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Immune Checkpoint Target

Regulators of the immune system, these pathways are vital for self-tolerance, which prevents the immune system from attacking cells indiscriminately

Introduction
Application
Key Features

Biotin-labeled Immune Checkpoint Proteins
Llama IgG Fc tagged Immune Checkpoint Proteins
The Next Wave of Immune Checkpoint Targets
Inhibitor Screening Kit
Reference

Tumour Cells   Immune Cells Tumour Cells - Immune Cells
PD-L1 - PD-1 B7-H3 - ?
PD-L2 - PD-1 B7-H3 (4lg) - ?
B7-2 - CTLA-4 B7-H4 - ?
B7-1 - CTLA-4 B7-H5 - ?
B7-1 - CD28 B7-H6 - ?
B7-2 - CD28 B7-H7 - CD28H
HVEM - BTLA CD47 - SIRP alpha
Nectin-1 - CD96 CD47 - SIRP gamma
CD155 - CD96 GITR Ligand - GITR
CD155 - DNAM-1 B7-H2 - ICOS
CD155 - TIGIT 4-1BB Ligand - 4-1BB
Nectin-3 - TIGIT CD27 Ligand - CD27
Nectin-2 - TIGIT OX40 Ligand - OX40
Nectin-2 - PVRIG CD48 - 2B4
Nectin-2 - DNAM-1 HVEM - CD160
Galectin-9 - TIM-3 HVEM - CD4 Ligand
MHCII - LAG-3 CD40 - CD30

INTRODUCTION

In recent years, immune checkpoint proteins have attracted increasing attention due to their involvement in cancer development.Often times, tumor cells can hijack the checkpoint pathways to avoid attacks from the immune system.Therefore, many believe that disabling immune checkpoints would help re-engage the body’s immune system to fight against cancers.

Intriguingly,anti-CTLA4 and anti-PD1 monoclonal antibodies have already demonstrated anti-tumor activities in patients. Encouraged by this early success, researchers have expanded their investigations into other immune checkpoint proteins in order to find better treatments of cancer.

To support these efforts, ACROBiosystems has developed an exclusive immune checkpoint protein collection, including Fc-tagged, His-tagged, mouse Fc-tagged, Strep-tagged, and/or tag free proteins.In addition to human proteins, mouse, rat, rabbit, rhesus macaque, and cynomolgus proteins are also available for your animal studies. The consistency and excellent performance of our proteins are confirmed by a variety of assays.

APPLICATION

KEY FEATURES

  • High quality - The consistency in purity and bioactivity is essential for recombinant proteins.All our proteins have to pass a variety of purity and bioactivity assessments.
  • High batch to batch consistency

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

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 contains 19 ligands and 29 receptors, including TNFSF4 (OX40 Ligand) , TNFSF7 (CD27 Ligand) , TNFSF5 (CD40 Ligand) , among others.The endogenous TNF superfamily ligands often form trimer (Russell et al., 2018)

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

>>>CD47&SIRP alpha

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

ACROBiosystems provides a unique set of CD47-relevant products, including mutilple avi tag pre-biotinylated proteins, for rapid high throughput screening.In addition, we also provide CD47 and SIRP alpha from other commonly used experimental animal species, including cynomolgus, rhesus macaque, mouse, rat, rabbit.

Figure 4. Immobilized Human SIRP alpha, Fc Tag (Cat.No.SIA-H5251) at 5μg/mL (100μL/well) can bind Human CD47, His Tag (Cat.No.CD7-H5227) with a linear range of 2-13 ng/mL (QC 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) .

 

 

 

 

 

 

 

 

 

 

 

 

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 ug/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 ug/ml.IC50=0.2257 ug/ml (Routinely tested).

>>>TIGIT Signaling Axis

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

ACROBiosystems carries a large collection of recombinant proteins from TIGIT signaling axis, including TIGIT, CD155, DNAM-1, CD96, Nectin1 (CD111), Nectin2 (CD112), PVRIG (CD112R), Nectin3 (CD113), Nectin4. In addition to the commonly used his, Fc and mouse Fc tagged proteins, we also carry multiple pre-biotinylated.In addition, we also provide TIGIT signaling axis proteins from other commonly used experimental animal species, including Cynomolgus, Rhesus macaque, mouse, rat, rabbit.

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

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 1μg/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 1μg/ml.The IC50 is 0.201μg/ml (Routinely tested) .

Biotin-labeled Immune Checkpoint Proteins

ACROBiosystems provides a variety of ready-to-use biotinylated checkpoint proteins with Low background, highly specific, high bioactivity and minimal batch-to-batch variation.These proteins which simplify your assay development can be used for ELISA, AlphaLISA, SPR, and FACS.

Llama IgG Fc tagged Immune Checkpoint Proteins

ACROBiosystems provides a unique series of llama lgG Fc tagged ImmuneCheckpoint Proteins with enhancing target protein immunogenicity, low tag immunogenicity, Long half-life and good stability, low endotoxin (0.01 EU perμg) , high purity and high activity for llama immunization.

The Next Wave of Immune Checkpoint Targets

Beyond well-characterized immune checkpoint proteins as described above, recent studies also identified a series of novel immune regulators.These proteins hold the promise of becoming 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

We are now offering a PD1-PD-L1 inhibitor screening assay kit (Cat.No.EP-101) for rapid and high throughput screening of candidate inhibitory antibodies or small molecules of the PD1 pathway.This assay employs a simple colorimetric ELISA platform, which measures the binding between immobilized human PD-L1 and in-house developed biotinylated PD-1 protein.

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.

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