Alan Wilhelm

A New Class of Antibody-Drug Conjugates with Potent DNA Alkylating Activity.

The promise of tumor-selective delivery of cytotoxic agents in the form of antibody–drug conjugates (ADC) has now been realized, evidenced by the approval of two ADCs, both of which incorporate highly cytotoxic tubulin-interacting agents, for cancer therapy. An ongoing challenge remains in identifying potent agents with alternative mechanisms of cell killing that can provide ADCs with high therapeutic indices and favorable tolerability. Here, we describe the development of a new class of potent DNA alkylating agents that meets these objectives. Through chemical design, we changed the mechanism of action of our novel DNA cross-linking agent to a monofunctional DNA alkylator. This modification, coupled with linker optimization, generated ADCs that were well tolerated in mice and demonstrated robust antitumor activity in multiple tumor models at doses 1.5% to 3.5% of maximally tolerated levels. These properties underscore the considerable potential of these purpose-created, unique DNA-interacting conjugates for broadening the clinical application of ADC technology. Mol Cancer Ther; 15(8); 1870–8. ©2016 AACR.

Abstract 2644: Antibody-drug conjugates (ADCs) with a novel DNA-alkylating agent, DGN462, are highly potent in vitro and in vivo against human cancer models

Most ADCs in the clinic utilize a tubulin-binding small molecule as the cytotoxic payload. Although many cancers are sensitive to tubulin agents, some are more responsive to DNA-interacting agents. We have developed a new class of highly potent cytotoxic agents, IGNs, for use in ADCs that consists of indolino-benzodiazepine dimers with a novel DNA-alkylating mechanism of action. We found that IGNs containing a di-imine moiety act via DNA-crosslinking and -alkylating, whereas IGNs with a mono-imine induce DNA alkylation only. Although ADCs with either form of IGN were highly active, ADCs containing di-imine IGNs caused delayed toxicity in mice, leading to mortality. The mono-imine IGN, DGN462, was selected as our lead IGN as, when conjugated to an antibody via a cleavable linkage, the resultant ADC had favorable tolerability in mice (maximally tolerated dose of 40 mg/kg; 700 µg/kg DGN462 dose) without delayed toxicity. The ability of DGN462 to alkylate DNA was demonstrated by measurement of DNA adduct formation with double-stranded DNA and in cells. DGN462 formed covalent DNA adducts through alkylation of the C2- amino group of guanine. Consistent with the activity of other DNA alkylating agents, DGN462-treated cells progressed slowly through S-phase and were arrested in G2/M phase of the cell cycle in a dose dependent manner. ADCs consisting of ∼3 molecules of DGN462 per antibody, attached via a cleavable linker, had desirable biochemical characteristics, including high monomer content (> 97%), low free drug ( A CD33-targeting DGN462 ADC was highly active against AML xenografts, with a minimal efficacious dose (MED) of 0.6 mg/kg (conjugate dose), while a non-targeting control was inactive. A DGN462 ADC targeting epidermal growth factor receptor was highly active and antigen-specific against a head and neck squamous cell carcinoma model, with a 1.6 mg/kg MED. The intact conjugate half-life was approximately 90 hours in mice, and bioactivity of conjugate was maintained at 72 hours post dosing, indicating that intact conjugate remains active in vivo. DGN462 ADCs have an acceptable safety profile and potent antigen-specific DNA-alkylating mechanism of action with the potential for activity in tumors with low sensitivity to tubulin agents, heterogeneous antigen expression and/or PgP-mediated drug resistance. Citation Format: Kathleen Whiteman, Charlene Audette, Andre Dandeneau, Megan Ellis, Nathan Fishkin, Lauren Harvey, Holly Johnson, Yelena Kovtun, Erin Maloney, Michael Miller, Alan Wilhelm, Ravi Chari. Antibody-drug conjugates (ADCs) with a novel DNA-alkylating agent, DGN462, are highly potent in vitro and in vivo against human cancer models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2644. doi:10.1158/1538-7445.AM2014-2644

A CD123-targeting antibody-drug conjugate, IMGN632, designed to eradicate AML while sparing normal bone marrow cells

The outlook for patients with refractory/relapsed acute myeloid leukemia (AML) remains poor, with conventional chemotherapeutic treatments often associated with unacceptable toxicities, including severe infections due to profound myelosuppression. Thus there exists an urgent need for more effective agents to treat AML that confer high therapeutic indices and favorable tolerability profiles. Because of its high expression on leukemic blast and stem cells compared with normal hematopoietic stem cells and progenitors, CD123 has emerged as a rational candidate for molecularly targeted therapeutic approaches in this disease. Here we describe the development and preclinical characterization of a CD123-targeting antibody-drug conjugate (ADC), IMGN632, that comprises a novel humanized anti-CD123 antibody G4723A linked to a recently reported DNA mono-alkylating payload of the indolinobenzodiazepine pseudodimer (IGN) class of cytotoxic compounds. The activity of IMGN632 was compared with X-ADC, the ADC utilizing the G4723A antibody linked to a DNA crosslinking IGN payload. With low picomolar potency, both ADCs reduced viability in AML cell lines and patient-derived samples in culture, irrespective of their multidrug resistance or disease status. However, X-ADC exposure was >40-fold more cytotoxic to the normal myeloid progenitors than IMGN632. Of particular note, IMGN632 demonstrated potent activity in all AML samples at concentrations well below levels that impacted normal bone marrow progenitors, suggesting the potential for efficacy in AML patients in the absence of or with limited myelosuppression. Furthermore, IMGN632 demonstrated robust antitumor efficacy in multiple AML xenograft models. Overall, these findings identify IMGN632 as a promising candidate for evaluation as a novel therapy in AML.

Abstract 647: SeriMabs: N-terminal serine modification enables modular, site-specific payload incorporation into antibody-drug conjugates (ADCs)

Site-specific incorporation of cell-killing agents into cancer-targeting antibodies is an active area of innovation in the field of ADCs. We have developed a highly modular site-specific conjugation platform employing N-terminal serine engineered antibodies (SeriMabs), using ligation chemistry orthogonal to lysine and cysteine modification which typically employs maleimide containing linkers. Humanized IgG1 antibodies were engineered with N-terminal serine residues on either the light or heavy chain, with the precise position optimized for quantitative conversion to the corresponding glyoxyl aldehyde in the presence of sodium periodate. The aminooxy functional group of the heterobifunctional linker N-[(aminooxy)acetyl]-3-[(3-nitro-2-pyridinyl)dithio]-L-alanine was condensed with the aldehyde group on the antibody using 4-amino phenethyl alcohol catalyst, yielding a stable oxime bond under conditions that fully maintain antibody integrity. The dithiopyridine groups were then reacted with thiol-containing maytansinoid or IGN cell-killing agents, yielding disulfide-linked SeriMab ADCs with exactly 2 payload molecules per antibody, as determined by MS analysis, in > 90% yield and with a monomer content of > 98%. Conjugation of payload was found to be selective for the N-terminal serine based on MS/MS analysis. The site-specific ADCs were found to bind to their target antigens with affinity similar to the corresponding lysine-conjugated ADCs, and FcRn binding (pH 5.8) of the SeriMab ADCs were comparable to lysine-conjugated controls. DGN462, our proprietary DNA-acting IGN payload used in our preclinical candidate IMGN779, was conjugated to SeriMabs against two targets, and in both cases, high, antigen-specific in vitro potency was noted. The oxime linkage used in the serine site-specific conjugates was found to be stable at pH 5.5 and pH 7.4 in buffer, as little detectable payload was released over 4 days at 37°C. SeriMab antiFRα-DGN462 (2 DGN462 molecules per Ab) demonstrated potent, dose-dependent antitumor activity against NCI-H2110 non-small cell lung cancer xenografts in SCID mice. SeriMab antiFRα-DGN462 was highly active at a single injection dose of 50 and 25 μg/kg (DGN462 dose) with a minimal efficacious dose (MED) of 10 μg/kg. We have developed a modular method for preparing site-specific, disulfide-linked ADCs with good biochemical characteristics, through an optimized N-terminal serine engineered IgG, using the same thiol containing effector payloads employed in our lysine-conjugation platform. Citation Format: Luke Harris, Daniel Tavares, Lingyun Rui, Erin Maloney, Alan Wilhelm, Juliet Costoplus, Katie Archer, Megan Bogalhas, Lauren Harvey, Rui Wu, Xuan Chen, Xiangyang Xu, Sonia Connaughton, Lintao Wang, Kathleen Whiteman, Olga Ab, Erica Hong, Wayne Widdison, Manami Shizuka, Michael Miller, Jan Pinkas, Thomas Keating, Ravi Chari, Nathan Fishkin. SeriMabs: N-terminal serine modification enables modular, site-specific payload incorporation into antibody-drug conjugates (ADCs). [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 647. doi:10.1158/1538-7445.AM2015-647

A DNA-Interacting Payload Designed to Eliminate Cross-Linking Improves the Therapeutic Index of Antibody–Drug Conjugates (ADCs)

Tumor-selective delivery of cytotoxic agents in the form of antibody–drug conjugates (ADCs) is now a clinically validated approach for cancer treatment. In an attempt to improve the clinical success rate of ADCs, emphasis has been recently placed on the use of DNA–cross-linking pyrrolobenzodiazepine compounds as the payload. Despite promising early clinical results with this class of ADCs, doses achievable have been low due to systemic toxicity. Here, we describe the development of a new class of potent DNA-interacting agents wherein changing the mechanism of action from a cross-linker to a DNA alkylator improves the tolerability of the ADC. ADCs containing the DNA alkylator displayed similar in vitro potency, but improved bystander killing and in vivo efficacy, compared with those of the cross-linker. Thus, the improved in vivo tolerability and antitumor activity achieved in rodent models with ADCs of the novel DNA alkylator could provide an efficacious, yet safer option for cancer treatment. Mol Cancer Ther; 17(3); 650–60. ©2018 AACR.

Abstract 75: Comparison of site-specific and lysine-linked indolino-benzodiazepine antibody-drug conjugates (ADCs)

ADCs are a promising modality for cancer therapy enabled by chemical conjugation of potent cytotoxic compounds to monoclonal antibodies. While many ADCs in clinical evaluation employ heterogeneous conjugation chemistries where the payload is linked through lysine or endogenous cysteine residues, there has recently been considerable interest in site-specific conjugation. ADCs prepared using site-specific methods are believed to have a wider therapeutic index compared to heterogeneous ADCs. We have previously shown that site-specific ADCs incorporating the maytansinoid-based tubulin inhibitor DM1 were less efficacious in vivo when compared to analogous lysine-linked conjugates (Yoder et al AACR 2015 Abstract #545). More recently, we presented results from the evaluation of 2.5-3.0 drug-to-antibody ratio (DAR) heterogeneous lysine-linked and 2 DAR site-specific ADCs using antibodies with engineered cysteines at position 442 in the heavy chains (known as CYSMABTM). These ADCs used the peptide-linked indolino-benzodiazepine DNA alkylator DGN549 (also known as IGN-P1) as the effector. Unlike the DM1 case, site-specific DGN549 ADCs were at least as active in vivo when compared to lysine-linked ADCs (Yoder et al AACR 2016 Abstract #2960). We have made further pharmacological comparisons between CYSMAB and lysine-linked DGN549 ADCs at matched payload doses using two different antibodies targeting distinct cell surface receptors. In the case of mAb1, CYSMAB and lysine-linked ADCs were comparably active. For mAb2, the CYSMAB ADC was more active than the lysine-linked ADC in some models and similarly active in others. The mAb1 CYSMAB ADC exhibited a significantly higher maximum tolerated dose (MTD) compared to the lysine-linked ADC. In contrast, the MTDs of the mAb2 ADCs were similar. However, the mAb2 CYSMAB conjugate was better tolerated in terms of median lethal dose. The mechanism for the improved tolerability of the mAb1 CYSMAB conjugate is not apparent. In an effort to understand whether it is a consequence of conjugation chemistry or DAR, we compared the tolerability of the mAb1 CYSMAB conjugate to that of a ~4 DAR site-specific analog and found that both factors contribute. To determine if our observations can be rationalized in terms of in vivo disposition, we compared the pharmacokinetics of mAb1 CYSMAB and lysine-linked ADCs at matched antibody doses. Intriguingly, the CYSMAB ADC showed slightly greater exposure. These results, along with our previous work on site-specific DM1 ADCs, indicate that in some cases site-specific conjugation can lead to improved efficacy and tolerability. However, generalizations cannot be made across different combinations of antibody, linker, and payload. We conclude that the advantages and disadvantages of site-specific conjugation should be carefully considered for every candidate ADC. Citation Format: Chen Bai, Nicholas C. Yoder, Alan Wilhelm, Sharlene Adams, Kathleen Whiteman, Jenny Lee, Katie O9Callaghan, Erin Maloney, Manami Shizuka, Yelena Kovtun, Thomas A. Keating. Comparison of site-specific and lysine-linked indolino-benzodiazepine antibody-drug conjugates (ADCs) [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 75. doi:10.1158/1538-7445.AM2017-75

Abstract 2965:In vitroandin vivoactivity of a site-specific SeriMab antibody-drug conjugate (ADC) using an indolino-benzodiazepine DNA-alkylating agent

Previously we have described the characterization of a proprietary class of indolino-benzodiazepine dimers, IGNs, with high potency against many cancer lines. Antibody-drug conjugates (ADCs) made with mono-imine containing IGNs were designed to only alkylate DNA and not cause DNA crosslinking. ADCs with the IGN linked via lysine residues of the antibody were shown to be highly potent and antigen specific. Here we apply our SeriMab site-specific technology platform, which employs N-terminal conjugatable aldehydes derived from oxidation of a serine residue, to link an IGN molecule to the antibody using a peptide linker. This ADC (SeriMab-IGN-P1) has exactly 2 IGN molecules per antibody conjugated to the N-termini of the heavy chains, as determined by MS analysis. Serimab-IGN-P1 was found to maintain binding to the target antigen with comparable affinity to the unconjugated antibody by FACS analysis (Kd = 300 pM). The conjugate was highly potent (IC50 = 4 pM) against a cancer cell line with high target expression (3 × 106 antibodies bound per cell) as well as a cell line with much lower target expression (2 × 104 antibodies bound per cell, IC50 = 22 pM). SeriMab-IGN-P1 also demonstrated strong bystander killing activity against proximal target-negative cancer cells, likely due to a cell permeable metabolite identified in cells expressing the target antigen. The unique oxime linkage in SeriMab-IGN-P1 was found to be stable in circulation in mice for 3 days, as determined by affinity capture LC-MS; the conjugate species with 2 IGNs per antibody remained predominant over this period with an estimated t1/2 of >10 days for payload release. In vivo, SeriMab-IGN-P1 was found to be effective against human lung cancer xenografts (NCI-H2110), with anti-tumor activity observed at single doses as low as 2 μg/kg (payload dose), and complete responses observed at 5 μg/kg. This conjugate was also well tolerated in CD-1 mice at doses significantly higher than the minimum effective dose that produced tumor regression. These data are highly encouraging for new ADC therapies using the SeriMab site-specific conjugation platform with potent DNA-acting payloads. Citation Format: Dilrukshi Vitharana, Alan Wilhelm, Luke Harris, Katie Archer, Manami Shizuka, Erin Maloney, Olga Ab, Rassol Laleau, Xiuxia Sun, Jan Pinkas, Michael Miller, Ravi Chari, Thomas Keating, Nathan Fishkin. In vitro and in vivo activity of a site-specific SeriMab antibody-drug conjugate (ADC) using an indolino-benzodiazepine DNA-alkylating agent. [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 2965.

Abstract 2960: Potentin vivoactivity of site-specific indolino-benzodiazepine antibody-drug conjugates (ADCs) generated via engineered cysteine conjugation

ADCs are widely studied for cancer therapy, with numerous agents in preclinical and clinical development embodying a wide array of targets, linker chemistries, and cytotoxic effector classes. A fourth element of ADC design that has received much attention recently is the site of conjugation of the cytotoxic molecule to the antibody. Historically, lysine- or interchain cysteine-directed conjugation has been used, but site-specific chemistries have become increasingly popular. Our previous evaluation of site-specific and lysine-linked ADCs utilizing a tubulin-acting maytansinoid effector molecule found the lysine-linked version was more active in vivo (Yoder et al., AACR 2015 #645). Here we present a comparison of engineered cysteine site-specific and lysine-linked ADCs utilizing the previously described indolino-benzodiazepine (henceforth referred to as IGN) effector IGN-P1 (Miller et al., AACR 2015 #652) which is designed to undergo proteolytic cleavage upon cell uptake to release a potently cytotoxic DNA alkylator. We show that HC-S442C mutants of human IgG1 can be conjugated via maleimide chemistry to IGN-P1 to give stable, potent, and homogeneous ADCs with drug to antibody ratio (DAR) of 2. The in vitro potency of engineered-cysteine IGN-P1 ADCs is largely dependent on the DAR of the ADC, although some difference is observed between HC-S442C and other cysteine mutants used for conjugation. Pharmacokinetic study of C442 maleimide conjugates suggests that the chemical linkage between effector and antibody is stable upon administration in mice. Further, and in contrast to our previous observations utilizing maytansinoid ADCs, the site-specific and Lys-linked IGN-P1 ADCs showed comparable efficacy in vivo on a molar drug basis. This effect was observed across two different antibodies targeting two different cell surface antigens. These results suggest that, in certain cases, site-specific conjugation chemistry can offer comparable activity to heterogeneous conjugation at well-tolerated doses. Citation Format: Nicholas C. Yoder, Chen Bai, Alan Wilhelm, Erin K. Maloney, Olga Ab, Emily E. Reid, Manami Shizuka, Daniel Tavares, Rassol Laleau, Xiuxia Sun, Megan E. Bogalhas, Lintao Wang, Jan Pinkas, Michael L. Miller, Ravi Chari, Thomas A. Keating. Potent in vivo activity of site-specific indolino-benzodiazepine antibody-drug conjugates (ADCs) generated via engineered cysteine conjugation. [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 2960.

Abstract 5325: A c-Kit targeting antibody-drug conjugate is efficiently metabolized and activated inside cancer cell lines and xenograft tumors

We have developed an antibody-drug conjugate (ADC) targeting the c-Kit receptor tyrosine kinase, which is selectively expressed on the cell surface of various tumors including gastrointestinal tumors (GIST), small cell lung cancer (SCLC) and acute myelogenous leukemia (AML). The ADC consists of a humanized c-Kit-binding monoclonal antibody (c-Kit-Ab) covalently attached to a maytansinoid tubulin inhibitor, DM1, via a non-cleavable linker, SMCC. Like other ADCs, the c-Kit ADC is a pro-drug that is activated upon metabolism inside cells. Its target-mediated internalization and lysosomal degradation generates active maytansinoid metabolites that bind and inhibit microtubule dynamics. To understand the activation of the ADC, we studied the kinetics and efficiency of its metabolism in cancer cells and xenograft tumor models. Using radiolabeled ADC and c-Kit-Ab, we followed the formation of metabolites in SCLC and AML cells and in SCLC tumors, as well as in engineered cell lines that express various mutant forms of c-Kit. We found that the ADC and c-Kit-Ab are efficiently metabolized in c-Kit-positive cancer cells irrespective of the mutational status or the activation state of c-Kit. The degradation half-life of c-Kit-Ab is 3-fold lower than that observed for T-DM1 and SGN35, indicating a faster rate of activation. Moreover, cell surface repopulation with c-Kit is highly efficient after ADC-mediated internalization, allowing multiple rounds of ADC recruitment and activation. Interestingly, cell surface repopulation of c-Kit is more rapid for cells with a c-Kit mutation than those with wild-type c-Kit, leading to generation of greater levels of active metabolites in cells with mutant c-Kit. This may lead to better targeting of the c-Kit-mutated cancer cells over normal cells with wild-type c-Kit, which may improve the safety of the c-Kit ADC. Consistent with the in vitro findings, the ADC was found to undergo efficient metabolism in tumors in vivo. While tumor localization of the ADC over a non-targeting conjugate is similar to that observed with T-DM1 and SAR3419, its tumor-specific formation of active metabolites is 3-fold greater. Thus, the c-Kit-targeting ADC is particularly efficient at achieving high levels of active metabolites inside c-Kit-positive tumor cells, consistent with its robust efficacy in mouse xenograft models. Citation Format: Erica Hong, Qifeng Qiu, Rui Wu, Alan Wilhelm, Kathleen Whiteman, Jan Pinkas, Hans Erickson, Tinya Abrams, Siew Schleyer. A c-Kit targeting antibody-drug conjugate is efficiently metabolized and activated inside cancer cell lines and xenograft tumors. [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 5325. doi:10.1158/1538-7445.AM2015-5325

Abstract 652: Antibody-drug conjugates (ADCs) of indolino-benzodiazepine DNA-alkylating agents

There are over thirty ADCs in the clinic today, reflecting the interest in ADCs for the treatment of cancer. Most of these ADCs utilize a tubulin-interacting small molecule as their cytotoxic payload. In order to continue to extend the utility of ADCs to more types of cancers, there is a strong interest in developing cytotoxic payloads with other mechanisms of action. Previously we have described the development of a class of indolino-benzodiazepine dimers (IGNs) with high potency and specificity. ADCs made with a mono-imine containing IGN, which only alkylate DNA, were found to display improved tolerability, along with the absence of delayed toxicity, compared to their DNA-cross-linking di-imine counterparts. Additional structure-activity relationship studies with mono-imine containing IGNs have resulted in two promising new leads, IGN-D1 and IGN-P1, which are conjugated to an antibody via either a disulfide or peptide linker, respectively. ADCs of IGN-D1 and IGN-P1 were prepared for evaluation using a folate receptor α (FRα)-binding antibody, anti-FRα, and EGFR-binding antibody, anti-EGFR. Anti-FRα-IGN-D1 and anti-FRα-IGN-P1 were found to be highly potent against the FRα-expressing KB cell line (IC50 values of 8 pM and 4 pM, respectively) and also demonstrated strong activity against nearby target-negative cancer cells. The addition of excess unconjugated antibody blocked the cytotoxic effect of these conjugates, thus demonstrating that their activity is antigen specific. In addition, both anti-FRα-IGN conjugates were found to be highly potent (IC50 values of 100 pM and 30 pM, respectively) against the breast ductal carcinoma cell line, T47D, despite only a moderate level of antigen expression on the target cells. Similarly, anti-EGFR-IGN-D1 and anti-EGFR-IGN-P1 were found to have IC50 values in the picomolar range against a panel of EGFR-expressing cell lines. In vivo, anti-FRα-IGN-D1 and anti-FRα-IGN-P1 were found to be effective against NCI-H2110 non-small cell lung cancer xenografts, with anti-tumor activity observed at single doses as low as 3 μg/kg (payload dose, equivalent to 0.15 mg/kg Ab dose). These data support evaluation of new ADC therapies with these two new IGNs for solid tumor indications as well as tumors with low antigen expression. Citation Format: Michael L. Miller, Manami Shizuka, Nathan Fishkin, Emily Reid, Katie Archer, Erin Maloney, Chen Bai, Olga Ab, Nick C. Yoder, Rui Wu, Erica Hong, Megan Bogalhas, Alan Wilhelm, Kathleen Whiteman, Ravi Chari. Antibody-drug conjugates (ADCs) of indolino-benzodiazepine DNA-alkylating agents. [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 652. doi:10.1158/1538-7445.AM2015-652