Methods for improved therapeutic use of recombinant aav

Abstract

To provide a method for improved therapeutic use of recombinant AAV.SOLUTION: Provided herein are methods for managing host immune responses to improve therapeutic outcomes in adeno-associated virus (AAV)-mediated gene therapy. Such methods include administering a recombinant adeno-associated virus (rAAV) to a subject after administration of a CD19 inhibitor, for example, an anti-CD19 antibody. The methods described herein can facilitate improvement of transgene expression, help overcome existing NAbs, and / or enable redosing with the same or substantially similar rAAV or transgene.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 910,790, filed on October 4, 2019, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes. Sequence Listing

[0002] This application includes a sequence listing that was electronically submitted in ASCII format, the entire disclosure of which is incorporated herein by reference. The name of the above ASCII copy created on September 3, 2020 is ULP - 006WO_SL_ST25.txt, and the size is 23,765 bytes. Technical Field of the Invention

[0003] The present disclosure generally relates to methods for managing host immune responses to improve therapeutic outcomes in adeno - associated virus (AAV) - mediated gene therapy.

Background Art

[0004] Background of the Invention A major challenge for the success of AAV - mediated gene therapy is the AAV capsid, vector DNA, and transgene product (Corti et al., 2014, Mol Ther Methods Induction of innate and adaptive immune responses against Clin Dev 1, 14033 (see reference). The innate immune response against gene therapy products (e.g., recombinant AAV, i.e., rAAV) can eliminate the product and transfected cells, and reduce both the intensity and duration of transgene expression (see Bessis et al., 2004, Gene Therapy 11, S10 - S17). The adaptive immune response occurs later and plays an important role - the adaptive immune response includes a humoral response characterized by the production of neutralizing antibodies (NAb) specific to the capsid or the expressed transgene, and a cell-mediated response including T cells and NK cells (see the same reference). Adaptive immunity not only contributes to the elimination of AAV products and transfected cells from the body, but also brings about a memory response that prevents the delivery of the same rAAV or transgene (see the same reference). Finally, some individuals are naturally exposed to AAV at an early age, and AAV NAb may be induced, which may render AAV - based gene therapy unusable in these patients. For the reasons described above, it is necessary to manage the host immune response to promote improved transgene expression, to overcome existing NAb, and / or to enable readministration with the same rAAV or transgene. The present invention addresses this need through pharmacological modulation of the humoral immune response using a CD19 inhibitor, such as an anti - CD19 antibody.

Prior Art Documents

Non - Patent Documents

[0005]

Non - Patent Document 1

Non - Patent Document 2

Summary of the Invention

Means for Solving the Problems

[0006] SUMMARY OF THE INVENTION The present invention provides a method for managing the host immune response to improve the therapeutic outcome in AAV gene therapy. More specifically, provided herein are methods comprising administering a CD19 inhibitor prior to administration of an AAV gene therapy product.

[0007] In a first aspect, the present disclosure provides a method for administering a recombinant adeno-associated virus (rAAV) to a subject, the method comprising first administering a CD19 inhibitor to the subject and then administering the rAAV to the subject.

[0008] In one embodiment, the CD19 inhibitor is administered to the subject at least about 12 hours before administration of the rAAV. In another embodiment, the CD19 inhibitor is administered to the subject at least about 24 hours before administration of the rAAV. In yet another embodiment, the CD19 inhibitor is administered to the subject at least about 2 days before administration of the rAAV. In yet another embodiment, the CD19 inhibitor is administered to the subject at least about 3, 4, 5, 6, 7 days, or more days before administration of the rAAV. In yet another embodiment, the CD19 inhibitor is administered to the subject at least about 7, 14, 21 days, or more days before administration of the rAAV. In an exemplary embodiment, the CD19 inhibitor is administered to the subject 7 days or about 7 days before administration of the rAAV.

[0009] In one embodiment, the CD19 inhibitor is administered once before administration of the rAAV. In another embodiment, the CD19 inhibitor is administered twice before administration of the rAAV. In yet another embodiment, the CD19 inhibitor is administered 3, 4, 5 times or more times before administration of the rAAV. In an exemplary embodiment, the CD19 inhibitor is administered once before administration of the rAAV.

[0010] In one embodiment, the CD19 inhibitor is selected from an anti-CD19 antibody, an anti-CD19 single-chain variable fragment (scFv), a CD19 antisense oligonucleotide, a CD19 small interfering RNA (siRNA), and a small molecule inhibitor of CD19. In an exemplary embodiment, the CD19 inhibitor is an anti-CD19 antibody.

[0011] In various embodiments described herein, the CD19 inhibitor can be an anti-CD19 antibody. In some embodiments, the anti-CD19 antibody can be selected from inebilizumab, tafasitamab, and FMC63. In an exemplary embodiment, the anti-CD19 antibody is inebilizumab.

[0012] In certain embodiments where the CD19 inhibitor is an anti-CD19 antibody, the anti-CD19 antibody can be administered at a dose of about 10 mg to about 3000 mg. In some embodiments, the anti-CD19 antibody can be administered at a dose of about 50 mg to about 2000 mg. In some embodiments, the anti-CD19 antibody can be administered at a dose of about 100 mg to about 1000 mg. In some embodiments, the anti-CD19 antibody can be administered at a dose of about 200 mg to about 500 mg. In some embodiments, the anti-CD19 antibody can be administered at a dose of about 250 mg to about 350 mg. In an exemplary embodiment, the anti-CD19 antibody can be administered at a dose of about 300 mg.

[0013] In certain embodiments where the CD19 inhibitor is an anti-CD19 antibody, the anti-CD19 antibody can be administered at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg. In an exemplary embodiment, the anti-CD19 antibody is inebilizumab and is administered at a dose of 300 mg.

[0014] In certain embodiments where the CD19 inhibitor is an anti-CD19 antibody, the anti-CD19 antibody can be administered at a dose of about 0.1 mg / kg to about 50 mg / kg. In some embodiments, the anti-CD19 antibody can be administered at a dose of about 1 mg / kg to about 10 mg / kg. In some embodiments, the anti-CD19 antibody can be administered at a dose of about 2 mg / kg to about 8 mg / kg. In some embodiments, the anti-CD19 antibody can be administered at a dose of about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg or about 8 mg / kg.

[0015] In certain embodiments where the CD19 inhibitor is an anti-CD19 antibody, the anti-CD19 antibody can be formulated into a pharmaceutical composition comprising the anti-CD19 antibody and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition is formulated for subcutaneous, intramuscular, intradermal, intraperitoneal or intravenous administration. In an exemplary embodiment, the pharmaceutical composition is formulated for intravenous administration.

[0016] In some embodiments, the CD19 inhibitor is administered subcutaneously, intramuscularly, intradermally, intraperitoneally or intravenously. In an exemplary embodiment, the CD19 inhibitor is administered intravenously.

[0017] In some embodiments, a method for administering recombinant adeno-associated virus (rAAV) to a subject can further comprise administering intravenous immunoglobulin (IVIg) before, at the same time as, or after administration of the CD19 inhibitor. In some embodiments, the CD19 inhibitor and IVIg can be administered simultaneously. In some embodiments, the CD19 inhibitor and IVIg can be administered sequentially. In some embodiments, the CD19 inhibitor and IVIg can be administered separately.

[0018] In some embodiments, a method for administering to a subject recombinant adeno-associated virus (rAAV) may further comprise administering a corticosteroid before, at the same time as, or after administration of a CD19 inhibitor. In some embodiments, the CD19 inhibitor and the corticosteroid may be administered simultaneously. In some embodiments, the CD19 inhibitor and the corticosteroid may be administered sequentially. In some embodiments, the CD19 inhibitor and the corticosteroid may be administered separately. In some embodiments, the corticosteroid is selected from prednisone, dexamethasone, hydrocortisone, methylprednisolone, betamethasone, cortisone, prednisolone, budesonide, and triamcinolone. In some embodiments, the corticosteroid is prednisolone. In some embodiments, the corticosteroid is administered at 60 mg / day. In some embodiments, prednisolone is administered about 5 days before administration of rAAV. In some embodiments, the corticosteroid is administered at 60 mg / day for 4 weeks, with the first dose being administered 5 days before administration of rAAV.

[0019] In some embodiments, the method for administering a recombinant adeno-associated virus (rAAV) may further comprise administering the agent before, simultaneously with, or after administration of the CD19 inhibitor. In some embodiments, the CD19 inhibitor and the agent may be administered simultaneously. In some embodiments, the CD19 inhibitor and the agent may be administered sequentially. In some embodiments, the CD19 inhibitor and the agent may be administered separately. In some embodiments, the agent is selected from a proteasome inhibitor, a TLR antagonist, an immunosuppressive macrocyclic molecule, an antimetabolite, a cGAS-STING antagonist, IL-2, an IL-2 mutein, an anti-CD38 antibody, an immunomodulatory drug (IMiD), an anti-B cell maturation antigen (BCMA) agent, an anti-SLAM family member 7 (SLAMF7) antibody, a mammalian target of rapamycin (mTOR) inhibitor, SEL-212, cyclophosphamide, mycophenolate mofetil, a phosphoinositide 3-kinase inhibitor, a Bruton's tyrosine kinase inhibitor, a sphingosine-1-phosphate receptor modulator, an anti-B cell activating factor (BAFF, also known as tumor necrosis factor ligand superfamily member 13B) inhibitor, or an IgG-degrading protease. In some embodiments, the proteasome inhibitor is selected from bortezomib, carfilzomib, ixazomib, oprozomib, delanzomib, and marizomib. In an exemplary embodiment, the proteasome inhibitor is bortezomib. In some embodiments, the IgG-degrading protease is IdeS of Streptococcus pyogenes or an engineered variant thereof. In some embodiments, the IgG-degrading protease is IdeZ of Streptococcus equi or an engineered variant thereof.

[0020] In some embodiments, the recombinant adeno-associated virus (rAAV) administered according to the methods of the present application comprises an AAV capsid and a vector genome packaged therein.

[0021] In some embodiments, the AAV capsid is derived from AAV of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, rh10, hu37 (i.e., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, AAV12, AAVrh10, AAVhu37) or engineered variants thereof. In an exemplary embodiment, the AAV capsid is an AAV serotype 9 (AAV9) capsid, an AAV9 variant capsid, an AAV serotype 8 (AAV8) capsid, an AAV8 variant capsid, an AAV serotype 2 (AAV2) capsid, or an AAV serotype hu37 (AAVhu37) capsid.

[0022] In some embodiments, the vector genome packaged in rAAV contains the coding sequence of a protein transduction gene. In one embodiment, the coding sequence is a native coding sequence. In another embodiment, the coding sequence is a codon-optimized coding sequence. In some embodiments, the coding sequence is ornithine transcarbamylase (OTC), glucose 6-phosphatase (G6Pase), factor VIII, factor IX, ATP7B, phenylalanine hydroxylase (PAH), argininosuccinate synthetase, cyclin-dependent kinase-like 5 (CDKL5), propionyl-CoA carboxylase subunit α (PCCA) and propionyl-CoA carboxylase subunit β (PCCB), survival motor neuron (SMN), iduronate-2-sulfatase (IDS), α-1-iduronidase (IDUA), tripeptidyl peptidase 1 (TPP1), low density lipoprotein receptor (LDLR), myotubularin 1, acid α-glucosidase (GAA), myotonic dystrophy protein kinase (DMPK), N-sulfo-glucosamine sulfohydrolase (SGSH), fibroblast growth factor-4 (FGF-4), rab escort protein 1 (REP1), carbamoyl synthetase 1 (CPS1), argininosuccinate lyase (ASL), arginase, fumarylacetoacetate hydrolase, α-1 antitrypsin, methylmalonyl-CoA mutase, cystic fibrosis transmembrane conductance regulator (CFTR) protein, and dystrophin gene product (e.g., minidystrophin or microdystrophin).

[0023] In some embodiments, rAAV is administered subcutaneously, intramuscularly, intradermally, intraperitoneally, intrathecally, intraventricularly, or intravenously. In an exemplary embodiment, rAAV is administered intravenously. In some embodiments, rAAV is administered at a dose of about 1×10 11 ~ about 1×10 14 genome copies (GC) / kg. In a further embodiment, rAAV is about 1×10 12 ~ about 1×10 13It is administered at a dose of [[number]] genomic copies (GC) / kg. In some embodiments, the rAAV is administered as a single dose. In other embodiments, the rAAV is administered as multiple doses.

[0024] In some embodiments, the subject is human. In one embodiment, the human subject is an adult subject, i.e., a human subject over 18 years old. In one embodiment, the human subject is a pediatric subject, i.e., a human subject from 0 to 18 years old (including both ends).

[0025] In a second aspect, the present disclosure is a method for administering at least two doses of recombinant adeno-associated virus (rAAV) to a subject, the method comprising: (a) administering a first dose of a CD19 inhibitor to the subject and then administering a first rAAV to the subject; (b) administering a second dose of a CD19 inhibitor to the subject and then administering a second rAAV to the subject. and including.

[0026] In some embodiments according to this second aspect, the first and second doses of the CD19 inhibitor are the same CD19 inhibitor. Alternatively, the first dose of the CD19 inhibitor may be a different CD19 inhibitor compared to the second dose.

[0027] In some embodiments according to this second aspect, the first rAAV and the second rAAV are the same rAAV. Alternatively, in some embodiments, the first rAAV and the second rAAV are different.

[0028] In some embodiments according to this second aspect, the first rAAV and the second rAAV contain the same vector capsid. In another embodiment, the first rAAV and the second rAAV contain different vector capsids.

[0029] In some embodiments according to this second aspect, the first rAAV and the second rAAV express the same transgene. In another embodiment, the first rAAV and the second rAAV express different transgenes.

[0030] In some embodiments according to this second aspect, the administration of the first dose of the CD19 inhibitor is performed 12 months prior to the administration of the second dose of the CD19 inhibitor. In one embodiment, the administration of the first dose of the CD19 inhibitor is performed 2 years prior to the administration of the second dose of the CD19 inhibitor. In another embodiment, the administration of the first dose of the CD19 inhibitor is performed 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 years prior to the administration of the second dose of the CD19 inhibitor.

[0031] In some embodiments according to this second aspect, the first dose of the CD19 inhibitor is an anti-CD19 antibody. In some embodiments according to this second aspect, the second dose of the CD19 inhibitor is an anti-CD19 antibody. In some embodiments according to this second aspect, the first and second doses of the CD19 inhibitor are anti-CD19 antibodies. In one embodiment, the anti-CD19 antibody is selected from inebilizumab, tafasitamab and FMC63. In an exemplary embodiment, the anti-CD19 antibody is inebilizumab.

[0032] In some embodiments according to this second aspect, the amounts of the CD19 inhibitor administered in the first and second doses are the same. Alternatively, the amount of the CD19 inhibitor administered in the first dose may be different from the amount of the CD19 inhibitor administered in the second dose. In an exemplary embodiment, the amounts of the CD19 inhibitor administered in the first and second doses are the same. In another exemplary embodiment, the CD19 inhibitor is inebilizumab, the first dose is 300 mg, and the second dose is 300 mg.

[0033] These and other aspects and features of the invention are described in the following sections of this application. In certain embodiments, for example, the following items are provided. (Item 1) A method for administering a recombinant adeno-associated virus (rAAV) to a subject, said method A method comprising first administering a CD19 inhibitor to the subject and then administering the rAAV to the subject. (Item 2) The method according to item 1, wherein the CD19 inhibitor is administered to the subject at least about 12 hours before the administration of the rAAV. (Item 3) The method according to item 1, wherein the CD19 inhibitor is administered to the subject at least about 24 hours before the administration of the rAAV. (Item 4) The method according to item 1, wherein the CD19 inhibitor is administered to the subject 7 days or about 7 days before the administration of the rAAV. (Item 5) The method according to item 1, wherein the CD19 inhibitor is administered to the subject 14 days or about 14 days before the administration of the rAAV. (Item 6) The method according to item 1, wherein the CD19 inhibitor is administered to the subject 21 days or about 21 days before the administration of the rAAV. (Item 7) The method according to item 1, wherein the CD19 inhibitor is administered once before the administration of the rAAV. (Item 8) The method according to item 1, wherein the CD19 inhibitor is administered twice before the administration of the rAAV. (Item 9) The method according to any of the preceding items, wherein the CD19 inhibitor is selected from an anti-CD19 antibody, an anti-CD19 scFv, a CD19 antisense oligonucleotide, a CD19 siRNA, and a small molecule inhibitor of CD19. (Item 10) The method according to item 9, wherein the CD19 inhibitor is an anti-CD19 antibody. (Item 11) The method according to item 10, wherein the anti-CD19 antibody is selected from inebilizumab, tafasitamab, and FMC63. (Item 12) The method according to item 11, wherein the anti-CD19 antibody is inebilizumab. (Item 13) The method according to item 10, wherein the anti-CD19 antibody is administered at a dose of about 10 mg to about 3000 mg. (Item 14) The method according to item 10, wherein the anti-CD19 antibody is administered at a dose of about 50 mg to about 2000 mg. (Item 15) The method according to item 10, wherein the anti-CD19 antibody is administered at a dose of about 100 mg to about 1000 mg. (Item 16) The method according to item 10, wherein the anti-CD19 antibody is administered at a dose of about 200 mg to about 500 mg. (Item 17) The method according to item 10, wherein the anti-CD19 antibody is administered at a dose of about 300 mg. (Item 18) The method according to item 10, wherein the anti-CD19 antibody is administered at a dose of about 1 mg / kg to about 10 mg / kg. (Item 19) The method according to item 10, wherein the anti-CD19 is administered at a dose of about 2 mg / kg to about 8 mg / kg. (Item 20) The method according to item 10, wherein the CD19 antibody is formulated into a pharmaceutical composition comprising the anti-CD19 antibody and a pharmaceutically acceptable carrier or excipient. (Item 21) The method according to item 20, wherein the pharmaceutical composition is formulated for subcutaneous, intramuscular, intradermal, intraperitoneal or intravenous administration. (Item 22) The method according to item 21, wherein the pharmaceutical composition is formulated for intravenous administration. (Item 23) The method according to item 1, wherein the CD19 inhibitor is administered subcutaneously, intramuscularly, intradermally, intraperitoneally or intravenously. (Item 24) The method according to item 23, wherein the CD19 inhibitor is administered intravenously. (Item 25) The method according to any of the preceding items, further comprising administering intravenous immunoglobulin (IVIg) before, simultaneously with, or after administration of the CD19 inhibitor. (Item 26) The method according to any one of the preceding items, further comprising administering a corticosteroid before, simultaneously with, or after administration of the CD19 inhibitor. (Item 27) The method according to item 26, wherein the corticosteroid is selected from prednisolone, dexamethasone, hydrocortisone, methylprednisolone, betamethasone, cortisone, prednisone, budesonide, and triamcinolone. (Item 28) The method according to item 27, wherein the corticosteroid is prednisolone and is administered at a dose of 60 mg / day. (Item 29) The method according to item 27, wherein prednisolone is administered about 5 days before administration of the rAAV. (Item 30) The method according to any one of the preceding items, further comprising administering, before, simultaneously with, or after administration of the CD19 inhibitor, a drug selected from a proteasome inhibitor, a TLR antagonist, an immunosuppressive macrocyclic molecule, an antimetabolite, a cGAS-STING antagonist, IL-2, an IL-2 mutein, an anti-CD38 antibody, an immunomodulatory drug (IMiD), an anti-B cell maturation antigen (BCMA) drug, an anti-SLAM family member 7 (SLAMF7) antibody, a mammalian target of rapamycin (mTOR) inhibitor, SEL-212, cyclophosphamide, mycophenolate mofetil, a phosphoinositide 3-kinase inhibitor, a Bruton's tyrosine kinase inhibitor, a sphingosine-1-phosphate receptor modulator, an anti-B cell activating factor (BAFF, also known as tumor necrosis factor ligand superfamily member 13B) inhibitor, or an IgG-degrading protease. (Item 31) The method according to item 30, wherein the proteasome inhibitor is selected from bortezomib, carfilzomib, ixazomib, oprozomib, delanzomib, and marizomib. (Item 32) The method according to item 31, wherein the proteasome inhibitor is bortezomib. (Item 33) The method according to item 30, wherein the IgG-degrading protease is IdeS of Streptococcus pyogenes or a engineered variant thereof. (Item 34) The method according to item 30, wherein the IgG-degrading protease is IdeZ of Streptococcus equi or a engineered variant thereof. (Item 35) The method according to any of the preceding items, wherein the rAAV comprises an AAV capsid and a vector genome packaged within the capsid. (Item 36) The method according to item 35, wherein the AAV capsid is derived from AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, rh10, hu37, or a engineered variant thereof. (Item 37) The method according to item 36, wherein the AAV capsid is an AAV9 capsid. (Item 38) The method according to item 36, wherein the AAV capsid is an AAV8 capsid. (Item 39) The method according to item 36, wherein the AAV capsid is a hu37 capsid. (Item 40) The method according to item 35, wherein the vector genome comprises a coding sequence of a protein transduction gene. (Item 41) The method according to item 40, wherein the coding sequence is a natural coding sequence. (Item 42) The method according to item 40, wherein the coding sequence is a codon-optimized coding sequence. (Item 43) The method according to item 40, wherein the coding array expresses a protein-introducing gene selected from ornithine transcarbamylase (OTC), glucose 6-phosphatase (G6Pase), factor VIII, factor IX, ATP7B, phenylalanine hydroxylase (PAH), argininosuccinate synthetase, cyclin-dependent kinase-like 5 (CDKL5), propionyl-CoA carboxylase subunit α (PCCA) and propionyl-CoA carboxylase subunit β (PCCB), survival motor neuron (SMN), iduronate-2-sulfatase (IDS), α-1-iduronidase (IDUA), tripeptidyl peptidase 1 (TPP1), low density lipoprotein receptor (LDLR), myotubularin 1, acid α-glucosidase (GAA), myotonic dystrophy protein kinase (DMPK), N-sulfo-glucosamine sulfohydrolase (SGSH), fibroblast growth factor-4 (FGF-4), rab escort protein 1 (REP1), carbamoyl synthetase 1 (CPS1), argininosuccinate lyase (ASL), arginase, fumarylacetoacetate hydrolase, α-1 antitrypsin, methylmalonyl-CoA mutase, cystic fibrosis transmembrane conductance regulator (CFTR) protein, and dystrophin gene product. (Item 44) The method according to any one of the preceding items, wherein the rAAV is administered subcutaneously, intramuscularly, intradermally, intraperitoneally, intrathecally, intracerebroventricularly, or intravenously. (Item 45) The method according to item 44, wherein the rAAV is administered intravenously. (Item 46) The rAAV is administered at a dose of about 1×10 11 ~ about 1×10 14 genomic copies (GC) / kg, according to any one of the preceding items. (Item 47) The method according to any one of the preceding items, wherein the subject is human. (Item 48) The method according to item 47, wherein the human subject is an adult subject. (Item 49) The method according to item 47, wherein the human subject is a pediatric subject. (Item 50) A method for administering at least two doses of recombinant adeno-associated virus (rAAV) to a subject, the method comprising: (a) administering a first dose of a CD19 inhibitor to the subject and then administering a first rAAV to the subject; and (b) administering a second dose of a CD19 inhibitor to the subject and then administering a second rAAV to the subject. A method comprising the above. (Item 51) The method according to item 50, wherein the first and second doses of the CD19 inhibitor are the same CD19 inhibitor. (Item 52) The method according to item 50, wherein the first dose of the CD19 inhibitor is a different CD19 inhibitor compared to the second dose. (Item 53) The method according to item 50, wherein the first rAAV and the second rAAV are the same rAAV. (Item 54) The method according to item 50, wherein the first rAAV and the second rAAV are different rAAVs. (Item 55) The method according to item 50, wherein the first rAAV and the second rAAV contain the same AAV capsid. (Item 56) The method according to item 50, wherein the first rAAV and the second rAAV contain different AAV capsids. (Item 57) The method according to item 50, wherein the first rAAV and the second rAAV express the same transgene. (Item 58) The method according to item 50, wherein the first rAAV and the second rAAV express different transgenes. (Item 59) The method according to item 50, wherein the administration of the first dose of the CD19 inhibitor is performed 12 months ahead of the administration of the second dose of the CD19 inhibitor. (Item 60) The method according to item 50, wherein the administration of the CD19 inhibitor at the first dose is performed 2 years ahead of the administration of the CD19 inhibitor at the second dose. (Item 61) The method according to item 50, wherein the administration of the CD19 inhibitor at the first dose is performed 10 years ahead of the administration of the CD19 inhibitor at the second dose. (Item 62) The method according to any one of items 50 to 61, wherein the CD19 inhibitor at the first dose is an anti-CD19 antibody. (Item 63) The method according to any one of items 50 to 61, wherein the CD19 inhibitor at the second dose is an anti-CD19 antibody. (Item 64) The method according to any one of items 50 to 61, wherein the CD19 inhibitors at the first and second doses are anti-CD19 antibodies. (Item 65) The method according to any one of items 62 to 64, wherein the anti-CD19 antibody is selected from inebilizumab, tafasitamab, and FMC63. (Item 66) The method according to item 65, wherein the anti-CD19 antibody is inebilizumab. (Item 67) The method according to item 50, wherein the amounts of the CD19 inhibitors administered at the first and second doses are the same. (Item 68) The method according to item 50, wherein the amounts of the CD19 inhibitors administered at the first and second doses are different.

Mode for Carrying Out the Invention

[0034] Detailed Description of the Invention The present invention provides a method for managing the host immune response to improve the therapeutic outcome in adeno-associated virus (AAV)-mediated gene therapy. In some embodiments, the methods described herein facilitate improved transgene expression, help overcome existing neutralizing antibodies (NAbs), and / or may allow readministration with the same or substantially similar rAAV or transgene. In some embodiments, the methods provided by the present disclosure include administering recombinant adeno-associated virus (rAAV) to a subject after administration of a CD19 inhibitor, such as an anti-CD19 antibody. Such methods may be useful for the prevention, treatment, or amelioration of various diseases and disorders as further described herein.

[0035] Unless otherwise specified, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0036] To facilitate the review of the various embodiments of the present disclosure, the following explanations of certain terms are provided.

[0037] Adeno-associated virus (AAV): A small replication-defective non-enveloped virus that infects humans and several other primate species. AAV is not known to cause disease and induces a very mild immune response. Gene therapy vectors utilizing AAV can infect both dividing and quiescent cells and can persist episomally without integrating into the host cell genome. These characteristics make AAV an attractive viral vector for gene therapy. Currently, there are 12 recognized serotypes of AAV (AAV1 - 12).

[0038] Administer / Administration: To provide or give a drug, such as a therapeutic agent (e.g., recombinant AAV or a CD19 inhibitor), to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, intratracheal, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0039] Antibody: The term “antibody” or “antibodies” includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single-chain Fv (scFv), single-chain antibodies, single-domain antibodies, domain antibodies, Fab fragments, F(ab’)2 fragments, antibody fragments exhibiting the desired biological activity, disulfide-bonded Fv (sdFv), and anti-idiotype (anti-Id) antibodies (e.g., including anti-Id antibodies to the antibodies used in any method of the present disclosure), intrabodies, and any epitope-binding fragment of the foregoing. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing an antigen-binding site. The immunoglobulin molecule can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0040] Antibody-dependent cell-mediated cytotoxicity: "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which non-specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize an antibody bound to a target cell and subsequently cause lysis of the target cell.

[0041] CD19 inhibitor: "CD19 inhibitor" refers to either (a) an agent that depletes (i.e., reduces the number or amount of) cells expressing CD19, such as B cells or plasma cells, or (b) an agent that directly or indirectly reduces, blocks, inhibits, suppresses, or interferes with the signal transduction resulting from the interaction of CD19 with one or more of its binding partners. Such agents include, but are not limited to, anti-CD19 antibodies, anti-CD19 scFv, CD19 antisense oligonucleotides, CD19 siRNA, and small molecule inhibitors of CD19.

[0042] Coding sequence: "Coding sequence" means a nucleotide sequence that encodes a polypeptide in vitro or in vivo when operably linked to appropriate regulatory sequences. The coding sequence may or may not include regions before and after the coding region, such as 5' untranslated (5'UTR) and 3' untranslated (3'UTR) sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0043] Codon optimization: "Codon optimization" nucleic acid refers to a nucleic acid sequence in which the codons have been altered to be optimal for expression in a particular system (such as a particular species or group of species). For example, a nucleic acid sequence can be optimized for expression in mammalian cells or a particular mammalian species (such as human cells). Codon optimization does not alter the amino acid sequence of the encoded protein.

[0044] Complement-dependent cytotoxicity: "Complement-dependent cytotoxicity" and "CDC" refer to the ability of a molecule to initiate complement activation and lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen.

[0045] Enhancer: A nucleic acid sequence that increases the transcription rate by increasing the activity of a promoter.

[0046] Intron: A stretch of DNA within a gene that does not contain protein-coding information. Introns are removed before the translation of messenger RNA.

[0047] Inverted terminal repeat (ITR): A symmetric nucleic acid sequence in the genome of adeno-associated virus that is required for efficient replication. The ITR sequences are located at each end of the AAV DNA genome. The ITR functions as an origin of replication for viral DNA synthesis and is an essential cis component for generating AAV integration vectors.

[0048] Isolated: An "isolated" biological component (e.g., a nucleic acid molecule, a protein, a virus, or a cell) is substantially separated or purified from the cells or tissues of the organism in which the component naturally occurs or from other biological components in the organism itself, such as other chromosomes and extrachromosomal DNA and RNA, proteins, and cells. "Isolated" nucleic acid molecules and proteins include those purified by standard purification methods. The term also encompasses nucleic acid molecules and proteins prepared by recombinant expression in host cells, as well as nucleic acid molecules and proteins chemically synthesized.

[0049] Operably linked: When a first nucleic acid sequence is in a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Generally, operably linked DNA sequences are adjacent and, when two protein-coding regions need to be linked, are within the same reading frame.

[0050] Pharmaceutically acceptable carrier: Pharmaceutically acceptable carriers (vehicles) useful in the present disclosure are conventional. Remington’s Pharmaceutical Science, by E.W. Martin, Mack Publishing Co., (Easton, Pennsylvania), 15th Edition (1975) describes compositions and formulations suitable for the pharmaceutical delivery of one or more therapeutic compounds, molecules or agents.

[0051] Generally, the nature of the carrier depends on the particular mode of administration employed. For example, parenteral formulations usually include injectable fluids that contain pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, etc. as vehicles. In the case of solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers can include, for example, pharmaceutical grade mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered can contain small amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents, such as sodium acetate or sorbitan monolaurate.

[0052] Prevention, treatment or amelioration of a disease: "Preventing" a disease refers to inhibiting the full development of the disease. "Treating" refers to a therapeutic intervention that improves the signs or symptoms after a disease or pathological condition has begun to develop. "Ameliorating" refers to a decrease in the number or severity of the signs or symptoms of a disease.

[0053] Promoter: A region of DNA that directs / initiates transcription of a nucleic acid (e.g., a gene). A promoter contains the necessary nucleic acid sequences near the transcription start site. Many promoter sequences are known to those skilled in the art, and even combinations of different promoter sequences in artificial nucleic acid molecules are possible. As used herein, a gene-specific endogenous promoter refers to a natural promoter element that regulates the expression of an endogenous gene of interest.

[0054] Purified: The term "purified" does not require absolute purity but is rather intended as a relative term. Thus, for example, a purified peptide, protein, virus, or other active compound is one that has been isolated, in whole or in part, from naturally associated proteins and other contaminants. In certain embodiments, the term "substantially purified" refers to a peptide, protein, virus, or other active compound that has been isolated from cells, cell culture medium, or other crude preparations and subjected to fractionation to remove various components of the initial preparation, such as proteins, cell debris, and other components.

[0055] Recombinant: A recombinant nucleic acid molecule is one that has a sequence not found in nature or that has a sequence created by an artificial combination of two separated sequence segments. This artificial combination can be achieved by chemical synthesis or by artificial manipulation of isolated segments of nucleic acid molecules, such as by genetic engineering techniques.

[0056] Similarly, a recombinant virus is a virus that contains a sequence (such as a genomic sequence) that does not exist in nature or that has been created by an artificial combination of at least two sequences of different origins. The term "recombinant" also includes nucleic acids, proteins, and viruses that have been modified only by the addition, substitution, or deletion of a portion of a natural nucleic acid molecule, protein, or virus. As used herein, "recombinant AAV" refers to an AAV particle in which a recombinant nucleic acid molecule, such as a recombinant nucleic acid molecule encoding a transgene, is packaged.

[0057] Serotype: A group of closely related microorganisms (such as viruses) distinguished by a characteristic set of antigens.

[0058] Stuffer sequence: Refers to the sequence of nucleotides contained within a larger nucleic acid molecule (such as a vector) that is typically used to create a desired spacing between two nucleic acid features (such as between a promoter and a coding sequence) or to extend a nucleic acid molecule to a desired length. A stuffer sequence does not contain protein-coding information, may be of unknown origin / synthetic, and / or may not be related to other nucleic acid sequences within the larger nucleic acid molecule.

[0059] Subject: A category that includes living multicellular vertebrate organisms, humans and non-human mammals. In some embodiments, the subject is human. In one embodiment, the human subject is an adult subject, i.e., a human subject over 18 years old. In one embodiment, the human subject is a pediatric subject, i.e., a human subject from 0 to 18 years old (including both ends).

[0060] Synthetic: Means produced by artificial means in a laboratory. For example, synthetic nucleic acids can be chemically synthesized in a laboratory.

[0061] Therapeutically effective amount: The amount of a particular pharmaceutical or therapeutic agent (such as a recombinant AAV or a CD19 inhibitor) sufficient to achieve a desired effect in a subject or cell being treated with the agent. The effective amount of the agent depends on several factors, such as, but not limited to, the subject or cell being treated and the mode of administration of the therapeutic composition.

[0062] Transgene: The term "transgene" refers to a polynucleotide that is introduced into a cell, transcribed into RNA, and can be translated and / or expressed under appropriate conditions as needed. The term "transgene" can also be used with respect to the expressed polypeptide, i.e., the protein transgene. A transgene can confer a desired property on the cell into which it is introduced or, alternatively, can result in a desired therapeutic outcome. In some examples, a transgene can be transcribed and translated to provide a functionally active protein transgene to a subject suffering from a disorder associated with a deficiency of an active protein. Any number of protein transgenes can be delivered in conjunction with the present invention, including, but not limited to, ornithine transcarbamylase (OTC), glucose 6-phosphatase (G6Pase), factor VIII, factor IX, ATP7B, phenylalanine hydroxylase (PAH), argininosuccinate synthetase, cyclin-dependent kinase-like 5 (CDKL5), propionyl-CoA carboxylase subunit α (PCCA), and propionyl-CoA carboxylase subunit β (PCCB). In some embodiments, one or more protein transgenes delivered in conjunction with the present invention are variants of any of the protein transgenes including, but not limited to, variants of ornithine transcarbamylase (OTC), variants of glucose 6-phosphatase (G6Pase), variants of factor VIII, variants of factor IX, variants of ATP7B, variants of phenylalanine hydroxylase (PAH), variants of argininosuccinate synthetase, variants of cyclin-dependent kinase-like 5 (CDKL5), variants of propionyl-CoA carboxylase subunit α (PCCA), and variants of propionyl-CoA carboxylase subunit β (PCCB).

[0063] Vector: A vector is a nucleic acid molecule that allows the insertion of foreign nucleic acids without disrupting the ability of the vector to replicate and / or integrate within a host cell. The vector may contain nucleic acid sequences that enable replication within the host cell, such as an origin of replication. The vector may also contain one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains regulatory sequences necessary to enable transcription and translation of one or more inserted genes. In some embodiments herein, the vector is an AAV vector.

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. "Comprising A or B" means including A or B, or including A and B. Further, all base sizes or amino acid sizes, and all molecular weight or molecular mass values given for nucleic acids or polypeptides are approximate values and are to be understood as being provided for illustrative purposes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. Further, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0065] The present invention provides methods for managing host immune responses to improve therapeutic outcomes in AAV gene therapy. More specifically, methods are provided herein that include administering an rAAV gene therapy product after administration of an inhibitor of CD19. Such methods may be useful for suppressing the development of long-lasting humoral immunity that may otherwise occur against the rAAV gene therapy product or the expressed transgene. As described herein, methods that utilize an inhibitor of CD19 represent an improvement over current approaches that employ anti-CD20 antibodies, such as those employed in clinical trial number NCT02240407 (“Re-administration of Intramuscular AAV9 in Patients with Late-Onset Pompe Disease (AAV9-GAA_IM)’’”) and clinical trial number NCT02525172 (“Immune Modulation Therapy for Pompe Disease”).

[0066] The mechanisms underlying the maintenance of long-term humoral immunity are not fully understood, but it is well established that antigen-specific antibody responses can persist throughout life (see Amanna et al., 2007, NEJM 357(19):1903-15 and Crotty et al., 2003, The Journal of Immunology, 171:4969-4973). Upon encountering an antigen, the B cells of interest proliferate, giving rise to memory B cells and clonally related plasma cells, and the memory B cells give rise to additional plasma cells upon re-encountering the antigen. Interestingly, B cell depletion studies using anti-CD20 antibodies have suggested that at least some plasma cells are maintained independently of B cell replenishment (see Bhoj et al., 2016, Blood 12:360-370 and Owczarczyk et al., 2011, Science Translational Medicine, Vol 3 Issue 101, 101ra92). This suggests that long-lived humoral immunity can be theoretically maintained by plasma cells and that such plasma cells are not affected by existing B cell depletion strategies employing anti-CD20 antibodies such as rituximab. Therefore, there is a need for a new approach to further deplete specific plasma cells that may otherwise prevent readministration of the same rAAV or AAV expressing the same or substantially similar transgenes.

[0067] Humoral immunity has been proposed to depend on distinct subsets of plasma cells with complementary functions and different kinetics (see Mei et al., 2015, Blood, 125:1739-1748). Recent studies have shown that some long-lived plasma cells in human bone marrow express CD19 and can persist for decades after antigen encounter (see Brynjolfsson et al., 2017, Blood Adv. 1:835-838). Human CD19 is a 95-kilodalton transmembrane glycoprotein belonging to the immunoglobulin (Ig) superfamily. CD19 is classified as a type I transmembrane protein with a single transmembrane domain, a cytoplasmic C-terminus, and an extracellular N-terminus. CD19 is specifically expressed in normal and neoplastic B cells as well as follicular dendritic cells. During B lymphocyte production, surface expression of CD19 begins during immunoglobulin gene rearrangement. The surface density of CD19 is highly regulated throughout B cell development and maturation until expression is lost during the terminal differentiation of plasma cells. Considering the expression of CD19 on B cells and plasma cells, CD19 can be a better target than CD20.

[0068] In a first aspect, the present disclosure provides a method for administering a recombinant adeno-associated virus (rAAV) to a subject, the method comprising first administering a CD19 inhibitor to the subject and then administering the rAAV to the subject.

[0069] In one embodiment, the CD19 inhibitor is selected from an anti-CD19 antibody, an anti-CD19 scFv, a CD19 antisense oligonucleotide, a CD19 siRNA, and a small molecule inhibitor of CD19.

[0070] In one embodiment, the CD19 inhibitor is an anti-CD19 antibody. In some embodiments, the anti-CD19 antibody can be selected from inebilizumab, tafasitamab, and FMC63.

[0071] In one embodiment, the anti-CD19 antibody is inebilizumab as described in U.S. Patent Nos. 8,323,653, 8,883,992 and 9,896,505, the disclosures of which are incorporated herein by reference.

[0072] In one embodiment, the anti-CD19 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 1. In one embodiment, the anti-CD19 antibody comprises a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2. In one embodiment, the anti-CD19 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0073] In one embodiment, the anti-CD19 antibody comprises a heavy chain variable region (VH) comprising at least one CDR having an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. In one embodiment, the anti-CD19 antibody comprises a light chain variable region (VL) comprising at least one CDR having an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. In one embodiment, the anti-CD19 antibody comprises a heavy chain variable region (VH) comprising at least one CDR having an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 and a light chain variable region (VL) comprising at least one CDR having an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0074] In one embodiment, the anti-CD19 antibody comprises a heavy chain variable region (VH) comprising CDRs having the amino acid sequences of SEQ ID NO: 3 (CDR1), SEQ ID NO: 4 (CDR2), and SEQ ID NO: 5 (CDR3). In one embodiment, the anti-CD19 antibody comprises a light chain variable region (VL) comprising CDRs having the amino acid sequences of SEQ ID NO: 6 (CDR1), SEQ ID NO: 7 (CDR2), and SEQ ID NO: 8 (CDR3). In one embodiment, the anti-CD19 antibody comprises a heavy chain variable region (VH) comprising CDRs having the amino acid sequences of SEQ ID NO: 3 (CDR1), SEQ ID NO: 4 (CDR2), and SEQ ID NO: 5 (CDR3), and a light chain variable region (VL) comprising CDRs having the amino acid sequences of SEQ ID NO: 6 (CDR1), SEQ ID NO: 7 (CDR2), and SEQ ID NO: 8 (CDR3).

[0075] In one embodiment, the anti-CD19 antibody is tafasitamab, which is described in U.S. Patent Nos. 8,524,867, 9,803,020, and International Publication No. 2018 / 002031, the disclosures of which are incorporated herein by reference.

[0076] In one embodiment, the anti-CD19 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 9. In one embodiment, the anti-CD19 antibody comprises a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 10. In one embodiment, the anti-CD19 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 10.

[0077] In one embodiment, the anti-CD19 antibody comprises a heavy chain variable region (VH) comprising at least one CDR having an amino acid sequence selected from SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. In one embodiment, the anti-CD19 antibody comprises a light chain variable region (VL) comprising at least one CDR having an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. In one embodiment, the anti-CD19 antibody comprises a heavy chain variable region (VH) comprising at least one CDR having an amino acid sequence selected from SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, and a light chain variable region (VL) comprising at least one CDR having an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

[0078] In one embodiment, the anti-CD19 antibody comprises a heavy chain variable region (VH) comprising CDRs having the amino acid sequences of SEQ ID NO: 11 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 13 (CDR3). In one embodiment, the anti-CD19 antibody comprises a light chain variable region (VL) comprising CDRs having the amino acid sequences of SEQ ID NO: 14 (CDR1), SEQ ID NO: 15 (CDR2), and SEQ ID NO: 16 (CDR3). In one embodiment, the anti-CD19 antibody comprises a heavy chain variable region (VH) comprising CDRs having the amino acid sequences of SEQ ID NO: 11 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 13 (CDR3), and a light chain variable region (VL) comprising CDRs having the amino acid sequences of SEQ ID NO: 14 (CDR1), SEQ ID NO: 15 (CDR2), and SEQ ID NO: 16 (CDR3).

[0079] In some embodiments, the anti-CD19 antibody can be selected from anti-CD19 antibodies described in any of the following references, the disclosures of which are incorporated herein by reference: WO 2005 / 012493 (Immunomedics, Inc.), WO 2006 / 089133 (Duke University), WO 2007 / 002223 (Medarex, Inc.), WO 2007 / 076950 (Merck Patent Gmbh), WO 2007 / 082715 (Friedrich-Alexander-Universitat Erlangen-Nurnberg), WO 2008 / 022152 (Xencor, Inc.), WO 2008 / 031056 (Medimmune, LLC), WO 2009 / 052431 (Seattle Genetics, Inc.), WO 2009 / 054863 (Medarex, Inc.), WO 2010 / 053716 (Immunomedics, Inc.), WO 2010 / 095031 (Glenmark Pharmaceuticals S.A.), WO 2010 / 102276 (Medimmune, LLC), WO 2011 / 147834 (Roche Glycart AG), WO 2012 / 010561 (Universite Claude Bernard Lyon), WO 2012 / 057765 (The Board of Regents of the University of Texas System), WO 2016 / 033570 (Juno Therapeutics, Inc.), WO 2017 / 015783 (Innovative Cellular Therapeutics Co., Ltd.), WO 2017 / 055328 (F. Hoffmann-La Roche (AG), International Publication No. 2017 / 066136 (Eureka Therapeutics, Inc.), International Publication No. 2018 / 002031 (Morphosys AG), International Publication No. 2018 / 108106 (Carsgen Theraeputics, Ltd.), International Publication No. 2018 / 126369 (Shanghai Sidansai Biotechnology Co., Ltd.) and International Publication No. 2019 / 057100 (Wuxi Biologics Co., Ltd.).

[0080] In some embodiments, the anti-CD19 antibody mediates antigen-dependent cell-mediated cytotoxicity (ADCC) of cells expressing CD19. In some embodiments, the anti-CD19 antibody mediates complement-dependent cell-mediated cytotoxicity (CDC) of cells expressing CD19. In some embodiments, the anti-CD19 antibody mediates apoptosis of cells expressing CD19. In some embodiments, the anti-CD19 antibody inhibits IgM / CpG-stimulated B cell proliferation.

[0081] In one embodiment, the anti-CD19 antibody can deplete circulating B cells, blood B cells, splenic B cells, marginal zone B cells, follicular B cells, peritoneal B cells, and / or bone marrow B cells after administration.

[0082] In one embodiment, the anti-CD19 antibody can deplete progenitor B cells, early pro-B cells, late pro-B cells, large pre-B cells, small pre-B cells, immature B cells, mature B cells, antigen-stimulated B cells, and / or plasma cells after administration.

[0083] In one embodiment, the anti-CD19 antibody can deplete B cells in a human subject after administration. In a specific embodiment, the anti-CD19 antibody administered according to the present invention can achieve at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% B cell depletion. In another embodiment, the anti-CD19 antibody can deplete a subset of B cells in a human subject after administration. In a specific embodiment, the anti-CD19 antibody can deplete circulating B cells, blood B cells, splenic B cells, marginal zone B cells, follicular B cells, peritoneal B cells, and / or bone marrow B cells after administration. CD19 is present on the surface of B cells at all developmental stages. Thus, the anti-CD19 antibody can deplete B cells at all developmental stages after administration. In a specific embodiment, the anti-CD19 antibody can achieve depletion of progenitor B cells, early pro-B cells, late pro-B cells, large pre-B cells, small pre-B cells, immature B cells, mature B cells, antigen-stimulated B cells, and / or plasma cells after administration. B cell depletion can persist for a long time. In one embodiment, B cell depletion by the anti-CD19 antibody administered according to the present invention can persist for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 15 days, at least 20 days, at least 25 days, or at least 30 days. In another embodiment, B cell depletion by the anti-CD19 antibody administered according to the present invention can persist for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, or at least 10 weeks. In a further embodiment, B cell depletion by the anti-CD19 antibody administered according to the present invention can persist for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months.

[0084] In one embodiment, the anti-CD19 antibody depletes at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of circulating B cells after administration. In one embodiment, the anti-CD19 antibody depletes at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of blood B cells after administration. In one embodiment, the anti-CD19 antibody depletes at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of splenic B cells after administration. In one embodiment, the anti-CD19 antibody depletes at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of marginal zone B cells after administration. In one embodiment, the anti-CD19 antibody depletes at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of follicular B cells after administration. In one embodiment, the anti-CD19 antibody depletes at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of peritoneal B cells after administration. In one embodiment, the anti-CD19 antibody depletes at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of bone marrow B cells after administration.In one embodiment, the anti-CD19 antibody depletes at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of precursor B cells after administration. In one embodiment, the anti-CD19 antibody depletes at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of early pro-B cells after administration. In one embodiment, the anti-CD19 antibody depletes at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of late pro-B cells after administration. In one embodiment, the anti-CD19 antibody depletes at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of large pre-B cells after administration. In one embodiment, the anti-CD19 antibody depletes at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of small pre-B cells after administration. In one embodiment, the anti-CD19 antibody depletes at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of immature B cells after administration. In one embodiment, the anti-CD19 antibody depletes at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of mature B cells after administration.In one embodiment, the anti-CD19 antibody depletes at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of antigen-stimulated B cells after administration. In one embodiment, the anti-CD19 antibody depletes at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of plasma cells after administration. The depletion of B cells and / or plasma cells can persist for a long time. In one embodiment, the B cell depletion and / or plasma cell depletion by the anti-CD19 antibody persists for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 15 days, at least 20 days, at least 25 days, or at least 30 days. In another embodiment, the B cell depletion and / or plasma cell depletion by the anti-CD19 antibody persists for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, or at least 10 weeks. In a further embodiment, the B cell depletion and / or plasma cell depletion by the anti-CD19 antibody persists for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months.

[0085] In one embodiment, the CD19 inhibitor is administered to the subject at least about 12 hours before administration of rAAV. In another embodiment, the CD19 inhibitor is administered to the subject at least about 24 hours before administration of rAAV. In yet another embodiment, the CD19 inhibitor is administered to the subject at least about 2 days before administration of rAAV. In yet another embodiment, the CD19 inhibitor is administered to the subject at least about 3, 4, 5, 6, 7 days, or more days before administration of rAAV. In yet another embodiment, the CD19 inhibitor is administered to the subject at least about 7, 14, 21 days, or more days before administration of rAAV. In yet another embodiment, the CD19 inhibitor is administered to the subject at least about 1 month before, at least about 2 months before, or at least about 3 months before administration of rAAV.

[0086] In one embodiment, the CD19 inhibitor is administered once before administration of rAAV. In another embodiment, the CD19 inhibitor is administered twice before administration of rAAV. In yet another embodiment, the CD19 inhibitor is administered 3, 4, 5 times or more times before administration of rAAV.

[0087] Administration of a CD19 inhibitor to a human subject can be by any route including, but not limited to, intravenous, intradermal, transdermal, subcutaneous, intramuscular, inhalation (e.g., via aerosol), buccal (e.g., sublingual), topical (i.e., both skin and mucosal surfaces including airway surfaces), intrathecal, intra-articular, intrapleural, intracerebral, intra-arterial, intraperitoneal, oral, intralymphatic, intranasal, rectal, or vaginal administration, perfusion via a local catheter, or direct injection into a lesion. In one embodiment, the CD19 inhibitor is administered by an intravenous push or intravenous infusion given over a defined period (e.g., 0.5 - 2 hours). The CD19 inhibitor can be delivered by peristaltic means or in depot form, but the most appropriate route for any given case depends on factors such as the species, age, sex and general condition of the subject, the nature and severity of the condition being treated, and / or the nature of the particular composition being administered (i.e., dosage, formulation). In some embodiments, the route of administration is by bolus or continuous infusion over a period of time.

[0088] In certain embodiments, the CD19 inhibitor is administered subcutaneously, intramuscularly, intradermally, intraperitoneally or intravenously. In an exemplary embodiment, the CD19 inhibitor is administered intravenously.

[0089] In certain embodiments, the dosage of a composition comprising a CD19 inhibitor, e.g., an anti-CD19 antibody, is measured in units of mg per kg of the subject's body weight. In other embodiments, the dosage of a composition comprising a CD19 inhibitor, e.g., an anti-CD19 antibody, is measured in units of mg per dosage administered to the subject. Any measure of dosage can be used in conjunction with the compositions and methods of the present invention, and dosage units can be converted by standard means in the art.

[0090] In certain embodiments where the CD19 inhibitor is an anti-CD19 antibody, the anti-CD19 antibody can be administered at a dose of about 10 mg to about 3000 mg. In some embodiments, the anti-CD19 antibody is administered at a dose of about 50 mg to about 2000 mg. In some embodiments, the anti-CD19 antibody is administered at a dose of about 100 mg to about 1000 mg. In some embodiments, the anti-CD19 antibody is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the anti-CD19 antibody is administered at a dose of about 250 mg to about 350 mg. In an exemplary embodiment, the anti-CD19 antibody is administered at a dose of about 300 mg.

[0091] In certain embodiments where the CD19 inhibitor is an anti-CD19 antibody, the anti-CD19 antibody can be administered at a dose of about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 2000 mg, about 2500 mg, or about 3000 mg.

[0092] In certain embodiments where the CD19 inhibitor is an anti-CD19 antibody, the anti-CD19 antibody can be administered at a dose of about 0.1 mg / kg to about 50 mg / kg based on the body weight of the subject. In some embodiments, the anti-CD19 antibody is administered at a dose of about 1 mg / kg to about 10 mg / kg. In some embodiments, the anti-CD19 antibody is administered at a dose of about 2 mg / kg to about 8 mg / kg. In some embodiments, the anti-CD19 antibody is administered at a dose of about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg or about 8 mg / kg.

[0093] In certain embodiments where the CD19 inhibitor is an anti-CD19 antibody, the anti-CD19 antibody can be administered at a dose of at least about 0.1 mg / kg, at least about 0.2 mg / kg, at least about 0.3 mg / kg, at least about 0.4 mg / kg, at least about 0.5 mg / kg, at least about 0.6 mg / kg, at least about 0.7 mg / kg, at least about 0.8 mg / kg, at least about 0.9 mg / kg, at least about 1 mg / kg, at least about 1.5 mg / kg, at least about 2 mg / kg, at least about 2.5 mg / kg, at least about 3 mg / kg, at least about 3.5 mg / kg, at least about 4 mg / kg, at least about 4.5 mg / kg, at least about 5 mg / kg, at least about 5.5 mg / kg, at least about 6 mg / kg, at least about 6.5 mg / kg, at least about 7 mg / kg, at least about 7.5 mg / kg, at least about 8 mg / kg, at least about 8.5 mg / kg, at least about 9 mg / kg, at least about 9.5 mg / kg, at least about 10 mg / kg, at least about 10.5 mg / kg, at least about 11 mg / kg, at least about 11.5 mg / kg, at least about 12 mg / kg, at least about 12.5 mg / kg, at least about 13 mg / kg, at least about 13.5 mg / kg, at least about 14 mg / kg, at least about 14.5 mg / kg, at least about 15 mg / kg, at least about 15.5 mg / kg, at least about 16 mg / kg, at least about 16.5 mg / kg, at least about 17 mg / kg, at least about 17.5 mg / kg, at least about 18 mg / kg, at least about 18.5 mg / kg, at least about 19 mg / kg, at least about 19.5 mg / kg, at least about 20 mg / kg, at least about 25 mg / kg, at least about 30 mg / kg, at least about 35 mg / kg, at least about 40 mg / kg, at least about 45 mg / kg, or at least about 50 mg / kg based on the body weight of the subject.

[0094] In certain embodiments where the CD19 inhibitor is an anti-CD19 antibody, the anti-CD19 antibody can be formulated into a pharmaceutical composition comprising the anti-CD19 antibody and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition is formulated for subcutaneous, intramuscular, intradermal, intraperitoneal, or intravenous administration. In an exemplary embodiment, the pharmaceutical composition is formulated for intravenous administration.

[0095] In some embodiments, a method for administering recombinant adeno-associated virus (rAAV) to a subject can further comprise administering intravenous immunoglobulin (IVIg) before, simultaneously with, or after administration of the CD19 inhibitor. In some embodiments, the CD19 inhibitor and IVIg can be administered simultaneously. In some embodiments, the CD19 inhibitor and IVIg can be administered sequentially. In some embodiments, the CD19 inhibitor and IVIg can be administered separately.

[0096] In some embodiments, the IVIg can be selected from Bivigam, Clairyg, Flebogam, Flebogammadit, Gammagard Liquid 10%, Gammaplex, Gammunex, IG Vena, Intratecth, Kiovig, Nanogam, Octagam, Octagam 10%, Polyglobin N 10%, Sandoglobulin NF solution, and Vigam. In an exemplary embodiment, the IVIg is Gammagard Liquid 10% (Baxter Healthcare International), a sterile liquid formulation of highly purified and concentrated immunoglobulin G (IgG) antibodies. A non-limiting list of commercially available immunoglobulin formulations that can be used in the methods of the present disclosure as IVIg is provided in U.S. Patent Application Publication No. 2017 / 0021114, the disclosure of which is incorporated herein by reference.

[0097] In some embodiments, the method for administration to a subject of recombinant adeno-associated virus (rAAV) may further comprise administering a corticosteroid before, simultaneously with, or after administration of the CD19 inhibitor. In some embodiments, the CD19 inhibitor and the corticosteroid may be administered simultaneously. In some embodiments, the CD19 inhibitor and the corticosteroid may be administered sequentially. In some embodiments, the CD19 inhibitor and the corticosteroid may be administered separately.

[0098] In some embodiments, the corticosteroid is selected from prednisolone, dexamethasone, hydrocortisone, methylprednisolone, betamethasone, cortisone, prednisone, budesonide, and triamcinolone. In certain exemplary embodiments, the corticosteroid is prednisolone.

[0099] In some embodiments, the corticosteroid is administered at a dose of about 10 mg / day to about 100 mg / day. In some embodiments, the corticosteroid is administered at a dose of about 20 mg / day to about 80 mg / day. In some embodiments, the corticosteroid is administered at a dose of about 30 mg / day to about 70 mg / day. In certain exemplary embodiments, the corticosteroid is administered at a dose of about 60 mg / day.

[0100] In some embodiments, the corticosteroid is administered at a dose of about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, or about 500 mg.

[0101] In some embodiments, the corticosteroid can be administered at a dose of about 0.1 mg / kg / day to about 10 mg / kg / day based on the subject's body weight. In some embodiments, the corticosteroid is administered at a dose of about 0.5 mg / kg / day to about 5 mg / kg / day. In some embodiments, the corticosteroid is administered at a dose of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5 mg / kg / day.

[0102] In some embodiments, the corticosteroid can be administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, or more days prior to the total administration of rAAV. For example, in certain exemplary embodiments, the corticosteroid can be administered at 60 mg / day for 5 days prior to the administration of rAAV.

[0103] In some embodiments, the corticosteroid can be administered at 60 mg / day for 4 weeks, with the first administration occurring 5 days prior to the administration of rAAV. In some embodiments, the corticosteroid is administered at 60 mg / day for 4 weeks, the first administration occurs 5 days prior to the administration of rAAV, and subsequently the corticosteroid can be tapered off for an additional 4 weeks.

[0104] In some embodiments, methods for administering to a subject recombinant adeno-associated virus (rAAV) include a proteasome inhibitor (e.g., bortezomib, carfilzomib, ixazomib, oprozomib, delanzomib, or marizomib), a toll-like receptor (TLR) antagonist (e.g., a TLR7 antagonist or a TLR9 antagonist, e.g., chloroquine, hydroxychloroquine, or quinacrine, see AT791 or E6446 (Lamphier et al., 2014, Mol Pharmacol., 85(3):429-40)), an immunosuppressive macrocyclic molecule (e.g., sirolimus, cyclosporine A, tacrolimus, or a rapamycin analog such as everolimus, biolimus, and temsirolimus), a metabolic antagonist (e.g., azathioprine, mercaptopurine, thiopurine, or methotrexate), an antagonist of the cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase stimulator of the interferon gene (cGAS-STING), interleukin-2 (IL-2), an IL-2 mutein (e.g., AMG 592, NKTR-358 or DEL106), anti-CD38 antibodies (e.g., daratumumab, isatuximab, MOR202 or TAK-079), immunomodulatory drugs (IMiDs) (e.g., lenalidomide, pomalidomide or apremilast), anti-B cell maturation antigen (BCMA) agents (e.g., chimeric antigen receptor T cells (CAR-T) (e.g., CT053 or bb2121), antibody-drug conjugates (ADCs) (e.g., GSK2857916), bispecific T cell engagers (BiTE), or anti-BCMA antibodies), anti-SLAM family member 7 (SLAMF7) antibodies (e.g., elotuzumab), mammalian target of rapamycin (mTOR) inhibitors (e.g., ABI-009 nab-rapamycin), synthetic vaccine particles encapsulating rapamycin (SVP-R), a combination of SVP-R and pegylated uricase (pegadricase) (e.g., SEL-212), cyclophosphamide, mycophenolate mofetil, phosphoinositide 3 kinase inhibitors (e.g., idelalisib, copanlisib, duvelisib, or alpelisib), Bruton's tyrosine kinase inhibitors (e.g., ibrutinib, zanubrutinib, acalabrutinib, evobrutinib, HM71224, BGB-3111, spebrutinib, or ONO-4059), sphingosine-1-phosphate receptor modulators (e.g., fingolimod or ozanimod), anti-B cell activating factor (BAFF or also known as tumor necrosis factor ligand superfamily member 13B) inhibitors (e.g., belimumab, atacicept or blisibimod), or IgG-degrading proteases (e.g., IdeS from Streptococcus pyogenes, engineered IdeS variants, IdeZ from Streptococcus equi, or IgdE enzymes from Streptococcus suis, Streptococcus porcinus, or Streptococcus equi). Some embodiments may further include administration of an agent selected from the group consisting of: In some embodiments, the agent may be administered before, simultaneously with, or after administration of the CD19 inhibitor. In some embodiments, the CD19 inhibitor and the agent may be administered simultaneously.In some embodiments, the CD19 inhibitor and the agent can be administered sequentially. In some embodiments, the CD19 inhibitor and the agent can be administered separately.

[0105] In some embodiments, the agent is a proteasome inhibitor. In some embodiments, the proteasome inhibitor is selected from bortezomib, carfilzomib, ixazomib, oprozomib, delanzomib, or marizomib. In an exemplary embodiment, the proteasome inhibitor is bortezomib. Thus, in some embodiments, the present disclosure provides a method for administering recombinant adeno-associated virus (rAAV) to a subject, the method comprising first administering a CD19 inhibitor and a proteasome inhibitor to the subject and then administering the rAAV to the subject. In some embodiments, the CD19 inhibitor is inebilizumab and the proteasome inhibitor is bortezomib. Thus, in some embodiments, the present disclosure provides a method of administering rAAV after administration of an anti-CD19 antibody such as inebilizumab and a proteasome inhibitor such as bortezomib.

[0106] In some embodiments, the agent is an IgG-degrading protease. Examples of proteases that can be used in the present invention include, but are not limited to, those described in International Publication No. WO 2020 / 016318 and / or International Publication No. WO 2020 / 159970, and include, for example, cysteine proteases derived from Streptococcus pyogenes, Streptococcus equi, Mycoplasma canis, Streptococcus agalactiae, Streptococcus pseudoporcinus, or Pseudomonas putida.

[0107] In certain embodiments, the IgG-degrading protease is IdeS from Streptococcus pyogenes (SEQ ID NO: 20), or a protease that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 20. In some embodiments, the protease is a engineered variant of SEQ ID NO: 20. Examples of engineered IdeS proteases are described in International Publication No. WO 2020 / 016318 and U.S. Patent Application Publication Nos. 20180023070 and 20180037962. In some embodiments, the engineered IdeS variant can have 1, 2, 3, 4, 5, or more amino acid modifications compared to SEQ ID NO: 20.

[0108] In certain embodiments, the IgG-degrading protease is IdeZ from Streptococcus equi (SEQ ID NO: 21), or a protease that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21. In some embodiments, the protease is a engineered variant of SEQ ID NO: 21. Examples of engineered IdeZ proteases are described in International Publication No. WO 2020 / 016318. In some embodiments, the engineered IdeZ variant can have 1, 2, 3, 4, 5, or more amino acid modifications compared to SEQ ID NO: 21.

[0109] Other proteases that can be used in the present invention include, for example, but not limited to, the IgdE enzymes from Streptococcus suis, Streptococcus porcinus, and Streptococcus equi described in International Publication No. WO 2017 / 134274.

[0110] In some embodiments, the IgG-degrading protease can be encapsulated within or conjugated to liposomes, nanoparticles, lipid nanoparticles (LNP), polymers, microparticles, microcapsules, micelles, or extracellular vesicles.

[0111] In some embodiments, the recombinant adeno-associated virus (rAAV) administered according to the methods of the present disclosure comprises an AAV capsid and a vector genome packaged therein.

[0112] In some embodiments, the packaged vector genome comprises AAV 5' inverted terminal repeat (ITR), a promoter sequence, a protein transduction gene or a functional fragment thereof, a partial or complete coding sequence of a functional variant or isoform, and an AAV 3' inverted terminal repeat (ITR).

[0113] In some embodiments, the packaged genome may further comprise an enhancer, an intron, a consensus Kozak sequence, and / or a polyadenylation signal. In some embodiments, the recombinant vector may further comprise one or more stuffer nucleic acid sequences. In one embodiment, the stuffer nucleic acid sequence is located between the intron and the partial or complete coding sequence of the protein transduction gene.

[0114] In various embodiments described herein, the rAAV comprises an AAV capsid. Any serotype of AAV capsid can be used in the present invention, and the selection of the AAV serotype depends in part on the cell type(s) targeted by the gene therapy. The AAV capsid can be of AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, rh10, hu37 (i.e., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10, AAVhu37), or any one of more than 100 variants isolated from human and non-human primate tissues (e.g., Choi et al., 2005, Curr Gene Ther. 5:299-310, 2005 and Gao et al., 2005, Curr Gene Ther. 5:285-297).

[0115] In addition to the foregoing capsids, variant AAV capsids engineered to have one or more beneficial therapeutic properties (e.g., improved targeting to selected tissues, increased ability to avoid immune responses, reduced stimulation of neutralizing antibodies, etc.) are also within the scope of the present disclosure. Non-limiting examples of such engineered variant capsids are described in U.S. Patent Nos. 9,506,083, 9,585,971, 9,587,282, 9,611,302, 9,725,485, 9,856,539, 9,909,142, 9,920,097, 10,011,640, 10,081,659, 10,179,176, 10,202,657, 10,214,566, 10,214,785, 10,266,845, 10,294,281, 10,301,648, 10,385,320, and 10,392,632 and PCT Publication Nos. WO 2017 / 165859, WO 2018 / 022905, WO 2018 / 156654, WO 2018 / 222503, and WO 2018 / 226602, the disclosures of which are incorporated herein by reference.

[0116] In certain exemplary embodiments, the rAAV administered according to the present disclosure comprises an AAV9 capsid. The AAV9 capsid is a self-assembling AAV capsid composed of multiple AAV9vp proteins. The AAV9vp protein typically encodes the vp1 amino acid sequence of SEQ ID NO: 19 (GenBank accession: AAS99264), the nucleic acid sequence of SEQ ID NO: 18, or is expressed as an alternatively spliced variant encoded by a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical thereto. These spliced variants result in proteins of different lengths of SEQ ID NO: 19. In certain embodiments, the AAV9 capsid comprises an AAV having an amino acid sequence that is 99% identical to AAS99264 or 99% identical to SEQ ID NO: 19. See also U.S. Patent No. 7,906,111 and International Publication No. 2005 / 033321. As used herein, AAV9 variants include, for example, those described in International Publication No. 2016 / 049230, U.S. Patent No. 8,927,514, U.S. Patent Application Publication No. 2015 / 0344911, and U.S. Patent No. 8,734,809.

[0117] As shown herein, the rAAV administered according to the present invention may, in some embodiments, comprise an AAV9 capsid. However, in other embodiments, another AAV capsid is selected. Tissue specificity is determined by the capsid type. AAV serotypes that transduce an appropriate target (e.g., liver, muscle, lung or CNS) can be selected as a source for the capsids of AAV viral vectors including, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAVrh64Rl, AAVrh64R2, AAVrh8. See, for example, U.S. Patent Application Publication No. 2007 / 0036760; U.S. Patent Application Publication No. 2009 / 0197338; and European Patent No. 1310571. See also International Publication No. 2003 / 042397 (AAV7 and other simian AAV), U.S. Patents Nos. 7,282,199 and 7,790,449 (AAV8). Further, AAVs not yet discovered, or recombinant AAVs based thereon, may be used as a source of AAV capsids. These documents also describe other AAVs that can be selected to generate AAV and are incorporated by reference. In some embodiments, the AAV capsid for use in a viral vector can be generated by mutagenesis (i.e., by insertion, deletion or substitution) of one of the foregoing AAV capsids or their coding nucleic acids. In some embodiments, the AAV capsid is a chimera that includes domains derived from two or three or four or more of the foregoing AAV capsid proteins. In some embodiments, the AAV capsid is a mosaic of Vpl, Vp2 and Vp3 monomers from two or three different AAVs or recombinant AAVs. In some embodiments, the rAAV composition comprises more than one of the foregoing capsids.

[0118] In some embodiments, the rAAV contains a packaged vector genome that includes an AAV ITR sequence that functions as both an origin of vector DNA replication and a packaging signal for the vector genome when AAV and adenovirus helper functions are provided in trans. Further, the ITR functions as a target for single-stranded endonucleatic nicking by the large Rep protein, separating individual genomes from the replication intermediate.

[0119] In some embodiments, the 5'-ITR sequence is derived from AAV2. In some embodiments, the 3'-ITR sequence is derived from AAV2. In some embodiments, both the 5'-ITR sequence and the 3'-ITR sequence are derived from AAV2. In some embodiments, the 5'-ITR sequence and / or the 3'-ITR sequence is derived from AAV2 and comprises or consists of SEQ ID NO: 17. In other embodiments, the 5'-ITR sequence and / or the 3'-ITR sequence is derived from a non-AAV2 source.

[0120] In various aspects described herein, the rAAV used in the methods disclosed herein can include a packaged genome that contains a promoter sequence useful for driving and regulating transgene expression, such as expression of a protein transgene. In an exemplary embodiment, the promoter sequence is located between a selected 5' ITR sequence and a partial or complete coding sequence for the protein transgene. In some embodiments, the promoter sequence is located downstream of an enhancer sequence. In some embodiments, the promoter sequence is located upstream of an intron sequence.

[0121] In some embodiments, the promoter is selected from the chicken β-actin (CBA) promoter, the cytomegalovirus immediate early gene (CMV) promoter, the transthyretin (TTR) promoter, the thyroxine binding globulin (TBG) promoter, the α-1 antitrypsin (A1AT) promoter, and the CAG promoter.

[0122] In addition to the promoter, the vector may contain other suitable transcription initiation, termination, enhancer sequences, and efficient RNA processing signals. Such sequences include splicing and polyadenylation (polyA) signals, regulatory elements that enhance expression (i.e., WPRE), sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak consensus sequence), and sequences that enhance protein stability.

[0123] In various aspects described herein, the rAAV used in the methods disclosed herein may contain a packaged genome comprising an AAV5' inverted terminal repeat (ITR), a promoter sequence, a protein transduction gene or a functional fragment thereof, a partial or complete coding sequence of a functional variant or isoform, and an AAV3' inverted terminal repeat (ITR).

[0124] In one embodiment, the partial or complete coding sequence of the protein transduction gene is a wild-type coding sequence. As used herein, the term "wild-type" refers to the same biopolymer (e.g., polypeptide sequence or polynucleotide sequence) as a naturally occurring biopolymer (e.g., polypeptide sequence or polynucleotide sequence).

[0125] In another embodiment, the partial or complete coding sequence of the protein transduction gene is a codon-optimized coding sequence. In one embodiment, the partial or complete coding sequence of the protein transduction gene is codon-optimized for expression in humans.

[0126] In various embodiments described herein, rAAV is provided that includes a packaged genome comprising the coding sequence of a transgene. Polypeptides delivered in conjunction with the rAAV described herein include polypeptides that may be useful for the treatment of mammals, including humans. Such polypeptides include, but are not limited to, ornithine transcarbamylase (OTC), glucose 6-phosphatase (G6Pase), Factor VIII, Factor IX, ATP7B, phenylalanine hydroxylase (PAH), argininosuccinate synthetase, cyclin-dependent kinase-like 5 (CDKL5), propionyl-CoA carboxylase subunit α (PCCA) and propionyl-CoA carboxylase subunit β (PCCB), survival motor neuron (SMN), iduronate-2-sulfatase (IDS), α-1-iduronidase (IDUA), tripeptidyl peptidase 1 (TPP1), low density lipoprotein receptor (LDLR), myotubularin 1, acid α-glucosidase (GAA), myotonic dystrophy protein kinase (DMPK), N-sulfo-glucosamine sulfohydrolase (SGSH), fibroblast growth factor-4 (FGF-4), rab escort protein 1 (REP1), carbamoyl synthetase 1 (CPS1), argininosuccinate lyase (ASL), arginase, fumarylacetoacetate hydrolase, α-1 antitrypsin, methylmalonyl-CoA mutase, cystic fibrosis transmembrane conductance regulator (CFTR) protein, and dystrophin gene products (e.g., minidystrophin or microdystrophin). A non-limiting list of suitable transgenes can be found in International Publication No. WO 2019 / 168961, the disclosure of which is incorporated herein by reference.

[0127] In some embodiments, the present invention can be used to deliver a fragment of the aforementioned transgene protein that contains at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, or at least 600 amino acid residues and retains one or more activities associated with the full-length polypeptide (e.g., catalytic activity in the case of an enzyme). Such fragments can be obtained by recombinant techniques that are well known in the art on a routine basis. Further, such fragments can be tested for catalytic activity by routine in vitro assays known to those skilled in the art.

[0128] In some embodiments, the present invention can be used to deliver variants of the aforementioned transgene proteins. In some embodiments, the variant protein can be at least 80% identical (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the wild-type therapeutic protein. In some embodiments, the variant transgene protein can have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 different residues compared to their respective wild-type proteins. Such variants can be obtained by recombinant techniques routine and well known in the art. Further, such variants can be tested for catalytic activity by routine in vitro assays known to those of skill in the art.

[0129] In some embodiments, the rAAV administered according to the methods of the present disclosure can be formulated as a pharmaceutical composition comprising the rAAV and a pharmaceutically acceptable carrier or excipient. Pharmaceutical formulations suitable for administration of rAAV can be found, for example, in U.S. Patent Application Publication No. 2012 / 0219528. Pharmaceutically acceptable carriers (vehicles) useful in the present disclosure are conventional. Remington’s Pharmaceutical Science, by E.W. Martin, Mack Publishing Co., (Easton, Pennsylvania), 15th Edition (1975) describes compositions and formulations suitable for the pharmaceutical delivery of one or more therapeutic compounds, molecules or agents. In some embodiments, the pharmaceutical composition comprising rAAV is formulated for subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intracerebroventricular, or intravenous administration. In an exemplary embodiment, the pharmaceutical composition is formulated for intravenous administration.

[0130] In some embodiments, the rAAV is formulated in a buffer / carrier suitable for injection into a human subject. The buffer / carrier should contain components that prevent the rAAV from sticking to the injection tube but do not interfere with rAAV binding activity in vivo. Various suitable solutions can include buffered saline, surfactants, and physiologically compatible salts or mixtures of salts adjusted to an ionic strength corresponding to about 100 mM sodium chloride (NaCl) to about 250 mM sodium chloride, or one or more of physiologically compatible salts adjusted to an equivalent ionic concentration. The pH can be in the range of 6.5 to 8.5, or 7 to 8.5, or 7.5 to 8. Suitable surfactants, or combinations of surfactants, can be selected from poloxamers, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene 10 (poly(propylene oxide)) sandwiched between two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (macrogol-15 hydroxystearate), LABRASOL (glyceryl polyoxycaprylate), polyoxy 10 oleyl ether, TWEEN® (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol.

[0131] In some embodiments, the viral particles (e.g., recombinant AAV) can be encapsulated or complexed with liposomes, nanoparticles, lipid nanoparticles (LNP), polymers, microparticles, microcapsules, micelles, or extracellular vesicles.

[0132] In yet another aspect, the present disclosure provides a method for administering a nucleic acid (e.g., DNA, siRNA, or mRNA) to a subject, the method comprising first administering a CD19 inhibitor to the subject and then administering a nucleic acid (e.g., DNA, siRNA, or mRNA) to the subject. In an exemplary embodiment, the nucleic acid is an mRNA encoding a protein. Any therapeutic protein of interest (e.g., a human wild-type protein) is suitable for use in the methods described herein. An exemplary list of human proteins can be found in International Publication No. WO 2020 / 118239. In a specific embodiment, the protein is selected from ornithine transcarbamylase (OTC), glucose-6-phosphatase (G6Pase), factor VIII, factor IX, ATP7B, phenylalanine hydroxylase (PAH), argininosuccinate synthetase, cyclin-dependent kinase-like 5 (CDKL5), propionyl-CoA carboxylase subunit α (PCCA) and propionyl-CoA carboxylase subunit β (PCCB), survival motor neuron (SMN), iduronate-2-sulfatase (IDS), α-1-iduronidase (IDUA), tripeptidyl peptidase 1 (TPP1), low density lipoprotein receptor (LDLR), myotubularin 1, acid α-glucosidase (GAA), myotonic dystrophy protein kinase (DMPK), N-sulphoglucosamine sulphotransferase (SGSH), fibroblast growth factor-4 (FGF-4), rab escort protein 1 (REP1), carbamoyl phosphate synthetase 1 (CPS1), argininosuccinate lyase (ASL), arginase, fumarylacetoacetate hydrolase, α-1 antitrypsin, methylmalonyl-CoA mutase, cystic fibrosis transmembrane conductance regulator (CFTR) protein, and dystrophin gene products (e.g., minidystrophin or microdystrophin).

[0133] In certain embodiments, the nucleic acid (e.g., DNA, siRNA or mRNA) is encapsulated or complexed with liposomes, nanoparticles, lipid nanoparticles (LNP), polymers, microparticles, microcapsules, micelles, or extracellular vesicles.

[0134] In some embodiments, the nucleic acid (e.g., DNA, siRNA or mRNA) is encapsulated in lipid nanoparticles (LNP). Examples of LNP capable of delivering nucleic acids are described in International Patent Publication Nos. WO 2015 / 074085, WO 2016 / 081029, WO 2017 / 117530, WO 2018 / 118102, WO 2018 / 119163, WO 2018 / 222926, WO 2019 / 191780, WO 2020 / 016318 and WO 2020 / 154746.

[0135] In one embodiment, the lipid nanoparticles for use in the present invention comprise (a) a nucleic acid (e.g., DNA, siRNA or mRNA), (b) a cationic lipid, (c) an aggregation reducer (such as a PEG-lipid), (d) optionally a non-cationic lipid (such as a neutral lipid), and (e) optionally a sterol. In one embodiment, the lipid nanoparticles comprise (i) at least one cationic lipid, (ii) a neutral lipid, such as DSPC, (iii) a sterol, such as cholesterol, and (iv) a PEG-lipid in a molar ratio of about 20-65% cationic lipid, 5-25% neutral lipid, 25-55% sterol, 0.5-15% PEG-lipid. In some embodiments, the cationic lipid is selected from ATX-002, ATX-081, ATX-095 or ATX-126 as described in International Publication No. WO 2018 / 222926.

[0136] In yet another aspect, the present disclosure provides a method for treating a disorder of interest, the method comprising first administering a CD19 inhibitor to the subject and then administering to the subject a therapeutically effective amount of recombinant adeno-associated virus (rAAV). As used herein, "disease", "disorder" and "condition" are used interchangeably to refer to an abnormal condition of a subject.

[0137] As will be appreciated by those skilled in the art with the present disclosure, individuals suffering from one or more various disorders can benefit from the methods of therapeutic recombinant AAV administration described herein. In some embodiments, the disorder can be selected from OTC deficiency, glycogen storage disease type 1a (GSD1a), hemophilia A, hemophilia B, Wilson's disease, phenylketonuria, citrullinemia, CDKL5 deficiency disorder (CDD), propionic acidemia, spinal muscular atrophy, mucopolysaccharidosis type I, mucopolysaccharidosis type II, CLN2 disease, homozygous familial hypercholesterolemia, Pompe disease, X-linked myotubular myopathy, myotonic dystrophy, Duchenne muscular dystrophy, cystic fibrosis, methylmalonic acidemia, Parkinson's disease, Alzheimer's disease, and Huntington's disease.

[0138] Any suitable method or route can be used to administer the rAAV or rAAV-containing composition described herein. Routes of administration include, for example, systemic, oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intravenous, intrathecal, intracerebroventricular, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration. In some embodiments, the rAAV or composition comprising rAAV is administered intravenously.

[0139] The specific dose administered can be a uniform dose per subject, e.g., 1.0×10 11 ~1.0×10 14It can be a virus with a single genomic copy (GC). Alternatively, the dose for the subject can be adjusted according to the approximate body weight or surface area of the subject. Other factors in determining the appropriate dosage can include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, as well as the age, gender and medical condition of the subject. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely done by those skilled in the art, particularly in light of the dosage information and assays disclosed herein. The dosage can also be determined by using known assays for determining the dosage to be used in conjunction with appropriate dose-response data. The dosage for an individual subject can also be adjusted while monitoring the progression of the disease.

[0140] In some embodiments, rAAV is measured by qPCR or digital droplet PCR (ddPCR) and is, for example, about 1.0×10 11 genomic copies per kilogram of subject body weight (GC / kg) to about 1×10 14 GC / kg, about 5×10 11 genomic copies per kilogram of subject body weight (GC / kg) to about 5×10 13 GC / kg, or about 1×10 12 to about 1×10 13 GC / kg and is administered at a dose of. In some embodiments, rAAV is administered at a dose of about 2×10 12 GC / kg. In some embodiments, rAAV is administered at a dose of about 6×10 12 GC / kg. In some embodiments, rAAV is administered at a dose of about 1×10 13 GC / kg

[0141] In a further aspect, the disclosure is a method for administering at least two doses of recombinant adeno-associated virus (rAAV) to a subject, the method comprising (a) administering a first dose of a CD19 inhibitor to the subject and then administering a first rAAV to the subject; and (b) administering a second dose of a CD19 inhibitor to the subject and then administering a second rAAV to the subject comprises.

[0142] In some embodiments according to this second aspect, the first and second doses of the CD19 inhibitor are the same CD19 inhibitor. Alternatively, the first dose of the CD19 inhibitor may be a different CD19 inhibitor compared to the second dose.

[0143] In some embodiments according to this second aspect, the first rAAV and the second rAAV are the same rAAV. Alternatively, the first rAAV and the second rAAV are different.

[0144] In some embodiments according to this second aspect, the first rAAV and the second rAAV comprise the same vector capsid. In another embodiment, the first rAAV and the second rAAV comprise different vector capsids.

[0145] In some embodiments according to this second aspect, the first rAAV and the second rAAV express the same transgene. In another embodiment, the first rAAV and the second rAAV express different transgenes.

[0146] In some embodiments according to this second aspect, administration of the first dose of the CD19 inhibitor is performed 12 months ahead of administration of the second dose of the CD19 inhibitor. In one embodiment, administration of the first dose of the CD19 inhibitor is performed 2 years ahead of administration of the second dose of the CD19 inhibitor. In another embodiment, administration of the first dose of the CD19 inhibitor is performed more than 3 years, more than 4 years, more than 5 years, more than 6 years, more than 7 years, more than 8 years, more than 9 years, more than 10 years, more than 15 years, or more than 20 years ahead of administration of the second dose of the CD19 inhibitor.

[0147] In some embodiments according to this second aspect, the first dose of the CD19 inhibitor is an anti-CD19 antibody. In some embodiments according to this second aspect, the second dose of the CD19 inhibitor is an anti-CD19 antibody. In some embodiments according to this second aspect, the first and second doses of the CD19 inhibitor are anti-CD19 antibodies. In one embodiment, the anti-CD19 antibody is selected from inebilizumab, tafasitamab, and FMC63. In an exemplary embodiment, the anti-CD19 antibody is inebilizumab.

[0148] In some embodiments according to this second aspect, the amounts of the CD19 inhibitor administered at the first and second doses are the same. Alternatively, the amount of the CD19 inhibitor administered at the first dose may be different from the amount of the CD19 inhibitor administered at the second dose. In an exemplary embodiment, the amounts of the CD19 inhibitor administered at the first and second doses are the same. In another exemplary embodiment, the CD19 inhibitor is inebilizumab, the first dose is 300 mg, and the second dose is 300 mg.

[0149] In some embodiments according to this second aspect, the regimen of administering the CD19 inhibitor followed by administering rAAV may be repeated 3, 4, 5 times or more. Thus, in some embodiments according to this second aspect, the method may further comprise step (c) of administering a third dose of the CD19 inhibitor to the subject and then administering a third rAAV to the subject.

[0150] Throughout this specification, when a composition is described as having, including, or comprising a particular component, or a process and method are described as having, including, or comprising a particular step, it is also contemplated that there are compositions of the invention consisting essentially of, or consisting of, the recited components, and processes and methods according to the invention consisting essentially of, or consisting of, the recited process steps.

[0151] In this application, when an element or constituent is said to be included in a list of elements or constituents that are recited and / or selected from a list of recited elements or constituents, it should be understood that the element or constituent can be any one of the recited elements or constituents, or the element or constituent can be selected from a group consisting of two or more of the recited elements or constituents.

[0152] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein can be combined in various ways, whether explicitly or implicitly in this specification, without departing from the spirit and scope of the invention. For example, when referring to a particular compound, that compound can be used in various embodiments of the compositions of the invention and / or in the methods of the invention, unless otherwise understood from the context. In other words, in this application, while embodiments have been described and depicted in a way that enables clear and concise description and delineation of their application, it is intended and understood that the embodiments can be variously combined or separated without departing from the teachings and the invention(s). For example, it will be understood that all features described and depicted herein can be applicable to all aspects of the invention(s) described and illustrated herein.

[0153] The expression "at least one of" should be understood to individually include each of the recited objects following such notation, and various combinations of two or more of the recited objects, unless otherwise understood from the context and use. The expression "and / or" in relation to three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0154] The use of the terms "include", "includes", "including", "have", "has", "having", "contain", "contains", or "containing", including their grammatical equivalents, should generally be understood as non-limiting and inclusive. For example, unless otherwise specified or understood from the context, it does not exclude additional elements or steps that are not listed.

[0155] When the term "about" is used before a quantitative value, the present invention includes the specific quantitative value itself, unless otherwise specified. As used herein, the term "about" refers to a variation of ±10% from the nominal value, unless otherwise indicated or inferred.

[0156] It should be understood that, as long as the present invention remains practicable, the order of steps or the order for performing a particular operation is not important. Furthermore, two or more steps or operations may be performed simultaneously.

[0157] Any examples or illustrative language in this specification, such as the use of "such as" or "including", are merely intended to better explain the present invention and do not limit the scope of the present invention unless claimed. No language in this specification should be construed as indicating an essential but unclaimed element for the practice of the present invention. Sequence Listing SEQ ID NO:1 (Heavy chain variable region - VH)

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Claims

【Claim 1】 The invention described in the specification.

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