International Journal of
Molecular Sciences Review
Methods to Design and Synthesize Antibody-Drug Conjugates (ADCs) Houzong Yao 1,† , Feng Jiang 1,2,† , Aiping Lu 1, * and Ge Zhang 1, * 1 2
* †
Institute for Advancing Translational Medicine in Bone & Joint Diseases, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China;
[email protected] (H.Y.);
[email protected] (F.J.) Faculty of Materials Science and Chemical Engineering, the State Key Laboratory Base of Novel Functional Materials and Preparation Science, Ningbo University, Ningbo 315211, Zhejiang, China Correspondence:
[email protected] (A.L.);
[email protected] (G.Z.); Tel.: +852-3411-2456 (A.L.); +852-3411-2958 (G.Z.) These authors contributed equally to this work.
Academic Editor: Már Másson Received: 11 January 2016; Accepted: 28 January 2016; Published: 2 February 2016
Abstract: Antibody-drug conjugates (ADCs) have become a promising targeted therapy strategy that combines the specificity, favorable pharmacokinetics and biodistributions of antibodies with the destructive potential of highly potent drugs. One of the biggest challenges in the development of ADCs is the application of suitable linkers for conjugating drugs to antibodies. Recently, the design and synthesis of linkers are making great progress. In this review, we present the methods that are currently used to synthesize antibody-drug conjugates by using thiols, amines, alcohols, aldehydes and azides. Keywords: antibody-drug conjugates (ADCs); targeted therapy; monoclonal antibodies (mAbs); drugs; linkers
1. Introduction Cancer is still one of the major threats to human health. However, cancer therapies used today always have more or less adverse side effects to normal tissues. Targeted therapy is a promising strategy to address this challenge. The pioneer of targeted therapy is Paul Ehrlich who introduced the principle “magic bullet” at the beginning of the 20th century [1]. To avoid side effects, drugs should be specifically delivered to cancer cells via binding to ligands that can specifically recognize the cancer-associated biomarkers such as antigens. Among the ligands for targeted therapy, antibodies are excellent candidates because of their specific recognitions and high affinities. Nowadays, antibody-drug conjugates (ADCs) are attracting tremendous attention for targeted cancer therapy. Antibody-drug conjugates are biotherapeutics that consist of monoclonal antibodies, potent cytotoxic drugs and linkers between them (Figure 1). The monoclonal antibodies lead the drug precursors to the target cancer cells, in which the prodrugs can be chemically or enzymatically converted to drugs in their active forms [2]. Conjugating cytotoxins to monoclonal antibodies that specifically tie to tumor cell surface antigens enables the drugs to be target-delivered to cancer cells and leaves normal cells unaffected. More important, many of the cytotoxic drugs that are too toxic for use in traditional chemotherapy can also be used in the construction of antibody-drug conjugates [3,4]. The linkers are also essential parts of antibody-drug conjugates, which account for stability in circulation, good pharmacokinetics and efficient release of toxic drugs in the tumor cells. The selection of antibody, drug, and linker has recently been summarized in a few excellent reviews [5–11]. In this review, we mainly describe the linking methods to design and synthesize ADCs, including those that are not discussed in the reviews mentioned above. Int. J. Mol. Sci. 2016, 17, 194; doi:10.3390/ijms17020194
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The selection of antibody, drug, and linker has recently been summarized in a few excellent The [5–11]. selection drug, and linker hasthe recently been summarized in aand fewsynthesize excellent reviews In of thisantibody, review, we mainly describe linking methods to design reviews [5–11]. In this review, we mainly describe the linking methods to design and synthesize ADCs, including those Int. J. Mol. Sci. 2016, 17, 194 that are not discussed in the reviews mentioned above. 2 of 16 ADCs, including those that are not discussed in the reviews mentioned above.
Figure 1. Schematic representation of an antibody-drug conjugate (ADC). Reprinted with permission Figure 1. from Reference [2]. representation Figure 1. Schematic Schematic representation of of an an antibody-drug antibody-drug conjugate conjugate (ADC). (ADC). Reprinted Reprinted with with permission permission from [2]. from Reference Reference [2].
2. Conjugation via Various Functional Groups to Synthesize Antibody-Drug Conjugates (ADCs) 2. 2. Conjugation Conjugationvia viaVarious VariousFunctional FunctionalGroups Groups to to Synthesize Synthesize Antibody-Drug Antibody-Drug Conjugates Conjugates (ADCs) (ADCs) 2.1. Conjugation via Thiols 2.1. Conjugation via Thiols Employing the thiols of interchain cysteine residues in monoclonal antibodies as attachment the thiols thiolsisofof interchain cysteine residues in monoclonal as attachment the interchain cysteine residues in monoclonal as attachment sites Employing for drug molecules one of the most used conjugation methods.antibodies In antibodies a human IgG1, theresites are sites for drug molecules isof one ofmost the used methods. In a [12]. human there are for molecules is one the used conjugation methods. In a sites human IgG1, there are four fourdrug interchain disulfide bonds that canmost be used as conjugation potential conjugation TheIgG1, four interchain four interchain disulfide bonds be used potential conjugation sites[12]. [12]. The four four interchain interchain disulfide thatthat can be used as as potential conjugation sites The disulfide bonds can bonds be reduced bycan tris(2-carboxyethyl) phosphine (TCEP) or dithiothreitol (DTT), disulfide bonds can be reduced by tris(2-carboxyethyl) phosphine (TCEP) or dithiothreitol (DTT), or dithiothreitol (DTT), which results in eight thiol groups that are available for phosphine conjugating(TCEP) drug molecules. Through this are available available for conjugating conjugating drug molecules. molecules. which results in eight thiol groupsratio that are for drug Through this method, different drug antibody (DAR) conjugates will be obtained when targeting typical method, drug antibody antibody-drug ratio (DAR) conjugates be obtained when targeting typical DARs of different 2–4 [13,14]. In addition, conjugatewill at each drug antibody ratio has several DARs of 2–4 [13,14]. In addition, antibody-drug conjugate at each drug antibody ratio has several DARs of Thus, 2–4 [13,14]. In addition, antibody-drug each drug antibody ratio has several isomers. over a hundred different species areconjugate present inatthe antibody-drug conjugate. Although isomers. Thus, over a hundred different species are present in the antibody-drug conjugate. Although isomers. Thus, over a hundred different species are present in the antibody-drug conjugate. Although conventional methods that employ cysteine residues as conjugation sites are highly heterogeneous, conventional that conventional methods that employ cysteine residues residues as as conjugation conjugation sites sites are are highly highly heterogeneous, heterogeneous, Adcetris® wasmethods approved by employ FDA in cysteine 2011. ® ® Adcetris was by 2011. Adcetris was approved approved by FDA FDA in in conjugates 2011. Homogeneous antibody-drug can be produced through cysteine residues when all Homogeneous conjugates can through cysteine residues when all Homogeneous antibody-drug conjugates can be be produced produced through cysteine residues when all interchain cysteines antibody-drug are coupled to drugs. For example, Senter and coworkers [15,16] developed such interchain cysteines are coupled to drugs. For example, Senter and coworkers [15,16] developed such interchain cysteines coupledof to cAC10, drugs. For Senter and coworkers [15,16] developed such a conjugate which are consisted an example, anti-CD30 monoclonal antibody, and monomethyl aaauristatin conjugate which consisted of cAC10, anconjugate anti-CD30 monoclonal antibody, and monomethyl conjugate consisted of cAC10, an anti-CD30 monoclonal antibody, and per monomethyl auristatin Ewhich (MMAE). This cAC10-vcMMAE contains eight drugs antibody, which is auristatin (MMAE). This ratio cAC10-vcMMAE conjugate contains eight drugs antibody, is E (MMAE). cAC10-vcMMAE conjugate contains eight drugs per antibody, which iscysteines thewhich highest the highestE This drug antibody (DAR) that can be obtained through using per interchain as the highest drug antibody ratio (DAR) that can be obtained through using interchain cysteines as drug antibody ratioHowever, (DAR) that can be obtained through with usingfour interchain as conjugation sites. conjugation sites. antibody-drug conjugates drugs cysteines per antibody generally have conjugation sites. However, antibody-drug conjugates with four drugs per antibody generally However, antibody-drug conjugates with four per antibody improved inhave vivo improved in vivo performance [17]. McDonagh etdrugs al. [18] developed agenerally method tohave control the conjugate improved in vivo performance [17]. McDonagh et al. [18] developed a method to control the conjugate performance [17]. McDonagh [18]interchain developedcysteines a methodtotoserines, control the conjugate sites by mutating sites by mutating four or sixetofal.the therefore leaving four or two sites bysix mutating four or six of the(Scheme interchain cysteines to serines, leaving fourmutated or two four or of the interchain cysteines to serines, leaving four or two cysteines for cysteines accessible for conjugating 1). therefore After reduction of thetherefore disulfide bonds,accessible the cysteines accessible for conjugating (Scheme 1). After reduction of the disulfide bonds, the mutated conjugating (Scheme 1). After reduction of the disulfide bonds, the mutated monoclonal antibodies monoclonal antibodies with the reduced number of interchain cysteines were conjugated with the monoclonal antibodies with the reduced numberwere ofantibody-drug interchain cysteines conjugated with the with reduced number of interchain cysteines conjugated with thewere drug vcMMAE. Through drug the vcMMAE. Through this method, homogenous conjugates with clear attachment drug vcMMAE. Through this method, homogenous conjugates clear this antibody-drug conjugatesantibody-drug with clear attachment siteswith could be attachment produced. sitesmethod, could behomogenous produced. sites could be produced.
Scheme 1. 1. Interchain conjugate to to the the remaining remaining Scheme Interchain cysteine cysteine to to serine serine mutagenesis mutagenesis enables enables drugs drugs to to conjugate Scheme 1. Interchain cysteine to serine mutagenesis enables drugs to conjugate to the remaining cysteines. Adapted from reference [18]. cysteines. Adapted from reference [18]. cysteines. Adapted from reference [18].
Reducing the disulfide bonds of a monoclonal antibody should not affect its functions [19]. What Reducing the disulfide bonds of monoclonal antibody should not affect functions [19]. What Reducing the disulfide bonds of aaare monoclonal antibody should affect its itsdisulfide functions [19]. What is more, interchain disulfide bonds easier to be reduced than not intrachain bonds [20]. is more, interchain disulfide bonds are easier to be reduced than intrachain disulfide bonds [20]. These is more, interchain disulfide bonds are easier to be reduced than intrachain disulfide bonds [20]. allow free thiol groups to be generated under mild reducing conditions while leaving the antibody intact at the same time. Liu et al. [21] took advantage of the fact that different disulfide bonds in a
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These allow allow free free thiol thiol groups groups to to be be generated generated under under mild mild reducing reducing conditions conditions while while leaving leaving the the These antibody intact at the same time. Liu et al. [21] took advantage of the fact that different disulfide bonds antibody intact at the same time. Liu et al. [21] took advantage of the fact that different disulfide bonds in aa monoclonal monoclonal antibody have different different susceptibilities towards reduction and developed developed another monoclonal antibody have different susceptibilities towards reduction and developed another strategy in antibody have susceptibilities towards reduction and another strategy to tightly control the site of conjugation. Limited reduction with TCEP or DTT to tightly control the site of conjugation. Limited reduction with TCEP or DTT predominantly yielded strategy to tightly control the site of conjugation. Limited reduction with TCEP or DTT predominantly yielded conjugates in which drugs were attached to heavy-light chain disulfides; conjugates in which drugs were attached to heavy-light chain disulfides; partial re-oxidation of fully predominantly yielded conjugates in which drugs were attached to heavy-light chain disulfides; 1 -dithiobis partial re-oxidation of fully reduced antibodies with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) reduced antibodies with 5,5 (2-nitrobenzoic acid) (DTNB) yielded conjugates that drugs partial re-oxidation of fully reduced antibodies with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) yielded conjugates that drugs were mainly attached to by heavy-heavy chain disulfides [13]. were mainly attached to by heavy-heavy chain disulfides [13]. yielded conjugates that drugs were mainly attached to by heavy-heavy chain disulfides [13]. 2.1.1. Addition to 2.1.1. Addition to Maleimides Maleimides Classically, Classically, cysteine cysteine residues residues can can be be modified modified through through addition addition of of thiols thiols to Classically, cysteine residues can be modified through addition of thiols to electrophiles electrophiles such such as as maleimides (Scheme 2) [22–25]. The conjugate could be achieved by reducing the disulfide bonds maleimides (Scheme 2) [22–25]. The conjugate could be achieved by reducing the disulfide bonds of of the then adding adding to to maleimides. maleimides. Addition most common common method method the antibody antibody and and then then adding to maleimides. Addition to to maleimides maleimides is is the the most most common method ® which was approved by ® for for the attaching drugs to antibodies. antibodies. Adcetris which was was approved approved by by the the FDA FDA for for attaching attaching drugs to to antibodies. Adcetris®,,, which the treatment treatment of of patients with Hodgkin’s lymphoma after failed autologous stem cell transplantation or patients with patients with patients with Hodgkin’s lymphoma after failed autologous stem cell transplantation or patients with systemic large-cell lymphoma after the failure at leastof systemic anaplastic anaplastic large-cell lymphoma after the of failure ofoneat atprior leastmulti-agent one prior priorchemotherapy multi-agent systemic anaplastic large-cell lymphoma after the failure least one multi-agent regimen, was produced by this method in which a maleimide-functionalized drug was conjugated chemotherapy regimen, regimen, was was produced produced by by this this method method in in which which aa maleimide-functionalized maleimide-functionalized drug chemotherapy drug to the interchain cysteine residues of an anti-CD30 antibody [15]. Maleimide-based antibody-drug was conjugated to the interchain cysteine residues of an anti-CD30 antibody [15]. Maleimide-based was conjugated to the interchain cysteine residues of an anti-CD30 antibody [15]. Maleimide-based conjugates wereconjugates recently found have limited blood circulation which would lower antibody-drug conjugates weretorecently recently foundstability to have haveinlimited limited stability in in[26], blood circulation [26], antibody-drug were found to stability blood circulation [26], the efficacy of the conjugates and damage healthy tissue. Succinimide or maleimide hydrolysis or is which would lower the efficacy efficacy of the the conjugates conjugates and damage damage healthy tissue. Succinimide Succinimide or which would lower the of and healthy tissue. amaleimide promisinghydrolysis method tois around this problem. Once hydrolyzed, the antibody-drug conjugates maleimide hydrolysis isget a promising method to get around this problem. Once hydrolyzed, the a promising method to get around this problem. Once hydrolyzed, the were no longer conjugates subject to elimination reactions of maleimides through retro-Michael reactions, thus antibody-drug conjugates were no no longer longer subject to elimination elimination reactions of maleimides maleimides through antibody-drug were subject to reactions of through improving thereactions, stabilities thus and potencies ADCs [27–29]. retro-Michael reactions, thus improvingofthe the stabilities and potencies potencies of of ADCs ADCs [27–29]. [27–29]. retro-Michael improving stabilities and
Scheme 2. 2. The The synthesis of antibody-drug conjugates (ADCs) through addition of of thiols thiols to to Scheme The synthesis synthesis of of antibody-drug antibody-drug conjugates conjugates (ADCs) (ADCs) through through the the addition Scheme 2. maleimides. Adapted from reference [23]. maleimides. Adapted from reference [23]. maleimides. Adapted from reference [23].
2.1.2. Disulfide-Thiol Disulfide-Thiol Exchange Exchange 2.1.2. The approach approach disulfide-thiol disulfide-thiol exchange exchange could could also also be be used used to to synthesis synthesis ADCs ADCs by by forming forming aa new new The disulfide bond between drugs and antibodies [30,31]. Ojima et al. [30] designed and synthesized novel disulfide bond bond between between drugs drugs and and antibodies antibodies [30,31]. [30,31]. Ojima Ojima et al. [30] designed and synthesized novel disulfide antibody-taxoid conjugates that include highly cytotoxic taxoid drug drug and and monoclonal monoclonal antibodies antibodies that that antibody-taxoid conjugates conjugates that include highly cytotoxic taxoid antibody-taxoid could recognize the EGFR expressed in cancer cells. In this study, taxoid bearing a free thiol group could recognize the EGFR expressed in cancer cells. In this study, taxoid bearing a free thiol group was attached attachedtoto tothe thepyridyldithio pyridyldithiogroups groups of the modified anti-EGFR antibodies through disulfidewas attached the pyridyldithio groups modified anti-EGFR antibodies through disulfidewas of of thethe modified anti-EGFR antibodies through disulfide-thiol thiol exchange exchange (Scheme 3). The The resulting conjugates possessremarkable remarkableantitumor antitumoractivities activities against against thiol (Scheme 3). resulting conjugates possess remarkable antitumor activities exchange (Scheme 3). The resulting conjugates possess EGFR-expressing A431 (human epidermoid) tumor xenografts in immune deficient mice. (human epidermoid) epidermoid) tumor tumor xenografts xenografts in in immune immune deficient deficientmice. mice. EGFR-expressing A431 (human
Scheme 3. 3. Preparation Preparation of of antibody-taxoid antibody-taxoid conjugates conjugates via via disulfide-thiol disulfide-thiol exchange. exchange. Adapted Adapted from from Scheme Scheme 3. Preparation of antibody-taxoid conjugates via disulfide-thiol exchange. Adapted from reference [30]. reference [30]. [30]. reference
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2.1.3. Addition to Alkynes
Addition to Alkynes To 2.1.3. avoid the maleimide instability issue, Kolodych et al. [32] developed a heterobifunctional To avoid the maleimide instability issue, Kolodych et al. [32] developed a heterobifunctional reagent, sodium 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF), for reagent, sodium 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF), for amine-to-thiol coupling (Scheme 4). This reagent comprises a 3-arylpropionitrile (APN)4 of group that Int. J. Mol. Sci. 2016, 17, 194 16 amine-to-thiol coupling (Scheme 4). This reagent comprises a 3-arylpropionitrile (APN) group that replaces the maleimide and allows for the preparation of remarkably stable conjugates. Addition replaces the maleimide and allows for the preparation of remarkably stable conjugates. Addition of to Alkynes of thiols2.1.3. in Addition the 3-arylpropionitriles predominantly produced Z-isomers thiols in theantibodies antibodies totothethe 3-arylpropionitriles predominantly produced Z-isomers of the addition of the To avoid the maleimide instability issue, Kolodych et al. [32] developed a heterobifunctional additionproducts. products. reagent, sodium 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF), for amine-to-thiol coupling (Scheme 4). This reagent comprises a 3-arylpropionitrile (APN) group that replaces the maleimide and allows for the preparation of remarkably stable conjugates. Addition of thiols in the antibodies to the 3-arylpropionitriles predominantly produced Z-isomers of the addition products.
Scheme
4.
The
conjugation
of
amine-drug
to
trastuzumab
by
using
sodium
4-((4-
Scheme 4. The conjugation of amine-drug to trastuzumab by using sodium (cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF). Reprinted with permission 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF). Reprinted with from reference [32]. permission from reference [32]. 2.1.4. Disulfide Re-Bridging Scheme 4. The conjugation of amine-drug to trastuzumab by using sodium 4-((42.1.4. Disulfide Re-Bridging Recently, various novel cysteine-relied conjugation methods have been developed [33]. Godwin (cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF). Reprinted with permission
and coworkers [34–36] developed a thiol conjugation approach in which interchain disulfide bonds
from referencenovel [32]. cysteine-relied conjugation methods have been developed [33]. Godwin Recently, various of the cysteines were partially reduced, followed by bis-alkylation (including Michael addition and and coworkers [34–36] developed a thiol conjugation approach in5). which interchain elimination) to introduce thiols of two cysteines to the drug (Scheme Depending on the disulfide reduction bonds 2.1.4. Disulfide Re-Bridging of the cysteines partially reduced, followedcan bybe bis-alkylation Michael and degree, thewere numbers of cysteines for conjugation eight or four to (including generate drug antibodyaddition ratios Recently, novel cysteine-relied methods havethe been developed [33]. Godwin (DARs) fourvarious and two, respectively. Theyconjugation also that thiobridge ADCs are more elimination) toofintroduce thiols of two cysteines todemonstrated the drug (Scheme 5). Depending on the reduction and coworkers [34–36]ADCs developed ahuman thiol conjugation approach in which interchain disulfide bonds than maleimide in serum. degree, stable the numbers of cysteines fortheconjugation can be eight or four to generate drug antibody ratios of theBehrens cysteines were partially reduced, followed by bis-alkylation (including Michaelresidues, additionthen and et al. [37] reduced all the disulfide bonds, exposing eight cysteine (DARs)elimination) of four and two, respectively. They alsotodemonstrated that the thiobridge ADCs are more to introduce thiols of two cysteines the drug 5). groups Depending on antibody the reduction similarly used dibromomaleimide (DBM) to react with the(Scheme free thiol of the and stable than maleimide ADCs in the human serum. cytotoxic degree, theanumbers of cysteines for conjugation be eightdrugs or fourwith to generate drug antibody produced dithiomaleimide (DTM) ADC. Fourcan this functional linker ratios were Behrens et al. [37] reduced all the disulfide bonds, eight cysteine residues, then similarly (DARs) and two, respectively. They also demonstrated that thecysteine thiobridge ADCs are more attachedof tofour the monoclonal antibodies conveniently byexposing linking with the residues. stable than maleimide ADCs in the human serum. used dibromomaleimide to [27,38–40] react with the freea thiol groups of the antibody and produced a Chudasama and(DBM) coworkers presented significant method towards next-generation Behrens et therapeutics al. [37] reduced all disulfide the disulfide bonds, exposing eight residues, then to the antibody-based through re-bridging. their works, thecysteine reduction of disulfides dithiomaleimide (DTM) ADC. Four cytotoxic drugs with In this functional linker were attached similarly used dibromomaleimide (DBM) to react with the free thiol groups of the antibody and and disulfide re-bridging could by belinking achieved in the one cysteine step by residues. the use of a single reagent: monoclonal antibodies conveniently with produced a dithiomaleimide (DTM) ADC. Four cytotoxic drugs with this functional linker were dithioaryl(TCEP)pyridazinedione [38]. Disulfide re-bridging through the use of Chudasama andmonoclonal coworkers [27,38–40] presented significant method towards next-generation attached to the antibodies conveniently by alinking with the cysteine residues. dibromopyridazinedione derivatives after disulfide reduction by TCEP was another strategy for the antibody-based therapeutics through disulfide re-bridging. InThe their works, the reduction of Chudasama and coworkers [27,38–40] presented a significant method towards next-generation construction of antibody-based therapeutics in their studies [39,40]. resulting conjugates were antibody-based therapeutics through disulfide re-bridging. In their the reduction disulfides disulfides and disulfide re-bridging could be achieved in works, one step by the ofuse of a single highly stable and had potent cytotoxicites against tumor cells. disulfide re-bridging could be achieved step by re-bridging the use of a through single reagent: reagent:anddithioaryl(TCEP)pyridazinedione [38].in one Disulfide the use of dithioaryl(TCEP)pyridazinedione [38]. Disulfide re-bridging through the use of dibromopyridazinedione derivatives after disulfide reduction by TCEP was another strategy for dibromopyridazinedione derivatives after disulfide reduction by TCEP was another strategy for the the construction of antibody-based therapeutics in their studies [39,40]. The resulting conjugates were construction of antibody-based therapeutics in their studies [39,40]. The resulting conjugates were highly stable cytotoxicites cells. highly and stablehad andpotent had potent cytotoxicitesagainst against tumor tumor cells.
Scheme 5. Three approaches to make ADCs through disulfide re-bridging: thiobridge, dibromomaleimide and pyridazinedione. Adapted from reference [35,37,40].
Scheme 5. Three approaches to make ADCs through disulfide re-bridging: thiobridge,
Scheme 5. Three approaches to make ADCs through disulfide re-bridging: dibromomaleimide and pyridazinedione. Adapted from reference [35,37,40]. dibromomaleimide and pyridazinedione. Adapted from reference [35,37,40].
thiobridge,
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Ligation (EPL) (EPL) and Alkylguanine-DNA-Alkyl Alkylguanine-DNA-Alkyl Transferase(AGT) (AGT) Reaction 2.1.5. 2.1.5. Expressed Expressed Protein Protein Ligation Ligation (EPL) and and Alkylguanine-DNA-Alkyl Transferase Transferase (AGT)Reaction Reaction Another Another strategy strategy for for making making homogeneous homogeneous ADCs ADCs was was inserting inserting entire entire domains domains or or proteins proteins into into The most well known such method isisexpressed protein ligation (EPL), which relied a antibodies. The most well known such method expressed protein ligation (EPL), which reliedon antibodies. The most well known such method is expressed protein ligation (EPL), which relied onona self-splicing intein to activate the C-terminal of the target protein and thus formed a new amide bond aself-splicing self-splicing intein activate C-terminal of the target protein formed new amide intein to to activate thethe C-terminal of the target protein and and thusthus formed a newa amide bond with with a small molecule, peptide or or protein. EPL followed the mechanism: (1) C-terminal thioester thioester bond a small molecule, peptide protein. EPL followed the mechanism: (1) with a small molecule, peptide or protein. EPL followed the mechanism: (1) C-terminal thioester through rearrangement an selected thiol displacement of formation through the the spontaneous spontaneousN Nto toS rearrangementof anintein; intein;(2) selected thiol displacement formation through the spontaneous N to SSrearrangement ofofan intein; (2)(2)selected thiol displacement of the intein sequence to give an activated thioester; (3) thiol exchange of the thioester with a β-amino of the intein sequence to give an activated thioester; (3) thiol exchange of the thioester a β-amino the intein sequence to give an activated thioester; (3) thiol exchange of the thioester with a β-amino ligand oror protein); andand (4) spontaneous N toNCto rearrangement to form mercapto ligand(small (smallmolecule, molecule,peptide peptide protein); spontaneous C rearrangement to mercapto ligand (small molecule, peptide or protein); and (4) (4) spontaneous N to C rearrangement to form an stable amideamide bond that links the antibody of interest to the drug with a inserted cysteine (Scheme 6) form an stable bond that links the antibody of interest to the drug with a inserted cysteine an stable amide bond that links the antibody of interest to the drug with a inserted cysteine (Scheme 6) [34]. (Scheme 6) [34]. [34].
Scheme 6. 6. Amino mercapto-derivitized mercapto-derivitized drug can be to Scheme be attached attached to an an antibody antibody through through intein intein splicing. splicing. Scheme 6. Amino Amino mercapto-derivitized drug drug can can be attached to an antibody through intein splicing. Reprinted with permission from reference [34]. Reprinted with permission from reference [34]. Reprinted with permission from reference [34].
Proteins that do not interfere with the function of an insertion can take advantage of the human interfere with with the function of an insertion can take advantage of the human Proteins that do not interfere O666-alkylguanine-DNA alkyltransferase (hAGT) reaction in which the guanine attached to the O666 O -alkylguanine-DNA alkyltransferase to the the O O -alkylguanine-DNA alkyltransferase (hAGT) (hAGT) reaction reaction in in which the guanine attached to benzyl group is attacked by the cysteine of hAGT and thus transferred to the drug-AGT conjugate attacked by the the cysteine cysteine of of hAGT hAGT and and thus thus transferred transferred to to the the drug-AGT drug-AGT conjugate conjugate benzyl group is attacked (Scheme 7). To realize this reaction, hAGT was directly evolved to possess comparable kinetics to the (Scheme 7). 7). To To realize realize this this reaction, reaction, hAGT hAGT was was directly directly evolved evolved to to possess possess comparable comparable kinetics kinetics to the (Scheme wild type hAGT, while retaining the substrate tolerance for the O-benzyl moiety [41]. wild type type hAGT, hAGT,while whileretaining retainingthe thesubstrate substratetolerance tolerancefor forthe theO-benzyl O-benzylmoiety moiety[41]. [41]. wild
Scheme 7. Human O66-alkylguanine-DNA alkyltransferase (hAGT) used guanine as a leaving group, Scheme 7. 7. Human Human O O6-alkylguanine-DNA Scheme -alkylguanine-DNA alkyltransferase alkyltransferase (hAGT) (hAGT) used used guanine guanine as as aa leaving leaving group, group, forming a thioether bond to a benzyl-derivitized drug. Adapted from Reference [41]. forming a thioether bond to a benzyl-derivitized drug. Adapted from Reference [41]. forming a thioether bond to a benzyl-derivitized drug. Adapted from Reference [41].
2.2. Conjugation via Amines 2.2. Conjugation Conjugation via via Amines Amines 2.2. 2.2.1. Formation of Amides 2.2.1. Formation Formation of of Amides Amides 2.2.1. Forming amide is one of the most important reactions for the nucleophilic amines. Amines could Forming amide of theofmost reactions reactions for the nucleophilic Amines could Forming amideis one is one the important most important for the amines. nucleophilic amines. typically be acylated by carboxyl via some familiar activating reagents, such as N-hydroxysuccinimide typically be acylated by carboxyl via some familiar activating reagents, such as N-hydroxysuccinimide Amines could typically be acylated by carboxyl via some familiar activating reagents, such as (NHS), 2-Succinimido-1,1,3,3-tetra-methyluronium tetrafluoroborate (TSTU), and Benzotriazol-1-yl(NHS), 2-Succinimido-1,1,3,3-tetra-methyluronium tetrafluoroborate (TSTU), and Benzotriazol-1-ylN-hydroxysuccinimide (NHS), 2-Succinimido-1,1,3,3-tetra-methyluronium tetrafluoroborate (TSTU), oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP). oxytripyrrolidinophosphonium hexafluorophosphate hexafluorophosphate (PyBOP). and Benzotriazol-1-yl-oxytripyrrolidinophosphonium (PyBOP). Amines of the antibodies can react with the carboxyls that derived from the drugs in the effect Amines of of the theantibodies antibodiescan canreact reactwith withthe thecarboxyls carboxylsthat thatderived derived from drugs effect Amines from thethe drugs in in thethe effect of of the NHS to give antibody-drug conjugates (Scheme 8) [22,23,42]. Amines of lysines are commonly of the NHS to give antibody-drug conjugates (Scheme 8) [22,23,42]. Amines of lysines are commonly the NHS to give antibody-drug conjugates (Scheme 8) [22,23,42]. Amines of lysines are commonly used used for linking drugs to antibodies because lysines are usually exposed on the surface of the used for linking drugs to antibodies lysines areexposed usually on exposed on the surface of the for linking drugs to antibodies becausebecause lysines are usually the surface of the antibodies antibodies and therefore easily accessible. Antibodies contain up to 80 lysines [43] and, as a result, antibodies and therefore easily accessible. Antibodies contain up to 80 lysines [43] and, as a result, and therefore easily accessible. Antibodies contain up to 80 lysines [43] and, as a result, conjugation conjugation through lysine residues inevitably leads to twofold heterogeneity: (1) different number conjugation through lysine residues inevitably leads to twofold heterogeneity: (1) different number through lysine residues inevitably leads to twofold heterogeneity: (1) different number of drugs per of drugs per antibody; and (2) antibodies with the same number of drugs attached at different sites of drugs per antibody; and (2) antibodies with the same number of drugs attached at different sites antibody; and (2) antibodies with the same number of drugs attached at different sites [31,44]. The [31,44]. The heterogeneity with respect to DARs can be restricted to a certain extent by adjusting the [31,44]. The heterogeneity respect can to bearestricted to a certain extentthe bystoichiometry adjusting the heterogeneity with respect with to DARs can to be DARs restricted certain extent by adjusting stoichiometry of drug and antibody used in the reaction; and with respect to site-specificity, the stoichiometry of drugused andinantibody usedand in with the reaction; with respect site-specificity, of drug and antibody the reaction; respect toand site-specificity, thetoheterogeneity canthe be heterogeneity can be limited by the chemical accessibility of reactive groups [45,46]. Mylotarg®® was ® heterogeneity can be limited by the chemical accessibility of reactive groups [45,46]. Mylotarg was limited by the chemical accessibility of reactive groups [45,46]. Mylotarg was the first antibody-drug the first antibody-drug conjugate on the market by lysine-coupling. In the conjugate, a semi-synthetic the first antibody-drug conjugate on the market by lysine-coupling. In the conjugate, a semi-synthetic calicheamicin derivative was activated with NHS, and then attached to the lysines of a humanized calicheamicin derivative was activated with NHS, and then attached to the lysines of a humanized
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conjugate on the market by lysine-coupling. In the conjugate, a semi-synthetic calicheamicin derivative Int. J. Mol. Sci. 2016, 17, 194 6 of 16 was with and then attached to the lysines of a humanized IgG4 [47]. However, Int.activated J. Mol. Sci. 2016, 17, NHS, 194 6 of 16 ® ® Mylotarg was withdrawn from the in 2010 duethe to the lack in of 2010 benefit improvement patients. Int. J. Mol. Sci. 2016, 17, 194Mylotarg 6 of 16 IgG4 [47]. However, wasmarket withdrawn from market due to the lack oftobenefit ® was withdrawn from the market in 2010 due to the lack of benefit IgG4 [47]. However, Mylotarg Recently, there was new clinically relevant conjugate generated by lysine modification: improvement to apatients. Recently, there antibody-drug was a new clinically relevant antibody-drug conjugate ® was withdrawn improvement to patients. Recently, there® was newthe clinically antibody-drug conjugate ® [48]. IgG4 [47]. However, Mylotarg market relevant in 2010 due to the lack of benefit Kadcyla generated by lysine modification: Kadcyla [48]. afrom generated by lysine modification: Kadcyla [48].a new clinically relevant antibody-drug conjugate improvement to patients. Recently, there ®was generated by lysine modification: Kadcyla® [48].
Scheme 8. Amines of the reacted with the carboxyls that derived from thefrom drugsthe in the Scheme 8. Amines of antibodies the antibodies reacted with the carboxyls that derived Scheme 8. Amines of the antibodies reacted with the carboxyls that derived from the effect of the N-hydroxysuccinimide (NHS). Adapted from reference [23]. drugs in the effect of the N-hydroxysuccinimide (NHS). Adapted from reference [23]. drugs in8.the effect of the N-hydroxysuccinimide Adaptedthat fromderived reference [23]. Scheme Amines antibodies reacted with(NHS). the carboxyls from the
drugs in the of the N-hydroxysuccinimide (NHS). Adapted from reference [23]. Hong al.[49] [49]effect developed an approach attach thethe anticancer drugdrug doxorubicin to Hong etetal. developed approachtotocovalently covalently attach anticancer doxorubicin Hong et al. [49] developed an(Fab’) approach to covalently attach the anticancer drug doxorubicin to an anti-EGFR antibody fragment through a polyethylene glycol (PEG) linker. In this work, to an anti-EGFR antibody fragment (Fab’) through a polyethylene glycol (PEG) linker. In this work, an anti-EGFR fragment through a polyethylene glycol (PEG) drug linker.doxorubicin In this work, Hong et al.antibody [49] developed an (Fab’) approach to covalently attach the anticancer to CIT–(CH 2)5–PEG24–CO2H was activated by TSTU or PyBOP and then coupled in situ with the CIT–(CH 2 )5 –PEG24 –CO2 H was activated by TSTU or PyBOP and then coupled in situ with the CIT–(CH 2)5–PEG 24–CO2H was activated by TSTU or PyBOP and then coupled in situ with the an anti-EGFR antibody fragment (Fab’) through a polyethylene glycol (PEG) linker. In this work, 1 -amine ofof DOXtotogive giveCIT–(CH CIT–(CH)2)5–PEG –PEG24–DOX, which was then conjugated with the cysteine C3C3′-amine DOX 2 5 24 –DOX, which was then conjugated with the cysteine C3′-amine of DOX to 2give CIT–(CH 2)5–PEG 24 –DOX, which wasand then conjugated thewith cysteine CIT–(CH 2)5–PEG 24–CO H Fab’. was activated by of TSTU or PyBOP then coupled with in situ the residues of an anti-EGFR Introduction PEG increased the drug, which residues of an anti-EGFR Fab’. Introduction of PEG increasedaqueous aqueoussolubility solubilityofof the drug, which residues of an anti-EGFR Fab’. Introduction of PEG increased aqueous solubility of the drug, which C3′-amine of DOX to give CIT–(CH 2)5–PEG24 –DOX, which was then conjugated with the cysteine a yield improvementofofthe theconjugation conjugation reaction reaction with with the ledled to to a yield improvement the Fab’. Fab’. led to a yield improvement of the conjugation withwith the Fab’. residues of anfamiliar anti-EGFR Fab’. Introduction of reaction PEG increased aqueous solubility of their the drug, which Besides activating reagent, amines could react carboxyl acids and derivatives Besides familiar activating reagent, amines could react with carboxyl acids and their derivatives to Besides familiar activating reagent, amines could react with carboxyl acids and their derivatives led to a yield improvement of the conjugation reaction with the Fab’. to form the amides under the influence of the BTG, SrtA and BirA. For example, Jeger et al. [50] form the amides under the influence of the BTG, SrtA and BirA. For example, Jeger et al. [50] observed to form the amides under the reagent, influence ofbythe BTG, SrtA with and BirA. Foracids example, Jeger et al. [50] Besides activating amines could react carboxyl andflexible their derivatives observed thefamiliar selective acylation of amines the glutamines in the heavy chain’s regions of theto selective acylation ofacylation amines byamines the glutamines inSrtA the and heavy chain’s flexibleflexible regions of IgG observed the selective of by the glutamines in the heavy chain’s regions of form the amides under the influence of the BTG, BirA. For example, Jeger et al. an [50] an IgG where the asparagines (N) were mutated to glutamines (Q). By using bacterial where (N) were mutated towere glutamines (Q).toByin using bacterial (BTG) an the IgGasparagines where the asparagines (N)they glutamines (Q).transglutaminase Byflexible using bacterial observed the selective acylation amines by themutated glutamines the heavy chain’s regions of transglutaminase (BTG) at theseofsites, synthesized homogeneous conjugates that were tumorat an these sites, they synthesized homogeneous conjugates that were tumor-uptake selective in vivo transglutaminase (BTG) at these sites, they synthesized homogeneous conjugates that were tumorIgGselective where inthe uptake vivoasparagines (Scheme 9). (N) were mutated to glutamines (Q). By using bacterial (Scheme uptake9). selective in(BTG) vivo (Scheme transglutaminase at these 9). sites, they synthesized homogeneous conjugates that were tumoruptake selective in vivo (Scheme 9).
Scheme 9. The reaction of the amine-drugs and antibodies under the influence of bacterial Scheme The reaction reaction of the the from amine-drugs transglutaminase (BTG). Adapted reference and [50]. antibodies Scheme 9. 9. The of amine-drugs and antibodies under underthe theinfluence influenceofofbacterial bacterial transglutaminase (BTG). Adapted from reference [50]. Scheme 9. The reaction of the amine-drugs and antibodies under the influence of bacterial transglutaminase (BTG). Adapted from reference [50]. transglutaminase Adapted fromsortases referencefound [50]. in Gram-positive bacteria, is a transpeptidase Sortase A (SrtA),(BTG). one of the many
Sortase A (SrtA), one of the many sortases found in Gram-positive bacteria, is a transpeptidase [51] that can recognize sequence, break theinTG bond and facilitate theisformation of a new Sortase A (SrtA), oneaaofLPXTG the many sortases found Gram-positive bacteria, a transpeptidase [51] [51] that can recognize LPXTG sequence, break the TG bond and facilitate the formation of a new Sortase A (SrtA), one of the many sortases found in Gram-positive bacteria, is a transpeptidase amide bond with the amine of glycine-derivitized drug. This was demonstrated through conjugating that can recognize a LPXTG sequence, break the TG bond and facilitate the formation of a new amide amide bond the amine of glycine-derivitized drug. was demonstrated [51] that can with recognize a LPXTG sequence, break the TGThis bond and facilitate thethrough formation of a new folate, biotin, rhodamine and so on [52,53]. The leaving glycine could return as a conjugating competing bond with the amine of glycine-derivitized drug. This was demonstrated through conjugating folate, folate, biotin, rhodamine and so on [52,53]. The leaving glycine could return as a competing amide bond with the amine of glycine-derivitized drug. This was demonstrated through conjugating nucleophile, which reversed the reaction, unless more than 10 equivalents of glycine-derivitized biotin, rhodamine and so on [52,53]. The leaving glycine could return as a competing nucleophile, nucleophile, which reversed the unless than 10 equivalents ofsolution glycine-derivitized folate, biotin, rhodamine and[54]. so reaction, on [52,53]. The leaving glycine return as atocompeting drugs were used (Scheme 10) Williamson et al.more [55] put forward acould creative solve this which reversed the (Scheme reaction, unless more than 10 equivalents of glycine-derivitized drugs werethis used drugs were used 10) [54]. Williamson et al. [55] put forward a creative solution to solve nucleophile, which reversed the reaction, unless more than 10 equivalents of glycine-derivitized problem: they used threonine esters as substrates and the product glycolic acids were far less (Scheme 10)they [54]. Williamson al. [55] as putsubstrates forward aand creative solution to solve thiswere problem: they problem: used threonine esters the product glycolic acids far this less drugs were used (Scheme 10) et [54]. Williamson et al. [55] put forward a creative solution to solve nucleophilic, enabling conjugation with a 1:1 stoichiometry. used threonine esters as substrates and the product glycolic acids were far less nucleophilic, enabling nucleophilic, enabling conjugation with a 1:1 stoichiometry. problem: they used threonine esters as substrates and the product glycolic acids were far less conjugation with a 1:1 stoichiometry. nucleophilic, enabling conjugation with a 1:1 stoichiometry.
Scheme 10. The sortase-mediated conjugation of glycine-derivitized drugs and the antibodies. Scheme 10. sortase-mediated conjugation of glycine-derivitized and the antibodies. Scheme 10. from TheThe sortase-mediated conjugation of glycine-derivitized drugsdrugs and the antibodies. Adapted Adapted reference [54]. Adapted from reference [54]. Scheme 10. The from reference [54]. sortase-mediated conjugation of glycine-derivitized drugs and the antibodies. Adapted from reference [54].could recognize and acylate the Avi-tag, which is a 15-amino acid The biotin ligase (BirA)
The biotin ligase (BirA) could recognize acylate the Avi-tag, which is analogs a 15-amino acid GLNDIFEAQKIEWHE sequence. Chen et al. [56]and found that BirA could accept keto of biotin GLNDIFEAQKIEWHE sequence. Chen et al. [56] found that BirA could accept keto analogs of biotin The biotin ligase (BirA) could recognize and acylate the Avi-tag, which is a 15-amino acid GLNDIFEAQKIEWHE sequence. Chen et al. [56] found that BirA could accept keto analogs of biotin
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The biotin ligase (BirA) could recognize and acylate the Avi-tag, which is a 15-amino acid GLNDIFEAQKIEWHE sequence. et al. [56] found thatthat BirAcould couldconjugate accept keto analogs of biotin as substrates substrates to to react react with with Avi-tagChen and form form intermediates that could conjugate with the oxyamines oxyamines as Avi-tag and intermediates with the as to react and form intermediates that could conjugate with the oxyamines of of substrates drugs (Scheme (Scheme 11).with BirAAvi-tag has aa similar similar function as formylglycine-generating formylglycine-generating enzyme (FGE), which which of drugs 11). BirA has function as enzyme (FGE), drugs (Scheme 11). BirA has a similar function as formylglycine-generating enzyme (FGE), which can can site-specifically site-specifically insert insert an an aldehyde aldehyde group group into into an an antibody. antibody. In In this this case, case, aa keto keto group group was was can site-specifically an aldehyde groupBirA into requires an antibody. In this case, ait group was introduced introduced into intoinsert the antibody. antibody. Although BirA requires long sequence, itketo is far far less restricted restricted in the the introduced the Although aa long sequence, is less in into the antibody. Although BirA requires a long sequence, it is far less restricted in the location along location along the antibody. They demonstrated the reaction with the installed carbonyl using location along the antibody. They demonstrated the reaction with the installed carbonyl using the antibody. They demonstrated the reaction with the installed carbonyl using fluorescein hydrazide. fluorescein hydrazide. fluorescein hydrazide.
Scheme 11. 11. The The BirA-mediated BirA-mediated conjugation conjugation of biotin-like biotin-like ketones, oxyamine-drugs and antibody. Scheme BirA-mediated conjugation of biotin-like ketones, ketones, oxyamine-drugs oxyamine-drugs and and antibody. antibody. 11. Adapted from from Reference [56]. Adapted Reference [56]. from Reference [56].
2.2.2. Formation Formation of of Carbamates Carbamates 2.2.2. Amines of of the the drugs could react with the hydroxyls that derived from the linkers in in thethe effect of the linkers in the effect of Amines the drugs drugs could couldreact reactwith withthe thehydroxyls hydroxylsthat thatderived derivedfrom from the linkers effect phosgene, 4-nitrophenyl chloroformate, etc. and form the carbamate containing drug-linkers phosgene, 4-nitrophenyl chloroformate, drug-linkers of phosgene, 4-nitrophenyl chloroformate,etc. etc.and andform formthe the carbamate carbamate containing containing drug-linkers (Scheme 12), which was then coupled to antibodies [57,58]. For instance, Jeffrey et al. [57] prepared (Scheme 12), which was then coupled to antibodies [57,58]. For instance, Jeffrey et al. [57] prepared antibody-drug conjugates conjugates in in which which the the amino-CBI amino-CBI drug, drug, aa DNA DNA minor minor groove groove binder binder drug drug (MGBs), (MGBs), antibody-drug was attached attached to to monoclonal antibodies through peptide linkers that designed to release drugs in the the was monoclonal antibodies through peptide linkers that designed to release drugs in to monoclonal antibodies through peptide linkers that designed to release drugs in lysosomes of target cells. In this study, the amino-CBI drug reacted with phosgene to form the lysosomes of target cells. In In thisthis study, the the lysosomes of target cells. study, theamino-CBI amino-CBIdrug drugreacted reactedwith withphosgene phosgene to to form the corresponding isocyanate isocyanate and and then then the the linker linker with with aa hydroxyl hydroxyl was was added added to to form form the the carbamate. carbamate. corresponding carbamate. Dubowchik et al. [59] linked the anticancer drug doxorubicin to chimeric BR96, an internalizing linked the the anticancer anticancer drug drug doxorubicin doxorubicin to to chimeric chimeric BR96, BR96, an an internalizing internalizing Dubowchik et al. [59] linked monoclonal antibody, antibody, through throughlysosomally lysosomallycleavable cleavable dipeptides. dipeptides. In In this this case, case, the the carbamate carbamate between between antibody, through lysosomally cleavable dipeptides. In monoclonal drug and and antibody antibody was was prepared prepared with with 4-nitrophenyl 4-nitrophenyl chloroformate. chloroformate. These These antibody-drug antibody-drug conjugates conjugates chloroformate. drug usually insert a spacer such as para-aminobenzyl carbamate (PABC) between the peptide linkers and usually insert a spacer such as para-aminobenzyl carbamate (PABC) between the peptide linkers and the drugs drugs to to minimize minimize the the steric steric interaction interaction effects. effects. This This approach approach has has previously previously been been used used to to release release the doxorubicin [60], [60], MMAE MMAE [17] [17] and and camptothecin camptothecin [61] [61] from from antibody-drug antibody-drugconjugates. conjugates. doxorubicin camptothecin [61] from antibody-drug conjugates.
Scheme 12. 12. The The reaction reaction of of amines amines of of the the drug drug and and hydroxyls hydroxyls of of the the linkers linkers in the the effect effect of of phosgene phosgene Scheme Scheme 12. The reaction of amines of the drug and hydroxyls of the linkers inin the effect of phosgene or or 4-nitrophenyl 4-nitrophenyl chloroformate. chloroformate. Adapted Adapted from from Reference Reference [57,58]. [57,58]. or 4-nitrophenyl chloroformate. Adapted from Reference [57,58].
Another carbamate carbamate was was designed designed for for the the hydroxy hydroxy containing containing drug. drug. For For instance, instance, antibody-drug antibody-drug Another Another carbamate was designed for the hydroxy containing drug. For instance, antibody-drug conjugate SYD985 SYD985 consists consists of of seco-DUBA seco-DUBA drug, drug, self-elimination self-elimination spacer, spacer, cleavable cleavable peptides peptides linker linker and and conjugate conjugate SYD985 consists of seco-DUBA drug, to self-elimination spacer,spacer cleavable peptides linker trastuzumab. The seco-DUBA seco-DUBA drug was was linked linked the self-elimination self-elimination via carbamate carbamate bond and that trastuzumab. The drug to the spacer via bond that trastuzumab. seco-DUBATreatment drug was linked to the self-elimination spacer via carbamate bond that derived from fromThecarbonate. carbonate. of MOM MOM protected duocarmycin duocarmycin with 4-nitrophenyl 4-nitrophenyl derived Treatment of protected with derived from carbonate. of MOMcarbonate. protected duocarmycin 4-nitrophenyl chloroformate chloroformate gave the theTreatment corresponding Commerciallywith available tert-butyl methyl(2chloroformate gave corresponding carbonate. Commercially available tert-butyl methyl(2gave the corresponding carbonate. Commercially available tert-butyl methyl(2-(methylamino) (methylamino) ethyl)carbamate was then used to synthesize the carbamate. Removal of the Boc and and (methylamino) ethyl)carbamate was then used to synthesize the carbamate. Removal of the Boc ethyl)carbamate was then used toacid synthesize the carbamate. Removal of the Boc and MOM groups MOM groups with trifluoroacetic (TFA) provided cyclization spacer-duocarmycin as a TFA salt. MOM groups with trifluoroacetic acid (TFA) provided cyclization spacer-duocarmycin as a TFA salt. with trifluoroacetic acid (TFA) provided cyclization TFA salt. Cyclization Cyclization spacer-duocarmycin was reacted reacted with spacer-duocarmycin the activated activated linker linker as toa synthesis synthesis drug-linker Cyclization spacer-duocarmycin was with the to drug-linker spacer-duocarmycin was reacted with the activated linker to synthesis drug-linker module under module under slightly basic conditions [62]. module under slightly basic conditions [62]. slightly basic conditions [62].
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2.3. Conjugation 2.3. Conjugation via via Alcohols Alcohols 2.3.1. Formation of Carbonates
Similar with amines, alcohols can react with chloroformates to form carbonates. For example, et al. al. [63] [63]conjugated conjugated7-ethyl-10-hydroxycamptothecin 7-ethyl-10-hydroxycamptothecin(SN-38) (SN-38)derivatives derivativesto tohMN-14, hMN-14, Moon et a a humanizedanti-CEACAM5 anti-CEACAM5mAb, mAb,via viaa acarbonate carbonatebond. bond. To construct the carbonate bond, humanized BOC-SN-38 [64] was firstly converted to its 20-O-chloroformate, and then reacted in situ with the linker, MC-Phe-Lys(MMT)-PABOH MC-Phe-Lys(MMT)-PABOH[60]. [60]. known linker, 2.3.2. Formation of Ether Bonds Jeffrey et al. [57] described described a method method to to conjugate conjugate amino-CBI drug to the monoclonal antibody by formation of carbamates (see Section 2.2.2). Another approach for attaching a DNA minor groove binder drug (MGB) derivative to mAb involved O-alkylation of the hydroxy in aza-CBI to form ether bond. In this approach, approach, a para-aminobenzyl ether (PABE) (PABE) group group was used as a self- elimination spacer between the drug and the peptides [57]. An important step in the synthesis of this antibody-MGB peptides antibody-MGB conjugate was the formation of an ether bond through the O-alkylation of aza-CBI by bromide and potassium carbonate (Scheme 13). This work also showed that the conjugate could cleave via amide bond hydrolysis and lead to the release of free phenolic drug [65]. This approach should be broadly applied to drugs that have a phenolic phenolic hydroxyl hydroxyl group group as as the the conjugate conjugate site site [66]. [66].
Scheme 13. 13. The through the the O-alkylation O-alkylation of aza-CBI by by bromide bromide and and Scheme The formation formation of of an an ether ether bond bond through of aza-CBI potassium carbonate. Adapted from reference [57]. potassium carbonate. Adapted from reference [57].
2.3.3. Formation of Ester Bond 2.3.3. Formation of Ester Bond In 2010, Quiles et al. [67] developed paclitaxel-monoclonal antibody (PTX-mAb) conjugates that In 2010, Quiles et al. [67] developed paclitaxel-monoclonal antibody (PTX-mAb) conjugates that could deliver significant doses of drugs to the tumor cells using the ester bond. The conjugates used could deliver significant doses of drugs to the tumor cells using the ester bond. The conjugates used PEG as linker, and the paclitaxel was attached to the linker with glutarate (GL) or succinate (SX) PEG as linker, and the paclitaxel was attached to the linker with glutarate (GL) or succinate (SX) through the ester bond, the resulting PTX–L–Lys[(PEG12)3–PEG4]–PEG6–CO2NHS (L = GL or SX) was through the ester bond, the resulting PTX–L–Lys[(PEG12 )3 –PEG4 ]–PEG6 –CO2 NHS (L = GL or SX) was then conjugated to C225, an antiepidermal growth factor receptor (anti-EGFR) monoclonal antibody, then conjugated to C225, an antiepidermal growth factor receptor (anti-EGFR) monoclonal antibody, producing completely soluble conjugates. producing completely soluble conjugates. 2.4. Conjugation 2.4. Conjugation via via Aldehydes Aldehydes 2.4.1. Conjugation via FGE
is another method method for linkingfor drugs to antibodies. Conjugation via viaaldehydes aldehydes is another linking drugs Formylglycineto antibodies. generating enzymes (FGEs),enzymes which recognize modify a shortand CXPXR (where X is any amino acid) Formylglycine-generating (FGEs), and which recognize modify a short CXPXR (where sequence, can beacid) usedsequence, to modify of antibodies to aldehyde-containing X is any amino canthe be cysteine used to residues modify the cysteine residues of antibodies to formylglycine (FGly)formylglycine residues. This method was applied to generate site-specific antibody-drug aldehyde-containing (FGly) residues. This method was applied to generate site-specific conjugates via incorporating cytotoxic drugs into monoclonal with a formylglycine [68]. antibody-drug conjugates via incorporating cytotoxic drugs antibodies into monoclonal antibodies with a Following the [68]. production of modified antibody, a chemical methoda can be used to conjugate a drug formylglycine Following the production of modified antibody, chemical method can be used to to the aldehyde formylglycine. or hydrazide drugs were attached the modified conjugate a druggroup to the of aldehyde group ofOxyamine formylglycine. Oxyamine or hydrazide drugstowere attached antibodies successfully (Scheme 14) [69]. Recently, this kind of aldehyde was to the modified antibodies successfully (Scheme 14) [69]. Recently, this kindconjugation of aldehydestrategy conjugation further developed Agarwal by et al. [70], who used (HIPS) chemistry strategy was furtherbydeveloped Agarwal et al. [70],hydrazino-iso-Pictet-Spengler who used hydrazino-iso-Pictet-Spengler (HIPS) to attach maytansine to the aldehyde-containing trastuzumab. The HIPS resulted in the chemistry to attach maytansine to the aldehyde-containing trastuzumab. Thechemistry HIPS chemistry resulted formation of a covalent C–C bond, which was more stable than oxime or hydrazone ligation products in physiological condition. What is more, this study showed that the aldehyde group can be
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in formation of194 a covalent C–C bond, which was more stable than oxime or hydrazone ligation Int.the J. Mol. Mol. Sci. 2016, 2016, 17, 17, of 16 16 Int. J. Sci. 194 99 of products in physiological condition. What is more, this study showed that the aldehyde group can be introduced many locations theantibody antibodywithout withoutaffecting affectingthe thestability stability and and activity activity of the introduced in in many locations of of the introduced in many locations of the antibody without affecting the stability and activity of the obtained antibody-drug conjugates. antibody-drug conjugates. conjugates. obtained antibody-drug O O N N H2N N H 2 O O
SH SH CTPHSR CTPHSR
FGE FGE
fGTPHSR fGTPHSR
CHO CHO fGTPHSR fGTPHSR
H H N N O O
N N
O O N N
N N fGTPHSR fGTPHSR
Scheme 14. 14. The The conversion conversion of of the the cysteine cysteine thiol thiol to to an an aldehyde aldehyde group group by by formylglycine-generating formylglycine-generating Scheme 14. cysteine thiol to an aldehyde group by formylglycine-generating enzymes (FGE) (FGE) enables enables reactions reactions with with oxyamine oxyamine drugs. drugs. Adapted Adapted from from reference reference [70]. [70]. enzymes reactions with oxyamine drugs. Adapted from reference [70].
2.4.2. Conjugation Conjugation via via aaRS aaRS 2.4.2. The tRNA/aminoacyl-tRNA tRNA/aminoacyl-tRNA synthetase synthetase (aaRS) pair pair can can site-specifically incorporate an an unnatural unnatural The synthetase (aaRS) can site-specifically site-specifically incorporate amino acid (e.g., p-acetylphenylalanine, pAcPhe) into antibody [71]. Recently, the genetic amino acid acid(e.g., (e.g., p-acetylphenylalanine, pAcPhe) into antibody [71]. the Recently, the genetic amino p-acetylphenylalanine, pAcPhe) into antibody [71]. Recently, genetic incorporation incorporation of unnatural unnatural amino acids into into antibodies had become useful tooldesign in the the [72,73]. ADC design design incorporation of amino acids antibodies become aa useful tool in ADC of unnatural amino acids into antibodies had become ahad useful tool in the ADC This [72,73]. This method was successfully realized by Axup et al. [74], who developed site-specific [72,73]. was Thissuccessfully method was successfully realized bywho Axup et al. [74], who developed method realized by Axup et al. [74], developed site-specific auristatinsite-specific conjugates auristatin conjugates of trastuzumab. trastuzumab. A A was p-acetylphenylalanine was codon loadedofonto onto thebyamber amber codon of auristatin conjugates of p-acetylphenylalanine was loaded the of of trastuzumab. A p-acetylphenylalanine loaded onto the amber tRNA aaRS codon and then tRNA by by aaRS aaRS and then then specifically specifically incorporated into the amber amber site site of the the trastuzumab heavy chain. chain. tRNA and incorporated into the of trastuzumab heavy specifically incorporated into the amber site of the trastuzumab heavy chain. After purification, the After purification, the antibody was coupled to the monomethyl auristatin F (MMAF) derivative that After purification, the antibody was coupled to the monomethyl auristatinthat F (MMAF) derivative that antibody was coupled to the monomethyl auristatin F (MMAF) derivative contains an oxyamine contains an oxyamine groupwith by an an oxime ligation with(Scheme the pAcPhe pAcPhe residues (Scheme 15). The The analysis analysis contains group by oxime ligation with the (Scheme 15). group byan anoxyamine oxime ligation the pAcPhe residues 15). residues The analysis of antitumor activity of antitumor antitumor activity activity and pharmacokinetics pharmacokinetics of of this this site-specific site-specific antibody-drug antibody-drug conjugate confirmed confirmed of and pharmacokineticsand of this site-specific antibody-drug conjugate confirmed its conjugate efficacy and stability its efficacy efficacy and stability in serum. its in serum. and stability in serum.
Scheme 15. 15. Incorporation Incorporation of of p-acetylphenylalanine p-acetylphenylalanine (pAcPhe) (pAcPhe) allows allows for for site-specific site-specific conjugation conjugation of of Scheme Scheme 15. Incorporation of p-acetylphenylalanine (pAcPhe) allows for site-specific conjugation of drugs to to the the modified modified antibodies. antibodies. Adapted Adapted from from reference reference [74]. [74]. drugs drugs to the modified antibodies. Adapted from reference [74].
2.4.3. Oxidation Oxidation of of Sialic Sialic Acids Acids 2.4.3. 2.4.3. Oxidation of Sialic Acids Glycoengineering has has been been employed employed to to synthesize synthesize site-specific site-specific antibody-drug antibody-drug conjugates, conjugates, in in Glycoengineering Glycoengineering has been employed to synthesize site-specific antibody-drug conjugates, in which sialic acids were used as chemical handles for selective conjugations. This was achieved by which sialic sialic acids acids were were used used as as chemical chemical handles handles for for selective selective conjugations. This was achieved achieved by by which This was incorporating sialic acids acids into into the the native native glycans glycans of of trastuzumab trastuzumabconjugations. through β-1,4-galactosyltransferase β-1,4-galactosyltransferase incorporating sialic through incorporating sialic acids into the native through β-1,4-galactosyltransferase (Gal T) T) and and α-2,6-sialyltransferase α-2,6-sialyltransferase (Sial glycans T). Prior Priorofto totrastuzumab reaction with with the oxyamine oxyamine drugs, the the alcohol alcohol (Gal (Sial T). reaction the (Gal T) and α-2,6-sialyltransferase (Sial T). Prior to reaction with the oxyamine drugs,drugs, the alcohol groups groups of sialic acids were oxidated to aldehyde groups. The resulting antibodies could react with groups of sialic acids were to oxidated to groups. aldehyde groups. The resultingcould antibodies could react with of sialic acids were oxidated aldehyde The resulting antibodies react with the cytotoxic the cytotoxic cytotoxic drugs drugs via via an an oxime oxime ligation ligation (Scheme (Scheme 16). 16). This This method method was was evaluated evaluated by by conjugating conjugating the drugs via an oxime ligation (Scheme 16). This method was evaluated by conjugating trastuzumab trastuzumab with two drugs, monomethyl auristatin E (MMAE) and dolastatin 10. with The trastuzumab with two drugs, monomethyl auristatin E (MMAE) and dolastatin 10. The glycoengineered antibody-drug conjugates exhibited comparable antitumor activities to the glycoengineered antibody-drug conjugates exhibited comparable antitumor activities to the conventional analogs analogs [75]. [75]. conventional
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two drugs, monomethyl auristatin E (MMAE) and dolastatin 10. The glycoengineered antibody-drug conjugates Int. J. Mol. Sci.exhibited 2016, 17, 194comparable antitumor activities to the conventional analogs [75]. 10 of 16 Int. J. Mol. Sci. 2016, 17, 194 10 of 16
Scheme Scheme 16. 16. The The modification modification of of glycans glycans of of trastuzumab trastuzumab by by Gal Gal T T and and Sial Sial T T leads leads to to the the incorporation incorporation Scheme 16. The modification of glycans of trastuzumab by Gal T and Sial T leads to the incorporation of sialic acids, which were oxidated and coupled to the drugs through oxime ligation. Adapted of sialic acids, which were oxidated and coupled to the drugs through oxime ligation. Adapted from from reference reference [75]. [75].
2.4.4. Reagent 2.4.4. Conjugation Conjugation via via Transamination Transamination Reagent Witus [76,77] introduced carbonyl groups by reagent pyridoxal [76,77] introduced carbonyl groups by transamination reagent pyridoxal 51 -phosphate Witus etet etal. al. al. [76,77] introduced carbonyl groups by transamination transamination reagent pyridoxal 5′-phosphate (PLP) at the N-terminus of antibodies, which could be used as unique attachment (PLP) at the (PLP) N-terminus of antibodies, which could be used sites forsites the 5′-phosphate at the N-terminus of antibodies, which couldas beunique used asattachment unique attachment sites for the conjugation formation (Scheme 17). However, the reaction yields were not very high and high conjugation formation (Scheme 17). However, the reaction yields were not very high and high for the conjugation formation (Scheme 17). However, the reaction yields were not very high and temperatures application of of this this method. method. To To solve solve these these problems, problems, temperatures were were required, required, which which limited limited the the application application of this method. To solve these problems, they developed a combinatorial peptide library screening platform and found a new they developed a combinatorial peptide library screening platform and found a new transamination transamination reagent, N-methylpyridinium-4-carboxaldehyde [78]. Antibodies benzenesulfonate salt salt (RS) Antibodies with reagent, N-methylpyridinium-4-carboxaldehyde N-methylpyridinium-4-carboxaldehyde benzenesulfonate benzenesulfonate salt (RS) [78]. Antibodies with glutamate-rich sequences were particularly reactive substrates for this reagent [79]. glutamate-rich sequences were particularly reactive reactive substrates substrates for for this this reagent reagent [79]. [79].
R R
PLP or RS PLP or RS
NH2 NH2
R1ONH2 R1ONH2
R R
R1 R N O 1 N O
O O O O
OH O OH O N N PLP PLP
R R
H H OPO322 OPO3
N N
H H O3S O3S RS RS
Scheme oxidation of amines to aldehyde groups which could react with oxyaminedrugs using Scheme 17. 17. The The The oxidation oxidation of of amines amines to toaldehyde aldehyde groups groups which which could could react react with with oxyaminedrugs oxyaminedrugs using transamination reagents pyridoxal 5′-phosphate (PLP) or N-methylpyridinium-4-carboxaldehyde 1 transamination or N-methylpyridinium-4-carboxaldehyde N-methylpyridinium-4-carboxaldehyde transamination reagents pyridoxal 55′-phosphate -phosphate (PLP) or benzenesulfonate salt (RS). (RS). Adapted Adapted from from reference reference [79]. benzenesulfonate salt reference [79]. [79].
2.5. Conjugation via Azides 2.5. 2.5. Conjugation Conjugation via via Azides Azides 2.5.1. 2.5.1. Click Click Reactions Reactions with with DBCO DBCO Azides can react with alkynes to triazoles through click such alkynes to form triazoles through click chemistries, such as copper-catalyzed Azides can canreact reactwith with alkynes to form form triazoles through click chemistries, chemistries, such as as coppercoppercatalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition azide-alkyne cycloaddition (CuAAC) (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) catalyzed azide-alkyne cycloaddition and strain-promoted azide-alkyne cycloaddition (SPAAC) (Scheme This approach used to antibody-drug (Scheme [80,81]. 18) This[80,81]. approach was recently was used recently to construct antibody-drug For (SPAAC) 18) (Scheme 18) [80,81]. This approach was recently used to construct construct conjugates. antibody-drug conjugates. For et conjugated drugs antibodies using In example, Zhou et al. [81]Zhou conjugated drugs to antibodies this method. Inmethod. this study, an conjugates. For example, example, Zhou et al. al. [81] [81] conjugated drugs to tousing antibodies using this this method. In this this study, an azide-containing reagent, sodium (difluoroalkylazido)sulfinate (DAAS-Na), allowed azide azide-containing reagent, sodium (DAAS-Na), allowedallowed azide groups study, an azide-containing reagent,(difluoroalkylazido)sulfinate sodium (difluoroalkylazido)sulfinate (DAAS-Na), azide groups to to and products then to to be linked heteroaromatics, and the products thencould be attached monoclonal antibodies groups to be betolinked linked to heteroaromatics, heteroaromatics, and the the could products could then be betoattached attached to monoclonal monoclonal antibodies by reactions. DAAS-Na used heteroarene reaction, by click reactions. used inwas the heteroarene reaction, in which ZnClin antibodies by click clickDAAS-Na reactions.was DAAS-Na was used in in the the functionalization heteroarene functionalization functionalization reaction, in2 which ZnCl 2 and TsOH·H2O were acid additives and tBuOOH was an oxidant. The resulting azideand TsOH¨ acid2O additives andadditives tBuOOHand wastBuOOH an oxidant. resultingThe azide-linked drugs which ZnClH 2 and TsOH·H were acid wasThe an oxidant. resulting azide2 O were linked linked drugs drugs could could react react with with aa dibenzylcyclooctyne dibenzylcyclooctyne (DBCO) (DBCO) containing containing antibody antibody through through aa CuAAC CuAAC reaction. This strategy expands the extent of bioactive drugs that can be linked to monoclonal reaction. This strategy expands the extent of bioactive drugs that can be linked to monoclonal antibodies. antibodies.
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could react with a dibenzylcyclooctyne (DBCO) containing antibody through a CuAAC reaction. This Int. J. Mol. Sci. 2016, 17, 194 11 of 16 strategy expands the Int. J. Mol. Sci. 2016, 17, 194extent of bioactive drugs that can be linked to monoclonal antibodies. 11 of 16
Scheme 18. The synthesis of ADCs through copper-catalyzed azide-alkyne cycloaddition (CuAAC) Scheme 18. The The synthesis synthesis of ADCs ADCs cycloaddition through copper-catalyzed copper-catalyzed azide-alkyne cycloaddition Scheme 18. of through azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne (SPAAC) click reactions. Adapted from (CuAAC) reference and strain-promoted azide-alkyne cycloaddition (SPAAC) click reactions. Adapted from reference [81]. [81].strain-promoted azide-alkyne cycloaddition (SPAAC) click reactions. Adapted from reference [81].
Microbial transglutaminase cancan catalyze the formation of an isopeptide bond between Microbial transglutaminase(MTGase) (MTGase) catalyze the formation of an isopeptide bond Microbial transglutaminase (MTGase) can catalyze the formation of an isopeptide bond between the aminethe group of glutamine and the primary amine of lysine while simultaneously releasing an between amine group of glutamine and the primary amine of lysine while simultaneously the amine gas group of glutamine and activity the primary amine ofwas lysine whiletosimultaneously releasing an ammonia [82]. The coupling of MTGase applied synthesize antibody-drug releasing an ammonia gas [82]. The coupling activity of MTGase was applied to synthesize ammonia [82]. For Theexample, couplingDennler activity etofal.MTGase was applied tohomogeneous synthesize antibody-drug conjugatesgas [83–85]. [83] afforded highly trastuzumabantibody-drug conjugates [83–85]. For example, Dennler et al.a[83] afforded a highly homogeneous conjugates [83–85]. For example, Dennler et al. [83] afforded a highly homogeneous trastuzumabMMAE conjugate with DAR of 2 using this enzymatic conjugation strategy. In this work, azidetrastuzumab-MMAE conjugate with DAR of 2 using this enzymatic conjugation strategy.anIn this MMAE conjugate with DAR of 2 using this enzymatic conjugation strategy. In this work, an azidecontaining linker, which involves a primary amine, was coupled to Q295 of the deglycosylated work, an azide-containing linker, which involves a primary amine, was coupled to Q295 of the containing which involves a primary amine, was coupled to Q295 of thewith deglycosylated antibody bylinker, MTGase. This enzymatic reaction was followed by afollowed SPAAC reaction the DBCOdeglycosylated antibody by MTGase. This enzymatic reaction was by a SPAAC reaction with antibody by MTGase. This enzymatic reaction was followed by a SPAAC reaction with the DBCOcontaining auristatin drug. the DBCO-containing auristatin drug. containing auristatin Cell-free proteindrug. synthesis (CFPS) (CFPS) system system was was also also efficiently efficiently applied applied to to produce produce monoclonal monoclonal Cell-free protein synthesis Cell-free protein synthesis (CFPS) system was also efficiently applied to produce monoclonal antibodies that that contain contain unnatural unnatural amino amino acids acids for antibodies for antibody-drug antibody-drug conjugate conjugate generations. generations. For For example, example, antibodies that contain unnaturala amino acids antibody-drug for antibody-drug conjugate generations. For example, Zimmerman et al. [86] prepared site-specific conjugate via CFPS system using new Zimmerman et al. [86] prepared a site-specific antibody-drug conjugate via CFPS system using aa new Zimmerman et al. [86] prepared a site-specific antibody-drug conjugate via CFPS system using a new synthetase totoincorporate incorporate a para-azidomethylphenylalanine (pAMF) the monoclonal antibody, synthetase a para-azidomethylphenylalanine (pAMF) to thetomonoclonal antibody, which synthetase to incorporate aDBCO-functionalized para-azidomethylphenylalanine (pAMF) to the monoclonal antibody, which was then linked to a MMAF drug by SPAAC reaction. was then linked to a DBCO-functionalized MMAF drug by SPAAC reaction. which was then linked to a DBCO-functionalized MMAF drug by SPAAC reaction. Terminal Alkynes Alkynes 2.5.2. Click Reactions with Terminal 2.5.2. Click Reactions with Terminal Alkynes In 2014, Bryden Bryden et et al. al. [87] [87] described described the theattachment attachmentofofazide-functionalized azide-functionalizedporphyrins porphyrinstotoa In 2014, Bryden et al. [87] described the attachment of azide-functionalized porphyrins to in a atratuzumab tratuzumabvia viaa anovel novelconjugation conjugationmethod. method.InInthis thisstudy, study,Trastuzumab Trastuzumab was was treated with TCEP tratuzumab via a novel conjugation method. In this study, Trastuzumab was treated with TCEP in to reduce reduce the the interchain interchain disulfide disulfide bond. bond. Treatment Treatment with with N-propargyl-3,4-dibromomaleimide N-propargyl-3,4-dibromomaleimide order to order toalkyne-containing reduce the interchain disulfide which bond. then Treatment with N-propargyl-3,4-dibromomaleimide yielded trastuzumab, successfully reacted with porphyrin derivatives yielded alkyne-containing trastuzumab, which then successfully reacted with porphyrin derivatives afford trastuzumab-porphyrin trastuzumab-porphyrin conjugates conjugates (Scheme 19). This through the CuAAC reaction to afford (Scheme 19). method through the CuAAC reaction to afford trastuzumab-porphyrin conjugates (Scheme 19). This method realized using using SPAAC SPAACreaction reaction[39]. [39]. could also be realized could also be realized using SPAAC reaction [39].
Scheme 19. of trastuzumab-porphyrin conjugates through N-propargyl-3,4Scheme 19. The synthesis The synthesis of trastuzumab-porphyrin conjugates through Scheme 19. The and synthesis of trastuzumab-porphyrin conjugates through N-propargyl-3,4dibromomaleimide a click chemistry. Reprinted with permission from Reference [87]. N-propargyl-3,4-dibromomaleimide and a click chemistry. Reprinted with permission from dibromomaleimide and a click chemistry. Reprinted with permission from Reference [87]. Reference [87].
3. Conclusions 3. Conclusions 3. Conclusions A promising approach to improve the potency of drugs is to be conjugated to monoclonal A promising approach to improve the potency of drugs is to be conjugated to monoclonal antibodies that enable these to cytotoxic to be site-specifically to tumor cells while A promising approach improvedrugs the potency of drugs is todelivered be conjugated to monoclonal antibodies that enable these cytotoxic drugs to be site-specifically delivered to tumor cells while avoiding the toxicity ofthese drugscytotoxic on normal cells.toThe of antibody-drug antibodies that enable drugs be linkers site-specifically deliveredconjugates to tumor profoundly cells while avoiding the toxicity of drugs on normal cells. The linkers of antibody-drug conjugates profoundly impact their potency and safety. Recently, a variety of methods have been developed to conjugate impact potency In and safety. Recently, a variety of developed to conjugate drugs totheir antibodies. this review, we summarized themethods methodshave that been are currently used to design drugs to antibodies. In this review, we summarized the methods that are currently used to design and synthesize antibody-drug conjugates, including heterogeneous ADCs and homogeneous ADCs, and synthesize antibody-drug conjugates, including ADCsand andazides. homogeneous ADCs, via various functional groups such as thiols, amines, heterogeneous alcohols, aldehydes Heterogeneous via various functional groups such as thiols, amines, alcohols, aldehydes and azides. Heterogeneous
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avoiding the toxicity of drugs on normal cells. The linkers of antibody-drug conjugates profoundly impact their potency and safety. Recently, a variety of methods have been developed to conjugate drugs to antibodies. In this review, we summarized the methods that are currently used to design and synthesize antibody-drug conjugates, including heterogeneous ADCs and homogeneous ADCs, via various functional groups such as thiols, amines, alcohols, aldehydes and azides. Heterogeneous ADCs were usually synthesized through the thiols of cysteine residues and the amines of lysines, however, the heterogeneity diminished their activities and promoted antibody aggregations, and increased toxicities in the circulation. Homogeneous ADCs made through the catalysis of site-specific conjugation enzymes such as AGT, BTG, aaRS and Sial T are more stable and have comparable or even better activities than those conventional analogs in vivo. We believe that a growing number of methods will be developed to synthesize ADCs in the near future, and more and more ADCs, especially site-specifically modified ADCs, will be produced. Acknowledgments: We thank the other academic staff members in Aiping Lu and Ge Zhang’s group at Hong Kong Baptist University (Hong Kong, China). We also thank Hong Kong Baptist University (Hong Kong, China) for providing critical comments and technical support. This study was supported by the Hong Kong General Research Fund (HKBU12102914 to Ge Zhang) and the Faculty Research Grant of Hong Kong Baptist University (FRG2/12-13/027 to Ge Zhang). Author Contributions: Houzong Yao and Feng Jiang wrote the manuscript; and Aiping Lu and Ge Zhang revised and approved the manuscript. Conflicts of Interest: The authors declare no conflict of interest.
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