PD-L1 [28-8] antibody user guide
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Content
PD-L1 Antibody overview......................................................................................................... 1 What is anti-PD therapy?.................................................................................................1 The foundation of anti-PD therapy efficacy lies in diagnostic accuracy...................3 Concordance of PD-L1 IHC assay systems....................................................................4
PD-L1 Antibody [28-8] IHC staining examples......................................................... 5 Different PD-L1 expression in human lung cancer........................................................5 Evaluate and interpret PD-L1 antibody [28-8] stained slides.......................................6 PD-L1 IHC staining of human normal tissue....................................................................7 PD-L1 IHC staining of human cancer tissue...................................................................7
PD-L1 Antibody [28-8] IHC protocol and troubleshooting................................ 8 PD-L1 manual IHC protocol.............................................................................................8 PD-L1 manual IHC troubleshooting...............................................................................11 Non-specific staining......................................................................................................12 PD-L1 automatic IHC protocol......................................................................................13 Automatic IHC protocol - BioGenex i6000...................................................................13 Automated IHC protocol - Leica BOND RX.................................................................14 Automated IHC protocol - Ventana Ultra....................................................................15 Ventana Ultra Protocol...................................................................................................16 Automated IHC protocol - Dako Omnis.......................................................................16 PD-L1 antibody [28-8] frequently asked questions......................................................18
Cancer immunotherapy: immune checkpoint poster......................................20 Antibody panels for immune checkpoints...............................................................22
PD-L1 antibody overview
What is anti-PD therapy? Anti-PD therapy acts to inhibit the interaction between PD-L1 and PD-1 in the tumor microenvironment and kill the tumor by generating a highly inhibitory tumor microenvironment into an active inflammatory state with reduced adverse effects.1 Cancer immunotherapy targeting the PD-1 pathway has achieved a higher objective response rate in cancer patients with fewer immune-related adverse events(irAEs)1. Treatments with PD-1/PD-L1 inhibitors, alone or in combination with other types of drugs, have revolutionized cancer treatment. As of June 2020, the National Medical Products Administration (NMPA) has approved 8 agents, and the United States Food and Drug Association (FDA) has approved 6 agents. Currently, ten PD-1/PD-L1 inhibitors are available worldwide to treat various cancer types (Table 1) 2,3.
Name
Pharmaceutical Target Company
NMPA Approved Indications and Usage
Approval in China
Pembrolizumab
PD-1
Merck Sharp & Dohme
1. First-line treatment of advanced non-small Jul 20 2018 cell lung cancer with PD-L1 expression ≥ 1% and without abnormal EGFR or ALK gene 2. In combination with chemotherapy in firstline treatment of advanced nonsquamous non-small cell lung cancer without abnormal EGFR or ALK gene 3. In combination with chemotherapy in first-line treatment of advanced squamous non-small cell lung cancer 4. Second-line treatment of advanced melanoma.
Nivolumab
PD-1
Bristol-Myers Squibb
1. Second-line treatment of advanced nonsmall cell lung cancer 2. Second-line treatment of PD-L1 positive advanced head and neck squamous cell carcinoma 3. Third-line treatment of advanced gastric cancer or esophagogastric junction (EGJ) cancer
Aug 23 2019 Dec 22 2014
Durvalumab
PD-L1
AstraZeneca
Treatment of unresectable, phase 3 non-small cell lung cancer without disease progression despite definitive chemoradiotherapy
Dec 6 2019
May 1 2017
Atezolizumab
PD-L1
Roche
In combination with chemotherapy in first-line extensive-stage small cell lung cancer
Feb11 2020
May 18 2016
Avelumab
PD-L1
Pfizer, Merck
N/A
Not yet approved
Mar 23 2017
Cemiplimab
PD-1
Sanofi SA, Regeneron
N/A
Not yet approved
Sep 28 2018
Sintilimab
PD-1
Innovent Biologics
Third-line treatment of classical Hodgkin’s lymphoma.
Dec 24 2018 Not yet approved
Toripalimab
PD-1
Junshi Biosciences Treatment of locally advanced or metastatic melanoma that has failed previous systemic therapy
Oct 9 2019
FDA approval Sep 4 2014
Not yet approved
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Name
Pharmaceutical Target Company
NMPA Approved Indications and Usage
Approval in China
FDA approval
Camrelizumab
PD-1
Hengrui Medicine
1. Third-line treament of classical Hodgkin’s lymphoma. 2. Second-line treatment of advanced hepatocellular carcinoma.
May 29 2019 Not yet approved
Tislelizumab
PD-1
BeiGene
1. Treatment of patients with locally advanced Dec 26 2019 Not yet or metastatic urothelial carcinoma with high approved PD-L1 expression that progressed during or following platinum-containing chemotherapy or within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy. 2. Third-line treatment for classical Hodgkin’s lymphoma.
Table 1. Ten PD-1/PD-L1 inhibitors currently available for the treatment of various cancer types. Figures correct as of July 20 2020.
Researchers have found tumor cells express PD-L1 which results in tumor immune evasion, known as “adaptive resistance”.6 Tumor antigens can be presented by tumor stromal cells and hematopoietically derived infiltrating cells including dendritic cells (DCs), macrophages, neutrophils, and lymphocytes. Tumor-specific effector T cells enter the tumor microenvironment after lymph node activation to become tumor-infiltrating lymphocytes (TILs). Through specific recognition of tumor antigens via T cell receptors (TCRs), TILs release IFN-y and induce PD-L1 expression on tumor cells and antigen-presenting cells in the tumor microenvironment. PD-L1 then interacts with the PD-1 receptor expressed by T cells, iTreg cells, and DC cells6. The resulting PD-1/PD-L1 pathway can lead to a number of processes, including: 1. Inducing the differentiation of iTreg cells7,8, 2. Stimulating IL-10 production, an anti-inflammatory cytokine 9; 3. Inducing T cell apoptosis, anergy, and functional exhaustion10-12, 4. Mediating DC suppression13, which acts as a molecular shield on tumor cells, and protects them from lysis by cytotoxic T lymphocytes (CTLs)14.
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The foundation of anti-PD therapy efficacy lies in diagnostic accuracy According to the new classification of human tumors, there are four types of TIL and PD-L1 expression based on tumor immunity1,15-17: (i) TILs are absent from the tumor microenvironment, tumor cells do not express PD-L1. (ii) TILs are present in the tumor microenvironment, tumor cells express PD-L1. This type of activated TIL is over-regulated. (iii) TILs are present in the tumor microenvironment, but tumor cells do not express PD-L1. This type of tumor develops a tolerance to TILs. (iv) TILs are absent from the tumor microenvironment, but tumor cells express PD-L1. This type of tumor cells express PD-L1 through other cancer signaling pathways. Anti-PD therapy alone is not effective for all types of tumors, therefore it is essential to identify and diagnose the patient’s cancer type. For example, in types (i) and (iv), the tumor microenvironment lacks TILs, meaning anti-PD therapy alone might not be an effective treatment1. The PD-L1 immunohistochemistry (IHC) assay is a crucial method for assessing the therapeutic value of anti-PD therapy and features in multiple clinical trials. One example is CheckMate-057, a randomized, open-label phase III study comparing Nivolumab monotherapy versus Docetaxel in patients with advanced or metastatic non-squamous cell non-small cell lung cancer (NSCLC). This study included patients aged 18 or older with Eastern Cooperative Oncology Group (ECOG) statuses of zero or one and disease recurrence or progression during or after prior platinum-based doublet chemotherapy, which may include maintenance therapy. . The control group was Docetaxel (n=290), and the experimental group was Nivolumab intravenous infusion (n=292). The primary endpoint of each study was overall survival (OS), and secondary endpoints were objective response rate (ORR) and progressionfree survival (PFS). Researchers classified PD-L1 expression levels in tumors into 1%, 5%, and 10%. Although non-squamous NSCLC patients with more than1% PD-L1 expression also benefited from Nivolumab treatment compared to Docetaxel, a higher, positive rate of PD-L1 was associated with greater OS benefit from Nivolumab treatment. The Hazard Ratio (HR) for OS was 0.38 (95% confidence interval (CI), 0.24 to 0.60,Nivolumab group n = 66, Docetaxel group n = 46) in non-squamous NSCLC patients with more than 50% PD-L1 expression (minimum of two years’ follow-up)18. After at least three years’ follow-up (minimum 40.3 months’ followup), Nivolumab continued to show an OS clinical benefit and improved PFS compared with Docetaxel, with a hazard ratio of 0.73 (95% CI: 0.62, 0.88) for OS and 0.89 (95% CI: 0.74, 1.06) for PFS. Three-year OS rates were 18% in Nivolumab monotherapy group versus 9% in Docetaxel group. Three-year PFS rates were 10% in Nivolumab monotherapy group and less than1% in Docetaxel group. CheckMate-017 is another randomized, open-label phase III study of Nivolumab monotherapy in advanced or metastatic squamous NSCLC which used a PD-L1 IHC assay. Pooled clinical trial analysis of previously mentioned CheckMate-057 and CheckMate-017 demonstrated that higher PD-L1 expression was associated with greater OS benefit in patients with squamous and non-squamous NSCLC with Nivolumab. The HR of OS was 0.42 (95% CI, 0.28 to 0.63) in nonsquamous NSCLC patients with 50% or more PD-L1 expression. Finally, in the CheckMate-078 randomized, open-label phase III study, researchers evaluated Nivolumab monotherapy’s safety and efficacy in a predominantly Chinese patient population with advanced or metastatic squamous and non-squamous NSCLC. A total sample size of approximately 504 patients (451 of them were Chinese patients) was randomly assigned to the Docetaxel control group and Nivolumab intravenous infusion experimental group in a 2:1 ratio. Among patients with more than 1%, PD-L1 expression, the median OS was 12.3 months with Nivolumab versus 7.9 months with Docetaxel (HR: 0.62; 95% CI: 0.45–0.87). In the subgroup with less than 1% PD-L1 expression, median OS was 11.4 months with Nivolumab and 10.2 months with Docetaxel (HR: 0.75; 95% CI: 0.52–1.09)19. NMPA Technical Review Guideline JSZ1800126 states that the PD-L1 IHC 28-8 pharmDx kit from Dako could be used as qualitative IHC assay
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for detecting PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) non-squamous nonsmall cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN) and melanoma tissues using EnVision FLEX visualization system on Autostainer Link 4820. Less than 1% PD-L1 expression detected in samples from non-squamous NSCLC patients may be associated with prolonged survival with the use of Nivolumab21.
Concordance of PD-L1 IHC assay systems Since the FDA approved the PD-L1 antibody [clone 28-8] for use as a complementary diagnostic to Nivolumab therapy, pharmaceutical and diagnostic companies have followed the “one drug. one diagnostic test” model when collaborating on the development of PD-L1 diagnostic and therapies. This has resulted in several PD-L1 companion and complementary diagnostic IHC assays. As of June 2020, NMPA has approved PD-L1 IHC assays; 28-8, 22C3, and SP263, based on antibody and clone type. The FDA has also approved PD-L1 IHC assays featuring clones 28-8, 22C3, SP263, and SP14223. See table 2 for more detailed information.
PD-L1 clone no.
Companion/ complementary Antibody diagnostic assay type
SP263
Ventana PD-L1 (SP263) Assay
Rabbit monoclonal
Antigen epitope
Complementary medicine: Manufacturer
Extracellular
Durvalumab (DurImfinziTM): Complementary therapy AstraZeneca for urothelial carcinoma
US FDA medical device registration/indications
NMPA imported
Nivolumab (OPDIVO®): Bristol-Myers Squibb Pembrolizumab (Keytruda®): MSD Class III registration for locally advanced or metastatic urothelial carcinoma
Tislelizumab (百泽安®): BeiGene
22C3
PD-L1 IHC 22C3 PharmDx
Mouse monoclonal
Extracellular
Pembrolizumab (Keytruda®): MSD
Complementary therapy for non-small cell lung cancer, gastric cancer, esophagogastric junction (EGJ) cancer, cervical cancer, urothelial carcinoma, squamous cell carcinoma of the head and neck, and esophageal squamous cell carcinoma
Class III registration for assisted in identifying non-small cell lung cancer
28-8
PD-L1 IHC 28-8 PharmDx
Rabbit monoclonal
Extracellular
Nivolumab (OPDIVO®): Bristol-Myers Squibb
Complementary therapy for nonsquamous small cell lung cancer and melanoma
Class III registration for nonsquamous small cell lung cancer
73-10
N/A
Rabbit monoclonal
Extracellular
Avelumab (Bavencio®): Pfizer, Merck
SP142
Ventana PD-L1 (SP263) Assay
Rabbit monoclonal
Extracellular
Atezolizumab (Tecentriq®): Complementary therapy Roche for urothelial carcinoma and triple-negative breast cancer
Table 2. PD-L1 IHC assays approved by the FDA and/or NMPA
Differences in the tumor microenvironment between cancer types has led to the development of multiple PD1/PD-L1 inhibitors, each corresponding to their own PD-L1 IHC assay system involving primary and secondary antibodies as well as automated IHC platforms. This means each inhibitor will subsequently result in separate interpretation methods and threshold values, which has created a need to normalize and standardize the available PD-L1 immunohistochemical assay methods. The International Association for the Study of Lung Cancer (IASLC) has released the phase I and II blueprint for joint research between academia and industry. 28-8, SP263, 22C3 assays showed a high level of concordance in tumor cell staining performance. SP142 assay exhibited less sensitivity, while 73-10 displayed higher sensitivity. Therefore the interpretation differences in each PD-L1 antibody are more significant than tumor cell staining24,25.
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PD-L1 antibody [28-8] IHC staining examples
Different PD-L1 expression in human lung cancer In the CheckMate-057 clinical study, researchers classified PD-L1 expression according to prespecified levels; more than 1%, more than 5%, and more than 10% for preplanned efficacy analysis. With OS as the primary endpoint, they compare Nivolumab’s efficacy with Docetaxel’s in patients with non-squamous NSCLC. The study showed that the OS time was longer with Nivolumab than with Docetaxel. The prespecified PD-L1 expression level cut-offs have similar predictive utility to identify non-squamous NSCLC patients who would benefit from Nivolumab. Figures 1 to 5 show the IHC results of recombinant anti-PL1 antibody [28-8] staining PD-L1 in human lung cancer tissues with different expression levels.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 1. Human lung cancer tissue PD-L1 antibody [28-8] IHC staining,（less than 1.0% expression Figure 2. Human lung cancer tissue PD-L1 antibody [28-8] IHC staining,（1% - 4.9% expression Figure 3. Human lung cancer tissue PD-L1 antibody [28-8] IHC staining,（5% - 9.9% expression Figure 4. Human lung cancer tissue PD-L1 antibody [28-8] IHC staining, 10%-49.9% expression Figure 5. Human lung cancer tissue PD-L1 antibody [28-8] IHC staining,（more than 50% expression
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Evaluate and interpret PD-L1 antibody [28-8] stained slides According to the NMPA Technical Review Guideline JSZ1800126, specific sequences and guidelines are followed when using the PD-L1 IHC 28-8 pharmDx kit from Dako to evaluate the slides21. They are summarized below: 1. Evaluate hematoxylin and eosin (H&E) staining of consecutive slides of the same tissue block to make sure the tissue sample is complete, well preserved, and needed to validate tumor indications. 2. Evaluate negative and positive control tissue for stain quality control to ensure that all reagents are working properly. 3. Evaluate two positive tissue controls. Select the control tissue from fresh biopsy/surgical specimens with the same tumor indication as the patient specimen. Fix, process, and embed the control tissue in the same way as the patient’s specimen as soon as possible. Stain positive control tissue slides with PD-L1 IHC 28-8 pharmDx kit and with negative control reagents to monitor appropriate tissue handling and reagents. 4. Evaluate two negative tissue controls. Select the control tissue from fresh biopsy/surgical specimens with the same tumor indication as the patient’s specimen. Fix, process, and embed the control tissue in the same way as the patient specimen as soon as possible. Stain negative control tissue slides with PD-L1 IHC 28-8 pharmDx kit and with negative control reagents to monitor appropriate tissue handling and reagents. 5. Evaluate negative control reagent staining of patient tissue slides to help interpret the specific staining pattern of the antigen. 6. Evaluate the specific staining pattern of the antigen with the PD-L1 primary antibody. The requirements for scoring stained slides are as follows21: 1. Evaluate all of the viable tumor cells on the PD-L1 stained slide. For a specimen to considered adequate for PD-L1 evaluation to determine the percentage of stained tumor cells, a minimum of 100 viable tumor cells must be present. For non-squamous NSCLC specimens, record the percentage of viable tumor cells showing circular and/or partial linear membrane staning at any intensity. 2. Exclude cytoplasmic staining, immune cells, necrotic cells, normal cells, and carcinoma in situ from scoring. 3. Determine the percentage of stained tumor cells in the entire sample, with the numerator as the number of stained viable tumor cells stained and the denominator as the number of viable tumor cells in the sample. 4. As for non-squamous NSCLC, set 1% PD-L1 expression as threshold.
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PD-L1 IHC staining of human normal tissue While earlier studies have reported PD-L1 expression in a small subset of macrophage-like cells in human tonsil, placenta, lung, and liver4,5, a more detailed summary of normal tissue reactivity is provided in the NMPA Technical Review Guideline JSZ1800126 for the PD-L1 IHC assay kit . A total of 30 normal tissues were tested in this study. Bone marrow cells in the adrenal gland, megakaryocytes, renal tubular epithelial cells, follicular macrophages, pancreatic epithelial cells (mainly islet cells), parathyroid cells, splenic sinuses cells, thymus bone marrow epithelium, supraorbital foramen, and tonsillar growth centers (immune cells) display membrane staining. Cytoplasmic staining can be observed in some tissue cells21. Figures 6 and 7 show IHC images of recombinant anti-PD-L1antibody [28-8] staining PD-L1 in human tonsil and placental tissues.
Figure 6
Figure 7
Figure 6. Human tonsil tissue PD-L1 antibody [28-8] IHC staining Figure 7. Human placental tissue PD-L1 antibody [28-8] IHC staining
PD-L1 IHC staining of human cancer tissue PD-L1 expression is heterogeneous in different cancers or patients with the same cancer. Figures 8 and 9 show IHC images of recombinant anti-PD-L1 antibody [28-8] staining PD-L1 in human cervical and gastric cancer tissues.
Figure 8
Figure 9
Figure 8. Human cervical cancer tissue PD-L1 antibody [28-8] IHC staining Figure 9. Human gastric cancer tissue PD-L1 antibody [28-8] IHC staining
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Anti-PD-L1 antibody [28-8] IHC protocol and troubleshooting
PD-L1 manual IHC protocol Materials – PAP pen (ab2601)
– Methanol
– Two pairs of sharp tweezers
– Hydrochloric acid solution (1ml concentrated hydrochloric acid+ 400ml ethanol)
– Fresh paraffin slices cut the day before the IHC assay (and already baked dry, at 42°C or 37°C overnight) – Kimwipes Delicate Task Wipers – 1X TBST wash buffer (0.1% Tween 20 in TBS) 2L or 1x TBS-T with Tween 20 (diluted from ab64204) 2L – Hematoxylin Solution (ab220365) – Neutral balsam – 30% Hydrogen peroxide (Analytical Reagent) or Hydrogen Peroxide Blocking Reagent (ab64218) – Goat serum – Two containers for 100% ethanol, one each of 95% ethanol, 85% ethanol, 75% ethanol, 50% ethanol, and two containers for xylene. The reagents should be made up fresh as possible. It is strongly recommended that do not reuse the reagents multiple times.
– A4 white paper – 1 ml, 200 μl, 100 μl, 20 μl, and 10 μl accurately calibrated pipettes and pipette tips – Coverslip – Rabbit-specific IHC polymer detection kit HRP/DAB (ab209101) – Recombinant Anti-PD-L1 antibody [28-8] (ab205921) – 10X Universal HIER antigen retrieval reagent (ab208572) – DAB chromogen (ab64238) – Pressure cooker – Coplin jar – Ultrapure water 2 L – Rabbit IgG, monoclonal antibodies [EPR25A] (ab172730)
Reagents 10X TBS solution
1X TBST wash buffer
– 24.23g Tris
Dilute 10X TBS to 1X and add final concentration of 0.1% Tween 20 and mix well.
– 80.06g NaCl – Add 800 mL of ddH2O and mix well. – Adjust the pH to 7.6 with concentrated hydrochloric acid then add ultrapure water up to 1 L and mix well.
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Method 1. The day before conducting the IHC analysis, bake paraffin sections overnight at 37⁰C or 42⁰C. 2. Perform the following washes: a. Xylene; 2 x 3min b. 100% ethanol; 2 x 3 min c. 95% ethanol 3 min d. 85% ethanol 3 min e. 75% ethanol 3 min f. 50% ethanol 3 min g. ddH2O 5min twice 3. Dilute 10X Universal HIER antigen retrieval reagent to 1X with ddH20. 4. Use pressure cooker to steam the slides for 2 min (or 30 sec at 120⁰C, or 15 min at 110⁰C). Do not secure the lid of the pressure cooker at this point. Bring antigen retrieval solution to the boil and add the section once boiling. Secure the lid and air valve. Let the pressure cooker reach full pressure. and then maintain 2 min. 5. Turn off the hotplate and remove the pressure cooker. Run cold water to cool the cooker until it can be handled. Open the lid and allow the sections to cool at room temperature. Gradually add ddH20 to the antigen retrieval solution and finally put the sections in ddH20. 6. TBST rinse sections 2 x 5 min. 7. 0.3% Hydrogen peroxide in TBST for 15min (normal tissue) or Hydrogen Peroxide Blocking Reagent ab64218 for 10 min. For blood or liver tissue, 0.3% hydrogen peroxide in methanol for 15 min. If the background is too high, increase the hydrogen peroxide to 3% and treatment time to 30 min. 8. TBST wash sections twice for 10 min.
11. TBST rinse sections four times for 10 min. 12. Incubate amplifier (ab209101) for 10 min at room temperature. 13. TBST rinse sections 3 times for 10 min. 14. Incubate ab209101 Detector for 10 min at room temperature. 15. TBST rinse sections 3 times for 10 min. 16. Mix ab64238 DAB Chromogen and DAB substrate in the ratio of 1:50. Mix well and incubate the sections for about 5 min. Place on A4 paper or under microscope to see the staining. When it gets to acceptable staining intensity, immerse the sections in a coplin jar with ddH20. 17. TBST rinse sections 3 times for 10 min. 18. Stain the sections with Hematoxylin Solution for 4 min. Rinse the sections carefully in coplin jar with running water; repeat once. 19. Rinse with a few drops of ethanol hydrochloride (1% HCl in 70% ethanol and concentrated hydrochloric acid). 20. Rinse carefully with running water for 7 min in a coplin jar. 21. ddH2O in orbital shaker incubator for 5 min. 22. Perform the following washes: a. 50% ethanol for 3 min. b. 75% ethanol for 3 min. c. 85% ethanol for 3min. d. 95% ethanol for 3 min. e. 100% ethanol 2 x 3 min. f. Xylene 2 x 3 min. 23. Seal the sections with neutral balsam. Be careful to avoid getting air bubble, then, air-dry with nail polish on the corners of the sections to seal.
9. Block with 5% Goat serum for 1 h at room temperature. 10. Dilute PD-L1 antibody 28-8 1:500 (final concentration 2 μg/ml) or recombinant rabbit monoclonal IgG isotype control (ab172730) (final concentration 2 μg/ ml) in 2% goat serum in TBST. Incubate primary antibody for an hour at room temperature.
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Supplementary notes – This protocol is for manual PD-L1 antibody 28-8 IHC experiment for research purposes only. It cannot be used for diagnostic or therapeutic purposes. For hospital pathology reports, please contact a qualified pathologist. – Recombinant Anti-PD-L1 antibody [28-8] (ab205921) must be combined with 10X Universal HIER antigen retrieval reagent (ab208572) and rabbit-specific IHC polymer. Detection kits HRP/DAB (ab209101), along with high-pressure, heat-induced epitope retrieval (2 min steam time) to produce optimal staining results. – The oxygen in the air will oxidize the antigen and result in loss of staining signal. It is strongly recommended to use fresh paraffin sections for staining. Please do not use sections that have been stored at room temperature for more than a week or at 4°C for more than a month for IHC staining. – The slides’ quality, dryness of the sections, and tissue fixation procedure will cause the sections to detach during heat-induced epitope retrieval. We recommend using positively charged microscope slides (such as Labserv, Thermo superfrost plus, and Dako FLEX IHC microscope slides). If the color of the tissue is similar to paraffin, it means that the sections are dry. If the tissue is white in color, the slide is not sufficiently dried and will easily detach.
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– Incubation of primary antibodies can also be done overnight at 4⁰C. – The samples must not dry out after adding xylene in the procedure as it may lead to high background staining. – Tween 20 in TBST is used to moisturize and reduce the background. – We recommend checking the PAP pen (ab2601) for leakage and tightness of the circles drawn on a spare slide. Leakage will cause the staining to be uneven. Please refer to the PAP pen (ab2601) datasheet for more information. – PD-L1 expression is localized in the membrane and endomembrane system. Human tonsil with hyperreactive changes can be used as postive controls. Screening of hyper-reactive tonsils is recommended to find tonsil with the highest expression of PD-L1 in crypt epithelium, macrophages homing the germinal centers and interfollicular mononuclear leukocytes. – It is necessary to use negative control to indicate non-specific background staining. We recommend using recombinant rabbit IgG, monoclonal [EPR25A] (ab172730) as an isotype control.
PD-L1 manual IHC troubleshooting No staining Source
Recommendation
No PD-L1 expression on the tissue specimen
Use human tonsil, squamous cell carcinoma of the head and neck, or placental tissue as positive controls.
Deparaffinization may be insufficient
Replace frequently used reagents with fresh xylene and ethanol as possible.
Over or poor fixation
Fix 15-20 times the volume of 10% NBF for 6-72 hours at 15-25°C.
Oxidation of antigens
Try to use fresh paraffin sections or sections stored at room temperature for less than a week, or stored at 4°C for less than a month.
Primary and secondary antibodies or amplification kits fail due to inadequate storage, inappropriate dilution, or multiple repeated freeze-thaw cycles
Use human tonsil, squamous cell carcinoma of the head and neck, or placental tissue as positive controls.
Insufficient antigen retrieval
Use 1X Universal HIER antigen retrieval reagent (ab208572) as antigen retrieval solution. Perform heat-induced epitope retrieval by steaming for 2 min.
Wash buffer contains sodium azide
Use TBST wash buffer.
Used conventional enzyme-linked secondary antibody
Use HRP polymer secondary antibody (ab209101).
High levels of endogenous peroxidase in the sample, which caused incomplete inactivation
For tissues with high endogenous peroxidase levels, increase the hydrogen peroxide to 3% and treatment time to 30 min.
Antibody binds to non-specific tissue
We recommend using recombinant rabbit IgG, monoclonal [EPR25A] (ab172730) as an isotype control to evaluate the expression.
Incubation temperature may be too high
Incubate primary and secondary antibodies at room temperature.
Primary antibody concentration may be too high
Titrate the antibody to the optimal concentration for the optimal result.
Tissue not washed enough after fixation; fixative still present
Rinse sections with orbital shaker incubator after fixation at least three times, 10 min each.
Phosphate buffered saline (PBS) residue react with DAB
Use TBST to rinse sections.
DAB staining took a long time
Observe the staining under the microscope or a piece of white A4 paper. As it gets to acceptable staining intensity, immerse the sections in ddH20.
Permeabilization has damaged the membrane and removed the membrane protein
There is no need to use any specific, intense permeabilization in the IHC assay using anti-PD-L1 28-8 antibody clone.
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Non-specific staining Source
Recommendation
The sections have dried out after deparaffinization
Incubate the sections in a moist chamber and use a high-quality PAP pen. Add Tween 20 with a final concentration of 0.1%.
High levels of endogenous peroxidase in the sample, which caused incomplete inactivation
For tissues with high endogenous peroxidase levels, increase the hydrogen peroxide to 3% and treatment time to 30 min.
Antibody binds to non-specific tissue
We recommend using recombinant rabbit IgG, monoclonal [EPR25A] (ab172730) as an isotype control to evaluate the expression.
High background Source
Recommendation
Antibody binds to non-specific tissue
The blocking step is performed with 5% normal serum or 5% BSA for 1 h at room temperature. We suggest increasing the serum concentration and/or blocking time.
Incubation temperature may be too high
Incubate primary and secondary antibodies at room temperature.
Primary antibody concentration may be too high
Titrate the antibody to the optimal concentration for the optimal result.
Tissue not washed enough after fixation; fixative still present
Rinse sections with orbital shaker incubator after fixation at least three times, 10 min each.
Phosphate buffered saline (PBS) residue react with DAB
Use TBST to rinse sections.
DAB staining took a long time
Observe the staining under the microscope or a piece of white A4 paper. As it gets to acceptable staining intensity, immerse the sections in ddH20.
Permeabilization has damaged the membrane and removed the membrane protein
There is no need to use any specific, intense permeabilization in the IHC assay using anti-PD-L1 28-8 antibody clone.
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PD-L1 automatic IHC protocol Automatic IHC protocol - BioGenex i6000 Materials – Xylene (EM Science, Cat # UN1307)
– Protein block (Dako Cat# X0909)
– Ethanol (AAPER alcohol and Chemical Co)
– Antibody Diluent (Abcam, ab64211)
– Wash buffer (DAKO, Cat# S3006)
– DAB (DAKO Cat# K3468) or DAB Substrate Kit (Abcam, ab64238)
– Target Retrieval Solution, AR6, 10X (DAKO, Cat#S1699) or Universal HIER antigen retrieval reagent (Abcam, ab208572)
– Recombinant Anti-PD-L1 antibody [28-8] (Abcam, ab205921, final concentration 2 μg/ml)
– Novolink Max Polymer Detection System (Leica, Cat# RE7260-CE)
– Isotype control rabbit monoclonal antibody (Abcam, ab172730, final concentration 2 μg/ml)
– Peroxidase block (Dako Cat# S2003) Equipment – BioGenex i6000 Autostainer – BioCare Medical Decloaking ChamberTM Plus
– Leica autostainer ST5020 (For deparaffinization and dehydration) – Leica Auto Coverslipper CV5030
Heat-induced antigen retrieval method 1.Perform deparaffinization and rehydration with the Leica ST5020 Multistainer using the following program: a.Wash with xylene 2 x 5 min; b.Wash with 100% ethanol 2 x 2 min; c.Wash with 95% ethanol 2 x 2 min; d.Wash with 70% ethanol 2 x 2 min; e.Wash with ddH2O for 2 min
2 .Perform antigen retrieval on Biocare Medical Decloaking Chamber Plus Dako AR6. Heat target retrieval solution to 110°C (P1) for 10 min, then move to the next step with P2. (fan on 95°C; fan off 90°C) 3. Cool slide at room temperature for 15 min. Rinse with dH2O for approximately 1 min.
IHC method 1. Apply Peroxidase Block for10 min
8. Wash three times with IHC wash buffer
2. Wash three times with wash buffer
9. Add the DAB chromogen substrate and develop for 5 min
3. Apply the Protein Block to the slides and incubate for 20 min at room temperature 4. Wash three times with wash buffer 5. Apply the pre-diluted antibodies to the slides and incubate for 1 h at room temperature
10. Wash slides five times with ddH2O at room temperature 11. Counterstain with hematoxylin for 1 min 12. Wash slides five times with ddH2O at room temperature
6. Wash three times with wash buffer 7. Add the Post Primary Block (NovoLink Kit) to the slides and incubate for 30 min
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Automatic IHC protocol - BioGenex i6000 (continued) Dehydration and coverslipping 1. Remove the slides from the BioGenex i6000 Autostainer
4. Wash with xylene three times, each for 2 min
2. Set up slides on the Leica Multistainer ST5020
5. Place coverslip with Cytoseal Mounting Medium in the Leica Auto Coverslipper CV5030
3. Dehydrate using the following program: a. 1 x 2 min wash with 70% ethanol b. 1 x 2 min wash with 95% ethanol c. 3 x 2 min wash with 100% ethanol
Automated IHC protocol - Leica BOND RX Materials – Bond Dewax Solution (Leica, Cat# AR9222)
– Antibody Diluent (Abcam, ab64211)
– Bond Epitope Retrieval 1 (Leica, Cat# AR9961) or Universal HIER antigen retrieval reagent (Abcam, ab208572)
– Recombinant Anti-PD-L1 antibody [28-8] (Abcam, ab205921, final concentration 2 μg/ml)
– Bond Wash Solution (Leica, Cat# AR9590)
– Isotype control rabbit monoclonal antibody (Abcam, ab172730, final concentration 2 μg/ml)
– Leica BOND Polymer Refine Detection kit (Leica, Cat# DS9800)
Equipment – Leica BOND RX autostainer IHC method 1. Heat and de-wax on Leica BOND RX autostainer 2. Performs antigen retrieval on Leica BOND RX autostainer at 100°C for 30 min using ER1 (Citric acid buffer from Leica) 3. Apply the “Peroxidase block” (Refine Kit) for 10 min and then rinse three times with wash buffer 4. Apply the diluted antibodies to the slides (see reagents) and incubate for 1 hour at room temperature 5. Wash three times with IHC wash buffer 6. Add the “Post Primary Block “ (Bond™ Polymer Refine Detection kit) to the slides, incubate for 30 min.
7. Wash three times with IHC wash buffer 8. Add the “NovoLink Polymer”(Refine Kit) to the slides, incubate for 30 min 9. Wash three times with IHC wash buffer 10. Add the DAB chromogen substrates (Refine Kit), develop for 10 min 11. Wash five times with ddH2O at room temperature 12. Counterstain with hematoxylin (Refine Kit) for eight min 13. Wash five times with ddH2O at room temperature
Dehydration and coverslipping 1. Remove the slides from the Leica BOND RX autostainer.
b. 1 x 2 min wash with 95% ethanol c. 2 x 2 min wash with 100% ethanol
2. Set up slides on the Leica ST5020 Multistaine.
4. Wash with xylene twice and, each time for 2 min;
3. Dehydrate using the following program:
5. Place coverslip with Cytoseal Mounting Medium in Leica coverslipper CV5030
a. 1 x 2 min wash with 70% ethanol
14
Automated IHC protocol - Ventana Ultra Materials – Xylene (EM Science, Cat # UN1307) – Ethanol (AAPER alcohol and Chemical Co) – Universal HIER antigen retrieval reagent (Abcam, ab208572) – Antibody Diluent (Abcam, ab64211)
– Ventana kit: ChromoMap DAB kit: Cat # 760-159 Anti-Rabbit HQ: Cat # 760-4815 Anti-HQ HRP: Cat # 760-4820 Hematoxylin II: Cat # 790-2208 Bluing Reagent: Cat # 760-2037 – Recombinant Anti-PD-L1 antibody [28-8] (ab205921)
Equipment – Ventana Ultra
– Biocare Decloaker
– Leica Autostainer (for baking, deparaffinization, and dehydration)
IHC method 1. Bake and deparaffinize on the Leica ST5020 Multistainer using the following program: a. Bake at 65°C for 10 min b. 3 x 3 min with xylene c. 2 x 2 min with 100% ethanol d. 1 x 1 min with 100% ethanol e. 1 X 1 min with 95% ethanol f. 1 X 1 min with 70% ethanol g. 1 X 3 min with PBS 2. Perform antigen retrieval on BioCare Medical Decloaking Chamber™ Plus using the Universal HIER antigen retrieval reagent (Abcam, ab208572) at 110°C for 10 min
4. Primary Antibody- Antibody Diluent (Abcam, ab64211): hand apply and incubate for 1h; PD-L1 antibody 28-8 ( ab205921) (final concentration is 7.5 μg/ mLl): Incubate the slide at 37°C. 5. Secondary and tertiary antibody incubation: incubate the slide with antiRabbit HQ (bridging antibody) 16 min at 37°C; then incubate HRP conjugated anti-HQ antibody 16 min at 37°C. 6. Add ChromoMap DAB and develop 7. Counterstain with hematoxylin II in reaction buffer and then apply bluing reagent
3. Load slides onto the Ventana Ultra (setup protocol as listed below)
15
Ventana Ultra Protocol Materials 1. Select antibody
9. Select – Enzyme conjugate
2. Select – 1st Antibody Manual Application
10. Apply one drop of Anti-HQ HRP (conjugate number one) and incubate for 16 min
3. Warm up slide to 37°C from very low temperatures (primary antibody) 4. Hand apply the primary antibody and incubate for 60 min 5. Select – Bridging Antibody 6. Select – Secondary Antibody 7. Warm up slide to 37°C from very low temperatures (secondary antibody) 8. Apply one drop of Anti-Rabbit HQ HRP (detection number one) and incubate for 16 min
11. Select – DAB 12. Select – Counterstain 13. Select – Use RB for Counterstain 14. Apply one drop of Hematoxylin II (counterstain), and incubate for eight min 15. Select – Post Counterstain 16. Select – Use RB for Post Counterstain 17. Apply one drop of bluing reagent (post counterstain) and incubate for four min
Dehydration and coverslipping 1. Remove the slides from the Ventana Ultra
5. Place coverslip with Cytoseal Mounting Medium manually
2. Wash slides with dawn soap 3. Rinse with deionized water 4. Set up slides on Leica ST5020 Multistainer and use the following program: a. Dehydrate with 70% ethanol for 2 min, 95% ethanol for 2 min, 3 x 2 min with 100% ethanol b. Rrinse with xylene 3 x 2 min
Automated IHC protocol - Dako Omnis Deparaffinization Two-Phase-Deparaffinization-IHC
Two-Phase-Deparaffinization-Wash-IHC
– Solvent: Clearify clearing agent
Reagent: DI water
– Transport liquid: DI water
Incubation: 5 seconds
– Temperature: 25°C
# cycles: 1
– Incubation(top):10 seconds – Incubation(bottom):1 minute – # cycles:1 Antigen retrieval Demasking-IHC – Reagent: EnVision FLEX TRS, Low pH – Temperature: 97°C – Incubation: 30 min – Cooling liquid: DI water
16
Automated IHC protocol - Dako Omnis (continued) Staining Step
Reagent
Incubation
Wash
Wash-buffer
2:40 min
# cycles
Primary Antibody
PD-L1 (1:400)
1 hour
Wash
Wash-buffer
2 min
Endogenic Enzyme Blocking
EnV FLEX Peroxidase-Blocking reagent
3 min
Wash
Wash-buffer
2 min
Secondary Reagent
EnV FLEX + Rabbit Linker
10 min
Wash
Wash-buffer
2 min
Labeled Polymer
EnV FLEX/HRP
20 min
Wash
Wash-buffer
2 min
10
Wash
Wash-buffer
2 min
10
Wash
DI Water
31 seconds
1
Wash
Wash-buffer
2 min
10
Substrate-Chromogen
EnV FLEX Substrate Working Solution
5 min
Wash
Wash-buffer
2 min
Wash
DI Water
31 seconds
1
Wash
Wash-buffer
2 min
10
2 10 10 10
10
Counterstaining Step
Reagent
Incubation
Staining
Hematoxylin
3 min
# cycles
Wash
Wash-buffer
2 min
10
Wash
Wash-buffer
2 min
10
17
PD-L1 antibody [28-8] frequently asked questions
Q1: Can I use PD-L1 antibody [28-8] (ab205921) to diagnose clinical patients? PD-L1 antibody [28-8] (ab205921) is for research use only and not for use in diagnostic purposes. If you wish to get a pathology report, please contact a qualified pathologist.
Q2: What do 28-8, SP142, and 73-10 stand for? These are clone numbers, the number of the hybridoma cell line from which the monoclonal antibody originated. Only monoclonal antibodies have clone numbers. Antibodies with identical clone numbers have identical antigen epitopes. Q3: Do I have to perform heat-induced epitope retrieval for PD-L1 antibody [28-8] (ab205921) manual IHC assay? What about a heated bath? For manual IHC staining with anti-PD-L1 antibody [28-8] (ab205921) heat-induced epitope retrieval must be performed by steaming for 2 min. We came up with this ideal method after extensive experiments. Other retrieval methods might result in weak signals or high background. Q4: Can I use other brands of self-made antigen retrieval solutions such as Citric acid pH6.0 buffer or Tris-EDTA pH9.0 buffer in a PD-L1 antibody [28-8] (ab205921) manual IHC assay? Can it be used with conventional non-HRP-polymer secondary antibodies or other secondary antibody staining systems? No. We found the PD-L1 antibody [28-8] in combination with the HIER antigen retrieval reagent (ab208572), and the rabbit specific IHC polymer detection kit HRP/DAB ( ab209101) can give the best result. Other reagents may result in a weak signal or high background. Q5: What is the concordance among the different PD-L1 antibodiesy, 28-8, SP142, SP263, 22C3, and & 73-10? The International Association for the Study of Lung Cancer (IASLC) has released the phase I and II blueprint for joint research between academia and industry. 28-8, SP263, 22C3 assays showed a high level of concordance in tumor cell staining performance. The SP142 assay exhibited less sensitivity, while 73-10 displayed higher sensitivity. Variation of the scoring on PD-L1 staining in immune cells is higher than that in tumor cells.24,25. Q6: Is it possible to skip the protein blocking step in for PD-L1 antibody [28-8] IHC staining? Does the 2% goat serum when incubating thein primary antibody incubation also act as a protein blocking reagent? No, you cannot skip the protein blocking step. The 2% goat serum in the primary antibody will significantly increase the background. Therefore, we recommend using 5% goat serum as a blocking reagent for 1 hr at room temperature for manual IHC staining.
18
Q7: Any other optimization tips? – You can draw a circle as small as possible with the PAP pen. However, do not let the pen come to direct contact with the tissue, causing non-specific signals. Add incubation reagents (mainly antibody) right above the tissue. Incubate the tissue in the wet box to prevent slides from drying out. – If you need to save the amount of antigen retrieval solution, place the slides in a coplin jar with antigen retrieval solution, or place the sections horizontally in a heat resistant plastic box with antigen retrieval solution.
19
Cancer immunotherapy: immune checkp T cell/dendritic cell interactions Neoantigens released by cancer cells in the tumor microenvironment are processed by DCs and presented on their cell surface. These antigen presenting cells (APCs) are recognized by T cells, resulting in T cell priming and activation. DCs may also exhibit inhibitory receptors which act to balance the immune response, preventing autoimmune inflammation.
Maintenance of T cell activation by stimulatory (eg (eg CTLA4 and PD-1) fine-tune the immune response or immune deficiency. In cancer, the balance betw immunosuppression leading to cancer progression.
T cell activation by a dendritic cell
T cell activation by a dendritic cell Naive T cell OX40
OX40L
ICOS
ICOSL
CD28
CD80
TCR
MHC I
T cell activation
Tc T cell priming
Activated T cell
Dendritic cell Dendritic cell
T cellby inhibition bycell a dendritic cell T cell inhibition a dendritic
Antigen capture
Tumor infiltration
Antigens
CTLA4
CD86
Tumor apoptosis Macrophage
PD1
PDL1
Inhibited T cell
Dendritic cell
®
RabMAb Knockout-validate antibodies
Regulators of T cell activation Antigen Presenting Cell
T Cell
T Cell Regulation
PD-L1
PD-1
Inhibition
PD-L2
PD-1
n/a
CD80 / CD86
CD28
Activation
CD80 / CD86
CTLA4
Inhibition
ICOSL
ICOS
Activation
CD276
n/a
Inhibition
B7-H4
n/a
Inhibition
B7-H5
CD28H
Activation
n/a
VISTA
Inhibition
HVEM
BTLA
Inhibition
CD40
CD40L
Activation
OX40L
OX40
Activation
CD137L
CD137
Activation
CD70
CD27
Activation
GAL9
TIM3
Inhibition
GITRL
GITR
Activation
MHC-II
LAG-3
Inhibition
20
PD-L1 [28-8]
B-CAP Cells – high
CXCR
HCC70 Cells – medium ES-2 Ce Cancer cell lines with varying lev
PD-L1 [28-8] - Highly specific for human PD-L1; no cross-re - Generated using the extracellular domain membrane specific staining
CXCR4 [EPUMBR3] - Specific IHC-P signals demonstrated in FFP embryonic tissues.
Key features - Available as Azide-free variants - Knockout (KO) cell line validated in key ap - Available as Alexa Fluor® conjugated prim
point poster T cell/tumor cell interactions
TCR and OX40) and inhibitory regulators e to prevent autoimmune inflammation ween these inputs is tipped toward
Tumor cells and non-transformed cells within the tumor microenvironment will exploit the immune checkpoint pathway inhibiting the anti-tumor immune response. This leads to cancer progression. They achieve this by over-expressing inhibitory immune checkpoint molecules such as PD-L1 on their cell surface. These bind to inhibitory receptors on the T cell surface causing the immune cells to deactivate. T cell inhibition by tumor cell
T cell inhibition by tumor cell TIM 3
cell proliferation
Galectin 9
TCR MHC I
LAG 3
Activated T cell MHC II PD1 PDL1
MDSC cell
Inhibited T cell
Tumor cell
Mast cell
Tumor T cell
CTL
Immunotherapy Inhibitors which target negative regulators of immune cell function such as CTLA4, PD-L1, and CXCR4 have great potential for use as immunotherapies. Blocking the T cell inhibitory signal results in an active immune response leading to tumor cell death. T cell activation by immunotherapy
ed immuno-oncology Tumor cell
R4 [EPUMBR3]
ells – low COLO205 Cells – none vels of PD-L1 expression)
eactivity with human PD-L2 n of PD-L1 protein – observed
PDL1
Activated T cell
PD1 Anti-PD-1 PD-1 inhibitors
Anti-CTLA4 CTLA4 inhibitors
CXCR4 CTLA4
CD86 CXCR4 inhibitors
Dendritic cell
PE CXCR4 -/- and WT E14 mouse
pplications: IHC, FC, WB mary antibodies
www.abcam.com/cancer Copyright © 2018 Abcam, All rights reserved. RabMAb® is a registered trademark of Abcam. *Adapted from Pardoll, et al., 2012
21
Antibody panels for immune checkpoints
Discover our range of hand-picked antibody panels to key immuno-oncology targets designed to save you time and provide you with easy access to the best selection of clones on the market. Each panel contains a selection of monoclonal antibodies that detect the same protein to increase your chances of finding a compatible clone for your assay with just one purchase.
Product code
ab252195
ab252193
ab252192
ab239749
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Component Panel name product code Size
GITR
Tested applications
Species reactivity
ab223841
20 µl
Anti-GITR antibody [EPR20566]
ICC/IF, IHC-P, WB
Hu
ab237714
20 µl
Anti-GITR antibody [CAL8]
Flow Cyt, IHC-P
Hu
ab237713
20 µl
Anti-GITR antibody [CAL52]
Flow Cyt, ICC/IF, IHC-P, IP
Hu
ab209236
20 µl
Anti-LAG-3 antibody [EPR20261]
Flow Cyt, ICC/IF, IHC-P, IP, WB
Hu
ab180187
20 µl
Anti-LAG-3 antibody [EPR4392(2)] - C-terminal
IHC-P
Hu
ab227706
20 µl
Anti-LAG-3 antibody [SP346] - C-terminal
IHC-P
Hu
ab237720
20 µl
Anti-LAG-3 antibody [CAL77]
Flow Cyt, IHC-P
Hu,Ms
ab237718
20 µl
Anti-LAG-3 antibody [CAL25]
Flow Cyt, IHC-P, IP, WB
Hu
ab237719
20 µl
Anti-LAG-3 antibody [CAL26]
IHC-P
Hu
ab237728
20 µl
Anti-PD1 antibody [CAL20]
ICC/IF, IHC-P, WB
Hu
ab137132
20 µl
Anti-PD1 antibody [EPR4877(2)]
IHC-P
Hu
ab227681
20 µl
Anti-PD1 antibody [SP269]
Flow Cyt, IHC-P
Hu
ab52587
20 µl
Anti-PD1 antibody [NAT105]
Flow Cyt, ICC/IF, IHC-P, IHC-Fr, IP, WB
Hu
ab216352
20 µl
Anti-PD1 antibody [NAT105EPR21106] - Chimeric
Flow Cyt, IHC-P, IP, WB
Hu
ab228415
1 x 10 µl
Anti-PD-L1 antibody [73-10]
Flow Cyt, ICC/IF, IHC-P, IP, WB
Hu
ab205921
1 x 10 µl
Anti-PD-L1 antibody [28-8]
Flow Cyt, ICC/IF, IHC-P, WB
Hu
ab228462
1 x 25 µl
Anti-PD-L1 antibody [SP142] - C-terminal
ICC/IF, IHC-P
ChHm, Hu
ab237726
1 x 10 µl
Anti-PD-L1 antibody [CAL10]
ELISA, ICC/IF, IHC-P, IP, WB
Hu
LAG-3
PD1
Component name
PD-L1
Product code
ab269812
ab252191
Component Panel name product code Size
PD-L1 / PD1 Multiplex IHC-IF
TIM 3
Component name
Tested applications
Species reactivity
ab16669
1 x 50 µl
Anti-CD3 antibody [SP7]
Flow Cyt, IHC-P, mIHC, WB
Hu,Ms, Rat
ab7753
1 x 50 µg
Anti-pan Cytokeratin antibody [C-11]
Flow Cyt, IHC-P, mIHC, WB
Cm, Goat, Hu,Rat
ab192847
1 x 50 µl
Recombinant Anti-CD68 antibody [SP251]
Flow Cyt, IHC-P, mIHC
Hu
ab16667
1 x 50 µl
Recombinant Anti-Ki67 antibody [SP6]
Flow Cyt, ICC, IHC-P, mIHC, WB
Hu,Ms, Rat
ab237728
1 x 50 µl
Recombinant Anti-PD1 antibody [CAL20]
ICC/IF,I HC-P, mIHC, WB
Hu
ab237726
1 x 50 µl
Recombinant Anti-PD-L1 antibody [CAL10]
ELISA, ICC/IF, IHC-P, IP, mIHC, WB
Hu
ab241332
20 µl
Anti-TIM 3 antibody [EPR22241]
IHC-P, IP, WB
Hu,Ms
ab246851
20 µl
Anti-TIM 3 antibody [CAL68]
IHC-P
Hu
ab252533
20 µl
Anti-TIM 3 antibody [BLR033F]
ICC, IHC-P, WB
Hu
ChHm: Chinese Hamster; Cm: Common marmoset; Goat: Goat; Hu: Human; Ms: Mouse; Rat: Rat. ELISA: ELISA; Flow Cyt: Flow Cytometry; ICC: Immunocytochemistry; ICC/IF: Immunocytochemistry / Immunofluorescence; IF: Immunofluorescence; IHC: Immunofluorescence; IHC-Fr: Immunohistochemistry (Frozen sections); IHC-P: Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections); IP: Immunoprecipitation; mIHC: Fluorescent multiplex immunohistochemistry; WB: Western blot
23
References
1. Sanmamed, M. F. & Chen, L. A Paradigm Shift in Cancer Immunotherapy: From Enhancement to Normalization. Cell 175, 313-326, doi:10.1016/j.cell.2018.09.035 (2018). 2. Xin Yu, J. et al. Trends in clinical development for PD-1/PD-L1 inhibitors. Nature reviews. Drug discovery 19, 163-164, doi:10.1038/d41573-019-00182-w (2020). 3. Cancer Research website, <www.cancerresearch.org/scientists/immuno-oncologylandscape/pd-1-pd-l1-landscape> 4. Dong, H. et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nature medicine 8, 793-800, doi:10.1038/nm730 (2002). 5. Petroff, M. G., Chen, L., Phillips, T. A. & Hunt, J. S. B7 family molecules: novel immunomodulators at the maternal-fetal interface. Placenta 23 Suppl A, S95-101, doi:10.1053/plac.2002.0813 (2002). 6. Chen, L. & Han, X. Anti-PD-1/PD-L1 therapy of human cancer: past, present, and future. The Journal of clinical investigation 125, 3384-3391, doi:10.1172/JCI80011 (2015). 7. Francisco, L. M. et al. PD-L1 regulates the development, maintenance, and function of induced regulatory T cells. The Journal of experimental medicine 206, 3015-3029, doi:10.1084/jem.20090847 (2009). 8. Amarnath, S. et al. The PDL1-PD1 axis converts human TH1 cells into regulatory T cells. Science translational medicine 3, 111ra120, doi:10.1126/scitranslmed.3003130 (2011). 9. Dong, H., Zhu, G., Tamada, K. & Chen, L. B7-H1, a third member of the B7 family, costimulates T-cell proliferation and interleukin-10 secretion. Nature medicine 5, 1365-1369, doi:10.1038/70932 (1999). 10. Selenko-Gebauer, N. et al. B7-H1 (programmed death-1 ligand) on dendritic cells is involved in the induction and maintenance of T cell anergy. Journal of immunology 170, 3637-3644, doi:10.4049/jimmunol.170.7.3637 (2003). 11. Tsushima, F. et al. Interaction between B7-H1 and PD-1 determines initiation and reversal of T-cell anergy. Blood 110, 180-185, doi:10.1182/blood-2006-11-060087 (2007). 12. Goldberg, M. V. et al. Role of PD-1 and its ligand, B7-H1, in early fate decisions of CD8 T cells. Blood 110, 186-192, doi:10.1182/blood-2006-12-062422 (2007). 13. Yao, S. et al. PD-1 on dendritic cells impedes innate immunity against bacterial infection. Blood 113, 5811-5818, doi:10.1182/blood-2009-02-203141 (2009). 14. Azuma, T. et al. B7-H1 is a ubiquitous antiapoptotic receptor on cancer cells. Blood 111, 3635-3643, doi:10.1182/blood-2007-11-123141 (2008). 15. Sznol, M. & Chen, L. Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer--response. Clinical cancer research : an official journal of the American Association for Cancer Research 19, 5542, doi:10.1158/1078-0432.CCR-13-2234 (2013). 16. Taube, J. M. et al. Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Science translational medicine 4, 127ra137, doi:10.1126/scitranslmed.3003689 (2012).
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17. Zhang, Y. & Chen, L. Classification of Advanced Human Cancers Based on Tumor Immunity in the MicroEnvironment (TIME) for Cancer Immunotherapy. JAMA oncology 2, 1403-1404, doi:10.1001/jamaoncol.2016.2450 (2016). 18. Horn, L. et al. Nivolumab Versus Docetaxel in Previously Treated Patients With Advanced Non-Small-Cell Lung Cancer: Two-Year Outcomes From Two Randomized, Open-Label, Phase III Trials (CheckMate 017 and CheckMate 057). Journal of clinical oncology : official journal of the American Society of Clinical Oncology 35, 3924-3933, doi:10.1200/ JCO.2017.74.3062 (2017). 19. Wu, Y. L. et al. Nivolumab Versus Docetaxel in a Predominantly Chinese Patient Population With Previously Treated Advanced NSCLC: CheckMate 078 Randomized Phase III Clinical Trial. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 14, 867-875, doi:10.1016/j.jtho.2019.01.006 (2019). 20. Agilent website <https://www.agilent.com/en/product/pharmdx/pd-l1-ihc-28-8-pharmdx/ pd-l1-ihc-28-8-pharmdx-for-autostainer-link-48-76917> 21. PD-L1 检测试剂盒（免疫组织化学法）体外诊断试剂产品注册技术审评报告. 22. NMPA website, <www.nmpa.gov.cn/WS04/CL2042> 23. FDA website, <www.fda.gov/medical-devices/vitro-diagnostics> 24. Hirsch, F. R. et al. PD-L1 Immunohistochemistry Assays for Lung Cancer: Results from Phase 1 of the Blueprint PD-L1 IHC Assay Comparison Project. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 12, 208-222, doi:10.1016/j.jtho.2016.11.2228 (2017). 25. Tsao, M. S. et al. PD-L1 Immunohistochemistry Comparability Study in Real-Life Clinical Samples: Results of Blueprint Phase 2 Project. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 13, 1302-1311, doi:10.1016/j.jtho.2018.05.013 (2018).
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