Research tools in immune checkpoint therapies

Cancer is a major global health concern. In line with GLOBOCAN 2020 estimates of cancer incidence and mortality from the International Agency for Research on Cancer, it was reported that 19.3 million new cancer cases and almost 10 million cancer deaths occurred worldwide in 2020.1

There are many cancer treatments currently available; immunotherapy uses the immune system to combat the cancer. In a number of solid tumors, immunotherapy is the first line of treatment.2 The proteins that act as immune system checkpoints can be considered effective therapeutic targets.3

FDA approved therapeutic antibodies that target immune checkpoint PD-1 (nivolumab (Opdivo®) and pembrolizumab (Keytruda®)), PDL1 (atezolizumab (Tecentriq®), durvalumab (Imfinzi®) and avelumab (Bavencio®)) and CTLA-4 (Ipilimumab(Yervoy®)) have been successful in the treatment of various cancers.4

Recently identified targets TIGIT and PVRIG are now under investigation. In January 2021, Roche declared that Tiragolumab (Anti-TIGIT) plus atezolizumab has demonstrated promising efficacy and safety in PD-L1-positive metastatic NSCLC, according to the data from the Phase II CITYSCAPE trial.5

Sino Biological provides complete products to assist with immune checkpoint therapeutic antibodies research.

Recombinant immune checkpoint proteins

Immunization, hybridoma hits identification, biopanning and affinity determination should be targeted as key steps in the advancement of antibodies. Sino Biological offer Tag-free, His tagged, Fc tagged or mouse Fc tagged proteins with high-levels of purity (Figure 1A, 1B) and verified bioactivity (Figure 1C).

Examples of recombinant checkpoint proteins from Sino Biological (A) The purity of recombinant human CD47 his tag protein (Cat: 12283-H08H) is greater than 95 % as determined by SDS-PAGE. (B) The purity of recombinant human LAG3 tag free protein (Cat: HPLC-16498-HNAH) is greater than 95 % as determined by SEC-HPLC. (C) Bioactivity assay of recombinant human CD47 his tag protein. Immobilized Human SIRP alpha hFc (Cat:11612-H02H1) at 2 μg/ml (100 μl/well) can bind Human CD47 His (Cat:12283-H08H).

Figure 1. Examples of recombinant checkpoint proteins from Sino Biological (A) The purity of recombinant human CD47 his tag protein (Cat: 12283-H08H) is greater than 95 % as determined by SDS-PAGE. (B) The purity of recombinant human LAG3 tag free protein (Cat: HPLC-16498-HNAH) is greater than 95 % as determined by SEC-HPLC. (C) Bioactivity assay of recombinant human CD47 his tag protein. Immobilized Human SIRP alpha hFc (Cat:11612-H02H1) at 2 μg/ml (100 μl/well) can bind Human CD47 His (Cat:12283-H08H). Image Credit: Sino Biological Inc.

Tumor cells may evade immune cell recognition by stimulating the ligand-receptor pathway. These receptor-ligand interactions impede anti-tumor immunity.6 For instance, in a number of cancer types, the binding of CD47 to SIRPα drives an inhibitory signaling pathway that results in the escape of malignant cells from phagocytosis by macrophages.7

Examples of biotinylated and unconjugated proteins from Sino Biological. (A) Bioactivity assay of biotinylated recombinant human TIGIT Fc tag protein (Cat: 10917-H02H-B). Immobilized human CD155 hFc tag protein(Cat:10109-H02H) at 2 μg/mL (100 μL/well) can bind Biotinylated human TIGIT hFc tag (Cat:10917-H02H-B). (B) Recombinant human CTLA-4 His tag protein (Cat: 11159-H08H) inhibits IL-2 secretion by stimulated Jurkat cells.

Figure 2. Examples of biotinylated and unconjugated proteins from Sino Biological. (A) Bioactivity assay of biotinylated recombinant human TIGIT Fc tag protein (Cat: 10917-H02H-B). Immobilized human CD155 hFc tag protein(Cat:10109-H02H) at 2 μg/mL (100 μL/well) can bind Biotinylated human TIGIT hFc tag (Cat:10917-H02H-B). (B) Recombinant human CTLA-4 His tag protein (Cat: 11159-H08H) inhibits IL-2 secretion by stimulated Jurkat cells. Image Credit: Sino Biological Inc.

Some immune checkpoint therapeutic antibodies boost anti-tumor activity by impeding the receptor-ligand pathway. For instance, therapeutic antibodies blocking PD-1 and its ligand PD-L1 have already demonstrated clinical efficacy.8

Anti-CD47 antibodies improve the phagocytosis of tumor cells by macrophages by inhibiting the binding of CD47 to SIRPα.9 So Sino Biological typically develops ligand-receptor pair proteins (Figure 3) in parallel.

Examples of ligand-receptor pair proteins from Sino Biological. (A) The binding assay of CD47 and SIRP alpha (Cat: 11612-H27H-B). Immobilized CD47 hFc tag protein (Cat:12283-H02H) at 10 μg/mL (100 μL/well) can bind Biotinylated SIRP alpha avi-his tag protein (Cat:11612-H27H-B). (B) The binding assay of CD96 (Cat: 11202-H08H-B) and CD155. Immobilized human CD155/PVR hFc tag protein(Cat:10109-H02H)at 2 μg/mL (100 μL/well) can bind Biotinylated human CD96 His tag protein (Cat:11202-H08H-B).

Figure 3. Examples of ligand-receptor pair proteins from Sino Biological. (A) The binding assay of CD47 and SIRP alpha (Cat: 11612-H27H-B). Immobilized CD47 hFc tag protein (Cat:12283-H02H) at 10 μg/mL (100 μL/well) can bind Biotinylated SIRP alpha avi-his tag protein (Cat:11612-H27H-B). (B) The binding assay of CD96 (Cat: 11202-H08H-B) and CD155. Immobilized human CD155/PVR hFc tag protein(Cat:10109-H02H)at 2 μg/mL (100 μL/well) can bind Biotinylated human CD96 His tag protein (Cat:11202-H08H-B). Image Credit: Sino Biological Inc.

Sino Biological also offers Cynomolgus, Rhesus, Mouse and Rat proteins (Figure 4) for cross-reactivity and animal studies.

Examples of proteins used for cross-reactivity and animal studies from Sino Biological. (A) Bioactivity assay of recombinant Cynomolgus TIGIT Fc tag protein (Cat: 90890-C02H). Immobilized human CD155/PVR his tag protein (Cat:10109-H08H) at 2 μg/mL (100 μL/well) can bind Cynomolgus TIGIT hFc tag protein (Cat:90890-C02H). (B) Recombinant mouse CTLA-4 Fc tag protein (Cat: 50503-M02H) inhibits IL-2 secretion by stimulated Jurkat cells.

Figure 4. Examples of proteins used for cross-reactivity and animal studies from Sino Biological. (A) Bioactivity assay of recombinant Cynomolgus TIGIT Fc tag protein (Cat: 90890-C02H). Immobilized human CD155/PVR his tag protein (Cat:10109-H08H) at 2 μg/mL (100 μL/well) can bind Cynomolgus TIGIT hFc tag protein (Cat:90890-C02H). (B) Recombinant mouse CTLA-4 Fc tag protein (Cat: 50503-M02H) inhibits IL-2 secretion by stimulated Jurkat cells. Image Credit: Sino Biological Inc.

Cytokine related products

When developing antibodies, functional activity needs to be tested on cells. Sino Biological offers cytokine-related products for cell-based functional assay, in particular cytokines proteins (Figure 5), ELISA kits and pair sets.

Cytokines proteins from Sino Biological. (A) Recombinant human IL12 His tag protein (Cat: CT011-H08H) induces IFN-γ secretion by NK-92 cells. (B) Recombinant human IL18 tag free protein (Cat: 10119-HNCE) induces IFN-γ secretion by KG 1 cells in the presence of TNF-alpha. (C) Recombinant human GM-CSF tag free Protein (Cat: 10015-HNAH) stimulates TF-1 cells proliferation. (D) Recombinant human IL-4 tag free Protein (Cat: 11846-HNAE) stimulates TF-1 cells proliferation.

Figure 5. Cytokines proteins from Sino Biological. (A) Recombinant human IL12 His tag protein (Cat: CT011-H08H) induces IFN-γ secretion by NK-92 cells. (B) Recombinant human IL18 tag free protein (Cat: 10119-HNCE) induces IFN-γ secretion by KG 1 cells in the presence of TNF-alpha. (C) Recombinant human GM-CSF tag free Protein (Cat: 10015-HNAH) stimulates TF-1 cells proliferation. (D) Recombinant human IL-4 tag free Protein (Cat: 11846-HNAE) stimulates TF-1 cells proliferation. Image Credit: Sino Biological Inc.

ELISA kits and ELISA pair sets

The role of cells in the immune system, like T and NK cells, was evaluated by establishing the secretion of some cytokines.10 Suppression of cytokines production in a variety of immune checkpoints expressing on cells of the immune system was observed in these cells.

Once the target had been blocked, however, the secretions of cytokines increased. For instance, CD96 negatively controls IFN-γ secretion in NK cells.11 After blocking CD96 by Anti-CD96-specific mAbs, NK cell IFNγ production improved.12

The high expression level of TIGIT produces lower secretions of IFN-γ and TNF-α, and the secretions of CD107a, IFN-γ and TNF-α on NK, CD8+ T and CD4+ T cells are boosted in response to the TIGIT blockade.10

Presently, the double-antibody sandwich technique is the most frequently used immunological detection method.

Based on the well-known recombinant protein platform, antibody technology platform, and QC platform, Sino Biological, Inc. has prepared a wide range of ELISA Kits (Table 1) and ELISA antibody pair sets (Table 2) for the quantitative determination of cytokines, which can be employed for accurate quantification of cytokines in plasma, serum, cell culture supernatant and other biological samples.

Table 1. ELISA Kits – Ready to Use. Source: Sino Biological Inc.

Species Target Cat# Linear range (pg/mL) Sample
Human IL2 KIT11848 18.75-1200 S, C, P
Human IL4 KIT11846 10.94-700 C
Human IL5 KIT15673 4.69-300 C
Human IL6 KIT10395A 5.47-350 S, C
Human IL8 KIT10098 2.5-160 C
Human IL10 KIT10947A 18.75-1200 C
Human TNFα KIT10602 31.25-2000 C
Human IFNγ KIT11725A 23.44-1500 C

 

Table 2. ELISA Pair Sets – Cost-effective. Source: Sino Biological Inc.

Species Target Cat# Linear range (pg/mL)
Human IL4 SEK11846 7.81-500
Human IL5 SEKA15673 3.91-250
Human IL6 SEKB10395 9.38-600
Human IL8 SEK10098 11.72-750
Human IL10 SEKA10947 14.06-900
Human TNFα SEKA10602 39.06-2500
Mouse TNFα SEK50349 31.25-2000
Human IFNγ SEKA11725 21.88-1400

 

This article details the tools that Sino Biological has developed for immune checkpoint therapeutic antibodies research. The toolset is comprised of two main components: 1) Immune checkpoint proteins with multiple species, multiple tags, high purity and verified bioactivity and 2) High-quality cytokine proteins, ELISA kits and ELISA pair sets for cell-based functional assay.

Reference:

  1. Hyuna Sung et al., “Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries,” CA: A Cancer Journal for Clinicians n/a, no. n/a, accessed February 7, 2021, https://doi.org/10.3322/caac.21660.
  2. Raju K. Vaddepally et al., “Review of Indications of FDA-Approved Immune Checkpoint Inhibitors per NCCN Guidelines with the Level of Evidence,” Cancers 12, no. 3 (March 20, 2020), https://doi.org/10.3390/cancers12030738.
  3. P. Zatloukalová et al., “[The Role of PD-1/PD-L1 Signaling Pathway in Antitumor Immune Response],” Klinicka Onkologie: Casopis Ceske a Slovenske Onkologicke Spolecnosti 29 Suppl 4, no. Suppl 4 (Fall 2016): 72–77.
  4. Hyun Tae Lee, Sang Hyung Lee, and Yong-Seok Heo, “Molecular Interactions of Antibody Drugs Targeting PD-1, PD-L1, and CTLA-4 in Immuno- Oncology,” Molecules 24, no. 6 (March 26, 2019), https://doi.org/10.3390/molecules24061190.
  5. “Genentech’s Novel Anti-TIGIT Tiragolumab Granted FDA Breakthrough Therapy Designation in Combination With Tecentriq for PD-L1-High Non-Small Cell Lung Cancer,” January 5, 2021, https://www.businesswire.com/news/home/20210104005887/en/Genentech%E2%80%99s-Novel-Anti-TIGIT-Tir.
  6. H. Harjunpää and C. Guillerey, “TIGIT as an Emerging Immune Checkpoint,” Clinical & Experimental Immunology 200, no. 2 (2020): 108–19, https://doi.org/10.1111/cei.13407.
  7. Wenting Zhang et al., “Advances in Anti-Tumor Treatments Targeting the CD47/SIRPα Axis,” Frontiers in Immunology 11 (2020), https://doi.org/10.3389/ fimmu.2020.00018.
  8. Zatloukalová et al., “The Role of PD-1/PD-L1 Signaling Pathway in Antitumor Immune Response.”
  9. Zhang et al., “Advances in Anti-Tumor Treatments Targeting the CD47/SIRPα Axis.”
  10. Fanqiao Meng et al., “Overexpression of TIGIT in NK and T Cells Contributes to Tumor Immune Escape in Myelodysplastic Syndromes,” Frontiers in Oncology 10 (2020), https://doi.org/10.3389/fonc.2020.01595.
  11. Haoyu Sun and Cheng Sun, “The Rise of NK Cell Checkpoints as Promising Therapeutic Targets in Cancer Immunotherapy,” Frontiers in Immunology 10 (2019), https://doi.org/10.3389/fimmu.2019.02354.
  12. Deepak Mittal et al., “CD96 Is an Immune Checkpoint That Regulates CD8+ T-Cell Antitumor Function,” Cancer Immunology Research 7, no. 4 (April 2019): 559–71, https://doi.org/10.1158/2326-6066.CIR-18-0637.

About Sino Biological Inc.

Sino Biological is an international reagent supplier and service provider. The company specializes in recombinant protein production and antibody development. All of Sino Biological's products are independently developed and produced, including recombinant proteins, antibodies and cDNA clones. Sino Biological is the researchers' one-stop technical services shop for the advanced technology platforms they need to make advancements. In addition, Sino Biological offers pharmaceutical companies and biotechnology firms pre-clinical production technology services for hundreds of monoclonal antibody drug candidates.

Sino Biological's core business

Sino Biological is committed to providing high-quality recombinant protein and antibody reagents and to being a one-stop technical services shop for life science researchers around the world. All of our products are independently developed and produced. In addition, we offer pharmaceutical companies and biotechnology firms pre-clinical production technology services for hundreds of monoclonal antibody drug candidates. Our product quality control indicators meet rigorous requirements for clinical use samples. It takes only a few weeks for us to produce 1 to 30 grams of purified monoclonal antibody from gene sequencing.


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Last updated: Dec 1, 2021 at 6:25 AM

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