Nicotinamide phosphoribosyltransferase (NAMPT) regulates the biosynthesis of NAD from nicotinamide via a salvage biosynthetic pathway. Inhibition of NAMPT depletes cellular NAD levels leading to disruption of energy metabolism and cell death. Non-targeted small molecule NAMPT inhibitors have demonstrated poor tolerability in clinical trials and in preclinical models, including cardiac and retinal toxicities in rats. In an effort to improve the therapeutic window of this drug class, we pursued a targeted-delivery approach using antibody-drug conjugates. Through a medicinal chemistry effort, we identified novel NAMPT inhibitors that incorporate chemical functionality in the solvent-exposed terminus to allow construction of enzyme-cleavable drug linkers. Additionally, we applied a pyridinium-based linker strategy that allows for traceless linker attachment through a conserved nicotinamide-mimetic moiety of NAMPT inhibitors. Candidate molecules were evaluated for NAMPT binding affinity and cellular cytotoxicity as free drugs, and for cellular cytotoxicity as ADCs with the alternate linker strategies. Comparisons across inhibitors and linker strategies provide insight into optimal design of cleavable drug linkers for this class of drugs. In vitro, the ADCs deplete NAD and lead to downstream ATP depletion in a time-dependent manner. In vivo evaluation using human tumor xenografts shows translation of the pharmacodynamic effect resulting in tumor regression in models of Hodgkin lymphoma, non-Hodgkin lymphoma, and acute myeloid leukemia. Toxicology studies in Sprague Dawley rats demonstrate excellent tolerability at active doses, with no observable cardiac or retinal toxicities at the highest tested doses in single- and multi-dose regimens. These findings detail the development of a novel payload class and optimized linker strategy for use with antibody-drug conjugates, and demonstrate a preclinical efficacy and safety profile to support continued efforts toward clinical therapeutics.Citation Format: Chris Neumann, Kathleen C. Olivas, Kung Pern Wang, Andrew B. Waight, David W. Meyer, Luke V. Loftus, Margo C. Zaval, Martha E. Anderson, Steven Jin, Julia H. Cochran, Jessica K. Simmons, Paul G. Pittman, Fu Li, Michelle L. Ulrich, Abbie Wong, Weiping Zeng, Robert P. Lyon, Peter D. Senter. Antibody-drug conjugates of NAMPT inhibitors: Discovery, optimization, and preclinical characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 983.
To provide a better understanding of the pharmacokinetics-pharmacodynamics relationships of antibody-based drugs, we analyzed several chimeric and humanized monoclonal antibodies or antibody-drug conjugates (ADC) for PK and efficacy among four strains of mice. Notably, antibodies and ADCs displayed a dose-dependent drug disposition profile in the plasma of NSG mice. The increased clearance rate in NSG mice resulted in the reduction of antitumor activity of ADCs. Furthermore, we identified that the abnormal clearance was mediated by Fc-FcγR interaction by comparing antibodies that lack FcγR binding capacity. We also found a high percentage of FcγR-expressing macrophages in the bone marrow, spleen, and liver of NSG mice, which may be responsible for the abnormal distribution of antibodies. Overall, these findings suggest that preclinical evaluation of efficacy and pharmacokinetics of antibodies and ADCs need to consider mouse strain-induced variations.
Abstract The recent clinical success of antibody-drug conjugates (ADCs) has spawned an increased effort to identify new technologies, and the development of new drug-linker chemistry is vital to expand the scope of conjugatable payloads. The tertiary amine functional group is a common structural motif present in many bioactive compounds, including antimitotics of the auristatin and tubulysin classes. Traditionally, conjugation of tertiary amines required drug derivatization or modification to remove an N-alkyl group, thus creating a readily conjugatable secondary amine. However, identifying appropriate modifications that do not compromise the activity of the drug is frequently time consuming and often unsuccessful. To eliminate the need for such structural modifications, we sought a method for stable conjugation and facile release through the tertiary amine functional group by creating linkers with a quaternary amine point of attachment. To validate the linker strategy, quaternary amine-based cleavable linkers bearing auristatin E were synthesized and evaluated as ADCs. The conjugates were stable in rodent plasma, and were potent and immunologically specific both in vitro and in vivo in a Hodgkin lymphoma xenograft model. A second application of this technology has been demonstrated with tubulysins, another class of potent antimitotics containing a tertiary amine at the N-terminus. A cleavable quaternary amine linker containing a tubulysin analog was synthesized and ADCs were prepared and evaluated. The tubulysin conjugates were potent and immunologically specific across a panel of cancer cell lines, including multiple MDR-positive lines. Furthermore, the tubulysin conjugate displayed ‘bystander activity’ in an in vitro co-culture assay. The quaternary amine linkers represent an advance in linker technology and will enable the evaluation of drug classes previously inaccessible as ADCs. Citation Format: Patrick J. Burke, Joseph Z. Hamilton, Thomas A. Pires, Jocelyn R. Setter, Joshua H. Hunter, Julia H. Cochran, Brian E. Toki, Peter D. Senter, Robert P. Lyon, Scott C. Jeffrey. Development of quaternary amine linkers for ADCs: Application to auristatin E and tubulysin. [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 2056.
Abstract Despite a number of new therapies for multiple myeloma (MM) most patients relapse, require multiple lines of therapy, and ultimately succumb to disease. Therapies that are well tolerated and active remain an unmet need. Currently, successful MM therapies combine agents with different mechanisms of action and safety profiles. Therapeutic antibodies have recently altered the MM treatment paradigm, allowing patients to achieve deeper responses with minimal added toxicity. SEA-BCMA is a humanized afucosylated IgG1 antibody targeting BCMA, which shows preclinical evidence of encouraging activity and tolerability. BCMA is expressed at the surface of plasma cells, and induces proliferative signals through the binding of its ligands APRIL and BAFF. SEA-BCMA acts through three mechanisms of action. One, it engages in increased binding to FcγRIII through SEA technology leading to enhanced antibody dependent cellular cytotoxicity. Two, it mediates antibody dependent cellular phagocytosis. Three, it blocks the proliferative signals from BCMA ligand binding. This antibody shows activity in all seven tumor xenograft models tested, inducing tumor delays at doses as low as 0.1mg/kg, and generating prolonged survival and durable regressions with repeat dosing. SEA-BCMA is active on tumor xenografts expressing as few as 2000 surface copies of BCMA antigen. Both effector function and ligand blocking contribute to overall in vivo activity. In the absence of effector cell recruitment, ligand blocking alone can induce prolonged durable regressions. In addition, SEA-BCMA can target MM cells in the presence of soluble BCMA. SEA-BCMA is tolerated up to 100mg/kg in cynomolgus monkey, reflecting lack of toxicity from cellular interactions with the Fc portion of the Ab, since SEA-BCMA does not bind to the cynomolgus monkey antigen. SEA-BCMA displays a 12-day half-life in these animals. In vitro testing with human PBMCs or bone marrow mononuclear cells induced minimal cytokine production in the presence of BCMA target. In summary, SEA-BCMA is highly active and well tolerated in preclinical models and is a strong candidate for treatment of MM patients. Citation Format: Heather Van Epps, Martha Anderson, Changpu Yu, Kerry Klussman, Lori Westendorf, Chris Carosino, Luke Manlove, Julia Cochran, Jason Neale, Dennis Benjamin, Maureen Ryan. SEA-BCMA: A highly active enhanced antibody for multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3833.
<p>Supplemental tables and figures referenced in main text: Table S1 - Crystallography data collection and refinement statistics; Table S2 - ADC characterization data; Figure S1 - ADC plasma stability data; Figure S2 - Time course for ADC activity in vitro; Table S3 - additional in vitro potency data for ADCs targeting CD30, CD19, and CD123.</p>
<div>Abstract<p>We have developed a highly active and well-tolerated camptothecin (CPT) drug-linker designed for antibody-mediated drug delivery in which the lead molecule consists of a 7-aminomethyl-10,11-methylenedioxy CPT (CPT1) derivative payload attached to a novel hydrophilic protease-cleavable valine–lysine–glycine tripeptide linker. A defined polyethylene glycol stretcher was included to improve the properties of the drug-linker, facilitating high antibody–drug conjugate (ADC) drug loading, while reducing the propensity for aggregation. A CPT1 ADC with 8 drug-linkers/mAb displayed a pharmacokinetic profile coincident with parental unconjugated antibody and had high serum stability. The ADCs were broadly active against cancer cells <i>in vitro</i> and in mouse xenograft models, giving tumor regressions and complete responses at low (≤3 mg/kg, single administration) doses. Pronounced activities were obtained in both solid and hematologic tumor models and in models of bystander killing activity and multidrug resistance. Payload release studies demonstrated that two CPTs, CPT1 and the corresponding glycine analog (CPT2), were released from a cAC10 ADC by tumor cells. An ADC containing this drug-linker was well tolerated in rats at 60 mg/kg, given weekly four times. Thus, ADCs comprised of this valine–lysine–glycine linker with CPT drug payloads have promise in targeted drug delivery.</p></div>
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