Production Of Antibody-drug Conjugates Research Articles

Abstract 869: Meditope enablement and structural analysis of anti-CD33 antibodies

Abstract Meditope Biosciences’ SnAP (Site-specific novel Antibody Platform) technology provides a novel and specific way to create antibody drug conjugates (ADCs). Cyclic peptides, termed ‘meditopes’, have been designed to selectively bind to a ‘meditope binding site’ engineered into the Fab of an antibody. The process of conferring meditope binding is referred to as “meditope enablement”. Enabling antibodies for meditope binding does not interfere with antigen recognition or antibody integrity due to the unique location of the binding site. Meditope peptide binding can be utilized to attach cytotoxic payloads to an enabled antibody in a specific and consistent manner, and has the potential to dramatically improve the consistency of ADC production. For advanced hematological cancers such as CD33 positive acute myeloid leukemia (AML), using an anti-CD33 monoclonal antibody conjugated to a potent cytotoxic agent may be a useful tumor reduction strategy, especially when patients have failed other treatments. To this end, meditope enablement of lintuzumab and gentuzumab permits a consistent, site-specific attachment of cytotoxins with a highly controllable drug antibody ratio (DAR). Structural and biophysical characterization of the meditope enabled anti CD33 antibodies has provided key insight to the meditope enablement process. Experiments confirm that payload conjugation and meditope peptide binding does not interfere with antibody integrity or antigen recognition. The meditope peptide drug complexes were tested in cell-based assays for CD33-targeted cell killing. Using X-ray crystallographic analysis and guided docking, we propose modifications to further enhance affinity of the meditope peptide conjugate : CD33 antibody interaction. Citation Format: Calin D. Dumitru, Michael Matho, Elisabeth Gardiner, John C. Williams, Krzysztof Bzymek. Meditope enablement and structural analysis of anti-CD33 antibodies. [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 869.

RP-HPLC DAR Characterization of Site-Specific Antibody Drug Conjugates Produced in a Cell-Free Expression System

Antibody drug conjugates (ADCs) harness the target specificity of a monoclonal antibody (mAb) and the high cytotoxicity of a small molecule, enabling improved delivery of a potent antitumor agent compared to traditional chemotherapy for cancer therapy. Only two ADCs have been marketed, both of which are produced via nonsite-specific conjugation of the cytotoxic drug to either interchain cysteine (Adcetris) or lysine (Kadcyla). A growing body of evidence suggests that site-specific ADCs, because of their payload homogeneity, will improve pharmacokinetics and have wider therapeutic windows when compared to heterogeneous ADCs. Previously, we have demonstrated the use of a cell free expression system (Xpress CF+) for rapid production of site-specific ADCs. Here we report the generation of a variety of ADCs via conjugation between a site-specific incorporated non-natural amino acid (nnAA), para-azidomethyl-l-phenylalanine (pAMF), and dibenzocyclooctyl-(polyethylene glycol)4 (DBCO-(PEG)4) linked payloads using this platform. We developed a reversed phase HPLC method for drug to antibody ratio (DAR) characterization, which is applicable to both reduced and intact ADCs. We demonstrate that these ADCs are of near complete conjugation and exhibit potent cell killing activity and in vitro plasma stability. Moreover, we generated an ADC conjugated at both light and heavy chains, resulting in a DAR close to 4. With the increased number of payloads, the resultant DAR 4 ADC is potentially more efficacious than its DAR 2 counterparts, which could further improve its therapeutic index. These studies have demonstrated the competency of Xpress CF+ for site-specific ADC production and improved our understanding of the site-specific ADCs in general.

Antibody–drug conjugates for targeted anticancer drug delivery

Antibody–drug conjugates (ADCs) are tumor-targeted therapeutic agents that combine the specificity of monoclonal antibodies (mAbs) with the potent anti-tumor effects of cytotoxic drugs. Over the past few years, ADCs have become a powerful tool in the field of cancer chemotherapy. Recently, two ADC products, brentuximab vedotin (Adcetris®) and trastuzumab emtansine (Kadcyla®), have received FDA approval and there are more than 40 ADC candidates in clinical trials for the treatment of various cancers. Despite the success of some products, considerable interests for the next generation of ADCs have focused on the development of homogeneous conjugates because most of the current ADCs are highly heterogeneous with different drug-to-antibody ratios and drug conjugation sites. Recent studies have demonstrated that the site-specific conjugation of drugs to mAbs could produce homogeneous ADC with better pharmacokinetic properties and improved therapeutic index. A number of approaches, including the use of engineered cysteines, the insertion of unnatural amino acids, and enzymatic ligation, have addressed the challenging issues for the synthesis of homogeneous ADC. This review discusses the limitations of current ADC technologies and describes recent site-specific conjugation methods that can be used to prepare homogeneous ADCs for targeted anticancer drug delivery.

Antibody Format and Drug Release Rate Determine the Therapeutic Activity of Noninternalizing Antibody-Drug Conjugates.

The development of antibody-drug conjugates (ADC), a promising class of anticancer agents, has traditionally relied on the use of antibodies capable of selective internalization in tumor cells. We have recently shown that also noninternalizing antibodies, coupled to cytotoxic drugs by means of disulfide linkers that can be cleaved in the tumor extracellular environment, can display a potent therapeutic activity. Here, we have compared the tumor-targeting properties, drug release rates, and therapeutic performance of two ADCs, based on the maytansinoid DM1 thiol drug and on the F8 antibody, directed against the alternatively spliced Extra Domain A (EDA) domain of fibronectin. The antibody was used in IgG or in small immune protein (SIP) format. In both cases, DM1 was coupled to unpaired cysteine residues, resulting in a drug-antibody ratio of 2. In biodistribution studies, SIP(F8)-SS-DM1 accumulated in the tumor and cleared from circulation more rapidly than IgG(F8)-SS-DM1. However, the ADC based on the IgG format exhibited a higher tumor uptake at later time points (e.g., 33%IA/g against 8%IA/g at 24 hours after intravenous administration). In mouse plasma, surprisingly, the ADC products in IgG format were substantially more stable compared with the SIP format (half-lives >48 hours and <3 hours at 37°C, respectively), revealing a novel mechanism for the control of disulfide-based drug release rates. Therapy experiments in immunocompetent mice bearing murine F9 tumors revealed that SIP(F8)-SS-DM1 was more efficacious than IgG(F8)-SS-DM1 when the two products were compared either in an equimolar basis or at equal milligram doses.

Glycans of Antibodies as a Specific Site for Drug Conjugation Using Glycosyltransferases.

The therapeutic cargo molecules conjugated to a specific site on a monoclonal antibody (mAb), called antibody-drug conjugates (ADCs), are becoming powerful tools in cancer treatment. Generally, the cargo molecules conjugate at the cysteine or lysine residue of the mAb, which generally results in a highly heterogeneous ADC. Therapeutic cargo molecules need to be conjugated in a site-specific manner to the mAb so that the bioefficacy of these molecules is not compromised. The mAb (IgG1) are N-glycosylated at the conserved residue Asn(297), which is present in each heavy chain of the IgG1, near the CH2 domain of the Fc fragment. The mutant or wild-type glycosyltransferases transfer sugars with a chemical handle to the glycan molecule of IgG1, making the site-specific linking of cargo molecules possible via the chemical handle, and thus making the process an invaluable technique for the production of homogeneous ADCs.

Approaches to Interchain Cysteine-Linked ADC Characterization by Mass Spectrometry.

Therapeutic antibody-drug conjugates (ADCs) harness the cell-killing potential of cytotoxic agents and the tumor targeting specificity of monoclonal antibodies to selectively kill tumor cells. Recent years have witnessed the development of several promising modalities that follow the same basic principles of ADC based therapies but which employ unique cytotoxic agents and conjugation strategies in order to realize therapeutic benefit. The complexity and heterogeneity of ADCs present a challenge to some of the conventional analytical methods that industry has relied upon for biologics characterization. This current review will highlight some of the more recent methodological approaches in mass spectrometry that have bridged the gap that is created when conventional analytical techniques provide an incomplete picture of ADC product quality. Specifically, we will discuss mass spectrometric approaches that preserve and/or capture information about the native structure of ADCs and provide unique insights into the higher order structure (HOS) of these therapeutic molecules.

Abstract 660: Engineering strategies to control stoichiometry, site of conjugation, serum stability and effector functions of antibody-drug conjugates

Abstract A promising approach to enhance the antitumor activity of antibodies and reduce the systemic toxicity of small molecule anti-cancer drugs is to covalently attach the small molecule to the antibody thus forming antibody drug conjugates (ADCs). Conventionally, ADCs are produced by chemical conjugation of drugs to antibodies through either lysine side chain amines or native cysteine sulfhydryl groups generated by reduction of interchain disulfide bonds. Both of these methods yield heterogeneous conjugates with complex biophysical properties. To limit these liabilities, we have designed, characterized, and validated antibody variants, which allow precise control of the site of conjugation and the drug load; such as two and/or four drugs per antibody can be site-specific conjugated. These engineered antibody variants can be efficiently and site-specifically conjugated with drug payloads at milligram and gram scales to yield homogenous ADC products. Engineered antibody variants described here retain antigen-binding affinities, FcRn binding, and have biochemical, biophysical and manufacturability properties similar to their non-engineered parent antibodies. Upon conjugation to highly cytotoxic drugs with anti-mitotic and/or DNA alkylating activities, the antibody variants have potent in vitro and in vivo anti-tumor activity. Furthermore, we describe ADCs vehicles that provide efficient scaffolds for site-specific conjugation of drugs while offering the opportunity to minimize off-target toxicities often observed with ADCs as a result of their binding to cells expressing Fc-gamma receptors. Finally, we show the impact of the identified sites for drug conjugation on the in vivo pharmacokinetic and safety profile of ADCs in rats. Our strategies for engineering ADCs with defined sites for conjugation, controlled drug loads, potentially decreased off-target toxicity, and optimal serum stability are broadly applicable to any full-length IgG or Fc-containing therapeutics as they involve the Fc constant domain of the antibody. Citation Format: Nazzareno Dimasi, Ryan Fleming. Engineering strategies to control stoichiometry, site of conjugation, serum stability and effector functions of antibody-drug conjugates. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 660. doi:10.1158/1538-7445.AM2014-660

Aldehyde tag coupled with HIPS chemistry enables the production of ADCs conjugated site-specifically to different antibody regions with distinct in vivo efficacy and PK outcomes.

It is becoming increasingly clear that site-specific conjugation offers significant advantages over conventional conjugation chemistries used to make antibody–drug conjugates (ADCs). Site-specific payload placement allows for control over both the drug-to-antibody ratio (DAR) and the conjugation site, both of which play an important role in governing the pharmacokinetics (PK), disposition, and efficacy of the ADC. In addition to the DAR and site of conjugation, linker composition also plays an important role in the properties of an ADC. We have previously reported a novel site-specific conjugation platform comprising linker payloads designed to selectively react with site-specifically engineered aldehyde tags on an antibody backbone. This chemistry results in a stable C–C bond between the antibody and the cytotoxin payload, providing a uniquely stable connection with respect to the other linker chemistries used to generate ADCs. The flexibility and versatility of the aldehyde tag conjugation platform has enabled us to undertake a systematic evaluation of the impact of conjugation site and linker composition on ADC properties. Here, we describe the production and characterization of a panel of ADCs bearing the aldehyde tag at different locations on an IgG1 backbone conjugated using Hydrazino-iso-Pictet-Spengler (HIPS) chemistry. We demonstrate that in a panel of ADCs with aldehyde tags at different locations, the site of conjugation has a dramatic impact on in vivo efficacy and pharmacokinetic behavior in rodents; this advantage translates to an improved safety profile in rats as compared to a conventional lysine conjugate.

Production of Site-Specific Antibody–Drug Conjugates Using Optimized Non-Natural Amino Acids in a Cell-Free Expression System

Antibody-drug conjugates (ADCs) are a targeted chemotherapeutic currently at the cutting edge of oncology medicine. These hybrid molecules consist of a tumor antigen-specific antibody coupled to a chemotherapeutic small molecule. Through targeted delivery of potent cytotoxins, ADCs exhibit improved therapeutic index and enhanced efficacy relative to traditional chemotherapies and monoclonal antibody therapies. The currently FDA-approved ADCs, Kadcyla (Immunogen/Roche) and Adcetris (Seattle Genetics), are produced by conjugation to surface-exposed lysines, or partial disulfide reduction and conjugation to free cysteines, respectively. These stochastic modes of conjugation lead to heterogeneous drug products with varied numbers of drugs conjugated across several possible sites. As a consequence, the field has limited understanding of the relationships between the site and extent of drug loading and ADC attributes such as efficacy, safety, pharmacokinetics, and immunogenicity. A robust platform for rapid production of ADCs with defined and uniform sites of drug conjugation would enable such studies. We have established a cell-free protein expression system for production of antibody drug conjugates through site-specific incorporation of the optimized non-natural amino acid, para-azidomethyl-l-phenylalanine (pAMF). By using our cell-free protein synthesis platform to directly screen a library of aaRS variants, we have discovered a novel variant of the Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS), with a high activity and specificity toward pAMF. We demonstrate that site-specific incorporation of pAMF facilitates near complete conjugation of a DBCO-PEG-monomethyl auristatin (DBCO-PEG-MMAF) drug to the tumor-specific, Her2-binding IgG Trastuzumab using strain-promoted azide-alkyne cycloaddition (SPAAC) copper-free click chemistry. The resultant ADCs proved highly potent in in vitro cell cytotoxicity assays.