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Generation of a new therapeutic peptide that depletes myeloid-derived suppressor cells in tumor-bearing mice.

Qin H, Lerman B, Sakamaki I, Wei G, Cha SC, Rao SS, Qian J, Hailemichael Y, Nurieva R, Dwyer KC, Roth J, Yi Q, Overwijk WW, Kwak LW - Nat. Med. (2014)

Bottom Line: Peptibody treatment was associated with inhibition of tumor growth in vivo, which was superior to that achieved with Gr-1-specific antibody.Immunoprecipitation of MDSC membrane proteins identified S100 family proteins as candidate targets.Our strategy may be useful to identify new diagnostic and therapeutic surface targets on rare cell subtypes, including human MDSCs.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. [2] Center for Cancer Immunology Research, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. [3].

ABSTRACT
Immune evasion is an emerging hallmark of cancer progression. However, functional studies to understand the role of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment are limited by the lack of available specific cell surface markers. We adapted a competitive peptide phage display platform to identify candidate peptides binding MDSCs specifically and generated peptide-Fc fusion proteins (peptibodies). In multiple tumor models, intravenous peptibody injection completely depleted blood, splenic and intratumoral MDSCs in tumor-bearing mice without affecting proinflammatory immune cell types, such as dendritic cells. Whereas control Gr-1-specific antibody primarily depleted granulocytic MDSCs, peptibodies depleted both granulocytic and monocytic MDSC subsets. Peptibody treatment was associated with inhibition of tumor growth in vivo, which was superior to that achieved with Gr-1-specific antibody. Immunoprecipitation of MDSC membrane proteins identified S100 family proteins as candidate targets. Our strategy may be useful to identify new diagnostic and therapeutic surface targets on rare cell subtypes, including human MDSCs.

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Identification and characterization of MDSC-binding peptides(a) Identification of Gr-1+CD11b+ MDSC in spleens of C57BL/6 mice (n = 5) challenged subcutaneously with EL4 mouse lymphoma cells for 3 weeks. Double positive cells contain 2 distinct populations including Gr-1highCD11b+ granulocytic (P7) and Gr-1intCD11b+ monocytic (P10) MDSC subsets. (b–c) Biopanning with Ph.D.-12 peptide phage display library on Gr-1 and CD11b labeled splenocytes showed enriched phage eluted from sorted MDSC subsets. Biopanning enrichment was expressed in either “Number of plaques / 106 cells” or phage “Output / Input ratio” (× 10–8). (d) Binding of synthetic FITC-conjugated G3 and H6 peptides on Gr-1+CD11b+ gated MDSC from EL4-bearing C57BL/6 mice (n = 4), compared with Gr-1−CD11b− gated non-MDSC splenocytes. A non-specific peptide (irrel peptide) was used as a negative control to exclude non-specific binding. The data are representative of 3 identical experiments.
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Figure 1: Identification and characterization of MDSC-binding peptides(a) Identification of Gr-1+CD11b+ MDSC in spleens of C57BL/6 mice (n = 5) challenged subcutaneously with EL4 mouse lymphoma cells for 3 weeks. Double positive cells contain 2 distinct populations including Gr-1highCD11b+ granulocytic (P7) and Gr-1intCD11b+ monocytic (P10) MDSC subsets. (b–c) Biopanning with Ph.D.-12 peptide phage display library on Gr-1 and CD11b labeled splenocytes showed enriched phage eluted from sorted MDSC subsets. Biopanning enrichment was expressed in either “Number of plaques / 106 cells” or phage “Output / Input ratio” (× 10–8). (d) Binding of synthetic FITC-conjugated G3 and H6 peptides on Gr-1+CD11b+ gated MDSC from EL4-bearing C57BL/6 mice (n = 4), compared with Gr-1−CD11b− gated non-MDSC splenocytes. A non-specific peptide (irrel peptide) was used as a negative control to exclude non-specific binding. The data are representative of 3 identical experiments.

Mentions: MDSC frequency is low in naïve C57BL/6 mice; however, after transplantation of syngeneic EL4 thymomas MDSC are increased accounting for approximately 10% of total splenocytes5. Splenic MDSC from EL4-bearing mice consist of two distinct subpopulations characterized by Gr-1highCD11b+ granulocytic (P7) and Gr-1intCD11b+ monocytic (P10) staining (Fig. 1a). With the goal of selecting specifically binding peptide ligands by phage display, we separated Gr-1 and CD11b labeled MDSC from non-MDSC by cell sorting after incubation with a Ph.D.-12 peptide phage library. Phage eluted from granulocytic or monocytic MDSC were then expanded by 3 rounds of competitive biopanning. We analyzed enrichment by the number of phage eluted from 106 MDSC (Fig. 1b) and by phage output, normalized to the initial input of 2 × 1010 phage (Fig. 1c).


Generation of a new therapeutic peptide that depletes myeloid-derived suppressor cells in tumor-bearing mice.

Qin H, Lerman B, Sakamaki I, Wei G, Cha SC, Rao SS, Qian J, Hailemichael Y, Nurieva R, Dwyer KC, Roth J, Yi Q, Overwijk WW, Kwak LW - Nat. Med. (2014)

Identification and characterization of MDSC-binding peptides(a) Identification of Gr-1+CD11b+ MDSC in spleens of C57BL/6 mice (n = 5) challenged subcutaneously with EL4 mouse lymphoma cells for 3 weeks. Double positive cells contain 2 distinct populations including Gr-1highCD11b+ granulocytic (P7) and Gr-1intCD11b+ monocytic (P10) MDSC subsets. (b–c) Biopanning with Ph.D.-12 peptide phage display library on Gr-1 and CD11b labeled splenocytes showed enriched phage eluted from sorted MDSC subsets. Biopanning enrichment was expressed in either “Number of plaques / 106 cells” or phage “Output / Input ratio” (× 10–8). (d) Binding of synthetic FITC-conjugated G3 and H6 peptides on Gr-1+CD11b+ gated MDSC from EL4-bearing C57BL/6 mice (n = 4), compared with Gr-1−CD11b− gated non-MDSC splenocytes. A non-specific peptide (irrel peptide) was used as a negative control to exclude non-specific binding. The data are representative of 3 identical experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4048321&req=5

Figure 1: Identification and characterization of MDSC-binding peptides(a) Identification of Gr-1+CD11b+ MDSC in spleens of C57BL/6 mice (n = 5) challenged subcutaneously with EL4 mouse lymphoma cells for 3 weeks. Double positive cells contain 2 distinct populations including Gr-1highCD11b+ granulocytic (P7) and Gr-1intCD11b+ monocytic (P10) MDSC subsets. (b–c) Biopanning with Ph.D.-12 peptide phage display library on Gr-1 and CD11b labeled splenocytes showed enriched phage eluted from sorted MDSC subsets. Biopanning enrichment was expressed in either “Number of plaques / 106 cells” or phage “Output / Input ratio” (× 10–8). (d) Binding of synthetic FITC-conjugated G3 and H6 peptides on Gr-1+CD11b+ gated MDSC from EL4-bearing C57BL/6 mice (n = 4), compared with Gr-1−CD11b− gated non-MDSC splenocytes. A non-specific peptide (irrel peptide) was used as a negative control to exclude non-specific binding. The data are representative of 3 identical experiments.
Mentions: MDSC frequency is low in naïve C57BL/6 mice; however, after transplantation of syngeneic EL4 thymomas MDSC are increased accounting for approximately 10% of total splenocytes5. Splenic MDSC from EL4-bearing mice consist of two distinct subpopulations characterized by Gr-1highCD11b+ granulocytic (P7) and Gr-1intCD11b+ monocytic (P10) staining (Fig. 1a). With the goal of selecting specifically binding peptide ligands by phage display, we separated Gr-1 and CD11b labeled MDSC from non-MDSC by cell sorting after incubation with a Ph.D.-12 peptide phage library. Phage eluted from granulocytic or monocytic MDSC were then expanded by 3 rounds of competitive biopanning. We analyzed enrichment by the number of phage eluted from 106 MDSC (Fig. 1b) and by phage output, normalized to the initial input of 2 × 1010 phage (Fig. 1c).

Bottom Line: Peptibody treatment was associated with inhibition of tumor growth in vivo, which was superior to that achieved with Gr-1-specific antibody.Immunoprecipitation of MDSC membrane proteins identified S100 family proteins as candidate targets.Our strategy may be useful to identify new diagnostic and therapeutic surface targets on rare cell subtypes, including human MDSCs.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. [2] Center for Cancer Immunology Research, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. [3].

ABSTRACT
Immune evasion is an emerging hallmark of cancer progression. However, functional studies to understand the role of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment are limited by the lack of available specific cell surface markers. We adapted a competitive peptide phage display platform to identify candidate peptides binding MDSCs specifically and generated peptide-Fc fusion proteins (peptibodies). In multiple tumor models, intravenous peptibody injection completely depleted blood, splenic and intratumoral MDSCs in tumor-bearing mice without affecting proinflammatory immune cell types, such as dendritic cells. Whereas control Gr-1-specific antibody primarily depleted granulocytic MDSCs, peptibodies depleted both granulocytic and monocytic MDSC subsets. Peptibody treatment was associated with inhibition of tumor growth in vivo, which was superior to that achieved with Gr-1-specific antibody. Immunoprecipitation of MDSC membrane proteins identified S100 family proteins as candidate targets. Our strategy may be useful to identify new diagnostic and therapeutic surface targets on rare cell subtypes, including human MDSCs.

Show MeSH
Related in: MedlinePlus