Protein-Drug Conjugation

BOC Sciences is the industry leading expert in small molecular, protein engineering and drug conjugation. We provide comprehensive services for the conjugation and characterization of protein drug conjugates. We can design linkers with optimal size, appropriate release mechanisms, and compatible conjugate chemistry, develop payload derivatives with modified toxicity and special linkage sites, synthesize customized drug-linker complexes, and more. Our analytical testing services support the entire protein drug conjugates development cycle, from bioconjugation method selection, binding assays and method validation, to stability assessment.

What are Protein Drug Conjugates?

With the approval of antibody drug conjugation and more and more ADC entering clinical trials, targeted delivery of cytotoxic agents to cancer cells has been proved to be an effective way to treat cancer. Protein-drug conjugates are a drug design strategy that combines proteins and drugs to achieve more precise and efficient therapies. Compared to large full-length antibodies, the use of smaller protein domains as payload delivery carriers for targeted delivery provides many potential benefits, including increased tumor permeability, adaptability to protein engineering and site-specific binding, and improved tolerance.

Services for Protein Drug Conjugates

• Protein selection and optimization
• Small molecule drug selection and optimization
• Bioconjugation services: suitable chemical linkers, protein engineering and site-specific binding.
• Conjugates purification and characterization: chromatography, electrophoresis, binding assays.
• Pharmacodynamic evaluation: drug release, cytotoxicity, pharmacodynamic, and others.

Why Protein Drug Conjugates?

• Highly targeted - Protein-drug conjugates can recognize and bind to the target through the specificity of the protein, thus achieving a high degree of targeting of the disease target and reducing the impact on healthy tissues.
• Enhanced efficacy - Protein-drug conjugates can deliver drugs directly to the target, thereby enhancing efficacy and reducing dosage and side effects.
• Prolong drug half-life - Protein-drug conjugates can prolong the half-life of drugs through the stability and long life of proteins, reducing the frequency and dosage of drugs.
• Improve drug solubility - Protein-drug conjugates show improved solubility and stability and improve the bioavailability and absorption of drugs.

Albumin–drug conjugates

As of now, albumin is the most extensively explored protein for preparing drug–protein conjugates especially in cancer. Albumin conjugates can further be modified by PEGylation to impart hydrophilicity to improve the blood residence of conjugates. Additionally, one can also go for coupling of targeting ligands such as cyclic or acyclic RGD, lactosamine and folate to the albumin to impart targeting potential to the conjugates. The high endogenous availability of albumin in blood has been conveniently exploited to prepare in situ albumin–drug conjugates using chemically modified drugs with maleimide group which binds to serum albumin when injected in blood stream.

Recently, scientists presented the development of a novel and effective HSA-SN-38 conjugate (SSH20). They observed a significant decrease in SSH20 uptake through immunofluorescence assays and western blotting after silencing of Cav-1 expression using RNA interference. Cytotoxicity assays indicated a reduced drug sensitivity in Cav-1-depleted cells. Importantly, there was a significantly diminished sensitivity to SSH20 in Cav-1-silenced tumors compared to Cav-1-expressing tumors in vivo. Notably, SSH20 demonstrated significantly greater potent than irinotecan in vitro and in vivo. Together, they have developed a novel HSA-conjugated chemotherapy that is potent, effective, safe, and demonstrates improved efficacy in high levels of Cav-1.

Fig. 1 Effect of Cav-1 expression on SSH20 sensitivity[1]

Transferrin–drug conjugates

Transferrin is an endogenous iron-transporting protein which functions through transferrin receptor (TfR). Differential levels and heterogeneous expression of TfR have been reported for TfR in cancerous as well as normal cells. Due to an increased iron requirement in tumor tissues, there is a 10-fold higher expression of TfR in cancer cells as compared to normal cells. Various cancers showing higher expression of TfR include breast cancer, leukemia, colon cancer, skin cancer, glioblastoma, and lymphoma. Apart from cancer, the use of transferrin has also been explored for oral delivery of drugs, giving the expression of TfRs in GI tract. It has been reported that TfR are expressed in duodenal epithelial cells and play a crucial role in receptor-mediated endocytosis for absorption of TfR targeted drug delivery systems. This underscores the potential of transferrin as a targeting ligand for devising drug–transferrin conjugates.

Gelatin–drug conjugates

Gelatin is another protein that has been explored for development of drug delivery systems ranging from microspheres to nanoparticles. Gelatin serves as a substrate for matrix metalloproteinase enzymes, including MMP-2 (gelatinase A) and MMP-9 (gelatinase B). MMP-2 and/or MMP-9 is/are overexpressed in several cancer tissues. This forms the basis for the hypothesis that if gelatin is conjugated to drugs, it would be preferentially cleaved inside tumors and release active drugs avoiding toxicity to normal cells where expression of the aforementioned enzymes is low or absent. This approach negates the need for tumor-specific linkers as gelatin functions both as a carrier as well as a substrate releasing drugs specifically in tumors.

Other drug conjugates

Hemoglobin (Hb), fibrinogen, insulin, etc. are utilized in formulating protein–drug conjugates. Hb–drug conjugates leverage the natural Hb scavenging activity of serum haptoglobin directed towards bHb scavenger receptor CD163 located on monocyte and macrophages. Consequently, Hb acts as a natural ligand for targeting macrophages and monocytes involved in monocytic leukemias. Furthermore, liver macrophages and Kupffer cells are activated in viral hepatitis and are implicated in liver cirrhosis and in hepatocellular carcinoma. Therefore, the application of Hb–drug conjugates can also be extended to the treatment of hepatitis and hepatocellular carcinoma.

Linkers

Specific linkers between the cancer-recognizing molecule and the cytotoxic drug are designed to remain highly stable in circulation and release the cytotoxic payload only after protein-drug conjugations have been internalized by the tumor cells. The therapeutic efficiency, pharmacokinetics stability and off-target effects of protein-drug conjugations are largely dependent on the characteristics of the linker. The selection of linker is primarily influenced by the intended mechanism of action of the protein-drug conjugation, the cellular trafficking pathway of the protein-drug conjugate and the functional groups present in both the targeting protein and in the cytotoxic drug. Linkers used in protein-drug conjugations are categorized as either cleavable or non-cleavable based on their metabolic fate in cancer cells.

Cleavable linkers are predominantly used in protein-drug conjugations that are designed for efficient receptor-mediated endocytosis and lysosomal targeting, where linker processing inside lysosomes releases active form of drug inside cancer cell. However, non-internalizing protein-drug conjugations (where the drug is released in the tumor microenvironment after binding to a cancer marker stably exposed on the cancer cell surface) or those targeting cellular compartments other than lysosomes cellular often utilize cleavable linkers. Examples of cleavable linkers include pH-sensitive hydrazone linkers (cleaved in the acidic environment of lysosomes), reducible disulfide linkers (releasing the drug upon linker reduction) and peptide linkers (degraded by lysosomal proteases such as cathepsin B). In contrast to the cleavable linkers, the release of a PDC-conjugated cytotoxic drug via non-cleavable linkers depends on the lysosomal proteolytic degradation of the targeting molecule itself. This limits the application of non-cleavable linkers to targeting internalizing cancer markers. Highly potent cytotoxic drugs used in conjugates fall into two main types, microtubule inhibitors and DNA-damaging drugs.

Our Advantages

• Novel site-specific conjugation technologies and robust methods
• Advanced equipment and technique
• Experienced scientific team
• Wide categories of biomolecule cross linking
• Data analysis, detailed report with results and discussion
• Cost-effective and high-quality products