AU2016324021A1 - Antibodies for generating anti-inflammatory macrophage and related uses - Google Patents

Abstract

The invention provides specific anti-Cathepsin G antibodies that are capable of inducing formation of anti-inflammatory macrophage. The invention also provides methods for generating anti-inflammatory macrophages. The methods involve contacting bone marrow cells or monocytes with a Cathepsin G antibody described herein, and culturing the cell mixture under conditions to allow formation of M2 macrophage. Also provided in the invention are therapeutic methods of using a pharmaceutical composition containing a Cathepsin G antibody or induced M2 macrophage described herein for treating autoimmune diseases and other disorders associated with undesired immune responses.

Description

The invention provides specific anti-Cathepsin G antibodies that are capable of inducing formation of anti-inflammatory macrophage. The invention also provides methods for generating anti-inflammatory macrophages. The methods involve contacting bone marrow cells or monocytes with a Cathepsin G antibody described herein, and culturing the cell mixture under condi tions to allow formation of M2 macrophage. Also provided in the invention are therapeutic methods of using a pharmaceutical composition containing a Cathepsin G antibody or induced M2 macrophage described herein for treating autoimmune diseases and other disorders associated with undesired immune responses.

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Antibodies for Generating Anti-Inflammatory Macrophage and Related Uses

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The subject patent application claims the benefit of priority to U.S. Provisional Patent Application Number 62/218,785 (filed September 15, 2015). The full disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes.

BACKGROUND OF THE INVENTION [0002] The fundamental problem that the immune system has is that it must kill an offending organism or tumor with minimal damage to self. This is difficult because the target and surrounding tissue are, chemically speaking, essentially the same. Thus, we know of many tissue injuries that are the result of the “shrapnel” from an immunological attack - a kind of collateral damage. Nature has evolved at least three less than perfect solutions to the problem of protecting the integrity of the host during an immunological attack. The first is containment, the second is high specificity of the T cell- or antibody-mediated response, and the third is an elaborate system of checks and balances. Thus, macrophages engulf bacteria and contain them in vesicles into which highly toxic chemicals are pumped. On the cellular side, the system of checks and balances operates, for example, by balancing effector and regulatory T-cells and/or pro-inflammatory and anti-inflammatory macrophages. However, in a therapeutic setting, 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.

[0003] There is a need in the art for better means for upregulating anti-inflammatory macrophage and for suppressing undesired immune responses. The present invention is directed to this and other unmet needs.

SUMMARY OF THE INVENTION [0004] In one aspect, the invention provides isolated or purified anti-Cathepsin G (CTSG) antibodies. These antibodies have the same binding specificity as that of a reference

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PCT/US2016/051290 anti-CTSG antibody having (1) heavy chain CDR1, 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 CDR1, CDR2 and CDR3 sequences respectively shown in SGSIASNS (SEQ ID NO:7), ENN, and QSYDSNFHWV (SEQ ID NO:8). Some of the antibodies contain heavy chain CDR1, 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 CDR1, 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 CDR1, 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 CDR1, 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).

[0005] 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. In some embodiments, 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.

[0006] In another aspect, 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 CDR1, 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 CDR1, CDR2 and CDR3 sequences respectively shown in SGSIASNS (SEQ ID NO:7), ENN, and QSYDSNFHWV (SEQ ID NO:8). In some of these methods, the employed bone marrow cells are human bone marrow cells. In some methods, the contacting occurs in vitro by

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PCT/US2016/051290 culturing the population of bone marrow cells or monocytes in the presence of the antiCathepsin G antibody. In some embodiments, the cells are cultured with the antibody for about 4 to 20 days. Some of the methods further include isolating CDI 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. In some methods, contacting the cells with the antibody occurs in vivo in a subject. In some of these methods, 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:1. Some related embodiments of the invention provide populations of anti-inflammatory M2 macrophages produced by the methods of the invention.

[0007] In another aspect, 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 CDR1, 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 CDR1, CDR2 and CDR3 sequences respectively shown in SGSIASNS (SEQ ID NO:7), ENN, and QSYDSNFHWV (SEQ ID NO: 8). Some of the methods are directed to treating lupus. In some embodiments, the employed antibody has heavy chain CDR1, 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 CDR1, 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). In some methods, the employed antibody has heavy chain and light chain variable region sequences show in SEQ ID NO:2 and SEQ ID NO:3, respectively. In some embodiments, the employed antibody is a scFv having an amino acid sequence that is substantially identical to SEQ ID NO:1. In one embodiment, the employed anti-Cathepsin G antibody is a scFv fragment shown in SEQ ID NO:1.

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PCT/US2016/051290 [0008] In still another aspect, 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. In some embodiments, the library of antibodies are unbiased, e.g., unbiased scFv antibody library. In some other embodiments, the employed antibodies are biased, e.g., antibodies recognizing CTSG. In some methods, the antibodies are expressed from a lentiviral based vector. In some methods, the employed cells are CD34⁺ bone marrow cells. In some methods, selection of M2 macrophages is based on cell morphology. In some other methods, 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.

[0009] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims.

DESCRIPTION OF THE DRAWINGS [0010] 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 (10⁹ 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. (E) The experiment was repeated using the selected sequence incorporated into lentivirus. This antibody clone (identified as LKAb) again induced colonies with a similar morphology that contained cells with macrophage morphologies. (F)

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After 1 week incubation with the purified LKAb antibody, bone marrow cells differentiated in culture to cells with morphologies consistent with that of macrophages.

[0011] 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. This was followed by incubation with LKAb or M-CSF for 6 days. Cells were then stained with anti-CD 1 lb and anti-F4/80. FACS analysis showed that the induced macrophage populations were dramatically reduced when CTSG mRNA was silenced. However, M-CSF still induced macrophages when CTSG mRNA was silenced.

The percentages of macrophages are shown (right). *P<0.05 (Student's t-test).

[0012] Figure 3 shows that LKAb induced anti-inflammatory M2 macrophage differentiation. (A) Mouse bone marrow cells were incubated with LKAb at the indicated concentrations (l-100pg/ml) for 6 days. Cells were then stained with anti-CDl lb and antiCD1 lc 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 lbspecific magnetic beads, and the isolated CD1 lb-positive or -negative populations were incubated with LKAb or M-CSF for 6 days. Cells were then stained with anti-CDl lb and anti-F4/80 and analyzed by FACS. Macrophage differentiation was mainly induced from the CD 1 lb-negative population. (C) Mouse bone marrow was incubated with medium, LKAb or M-CSF for 6 days. Cells were then stained with anti-CDl6/32 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. Cells were harvested and total RNA was extracted for qRT-PCR analysis. IDO1 was used as a Ml specific gene and Arg-1 as a M2 specific gene. qRT-PCR showed high Arg-1 mRNA expression in macrophages induced by LKAb but only low levels of IDOL *** p<0.0005 (Student's t-test). (E) Macrophages induced by the LKAb

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PCT/US2016/051290 in vivo expressed M2 cytokines. LKAb and PBS were injected i.p. into C57BL/6 or BALB/c mice 3 times/week for 2 weeks and IL-10 levels in the sera were measured. Mice treated with LKAb showed dramatically increased IL-10 levels. IL-10 is one of the major antiinflammatory cytokines. *** p<0.0005 (Student's t-test).

[0013] Figure 4 shows that CTSG, CD14 and NAFAT play a key role in LKAbdependent M2 macrophage differentiation. (A) Bone marrow ceils from wild type, CTSG knockout, CD 14 knockout mice or NF AT 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. However, CD 14 and F4/80 expression was significantly reduced in both the CTSG and CD14 knockout mice. CD14 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).

[0014] Figure 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. As with mice, LKAb also induced human bone marrow to differentiate into anti-inflammatory macrophages.

[0015] Figure 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. (D) Kaplan-Meier plot representing survival rates of treated and control mice. Significant differences between

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LKAb treated and PBS control mice are indicated by * p<0.05, ** p<0.005, *** p<0.0005 (Student's t-test).

[0016] 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.

[0017] 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.

[0018] Figure 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 antiCD16/32. There is no macrophage differentiation in cells from CD14 (-/-) mice incubated with LKAb.

DETAILED DESCRIPTION OF THE INVENTION

I. Overview [0019] The recent discovery of many agonist antibodies that govern cell fates has opened the way to induce selectively a large variety of specific cells of the immune system from normal or malignant bone marrow or blood. Often these agonist antibodies induce cell differentiation along lineages expected from the known function of the receptor to which they bind. In other cases, they activate differentiation or trans-differentiation pathways that are different from what would be expected from the nature of the receptor with which they interact.

[0020] 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. Specifically, as detailed in the Examples herein, 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. 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

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PCT/US2016/051290 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. The target is unusual in that CTSG was classically thought to be an effector molecule secreted by activated PMNs. Importantly, 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).

[0021] 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.

[0022] In accordance with these studies, 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.

II. Definitions [0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1^st ed., 1992); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3^rd ed., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1^st ed., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994);

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Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt.

Ltd. (2002); and J Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4^th ed., 2000). In addition, the following definitions are provided to assist the reader in the practice of the invention.

[0024] The term antibody or antigen-binding fragment refers to polypeptide chain(s) which exhibit a strong monovalent, bivalent or polyvalent binding to a given antigen, epitope or epitopes. Unless otherwise noted, 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. Unless otherwise noted, the term “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., Serafmi, J Nucl. Med. 34:533-6, 1993).

[0025] An intact “antibody” typically comprises at least two heavy (H) chains (about 5070 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.

[0026] 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 _H3. 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.

[0027] The 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). Each Vh and Vl is

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PCT/US2016/051290 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., Rabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, U.S.

Government Printing Office (1987 and 1991).

[0028] 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. Examples of such antibody fragments include (i) a Fab fragment, a monovalent fragment consisting of the V_L, 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 al., Nature 341:544-546, 1989); and (vii) an isolated complementarity determining region (CDR).

[0029] 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. For example, a single-chain antibody may comprise a tether segment for linking to the encoding polynucleotide. As an example, a single chain variable region fragment (scFv) is a single-chain antibody. 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.

[0030] 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. The heavy chain variable (Vh) domain folds autonomously and functions independently as an antigen-binding unit. Its binding surface involves only three CDRs as compared to the six CDRs in classical antigen-binding

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PCT/US2016/051290 molecules (Fabs) or single chain variable fragments (scFvs). Camelid antibodies are capable of attaining binding affinities comparable to those of conventional antibodies.

[0031] 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). In particular, 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. Disulfidestabilized Fv fragments (dsFvs) can be made using methods described in, e.g., Reiter et al., Int. J. Cancer 67:113-23, 1996. Similarly, single domain antibodies (dAbs) 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. 11:500-515, 2002; Ghahroudi et al., FEBS Letters 414:521-526, 1997; and Bond et al., J Mol Biol. 332:643-55, 2003. Other types of antigen-binding fragments (e.g., 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.

[0032] 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.

[0033] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the

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PCT/US2016/051290 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. Optionally, 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.

[0034] Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c, 1970; by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; by the search for similarity method of Pearson and Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444, 1988; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI); or by manual alignment and visual inspection (see, e.g., Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003)).

Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively.

[0035] A ligand is a molecule that is recognized by a particular antigen, receptor or target molecule. Examples of 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. In some embodiments, the ligand is a molecule that specifically binds to a monocyte surface marker.

[0036] M2 macrophages (also termed “alternatively activated macrophages” or “antiinflammatory macrophages”) are one of two main groups of macrophages. The other group

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PCT/US2016/051290 of macrophages, Ml macrophages (or “classically activated macrophages”), are killer macrophages which are activated by LPS and IFN-gamma, and secrete high levels of IL-12 and low levels of IL-10. In contrast, 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 macrophages, and can be further elevated by IL-4. 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-γ, IL-4) also down regulate M2 and Ml activity, respectively. [0037] 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 macrophages 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 divide/differentiate into macrophages and dendritic cells to elicit an immune response. 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.

[0038] Unless otherwise noted, the term 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.

[0039] The term subject refers to human and non-human animals (especially nonhuman 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.

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PCT/US2016/051290 [0040] The term target, target molecule, or target cell 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.

[0041] 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. In prokaryotes, yeast, and mammalian cells for example, the transforming polynucleotide may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, 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. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming polynucleotide. 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.

III. Antibodies converting bone marrow cells into M2 microphages [0042] 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. As exemplified herein, 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 AREEEQQFSLDY (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).

[0043] The antibodies of the invention for inducing M2 macrophage are preferably monoclonal antibodies like the antibodies exemplified in the Examples below. In general, the antibodies have the same binding specificity as that of the LKAb agonist antibody and

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PCT/US2016/051290 compete with the LKAb antibody for binding to Cathepsin G. In addition to containing variable regions sequences derived from the LKAb antibody, some agonist antibodies of the invention can also contain other antibody sequences fused to the variable region sequences. For example, 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.

[0044] 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. In still some other embodiments, 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. In some other embodiments, other than the identical CDR sequences, 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. Relative to 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. Usually, antibodies incorporating such alterations exhibit substantial sequence identity to the LKAb antibody. For example, 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. Similarly, 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. In various embodiments, 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.

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PCT/US2016/051290 [0045] 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. General methods for preparation of monoclonal or polyclonal antibodies are well known in the art. See, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1998; Kohler & Milstein, Nature 256:495-497, 1975; Kozbor et al., Immunology Today 4:72, 1983; and Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, 1985.

IV. Converting monocytes or bone marrow cells into M2 macrophages [0046] The invention provides methods for generating M2 macrophages as well as therapeutic applications of M2 macrophages generated in vitro or in vitro. In some embodiments, 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-4Ralpha, 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-lOhigh, IL-121ow M2 profile with some features of tumor-associated macrophages (TAMs). 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.

[0047] Typically, 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. The monocyte-containing samples can be maintained and cultured in any physiologically-acceptable solution suitable for the collection and/or culture of the cells. For example, human bone marrow cells can be cultured in StemSpan serum-free media (STEMCELL Technologies) as exemplified herein. Other suitable culture media include, e.g., a saline solution (e.g., phosphate-buffered saline, Kreb's solution, modified

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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 bacteriocidally or bacteriostatically effective amount of an antibiotic. Examples of suitable antibiotics 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.

[0048] In some embodiments, the isolated or cultured monocyte or precursor cells can also be contacted with another agent that promotes monocyte differentiation into M2 macrophage. For example, the population of monocytes or precursors can be stimulated with the cytokines noted above for eliciting different M2 subtypes (e.g., IF-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 has been shown to polarize monocytes towards M2 macrophages (Jaguin et al., Cell Immunol. 281:51-61, 2013). In various embodiments, 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. The 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 10⁵ 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.

[0049] The in vitro induced M2 macrophage population can be further subject to analysis for expression of M2 markers and enrichment for M2 macrophages. For example, the cells can be examined for expression of “anti-inflammatory” cytokine expression profile, which includes high levels of IL-10 and IL-IRA 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. For example, M2a macrophages produce IL-10, TGFp and IL-Ira, M2b

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PCT/US2016/051290 macrophages produce IL-1, IL-6, IL-10 and TGFa, M2c macrophages produce IL-10 and TGFp, and M2d macrophages produce IL-10, IL-12, TNFa and TNFp. Moreover, M2 macrophages also have distinct chemokine and chemokine receptor profiles unlike that of Ml macrophages. For example, M2 macrophages expresses chemokines CCL17, CCL22 and CCL24 while Ml macrophages secret the TH1 cell-attracting chemokines CXCL9 and CXCL10.

[0050] 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. Typically, 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. In some embodiments, the antibody is contacted with the cells in vitro. In these embodiments, the cell population can be cultured at a concentration of about lxlO², lxlO³, lxlO⁴, lxlO⁵, lxlO⁶, lxlO⁷, 1x10^s cells/ml or higher. As exemplified herein, 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 pg/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. In some of these embodiments, the cells can be examined along the process for molecular markers and/or morphology indicative of the presence of M2 macrophages as exemplified herein. [0051] The cell culture may be performed under known culture conditions, and the conditions which are used in normal cell culture can be applied. For example, culture can be performed under the conditions of37°C and 5% CO₂. 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. In some embodiments, 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. In some embodiments, 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. For cell culture bags, a CO₂ gas permeable bag for cell culture can be used. When

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PCT/US2016/051290 a large amount of cells are being treated, use of a bioreactor is advantageous. Although 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.

[0052] In some embodiments, M2 macrophages can be generated in vitro from monocytes or bone marrow cells with a library of antibodies as exemplified herein. In some embodiments, 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). In these methods, the cells (e.g., CD34⁺ bone marrow 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). In these embodiments, the selected M2 macrophages are further manipulated to isolate the specific antibody expressed therein. By driving monocytic cells to become M2 macrophages, 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.

V. Therapeutic applications [0053] 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. As exemplified herein, treatment with the LKAb antibody resulted in therapeutic effects in subjects afflicted with systemic autoimmunity such as lupus (see, e.g., Example 6). 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. Accordingly, some embodiments of the invention provide

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PCT/US2016/051290 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. In some of these embodiments, 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. In some other embodiments, 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.

[0054] Pharmaceutical 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. Such 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. Examples of specific 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, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgiafibromyositis, grave's disease, guillain-barre, hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulin dependent diabetes (Type 1), juvenile arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis

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PCT/US2016/051290 nodosa, polychondritis, polyglancular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, sepsis, Sjogren's syndrome, stiff-man syndrome, systemic inflammatory response syndrome (SIRS), Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.

[0055] 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. In some preferred embodiments, the subject is a human patient. In some embodiments, the agonist antibody is contacted with monocytes or precursor cells in vivo upon being administered to the subject to induce formation of M2 macrophage. For example, a therapeutically effective amount of the antibody can be administered to a subject afflicted with an autoimmune disease or other inflammatory disorder. Upon contacting the antibody with monocytes or monocytes precursors in vivo, M2 macrophages can be induced which in turn can exert antiinflammatory activities. In some embodiments, 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). In some embodiments, a bispecific antibody comprised of the antigen-binding site of the agonist antibody and also the antigenbinding 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.

[0056] In some other embodiments, subjects with autoimmune diseases or other undesired immune responses can be treated ex vivo. In these methods, the pharmaceutical composition to be administered to a subject contains a population of M2 macrophages generated in vitro via the methods described herein. Specifically, 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. Upon conversion into M2 macrophages and enrichment, the induced M2 macrophages are then administered to the subject in a pharmaceutical composition. Preferably, the bone marrow

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PCT/US2016/051290 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. [0057] Some preferred embodiments of the invention are directed to treating human subjects afflicted with or suspected of having autoimmune disorders. Examples of 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, Behqet's disease, Coeiiac disease, Chagas disease, Chronic obstructive pulmonary disease, Crohn’s Disease, Dermatomyositis, Diabetes mellitus type 1, Endometriosis, Goodpasture's syndrome, Graves' disease, graft versus host disease (GVHD), Guillain-Barre syndrome (GBS), 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's granulomatosis.

[0058] Also provided in the invention are M2 macrophage populations generated in vitro by the methods described herein and therapeutic applications of such M2 macrophage populations. For therapeutic applications of the 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

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PCT/US2016/051290 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). The M2 macrophages can also be isolated or enriched by removal of cells other than M2 macrophages in the induced cell population. Thus, human M2 macrophages may be isolated or enriched by depletion of cells displaying non-M2 macrophage markers using, e.g., antibodies targeting such markers. Examples of such 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. The separation and enrichment of M2 macrophage can employ various routinely practiced techniques, e.g., flow cytometry, fluorescence-activated cell sorting (FACS) or magnetic cell sorting using microbeads conjugated with specific antibodies.

Upon enrichment, the 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.

[0059] The invention additionally provides 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. In some embodiments, 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.

[0060] The pharmaceutical 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. 116:3266-76, 2006; Iwasaki et al.,

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Jpn. J. Cancer Res. 88:861-6, 1997; Jespersen et al., Eur. Heart J. 11:269-74, 1990; and Martens, Resuscitation 27:177, 1994. For example, 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). 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. In some embodiments, administration of therapeutic composition is carried out by local or central injection of the cells into the subject. In some other embodiments, 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. See, e.g., Goodman & Gilman's The Pharmacological Bases of Therapeutics, Hardman et al., eds., McGraw-Hill Professional (10^th ed., 2001); Remington: The Science and Practice of Pharmacy, Gennaro, ed., Lippincott Williams & Wilkins (20^th ed., 2003); and Pharmaceutical Dosage Forms and Drug Delivery Systems, Ansel et al. (eds.), Lippincott Williams & Wilkins (7^th ed., 1999).

EXAMPLES [0061] The following examples are provided to further illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims.

Example 1. Selection system for identifying M2 macrophage-inducing antibodies [0062] We employed an unbiased intracellular combinatorial antibody library to simultaneously discover new targets and antibodies that induce proliferation and/or differentiation of cells that express them. The general selection scheme is illustrated in Fig. 1A. The unbiased antibody library in lentiviruses, which contains about 10⁸ 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.

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IE) and contained cells with a morphology that was similar to those selected after the first round of infection. To characterize the selected antibody (named LKAb), the encoding gene was transferred to a mammalian expression vector. When 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).

Example 2. Identification of binding target of antibody LKAb [0063] The purified 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). Classically, 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 overexpressing 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). Moreover, the protein fraction captured by our antibody was shown to have CTSG activity (Fig. 2C). Finally, when CTSG expression was silenced using shRNA, the ability of our antibody to induce macrophage formation was markedly reduced (Fig. 2D).

Example 3. Selective induction of M2 macrophages by antibody LKAb [0064] Mouse bone marrow cells were incubated with the selected antibody, and FACS analysis using CD1 lb and CD1 lc markers was carried out to confirm that LKAb acted in a dose dependent manner and that the induced cells were of macrophage, and not dendritic cell lineage (Fig. 3A). To determine which cell population was differentiated by our antibody, bone marrow cells were sorted according to the presence or absence of the CD1 lb marker. Only the CD1 lb-negative population differentiated to macrophages (Fig. 3B). By contrast, M-CSF induced macrophage differentiation in both populations (Fig. 3B). FACS analysis using the Ml markers CD16/32, CD86, and the M2 markers CD36 (Scavenger receptor),

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MHCII, and CD 14, showed that the macrophages induced by the antibody were mostly of the M2 type (Fig. 3C). The high selectivity of our antibody for induction of M2 macrophages was confirmed by qRT-PCR analysis comparing the ability of the cells induced by either LKAb or M-CSF to express the Ml marker IDO1 or the M2 marker ARG-1. Cells induced by our antibody displayed high expression of ARG-1 and limited expression of IDO-1, whereas cells induced with M-CSF showed high expression of IDO1 and only low expression of ARG-1 (Fig. 3D).

[0065] Expression of the cytokine IL-10 is another feature of M2 macrophages. To determine the in vivo effect of our antibody on IL-10 production, we injected 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). To study the activation of signaling pathways, fresh mouse bone marrow cells were treated with the antibody, and 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).

Example 4. Mechanistic studies [0066] 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.

[0067] Given its role in the innate immune system and the high concentration of CD 14 on the surface of monocytes, we assessed CD 14 involvement in differentiation of macrophages by our antibody. Bone marrow cells from CTSG (-/-), CD14 (-/-) and wildtype mice were incubated with LKAb, and FACS analysis using CD14 and F4/80 markers showed that this antibody increased CD14 expression in wild-type cells (Fig. 4A), presumably because the activity of CTSG was inhibited. However, no increase in CD 14 was observed in the CTSG (-/-) or CD14 (-/-) cells (Fig. 4A). In addition, the ability of the antibody to induce the general macrophage marker F4/80 was significantly decreased in

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PCT/US2016/051290 bone marrow cells from CTSG (-/-) or CD14 (-/-) mice (Fig. 4A). Consistent with these results, we found that LKAb inhibited CTSG activity by 50 % (Fig. 4B).

[0068] 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 CD14 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 NF AT signaling pathway that is dependent on CD14 but independent of TLR4. Therefore, we used NF AT 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. We found that macrophage differentiation was impaired in NFATcl (-/-) mice (Fig. 4A). As expected, NF AT inactivation in non-macrophage cells (such as CD4 expressing T cells and dendritic cells) had no consequence on macrophage differentiation by our antibody (Fig.

4A). Thus, we have a picture of CTSG’s role in macrophage polarization suggesting that this molecule operates through CD 14 and NF AT to regulate macrophage activation and differentiation (Fig. 4C).

Example 5. Inducing M2 macrophages from human bone marrow [0069] Since the human and mouse CTSG proteins are highly homologous, we wished to test the ability of our antibody to induce macrophage formation from human total bone marrow. As in the mouse, LKAb selectively induced M2 macrophages, as evidenced by a large increase in cells bearing the CD14, CD206 and CD200R markers (Fig. 5).

Example 6. LKAb treatment reduces systemic autoimmunity [0070] We next investigated whether this antibody could be used therapeutically in lupus, an autoimmune disease in which Ml macrophage infiltration appears to significantly contribute to end-organ pathology. For this experiment, we used MRL-Fas/lpr mice, which develop lupus-like manifestations with high titers of anti-nuclear autoantibodies, immune complex glomerulonephritis (GN), and early mortality primarily due to defective FasWO 2017/048629

PCT/US2016/051290 mediated T and B cell apoptosis. Treatments with LKAb (75 pg/mouse i.p. two times/week) were initiated at 6 weeks of age, and the experiment was terminated at 20 weeks of age, when >50% of the control (PBS-treated) mice were dead and the remaining exhibited signs of discomfort due to advanced disease. The results showed significant reductions in disease parameters in LKAb-treated mice compared to controls. Thus, titers of anti-chromatin IgG2a autoantibodies, the main isotype induced in this model, were reduced in the serum of treated mice (Fig. 6A). The treatment also significantly inhibited progression of lymphadenopathy (Fig. 6B). Moreover, at 20 weeks of age, treated mice had significantly reduced GN scores (Fig. 6C) and, accordingly, extended survival (Fig. 6D).

Example 7. Materials and methods [0071] Mouse strains and cell lines: The following mouse strains were used: C57BL/6J, BALB/c, C3H/HeJ, C3H/HeOuJ, C57BL/10ScNJ, B6.129S4-CD14^tmlfrm/J and MRL/MPJFas (lpr)/J (The Jackson laboratory). CTSG knockout mice were the generous gift of Dr. Christine Pham (Washington University, St. Louis, MO). NFATcl (-/-) and ΝΡΑΤε2^{ηοχ/ηοχ}: CD4^Cre mice were the generous gift of Dr. Anjana Rao (La Jolla institute for allergy and immunology). The HEK293T 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).

For inducing M2 macrophage with the LKAb antibody, human total bone marrow cells (AllCells) were cultured in StemSpan serum-free media with cytokine cocktail (STEMCELL Technologies) and the antibody (10 pg/ml) at 37°C. The mouse bone marrow cells 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.

[0072] Combinatorial antibody library and lentivirus: Single-Chain Fv (ScFv) genes were obtained from a naive human combinatorial antibody library (1 χ 10¹¹ 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 0.22WO 2017/048629

PCT/US2016/051290 pm 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.

[0073] 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 χ 10⁴ 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.

[0074] Purification of scFv-Fc proteins: For single antibodies, 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. [0075] 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 μΐ 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 (www.matrixscience.com) search engine.

[0076] Western blot: Bone marrow cells were washed with PBS and then lysed in lysis buffer (50 mM Hepes, pH 7.2, 150 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 10% glycerol, 1% Triton X-100). The lysates were then centrifuged at 20,000 χ g for 15 min at 4 °C. The proteins were denatured in Laemmli sample buffer (5 min at 95 °C), separated by SDS/PAGE, and transferred to nitrocellulose membranes using the iBlot blotting system

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PCT/US2016/051290 (Invitrogen). Membranes were blocked in phosphate buffered saline with Tween 20 (PBST) containing 5% BSA for 30 min before being incubated with antibodies for 3 h. Cathepsin G antibody (Santa Cruz Biotechnology) was used for identification. After washing the membranes several times with PBST, the blots were incubated with horseradish peroxidaseconjugated anti-goat or anti-rabbit antibody for 1 h. The membranes were then washed with PBST and developed by ECL. Phosphorylation was performed with phospho-AKT, ERK and p38 (Cell Signaling Technology).

[0077] 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.

[0078] Flow cytometry and cell sorting: Cells were stained with anti-mouse or human CD1 lb, CD1 lc, CD14, F4/80, CD16/32, CD36, CD86, MHCII, CD206, CD64, and CD200R (BD Biosciences). Stained cells were analyzed with a LSRII flow cytometer (Becton Dickinson). To obtain CD1 lb positive and negative bone marrow cells, CDllb⁺ microbeads (15 μΐ/ΐ0 cells; Miltenyi Biotech) were added to a suspension of 10 cells/mL of separation buffer and incubated for 15 minutes on ice followed by washing. The cells were resuspended in separation buffer (500 μΙ/IO⁸ cells), run through a SuperMACS II Separation Unit equipped with an MS column (Miltenyi Biotec), washed 3 times with buffer (500 μΐ), and flow-through cells were collected under pressure.

[0079] Real Time Quantitative RT-PCR: Macrophage were obtained from bone marrow cells cultured with LKAb antibody or M-CSF, RNA was extracted (Qiagen) and cDNA synthesized (Invitrogen). PCR was performed in triplicate using 400 ng cDNA, the RT SYBR Green supermix (Bio-Rad Laboratories), primer sets specific for mouse Arg-1 or IDO1 gene sequences, and a Cl000 Thermal cycler (Bio-Rad Laboratories).

[0080] 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.

[0081] Cytokine assay: Cytokines were quantified in serum collected from individual mice using a mouse cytokine magnetic bead panel (EMD Millipore) according to manufacturer’s instructions.

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PCT/US2016/051290 [0082] Treatment with isolated antibody: MRL-Fas^lpr mice were treated i.p. with LKAb (75pg/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. Standard curves were generated using calibrated mouse serum (Accurate Chemical and Scientific Company).

[0083] 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.

[0084] LKAb antibody amino acid sequence [0085] scFv sequence (SEQ ID NO: 1) [0086] MAQVQLVQSG AEVKKPGASV KVSCKASGYT FTSYYMHWVR QAPGQGLEWM GGIIPIFGTG NYAQKFQGRV TITADESTST AYMELSSLRS EDTAVYYCAR EEEQQFSLDY WGQGTLVTVS SGGGGGLS NF MLTQPHSVSE SPGKTVTISC TRSSGSIASN SVHWYQQRPG SAPTTVISEN NQRPSGVPDR FSGSIDSSSN SASLTISGLK TEDEADYYCQ SYDSNFHWVF GGGTQLTVLG [0087] Heavy chain variable region sequence (SEQ ID NO:2) [0088] MAQVQLVQSG AEVKKPGASV KVSCKASGYT FTSYYMHWVR QAPGQGLEWM GGIIPIFGTG NYAQKFQGRV TITADESTST AYMELSSLRS EDTAVYYCAR EEEQQFSLDY WGQGTLVTVS S [0089] Light chain variable region sequence (SEQ ID NO:3) [0090] NF MLTQPHSVSE SPGKTVTISC TRSSGSIASN SVHWYQQRPG SAPTTVISEN NQRPSGVPDR FSGSIDSSSN SASLTISGLK TEDEADYYCQ SYDSNFHWVF GGGTQLTVLG

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PCT/US2016/051290 [0091] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0092] All publications, databases, GenBank sequences, patents, and patent applications cited in this specification are herein incorporated by reference as if each was specifically and individually indicated to be incorporated by reference.

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Claims (38)

1. WE CLAIM:

   1. An isolated or purified anti-Cathepsin G (CTSG) antibody, which has the same binding specificity as that of a reference antibody comprising (1) heavy chain CDR1, 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 CDR1, CDR2 and CDR3 sequences respectively shown in SGSIASNS (SEQ ID NO:7), ENN, and QSYDSNFHWV (SEQ ID NO:8).
2. 2. The antibody of claim 1, comprising heavy chain CDR1, 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 CDR1, CDR2 and CDR3 sequences that are substantially identical to SGSIASNS (SEQ ID NO:7), ENN, and QSYDSNFHWV (SEQ ID NO:8), respectively.
3. 3. The antibody of claim 1, comprising heavy chain CDR1, 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 CDR1, 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).
4. 4. The antibody of claim 1, comprising heavy chain and light chain variable region sequences that are substantially identical to SEQ ID NO:2 and SEQ ID NO:3, respectively.
5. 5. The antibody of claim 1, comprising 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.
6. 6. The antibody of claim 1, comprising heavy chain and light chain variable region sequences show in SEQ ID NO:2 and SEQ ID NO:3, respectively.

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   The antibody of claim 1, comprising an amino acid sequence that is
7. 7.

   substantially identical to SEQ ID NO:1.
8. 8.

   The antibody of claim 1, comprising an amino acid sequence that is at least 90% or 95% identical to SEQ ID NO: 1.
9. 9.

   The antibody of claim 1, comprising a scFv antibody fragment shown in

   SEQ ID NO:1.
10. 10.

    A method for inducing formation of anti-inflammatory M2 macrophage, comprising contacting a population of bone marrow cells or monocytes with an antiCathepsin 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 CDR1, 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 CDR1, CDR2 and CDR3 sequences respectively shown in SGSIASNS (SEQ ID NO:7), ENN, and QSYDSNFHWV (SEQ ID NO:8).
11. 11. The method of claim 10, wherein the bone marrow cells are human bone marrow cells.
12. 12. The method of claim 10, wherein the contacting occurs in vitro by culturing the population of bone marrow cells or monocytes in the presence of the antiCathepsin G antibody.
13. 13. The method of claim 12, wherein culturing is for about 4 to 20 days.
14. 14. The method of claim 12, further comprising isolating CD1 lb- cells from the cultured cell population.
15. 15. The method of claim 12, further comprising detecting in the cultured cell population at least one cellular marker expressed by M2 macrophage.

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16. 16. The method of claim 15, wherein the at least one cellular marker expressed by M2 macrophage is CD36, MHCII, CD14, or ARG-1.
17. 17. The method of claim 10, wherein the contacting occurs in vivo in a subject afflicted with an autoimmune disease.
18. 18. The method of claim 10, wherein the anti-Cathepsin G antibody comprises heavy chain CDR1, 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 CDR1, 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).
19. 19. The method of claim 10, wherein the anti-Cathepsin G antibody comprises heavy chain and light chain variable region sequences that are substantially identical to SEQ ID NO:2 and SEQ ID NO:3, respectively.
20. 20. The method of claim 10, wherein the anti-Cathepsin G antibody comprises heavy chain and light chain variable region sequences show in SEQ ID NO:2 and SEQ ID NO:3, respectively.
21. 21. The method of claim 10, wherein the anti-Cathepsin G antibody comprises an amino acid sequence that is substantially identical to SEQ ID NO: 1.
22. 22. The method of claim 10, wherein the anti-Cathepsin G antibody comprises a scFv antibody fragment shown in SEQ ID NO: 1.
23. 23. A population of anti-inflammatory M2 macrophage produced by the method of claim 10.
24. 24. A method for treating or ameliorating the symptoms of an autoimmune disease in a subject, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an anti-Cathepsin G antibody, thereby treating or ameliorating the symptoms of the autoimmune disease in the subject, wherein the anti-Cathepsin G antibody has the same binding specificity as that of a reference antibody

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    PCT/US2016/051290 comprising (a) heavy chain CDR1, CDR2 and CDR3 sequences respectively shown in

    GYTFTSYY (SEQ ID NO:4), IIPIFGTG (SEQ ID NOD), and AREEEQQFSLDY (SEQ ID

    NO:6); and (b) light chain CDR1, CDR2 and CDR3 sequences respectively shown in

    SGSIASNS (SEQ ID NOD), ENN, and QSYDSNFHWV (SEQ ID NO:8).
25. 25. The method of claim 24, wherein the autoimmune disease is lupus.
26. 26. The method of claim 24, wherein the anti-Cathepsin G antibody comprises heavy chain CDR1, CDR2 and CDR3 sequences that are respectively identical to GYTFTSYY (SEQ ID NOD), IIPIFGTG (SEQ ID NO:5), and AREEEQQFSLDY (SEQ ID NO:6); and light chain CDR1, CDR2 and CDR3 sequences that are respectively identical to SGSIASNS (SEQ ID NO:7), ENN, and QSYDSNFHWV (SEQ ID NO:8) (SEQ ID NOD).
27. 27. The method of claim 24, wherein the anti-Cathepsin G antibody comprises heavy chain and light chain variable region sequences that are substantially identical to SEQ ID NO:2 and SEQ ID NOD, respectively.
28. 28. The method of claim 24, wherein the anti-Cathepsin G antibody comprises heavy chain and light chain variable region sequences show in SEQ ID NOD and SEQ ID NOD, respectively.
29. 29. The method of claim 24, wherein the anti-Cathepsin G antibody comprises an amino acid sequence that is substantially identical to SEQ ID NO:1.
30. 30. The method of claim 24, wherein the anti-Cathepsin G antibody comprises a scFv antibody fragment shown in SEQ ID NO: 1.
31. 31. A method for obtaining anti-inflammatory M2 macrophages from a population of bone marrow cells or monocytes, comprising (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; thereby obtaining M2 macrophages from the population of bone marrow cells or monocytes.

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32. 32. The method of claim 31, wherein the library of antibodies are unbiased.
33. 33. The method of claim 31, wherein the antibodies are scFvs.
34. 34. The method of claim 31, wherein the vectors are lentiviral vectors.
35. 35. The method of claim 31, wherein the cells are CD34⁺ bone marrow cells.
36. 36. The method of claim 31, wherein the M2 macrophages are selected based on morphology.
37. 37. The method of claim 31, wherein the M2 macrophages are selected based on expression of surface markers.
38. 38. The method of claim 31, further comprising isolating the antibodyexpressing vectors from the selected M2 macrophages.

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    Fig. 1.

    OO

    1 repeat from ? colonies

    Human ScFv Phage Library

    Human ScFv Lentjviral Library

    Leutiviius Transduction to Mouse Bone matron*

    Isolate DNA from colony

    ScFv Ab induction t

    Macrophage

    Fig. 2.

    anti-CTSGAb

    B IP : LKAb

    WB: anti-CTSGAb

    CTSG

    28KDi

    LKAb sbRN'A+LKAb shRNA+M-CSF

    F4/80

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    2/6

    Fig. 3.

    CDI4_ CD 36_ _CD36_ ~ CD lib ^W CDllb lpg/niL

    10pg/inL lOOgg/mL

    If’ 33.3 46.1 75.4-^ , ' J w r Ir f-fei

    -.- , • , „· ,,· ,.· .,- , ,.- CDllc LKAb M-CSF CDllb sort + CDllb sort +

    Control LKAb M-CSF 25.3 81.3 28.1 ·.,/-+ is’ \ 1 . ..sted j ' uvFai,*·'· / ,.- -¾ MHC π ,.- ^,5·¹ /W < ii, : ²“ /'N:,.. / . > \ id A . .sv Wv ,.-- VXsi'-i-' CD16/32 ,.- 3.18 26.0 8.40 / · • Ό _w° »’ .o^: ,? «* ,f^a ,? .ri V If¹ ,? io >o*

    CD86

    C57BL6

    BALB/C

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    Fig- 4.

    A

    Control LKAb MCSF 43.7 if,’ ί®»⁴? ' ** 3.44 . ·. / · f , <sA^f” U I '' i ========= 1 [ 1 i 1 .,,.:1 isE 1 i AS/ ifi· feB I

    CTSG(-/-)

    CD14(-/-)

    NFATcl(-/-)

    NFATc2^FloiZF,0i :CD4^Cre

    Macrophage precursor <u_ .s'

    WiM

    C7SG{·/-) CDI4P·) NFATtJH·) >TATc2:CD4

    M2 Macrophage

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    CD200R CD206 CD14

    Fig. 5.

    Control LKAb M-CSF

    CDllb

    “· ⁸ τ 5.63Γ'?“η i 4.27 j- 1 v V J VxV-U rr / .

    CD64

    11.0 33.8 24,0 cm Cf ^?

    CD64

    Fig. 6.

    lgG2a

    Anti-Chromatin /4 0 ΙΑ,ί/<Α

    Salivary LNs

    12 13 14 15 16

    Age (weeks)

    Kidney Disease (20 wks)

    «( * PBS LKAb 100 75 & « . · > 50 “7“ £ 3 IS “·» > . · - 20 pm 20 pm <0 (

    | LKAb I PBS p=0.0339

    Age (days)

    PBS LKAb

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    Fig. 7.

    IP Control

    Hit name Hit# CTSG preproprotein 5 Neutrophil elastase 5 Myeloid secondary granule protein 2 Myeloperoxidase 2

    Fig. 8.

    p-AKT

    AKT p-ERK(p44/42)

    ERK(p44/42) p-P38

    P38

    PCT/US2016/051290

    CD16/32