Daniel Tavares

IMGN853, a Folate Receptor-α (FRα)–Targeting Antibody–Drug Conjugate, Exhibits Potent Targeted Antitumor Activity against FRα-Expressing Tumors

A majority of ovarian and non–small cell lung adenocarcinoma cancers overexpress folate receptor α (FRα). Here, we report the development of an anti-FRα antibody–drug conjugate (ADC), consisting of a FRα-binding antibody attached to a highly potent maytansinoid that induces cell-cycle arrest and cell death by targeting microtubules. From screening a large panel of anti-FRα monoclonal antibodies, we selected the humanized antibody M9346A as the best antibody for targeted delivery of a maytansinoid payload into FRα-positive cells. We compared M9346A conjugates with various linker/maytansinoid combinations, and found that a conjugate, now denoted as IMGN853, with the N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB) linker and N2′-deacetyl-N2′-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4) exhibited the most potent antitumor activity in several FRα-expressing xenograft tumor models. The level of expression of FRα on the surface of cells was a major determinant in the sensitivity of tumor cells to the cytotoxic effect of the conjugate. Efficacy studies of IMGN853 in xenografts of ovarian cancer and non–small cell lung cancer cell lines and of a patient tumor-derived xenograft model demonstrated that the ADC was highly active against tumors that expressed FRα at levels similar to those found on a large fraction of ovarian and non-small cell lung cancer patient tumors, as assessed by immunohistochemistry. IMGN853 displayed cytotoxic activity against FRα-negative cells situated near FRα-positive cells (bystander cytotoxic activity), indicating its ability to eradicate tumors with heterogeneous expression of FRα. Together, these findings support the clinical development of IMGN853 as a novel targeted therapy for patients with FRα-expressing tumors. Mol Cancer Ther; 14(7); 1605–13. ©2015 AACR.

Abstract 4565: IMGN529: A therapeutic maytansinoid conjugate of an anti-CD37 antibody with multiple mechanisms of action for B-cell lymphoma and leukemia

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL CD37 is a B-cell surface antigen that is an attractive target for antibody and antibody-drug conjugate mediated therapies due to its restricted expression profile. It is expressed on malignant B-cells in NHL and CLL, but on normal tissue its expression is highly restricted to B-cells present in blood and lymphoid tissues. A large panel of anti-CD37 murine monoclonal antibodies were generated and screened for their specific CD37 binding affinity, direct anti-proliferative activity and pro-apoptotic activity against lymphoma cell lines. Selected antibodies were humanized by variable domain resurfacing and one antibody, designated K7153A, demonstrated the best overall activity in terms of direct antibody activity as well as effector function. K7153A demonstrated much stronger pro-apoptotic activity against Ramos and Raji cells than either of two reference compounds, the anti-CD37 SMIP TRU-016 or the anti-CD20 antibody rituximab, and did not require cross-linking to achieve this effect. The antibody-maytansinoid conjugate, IMGN529, was produced by conjugation of K7153A with the potent maytansinoid, DM1, via the non-cleavable linker, SMCC. IMGN529 retains the high specific binding affinity of the K7153A antibody, with an EC50 of 0.5 nM. IMGN529 also demonstrated the same strong pro-apoptotic activity as the K7153A antibody against Ramos cells, with an EC50 of 0.1 nM. Antibody-dependent cell-mediated cytotoxicity (ADCC) assays, using purified human NK cells as effector cells, showed that K7153A and IMGN529 have similar potent ADCC activity against Ramos and Daudi cells with an EC50 of less than 10 pM. In addition, both K7153A and IMGN529 demonstrated comparable complement-dependent cytotoxicity (CDC) in the presence of human complement against Ramos cells. These results indicate that IMGN529 retains the intrinsic functions of the K7153A antibody. IMGN529 was highly cytotoxic in vitro against NHL cell lines such as Daudi, BJAB, Namalwa and SU-DHL-4 with a greater degree of cell killing and lower EC50 value (19 – 36 pM) than the K7153 antibody alone. In contrast, TRU-016 showed no effect on any of these cell lines and rituximab was only active against SU-DHL-4 cells. In vivo, IMGN529 showed markedly higher efficacy against established SU-DHL-4 and BJAB xenograft tumors than the K7153A antibody alone, with significant anti-tumor activity at single doses of 5 mg/kg or lower. Together, these results demonstrate that IMGN529 combines the strong pro-apoptotic activity, CDC and ADCC activity of its anti-CD37 antibody component with the potent cytotoxic activity provided by the targeted delivery of its maytansinoid payload. IMGN529 is a highly active antibody-drug conjugate with a unique combination of anti-tumor activities and is therefore a promising therapeutic candidate for the treatment of CD37-positive lymphomas and leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4565. doi:10.1158/1538-7445.AM2011-4565

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

Abstract 2967: In vitro and in vivo activity of site-specific antibody-drug conjugates (ADCs) with 2 and 4 maytansinoid molecules per antibody prepared through conjugation to SeriMabs (N-terminal serine engineered Abs)

Site-specific attachment of cell-killing agents to antibodies directed against tumor-associated antigens has continued to be an active area of innovation in the field of ADCs. Most reports focus on homogeneous ADCs that have a DAR (cytotoxic molecules per antibody ratio) of 2. Here we describe the preparation, biochemical characterization, and biological evaluation of ADCs made through conjugation of maytansinoids (DM1, DM4) to aldehydes derived from chemically oxidized N-terminal serines (SeriMab) engineered onto the antibody heavy chain (2 DAR) or both light and heavy chain simultaneously (4 DAR). ADCs prepared with a non-cleavable linker or a cleavable disulfide linker were homogeneous 2 or 4 DAR by MS analysis, and were produced in high yield with a monomer content of >98%. Despite conjugation at the N-termini of both the light and heavy chain variable regions, FACS analysis showed the 4 DAR SeriMab conjugates maintained binding to the target antigen. The ADCs showed antigen-specific potency in vitro on a panel of target-expressing cancer cell lines. In the disulfide cleavable linker series, the 2 DAR SeriMab conjugate was 2-5 fold less active than lysine-conjugated Ab-SPDB-DM4 (3.4 DAR), while the 4 DAR SeriMab conjugate was comparably active on an antibody basis. The SeriMab conjugates also displayed strong bystander killing. Surprisingly, in the non-cleavable linker series, the 2 DAR SeriMab conjugate was up to 17-fold more active (depending on cell line) than lysine-conjugated Ab-SMCC-DM1 (3.5 DAR), and the 4 DAR SeriMab conjugate was up to 100-fold more potent than the SMCC-DM1 conjugate on an antibody concentration basis. In a P-gp-positive multi-drug resistant cell line, the non-cleavable 4 DAR SeriMab-maytansinoid conjugate was highly active while the 2 DAR SeriMab ADCs and lysine-conjugated maytansinoid ADCs were >100-fold less potent. The unique oxime bond formed with the non-cleavable SeriMab-maytansinoid conjugate was found to be stable in circulation in mice for >3 days as assayed by affinity capture LC-MS. Polar carboxylic acid containing metabolites were identified which may lead to high cellular retention of maytansinoid species in cancer cells, yielding higher in vitro potency than lysine-linked ADCs in some cell lines. The in vivo anti-tumor activity of disulfide cleavable 2 and 4 DAR SeriMab-DM4 ADCs was evaluated in a clinically relevant cancer xenograft model. The 4 DAR conjugate was active at 60 μg/kg (maytansinoid payload dose) and was more active than the 2 DAR conjugate at this same payload dose. Using the SeriMab conjugation platform we show that in vitro and in vivo activity of site-specific ADCs can be dependent on amount of cytotoxic agent attached per antibody. Citation Format: Luke Harris, Leanne Lanieri, Jose Ponte, Erin Maloney, Laura Bartle, Olga Ab, Juliet Costoplus, Lingyun Rui, Jan Pinkas, Ravi Chari, Thomas Keating, Daniel Tavares, Nathan Fishkin. In vitro and in vivo activity of site-specific antibody-drug conjugates (ADCs) with 2 and 4 maytansinoid molecules per antibody prepared through conjugation to SeriMabs (N-terminal serine engineered Abs). [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 2967.

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 653: Antibody-drug conjugates: engineered N-terminal serine residues as a novel approach for site-specific conjugation

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Emerging data from the growing number of antibody-drug conjugates (ADCs) in clinical studies reflects the critical contributions from each component of the ADC. The tumor-targeting antibody and the cytotoxic effector molecule are recognized as key elements of an ADC, but increasingly the linkers and linkage mechanisms are being seen as critical for optimizing the design of an ADC based on the specific properties of the target antigen, tumor types, and clinical indications. Traditionally, antibodies have been conjugated to their cytotoxic payloads either at their lysine residues or through cysteine residues accessed through reduction of intrachain disulfide bonds. Recently a number of site-specific conjugation methods have been reported. Here we describe the construction of antibodies and antibody fragments with engineered N-terminal serine residues to facilitate a novel approach for site-specific antibody conjugation. Serine residues can be engineered into at least four different N-terminal positions on an antibody. Serines are found at the N-termini of some naturally occurring human antibodies, but in most cases they must be introduced by either replacing the native N-terminal residue or by including an additional serine residue at the N-terminus. These modifications can be made on the light chain, heavy chain, or both to provide as many as 4 potential linkage sites per antibody. Modification at the N-terminus situates the linked cytotoxic molecule well outside of the antibodys target binding sites in the variable region complementarity determining regions (CDRs). Molecular modeling demonstrates that this is the case whether the linkage is on either the light or heavy chain N-termini. ELISA and flow cytometry binding data demonstrated that changing the N-terminal residue to serine itself had no impact on the binding properties of the parent antibody. Likewise, ADCs utilizing the N-terminal serine for conjugation had similar binding properties compared with lysine or engineered cysteine conjugates of the same antibody. Finally, in vitro cytotoxicity assays demonstrated that N-terminal serine conjugates were at least as potent as conventional ADCs against antigen-expressing tumor cells. The utilization of a variable region framework residue means that the same N-terminal serines used for antibody conjugation can be applied to antibody fragments. Due to the size and residue usage limitations of antibody fragments, conjugation of hydrophobic, cytotoxic agents to these molecules can be challenging. We have built and tested N-terminal serine conjugates with Fabs, scFvs, and sdAbs, establishing the utility of this approach across a wide range of antibody fragment formats. In conclusion, conjugation of engineered antibody N-terminal serine residues represents a promising approach to site-specific ADCs that is worthy of further development. Citation Format: Daniel Tavares, Lingyun Rui, Olga Ab, Luke Harris, Erin Maloney, Thomas Keating, Thomas Chittenden, Nathan Fishkin. Antibody-drug conjugates: engineered N-terminal serine residues as a novel approach for site-specific conjugation. [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 653. doi:10.1158/1538-7445.AM2015-653

Abstract 645: Stability and efficacy comparison of site-specific and lysine-linked maytansinoid antibody-drug conjugates

As with any therapeutic molecule, antibody-drug conjugates (ADCs) exhibit structure-activity relationships, and medicinal chemistry efforts in this field strive to optimize structure to give the maximum therapeutic index. Recent interest in ADCs as cancer therapy has led to a number of different combinations of linker, payload, and conjugation chemistry.In particular, site-specific methods of payload conjugation have been suggested to generally improve therapeutic properties as compared with more established approaches directed toward lysines or endogenous cysteines.We have investigated the preparation, stability, and activity of anti-folate receptor alpha (FRα) ADCs carrying the microtubule inhibitor, DM1, and conjugated to engineered cysteine mutants utilizing different sites, and compared these ADCs with lysine-directed heterogeneous conjugates. In both embodiments, the DM1 is linked with a protease-cleavable linker. We show that highly homogeneous DM1 ADCs can be produced using engineered cysteine chemistry, enabling assessment of the effects of site-specific conjugation in cells and in animal models. We find that in vitro potency of both lysine-linked and engineered cysteine-linked ADCs against FRα-positive KB cells scales with the total DM1 delivered to cells. Buffer stability experiments in the presence of excess thiol suggest that most engineered cysteine conjugates are comparable in stability to the lysine-linked ADC. A notable exception shows about twice as much fractional DM1 loss upon 3 days of incubation as the other conjugates. Comparison of in vivo activity of two site-specific DM1 ADCs in a KB xenograft model shows measurable activity differences between different conjugation sites. However, a lysine-linked conjugate using almost identical linker chemistry shows approximately 2-fold superior activity to either site-specific construct on a molar DM1 basis. We conclude that, while site-specific conjugation of ADCs may provide a benefit in certain contexts, in other contexts, it may lead to decreased activity, such as in the anti-FRα/KB model examined here. We also observe that different conjugation sites may offer significant differences in activity. It is therefore advisable to evaluate each unique combination of payload, linker, drug:antibody ratio, conjugation site(s), and antibody to the maximum extent possible. Citation Format: Nicholas C. Yoder, Chen Bai, Daniel Tavares, Wayne C. Widdison, Olga Ab, Kathleen R. Whiteman, Alan Wilhelm, Erin K. Maloney, Hans K. Erickson, Thomas A. Keating. Stability and efficacy comparison of site-specific and lysine-linked maytansinoid antibody-drug conjugates. [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 645. doi:10.1158/1538-7445.AM2015-645

Abstract 4324: Maytansinoid conjugates of a novel type III anti-CD20 antibody with potent antitumor activity.

Previously identified anti-CD20 antibodies have been classified as either Type I antibodies with lipid raft and complement-dependent cytotoxicity (CDC) activity or Type II antibodies with strong pro-apoptotic activity, but not both. We describe here a novel anti-CD20 antibody with a unique combination of functional properties that defines a new Type III anti-CD20 antibody classification. In addition, when conjugated to the potent, microtubule-acting maytansinoids DM1 or DM4, it demonstrated enhanced cell-killing activity. Novel anti-CD20 antibodies were generated by immunizing mice with CD20-positive cells. One of these murine monoclonal anti-CD20 antibodies demonstrated very strong pro-apoptotic activity against Ramos and Raji lymphoma cells in the absence of cross-linking agents, similar to the activity of Type II antibodies. The humanized version of the antibody, termed D1302A, had a stronger pro-apoptotic effect against Ramos cells than other CD20 antibodies tested including rituximab, tositumumab (B1) and GA101-NG (non-glycoengineered GA101/afutuzumab). Strikingly, D1302A also induced CD20-redistribution to lipid rafts and showed CDC activity similar to the Type I antibody rituximab, while the Type II antibodies tositumumab and GA101-NG lacked these activities. D1302A, rituximab, and ofatumumab demonstrated comparable antibody-dependent cell mediated cytotoxicity (ADCC) activity on Ramos cells with human natural killer effector cells. In vivo, D1302A showed strong efficacy in an SU-DHL-4 xenograft model (at 1 or 10 mg/kg x3) and was more active than rituximab. D1302A was conjugated to DM1 via the non-cleavable SMCC thioether linker and to DM4 via the sulfo-SPDB disulfide linker. The resulting antibody-drug conjugates retained all the functional activities of the unconjugated antibody including binding affinity, pro-apoptotic, CDC and ADCC activities, and showed enhanced and specific in vitro cytotoxicity against Granta-519 and Farage lymphoma cells. In vivo, single-dose treatment with maytansinoid conjugates of D1302A elicited enhanced activity in Daudi and SU-DHL-4 xenograft models as compared to the antibody alone and showed comparable efficacy as standard of care treatment regimens such as rituximab or rituximab + chemotherapy (R-CHOP). D1302A is a novel Type III anti-CD20 antibody that has the functional properties of both Type I (potent CDC activity) and Type II (strong pro-apoptotic activity) antibodies, together with ADCC activity. Conjugation of D1302A with maytansinoids further increases anticancer activity in vitro and in vivo, combining the potent antibody-produced activities with a fourth cytotoxic mechanism distinct from that of other CD20 targeted agents. Therefore, D1302A-maytansinoid conjugates provide a unique combination of anti-tumor activities and are promising therapeutic candidates for the treatment of CD20+ lymphomas and leukemias. Citation Format: Jutta Deckert, Peter U. Park, Yong Yi, Sharon Chicklas, Min Li, Erin K. Maloney, Rui Wu, Leanne Lanieri, Jennifer A. Coccia, Joe Ponte, Lingyun Rui, Daniel J. Tavares, Jan Pinkas, Thomas Chittenden. Maytansinoid conjugates of a novel type III anti-CD20 antibody with potent antitumor activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4324. doi:10.1158/1538-7445.AM2013-4324