A pharmaceutical composition and uses thereof

Abstract

A composition comprising an immunoglobulin-degrading enzyme and a functional molecule capable of simultaneously targeting and binding immunoglobulins and ASGPR, wherein application of the composition can effectively eliminate neutralizing antibodies or autoimmune antibodies.

Description

A pharmaceutical composition and its application

[0001] Priority Declaration

[0002] This disclosure claims priority to Chinese patent application No. 2024100154234 and PCT patent application No. PCT / CN2024 / 112587, and the technical solutions of the Chinese patent application and PCT patent application are fully incorporated into this disclosure. Technical Field

[0003] The present disclosure relates to the field of immunotherapy. More specifically, the present disclosure relates to suppressing the body's immune response using a pharmaceutical composition that reduces immunoglobulin levels in the body. Background Art

[0004] Before the immune system can protect the body from foreign or dangerous substances, it must first identify them. These substances include bacteria, viruses, parasites (such as worms), certain cancer cells, and even transplanted organs and tissues. These substances have molecules that the immune system recognizes and stimulates a response. These molecules are called antigens.

[0005] Adeno-Associated Virus (AAV) is the most common delivery vector in the gene therapy industry. Currently, two products have been approved for marketing, and nearly hundreds of clinical trials are underway. Humans are naturally infected with AAV from infancy, which leads to a humoral response to the virus in the body. When this group of people receive gene therapy using AAV as a vector, the delivered AAV will be neutralized by pre-existing antibodies before entering the cells, affecting the treatment effect and increasing safety risks. Due to the presence of neutralizing antibodies, many patients are excluded from gene therapy. Reducing the level of neutralizing antibodies in the body can reduce the safety risks of treatment and expand the treatment population.

[0006] Furthermore, under normal circumstances, the immune system only responds to foreign or dangerous substances and does not react to antigens of one's own tissues. However, sometimes immune dysfunction occurs, and one's own tissues are mistaken for foreign, producing antibodies (called autoimmune antibodies) or immune cells that attack one's own cells or tissues. This reaction is called an autoimmune reaction, which causes inflammation and tissue damage. In severe cases, the inflammation and tissue damage caused by it can lead to pain, joint deformities, weakness, jaundice, itching, difficulty breathing, fluid accumulation (edema), and even death. Autoimmune reactions may lead to autoimmune diseases.

[0007] Therefore, there is a need in the art for a method of reducing neutralizing antibodies or autoimmune antibodies against immunogenic substances. Summary of the Invention

[0008] Embodiments of some aspects of the present disclosure include providing a pharmaceutical composition, wherein after administration of the composition, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, at least 99.5%, at least 99.9%, at least 99.99% or at least 99.999% of the immunoglobulins in the subject are degraded.

[0009] Embodiments of some aspects of the present disclosure include providing a pharmaceutical composition, wherein after administration of the composition, the neutralizing antibody or autoimmune antibody titer in the subject is less than about 1:10,000, or less than about 1:1000, or less than about 1:100, or less than about 1:50, or less than about 1:40, or less than about 1:30, or less than about 1:20, or less than about 1:10, or less than about 1:5, or less than about 1:2.

[0010] In some embodiments, the composition comprises a first molecule and a second molecule, wherein the first molecule is capable of degrading immunoglobulin, and the second molecule is a functional molecule capable of binding to immunoglobulin and lysosomal targeting receptor; in one embodiment, the immunoglobulin is a pre-existing antibody in the subject, an anti-drug antibody caused by the administration of an immunogenic substance, or an autoimmune antibody caused by an autoimmune disease in the subject.

[0011] In some embodiments, the first molecule in the composition described in the present disclosure is an immunoglobulin degrading enzyme. In one embodiment, the immunoglobulin degrading enzyme is selected from IgG degrading enzyme, IgE degrading enzyme, IgM degrading enzyme, IgD degrading enzyme or IgA degrading enzyme.

[0012] In some embodiments, the second molecule in the composition described in the present disclosure is a functional molecule that binds to an immunoglobulin and a lysosomal targeting receptor; the immunoglobulin is IgG, IgE, IgM, IgD or IgA; in one embodiment, the immunoglobulin is an IgG antibody.

[0013] In some embodiments, the lysosomal targeting receptor in the composition described herein is ASGPR or M6PR; in one embodiment, the lysosomal targeting receptor is ASGPR.

[0014] In some embodiments, the second molecule in the pharmaceutical composition of the present disclosure has a structure of formula (I): f -LT a (I)

[0015] Where T f For immunoglobulin binding ligand, T ais an affinity ligand for ASGPR and / or M6PR, and L is a linker.

[0016] In some embodiments, the T f Affinity K for immunoglobulins D Value is less than 1×10 -7 M, 1×10 -8 M, 1×10 -9 M or 1×10 -10 M, or EC 50 <1000nM, 100nM, 10nM or 1nM; Optionally, the T f Binding affinity K of immunoglobulin D Value is less than 1×10 -8 M; or EC 50 <100nM.

[0017] In some embodiments, the T a Affinity K for ASGPR and / or M6PR D Value is less than 1×10 -7 M, 1×10 -8 M, 1×10 -9 M or 1×10 -10 M. Optionally, the T a Affinity K for ASGPR and / or M6PR D The value is 1×10 -7 M~1×10 -10 Between M.

[0018] In some embodiments, the T f It is a polypeptide or fragment thereof that specifically binds to IgG, IgE, IgM, IgD or IgA; in some embodiments, the polypeptide specifically binds to the constant region or variable region of IgG, IgE, IgM, IgD or IgA; in some embodiments, the polypeptide is selected from protein A, Z33, protein G, FcR, or an antibody or antigen-binding fragment thereof that specifically binds to the constant region or variable region of IgG, IgE, IgM, IgD or IgA; optionally, the antigen-binding fragment of the antibody that specifically binds to IgG, IgE, IgM, IgD or IgA is selected from VHH or scFv, affibody, Fab, or F(ab')2 fragment.

[0019] In some embodiments, the T aA residue selected from a polypeptide or fragment thereof that specifically binds to ASGPR, asialoglycoprotein, N-acetylgalactosamine (GalNac), triGalNAc, galactose, galactoside, galactosamine, or galactan; optionally, the polypeptide that specifically binds to ASGPR is an anti-ASGPR antibody or an antigen-binding fragment thereof; optionally, the antigen-binding fragment of the anti-ASGPR antibody comprises a VHH, scFv, affibody, Fab, or F(ab')2 fragment; optionally, the ASGPR affinity ligand comprises:

[0020] residues.

[0021] In some embodiments, L is a bond, a peptide linker, or a non-peptide linker.

[0022] In some embodiments, the first molecule in the pharmaceutical composition of the present disclosure is selected from: at least one immunoglobulin degrading enzyme as shown in SEQ ID NOs: 1 to 13.

[0023] In some embodiments, the T in the pharmaceutical composition of the present disclosure is f is selected from HFC-1, FcRn, ProteinA, ProteinG, immunoglobulin constant region binding peptide or Z33, and can also be selected from mutants with immunoglobulin binding activity derived from the aforementioned proteins or polypeptides, or fragments with immunoglobulin binding activity derived from the aforementioned proteins, polypeptides or mutants; optionally, the T f It comprises a polypeptide as shown in any one of SEQ ID NOs: 19 to 28.

[0024] In some embodiments, the T in the pharmaceutical composition of the present disclosure is a The fragment is selected from the ASGPR binding fragment comprising the polypeptide shown in any one of SEQ ID NOs: 14 to 16.

[0025] In some embodiments, the T of the second molecule in the pharmaceutical composition of the present disclosure is a is selected from the group consisting of a polypeptide as shown in any one of SEQ ID NOs: 14 to 16, wherein the second molecule T f The polypeptide comprises any one of SEQ ID NOs: 19 to 29, and L comprises a polypeptide as shown in SEQ ID NOs: 17 or 18. For example, an optional combination includes a polypeptide as shown in any one of SEQ ID NOs: 30 to 36.

[0026] In some embodiments, the pharmaceutical composition of the present disclosure comprises a first molecule and a second molecule, wherein the first molecule is selected from an immunoglobulin degrading enzyme as shown in any one of SEQ ID NOs: 1 to 13; and the second molecule is selected from a functional molecule as shown in any one of SEQ ID NOs: 30 to 36.

[0027] In some embodiments, the pharmaceutical composition described in the present disclosure further comprises a pharmaceutically acceptable carrier.

[0028] In some embodiments, the pharmaceutical composition of the present disclosure further comprises an immunogenic substance, including a viral vector or a non-viral vector; preferably an adeno-associated virus (AAV) vector.

[0029] The second aspect of the present disclosure provides a functional molecule having a structure of formula (I):

[0030] T f -LT a (I)

[0031] Where T f For immunoglobulin binding ligand, T a is an affinity ligand for ASGPR and / or M6PR, and L is a linker.

[0032] In some embodiments, the T f Affinity K for immunoglobulins D Value is less than 1×10 -7 M, 1×10 -8 M, 1×10 -9 M or 1×10 -10 M, or EC 50 <1000nM, 100nM, 10nM or 1nM; Optionally, the T f Binding affinity K of immunoglobulin D Value is less than 1×10 -8 M; or EC 50 <100nM.

[0033] In some embodiments, the T a Affinity K for ASGPR and / or M6PR D Value is less than 1×10 -7 M, 1×10 -8 M, 1×10 -9 M or 1×10 -10 M. Optionally, the T a Affinity K for ASGPR and / or M6PR D The value is 1×10 -7 M~1×10 -10Between M.

[0034] In some embodiments, the T in the functional molecules described in the present disclosure is f It is a polypeptide or fragment thereof that specifically binds to IgG, IgE, IgM, IgD or IgA; optionally, the polypeptide specifically binds to the constant region or variable region of IgG, IgE, IgM, IgD or IgA; optionally, the polypeptide is selected from protein A, Z33, protein G, FcR, or an antibody or antigen-binding fragment thereof that specifically binds to the constant region or variable region of IgG, IgE, IgM, IgD or IgA; optionally, the antigen-binding fragment of the antibody that specifically binds to IgG, IgE, IgM, IgD or IgA includes a VHH or scFv, affibody, Fab, or F(ab')2 fragment.

[0035] In some embodiments, the T a A residue selected from a polypeptide or fragment thereof that specifically binds to ASGPR, asialoglycoprotein, N-acetylgalactosamine (GalNac), triGalNAc, galactose, galactoside, galactosamine, or galactan; optionally, the polypeptide that specifically binds to ASGPR is an anti-ASGPR antibody or an antigen-binding fragment thereof; optionally, the antigen-binding fragment of the anti-ASGPR antibody comprises a VHH or scFv, affibody, Fab, or F(ab')2 fragment; optionally, the ASGPR affinity ligand comprises:

[0036] residues.

[0037] In some embodiments, L is a bond, a peptide linker, or a non-peptide linker.

[0038] In some embodiments, the T in the pharmaceutical composition of the present disclosure is f is selected from HFC-1, FcRn, ProteinA, ProteinG, immunoglobulin constant region binding peptide or Z33, and can also be selected from mutants with immunoglobulin binding activity derived from the aforementioned proteins or polypeptides, or fragments with immunoglobulin binding activity derived from the aforementioned proteins, polypeptides or mutants; optionally, the T f It comprises a polypeptide as shown in any one of SEQ ID NOs: 19 to 28.

[0039] In some embodiments, the T in the functional molecules described in the present disclosure is a The fragment is selected from the ASGPR binding fragment comprising the polypeptide shown in any one of SEQ ID NOs: 14 to 16.

[0040] In some embodiments, Ta of the functional molecule described in the present disclosure is selected from a polypeptide as shown in any one of SEQ ID NOs: 14 to 16, Tf of the second molecule comprises a polypeptide as shown in any one of SEQ ID NOs: 19 to 28, and L comprises a polypeptide as shown in SEQ ID NO: 17 or 18; optionally, the second molecule comprises a polypeptide as shown in any one of SEQ ID NOs: 30 to 36.

[0041] Embodiments of other aspects of the present disclosure include providing a method for reducing the amount of neutralizing antibodies in a subject, comprising administering to the subject a pharmaceutically effective amount of the pharmaceutical composition of the present disclosure or the functional molecule of the present disclosure; optionally, the administration comprises administering the second molecule and the first molecule simultaneously, or administering the first molecule first and then the second molecule, or administering the second molecule first and then the first molecule.

[0042] Embodiments of other aspects of the present disclosure include providing a method for improving the efficacy of gene therapy treatment in a subject, comprising administering to the subject a pharmaceutically effective amount of the pharmaceutical composition of the present disclosure or the functional molecule of the present disclosure; optionally, the administration comprises administering the second molecule and the first molecule as an immunoglobulin degrading enzyme simultaneously, or administering the first molecule first and then the second molecule, or administering the second molecule first and then the first molecule.

[0043] Embodiments of other aspects of the present disclosure include providing a method for treating an autoimmune disease or antibody-mediated organ transplant rejection in a subject, comprising administering to the subject a pharmaceutically effective amount of the pharmaceutical composition of the present disclosure or the functional molecule of the present disclosure; optionally, the administration comprises administering the second molecule and the first molecule as an immunoglobulin degrading enzyme simultaneously, or administering the first molecule first and then the second molecule, or administering the second molecule first and then the first molecule.

[0044] Other aspects of the present disclosure include providing a use of the pharmaceutical composition or the functional molecule described in the present disclosure in the preparation of a drug, wherein the drug can reduce the amount of pre-existing antibodies, autoimmune antibodies, neutralizing antibodies and / or anti-drug antibodies in a subject.

[0045] Other aspects of the present disclosure include providing a use of the pharmaceutical composition in the preparation of a medicament for improving the efficacy of gene therapy treatment in a subject.

[0046] Other aspects of the present disclosure include providing a use of the pharmaceutical composition in the preparation of a medicament for treating an autoimmune disease or antibody-mediated organ transplant rejection in a subject.

[0047] Another aspect of the present disclosure provides a VHH antibody domain or a fragment thereof, comprising CDR1 shown in SEQ ID NO: 37, CDR2 shown in SEQ ID NO: 38, and CDR3 shown in SEQ ID NO: 39.

[0048] In some embodiments, the VHH antibody domain or fragment thereof specifically binds IgM.

[0049] In some embodiments, the VHH antibody domain or fragment thereof is humanized.

[0050] In some embodiments, the VHH antibody domain or fragment thereof has the amino acid sequence shown in SEQ ID NO:27.

[0051] In some embodiments, the VHH antibody domain or fragment thereof is used to prepare an IgM affinity matrix. The IgM affinity matrix can be used for IgM separation and purification, or for binding IgM, transporting IgM, promoting reactions using IgM as a substrate, etc.

[0052] In some embodiments, the VHH antibody domain or fragment thereof or the IgM affinity matrix is ​​used for separation and purification of IgM in plasma and / or serum, or for separation and purification of monoclonal IgM in cell culture.

[0053] In some embodiments, the separation and purification is efficient, high flow rate, and high yield, and the obtained IgM has high activity.

[0054] Based on the above-mentioned VHH antibody domain or fragment thereof, another aspect of the present disclosure provides a functional molecule having the structure of formula (II):

[0055] T a -L-VHH(Ⅱ); wherein VHH is the VHH antibody domain or fragment thereof according to any one of the aforementioned embodiments, T a is an affinity ligand for ASGPR and / or M6PR, and L is a linker.

[0056] In some embodiments, the affinity K of the VHHs described herein to immunoglobulins is D Value is less than 1×10 -7 M, 1×10 -8 M, 1×10 -9 M or 1×10 -10 M, or EC 50 <1000nM, 100nM, 10nM or 1nM; Optionally, the T f Binding affinity K of immunoglobulin D Value is less than 1×10 -8 M; or EC50 <100nM.

[0057] In some embodiments, the T a Affinity K for ASGPR and / or M6PR D Value is less than 1×10 -7 M, 1×10 -8 M, 1×10 -9 M or 1×10 -10 M. Optionally, the T a Affinity K for ASGPR and / or M6PR D The value is 1×10 -7 M~1×10 -10 Between M.

[0058] In some embodiments, the T a A residue selected from a polypeptide or fragment thereof that specifically binds to ASGPR, asialoglycoprotein, N-acetylgalactosamine (GalNac), triGalNAc, galactose, galactoside, galactosamine, or galactan; optionally, the polypeptide that specifically binds to ASGPR is an anti-ASGPR antibody or an antigen-binding fragment thereof; optionally, the antigen-binding fragment of the anti-ASGPR antibody comprises a VHH or scFv, affibody, Fab, or F(ab')2 fragment; optionally, the ASGPR affinity ligand comprises:

[0059] residues.

[0060] In some embodiments, L is a bond, a peptide linker, or a non-peptide linker.

[0061] In some embodiments, the T in the functional molecules described in the present disclosure is a The fragment is selected from the ASGPR binding fragment comprising the polypeptide shown in any one of SEQ ID NOs: 14 to 16.

[0062] In some embodiments, the functional molecule described in the present disclosure comprises a polypeptide as shown in SEQ ID NO:34.

[0063] Based on the above functional molecules, another aspect of the present disclosure provides a pharmaceutical composition comprising an immunoglobulin degrading enzyme and the functional molecule having any one of the structures of formula (II) in the above embodiments.

[0064] In some embodiments, the immunoglobulin degrading enzyme comprises an IgG degrading enzyme, an IgE degrading enzyme, an IgM degrading enzyme, an IgD degrading enzyme, or an IgA degrading enzyme.

[0065] In some embodiments, the immunoglobulin degrading enzyme includes at least one immunoglobulin degrading enzyme shown in SEQ ID NOs: 1-13.

[0066] In some embodiments, the pharmaceutical composition comprises an immunoglobulin degrading enzyme comprising a sequence shown in at least one of SEQ ID NOs: 1 to 13 and a functional molecule comprising a sequence shown in SEQ ID NO: 34.

[0067] In order to make the technical solutions described in the present disclosure clearer, the present disclosure is further described below in conjunction with the accompanying drawings and specific implementation methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] FIG1 shows the ASGPR and IVIG binding activity test results of functional molecules 1 and 5;

[0069] FIG2 shows the in vitro activity test results of functional molecules 1, 6 and 7;

[0070] FIG3 shows the results of the degradation of human IgG by functional molecule 1 in mice;

[0071] FIG4 shows the results of the detection of the degradation effect of functional molecule 5 on human IgM in mice;

[0072] FIG5 shows the degradation effect of IdeE-mut on human IgG;

[0073] Figure 6 is a comparison of the degradation effects of IdeE-mut and Imlifidase on pre-existing antibody populations;

[0074] Figure 7a is a comparison of the degradation effects of IdeE-mut on the Chinese population with pre-existing antibodies;

[0075] Figure 7b is a comparison of the degradation effect of IdeE-mut on the New Zealand pre-existing antibody population;

[0076] Figure 8a shows the degradation results of total IgG in serum by IdeE-mut;

[0077] Figure 8b shows the degradation effect of IdeE-mut on AAV2 neutralizing antibodies;

[0078] Figure 8c shows the degradation effect of IdeE-mut on AAV8 neutralizing antibodies;

[0079] FIG9 shows the degradation effect of IdeE-mut on AAV2 neutralizing antibodies of different titers;

[0080] Figure 10 shows the changes in F(ab')2 during the degradation of AAV2 neutralizing antibodies by IdeE-mut;

[0081] Figure 11 shows the changes in F(ab')2 during the degradation of AAV8 neutralizing antibodies by IdeE-mut;

[0082] Figure 12 is a comparison of the changes in AAV2 and AAV8 with different neutralizing antibody titers after degradation by IdeE-mut;

[0083] FIG13 is a comparison of the degradation effects of IVIg before and after the combined use of IdeE-mut and functional molecule 6. DETAILED DESCRIPTION

[0084] I. Detailed Description of the Invention

[0085] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in this detailed description is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0086] All publications, patent applications, patents, GenBank or other accession numbers and other references mentioned herein are incorporated by reference in their entirety.

[0087] It is specifically contemplated that the various features described herein can be used in any combination unless the context dictates otherwise.

[0088] The following terms are used in this specification and the appended claims:

[0089] The singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0090] In addition, when referring to a measurable value (e.g., the amount of length of a polynucleotide or polypeptide sequence, dosage, time, temperature, etc.), the term "about" as used herein is intended to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0091] Additionally, as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted in the alternative ("or").

[0092] Recitation of numerical ranges herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is recited as 1% to 50%, it is intended that values ​​such as 2% to 40%, 10% to 30%, or 1% to 3% be expressly recited in this specification. These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between and including the lowest and highest values ​​recited are to be considered to be expressly stated in this disclosure.

[0093] The term "immunoglobulin" refers to a class of globulins present in human and animal blood (serum), tissue fluid, and other exocrine fluids that have antibody activity and a chemical structure similar to that of antibody molecules. In some embodiments, immunoglobulins can be interpreted as antibodies, further including IgG, IgE, IgM, IgD, or IgA antibodies.

[0094] The term "lysosome-targeting receptors (LTRs)" refers to cell membrane surface receptors required for protein transport to lysosomes, which facilitate protein transport to lysosomes. As used herein, in certain embodiments, the term "lysosome-targeting receptors" refers to CI-M6PR, IGF2R, ASGPR (Zhu L et al., Bioorg Chem. 2023; 141: 106908).

[0095] The term "immunoglobulin degrading enzyme" refers to an enzyme that can cleave immunoglobulins. In some embodiments, the "immunoglobulin degrading enzyme" is an "IgG degrading enzyme", specifically an IgG-specific degrading protease whose cleavage site is between IgG positions G236 and G237 (EU numbering). Currently known IgG degrading enzymes include those disclosed in the literature (Lannergard, J. and B. Guss. 2006. FEMS Microbiol Lett 262(2): 230-235; Hulting, G. et al. (2009). FEMS Microbiol Lett 298(1): 44-50), and immunoglobulin degrading enzymes disclosed in WO2016128559A1, WO2016128558A1, WO2022223818A1, WO2021254479A1, and WO2023116817A1, as well as all enzymes having the activity of cleaving immunoglobulins as described above and their variants. Variants of these known enzymes can also play the same role. In some embodiments, the IgG degrading enzyme comprises SEQ ID NO: 8-15.

[0096] It should be understood that the IgG degrading enzyme can be a derivative of any of the above-mentioned wild-type IgG degrading enzymes, such as a truncated derivative, a derivative comprising more amino acids than the corresponding wild-type IgG degrading enzyme, a derivative comprising one or more amino acid substitutions (e.g., one or more conservative substitutions, one or more non-conservative substitutions, substitutions of natural amino acids with non-natural amino acids, and / or the like), etc. The derivative retains at least a portion of the hydrolase activity of the parent wild-type IgG degrading enzyme.

[0097] In some embodiments, the "immunoglobulin degrading enzyme" may be an IgA degrading enzyme, an IgD degrading enzyme, an IgE degrading enzyme, or an IgM degrading enzyme, including IgA1 protease, IgM protease, IgMBRAZOR, etc. Further, the immunoglobulin degrading enzyme may be selected from CN116568329A.

[0098] "Vector" refers to a compound used as a vehicle to carry exogenous genetic material into another cell, where the exogenous genetic material can be replicated and / or expressed. Cloning vectors containing exogenous nucleic acids are called recombinant vectors. Examples of nucleic acid vectors are plasmids, viral vectors, cosmids, expression cassettes, and artificial chromosomes. Recombinant vectors typically contain an origin of replication, a multiple cloning site, and a selective marker. The nucleic acid sequence typically consists of an insert (recombinant nucleic acid or transgene) and a larger sequence that serves as the vector's "skeleton." The purpose of a vector for transferring genetic information to another cell is typically to separate, propagate, or express the insert in the target cell. Expression vectors (expression constructs or expression cassettes) are used for the expression of exogenous genes in target cells and typically have promoter sequences that drive the expression of exogenous genes / ORFs. Inserting a vector into a target cell refers to the transformation or transfection of bacteria and eukaryotic cells, although inserting a viral vector is typically referred to as transduction. The term "vector" can also be generally used to describe a substance used to carry exogenous genetic material into another cell, such as, but not limited to, transformed cells or nanoparticles.

[0099] As used herein, in certain embodiments, the terms "vector," "viral vector," "delivery vector" (and similar terms) generally refer to a viral particle that functions as a nucleic acid delivery vehicle and includes a viral nucleic acid (i.e., a vector genome) packaged within the viral particle. A viral vector according to the present disclosure includes a chimeric AAV capsid according to the present disclosure and is capable of packaging an AAV or rAAV genome or any other nucleic acid including a viral nucleic acid. Alternatively, in some contexts, the terms "vector," "viral vector," "delivery vector" (and similar terms) may be used to refer to a vector genome (e.g., vDNA) in the absence of a viral particle and / or to a viral capsid that acts as a transporter to deliver molecules tethered to or packaged within the capsid.

[0100] It further comprises a therapeutic viral vector or non-viral vector; preferably a recombinant lentiviral vector, a recombinant adenoviral vector or a recombinant adeno-associated virus (AAV) vector.

[0101] In certain embodiments, the recombinant AAV vector comprises or consists of a capsid protein that is at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to one or more of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rAAVrh74, AAVrh10, AAV-2i8, SPK1, SPK2 capsid proteins (VP1, VP2 and / or VP3 sequences). In certain embodiments, the recombinant AAV vector comprises or consists of a sequence that is at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rAAVrh74, AAVrh10, or AAV-2i8, ITRs.

[0102] In certain embodiments, recombinant AAV vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10, rAAVrh74, and AAV-2i8 variants thereof (e.g., ITR and capsid variants, such as amino acid insertions, additions, substitutions, and deletions), e.g., as described in WO 2013 / 158879, WO 2015 / 013313, and US2013 / 0059732.

[0103] Additionally, viral capsid or genomic elements may contain other modifications, including insertions, deletions, and / or substitutions.

[0104] As used herein, the term "antibody" refers to a polypeptide comprising at least a light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen. The term encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, single-chain antibodies or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), fully human antibodies, chimeric antibodies, or humanized antibodies, full-length antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0105] As used herein, the term "neutralizing antibody" refers to an antibody that specifically binds to an immunogenic substance and inhibits one or more biological activities of the immunogenic substance at the level present in a subject. These antibodies may be pre-existing and may inhibit or reduce the level of recombinant viral vector cellular transduction in a subject prior to administration of the viral vector. Alternatively, the antibodies may be produced in a subject following exposure to the virus upon which the recombinant viral vector is based. Furthermore, they may be produced in a subject after administration of the recombinant viral vector.

[0106] The term "anti-drug antibodies (ADAs)" refers to antibodies produced by the human immune system in response to foreign drugs, such as biologic drugs. These antibodies may affect the efficacy and safety of drugs.

[0107] The term “pre-existing antibodies” refers to a persistent memory immune response that may result from the host’s previous exposure to a component of a foreign substance, such as a biologic drug, and from the initial exposure.

[0108] In some embodiments, the immunogenic substance can be a nucleic acid delivery vector, for example, a viral vector or a non-viral vector. In some embodiments, the viral vector is an adeno-associated virus.

[0109] In some embodiments, the immunogenic substance is a gene editing complex, for example, a CRISPR complex, which refers to a complex formed by the binding or modification of Cas protein, gRNA and target nucleic acid in the CRISPR system.

[0110] In some embodiments, the immunogenic substance is a protein or nucleic acid. In some embodiments, the protein is an enzyme, a regulatory protein, or a structural protein, such as one that can replace a missing or defective protein in a subject. In some embodiments, the nucleic acid is a functional nucleic acid, such as an antisense nucleic acid or an inhibitory RNA.

[0111] The term "affinity" or "binding affinity" refers to the strength of the binding interaction between a single biomolecule (e.g., protein or DNA) and its ligand / binding partner (e.g., drug or inhibitor). Binding affinity is generally measured by the equilibrium dissociation constant (K D ) is measured and reported, and this constant is used to assess and rank the strength of bimolecular interactions. K D The smaller the value, the greater the binding affinity of the ligand for its target. DThe larger the value, the weaker the mutual attraction and binding between the target molecule and the ligand. Methods for measuring binding affinity include methods that require labeling of interactors and label-free methods. The main label-based qualitative method is enzyme-linked immunosorbent assay (ELISA). Key label-free quantitative methods include spectrometry, isothermal titration calorimetry (ITC) or optical biosensors such as surface plasmon resonance (SPR), biolayer interferometry (BLI) and grating coupled interferometry (GCI). In this article, EC can also be used. 50 Expresses affinity.

[0112] The term "antigen-binding fragment" of an antibody (used interchangeably herein with "antibody fragment" and "antigen-binding portion") refers to a molecule that is not a complete antibody, which comprises a portion of a complete antibody that is used to bind to the antigen to which the complete antibody binds. As will be appreciated by those skilled in the art, the antigen-binding portion of an antibody typically comprises amino acid residues from a "complementarity determining region" or "CDR". Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of complete antibodies. Antigen-binding fragments include, but are not limited to, Fab, scFab, Fab', F(ab')2, Fab'-SH, Fv, single-chain Fv, diabody, triabody, tetrabody, minibody, and single-domain antibody VHH. For a more detailed description of antibody fragments, see: Fundamental Immunology, WE Paul, ed., Raven Press, NY (1993); Shao Rongguang et al. (eds.), Antibody Drug Research and Application, People's Medical Publishing House (2013); Hollinger et al., PNAS USA 90:6444-6448 (1993); Hudson et al., Nat. Med. 9:129-134 (2003).

[0113] The term "constant region" refers to a region on an antibody in which the composition and arrangement order of amino acid residues are relatively stable, including a heavy chain constant region comprising a CH1 region, a hinge region, and an Fc region; and a light chain constant region comprising a CL region.

[0114] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three complementarity determining regions (see, e.g., Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains from antibodies that bind to a specific antigen can be used to isolate antibodies that bind to the antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0115] “T f "" refers to the residues of a polypeptide that can specifically bind to an antibody (such as IgG, IgE, IgM, IgD or IgA) through non-covalent binding. f The specific binding method of the antibody can include specific binding to the constant region of the antibody (including the C H region (including Fc region, CH1 region, hinge region) and / or C L region), variable region (including antibody V H Area V L Region) proteins, polypeptides, antibodies and antibody binding fragments, including but not limited to protein A, protein G, Fc receptors (FcR), other Fc binding peptides, and Fc binding fragments of the above proteins or polypeptides and antibodies or antibody binding fragments that can specifically bind to the constant region and variable region of the antibody. The Fc binding proteins or fragments thereof disclosed in the article published by Weonu Choe et al. in 2016 (Weonu Choe et al., Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides. Materials (Basel). 2016 Dec; 9 (12): 994.) are all introduced into the present disclosure. T f The method of specifically binding to an antibody may also include specifically binding to the variable region of an antibody, for example, by screening antibodies to obtain an antibody or antibody binding fragment that specifically binds to the variable region of an IgG antibody.

[0116] "Protein A," also known as Streptococcal protein A (SPA), refers to a cell wall-anchored surface protein derived from Staphylococcus aureus that enables the bacterium to evade innate and adaptive immune responses. Protein A binds to the Fc portion of immunoglobulins. Prior art, such as protein A and its Fc-binding fragments disclosed in patent documents such as CN106422418A and EP2655404A1, are incorporated into the present disclosure. An exemplary Fc-binding fragment of protein A, such as Z33, has an amino acid sequence as shown in SEQ ID NO:21, which is a fragment of protein A that binds to Fc and is capable of binding to Fc when present alone. Fc-binding peptides such as those shown in SEQ ID NOs:14-16 are other polypeptides capable of binding to Fc.

[0117] Protein G, also known as Streptococcal Protein G (SPG), is a protein isolated from the cell wall and culture supernatant of Streptococci that binds to the Fc region of antibodies. Prior art Protein G and its Fc-binding fragments, such as those disclosed in patents such as US5108894A and EP1054061A1, are incorporated into the present disclosure.

[0118] FcR is a type of cell surface protein that specifically binds to the Fc fragment of an antibody. FcRs that bind to IgG antibodies include FcγR and FcRn.

[0119] The effective amount of the compositions of the present disclosure is the amount of the inhibition of immunogenic substances at least partially blocked by neutralizing antibodies. In some embodiments, the effective amount of the compositions of the present disclosure is enough to suppress neutralizing antibodies at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99% or 99.999% amount. Total immunoglobulin can be total serum immunoglobulin (for example, for systemic administration of the compositions of the present disclosure). Total immunoglobulin can be the total level in local fluid or tissue (for example, for the specific delivery of eyes, ears, lungs, brain, muscles, joints, etc.). Total immunoglobulin can be measured by technology known in the art, for example, by using antibodies in conjunction with immunoglobulin Fc regions or by using protein A or G in conjunction with immunoglobulin to carry out ELISA to serum. In addition, for mice, it is known that its serum contains 5mg / ml to 10mg / ml immunoglobulin. The normal range of serum immunoglobulin in humans is 8-10 mg / ml. For in vivo estimates, the high end of 10 mg / ml can be used to calculate the ratio. Local immunoglobulin content can be estimated based on tissue weight (40 mL serum per 1 kg weight), or the concentration of Ig in a specific body fluid and the volume of the fluid in that organ if the volume of the fluid in that organ is lower than serum (e.g., eye, cerebrospinal fluid).

[0120] The present disclosure f Affinity K for immunoglobulins D Value is less than 1×10 -7 M, 1×10 -8 M, 1×10 -9 M or 1×10 -10 M, or EC 50 <1000nM, 100nM, 10nM or 1nM; Optionally, the T f Binding affinity K of immunoglobulin D Value is less than 1×10 -8 M; or EC 50 <100 nM. In some embodiments, the T a Affinity K for ASGPR and / or M6PR D Value is less than 1×10 -7 M, 1×10 -8 M, 1×10 -9 M or 1×10 -10 M. Optionally, the T a Affinity K for ASGPR and / or M6PR D The value is 1×10 -7 M~1×10 -10 Between M.

[0121] "ASGPR affinity ligand" refers to a ligand that can bind to ASGPR with affinity, including anti-ASGPR antibodies or antigen-binding fragments thereof, asialoglycoprotein, N-acetylgalactosamine (GalNac), triGalNAc, galactose, galactoside, galactosamine, galactan; optionally, the antigen-binding fragment of the anti-ASGPR antibody is selected from VHH or scFv, Fab, or F(ab')2 fragment. For example, ASGPR ligands capable of binding to ASGPR disclosed in CN106471010A, CN105713881A, CN112236169A, US20210324085A1, CN115335081A, CN116234582A, WO2023288033, WO2021155317A, WO2022157626A, WO2021142377A, WO2022192478A, WO2019199634A, and WO2020132100A are all incorporated into the present disclosure. The ASGPR ligands also include: ATACs, AVI-1, AVI-2, and AVI-3. Some of the ligand structures are as follows:

[0122] It should be noted that, for the functional molecule having the structure of formula (I) provided in the present disclosure: T f -LT a (I), when T f and T a When both are peptides or proteins, T f Can be located at the N end of L, accordingly, T a Located at the C-terminus of L; T f It can also be located at the C-terminus of L. Accordingly, T a Located at the N-terminus of L. Similarly, for the functional molecule having the structure of formula (II): T a -L-VHH(Ⅱ), when T a When it is a polypeptide or protein, VHH can be located at the N-terminus or the C-terminus of L.

[0123] Any of the compositions disclosed herein for reducing neutralizing antibodies against immunogenic substances can be administered by injection, infusion, or a combination thereof. A pharmaceutical composition comprising any of the compositions disclosed herein can be administered at a dose of about 1.1 mg / kg to about 750 mg / kg, wherein the dose of the IgG degrading enzyme administered is, for example, about 0.01 to 20 mg / kg, and the dose of the bifunctional molecule capable of binding to an immunoglobulin and an ASGPR is about 1 to 1000 mg / kg, including all values ​​and subranges therebetween.

[0124] The first molecule and the second molecule included in the composition of the present disclosure can be administered to the subject on any schedule. In some embodiments, the first molecule of the present disclosure is administered to the subject before the second molecule is administered. In some embodiments, the second molecule of the composition of the present disclosure is administered to the subject while the immunogenic substance is administered. As used herein, the term "simultaneously" refers to being close enough in time to produce a combined effect (i.e., simultaneously can be simultaneous, or can be two or more events that occur within a short period of time before or after each other).

[0125] The compositions of the present disclosure can be administered to a subject by finding any schedule that effectively blocks the inhibition of the immunogenic substance by neutralizing antibodies. In some embodiments, the compositions of the present disclosure are administered to the subject before the immunogenic substance is administered. In some embodiments, the compositions of the present disclosure are administered to the subject while the immunogenic substance is administered. As used herein, the term "simultaneously" refers to being close enough in time to produce a combined effect (i.e., simultaneously, it can be simultaneous, or it can be two or more events that occur within a short time before or after each other).

[0126] In some embodiments, it may be necessary to administer the immunogenic substance and / or the composition of the present disclosure to the subject more than once to provide a treatment or other beneficial effect. The composition of the present disclosure may be administered, for example, 1, 2, 3, 4 or more times. In some embodiments, each time the immunogenic substance is administered to the subject, the composition of the present disclosure is administered to the subject, for example, in the same manner as described above, for example, before or at the same time as the immunogenic drug is administered. Using the composition of the present disclosure each time the immunogenic drug is administered can inhibit the effect of neutralizing antibodies on the immunogenic drug, which is often a problem when the drug is administered again. In some embodiments, the same composition of the present disclosure is administered each time. In other embodiments, a different composition of the present disclosure is administered each time. Without being bound by theory, it is believed that using a different composition of the present disclosure for each administration can limit the effect of inhibitory antibodies against the composition of the present disclosure, which may occur when the same protein is administered again.

[0127] The capabilities of the disclosed compositions may be advantageously employed in other approaches where inhibition of neutralizing antibodies from binding their antigen is beneficial, for example, where immunosuppression is desired or where excess antibodies are present.

[0128] Another aspect of the present disclosure relates to a method of treating an autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition of the present disclosure, thereby treating the autoimmune disease.

[0129] The term "autoimmune disease" as used herein refers to any disease associated with an autoimmune response caused by antibodies. Examples include, but are not limited to, autoimmune diseases or conditions mediated by pathogenic antibodies (including IgG, IgE, IgM, IgD, or IgA).

[0130] The term "antibody-mediated organ rejection," also known as "antibody-mediated rejection," refers to the immune damage caused by the involvement of multiple humoral immune effectors, such as antibodies and complement. AMR plays a significant pathogenic role in hyperacute, acute, and chronic rejection. It often occurs after organ transplantation (Tx). In some embodiments, antibody-related organ transplant rejection includes, but is not limited to, graft rejection during organ transplantation.

[0131] Unless otherwise indicated, the term "polypeptide" as used herein includes peptides and proteins.

[0132] The term "nucleotide" or "polynucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single or double stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.). Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. In particular, degenerate codon substitutions can be achieved by generating sequences in which position 3 of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al., (1992); Rossolini et al., Mol Cell. Probes 8:91-98 (1994)).

[0133] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description directed to a polypeptide applies equally to describing a peptide and describing a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally encoded amino acid. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked via covalent peptide bonds.

[0134] The term "host cell" means a cell comprising a nucleic acid of the present disclosure, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector, such as a plasmid, or may be integrated into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell.

[0135] The term "transformation" refers to a process by which a heterologous DNA sequence is introduced into a host cell or organism.

[0136] The term "expression" means the transcription and / or translation of an endogenous gene or a transgene in a cell.

[0137] The compositions and methods disclosed herein can be used for veterinary and medical applications. Suitable subjects include mammals. As used herein, the term "mammal" includes, but is not limited to, humans, non-human primates, cattle, sheep, goats, horses, cats, dogs, rabbits, etc. Human subjects include neonates, infants, teenagers, adults, and elderly subjects. In some embodiments, the human subject may be less than 6 months, less than 2 years old, less than 5 years old, less than 10 years old, 10-18 years old, 19-29 years old, 30-35 years old, 36-40 years old, or greater than 40 years old. In representative embodiments, the subject "needs" the methods described herein. The terms "subject" and "patient" are used interchangeably herein to refer to a human subject.

[0138] The terms "treat," "treating," or "treatment of" (and grammatical variations thereof) mean that the severity of a subject's condition is reduced, at least partially improved, or stabilized and / or that at least one clinical symptom is alleviated, relieved, reduced, or stabilized to some extent and / or that the progression of a disease or disorder is delayed.

[0139] As used herein, the term "amino acid" includes any naturally occurring amino acid, modified forms thereof, and synthetic amino acids.

[0140] Furthermore, non-natural amino acids can be "unnatural" amino acids (as described in Wang et al., Annu Rev Biophys Biomol Struct. 35:225-49 (2006)). These unnatural amino acids can be advantageously used to chemically link the target molecule to the AAV capsid protein.

[0141] The term "domain" is intended to encompass a portion of a protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Domains are able to form compact three-dimensional structures and are generally independently stable and foldable. A domain can occur in a variety of evolutionarily related proteins. The length of a domain varies from about 25 amino acids to about 500 amino acids. A "domain" can also include a domain from a wild-type protein that has one or more amino acid residues replaced by conservative substitutions. Because they are self-stabilizing in the protein environment, domains can be "exchanged" between one protein and another to form chimeric proteins through genetic engineering.

[0142] The term "gene therapy" refers to a method for changing the expression of endogenous genes by exogenous administration of genes. As used herein, "gene therapy" also refers to replacing the defective gene encoding the defective protein or replacing the missing gene by introducing a functional gene corresponding to the defective or missing gene into the somatic cells or stem cells of an individual in need. Gene therapy can be accomplished by an ex vivo method, in which differentiated or stem cells are removed from the individual's body and a normal copy of the defective gene is subsequently introduced into the explanted cells using a viral vector as a gene delivery vehicle. In addition, direct gene transfer technology in vivo allows the use of a wide range of viral vectors, liposomes, protein-DNA complexes or naked DNA to transfer genes in situ into the cells of an individual to achieve a therapeutic effect. The term "gene therapy" also refers to replacing the defective gene encoding the defective protein by introducing a polynucleotide whose function is substantially the same as that of the defective gene or protein when it is not defective into the somatic cells or stem cells of an individual in need.

[0143] The term "gene editing" refers to the insertion, deletion, or replacement of DNA at a specific site in the genome of an organism or cell. Gene editing can be performed using one or more targeted nuclease systems, such as PRIME, the CRISPR / Cas system, the CRISPR / Cpf1 system, Zn finger nucleases, TALENs, homing endonucleases, and others.

[0144] In some embodiments, compositions disclosed herein also include at least one pharmaceutically acceptable carrier, excipient and / or medium, for example, solvent, buffer, solution, dispersion medium, coating, antibacterial and antifungal agent, isotonic agent and absorption delay agent. In some embodiments, pharmaceutically acceptable carrier, excipient and / or medium may include saline, buffered saline, glucose, water, glycerol, sterile isotonic aqueous buffer, phosphate buffer solution, amino acid buffer, bicarbonate buffer solution and combination thereof. In some embodiments, pharmaceutically acceptable carrier, excipient and / or medium include phosphate buffered saline, sterile saline, lactose, sucrose, calcium phosphate, dextran, agar, pectin, peanut oil, sesame oil, medical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) or its suitable mixture. In some embodiments, compositions disclosed herein also include a small amount of emulsifying or wetting agent or pH buffer. Formulations of the compositions disclosed herein can be prepared for storage by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions, or suspensions.

[0145] In some embodiments, the composition is in solid form such as a lyophilized powder, liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder suitable for reconstitution. In some embodiments, the composition can be formulated for delivery using liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, liposomes, nanospheres, nanoparticles, etc.

[0146] Provided herein are methods for reducing neutralizing antibodies against an immunogenic substance in a subject, comprising administering to the subject a therapeutically effective amount of a recombinant biological or pharmaceutical entity and any one of the compositions disclosed herein that promotes degradation or reduction of antibodies against the immunogenic substance.

[0147] In some embodiments, a method of reducing the amount of neutralizing antibodies against a recombinant adeno-associated viral (AAV) vector in a subject comprises administering to the subject a therapeutically effective amount of a composition that promotes degradation of neutralizing antibodies.

[0148] In some embodiments, the neutralizing antibodies to be reduced and / or degraded include IgG, IgM, IgE, IgD and / or IgA. In some embodiments, the anti-drug antibodies include IgG.

[0149] Another aspect of the present disclosure provides a kit for reducing and / or eliminating neutralizing antibodies and / or immunoglobulins against an immunogenic substance in a subject, the kit comprising, consisting of, or consisting essentially of any one of the compositions described herein, the means for administering the composition, and instructions for use.

[0150] Another aspect of the present disclosure provides an IgM affinity matrix containing VHHs, comprising a resin matrix coupled to the VHHs described in the aforementioned embodiments. For example, the resin matrix can be a cross-linked styrene-divinylbenzene polymer. Optionally, the coupling method includes epoxide coupling.

[0151] In one embodiment, the IgM affinity matrix is ​​a granular filler, and the particle size of the particles is 10 to 100 μm, including but not limited to 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm or 100 μm.

[0152] In one embodiment, the IgM affinity matrix is ​​a slurry filler containing resin particles, and the slurry solvent may be ethanol.

[0153] In one embodiment, the IgM affinity matrix is ​​used to adsorb IgM antibodies in plasma or serum.

[0154] In one embodiment, after the IgM affinity matrix adsorbs the IgM antibody, the IgM antibody can be recovered by acid elution; optionally, the eluent for the acidic elution can be a glycine solution, and further optionally, the concentration of the glycine solution can be 0.01 to 1 M, including but not limited to 0.01 M, 0.015 M, 0.02 M, 0.025 M, 0.03 M, 0.035 M, 0.04 M, 0.045 M, 0.05 M, 0.06 M, 0.07 M, 0.08 M, 0.10 M, 0.11 M, 0.12 M, 0.13 M, 0.14 M, 0.15 M, 0.16 M, 0.17 M, 0.18 M, 0.19 M, 0.20 M, 0.21 M, 0.22 M, 0.23 M, 0.24 M, 0.25 M, 0.26 M, 0.27 M, 0.28 M, 0.29 M, 0.30 M, 0.31 M, 0.32 M, 0.33 M, 0.34 M, 0.35 M, 0.36 M, 0.37 M, 0.38 M, 0.39 M, 0.40 M, 0.41 M, 0.42 M, 0.43 M, 0.44 M, 0.45 055M, 0.06M, 0.065M, 0.07M, 0.075M, 0.08M, 0.085M, 0.09M, 0.095M, 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0.95M or 1M.

[0155] In one embodiment, the present disclosure provides a container containing the above-mentioned IgM affinity matrix. Those skilled in the art can select the material and shape of the container according to actual needs, such as a glass bottle, a plastic bottle or a resin bottle. The capacity of the container can be 1 ml to 2000 ml, including but not limited to 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, 200 ml, 300 ml, 400 ml, 500 ml, 600 ml, 700 ml, 800 ml, 900 ml, 1000 ml, 1200 ml, 1400 ml, 1600 ml, 1800 ml or 2000 ml.

[0156] The advantage of the technical solution provided by the present disclosure is that it provides a pharmaceutical composition that can further degrade the level of immunoglobulins in the body, such as neutralizing antibodies or autoimmune antibodies, by combining immunoglobulin degrading enzymes with functional molecules that simultaneously target and bind to immunoglobulins and ASGPRs, thereby solving clinical problems caused by the possible or existing neutralizing antibodies against gene therapy vectors or anti-drug antibodies / autoimmune antibodies against autoimmune diseases / organ transplants.

[0157] The beneficial effects of the present disclosure are at least that: IgG degrading enzyme alone cannot immediately eliminate the impact of neutralizing antibodies or autoimmune antibodies in the body on treatment, the decline in the titer of neutralizing antibodies in the patient's body is slow, and the body will also produce new neutralizing antibodies. For AAV administration, the above performance will affect the efficacy of gene therapy, especially for patients with high titer neutralizing antibodies or autoimmune antibodies in the body. The solution provided by the present disclosure can quickly reduce the level of immunoglobulins in the body, further reduce the titer of neutralizing antibodies or autoimmune antibodies, further eliminate residual or newly generated neutralizing antibodies or autoimmune antibodies, significantly inhibit the occurrence of undesirable immune responses, and reduce or even eliminate the negative impact on the "immunogenic substance" that needs to be administered to the subject.

[0158] II. Specific Examples

[0159] The technical solutions of the present disclosure are further illustrated below by specific embodiments. Those skilled in the art should understand that the embodiments described herein are merely intended to aid understanding of the present disclosure and should not be construed as limiting the present disclosure. Experimental methods in the following examples, where specific conditions are not specified, were performed according to conventional methods and conditions, or as selected from commercial product specifications.

[0160] Example 1. Protein construction, expression and preparation

[0161] According to the amino acid sequence in Table 1, SEQ ID NO: 14-16, 19-20, 27-36 were synthesized after codon optimization, and the corresponding light chain or heavy chain plasmid was obtained by plasmid extraction. The plasmid DNA containing the target protein coding sequence was transfected into ExpiCHO-S cell line (Gibco) using the transient transfection kit of The transfection kit instructions were followed. Cell lines expressing the various target proteins were obtained. Each cell line was expanded to a volume of 100 ml using CD2 basal medium and cultured in a fed-batch format. Starting on day 3, 2% Feed4 was added daily for 12 days of suspension culture. The culture supernatants were collected and protein samples were purified. Purified samples were sterile-filtered using a 0.22 μm membrane and stored at 2–8°C.

[0162] After codon optimization, polynucleotide sequences corresponding to SEQ ID NOs: 1-36 were synthesized and a C-terminal 6×histidine tag was added. After sequence synthesis, the sequences were inserted into the pET32a expression vector. After correct sequencing, recombinant plasmids for expression were obtained. The mutant recombinant plasmids were electroporated into Escherichia coli BL21 Star (DE3) and plated on LB agarose plates containing 100 μg / ml ampicillin. The plates were cultured overnight at 37°C until colonies emerged. A single colony was selected and inoculated into 3 ml of LB medium containing 100 μg / ml ampicillin and incubated overnight at 37°C at 250 rpm. 500 μl of the overnight culture was inoculated into 50 ml of LB medium containing 100 μg / ml ampicillin. After incubation at 37°C for 4 hours, 0.1 mM IPTG was added for induction, and the culture was continued overnight. The supernatant of the overnight induction culture was collected by centrifugation and purified by Ni affinity chromatography to obtain a protein sample. After purification, the samples were sterilized by filtration using a 0.22 μm membrane and stored at 2–8 °C.

[0163] Table 1. Amino acid sequences of enzymes, peptides, and fusion proteins

[0164] Example 2. In vitro affinity detection of functional molecules 1, 6, and 7

[0165] The BLI method was used to test the affinity of functional molecules 1, 6, and 7 for the target protein IVIg. The detection process is as follows: 5 μg / ml IVIg was immobilized using the proL sensor (approximately 0.5 nm). Then, the binding and dissociation of functional molecules 1, 6, and 7 at concentrations of 500, 250, 125, 62.5, and 31.25 nM were measured. The final affinity data between different functional molecules and IVIg were calculated and statistically analyzed as follows:

[0166] Table 2 Affinity between functional molecules and IVIg

[0167] The results showed that functional molecule 1 has extremely high affinity. The characteristic of this molecule is that it does not dissociate after binding, and the dissociation constant can reach 3.26E-07; functional molecule 6 has relatively high affinity, and the dissociation constant can reach 3.07E-09; functional molecule 7 has weaker affinity than functional molecules 1 and 6, with a dissociation constant of 4.81E-08. This molecule is characterized by fast dissociation, and the dissociation constant is only 1.51E-03.

[0168] The affinity of functional molecules 1, 6, and 7 for binding to ASGPR was detected using BLI. The detection process was as follows: 5 μg / ml Fc-ASGPR was immobilized using proL (approximately 0.5 nm). Then, 300, 150, 75, 32.5, and 16.3 nM of functional molecules 1, 6, and 7 were bound and dissociated. Finally, the affinity data of different functional molecules to mouse ASGPR were calculated and statistically analyzed as follows:

[0169] Table 3 Affinity of functional molecules to mouse ASGPR

[0170] *: The detection method may cause non-specific binding and inaccurate test results

[0171] The results showed that functional molecules 6 and 7 bind well to ASGPR and human IgG. The affinity of functional molecule 1 was too high due to its ability to bind to the Fc structure of the Fc-ASGPR molecule. However, it is estimated that functional molecules 1, 6, and 7 all bind to ASGPR at approximately 1E-08.

[0172] Example 3. Detection of binding activity of functional molecule 5

[0173] To test the differential binding of functional molecule 5 to human and mouse IgM, plates were coated with either human or mouse IgM. Different concentrations of functional molecule 5 were then added and tested using anti-his-HRP antibody. The results are shown in the table below. The data show that the binding affinity for human IgM is 100-fold higher than that for mouse IgM, suggesting that the degradation effect on mouse IgM is weaker. (Figure 1, a)

[0174] Table 4 Binding differences of functional molecule 5 to human and mouse IgM

[0175] Comparison of the binding activity of functional molecules 1 and 5 to mouse ASGPR: Mouse ASGPR-his was diluted to 2 μg / ml in coating buffer, and 100 μl / well was added to a 96-well microtiter plate and coated overnight at 2-8°C. After blocking, 100 μl / well of diluted functional molecule 5 or functional molecule 1 was added and incubated at 37°C for 1 hour. Both protein concentrations started at 100 μg / ml. After washing, 2 μg / ml of diluted human IgM or human IVIG was added and incubated at 37°C for 1 hour. After washing, an enzyme-linked secondary antibody (HRP-anti-Human IgM (μ)) was added to recognize the μ chain of human IgM, or an enzyme-linked secondary antibody (HRP-anti-Human κ) was added to recognize human IVIG. The plate was incubated at 37°C for 1 hour before colorimetric reading.

[0176] The results showed that functional molecule 5 could bind well to ASGPR and human IgM, while functional molecule 1 could bind well to ASGPR and human IgG.

[0177] (Figure 1)

[0178] Example 4 In vitro activity data

[0179] Incycute and flow cytometry data showed no targeted degradation of functional molecule 7. Trastuzumab was pre-labeled with CY5 as a fluorescent detection antibody. Then, varying concentrations of trastuzumab-CY5 were mixed with 50 nM of the functional protein molecule and incubated with HepG2 cells for 24 hours. Finally, flow cytometry was used to detect the intracellular fluorescence signal.

[0180] The results showed that, as shown in FIG2 , 50 nM of functional molecules 1 (a) and 6 (b) could effectively internalize the trastuzumab-cy5 antibody, while functional molecule 7 (c) could not internalize the trastuzumab antibody.

[0181] Example 5. Degradation of human IgG by functional molecule 1 in mice

[0182] 10-week-old healthy C57BL / 6 female mice were intraperitoneally injected with 200 mg / kg of IVIg 16 hours beforehand. The next day, blood was collected 15 minutes beforehand as the starting concentration of IVIg. Following this, different doses of functional protein 1 (1 times the molar dose of IVIg: 55.6 mg / kg; 10 times the molar dose of IVIg: 166.8 mg / kg; PBS was used as a negative control) were intraperitoneally injected. 50 μl of mouse serum was collected 1, 2, 4, and 8 hours after administration and subsequently analyzed by ELISA.

[0183] Dilute anti-human IgGγ at a dilution of 1:1000 with coating solution, add 100μl / well to the ELISA plate, and coat overnight at 2-8°C. Block at 37°C for 2 hours. After washing, add 100μl / well of the gradient diluted standard and the sample to be tested. The standard concentration starts at 200ng / ml and is diluted 2-fold with a total of 10 concentration points, including a 0 concentration point. The serum sample to be tested is diluted 100,000 times and added to the ELISA plate at a dilution of 100μl / well. Incubate at 37°C and 300rpm for 1 hour. After washing, add the enzyme-labeled secondary antibody diluted at 1:5000. Incubate at 37°C and 300rpm for 1 hour. After washing, develop the color for 4-5 minutes and terminate the detection.

[0184] The results showed that functional molecule 1 could significantly eliminate IVIg in the body in a dose-dependent manner (as shown in FIG3 ).

[0185] Example 6. Degradation of human IgM by functional molecule 5 in mice

[0186] Ten-week-old healthy C57BL / 6 female mice were injected intravenously with human IgM 15 minutes before administration. Functional molecule 5 was then administered intravenously at 0:00. The IgM concentration at administration was approximately 1.5 g / L. The doses of functional molecule 5 were 3, 9, and 30 mg / kg, respectively. The molar ratios of functional molecule 5 to human IgM were approximately 1:1, 3:3, and 10:1, respectively. Approximately 50 μl of mouse serum was collected 15, 30, 1, 2, and 4 hours after administration and subsequently analyzed by ELISA.

[0187] Human IgM antigen (L4) was diluted to 1 μg / ml using coating solution, and 100 μl / well was added to the ELISA plate and coated overnight at 2-8°C. After blocking and washing, a gradient dilution of the standard (human IgM) was added. The standard was prepared at a concentration of 2000 ng / ml and diluted 3-fold to a total of 10 concentration points, including a concentration point of 0. 100 μl / well was added to the ELISA plate. After incubation and washing, the enzyme-labeled secondary antibody HRP-Anti-Human IgM (μ) was added at a dilution of 1:10,000 in the coating solution diluted at 2 μg / ml. After incubation and washing, the plate was colorimetrically detected. As shown in Figure 4, functional molecule 5 can effectively degrade human IgM. However, due to the short half-life of IgM in mice, the degradation of functional molecule 5 can only be clearly distinguished within 0.25 to 1 hour.

[0188] Example 7. IdeE-mut degrades IVIg in vivo to reduce low- to medium-level neutralizing antibody titers

[0189] The safety and tolerability, pharmacokinetic profile, pharmacodynamic profile, and immunogenicity of IdeE-mut in humans were evaluated. A total of 68 healthy subjects were enrolled in the study, with specific groups shown in Table 5. All subjects completed the study. The results of the Phase I clinical trials conducted in China and New Zealand were consistent, demonstrating that IdeE-mut was safe and well-tolerated in healthy subjects. The PK profile of IdeE-mut was well-characterized across all dose groups, conforming to a two-compartment model, with an optimal dose response achieved at a dose of 0.25 mg / kg. IdeE-mut efficiently, rapidly, and specifically cleaves human IgG. A 0.25 mg / kg dose of IdeE-mut cleaves over 90% of IgG within 45 minutes of administration, and IgG levels remain low for one week (average reduction of over 70%). The prevalence and titer of pre-existing anti-IdeE-mut antibodies were both low, demonstrating a significant clinical advantage over the comparable foreign product, Imlifidase. As shown in Figure 5, the use of IdeE-mut improves clinical safety and efficacy.

[0190] Furthermore, as shown in Figure 6, before dosing, the number of volunteers from both countries with pre-existing antibodies to IdeE-mut (Figure 6, left panel, sample population used in this clinical trial) was far lower than the number of volunteers with pre-existing antibodies to IdeS (anti-IdeS) (Figure 6, right panel, sample population from the Phase I clinical trial of Imlifidase). Two weeks and two months after IdeE-mut administration, Chinese volunteers showed a wider range of ADA titers in the 0.25 mg / kg and 0.40 mg / kg dose groups than New Zealand volunteers. There were no significant differences in the median and range of ADA titers between participants from both countries before IdeE-mut administration and one week and six months after administration. Most volunteers in both studies began to experience changes in ADA levels around day 14 after IdeE-mut administration, peaked around two weeks, and then gradually declined. After six months of IdeE-mut administration, ADA levels returned to baseline in 56.86% (29 / 51) of the volunteers (Figures 7a and 7b).

[0191] Table 5 Clinical trial design

[0192] Thirty-two subjects from China were selected to detect the pre-existing titer of AAV neutralizing antibodies (Nab) in the subjects' sera, defined as 100%. The AAV neutralizing antibody titer and total IgG, as well as the titer of F(ab')2 derived from IgG, were then detected 6 hours, 2 days, 3 days, 4 days, 7 days, 14 days, and 21 days after administration of IdeE-mut to explore the optimal time window for AAV gene therapy after administration of IdeE-mut. This study selected AAV2 and AAV8 serotypes as indicators for detecting neutralizing antibody titers. These two serotypes are widely used in gene therapy and are known to have relatively high pre-existing neutralizing antibodies in more than 50% of the population.

[0193] By cleaving IgG into F(ab')2 and Fc fragments, IdeE-mut reduced serum levels of total IgG, AAV2, and AAV8 neutralizing antibodies (Nab) by over 90% (Figures 8a-8c). The initial pre-existing AAV2 neutralizing antibody titers for all subjects in the 0.25 mg / kg and 0.40 mg / kg AAV2 serotype groups were divided into two groups (>1:1000, 5 subjects; 1:100-1:1000, 4 subjects) to further analyze the ability of IdeE-mut to reduce neutralizing antibody levels. The results are shown in Figure 9.

[0194] To assess how IdeE-mut altered AAV2 antibody titers and the potential for a second dose, this example subsequently tested changes in F(ab')2 in these nine volunteers ( Figure 10 ). IdeE-mut's degradation of IgG led to rapid production of F(ab')2. F(ab')2 was gradually metabolized and nearly eliminated by day 7. The trends in AAV8 antibody titers mirrored those for AAV2 antibodies, as shown in Figure 11 .

[0195] The changes in serum AAV neutralizing antibodies (Nab) were compared after administration of IdeE-mut to AAV2 and AAV8 serotype samples with different neutralizing antibody titers. The results are shown in Figure 12. A single administration of IdeE-mut can effectively and significantly reduce the neutralizing antibodies of AAV2 and AAV8. With the Nab cutoff value of AAV2 at 1:100, a single administration of IdeE-mut can reduce the neutralizing antibody titer of 1:1000 or less to below the cutoff value. With the Nab cutoff value of AAV8 at 1:5, a single administration of IdeE-mut can reduce the neutralizing antibody titer of 1:50 or less to below the cutoff value.

[0196] Example 8. Changes in IgG degradation levels in vivo by IdeE-mut and functional molecule 6

[0197] The experiment used a strain of immunodeficient (SCID) mice, which do not contain mouse IgG. IVIg was administered 16 hours in advance, so that the IVIg concentration in the blood reached above 4g / L at 0 hours. To verify the two-dose regimen for complete IVIg elimination, the first dose was 3mg / kg IdeE-mut, corresponding to a clinical dose of 0.25mg / kg. The second dose was designed to be a high or low dose of functional molecule 6, 20mg / kg and 50mg / kg, respectively. The first dose was administered at 0 hours, and the second dose was administered 8 hours after the first dose. Sampling time points were -15 minutes, 7.5 hours, 10 hours, 12 hours, and 24 hours. The changes in IVIg concentration in the blood were determined by ELISA.

[0198] Table 6 Experimental design table

[0199] ELISA method for IVIg detection: Dilute anti-Human IgGγ at a 1:1000 dilution using coating solution, add 100 μl / well to the ELISA plate, and coat overnight at 2-8°C. Block at 37°C for 2 hours. After washing, add 100 μl / well of serially diluted standards and test samples. The standard concentration starts at 100 ng / ml and is serially diluted 2-fold for a total of 8 concentration points. After diluting the test serum sample 20,000-200,000 times, add 100 μl / well to the ELISA plate. Incubate at 37°C, 300 rpm for 1 hour. After washing, add enzyme-linked secondary antibody at a 1:5000 dilution. Incubate at 37°C, 300 rpm for 1 hour. After washing, stop color development and perform detection.

[0200] The results are shown in Figure 13. IdeE-mut can significantly reduce IVIg in mice, and the combination of functional molecules 6 can further reduce IVIg in the body.

[0201] Example 9 ProAnM in vitro experiment

[0202] ProAnM was diluted to 2 μg / ml in coating buffer and added to a 96-well microtiter plate for overnight coating. After washing, blocking buffer was added and incubated at 37°C for 2 hours. After washing, monkey serum samples were diluted 10-fold, 100-fold, 1000-fold, and 10,000-fold in PBST, added to the 96-well microtiter plate, and incubated at 37°C.

[0203] Dilute the enzyme-conjugated secondary antibody, anti-Human IgM(μ)HRP, in PBST and add to the sample. Incubate at 37°C, 300 rpm for 1 hour. Discard any liquid in the wells and wash the plate with PBST. Add the colorimetric solution and develop at room temperature in the dark for an appropriate time. Add the stop solution to terminate the colorimetric reaction and vortex to mix. Place the plate in a microplate reader and read at a dual wavelength of 450 nm as the detection wavelength and 630 nm as the reference wavelength.

[0204] Table 7 ProAnM ELISA detection binding

[0205] Example 10. IdeE-mut degrades IVIg in vivo to reduce low- to medium-level neutralizing antibody titers

[0206] After intraperitoneal injection of IVIg into mice, the neutralizing antibody titer was approximately 1:160 (this does not represent samples with high neutralizing antibody titers (CN112689515A)). Human neutralizing antibody Nab (neutralizing antibody) IgG1 was purchased and mixed with IVIg at different ratios for research.

[0207] C57BL / 6 mice were used to evaluate the effect of IgG degrading enzymes on AAV9 infection in vivo. Human IVIg with different neutralizing antibody titers was intraperitoneally injected into C57BL / 6 mice, with 6 mice per group. On day -1, human IVIg was injected intraperitoneally at a dose of 1g / kg. 24 hours after human IVIg injection, on day 0, IdeE-mut protease was injected intravenously at a dose of 5mg / kg to 20mg / kg. On day 1, AAV9-Luc was injected at a dose of 5×10 10 vg / mouse, and mouse fluorescence was monitored on days 6 and 10. IdeE-mut effectively cleaved IVIg in mice, cleaving over 90% of IVIg within 2 hours of administration. However, IdeE-mut only reduced low- to medium-titer neutralizing antibodies to lower levels, not high-titer neutralizing antibodies.

[0208] Table 8. Experiment on IdeE-mut degradation of IVIg in vivo

[0209] Detection of IVIg content in serum: dilute anti-human IgGγ at a dilution of 1:1000 with coating solution, add 100μl / well to the ELISA plate, and coat overnight at 2-8℃. Block at 37℃ for 2 hours. After washing, add 100μl / well of gradient diluted standard and test sample. The standard concentration starts from 200ng / ml and is diluted 2-fold with a total of 10 concentration points, including a 0 concentration point. The serum sample to be tested is diluted 10,000 times, and 100μl / well is added to the ELISA plate. Incubate at 37℃ and 300rpm for 1 hour. After washing, add enzyme-labeled secondary antibody diluted at 1:5000. Incubate at 37℃ and 300rpm for 1 hour. After washing, color development for 4-5 minutes to terminate the detection.

[0210] Example 11. Effect of combined use of IdeE-mut and functional molecule 1 fusion protein on AAV infection in vivo

[0211] C57BL / 6 mice were used to evaluate the effect of IgG degrading enzyme on AAV9 infection in vivo. A mixture of human IVIg and human Nab IgG1 with different neutralizing antibody titers was intraperitoneally injected into C57BL / 6 mice at a dose of 1g / kg, with 6 mice in each group. Human IVIg was injected intraperitoneally 2 days in advance at a dose of 1g / kg; one day in advance (Day-1), IdeE-mut protease was intravenously injected into mice at a dose of 5-20mg / kg. Different doses of functional molecule 1 were injected on day 0 (Day 0). On day 1, AAV9-Fluc was injected at a dose of 5×10 10 vg / mouse, and mouse fluorescence was monitored on days 6 and 10. Administration of either IgG degrading enzyme or functional molecule 1 alone failed to degrade high-titer neutralizing antibodies. However, administration of IgG degrading enzyme combined with functional molecule 1 further effectively eliminated neutralizing antibodies within 24 hours of functional molecule 1 administration, eliminating the interference of high-titer neutralizing antibodies with AAV9 viral infection.

[0212] Table 9. Dosage regimen

[0213] Example 12. Effect of combined use of IdeE-mut and peptide3-4F3-peptide3 fusion proteins on AAV infection in vivo

[0214] C57BL / 6 mice were used to evaluate the effect of IgG degrading enzyme on AAV9 infection in vivo. A mixture of human IVIg and human Nab IgG1 with different neutralizing antibody titers was intraperitoneally injected into C57BL / 6 mice at a dose of 1g / kg, with 6 mice in each group. Human IVIg was injected intraperitoneally 2 days in advance at a dose of 1g / kg; one day in advance (Day-1), IdeE-mut protease was intravenously injected into mice at a dose of 5-20mg / kg. Different doses of peptide3-4F3-peptide3 were injected on Day 0. On Day 1, AAV9-Flu was injected at a dose of 5×10 10 vg / mouse, and the mouse fluorescence and liver gene copy number were measured on days 6 and 10. Administration of either IgG degrading enzyme or peptide3-4F3-peptide3 alone failed to degrade high-titer neutralizing antibodies. However, administration of IgG degrading enzyme combined with peptide3-4F3-peptide3 further effectively eliminated neutralizing antibodies within 24 hours of peptide3-4F3-peptide3 administration, eliminating the interference of high-titer neutralizing antibodies with AAV9 viral infection.

[0215] Table 10. Dosage regimen

[0216] Example 13. Re-dose study in cynomolgus monkeys

[0217] To evaluate the ability of IdeE-mut and peptide3-4F3-peptide3 combination to degrade or reduce the effects of NAbs on AAV capsids in a large animal model, a study was conducted in cynomolgus monkeys (Macaca fascicularis). The monkeys were first screened for the presence of pre-existing NAbs against AAV capsids. NAb-positive animals may be due to exposure to naturally occurring AAV in the wild or in group settings. Animals were grouped based on negative or positive NAb titers, and if positive, they were grouped based on how high the pre-existing NAb titer was.

[0218] First, cynomolgus monkeys were injected with 2×10 12 vg / kg AAV virus. After the generation of NAb titers, animals are infused with single or multiple ascending doses of 0, 5 and 20 mg / kg (and higher doses) of IdeE-mut by intravenous, subcutaneous, intraperitoneal or other routes of administration within 3-5 weeks of vector infusion. Different doses of peptide3-4F3-peptide3 are given 24 hours or 48 hours after IdeE-mut administration, and animals are followed up by measuring anti-AAV capsid IgG and / or NAbs against AAV. When neutralizing antibody titers are sufficiently reduced, they are infused with 2×10 12 vg / kg AAV-Flu virus. Following transduction, in addition to measuring NAb production against AAV capsids using anti-AAV IgG ELISA or cell-based NAb assays, animals are monitored for gene copy number and fluorescence to determine the achieved transduction level. Combining these assays can better eliminate neutralizing antibodies.

[0219] Although the specific embodiments of the present disclosure have been described above, those skilled in the art will appreciate that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present disclosure. Therefore, the scope of protection of the present disclosure is defined by the appended claims.

Claims

1. A pharmaceutical composition, wherein, After administration of the pharmaceutical composition, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, at least 99.5%, 99.9%, 99.99%, or at least 99.999% of the immunoglobulins in the subject are degraded.

2. A pharmaceutical composition, wherein, After administration of the pharmaceutical composition, the titer of neutralizing antibodies and / or autoantibodies present in the subject's body is less than about 1:10000, or less than about 1:1000, or less than about 1:100, or less than about 1:50, or less than about 1:40, or less than about 1:30, or less than about 1:20, or less than about 1:10, or less than about 1:5, or less than about 1:

2.

3. The pharmaceutical composition according to claim 1 or 2, which comprises a first molecule and a second molecule, wherein the first molecule is capable of degrading immunoglobulins, and the second molecule is a functional molecule capable of binding immunoglobulins and lysosomal targeting receptors; Optionally, the immunoglobulin is a pre-existing antibody in the subject, a drug-resistant antibody caused by administration of an immunogenic substance, or an autoantibody causing an autoimmune disease in the subject.

4. The pharmaceutical composition according to claim 3, wherein, The first molecule is an immunoglobulin-degrading enzyme; Optionally, the immunoglobulin-degrading enzyme comprises an IgG-degrading enzyme, an IgE-degrading enzyme, an IgM-degrading enzyme, an IgD-degrading enzyme, or an IgA-degrading enzyme.

5. The pharmaceutical composition according to claim 3 or 4, wherein, The immunoglobulins include IgG, IgE, IgM, IgD, or IgA; Optionally, the immunoglobulin is IgG.

6. The pharmaceutical composition according to any one of claims 3 to 5, wherein, The lysosomal targeting receptors include ASGPR and / or M6PR; Optionally, the lysosomal targeting receptor is ASGPR.

7. The pharmaceutical composition according to any one of claims 3 to 6, wherein, The second molecule has the structure of formula (I): T f -L-T a (I); Among them, the T f is an immunoglobulin-binding ligand, and T a is an ASGPR and / or M6PR affinity ligand, and L is a linker.

8. The pharmaceutical composition according to claim 7, wherein, The said T f has an affinity K D with immunoglobulin less than 1×10 -7 M, 1×10 -8 M, 1×10 -9 M or 1×10 -10 M, or an EC 50 <1000 nM, 100 nM, 10 nM or 1 nM.

9. The pharmaceutical composition according to claim 7 or 8, wherein, The said T f combined with the affinity K of the immunoglobulin D value is less than 1×10 -8 M; Or EC 50 < 100 nM.

10. The pharmaceutical composition according to any one of claims 7 to 9, wherein, The T a affinity K with ASGPR and / or M6PR D value is less than 1×10 -7 M, 1×10 -8 M, 1×10 -9 M or 1×10 -10 M.

11. The pharmaceutical composition according to any one of claims 7 to 10, wherein The said T a has an affinity K D value with ASGPR and / or M6PR in the range of 1×10 -7 M to 1×10 -10 M.

12. The pharmaceutical composition according to any one of claims 7 to 11, wherein The said T f is a polypeptide or its fragment that specifically binds to IgG, IgE, IgM, IgD or IgA; Optionally, the polypeptide specifically binds to the constant region or variable region of IgG, IgE, IgM, IgD, or IgA; Optionally, the polypeptide is selected from Protein A, Z33, Protein G, FcR, or an antibody or an antigen-binding fragment thereof that specifically binds to the constant region or variable region of IgG, IgE, IgM, IgD, or IgA; Optionally, the antigen-binding fragment of the antibody that specifically binds to IgG, IgE, IgM, IgD, or IgA comprises VHH, scFv, affibody, Fab, or F(ab')2; The said T a comprises a polypeptide or a fragment thereof that specifically binds to ASGPR, asialoglycoprotein, N-acetylgalactosamine (GalNac), triGalNAc, galactose, galactoside, galactosamine or a residue of galactan; Optionally, the polypeptide that specifically binds to ASGPR is an anti-ASGPR antibody or an antigen-binding fragment thereof; Optionally, the antigen-binding fragment of the anti-ASGPR antibody comprises VHH, scFv, affibody, Fab, or F(ab')2 fragment; Optionally, the ASGPR affinity ligand comprises: of residue; The L is a bond, a peptide linker, or a non-peptide linker.

13. The pharmaceutical composition according to any one of claims 3 to 12, wherein, The first molecule is selected from the group consisting of at least one immunoglobulin-degrading enzyme shown in SEQ ID NO: 1 to 13.

14. The pharmaceutical composition according to any one of claims 7 to 13, wherein The said T f Selected from any one of the following: (a) HFC-1, FcRn, Protein A, Protein G, an immunoglobulin constant region-binding peptide, or Z33; (b) The mutant of (a); (c) A fragment derived from (a) or (b); Optionally, the T f comprises a polypeptide as shown in any one of SEQ ID NOs: 19 to 29.

15. The pharmaceutical composition according to any one of claims 7 to 14, wherein, The T a is selected from polypeptides comprising any one of the polypeptides shown in SEQ ID NO: 14 to 16.

16. The pharmaceutical composition according to any one of claims 7 to 15, wherein, The T of the second molecule a selected from the polypeptides shown in any one of SEQ ID NO: 14 to 16, the T of the second molecule f comprises the polypeptides shown in any one of SEQ ID NO: 19 to 29, and the L comprises the functional molecule shown in SEQ ID NO: 17 or 18; Optionally, the second molecule is selected from polypeptides comprising any one of SEQ ID NOs: 30 to 36.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein, The first molecule is selected from immunoglobulin-degrading enzymes comprising any one of SEQ ID NOs: 1 to 13; the second molecule is selected from polypeptides comprising any one of SEQ ID NOs: 30 to 36.

18. The pharmaceutical composition according to any one of claims 1 to 17, further comprising a pharmaceutically acceptable carrier.

19. The pharmaceutical composition according to any one of claims 1 to 18, further comprising a therapeutic immunogenic substance, the therapeutic immunogenic substance comprising a viral vector or a non-viral vector; for example, an adeno-associated virus (AAV) vector.

20. A functional molecule having the structure of formula (I): T f -L-T a (I) wherein T f is an immunoglobulin-binding ligand, T a is an ASGPR and / or M6PR affinity ligand, and L is a linker.

21. The functional molecule according to claim 20, wherein The T f has an affinity K D value less than 1×10 -7 M, 1×10 -8 M, 1×10 -9 M or 1×10 -10 M, or an EC 50 <1000 nM, 100 nM, 10 nM or 1 nM.

22. The functional molecule according to claim 20 or 21, wherein The said T f Combined with the affinity K of immunoglobulin D The value is less than 1×10 -8 M; Or EC 50 < 100 nM.

23. The functional molecule according to any one of claims 20 to 22, wherein, The T a affinity K with ASGPR and / or M6PR D value is less than 1×10 -7 M, 1×10 -8 M, 1×10 -9 M or 1×10 -10 M.

24. The functional molecule according to any one of claims 20 to 23, wherein, The T a affinity K with ASGPR and / or M6PR D value is between 1×10 -7 M and 1×10 -10 M.

25. The functional molecule according to any one of claims 20 to 24, wherein The said T f is a polypeptide or its fragment that specifically binds to IgG, IgE, IgM, IgD or IgA; Optionally, the polypeptide specifically binds to the constant region or variable region of IgG, IgE, IgM, IgD or IgA; Optionally, the polypeptide is selected from Protein A, Z33, Protein G, FcR, or an antibody or an antigen-binding fragment thereof that specifically binds to the constant region or variable region of IgG, IgE, IgM, IgD or IgA; Optionally, the antigen-binding fragment of the antibody that specifically binds to IgG, IgE, IgM, IgD or IgA comprises a VHH, scFv, affibody, Fab, or F(ab’)2 fragment; Optionally, the T a comprises a polypeptide or a fragment thereof that specifically binds to ASGPR, asialoglycoprotein, N-acetylgalactosamine (GalNac), triGalNAc, galactose, galactoside, galactosamine, or a residue of galactan; Optionally, the polypeptide that specifically binds to ASGPR is an anti-ASGPR antibody or an antigen-binding fragment thereof; Optionally, the antigen-binding fragment of the anti-ASGPR antibody comprises a VHH, scFv, affibody, Fab, or F(ab’)2 fragment; Optionally, the ASGPR affinity ligand includes: of the residue; L is a bond, a peptide linker or a non-peptide linker.

26. The functional molecule according to any one of claims 20 to 25, wherein, The said T f Selected from any one of the following: (a) HFC-1, FcRn, Protein A, Protein G, an immunoglobulin constant region-binding peptide or Z33; (b) The mutant of (a); (c) A fragment derived from (a) or (b); Optionally, the T f comprises a polypeptide shown in any one of SEQ ID NO: 19 to 29.

27. The functional molecule according to any one of claims 20 to 26, wherein, The T a is selected from the polypeptides shown in any one of SEQ ID NO:14 to 16.

28. The functional molecule according to any one of claims 20 to 27, wherein, The T of the functional molecule a selected from the polypeptides shown in any one of SEQ ID NO: 14 to 16, the T of the second molecule f comprises the polypeptides shown in any one of SEQ ID NO: 19 to 29, and the L comprises the polypeptides shown in SEQ ID NO: 17 or 18; Optionally, the second molecule comprises a polypeptide comprising any one of SEQ ID NOs: 30 to 36.

29. A method for reducing the amount of neutralizing antibodies and / or autoantibodies in a subject, comprising administering to the subject a pharmaceutically effective amount of the pharmaceutical composition according to any one of claims 1 to 19 or the functional molecule according to any one of claims 19 to 28; Optionally, the administration comprises administering the second molecule and the first molecule simultaneously, or administering the first molecule first and then the second molecule, or administering the second molecule first and then the first molecule.

30. A method for enhancing the therapeutic efficacy of gene therapy in a subject, comprising administering to the subject a pharmaceutically effective amount of the pharmaceutical composition according to any one of claims 1 to 19 or the functional molecule according to any one of claims 19 to 28; Optionally, the administration includes co - administration of the second molecule and the first molecule which is an IgG - degrading enzyme, or administration of the first molecule followed by the second molecule, or administration of the second molecule followed by the first molecule.

31. A method for treating an autoimmune disease or antibody - mediated organ transplant rejection in a subject, comprising administering to the subject a pharmaceutically effective amount of the pharmaceutical composition according to any one of claims 1 - 19 or the functional molecule according to any one of claims 19 - 28; Optionally, the administration includes co - administration of the second molecule and the first molecule which is an IgG - degrading enzyme, or administration of the first molecule followed by the second molecule, or administration of the second molecule followed by the first molecule.

32. Use of the pharmaceutical composition according to any one of claims 1 - 19 or the functional molecule according to any one of claims 19 - 28 in the preparation of a medicament; The uses of the drug include: (a) for reducing the amount of pre - existing antibodies, auto - immune antibodies, neutralizing antibodies and / or anti - drug antibodies in a subject, (b) for enhancing the therapeutic efficacy of gene therapy in a subject, or (c) for treating an autoimmune disease or antibody - mediated organ transplant rejection in a subject.

33. A VHH antibody domain or a fragment thereof, comprising CDR1 shown in SEQ ID NO:37, CDR2 shown in SEQ ID NO:38, and CDR3 shown in SEQ ID NO:

39.

34. The VHH antibody domain or fragment thereof according to claim 33, wherein, The VHH antibody domain or a fragment thereof specifically binds to IgM.

35. The VHH antibody domain or fragment thereof according to claim 33 or 34, wherein, The VHH antibody domain or a fragment thereof is a humanized VHH antibody domain or a fragment thereof.

36. The VHH antibody domain or fragment thereof according to any one of claims 33 to 35, wherein, The VHH antibody domain or a fragment thereof has the amino acid sequence shown in SEQ ID NO:

27.

37. A functional molecule containing the VHH antibody domain or a fragment thereof according to any one of claims 33 to 36, which has the structure of formula (II): T a -L-VHH (II); Wherein VHH is the VHH antibody domain or a fragment thereof described in any one of claims 33 to 36, T a is an ASGPR and / or M6PR affinity ligand, and L is a linker.

38. The functional molecule according to claim 37, wherein, The affinity K of the VHH with immunoglobulin D value is less than 1×10 -7 M, 1×10 -8 M, 1×10 -9 M or 1×10 -10 M, or EC 50 <1000 nM, 100 nM, 10 nM or 1 nM; The T a affinity K with ASGPR and / or M6PR D value is less than 1×10 -7 M, 1×10 -8 M, 1×10 -9 M or 1×10 -10 M.

39. The functional molecule according to claim 37 or 38, wherein, The affinity K of the VHH for the immunoglobulin D value is less than 1×10 -8 M; or EC 50 < 100 nM.

40. The functional molecule according to any one of claims 37 to 39, wherein, The T a affinity K with ASGPR and / or M6PR D value is between 1×10 -7 M and 1×10 -10 M.

41. The functional molecule according to any one of claims 37 to 40, wherein, Said T a comprises a polypeptide or a fragment thereof that specifically binds to ASGPR, asialoglycoprotein, N-acetylgalactosamine (GalNac), triGalNAc, galactose, galactoside, galactosamine or a residue of galactan; Optionally, the polypeptide that specifically binds to ASGPR is an anti - ASGPR antibody or an antigen - binding fragment thereof; Optionally, the antigen - binding fragment of the anti - ASGPR antibody includes a VHH, scFv, affibody, Fab, or F(ab’)2 fragment; Optionally, the ASGPR affinity ligand comprises: residues; The L is a bond, a peptide linker or a non - peptide linker.

42. The functional molecule according to any one of claims 37 to 41, wherein The T a is selected from the polypeptides shown in any one of SEQ ID NO:14 to 16.

43. The functional molecule according to any one of claims 37 to 42, wherein, The functional molecule contains a polypeptide comprising as shown in SEQ ID NO:

34.

44. A pharmaceutical composition comprising an immunoglobulin - degrading enzyme and the functional molecule according to any one of claims 37 - 43.

45. The pharmaceutical composition according to claim 44, wherein, The immunoglobulin - degrading enzyme includes an IgG - degrading enzyme, an IgE - degrading enzyme, an IgM - degrading enzyme, an IgD - degrading enzyme or an IgA - degrading enzyme.

46. The pharmaceutical composition according to claim 44 or 45, wherein, The immunoglobulin - degrading enzyme includes at least one immunoglobulin - degrading enzyme shown in SEQ ID NO:1 - 13.

47. The pharmaceutical composition according to any one of claims 44 to 46, wherein, The pharmaceutical composition includes an immunoglobulin - degrading enzyme containing at least one sequence shown in SEQ ID NO:1 - 13 and a functional molecule containing the sequence shown in SEQ ID NO:34.

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