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[Fluidity One-W Serum] Fluidic Analytics 社의 MDS 와 SPR, MST, ITC 비교: PD-1/PD-L1 간의 친화도(Affinity)를 중심으로
인성크로마텍(주)
Date : 2021.08.09
분류 : Analytical Products > Fluidic Analytics

 

Affinity of PD-1/PD-L1 interaction—a comparison between SPR, MST, ITC and MDS

 

Published on 13 Nov 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. 

 

 

 

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.

 

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. 

 

 

 

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). 

 

 

42dcabaa062f7f8f28be6efc23019082.png


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.

 

 

189c6e80e886b50577e2a01006af2ce5.png


Figure 2: Binding curve for unlabelled PD-L1 to 500 nM Alexa Fluor™ 488-labeled PD-1. 

All measurements were performed intriplicate. 

KD was determined by non-linear least squares fitting of the data with 

Equation 1 setting the stoichiometry parameter to n = 1.

 

 


eb839497c0b139eb304c2a736b5fdddf.png

 

 Table 1: A comparison of binding affinity values for PD-1/PD-L1 from a range of biophysical techniques

 

 

 

 

 

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

 

1.  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. 

 

2.  Uniprot Consortium. Uniprot: a worldwide hub of protein knowledge. Nucleic Acids Research. 2019. 47, 506-515.

 

3.  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. 

 

4.  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. 

 

5.  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. 

 

6.  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.