WO2017048629A1 - Anticorps pour la production de macrophages anti-inflammatoires et utilisations associées - Google Patents

Anticorps pour la production de macrophages anti-inflammatoires et utilisations associées Download PDF

Info

Publication number
WO2017048629A1
WO2017048629A1 PCT/US2016/051290 US2016051290W WO2017048629A1 WO 2017048629 A1 WO2017048629 A1 WO 2017048629A1 US 2016051290 W US2016051290 W US 2016051290W WO 2017048629 A1 WO2017048629 A1 WO 2017048629A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antibody
cells
cathepsin
macrophages
Prior art date
Application number
PCT/US2016/051290
Other languages
English (en)
Inventor
Kyung Ho Han
Richard A. Lerner
Original Assignee
The Scripps Research Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Scripps Research Institute filed Critical The Scripps Research Institute
Priority to AU2016324021A priority Critical patent/AU2016324021A1/en
Priority to EP16847105.0A priority patent/EP3349768A4/fr
Publication of WO2017048629A1 publication Critical patent/WO2017048629A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4614—Monocytes; Macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation

Definitions

  • the system of checks and balances operates, for example, by balancing effector and regulatory T-cells and/or pro-inflammatory and anti-inflammatory macrophages.
  • one or the other arm of an otherwise balanced system may need to be favored.
  • Such control has become one of the major goals in immunology for the treatment of autoimmunity and cancer.
  • the invention provides isolated or purified anti-Cathepsin G
  • CTSG antibodies have the same binding specificity as that of a reference anti-CTSG antibody having (1) heavy chain CDRl, CDR2 and CDR3 sequences respectively shown in GYTFTSYY (SEQ ID NO:4), IIPIFGTG (SEQ ID NO:5), and AREEEQQFSLDY (SEQ ID NO:6); and (2) light chain CDRl, CDR2 and CDR3 sequences respectively shown in SGSIASNS (SEQ ID NO:7), EN , and QSYDSNFHWV (SEQ ID NO:8).
  • Some of the antibodies contain heavy chain CDRl , CDR2 and CDR3 sequences that are substantially identical to GYTFTSYY (SEQ ID NO:4), IIPIFGTG (SEQ ID NO:5), and AREEEQQFSLDY (SEQ ID NO:6), respectively; and light chain CDRl , CDR2 and CDR3 sequences that are substantially identical to SGSIASNS (SEQ ID NO: 7), ENN, and
  • QSYDSNFHWV (SEQ ID NO:8), respectively.
  • Some antibodies contain heavy chain CDRl, CDR2 and CDR3 sequences that are respectively identical to GYTFTSYY (SEQ ID NO:4), IIPIFGTG (SEQ ID NO:5), and AREEEQQFSLDY (SEQ ID NO:6); and light chain CDRl, CDR2 and CDR3 sequences that are respectively identical to SGSIASNS (SEQ ID NO:7), ENN, and QSYDSNFHWV (SEQ ID NO:8) (SEQ ID NO:8).
  • Some antibodies of the invention contain heavy chain and light chain variable region sequences that are substantially identical to SEQ ID NO:2 and SEQ ID NO:3, respectively. Some antibodies contain heavy chain and light chain variable region sequences that are at least 90% or 95% identical to SEQ ID NO:2 and SEQ ID NO:3, respectively. Some of the antibodies contain heavy chain and light chain variable region sequences show in SEQ ID NO:2 and SEQ ID NO:3, respectively.
  • the antibody is a scFv having an amino acid sequence that is substantially identical to SEQ ID NO: 1. Some of the scFvs have an amino acid sequence that is at least 90% or 95% identical to SEQ ID NO: 1. In one embodiment, the scFv has a sequence shown in SEQ ID NO: 1.
  • the invention provides methods for inducing formation of antiinflammatory M2 macrophages.
  • the methods entail contacting a population of bone marrow cells or monocytes with an anti-Cathepsin G antibody under conditions sufficient to induce formation of anti-inflammatory macrophage, wherein the anti-Cathepsin G antibody has the same binding specificity as that of a reference antibody comprising (a) heavy chain CDRl, CDR2 and CDR3 sequences respectively shown in GYTFTSYY (SEQ ID NO:4), IIPIFGTG (SEQ ID NO:5), and AREEEQQFSLDY (SEQ ID NO:6); and (b) light chain CDRl , CDR2 and CDR3 sequences respectively shown in SGSIASNS (SEQ ID NO:7), ENN, and
  • the employed bone marrow cells are human bone marrow cells.
  • the contacting occurs in vitro by culturing the population of bone marrow cells or monocytes in the presence of the anti- Cathepsin G antibody.
  • the cells are cultured with the antibody for about 4 to 20 days.
  • Some of the methods further include isolating CD1 lb- cells from the cultured cell population.
  • Some methods further include detecting in the cultured cell population at least one cellular marker expressed by M2 macrophage, e.g., CD36, MHCII, CD 14, or ARG-1.
  • contacting the cells with the antibody occurs in vivo in a subject.
  • the subject is afflicted with an autoimmune disease, and the antibody is administered to the subject via a pharmaceutical composition.
  • Some methods of the invention use an anti-Cathepsin G antibody having an amino acid sequence that is substantially identical to SEQ ID NO: l .
  • Some related embodiments of the invention provide populations of anti-inflammatory M2 macrophages produced by the methods of the invention.
  • the invention provides methods for treating or ameliorating the symptoms of an autoimmune disease in a subject.
  • the methods involve administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an anti-Cathepsin G antibody.
  • the anti-Cathepsin G antibody used in the methods typically has the same binding specificity as that of a reference antibody comprising (a) heavy chain CDRl, CDR2 and CDR3 sequences respectively shown in GYTFTSYY (SEQ ID NO:4), IIPIFGTG (SEQ ID NO: 5), and AREEEQQFSLDY (SEQ ID NO:6); and (b) light chain CDRl, CDR2 and CDR3 sequences respectively shown in SGSIASNS (SEQ ID NO:7), EN , and QSYDSNFHWV (SEQ ID NO: 8).
  • the employed antibody has heavy chain CDRl , CDR2 and CDR3 sequences that are respectively identical to GYTFTSYY (SEQ ID NO:4), IIPIFGTG (SEQ ID NO:5), and AREEEQQFSLDY (SEQ ID NO:6); and light chain CDRl , CDR2 and CDR3 sequences that are respectively identical to SGSIASNS (SEQ ID NO:7), ENN, and QSYDSNFHWV (SEQ ID NO:8) (SEQ ID NO:8).
  • the employed antibody has heavy chain and light chain variable region sequences show in SEQ ID NO:2 and SEQ ID NO:3, respectively.
  • the employed antibody is a scFv having an amino acid sequence that is substantially identical to SEQ ID NO: l .
  • the employed anti-Cathepsin G antibody is a scFv fragment shown in SEQ ID NO: l .
  • the invention provides methods for obtaining antiinflammatory M2 macrophages from a population of bone marrow cells or monocytes. These methods entail (a) introducing into the bone marrow cells or monocytes a plurality of vectors expressing a library of antibodies, (b) culturing the cells harboring the vectors under conditions to express the library of antibodies, and (c) selecting one or more macrophages from the cells expressing the library of antibodies.
  • the library of antibodies are unbiased, e.g., unbiased scFv antibody library.
  • the employed antibodies are biased, e.g., antibodies recognizing CTSG.
  • the antibodies are expressed from a lentiviral based vector.
  • the employed cells are CD34 + bone marrow cells.
  • selection of M2 macrophages is based on cell morphology.
  • selection of M2 macrophages is based on expression of one or more M2 surface markers.
  • Some methods of the invention further include isolating the antibody-expressing vectors from the selected M2 macrophages. These methods allow one to obtain novel M2 macrophage-inducing antibody agents that can be readily used in various therapeutic applications described herein, e.g., treating autoimmune diseases.
  • Figure 1 shows selection of an agonist antibody that induces macrophage cell differentiation.
  • A Scheme of the phenotype selection. The selection starts with a human scFv phage library (10 9 members). ScFv genes were transferred to a lentiviral vector to make lentiviral intra-body libraries. Total mouse bone marrow cells were infected with the antibody library and plated on soft agar.
  • B After 2 weeks of incubation, 7 colonies with compact morphologies had grown.
  • C, D These colonies were harvested and the antibody genes were recovered by PCR. One sequence was present in all colonies and was used for further studies. H&E staining of cells from the colonies showed cells with the classical morphologies of macrophages.
  • Figure 2 shows identification of a novel antigen recognized by LKAb.
  • A Both commercial anti-CTSG antibody and LKAb bound to a 28 kDa protein in Western blots of lysates from cells overexpressing CTSG.
  • B Lysates of mouse bone marrow cells were incubated with LKAb for immuno-purification. Eluates from these immune complexes bound to the commercial anti-CTSG antibody in Western blots.
  • C A CTSG enzymatic assay showed that CTSG enzyme activity was present in the eluates and mouse bone marrow total lysates.
  • D Mouse bone marrow was incubated for two days with lentiviruses containing CTSG shRNA.
  • FIG. 3 shows that LKAb induced anti-inflammatory M2 macrophage differentiation.
  • A Mouse bone marrow cells were incubated with LKAb at the indicated concentrations (l-10( ⁇ g/ml) for 6 days. Cells were then stained with anti-CDl lb and anti- CD1 l c and analyzed by FACS. Encircled shapes identify positive macrophage populations. LKAb induces differentiation of CD1 lb+ macrophages but not CD1 lc+ dendritic cells. This process is dose dependent.
  • B Mouse total bone marrow cells were separated using CD1 lb- specific magnetic beads, and the isolated CD 1 lb-positive or -negative populations were incubated with LKAb or M-CSF for 6 days.
  • C Mouse bone marrow was incubated with medium, LKAb or M-CSF for 6 days. Cells were then stained with anti-CDl 6/32 and anti-CD86 as Ml macrophage markers, and anti-CD36, anti-MHCII and anti-CD 14 as M2 macrophage markers. Encircled shapes mark M2 type specific populations. The macrophages induced by LKAb selectively expressed M2 type markers.
  • D Mouse bone marrow was induced by LKAb antibody or M-CSF for 6 days.
  • FIG. 4 shows that CTSG, CD14 and NAFAT play a key role in LKAb- dependent M2 macrophage differentiation.
  • A Bone marrow cells from wild type, CTSG knockout, CD 14 knockout mice or NFAT knockout mice were incubated with medium, LKAb or M-CSF for 6 days. Cells were then stained with anti-CD 14 and anti-F4/80. FACS analysis showed CD14 and F4/80 expression was increased by LKAb in normal mice.
  • CD 14 and F4/80 expression was significantly reduced in both the CTSG and CD 14 knockout mice.
  • CD 14 and F4/80 expression was also reduced in NFATcl, but not in NFATc2:CD4 Cre , knockout mice. The percentages of M2 macrophages are shown (bottom). *P ⁇ 0.05 (Student's t-test).
  • B Wild type mouse bone marrow cells were incubated with LKAb, a CTSG inhibitor, or both, and CTSG activity was determined. The antibody-induced reduction of CTSG activity was comparable to that of the inhibitor.
  • C Critical cell surface components and their downstream signaling pathways potentially involved in macrophage differentiation mediated by LKAb (pictured here as bound to CTSG).
  • FIG. 5 shows that LKAb induces human bone marrow cells to differentiate into M2 macrophages.
  • Human total bone marrow was incubated with medium, LKAb or M-CSF for 6 days.
  • Cells were then stained with anti-CDl lb and anti-CD64 as Ml type markers, and anti-CD 14, anti-CD206 and anti-CD200R as M2 markers. Encircled shapes mark the M2 type specific populations.
  • LKAb also induced human bone marrow to differentiate into anti-inflammatory macrophages.
  • FIG. 6 shows that treatment with LKAb reduced lupus-like disease in MRL-lpr mice.
  • Mice (6-7/group) were injected i.p. with LKAb or PBS (from the age of 6 weeks until termination of the experiment at 20 weeks) and followed for manifestations of disease.
  • A IgG2a anti-chromatin autoantibody levels determined by ELISA at 12 weeks of age.
  • B Progression of lymphadenopathy between 12 and 16 weeks of age assessed by palpation of axillary and salivary lymph nodes (LNs) and scored on a 0-4 scale.
  • C Kidney disease determined by histological examination for glomerulonephritis (GN) at 20 weeks of age. Representative images for treated and control mice are shown.
  • Figure 7 shows LKAb's target identification by mass spectrometry.
  • A Cell lysates of mouse bone marrow were incubated with LKAb for immunoprecipitation. Immuno-precipitated eluates were separated on SDS/PAGE gels that were silver-stained.
  • B, C Nano-LC-MS/MS analysis identified several candidates as antigen.
  • Figure 8 shows LKAb activated signaling pathways.
  • Mouse bone marrow cells were incubated with LKAb, M-CSF or GM-CSF and analyzed by Western blot with antibodies specific for phospho-AKT, ERK and p38.
  • FIG. 9 shows that LKAb-induced macrophage differentiation requires CD 14 but not TLR4.
  • Mouse bone marrow cells from C3H/HeOuJ (wild type), C3H/HeJ (TLR4 mutation) or C57BL/10ScNJ (TLR4 knockout) mice were incubated with LKAb, M-CSF or vehicle (C3H/HeOuJ only) for 6 days. Cells were then stained with anti-CD36 or anti- CD16/32. There is no macrophage differentiation in cells from CD14 (-/-) mice incubated with LKAb.
  • the present invention is predicated in part on the studies undertaken by the present inventors to obtain antibody agonists that can selectively induce formation of antiinflammatory M2 macrophages.
  • the inventors used an unbiased intracellular combinatorial library of antibodies and identified an antibody (termed “LKAb” herein) that induces bone marrow cells to differentiate into M2 macrophages.
  • LKAb an antibody that induces bone marrow cells to differentiate into M2 macrophages.
  • the inventors also found that the primary target of the antibody is Cathepsin G (CTSG), and that LKAb inhibited CTSG activity by 50%. It was additionally observed that the requirements for efficient macrophage polarization by this antibody include the presence of CTSG and CD 14 that appear to operate via p38, a classical marker for macrophage differentiation.
  • CTSG Cathepsin G
  • CTSG was classically thought to be an effector molecule secreted by activated PMNs.
  • the antibodies identified by the inventors were found to be therapeutically effective in blocking autoimmunity in a classic mouse model of spontaneous systemic lupus erythematosus (SLE).
  • CTSG The exact role of CTSG in the unanticipated function of inducing antiinflammatory M2 macrophages is not yet fully understood. However, the observations by the inventors suggest that this effect is dependent on inhibition of CTSG proteolytic activity, upregulation of CD14, and activation of NFAT-dependent signaling. The efficacy of this antibody in reducing nephritis in a mouse model of lupus suggests that a similar treatment can be applicable to several inflammatory syndromes in which inflammatory macrophages frequently predominate and mediate tissue damage in afflicted organs.
  • the present invention provides specific antibody agents that are capable of inducing formation of M2 macrophages from monocytes or bone marrow cells.
  • the invention also provides methods for generating M2 macrophages from bone marrow cells with a library of unbiased antibodies, CTSG biased antibodies, or the specific anti-CTSG antibodies exemplified herein.
  • the invention further provides methods of using the identified anti-CTSG antibodies and derivatives therefrom for treating autoimmunity and for suppressing undesired immune responses.
  • antibody refers to polypeptide chain(s) which exhibit a strong monovalent, bivalent or polyvalent binding to a given antigen, epitope or epitopes.
  • antibodies or antigen-binding fragments used in the invention can have sequences derived from any vertebrate, camelid, avian or pisces species. They can be generated using any suitable technology, e.g., hybridoma technology, ribosome display, phage display, gene shuffling libraries, semi-synthetic or fully synthetic libraries or combinations thereof.
  • antibody as used in the present invention includes intact antibodies, antigen-binding polypeptide fragments and other designer antibodies that are described below or well known in the art (see, e.g., Serafini, J Nucl. Med. 34:533-6, 1993).
  • An intact "antibody” typically comprises at least two heavy (H) chains (about 50- 70 kD) and two light (L) chains (about 25 kD) inter-connected by disulfide bonds.
  • the recognized immunoglobulin genes encoding antibody chains include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • Each heavy chain of an antibody is comprised of a heavy chain variable region (VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI , C H2 and C ⁇ 3 ⁇
  • Each light chain is comprised of a light chain variable region (VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system and the first component (Clq) of the classical complement system.
  • VH and VL regions of an antibody can be further subdivided into regions of hypervariability, also termed complementarity determining regions (CDRs), which are interspersed with the more conserved framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the locations of CDR and FR regions and a numbering system have been defined by, e.g., Kabat et ah, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, U.S. Government Printing Office (1987 and 1991).
  • Antibodies to be used in the invention also include antibody fragments or antigen-binding fragments which contain the antigen-binding portions of an intact antibody that retain capacity to bind the cognate antigen.
  • antibody fragments include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an intact antibody; (v) disulfide stabilized Fvs (dsFvs) which have an interchain disulfide bond engineered between structurally conserved framework regions; (vi) a single domain antibody (dAb) which consists of a V H domain (see, e.g., Ward et
  • Antibodies suitable for practicing the present invention also encompass single chain antibodies.
  • the term "single chain antibody” refers to a polypeptide comprising a VH domain and a VL domain in polypeptide linkage, generally linked via a spacer peptide, and which may comprise additional domains or amino acid sequences at the amino- and/or carboxyl-termini.
  • a single-chain antibody may comprise a tether segment for linking to the encoding polynucleotide.
  • a single chain variable region fragment (scFv) is a single-chain antibody.
  • a scFv Compared to the VL and VH domains of the Fv fragment which are coded for by separate genes, a scFv has the two domains joined (e.g., via recombinant methods) by a synthetic linker. This enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules.
  • Antibodies that can be used in the practice of the present invention also encompass single domain antigen-binding units which have a camelid scaffold.
  • Animals in the camelid family include camels, llamas, and alpacas.
  • Camelids produce functional antibodies devoid of light chains.
  • Fabs classical antigen-binding molecules
  • scFvs single chain variable fragments
  • the various antibodies or antigen-binding fragments described herein can be produced by enzymatic or chemical modification of the intact antibodies, or synthesized de novo using recombinant DNA methodologies, or identified using phage display libraries. Methods for generating these antibodies or antigen-binding molecules are all well known in the art. For example, single chain antibodies can be identified using phage display libraries or ribosome display libraries, gene shuffled libraries (see, e.g., McCafferty et al., Nature 348:552-554, 1990; and U.S. Pat. No. 4,946,778).
  • scFv antibodies can be obtained using methods described in, e.g., Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988.
  • Fv antibody fragments can be generated as described in Skerra and Pluckthun, Science 240: 1038-41 , 1988.
  • Disulfide- stabilized Fv fragments (dsFvs) can be made using methods described in, e.g., Reiter et al., Int. J. Cancer 67: 1 13-23, 1996.
  • single domain antibodies can be produced by a variety of methods described in, e.g., Ward et al., Nature 341 :544-546, 1989; and Cai and Garen, Proc. Natl. Acad. Sci. USA 93:6280-85, 1996.
  • Camelid single domain antibodies can be produced using methods well known in the art, e.g., Dumoulin et al., Nature Struct. Biol. 1 1 :500-515, 2002; Ghahroudi et al., FEBS Letters 414:521-526, 1997; and Bond et al., J Mol Biol. 332:643-55, 2003.
  • antigen-binding fragments e.g., Fab, F(ab') 2 or Fd fragments
  • Fab, F(ab') 2 or Fd fragments can also be readily produced with routinely practiced immunology methods. See, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1998.
  • the term "contacting" has its normal meaning and refers to combining two or more agents (e.g., polypeptides or phage), combining agents and cells, or combining two populations of different cells. Contacting can occur in vitro, e.g., mixing two polypeptides or mixing a population of antibodies with a population of cells in a test tube or growth medium. Contacting can also occur in a cell or in situ, e.g., contacting two polypeptides in a cell by coexpression in the cell of recombinant polynucleotides encoding the two
  • polypeptides or in a cell lysate.
  • Contacting can also occur in vivo inside a subject, e.g., via targeted delivery of an antibody to a specific group of cells.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same.
  • Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
  • a "ligand” is a molecule that is recognized by a particular antigen, receptor or target molecule.
  • ligands that can be employed in the practice of the present invention include, but are not restricted to, agonists and antagonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones, hormone receptors, polypeptides, peptides, enzymes, enzyme substrates, cofactors, drugs (e.g. opiates, steroids, etc.), lectins, sugars, polynucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.
  • the ligand is a molecule that specifically binds to a monocyte surface marker.
  • M2 macrophages are one of two main groups of macrophages.
  • M2 macrophages function in constructive processes like wound healing and tissue repair, and turn off damaging immune system activation by producing anti-inflammatory cytokines.
  • M2 is the phenotype of resident tissue
  • M2 macrophages produce high levels of IL-10, TGF-beta and low levels of IL-12.
  • the names Ml and M2 were chosen because Ml and M2 macrophages promote Thl and Th2 responses, respectively. Products of Thl and Th2 responses (e.g., IFN- ⁇ , IL-4) also down regulate M2 and Ml activity, respectively.
  • Monocytes are a type of leukocytes (white blood cells) and are part of the innate immune system of vertebrates including all mammals. Monocytes constitute 2% to 10% of all leukocytes in the human body. They constitute between three to eight percent of the leukocytes in the blood. Half of them are stored as a reserve in the spleen. Monocytes play multiple roles in immune function. Such roles include: (1) replenishing resident
  • Monocytes under normal states, and (2) in response to inflammation signals. Monocytes can move quickly (approx. 8-12 hours) to sites of infection in the tissues and
  • Monocytes are produced by the bone marrow from precursors called monoblasts, bipotent cells that differentiated from hematopoietic stem cells. Monocytes circulate in the bloodstream for about one to three days and then typically move into tissues throughout the body.
  • receptor broadly refers to a molecule that has an affinity for a given ligand. Receptors may-be naturally-occurring or manmade molecules. Also, they can be employed in their unaltered state or as aggregates with other species. Receptors may be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance. A typical example of receptors which can be employed in the practice of the invention is cell surface receptor.
  • subject refers to human and non-human animals (especially non- human mammals). In addition to human, it also encompasses other non-human animals such as cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys.
  • target refers to a molecule or biological cell of interest that is to be analyzed or detected, e.g., a ligand such as a cytokine or hormone, a polypeptide, a cellular receptor or a cell.
  • a cell has been "transformed” by exogenous or heterologous polynucleotide when such polynucleotide has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.
  • the transforming polynucleotide may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming polynucleotide has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • the invention provides novel M2 macrophage-inducing antibodies that can convert bone marrow cells (e.g., CD34 + cells) or monocytes into anti-inflammatory macrophage.
  • These agonist antibodies have the same binding specificity as that of a specific anti-CTSG scFv antibody (“LKAb") identified herein.
  • LKAb specific anti-CTSG scFv antibody
  • antibody LKAb can induce M2 macrophage formation from both human and mouse bone marrow cells (see, e.g., Figs. 3 and 5).
  • This scFv antibody has an amino acid sequence shown in SEQ ID NO: 1.
  • the sequences of the heavy chain and the light chain portions of the scFv are respectively shown in SEQ ID NOs:2 and 3.
  • the CDR sequences of the heavy chain variable region of this antibody are GYTFTSYY (SEQ ID NO:4), IIPIFGTG (SEQ ID NO:5), and AREEEQQF SLD Y (SEQ ID NO:6).
  • the CDR sequences of its light chain variable region are SGSIASNS (SEQ ID NO:7), ENN, and QSYDSNFHWV (SEQ ID NO:8).
  • the antibodies of the invention for inducing M2 macrophage are preferably monoclonal antibodies like the antibodies exemplified in the Examples below.
  • the antibodies have the same binding specificity as that of the LKAb agonist antibody and compete with the LKAb antibody for binding to Cathepsin G.
  • some agonist antibodies of the invention can also contain other antibody sequences fused to the variable region sequences.
  • the antibodies can contain an Fc portion of IgG.
  • the antibodies can also be conjugated, covalently or noncovalently, to another entity that specifically targets a surface antigen, receptor or marker on monocyte cells.
  • Some M2 macrophage-inducing agonist antibodies of the invention harbor variable region sequences that are substantially identical (e.g., at least 90% or 95% identical) to that of the LKAb antibody.
  • Some other agonist antibodies have all CDR sequences in their variable regions of the heavy chain and light chain that are respectively identical or substantially identical (e.g., at least 90% or 95% identical) to the corresponding CDR sequences of the LKAb agonist antibody.
  • the agonist antibody has its entire heavy chain and light chain variable region sequences respectively identical to the corresponding variable region sequences of the LKAb antibody.
  • the antibodies contain amino acid residues in the framework portions of the variable regions that are different from the corresponding amino acid residues of the LKAb antibody.
  • the agonist antibodies of the invention can undergo non-critical amino-acid substitutions, additions or deletions in the variable region without loss of binding specificity or effector functions, or other modifications that do not cause intolerable reduction of binding affinity or CTSG-inhibiting activities.
  • antibodies incorporating such alterations exhibit substantial sequence identity to the LKAb antibody.
  • the mature light chain variable regions of some of the agonist antibodies of the invention have at least 75%, at least 85% or at least 90% sequence identity to the sequence of the mature light chain variable region of the LKAb antibody.
  • the mature heavy chain variable regions of the antibodies typically show at least 75%, at least 85% or at least 90% sequence identity to the sequence of the mature heavy chain variable region of the LKAb antibody.
  • the antibodies typically have their entire variable region sequences that are substantial identical (e.g., at least 75%, 85%, 90%, 95%, or 99%) to the corresponding variable region sequences of the LKAb antibody.
  • Some M2 macrophage-inducing agonist antibodies of the invention have the same binding specificity but improved affinity or CTSG-inhibiting activities if compared with the LKAb antibody.
  • the agonist antibodies of the invention can be generated in accordance with routinely practiced immunology methods. Some of such methods are exemplified herein in the Examples.
  • M2 macrophages are generated from monocytes or bone marrow cells (e.g., CD34 + cells) with a specific M2 macrophage-inducing antibody. Any of the agonist antibodies described herein can be used in the therapeutic methods of the invention for generating M2 macrophages in vivo or in vitro.
  • M2 macrophages can be further divided into subsets M2a, M2b, and M2c based on gene expression profiles (Mantovani et al., Trends Immunol. 25:677-86, 2004).
  • the M2a subtype is elicited by IL-4 or IL-13 (common IL-4R- alpha, CD124).
  • the M2b is elicited by IL-1R ligands or exposure to immune complexes plus LPS.
  • the M2c subtype can be elicited by IL-10, TGF-beta and glucocorticoid hormones.
  • the fourth type of macrophage M2d is characterized by an IL-l Ohigh, IL-121ow M2 profile with some features of tumor-associated macrophages (TAMs).
  • TAMs tumor-associated macrophages
  • the M2d subtype has phenotypic and functional attributes similar to ovarian TAMs but distinct from M2a-c.
  • the M2 macrophages produced by methods of the invention can include one or more of the subsets of M2 macrophages.
  • the methods involve contacting a M2 macrophage-inducing antibody described herein with a population of monocytes or monocyte precursor cells (e.g., bone marrow cells) isolated from a subject under appropriate conditions as exemplified herein to facilitate the conversion.
  • monocytes or monocyte precursor cells e.g., bone marrow cells
  • the monocyte-containing samples can be maintained and cultured in any physiologically-acceptable solution suitable for the collection and/or culture of the cells.
  • human bone marrow cells can be cultured in StemSpan serum-free media (STEMCELL Technologies) as exemplified herein.
  • Suitable culture media include, e.g., a saline solution (e.g., phosphate-buffered saline, Kreb's solution, modified Kreb's solution, Eagle's solution, 0.9% NaCl, etc.), and a culture medium (e.g., DMEM, H.DMEM, etc.), and the like.
  • the solution can contain one or more tissue-degrading enzymes, e.g., a metalloprotease, a serine protease, a neutral protease, a hyaluronidase, an RNase, or a DNase, or the like.
  • Such enzymes include, but are not limited to, collagenases (e.g., collagenase I, II, III or IV, a collagenase from Clostridium histolyticum, etc.); dispase, thermolysin, elastase, trypsin, LIBERASE, hyaluronidase, and the like.
  • the solution can further include a bacteriocidal ly or bacteriostatically effective amount of an antibiotic.
  • antibiotics examples include a macrolide (e.g., tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime, cefprozil, cefaclor, cefixime or cefadroxil), a clarithromycin, an erythromycin, a penicillin (e.g., penicillin V) or a quinolone (e.g., ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, a streptomycin, and etc.
  • a macrolide e.g., tobramycin
  • cephalosporin e.g., cephalexin, cephradine, cefuroxime, cefprozil, cefaclor, cefixime or cefadroxil
  • a clarithromycin an erythromycin
  • a penicillin e.g., penicillin V
  • a quinolone e.g
  • the isolated or cultured monocyte or precursor cells can also be contacted with another agent that promotes monocyte differentiation into M2 macrophage.
  • the population of monocytes or precursors can be stimulated with the cytokines noted above for eliciting different M2 subtypes (e.g., IL-4 or IL-10).
  • M2 subtypes e.g., IL-4 or IL-10
  • a cytokine cocktail for inducing M2 macrophage from human bone marrow cells is exemplified herein.
  • the cells can also be stimulated with macrophage colony stimulating factor (M-CSF).
  • M-CSF macrophage colony stimulating factor
  • M-CSF has been shown to polarize monocytes towards M2 macrophages (Jaguin et al., Cell Immunol. 281 :51-61, 2013).
  • contacting the monocyte cell population with the stimulating cytokine such as M-CSF can be performed prior to, simultaneously with or subsequent to treatment with the antibody.
  • concentration of each of the cytokines or agents in the culture medium can be in the range of, e.g., about 0.01 to 1 x 10 s U/ml.
  • the culture medium can further contain serum or plasma.
  • the amount of serum or plasma can be present at concentration of, e.g., about 0 to 20% by volume, preferably more than 0 to 10% by volume.
  • the in vitro induced M2 macrophage population can be further subject to analysis for expression of M2 markers and enrichment for M2 macrophages.
  • the cells can be examined for expression of "anti-inflammatory" cytokine expression profile, which includes high levels of IL-10 and IL-1RA and low expression of IL-12.
  • M2 macrophages also express high levels of scavenger mannose and galactose receptors.
  • the cells can also be examined for cytokine profiles indicative of the different subtypes of M2 macrophages.
  • M2a macrophages produce IL-10, TGFp and IL-lra
  • M2b macrophages produce IL-1 , IL-6, IL-10 and TGFa
  • M2c macrophages produce IL-10 and TGFp
  • M2d macrophages produce IL-10, IL-12, TNFa and TNFp.
  • M2 macrophages also have distinct chemokine and chemokine receptor profiles unlike that of Ml macrophages.
  • M2 macrophages expresses chemokines CCL17, CCL22 and CCL24 while Ml macrophages secret the TH1 cell-attracting chemokines CXCL9 and CXCL10.
  • the culture of a cell population in the presence of the agonist antibody and/or other agents can be performed in accordance with standard cell culturing protocols well known in the art. Some specific procedures for generating M2 macrophages from bone marrow cells are exemplified herein.
  • the isolated bone marrow cells or monocyte population are contacted with an effective amount of an antibody having the same binding specificity as that of LKAb agonist antibody under appropriate conditions to facilitate the conversion.
  • the antibody is contacted with the cells in vitro.
  • the cell population can be cultured at a concentration of about lxl O 2 , lxlO 3 , lxl O 4 , lxlO 5 , lxlO 6 , lxlO 7 , lxlO 8 cells/ml or higher.
  • the bone marrow cells or monocytes can be cultured in the presence of an effective amount of the antibody, e.g., at a concentration of 0.1 , 0.25, 0.5, 1.0, 2.5, 5.0, 10, 25, 50, 100 ⁇ g/ml or higher.
  • the contacting can last for a sufficient period of time, e.g., at least 12 hours, 1 day, 2 days, 4 days, 6 days, 10 days, 25 days, 50 days, 75 days, 100 days or longer.
  • the cells can be examined along the process for molecular markers and/or morphology indicative of the presence of M2 macrophages as exemplified herein.
  • the cell culture may be performed under known culture conditions, and the conditions which are used in normal cell culture can be applied.
  • culture can be performed under the conditions of 37°C and 5% C0 2 .
  • Cells can be diluted by adding a fresh medium to a cell culture liquid at a suitable time interval, a medium can be exchanged with a fresh medium, or a cell culture instrument can be exchanged.
  • the culturing period for converting the bone marrow cells into M2 macrophages can be, e.g., from 1 to 100 days, 2 to 75 days, 3-50 days, or 4-25 days.
  • the bone marrow cells are cultured in the presence of the agonist antibody and other agents for a period of around 4 days, 5 days, 6 days, 7 days, or 8 days.
  • Any cell culture instrument can be used in the invention. These include, e.g., a petri dish, a flask, a bag, a bioreactor etc. can be used.
  • a C0 2 gas permeable bag for cell culture can be used.
  • use of a bioreactor is advantageous.
  • the cell culturing can be performed in either an open system or a closed system, it is preferable to perform the culturing in a closed system from a view point of safety of the resulting M2 macrophages.
  • M2 macrophages can be generated in vitro from monocytes or bone marrow cells with a library of antibodies as exemplified herein.
  • the employed antibodies are single chain antibodies such as scFvs.
  • the antibody library can be either an unbiased library of antibodies or a biased library of antibodies (e.g., CTSG biased antibodies).
  • the cells e.g., CD34 + bone marrow cells
  • the cells can be contacted with the antibodies by introducing vectors expressing the antibody library (e.g., lentiviral vectors) into the cells. After expressing the antibodies in the cells and culturing the cells under appropriate conditions, cells bearing M2 macrophage morphology and/or cellular markers can then be selected.
  • Some related embodiments of the invention are directed to identifying antibodies (e.g., intact immunoglobulin molecules or scFvs) from a library containing antibody variable fragments that alters the morphology and function of bone marrow cells (e.g., CD34 + bone marrow cells).
  • the selected M2 macrophages are further manipulated to isolate the specific antibody expressed therein.
  • these novel M2-inducing antibody agents can be used in various therapeutic applications described herein.
  • Detailed methods for introducing antibody-expressing vectors or viruses into monocytes or bone marrow cells and for selecting M2 macrophages can be based on routinely practiced protocols and the specific procedures exemplified herein.
  • the invention provides methods and compositions for treating autoimmune diseases and other diseases (e.g., some inflammatory disorders) via the use of agonist antibodies described herein to induce formation of M2 microphages.
  • autoimmune diseases and other diseases e.g., some inflammatory disorders
  • treatment with the LKAb antibody resulted in therapeutic effects in subjects afflicted with systemic autoimmunity such as lupus (see, e.g., Example 6).
  • systemic autoimmunity such as lupus
  • the ability to induce a population of monocytes or monocyte precursors (e.g., from bone marrow cells) to become M2 macrophages enables production of anti-inflammatory cytokines that are beneficial for therapeutic applications.
  • some embodiments of the invention provide pharmaceutical compositions containing a M2-inducing agonist antibody or a population of in vitro induced M2 macrophage as described herein. These pharmaceutical compositions are suitable for administration to patients so that the converted M2 macrophages can recognize and kill the neighboring diseased cells.
  • a recognition element such as an antibody can be expressed on the M2 macrophage surface to provide more specific and/or stronger cytotoxicity against target cells.
  • M2 macrophage populations induced with the agonist antibody and further enriched in vitro can be administered to subjects suffering from autoimmune diseases or other conditions with undesired immune responses.
  • the pharmaceutical composition of the invention can be used alone or in combination with other known agents in the art for treating autoimmune diseases or other inflammatory disorders.
  • compositions containing the M2 macrophage-inducing antibodies described herein or containing M2 macrophages produced by the method of the present invention may be used for treatment of various autoimmune diseases, inflammatory disorders and other conditions with undesired immune responses.
  • diseases or conditions include, e.g., asthma, autoimmune diseases, chronic inflammation, chronic prostatitis, gomerulonephritis, hypersensitivities, inflammatory bowel diseases, pelvic inflammatory diseases, reperfusion injury, rheumatoid arthritis, transplant rejection and vasculitis.
  • inflammatory disorders include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, acute respiratory distress syndrome (ARDS), alopecia areata, anklosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (alps), autoimmune
  • thrombocytopenic purpura ATP
  • Blehcet's disease bullous pemphigoid
  • cardiomyopathy celiac sprue-dermatitis
  • chronic fatigue syndrome immune deficiency syndrome (CFIDS)
  • chronic inflammatory demyelinating polyneuropathy cicatricial pemphigoid
  • cold agglutinin disease Crest syndrome, Crohn's disease
  • Dego's disease dermatomyositis
  • the therapeutic methods of the invention typically involve administering to a subject a pharmaceutical composition that contains an M2-inducing agonist antibody described herein or a population of M2 macrophages.
  • the subject is a human patient.
  • the agonist antibody is contacted with monocytes or precursor cells in vivo upon being administered to the subject to induce formation of M2 macrophage.
  • a therapeutically effective amount of the antibody can be administered to a subject afflicted with an autoimmune disease or other inflammatory disorder.
  • M2 macrophages can be induced which in turn can exert antiinflammatory activities.
  • the antibody can be conjugated to a moiety that specifically recognizes a surface marker on the monocytes or monocytes precursors to facilitate targeted delivery of the agonist antibody, e.g., a ligand for a monocyte specific surface marker that is well known in the art (e.g., CD14).
  • a bispecific antibody comprised of the antigen-binding site of the agonist antibody and also the antigen- binding site of a second antibody recognizing a monocyte specific surface marker can be used.
  • Such immune conjugates or bispecific antibodies can be generated using standard procedures routinely practiced in the art.
  • subjects with autoimmune diseases or other undesired immune responses can be treated ex vivo.
  • the pharmaceutical composition to be administered to a subject contains a population of M2 macrophages generated in vitro via the methods described herein.
  • a bone marrow sample or a population of monocytes can be first obtained from the subject in need of treatment.
  • the cells are then contacted in vitro with a M2-inducing antibody described herein.
  • the induced M2 macrophages are then administered to the subject in a pharmaceutical composition.
  • the bone marrow cells for inducing M2 macrophage formation with the agonist antibody are obtained from the same subject into whom the resulting M2 macrophage population will be administered.
  • Some preferred embodiments of the invention are directed to treating human subjects afflicted with or suspected of having autoimmune disorders.
  • autoimmune disorders and related diseases suitable for the methods of the invention include, e.g., Acute disseminated encephalomyelitis (ADEM), Addison's disease, Alopecia areata, Ankylosing spondylitis, Antiphospholipid antibody syndrome (APS), Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune inner ear disease, Bullous pemphigoid, Behcet's disease, Coeliac disease, Chagas disease, Chronic obstructive pulmonary disease, Crohn's Disease, Dermatomyositis, Diabetes mellitus type 1 ,
  • GVHD Guillain-Barre syndrome
  • GRS Guillain-Barre syndrome
  • MS Hashimoto's disease
  • Hidradenitis suppurativa Kawasaki disease
  • Idiopathic thrombocytopenic purpura Interstitial cystitis
  • Lupus erythematosus Mixed Connective Tissue Disease
  • Morphea Multiple sclerosis
  • MS Myasthenia gravis
  • Narcolepsy Neuromyotonia, Pemphigus vulgaris, Pernicious anaemia, Psoriasis, Psoriatic Arthritis, Polymyositis, Primary biliary cirrhosis, Rheumatoid arthritis, Schizophrenia, Scleroderma, Sjogren's syndrome, Stiff person syndrome, Temporal arteritis (aka "giant cell arteritis"), Ulcerative Colitis, Vasculitis, Vitiligo, Microscopic polyangiitis, Glomerulonephritis, and Wegener
  • M2 macrophage populations generated in vitro by the methods described herein and therapeutic applications of such M2 macrophage populations.
  • the induced cell population is preferably enriched for M2 macrophages.
  • M2 macrophages can be first isolated from the induced cell population noted above, and then further enriched in vitro before being administered to a subject in need of treatment of a disease or disorder (e.g., autoimmune diseases). Isolation and enrichment of M2 macrophages from the induced cell population can be readily carried out with methods well known in the art. For example, activated M2 macrophages can be isolated or selected for cells expressing M2 macrophage specific markers.
  • Such markers expressed by human M2 macrophage include, e.g., CD36 (aka “SR”, scavenger receptor), MHCII, ARG-1 (arginase- 1 ), CD 14, CD 163, CD206, TGM2 (transglutaminase 2), DecoyR, IL-1R II, CD86, VEGF, TLR1 and TLR8.
  • Enrichment can also include staining cells from the induced cell population or previously selected cells with antibodies recognizing M2 macrophage specific markers (e.g., CD36 and ARG-1), and selecting for cells with high expression of Ml macrophage specific markers (e.g., ARG-1) and low expression of markers for other type of cells (e.g., Ml macrophage markers such as IDO-1).
  • M2 macrophages can also be isolated or enriched by removal of cells other than M2 macrophages in the induced cell population.
  • human M2 macrophages may be isolated or enriched by depletion of cells displaying non-M2 macrophage markers using, e.g., antibodies targeting such markers.
  • markers include, e.g., CD1 lb, CD80, CD86, CD16/32, IL-1R I, TLR2, TLR4 and iNOS. Protocols reported in the literature can also be used and modified for isolation and enrichment of M2 macrophages in the methods of the present invention. See, e.g., Gabrilovich et al, Nat. Rev. Immunol. 12, 253-268, 2012, Kitani et al, Immonol. 4, 1 -7, 2014; and Yang et al, Mol. Endocrinol. 28:565-574, 2014.
  • M2 macrophage populations of the invention typically contain at least 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 98% or more of homogeneous or
  • heterogeneous M2 macrophages that express one or more of the specific M2 markers described herein.
  • kits or pharmaceutical combinations for converting bone marrow cells or monocyte cells into M2 macrophages.
  • the kits typically contain one or more agonist antibodies described herein, tools and materials for isolating bone marrow cells or monocyte populations from a subject, and reagents for co-culturing monocytes or precursor cells with the agonist antibody.
  • the kits can contain the agonist antibody and a cultured monocyte cell population for generating M2 macrophages that can be applied allogeneically to subjects afflicted with autoimmune diseases or other inflammatory disorders.
  • compositions containing an agonist antibody or a M2 macrophage population described herein can be administered to subjects in need of treatment in accordance with standard procedures of pharmacology.
  • Methods of administering the therapeutic compositions to a subject can be accomplished based on procedures routinely practiced in the art. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co, 20 th ed, 2000; Ritter et al, J. Clin. Invest. 1 16:3266-76, 2006; Iwasaki et al. Jpn. J. Cancer Res. 88:861-6, 1997; Jespersen et al., Eur. Heart J.
  • a composition containing the induced M2 macrophages are typically administered (e.g., via injection) in a physiologically tolerable medium, such as phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the isolated cells, or their engineered form as disclosed herein, should be administered to the subject in a number sufficient to inhibit the development of the disease in the subject.
  • administration of therapeutic composition is carried out by local or central injection of the cells into the subject.
  • the administration is via a systemic route such as peripheral administration. Additional guidance for preparation and administration of the pharmaceutical compositions of the invention are described in the art.
  • Example 1 Selection system for identifying M2 macrophage-inducing antibodies
  • Fig. 1A The unbiased antibody library in lentiviruses, which contains about 10 s unique members, was used to infect total mouse bone marrow cells. The infected cells were plated onto soft agar to observe the formation of colonies (Fig. IB). Large colonies were harvested, and the morphology of the cells therein was determined. All colonies contained cells with the classical morphology of macrophages (Fig. 1C, D). The antibody gene integrated into the cells was recovered by PCR. One sequence was repeated in 7 colonies and this antibody was chosen for further studies.
  • This antibody gene was reinserted into lentivirus and fresh cells were infected. These cells, now infected with a single lentivirus, also formed colonies (Fig. I E) and contained cells with a morphology that was similar to those selected after the first round of infection.
  • Fig. I E colonies
  • Fig. I E the encoding gene was transferred to a mammalian expression vector.
  • the purified antibody was incubated with bone marrow, a large percentage of the cells attached to the plate and developed a macrophage morphology (Fig. IF).
  • LKAb antibody was incubated with an extract of total mouse bone marrow cells and the immune complexes were captured on a protein A/G column. Proteins that reacted with the antibody were identified by silver staining of SDS gels and their identity determined by mass spectrometry (Fig. 7A, B). Four candidate proteins based on the number of peptide "hits" were tested for their ability to bind to antibody LKAb (Fig. 7C). Out of the candidate antigens, only Cathepsin G (CTSG) bound to LKAb as determined by Western blotting and ELISA analyses (data not shown).
  • CTSG Cathepsin G
  • CTSG is known to be secreted by activated leukocytes, but it has also been shown to be present on the surface of monocytic cells.
  • CTSG was confirmed as the target antigen by comparing LKAb to a commercial anti-CTSG antibody using Western blotting of an extract from cells over- expressing CTSG (Fig. 2A).
  • the commercial anti-CTSG antibody also reacted strongly with a protein from mouse bone marrow extracts that was first captured by our antibody (Fig. 2B).
  • the protein fraction captured by our antibody was shown to have CTSG activity (Fig. 2C).
  • Fig. 2D the ability of our antibody to induce macrophage formation was markedly reduced.
  • cytokine IL-10 is another feature of M2 macrophages.
  • LKAb i.p. into C57BL/6 and BALB/c mice and found significantly higher levels of IL-10 in treated compared to control mice (Fig. 3E).
  • cell lysates were assessed by Western blotting using anti-phospho p38, ERK and AKT. ERK and AKT were activated to some extent, but p38 was strongly activated, consistent with its known role in M2 macrophage polarization (Fig. 8).
  • CTSG While CTSG is located on the surface of macrophages and polymorphonuclear leukocytes, it lacks a signal transduction domain, and thus its role in cell activation is indirect. Nevertheless, there is an increasing awareness of the role that CTSG plays in the regulation of important cell surface receptors. In particular, CTSG has been reported to regulate monocyte activation by down-regulation of CD 14, which is a critical co-receptor for TLR4 in the innate immune system and is involved in differentiation of cells in the macrophage lineage.
  • TLR4 Since CD 14 and TLR4 are co-receptors, we studied whether TLR4 was also required for LKAb-mediated macrophage differentiation. Using bone marrow cells from wild-type and TLR4 (-/-) mice, we showed that the cell polarizing function of our antibody was TLR4-independent (Fig. 9). The finding that LKAb required CD 14 but not TLR4 to induce macrophage differentiation left open the question of the mechanism of signal transduction. Unlike TLR4, CD14 does not have a signal transduction domain. However, recently dendritic cells have been reported to be activated through a NFAT signaling pathway that is dependent on CD14 but independent of TLR4.
  • NFAT knockout mice to determine whether this pathway plays a role in macrophage differentiation induced by our antibody.
  • Bone marrow cells from NFATcl (-/-) and NFATc2 flox flox : CD4 Cre mice were incubated with LKAb and analyzed by FACS using the CD 14 and F4/80 markers.
  • NFATcl (-/-) mice Fig. 4A.
  • NFAT inactivation in non-macrophage cells such as CD4 expressing T cells and dendritic cells
  • Fig. 4C we have a picture of CTSG's role in macrophage polarization suggesting that this molecule operates through CD 14 and NFAT to regulate macrophage activation and differentiation.
  • Example 5 Inducing M2 macrophages from human bone marrow
  • Mouse strains and cell lines The following mouse strains were used: C57BL/6J, BALB/c, C3H/HeJ, C3H/HeOuJ, C57BL/10ScNJ, B6.129S4-CD14 tm l fm /J and MRL/MPJ- Fas (lpr)/J (The Jackson laboratory).
  • CTSG knockout mice were the generous gift of Dr. Christine Pham (Washington University, St. Louis, MO).
  • NFATcl (-/-) and ⁇ 2 ⁇ / ⁇ : CD4 Cre mice were the generous gift of Dr. Anjana Rao (La Jolla institute for allergy and immunology).
  • the F1EK293T cell line was maintained in DMEM medium containing 10% FCS, penicillin and streptomycin (Gibco-Invitrogen).
  • the HEK293F cell line was maintained in Freestyle 293 Expression Media with 4 mM Glutamax (Gibco-Invitrogen).
  • human total bone marrow cells All- Cells were cultured in StemSpan serum-free media with cytokine cocktail (STEMCELL Technologies) and the antibody (10 ⁇ g/ml) at 37°C.
  • mice were similarly cultured, except that the cells were in the DMEM/F12 medium containing 10% FCS, penicillin and streptomycin (Gibco-Invitrogen). Mice were housed and handled according to protocols approved by the Institutional Animal Care and Use Committee at The Scripps Research Institute.
  • Combinatorial antibody library and lentivirus Single-Chain Fv (ScFv) genes were obtained from a naive human combinatorial antibody library (1 ⁇ 10 1 1 library diversity). ScFv genes were sub-cloned into a lentiviral vector. Lentivirus was produced in HEK293T cells by co-transfection of lentiviral vectors with the pCMVD8.91 and pVSVg viral packaging vectors at a ratio of 1 : 1 : 1. Supernatants containing virus were collected at 48 h post-transfection. Cell debris was removed by centrifugation and filtration through a 0.22- ⁇ polyethersulfone membrane filter unit from Millipore. The titer of the lentivirus preparation was determined using a Lenti-X p24 ELISA (Clontech). The virus preparations were aliquoted and frozen at -80 °C.
  • Transduction and colony forming cell assay The bone marrow cells were incubated with lentivirus for 3 days at 37 °C. Selection of agonist antibodies was done by a Colony-Forming Cell Assay using Methylcellulose-Based Media. Bone marrow cells were transduced with the lentiviral antibody library at a multiplicity of infection of 2 and added to methylcellulose media at the final concentrations of 1.27% methylcellulose and ⁇ 3 ⁇ 10 4 cells/mL. A total of 1.5 mL of cell suspension was added to 35-mm-diameter dishes. The cells in soft agar were cultured for 2 wk. The colonies were harvested with the aid of a micromanipulator (Sutter Instruments). The antibody genes from each colony were amplified by PCR with primer pairs customized for our lentiviral vector. The amplified antibody genes were analyzed by electrophoresis and recovered.
  • the antibody expression vector was transfected into HEK293F cells. Antibodies from the pooled supernatants were purified using HiTrap Protein G HP columns with an AKTAxpress purifier (GE). The buffer was exchanged to Dulbecco's PBS (pH 7.4) and stored at 4 °C. The vector encoding the ScFv-Fc tag fusion protein was transfected into HEK293F cells for transient expression.
  • Immunoprecipitation and Mass Spectrometry For immunoprecipitation, mouse bone marrow cells were prepared and solubilized in lysis buffer. The lysates were incubated with LKAb for 2 hours at 4°C, followed by incubation with 50 ⁇ of protein G-Sepharose beads (Pierce). The eluent was introduced into the linear trap quadrupole mass spectrometer from a nano-ion source with a 2-kV electrospray voltage. The analysis method consisted of a full MS scan with a range of 400-2,000 m/z followed by data-dependent MS/MS on the three most intense ions from the full MS scan. The raw data from the linear trap quadrupole were searched using the IPI human FASTA database with the MASCOT
  • Silencing CTSG mRNA Mouse CTSG shRNAs (Santa Cruz Biotechnology) used for producing lentiviral particles contain three target-specific constructs designed to knock down gene expression. Mouse bone marrow cells were infected with Lentivirus CTSG shRNA and cultured with LKAb or M-CSF for 6 days.
  • CD200R (BD Biosciences). Stained cells were analyzed with a LSRII flow cytometer (Becton Dickinson). To obtain CD1 lb positive and negative bone marrow cells, CD1 lb +
  • CTSG enzymatic activity To monitor enzyme activities, we used an assay based on Cathepsin G-mediated cleavage of a specific substrate and release of the dye group pNA (4-Nitroaniline). This assay was performed with mouse bone marrow lysates that were incubated with LKAb or CTSG inhibitor.
  • Cytokine assay Cytokines were quantified in serum collected from individual mice using a mouse cytokine magnetic bead panel (EMD Millipore) according to
  • MRL-Fas lpr mice were treated i.p. with LKAb (75 ⁇ g/mouse) two times per week. Treatments were initiated at 6 weeks of age and the experiment was terminated at 20 weeks of age. Total and anti-chromatin serum IgG subclasses were assessed by ELISA using 96-well plates coated with goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) and mouse chromatin, respectively. Bound antibodies were detected using alkaline phosphatase-conjugated goat antibodies (Caltag Laboratories) to mouse IgG or IgG2a, the main autoantibody subclass in this model.
  • Statistical analysis The data are expressed as the means ⁇ SE. Statistical analysis was performed using the Student t test or by one-way analysis of variance and the post hoc test. The groups were analyzed by unpaired two-tailed Student's t test. Survival was analyzed by Kaplan-Meier plot and log rank test. P values of ⁇ 0.05 were considered significant.

Abstract

L'invention concerne des anticorps anti-cathepsine G spécifiques qui permettent d'induire la formation de macrophages anti-inflammatoires. L'invention concerne également des procédés permettant de produire des macrophages anti-inflammatoires. Les procédés consistent à mettre en contact des cellules de moelle osseuse ou des monocytes avec un anticorps de cathepsine G décrit dans la description de l'invention, et à mettre en culture le mélange de cellules dans des conditions permettant la formation de macrophages M2. L'invention concerne également des méthodes thérapeutiques d'utilisation d'une composition pharmaceutique contenant un anticorps de cathepsine G ou des macrophages M2 induits décrits dans la description de l'invention pour traiter des maladies auto-immunes et d'autres troubles associés à des réponses immunitaires indésirables.
PCT/US2016/051290 2015-09-15 2016-09-12 Anticorps pour la production de macrophages anti-inflammatoires et utilisations associées WO2017048629A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2016324021A AU2016324021A1 (en) 2015-09-15 2016-09-12 Antibodies for generating anti-inflammatory macrophage and related uses
EP16847105.0A EP3349768A4 (fr) 2015-09-15 2016-09-12 Anticorps pour la production de macrophages anti-inflammatoires et utilisations associées

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201562218785P 2015-09-15 2015-09-15
US62/218,785 2015-09-15

Publications (1)

Publication Number Publication Date
WO2017048629A1 true WO2017048629A1 (fr) 2017-03-23

Family

ID=58289788

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2016/051290 WO2017048629A1 (fr) 2015-09-15 2016-09-12 Anticorps pour la production de macrophages anti-inflammatoires et utilisations associées

Country Status (3)

Country Link
EP (1) EP3349768A4 (fr)
AU (1) AU2016324021A1 (fr)
WO (1) WO2017048629A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116445407A (zh) * 2023-06-16 2023-07-18 广州正源生物技术有限公司 一种胎盘巨噬细胞的提取和扩增培养方法
WO2023146860A1 (fr) * 2022-01-28 2023-08-03 The George Washington University Compositions et méthodes de traitement d'un sujet ayant une inflammation

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060272041A1 (en) * 2001-09-24 2006-11-30 Wisotzkey Robert G Cathepsin G (CTSG) Disruptions, Compositions and Methods Relating Thereto
US7786269B2 (en) * 2001-12-04 2010-08-31 Dana-Farber Cancer Institute, Inc. Antibody to latent membrane proteins and uses thereof
US20110117086A1 (en) * 2009-10-16 2011-05-19 Bioréalités S.A.S. Monoclonal antibodies to progastrin and their uses
US20130142760A1 (en) * 2010-08-18 2013-06-06 Cedars-Sinai Medical Center Atherosclerosis inhibition via modulation of monocyte-macrophage phenotype using apo a-i milano gene transfer
US20140114054A1 (en) * 2011-05-09 2014-04-24 University Of Miyazaki Antibody capable of specifically recognizing transferrin receptor

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060272041A1 (en) * 2001-09-24 2006-11-30 Wisotzkey Robert G Cathepsin G (CTSG) Disruptions, Compositions and Methods Relating Thereto
US7786269B2 (en) * 2001-12-04 2010-08-31 Dana-Farber Cancer Institute, Inc. Antibody to latent membrane proteins and uses thereof
US20110117086A1 (en) * 2009-10-16 2011-05-19 Bioréalités S.A.S. Monoclonal antibodies to progastrin and their uses
US20130142760A1 (en) * 2010-08-18 2013-06-06 Cedars-Sinai Medical Center Atherosclerosis inhibition via modulation of monocyte-macrophage phenotype using apo a-i milano gene transfer
US20140114054A1 (en) * 2011-05-09 2014-04-24 University Of Miyazaki Antibody capable of specifically recognizing transferrin receptor

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HAN, KH ET AL.: "An Agonist Antibody that Blocks Autoimmunity by Inducing Anti-Inflammatory Macrophages.", FASEB JOURNAL., vol. 30, no. 2, 19 October 2015 (2015-10-19), pages 738 - 747, XP055368761 *
ITALIANI, P ET AL.: "From Monocytes to M1/M2 Macrophages: Phenotypical vs. Functional Differentiation.", FRONTIERS IN IMMUNOLOGY., vol. 5, no. 514, 17 October 2014 (2014-10-17), pages 1 - 22, XP055368757 *
MIA, S ET AL.: "An optimized Protocol for Human M2 Macrophages using M-CSF and . IL -4/ IL -10/TGF-beta Yields a Dominant Immunosuppressive Phenotype.", SCANDINAVIAN JOURNAL OF IMMUNOLOGY ., vol. 79, no. 5, May 2014 (2014-05-01), pages 305 - 314, XP055163193 *
See also references of EP3349768A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023146860A1 (fr) * 2022-01-28 2023-08-03 The George Washington University Compositions et méthodes de traitement d'un sujet ayant une inflammation
CN116445407A (zh) * 2023-06-16 2023-07-18 广州正源生物技术有限公司 一种胎盘巨噬细胞的提取和扩增培养方法
CN116445407B (zh) * 2023-06-16 2023-09-08 广州正源生物技术有限公司 一种胎盘巨噬细胞的提取和扩增培养方法

Also Published As

Publication number Publication date
EP3349768A1 (fr) 2018-07-25
EP3349768A4 (fr) 2019-01-23
AU2016324021A1 (en) 2018-04-12

Similar Documents

Publication Publication Date Title
US20210095012A1 (en) Anti-semaphorin 3a antibody and treatment of alzheimer's disease and inflammatory immune diseases using same
KR102362609B1 (ko) 항garp 단백질 항체와 그 용도
EP3085773B1 (fr) Utilisation d'anticorps il-1 alpha
JP2021184710A (ja) 重症熱性血小板減少症候群ウイルスの外膜糖タンパク質に結合する抗体及びその用途
US9944700B2 (en) Notch2 binding molecules for treating respiratory diseases
EP3024849A2 (fr) Nouveaux anticorps pour le diagnostic et le traitement de la polyarthrite rhumatoïde
CN114075289A (zh) 抗cd73的抗体及其用途
WO2016090035A2 (fr) Modulateurs de l'activine et procédés de modulation de réponses immunitaires et de cellules t auxiliaires folliculaires
Lameris et al. Generation and characterization of CD1d‐specific single‐domain antibodies with distinct functional features
EP3349768A1 (fr) Anticorps pour la production de macrophages anti-inflammatoires et utilisations associées
WO2016205784A1 (fr) Procédés et compositions pour produire des cellules tueuses naturelles activées, et utilisations associées
JP6742581B2 (ja) 標的細胞障害活性を有するｉＰＳ／ＥＳ細胞特異的抗体及びその用途
CN107849134B (zh) 用抗ccr4抗体介导细胞因子表达的方法
CN115724971A (zh) 重组抗人cd25抗体及其应用
US20190048086A1 (en) Anti-citrullinated hla polypeptide antibodies and uses thereof
CN114258402A (zh) 抗cd123抗体、抗cd123嵌合抗原受体和抗cd123嵌合抗原受体t细胞
US20200140851A1 (en) Methods and Compositions Related to In Vivo Selection of Functional Molecules
US20230159652A1 (en) Transferrin receptor 1 targeting for carcinogenesis prevention
CN116948023B (zh) Tau蛋白抗体及其应用
CN113698480B (zh) 一组il-23单克隆抗体及其医药用途
CN112062848B (zh) 抗cd47单克隆抗体及其应用
JP7181523B2 (ja) 糖鎖をエピトープとして特異的に認識する新規抗体及びその用途
WO2022246021A1 (fr) Compositions et méthodes pour le traitement du cancer
Noto The Role of Type 2 Cytokines and Mast Cells in the Development of Gastric Metaplasia
KR20240038198A (ko) 인슐린 분비 베타세포 분화 유도 항체 및 이를 유효성분으로 포함하는 대사성 질환 예방 또는 치료용 조성물

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16847105

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2016324021

Country of ref document: AU

Date of ref document: 20160912

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2016847105

Country of ref document: EP