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

Trans-activator of transcription (Tat) with nuclear translocation signal peptide drug candidate

Endocytosis TAT Peptide

Endocytosis TAT Peptide

Nuclear translocation of annexin A1 (ANXA1) has been reported to participate in diverse cellular processes. It was found that the amino-acid residues from R228 to F237 function as a unique nuclear translocation signal (NTS) and are required for nuclear translocation of ANXA1.

Trans-activator of transcription (Tat) is a cell-penetrating peptide. The peptide can translocate numerous proteins, peptides, DNA, RNA, and small drugs into the cytoplasm with high efficiency. The Tat-NTS (NH2-YGRKKRRQRRR-RSFPHLRRVF-CONH2) peptide was found to specifically block the interaction of ANXA1 with importin β. This blocking would inhibit the nuclear translocation of ANXA1, consequently protecting neurons from ischemic stroke damage. So the Tat-NTS peptide can be used as a novel and potentially promising new therapeutic candidate for the treatment of ischemic stroke.

From the experiment, the Tat-NTS peptide-treated mice displayed remarkable cognitive improvement. In addition, the Tat-NTS peptide had no obvious effect on neuronal apoptosis. So the administration of Tat-NTS peptide to dissociation of ANXA1–importin β interaction may be a new drug in ischemic stroke therapy.

The Tat sequence is originally from a crucial non-structural protein of human immunodeficiency virus (HIV). The function of Tat is to bind to the viral long terminal repeat (LTR) and activate cellular transcription machinery to initiate transcription of the viral proteins. Later, it was found that this 11-amino acid positively charged peptide can pull diverse molecules across cell membranes in vitro and in vivo. Fusion proteins constructed with TAT rapidly enter and exit cells and cross intracellular membranes. Electrostatic interactions between TAT and the cell membrane have been implicated as a part of the mechanism of transduction. Neither apoptosis nor necrosis is induced in cells after exposure to TAT.


Molecular grafting of macrocyclic disulfide-rich peptides

Bioactive peptides have potential as drug leads. However, one of the peptides’ limitations is their poor stability.

One solution is to graft peptides onto suitable molecular scaffolds. When the peptide scaffolds are rich in disulfide bonds and have no free ends for proteases to attack, the peptide drug would be a stable candidate.

Cyclic disulfide-rich peptides: Stability Matters
Many natural peptides have stable and conserved cyclization. The cyclic chlorotoxin can preferentially bind to tumor cells. The defensin, an antimicrobial peptide, is an 18 amino acid peptide with three disulfide bonds in a laddered arrangement. The cyclotide has about 30 amino acids with three disulfide bonds in a knotted configuration. The cyclic conotoxin cVc1.1 has potent analgesic activity.

disulfide rich cyclic natural product

disulfide-rich cyclic natural product

Molecular grafting
The purpose of molecular grafting is to make a linear peptide sequence into a disulfide-rich peptide with desired stability or oral bioavailability. It was found that the stability of linear peptide epitopes in human serum can be enhanced by grafting them onto a stable scaffold. For example, a crafted peptide comprising an epitope from myelin oligodendrocyte glycoprotein (MOG) is resistant to degradation in human serum (>24 h) and remained intact in strong acid after 24 hours. In the MOG study, the epitope was grafted into the β-turn of Kalata B1 to mimic its native conformation and it turned out to have the increased bioactivity and improved affinity and receptor selectivity. A grafted peptide based on the cell-penetrating peptide cyclotide MCoTI-I demonstrated the delivery of the bioactive peptide across the cell membrane and into the intracellular space.

Cyclotide Peptide Synthesis

Cyclotide Peptide Synthesis

How to make molecular grafting
1. Selection of a suitable epitope and scaffold pair. The termini of the epitope should not be essential for its activity. The size of the epitope is an important consideration. Epitopes ranging from 10 amino acids to 20 amino acids are frequently used. Here are a few examples of the epitope size from the disulfide-rich peptide scaffolds by chemical design: cyclic conotoxin (6 amino acids), defensin (6, 7, or 12 amino acids), Kalata B1 (3, 6, or 9 amino acids), McoTI (3, 7, 9, 16, 18, or 21 amino acids), Cyclic chlorotoxin (10 amino acids).

2.Structural and functional characterization.
The peptide epitope should be from the fragments of an interacting protein or functional and bioactive known domains from the screening assays such as a phage display library.

Then where should the epitope be grafted onto the scaffold?There are a few possible suggestions: insertion between two existing residues; substitution of one or more residues in a single loop; replacement of residues that span across connected loops; or replacement of most of the native residues of the scaffold.

3. How to make the grafted peptides?
The grafted peptides have been made using solid-phase chemical peptide synthesis. First, the reduced linear precursors are assembled. They are the sequences from the scaffold and epitope. Then oxidization and cyclization are performed to form the final product. However, synthesis of more complex peptides with multiple disulfide bonds can be challenging. A peptide comprising just three disulfide bonds would have 15 different connectivities. In addition, grafting of an epitope onto a scaffold may not be folded correctly, or it does not have the desired activity.

Future perspectives
It is encouraging that many grafted peptides have exhibited oral activity. The cyclic disulfide-rich scaffolds have enhanced stability. However, not all grafted peptides fold into the desired conformation. So a more detailed understanding of how grafting affects folding of disulfide-rich peptides would be beneficial.


disulfide formation

disulfide formation

Simple method to prepare antibody-peptide, antibody-oligonucleotide or antibody-compound conjugates

We describe a simple method for preparing antibody-peptide, antibody-oligonucleotide or antibody-compound conjugates and discuss its applications in drug delivery and new drug design. Conjugation is based on alkyne-azide cycloaddition. This Cu-free click reaction starts from the dibenzocyclooctyne (DBCO) moiety-activated antibodies and subsequently linked covalently with an azide-modified peptide, oligonucleotide or compounds. The reaction is performed under physiological conditions and has no adverse effects on antibodies or proteins. This can also be used as the click chemistry fluorescence labeling and the click chemistry in peptide-based drug design.

However, the copper-catalyzed alkyne-azide cycloaddition (CuAAC) is not suitable for applications involving functional biomolecules because copper ions can cause protein denaturation.

Measuring the protein levels directly is challenging. However, the signals can be amplified by immuno-PCR using oligonucleotide-attached antibodies to detect protein indirectly.



1. Conjugation of DBCO to the Antibody. The DBCO-PEG5-NHS was used to react with the NH2 groups on the antibody. The inclusion of a PEG5 linker improves the water solubility of the hydrophobic DBCO, introduces a spacer and flexibility between the antibody molecule and the peptide/oligonucleotide or compounds. This will alleviate the steric effect of the antibody on the enzymatic reactions.

2. Prepare the azido-Peptide or azido-oligonucleotide. LifeTein provides click chemistry modified peptide synthesis: N-terminal azide-peptide/oligo or C-terminal peptide/oligo-azide.

3. Covalent attachment of the peptide/oligonucleotide to the antibody. The reaction between DBCO and azide is slow compared to CuAAC reaction. The reaction time of 16–18 h in PBS at 4 °C is ideal to increase the final product yield. The DBCO-antibody in the intermediate reaction is stable.