Tools for Immune Checkpoint Therapeutic Antibodies Research

Introduction

Cancer is a global health problem. According to GLOBOCAN 2020 estimates of cancer incidence and mortality from the International Agency for Research on Cancer, approximately 19.3 million new cancer cases and almost 10 million cancer deaths occurred around the world in 20201. There are many cancer treatments now, and Immunotherapy is one of the cancer treatments that use your immune system to fight cancer. In some solid tumors, immunotherapy is the first line of treatment2. The checkpoint proteins of the immune system can be considered as effective therapeutic targets3. FDA approved therapeutic antibodies targeting immune checkpoint PD-1(nivolumab (Opdivo®) and pembrolizumab (Keytruda®)), PDL1(atezolizumab (Tecentriq®), durvalumab (Imfinzi®)and avelumab(Bavencio®)) and CTLA-4 (Ipilimumab(Yervoy®)) have shown success in the treatment of several cancers4. New targets TIGIT and PVRIG are being investigated. In January 2021, Roche announced that Tiragolumab (Anti-TIGIT) plus atezolizumab has shown encouraging efficacy and safety in PD-L1-positive metastatic NSCLC based on data from the Phase II CITYSCAPE trial5.

Sino Biological offers comprehensive products to support immune checkpoint therapeutic antibodies research.

Immunization, hybridoma hits identification, biopanning, affinity determination should be important steps in the development of antibodies. We offer Tag-free, His tagged, Fc tagged or mouse Fc tagged proteins with high purity (Figure 1A, 1B) and validated bioactivity (Figure 1C).

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).

And biotinylated and unconjugated (Figure 2)proteins are also available for multiple applications.

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.

Tumor cells could escape immune cell recognition by activating the ligand-receptor pathway. These receptor–ligand interactions impair anti-tumor immunity6. For example, in many cancer types, the binding of CD47 to SIRPα triggers an inhibitory signaling pathway that leads to the evasion of malignant cells from phagocytosis by macrophages7. Some immune checkpoint therapeutic antibodies increase antitumor activity by blocking the receptor-ligand pathway. For example, therapeutic antibodies blocking PD-1 and its ligand PD-L1 have already shown clinical efficacy8. And Anti-CD47 antibodies enhance the phagocytosis of tumor cells by macrophages by blocking the binding of CD47 to SIRPα9. So we usually develop ligand-receptor pair proteins (Figure 3) together.

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).

We also offer Cynomolgus, Rhesus, Mouse, and Rat proteins (Figure 4) for cross-reactivity and animal studies.

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 2μg/mL (100μL/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.

In the development of antibodies, the functional activities of antibodies need to be tested on cells. We offer cytokine-related products for cell-based functional assay, including cytokines proteins (Figure 5), ELISA kits and pair sets.

Cytokines Proteins

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.

ELISA Kits and ELISA Pair Sets

The functions of cells of immune systems, like T and NK cells, were analyzed by determining the secretion of some cytokines10. Some immune checkpoints expressing on cells of the immune system could suppress cytokines production of these cells. But after blocking the target, the secretions of cytokines are increased. For example, CD96 negatively regulates IFN-γ secretion in NK cells11. After blocking CD96 by Anti-CD96-specific mAbs, NK cell IFNγ production is enhanced12. The high expression level of TIGIT leads to 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 increased in response to TIGIT blockade10.

So far, the double-antibody sandwich method is the most commonly used immunological detection method. Based on the well-established recombinant protein platform, antibody technology platform, and QC platform, Sino Biological Inc. has developed a variety of ELISA Kits (Table 1) and ELISA antibody pair sets (Table 2) for the quantitative detection of cytokines, which can be used to accurately quantify cytokines in plasma, serum, cell culture supernatant, and other biological samples.

Table 1: ELISA Kits – Ready to Use

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

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

Summary

This application note summarizes the tools developed by Sino Biological for immune checkpoint therapeutic antibodies research. The tool set includes (1) Immune checkpoint proteins with multiple tags, multiple species, high purity and validated bioactivity. (2) High-quality cytokine proteins, ELISA kits and ELISA pair sets for cell-based functional assay.

Reference:

1Hyuna 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.
2Raju 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.
3P. 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.
4Hyun 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.
6H. 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.
7Wenting 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.
8Zatloukalová et al., “The Role of PD-1/PD-L1 Signaling Pathway in Antitumor Immune Response.”
9Zhang et al., “Advances in Anti-Tumor Treatments Targeting the CD47/SIRPα Axis.”
10Fanqiao 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.
11Haoyu 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.
12Deepak 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.

By Molecule Initial