Antibody-Drug Conjugates with Indolinobenzodiazepine Dimer Payloads: DNA-Binding Mechanism of Indolinobenzodiazepine Dimer Catabolites in Target Cancer Cells.

DNA-targeting indolinobenzodiazepine dimer (IGN) payloads are used in several clinical-stage antibody-drug conjugates. IGN drugs alkylate DNA through the single imine moiety present in the dimer in contrast to the pyrrolobenzodiazepine dimer drugs, such as talirine and tesirine, which contain two imine moieties per dimer and cross-link DNA. This study explored the mechanism of binding of IGN to DNA in cells and to synthetic duplex and hairpin oligonucleotides. New, highly sensitive IGN-DNA binding enzyme-linked immunosorbent assay methods were developed using biotinylated IGN analogues (monoimine, diimine, and diamine IGNs) and digoxigenin-labeled duplex oligonucleotides, which allowed the measurement of drug-DNA adducts in viable cells at concentrations below IC50. Furthermore, the release of free drug from the IGN-DNA adduct upon treatment with nuclease ex vivo was tested under physiological conditions. The monoimine IGN drug formed a highly stable adduct with DNA in cells, with stability similar to that of the diimine drug analogue. Both monoimine and diimine IGN-DNA adducts released free drugs upon DNA cleavage by nuclease at 37 °C, although more free drug was released from the monoimine compared to the diimine adduct, which presumably was partly cross-linked. The strong binding of the monoimine IGN drug to duplex DNA results from both the noncovalent IGN-DNA interaction and the covalent bond formation between the 2-amino group of a guanine residue and the imine moiety in IGN.

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.

Memory Retrieval Has a Dynamic Influence on the Maintenance Mechanisms That Are Sensitive to ζ-Inhibitory Peptide (ZIP).

In neuroscientists' attempts to understand the long-term storage of memory, topics of particular importance and interest are the cellular and system mechanisms of maintenance (e.g., those sensitive to ζ-inhibitory peptide, ZIP) and those induced by memory retrieval (i.e., reconsolidation). Much is known about each of these processes in isolation, but less is known concerning how they interact. It is known that ZIP sensitivity and memory retrieval share at least some molecular targets (e.g., recycling α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA, receptors to the plasma membrane); conversely, the fact that sensitivity to ZIP emerges only after consolidation ends suggests that consolidation (and by extension reconsolidation) and maintenance might be mutually exclusive processes, the onset of one canceling the other. Here, we use conditioned taste aversion (CTA) in rats, a cortically dependent learning paradigm, to test this hypothesis. First, we demonstrate that ZIP infusions into gustatory cortex begin interfering with CTA memory 43-45 h after memory acquisition-after consolidation ends. Next, we show that a retrieval trial administered after this time point interrupts the ability of ZIP to induce amnesia and that ZIP's ability to induce amnesia is reengaged only 45 h after retrieval. This pattern of results suggests that memory retrieval and ZIP-sensitive maintenance mechanisms are mutually exclusive and that the progression from one to the other are similar after acquisition and retrieval. They also reveal concrete differences between ZIP-sensitive mechanisms induced by acquisition and retrieval: the latency with which ZIP-sensitive mechanisms are expressed differ for the two processes. SIGNIFICANCE STATEMENT: Memory retrieval and the molecular mechanisms that are sensitive to ζ-inhibitory peptide (ZIP) are the few manipulations that have been shown to effect memory maintenance. Although much is known about their effect on maintenance separately, it is unknown how they interact. Here, we describe a model for the interaction between memory retrieval and ZIP-sensitive mechanisms, showing that retrieval trials briefly (i.e., for 45 h) interrupt these mechanisms. ZIP sensitivity emerges across a similar time window after memory acquisition and retrieval; the maintenance mechanisms that follow acquisition and retrieval differ, however, in the latency with which the impact of ZIP is expressed.

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.

Sensory Cortical Activity Is Related to the Selection of a Rhythmic Motor Action Pattern.

UNLABELLED: Rats produce robust, highly distinctive orofacial rhythms in response to taste stimuli-responses that aid in the consumption of palatable tastes and the ejection of aversive tastes, and that are sourced in a multifunctional brainstem central pattern generator. Several pieces of indirect evidence suggest that primary gustatory cortex (GC) may be a part of a distributed forebrain circuit involved in the selection of particular consumption-related rhythms, although not in the production of individual mouth movements per se. Here, we performed a series of tests of this hypothesis. We first examined the temporal relationship between GC activity and orofacial behaviors by performing paired single-neuron and electromyographic recordings in awake rats. Using a trial-by-trial analysis, we found that a subset of GC neurons shows a burst of activity beginning before the transition between nondistinct and taste-specific (i.e., consumption-related) orofacial rhythms. We further showed that shifting the latency of consumption-related behavior by selective cueing has an analogous impact on the timing of GC activity. Finally, we showed the complementary result, demonstrating that optogenetic perturbation of GC activity has a modest but significant impact on the probability that a specific rhythm will be produced in response to a strongly aversive taste. GC appears to be a part of a distributed circuit that governs the selection of taste-induced orofacial rhythms. SIGNIFICANCE STATEMENT: In many well studied (typically invertebrate) sensorimotor systems, top-down modulation helps motor-control regions "select" movement patterns. Here, we provide evidence that gustatory cortex (GC) may be part of the forebrain circuit that performs this function in relation to oral behaviors ("gapes") whereby a substance in the mouth is rejected as unpalatable. We show that GC palatability coding is well timed to play this role, and that the latency of these codes changes as the latency of gaping shifts with learning. We go on to show that by silencing these neurons, we can change the likelihood of gaping. These data help to break down the sensory/motor divide by showing a role for sensory cortex in the selection of motor behavior.

Dynamic taste responses of parabrachial pontine neurons in awake rats.

The parabrachial nuclei of the pons (PbN) receive almost direct input from taste buds on the tongue and control basic taste-driven behaviors. Thus it is reasonable to hypothesize that PbN neurons might respond to tastes in a manner similar to that of peripheral receptors, i.e., that these responses might be narrow and relatively "dynamics free." On the other hand, the majority of the input to PbN descends from forebrain regions such as gustatory cortex (GC), which processes tastes with "temporal codes" in which firing reflects first the presence, then the identity, and finally the desirability of the stimulus. Therefore a reasonable alternative hypothesis is that PbN responses might be dominated by dynamics similar to those observed in GC. Here we examined simultaneously recorded single-neuron PbN (and GC) responses in awake rats receiving exposure to basic taste stimuli. We found that pontine taste responses were almost entirely confined to canonically identified taste-PbN (t-PbN). Taste-specificity was found, furthermore, to be time varying in a larger percentage of these t-PbN responses than in responses recorded from the tissue around PbN (including non-taste-PbN). Finally, these time-varying properties were a good match for those observed in simultaneously recorded GC neurons-taste-specificity appeared after an initial nonspecific burst of action potentials, and palatability emerged several hundred milliseconds later. These results suggest that the pontine taste relay is closely allied with the dynamic taste processing performed in forebrain.

Inactivation of basolateral amygdala specifically eliminates palatability-related information in cortical sensory responses.

Evidence indirectly implicates the amygdala as the primary processor of emotional information used by cortex to drive appropriate behavioral responses to stimuli. Taste provides an ideal system with which to test this hypothesis directly, as neurons in both basolateral amygdala (BLA) and gustatory cortex (GC)-anatomically interconnected nodes of the gustatory system-code the emotional valence of taste stimuli (i.e., palatability), in firing rate responses that progress similarly through "epochs." The fact that palatability-related firing appears one epoch earlier in BLA than GC is broadly consistent with the hypothesis that such information may propagate from the former to the latter. Here, we provide evidence supporting this hypothesis, assaying taste responses in small GC single-neuron ensembles before, during, and after temporarily inactivating BLA in awake rats. BLA inactivation (BLAx) changed responses in 98% of taste-responsive GC neurons, altering the entirety of every taste response in many neurons. Most changes involved reductions in firing rate, but regardless of the direction of change, the effect of BLAx was epoch-specific: while firing rates were changed, the taste specificity of responses remained stable; information about taste palatability, however, which normally resides in the "Late" epoch, was reduced in magnitude across the entire GC sample and outright eliminated in most neurons. Only in the specific minority of neurons for which BLAx enhanced responses did palatability specificity survive undiminished. Our data therefore provide direct evidence that BLA is a necessary component of GC gustatory processing, and that cortical palatability processing in particular is, in part, a function of BLA activity.

Synthesis and biological evaluation of antibody conjugates of phosphate prodrugs of cytotoxic DNA alkylators for the targeted treatment of cancer.

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.