Conjugatable Ligands: Methods Overview

Conjugatable ligands are chemically modified to allow their attachment to a peptide/protein of interest (POI) with a chemical linker. Their assembly generates a bioconjugate. Successful applications for bioconjugates (e.g. tumor targeting, vaccination) is dependent on an appropriate and effective conjugation method. In theory, the process is possible via most amino acid residues on the POI and specific functional groups on the ligand. Multiple strategies for protein bioconjugation have been described [1,2] for which key points apply:

• The choice of the conjugation method is based on the presence of specific amino acid residues on the POI that can be chemoselectively modified without affecting its conformation or function.
• Cysteine and Lysine residues are the most commonly used amino acids in the bioconjugation of peptides/proteins.
• The conjugatable ligand features a chemoselective functional group that is complementary to the targeted amino acid residues on the POI.

Here we summarize the cysteine- and lysine-based coupling principles in the context of bioconjugation to a monoclonal antibody (mAb) using InvivoGen's ready-to-use conjugatable ligands. We also introduce the concept of click chemistry-based coupling using InvivoGen's conjugatable ligands designed for flexible bioconjugation. Finally, we provide a calculation method to determine the ligand (or drug)/antibody ratio (DAR) using a spectrophotometer.

Cysteine based coupling

Cysteine-based conjugation relies on a chemical reaction between cysteine residues within the POI and a thiol-reactive functional group (e.g. maleimide) installed on the ligand (or linker + ligand). This method is commonly used for the production of Antibody-Drug Conjugates (ADCs), a therapeutic class of bioconjugates allowing targeted delivery of highly cytotoxic agents (i.e. to target cancer cells) [2].

Antibodies generally do not possess free thiols, therefore cysteine-based coupling relies on the reduction of disulfide bonds within the POI.

• Interchain cysteine-based conjugation to antibodies:

This strategy relies on the partial reduction of the 8 cysteine residues forming the 4 interchain disulfide bonds (which have higher solvent accessibility than the intrachain disulfides). This presents two main advantages:
- the immunoglobulin conformation is conserved as the interchain disulfide bonds are not critical for structural antibody stability,
- the degree of conjugation is controlled as the disulfide bonds can be selectively reduced under mild conditions to give 2, 4, 6, or 8 free thiols.

The interchain cysteine-based method generates antibody conjugates with heterogeneous conjugation sites and a different number of ligands attached, resulting in a DAR of 0, 2, 4, 6, or 8 (see illustration).

• THIOMAB™ cysteine-based conjugation to antibodies:

This strategy was developed to improve the homogeneity of the preparation and control the number of ligands attached. It relies on the introduction of disulfide-capped cysteine residues into specific sites on the backbone of the antibody. After reduction of the interchain and engineered disulfide cysteines and a re-oxidation step to re-connect the interchain bonds, the conjugation takes place only at the 2 non-native cysteine residues. This valuable strategy allows the generation of antibody conjugates with a DAR of 2 and >90% homogeneity [2].

InvivoGen's ready-to-use conjugatable ligands, which feature an activated maleimide functional group, are suited for both interchain reduction or THIOMAB™ conjugation strategies.

Lysine based coupling

Lysine-based conjugation relies either on a chemical reaction between lysine residues within the POI and a functional group installed on the ligand (or linker + ligand), either directly or after lysine modification. This method is also commonly used for the production of ADCs. It takes advantage of the numerous (>80) lysine residues in the immunoglobulin scaffold, most of which (>20) are exposed at the antibody surface [2].

• Direct lysine-based conjugation to antibodies:

This strategy relies on reactive amine side chains of lysine residues due to their good nucleophilicity.  Generally, ligands (or 'linker+ligand' segments) bearing activated esters (e.g. N-hydroxysuccinimide (NHS)) readily react with the antibody lysine residues to generate amide bonds.

• Modified lysine-based conjugation to antibodies:

This method relies on the replacement of free amine groups with sulfhydryl groups on the lysine residues. This thiolation step can be achieved using Traut’s Reagent [3] or SATA Reagent [4] (see illustration). InvivoGen's ready-to-use conjugatable ligands, which feature a maleimide functional group, are suited for this modified lysine conjugation strategy.

While lysine-based coupling is easy, it allows less precise control of the conjugation sites and the number of ligands attached. Lysine-directed conjugations have been estimated to occur on both the heavy and light chains, and theoretically, the DAR can range from 0 to 8 (see illustration).

Click chemistry-based coupling

"Click Chemistry" is a term used to describe reactions that can occur spontaneously (i.e. without requiring a functional group to be activated) and can be conducted in easily removable or benign solvents (e.g. water). This represents a great advantage for generating conjugates with biocompatibility

InvivoGen has developed conjugatable PRR ligands featuring an azido group, which allow a flexible choice among commercially available linkers containing an azido-reactive functional group (e.g. alkyne, BCN,  DBCO, or TCO).

Note: InvivoGen does not provide linkers for click chemistry.

Ligand (drug) to antibody ratio (DAR)

Each bioconjugate has an optimal DAR with the right balance of efficacy, clearance, and toxicity. It differs for each immunoglobulin (depending on its size and glycosylation) and the application for which the conjugate is generated. The user should assess different molar ratios of antibody and ligand to determine the optimal DAR. Importantly, this characterization also allows controlling batch-to-batch variability.
The final DAR can be determined by spectrophotometry or mass spectrometry.

Below is a method to determine the final DAR using a spectrophotometer (i.e. Nanodrop) [5]:

1. Harvest samples throughout the conjugation procedure
Sample 1 (S1): Antibody of interest solution
Sample 2 (S2): Conjugatable ligand solution
Sample 3 (S3): Desalted antibody conjugate
Note: All samples must be diluted using the same buffer for optical density (OD) measures.

2. Measure OD315nm and OD280nm in triplicates for S1, S2, and S3. Note: All samples must be in the linear range of the spectrophotometer.

3a. For a conjugated TLR7 agonist-mAb: calculate the ratio (R) of OD315nm to OD280nm for each sample (R1, R2, and R3).
3b. For a conjugated STING agonist-mAb: calculate the ratio (R) of OD260nm to OD280nm for each sample (R1, R2, and R3).

4. Determine the molar extinction coefficient (ε) at 280nm for the antibody of interest using its amino acid sequence. Note: The average molar extinction coefficient (ε) at 280nm for an IgG1 mAb is 210,000 M^-1 cm^-1.

5. The molar extinction coefficient (ε) at 280nm for each InvivoGen's conjugatable ligand is indicated in their technical data sheet (TDS).

6. Calculate the DAR:

In the context of conjugation to a peptide or protein to generate an antigen-adjuvant conjugate, the final ligand (drug) to protein ratio can also be determined by spectrophotometry or mass spectrometry. If using a spectrophotometer, it is necessary to determine the molar extinction coefficient (ε) at 280nm for the peptide/protein of interest using its amino acid sequence.

References

1. Gauthier M.A & Klok H.A., 2008. Peptide/protein-polymer conjugates: synthetic strategies and design concepts. Chem Commun. 23:2591-2611.
2. Yamada K. & Ito Y., 2019. Recent chemical approaches for site-specific conjugation of native antibodies: technologies toward next-generation antibody-drug conjugates. ChemBioChem. 20(21):2729-2737.
3. Brückner M., et al. 2021. The conjugation strategy affects antibody orientation and targeting properties of nanocarriers. Nanoscale. 13(21):9816-9824.
4. Ackerman S.E, et al. 2021. Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity. Nat Cancer. 2(1):18.
5. Ou J, et al. 2018. Bioprocess development of antibody-drug conjugate production for cancer treatment. PlosOne 13(10): e0206246.