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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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Generation and characterization of MDSC-specific peptibodies(a) Schematic representation of peptibody construction. (b) Characterization of recombinant peptibodies (Pep-H6, Pep-G3 and a control Pep-irrel) that were purified using Protein A chromatography. The identity of peptibodies was verified by Western blot using HRP-conjugated anti-mouse IgG (left) or anti-His tag antibodies (right). (c) Binding of FITC-conjugated Pep-H6 or Pep-G3 on CD11b+Ly6G+Ly6Cint/low gated granulocytic MDSC and CD11b+Ly6G−Ly6Chigh gated monocytic MDSC in splenocytes from EL4-bearing C57BL/6 mice (n = 5). (d) Binding of the peptibodies with granulocytic (upper panel) and monocytic (lower panel) MDSC subsets in splenocytes from different species of mice (C57BL/6 and Balb/c) challenged with various tumors (n = 3 for each tumor type). (e) Characterization of binding specificity of the peptibodies on Ly6G + CD11c − gated granulocytic MDSC (gMDSC) versus CD11c+Ly6G− gated DC in splenocytes pooled from EL4-bearing C57BL/6 mice (n = 5). (f) Identification of peptibody binding on CD11b+Gr-1+ immature myeloid cells in the bone marrow from EL4-bearing C57BL/6 mice (n = 5). (g) Co-staining of APC-labeled Pep-G3 and FITC-labeled Pep-H6 with CD11b+Gr-1+ MDSC in splenocytes pooled from EL4-bearing C57BL/6 mice (n = 5). The data represent 3 independent experiments.
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Figure 2: Generation and characterization of MDSC-specific peptibodies(a) Schematic representation of peptibody construction. (b) Characterization of recombinant peptibodies (Pep-H6, Pep-G3 and a control Pep-irrel) that were purified using Protein A chromatography. The identity of peptibodies was verified by Western blot using HRP-conjugated anti-mouse IgG (left) or anti-His tag antibodies (right). (c) Binding of FITC-conjugated Pep-H6 or Pep-G3 on CD11b+Ly6G+Ly6Cint/low gated granulocytic MDSC and CD11b+Ly6G−Ly6Chigh gated monocytic MDSC in splenocytes from EL4-bearing C57BL/6 mice (n = 5). (d) Binding of the peptibodies with granulocytic (upper panel) and monocytic (lower panel) MDSC subsets in splenocytes from different species of mice (C57BL/6 and Balb/c) challenged with various tumors (n = 3 for each tumor type). (e) Characterization of binding specificity of the peptibodies on Ly6G + CD11c − gated granulocytic MDSC (gMDSC) versus CD11c+Ly6G− gated DC in splenocytes pooled from EL4-bearing C57BL/6 mice (n = 5). (f) Identification of peptibody binding on CD11b+Gr-1+ immature myeloid cells in the bone marrow from EL4-bearing C57BL/6 mice (n = 5). (g) Co-staining of APC-labeled Pep-G3 and FITC-labeled Pep-H6 with CD11b+Gr-1+ MDSC in splenocytes pooled from EL4-bearing C57BL/6 mice (n = 5). The data represent 3 independent experiments.

Mentions: We genetically fused sequences encoding H6 and G3 peptides with the Fc portion of mouse IgG2b to generate peptibodies (Pep-H6 and -G3, respectively) (Fig. 2a). Control peptibodies (Pep-irrel) were also made using non-specific sequences. Then we produced recombinant peptibodies from 293T mammalian cells, followed by purification of the peptibodies by Protein A chromatography and characterization by Western blot using anti-mouse IgG and anti-His tag antibodies (Fig. 2b). These peptibodies were conjugated with fluorescein isothiocyanate (FITC) and analyzed for binding specificity on CD11b+Ly6G−Ly6Chigh monocytic MDSC and CD11b+Ly6G+Ly6Cint/low granulocytic MDSC from EL4-bearing mice. Gating on these well-defined subpopulations, we conclusively showed that Pep-H6 and Pep-G3 bind both monocytic and granulocytic MDSC (Fig. 2c), (Supplementary Fig.1a). However, the expression of the Pep-G3 target seems to be lower in granulocytic MDSC than in monocytic MDSC.


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)

Generation and characterization of MDSC-specific peptibodies(a) Schematic representation of peptibody construction. (b) Characterization of recombinant peptibodies (Pep-H6, Pep-G3 and a control Pep-irrel) that were purified using Protein A chromatography. The identity of peptibodies was verified by Western blot using HRP-conjugated anti-mouse IgG (left) or anti-His tag antibodies (right). (c) Binding of FITC-conjugated Pep-H6 or Pep-G3 on CD11b+Ly6G+Ly6Cint/low gated granulocytic MDSC and CD11b+Ly6G−Ly6Chigh gated monocytic MDSC in splenocytes from EL4-bearing C57BL/6 mice (n = 5). (d) Binding of the peptibodies with granulocytic (upper panel) and monocytic (lower panel) MDSC subsets in splenocytes from different species of mice (C57BL/6 and Balb/c) challenged with various tumors (n = 3 for each tumor type). (e) Characterization of binding specificity of the peptibodies on Ly6G + CD11c − gated granulocytic MDSC (gMDSC) versus CD11c+Ly6G− gated DC in splenocytes pooled from EL4-bearing C57BL/6 mice (n = 5). (f) Identification of peptibody binding on CD11b+Gr-1+ immature myeloid cells in the bone marrow from EL4-bearing C57BL/6 mice (n = 5). (g) Co-staining of APC-labeled Pep-G3 and FITC-labeled Pep-H6 with CD11b+Gr-1+ MDSC in splenocytes pooled from EL4-bearing C57BL/6 mice (n = 5). The data represent 3 independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4048321&req=5

Figure 2: Generation and characterization of MDSC-specific peptibodies(a) Schematic representation of peptibody construction. (b) Characterization of recombinant peptibodies (Pep-H6, Pep-G3 and a control Pep-irrel) that were purified using Protein A chromatography. The identity of peptibodies was verified by Western blot using HRP-conjugated anti-mouse IgG (left) or anti-His tag antibodies (right). (c) Binding of FITC-conjugated Pep-H6 or Pep-G3 on CD11b+Ly6G+Ly6Cint/low gated granulocytic MDSC and CD11b+Ly6G−Ly6Chigh gated monocytic MDSC in splenocytes from EL4-bearing C57BL/6 mice (n = 5). (d) Binding of the peptibodies with granulocytic (upper panel) and monocytic (lower panel) MDSC subsets in splenocytes from different species of mice (C57BL/6 and Balb/c) challenged with various tumors (n = 3 for each tumor type). (e) Characterization of binding specificity of the peptibodies on Ly6G + CD11c − gated granulocytic MDSC (gMDSC) versus CD11c+Ly6G− gated DC in splenocytes pooled from EL4-bearing C57BL/6 mice (n = 5). (f) Identification of peptibody binding on CD11b+Gr-1+ immature myeloid cells in the bone marrow from EL4-bearing C57BL/6 mice (n = 5). (g) Co-staining of APC-labeled Pep-G3 and FITC-labeled Pep-H6 with CD11b+Gr-1+ MDSC in splenocytes pooled from EL4-bearing C57BL/6 mice (n = 5). The data represent 3 independent experiments.
Mentions: We genetically fused sequences encoding H6 and G3 peptides with the Fc portion of mouse IgG2b to generate peptibodies (Pep-H6 and -G3, respectively) (Fig. 2a). Control peptibodies (Pep-irrel) were also made using non-specific sequences. Then we produced recombinant peptibodies from 293T mammalian cells, followed by purification of the peptibodies by Protein A chromatography and characterization by Western blot using anti-mouse IgG and anti-His tag antibodies (Fig. 2b). These peptibodies were conjugated with fluorescein isothiocyanate (FITC) and analyzed for binding specificity on CD11b+Ly6G−Ly6Chigh monocytic MDSC and CD11b+Ly6G+Ly6Cint/low granulocytic MDSC from EL4-bearing mice. Gating on these well-defined subpopulations, we conclusively showed that Pep-H6 and Pep-G3 bind both monocytic and granulocytic MDSC (Fig. 2c), (Supplementary Fig.1a). However, the expression of the Pep-G3 target seems to be lower in granulocytic MDSC than in monocytic MDSC.

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