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

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 spare the healthy ones. These include mainly diverse nanoparticles, liposomes, and protein-based drug carriers.

Cancer cells often expose elevated levels of specific marker proteins on their surface. Antibody drug conjugates (ADCs) are novel protein-based anti-cancer therapeutics, which combine in one molecule extreme specificity of monoclonal antibodies and high cytotoxic potency of drugs. Once bound to the cancer cell surface, ADC utilize receptor-mediated endocytosis to enter the cell. Subsequently, ADCs are transported via endosomal compartments to the lysosomes, where proteases degrade ADCs and release active cytotoxic drugs. Currently, there are eight ADCs approved in the US for the treatment of diverse tumors, including triple negative breast cancer, HER2 positive breast cancer, urothelial cancer, diffuse large B-cell lymphoma, hematological malignancy, Hodgkin lymphoma and acute myeloid leukemia. Large number of ADCs are also either under development or await approval¹. Noteworthy, protein engineering approaches have led to the development of diverse alternative to monoclonal antibodies protein scaffolds and their cytotoxic conjugates. These include different antibody fragments^2,3, DARPins^4,5, knottins^6,7, centyrins⁸, affibodies^9,10, or engineered receptor ligands^11,12.

There are several critical requirements that have to be met by a successful protein-based cytotoxic conjugate, namely the conjugate stability, extraordinary specificity, high affinity of the conjugate towards cancer-specific marker, rapid internalization of the conjugate into the cancer cell interior, its efficient transport to the lysosomes and effective intracellular release of the active payload. Another important feature is conjugates homogeneity, which largely depends on the applied strategy for the attachment of the payload to the targeting proteins. There are several methods available for site-specific conjugation of proteins with cytotoxic drugs, like modification of protein side-chain cysteine or lysine residues, attachment of the drug to unnatural amino acids incorporated into the targeting proteins, or enzymatic modifications of the targeting proteins (e.g., with transglutaminase, glycosyltransferase, formylglycine-generating enzyme, sortase A). In most cases site-specific conjugation methods require modifications of the targeting molecules (e.g., via cysteine engineering or introduction of short peptide tags), but in turn result in an efficient production of homogenous conjugate of interest.

Here we provide protocols for highly efficient site-specific conjugation of targeting proteins with cytotoxic drugs. As exemplary proteins we used two different molecules: the fragment crystallizable (Fc) of human IgG and an engineered variant of human fibroblast growth factor 2 (FGF2). The Fc fragment constitutes integral part of typical ADCs, but it is also present in other types of conjugates like cytotoxic peptibodies or conjugates of antibody fragments. FGF2 is a natural fibroblast growth factor receptor (FGFR) ligand that was successfully engineered to yield a selective cytotoxic conjugate targeting FGFR-overproducing cancer cells.

We present two distinct conjugation strategies allowing for the site-specific incorporation of cytotoxic drugs. First, the protocol for conjugation to the cysteine side chains of the Fc fragment via maleimide-thiol chemistry based on Hermanson’s protocol¹³ is provided (Figure 1A,B). In this protocol two disulfide linkages are initially reduced with tris(2-carboxyethyl)phosphine (TCEP) and resulting free thiol groups are subjected to conjugation with monomethyl auristatin E (MMAE) via maleimide-thiol chemistry (Figure 1B). Due to the interaction between constant heavy chain domains 2 and 3 (CH2 and CH3) the dimeric structure of the drug-linked Fc is preserved. Secondly, the strategy for the generation of double warhead FGF2 conjugate is presented that combines cysteine engineering and sortase A-mediated ligation for incorporation of two distinct drugs into FGF2 in a site-specific manner (Figure 1A,C). The cysteine-free variant of FGF2 bearing additional N-terminal KCKSGG with single exposed cysteine residue and C-terminal LPETGG short peptide tag is used¹². The maleimide-thiol reaction allows for the conjugation of MMAE to cysteine within KCKSSG linker designed by our group^14,15. Sortase A-dependent step (based on Chen et al.)¹⁶ mediates the ligation of methotrexate (MTX)-linked tetraglycine peptide GGGG-MTX to the C-terminal LPETGG sequence, yielding two types of single warhead conjugates (Figure 1C). Sortase A is a cysteine protease that catalyzes the transpeptidation reaction between LPETGG and GGGG motifs. The enzyme binds to the LPETGG motif at the C-terminus of the protein, then the amide bond between the threonine and the glycine is hydrolyzed to form an enzyme-substrate complex. The next step is the aminolysis of the thioester enzyme-substrate bond, where the donor of a primary amino group is the glycine residue of the tetraglycine motif¹⁷. Combination of these two approaches generates site-specific FGF2 double warhead conjugates (Figure 1C). In principle, provided conjugation protocols can be successfully applied to any engineered targeting protein of interest to generate selective cytotoxic conjugates. Moreover, versatility of this approach makes it suitable for many other protein-protein and protein-peptide ligation purposes, as well as for the attachment of lipids, polymers, nucleic acids and fluorophores to proteins with available sulfhydryl group (or generated by reduction of native disulfide bonds) and/or with introduced small peptide tag.