Katharine C. Lai

Antibody-Maytansinoid Conjugates Designed to Bypass Multidrug Resistance

Conjugation of cytotoxic compounds to antibodies that bind to cancer-specific antigens makes these drugs selective in killing cancer cells. However, many of the compounds used in such antibody-drug conjugates (ADC) are substrates for the multidrug transporter MDR1. To evade the MDR1-mediated resistance, we conjugated the highly cytotoxic maytansinoid DM1 to antibodies via the maleimidyl-based hydrophilic linker PEG(4)Mal. Following uptake into target cells, conjugates made with the PEG(4)Mal linker were processed to a cytotoxic metabolite that was retained by MDR1-expressing cells better than a metabolite of similar conjugates prepared with the nonpolar linker N-succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). In accord, PEG(4)Mal-linked conjugates were more potent in killing MDR1-expressing cells in culture. In addition, PEG(4)Mal-linked conjugates were markedly more effective in eradicating MDR1-expressing human xenograft tumors than SMCC-linked conjugates while being tolerated similarly, thus showing an improved therapeutic index. This study points the way to the development of ADCs that bypass multidrug resistance.

Disulfide-Linked Antibody−Maytansinoid Conjugates: Optimization of In Vivo Activity by Varying the Steric Hindrance at Carbon Atoms Adjacent to the Disulfide Linkage

In this report, we describe the synthesis of a panel of disulfide-linked huC242 (anti-CanAg) antibody maytansinoid conjugates (AMCs), which have varying levels of steric hindrance around the disulfide bond, in order to investigate the relationship between stability to reduction of the disulfide linker and antitumor activity of the conjugate in vivo. The conjugates were first tested for stability to reduction by dithiothreitol in vitro and for plasma stability in CD1 mice. It was found that the conjugates having the more sterically hindered disulfide linkages were more stable to reductive cleavage of the maytansinoid in both settings. When the panel of conjugates was tested for in vivo efficacy in two human colon cancer xenograft models in SCID mice, it was found that the conjugate with intermediate disulfide bond stability having two methyl groups on the maytansinoid side of the disulfide bond and no methyl groups on the linker side of the disulfide bond (huC242-SPDB-DM4) displayed the best efficacy. The ranking of in vivo efficacies of the conjugates was not predicted by their in vitro potencies, since all conjugates were highly active in vitro, including a huC242-SMCC-DM1 conjugate with a noncleavable linkage which showed only marginal activity in vivo. These data suggest that factors in addition to intrinsic conjugate potency and conjugate half-life in plasma influence the magnitude of antitumor activity observed for an AMC in vivo. We provide evidence that bystander killing of neighboring nontargeted tumor cells by diffusible cytotoxic metabolites produced from target cell processing of disulfide-linked antibody-maytansinoid conjugates may be one additional factor contributing to the activity of these conjugates in vivo.

A novel anti-CD37 antibody-drug conjugate with multiple anti-tumor mechanisms for the treatment of B-cell malignancies

CD37 has gathered renewed interest as a therapeutic target in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL); however, CD37-directed antibody-drug conjugates (ADCs) have not been explored. Here, we identified a novel anti-CD37 antibody, K7153A, with potent in vitro activity against B-cell lines through multiple mechanisms including apoptosis induction, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity. The antibody was conjugated to the maytansinoid, DM1, a potent antimicrotubule agent, via the thioether linker, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and the resulting ADC, IMGN529, retained the intrinsic antibody activities and showed enhanced cytotoxic activity from targeted payload delivery. In lymphoma cell lines, IMGN529 induced G2/M cell cycle arrest after internalization and lysosomal processing to lysine-N(ε)-SMCC-DM1 as the sole intracellular maytansinoid metabolite. IMGN529 was highly active against subcutaneous B-cell tumor xenografts in severe combined immunodeficient mice with comparable or better activity than rituximab, a combination of cyclophosphamide, vincristine, and prednisone, or bendamustine. In human blood cells, CD37 is expressed in B cells at similar levels as CD20, and IMGN529 resulted in potent and specific depletion of normal and CLL B cells. These results support evaluation of the CD37-targeted ADC, IMGN529, in clinical trials in patients with B-cell malignancies including NHL and CLL.

Evaluation of Targets for Maytansinoid ADC Therapy Using a Novel Radiochemical Assay.

PurposeMany antibody-drug conjugates (ADCs) become active only after antigen-mediated internalization and release of the cytotoxic agent via antibody degradation. Quantifying these processes can provide critical information on the suitability of a particular receptor target or antibody for ADC therapy by providing insight into the amount of cytotoxic agent released. We describe a simple and inexpensive radiolabel assay to monitor this process in cultured cancer cells.MethodsMonoclonal antibodies were trace-labeled at their lysine residues by treatment with the N-hydroxysuccinimide ester of [3H]propionic acid. Human cancer cell cultures were treated with the labeled antibody at concentrations sufficient to saturate the targeted antigen. After washing to remove unbound antibody, cells were incubated and analyzed for antigen expression, conjugate degradation and catabolite formation. Results were compared with data obtained from similar assays run with radiolabeled antibody-[3H]maytansinoid conjugates ([3H]AMCs). To exemplify the method, studies were conducted with a panel of [3H]propionamide-antibodies to evaluate processing efficiency in EGFR-expressing SCCHN cell lines, and in NHL cell lines expressing the B-cell targets CD19, CD20, CD22 and CD37.ResultsUse of the [3H]propionamide-antibody assay yielded cell-mediated processing results similar to those obtained with corresponding maytansinoid ADCs. Further exploration allowed comparison of expression levels, antigen-dependent degradation, and catabolite formation across a panel of EGFR-expressing SCCHN cell lines, and for multiple targets in various B-cell cancer indications.ConclusionsThe [3H]propionamide-antibody assay described here is a sensitive, facile method which enables rapid and robust assessment of relative antibody processing amounts for target, antibody, and cell line evaluation.

A new, triglycyl peptide linker for antibody-drug conjugates (ADCs) with improved targeted killing of cancer cells

A triglycyl peptide linker (CX) was designed for use in antibody–drug conjugates (ADC), aiming to provide efficient release and lysosomal efflux of cytotoxic catabolites within targeted cancer cells. ADCs comprising anti-epithelial cell adhesion molecule (anti-EpCAM) and anti-EGFR antibodies with maytansinoid payloads were prepared using CX or a noncleavable SMCC linker (CX and SMCC ADCs). The in vitro cytotoxic activities of CX and SMCC ADCs were similar for several cancer cell lines; however, the CX ADC was more active (5–100-fold lower IC50) than the SMCC ADC in other cell lines, including a multidrug-resistant line. Both CX and SMCC ADCs showed comparable MTDs and pharmacokinetics in CD-1 mice. In Calu-3 tumor xenografts, antitumor efficacy was observed with the anti-EpCAM CX ADC at a 5-fold lower dose than the corresponding SMCC ADC in vivo. Similarly, the anti-EGFR CX ADC showed improved antitumor activity over the respective SMCC conjugate in HSC-2 and H1975 tumor models; however, both exhibited similar activity against FaDu xenografts. Mechanistically, in contrast with the charged lysine-linked catabolite of SMCC ADC, a significant fraction of the carboxylic acid catabolite of CX ADC could be uncharged in the acidic lysosomes, and thus diffuse out readily into the cytosol. Upon release from tumor cells, CX catabolites are charged at extracellular pH and do not penetrate and kill neighboring cells, similar to the SMCC catabolite. Overall, these data suggest that CX represents a promising linker option for the development of ADCs with improved therapeutic properties. Mol Cancer Ther; 15(6); 1311–20. ©2016 AACR.

The Antitumor Activity of IMGN529, a CD37-Targeting Antibody-Drug Conjugate, Is Potentiated by Rituximab in Non-Hodgkin Lymphoma Models

Naratuximab emtansine (IMGN529) is an investigational antibody-drug conjugate consisting of a CD37-targeting antibody conjugated to the maytansine-derived microtuble disruptor, DM1. IMGN529 has shown promising preclinical and clinical activity in non-Hodgkin lymphoma, including diffuse large B-cell lymphoma (DLBCL). Due to the aggressive nature of the disease, DLBCL is often treated with combination therapies to maximize clinical outcomes; therefore, we investigated the potential of combining IMGN529 with both standard-of-care and emerging therapies against multiple oncology-relevant targets and pathways. The strongest enhancement in potency was seen with anti-CD20 antibodies, including rituximab. The combination of IMGN529 and rituximab was more potent than either agent alone, and this combinatorial benefit was associated with increased apoptotic induction and cell death. Additional studies revealed that rituximab treatment increased the internalization and degradation of the CD37-targeting antibody moiety of IMGN529. The combination of IMGN529 and rituximab was highly efficacious in multiple xenograft models, with superior antitumor efficacy seen compared to either agent alone or treatment with R-CHOP therapy. These findings suggest a novel mechanism whereby the potency of IMGN529 can be enhanced by CD20 binding, which results in the increased internalization and degradation of IMGN529 leading to the generation of greater amounts of cytotoxic catabolite. Overall, these data provide a biological rationale for the enhanced activity of IMGN529 in combination with rituximab and support the ongoing clinical evaluation of IMGN529 in combination with rituximab in patients with relapsed and/or refractory DLBCL.

Abstract 2830: Antibody and linker selection for the anti-CD37 antibody-maytansinoid conjugate IMGN529 for the treatment of B-cell malignancies

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL CD37 represents an attractive target for an antibody-maytansinoid conjugate (AMC) due to its prevalence in B-cell malignancies, such as non-Hodgkins lymphoma (NHL) and chronic lymphocytic leukemia (CLL), and its restricted expression on normal tissue, where it is mainly found on B-cells in blood and lymphoid tissues. Additionally, since antibodies to CD37 have been described to have anti-tumor activity, this target has potential for the development of an AMC containing a functional antibody. To select the antibody for this AMC, a large panel of anti-CD37 antibodies was generated by immunizing mice with CD37+ cells. Anti-CD37 antibodies were selected based on their superior ability to induce apoptosis in Ramos and Raji cells in comparison to the anti-CD20 antibody, rituximab, and the anti-CD37 SMIP, TRU-016. Surprisingly, unlike TRU-016, these antibodies had potent apoptotic activity in the absence of cross-linking agent. After humanization by variable domain re-surfacing, the selected antibodies retained high affinity binding to CD37+ B-cells with an EC50 of < 1 nM. They had much stronger pro-apoptotic activity than rituximab against Ramos cells, with the K7153A antibody among those with the best EC50. They all had antibody-dependent cell-mediated cytotoxicity (ADCC) activity, with K7153A having the most potent activity against Daudi cells. When SMCC-DM1 conjugates of humanized antibodies were compared, the K7153A-SMCC-DM1 conjugate had the most potent specific cytotoxicity against Daudi and Granta-519 cells in vitro. Therefore, the K7153A anti-CD37 antibody provided the best overall anti-tumor activity in terms of its direct pro-apoptotic activity, effector function and potency when used in an AMC. To determine the most effective linker design, maytansinoid conjugates of K7153A were prepared with either hindered disulfide (SPP-DM1) or thioether (SMCC-DM1) linker chemistries. Both conjugates were highly active against lymphoma cells in vitro, with the SMCC-DM1 conjugate being somewhat more potent. In vivo, a single dose of either 10 mg/kg of K7153A-SMCC-DM1 or 5 mg/kg of K7153A-SPP-DM1 was highly active against established SU-DHL-4 sc xenograft tumors. Both treatments resulted in >50% tumor-free survivors at study end. Similarly, the same treatment dose and schedule resulted in good efficacy with both conjugates in a BJAB sc xenograft model. Thus, the K7153A-SMCC-DM1 conjugate was highly active against lymphoma xenograft tumors and, based on preclinical experience, is expected to have comparable, if not better, therapeutic index to that of the SPP-linked conjugate. Taken together, these data support the selection of the K7153A antibody and the SMCC-DM1 design as the optimal anti-CD37 antibody-maytansinoid conjugate for clinical development (designated IMGN529). 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 2830. doi:10.1158/1538-7445.AM2011-2830

IMGN779, a Novel CD33-Targeting Antibody–Drug Conjugate with DNA-Alkylating Activity, Exhibits Potent Antitumor Activity in Models of AML

The myeloid differentiation antigen CD33 has long been exploited as a target for antibody-based therapeutic approaches in acute myeloid leukemia (AML). Validation of this strategy was provided with the approval of the CD33-targeting antibody–drug conjugate (ADC) gemtuzumab ozogamicin in 2000; the clinical utility of this agent, however, has been hampered by safety concerns. Thus, the full potential of CD33-directed therapy in AML remains to be realized, and considerable interest exists in the design and development of more effective ADCs that confer high therapeutic indices and favorable tolerability profiles. Here, we describe the preclinical characterization of a novel CD33-targeting ADC, IMGN779, which utilizes a unique DNA-alkylating payload to achieve potent antitumor effects with good tolerability. The payload, DGN462, is prototypical of a novel class of purpose-created indolinobenzodiazeprine pseudodimers, termed IGNs. With low picomolar potency, IMGN779 reduced viability in a panel of AML cell lines in vitro. Mechanistically, the cytotoxic activity of IMGN779 involved DNA damage, cell-cycle arrest, and apoptosis consistent with the mode of action of DGN462. Moreover, IMGN779 was highly active against patient-derived AML cells, including those with adverse molecular abnormalities, and sensitivity correlated to CD33 expression levels. In vivo, IMGN779 displayed robust antitumor efficacy in multiple AML xenograft and disseminated disease models, as evidenced by durable tumor regressions and prolonged survival. Taken together, these findings identify IMGN779 as a promising new candidate for evaluation as a novel therapeutic in AML. Mol Cancer Ther; 17(6); 1271–9. ©2018 AACR.

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 45:In vitroandin vivoactivity of a novel c-Met-targeting antibody-drug conjugate using a DNA-alkylating, indolinobenzodiazepine payload

Purpose: c-Met dysregulation and/or overexpression are associated with tumor progression, metastasis and poor prognosis in numerous cancers. Despite strong pre-clinical evidence that blocking c-Met activity inhibits tumor cell growth and metastasis, targeted therapies have thus far failed to deliver an effective treatment option to the majority of patients. To address patients with both c-Met over-expressing and MET amplified tumors, we designed an antibody-drug conjugate (ADC) comprised of a humanized anti-c-Met monoclonal antibody linked to a highly potent indolinobenzodiazepine DNA-alkylating payload (DGN549) to enable activity against not only MET amplified but also c-Met over-expressing tumors. Experimental Design: Panels of monoclonal antibodies (Abs) against c-Met were generated and screened for antagonistic and agonistic activity in the presence or absence of the c-Met ligand, HGF. Lead Abs were humanized and conjugated to DGN549 either through lysine (Drug-to-Ab ratio (DAR) = 2.5) or engineered cysteine residues (DAR 2.0). Abs were also conjugated via lysine residues to the potent anti-microtubule maytansine derivative, DM4, using a sulfo-SPDB linker (DAR 3.5). Binding and cytotoxicity of ADCs were tested in vitro on normal and cancer cell lines with varying c-Met levels. Expression of c-Met was evaluated in patient tumors and xenografts along with normal human tissues using the CONFIRM immunohistochemistry assay. In vivo efficacy of anti-c-Met-DGN549 and anti-c-Met-DM4 ADCs was tested in both MET amplified and c-Met over-expressed (but non-amplified) xenograft tumor models. Results: A humanized anti-c-Met antibody, hucMet27, was identified which exhibits low c-Met agonist activity. Conjugates of hucMet27 were prepared with two different payloads, DGN549 and DM4, and in vitro and in vivo activity were determined. Both DGN549 and DM4 conjugates of hucMet27 bound with similar sub-nanomolar affinity to c-Met-expressing cells. hucMet27-DGN549 conjugates exhibited potent cytotoxicity against a large panel of c-Met expressing cell lines. By contrast, the potency of the hucMet27-DM4 conjugate was restricted mainly to cell lines harboring MET amplification, despite all cell lines demonstrating sensitivity to the unconjugated payload. When tested in mice bearing human xenograft tumors, both hucMet27-DGN549 and hucMet-DM4 conjugates were highly active in a MET amplified model, whereas hucMet27-DGN549 was more potent in inducing regressions in a model with c-Met over-expression without MET amplification. Conclusion: hucMet27-DGN549 exhibits compelling c-Met targeted anti-cancer activity in vitro and in vivo, and represents a promising therapeutic strategy to deliver a potent cytotoxic agent to tumor cells bearing a wide range of c-Met expression. Citation Format: Katharine C. Lai, Asli Muvaffak, Min Li, Marian Themeles, Surina Sikka, Kerry Donahue, Stuart W. Hicks, Angela Romanelli, Thomas Chittenden. In vitro and in vivo activity of a novel c-Met-targeting antibody-drug conjugate using a DNA-alkylating, indolinobenzodiazepine payload [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 45. doi:10.1158/1538-7445.AM2017-45