Site-specific chemical modification of antibody fragments using traceless cleavable linkers


Antibody-drug conjugates (ADCs) are promising agents for the selective delivery of cytotoxic drugs to specific cells (for example, tumors). In this protocol, we describe two strategies for the precise modification at engineered C- or N-terminal cysteines of antibodies in IgG, diabody and small immunoprotein (SIP) formats that yield homogenous ADCs. In this protocol, cemadotin derivatives are used as model drugs, as these agents have a potent cytotoxic activity and are easy to synthesize. However, other drugs with similar functional groups could be considered. In the first approach, a cemadotin derivative containing a sulfhydryl group results in a mixed disulfide linkage. In the second approach, a cemadotin derivative containing an aldehyde group is joined via a thiazolidine linkage. The procedures outlined are robust, enabling the preparation of ADCs with a defined number of drugs per antibody in a time frame between 7 and 24 h.

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Figure 1: Site-specific conjugation of cytotoxic payloads to antibodies using traceless cleavable linkers.
Figure 2: Construction of a traceless chemically defined tumor-vascular targeting antibody-drug conjugate based on disulfide linkage.
Figure 3: Construction of a traceless chemically defined tumor-vascular targeting antibody-drug conjugate based on thiazolidine linkage.
Figure 4: Characterization of purified SIP(L19)-SS-CH2Cem.
Figure 5: Characterization of purified Db(F8)-thz-Cem.


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G.J.L.B. thanks the European Molecular Biology Organization (EMBO) and the Novartis Foundation for generous funding. We thank N. Huguenin-Dezot and S. Trüssel for their contributions during these projects. We also thank S. Wulhfard, A. Villa and K. Schwager for their help in protein engineering and expression. Financial contributions from ETH Zürich, the Swiss National Science Foundation, SwissBridge/Stammbach Stiftung, Kommission für Technologie und Innovation (KTI), Philochem AG and Philogen SpA are gratefully acknowledged.

Author information

G.J.L.B., M.S., I.H. and G.C. carried out the experiments. G.J.L.B., G.C. and D.N. designed the research and interpreted the data. G.J.L.B., G.C. and D.N. wrote the manuscript.

Correspondence to Dario Neri.

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Competing interests

G.C. is employed at Philochem AG. D.N. is a co-founder and shareholder of Philogen SpA.

Integrated supplementary information

Supplementary Figure 1 A typical analysis of a conjugation reaction by LC-MS.

The total ion chromatogram, combined ion series and deconvoluted spectra are shown for the starting material and product of the reaction of SIP(F8)-Ellman's with CemCH2-SH. Identical analyses were carried out for all the conjugation reactions performed in this work.

Supplementary Figure 2 Characterization of purified SIP(F8)-SS-CH2Cem.

a) ESI-MS spectrum; b) SDS-PAGE; M, molecular marker; 1, SIP(F8) non-reducing conditions; 2, SIP(F8) reducing conditions; 3, purified SIP(F8)-SS-Ellman's; 4, purified SIP(F8)-SS-CH2Cem c) size-exclusion chromatography; arrows indicate standard proteins (11 mL: ferritin 440 kDa; 14.1 mL: BSA 67 kDa; 15.4 mL: lactoglobulin 35 kDa); and d) Biacore analysis towards recombinant 11A12 fibronectin. Data previously described in [Bernardes, G.J.L. et al., A traceless vascular-targeting antibody–drug conjugate for cancer therapy. Angew. Chem. Int. Ed. 51, 941-944 (2012)]. A single peak after deconvolution of the multiply-charged ions spectrum should be detected by LCMS analysis of SIP(F8)-SS-CH2Cem at 39415 Da; SDS-PAGE analysis should give a single major band at ~ 39 kDa; in some cases, SDS-PAGE shows a very minor band at 75 kDa (see TROUBLESHOOTING); a peak eluting at a retention volume of 15.3 mL corresponding to the noncovalent homodimeric form of SIP(F8)-SS-CH2Cem should be detected by gel filtration; and finally, SIP(F8)-SS-CH2Cem should remain immunoreactive towards recombinant 11A12 fibronectin as observed by direct comparison of the sensorgram profiles of modified and nonmodified antibody fragments.

Supplementary information

Supplementary Figure 1

A typical analysis of a conjugation reaction by LC-MS. (PDF 288 kb)

Supplementary Figure 2

Characterization of purified SIP(F8)-SS-CH2Cem. (PDF 372 kb)

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Bernardes, G., Steiner, M., Hartmann, I. et al. Site-specific chemical modification of antibody fragments using traceless cleavable linkers. Nat Protoc 8, 2079–2089 (2013).

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