Epitope mapping using synthetic peptides

Epitope mapping identifies antigen regions that serve as binding sites for antibodies. The overlapping linear peptides derived from the primary sequence of the antigen is frequently used for the epitope screening. Individual peptides can be divided into several fragments that overlap. The resulting overlapping peptide libraries can then be used for processes including continuous and linear epitope mapping.

For example, to map the epitope of an antibody, a few overlapping fragments spanning the target regions are constructed in an expression vector. These constructs are transiently transfected in cells and whole cell lysates collected after 48 hours are subjected to Western blotting with the antibody. To further map the region of this fragment, a series of overlapping peptides are synthesized. These peptides and lysates from the cells expressing target full-length gene or empty vector (negative) are performed by the dot blot.

Mapping epitopes quickly and accurately are challenging because the epitopes tend to be nonlinear on antigens. Combining binding specifically toward two distinct epitopes into a single molecule can significantly enhance the immunotherapeutic properties of monoclonal antibodies. Multivalent interactions are the most efficient at driving IgE receptor signaling pathways.

There are several useful tools for studying the antigen-antibody interactions.

  1. Use the MAPs as the immunogens. Multiple Antigenic Peptides (MAPs) are peptides that are branched artificially, in which Lys residues are used as the scaffolding core to support the formation of 8 branches with varying or the same peptide sequences. For example, one goal is to include different epitopes from different virus proteins in a single unit. The epitopes showing the right prediction of antigenicity and conserved in most serotypes of a virus are selected and assembled as the MAP.
  2. Screening combinatorial peptide libraries to optimize enzyme substrates and create high-affinity protein ligands. A critical biological application of custom peptide libraries is the characterization of the binding events that occur between specific proteins and their peptide ligands. A series of Overlapping Peptide, Truncation Peptide, Alanine Peptide Scanning, Scrambled Peptide, or Positional Peptide can be used for mapping and validating epitopes, the characterization of therapeutic antibodies, studying anti-antibody and neutralizing antibody actions in vitro.
Synthetic Peptide Library

Cell Penetration Peptide Synthesis

Tat, the transcription activator of the human immunodeficiency virus type 1 (HIV-1) viral genome, enters cells in a non-toxic and highly efficient manner. Tat is the first known cell-penetrating peptide.

CPPs have been used as a carrier to deliver proteins or genes into cells and tissues. In this study, a CPP library composed of 55 CPPs were used to deliver genes into plant cells. Many CPPs showed efficient penetration into plant cells. The Lys-containing CPPs have higher penetration efficiency in the plant than in animal cells. This could be due to differences in lipid composition and surface charge of the cell membranes. No correlations were detected between the penetration efficiency and the cationic, amphipathic, or hydrophobic properties of peptides.

D-R9 is composed of D-form amino acids. D-R9 bound preferentially to the membrane and did not penetrate the cytosol or vacuole. In mammalian cells, poly-Lysine-based CPPs are efficient and interact with membrane lipid head groups to induce wrapping of the membrane monolayers. Arg-rich peptides, such as the Tat peptide, are among the most efficient CPPs. Arg-rich CPPs may generate negative Gaussian membrane curvature to form pores or protrusions from endocytosis. The cell penetration efficiency of CPPs containing poly-Arg is higher than those containing poly-Lys. However, in a plant, Arg-rich CPPs are not the most efficient at penetrating plant cell.

CPP peptide property

CPP peptide property

How to do competition binding assay?

Endocrine fibroblast growth factors (FGFs) require Klotho transmembrane proteins as necessary co-receptors to activate FGF receptor (FGFR) signaling.

A series of peptides were synthesized by LifeTein and used for the competition binding assay. Both the KL1 and KL2 domains of β-Klotho participates in ligand interaction. The FGF19 peptide was used for alanine scanning mutagenesis. It was found that a single amino acid mutations in either region were sufficient to abolish β-Klotho binding. FGF19 and FGF21 function through β-Klotho to regulate glucose and lipid metabolism.

How to perform the solid-phase binding assay

1. The 96-well plates were coated overnight at 4 °C with 2 µg/mL of antibody in PBS.

2. Plates were washed twice with PBST and blocked with 3% (w/v) BSA in PBS for 1.5 hours at room temperature.

3. The conditioned media containing β-Klotho were added to the plates and incubated for 1.5 hours at room temperature.

4. Plates were washed a few times.

5. The peptide mutation FGF21 and an anti-β-Klotho antibody were biotinylated with EZ-Link Sulfo-NHS-LC-Biotin at the indicated concentrations.

6. After wash, streptavidin-HRP was used for detection.

7. EC50 values were determined.

How to do competition binding assay?

1. The WT and mutant peptides were custom synthesized and purified (>95% purity) by LifeTein.

2. Binding of FGF19 and FGF21 peptides to β-Klotho was assessed.

3. The β-Klotho ECD 6 × His, varying amounts of FGF19 and 21 peptides, and biotinylated human FGF19 or FGF21 protein were prepared.

4. The streptavidin donor beads and nickel chelate acceptor beads were added to the plates.

5.Plates were incubated for 3 hours at room temperature protected from light and read on the Plate Reader.

Reference: Scientific Reports, volume 8, Article number: 11045 (2018)




Tumor Targeting of Conjugated Synthetic Peptides

The folate receptor alpha (FRα) is highly expressed in ovarian cancer and not in normal tissues. An FRα binding peptide C7 (Met-His-Thr-Ala-Pro-Gly-Trp-Gly-Tyr-Arg-Leu-Ser, MHTAPGWGYRLS)  was found to bind to FRα expressing cells. This tumor-targeting peptide was proved by both phage homing experiment and fluorescence imaging.

Tumor Targeting of Conjugated Synthetic Peptides
1. The FITC conjugated peptide FITC-MHTAPGWGYRLS was dissolved in PBS.
2. The peptide was injected intravenously into a tumor-bearing nude mouse.
3. After 2 h, tumor and other organ tissues were harvested and analyzed using a fluorescence imaging system.

Cell internalization of Synthesized Peptide
1. Cells were seeded in 24 well plates containing coverslips and incubated for 24 hours in medium with FBS.
2. FITC conjugated peptide was incubated with the cells for 4 hours at 37 °C.
3. The cells were washed once with PBS and fixed in 4% paraformaldehyde.
4. Cells were washed three times with PBS and stained with DAPI for 20 min at room temperature.
5. Internalized fluorescent signals were imaged with a confocal microscope.

This tumor-specific peptide could be a potent and selective ligand for FRα. It has a great potential for delivery of cancer therapeutics or imaging agents to FRα expressing tumors.

Reference: Scientific Reports, volume 8, Article number: 8426 (2018)

tumor targeting peptides

tumor targeting peptides

Cell Penetration Peptide Targeting Cancer

Peptide Targeting Cancer

Cancer is expected to surpass the current number one, cardiovascular diseases by 2030 as the leading cause of death.

The targeting peptides can be considered as an alternative vehicle for the delivery of anti-cancer drugs because of their lower molecular weight and excellent tolerability by human bodies.

The following modification can prolong the half-life of the peptides from the degradation by blood proteases: forming cyclization within a peptide, blocking of the C- and N- terminus, replacement of standard L-type amino acids by their D-amino acid counterparts, using of unnatural amino acids incompatible with endogenous proteases.

About targeting peptides:
1. Somatostatin (SST) derivatives: Binding of the natural ligand somatostatin peptide to the receptors leads to inhibition of overexpressed SSTR2 and 5 in breast cancer. For example, cyclic SSTR agonist octreotide (fCfDWKTCT), selectively binds SSTR2 and 5.

2. Peptide-derivatives of gastrin-releasing peptide (GRP)

The gastrin-releasing peptide receptor is associated with the prostate and breast cancer. It was found that bombesin (YQRLGNQWAVGHLM) and its derivatives could be used as targeting peptide for detection of prostate cancer via PET and CT screening.

3. Peptides targeting tumor microenvironment

The glycoprotein prosaposin (PSAP) can inhibit metastases from breast and lung cancer in preclinical models. A cyclic PSAP peptide (DWLPK) could hinder metastatic spread and restrain tumor development. The synthetic antagonist of CXCR4 called NT21MP (LGASWHRPDKCCLGYQKRPLP) exhibits anti-tumor activities through decreased adhesion and migration of breast cancer cells. The peptide R (RACRFFC) targeting of CXCR4, showed capacities to remodel the tumor stroma.

4. Peptides targeting the tumor pH and temperature

The pHLIP (pH-Low Insertion Peptide), ACEQNPIYWARYADWLFTTPLLLLDLALLVDADET, can insert into cell membranes as an a-helix under low pH conditions. The pHLIP peptide increases the uptake of the peptide-coated drug to tumors compared to the naked particles. Thus, the local tumor microenvironment can be used to trigger peptide drug formulations to respond accordingly.

The elastin-like polypeptides (ELP) is conjugated to the cell penetrating peptide Bac (RRIRPRPPRLPRPRPRPLPFPRPG) for improved cellular penetration to deliver gemcitabine for pancreatic cancer.

5. Peptides targeting tumor tissues

A widely used endothelial binding peptide is The tripeptide arginine-glycine-aspartic acid (RGD), an endothelial-binding peptide, has high specificity towards integrins for anti-tumor and anti-angiogenic treatments.

The radiolabeled, PEGylated RGD has been used as a PET-probe to detect gliomas, and the iron-oxide nanoparticles coupled RGD was used for the MR imaging of brain tumors.

The cyclic iRGD (CRGDKGPDC) is a prototypic tumor-penetrating peptide binding integrins. The iRGD peptide increased the tumor tissue penetration and the delivery of drugs, nanoparticles, or antibodies in vivo.

The cRGD (RGDdYK) and cilengitide (cRGDf [N-Me]V) with the combination therapy of temozolomide (TMZ) radiochemotherapy were used in the clinical trials.

A linear targeting peptide called CooP (CGLSGLGVA) binds the mammary-derived growth inhibitor (MDGI). The MDGI is a fatty acid binding protein that is highly expressed at the cell membrane of malignant glioma cells.

The coop peptide is a homing peptide targeting glioma cells and tumor-associated blood vessels. The chemotherapeutic drug conjugated CooP peptide can reduce the number of invasive tumor cells.

The cell penetration peptides are the cure for the difficult-to-access cancers such as brain tumors. This endothelial-specific peptide with enhanced penetrance would allow better passage of the drug conjugates through the blood-brain barrier.

The peptide drugs have the benefits of high specificity, low antigenicity, low cost and simple production. The peptides have the potentials for the development of therapy options for various tumors in the field of personalized medicine of cancer.



How to analyze the cell viability with peptides?

  1. Cells were seeded on 96-well culture plates (10000 cells/well) and incubated in 100 μL of DMEM containing 10% FBS.
  2. The medium was then replaced with fresh medium containing 10% FBS, and a peptide solution was added to each well at an appropriate concentration (for example 0.5uM, 1uM, 1.5uM, 2uM).
  3. After a 2-h incubation, Cell counting kit-8 was used according to the manufacturer’s protocol. Cell Counting Kit-8 (CCK-8) allows sensitive colorimetric assays for the determination of cell viability in cell proliferation and cytotoxicity assays.
  4. Cell viability was evaluated by the absorbance of formazan from each well, and 100% cell viability was calculated from the wells without peptides.
  5. The results are presented as the mean and standard deviation obtained from 5 samples.


How to measure the cellular uptake of peptides?

TMR/Cy3/Cy5 was introduced for the fluorescent label with peptides to evaluate the cell-penetrating ability and intracellular distribution of each peptide.

  1. Cells (HeLa or Huh-7) were seeded on 24-well culture plates (40,000 cells/well) and incubated in 400 μL of DMEM containing 10% fetal bovine serum (FBS).
  2. The medium was then replaced with fresh medium containing 10% FBS, and a Tetramethylrhodamine carboxylic acid (TMR)-labeled peptides
  3. The solution was added to each well at an appropriate concentration (for example 0.5uM, 1uM, 1.5uM, 2uM).
  4. After a 1, 2, 3 or 4 hours incubation, the medium was removed, and cells were washed with ice-cold PBS and trypsin.
  5. After the addition of medium containing 10% FBS, cells were centrifuged at 1600 rpm for 3 min at 4 °C. The cell pellets obtained were suspended in ice-cold PBS, centrifuged at 1600 rpm for 3 min at 4 °C, and then treated with Cell lysis buffer.
  6. The fluorescence intensity of each lysate was measured using a spectrofluorometer. The amount of protein in each well was concomitantly determined using the BCA protein assay.
  7. The results are presented as the mean and standard deviation obtained from 3 samples.

Reference: https://www.nature.com/articles/srep19913#s1

Cy5 labeled peptide in cells

Cy5 labeled peptide in cells

Peptide Antigens from Tumor Cells: Potential Peptide Vaccines for Cancer

Check the cancer peptide database for a list of tumor peptides.

Tumor antigens can be classified into two categories based on their pattern of expression: tumor-specific antigens (TSA) and tumor-associated antigens (TAA).

Targeting tumor-associated antigens (TAAs) is a promising approach for cancer immunotherapy. Neoantigens are tumor-specific antigens that originate from somatic mutations in cancer cells but not in healthy tissues. So the TAAs are considered as ideal targets for novel immunotherapies. Antigens of three classes can induce tumor-specific T cell responses.

1. Antigens derived from viral proteins: Viral proteins are produced inside the tumor cells. So the antigenic peptides can be detected by T cells.

2. Antigens derived from point mutations: Many CTL isolated from the tumors were found to recognize antigens that arise from point mutations in ubiquitously expressed genes. These mutations are passenger mutations and the corresponding antigenic peptides are unique to the tumors in which they were identified.

3. Antigens encoded by cancer-germline genes: Cancer-germline genes are expressed in many cancer types and not in normal tissues except germline and trophoblastic cells. The tumor-specific pattern of expression results from the genome-wide demethylation in male germ cells.

A large number of antigenic peptides recognized by antitumor CTL have been identified. Candidate peptides can be synthesized and tested for HLA binding in vitro. The elution of antigenic peptides from MHC class I molecules immunopurified from the surface of tumor cells can be used to identify the antigens. TAAs can be targeted using peptide vaccines or by cellular approaches. The delivery of new peptide drugs might show great promises for future therapies.

Peptide Antigens

Tumor-associated peptide antigens

LifeTein can customize a discovery and development path to fit your exact needs for peptide synthesis.


Polypeptides pegylation as new drug candidates

Polypeptides are used as new drug candidates to target specific disease symptoms. However, peptide drugs are rapidly degraded by proteolytic enzymes and neutralized by antibodies. Pegylation of polypeptide drugs improves their pharmacodynamic and pharmacokinetic profiles. Pegylating site-specifically can minimize the loss of biological activity and reduce immunogenicity. LifeTein offers peptide pegylation service and the PEG-modification of peptides through primary amines and sulfhydryl groups.

A significant limitation of the present PEGylated peptides is their heterogeneous nature because PEG is conjugated at many different nucleophilic amine residues. LifeTein’s approach to peptide PEGylation can address the fundamental issues of site-specific conjugation and high-efficiency conjugation. The click chemistry is widely used in the pegylation process.

The efficient ratio of 1:1 PEGylation of a peptide can be completed in 24 hours and purification of the PEG-protein conjugate in another three h, without destroying their tertiary structure or abolishing their biological activity.

LifeTein’s improved technology is the use of branched structures, in contrast to the linear structures. Branched PEGs have increased molecular masses of up to 60 kDa or more, which is good at cloaking the attached polypeptide drug from the immune system and proteolytic enzymes.

Pegylation is the established method for improving the pharmacokinetics and pharmacodynamics of peptide pharmaceuticals.

New frontiers for the technology are now emerging for PEG-based hydrogels and PEG-modified liposomes, small-molecule modification, and the primary targets for pegylation of small-molecule drugs, oligonucleotides, lipids, cofactors, antibodies, saccharides, and nanoparticles.

Pegylation service from LifeTein

Pegylation service from LifeTein

Cyclic peptides as broad-spectrum antiviral agents

Cyclic peptides as broad-spectrum antiviral agent

Cyclic peptides as broad-spectrum antiviral agents

Antiviral drugs and vaccines are the most powerful tools to combat viral diseases. Most drugs and vaccines only target a single virus. However, the broad-spectrum antivirals can be used for rapid management of new or drug-resistant viral strains. Cyclized peptides and peptide analogs are excellent examples of broad-spectrum antivirals.

An artificial peptide molecule was found to neutralize a broad range of group 1 influenza A viruses, including H5N1. The peptide design was based on complementarity determining region (CDR) loops have been reported for other viral targets. The optimized peptides bind to the highly conserved stem epitope and block the low pH-induced conformational rearrangements associated with membrane fusion.

These peptidic compounds and their advantageous biological properties should accelerate development of novel small molecule and peptide-based therapeutics against influenza virus.

The linear peptide is Suc-SQLRSLEYFEWLSQ-NH2. Three cyclization strategies were used: head to tail, side chain to side chain and side chain to tail. An ornithine (Orn) side chain was fused with the carboxyl terminus of β-alanine for lactam formation.

Check here for more details: Potent peptidic fusion inhibitors of influenza virus, Science 28 Sep 2017, DOI: 10.1126/science.aan0516

Lately, more broad-spectrum antiviral agents were found to target viruses. It was found that 55 compounds can target eight different RNA and DNA viruses. Dalbavancin is a novel lipo-glycopeptide antibiotic. The lipoglycopeptide disrupts bacterial cell wall formation by binding to
the terminal d-alanyl-d-alanine peptidoglycan sequence in Gram-positive bacteria in a linear, concentration-dependent manner. The dalbavancin has effects on echovirus 1, ezetimibe against HIV1 and Zika virus.

More details: https://www.ncbi.nlm.nih.gov/pubmed/29698664