WO2021258040A2

WO2021258040A2 - Compositions and methods for modulating flrt3 mediated signal transduction

Info

Publication number: WO2021258040A2
Application number: PCT/US2021/038201
Authority: WO
Inventors: Dallas Benjamin Flies
Original assignee: Nextcure, Inc.
Priority date: 2020-06-18
Filing date: 2021-06-21
Publication date: 2021-12-23
Also published as: CA3183242A1; CN116322748A; JP2023530489A; AU2021293287A1; WO2021258040A3

Abstract

Compositions and methods of use thereof for modulating FLRT3 mediated signaling are provided. For example, immunomodulatory agents are provided that reduce FLRT3 expression, ligand binding, crosslinking, FLRT3 mediated signaling, or a combination thereof. In another embodiment, immunomodulatory agents are provided that enhance or promote FLRT3 expression, ligand binding, crosslinking, FLRT3 mediated signaling, or a combination thereof. Such agents can be used to modulate an immune response in a subject in need thereof.

Description

COMPOSITIONS AND METHODS FOR MODULATING FLRT3 MEDIATED SIGNAL TRANSDUCTION

FIELD OF THE INVENTION

The invention is generally related to the field of immunomodulation, and more particularly to compositions and methods for modulating immune responses in a subject.

BACKGROUND OF THE INVENTION Immunotherapies have made significant advances in the treatment of diseases such as cancer (Iwai, Y., et al., Journal of Biomedical Science, 24:26 (2017). Early immunotherapies accelerated T-cell activity. Current immune-checkpoint inhibitors take the brakes off the anti-tumor immune responses. Successful clinical trials with programmed cell death protein 1 (PD-1) monoclonal antibodies and other immune-checkpoint inhibitors have opened new avenues in cancer immunology. The failure of a large subset of cancer patients to respond to these new immunotherapies has led to intensified research to find new therapies.

FLRT3 (fibronectin and leucine-rich transmembrane protein-3) is a member of the fibronectin leucine-rich repeat transmembrane protein family and has a FN (fibronectin) type III domain and leucine-rich repeats. FLRT3 is expressed in various tissues including kidney, skeletal muscle, brain and lung. FLRT3 has been reported to have crucial functions during early embryonic development. Most of the literature describe functions in neurons: neuronal development, migration, axonal guidance, neurite outgrowth. Expression of FLRT3 has been shown to have prognostic value in certain cancers. In renal clear cell carcinoma, higher expression level of FLRT3 was associated with a better survival of the patients during a 5 -years follow-up time (The Human Protein Atlas, 2018a), while in one of the less vascularized cancers, pancreatic cancer (Longo et al., 2016), a lower expression level of FLRT3 associates with better prognosis (The Human Protein Atlas, 2018b). LPHN3, UNC5B and UNC5D have been reported binding partners of FLRT3. FLRTs, LPHNs, and UNC5s are families of interacting neuronal cell-surface receptors that mediate brain development. The LPHN3/FLRT3 structure reveals that LPHN3 binds to FLRT3 at a site distinct from UNC5. FLRT3 simultaneously binds to LPHN3 and UNC5, and forms a trimeric complex. UNC5B mediates apoptosis in tumors in the absence of netrin through the activation of DAP kinase and is involved in leukocyte migration inhibition. UNC5B has been shown to be expressed on thymocytes and T cells. Expression of UNC5B correlates with bladder cancer stage and the receptor is a potential predictor of both bladder and colorectal cancer prognosis and possible disease recurrence.

FLRT3 is positively regulated by both FGF and TGF-b suggesting that it is involved in the regulation of FGF signaling in many tissues during development. Therefore, it is an object of the invention to provide compositions that modulate leucine-rich repeat transmembrane protein FLRT3 (FLRT3) mediated signal transduction thereby promoting a suppressive immunological response. Such compositions are useful for the treatment of inflammatory diseases and disorders and autoimmune diseases. It is also an object of the invention to provide compositions that modulate FLRT3 mediated signal transduction by blocking FLRT3 interacting with its ligands. Such compositions are useful for the treatment of cancer.

SUMMARY OF THE INVENTION

Compositions and methods of their use for modulating FLRT3 mediated signal transduction are provided. One embodiment provides compositions and methods that induce, promote, or enhance FLRT3 mediated signal transduction. For example, immunomodulatory agents are provided that induce, promote, or enhance FLRT3 expression, ligand binding, crosslinking, signal transduction, or a combination thereof.

Exemplary anti-FLRT3 antibody or antigen-binding fragment thereof including the following light and heavy chain variable regions are disclosed: a) a light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 19, 21, 23, 25, 27, and 29, and b) a heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, and 98, wherein the antibody of antigen-binding fragment thereof binds to FLRT3.

In one embodiment, the antibody or antigen-binding fragment further includes one or more constant domains from an immunoglobulin constant region (Fc). In another embodiment, the antibody or antigen-binding fragment is humanized.

Other embodiments provide exemplary anti-FLRT3 antibody or antigen-binding fragments thereof that have a heavy chain variable region and a light chain variable region selected from the group consisting of: a) heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO:209, and light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO:206; b) heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO:200, and light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 195; c) heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 107, and a light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 102; d) heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 119, and a light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 114; e) heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 128, and a light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 126;

1) heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 136, and a light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 134; g) heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 144, and a light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 141; h) heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 149, and a light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 134; i) heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 157, and a light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 153; j) heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 167, and a light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 162; k) heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 177, and a light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 172; and l) heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 188, and a light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 183.

In one embodiment, the antibody or antigen-binding fragment thereof has one or more constant domains from an immunoglobulin constant region (Fc). In another embodiment, the antibody or antigen-binding fragment is humanized.

Another embodiment provides an anti-FLRT3 antibody produced by a hybridoma selected from the group consisting of 14B7, 17A1, 15G11, 18A7, 9F3, 1H5, 2F7, 5C2, 7H7, 10C6, 12A4, 13B2, 14D3, and 20C3.

Other embodiments provide a pharmaceutical composition including any one of the disclosed antibodies or antigen-binding fragments, and a pharmaceutically acceptable excipient.

One embodiment provides a fusion protein or an antigen binding fragment thereof having 80%, 85%, 90%, 95%, 99%, or 100% to SEQ ID NO:215, 216, or 217.

Also provided are methods for treating a tumor in a subject in need thereof by administering a therapeutically effective amount of any one of the disclosed antibody or antigen-binding fragments or fusion proteins to the subject to reduce tumor burden in the subject. The tumor can be a colorectal tumor, a lymphoma tumor, or an ovarian tumor.

Another embodiment provides a method for promoting an immune response in a subject in need thereof by administering a therapeutically effective amount of any one of the disclosed antibody or antigen-binding fragments or fusion proteins in an amount effective to promote an immune response in the subject. The promoted immune response retards or prevents tumor growth, inhibits tumor-mediated immune suppression, eliminates tumors, depletes or blocks the activity of tumor-associated macrophages (TAMs), decreases TAM-mediated immune suppression, reduces or reverses T cell suppression, increases T cell priming, activation, proliferation and effector function, or a combination thereof.

Yet another embodiment provides a method of treating an autoimmune disease is a subject in need thereof by administering a therapeutically effective amount of any one of the disclosed antibody or antigen-binding fragments or fusion proteins to treat the autoimmune disease.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1D show that FLRT3’s immunosuppression in vitro is comparable to PD-L1. Fig. 1A is an illustration of NFKB activation in human Jurkat T cells. Fig. IB is a bar graph showing the effect of FLRT3, PD-L1 and CD80 on NFKB activation in human Jurkat GFP reporter cells. Fig. 1C is an illustration of IFNy production in human primary T cells. Fig. ID is a bar graph showing the effect of FLRT3, PD-L1 and CD80 on the production of IFNy.

Figures 2A-2B shows FLRT3 is expressed in mouse and human cancers. Fig. 2A is a bar graph showing expression levels of FLRT3 in mouse cancer cells. Fig. 2B is a graph showing FLRT3 expression levels in human cancer cells.

Figure 3 shows FLRT3 expression is significantly increased in several human cancer types. Fig. 3 is a box plot comparing TCGA tumor tissue (red) + TCGA normal and GTEx normal tissues (grey).

Figures 4A-4C show FLRT3 high expression correlates with reduced overall survival in kidney (A4), pancreatic (4B) and lung (4C) cancers.

Figures 5A-5I plots showing FLRT3 expression analyses on cell lines using FLRT3 (monoclonal antibody) mAh 14B7.

Figures 6A-6C show FLRT3 overexpression increased tumor growth in the NSG mouse model. Fig. 6A is an illustration of the protocol used to inject the mice and determine tumor measurements. Figs. 6B-6C are line graphs showing the change in tumor volume after 52 days of inoculating the mice.

Figures 7A-7B show soluble FLRT3 protects against GvHD. Fig. 7A is the illustration showing the mice treatment protocol. Fig. 7B is a line graph showing clinical GvHD scores up to 58 days post tumor challenge with FLRT3 and control.

Figures 8A-8F show soluble FLRT3 increased tumor growth in humanized models and inhibits immune function. Figs. 8A is an illustration of the protocol used to treat mice to determine tumor measurements. Fig. 8B is a line graph showing the effect of FLRT3 on tumor growth in the HT-29 mouse tumor model. Fig. 8C-8J are flow cytometry and bar graphs showing the effect of FLRT3 Fc on cytokine production. Fig. 8K-8P are bar graphs showing the effects of FLRT3 Fc on cytokine production. Figs. 8Q is an illustration of the protocol used to treat mice to determine tumor measurements. Fig. 8R is a line graph showing the effect of soluble FLRT3 on HT29-OKT3 tumor growth. Figs. 8S-8T are dot plots showing the effect of FLRT3 Fc on human CD45+ cells (8S) and T cell subsets (8T).

Figures 9A-9B show FLRT3 promotes growth of 624Mel in a humanized mouse model. Fig. 9A is an illustration of the treatment of NSG mice. Fig. 9B is a line graph showing the effect of FLRT3 on tumor size. Fig. 9C is a line graph showing the effect of FLRT3 on tumor size. Fig. 9D is a bar graph showing levels of soluble FLRT3 in the 624Mel tumor model.

Figures 10 are line graphs showing monoclonal antibodies identified by binding to hFLRT3 on 293T-OKT3 cells (13A) as compared to EV-293T- OKT3 (13B).

Figure 11 is an illustration showing that FLRT3 antibodies fall into three bins.

Figures 12A-12B show binding EC50 values of top binders to 293T- hFLRT3 cells. Figs. 12A-12B are a line graph (12A) and non-linear regression fit graph (12B) showing the maximal response of the top binders. Figure 13 is a line graph showing 14B3 binds to mouse FLRT3 on 239T-OKT3-mFLRT3 cells.

Figures 14A-14C show 14B7 disrupts Unc5B-FLRT3 interaction in ELISA assays. Fig. 14A is an illustration of the Unc5B-FLRT3 interaction. Fig. 14B shows the fluorescence intensity of Unc5B-Fc binding to FLRT3 Fc relative to control Fc. Fig. 14C is a line graph showing the binding of FLRT3 to Unc5B-FC.

Figures 15A-15B show 14B7 disrupts FLRT3-Unc5B interaction in cell-based assays. Figs. 15A-15B are line graphs showing the treatment of 14B7 and 14D3 on FLRT3-Fc (15A) and antibody concentrations (15B).

Figures 16A-16E show 14B7 blocks other FLRT3-Unc5 interactions. Fig. 16A is a bar graph showing FLRT3-Fc binding on coated Unc5 proteins. Figs. 16B-16E show the effect of 14B7 on FLRT3-Fc binding to Unc5 proteins.

Figures 17A-17C show 14B7 disrupts Unc5B-FLRT3 interaction in ELISA assays. Fig. 17A is an illustration of binding of Unc5B-Fc biotin to FLRT3-Fc in presence of increasing of antibodies. Fig. 17B is a bar graph showing the mean fluorescence intensity of Unc5B Fc binding to FLRT3 Fc in comparison to control Fc. Fig. 17C is a line graph showing the effect of 14B7 on Unc5B-FC binding to FLRT3 Fc.

Figure 18 shows binding curves of FLRT3 antibodies to FLRT3 monomer by octet.

Figure 19 is a line graph showing FLRT3 antibodies bind to A549 cells that express endogenous hFLRT3.

Figures 20A-20B show binding EC50 values for A549 cells. Fig. 20A is a line graph and Fig. 20B is non-linear regression fit graph of normalized data for the top binders.

Figures 21A-21I show FLRT3 inhibition of T cells. Fig. 21A is an illustration of the assay. Fig. 21B-21E are bar graphs showing FLRT3 suppresses IFN-g production, and Fig. 21F-21I are bar graphs showing FLRT3 inhibits T cell proliferation in total PBMCs for donors 46 (21B, 25F), 66 (21 C, 21G), 67 (21D, 21H), and 68 (21E, 211). Figures 22A-22D show FLRT3 antibodies reverse cell death and NF- kB-GFP reporter signaling in Jurkat T cells. Fig. 22A is an illustration of the assay and Fig. 22B is bar graph showing FLRT3 increased Jurkat T cell death and reversal by 14B7 antibody. Fig. 22C is and illustration of the assay and Fig. 22D is a bar graph showing FLRT3 inhibits NF-kB-GFP signaling and reversal by 14B7 antibody.

Figures 23A-23B show FLRT3 antibodies reverse inhibition of T cells. Figs. 23A-23B are bar graphs showing total PBMCs from Donor 68 (23A) and Donor 67 (23B) in the presence of empty vector or FLRT3- HMC3-OKT3 +/- FLRT3 Abs and reversal of primary T cell inhibition by FLRT3 antibodies.

Figures 24A-24J show 14B7 promotes T cell priming and activation. Figure 24A is an illustration of the assay. Figs. 24B-24D are bar graphs showing 14B7 increased IFN-g production in co-culture of 293T-FLRT3 cells with PBMC donors 68, 76 and 1805E. Figs. 24E-2G are bar graphs showing 14B7 increased IFN-g production in co-culture of A375 melanoma cells with PBMC donors 68, 76 and 1805E. Figs. 24H-24J are bar graphs showing 14B7 increased IFN-g production in co-culture of SKOV3 cells with PBMC donors 68, 76 and 1805E.

Figures 25A-25D show 14B7 promotes T cell effector function. Figure 25A is an illustration of the assay. Fig. 25B-25D are bar graphs showing 14B7 increased IFN-g production in co-culture of SKOV3 cells with PBMC donors 81, 83 and 97.

Figures 26A-26C show 14B7 inhibits growth of FLRT3+ 624-Mel tumors. Fig. 26A illustrates the treatment protocol of using NSG mice. Fig. 26B is a line graph showing the effect of 14B7 in tumor growth post treatment. Fig. 26C is a survival probability graph showing the effect of 14B7 on survival post treatment.

Figures 27A-27C shows 14B7 inhibits growth of A549 tumors that naturally express FLRT3. Fig. 27A is an illustration of the treatment protocol with NSG mice. Fig. 27B is a line graph showing the effect of 14B7 in tumor growth post treatment. Fig. 27C is a dot plot showing the effect of 14B7 on tumor volume.

Figures 28A-28B shows 14B7 inhibits growth of CT26 tumors that are transduced to overexpress FLRT3. Fig. 28A is an illustration of the treatment protocol with Balb/c mice. Fig. 28B is a bar graph showing the effect of 14B7 in tumor growth on day 21 after tumor injection.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

As used herein, the terms “immunomodulatory agent” and “binding moiety” are used interchangeably.

As used herein, the term “FLRT3 immunomodulatory agent” refers to leucine-rich repeat transmembrane protein FLRT3 (FLRT3) binding moieties including, but not limited to antibodies and antigen binding fragments thereof, and FLRT3 fusion proteins and binding fragments thereof. In one embodiment, FLRT3 has an amino acid sequence according to UniProtKB - Q9NZU0 (FLRT3_HUMAN) which is incorporated by reference in its entirety. Other names for FLRT3 include fibronectin-like domain-containing leucine-rich transmembrane protein 3.

As used herein, a molecule is said to be able to “immunospecifically bind” a second molecule if such binding exhibits the specificity and affinity of an antibody to its cognate antigen. Antibodies are said to be capable of immunospecifically binding to a target region or conformation (“epitope”) of an antigen if such binding involves the antigen recognition site of the immunoglobulin molecule. An antibody that immunospecifically binds to a particular antigen may bind to other antigens with lower affinity if the other antigen has some sequence or conformational similarity that is recognized by the antigen recognition site as determined by, e.g., immunoassays, BIACORE® assays, or other assays known in the art, but would not bind to a totally unrelated antigen. In some embodiments, however, antibodies (and their antigen binding fragments) will not cross-react with other antigens. Antibodies may also bind to other molecules in a way that is not immunospecific, such as to FcR receptors, by virtue of binding domains in other regions/domains of the molecule that do not involve the antigen recognition site, such as the Fc region.

As used herein, a molecule is said to “physiospecifically bind” a second molecule if such binding exhibits the specificity and affinity of a receptor to its cognate binding ligand. A molecule can be capable of physiospecifically binding to more than one other molecule.

As used herein, the term “antibody” is intended to denote an immunoglobulin molecule that possesses a “variable region” antigen recognition site. The term “variable region” is intended to distinguish such domain of the immunoglobulin from domains that are broadly shared by antibodies (such as an antibody Fc domain). The variable region includes a “hypervariable region” whose residues are responsible for antigen binding. The hypervariable region includes amino acid residues from a “Complementarity Determining Region” or “CDR” (i.e., typically at approximately residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and at approximately residues 27-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Rabat et ctl, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from a “hypervariable loop” (i.e., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (HI), 53-55 (H2) and 96- 101 (H3) in the heavy chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined. The term antibody includes monoclonal antibodies, multi- specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (See e.g., Muyldermans et al., 2001, Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech. 1:253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231:25; International Publication Nos. WO 94/04678 and WO 94/25591; U.S. Patent No. 6,005,079), single-chain Fvs (scFv) (see, e.g., see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994)), single chain antibodies, disulfide-linked Fvs (sdFv), intrabodies, and anti-idiotypic (anti-id) antibodies (including, e.g., anti-id and anti-anti -Id antibodies to antibodies). In particular, such antibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g. , IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

As used herein, the term “antigen binding fragment” of an antibody refers to one or more portions of an antibody that contain the antibody’s Complementarity Determining Regions (“CDRs”) and optionally the framework residues that include the antibody’s “variable region” antigen recognition site, and exhibit an ability to immunospecifically bind antigen. Such fragments include Fab', F(ab')2, Fv, single chain (ScFv), and mutants thereof, naturally occurring variants, and fusion proteins including the antibody’s “variable region” antigen recognition site and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor or receptor ligand, etc.).

As used herein, the term “fragment” refers to a peptide or polypeptide including an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

As used herein the term “modulate” relates to a capacity to alter an effect, result, or activity (e.g., signal transduction). Such modulation can be agonistic or antagonistic. Antagonistic modulation can be partial (i.e., attenuating, but not abolishing) or it can completely abolish such activity (e.g., neutralizing). Modulation can include internalization of a receptor following binding of an antibody or a reduction in expression of a receptor on the target cell. Agonistic modulation can enhance or otherwise increase or enhance an activity (e.g., signal transduction). In a still further embodiment, such modulation can alter the nature of the interaction between a ligand and its cognate receptor so as to alter the nature of the elicited signal transduction. For example, the molecules can, by binding to the ligand or receptor, alter the ability of such molecules to bind to other ligands or receptors and thereby alter their overall activity. In some embodiments, such modulation will provide at least a 10% change in a measurable immune system activity, at least a 50% change in such activity, or at least a 2-fold, 5- fold, 10-fold, or at least a 100-fold change in such activity.

The term “substantially,” as used in the context of binding or exhibited effect, is intended to denote that the observed effect is physiologically or therapeutically relevant. Thus, for example, a molecule is able to substantially block an activity of a ligand or receptor if the extent of blockage is physiologically or therapeutically relevant (for example if such extent is greater than 60% complete, greater than 70% complete, greater than 75% complete, greater than 80% complete, greater than 85% complete, greater than 90% complete, greater than 95% complete, or greater than 97% complete). Similarly, a molecule is said to have substantially the same immunospecificity and/or characteristic as another molecule, if such immunospecificities and characteristics are greater than 60% identical, greater than 70% identical, greater than 75% identical, greater than 80% identical, greater than 85% identical, greater than 90% identical, greater than 95% identical, or greater than 97% identical).

As used herein, the “activating” or “stimulatory” signals encompass signals that result in enhancing an activity or enhancing signal transduction.

As used herein, “suppressive” signals refer to signals that suppress immune activity.

The term “derivative” refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to the same target of a parent or reference antibody but which differs in amino acid sequence from the parent or reference antibody or antigen binding fragment thereof by including one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to the parent or reference antibody or antigen binding fragment thereof. In some embodiments, such derivatives will have substantially the same immunospecificity and/or characteristics, or the same immunospecificity and characteristics as the parent or reference antibody or antigen binding fragment thereof. The amino acid substitutions or additions of such derivatives can include naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. The term “derivative” encompasses, for example, chimeric or humanized variants, as well as variants having altered CHI, hinge, CH2, CH3 or CH4 regions, so as to form, for example antibodies, etc., having variant Fc regions that exhibit enhanced or impaired effector or binding characteristics.

As used herein, a “chimeric antibody” is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies having a variable region derived from a non human antibody and a human immunoglobulin constant region.

As used herein, the term “humanized antibody” refers to an immunoglobulin including a human framework region and one or more CDR’s from a non-human (usually a mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDR's is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor.” Constant regions need not be present, but if they are, they should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-99%, or about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDR’s, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody is an antibody including a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody would not encompass a typical chimeric antibody, because, e.g., the entire variable region of a chimeric antibody is non-human.

The term “endogenous concentration” refers to the level at which a molecule is natively expressed (i.e., in the absence of expression vectors or recombinant promoters) by a cell (which cell can be a normal cell, a cancer cell or an infected cell).

As used herein, the terms “treat,” “treating,” “treatment” and “therapeutic use” refer to the elimination, reduction or amelioration of one or more symptoms of a disease or disorder. As used herein, a “therapeutically effective amount” refers to that amount of a therapeutic agent sufficient to mediate a clinically relevant elimination, reduction or amelioration of such symptoms. An effect is clinically relevant if its magnitude is sufficient to impact the health or prognosis of a recipient subject. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g., delay or minimize the spread of cancer. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.

As used herein, the term “prophylactic agent” refers to an agent that can be used in the prevention of a disorder or disease prior to the detection of any symptoms of such disorder or disease. A “prophylactically effective” amount is the amount of prophylactic agent sufficient to mediate such protection. A prophylactically effective amount may also refer to the amount of the prophylactic agent that provides a prophylactic benefit in the prevention of disease.

As used herein, the term “cancer” refers to a neoplasm or tumor resulting from abnormal uncontrolled growth of cells. The term “cancer” refers to a disease involving cells that have the potential to metastasize to distal sites and exhibit phenotypic traits that differ from those of non-cancer cells, for example, formation of colonies in a three-dimensional substrate such as soft agar or the formation of tubular networks or web-like matrices in a three-dimensional basement membrane or extracellular matrix preparation. Non-cancer cells do not form colonies in soft agar and form distinct sphere like structures in three-dimensional basement membrane or extracellular matrix preparations.

As used herein, an “immune cell” refers to any cell from the hemopoietic origin including, but not limited to, T cells, B cells, monocytes, dendritic cells, and macrophages.

As used herein, “inflammatory molecules” refer to molecules that result in inflammatory responses including, but not limited to, cytokines and metalloproteases such as including, but not limited to, IL-Ib, TNF-a, TGF- beta, IFN-g, IL-18, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs.

As used herein, “valency” refers to the number of binding sites available per molecule.

As used herein, the terms “immunologic,” “immunological” or “immune” response is the development of a beneficial humoral (antibody mediated) and/or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against a peptide in a recipient patient. Such a response can be an active response induced by administration of immunogen or a passive response induced by administration of antibody or primed T-cells. A cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules to activate antigen-specific CD4⁺ T helper cells and/or CD8⁺ cytotoxic T cells. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils, activation or recruitment of neutrophils or other components of innate immunity. The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4⁺ T cells) or CTL (cytotoxic T lymphocyte) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating antibodies and T-cells from an immunized syngeneic animal and measuring protective or therapeutic effect in a second subject. An “immunogenic agent” or “immunogen” is capable of inducing an immunological response against itself on administration to a mammal, optionally in conjunction with an adjuvant.

As used herein, the terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, humans, rodents, such as mice and rats, and other laboratory animals.

As used herein, the term “polypeptide” refers to a chain of amino acids of any length, regardless of modification (e.g., phosphorylation or glycosylation). The term polypeptide includes proteins and fragments thereof. The polypeptides can be “exogenous,” meaning that they are “heterologous,” i.e., foreign to the host cell being utilized, such as human polypeptide produced by a bacterial cell. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (lie, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).

As used herein, the term “variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

Modifications and changes can be made in the structure of the polypeptides of the disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide’s biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties.

In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (- 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 ± 1); threonine (-0.4); alanine (-0.5); histidine (- 0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (lie: Leu, Val), (Leu: lie, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Trp: Tyr), (Tyr: Trp, Phe), and (Val: He, Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the polypeptide of interest. The term “percent (%) sequence identity” is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

For purposes herein, the % sequence identity of a given nucleotides or amino acids sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given sequence C that has or comprises a certain % sequence identity to, with, or against a given sequence D) is calculated as follows:

100 times the fraction W/Z, where W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program in that program’s alignment of C and D, and where Z is the total number of nucleotides or amino acids in D. It will be appreciated that where the length of sequence C is not equal to the length of sequence D, the % sequence identity of C to D will not equal the % sequence identity of D to C.

As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents.

II. Compositions

A. FLRT3 Polypeptides

Fibronectin leucine-rich repeat transmembrane protein FLRT3 is a member of the FLRT family of proteins which structurally resemble small leucine-rich proteoglycans found in the extracellular matrix. FLRT3 functions in cell-cell adhesion, cell migration, and axon guidance, exerting either an attractive or repulsive role depending on its interaction partner. Interaction with ADGR3 on adjacent cells mediates cell-cell adhesion. FLRT3 interaction with the intracellular domain of ROBOl mediates axon attraction towards cells expressing NTN1. Interaction with UNC5B mediates axon growth cone collapse and plays a repulsive role in neuron guidance. FLRT3 also plays a role in fibroblast growth factor-mediated signaling cascades.

FLRT3 is expressed in the kidney, brain, pancreas, skeletal muscle, lung, liver, placenta, and heart. Down regulation of FLRT3 has been detected in lung transplant patients with primary graft dysfunction. Mutations in FLRT3 are associated with hypogonadotropic hypogonadism 21 with or without anosmia (HH21).

1. Human FLRT3

Sequences for human FLRT3 are known in the art. For example, the nucleic acid sequence for human FLRT3 transcript variant 1 is as follows:

Sequence: NM_013281.3 which is incorporated by reference in its entirety.)

The consensus amino acid sequence for FLRT3 is:

The underlined sequence is the signal sequence. The bolded sequence is the transmembrane sequence, and the double underlined sequence is the intracellular domain. In one embodiment, the FLRT3 protein does not contain the signal sequence.

In one embodiment, the FLRT3 amino acid sequence has 85%, 90%, 95%, 99%, 100% sequence identity to SEQ ID NO:2.

One embodiment provides an immunomodulatory agent that specifically binds to SEQ ID NO:2 or a functional fragment thereof and modulates FLRT3 mediated signal transduction.

Amino acids 29-528 of SEQ ID NO: 2 represent the extracellular domain of human FLRT3 and has the following sequence:

One embodiment provides an immunomodulatory agent that specifically binds to SEQ ID NO:3 or a functional fragment thereof and modulates FLRT3 mediated signal transduction.

2. Murine FLRT3

The amino acid sequence for murine FLRT3 is:

One embodiment provides an immunomodulatory agent that specifically binds to SEQ ID NO:4 or a functional fragment thereof and modulates FLRT3 mediated signal transduction .

Amino acids 29-528 of SEQ ID NO: 2 represent the extracellular domain of murine FLRT3 and has the following sequence:

One embodiment provides an immunomodulatory agent that specifically binds to SEQ ID NO:5 or a functional fragment thereof and modulates FLRT3 mediated signal transduction.

B. Binding Partners

One embodiment provides immunomodulatory agents that specifically bind to a binding partner of FLRT3 and modulates FLRT3 mediated signal transduction. Exemplary binding partners of FLRT3 include but are not limited to ROBOl, members of the latrophibn family (such as ADGRL3), and members of the UNC-5 family (such as UNC5B).

Roundabout homolog 1 (ROBOl) interacts with the intracellular domain of FLRT3 to mediate axonal attraction. The consensus amino acid sequence for ROBOl is as follows:

j i

I I

One embodiment provides an immunomodulatory agent that specifically binds to SEQ ID NO:6 or a functional fragment thereof and modulates FLRT3 mediated signal transduction.

In one embodiment, the functional fragment is an isoform of ROBOl. Several isoforms are recognized in the art including but not limited to isoform 2, isoform 3, isoform 4, isoform 5, and isoform 6.

One embodiment provides an immunomodulatory agent that specifically binds to any one of the isoforms of ROBOl or a functional fragment thereof and modulates FLRT3 mediated signal transduction.

Adhesion G protein-coupled receptor L3 (ADGRL3) interacts with the extracellular domain of FLRT3 to mediate cell-cell adhesion and neuron guidance. The consensus amino acid sequence for ADGRL3 is as follows:

One embodiment provides an immunomodulatory agent that specifically binds to SEQ ID NO:7 or a functional fragment thereof and modulates FLRT3 mediated signal transduction.

One embodiment provides an immunomodulatory agent that specifically binds to any one of the isoforms of ADGRL1 or a functional fragment thereof and modulates FLRT3 mediated signal transduction. Netrin receptor UNC5B (UNC5B) interacts with the extracellular domain of FLRT3 to mediate axon growth cone collapse and play a repulsive role in neuron guidance. The consensus amino acid sequence for UNC5B is as follows:

One embodiment provides an immunomodulatory agent that specifically binds to SEQ ID NO: 8 or a functional fragment thereof and modulates FLRT3 mediated signal transduction.

C. Immunomodulatory Agents or Binding Moieties Immunomodulatory agents or binding moieties including agonists and antagonists of FLRT3 are provided. An agonist of FLRT3 typically induces, promotes, or enhances FLRT3 mediated signaling. An antagonist of FLRT3 typically inhibits, reduces, or blocks FLRT3 mediated signaling. The disclosed compositions and methods can be used to modulate FLRT3 and/or counter-receptor signaling on, for example, immune cells including but not limited to monocytes, Tregs, tumor-associated macrophages (TAMs), Myeloid Derived Suppressor Cells (MDSC), T cells, Th2 cells, myeloid cells including antigen-presenting cells (e.g., monocyte, macrophage, or dendritic cell), T cells, Natural Killer (NK) cells, or a combination thereof. In some embodiments, the compositions are specifically targeted to one or more cell types. In some embodiments, the disclosed compositions can be used on tumor cells.

In some embodiments, the anti-FLRT3 agonists induce, promote, or enhance FLRT3 mediated signaling through a known ligand or unknown counter-receptor through FLRT3 interaction with said known or unknown counter-receptor. For example, in some embodiments, the FLRT3 agonist binds to, induces, promotes or creates a conformational change, or otherwise promotes FLRT3 mediated signal transduction.

In some embodiments, the anti-FLRT3 antagonists inhibit, reduce, block, or otherwise disrupt signaling through a known or unknown counter receptor through blockade of FLRT3 interaction with said known or unknown counter-receptor. For example, in some embodiments, the FLRT3 antagonist binds to FLRT3 or a ligand thereof and inhibits, blocks, creates a conformational change, or otherwise interferes with FLRT3 mediated signal transduction.

1. Antibodies

The sequences of light and heavy chain variable regions for monoclonal antibodies produced by 14 hybridomas, referred to herein as 14B7, 17A1, 15G11, 18A7, 9F3, 1H5, 2F7, 5C2, 7H7, 10C6, 12A4, 13B2, 14D3, and 20C3 are provided below. CDRs are underlined and bolded in the context of the light and heavy chain sequences.

One embodiment provides an anti-FLRT3 antibody produced by a hybridoma selected from the group consisting of 14B7, 17A1, 15G11, 18A7, 9F3, 1H5, 2F7, 5C2, 7H7, 10C6, 12A4, 13B2, 14D3, and 20C3.

Another embodiment provides an anti-FLRT3 antibody having at least one light chain or at least one heavy chain of the antibody produced by one or more of the hybridomas selected from the group consisting of 14B7, 17A1, 15G11, 18A7, 9F3, 1H5, 2F7, 5C2, 7H7, 10C6, 12A4, 13B2, 14D3, and 20C3. a. 14B7 i. Light chain

One embodiment provides a humanized antibody or antigen binding fragment with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:

One embodiment provides a humanized antibody or antigen binding fragment with a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity with the following:

One embodiment provides an anti-FLRT3 antibody of antigen binding fragment having a light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 19, 21, 23, 25, 27, and 29, and a heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, and 98,

In some embodiments, the antibody is humanized b. 18A7 i. Light Chain

ii. Heavy Chain

One embodiment provides an anti-FLRT3 antibody with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity to the amino acid according to SEQ ID NO: 102, and a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity to the amino acid according to SEQ ID NO: 107.

In one embodiment, the antibody is humanized c. 1H5 i. Light chain

The light chain CDRs of 1H5 are bolded and are as follows:

In another embodiment, SEQ ID NO: 114 includes the sequence of human kappa constant domain:

ii. Heavy chain

Q

In another embodiment, SEQ ID NO: 119 includes the sequence of human IgGl constant domain:

One embodiment provides an anti-FLRT3 antibody with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity to the amino acid according to SEQ ID NO: 114, and a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity to the amino acid according to SEQ ID NO: 119.

In one embodiment, the antibody is humanized.

In another embodiment, SEQ ID NO:209 includes the sequence of human IgGl constant domain, G1FES:

One embodiment provides an anti-FLRT3 antibody with a light chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity to the amino acid according to SEQ ID NO: 206, and a heavy chain variable region having at least 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% sequence identity to the amino acid according to SEQ ID NO:209.

In one embodiment, the antibody is humanized.

The activity of an antibody or antigen binding fragment thereof that is specific for FLRT3 can be determined using functional assays that are known in the art, and include the assays discussed below. Typically, the assays include determining if the antibody or antigen binding fragment thereof increases (i.e., agonist) or decreases (i.e., antagonist) signaling through FLRT3.

In some embodiments, the disclosed antibodies and antigen binding fragments thereof immunospecifically bind to human or mouse FLRT3. In some embodiments, the antibody binds to an extracellular domain of human or mouse FLRT3.

For example, molecules are provided that can immunospecifically bind to FLRT3:

(I) arrayed on the surface of a cell (especially a live cell);

(II) arrayed on the surface of a cell (especially a live cell) at an endogenous concentration;

(III) arrayed on the surface of a live cell, and modulates binding between FLRT3 and a ligand thereof;

(IV) arrayed on the surface of a live cell, and reduces or inhibits immune response by FLRT3;

(V) arrayed on the surface of a live cell, wherein the cell is a tumor cell;

(VI) combinations ofl-IV and V;

(VII) combinations of I-III and V; and

(VIII) arrayed on the surface of a live myeloid or lymphoid derived cancer cells (AML or ALL), and enhances apoptosis and differentiation resulting in reduced self-renewal of cancer stem cells.

To prepare an antibody or antigen binding fragment thereof that specifically binds to FLRT3 purified proteins, polypeptides, fragments, fusions, or epitopes to FLRT3 or polypeptides expressed from nucleic acid sequences thereof, can be used. The antibodies or antigen binding fragments thereof can be prepared using any suitable methods known in the art such as those discussed in more detail below. a. Human and Humanized Antibodies In some embodiments, the antibodies are humanized antibodies. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.

Transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production can be employed. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge.

Optionally, the antibodies are generated in other species and “humanized” for administration in humans. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)₂, or other antigen binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all, of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Humanization can be essentially performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, a humanized form of a nonhuman antibody (or a fragment thereof) is a chimeric antibody or fragment, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important in order to reduce antigenicity. According to the “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies.

It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences. Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i. e.. the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

The antibody can be bound to a substrate or labeled with a detectable moiety or both bound and labeled. The detectable moieties contemplated with the present compositions include fluorescent, enzymatic and radioactive markers. b. Single-Chain Antibodies

In some embodiments, the antibodies are single-chain antibodies. Methods for the production of single-chain antibodies are well known to those of skill in the art. A single chain antibody is created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain via a 15 to 25 amino acid peptide or linker have been developed without significantly disrupting antigen binding or specificity of the binding. The linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation. These Fvs lack the constant regions (Fc) present in the heavy and light chains of the native antibody. c. Monovalent Antibodies

In some embodiments, the antibodies are monovalent antibodies. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment, called the F(ab’)₂ fragment, that has two antigen combining sites and is still capable of cross-linking antigen.

The Fab fragments produced in the antibody digestion also contain the constant domains of the light chain and the first constant domain of the heavy chain. Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain domain including one or more cysteines from the antibody hinge region. The F(ab’)2 fragment is a bivalent fragment comprising two Fab’ fragments linked by a disulfide bridge at the hinge region. Fab’-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains bear a free thiol group. Antibody fragments originally were produced as pairs of Fab’ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. d. Hybrid Antibodies

In some embodiments, the antibodies are hybrid antibodies. In hybrid antibodies, one heavy and light chain pair is homologous to that found in an antibody raised against one epitope, while the other heavy and light chain pair is homologous to a pair found in an antibody raised against another epitope. This results in the property of multi-functional valency, i.e., ability to bind at least two different epitopes simultaneously. Such hybrids can be formed by fusion of hybridomas producing the respective component antibodies, or by recombinant techniques. Such hybrids may, of course, also be formed using chimeric chains. e. Conjugates or Fusions of Antibody Fragments

In some embodiments, the antibodies are conjugates or fusions of antibody fragments. The targeting function of the antibody can be used therapeutically by coupling the antibody or a fragment thereof with a therapeutic agent. Such coupling of the antibody or fragment (e.g., at least a portion of an immunoglobulin constant region (Fc)) with the therapeutic agent can be achieved by making an immunoconjugate or by making a fusion protein, comprising the antibody or antibody fragment and the therapeutic agent.

Such coupling of the antibody or fragment with the therapeutic agent can be achieved by making an immunoconjugate or by making a fusion protein, or by linking the antibody or fragment to a nucleic acid such as an siRNA, comprising the antibody or antibody fragment and the therapeutic agent.

In some embodiments, the antibody is modified to alter its half-life. In some embodiments, it is desirable to increase the half-life of the antibody so that it is present in the circulation or at the site of treatment for longer periods of time. For example, it may be desirable to maintain titers of the antibody in the circulation or in the location to be treated for extended periods of time. Antibodies can be engineered with Fc variants that extend half-life, e.g., using Xtend™ antibody half-life prolongation technology (Xencor, Monrovia, CA). In other embodiments, the half-life of the anti- DNA antibody is decreased to reduce potential side effects. The conjugates disclosed can be used for modifying a given biological response. The drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin.

2. Proteins and Polypeptides a. Protein and Polypeptide Compositions

The immunomodulatory or binding agent can be a FLRT3 protein, polypeptide, or fusion protein. For example, the immunomodulatory agent or binding moiety can be an isolated or recombinant protein or polypeptide, or functional fragment, variant, or fusion protein thereof of FLRT3.

The FLRT3 protein or polypeptide, or functional fragment, variant, or fusion protein thereof can be an agonist or an antagonist. For example, in some embodiments an antagonist of FLRT3 is a FLRT3 polypeptide or a fragment or fusion protein thereof that binds to a ligand of FLRT3. The polypeptide can be a soluble fragment, for example the extracellular domain of FLRT3, or a functional fragment thereof, or a fusion protein thereof. In some embodiments, a soluble ligand of FLRT3 may serve as an antagonist, decreasing FLRT3 mediated signal transduction.

The activity of a protein or polypeptide of FLRT3, or any fragment, variant or fusion protein thereof can be determined using functional assays that are known in the art, and include the assays discussed below. Typically, the assays include determining if the protein, polypeptide or fragment, variant or fusion protein thereof increases (i.e., agonist) or decreases (i.e., antagonist) signaling through the FLRT3 receptor. In some embodiments, the assay includes determining if the protein, polypeptide or fragment, variant, or fusion protein thereof increases (i.e., agonist) or decreases (i.e., antagonist) the immune response associated with FLRT3. Typically, the assays include determining if the protein, polypeptide or fragment, variant, or fusion protein thereof increases (i.e., agonist) or decreases (i.e., antagonist) signaling through FLRT3. In some embodiments, the assay includes determining if the protein, polypeptide or fragment, variant, or fusion protein thereof decreases (i.e., agonist) or increases (i.e., antagonist) an immune response regulated by FLRT3. In some embodiments, the assay includes determining if the protein, polypeptide or fragment, variant, or fusion protein thereof increases (i.e., antagonist) the apoptosis and differentiation of acute myeloid leukemia cells and acute lymphoblastic leukemia cells resulting in reduced self-renewal capacity of AML and ALL stem cells.

Nucleic acid and polypeptide sequences for FLRT3 are known in the art and exemplary protein and peptide sequences are provided above. The sequences can be used, as discussed in more detail below, by one of skill in the art to prepare any protein or polypeptide of FLRT3, or any fragment, variant, or fusion protein thereof. Generally, the proteins, polypeptides, fragments, variants, and fusions thereof of FLRT3 are expressed from nucleic acids that include sequences that encode a signal sequence. The signal sequence is generally cleaved from the immature polypeptide to produce the mature polypeptide lacking the signal sequence. The signal sequence can be replaced by the signal sequence of another polypeptide using standard molecule biology techniques to affect the expression levels, secretion, solubility, or other property of the polypeptide FLRT3 proteins with and without a signal sequence are disclosed. It is understood that in some cases, the mature protein as it is known or described in the art, i.e., the protein sequence without the signal sequence, is a putative mature protein. During normal cell expression, a signal sequence can be removed by a cellular peptidase to yield a mature protein. The sequence of the mature protein can be determined or confirmed using methods that are known in the art. i. Fragments

As used herein, a fragment of FLRT3 refers to any subset of the polypeptide that is at least one amino acid shorter than full length protein. Useful fragments include those that retain the ability to bind to their natural ligand or ligands. A polypeptide that is a fragment of any full-length FLRT3 typically has at least 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 98 percent, 99 percent, 100 percent, or even more than 100 percent of the ability to bind its natural ligand respectively as compared to the full-length protein. Fragments of FLRT3 include cell free fragments. Cell free polypeptides can be fragments of full-length, transmembrane, polypeptides that may be shed, secreted or otherwise extracted from the producing cells. Cell free fragments of polypeptides can include some or all of the extracellular domain of the polypeptide, and lack some or all of the intracellular and/or transmembrane domains of the full-length protein. In one embodiment, polypeptide fragments include the entire extracellular domain of the full-length protein. In other embodiments, the cell free fragments of the polypeptides include fragments of the extracellular domain that retain biological activity of full-length protein. The extracellular domain can include 1, 2, 3, 4, or 5 contiguous amino acids from the transmembrane domain, and/or 1, 2, 3, 4, or 5 contiguous amino acids from the signal sequence. Alternatively, the extracellular domain can have 1, 2, 3, 4, 5 or more amino acids removed from the C-terminus, N-terminus, or both. In some embodiments the extracellular domain is the only functional domain of the fragment (e.g., the ligand binding domain). ii. Variants

Variants of FLRT3, and fragments thereof are also provided. In some embodiments, the variant is at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99 percent identical to any one of SEQ ID NO:2-5. Useful variants include those that increase biological activity, as indicated by any of the assays described herein, or that increase half-life or stability of the protein. The protein and polypeptides of FLRT3, and fragments, variants, and fusion proteins thereof can be engineered to increase biological activity. For example, in some embodiments, a FLRT3 polypeptide, protein, or fragment, variant or fusion thereof has been modified with at least one amino acid substitution, deletion, or insertion that increases a function thereof.

Finally, variant polypeptides can be engineered to have an increased half-life relative to wild type. These variants typically are modified to resist enzymatic degradation. Exemplary modifications include modified amino acid residues and modified peptide bonds that resist enzymatic degradation. Various modifications to achieve this are known in the art. The variants can be modified to adjust for effects of affinity for the receptor on the half-life of proteins, polypeptides, fragments, or fusions thereof at serum and endosomal pH. iii. Fusion Proteins

Fusion polypeptides have a first fusion partner including all or a part of a human or mouse FLRT3 polypeptide fused to a second polypeptide directly or via a linker peptide sequence that is fused to the second polypeptide. In one embodiment, the ECD of human or mouse FLRT3 or a fragment thereof is fused to a second polypeptide. The fusion proteins optionally contain a domain that functions to dimerize or multimerize two or more fusion proteins. The peptide/polypeptide linker domain can either be a separate domain, or alternatively can be contained within one of the other domains (first polypeptide or second polypeptide) of the fusion protein. Similarly, the domain that functions to dimerize or multimerize the fusion proteins can either be a separate domain, or alternatively can be contained within one of the other domains (first polypeptide, second polypeptide or peptide/polypeptide linker domain) of the fusion protein. In one embodiment, the dimerization/multimerization domain and the peptide/polypeptide linker domain are the same.

Fusion proteins disclosed herein are of formula I:

N-R1-R2-R3-C wherein “N” represents the N-terminus of the fusion protein, “C” represents the C-terminus of the fusion protein. In some embodiments, “Ri” is a polypeptide or protein of FLRT3 or fragment or variant thereof, “R2” is an optional peptide/polypeptide linker domain, and “R3” is a second polypeptide. Alternatively, R3 may be a polypeptide or protein of FLRT3, or fragment or variant thereof and Ri may be a second polypeptide. In some embodiments, the FLRT3 polypeptide is the extracellular domain.

Dimerization or multimerization can occur between or among two or more fusion proteins through dimerization or multimerization domains. Alternatively, dimerization or multimerization of fusion proteins can occur by chemical crosslinking. The dimers or multimers that are formed can be homodimeric/homomultimeric or heterodimeric/heteromultimeric.

In some embodiments, the fusion protein includes the extracellular domain of FLRT3, or a fragment or variant thereof, fused to an Ig Fc region. Recombinant Ig fusion proteins can be prepared by fusing the coding region of the extracellular domain or a fragment or variant thereof to the Fc region of human IgGl, IgG2, IgG3 or IgG4 or mouse IgG2a, or other suitable Ig domain, as described previously (Chapoval, et al., Methods Mol Med., 45:247-255 (2000)). One embodiment provides a fusion protein having 80%, 85%, 90%,

95%, 99%, or 100% sequence identity to FLRT3 Fc (IgGl Fc; wild type) (SEQ ID NO:215):

The underlined sequence is the signal sequence. The bolded sequence is a linker, and the double underlined sequence is the Fc domain of wild type IgGl. In one embodiment, the fusion protein does not have a signal sequence. One embodiment provides a fusion protein having 80%, 85%,

90%, 95%, 99%, or 100% sequence identity to FLRT3 Fc (IgGl Fc; FES)

The underlined sequence is the signal sequence. The bolded sequence is a linker, and the double underlined sequence is the Fc domain of mutated IgGl (IgGl-FES). In one embodiment, the fusion protein does not have a signal sequence.

One embodiment provides a fusion protein having 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to FLRT3 Fc (IgG4 Fc; G4P) (SEQ ID NO:217):

The underlined sequence is the signal sequence. The double underlined sequence is hG4P Fc (G4P). iv. Polypeptide Modifications The polypeptides and fusion proteins may be modified by chemical moieties that may be present in polypeptides in a normal cellular environment, for example, phosphorylation, methylation, amidation, sulfation, acylation, glycosylation, sumoylation and ubiquitylation. Fusion proteins may also be modified with a label capable of providing a detectable signal, either directly or indirectly, including, but not limited to, radioisotopes and fluorescent compounds.

The polypeptides and fusion proteins may also be modified by chemical moieties that are not normally added to polypeptides in a cellular environment. For example, the disclosed fusion proteins may also be modified by covalent attachment of polymer chains, including, but not limited to, polyethylene glycol polymer (PEG) chains (i.e., pegylation). Conjugation of macromolecules to PEG has emerged recently as an effective strategy to alter the pharmacokinetic (PK) profiles of a variety of drugs, and thereby to improve their therapeutic potential. PEG conjugation increases retention of drugs in the circulation by protecting against enzymatic digestion, slowing filtration by the kidneys and reducing the generation of neutralizing antibodies. In addition, PEG conjugates can be used to allow multimerization of the fusion proteins.

Modifications may be introduced into the molecule by reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Another modification is cyclization of the protein.

Examples of chemical derivatives of the polypeptides include lysinyl and amino terminal residues derivatized with succinic or other carboxylic acid anhydrides. Derivatization with a cyclic carboxylic anhydride has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; 0-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate. Carboxyl side groups, aspartyl or glutamyl, may be selectively modified by reaction with carbodiimides (R — N=C=N— R') such as l-cyclohexyl-3-(2-morpholinyl-(4- ethyl)carbodiimide or l-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonia. Fusion proteins may also include one or more D-amino acids that are substituted for one or more L-amino acids. v. Modified Binding Properties

Binding properties of the proteins, polypeptides, fragments, variants and fusions thereof are relevant to the dose and dose regimen to be administered. In one embodiment the disclosed proteins, polypeptides, fragments, variants and fusions thereof have binding properties to FLRT3 or an FLRT3 ligand that demonstrate a higher term, or higher percentage, of occupancy of a binding site (e.g., on the ligand) relative to other receptor molecules that bind thereto. In other embodiments, the disclosed proteins, polypeptides, fragments, variants and fusions thereof have reduced binding affinity to FLRT3 relative to wild type protein.

In some embodiments the proteins, polypeptides, fragments, variants and fusions thereof have a relatively high affinity for FLRT3 and may therefore have a relatively slow off rate. In other embodiments, the proteins polypeptides, fragments, variants and fusions thereof are administered intermittently over a period of days, weeks or months to dampen immune responses which are allowed to recover prior to the next administration, which may serve to alter the immune response without completely turning the immune response on or off and may avoid long term side effects.

3. Isolated Nucleic Acid Molecules

Isolated nucleic acid sequences encoding the FLRT3 proteins, polypeptides, fragments, variants and fusions thereof are disclosed herein. As used herein, “isolated nucleic acid” refers to a nucleic acid that is separated from other nucleic acid molecules that are present in a mammalian genome, including nucleic acids that normally flank one or both sides of the nucleic acid in a mammalian genome. The term “isolated” as used herein with respect to nucleic acids also includes the combination with any non- naturally-occurring nucleic acid sequence, since such non-naturally- occurring sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome.

An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment), as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, a cDNA library or a genomic library, or a gel slice containing a genomic DNA restriction digest, is not to be considered an isolated nucleic acid.

Nucleic acids encoding the proteins, polypeptides, fragments, variants and fusions thereof may be optimized for expression in the expression host of choice. Codons may be substituted with alternative codons encoding the same amino acid to account for differences in codon usage between the mammal from which the nucleic acid sequence is derived and the expression host. In this manner, the nucleic acids may be synthesized using expression host-preferred codons.

Nucleic acids can be in sense or antisense orientation, or can be complementary to a reference sequence encoding a polypeptide or protein of FLRT3. Nucleic acids can be DNA, RNA, or nucleic acid analogs. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone. Such modification can improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety can include deoxyuridine for deoxythymidine, and 5-methyl-2’- deoxycytidine or 5-bromo-2’-deoxycytidine for deoxycytidine. Modifications of the sugar moiety can include modification of the 2’ hydroxyl of the ribose sugar to form 2’ -O-methyl or 2’-0-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7:187-195; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5-23. In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.

Nucleic acids encoding polypeptides can be administered to subjects in need thereof. Nucleic delivery involves introduction of “foreign” nucleic acids into a cell and ultimately, into a live animal. Compositions and methods for delivering nucleic acids to a subject are known in the art (see Understanding Gene Therapy, Lemoine, N.R., ed., BIOS Scientific Publishers, Oxford, 2008).

4. Vectors and Host Cells

Vectors encoding the proteins, polypeptides, fragments, variants and fusions thereof are also provided. Nucleic acids, such as those described above, can be inserted into vectors for expression in cells. As used herein, a “vector” is a replicon, such as a plasmid, phage, virus or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Vectors can be expression vectors. An “expression vector” is a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence. Nucleic acids in vectors can be operably linked to one or more expression control sequences. As used herein, “operably linked” means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II). To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer also can be located downstream from the transcription initiation site. A coding sequence is “operably linked” and “under the control” of expression control sequences in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence.

Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalo virus, retroviruses, vaccinia viruses, adenoviruses, and adeno-associated viruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

An expression vector can include a tag sequence. Tag sequences, are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, Flag™ tag (Kodak, New Haven, CT), maltose E binding protein and protein A. In one embodiment, a nucleic acid molecule encoding one of the disclosed polypeptides is present in a vector containing nucleic acids that encode one or more domains of an Ig heavy chain constant region, for example, having an amino acid sequence corresponding to the hinge, CH2 and CH3 regions of a human immunoglobulin Cyl chain.

Vectors containing nucleic acids to be expressed can be transferred into host cells. The term “host cell” is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used herein, “transformed” and “transfected” encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of a number of techniques. Although not limited to a particular technique, a number of these techniques are well established within the art. Prokaryotic cells can be transformed with nucleic acids by, for example, electroporation or calcium chloride mediated transformation. Nucleic acids can be transfected into mammalian cells by techniques including, for example, calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, or microinjection. Host cells (e.g., a prokaryotic cell or a eukaryotic cell such as a CHO cell) can be used to, for example, produce the proteins, polypeptides, fragments, variants and fusions thereof described herein.

The vectors described can be used to express the proteins, polypeptides, fragments, variants and fusions thereof in cells. An exemplary vector includes, but is not limited to, an adenoviral vector. One approach includes nucleic acid transfer into primary cells in culture followed by autologous transplantation of the ex vivo transformed cells into the host, either systemically or into a particular organ or tissue. Ex vivo methods can include, for example, the steps of harvesting cells from a subject, culturing the cells, transducing them with an expression vector, and maintaining the cells under conditions suitable for expression of the encoded polypeptides. These methods are known in the art of molecular biology. The transduction step can be accomplished by any standard means used for ex vivo gene therapy, including, for example, calcium phosphate, lipofection, electroporation, viral infection, and biolistic gene transfer. Alternatively, liposomes or polymeric microparticles can be used. Cells that have been successfully transduced then can be selected, for example, for expression of the coding sequence or of a drug resistance gene. The cells then can be lethally irradiated (if desired) and injected or implanted into the subject. In one embodiment, expression vectors containing nucleic acids encoding fusion proteins are transfected into cells that are administered to a subject in need thereof.

In vivo nucleic acid therapy can be accomplished by direct transfer of a functionally active DNA into mammalian somatic tissue or organ in vivo. For example, nucleic acids encoding polypeptides disclosed herein can be administered directly to lymphoid tissues. Alternatively, lymphoid tissue specific targeting can be achieved using lymphoid tissue-specific transcriptional regulatory elements (TREs) such as a B lymphocyte-, T lymphocyte-, or dendritic cell-specific TRE. Lymphoid tissue specific TREs are known in the art.

Nucleic acids may also be administered in vivo by viral means. Nucleic acid molecules encoding fusion proteins may be packaged into retrovirus vectors using packaging cell lines that produce replication- defective retroviruses, as is well-known in the art. Other virus vectors may also be used, including recombinant adenoviruses and vaccinia virus, which can be rendered non-replicating. In addition to naked DNA or RNA, or viral vectors, engineered bacteria may be used as vectors.

Nucleic acids may also be delivered by other carriers, including liposomes, polymeric micro- and nanoparticles and polycations such as asialoglycoprotein/polylysine.

In addition to virus- and carrier-mediated gene transfer in vivo, physical means well-known in the art can be used for direct transfer of DNA, including administration of plasmid DNA and particle-bombardment mediated gene transfer.

5. Small Molecules The immunomodulatory agent can be a small molecule. Small molecules agonists and antagonists FLRT3 are known in the art or can be identified using routine screening methods.

In some embodiments, screening assays can include random screening of large libraries of test compounds. Alternatively, the assays may be used to focus on particular classes of compounds suspected of modulating the level of FLRT3. Assays can include determinations of FLRT3 mediated signaling activity. Other assays can include determinations of nucleic acid transcription or translation, mRNA levels, mRNA stability, mRNA degradation, transcription rates, and translation rates.

D. Pharmaceutical Compositions Pharmaceutical compositions including the disclosed immunomodulatory agents are provided. Pharmaceutical compositions containing the immunomodulatory agent can be for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.

In some in vivo approaches, the compositions disclosed herein are administered to a subject in a therapeutically effective amount. As used herein the term “effective amount” or “therapeutically effective amount” means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated or to otherwise provide a desired pharmacologic and/or physiologic effect. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g, age, immune system health, etc.), the disease, and the treatment being affected.

For the disclosed immunomodulatory agents, as further studies are conducted, information will emerge regarding appropriate dosage levels for treatment of various conditions in various patients, and the ordinary skilled worker, considering the therapeutic context, age, and general health of the recipient, will be able to ascertain proper dosing. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired. For the disclosed immunomodulatory agents, generally dosage levels of 0.001 to 20 mg/kg of body weight daily are administered to mammals. Generally, for intravenous injection or infusion, dosage may be lower.

In certain embodiments, the immunomodulatory agent is administered locally, for example by injection directly into a site to be treated. Typically, the injection causes an increased localized concentration of the immunomodulatory agent composition which is greater than that which can be achieved by systemic administration. The immunomodulatory agent compositions can be combined with a matrix as described above to assist in creating an increased localized concentration of the polypeptide compositions by reducing the passive diffusion of the polypeptides out of the site to be treated.

1. Formulations for Parenteral Administration

In some embodiments, compositions disclosed herein, including those containing peptides and polypeptides, are administered in an aqueous solution, by parenteral injection. The formulation may also be in the form of a suspension or emulsion. In general, pharmaceutical compositions are provided including effective amounts of a peptide or polypeptide, and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions optionally include one or more for the following: diluents, sterile water, buffered saline of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and additives such as detergents and solubilizing agents (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and com oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations may be lyophilized and redissolved/resuspended immediately before use. The formulation may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.

2. Formulations for Oral Administration

In some embodiments the compositions are formulated for oral delivery. Oral solid dosage forms are described generally in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, pellets, powders, or granules or incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the disclosed. See, e.g.. Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712, which are incorporated herein by reference. The compositions may be prepared in liquid form, or may be in dried powder (e.g., lyophilized) form. Liposomal or proteinoid encapsulation may be used to formulate the compositions. Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Patent No. 5,013,556). See also Marshall, K. In: Modem Pharmaceutics Edited by G. S. Banker and C. T. Rhodes Chapter 10, 1979. In general, the formulation will include the peptide (or chemically modified forms thereol) and inert ingredients which protect peptide in the stomach environment, and release of the biologically active material in the intestine.

The agents can be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where the moiety permits uptake into the blood stream from the stomach or intestine, or uptake directly into the intestinal mucosa. Also desired is the increase in overall stability of the component or components and increase in circulation time in the body. PEGylation is an exemplary chemical modification for pharmaceutical usage. Other moieties that may be used include: propylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyproline, poly-1, 3-dioxolane and poly-1, 3, 6-tioxocane [see, e.g., Abuchowski and Davis (1981) "Soluble Polymer-Enzyme Adducts," in Enzymes as Drugs. Hocenberg and Roberts, eds. (Wiley-Interscience: New York, N.Y.) pp. 367-383; and Newmark, et al. (1982) J. Appl. Biochem. 4:185-189]

Another embodiment provides liquid dosage forms for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may contain other components including inert diluents; adjuvants such as wetting agents, emulsifying and suspending agents; and sweetening, flavoring, and perfuming agents.

Controlled release oral formulations may be desirable. The agent can be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms, e.g., gums. Slowly degenerating matrices may also be incorporated into the formulation. Another form of a controlled release is based on the Oros therapeutic system (Alza Corp.), i. e.. the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects.

For oral formulations, the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine. In some embodiments, the release will avoid the deleterious effects of the stomach environment, either by protection of the agent (or derivative) or by release of the agent (or derivative) beyond the stomach environment, such as in the intestine. To ensure full gastric resistance a coating impermeable to at least pH 5.0 is essential. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D™, Aquateric™, cellulose acetate phthalate (CAP), Eudragit L™, Eudragit S™, and Shellac™. These coatings may be used as mixed films.

3. Formulations for Topical Administration

The disclosed immunomodulatory agents can be applied topically. Topical administration does not work well for most peptide formulations, although it can be effective especially if applied to the lungs, nasal, oral (sublingual, buccal), vaginal, or rectal mucosa.

Compositions can be delivered to the lungs while inhaling and traverse across the lung epithelial lining to the blood stream when delivered either as an aerosol or spray dried particles having an aerodynamic diameter of less than about 5 microns.

A wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are the Ultravent nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn II nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler powder inhaler (Fisons Corp., Bedford, Mass.). Nektar, Alkermes and Mannkind all have inhalable insulin powder preparations approved or in clinical trials where the technology could be applied to the formulations described herein.

Formulations for administration to the mucosa will typically be spray dried drug particles, which may be incorporated into a tablet, gel, capsule, suspension or emulsion. Standard pharmaceutical excipients are available from any formulator.

Transdermal formulations may also be prepared. These will typically be ointments, lotions, sprays, or patches, all of which can be prepared using standard technology. Transdermal formulations may require the inclusion of penetration enhancers.

4. Controlled Delivery Polymeric Matrices The immunomodulatory agents disclosed herein can also be administered in controlled release formulations. Controlled release polymeric devices can be made for long term release systemically following implantation of a polymeric device (rod, cylinder, film, disk) or injection (microparticles). The matrix can be in the form of microparticles such as microspheres, where the agent is dispersed within a solid polymeric matrix or microcapsules, where the core is of a different material than the polymeric shell, and the peptide is dispersed or suspended in the core, which may be liquid or solid in nature. Unless specifically defined herein, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer may be cast as a thin slab or film, ranging from nanometers to four centimeters, a powder produced by grinding or other standard techniques, or even a gel such as a hydrogel.

Either non-biodegradable or biodegradable matrices can be used for delivery of fusion polypeptides or nucleic acids encoding the fusion polypeptides, although in some embodiments biodegradable matrices are preferred. These may be natural or synthetic polymers, although synthetic polymers are preferred in some embodiments due to the better characterization of degradation and release profiles. The polymer is selected based on the period over which release is desired. In some cases linear release may be most useful, although in others a pulse release or “bulk release” may provide more effective results. The polymer may be in the form of a hydrogel (typically in absorbing up to about 90% by weight of water), and can optionally be crosslinked with multivalent ions or polymers.

The matrices can be formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any of the methods developed for making microspheres for drug delivery, for example, as described by Mathiowitz and Langer, J. Controlled Release, 5:13-22 (1987); Mathiowitz, et ak, Reactive Polymers, 6:275-283 (1987); and Mathiowitz, et ah, J. Appl. Polymer ScL, 35:755-774 (1988). The devices can be formulated for local release to treat the area of implantation or injection - which will typically deliver a dosage that is much less than the dosage for treatment of an entire body - or systemic delivery. These can be implanted or injected subcutaneously, into the muscle, fat, or swallowed.

III. Methods of Manufacture

A. Methods of Making Antibodies

The disclosed antibodies can be generated in cell culture, in phage, or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, and apes. Therefore, in one embodiment, an antibody is a mammalian antibody. Phage techniques can be used to isolate an initial antibody or to generate variants with altered specificity or avidity characteristics. Such techniques are routine and well known in the art. In one embodiment, the antibody is produced by recombinant means known in the art. For example, a recombinant antibody can be produced by transfecting a host cell with a vector comprising a DNA sequence encoding the antibody. One or more vectors can be used to transfect the DNA sequence expressing at least one VL and one VH region in the host cell. Exemplary descriptions of recombinant means of antibody generation and production include Delves, Antibody Production: Essential Techniques (Wiley, 1997); Shephard, et ak, Monoclonal Antibodies (Oxford University Press, 2000); Goding, Monoclonal Antibodies: Principles And Practice (Academic Press, 1993); Current Protocols In Immunology (John Wiley & Sons, most recent edition).

FLRT3 deficient (“knockout) mice or wild type mice can be utilized for the generation of high affinity mAbs against FLRT3 using proprietary immunization techniques.

The disclosed antibodies can be modified by recombinant means to increase greater efficacy of the antibody in mediating the desired function. Thus, it is within the scope of the invention that antibodies can be modified by substitutions using recombinant means. Typically, the substitutions will be conservative substitutions. For example, at least one amino acid in the constant region of the antibody can be replaced with a different residue. See, e.g., U.S. Pat. No. 5,624,821, U.S. Pat. No. 6,194,551, Application No. WO 9958572; and Angal, et al., Mol Immunol. 30:105-08 (1993). The modification in amino acids includes deletions, additions, and substitutions of amino acids. In some cases, such changes are made to reduce undesired activities, e.g., complement-dependent cytotoxicity. Frequently, the antibodies are labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. These antibodies can be screened for binding to proteins, polypeptides, or fusion proteins of FLRT3. See, e.g., Antibody Engineering: A Practical Approach (Oxford University Press, 1996).

For example, suitable antibodies with the desired biologic activities can be identified using in vitro assays including but not limited to: proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, signal transduction, and in vivo assays such as the inhibition of tumor growth. The antibodies provided herein can also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for the ability to bind to the specific antigen without inhibiting the receptor-binding or biological activity of the antigen. As neutralizing antibodies, the antibodies can be useful in competitive binding assays.

Antibodies that can be used in the disclosed compositions and methods include whole immunoglobulin (i.e., an intact antibody) of any class, fragments thereof, and synthetic proteins containing at least the antigen binding variable domain of an antibody. The variable domains differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies.

Also disclosed are fragments of antibodies which have bioactivity. The fragments, whether attached to other sequences or not, include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the non- modified antibody or antibody fragment.

Techniques can also be adapted for the production of single-chain antibodies specific to an antigenic peptide. Methods for the production of single-chain antibodies are well known to those of skill in the art. A single chain antibody can be created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain via a 15 to 25 amino acid peptide or linker have been developed without significantly disrupting antigen binding or specificity of the binding. The linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation.

Divalent single-chain variable fragments (di-scFvs) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs. ScFvs can also be designed with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Diabodies have been shown to have dissociation constants up to 40-fold lower than corresponding scFvs, meaning that they have a much higher affinity to their target. Still shorter linkers (one or two amino acids) lead to the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced. They exhibit an even higher affinity to their targets than diabodies.

A monoclonal antibody is obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. Monoclonal antibodies include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.

Monoclonal antibodies can be made using any procedure which produces monoclonal antibodies. In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

Antibodies may also be made by recombinant DNA methods. DNA encoding the disclosed antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques.

Methods of making antibodies using protein chemistry are also known in the art. One method of producing proteins comprising the antibodies is to link two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA). One skilled in the art can readily appreciate that a peptide or polypeptide corresponding to the antibody, for example, can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of an antibody can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof. Alternatively, the peptide or polypeptide is independently synthesized in vivo as described above. Once isolated, these independent peptides or polypeptides may be linked to form an antibody or antigen binding fragment thereof via similar peptide condensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains. Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two-step chemical reaction. The first step is the chemoselective reaction of an unprotected synthetic peptide- alpha-thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site.

B. Methods for Producing Proteins

The disclosed proteins, polypeptides, fragments, variants and fusions thereof can be manufactured using conventional techniques that are known in the art. Isolated fusion proteins can be obtained by, for example, chemical synthesis or by recombinant production in a host cell. To recombinantly produce a protein, polypeptide, fragment, variant or fusion thereof, a nucleic acid containing a nucleotide sequence encoding the protein, polypeptide, fragment, variant or fusion thereof can be used to transform, transduce, or transfect a bacterial or eukaryotic host cell (e.g., an insect, yeast, or mammalian cell). In general, nucleic acid constructs include a regulatory sequence operably linked to a nucleotide sequence encoding the protein, polypeptide, fragment, variant or fusion thereof. Regulatory sequences (also referred to herein as expression control sequences) typically do not encode a gene product, but instead affect the expression of the nucleic acid sequences to which they are operably linked.

Useful prokaryotic and eukaryotic systems for expressing and producing polypeptides are well known in the art include, for example, Escherichia coli strains such as BL-21, and cultured mammalian cells such as CHO cells.

In eukaryotic host cells, a number of viral-based expression systems can be utilized to express fusion proteins. Viral based expression systems are well known in the art and include, but are not limited to, baculoviral, SV40, retroviral, or vaccinia based viral vectors.

Mammalian cell lines that stably express proteins, polypeptides, fragments, variants or fusions thereof, can be produced using expression vectors with appropriate control elements and a selectable marker. For example, the eukaryotic expression vectors pCR3.1 (Invitrogen Life Technologies) and p91023(B) (see Wong et al. (1985) Science 228:810-815) are suitable for expression of proteins, polypeptides, fragments, variants or fusions thereof, in, for example, Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCK cells, and human vascular endothelial cells (HUVEC). Additional suitable expression systems include the GS Gene Expression System™ available through Lonza Group Ltd. Following introduction of an expression vector by electroporation, lipofection, calcium phosphate, or calcium chloride co-precipitation, DEAE dextran, or other suitable transfection method, stable cell lines can be selected (e.g., by metabolic selection, or antibiotic resistance to G418, kanamycin, or hygromycin). The transfected cells can be cultured such that the polypeptide of interest is expressed, and the polypeptide can be recovered from, for example, the cell culture supernatant or from lysed cells. Alternatively, a protein, polypeptide, fragment, variant or fusion thereof, can be produced by (a) ligating amplified sequences into a mammalian expression vector such as pcDNA3 (Invitrogen Life Technologies), and (b) transcribing and translating in vitro using wheat germ extract or rabbit reticulocyte lysate.

Proteins, polypeptides, fragments, variants or fusions thereof, can be isolated using, for example, chromatographic methods such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, DEAE ion exchange, gel filtration, and hydroxylapatite chromatography. In some embodiments, proteins, polypeptides, fragments, variants or fusions thereof can be engineered to contain an additional domain containing amino acid sequence that allows the polypeptides to be captured onto an affinity matrix. For example, an Fc-fusion polypeptide in a cell culture supernatant or a cytoplasmic extract can be isolated using a protein A column. In addition, a tag such as c-myc, hemagglutinin, polyhistidine, or Flag™ (Kodak) can be used to aid polypeptide purification. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus. Other fusions that can be useful include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase. Immunoaffmity chromatography also can be used to purify polypeptides. Fusion proteins can additionally be engineered to contain a secretory signal (if there is not a secretory signal already present) that causes the Proteins, polypeptides, fragments, variants or fusions thereof to be secreted by the cells in which it is produced. The secreted Proteins, polypeptides, fragments, variants or fusions thereof can then conveniently be isolated from the cell media.

C. Methods for Producing Isolated Nucleic Acid Molecules

Isolated nucleic acid molecules can be produced by standard techniques, including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid encoding a variant polypeptide. PCR is a technique in which target nucleic acids are enzymatically amplified. Typically, sequence information from the ends of the region of interest or beyond can be employed to design oligonucleotide primers that are identical in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers typically are 14 to 40 nucleotides in length but can range from 10 nucleotides to hundreds of nucleotides in length. General PCR techniques are described, for example in PCR Primer: A Laboratory Manual ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. When using RNA as a source of template, reverse transcriptase can be used to synthesize a complementary DNA (cDNA) strand. Ligase chain reaction, strand displacement amplification, self-sustained sequence replication or nucleic acid sequence-based amplification also can be used to obtain isolated nucleic acids. See, for example, Lewis (1992) Genetic Engineering News 12:1; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878; and Weiss (1991 ) Science 254:1292-1293.

Isolated nucleic acids can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides (e.g., using phosphoramidite technology for automated DNA synthesis in the 3’ to 5’ direction). For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase can be used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector. Isolated nucleic acids can also obtained by mutagenesis. Protein-encoding nucleic acids can be mutated using standard techniques, including oligonucleotide- directed mutagenesis and/or site-directed mutagenesis through PCR. See, Short Protocols in Molecular Biology. Chapter 8, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al, 1992.

IV. Assays and Antibody Screening

One embodiment provides assays for antibody screening. Assays for antibody screening include:

1. Analysis of binding affinity of FLRT3 -Fc to ligands in comparison to FLRT3.

2. Functional assays to confirm FLRT3 -Fc prevents signaling by FLRT3 expressing cells. Reporter cells may be utilized for these assays, or primary FLRT3 + cells are another option.

A. Phase screening

1. Phase I screening: screen for mAb binding to cell lines transfected to express cell surface FLRT3. Additionally, mAbs should have the capacity to bind endogenously expressed FLRT3 on the surface of primary human cell subsets, or endogenously expressed. These mAbs should be highly specific for FLRT3.

2. Phase II screening: FLRT3 specific mAbs should block the binding of FLRT3 to its ligands.

3. Phase III screening: Functional assays to confirm that FLRT3 mAbs or combination of mAbs modulate FLRT3 mediated signaling. These assays will utilize cell lines that express endogenous FLRT3, or primary cells such as human monocytes, macrophages and dendritic cell subsets or any other leukocyte populations that express FLRT3 to assess function in the presence of FLRT3 mAbs. Additionally, reporter cells lines may be used to determine if signaling pathways such as NF-kB (NF-kB reporter) or NFAT (NFAT reporter) are altered following culture with FLRT3 mAbs. 4. Phase IV screening: Functional assays to determine if FLRT3 mAbs are capable of inducing antibody dependent cell cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) or cellular apoptosis through other mechanisms, of FLRT3 expressing cell lines. In particular, FLRT3 mAbs will be tested for the ability to deplete through one of these methods leukemia cell lines, known to express FLRT3 on the cell surface. FLRT3 mAbs may also be engineered to deplete FLRT3 expressing cells and tested as described later in this document through known methods.

5. Phase V screening: Functional assays to determine if FLRT3 mAbs are capable of delivering or inducing a positive or negative signal (agonist) via FLRT3 into FLRT3 expressing cells to stimulate or inhibit cellular function, respectively. Cell lines that endogenously express FLRT3, or transfectants of cell lines will be assessed for activation or inhibition following culture with FLRT3. In other assays, reporter cell lines will be used to determine whether FLRT3 mAbs enhance or dampen positive signaling pathways such as NF-kB (NF-kB reporter) or other known cell signaling reporters. Induction of apoptosis in cell lines will also be evaluated

Phase II and III assays can be used to predict the concentrations of FLRT3 mAb(s) required to block physiological levels of ligands in vivo.

V. Method of Use

The disclosed antagonists or agonists of FLRT3 mediated signaling can be used to modulate immune responses in subjects in need of such treatment.

In one embodiment, the FLRT3 binding moieties induce, promote, or enhance ligand binding to FLRT3 and induce, promote, or enhance proliferation or activation of FLRT3+ immunosuppressive cells or cause depletion of these cells.

In one embodiment, the FLRT3 binding moieties inhibit, reduce or block ligand binding to FLRT3 and inhibit, reduce, or block FLRT3+ immunosuppressive cells or cause depletion of these cells.

Exemplary methods are discussed in more detail below. A. Immune Response Stimulation 1. Therapeutic Strategies

Methods of inducing or enhancing an immune response in a subject are provided. Typically, the methods include administering a subject an effective amount of a FLRT3 immunomodulatory agent or binding moiety, or cells primed ex vivo with the FLRT3 immunomodulatory agent or binding moiety. The immune response can be, for example, inhibition of suppressive immune signals from for example, Treg and MDSC at a tumor site.

Alternatively, the immunomodulatory agent can stimulate signal transduction through FLRT3 and promote or enhance an immune response.

In some embodiments, the FLRT3 immunomodulatory agents or binding moieties can be used to block suppressive immune cells to tumor microenvironments. In another embodiment, the FLRT3 immunomodulatory agent or binding moieties can be used to inhibit, reduce, or block tumor metastasis. In some embodiments, the agent can reduce or inhibit the activity of Tregs, reduce the production of cytokines such as IL-10 from Tregs, reduce the differentiation of Tregs, reduce the number of Tregs, reduce the ratio of Tregs within an immune cell population, or reduce the survival of Tregs. The immunomodulatory agent or binding moiety can be administered to a subject in need thereof in an effective amount to overcome T cell exhaustion and/or T cell anergy. Overcoming T cell exhaustion or T cell anergy can be determined by measuring T cell function using known techniques.

The methods can be used in vivo or ex vivo to inhibit, reduce, or block suppressive immune responses and thereby have a stimulating therapeutic effect.

In some embodiments, the agent, or nucleic acid encoding the agent, is administered directly to the subject. In some embodiments, the agent or nucleic acid encoding the agent, is contacted with cells (e.g., immune cells) ex vivo, and the treated cells are administered to the subject (e.g., adoptive transfer). The agents can enable a more robust immune response to be possible. The disclosed compositions are useful to stimulate or enhance immune responses involving T cells by inhibiting, reducing or blocking suppressive immune signal transduction through FLRT3.

The immunomodulatory agents utilized for increasing an immune response are typically those that reduce FLRT3 expression, ligand binding, crosslinking, suppressive signaling, or a combination thereof. For example, the agent can be an antagonist of FLRT3, such as an antagonist (blocking) anti- FLRT3 antibody or antigen binding fragment thereof. The agent can also be a FLRT3 polypeptide, for example, a soluble polypeptide, or fusion protein thereof that can serve as a decoy receptor for one or more FLRT3 ligands or receptors.

FLRT3 blockade, for example using function blocking anti- FLRT3 antibodies, can be an alternative agent or complementary agent to soluble FLRT3 polypeptides and fusion proteins. For example, in some embodiments, FLRT3 blockade is combined with a decoy receptor such as soluble FLRT3 or fusion protein thereof. The combined treatment (e.g., FLRT3 -Fc and FLRT3 blockade) may be complementary.

In some embodiments, immune response stimulating therapy (e.g., in the treatment of cancer or infections) includes depletion of FLRT3 + cells.

Development and identification of FLRT3 depleting mAbs can be carried out according to known construction and screening methods including those discussed herein. See, for example, Reff, et al, Blood. Vol83, No 2, 1994: pp 435-445, which describes preparation of an anti- CD20 chimeric antibody that binds to human Clq, and mediates complement-dependent cell lysis (CDCC) in the presence of human complement, and anti-body-dependent cellular cytotoxicity (ADCC) with human effector cells. Rituximab destroys B cells and is therefore used to treat diseases which are characterized by overactive, dysfunctional, or excessive numbers of B cells. Other B cell-depleting antibodies include ocrelizumab and ofatumumab. In another example, CD3 Abs can preferentially target and deplete activated effector T cells while preserving CD4+Foxp3+ Tregs. The antibodies transiently deplete T cells although they display no or little complement-dependent and antibody-dependent cellular cytotoxicity. Redirected cell lysis due to the ability to crosslink CD3 molecules expressed by two different cells (cytotoxic CD8+ T cells on one side and other target T cells on the other side) has been shown, however, T cell depletion mostly results from AICD (reviewed in You, Front Immunol. 2015; 6: 242).

2. Subjects to be Treated a. Treatment of Cancer

The disclosed compositions and methods can be used to treat cancer. Generally, the agents are used to stimulate or enhance an immune response to cancer in the subject by administering to the subject an amount of an immunomodulatory agent, for example any one of the disclosed antibodies or antigen-binding fragments or fusion proteins that inhibits, reduces, or blocks FLRT3 expression, ligand binding, crosslinking, suppressive signaling, or a combination thereof. The immunomodulatory agent can bind FLRT3 and promote or enhance an immune response by stimulating signal transduction through FLRT3. The method can reduce or more symptoms of the cancer.

In one embodiment the FLRT3 immunomodulatory agents or binding moieties inhibit, reduce, or block FLRT3 and ligand binding and thereby inhibit, reduce, or block Treg and MDSC suppressive functions at a tumor site.

In another embodiment, the FLRT3 immunomodulatory agents or binding moieties inhibit, reduce, or block FLRT3 and deplete the suppressive immune cells, for example in a tumor microenvironment.

In another embodiment, the FLRT3 immunomodulatory agents or binding moieties inhibit, reduce, or block FLRT3 and ligand binding and thereby inhibit, reduce, or block trafficking of suppressive immune cells to a tumor microenvironment and thereby inhibit, reduce, or block tumor metastasis.

Cancer cells acquire a characteristic set of functional capabilities during their development through various mechanisms. Such capabilities include evading apoptosis, self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion/metastasis, limitless replicative potential, and sustained angiogenesis. The term “cancer cell” is meant to encompass both pre-malignant and malignant cancer cells. In some embodiments, cancer refers to a benign tumor, which has remained localized. In other embodiments, cancer refers to a malignant tumor, which has invaded and destroyed neighboring body structures and spread to distant sites. In yet other embodiments, the cancer is associated with a specific cancer antigen (e.g., pan-carcinoma antigen (KS 1/4), ovarian carcinoma antigen (CA125), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), CD19, CD20, HER2/neu, etc.).

The methods and compositions disclosed herein are useful in the treatment or prevention of a variety of cancers or other abnormal proliferative diseases, including (but not limited to) the following: carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin; including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Berketts lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscarama, and osteosarcoma; and other tumors, including melanoma, xenoderma pegmentosum, keratoactanthoma, seminoma, thyroid follicular cancer and teratocarcinoma.

Cancers caused by aberrations in apoptosis can also be treated by the disclosed methods and compositions. Such cancers may include, but are not limited to, follicular lymphomas, carcinomas with p53 mutations, hormone dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes. In specific embodiments, malignancy or dysproliferative changes (such as metaplasias and dysplasias), or hyperproliferative disorders, are treated or prevented by the methods and compositions in the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus. In other specific embodiments, sarcoma, melanoma, or leukemia is treated or prevented by the methods and compositions.

The disclosed compositions and methods are particularly useful for the treatment of cancers that are associated with cells that express abnormally high levels of FLRT3 or FLRT3 specific binding partner, including a ligand or counter-receptor.

Specific cancers and related disorders that can be treated or prevented by methods and compositions disclosed herein include, but are not limited to, leukemias including, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as, but not limited to, Hodgkin's disease or non-Hodgkin's disease lymphomas (e.g., diffuse anaplastic lymphoma kinase (ALK) negative, large B-cell lymphoma (DLBCL); diffuse anaplastic lymphoma kinase (ALK) positive, large B-cell lymphoma (DLBCL); anaplastic lymphoma kinase (ALK) positive, ALK+ anaplastic large-cell lymphoma (ALCL), acute myeloid lymphoma (AML)); multiple myelomas such as, but not limited to, smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas such as, but not limited to, bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft- tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors including but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nongbal tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer including, but not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, and inflammatory breast cancer; adrenal cancer, including but not limited to, pheochromocytom and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer, including but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers including but not limited to, Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipius; eye cancers including, but not limited to, ocular melanoma such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers, including, but not limited to, squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, including but not limited to, squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers including, but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers including, but not limited to, endometrial carcinoma and uterine sarcoma; ovarian cancers including, but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers including, but not limited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers including, but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers; liver cancers including, but not limited to, hepatocellular carcinoma and hepatoblastoma, gallbladder cancers including, but not limited to, adenocarcinoma; cholangiocarcinomas including, but not limited to, papillary, nodular, and diffuse; lung cancers including but not limited to, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; testicular cancers including, but not limited to, germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers including, but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers including, but not limited to, squamous cell carcinoma; basal cancers; salivary gland cancers including, but not limited to, adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers including, but not limited to, squamous cell cancer, and verrucous; skin cancers including, but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney cancers including, but not limited to, renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer); Wilms' tumor; bladder cancers including, but not limited to, transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas (for a review of such disorders, see Fishman et ak, 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A., Inc., United States of America). b. T reatment of Infections

The disclosed compositions and methods can be used to treat infections and infectious diseases. Generally, the agents are used to stimulate or enhance an immune response to infection in the subject by administering to the subject an amount of an immunomodulatory agent that modulates FLRT3 expression, ligand binding, crosslinking, suppressive signaling, or a combination thereof including but not limited to any one of the disclosed antibodies or antigen-binding fragments or fusion proteins. In one embodiment, the immunomodulatory agent inhibits, reduces, or blocks a suppressive immune signal transduction through FLRT3. In another embodiment, the immunomodulatory agent induces, promotes, or enhances an immune response by inducing, promoting, or enhancing signal transduction through FLRT3. The method can reduce one or more symptoms of the infection.

The infection or disease can be caused by a bacterium, virus, protozoan, helminth, or other microbial pathogen that enters intracellularly and is attacked, i. e.. by cytotoxic T lymphocytes.

The infection or disease can be acute or chronic. An acute infection is typically an infection of short duration. During an acute microbial infection, immune cells begin expressing immunomodulatory receptors. Accordingly, in some embodiments, the method includes increasing an immune stimulatory response against an acute infection.

The infection can be caused by, for example, but not limited to Candida albicans, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria meningitidis, Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, or Mycobacterium.

In some embodiments, the disclosed compositions are used to treat chronic infections, for example infections in which T cell exhaustion or T cell anergy has occurred causing the infection to remain with the host over a prolonged period of time. Exemplary infections to be treated are chronic infections cause by a hepatitis virus, a human immunodeficiency virus (HIV), a human T- lymphotrophic virus (HTLV), a herpes virus, an Epstein-Barr virus, or a human papilloma virus.

Because viral infections are cleared primarily by T cells, an increase in T-cell activity would be therapeutically useful in situations where more rapid or thorough clearance of an infective viral agent would be beneficial to an animal or human subject. Thus, the disclosed compositions can be administered for the treatment of local or systemic viral infections, including, but not limited to, immunodeficiency (e.g., HIV), papilloma (e.g, HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., human influenza virus A), and common cold (e.g., human rhinovirus) and other viral infections, caused by, for example, HTLV, hepatitis virus, respiratory syncytial virus, vaccinia virus, and rabies virus. The molecules can be administered topically to treat viral skin diseases such as herpes lesions or shingles, or genital warts. The molecules can also be administered systemically to treat systemic viral diseases, including, but not limited to, AIDS, influenza, the common cold, or encephalitis.

Representative infections that can be treated, include but are not limited to infections cause by microorganisms including, but not limited to,

Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus influenza type B (HIB), Hyphomicrobium, Legionella, Leptspirosis, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, and Treponema, Vibrio, Yersinia, Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis and Schistosoma mansoni.

Other microorganisms that can be treated using the disclosed compositions and methods include, bacteria, such as those of Klebsiella, Serratia, Pasteurella; pathogens associated with cholera, tetanus, botulism, anthrax, plague, and Lyme disease; or fungal or parasitic pathogens, such as Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizophus), Sporothrix (schenkii), Blastomyces (dermatitidis), Paracoccidioides (brasiliensis), Coccidioides (immitis) and Histoplasma (capsulatuma), Entamoeba, histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondi, etc.), Sporothrix, Blastomyces, Paracoccidioides, Coccidioides, Histoplasma, Entamoeba, Histolytica, Balantidium, Naegleria, Acanthamoeba, Giardia, Cryptosporidium, Pneumocystis, Plasmodium, Babesia, or Trypanosoma, etc.

B. Immune Response Inhibiting 1. Therapeutic Strategies

Methods of reducing or inhibiting an immune response in a subject are provided. Typically, the methods include administering a subject an effective amount of any one of the disclosed antibodies or antigen-binding fragments or fusion proteins, or cells primed ex vivo with these immunomodulatory agents. The immune response can be, for example, promoting or enhancing a suppressive immune response. In one embodiment, the disclosed compositions promote, enhance, or activate Tregs, increase the production of cytokines such as IL-10 from Tregs, increase the differentiation of Tregs, increase the number of Tregs, increase the ratio of Tregs within an immune cell population, or increase the survival of Tregs.to provide an immune suppressive response. In another embodiment, the immunomodulatory agent promotes a suppressive immune response by inducing, promoting, or enhancing signal transduction through FLRT3.

The methods can be used in vivo or ex vivo as immune response- inhibiting therapeutic applications. Thus, in some embodiments, the agent, or nucleic acid encoding the agent, is administered directly to the subject. In some embodiments, the agent or nucleic acid encoding the agent, is contacted with cells (e.g., immune cells) ex vivo, and the treat cells are administered to the subject (e.g. adoptive transfer). In general, the disclosed immunomodulatory agents can be used for treating a subject having or being predisposed to any disease or disorder to which the subject's immune system mounts an overactive or inappropriate immune response. The agents can enable a less robust immune response to be possible. The disclosed compositions are useful to reduce or inhibit immune responses involving T cells.

The immunomodulatory agents utilized for reducing an immune response are typically those that increase FLRT3 expression, ligand binding, crosslinking, FLRT3 mediated signaling, or a combination thereof. For example, the agent can be an agonist of FLRT3, such as an agonist (stimulating) anti- FLRT3 antibody or antigen binding fragment thereof a. Inflammatory Responses

The disclosed compositions and methods can be used to treat inflammation. Generally, the agents are used to reduce or inhibit an immune response in the subject by administering to the subject an amount of an immunomodulatory agent that modulates FLRT3 expression, ligand binding, crosslinking, suppressive signaling, or a combination thereof. The method can reduce or more symptoms of the inflammation. In inflammation can be acute, chronic, or persistent inflammation.

In some embodiments, the immunomodulatory agents slow down the immune system. For example, agent can be used to control hyper- inflammatory response causing damage healthy tissues. Accordingly, in some embodiments, the agents are administered to a subject undergoing a hyper-inflammatory response. In such cases, controlling excessive immune responses can be beneficial to the subject.

A method for treating an inflammatory response in a subject in need thereof comprising administering an effective amount of any one of the antibodies or antigen-binding fragments or fusion proteins to treat the inflammatory response. b. Inflammatory and Autoimmune Diseases/ disorders

Agents that modulate FLRT3 expression, ligand binding, crosslinking, suppressive signaling, or a combination thereof can also be used to treat inflammatory or autoimmune diseases and disorders. Representative inflammatory or autoimmune diseases/disorders include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison’s disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (alps), autoimmune thrombocytopenic purpura (ATP), Bechet’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, fibromyalgia - fibromyositis, Graft versus host disease, grave’s disease, Guillain-Barre, Hashimoto’s thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulin dependent diabetes (Type I), juvenile arthritis, Meniere’s disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud’s phenomenon, Reiter’s syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren’s syndrome, stiff-man syndrome, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener’s granulomatosis.

In some embodiments the inflammation or autoimmune disease is caused by a pathogen or is the result of an infection.

One embodiment provides a method of treating inflammation in a subject in need thereof comprising administering an effective amount of any one of the disclosed antibodies or antigen-binding fragments or fusion proteins to treat the inflammation.

A method for treating an autoimmune disease in a subject in need thereof comprising administering an effective amount of any one of the disclosed antibodies or antigen-binding fragments or fusion proteins to treat the autoimmune disease.

VI. Combination Therapies

The disclosed immunomodulatory agents can be administered to a subject in need thereof alone or in combination with one or more additional therapeutic agents. In some embodiments, the immunomodulatory agent and the additional therapeutic agent are administered separately, but simultaneously. The immunomodulatory agent and the additional therapeutic agent can also be administered as part of the same composition. In other embodiments, the immunomodulatory agent and the second therapeutic agent are administered separately and at different times, but as part of the same treatment regime.

The subject can be administered a first therapeutic agent 1, 2, 3, 4, 5, 6, or more hours, or 1, 2, 3, 4, 5, 6, 7, or more days before administration of a second therapeutic agent. In some embodiments, the subject can be administered one or more doses of the first agent every 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, or 48 days prior to a first administration of second agent. The immunomodulatory agent can be the first or the second therapeutic agent.

The immunomodulatory agent and the additional therapeutic agent can be administered as part of a therapeutic regimen. For example, if a first therapeutic agent can be administered to a subject every fourth day, the second therapeutic agent can be administered on the first, second, third, or fourth day, or combinations thereof. The first therapeutic agent or second therapeutic agent may be repeatedly administered throughout the entire treatment regimen.

Exemplary molecules include, but are not limited to, cytokines, chemotherapeutic agents, radionuclides, other immunotherapeutic, enzymes, antibiotics, antivirals (especially protease inhibitors alone or in combination with nucleosides for treatment of HIV or Hepatitis B or C), anti-parasites (helminths, protozoans), growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and bioactive fragments thereof (including humanized, single chain, and chimeric antibodies), antigen and vaccine formulations (including adjuvants), peptide drugs, anti-inflammatories, ligands that bind to Toll-Like Receptors (including but not limited to CpG oligonucleotides) to activate the innate immune system, molecules that mobilize and optimize the adaptive immune system, other molecules that activate or up-regulate the action of cytotoxic T lymphocytes, natural killer cells and helper T-cells, and other molecules that deactivate or down- regulate suppressor or regulatory T-cells.

The additional therapeutic agents are selected based on the condition, disorder or disease to be treated. For example, the immunomodulatory agent can be co-administered with one or more additional agents that function to enhance or promote an immune response or reduce or inhibit an immune response.

A. Increasing Immune Responses 1. Antimicrobials

For example, FLRT3 immunomodulatory agents can be used in a preventive or prophylactic role in the treatment and prevention of disease as discussed above, and also in the context of severe trauma injuries like major bum, open bone fracture, accidental amputation or other wounds. Therefore, the FLRT3 immunomodulatory agents can be administered to the subject in combination with an antimicrobial such as an antibiotic, an antifungal, an antiviral, an antiparasitic, or essential oil. In some embodiments, the subject is administered the FLRT3 immunomodulatory agent and/or the antimicrobial at time of admission to the hospital to prevent further bacterial, fungal or viral complications. The antibiotic can target pathogens and the FLRT3 immunomodulatory agent can stimulate the immune system to provide an enhanced response to treat or prevent further infection or disease.

2. Chemotherapeutic Agents

The FLRT3 immunomodulatory agents can be combined with one or more chemotherapeutic agents and pro-apoptotic agents. Representative chemotherapeutic agents include, but are not limited to amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, tioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or a combination thereof. Representative pro-apoptotic agents include, but are not limited to fludarabinetaurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2) and combinations thereof.

3. Other Immunomodulators a. PD-1 antagonists

In some embodiments, FLRT3 immunomodulatory agents are co administered with a PD-1 antagonist. Programmed Death- 1 (PD-1) is a member of the CD28 family of receptors that delivers a negative immune response when induced on T cells. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that decreases T cell multiplication and/or the strength and/or duration of a T cell response. Suitable PD-1 antagonists are described in U.S. Patent Nos. 8,114,845, 8,609,089, and 8,709,416, which are specifically incorporated by reference herein in their entities, and include compounds or agents that either bind to and block a ligand of PD-1 to interfere with or inhibit the binding of the ligand to the PD-1 receptor, or bind directly to and block the PD-1 receptor without inducing inhibitory signal transduction through the PD-1 receptor.

In some embodiments, the PD-1 receptor antagonist binds directly to the PD-1 receptor without triggering inhibitory signal transduction and also binds to a ligand of the PD-1 receptor to reduce or inhibit the ligand from triggering signal transduction through the PD-1 receptor. By reducing the number and/or amount of ligands that bind to PD-1 receptor and trigger the transduction of an inhibitory signal, fewer cells are attenuated by the negative signal delivered by PD-1 signal transduction and a more robust immune response can be achieved.

It is believed that PD-1 signaling is driven by binding to a PD-1 ligand (such as B7-H1 or B7-DC) in close proximity to a peptide antigen presented by major histocompatibility complex (MHC) (see, for example, Freeman, Proc. Natl. Acad. Sci. U. S. A, 105:10275-10276 (2008)). Therefore, proteins, antibodies or small molecules that prevent co-ligation of PD-1 and TCR on the T cell membrane are also useful PD-1 antagonists.

In some embodiments, the PD-1 receptor antagonists are small molecule antagonists or antibodies that reduce or interfere with PD-1 receptor signal transduction by binding to ligands of PD-1 or to PD-1 itself, especially where co-ligation of PD-1 with TCR does not follow such binding, thereby not triggering inhibitory signal transduction through the PD- 1 receptor.

Other PD-1 antagonists contemplated by the methods of this invention include antibodies that bind to PD-1 or ligands of PD-1, and other antibodies.

Suitable anti-PD-1 antibodies include, but are not limited to, those described in the following US Patent Nos: 7,332,582, 7,488,802, 7,521,051, 7,524,498, 7,563,869, 7,981,416, 8,088,905, 8,287,856, 8,580,247,

8,728,474, 8,779,105, 9,067,999, 9,073,994, 9,084,776, 9,205,148, 9,358,289, 9,387,247, 9,492,539, 9,492,540, all of which are incorporated by reference in their entireties.

See also Berger et al., Clin. Cancer Res., 14:3044-3051 (2008).

Exemplary anti-B7-Hl (also referred to as anti-PD-Ll) antibodies include, but are not limited to, those described in the following US Pat Nos: 8,383,796, 9,102,725, 9,273,135, 9,393,301, and 9,580,507 all of which are specifically incorporated by reference herein in their entirety.

For anti-B7-DC (also referred to as anti-PD-L2) antibodies see US Pat. Nos.: 7,411,051, 7,052,694, 7,390,888, 8,188,238, and 9,255,147 all of which are specifically incorporated by reference herein in their entirety.

Other exemplary PD-1 receptor antagonists include, but are not limited to B7-DC polypeptides, including homologs and variants of these, as well as active fragments of any of the foregoing, and fusion proteins that incorporate any of these. In some embodiments, the fusion protein includes the soluble portion of B7-DC coupled to the Fc portion of an antibody, such as human IgG, and does not incorporate all or part of the transmembrane portion of human B7-DC.

The PD-1 antagonist can also be a fragment of a mammalian B7-H1, for example from mouse or primate, such as a human, wherein the fragment binds to and blocks PD-1 but does not result in inhibitory signal transduction through PD-1. The fragments can also be part of a fusion protein, for example an Ig fusion protein.

Other useful polypeptides PD-1 antagonists include those that bind to the ligands of the PD-1 receptor. These include the PD-1 receptor protein, or soluble fragments thereof, which can bind to the PD-1 ligands, such as B7- H1 or B7-DC, and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction. B7-H1 has also been shown to bind the protein B7.1 (Butte et al., Immunity, Vol. 27, pp. 111-122, (2007)). Such fragments also include the soluble ECD portion of the PD-1 protein that includes mutations, such as the A99L mutation, that increases binding to the natural ligands (Molnar et al., PNAS, 105:10483-10488 (2008)). B7-1 or soluble fragments thereof, which can bind to the B7-H1 ligand and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction, are also useful.

PD-1 and B7-H1 anti-sense nucleic acids, both DNA and RNA, as well as siRNA molecules can also be PD-1 antagonists. Such anti-sense molecules prevent expression of PD-1 on T cells as well as production of T cell ligands, such as B7-H1, PD-L1 and/or PD-L2. For example, siRNA (for example, of about 21 nucleotides in length, which is specific for the gene encoding PD-1, or encoding a PD-1 ligand, and which oligonucleotides can be readily purchased commercially) complexed with carriers, such as polyethyleneimine (see Cubillos-Ruiz et al., J. Clin. Invest. 119(8): 2231- 2244 (2009), are readily taken up by cells that express PD-1 as well as ligands of PD-1 and reduce expression of these receptors and ligands to achieve a decrease in inhibitory signal transduction in T cells, thereby activating T cells. b. CTLA4 antagonists

Other molecules useful in mediating the effects of T cells in an immune response are also contemplated as additional therapeutic agents. In some embodiments, the molecule is an antagonist of CTLA4, for example an antagonistic anti-CTLA4 antibody. An example of an anti-CTLA4 antibody contemplated for use in the methods of the invention includes an antibody as described in PCT/US2006/043690 (Fischkoff et al., WO/2007/056539).

Dosages for anti-PD-1, anti-B7-Hl, and anti-CTLA4 antibody, are known in the art and can be in the range of, for example, 0.1 to 100 mg/kg, or with shorter ranges of 1 to 50 mg/kg, or 10 to 20 mg/kg. An appropriate dose for a human subject can be between 5 and 15 mg/kg, with 10 mg/kg of antibody (for example, human anti-PD-1 antibody) being a specific embodiment.

Specific examples of an anti-CTLA4 antibody useful in the methods of the invention are Ipilimumab, a human anti-CTLA4 antibody, administered at a dose of, for example, about 10 mg/kg, and Tremelimumab a human anti-CTLA4 antibody, administered at a dose of, for example, about 15 mg/kg. See also Sammartino, et al., Clinical Kidney Journal, 3(2): 135- 137 (2010), published online December 2009.

In other embodiments, the antagonist is a small molecule. A series of small organic compounds have been shown to bind to the B7-1 ligand to prevent binding to CTLA4 (see Erbe et al., J. Biol. Chem., 277:7363-7368 (2002). Such small organics could be administered alone or together with an anti-CTLA4 antibody to reduce inhibitory signal transduction of T cells.

4. Potentiating Agents

In some embodiments, additional therapeutic agents include a potentiating agent. The potentiating agent acts to increase efficacy the immune response up-regulator, possibly by more than one mechanism, although the precise mechanism of action is not essential to the broad practice of the present invention.

In some embodiments, the potentiating agent is cyclophosphamide. Cyclophosphamide (CTX, Cytoxan^®, or Neosar^®) is an oxazahosphorine drug and analogs include ifosfamide (IFO, Ifex), perfosfamide, trophosphamide (trofosfamide; Ixoten), and pharmaceutically acceptable salts, solvates, prodrugs and metabolites thereof (US patent application 20070202077 which is incorporated in its entirety). Ifosfamide (MITOXANA^®) is a structural analog of cyclophosphamide, and its mechanism of action is considered to be identical or substantially similar to that of cyclophosphamide. Perfosfamide (4-hydroperoxycyclophosphamide) and trophosphamide are also alkylating agents, which are structurally related to cyclophosphamide. For example, perfosfamide alkylates DNA, thereby inhibiting DNA replication and RNA and protein synthesis. New oxazaphosphorines derivatives have been designed and evaluated with an attempt to improve the selectivity and response with reduced host toxicity (Liang J, Huang M, Duan W, Yu XQ, Zhou S. Design of new oxazaphosphorine anticancer drugs. Curr Pharm Des. 2007;13(9):963-78. Review). These include mafosfamide (NSC 345842), glufosfamide (D 19575, beta-D-glucosylisophosphoramide mustard), S-(-)-bromofosfamide (CBM-11), NSC 612567 (aldophosphamide perhydrothiazine) and NSC 613060 (aldophosphamide thiazolidine). Mafosfamide is an oxazaphosphorine analog that is a chemically stable 4-thioethane sulfonic acid salt of 4-hydroxy-CPA. Glufosfamide is IFO derivative in which the isophosphoramide mustard, the alkylating metabolite of IFO, is glycosidically linked to a beta-D-glucose molecule. Additional cyclophosphamide analogs are described in US patent 5,190,929 entitled “Cyclophosphamide analogs useful as anti-tumor agents” which is incorporated herein by reference in its entirety.

While CTX itself is nontoxic, some of its metabolites are cytotoxic alkylating agents that induce DNA crosslinking and, at higher doses, strand breaks. Many cells are resistant to CTX because they express high levels of the detoxifying enzyme aldehyde dehydrogenase (ALDH). CTX targets proliferating lymphocytes, as lymphocytes (but not hematopoietic stem cells) express only low levels of ALDH, and cycling cells are most sensitive to DNA alkylation agents.

Low doses of CTX (< 200 mg/kg) can have immune stimulatory effects, including stimulation of anti-tumor immune responses in humans and mouse models of cancer (Brode & Cooke Crit Rev. Immunol. 28:109-126 (2008)). These low doses are sub-therapeutic and do not have a direct anti tumor activity. In contrast, high doses of CTX inhibit the anti-tumor response. Several mechanisms may explain the role of CTX in potentiation of anti-tumor immune response: (a) depletion of CD4+CD25+FoxP3+ Treg (and specifically proliferating Treg, which may be especially suppressive), (b) depletion of B lymphocytes; (c) induction of nitric oxide (NO), resulting in suppression of tumor cell growth; (d) mobilization and expansion of CDllb+Gr-l+ MDSC. These primary effects have numerous secondary effects; for example following Treg depletion macrophages produce more IFN-g and less IL-10. CTX has also been shown to induce type I IFN expression and promote homeostatic proliferation of lymphocytes.

Treg depletion is most often cited as the mechanism by which CTX potentiates the anti-tumor immune response. This conclusion is based in part by the results of adoptive transfer experiments. In the AB1-HA tumor model, CTX treatment at Day 9 gives a 75% cure rate. Transfer of purified Treg at Day 12 almost completely inhibited the CTX response (van der Most et al. Cancer Immunol. Immunother. 58:1219-1228 (2009). A similar result was observed in the HHD2 tumor model: adoptive transfer of CD4+CD25+ Treg after CTX pretreatment eliminated therapeutic response to vaccine (Taieb, J. J. Immunol. 176:2722-2729 (2006)).

Numerous human clinical trials have demonstrated that low dose CTX is a safe, well-tolerated, and effective agent for promoting anti-tumor immune responses (Bas, & Mastrangelo Cancer Immunol. Immunother. 47:1-12 (1998)).

The optimal dose for CTX to potentiate an anti-tumor immune response, is one that lowers overall T cell counts by lowering Treg levels below the normal range but is subtherapeutic (see Machiels et al. Cancer Res. 61:3689-3697 (2001)).

In human clinical trials where CTX has been used as an immunopotentiating agent, a dose of 300 mg/m² has usually been used. For an average male (6 ft, 170 pound (78 kg) with a body surface area of 1.98 m²), 300 mg/m² is 8 mg/kg, or 624 mg of total protein. In mouse models of cancer, efficacy has been seen at doses ranging from 15 - 150 mg/kg, which relates to 0.45 - 4.5 mg of total protein in a 30g mouse (Machiels et al. Cancer Res. 61:3689-3697 (2001), Hengst et al Cancer Res. 41:2163-2167 (1981), Hengst Cancer Res. 40:2135-2141 (1980)).

For larger mammals, such as a primate, such as a human, patient, such mg/m² doses may be used but unit doses administered over a finite time interval may also be used. Such unit doses may be administered on a daily basis for a finite time period, such as up to 3 days, or up to 5 days, or up to 7 days, or up to 10 days, or up to 15 days or up to 20 days or up to 25 days, are all specifically contemplated by the invention. The same regimen may be applied for the other potentiating agents recited herein.

In other embodiments, the potentiating agent is an agent that reduces activity and/or number of regulatory T lymphocytes (T-regs), such as Sunitinib (SUTENT^®), anti-TGF or Imatinib (GLEEVAC^®). The recited treatment regimen may also include administering an adjuvant.

Useful potentiating agents also include mitosis inhibitors, such as paclitaxol, aromatase inhibitors (e.g. Letrozole) and angiogenesis inhibitors (VEGF inhibitors e.g. Avastin, VEGF-Trap) (see, for example, Li et al., Vascular endothelial growth factor blockade reduces intratumoral regulatory T cells and enhances the efficacy of a GM-CSF -secreting cancer immunotherapy. Clin Cancer Res. 2006 Nov 15; 12(22):6808-16.), anthracy dines, oxaliplatin, doxorubicin, TLR4 antagonists, and IL-18 antagonists.

B. Reducing Immune Responses

1. Immunosuppressive Agents

In some embodiments, the immune response, or inflammatory/autoimmune disease/disorder is treated by administering to the subject a FLRT3 immunomodulatory agent and a second agent that is an immune suppressant. Immunosuppressive agents include, but are not limited to antibodies against other lymphocyte surface markers (e.g., CD40, alpha-4 integrin) or against cytokines), fusion proteins (e.g., CTLA-4-Ig (Orencia®), TNFR-Ig (Enbrel®)), TNF-a blockers such as Enbrel, Remicade, Cimzia and Humira, cyclophosphamide (CTX) (i.e., Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune™), methotrexate (MTX) (i.e., Rheumatrex®, Trexall®), belimumab (i.e., Benlysta®), or other immunosuppressive drugs (e.g., cyclosporin A, FK506-like compounds, rapamycin compounds, or steroids), anti-proliferatives, cytotoxic agents, or other compounds that may assist in immunosuppression.

The therapeutic agent can be a CTLA-4 fusion protein, such as CTLA-4-Ig (abatacept). CTLA-4-Ig fusion proteins compete with the co stimulatory receptor, CD28, on T cells for binding to CD80/CD86 (B7-1/B7- 2) on antigen presenting cells, and thus function to inhibit T cell activation. In another embodiment, the therapeutic agent is a CTLA-4-Ig fusion protein known as belatacept. Belatacept contains two amino acid substitutions (L104E and A29Y) that markedly increase its avidity to CD86 in vivo. In another embodiment, the therapeutic agent is Maxy-4.

In another embodiment, the therapeutic agent is cyclophosphamide (CTX). Cyclophosphamide (the generic name for Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune™), also known as cytophosphane, is a nitrogen mustard alkylating agent from the oxazophorines group. It is used to treat various types of cancer and some autoimmune disorders. Cyclophosphamide (CTX) is the primary drug used for diffuse proliferative glomerulonephritis in patients with renal lupus.

The therapeutic agent can be administered in an effective amount to reduce the blood or serum levels of anti-double stranded DNA (anti-ds DNA) auto antibodies and/or to reduce proteinuria in a patient in need thereof.

In another embodiment, the therapeutic agent increases the amount of adenosine in the serum, see for example, WO 08/147482. For example, the second therapeutic agent can be CD73-Ig, recombinant CD73, or another agent (e.g, a cytokine or monoclonal antibody or small molecule) that increases the expression of CD73, see for example WO 04/084933. In another embodiment the therapeutic agent is Interferon-beta.

The therapeutic agent can be a small molecule that inhibits or reduces differentiation, proliferation, activity, and/or cytokine production and/or secretion by Thl, Thl7, Th22, and/or other cells that secrete, or cause other cells to secrete, inflammatory molecules, including, but not limited to, IL-Ib, TNF-a, TGF-beta, IFN-g, IL-18 IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs. In another embodiment, the therapeutic agent is a small molecule that interacts with Tregs, enhances Treg activity, promotes or enhances IL-10 secretion by Tregs, increases the number of Tregs, increases the suppressive capacity of Tregs, or combinations thereof.

In some embodiments, the composition increases Treg activity or production. Exemplary Treg enhancing agents include but are not limited to glucocorticoid fluticasone, salmeteroal, antibodies to IL-12, IFN-g, and IL-4; vitamin D3, and dexamethasone, and combinations thereof. In some embodiments, the therapeutic agent is an antibody, for example, a functions blocking antibody against a proinflammatory molecule such as IL-6, IL-23, IL-22 or IL-21.

As used herein the term “rapamycin compound” includes the neutral tricyclic compound rapamycin, rapamycin derivatives, rapamycin analogs, and other macrolide compounds which are thought to have the same mechanism of action as rapamycin (e.g., inhibition of cytokine function). The language “rapamycin compounds” includes compounds with structural similarity to rapamycin, e.g., compounds with a similar macrocyclic structure, which have been modified to enhance their therapeutic effectiveness. Exemplary Rapamycin compounds are known in the art (See. e.g. W095122972, WO 95116691, WO 95104738, U.S. Patent No. 6,015,809; 5,989,591; U.S. Patent No. 5,567,709; 5,559,112; 5,530,006; 5,484,790; 5,385,908; 5,202,332; 5,162,333; 5,780,462; 5,120,727).

The language “FK506-like compounds” includes FK506, and FK506 derivatives and analogs, e.g., compounds with structural similarity to FK506, e.g., compounds with a similar macrocyclic structure which have been modified to enhance their therapeutic effectiveness. Examples of FK506- like compounds include, for example, those described in WO 00101385. In some embodiments, the language “rapamycin compound” as used herein does not include FK506-like compounds.

2. Anti-inflammatories

Other suitable therapeutic agents include, but are not limited to, anti inflammatory agents. The anti-inflammatory agent can be non-steroidal, steroidal, or a combination thereof. One embodiment provides oral compositions containing about 1% (w/w) to about 5% (w/w), typically about 2.5 % (w/w) or an anti-inflammatory agent. Representative examples of non-steroidal anti-inflammatory agents include, without limitation, oxicams, such as piroxicam, isoxicam, tenoxicam, sudoxicam; salicylates, such as aspirin, disalcid, benorylate, trilisate, safapryn, solprin, diflunisal, and fendosal; acetic acid derivatives, such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acematacin, fentiazac, zomepirac, clindanac, oxepinac, felbinac, and ketorolac; fenamates, such as mefenamic, meclofenamic, flufenamic, niflumic, and tolfenamic acids; propionic acid derivatives, such as ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indopropfen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, and tiaprofenic; pyrazoles, such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone, and trimethazone. Mixtures of these non-steroidal anti-inflammatory agents may also be employed.

Representative examples of steroidal anti-inflammatory drugs include, without limitation, corticosteroids such as hydrocortisone, hydroxyl- triamcinolone, alpha-methyl dexamethasone, dexamethasone-phosphate, beclomethasone dipropionates, clobetasol valerate, desonide, desoxymethasone, desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluclorolone acetonide, fludrocortisone, flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortine butylesters, fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolone, fludrocortisone, diflurosone diacetate, fluradrenolone acetonide, medrysone, amcinafel, amcinafide, betamethasone and the balance of its esters, chloroprednisone, chlorprednisone acetate, clocortelone, clescinolone, dichlorisone, diflurprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone, and mixtures thereof.

VII. Kits

The disclosed FLRT3 immunomodulatory agents can be packaged in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity. The agent can be supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline to the appropriate concentration for administration to a subject. For example, the agent can be supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg, or at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg. The lyophilized agent can be stored at between 2 and 8°C in their original container and are typically administered within 12 hours, or within 6 hours, or within 5 hours, or within 3 hours, or within 1 hour after being reconstituted.

In an alternative embodiment, agent can be supplied in liquid form in a hermetically sealed container indicating the quantity and concentration. In some embodiments, the liquid form of the agent supplied in a hermetically sealed container including at least 1 mg/ml, or at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 50 mg/ml, at least 100 mg/ml, at least 150 mg/ml, at least 200 mg/ml of the agent.

Pharmaceutical packs and kits including one or more containers filled with agent are also provided. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of a disease can also be included in the pharmaceutical pack or kit. The pharmaceutical pack or kit can also include one or more containers filled with one or more of the ingredients of the disclosed pharmaceutical compositions. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Kits designed for the above-described methods are also provided. Embodiments typically include one or more FLRT3 immunomodulatory agents. In particular embodiments, a kit also includes one or more other prophylactic or therapeutic agents useful for the treatment of cancer, in one or more containers. In other embodiments, a kit also includes one or more anti-inflammatory agents useful for the treatment inflammatory and autoimmune diseases, in one or more containers.

EXAMPLES

Example 1: FLRT3’s Immunosuppression In Vitro is Comparable to PD-L1

To determine if FLRT3 is as immunosuppressive as PD-L1, cell- based assays were performed to determine NF-KB activation or IFN-g secretion in the presence of cells expressing human CD3 antibody single chain variable fragment (OKT3) and FLRT3, PD-L1, empty vector negative control or CD80 positive control (Fig. 1A and C). FLRT3 expression results in a comparable decrease in NF-KB -GFP -reporter fluorescence compared to PD-L1 in Jurkat T cells expressing the FLRT3 binding partner UNC5B (Fig. IB). FLRT3 expression also results in a comparable decrease in IFNy production compared to PD-L1 when using purified primary healthy donor T cells (Fig. ID).

Example 2: FLRT3 is Expressed in Mouse and Human Cancers

FLRT3 was shown to be expressed in mouse and human cancers. Figure 2 shows FLRT3 RNA sequence data from mouse cancer passages in immunodeficient mice. The top mouse tumors expressing FLRT3 were breast (4T1, TC, EMT6) and pancreatic (Pan02) tumors (Fig. 2A), while the top human tumors expressing FLRT3 were pancreatic, gastric, H&N, liver, lung, and several others tumors (Fig. 2B). Figures 3 and 4 show FLRT3 RNA expression data from the human cancer patients. Significantly (p < 0.01) increased expression of FLRT3 was also shown in esophageal carcinoma, head and neck squamous, kidney renal papillary carcinoma, pancreatic adenocarcinoma, pheochromocytoma, thyroid carcinoma, thymoma, and others Fig. 3). Overall survival studies showed that high expression of FLRT3 Correlates with reduced overall survival in kidney chromophobe, pancreatic adedocarcinoma and lung squamous cell carcinoma (Fig. 4A-4C). Furthermore, cancer cell lines stained with the 14B7 antibody were assessed for expression of FLRT3. A human kidney and colon cell lines engineered to stably overexpress membrane OKT3 (anti-CD3 monoclonal antibody) scFV sequence, HEK293T-OKT3 (Fig. 5A) and HT29-OKT3 (Fig. 5C) respectively, were shown to express low levels of FLRT3, whereas there was no expression in microglial HMC3 cells (Fig. 5B). A549 (lung, Fig. 5E), MCF7 (breast, Fig. 5G), SKOV3 (ovarian, Fig. 5F) were shown to have relatively higher expression of FLRT3. This data is in line with the RNA database.

Example 3: FLRT3 Overexpression Increases Tumor Growth in NSG mouse model

Tumor volumes were measured every two to three days post inoculation with empty vector (EV) or FLRT3-HT29-OKT3 in immunodeficient NSG (NOD scid gamma) mice to determine the effect of FLRT3 overexpression on human PBMCs (Fig. 6A). In the HT29-OKT3 + NSG model, FLRT3 augments in vivo tumor growth in the presence of 5e6 (Fig. 6B) and 10e6 (Fig. 6C) human peripheral blood mononuclear cells (PBMC). The strong immune response seen in the 10e6 PBMCs group partially masked the inhibitory effects of FLRT3.

Example 4: Soluble FLRT3 Increased Tumor Growth in Humanized Models and Inhibits Immune Function.

Mice inoculated with HT29-OKT3 were treated with hPBMCs and soluble FLRT3 (FLRT3-Fc) and tumor volume was measured twice a week for up to 60 days (Fig. 7A). Clinical graft versus host disease (GvHD) scores showed that soluble FLRT3 protects the NSG mice against GvHD (Fig. 7B). Treatment of HT-29 colorectal cancer tumor bearing mice with soluble FLRT3 Fc fusion protein (Fig 8A) resulted in increased tumor growth (Fig 8B). Immune cell analysis showed that soluble FLRT3 Fc fusion protein caused immune suppression of IFN-g and TNF production in CD8 T cells by intracellular cytokine staining analysis (Fig. 8C-8J). Analysis of serum from mice also demonstrated inhibition of cytolytic cytokines including TNF, Granzyme B, Perforin and Granulysin (Fig. 8K-8P). In a similar repeat study of the HT-29 colorectal tumor model (Fig 8Q), FLRT3 overexpression also increased tumor growth (Fig. 8R) that correlated with decreased human CD45+ cells (Fig. 8S) and decreased CD8 T cells (Fig 8T) in the blood. Example 5: FLRT3 Promotes Tumor Growth in Humanized in Vivo Models

Tumor measurements of mice inoculated with empty vector control or FLRT3 overexpressing624Mel tumor cells and hPBMCs (Fig. 9A) showed that FLRT3 promotes growth of 624Mel in a humanized model (Fig. 9B). In another model with 624Mel overexpressing FLRT3 or empty vector control, soluble FLRT3 was detected in mice 624Mel-FLRT3 implanted mice (Figs. 9C and 9D). This data suggests that soluble FLRT3 may promote tumor growth and restrict immune activity locally and systemically. Example 6: FLRT3 Antibodies Identified by Binding to hFLRT3 on Cells

Thirteen FLRT3 antibodies were isolated and binding was assessed in a FACS assay. Eight out of 13 antibodies bound specifically to FLRT3 in a dose-dependent manner (Fig. 10). Out of the eight antibodies, three bound more strongly than the other five. Binding activity was comparable or better than the commercial FLRT3 antibody. Table 1 shows the K_D values for the isolated FLRT3 recombinant monoclonal antibodies. The 14B7 antibody shows the highest affinity of all the antibodies shown. Table 1: Affinity measurements with FLRT3-His monomeric fusion protein.

Further analysis of the binding studies indicated that FLRT3 antibodies fall into 3 bins. As indicated in Table 2, 1H5 and 2F7 share a bin, 14D3 and 15G11 share a bin and 14B7 is in its own bin (Fig.11). Table 2: FLRT3 monoclonal antibodies fall into three bins.

A binding curve for the top 3 mAb was performed for binding to human FLRT3 transfected cells 293T cells (Fig 12A), and EC50 values for top 3 binders were calculated based on non-linear regression fit (Fig. 12B). MFI values of each group were normalized within that group- i.e. largest value 100% and smallest value 0%. EC50 values for 14B7 < 1.5 nM, for 14D3, 15G11 < 5 nM in two separate experiments (Table 3).

Table 3: EC 50 values of top binders.

Binding of antibodies to mFLRT3 was tested on transiently transfected 293T cells. The antibody 14B7 showed strong binding to mouse FLRT3 (Fig. 13). Antibodies 14D3 and 15G11 showed weak binding at higher antibody concentrations.

Example 8: 14B7 Disrupts Unc5B-FLRT3 Interaction

The FLRT3 antibodies were tested for their ability to block FLRT3- Unc5B interaction in ELISA assays (Fig. 14A). The binding of FLRT3-Fc biotin to Unc5B-Fc was confirmed in the absence of blocking mAbs (Fig. 14B). Testing FLRT3 Fc binding to UNC5B Fc was performed in presence of increasing concentrations of antibodies and 14B7 was shown to block the interaction of Unc5B-FLRT3 at 5 andlO μg/mL (Fig. 14C).

The titration of increasing concentrations of soluble FLRT3 showed that 14B7 blocked FLRT3-Unc5B binding in ELISA assays. The data was confirmed by flow cytometry at titrating concentrations of incubated FLRT3- Fc (Fig. 15A). The 14B7 antibody blocked Unc5B-FLRT3 binding in this assay at as low as 0.9 pg/mL concentration (Fig. 15B). FLRT3 was shown to also bind to Unc5A, Unc5C, and Unc5D in ELISA assay (Fig. 16A). Antibody 14B7 blocked FLRT3 binding to all Unc5 proteins (Fig. 16B-16E). Example 9: 14B7 disrupts Binding of Unc5B-Fc Biotin to FLRT3-Fc

Similar to example 8, but in reverse orientation (Fig. 17A), binding of Unc5B-Fc biotin to FLRT3-Fc was confirmed (Fig. 17B), and tested in presence of increasing concentrations of antibodies (Fig. 17C). ELISA assays showed that 14B7 disrupted Unc5B-FLRT3 interaction in at all concentrations tested (Fig. 17C). In an Octet analysis, the binding curves of FLRT3 antibodies to FLRT3 monomer showed that 14B7 was consistently a top binder (Fig 18).

Example 10: FLRT3 Antibodies Bind to A549 Cells That Express Endogenous hFLRT3

Increasing concentrations of FLRT3 antibodies bound to A549 cells that express endogenous hFLRT3 (Fig. 19). Binding curves for the top three antibodies were performed (Fig. 20A). MFI values of each group were normalized within that group- i.e. largest value 100% and smallest value 0% (Fig. 20A). EC50 values were calculated based on non-linear regression fit (Fig. 20B). EC50 value of 14B7 was less thanl nM demonstrating stronger binding than the other antibodies (Table 4).

Table 4. EC50 values of top binders in A549 cells.

Example 11: FLRT3 Suppresses IFN-g Production in 293T-OKT3- FLRT3 Assay Similar to the assays described in Example 1, FLRT3, PDL1 inhibitory control, empty vector negative control and CD80 stimulatory control was expressed on 293T-OKT3 cells and co-cultured with total PBMCs (Fig. 21A) from multiple donors for evaluation of T cell IFN-g secretion (Fig. 21B-E) and T cell proliferation based on CFSE dilution (Fig. 21F-I). These data demonstrated that FLRT3 suppressed T cell effector function based on inhibition of IFN-g secretion similarly to PD-L1. Whereas, T cell proliferation was less inhibited, once again similar to PD-L1 effects. Example 12: FLRT3 Antibodies Reverse Inhibition of T cells

To determine if FLRT3 antibodies could reverse T cell mediated suppression, assays were performed similar to Example 1. Jurkat T cells that were transduced to express Unc5B and contained an NF-kB-GFP reporter were co-cultured with 293T-OKT3 cells overexpressing either FLRT3 or empty vector control (EV), and with FLRT3 antibody 14B7 or control antibody (Fig 22A-22D). These assays demonstrated the FLRT3 expression reduced Jurkat T cell viability and NF-kB-GFP reporter activity (Fig. 22A- 22D). When 14B7 antibody was added to the culture, Jurkat T cell viability and NF-kB-GFP reporter activity were restored (Fig. 22A-22D).

Total PBMCs from two different donors, Donor 68 (Fig. 23A) and Donor 67 (Fig. 23B) were activated in the presence of HMC3-OKT3 cells overexpressing FLRT3 or empty vector control, and with FLRT3 antibodies or control antibodies. Four days later, supernatants were collected and IFN-g production was analyzed by ELISA. Antibody 14B7 reversed the FLRT3 mediated inhibitory effects on IFN-g in both donors in comparison to the control antibody.

Additional donors PBMCs were co-cultured with FLRT3 overexpressing or endogenously expressing cell lines with FLRT3 antibody 14B7 or control to determine if blockade of FLRT3 robustly promoted T cell activity during T cell priming and activation (Fig. 24 A). Donors 68, 76 and 1805E were culture with 293T-FLRT overexpressing cells (without OKT3) (Fig. 24B-24D), A549 lung cancer cells that endogenously express FLRT3 (Fig. 24E-24G), and SKOV3 ovarian cancer cells that endogenously express FLRT3 (Fig. 24H-24J). Following co-culture, supernatant levels of IFN-g were determined by ELISA. These results indicate that 14B7 blockade of FLRT3 enhanced T cell priming, activation, and IFN-g secretion.

In a similar assay as above but modified to test whether FLRT3 blockade by 14B7 augments T cell activity during the effector phase, T cells were pre-activated, then re-stimulated with SKOV3 ovarian cancer cells that endogenously express FLRT3 in the presence of anti-CD3+anti-CD28 immunocult beads in the presence of 14B7 or control antibodies (Fig. 25A). Three different donors were evaluated and all three demonstrated an increased in IFN-g secretion in the presence of 14B7 in comparison to control antibody (Fig. 25B-25D). Example 13: 14B7 Inhibits Growth of FLRT3+ Tumors and Cancer Cells Naturally Expressing FLRT3.

Tumor kinetics studies of NSG mice with FLRT3 overexpressing- 624Mel-tumors and HLA-matched hPBMCs admixed and implanted were analyzed up to 40 days post treatment (Fig. 26A). Antibody 14B7 decreased tumor growth of FLRT3+ 624Mel tumors (Fig. 26B) and increased the probability of survival for the mice (Fig. 26C). Tumor kinetics studies of NSG mice with A549 WT cells that endogenously express FLRT3, plus HLA-mismatched human PBMCs administered 7 days later, showed that 14B7 decreased tumor growth in A549 tumors naturally expressing FLRT3 (Figs. 27A-27C). A tumor growth study in syngeneic Balb/c mice that are fully immune competent were implanted with CT26 tumor cells that were transduced to overexpress FLRT3, and treated with 14B7 or control antibody (Fig. 28A). Tumor growth assessed on day 21 showed a significant reduction in CT26 tumor growth in the presence of 14B7 FLRT3 blockade (Fig. 28B).

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We Claim:

1. An anti-FLRT3 antibody or antigen-binding fragment thereof comprising: a) a light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 19, 21, 23, 25, 27, and 29, and b) a heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, and 98, wherein the antibody of antigen-binding fragment thereof binds to FLRT3.

2. The antibody or antigen-binding fragment of claim 1, further comprising one or more constant domains from an immunoglobulin constant region (Fc).

3. The antibody or antigen-binding fragment of claim 1, wherein the antibody is humanized.

4. A method for treating a tumor in a subject in need thereof, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof of claim 1 to the subject to reduce tumor burden in the subject.

5. The method of claim 4 wherein the tumor is a colorectal tumor, a lymphoma tumor, or an ovarian tumor.

6. A method for promoting an immune response in a subject in need thereof, comprising administering an effective amount of the monoclonal antibody of claim 1 in an amount effective to promote an immune response in the subject.

7. The method of claim 6, wherein the promoted immune response retards or prevents tumor growth, inhibits tumor-mediated immune suppression, eliminates tumors, depletes or blocks the activity of tumor- associated macrophages (TAMs), decreases TAM-mediated immune suppression, reduces or reverses T cell suppression, increases T cell proliferation, or a combination thereof.

8. A method of treating an autoimmune disease is a subject in need thereof, comprising administering an effective amount of the antibody or antigen-binding fragment of claim 1 to treat the autoimmune disease.

9. An anti-FLRT3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region selected from the group consisting of: a) heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO:209, and light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO:206; b) heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO:200, and light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 195; c) heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 107, and a light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 102; d) heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 119, and a light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 114; e) heavy chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 128, and a light chain variable region having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 126;

10. The antibody or antigen-binding fragment of claim 9, further comprising one or more constant domains from an immunoglobulin constant region (Fc).

11. The antibody or antigen-binding fragment of claim 9, wherein the antibody is humanized.

12. A method for treating a tumor in a subject in need thereof, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof of claim 9 to the subject to reduce tumor burden in the subject.

13. The method of claim 12, wherein the tumor is a colorectal tumor, a lymphoma tumor, or an ovarian tumor.

14. A method for promoting an immune response in a subject in need thereof, comprising administering an effective amount of the monoclonal antibody of claim 9 in an amount effective to promote an immune response in the subject.

15. The method of claim 14, wherein the promoted immune response retards or prevents tumor growth, inhibits tumor-mediated immune suppression, eliminates tumors, depletes or blocks the activity of tumor- associated macrophages (TAMs), decreases TAM-mediated immune suppression, reduces or reverses T cell suppression, increases T cell proliferation, or a combination thereof.

16. A method of treating an autoimmune disease is a subject in need thereof, comprising administering an effective amount of the antibody or antigen-binding fragment of claim 9 to treat the autoimmune disease.

17. An anti-FLRT3 antibody produced by a hybridoma selected from the group consisting of 14B7, 17A1, 15G11, 18A7, 9F3, 1H5, 2F7, 5C2, 7H7, 10C6, 12A4, 13B2, 14D3, and 20C3.

18. A pharmaceutical composition comprising the antibody or antigen binding fragment thereof according to claim 1, and a pharmaceutically acceptable excipient.

19. A fusion protein or an antigen binding fragment thereof having 80%, 85%, 90%, 95%, 99%, or 100% to SEQ ID NO:215, 216, or 217.

20. A method for treating a tumor in a subject in need thereof, comprising administering a therapeutically effective amount of fusion protein of claim 19 to the subject to reduce tumor burden in the subject.

21. A method of treating inflammation in a subject in need thereof, comprising administering an effective amount of the fusion protein of claim 19 to treat the inflammation.