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WO2015048748A1 - Peptides spécifiques dirigés contre les cellules myéloïdes suppressives (mdsc), à usage diagnostique et thérapeutique - Google Patents

Peptides spécifiques dirigés contre les cellules myéloïdes suppressives (mdsc), à usage diagnostique et thérapeutique Download PDF

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Publication number
WO2015048748A1
WO2015048748A1 PCT/US2014/058318 US2014058318W WO2015048748A1 WO 2015048748 A1 WO2015048748 A1 WO 2015048748A1 US 2014058318 W US2014058318 W US 2014058318W WO 2015048748 A1 WO2015048748 A1 WO 2015048748A1
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Prior art keywords
peptibody
mdsc
cancer
cell
peptide
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Inventor
Larry W. Kwak
Hong QIN
Soungchul CHA
Beatricia LERMAN
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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    • G01N33/57585—
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates generally to the fields of immunology and cancer biology. More particularly, it concerns peptides and peptibodies for use in depleting myeloid-derived suppressor cells in a subject. 2. Description of Related Art
  • MDSC are potent inhibitors of T-cells in mice (Bronte and Zanovello, 2005; Rodriguez et al, 2002; Ochoa et al, 2007; Ugel et al, 2009), but more specific surface markers that would allow isolation of viable cells would facilitate additional studies to precisely understand tumor-MDSC interactions in the microenvironment.
  • identification of MDSC has relied on a combination of cell surface molecules, none of which is MDSC specific. Lack of MDSC- specific markers makes it difficult to deplete those immune suppressive cells that contribute to the immune escape mechanism of tumor cells. Therefore, there is a need for MDSC- specific peptides and peptibodies for identifying and depleting of MDSC. New specific markers are also needed for targeting MDSC in vivo to test the hypothesis that MDSC inhibition enhances antitumor immunity (Marigo et ah, 2008; Ugel et ah, 2009).
  • MDSC myeloid- derived suppressor cells
  • a peptibody that comprises a peptide that specifically binds to a myeloid-derived suppressor cell (MDSC), said peptide being fused to an Fc portion of an IgG antibody.
  • the Fc may be from an IgGl, IgG2, or IgG3 antibody.
  • the Fc portion is derived from a human or a mouse IgG antibody.
  • the peptibody may be conjugated to an imaging agent (e.g., fluorochrome) or a radioligand.
  • the MDSC may be a human MDSC and the peptide may specifically bind to said human MDSC.
  • the MDSC may be a mouse MDSC.
  • the peptide that specifically binds to a mouse MDSC may have a sequence according to either SEQ ID NOs: 1 or 2.
  • this peptide may be comprised in a peptibody having a sequence that is at least 90%, 95%, 98%, or 99% identical to the sequence of either SEQ ID NOs: 3 or 4.
  • the peptibody may have a sequence that is the sequence of SEQ ID NOs: 3 or 4.
  • nucleic acid comprising a nucleotide sequence that encodes a peptibody of the present embodiments.
  • said nucleic acid may be comprised within an expression vector.
  • said expression vector may be comprised within a host cell (e.g., bacterial cell, a fungal cell, an insect cell, or a mammalian cell).
  • a method of producing a peptibody of the present embodiments comprising the peptibody in a host cell (e.g., bacterial cell, a fungal cell, an insect cell, or a mammalian cell) and purifying the polypeptide therefrom.
  • a pharmaceutical formulation comprising a peptibody of the present embodiments in a pharmaceutically acceptable carrier.
  • a method of depleting MDSC in a subject comprising administering to the subject a formulation of the present embodiments, or an agent that specifically binds to a cell surface SlOO protein on a MDSC.
  • the method is a method of inhibiting tumor growth.
  • the method is a method of treating cancer.
  • the cancer may be a breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, adrenal cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, blood cancer, or skin cancer.
  • the cancer may be metastatic, recurrent, or multi-drug resistant.
  • the subject may be a human patient.
  • the subject may be a non-human mammal.
  • the formulation may be administered systemically, intravenously, intradermally, intraperitoneally, intramuscularly, subcutaneously, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, or via a lavage.
  • the method may further comprise administering at least a second therapy to the subject, wherein the second therapy is an anticancer therapy (e.g., a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy or cytokine therapy).
  • an anticancer therapy e.g., a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy or cytokine therapy.
  • the formulation may be administered at least a second time. In one aspect, the formulation may be administered over a period of 1 week to 6 months.
  • a method is provided for identifying a MDSC in a patient sample. The method comprises (a) obtaining a sample from a subject; (b) contacting the sample with an agent that specifically binds to a cell surface SlOO protein on a MDSC; and (c) detecting binding of the agent to a cell in the sample, wherein said binding identifies the cell as a MDSC.
  • an agent that specifically binds to a cell surface SI 00 protein on a MDSC may be a peptide.
  • the peptide may have a sequence according to either SEQ ID NOs: 1 or 2.
  • the peptide may be conjugated to an imaging agent or radioligand.
  • detecting binding may comprise detecting binding of the imaging agent or radioligand to the cell.
  • the peptide may be comprised within a peptibody of the embodiments.
  • an agent that specifically binds to a cell surface S100 protein on a MDSC may be an antibody, such as IgG, IgM, IgA, IgD, IgE, or a polypeptide comprising an antibody CDR domain that retains S lOO-binding activity.
  • the antibody may be a chimeric antibody, an affinity matured antibody, a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, or an antigen- binding antibody fragment or a natural or synthetic ligand.
  • antibody fragments suitable for the present embodiments include, without limitation: (i) the Fab fragment, consisting of VL, VH, CL, and CHI domains; (ii) the "Fd” fragment consisting of the VH and CHI domains; (iii) the "Fv” fragment consisting of the VL and VH domains of a single antibody; (iv) the "dAb” fragment, which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments; (vii) single chain Fv molecules ("scFv”), wherein a VH domain and a VL domain are linked by a peptide linker that allows the two domains to associate to form a binding domain; (viii) bi-specific single chain Fv dimers; and (ix) diabodies, multivalent or multispecific fragments constructed by gene fusion.
  • Fv, scFv, or diabody molecules may be stabilized by
  • a method for identifying a MDSC in a patient sample. The method comprises (a) obtaining a sample from a subject; (b) contacting the sample with a peptide that specifically binds to a MDSC; and (c) detecting binding of the peptide to a cell in the sample, wherein said binding identifies the cell as a MDSC.
  • the peptide may have a sequence according to either SEQ ID NOs: 1 or 2.
  • the peptide may be conjugated to an imaging agent or radioligand.
  • detecting may comprise detecting binding of the imaging agent or radioligand to the cell.
  • the peptide may be comprised within a peptibody of the embodiments.
  • a method of identifying an MDSC comprising (a) obtaining a sample from a subject; (b) contacting the sample with a peptibody of the embodiments; and (c) detecting binding of the peptibody to a cell in the sample, wherein said binding identifies the cell as a MDSC.
  • the subject is a human patient.
  • the sample is a tumor sample, a spleen sample, or a serum sample.
  • kits comprising a peptide or peptibody of the present embodiments.
  • a composition comprising a peptibody or a nucleic acid encoding a peptibody is provided for use in the treatment of a cancer in a subject.
  • the use of a peptibody or a nucleic acid encoding a peptibody in the manufacture of a medicament for the treatment of a tumor is provided.
  • Said peptibody may be any peptibody of the embodiments.
  • a composition comprising a peptide or peptibody is provided for use in identifying a MDSC in a sample from a subject.
  • the peptide or peptibody may be conjugated to an imaging agent or radioligand.
  • Said peptibody may be any peptibody of the embodiments.
  • Embodiments discussed in the context of methods and/or compositions of the invention may be employed with respect to any other method or composition described herein. Thus, an embodiment pertaining to one method or composition may be applied to other methods and compositions of the invention as well.
  • FIGs. la-d Identification and characterization of MDSC-binding peptides.
  • FIGs. lb-c Biopanning with PLD.-12 peptide phage display library on Gr-1- and CD 1 lb-labeled splenocytes showed enriched phage eluted from sorted MDSC subsets. Biopanning enrichment was expressed in either "Number of plaques / 10 6 cells" or phage "Output / Input ratio" (x 10 ⁇ 8 ). (FIG.
  • FIGs. 2a-g Generation and characterization of MDSC-specific peptibodies.
  • FIG. 2a Schematic representation of peptibody construction.
  • FIG. 2b 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).
  • FIG. 2a Schematic representation of peptibody construction.
  • FIG. 2b 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
  • FIGs. 3a-h Peptibodies specifically depleted tumor-induced MDSC in multiple tumor models and inhibited tumor growth in vivo.
  • Control mice received Gr-1 -depleting mAb (positive control), irrelevant control peptibody (Pep-irrel), or PBS.
  • Plots are shown for individual representative mice (FIG. 3a) and composite results (FIG.
  • FIGs. 4a-f Peptibodies recognize extracellular S100 family proteins on the surface of MDSC.
  • FIG. 4a Schematic representation of a strategy for peptibody-based isolation of candidate cell type-specific surface markers.
  • FIG. 4b Proteomic analysis from sorted Gr-1 CDl lb splenic MDSC from EL4-bearing C57BL/6 mice revealing predominant peptides with homology to S100A9. The data are representative of two independent experiments.
  • FIG. 4c 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).
  • FIG. 4d Detection of both S100A9 and S 100A8 proteins in Protein A eluates of Pep-H6-bound, sorted MDSC lysate by Western blot. All Western blot data shown are representative of three individual experiments.
  • FIGs. 5a-b Peptibodies recognized MDSC in tumor-bearing mice.
  • a non-specific peptibody (Pep-irrel) was used as a negative control.
  • FIG. 5b Increase of MDSC in tumor-challenged mice.
  • Splenocytes from naive mice or mice challenged with various tumors were stained for CDl lb and Gr-1 to identify MDSC. Frequencies of splenic MDSC are shown as mean ⁇ s.e.m. (** P ⁇ 0.01 compared with naive mice by two-tailed student's t-test).
  • FIG. 7 Depletion of intratumoral MDSC in EL4 tumor-challenged mice by peptibody treatment. Groups of five C57BL/6 mice were challenged s.c. with EL4 tumor cells followed by peptibody treatment as in FIG. 3 a. The percentage of Gr-1 + CD1 lb + MDSC from single cell suspensions prepared from tumors harvested on Day 20 is shown as mean ⁇ s.e.m. (* P ⁇ 0.01 compared with PBS by two-tailed Student's Mest).
  • FIG. 8 Long-term administration did not diminish peptibody-induced MDSC depletion.
  • EL4-bearing C57BL/6 mice were treated with peptibodies every other day for two weeks.
  • splenocytes were harvested and stained for Gr-1 + CD1 lb + MDSC, shown as the mean ⁇ s.e.m. of five mice per group.
  • Plots are shown with frequency of splenic MDSC for individual representative mice. Differences between groups were analyzed by two-tailed Student's Mest. The data represent two independent experiments.
  • FIGs. lOa-b Peptibodies did not cause bone marrow toxicity. Frequencies of Grl + CDl lb + splenic MDSC (FIG. 10a) or bone marrow immature myeloid cells (FIG. 10b) from naive C57BL/6 mice treated with peptibodies for three consecutive days.
  • FIGs. lla-c Identification of S100A9 on tumor-infiltrated human MDSC in a lymphoma patient. Staining of human splenic lymphoma samples identified a HLA- DR CD33 + CDl lb + subpopulation that contained both CD 15 + (92%) and CD14 + (8%) cells, suggesting granulocytic and monocytic MDSC, respectively (FIG. 11a). These tumor- infiltrated human MDSC suppressed in vitro activation of HLA-matched, allogeneic antigen- specific T cells (FIG. l ib). The FACS data revealed the presence of S100A9 on the surface of these tumor-infiltrated human MDSC (FIG. 1 lc).
  • the epitope(s) recognized may be S 100 protein-derived, because the protocol used viable cells to screen the peptide library, combinatorial native conformational epitopes comprised of the SlOO-receptor complex would have also been preserved.
  • the cell surface receptor for S100A9 and S100A8 on MDSC is as yet unidentified or different from those on DC, such as RAGE and TLR4.
  • a MDSC-depleting peptibody could cause tumor regression by (1) depleting MDSC by complement-dependent cytotoxicity or ADCC, (2) inducing apoptosis by blocking the binding of S 100 family protein with its receptor(s) on MDSC or on tumor cells (Ichikawa et at, 201 1; Kallberg et ah, 2012) and interfering with SlOO-induced survival signals, and (3) inducing direct cytotoxicity against tumor cells (Ichikawa et al, 201 1; Kallberg et al, 2012).
  • S100A9 was recently reported on human MDSC isolated from patients with colon cancer (Zhao et al, 2012).
  • MDSC-specific binding affinity in vivo MDSC-depletion, and low toxicity make the present peptibodies an attractive adjuvant for cancer immunotherapy, as amelioration of tumor suppressive microenvironment is required for maximizing the therapeutic benefits of cancer immunotherapy (Corzo et al, 2010; Marigo et al, 2010; Fernandez et al, 2011).
  • its high binding affinity can also be utilized to develop therapeutic reagents, such as a cytotoxic radiolabeled peptibody.
  • the MDSC-specific binging peptides may also serve as MDSC-targeting agents for any conjugated cargo, preferably cytotoxic cargo or a reporter.
  • the peptides and peptibodies also provide new diagnostic markers for MDSC.
  • MDSC myeloid derived suppressor cell
  • MSCs myeloid suppressor cells
  • Human MDSCs are characterized by at least the expression of the cell markers CD l ib and CD33. Human MDSCs may also express the markers CD 15 and/or CD14. Murine MDSCs at least express the markers CD l lb and Gr-1.
  • Murine MDSCs may also express CD1 15 and/or F4/80 (see Li et al, 2004).
  • Murine MDSCs may also express CD31, c-kit, vascular endothelial growth factor (VEGF)-receptor, or CD40 (Bronte et al, 2000).
  • MDSCs may further differentiate into several cell types, including macrophages, neutrophils, dendritic cells, Langerhand cells, monocytes or granulocytes. MDSCs may be found naturally in normal adult bone marrow of human and animals or in sites of normal hematopoiesis, such as the spleen in newborn mice.
  • MDSCs Upon distress, MDSCs may be found in the adult spleen. MDSCs can suppress the immunological response of T cells, induce T regulatory cells, and produce T cell tolerance. Morphologically, MDSCs usually have large nuclei and a high nucleus-to- cytoplasm ratio. MDSCs can secrete TFG- ⁇ and IL-10 and produce nitric oxide (NO) in the presence of IFN- ⁇ or activated T cells. MDSCs may form dendriform cells; however, MDSCs are distinct from dendritic cells (DCs) in that DCs are smaller and express CDl lc; MDSCs do not express CD1 lc, or express only a low level of CD1 lc.
  • DCs dendritic cells
  • T cell inactivation by MDSCs in vitro can be mediated through several mechanisms: IFN-y-dependent nitric oxide production (Kusmartsev et ah, 2000); Th2-mediated-IL-4/IL-13-dependent arginase 1 synthesis (Bronte et ah, 2003); loss of CDt ' , signaling in T cells (Rodriguez et ah, 2003); and suppression of the T cell response through reactive oxygen species (Bronte et ah, 2003; Bronte et ah, 2003; Kusmartsev et al, 2004; Schmielau and Finn, 2001).
  • a peptide that binds to at least a portion of an MDSC- specific epitope, fused either directly or indirectly to another molecule, such as an Fc domain of an antibody (e.g., a peptibody), and identifies and/or depletes MDSC and its associated use in treatment and diagnosis of diseases are contemplated.
  • an antibody e.g., a peptibody
  • the MDSC-specific epitope-binding peptibody is humanized.
  • peptibodies may be created that are specific to a MDSC-specific antigen, one or more of its respective epitopes, or conjugates of any of the foregoing, whether such antigens or epitopes are isolated from natural sources or are synthetic derivatives or variants of the natural antigen or epitope.
  • fusion protein refers to a chimeric protein containing proteins or protein fragments operably linked in a non-native way.
  • a peptide or peptibody can be chemically conjugated to, or expressed as, a fusion protein with other proteins.
  • all such fused proteins are included in the definition of peptibodies.
  • the term "peptibody” refers to a molecule comprising peptide(s) fused either directly or indirectly to other molecules such as an Fc domain of an antibody, where the peptide moiety specifically binds to a desired target.
  • the peptide(s) may be fused to either an Fc region or inserted into an Fc-Loop. Fc-Loops are described in U.S. Patent Application Publication No. US2006/0140934, incorporated herein by reference in its entirety.
  • the invention includes such molecules comprising an Fc domain modified to comprise a peptide as an internal sequence of the Fc domain.
  • the Fc internal peptide molecules may include more than one peptide sequence in tandem in a particular internal region, and they may include further peptides in other internal regions. While the putative loop regions are exemplified, insertions in any other non-terminal domains of the Fc are also considered part of this invention.
  • a peptibody does not comprise an antigen-binding antibody Fab domain.
  • Fc refers to a molecule or sequence comprising the sequence of a non-antigen-binding fragment resulting from digestion of a whole antibody, whether in monomeric or multimeric form. Typically, a Fc comprises a CH2 and CH3 domain.
  • the original immunoglobulin source of the Fc is in one aspect of human origin and may be any of the immunoglobulins.
  • a Fc is a monomeric polypeptide that may be linked into dimeric or multimeric forms by covalent association (i.e., disulfide bonds), non-covalent association or a combination of both.
  • the number of intermolecular disulfide bonds between monomeric subunits of Fc molecules ranges from one to four depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgGl, IgG2, IgG3, IgAl , IgGA2).
  • class e.g., IgG, IgA, IgE
  • subclass e.g., IgGl, IgG2, IgG3, IgAl , IgGA2
  • Fc disulfide-bonded dimer resulting from papain digestion of an IgG (Ellison et al, 1982).
  • the term "Fc" as used herein is generic to the monomeric, dimeric, and multimeric forms.
  • Fc variant refers to a molecule or sequence that is modified from a native Fc, but still comprises a binding site for the salvage receptor, FcRn.
  • International PCT Application Publications WO 97/34631 and WO 96/32478 describe exemplary Fc variants, as well as interaction with the salvage receptor, and are hereby incorporated by reference.
  • the term “Fc variant” comprises a molecule or sequence that is humanized from a non-human native Fc.
  • a native Fc comprises sites that may be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention.
  • Fc variant comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody- dependent cellular cytotoxicity (ADCC).
  • ADCC antibody- dependent cellular cytotoxicity
  • Fc domain encompasses native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fes, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means.
  • Fc sequences are known in the art and are contemplated for use in the invention. For example, Fc IgGl (GenBank Accession No. POl 857), Fc IgG2 (GenBank Accession No. POl 859), Fc IgG3 (GenBank Accession No. POl 860), Fc IgG4 (GenBank Accession No. POl 861), Fc IgAl (GenBank Accession No.
  • Fc IgA2 GenBank Accession No. POl 877
  • Fc IgD GenBank Accession No. POl 880
  • Fc IgM GenBank Accession No. P01871
  • Fc IgE GenBank Accession No. POl 854
  • multimer as applied to Fc domains or molecules comprising Fc domains refers to molecules having two or more polypeptide chains associated covalently, noncovalently, or by both covalent and non-covalent interactions.
  • IgG molecules typically form dimers; IgM, pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, or tetramers. Multimers may be formed by exploiting the sequence and resulting activity of the native Ig source of the Fc or by derivatizing such a native Fc.
  • Embodiments provide peptibodies against MDSC-specific antigens, polypeptides and peptides that are linked to at least one agent to form a peptibody conjugate or payload.
  • it is conventional to link or covalently bind or complex at least one desired molecule or moiety.
  • a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule.
  • Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity.
  • Non-limiting examples of effector molecules that may be attached to peptibodies include toxins, therapeutic enzymes, antibiotics, radio-labeled nucleotides and the like.
  • a reporter molecule is defined as any moiety that may be detected using an assay.
  • Non-limiting examples of reporter molecules that may be conjugated to peptibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, such as biotin.
  • a metal chelate complex employing, for example, an organic chelating agent such as diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N- chloro-p-toluenesulfonamide; and/or tetrachloro-3-6-diphenylglycouril attached to the peptibody.
  • DTPA diethylenetriaminepentaacetic acid anhydride
  • Peptibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate.
  • Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.
  • peptide refers to molecules of 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13 , 14 , 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38 ,39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58,
  • Peptides typically contain random and/or flexible conformations, such as random coils; and typically lack stable conformations, such as those observed in larger proteins/polypeptides, typically via secondary and tertiary structures.
  • numerous size ranges of peptides are contemplated herein, such from about: 3- 90, 3-80, 3-70, 3-60, 3-50; 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30; 10-90, 10-80, 10-70, 10-
  • the peptides used herein are no more than 100, 90, 80, 70, 60, 50, 40, 30, or 20 amino acids in length.
  • Exemplary peptides may be generated by any of the methods set forth herein, such as carried in a peptide library (e.g., a phage display library), generated by chemical synthesis, derived by digestion of proteins, or generated using recombinant DNA techniques.
  • Peptides include D and L form, either purified or in a mixture of the two forms.
  • protein and “polypeptide” refer to compounds comprising amino acids residues joined via peptide bonds and are used interchangeably.
  • a protein or peptide generally refers, but is not limited to, a protein of greater than about 200 amino acids, up to a full length sequence translated from a gene; a polypeptide of greater than about 100 amino acids; and/or a peptide of from about 3 to about 100 amino acids.
  • protein polypeptide
  • peptide are used interchangeably herein.
  • amino acid residue refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimic known in the art.
  • the residues of the protein or peptide are sequential, without any non-amino acids interrupting the sequence of amino acid residues.
  • the sequence may comprise one or more non-amino acid moieties.
  • the sequence of residues of the protein or peptide may be interrupted by one or more non-amino acid moieties.
  • protein or peptide encompasses amino acid sequences comprising at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge.
  • Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
  • substitutions may be non-conservative such that a function or activity of the polypeptide is affected.
  • Non-conservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.
  • Proteins or peptides may be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides, or peptides through standard molecular biological techniques, the isolation of proteins or peptides from natural sources, or the chemical synthesis of proteins or peptides. Proteins may be recombinant, or synthesized in vitro. Alternatively, a non-recombinant or recombinant protein may be isolated from bacteria. It is also contemplated that a bacterium containing such a variant may be implemented in compositions and methods. Consequently, a protein need not be isolated.
  • nucleotide and protein, polypeptide, and peptide sequences corresponding to various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art.
  • One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (available on the world wide web at ncbi.nlm.nih.gov/).
  • Genbank and GenPept databases available on the world wide web at ncbi.nlm.nih.gov/.
  • the coding regions for known genes may be amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.
  • various commercial preparations of proteins, polypeptides, and peptides are known to those of skill in the art.
  • Protein purification techniques are well known to those of skill in the art.
  • the protein or polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity) unless otherwise specified.
  • Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, gel exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography, and isoelectric focusing.
  • a particularly efficient method of purifying peptides is fast-performance liquid chromatography (FPLC) or even high-performance liquid chromatography (HPLC).
  • a purified protein or peptide is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state.
  • An isolated or purified protein or peptide therefore, also refers to a protein or peptide free from the environment in which it may naturally occur.
  • purified will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity.
  • substantially purified this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the proteins in the composition.
  • Various methods for quantifying the degree of purification of the protein or peptide are known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis.
  • a preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity therein, assessed by a "-fold purification number.”
  • the actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification, and whether or not the expressed protein or peptide exhibits a detectable activity.
  • Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "- fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
  • a protein or peptide may be isolated or purified, for example, a peptibody or a fusion protein containing the peptibody.
  • a His tag or an affinity epitope may be comprised in such a peptibody to facilitate purification.
  • Affinity chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule to which it can specifically bind. This is a receptor- ligand type of interaction.
  • the column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution.
  • Elution occurs by changing the conditions to those in which binding will not occur (e.g., altered pH, ionic strength, temperature, etc.).
  • the matrix should be a substance that does not adsorb molecules to any significant extent and that has a broad range of chemical, physical, and thermal stability.
  • the ligand should be coupled in such a way as to not affect its binding properties.
  • the ligand should also provide relatively tight binding. It should be possible to elute the substance without destroying the sample or the ligand.
  • Size exclusion chromatography is a chromatographic method in which molecules in solution are separated based on their size, or in more technical terms, their hydrodynamic volume. It is usually applied to large molecules or macromolecular complexes, such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase.
  • the underlying principle of SEC is that particles of different sizes will elute (filter) through a stationary phase at different rates. This results in the separation of a solution of particles based on size. Provided that all the particles are loaded simultaneously or near simultaneously, particles of the same size should elute together.
  • Each size exclusion column has a range of molecular weights that can be separated. The exclusion limit defines the molecular weight at the upper end of this range and is where molecules are too large to be trapped in the stationary phase. The permeation limit defines the molecular weight at the lower end of the range of separation and is where molecules of a small enough size can penetrate into the pores of the stationary phase completely and all molecules below this molecular mass are so small that they elute as a single band.
  • High-performance liquid chromatography is a form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds.
  • HPLC utilizes a column that holds chromatographic packing material (stationary phase), a pump that moves the mobile phase(s) through the column, and a detector that shows the retention times of the molecules. Retention time varies depending on the interactions between the stationary phase, the molecules being analyzed, and the solvent(s) used.
  • compositions and methods of the present invention may comprise peptibodies conjugated with heterologous peptide segments or polymers, such as polyethylene glycol.
  • the peptibodies may be linked to PEG to increase the hydrodynamic radius of the enzyme and hence increase the serum persistence.
  • peptibodies are disclosed.
  • the peptibodies may be PEGylated in accordance with the methods disclosed herein.
  • PEGylated refers to conjugation with polyethylene glycol (PEG), which has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification.
  • PEG polyethylene glycol
  • PEG can be coupled (e.g., covalently linked) to active agents through the hydroxy groups at the end of the PEG chain via chemical methods; however, PEG itself is limited to at most two active agents per molecule.
  • copolymers of PEG and amino acids have been explored as novel biomaterial that would retain the biocompatibility of PEG, but that would have the added advantage of numerous attachment points per molecule (thus providing greater drug loading), and that can be synthetically designed to suit a variety of applications.
  • PEGylation is the process of covalent attachment of poly(ethylene glycol) polymer chains to another molecule, normally a drug or therapeutic protein. PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target macromolecule.
  • the covalent attachment of PEG to a drug or therapeutic protein can "mask" the agent from the host's immune system (reduced immunogenicity and antigenicity) or increase the hydrodynamic size (size in solution) of the agent, which prolongs its circulatory time by reducing renal clearance.
  • PEGylation can also provide water solubility to hydrophobic drugs and proteins.
  • compositions there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and/or protein per ml.
  • concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or any range derivable therein).
  • about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% may be a MDSC antigen-specific peptibody.
  • nucleic acid sequences encoding a peptide or peptibody may be disclosed.
  • nucleic acid sequences can be selected based on conventional methods. For example, if the peptibody is derived from a mammalian Fc and contains multiple codons that are rarely utilized in E. coli, then that may interfere with expression. Therefore, the respective genes or variants thereof may be codon optimized for E. coli expression.
  • Various vectors may be also used to express the protein of interest, such a peptibody. Exemplary vectors include, but are not limited, plasmid vectors, viral vectors, transposon, or liposome-based vectors.
  • Host cells may be any that may be transformed to allow the expression and secretion of peptibody and conjugates thereof.
  • the host cells may be bacteria, mammalian cells, yeast, or filamentous fungi.
  • Various bacteria include Escherichia and Bacillus.
  • Yeasts belonging to the genera Saccharomyces, Kiuyveromyces, Hansenula, or Pichia would find use as an appropriate host cell.
  • filamentous fungi may be used as expression hosts, including the following genera: Aspergillus, Trichoderma, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, and Pyricularia.
  • Examples of usable host organisms include bacteria, e.g., Escherichia coli MCI 061, derivatives of Bacillus subtilis BRB1 (Sibakov et al, 1984), Staphylococcus aureus SAI123 (Lordanescu, 1975) or Streptococcus lividans (Hopwood et al, 1985); yeasts, e.g., Saccharomyces cerevisiae AH 22 (Mellor et al., 1983) or Schizosaccharomyces pombe; and filamentous fungi, e.g., Aspergillus nidulans, Aspergillus awamori (Ward, 1989), or Trichoderma reesei (Penttila et al., 1987; Harkki et al., 1989).
  • bacteria e.g., Escherichia coli MCI 061, derivatives of Bacillus subtilis BRB1 (Sibakov
  • Examples of mammalian host cells include Chinese hamster ovary cells (CHO-K1 ; ATCC CCL61), rat pituitary cells (GH1 ; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells ( ⁇ -4- ⁇ - ⁇ ; ATCC CRL 1548), SV40-transformed monkey kidney cells (COS-1 ; ATCC CRL 1650), and murine embryonic cells ( IH-3T3; ATCC CRL 1658).
  • CHO-K1 Chinese hamster ovary cells
  • GH1 rat pituitary cells
  • ATCC CCL2.2 HeLa S3 cells
  • rat hepatoma cells ⁇ -4- ⁇ - ⁇
  • ATCC CRL 1548 rat hepatoma cells
  • COS-1 SV40-transformed monkey kidney cells
  • IH-3T3 ATCC CRL 1658
  • Mammalian host cells expressing the peptibody and/or a fusion protein thereof are cultured under conditions typically employed to culture the parental cell line. Generally, cells are cultured in a standard medium containing physiological salts and nutrients, such as standard RPMI, MEM, IMEM, or DMEM, typically supplemented with 5%-10% serum, such as fetal bovine serum. Culture conditions are also standard, e.g., cultures are incubated at 37 °C in stationary or roller cultures until desired levels of the proteins are achieved. V. Treatment and Diagnosis of Diseases
  • Certain aspects of the present embodiments can be used to prevent or treat a disease or disorder associated with MDSC-mediated immune suppression.
  • MDSCs may be depleted by any suitable means to induce, for example, tumor regression.
  • such substances would be a peptibody against a MDSC-specific epitope, such as, for example, an anti-cell surface S 100 peptibody.
  • Treatment refers to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.
  • a treatment may include administration of a pharmaceutically effective amount of an MDSC-depleting peptibody.
  • Subject and “patient” refer to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human.
  • terapéuticaally effective amount refers to an amount of a therapeutic composition (such as a therapeutic polynucleotide and/or therapeutic polypeptide) that is employed in methods to achieve a therapeutic effect.
  • therapeutic benefit or “therapeutically effective” as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.
  • treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis.
  • Treatment of cancer may also refer to prolonging survival of a subject with cancer.
  • An MDSC-depleting peptibody may be administered to treat a cancer.
  • the cancer may be a solid tumor, metastatic cancer, or non-metastatic cancer.
  • the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; small cell lung cancer; non-small cell lung cancer; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocar
  • a peptibody may be used herein as an antitumor agent in a variety of modalities for depleting and/or detecting MDSC where MDSC depletion and/or detection are considered desirable.
  • the depletion is accomplished by administering, by intravenous or intraperitoneal injection, a therapeutically effective amount of a physiologically tolerable composition comprising a peptibody of this invention to a patient, thereby depleting MDSC in the patient.
  • a therapeutically effective amount of a peptibody is a predetermined amount calculated to achieve the desired effect, i.e., to deplete MDSC.
  • the dosage ranges for the administration of peptibodies of the invention are those large enough to produce the desired effect in which the symptoms of tumor cell division and cell cycling are reduced.
  • the dosage should not be so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like.
  • the dosage will vary with age of, condition of, sex of, and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication.
  • a therapeutically effective amount of a peptibody may be an amount such that when administered in a physiologically tolerable composition is sufficient to achieve an intravascular (plasma) or local concentration of from about 0.001 to about 100 units (U) per mL, preferably above about 0.1 U, and more preferably above 1 U peptibody per mL.
  • Typical dosages can be administered based on body weight, and are in the range of about 5-1000 U/kilogram (kg)/day, preferably about 5-100 U/kg/day, more preferably about 10-50 U/kg/day, and more preferably about 20-40 U/kg/day.
  • the peptibody can be administered parenterally by injection or by gradual infusion over time.
  • the peptibody can be administered intravenously, intraperitoneally, orally, intramuscularly, subcutaneously, intracavity, transdermally, dermally, can be delivered by peristaltic means, can be injected directly into the tissue containing the tumor cells, or can be administered by a pump.
  • the therapeutic compositions containing peptibodies are conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic composition refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier, or vehicle.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • quantity to be administered depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for initial administration and booster shots are also contemplated and are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration.
  • Exemplary multiple administrations are described herein and are particularly preferred to maintain continuously high serum and tissue levels of peptibodies and conversely low serum and tissue levels of MDSC.
  • continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.
  • Some embodiments disclosed herein concern diagnostic and prognostic methods for the detection of inflammation and/or cancer. Such detection methods may be used, for example, for early diagnosis of the disease, to determine whether a tumor is malignant or benign, to monitor the progress of the disease or the progress of treatment protocols, or to assess the grade of the cancer. The detection can occur in vitro or in vivo.
  • the presence of a cancer is determined when the percentage of myeloid cells is at least 2% MDSC, at least 3% MDSC, at least 4% MDSC, at least 5% MDSC, at least 6% MDSC, at least 7% MDSC, at least 8% MDSC, at least 9% MDSC, at least 10% MDSC, at least 11% MDSC, at least 12% MDSC, at least 13% MDSC, at least 14% MDSC, or at least 15% MDSC.
  • Useful methods include, but are not limited to, flow cytometry, an enzyme immune assay (EIA) such as enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay, or Western blot detection.
  • compositions may comprise, for example, at least about 0.1% of an active compound.
  • an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • compositions of the present embodiments are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified.
  • phrases "pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate.
  • the preparation of a pharmaceutical composition comprising an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure.
  • animal (e.g., human) administration it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.
  • aqueous solvents e.g., water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc.
  • non-aqueous solvents e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate
  • dispersion media coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art.
  • the pH and exact concentration e.g., water, alcoholic/aqueous solutions,
  • unit dose refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen.
  • the quantity to be administered both according to number of treatments and unit dose, depends on the effect desired.
  • the actual dosage amount of a composition of the present embodiments administered to a patient or subject can be determined by physical and physiological factors, such as body weight, the age, health, and sex of the subject, the type of disease being treated, the extent of disease penetration, previous or concurrent therapeutic interventions, idiopathy of the patient, the route of administration, and the potency, stability, and toxicity of the particular therapeutic substance.
  • a dose may also comprise from about 1 ⁇ g/kg/body weight to about 1000 mg/kg/body weight (this such range includes intervening doses) or more per administration, and any range derivable therein.
  • a range of about 5 ⁇ g/kg/body weight to about 100 mg/kg/body weight, about 5 ⁇ g/kg/body weight to about 500 mg/kg/body weight, etc. can be administered.
  • the practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • the active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes.
  • parenteral administration e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes.
  • such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the proteinaceous compositions may be formulated into a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • a pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Solutions of therapeutic compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • compositions of the present invention are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified.
  • a typical composition for such purpose comprises a pharmaceutically acceptable carrier.
  • the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline.
  • Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters, such as ethyloleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc.
  • Intravenous vehicles include fluid and nutrient replenishers.
  • Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well known parameters.
  • Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like.
  • the compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
  • the therapeutic compositions of the present invention may include classic pharmaceutical preparations. Administration of therapeutic compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Topical administration may be particularly advantageous for the treatment of skin cancers, to prevent chemotherapy-induced alopecia or other dermal hyperproliferative disorder. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. For treatment of conditions of the lungs, or respiratory tract, aerosol delivery can be used. Volume of the aerosol is between about 0.01 mL and 0.5 mL.
  • an effective amount of the therapeutic composition is determined based on the intended goal. For example, one skilled in the art can readily determine an effective amount of an antibody of the invention to be administered to a given subject, by taking into account factors such as the size and weight of the subject; the extent of the neovascularization or disease penetration; the age, health and sex of the subject; the route of administration; and whether the administration is regional or systemic.
  • the term "unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen.
  • the quantity to be administered both according to number of treatments and unit dose, depends on the protection or effect desired.
  • Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are particular to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.
  • compositions and methods of the present embodiments involve a peptibody against a MDSC-specific epitope to deplete MDSCs, in combination with a second or additional therapy.
  • Such therapy can be applied in the treatment of any disease that is associated with MDSCs.
  • the disease may be cancer.
  • Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect, such as the killing of a cancer cell and/or the inhibition of cellular hyperproliferation.
  • This process may involve contacting the cells with both a peptibody and a second therapy.
  • a tissue, tumor, or cell can be contacted with one or more compositions or pharmacological formulation(s) comprising one or more of the agents (i.e., peptibody or an anti-cancer agent), or by contacting the tissue, tumor, and/or cell with two or more distinct compositions or formulations, wherein one composition provides 1) a peptibody, 2) an anti-cancer agent, or 3) both a peptibody and an anti-cancer agent.
  • a combination therapy can be used in conjunction with chemotherapy, radiotherapy, surgical therapy, or immunotherapy.
  • contacted and “exposed,” when applied to a cell are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell.
  • both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.
  • a peptibody may be administered before, during, after, or in various combinations relative to an anti-cancer treatment.
  • the administrations may be in intervals ranging from concurrently to minutes to days to weeks.
  • the peptibody is provided to a patient separately from an anti-cancer agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient.
  • a course of treatment will last 1-90 days or more (this such range includes intervening days). It is contemplated that one agent may be given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof, and another agent is given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof. Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no anti-cancer treatment is administered.
  • This time period may last 1-7 days, and/or 1-5 weeks, and/or 1-12 months or more (this such range includes intervening days), depending on the condition of the patient, such as their prognosis, strength, health, etc. It is expected that the treatment cycles would be repeated as necessary.
  • chemotherapeutic agents may be used in accordance with the present embodiments.
  • the term "chemotherapy” refers to the use of drugs to treat cancer.
  • a "chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer.
  • agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle.
  • an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.
  • chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); do
  • DNA damaging factors include what are commonly known as ⁇ -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells.
  • Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287), and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes.
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
  • immunotherapeutics generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
  • Rituximab (RITUXAN®) is such an example.
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing.
  • the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
  • the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
  • Various effector cells include cytotoxic T cells and NK cells.
  • the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
  • Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B, and pl55.
  • An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects.
  • Immune stimulating molecules also exist including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand.
  • cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN
  • chemokines such as MIP-1, MCP-1, IL-8
  • growth factors such as FLT3 ligand.
  • immunotherapies currently under investigation or in use are immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patents 5,801,005 and 5,739, 169; Hui and Hashimoto, 1998; Christodoulides et al, 1998); cytokine therapy, e.g., interferons ⁇ , ⁇ , and ⁇ , IL-1, GM-CSF, and TNF (Bukowski et al, 1998; Davidson et al, 1998; Hellstrand et al, 1998); gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al, 1998; Austin- Ward and Villaseca, 1998; U.S.
  • immune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds
  • cytokine therapy
  • Patents 5,830,880 and 5,846,945) ; and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-pl 85 (Hollander, 2012; Hanibuchi et al, 1998; U.S. Patent 5,824,31 1). It is contemplated that one or more anti-cancer therapies may be employed with the antibody therapies described herein.
  • Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies.
  • Tumor resection refers to physical removal of at least part of a tumor.
  • treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs' surgery).
  • a cavity may be formed in the body.
  • Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, or 12 months. These treatments may be of varying dosages as well.
  • agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment.
  • additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population.
  • cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatments.
  • Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments.
  • Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy.
  • kits are envisioned containing therapeutic agents and/or other therapeutic and delivery agents.
  • a kit is contemplated for preparing and/or administering a therapy of the embodiments.
  • the kit may comprise one or more sealed vials containing any of the pharmaceutical compositions of the present embodiments.
  • the kit may include, for example, at least one MDSC-specific peptide and/or peptibody as well as reagents to prepare, formulate, and/or administer the components of the embodiments or perform one or more steps of the inventive methods.
  • the kit may also comprise a suitable container, which is a container that will not react with components of the kit, such as an eppendorf tube, an assay plate, a syringe, a bottle, or a tube.
  • a suitable container which is a container that will not react with components of the kit, such as an eppendorf tube, an assay plate, a syringe, a bottle, or a tube.
  • the container may be made from sterilizable materials such as plastic or glass.
  • the kit may further include an instruction sheet that outlines the procedural steps of the methods set forth herein, and will follow substantially the same procedures as described herein or are known to those of ordinary skill in the art.
  • the instruction information may be in a computer readable media containing machine-readable instructions that, when executed using a computer, cause the display of a real or virtual procedure of delivering a pharmaceutically effective amount of a therapeutic agent. VII. Examples
  • Example 1 Generation of novel therapeutic peptides that deplete MDSC in tumor- bearing mice
  • MDSC frequency is low in naive C57BL/6 mice; however, after transplantation of syngeneic EL4 thymomas, MDSC are increased accounting for approximately 10% of total splenocytes (Gabrilovich and Nagaraj, 2009).
  • Splenic MDSC from EL4-bearing mice consist of two distinct subpopulations characterized by Gr-l high CDl lb + granulocytic (P7) and Gr-l int CDl lb + monocytic (P10) staining (FIG. la).
  • Gr-1 and CD l ib labeled MDSC were separated 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 three rounds of competitive biopanning. Enrichment was analyzed by the number of phage eluted from 10 6 MDSC (FIG. lb) and by phage output, normalized to the initial input of 2 x 10 10 phage (FIG. lc).
  • peptibodies were conjugated with fluorescein isothiocyanate (FITC) and analyzed for binding specificity on CDl lb Ly6G Ly6C high monocytic MDSC and CD1 lb + Ly6G + Ly6C int/low granulocytic MDSC from EL4- bearing mice. Gating on these well-defined subpopulations, the inventors conclusively showed that Pep-H6 and Pep-G3 bind both monocytic and granulocytic MDSC (FIGs. 2c and 5a). However, the expression of the Pep-G3 target seems to be lower in granulocytic MDSC than in monocytic MDSC.
  • FITC fluorescein isothiocyanate
  • Peptibodies deplete MDSC in vivo and retard tumor growth.
  • the effect of peptibody treatment on MDSC in vivo was determined.
  • Intravenous injection of 50 ⁇ g Pep-H6 and Pep-G3 completely depleted MDSC in blood and spleens of EL4 tumor- bearing mice, compared with control peptibody (Pep-irrel) or untreated mice (FIGs. 3 a and 3b).
  • peptibodies depleted both monocytic and granulocytic MDSC subsets
  • Gr-1 mAb depleted granulocytic, but not monocytic, MDSC in both EL4 and EG.7 models.
  • peptibody treatment depleted intratumoral MDSC in both EG.7 (FIGs. 3c and 3d) and EL4 models (FIG. 7).
  • Peptibodies also depleted blood and splenic MDSC in mice bearing A20 lymphomas (FIG. 3e).
  • peptibodies were administered to EL4-bearing mice every other day to achieve a continuous depletion of systemic MDSC.
  • treatment with Pep- H6 or Pep-G3 alone significantly delayed tumor development, measured by tumor size (FIG. 3g) or tumor mass (FIG. 3h), compared with untreated or Pep-irrel-treated control mice.
  • FIG. 3g tumor size
  • FIG. 3h tumor mass
  • peptibodies clearly inhibited tumor growth in vivo
  • Gr-1 mAb treatment was associated with less consistent inhibition of tumor growth. This effect of peptibody treatment correlated with MDSC depletion at the end of the two-week treatment (FIG. 8).
  • the inventors To evaluate potential off-target activity of our specific peptibodies, the inventors also assessed potential depletion of normal cell types, including those of myeloid origin. With the exception of transient reduction of blood neutrophils, and possibly monocytes, no depletion of DC, T, B, NK cells, or Grl CDl lb + immature myeloid precursor cells in the bone marrow was observed in tumor-bearing mice. The inventors also observed that in naive mice, peptibody treatment reduced circulating Gr-l CDl lb + splenocytes (FIG. 10a), but did not affect immature myeloid cells in the bone marrow (FIG.
  • Example 2 Generation of a novel therapeutic peptide that depletes human MDSC
  • Identification and characterization of human MDSC By staining splenic lymphoma samples, the inventors identified a HLA-DR CD 33 + CD1 lb + subpopulation that contained both CD15 + (92%) and CD 14 + (8%) cells, suggesting granulocytic and monocytic MDSC, respectively (FIG. 11a). These tumor-infiltrated human MDSC suppressed in vitro activation of HLA-matched, allogeneic antigen-specific T cells (FIG. l ib).
  • HLA-A2 + lymphoma idiotype-peptide specific T-cell clones were mixed with peptide-pulsed T2 cells, with or without HLA-DR CD 1 lb + CD33 + sorted human MDSC isolated from (a) at a 1 : 1 ratio. After a 24 h incubation, culture medium was collected and assayed for TNFg by ELISA. Negative controls included T cells + T2 cells pulsed with irrelevant or no peptide. The FACS data revealed the presence of S100A9 on the surface of these tumor-infiltrated human MDSC (FIG. 1 lc).
  • Expression of S100A9 has also been screened on the cell surface of human MDSC isolated from the blood of five additional patients with a range of hematological tumors, including follicular lymphoma and multiple myeloma, or directly from the tumors as above. Gating on HLA-DR-/CD1 lb+/CD33+ human MDSC cells, a significant proportion of cells was identified as being positively stained for human S100A9, compared with minimal to no staining of gated cells from normal human donors, which supports the hypothesis that S100A9 can be targeted for human MDSC depletion therapy.
  • [00148] Generation of human MDSC-specific peptibodies Sequences encoding the identified peptides will be genetically fused with the Fc portion of human IgG to generate peptibodies. Recombinant peptibodies will be produced in 293T mammalian cells, followed by purification of the peptibodies by Protein A chromatography and characterization by Western blot. These peptibodies will be conjugated with fluorescein isothiocyanate (FITC) and analyzed for binding specificity to human MDSC.
  • FITC fluorescein isothiocyanate
  • Phage display Total splenocytes from EL4 tumor-bearing C57BL/6 mice were blocked with formaldehyde-inactivated M13 phage and 2.4G2 antibody (BD Biosciences, San Jose, CA), followed by staining with anti-CD 1 lb-APC and anti-Gr- 1 -FITC (BD Biosciences). Gr-1- and CD1 lb-labeled splenocytes were then incubated with 2 x 10 10 Ph.D. -12 peptide phage display library (New England BioLabs Inc., Ipswich, MA) for 1 h at 4 °C.
  • FITC-conjugated H6 or G3 synthetic peptides were made by Pi Proteomics, LLC (Huntsville, AL). After blocking with 2.4G2 antibody, splenocytes from EL4-bearing C57BL/6 mice were co-stained with anti- CDl lb-APC, anti-Gr-l-PE, and FITC-conjugated peptides. H6 and G3 peptides were analyzed for their binding to Gr-1 CD 1 lb + gated MDSC, compared with Gr-1 CD l lb gated non-MDSC splenocytes. A non-specific peptide (irrel peptide) was used as a negative control to exclude non-specific binding. FACS analysis was performed using LSRFortessa cell analyzer (BD Biosciences) and results were analyzed using Flow Jo software.
  • Peptibodies excreted into growth media were purified using Protein A chromatography (GE Healthcare Life Sciences, Pittsburgh, PA). The identity of peptibodies was verified by Western blot using anti-His-HRP (BD Biosciences) or anti-mouse IgG-HRP antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) (Table 3). Recombinant peptibodies used in all in vivo studies were produced by Aldevron (Madison, WI).
  • mice used for in vivo studies Six-week-old C57BL/6 and Balb/c female mice were purchased from US National Cancer Institute. S100A9-deficient female mice were a generous gift from Dr. Donna Kusewitt (M.D. Anderson Cancer Center, Smithville, TX) by permission of Dr. Johannes Roth (University of Muenster, Germany) who originally generated the model. Mice were maintained in a pathogen-free mouse facility according to institutional guidelines. All animal studies were approved by the Institutional Animal Care and Use Committee at MD Anderson Cancer Center.
  • splenocytes from EL4- bearing C57BL/6 mice, EG.7-bearing C57BL/6 mice, B16-bearing C57BL/6 mice, and A20- bearing Balb/c mice were co-stained with anti-CD 11 b-APC, anti-Ly6G-PE, anti-Ly6C- PerCP, and FITC-conjugated peptibodies.
  • Peptibody binding was analyzed on CDl lb + Ly6G + Ly6C int/low gated granulocytic MDSC and CDl lb + Ly6GXy6C high gated monocytic MDSC, respectively.
  • splenocytes from EL4-bearing C57BL/6 mice were co-stained with anti-CD 1 1c- PerCP, anti-Ly6G-PE, and peptibody-FITC.
  • CDl lc Ly6G DC were analyzed for peptibody binding.
  • splenocytes from EL4-bearing C57BL/6 mice were co- stained for CD 19-APC, CD3-PerCP, CD49b-PE, and peptibody-FITC.
  • CD3 + T cells, CD19 + B cells, and CD3 ⁇ CD49b NK cells were gated and analyzed for peptibody binding.
  • bone marrow cells from EL4-bearing C57BL/6 mice were isolated and co-stained with anti-CD 11 b-APC, anti-Gr-l-PE, and FITC-conjugated peptibodies.
  • CDl lb + Gr-l + immature myeloid cells were analyzed for their binding to peptibodies.
  • Pep-H6-bound population would be overlapped with Pep-G3-bound populations
  • splenocytes from EL4-bearing C57BL/6 mice were co-stained with anti-Gr-l-PE, anti- CD l lb-PerCP, and either APC-labeled Pep-G3 or FITC-labeled Pep-H6, or both.
  • CD 1 lb Gr-1 gated MDSC were analyzed for peptibody binding.
  • mice In vivo MDSC depletion. Groups of five C57BL/6 mice were challenged s.c. with 10 6 EL4 or EG.7 mouse thymoma tumor cells on day 0. Tumor-bearing mice were treated i.v. with 50 ⁇ g of peptibodies per day for three consecutive days (days 17, 18 and 19), and then sacrificed on day 20 to harvest blood and spleens. Control mice received Gr-1 monoclonal antibodies (clone 1A8, BioXcell, West Riverside, NH), irrelevant control peptibody (Pep-irrel), or PBS. Ficolled blood and splenocytes were stained for Gr-1- PE and CDl lb-APC to identify MDSC.
  • Gr-1 monoclonal antibodies clone 1A8, BioXcell, West Riverside, NH
  • Pep-irrel irrelevant control peptibody
  • PBS Ficolled blood and splenocytes were stained for Gr-1- PE and CD
  • EL4 or EG.7 subcutaneous tumors were cut into small pieces of 2-4 mm and digested with the enzyme mix (40 min at 37 °C) provided in "Tumor Dissociation Kit” (Miltenyi Biotec Inc., Auburn, CA). Tumors were dissociated into single cell suspension with a GENTLEMACSTM Dissociator (Milteny Biotec Inc.). The tumor cells were then stained for Gr-l-PE and CD1 lb-APC to identify intratumoral MDSC.
  • splenocytes from peptibody-treated, EL4-bearing C57BL/6 mice were co-stained for CDl lb-APC, CDl lc-PerCp, and Ly6G-PE and myeloid cells were gated based on forward and side scatter profile.
  • Ly6G CDl lc DC were enumerated and their representative frequencies were calculated by multiplying the myeloid cell frequency in total splenocytes.
  • Splenocytes were also stained for CD19-APC, CD3-PerCP, and CD49b-PE, and frequencies of CD3 + T cells, CD19 + B cells, and CD3 CD49b + NK cells in total splenocytes were analyzed. Bone marrow cells were stained for CD1 lb-APC and Gr-l-PE. Double positive immature myeloid cells were enumerated.
  • mice were challenged with 10 6 EL4 tumor cells on Day 0. Twenty-four hours later, mice started to receive 50 ⁇ g of peptibodies per day, every other day for two weeks. Control mice received Gr-1 monoclonal antibodies (BioXcell), Pep-irrel, or PBS. Tumor dimensions were measured daily with calipers to monitor growth. On day 14, mice were sacrificed and spleens and subcutaneous tumors were isolated. Tumor mass was measured on an analytical balance. Splenocytes were stained for MDSC using anti-Gr-l-PE and anti-CD 1 lb-APC antibodies.
  • S100A9-deficient mouse studies Groups of three S 100A9-deficient C57BL/6 mice were challenged s.c. with 10 6 EL4 tumors on day 0. Three weeks later, splenocytes were harvested and co-stained with anti-CD l lb-APC, anti-Gr-l-PE, and peptibody-FITC. CDl lb Gr-1 MDSC were analyzed for peptibody binding.
  • EL4-bearing S 100A9-deficient C57BL/6 mice were treated i.p. with 50 ⁇ g of peptibodies for three consecutive days (days 17, 18 and 19). Splenocytes were harvested on day 20 and stained for MDSC, as above.
  • HIF-1 alpha regulates function and differentiation of myeloid-derived suppressor cells in the tumor microenvironment, J. Exp. Med., 207:2439-2453, 2010.
  • Zhao et al, S 100A9 a new marker for monocytic human myeloid-derived suppressor cells.

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Abstract

L'invention concerne de nouveaux réactifs de fusion MDSC-peptide spécifique-domaine Fc (pepticorps) à usage diagnostique et thérapeutique pour épuiser les MDSC intratumorales, du sang et de la rate .
PCT/US2014/058318 2013-09-30 2014-09-30 Peptides spécifiques dirigés contre les cellules myéloïdes suppressives (mdsc), à usage diagnostique et thérapeutique Ceased WO2015048748A1 (fr)

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USRE49435E1 (en) 2014-11-12 2023-02-28 Seagen Inc. Glycan-interacting compounds and methods of use
US11028181B2 (en) 2015-11-12 2021-06-08 Seagen Inc. Glycan-interacting compounds and methods of use
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US11401330B2 (en) 2016-11-17 2022-08-02 Seagen Inc. Glycan-interacting compounds and methods of use
US11253609B2 (en) 2017-03-03 2022-02-22 Seagen Inc. Glycan-interacting compounds and methods of use
WO2024008877A1 (fr) * 2022-07-07 2024-01-11 Rigshospitalet Médicaments antifongiques personnalisés et outils de diagnostic des infections fongiques invasives
WO2025132452A1 (fr) 2023-12-19 2025-06-26 Rigshospitalet Médicaments antifongiques personnalisés et outils de diagnostic des infections fongiques invasives

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