Use of the Npys thiol protection in solid phase peptide synthesis. Application to direct peptide-protein conjugation through cysteine residues

The protection of the thiol function of cysteine with the 3-nitro-2-pyridylsulfenyl (Npys) group has been successfully applied in the solid phase synthesis of nine peptides. A reexamination of the chemical stability of the protecting group has shown that, while Npys is essentially suitable for standard Boc/benzyl synthesis conditions, it is inadequate for the Fmoc strategy. Its proven stability to "high" HF acidolysis can not be extended to "low-high" conditions without significant thiol deprotection. On the other hand, the Npys group is quite compatible with standard photolytical cleavage conditions. The stability of Npys to HF and its thiol-activating character allow its application in peptide-carrier protein conjugation reactions by specific coupling through cysteine residues in the peptide.     

Comparison of Methods for Chemical Conjugation of an Influenza Peptide to Wild-Type and Cysteine-Mutant Virus-Like Particles Expressed in <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis>

Chemical conjugation of the influenza peptide antigen M2E to different variants of virus-like particles (VLPs) was investigated. Wild-type cowpea chlorotic mottle virus (CCMV) and two novel cysteine mutants of CCMV, all expressed in Pseudomonas fluorescens, were utilized in this study. Two different conjugation schemes, primary amine-directed and cysteine-directed, were tested and compared. Both strategies were successfully used to attach M2E peptides to the surface of these VLPs. Ultimately, the cysteine-directed conjugation strategy using the CCMV cysteine mutant particles displayed key advantages over the primary amine-directed strategy.     

The subunit structure of rabbit skeletal-muscle phosphofructokinase and the amino acid sequence of the tryptic peptide containing the highly reactive thiol group.

1. The single highly reactive (class I) thiol group per 80000-mol.wt. subunit of skeletal-muscle phosphofructokinase was specifically carboxymethylated with iodo[2-14C]acetate, and after denaturation the remaining thiol groups were carboxymethylated with bromo[2-3H]acetate. After tryptic digestion and peptide 'mapping' it was found that the 14C radioactivity was in a spot that did not contain significant amounts of 3H radioactivity, so it is concluded that there is not a second, 'buried' cysteine residue within a sequence identical with that of the class-I cysteine peptide. 2. The total number of tryptic peptides as well as the number of those containing cysteine, histidine or tryptophan were inconsistent with the smallest polypeptide chain of phosphofructokinase (mol.wt. about 80000) being composed of two identical amino acid sequences. 3. The amino acid sequence of the tryptic peptide containing the class-I thiol group was shown to be Cys-Lys-Asp-Phe-Arg. This sequence is compared with part of the sequence containing the highly reactive thiol group of phosphorylase.     

Integrin binding human antibody constant domains--probing the C-terminal structural loops for grafting the RGD motif

Recently, it has been demonstrated that loops of the crystallizable fragment of IgG1 (IgG1-Fc) can be engineered to form antigen-binding sites. In this work C-terminal structural loops in the CH3 domains of homodimeric IgG1-Fc have been functionalized to form integrin-binding sites in order to probe the effect of engineering on structural integrity and thermal stability of IgG1-Fc as well as on binding to the ligands Protein A, CD16 and FcRn, respectively. The peptide sequence GCRGDCL - a disulfide-bridged cyclic heptapeptide that confers binding to human αvβ3 integrin was introduced into AB, CD and/or EF loops and single and double mutants were heterologously expressed in Pichia pastoris. Integrin binding of engineered IgG-Fc was tested using both binding to coated αvβ3 integrin in ELISA or to αvβ3-expressing K562 cells in FACS analysis. Additionally, blocking of αvβ3-mediated cell adhesion to vitronectin was investigated. The data presented in this report demonstrate that bioactive integrin-binding peptide(s) can be grafted on the C-terminal loops of IgG-Fc without impairing binding to effector molecules. Observed differences between the investigated variants in structural stability and integrin binding are discussed with respect to the known structure of IgG-Fc and its structural loops.     

Systematic modulation of Michael-type reactivity of thiols through the use of charged amino acids.

A quantitative structure-reactivity relationship for the Michael-type addition of thiols onto acrylates was determined. Several thiol-containing peptides were investigated by examining the correlation between the second-order rate constant of their addition onto PEG-diacrylate and the pK(a) of the thiols within a peptide. By introducing charged amino acids in close proximity to a cysteine, the pK(a) of the thiol was systematically modulated by electrostatic interactions. Positive charges from the amino acid arginine decreased the pK(a) of the thiol and accelerated the reaction with acrylates while negative charges from aspartic acids showed the opposite effect. A linear correlation between thiolate concentrations and kinetic constants was found, confirming the role of thiolates as the reactive species in this Michael-type reaction. The relevant factors influencing the reactivity were the sign and the number of the neighboring charges, while the position of these charges had little effect on reactivity. These results provide a basis for the rational design of peptides, where the kinetics and thus selectivity of protein/peptide conjugation with polymeric structures via Michael-type addition reactions can be controlled.     

Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates

The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.     

Use of methanethiolation to investigate the catalytic role of sulphydryl groups in rabbit skeletal muscle pyruvate kinase.

Incubation of rabbit skeletal muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) with methyl methanethiosulphonate resulted in the time- and inhibitor concentration-dependent loss of enzyme activity. Substrates or products of the catalytic reaction prevented the loss of activity caused by methanethiolation. Their effectiveness as protecting agents was placed in the order ADP greater than ATP greater than Mg2+ greater than phosphoenolpyruvate greater than pyruvate. The essential catalytic cation, K+, had no effect on the methanethiolation reaction. [Me-3H]Methanethiosulphonate modified all the available cysteine thiol groups which correlated to the incorporation of four SC3H3 groups per protomer. Four radioactive peptides were obtained on tryptic peptide mapping. When methanethiolation was carried out in the presence of Mg2+ alone or with Mg2+ and ATP together, then only three SC3H3 groups were incorporated into each subunit. If MgATP protected methanethiolated pyruvate kinase was reacted with iodo[2-3H]acetic acid then 1.37 +/- 0.2 groups per protomer were carboxymethylated. 70% of the radioactivity was located in a single peptide on tryptic peptide mapping. This peptide was isolated and contained the segment carboxymethyl cysteine (Glx, Asx, Ser) Arg. Collectively these data indicate that although all thiol groups are equally accessible to methyl methanethiosulphonate, only a single thiol group participates in the catalytic event. An additional role in the maintenance of structure for this thiol group was also shown in studied of reduction and thermal denaturation of the enzyme.     

Antibody directed against the 142-148 sequence of the myosin heavy chain interferes with myosin-actin interaction.

It has been reported recently that the isolated and renatured 23-kDa N-terminal fragment of rabbit skeletal muscle myosin binds tightly to F-actin in an ATP-dependent manner [Muhlrad, A. (1989) Biochemistry 28, 4002-4010]. The binding to actin is of electrostatic nature and may involve a positively charged cluster of residues on the 23-kDa fragment stretching from Arg-143 to Arg-147. An octapeptide containing this positive cluster was synthesized and coupled to BSA through a cysteine residue added to the N-terminus of the peptide. Polyclonal antibody was raised against the BSA-coupled peptide in rabbits which recognized the N-terminal 23-kDa fragment of rabbit skeletal myosin subfragment 1, and a peptide comprised of residues 122-204 of the 23K fragment in Western blots. The purified antibody [IgG and F(ab)] inhibited the actin-activated ATPase activity of S1 without affecting its Mg2(+)- and K+(EDTA)-modulated ATPase activity. Both IgG and F(ab) decreased the binding of S1 to F-actin in a sedimentation assay, and actin inhibited the binding of both IgG and F(ab) to S1 in a competitive binding assay. The cysteine thiol of the synthetic octapeptide was labeled by the fluorescent thiol reagent monobromobimane, and the labeled peptide was found to bind to actin in a sedimentation assay. The results support the possibility that the positively charged Arg-143 to Arg-147 stretch of residues on the 23-kDa fragment participates in actin binding of myosin and may represent an essential constituent of the actin-S1 interface.     

Boron-enriched streptavidin potentially useful as a component of boron carriers for neutron capture therapy of cancer.

A boron-enriched streptavidin has been prepared by chemical conjugation of a boron-rich compound, B(12)H(11)SH(2)(-) (BSH), to a genetically engineered streptavidin variant. The streptavidin variant used has 20 cysteine residues per molecule, derived from a C-terminal cysteine stretch consisting of five cysteine residues per subunit. Because natural streptavidin has no cysteine residues, the reactive sulfhydryl groups of the cysteine stretch serve as unique conjugation sites for sulfhydryl chemistry. BSH was conjugated irreversibly to the sulfhydryl groups of the streptavidin variant via a sulfhydryl-specific homobifunctional chemical cross-linker. Quantitative boron analysis indicates that the resulting streptavidin-BSH conjugate carries approximately 230 boron atoms/molecule. This indicates that the chemical conjugation of BSH to the streptavidin variant was highly specific and efficient because this method should allow the conjugation of a maximum of 240 boron atoms/streptavidin molecule. This boron-enriched streptavidin retained both full biotin-binding ability and tetrameric structure, suggesting that the conjugation of BSH has little, if any, effect on the fundamental properties of streptavidin. This boron-enriched streptavidin should be very useful as a component of targetable boron carriers for neutron capture therapy of cancer. For example, a monoclonal antibody against a tumor-associated antigen can be attached tightly to the boron-enriched streptavidin upon simple biotinylation, and the resulting conjugate could be used to target boron to tumor cells on which the tumor-associated antigen is overexpressed.     

Improving the stability of thiol–maleimide bioconjugates via the formation of a thiazine structure

Linker stability is critically important for the efficacy and safety of peptide and protein conjugates used for biological applications. One common conjugation strategy, thiol–maleimide coupling, generates a succinimidyl thioether linker with limited stability under physiological conditions. We have shown in previous work that when a peptide with an N-terminal cysteine is conjugated to a maleimide reagent, a thiazine structure is formed via a chemical rearrangement. Our preliminary work indicated that the thiazine linker has favorable stability. Here, we report the evaluation of a thiazine linker as an alternative to the widely used succinimidyl thioether linker for thiol–maleimide bioconjugation. The stability of the thiazine conjugate in comparison to the thioether conjugate was assessed across a broad pH range. Additionally, the propensity for retro-Michael reaction and cross-reactivity with other thiols was evaluated by treating conjugates in the presence of glutathione. The studies indicated that the thiazine linker degrades markedly slower than the thioether conjugate. In addition, the thiazine linker is over 20 times less susceptible to glutathione adduct formation. The NMR study of the thiazine structure confirmed that the formation of the thiazine linker is a stereoselective process that yields a single diastereomer. In summary, we propose the use of the thiazine linker obtained by conjugation of maleimide-containing reagents with peptides or proteins presenting an N-terminal cysteine as a novel approach for bioconjugation. The advantages of this approach are the formation of a linker with a well-defined stereochemical configuration, increased stability at physiological pH, and a strongly reduced propensity for thiol exchange.     

Cysteinylated protein as reactive disulfide: an alternative route to affinity labeling

Engineering the permanent formation of a receptor-ligand complex has a number of promising applications in chemistry, biology, and medicine. Antibodies and other proteins can be excellent receptors for synthetic ligands such as probes or drugs. Because proteins possess an array of nucleophilic sites, the placement of an electrophile on the synthetic ligand to react with a nucleophile on the macromolecule is a standard practice. Previously, we have used the site-directed incorporation of cysteine nucleophiles at the periphery of an antibody's binding site, paired with the chemical design of weakly electrophilic ligands, to produce receptor-ligand pairs that conjugate specifically and permanently (Corneillie et al. (2004) Bioconjugate Chem. 15, 1392-1402 and references therein). After protein expression in Drosophila S2 cells, we found, as is frequently observed, that the engineered cysteine was reversibly blocked by disulfide linkage to a cysteine monomer (cysteinylated). Removal of the cysteine monomer requires some care because of the need to preserve other disulfide linkages in the protein. Here, we report that cysteinylation can be used to advantage by treating the cysteine monomer as a leaving group and the protein disulfide as an electrophile with special affinity for thiols. Two ligands bearing thiol side chains were synthesized and incubated with the cysteinylated antibody Fab fragment 2D12.5 G54C, with the finding that both ligands become covalently attached within a few minutes under physiological conditions. The attachment is robust even in the presence of excess thiol reagents. This rapid, specific conjugation is particularly interesting for biomedical applications.     

Synthesis and nucleophilic reactivity of a series of glutathione analogues, modified at the gamma-glutamyl moiety.

A series of GSH analogues with modifications at the gamma-glutamyl moiety was synthesized and purified by following peptide chemistry methodology. Benzyl, benzyloxycarbonyl and t-butyloxycarbonyl protective groups were used to protect individual amino acid functional groups. The formation of peptide bonds was accomplished through coupling of free amino groups with active esters, generated by reaction of the carboxylate functions with dicyclohexylcarbodi-imide and 1-hydroxybenzotriazole. The protecting groups in the tripeptides were removed in a single step by using Na in liquid NH3. Precautions were taken in order to prevent oxidation of the thiol function in the cysteine residue. Thus GSH analogues containing both L- and D-glutamic acid and L- and D-aspartic acid, coupled to cysteinylglycine through both the alpha- and the omega-carboxylate group, were synthesized. Also, decarboxy-GSH and deamino-GSH, lacking one functional group in the glutamate moiety, were prepared. The spontaneous non-enzyme-catalysed nucleophilic reaction of these GSH analogues with the electrophilic model substrate 1-chloro-2,4-dinitrobenzene showed appreciable rate differences, indicating the importance of intramolecular interactions in determining the nucleophilic reactivity of the thiol function in the cysteine residue. In particular, the free amino group in the gamma-L-glutamic acid residue appears to play a crucial role in activating the thiol group in GSH. In an adjacent paper [Adang, Brussee, Meyer, Coles, Ketterer, van der Gen & Mulder (1988) Biochem. J. 255, 721-724] these results are compared with those obtained in a study on the ability of these GSH analogues to act as a co-substrate in the glutathione S-transferase-catalysed conjugation reaction with 1-chloro-2,4-dinitrobenzene.     

DNA binding to protein-gold nanocrystal conjugates

The E. coli DNA binding protein lac repressor (LacI) and a derivative with a designed thiol (T334C) were developed as gold nanocrystal conjugates to assess the effects of conjugation on DNA binding function. The designed derivative was engineered with a solvent-accessible thiol to promote oriented conjugation, avoiding obstruction of the DNA-binding domain by the nanocrystal. Analytical ultracentrifugation (AU) and electrophoretic mobility shift assays (EMSA) were used to evaluate the ability of conjugated repressors to bind the natural operator DNA sequence O(1). The results show that LacI does not retain significant DNA binding function when conjugated to gold nanocrystals, presumably because the basic DNA-binding domain is the site for nonspecific conjugation. T334C, with the potential for both directed and nonspecific conjugation, shows enhanced interaction with O(1) when conjugated. Interestingly, the order of component addition is a key factor in producing functional lac repressor conjugates.     

Self-assembled "dock and lock" system for linking payloads to targeting proteins

Random conjugation of therapeutic or diagnostic payloads to targeting proteins generates functionally heterogeneous products. Conjugation of payloads to an adapter that binds to a peptide tag engineered into a targeting protein provides an alternative strategy. To progress into clinical development, an adapter/docking tag system should include humanized components and be stable in circulation. We describe here an adapter/docking tag system based on mutated fragments of human RNase I that spontaneously bind to each other and form a conjugate with a disulfide bond between complimentary cysteine residues. This self-assembled "dock and lock" system utilizes the previously described fusion C-tag, a 1-15 aa fragment of human RNase I with the R4C amino acid substitution, and a newly engineered adapter protein (Ad-C), a 21-127-aa fragment of human RNase I with the V118C substitution. Two vastly different C-tagged recombinant proteins, human vascular endothelial growth factor (VEGF) and a 254-aa long N-terminal fragment of anthrax lethal factor (LFn), retain functional activities after spontaneous conjugation of Ad-C to N-terminal or C-terminal C-tag, respectively. Ad-C modified with pegylated phospolipid and inserted into the lipid membrane of drug-loaded liposomes (Doxil) retained the ability to conjugate C-tagged proteins, yielding targeted liposomes decorated with functionally active proteins. To further optimize the system, we engineered an adapter with an additional cysteine residue at position 88 for site-specific modification, conjugated it to C-tagged VEGF, and labeled with a near-infrared fluorescent dye Cy5.5, yielding a unique functionally active probe for in vivo molecular imaging. We expect that this self-assembled "dock and lock" system will provide new opportunities for using functionally active proteins for biomedical purposes.     

Apolipoprotein A-I(Milano) and apolipoprotein A-I(Paris) exhibit an antioxidant activity distinct from that of wild-type apolipoprotein A-I

Apolipoprotein A-I(Milano) (apoA-I(Milano)) and apoA-I(Paris) are rare cysteine variants of apoA-I that produce a HDL deficiency in the absence of cardiovascular disease in humans. This paradox provides the basis for the hypothesis that the cysteine variants possess a beneficial activity not associated with wild-type apoA-I (apoA-I(WT)). In this study, a unique antioxidant activity of apoA-I(Milano) and apoA-I(Paris) is described. ApoA-I(Milano) was twice as effective as apoA-I(Paris) in preventing lipoxygenase-mediated oxidation of phospholipids, whereas apoA-I(WT) was poorly active. Antioxidant activity was observed using the monomeric form of the variants and was equally effective before and after initiation of oxidative events. ApoA-I(Milano) protected phospholipid from reactive oxygen species (ROS) generated via xanthine/xanthine oxidase (X/Xo) but failed to inhibit X/Xo-induced reduction of cytochrome c. These results indicate that apoA-I(Milano) was unable to directly quench ROS in the aqueous phase. There were no differences between lipid-free apoA-I(Milano,) apoA-I(Paris), and apoA-I(WT) in mediating the efflux of cholesterol from macrophages, indicating that the cysteine variants interacted normally with the ABCA1 efflux pathway. The results indicate that incorporation of a free thiol within an amphipathic alpha helix of apoA-I confers an antioxidant activity distinct from that of apoA-I(WT). These studies are the first to relate gain of function to rare cysteine mutations in the apoA-I primary sequence.     

Enzymatic deglutathionylation to generate interleukin-4 cysteine muteins with free thiol

Interleukin-4 (IL-4) is a prototypical regulator protein of the immune system that is crucial for the pathogenesis and maintenance of asthma and other atopic diseases. It, together with IL-13, uses the IL-4 receptor α chain (IL-4Rα) to signal into immune and other cells. An IL-4 mutein acting as a dual IL-4/IL-13 receptor antagonist is in clinical development. Here, it is described how IL-4 muteins containing a single engineered cysteine with a free thiol can be prepared and used for site-specific chemical modification. The muteins were initially expressed in E. coli, refolded, and purified, but not in a fully reduced nonconjugated form. Attempts to reduce the cysteine chemically failed because the native disulfide bonds of IL-4 were also reduced under similar conditions. Therefore, an enzymatic procedure was developed to reduce glutathionylated IL-4 cysteine muteins employing glutaredoxin and reduced glutathione. Cysteine muteins engineered at four different positions around the IL-4Rα binding site were enzymatically reduced at different rates. All muteins were prepared with free thiol in reasonable yield and were modified by N-ethylmaleimide (NEM) or maleimido-PEG. The effect on IL-4Rα binding of cysteine substitution and of the site-specific modification by glutathione, N-ethylmaleimide (NEM), or a branched 2.36 kDa poly(ethylene glycol) (PEG) will be discussed.     

Impact of linker and conjugation chemistry on antigen binding,Fc receptor binding and thermal stability of model antibody-drug conjugates

Antibody-drug conjugates (ADCs) with biotin as a model cargo tethered to IgG1 mAbs via different linkers and conjugation methods were prepared and tested for thermostability and ability to bind target antigen and Fc receptor. Most conjugates demonstrated decreased thermostability relative to unconjugated antibody, based on DSC, with carbohydrate and amine coupled ADCs showing the least effect compared with thiol coupled conjugates. A strong correlation between biotin-load and loss of stability is observed with thiol conjugation to one IgG scaffold, but the stability of a second IgG scaffold is relatively insensitive to biotin load. The same correlation for amine coupling was less significant. Binding of antibody to antigen and Fc receptor was investigated using surface plasmon resonance. None of the conjugates exhibited altered antigen affinity. Fc receptor FcγIIb (CD32b) interactions were investigated using captured antibody conjugate. Protein G and Protein A, known inhibitors of Fc receptor (FcR) binding to IgG, were also used to extend the analysis of the impact of conjugation on Fc receptor binding. H10_NPEG4 was the only conjugate to show significant negative impact to FcR binding, which is likely due to higher biotin-load compared with the other ADCs. The ADC aHIS_NLC and aHIS_TPEG8 demonstrated some loss in affinity for FcR, but to much lower extent. The general insensitivity of target binding and effector function of the IgG1 platform to conjugation highlight their utility. The observed changes in thermostability require consideration for the choice of conjugation chemistry, depending on the system being pursued and particular application of the conjugate.     

A reversible conformational transition in muscle actin is caused by nucleotide exchange and uncovers cysteine in position 10

ATP-G-actin in the absence of excess ATP and divalent metal ions was treated with ADP in amounts large enough to ensure complete formation of ADP-G-actin. Under these conditions the monomer undergoes a very slow structural transition as seen by the exposure of 2.0 +/- 0.2 thiol groups per actin molecule. Once exposed, the second thiol group reacts with 5,5'-dithiobis-(2-nitrobenzoic acid) at a rate approximately 10-fold higher than that of cysteine 374. Labeling experiments with 2,4-dinitrophenyl [1-14C]cysteinyl disulfide followed by digestion and peptide analysis showed (besides reaction with cysteine 374) nearly exclusive labeling of cysteine 10. Since this residue is completely shielded in ATP-G-actin, exchange of ATP for ADP must have caused a partial unfolding of the protein uncovering the side chain of this cysteine. The transition is reversible, because addition of ATP or of excess divalent metal ions restored the conformation with only cysteine 374 exposed. Reversibility of the transition allowed us to directly determine the relative affinities of ATP and ADP to monomeric actin in the absence of Me2+ ions. By determination of the 50% exposure value of cysteine 10 from either side of the equilibrium we found a value of KATP/KADP = 30. The rate of uncovering of the thiol of cysteine 10 at 0 degree C was distinctly slower (t1/2 = 9 h) than its reshielding by the addition of ATP (t1/2 = 3 h). The structural change was accompanied by a decrease in polymerization rate. Relative polymerization rates were determined as ATP-G(1S)-actin:ADP-G(approximately 1S)-actin:ADP-G(2S)-actin = 1.0:0.35:0.1. From the data presented here we conclude that preparations of ADP-G-actin remain undefined unless the number of thiol groups exposed has been determined.     

Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index

Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.     

Impact of linker and conjugation chemistry on antigen binding,Fc receptor binding and thermal stability of model antibody-drug conjugates

Antibody-drug conjugates (ADCs) with biotin as a model cargo tethered to IgG1 mAbs via different linkers and conjugation methods were prepared and tested for thermostability and ability to bind target antigen and Fc receptor. Most conjugates demonstrated decreased thermostability relative to unconjugated antibody, based on DSC, with carbohydrate and amine coupled ADCs showing the least effect compared with thiol coupled conjugates. A strong correlation between biotin-load and loss of stability is observed with thiol conjugation to one IgG scaffold, but the stability of a second IgG scaffold is relatively insensitive to biotin load. The same correlation for amine coupling was less significant. Binding of antibody to antigen and Fc receptor was investigated using surface plasmon resonance. None of the conjugates exhibited altered antigen affinity. Fc receptor FcγIIb (CD32b) interactions were investigated using captured antibody conjugate. Protein G and Protein A, known inhibitors of Fc receptor (FcR) binding to IgG, were also used to extend the analysis of the impact of conjugation on Fc receptor binding. H10_NPEG4 was the only conjugate to show significant negative impact to FcR binding, which is likely due to higher biotin-load compared with the other ADCs. The ADC aHIS_NLC and aHIS_TPEG8 demonstrated some loss in affinity for FcR, but to much lower extent. The general insensitivity of target binding and effector function of the IgG1 platform to conjugation highlight their utility. The observed changes in thermostability require consideration for the choice of conjugation chemistry, depending on the system being pursued and particular application of the conjugate.