Cells were seeded on 96-well culture plates (10000 cells/well) and incubated in 100 μL of DMEM containing 10% FBS.
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).
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.
Cell viability was evaluated by the absorbance of formazan from each well, and 100% cell viability was calculated from the wells without peptides.
The results are presented as the mean and standard deviation obtained from 5 samples.
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.
