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Site-specific chemical conjugation of antibodies by using affinity peptide for the development of therapeutic antibody format Satoshi Kishimoto, Yuichi Nakashimada, Riri Yokota, Takaaki Hatanaka, Motoyasu Adachi, and Yuji Ito Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00865 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 4, 2019
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Bioconjugate Chemistry
Site-Specific Chemical Conjugation of Antibodies by Using Affinity Peptide for the Development of Therapeutic Antibody Format Satoshi Kishimoto,†,‡ Yuichi Nakashimada,†,‡ Riri Yokota,†,‡ Takaaki Hatanaka,†,‡ Motoyasu Adachi,# and Yuji Ito*,†,‡ †Graduate
School of Science and Engineering, Kagoshima University, Kagoshima 890-0065, Japan Quantum Beam Science Center, National Institutes for Quantum and Radiological Science and Technology, 2-4 Shirakata, Tokai, Ibaraki 319-1106, Japan #Tokai
ABSTRACT: Artificially modified IgG molecules are increasingly utilized in industrial and clinical applications. In the present study, the method of chemical conjugation by affinity peptide (CCAP) for site-specific chemical modification has been developed by using a peptide that bound with high affinity to human IgG-Fc. This method enabled a rapid modification of a specific residue (Lys248 on Fc) in a one-step reaction under mild condition to form a stable amide bond between the peptide and Fc. The monovalent peptide-IgG conjugate not only maintained complete antigen binding but also bound to Fc receptors (FcRn, FcγRI, and FcγRIIIa), indicating that it is a suitable conjugate form that can be further developed into highly functional antibody therapeutics. CCAP was applied for the preparation of an antibody-drug conjugate and a bispecific antibody to demonstrate the usefulness of this method.
In recent years, various groups have reported sitespecific modifications of antibodies8,9 made to avoid the adverse effects of such modifications on antigen binding and stability of antibodies. In particular, a ketone group was introduced to the end of the antibody sugar chain by means of glycotransferase,10–12 enabling the attachment of alkoxyamine-containing molecules by oxime bond.12 Furthermore, several methods to modify Gln295 of IgG have been reported that involved acyl transfer of a lysine substrate to a glutamine residue located in a flexible region of the protein by transglutaminase.13,14 In another approach, Cys residues were introduced to a conjugate by genetic engineering to react with a thiol reactive agent.15 However, the latter mutation affected antibody stability, and the introduced thiol-reactive linker was lost from the antibody due to the exchange of maleimide moiety with reactive thiol in human serum albumin (HSA),16 depending on the location of the introduced Cys residue. Introduction of unnatural amino acids to attach alkoxyamine-containing
INTRODUCTION Development of technology for modification of IgG molecules has an important role in expanding the industrial and pharmaceutical applications of antibodies. Antibody-drug conjugates (ADCs)1,2 and radionuclidemodified antibodies3,4 have been developed that enabled target-specific transport of anticancer agents and radionuclides. In general, chemical modifications of antibodies have been made by random reactions with activated carboxyl groups of N-hydroxysuccinimide (NHS) esters toward Lys residues or by reactions of thiol-specific reagents, like maleimide, with cysteines5. Representative ADCs prepared by these conventional modifications include trastuzumab emtansine (T-DM1), which is a conjugate of the anti-cancer drug DM1 with the anti-HER2 antibody therapeutic trastuzumab6 or brentuximab vedotin, a conjugate of monomethyl auristatin E with an anti-CD30 antibody.7
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molecules by oxime bond or alkyne by click chemistry has also been carried out.17,18 However, these approaches require genetic engineering of antibodies that delays the development of antibody drugs due to time-consuming process and high production cost. Park et al. succeeded in the site-specific modification of Met252 in human IgG1-Fc through a peptide-directed photo-cross-linking reaction with the photoreactive unnatural amino acid p-benzoylphenylalanine introduced in the IgG-binding peptide FcIII.19 Although this reaction was highly site-specific for IgG1-Fc, it required a long (i.e., 2 h) UV exposure to proceed, which might chemically damage IgG. More recently, a modification based on the IgG-Fc binding protein Z33 derived from protein A was reported by Yu et al.20 They achieved successful sitespecific conjugation between IgG and Z33, which was introduced by using the non-canonical amino acid 4fluorophenyl carbamate lysine due to the proximal effect between the corresponding residues.24 Although it enabled modifications of both the murine and human IgG molecules, due to the broad specificity of Z33, the molecular weight of Z33 (4.1 kDa) may be too large for use in pharmaceutical applications due to potential antigenicity. Previously, by using biopanning against human IgG1 from random peptide library constructed on a T7 phage display system, we identified the peptide GPDCAYHRGELVWCTFH, consisting of 17 amino acids.21 This peptide, IgG-BP, which was intramolecularly crosslinked with a disulfide bond, bound to the Fc site of the human IgG antibody with a Kd of 10 nM, showed specificity to human IgG1, IgG2, and IgG4, and functioned as an alternative affinity ligand instead of protein A for IgG antibody purification.22 This peptide is similar to FcIII peptide reported previously23 but, in contrast to the latter, has W6Y and L8R substitutions. The crystal structure analysis of the complex of IgG-BP and human IgG1-Fc revealed that Arg8 of IgG-BP is directed to and lies within 10 Å of IgG1-Fc Lys248 residue. This observation inspired us to make the chemical connection between these sites after the replacement of Arg8 in IgGBP with Lys. In the present study, we report efficient and site-specific chemical modification of IgG by using IgG-BP-derived peptide reagent. This conjugation reaction proceeded rapidly (
