TMR/Cy3/Cy5 was introduced for the fluorescent label with peptides to evaluate the cell-penetrating ability and intracellular distribution of each peptide.
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).
The medium was then replaced with fresh medium containing 10% FBS, and a Tetramethylrhodamine carboxylic acid (TMR)-labeled peptides
The solution was added to each well at an appropriate concentration (for example 0.5uM, 1uM, 1.5uM, 2uM).
After 1, 2, 3, or 4 hours of incubation, the medium was removed, and cells were washed with ice-cold PBS and trypsin.
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.
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.
The results are presented as the mean and standard deviation obtained from 3 samples.
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.
Tumor-associated peptide antigens
LifeTein can customize a discovery and development path to fit your exact needs for peptide synthesis.
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 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 hours, 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.
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.
Transactivator 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.
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
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 remains 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 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
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 the folding of disulfide-rich peptides would be beneficial.
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.
Antibody-Conjugate
Preparing Antibody-Peptide, Antibody Oligonucleotide or Antibody-Compound Conjugates
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.
In the realm of peptide synthesis and bioconjugation, LifeTein stands at the forefront, offering innovative solutions for linking peptides to other biomolecules. Typically, peptides have three biological functional groups available for conjugation: amino (–NH2), carboxyl (–COOH), and thiol (–SH). Among these, the thiol group, particularly from cysteine residues, is often the most effective for bioconjugation. The reaction between maleimides and thiols is a widely recognized method for the bioconjugation and labeling of biomolecules, and LifeTein has mastered this technique to offer superior results.
LifeTein has embraced Click Chemistry, an efficient method for conjugating peptides with various biomolecules. This technique involves modifying the peptide with azide groups (–N3). A standout feature in LifeTein’s arsenal is the novel Copper-free Click Chemistry, which is based on the reaction of a diaryl cyclooctene moiety (DBCO) with an azide-modified peptide. This reaction is not only rapid at room temperature but also avoids the use of cytotoxic Cu(I) catalysts, leading to almost quantitative yields of stable triazoles.
The DBCO component allows copper-free click chemistry to be safely employed with live cells, whole organisms, and non-living samples, which is a significant advantage in various biological applications. Importantly, within physiological temperature and pH ranges, the DBCO group does not react with amines or hydroxyls, which are abundantly present in many biomolecules. The reaction of the DBCO group with the azide group is notably faster than with the sulfhydryl group (–SH, thiol), making it a preferred choice for many of LifeTein’s clients.
Practical Applications: Peptide Drug Conjugations
A prime example of the application of these techniques is in the creation of antibody-biomolecule conjugates. LifeTein’s protocol for Click chemistry of antibody-DNA conjugation is straightforward and efficient:
Pre-conjugation Preparations: Remove all additives from antibody solutions using methods like dialysis or desalting. It’s crucial to eliminate BSA and gelatin from these solutions and concentrate the antibody post-purification.
Activation with DBCO-NHS Ester: The antibody is mixed with a 20-30 fold molar excess of DBCO-NHS ester dissolved in DMSO and incubated at room temperature or on ice.
Quenching the Activation Reaction: This step involves adding Tris-HCl (50-100mM, pH 8) to the reaction mixture, followed by incubation at room temperature or on ice to stabilize the reaction.
Equilibration and Removal of Non-reactive DBCO-NHS Ester: This is achieved using a Zeba column, following the manufacturer’s instructions to ensure precision and effectiveness.
Copper-Free Click Reaction: The DBCO-NHS ester labeled antibody is then mixed with a 2-4 times molar excess of azide-modified oligos. This mixture is incubated overnight at 4°C or for a few hours at room temperature, facilitating the conjugation process.
Validation and Purification: The final step involves validating the conjugation and purifying the product using HPLC, ensuring the high quality and efficacy of the conjugate.
LifeTein’s expertise in peptide synthesis and conjugation is further exemplified by their application of Click Chemistry and thiol-maleimide bioconjugation techniques. These methods are not only efficient but also versatile, opening up new possibilities in the field of peptide-based therapeutics and research.
Selected References:
Simon et al. (2012). Facile Double-Functionalization of Designed Ankyrin Repeat Proteins using Click and Thiol Chemistries. Bioconjugate Chem. 23(2):279.
Arumugam et al. (2011). [18F]Azadibenzocyclooctyne ([18F]ADIBO): A biocompatible radioactive labeling synthon for peptides using catalyst-free [3+2] cycloaddition. Bioorg. Med. Chem. Lett. 21:6987.
Campbell-Verduyn et al. (2011). Strain-Promoted Copper-Free Click Chemistry for 18F Radiolabeling of Bombesin. Angew. Chem. Int. Ed. 50:11117.
Through these advanced techniques, LifeTein continues to be a leader in the field of peptide synthesis and bioconjugation, contributing significantly to the advancement of biomedical research and therapeutic development.
One example of peptide drug conjugations is the antibody-biomoleule conjugate.
click chemistry: DBCO-azide
A simple protocol: Click chemistry of antibody-DNA conjugation
Pre-conjugation considerations
Remove all additives from antibody solutions using dialysis or desalting.
Remove BSA and gelatin from antibody solutions.
Concentrate the antibody after dialysis or purification.
Activation of antibodies with DBCO-NHS ester
Mix antibody with 20-30 fold molar excess over antibody of DBCO-NHS ester dissolved in DMSO.
Incubates at room temperature for 30 min or 2 hours on ice.
Quenching activation reaction
Add Tis-Hcl (50-100mM, pH 8) to the reaction.
Incubate at RT for 5 min or 15 minutes on ice.
Equilibration and removal of non-reactive DBCO-NHS ester by Zeba column (Follow the manufacturer’s instruction)
Copper-Free click reaction
Mix DBCO-NHS ester labeled antibody with 2-4 times molar excess of azide-modified Oligos.
Incubated overnight (around 10-12 hours) at 4°C or 3-4 hours at room temperature.
Validation of conjugation and purification by HPLC
The reaction of maleimides with thiols is widely used for bioconjugation and labeling of biomolecules such as proteins and peptides. Maleimides are electrophilic compounds which show high selectivity towards thiols.
The Reaction of Maleimides With Thiols
1. Dissolve the peptide or other biomolecules containing thiol in degassed buffer (PBS, Tris, or HEPES) at pH 7-7.5. 2. Add a 100x molar excess of TCEP (tris-carboxyethyl phosphine) reagent to reduce disulfide bonds. 3. Dissolve maleimide in DMSO or fresh DMF (1-10mg in 100uL). 4. Add dye solution such as cy5 maleimide to thiol solution (20x fold excess of dye), flush with an inert gas, and close tightly. 5. Mix thoroughly and keep at room temperature or 4C overnight. 6. Purify by gel filtration, HPLC, FPLC, or electrophoresis.
Interest in personalized treatment has been fuelled by the concept to tailor therapy with the best response and highest safety margin to ensure better patient care. Personalized medicine holds promise for improving health care while also lowering costs.
Synthetic Peptides for Personalized Treatment
An immunogenic personal neoantigen vaccine for melanoma patients using the synthetic peptides provides an opportunity to develop agents that are targeted to patient groups that do not respond to medications as intended and for whom the traditional health systems have otherwise failed.
The T cell epitopes with tumor-specific expression arising from non-silent somatic mutations are not expressed in normal tissues. These neoantigens are mutated peptides with the high-affinity binding of autologous HLA molecules.
The vaccination with neoantigens can induce new T cell specificities in cancer patients. Using the synthetic peptides as a personalized vaccine, researchers found that of 6 vaccinated patients, 4 had no recurrence at 25 months post-vaccination.
The T cells discriminated mutated from wildtype peptide antigens, and directly recognized autologous tumor. From this study, immunizing peptides were selected based on HLA binding predictions. Each patient received up to 20 long peptides in 4 pools.
Some fusion protein or chimeric proteins could never be produced from the e.coli expression system, especially when several hydrophobic sequences are involved in the functional domains. Obtaining peptides sized 100–200 amino acids using chemical synthesis is much faster and cheaper than cloning and overexpressing in Escherichia coli. In addition, the resulting peptide is always correct. Chemical synthesis can be used to incorporate non-genetically encoded structures, such as D-amino acids, into the protein in a completely regular fashion. Synthetic peptides eliminate problems such as poor or no expression, cloning errors, tags like FLAG or 6-His, or the mistranslation of non-preferred codons in prokaryotic hosts. Artificial amino acids that have isosteric side chains can be used to investigate the functional importance of specific residues. All these chimeric proteins can be achieved by the peptide design and synthesis using the click chemistry.
