USE OF FOLIC ACID AND FOLATE MODIFICATION IN INDUCING B-CELL IMMUNE TOLERANCE AND TARGETING mIgM-POSITIVELY-EXPRESSED B-CELL LYMPHOMA

Abstract

Generally, the present invention relates to the use of folic acid and folate modification in inducing B-cell immune tolerance and targeting mIgM-positively-expressed B-cell lymphoma. The present invention also relates to a method, a pharmaceutical composition and a combination for inducing B-cell immune tolerance and for targeting mIgM-positively-expressed B-cell lymphoma.

Claims

1. A method for inducing B-cell immune tolerance, especially for reducing the production of the antibodies against an immunogenic substance, and/or for the treatment or prevention of a disease or disorder that may be mediated by B-cell immune tolerance, comprising administrating an effective amount of folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof to a subject.

2. The method according to claim 1, wherein the folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof is selected from the group consisting of free folic acid, a pharmaceutically acceptable salt of folic acid, a pharmaceutically acceptable ester of folic acid, and a pharmaceutically acceptable conjugate of folic acid (e.g., folate-albumin conjugate, folate-polyethylene glycol conjugate, etc.), or any combination thereof.

3. The method according to claim 1, wherein the B-cell is the one expressing mIgM, especially the one highly expressing mIgM, more especially spleen B-cell or lymph node B-cell.

4. The method according to claim 1, wherein the immunogenic substance is a biomacromolecular active agent, and the medicament is administered in combination with the biomacromolecular active agent, e.g., before and/or during the administration of the biomacromolecular active agent, to reduce the production of Anti-Drug Antibodies against the biomacromolecule active agent; preferably, the biomacromolecule active agent is selected from the group consisting of: polypeptide drugs, e.g., p53 activating peptide, melittin, scorpion venom peptide, antibacterial peptide, or insulin; or protein drugs, e.g., antibody drugs and especially monoclonal or polyclonal antibody drugs, interferons, growth factors, growth factor inhibitors, enzymes, or albumins, e.g., human serum albumin or ovalbumin, including murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, or fully human monoclonal antibodies, e.g., Tumor Necrosis Factor α (TNFα) mAbs (e.g., adalimumab, etanercept or infliximab), PD1/PD-L1 mAbs (e.g., nivolumab, pembrolizumab, atezolizumab, sintilimab, toripalimab, or camrelizumab); HER2 mAbs (e.g., trastuzumab, pertuzumab, or lapatinib); CD20 mAbs (e.g., rituximab, ibritumomab tiuxetan, or tositumomab); Vascular Endothelial Growth FactorNascular Endothelial Growth Factor Receptor (VEGFNEGFR) mAbs (e.g., bevacizumab, ranibizumab, aflibercept, or ramucirumab); and Epidermal Growth Factor Receptor (EGFR) mAbs (e.g., cetuximab, panitumumab, or necitumumab); more preferably, the biomacromolecule active agent is selected from the group consisting of adalimumab, infliximab, atezolizumab, sintilimab, toripalimab, trastuzumab, albumins and insulin.

5. The method according to claim 1, wherein the immunogenic substance is an immunogenic drug delivery system, e.g., a microcarrier drug delivery system, preferably selected from the group consisting of liposomes, microspheres, microcapsules, nanoparticles, nanocapsules, lipid nanodiscs or polymer micelles, and wherein the medicament is administered in combination with the drug delivery system, e.g., before and/or during the administration of the drug delivery system, to reduce the production of antibodies against the immunogenic drug delivery system; preferably, the drug delivery system is loaded with the biomacromolecule active agent as defined in claim 4.

6. The method according to claim 1, for the treatment or prevention of a disease or disorder that may be mediated by B-cell immune tolerance, e.g., hypersensitivity, autoimmune disease or transplant rejection; preferably, the hypersensitivity is selected from the group consisting of anaphylactic shock, respiratory hypersensitivity (e.g., allergic asthma or allergic rhinitis) and gastrointestinal tract hypersensitivity; the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, Hashimoto's thyroiditis, toxic diffuse goiter, ankylosing spondylitis, autoimmune encephalomyelitis, neuromyelitis optica spectrum disorders, anticardiolipin syndrome, hemophilia and psoriasis; and the transplant rejection is selected from the group consisting of organ transplant rejection, tissue transplant rejection (e.g., bone marrow transplant rejection) and recurrent miscarriage.

7. A method of for inducing B-cell immune tolerance, especially for reducing the production of the antibodies against an immunogenic substance, and/or for the treatment or prevention of a disease or disorder that may be mediated by B-cell immune tolerance, comprising administrating an effective amount of a folate-modified immunogenic substance to a subject.

8. The method according to claim 7, wherein the B-cell is the one expressing mIgM, especially the one highly expressing mIgM, more especially spleen B-cell or lymph node B-cell.

9. The method according to claim 7, wherein the immunogenic substance is a biomacromolecule active agent; preferably, the biomacromolecule active agent is selected from the group consisting of: polypeptide drugs, e.g., p53 activating peptide, melittin, scorpion venom peptide, antibacterial peptide, or insulin; or protein drugs, e.g., antibody drugs and especially monoclonal or polyclonal antibody drugs, interferons, growth factors, growth factor inhibitors, enzymes, or albumins, e.g., human serum albumin or ovalbumin, including murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, or fully human monoclonal antibodies, e.g., Tumor Necrosis Factor α (TNFα) mAbs (e.g., adalimumab, etanercept or infliximab), PD1/PD-L1 mAbs (e.g., nivolumab, pembrolizumab, atezolizumab, sintilimab, toripalimab, or camrelizumab); HER2 mAbs (e.g., trastuzumab, pertuzumab, or lapatinib); CD20 mAbs (e.g., rituximab, ibritumomab tiuxetan, or tositumomab); Vascular Endothelial Growth FactorNascular Endothelial Growth Factor Receptor (VEGFNEGFR) mAbs (e.g., bevacizumab, ranibizumab, aflibercept, or ramucirumab); and Epidermal Growth Factor Receptor (EGFR) mAbs (e.g., cetuximab, panitumumab, or necitumumab); more preferably, the biomacromolecule active agent is selected from the group consisting of adalimumab, infliximab, atezolizumab, sintilimab, toripalimab, trastuzumab, albumins and insulin.

10. The method according to claim 7, wherein the immunogenic substance is a pathogenic antigenic substance causing an abnormal immune response in the body, preferably a pathogenic antigenic substance causing hypersensitivity, autoimmune disease or transplant rejection, e.g., ovalbumin, proteolipid protein polypeptide, Dby polypeptide, Uty, myelin basic protein, denatured IgG, thyroglobulin, thyrotropin receptor, histone, acetylcholine receptor, major histocompatibility antigens (including HLA-I and HLA-II), minor histocompatibility antigens, blood group antigens, tissue-specific antigens (e.g., vascular endothelial cell antigens, skin SK antigens).

11. The method according to claim 10, for the treatment or prevention of hypersensitivity, autoimmune disease or transplant rejection; preferably, the hypersensitivity is selected from the group consisting of anaphylactic shock, respiratory hypersensitivity (e.g., allergic asthma or allergic rhinitis) and gastrointestinal tract hypersensitivity; the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, Hashimoto's thyroiditis, toxic diffuse goiter, ankylosing spondylitis, autoimmune encephalomyelitis, neuromyelitis optica spectrum disorders, anticardiolipin syndrome, hemophilia and psoriasis; and the transplant rejection is selected from the group consisting of organ transplant rejection, tissue transplant rejection (e.g., bone marrow transplant rejection) and recurrent miscarriage.

12. A method for the targeted diagnosis, treatment or prevention of mIgM-positively-expressed B-cell lymphoma, comprising administrating an effective amount of folate-modified antitumor agent or folate-modified probe to a subject.

13. The method according to claim 12, wherein the antitumor agent in the folate-modified antitumor agent is an antitumor agent useful for treating or preventing mIgM-positively-expressed B-cell lymphoma, preferably anthracyclines, e.g., adriamycin or epirubicin; taxanes, e.g., paclitaxel, docetaxel or cabazitaxel; camptothecins, e.g., camptothecin, hydroxycamptothecin, 9-nitrocamptothecin or irinotecan; vinblastine drugs, e.g., vincristine or vinorelbine; proteasome inhibitors, e.g., bortezomib or carfilzomib; lactone drugs, e.g., parthenolide; cyclophosphamides, etoposide, gemcitabine, cytarabine, 5-fluorouracil, teniposide, mubritinib, epothilone, actinomycin D, mitoxantrone, mitomycin, bleomycin, cisplatin, oxaliplatin, p53 activating peptide, melittin, scorpion venom peptide, triptolide, bevacizumab or trastuzumab; and wherein the probe in the folate-modified probe is a contrast agent, preferably fluorescent substance, e.g., fluorescein, carboxyfluorescein (FAM), fluorescein isothiocyanate (FITC), hexachlorofluorescein (HEX), coumarin 6, near-infrared dyes Cy5, Cy5.5, Cy7, ICG, IR820, DiR or DiD; or radioactive substance, e.g., magnetic resonance contrast agent, e.g., Gd-DTPA or radiocontrast agent, e.g., 99mTc-DTPA.

14. (canceled)

15. (canceled)

16. The method according to claim 7, wherein the folate-modified immunogenic substance is folate-modified human serum albumin, folate-modified insulin, folate-modified adalimumab, folate-modified infliximab, folate-modified atezolizumab or folate-modified trastuzumab.

17. The method according to claim 12 wherein the folate-modified antitumor agent is folate-modified cabazitaxel or folate-modified triptolide.

18. A pharmaceutical composition comprising an effective amount of folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof and/or folate-modified immunogenic substance and/or folate-modified antitumor agent or folate-modified probe, and optionally one or more pharmaceutically acceptable excipients.

19. A pharmaceutical composition according to claim 18, comprising combination of folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof with an immunogenic substance, wherein the immunogenic substance is preferably a biomacromolecule active agent or an immunogenic drug delivery system; wherein, more preferably, the biomacromolecule active agent is selected from the group consisting of: polypeptide drugs, e.g., p53 activating peptide, melittin, scorpion venom peptide, antibacterial peptide, or insulin; or protein drugs, e.g., antibody drugs and especially monoclonal or polyclonal antibody drugs, interferons, growth factors, growth factor inhibitors, enzymes, or albumins, e.g., human serum albumin or ovalbumin, including murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, or fully human monoclonal antibodies, e.g., Tumor Necrosis Factor α (TNFα) mAbs (e.g., adalimumab, etanercept, or infliximab), PD1/PD-L1 mAbs (e.g., nivolumab, pembrolizumab, atezolizumab, sintilimab, toripalimab, or camrelizumab); HER2 mAbs (e.g., trastuzumab, pertuzumab, or lapatinib); CD20 mAbs (e.g., rituximab, ibritumomab tiuxetan, or tositumomab); Vascular Endothelial Growth FactorNascular Endothelial Growth Factor Receptor (VEGFNEGFR) mAbs (e.g., bevacizumab, ranibizumab, aflibercept or ramucirumab); and Epidermal Growth Factor Receptor (EGFR) mAbs (e.g., cetuximab, panitumumab or necitumumab); still more preferably, the biomacromolecule active agent is selected from the group consisting of adalimumab, infliximab, atezolizumab, sintilimab, toripalimab, trastuzumab, albumins and insulin; or wherein, more preferably, the immunogenic drug delivery system is microcarrier drug delivery system, e.g., liposome, microsphere, microcapsule, nanoparticle, nanocapsule, lipid nanodisc or polymer micelle.

Description

BRIEF DESCRIPTION OF FIGURES

[0114] FIG. 1 shows the nuclear magnetic resonance spectrum of folate-OVA complex of Example 1.

[0115] FIG. 2 shows a quantitative standard curve for the folic acid molecule.

[0116] FIG. 3 shows the nuclear magnetic resonance spectrum of folate-PEG.sub.2000-Cy5 of Example 3.

[0117] FIG. 4 shows the binding mode of folate with mouse mIgM in Example 4 (a: binding site of folate with mIgM; b: analysis on the binding force of folate to mIgM).

[0118] FIG. 5 shows the results of flow cytometric analysis of the binding of folate to mouse mIgM in Example 4 (a: the effect of anti-IgM antibody to eliminate mIgM on the surface of B-cell; b: the results of the uptake of folate by B-cell under different anti-IgM antibody concentrations: c: Linear fitting curve of B-cell uptake of folate and mIgM expression level).

[0119] FIG. 6 shows confocal micrographs of folate binded to mouse mIgM in Example 4.

[0120] FIG. 7 shows the distribution of FA-PEG.sub.2000-Cy5 or PEG.sub.2000-Cy5 in the spleen 1 hours (a) and 4 hours (b) after injection via tail vein in Example 5. (* p<0.05. **p<0.01, T test) FIG. 8 shows the flow cytometry graphs (a: 1 h; c: 4 h) and statistical analysis (b: 1 h; d: 4 h) of spleen lymphocytes 1 hours and 4 hours after injection of FA-PEG.sub.2000-Cy5 or PEG.sub.2000-Cy5 via tail vein in Example 5. (ns: no statistical difference, ***p<0.001, T test) FIG. 9 shows the immunofluorescence photographs of the distribution of FA-PEG.sub.2000-Cy5 or PEG.sub.2000-Cy5 in the spleens 1 hour after injection via tail vein in Example 5.

[0121] FIG. 10 shows the immunofluorescence photographs of the binding of FA-PEG.sub.2000-Cy5 to IgM-positively-expressed marginal B-cell in the spleen 1 hour after injection via tail vein in Example 5.

[0122] FIG. 11 shows the B-cell proliferation (a, b) and differentiation (c, d) results obtained by flow cytometry after stimulating the mice with FA-OVA. OVA and physiological saline respectively for three times in Example 6. (*p<0.05, ***p<0.001, one-way ANOVA)

[0123] FIG. 12 shows the expression of serum OVA-specific antibodies after stimulating the mice with FA-OVA, OVA and physiological saline respectively for three times in Example 6 (a: IgE; b: IgG; c: IgG1; d: IgG2a).

[0124] FIG. 13 shows the expression of serum FVIII-specific antibodies after stimulating the mice with FVIII, folate-FVIII and physiological saline respectively for three times in Example 6.

[0125] FIG. 14 shows the expression of OVA-specific IgG antibodies in mouse serum after oral administration of folic acid in Example 7 (a: day 14; b: day 21, c: day 28).

[0126] FIG. 15 shows the expression of KLH-specific IgG antibodies in mouse serum after oral administration of folic acid in Example 7 (a: day 21; b: day 28: c: day 35).

[0127] FIG. 16 shows the expression of serum-specific antibodies after stimulating the mice in Example 8 (a: adalimumab as a model mAb; b: infliximab as a model mAb; c: trastuzumab as a model mAb).

[0128] FIG. 17 shows the expression of serum sLip-specific antibodies after stimulating the mice with sLip, sLip+FA (10 μg), sLip+FA (50 μg) and physiological saline in Example 9, respectively.

[0129] FIG. 18 shows the body temperature change curves of the mice which was treated with FA-OVA, OVA and physiological saline respectively and then was challenged with OVA in Example 10.

[0130] FIG. 19 shows the level of the serum OVA-specific antibodies in the mice which was treated with FA-OVA, OVA and physiological saline respectively and then was challenged with OVA in Example 10 (a: IgE; b: IgG.sub.1; c: IgG.sub.2a).

[0131] FIG. 20 shows the body temperature change curves of the sensitized mice which was treated with FA-OVA, OVA and physiological saline respectively and then was challenged with OVA in Example 11 (ns: no statistical difference, **p<0.01, ***p<0.001, one-way ANOVA) FIG. 21 shows the levels of serum OVA-specific antibodies in the sensitized mice which was treated with FA-OVA. OVA and physiological saline respectively and then was challenged with OVA in Example 11 (a: IgE: b: IgG.sub.2a; c: IgG.sub.1) (ns: no statistical difference, ***p<0.001, one-way ANOVA).

EXAMPLES

[0132] The present invention is further illustrated below in combination with the specific examples. It should be understood that these examples are only illustrative and are not intended to limit the scope of the present invention. The experimental materials and reagents are commercially available or are parred according to methods known in the art, unless otherwise specified. Abbreviations used herein have the meanings commonly understood in the art, unless otherwise indicated.

[0133] Particularly, the abbreviations herein have the following meanings:

TABLE-US-00001 abbreviations meanings BSA bovine serum albumin Chol cholesterol DAPI fluorescent dye 4′,6-diamidino-2-phenylindole Dby NAGFNSNRANSSRSS DCC dicyclohexyl carbodiimide DMSO dimethyl sulfoxide DSPE distearoylphosphatidylethanolamine EDC carbodiimide EDTA ethylenediamine tetraacetic acid FA folic acid FVIII coagulation factor VIII h hour(s) HPLC high-performance liquid chromatography HSPC hydrogenated soybean phospholipid mPEG polyethylene glycol monomethyl ether NHS N-hydroxysuccinimide OVA ovalbumin PBS phosphate buffer solution, pH = 7.4 PEG polyethylene glycol PLGA poly(lactic-co-glycolic acid) PLP Proteolipid protein POPC 1-palmitoyl-2-oleoyl lecithin TEA triethanolamine TLC thin-layer chromatography

Example 1: Preparation of Folate-Protein Complex

[0134] Folate-OVA was synthesized as an example of folate-modified protein complexes. Folic acid (4.4 mg) was dispersed in 3 mL PBS (pH=7.4), and carbodiimide (EDC) (15 mg) was added. The mixture was stirred for 3 h in dark to activate folic acid. Ovalbumin (OVA) (4.3 mg) was fully dissolved in 0.2 mL PBS. Then, the activated folic acid solution was added dropwise into the OVA solution. The mixture was stirred for 3 h in dark, and shaken overnight at 4° C. The next day, the mixture was dialyzed against 10 mM PBS for 72 h, changing the dialysate every 12 h. The product was obtained by lyophilization.

[0135] Folic acid itself is insoluble in water. Upon linking with OVA, the folate-OVA complex is easily soluble in water due to the good water solubility of the protein, suggesting that folate was successfully linked to OVA. FIG. 1 provides the .sup.1HNMR of the obtained folate-OVA complex, wherein the signals δ7.45 and δ6.45 are the signal peaks of a group of symmetrical “AA‘BB’” spin systems. Thus, it can be determined that said signals are the two groups of proton signal peaks of the para-disubstituted benzene ring in the folic acid molecule, suggesting that the molecule of the product contains the structure of folate. The structure of the product is further determined to be folate-OVA. The OD.sub.365 nm of the obtained folate complex was measured by an ultraviolet spectrophotometer. FIG. 2 provides the standard curve to quantify the degree of folate modification. As shown in Table 1, the obtained folate-OVA has a degree of folate modification of about 8.66. The degree of folate modification means the ratio of folate in the folate complex to the folate complex.

[0136] Folate-coagulation factor VIII (FA-FVIII) was synthesized as another example of folate-modified protein complex. FVIII stock solution (20 IU/mL, obtained from Shanghai RAAS) was half-diluted with 0.1 M carbonate buffer solution (pH=9.5). The 50-fold equivalents of folic acid and 60-fold equivalents of EDC (both dissolved in 0.1 M carbonate buffer) were added. Within 15 minutes after the addition, 2 M hydrochloric acid solution was added dropwise to adjust the pH of the reaction solution to 6.6. The reaction was shaken at room temperature overnight, then dialyzed for 72 h, to obtain the product. As shown in Table 1, the calculated degree of folate modification of FA-FVIII is about 8.03.

Example 2: Preparation of Folate-Monoclonal Antibody Complex

[0137] Folate-adalimumab complex (FA-Adalimumab) and folate-trastuzumab complex (FA-Trastuzumab) were prepared as examples of folate-monoclonal antibody complexes. A PBS solution containing 1 mg/mL each mAbs was prepared respectively, and tris(2-carboxyethyl)phosphine and EDTA were added to a concentrations of 10 mM and 5 mM, respectively. The reaction was shaken at room temperature for 90 minutes, and then was mixed with 15 equivalents of folate-PEG.sub.400-Mal (Folate-PEG.sub.400-Maleimide, purchased from Shanghai Yisheng Biotechnology Co., Ltd.). The reaction was shaked at room temperature for 20 minutes. In order to block the unreacted sulfhydryl groups, the solution was further reacted with 15 equivalents of maleimide at room temperature for 20 minutes under shaking. Subsequently, the reaction solution was dialyzed against PBS for 72 h to remove small molecules from the system, to obtain folate-monoclonal antibody complex solution. Table 1 provides the degree of folate modification in the folate-mAb complexs.

TABLE-US-00002 TABLE 1 The results of the degree of folate modification in each folate-modified biomacromolecule prepared in Examples 1-2 concentration Concentration of Degree of sample of sample OD.sub.365 nm folate in the complex Modification Folate-OVA 1 mM 0.41 8.66 mM 8.66 (per molecule) Folate-FVIII 1 IU/I 0.35 14.20 IU/I 14.20 (per IU) Folate-Adalimumab 2 mM 0.40 16.068 mM 8.03 (per molecule) Folate-Trastuzumab 2 mM 0.53 21.35 mM 10.68 (per molecule)

Example 3: Preparation of Folate-Contrast Agent Complexs

[0138] Folate-PEG.sub.2000-Cy5 (i.e., FA-PEG.sub.2000-Cy5) was prepared as an example of folate-contrast agent complexs. Folic acid (0.57 g, 1.28 mmol), NHS (0.37 g, 2.8 mmol) and DCC (0.6 g, 2.8 mmol) were added into a 250 mL round-bottom flask, and then DMSO (75 mL) was added. Reaction was performed under nitrogen protection at 25° C. After the substantial activation of folic acid monitored by TLC, the reaction was passed through a 0.22 μm organic filter membrane, and NH.sub.2-PEG.sub.2000-Cy5 (3 g, 1.15 mmol, purchased from Xi'an Ruixi Biological Technology Co., Ltd) and TEA (350 μl) were added. After 2 h, the reaction was complete as monitored by HPLC. The reaction solution was purified by G50 silica gel column (eluent: 0.01M PBS, pH 8.0) to obtain FA-PEG.sub.2000-Cy5.

[0139] Folic acid itself is insoluble in water, and the introduction of PEG.sub.2000 improves the water solubility of folic acid. FIG. 3 provides the .sup.1HNMR of the obtained FA-PEG.sub.2000-Cy5, wherein the signals δ7.65 and δ6.65 are the signal peaks of a group of symmetrical “AA‘BB’” spin systems. Thus, it can be determined that said signals are the two groups of proton signal peaks of the para-disubstituted benzene ring in the folic acid molecule, suggesting that the molecule of the product contains the structure of folate. The signal δ3.51 is a typical signal peak of PEG, further determining that the structure of the product is FA-PEG.sub.2000-Cy5.

Example 4: Specific Binding of Folate to mIgM on the Surface of B-Cell

[0140] The binding mode of folate in the murine IgM Fc domain was investigated by homology modeling and molecular docking. Molecular docking studies were performed with Schrodinger 2015 software to investigate the binding mode of folate in IgM Fc. FIG. 4a shows the binding mode of folate to murine mIgM. It can be seen that the binding pocket of folate to mIgM is located between Cμ3 and Cμ4 of the Fc fragment. FIG. 4b further provides an analysis on the binding force of folate to murine IgM monomers, wherein the binding pocket of folate is located at the junction of the two heavy chains (chain A and chain B) of the IgM monomer: the pteridine fragment in the folate structure may form hydrogen bonds with Phe287, Tyr288 and Pro285 in chain A and Glu52 in chain B; Phe55 in chain B forms a n-n interaction with the benzene fragment of folate: Lys25 in chain B forms an ionic interaction with the carboxyl group. The predicted binding energy between folate and Fc fragment of IgM is −10.929 kcal/mol, and the affinity reaches a level of 104 mol.

[0141] The binding of folate to mIgM was investigated with mIgM-positively-expressed spleen B-cell. BALB/c mouse spleen lymphocytes were extracted from mouse spleen lymphocyte isolates (purchased from Solarbo Technology Co., Ltd.), counted to 2×10.sup.6/mL, and plated on a 24-well culture plate (1 mL/well). Anti-IgM (ab97230, 1 mg/mL) was added to each well to the working concentrations of 0, 2.5, 5, 12.5 μg/mL. After being incubated at 37° C. for 24 h, the cells were divided into FA-PEG-Cy5 group and PEG-Cy5 group (n=3-4), 10 μL of FA-PEG-Cy5 (0.1 mM, prepared according to Example 3) or PEG-Cy5 (0.1 mM, purchased from Xi'an Ruixi Biological Co., Ltd.) were added, and the uptake was performed at 37° C. for 0.5 h. After centrifugation and washing of the cells, FITC-IgM and CD19-PE were added and the mixture was incubated at 4° C. for 1 h to label mIgM and CD19 on the cell surface. The expression level of mIgM in mouse spleen B-cell and the uptake of FA-PEG-Cy5 by cell were measured with flow cytometry. FIG. 5 provides the results of flow cytometric analysis of the binding of folate to mIgM. The results in FIG. 5a show that the mouse spleen B-cell mIgM can be effectively reduced with the increase of the concentrations of anti-IgM antibodies. The results in FIG. 5b show that the uptake of FA-PEG-Cy5 gradually decreased with the increase of anti-IgM concentrations, while the uptake of PEG-Cy5 does not vary much. As shown in FIG. 5c, by linearly fitting the expression level of mIgM to the uptake of FA-PEG-Cy5, the correlation coefficient R.sup.2 is 0.82, indicating that the uptake ability of FA-PEG-Cy5 is positively correlated with the expression level of mIgM.

[0142] The binding of folate to mIgM was further investigated by a confocal microscope. The extracted BALB/c mouse spleen lymphocytes were counted to 2×10.sup.6/mL. Anti-IgM antibodies (anti-mouse IgM, ab97230, 10 μg/mL), free folic acid molecule (0.5M), complement inactivated serum (serum was heat-treated at 56° C. for 30 min) or bovine serum albumin (5%) was added to the cells respectively, and the mixture was incubated at room temperature for 1 h. Then, the mixture was co-incubated with FA-PEG.sub.2000-Cy5 (prepared according to Example 3) at room temperature for 1 h. Similarly, the cells were incubated with bovine serum albumin (5%) at room temperature for 1 h and then co-incubated with PEG.sub.2000-Cy5 (purchased from Xian Ruixi Biological Co., Ltd.) for 1 h at room temperature. B-cells were screened for CD19 marker, and the binding of folate to mIgM was observed by confocal laser microscopy. FIG. 6 provides confocal laser micrographs (Carl Zeiss LSM710, Germany) of folate binded to mIgM, showing that the binding of folate (FA-PEG.sub.2000-Cy5) to B-cell is much higher than that of PEG.sub.2000-Cy5 and can be inhibited by anti-IgM antibodies and free folic acid molecules, independent of serum complement proteins, i.e., independent of complement receptors. The above results show that folate can specifically bind and target to mIgM of B-cell.

Example 5: Evaluation of Targeting of Folate to Spleen Marginal Region B-Cell

[0143] BALB/c mice (male, 6 weeks old, Slac) were injected with folate-contrast agent complex FA-PEG.sub.2000-Cy5 (prepared according to Example 3) or PEG.sub.2000-Cy5 (as a control) via tail vein in 110 nmol/mouse, with three in each group. Mice were sacrificed at 1 h and 4 h respectively, and spleens were isolated. The tissue was homogenated. According to the fluorescence standard curve of FA-PEG.sub.2000-Cy5 or PEG.sub.2000-Cy5, the folate concentration in the spleen homogenate was measured with a fluorence microplate reader, and the data were shown in FIG. 7 (a: 1 hour; b: 4 hours, *p<0.05, **p<0.01, ***p<0.001, one-way ANOVA). FIG. 7 shows that: 1 hour after injection, the spleen accumulations of FA-PEG.sub.2000-Cy5 and PEG.sub.2000-Cy5 were 9.35±2.10 nmol/g tissue and 4.57±1.19 nmol/g tissue, respectively; 4 hours after injection, the spleen accumulations of FA-PEG.sub.2000-Cy5 and PEG.sub.2000-Cy5 were 10.20±2.44 nmol/g tissue and 7.07±1.44 nmol/g tissue, respectively. The accumulation of FA-PEG.sub.2000-Cy5 in the spleen was significantly higher than that of the control PEG.sub.2000-Cy5, suggesting the spleen-targeting ability of FA-PEG.sub.2000-Cy5.

[0144] In addition, the spleens of mice sacrificed at 1 h or 4 h were collected, and the spleen lymphocytes were isolated for flow cytometric analysis (CytoFlex S. Beckman). FIG. 8 shows the flow sorting graphs (a: 1 h: c: 4 h) and statistical analysis results (b: 1 h; d: 4 h) of the spleen lymphocytes of mice sacrificed at 1 h or 4 h after administration of FA-PEG.sub.2000-Cy5 or PEG.sub.2000-Cy5, respectively. The results show that folate has a significant B-cell targeting ability.

[0145] The spleens from the mice sacrificed at 1 h were subjected to cryosection analysis. FIG. 9 shows the immunofluorescence photographs of the distribution of FA-PEG.sub.2000-Cy5 (a) and PEG.sub.2000-Cy5 (b) in the spleens, wherein the blue is the cell nucleus labeled with DAPI, the green is the B-cells labeled with CD19-PE antibody (553786, BD Pharmingen), the red is Cy5, and the yellow is the overlapping color of green and red. The results showed that folate was mainly distributed in the marginal area of the spleen, and significantly overlap the marginal B-cell. That is, folate targets the marginal B-cell of the spleen.

[0146] Since the splenic marginal B-cell highly expresses mIgM, the marginal B-cell was labeled with anti-IgM-FITC antibody (148445, Jackson). FIG. 10 provides the immunofluorescence photographs of the distribution of FA-PEG.sub.2000-Cy5 in the splenic marginal B-cell, wherein the blue is cell nucleus labeled with DAPI, the green is the marginal B-cell labeled with anti-IgM-FITC, the red is Cy5, and the yellow is the overlapping color of green and red. The results showed that folate significantly overlap the marginal B-cell. That is, folate targets the marginal B-cell of the spleen.

Example 6: The Role of Folate Modification in Mediating Humoral Immunosuppression and in Reducing Anti-Drug Antibodies of Protein Biomacromolecular Drugs

[0147] The role of folate modification in mediating humoral immunosuppression and in reducing Anti-Drug Antibodies (ADA) of biomacromolecular drugs was investragated by using ovalbumin (OVA) as a model protein. OVA has strong immunogenicity and can induce the body to produce an immune response. Repeated stimulation of mice by OVA can cause the mice to produce lots of specific antibodies. The BALB/c female mice (5 weeks old) were randomly divided into FA-OVA (folate-OVA complex. Example 1) group, OVA group and physiological saline control group, with 6 mice in each group. FA-OVA, OVA and physiological saline were administered to mice via tail vein injection at a dose of 10 μg/mouse, once a week, three times in total.

[0148] One week after the last administration, the spleen lymphocyte suspension was collected and subjected to flow cytometry for analysis of proliferation and differentiation of splenic B lymphocytes. CD86 was used as a marker for cell proliferation, and GL-7 was used as germinal center B-cell surface marker. FIGS. 11a and 11b provide the CD86 flow sorting graphs and statistical analysis results of mouse spleen lymphocytes, respectively. FIGS. 11c and 11d provide the GL7 flow sorting graphs and statistical analysis results of mouse spleen lymphocytes, respectively. It can be seen from FIG. 11 that the B-cell in the OVA group mice proliferated significantly and differentiated into the germinal center B-cell, while the proliferation of B-cell and the differentiation into germinal center B-cell were significantly inhibited in the FA-OVA group mice, suggesting the efficacy of folate modification in inhibiting B-cell proliferation and differentiation, which results in B-cell anergy.

[0149] One week after the last administration, the serum of the mice was collected and was measured by ELISA for the expression of OVA-specific antibodies in the serum. The results are shown in FIG. 12, wherein FIGS. 12a, 12b, 12c and 12d show the results of OVA-specific IgE, IgG, IgG.sub.1 and IgG.sub.2a, respectively. The data showed that repeated injections of OVA induced the body to produce a large amount of OVA-specific antibodies, while the expression of antibodies in FA-OVA group mice was significantly inhibited, which was comparable to the level in the physiological saline control group. This shows that the folate modification can significantly reduce the immunogenicity of antigenic substances. e.g., OVA, effectively inhibit humoral immunity, and reduce the production of Anti-Drug Antibodies of biomacromolecular protein drugs.

[0150] In addition, the whole blood of the mice was collected one week after the last administration for blood routine analysis. The results showed that there were no obvious abnormalities in the blood routine indicators of the mice in each group, indicating that the folate modification did not produce obvious side effects. The data are shown in Table 2 below.

TABLE-US-00003 TABLE 2 physiological FA-OVA OVA saline WBC (10*9/L) 3.77 ± 0.42 3.43 ± 0.51  4.35 ± 0.70 RBC (10*12/L) 9.96 ± 0.89 10.55 ± 1.13  10.56 ± 0.40 HGB (g/L) 161.50 ± 9.26  169.25 ± 13.45  172.00 ± 4.00  HCT (%) 49.83 ± 4.53  52.15 ± 5.97  53.37 ± 1.55 MCV (fL) 50.075 ± 1.37  49.43 ± 0.43  50.53 ± 0.45 MCH (pg) 16.28 ± 0.59  16.08 ± 0.67  16.30 ± 0.20 MCHC (g/L) 325.00 ± 12.35  325.75 ± 16.50  322.33 ± 1.53  PLT (10*9/L) 586.50 ± 59.29  635.00 ± 36.59  681.00 ± 82.31 RDW-CV (%) 14.78 ± 0.50  14.63 ± 0.33  15.10 ± 0.17 PDW (fL)  5.58 ± 0.096 5.53 ± 0.15  5.57 ± 0.31 MPV (fL)  7.05 ± 0.058 7.10 ± 0.14 7.067 ± 0.21 P-LCR (%) 4.35 ± 1.01 5.15 ± 1.48  5.37 ± 1.06 PCT (%)  0.41 ± 0.042  0.45 ± 0.016  0.49 ± 0.067 NRBC (10*9/L) 0.015 ± 0.013 0.025 ± 0.013  0.030 ± 0.020 NEYT (10*9/L) 0.94 ± 0.23 0.80 ± 0.21  0.82 ± 0.27 LYMPH (10*9/L) 2.78 ± 0.19 2.58 ± 0.29  3.42 ± 0.52 MONO (10*9/L)  0.035 ± 0.0058 0.035 ± 0.017  0.033 ± 0.011 BASO (10*9/L) 0 0 0

[0151] The role of folate modification in reducing Anti-Drug Antibodies (ADAs) of protein biomacromolecular drugs was investragated by using coagulation factor VIII (FVIII) as a model protein. The BALB/c female mice (5 weeks old) were randomly divided into FVIII group, FA-FVIII group and physiological saline control group, with 5 mice in each group. FVIII, FA-FVIII (prepared according to Example 1) and physiological saline were administered to mice via tail vein injection at a dose of 0.5 IU/mouse, once a week, three times in total. One week after the last administration, the serum of the mice was collected and was measured by ELISA for the expression of FVIII-specific antibodies in the serum. The results in FIG. 13 show that the antibody level in the FA-FVIII group was significantly lower than that in the FVIII group, suggesting that folate modification effectively reduces the production of Anti-Drug Antibodies of protein drugs.

Example 7: Effect of Folic Acid in Mediating Humoral Immunosuppression

[0152] The BALB/c female mice (5 weeks old) were randomly divided into 4 groups, with 6 mice in each group, and were treated as follows: (1) FA(ig) group: each mouse was administered with 3 mg free folic acid by gavage every day for two weeks: (2) FA(ig)+OVA group: each mouse was administered with 3 mg free folic acid by gavage every day for two weeks, and was administered with OVA via tail vein injection on day 0 and day 7 respectively (10 μg/mouse): (3) OVA group: each mouse was administered with OVA via tail vein injection on days 0 and day 7 respectively (10 μg/mouse); (4) Physiological saline group: each mouse was administered with physiological saline via tail vein injection on day 0 and day 7 respectively. Then, on days 14, 21 and 28, all mice were administered with 100 μl mixed solution of OVA and KLH (hemocyanin) in water (100 μg/mL OVA and 100 ug/mL KLH). The serum was collected before the administration of the mixed solution on days 14, 21 and 28 and on day 35. The specific antibodies in the serum were detected by ELISA.

[0153] FIG. 14 shows the levels of OVA-specific antibodies in mouse serum on day 14 (a), day 21 (b) and day 28 (c), respectively. The results showed that, on days 14 and 21, the level of OVA-specific IgG in the serum of the FA(ig)+OVA group mice was significantly lower than that in the OVA group, suggesting that folate exerts an effect of humoral immunosuppression and the combination of FA and OVA can reduce the production of OVA-specific antibodies. On day 28, the level of OVA-specific IgG in the serum of the FA(ig)+OVA group mice was comparable to that in the OVA group, suggesting that the immunosuppressive effect of FA is reversible and non-persistent. The immune function of the body recovered when the administration of FA is discontinued.

[0154] FIG. 15 shows the levels of KLH-specific antibodies in mouse serum on day 21 (a), day 28 (b) and day 35 (c), respectively. The results showed that the levels of KLH-specific antibodies in FA(ig) group and physiological saline group were similar, and both increased with the increase of injection times. The results also confirmed that the immune function of the body recovered when the administration of FA is discontinued.

Example 8: Efficacy of Folic Acid and Folate Modification in Reducing the Production of Anti-Drug Antibodies of Antibodies Biomacromolecular Drugs

[0155] Adalimumab and infliximab are humanized monoclonal antibodies against human tumor necrosis factor (TNF), and trastuzumab is a humanized monoclonal antibody against human epidermal growth factor receptor-2 (HER2). Their repeated injections are very likely to induce the body to produce antibodies against said mAbs, affecting the efficacy of the mAbs. The efficacy of folic acid and folate modification in reducing the Anti-Drug Antibodies of antibodies drugs was investigated with infliximab and trastuzumab as model mAbs.

[0156] The BALB/c female mice (6 weeks old) were randomly divided into Adalimumab group, FA-Adalimumab group (prepared according to Example 2) and physiological saline group, with mice in each group. For the Adalimumab group and the FA-Adalimumab group, a dose of 30 μg antibody/mouse was administrated by subcutaneous injection on days 0, 7 and 14. For the physiological saline group, the mice were subcutaneously injected with physiological saline on days 0, 7 and 14. One week after the last administration (day 21), the serum was collected from the mice, and was measured by ELISA for the antibodies against Adalimumab. The results are shown in FIG. 16a. The results showed that the level of antibodies in the FA-Adalimumab group was significantly lower than that in the Adalimumab group, suggesting that folate modification effectively reduced the production of Anti-Drug Antibodies of the antibody drugs.

[0157] The BALB/c female mice (6 weeks old) were randomly divided into infliximab group, FA gavage+infliximab group and physiological saline group, with 5 mice in each group. For the infliximab group, a dose of 60 μg antibody/mouse was administrated via tail vein injection on days 0, 7 and 14. For the FA gavage+infliximab group, each mouse was administered with 3 mg free folic acid by gavage per day for two weeks, and infliximab was administered via tail vein injection at a dose of 60 μg antibody/mouse on days 0, 7 and 14. For the physiological saline group, the mice were injected with physiological saline via tail vein on days 0, 7 and 14. One week after the last administration (day 21), the serum was collected from the mice, and was measured by ELISA for the antibodies against infliximab. The results are shown in FIG. 16b. The results showed that the level of antibodies in the FA gavage+infliximab group was significantly lower than that in the infliximab group, suggesting that FA effectively reduced the production of Anti-Drug Antibodies of the antibody drugs.

[0158] The BALB/c female mice (6 weeks old) were randomly divided into trastuzumab group, folate-trastuzumab group (obtained according to Example 2), FA gavage+trastuzumab group and physiological saline group, with 5 mice in each group. For the trastuzumab group and the folate-trastuzumab group, a dose of 60 μg antibody/mouse was administrated via tail vein injection on days 0, 7 and 14. For the FA gavage+trastuzumab group, each mouse was administered by gavage with 3 mg free folic acid per day for two weeks, and trastuzumab was administered via tail vein injection at a dose of 60 μg antibody/mouse on days 0, 7 and 14. For the physiological saline group, the mice were injected with physiological saline via tail vein on days 0, 7 and 14. One week after the last administration (day 21), the serum was collected from the mice, and was measured by ELISA for the antibodies against trastuzumab. The results are shown in FIG. 16c. The results showed that the levels of antibodies in the FA gavage+trastuzumab group and in the folate-trastuzumab group were significantly lower than that in the trastuzumab group, suggesting that FA and folate modification effectively reduced the production of Anti-Drug Antibodies of the antibody drugs.

Example 9: Efficacy of Folic Acid in Reducing the Production of Antibodies Against Drug Delivery Systems

[0159] The efficacy of folic acid in reducing the production of antibodies aginst drug delivery systems was investigated with liposome as an example.

[0160] Liposomes (sLip) were prepared firstly. HSPC. Chol and mPEG.sub.2000-DSPE (all purchased from AVT (Shanghai) Pharmaceutical Tech Co., Ltd.) were weighted in a molar ratio of 52:43:5. The film materials were dissolved in 5 mL chloroform, and the mixture was subjected to rotary evaporation under reduced pressure to remove chloroform, to obtain a uniform lipid film. Residual organic solvent was removed by vacuum drying overnight. Shaking in a water bath at 60° C. was performed until the liposome membrane is completely hydrated to obtain white liposome suspension. The liposome suspension went through 200 and 100 nuclear pore membrane by a micro-extruder sequentially, to obtain a liquid with pale blue opalescence, i.e., a liposome (sLip) solution.

[0161] The BALB/c female mice (6 weeks old) were randomly divided into sLip group, sLip+FA (10 μg) group, sLip+FA (50 μg) group and physiological saline group, with 4 mice in each group. For the sLip group, the liposome solution (5 mg liposome/kg mouse body weight) was administered in a single dose via tail vein. For sLip+FA(0 μg) group, a mixed solution of liposome (5 mg liposome/kg mouse body weight) and 10 μg FA was administered in a single dose via tail vein. For sLip+FA(50 μg) group, a mixed solution of liposome (5 mg liposome/kg mouse body weight) and 50 μg FA was administered in a single dose via tail vein. For the physiological saline group, each mouse was injected with the same volume of physiological saline via tail vein. Serum was collected from all mice 5 days after administration, and was measured by ELISA for the concentrations of the antibodies against sLip. The results are shown in FIG. 17. The results show that the combination of folic acid and liposomes can effectively reduce the production of the antibodies against liposomes, and the effect increases as the concentration of folic acid increases.

Example 10: Efficacy of Folate Modification in the Prevention of Anaphylactic Shock

[0162] The BALB/c female mice (5 weeks old) were randomly divided into FA-OVA group, OVA group and physiological saline group, with 10 mice in each group. FA-OVA or OVA or physiological saline of an equal volume was administered to the mice via tail vein injection at a dose of 10 μg OVA/mouse, once a week for a total of three times. One week after the last administration, each mouse was injected with 10 μg OVA (allergen) via tail vein. Changes in body temperature of the mice within 2 hours after OVA stimulation were recorded (FIG. 18). In the meanwhile, the serum was collected from the mice, and was measured by ELISA for the expression of OVA-specific antibodies in the serum (FIG. 19). FIG. 18 shows that the body temperature of the FA-OVA group mice did not change significantly, which was substantially comparable to that in the physiological saline group. However, the OVA group mice experienced severe anaphylactic shock, their body temperature dropped significantly, and two mice died. FIG. 19 shows that the FA-OVA group is substantially comparable to the physiological saline group, while the OVA group mice produced a large number of OVA-specific antibodies, suggesting that folate modification can significantly reduce the production of the antibodies caused by OVA. Therefore, folate modification can effectively prevent anaphylactic shock induced by allergens.

Example 11: Efficacy of Folate Modification in the Treatment of Anaphylactic Shock

[0163] The BALB/c female mice (5 weeks old) were subcutaneously injected with 10 μg OVA for sensitization, once a week, three times in total. One week after the last sensitization, the OVA-sensitized mice were injected with FA-OVA, OVA and physiological saline via tail vein at a dose of 4 μg/mouse, once every two days for three times in total. After the last administration, the mice were injected with 25 μg OVA (allergen) via tail vein. Changes in body temperature of the mice within 2 hours after the injection were recorded (FIG. 20). In the meanwhile, the serum was collected from the mice and was measured by ELISA for the level of the OVA-specific antibodies in the serum (FIG. 21). The mice without OVA sensitization but stimulated by tail vein injection of OVA served as the Naïve control group.

[0164] FIG. 20 shows the body temperature change curves of the sensitized mice which was treated with FA-OVA, OVA and physiological saline respectively and then was challenged with OVA. It can be seen that FA-OVA treatment showed a desensitizing effect, and the decrease in body temperature of the mice was controlled and returned to normal within 2 hours.

[0165] FIG. 21 shows the levels of serum OVA-specific antibodies in the sensitized mice which was treated with FA-OVA, OVA and physiological saline respectively and then was challenged with OVA. It can be seen that the expressions of OVA-specific antibodies IgE (a), IgG.sub.1 (b) and IgC.sub.2a(c) in the FA-OVA group were comparable to those in the physiological saline group and significantly decreased compared with those in the OVA group, suggesting the efficacy of folate modification in the treatment of antigen allergic diseases.

Example 12: Efficacy of Folate Modification in Autoimmune Encephalomyelitis

[0166] The efficacy of folate modification in autoimmune encephalomyelitis was investigated using an experimental autoimmune encephalomyelitis (EAE) model. The model was established as follows: the animals were injected with whole protein or protein fragment (e.g., PLP polypeptide) that induces nerve cell myelin sheath and peripheral protein, causing inflammation and demyelination symptoms and inducing the autoimmune response of the animal: thereby the injected self-proteins are regarded as foreign objects and cause the immune system to attack the self myelin sheath.

[0167] The female C57/BL6 mice were intravenously injected with PLP polypeptide (amino acid sequence. HSLGKWLGHPDKF) or folate-PLP complex (FA-PLP) at a dose of 10 μg/mouse, once a week for three times in total. One week later, the emulsion of PLP polypeptide in Freund's complete adjuvant was prepared, the concentration of PLP being 1 μg/μL. Four points were set up on both sides of the back of the mouse, and 0.05 mL PLP emulsion was injected subcutaneously at each point to induce inflammation, 2 and 24 hours after PLP injection, 150 ng pertussis toxin was injected intraperitoneally. The incidence in the mice was observed every day, and the mice were scored according to the 5-point scoring standard (Kono method). The incidence scoring curve was plotted. The results show that the modification of PLP by folate causes the body's immune tolerance to PLP and contributes to reducing the inflammatory response.

Example 13: Efficacy of Folate Modification in Bone Marrow Transplant Rejection

[0168] The CD45.1 mice donated bone marrow and the CD45.2 mice received bone marrow transplantation, 7 days before and 1 day after bone marrow transplantation, the CD45.2 mice were injected with FA-Dby (folate-modified Dby polypeptide: the amino acid sequence of Dby is NAGFNSNRANSSRSS) or FA+Dby (a physical mixture of folic acid and Dby in water) via tail vein at a dose of 10 μg/mouse. One day before bone marrow transplantation, each CD45.2 mouse received 200 cGy low-dose radiation. The donor mice were sacrificed, the femur and tibia were cut on an ultra-clean bench, and the cavity was purged repeatedly. The bone marrow cells were extracted, and washed with cPBS. The Recipient mice were injected intravenously with 5×10.sup.6 cells. After bone marrow transplantation, blood was collected from the mice once a week, and the chimerism of lymphocytes was measured by flow cytometry. Purebred CD45.1 mice were used as the control. The measurement indicators are CD45.1/CD45.2 and CD45.1/CD90.2.

Example 14: Efficacy of Folate-Human Serum Albumin Conjugate in Reducing the Immunogenicity of Biomacromolecule and Drug Delivery System

[0169] The efficacy of folate conjugates in reducing the immunogenicity of biomacromolecule (OVA, trastuzumab) and drug delivery system (sLip) was investigated with folate-human serum albumin (FA-HSA, purchased from Xi'an Ruixi Biological Technology Co., Ltd) as a representative of folate conjugates.

[0170] The BALB/c female mice (5 weeks old) were randomly divided into OVA group, FA-HSA+OVA group and physiological saline control group, with 6 mice in each group. The following treatments were performed respectively: (1) OVA group: each mouse was injected with 10 μg OVA via tail vein on days 0, 7 and 14: (2) FA-HSA+OVA group: each mouse was injected with a mixed solution of 10 μg OVA and 10 μg FA-HAS via tail vein on days 0.7 and 14; (3) Physiological saline group: each mouse was injected with the same volume of physiological saline via tail vein on days 0, 7 and 14. Serum was collected from all mice on day 21, and was measured by ELISA for the OVA-specific antibody (IgG) titer.

[0171] The above experiment was repeated with trastuzumab instead of OVA, adjusting the dose to 60 μg antibody/mouse.

[0172] The BALB/c female mice (6 weeks old) were randomly divided into sLip group (prepared according to Example 9), sLip+FA-HSA group and physiological saline group, with 6 mice in each group. For the sLip group, the mice were injected with liposome solution (5 mg liposome/kg mouse body weight) in a single dose via tail vein. For the sLip+FA-HSA group, the mice were injected with a mixed solution of liposome (5 mg liposome/kg mouse body weight) and 10 μg FA-HSA in a single dose via tail vein. For the physiological saline group, the mouse was injected with the same volume of physiological saline via tail vein. Five days after administration, the serum was collected from all mice, and was measured by ELISA for the antibody (IgM) titer against sLip in the serum.

Example 15: Efficacy of Folate Conjugates in Reducing the Immunogenicity of Biomacromolecule and Drug Delivery System

[0173] Similarly to Example 14, the efficacy of folate-polyethylene glycol.sub.40 kDa (FA-PEG.sub.40 kDa, purchased from Xi'an Ruixi Biological Technology Co., Ltd) in reducing the immunogenicity of biomacromolecule (OVA, trastuzumab) and drug delivery system (sLip) is investigated with FA-PEG.sub.40 kDa instead of FA-HAS. FA-PEG.sub.40 KDa is administrated at a dose of 100 nM/mouse.

Formulation Examples

Example A

[0174] Injectable solutions containing the agent according to the invention can be prepared in a conventional manner:

TABLE-US-00004 Ingredients Amount (per mL) Agent according to the invention 5.0 mg Polyethylene glycol 400 150.0 mg Acetic acid qs to pH 5.0 Water for Injection add to 1.0 mL

[0175] The agent according to the invention is dissolved in a mixture of polyethylene glycol 400 and water for injection (part). Acetic acid is added to adjust pH to 5.0. The volume is made up to 1.0 mL by adding the remaining amount of water. The solution is filtered, filled into vials in appropriate overdose, and sterilized.

Example B

[0176] Oral tablets containing the following ingredients can be prepared in a conventional manner:

TABLE-US-00005 Ingredients per tablet Agent according to the invention 10 mg Corn starch 80 mg Lactose 95 mg Magnesium stearate 5 mg

[0177] The above ingredients are mixed homogeneously and are compressed into tablets.

Example C

[0178] Oral capsules containing the following ingredients can be prepared in a conventional manner:

TABLE-US-00006 Ingredients per capsule Agent according to the invention 10.0 mg Lactose 95.0 mg Corn starch 20.0 mg Talc 5.0 mg

[0179] The ingredients are sieved, blended and filled into capsule shells.

Example D

[0180] Aerosol containing the following ingredients can be produced in a conventional manner:

TABLE-US-00007 Ingredients Content (per 100 mL) Agent according to the invention 80.0 mg Oleic acid Appropriate amount Dichlorodifluoromethane Appropriate amount

[0181] The agent according to the invention is mixed with oleic acid, filled into an aerosol bottle in doses, and injected with dichlorodifluoromethane under pressure.

[0182] The invention has been illustrated through description and examples for the purposes of clarity and understanding. It can be understood that the above description and examples are only illustrative rather than restrictive, and various modifications and changes may be made within the scope of the present invention. Accordingly, the scope of the present invention should not be limited to the above description and examples, but should depend on the accompanying claims together with the full scope of equivalents to satisfy these claims.

[0183] Patents, patent applications, and scientific literatures cited herein are incorporated herein in their entirety to the same extent as if each were specifically and individually indicated. Any discrepancy between any reference cited herein and the specific teachings of the specification shall be resolved in favor of the latter. Likewise, any discrepancy between the definition of a word or expression as understood in the art and the definition of that word or expression specifically taught in the specification shall also be resolved in favor of the latter.