Challenges in Antibody–Drug Conjugate Discovery: A Bioconjugation and Analytical Perspective
2015; Future Science Ltd; Volume: 7; Issue: 13 Linguagem: Inglês
10.4155/bio.15.81
ISSN1757-6199
AutoresChetana Rao, Vangipuram S. Rangan, Shrikant Deshpande,
Tópico(s)Biosimilars and Bioanalytical Methods
ResumoBioanalysisVol. 7, No. 13 EditorialFree AccessChallenges in antibody–drug conjugate discovery: a bioconjugation and analytical perspectiveChetana Rao, Vangipuram S Rangan & Shrikant DeshpandeChetana Rao*Author for correspondence: E-mail Address: chetana.rao@bms.com Biologics Discovery California, Bristol Myers Squibb, 700 Bay Road, Redwood City, CA, USA, Vangipuram S Rangan Biologics Discovery California, Bristol Myers Squibb, 700 Bay Road, Redwood City, CA, USA & Shrikant Deshpande Biologics Discovery California, Bristol Myers Squibb, 700 Bay Road, Redwood City, CA, USAPublished Online:30 Jul 2015https://doi.org/10.4155/bio.15.81AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: antibody–drug conjugateconjugationdrug–antibody ratiopayloadpayload linkerWith the approval of Adcetris to treat Hodgkin's lymphoma and Kadcyla to treat Her2 positive advanced breast cancer, antibody–drug conjugates (ADCs) are fast becoming a well-proven strategy to treat cancer. For successful development of ADCs, several parameters at early stages in research need careful exaanmination. The scope of this editorial is to examine the detailed physicochemical characterization in early discovery phase processes of ADC generation. In particular, here we describe and discuss analytical assays that test the stability of the antibody, of the payload linker, of the ADC during the conjugation process and of the final ADC product.The notion of generating an ADC, although simple, has many moving parts. It is composed of multipart Lego building blocks where an antibody, payload with linker and the mode of attachment of the payload linker to the antibody must have a perfect fit for its success. During the discovery process, the properties of an antibody are carefully chosen for thermal and chemical stabilities, and for binding affinities. An antibody that is nominated for an ADC platform can be screened for internalization properties using established assays such as HumZap or FabZap [1]. Sequence liabilities such as deamidation, oxidation, isomerization, etc., that might present challenges during manufacturing, can be circumvented by a site-directed mutagenesis approach. A careful sequence analysis should be performed to confirm that the generation of these mutants does not compromise the properties including affinity, stability and internalization which can be evaluated using surface plasmon resonance as a tool as well as FACS or ELISA binding to cells or soluble antigen, respectively [2].A payload, the cytotoxic component of an ADC molecule, should be selected with biology in mind. The potency of the payload should be sufficiently high to bring about cell killing in the tumor cell. Tubulin polymerization agents such as auristatins, maytansines [3,4] and DNA strand breakers such as calicheamycin [5] are clinically validated payloads. Other classes, for example, DNA alkylating agents which bind to the minor groove of DNA, such as duocarmycins and pyrrolobenzodiazepines, have shown preclinical efficacy in mouse models [6,7]. The mechanism of action of the payload will determine which end point is preferable to measure the potency of the naked payload.Another part of an ADC, the linker moiety that is attached to the payload, has a considerable effect on the efficacy and the safety profile of the ADC in vivo. The stability in circulation and the susceptibility to cleavage in the tumor microenvironment are prerequisites of a successful linker. Linkers can be of different types such as hydrazone, disulfide linkers and cleavable or noncleavable peptide linkers [8–11]. The mode of release of the active payload from these linkers differs markedly in vivo. Hydrazone linkers are pH sensitive and are cleaved at lower pH in compartments such as endosome and/or lysosome by releasing the active payload. Disulfide linkers take advantage of the reducing environment of the intracellular milieu to release the active payload [12]. Cleavable peptide linkers are known to be cleaved by lysosomal proteases, whereas the noncleavable linkers release the active payload by degradation of antibody in lysosomal compartment. Peptide-based linkers (cleavable and noncleavable) have found more usage in recent times because they exhibit higher plasma stability and improved activity [12]. Evaluation of the stability of the linker is therefore imperative prior to conjugation efforts. Analytical tests such as following UV/VIS spectra by reverse phase HPLC and ESI-ToF-MS can pinpoint any potential instabilities of the payload linker in serum as well as under conjugation conditions [13]. In addition, lysosomal extracts can be used to monitor the efficiency of payload release from the linker [14].Conjugating a hydrophobic payload linker to a hydrophilic antibody is a challenging process. How can a hydrophobic payload linker be favorably attached to a hydrophilic antibody without compromising the biophysical properties of the individual molecules? Understanding the solubility of the antibody and the payload linker under different buffer conditions in the presence or absence of solvents is the first step toward answering the question. Assessing the solubilities of the antibody and the payload linker in a variety of buffer systems at different pH conditions in the absence of any added solvent would be a good starting point. The amount of antibody as measured by size-exclusion chromatography (SEC)-HPLC and payload linker as measured on a reverse phase (RP)-HPLC is determined. A marked reduction in peak area on the two independent analytical HPLC procedures points to loss of the antibody and/or the payload linker. Instability, in this case, could be due to aggregation/precipitation of the antibody or just precipitation in case of the payload linker.Upon identification of pain points, the solubility of the payload linker can be increased by the addition of 5–50% of organic solvents such as dimethlysulfoxide (DMSO), ethylene glycol dimethyl ether (EG-DME), N-methylpyrrolidone (NMP), ethanol, methanol and propylene glycol. Furthermore, the compatibility of the antibody in the presence of these solvents should be evaluated by measuring its aggregation/precipitation propensity in the presence of solvents measured either by SEC-HPLC (or by SEC-MALS for a more comprehensive assay). The nature of the linker should be considered during the conjugation process. For example, for a hydrazone linker, it is prudent to use neutral pH buffers rather than acidic buffers to maintain the integrity of the hydrazone linker. In case of cleavable peptide linkers, the buffers should be free of any contaminating proteases that impact the stability of the payload linker.Several parameters should be evaluated during the conjugation reaction. For example, if maleimide chemistry is used to attach the payload linker to the antibody either via hinge disulfides or using modified lysines via 2-iminothiolane or SMCC, it is desirable to work in a pH range of <7.5 for maintaining the integrity of the maleimide component, which can be hydrolyzed at higher pH. On the other hand, if other chemistries such as oxime chemistry are employed to generate site-specific ADCs, a low pH and presence of a catalyst for conjugation is required [15]. Different catalysts can be evaluated to optimize the conjugation efficiency. Enzyme assisted ligation of payload linker (e.g., bacterial transglutaminase), which results in a homogeneous site-specific ADC, should be carried out at the pH optimal for the enzyme for maximal conjugation. ADCs generated by modifying lysines result in heterogeneous populations making analysis of drug distribution by chromatographic methods, such as hydrophobic interaction, quite challenging. However, for cysteine conjugated or site-specific ADCs, HIC as well as LC–MS are powerful tools to evaluate drug distribution [14,16].During the entire course of the ADC optimization throughout the discovery phase, analytical tests should be in place to evaluate the product generated at every step. Drug–antibody ratio (DAR) is the most critical attribute of an ADC. Therefore, methods employed to measure DAR should have a high level of confidence. UV/VIS spectroscopy methods are commonly used to measure the concentration of the antibody and the DAR. It is of utmost importance to monitor the presence of any free unconjugated payload linker, if any, by precipitation methods using organic solvents and determining the amounts in the supernatant by RP-HPLC or MS. The presence of any unconjugated payload linker in the product can overestimate the reported DAR and is moreover indicative of gaps in purification methods for removal of unconjugated payload linker. In our view, complementary analytical methods for determining DAR should be in place to ensure reliability of the DAR number in the final product. For example, competition ELISA methods can be utilized if an antipayload linker antibody is available. Cleavage followed by the measurement of the active payload from the ADC prep is additionally a reliable method to measure DAR [17,18].A well established SEC or SEC-MALS method aids in keeping track of aggregation during the conjugation process and the final ADC generation. Additionally, SDS-PAGE or CE-SDS can be utilized for estimating purity and identity. Peptide mapping can provide insights regarding the positions where the payload is attached as well as antibody identity. This can be quite challenging for the random lysine generated ADCs. cIEF can be used for evaluating isoforms of the ADC [16]. In addition, HIC can be applied for measuring unconjugated antibody for lysine modified ADC or for drug distribution for cysteine modified and site specific ADCs. For monitoring product heterogeneity, MS methods are also useful [16,19].Does the conjugation process alter the binding ability of the antibody? This is an elementary question that should be raised during the discovery phase for ADC generation. To this point, antigen binding ELISA to measure the binding potency, when the target is a membrane protein, and FACS binding to cell lines expressing the target protein would be useful. To ensure the presence of drug component on the ADC, an ADC binding ELISA using antidrug antibody should also be in place [20].Analytical methods need to distinguish the primary structure of an ADC. This can be done by analyzing intact mass using mass spectrometry, peptide mapping and FTIR, to name a few. Adding a payload linker on the antibody may alter the secondary structure. Analytical tests such as circular dichroism and DSC need to be performed to validate the secondary structure and stability of ADCs [16]. Results from all these analytical methods render confidence in choosing the right molecule. The lessons learnt by employing analytical methods during the ADC discovery phase could be extended to ease the development process and manufacturing of ADCs.ConclusionThe promise of ADCs for treating cancer will continue to be investigated in the future. As the field continues to mature, we as scientists should strive in producing well defined molecules, with rigor, in early discovery space to enhance a smooth transition of these molecules into development and finally to the clinic. Although there are two US FDA approved products in the market, and several others are in clinical development, challenges in the conjugation process and limitations in product characterizations still exist. This is especially true for random conjugated derived ADCs. Due to the inherent heterogeneity of the ADC, characterization of drug load and drug distribution becomes challenging. This heterogeneity of ADCs can be circumvented with the production of site-specific ADCs which result in a homogeneous product, making the physicochemical characterization simpler. However, although there is a huge advantage in producing a homogeneous ADC from a CMC perspective, the clinical proof of activity of such an ADC still remains to be seen. 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This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download
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