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Proximity-induced Site-specific Antibody Conjugation Han Xiao, Chenfei Yu, Juan Tang, Axel Loredo, Yuda Chen, Sung Yun Jung, Antrix Jain, and Aviva Gordon Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00680 • Publication Date (Web): 29 Oct 2018 Downloaded from http://pubs.acs.org on October 29, 2018
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Proximity-induced Site-specific Antibody Conjugation Chenfei Yu,†⊥ Juan Tang,†⊥ Axel Loredo,† Yuda Chen,† Sung Yun Jung,‡ Antrix Jain,‡ Aviva Gordon,† Han Xiao†§#* †Department ‡Baylor
of Chemistry, Rice University, 6100 Main Street, Houston, Texas, 77005, United States
College of Medicine, 1 Baylor Plaza, Houston, TX, 77030, United States
§Department
of Biosciences, Rice University, 6100 Main Street, Houston, Texas, 77005, United States
#Department
of Bioengineering, Rice University, 6100 Main Street, Houston, Texas, 77005, United States
Supporting Information. ABSTRACT: Site-specific antibody conjugates with a well-defined structure and superb therapeutic index are of great interest for basic research, disease diagnostics, and therapy. Here, we develop a novel proximity-induced antibody conjugation strategy enabling site-specific covalent bond formation between functional moieties and native antibodies without antibody engineering or additional UV/chemical treatment. A high conjugation efficiency and specificity was achieved with IgGs from different species and subclasses. The utility of this approach was demonstrated by site-specific conjugation of the smallmolecule fluorophore to a native antibody and in vitro characterization of their activities.
INTRODUCTION Monoclonal antibodies with excellent selectivity and a broad collection of targets are extensively used as affinity reagents in many biological applications, from in vitro assays to disease diagnostics to targeted therapies. These applications often require the modification of antibodies by various chemical molecules (e.g., fluorophores, drugs, nanoparticles) or biological reagents (e.g., enzymes, cytokines, antibodies).1–5 To covalently label antibodies, various methods have been developed, most commonly involving nonspecific acylation of lysine residues with highly reactive esters and alkylation of cysteine residues with maleimides.6,7 The resulting products are heterogeneous antibody conjugates that cannot be further purified. Antibodies derived from such heterogeneous modification may suffer from diminished binding affinity and therapeutic index due to a lack of control over the modification ratio and site.8–10 With advances in the fields of bioorthogonal chemistry and protein engineering, several strategies have been developed for preparing sitespecific antibody conjugates.11 These include THIOMABTM, which affords ultra-reactive cysteine residues for conjugation;12 SMARTagTM, which genetically encodes a peptide tag for further enzymatic modification;13–15 and the SiteClickTM labeling system, which introduces an unnatural
sugar and noncanonical amino acid (ncAA) technology that enables site-specific incorporation of the 21st amino acid with a distinct reactive moiety.16–19 In general, current site-specific antibody-labeling methods first require the site-specific introduction of a unique reactive moiety into antibodies, followed by selective modification using bioorthogonal chemistry. However, the site-specific installation of a bioorthogonal functionality always requires a certain amount of antibody engineering, which is time-consuming, expensive, and may result in low yield. To carry out a site-specific labeling of native antibodies without engineering the antibody, photocrosslinker was introduced into antibody-affinity peptides.20–23,22 Upon the UV irradiation, the photocrosslinker can crosslink affinity peptides to the antibody. However, the lack of chemical selectivity of this method can result in non-specific crosslinking and a 30 60 mins exposure to UV irradiation was known to cause protein damage.24 To avoid the use of UV irradiation, a metallopeptide catalyst and sulfonyl acrylate reagents were recently used to label native proteins.25,26 Well-defined antibody conjugates were prepared using these strategies, but an additional biotin-based affinity purification or a sequence-based computational design were required to obtain the conjugate, respectively.
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Figure 1. Preparation and characterization of the Tras-FB conjugate. (a) FPheK allowing for crosslinking to a proximal lysine residue will be introduced into FB protein derived from protein A. The resulting adaptive peptides can be used to label native antibodies upon binding. (b) Structures of lysine analogs that can crosslink with the lysine residue. (c) The structure of the B domain from Staphylococcus protein A (FB protein) interaction with the Fc fragment is also shown (PDB: 1FC2). (d) SDS-PAGE analysis of crosslinked mixtures in the absence (left) and presence (right) of the reducing reagents.
Here, we develop a new platform for efficient and sitespecific labeling of native antibodies based on proximityinduced reactivity between an ncAA and a nearby antibody lysine residue. The resulting proximity-induced conjugation technology, named pClick, does not require any antibody engineering, UV/chemical treatment, thus enabling attachment of various functional molecules, including small molecules and enzymes, to most antibodies used for research and therapy.
RESULT AND DISCUSSION To selectively react with an adjacent native amino acid, such as cysteine or lysine, a number of ncAAs with reactive halide, aryl ketone, Michael acceptor, aryl isothiocyanate, or aryl carbamate side chains have been developed.27–32 These ncAAs have been genetically incorporated into various proteins to enhance reactivity between proteins and small molecules or to capture transient protein–protein interactions. Because of the high efficiency and selectivity of these crosslinking reactions, we envisioned that proximity-enhanced bio-reactivity could be used to site-specifically label native antibodies without antibody engineering (Fig. 1a). Specifically, ncAAs allowing crosslinking to a proximal lysine residue will be introduced into specific sites of peptides that have wellcharacterized binding sites at the fragment crystallizable (Fc) or antigen-binding (Fab) fragment of antibodies (Fig.
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To avoid the potential disruption of the Fab-binding site and the Fc receptor-binding site, we used the B domain of protein A (FB protein) from Staphylococcus aureus.33 The FB protein is a small and stable protein that binds to the CH2–CH3 junction of the immunoglobulin G (IgG) antibody with a 10-100 µM dissociation constant (KD).34 The co-crystal structure (PDB: 1FC2) reveals that Leu18 and His19 in the FB protein come into close proximity to Lys316 in the human IgG and that FB residues Glu25, Glu26, Arg28, and Asn29 are close to Lys337 in the IgG (Fig. 1c). To test if an electrophilic ncAA-containing FB domain could be used to site-specifically label antibodies, 4-fluorophenyl carbamate lysine (FPheK, Fig. 1b), an ncAA that can react with a proximal lysine to form a stable crosslink, was site-specifically incorporated into the above six residues of the FB protein using the ncAA technology reported previously.30 All FB mutants containing FPheK were purified by Ni-NTA chromatography and fully characterized by SDS-PAGE and ESI-MS. We incubated trastuzumab (Tras), the native human epidermal growth factor receptor 2 (HER2)-specific antibody, with 8 equiv of each FB mutant separately in PBS buffer (pH 8.5) at 37°C for 48 h. Most of the mutants showed no or very weak crosslinking to Tras, with the exception being the variant
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1b). Upon binding to the antibody, the crosslinking ncAAcontaining peptide will enable proximity-induced covalent attachment of the crosslinking ncAA to the nearby lysine residue of the antibody. Because of the high labeling selectivity and mild crosslinking conditions, we reasoned that this method would be able to covalently attach a variety of functional reagents to the large library of existing antibodies.
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Figure 2. Proximity-induced ligation between FB proteins and Tras. a) Reducing SDS-PAGE analysis of a reaction of FB-E25FPheK with Tras at 1:8 ratio for 0, 6, 12, 24, and 48 h (left) and at 2:1, 1:1, 1:2, 1:4, and 1:8 ratios for 48 h (right). b) Reducing SDS-PAGE analysis of the reaction of Tras and FB-E25FPheK, FB-E25AcrK and FB-E25BrC6K mutants. c) SDS-PAGE analysis of the FB-E25FPheK mutant, Tras, and Tras/FB-E25FPheK (conj.) under non-reducing conditions, visualized by Coomassie staining. d) Mass spectrometry analysis of Tras and Tras/FB-E25FPheK.
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Figure 3. (a) Reducing SDS-PAGE analysis of various IgG subclasses alone or after conjugation with the FB-E25FPheK mutant. (b) Preparation of the Tras-FL conjugate. (c) SDS-PAGE analysis of Tras and Tras-FL, visualized by Coomassie staining (left) and UV transillumination (right). (d) Binding of Tras in BT-474, SK-BR-3, and MDA-MB-468 cells visualized by confocal microscopy. Cells were incubated with 30 nM Tras-FL (green fluorescence) in media for 30 min at 37C and stained with DilC18 (red fluorescence) and Hoechst nuclear stain (blue fluorescence). Scale bar = 20 m.
with FPheK at position 25 (Fig. 1d). Reducing SDS-PAGE analysis of Tras incubated with the FB containing an E25FPheK mutation (FB-E25FPheK) revealed a new band of 57 kDa, consistent with the formation of the FB–heavy chain complex. Next, we sought to optimize the conjugation conditions using Tras and the FB-E25FPheK mutant as model substrates. Based on SDS-PAGE analysis, the modification efficiency improved with both longer reaction time and an increased amount of the FB mutant (Fig. 2a). The conjugation reaction with 8 equiv of FBE25FPheK mutant for 48 h afforded the Tras-FB conjugate at greater than 95% conjugation yield. Upon reaction completion, the resulting conjugate was purified by a protein-L column to remove excess FB proteins. Analysis by ESI-MS of the Tras-FB conjugate revealed a mass difference of 7631 Da between the unconjugated (49,123 Da) and FB-conjugated heavy chain (56,754 Da, Fig. 2d). Unreacted IgG heavy chain or degradation products were undetectable by SDS-PAGE or ESI-MS (Fig. 2c and d). We further confirmed that FB-E25FPheK formed the crosslinking with Lys337 in the high chain of native Tras by MS/MS sequencing (Fig. S2). To explore the efficiencies of various electrophilic moieties, two other lysine analogs, N-acryloyl-lysine (AcrK) and 2-amino-6-(6-bromohexanamido)hexanoic acid (BrC6K), were synthesized and incorporated into the Glu25 residue that exhibited the best crosslinking efficiency (Fig. 1b).29,32 The substitution of Glu25 with AcrK or BrC6K showed 20–40% conjugation efficiency, which was significantly lower than that of FPheK (95%) (Fig. 2b). Thus, FPheK was used in the latter conjugation experiment. Antibodies, particularly of the IgG isotype, are widely used as affinity reagents in many research applications, disease diagnostics, and therapies. To demonstrate the generality of the conjugation method developed above, the
conjugation efficiency and specificity of IgGs from different species and subclasses were tested. The FBE25FPheK protein was crosslinked to human IgG1 and IgG2 and mouse IgG1, IgG2a, and IgG2b with efficiencies of 96%, 99%, 99%, 91%, and 99%, respectively (Fig. 3a). We further tested the stability of antibody-FB conjugate in human serum with ELISA. The Figure S4 shows that after 2-hour incubation in human serum, no significant degradation of antibody conjugate was observed. Human IgG1 is the predominant antibody subclass used today for antibody therapy, whereas mouse IgG1 is the most employed antibody subclass for biotechnological applications.35 The successful site-specific labeling of these antibody subclasses suggests a high potential for application of the developed proximity-induced antibody conjugation method. Fluorophore-labeled monoclonal antibodies provide a powerful tool for disease detection, intraoperative imaging, and pharmacokinetic characterization of therapeutic reagents. Attaching a fluorophore to the mutant FB protein should allow for site-specific introduction of an imaging reagent to native antibodies without antibody engineering. To explore this possibility, we first expressed a FB protein with E25FPheK and F6C mutations, followed by functionalizing the protein with a fluorescein maleimide by site-specific conjugation to the cysteine residue (Fig. 3b). After overnight reaction, the fluorescein-labeled FB mutant was buffer-exchanged into pH 8.5 PBS buffer and added to the native trastuzumab antibody. The resulting fluorecein-labeled trastuzumab (Tras-FL) was further washed in an Amicon 100,000 molecular-weight-cutoff protein concentrator to remove unreacted FB proteins (Fig. 3c). The conjugation reaction afforded Tras-FL in greater than 98% conjugation yield, as observed by SDS-PAGE analysis (Fig. 3c). A band was observed only for the Tras-FL under UV transillumination, indicating the successful incorporation of the fluorophore (Fig. 3c). With the fluorophore-labeled Tras in hand, we
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tested its utility for visualizing antigens on the breast cancer cell surfaces. HER2-positive BT-474 and SK-BR-3 cells and HER2-negative MDA-MB-468 cells were treated for 30 min with Tras-FL prepared above. Confocal fluorescent imaging indicated that cell-surface–associated fluorescence was exhibited only by BT-474 and SK-BR-3 cells, while HER2-negative MDA-MB-468 cells did not exhibit any associated fluorescence (Fig. 3d). These results indicated that antibodies site-specifically modified with a functionalized FB-E25FPheK protein retain their antigenbinding ability. To show the versatility of pClick, we also prepared the fluorophore-labeled FB protein using Nhydroxysuccinimide (NHS)-ester chemistry. The resulting Alexa FluorTM 488-labeled FB protein was able to conjugate to the native trastuzumab antibody with a more than 95% conjugation efficiency (Fig. S5).
Texas (CPRIT RR170014) and the Robert A. Welch Foundation (C-1970).
CONCLUSION
(5)
In conclusion, we have developed a novel proximityinduced antibody conjugation platform for the preparation of site-specific antibody conjugate without the need for antibody engineering. With the introduction of a crosslinking ncAA allowing covalent bond formation with a proximal lysine, affinity peptides with various functional moieties can be site-specifically conjugated to native antibodies. This platform will enable rapid, site-specific, efficient conjugation to the existing native antibody and further facilitate antibody conjugate discovery and design.
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ASSOCIATED CONTENT Supporting Information (10)
The Supporting Information is available free of charge on the ACS Publications website.
(11)
AUTHOR INFORMATION Corresponding Author * E-mail:
[email protected] (12)
ORCID:
Han Xiao: 0000-0002-4311-971X Yuda Chen: 0000-0002-5399-1720
(13)
Axel Loredo: 0000-0003-0144-8192 (14)
Author Contributions ⊥These
authors contributed equally. (15)
Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT We thank Prof. Peter G. Schultz for kindly providing the plasmids pUltra-MbPylRS, and pBK-PheKRS1. This work was supported by the Cancer Prevention Research Institute of
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