Immune checkpoint proteins are considered to be the main regulators of the immune response against threats and maintain self-tolerance. The classic immune checkpoint includes co-inhibitory receptors (CTLA-4, PD-1, TIGIT, LAG-3) that suppress T-cell function and co-stimulatory receptors (GITR, 4-1BB, OX40, CD40) that tend to trigger T cell activation.
Analysis on the co-inhibitory receptor CTLA-4 (cytotoxic T-lymphocyte antigen 4) and PD-1 (programmed cell death protein 1) demonstrated considerably enhanced survival rates of patients with metastatic solid tumors following the suppression of such receptor pathways. Therefore, antibody drugs against immune checkpoints for tumors, cancer and other disease therapeutics are a primary mode in cancer therapy.
Pathways of immune checkpoint
Image Credit: ACROBiosystems
- High purity verified by HPLC or MALS
- High bioactivity verified by ELISA/SPR/FACS, etc.
- High batch-to-batch consistency ensured by stringent QC testing
- Antibody binding assay
- Antibody affinity assay
- Neutralizing antibody binding assay
- Cell-based assay
TNFSF proteins. Image Credit: ACROBiosystems
CD47 proteins. Image Credit: ACROBiosystems
CTLA-4 homodimer proteins. Image Credit: ACROBiosystems
B7-CD28 family proteins. Image Credit: ACROBiosystems
Authentic structure verified by SEC-MALS
The purity of Human CD27 Ligand, His, Flag Tag (active trimer) (MALS verified) (Cat. No. CDL-H52Da) was more than 95% in HP-SEC, and around 55-70 kDa verified by SEC-MALS. Image Credit: ACROBiosystems
High batch-to-batch consistency
The uniformity in purity and bioactivity is vital for recombinant proteins. All the proteins are in need to pass a range of purity and bioactivity assessments.
Biotinylated Human PD-1 / PDCD1 Protein, Avitag™, His Tag (recommended for biopanning) (MALS verified)（Cat. No. PD1-H82E4. Image Credit: ACROBiosystems
Application 1: Development and screening of antibody drugs
Antibody binding assay
Immobilized Human PD-1, His Tag (Cat. No. PD1-H5221) at 2 μg/mL (100 μL/well) can bind Nivolumab with a linear range of 0.1-3 ng/mL (Routinely tested). Image Credit: ACROBiosystems
Serial dilutions of nivolumab were added into Human PD-1, Fc Tag, low endotoxin (Cat. No. PD1-H5257): Biotinylated Human PD-L1, Fc, Avitag, His Tag (Cat. No. PD1-H82F3) binding reactions. The half maximal inhibitory concentration (IC50) is 0.5381 μg/mL (Routinely tested). Image Credit: ACROBiosystems
Moreover, ACROBiosystems have also developed PD1: PD-L1 (Cat. No. EP-101) and CD47: SIRP alpha (Cat. No. EP-102) inhibitor screening assay kits that can perform high-throughput screening of neutralizing antibodies or small molecules.
Antibody drug affinity assay
Anti-LAG-3 MAb (Human IgG1) captured on CM5 chip via Anti-Human IgG Fc antibodies surface, can bind Human LAG-3, His Tag (Cat. No. LA3-H5222) with an affinity constant of 4.57 nM as determined in a SPR assay (Biacore T200) (Routinely tested). Image Credit: ACROBiosystems
Loaded Anti-LAG-3 MAb (Human IgG1) on AHC Biosensor, can bind Human LAG-3, His Tag (Cat. No. LA3-H5222) with an affinity constant of 7.47 nM as determined in BLI assay (ForteBio Octet Red96e) (Routinely tested). Image Credit: ACROBiosystems
Neutralizing antibody binding assay
Serial dilutions of Human TIGIT Neutralizing antibody were added into Human TIGIT, Fc Tag (Cat. No. TIT-H5254): Biotinylated Human CD155, Fc, Avitag (Cat. No. CD5-H82F6) binding reactions. The half maximal inhibitory concentration (IC50) is 0.55834 μg/mL (Routinely tested). Image Credit: ACROBiosystems
FACS analysis shows that the binding of Human CD47 to ACHN expressing SIRP-a was inhibited by increasing concentration of neutralizing SIRP-a antibody. The concentration of Human CD47 used is 20 μg/mL. IC50 = 9.334 μg/mL. Image Credit: ACROBiosystems
Human 4-1BB Ligand (71-254), Fc Tag, active trimer (Cat. No. 41L-H5265) induce IL-8 secretion in HT1080 human CD137 cell line. The EC50 for this effect is 0.079 μg/mL (Routinely tested). Image Credit: ACROBiosystems
Pharmacokinetics (PK) analysis
Immobilized Human CTLA-4, His Tag, active dimer (Cat. No. CT4-H52H9) at 2 μg/mL, add increasing concentrations of CTLA-4 x OX40 Bispecific Antibody in 50% Human serum and then add Biotinylated Human OX40, Avitag, His Tag(Cat. No. TN4-H82E4) at 1 μg/mL. Detection was performed using HRP-conjugated streptavidin with sensitivity of 4 ng/mL (Intact assay, Routinely tested). Image Credit: ACROBiosystems