Immune Checkpoint Proteins - Characteristics and Applications

Over the past few years, immune checkpoint proteins have attracted a great deal of attention due to their involvement in cancer development. The checkpoint pathways are frequently seized by tumor cells to avoid attacks from the immune system. For this reason, many believe that disabling immune checkpoints would help to re-engage the body’s immune system to fight against cancers.

Interestingly, anti-PD1 and anti-CTLA4 monoclonal antibodies have previously shown anti-tumor activities in patients. Encouraged by this early success, scientists have expanded their research to include other immune checkpoint proteins so as to discover and potentially develop new and improved cancer treatments.

ACROBiosystems has created a special immune checkpoint protein collection, including mouse Fc-tagged, Strep-tagged, His-tagged, Fc-tagged, and/or tag-free proteins to support these efforts. Apart from human proteins, rabbit, rat, mouse, cynomolgus, and rhesus macaque proteins are also available for animal studies. The excellent performance and consistency of ACROBiosystems’ proteins have been verified by a variety of different assays.

Application

Key Features

High Quality

The consistency in purity and bioactivity is necessary for recombinant proteins. All proteins must pass different bioactivity and purity evaluations.

High Batch-to-Batch Consistency

In the above ELISA analysis, three different lots of Human TIGIT His tag (Cat.No.TIT-H52H3) were used against Human CD155 Protein, Fc Tag (Cat.No.CD5-H5251). The result showed that the batch variation among the tested samples is negligible.Figure 1. In the above ELISA analysis, three different lots of Human TIGIT His tag (Cat.No.TIT-H52H3) were used against Human CD155 Protein, Fc Tag (Cat.No.CD5-H5251). The result showed that the batch variation among the tested samples is negligible.

Purity Greater than 90% by SEC-HPLC

The purity of ACROBiosystems Human LAG-3, Fc Tag (Cat.No.LA3-H5255) is determined to be greater than 94% (A), while the purity of a comparable product from company R is determined to be greater than 76% (B).Figure 2. The purity of ACROBiosystems Human LAG-3, Fc Tag (Cat.No.LA3-H5255) is determined to be greater than 94% (A), while the purity of a comparable product from company R is determined to be greater than 76% (B).

Trimer Proteins Validated in SEC-HPLC

The tumor necrosis factor (TNF) superfamily comprises of 29 receptors and 19 ligands, including TNFSF5 (CD40 Ligand), TNFSF7 (CD27 Ligand), and TNFSF4 (OX40 Ligand), among others. The endogenous TNF superfamily ligands usually form a trimer (Russell et al., 2018).

The purity of trimer Human OX40 ligand (Cat.No.OXL-H52Q8) was />90% as determined by SEC-HPLC.Figure 3. The purity of trimer Human OX40 ligand (Cat.No.OXL-H52Q8) was >90% as determined by SEC-HPLC.

Bioactivity Validated in Functional ELISA, SPR and/or FACS

Bioactivity of ACROBiosystems’ Immune Checkpoint Proteins and their ligands are confirmed in a series of assay, including ELISA (Figures 4 and 8), SPR (Figures 5 and 9) and FACS, including binding assay (Figures 6 and 10) and/or neutralizing assay (Figures 7 and 11).

CD47&SIRP Alpha

The interaction between CD47 and SIRPalpha (Figures 4 and 6) plays an inhibitory role in phagocytosis (Liu et al., 2017). For this reason, blocking the SIRPalpha-CD47 interaction with a monoclonal antibody (Figure 7) provides an effective anti-cancer therapy by increasing the elimination of cancer cells by the innate immunity (Huang et al., 2017).

ACROBiosystems offers a distinctive set of CD47-relevant products, including multiple avi tag pre-biotinylated proteins, to enable fast high throughput screening. Additionally, the company also offers SIRP alpha and CD47 from other generally used experimental animal species, including rhesus macaque, rat, cynomolgus, mouse, and rabbit.

Immobilized Human SIRP alpha, Fc Tag (Cat.No.SIA-H5251) at 5 μg/mL (100 μL/well) can bind Human CD47, His Tag (Cat.No.CD7-H5227) with a linear range of 2–13 ng/mL (QC tested).

Figure 4. Immobilized Human SIRP alpha, Fc Tag (Cat.No.SIA-H5251) at 5 μg/mL (100 μL/well) can bind Human CD47, His Tag (Cat.No.CD7-H5227) with a linear range of 213 ng/mL (QC tested).

Anti-Human CD47 MAb (HumanIgG4) captured on CM5 chip via Anti-Human IgGFc antibodies surface, can bind Human CD47, His Tag (Cat.No.CD7-H5227) with an affinity constant of 1.66 nM as determined in a SPR assay (Biacore T200) (Routinely tested).

Figure 5. Anti-Human CD47 MAb (HumanIgG4) captured on CM5 chip via Anti-Human IgGFc antibodies surface, can bind Human CD47, His Tag (Cat.No.CD7-H5227) with an affinity constant of 1.66 nM as determined in a SPR assay (Biacore T200) (Routinely tested).

FACS assay shows that recombinant Human SIRP alpha, His Tag (HPLC-verified) (Cat.No.SIA-H5225) can bind to Jurkat cell expressing CD47. The concentration of SIRP alpha used is 1 µg/ml (Routinely tested).Figure 6. FACS assay shows that recombinant Human SIRP alpha, His Tag (HPLC-verified) (Cat.No.SIA-H5225) can bind to Jurkat cell expressing CD47. The concentration of SIRP alpha used is 1 µg/ml (Routinely tested).

FACS analysis shows that the binding of Human SIRP alpha, His Tag (HPLC-verified) (Cat.No.SIA-H5225) to Jurkat expressing CD47 was inhibited by increasing concentration of neutralizing anti-CD47 antibody. The concentration of SIRP alpha used is 1 µg/ml.IC50=0.2257 µg/ml (Routinely tested).Figure 7. FACS analysis shows that the binding of Human SIRP alpha, His Tag (HPLC-verified) (Cat.No.SIA-H5225) to Jurkat expressing CD47 was inhibited by increasing concentration of neutralizing anti-CD47 antibody. The concentration of SIRP alpha used is 1 µg/ml.IC50=0.2257 µg/ml (Routinely tested).

TIGIT Signaling Axis

The TIGIT-CD155 interaction (Figures 8 and 10) activates the immunosuppressive mechanism in melanoma (Mahnke and Enk, 2016). Therefore, blocking the TIGIT-CD155 interaction with a monoclonal antibody (Figure 11) enhances anticancer therapy by inhibitory signals.

ACROBiosystems has a huge collection of recombinant proteins from TIGIT signaling axis, such as TIGIT, CD155, DNAM-1, CD96, Nectin1 (CD111), Nectin2 (CD112), PVRIG (CD112R), Nectin3 (CD113), and Nectin4. Along with the oft-used Fc, His, and mouse Fc tagged proteins, the company also carries multiple pre-biotinylated proteins. Additionally, ACROBiosystems also offers TIGIT signaling axis proteins from other commonly used experimental animal species, including Rhesus macaque, rat, mouse, rabbit, and Cynomolgus.

Immobilized Human TIGIT, His Tag (Cat.No.TIT-H52H3) at 2 μg/mL (100 μL/well) can bind Human CD155, Fc Tag (Cat.No.CD5-H5251) with a linear range of 8–128 ng/mL (QC tested).Figure 8. Immobilized Human TIGIT, His Tag (Cat.No.TIT-H52H3) at 2 μg/mL (100 μL/well) can bind Human CD155, Fc Tag (Cat.No.CD5-H5251) with a linear range of 8128 ng/mL (QC tested).

Anti-Human TIGIT MAb (Mouse IgG1) captured on CM5 chip via Anti-Mouse antibodies surface, can bind Human TIGIT, His Tag (Cat.No.TIT-H52H3) with an affinity constant of 3.93 nM as determined in a SPR assay (Biacore T200) (Routinely tested).Figure 9. Anti-Human TIGIT MAb (Mouse IgG1) captured on CM5 chip via Anti-Mouse antibodies surface, can bind Human TIGIT, His Tag (Cat.No.TIT-H52H3) with an affinity constant of 3.93 nM as determined in a SPR assay (Biacore T200) (Routinely tested).

FACS assay shows that Biotinylated Human CD155, Fc Tag, Avi Tag (Cat.No.CD5-H82F6) can bind to 293T cell overexpressing human TIGIT. The concentration of CD155 is 1 μg/ml (Routinely tested).Figure 10. FACS assay shows that Biotinylated Human CD155, Fc Tag, Avi Tag (Cat.No.CD5-H82F6) can bind to 293T cell overexpressing human TIGIT. The concentration of CD155 is 1 μg/ml (Routinely tested).

FACS analysis shows that the binding of Biotinylated Human CD155, Fc Tag, Avi Tag (Cat.No.CD5-H82F6) to 293T overexpressing TIGIT was inhibited by increasing concentration of neutralizing Anti-Human TIGIT MAb. The concentration of CD155 used is 1 μg/ml. The IC50 is 0.201 μg/ml (Routinely tested).Figure 11. FACS analysis shows that the binding of Biotinylated Human CD155, Fc Tag, Avi Tag (Cat.No.CD5-H82F6) to 293T overexpressing TIGIT was inhibited by increasing concentration of neutralizing Anti-Human TIGIT MAb. The concentration of CD155 used is 1 μg/ml. The IC50 is 0.201 μg/ml (Routinely tested).

Fluorescence-Activated Cell Sorting (FACS) Tested Immune Checkpoint Proteins.

Molecule Sign Species Tag  
B7-2 CD6-H5257 Human C-Fc Request FACS Protocol
CD6-H82F5 Human C-Fc&C-Avi Request FACS Protocol
CD155 CD5-H5251 Human C-Fc Request FACS Protocol
CD5-H82F6 Human C-Fc&C-Avi Request FACS Protocol
CD5-H5254 Human C-mFc (mIgG2a) Request FACS Protocol
CD40 CD0-H5253 Human C-Fc Request FACS Protocol
DNAM-1 DN1-H52H6 Human C-10×His Request FACS Protocol
DN1-H5257 Human C-Fc Request FACS Protocol
DN1-H82F9 Human C-Fc&C-Avi Request FACS Protocol
LAG-3 LA3-C5252 Cynomolgus C-Fc Request FACS Protocol
LA3-H52Aa Human C-mFc (mIgG2a) Request FACS Protocol
LA3-H82F3 Human C-mFc (mIgG2a) &C-Avi Request FACS Protocol
OX40 OX0-H5224 Human C-10×His Request FACS Protocol
OX0-H5255 Human C-Fc Request FACS Protocol
OX0-H82F7 Human C-Fc&C-Avi Request FACS Protocol
TN4-H82E4 Human C-Avi&C-6×His Request FACS Protocol
OX0-H5252 Human C-mFc (mIgG2a) Request FACS Protocol
OX40 Ligand OXL-H52Q8 Human N-6×His Request FACS Protocol
OXL-H526x Human N-Fc Request FACS Protocol
OXL-H5250 Human C-mFc (mIgG2a) Request FACS Protocol
PD-1 PD1-H5257 Human C-Fc Request FACS Protocol
PD1-H82F2 Human C-Fc&C-Avi&C-6×His Request FACS Protocol
PD-L1 PD1-H5258 Human C-Fc Request FACS Protocol
PD1-H82F3 Human C-Fc&C-Avi&C-6×His Request FACS Protocol
PD1-H52A3 Human C-mFc (mIgG1) Request FACS Protocol
PD-L2 PD2-H5251 Human C-Fc Request FACS Protocol
PD2-H52A5 Human C-mFc (mIgG1) Request FACS Protocol
SIRP alpha SIA-H5225 Human C-9×His Request FACS Protocol
SIA-H5251 Human C-Fc Request FACS Protocol
SIA-H52A8 Human C-mFc (mIgG1) Request FACS Protocol
SIA-H5351 Human C-Fc Request FACS Protocol
CDA-H82F2 Human C-Fc&C-Avi Request FACS Protocol
TIGIT TIT-H5254 Human C-Fc Request FACS Protocol
TIT-H52H3 Human C-10×His Request FACS Protocol
TIT-H82F1 Human C-Fc&C-Avi Request FACS Protocol
TIT-H82E5 Human C-Avi&C-10×His Request FACS Protocol
TIT-H5253 Human C-mFc (mIgG2a) Request FACS Protocol

 

Biotin-Labeled Immune Checkpoint Proteins

ACROBiosystems offers different ready-to-use biotinylated checkpoint proteins with highly specific, high bioactivity, low background, and minimal batch-to-batch variation. These proteins will ease assay development and can be employed for AlphaLISA, FACS, ELISA, and SPR.

Llama IgG Fc Tagged Immune Checkpoint Proteins

ACROBiosystems offers an exclusive series of llama IgG Fc tagged Immune Checkpoint Proteins with low tag immunogenicity, target protein immunogenicity, excellent stability, long half-life, high purity, low endotoxin (0.01 EU per μg), and high activity for llama immunization.

The Next Wave of Immune Checkpoint Targets

As well as the immune checkpoint proteins characterized above, new studies have also detected a series of innovative immune regulators. These proteins could have the potential to become the next wave of cancer drug targets.

LILRA&B
LILRA1 LILRA2 LILRA3 LILRA5 LILRA6
LILRB1 LILRB2 LILRB3 LILRB4 LILRB5
LILRA&2
LAIR-1 LAIR-2 LILRA3 LILRA5 LILRA6
SLAMF
SLAMF1 SLAMF2 (CD48) SLAMF3 (CD229) SLAMF4 (2B4) SLAMF5 (CD84)
SLAMF5 (NTB-A) SLAMF7 SLAMF9 (CD2F-10)    
Butyrophilins
BTN1A1 BTN3A1 BTN3A2 BTN3A3 BTNL3
BTNL9        

 

Inhibitor Screening Kit

ACROBiosystems is currently providing a PD1-PD-L1 inhibitor screening assay kit (Cat.No.EP-101) for fast and high throughput screening of candidate inhibitory antibodies or small molecules of the PD1 pathway. This assay uses a simple colorimetric ELISA platform that measures the binding between the in-house developed biotinylated PD-1 protein and the immobilized human PD-L1.

Reference

  • Russell, M.S., Muralidharan, A., Larocque, L., Cao, J., Deschambault, Y., Varga, J., Thulasi Raman, S.N., and Li, X. (2018) .Identification and characterisation of the CD40-ligand of Sigmodon hispidus.PLoS ONE 13.
  • Liu, X., Kwon, H., Li, Z., and Fu, Y. (2017) .Is CD47 an innate immune checkpoint for tumor evasion?J Hematol Oncol 10.
  • Huang, Y., Ma, Y., Gao, P., and Yao, Z. (2017) .Targeting CD47:the achievements and concerns of current studies on cancer immunotherapy.J.Thorac.Dis.9, E168–E174.
  • Mahnke, K., and Enk, A.H. (2016) .TIGIT-CD155 Interactions in Melanoma:A Novel Co-Inhibitory Pathway with Potential for Clinical Intervention.J.Invest.Dermatol.136, 9–11.

ACROBiosystems

ACROBiosystems is an internationally recognized manufacturer of recombinant proteins committed to supporting cancer immunotherapy. We specialize in mammalian cell-based recombinant protein production and process development.

Our goal is to support professionals from pharmaceutical companies, CROs and research institutes who are working on the cancer immunotherapy area by providing high-quality proteins, antibodies and assay kits.

We have multiple offices and branches in North America, Europe, and Asia, and we are proud of serving customers from over 50 countries.


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Last updated: Dec 17, 2018 at 10:53 AM

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