Edmund I. Graziani

Mild Method for Succinimide Hydrolysis on ADCs: Impact on ADC Potency, Stability, Exposure, and Efficacy

The stability of the connection between the antibody and the toxin can have a profound impact on ADC safety and efficacy. There has been increasing evidence in recent years that maleimide-based ADCs are prone to payload loss via a retro-Michael type reaction. Herein, we report a mild method for the hydrolysis of the succinimide-thioether ring which results in a ring-opened linker. ADCs containing this hydrolyzed succinimide linker show equivalent cytotoxicity, improved in vitro stability, improved PK exposure, and improved efficacy as compared to their nonhydrolyzed counterparts. This method offers a simple way to improve the stability, exposure, and efficacy of maleimide-based ADCs.

Tumor Cells Chronically Treated with a Trastuzumab–Maytansinoid Antibody–Drug Conjugate Develop Varied Resistance Mechanisms but Respond to Alternate Treatments

Antibody–drug conjugates (ADC) are emerging as clinically effective therapy. We hypothesized that cancers treated with ADCs would acquire resistance mechanisms unique to immunoconjugate therapy and that changing ADC components may overcome resistance. Breast cancer cell lines were exposed to multiple cycles of anti-Her2 trastuzumab–maytansinoid ADC (TM-ADC) at IC80 concentrations followed by recovery. The resistant cells, 361-TM and JIMT1-TM, were characterized by cytotoxicity, proteomic, transcriptional, and other profiling. Approximately 250-fold resistance to TM-ADC developed in 361-TM cells, and cross-resistance was observed to other non–cleavable-linked ADCs. Strikingly, these 361-TM cells retained sensitivity to ADCs containing cleavable mcValCitPABC-linked auristatins. In JIMT1-TM cells, 16-fold resistance to TM-ADC developed, with cross-resistance to other trastuzumab-ADCs. Both 361-TM and JIMT1-TM cells showed minimal resistance to unconjugated mertansine (DM1) and other chemotherapeutics. Proteomics and immunoblots detected increased ABCC1 (MRP1) drug efflux protein in 361-TM cells, and decreased Her2 (ErbB2) in JIMT1-TM cells. Proteomics also showed alterations in various pathways upon chronic exposure to the drug in both cell models. Tumors derived from 361-TM cells grew in mice and were refractory to TM-ADC compared with parental cells. Hence, acquired resistance to trastuzumab–maytansinoid ADC was generated in cultured cancer cells by chronic drug treatment, and either increased ABCC1 protein or reduced Her2 antigen were primary mediators of resistance. These ADC-resistant cell models retain sensitivity to other ADCs or standard-of-care chemotherapeutics, suggesting that alternate therapies may overcome acquired ADC resistance. Mol Cancer Ther; 14(4); 952–63. ©2015 AACR.

Development of Solid-Phase Site-Specific Conjugation and Its Application toward Generation of Dual Labeled Antibody and Fab Drug Conjugates.

The focus of the antibody-drug conjugate (ADC) field is shifting toward development of site-specific, next-generation ADCs to address the issue of heterogeneity, metabolic instability, conjugatability, and less than ideal therapeutic index associated with the conventional (heterogeneous) ADCs. It is evident from the recent literature that the site of conjugation, the structure of the linker, and the physicochemical properties of the linker-payload all have a significant impact on the safety and efficacy of the resulting ADCs. Screening multiple linker-payloads on multiple sites of an antibody presents a combinatorial problem that necessitates high-throughput conjugation and purification methodology to identify ADCs with the best combination of site and payload. Toward this end, we developed a protein A/L-based solid-phase, site-specific conjugation and purification method that can be used to generate site-specific ADCs in a 96-well plate format. This solid-phase method has been shown to be versatile because of its compatibility with various conjugation functional handles such as maleimides, haloacetamides, copper free click substrates, and transglutaminase substrates. The application of this methodology was further expanded to generate dual labeled, site-specific antibody and Fab conjugates.

Amphotericin B, identified from a natural product screen, antagonizes CNS inhibitors to promote axon growth via activation of an Akt pathway in neurons

J. Neurochem. (2010) 113, 1331–1342.

Abstract 4629: Tumor cells selected for resistance to an antibody-drug conjugate retain sensitivity to ADCs with modified linkers and payloads.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnAntibody-drug conjugates (ADCs) enable targeted delivery of therapeutics to cancer cells and offer potential for more selective therapy. Several ADCs are demonstrating promising clinical efficacy, however due to the complexity of human cancer, tumors become refractory to most drug treatments. We hypothesized that cultured tumor cells chronically treated with an ADC would acquire mechanisms of resistance unique to ADC-based therapy. Human breast cancer cell lines were exposed to multiple cycles of an anti-Her2 trastuzumab-maytansinoid conjugate (TM) at IC80 concentrations for 3 days followed by ∼1 week without treatment to simulate a maximally tolerated dose followed by recovery. After ∼2 months, significant resistance developed in JIMT1 and MDA-MB-361 cell lines. The potency of TM conjugate on drug-selected cell lines was reduced to the activity observed on Her2-negative cells (>20 & >270X in JIMT1 & 361 cell models, respectively). Flow cytometry revealed 58% and 25% decreases in Her2 receptor number in JIMT-TM & 361-TM, respectively. Proteomic profiling of surface proteins in JIMT-TM cells demonstrated significant increases in proteins involved in post-translational modification (e.g., ubiquitinating enzymes, kinases, and phosphatases), as well as elevated levels of endosomal and vesicle proteins (e.g., RAB family members), and proteins mediating microtubule and actin dynamics. Notably, ABC drug transporters were not altered in JIMT-TM cells. In 361-TM cells, an increase in ABCC1 (MRP1) was observed, but no changes in ABCB1 (MDR1) which typically effluxes tubulin inhibitors. These data suggest the acquisition of complex resistance mechanisms upon ADC treatment. The cross-resistance profile of these ADC refractory models was evaluated. Minimal or no resistance (1 - 6X) was observed to free drugs, including maytansine or other standard-of-care tubulin or DNA targeted therapeutics. In JIMT-TM cells, cross-resistance was observed to other trastuzumab-based ADCs, including those containing either non-cleavable or cleavable linkers, and delivering payloads with various biological mechanisms of action. In contrast, 361-TM cells (which were made resistant to an ADC delivering a tubulin inhibitor via a non-cleavable linker) retained significant sensitivity to ADCs containing cleavable linkers, even those containing other tubulin inhibitor-based payloads. Moreover, both 361-TM and JIMT-TM resistant cell lines retained sensitivity to ADCs delivering payloads with alternative (non-tubulin) mechanisms of action. Therefore, despite the reduction in antigen levels observed in both cell lines, modification of the linker and/or the payload was able to overcome resistance mediated by the initial ADC therapy. These data offer the potential to treat refractory tumors with ADCs containing the same antibody vehicle, but delivering alternative linkers or payloads.nnCitation Format: Xingzhi Tan, Guixian Jin, Jeremy Myers, Veronica Diesl, Max Follettie, My-Hanh Lam, Sylvia Musto, Kiran Khandke, Manoj Charati, Edmund Graziani, Andreas Maderna, Chakrapani Subramanyam, Frank Koehn, Russell Dushin, Kim Arndt, Christopher J. ODonnell, Hans-Peter Gerber, Frank Loganzo. Tumor cells selected for resistance to an antibody-drug conjugate retain sensitivity to ADCs with modified linkers and payloads. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4629. doi:10.1158/1538-7445.AM2013-4629

Diversity oriented biosynthesis via accelerated evolution of modular gene clusters

Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes.Reengineering polyketide synthase encoding genes to produce analogues of natural products can be slow and low-yielding. Here the authors use accelerated evolution to recombine the gene cluster for rapid production of rapamycin-related products.

Site Selection: a Case Study in the Identification of Optimal Cysteine Engineered Antibody Drug Conjugates

As the antibody drug conjugate (ADC) community continues to shift towards site-specific conjugation technology, there is a growing need to understand how the site of conjugation impacts the biophysical and biological properties of an ADC. In order to address this need, we prepared a carefully selected series of engineered cysteine ADCs and proceeded to systematically evaluate their potency, stability, and PK exposure. The site of conjugation did not have a significant influence on the thermal stability and in vitro cytotoxicity of the ADCs. However, we demonstrate that the rate of cathepsin-mediated linker cleavage is heavily dependent upon site and is closely correlated with ADC hydrophobicity, thus confirming other recent reports of this phenomenon. Interestingly, conjugates with high rates of cathepsin-mediated linker cleavage did not exhibit decreased plasma stability. In fact, the major source of plasma instability was shown to be retro-Michael mediated deconjugation. This process is known to be impeded by succinimide hydrolysis, and thus, we undertook a series of mutational experiments demonstrating that basic residues located nearby the site of conjugation can be a significant driver of succinimide ring opening. Finally, we show that total antibody PK exposure in rat was loosely correlated with ADC hydrophobicity. It is our hope that these observations will help the ADC community to build “design rules” that will enable more efficient prosecution of next-generation ADC discovery programs.

Chemistry and Pharmacology of Rapamycin and Its Derivatives

I. Abstract Rapamycin is a bacterially derived natural product with a remarkable history as both a chemical probe for studies of cell growth control-related pathways, and a bona fide drug with established or predicted clinical activities in a variety of disease settings. Rapamycin was first noted as a potent antifungal and immunosuppressive agent, and studies of this drugs mechanism of action revealed that rapamycin was a surgically precise inhibitor of a novel protein serine–threonine kinase, appropriately termed the target of rapamycin (TOR). Intensive research efforts have revealed that the TOR orthologs function in a highly conserved pathway of eukaryotic cell growth control. This review focuses on the impact of rapamycin exposure on the TOR ortholog (termed mTOR) expressed in mammalian cells. We briefly describe the biosynthesis of rapamycin and the chemical modifications of the parent compound that yielded several of the experimentally useful and/or clinically active derivatives, collectively termed rapalogs. We then review in some detail the pharmacology of rapamycin, particularly as it relates to the growth and proliferation of cancer cells. The opportunities and challenges associated with the development of rapalogs as anticancer agents are then discussed. Finally, we briefly review some provocative recent insights into the effects of rapamycin on the immune system and on organismal aging.

Abstract 868: Creating a superior, site-specific anti-HER2 antibody-drug conjugate (NG-HER2 ADC) for treatment of solid tumors

Antibody-drug conjugates (ADCs) have emerged as an important class of cancer therapeutics. The FDA approval of Kadcyla (T-DM1), a single agent for treatment of HER2-positive advanced metastatic breast cancer, was a significant milestone in the field of targeted therapy, as the first and only ADC for treatment of solid tumors. Despite the 3-month improvement over standard of care in the median survival, almost all the patients eventually became refractory to T-DM1. We have identified several possible areas for improvement: 1) The potency of T-DM1 as confirmed by the Phase III clinical data is restricted to high HER2 tumors which leaves moderate or low HER2 expressing patients without access to T-DM1 treatment; 2) The 48% overall response rate is indicative of intrinsic resistance to T-DM1 and all T-DM1 treated patients eventually relapse. 3) The randomized lysine conjugation in T-DM1 generates heterogeneity of the product. We have developed a novel, site-specific anti-HER2 ADC (NG-HER2 ADC) and evaluated it in comparative preclinical studies with T-DM1. The results show that the NG-HER2 ADC is ∼ 10 fold more potent than T-DM1 in HER2 3+ xenograft models of breast and gastric cancers. Our proprietary cleavable and permeable linker-payload can mediate bystander effect and this enables potent anti-tumor activity in non-HER2 amplified breast cancer and heterogeneous low HER2 NSCLC PDX models, where T-DM1 is ineffective. Our ADC can overcome T-DM1 resistance in in vitro and in vivo models.. Our site-specific ADC at HNSTD of 9 mg/kg in cynomolgus monkeys showed high AUC, long half-life and had normal clinical observations with no marked neutropenia. On the contrary, conventional conjugates with cleavable linker payloads typically have severe bone marrow toxicity as DLT above 5 mg/kg. The therapeutic index for NG-HER2 ADC is significantly greater than T-DM1 in all models tested. NG-HER2 ADC has a projected clinical efficacious dose of ∼1 mg/kg, compared to 3-5 mg/kg for T-DM1, based on PK/PD modeling. In addition, the activity of the NG-HER2 ADC shows increased infiltration of CD8 positive effector cells, an essential component for immuno-oncology (IO) efficacy, in a syngeneic HER2 overexpressing model. This property potentially allows the combination of the ADC with IO drugs to improve the long-term, overall survival. Our data provides preclinical proof of concept for NG-HER2 ADC with best-in-class potential and is currently being tested in preparation for clinical trials for treatment of HER2 solid tumors. Citation Format: Dangshe Ma, Bitha Narayanan, Kim Marquette, Edmund Graziani, Frank Loganzo, Manoj Charati, Nadira Prashad, Nathan Tumey, Jon Golas, Christine Hosselet, George Hu, Frank Barletta, Alison Betts, Judy Lucas, Chris O’Donnell, Lioudmila Tchistiakova, Hans-Peter Gerber, Puja Sapra. Creating a superior, site-specific anti-HER2 antibody-drug conjugate (NG-HER2 ADC) for treatment of solid tumors. [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 868.

Abstract 644: Impact of conjugation site on pharmacokinetics and off-target toxicity of site-specific antibody drug conjugates

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnSite-specific conjugation of toxic payload to antibody yields homogeneous ADCs resulting in improved therapeutic index over conventional conjugation. To understand the role of conjugation site on pharmacokinetics, off-target toxicity, cysteine(cys) was engineered into a humanized anti-IL13Rα2 antibody, hAB08, at various sites within the IgG1/k constant domains. Single cys mutants L443C and Q347C with DAR 2, and double cys mutants K392C+L443C, L443C/kK183C and Q347C/kK183C with DAR 4 were conjugated using auristatin 0101 via maleimidocapronic - valine-citruline-p-aminobenzyloxycarbonyl (vc, cleavable linker). All cys mutants and their conjugates maintained their binding properties compared to parental hAb08. Cysteine substitution sites were selected based on conjugation efficiency and in vitro stability. In vivo efficacy was evaluated in mouse xenograph tumor models. Pharmacokinetics (PK) and off target toxicity (TOX) were assessed in both mouse and rat in vivo studies. Our data demonstrate that that all site specific conjugates show improved in vitro stability, comparable or better in vivo efficacy in mice xenograph models and in vivo stability (ratio of ADC-AUC/Ab-AUC) compared to conventional conjugates. However PK and off-target toxicity of antibody drug conjugates had been significantly impacted by conjugation site and animal species. L443 mutants show higher ADC-AUC, lower clearance and longer half life than Q347 mutants in mice while a reverse TK behavior was observed in rat. All site specific conjugates show higher dose tolerance and less off-target toxicity relative to conventional conjugates in rat. This off-target toxicity improvement depends on sites of conjugation. The L443 mutants exhibited a better off-target TOX profile relative to Q347 mutants. Taken together, site-specific conjugation improved in vivo efficacy and off-target toxicity relative to conventionally conjugated ADC. PK differences and off-target toxicity improvements depend on conjugation sites and animal species.nnNote: This abstract was not presented at the meeting.nnCitation Format: Dangshe Ma, Fang Jin, Frank Barletta, George Hu, Nathan Tumey, Haige Zhang, Tao He, Eric Sousa, Manoj Charti, Kiran Khadke, Judy Lucas, Darren Ferguson, Christoper Brown, Weijun Ma, Scott Gatto, William Brady, Edmund Graziani, Hans-Peter Gerber, Puja Sapra, Lioudmila Tchistikova. Impact of conjugation site on pharmacokinetics and off-target toxicity of site-specific antibody drug conjugates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 644. doi:10.1158/1538-7445.AM2015-644