Peptide antigens

The use of synthetic peptide antigens for the generation of custom antibodies has markedly increased over the last years as a viable alternative to the use of full length proteins as immunogens.  From experience, the use of relatively short peptides mimicking specific parts of the target protein has shown to have a number of advantages in various situations.

Advantages of using Peptide antigens

First of all, peptide antigens offer a smart solution for antibody generation in cases where the target protein’s amino acid sequence is known, but the protein itself is not available, or structurally too complex to be obtained as a structurally stable and intact protein (e.g. certain transmembrane receptors, ion channels, etc.).  Moreover, the peptide immunogen approach offers flexibility in selecting and designing antigens for domains of the target protein as an additional advantage. Peptide antigens are also highly useful for generating antibodies against post-translationally modified binding sites on target proteins.

Other advantages are:

• the epitope is well-defined from the start
• the protein sequence  is all that is needed
• a much lower chance of cross-reactivity
• the antigen is fully synthetic, which is cost-effective with usually very short supply times

Pepscan’s unique and proven protein mimicry technologies, in combination with our thorough expertise in peptide synthesis allow the creation of superior peptide antigens for monoclonal antibody generation. We have developed leading expertise in design and synthesis of complex peptide antigens and immunogens.  Therefore, we can also address your need for conformationally-constrained peptide constructs, incorporation of PTM’s, such as phosphorylation or sulphation, or conjugation to carrier proteins (KLH, BSA, etc.).

With over 25 years of experience, our staff members are more than happy to assist you in developing a plan that is tailored to meet your specific needs.

Design Strategies for Peptide Antigens

Although many peptide sequences can be immunogenic, not all are equally effective in generating antibodies with reactivity against the target protein. Successful antibody generation with peptide antigens depends on several factors that need to be considered in the design, including:

• the accuracy of the target protein’s amino acid sequence
• the predicted 2ary / 3airy  structure of the intact protein
• the selection of domains/regions of the protein to be mimicked by the peptide antigen
• the need of conjugating peptide antigens to a larger carrier protein
• the ease of synthesis of certain peptide sequences

Therefore, the following general recommendations apply to the design of peptide antigens:

1. Ensure that the correct species and protein sequence have been identified.
2. Select the peptide antigen from a solvent-accessible region of the native protein. Often, these are regions exposed at the protein surface that are in contact with the aqueous (hydrophilic) environment of the solvent. Certain areas of the protein may be inaccessible to antibodies, such as transmembrane regions or the centre of a globular protein.
3. An important advantage is that peptide antigens can be designed to cover a specific epitope of interest. Peptide antigen candidates are screened against a specific protein database. Certain domains may be also present in other proteins: these sequences should be avoided in order to minimize the likelihood of unwanted cross-reactivity and thus optimize your overall antibody-specificity. Sequences with minimal sequence homology are best selected in order to reduce unwanted off-target protein binding.
4. Depending on the 3D-structure of the targeted domain in the intact protein, constraining techniques may be required to optimally mimic the tertiary structure of the antigen. As one of the specialist in protein surface mimicry, Pepscan has available various (partly proprietary) technologies for the design and synthesis of conformationally-constrained peptide antigens(¹, ²).
5. Peptide antigens are often too small to generate a significant immune response without prior conjugation to a carrier protein such as KLH, BSA or OVA. Usually the use of peptide – carrier protein conjugates is therefore recommended.

Synthesis of Peptide Antigens

For the design of the peptide antigens the ease of synthesis of specific peptide sequences should also be considered.  Both hydrophobic and hydrophilic residues should be included, and it is favorable for the peptide to incorporate amino acids that are immunogenicity-promoting (such as basic and aromatic amino acids). Hydrophobic amino acid content should best be kept below 50%, and long chains of hydrophobic residues should preferably also be avoided. For peptide solubility, at least one charged residue (arginine, glutamine, aspartic acid, or lysine) should be incorporated within every five amino acids. Peptide solubility can also be improved via conservative replacements or addition of polar residues to the N- or C-terminus. In designing the antigens preferably avoid complex regions, such as beta-sheets or alpha-helices, but instead aim for flexible regions.

Pepscan applies its state-of-the-art capabilities and expertise for the synthesis of peptide antigens, including (if required) conformational constraints, PTM’s, conjugation or biotinylation. For most antibody generation projects a purity at least 85% is recommended for any peptide antigen. However, if desired we can provide antigens with higher purities also. We generally will synthesize approximately 10 mgs of peptide, which is usually sufficient for protein-conjugation, ELISA-screening and constructing an affinity matrix chromatography setup if desired.

References:

1. R.S. Boshuizen et al: A combination of in vitro techniques for efficient discovery of functional monoclonal antibodies against human CXC chemokine receptor-2 (CXCR2). mAbs 6 (6), 1415–1424; 2014
2. L.E.J. Smeenk et al.: Reconstructing the discontinuous and conformational β1/β3-loop binding site on hFSH/hCG by using highly constrained multicyclic peptides.  ChemBioChem 16 (1), 91-99; 2015