Site-Specific Conjugation of the Indolinobenzodiazepine DGN549 to Antibodies Affords Antibody-Drug Conjugates with an Improved Therapeutic Index as Compared with Lysine Conjugation.

Antibody-drug conjugates have elicited great interest recently as targeted chemotherapies for cancer. Recent preclinical and clinical data have continued to raise questions about optimizing the design of these complex therapeutics. Biochemical methods for site-specific antibody conjugation have been a design feature of recent clinical ADCs, and preclinical reports suggest that site-specifically conjugated ADCs generically offer improved therapeutic indices (i.e., the fold difference between efficacious and maximum tolerated doses). Here we present the results of a systematic preclinical comparison of ADCs embodying the DNA-alkylating linker-payload DGN549 generated with both heterogeneous lysine-directed and site-specific cysteine-directed conjugation chemistries. Importantly, the catabolites generated by each ADC are the same regardless of the conjugation format. In two different model systems evaluated, the site-specific ADC showed a therapeutic index benefit. However, the therapeutic index benefit is different in each case: both show evidence of improved tolerability, though with different magnitudes, and in one case significant efficacy improvement is also observed. These results support our contention that conjugation chemistry of ADCs is best evaluated in the context of a particular antibody, target, and linker-payload, and ideally across multiple disease models.

Optimizing Lysosomal Activation of Antibody-Drug Conjugates (ADCs) by Incorporation of Novel Cleavable Dipeptide Linkers.

Although peptide linkers are used in multiple clinical-stage ADCs, there are only few reports on optimizing peptide linkers for efficient lysosomal proteolysis and for stability in circulation. We screened multiple dipeptide linkers for efficiency of proteolysis and compared them to the dipeptide linkers currently being evaluated in the clinic: Val-Cit, Val-Ala, and Ala-Ala. Lead dipeptide linkers selected from the initial screen were incorporated into ADCs with indolinobenzodiazepine dimer (IGN) payloads to evaluate cellular processing, in vitro cytotoxic activity, plasma stability, and in vivo efficacy. ADCs with several dipeptide linkers bearing l-amino acids showed faster lysosomal processing in target cancer cells compared to the l-Ala-l-Ala linked ADC. These variances in linker processing rates did not result in different in vitro and in vivo activities among peptide linker ADCs, presumably due to accumulation of threshold cytotoxic catabolite levels for ADCs of several peptide linkers in the cell lines and xenografts tested. ADCs with l-amino acid dipeptide linkers exhibited superior in vitro cytotoxic potencies in multiple cell lines compared to an ADC with a d-Ala-d-Ala dipeptide linker and an ADC with a noncleavable linker. This work adds to the toolbox of stable, lysosomally cleavable peptide linkers for ADCs.

Effect of Linker Stereochemistry on the Activity of Indolinobenzodiazepine Containing Antibody-Drug Conjugates (ADCs).

Antibody-drug conjugates (ADCs) that incorporate potent indolinobenzodiazepine DNA alkylators as the payload component are currently undergoing clinical evaluation. In one ADC design, the payload molecules are linked to the antibody through a peptidase-labile l-Ala-l-Ala linker. In order to determine the role of amino acid stereochemistry on antitumor activity and tolerability, we incorporated l- and d-alanyl groups in the dipeptide, synthesized all four diastereomers, and prepared and tested the corresponding ADCs. Results of our preclinical evaluation showed that the l-Ala-l-Ala configuration provided the ADC with the highest therapeutic index (antitumor activity vs toxicity).

Synthesis of Highly Potent N-10 Amino-Linked DNA-Alkylating Indolinobenzodiazepine Antibody-Drug Conjugates (ADCs).

Indolinobenzodiazepine DNA alkylators (IGNs) are the cytotoxic payloads in antibody-drug conjugates (ADCs) currently undergoing Phase I clinical evaluation (IMGN779, IMGN632, and TAK164). These ADCs possess linkers that have been incorporated into a central substituted phenyl spacer. Here, we present an alternative strategy for the IGNs, linking through a carbamate at the readily available N-10 amine present in the monoimine containing dimer. As a result, we have designed a series of N-10 linked IGN ADCs with a wide range of in vitro potency and tolerability, which may allow us to better match an IGN with a particular target based on the potential dosing needs.

Design, synthesis and evaluation of novel, potent DNA alkylating agents and their antibody-drug conjugates (ADCs).

Antibody-drug conjugates (ADCs) incorporating potent indolinobenzodiazepine (IGN) DNA alkylators as the cytotoxic payload are currently undergoing clinical evaluation. The optimized design of these payloads consists of an unsymmetrical dimer possessing both an imine and an amine effectively eliminating DNA crosslinking and demonstrating improved tolerability in mice. Here we present an alternate approach to generating DNA alkylating ADCs by linking the IGN monomer with a biaryl system which has a high DNA binding affinity to potentially enhance tolerability. These BIA ADCs were found to be highly cytotoxic in vitro and demonstrated potent antitumor activity in vivo.

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.

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.