Anti-PDCD1 Recombinant Antibody (Nivolumab) (CAT#: TAB-770)

Figure 1 Limited PD-1 expression in normal human tissues.

Immunohistochemistry in positive control tissue, hyperplastic tonsil (A), using FITC-conjugated nivolumab. Strong immunoreactivity was distributed in subsets of lymphocytes primarily in germinal center of the tonsil; FITC-conjugated human IgG4 was used as an isotype control in tonsil (B). No specific staining was observed in cerebellum (C), heart (D), lung (E), or kidney (F). Positive staining was revealed in a very small number of scattered endocrine cells (G–I) in four of five pituitary samples. G and H represent two positive samples, and I represents negative pituitary tissue. Insets in G and H are high-power views showing strong immunoreactivity in large cytoplasmic spherical organelles, enigmatic body-like structures, with weak cytoplasmic staining. Br, bronchiole of the lung; GC, germinal center of the tonsil; Gl, glomerulus of the kidney; GL, granular layer of the cerebellum cortex; ML, molecular layer of the cerebellum cortex; MZ, mantle zone of the tonsil.

Wang, C., Thudium, K. B., Han, M., Wang, X. T., Huang, H., Feingersh, D., ... & Singh, S. (2014). In vitro characterization of the anti-PD-1 antibody nivolumab, BMS-936558, and in vivo toxicology in non-human primates. Cancer immunology research.

Figure 2 PD-1 blockade enhances T-cell function.

A, 105 purified CD4+ T cells were cocultured with 104 allogeneic monocyte-derived DCs in the presence of a titration of nivolumab or isotype control antibody in triplicates for 6 days. Supernatants were collected at day 5 and measured for IFNγ production by ELISA. The cultured cells were labeled with 1 μCi 3H-thymidine for another 18 hours before being analyzed for proliferation. Representative data from multiple donor DC/T-cell pairs are shown. CPM, counts per minute. B, 105 PBMCs were stimulated with serial dilutions of SEB in the presence of a fixed amount of nivolumab or isotype control antibody in solution (20 μg/mL). Supernatants were collected after 3 days for measurement of IL-2 by ELISA. Representative data from multiple healthy donors (n = 18) are shown. C, 2 × 105 PBMCs from a CMV-positive donor were stimulated with lysate from CMV-infected cells in the presence of nivolumab or isotype control. Supernatants were collected after 4 days and assayed for IFNγ secretion by ELISA. D, 5 × 104 CD4+CD25+ Tregs were cocultured with 105 CD4+CD25− responder T cells and 2 × 104 DCs in the presence of 20 μg/mL of nivolumab or isotype control antibody in an allogeneic MLR for 6 days. IFNγ was analyzed from the supernatants collected at day 5 and proliferation was measured at day 6 after 18 hours of 3H-thymidine labeling.

Wang, C., Thudium, K. B., Han, M., Wang, X. T., Huang, H., Feingersh, D., ... & Singh, S. (2014). In vitro characterization of the anti-PD-1 antibody nivolumab, BMS-936558, and in vivo toxicology in non-human primates. Cancer immunology research.

Figure 3 The absence of ADCC by nivolumab in vitro.

IL-2–activated human PBMCs (effector cells) were incubated with activated human CD4+ T cells (target cells) in an effector-to-target cell ratio of 50:1 in the presence of serial dilutions of nivolumab or a positive control anti-MHC class I antibody for 3 hour at 37°C. A–C, data from three individual ADCC assays using cells from different donors are shown. Purified CD4+ T cells were activated by coated anti-CD3 antibody (4 μg/mL) plus soluble anti-CD28 antibody (1 μg/mL) and IL-2 (100 U/mL) for 3 days. PD-1 expression on activated CD4+ T cells in each of the ADCC assays is shown in the right (solid line for PD-1, gray line for isotype control).

Wang, C., Thudium, K. B., Han, M., Wang, X. T., Huang, H., Feingersh, D., ... & Singh, S. (2014). In vitro characterization of the anti-PD-1 antibody nivolumab, BMS-936558, and in vivo toxicology in non-human primates. Cancer immunology research.

Figure 4 Pathological Assessment of Response to Neoadjuvant Blockade of Programmed Death 1 (PD-1).

Panel A shows pathological regression in the resected primary lung tumor after neoadjuvant administration of nivolumab, according to the percentage of remaining viable tumor cells, for each of the 20 patients who underwent surgical resection. The gray horizontal line indicates the threshold for a major pathological response (90% regression). Clinical and pathological features that include the presence or absence of lymph-node (LN) metastases in the surgical specimen and preoperative radiologic response (according to Response Evaluation Criteria in Solid Tumors [RECIST]) are annotated for each patient. AC denotes adenocarcinoma, SCC squamous-cell carcinoma, PR partial response, SD stable disease, and PD-L1 programmed death ligand 1. Also shown are biopsy specimens obtained before (Panel B) and after (Panel C) neoadjuvant administration of nivolumab in a patient (MD043-008) who had a major pathological response (multiplex immunofluorescence staining). With this staining technique, visible structures include cytokeratin-positive tumor cells (orange), CD68+ macrophages (magenta), FoxP3+ regulatory T cells (yellow), CD8+ T cells (green), PD-1+ cells (red), and PD-L1+ cells (white). In the pretreatment specimen, only a few intratumoral macrophages are seen expressing PD-L1. However, there are multiple foci where PD-L1 and PD-1 are expressed in close proximity to each other (inset with white circle) in the pretreatment specimen. Focal, geographic tumor-cell PD-L1 expression was observed in an adaptive pattern (not shown in this image). After two doses of nivolumab, the tumor is infiltrated by liquid containing CD8+ and PD-1+ immune cells. Some of the infiltrating immune cells express PD-L1, which is consistent with an adaptive immune resistance mechanism.

Forde, P. M., Chaft, J. E., Smith, K. N., Anagnostou, V., Cottrell, T. R., Hellmann, M. D., ... & Battafarano, R. J. (2018). Neoadjuvant PD-1 blockade in resectable lung cancer. New England Journal of Medicine, 378(21), 1976-1986.

Figure 5 Identification of Mutation-Associated, Neoantigen-Specific T Cells after Neoadjuvant Treatment with Nivolumab.

An antigen-recognition assay that evaluates in vitro expansion of T-cell clones after peptide stimulation was performed with the use of 47 algorithm-predicted, candidate mutation–associated neoantigens on peripheral-blood T cells obtained from Patient 1 before the administration of nivolumab (on day −28) and after administration (on day 44 after surgical resection) to determine the repertoire of functional mutation-associated neoantigen recognition. Seven mutation-associated neoantigens were recognized at both time points. The 47 peptides that were chosen for analysis represented the top 10 ImmunoSelect-R pipeline (Personal Genome Diagnostics) peptides that were predicted to bind specifically to each of the patient's five HLA class I alleles. Three peptides were excluded because they were predicted to bind multiple HLA alleles. Three T-cell clones from a peripheral-blood sample obtained on day 44 that specifically expanded in culture to mutation-associated neoantigen (MANA) number 7 were found in the pretreatment tumor-biopsy specimen and the resection specimen (Panel A). Also shown are the frequencies of the three T-cell clones in longitudinal analysis of peripheral-blood T cells before and after treatment (Panel B) and in a tumor-biopsy sample before treatment, in resected tumor, in normal lung, and in tumor-involved and tumor-uninvolved lymph nodes at resection (Panel C). TCR Vβ CDR3 AA denotes T-cell receptor Vβ complementarity-determining region 3 amino acid.

Forde, P. M., Chaft, J. E., Smith, K. N., Anagnostou, V., Cottrell, T. R., Hellmann, M. D., ... & Battafarano, R. J. (2018). Neoadjuvant PD-1 blockade in resectable lung cancer. New England Journal of Medicine, 378(21), 1976-1986.

Figure 1 Anti-PDCD1 Recombinant Antibody (TAB-770) in ELISA

ELISA analysis of TAB-770 was performed by coating with PD-1 Protein, Human, Recombinant (His Tag).

Figure 2 Anti-PDCD1 Recombinant Antibody (TAB-770) in WB

WB analysis of TAB-770 was performed by with PD-1 Protein, Human, Recombinant (His Tag).
Lane 1: TAB-770 antibody
Lane 2: Negative control
Lane 3: 100ng PDCD1 antigen
Lane 4: 200ng PDCD1 antigen
Lane 5: 400ng PDCD1 antigen

Figure 3 SDS-PAGE analysis of anti-PD-1 antibody (Cat# TAB-770)

The purified anti-PD-1 antibody (Cat# TAB-770) was loaded in SDS-PAGE gels in β-mercaptoethanol-reduced (Lane 1) and non-reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result of different β-mercaptoethanol-reduced proteins (Heavy chain and Light chain) migrate as about 50 kDa and 25 kDa, respectively.

Figure 4 SEC-HPLC analysis of anti-PD-1 antibody (Cat# TAB-770)

Size exclusion chromatography (SEC-HPLC) is a technique of purity analysis and macromolecular separation based on molecule size. The purity of anti-PD-1 antibody (Cat# TAB-770) was determined by this method with Agilent 1200 Series.

Figure 5 ELISA analysis of anti-PD-1 antibody (Cat# TAB-770)

ELISA analysis of anti-PD-1 antibody (Cat# TAB-770) was performed by coating with recombinant human PD-1 protein (His tag). Then blocked with BSA and incubated with anti-PD-1 antibodies. The HRP-conjugated goat anti-human IgG was used as a secondary antibody. Detection was performed using TMB substrate and stopped with sulfuric acid. The absorbances were read on a spectrophotometer at 450 nm.

Figure 6 Western blot analysis of anti-PD-1 antibody (Cat# TAB-770)

Western blot analysis of anti-PD-1 antibody (Cat# TAB-770) was performed by loading human PD-1 protein (His tag) onto a 12% Tris-HCl polyacrylamide gel. Proteins were transferred to a NC membrane and blocked with 5% skim milk for at least one hour. Membranes were probed with anti-PD-1 antibodies and HRP goat anti-human IgG was used as a secondary antibody (1/2000).
Lane 1: Reduced antigen (0.1 μg)
Lane 2: Reduced antigen (0.3 μg)
Lane 3: Reduced antigen (0.6 μg)

Figure 7 Dot blot analysis of anti-PD-1 antibody (Cat# TAB-770)

Dot blot analysis of anti-PD-1 antibody (Cat# TAB-770) was performed by coating with human PD-1 protein (His tag).
The anti-PD-1 antibody incubation concentration: 2 μg/mL.
HRP-conjugated goat anti-human IgG was used as a secondary antibody (1/2000).

Figure 8 Sensorgram fit of human PD-1 analyte-binding anti-PD-1 antibody ligand (Cat# TAB-770)

Dilute the antibody ligand (5 μg/mL) and antigen analyte with running buffer. The ligand was injected to sample channel (Fc2) at a flow rate of 10 μL/min to reach a capture level of about 400 RU. The analyte was injected to Fc1-Fc2 of the channel at a flow rate of 30 μL/min for an association phase of the corresponding time. The association and dissociation process were all handling in the running buffer. Repeat 6 cycles of analyte according to analyte concentrations in ascending order.

Figure 9 PD-L1 binding to PD-1 was blocked by anti-PD-1 antibody (Cat# TAB-770), IC₅₀ = 246.6 ng/mL.

In a functional ELISA, the recombinant human PD‑1 protein (His tag) was coated at 2 µg/mL, 100 µL/well. Anti-PD-1 antibody (Cat# TAB-770) were 3-fold serial diluted from 20 µg/mL, 100 µL/well. Coated PD-1 protein was incubated with the human PD-L1 mFc chimera (5 µg/mL) and anti-PD-1 antibody (Cat# TAB-770). The PD-L1 binding to PD-1 was detected by HRP-goat anti-mouse IgG secondary antibody. Human IgG4 antibody (Cat# MOB-0408ZL, Creative Biolabs) was used as an isotype control.

Figure 10 Blockade of PD-1/PD-L1 interaction by anti-PD-1 antibody (Cat# TAB-770) resulted in an increase of IFN-γ production.

PBMCs were stimulated with 2 μg/mL anti-CD3 antibody (Cat# TAB-0416CL, Creative Biolabs) and cultured for 144 hours with PD-1 antibody (Cat# TAB-770, Creative Biolabs). Human IgG4 antibody (Cat# MOB-0408ZL, Creative Biolabs) was used as an isotype control. After cultivation, the supernatant was collected and the IFN-γ secretion level was analyzed using a human IFN gamma ELISA kit. The assay was performed in three parallel wells.