Application Note Affinity of PD-1/PD-L1 interaction—a comparison between SPR, MST, ITC and MDS Published on November 13th, 2019 Authors: Sebastian Fiedler, Monika Piziorska, Haris Choudhery and Sean Devenish The binding affinity of PD‑1/PD‑L1, determined by microfluidic diffusional sizing (MDS) on the Fluidity One‑W, is in good agreement with values obtained by SPR, MST and ITC. Further, MDS analysis allowed the absolute size of PD‑1 and PD‑1/PD‑L1 complex to be measured, from which the stoichiometry of the complex was inferred. Thus, the Fluidity One‑W can determine binding affinity and stoichiometry of protein interactions without prior knowledge of the structure of the protein complex. DOWNLOAD APPLICATION NOTE Introduction The PD‑1/PD‑L1 interaction functions as an immune checkpoint that prevents autoimmune responses. This immunosuppressive effect of PD‑1/PD‑L1 is employed by many types of cancer cells to evade recognition and destruction by T‑cells. Consequently, inhibition of this interaction is a major target in cancer immunotherapy, and several monoclonal antibodies are being used successfully in the clinic against a variety of cancers.1 Programmed cell death protein 1 (PD-1) is expressed on many types of immune cells including T‑cells, B‑cells and macrophages. When programmed death ligand 1 (PD‑L1) binds to PD‑1, regulatory T‑cell apoptosis is suppressed while apoptosis of antigen-specific T‑cells is promoted. Here, the binding affinity (KD) between PD‑1/PD‑L1 was measured using microfluidic diffusional sizing (MDS) on the Fluidity One‑W. This KD was compared with literature values from other biophysical methods: surface plasmon resonance (SPR), microscale thermophoresis (MST) and isothermal titration calorimetry (ITC). In addition to measuring binding affinity, the Fluidity One‑W reports the absolute size of free PD‑1 and PD‑L1 as well as the PD‑1/PD‑L1 complex. These data were used to infer the stoichiometry of the protein complex. Webinar – Comprehensive profiling of SARS-CoV-2 antibodies – All antibodies are not created equal WATCH WEBINAR Methods Sample preparation PD-1 (R&D Systems) was reconstituted into PBS at pH 7.4 resulting in a final concentration of 24 µM, diluted into labelling buffer (0.2 M NaHCO3, pH 8.3) and mixed with Alexa Fluor™ 488 NHS ester (Thermo Fisher Scientific) at a dye-to-protein ratio of 3:1. The protein–dye mixture was incubated at 4 °C overnight and purified with a 1 mL Pierce® Desalting Column (Thermo Fisher Scientific) using PBS-T (PBS with 0.05% Tween 20) at pH 7.4 as a buffer. PD-L1 (R&D Systems) was reconstituted into PBS (pH 7.4) at a final concentration of 250 μM. To determine Rh of unbound PD-L1, PD-L1 carrying a C-terminal His tag was mixed with HIS-Lite™ OG488-Tris NTA-Ni complex (Stratech) in PBS-T buffer (pH 7.4) giving final concentrations of 2 μM and 1 μM, respectively. The mixture was incubated for 15 min at 4 °C and diluted to a PD-L1 concentration of 400 nM. Samples were measured on the Fluidity One-W at the medium flow rate in triplicate. Experimental protocol Alexa Fluor™ 488-labeled PD-1 was measured on the Fluidity One-W at 500 nM using the medium flow-rate setting to obtain the size of the unbound protein. To obtain binding curves, 500 nM Alexa Fluor™ 488-labeled PD1 was mixed with unlabeled PD-L1 samples between 0 – 225 μM and equilibrated at 4 °C overnight. Samples were then measured on the Fluidity One-W using the medium flow-rate setting and the KD was automatically determined by the Fluidity One-W utilizing non-linear least squares fitting to Equation 1 (see Appendix). Results The absolute sizes (hydrodynamic radii, Rh) of unbound PD-1, unbound PD-L1 and the PD-1/PD-L1 complex were measured on the Fluidity One-W, allowing the stoichiometry of the interaction to be inferred. As depicted in Figure 1, the experimental Rh of the PD-1/PD-L1 complex is shown to be consistent with a hypothetical Rh derived from Rh values of the unbound binding partners—showing that the interaction has a 1:1 binding stoichiometry. Due to glycosylation, the individual Rh values of both unbound PD-1 and unbound PD-L1 are considerably larger than expected based on their nominal molecular weights (UniProt: Q15116 and Q9NZQ7).2 Figure 2 shows an equilibrium binding curve obtained by the addition of unlabeled PD-L1 to a constant concentration of Alexa Fluor™ 488-labeled PD-1. From this, the KD of the PD-1/PD-L1 interaction was determined to be 4 μM which agrees well with literature data obtained using other biophysical methods (Table 1). Figure 1: Absolute size measurement of PD-1, PD-L1 and complex. Experimentally determined hydrodynamic radii (Rh) of PD-1, PD-L1 and the PD-1/PD-L1 complex. Here, the PD-1/PD-L1 experimental Rh is consistent with the hypothetical Rh of a 1:1 complex derived from absolute size measurements of the individual proteins. Figure 2: Binding curve for unlabeled PD-L1 to 500 nM Alexa Fluor™ 488-labeled PD-1. All measurements were performed in triplicate. KD was determined by non-linear least squares fitting of the data with Equation 1 setting the stoichiometry parameter to n = 1. Table 1: A comparison of binding affinity values for PD-1/PD-L1 from a range of biophysical techniques MethodKD (μM)Reference Fluidity One-W4.0Present work ITC2.23 SPR equilibrium3.94 SPR equilibrium0.75 MST7.26 Conclusion Using the Fluidity One-W, the KD of the PD-1/PD-L1 interaction was accurately determined and agrees with literature values derived from ITC, SPR and MST. Crucially, because the Fluidity One-W performs absolute size measurements in solution, it provides key information on the quality of proteins as well as the stoichiometry of protein complexes. This type of data gives researchers vital additional information about the protein complexes they are studying without the need for detailed structural information. References Lee, H.T., Lee, S.H. and Heo, Y.S. Molecular interactions of antibody drugs targeting PD-1, PD-L1, and CTLA-4 in immuno-oncology. Molecules. 2019. 24, 1190. DOI: 10.3390/molecules24061190 Uniprot Consortium. Uniprot: a worldwide hub of protein knowledge. Nucleic Acids Research. 2019. 47, 506-515. DOI: 10.1093/nar/gky1049 Cheng, X., Veverka, V., Radhakrishnan, A., Waters, L.C., Muskett, F.W., Morgan, S.H., Huo, J., Yu, C., Evans, E.J., Leslie, A.J. and Griffiths, M. Structure and interactions of the human programmed cell death 1 receptor. Journal of Biological Chemistry, 2013, 288, 11771-11785. DOI: 10.1074/jbc.M112.448126 Zhang, X., Schwartz, J.C.D., Guo, X., Bhatia, S., Cao, E., Chen, L., Zhang, Z.Y., Edidin, M.A., Nathenson, S.G. and Almo, S.C. Structural and functional analysis of the costimulatory receptor programmed death-1. Immunity, 2004, 20, 337-347. DOI: 10.1016/S1074-7613(04)00051-2 Latchman, Y., Wood, C.R., Chernova, T., Chaudhary, D., Borde, M., Chernova, I., Iwai, Y., Long, A.J., Brown, J.A., Nunes, R. and Greenfield, E.A. PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nature Immunology, 2001, 2, 261–268.DOI: 10.1038/85330 Magnez, R., Thiroux, B., Taront, S., Segaoula, Z., Quesnel, B. and Thuru, X. PD-1/PD-L1 binding studies using microscale thermophoresis. Scientific Reports, 2017, 7, 17623. DOI: https://doi.org/10.1038/s41598-017-17963-1 Watch our short webinar on Affinity of PD-1/PD-L1 interaction—a comparison between SPR, MST, ITC and MDS: Download application note Fill out the form below to download the "Affinity of PD-1/PD-L1 interaction—a comparison between SPR, MST, ITC and MDS" as a PDF. You can print it, share it with friends and family or just use it as light reading! 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