapamycin (and its analogues) has shown activity in a variety of tumor cell lines primarily through induction of cell cycle arrest. Activity has also been shown clinically in mantle cell lymphoma and advanced renal cell carcinoma. Here, we show that treatment of the follicular lymphoma lines DoHH-2 and SuDHL-4 with 100 nmol/L rapamycin induces substantial G0-G1 arrest. Addition of as little as 39 nmol/L ABT-263 to the rapamycin regimen induced a 3-fold increase in sub-G0 cells. Combination of these agents also led to a significant increase in Annexin V staining over ABT-263 alone. In xenograft models of these tumors, rapamycin induced a largely cytostatic response in the DoHH-2 and SuDHL-4 models. Coadministration with ABT-263 induced significant tumor regression, with DoHH-2 and SuDHL-4 tumors showing 100% overall response rates. Apoptosis in these tumors was significantly enhanced by combination therapy as measured by staining with an antibody specific for cleaved caspase-3. These data suggest that combination of ABT-263 and rapamycin or its analogues represents a promising therapeutic strategy for the treatment of lymphoma. [Mol Cancer Ther 2008;7(10):3265–74]

Purpose: The purpose of this study was to characterize the activity of the Bcl-2 protein family inhibitor ABT-263 in a panel of small cell lung cancer (SCLC) xenograft models. Experimental Design: A panel of 11 SCLC xenograft models was established to evaluate the efficacy of ABT-263. Single agent activity was examined on a continuous dosing schedule in each of these models. The H146 model was used to further evaluate dose and schedule, comparison to standard cytotoxic agents, and induction of apoptosis. Results: ABT-263 exhibited a range of antitumor activity, leading to complete tumor regression in several models. Significant regressions of tumors as large as 1 cc were also observed. The efficacy of ABT-263 was also quite durable; in several cases, minimal tumor regrowth was noted several weeks after the cessation of treatment. Antitumor effects were equal or superior to that of several clinically approved cytotoxic agents. Regression of large established tumors was observed through several cycles of therapy and efficacy was retained in a Pgp-1 overexpressing line. Significant efficacy was observed on several dose and therapeutic schedules and was associated with significant induction of apoptosis. Conclusions: ABT-263 is a potent, orally bioavailable inhibitor of Bcl-2 family proteins that has recently entered clinical trials. The efficacy data reported here suggest that SCLC is a promising area of clinical investigation with this agent.

The Akt kinases are central nodes in signal transduction pathways that are important for cellular transformation and tumor progression. We report the development of a series of potent and selective indazole-pyridine based Akt inhibitors. These compounds, exemplified by A-443654 (Ki = 160 pmol/L versus Akt1), inhibit Akt-dependent signal transduction in cells and in vivo in a dose-responsive manner. In vivo, the Akt inhibitors slow the progression of tumors when used as monotherapy or in combination with paclitaxel or rapamycin. Tumor growth inhibition was observed during the dosing interval, and the tumors regrew when compound administration was ceased. The therapeutic window for these compounds is narrow. Efficacy is achieved at doses ∼2-fold lower than the maximally tolerated doses. Consistent with data from knockout animals, the Akt inhibitors induce an increase in insulin secretion. They also induce a reactive increase in Akt phosphorylation. Other toxicities observed, including malaise and weight loss, are consistent with abnormalities in glucose metabolism. These data show that direct Akt inhibition may be useful in cancer therapy, but significant metabolic toxicities are likely dose limiting.