Enzyme-Based Labeling Strategies for Antibody–Drug Conjugates and Antibody Mimetics
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antibodies Review Enzyme-Based Labeling Strategies for Antibody–Drug Conjugates and Antibody Mimetics Georg Falck ID and Kristian M. Müller * ID Cellular and Molecular Biotechnology, Faculty of Technology, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany; [email protected] * Correspondence: [email protected]; Tel.: +49-521-1066323 Received: 1 November 2017; Accepted: 18 December 2017; Published: 4 January 2018 Abstract: Strategies for site-specific modification of proteins have increased in number, complexity, and specificity over the last years. Such modifications hold the promise to broaden the use of existing biopharmaceuticals or to tailor novel proteins for therapeutic or diagnostic applications. The recent quest for next-generation antibody–drug conjugates (ADCs) sparked research into techniques with site selectivity. While purely chemical approaches often impede control of dosage or locus of derivatization, naturally occurring enzymes and proteins bear the ability of co- or post-translational protein modifications at particular residues, thus enabling unique coupling reactions or protein fusions. This review provides a general overview and focuses on chemo-enzymatic methods including enzymes such as formylglycine-generating enzyme, sortase, and transglutaminase. Applications for the conjugation of antibodies and antibody mimetics are reported. Keywords: chemo-enzymatic labeling; armed antibody; antibody coupling; antibody conjugation 1. Introduction 1.1. Antibody–Drug Conjugates In the fight against cancer, antibody–drug conjugates (ADC) have gained considerable attention, especially since the market release of Kadcyla and Adcetris, which have doubled in yearly sales since 2013, closing in on a billion dollars [1,2]. A vast number of new ADCs are in the pipeline, with seven of them in pivotal clinical trials [3]. A factor fueling the success of ADCs is the promising expansion of the therapeutic window compared to “naked” therapeutic monoclonal antibodies (mABs) and classical chemotherapy. Furthermore, this approach could revive antibodies with suitable affinities yet insufficient cytotoxicity, and highly potent drugs unfavorable for unspecific systemic application. Improvement of therapeutic efficacy has been proven for the trastuzumab–maytansine conjugate T-DM1 on tumor models refractory to trastuzumab [4]. However, said potential therapeutic window is limited by the heterogeneity, stability, and pharmacokinetics of current ADCs and could be restored by site-specific conjugation strategies [5,6]. The drug to antibody ratio (DAR) emerged as a key term in the discussion of the ideal ADC, which has to be high enough to provide sufficient therapeutic potency as well as low enough to not generate heavily drug-modified conjugates with impaired binding properties, stability, and circulation half-life [7,8]. Depending on the properties of the drug, the ideal DAR for most ADCs is between 2 and 4 [7,9]. Coupling strategies for first generation ADCs have been exclusively chemical with random coupling of thiol groups on reduced cysteines, of side chain amine groups on lysines and aldehyde groups on oxidized glycostructures [10]. The abundance of such modification targets leads to a broad distribution of DARs. Subsequent strategies therefore focused on altering interchain disulfide bonds, changing respective cysteines to serines or introducing new free cysteines [11], as utilized for the THIOMAB conjugation strategy [12,13], or modulating the Antibodies 2018, 7, 4; doi:10.3390/antib7010004 www.mdpi.com/journal/antibodies Antibodies 2018, 7, 4 2 of 19 Antibodies 2018, 7, 4 2 of 18 reactivitycysteine residues of specific for cysteine the π-clamp residues strategy for the [14].π-clamp In contrast, strategy this [14 review]. In contrast, will focus this on review enzyme-based will focus onstrategies enzyme-based to createstrategies homogenous to create antibody–drug homogenous conjugates antibody–drug (Figure 1). conjugates (Figure1). FigureFigure 1. 1.Depiction Depiction ofof unspecificunspecific chemicalchemical couplingcoupling and controlled controlled chemo-enzymatic chemo-enzymatic coupling. coupling. 1.2. Antibody Fragments and Mimetics 1.2. Antibody Fragments and Mimetics Antibody fragments and mimetics were created to generate economical and more convenient high- Antibody fragments and mimetics were created to generate economical and more convenient affinity proteins with alternative properties, though in part similar to those of full antibodies. Intended high-affinity proteins with alternative properties, though in part similar to those of full antibodies. differences to full antibodies are their smaller size and the possibility to be produced in prokaryotic Intended differences to full antibodies are their smaller size and the possibility to be produced in cells. Some of these mimetics are directly derived from their full antibody counterparts, like antigen- prokaryotic cells. Some of these mimetics are directly derived from their full antibody counterparts, like binding fragments (Fab), single-chain variable fragments (scFv) or domain antibodies, such as VHH antigen-binding fragments (Fab), single-chain variable fragments (scFv) or domain antibodies, such as domains (also termed Nanobodies). Nanobodies are derived from camelid heavy–chain–only VantibodiesHH domains and (alsohave termedbeen used Nanobodies). extensively Nanobodiesfor imaging areand derived even therapeutic from camelid applications heavy–chain–only [15]. They antibodiesinhibited antigen and have binding been or used were extensively loaded with for toxi imagingns and and thereby even delayed therapeutic tumor applications growth [16,17]. [15]. TheyScFv inhibitedantibodies antigen for therapy binding are orhuman were loadedor humanized with toxins light and and thereby heavy chain delayed variable tumor fragments growth [16 fused,17]. ScFvby a antibodiesflexible (glycine-serine) for therapy arelinker. human They or have humanized shown in lightcreased and distribution heavy chain across variable tumors fragments while retaining fused by aantigen flexible affinity. (glycine-serine) Their potential linker. They use havefor cancer shown th increasederapy has distribution been discussed across tumors[18–20] whilewith retainingBlincyto antigen(blinatumomab, affinity. anti Their CD3 potential scFv–anti use CD19 for cancer scFv) being therapy an hasapproved been discussed drug. Other [18 –antibody20] with mimetics Blincyto (blinatumomab,consist of conserved anti CD3protein scFv–anti scaffolds CD19 with scFv) variab beingle affinity an approved regions drug.mimicking Other the antibody complementary mimetics consistdetermining of conserved regions proteinof antibodies. scaffolds Designed with variable ankyrin affinity repeat regions proteins mimicking (DARPins) the complementary showed high determiningspecificity and regions good tumor of antibodies. penetration Designed due to their ankyrin small size repeat and proteinsstability [21]. (DARPins) They consist showed of linker high specificityconnected andturn–helix–helix good tumor penetrationmotifs, typically due tothree their plus small two size capping and stability domains, [21 which]. They form consist the ofantigen linker connectedbinding surface. turn–helix–helix Repebodies, motifs, similar typically to DARPins, three are plus composed two capping of repetitive domains, motifs which called form theleucin-rich antigen bindingrepeats organized surface. Repebodies, in a β–strand–turn– similar toα DARPins,–helix structure are composed [22]. Derived of repetitive from the motifs α-helical called Z leucin-richdomain of repeatsstaphylococcus organized protein in a β A,–strand–turn– Affibodies, αthe–helix smallest structure of the [22 antibody]. Derived mimetics from the withα-helical around Z 6 domain kDa, are of staphylococcusmostly used for proteinimaging A, [23]. Affibodies, A lot of the the pros smallest and cons of the of antibodyantibody mimeticsfragments with mimetics around are 6 common, kDa, are mostlysuch as usedtheir forability imaging to penetrate [23]. A tumors, lot of the but pros also and their cons relatively of antibody short fragmentshalf-life in mimeticsblood circulation are common, and, suchdue to as the their lack ability of an to Fc penetrate part, their tumors, missing but antibo alsody-dependent their relatively cell-mediated short half-life cytotoxicity in blood circulation (ADCC) and,and duecomplement-dependent to the lack of an Fc part,cytotoxicity their missing (CDC antibody-dependent[24,25]. While the shorter cell-mediated circulation cytotoxicity time of antibody (ADCC) andmimetics complement-dependent narrows the therapeutic cytotoxicity window (CDC for cancer [24,25 ].th Whileerapy, theit is shorter otherwise circulation beneficial time for of diagnostic antibody mimeticsapproaches, narrows as the thepatient’s therapeutic exposure window to substances for cancer such therapy, as radiolabel it is otherwises is shorter. beneficial The enhanced for diagnostic tumor approaches,penetration, asdue the to patient’s smaller size exposure and typically to substances lower suchaffinities, as radiolabels can be advantageous is shorter. The in enhancedthe treatment tumor of penetration,solid tumors. due Whereas to smaller whole size antibo anddies typically usually lower accumulate affinities, between can be advantageoustumor interstitium in the and treatment tumor ofsurface solid cells, tumors. antibody Whereas fragments whole and antibodies mimetics are usually able to accumulate