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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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Peptibodies recognize extracellular S100 family proteins on the surface of MDSC(a) Schematic representation of a strategy for peptibody-based isolation of candidate cell type-specific surface markers. (b) Proteomic analysis from sorted Gr-1 + CD11b + splenic MDSC from EL4-bearing C57BL/6 mice revealing predominant peptides with homology to S100A9. The data are representative of 2 independent experiments. (c) Identification of S100A9 protein in Protein A eluates of Pep-H6-bound, sorted MDSC lysate (without biotinylation) by Western blot. Recombinant mouse S100A9 protein served as a positive control (left panel), and lysates from unbound MDSC were negative controls (right panel). Input lysates were blotted with actin as an internal control. (d) Detection of both S100A9 and S100A8 proteins in Protein A eluates of Pep-H6-bound, sorted MDSC lysate by Western blot. All Western blot data shown are representative of 3 individual experiments. (e) Binding of Pep-H6 and Pep-G3 peptibodies with CD11b+Gr-1+ gated splenic MDSC form EL4-bearing, S100A9-deficient C57BL/6 mice (n = 3). The data are representative of 2 independent experiments. (f) Frequencies of CD11b+Gr-1+ splenic MDSC from EL4-bearing, S100A9-deficient C57BL/6 mice (n = 3) after peptibody treatment as in (3a).
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Figure 4: Peptibodies recognize extracellular S100 family proteins on the surface of MDSC(a) Schematic representation of a strategy for peptibody-based isolation of candidate cell type-specific surface markers. (b) Proteomic analysis from sorted Gr-1 + CD11b + splenic MDSC from EL4-bearing C57BL/6 mice revealing predominant peptides with homology to S100A9. The data are representative of 2 independent experiments. (c) Identification of S100A9 protein in Protein A eluates of Pep-H6-bound, sorted MDSC lysate (without biotinylation) by Western blot. Recombinant mouse S100A9 protein served as a positive control (left panel), and lysates from unbound MDSC were negative controls (right panel). Input lysates were blotted with actin as an internal control. (d) Detection of both S100A9 and S100A8 proteins in Protein A eluates of Pep-H6-bound, sorted MDSC lysate by Western blot. All Western blot data shown are representative of 3 individual experiments. (e) Binding of Pep-H6 and Pep-G3 peptibodies with CD11b+Gr-1+ gated splenic MDSC form EL4-bearing, S100A9-deficient C57BL/6 mice (n = 3). The data are representative of 2 independent experiments. (f) Frequencies of CD11b+Gr-1+ splenic MDSC from EL4-bearing, S100A9-deficient C57BL/6 mice (n = 3) after peptibody treatment as in (3a).

Mentions: To explore the target of our peptibody, we developed a strategy to identify peptibody-bound proteins on the surface of MDSC (Fig. 4a). Specifically, surface proteins on sorted Gr-1+CD11b+ MDSC were biotinylated and subsequently captured by monomeric avidin after cell lysis. Then, we used Pep-H6 that was immobilized on Protein A to immunoprecipitate the surface protein of interest. Proteomic analysis of eluted proteins suggested that S100A9 was the source protein, with sequence coverage above 40% (Fig. 4b). Consistent with these results, immunoprecipitation studies (without prior biotinylation) showed that eluted proteins from Pep-H6-bound, sorted MDSC co-migrated with recombinant S100A9 (6 × His tagged) and was recognized by S100A9 antibodies (Fig. 4c), but no signal was detected when using lysates from MDSC without peptibody, excluding contamination by intracellular S100A9 (Fig. 4c). Furthermore, such direct immunoprecipitation of MDSC cell surface proteins with Pep-H6 revealed a protein band that more closely co-migrated with native S100A9 identified by immunoblotting of MDSC total cell lysates (Fig. 4d). Immunoprecipitation experiments suggested that the peptibody also recognizes S100A8, consistent with the formation of S100A9 and S100A8 dimers (Fig. 4d). Immunoprecipitation with Pep-G3 also suggested binding to S100A9 and S100A8 proteins (Supplementary Fig. 5). These results suggested either cross-reactivity with S100A8 or perhaps recognition of a combinatorial determinant on the S100A9 and S100A8 complex.


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)

Peptibodies recognize extracellular S100 family proteins on the surface of MDSC(a) Schematic representation of a strategy for peptibody-based isolation of candidate cell type-specific surface markers. (b) Proteomic analysis from sorted Gr-1 + CD11b + splenic MDSC from EL4-bearing C57BL/6 mice revealing predominant peptides with homology to S100A9. The data are representative of 2 independent experiments. (c) Identification of S100A9 protein in Protein A eluates of Pep-H6-bound, sorted MDSC lysate (without biotinylation) by Western blot. Recombinant mouse S100A9 protein served as a positive control (left panel), and lysates from unbound MDSC were negative controls (right panel). Input lysates were blotted with actin as an internal control. (d) Detection of both S100A9 and S100A8 proteins in Protein A eluates of Pep-H6-bound, sorted MDSC lysate by Western blot. All Western blot data shown are representative of 3 individual experiments. (e) Binding of Pep-H6 and Pep-G3 peptibodies with CD11b+Gr-1+ gated splenic MDSC form EL4-bearing, S100A9-deficient C57BL/6 mice (n = 3). The data are representative of 2 independent experiments. (f) Frequencies of CD11b+Gr-1+ splenic MDSC from EL4-bearing, S100A9-deficient C57BL/6 mice (n = 3) after peptibody treatment as in (3a).
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Figure 4: Peptibodies recognize extracellular S100 family proteins on the surface of MDSC(a) Schematic representation of a strategy for peptibody-based isolation of candidate cell type-specific surface markers. (b) Proteomic analysis from sorted Gr-1 + CD11b + splenic MDSC from EL4-bearing C57BL/6 mice revealing predominant peptides with homology to S100A9. The data are representative of 2 independent experiments. (c) Identification of S100A9 protein in Protein A eluates of Pep-H6-bound, sorted MDSC lysate (without biotinylation) by Western blot. Recombinant mouse S100A9 protein served as a positive control (left panel), and lysates from unbound MDSC were negative controls (right panel). Input lysates were blotted with actin as an internal control. (d) Detection of both S100A9 and S100A8 proteins in Protein A eluates of Pep-H6-bound, sorted MDSC lysate by Western blot. All Western blot data shown are representative of 3 individual experiments. (e) Binding of Pep-H6 and Pep-G3 peptibodies with CD11b+Gr-1+ gated splenic MDSC form EL4-bearing, S100A9-deficient C57BL/6 mice (n = 3). The data are representative of 2 independent experiments. (f) Frequencies of CD11b+Gr-1+ splenic MDSC from EL4-bearing, S100A9-deficient C57BL/6 mice (n = 3) after peptibody treatment as in (3a).
Mentions: To explore the target of our peptibody, we developed a strategy to identify peptibody-bound proteins on the surface of MDSC (Fig. 4a). Specifically, surface proteins on sorted Gr-1+CD11b+ MDSC were biotinylated and subsequently captured by monomeric avidin after cell lysis. Then, we used Pep-H6 that was immobilized on Protein A to immunoprecipitate the surface protein of interest. Proteomic analysis of eluted proteins suggested that S100A9 was the source protein, with sequence coverage above 40% (Fig. 4b). Consistent with these results, immunoprecipitation studies (without prior biotinylation) showed that eluted proteins from Pep-H6-bound, sorted MDSC co-migrated with recombinant S100A9 (6 × His tagged) and was recognized by S100A9 antibodies (Fig. 4c), but no signal was detected when using lysates from MDSC without peptibody, excluding contamination by intracellular S100A9 (Fig. 4c). Furthermore, such direct immunoprecipitation of MDSC cell surface proteins with Pep-H6 revealed a protein band that more closely co-migrated with native S100A9 identified by immunoblotting of MDSC total cell lysates (Fig. 4d). Immunoprecipitation experiments suggested that the peptibody also recognizes S100A8, consistent with the formation of S100A9 and S100A8 dimers (Fig. 4d). Immunoprecipitation with Pep-G3 also suggested binding to S100A9 and S100A8 proteins (Supplementary Fig. 5). These results suggested either cross-reactivity with S100A8 or perhaps recognition of a combinatorial determinant on the S100A9 and S100A8 complex.

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