WO2018195773A1

WO2018195773A1 - Use of b7-dc mutant in treatement of asthma

Info

Publication number: WO2018195773A1
Application number: PCT/CN2017/081845
Authority: WO
Inventors: Lieping Chen; Xinxin NIE
Original assignee: Sun Yat-Sen University
Priority date: 2017-04-25
Filing date: 2017-04-25
Publication date: 2018-11-01

Abstract

Disclosed is a method for prophylaxis or treatment of asthma in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a B7-DC mutant with an amino acid sequence as shown in SEQ ID NO. 1 or 2, or an amino acid sequence having at least 80% identity to SEQ ID NO. 1 or 2 and a K113S mutation.

Description

USE OF B7-DC MUTANT IN THE TREATMENT OF ASTHMA

TECHNICAL FIELD

The present invention is related to a method for prophylaxis or treatment of asthma in a subject. The instant invention also concerns use of a B7-DC mutant in the preparation of a pharmaceutical compositions for prophylaxis or treatment of asthma in a subject.

BACKGOUND

Asthma is a chronic small airway disorder characterized by airway obstruction, wheezing airway hyper-responsiveness and inflammation recurrently. Most of this disease arises from exposure to allergens such as pollen, house dust mites, animal dander fungi, molds, air irritants and infections. Different types of immune cells participate in the pathogenesis of asthma, including eosinophils, macrophages, T cells, neutrophils and mast cells. The CD4+ T helper 2 subset (Th2) is an important element in asthma progression. When an allergen stimulates the immune system through the airway of the lung, Th2 cells produce high levels of IL-5, IL-13 and IL-4, which subsequently promote allergen-specific IgE and airway smooth muscle mediators from mast cells. IL-5 can promote eosinophil development, activation, and recruitment to the airway of the lung. IL-13 is involved in airway remodeling and airway hyper-responsiveness (AHR) to promote asthma disease. T helper 1 subset, however, often suppresses Th2-mediated response and ample evidence indicates that the Th1/Th2 balance are critical for asthma development. The Th1 can mediate suppression of Th2 responses via the production of interferon-γ (IFN-γ) and IL-12.

B7-DC (also called PD-L2, CD273) has been reported as a ligand for programmed death one (PD-1) , but its function in the regulation of T cell responses remains elusive. While several studies support a role for B7-DC in the suppression of T cell responses via PD-1 engagement on T cells, others have shown that B7-DC may co-stimulate T cell responses and enhance immune response. Using site-directed mutagenesis, a B7-DC variant (K113S, a replacement of lysine at positive 113 with serine) lost their binding to PD-1, but retained its function in the stimulation of T cell responses. Based on these findings, the existence of a new costimulatory receptor for B7-DC was proposed.

Recently, B7-DC was found to bind repulsive guidance molecule family member b (RGMb, also called DRAGON) . RGMb, a GPI-linked membrane-associated protein, contains an N-terminal 50 amino acid signal peptide, C-terminal 35 amino acid GPI attachment signal, RGM N-terminus domain, RGM C-terminus domain and a von Willebrand factor type-D domain in the middle of the molecule. RGMb mRNA is widespread in the spinal cord, brain (midbrain, hindbrain and forebrain) , liver, kidney, optic nerve and reproductive tract. Studies show that RGMb is a coreceptor of BMP2/4 (bone

morphogenetic protein

2 and 4, BMP2/4) to enhance Smad phosphorylation responses. RGMb mRNA is also found in epithelial cells, macrophages and dendritic cells. Normal lung interstitial macrophages and alveolar epithelial cells were shown to express high levels of RGMb mRNA. In a recent study, blockade of the RGMb/PD-L2 interaction by a monoclonal antibody (mAb) impaired the development of respiratory tolerance. The role of RGMb on T cell response to antigens, however, remains unknown. RGMb knockout (KO) mice die in about two weeks after birth, largely due to its requirement in neural development. This prevents the use of this strain for further dissection of B7-DC/RGMb function.

SUMMARY

In the instant invention, we identified K113S, a B7-DC variant with selective binding capacity to RGMb but not PD-1. We show that K113S triggers RGMb to costimulate CD4+ T cell growth with bias to the Th1 response, as evidenced by increased IFN-γ production in the Th1 differentiation condition； and

T cells constitutively express RGMb on their cell surface. Finally, K113S treatment of mice in an experimental asthma model enhanced IFN-γ production in BALF, accompanied by suppressed IL-5 and IL-13 production. Ultimately, our results support a role for RGMb as a costimulatory molecule toward CD4+ Th1 responses.

In one aspect of the invention, a method for prophylaxis or treatment of asthma in a subject is provided. The method comprises administering to the subject in need thereof a therapeutically effective amount of a B7-DC mutant with an amino acid sequence as shown in SEQ ID NO. 1 or 2, or an amino acid sequence having at least 80％identity to SEQ ID NO. 1 or 2 and a K113S mutation.

In another aspect of the invention, provided is use of the B7-DC mutant in the preparation of a pharmaceutical composition for prophylaxis or treatment of asthma in a subject.

In some embodiments of the invention, the asthma is a Th2-mediated asthma. In some embodiments of the invention, the administration is carried out intravenously. In some embodiments of the invention, the B7-DC mutant is contained in a vector, preferably a plasmid. In some embodiments of the invention, the B7-DC mutant is fused with a human IgG Fc fragment, e.g., a human IgG1 Fc fragment.

The present invention demonstrated that recombinant K113S protein interacts with RGMb in a similar affinity as wild type B7-DC. More importantly, K113S costimulates CD4+ T cell responses via RGMb and promotes Th1 polarization. RGMb was found to express on the surface of

mouse T cells, macrophages, neutrophils and dendritic cells. Finally, K113S/RGMb costimulation suppresses Th2-mediated asthma and ameliorates small airway inflammation and lung pathology in an experimental mouse model.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1. Binding of B7-DC variants to PD-1 or RGMb. (A) CHO cell line transfected to overexpress mouse PD-1 (PD-1+ CHO) were stained with indicated concentrations of control Ig (Flag-hIg) , B7-DC-hIg or its variants and subsequently analyzed by flow cytometry. (B) 293 cell line transfected to overexpress mouse RGMb (RGMb+ 293T) were stained with indicated concentrations of control Ig (Flag-hIg) , B7-DC-hIg or its variant and subsequently analyzed by flow cytometry. The second antibody used for staining was mouse anti-human IgG Fc-_{Alexa Fluor 647}. All data are representatives of three or more independent experiments.

Figure 2. The binding affinity of B7-DC and its variants to RGMb. (A/B) Biacore analysis of the surface plasmon resonance of B7-DC-hIg (A) or K113S-hIg (B) interactions with mouse RGMb which was coated on the CM5 biosensor chip at various concentrations (3.906-500nM) . (C/D) Biacore analysis of the surface plasmon resonance of R56S-hIg (C) or E71S-hIg (D) interactions with RGMb which was coated on the chip at various concentrations (0.1094-7 μM) . (E) The response units of surface plasmon resonance analysis of RGMb interactions with I105A-hIg, D111S-hIg, R101S-hIg and control Flag-hIg at 10 μM. (F) 293T cell line transfected to overexpress mouse RGMb was stained at the indicated concentrations of anti-mRGMb-biotin (second antibody SA-APC) and 1 μg B7-DC-hIg or K113S-hIg (second antibody goat anti-human Fc-PE) and subsequently analyzed by flow cytometry. All data are representatives of three or more independent experiments.

Figure 3. Expression of RGMb and B7-DC on mouse myeloid and lymphoid cells. (A) RGMb cell surface expression was examined by specific polyclonal antibody (

Minneapolis, MN) by flow cytometry analysis on alveolar macrophage (AM) , peritoneal macrophage (pMφ) , bone marrow-derived dendritic cells (BMDC, immature DC, blue line

mature DC, red line) , macrophage cell line RAW264.7 and freshly isolated spleen neutrophils (PMN) , T, B and NK cells. (B) B7-DC cell surface expression was tested by flow cytometry on alveolar macrophages (AM) , peritoneal macrophages (pMφ) , bone marrow-derived dendritic cells (BMDC in both immature and mature DC, blue and red line respectively) and macrophage cell line RAW264.7. All data are representatives of three or more experiments.

Figure 4. Effect of K113S variant in OVA-induced mouse model of asthma. (A) Sensitization and challenge protocol of OVA-induced asthma and treatment. BALB/c mice were inoculated intraperitoneally (i.p. ) with 10 μg grade V OVA and 1 mg alum gel in PBS on

day

0 and 5. Mice were subsequently challenged by inhalation of 1％OVA in PBS for 30 min on day 11-13. Plasmids (Flag-hIg and K113S-hIg) were injected intravenously (i.v. ) via tail veins at 20ug/mouse in 2ml PBS within 5-10 seconds one day before the first OVA immunization. (B) Total cell and eosinophil number of BALF from mice after asthma induction with Flag-hIg and K113S-hIg treatment. (C) Cells in BALF from Flag-hIg or K113S-hIg treated mice were stained with Diff-Quick dye liquor. (D) Histology of lung from mice treated with Flag-hIg or K113S-hIg. Mice were sacrificed at day 14 and lung tissues were processed and stained with

to show cell infiltration around the small airway. (E) Airway hyper-responsiveness to methacholine challenge. Mice were treated with Flag-hIg and K113S-hIg and were measured for airway resistance and Penh of lung. Data are shown as the mean ± SEM and represent three independent experiments, 5 mice in each group. NS, not significant difference； ^＊: p<0.05； ^＊＊: p<0.01； ^＊＊＊: p<0.001.

Figure 5. Effect of K113S on CD4+ T cell costimulation and Th1/Th2 cytokine production. (A/B) Purified CD4⁺ T cells from

BALB/c mice were labeled with CFSE and stimulated under Th1 differentiation conditions (IL-2 10ng/ml, IL-12 10ng/ml, anti-IL-4 10 μg/ml) in the absence (A) or presence (B) of pMφ on pre-coated plates with anti-CD3 at 2.5 μg/ml and soluble anti-CD28 at 1 μg/ml for 3 days. CFSE dilution and IFN-γ intracellular staining were evaluated by flow cytometry. (C) Intracellular IL-2, IFN-γ and IL-4 were assayed by specific mAb by flow cytometry in CD4 cells from BALF upon treatment by Flag-hIg or K113S-hIg during OVA-induced asthma. On day 14 after asthma induction, cells from BALF were stimulated with PMA/ionomycin/brefeldin A for 4-6h and harvested for flow cytometry analysis. (D/E) Intracellular IL-5 (D) and IL-13 (E) were assayed by specific mAb with flow cytometry in CD4 cells from BALF upon treatment by Flag-hIg or K113S-hIg during OVA-induced asthma. Data were representative of at least three independent experiments, 5 mice in each group. NS, not significant difference； ^＊: p<0.05.

Figure 6. K113S-hIg protein levels in BALF and sera upon hydrodynamic injection. (A) Flag-hIg and K113S-hIg levels in BALF at day 14 after the immunization. (B) Flag-hIg and K113S-hIg levels in sera at the indicated days after hydrodynamic injection. The data were shown as mean ± SEM and samples were obtained from 5 individual mice from each group. Data were representatives of at least three independent experiments.

Figure 7. The levels of IL-4 in BALF and OVA-specific IgE in sera upon K113S treatment. (A) IL-4 levels were tested in BALF by specific ELISA and (B) OVA specific IgE was analyzed in sera from the asthma model which were treated with Flag-hIg/K113S or hIg. Mean ± SEM and represent three individual experiments. NS, not significant. Data were representative from three experiments, with 5 mice in each group.

DETAILED DESCRIPTION

Definitions

As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total) , whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal, ” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on. The subject herein is preferably a human.

As used herein, phrases such as “to a patient in need of treatment” or “asubject in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of an polypeptide or composition of the present disclosure used, e.g., for detection, for a diagnostic procedure and/or for treatment.

Met hods and Therapies

An aspect of the disclosure provides a method for prophylaxis or treatment of asthma in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a B7-DC mutant having an amino acid sequence as shown in SEQ ID NO. 1 or 2 or an amino acid sequence having at least 80％sequence identity to SEQ ID NO. 1 or 2 and a K113S mutation, or a pharmaceutical composition comprising the B7-DC mutant. Equally, the disclosure provides the B7-DC mutant as described above for use in a method for treating or alleviating symptoms involved with asthma.

In certain embodiments, the B7-DC mutant or the pharmaceutical composition is administered parenterally, e.g. intravenously, Jintramuscularly， percutaneously or intracutaneously.

In some embodiments, it may be desirable to combine a B7-DC mutant with other agents effective in the treatment of asthma. For example, the treatment of asthma may be implemented with a B7-DC mutant and other anti-asthma therapies, such as β2 receptor agonists available in the market.

In certain embodiments, the methods of treating asthma prevent the progression of the asthma and/or the onset of disease caused by asthma. Thus, in some embodiments, a method for preventing the progression of asthma and/or the onset of disease caused by asthma, comprises administering an effective amount of a B7-DC mutant to a subject in need thereof. In certain embodiments, the methods of treating asthma prevent the onset, progression and/or recurrence of a symptom associated with asthma. Thus, in some embodiments, a method for preventing a symptom associated with asthma in a subject, comprises administering an effective amount of a B7-DC mutant to a subject in need thereof.

Compositions

An aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of B7-DC mutant and a pharmaceutically acceptable carrier. The pharmaceutical composition is useful for prophylaxis or treatment of asthma in a subject. The B7-DC mutant may be prepared in a suitable pharmaceutically acceptable carrier or excipient.

As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions； solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.

Examples

Materials and Methods

Mice. Female BALB/c mice were purchased from and housed in a specific pathogen-free facility in the Experimental Animal Center of Sun Yat-Sen University. All mouse experiments were carried out in accordance with the Sun Yat-Sen University Laboratory Animal Center guidelines and were approved by the Institutional Animal Care and Use Committee.

Cell lines. Chinese hamster ovary (CHO) line expressing murine PD-1 was cultured in Ham’s F12 medium (GIBCO) with 1％FBS. The 293T cell line (ATCC) was cultured in DMEM (Cellgro) with 10％FBS and was transfected with lipofectamine 3000 and mouse RGMb cDNA in a peGFP-N3 expression vector. The cell surface expression of RGMb was verified by staining with RGMb antibody (

Minneapolis, MN) and eGFP fluorescence.

Recombinant fusion proteins, antibodies and cytokines. The plasmids encoding fusion protein of mouse B7-DC-human IgG1 Fc (B7-DC-hIg) and its variants were described previously (Wang, S., et al. Molecular modeling and functional mapping of B7-H1 and B7-DC uncouple costimulatory function from PD-1 interaction. J Exp Med 197, 1083-1091 (2003) ) . Recombinant proteins were produced by 293T cell transfection and purified by protein A affinity column (GE) . Anti-hIg was purchased from Jackson ImmunoResearch (West Grove, PA) , His-tagged recombinant mouse RGMb and anti-murine RGMb-biotin were purchased from

Streptavidin-PE was purchased from BioLegend (San Diego, CA) . Anti-CD3e-PE, anti-CD4-PE, anti-CD8-PE, anti-CD11c-PE, anti-Gr1-PE, anti-B220-PE, anti-CD49b-PE, anti-F4/80-PE, anti-mB7-DC-APC, anti-IL-2-FITC, anti-IFN-γ-PE, anti-IL-4-APC monoclonal antibodies (mAb) and mouse IL-4, IL-5, IL-13 ELISA kits were purchased from eBioscience (San Diego, CA) . Fisher HealthCare^TM PROTOCOL^TM Hema 3^TM Fixative and Solutions, Inject Alum and Carboxyfluorescein succinimidyl ester (CFSE) staining dye were purchased from Thermo Scientific (Waltham, MA) . Albumin from chicken egg Grade V (OVA) and methacholine were purchased from Sigma-Aldrich (St. Louis, MO) . Anti-IgE-biotin, Cytofix-CytoPerm with Golgiplug kit and anti-CD4-BV421 were purchased from BD (Franklin Lakes, NJ) .

Surface plasmon resonance (SPR) analysis. The affinity of murine RGMb with B7-DC and its variants were analyzed on Biacore T100 instrument as described previously (Wang, S., et al. Molecular modeling and functional mapping of B7-H1 and B7-DC uncouple costimulatory function from PD-1 interaction. J Exp Med 197, 1083-1091 (2003) ) . In brief, his-tagged RGMb was immobilized on a CM5 senor chip (GE healthcare) at 2000 RUs and the control flow cell was similarly prepared. Purified Flag-hIg, mB7-DC-hIg and variant fusion proteins were diluted in HEPES buffer at the indicated concentrations (mB7-DC-hIg and K113S-hIg from 3.9 nM to 500 nM, R56S-hIg and E71S-hIg from 109.4 nM to 7 μm, I105A-hIg, D111S-hIg, R101S-hIg and Flag-hIg at 10 μM) . The proteins were injected at a flow rate of 20 μl/min for 3min, and buffer was passed over the surface for 5 min for dissociation. Data were analyzed using the Biacore T100 software.

Experimental asthma mouse model. The OVA-induced asthma in mouse model was described previously. Briefly, 6-8 weeks old BALB/c mice were immunized by i. p. injection of 10 μg OVA protein (Sigma-Aldrich) mixed to 4mg aluminum hydroxide Gel (Inject Alum, Thermo) on day 0 and day 5. Mice were challenged nasally with 1％OVA in PBS using a nebulizer (Yuwell 402AI, Jiangsu, China) for 30 min on days 11 to 13. Mice were anesthetized on day 14. Blood was collected from the inferior vena cava and sera were prepared for OVA-specific IgE detection. Lungs were lavaged with 0.5ml of warm PBS via a tracheal cannula three times with a total 1 ml PBS. The bronchoalveolar lavage fluids (BALFs) were centrifuged and the supernatants collected for cytokine assays (below) . A coulter counter (Beckman Coulter, San Diego, CA) was used to count total number of cells. The lungs were also fixed with 10％Formalin and tissue sections were stained by H/E for histological evaluation. For hydrodynamic injection of plasmids in vivo, control Flag-hIg or K113S-hIg plasmid at 20 μg in 2ml volume of PBS were injected within 5-10 seconds via tail vein, as described previously. Human IgG protein levels in sera and BALF upon the hydrodynamic injections were tested by specific sandwich ELISA at indicated time points (Fig. 6) .

Airway hyper-reactivity (AHR) assays. AHR to methacholine was evaluated 24h after the last 1％OVA challenge by both invasive and non-invasive assays. The invasive assay was determined by FinePointe Resistance and Compliance System (Buxco Electronics, NY) . Briefly, mice were anesthetized, and tracheotomy was performed. PBS, followed by methacholine (12.5, 25mg/ml) , was nebulized and flowed into the trachea of mice for 30 seconds. Mice respiratory flow and lung pressure was directly measured. Airway resistance (R_L) was defined as the pressure driving respiration divided by flow, which was computed using Buxco FinePointe Software. The non-invasive assay was determined using FinePointe for Whole Body Plethysmography System (Buxco Electronics, NY, which relies on the 4 designed chambers to connect a sensitive pressure transducer to measure pressure changes and flow inside the chamber and transmit information to the Buxco FinePointe Software. The software calculates several flow-derived parameters, including respiratory rate, lung volume, peak flow and time intervals. It reported data as “enhanced pause” (Penh) . Penh＝ (PEP/PIP) x ( (Te-Tr) /Tr) . Te, expiratory time (s) ； Tr, relaxation time (s) ； PEP, peak expiratory pressure (ml/s) ； PIP, peak inspiratory pressure (ml/s) .

Mice were placed individually into each chamber and allowed to acclimate for several minutes to recording baseline periods of the chamber. After baseline measurements, increasing concentrations of methacholine in PBS (0, 3.125, 6.25, 12.5, 25, 50 mg/ml) were nebulized for 1min in the main chamber through the inlet. Then the airway responses were recorded for 5 min. The Penh was computed for each group for the increasing concentration of methacholine.

Th1 polarization assay in vitro. For Th1 generation, CD4⁺ T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE, Thermo Fisher Scientific, MA) at 2 μM and stimulated with anti-CD3 antibody (clone 145-2C11) at 2.5 μg/ml and the indicated fusion protein at 5 μg/ml coated on the plate in the presence of 10 μg/ml anti-IL-4 and 10ng/ml IL-12 for 3 days. In the last 6h, 10ng/ml phorbol 12-myristate 13-acetate (PMA) , 1 μg/ml ionomycin and 10 μg/ml brefeldin A were added to the culture and cells were stained with anti-CD4mAb. Upon fixation and permeabolization using the Cytofix/Cytoperm Kit (BD Bioscience, NJ) , cells were stained by mAb to IFN-γ and subjected to flow cytometry analysis. Similar methods were also used to detect IFN-γ, IL-2 and IL-4 positive cells in BALFs. Cytokines in BALF were also measured using an ELISA kit (eBioscience, CA) following the protocol.

Statistical analysis. Each experiment presented in this report was repeated at least three times and the numbers of animals were stated in the figure legends. The data are presented as the mean ±SEM, followed by the significant difference determined using a two-tailed student's t-test or analysis of variance (ANOVA) test. ^＊: P < 0.05, ^＊＊: P < 0.01 and ^＊＊＊: P < 0.001.

Results

Identification of a B7-DC K113S variant that selectively binds RGMb but not PD-1

We showed previously the generation of several B7-DC variants with mutations in the binding domain of B7-DC and PD-1. A variant K113S, in which the lysine in amino acid position 113 was replaced by serine, completely lost binding capacity to PD-1. Interestingly, K113S remained costimulatory for lymphocytes in vitro. This finding suggests that B7-DC may function via a receptor other than PD-1 for its costimulatory function. To determine if K113S interacts with RGMb, we prepared recombinant fusion proteins encoding a set of variants (R56S-hIg, E71S-hIg, R101S-hIg, I105A-hIg, D111S-hIg, K113S-hIg) and tested their binding capacity to RGMb. Using flow cytometry, we showed that I105A, D111S, K113S variants did not bind PD-1+ CHO cells at all tested concentrations, while R56S, E71S, and R101S variants, and wild type B7-DC bound PD-1 significantly (Fig. 1A) , a result similar to our previous finding. Interestingly, K113S-hIg bound RGMb+ 293T cells, similar to wild type B7-DC-hIg (Fig. 1 B) while R101S, I105A and D111S completely lost binding to RGMb. R56S and E71S appear to have weak binding to RGMb at high protein concentrations. The above data confirm that B7-DC interacts with both RGMb and PD-1； while, as a new finding, the K113S selectively binds to RGMb, but not to PD-1.

K113S binds RGMb at a similar affinity to wild type B7-DC

We determined the binding affinity for K113S using SPR analysis with the Biacore instrument and the affinity was calculated using Biacore T100 software. K113S bound RGMb with an affinity similar to B7-DC at nM levels (B7-DC KD ＝ 1.82E-07 M, K113S KD ＝ 1.28E-07) (Table 1, Figure 2A, B) ； while R56S, E71S, I105A and D111S bound RGMb at μM levels (Fig. 2C, D and E) and R101S at a similar level as the Flag-hIg control (Fig. 2E) .

Table 1. Affinity measurement of B7-DC variants

The affinity (KD) was calculated based on Biacore T100 software from “ka” (association) and “kd” (dissociation) .

To further validate the interaction between K113S and RGMb, we tested capacity of RMGb antibody to compete the binding with K113S and B7-DC for RGMb. RGMb antibody at various concentrations up to 625 ng/ml were included in the culture to compete for B7-DC or K113S fusion protein at 1 μg/ml to bind RGMb+ 293T cells using flow cytometry. Consistent with the Biocore analysis, the RGMb antibody was slightly less efficient to compete K113S-hIg than B7-DC-hIg to bind RGMb+ 293T cells in the most of tested concentrations (Fig 2F) . Therefore, lysine at amino acid 113, while is critically involved in PD-1 binding, plays minimal role in the interaction with RGMb. Our study thus identified a highly selective RGMb ligand without PD-1 interference.

Constitutive expression of RGMb on murine lymphocytes and myeloid cells

Using RGMb antibody, we analyzed RGMb protein expression on various immune cells. RGMb appears to constitutively express at high levels on myeloid cells, including freshly isolated alveolar macrophages (AMs) , peritoneal macrophages (pMφs) , bone marrow dendritic cells (BMDCs) and neutrophils (PMNs) and a macrophage-like cell line RAW264.7 with the highest expression on pMφ(Fig. 3A) . Interestingly, RGMb protein could also be detected in low levels on the surface of resting CD3⁺, CD4⁺, CD8⁺ T cells and B cells of splenocytes, while NK cells are negative (Fig. 3A) . The expression of B7-DC, however, was limited to bone marrow-derived dendritic cells (BMDCs) while its expression levels on AMs and RAW264.7 were minimal (Fig. 3B) , a result similar to that described previously.

Engagement of RGMb suppressed lung inflammation and airway hypersensitivity

Taking advantage of RGMb-specific binding by K113S, we examined the function of RGMb in an OVA-induced asthma model in which mice were first immunized and challenged later with OVA (Fig. 4A) . In this model, mice developed Th2-like inflammatory responses with heavy infiltration of eosinophils. One day before immunization, mice were injected i.v. with K113S-hIg plasmid under high pressure (hydrodynamic injection) to force plasmid expression, mainly in the liver. As expected, total infiltrating cell numbers in the BALF was dramatically increased upon challenge with OVA. In contrast, mice treated with K113S-hIg had significantly decreased total cell numbers (Fig. 4B) . The number of eosinophils, the primary cell type during OVA-induced asthma, was also significantly suppressed by K113S-hIg (Fig. 4B and 4C) . Histological analysis of lung showed K113S-hIg treated mice had significantly less inflammatory infiltration around small airways compared with those treated with control plasmid (Fig. 4D) .

Airway hypersensitivity (AHR) was accessed by resistance of lung (R_L) and Penh, an index of airway hypersensitivity, in this model, as described in Materials and Methods. Baseline R_L had no significant differences among mice treated with the control or K113S plasmids. However, The R_L of 12.5 mg/ml methacholine challenge was decreased in K113S-hIg treated mice vs. Flag-hIg treated mice (Fig. 4E) . Furthermore, the Penh was significantly lower in mice treated with K113S-hIg than Flag-hIg (Fig 4E) . These results support that triggering of RGMb by K113S-hIg suppresses OVA-induced asthma inflammation and improves lung function in this experimental asthma model.

K113S costimulates Th1 response via RGMb

We hypothesized that K113S in suppressing asthma inflammation is caused by costimulation of Th1 T cell response which subsequently suppresses Th2-like inflammation. To test this, we first tested the ability of K113S to stimulate Th1 response during

CD4⁺ T cell differentiation to Th1 in vitro. In this system, purified

T cells were cultured in Th1 polarization conditions (anti-CD3 + IL-12/anti-IL-4) for 3 days； Th1 CD4+ T cells were identified as IFN-γ producing cells by intracellular staining. In this culture system, pMφ were also added to closely mimic lung environment in vivo. In the presence of K113S-hIg, IFN-γ⁺ T cells increased significantly in comparison with that of control Ig (Fig. 5A-5B) . To confirm this finding, cytokines in BALF from mice with OVA-induced asthma were measured using specific ELISA. K113S treatment significantly increased BALF IFN-γ levels while the changes in IL-2 and IL-4 were negligible (Fig. 5C) . In an OVA-induced asthma model, IL-5 and IL-13 play an essential role in initiating and maintaining inflammatory responses in airways. Therefore, we also measured these cytokines in BALF in this model. Mice treated with K113S-hIg plasmid had significantly decreased IL-5 and IL13, while IL-4 production was not significant (Fig. 5D-E, Fig. 7) . Allergen-specific IgE may also play a role in asthma pathogenesis. In our model, however, OVA specific IgE was no different between the mice treated with the K113S vs. the control plasmids (Fig. 7) . Our results indicate that RGMb engagement by K113S selectively stimulate IFN-γ producing Th1 T cells accompanied with reduced IL-5 and IL-13 to suppress symptoms of asthma in this model.

The role of B7-DC in T cell responses remains controversial because both coinhibitory and costimulatory functions were reported in various experimental systems in vitro and in vivo. In addition to interacting with the coinhibitory receptor PD-1, B7-DC was also shown to bind repulsive guidance molecule b (RGMb) . Our findings provide an explanation for contradictory data on B7-DC studies. During the priming phase, B7-DC on dendritic cells may costimulate

CD4+ T cells via RGMb, favoring Th1 T cell proliferation and differentiation. Upon costimulation and activation, T cells are induced to express PD-1 which, upon engagement with B7-DC, transmits a suppressive signal to T cells. In the context of constitutive expression of B7-DC by dendritic cells, the costimulatory function of B7-DC may be quickly attenuated by its PD-1 engagement, which may constitute a safeguard to prevent further expansion and over-activation of T cells. Therefore, dependent on the timing and levels of RGMb and PD-1 receptors, B7-DC may display different, and sometime opposite effects on T cell responses. While it is yet to be tested if human RGMb has similar expression pattern, and more importantly, could also be costimulated upon engagement by B7-DC, our study provides a rationale to explore this possibility.

While the mechanism of RGMb-mediated costimulation has yet to be fully elucidated, our findings in the OVA-induced asthma model indicate that K113S could attenuate the asthma response and improve lung function by reducing respiratory inflammation and resistance. Both total inflammatory cell number and eosinophils in the BALF were alleviated after K113S treatment. Furthermore, pathological inflammation around small airways in the lung was also abated by this treatment. While it is possible that triggering RGMb directly suppresses the Th2 response, including IL-5 and IL-13 production, eosinophil infiltration, and airway remodeling during asthma progression； increased IFN-γ was detected in BALF. These findings supports the costimulation of the Th1 response. The role of the Th1 response in the suppression of Th2-mediated asthma is well documented. Therefore, it is highly likely that costimulation via RGMb deviates the balance of Th1/Th2, leading to suppression of the Th2 response. It is worthwhile to mention that the effect of RGMb is somewhat unique and may not be considered as a typical “Th1-like” response. In our study, although IFN-γ production increased significantly, there was minimal change in the levels of IL-2, another important Th1 cytokine. On the other hand, the inhibitory effect of RGMb on the “Th2-like” response is somewhat partial, as was indicated, mainly by the suppression of IL-5 and IL-13 but not IL-4. In addition, the effect of RGMb on the suppression of OVA-specific IgE is also marginal (Fig. 5) . Therefore, the role of RGMb on Th1 polarization may be unique with only a partial effect.

Currently, specific agonists for RGMb (either antibody or small molecule) are not yet available, and the RGMb KO is lethal in mice, which limits further exploration of RGMb function. Selective binding of K113S to RGMb but not PD-1 represents a new opportunity to conduct such studies. Development of such an agonist is also useful for potential therapeutic manipulation of human disease, including asthma.

It should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the disclosures embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure.

The disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the disclosure with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control. The disclosures illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising, ” “including, ” containing, ” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed.

Claims

A method for prophylaxis or treatment of asthma in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a B7-DC mutant with an amino acid sequence as shown in SEQ ID NO. 1 or 2, or an amino acid sequence having at least 80％identity to SEQ ID NO. 1 or 2 and a K113S mutation.
The method of claim 1, wherein the asthma is a Th2-mediated asthma.
The method of claim 1, wherein the administration is carried out intravenously.
The method of claim 1, wherein the B7-DC mutant is contained in a vector.
The method of claim 4, wherein the vector is a plasmid.
The method of claim 1, wherein the B7-DC mutant is fused with a human IgG Fc fragment.
The method of claim 6, wherein the human IgG Fc fragment is a human IgG1 Fc fragment.
The method of claim 1, wherein the subject is a human being.
Use of a B7-DC mutant in the preparation of a pharmaceutical composition for prophylaxis or treatment of asthma in a subject, wherein the B7-DC mutant has an amino acid sequence as shown in SEQ ID NO. 1 or 2, or an amino acid sequence having at least 80％identity to SEQ ID NO. 1 or 2 and a K113S mutation.
[Rectified under Rule 91, 03.08.2017]
The use of claim 9, wherein the asthma is a Th2-mediated asthma.
[Rectified under Rule 91, 03.08.2017]
The use of claim 9, wherein the B7-DC mutant is contained in a vector.
[Rectified under Rule 91, 03.08.2017]
The use of claim 11, wherein the vector is a plasmid.
[Rectified under Rule 91, 03.08.2017]
The use of claim 9, wherein the B7-DC mutant is fused with a human IgG Fc fragment.
[Rectified under Rule 91, 03.08.2017]
The use of claim 13, wherein the human IgG Fc fragment is a human IgG1 Fc fragment.
[Rectified under Rule 91, 03.08.2017]
The use of claim 9, wherein the subject is a human being.