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 DNAinteracting conjugates for broadening the clinical application of ADC technology. Mol Cancer Ther; 15(8); 1870-8. Ó2016 AACR.

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 to those of the cross-linker. Thus, the improved in vivo tolerability and anti-tumor activity achieved in rodent models with ADCs of the novel DNA alkylator could provide an efficacious, yet safer option for cancer treatment.on May 9, 2018.

The synthesis and biological evaluation of phosphate prodrugs of analogues of 1 (CC-1065) and their conjugates with antibodies are described. The phosphate group on the 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) portion of the compounds confers enhanced solubility and stability in aqueous solutions. In the presence of phosphatases, these compounds convert into active DNA-alkylating agents. The synthesis of the prodrugs was achieved sequentially through coupling of CBI with a bis-indolyl moiety, followed by attachment of a thiol-containing linker, and conversion of the hydroxyl group of CBI into a phosphate prodrug. The linkers incorporated into the prodrugs enable conjugation to an antibody via either a stable disulfide or thioether bond, in aqueous buffer solutions containing as little as 5% organic cosolvent, resulting in exclusively monomeric and stable antibody-cytotoxic prodrug conjugates. Two disulfide-containing linkers differing in the degree of steric hindrance were used in antibody conjugates to test the effect of different rates of intracellular disulfide cleavage and effector release on biological activity. The prodrugs can be converted to the active cytotoxic compounds through the action of endogenous phosphatases. Antibody-prodrug conjugates displayed potent antigen-selective cytotoxic activity in vitro and antitumor activity in vivo.

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.

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.

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).

We investigate the interaction of power, gender, and language use in the Enron email corpus. We present a freely available extension to the Enron corpus, with the gender of senders of 87% messages reliably identified. Using this data, we test two specific hypotheses drawn from the sociolinguistic literature pertaining to gender and power: women managers use face-saving communicative strategies, and women use language more explicitly than men to create and maintain social relations. We introduce the notion of "gender environment" to the computational study of written conversations; we interpret this notion as the gender makeup of an email thread, and show that some manifestations of power differ significantly between gender environments. Finally, we show the utility of gender information in the problem of automatically predicting the direction of power between pairs of participants in email interactions.

The clinical support for ADC therapeutics has expanded as more highly-engineered ADCs advance in human clinical testing. Most of the ADCs now in clinical testing contain a tubulin-acting compound (a maytansine or dolastatin derivative) as the cytotoxic agent. While tubulin-acting agents can be effective against many different types of cancers, some cancers are more responsive to DNA-acting agents. To expand the therapeutic potential for ADCs, we sought to develop a new class of cytotoxic agents with a novel, DNA-acting mechanism of action for use with tumor-targeting antibodies. Herein, we report the development of our IGN family of cytotoxic agents. These IGN agents comprise indolino-benzodiazepine dimers that are highly potent by virtue of their ability to alkylate and crosslink DNA. This novel class of compounds demonstrated sequence-selective DNA adduct formation in vitro and cytotoxicity in the picomolar range towards cultured human cancer cells. The intense potency of these compounds, along with their desired aqueous solubility and stability, make them ideally suited for use in ADCs. A lead compound from this class was conjugated to an EpCAM-binding antibody, B38.1, and to a CD33-binding antibody, huMy9-6, through amide bonds. The B38.1-IGN conjugate was highly potent against three different EpCAM-expressing cell lines - COLO 205, LoVo and OVCAR-3 - with IC50 values of 1 pM, 5 pM and 18 pM, respectively. The addition of excess unconjugated B38.1 antibody abolished this cytotoxic effect, demonstrating that the activity of the conjugate is antigen specific. The B38.1-IGN conjugate was considerably less potent towards the antigen-negative Namalwa cell line, with an IC50 value of >1 nM, further demonstrating antigen specificity. Similar potent cytotoxicity was seen with a huMy9-6-IGN conjugate targeting the CD33-positive human promyelocytic leukemia cell line, NB4 (IC50 ∼4pM), in spite of the low antigen expression level (∼10,000 molecules/cell) in this cell line. Of particular interest, the B38.1-IGN conjugate also was potent towards multidrug resistant cancer cells. B38.1-IGN had a IC50 value of 14 pM for COLO 205MDR, a COLO 205 clone engineered to overexpress MDR1 transporter, and 7 pM for HCT-15, an EpCAM-expressing cell line that naturally expresses MDR1. Antibody-IGN conjugates demonstrated dose-dependent activity in multiple human tumor xenograft models in mice, with anti-tumor activity observed at non-toxic doses. The unique mechanism of action of the IGN class of compounds, and the high antigen-specific potency of antibody-IGN conjugates seen in vitro and in vivo, provides a promising new cytotoxic agent for use in the development of new ADCs. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B126.