Supplementary Data from Peptide Blocking of PD-1/PD-L1 Interaction for Cancer Immunotherapy

Li, Chunlin; Zhang, Nengpan; Zhou, Jundong; Ding, Chen; Jin, Yaqing; Cui, Xueyuan; Pu, Kefeng; Zhu, Yimin

doi:10.1158/2326-6066.22540007.v1

Supplementary Data from Peptide Blocking of PD-1/PD-L1 Interaction for Cancer Immunotherapy

journal contribution

posted on 2023-04-03, 23:28 authored by Chunlin Li, Nengpan Zhang, Jundong Zhou, Chen Ding, Yaqing Jin, Xueyuan Cui, Kefeng Pu, Yimin Zhu

Fig. S1. The binding specificity of consensus sequence CWCWR to MDA-MB-435 and MDA-MB-231 cells. Fig. S2. A, The schematic illustration of construction the focused library. B, Ten clones were randomly selected for sequencing to examine the diversity of the focused library. Fig. S3. Binding properties of peptides displayed on the bacteria surface. Fig. S4. The binding properties of TPP-1 and SPP-1 to PD-L1 were determined by the ELISA (n=3). Fig. S5. A, The PD-L1 expression of CHO-K1/PD-L1, MDA-MB-231 and MDA-MB-435 cell lines were determined by anti-PD-L1 antibody and flow cytometer. B, The binding properties of TPP-1 to MDA-MB-231 and MDA-MB-435 cell lines. Fig. S6. The bright field (A) and fluorescence microscopic image (B) of TPP-1 to CHO-K1. The bright field (C) and fluorescence microscopic image (D) of TPP-1 to CHO-K1/PD-L1. Fig. S7. A, The ED50 of PD-L1 to block the activated T cells (n=3). B, The TPP-1 peptide or PD-L1 plus TPP-1 peptide did not activate the T cells without the CD3 antibody. C, TPP-1 could effectively restore T cells proliferation which were inhibited by PD-L1 evaluated by T cell activation assay (n=3). Data were presented as mean {plus minus} SEM, *P<0.05, **P<0.01, unpaired t-test. Fig. S9. A, The bioluminescence signals of H460-Luc cells were determined by Cytation 3. B, The expression of PD-L1 on H460 was detected with flow cytometry. Fig. S8. A, The PD-L1 expression in matured DCs, the dotted line represented the isotype mAb and filled line represented the anti-PD-L1 mAb. B, The proliferation of T cells in MLR assay (n=3). Fig. S9. A, The bioluminescence signals of H460-Luc cells were determined by Cytation 3. B, The expression of PD-L1 on H460 was detected with flow cytometry. Fig. S10. A, In vivo fluorescence images of mice taken at different time points post s.c. injection of FITC labeled TPP-1. B, The statistical result of the relative fluorescence unit. Fig. S11. A, The location of CWCWR (yellow) in TPP-1 peptide. B, The possible binding location of TPP-1 (blue) peptide to PD-L1 (PDB ID of PD-L1: 3BIS). C, The crystal structures of PD-1 (light sea green) and PD-L1 (dark khaki) complex (PDB ID of the complex: 3BIK).

Funding

National Natural Science Foundation of China

Natural Science Foundation of Jiangsu Province

Science and Technology Bureau of Suzhou

History

ARTICLE ABSTRACT

Immunotherapy has become a promising alternative therapeutic approach for cancer patients. Interruption of immune checkpoints, such as CTLA-4 and PD-1, has been verified to be a successful means for cancer therapy in clinical trials. mAb targeting PD-L1 has been approved to treat urothelial carcinoma, non–small cell lung cancer, or Merkel cell carcinoma by the FDA. However, the high cost of the antibody can limit its application. In our study, targeting PD-L1 peptide (TPP-1), which specifically binds to PD-L1 with high affinity, was identified through bacterial surface display methods. Using a T-cell activation assay and mixed lymphocyte reaction, TPP-1 was verified to interfere with the interaction of PD-1/PD-L1. To examine the inhibitory effect of TPP-1 on tumor growth in vivo, a xenograft mouse model using H460 cells was established. The growth rate of tumor masses in TPP-1 or PD-L1 antibody–treated mice was 56% or 71% lower than that in control peptide–treated mice, respectively, indicating that TPP-1 inhibits, or at least retards, tumor growth. IHC of the tumors showed that IFNγ and granzyme B expression increased in the TPP-1 or PD-L1 antibody–treated groups, indicating that TPP-1 attenuates the inhibitory effect of PD-L1 on T cells and that T cells may get reactivated. On the basis of our data, TPP-1 peptide could work as an alternative to antibodies for tumor immunotherapy. Cancer Immunol Res; 6(2); 178–88. ©2017 AACR.