Preparation of Site-Specific Cytotoxic Protein Conjugates via Maleimide-thiol Chemistry and Sortase A-Mediated Ligation

Summary

Here we provide detailed protocols for a site-specific labeling of proteins with cytotoxic drugs using maleimide-thiol reaction and sortase A-mediated ligation.

Abstract

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as growth factor receptors on their surface. This feature enables development of highly selective therapeutics, the protein bioconjugates, composed of targeting proteins (antibodies or receptor ligands) connected to highly cytotoxic drugs by a specific linker. Due to very high affinity and selectivity of targeting proteins the bioconjugates recognize marker proteins on the cancer cells surface and utilize receptor-mediated endocytosis to reach the cell interior. Intracellular vesicular transport system ultimately delivers the bioconjugates to the lysosomes, where proteolysis separates free cytotoxic drugs from the proteinaceous core of the bioconjugates, triggering drug-dependent cancer cell death. Currently, there are several protein bioconjugates approved for cancer treatment and large number is under development or clinical trials.

One of the main challenges in the generation of the bioconjugates is a site-specific attachment of the cytotoxic drug to the targeting protein. Recent years have brought a tremendous progress in the development of chemical and enzymatic strategies for protein modification with cytotoxic drugs. Here we present the detailed protocols for the site-specific incorporation of cytotoxic warheads into targeting proteins using a chemical method employing maleimide-thiol chemistry and an enzymatic approach that relies on sortase A-mediated ligation. We use engineered variant of fibroblast growth factor 2 and fragment crystallizable region of human immunoglobulin G as an exemplary targeting proteins and monomethyl auristatin E and methotrexate as model cytotoxic drugs. All the described strategies allow for highly efficient generation of biologically active cytotoxic conjugates of defined molecular architecture with potential for selective treatment of diverse cancers.

Introduction

Decades of scientific efforts have led to an enormous advancement in our knowledge about the molecular mechanisms governing cancer development and progression. At the same time, the therapeutic possibilities are still largely limited due to the adverse effects of drugs caused by their lack of selectivity, the great variability of tumors and drug-resistance developed after prolonged treatment. Targeted anti-cancer therapies have been gaining attention in recent years as novel and highly promising approaches for treatment of diverse tumors. Targeted therapies rely on sophisticated drug delivery systems that precisely deliver the cytotoxic payload to the cancer cells and....

Protocol

1. Conjugation of the Fc-domain with MMAE

NOTE: Prior to site-specific conjugations prepare key reagents: highly pure protein of interest (in this case the Fc fragment and engineered FGF2 variant, as a starting point 1-5 mg of recombinant protein, prepared according to Sokolowska-Wedzina¹⁸), maleimidocaproyl-Val-Cit-p-aminobenzyl alcohol (PABC)-monomethyl auristatin E (MMAE) (CAUTION, highly cytotoxic agent, handle with care), Tris(2-carboxyethyl)phosphine‎.......

Discussion

Due to the high interest in the design of selective therapeutics against diverse cancer types there is an urgent need for strategies allowing for site-specific attachment of distinct cargoes to the targeting proteins. The site-specific modification of targeting proteins is critical as it ensures homogeneity of developed bioactive conjugates, a prerequisite for modern therapeutics. There are several methods, both chemical and enzymatic allowing for site-specific attachment of cargo to the protein of choice. In most cases .......

Materials

Name	Company	Catalog Number	Comments
CM-Sepharose column	Sigma-Aldrich, Saint Louis, MO, USA	CCF100
Heparin Sepharose column	GE Healthcare, Chicago, IL, USA	GE17-0407-01
HiTrap Desalting column	GE Healthcare, Chicago, IL, USA	GE17-1408-01
HiTrap MabSelect SuRe column	GE Healthcare, Chicago, IL, USA	GE11-0034-93
maleimidocaproyl-Val-Cit-PABC-monomethyl auristatin E (MMAE)	MedChemExpress, Monmouth Junction, NJ, USA	HY-100374	Toxic
N,N-Dimethylacetamide (DMAc)	Sigma-Aldrich, Saint Louis, MO, USA	185884
Tris(2-carboxyethyl)phosphine (TCEP)	Sigma-Aldrich, Saint Louis, MO, USA	646547