ACROBiosystems – Immune Checkpoint Proteins|Tokyo Future Style is a bio-tech trading company

APPLICATIONFEATURESCD47 & SIRPalphaTIGIT SIGNALNEXT TARGET

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.

Related page: Introduction of ACROBiosystems
Related Page: Bispecific antibody targets

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-MALS

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 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.

Bioactivity validated in functional ELISA, SPR & 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) .

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CD47&SIRP alpha

The CD47-SIRPalpha interaction 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. Immobilized Anti-CD47 MAb, Human IgG4 at 2 μg/mL (100 μL/well) can bind Unconjugated Human CD47, His,Avitag (Cat. No. CD7-HA2E9) with a linear range of 0.05-2 ng/mL (QC tested).
Figure 6.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 7. Anti-Human CD47 MAb (Human IgG4) captured on CM5 chip via Anti-Human IgG Fc antibodies surface, can bind Human CD47. Loaded Anti-Human CD47 MAb (Human IgG4) on AHC Biosensor, can bind Human CD47, His Tag (Cat. No. CD7-H5227) with an affinity constant of 6.18 nM as determined in BLI assay (ForteBio Octet Red96e) (Routinely tested).
Figure 8.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 9.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) .

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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 10.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 11.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 12.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 13.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) .

Fluorescence-activated cell sorting (FACS) tested immune checkpoint proteins

Cat. No.	Molecule	Species	Tag
CD6-H5257	B7-2	Human	C-Fc
CD6-H82F5	B7-2	Human	C-Fc&C-Avi
CD5-H5251	CD155	Human	C-Fc
CD5-H82F6	CD155	Human	C-Fc&C-Avi
CD5-H5254	CD155	Human	C-mFc (mIgG2a)
CD0-H5253	CD40	Human	C-Fc
DN1-H52H6	DNAM-1	Human	C-10×His
DN1-H5257	DNAM-1	Human	C-Fc
DN1-H82F9	DNAM-1	Human	C-Fc&C-Avi
LA3-C5252	LAG-3	Cynomolgus	C-Fc
LA3-H52Aa	LAG-3	Human	C-mFc (mIgG2a)
LA3-H82F3	LAG-3	Human	C-mFc (mIgG2a) &C-Avi
OX0-H5224	OX40	Human	C-10×His
OX0-H5255	OX40	Human	C-Fc
OX0-H82F7	OX40	Human	C-Fc&C-Avi
TN4-H82E4	OX40	Human	C-Avi&C-6×His
OX0-H5252	OX40	Human	C-mFc (mIgG2a)
OXL-H52Q8	OX40 Ligand	Human	N-6×His
OXL-H5250	OX40 Ligand	Human	C-mFc (mIgG2a)
PD1-H5257	PD-1	Human	C-Fc
PD1-H5258	PD-L1	Human	C-Fc
PD1-H82F3	PD-L1	Human	C-Fc&C-Avi&C-6×His
PD1-H52A3	PD-L1	Human	C-mFc (mIgG1)
PD2-H5251	PD-L2	Human	C-Fc
PD2-H52A5	PD-L2	Human	C-mFc (mIgG1)
SIA-H5225	SIRP alpha	Human	C-9×His
SIA-H5251	SIRP alpha	Human	C-Fc
SIA-H52A8	SIRP alpha	Human	C-mFc (mIgG1)
CDA-H82F2	SIRP alpha	Human	C-Fc&C-Avi
TIT-H5254	TIGIT	Human	C-Fc
TIT-H52H3	TIGIT	Human	C-10×His
TIT-H82F1	TIGIT	Human	C-Fc&C-Avi
TIT-H82E5	TIGIT	Human	C-Avi&C-10×His
TIT-H5253	TIGIT	Human	C-mFc (mIgG2a)

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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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