EA047128B1 - THERAPEUTIC DRUGS BASED ON COMPLETELY HUMAN, POST-TRANSLATIONAL MODIFIED ANTIBODIES - Google Patents

Description

This application contains a Sequence Listing which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy in question, created on October 17, 2018, is called 26115_105004_SL.txt and is 400185 bytes in size.

1. Introduction

Described are compositions and methods for delivering a fully human post-translationally modified (HuPTM) therapeutic monoclonal antibody (mAb) or an antigen-binding fragment of a HuPTM therapeutic mAb—for example, a fully glycosylated (HuGly) Fab of a human therapeutic mAb—to a human subject diagnosed with a disease or condition for which Treatment with therapeutic mAb is indicated.

2. Field of technology

Therapeutic mAbs have been shown to be effective in treating a number of diseases and conditions. However, because these agents are only effective for a short period of time, repeated injections are often required over long periods of time, placing significant burden on patients.

3. Essence of the invention

Compositions and methods are described for delivering a HuPTM mAb or an antigen binding fragment of a HuPTM therapeutic mAb (e.g., a fully human glycosylated Fab (HuGlyFab) therapeutic mAb) to a patient (human subject) diagnosed with a disease or condition for which treatment with a therapeutic mAb is indicated. Such antigen binding fragments of therapeutic mAbs include Fab, F(ab') ₂ or scFv (single chain variable fragment) (collectively referred to herein as antigen binding fragment). The HuPTM Fab as used herein may include other antigen binding mAb fragments. In an alternative embodiment, full-length mAbs may be used. Delivery may advantageously be accomplished by gene therapy—for example, by administering a viral vector or other DNA expression construct encoding a therapeutic mAb or an antigen-binding fragment thereof (or a hyperglycosylated derivative of both) to a patient (human subject) diagnosed with a condition, to which treatment with a therapeutic mAb is indicated - to create a permanent depot in the patient's tissue or organ that continuously supplies HuPTM mAb or an antigen-binding fragment of the therapeutic mAb, i.e. glycosylated human transgene product into the target tissue, where the mAb or its antigen-binding fragment exerts its therapeutic effect.

A HuPTM mAb or HuPTM antigen binding fragment encoded by a transgene may include, without limitation, a full-length or antigen binding fragment of a therapeutic antibody that binds to:

nervous system targets, including amyloid beta peptides (Ae or Abeta) derived from amyloid precursor protein (APP) involved in Alzheimer's disease, including, but not limited to, aducanumab, crenezumab, gantenerumab and BAN2401, indicated for the treatment of the disease Alzheimer's (see FIGS. 2A-2C and 2F); may protein is involved in tauopathies, including Alzheimer's disease, progressive supranuclear palsy, frontotemporal dementia, chronic traumatic encephalopathy, Pick's complex, primary age-related tauopathy, including, but not limited to, aTAU (see Fig. 2D) for treatment of tauopathies; and the CGRP receptor involved in migraines and cluster headaches, including, but not limited to, erenumab (AIMOVIG™) (see Fig. 2E), eptinezumab, fremanezumab and galcanezumab for the treatment of migraines and cluster headaches;

interleukins or interleukin receptors, including, but not limited to, IL4R, such as dupilumab (see Fig. 3A), indicated for the treatment of atopic dermatitis; IL17A, such as ixekizumab (TALTZ®) or secukinumab (COSENTYX®) (see Figs. 3B and 3C), indicated for the treatment of plaque psoriasis, psoriatic arthritis and ankylosing spondylitis; IL-5, such as mepolizumab (NUCALA®) (see Fig. 3D), indicated for the treatment of asthma; and IL12/IL23, such as ustekinumab (STELARA®) (see FIG. 3E), indicated for the treatment of psoriasis and Crohn's disease;

integrin, including, but not limited to, vedolizumab (ENTYVIO®), indicated for the treatment of ulcerative colitis and Crohn's disease (see FIG. 4A), and natalizumab (anti-integrin alpha 4) for the treatment of multiple sclerosis and Crohn's disease (see FIG. 4B);

hypercholesterolemia and cardiovascular disease targets such as PCSK9, including, but not limited to, alirocumab (PRALUENT®) and evolocumab (REPATHA®), indicated for the treatment of HeFH and HoFH (see FIGS. 5A and 5B); or ANGPTL3, including, but not limited to, evinacumab (see Fig. 5C), indicated for the treatment of HoFH and severe forms of dyslipidemia, and pro-inflammatory/pro-atherogenic phospholipids, including, without limitation, E06-scFv for the treatment of cardiovascular diseases, including including atherosclerosis (see Fig. 5D);

RANKL, including but not limited to denosumab (XGEVA® and PROLIA®), indicated for le

- 1047128 for the treatment of osteoporosis, increased bone mass in patients with breast and prostate cancer, and for the prevention of skeletal-related events due to bone metastases (see FIG. 6);

PD-1 or PD-L1 or PD-L2 (these antibodies are sometimes referred to herein as PD-1 blockers), including, but not limited to, nivolumab (OPDIVO®) and pembrolizumab (KEYTRUDA®), indicated for the treatment of metastatic melanoma, lymphomas and non-small cell lung carcinomas (see FIGS. 7A and 7B);

BLyS (B-lymphocyte stimulator, also known as B-cell activating factor (BAFF)), including, but not limited to, belimumab (BENLYSTA®), indicated for the treatment of systemic lupus erythematosus (SLE) (see Fig. 8E);

ocular targets, including, but not limited to, VEGF (vascular endothelial growth factor), including, without limitation, ranibizumab (LUCENTIS®), bevacizumab (AVASTEST®), and brolucizumab, indicated for the treatment of neovascular age-related macular degeneration (eg, wet AMD forms) (see Figs. 8A, 8B and 8D); factor D, including, but not limited to, lampalizumab, for the treatment of dry AMD (see Fig. 8C); and matrix metalloproteinase 9 (MMP9), including, but not limited to, andecaliximab, for the treatment of dry AMD (Fig. 8G);

TNF-alpha, including, but not limited to, adalimumab (HUMIRA®) and infliximab (REMICADE®), indicated for the treatment of rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, plaque psoriasis and ulcerative colitis (Fig. 9A for adalimumab and Fig. 9B for infliximab); and plasma protein targets, such as human complement proteins, including, but not limited to, anti-C5 and C5a complement proteins, such as eculizumab (SOLIRIS®), for the treatment of patients with paroxysmal nocturnal hemoglobinuria (PNH) to reduce hemolysis or treat atypical hemolytic uremic syndrome (aHUS) to inhibit complement-mediated thrombotic microangiopathy (Fig. 8F); and plasma kallikrein, including, but not limited to, lanadelumab for the treatment of hereditary angioedema (see Fig. 8H);

or such mAbs or antigen binding fragments designed to contain additional glycosylation sites in the Fab domain (for example, see Courtois et al., 2016, mAbs 8: 99-112, which is incorporated herein by reference in its entirety, for descriptions of antibody derivatives that are hyperglycosylated in the Fab domain of the full-length antibody).

The recombinant vector used to deliver the transgene includes non-replicating recombinant adeno-associated virus (rAAV) vectors. However, other viral vectors can be used, including, but not limited to, lentiviral vectors; vaccinia viral vectors or non-viral expression vectors called naked DNA constructs. Transgene expression can be controlled by constitutive or tissue-specific expression control elements.

Gene therapy constructs are designed such that both heavy and light chains are expressed. The coding sequences for the heavy and light chains can be constructed in a single construct in which the heavy and light chains are separated by a cleavable linker or IRES such that separate heavy and light chain polypeptides are expressed. In some embodiments, the coding sequences encode Fab or F(ab') ₂ or scFv. In other embodiments, the constructs express a scFv in which the heavy and light chain variable domains are connected via a flexible, non-cleavable linker. In some embodiments, the construct is expressed from the N terminus of NH ₂ -V _L -linker-V _H -COOH or NH ₂ -V _H -linker Vl-COOH.

Therapeutic antibodies delivered by gene therapy have several advantages over injected or infused therapeutic antibodies, which dissipate over time, resulting in peak and trough levels. Continuous expression of the antibody of a transgene product, as opposed to repeated administration of the antibody, ensures a more constant level of antibody present at the site of action and is less risky and more convenient for patients since fewer injections are required. In addition, antibodies expressed from transgenes are post-translationally modified in a different way than those directly introduced due to the different microenvironments present during and after translation. Without being bound by any particular theory, this results in antibodies that have different diffusion, bioactivity, distribution, affinity, pharmacokinetics and immunogenicity characteristics such that the antibodies delivered to the site of action are biologically superior to those administered directly.

In addition, antibodies expressed from transgenes in vivo are unlikely to contain degradation products associated with antibodies produced by recombinant technologies, such as protein aggregation and protein oxidation. Aggregation is a problem associated with protein production and storage due to high protein concentrations, surface interaction with production equipment and containers, and purification with certain buffer systems. These conditions which

- 2 047128 promote aggregation, do not exist when transgenes are expressed in gene therapy. Oxidation, such as the oxidation of methionine, tryptophan, and histidine, is also associated with protein production and storage and is caused by stressful cell culture conditions, metal-air contact, and impurities in buffers and excipients. Proteins expressed by transgenes in vivo can also be oxidized under unfavorable conditions. However, humans and many other organisms are equipped with an antioxidant defense system that not only reduces oxidative stress, but sometimes also restores and/or reverses the oxidation process. Thus, proteins produced in vivo are unlikely to be in oxidized form. Both aggregation and oxidation can affect activity, pharmacokinetics (clearance), and immunogenicity.

Pharmaceutical compositions suitable for administration to human subjects contain a suspension of the recombinant vector in a formulation buffer containing a physiologically compatible aqueous buffer, a surfactant and optional excipients.

The present invention is based, in part, on the following principles.

(i) mAb-based therapeutics currently available on the market are characterized by immunoglobulin G (IgG) isotypes such as IgG1, IgG2 and IgG4, which are generally characterized by pharmacokinetic (PK) characteristics such as slow clearance, prolonged half-life and limited tissue distribution. Following intravenous administration, typical serum PK mAb profiles are biphasic with a rapid distribution phase and a slower elimination phase; thus, repeated administration is required to maintain doses needed to treat chronic conditions. In addition, the distribution of mAbs is typically limited to vascular and interstitial spaces due to their large size and hydrophilicity. The distribution of mAbs from the bloodstream into most tissues typically ranges from 5 to 15%, with the exception of the brain, where it is much lower. (See, for example, Kamath, 2016, Drug Discovery Today: Technologies 21-22: 75-83, which is incorporated herein by reference in its entirety.) Continuous in situ production of HuPTM mAb or HuPTM Fab avoids re-administration and allows Fabs that would otherwise have too short a systemic half-life to be used to achieve efficacy; and the described methods of administration provide direct access to target tissues, such as the brain, and delivery of higher doses to such tissues can be achieved.

(ii) The Fab region of a number of therapeutic mAbs possesses glycosylation sites. For example, see FIG. 2A-2F, 3A-3E, 4A-4B, 5A-5D, 6, 7A-7B, 8A-8H and 9A-9B, which identify and highlight in blue and green, respectively, consensus and non-consensus glycosylation sites at asparagine (N) , as well as glutamine (Q) residues, which are glycosylation sites in the Fab region of certain therapeutic mAbs. (See, for example, Valliere-Douglass et al., 2009, J. Biol. Chem. 284: 32493-32506 and Valliere-Douglass et al., 2010, J. Biol. Chem. 285: 16012-16022, each of which is incorporated by reference in its entirety to identify N-linked glycosylation sites in antibodies). Additionally, O-glycosylation involves the addition of N-acetylgalactosamine to serine or threonine residues by an enzyme. It has been demonstrated that amino acid residues present in the hinge region of antibodies can be O-glycosylated. The ability to glycosylate provides another advantage to the therapeutic antibodies presented herein over, for example, antigen binding moieties produced in E. coli, again since E. coli does not naturally contain a mechanism equivalent to that used for O-glycosylation in of people. (Instead, O-glycosylation in E. coli has only been demonstrated when the bacteria are modified to contain specific O-glycosylation machinery. See, e.g., Farid-Moayer et al., 2007, J. Bacteriol. 189:8088-8098) . In addition, the amino acid sequence of a Fab can be modified to construct hyperglycosylated variants (e.g., see the amino acid substitutions that can be made to construct hyperglycosylated Fab regions of therapeutic antibodies shown in FIGS. 11A and 11B; and Courtois et al., 2016 , mAbs 8: 99-112, which is incorporated herein by reference in its entirety to describe antibody derivatives that are hyperglycosylated in the Fab domain of the full-length antibody).

(iii) In addition to glycosylation sites, Fab regions may contain tyrosine (Y) sulfation sites in or near the CDRs; see fig. 2A-2F, 3A-3E, 4A-4B, 5A-5D, 6, 7A-7B, 8A-8H, and 9A9B, which identify tyrosine-O-sulfation sites in the Fab region of certain therapeutic mAbs, as highlighted in yellow (See, for example, Yang et al., 2015, Molecules 20: 2138-2164 (particularly at 2154), which is incorporated by reference in its entirety for the analysis of amino acids surrounding tyrosine residues subjected to protein tyrosine sulfation). The rules can be summarized as follows: Y residues with E or D within the +5 to -5 position of Y, and where the -1 position of Y is a neutral or acidic charged amino acid, but not a basic amino acid, such as R, K or H , which eliminates sulfation.

(iv) Glycosylation of Fab regions such as those shown in FIG. 2A-2F, 3A-3E, 4A-4B, 5A5D, 6, 7A-7B, 8A-8H and 9A-9B, human cells will lead to the addition of glycans that can

- 3 047128 can improve the stability, half-life and reduce undesirable aggregation and/or immunogenicity of the transgene product. (See, e.g., Bovenkamp et al., 2016, J. Immunol. 196: 1435-1441, for a review of the growing importance of Fab glycosylation; and Fig. 10, which identifies glycans that can be attached to HuGlyFab (adapted from Bondt et al., 2014, Mol & Cell Proteomics 13.1: 3029-2029)). Fab and Fc fragments of antibodies have been shown to have different glycosylation patterns, with Fab glycans having high levels of galactosylation, sialylation, and bisection (e.g., by GlcNAc fission) but low fucosylation relative to Fc glycans.

(For example, see Bondt et al., 2014, Mol. & Cell. Proteomics 13.11:3029-3039, incorporated herein by reference in its entirety for the disclosure of Fab-associated Nglycans).

(v) It is significant that the glycans that are added to the HuGlyFab of the present invention are highly processed N-glycans of the complex type that contain 2,6 sialic acid. Such glycans are not present in (a) therapeutic mAbs produced in E. coli (which are not glycosylated at all); (b) in therapeutic antibodies produced in CHO cells that lack the 2,6-sialyltransferase required to add 2,6-sialic acid during glycosylation; or (c) in therapeutic antibodies produced either in CHO or in murine cell lines that add N-glycolylneuraminic acid (Neu5Gc or NeuGc), which is not naturally occurring in humans (and potentially immunogenic), instead of N-acetylneuraminic acid (Neu5Ac) - predominant human sialic acid. See, for example, Dumont et al., 2015, Crit. Rev. Biotechnol. 36(6):1110-1122; Huang et al., 2006, Anal. Biochem. 349:197-207 (NeuGc is the predominant sialic acid in murine cell lines such as SP2/0 and NS0) and Song et al., 2014, Anal. Chem. 86:5661-5666, each of which is incorporated herein by reference in its entirety.

(vi) The human glycosylation pattern of the HuGlyFab of the present invention should reduce the immunogenicity of the transgene product and increase the efficiency. Importantly, when the antigen binding fragments used in accordance with the methods described herein are expressed in human target cells, the need for in vitro production in prokaryotic host cells (eg, E. coli) or eukaryotic host cells (eg, CHO cells or mouse NS0 or SP2/0 cells). Instead, as a result of the methods described herein (eg, using human target cells to express antigen binding fragments), the N-glycosylation sites of the antigen binding fragments preferentially contain glycans suitable and useful for treating humans. Such an advantage is not achievable when CHO cells, murine cells, or E. coli are used in the production of antibodies/antigen-binding fragments because, for example, (a) CHO cells lack the components necessary to add certain glycans (e.g., 2,6-sialic acid and halving GlcNAc); (b) CHO cells and mouse cells (NS0 and SP2/0 cells) add Neu5Gc as non-human sialic acid instead of Neu5Ac; (c) CHO cells can also produce an immunogenic glycan, α-Gal antigen, which reacts with anti-α-Gal antibodies present in most people, which at high concentrations can cause anaphylaxis (see, for example, Bosques, 2010, Nat Biotech 28 :1153-1156); and (d) E. coli does not naturally contain the components required for N-glycosylation. (vii) tyrosine sulfation of Fab regions such as those shown in FIG. 2A2F, 3A-3E, 4A-4B, 5A-5D, 6, 7A-7B, 8A-8H and 9A-9B, a powerful post-translational process in many human cells, should result in transgene products with increased avidity for their molecular targets. Indeed, tyrosine sulfation of Fab antibodies has been shown to dramatically increase antigen avidity and activity. (See, for example, Loos et al., 2015, PNAS 112: 12675-12680 and Choe et al., 2003, Cell 114: 161-170). Such post-translational modifications are not present on therapeutic antibodies produced in E. coli (a host that does not possess the enzymes required for tyrosine sulfation) and are, at best, underrepresented in therapeutic mAbs produced in CHO cells. CHO cells are not secretory cells and have a limited ability to post-translationally sulfate tyrosine. (See, for example, Loos et al., 2015, PNAS 112: 12675-12680 and Choe et al., 2003, Cell 114: 161-170, especially the discussion on page 1537).

For the foregoing reasons, production of a HuPTM mAb or HuPTM Fab should result in a biologically improved molecule for the treatment of a disease via gene therapy - for example, by administering a viral vector or other DNA expression construct encoding a full-length or HuPTM Fab therapeutic mAb to a human subject. who has been diagnosed with a disease symptom for that mAb, to establish in the subject a permanent depot that continuously supplies the human glycosylated sulfated transgene product produced by the subject's transduced cells. The cDNA design for HuPTMmAb or HuPTM Fab must include a signal peptide that ensures proper co- and post-translational processing (protein glycosylation and sulfation) by transduced human cells.

- 4 047128

As an alternative or complementary to gene therapy treatment, full-length or HuPTM Fab can be produced in human cell lines using recombinant DNA technology and the glycoprotein can be administered to patients.

Combination therapies providing delivery of full-length or HuPTM Fab to a patient, accompanied by the administration of other available treatments, are covered by the methods of the present invention. Additional treatments may be given before, simultaneously, or after gene therapy treatment. Such additional treatments may include, without limitation, co-therapy with a therapeutic mAb.

Methods for making viral vectors, in particular AAV-based viral vectors, are also provided. In specific embodiments, methods are provided for producing recombinant AAVs comprising culturing a host cell containing an artificial genome comprising a cis-expression cassette flanked by an AAV ITR, wherein the cis-expression cassette contains a transgene encoding a therapeutic antibody operably linked to expression control elements that will control transgene expression in human cells; a trans-expression cassette lacking the AAV ITRs, wherein the trans-expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that direct expression of the AAV rep and capsid proteins in a host cell in culture and provide the rep and cap proteins in trans ; sufficient auxiliary functions of the adenovirus for replication and packaging of the artificial genome with AAV capsid proteins; and recovery of recombinant AAV encapsulating the artificial genome from cell culture.

3.1. Exemplary Embodiments

Composition.

1. A pharmaceutical composition for the treatment of Alzheimer's disease, migraines, cluster headaches or tauopathies, including chronic traumatic encephalopathy, progressive supranuclear palsy and frontotemporal dementia in a human subject in need thereof, comprising an adeno-associated virus (AAV) vector containing:

(a) a viral capsid that is at least 95% identical to the amino acid sequence of the AAV9 capsid (SEQ ID NO: 79) or the AAVrh10 capsid (SEQ ID NO: 80), and (b) an artificial genome containing an expression cassette flanked by inverted terminals AAV repeats (ITR), wherein the expression cassette contains a transgene encoding an anti-amyloid beta, anti-tau or anti-CGRPR mAb or an antigen binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in human CNS cells;

wherein said AAV vector is formulated for intrathecal administration into the CNS of said subject.

2. The pharmaceutical composition according to claim 1, in which the mAb to amyloid β is aducanumab, crenezumab, gantenumab or BAN2401, and the mAb to tau is aTAU, and the mAb to CGRPR is erenumab, eptinezumab, fremanezumab or galcanezumab.

3. The pharmaceutical composition according to claim 1 or 2, wherein the antigen binding fragment is a Fab, F(ab') ₂ or single chain variable domain (scFv).

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antigen binding fragment comprises a heavy chain with the amino acid sequence SEQ ID NO: 1 and a light chain with the amino acid sequence SEQ ID NO: 2 or a heavy chain with the amino acid sequence SEQ ID NO: 3 and a light chain with the amino acid sequence SEQ ID NO: 4; or a heavy chain having the amino acid sequence SEQ ID NO: 5 and a light chain having the amino acid sequence SEQ ID NO: 6; or a heavy chain having the amino acid sequence of SEQ ID NO: 53 and a light chain having the amino acid sequence of SEQ ID NO: 54; a heavy chain having the amino acid sequence SEQ ID NO: 55 and a light chain having the amino acid sequence SEQ ID NO: 56; or a heavy chain having the amino acid sequence of SEQ ID NO: 57 and a light chain having the amino acid sequence of SEQ ID NO: 58.

5. The pharmaceutical composition according to claim 4, in which the transgene contains the nucleotide sequence SEQ ID NO: 101 encoding the heavy chain, and the nucleotide sequence SEQ ID NO: 102 encoding the light chain; or the nucleotide sequence SEQ ID NO: 103 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 104 encoding the light chain; or the nucleotide sequence SEQ ID NO: 105 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 106 encoding the light chain; or a heavy chain having the nucleotide sequence of SEQ ID NO: 153 and a light chain having the nucleotide sequence of SEQ ID NO: 154; a heavy chain having the nucleotide sequence SEQ ID NO: 155 and a light chain having the nucleotide sequence SEQ ID NO: 156, or a heavy chain having the nucleotide sequence SEQ ID NO: 157 and a light chain having the nucleotide sequence SEQ ID NO: 158.

6. Pharmaceutical composition according to any one of claims 1 to 4, in which the antibody or antigen-binding fragment thereof is a hyperglycosylated mutant.

7. Pharmaceutical composition according to any one of claims 1 to 6, in which the transgene encodes a signal

- 5 047128 sequence at the N-terminus of the heavy chain and light chain of the specified antigen-binding fragment, which directs secretion and post-translational modification in the specified cells of the human CNS.

8. The pharmaceutical composition according to claim 7, in which the specified signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a signal sequence from table. 1.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the AAV capsid is AAV9.

10. A pharmaceutical composition for the treatment of atopic dermatitis in a human subject in need thereof, comprising an AAV vector containing:

(a) a viral capsid that is at least 95% identical to the amino acid sequence of the AAV8 capsid (SEQ ID NO: 78) or the AAV9 capsid (SEQ ID NO: 79); and (b) an artificial genome containing an expression cassette flanked by an AAV ITR, wherein the expression cassette contains a transgene encoding an anti-IL4R mAb or an antigen-binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in human liver cells or muscle cells person;

wherein said AAV vector is formulated for intravenous administration into the liver or muscle of said subject.

11. The pharmaceutical composition according to claim 10, wherein the anti-IL4R mAb is dupilumab.

12. The pharmaceutical composition according to claim 10 or 11, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

13. The pharmaceutical composition according to any one of claims 10 to 12, wherein the antigen binding fragment contains a heavy chain with the amino acid sequence SEQ ID NO: 7 and a light chain with the amino acid sequence SEQ ID NO: 8.

14. The pharmaceutical composition according to claim 13, wherein the transgene contains a nucleotide sequence of SEQ ID NO: 107 encoding a heavy chain and a nucleotide sequence of SEQ ID NO: 108 encoding a light chain.

15. Pharmaceutical composition according to any one of claims 10-13, in which the antibody or antigen-binding fragment thereof is a hyperglycosylated mutant.

16. The pharmaceutical composition according to any one of claims 10 to 15, wherein the transgene encodes a signal sequence at the N-terminus of the heavy chain and the light chain of said antigen-binding fragment, which directs secretion and post-translational modification in said human liver cells or human muscle cells.

17. The pharmaceutical composition according to claim 16, in which the specified signal sequence is selected from the signal sequences in table. 2 or 3.

18. The pharmaceutical composition according to any one of claims 10 to 17, wherein the AAV capsid is AAV8.

19. A pharmaceutical composition for the treatment of psoriasis, psoriatic arthritis, ankylosing spondylitis or Crohn's disease in a human subject in need thereof, comprising an AAV vector containing:

(a) a viral capsid that is at least 95% identical to the amino acid sequence of the AAV8 capsid (SEQ ID NO: 78) or the AAV9 capsid (SEQ ID NO: 79); and (b) an artificial genome containing an expression cassette flanked by an AAV ITR, wherein the expression cassette contains a transgene encoding an anti-IL17A or anti-IL12/IL23 mAb or an antigen-binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in cells human liver or human muscle cells;

wherein said AAV vector is formulated for intravenous administration into the liver or muscle of said subject.

20. The pharmaceutical composition according to claim 19, in which the mAb to IL17A or to IL12/IL23 is ixekizumab, secukinumab or ustekinumab.

21. The pharmaceutical composition according to claim 19 or 20, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

22. The pharmaceutical composition according to any one of claims 19 to 21, wherein the antigen binding fragment comprises a heavy chain with the amino acid sequence SEQ ID NO: 9 and a light chain with the amino acid sequence SEQ ID NO: 10 or a heavy chain with the amino acid sequence SEQ ID NO: 11 and a light chain with the amino acid sequence SEQ ID NO: 12; or a heavy chain having the amino acid sequence of SEQ ID NO: 13 and a light chain having the amino acid sequence of SEQ ID NO: 14.

23. The pharmaceutical composition according to claim 22, in which the transgene contains the nucleotide sequence SEQ ID NO: 109 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 110 encoding the light chain; or the nucleotide sequence SEQ ID NO: 111 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 112 encoding the light chain;

- 6 047128 or the nucleotide sequence SEQ ID NO: 113 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 114 encoding the light chain.

24. The pharmaceutical composition according to any one of claims 19 to 22, wherein the antibody or antigen-binding fragment thereof is a hyperglycosylated mutant.

25. The pharmaceutical composition according to any one of claims 19 to 24, wherein the transgene encodes a signal sequence at the N-terminus of the heavy chain and the light chain of said antigen-binding fragment, which directs secretion and post-translational modification in said human liver cells or human muscle cells.

26. The pharmaceutical composition according to claim 25, in which the specified signal sequence is selected from the signal sequences in table. 2 or 3.

27. The pharmaceutical composition according to any one of claims 19 to 26, wherein the AAV capsid is AAV8.

28. A pharmaceutical composition for the treatment of asthma in a human subject in need thereof, comprising an AAV vector comprising:

(a) a viral capsid that is at least 95% identical to the amino acid sequence of the AAV8 capsid (SEQ ID NO: 78) or the AAV9 capsid (SEQ ID NO: 79); and (b) an artificial genome containing an expression cassette flanked by an AAV ITR, wherein the expression cassette contains a transgene encoding an anti-IL-5 mAb or an antigen-binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in human liver cells or human muscle cells;

wherein said AAV vector is formulated for intravenous administration into the liver or muscle of said subject.

29. The pharmaceutical composition according to claim 28, wherein the anti-IL-5 mAb is mepolizumab.

30. The pharmaceutical composition according to claim 28 or 29, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

31. The pharmaceutical composition according to any one of claims 28 to 30, wherein the antigen binding fragment comprises a heavy chain with the amino acid sequence SEQ ID NO: 15 and a light chain with the amino acid sequence SEQ ID NO: 16.

32. The pharmaceutical composition according to claim 31, wherein the transgene contains a nucleotide sequence of SEQ ID NO: 115 encoding a heavy chain and a nucleotide sequence of SEQ ID NO: 116 encoding a light chain.

33. The pharmaceutical composition according to any one of claims 28-31, in which the antibody or antigen-binding fragment thereof is a hyperglycosylated mutant.

34. The pharmaceutical composition according to any one of claims 28 to 33, wherein the transgene encodes a signal sequence at the N-terminus of the heavy chain and the light chain of said antigen binding fragment, which directs secretion and post-translational modification in said human liver cells or human muscle cells.

35. The pharmaceutical composition according to claim 34, in which the specified signal sequence is selected from the signal sequences in table. 2 or 3.

36. The pharmaceutical composition according to any one of claims 28 to 35, wherein the AAV capsid is AAV8.

37. A pharmaceutical composition for the treatment of multiple sclerosis, ulcerative colitis or Crohn's disease in a human subject in need thereof, comprising an AAV vector containing:

(a) a viral capsid that is at least 95% identical to the amino acid sequence of the AAV8 capsid (SEQ ID NO: 78), the AAV9 capsid (SEQ ID NO: 79) or the AAVrh10 capsid (SEQ ID NO: 80); and (b) an artificial genome containing an expression cassette flanked by an AAV ITR, wherein the expression cassette contains a transgene encoding an anti-integrin mAb or an antigen-binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in human liver cells, muscle cells human or human CNS cells;

wherein said AAV vector is formulated for intravenous administration into the liver or muscle of said subject or for intrathecal administration into the CNS of said subject.

38. The pharmaceutical composition according to claim 37, wherein the anti-integrin mAb is vedolizumab or natalizumab.

39. The pharmaceutical composition according to claim 37 or 38, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

40. The pharmaceutical composition according to any one of claims 37 to 39, wherein the antigen binding fragment comprises a heavy chain with the amino acid sequence SEQ ID NO: 17 and a light chain with the amino acid sequence SEQ ID NO: 18 or a heavy chain with the amino acid sequence SEQ ID NO: 19 and a light chain having the amino acid sequence SEQ ID NO: 20.

41. The pharmaceutical composition according to claim 40, in which the transgene contains a nucleotide sequence

- 7 047128 the activity of SEQ ID NO: 117, encoding the heavy chain, and the nucleotide sequence of SEQ ID NO: 118, encoding the light chain; or the nucleotide sequence SEQ ID NO: 119 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 120 encoding the light chain.

42. The pharmaceutical composition according to any one of claims 37-41, in which the antibody or antigen-binding fragment thereof is a hyperglycosylated mutant.

43. The pharmaceutical composition according to any one of claims 37 to 42, wherein the transgene encodes a signal sequence at the N-terminus of the heavy chain and the light chain of said antigen-binding fragment, which directs secretion and post-translational modification in said human liver cells or human muscle cells.

44. The pharmaceutical composition according to claim 43, in which the specified signal sequence is selected from the signal sequences in table. 1, 2 or 3.

45. The pharmaceutical composition according to any one of claims 37 to 44, wherein the AAV capsid is AAV8.

46. A pharmaceutical composition for the treatment of HeFH, HoFH, dyslipidemia, cardiovascular diseases, including atherosclerotic cardiovascular disease (ACD), atherosclerotic plaque formation, abnormally high levels of non-HDL cholesterol and LDL, aortic stenosis, liver stenosis or hypercholesterolemia, in human subject in need, containing an AAV vector containing:

(a) a viral capsid that is at least 95% identical to the amino acid sequence of the AAV8 capsid (SEQ ID NO: 78) or the AAV9 capsid (SEQ ID NO: 79); and (b) an artificial genome containing an expression cassette flanked by an AAV ITR, wherein the expression cassette contains a transgene encoding an anti-PCSK9, anti-ANGPTL3, or anti-OxPL mAb, or an antigen-binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in human liver cells or human muscle cells; wherein said AAV vector is formulated for intravenous administration into the liver or muscle of said subject.

47. The pharmaceutical composition according to claim 46, in which the mAb to PCSK9 or to ANGPTL3 is alirocumab, evolocumab or evinacumab or to OxPL is E06.

48. The pharmaceutical composition according to claim 46 or 47, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

49. The pharmaceutical composition according to any one of claims 46 to 48, wherein the antigen binding fragment comprises a heavy chain having the amino acid sequence SEQ ID NO: 21 and a light chain having the amino acid sequence SEQ ID NO: 22 or a heavy chain having the amino acid sequence SEQ ID NO: 23 and a light chain with the amino acid sequence SEQ ID NO: 24; a heavy chain having the amino acid sequence SEQ ID NO: 25 and a light chain having the amino acid sequence SEQ ID NO: 26; or a heavy chain having the amino acid sequence SEQ ID NO: 59 and a light chain having the amino acid sequence SEQ ID NO: 60.

50. The pharmaceutical composition according to claim 49, wherein the transgene contains the nucleotide sequence SEQ ID NO: 121 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 122 encoding the light chain; or the nucleotide sequence SEQ ID NO: 123 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 124 encoding the light chain; the nucleotide sequence SEQ ID NO: 125 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 126 encoding the light chain; or the nucleotide sequence SEQ ID NO: 159 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 160 encoding the light chain.

51. The pharmaceutical composition according to any one of claims 44-50, in which the antibody or antigen-binding fragment thereof is a hyperglycosylated mutant.

52. The pharmaceutical composition according to any one of claims 44 to 51, wherein the transgene encodes a signal sequence at the N-terminus of the heavy chain and the light chain of said antigen binding fragment, which directs secretion and post-translational modification in said human liver cells or human muscle cells.

53. The pharmaceutical composition according to claim 52, in which the specified signal sequence is selected from the signal sequences in table. 2 or 3.

54. The pharmaceutical composition according to any one of claims 44 to 53, wherein the AAV capsid is AAV8.

55. A pharmaceutical composition for the treatment of osteoporosis in a human subject in need thereof, comprising an AAV vector containing:

(a) a viral capsid that is at least 95% identical to the amino acid sequence of the AAV8 capsid (SEQ ID NO: 78) or the AAV9 capsid (SEQ ID NO: 79); and (b) an artificial genome containing an expression cassette flanked by an AAV ITR, wherein the expression cassette contains a transgene encoding an anti-RANKL mAb or an antigen-binding fragment thereof operably linked to one or more regulatory sequences that

- 8 047128 which control the expression of the transgene in human liver cells or human muscle cells;

wherein said AAV vector is formulated for intravenous administration into the liver or muscle of said subject.

56. The pharmaceutical composition according to claim 55, wherein the mAb to RANLK is denosumab.

57. The pharmaceutical composition according to claim 55 or 56, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

58. The pharmaceutical composition according to any one of claims 55 to 57, wherein the antigen binding fragment comprises a heavy chain having the amino acid sequence SEQ ID NO: 27 and a light chain having the amino acid sequence SEQ ID NO: 28.

59. The pharmaceutical composition according to claim 58, wherein the transgene contains a nucleotide sequence of SEQ ID NO: 127 encoding a heavy chain and a nucleotide sequence of SEQ ID NO: 128 encoding a light chain.

60. The pharmaceutical composition according to any one of claims 55-59, in which the antibody or antigen-binding fragment thereof is a hyperglycosylated mutant.

61. The pharmaceutical composition according to any one of claims 55 to 60, wherein the transgene encodes a signal sequence at the N-terminus of the heavy chain and the light chain of said antigen binding fragment, which directs secretion and post-translational modification in said human liver cells or human muscle cells.

62. The pharmaceutical composition according to claim 61, in which the specified signal sequence is selected from the signal sequences in table. 2 or 3.

63. The pharmaceutical composition according to any one of claims 55 to 62, wherein the AAV capsid is AAV8.

64. A pharmaceutical composition for the treatment of metastatic melanoma, lymphoma or non-small cell lung carcinoma in a human subject in need thereof, comprising an AAV vector comprising:

(a) a viral capsid that is at least 95% identical to the amino acid sequence of the AAV8 capsid (SEQ ID NO: 78) or the AAV9 capsid (SEQ ID NO: 79); and (b) an artificial genome containing an expression cassette flanked by an AAV ITR, wherein the expression cassette contains a transgene encoding a PD-1 blocker mAb or an antigen binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in human liver cells or human muscle cells;

wherein said AAV vector is formulated for intravenous administration into the liver or muscle of said subject.

65. The pharmaceutical composition according to claim 64, wherein the PD-1 blocker mAb is nivolumab or pembrolizumab.

66. The pharmaceutical composition according to claims 64 or 65, wherein the antigen binding fragment is a Fab, F(ab')2 or scFv.

67. The pharmaceutical composition according to any one of claims 64 to 66, wherein the antigen binding fragment comprises a heavy chain having the amino acid sequence SEQ ID NO: 29 and a light chain having the amino acid sequence SEQ ID NO: 30 or a heavy chain having the amino acid sequence SEQ ID NO: 31 and a light chain having the amino acid sequence SEQ ID NO: 32.

68. The pharmaceutical composition according to claim 67, wherein the transgene contains the nucleotide sequence SEQ ID NO: 129 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 130 encoding the light chain; or the nucleotide sequence SEQ ID NO: 131 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 132 encoding the light chain.

69. The pharmaceutical composition according to any one of claims 64 to 68, wherein the antibody or antigen-binding fragment thereof is a hyperglycosylated mutant.

70. The pharmaceutical composition according to any one of claims 64 to 69, wherein the transgene encodes a signal sequence at the N-terminus of the heavy chain and the light chain of said antigen binding fragment, which directs secretion and post-translational modification in said human liver cells or human muscle cells.

71. The pharmaceutical composition according to claim 70, in which the specified signal sequence is selected from the signal sequences in table. 2 or 3.

72. The pharmaceutical composition according to any one of claims 64 to 71, wherein the AAV capsid is AAV8.

73. A pharmaceutical composition for the treatment of systemic lupus erythematosus (SLE) in a human subject in need thereof, comprising an AAV vector containing:

(a) a viral capsid that is at least 95% identical to the amino acid sequence of the AAV8 capsid (SEQ ID NO: 78) or the AAV9 capsid (SEQ ID NO: 79); and (b) an artificial genome containing an expression cassette flanked by an AAV ITR, wherein the expression cassette contains a transgene encoding an anti-BLyS mAb or an antigen-binding fragment thereof operably linked to one or more regulatory sequences that

- 9 047128 control transgene expression in human liver cells or human muscle cells;

wherein said AAV vector is formulated for intravenous administration into the liver or muscle of said subject.

74. The pharmaceutical composition according to claim 73, wherein the anti-BLyS mAb is belimumab.

75. The pharmaceutical composition according to claims 73 or 74, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

76. The pharmaceutical composition according to any one of claims 73 to 75, wherein the antigen binding fragment comprises a heavy chain with the amino acid sequence SEQ ID NO: 41 and a light chain with the amino acid sequence SEQ ID NO: 42.

77. The pharmaceutical composition according to claim 76, wherein the transgene contains a nucleotide sequence of SEQ ID NO: 141 encoding a heavy chain and a nucleotide sequence of SEQ ID NO: 142 encoding a light chain.

78. The pharmaceutical composition according to any one of claims 73-77, in which the antibody or antigen-binding fragment thereof is a hyperglycosylated mutant.

79. The pharmaceutical composition according to any one of claims 73 to 78, wherein the transgene encodes a signal sequence at the N-terminus of the heavy chain and light chain of said antigen binding fragment, which directs secretion and post-translational modification in said human liver cells or human muscle cells.

80. The pharmaceutical composition according to claim 79, in which the specified signal sequence is selected from the signal sequences in table. 2 or 3.

81. The pharmaceutical composition according to any one of claims 73 to 80, wherein the AAV capsid is AAV8.

82. A pharmaceutical composition for the treatment of eye disorders, including age-related macular degeneration, in a human subject in need thereof, comprising an AAV vector containing:

(a) a viral capsid that is at least 95% identical to the amino acid sequence of the AAV8 capsid (SEQ ID NO: 78) or the AAV9 capsid (SEQ ID NO: 79); and (b) an artificial genome containing an expression cassette flanked by an AAV ITR, wherein the expression cassette contains a transgene encoding an anti-MMP9, anti-VEGF or anti-fD mAb, or an antigen-binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in human retinal cells;

wherein said AAV vector is formulated for subretinal, intravitreal or suprachoroidal administration into the eye of said subject.

83. The pharmaceutical composition according to claim 82, wherein the anti-VEGF mAb is ranibizumab, bevacizumab or brolucizumab, the anti-Fd mAb is lampalizumab, or the anti-MMP9 mAb is andecaliximab.

84. The pharmaceutical composition according to claim 82 or 83, wherein the antigen binding fragment is a Fab, F(ab')2 or scFv.

85. The pharmaceutical composition according to any one of claims 82 to 84, wherein the antigen binding fragment comprises a heavy chain having the amino acid sequence SEQ ID NO: 33 and a light chain having the amino acid sequence SEQ ID NO: 34 or a heavy chain having the amino acid sequence SEQ ID NO: 35 and a light chain with the amino acid sequence SEQ ID NO: 36; or a heavy chain having the amino acid sequence of SEQ ID NO: 37 and a light chain having the amino acid sequence of SEQ ID NO: 38; or a heavy chain having the amino acid sequence of SEQ ID NO: 39 and a light chain having the amino acid sequence of SEQ ID NO: 40; or a heavy chain having the amino acid sequence of SEQ ID NO: 45 and a light chain having the amino acid sequence of SEQ ID NO: 46.

86. The pharmaceutical composition according to claim 85, wherein the transgene contains the nucleotide sequence SEQ ID NO: 133 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 134 encoding the light chain; or the nucleotide sequence SEQ ID NO: 135 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 136 encoding the light chain; or the nucleotide sequence SEQ ID NO: 137 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 138 encoding the light chain; or the nucleotide sequence SEQ ID NO: 139 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 140 encoding the light chain; or the nucleotide sequence SEQ ID NO: 145 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 146 encoding the light chain.

87. The pharmaceutical composition according to any one of claims 82-85, in which the antibody or antigen-binding fragment thereof is a hyperglycosylated mutant.

88. The pharmaceutical composition according to any one of claims 82 to 87, wherein the transgene encodes a signal sequence at the N-terminus of the heavy chain and light chain of said antigen-binding fragment, which directs secretion and post-translational modification in said human retinal cells.

89. The pharmaceutical composition according to claim 88, in which the specified signal sequence

- 10 047128 selected from the signal sequences in the table. 1.

90. The pharmaceutical composition according to any one of claims 82 to 89, wherein the AAV capsid is AAV8.

91. A pharmaceutical composition for the treatment of rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, plaque psoriasis or ulcerative colitis in a human subject in need thereof, comprising an AAV vector containing:

(a) a viral capsid that is at least 95% identical to the amino acid sequence of the AAV8 capsid (SEQ ID NO: 78) or the AAV9 capsid (SEQ ID NO: 79); and (b) an artificial genome containing an expression cassette flanked by an AAV ITR, wherein the expression cassette contains a transgene encoding an anti-TNF antibody or an antigen-binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in human liver cells or muscle cells person;

wherein said AAV vector is formulated for intravenous administration to said subject.

92. The pharmaceutical composition according to claim 91, wherein the mAb to TNF-alpha is adalimumab or infliximab.

93. The pharmaceutical composition according to claim 91 or 92, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

94. The pharmaceutical composition according to any one of claims 91 to 93, wherein the antigen binding fragment comprises a heavy chain having the amino acid sequence SEQ ID NO: 49 and a light chain having the amino acid sequence SEQ ID NO: 50 or a heavy chain having the amino acid sequence SEQ ID NO: 51 and a light chain having the amino acid sequence SEQ ID NO: 52.

95. The pharmaceutical composition according to claim 94, in which the transgene contains the nucleotide sequence SEQ ID NO: 149 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 150 encoding the light chain; or the nucleotide sequence SEQ ID NO: 151 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 152 encoding the light chain.

96. The pharmaceutical composition according to any one of claims 91-94, in which the antibody or antigen-binding fragment thereof is a hyperglycosylated mutant.

97. The pharmaceutical composition according to any one of claims 91 to 96, wherein the transgene encodes a signal sequence at the N-terminus of the heavy chain and the light chain of said antigen binding fragment, which directs secretion and post-translational modification in said human liver cells or human muscle cells.

98. The pharmaceutical composition according to claim 97, in which the specified signal sequence is selected from the signal sequences in table. 2 or 3.

99. The pharmaceutical composition according to any one of claims 91 to 98, wherein the AAV capsid is AAV8.

100. A pharmaceutical composition for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) or atypical hemolytic uremic syndrome (aHUS) in a human subject in need thereof, comprising an AAV vector containing:

(a) a viral capsid that is at least 95% identical to the amino acid sequence of the AAV8 capsid (SEQ ID NO: 78) or the AAV9 capsid (SEQ ID NO: 79); and (b) an artificial genome containing an expression cassette flanked by an AAV ITR, wherein the expression cassette contains a transgene encoding a mAb to the complement protein C5 or C5a or an antigen-binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in liver cells person;

wherein said AAV vector is formulated for intravenous administration to said subject.

101. The pharmaceutical composition according to claim 100, in which the mAb to complement protein C5 or C5a is eculizumab.

102. The pharmaceutical composition according to claim 100 or 101, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

103. The pharmaceutical composition according to any one of claims 100-102, in which the antigen-binding fragment contains a heavy chain with the amino acid sequence SEQ ID NO: 43 and a light chain with the amino acid sequence SEQ ID NO: 44.

104. The pharmaceutical composition according to claim 103, wherein the transgene contains a nucleotide sequence of SEQ ID NO: 143 encoding a heavy chain and a nucleotide sequence of SEQ ID NO: 144 encoding a light chain.

105. The pharmaceutical composition according to any one of claims 101-104, in which the antibody or antigen-binding fragment thereof is a hyperglycosylated mutant.

106. The pharmaceutical composition according to any one of claims 100 to 105, wherein the transgene encodes a signal sequence at the N-terminus of the heavy chain and the light chain of said antigen-binding fragment, which directs secretion and post-translational modification in said human liver cells.

107. The pharmaceutical composition according to claim 106, in which the specified signal sequence

- 11 047128 selected from the signal sequences in the table. 3.

108. The pharmaceutical composition according to any one of claims 101 to 107, wherein the AAV capsid is AAV8.

109. A pharmaceutical composition for the treatment of hereditary angioedema in a human subject in need thereof, comprising an AAV vector, comprising:

(a) a viral capsid that is at least 95% identical to the amino acid sequence of the AAV8 capsid (SEQ ID NO: 78) or the AAV9 capsid (SEQ ID NO: 79); and (b) an artificial genome containing an expression cassette flanked by an AAV ITR, wherein the expression cassette contains a transgene encoding a plasma kallikrein mAb or an antigen-binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in human liver or muscle cells human cells; wherein said AAV vector is formulated for intravenous administration to said subject.

110. The pharmaceutical composition according to claim 109, wherein the mAb to plasma kallikrein is lanadelumab.

111. The pharmaceutical composition according to claim 109 or 111, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

112. The pharmaceutical composition according to any one of claims 109-111, in which the antigen-binding fragment contains a heavy chain with the amino acid sequence SEQ ID NO: 47 and a light chain with the amino acid sequence SEQ ID NO: 48.

113. The pharmaceutical composition according to claim 112, wherein the transgene contains the nucleotide sequence SEQ ID NO: 147 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 148 encoding the light chain.

114. The pharmaceutical composition according to any one of claims 110-113, in which the antibody or antigen-binding fragment thereof is a hyperglycosylated mutant.

115. The pharmaceutical composition according to any one of claims 109 to 114, wherein the transgene encodes a signal sequence at the N-terminus of the heavy chain and the light chain of said antigen-binding fragment, which directs secretion and post-translational modification in said human liver cells.

116. The pharmaceutical composition according to claim 115, in which the specified signal sequence is selected from the signal sequences in table. 3.

117. The pharmaceutical composition according to any one of claims 110 to 116, wherein the AAV capsid is AAV8.

Method of treatment.

118. A method of treating Alzheimer's disease, migraines, cluster headaches or tauopathies, including chronic traumatic encephalopathy, progressive supranuclear palsy and frontotemporal dementia, in a human subject in need thereof, comprising delivery into the cerebrospinal fluid (CSF) of said subject - a human therapeutically effective amount of an anti-amyloid beta, anti-tau, or anti-CGRPR mAb or an antigen-binding fragment thereof produced by cells of the human central nervous system (CNS).

119. A method of treating Alzheimer's disease, migraines, cluster headaches or tauopathies, including chronic traumatic encephalopathy, progressive supranuclear palsy and frontotemporal dementia in a human subject in need thereof, comprising: administering therapeutically into the posterior cerebellomedullary cistern of said subject an effective amount of a recombinant nucleotide expression vector containing a transgene encoding an anti-amyloid beta, anti-tau, or anti-CGRPR mAb, or an antigen-binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in human CNS cells such that a depot is formed that releases a human post-translationally modified (HuPTM) form of said mAb or antigen-binding fragment thereof.

120. The method of claim 118 or 119, wherein the anti-amyloid beta mAb is aducanumab, crenezumab, gantenumab or BAN2401, or wherein the anti-tau mAb is aTAU, or wherein the anti-CGRPR is erenumab, eptinezumab, fremanezumab or galcanezumab.

121. The method according to any one of claims 118-120, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

122. The method according to any one of claims 118 to 121, wherein the antigen binding fragment comprises a heavy chain having the amino acid sequence SEQ ID NO: 1 and a light chain having the amino acid sequence SEQ ID NO: 2 or a heavy chain having the amino acid sequence SEQ ID NO: 3 and light chain with amino acid sequence SEQ ID NO: 4; or a heavy chain having the amino acid sequence SEQ ID NO: 5 and a light chain having the amino acid sequence SEQ ID NO: 6; or a heavy chain having the amino acid sequence of SEQ ID NO: 53 and a light chain having the amino acid sequence of SEQ ID NO: 54; a heavy chain having the amino acid sequence SEQ ID NO: 55 and a light chain having the amino acid sequence SEQ ID NO: 56; or heavier

- 12 047128 light chain with amino acid sequence SEQ ID NO: 57 and light chain with amino acid sequence SEQ ID NO: 58.

123. The method according to claim 122, wherein the transgene contains the nucleotide sequence SEQ ID NO: 101 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 102 encoding the light chain; or the nucleotide sequence SEQ ID NO: 103 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 104 encoding the light chain; or the nucleotide sequence SEQ ID NO: 105 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 106 encoding the light chain; or a heavy chain having the nucleotide sequence of SEQ ID NO: 153 and a light chain having the nucleotide sequence of SEQ ID NO: 154; a heavy chain having the nucleotide sequence SEQ ID NO: 155 and a light chain having the nucleotide sequence SEQ ID NO: 156; or a heavy chain having the nucleotide sequence of SEQ ID NO: 157 and a light chain having the nucleotide sequence of SEQ ID NO: 158.

124. The method according to any one of claims 118-122, wherein the mAb or antigen-binding fragment thereof is a hyperglycosylated mutant.

125. The method according to any one of claims 118-124, wherein the mAb or antigen-binding fragment thereof comprises an alpha-2,6-sialylated glycan.

126. The method according to any one of claims 118-125, wherein the mAb or antigen binding fragment thereof is glycosylated but does not contain detectable NeuGc and/or α-Gal.

127. The method according to any one of claims 118-126, wherein the mAb or antigen-binding fragment thereof comprises tyrosine sulfation.

128. The method according to any one of claims 119-127, wherein the recombinant expression vector is AAV9 or AAVrh10.

129. The method according to any one of claims 119-128, wherein the production of the specified HuPTM form of the specified mAb or an antigen-binding fragment thereof is confirmed by transducing human CNS cells in culture using the specified recombinant nucleotide expression vector and the expression of the specified mAb or an antigen-binding fragment thereof.

130. A method of treating psoriasis, psoriatic arthritis, ankylosing spondylitis or Crohn's disease in a human subject in need thereof, comprising delivering into the bloodstream of said human subject a therapeutically effective amount of an anti-IL17A or anti-IL12/IL23 mAb or an antigen-binding fragment thereof produced by human liver cells or human muscle cells.

131. A method of treating psoriasis, psoriatic arthritis, ankylosing spondylitis or Crohn's disease in a human subject in need thereof, comprising:

administering into the liver or muscle of said subject a therapeutically effective amount of a recombinant nucleotide expression vector containing a transgene encoding an anti-IL17A or anti-IL12/IL23 mAb or an antigen-binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in human liver cells, or human muscle cells such that a depot is formed that releases the HuPTM form of the specified mAb or antigen binding fragment thereof.

132. The method of claim 130 or 131, wherein the anti-IL17A or anti-IL12/IL23 mAb is ixekizumab, secukinumab or ustekinumab.

133. The method according to any one of claims 130-132, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

134. The method according to any one of claims 130-133, wherein the antigen binding fragment comprises a heavy chain having the amino acid sequence SEQ ID NO: 9 and a light chain having the amino acid sequence SEQ ID NO: 10 or a heavy chain having the amino acid sequence SEQ ID NO: 11 and light chain with amino acid sequence SEQ ID NO: 12; or a heavy chain having the amino acid sequence of SEQ ID NO: 13 and a light chain having the amino acid sequence of SEQ ID NO: 14.

135. The method of claim 134, wherein the transgene comprises a nucleotide sequence of SEQ ID NO: 109 encoding a heavy chain and a nucleotide sequence of SEQ ID NO: 110 encoding a light chain; or the nucleotide sequence SEQ ID NO: 111 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 112 encoding the light chain; or the nucleotide sequence SEQ ID NO: 113 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 114 encoding the light chain.

136. The method according to any one of claims 132-134, wherein the mAb or antigen-binding fragment thereof is a hyperglycosylated mutant.

137. The method according to any one of claims 132-136, wherein the mAb or antigen-binding fragment thereof contains an alpha-2,6-sialylated glycan.

138. The method according to any one of claims 132-137, wherein the mAb or antigen binding fragment thereof is glycosylated but does not contain detectable NeuGc or α-Gal.

- 13 047128

139. The method according to any one of claims 132-138, wherein the mAb or antigen-binding fragment thereof comprises tyrosine sulfation.

140. The method according to any one of claims 133-139, wherein the recombinant expression vector is AAV8 or AAV9.

141. The method according to any one of claims 133-140, wherein the production of the specified form of HuPTM mAb or its antigen-binding fragment is confirmed by transduction of liver cells or human muscle cells in culture with the specified recombinant nucleotide expression vector and the expression of the specified mAb or its antigen-binding fragment.

142. A method of treating multiple sclerosis, ulcerative colitis, or Crohn's disease in a human subject in need thereof, comprising delivering into the CSF or bloodstream of said human subject a therapeutically effective amount of an anti-integrin mAb or antigen-binding fragment thereof produced by human CNS cells, human liver cells, or muscle cells person.

143. A method of treating multiple sclerosis, ulcerative colitis or Crohn's disease in a human subject in need thereof, comprising:

administering into the CNS, liver, or muscle of said human subject a therapeutically effective amount of a recombinant nucleotide expression vector containing a transgene encoding an anti-integrin mAb or an antigen-binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in human CNS cells, cells human liver or human muscle cells such that a depot is formed which releases the HuPTM form of said mAb or antigen binding fragment.

144. The method of claim 142 or 143, wherein the anti-integrin mAb is natalizumab or vedolizumab.

145. The method according to any one of claims 142-144, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

146. The method according to any one of claims 142 to 145, wherein the antigen binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO: 17 and a light chain having the amino acid sequence of SEQ ID NO: 18 or a heavy chain having the amino acid sequence of SEQ ID NO: 19 and light chain with amino acid sequence SEQ ID NO: 20.

147. The method of claim 146, wherein the transgene comprises a nucleotide sequence of SEQ ID NO: 117 encoding a heavy chain and a nucleotide sequence of SEQ ID NO: 118 encoding a light chain; or the nucleotide sequence SEQ ID NO: 119 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 120 encoding the light chain.

148. The method according to any one of claims 142-145, wherein the antibody or antigen-binding fragment thereof is a hyperglycosylated mutant.

149. The method according to any one of claims 142-148, wherein the mAb or antigen-binding fragment thereof contains an alpha-2,6-sialylated glycan.

150. The method of any one of claims 142 to 149, wherein the mAb or antigen binding fragment thereof is glycosylated but does not contain detectable NeuGc or α-Gal.

151. The method according to any one of claims 142-150, wherein the mAb or antigen-binding fragment thereof comprises tyrosine sulfation.

152. The method according to any one of claims 143-151, wherein the recombinant expression vector is AAV8, AAV9 or AAVrh10.

153. The method according to any one of claims 143-152, wherein the production of the HuPTM mAb or an antigen-binding fragment thereof is confirmed by transducing human CNS cells, human liver cells or muscle cells in culture with the specified recombinant nucleotide expression vector and the expression of the specified mAb or an antigen-binding fragment thereof .

154. A method of treating atopic dermatitis in a human subject in need thereof, comprising delivering into the bloodstream of said human subject a therapeutically effective amount of an anti-IL4R mAb or an antigen-binding fragment thereof produced by human liver cells or human muscle cells.

155. A method of treating atopic dermatitis in a human subject in need, comprising:

administering into the liver or muscle of said human subject a therapeutically effective amount of a recombinant nucleotide expression vector containing a transgene encoding an anti-IL4R mAb or an antigen-binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in human liver cells or human muscle cells , so that a depot is formed which releases the HuPTM form of said mAb or antigen binding fragment thereof.

156. The method of claim 154 or 155, wherein the anti-IL-4R mAb is dupilumab.

157. The method according to any one of claims 154-156, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

- 14 047128

158. The method according to any one of claims 154 to 157, wherein the antigen binding fragment comprises a heavy chain having the amino acid sequence SEQ ID NO: 7 and a light chain having the amino acid sequence SEQ ID NO: 8.

159. The method of claim 158, wherein the transgene comprises a nucleotide sequence of SEQ ID NO: 107 encoding a heavy chain and a nucleotide sequence of SEQ ID NO: 108 encoding a light chain.

160. The method according to any one of claims 154-158, wherein the mAb or antigen-binding fragment thereof is a hyperglycosylated mutant.

161. The method according to any one of claims 154-160, wherein the mAb or antigen-binding fragment thereof contains an alpha-2,6-sialylated glycan.

162. The method according to any one of claims 154-161, wherein the mAb or antigen binding fragment thereof is glycosylated but does not contain detectable NeuGc or α-Gal.

163. The method according to any one of claims 154-162, wherein the mAb or antigen-binding fragment thereof comprises tyrosine sulfation.

164. The method according to any one of claims 155-163, wherein the recombinant expression vector is AAV8 or AAV9.

165. The method according to any one of claims 155-164, wherein the production of the HuPTM form of said mAb or an antigen-binding fragment thereof is confirmed by transducing liver cells or human muscle cells in culture with said recombinant nucleotide expression vector and expression of said mAb or an antigen-binding fragment thereof.

166. A method of treating asthma in a human subject in need thereof, comprising delivering into the bloodstream of said human subject a therapeutically effective amount of an anti-IL-5 mAb or an antigen-binding fragment thereof produced by human liver cells or human muscle cells.

167. A method of treating asthma in a human subject in need, comprising:

administering into the liver or muscle of said human subject a therapeutically effective amount of a recombinant nucleotide expression vector containing a transgene encoding an anti-IL-5 mAb or an antigen-binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in human liver or muscle cells human cells such that a depot is formed that releases the HuPTM form of the specified mAb or antigen binding fragment thereof.

168. The method of claim 166 or 167, wherein the anti-IL-5 mAb is mepolizumab.

169. The method according to any one of claims 166-168, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

170. The method according to any one of claims 166 to 159, wherein the antigen binding fragment comprises a heavy chain having the amino acid sequence SEQ ID NO: 15 and a light chain having the amino acid sequence SEQ ID NO: 16.

171. The method of claim 170, wherein the transgene comprises a nucleotide sequence of SEQ ID NO: 115 encoding a heavy chain and a nucleotide sequence of SEQ ID NO: 116 encoding a light chain.

172. The method according to any one of claims 166-170, wherein the antibody or antigen-binding fragment thereof is a hyperglycosylated mutant.

173. The method according to any one of claims 166-172, wherein the mAb or antigen-binding fragment thereof comprises an alpha-2,6-sialylated glycan.

174. The method of any one of claims 166 to 173, wherein the mAb or antigen binding fragment thereof is glycosylated but does not contain detectable NeuGc or α-Gal.

175. The method according to any one of claims 166 to 174, wherein the mAb or antigen binding fragment thereof comprises tyrosine sulfation.

176. The method according to any one of claims 167-175, wherein the recombinant expression vector is AAV8 or AAV9.

177. The method according to any one of claims 167-176, wherein the production of said HuPTM form of said mAb or an antigen-binding fragment thereof is confirmed by transducing liver cells or human muscle cells in culture with said recombinant nucleotide expression vector and expression of said mAb or an antigen-binding fragment thereof.

178. A method of treating HeFH, HoFH, dyslipidemia, cardiovascular diseases including atherosclerotic cardiovascular disease (ACD), atherosclerotic plaque formation, abnormally high levels of non-HDL cholesterol and LDL, aortic stenosis, hepatic stenosis or hypercholesterolemia and dyslipidemia in a human subject in need thereof, comprising delivering into the bloodstream of said human subject a therapeutically effective amount of an anti-PCSK9, anti-ANGPTL3, anti-OxPL mAb, or an antigen-binding fragment thereof, produced by human liver cells or human muscle cells.

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179. A method of treating HeFH, HoFH, dyslipidemia, cardiovascular diseases, including atherosclerotic cardiovascular disease (ACD), atherosclerotic plaque formation, abnormally high levels of non-HDL cholesterol and LDL, aortic stenosis, hepatic stenosis or hypercholesterolemia, in human subject in need, providing:

administering into the liver or muscle of said human subject a therapeutically effective amount of a recombinant nucleotide expression vector containing a transgene encoding an anti-PCSK9, anti-OxPL, or anti-ANGPTL3 mAb, or an antigen-binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in cells human liver or human muscle cells such that a depot is formed that releases a form of the HuPTM mAb or an antigen-binding fragment thereof.

180. Method according to paragraphs. 178 or 179, wherein the anti-PCSK9 mAb is alirocumab or evolocumab or the anti-ANGPTL3 mAb is evinacumab or the anti-OxPL mAb is E06.

181. The method according to any one of claims 178-180, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

182. The method according to any one of claims 178 to 181, wherein the antigen binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO: 21 and a light chain having the amino acid sequence of SEQ ID NO: 22 or a heavy chain having the amino acid sequence of SEQ ID NO: 23 and light chain with amino acid sequence SEQ ID NO: 24; a heavy chain having the amino acid sequence SEQ ID NO: 25 and a light chain having the amino acid sequence SEQ ID NO: 26; or a heavy chain having the amino acid sequence SEQ ID NO: 59 and a light chain having the amino acid sequence SEQ ID NO: 60.

183. The method of claim 182, wherein the transgene comprises a nucleotide sequence of SEQ ID NO: 121 encoding a heavy chain and a nucleotide sequence of SEQ ID NO: 122 encoding a light chain; or the nucleotide sequence SEQ ID NO: 123 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 124 encoding the light chain; the nucleotide sequence SEQ ID NO: 125 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 126 encoding the light chain; or a heavy chain having the nucleotide sequence of SEQ ID NO: 159 and a light chain having the nucleotide sequence of SEQ ID NO: 160.

184. The method according to any one of claims 178-182, wherein the mAb or antigen-binding fragment thereof is a hyperglycosylated mutant.

185. The method according to any one of claims 178-184, wherein the mAb or antigen-binding fragment thereof contains an alpha-2,6-sialylated glycan.

186. The method according to any one of claims 178-185, wherein the mAb or antigen binding fragment thereof is glycosylated but does not contain detectable NeuGc or α-Gal.

187. The method according to any one of claims 178-186, wherein the mAb or antigen-binding fragment thereof comprises tyrosine sulfation.

188. The method according to any one of claims 179-187, wherein the recombinant expression vector is AAV8 or AAV9.

189. The method according to any one of claims 179-188, wherein the production of the specified form of HuPTM mAb or an antigen-binding fragment thereof is confirmed by transduction of human liver cells or human muscle cells in culture with the specified recombinant nucleotide expression vector and the expression of the specified mAb or an antigen-binding fragment thereof.

190. A method of treating osteoporosis, increasing bone mass in patients with breast or prostate cancer, or preventing skeletal-related events due to bone metastasis in a human subject in need thereof, comprising delivering a therapeutically effective amount of a mAb into the bloodstream of said human subject to RANKL or its antigen-binding fragment produced by human liver cells or human muscle cells.

191. A method of treating osteoporosis, increasing bone mass in patients with breast or prostate cancer, or preventing skeletal-related events due to bone metastasis in a human subject in need thereof, comprising administering to the liver or muscle of said human subject a therapeutically effective a quantity of a recombinant nucleotide expression vector containing a transgene encoding an anti-RANKL mAb or an antigen-binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in human liver cells or human muscle cells such that a depot is formed that releases a form of the HuPTM mAb or an antigen-binding fragment thereof.

192. The method of claim 190 or 191, wherein the anti-RANKL mAb is denosumab.

193. The method according to any one of claims 190-192, wherein the antigen binding fragment is a Fab, F(ab')2 or scFv.

194. The method according to any one of claims 190 to 193, wherein the antigen binding fragment comprises a heavy chain having the amino acid sequence SEQ ID NO: 27 and a light chain having the amino acid sequence SEQ ID NO: 28.

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195. The method of claim 194, wherein the transgene comprises a nucleotide sequence of SEQ ID NO: 128 encoding a heavy chain and a nucleotide sequence of SEQ ID NO: 127 encoding a light chain.

196. The method according to any one of claims 190-194, wherein the mAb or antigen-binding fragment thereof is a hyperglycosylated mutant.

197. The method according to any one of claims 190-196, wherein the mAb or antigen-binding fragment thereof comprises an alpha-2,6-sialylated glycan.

198. The method of any one of claims 190 to 197, wherein the mAb or antigen binding fragment thereof is glycosylated but does not contain detectable NeuGc or α-Gal.

199. The method according to any one of claims 190-198, wherein the mAb or antigen-binding fragment thereof comprises tyrosine sulfation.

200. The method according to any one of claims 191-199, wherein the recombinant expression vector is AAV8 or AAV9.

201. The method according to any one of claims 191-200, wherein the production of said HuPTM form of said mAb or an antigen-binding fragment thereof is confirmed by transducing liver cells or human muscle cells in culture with said recombinant nucleotide expression vector and expression of said mAb or an antigen-binding fragment thereof.

202. A method of treating metastatic melanoma, lymphoma, non-small cell lung carcinoma, squamous cell carcinoma of the head and neck, urothelial carcinoma, cancer with high microsatellite instability, gastric cancer, renal cell carcinoma, metastatic colon cancer with mismatch repair deficiency and hepatocellular carcinoma in a needy person wherein a human subject comprising delivering into the bloodstream of said human subject a therapeutically effective amount of a PD-1 blocker mAb or an antigen binding fragment thereof produced by human liver cells or human muscle cells.

203. A method of treating metastatic melanoma, lymphoma, non-small cell lung carcinoma, squamous cell carcinoma of the head and neck, urothelial carcinoma, cancer with high microsatellite instability, gastric cancer, renal cell carcinoma, metastatic colon cancer with mismatch repair deficiency and hepatocellular carcinoma in a needy person in this subject-person, providing:

administering into the liver or muscle of said human subject a therapeutically effective amount of a recombinant nucleotide expression vector containing a transgene encoding a PD-1 mAb blocker or an antigen binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in human liver cells or muscle cells human, such that a depot is formed which releases the HuPTM form of said mAb or antigen binding fragment thereof.

204. The method of claim 202 or 203, wherein the PD-1 blocking mAb is nivolumab or pembrolizumab.

205. The method according to any one of claims 202-204, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

206. The method according to any one of claims 202 to 205, wherein the antigen binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO: 29 and a light chain having the amino acid sequence of SEQ ID NO: 30 or a heavy chain having the amino acid sequence of SEQ ID NO: 31 and light chain with amino acid sequence SEQ ID NO: 32.

207. The method of claim 206, wherein the transgene comprises a nucleotide sequence of SEQ ID NO: 129 encoding a heavy chain and a nucleotide sequence of SEQ ID NO: 130 encoding a light chain; or the nucleotide sequence SEQ ID NO: 131 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 132 encoding the light chain.

208. The method according to any one of claims 202-206, wherein the mAb or antigen-binding fragment thereof is a hyperglycosylated mutant.

209. The method according to any one of claims 202-208, wherein the mAb or antigen-binding fragment thereof comprises an alpha-2,6-sialylated glycan.

210. The method of any one of claims 202 to 209, wherein the mAb or antigen binding fragment thereof is glycosylated but does not contain detectable NeuGc or α-Gal.

211. The method according to any one of claims 202-210, wherein the mAb or antigen-binding fragment thereof comprises tyrosine sulfation.

212. The method according to any one of claims 203-211, wherein the recombinant expression vector is AAV8 or AAV9.

213. The method according to any one of claims 203-212, wherein the production of said HuPTM form of said mAb or an antigen-binding fragment thereof is confirmed by transducing liver cells or human muscle cells in culture with said recombinant nucleotide expression vector and expression of said mAb or antigen-binding fragment thereof.

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214. A method of treating systemic lupus erythematosus (SLE) in a human subject in need thereof, comprising delivering into the bloodstream of said human subject a therapeutically effective amount of an anti-BLyS mAb or an antigen-binding fragment thereof produced by human liver cells or human muscle cells.

215. A method of treating SLE in a human subject in need, comprising:

administering to the liver or muscle of said subject a therapeutically effective amount of a recombinant nucleotide expression vector containing a transgene encoding an anti-BLyS mAb or an antigen-binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in human liver cells or human muscle cells, so that a depot is formed which releases the HuPTM form of the specified mAb or an antigen binding fragment thereof.

216. The method of claim 214 or 215, wherein the anti-BLyS mAb is belimumab.

217. The method according to any one of claims 214-216, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

218. The method according to any one of claims 214 to 217, wherein the antigen binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO: 41 and a light chain having the amino acid sequence of SEQ ID NO: 42.

219. The method of claim 218, wherein the transgene comprises a nucleotide sequence of SEQ ID NO: 139 encoding a heavy chain and a nucleotide sequence of SEQ ID NO: 142 encoding a light chain.

220. The method according to any one of claims 214-218, wherein the mAb or antigen-binding fragment thereof is a hyperglycosylated mutant.

221. The method according to any one of claims 214-220, wherein the mAb or antigen-binding fragment thereof comprises an alpha-2,6-sialylated glycan.

222. The method of any one of claims 214 to 221, wherein the mAb or antigen binding fragment thereof is glycosylated but does not contain detectable NeuGc or α-Gal.

223. The method according to any one of claims 214-222, wherein the mAb or antigen-binding fragment thereof comprises tyrosine sulfation.

224. The method according to any one of claims 215-223, wherein the recombinant expression vector is AAV8 or AAV9.

225. The method according to any one of claims 215-224, wherein the production of said HuPTM form of said mAb or an antigen-binding fragment thereof is confirmed by transducing liver cells or human muscle cells in culture with said recombinant nucleotide expression vector and expression of said mAb or an antigen-binding fragment thereof.

226. A method of treating an eye disorder including neovascular age-related macular degeneration (nAMD), dry AMD, diabetic retinopathy, diabetic macular edema (DME), central retinal vein occlusion (RVO), pathological myopia, or polypoid vascular vasculopathy, in a patient in need of of a human subject, comprising delivering to the retina of said human subject a therapeutically effective amount of an anti-MMP9, anti-VEGF or anti-fD mAb, or an antigen-binding fragment thereof, produced by human retinal cells.

227. A method of treating an eye disorder, including nAMD, dry AMD, diabetic retinopathy, DME, RVO, pathological myopia, or polypoidal choroidal vasculopathy, in a human subject in need thereof, comprising:

administering subretinal, intravitreal, or suprachoroidal to a specified human subject a therapeutically effective amount of a recombinant nucleotide expression vector containing a transgene encoding an anti-MMP9, anti-VEGF, or anti-fD mAb, or an antigen-binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in human retinal cells such that a depot is formed that releases the HuPTM form of the specified mAb or antigen binding fragment thereof.

228. The method of claim 226 or 227, wherein the anti-MMP9, anti-VEGF or anti-fD mAb is andecaliximab, ranibizumab, bevacizumab, brolucizumab or lampalizumab.

229. The method according to any one of claims 226-228, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

230. The method according to any one of claims 226 to 229, wherein the antigen binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO: 33 and a light chain having the amino acid sequence of SEQ ID NO: 34 or a heavy chain having the amino acid sequence of SEQ ID NO: 35 and light chain with amino acid sequence SEQ ID NO: 36; or a heavy chain having the amino acid sequence of SEQ ID NO: 37 and a light chain having the amino acid sequence of SEQ ID NO: 38; or a heavy chain having the amino acid sequence of SEQ ID NO: 39 and a light chain having the amino acid sequence of SEQ ID NO: 40; or a heavy chain having the amino acid sequence of SEQ ID NO: 45 and a light chain having the amino acid sequence of SEQ ID NO: 46.

231. The method of claim 230, wherein the transgene contains the nucleotide sequence of SEQ ID

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NO: 133, encoding the heavy chain, and the nucleotide sequence of SEQ ID NO: 134, encoding the light chain; or the nucleotide sequence SEQ ID NO: 135 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 136 encoding the light chain; or the nucleotide sequence SEQ ID NO: 137 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 138 encoding the light chain; or the nucleotide sequence SEQ ID NO: 139 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 140 encoding the light chain; or the nucleotide sequence SEQ ID NO: 145 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 146 encoding the light chain.

232. The method according to any one of claims 226-230, wherein the mAb or antigen-binding fragment thereof is a hyperglycosylated mutant.

233. The method according to any one of claims 226-232, wherein the mAb or antigen-binding fragment thereof comprises an alpha-2,6-sialylated glycan.

234. The method of any one of claims 226 to 233, wherein the mAb or antigen binding fragment thereof is glycosylated but does not contain detectable NeuGc or α-Gal.

235. The method according to any one of claims 226-234, wherein the mAb or antigen-binding fragment thereof comprises tyrosine sulfation.

236. The method according to any one of claims 227-235, wherein the recombinant expression vector is AAV8 or AAV9.

237. The method according to any one of claims 227-236, wherein the production of said HuPTM form of said mAb or an antigen-binding fragment thereof is confirmed by transducing human retinal cells in culture with said recombinant nucleotide expression vector and expression of said mAb or an antigen-binding fragment thereof.

238. A method of treating cystic fibrosis (CF), rheumatoid arthritis (RA), UC, CD, solid tumors, pancreatic adenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, esophagogastric adenocarcinoma, gastric cancer, colorectal cancer or breast cancer in a person in need of of a human subject, comprising delivering into the bloodstream of said human subject a therapeutically effective amount of an anti-MMP9 mAb or an antigen-binding fragment thereof produced by human liver cells or human muscle cells.

239. A method of treating cystic fibrosis (CF), rheumatoid arthritis (RA), UC, CD, solid tumors, pancreatic adenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, esophagogastric adenocarcinoma, gastric cancer, colorectal cancer or breast cancer in a person in need of this human subject, providing:

administering to the liver or muscle of said subject a therapeutically effective amount of a recombinant nucleotide expression vector containing a transgene encoding an anti-MMP9 mAb or an antigen-binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in human liver cells or human muscle cells, so that a depot is formed which releases the HuPTM form of the specified mAb or an antigen binding fragment thereof.

240. The method of claim 238 or 239, wherein the anti-MMP9 mAb is andecaliximab.

241. The method according to any one of claims 238-240, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

242. The method according to any one of claims 238-224, wherein the antigen binding fragment comprises a heavy chain having the amino acid sequence SEQ ID NO: 45 and a light chain having the amino acid sequence SEQ ID NO: 46.

243. The method of claim 242, wherein the transgene comprises a nucleotide sequence of SEQ ID NO: 145 encoding a heavy chain and a nucleotide sequence of SEQ ID NO: 146 encoding a light chain.

244. The method according to any one of claims 238-242, wherein the mAb or antigen-binding fragment thereof is a hyperglycosylated mutant.

245. The method according to any one of claims 238-244, wherein the mAb or antigen-binding fragment thereof comprises an alpha-2,6-sialylated glycan.

246. The method of any one of claims 238 to 245, wherein the mAb or antigen binding fragment thereof is glycosylated but does not contain detectable NeuGc or α-Gal.

247. The method according to any one of claims 238-246, wherein the mAb or antigen-binding fragment thereof comprises tyrosine sulfation.

248. The method according to any one of claims 239-247, wherein the recombinant expression vector is AAV8 or AAV9.

249. The method according to any one of claims 239-248, wherein the production of said HuPTM form of said mAb or an antigen-binding fragment thereof is confirmed by transducing liver cells or human muscle cells in culture with said recombinant nucleotide expression vector and expression of said mAb or an antigen-binding fragment thereof.

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250. A method of treating hereditary angioedema in a human subject in need thereof, comprising delivering into the bloodstream of said human subject a therapeutically effective amount of an anti-kallikrein mAb or an antigen-binding fragment thereof produced by human liver cells or human muscle cells.

251. A method of treating hereditary angioedema in a human subject in need thereof, comprising administering into the liver or muscle of said subject a therapeutically effective amount of a recombinant nucleotide expression vector containing a transgene encoding an anti-kallikrein mAb or an antigen-binding fragment thereof operably linked to one or more regulatory sequences, which control the expression of the transgene in human liver cells or in human muscle cells so that a depot is formed that releases the HuPTM form of the specified mAb or antigen binding fragment thereof.

252. The method of claim 250 or 251, wherein the anti-kallikrein mAb is lanadelumab.

253. The method according to any one of claims 250-252, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

254. The method according to any one of claims 250-253, wherein the antigen binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO: 47 and a light chain having the amino acid sequence of SEQ ID NO: 48.

255. The method of claim 254, wherein the transgene comprises a nucleotide sequence of SEQ ID NO: 147 encoding a heavy chain and a nucleotide sequence of SEQ ID NO: 148 encoding a light chain.

256. The method according to any one of claims 250-255, wherein the mAb or antigen-binding fragment thereof is a hyperglycosylated mutant.

257. The method according to any one of claims 250-256, wherein the mAb or antigen-binding fragment thereof comprises an alpha-2,6-sialylated glycan.

258. The method of any one of claims 250 to 257, wherein the mAb or antigen binding fragment thereof is glycosylated but does not contain detectable NeuGc or α-Gal.

259. The method according to any one of claims 250-258, wherein the mAb or antigen-binding fragment thereof comprises tyrosine sulfation.

260. The method according to any one of claims 251-259, wherein the recombinant expression vector is AAV8 or AAV9.

261. The method according to any one of claims 251-260, wherein the production of said HuPTM form of said mAb or an antigen-binding fragment thereof is confirmed by transducing liver cells or human muscle cells in culture with said recombinant nucleotide expression vector and expression of said mAb or an antigen-binding fragment thereof.

262. A method of treating rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, plaque psoriasis or ulcerative colitis in a human subject in need thereof, comprising delivering into the bloodstream of said human subject a therapeutically effective amount of an anti-TNF-alpha mAb or an antigen-binding fragment thereof produced by the cells human liver or human muscle cells.

263. A method of treating rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, plaque psoriasis or ulcerative colitis in a human subject in need thereof, comprising administering to the liver or muscle of said subject a therapeutically effective amount of a recombinant nucleotide expression vector containing a transgene encoding a mAb to TNFalpha or an antigen binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in human liver cells or human muscle cells such that a depot is formed that releases the HuPTM form of the mAb or antigen binding fragment thereof.

264. The method of claim 262 or 263, wherein the anti-TNF-alpha mAb is adalimumab or infliximab.

265. The method according to any one of claims 262-264, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

266. The method of any one of claims 262 to 265, wherein the antigen binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO: 49 and a light chain having the amino acid sequence of SEQ ID NO: 50 or a heavy chain having the amino acid sequence of SEQ ID NO: 51 and light chain with amino acid sequence SEQ ID NO: 52.

267. The method according to claim 26, wherein the transgene contains the nucleotide sequence SEQ ID NO: 149 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 150 encoding the light chain; the nucleotide sequence SEQ ID NO: 151 encoding the heavy chain, and the nucleotide sequence SEQ ID NO: 152 encoding the light chain.

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268. The method according to any one of claims 262-266, wherein the mAb or antigen-binding fragment thereof is a hyperglycosylated mutant.

269. The method according to any one of claims 262-268, wherein the mAb or antigen-binding fragment thereof comprises an alpha-2,6-sialylated glycan.

270. The method of any one of claims 262 to 269, wherein the mAb or antigen binding fragment thereof is glycosylated but does not contain detectable NeuGc or α-Gal.

271. The method according to any one of claims 262-270, wherein the mAb or antigen-binding fragment thereof comprises tyrosine sulfation.

272. The method according to any one of claims 263-271, wherein the recombinant expression vector is AAV8 or AAV9.

273. The method according to any one of claims 263-272, wherein the production of said HuPTM form of said mAb or an antigen-binding fragment thereof is confirmed by transducing liver cells or human muscle cells in culture with said recombinant nucleotide expression vector and expression of said mAb or an antigen-binding fragment thereof.

274. A method of treating PNH or aHUS in a human subject in need thereof, comprising delivering into the bloodstream of said human subject a therapeutically effective amount of a mAb to the C5 or C5a protein, or an antigen-binding fragment thereof, produced by human liver cells.

275. A method of treating PNH or aHUS in a human subject in need thereof, comprising administering to the liver of said subject a therapeutically effective amount of a recombinant nucleotide expression vector containing a transgene encoding an mAb to the C5 or C5a protein or an antigen binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in human liver cells such that a depot is formed that releases the HuPTM form of the mAb or antigen binding fragment thereof.

276. The method according to claims 274-275, wherein the mAb to the C5 or C5a protein or antigen binding fragment is eculizumab.

277. The method according to any one of claims 274-276, wherein the antigen binding fragment is a Fab, F(ab') ₂ or scFv.

278. The method of any one of claims 274 to 277, wherein the antigen binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO: 43 and a light chain having the amino acid sequence of SEQ ID NO: 44.

279. The method of claim 28, wherein the transgene comprises the nucleotide sequence SEQ ID NO: 143 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 144 encoding the light chain.

280. The method according to any one of claims 274-279, wherein the mAb or antigen-binding fragment thereof is a hyperglycosylated mutant.

281. The method according to any one of claims 274-280, wherein the mAb or antigen-binding fragment thereof comprises an alpha-2,6-sialylated glycan.

282. The method of any one of claims 274 to 281, wherein the mAb or antigen binding fragment thereof is glycosylated but does not contain detectable NeuGc or α-Gal.

283. The method according to any one of claims 274-282, wherein the mAb or antigen-binding fragment thereof comprises tyrosine sulfation.

284. The method according to any one of claims 275-28, wherein the recombinant expression vector is AAV8 or AAV9.

285. The method according to any one of claims 275-284, wherein the production of said HuPTM form of said mAb or an antigen-binding fragment thereof is confirmed by transducing liver cells or human muscle cells in culture with said recombinant nucleotide expression vector and expression of said mAb or an antigen-binding fragment thereof.

Preparation method.

286. A method for producing recombinant AAVs, comprising (a) culturing a host cell containing (i) an artificial genome containing a cis-expression cassette flanked by an AAV ITR, wherein the cis-expression cassette contains a transgene encoding a therapeutic antibody operably linked to control elements expressions that will control the expression of the transgene in human cells;

(ii) a trans-expression cassette lacking the AAV ITRs, wherein the trans-expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that direct expression of the AAV rep and capsid proteins in a host cell in culture and provide proteins rep and cap in trans;

(iii) sufficient accessory functions of the adenovirus for replication and packaging of the artificial genome by AAV capsid proteins and (b) recovery of recombinant AAV encapsulating the artificial genome from culture

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287. The method of claim 286, wherein the transgene encodes an mAb or an antigen-binding fragment thereof that contains the heavy and light chain variable domains of aducanumab, crenezumab, gantenerumab, BAN2401, aTAU, erenumab, eptinezumab, fremanezumab, or galcanezumab.

288. The method of claim 286 or 287, wherein the AAV capsid protein is an AAV9 or AAVrh10 capsid protein.

289. The method of claim 286, wherein the transgene encodes a mAb or antigen-binding fragment that contains the heavy and light chain variable domains of ixekizumab, secukinumab, or ustekinumab.

290. The method of claim 286, wherein the transgene encodes a mAb or antigen-binding fragment that contains the heavy and light chain variable domains of natalizumab or vedolizumab.

291. The method of claim 286, wherein the transgene encodes a mAb or antigen binding fragment that contains the heavy and light chain variable domains of dupilumab.

292. The method of claim 286, wherein the transgene encodes a mAb or antigen binding fragment that contains the variable domains of the heavy and light chains of mepolizumab.

293. The method of claim 286, wherein the transgene encodes a mAb or antigen-binding fragment that contains the heavy and light chain variable domains of alirocumab, evolocumab, evinacumab, or E06.

294. The method of claim 286, wherein the transgene encodes a mAb or antigen binding fragment that contains the variable domains of the denosumab heavy and light chains.

295. The method of claim 286, wherein the transgene encodes a mAb or antigen binding fragment that contains the heavy and light chain variable domains of nivolumab or pembrolizumab.

296. The method of claim 286, wherein the transgene encodes a mAb or antigen binding fragment that contains the belimumab heavy and light chain variable domains.

297. The method of claim 286, wherein the transgene encodes a mAb or antigen-binding fragment that contains the heavy and light chain variable domains of ranibizumab, bevacizumab, or lampalizumab.

298. The method of claim 286, wherein the transgene encodes a mAb or antigen-binding fragment that contains the variable domains of the heavy and light chains of andecaliximab.

299. The method of claim 286, wherein the transgene encodes a mAb or antigen binding fragment that contains the heavy and light chain variable domains of lanadelumab.

300. The method of claim 286, wherein the transgene encodes a mAb or antigen-binding fragment that contains the heavy and light chain variable domains of adalimumab or infliximab.

301. The method of claim 286, wherein the transgene encodes a mAb or antigen binding fragment that contains the variable domains of the eculizumab heavy and light chains.

302. The method of any one of claims 286 or 289-301, wherein the AAV capsid protein is an AAV8 or AAV9 capsid protein.

4. Brief description of drawings

The patent or application file contains at least one graphic material in color. Copies of this patent or patent application publication with color graphics will be made available by the Office upon request and payment of the required fee.

Fig. 1. Scheme of the genome construction of the rAAV vector containing an expression cassette encoding the heavy and light chains of the Fab region of the therapeutic mAb, controlled by expression elements flanked by the AAV ITR.

Fig. 2A-F. Amino acid sequence of the transgene construct for the Fab region of therapeutic antibodies to CNS targets: to Av, Fab of aducanumab (Fig. 2A); k Av, crenezumab Fab (Fig. 2B); to Av, Fab of gantenerumab (Fig. 2C), to tau, Fab aTAU (Fig. 2D) and to CGRPR, Fab of erenumab (Fig. 2E) and to Av of BAN2401 (Fig. 2F). Glycosylation sites are shown in bold. Glycosylation sites for glutamine are highlighted in green; glycosylation sites at asparagine (N) are highlighted in purple; and non-consensus glycosylation sites at asparagine (N) are highlighted in blue; Tyrosine O-sulfation sites (italics) are highlighted in yellow. The heavy chain loop regions are highlighted in grey.

Fig. 3 A-E. Amino acid sequence of the transgene construct for the Fab region of therapeutic antibodies to interleukins: to IL4R, dupilumab (Fig. 3A); to IL-17, ixekizumab (Fig. 3B); secukinumab (Fig. 3C); to IL-12/IL-23, ustekinumab (Fig. 3D) and to IL5, mepolizumab (Fig. 3E). Glycosylation sites are shown in bold. Glycosylation sites at glutamine are highlighted in green, and non-consensus glycosylation sites at asparagine (N) are highlighted in blue; Tyrosine O-sulfation sites (italics) are highlighted in yellow. The heavy chain loop regions are highlighted in grey.

Fig. 4A, B. Amino acid sequence of the transgene construct for the Fab region of therapeutic antibodies to integrin: vedolizumab (Fig. 4A) and natalizumab (Fig. 4B). Glycosylation sites are shown in bold. Glycosylation sites for glutamine are highlighted in green, and non-consensus

- 22 047128 glycosylation sites at asparagine (N) are highlighted in blue; Tyrosine O-sulfation sites (italics) are highlighted in yellow. The heavy chain loop regions are highlighted in grey.

Fig. 5A-D. Amino acid sequence of the transgene construct for the Fab region of therapeutic antibodies to PCSK9: alirocumab (Fig. 5A); evolocumab (Fig. 5B); ANGPTL3: evinacumab (Fig. 5C), OxPL: E06-scFv. Glycosylation sites are shown in bold. Glycosylation sites at glutamine are highlighted in green, and non-consensus glycosylation sites at asparagine (N) are highlighted in blue; Tyrosine O-sulfation sites (italics) are highlighted in yellow. Loop areas are highlighted in grey.

Fig. 6. Amino acid sequence of the transgene construct for the Fab region of denosumab, a therapeutic antibody to RANKL. Glycosylation sites are shown in bold. Glycosylation sites at glutamine are highlighted in green, and non-consensus glycosylation sites at asparagine (N) are highlighted in blue; Tyrosine O-sulfation sites (italics) are highlighted in yellow. The hinge area is highlighted in grey.

Fig. 7A and B. Amino acid sequence of the transgene construct for the Fab region of therapeutic antibodies that are PD-1 blockers: nivolumab (Fig. 7A) and pembrolizumab (Fig. 7B). Glycosylation sites are shown in bold. Glycosylation sites at glutamine are highlighted in green, and non-consensus glycosylation sites at asparagine (N) are highlighted in blue; Tyrosine O-sulfation sites (italics) are highlighted in yellow. Loop areas are highlighted in grey.

Fig. 8A-N. Amino acid sequence of the transgene construct for the Fab region of therapeutic antibodies directed against the biological factors: VEGF, ranibizumab (Fig. 8A), bevacizumab (Fig. 8B) and brolucizumab (Fig. 8D); k fD, lampalizumab (Fig. 8C); k BLyS, belimumab (Fig. 8E); to human complement protein C5, eculizumab (Fig. 8F); to MMP 9, andecaliximab (Fig. 8G) and to kallikrein, lanadelumab (Fig. 8H). Glycosylation sites are shown in bold. Glycosylation sites at glutamine are highlighted in green, and non-consensus glycosylation sites at asparagine (N) are highlighted in blue; Tyrosine O-sulfation sites (italics) are highlighted in yellow. Loop areas are highlighted in grey.

Fig. 9A and B. Amino acid sequence of the transgene construct for the Fab region of the TNF-alpha therapeutic antibody: adalimumab (Fig. 9A) and infliximab (Fig. 9B). Glycosylation sites are shown in bold. Glycosylation sites at glutamine are highlighted in green, and non-consensus glycosylation sites at asparagine (N) are highlighted in blue; Tyrosine O-sulfation sites (italics) are highlighted in yellow. Loop areas are highlighted in grey.

Fig. 10. Glycans that can be attached to the HuGlyFab regions of full-length mAbs or antigen binding domains. (Adapted from Bondt et al., 2014, Mol & Cell Proteomics 13.1: 30293039).

Fig. 11A and B. Alignment of the amino acid sequences of the heavy chain (Fig. 11A) (SEQ ID NOs: 283-299, 59, 300-302, 313, 303, 39 and 304-310, respectively, in order) and the light chain (Fig. 11B) (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 60, 58 36, 54, 311, 314, 312, 38, 234, 42, 44, 48, 247 and 235, respectively, in order) of the Fab fragments of the therapeutic antibodies disclosed herein. Positions that can be substituted to produce hyperglycosylated variant Fab regions are highlighted in green. Four substitutions (one in the heavy chain and three in the light chain) that would result in hyperglycosylation of the Fab region in human cells are indicated above the positions of the amino acid residues. (For the construction of mAbs or antigen-binding fragments containing additional glycosylation sites in the Fab domain, see, for example, Courtois et al., 2016, mAbs 8: 99-112, for a description of antibody derivatives that are hyperglycosylated in the Fab domain of a full-length antibody).

Fig. 12. Clustal multiple alignment of AAV capsid 1-9 sequences. Amino acid substitutions (shown in bold in the bottom rows) can be made for AAV9 and AAV8 capsids by recruiting amino acid residues from the corresponding position of other aligned AAV capsids. The sequence shown in red represents the hypervariable regions. The amino acid sequences of AAV capsids are assigned SEQ ID NO: as follows: AAV1 is SEQ ID NO: 71; AAV2 is SEQ ID NO: 72; AAV3-3 is SEQ ID NO: 73; AAV4-4 is SEQ ID NO: 74; AAV5 is SEQ ID NO: 75; AAV6 is SEQ ID NO: 76; AAV7 is SEQ ID NO: 77; AAV8 is SEQ ID NO: 78; AAV9 is SEQ ID NO: 79; hu31 is SEQ ID NO: 81 and hu32 is SEQ ID NO: 82.

5. Detailed description of the invention

Compositions and methods are described for delivering a fully human post-translationally modified (HuPTM) therapeutic monoclonal antibody (mAb) or an antigen binding fragment of a HuPTM therapeutic mAb (e.g., a fully human glycosylated Fab (HuGlyFab) therapeutic mAb) to a patient (human subject) who has been diagnosed with a disease or a condition for which treatment with a therapeutic mAb is indicated. Delivery may be pre

- 23 047128 is essentially carried out by means of gene therapy, for example, by introducing a viral vector or other DNA expression construct encoding a therapeutic mAb or an antigen-binding fragment thereof (or a hyperglycosylated derivative of both), to a patient (human subject) diagnosed with the condition for which it is indicated treatment with a therapeutic mAb to create a permanent depot in a patient's tissue or organ that continuously delivers the HuPTM mAb or antigen binding fragment of the therapeutic mAb, e.g., a human glycosylated transgene product, to the target tissue in which the mAb or antigen binding fragment exerts its therapeutic effect.

A HuPTM mAb or HuPTM antigen binding fragment encoded by a transgene may include, without limitation, a full-length or antigen binding fragment of a therapeutic antibody that binds to:

targets of the nervous system, including amyloid beta peptides (Ae or Abeta), may protein and the CGRP receptor, interleukins or interleukin receptors, including IL4R, IL17A, IL-5 and IL12/IL23, integrins, including integrin- alpha-4,

PCSK9, ANGPTL3 or oxidized phospholipids such as OxPL,

RANKL,

PD-1 or PD-L1 or PD-L2,

BLyS (B-lymphocyte stimulator, also known as B-cell activating factor (BAFF)), ocular targets including VEGF, fD and matrix metalloproteinase 9 (MMP9), TNF-alpha and plasma target proteins such as complement proteins human complement proteins, including complement proteins C5 and C5a and plasma kallikrein, or such mAbs or antigen-binding fragments designed to contain additional glycosylation sites in the Fab domain (for example, see Courtois et al., 2016, mAbs 8: 99-112, which is incorporated herein by reference in its entirety to describe antibody derivatives that are hyperglycosylated in the Fab domain of the full-length antibody). The amino acid sequences of the heavy and light chains of the above antigen-binding fragments are presented in table. 4, and the codon-optimized nucleotide sequences encoding the heavy and light chains of these antigen-binding fragments are presented in Table. 5.

The recombinant vector used to deliver the transgene includes non-replicating recombinant adeno-associated virus (rAAV) vectors. rAAVs are particularly attractive vectors for a number of reasons - they can transduce non-replicating cells and therefore can be used to deliver the transgene to tissues where cell division occurs at low levels, such as the CNS; they can be modified to preferentially target a specific organ of choice; and there are hundreds of capsid serotypes to choose from to achieve the desired tissue specificity and/or to avoid neutralization by the patient's pre-existing antibodies to some AAVs. Such rAAVs include, but are not limited to, AAV-based vectors containing capsid components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, or AAVrh20. In preferred embodiments, the AAV-based vectors provided herein comprise capsids from one or more serotypes AAV8, AAV9, AAV10, AAV11, AAVrh10, or AAVrh20.

However, other viral vectors may be used, including, but not limited to, lentiviral vectors; vaccinia viral vectors or non-viral expression vectors called naked DNA constructs. Transgene expression can be controlled by constitutive or tissue-specific expression control elements.

Gene therapy constructs are designed such that both heavy and light chains are expressed. More specifically, the heavy and light chains should be expressed in approximately equal amounts, that is, the heavy and light chains are expressed in an approximately 1:1 ratio of heavy chains to light chains. The coding sequences for the heavy and light chains can be constructed in a single construct in which the heavy and light chains are separated by a cleavable linker or IRES such that separate heavy and light chain polypeptides are expressed. In some embodiments, the coding sequences encode Fab or F(ab') ₂ or scFv.

In some embodiments, nucleic acids (e.g., polynucleotides) and nucleic acid sequences disclosed herein can be codon optimized, for example, using any codon optimization method known to one of ordinary skill in the art (see, for example, review of Quax et al., 2015, Mol Cell 59:149-161). The codon-optimized nucleotide sequences of the variable domains of the heavy and light chains of therapeutic antibodies are disclosed in table. 5. Each heavy and light chain requires a leader to ensure proper post-translational processing and secretion (unless expressed as scFv, in which only the N-terminal chain requires a leader sequence). Applicable leader after

- 24 047128 conditions for the expression of heavy and light chains of therapeutic antibodies in human cells are disclosed herein. An exemplary recombinant expression construct is shown in FIG. 1.

Production of a HuPTMmAb or HuPTM Fab (including a HuPTM scFv) should result in a biologically improved molecule for treating a disease via gene therapy - for example, by introducing a viral vector or other DNA expression construct encoding a full-length or HuPTM Fab or other antigen-binding fragment , such as a scFv, therapeutic mAb to a patient (human subject) diagnosed with a disease for that mAb, to establish in the subject a permanent depot that continuously supplies the human glycosylated sulfated transgene product produced by the subject's transduced cells. The cDNA design for HuPTMmAb or HuPTM Fab or HuPTM scFv must include a signal peptide that ensures proper co- and post-translational processing (protein glycosylation and sulfation) by transduced human cells.

Pharmaceutical compositions suitable for administration to human subjects contain a suspension of the recombinant vector in a formulation buffer containing a physiologically compatible aqueous buffer, a surfactant and optional excipients. Such a formulation buffer may contain one or more of polysaccharides, surfactants, polymer or oil.

As an alternative or complementary treatment to gene therapy, a full-length or HuPTM Fab or other antigen-binding fragment thereof can be produced in human cell lines using recombinant DNA technology and the glycoprotein can be administered to patients. Human cell lines that can be used to produce such a recombinant glycoprotein include, but are not limited to, human embryonic kidney 293 (HEK293), HT-1080 fibrosarcoma, HKB-11, CAP, HuH-7 and retinal cell lines, PER. C6 or RPE, to name a few (for example, see Dumont et al., 2015, Crit. Rev. Biotechnol. 36(6): 1110-1122, which is incorporated by reference in its entirety for a review of human cell lines that can be used for recombinant production of HuPTM Fab or HuPTM scFv, e.g. HuPTM Fab glycoprotein). To ensure complete glycosylation, especially sialylation and tyrosine sulfation, the cell line used for production can be enhanced by engineering host cells to coexpress α-2,6-sialyltransferase (or both α-2,3- and α-2 ,6sialyltransferase) and/or the enzymes TPST-1 and TPST-2, responsible for tyrosine sulfation in human cells.

It is not necessary that every molecule resulting from gene or protein therapy be fully glycosylated and sulfated. Rather, the population of glycoproteins produced must have sufficient glycosylation (including 2,6-sialylation) and sulfation to demonstrate efficacy. The goal of the gene therapy of the present invention is to slow or stop the progression of the disease.

Combination treatments involving delivery of a full-length or HuPTM Fab or an antigen-binding fragment thereof to a patient, accompanied by the administration of other available treatments, are covered by the methods of the present invention. Additional treatments may be given before, simultaneously, or after gene therapy treatment. Such additional treatments may include, without limitation, co-therapy with a therapeutic mAb.

Methods for making viral vectors, in particular AAV-based viral vectors, are also provided. In specific embodiments, methods are provided for producing recombinant AAVs comprising culturing a host cell containing an artificial genome containing a cis-expression cassette flanked by an AAV ITR, wherein the cis-expression cassette contains a transgene encoding a therapeutic antibody operably linked to expression control elements that will control transgene expression in human cells; a trans-expression cassette lacking the AAV ITRs, wherein the trans-expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that direct expression of the AAV rep and capsid proteins in a host cell in culture and delivers the rep and cap proteins in trans ; sufficient auxiliary functions of the adenovirus for replication and packaging of the artificial genome with AAV capsid proteins; and recovery of recombinant AAV encapsulating the artificial genome from cell culture.

5.1. Constructions

Provided herein are viral vectors or other DNA expression constructs encoding HuPTMmAb or an antigen binding fragment thereof, particularly HuGlyFab, or a hyperglycosylated derivative of the HuPTMmAb antigen binding fragment. Viral vectors and other DNA expression constructs provided herein provide any suitable method for delivering a transgene to a target cell.

Transgene delivery vehicles include viral vectors, liposomes, other lipid-containing complexes, other macromolecular complexes, synthetically modified mRNA,

- 25 047128 unmodified mRNA, small molecules, non-biologically active molecules (eg gold particles), polymerized molecules (eg dendrimers), naked DNA, plasmids, phages, transposons, cosmids or episomes. In some embodiments, the vector is a targeting vector, such as a vector targeting retinal pigment epithelial cells, CNS cells, muscle cells, or liver cells.

In some aspects, the present disclosure provides a nucleic acid for use, wherein the nucleic acid comprises a nucleotide sequence that encodes a HuPTMmAb or HuGlyFab, or another antigen binding fragment thereof, as a transgene described herein operably linked to a promoter selected for expression in the tissue targeted for transgene expression, for example, without limitation, CB7 promoter (see Fig. 1), cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, GFAP (glial fibrillary acidic protein) promoter, MBP (myelin basic protein) promoter, MMT promoter, EF-1 alpha promoter, UB6 promoter, avian beta actin promoter, CAG promoter, RPE65 promoter and opsin promoter, liver specific promoters such as TBG (thyroxine binding globulin) promoter, APOA2 promoter, SERPINA1 (hAAT) promoter or the mIR22 promoter, or a muscle-specific promoter such as the human desmin promoter or the Pitx3 promoter, inducible promoters such as the hypoxia-inducible promoter or the rapamycin-inducible promoter.

In certain embodiments, provided herein are recombinant vectors that contain one or more nucleic acids (eg, polynucleotides). Nucleic acids may contain DNA, RNA, or a combination of DNA and RNA. In certain embodiments, the DNA comprises one or more sequences selected from the group consisting of promoter sequences, sequences of a gene of interest (a transgene, e.g., nucleotide sequences encoding the heavy and light chains of HuPTMmAb or HuGlyFab or other antigen-binding fragment), untranslated regions, and termination sequences. In certain embodiments, viral vectors provided herein comprise a promoter operably linked to a gene of interest.

In certain embodiments, nucleic acids (e.g., polynucleotides) and nucleic acid sequences disclosed herein can be codon optimized, for example, using any codon optimization method known to one of ordinary skill in the art (see, for example, review of Quax et al., 2015, Mol Cell 59:149-161). Codon optimized nucleotide sequences for expression in human cells are presented herein for the HuGlyFab heavy and light chains in Table 1. 5.

In a specific embodiment, the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank an expression cassette; (2) one or more control elements, b) avian β-actin intron and c) rabbit poly-A β-globin signal; and (3) nucleic acid sequences encoding the heavy and light chains of the VEGF antigen-binding fragment, separated by a self-cleaving furin (F)/F2A linker, allowing for the expression of equal amounts of heavy and light chain polypeptides. An exemplary structure is shown in FIG. 1.

5.1.1. mRNA vectors

In certain embodiments, as an alternative to DNA vectors, the vectors provided herein are modified mRNA encoding a gene of interest (eg, a transgene, such as HuPTMmAb or HuGlyFab or another antigen-binding fragment thereof). The synthesis of modified and unmodified mRNA for transgene delivery to retinal pigment epithelial cells is described, for example, in Hansson et al., J. Biol. Chem., 2015, 290(9):5661-5672, which is incorporated herein by reference in its entirety. In some embodiments, provided herein is a modified mRNA encoding a HuPTMmAb or a HuPTM Fab, or a HuPTM scFv.

5.1.2. Viral vectors

Viral vectors include adenovirus, adeno-associated virus ( ^AAV , e.g., AAV8, AAV9, AAVrh10), lentivirus, helper-dependent adenovirus, herpes simplex virus, poxvirus, hemagglutinin virus of Japan (HVJ), alpha virus, vaccinia virus smallpox and retrovirus. Retroviral vectors include murine leukemia virus (MLV) and human immunodeficiency virus (HIV) vectors. Alpha viral vectors include Semliki Forest virus (SFV) and Sindbis virus (SIN). In certain embodiments, the viral vectors provided herein are recombinant viral vectors. In certain embodiments, the viral vectors provided herein are modified such that they are replication deficient in humans. In some embodiments, the viral vectors are hybrid vectors, for example, an AAV vector housed within a helpless adenoviral vector. According to certain options

- 26 047128 embodiments provided herein are viral vectors comprising a viral capsid from a first virus and viral envelope proteins from a second virus. In specific embodiments, the second virus is vesicular stomatitis virus (VSV). In more specific embodiments, the envelope protein is a VSV-G protein.

In certain embodiments, the viral vectors provided herein are HIV-based viral vectors. In some embodiments, HIV-based vectors provided herein comprise at least two polynucleotides, the gag and pol genes being derived from the HIV genome and the env gene being derived from another virus.

In certain embodiments, the viral vectors provided herein are herpes simplex virus-based viral vectors. In certain embodiments, the herpes simplex virus vectors provided herein are modified to lack one or more immediate early (IE) genes, rendering them noncytotoxic.

In certain embodiments, the viral vectors provided herein are MLV-based viral vectors. In certain embodiments, MLV-based vectors provided herein contain up to 8 kb. heterologous DNA instead of viral genes.

In certain embodiments, the viral vectors provided herein are lentivirus-based viral vectors. In certain embodiments, the lentiviral vectors provided herein are derived from human lentiviruses. In certain embodiments, the lentiviral vectors provided herein are derived from non-human lentiviruses. In certain embodiments, the lentiviral vectors provided herein are packaged within a lentiviral capsid. In some embodiments, lentiviral vectors provided herein comprise one or more of the following elements: long terminal repeats, a primer binding site, a polypurine tract, att sites, and an encapsidation site.

In certain embodiments, the viral vectors provided herein are alpha virus-based viral vectors. In certain embodiments, the alphavirus vectors provided herein are recombinant, replication-defective alpha viruses. In certain embodiments, the alpha virus replicons in the alpha virus vectors provided herein are targeted to specific cell types by displaying a functional heterologous ligand on their virion surface.

In certain embodiments, the viral vectors provided herein are AAV-based viral vectors. In some embodiments, the AAV-based vectors provided herein do not encode the AAV rep gene (required for replication) and/or the AAV cap gene (required for the synthesis of capsid proteins) (rep and cap proteins may be provided by packaging cells in trans). Several serotypes of AAV have been identified. In certain embodiments, the AAV-based vectors provided herein contain components of one or more AAV serotypes. In certain embodiments, the AAV-based vectors provided herein comprise capsid components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAVrh10. In preferred embodiments, the AAV-based vectors provided herein contain components from one or more serotypes AAV8, AAV9, AAV10, AAV11, or AAVrh10. Provided are viral vectors in which the capsid protein is a variant of the AAV8 capsid protein (SEQ ID NO: 78), the AAV9 capsid protein (SEQ ID NO: 79) or the AAVrh10 capsid protein (SEQ ID NO: 80), more specifically at least 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the amino acid sequence of AAV8 capsid protein (SEQ ID NO: 78), AAV9 capsid protein (SEQ ID NO: 79) or AAVrh10 capsid protein (SEQ ID NO: 80), while maintaining the biological function of the native capsid. In certain embodiments, the encoded AAV capsid is characterized by the sequence SEQ ID NO: 78, 79, or 80 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions and preservation of the biological function of the AAV8 AAV9 or AAVrh10 capsid. In fig. 12 shows a comparative alignment of the amino acid sequences of the capsid proteins of various AAV serotypes with potential amino acids that may be substituted at specific positions in the aligned sequences based on comparisons in the series labeled SUBS. Accordingly, in specific embodiments, the AAV vector contains a variant capsid of AAV8, AAV9, or AAVrh10 that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions that are not present at this position in the native AAV capsid sequence as indicated in the SUBS line of FIG. 12. The sequence for AAVrh10 is given in

- 27 047128 table. 4.

In some embodiments, the AAV used in the compositions and methods described herein is Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated by reference in its entirety. In some embodiments, the AAV that is used in the methods described herein contains one of the following amino acid inserts: LGETTRP (SEQ ID NO: 162) or LALGETTRP (SEQ ID NO: 163), as described in US patent No. 9193956; 9458517 and 9587282 and US Patent Application Publication No. 2016/0376323, each of which is incorporated herein by reference in its entirety. In certain embodiments, the AAV that is used in the methods described herein is AAV.7m8 (including variants) as described in US Pat. No. 9,193,956; 9458517 and 9587282, US Patent Application No. 2016/0376323 and International Publication WO 2018/075798, each of which is incorporated herein by reference in its entirety. In certain embodiments, the AAV that is used in the methods described herein is any AAV disclosed in US Pat. No. 9,585,971, such as AAV-PHP.B. In certain embodiments, the AAV used in the compositions and methods described herein is an AAV2/Rec2 or AAV2/Rec3 vector that contains hybrid capsid sequences derived from AAV8 capsids and capsids of serotypes Cy5, rh20, or rh39, as described in the publication Charbel Issa et al., 2013, PLoS One 8(4): e60361, which is incorporated herein by reference for these vectors. In certain embodiments, the AAV that is used in the methods described herein is the AAV disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: US Pat. No. 7,906,111; 8524446; 8999678; 8628966; 8927514; 8734809; US 9284357; 9409953; 9169299; 9193956; 9458517 and 9587282, US Patent Application Publication No. 2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257 and international patent applications No. PCT/US2015/034799; PCT/EP2015/053335.

Some of the methods described herein use viral vectors based on AAV8, AAV9 and AAVrh10. The nucleotide sequences of AAV-based viral vectors and methods for producing recombinant AAV and AAV capsids are described, for example, in US Patent No. 7282992 B2, US Patent No. 7790449 B2, US Patent No. 8318480 B2, US Patent No. 8982322 B2 and International Patent Application No. PCT /EP2014/076466, each of which is incorporated herein by reference in its entirety. In one aspect, provided herein are AAV-based viral vectors (eg, AAV8, AAV9, or AAVrh10) encoding a transgene (eg, HuPTM Fab). The amino acid sequences of AAV capsids, including AAV8, AAV9 and AAVrh10, are shown in FIG. 12 and in table. 4.

In certain embodiments above, single chain AAV (ssAAV) can be used. In some embodiments, a self-complementary vector, such as scAAV, may be used (see, for example, Wu, 2007, Human Gene Therapy, 18(2): 17182, McCarty et al., 2001, Gene Therapy, Vol 8, Number 16, Pages 1248-1254 and US Patent Nos. 6596535; 7125717 and 7456683, each of which is incorporated herein by reference in its entirety).

In certain embodiments, the viral vectors used in the methods described herein are adenovirus-based viral vectors. The recombinant adenoviral vector can be used for transfer into a transgene encoding HuPTMmAb or HuGlyFab or an antigen binding fragment. The recombinant adenovirus may be a first generation vector with an E1 deletion, with or without an E3 deletion, and with an expression cassette inserted into any deleted region. The recombinant adenovirus may be a second generation vector that contains complete or partial deletions of the E2 and E4 regions. Helper-dependent adenovirus retains only the adenovirus inverted terminal repeats and the packaging signal (phi). The transgene is inserted between the packaging signal and the 3'ITR, with or without sequences, to maintain the genome close to the wild type size of approximately 36 kb. An exemplary protocol for producing adenoviral vectors can be found in Alba et al., 2005, Gutless adenovirus: last generation adenovirus for gene therapy.

Gene Therapy 12:S18-S27, which is incorporated herein by reference in its entirety.

In some embodiments, the viral vectors used in the methods described herein are lentivirus-based viral vectors. A recombinant lentiviral vector can be used to carry a transgene encoding the antigen binding fragment of the HuPTM mAb. To create the construct, four plasmids are used: a Gag/pol sequence-containing plasmid, a Rev sequence-containing plasmid, a coat protein-containing plasmid (i.e., VSV-G), and a Cis plasmid with packaging elements and the anti-VEGF antigen-binding fragment gene.

To produce a lentiviral vector, the four plasmids are cotransfected into cells (i.e., HEK293-based cells), whereby polyethylenimine or calcium phosphate can be used to

- 28 047128 as transfection agents, among other things. The lentivirus is then collected in the supernatant (lentiviruses must be released from cells to be active, so the cells do not/should not be collected). The supernatant is filtered (0.45 µm) and then magnesium chloride and benzonase are added. Further downstream processes can vary widely, with the use of TFF and column chromatography being the most GMP compliant. Others use ultracentrifugation with or without column chromatography. Exemplary protocols for the production of lentiviral vectors can be found in Lesch et al., 2011, Production and purification of lentiviral vector generated in 293T suspension cells with baculoviral vectors, Gene Therapy 18:531-538 and Ausubel et al., 2012, Production of CGMP- Grade Lentiviral Vectors, Bioprocess Int. 10(2):32-43, both of which are incorporated herein by reference in their entirety.

In a specific embodiment, the vector for use in the methods described herein is a vector that encodes an antigen binding fragment of a HuPTM mAb, such as HuGlyFab, such that when the vector is introduced into a suitable cell, a glycosylated and/or tyrosine sulfated version of the antigen binding fragment of the HuPTM mAb or HuGlyFab expressed by the cell.

5.1.3. Promoters and modifiers of gene expression

In some embodiments, the vectors provided herein contain components that modulate gene delivery or gene expression (eg, expression control elements). In certain embodiments, the vectors provided herein contain components that modulate gene expression. In certain embodiments, the vectors provided herein contain components that affect binding or targeting to cells. In certain embodiments, the vectors provided herein contain components that influence the localization of a polynucleotide (eg, a transgene) in a cell after uptake. In certain embodiments, the vectors provided herein contain components that can be used as detectable or selectable markers, for example, to detect or select cells that have taken up a polynucleotide.

In certain embodiments, the viral vectors provided herein contain one or more promoters that control expression of the transgene. In certain embodiments, the promoter is a constitutive promoter. In certain embodiments, the promoter is a CB7 promoter (Dinculescu et al., 2005, Hum Gene Ther 16:649-663, incorporated herein by reference in its entirety). In some embodiments, the CB7 promoter includes other expression control elements that enhance expression of the transgene driven by the vector. In certain embodiments, other expression control elements include an avian β-actin intron and/or a rabbit β-globin polA signal. In certain embodiments, the promoter comprises a TATA box. In certain embodiments, a promoter comprises one or more elements. In certain embodiments, one or more promoter elements may be inverted or moved relative to each other. In certain embodiments, promoter elements are arranged to function together. In certain embodiments, the promoter elements are arranged to function independently. In certain embodiments, the viral vectors provided herein comprise one or more promoters selected from the group consisting of the human CMV immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat (RS), and the rat insulin promoter. In certain embodiments, the vectors provided herein contain one or more long terminal repeat (LTR) promoters selected from the group consisting of AAV, MLV, MMTV, SV40, RSV, HIV-1, and HIV-2 LTRs. In certain embodiments, the vectors provided herein contain one or more tissue-specific promoters (eg, a retinal epithelial cell-specific promoter, a CNS-specific promoter, a liver-specific promoter, or a muscle-specific promoter). In certain embodiments, the viral vectors provided herein comprise an RPE65 promoter or an opsin promoter (retinal/CNS cell-specific promoter). In certain embodiments, the viral vectors provided herein comprise a liver cell-specific promoter, such as a TBG (thyroxine-binding globulin) promoter, an APOA2 promoter, a SERPINA1 (hAAT) promoter, or a MIR122 promoter. In certain embodiments, the viral vector provided herein comprises a muscle-specific promoter, such as the human desmin promoter (Jonuschies et al., 2014, Curnrn. Gene Ther. 14:276-288) or the Pitx3 promoter (Coulon et al., 2007 , JBC 282:33192). In other embodiments, the viral vector contains a VMD2 promoter.

In certain embodiments, the promoter is an inducible promoter. In certain embodiments, the promoter is an inducible

- 29 047128 hypoxia-sensitive promoter. In certain embodiments, the promoter comprises a hypoxia-inducible factor (HIF) binding site. In certain embodiments, the promoter comprises a HIF-1a binding site. In certain embodiments, the promoter comprises a HIF-2a binding site. In certain embodiments, the HIF binding site comprises an RCGTG motif. For details regarding the location and sequence of HIF binding sites, see, for example, Schodel, et al., Blood, 2011, 117(23):e207-e217, which is incorporated herein by reference in its entirety. In certain embodiments, the promoter comprises a binding site for a hypoxia-inducible transcription factor other than the HIF transcription factor. In certain embodiments, viral vectors provided herein contain one or more IRES sites that are preferentially translated under hypoxia. For ideas regarding hypoxia-induced gene expression and factors involved therein, see, for example, Kenneth and Rocha, Biochem J., 2008, 414:19-29, which is incorporated herein by reference in its entirety. In specific embodiments, the hypoxia-inducible promoter is a human N-WASP promoter, see, for example, Salvi, 2017, Biochemistry and Biophysics Reports 9: 13-21 (incorporated by reference for the N-WASP promoter idea) or is a hypoxia-inducible human Epo promoter, see Tsuchiya et al., 1993, J. Biochem. 113:395-400 (incorporated by reference to disclose the hypoxia-inducible Epo promoter). In other embodiments, the promoter is a drug-inducible promoter, for example, a promoter that is induced by administration of rapamycin or analogs thereof. See, for example, the disclosure of rapamycin-inducible promoters in PCT publications WO 94/18317, WO 96/20951, WO 96/41865, WO 99/10508, WO 99/10510, WO 99/36553 and WO 99/41258 and US 7067526, which are incorporated herein by reference in their entirety to disclose drug-inducible promoters.

In certain embodiments, viral vectors provided herein contain one or more regulatory elements other than a promoter. In certain embodiments, viral vectors provided herein comprise an enhancer. In certain embodiments, the viral vectors provided herein contain a repressor. In certain embodiments, the viral vectors provided herein contain an intron or a chimeric intron. In certain embodiments, viral vectors provided herein contain a polyadenylation sequence.

5.1.4. Signal peptides

In certain embodiments, the vectors provided herein contain components that modulate protein delivery. In certain embodiments, the viral vectors provided herein contain one or more signal peptides. Signal peptides may also be referred to herein as leader sequences or leader peptides. In certain embodiments, the signal peptides allow the transgene product to achieve proper packaging (eg, glycosylation) in the cell. In certain embodiments, the signal peptides allow the transgene product to achieve proper localization in the cell. In certain embodiments, the signal peptides allow the transgene product to achieve secretion from the cell.

There are two main approaches to selecting a signal sequence for protein production in the context of gene therapy or cell culture. One approach is to use a signal peptide from proteins homologous to the protein being expressed. For example, a human antibody signal peptide can be used to express IgG in CHO or other cells. Another approach is to identify signal peptides optimized for the specific host cells used for expression. Signal peptides can be interchangeable between different proteins, or even between proteins from different organisms, but typically the signal sequences of the most abundant secreted proteins of that cell type are used for protein expression. For example, human albumin signal peptide, the most abundant protein in plasma, was found to significantly increase the yield of protein production in CHO cells. However, certain signal peptides may retain function and exhibit activity after cleavage from the expressed protein as post-targeting functions. Thus, in certain embodiments, the signal peptide is selected from the signal peptides of the most abundant proteins secreted by the cells used for expression to avoid post-targeting functions. In a preferred embodiment, the signal sequence is fused to both the heavy and light chain sequences. The preferred sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) (see FIGS. 2-9). Alternatively, signal sequences that are suitable for expression of HuPTM mAb or Fab in

- 30 047128 eye (including the central nervous system), muscles or liver, are presented in table. 1, 2 and 3, respectively, below.

Table 1

Signal peptides for expression in ocular/CNS tissue

Signal peptide source SEQID NO: Subsequence VEGFA signal peptide 164 MNFLLSWVHWSLALLLYLHHAKWSQA Fibulin-1 signaling peptide 165 MERAAPSRRVPLPLLLLGGLALLAAGVDA Vitronectin signal peptide 166 MAPLRPLLILALLAWVALA Complement factor H signal peptide 167 MRLLAKIICLMLWAICVA Signal peptide opticin 168 MRLLAFLSLLALVLQETGT Albumin signal peptide 169 MKW VTFISLLFLF S S AYS Chymotrypsinogen signal peptide 170 MAFLWLLSCWALLGTTFG Interleukin-2 signal peptide 171 MYRMQLLSCIALILALVTNS Trypsinogen signal peptide-2 172 MNLLLILTFVAAAVA

table 2

Signal peptide for expression in muscle cells

Signal peptide source SEQID NO: Subsequence Human SPARC 173 MRAWIFFLLCLAGRALA Human alpha-1(1) collagen 174 MFSFVDLRLLLLLAATALLTHG Human lactotransferrin 175 MKLVFLVLLFLGALGLCLA Human SZ complement 176 MGPTSGPSLLLLLLTHLPLALG Human lumican 177 MSLSAFTLFLALIGTSG Human gelsolin isoform 1 178 MAPHRPAPALLCALSLALCALSLPVRA Human procathepsin N 179 MWATLPLLCAGAWLLGVPVCGA Human SERPINF1 180 MQ ALVLLLCIGALLGHS SC SERPINE1 person 181 MQMSPALTCLVLGLALVFGEGSA Human cathepsin D 182 MQPSSLLPLALCLLAAPASA human TIMP1 183 MAPFEPLASGILLLLWLIAPSRA Human fibronectin 184 MLRGPGPGLLLLAVQCLGTAVPSTGASKSKR Human complement subcomponent Cis 185 MWCIVLF SLL AW VY A Human cathepsin L1 186 MNPTLILAAFCLGIASA Cathepsin B human 187 MWQLWASLCCLLVLANA Proline-rich acidic phosphoprotein 1/2 of human saliva 188 MLLILLSVALLAFSSA Human follistatin-related protein 1 189 MWKRWLALALALVAVAWVRA

- 31 047128

Table 3

Signal peptides for expression in liver cells

Signal peptide SEQID NO: Subsequence Human serum albumin 169 MKW VTFISLLFLF S S AYS Human antitrypsin a-1 (SERPINA1) 190 MPSSVSWGILLLAGLCCLVPVSLA Human apolipoprotein A-1 191 MKAAVLTLAVLFLTGSQA Human apolipoprotein A-2 192 MKLLAATVLLLTICSLEG Human apolipoprotein B-100 193 MDPPRPALLALLALPALLLLLLLAGARA Human coagulation factor IX 194 MQRVNMIMAESPGLITICLGYLLSAEC Human complement C2 195 MGPLMVLFCLLFLYPGLADS Human complement factor H-related protein 2 (CFHR2) 196 MWLL VS VILISRIS S VGG Human complement factor H-related protein 5 (CFHR5) 197 MLLLFSVILISWVSTVGG human fibrinogen a-chain (FGA) 198 MFSMRIVCLVLSVVGTAWT Human fibrinogen β chain (FGB) 199 MKRMVSWSFHKLKTMKHLLLLLLCVFLVKS Human fibrinogen γ chain (FGG) 200 MSWSLHPRNLILYF YALLFLS STC VA human a-2-H8-glycoprotein (AHSG) 201 MKSLVLLLCLAQLWGCHS Human hemopexin (HPX) 202 MARVLGAPVALGLWSLCWSLAIA Human kininogen-1 203 MKLITILFLC SRLLLSLT Human mannose binding protein C (MBL2) 204 MSLFPSLPLLLLSMVAASYS Human plasminogen (PLMN) 205 MENKE VVLLLLLLFLKSGQG Human prothrombin (coagulation factor II) 206 MAHVRGLQLPGCL AL AALC SLVHS Human secreted phosphoprotein 24 207 MISRMEKMTMMMKILIMFALGMNYWSCSG Human antithrombin-III (SERPINC1) 208 MYSNVIGTVTSGKRKVYLLSLLLIGFWDCVTC Human serotransferrin (TF) 209 MRLAVGALLVCAVLGLCLA

5.1.5. Polycistronic mRNAs - IRES and F2A linkers and scFv constructs

Internal ribosome landing sites. One construct can be designed to encode both heavy and light chains separated by a cleavable linker or IRES such that separate heavy and light chain polypeptides are expressed by the transduced cells. In certain embodiments, viral vectors provided herein provide polycistronic (eg, bicistronic) mRNAs. For example, a viral construct may encode heavy and light chains separated by internal ribosomal entry site (IRES) elements (for examples of the use of IRES elements to create bicistronic vectors, see, for example, Gurtu et al., 1996, Biochem. Biophys. Res. Comm 229(1):295-8, which is incorporated herein by reference in its entirety). IRES elements bypass the ribosome scanning model and begin translation at internal sites. The use of IRES in AAV is described, for example, in Furling et al., 2001, Gene Ther 8(11): 854-73, which is incorporated herein by reference in its entirety. In some embodiments, the bicistronic mRNA is contained in a viral vector with a limit on the size of the polynucleotide(s) therein. In certain embodiments, the bicistronic mRNA is contained within an AAV virus-based vector (eg, an AAV8-based, AAV9-based, or AAVrh10-based vector).

Linkers (()urin-1'2A. In other embodiments, viral vectors provided herein encode heavy and light chains separated by a cleavable linker,

- 32 047128 such as self-cleaving fury linkers! i/F2A (F/F2A) (published by Fang et al., 2005, Nature Biotechnology 23: 584-590 and Fang, 2007, Mol Ther 15: 1153-9, each of which is incorporated herein by reference in its entirety). For example, a furin^2A linker can be included in an expression cassette to separate the heavy and light chain coding sequences, resulting in a construct with the structure:

Leader - Heavy chain - Furin site - F2A site - Leader - Light chain - PolyA. The F2A site with the amino acid sequence LLNFDLLKLAGDVESNPGP (SEQ ID NO: 210) is self-processing, resulting in cleavage between the terminal amino acid residues G and P. Additional linkers that may be used include, but are not limited to:

T2A:(GSG)EGRGSLLTCGDVEENPGP (SEQ Ш NO: 211);

P2A: (GSG) AT NF SLLKQ AGD V E E N P G P (SEQ ID NO: 212);

E2A: (GSG) QCTNYALLKLAGDVESNPGP (SEQ Ш NO: 213);

F2A: (GSG) VKQTLNFDLLKLAGDVESNPGP (SEQ Ш NO: 214).

The peptide bond is skipped when the ribosome encounters the F2A sequence in the open reading frame, resulting in termination of translation or continued translation of the subsequent sequence (light chain). This self-processing sequence results in a number of additional amino acids at the C-terminal end of the heavy chain. However, such additional amino acids are then cleaved by host cell furin at furin sites located immediately upstream of the F2A site and after the heavy chain sequence, and are further cleaved by carboxypeptidases. The resulting heavy chain may contain one, two, three or more additional amino acids included at the C-terminus, or may contain no such additional amino acids depending on the sequence of the furin linker used and the carboxypeptidase that cleaves the linker in vivo (see, for example, Fang et al., 17 April 2005, Nature Biotechnol. Advance Online Publication; Fang et al., 2007, Molecular Therapy 15(6): 1153-1159; Luke, 2012, Innovations in Biotechnology, Ch. 8, 161-186). Furin linkers that can be used contain a series of four basic amino acids, for example, RKRR (SEQ ID NO: 215), RRRR (SEQ ID NO: 216), RRKR (SEQ ID NO: 217) or RKKR (SEQ ID NO: 218). Once this linker is cleaved by carboxypeptidase, additional amino acids may remain, so that at the C-terminus of the heavy chain there may be an additional zero, one, two, three or four amino acids left, for example, R, RR, RK, RKR, RRR, RRK, RKK, RKRR (SEQ ID NO: 215), RRRR (SEQ ID NO: 216), RRKR (SEQ ID NO: 217) or RKKR (SEQ ID NO: 218). In certain embodiments, one linker is cleaved by carboxypeptidase, leaving no additional amino acids. In certain embodiments, from 0.5% to 1%, 1% to 2%, 5%, 10%, 15%, or 20% of the antibody, e.g., antigen binding fragment, population produced by the constructs for use in the methods described herein one, two, three or four amino acids remain at the C-terminus of the heavy chain after cleavage. In some embodiments, the furin linker is characterized by the sequence RXK/RR such that the additional amino acids at the C-terminus of the heavy chain are R, RX, RXK, RXR, RXKR, or RXRR, where X is any amino acid, such as alanine (A). In certain embodiments, no additional amino acids may remain at the C-terminus of the heavy chain.

Flexible peptide linker. In some embodiments, a single construct can be designed to encode both the heavy and light chains (preferably the heavy and light chain variable domains) separated by a flexible peptide linker, such as those encoding scFvs. The flexible peptide linker may be composed of flexible residues such as glycine and serine such that adjacent heavy chain and light chain domains can move freely relative to each other. The design can be arranged such that the heavy chain variable domain is at the N-terminus of the scFv, followed by a linker and then a light chain variable domain. Alternatively, the construct can be designed such that the light chain variable domain is at the N-terminus of the scFv, followed by a linker and then a heavy chain variable domain. That is, the components can be arranged as NH ₂ -VL-linker-V _H -COOH or NH ₂ -V _H -linker-VL-COOH.

In certain embodiments, an expression cassette described herein is contained in a viral vector with a limit on the size of the polynucleotide(s) therein. In certain embodiments, the expression cassette is contained in an AAV virus vector. Due to size limitations of certain vectors, the vector may or may not contain coding sequences for the complete heavy and light chains of a therapeutic antibody, but may contain coding sequences for the heavy and light chains of antigen binding fragments, such as Fab or F(ab' fragment heavy and light chains ) ₂ or scFv. In particular, the AAV vectors described herein can accommodate a transgene of approximately 4.7 kb. For designs such as those in FIG. 1, which contains the CB7 promoter, the avian β-actin intron, the rabbit β-globin polyA signal, and the ITR, the encoded therapeutic antibody may contain approximately 752 amino acids

- 33 047128 lots. Replacement of smaller expression elements would allow the expression of larger protein products, such as full-length therapeutic antibodies.

5.1.6. Untranslated areas

In some embodiments, viral vectors provided herein contain one or more untranslated regions (UTRs), such as 3' and/or 5' UTRs. In certain embodiments, the UTRs are optimized for the desired level of protein expression. In certain embodiments, the UTRs are optimized for the half-life of the transgene mRNA. In certain embodiments, the UTRs are optimized for transgene mRNA stability. In certain embodiments, the UTRs are optimized for the secondary structure of the transgene mRNA.

5.1.7. Inverted terminal repeats

In certain embodiments, viral vectors provided herein comprise one or more inverted terminal repeat (ITR) sequences. ITR sequences can be used to package a recombinant gene expression cassette into a viral vector virion. In certain embodiments, the ITR is derived from an AAV, such as AAV8 or AAV2 (see, e.g., Yan et al., 2005, J. Virol., 79(1):364-379; US Pat. No. 7282199 B2, US Pat. No. 7790449 B2, US Patent No. 8318480 B2, US Patent No. 8962332 B2 and International Patent Application No. PCT/EP2014/076466, each of which is incorporated herein by reference in its entirety).

In certain embodiments, modified ITRs can be used to produce a self-complementary vector, such as scAAV (see, for example, Wu, 2007, Human Gene Therapy, 18(2): 171-82, McCarty et al., 2001, Gene Therapy , Vol 8, Number 16, Pages 1248-1254 and US Patent Nos. 6,596,535; 7,125,717 and 7,456,683, each of which is incorporated herein by reference in its entirety).

5.1.8. Transgenes

The transgenes encode a HuPTM mAb as a full-length antibody or an antigen-binding fragment thereof, preferably a Fab (HuGlyFab) or F(ab') ₂ fragment or scFv based on a therapeutic antibody disclosed herein. In certain embodiments, the HuPTM mAb or antigen binding fragment, particularly HuGlyFab, is designed to contain additional glycosylation sites in the Fab domain (e.g., see Courtois et al., 2016, mAbs 8: 99-112, which is incorporated in its entirety in this document by reference to describe hyperglycosylation sites on the Fab domain). In fig. 11 shows alignments of the Fab heavy and light chains of therapeutic antibodies disclosed herein, highlighting in green residues that may be replaced by asparagine or, in some cases, serine, resulting in hyperglycosylation.

In certain embodiments, the transgenes encode either a full-length antibody or an antigen-binding fragment thereof with a heavy and light chain coding sequence. When using a full-length antibody, a construct encoding a modified mAb can be used. For example, C-terminal lysines (-K), conserved in the heavy chain genes of all human IgG subclasses, are generally absent from circulating antibodies - C-terminal lysines are cleaved off in the bloodstream, resulting in a heterogeneous population of circulating IgG (van den Bremer et al., 2015, mAbs 7:672-680). In vector constructs for full-length mAbs, the DNA encoding the C-terminal lysine (-K) or glycine-lysine (-GK) of the Fc terminus can be removed to produce a more homogeneous antibody product in situ. (See Hu et al., 2017 Biotechnol. Prog. 33: 786-794, which is incorporated by reference in its entirety.)

Alternatively, antigen binding fragments are advantageously used. In fig. 2A-2F, 3A-3E, 4A-4B, 5A-5D, 6, 7A, 7B, 8A-8H and 9A-9B provide the amino acid sequences of the heavy and light chains of Fab and scFv fragments of therapeutic antibodies (see also Table 4, which provides the amino acid sequences of the heavy and light chains of therapeutic antibodies). The transgene may contain nucleotide sequences encoding heavy and light chain sequences, using nucleotide sequences that encode the Fab portion of the heavy chain plus a portion of the heavy chain constant domain for the appropriate isotype, as further described herein, and the light chain. Nucleotide sequences, codon optimized for expression in human cells, encoding parts of the Fab fragments of the heavy and light chains of the therapeutic antibodies disclosed herein are presented in table. 5. The transgene may encode a Fab fragment using nucleotide sequences encoding the sequences shown in FIG. 2A-2F, 3A-3E, 4A-4B, 5A-5C, 6, 7A, 7B, 8A-8H, and 9A-9B, but not including the portion of the hinge region on the heavy chain that forms interchain disulfide bonds (i.e. e. part containing the sequence CPPCPA (SEQ ID NO: 219)). Heavy chain variable domain sequences that do not contain the CPPCP sequence (SEQ ID NO: 220) of the hinge region at the C-terminus will not form intrachain disulfide bonds and thus will form Fab fragments with the corresponding sequential

- 34 047128 light chain variable domain sequences, whereas these heavy chain variable domain sequences with part of the hinge region at the C-terminus containing the sequence CPPCP (SEQ ID NO: 220) will form intrachain disulfide bonds and thus will form fragments Fab2. For example, in some embodiments, the transgene may encode a scFv comprising a light chain variable domain and a heavy chain variable domain connected by a flexible linker therebetween (where the heavy chain variable domain may be at either the N-terminal end or the C-terminal end of the scFv), e.g. as shown for brolucizumab in FIG. 8D and E06 in FIG. 5D. Alternatively, in other embodiments, the transgene may encode F(ab') ₂ fragments comprising a nucleotide sequence that encodes a light chain and a heavy chain sequence that includes at least a CPPCA (SEQ ID NO: 221) hinge region sequence, as shown in Fig. 2A-2F, 3A-3E, 4A-4B, 5A-5C, 6, 7A, 7B, 8A-8C, 8E-8H, and 9A-9B, which depict various regions of the hinge region that may be included in the C-terminal sequence heavy chain. Pre-existing anti-hinge antibodies (AHA) may cause immunogenicity and reduce efficacy. Thus, in certain embodiments of the IgG1 isotype, the C terminus ends with D221 or ends with a T225L or L242 mutation, which may reduce binding to AHA. (See, for example, Brezski, 2008, J Immunol 181: 3183-92 and Kim, 2016, 8: 1536-1547). For IgG2, the risk of AHA is lower because the hinge region of IgG2 is not as susceptible to the enzymatic degradation required to produce endogenous AHA. (See, for example, Brezski, 2011, MAbs 3: 558-567).

In certain embodiments, the viral vectors provided herein contain the following elements in the following order: a) a constitutive or inducible (eg, hypoxia-inducible or rifamycin-inducible) promoter sequence and b) a transgene-encoding sequence (eg, HuGlyFab). In certain embodiments, the transgene coding sequence comprises multiple ORFs separated by IRES elements. In certain embodiments, the ORFs encode the heavy and light chain domains of HuGlyFab. In certain embodiments, the transgene coding sequence comprises multiple subunits in a single ORF separated by F/F2A sequences. In certain embodiments, the transgene-containing sequence encodes the HuGlyFab heavy and light chain domains separated by the F/F2A sequence. In certain embodiments, the transgene-containing sequence encodes the HuGlyFab heavy and light chain variable domains separated by a flexible peptide linker. In certain embodiments, the viral vectors provided herein comprise the following elements in the following order: a) a constitutive or inducible promoter sequence and b) a transgene encoding sequence (e.g., HuGlyFab), wherein the transgene comprises a nucleotide sequence encoding a signal peptide, a light chain, and Fab portion of the heavy chain separated by an IRES element. In some embodiments, the viral vectors provided herein comprise the following elements in the following order: a) a constitutive or hypoxia-inducible promoter sequence and b) a sequence encoding a transgene containing a signal peptide, a light chain and a heavy chain sequence separated by an F/F2A cleavable sequence or a flexible peptide linker.

In certain embodiments, the viral vectors provided herein contain the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a constitutive or inducible promoter sequence, d) a second linker sequence, e) an intronic sequence, f) third linker sequence, g) first UTR sequence, h) transgene coding sequence (eg HuGlyFab), i) second UTR sequence, j) fourth linker sequence, k) poly A sequence, l) fifth linker sequence and m) second sequence ITR.

In certain embodiments, the viral vectors provided herein contain the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a constitutive or inducible promoter sequence, d) a second linker sequence, e) an intronic sequence, f) third linker sequence, g) first UTR sequence, h) transgene coding sequence (eg HuGlyFab), i) second UTR sequence, j) fourth linker sequence, k) poly A sequence, l) fifth linker sequence and m) second sequence ITR, wherein the transgene contains a signal and wherein the transgene encodes a light chain and a heavy chain sequence separated by an F/F2A cleavable sequence.

5.1.9. Making and testing vectors

The viral vectors provided herein can be produced using host cells. The viral vectors provided herein can be produced using mammalian host cells, for example, A549, WEHI, 10T1/2, BHK, MDCK, C0S1, C0S7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, 293, Saos, C2C12, L, HT1080, HepG2, primary 35 047128 fibroblasts, hepatocytes and myoblasts. The viral vectors provided herein can be made using host cells from human, monkey, mouse, rat, rabbit or hamster.

Host cells are stably transformed by sequences encoding the transgene and associated elements (eg, the vector genome), and means of virus production in the host cells, such as replication and capsid genes (eg, AAV rep and cap genes). For a method of producing recombinant AAV vectors with AAV8 capsids, see section IV of the detailed specification of US Pat. No. 7,282,992 B2, which is incorporated herein by reference in its entirety. The genome copy titers of these vectors can be determined, for example, by TAQMAN® analysis. Virions can be isolated, for example, by CsCl ₂ precipitation.

Alternatively, baculovirus expression systems in insect cells can be used to produce AAV vectors. For a review, see Aponte-Ubillus et al., 2018, Appl. Microbiol. Biotechnol. 102:1045-1054, which is incorporated herein by reference in its entirety for manufacturing techniques.

In vitro assays, such as cell culture assays, can be used to measure transgene expression from a vector described herein, indicating, for example, the potency of the vector. For example, the PER.C6® cell line (Lonza), a cell line derived from human embryonic retinal cells or retinal pigment epithelial cells, such as the hTERT RPE-1 retinal pigment epithelial cell line (available from ATCC®), can be used to assess expression transgene Following expression, the characteristics of the expressed product can be determined, including determination of the glycosylation and tyrosine sulfation patterns associated with HuGlyFab. Patterns of glycosylation and methods for its determination are discussed in section 5.2.1, and patterns of tyrosine sulfation and methods for its determination are discussed in section 5.2.2. In addition, benefits resulting from glycosylation/sulfation of cell-expressed HuGlyFab can be determined using assays known in the art, for example, the methods described in sections 5.2.1 and 5.2.2.

5.1.10. Compositions

Pharmaceutical compositions suitable for administration to human subjects contain a suspension of the recombinant vector in a formulation buffer containing a physiologically compatible aqueous buffer, a surfactant and optional excipients. Such a formulation buffer may contain one or more of polysaccharides, surfactants, polymer or oil.

5.2. N-glycosylation, tyrosine sulfation and O-glycosylation

The amino acid sequence (primary sequence) of the HuGlyFab and HuPTM scFv disclosed herein contains at least one site at which Nglycosylation or tyrosine sulfation occurs (see Figs. 2A-2F, 3A-3E, 4A-4B, 5A-5D, 6 , 7A-7B, 8A8H and 9A-9B for glycosylation and/or sulfation positions in the amino acid sequences of Fab fragments of therapeutic antibodies).

5.2.1. N-glycosylation

Reverse glycosylation sites.

The canonical N-glycosylation sequence is known in the art as Asn-X-Ser (or Thr), where X can be any amino acid other than Pro. However, it has recently been demonstrated that asparagine (Asn) residues of human antibodies can be glycosylated in the context of the reverse consensus motif Ser (or Thr)-X-Asn, where X can be any amino acid except Pro. See Valliere-Douglass et al., 2009, J. Biol. Chem. 284:32493-32506; and Valliere-Douglass et al., 2010, J. Biol. Chem. 285:16012–16022. As disclosed herein, certain HuGlyFab and HuPTM scFv disclosed herein contain such reverse consensus sequences.

Non-consensus glycosylation sites.

In addition to reverse N-glycosylation sites, it has recently been demonstrated that glutamine (Gln) residues of human antibodies can be glycosylated in the context of a non-consensus Gln-Gly-Thr motif. See Valliere-Douglass et al., 2010, J. Biol. Chem. 285:16012–16022. It is surprising that some of the HuGlyFab fragments disclosed herein contain such non-consensus sequences. Additionally, O-glycosylation involves the addition of an N-acetylgalactose amine to serine or threonine residues via an enzyme. It has been demonstrated that amino acid residues present in the hinge region of antibodies can be O-glycosylated. The ability to O-glycosylate provides another advantage to the therapeutic antibodies presented herein compared to, for example, antigen binding moieties produced in E. coli, again since E. coli does not naturally contain a mechanism equivalent to that used in O-glycosylation person. (Instead, O-glycosylation in E. coli has only been demonstrated when the bacteria are modified to contain specific

- 36 047128 mechanisms of O-glycosylation. See, for example, Farid-Moayer et al., 2007, J. Bacteriol. 189:8088-8098).

Designed N-glycosylation sites.

In some embodiments, the nucleic acid encoding HuGlyFab or HuTPM scFv is modified to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Nglycosylation sites (including the canonical consensus Nglycosylation sequence, reverse N-glycosylation site, and non-consensus N-glycosylation sites) that are typically associated with HuGlyFab or HuPTM scFv (e.g., relative to the number of N-glycosylation sites associated with HuGlyFab or HuPTM scFv in the unmodified state). In certain embodiments, the introduction of glycosylation sites is accomplished by inserting N-glycosylation sites (including the canonical consensus N-glycosylation sequence, the reverse N-glycosylation site, and non-consensus N-glycosylation sites) into any part of the primary structure of the antigen binding fragment, provided that the introduction is not influences the binding of the antigen-binding fragment to its antigen. The introduction of glycosylation sites can be accomplished, for example, by adding new amino acids to the primary structure of the antigen binding fragment or antibody from which the antigen binding fragment is derived (i.e., glycosylation sites are added in whole or in part) or by mutating existing amino acids in the antigen binding fragment or antibody from from which the antigen-binding fragment is obtained to create N-glycosylation sites (ie, no amino acids are added to the antigen-binding fragment/antibody, but selected amino acids of the antigen-binding fragment/antibody are mutated to form N-glycosylation sites). Those skilled in the art will appreciate that the amino acid sequence of a protein can be easily modified using approaches known in the art, for example, recombinant approaches that involve modification of the nucleic acid sequence encoding the protein.

In a specific embodiment, the HuGlyMab or antigen binding fragment is modified such that when expressed in mammalian cells, such as retina, CNS, liver, or muscle cells, it can be hyperglycosylated. See Courtois et al., 2016, mAbs 8:99-112, which is incorporated herein by reference in its entirety.

N-glycosylation of HuPTM antigen-binding fragments.

Unlike small molecule drugs, biologics typically contain a mixture of many variants with different modifications or forms that may have different potencies, pharmacokinetics, and/or safety profiles. It is not necessary that every molecule produced in a gene or protein therapy be fully glycosylated and sulfated. Rather, the population of glycoproteins produced must have sufficient glycosylation (including 2,6-sialylation) and sulfation to demonstrate efficacy. The goal of a gene therapy treatment provided herein may be, for example, to slow or stop the progression of a disease or abnormal condition or to reduce the severity of one or more symptoms associated with a disease or abnormal condition.

When HuGlyFab or HuPTM scGv is expressed in a human cell, the N-glycosylation sites of the antigen binding moiety can be glycosylated by various glycans. The N-glycans of the antigen binding fragments have been characterized in the art. For example, in the publication Bondt et al., 2014, Mol. & Cell. Proteomics 13.11:3029-3039 (incorporated herein by reference in its entirety for disclosure of Fab-associated N-glycans; see also FIG. 10) characterized Fab-associated glycans and demonstrated that the Fab and Fc portions of antibodies contain distinct glycosylation patterns , with Fab glycans exhibiting high galactosylation, sialylation, and bisection (eg, GlcNAc bisection) but low fucosylation relative to Fc glycans. Like Bondt, the authors of Huang et al., 2006, Anal. Biochem. 349:197-207 (incorporated herein by reference in its entirety to disclose Fab-associated N-glycans) found that most Fab glycans are sialylated. However, in the Fab antibody studied by Huang (which was generated in a murine cell background), the sialic residues identified were N-glycolylneuraminic acid (Neu5Gc or NeuGc) (which is not naturally occurring in humans) instead of N-acetylneuraminic acid (Neu5Ac, the predominant human sialic acid ). In addition, Song et al., 2014, Anal. Chem. 86:56615666 (incorporated herein by reference in its entirety for the disclosure of Fab-associated N-glycans) describes a library of N-glycans coupled to commercially available antibodies.

Importantly, when HuGlyFab or HuPTM scFv is expressed in human cells, the need for in vitro production in prokaryotic host cells (eg, H. coli) or eukaryotic host cells (eg, CHO cells or NS0 cells) is eliminated. Instead, the methods described herein result in the N-glycosylation sites of HuGlyFab or HuPTM scFv being advantageously decorated with glycans suitable and useful for human therapy. Such a pre

- 37 047128 property is not available when CHO cells, NS0 cells or E. coli are used in the production of antibodies/antigen binding fragments because, for example, CHO cells (1) do not express 2,6 sialyltransferase and therefore cannot add 2,6- sialic acid during Nglycosylation; (2) can add Neu5Gc as sialic acid instead of Neu5Ac and (3) can also produce an immunogenic glycan, α-Gal antigen, which reacts with the anti-α-Gal antibodies present in most individuals, which at high concentrations can cause anaphylaxis; and because (4) E. coli does not naturally contain the components required for Nglycosylation.

Assays for determining the glycosylation pattern of antibodies, including antigen binding fragments, are known in the art. For example, hydrazinolysis can be used to analyze glycans. First, the polysaccharides are released from their associated protein by incubation with hydrazine (the Ludger Liberate Hydrazinolysis Glycan Release Kit, Oxfordshire, UK can be used). Nucleophilic hydrazine attacks the glycosidic bond between the polysaccharide and the carrier protein and allows the release of bound glycans. N-acetyl groups are lost during this treatment and must be restored by repeated N-acetylation. Glycans can also be released using enzymes such as glycosidases or endoglycosidases such as PNGase F and Endo H, which are broken down cleanly and with fewer side reactions than hydrazines. Free glycans can be purified on carbon columns and then labeled at the reducing end with fluorophore-2-aminobenzamide. Labeled polysaccharides can be separated on a GlycoSep-N column (GL Sciences) according to the HPLC protocol of Royle et al., Anal Biochem 2002, 304(1):70-90. The resulting fluorescence chromatogram shows the length of the polysaccharide and the number of repeating units. Structural information can be collected by collecting individual peaks and then performing MS/MS analysis. In this way, the monosaccharide composition and the repeat unit sequence can be confirmed, and further, the homogeneity of the polysaccharide composition can be identified. Specific low or high molecular weight peaks can be analyzed by MALDI-MS/MS and the result used to confirm the glycan sequence. Each peak in the chromatogram corresponds to a polymer, such as a glycan, consisting of a certain number of repeating units and fragments, such as their sugar residues. Thus, the chromatogram allows the measurement of the length distribution of a polymer, such as a glycan. Elution time is an indicator of polymer length, while fluorescence intensity correlates with the molar content of the corresponding polymer, such as glycan. Other methods for assessing glycans associated with antigen-binding moieties include those described by Bondt et al., 2014, Mol. & Cell. Proteomics 13.11:3029-3039, Huang et al., 2006, Anal. Biochem. 349:197-207 and/or Song et al., 2014, Anal. Chem. 86:5661–5666.

The homogeneity or heterogeneity of glycan patterns associated with antibodies (including antigen binding moieties), as it relates to both the length or size of the glycan and the number of glycans present at glycosylation sites, can be assessed using methods known in the art. for example, methods for measuring glycan length or size and hydrodynamic radius. HPLC, such as size exclusion, normal phase, reverse phase, and anion exchange HPLC, as well as capillary electrophoresis, can measure the hydrodynamic radius. A higher number of glycosylation sites in a protein results in a larger change in hydrodynamic radius compared to a carrier with fewer glycosylation sites. However, when individual glycan chains are analyzed, they can be more uniform due to their more controlled length. Glycan length can be measured using hydrazinolysis, SDS-PAGE and capillary gel electrophoresis. Additionally, homogeneity may also mean that certain glycosylation site usage patterns vary over a wider/narrower range. These factors can be measured using glycopeptide LC-MS/MS.

In certain embodiments, the HuPTM mAbs or antigen binding fragments thereof also do not contain detectable NeuGc and/or α-Gal. The term NeuGc detectable or α-Gal detectable or does not contain or lack NeuGc or α-Gal means as used herein that the HuPTM mAb or antigen binding fragment does not contain NeuGc or αGal fragments detectable using standard assay methods known in the art. For example, NeuGc can be detected by HPLC according to Hara et al., 1989, Highly Sensitive Determination of N-Acetyl-and N-Glycolylneuraminic Acids in Human Serum and Urine and Rat Serum by Reversed-Phase Liquid Chromatography with Fluorescence Detection . J. Chromatogr., B: Biomed. 377, 111-119, which is hereby incorporated by reference for a method for detecting NeuGc. Alternatively, NeuGc can be detected using mass spectrometry. A-Gal can be detected using ELISA, see, for example, Galili et al., 1998, A sensitive assay for measuring α-Gal epitope expression on cells by a monoclonal anti-Gal antibody. Transplantation. 65(8): 1129-32, or using mass spectrometry, see, for example, Ayoub et al., 2013, Correct primary structure assessment

- 38 047128 and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques. Landes Bioscience. 5(5):699-710. See also references cited in Platts-Mills et al., 2015, Anaphylaxis to the Carbohydrate Side-Chain Alpha-gal Immunol Allergy Clin North Am. 35(2): 247-260.

Advantages of N-glycosylation.

N-glycosylation provides numerous advantages to the HuGlyFab or HuPTM scFv described herein. Such advantages are not achievable by producing antigen-binding fragments in E. coli because E. coli does not naturally possess the components required for N-glycosylation. Additionally, some benefits are not achievable due to antibody production, such as in CHO cells (or murine cells such as NS0 cells), since CHO cells lack the components needed to add certain glycans (e.g., 2,6 sialic acid and dissection GlcNAc), and because either CHO or mouse cell lines add NNglycolylneuraminic acid (Neu5Gc or NeuGc), which is not naturally occurring in humans (and potentially immunogenic), instead of N-acetylneuraminic acid (Neu5Ac), the predominant human sialic acid. See, for example, Dumont et al., 2015, Crit. Rev. Biotechnol. 36(6): 1110-1122; Huang et al., 2006, Anal. Biochem. 349:197-207 (NeuGc is the predominant sialic acid in murine cell lines such as SP2/0 and NS0) and Song et al., 2014, Anal. Chem. 86:56615666, each of which is incorporated herein by reference in its entirety. In addition, CHO cells can also produce an immunogenic glycan, α-Gal antigen, which reacts with α-Gal antibodies present in most people, which at high concentrations can cause anaphylaxis. See, for example, Bosques, 2010, Nat. Biotech. 28:1153–1156. The glycosylation pattern of the human HuGlyFab HuPTM scFv described herein should reduce the immunogenicity of the transgene product and increase potency.

Although non-canonical glycosylation sites typically result in low levels of glycosylation (e.g., 1-5%) of the antibody population, the functional benefits can be significant (see, e.g., van de Bovenkamp et al., 2016, J. Immunol. 196: 1435-1441). For example, Fab glycosylation can affect the stability, half-life, and binding characteristics of an antibody. To determine the effect of Fab glycosylation on the affinity of an antibody for its target, any method known to one skilled in the art, such as enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR), can be used. To determine the effect of Fab glycosylation on the half-life of an antibody, any method known to one skilled in the art may be used, for example, by measuring levels of radioactivity in the blood or organs of a subject administered a radiolabeled antibody. To determine the effect of Fab glycosylation on the stability, e.g., levels of aggregation or unfolding of the antibody protein, any method known to one of ordinary skill in the art may be used, e.g., differential scanning calorimetry (DSC), high performance liquid chromatography (HPLC), e.g. liquid chromatography (SEC-HPLC), capillary electrophoresis, mass spectrometry or turbidity measurement.

The presence of sialic acid on HuGlyFab or HuPTM scFv used in the methods described herein may affect the rate of clearance of HuGlyFab or HuPTM scFv. Accordingly, HuGlyFab or HuPTM scFv sialic acid samples can be used to create a therapeutic agent having an optimized clearance rate. Methods for assessing the rate of clearance of antigen binding fragments are known in the art. See, for example, Huang et al., 2006, Anal. Biochem. 349:197-207.

In another specific embodiment, the benefit provided by Nglycosylation is reduced aggregation. Occupied N-glycosylation sites may mask aggregation-prone amino acid residues, resulting in decreased aggregation. Such N-glycosylation sites may be native to the antigen binding fragment used herein, or may be incorporated into the antigen binding fragment used herein, causing the HuFlyFab or HuPTM scFv to be less prone to aggregation when expressed, e.g. human cells. Methods for assessing antibody aggregation are known in the art. See, for example, Courtois et al., 2016, mAbs 8:99-112, which is incorporated herein by reference in its entirety.

In another specific embodiment, the benefit provided by Nglycosylation is reduced immunogenicity. Such N-glycosylation sites may be native to the antigen binding fragment used herein or may be incorporated into the antigen binding fragment used herein, resulting in a HuGlyFab or HuPTM scFv that is less likely to be immunogenic when expressed, e.g., expressed in human retinal cells, human CNS cells, human liver cells or human muscle cells.

In another specific embodiment, the advantage of N-glycosylation is protein stability. It is well known that N-glycosylation of proteins imparts stability to them, and

- 39 047128 The present art knows methods for assessing the stability of a protein resulting from Nglycosylation. See, for example, Sola and Griebenow, 2009, J Pharm Sci., 98(4): 1223-1245.

In another specific embodiment, the benefit provided by Nglycosylation is altered binding affinity. It is known in the art that the presence of N-glycosylation sites in the variable domains of an antibody can increase the affinity of the antibody for its antigen. See, for example, Bovenkamp et al., 2016, J. Immunol. 196:1435–1441. Assays for measuring the binding affinity of antibodies are known in the art. See, for example, Wright et al., 1991, EMBO J. 10:2717-2723 and Leibiger et al., 1999, Biochem. J. 338:529–538.

5.2.2. Tyrosine sulfation

Tyrosine sulfation occurs at tyrosine (Y) residues with glutamate (E) or aspartate (D) within the +5 to -5 position of Y, and where the -1 Y position is a neutral or acidic charged amino acid, but not a basic amino acid, for example, arginine (R), lysine (K) or histidine (H), which eliminates sulfation. Surprisingly, the HuGlyFab and HuPTM scFv described herein contain tyrosine sulfation sites (see Figures 2A-2F, 3A-3E, 4A-4B, 5A5D, 6, 7A-7B, 8A-8H and 9A-9B).

It is important to note that antigen-binding moieties with tyrosine sulfation cannot be produced in E. coli, which does not naturally possess the enzymes required for tyrosine sulfation. In addition, CHO cells are deficient in tyrosine sulfation - they are not secretory cells and have a limited ability to post-translationally sulfate tyrosine. See, for example, Mikkelsen & Ezban, 1991, Biochemistry 30: 1533-1537. Advantageously, the methods presented herein require the expression of Fab HuPTM in human cells that are secretory and have the ability to sulfate tyrosine.

Sulfation of tyrosine is beneficial for several reasons. For example, tyrosine sulfation of the antigen-binding moiety of therapeutic antibodies against targets has been shown to dramatically increase antigen avidity and activity. See, for example, Loos et al., 2015, PNAS 112: 12675-12680 and Choe et al., 2003, Cell 114: 161-170. Assays for detecting tyrosine sulfation are known in the art. See, for example, Yang et al., 2015, Molecules 20:2138–2164.

5.2.3. O-glycosylation

O-glycosylation involves the addition of N-acetylgalactosamine to serine or threonine residues by an enzyme. It has been demonstrated that amino acid residues present in the hinge region of antibodies can be O-glycosylated. In certain embodiments, HuGlyFabs contain all or part of their hinge region and are thus capable of glycosylation when expressed in human cells. The possibility of O-glycosylation provides another advantage to HuGlyFab provided herein compared to, for example, antigen-binding fragments produced in E. coli, again because E. coli in nature does not contain a mechanism equivalent to that used in human O-glycosylation. (Instead, O-glycosylation in E. coli has only been demonstrated when the bacteria are modified to contain specific O-glycosylation machinery. See, e.g., Farid Moayer et al., 2007, J. Bacteriol. 189:8088-8098. O-glycosylated HuGlyFab, due to the presence of glycans, shares characteristics with N-glycosylated HuGlyFab (as discussed above).

5.3. Vector-based therapeutic antibodies

5.3.1. Anti-ABeta HuPTM designs and formulations for the treatment of Alzheimer's disease

Described are compositions and methods for delivering HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fab, that bind to amyloid beta (Ab or Abeta) peptides derived from amyloid precursor protein, which may be beneficial in the treatment of Alzheimer's disease (AD), etc. .P. In specific embodiments, the HuPTM mAb is aducanumab, crenezumab, gantenerumab, or BAN2401 or an antigen binding fragment of one of the above. The amino acid sequences of the Fab fragments of these antibodies are presented in Fig. 2A2C and 2F. Delivery may be accomplished by gene therapy—for example, by administering a viral vector or other DNA expression construct encoding an Ab-binding HuPTM mAb (or an antigen-binding fragment thereof and/or a hyperglycosylated derivative or other derivative thereof) to human patients diagnosed with or the presence of one or more symptoms of AD, to create a permanent depot that continuously supplies human PTM, for example, glycosylated human, transgenic product.

Transgenes.

Provided are recombinant vectors containing a transgene encoding a HuPTM mAb or a HuPTM Fab (or other antigen binding fragment of a HuPTM mAb) that binds to Ab, which can be administered to deliver the HuPTM mAb or antigen binding fragment to a patient. A transgene is a nucleic acid containing nucleotide sequences encoding an antigen-binding fragment of an antibody that binds to Av, such as aducanumab, crenezumab,

- 40 047128 gantenerumab or BAN2401 or variants thereof, as described in detail herein. The transgene may also encode an anti-Ab antigen-binding fragment that contains additional glycosylation sites (for example, see Courtois et al., 2016, mAbs 8: 99-112, which is incorporated herein by reference in its entirety).

In certain embodiments, the anti-Ab antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of aducanumab (having amino acid sequences SEQ ID NO: 1 and 2, respectively, see Table 4 and FIG. 2A). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 101 (encoding the Fab portion of the aducanumab heavy chain) and SEQ ID NO: 102 (encoding the Fab portion of the aducanumab light chain ), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular human CNS cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or one of the sequences presented in table. 1 above.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-Ab antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 1 with an additional hinge region sequence beginning after the C-terminal aspartate (D) comprising all or part of the amino acid sequence KTHTCPPCPAPELLGG (SEQ ID NO: 222) and specifically, KTHL (SEQ ID NO: 223), KTHT (SEQ ID NO: 224), KTHTCPHCHA (SEQ ID NO: 225), KTHLCPPCPA (SEQ ID NO: 226), KTHTCPPCPAPELLGGPSVFL (SEQ ID NO: 227) or KTHLCPPCPAPELLGGPSVFL (SEQ ID NO: 228) as shown in FIG. 2A. These hinge regions can be encoded by the nucleotide sequences at the 3' end of SEQ ID NO: 1 hinge region coding sequences presented in table. 4 (SEQ ID NO: 101).

In certain embodiments, an anti-Ab antigen-binding fragment transgene encodes an Ab antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 2. In certain embodiments, the anti-Ab antigen binding fragment transgene encodes an antigen binding Ab fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 1. In certain embodiments, the anti-Ab antigen-binding fragment transgene encodes an antigen-binding Ab fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 2, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 1. In certain embodiments, the antigen binding Ab fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 with 1, 2, 3, 4, 5 , 6, 7 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. in those areas outside the CDR, which are highlighted in FIG. 2A) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In certain embodiments, the antigen binding Ab fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 2 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (ie, those regions outside the CDRs that are underlined in Fig. 2A) or are substitutions to an amino acid present at that position in light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In some embodiments, the anti-Ab antigen-binding fragment transgene encodes a hyperglycosylated aducanumab Fab comprising a heavy chain and a light chain of SEQ ID NO: 1 and 2, respectively, with one or more of the following mutations: T119N (heavy chain), Q160N, or Q160S (light chain) and/or E195N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-Ab antigen binding fragment transgene encodes an antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of aducanumab, which are highlighted in the variable domain sequences

- 41 047128 heavy and light chain in FIG. 2A, which are spaced between framework regions, typically human framework regions, and linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-Ab antibody or antigen binding agent thereof fragment.

In some embodiments, the anti-Ab antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of crenezumab (having amino acid sequences SEQ ID NO: 3 and 4, respectively, see Table 4 and FIG. 2B). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 103 (encoding the Fab portion of the crenezumab heavy chain) and SEQ ID NO: 104 (encoding the Fab portion of the crenezumab light chain ), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular human CNS cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or the signal sequence shown in table. 1.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequences. In specific embodiments, the anti-Ab antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 3 with an additional hinge region sequence beginning after the C-terminal tyrosine (Y) containing all or part of the amino acid sequence GPPCPPCPA (SEQ ID NO: 229) or GPPCPPCPAPEFLGGPSVFL (SEQ ID NO: 230) as shown in FIG. 2B. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 3 hinge region encoding sequences presented in table. 5 (SEQ ID NO: 103).

In certain embodiments, an anti-Ab antigen-binding fragment transgene encodes an Ab antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-Ab antigen binding fragment transgene encodes an antigen binding Ab fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-Ab antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86% identical to 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 4, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 3. In certain embodiments, the antigen binding Ab fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 with 1, 2, 3, 4, 5, 6 , 7 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. in those areas outside the CDR, which are highlighted in FIG. 2B) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In certain embodiments, the antigen binding Ab fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 4 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e., those regions outside the CDRs that are underlined in Fig. 2B) or are substitutions to an amino acid present at that position in light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In some embodiments, the anti-Ab antigen-binding fragment transgene encodes a hyperglycosylated crenezumab Fab containing a heavy chain and a light chain of SEQ ID NO: 3 and 4, respectively, with one or more of the following mutations: T107N (heavy chain), Q165N, or Q165S (light chain) and/or E200N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-Ab antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of crenezumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 2B, which are located between framework regions, typically human framework regions, and are associated with constant domains depending on the shape of the antigen binding molecule, as is known in the art to form a variable

- 42 047128 heavy and/or light chain domain of an anti-Αβ antibody or an antigen-binding fragment thereof.

In some embodiments, the anti-Ab antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of gantenerumab (having amino acid sequences SEQ ID NOs: 5 and 6, respectively, see Table 4 and FIG. 2C). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 105 (encoding the Fab portion of the gantenerumab heavy chain) and SEQ ID NO: 106 (encoding the Fab portion of the gantenerumab light chain), as presented in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular human CNS cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or the signal sequence presented in table. 1.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-Ab antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 5 with an additional hinge region sequence beginning at the C-terminal aspartate (D) containing all or part of the amino acid sequence KTHTCPPCPAPELLGG (SEQ ID NO: 222) and specifically, KTHL (SEQ ID NO: 223), KTHT (SEQ ID NO: 224), KTHTCPPCPA (SEQ ID NO: 225), KTHLCPPCPA (SEQ ID NO: 226), KTHTCPPCPAPELLGGPSVFL (SEQ ID NO: 227) or KTHLCPPCPAPELLGGPSVFL (SEQ ID NO: 228) as shown in FIG. 2C. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 5 hinge region encoding sequences presented in table. 5 (SEQ ID NO: 105).

In certain embodiments, an anti-Ab antigen-binding fragment transgene encodes an Ab antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 6. In certain embodiments, the anti-Ab antigen binding fragment transgene encodes an antigen binding Ab fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 5. In certain embodiments, the anti-Ab antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86% identical to 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 6, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 5. In certain embodiments, the antigen binding Ab fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 5 with 1, 2, 3, 4, 5, 6 , 7 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. in those areas outside the CDR, which are highlighted in FIG. 2C) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the antigen binding Ab fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 6 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e., those regions outside the CDRs that are underlined in Fig. 2C) or are substitutions to an amino acid present at that position in light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In some embodiments, the anti-Ab antigen binding fragment transgene encodes a hyperglycosylated Fab of gantenerumab comprising a heavy chain and a light chain of SEQ ID NO: 5 and 6, respectively, with one or more of the following mutations: L121N (heavy chain), Q161N, or Q161S (light chain) and/or E196N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In some embodiments, the anti-Ab antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of gantenerumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 2C, which are located between framework regions, generally human framework regions, and are associated with constant domains depending on the shape of the antigen binding molecule, as known in the art, to form a heavy and/or light chain variable domain of an anti-Ab antibody or an antigen binding fragment thereof .

- 43 047128

In certain embodiments, the anti-Aβ antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the BAN2401 Fab portion (having amino acid sequences SEQ ID NOs: 57 and 58, respectively, see Table 4 and FIG. 2F). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 157 (encoding the Fab portion of the heavy chain of BAN2401) and SEQ ID NO: 158 (encoding the Fab portion of the light chain of BAN2401 ), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular human CNS cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or one of the sequences presented in table. 1 above.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-Ab antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 57 with an additional hinge region sequence beginning after the C-terminal aspartate (D) containing all or part of the amino acid sequence KTHTCPPCPAPELLGG (SEQ ID NO: 222) or KTHLCPPCPAPELLGG (SEQ ID NO: 239) and in particular KTHL (SEQ ID NO: 223), KTHT (SEQ ID NO: 224), KTHTCPPCPA (SEQ ID NO: 225) or KTHLCPPCPA (SEQ ID NO: 226), as shown in Fig. 2F. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 57 hinge region encoding sequences presented in table. 4 (SEQ ID NO: 157).

In certain embodiments, an anti-Ab antigen-binding fragment transgene encodes an Ab antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 58. In certain embodiments, the anti-Ab antigen binding fragment transgene encodes an antigen binding Ab fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 57. In certain embodiments, the anti-Ab antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86% identical to 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 58, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 57. In certain embodiments, the antigen binding fragment Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 57 with 1, 2, 3, 4, 5, 6 , 7 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. in those areas outside the CDR, which are highlighted in FIG. 2F) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In certain embodiments, the antigen binding Ab fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 58 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e., those non-CDR regions that are underlined in Fig. 2F) or are substitutions to an amino acid present at that position in the lung chains of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-Ab antigen binding fragment transgene encodes a hyperglycosylated BAN2401Fab containing a heavy chain and a light chain of SEQ ID NOs: 57 and 58, respectively, with one or more of the following mutations: T119N (heavy chain), Q165N, or Q165S (light chain ) and/or E200N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-Ab antigen binding fragment transgene encodes an antigen binding fragment and comprises nucleotide sequences encoding the six BAN2401 CDRs that are highlighted in the heavy and light chain variable domain sequences in FIG. 2F, which are spaced between framework regions, typically human framework regions, and linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art, to form the heavy and/or light chain variable domain of an anti-Ab antibody or antigen binding agent thereof fragment.

- 44 047128

Methods of gene therapy.

Methods for treating people with AD by introducing a viral vector containing a transgene encoding an antibody to Av or an antigen-binding fragment thereof are presented. The antibody may be aducanumab, crenezumab, gantenerumab or BAN2401 and is preferably a Fab fragment thereof or another antigen binding fragment thereof. In certain embodiments, the patient has been diagnosed and/or has symptoms associated with prodromal AD, i.e. with mild cognitive impairment associated with early AD or even pre-AD. Recombinant vectors used for transgene delivery are described in section 5.4.1 and shown in FIG. 2A-C. Such vectors should have tropism for human CNS cells and may include non-replicating rAAVs, especially those carrying the AAV9, AAVrh10, AAVrh20, AAVrh39 or AAVcy5 capsid. Recombinant vectors can be administered by any method such that the recombinant vector enters the CNS, preferably by introducing the recombinant vector into the cerebrospinal fluid (CSF). See section 5.5.1 for details of treatment options.

Subjects to receive such gene therapy may be those who respond to anti-Av therapy. In specific embodiments, the methods include treating patients who have been diagnosed with AD or have one or more symptoms associated therewith and who are determined to respond to anti-Av antibody treatment or are considered good candidates for anti-Av antibody therapy. In specific embodiments, patients have previously been treated with aducanumab, crenezumab, gantenerumab, or BAN2401 and have been found to be sensitive to one or more of aducanumab, crenezumab, gantenerumab, or BAN2401. To determine sensitivity, a transgene product of an anti-Ab antibody or antigen-binding fragment (eg, produced in human cell culture, bioreactors, etc.) can be administered directly to the subject.

Post-translationally modified human antibodies

Production of a HuPTM anti-Ab mAb or a HuPTM Fab should result in a biologically improved molecule for the treatment of AD via gene therapy—for example, by administering a viral vector or other DNA expression construct encoding the anti-Ab HuPTM Fab intrathecally, especially via intracisternal or lumbar administration, or intravenous administration to human subjects (patients) who have been diagnosed with AD or have one or more symptoms of AD, to establish a permanent depot in the CNS that continuously supplies the fully human post-translationally modified, e.g., human glycosylated, sulfated transgene product produced transduced CNS cells.

The cDNA design for HuPTMmAb anti-Ab or anti-Ab HuPTM Fab must include a signal peptide that ensures proper co- and post-translational processing (protein glycosylation and sulfation) by transduced CNS cells. For example, the signal sequence may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161).

As an alternative or complementary to gene therapy treatment, HuPTM anti-Ab mAb or HuPTM Fab can be produced in human cell lines using recombinant DNA technology and administered to patients diagnosed with AD or those for whom AD therapy is considered appropriate.

In specific embodiments, the anti-Ab HuPTM mAb OR its antigen binding fragment comprises heavy and light chains with the amino acid sequences of the Fab portions of the aducanumab heavy and light chains, as shown in FIG. 2A (with non-consensus asparagine (N) glycosylation sites in green, glutamine (Q) glycosylation sites in blue, and Y sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions N166 of the heavy chain (SEQ ID NO: 1) or N158 and/or N210 of the light chain (SEQ ID NO: 2). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the aducanumab heavy and light chain variable domain sequences contains a sulfation group at Y94 and/or Y95 of the heavy chain (SEQ ID NO: 1) and/or Y86 and/or Y87 of the light chain (SEQ ID NO: 2). In other embodiments, the HuPTM anti-Ab mAb or antigen-binding fragment thereof does not contain any detectable (e.g., detected by assays known in the art, such as those described in section 5.2 below) NeuGc fragments and/or does not contain any detectable ( for example, detected using assays known in the art, such as those described in section 5.2 below) alpha-Gal fragments.

In specific embodiments, the HuPTM anti-Ab mAb or antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the crenezumab heavy and light chains, as shown in FIG. 2B (with non-consensus asparagine (N) glycosylation sites in green, glutamine (Q) glycosylation sites in blue, and Y sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions

- 45 047128 N52, Q104, N154 and/or N196 heavy chain (SEQ ID NO: 3) or Q105, N163 and/or N215 light chain (SEQ ID NO: 4). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the crenezumab heavy and light chain variable domain sequences has a sulfation group at Y94 and/or Y95 of the heavy chain (SEQ ID NO: 3) and/or Y91 and/or Y92 of the light chain (SEQ ID NO: 4). In other embodiments, the HuPTM anti-Ab mAb or antigen binding fragment thereof does not contain any detectable NeuGc fragments and/or does not contain any detectable alpha-Gal fragments.

In specific embodiments, the HuPTM anti-Ab mAb or antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the gantenerumab heavy and light chains, as shown in FIG. 2C (with asparagine (N) glycosylation sites in purple, non-consensus asparagine (N) glycosylation sites in green, glutamine (Q) glycosylation sites in blue, and Y-sulfation sites in yellow) , is characterized by glycosylation, in particular 2,6-sialylation at one or more amino acid positions N52, N77, Q118 and/or N168 of the heavy chain (SEQ ID NO: 5) or Q101, N159 and/or N211 of the light chain (SEQ ID NO : 6). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with gantenerumab heavy and light chain variable domain sequences has a sulfation group at Y94 and/or Y95 of the heavy chain (SEQ ID NO: 5) and/or Y87 and/or Y88 of the light chain (SEQ ID NO: 6). In other embodiments, the HuPTM anti-Ab mAb or antigen binding fragment thereof does not contain any detectable NeuGc fragments and/or does not contain any detectable alpha-Gal fragments.

In specific embodiments, the HuPTM anti-Ab mAb or antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the BAN2401 heavy and light chain, as shown in FIG. 2F (with non-consensus asparagine (N) glycosylation sites in green, glutamine (Q) glycosylation sites in blue, and Y sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions Q116 and/or N166 of the heavy chain (SEQ ID NO: 57) or N163 and/or N215 of the light chain (SEQ ID NO: 58). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with heavy and light chain variable domain sequences BAN2401 has a sulfation group at Y94 and/or Y95 of the heavy chain (SEQ ID NO: 57) and/or Y91 of the light chain (SEQ ID NO: 58) . In other embodiments, the HuPTM anti-Ab mAb or antigen binding fragment thereof does not contain any detectable NeuGc fragments and/or does not contain any detectable alpha-Gal fragments.

In certain embodiments, the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1%, or 2% 2,6-sialylated and/or sulfated and may be at least 5%, 10%, or even 50% or 100% glycosylated by 2,6-sialylation and/or sulfated. The goal of gene therapy treatment presented herein is to slow or stop the progression of AD, particularly cognitive impairment.

Efficacy may be monitored by measuring reduction in plaque formation and/or improvement in cognitive function or reduction in cognitive decline.

Combinations of delivery of HuPTM mAb to Av or an antigen-binding fragment thereof to the CNS, accompanied by delivery of other available treatments, are covered by the methods presented herein. Additional treatments may be given before, simultaneously, or after gene therapy treatment. Available treatments for AD that can be combined with the gene therapy presented herein include, but are not limited to, ARICEPT® (donepezil), RAZADYNE® (galantamine), NAMENDA® (rivastigmine) and NAMZARIC® (donepezil and memantine) to to name a few, and administration with anti-Av agents, including but not limited to aducanumab, crenezumab, gantenerumab or BAN2401, or anti-Tau agents such as aTAU.

5.3.2. Anti-tau HuPTM designs and formulations for tauopathies such as Alzheimer's disease, chronic traumatic encephalopathy, progressive supranuclear palsy or frontotemporal dementia

Compositions and methods are described for delivering HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fab, that bind to tau protein (Tau), such as monomeric tau, oligomeric tau, unphosphorylated tau, and phosphorylated tau, which may be useful in the treatment of Alzheimer's disease ( AD), chronic traumatic encephalopathy (CTE), Pick complex, primary age-related tauopathies, progressive supranuclear palsy (PSP), frontotemporal dementia (FD) and other tauopathies. In specific embodiments, the HuPTM mAb is an antibody having the Fab fragments shown in FIG. 2D (referred to herein as aTAU), or an antigen binding fragment thereof. Delivery can be carried out using gene therapy - for example, by introducing a viral vector or other DNA expression con

- 46 047128 a construct encoding a tau-binding HuPTM mAb (or an antigen-binding fragment thereof and/or a hyperglycosylated derivative or other derivative thereof), to human patients diagnosed with or having one or more symptoms of AD, CTE, PSP, FD or other tauopathies, to create a permanent depot that continuously supplies human PTM, for example, glycosylated human, transgenic product.

Transgenes.

Provided are recombinant vectors containing a transgene encoding a HuPTM mAb or a HuPTM Fab (or other antigen binding fragment of a HuPTM mAb) that binds to tau, which can be administered to deliver the HuPTM mAb or antigen binding fragment to a patient. A transgene is a nucleic acid containing nucleotide sequences encoding an antigen binding fragment of an antibody that binds to tau, such as aTAU or variants thereof, as described in detail herein. The transgene may also encode an anti-tau antigen binding fragment that contains additional glycosylation sites (for example, see Courtois et al., 2016, mAbs 8: 99-112, which is incorporated herein by reference in its entirety).

In some embodiments, the anti-tau antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of aTAU (having amino acid sequences SEQ ID NOs: 53 and 54, respectively, see Table 4 and FIG. 2D). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 153 (encoding the Fab portion of the aTAU heavy chain) and SEQ ID NO: 154 (encoding the Fab portion of the aTAU light chain ), as shown in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular human CNS cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or one of the sequences presented in table. 1 above.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-Tau antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 53 with an additional hinge region sequence beginning after the C-terminal aspartate (D) comprising all or part of the amino acid sequence GPPCPPCPAPEFLGG (SEQ ID NO: 231) and specifically, GPPCPPCPA (SEQ ID NO: 229) or GPPCPPCPAPEFLGGPSVFL (SEQ ID NO: 230), as shown in FIG. 2D. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 53 hinge region encoding sequences presented in table. 4 (SEQ ID NO: 153).

In certain embodiments, the anti-tau antigen binding fragment transgene encodes a tau antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 54. In certain embodiments, the anti-tau antigen binding fragment transgene encodes a tau antigen binding fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 53. In certain embodiments, the anti-tau antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86% identical to 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 54, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 53. In certain embodiments, the tau antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 53 with 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. those areas outside the CDR that are highlighted in FIG. 2D) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In certain embodiments, the tau antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 54 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e., those regions outside the CDRs that are underlined in Fig. 2D) or are substitutions to an amino acid present in it

- 47047128 position in the light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-tau antigen binding fragment transgene encodes a hyperglycosylated aTAU Fab containing a heavy chain and a light chain of SEQ ID NO: 53 and 54, respectively, with one or more of the following mutations: T110N (heavy chain), Q164N, or Q164S (light chain) and/or E199N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-tau antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six aTAU CDRs that are highlighted in the heavy and light chain variable domain sequences in FIG. 2D, which are located between framework regions, generally human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art to form a heavy and/or light chain variable domain of an anti-tau antibody or an antigen binding fragment thereof .

Methods of gene therapy.

Methods are provided for treating human subjects with AD, CTE, PSP, FD, or other tauopathies by administering a viral vector containing a transgene encoding an anti-tau antibody or an antigen-binding fragment thereof. The antibody may be aTAU and is preferably a Fab fragment thereof or another antigen binding fragment thereof. In certain embodiments, the patient has been diagnosed and/or has symptoms associated with prodromal AD, i.e. with mild cognitive impairment associated with early AD or even pre-AD. The recombinant vector used to deliver the transgene is described in section 5.4.1 and shown in FIG. 2D. Such vectors should have tropism for human CNS cells and may include non-replicating rAAVs, especially those carrying the AAV9, AAVrh10, AAVrh20, AAVrh39 or AAVcy5 capsid. Recombinant vectors can be administered by any method such that the recombinant vector enters the CNS, preferably by introducing the recombinant vector into the cerebrospinal fluid (CSF). See section 5.5.1 for details of treatment options.

Subjects to receive such gene therapy may be those who respond to anti-tau therapy. In specific embodiments, the methods include treating patients who have been diagnosed with AD, PSP, or FD, or who have one or more symptoms associated therewith, and who are determined to respond to anti-tau antibody treatment or are considered good candidates for anti-tau antibody therapy. In specific embodiments, the patients have previously been treated with aTAUs and have been found to be sensitive to one or more of the aTAUs. To determine sensitivity, a transgene product of an anti-tau antibody or antigen binding fragment (eg, produced in human cell culture, bioreactors, etc.) can be administered directly to the subject.

Post-translationally modified human antibodies.

Production of a HuPTM anti-tau mAb or HuPTM Fab should result in a biologically improved molecule for the treatment of AD, PSP or FD through gene therapy, e.g., by administering a viral vector or other DNA expression construct encoding the anti-tau HuPTM Fab intrathecally, esp. via intracisternal or lumbar administration, or intravenous administration to human subjects (patients) who have been diagnosed with or have one or more symptoms of AD, PSP or FD, to create a permanent depot in the CNS that continuously supplies fully human post-translationally modified, e.g., glycosylated human , a sulfated transgene product produced by transduced CNS cells.

The cDNA design for HuPTMmAb anti-tau or anti-tau HuPTM Fab must include a signal peptide that ensures proper co- and post-translational processing (protein glycosylation and sulfation) by transduced CNS cells. For example, the signal sequence may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161).

As an alternative or complementary to gene therapy treatment, HuPTM anti-tau mAb or HuPTM Fab can be produced in human cell lines using recombinant DNA technology and administered to patients diagnosed with AD PSP or FD or those for whom AD, PSP or FD therapy is considered appropriate.

In specific embodiments, the HuPTM mAb to tau or an antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the aTAU heavy and light chain, as shown in FIG. 2D (with non-consensus asparagine (N) glycosylation sites in green, glutamine (Q) glycosylation sites in blue, and Y-sulfation sites in yellow) is characterized by glycosylation, especially 2,6-sialylation, at one or more amino acid positions N57 and/or Q107 and/or N157 and/or N199 of the heavy chain (SEQ ID NO: 53), or N78 and/or Q104 and/or N162 and/or N214 of the light chain ( SEQ ID NO: 54) Alternatively or in addition to a HuPTM mAb or an antigen binding fragment thereof having heavy and light chain variable domain sequences

- 48 047128 aTAU has a sulfation group at Y96 and/or Y97 and/or Y104 of the heavy chain (SEQ ID NO: 53) and/or Y90 and/or Y91 of the light chain (SEQ ID NO: 54). In other embodiments, the HuPTM anti-tau mAb or antigen-binding fragment thereof does not contain any detectable (e.g., detected by assays known in the art, such as those described in section 5.2 below) NeuGc fragments and/or does not contain any detectable (eg, detected using assays known in the art, eg, those described in section 5.2 below) alpha-Gal fragments.

In certain embodiments, the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1%, or 2% 2,6-sialylated and/or sulfated and may be at least 5%, 10%, or even 50% or 100% glycosylated by 2,6-sialylation and/or sulfated. The goal of treatment with the gene therapy presented herein is to slow or stop the progression of AD, PSP or FD, particularly cognitive impairment, gross or fine motor skills or visual impairment. Efficacy may be monitored by measuring reduction in plaque formation and/or improvement in cognitive function, through motor skills or vision, or reduction in decline in cognitive function, motor skills or vision.

Combinations of delivering a HuPTM mAb to tau or an antigen-binding fragment thereof to the CNS, accompanied by delivery of other available treatments, are covered by the methods presented herein. Additional treatments may be given before, simultaneously, or after gene therapy treatment. Available treatments for AD, PSP or FD that can be combined with gene therapy provided herein include, but are not limited to, ARICEPT® (donepezil), RAZADYNE® (galantamine), NAMENDA® (rivastigmine) and NAMZARIC® (donepezil and memantine), to name a few, and administration with anti-Tau agents, including, but not limited to, aTAU and anti-Av agents such as, but not limited to, aducanumab, crenezumab, and gantenerumab. 5.3.3. Anti-CGRPR HuPTM designs and formulations for the treatment of migraines and cluster headaches.

Compositions and methods are described for the delivery of HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fab, that bind to the calcitonin gene-related peptide receptor (CGRPR), which may be useful in the treatment of migraines and cluster headaches (collectively referred to as headaches). ). In specific embodiments, the HuPTM mAb is erenumab, eptinezumab, fremanezumab, galcanezumab, or an antigen binding fragment of one of the above. The amino acid sequence for erenumab Fab fragments is shown in FIG. 2E. Delivery may be through gene therapy - for example, by administering a viral vector or other DNA expression construct encoding a CGRPR-binding HuPTM mAb (or an antigen-binding fragment thereof and/or a hyperglycosylated derivative or other derivative thereof) to human patients diagnosed with or the presence of one or more symptoms of migraines and cluster headaches, to create a permanent depot that continuously supplies the person with RTM, for example, a glycosylated human transgenic product.

Transgenes.

Provided are recombinant vectors containing a transgene encoding a HuPTM mAb or a HuPTM Fab (or other antigen binding fragment of a HuPTM mAb) that binds to CGRPR, which can be administered to deliver the HuPTM mAb or antigen binding fragment to a patient. A transgene is a nucleic acid containing nucleotide sequences encoding an antigen binding fragment of an antibody that binds to CGRPR, such as erenumab, eptinezumab, fremanezumab, galcanezumab, or variants thereof, as described in detail herein or in accordance with the details set forth herein. The transgene may also encode an anti-CGRPR antigen binding fragment that contains additional glycosylation sites (for example, see Courtois et al., 2016, mAbs 8: 99-112, which is incorporated herein by reference in its entirety).

In some embodiments, the anti-CGRPR antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of erenumab (having amino acid sequences SEQ ID NOs: 55 and 56, respectively, see Table 4 and FIG. 2E). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 155 (encoding the Fab portion of the erenumab heavy chain) and SEQ ID NO: 156 (encoding the Fab portion of the erenumab light chain ), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular human CNS cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or one of the sequences presented in table. 1 above.

In addition to the heavy and light chain variable domain sequences, transgenes

- 49 047128 may contain at the C-terminus of the heavy chain variable domain sequence all or part of the hinge region. In specific embodiments, the anti-CGRPR antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 55 with an additional hinge region sequence beginning after the C-terminal aspartate (D) comprising all or part of the amino acid sequence CPPCPAPPVAGG (SEQ ID NO: 232) and specifically, CPPCPA (SEQ ID NO: 219) or CPPCPAPPVAG (SEQ ID NO: 233), as shown in FIG. 2E. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 55 hinge region encoding sequences presented in table. 4 (SEQ ID NO: 155).

In certain embodiments, the anti-CGRPR antigen binding fragment transgene encodes a CGRPR antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 56. In certain embodiments, the anti-CGRPR antigen binding fragment transgene encodes a CGRPR antigen binding fragment containing severe a chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 55. In certain embodiments, the anti-CGRPR antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 56, and severe a chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 55. In certain embodiments, the CGRPR antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 55 with 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. in those areas outside the CDR, which are highlighted in FIG. 2E) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In certain embodiments, the tau antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 56 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (ie, those regions outside the CDRs that are underlined in Fig. 2E) or are substitutions to an amino acid present at that position in light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-CGRPR antigen binding fragment transgene encodes a hyperglycosylated erenumab Fab containing a heavy chain and a light chain of SEQ ID NOs: 55 and 56, respectively, with one or more of the following mutations: T125N (heavy chain) and/or Q198N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-CGRPR antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of erenumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 2E, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen-binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-tau antibody or antigen-binding antibody thereof fragment.

Methods of gene therapy.

Methods are provided for treating human subjects for migraines and cluster headaches by administering a viral vector containing a transgene encoding an anti-CGRPR antibody or an antigen-binding fragment thereof. The antibody may be erenumab, eptinezumab, fremanezumab or galcanezumab and is preferably a Fab fragment thereof or another antigen binding fragment thereof. In certain embodiments, the patient has been diagnosed with and/or has symptoms associated with episodic migraines or chronic migraines. In certain embodiments, the patient has been diagnosed and/or has symptoms associated with episodic cluster headaches or chronic cluster headaches. The recombinant vector used to deliver the transgene is described in section 5.4.1 and shown in FIG. 2E. Such vectors should have tropism for human CNS cells and may include non-replicating rAAVs, especially those carrying the AAV9, AAVrh10, AAVrh20, AAVrh39 or AAVcy5 capsid. Recombinant vectors can be administered by any method such that the recombinant vector enters the CNS, preferably by introducing the recombinant vector into the cerebrospinal fluid (CSF). See section 5.5.1 for details of treatment options.

- 50 047128

Subjects to receive such gene therapy may be those who respond to anti-CGRPR therapy. In specific embodiments, the methods include treating patients who have been diagnosed with, or have one or more symptoms associated with, migraines or cluster headaches and who are determined to respond to treatment with an anti-CGRPR antibody or are considered good candidates for anti-CGRPR antibody therapy. In specific embodiments, the patients have previously been treated with erenumab, eptinezumab, fremanezumab, or galcanezumab and have been found to be sensitive to one or more of erenumab, eptinezumab, fremanezumab, and galcanezumab. To determine sensitivity, a transgene product of an anti-CGRPR antibody or antigen binding fragment (eg, produced in human cell culture, bioreactors, etc.) can be administered directly to the subject.

Post-translationally modified human antibodies.

Production of the HuPTM anti-CGRPR mAb or HuPTM Fab should result in a biologically enhanced molecule for the treatment of migraines or cluster headaches through gene therapy - for example, by administering a viral vector or other DNA expression construct encoding the HuPTM Fab anti-CGRPR construct intrathecally, in particular, by intracisternal or lumbar administration, or intravenous administration to human subjects (patients) diagnosed with or having one or more symptoms of migraines or cluster headaches, to create a permanent depot in the CNS that continuously supplies fully human post-translationally modified, e.g., glycosylated human , a sulfated transgene product produced by transduced CNS cells.

The cDNA design for the HuPTM anti-CGRPR mAb or anti-CGRPR HuPTM Fab must include a signal peptide that ensures proper co- and post-translational processing (protein glycosylation and sulfation) by transduced CNS cells. For example, the signal sequence may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161).

As an alternative or complementary to gene therapy treatment, HuPTM anti-CGRPR mAb or HuPTM Fab can be produced in human cell lines using recombinant DNA technology and administered to patients diagnosed with migraine or cluster headaches, or to those receiving therapy for migraine or cluster headaches is considered appropriate.

In specific embodiments, the HuPTM mAb to CGRPR or an antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the erenumab heavy and light chain, as shown in FIG. 2E (with non-consensus asparagine (N) glycosylation sites in green, glutamine (Q) glycosylation sites in blue, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation , at one or more amino acid positions N77 and/or Q122 and/or N172 and/or N205 and/or N214 of the heavy chain (SEQ ID NO: 55) or N28 and/or N174 of the light chain (SEQ ID NO: 56 ). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the erenumab heavy and light chain variable domain sequences contains a sulfation group at Y94 and/or Y95 of the heavy chain (SEQ ID NO: 55) and/or Y87 and/or Y88 of the light chain (SEQ ID NO: 56). In other embodiments, the HuPTM anti-CGRPR mAb or antigen binding fragment thereof does not contain any detectable (e.g., detected by assays known in the art, such as those described in section 5.2 below) NeuGc fragments and/or does not contain any detectable (eg, detected using assays known in the art, eg, described in section 5.2 below) alpha-Gal fragments.

In certain embodiments, the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1%, or 2% 2,6 sialylated and/or sulfated and may be at least 5%, 10%, or even 50% , or 100% glycosylated by 2,6-sialylation and/or sulfated. The goal of the gene therapy treatment presented herein is to prevent or reduce the intensity or frequency of migraines, cluster headaches, or one or more associated symptoms, including nausea, light sensitivity, sound sensitivity, red eyes, swelling of the eyelids, forehead and facial sweating, watery eyes (lacrimation), abnormally small pupil size (miosis), nasal congestion, runny nose (rhinorrhea), and drooping eyelid (ptosis). Effectiveness may be monitored by measuring a decrease in the intensity or frequency of migraines or cluster headaches or a decrease in the number of acute migraine-specific medications used over a period of time.

Combinations of delivery of HuPTM mAb to CGRPR or an antigen-binding fragment thereof to the CNS, accompanied by delivery of other available treatments, are covered by the methods presented herein. Additional treatments may be given before, at the same time, or after gene therapy treatment. Available treatments for cluster headaches or migraines that may be combined with the gene therapy presented herein include, but are not limited to, triptans, ergotamine derivatives and NSAIDs, to name a few, and administration of anti-CGRPR agents, including but not limited to , erenumab, eptinezumab, fremanezumab and galcanezumab.

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5.3.4. HuPTM constructs and compounds for interleukin and interleukin receptor for autoimmune disorders

Compositions and methods are described for delivery of HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fab, that bind to interleukins (ILs) or interleukin receptors (ILRs) (e.g., IL4R, IL17A, IL12/IL23, or IL-5) derived from anti -IL or anti-ILR indicated for the treatment of one or more autoimmune diseases such as atopic dermatitis, psoriasis (eg, plaque psoriasis, pustular psoriasis, and erythrodermic psoriasis), arthritis (eg, psoriatic arthritis and alkylating spondylitis), Crohn's disease, or asthma (hereinafter collectively referred to as AI-D claims). In specific embodiments, the HuPTM mAb is characterized by the amino acid sequence of dupilumab, ixekizumab, secukinumab, ustekinumab, or mepolizumab or an antigen binding fragment of one of the above. The amino acid sequences of the Fab fragments of these antibodies are presented in Fig. 3A-3E, respectively. Delivery may be accomplished by gene therapy, e.g., by administering a viral vector or other DNA expression construct encoding an IL/ILR-binding HuPTM mAb (or an antigen-binding fragment thereof and/or a hyperglycosylated derivative or other derivative thereof) to human subjects. with a diagnosis of or presence of one or more symptoms of atopic dermatitis, psoriasis (eg, plaque psoriasis), arthritis (eg, psoriatic arthritis and alkylating spondylitis), Crohn's disease or asthma, to create a permanent depot that continuously supplies human RTM, e.g. , a transgenic product.

Transgenes.

Provided are recombinant vectors containing a transgene encoding a HuPTM mAb or a HuPTM Fab (or other antigen binding fragment of a HuPTM mAb) that binds to IL/ILR, which can be administered to deliver the HuPTM mAb or antigen binding fragment to a patient. A transgene is a nucleic acid containing nucleotide sequences encoding an antigen binding fragment of an IL/ILR binding antibody, such as dupilumab, ixekizumab, secukinumab, ustekinumab, mepolizumab, or variants thereof, as described in detail herein. The transgene may also encode an anti-IL/ILR antigen-binding fragment that contains additional glycosylation sites (eg, see Courtois et al.).

In certain embodiments, the anti-IL4R antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of dupilumab (having amino acid sequences SEQ ID NO: 7 and 8, respectively, see Table 4 and FIG. 3A). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 107 (encoding the Fab portion of the heavy chain of dupilumab) and SEQ ID NO: 108 (encoding the Fab portion of the light chain of dupilumab), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular human liver cells (eg, hepatocytes) or human muscle cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-integrin antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 7 with an additional hinge region sequence beginning after the C-terminal tyrosine (Y) containing all or part of the amino acid sequence GPPCPPCPAPEFLGG (SEQ ID NO: 231) and specifically, GPPCPPCPA (SEQ ID NO: 229) or GPPCPPCPAPEFLGGPSVFL (SEQ ID NO: 230), as shown in FIG. 3A. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 7 hinge region encoding sequences presented in table. 5.

In certain embodiments, an anti-IL4R antigen binding fragment transgene encodes an IL4R antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 8. In certain embodiments, the anti-IL4R antigen binding fragment transgene encodes an IL4R antigen binding fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 7. In certain embodiments, the anti-IL4R antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86% identical to , 87%, 88%, 89%, 90%, 91%, 92%,

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93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 8, and a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 7. According to In specific embodiments, the IL4R antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. those regions outside the CDRs that are underlined in Fig. 3A) or are substitutions to an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the IL4R antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 8 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (ie, those regions outside the CDRs that are underlined in Fig. 3A) or are substitutions to an amino acid present at that position in light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-IL4R antigen binding fragment transgene encodes a hyperglycosylated Dupilumab Fab comprising a heavy chain and a light chain of SEQ ID NO: 7 and 8, respectively, with one or more of the following mutations: T120N (heavy chain), Q165N, or Q165S (light chain) and/or E200N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-IL4R antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of dupilumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 3A, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-IL4R antibody or antigen binding antibody thereof fragment.

In certain embodiments, the anti-IL17A antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of ixekizumab (having amino acid sequences SEQ ID NO: 9 and 10, respectively, see Table 4 and FIG. 3B). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 109 (encoding the Fab portion of the heavy chain of ixekizumab) and SEQ ID NO: 110 (encoding the Fab portion of the light chain of ixekizumab ), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular human liver cells (eg, hepatocytes) or muscle cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequences. In specific embodiments, the anti-integrin antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 9 with an additional hinge region sequence beginning after the C-terminal tyrosine (Y) containing all or part of the amino acid sequence GPPCPPCPAPEFLGG (SEQ ID NO: 231) and specifically, GPPCPPCPA (SEQ ID NO: 229) or GPPCPPCPAPEFLGGPSVFL (SEQ ID NO: 230), as shown in FIG. 3B. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 9 hinge region encoding sequences presented in table. 5.

In certain embodiments, the anti-IL17A antigen binding fragment transgene encodes an IL17A antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 10. In certain embodiments, the anti-IL17A antigen binding fragment transgene encodes an IL17A antigen binding fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 9. In certain embodiments, the anti-IL17A antigen binding fragment transgene encodes an IL17A antigen binding fragment comprising a light chain comprising

- 53 047128 amino acid sequence which is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 10, and a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 9. In certain embodiments, the IL17A antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 with 1, 2, 3, 4, 5, 6 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and substitutions, insertions or deletions are preferably performed in framework regions (i.e., those regions outside the CDRs that are underlined in Fig. 3B) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown alignment in Fig. 11A. In specific embodiments, the IL17A antigen binding fragment comprises a light chain comprising the amino acid sequence SEQ ID NO: 10 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e., those regions outside the CDRs that are underlined in Fig. 3B) or are substitutions to an amino acid present at that position in the light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-IL-17A antigen binding fragment transgene encodes a hyperglycosylated ixekizumab Fab containing a heavy chain and a light chain of SEQ ID NO: 9 and 10, respectively, with one or more of the following mutations: L114N (heavy chain), Q165N, or Q165S (light chain) and/or E200N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-II7A antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of ixekizumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 3B, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-IL17A antibody or antigen binding agent thereof fragment.

In certain embodiments, the anti-IG17L antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the secukinumab Fab portion (having amino acid sequences SEQ ID NOs: 11 and 12, respectively, see Table 4 and FIG. 3C). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 111 (encoding the Fab portion of the secukinumab heavy chain) and SEQ ID NO: 112 (encoding the Fab portion of the secukinumab light chain ), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular human liver cells (eg, hepatocytes) or muscle cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences listed in table. 2 or 3, which correspond to secreted proteins of myocytes or hepatocytes, respectively.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequences. In specific embodiments, the anti-integrin antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 11 with an additional hinge region sequence beginning after a C-terminal aspartic acid (D) containing all or part of the amino acid sequence KTHT CPPCPAPELLGGPSVFL (SEQ ID NO: 227 ) and in particular KTHT (SEQ ID NO: 224), KTHL (SEQ ID NO: 223), KTHTCPPCPA (SEQ ID NO: 225), KTHLCPPCPA (SEQ ID NO: 226), KTHTCPPCPAPELLGGPSVFL (SEQ ID NO: 227) or KTHLCPPCPAPELLGGPSVFL (SEQ ID NO: 228) as shown in FIG. 3C. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 11 hinge region encoding sequences presented in table. 5.

In certain embodiments, the anti-GG17A antigen binding fragment transgene encodes an IL17A antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 12. In certain embodiments, the anti-III17A antigen binding fragment transgene encodes an IL17A antigen binding fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,

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92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 11. In certain embodiments, the anti-IL17A antigen binding fragment transgene encodes an antigen binding fragment containing a lung a chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 12, and a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 11. In specific embodiments, the IL17A antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 with 1, 2, 3, 4, 5, 6 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and substitutions, insertions or deletions are preferably performed in frame areas (i.e. in those areas outside the CDR that are highlighted in FIG. 3C) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the IL17A antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 12 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e., those regions outside the CDRs that are underlined in Fig. 3C) or are substitutions to an amino acid present at that position in light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-IL17A antigen binding fragment transgene encodes a hyperglycosylated secukinumab Fab containing a heavy chain and a light chain of SEQ ID NOs: 11 and 12, respectively, with one or more of the following mutations: L122N (heavy chain), Q161N, or Q161S (light chain) and/or E196N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-IL17A antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of secukinumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 3C, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art to form an anti-IL/ILR antibody heavy and/or light chain variable domain or its antigen-binding fragment.

In certain embodiments, the anti-IL-12/IL-23 antigen binding fragment transgene comprises nucleotide sequences encoding the Fab heavy and light chains of ustekinumab (having amino acid sequences SEQ ID NOs: 13 and 14, respectively, see Table 4 and FIG. 3D ). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 113 (encoding the Fab portion of the ustekinumab heavy chain) and SEQ ID NO: 114 (encoding the Fab portion of the ustekinumab light chain ), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular human liver cells (eg, hepatocytes) or muscle cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-integrin antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 13 with an additional hinge region sequence beginning after the C-terminal aspartic acid (D) containing all or part of the amino acid sequence KTHT CPPCPAPELLGGPSVFL (SEQ ID NO: 227 ) and in particular KTHT (SEQ ID NO: 224), KTHL (SEQ ID NO: 223), KTHTCPPCPA (SEQ ID NO: 225), KTHLCPPCPA (SEQ ID NO: 226), KTHTCPPCPAPELLGGPSVFL (SEQ ID NO: 227) or KTHLCPPCPAPELLGGPSVFL (SEQ ID NO: 228) as shown in FIG. 3D. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 13 hinge region encoding sequences presented in table. 5.

In certain embodiments, the anti-IL12/IL23 antigen binding fragment transgene encodes an IL/ILR antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence presented in SEQ ID

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NO: 14. In certain embodiments, the aHTu-IL12/IL23 antigen binding fragment transgene encodes an IL12/IL23 antigen binding fragment comprising a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 13. In certain embodiments, the anti-IL12 transgene /IL23 antigen binding fragment encodes an antigen binding fragment containing a light chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 14, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88% identical 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 13. In specific embodiments, the binding antigen The IL12/IL23 fragment contains a heavy chain containing the amino acid sequence SEQ ID NO: 13 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions , insertions or deletions, and substitutions, insertions or deletions are preferably made in framework regions (i.e. those areas outside the CDR that are highlighted in FIG. 3D) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the IL12/IL23 antigen binding fragment comprises a light chain comprising the amino acid sequence SEQ ID NO: 14 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (ie, those regions outside the CDRs that are underlined in Fig. 3D), or are substitutions to an amino acid present at that position in the light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti4L2/III23 antigen binding fragment transgene encodes a hyperglycosylated ustekinumab Fab containing a heavy chain and a light chain of SEQ ID NOs: 13 and 14, respectively, with one or more of the following mutations: L114N (heavy chain), Q160N, or Q160S (light chain) and/or E195N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti4N2/III23 antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of ustekinumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 3D, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-IL12/IL23 antibody or its antigen-binding fragment.

In certain embodiments, the anti-III-5 antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of mepolizumab (having amino acid sequences SEQ ID NOs: 15 and 16, respectively, see Table 4 and FIG. 3E). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 115 (encoding the Fab portion of the heavy chain of mepolizumab) and SEQ ID NO: 116 (encoding the Fab portion of the light chain of mepolizumab ), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular liver cells (eg, hepatocytes) or human muscle cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-integrin antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 15 with an additional hinge region sequence beginning after the C-terminal aspartic acid (D) containing all or part of the amino acid sequence KTHT CPPCPAPELLGGPSVFL (SEQ ID NO: 227 ) and in particular KTHT (SEQ ID NO: 224), KTHL (SEQ ID NO: 223), KTHTCPPCPA (SEQ ID NO: 225), KTHLCPPCPA (SEQ ID NO: 226), KTHTCPPCPAPELLGGPSVFL (SEQ ID NO: 227) or KTHLCPPCPAPELLGGPSVFL (SEQ ID NO: 228) as shown in FIG. 3E. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 15 hinge region encoding sequences presented in table. 5.

In certain embodiments, the anti-III-5 antigen binding fragment transgene encodes an IL-5 antigen binding fragment containing a light chain containing an amino acid

- 56 047128 lot sequence which is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 16. In certain embodiments, the anti-IL-3 antigen binding fragment transgene encodes an IL-5 antigen binding fragment containing a heavy chain containing an amino acid sequence that is at least 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 15. In certain embodiments, the anti-IL-5 antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence presented in SEQ ID NO: 16, and a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence presented in SEQ ID NO: 15. In specific embodiments, the IL-5 antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. in those areas outside the CDR, which are highlighted in FIG. 3E) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In certain embodiments, the IL-5 antigen binding fragment comprises a light chain comprising the amino acid sequence SEQ ID NO: 16 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (ie, those regions outside the CDRs that are underlined in Fig. 3E) or are substitutions to an amino acid present in it position in the light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-IL-5 antigen binding fragment transgene encodes a hyperglycosylated mepolizumab Fab containing a heavy chain and a light chain of SEQ ID NO: 15 and 16, respectively, with one or more of the following mutations: T114N (heavy chain), Q166N, or Q166S (light chain) and/or E201N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-IL-5 antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of mepolizumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 3E, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art to form an anti-IL-5 antibody heavy and/or light chain variable domain or its antigen-binding fragment.

Methods of gene therapy.

Methods are provided for treating human subjects with one or more of the claimed AI-Ds by administering a viral vector containing a transgene encoding an anti-IL/ILR antibody or an antigen-binding fragment thereof. The antibody may be dupilumab, ixekizumab, secukinumab, ustekinumab or mepolizumab and is preferably a Fab fragment thereof or another antigen binding fragment thereof. In embodiments, the patient has been diagnosed with and/or has symptoms associated with one or more of the claimed AI-Ds. Recombinant vectors used for transgene delivery are described in section 5.4.2. Such vectors should have tropism for human liver or muscle cells and may include non-replicating rAAVs, especially those carrying the AAV8 or AAV9 capsid. Recombinant vectors such as those shown in FIG. 3A-3E can be administered by any route such that the recombinant vector enters the liver or muscle tissue, preferably by introducing the recombinant vector into the bloodstream (or in an alternative embodiment into the liver bloodstream, eg via the hepatic artery). See section 5.5.2 for details of treatment options.

Subjects to whom such gene therapy is administered may be those who respond to anti-IL/ILR therapy. In specific embodiments, the methods include treating patients who have been diagnosed with one or more of the claimed AI-Ds or who have one or more symptoms associated therewith and who are identified as being sensitive to treatment with an anti-IL/ILR antibody or are considered good candidates for anti-IL/ILR antibody therapy. In specific embodiments, the patients have previously been treated with dupilumab, ixekizumab, secukinumab, ustekinumab, or mepolizumab and have been found to be sensitive to dupilumab, ixekizumab, secukinumab, ustekinumab, or mepolizumab. To determine sensitivity, a transgene product of an anti-IL/ILR antibody or antigen-binding fragment (eg, produced in human cell culture, bioreactors, etc.) can be administered directly to the subject.

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Post-translationally modified human antibodies.

Production of a HuPTM anti-IL/ILR mAb or HuPTM Fab should result in a biologically improved molecule for the treatment of one or more of the claimed AI-Ds through gene therapy - for example, by introducing a viral vector or other DNA expression construct encoding anti-IL/ILR ILR HuPTM Fab, subcutaneously, intramuscularly or intravenously to human subjects (patients) diagnosed with or having one or more symptoms of one or more of the claimed AI-Ds, to establish a permanent depot in the liver or muscle tissue that continuously supplies fully human post-translationally modified, for example, human glycosylated, sulfated transgenic product produced by transduced liver or muscle cells.

The cDNA design for anti-IL/ILR HuPTMmAb or anti-IL/ILR HuPTM Fab must include a signal peptide that ensures proper co- and post-translational processing (protein glycosylation and sulfation) by transduced liver or muscle cells. For example, the signal sequence may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

As an alternative or complementary to gene therapy treatment, HuPTM anti-IL/ILR mAb or HuPTM Fab can be generated in human cell lines using recombinant DNA technology and administered to patients diagnosed with one or more of the reported AI-Ds or for whom therapy for one or more more of the declared AI-D is considered suitable.

In specific embodiments, the anti-IL4R HuPTM mAb or antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the heavy and light chain of dupilumab, as shown in FIG. 3A (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions N77, N167 and/or Q117 of the heavy chain (SEQ ID NO: 7) or Q105, N163 and/or N215 of the light chain (SEQ ID NO: 8). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the heavy and light chain variable domain sequences of dupilumab contains a sulfation group at Y94 and/or Y95 of the heavy chain (SEQ ID NO: 7) and/or Y91 and/or Y92 of the light chain (SEQ ID NO: 8). In other embodiments, the anti-IL4R HuPTM mAb or antigen binding fragment thereof does not contain any detectable NeuGc fragments and/or does not contain any detectable alpha-Gal fragments.

In specific embodiments, the anti-IL17A HuPTM mAb or antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the ixekizumab heavy and light chain Fab portions as shown in FIG. 3B (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions Q111, N161 and/or N203 of the heavy chain (SEQ ID NO: 9) or Q105, N163 and/or N215 of the light chain (SEQ ID NO: 10). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the ixekizumab heavy and light chain variable domain sequences contains a sulfation group at Y94 and/or Y95 of the heavy chain (SEQ ID NO: 9) and/or Y91 and/or Y92 of the light chain (SEQ ID NO: 10). In other embodiments, the anti-IL17A HuPTM mAb or antigen binding fragment thereof does not contain any detectable NeuGc fragments and/or does not contain any detectable α-Gal fragments.

In specific embodiments, the anti-IL17A HuPTM mAb or antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the secukinumab heavy and light chain Fab portions as shown in FIG. 3C (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions N169 of the heavy chain (SEQ ID NO: 11) or Q101, N159 and/or N211 of the light chain (SEQ ID NO: 12). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the secukinumab heavy and light chain variable domain sequences contains a sulfation group at Y94 and/or Y95 and/or Y107 and/or Y108 of the heavy chain (SEQ ID NO: 11) and/or Y87 and/or Y88 light chain (SEQ ID NO: 12). In other embodiments, the anti-IL17A HuPTM mAb or antigen binding fragment thereof does not contain detectable NeuGc fragments and/or does not contain detectable α-Gal fragments.

In specific embodiments, HuPTM mAb to IL12/IL23 or antigen binding thereof

- 58 047128 This fragment contains heavy and light chains with the amino acid sequences of the Fab portions of the heavy and light chains of ustekinumab, as shown in FIG. 3D (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions Q111 and/or N161 of the heavy chain (SEQ ID NO: 13) or Q100, N158 and/or N210 of the light chain (SEQ ID NO: 14). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the ustekinumab heavy and light chain variable domain sequences contains a sulfation group at Y86 and/or Y87 of the light chain (SEQ ID NO: 14). In other embodiments, the anti-IL12/IL23 HuPTM mAb or antigen binding fragment thereof does not contain detectable NeuGc fragments and/or does not contain detectable α-Gal fragments.

In specific embodiments, the IL-5 mAb HuPTM or an antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the heavy and light chain of mepolizumab, as shown in FIG. 3E (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions N76 and/or N161 of the heavy chain (SEQ ID NO: 15) or N22, N34, N164 and/or N216 of the light chain (SEQ ID NO: 16). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the mepolizumab heavy and light chain variable domain sequences contains a sulfation group at Y93 and/or Y94 of the heavy chain (SEQ ID NO: 15) and/or Y92 and/or Y93 of the light chain (SEQ ID NO: 16). In other embodiments, the anti-IL-5 HuPTM mAb or antigen binding fragment thereof does not contain detectable NeuGc fragments and/or does not contain detectable alpha-Gal fragments.

In certain embodiments, the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1%, or 2% 2,6-sialylated and/or sulfated and may be at least 5%, 10%, or even 50% 2,6-sialylated and/or sulfated. % or 100% glycosylated and/or sulfated. The goal of treatment with the gene therapy presented herein is to slow or stop the progression of AI-D in a subject.

Effectiveness can be monitored by assessing symptoms or the degree of inflammation in the affected tissue or area of the body, such as the skin, colon or joints. For example, with regard to CD, effectiveness can be monitored by assessing the Crohn's Disease Activity Index [CDAI] during the course of treatment (eg, see Best WR et al. (1976) Gastroenterology 70(3):439-44, Development of a Crohn's disease activity index. National Cooperative Crohn's Disease Study. For psoriasis and atopic dermatitis, effectiveness can be monitored by assessing changes in the affected skin or the patient's quality of life over the course of treatment. One or more standardized assessments may be used to assess change. (See, for example, Feldman & Krueger, (2005) Ann. Rheum. Dis. 64(Suppl II):ii65-ii68: Psoriasis assessment tools in clinical trials, which describes standardized assessments, including the Area and Severity Index Psoriasis (PASI), Physician Global Assessment (PGA), Grid System, NPF Psoriasis Score (NPF-PS), 36-Item Short Form Health Survey (SF-36), Euro QoL, Dermatologic Quality of Life Index diseases (DLQI) and Skindex; Schram et al. (2012) Allergy; 67: 99-106: EASI, (objective) SCORAD and POEM for atopic eczema: responsiveness and minimal clinically important difference, which describes standardized assessments, including Eczema Area and Severity Index (EASI) and Severity of Atopic Dermatitis Index (SCORAD)). For arthritis, effectiveness can be monitored by assessing one or more disease activities, the patient's level of function, or the degree of structural damage to the patient's joints (eg, see Zockling & Braun (2005) Clin. Exp. Rheumatol 23 (Suppl. 39) S133-S141 : Assessment of ankylosing spondylitis, which describes a standardized assessment of ankylosing spondylitis, see also Coates et al. (2011) J. Rheumatol. 38(7): 1496-1501: Development of a disease severity and responder index for psoriatic arthritis ( PsA)-report of the OMERACT 10 PsA special interest group, which describes standardized assessments of psoriatic arthritis).

Combinations of delivering a HuPTM mAb to IL/ILR or an antigen-binding fragment thereof to the liver or muscle, accompanied by delivery of other available treatments, are covered by the methods presented herein. Additional treatments may be given before, simultaneously, or after gene therapy treatment. Available treatments for the claimed AI-Ds that can be combined with the gene therapy provided herein include, but are not limited to, phototherapy for psoriasis, aminosalicylates, immunomodulatory agents (e.g., azathioprine (AZA), 6-mercaptopurine (6-MP), methotrexate (MTX)), oral or topical corticosteroids (eg, prednisone or budesonide), topical calcineurin inhibitors, inhaled corticosteroids for the treatment of asthma and/or antibiotics for the treatment of Crohn's disease and administration of anti-IL/ILR agents, including but not limited to , dupilumab, ixekizumab, secukinumab, ustekinumab or me

- 59 047128 polyzumab.

5.3.5. HuPTM constructs and compounds targeting integrin in IBD or multiple sclerosis

Described are compositions and methods of delivering HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fab, that bind to integrin (e.g., α4 or α4β7 integrin) derived from anti-a4 integrin or anti-a4b7 integrin and are indicated for the treatment of inflammatory bowel diseases ( IBD), such as ulcerative colitis (UC) or Crohn's disease (CD), and multiple sclerosis (MS). In specific embodiments, the HuPTM mAb is characterized by the amino acid sequence of vedolizumab, natalizumab, or an antigen binding fragment of one of the above. The amino acid sequences of the Fab fragments of vedolizumab and natalizumab are presented in FIG. 4A and 4B, respectively. Delivery may be accomplished by gene therapy—for example, by administering a viral vector or other DNA expression construct encoding an integrin-binding HuPTM mAb (or an antigen-binding fragment thereof and/or a hyperglycosylated derivative or other derivative thereof) to human patients diagnosed with or the presence of one or more symptoms of IBD or MS, to create a permanent depot that continuously supplies human PTM, for example, glycosylated human, transgenic product.

Transgenes.

Recombinant vectors are provided containing a transgene encoding a HuPTM mAb or a HuPTM Fab (or other antigen binding fragment of a HuPTM mAb) that binds to an integrin that can be administered to deliver the HuPTM mAb or antigen binding fragment to a patient. A transgene is a nucleic acid containing nucleotide sequences encoding an antigen binding fragment of an antibody that binds to an integrin, such as vedolizumab, natalizumab, or variants thereof, as described in detail herein. The transgene may also encode an anti-integrin antigen-binding fragment that contains additional glycosylation sites (eg, see Courtois et al.).

In some embodiments, the anti-integrin antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of vedolizumab (having amino acid sequences SEQ ID NOs: 17 and 18, respectively, see Table 4 and FIG. 4A). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 117 (encoding the Fab portion of the heavy chain of vedolizumab) and SEQ ID NO: 118 (encoding the Fab portion of the light chain of vedolizumab ), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular human liver cells (eg, hepatocytes) or muscle cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-integrin antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 17 with an additional hinge region sequence beginning after the C-terminal aspartate (D) containing all or part of the amino acid sequence KTHTCPPCPAPELAGA (SEQ ID NO: 236) and specifically KTHL (SEQ ID NO: 223), KTHT (SEQ ID NO: 224), KTHTCPPCPA (SEQ ID NO: 225), KTHLCPPCPA (SEQ ID NO: 226), KTHTCPPCPAPELAGAPSVFL (SEQ ID NO: 237) or KTHLCPPCPAPELAGAPSVFL (SEQ ID NO: 238) as shown in FIG. 4A. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 117 hinge region encoding sequences presented in table. 5.

In certain embodiments, an anti-integrin antigen binding fragment transgene encodes an antigen binding integrin fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 18. In certain embodiments, the anti-integrin antigen binding fragment transgene encodes an antigen binding integrin fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 17. In certain embodiments, the anti-integrin antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain containing an amino acid sequence that is at least 85%, 86% identical to 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID

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NO: 18, and a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 17. In certain embodiments, the antigen binding integrin fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 with 1, 2, 3, 4 , 5 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e., in those regions which are outside the CDRs that are underlined in FIG. 4A) or are substitutions to an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the antigen binding integrin fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 18 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (ie, those regions outside the CDRs that are underlined in Fig. 4A) or are substitutions to an amino acid present at that position in light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-integrin antigen binding fragment transgene encodes a hyperglycosylated vedolizumab Fab comprising a heavy chain and a light chain of SEQ ID NOs: 17 and 18, respectively, with one or more of the following mutations: L116N (heavy chain), Q165N, or Q165S (light chain) and/or E200N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-integrin antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of vedolizumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 4A, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-integrin antibody or antigen binding antibody thereof fragment.

In certain embodiments, the anti-integrin antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of natalizumab (having amino acid sequences SEQ ID NOs: 19 and 20, respectively, see Table 4 and FIG. 4B). The nucleotide sequences may represent codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 119 (encoding the Fab portion of the natalizumab heavy chain) and SEQ ID NO: 120 (encoding the Fab portion of the natalizumab light chain) , as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular liver cells (eg, hepatocytes) or human muscle cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively. Alternatively, especially for the treatment of MS, the heavy and light chains contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human CNS cells, for example, any of the signal sequences listed in table. 1.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequences. In specific embodiments, the anti-integrin antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 19 with an additional hinge region sequence beginning after the C-terminal aspartate (D) comprising all or part of the amino acid sequence GPPCPPCPAPEFLGG (SEQ ID NO: 231) and specifically, GPPCPPCPA (SEQ ID NO: 229) or GPPCPPCPAPEFLGGPSVFL (SEQ ID NO: 230), as shown in FIG. 4B. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 119 hinge region encoding sequences presented in table. 5.

In certain embodiments, an anti-integrin antigen binding fragment transgene encodes an antigen binding integrin fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 20. In certain embodiments, the anti-integrin antigen binding fragment transgene encodes an antigen binding integrin fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% identical to the sequence presented in

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SEQ ID NO: 19. In certain embodiments, the anti-integrin antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence presented in SEQ ID NO: 20, and a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 19. In specific embodiments, the antigen binding integrin fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 with 1, 2, 3, 4, 5 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e., those regions outside the CDRs that are underlined in FIG. 4B) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the antigen binding integrin fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e., those non-CDR regions that are underlined in Fig. 4B) or are substitutions to an amino acid present at that position in the lung chains of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-integrin antigen binding fragment transgene encodes a hyperglycosylated natalizumab Fab containing a heavy chain and a light chain of SEQ ID NOs: 19 and 20, respectively, with one or more of the following mutations: L118N (heavy chain), Q159N, or Q159S (light chain) and/or E194N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-integrin antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of natalizumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 4B, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-integrin antibody or antigen binding antibody thereof fragment.

In specific embodiments, AAV vectors are provided comprising a viral capsid that is at least 95% identical to the amino acid sequence of an AAV8 capsid (SEQ ID NO: 78), an AAV9 capsid (SEQ ID NO: 79), or an AANrh10 capsid (SEQ ID NO: 80 ); and an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette contains a transgene encoding an anti-integrin mAb or an antigen-binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in liver or muscle cells person.

Methods of gene therapy.

Methods are provided for treating people with IBD or MS by administering a viral vector containing a transgene encoding an anti-integrin antibody or an antigen-binding fragment thereof. The antibody may be vedolizumab or natalizumab and is preferably a Fab fragment thereof or another antigen binding fragment thereof. In embodiments, the patient has been diagnosed with and/or has symptoms associated with IBD, such as UC or CD, or MS. In certain embodiments, the IBD may be moderately or strongly active. The recombinant vector used to deliver the transgene is described in Sections 5.4.1 and 5.4.2. In some embodiments, such vectors must have tropism for human liver cells and may include non-replicating rAAV, especially those carrying an AAV8 or AAV9 capsid. Recombinant vectors such as those shown in FIG. 4A and 4B may be administered by any method such that the recombinant vector enters the liver or muscle tissue, preferably by introducing the recombinant vector into the bloodstream. See 5.5.2 for details regarding treatment options. In other embodiments, such vectors must have tropism for human CNS cells and may include non-replicating rAAV, especially those carrying the capsid of AAV9, AAVrh10, AAVrh20, AAVrh39, or AAVcy5. A recombinant vector such as that shown in FIG. 4B may be administered by any method such that the recombinant vector enters the CNS, preferably by introducing the recombinant vector into the cerebrospinal fluid (CSF). See section 5.5.1 for details of treatment options.

Subjects to receive such gene therapy may be those who respond to anti-integrin therapy. In specific embodiments, the methods include treating patients who have been diagnosed with IBD, MS, or who have one or more symptoms associated therewith, and

- 62 047128 which are determined to respond to anti-integrin antibody treatment or are considered good candidates for anti-integrin antibody therapy. In specific embodiments, patients have been previously treated with vedolizumab and/or natalizumab and have been found to respond to vedolizumab or natalizumab. To determine sensitivity, an anti-integrin antibody transgene product or antigen-binding fragment (eg, produced in cell culture, bioreactors, etc.) can be administered directly to the subject.

Post-translationally modified human antibodies.

Production of a HuPTM mAb or HuPTM Fab to an integrin should result in a biologically improved molecule for the treatment of IBD or MS via gene therapy, e.g., by administering a viral vector or other DNA expression construct encoding a HuPTM Fab to an integrin, subcutaneously, intramuscularly, or intravenously to subjects -in humans (patients) who have been diagnosed with or have one or more symptoms of IBD or MS, to establish a permanent depot in the liver, muscle or CNS tissue that continuously supplies the fully human post-translationally modified, such as human glycosylated, sulfated transgene product produced by the transduced cells of the liver, muscles or central nervous system.

The cDNA design for HuPTMmAb anti-integrin or anti-integrin HuPTM Fab must include a signal peptide that ensures proper co- and post-translational processing (protein glycosylation and sulfation) by transduced liver or muscle cells. For example, the signal sequence may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, in some embodiments, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences listed in table. 1, 2 or 3, which correspond to proteins secreted by CNS cells, myocytes or hepatocytes, respectively.

As an alternative or complementary to gene therapy treatment, HuPTM anti-integrin mAb or HuPTM Fab can be generated in human cell lines using recombinant DNA technology and administered to patients diagnosed with IBD or MS, or for whom therapy for IBD or MS is considered appropriate.

In specific embodiments, the HuPTM integrin mAb or antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the heavy and light chains of vedolizumab, as shown in FIG. 4A (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions Q113 and/or N163 of the heavy chain (SEQ ID NO: 17) or Q105 and/or N163 and/or N215 of the light chain (SEQ ID NO: 18). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the vedolizumab heavy and light chain variable domain sequences contains a sulfation group at Y94, Y95 and/or Y106 of the heavy chain (SEQ ID NO: 17) and/or Y91 and/or Y92 of the light chain ( SEQ ID NO: 18). In other embodiments, the HuPTM integrin mAb or antigen binding fragment thereof does not contain detectable NeuGc fragments and/or does not contain detectable alpha-Gal fragments.

In specific embodiments, the HuPTM integrin mAb or antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the heavy and light chains of natalizumab, as shown in FIG. 4B (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions Q115, N165 and/or N207 of the heavy chain (SEQ ID NO: 19) or Q99, N157 and/or N209 of the light chain (SEQ ID NO: 20). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the natalizumab heavy and light chain variable domain sequences contains a sulfation group at Y94 and/or Y95 of the heavy chain (SEQ ID NO: 19) and/or Y86 and/or Y87 of the light chain (SEQ ID NO: 20). In other embodiments, the HuPTM integrin mAb or antigen binding fragment thereof does not contain detectable NeuGc fragments and/or does not contain detectable alpha-Gal fragments.

In certain embodiments, the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1%, or 2% glycosylated and can be at least 5%, 10%, or even 50% or 100% glycosylated and/or sulfated. The goal of treatment with the gene therapy presented herein is to slow or stop the progression of IBD or MS, particularly reducing pain and discomfort for the patient and/or in the case of MS, improving mobility. Effectiveness can be monitored by assessing symptoms or the degree of inflammation in the affected tissue. For example, for UC, effectiveness can be monitored by assessing the Mayo score and endoscopic Mayo score over the course of treatment (eg, see Lobaton et al. (2015) J. Crohns Colitis. 2015

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Oct;9(10):846-52, The Modified Mayo Endoscopic Score (MMES): A New Index for the Assessment of Extension and Severity of Endoscopic Activity in Ulcerative Colitis Patients. With regard to CD, effectiveness can be monitored by assessing the Crohn's Disease Activity Index [CDAI] during the course of treatment (eg, see Best WR et al. (1976) Gastroenterology, Mar;70(3):439-44, Development of a Crohn's disease activity index. National Cooperative Crohn's Disease Study. For example, for MS, effectiveness can be monitored by assessing relapse rates (eg, annual relapse rate), physical disability status (eg, Kurtzke's Extended Disability Scale (EDSS) disability score), and biological markers, including brain scans using MRI (eg, assessing T1-weighted gadolinium (Gd)-enhancing lesions and T2 hyperintense lesions using magnetic resonance imaging).

Combinations of delivery of an integrin HuPTM mAb or an antigen-binding fragment thereof to the CNS, liver or muscle, accompanied by delivery of other available treatments, are covered by the methods presented herein. Additional treatments may be given before, simultaneously, or after gene therapy treatment. Available treatments for IBD that can be combined with the gene therapy presented herein include, but are not limited to, aminosalicylates, corticosteroids and immunomodulators (eg, azathioprine, 6-mercaptopurine and/or methotrexate) and administration with anti-integrin agents, including, without restrictions, vedolizumab or natalizumab. Available treatments for multiple sclerosis that can be combined with gene therapy provided herein include, but are not limited to, interferon beta, interferon beta 1a, glatiramer acetate, cyclophosphamide, corticosteroids, immunomodulators (eg, azathioprine, 6-mercaptopurine and/or methotrexate), and mitoxantrone and administration with anti-integrin agents, including, but not limited to, natalizumab.

5.3.6. HuPTM mAb to PCSK9 and ANGPTL3

Compositions and methods of delivery of HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fab, that bind to proprotein convertase subtilisin/kexin type 9 (PCSK9), derived from anti-PCSK9 or angiopoietin-like 3 (ANGPTL3), indicated for the treatment of heterozygous familial hypercholesterolemia are described. (HeFH), homozygous familial hypercholesterolemia (HoFH) or atherosclerotic cardiovascular disease (ACD), lowering low-density lipoprotein cholesterol (LDL-C), triglyceride (TG) levels and/or total cholesterol and/or reducing or slowing the formation of atherosclerotic plaques. In specific embodiments, the HuPTM mAb is characterized by the amino acid sequence of alirocumab, evolocumab, evinacumab, or an antigen binding fragment of one of the above. The amino acid sequences of the Fab fragments of alirocumab, evolocumab and evinacumab are presented in Fig. 5A-5C, respectively. Delivery can be accomplished by gene therapy - for example, by administering a viral vector or other DNA expression construct encoding a PCSK9 or HuPTM binding mAb to ANGPTL3 (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative thereof) to human patients with diagnosis or presence of one or more symptoms of HeFH, HoFH or ACD; abnormally high levels of LDL-C, TG and/or TC or abnormal atherosclerotic plaque to create a permanent depot that continuously supplies human PTM, eg human glycosylated, transgenic product.

Transgenes.

Recombinant vectors are provided containing a transgene encoding a HuPTM mAb or a HuPTM Fab (or other antigen binding fragment of a HuPTM mAb) that binds to PCSK9 or ANGPTL3, which can be administered to deliver the HuPTM mAb or antigen binding fragment to a patient. A transgene is a nucleic acid containing nucleotide sequences encoding an antigen binding fragment of an antibody that binds to PCSK9 or ANGPTL3, such as alirocumab, evolocumab, evinacumab, or variants thereof, as described in detail herein. The transgene may also encode an anti-PCSK9 or anti-ANGPTLS antigen-binding fragment that contains additional glycosylation sites (eg, see Courtois et al.).

In certain embodiments, the anti-PCSK9 antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of alirocumab (having amino acid sequences SEQ ID NOs: 21 and 22, respectively, see Table 4 and FIG. 5A). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 121 (encoding the Fab portion of the alirocumab heavy chain) and SEQ ID NO: 122 (encoding the Fab portion of the alirocumab light chain ), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular human liver cells (eg, hepatocytes) or human muscle cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-PCSK9 antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 21 with an additional hinge region sequence beginning after the C-terminal aspartic acid (D) containing all or part of the amino acid sequence KTHTCPPCPAPELLGG (SEQ ID NO: 222) and in particular KTHT (SEQ ID NO: 224), KTHL (SEQ ID NO: 223), KTHTCPPCPA (SEQ ID NO: 225), KTHLCPPCPA (SEQ ID NO: 226), KTHTCPPCPAPELLGGPSVFL (SEQ ID NO: 227) or KTHLCPPCPAPELLGGPSVFL (SEQ ID NO: 228), as shown in FIG. 5A. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 21 hinge region encoding sequences presented in table. 5.

In certain embodiments, the anti-PCSK9 antigen binding fragment transgene encodes a PCSK9 antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 22. In certain embodiments, the anti-PCSK9 antigen binding fragment transgene encodes a PCSK9 antigen binding fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 21. In certain embodiments, the anti-PCSK9 antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86% identical to 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 22, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 21. In certain embodiments, the PCSK9 antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21 with 1, 2, 3, 4, 5, 6 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. in those areas outside the CDR that are highlighted in FIG. 5A) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the PCSK9 antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 22 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (ie, those regions outside the CDRs that are underlined in Fig. 5A) or are substitutions to an amino acid present at that position in the light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-PCSK9 antigen binding fragment transgene encodes a hyperglycosylated alirocumab Fab containing a heavy chain and a light chain of SEQ ID NOs: 21 and 22, respectively, with one or more of the following mutations: L113N (heavy chain), Q166N, or Q166S (light chain) and/or E201N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-PCSK9 antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of alirocumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 5A, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen-binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-PCSK9 antibody or antigen-binding antibody thereof fragment.

In certain embodiments, the anti-PCSK9 antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of evolocumab (having amino acid sequences SEQ ID NOs: 23 and 24, respectively, see Table 4 and FIG. 5B). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 123 (encoding the Fab portion of the heavy chain of evolocumab) and SEQ ID NO: 124 (encoding the Fab portion of the light chain of evolocumab ), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression

- 65 047128 and secretion in human cells, in particular in liver cells (eg hepatocytes) or human muscle cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-PCSK9 antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 23 with an additional hinge region sequence beginning after the C-terminal glutamic acid (E) containing all or part of the amino acid sequence KTHTCPPCPAPELLGG (SEQ ID NO: 222) and in particular KTHT (SEQ ID NO: 224), KTHL (SEQ ID NO: 223), KTHTCPPCPA (SEQ ID NO: 225), KTHLCPPCPA (SEQ ID NO: 226), KTHTCPPCPAPELLGGPSVFL (SEQ ID NO: 227) or KTHLCPPCPAPELLGGPSVFL (SEQ ID NO: 228), as shown in FIG. 5V. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 23 hinge region encoding sequences presented in table. 5.

In certain embodiments, the anti-PCSK9 antigen binding fragment transgene encodes a PCSK9 antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 24. In certain embodiments, the anti-PCSK9 antigen binding fragment transgene encodes a PCSK9 antigen binding fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 23. In certain embodiments, the anti-PCSK9 antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86% identical to 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 24, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 23. In certain embodiments, the PCSK9 antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 23 with 1, 2, 3, 4, 5, 6 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. in those areas outside the CDR that are highlighted in FIG. 5B) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the PCSK9 antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 24 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (ie, those regions outside the CDRs that are underlined in Fig. 5B) or are substitutions to an amino acid present at that position in light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-PCSK9 antigen binding fragment transgene encodes a hyperglycosylated evolocumab Fab containing a heavy chain and a light chain of SEQ ID NOs: 23 and 24, respectively, with one or more of the following mutations: T110N (heavy chain) and/or Q197N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-PCSK9 antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of evolocumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 5B, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen-binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-PCSK9 antibody or antigen-binding antibody thereof fragment.

In certain embodiments, the anti-ANGPTL3 antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chain variable domains of evinacumab (having amino acid sequences SEQ ID NOs: 25 and 26, respectively, see Table 4 and FIG. 5C). They can be fused to the heavy chain constant domain C1 and/or the light chain constant domain to form a Fab fragment. Nucleotide sequences may be codons optimized for expression in human cells and may,

- 66 047128 example, contain the nucleotide sequences SEQ ID NO: 25 (encoding the variable domain of the heavy chain of evinacumab) and SEQ ID NO: 26 (encoding the variable domain of the light chain of evinacumab), as presented in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular liver cells (eg, hepatocytes) or human muscle cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain sequence. In specific embodiments, the αanti-ANGPTL3 antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 25 with an additional hinge region sequence beginning after the C-terminal aspartic acid (D) containing all or part of the amino acid sequence KTHT CPPCPAPELLGGPSVFL (SEQ ID NO: 227 ) and in particular KTHT (SEQ ID NO: 224), KTHL (SEQ ID NO: 223), KTHTCPPCPA (SEQ ID NO: 225), KTHLCPPCPA (SEQ ID NO: 226), KTHTCPPCPAPELLGGPSVFL (SEQ ID NO: 227) or KTHLCPPCPAPELLGGPSVFL (SEQ ID NO: 228) as shown in FIG. 5C. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 25 hinge region encoding sequences presented in table. 5.

In certain embodiments, the anti-ANGPTL3 antigen binding fragment transgene encodes an ANGPTL3 antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 26. In certain embodiments, the anti-ANGPTL3 antigen binding fragment transgene encodes an ANGPTL3 antigen binding fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 25. In certain embodiments, the anti-ANGPTL3 antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86% identical to 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 26, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 25. In certain embodiments, the ANGPTL3 antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 with 1, 2, 3, 4, 5 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. in those areas outside the CDR, which are highlighted in FIG. 5C) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the ANGPTL3 antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 26 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e., those non-CDR regions that are underlined in Fig. 5C) or are substitutions to an amino acid present at that position in the lung chains of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-ANGPTL3 antigen binding fragment transgene encodes a hyperglycosylated evinacumab Fab containing a heavy chain and a light chain of SEQ ID NOs: 25 and 26, respectively, with one or more of the following mutations: M121N (heavy chain), Q160N, or Q160S (light chain) and/or E195N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-ANGPTL3 antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of evinacumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 5C, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-ANGPTL3 antibody or antigen binding antibody thereof fragment.

Methods of gene therapy.

- 67 047128

Methods are provided for treating subjects with HeFH, HoFH, or ADC by administering a viral vector containing a transgene encoding an anti-PCSK9 or anti-ANGPTL3 mAb or an antigen-binding fragment thereof. The antibody may be alirocumab, evolocumab or evinacumab and is preferably a Fab fragment thereof or another antigen binding fragment thereof. In embodiments, the patient has been diagnosed with HeFH, HoFH, or ADC disease and/or has symptoms associated with HeFH, HoFH, or ADC. Recombinant vectors used for transgene delivery are described in section 5.4.2. Such vectors should have tropism for human liver or muscle cells and may include non-replicating rAAVs, especially those carrying the AAV8 or AAV9 capsid. Recombinant vectors such as those shown in FIG. 5A-5C can be administered by any route so that the recombinant vector reaches the liver or muscle tissue, preferably by introducing the recombinant vector into the bloodstream. See Section 5.5.2 for details of treatment options.

Subjects to whom such gene therapy is administered may be those who respond to anti-PCSK9 or anti-ANGPTL3 therapy. In specific embodiments, the methods include treating patients who have been diagnosed with HeFH, HoFH, or ADC, or have one or more symptoms associated therewith, and who are determined to respond to treatment with an anti-PCSK9 or anti-ANGPTL3 antibody, or are considered good candidates for anti-PCSK9 or anti-ANGPTL3 antibody therapy. PCSK9 or to ANGPTL3. In specific embodiments, the patients have previously been treated with alirocumab, evolocumab, or evinacumab and have been found to be sensitive to alirocumab, evolocumab, or evinacumab. To determine sensitivity, a transgene product of an anti-PCSK9 or anti-ANGPTL3 antibody or antigen-binding fragment (eg, produced in cell culture, bioreactors, etc.) can be administered directly to the subject.

Post-translationally modified human antibodies.

Production of a HuPTM mAb to PCSK9 or to ANGPTL3 or a HuPTM Fab should result in a biologically improved molecule for treating HeFH, HoFH or ADC using gene therapy, for example, by introducing a viral vector or other DNA expression construct encoding a HuPTM Fab to PCSK9 or to ANGPTL3, subcutaneously, intramuscularly, or intravenously in human subjects (patients) diagnosed with or having one or more symptoms of HeFH, HoFH, or ADC, to establish a permanent depot in the liver or muscle tissue that continuously supplies a fully human post-translationally modified, e.g., human glycosylated, sulfated transgene product, produced by transduced liver or muscle cells.

In specific embodiments, the HuPTM mAb to PCSK9 or an antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the alirocumab heavy and light chain Fab portions as shown in FIG. 5A (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions N30, N59 and/or N160 of the heavy chain (SEQ ID NO: 21) or N22, N35, Q106, N164 and/or N216 of the light chain (SEQ ID NO: 22). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the alirocumab heavy and light chain variable domain sequences contains a sulfation group at Y94 and/or Y95 of the heavy chain (SEQ ID NO: 21) and/or Y92 and/or Y93 of the light chain (SEQ ID NO: 22). In other embodiments, the anti-PCSK9 HuPTM mAb or antigen binding fragment thereof does not contain detectable NeuGc fragments and/or does not contain detectable alpha-Gal fragments.

In specific embodiments, the anti-PCSK9 HuPTM mAb or antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the heavy and light chains of evolocumab, as shown in FIG. 5B (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions Q107 and/or N157 and/or N190 and/or N199 of the heavy chain (SEQ ID NO: 23), or N71 and/or N173 of the light chain (SEQ ID NO: 24). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the heavy and light chain variable domain sequences of evolocumab contains a sulfation group at Y94 and/or Y95 of the heavy chain (SEQ ID NO: 23) and/or Y88 and/or Y89 of the light chain (SEQ ID NO: 24). In other embodiments, the anti-PCSK9 HuPTM mAb or antigen binding fragment thereof does not contain detectable NeuGc fragments and/or does not contain detectable alpha-Gal fragments.

In specific embodiments, the anti-ANGPTL3 HuPTM mAb or antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the heavy and light chains of evinacumab, as shown in FIG. 5C (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow),

- 68 047128 is characterized by glycosylation, in particular 2,6-sialylation, at one or more amino acid positions N77, Q118 and/or N168 of the heavy chain (SEQ ID NO: 25) or Q100, N158 and/or N210 of the light chain (SEQ ID NO : 26). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the heavy and light chain variable domain sequences of evinacumab contains a sulfation group at Y95 of the heavy chain (SEQ ID NO: 25) and/or Y86 and/or Y87 of the light chain (SEQ ID NO: 26) . In other embodiments, the anti-PCSK9 HuPTM mAb or antigen binding fragment thereof does not contain detectable NeuGc fragments and/or does not contain detectable alpha-Gal fragments.

In some embodiments, the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1%, or 2% glycosylated and/or sulfated, and may be at least 5%, 10%, or even 50% or 100% glycosylated. % glycosylated and/or sulfated. The goal of the gene therapy treatment presented herein is to slow or stop the progression of HeFH, HoFH, or ADC and/or reduce low-density lipoprotein (LDL-C) levels. Efficacy can be monitored by monitoring LDL-C levels. For example, effectiveness can be monitored by assessing the average percentage change in LDL-C from baseline.

Combinations of delivery of HuPTM mAb to PCSK9 or to ANGPTL3 or an antigen-binding fragment thereof to the liver or muscle, accompanied by delivery of other available treatments, are covered by the methods presented herein. Additional treatments may be given before, simultaneously, or after gene therapy treatment. Available treatments for HeFH, HoFH or ACD that can be combined with the gene therapy presented herein include, but are not limited to, diet, statins, ezetimibe and LDL apheresis, as well as administration with anti-PCSK9 or anti-ANGPTL3 agents, including including, without limitation, alirocumab, evolocumab or evinacumab.

5.3.7. HuPTM mAb to OxPL

Compositions and methods are described for the delivery of HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fab, that bind to oxidized phospholipids (OxPL), indicated for the treatment and/or reduction and/or delay of cardiovascular diseases, including atherosclerotic cardiovascular disease disease (ACD), atherosclerotic plaque formation, abnormally high levels of non-HDL cholesterol and LDL, aortic stenosis, hepatic stenosis, or hypercholesterolemia. In certain embodiments, the HuPTM mAb is characterized by the amino acid sequence of E06-scFv or an antigen binding fragment thereof. The amino acid sequences of E06-scFv are shown in FIG. 5D. Delivery may be accomplished by gene therapy—for example, by administering a viral vector or other DNA expression construct encoding an OxPL-binding HuPTM mAb (or an antigen-binding fragment thereof and/or a hyperglycosylated derivative or other derivative thereof) to human patients diagnosed with or the presence of one or more symptoms of cardiovascular disease, ACD, hypercholesterolemia, abnormally high levels of non-HDL cholesterol and/or LDL, aortic stenosis, hepatic stenosis and/or abnormal atherosclerotic plaque to create a permanent depot that continuously supplies human RTM, e.g. , human glycosylated, transgenic product.

Transgenes.

Recombinant vectors containing a transgene encoding are presented.

A HuPTM mAb or HuPTM Fab (or other antigen binding fragment of a HuPTM mAb) that binds to OxPL, which can be administered to deliver the HuPTM mAb or antigen binding fragment to a patient. A transgene is a nucleic acid containing nucleotide sequences encoding an antigen binding fragment of an antibody that binds to OxPL, such as E06-scFv or variants thereof, as described in detail herein. The transgene may also encode an anti-OxPL antigen-binding fragment that contains additional glycosylation sites (eg, see Courtois et al.).

In certain embodiments, the anti-OxPL antigen binding fragment transgene comprises nucleotide sequences encoding the E06-scFv heavy and light chain variable domains (having amino acid sequences SEQ ID NOs: 59 and 60, respectively, see Table 4 and FIG. 5D). E06-scFv is a scFv molecule and thus contains the heavy and light chain variable domains of an anti-OxPL mAb connected by a flexible linker. The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 159 (encoding the E06-scFv heavy chain variable domain) and SEQ ID NO: 160 (encoding the E06 light chain variable domain -scFv), as indicated in table. 5. E06 is a scFv, and thus the scFv is expressed as a single protein chain with a linker between the light and heavy chains. ScFv contains a leader sequence at the N-terminus for appropriate expression and secretion in human cells, in particular human liver cells (such as hepatocytes) or human muscle cells. In other embodiments, wherein the heavy and light chains are expressed as separate

- 69 047128 nal proteins, both containing a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular human liver cells (eg hepatocytes) or human muscle cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

In addition to the heavy and light chain variable domain sequences, transgenes may contain a flexible peptide linker at the C-terminus of the light chain variable domain sequence. The flexible peptide linker sequence may contain flexible residues such as glycine (G) or serine (S). In some embodiments, the flexible peptide linker may contain 10-30 residues of either G, S, or both G and S. Charged residues, such as E and K, can be used and interspersed to enhance solubility. The flexible peptide linker sequence may be characterized by the amino acid sequence (GGGGS)n, where n may be 1, 2, 3, 4, 5 or 6 (SEQ ID NO: 243). In this case, the signal sequence is fused to the N-terminus of the scFv heavy or light chain variable domain sequence, as appropriate.

In certain embodiments, the anti-OxPL antigen binding fragment transgene encodes an OxPL antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 60. In certain embodiments, the anti-OxPL antigen binding fragment transgene encodes an OxPL antigen binding fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 59. In certain embodiments, the anti-OxPL antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86% identical to 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 60, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 59. In certain embodiments, the OxPL antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 59 with 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. those areas outside the CDR that are highlighted in FIG. 5D) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In certain embodiments, the OxPL antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 60 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e., those regions outside the CDRs that are underlined in Fig. 5D) or are substitutions to an amino acid present at that position in light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-OxPL antigen binding fragment transgene encodes a hyperglycosylated Fab E06-scFv containing a heavy chain and a light chain of SEQ ID NOs: 59 and 60, respectively, with an optional T118N (heavy chain) mutation (see FIG. 11A (heavy chain) ) and B (light chain)).

In certain embodiments, the anti-OxPL antigen binding fragment transgene encodes an antigen binding fragment and comprises nucleotide sequences encoding the six E06-scFv CDRs that are highlighted in the heavy and light chain variable domain sequences in FIG. 5D, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen-binding molecule, as is known in the art, to form the heavy and/or light chain variable domain of an anti-OxPL antibody or antigen-binding antibody thereof fragment.

Methods of gene therapy.

Methods are provided for treating human subjects for cardiovascular disease, ACD, hypercholesterolemia, abnormally high levels of non-HDL cholesterol and/or LDL, aortic stenosis, hepatic stenosis, and/or abnormal atherosclerotic plaque by administering a viral vector containing a transgene encoding a mAb to OxPL or its antigen-binding fragment. The antibody may be E06-scFv and is preferably a Fab fragment thereof or another

- 70 047128 antigen-binding fragment. In embodiments, the patient has been diagnosed with and/or has symptoms associated with cardiovascular disease, ACD, hypercholesterolemia, abnormally high levels of non-HDL cholesterol and/or LDL, aortic stenosis, hepatic stenosis, and/or abnormal atherosclerotic plaque. Recombinant vectors used for transgene delivery are described in section 5.4.2. Such vectors should have tropism for human liver or muscle cells and may include non-replicating rAAVs, especially those carrying the AAV8 or AAV9 capsid. Recombinant vectors such as those shown in FIG. 5D may be administered by any route such that the recombinant vector enters the liver or muscle tissue, preferably by introducing the recombinant vector into the bloodstream. See Section 5.5.2 for details of treatment options.

Subjects to receive such gene therapy may be those who respond to anti-OxPL therapy. In specific embodiments, the methods include treating patients who have been diagnosed with cardiovascular disease, ACD, hypercholesterolemia, abnormally high levels of non-HDL cholesterol and/or LDL, aortic stenosis, hepatic stenosis, and/or abnormal atherosclerotic plaque, or have one or more related are symptomatic and have been identified as responding to anti-OxPL antibody treatment or are considered good candidates for anti-OxPL antibody therapy. In specific embodiments, patients have previously been treated with E06-scFv or E06 and have been found to respond to E06-scFv or E06. To determine sensitivity, a transgene product of an anti-OxPL antibody or antigen binding fragment (eg, produced in cell culture, bioreactors, etc.) can be administered directly to the subject.

Post-translationally modified human antibodies.

Production of HuPTM mAb to OxPL or HuPTM Fab should result in the formation of a biologically improved molecule for the treatment of cardiovascular disease, ACD, hypercholesterolemia, abnormally high levels of non-HDL cholesterol and/or LDL, aortic stenosis, hepatic stenosis and/or abnormal atherosclerotic plaque, administered via gene therapy - for example, by administering a viral vector or other DNA expression construct encoding a HuPTM Fab to OxPL subcutaneously, intramuscularly or intravenously to human subjects diagnosed with or having one or more symptoms of cardiovascular disease, ACD, hypercholesterolemia, abnormally high levels of non-HDL cholesterol and/or LDL, aortic stenosis, hepatic stenosis and/or abnormal atherosclerotic plaque, to create a permanent depot in the liver or muscle tissue that continuously supplies a fully human post-translationally modified, e.g., human glycosylated, sulfated transgene product , produced by transduced liver or muscle cells.

In specific embodiments, the HuPTM mAb to OxPL or an antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the E06-scFv heavy and light chain Fab portions as shown in FIG. 5D (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, especially 2,6-sialylation, at one or more amino acid positions N53 of the heavy chain (SEQ ID NO: 59). Alternatively or in addition, HuPTM scFv or another antigen binding fragment thereof with heavy and light chain variable domain sequences E06scFv contains a sulfation group at Y58 and/or Y62 and/or Y96 and/or Y97 of the heavy chain (SEQ ID NO: 59) and/ or Y42 light chain (SEQ ID NO: 60). In other embodiments, the HuPTM mAb to OxPL or antigen binding fragment thereof does not contain detectable NeuGc fragments and/or does not contain detectable alpha-Gal fragments.

In some embodiments, the HuPTM mAb or Fab scFv is therapeutically effective and is at least 0.5%, 1%, or 2% glycosylated and/or sulfated, and may be at least 5%, 10%, or even 50% or 100% glycosylated. % glycosylated and/or sulfated. The goal of treatment with gene therapy presented herein is to treat, slow and/or stop the progression of cardiovascular disease, ACD, hypercholesterolemia, abnormally high levels of non-HDL cholesterol and/or LDL, aortic stenosis, hepatic stenosis and/or abnormal atherosclerotic plaque. Efficacy may be monitored by monitoring LDL levels, markers of inflammation, or changes in the degree of aortic stenosis, such as monitoring changes in aortic valve region, peak and mean transvalvular gradients, and/or maximum aortic velocity. For example, effectiveness can be monitored by assessing the average percentage change in LDL from baseline.

Combinations of delivery of HuPTM mAb to OxPL or an antigen binding fragment thereof to the liver or muscle, accompanied by delivery of other available treatments, are covered by the methods presented herein. Additional treatments may be given before, simultaneously, or after gene therapy treatment. Available treatments for cardiovascular disease, ACD, hypercholesterolemia, abnormally high levels of non-HDL cholesterol and/or LDL, aortic stenosis, liver stenosis and/or abnormal atherosclerotic plaque that can be combined with the gene therapy presented herein , include, but are not limited to, diet, statins, ezetimibe and LDL apheresis, as well as the use of anti-OxPL agents, including, but not limited to, E06-scFv.

5.3.8. Anti-RANKL HuPTM designs and formulations for osteoporosis

Compositions and methods are described for the delivery of HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fab, that bind to receptor activator of nuclear factor kappa B ligand (RANKL) derived from an anti-RANKL antibody, such as denosumab (Fig. 6), and are shown for the treatment of osteoporosis or abnormal bone loss or bone weakness (eg, treatment of giant cell tumor of bone, treatment of drug-induced bone loss, slowing the loss (or gain) of bone mass in breast and prostate cancer patients, preventing skeletal-related events from - for bone metastasis or to reduce bone resorption and remodeling. In specific embodiments, the HuPTM mAb is characterized by the amino acid sequence of denosumab or an antigen binding fragment thereof. The amino acid sequence of the Fab fragment of this antibody is shown in FIG. by administering a viral vector or other DNA expression construct encoding a RANKL-binding HuPTM mAb (or an antigen-binding fragment and/or a hyperglycosylated derivative or other derivative thereof) to human patients diagnosed with osteoporosis or experiencing bone loss to create permanent a depot that continuously supplies human PTM, such as glycosylated human, transgenic product.

Transgenes.

Provided are recombinant vectors containing a transgene encoding a HuPTM mAb or a HuPTM Fab (or other antigen binding fragment of a HuPTM mAb) that binds to RANKL, which can be administered to deliver the HuPTM mAb or antigen binding fragment to a patient. A transgene is a nucleic acid containing nucleotide sequences encoding an antigen binding fragment of an antibody that binds to RANKL, such as denosumab or variants thereof, as described in detail herein. The transgene may also encode an anti-RANKL antigen-binding fragment that contains additional glycosylation sites (eg, see Courtois et al.).

In certain embodiments, the anti-RANKL antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of denosumab (having amino acid sequences SEQ ID NO: 27 and 28, respectively, see Table 4 and FIG. 6). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 127 (encoding the Fab portion of the denosumab heavy chain) and SEQ ID NO: 128 (encoding the Fab portion of the denosumab light chain), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular liver cells (eg, hepatocytes) or human muscle cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-RANKL antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 27 with an additional hinge region sequence beginning after the C-terminal aspartate (D) containing all or part of the amino acid sequence KTHTCPPCPAPELLGG (SEQ ID NO: 222) and specifically, KTHL (SEQ ID NO: 223), KTHT (SEQ ID NO: 224), KTHTCPPCPA (SEQ ID NO: 225), KTHLCPPCPA (SEQ ID NO: 226), KTHTCPPCPAPELLGGPSVFL (SEQ ID NO: 227) or KTHLCPPCPAPELLGGPSVFL (SEQ ID NO: 228) as shown in FIG. 6. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 27 hinge region encoding sequences presented in table. 5.

In certain embodiments, the anti-RANKL antigen binding fragment transgene encodes a RANKL antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 28. In certain embodiments, the anti-RANKL antigen binding fragment transgene encodes a RANKL antigen binding fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,

- 72 047128

92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 27. In certain embodiments, the anti-RANKL antigen binding fragment transgene encodes an antigen binding fragment containing a lung a chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 28, and a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 27. In certain embodiments, the RANKL antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 27 with 1, 2, 3, 4, 5, 6 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and substitutions, insertions or deletions are preferably performed in frame areas (i.e. in those areas outside the CDR that are highlighted in FIG. 6) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In certain embodiments, the RANKL antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 28 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e., those regions outside the CDRs that are underlined in FIG. 6) or are substitutions to an amino acid present at that position in light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-RANKL antigen binding fragment transgene encodes a hyperglycosylated denabumab Fab containing a heavy chain and a light chain of SEQ ID NOs: 27 and 28, respectively, with one or more of the following mutations: L117N (heavy chain), Q161N, or Q161S (light chain) and/or E196N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-RANKL antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of denosumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 6, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen-binding molecule, as is known in the art, to form the heavy and/or light chain variable domain of an anti-RANKL antibody or antigen-binding antibody thereof fragment.

Methods of gene therapy.

Methods are provided for treating people with osteoporosis or abnormal bone loss (eg, in patients with breast or prostate cancer or due to bone metastases) by administering a viral vector containing a transgene encoding an anti-RANKL antibody or an antigen-binding fragment thereof. The antibody may be denosumab and is preferably a Fab fragment thereof or another antigen binding fragment thereof. In embodiments, the patient has been diagnosed and/or has symptoms associated with osteoporosis or abnormal bone loss. Recombinant vectors used for transgene delivery are described in section 5.4.2. Such vectors should have tropism for human liver or muscle cells and may include non-replicating rAAVs, especially those carrying the AAV8 or AAV9 capsid. A recombinant vector such as that shown in FIG. 6 may be administered by any method such that the recombinant vector enters the liver or muscle tissue, preferably by introducing the recombinant vector into the bloodstream. See Section 5.5.2 for details of treatment options.

Subjects to receive such gene therapy may be those who respond to anti-RANKL therapy. In specific embodiments, the methods include treating patients who have been diagnosed with, or have one or more symptoms associated with, osteoporosis or abnormal bone loss and who are determined to respond to treatment with an anti-RANKL antibody or are considered good candidates for therapy with an anti-RANKL antibody . In specific embodiments, the patients have previously been treated with denosumab and have been found to be sensitive to denosumab. To determine sensitivity, a transgene product of an anti-RANKL antibody or antigen binding fragment (eg, produced in cell culture, bioreactors, etc.) can be administered directly to the subject.

Post-translationally modified human antibodies.

Production of an anti-RANKL HuPTM mAb or a HuPTM Fab should result in a biologically improved molecule for the treatment of osteoporosis or bone loss through gene therapy - for example, by administering a viral vector or other DNA expression construct encoding the anti-RANKL HuPTM Fab intravenously to subjects. people (patients) diagnosed with or experiencing one or more symptoms of osteoporosis or bone loss, to establish a permanent depot in the liver or muscle tissue that continuously supplies the fully human post-translationally modified, e.g., human glycosylated, sulfated transgene product produced transduced liver or muscle cells.

The cDNA design for the HuPTMmAb anti-RANKL or anti-RANKL HuPTM Fab must include a signal peptide that ensures proper co- and post-translational processing (protein glycosylation and sulfation) by the transduced liver or muscle cells. For example, the signal sequence may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

As an alternative or complementary to gene therapy treatment, HuPTM mAb to RANKL or HuPTM Fab can be produced in human cell lines using recombinant DNA technology and administered to patients diagnosed with osteoporosis or bone loss, or to those undergoing therapy for osteoporosis or bone loss is considered appropriate.

In specific embodiments, the HuPTM mAb to RANKL or an antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the denosumab heavy and light chains, as shown in FIG. 6 (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) are characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions N77 and/or N164 and/or Q114 of the heavy chain (SEQ ID NO: 27) or N159 and/or N211 and/or Q101 of the light chain (SEQ ID NO: 28). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the denosumab heavy and light chain variable domain sequences contains a sulfation group at Y94 and/or Y95 of the heavy chain (SEQ ID NO: 27) and/or Y88 of the light chain (SEQ ID NO: 28) . In other embodiments, the HuPTM mAb to RANKL or an antigen binding fragment thereof does not contain detectable NeuGc fragments and/or does not contain detectable alpha-Gal fragments.

In some embodiments, the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1%, or 2% glycosylated and/or sulfated, and may be at least 5%, 10%, or even 50% or 100% glycosylated. % glycosylated and/or sulfated. The goal of treatment with the gene therapy presented herein is to slow or stop the progression of osteoporosis or bone loss. Efficacy can be monitored by assessing bone tissue or skeletal events or the absence of skeletal events. For example, for osteoporosis, effectiveness can be monitored by assessing bone mineral content, assessing radiographs for vertebral fractures, or diagnostic imaging to confirm clinical fractures.

Combinations of delivery of HuPTM mAb to RANKL or an antigen-binding fragment thereof to the liver or muscle, accompanied by delivery of other available treatments, are covered by the methods presented herein. Additional treatments may be given before, simultaneously, or after gene therapy treatment. Available treatments for osteoporosis or bone loss that may be combined with gene therapy provided herein include, but are not limited to, bisphosphonates (eg, zoledronic acid), parathyroid hormone (eg, teriparatide [PTH 1-34] and/or or full-length PTH 1-84), calcium, vitamin D, and chemotherapy, cryotherapy, or radiation therapy in patients diagnosed with cancer, as well as anti-RANKL agents, including, but not limited to, denosumab.

5.3.9. HuPTM designs and formulations PD blockers in cancer and lymphoma

Compositions and methods of delivering HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fab, that bind to programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1) or programmed death ligand 2 (PD-L2) are described. , derived from PD-1 blockers (eg, anti-PD-1, anti-PD-L1, or anti-PD-L2), indicated for the treatment of unresectable/metastatic melanoma, lymphomas (eg, Hodgkin's lymphoma), and carcinomas (eg, renal - cell carcinoma, squamous cell carcinoma and non-small cell lung carcinoma). In specific embodiments, the HuPTM mAb is characterized by the amino acid sequence of nivolumab, pembrolizumab, or an antigen binding fragment of one of the above. The amino acid sequences of Fab fragments of nivolumab and pembrolizumab are presented in Fig. 7A and 7B, respectively. Delivery may be via gene therapy - for example, by administering a viral vector or other DNA expression construct encoding a PD-1/PD-L1/PD-L2 binding HuPTM mAb or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative thereof) to patients ( human subjects) diagnosed with or presenting with one or more symptoms of melanoma, carcinomas or lymphomas, to create a permanent depot that continuously supplies human PTM, for example, glycosylated human, transgenic product.

Transgenes.

- 74 047128

Provided are recombinant vectors containing a transgene encoding a HuPTM mAb or a HuPTM Fab (or other antigen binding fragment of a HuPTM mAb) that binds to PD-1/PDL1/PD-L2, which can be administered to deliver the HuPTM mAb or antigen binding fragment to a patient. A transgene is a nucleic acid containing nucleotide sequences encoding an antigen binding fragment of an antibody that binds to PD-1/PD-L1/PD-L2, such as nivolumab, pembrolizumab, or variants thereof, as described in detail herein. The transgene may also encode an anti-PD-i, anti-PD-LT, or anti-PD-L2 antigen-binding fragment that contains additional glycosylation sites (eg, see Courtois et al.).

In some embodiments, the anti-PD-1 antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of nivolumab (having amino acid sequences SEQ ID NO: 29 and 30, respectively, see Table 4 and FIG. 7A). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 129 (encoding the Fab portion of the heavy chain of nivolumab) and SEQ ID NO: 130 (encoding the Fab portion of the light chain of nivolumab), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular human liver cells (eg, hepatocytes) or human muscle cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-integrin antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 29 with an additional hinge region sequence beginning after the C-terminal tyrosine (Y) containing all or part of the amino acid sequence GPPCPPCPAPEFLG (SEQ ID NO: 240) and specifically, GPPCPPCPA (SEQ ID NO: 229) or GPPCPPCPAPEFLGPSVFL (SEQ ID NO: 241), as shown in FIG. 7A. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 29 hinge region encoding sequences presented in table. 5.

In certain embodiments, the anti-PD-1 antigen binding fragment transgene encodes a PD-1 antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 30. In certain embodiments, the anti-PD-1 antigen binding fragment transgene encodes a PD-1 antigen binding fragment containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% is 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 29. In certain embodiments, the anti-PD-1 antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence, which is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequence set forth in SEQ ID NO: 30, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 29. In certain embodiments, the PD-1 antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (those. in those areas outside the CDR, which are highlighted in FIG. 7A) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the PD-1 antigen binding fragment comprises a light chain comprising the amino acid sequence SEQ ID NO: 30 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (ie, those non-CDR regions that are underlined in Fig. 7A) or are substitutions to an amino acid present at that position in the light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In some embodiments, the anti-PD-1 antigen binding fragment transgene encodes a hyperglycosylated nivolumab Fab comprising a heavy chain and a light chain of SEQ ID NO: 29 and 30, respectively, with one or more of the following mutations: L108N (heavy chain), Q160N, or Q160S (light chain) and/or E195N (light chain) (see Fig. 11A (heavy chain) and B (light

- 75 047128 chain)).

In some embodiments, the anti-PD-1 antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of nivolumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 7A, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art to form an anti-PD-1 antibody heavy and/or light chain variable domain or its antigen-binding fragment.

In some embodiments, the anti-PD-1 antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of pembrolizumab (having amino acid sequences SEQ ID NOs: 31 and 32, respectively, see Table 4 and FIG. 7B). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 131 (encoding the Fab portion of the heavy chain of pembrolizumab) and SEQ ID NO: 132 (encoding the Fab portion of the light chain of pembrolizumab ), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular liver cells (eg, hepatocytes) or human muscle cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequences. In specific embodiments, the anti-integrin antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 31 with an additional hinge region sequence beginning after the C-terminal tyrosine (Y) containing all or part of the amino acid sequence GPPCPPCPAPEFLG (SEQ ID NO: 240) and specifically, GPPCPPCPA (SEQ ID NO: 229) or GPPCPPCPAPEFLGPSVFL (SEQ ID NO: 241), as shown in FIG. 7B. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 31 hinge region coding sequences presented in table. 5.

In certain embodiments, the anti-PD-1 antigen binding fragment transgene encodes a PD-1 antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 32. In certain embodiments, the anti-PD-1 antigen binding fragment transgene encodes a PD-1 antigen binding fragment comprising a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 31. In certain embodiments, the anti-PD-1 antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence, presented in SEQ ID NO: 32, and a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 31. In certain embodiments, the PD-1 antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 with 1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. .e. in those areas outside the CDR, which are highlighted in FIG. 7B) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the PD-1 antigen binding fragment comprises a light chain comprising the amino acid sequence SEQ ID NO: 32 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (ie, those regions outside the CDRs that are underlined in Fig. 7B) or are substitutions to an amino acid present in it position in the light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the αanti-PD-1 antigen binding fragment transgene encodes a hyperglycosylated Fab of pembrolizumab containing a heavy chain and a light chain from SEQ ID NOs: 31 and 32, respectively, with one or more of the following mutations: T115N (heavy

- 76 047128 chain), Q164N or Q164S (light chain) and/or E199N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-PD-i antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of pembrolizumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 7B, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art to form an anti-PD-1 antibody heavy and/or light chain variable domain or its antigen-binding fragment.

Methods of gene therapy.

Methods are provided for treating melanomas, carcinomas or lymphomas in human subjects by administering a viral vector containing a transgene encoding one or more of anti-PD-1, anti-PD-L1 and anti-PD-L2 antibodies or an antigen-binding fragment thereof. In particular, methods are provided for treating metastatic melanoma, lymphoma, non-small cell lung carcinoma, squamous cell carcinoma of the head and neck, urothelial carcinoma, cancer with high microsatellite instability, gastric cancer, renal cell carcinoma, metastatic colon cancer with mismatch repair deficiency or hepatocellular carcinoma by administering a viral vector containing a transgene encoding one or more antibodies to PD-1, to PD-L1 and to PD-L2 or an antigen-binding fragment thereof. The antibody may be nivolumab or pembrolizumab and is preferably a Fab fragment thereof or another antigen binding fragment thereof. In embodiments, the patient has been diagnosed with a disease and/or has symptoms associated with melanoma, carcinoma, or lymphoma. Recombinant vectors used for transgene delivery are described in section 5.4.2. Such vectors should have tropism for human liver or muscle cells and may include non-replicating rAAVs, especially those carrying the AAV8 or AAV9 capsid. Recombinant vectors such as those shown in FIG. 7A and 7B may be administered by any method such that the recombinant vector enters the liver or muscle tissue, preferably by introducing the recombinant vector into the bloodstream. See Section 5.5.2 for details of treatment options.

Subjects to whom such gene therapy is administered may be patients responding to anti-PD-1, anti-PD-Lt or anti-PD-L2 therapy. In specific embodiments, the methods include treating patients who have been diagnosed with, or have one or more symptoms associated with, melanoma, carcinoma, or lymphoma and who are determined to respond to treatment with an anti-PD-1, anti-PD-L1, or anti-PD-L2 antibody, or are considered good candidates for anti-PD-1, anti-PD-L1, or anti-PD-L2 antibody therapy. In specific embodiments, patients have previously been treated with nivolumab or pembrolizumab and have been found to respond to nivolumab or pembrolizumab. To determine sensitivity, a transgene product of an anti-PD1, anti-PD-L1, or anti-PD-L2 antibody or antigen-binding fragment (eg, produced in cell culture, bioreactors, etc.) can be administered directly to the subject.

Post-translationally modified human antibodies.

Production of HuPTM mAbs against PD-1, against PD-L1 and/or against PD-L2 or HuPTM Fab should lead to the formation of a biologically improved molecule for the treatment of melanoma, carcinomas or lymphomas, carried out using gene therapy - for example, by introducing a viral vector or another DNA expression construct encoding HuPTM Fab to PD-1, to PD-L1 and/or to PD-L2, subcutaneously, intramuscularly or intravenously to human subjects diagnosed with or having one or more symptoms of melanoma, carcinomas, lymphomas or other cancers to establish a permanent depot in the liver or muscle tissue that continuously supplies the fully human post-translationally modified, e.g., glycosylated human, sulfated transgene product produced by the transduced liver or muscle cells.

DNA constructs for HuPTMmAb or HuPTM Fab must include a signal peptide that ensures proper co- and post-translational processing (protein glycosylation and sulfation) by transduced liver or muscle cells. For example, the signal sequence may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

As an alternative or complementary to gene therapy treatments, HuPTM anti-PD-1, anti-PD-L1 or anti-PD-L2 mAbs or HuPTM Fab can be generated in human cell lines using recombinant DNA technology and administered to patients diagnosed with metastatic melanoma, lymphoma , non-small cell lung carcinoma, squamous cell carcinoma of the head and neck, urothelial carcinoma, cancer with high microsatellite instability, gastric cancer, renal cell carcinoma, metastatic colon cancer with mismatch repair deficiency or hepatocellular carcinoma, or those for whom therapy for metastatic melanoma, lymphoma, non-small cell lung carcinoma, squamous cell carcinoma of the head and neck, urothelial carcinoma, cancer with high microsatellite instability, gastric cancer, renal cell carcinoma, metastatic colon cancer with mismatch repair deficiency or hepatocellular carcinoma is considered appropriate.

In specific embodiments, the anti-PD-1 mAb HuPTM or an antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the heavy and light chain of nivolumab, as shown in FIG. 7A (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions N77, Q105 and/or N155 of the heavy chain (SEQ ID NO: 29) or N93, Q100, N158 and/or N210 of the light chain (SEQ ID NO: 30). Alternatively or in addition, mAb HuPTM or an antigen binding fragment thereof with the nivolumab heavy and light chain variable domain sequences contains a sulfation group at Y94 and/or Y95 of the heavy chain (SEQ ID NO: 29) and/or Y86 and/or Y87 of the light chain (SEQ ID NO: 30). In other embodiments, the HuPTM PD-1 mAb or antigen binding fragment thereof does not contain detectable NeuGc fragments and/or does not contain detectable alpha-Gal fragments.

In specific embodiments, the HuPTM PD-1 mAb or antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the heavy and light chains of pembrolizumab, as shown in FIG. 7B (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions Q112, N162 and/or N204 of the heavy chain (SEQ ID NO: 31) or N162 and/or N214 of the light chain (SEQ ID NO: 32). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with pembrolizumab heavy and light chain variable domain sequences is characterized by the presence of a sulfation group at Y94 and/or Y95 of the heavy chain (SEQ ID NO: 31) and/or Y90 and/or Y91 of the light chain (SEQ ID NO: 32). In other embodiments, the HuPTM anti-PD-1 mAb or antigen binding fragment thereof does not contain detectable NeuGc fragments and/or does not contain detectable alpha-Gal fragments.

In some embodiments, the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1%, or 2% glycosylated and/or sulfated, and may be at least 5%, 10%, or even 50% or 100% glycosylated. % glycosylated and/or sulfated. The goal of treatment with the gene therapy presented herein is to slow or stop the progression of metastatic melanoma, lymphoma, non-small cell lung carcinoma, head and neck squamous cell carcinoma, urothelial carcinoma, cancer with high microsatellite instability, gastric cancer, renal cell carcinoma, metastatic colon cancer with mismatch repair deficiency or hepatocellular carcinoma. Efficacy may be monitored by one or more oncology endpoints, including overall survival, progression-free survival, time to progression, time to treatment failure, event-free survival, time to next treatment, objective response rate, or duration of response (see eg, US Department of Health and Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research, Guidance for Industry: Clinical Trial Endpoints for Cancer Drug and Treatment Approval https:/. /www.fda.gov/downloads/Drugs/Guidances/ucm071590.pdf. Accessed October 13, 2017; Oncology Endpoints in a Changing Landscape 2016;

Combinations of delivery of one or more anti-PD-1, anti-PD-L1, and anti-PD-L2 HuPTM mAbs or antigen binding fragments thereof to the liver or muscle, accompanied by delivery of other available treatments, are covered by the methods presented herein. Additional treatments may be given before, simultaneously, or after gene therapy treatment. Available treatments for metastatic melanoma, lymphoma, non-small cell lung carcinoma, squamous cell carcinoma of the head and neck, urothelial carcinoma, cancer with high microsatellite instability, gastric cancer, renal cell carcinoma, metastatic colon cancer with mismatch repair deficiency or hepatocellular carcinoma, which may be combined with gene therapies provided herein include, but are not limited to, chemotherapy (e.g., cisplatin, gemcitabine, pemetrexed, carboplatin, and/or paclitaxel), radiation therapy, cryotherapy, targeted small molecule therapies, other antibodies, and vaccine therapy and administration with one or more anti-PD-1, anti-PD-L1 and anti-PD-L2 agents, including, but not limited to, nivolumab and pembrolizumab.

5.3.10. Anti-VEGF or anti-fD HuPTM designs and formulations for eye diseases

Compositions and methods of delivering HuPTM mAb and antigen-binding fragments thereof, such as HuPTM Fab, that bind to vascular endothelial growth factor (VEGF) or complement (e.g., factor D (fD)) derived from anti-VEGF or anti-Ab are described. -complement (eg, anti-fD), respectively, indicated for the treatment of one or more retinal diseases, including diabetic retinopathy, myopic choroidal neovascularization (mCNV), macular degeneration (eg, neovascular (wet) age-related macular degeneration (AMD)) , macular edema (eg, retinal vein occlusion (RVO) macular edema or diabetic macular edema (DME)); to suppress angiogenesis; or, if derived from anti-VEGF, for the treatment of one or more types of cancer, including epithelial ovarian cancer, fallopian tube cancer, abdominal cancer, cervical cancer, metastatic colorectal cancer, metastatic HER2-negative breast cancer, metastatic renal cell carcinoma, glioblastoma, non-small cell lung cancer (NSCLC). In specific embodiments, the HuPTM mAb is characterized by the amino acid sequence of ranibizumab, bevacizumab, lampalizumab, brolucizumab, or an antigen binding fragment of one of the above. The amino acid sequences of the Fab fragments of ranibizumab, bevacizumab and lampalizumab and the scFv of brolucizumab are presented in FIG. 8A-8D, respectively. Delivery can be accomplished by gene therapy - for example, by administering a viral vector or other DNA expression construct encoding a VEGF or factor D binding HuAPTM mAb (or an antigen binding fragment thereof and/or a hyperglycosylated derivative or other derivative thereof, including scFv) to patients (human subjects) diagnosed with or having one or more symptoms of a retinal disorder (eg, diabetic retinopathy, mCNV, macular degeneration or macular edema) or cancer (eg, ovarian epithelial cancer, fallopian tube cancer, abdominal cancer, cervical cancer, metastatic colorectal cancer, metastatic HER2-negative breast cancer, metastatic renal cell carcinoma, glioblastoma or NSCLC) to create a permanent depot that continuously supplies human RTM, eg human glycosylated, transgenic product.

Recombinant vectors are provided containing a transgene encoding a HuPTM mAb or a HuPTM Fab (or other antigen binding fragment of a HuPTM mAb) that binds to VEGF or fD, which can be administered to deliver the HuPTM mAb or antigen binding fragment to a patient. A transgene is a nucleic acid containing nucleotide sequences encoding an antigen binding fragment of an antibody that binds to VEGF or fD, such as ranibizumab, bevacizumab, lampalizumab, brolucizumab, or variants thereof, as described in detail herein. The transgene may also encode an anti-VEGF or anti-fD antigen-binding fragment that contains additional glycosylation sites (eg, see Courtois et al.).

In some embodiments, the anti-VEGF antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of ranibizumab (having amino acid sequences SEQ ID NOs: 33 and 34, respectively, see Table 4 and FIG. 8A). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 133 (encoding the Fab portion of the ranibizumab heavy chain) and SEQ ID NO: 134 (encoding the Fab portion of the ranibizumab light chain ), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular one or more cells forming the retina. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences listed in table. 1, which correspond to proteins secreted by one or more cells forming the retina. Alternatively, the signal sequence may be suitable for expression in muscle or liver cells, such as those listed in table. 2 and 3 below.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-VEGF antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 33 with an additional hinge region sequence beginning after the C-terminal aspartic acid (D) containing all or part of the amino acid sequence KTHTCPPCPAPELLGG (SEQ ID NO: 222) and in particular KTHT (SEQ ID NO: 224), KTHL (SEQ ID NO: 223), KTHTCPPCPA (SEQ ID NO: 225), KTHLCPPCPA (SEQ ID NO: 226), KTHTCPPCPAPELLGGPSVFL (SEQ ID NO: 227) or KTHLCPPCPAPELLGGPSVFL (SEQ ID NO: 228), as shown in FIG. 8A. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 33 hinge region encoding sequences presented in table. 5.

In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes a VEGF antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,

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92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 34. In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes a VEGF antigen binding fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 33. In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86% identical to 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 34, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 33. In certain embodiments, the VEGF antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 with 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. those areas outside the CDR that are highlighted in FIG. 8A) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the VEGF antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 34 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (ie, those regions outside the CDRs that are underlined in Fig. 8A) or are substitutions to an amino acid present at that position in light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes a hyperglycosylated ranibizumab Fab containing the heavy chain and light chain of SEQ ID NOs: 33 and 34, respectively, with one or more of the following mutations: L118N (heavy chain), Q160N, or Q160S ( light chain) and/or E195N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of ranibizumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 8A, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-VEGF antibody or antigen binding agent thereof fragment.

In certain embodiments, the anti-VEGF antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of bevacizumab (having amino acid sequences SEQ ID NOs: 35 and 36, respectively, see Table 4 and FIG. 8B). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 135 (encoding the Fab portion of the bevacizumab heavy chain) and SEQ ID NO: 136 (encoding the Fab portion of the bevacizumab light chain ), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular one or more types of retinal cells or liver cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences listed in table. 1 or 3, which correspond to proteins secreted by retinal cells or liver cells, respectively.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-integrin antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 35 with an additional hinge region sequence beginning after the C-terminal aspartic acid (D) containing all or part of the amino acid sequence KTHTCPPCPAPELLGG (SEQ ID NO: 222) and in particular KTHT (SEQ ID NO: 224), KTHL (SEQ ID NO: 223), KTHTCPPCPA (SEQ ID NO: 225), KTHLCPPCPA (SEQ ID NO: 226), KTHTCPPCPAPELLGGPSVFL (SEQ ID NO: 227) or KTHLCPPCPAPELLGGPSVFL (SEQ ID NO: 228), as shown in FIG. 8B. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 35 hinge region encoding sequences presented in table. 5.

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In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes a VEGF antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 36. In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes a VEGF antigen binding fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 35. In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86% identical to 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 36, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 35. In certain embodiments, the VEGF antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 with 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. in those areas outside the CDR, which are highlighted in FIG. 8B) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the VEGF antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 36 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (ie, those regions outside the CDRs that are underlined in Fig. 8B) or are substitutions to an amino acid present at that position in light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes a hyperglycosylated bevacizumab Fab comprising a heavy chain and a light chain of SEQ ID NO: 35 and 36, respectively, with one or more of the following mutations: L118N (heavy chain) and/or Q160N or Q160S (light chain) and/or E195N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of bevacizumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 8B, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-VEGF antibody or antigen binding agent thereof fragment.

In certain embodiments, the anti-fD antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of lampalizumab (having amino acid sequences SEQ ID NOs: 37 and 38, respectively, see Table 4 and FIG. 8C). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 137 (encoding the Fab portion of the lampalizumab heavy chain) and SEQ ID NO: 138 (encoding the Fab portion of the lampalizumab light chain ), as indicated in table. 5. Both the heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular one or more human cells forming the retina. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 1, which correspond to proteins secreted by the cells that form the retina.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-integrin antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 37 with an additional hinge region sequence beginning after a C-terminal aspartic acid (D) containing all or part of the amino acid sequence KTHT CPPCPAPELLGGPSVFL (SEQ ID NO: 227 ) and in particular KTHT (SEQ ID NO: 224), KTHL (SEQ ID NO: 223), KTHTCPPCPA (SEQ ID NO: 225), KTHLCPPCPA (SEQ ID NO: 226), KTHTCPPCPAPELLGGPSVFL (SEQ ID NO: 227) or

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KTHLCPPCPAPELLGGPSVFL (SEQ ID NO: 228), as shown in FIG. 8C. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 37 hinge region encoding sequences presented in table. 5.

In certain embodiments, an anti-fD antigen binding fragment transgene encodes an fD antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 38. In certain embodiments, the anti-fD antigen binding fragment transgene encodes an fD antigen binding fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 37. In certain embodiments, the anti-fD antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86% identical 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 38, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 37. In certain embodiments, the fD antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 37 with 1, 2, 3, 4, 5, 6, 7 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. in those areas outside the CDR, which are highlighted in FIG. 8C) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the fD antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 38 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e., those non-CDR regions that are underlined in Fig. 8C) or are substitutions to an amino acid present at that position in the lung chains of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-fD antigen binding fragment transgene encodes a hyperglycosylated lampalizumab Fab containing the heavy chain and light chain of SEQ ID NOs: 37 and 38, respectively, with one or more of the following mutations: L110N (heavy chain), Q160N, or Q160S ( light chain) and/or E195N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-fD antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of lampalizumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 8C, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art, to form a heavy and/or light chain variable domain of the fD antigen or an antigen binding fragment thereof .

In certain embodiments, the anti-VEGF antigen binding fragment transgene comprises nucleotide sequences encoding the brolucizumab heavy and light chain variable domains (having amino acid sequences SEQ ID NOs: 39 and 40, respectively, see Table 4 and FIG. 8D). Brolucizumab is a scFv molecule and thus contains the heavy and light chain variable domains of an anti-VEGF mAb connected by a flexible linker. The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 139 (encoding part of the heavy chain variable domain of brolucizumab) and SEQ ID NO: 142 (encoding part of the variable domain of brolucizumab light chain ), as indicated in table. 5. Even the heavy and light chain variable domains are expressed as separate proteins, each of the heavy and light chain sequences having a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular in one or more cells, forming the retina. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences listed in table. 1, which correspond to proteins secreted by one or more cells forming the retina.

In addition to the heavy and light chain variable domain sequences, transgenes may contain a flexible peptide linker at the C-terminus of the light chain variable domain sequence. The flexible peptide linker sequence may contain flexible residues such as

- 82 047128 glycine (G) or serine (S). In some embodiments, the flexible peptide linker may contain 10-30 residues of either G, S, or both G and S. Charged residues such as E and K may be used and interspersed to enhance solubility. The flexible peptide linker sequence may be characterized by the amino acid sequence (GGGGS) _n , where n may be 1, 2, 3, 4, 5 or 6 (SEQ ID NO: 243). In this case, the signal sequence is fused to the N-terminus of the scFv, the heavy or light chain variable domain sequence, as appropriate.

In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes a VEGF antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 40. In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes a VEGF antigen binding fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 39. In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86% identical to 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 40, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 39. In certain embodiments, the VEGF antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 39 with 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. those areas outside the CDR that are highlighted in FIG. 8D) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the VEGF antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 40 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (ie, those regions outside the CDRs that are underlined in Fig. 8D) or are substitutions to an amino acid present at that position in light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes a hyperglycosylated brolucizumab scFv containing one chain of SEQ ID NOs: 39 and 40, respectively, with the following mutation: L115N (heavy chain) (see FIG. 11A (heavy chain).

In certain embodiments, the anti-VEGF antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of brolucizumab, which are highlighted in the single chain variable domain sequences in FIG. 8D, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-VEGF antibody or antigen binding agent thereof fragment.

Methods of gene therapy.

Methods are provided for treating human subjects with one or more retinal diseases (such as diabetic retinopathy, mCNV, macular degeneration or macular edema) or cancer (such as ovarian epithelial cancer, fallopian tube cancer, abdominal cancer, cervical cancer, metastatic colorectal cancer , metastatic HER2-negative breast cancer, metastatic renal cell carcinoma, glioblastoma, or NSCLC) by introducing a viral vector containing a transgene encoding an anti-VEGF antibody or antigen-binding fragment thereof. The antibody or Fab fragment thereof may be ranibizumab, bevacizumab or brolucizumab. In embodiments, the patient has been diagnosed with and/or has symptoms associated with one or more of the various retinal diseases or cancers listed above.

Also provided are methods of treating human subjects with one or more retinal diseases (such as diabetic retinopathy, mCNV, macular degeneration, or macular edema) by administering a viral vector containing a transgene encoding an anti-fD antibody or an antigen-binding fragment thereof. The antibody may be lampalizumab and is preferably a Fab fragment thereof or another antigen binding fragment thereof. In embodiments, the patient has been diagnosed with a disease and/or has symptoms associated with one or more of the various retinal diseases listed above.

The recombinant vector used to deliver the transgene is described in section 5.4.3. Such centuries

- 83 047128 tori must have tropism for cells such as the human retina and may include non-replicating rAAV, especially those that carry the AAV8 capsid. Alternatively, vectors containing the AAV.7m8 capsid can be used for ophthalmic indications. Recombinant vectors such as those shown in FIG. 8A-8D can be administered by any method so that the recombinant vector enters the retina, preferably by introducing the recombinant vector into the eye. See Section 5.5.3 for details of treatment options. For example, for liver delivery for cancer treatment, the recombinant vector used to deliver the transgene is described in section 5.4.2. Such vectors should have tropism for human liver cells and may include non-replicating rAAVs, especially those carrying the AAV8 or AAV9 capsid. Recombinant vectors such as those shown in FIG. 8A-8C can be administered by any route so that the recombinant vector reaches the liver, preferably by introducing the recombinant vector into the bloodstream. See Section 5.5.2 for details of treatment options.

Subjects to whom such gene therapy is administered may be subjects responding to anti-VEGF or anti-fD therapy. In specific embodiments, the methods include treating patients who have been diagnosed with or are considered to have responded to treatment with an anti-VEGF antibody or an anti-fD antibody or who have one or more symptoms associated with one or more retinal disorders or types of cancer. good candidates for anti-VEGF or anti-fD therapy. In specific embodiments, the patients have previously been treated with ranibizumab, bevacizumab, lampalizumab, or brolucizumab and have been found to be sensitive to ranibizumab, bevacizumab, lampalizumab, or brolucizumab. To determine sensitivity, a transgene product of an anti-VEGF or anti-fD antibody or antigen binding fragment (eg, produced in cell culture, bioreactors, etc.) can be administered directly to the subject.

Post-translationally modified human antibodies.

Production of an anti-VEGF or anti-fD HuPTM mAb or HuPTM Fab should result in a biologically improved molecule for the treatment of one or more retinal diseases or cancers through gene therapy - for example, by introducing a viral vector or other DNA expression construct encoding anti- VEGF or anti-fD HuPTM Fab, subretinal, intravitreal or suprachoroidal to human subjects diagnosed with or having one or more symptoms of one or more retinal disorders, or by administration of a viral vector or other DNA expression construct encoding an anti-VEGF HuPTM Fab, subcutaneously, intramuscularly, or intravenously to human subjects (patients) diagnosed with cancer to establish a permanent depot in the retina or liver that continuously supplies the fully human post-translationally modified, e.g., human glycosylated, sulfated transgene product produced by the transduced retinal or liver cells.

As an alternative or complementary treatment to gene therapy, HuPTM anti-VEGF or anti-fD mAbs or HuPTM Fab can be generated in human cell lines using recombinant DNA technology and administered to patients diagnosed with retinal disease or cancer for which therapy for the retinal disorder or cancer is considered appropriate.

In specific embodiments, the HuPTM anti-VEGF mAb or antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the ranibizumab heavy and light chains, as shown in FIG. 8A (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions Q115 and/or N165 of the heavy chain (SEQ ID NO: 33) or Q100, N158 and/or N210 of the light chain (SEQ ID NO: 34). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the ranibizumab heavy and light chain variable domain sequences contains a sulfation group at Y94 and/or Y95 of the heavy chain (SEQ ID NO: 33) and/or Y86 and/or Y87 of the light chain (SEQ ID NO: 34). In other embodiments, the HuPTM VEGF mAb or antigen binding fragment thereof does not contain detectable NeuGc fragments and/or does not contain detectable alpha-Gal fragments.

In specific embodiments, the HuPTM anti-VEGF mAb or antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the bevacizumab heavy and light chains, as shown in FIG. 8B (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions Q115 and/or N165 of the heavy chain (SEQ ID NO: 35) or Q100, N158 and/or N210 of the light chain (SEQ ID NO: 36). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the bevacizumab heavy and light chain variable domain sequences contains a sulfation group at Y94 and/or Y95 of the heavy chain (SEQ ID NO: 35) and/or Y86 and/or Y87 of the light chain (SEQ

- 84 047128

ID NO: 36). In other embodiments, the HuPTM VEGF mAb or antigen binding fragment thereof does not contain detectable NeuGc fragments and/or does not contain detectable alpha-Gal fragments.

In specific embodiments, the HuPTM mAb to fD or an antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the lampalizumab heavy and light chains, as shown in FIG. 8C (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions Q107 and/or N157 of the heavy chain (SEQ ID NO: 37) or Q100 and/or N158 and/or N210 of the light chain (SEQ ID NO: 38). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the lampalizumab heavy and light chain variable domain sequences contains a sulfation group at Y60 and/or Y94 and/or Y95 of the heavy chain (SEQ ID NO: 37) and/or Y86 and/or Y87 of the light chain circuits (SEQ ID NO: 38). In other embodiments, the HuPTM mAb to fD or an antigen binding fragment thereof does not contain detectable NeuGc fragments and/or does not contain detectable alpha-Gal fragments.

In specific embodiments, the HuPTM VEGF mAb or antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the brolucizumab heavy and light chain variable domains as shown in FIG. 8D (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions N77 and/or Q112 of the heavy chain (SEQ ID NO: 39) or N97 and/or Q103 of the light chain (SEQ ID NO: 40). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the brolucizumab heavy and light chain variable domain sequences contains a sulfation group at Y32 and/or Y33 and/or Y34 and/or Y59 and/or Y60 and/or Y94 and /or Y95 heavy chain (SEQ ID NO: 39) and/or Y86 and/or Y87 light chain (SEQ ID NO: 40). In other embodiments, the HuPTM anti-VEGF mAb or antigen binding fragment thereof does not contain detectable NeuGc fragments and/or does not contain detectable alpha-Gal fragments.

In some embodiments, the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1%, or 2% glycosylated and/or sulfated, and may be at least 5%, 10%, or even 50% or 100% glycosylated. % glycosylated and/or sulfated. The goal of treatment of the gene therapy proposed herein is to slow or stop the progression of a retinal disease or type of cancer and/or suppress angiogenesis. In the case of retinal disorders, effectiveness can be monitored by monitoring visual acuity. For example, effectiveness can be monitored by assessing change in visual acuity from baseline. For cancer, efficacy can be monitored by assessing one or more oncologic endpoints, including survival, progression-free survival, time to progression, time to treatment failure, event-free survival, time to next treatment, objective response rate, or duration of response. (See, for example, US Department of Health and Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research. Guidance for Industry: Clinical Trial Endpoints for the Approval of Drugs and Cancer Treatments. https://www.fda.gov/downloads/Drugs/Guidances/ucm071590.pdf. Accessed October 13, 2017; Oncology Endpoints in a Changing Landscape. 2016; 12).

Combinations of delivery of an anti-VEGF or anti-fD HuPTM mAb or an antigen-binding fragment thereof to the retina or liver, accompanied by delivery of other available treatments, are covered by the methods presented herein. Additional treatments may be given before, simultaneously, or after gene therapy treatment. Available treatments for diabetic retinopathy, mCNV, macular degeneration, or macular edema that may be combined with the gene therapy provided herein include, but are not limited to, laser photocoagulation, photodynamic therapy with verteporfin, aflibercept, and/or intravitreal steroids, and administration of anti-inflammatory drugs. VEGF or anti-fD agents, including, but not limited to, ranibizumab, bevacizumab, lampalizumab or brolucizumab. Available treatments for ovarian epithelial cancer, fallopian tube cancer, abdominal cancer, cervical cancer, metastatic colorectal cancer, metastatic HER2-negative breast cancer, metastatic renal cell carcinoma, glioblastoma or NSCLC that can be combined with the gene presented herein therapies include, but are not limited to, chemotherapy (eg, cisplatin, gemcitabine, pemetrexed, 5-fluorouracil, carboplatin, irinotecan, interferon alfa, oxaliplatin, pegylated paclitaxel, liposomal doxorubicin and/or topotecan), chemotherapeutic protective drugs (eg, leucovorin ), radiation therapy, cryotherapy, targeted small molecule therapy, other antibodies, afilbercept and/or vaccine therapy, and anti-VEGF administration, including, but not limited to, ranibizumab or bevacizumab.

5.3.11. Anti-BLyS HuPTM designs and formulations for systemic lupus erythematosus

Compositions and methods are described for the delivery of HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fab, which bind to B-lymphocyte stimulator (BLyS) derived from an anti-BLyS antibody, such as belimumab (Fig. 8E) and are indicated for the treatment of systemic lupus erythematosus (SLE) and decreased levels of autoreactive B cells and plasma immunoglobulin-producing cells. In certain embodiments, the HuPTM mAb is characterized by the amino acid sequence of belimumab or an antigen binding fragment thereof. The amino acid sequence of the Fab fragment of this antibody is shown in FIG. 8E. Delivery may be accomplished by gene therapy, e.g., by administering a viral vector or other DNA expression construct encoding the BLyS-binding mAb HuPTM (or an antigen-binding fragment thereof and/or a hyperglycosylated derivative or other derivative thereof) to human subjects diagnosed with SLE to create a permanent depot that continuously supplies human PTM, eg human glycosylated, transgenic product.

Transgenes.

Recombinant vectors are provided containing a transgene encoding a HuPTM mAb or a HuPTM Fab (or other antigen binding fragment of a HuPTM mAb) that binds to BLyS, which can be administered to deliver the HuPTM mAb or antigen binding fragment to a patient. A transgene is a nucleic acid containing nucleotide sequences encoding an antigen binding fragment of an antibody that binds to BLyS, such as belimumab or variants thereof, as described in detail herein. The transgene may also encode an anti-BLyS antigen-binding fragment that contains additional glycosylation sites (eg, see Courtois et al.).

In some embodiments, the anti-BLyS antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of belimumab (having amino acid sequences SEQ ID NOs: 41 and 42, respectively, see Table 4 and FIG. 8E).

The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 141 (encoding the Fab portion of the belimumab heavy chain) and SEQ ID NO: 142 (encoding the Fab portion of the belimumab light chain ), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular liver cells (eg, hepatocytes) or human muscle cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-BLyS antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 41 with an additional hinge region sequence beginning after the C-terminal aspartate (D) containing all or part of the amino acid sequence KTHTCPPCPAPELLGG (SEQ ID NO: 222) and specifically, KTHL (SEQ ID NO: 223), KTHT (SEQ ID NO: 224), KTHTCPPCPA (SEQ ID NO: 225), KTHLCPPCPA (SEQ ID NO: 226), KTHTCPPCPAPELLGGPSVFL (SEQ ID NO: 227) or KTHLCPPCPAPELLGGPSVFL (SEQ ID NO: 228) as shown in FIG. 8E. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 41 hinge region coding sequences presented in table. 5.

In certain embodiments, the anti-BLyS antigen binding fragment transgene encodes a BLyS antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 42. In certain embodiments, the anti-BLyS antigen binding fragment transgene encodes a BLyS antigen binding fragment containing severe a chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 41. In certain embodiments, the anti-BLyS antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO:

- 86 047128

42, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 41. In certain embodiments, the BLyS antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 41 with 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. those regions that are outside the CDRs that are underlined in FIG. 8E) or are substitutions to an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the BLyS antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 42 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e., those non-CDR regions that are underlined in Fig. 8E) or are substitutions to an amino acid present at that position in the lung chains of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-BLyS antigen binding fragment transgene encodes a hyperglycosylated belimumab Fab containing a heavy chain and a light chain of SEQ ID NOs: 41 and 42, respectively, with one or more of the following mutations: Ml 18N (heavy chain) and/or Q196N ( light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-BLyS antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of belimumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 8E, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen-binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-BLyS antibody or antigen-binding antibody thereof fragment.

Methods of gene therapy.

Methods are provided for treating people with SLE by administering a viral vector containing a transgene encoding an anti-BLyS antibody or an antigen-binding fragment thereof. The antibody may be belimumab and is preferably a Fab fragment thereof or another antigen binding fragment thereof. In embodiments, the patient has been diagnosed with SLE and/or has symptoms associated with SLE. Recombinant vectors used for transgene delivery are described in section 5.4.2. Such vectors should have tropism for human liver or muscle cells and may include non-replicating rAAVs, especially those carrying the AAV8 or AAV9 capsid. Recombinant vectors such as those shown in FIG. 8E can be administered by any route so that the recombinant vector reaches the liver or muscle tissue, preferably by introducing the recombinant vector into the bloodstream. See Section 5.5.2 for details of treatment options.

Subjects to receive such gene therapy may be those who respond to anti-BLyS therapy. In specific embodiments, the methods include treating patients who have been diagnosed with or have one or more symptoms associated with SLE and who are determined to respond to anti-BLyS antibody treatment or are considered good candidates for anti-BLyS antibody therapy. In specific embodiments, the patients have previously been treated with belimumab and have been found to be sensitive to belimumab. To determine sensitivity, a transgene product of an anti-BLyS antibody or antigen-binding fragment (eg, produced in cell culture, bioreactors, etc.) can be administered directly to the subject.

Post-translationally modified human antibodies

Production of the HuPTM anti-BLyS mAb or HuPTM Fab should result in a biologically enhanced molecule for the treatment of SLE via gene therapy - for example, by administering a viral vector or other DNA expression construct encoding the HuPTM Anti-BLyS Fab intravenously to human subjects (patients) with a diagnosis or presence of one or more symptoms of SLE, to establish a permanent depot in the liver or muscle tissue that continuously supplies a fully human post-translationally modified, e.g., human glycosylated, sulfated transgene product produced by transduced liver or muscle cells.

The cDNA design for HuPTMmAb to BLyS or HuPTM Fab to BLyS must include a signal peptide that ensures proper co- and post-translational processing (protein glycosylation and sulfation) by transduced liver or muscle cells. For example, the signal sequence may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3,

- 87 047128 which correspond to proteins secreted by myocytes or hepatocytes, respectively.

As an alternative or complementary treatment to gene therapy,

HuPTM mAb to BLyS or HuPTM Fab can be produced in human cell lines using recombinant DNA technology and administered to patients diagnosed with SLE or for whom SLE therapy is considered appropriate.

In specific embodiments, the HuPTM mAb to BLyS or an antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the belimumab heavy and light chains, as shown in FIG. 8E (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions N30 and/or N63 and/or N165 of the heavy chain (SEQ ID NO: 41) or N68 and/or N95 of the light chain (SEQ ID NO: 42). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the belimumab heavy and light chain variable domain sequences contains a sulfation group at Y94 and/or Y95 of the heavy chain (SEQ ID NO: 41) and/or Y85 and/or Y86 of the light chain (SEQ ID NO: 42). In other embodiments, the HuPTM mAb to BLyS or an antigen binding fragment thereof does not contain detectable NeuGc fragments and/or does not contain detectable alpha-Gal fragments.

In certain embodiments, the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1%, or 2% glycosylated and/or sulfated, and may be at least 5%, 10%, or even 50% or 100% glycosylated. % glycosylated and/or sulfated. The goal of treatment with the gene therapy presented herein is to slow or stop the progression of SLE, reduce levels of pain or discomfort for the patient, or reduce the content of autoreactive B cells and immunoglobulin-producing plasma cells. Effectiveness may be monitored by assessing the function, symptoms, or degree of inflammation in the affected tissue or area of the body, such as the skin, joints, kidneys, lungs, blood cells, heart, and brain. For example, effectiveness may be monitored by monitoring the presence, extent or frequency of one or more symptoms, including seizures, psychosis, organic brain syndrome, visual impairment, other neurological problems, alopecia, skin rash, muscle weakness, arthritis, inflammation of the blood vessels, ulcers mucous membranes, chest pain, aggravated by deep breathing, and manifestations of pleurisy and/or pericarditis and fever. Standardized disease indices can be used, such as the Systemic Lupus Erythematosus Study National Study of Estrogen Safety in Systemic Lupus Erythematosus Activity Index, the British Systemic Lupus Erythematosus Study Group (BILAG) Lupus Erythematosus Erythematosus Activity Index (SELENA-SLEDAI) A, BILAG B, Activity Index systemic lupus erythematosus (SLAM) or PGA assessment. (See, for example, Liang MH et al. (1988) Measurement of systemic lupus erythematosus activity in clinical research, Arthritis Rheum. 31:817-25; Diaz et al. (2011) Measures of adult systemic lupus erythematosus: updated version of British Isles Lupus Assessment Group (BILAG 2004), European Consensus Lupus Activity Measurements (ECLAM), Systemic Lupus Activity Measure, Revised (SLAM-R), Systemic Lupus Activity Questionnaire for Population Studies (SLAQ), Systemic Lupus Erythematosus Disease Activity Index 2000 ( SLEDAI-2 K), and Systemic Lupus, International Collaborating Clinics/American College of Rheumatology Damage Index (SDI) Arthritis Care Res 63:S37-46).

Combinations of delivery of HuPTM mAb to BLyS or an antigen-binding fragment thereof to the liver or muscle, accompanied by delivery of other available treatments, are covered by the methods presented herein. Additional treatments may be given before, at the same time, or after gene therapy treatment. Available treatments for SLE that can be combined with gene therapy presented herein include, but are not limited to, corticosteroids, antimalarials, NSAIDs and immunosuppressants, as well as administration with anti-BLyS agents, including but not limited to belimumab.

5.3.12. Anti-CP-C5 HuPTM designs and formulations for paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome

Compositions and methods are described for delivery of HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fab, that bind to complementary protein C5 (or C5a) (CP-C5) derived from an anti-CP-C5 antibody, such as eculizumab (Figure 8F) , and are indicated for the treatment of paroxysmal nocturnal hemoglobinuria (PNH), the treatment of atypical hemolytic uremic syndrome (aHUS), reducing the destruction of blood cells and/or reducing the need for blood transfusions. In certain embodiments, the HuPTM mAb is characterized by the amino acid sequence of eculizumab or an antigen binding fragment thereof. The amino acid sequence of the Fab fragment of this antibody is shown in FIG. 8F. Delivery may be accomplished by gene therapy - for example, by administering a viral vector or other DNA expression construct encoding a CP-C5 binding HuPTM mAb (or an antigen binding fragment thereof and/or a hyperglycosylated derivative or other derivative thereof) to human subjects.

- 88 047128 diagnosed with PNH or aHUS to create a permanent depot that continuously supplies human

RTM, for example, glycosylated human, transgenic product.

Transgenes.

Recombinant vectors are provided containing a transgene encoding a HuPTM mAb or a HuPTM Fab (or other antigen binding fragment of a HuPTM mAb) that binds to CP-C5, which can be administered to deliver the HuPTM mAb or antigen binding fragment to a patient. A transgene is a nucleic acid containing nucleotide sequences encoding an antigen binding fragment of an antibody that binds to CP-C5, such as eculizumab or variants thereof, as described in detail herein. The transgene may also encode an anti-CP-C5 antigen-binding fragment that contains additional glycosylation sites (eg, see Courtois et al.).

In some embodiments, the anti-CP-C5 antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the eculizumab Fab portion (having amino acid sequences SEQ ID NOs: 43 and 44, respectively, see Table 4 and FIG. 8F). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 143 (encoding the Fab portion of the eculizumab heavy chain) and SEQ ID NO: 144 (encoding the Fab portion of the eculizumab light chain ), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular human liver cells (eg, hepatocytes). The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences listed in table. 3, which correspond to proteins secreted by hepatocytes.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-CP-C5 antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 43 with an additional hinge region sequence beginning after the C-terminal glutamic acid (E) containing all or part of the amino acid sequence CPPCPAPPVAGG (SEQ ID NO: 232) and in particular CPPCPA (SEQ ID NO: 219) or CPPCPAPPVAG (SEQ ID NO: 233), as shown in FIG. 8F. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 43 hinge region encoding sequences presented in table. 5.

In certain embodiments, the anti-CP-C5 antigen binding fragment transgene encodes a CP-C5 antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 44. In certain embodiments, the anti-CP-C5 antigen binding fragment transgene encodes a CP-C5 antigen binding fragment containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 43. In certain embodiments, the anti-CP-C5 antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence, which is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequence set forth in SEQ ID NO: 44, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 43. In certain embodiments, the CP-C5 antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 43 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions ( those. in those areas outside the CDR, which are highlighted in FIG. 8F) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the CP-C5 antigen binding fragment comprises a light chain comprising the amino acid sequence SEQ ID NO: 44 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (ie, those non-CDR regions that are underlined in Fig. 8F) or are substitutions to an amino acid present at that position in the light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-CP-C5 antigen-binding transgene

- 89 047128 fragment encodes a hyperglycosylated eculizumab Fab containing a heavy chain and a light chain from SEQ ID NO: 43 and 44, respectively, with one or more of the following mutations: L117N (heavy chain), Q160N or Q160S (light chain) and/or E195N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-CP-C5 antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six eculizumab CDRs that are highlighted in the heavy and light chain variable domain sequences in FIG. 8F, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-CP-C5 antibody or its antigen-binding fragment.

Methods of gene therapy.

Methods are provided for treating human subjects with PNH or aHUS by administering a viral vector containing a transgene encoding an anti-CP-C5 antibody or an antigen-binding fragment thereof. The antibody may be eculizumab and is preferably a Fab fragment thereof or another antigen binding fragment thereof. In embodiments, the patient has been diagnosed with a disease and/or has symptoms associated with PNH or aHUS. Recombinant vectors used for transgene delivery are described in section 5.4.2. Such vectors should have tropism for human liver cells and may include non-replicating rAAVs, especially those carrying the AAV8 or AAV9 capsid. Recombinant vectors such as those shown in FIG. 8F can be administered by any route so that the recombinant vector reaches the liver, preferably by introducing the recombinant vector into the bloodstream. See Section 5.5.2 for details of treatment options.

Subjects to receive such gene therapy may be those who respond to anti-CPC5 therapy. In specific embodiments, the methods include treating patients who have been diagnosed with PNH or aHUS or who have one or more symptoms associated therewith and who are determined to respond to treatment with an anti-CP-C5 antibody or are considered good candidates for anti-CP-C5 antibody therapy. In specific embodiments, the patients have previously been treated with eculizumab and have been found to be sensitive to eculizumab. To determine sensitivity, a transgene product of an anti-CP-C5 antibody or antigen binding fragment (eg, produced in cell culture, bioreactors, etc.) can be administered directly to the subject.

Post-translationally modified human antibodies.

Production of a HuPTM mAb to CP-C5 or a HuPTM Fab should result in a biologically improved molecule for the treatment of PNH or aHUS through gene therapy, e.g., by administering a viral vector or other DNA expression construct encoding a HuPTM Fab to CP-C5 intravenously to subjects -in humans (patients) diagnosed with or presenting with one or more symptoms of PNH or aHUS, to establish a permanent depot in liver tissue that continuously supplies a fully human post-translationally modified, e.g., human glycosylated, sulfated transgene product produced by transduced liver cells.

The cDNA design for HuPTMmAb to CP-C5 or HuPTM Fab to CP-C5 should include a signal peptide that ensures proper co- and post-translational processing (protein glycosylation and sulfation) by transduced liver cells. For example, the signal sequence may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences listed in table. 3, which correspond to proteins secreted by hepatocytes.

As an alternative or complementary to gene therapy treatment, HuPTM mAb to CP-C5 or HuPTM Fab can be generated in human cell lines using recombinant DNA technology and administered to patients diagnosed with PNH or aHUS or those for whom therapy for PNH or aHUS is considered expedient.

In specific embodiments, the HuPTM mAb to CP-C5 or an antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the eculizumab heavy and light chain Fab portions as shown in FIG. 8F (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions N63 and/or Q114 and/or N164 and/or N197 and/or N206 of the heavy chain (SEQ ID NO: 43) or N28 and/or Q100 and/or N158 and/or N210 light chain (SEQ ID NO: 44). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with eculizumab heavy and light chain variable domain sequences contains a sulfation group at Y94 and/or Y95 of the heavy chain

- 90 047128 (SEQ ID NO: 43) and/or Y86 and/or Y87 light chain (SEQ ID NO: 44). In other embodiments, the anti-CP-C5 HuPTM mAb or antigen binding fragment thereof does not contain any detectable NeuGc fragments and/or does not contain any detectable alpha-Gal fragments.

In certain embodiments, the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1%, or 2% glycosylated and/or sulfated, and may be at least 5%, 10%, or even 50% or 100% glycosylated. % glycosylated and/or sulfated. The goal of treatment for the gene therapy presented here is to slow or stop the progression of PNH or aHUS, reduce the need for blood transfusions, or reduce the destruction of red blood cells. Efficacy may be monitored by measuring stabilization of hemoglobin and/or number of red blood cell units transfused or assessing the level of fatigue and/or health-related quality of life over the course of treatment.

Combinations of delivery of HuPTM mAb to CP-C5 or an antigen-binding fragment thereof to the liver accompanied by delivery of other available treatments are covered by the methods presented herein. Additional treatments may be given before, at the same time, or after gene therapy treatment. Available treatments for PNH or aHUS that can be combined with the gene therapy provided herein include, but are not limited to, treatment with anticoagulants and steroids/immunosuppressants and administration of anti-CP-C5 agents, including but not limited to eculizumab.

5.3.13. Anti-MMP9 HuPTM constructs and formulations for ocular disorders, cystic fibrosis, rheumatoid arthritis, inflammatory bowel disease and cancer.

Compositions and methods are described for delivery of HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fab, that bind to anti-MMP9-derived matrix metalloproteinase 9 (MMP9) indicated for the treatment of one or more retinal diseases, including macular degeneration (eg , dry age-related macular degeneration (AMD), cystic fibrosis (CF), rheumatoid arthritis (RA), IBD (eg, UC and CD), and one or more types of cancer (eg, solid tumors, pancreatic adenocarcinoma, lung adenocarcinoma, squamous cell lung carcinoma, esophagogastric adenocarcinoma, gastric cancer, colorectal cancer or breast cancer) or to inhibit extracellular matrix degradation. In specific embodiments, the HuPTM mAb is characterized by the amino acid sequence of andecaliximab or an antigen binding fragment thereof. The amino acid sequence of andecaliximab Fab fragments is shown in FIG. 8G. Delivery can be accomplished by gene therapy - for example, by administering a viral vector or other DNA expression construct encoding an MMP9-binding HuPTM mAb (or an antigen-binding fragment thereof and/or a hyperglycosylated derivative or other derivative thereof) to human patients with diagnosis or presence of one or more symptoms of a retinal disorder (such as macular degeneration), RA, CF, IBD (such as UC or CD) or one or more cancers (such as those listed above) to create a permanent depot that continuously supplies human RTM , for example, human glycosylated, transgenic product.

Provided are recombinant vectors containing a transgene encoding a HuPTM mAb or a HuPTM Fab (or other antigen binding fragment of a HuPTM mAb) that binds to MMP9, which can be administered to deliver the HuPTM mAb or antigen binding fragment to a patient. A transgene is a nucleic acid containing nucleotide sequences encoding an antigen binding fragment of an antibody that binds to MMP9, such as andecaliximab or variants thereof, as described in detail herein. The transgene may also encode an anti-MMP9 antigen-binding fragment that contains additional glycosylation sites (eg, see Courtois et al.).

In certain embodiments, the anti-MMP9 antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of andecaliximab (having amino acid sequences SEQ ID NOs: 45 and 46, respectively, see Table 4 and FIG. 8G). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 145 (encoding the Fab portion of the andecaliximab heavy chain) and SEQ ID NO: 146 (encoding the Fab portion of the andecaliximab light chain ), as indicated in table. 5. In the case of treating eye diseases, the heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular one or more cells forming the retina. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences listed in table. 1, which correspond to proteins secreted by one or more cells forming the retina. For the treatment of non-ocular diseases, the heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, particularly liver cells (eg, hepatocytes) or human muscle cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID

NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table.

or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-MMP9 antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 45 with an additional hinge region sequence beginning after the C-terminal aspartic acid (D) containing all or part of the amino acid sequence GPPCPPCPAPEFLGG (SEQ ID NO: 231) and in particular GPPCPPCPA (SEQ ID NO: 229) or GPPCPPCPAPEFLGGPSVFL (SEQ ID NO: 230) as shown in FIG. 8G. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 45 hinge region encoding sequences presented in table. 5.

In certain embodiments, the anti-MMP9 antigen binding fragment transgene encodes an MMP9 antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 46. In certain embodiments, the anti-MMP9 antigen binding fragment transgene encodes an MMP9 antigen binding fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 45. In certain embodiments, the anti-MMP9 antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86% identical to 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 46, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 45. In certain embodiments, the MMP9 antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 45 with 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. those areas outside the CDR that are highlighted in FIG. 8G) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the MMP9 antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 46 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (ie, those regions outside the CDRs that are underlined in Fig. 8G) or are substitutions to an amino acid present at that position in light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-MMP9 antigen binding fragment transgene encodes a hyperglycosylated andecaliximab Fab containing a heavy chain and a light chain of SEQ ID NOs: 45 and 46, respectively, with one or more of the following mutations: L110N (heavy chain), Q160N, or Q160S (light chain) and/or E195N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-MMP9 antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of andecaliximab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 8G, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art, to form a heavy and/or light chain variable domain of an MMP9 antibody or an antigen binding fragment thereof .

Methods of gene therapy.

Methods are provided for treating human subjects with one or more retinal diseases (such as macular degeneration), IBD, CF, RA, or cancer by administering a viral vector containing a transgene encoding an anti-MMP9 antibody or an antigen-binding fragment thereof. The antibody may be andecaliximab and is preferably a Fab fragment thereof or another antigen binding fragment thereof. In embodiments, the patient has been diagnosed with a disease and/or has symptoms associated with one or more retinal diseases, IBD, CF, RA, or forms of cancer.

- 92 047128

The recombinant vector used to deliver the transgene is described in section 5.4.3. Such vectors should have tropism for human retinal-type cells and may include non-replicating rAAV, especially those carrying the AAV8 capsid. Recombinant vectors such as those shown in FIG. 8G can be administered by any method such that the recombinant vector enters the retina, preferably by introducing the recombinant vector into the eye. See Section 5.5.3 for details of treatment options. For example, for liver delivery for cancer treatment, the recombinant vector used to deliver the transgene is described in section 5.4.2.

Such vectors should have tropism for human liver cells and may include non-replicating rAAVs, especially those carrying the AAV8 or AAV9 capsid. Recombinant vectors such as those shown in FIG. 8G may be administered by any route such that the recombinant vector reaches the liver, preferably by introducing the recombinant vector into the bloodstream. See Section 5.5.2 for details of treatment options.

Subjects to receive such gene therapy may be those who respond to anti-MMP9 therapy. In specific embodiments, the methods include treating patients who have been diagnosed with one or more retinal disorders or types of cancer, or who have one or more symptoms associated therewith, and who are determined to respond to anti-MMP9 antibody treatment or are considered good candidates for anti-MMP9 antibody therapy. MMP9. In specific embodiments, the patients have previously been treated with andecaliximab and have been found to be sensitive to andecaliximab. To determine sensitivity, a transgene product of an anti-MMP9 antibody or antigen binding fragment (eg, produced in cell culture, bioreactors, etc.) can be administered directly to the subject.

Post-translationally modified human antibodies.

The production of a HuPTM mAb to MMP9 or a HuPTM Fab should result in a biologically improved molecule for the treatment of one or more retinal diseases, CF, RA, IBD or forms of cancer administered by gene therapy - for example, by introducing a viral vector or other DNA expression construct, encoding HuPTM Fab to MMP9, subretinal, intravitreal or suprachoroidal to human subjects (patients) diagnosed with or having one or more symptoms of one or more retinal disorders, or by administering a viral vector or other DNA expression construct encoding HuPTM Fab to MMP9, subcutaneously, intramuscularly or intravenously in human subjects (patients) diagnosed with RA, CF, IBD, or cancer to establish a permanent retinal or liver depot that continuously supplies fully human post-translationally modified, e.g., human glycosylated, sulfated transgene product produced by transduced retinal cells or liver.

The cDNA design for an anti-MMP9 HuPTMmAb or an anti-MMP9 HuPTM Fab must include a signal peptide that ensures proper co- and post-translational processing (protein glycosylation and sulfation) by transduced retinal or liver cells. For example, the signal sequence may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences listed in table. 1 or 3, which correspond to proteins secreted by retinal or liver cells, respectively.

As an alternative or complementary to gene therapy treatment, HuPTM mAb to MMP9 or HuPTM Fab can be produced in human cell lines using recombinant DNA technology and administered to patients diagnosed with retinal disease or cancer who are undergoing therapy for retinal disorders, IBD, CF, RA or cancer is considered suitable.

In specific embodiments, the HuPTM mAb to MMP9 or an antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the heavy and light chains of andecaliximab, as shown in FIG. 8G (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions N58, N76, Q107, N157 and/or N199 of the heavy chain (SEQ ID NO: 45) or N158 and/or N210 of the light chain (SEQ ID NO: 46). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with the variable domain sequences of the heavy and light chain of andecaliximab contains a sulfation group at Y93 and/or Y94 of the heavy chain (SEQ ID NO: 45) and/or Y86 and/or Y87 of the light chain (SEQ ID NO: 46). In other embodiments, the HuPTM mAb to MMP9 or antigen binding fragment thereof does not contain detectable NeuGc fragments and/or does not contain detectable alpha-Gal fragments.

In some embodiments, the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1%, or 2% glycosylated and/or sulfated, and may be at least 5%, 10%, or even 50% or 100% glycosylated. glycosylated and/or sulfated. The goal of treatment with gene therapy presented herein is to slow or stop the progression of the disease being treated, or to attenuate one or more

- 93 047128 its symptoms. In the case of retinal disorders, effectiveness can be monitored by monitoring visual acuity. For example, effectiveness can be monitored by assessing changes in visual acuity from baseline. For cancer, efficacy can be monitored by assessing one or more oncologic endpoints, including overall survival, progression-free survival, time to progression, time to treatment failure, event-free survival, time to next treatment, objective response rate, or duration response (see, e.g., U.S. Department of Health and Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research. Guidance for Industry: Clinical Trial Endpoints for the Approval of Drugs and Cancer Treatments . https://www.fda.gov/downloads/Drugs/Guidances/ucm071590.pdf. Accessed October 13, 2017; -12). For RA, effectiveness can be monitored by assessing one or more of (1) number of swollen joints, (2) number of tender joints, (3) physician's global assessment of disease activity, (4) patient self-report of functional status, (5) patient self-report pain, (6) a global assessment of the patient's disease activity, (7) laboratory measurements of CRP and erythrocyte sedimentation rate, and (8) radiographic progression. (See, for example, Smolen JS, Aletaha D. Assessment of rheumatoid arthritis activity in clinical trials and clinical practice UptoDate.com Wolters Kluwer Health. Available at: www.uptodate.com, December 2017) For example, regarding CD effectiveness can be monitored by assessing the Crohn's disease activity index [CDAI] over the course of treatment (eg, see Best WR et al. (1976) Gastroenterology, Mar;70(3):439-44, Development of a Crohn's disease activity index. National Cooperative Crohn's Disease Study.). For UC, effectiveness can be monitored by assessing Mayo Endoscopic Score and Mayo Endoscopic Score over the course of treatment (eg, see Lobaton et al., The Modified Mayo Endoscopic Score (MMES): A New Index for the Assessment of Extension and Severity of Endoscopic Activity in Ulcerative Colitis Patients, J. Crohns Colitis 2015 Oct:9(10):846-52). For MB, effectiveness can be monitored by assessing forced expiratory volume in 1 s (FEV1), reduced incidence of pulmonary exacerbations, improved quality of life (QoL), and, for younger patients, improved growth (eg, see VanDevanter and Konstan, Outcome measurement for clinical trials assessing treatment of cystic fibrosis lung disease, Clin. Investig. 2(2): 163-175 (2012).

Combinations of delivery of HuPTM mAb to MMP9 or an antigen binding fragment thereof to the retina or liver, accompanied by delivery of other available treatments, are covered by the methods presented herein.

Additional treatments may be given before, simultaneously, or after gene therapy treatment. Available treatments for macular degeneration that can be combined with the gene therapy provided herein include, but are not limited to, laser photocoagulation, photodynamic therapy with verteporfin, aflibercept and/or intravitreal steroids, and administration of anti-MMP9 agents, including but not limited to , andecaliximab. Available treatments for one or more of the cancers listed above that can be combined with gene therapy provided herein include, but are not limited to, chemotherapy (e.g., cisplatin, gemcitabine, pemetrexed, 5-fluorouracil, carboplatin, irinotecan, interferon alfa, oxaliplatin, paclitaxel pegylated liposomal doxorubicin and/or topotecan), chemotherapeutic protective drugs (eg, leucovorin), radiation therapy, cryotherapy, targeted small molecule therapy, other antibodies, afilbercept and/or vaccine therapy and anti-MMP9 administration, including, without limitation, andecaliximab. Available treatments for RA that can be combined with gene therapy presented herein include, but are not limited to, bisphosphonates, non-steroidal anti-inflammatory drugs (eg, celecoxib, naproxen, aspirin, indomethacin, sulfasalazine and ketoprofen), steroids (eg, prednisone) , disease-modifying antirheumatic drugs and other immunosuppressants (eg, leflunomide, methotrexate, tofactinib, azathioprine, mycophenolate, cyclosphamide, cyclosporine), hydroxychloroquine, abatacept, anakinra, apremilast, TNF inhibitors, other antibodies (eg, tocilizumab, secukinimab, rituximab) and administration of anti-MMP9, including, without limitation, andecaliximab. Available treatments for IBD that can be combined with the gene therapy presented herein include, but are not limited to, non-steroidal anti-inflammatory drugs (eg, mesalamine, sulfasalazine), steroids (eg, hydrocortisone, prednisone, budesonide), immunosuppressants (eg, methotrexate , mercaptopurine, azathioprine), vitamins (eg, iron, cholecalciferol), antibiotics (eg, aminosalicylic acid, metronidazole), other antibodies (eg, infliximab, adalimumab) and anti-MMP9 administration, including, but not limited to, andecaliximab. Available treatments for CF that can be combined with the gene therapy presented herein include, but are not limited to, antibiotics, vaccines and cough medications (eg, acetylcysteine and Dornas alfa) and anti-MMP9 administration, including but not limited to , andecaliximab.

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5.3.14. Anti-pKal HuPTM designs and formulations for angioedema

Compositions and methods are described for the delivery of HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fab, which bind to kallikrein (pKal), derived from an anti-pKal antibody, and are indicated for the treatment of angioedema, such as hereditary angioedema. In specific embodiments, the HuPTM mAb is characterized by the amino acid sequence of lanadelumab or an antigen binding fragment thereof. The amino acid sequence of the Fab fragment of this antibody is shown in FIG. 8N. Delivery may be accomplished by gene therapy, for example, by administering a viral vector or other DNA expression construct encoding a pKal binding HuPTM mAb (or an antigen binding fragment thereof and/or a hyperglycosylated derivative or other derivative thereof) to human patients. diagnosed with angioedema, to create a permanent depot that continuously supplies human PTM, for example, glycosylated human, transgenic product.

Transgenes.

Recombinant vectors are provided containing a transgene encoding a HuPTM mAb or a HuPTM Fab (or other antigen binding fragment of a HuPTM mAb) that binds to pKal, which can be administered to deliver the HuPTM mAb or antigen binding fragment to a patient. A transgene is a nucleic acid containing nucleotide sequences encoding an antigen binding fragment of an antibody that binds to pKal, such as lanadelumab or variants thereof, as described in detail herein. The transgene may also encode an anti-pKal antigen-binding fragment that contains additional glycosylation sites (eg, see Courtois et al.).

In certain embodiments, the anti-pKal antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of lanadelumab (having amino acid sequences SEQ ID NOs: 47 and 48, respectively, see Table 4 and FIG. 8H). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 147 (encoding the Fab portion of the heavy chain of lanadelumab) and SEQ ID NO: 148 (encoding the Fab portion of the light chain of lanadelumab ), as indicated in table. 5. The heavy and light chain sequences contain a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular liver cells (eg, hepatocytes) or human muscle cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequences. In specific embodiments, the anti-pKal antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 47 with an additional hinge region sequence beginning after the C-terminal aspartate (D) containing all or part of the amino acid sequence KTHTCPPCPAPELLGG (SEQ ID NO: 222) and specifically, KTHL (SEQ ID NO: 223), KTHT (SEQ ID NO: 224), KTHTCPPCPA (SEQ ID NO: 225), KTHLCPPCPA (SEQ ID NO: 226), KTHTCPPCPAPELLGGPSVFL (SEQ ID NO: 227) or KTHLCPPCPAPELLGGPSVFL (SEQ ID NO: 228) as shown in FIG. 8N. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 47 hinge region encoding sequences presented in table. 5.

In certain embodiments, the anti-pKal antigen binding fragment transgene encodes a pKal antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 48. In certain embodiments, the anti-pKal antigen binding fragment transgene encodes a pKal antigen binding fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 47. In certain embodiments, the anti-pKal antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain containing an amino acid sequence that is at least 85%, 86% identical to 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 48, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 47. In specific embodiments, the linkage

- 95 047128 antigen pKal fragment contains a heavy chain containing the amino acid sequence SEQ ID NO: 47 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (ie, those regions outside the CDRs that are underlined in Fig. 8H) or are substitutions to an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the pKal antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 48 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (ie, those regions outside the CDRs that are underlined in Fig. 8H) or are substitutions to an amino acid present at that position in light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-pKal antigen binding fragment transgene encodes a hyperglycosylated lanadelumab Fab containing a heavy chain and a light chain from SEQ ID NOs: 47 and 48, respectively, with one or more of the following mutations: M117N (heavy chain) and/or Q159N, Q159S and/or E194N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-pKal antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of lanadelumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 8H, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-pKal antibody or antigen binding antibody thereof fragment.

Methods of gene therapy.

Methods are provided for treating human subjects with angioedema by administering a viral vector containing a transgene encoding an anti-pKal antibody or an antigen-binding fragment thereof. The antibody may be lanadelumab and is preferably a Fab fragment thereof or another antigen binding fragment thereof. In embodiments, the patient has been diagnosed with and/or has symptoms associated with angioedema. Recombinant vectors used for transgene delivery are described in section 5.4.2. Such vectors should have tropism for human liver or muscle cells and may include non-replicating rAAVs, especially those carrying the AAV8 or AAV9 capsid. Recombinant vectors such as those shown in FIG. 8H can be administered by any route so that the recombinant vector reaches the liver or muscle tissue, preferably by introducing the recombinant vector into the bloodstream. See section 5.5.2 for details of treatment options.

Subjects to receive such gene therapy may be those who respond to anti-pKal therapy. In specific embodiments, the methods include treating patients who have been diagnosed with or have one or more symptoms associated with angioedema and who are determined to respond to or are considered good candidates for anti-pKal antibody treatment. In specific embodiments, the patients have previously been treated with lanadelumab and have been found to be sensitive to lanadelumab. To determine sensitivity, a transgene product of an anti-pKal antibody or antigen binding fragment (eg, produced in cell culture, bioreactors, etc.) can be administered directly to the subject.

Post-translationally modified human antibodies.

Production of the HuPTM anti-pKal mAb or HuPTM Fab should result in a biologically improved molecule for the treatment of angioedema via gene therapy - for example, by administering a viral vector or other DNA expression construct encoding the HuPTM Fab anti-pKal intravenously to human subjects (patients). ) with a diagnosis or presence of one or more symptoms of angioedema, to establish a permanent depot in the liver or muscle tissue that continuously supplies a fully human post-translationally modified, e.g., human glycosylated, sulfated transgene product produced by transduced liver or muscle cells.

In specific embodiments, the HuPTM mAb to pKal or an antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the heavy and light chain of lanadelumab, as shown in FIG. 8H (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions N77, Q114 and/or N164 of the heavy chain (SEQ ID NO: 47) or Q99, N157 and/or N209 of the light chain (SEQ ID NO: 48). Alternatively or in addition to HuPTM mAb or antigen binding fragment thereof

- 96 047128 with the variable domain sequences of the heavy and light chain of lanadelumab contains a sulfation group at Y94 and/or Y95 of the heavy chain (SEQ ID NO: 47) and/or Y86 and/or Y87 of the light chain (SEQ

ID NO: 48). In other embodiments, the HuPTM mAb to pKal or an antigen binding fragment thereof does not contain detectable NeuGc fragments and/or does not contain detectable alpha-Gal fragments.

In certain embodiments, the HuPTM mAb or Fab (or a hyperglycosylated derivative of both) is therapeutically effective and is at least 0.5%, 1%, or 2% glycosylated and/or sulfated and may be at least 5% 10 % or even 50% or 100% glycosylated and/or sulfated. The goal of treatment with the gene therapy presented herein is to slow or stop the progression of angioedema, reduce levels of pain or discomfort for the patient, or reduce the content of autoreactive B cells and immunoglobulin-producing plasma cells. Effectiveness may be monitored by assessing the function, symptoms, or degree of inflammation in the affected tissue or area of the body, such as the skin, joints, kidneys, lungs, blood cells, heart, and brain. For example, effectiveness can be monitored by assessing changes in the severity or frequency of attacks.

Combinations of delivery of HuPTM mAb to pKal or an antigen binding fragment thereof to the liver or muscle, accompanied by delivery of other available treatments, are covered by the methods presented herein. Additional treatments may be given before, simultaneously, or after gene therapy treatment. Available treatments for angioedema that can be combined with the gene therapy provided herein include, but are not limited to, danazol, a bradykinin receptor antagonist (eg, icatibant), a plasma kallikrein inhibitor (eg, ecallantide), a C1-esterase inhibitor, alpha-conestat, antifibrinolytic agents (eg, tranexamic acid), omalizumab and fresh frozen plasma transfusion, antihistamines and corticosteroids and administration with anti-pKal agents, including, but not limited to, lanadelumab.

5.3.15. Anti-TNFa HuPTM designs and formulations for various autoimmune diseases - adalimumab and infliximab

Compositions and methods are described for delivery of HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fab, that bind to tumor necrosis factor alpha (TNFa), derived from an anti-TNFa antibody, such as adalimumab (Figure 9A) or infliximab (Figure 9B). and are indicated for the treatment of one or more autoimmune disorders such as hytradenitis suppurativa (HS), atopic dermatitis, psoriasis (eg, plaque psoriasis, pustular psoriasis, and erythrodermic psoriasis), arthritis (eg, juvenile idiopathic arthritis, rheumatoid arthritis, psoriatic arthritis, and alkylating spondylitis) and/or IBD (eg, Crohn's disease and ulcerative colitis) (hereinafter collectively referred to as AI-Ds(2) subject). In certain embodiments, the HuPTM mAb is characterized by the amino acid sequence of adalimumab or an antigen binding fragment thereof. In other embodiments, the HuPTM mAb is characterized by the amino acid sequence of infliximab or an antigen binding fragment thereof. The amino acid sequences of the antibody Fab fragments are shown in FIG. 9A and 9B. Delivery may be accomplished by gene therapy - for example, by administering a viral vector or other DNA expression construct encoding a TNFa binding HuPTM mAb (or an antigen binding fragment thereof and/or a hyperglycosylated derivative or other derivative thereof) to human patients diagnosed with one or more of the claimed AI-D(2) to create a permanent depot that continuously supplies human PTM, eg human glycosylated, transgenic product.

Transgenes.

Provided are recombinant vectors containing a transgene encoding a HuPTM mAb or a HuPTM Fab (or other antigen binding fragment of a HuPTM mAb) that binds to TNFa, which can be administered to deliver the HuPTM mAb or antigen binding fragment to a patient. A transgene is a nucleic acid containing nucleotide sequences encoding an antigen binding fragment of an antibody that binds to TNFa, such as adalimumab or infliximab or variants thereof, as described in detail herein. The transgene may also encode an anti-TNFa antigen-binding fragment that contains additional glycosylation sites (eg, see Courtois et al.).

In certain embodiments, the anti-TNFa antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of adalimumab (having amino acid sequences SEQ ID NO: 49 and 50, respectively, see Table 4 and FIG. 9A). The nucleotide sequences may be codons optimized for expression in human cells and may, for example, comprise the nucleotide sequences SEQ ID NO: 149 (encoding the Fab portion of the adalimumab heavy chain) and SEQ ID NO: 150 (encoding the Fab portion of the adalimumab light chain ), as indicated in table. 5. Each of the heavy and light chain sequences contains a signal or leader sequence at the N-terminus suitable

- 97 047128 for expression and secretion in human cells, in particular in liver cells (eg hepatocytes) or human muscle cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-TNFa antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 49 with an additional hinge region sequence beginning after the C-terminal aspartate (D) containing all or part of the amino acid sequence KTHTCPPCPAPELLGG (SEQ ID NO: 222) and specifically, KTHL (SEQ ID NO: 223), KTHT (SEQ ID NO: 224), KTHTCPPCPA (SEQ ID NO: 225), KTHLCPPCPA (SEQ ID NO: 226), KTHTCPPCPAPELLGGPSVFL (SEQ ID NO: 227) or KTHLCPPCPAPELLGGPSVFL (SEQ ID NO: 228) as shown in FIG. 9A. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 49 hinge region encoding sequences presented in table. 5.

In certain embodiments, the anti-TNFa antigen binding fragment transgene encodes a TNFa antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 50. In certain embodiments, the anti-TNFa antigen binding fragment transgene encodes a TNFa antigen binding fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 49. In certain embodiments, the anti-TNFa antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain containing an amino acid sequence that is at least 85%, 86% identical to 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 50, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 49. In certain embodiments, the TNFa antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 with 1, 2, 3, 4, 5, 6 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. in those areas outside the CDR that are highlighted in FIG. 9A) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11A. In specific embodiments, the TNFa antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 50 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (ie, those regions outside the CDRs that are underlined in Fig. 9A) or are substitutions to an amino acid present at that position in light chain of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-TNFa antigen-binding fragment transgene encodes a hyperglycosylated adalimumab Fab containing a heavy chain and a light chain from SEQ ID NOs: 49 and 50, respectively, with one or more of the following mutations: L116N (heavy chain), Q160N, or Q160S ( light chain) and/or E195N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-TNFa antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of adalimumab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 9A, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-TNFa antibody or antigen binding agent thereof fragment.

In certain embodiments, the anti-TNFa antigen binding fragment transgene comprises nucleotide sequences encoding the heavy and light chains of the Fab portion of infliximab (having amino acid sequences SEQ ID NOs: 51 and 52, respectively, see Table 4 and FIG. 9B). Nucleotide sequences may be codon optimized

- 98 047128 baths for expression in human cells, and may, for example, contain the nucleotide sequences SEQ ID NO: 151 (encoding the Fab part of the infliximab heavy chain) and SEQ ID NO: 152 (encoding the Fab part of the infliximab light chain), as indicated in table 5. Each of the heavy and light chain sequences contains a signal or leader sequence at the N-terminus suitable for expression and secretion in human cells, in particular liver cells (eg, hepatocytes) or human muscle cells. The signal sequence may be characterized by the amino acid sequence MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Alternatively, the signal sequence may be characterized by an amino acid sequence selected from any of the signal sequences presented in table. 2 or 3, which correspond to proteins secreted by myocytes or hepatocytes, respectively.

In addition to the heavy and light chain variable domain sequences, transgenes may contain all or part of the hinge region at the C-terminus of the heavy chain variable domain sequence. In specific embodiments, the anti-TNFa antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 51 with an additional hinge region sequence beginning after the C-terminal aspartate (D) containing all or part of the amino acid sequence KTHTCPPCPAPELLGG (SeQ ID NO: 222) and specifically, KTHL (SEQ ID NO: 223), KTHT (SEQ ID NO: 224), KTHTCPPCPA (SEQ ID NO: 225), KTHLCPPCPA (SEQ ID NO: 226), KTHTCPPCPAPELLGGPSVFL (SEQ ID NO: 227) or KTHLCPPCPAPELLGGPSVFL (SEQ ID NO: 228) as shown in FIG. 9B. These hinge regions can be encoded by nucleotide sequences at the 3' end of SEQ ID NO: 51 hinge region encoding sequences presented in table. 5.

In certain embodiments, the anti-TNFa antigen binding fragment transgene encodes a TNFa antigen binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 52. In certain embodiments, the anti-TNFa antigen binding fragment transgene encodes a TNFa antigen binding fragment, containing a heavy chain containing an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 51. In certain embodiments, the anti-TNFa antigen binding fragment transgene encodes an antigen binding fragment comprising a light chain containing an amino acid sequence that is at least 85%, 86% identical to 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence presented in SEQ ID NO: 52, and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 51. In certain embodiments, the TNFa antigen binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 51 with 1, 2, 3, 4, 5, 6 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e. in those areas outside the CDR that are highlighted in FIG. 9B) or are substitutions for an amino acid present at that position in the heavy chain of one or more other therapeutic antibodies, for example, as determined by the alignment in FIG. 11A. In specific embodiments, the TNFa antigen binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 52 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are preferably made in framework regions (i.e., those non-CDR regions that are underlined in Fig. 9B) or are substitutions to an amino acid present at that position in the lung chains of one or more other therapeutic antibodies, for example, as shown by the alignment in FIG. 11B.

In certain embodiments, the anti-TNFa antigen-binding fragment transgene encodes a hyperglycosylated infliximab Fab containing a heavy chain and a light chain from SEQ ID NOs: 51 and 52, respectively, with one or more of the following mutations: T115N (heavy chain), Q160N, or Q160S ( light chain) and/or E195N (light chain) (see Fig. 11A (heavy chain) and B (light chain)).

In certain embodiments, the anti-TNFa antigen binding fragment transgene encodes the antigen binding fragment and comprises nucleotide sequences encoding the six CDRs of infliximab, which are highlighted in the heavy and light chain variable domain sequences in FIG. 9B, which are located between framework regions, typically human framework regions, and are linked to constant domains depending on the shape of the antigen binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-TNFa antibody or antigen binding agent thereof fragment.

- 99 047128

Methods of gene therapy.

Methods are provided for treating human subjects with one or more of the claimed AI-D(2) by administering a viral vector containing a transgene encoding an anti-TNFa antibody or an antigen-binding fragment thereof. The antibody may be adalimumab or infliximab and is preferably a Fab fragment thereof or another antigen binding fragment thereof. In embodiments, the patient has been diagnosed with and/or has symptom(s) associated with one or more of the claimed AI-D(2). Recombinant vectors used for transgene delivery are described in section 5.4.2. Such vectors should have tropism for human liver or muscle cells and may include non-replicating rAAVs, especially those carrying the AAV8 or AAV9 capsid. Recombinant vectors such as those shown in FIG. 9A and 9B may be administered by any method such that the recombinant vector enters the liver or muscle tissue, preferably by introducing the recombinant vector into the bloodstream. See section 5.5.2 for details of treatment options.

Subjects to whom such gene therapy is administered may be those who respond to anti-TNFa therapy. In specific embodiments, the methods include treating patients who have been diagnosed with, or who have one or more symptoms associated with, one or more of the claimed AI-D(2) and who are determined to respond to treatment with an anti-TNFa antibody or are considered good candidates for anti-TNFa antibody therapy. In specific embodiments, the patients have previously been treated with adalimumab or infliximab and have been found to be sensitive to adalimumab or infliximab. In other embodiments, the patients have previously been treated with an anti-TNFa antibody or fusion protein, such as etanercept, golimumab, or certolizumab, or another anti-TNFa agent. To determine sensitivity, a transgene product of an anti-TNFa antibody or antigen-binding fragment (eg, produced in cell culture, bioreactors, etc.) can be administered directly to the subject.

Post-translationally modified human antibodies.

Production of a HuPTM mAb to TNFa or HuPTM Fab should result in a biologically improved molecule for the treatment of one or more of the claimed AI-D(2) delivered by gene therapy - for example, by introducing a viral vector or other DNA expression construct encoding HuPTM Fab to TNFa, intravenously in human subjects (patients) diagnosed with or having one or more symptoms of one or more claimed AI-D(2), to establish a permanent depot in the liver or muscle tissue that continuously supplies fully human post-translationally modified, e.g., human glycosylated , a sulfated transgenic product produced by transduced liver or muscle cells.

In specific embodiments, the HuPTM TNFa mAb or antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the adalimumab heavy and light chain Fab portions as shown in FIG. 9A (with non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6-sialylation, at one or more amino acid positions N54 and/or N163 and/or Q113 of the heavy chain (SEQ ID NO: 49) or Q100 and/or N158 and/or N210 of the light chain (SEQ ID NO: 50). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with adalimumab heavy and light chain variable domain sequences contains a sulfation group at Y94 and/or Y95 and/or Y32 of the heavy chain (SEQ ID NO: 49) and/or Y86 and/or Y87 light chain (SEQ ID NO: 50). In other embodiments, the HuPTM TNFa mAb or antigen binding fragment thereof does not contain any detectable NeuGc fragments and/or does not contain any detectable alpha-Gal fragments.

In specific embodiments, the HuPTM anti-TNFa mAb or antigen binding fragment thereof comprises heavy and light chains with the amino acid sequences of the Fab portions of the infliximab heavy and light chain, as shown in FIG. 9B (with consensus and non-consensus asparagine (N) glycosylation sites in blue, glutamine (Q) glycosylation sites in green, and Y-sulfation sites in yellow) is characterized by glycosylation, particularly 2,6- sialylation, at one or more amino acid positions N57 and/or N101 and/or Q112 and/or N162 of the heavy chain (SEQ ID NO: 51) or N41 and/or N76 and/or N158 and/or N210 of the light chain (SEQ ID NO: 52). Alternatively or in addition, the HuPTM mAb or antigen binding fragment thereof with adalimumab heavy and light chain variable domain sequences contains a sulfation group at Y96 and/or Y97 of the heavy chain (SEQ ID NO: 51) and/or Y86 and/or Y87 of the light chain (SEQ ID NO: 52). In other embodiments, the HuPTM TNFa mAb or antigen binding fragment thereof does not contain any detectable NeuGc fragments and/or does not contain any detectable alpha-Gal fragments.

In some embodiments, the HuPTM mAb or Fab is therapeutically effective and is glycosylated and/or sulfated to at least 0.5%, 1%, or 2% and may be

- 100 047128 is at least 5%, 10% or even 50% or 100% glycosylated and/or sulfated. The goal of treatment with the gene therapy provided herein is to slow or stop the progression or amelioration of one or more symptoms of one or more of the claimed AI-D(2), such as reducing the level of pain or discomfort for the patient.

Effectiveness can be monitored by assessing symptoms or the degree of inflammation in the affected tissue or area of the body, such as the skin, colon or joints. For example, with regard to CD, effectiveness can be monitored by assessing the Crohn's Disease Activity Index [CDAI] during the course of treatment (eg, see Best WR et al. (1976) Gastroenterology, Mar;70(3):439-44, Development of a Crohn's disease activity index. National Cooperative Crohn's Disease Study.). For UC, effectiveness can be monitored by assessing Mayo score and Mayo endoscopic score over the course of treatment (eg, see Lobaton et al. (2015) J. Crohns Colitis. 2015 Oct;9(10):846- 52, The Modified Mayo Endoscopic Score (MMES): A New Index for the Assessment of Extension and Severity of Endoscopic Activity in Ulcerative Colitis Patients. For psoriasis, HS and atopic dermatitis, effectiveness can be monitored by assessing changes in the affected skin or the patient's quality of life over the course of treatment. One or more standardized assessments may be used to assess change (see, for example, Feldman & Krueger, (2005) Ann. Rheum. Dis. 64(Suppl II):ii65-ii68: Psoriasis assessment tools in clinical trials, in which describes standardized assessments including Psoriasis Area and Severity Index (PASI), Physician Global Assessment (PGA), Lattice System, NPF Psoriasis Score (NPF-PS), 36-Item Short Form Health Survey (SF- 36), Euro QoL, dermatological disease quality of life index (DLQI) and Skindex; Schram et al. (2012) Allergy 67: 99-106: EASI, (objective) SCORAD and POEM for atopic eczema: responsiveness and minimal clinically important difference, which describes standardized scores including the Eczema Area and Severity Index (EASI) and the Severity of Atopic Dermatitis Index (SCORAD). For arthritis, effectiveness can be monitored by assessing one or more disease activities, the patient's level of function, or the degree of structural damage to the patient's joints (eg, see Zockling & Braun (2005) Clin. Exp. Rheumatol 23 (Suppl. 39) S133-S141 : Assessment of ankylosing spondylitis, which describes a standardized assessment of ankylosing spondylitis, see also Coates et al. (2011) J. Rheumatol. 38(7): 1496-1501: Development of a disease severity and responder index for psoriatic arthritis ( PsA)-report of the OMERACT 10 PsA special interest group, which describes standardized assessments of psoriatic arthritis.

Combinations of delivery of HuPTM mAb to TNFa or an antigen-binding fragment thereof to the liver or muscle, accompanied by delivery of other available treatments, are covered by the methods presented herein. Additional treatments may be given before, simultaneously, or after gene therapy treatment. Available treatments for the claimed AI-D(2) that can be combined with the gene therapy provided herein include, but are not limited to, phototherapy for psoriasis, aminosalicylates, immunomodulatory agents (e.g., azathioprine (AZA), 6-mercaptopurine (6- MR), methotrexate (MTX)), oral or topical corticosteroids (eg, prednisone or budesonide), topical calcineurin inhibitors, antibiotics for IBD, and anti-TNFa agents including, but not limited to, adalimumab or infliximab.

5.4. Delivery of gene therapy constructs

5.4.1. Constructs for delivery to the central nervous system

Sections 5.3.1, 5.3.2, 5.3.3, and 5.3.4 describe recombinant vectors that contain a transgene encoding a HuPTM mAb or a HuPTM Fab (or other antigen-binding fragment of a HuPTM mAb) that binds to Ab, tau, CGRPR, and integrin, respectively. Such a recombinant vector used to deliver the transgene must have tropism for human CNS cells, such as glial and neuronal cells. Such vectors may include non-replicating recombinant adeno-associated viral vectors (rAAV), especially those carrying the AAV9, AAVrh10, AAVrh20, AAVrh39 or AAVcy5 capsid. However, other viral vectors can be used, including, but not limited to, lentiviral vectors, vaccinia viral vectors, or non-viral expression vectors called naked DNA constructs.

In specific embodiments, constructs for administering gene therapy to humans are provided, comprising an AAV vector that contains a viral capsid that is at least 95% identical to the amino acid sequence of the AAV9 capsid (SEQ ID NO: 79); and a viral or artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette contains a transgene encoding the heavy and light chains of a therapeutic antibody operably linked to one or more regulatory sequences that control expression of the transgene in human cells, which expresses and delivers a therapeutic antibody in a therapeutically appropriate manner as described herein, especially expressed from CNS cells. In certain embodiments, the encoded AAV9 capsid is characterized by the sequence SEQ ID

- 101 047128

NO: 79 s 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions, in particular substitutions to amino acid residues found at the corresponding position in other AAV capsids, for example, in the SUBS line in FIG. 12, which provides a comparison of the amino acid sequences of the capsid sequences of different AAVs, highlighting amino acids suitable for substitution at different positions within the capsid sequence.

In other specific embodiments, constructs for administering gene therapy to a human subject are provided, comprising an AAV vector that contains a viral capsid that is at least 95% identical to the amino acid sequence of the AAVrh10 capsid (SEQ ID NO: 80); and a viral or artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette contains a transgene encoding the heavy and light chains of a therapeutic antibody operably linked to one or more regulatory sequences that control expression of the transgene in human cells, which express and deliver a therapeutic antibody in a therapeutically appropriate manner as described herein, especially from CNS cells. In certain embodiments, the encoded AAVrh10 capsid is characterized by the sequence SEQ ID NO: 80 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions, in particular substitutions to amino acid residues found at the corresponding position in other AAV capsids, for example, in the SUBS line on fig. 12, which provides a comparison of the amino acid sequences of the capsid sequences of different AAVs, highlighting amino acids suitable for substitution at different positions within the capsid sequence.

Preferably, the HuPTM mAb or antigen binding fragment thereof, including the HuPTM Fab transgene, is controlled by appropriate expression control elements for expression of HuPTM Fab in human CNS cells, e.g., CB7 promoter (avian β-actin promoter and CMV enhancer), RSV promoter, GFAP promoter ( glial fibrillary acidic protein), MBP (myelin basic protein) promoter, MMT promoter, EF-1a promoter, U86 promoter, RPE65 promoter or opsin promoter, inducible promoter such as hypoxia inducible promoter or drug inducible promoter such as rapamycin inducible promoters and related agents and other expression control elements that enhance vector-driven transgene expression (eg, introns such as avian β-actin intron, mouse small virus (MVM) intron, human factor IX intron (eg, FIX truncated intron 1) , β-globin splice donor/immunoglobulin heavy chain splice acceptor intron, adenovirus splice donor/immunoglobulin splice acceptor intron, SV40 late splice donor/splice acceptor intron (19S/16S), and hybrid adenovirus splice donor/IgG splice acceptor intron and polyA signals, such as rabbit β-globin polyA signal, human growth hormone (hGH) polyA signal, SV40 late polyA signal, synthetic polyA (SPA) signal, and bovine growth hormone (bGH) polyA signal. See, for example, Powell and Rivera-Soto, 2015, Discov. Med., 19(102):49-57.

Gene therapy constructs are designed such that both heavy and light chains are expressed. More specifically, the heavy and light chains should be expressed in approximately equal amounts, that is, the heavy and light chains are expressed in an approximately 1:1 ratio of heavy chains to light chains. The coding sequences for the heavy and light chains can be constructed in a single construct in which the heavy and light chains are separated by a cleavable linker or IRES such that separate heavy and light chain polypeptides are expressed. The leader sequence for each of the heavy and light chains is preferably MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Section 5.1.5 above provides specific sequences of IRES, 2A and other linker sequences that can be used with the methods and compositions presented herein. In specific embodiments, the linker is a (|)urin-P2L linker

RKRRAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 242). In specific embodiments, the transgene is a nucleotide sequence that encodes the following: a signal sequence - a heavy chain Fab portion - a furin-F2A linker sequence a signal sequence - a light chain Fab portion. See, for example, FIG. 2A-2C and 2F for sequences for Fab expression of aducanumab, crenezumab, gantenerumab or BAN2401, respectively; fig. 2D for aTAU Fab expression sequence; fig. 2E for erenumab Fab expression sequence and FIG. 4B for the sequence for natalizumab Fab expression.

In a specific embodiment, the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank an expression cassette; (2) control elements that include a) the CB7 promoter containing the CMV enhancer/avian β-actin promoter, b) the avian β-actin intron, and c) the rabbit poly-A β-globin signal; and (3) nucleic acid sequences encoding the heavy and light chains of Aβ-102 047128 binding, tau-binding, CGRPR-binding, integrin-binding Fab, separated by a self-cleaving furin (F)/F2A linker, ensuring the expression of equal amounts of heavy and light polypeptides chains. An exemplary structure is shown in FIG. 1.

In specific embodiments, AAV vectors are provided comprising a viral capsid that is at least 95% identical to the amino acid sequence of the capsid of AAV9 (SEQ ID NO: 79) or AAVrh10 (SEQ ID NO: 80); and an artificial genome containing an expression cassette flanked by AAV inverted terminal repeats (ITR), wherein the expression cassette contains a transgene encoding an anti-Av, anti-tau, anti-CGRPR or anti-integrin mAb or an antigen-binding fragment thereof operably linked to one or more regulatory sequences, which control transgene expression in human CNS cells.

5.4.2. Constructs for delivery to liver or muscle cells

Sections 5.3.4, 5.3.5, 5.3.6, 5.3.7, 5.3.8, 5.3.9, 5.3.10, 5.3.11, 5.3.12, 5.3.13, 5.3.14, 5.3. 15 describe recombinant vectors that contain a transgene encoding a HuPTM mAb or a HuPTM Fab (or other antigen-binding fragment of a HuPTM mAb) that binds interleukins (IL) or interleukin receptors (ILRs), integrin, PCSK9, ANGPTL3, OxPL RANKL, PD-1 /PD-L1/PD-L2, VEGF, factor D (fD), BLyS, CP-C5, MMP9, pKal or TNFa. Such a recombinant vector used to deliver the transgene may be characterized by tropism for human liver or muscle cells. Such vectors may include non-replicating recombinant adeno-associated viral vectors (rAAV), particularly preferred are those carrying the AAV8 or AAV9 capsid. However, other viral vectors can be used, including, but not limited to, lentiviral vectors, vaccinia viral vectors, or non-viral expression vectors called naked DNA constructs.

In specific embodiments, constructs for administering gene therapy to humans are provided, comprising an AAV vector that contains a viral capsid that is at least 95% identical to the amino acid sequence of the AAV8 capsid (SEQ ID NO: 78); and a viral or artificial genome containing an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette contains a transgene encoding the heavy and light chains of a therapeutic antibody operably linked to one or more regulatory sequences that control expression of the transgene in human cells ( e.g., human muscle or liver cells) that express and deliver the therapeutic antibody in a therapeutically appropriate manner as described herein. In certain embodiments, the encoded AAV8 capsid is characterized by the sequence SEQ ID NO: 78 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions, in particular substitutions to amino acid residues found at the corresponding position in other AAV capsids, for example, in the SUBS line on fig. 12, which provides a comparison of the amino acid sequences of the capsid sequences of different AAVs, highlighting amino acids suitable for substitution at different positions within the capsid sequence.

In specific embodiments, constructs for administration by gene therapy to a human subject are provided, comprising an AAV vector that contains a viral capsid that is at least 95% identical to the amino acid sequence of the AAV9 capsid (SEQ ID NO: 79); and a viral or artificial genome containing an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette contains a transgene encoding the heavy and light chains of a therapeutic antibody operably linked to one or more regulatory sequences that control expression of the transgene in human cells ( e.g., human muscle or liver cells) that express and deliver the therapeutic antibody in a therapeutically appropriate manner as described herein. In some embodiments, the encoded AAV9 capsid is characterized by the sequence SEQ ID NO: 79 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions, in particular substitutions to amino acid residues found at the corresponding position in other AAV capsids, for example, in the SUBS line on fig. 12, which provides a comparison of the amino acid sequences of the capsid sequences of different AAVs, highlighting amino acids suitable for substitution at different positions within the capsid sequence.

Preferably, the HuPTM mAb or antigen binding fragment thereof, including the HuPTM Fab transgene, is controlled by appropriate expression control elements for expression of HuPTM Fab in human liver or muscle cells, e.g., the CB7 promoter (avian β-actin promoter and CMV enhancer), liver-specific promoters such as the TBG (thyroxine-binding globulin) promoter, the APOA2 promoter, the SERPINA1 (hAAT) promoter or the MIR122 promoter, or muscle-specific promoters such as the human desmin promoter or the human Pitx3 promoter or inducible promoters such as the hypoxia-inducible promoters. or rapamycin-inducible promoter, and may include other expression control elements that enhance expression of the vector-driven transgene (e.g., introns such as avian β-actin intron, mouse small virus (MVM) intron, human factor IX intron (e.g. truncated intron 1 FIX), β-globin splice donor/immunoglobulin heavy chain splice acceptor intron, adenovirus splice donor/immunoglobulin splice acceptor intron, SV40 late splice donor/splice acceptor intron (19S/16S) and hybrid adenovirus splice donor intron splice acceptor IgG and polyA signals, such as rabbit β-globin polyA signal, human growth hormone (hGH) polyA signal, SV40 late polyA signal, synthetic polyA signal (SPA), and bovine growth hormone (bGH) polyA signal. See, for example, Powell and Rivera-Soto, 2015, Discov. Med., 19(102):49-57.

Gene therapy constructs are designed such that both heavy and light chains are expressed. More specifically, the heavy and light chains should be expressed in approximately equal amounts, that is, the heavy and light chains are expressed in an approximately 1:1 ratio of heavy chains to light chains. The coding sequences for the heavy and light chains can be constructed in a single construct in which the heavy and light chains are separated by a cleavable linker or IRES such that separate heavy and light chain polypeptides are expressed. The leader sequence for each of the heavy and light chains is preferably MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Section 5.1.5 above provides specific sequences of IRES, 2A and other linker sequences that can be used with the methods and compositions presented herein. In specific embodiments, the linker is a (|)urin-E2L linker

RKRRAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 242). In certain embodiments, the transgene is a nucleotide sequence that encodes the following: a signal sequence - a heavy chain Fab portion - a furin-F2A linker sequence; a signal sequence - a light chain Fab portion. See, for example, FIG. 3A-3E for the Fab expression sequences of dupilumab, ixekizumab, secukinumab, ustekinumab and mepolizumab, respectively; fig. 4A and 4B for Fab expression sequences of vedolizumab and natalizumab, respectively; fig. 5A-5D for Fab expression sequences of alirocumab, evolocumab, evinacumab and E06-scFv, respectively; fig. 6 for denosumab Fab expression sequence; fig. 7A and 7B for the Fab expression sequences of nivolumab and pembrolizumab, respectively; fig. 8A-8C for the Fab expression sequences of ranibizumab, bevacizumab and lampalizumab, respectively; fig. 8E for belimumab Fab expression sequence; fig. 8F for eculizumab Fab expression sequence; fig. 8G for andecaliximab Fab expression sequence; fig. 8H for lanadelumab Fab expression sequence and FIG. 9A and 9B for sequences for adalimumab Fab expression and infliximab Fab expression, respectively.

In a specific embodiment, the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank an expression cassette; (2) control elements that include a) an inducible promoter, preferably a hypoxia inducible promoter, b) an avian β-actin intron, and c) a rabbit β-globin poly A signal; and (3) nucleic acid sequences encoding the heavy and light chains of IL/ILR-binding, integrin-binding, PCSK9-binding, LNGPTL3-binding, RANKL-binding, OxPL-binding, PD-1/PD-L1/PD-L2 β-binding, VabF-binding Fab, fD-binding, BLyS-binding, pKal-binding or TNFa-binding Fab separated by a furin (F)/F2A linker cleaving linker, allowing expression of equal amounts of heavy and light chain polypeptides. An exemplary structure is shown in FIG. 1.

In a specific embodiment, the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank an expression cassette; (2) control elements that include a) an inducible promoter, preferably a hypoxia inducible promoter, b) an avian β-actin intron, and c) a rabbit β-globin poly A signal; and (3) nucleic acid sequences encoding the heavy and light chains of IL/ILR-binding, integrin-binding, PCSK9-binding, ANGPTL3-binding, OxPL-binding, RANKL-binding, PD-1/PDL1/PD-L2-binding, VEGF -binding Fab, fD-binding, BLyS-binding, CP-C5-binding, MMP9-binding, pKal-binding, TNFa-binding Fab, separated by a self-cleaving furin (F)/F2A linker, ensuring the expression of equal amounts of heavy and light chain polypeptides. An exemplary structure is shown in FIG. 1.

In specific embodiments, AAV vectors are provided comprising a viral capsid that is at least 95% identical to the amino acid sequence of the AAV8 capsid (SEQ ID NO: 78); and an artificial genome containing an expression cassette flanked by AAV inverted terminal repeats (ITR), wherein the expression cassette contains a transgene encoding a MAb to IL/ILR, to integrin, to PCSK9, to ANGPTL3, to OxPL, to RANKL, to PD-1, to PD-L1,

- 104 047128 to PD-L2, to VEGF, to FD, to BLyS, to CP-C5, to MMP9, to pKal or to TNFa or an antigen-binding fragment operably linked to one or more regulatory sequences that control the expression of the transgene in cells human liver or muscles.

In specific embodiments, AAV vectors are provided comprising a viral capsid that is at least 95% identical to the amino acid sequence of AAV9 (SEQ ID NO: 79); and an artificial genome containing an expression cassette flanked by AAV inverted terminal repeats (ITR), wherein the expression cassette contains a transgene encoding mAbs to IL/ILR, to integrin, to PCSK9, to ANGPTL3, to RANKL, to RANKL, to OxPL, to PD -1, anti-PD-L1, anti-PD-L2, anti-VEGF, anti-fD, anti-BLyS, anti-pKal, or anti-TNFa, or an antigen-binding fragment thereof, operably linked to one or more regulatory sequences that control transgene expression in human muscle cells.

5.4.3. Constructs for delivery to retinal cell types

Sections 5.3.9 and 5.3.12 describe recombinant vectors that contain a transgene encoding a HuPTM mAb or a HuPTM Fab (or other antigen-binding fragment of a HuPTM mAb) that binds to VEGF, factor D (fD) or MMP9. Such recombinant vectors used to deliver the transgene may be tropic for one or more human retinal cell types. Such vectors may include non-replicating recombinant adeno-associated viral vectors (rAAV), particularly preferred are those carrying the AAV8 capsid. Alternatively, an AAV vector containing the AAV.7m8 capsid can be used. However, other viral vectors can be used, including, but not limited to, lentiviral vectors, vaccinia viral vectors, or non-viral expression vectors called naked DNA constructs.

In specific embodiments, constructs for administering gene therapy to a human subject are provided, comprising an AAV vector that contains a viral capsid that is at least 95% identical to the amino acid sequence of the AAV8 capsid (SEQ ID NO: 78); and a viral or artificial genome containing an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette contains a transgene encoding the heavy and light chains of a therapeutic antibody operably linked to one or more regulatory sequences that control expression of the transgene in human cells ( e.g., retinal cells or liver cells) that express and deliver the therapeutic antibody in a therapeutically appropriate manner as described herein. In some embodiments, the encoded AAV8 capsid is characterized by the sequence SEQ ID NO: 78 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions, in particular substitutions to amino acid residues found at the corresponding position in other AAV capsids, for example, in the SUBS line on fig. 12, which provides a comparison of the amino acid sequences of the capsid sequences of different AAVs, highlighting amino acids suitable for substitution at different positions within the capsid sequence.

Preferably, the HuPTM mAb or an antigen binding fragment thereof, including the HuPTM Fab transgene, is controlled by appropriate expression control elements for expression of HuPTM Fab in human retinal cells or liver cells, for example, the CB7 promoter (avian β-actin promoter and CMV enhancer), or tissue-specific promoters, such as RPE-specific promoters, for example, the RPE65 promoter, or cone-specific promoters, for example, the opsin promoter, or liver-specific promoters, such as the TBG (thyroxine-binding globulin) promoter, APOA2 promoter, SERPINA1 (hAAT) promoter or the MIR122 promoter, inducible promoters, such as hypoxia-inducible promoters, and drug-inducible promoters, such as promoters inducible by rapamycin and related drugs, and may include other expression control elements that enhance expression of the vector-driven transgene (eg, introns such such as avian β-actin intron, mouse small virus (MVM) intron, human factor IX intron (e.g. truncated FIX intron 1), β-globin splice donor/immunoglobulin heavy chain splice acceptor intron, adenovirus splice donor/immunoglobulin splice acceptor intron, SV40 late splice donor/splice acceptor intron (19S/16S) and hybrid adenovirus/splice acceptor splice donor intron IgG and polyA signals such as rabbit β-globin polyA signal, human growth hormone (hGH) polyA signal, SV40 late polyA signal, synthetic polyA signal (SPA) and bovine growth hormone (bGH) polyA signal. See, for example, Powell and Rivera-Soto, 2015, Discov. Med., 19(102):49-57.

Gene therapy constructs are designed such that both heavy and light chains are expressed. More specifically, the heavy and light chains should be expressed in approximately equal amounts, that is, the heavy and light chains are expressed in a ratio of approximately 1:1 heavy chains to light chains. The coding sequences for the heavy and light chains can be constructed in a single construct in which the heavy and light chains are separated by a cleavable linker or IRES such that separate heavy and light chain polypeptides are expressed. The leader sequence for each of the heavy and light chains is preferably MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). Section 5.1.5 above provides specific IRES, 2A and other linker sequences that can be used with the methods and compositions provided herein. In specific embodiments, the linker is a furin-B2A linker RKRRAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 242). In specific embodiments, the transgene is a nucleotide sequence that encodes the following: a signal sequence - a heavy chain Fab portion - a furin-B2A linker sequence; a signal sequence - a light chain Fab portion. See fig. 8A-8C for the Fab expression sequences of ranibizumab, bevacizumab and lampalizumab, respectively, and FIG. 8G for the sequence for andecaliximab Fab expression.

In a specific embodiment, the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank an expression cassette; (2) control elements that include a) the CB7 promoter containing the CMV enhancer/avian β-actin promoter, b) the avian β-actin intron, and c) the rabbit poly-A β-globin signal; and (3) nucleic acid sequences encoding the heavy and light chains of a VabF-binding, fD-binding, or MMP9-binding Fab, separated by a self-cleaving furin (F)/F2A linker, allowing expression of equal amounts of heavy and light chain polypeptides. An exemplary structure is shown in FIG. 1.

In another embodiment, the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank an expression cassette; (2) control elements that include a) the CB7 promoter containing the CMV enhancer/avian β-actin promoter, b) the avian β-actin intron, and c) the rabbit poly-A β-globin signal; and (3) nucleic acid sequences encoding the heavy and light chains of a VabF-binding, fD-binding, or MMP9-binding Fab separated by a flexible peptide linker to ensure proper folding and solubility.

In specific embodiments, AAV vectors are provided comprising a viral capsid that is at least 95% identical to the amino acid sequence of the AAV8 capsid (SEQ ID NO: 78); and an artificial genome containing an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette contains a transgene encoding an anti-VEGF, anti-fD, or anti-MMP9 mAb or an antigen-binding fragment thereof operably linked to one or more regulatory sequences that control expression transgene in one or more types of retinal cells (such as human photoreceptor cells (cone cells, rod cells); horizontal cells; bipolar cells; amarcrine cells; retinal ganglion cells (medium cell, umbrella cell, bistratified cell, retinal giant ganglion cell, photosensitive ganglion cell and müller glia) and retinal pigment epithelial cells).

5.5. Dose administration

5.5.1. Administration for CNS Delivery

Sections 5.3.1, 5.3.2, 5.3.3 and 5.3.4 describe recombinant vectors that contain a transgene encoding a HuPTM mAb or a HuPTM Fab (or other antigen-binding fragment of a HuPTM mAb) that binds to Ab, tau, CGRPR and integrin, respectively. Therapeutically effective doses of any such recombinant vector should be administered by any route such that the recombinant vector reaches the CNS, preferably by introducing the recombinant vector into the cerebrospinal fluid (CSF). In certain embodiments, the vector is administered intrathecally, specifically intracisternally (eg, into the cisterna magna) or, alternatively, via lumbar delivery. Alternatively, the recombinant vector can be administered intravenously. In particular, recombinant AAV9 vectors have been shown to cross the blood-brain barrier and, as such, may be useful for delivering anti-Av, anti-tau, anti-CGRPR or anti-integrin transgene product to the CNS. In particular, scAAV9 may be particularly useful for intravenous administration. Intrathecal, including intracisternal or lumbar, or intravenous administration should result in expression of a soluble transgene product in CNS cells. Expression of a transgene product (eg, an encoded anti-Ab, anti-tau, anti-CGRPR, or anti-integrin antibody) results in delivery and maintenance of the transgene product to the CNS. Since the transgenic product is continuously produced, maintaining lower concentrations may be effective. The concentration of the transgene product can be measured in samples from patients with CSF.

Pharmaceutical compositions suitable for intrathecal, intracisternal, lumbar or intravenous administration include a suspension of a recombinant vector containing a transgene encoding an anti-Ab, anti-tau, anti-CGRPR or anti-integrin antibody or an antigen-binding fragment thereof, in a formulation buffer containing a physiologically compatible water buffer. Buffer for

- 106 047128 composition may contain one or more of polysaccharides, surfactants, polymer or oil.

5.5.2. Administration for delivery to liver or muscle tissue

Sections 5.3.4, 5.3.5, 5.3.6, 5.3.7, 5.3.8, 5.3.9, 5.3.10, 5.3.11, 5.3.12, 5.3.13 and 5.3.14 describe recombinant vectors that contain a transgene encoding a HuPTM mAb or a HuPTM Fab (or other antigen-binding fragment of a HuPTM mAb) that binds to interleukins (IL) or interleukin receptors (ILRs), integrin, PCSK9, ANGPTL3, RANKL, PD-1/PD-L1/PD- L2, VEGF, factor D (fD), BLyS, CP-C5, MMP9, pKal or TNFa. Therapeutically effective doses of any such recombinant vector should be administered by any route such that the recombinant vector reaches the liver or muscle (eg, skeletal muscle), preferably by introducing the recombinant vector into the bloodstream. Alternatively, the vector can be administered directly to the liver through the hepatic bloodstream, for example, through the hepatic veins or through the hepatic artery. In specific embodiments, the vector is administered subcutaneously, intramuscularly, or intravenously. Intramuscular, subcutaneous, intravenous or hepatic administration should result in expression of a soluble transgene product in liver or muscle cells. Alternatively, the vector can be administered directly to the liver through the hepatic bloodstream, for example, through the hepatic veins or through the hepatic artery. Expression of a transgene product (e.g., encoded anti-IL/ILR, anti-integrin, anti-PCSK9, anti-ANGPTL3, anti-RANKL, anti-PD-1, anti-PD-L1, anti-PD-L2, anti-VEGF, anti-fD, anti-BLyS, anti- CP-C5, MMP9, pKal or TNFa) leads to the delivery and maintenance of the transgene product in the liver or muscle.

The concentration of the transgene product can be measured in patient serum samples. In specific embodiments, dosages are desired that maintain the concentration of the anti-IL/ILR antibody transgene product at a C _min of at least 2 μg/ml, such as a C _min of 5 to 30 μg/ml, 5 to 50 μg/ml, or 5 up to 80 μg/ml, or from 5 to 100 μg/ml, or from 5 to 200 μg/ml, depending on the mAb used. For example, to achieve a C _min of approximately 60 to 90 mcg/mL of dupilumab (comparable to biweekly dosing) or 170 to 200 mcg/mL of dupilumab (comparable to weekly dosing) or approximately 2 mcg/mL to 12 mcg/mL of ixekizumab, or approximately 13 mcg/mL to 50 mcg/mL secukinumab.

In certain embodiments, dosages are desired that maintain the concentration of the anti-TNFa antibody transgene product at a C _min of at least 0.5 μg/ml or at least 1 μg/ml (e.g., C _min of 1 to 10 μg/ml, 3 up to 30 µg/ml or from 5 to 15 µg/ml, or from 5 to 30 µg/ml).

In certain embodiments, dosages are desired that maintain the concentration of the anti-integrin antibody transgene product at a C _min of at least 10 μg/ml (eg, a C _min of 10 to 60 μg/ml).

In certain embodiments, dosages are desired that maintain the concentration of the anti-PCSK9 or anti-ANGPTL3 antibody transgene product at a C _min of at least 10 μg/ml, e.g., a C min of 10 to 80 μg/ml.

In certain embodiments, dosages that maintain the concentration of the anti-RANKL antibody transgene product at a Cmin of at least 10 μg/ml (eg, a Cmin of 10 to 50 μg/ml or 15 to 30 μg/ml) are desired.

In particular embodiments, dosages are desired that maintain the concentration of the anti-PD-1, anti-PD-L1, or anti-PD-L2 antibody transgene product at a C _min of at least 10 μg/mL, e.g., a C _min of 10 to 100 μg/mL is desired. or from 100 to 300 µg/ml, or from 300 to 600 µg/ml.

In specific embodiments, dosages are desired that maintain the concentration of the anti-BLyS antibody transgene product at a C _min of at least 70 μg/ml (e.g., a C _min of 70 to 150 μg/ml or 100 to 200 μg/ml or 200 to 350 µg/ml).

In specific embodiments, dosages that maintain the concentration of the anti-pKal antibody transgene product at a Cmin of at least 70 μg/mL are desired (e.g., a Cmin of 70 to 150 μg/mL or 100 to 200 μg/mL or 200 to 350 μg /ml).

Expression of a transgene product (eg, an encoded anti-VEGF antibody) results in delivery and maintenance of the transgene product in the liver. In some embodiments, dosages that maintain the concentration of the VEGF transgene product at a Cmin of at least 90 μg/ml are desired, e.g., a _Cmin of 90 μg/ml to 200 μg/ml. In certain embodiments, dosages are desired that maintain the concentration of the anti-CP-C5 antibody transgene product at a C _min of at least 30 μg/ml, e.g., a C _min of 30 to 300 μg/ml or 100 to 200 μg/ml.

However, in all cases, since the transgenic product is continuously produced, maintaining lower concentrations may be effective.

Although the transgenic product is continuously produced, maintaining lower concentrations may be effective. The concentration of the transgene product can be measured in patient serum samples.

- 107 047128

Pharmaceutical compositions suitable for intravenous, intramuscular, subcutaneous or hepatic administration include a suspension of a recombinant vector containing a transgene encoding an anti-IL/ILR, anti-integrin, anti-PCSK9, anti-ANGPTL3, anti-RANKL, anti-RANKL anti-PD-1 antibody, anti-PD-L1, anti-PD-L2, anti-VEGF, anti-FD, anti-BLyS, anti-CP-C5, anti-MMP9, anti-pKal, or anti-TNFa or an antigen-binding fragment thereof, in a formulation buffer containing a physiologically compatible aqueous buffer. The formulation buffer may contain one or more of polysaccharides, surfactants, polymer or oil.

5.5.3. Administration for delivery to retinal type cells

Therapeutically effective doses of the recombinant vector should be administered by any route such that the recombinant vector reaches the retina, preferably by administering the recombinant vector directly into the eye. In specific embodiments, the vector is administered subretinal (a surgical procedure performed by trained retinal surgeons that involves partial vitrectomy with the subject under local anesthesia and injection of gene therapy into the retina; see, for example, Campochiaro et al., 2016, Hum Gen Ther Sep 26 epub:doi: 10.1089/hum.2016.117, which is incorporated herein by reference in its entirety), either intravitreal or suprachoroidal, such as by microinjection or microcannulation. (See, for example, Patel et al., 2012, Invest Ophth & Vis Sci 53:4433-4441; Patel et al., 2011, Pharm Res 28:166-176; Olsen, 2006, Am J Ophth 142:777 -787, each of which is incorporated by reference in its entirety). Subretinal, intravitreal or suprachoroidal administration should result in expression of a soluble transgene product in one or more of the following retinal cell types: human photoreceptor cells (cone cells, rod cells); horizontal cells; bipolar cells; amarcrine cells; retinal ganglion cells (dwarf cell, umbrella cell, bistratified cell, retinal giant ganglion cell, photosensitive ganglion cell and müllerian glia); and retinal pigment epithelial cells. Expression of a transgene product (eg, an encoded anti-VEGF, anti-fD, anti-MMP9 antibody) results in delivery and maintenance of the transgene product in the retina.

The concentration of the transgene product can be measured in samples of the vitreous and/or anterior chamber of the treated eye of patients. In specific embodiments, dosages are desired that maintain a concentration of the anti-VEGF, anti-fD transgene product at a C _min of at least 0.33 μg/mL in the vitreous or 0.11 μg/mL in the aqueous humor (anterior chamber of the eye) in within three months; thereafter, vitreous _Cmin concentrations of the transgenic product should be maintained in the range of 1.70 to 6.60 μg/ml and/or aqueous humor _Cmin concentrations in the range of 0.567 to 2.20 μg/ml. However, since the transgenic product is continuously produced, maintaining lower concentrations may be effective. Alternatively, concentrations in vitreous humor can be assessed and/or monitored by measuring the concentrations of the transgene product in the patient's serum - the ratio of systemic to vitreal exposure to the transgene product is approximately 1:90,000. (For example, see vitreous ranibizumab concentrations and serum ranibizumab concentrations reported in Xu L, et al., 2013, Invest. Opthal. Vis. Sci. 54: 1616-1624, on page 1621 and in Table 5 on page 1623, which is incorporated herein by reference in its entirety). However, since the transgenic product is continuously produced, maintaining lower concentrations may be effective.

Suitable pharmaceutical compositions for administration include a suspension of a recombinant vector containing a transgene encoding an anti-VEGF, anti-fD or anti-MMP9 antibody or an antigen-binding fragment thereof, in a formulation buffer containing a physiologically compatible aqueous buffer. The formulation buffer may contain one or more of polysaccharides, surfactants, polymer or oil.

6. Examples

6.1. Example 1: Aducanumab Fab cDNA Vector

A vector based on aducanumab Fab cDNA is constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the aducanumab heavy and light chain sequences (amino acid sequences are SEQ ID NO: 1 and 2, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human CNS cells and may be the nucleotide sequence of SEQ ID NO: 101 and 102, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, in particular MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 2A for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.2. Example 2 Crenezumab Fab cDNA Vector

A vector based on crenezumab Fab cDNA is constructed, containing a transgene containing

- 108 047128 leotide sequences encoding the Fab portion of the crenezumab heavy and light chain sequences (amino acid sequences are SEQ ID NO: 3 and 4, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human CNS cells and may be the nucleotide sequence of SEQ ID NO: 103 and 104, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, in particular MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 2B for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.3. Example 3 Gantenerumab Fab cDNA Vector

A gantenerumab Fab cDNA vector is constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the gantenerumab heavy and light chain sequences (amino acid sequences are SEQ ID NO: 5 and 6, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human CNS cells and may be the nucleotide sequence of SEQ ID NO: 105 and 106, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, in particular MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 2C for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.4. Example 4 Dupilumab Fab cDNA Vector

A dupilumab Fab cDNA vector is constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the dupilumab heavy and light chain sequences (the amino acid sequences are SEQ ID NO: 7 and 8, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human liver or muscle cells and may be the nucleotide sequence of SEQ ID NO: 107 and 108, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a liver-specific signal sequence from table. 2 or muscle-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 3A for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.5. Example 5 Ixekizumab Fab cDNA Vector

A vector based on ixekizumab Fab cDNA is constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the ixekizumab heavy and light chain sequences (amino acid sequences are SEQ ID NO: 9 and 10, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human muscle or liver cells and may be the nucleotide sequence of SEQ ID NO: 109 and 110, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a liver-specific signal sequence from table. 2 or muscle-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 3B for the amino acid sequence of the transgene product. Optionally, the vector further contains a hypoxia-inducible promoter.

6.6. Example 6 Secukinumab Fab cDNA Vector

A secukinumab Fab cDNA vector is constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the secukinumab heavy and light chain sequences (amino acid sequences are SEQ ID NO: 11 and 12, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human liver or muscle cells and may be the nucleotide sequence of SEQ ID NO: 111 and 112, respectively. The transgene also contains nucleotide sequences that encode a signal peptide selected from the group presented in table. 2 or 3, respectively. Nucleotide sequences, encoding

- 109 047128 The light chain and heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a liver-specific signal sequence from table. 2 or muscle-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 3C for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.7. Example 7 Ustekinumab Fab cDNA Vector

A vector based on ustekinumab Fab DNA is constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the ustekinumab heavy and light chain sequences (amino acid sequences are SEQ ID NO: 13 and 14, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human liver or muscle cells and may be the nucleotide sequence of SEQ ID NO: 113 and 114, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a liver-specific signal sequence from table. 2 or muscle-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 3D for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.8. Example 8 Mepolizumab Fab cDNA Vector

A vector is constructed based on the mepolizumab Fab cDNA containing a transgene containing nucleotide sequences encoding the Fab portion of the mepolizumab heavy and light chain sequences (amino acid sequences are SEQ ID NO: 15 and 16, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human muscle or liver cells and may be the nucleotide sequence of SEQ ID NO: 115 and 116, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a liver-specific signal sequence from table. 2 or muscle-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 3E for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.9. Example 9 Vedolizumab Fab cDNA Vector

A vector is constructed based on the vedolizumab Fab cDNA containing a transgene containing nucleotide sequences encoding the Fab portion of the vedolizumab heavy and light chain sequences (amino acid sequences are SEQ ID NO: 17 and 18, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human liver or muscle cells and may be the nucleotide sequence of SEQ ID NO: 117 and 118, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a liver-specific signal sequence from table. 2 or muscle-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 4A for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.10. Example 10 Natalizumab Fab cDNA Vector

A natalizumab Fab cDNA vector is constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the natalizumab heavy and light chain sequences (amino acid sequences are SEQ ID NO: 19 and 20, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human liver or muscle cells and may be the nucleotide sequence of SEQ ID NO: 119 and 120, respectively. The transgene also contains nucleotide sequences that encode a signal peptide that can

- 110 047128 is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or is a liver-specific signal sequence from table. 2 or muscle-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 4B for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.11. Example 11 Alirocumab Fab cDNA Vector

A vector is constructed based on alirocumab Fab cDNA containing a transgene containing nucleotide sequences encoding the Fab portion of the alirocumab heavy and light chain sequences (amino acid sequences are SEQ ID NO: 21 and 22, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human liver or muscle cells and may be the nucleotide sequence of SEQ ID NO: 121 and 122, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a liver-specific signal sequence from table. 2 or muscle-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 5A for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.12. Example 12 Evolocumab Fab cDNA Vector

An evolocumab Fab cDNA vector is constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the evolocumab heavy and light chain sequences (amino acid sequences are SEQ ID NO: 23 and 24, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human liver or muscle cells and may be the nucleotide sequence of SEQ ID NO: 123 and 124, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a liver-specific signal sequence from table. 2 or muscle-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 5B for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.13. Example 13 Evinacumab Fab cDNA Vector

An evinacumab Fab cDNA vector is constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the evinacumab heavy and light chain sequences (the amino acid sequences are SEQ ID NOs: 25 and 26, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human liver or muscle cells and may be the nucleotide sequence of SEQ ID NO: 125 and 126, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a liver-specific signal sequence from table. 2 or muscle-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 5C for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.14. Example 14 Denosumab Fab cDNA Vector

A denosumab Fab cDNA vector is constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the denosumab heavy and light chain sequences (amino acid sequences are SEQ ID NOs: 27 and 28, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human liver or muscle cells and may be the nucleotide sequence of SEQ ID NO: 127 and 128, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a liver-specific signal sequence from table. 2 or muscle-specific signal sequence - 111 047128 from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. Cm.

fig. 6 for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.15. Example 15 Nivolumab Fab cDNA Vector

A nivolumab Fab cDNA vector is constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the nivolumab heavy and light chain sequences (amino acid sequences are SEQ ID NOs: 29 and 30, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human liver or muscle cells and may be the nucleotide sequence of SEQ ID NO: 129 and 130, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a liver-specific signal sequence from table. 2 or muscle-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 7A for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.16. Example 16 Pembrolizumab Fab cDNA Vector

A pembrolizumab Fab cDNA vector is constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the pembrolizumab heavy and light chain sequences (amino acid sequences are SEQ ID NOs: 31 and 32, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human liver or muscle cells and may be the nucleotide sequence of SEQ ID NO: 131 and 132, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a liver-specific signal sequence from table. 2 or muscle-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 7B for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.17. Example 17 Ranibizumab Fab cDNA Vector

A ranibizumab Fab cDNA vector is constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the ranibizumab heavy and light chain sequences (amino acid sequences are SEQ ID NO: 33 and 34, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human retinal or liver cells and may be the nucleotide sequence of SEQ ID NO: 133 and 134, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a retinal-specific signal sequence from table. 1 or liver-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 8A for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.18. Example 18 Bevacizumab Fab cDNA Vector

A bevacizumab Fab cDNA vector is constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the bevacizumab heavy and light chain sequences (amino acid sequences are SEQ ID NO: 35 and 36, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human retinal or liver cells and may be the nucleotide sequence of SEQ ID NO: 135 and 136, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a retinal-specific signal sequence from table. 1 or liver-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic

- 112 047128 vector. See fig. 8B for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.19. Example 19 Lampalizumab Fab cDNA Vector

A lampalizumab Fab cDNA vector is constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the lampalizumab heavy and light chain sequences (amino acid sequences are SEQ ID NO: 37 and 38, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human retinal cells and may be the nucleotide sequence of SEQ ID NO: 137 and 138, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a retinal-specific signal sequence from table. 1. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 8C for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.20. Example 20 Brolucizumab scFv cDNA vector

A brolucizumab scFv cDNA vector is constructed containing a transgene containing nucleotide sequences encoding the variable domain of the brolucizumab heavy and light chain sequences (amino acid sequences are SEQ ID NO: 39 and 40, respectively). The nucleotide sequence encoding the heavy and light chain variable domains is a codon optimized for expression in human retinal or liver cells and may be the nucleotide sequence of SEQ ID NO: 139 and 140, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a retinal-specific signal sequence from table. 1 or liver-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by a flexible peptide linker. See fig. 8D for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.21. Example 21 Belimumab Fab cDNA Vector

A belimumab Fab cDNA vector is constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the belimumab heavy and light chain sequences (amino acid sequences are SEQ ID NOs: 41 and 42, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human liver cells and may be the nucleotide sequence of SEQ ID NO: 141 and 142, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a liver-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 8E for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.22. Example 22 Eculizumab Fab cDNA Vector

A vector based on eculizumab Fab cDNA was constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the eculizumab heavy and light chain sequences (amino acid sequences are SEQ ID NO: 43 and 44, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human liver cells and may be the nucleotide sequence of SEQ ID NO: 143 and 144, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a liver-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 8F for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as the CB7 promoter.

6.23. Example 23: Andecaliximab Fab cDNA Vector

A vector is constructed based on the Fab cDNA of andecaliximab, containing a transgene containing nucleotide sequences encoding the Fab part of the heavy and light chain sequences

- 113 047128 andecaliximab (amino acid sequences are SEQ ID NO: 45 and 46, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human liver cells and may be the nucleotide sequence of SEQ ID NO: 145 and 146, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a liver-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 8G for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.24. Example 24 Lanadelumab Fab cDNA Vector

A lanadelumab Fab cDNA vector is constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the lanadelumab heavy and light chain sequences (amino acid sequences are SEQ ID NOs: 47 and 48, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human liver cells and may be the nucleotide sequence of SEQ ID NO: 147 and 148, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a liver-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 8H for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.25. Example 25 Adalimumab Fab cDNA Vector

A vector based on adalimumab Fab cDNA is constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the adalimumab heavy and light chain sequences (amino acid sequences are SEQ ID NO: 49 and 50, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human liver or muscle cells and may be the nucleotide sequence of SEQ ID NO: 149 and 150, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a liver- or muscle-specific signal sequence from Table 1. 2 or 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 9A for the amino acid sequence of the transgene product. The vector further includes an inducible promoter, such as a hypoxia inducible promoter.

6.26. Example 26 Infliximab Fab cDNA Vector

A vector is constructed based on the infliximab Fab cDNA containing a transgene containing nucleotide sequences encoding the Fab portion of the infliximab heavy and light chain sequences (amino acid sequences are SEQ ID NO: 51 and 52, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human liver cells and may be the nucleotide sequence of SEQ ID NO: 151 and 152, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a liver-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 9B for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as the CB7 promoter.

6.27. Example 27 Fab aTAU cDNA Vector

A Fab aTAU cDNA vector is constructed containing a transgene containing nucleotide sequences encoding the Fab portion of the aTAU heavy and light chain sequences (amino acid sequences are SEQ ID NO: 53 and 54, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human CNS cells and may be the nucleotide sequence of SEQ ID NO: 153 and 154, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, in particular MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bi-cistronic vector. See fig. 2D for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.28. Example 28 Erenumab Fab cDNA Vector

A vector is constructed based on the erenumab Fab cDNA containing a transgene containing nucleotide sequences encoding the Fab portion of the erenumab heavy and light chain sequences (amino acid sequences are SEQ ID NO: 55 and 56, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human CNS cells and may be the nucleotide sequence of SEQ ID NO: 155 and 156, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, in particular MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 2E for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.29. Example 29: Fab BAN2401 cDNA vector

A vector is constructed based on the BAN2401 Fab cDNA containing a transgene containing nucleotide sequences encoding the Fab portion of the BAN2401 heavy and light chain sequences (amino acid sequences are SEQ ID NO: 57 and 58, respectively). The nucleotide sequence encoding the Fab portion of the heavy and light chain is a codon optimized for expression in human CNS cells and may be the nucleotide sequence of SEQ ID NO: 157 and 158, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, in particular MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161). The nucleotide sequences encoding the light chain and the heavy chain are separated by IRES elements or 2A cleavage sites to create a bicistronic vector. See fig. 2F for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

6.30. Example 30 Fab E06-scFv cDNA vector

A Fab E06-scFv cDNA-based vector is constructed containing a transgene containing nucleotide sequences encoding the heavy and light chain variable domain sequences of E06-scFv (amino acid sequences are SEQ ID NO: 59 and 60, respectively). The nucleotide sequence encoding the heavy and light chain variable domains is codon optimized for expression in human liver or muscle cells and may be the nucleotide sequence of SEQ ID NO: 159 and 160, respectively. The transgene also contains nucleotide sequences that encode a signal peptide, which may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 161) or a liver-specific signal sequence from table. 2 or muscle-specific signal sequence from table. 3. The nucleotide sequences encoding the light chain and the heavy chain are separated by a flexible peptide linker. See fig. 5D for the amino acid sequence of the transgene product. The vector further includes a constitutive promoter, such as CB7, or an inducible promoter, such as a hypoxia-inducible promoter.

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Table 4

Fab fragment amino acid sequence table

mAb Chain/ SEQ GO NO. Subsequence Aducanumab Heavy/SEQID NO:1 XVQLVESGGG WQPGRSLRL SCAASGFAFS SYGMHWVRQA PGKGLEWVAV IWFDGTKKYY TDSVKGRFTI SRDNSKNTLY LQMNTLRAED TAVYYCARDR GIGARRGPYY MDVWGKGTTV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSS GLYSLS SWTVPSSSL GTQTYICNVN HKPSNTKVDK RVEPKSCD +/- KTNT (or KTHL) +/- SRRSPA +/- PELLGGPSVFL Aducanumab Light/ SEQID NO: 2 DIQMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYSTPLTFGG GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC Crenezumab Heavy/ SEQID NO: 3 EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYGMSWVRQA PGKGLELVAS INSNGGSTYY PDSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCASGD YWGQGTTVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT KTYTCNVDHK PSNTKVDKRV ESKY +/- GPPCPPCPA +/PEFLGGPSVFL

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Crenezumab Light / SEQ ID NO: 4 DIVMTQSPLS LPVTPGEPAS ISCRSSQSLV YSNGDTYLHW YLQKPGQSPQ LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCSQSTHVP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASWCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC G antenerumab Heavy/ SEQ GO NO: 5 QVELVESGGG LVQPGGSLRL SCAASGFTFS SYAMSWVRQA PGKGLEWVSA INASGTRTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGK GNTHKPYGYV RYFDVWGQGT LVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSG LYS LSSWTVPSS SLGTQTYICN VNHKPSNTKV DKKVEPKSCD + /- KTHT (or KTHL) +/- SRRSPA +/- PELLGGPSVFL G antenerumab Light / SEQ GO NO: 6 DIVLTQSPAT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGVP ARFSGSGSGT DFTLTISSLE PEDFATYYCL QIYNMPITFG QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASWCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEK H KVYACEVTHQ GLSSPVTKSF NRGEC Dupilumab Heavy / SEQ GO NO: 7 EVQLVESGGG LEQPGGSLRL SCAGSGFTFR DYAMTWVRQA PGKGLEWVSS ISGSGGNTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKDR LSITIRPRYY GLDVWGQGTT VTVSSASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSWTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKY +/- GPPCPPCPA +/- PEFLGGPSVFL Dupilumab Light / SEQ GO NO: 8 DIVMTQSPLS LPVTPGEPAS ISCRSSQSLL YSIGYNYLDW YLQKSGQSPQ LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGF YYCMQALQTP YTFGQGTKLE IKRTVAAPSV FIFPPSDEQL KSGTASWCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SST LTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC Ixekizumab Heavy/ SEQ GO NO: 9 QVQLVQSGAE VKKPGSSVKV SCKASGYSFT DYHIHWVRQA PGQGLEWMGV INPMYGTTDY NQRFKGRVTI TADESTSTAY MELSSLRSED TAVYYCARYD YFTGTGVYWG QGTLVTVSSA STKGPSVFPL APCSRSTSES TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LY SLSSVVTV PSSSLGTKTY TCNVDHKPSN TKVDKRVESK Y +/- GPPCPPCPA +/PEFLGGPSVFL Ixekizumab Light / SEQ GO NO: 10 DIVMTQTPLS LSVTPGQPAS ISCRSSRSLV HSRGNTYLHW YLQKPGQSPQ LLIYKVSNRF IGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCSQSTHLP FTFGQGTKLE IKRTVAAPSV FIFPPSDEQL KSGTASWCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC Secukinumab Heavy / SEQ GO NO: AND EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYWMNWVRQA PGKGLEWVAA INQDGSEKYY VGSVKGRFTI SRDNAKNSLY LQMNSLRVED TAVYYCVRDY YDILTDYYIH YWYFDLWGRG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF QSSGLY SLSSWTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC D +/- KTHT (or KTHL) +/- CPPCPA +/- PELLGGPSVFL

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Sekhu throw mab Light / SEQ ID NO: 12 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPCTFG QGTRLEIKRT VAAPSVFIFP PSDEQLKSGT ASWCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKAD YEKH KVYACEVTHQ GLSSPVTKSF NRGEC Ustekinumab Heavy/ SEQ GO NO: 13 EVQLVQSGAE VKKPGESLKI SCKGSGYSFT TYWLGWVRQM PGKGLDWIGI MSPVDSDIRY SPSFQGQVTM SVDKSITTAY LQWNSLKASD TAMYYCARRR PGQGYFDFWG QGTLVTVSSS STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQ SSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKRVEPK SCD +/- KTHT (or KTHL)+/- CPPCPA +/- PELLGGPSVFL Ustekinumab Light / SEQ GO NO: 14 DIQMTQSPSS LSASVGDRVT ITCRASQGIS SWLAWYQQKP EKAPKSLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YNIYPYTFGQ GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC Mepolizumab Heavy / SEQ GO NO: 15 QVTLRESGPA LVKPTQTLTL TCTVSGFSLT SYSVHWVRQP PGKGLEWLGV IWASGGTDYN SALMSRLSIS KDTSRNQWL TMTNMDPVDT ATYYCARDPP SSLLRLDYWG RGTPVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG VTV PSSSLGTQTY ICNVNHKPSN TKVDKRVEPK SCD +/- KTHT (or KTHL) +/- CPPCPA +/- PELLGGPSVFL Mepolizumab Light / SEQ GO NO: 16 DIVMTQSPDS LAVSLGERAT INCKSSQSLL NSGNQKNYLA WXQQKPGQPP KLLIYGASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQNVHSF PFTFGGGTKL EIKRTVAAPS VFIFPPSDEQ LKSGTASWC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS STLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC Vedolizumab Heavy / SEQ GO NO: 17 QVQLVQSGAE VKKPGASVKV SCKGSGYTFT SYWMHWVRQA PGQRLEWIGE IDPSESNTNY NQKFKGRVTL TVDISASTAY MELSSLRSED TAVYYCARGG YDGWDYAIDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS LYSLSSW TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE PKSCD +/- KTHT (or KTHL) +/- CPPCPA +/- PELAGAPSVFL Vedolizumab Light / SEQ GO NO: 18 DWMTQSPLS LPVTPGEPAS ISCRSSQSLA KSYGNTYLSW YLQKPGQSPQ LLIYGISNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCLQGTHQP YTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASWCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL S STLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC Natalizumab Heavy /SEQ GO NO: 19 QVQLVQSGAE VKKPGASVKV SCKASGFNIK DTYIHWVRQA PGQRLEWMGR IDPANGYTKY DPKFQGRVTI TADTSASTAY MELSSLRSED TAVYYCAREG YYGNYGVYAM DYWGQGTLVT VSSASTKGPS VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS WTVPSSSLG TKTYTCNVDH KPSNTKVDKR VESKY +/- GPPCPPCPA +/PEFLGGPSVFL

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Natalizumab Light / SEQ GO NO: 20 DIQMTQSPSS LSASVGDRVT ITCKTSQDIN KYMAWYQQTP GKAPRLLIHY TSALQPGIPS RFSGSGSGRD YTFTISSLQP EDIATYYCLQ YDNLWTFGQG TKVEIKRTVA APSVFIFPPS DEQLKSGTAS WCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TL SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC Alirocumab Heavy/ SEQID NO: 21 EVQLVESGGG LVQPGGSLRL SCAASGFTFN NYAMNWVRQA PGKGLDWVST ISGSGGTTNY ADSVKGRFII SRDSSKHTLY LQMNSLRAED TAVYYCAKDS NWGNFDLWGR GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSWTVP SSSLG TQTYI CNVNHKPSNT KVDKKVEPKS CD +/- KTHT (or KTHL) +/- CPPCPA +/- PELLGGPSVFL Alirocumab Light / SEQID NO: 22 DIVMTQSPDS LAVSLGERAT INCKSSQSVL YRSNNRNFLG WYQQKPGQPP NLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQQYYTT PYTFGQGTKL EIKRTVAAPS VFIFPPSDEQ LKSGTASWC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC Evolocumab Heavy/ SEQID NO: 23 EVQLVQSGAE VKKPGASVKV SCKASGYTLT SYGISWVRQA PGQGLEWMGW VSFYNGNTNY AQKLQGRGTM TTDPSTSTAY MELRSLRSDD TAVYYCARGY GMDVWGQGTT VTVSSASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSWTV PSSN FGTQTYTCNV DHKPSNTKVD KTVERKCCVE +/- CPPCPA +/- PPVAG Evolocumab Light / SEQID NO: 24 ESALTQPASV SGSPGQSITI SCTGTSSDVG GYNSVSWYQQ HPGKAPKLMI YEVSNRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYC NSYTSTSMVF GGGTKLTVLG QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA GVETTTPSKQ SNNKYAASSY LSLTPE QWKS HRSYSCQVTH EGSTVEKTVA PTECS Evinacumab Heavy / SEQID NO: 25 EVQLVESGGG VIQPGGSLRL SCAASGFTFD DYAMNWVRQG PGKGLEWVSA ISGDGGSTYY ADSVKGRFTI SRDNSKNSLY LQMNSLRAED TAFFYCAKDL RNTIFGVVIP DAFDIWGQGT MVTVSSASTK GPSVFPLAPC SRSTSESTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS L SSWTVPSS SLGTKTYTCN VDHKPSNTKV DKRVESKYGP P + /CPPCPA +/- PEFLGGPSVFL Evinacumab Light / SEQID NO: 26 DIQMTQSPST LSASVGDRVT ITCRASQSIR SWLAWYQQKP GKAPKLLIYK ASSLESGVPS RFSGSGSGTE FTLTISSLQP DDFATYYCQQ YNSYSYTFGQ GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC Denosumab Heavy / SEQID NO: 27 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYAMSWVRQA PGKGLEWVSG ITGSGGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKDP GTTVIMSWFD PWGQGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPS SSLGT QTYICNVNHK PSNTKVDKKV EPKSCD +/- KTHT (or KTHL) +/- CPPCPA +/- PELLGGPSVFL

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Denosumab Light / SEQ GO NO: 28 EIVLTQSPGT LSLSPGERAT LSCRASQSVR GRYLAWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGGT DFTLTISRLE PEDFAVFYCQ QYGSSPRTFG QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASWCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSK ADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC Nivolumab Heavy / SEQID NO: 29 T KTYTCNVDH KPSNTKVDKR VESKY +/- GPPCPPCPA + /PEFLGGPSVFL Nivolumab Light / SEQID NO: 30 EIVLTQSPAT LSLSPGERAT LSCRASQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSK ADYEKHK VYACEVTHQG LSSPVTKSFN RGEC Pembrolizum b Heavy/ SEQID NO: 31 QVQLVQSGVE VKKPGASVKV SCKASGYTFT NYYMYWVRQA PGQGLEWMGG INPSNGGTNF NEKFKNRVTL TTDSSTTTAY MELKSLQFDD TAVYYCARRD YRFDMGFDYW GQGTTVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSS WT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KY +/- GPPCPPCPA +/PEFLGGPSVFL Pembrolizum b Light / SEQID NO: 32 A LSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC Ranibizumab Heavy / SEQID NO: 33 EVQLVESGGG LVQPGGSLRL SCAASGYDFT HYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP YYYGTSHWYF DVWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL Q SSGLYSLSS WTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCD +/- KTHT (or KTHL) +/- CPPCPA +/- PELLGGPSVFL Ranibizumab Light / SEQID NO: 34 DIQLTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD LT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC Bevacizumab Heavy / SEQID NO: 35 EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL LYSLSS WTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCD +/- KTHT (KTHL) +/- CPPCPA +/- PELLGGPSVFL

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Bevacizumab Light / SEQ GO NO: 36 DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSSKD LT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC Lampalizumab Heavy / SEQID NO: 37 EVQLVQSGPE LKKPGASVKV SCKASGYTFT NYGMNWVRQA PGQGLEWMGW INTYTGETTY ADDFKGRFVF SLDTSVSTAY LQISSLKAED TAVYYCEREG GVNNWGQGTL VTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSWTVPS SS LGTQTYICNV NHKPSNTKVD KKVEPKSCD +/- KTHT (or KTHL) + /CPPCPA +/- PELLGGPSVFL Lampalizumab Light / SEQID NO: 38 DIQVTQSPSS LSASVGDRVT ITCITSTDID DDMNWYQQKP GKVPKLLISG GNTLRPGVPS RFSGSGSGTD FTLTISSLQP EDVATYYCLQ SDSLPYTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSK ADYEKHK VYACEVTHQG LSSPVTKSFN RGEC Brolucizumab Heavy / SEQID NO: 39 EVQLVESGGG LVQPGGSLRL SCTASGFSLT DYYYMTWVRQ APGKGLEWVG FIDPDDDPYY ATWAKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAGGD HNSGWGLDIW GQGTLVTVSS Brolucizumab Light / SEQID NO: 40 EIVMTQSPST LSASVGDRVI ITCQASEIIH SWLAWYQQKP GKAPKLLIYL ASTLASGVPS RFSGSGSGAE FTLTISSLQP DDFATYYCQN VYLASTNGAN FGQGTKLTVL G Belimumab Heavy / SEQID NO: 41 QVQLQQSGAE VKKPGSSVRV SCKASGGTFN NNAINWVRQA PGQGLEWMGG IIPMFGTAKY SQNFQGRVAI TADESTGTAS MELSSLRSED TAVYYCARSR DLLLFPHHAL SPWGRGTMVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSL SS WTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCD +/- KTHT (or KTHL) +/- CPPCPA +/-PELLGGPSVFL Belimumab Light / SEQID NO: 42 SSELTQDPAV SVALGQTVRV TCQGDSLRSY YASWYQQKPG QAPVLVIYGK NNRPSGIPDR FSGSSSGNTA SLTITGAQAE DEADYYCSSR DSSGNHWVFG GGTELTVLGQ PKAAPSVTLF PPSSEELQAN KATLVCLISD FYPGAVTVAW KADSSPVKAG VETTTPSKQS NNKYAASSYL SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP TECS Eculizumab Heavy/ SEQID NO: 43 QVQLVQSGAE VKKPGASVKV SCKASGYIFS NYWIQWVRQA PGQGLEWMGE ILPGSGSTEY TENFKDRVTM TRDTSTSTVY MELSSLRSED TAVYYCARYF FGSPNWYFD VWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSNFGT QTYTCNVDHK PSNTKVDKTV ERKCCVE +/- CPPCPA +/- PPVAG Eculizumab Light / SEQID NO: 44 DIQMTQSPSS LSASVGDRVT ITCGASENIY GALNWYQQKP GKAPKLLIYG ATNLADGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQN VLNTPLTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ

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ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC Andekalixim ab Heavy / SEQ GO NO: 45 QVQLQESGPG LVKPSETLSL TCTVSGFSLL SYGVHWVRQP PGKGLEWLGV IWTGGTTNYN SALMSRFTIS KDDSKNTVYL KMNSLKTEDT AIYYCARYYY GMDYWGQGTL VTVSSASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLY SL SSWTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKY +/- GPPCPPCPA + /PEFLGGPSVFL Andekalixim ab Light / SEQID NO: 46 DIQMTQSPSS LSASVGDRVT ITCKASQDVR NTVAWYQQKP GKAPKLLIYS SSYRNTGVPD RFSGSGSGTD FTLTISSLQA EDVAVYYCQQ HYITPYTFGG GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC Lanadelumab Heavy / SEQID NO: 47 EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR IGVPRRDEFD IWGQGTMVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLSV SSGLYSLSQ VTVPSSSLGT QTYICNVNHK PSNTKVDKRV EPKSCD +/- KTHT (or KTHL) +/- CPPCPA +/- PELLGGPSVFL Lanadelumab Light / SEQID NO: 48 DIQMTQSPST LSASVGDRVT ITCRASQSIS SWLAWYQQKP GKAPKLLIYK ASTLESGVPS RFSGSGSGTE FTLTISSLQP DDFATYYCQQ YNTYWTFGQG TKVEIKRTVA APSVFIFPPS DEQLKSGTAS WCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TLTL SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC Adalimumab Heavy/ SEQID NO: 49 EVQLVESGGG LVQPGRSLRL SCAASGFTFD DYAMHWVRQA PGKGLEWVSA ITWNSGHIDY ADSVEGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAKVS YLSTASSLDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TV PSSSLGTQ TYICNVNHKP SNTKVDKKVE PKSCD +/- KTHT (KTHL) +/- CPPCPA +/- PELLGGPSVFL Adalimumab Light / SEQID NO: 50 RFSGSGSGTD FTLTISSLQP EDVATYYCQR YNRAPYTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC Infliximab Heavy/ SEQID NO: 51 EVKLEESGGG LVQPGGSMKL SCVASGFIFS NHWMNWVRQS PEKGLEWVAE IRSKSINSAT HYAESVKGRF TISRDDSKSA VYLQMTDLRT EDTGVYYCSR NYYGSTYDYW GQGTTLTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLY SLSSWT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCD +/- KTHT (KTHL) +/- CPPCPA +/- PELLGGPSVFL Infliximab Light / SEQID NO: 52 DILLTQSPAI LSVSPGERVS FSCRASQFVG SSIHWYQQRT NGSPRLLIKY ASESMSGIPS RFSGSGSGTD FTLSINTVES EDIADYYCQQ SHSWPFTFGS GTNLEVKRTV AAPSVFIFPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADY EKHK VYACEVTHQG LSSPVTKSFN RGEC

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aTAU Heavy / SEQ GO NO: 53 EVKWESGGG LVQPGGSMKL SCWSGTFFS NYWVNWVRQA PGKGLEWVAQ IRLKSDNYAT HYEESVKGRF TISRDDSKSS VYLQMNNLRA EDSGIYYCTN WEDYWGQGTT VTVSSASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSW TVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKY +/- GPPCPPCPA + /PEFLGGPSVFL aTAU Light / SEQ GO NO: 54 DIVLTQSPDS LAVSLGERAT ISCRASQSVS TSRYSYIHWY QQKPGQPPKL LIKYASNLES GVPSRFSGSG SGTDFTLNIH PLEPEDFATY YCHHSWEIPL TFGQGTKLEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC Erenumab Heavy / SEQ GO NO: 55 QVQLVESGGG WQPGRSLRL SCAASGFTFS SFGMHWVRQA PGKGLEWVAV ISFDGSIKYS VDSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCARDR LNYYDSSGYY HYKYYGMAVW GQGTTVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV QSS GLYSLSSWT VPSSNFGTQT YTCNVDHKPS NTKVDKTVER KCCVE +/CPPCPA +/-PPVAG Erenumab Light / SEQ GO NO: 56 QSVLTQPPSV SAAPGQKVTI SCSGSSSNIG NNYVSWYQQL PGTAPKLLIY DNNKRPSGIP DRFSGSKSGT STTLGITGLQ TGDEADYYCG TWDSRLSAW FGGGTKLTVL GQPKANPTVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADGSPVK AGVETTKPSK QSNNKYAASS YLSLTPEQW K SHRSYSCQVT HEGSTVEKTV APTECS BAN2401 Heavy / SEQ GO NO: 57 EVQLVESGGG LVQPGGSLRL SCSASGFTFS SFGMHWVRQA PGKGLEWVAY ISSGSSTIYY GDTVKGRFTI SRDNAKNSLF LQMSSLRAED TAVYYCAREG GYYYGRSYYT MDYWGQGTTV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLY SLS SWTVPSSSL GTQTYICNVN HKPSNTKVDK KVEPKSCD +/- KTHT (KTHL) +/- CPPCPA +/- PELLGG BAN2401 Light / SEQ GO NO: 58 DWMTQSPLS LPVTPGAPAS ISCRSSQSIV HSNGNTYLEW YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLRI SRVEAEDVGI YYCFQGSHVP PTFGPGTKLE IKRTVAAPSV FIFPPSDEQL KSGTASWCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLS KAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC E06-scFV Heavy / SEQ GO NO: 59 EVKLVESGGG LVQPGGSLRL SCATSGFTFS DFYMEWVRQA PGKRLEWIAA SRNKANDYTT EYADSVKGRF IVSRDTSQSI LYLQMNALRA EDTAIYYCAR DYYGSSYWYF DVWGAGTTVT VSS EOB-scFV Light / SEQ GO NO: 60 DIVMTQSPSS LSVSAGKKVT ISCTASESLY SSKHKVHYLA WYQKKPEQSP KLLIYGASNR YIGVPDRFTG SGSGTDFTLT ISSVQVEDLT HYYCAQFYSY PLTFGAGTKL EIK AAVrhlO SEQ GO NO:80 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ AKKRVLEPLG LVEEGAKTAP GKKRPVEPSP QRSPDSSTGI GKKGQQPAKK QTGDS ESVPDPQPIG EPPAGPSGLG SGTMAAGGGA PMADNNEGAD GVGSSSGNWH CDSTWLGDRV ITTSTRTWAL PTYNNHLYKQ ISNGTSGGST NDNTYFGYST PWGYFDFNRF HCHFSPRDWQ RLINNNWGFR PKRLNFKLFN IQVKEVTQNE GTKTIANNLT STIQVFTDSE YQLPYVLGSA HQGCLPPFPA DVFMIPQYGY LTL NNGSQAV GRSSFYCLEY FPSQMLRTGN NFEFSYQFED VPFHSSYAHS QSLDRLMNPL IDQYLYYLSR TQSTGGTAGT QQLLFSQAGP NNMSAQAKNW LPGPCYRQQR VSTTLSQNNN SNFAWTGATK YHLNGRDSLV NPGVAMATHK DDEERFFPSS GVLMFGKQGA GKDNVDYSSV IKT TNPVATEQYG WADNLQQQN AAPIVGAVNS QGALPGMVWQ NRDVYLQGPI WAKIPHTDGN FHPSPLMGGF GLKHPPPQIL IKNTPVPADP PTTFSQAKLA SFITQYSTGQ VSVEIEWELQ KENSKRWNPE IQYTSNYYKS TNVDFAVNTD GTYSEPRPIG TRYLTRNL

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Table 5

Fab fragment nucleic acid sequence table

mAb Chain/ SEQ ID NO. Subsequence Aducanumab I'm heavy/ SEQ GO NO:101 gtgcagctgg tggagagcgg cggcggcgtg gtgcagcccg gcagaagcct gagactgagc tgcgccgcca gcggcttcgc cttcagcagc tacggcatgc actgggtgag acaggccccc ggcaagggcc tggagtgggt ggccgtgatc tggttcgacg gcaccaagaa gtactacacc gac agcgtga agggcagatt caccatcagc agagacaaca gcaagaacac cctgtacctg cagatgaaca ccctgagagc cgaggacacc gccgtgtact actgcgccag agacagaggc atcggcgcca gaagaggccc ctactacatg gacgtgtggg gcaagggcac caccgtgacc gtgagcagcg ccagcaccaa gggccccagc gtgttccccc tggcccccag cagcaagagc accagcggcg gcaccgccgc cctgggctgc ctggtgaagg actacttccc cgagcccgtg accgtgagct ggaacagcgg cgccctgacc agcggcgtgc acaccttccc cgccgtgctg cagagcagcg gcctgtacag cctgagcagc gtggtgaccg tgcccagcag cagcctgggc acccagacct acatctgcaa cgtgaaccac aagcccagca acaccaaggt ggacaagaga gtggagccca agagctgcga c +/- a agacccacacc (or aagacccacctg) + /tgccccccctgccccgcc +/cccgagctgctgggcggccccagcgtgttcctg Aducanumab Light / SEQID NO: 102 gacatccaga tgacccagg ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc atcacctgca gagccagcca gagcatcagc agctacctga actggtacca gcagaagccc ggcaaggccc ccaagctgct gatctacgcc gccagcagcc tgcagagcgg cgtgcccagc agattcagcg gcagc ggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc gaggacttcg ccacctacta ctgccagcag agctacagca cccccctgac cttcggcggc ggcaccaagg tggagatcaa gagaaccgtg gccgccccca gcgtgttcat cttccccccc agcgacgagc agctgaagag cggc accgcc agcgtggtgt gcctgctgaa caacttctac cccagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag

- 124 047128

gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggc ctgagcagcc ccgtgaccaa gagcttcaac agaggcgagt gc Crenezumab I am heavy / SEQ ID NO: 103 gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgagactg agctgcgccg ccagcggctt caccttcagc agctacggca tgagctgggt gagacaggcc cccggcaagg gcctggagct ggtggccagc atcaacagca acggcggcag cacctactac cccgacagc g tgaagggcag attcaccatc agcagagaca acgccaagaa cagcctgtac ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc cagcggcgac tactggggcc agggcaccac cgtgaccgtg agcagcgcca gcaccaaggg ccccagcgtg ttccccctgg ccctgcag c agaagcacc agcgagagca ccgccgccct gggctgcctg gtgaaggact acttccccga gcccgtgacc gtgagctgga acagcggcgc cctgaccagc ggcgtgcaca ccttccccgc cgtgctgcag agcagcggcc tgtacagcct gagcagcgtg gtgaccgtgc ccagcagcag cctgggcacc aagacctaca cctgcaacgt ggaccacaag cccagcaaca ccaaggtgga caagagagtg gagagcaagt ac +/- ggccccccctgccccccctgccccgcc + /cccgagttcct gggcggccccagcgtgttcctg Crenezumab Light / SEQID NO: 104 gacatcgtga tgacccagag ccccctgagc ctgcccgtga cccccggcga gcccgccagc atcagctgca gaagcagcca gagcctggtg tacagcaacg gcgacaccta cctgcactgg tacctgcaga agcccggcca gagcccccag ctgctgatct acaaggtgag caacagattc agcggcgtg c ccgacagatt cagcggcagc ggcagcggca ccgacttcac cctgaagatc agcagagtgg aggccgagga cgtgggcgtg tactactgca gccagagcac ccacgtgccc tggaccttcg gccagggcac caaggtggag atcaagagaa ccgtggccgc ccccagcgtg ttcatcttcc ccccca gcga cgagcagctg aagagcggca ccgccagcgt ggtgtgcctg ctgaacaact tctaccccag agaggccaag gtgcagtgga aggtggacaa cgccctgcag agcggcaaca gccaggagag cgtgaccgag caggacagca aggacagcac ctacagcctg agcagcaccc tgaccctgag caaggccgac tacgagaagc acaaggtgta cgcctgcgag gtgacccacc agggcctgag cagccccgtg accaagagct tcaacagagg cgagtgc G antenerumab I'm Heavy SEQ ID NO: 105 caggtggagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgagactg agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gagacaggcc cccggcaagg gcctggagtg ggtgagcgcc atcaacgcca gcggcaccag aacctactac gccgacagc g tgaagggcag attcaccatc agcagagaca acagcaagaa caccctgtac ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc cagaggcaag ggcaacaccc acaagcccta cggctacgtg agatacttcg acgtgtgggg ccagggcacc

- 125 047128

ctggtgaccg tgagcagcgc cagcaccaag ggccccagcg tgttccccct ggcccccagc agcaagagca ccagcggcgg caccgccgcc ctgggctgcc tggtgaagga ctacttcccc gagcccgtga ccgtgagctg gaacagcggc gccctgacca gcggcgtgca caccttcccc gccg tgctgc agagcagcgg cctgtacagc ctgagcagcg tggtgaccgt gcccagcagc agcctgggca cccagaccta catctgcaac gtgaaccaca agcccagcaa caccaaggtg gacaagaagg tggagcccaa gagctgcgac +/- aagacccacacc (or aagacccacctg) +/- tgccccccct gccccgcc +/ ccgagctgctgggcggccccagcgtgttcctg G antenerumab Light / SEQ ID NO: 106 gacatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gagagccacc ctgagctgca gagccagcca gagcgtgagc agcagctacc tggcctggta ccagcagaag cccggccagg cccccagact gctgatctac ggcgccagca gcagagccac cggcgtgccc gccagattca gcggcag cgg cagcggcacc gacttcaccc tgaccatcag cagcctggag cccgaggact tcgccaccta ctactgcctg cagatctaca acatgcccat caccttcggc cagggcacca aggtggagat caagagaacc gtggccgccc ccagcgtgtt catcttcccc cccagcgacg agcagctgaa gagcggcacc gccagcgt gg tgtgcctgct gaacaacttc taccccagag aggccaaggt gcagtggaag gtggacaacg ccctgcagag cggcaacagc caggagagcg tgaccgagca ggacagcaag gacagcacct acagcctgag cagcaccctg accctgagca aggccgacta cgagaagcac aaggtgtacg cctgcgaggt gacccaccag ggcctgagca gccccgtgac caagagcttc aacagaggcg agtgc Dupilumab I am heavy/SEQ ID NO: 107 gaggtgcagc tggtggagag cggcggcggc ctggagcagc ccggcggcag cctgagactg agctgcgccg gcagcggctt caccttcaga gactacgcca tgacctgggt gagacaggcc cccggcaagg gcctggagtg ggtgagcagc atcagcggca gcggcggcaa cacctactac gccgacag cg tgaagggcag attcaccatc agcagagaca acagcaagaa caccctgtac ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc caaggacaga ctgagcatca ccatcagacc cagatactac ggcctggacg tgtggggcca gggcaccacc gtgaccgtga gcagcgccag caccaagggc ccca gcgtgt tccccctggc cccctgcagc agaagcacca gcgagagcac cgccgccctg ggctgcctgg tgaaggacta cttccccgag cccgtgaccg tgagctggaa cagcggcgcc ctgaccagcg gcgtgcacac cttccccgcc gtgctgcaga gcagcggcct gtacagcctg agcagcgtgg tgaccgtgcc cagcagcagc ctgggcacca agacctacac ctgcaacgtg gaccacaagc ccagcaacac caaggtggac aagagagtgg agagcaagtac +/ggccccccctgccccccctgccccgcc +/cccgagttcctgggcggccccagcgtgttcctg

- 126 047128

Dupilumab Light / SEQ GO NO: 108 gacatcgtga tgacccagag ccccctgagc ctgcccgtga cccccggcga gcccgccagc atcagctgca gaagcagcca gagcctgctg tacagcatcg gctacaacta cctggactgg tacctgcaga agagcggcca gagcccccag ctgctgatct acctgggcag caacagagcc agcggcgtgc ccgacagatt cagcggcagc ggcagcggca ccgacttcac cctgaagatc agcagagtgg aggccgagga cgtgggcttc tactactgca tgcaggccct gcagaccccc tacaccttcg gccagggcac caagctggag atcaagagaa ccgtggccgc ccccagcgtg ttcatcttcc cccccagcga cga gcagctg aagagcggca ccgccagcgt ggtgtgcctg ctgaacaact tctaccccag agaggccaag gtgcagtgga aggtggacaa cgccctgcag agcggcaaca gccaggagag cgtgaccgag caggacagca aggacagcac ctacagcctg agcagcaccc tgaccctgag caaggccgac tacgagaagc acaaggtgta cgcctgcgag gtgacccacc agggcctgag cagccccgtg accaagagct tcaacagagg cgagtgc Ixekizumab I am heavy / SEQ GO NO: 109 caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcagcag cgtgaaggtg agctgcaagg ccagcggcta cagcttcacc gactaccaca tccactgggt gagacaggcc cccggccagg gcctggagtg gatgggcgtg atcaacccca tgtacggcac caccgactac aaccagagat tcaaggg cag agtgaccatc accgccgacg agagcaccag caccgcctac atggagctga gcagcctgag aagcgaggac accgccgtgt actactgcgc cagatacgac tacttcaccg gcaccggcgt gtactggggc cagggcaccc tggtgaccgt gagcagcgcc agcaccaagg gccccagcgt gttccccct g gccccctgca gcagaagcac cagcgagagc accgccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgagctgg aacagcggcg ccctgaccag cggcgtgcac accttccccg ccgtgctgca gagcagcggc ctgtacagcc tgagcagcgt ggtgaccgtg cccagcagca gcctgggcac caagacctac acctgcaacg tggaccacaa gcccagcaac accaaggtgg acaagagagt ggagagcaag tac + /ggccccccctgcc ccccgcc + /cccgagttcctgggcggccccagcgtgttcctg Ixekizumab Light / SEQID NO: 110 gacatcgtga tgacccagac ccccctgagc ctgagcgtga cccccggcca gcccgccagc atcagctgca gaagcagcag aagcctggtg cacagcagag gcaacaccta cctgcactgg tacctgcaga agcccggcca gagcccccag ctgctgatct acaaggtgag caacagattc atcggcgtgc ccgacagatt cagcggcagc ggcagcggca ccgacttcac cctgaagatc agcagagtgg aggccgagga cgtgggcgtg tactactgca gccagagcac ccacctgccc ttcaccttcg gccagggcac caagctggag atcaagagaa ccgtggccgc ccccagcgtg ttcatcttcc cgagcagctg aagagcggca ccgccagcgt ggtgtgcctg ctgaacaact tctaccccag agaggccaag gtgcagtgga aggtggacaa cgccctgcag agcggcaaca gccaggagag cgtgaccgag caggacagca

- 127 047128

aggacagcac ctacagcctg agcagcaccc tgaccctgag caaggccgac tacgagaagc acaaggtgta cgcctgcgag gtgacccacc agggcctgag cagccccgtg accaagagct tcaacagagg cgagtgc Secukinumab I'm heavy / SEQ IDNO: 111 gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgagactg agctgcgccg ccagcggctt caccttcagc aactactgga tgaactgggt gagacaggcc cccggcaagg gcctggagtg ggtggccgcc atcaaccagg acggcagcga gaagtactac gtgggcag cg tgaagggcag attcaccatc agcagagaca acgccaagaa cagcctgtac ctgcagatga acagcctgag agtggaggac accgccgtgt actactgcgt gagagactac tacgacatcc tgaccgacta ctacatccac tactggtact tcgacctgtg gggcagaggc accctggtga ccgtgagcag cacc aagggcccca gcgtgttccc cctggccccc agcagcaaga gcaccagcgg cggcaccgcc gccctgggct gcctggtgaa ggactacttc cccgagcccg tgaccgtgag ctggaacagc ggcgccctga ccagcggcgt gcacaccttc cccgccgtgc tgcagagcag cggcctgtac agcctgagca gcgtggtgac cgtgcccagc agcagcctgg gcacccagac ctacatctgc aacgtgaacc acaagcccag caacaccaag gtggacaaga gagtggagcc ca agagctgc gac +/- aagacccacacc (or aagacccacctg) +/- tgccccccctgccccgcc +/ ccgagctgctgggcggccccagcgtgttcctg Secukinumab Light / SEQ ID NO: 112 gagatcgtgc tgacccagag ccccggcacc ctgagcctga gccccggcga gagagccacc ctgagctgca gagccagcca gagcgtgagc agcagctacc tggcctggta ccagcagaag cccggccagg cccccagact gctgatctac ggcgccagca gcagagccac cggcatcccc gacagattca gcggcagcgg ca gcggcacc gacttcaccc tgaccatcag cagactggag cccgaggact tcgccgtgta ctactgccag cagtacggca gcagcccctg caccttcggc cagggcacca gactggagat caagagaacc gtggccgccc ccagcgtgtt catcttcccc cccagcgacg agcagctgaa gagcggcacc gccagcgtgg t gtgcctgct gaacaacttc taccccagag aggccaaggt gcagtggaag gtggacaacg ccctgcagag cggcaacagc caggagagcg tgaccgagca ggacagcaag gacagcacct acagcctgag cagcaccctg accctgagca aggccgacta cgagaagcac aaggtgtacg cctgcgaggt gacccaccag ggcctgagca gccccgtgac caagagcttc aacagaggcg agtgc Ustekinumab I am heavy/SEQ IDNO: FROM gaggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgagag cctgaagatc agctgcaagg gcagcggcta cagcttcacc acctactggc tgggctgggt gagacagatg cccggcaagg gcctggactg gatcggcatc atgagccccg tggacagcga catcagatac agccccagct gggcca ggtgaccatg agcgtggaca agagcatcac caccgcctac ctgcagtgga acagcctgaa ggccagcgac accgccatgt actactgcgc cagaagaaga cccggccagg gctacttcga

- 128 047128

cttctggggc cagggcaccc tggtgaccgt gagcagcagc agcaccaagg gccccagcgt gttccccctg gcccccagca gcaagagcac cagcggcggc accgccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgagctgg aacagcggcg ccctgaccag cgg cgtgcac accttccccg ccgtgctgca gagcagcggc ctgtacagcc tgagcagcgt ggtgaccgtg cccagcagca gcctgggcac ccagacctac atctgcaacg tgaaccacaa gcccagcaac accaaggtgg acaagagagt ggagcccaag agctgcgac + /aagacccaca cc (or aagacccacctg)+/tgccccccctgccccgcc +/- cccgagctgctgggcggccccagcgtgttcctg Stekinumab Light / SEQ GO NO: 114 gacatccaga tgacccagg ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc atcacctgca gagccagcca gggcatcagc agctggctgg cctggtacca gcagaagccc gagaaggccc ccaagagcct gatctacgcc gccagcagcc tgcagagcgg cgtgcccagc agattcagcg gcagc ggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc gaggacttcg ccacctacta ctgccagcag tacaacatct acccctacac cttcggccag ggcaccaagc tggagatcaa gagaaccgtg gccgccccca gcgtgttcat cttccccccc agcgacgagc agctgaagag cggcacc gcc agcgtggtgt gcctgctgaa caacttctac cccagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggc ctgagcagcc ccgtgaccaa gagcttcaac agaggcgagt gc Mepolizumab I am heavy / SEQ GO NO: 115 caggtgaccc tgagagagag cggccccgcc ctggtgaagc ccacccagac cctgaccctg acctgcaccg tgagcggctt cagcctgacc agctacagcg tgcactgggt gagacagccc cccggcaagg gcctggagtg gctgggcgtg atctgggcca gcggcggcac cgactacaac agcgccctga tgagcagact gagcatcagc aaggacacca gcagaaacca ggtggtgctg accatgacca acatggaccc cgtggacacc gccacctact actgcgccag agaccccccc agcagcctgc tgagactgga ctactggggc agaggcaccc ccgtgaccgt gagcagcgcc agcaccaagg gccccagcgt gttccccctg gcccccagca gcaagagcac cagcggcggc accgccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgagctgg aacagcggcg ccctgaccag cggcgtgcac accttccccg ccgtgctgca gagcagcggc ctgtacagcc tgagcagcgt ggtgaccgtg cccagcagca gcctgggcac ccagacctac atctgcaacg tgaaccacaa gcccagcaac accaaggtgg acaagagagt ggagcccaag agctgcgac +/aagacccacacc (or aagacccacctg) +/tgccccccctgccccgcc +/cccgagctgctgggcggccccagcgtgttcctg

- 129 047128

Mepolizumab Light / SEQ GO NO: 116 gacatcgtga tgacccagag ccccgacagc ctggccgtga gcctgggcga gagagccacc atcaactgca agagcagcca gagcctgctg aacagcggca accagaagaa ctacctggcc tggcagcaga agcccggcca gccccccaag ctgctgatct acggcgccag caccagagag agcggcgtgc ccgacag att cagcggcagc ggcagcggca ccgacttcac cctgaccatc agcagcctgc aggccgagga cgtggccgtg tactactgcc agaacgtgca cagcttcccc ttcaccttcg gcggcggcac caagctggag atcaagagaa ccgtggccgc ccccagcgtg ttcatcttcc cccccagcga cgag cagctg aagagcggca ccgccagcgt ggtgtgcctg ctgaacaact tctaccccag agaggccaag gtgcagtgga aggtggacaa cgccctgcag agcggcaaca gccaggagag cgtgaccgag caggacagca aggacagcac ctacagcctg agcagcaccc tgaccctgag caaggccgac tacgagaagc acaaggtgta cgcctgcgag gtgacccacc agggcctgag cagccccgtg accaagagct tcaacagagg cgagtgc Vedolizumab I am heavy / SEQ GO NO: 117 caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg agctgcaagg gcagcggcta caccttcacc agctactgga tgcactgggt gagacaggcc cccggccaga gactggagtg gatcggcgag atcgacccca gcgagagcaa caccaactac aaccagaagt tcaaggg cag agtgaccctg accgtggaca tcagcgccag caccgcctac atggagctga gcagcctgag aagcgaggac accgccgtgt actactgcgc cagaggcggc tacgacggct gggactacgc catcgactac tggggccagg gcaccctggt gaccgtgagc agcgccagca ccaagggccc cagcgtgttc cccctggccc ccagcagcaa gagcaccagc ggcggcaccg ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg agctggaaca gcggcgccct gaccagcggc gtgcacacct tccccgccgt gctgcagagc agcggcctgt acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gaaggtggag cccaagagct gcgac + /aagacccac acc (or aagacccacctg) + /tgccccccctgccccgcc +/cccgagctggccggcgcccccagcgtgttcctg Vedolizumab Light / SEQID NO: 118 gacgtggtga tgacccagag ccccctgagc ctgcccgtga cccccggcga gcccgccagc atcagctgca gaagcagcca gagcctggcc aagagctacg gcaacaccta cctgagctgg tacctgcaga agcccggcca gagcccccag ctgctgatct acggcatcag caacagattc agcggcgtgc cagatt cagcggcagc ggcagcggca ccgacttcac cctgaagatc agcagagtgg aggccgagga cgtgggcgtg tactactgcc tgcagggcac ccaccagccc tacaccttcg gccagggcac caaggtggag atcaagagaa ccgtggccgc ccccagcgtg ttcatcttcc cccccagcga cga gcagctg aagagcggca ccgccagcgt ggtgtgcctg ctgaacaact tctaccccag agaggccaag gtgcagtgga aggtggacaa cgccctgcag

- 130 047128

agcggcaaca gccaggagag cgtgaccgag caggacagca aggacagcac ctacagcctg agcagcaccc tgaccctgag caaggccgac tacgagaagc acaaggtgta cgcctgcgag gtgacccacc agggcctgag cagccccgtg accaagagct tcaacagagg cgagtgc Natalizumab I am heavy/SEQ GO NO: 119 caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg agctgcaagg ccagcggctt caacatcaag gacacctaca tccactgggt gagacaggcc cccggccaga gactggagtg gatgggcaga atcgaccccg ccaacggcta caccaagtac gaccccaagt tccaggcag a gtgaccatc accgccgaca ccagcgccag caccgcctac atggagctga gcagcctgag aagcgaggac accgccgtgt actactgcgc cagagaggc tactacggca actacggcgt gtacgccatg gactactggg gccagggcac cctggtgacc gtgagcagcg ccagcaccaa gggccccagc gtgttc cccc tggccccctg cagcagaagc accagcgaga gcaccgccgc cctgggctgc ctggtgaagg actacttccc cgagcccgtg accgtgagct ggaacagcgg cgccctgacc agcggcgtgc acaccttccc cgccgtgctg cagagcagcg gcctgtacag cctgagcagc gtggtgaccg tgcccagcag cagcctgggc accaagacct acacctgcaa cgtggaccac aagcccagca acaccaaggt ggacaagaga gtggagagca agtac +/ggccccccctg ccccccctgccccgcc +/cccgagttcctgggcggccccagcgtgttcctg Natalizumab Light\ SEQ GO NO: 120 gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc atcacctgca agaccagcca ggacatcaac aagtacatgg cctggtacca gcagaccccc ggcaaggccc ccagactgct gatccactac accagcgccc tgcagcccgg catccccagc agattcagcg gcagcggcag c ggcagagac tacaccttca ccatcagcag cctgcagccc gaggacatcg ccacctacta ctgcctgcag tacgacaacc tgtggacctt cggccagggc accaaggtgg agatcaagag aaccgtggcc gcccccagcg tgttcatctt cccccccagc gacgagcagc tgaagagcgg caccgccagc gtggtg tgcc tgctgaacaa cttctacccc agagaggcca aggtgcagtg gaaggtggac aacgccctgc agagcggcaa cagccaggag agcgtgaccg agcaggacag caaggacagc acctacagcc tgagcagcac cctgaccctg agcaaggccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg agcagccccg tgaccaagag cttcaacaga ggcgagtgc Alirocumab I am heavy/SEQ GO NO: 121 t gaagggcag attcatcatc agcagagaca gcagcaagca caccctgtac ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc caaggacagc aactggggca acttcgacct gtggggcaga ggcaccctgg tgaccgtgag cagcgccagc

- 131 047128

accaagggcc ccagcgtgtt ccccctggcc cccagcagca agagcaccag cggcggcacc gccgccctgg gctgcctggt gaaggactac ttccccgagc ccgtgaccgt gagctggaac agcggcgccc tgaccagcgg cgtgcacacc ttccccgccg tgctgcagag cagcggcctg cctga gcagcgtggt gaccgtgccc agcagcagcc tgggcaccca gacctacatc tgcaacgtga accacaagcc cagcaacacc aaggtggaca agaaggtgga gcccaagagc tgcgac + /aagacccacacc (or aagacccacctg) +/tgccccccctgccccgcc +/- cccgagctgctgggcggccccagcgtgttcctg Alirocumab Light / SEQ GO NO: 122 gacatcgtga tgacccagag ccccgacagc ctggccgtga gcctgggcga gagagccacc atcaactgca agagcagcca gagcgtgctg tacagaagca acaacagaaa cttcctgggc tggtaccagc agaagcccgg ccagcccccc aacctgctga tctactgggc cagcaccaga gagagcggcg tgccc gacag attcagcggc agcggcagcg gcaccgactt caccctgacc atcagcagcc tgcaggccga ggacgtggcc gtgtactact gccagcagta ctacaccacc ccctacacct tcggccaggg caccaagctg gagatcaaga gaaccgtggc cgcccccagc gtgttcatct tcccccccag cgacgagcag ctga agagcg gcaccgccag cgtggtgtgc ctgctgaaca acttctaccc cagagaggcc aaggtgcagt ggaaggtgga caacgccctg cagagcggca acagccagga gagcgtgacc gagcaggaca gcaaggacag cacctacagc ctgagcagca ccctgaccct gagcaaggcc gactacgaga agcacaaggt gtacgcctgc gaggtgaccc accaccgcct gagcagcccc gtgaccaaga gcttcaacag aggcgagtgc Evolocumab I'm heavy/SEQ GO NO: 123 gaggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg agctgcaagg ccagcggcta caccctgacc agctacggca tcagctgggt gagacaggcc cccggccagg gcctggagtg gatgggctgg gtgagcttct acaacggcaa caccaactac gcccagaagc tg cagggcag aggcaccatg accaccgacc ccagcaccag caccgcctac atggagctga gaagcctgag aagcgacgac accgccgtgt actactgcgc cagaggctac ggcatggacg tgtggggcca gggcaccacc gtgaccgtga gcagcgccag caccaagggc cccagcgtgt tccccctggc cccctgcagc a gaagcacca gcgagagcac cgccgccctg ggctgcctgg tgaaggacta cttccccgag cccgtgaccg tgagctggaa cagcggcgcc ctgaccagcg gcgtgcacac cttccccgcc gtgctgcaga gcagcggcct gtacagcctg agcagcgtgg tgaccgtgcc cagcagcaac ttcggcaccc agacctacac ctgcaacgtg gaccacaagc ccagcaacac caaggtggac aagaccgtgg agagaaagtg ctgcgtggag +/- tgccccccctgccccgcc +/- ccccccgtggccggc Evolocumab Light / SEQID NO: 124 gagagcgccc tgacccagcc cgccagcgtg agcggcagcc ccggccagag catcaccatc agctgcaccg gcaccagcag cgacgtgggc ggctacaaca gcgtgagctg gtaccagcag

- 132 047128

caccccggca aggcccccaa gctgatgatc tacgaggtga gcaacagacc cagcggcgtg agcaacagat tcagcggcag caagagcggc aacaccgcca gcctgaccat cagcggcctg caggccgagg acgaggccga ctactactgc aacagctaca ccagcaccag catggtgttc ggcggcggca ccaagctgac c gtgctgggc cagcccaagg ccgcccccag cgtgaccctg ttccccccca gcagcgagga gctgcaggcc aacaaggcca ccctggtgtg cctgatcagc gacttctacc ccggcgccgt gaccgtggcc tggaaggccg acagcagccc cgtgaaggcc ggcgtggaga ccaccacccc cagcaagcag agcaacaaca agtacgccgc cagcagctac ctgagcctga cccccgagca gtggaagagc cacagaagct acagctgcca ggtgacccac gagggcagca ccgtggagaa gaccgtggcc cccaccgagt gcagc Evinacumab I am heavy / SEQ GO NO: 125 gaggtgcagc tggtggagag cggcggcggc gtgatccagc ccggcggcag cctgagactg agctgcgccg ccagcggctt caccttcgac gactacgcca tgaactgggt gagacagggc cccggcaagg gcctggagtg ggtgagcgcc atcagcggcg acggcggcag cacctactac gccgaca gcg tgaagggcag attcaccatc agcagagaca acagcaagaa cagcctgtac ctgcagatga acagcctgag agccgaggac accgccttct tctactgcgc caaggacctg agaaacacca tcttcggcgt ggtgatcccc gacgccttcg acatctgggg ccagggcacc atggtgaccg tgagcagc gc cagcaccaag ggccccagcg tgttccccct ggccccctgc agcagaagca ccagcgagag caccgccgcc ctgggctgcc tggtgaagga ctacttcccc gagcccgtga ccgtgagctg gaacagcggc gccctgacca gcggcgtgca caccttcccc gccgtgctgc agagcagcgg cctgtacagc ctgagcagcg tggtgaccgt gcccagcagc agcctgggca ccaagaccta cacctgcaac gtggaccaca agcccagcaa caccaaggtg gacaagagg tggagagcaa gtacgg cccc ccc +/- tgccccccctgccccgcc + /cccgagttcctgggcggccccagcgtgttcctg Evinacumab Light / SEQ GO NO: 126 gacatccaga tgacccagag ccccagcacc ctgagcgcca gcgtgggcga cagagtgacc atcacctgca gagccagcca gagcatcaga agctggctgg cctggtacca gcagaagccc ggcaaggccc ccaagctgct gatctacaag gccagcagcc tggagagcgg cgtgcccagc agattcagcg gcagcggca g cggcaccgag ttcaccctga ccatcagcag cctgcagccc gacgacttcg ccacctacta ctgccagcag tacaacagct acagctacac cttcggccag ggcaccaagc tggagatcaa gagaaccgtg gccgccccca gcgtgttcatcat cttccccccc agcgacgagc agctgaagag cggcaccgcc a gcgtggtgt gcctgctgaa caacttctac cccagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggc

- 133 047128

ctgagcagcc ccgtgaccaa gagcttcaac agaggcgagt gc Denosumab I'm heavy/ SEQ IDNO: 127 gaggtgcagc tgctggagag cggcggcggc ctggtgcagc ccggcggcag cctgagactg agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gagacaggcc cccggcaagg gcctggagtg ggtgagcggc atcaccggca gcggcggcag cacctactac gccga cagcg tgaagggcag attcaccatc agcagagaca acagcaagaa caccctgtac ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc caaggacccc ggcaccaccg tgatcatgag ctggttcgac ccctggggcc agggcaccct ggtgaccgtg agcagcgcca gcaccaaggg ccccagcgtg ttccccctgg cccccagcag caagagcacc agcggcggca ccgccgccct gggctgcctg gtgaaggact acttccccga gcccgtgacc gtgagctgga acagcggcgc cctgaccagc ggcgtgcaca ccttccccgc cgtgctgcag agcagcggcc tgtacagcct gagcagcgtg gtgaccgtgc ccagcagcag cctgggcacc cagacctaca tctgcaacgt gaaccacaag cccagcaaca ccaaggtgga caagaaggtg gagcccaaga gctgcgac +/- aagacccacacc (aagacccacctg) +/tgccccccctgccccgcc +/cccgagctgctgggcggccccagcgtgttcctg Denosumab Light / SEQID NO: 128 gagatcgtgc tgacccagag ccccggcacc ctgagcctga gccccggcga gagagccacc ctgagctgca gagccagcca gagcgtgaga ggcagatacc tggcctggta ccagcagaag cccggccagg cccccagact gctgatctac ggcgccagca gcagagccac cggcatcccc gacagattca gcggcagcgg cggcacc gacttcaccc tgaccatcag cagactggag cccgaggact tcgccgtgtt ctactgccag cagtacggca gcagccccag aaccttcggc cagggcacca aggtggagat caagagaacc gtggccgccc ccagcgtgtt catcttcccc cccagcgacg agcagctgaa gagcggcacc gccagcgtgg t gtgcctgct gaacaacttc taccccagag aggccaaggt gcagtggaag gtggacaacg ccctgcagag cggcaacagc caggagagcg tgaccgagca ggacagcaag gacagcacct acagcctgag cagcaccctg accctgagca aggccgacta cgagaagcac aaggtgtacg cctgcgaggt gacccaccag ggcctgagca gccccgtgac caagagcttc aacagaggcg agtgc Nivolumab I'm heavy/SEQ IDNO: 129 caggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggcagaag cctgagactg gactgcaagg ccagcggcat caccttcagc aacagcggca tgcactgggt gagacaggcc cccggcaagg gcctggagtg ggtggccgtg atctggtacg acggcagcaa gagatactac gccgacagcg t gaagggcag attcaccatc agcagagaca acagcaagaa caccctgttc ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc caccaacgac gactactggg gccagggcac cctggtgacc gtgagcagcg ccagcaccaa gggccccagc gtgttccccc tggccccctg accagcgaga

- 134 047128

gcaccgccgc cctgggctgc ctggtgaagg actacttccc cgagcccgtg accgtgagct ggaacagcgg cgccctgacc agcggcgtgc acaccttccc cgccgtgctg cagagcagcg gcctgtacag cctgagcagc gtggtgaccg tgcccagcag cagcctgggc accaagacct acacctgcaa cgtggaccac aagcccagca acaccaaggt ggacaagaga gtggagagca agtac +/- ggccccccctgccccccctgccccgcc +/cccgagttcctgggcggccccagcgtgttcctg Nivolumab Light / SEQ GO NO: 130 gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gagagccacc ctgagctgca gagccagcca gagcgtgagc agctacctgg cctggtacca gcagaagccc ggccaggccc ccagactgct gatctacgac gccagcaaca gagccaccgg catccccgcc agattcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctggagccc gaggacttcg ccgtgtacta ctgccagcag agcagcaact ggcccagaac cttcggccag ggcaccaagg tggagatcaa gagaaccgtg gccgccccca gcgtgttcat cttccccccc agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac cccagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggc ctgagcagcc ccgtgaccaa gagcttcaac agaggcgagt gc Pembrolizum b I am heavy/SEQ GO NO: 131 caggtgcagc tggtgcagag cggcgtggag gtgaagaagc ccggcgccag cgtgaaggtg agctgcaagg ccagcggcta caccttcacc aactactaca tgtactgggt gagacaggcc cccggccagg gcctggagtg gatgggcggc atcaacccca gcaacggcgg caccaacttc aacgagaagt tcaagaac ag agtgaccctg accaccgaca gcagcaccac caccgcctac atggagctga agagcctgca gttcgacgac accgccgtgt actactgcgc cagaagac tacagattcg acatgggctt cgactactgg ggccagggca ccaccgtgac cgtgagcagc gccagcacca agggccccag cgtgttcccc ctgg ccccct gcagcagaag caccagcgag agcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgagc tggaacagcg gcgccctgac cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg caccaagacc tacacctgca acgtggacca caagcccagc aacaccaagg tggacaagag agtggagagc aagtac +/ggccccccctgccccccctgcc ccgcc +/cccgagttcctgggcggccccagcgtgttcctg Pembrolizum b Light/ SEQID NO: 132 gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gagagccacc ctgagctgca gagccagcaa gggcgtgagc accagcggct acagctacct gcactggtac cagcagaagc ccggccaggc ccccagactg ctgatctacc tggccagcta cctggagagc ggcgtgcccg gattcag

- 135 047128

cggcagcggc agcggcaccg acttcaccct gaccatcagc agcctggagc ccgaggactt cgccgtgtac tactgccagc acagcagaga cctgcccctg accttcggcg gcggcaccaa ggtggagatc aagagaaccg tggccgcccc cagcgtgttc atcttccccc ccagcgacga gcagctga ag agcggcaccg ccagcgtggt gtgcctgctg aacaacttct accccagaga ggccaaggtg cagtggaagg tggacaacgc cctgcagagc ggcaacagcc aggagagcgt gaccgagcag gacagcaagg acagcaccta cagcctgagc agcaccctga ccctgagcaa ggccgactac gagaagcaca aggtgtacgc ctgcgaggtg acccaccagg gcctgagcag ccccgtgacc aagagcttca acagaggcga gtgc Ranibizumab I'm heavy/SEQ IDNO: 133 gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgagactg agctgcgccg ccagcggcta cgacttcacc cactacggca tgaactgggt gagacaggcc cccggcaagg gcctggagtg ggtgggctgg atcaacacct acaccggcga gcccacctac gccgccgact tca agagaag attcaccttc agcctggaca ccagcaagag caccgcctac ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc caagtacccc tactactacg gcaccagcca ctggtacttc gacgtgtggg gccagggcac cctggtgacc gtgagcagcg ccagcaccaa gggccccagc gtgttccccc tggcccccag cagcaagagc accagcggcg gcaccgccgc cctgggctgc ctggtgaagg actacttccc cgagcccgtg accgtgagct ggaacagcgg cgccctgacc agcggcgtgc acaccttccc cgccgtgctg cagagcagcg gcctgtacag cctgagcagc gtggtgaccg tgcccagcag cagcctgggc acccagacct acatctgcaa cgtgaaccac aagcccagca acaccaaggt ggacaagaaggtggagcccaagagctgcgac +/- aaga cccacacc (or aagacccacctg) +/tgccccccctgccccgcc +/cccgagctgctgggcggccccagcgtgttcctg Ranibizumab Light / SEQ ID NO: 134 gacatccagc tgacccagg ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc atcacctgca gcgccagcca ggacatcagc aactacctga actggtacca gcagaagccc ggcaaggccc ccaaggtgct gatctacttc accagcagcc tgcacagcgg cgtgcccagc agattcagcg gca gcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc gaggacttcg ccacctacta ctgccagcag tacagcaccg tgccctggac cttcggccag ggcaccaagg tggagatcaa gagaaccgtg gccgccccca gcgtgttcat cttccccccc agcgacgagc agctgaagag cgg caccgcc agcgtggtgt gcctgctgaa caacttctac cccagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggc ctgagcagcc ccgtgaccaa gagcttcaac agaggcgagt gc

- 136 047128

Bevacizumab I'm heavy / SEQ IDNO: 135 gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgagactg agctgcgccg ccagcggcta caccttcacc aactacggca tgaactgggt gagacaggcc cccggcaagg gcctggagtg ggtgggctgg atcaacacct acaccggcga gcccacctac gccgccgact tca agagaag attcaccttc agcctggaca ccagcaagag caccgcctac ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc caagtacccc cactactacg gcagcagcca ctggtacttc gacgtgtggg gccagggcac cctggtgacc gtgagcagcg ccagcaccaa gggccccag c gtgttccccc tggcccccag cagcaagagc accagcggcg gcaccgccgc cctgggctgc ctggtgaagg actacttccc cgagcccgtg accgtgagct ggaacagcgg cgccctgacc agcggcgtgc acaccttccc cgccgtgctg cagagcagcg gcctgtacag cctgagcagc gtggtgaccg tgcccagcag cagcctgggc acccagacct acatctgcaa cgtgaaccac aagcccagca acaccaaggt ggacaagaag gtggagccca agagctgcga c +/- a agacccacacc (aagacccacctg) +/tgccccccctgccccgcc +/cccgagctgctgggcggccccagcgtgttcctg Bevacizumab Light / SEQ ID NO: 136 gacatccaga tgacccagg ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc atcacctgca gcgccagcca ggacatcagc aactacctga actggtacca gcagaagccc ggcaaggccc ccaaggtgct gatctacttc accagcagcc tgcacagcgg cgtgcccagc agattcagcg cggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc gaggacttcg ccacctacta ctgccagcag tacagcaccg tgccctggac cttcggccag ggcaccaagg tggagatcaa gagaaccgtg gccgccccca gcgtgttcat cttccccccc agcgacgagc agctgaagag cggc accgcc agcgtggtgt gcctgctgaa caacttctac cccagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggc ctgagcagcc ccgtgaccaa gagcttcaac agaggcgagt gc Lampalizumab I'm heavy/SEQ IDNO: 137 gaggtgcagc tggtgcagag cggccccgag ctgaagaagc ccggcgccag cgtgaaggtg agctgcaagg ccagcggcta caccttcacc aactacggca tgaactgggt gagacaggcc cccggccagg gcctggagtg gatgggctgg atcaacacct acaccggcga gaccacctac gccgacgact tcaagggcag attcgtgttc agcctggaca ccagcgtgag caccgcctac ctgcagatca gcagcctgaa ggccgaggac accgccgtgt actactgcga gagagaggc ggcgtgaaca actggggcca gggcaccctg gtgaccgtga gcagcgccag caccaagggc cccagcgtgt tccccctggc cagc aagagcacca gcggcggcac cgccgccctg ggctgcctgg tgaaggacta cttccccgag cccgtgaccg tgagctggaa cagcggcgcc

- 137 047128

ctgaccagcg gcgtgcacac cttccccgcc gtgctgcaga gcagcggcct gtacagcctg agcagcgtgg tgaccgtgcc cagcagcagc ctgggcaccc agacctacat ctgcaacgtg aaccacaagc ccagcaacac caaggtggac aagaaggtgg agcccaagag ctgcgac +/ - aagacccacacc (or aagacccacctg) +/- tgccccccctgccccgcc +/cccgagctgctgggcggccccagcgtgttcctg Lampalizumab Light / SEQ ID NO: 138 gacatccagg tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc atcacctgca tcaccagcac cgacatcgac gacgacatga actggtacca gcagaagccc ggcaaggtgc ccaagctgct gatcagcggc ggcaacaccc tgagacccgg cgtgcccagc agattcagcg gcag cggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc gaggacgtgg ccacctacta ctgcctgcag agcgacagcc tgccctacac cttcggccag ggcaccaagg tggagatcaa gagaaccgtg gccgccccca gcgtgttcat cttccccccc agcgacgagc agctgaagag c ggcaccgcc agcgtggtgt gcctgctgaa caacttctac cccagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggc ctgagcagcc ccgtgaccaa gagcttcaac agaggcgagt gc Brolucizumab Light / SEQ GO NO: 139 gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgagactg agctgcaccg ccagcggctt cagcctgacc gactactact acatgacctg ggtgagacag gcccccggca agggcctgga gtgggtgggc ttcatcgacc ccgacgacga cccctactac gccacctg gg ccaagggcag attcaccatc agcagagaca acagcaagaa caccctgtac ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc cggcggcgac cacaacagcg gctggggcct ggacatctgg ggccagggca ccctggtgac cgtgagcagc Brolucizumab I am heavy/SEQ GO NO: 140 gagatcgtga tgacccagag ccccagcacc ctgagcgcca gcgtgggcga cagagtgatc atcacctgcc aggccagcga gatcatccac agctggctgg cctggtacca gcagaagccc ggcaaggccc ccaagctgct gatctacctg gccagcaccc tggccagcgg cgtgcccagc agattcagcg gca gcggcag cggcgccgag ttcaccctga ccatcagcag cctgcagccc gacgacttcg ccacctacta ctgccagaac gtgtacctgg ccagcaccaa cggcgccaac ttcggccagg gcaccaagct gaccgtgctg ggc Belimumab I am heavy/SEQ GO NO: 141 caggtgcagc tgcagcagag cggcgcggaa gtgaaaaaac cgggcagcag cgtgcgcgtg agctgcaaag cgagcggcgg cacctttaac aacaacgcga ttaactgggt gcgccaggcg ccgggccagg gcctggaatg gatgggcggc attattccga tgtttggcac gaaatat agccagaact ttcagggccg cgtggcgatt accgcggatg aaagcaccgg caccgcgagc atggaactga gcagcctgcg cagcgaagat accgcggtgt

- 138 047128

attack ctggtgaaag attattttcc ggaaccggtg accgtgagca acagcggcgc gctgaccagc ggcgtgcata cctttccggc ggtgctgcag agcagcggcc tgtatagcct gagcagcgtg gtgaccgtgc cgagcagcag cctgggcacc cagacctata tttcaacgt gaaccataaa cc gagcaaca ccaaagtgga taaaaaagtg gaaccgaaaagctgcgat+/aaaacccatacc (or aaaacccatctg) + /tgcccgccgtgcccggcg +/ - ccggaactgctgggcggcccgagcgtgtttctg Belimumab Light / SEQ GO NO: 142 agcagcgaac tgacccagga tccggcggtg agcgtggcgc tgggccagac cgtgcgcgtg acctgccagg gcgatagcct gcgcagctat tatgcgagct ggtatcagca gaaaccgggc caggcgccgg tgctggtgat ttatggcaaa aacaaccgcc cgagcggcat tccggatcg c tttagcggca gcagcagcgg caacaccgcg agcctgacca ttaccggcgc gcaggcggaa gatgaagcgg attattattg cagcagccgc gatagcagcg gcaaccattg ggtgtttggc ggcggcaccg aactgaccgt gctgggccag ccgaaagcgg cgccgagcgt gaccctgttt gagca gcgaagaact gcaggcgaac aaagcgaccc tggtgtgcct gattagcgat ttttatccgg gcgcggtgac cgtggcgtgg aaagcggata gcagcccggt ggcgggcgtg gaaaccacca ccccgagcaa acagagcaac aacaaatatg cggcgagcag ctatctgagc ctgaccccgg aacagtggaa aagccatcgc agctatagct gccaggtgac ccatgaaggc agcaccgtgg aaaaaaccgt ggcgccgacc gaatgcagc Eculizumab I am heavy / SEQ GO NO: 143 caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg agctgcaagg ccagcggcta catcttcagc aactactgga tccagtgggt gagacaggcc cccggccagg gcctggagtg gatgggcgag atcctgcccg gcagcggcag caccgagtac accgagaact tcaaggacag agtgaccatg accagagaca ccagcaccag caccgtgtac atggagctga gcagcctgag aagcgaggac accgccgtgt actactgcgc cagatacttc ttcggcagca gccccaactg gtacttcgac gtgtggggcc agggcaccct ggtgaccgtg agcagcgcca gcaccaaggg cccca gcgtg ttccccctgg ccccctgcag cagaagcacc agcgagagca ccgccgccct gggctgcctg gtgaaggact acttccccga gcccgtgacc gtgagctgga acagcggcgc cctgaccagc ggcgtgcaca ccttccccgc cgtgctgcag agcagcggcc tgtacagcct gagcagcgtg gtgaccgtgc ccagcagcaa cttcggcacc cagacctaca cctgcaacgt ggaccacaag cccagcaaca ccaaggtgga caagaccgtg gagagaaagt gctgcgtgga g +/- tgccccccctgccccgcc +/- ccccccgtggccggc

- 139 047128

Eculizumab Light / SEQ GO NO: 144 gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc atcacctgcg gcgccagcga gaacatctac ggcgccctga actggtacca gcagaagccc ggcaaggccc ccaagctgct gatctacggc gccaccaacc tggccgacgg cgtgcccagc agattcagcg gcag cggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc gaggacttcg ccacctacta ctgccagaac gtgctgaaca cccccctgac cttcggccag ggcaccaagg tggagatcaa gagaaccgtg gccgccccca gcgtgttcat cttccccccc agcgacgagc agctgaagag cggc accgcc agcgtggtgt gcctgctgaa caacttctac cccagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggc ctgagcagcc ccgtgaccaa gagcttcaac agaggcgagt gc Andekalixim ab I am heavy / SEQ GO NO: 145 caggtgcagc tgcaggagag cggccccggc ctggtgaagc ccagcgagac cctgagcctg acctgcaccg tgagcggctt cagcctgctg agctacggcg tgcactgggt gagacagccc cccggcaagg gcctggagtg gctgggcgtg atctggaccg gcggcaccac caactacaac agcgcc ctga tgagcagatt caccatcagc aaggacaca gcaagaacac cgtgtacctg aagatgaaca gcctgaagac cgaggacacc gccatctact actgcgccag atactactac ggcatggact actggggcca gggcaccctg gtgaccgtga gcagcgccag caccaagggc cccagcgtgt tccccctggc cccctgcagc cca gcgagagcac cgccgccctg ggctgcctgg tgaaggacta cttccccgag cccgtgaccg tgagctggaa cagcggcgcc ctgaccagcg gcgtgcacac cttccccgcc gtgctgcaga gcagcggcct gtacagcctg agcagcgtgg tgaccgtgcc cagcagcagc ctgggcacca agacctacac ctgcaacgtg gaccacaagc ccagcaacac caaggtggac aagagagtgg agagcaagta c +/- ggccccccctgccccccctgccccgcc +/- cccgagttcctgggcggccccagcgtgttcctg Andekalixim ab Light / SEQID NO: 146 gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc atcacctgca aggccagcca ggacgtgaga aacaccgtgg cctggtacca gcagaagccc ggcaaggccc ccaagctgct gatctacagc agcagctaca gaaacaccgg cgtgcccgac agattcagcg gca gcggcag cggcaccgac ttcaccctga ccatcagcag cctgcaggcc gaggacgtgg ccgtgtacta ctgccagcag cactacatca ccccctacac cttcggcggc ggcaccaagg tggagatcaa gagaaccgtg gccgccccca gcgtgttcat cttccccccc agcgacgagc agctgaagag c ggcaccgcc agcgtggtgt gcctgctgaa caacttctac cccagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc ctgagcaagg ccgactacga

- 140 047128

gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc ctgagcagcc ccgtgaccaa gagcttcaac agaggcgagt gc Lanad elumab I'm heavy/SEQ IDNO: 147 gaggtgcagc tgctggagag cggcggcggc ctggtgcagc ccggcggcag cctgagactg agctgcgccg ccagcggctt caccttcagc cactacatca tgatgtgggt gagacaggcc cccggcaagg gcctggagtg ggtgagcggc atctacagca gcggcggcat caccgtgtac gcc gacagcg tgaagggcag attcaccatc agcagagaca acagcaagaa caccctgtac ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc ctacagaaga atcggcgtgc ccagaagaga cgagttcgac atctggggcc agggcaccat ggtgaccgtg agcagcgcca gcaccaaggg ccccagcgtg ttccccctgg cccccagcag caagagcacc agcggcggca ccgccgccct gggctgcctg gtgaaggact acttccccga gcccgtgacc gtgagctgga acagcggcgc cctgaccagc ggcgtgcaca ccttccccgc cgtgctgcag agcagcggcc tgtacagcct gagcagcgtg gtgaccgtgc ccagcagcag cctgggcacc cagacctaca tctgcaacgt gaaccacaag cccagcaaca ccaaggtgga caagagagtg gagcccaaga gctgcgac +/- aagacccacacc (or aagacccacctg) +/tgccccccctgccccgcc +/cccgagctgctgggcggccccagcgtgttcctg Lanad elumab Light / SEQID NO: 148 gacatccaga tgacccagag ccccagcacc ctgagcgcca gcgtgggcga cagagtgacc atcacctgca gagccagcca gagcatcagc agctggctgg cctggtacca gcagaagccc ggcaaggccc ccaagctgct gatctacaag gccagcaccc tggagagcgg cgtgcccagc agattcagcg gcagcgg cag cggcaccgag ttcaccctga ccatcagcag cctgcagccc gacgacttcg ccacctacta ctgccagcag tacaacacct actggacctt cggccagggc accaaggtgg agatcaagag aaccgtggcc gcccccagcg tgttcatctt cccccccagc gacgagcagc tgaagagcgg caccgccagc gtgg tgtgcc tgctgaacaa cttctacccc agagaggcca aggtgcagtg gaaggtggac aacgccctgc agagcggcaa cagccaggag agcgtgaccg agcaggacag caaggacagc acctacagcc tgagcagcac cctgaccctg agcaaggccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg agcagccccg tgaccaagag cttcaacaga ggcgagtgc Adalimumab I'm heavy/ SEQ IDNO: 149 gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcagaag cctgagactg agctgcgccg ccagcggctt caccttcgac gactacgcca tgcactgggt gagacaggcc cccggcaagg gcctggagtg ggtgagcgcc atcacctgga acagcggcca catcgactac gccgacagc g tggagggcag attcaccatc agcagagaca acgccaagaa cagcctgtac ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc caaggtgagc tacctgagca ccgccagcag cctggactac tggggccagg gcaccctggt gaccgtgagc agcgccagca ccaagggccc cagcg tgttc cccctggccc

- 141 047128

ccagcagcaa gagcaccagc ggcggcaccg ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg agctggaaca gcggcgccct gaccagcggc gtgcacacct tccccgccgt gctgcagagc agcggcctgt acagcctgag cagcgtggtg accgtgc cca gcagcagcct gggcacccag acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gaaggtggag cccaagagct gcgac + /aagacccacacc (aagacccacctg) +/tgccccccctgccccgcc +/ccgagctgctgggcggccccagcgtgttcctg Adalimumab Light / SEQ GO NO: 150 agattcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc gaggacgtgg ccacctacta ctgccagaga tacaacagag ccccctacac cttcggccag ggcaccaagg tggagatcaa gagaaccgtg gccgccccca gcgtgttcat cttccccccc agcgacgag c agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac cccagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc ctgagcagcc ccgtgaccaa gagcttcaac agaggcgagt gc Infliximab I am heavy / SEQ GO NO: 151 gaggtgaagc tggaggagag cggcggcggc ctggtgcagc ccggcggcag catgaagctg agctgcgtgg ccagcggctt catcttcagc aaccactgga tgaactgggt gagacagagc cccgagaagg gcctggagtg ggtggccgag atcagaagca agagcatcaa cagcgccacc cactacgccg aga gcgtgaa gggcagattc accatcagca gagacgacag caagagcgcc gtgtacctgc agatgaccga cctgagaacc gaggacaccg gcgtgtacta ctgcagcaga aactactacg gcagcaccta cgactactgg ggccagggca ccaccctgac cgtgagcagc gccagcacca agggccccag cgtgttcccc ctggccccca gcagcaagag caccagcggc ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgagc tggaacagcg gcgccctgac cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagaa ggtggagccc aagagctgcg ac +/aagacccacacc ( aagacccacctg) +/tgccccccctgccccgcc +/cccgagctgctgggcggccccagcgtgttcctg Infliximab Light/ SEQID NO: 152 gacatcctgc tgacccagag ccccgccatc ctgagcgtga gccccggcga gagagtgagc ttcagctgca gagccagcca gttcgtgggc agcagcatcc actggtacca gcagagaacc aacggcagcc ccagactgct gatcaagtac gccagcgaga gcatgagcgg catccccagc agattcagcg gcagc ggcag cggcaccgac ttcaccctga gcatcaacac cgtggagagc gaggacatcg ccgactacta ctgccagcag agccacagct

- 142 047128

ggcccttcac cttcggcagc ggcaccaacc tggaggtgaa gagaaccgtg gccgccccca gcgtgttcat cttccccccc agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac cccagagagg ccaaggtgca gtggaaggtg gacaacgccc t gcagagcgg caacagccag gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggc ctgagcagcc ccgtgaccaa gagcttcaac agaggcgagt gc aTAU I'm heavy/SEQ IDNO: 153 a gagcgtgaa gggcaggttc accatcagca gggacgacag caagagcagc gtgtacctgc agatgaacaa cctgagggcc gaggacagcg gcatctacta ctgcaccaac tgggaggact actggggcca gggcaccacc gtgaccgtga gcagcgccag caccaagggc cccagcgtgt tccccctggc cccctgcagc aggagcacca gcgagagcac cgccgccctg ggctgcctgg tgaaggacta cttccccgag cccgtgaccg tgagctggaa cagcggcgcc ctgaccagcg gcgtgcacac cttccccgcc gtgctgcaga gcagcggcct gtacagcctg agcagcgtgg tgaccgtgcc cagcagcagc ctgggcacca agacctacac ctgcaacgtg gaccacaagc ccagcaacac caaggtggac aagaggtgg agagcaagta c +/- ggccccccctgccccccctgccccgcc +/- cccgagttcctgggcggccccagcgtgttcctg aTAU Light / SEQ GO NO: 154 gacatcgtgc tgacccagag ccccgacagc ctggccgtga gcctgggcga gagggccacc atcagctgca gggccagcca gagcgtgagc accagcaggt acagctacat ccactggtac cagcagaagc ccggccagcc ccccaagctg ctgatcaagt acgccagcaa cctggagagc ggcgtgccca gca ggttcag cggcagcggc agcggcaccg acttcaccct gaacatccac cccctggagc ccgaggactt cgccacctac tactgccacc acagctggga gatccccctg accttcggcc agggcaccaa gctggagatc aagaggaccg tggccgcccc cagcgtgttc atcttccccc ccagcgacga gcagctgaag ag cggcaccg ccagcgtggt gtgcctgctg aacaacttct accccaggga ggccaaggtg cagtggaagg tggacaacgc cctgcagagc ggcaacagcc aggagagcgt gaccgagcag gacagcaagg acagcaccta cagcctgagc agcaccctga ccctgagcaa ggccgactac gagaagcaca aggtgtacgc ctgcgaggtg acccaccagg gcctgagcag ccccgtgacc aagagcttca acaggggcga gtgc Erenumab I'm heavy/SEQ IDNO: 155 caggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggcagaag cctgagactg agctgcgccg ccagcggctt caccttcagc agcttcggca tgcactgggt gagacaggcc cccggcaagg gcctggagtg ggtggccgtg atcagcttcg

- 143 047128

acggcagcat caagtacagc gtggacagcg tgaagggcag attcaccatc agcagagaca acagcaagaa caccctgttc ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc cagagacaga ctgaactact acgacagcag cggctactac cactacaagt actacggcat ggccgtgtgg ggcca gggca ccaccgtgac cgtgagcagc gccagcacca agggccccag cgtgttcccc ctggccccct gcagcagaag caccagcgag agcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgagc tggaacagcg gcgccctgac cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcaacttcgg cacccagacc tacacctgca acgtggacca caagcccagc aacaccaagg tggacaagac cgtggagaga aagtgctgcgt ggagtgcccc ccctgcccc gccccccccg tggccggc Erenumab Light / SEQ GO NO: 156 cagagcgtgc tgacccagcc ccccagcgtg agcgccgccc ccggccagaa ggtgaccatc agctgcagcg gcagcagcag caacatcggc aacaactacg tgagctggta ccagcagctg cccggcaccg cccccaagct gctgatctac gacaacaaca agagacccag cggcatcccc gacagattca gcggcagcaa gagcggcacc agcaccaccc tgggcatcac cggcctgcag accggcgacg aggccgacta ctactgcggc acctgggaca gcagactgag cgccgtggtg ttcggcggcg gcaccaagct gaccgtgctg ggccagccca aggccaaccc caccgtgacc ctgttccccc ccagcagcga ggagctgcag gccaacaagg ccaccctggt gtgcctgatc agcgacttct accccggcgc cgtgaccgtg gcctggaagg ccgacggcag ccccgtgaag gccggcgtgg agaccaccaa gcccagcaag cagagcaaca acaagtacgc cgccagcagc tacctgagcc tgacccccga gcagtggaag agccacagaa gctacagctg ccaggtgacc cacgagggca gcaccgtgga gaagaccgtg gcccccaccg agtgcagc BAN2401 I am heavy/SEQ GO NO: 157 gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg agctgcagcg ccagcggctt caccttcagc agcttcggca tgcactgggt gaggcaggcc cccggcaagg gcctggagtg ggtggcctac atcagcagcg gcagcagcac catctactac ggcgacaccg tgaagggcag gttcaccatc agcagggaca acgccaagaa cagcctgttc ctgcagatga gcagcctgag ggccgaggac accgccgtgt actactgcgc cagggaggc ggctactact acggcaggag ctactacacc atggactact ggggccaggg caccaccgtg accgtgagca gcgccagcac ca agggcccc agcgtgttcc ccctggcccc cagcagcaag agcaccagcg gcggcaccgc cgccctgggc tgcctggtga aggactactt ccccgagccc gtgaccgtga gctggaacag cggcgccctg accagcggcg tgcacacctt ccccgccgtg ctgcagagca gcggcctgta cagcctgagc agcgtggtga ccgtgcccag cagcagcctg ggcacccaga cctacatctg caacgtgaac cacaagccca gcaacaccaa ggtggacaag

- 144 047128

aaggtggagcccaagagctgcgac +/- aagacccacacc (or aagacccacctg) +/- tgccccccctgccccgcc +/ ccgagctgctgggcggc BAN2401 Light / SEQ GO NO: 158 gacgtggtga tgacccagag ccccctgagc ctgcccgtga cccccggcgc ccccgccagc atcagctgca ggagcagcca gagcatcgtg cacagcaacg gcaacaccta cctggagtgg tacctgcaga agcccggcca gagccccaag ctgctgatct acaaggtgag caacaggttc agcggcgt gc ccgacaggtt cagcggcagc ggcagcggca ccgacttcac cctgaggatc agcagggtgg aggccgagga cgtgggcatc tactactgct tccagggcag ccacgtgccc cccaccttcg gccccggcac caagctggag atcaagagga ccgtggccgc ccccagcgtg ttcatcttcc cga cgagcagctg aagagcggca ccgccagcgt ggtgtgcctg ctgaacaact tctaccccag ggaggccaag gtgcagtgga aggtggacaa cgccctgcag agcggcaaca gccaggagag cgtgaccgag caggacagca aggacagcac ctacagcctg agcagcaccc tgaccctgag caaggccgac tacgagaagc acaaggtgta cgcctgcgag gtgacccacc agggcctgag cagccccgtg accaagagct tcaacagggg cgagtgc EOB-scFV I am heavy / SEQ GO NO: 159 gaggtgaagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg agctgcgcca ccagcggctt caccttcagc gacttctaca tggagtgggt gaggcaggcc cccggcaaga ggctggagtg gatcgccgcc agcaggaaca aggccaacga ctacaccacc gagtacgcc g acagcgtgaa gggcaggttc atcgtgagca gggacaccag ccagagcatc ctgtacctgc agatgaacgc cctgagggcc gaggacaccg ccatctacta ctgcgccagg gactactacg gcagcagcta ctggtacttc gacgtgtggg gcgccggcac caccgtgacc gtgagcagc EOB-scFV Light/ SEQID NO: 160 gacatcgtga tgacccagg ccccagcagc ctgagcgtga gcgccggcaa gaaggtgacc atcagctgca ccgccagcga gagcctgtac agcagcaagc acaaggtgca ctacctggcc tggtaccaga agaagcccga gcagagcccc aagctgctga tctacggcgc cagcaacagg tacatcgg cg tgcccgacag gttcaccggc agcggcagcg gcaccgactt caccctgacc atcagcagcg tgcaggtgga ggacctgacc cactactact gcgcccagtt ctacagctac cccctgacct tcggcgccgg caccaagctg gagatcaag

Equivalents

While the present invention has been described in detail with reference to specific embodiments thereof, it should be understood that embodiments that are functionally equivalent are within the scope of the present invention. Indeed, various modifications of the present invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Those skilled in the art will recognize or be able to determine, using no more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Such equivalents are intended to be covered by the following claims.

All publications, patents and patent applications referenced herein are incorporated herein by reference in the specification to the same extent as if each individual publication, patent or patent application had been specifically and separately indicated to be incorporated herein by reference in its entirety .

Claims (16)

1. A pharmaceutical composition for intravenous administration into the liver or muscle of a human subject for the treatment of hereditary angioedema in a human subject in need thereof, comprising an AAV vector containing: (a) a viral capsid that is at least 95% identical to the amino acid sequence of the AAV8 capsid (SEQ ID NO: 78) or the AAV9 capsid (SEQ ID NO: 79); and (b) an artificial genome containing an expression cassette flanked by an AAV ITR, wherein the expression cassette contains a transgene encoding the antibody lanadelumab or an antigen binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in human muscle cells or human liver cells - 145 047128 catchers; and wherein the transgene comprises a nucleotide sequence encoding a heavy chain comprising a heavy chain CDR i) GFTFSHYIMM (amino acids 26-35 of SeQ ID NO: 47); ii) GIYSSGGITVYADSVKG (amino acids 50-66 of SEQ ID NO: 47); and iii) YRRIGVPRRDEFDI (amino acids 98-111 of SEQ ID NO: 47); wherein the transgene contains a nucleotide sequence encoding a light chain containing a light chain CDR i) SASVGDRVTITCRASQSISSWLA (amino acids 12-34 of SEQ ID NO: 48); ii) KASTLES (amino acids 50-56 of SEQ ID NO: 48); and iii) QQYNTYWT (amino acids 89-96 of SEQ ID NO: 48); wherein the nucleotide sequence encoding the heavy chain and the nucleotide sequence encoding the light chain are separated by a nucleotide sequence encoding a cleavable linker or IRES; wherein said antibody lanadelumab is expressed in said subject. 2. The pharmaceutical composition according to claim 1, wherein the antibody lanadelumab or an antigen-binding fragment thereof is a Fab, F(ab') ₂ or a full-length antibody. 3. The pharmaceutical composition according to claim 1, wherein the antibody lanadelumab or an antigen-binding fragment thereof contains a heavy chain with the amino acid sequence SEQ ID NO: 47 and a light chain with the amino acid sequence SEQ ID NO: 48. 4. The pharmaceutical composition according to claim 3, wherein the transgene contains the nucleotide sequence SEQ ID NO: 147 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 148 encoding the light chain. 5. The pharmaceutical composition according to any of the preceding claims, wherein the transgene encodes a signal sequence at the N-terminus of the heavy chain and at the N-terminus of the light chain of said antibody lanadelumab or an antigen-binding fragment thereof, which directs secretion and post-translational modification in said human liver cells. 6. The pharmaceutical composition according to claim 5, in which the specified signal sequence is selected from the signal sequences from table. 3. 7. The pharmaceutical composition according to claim 1, wherein the heavy chain comprises a heavy chain Fab fragment and a hinge region sequence at the C-terminus of the Fab. 8. The composition of any one of the preceding claims, wherein the cleavable linker is the nucleotide sequence of SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217 or SEQ ID NO: 218. 9. The pharmaceutical composition according to any one of the preceding claims, wherein the AAV capsid is AAV8. 10. The use of a pharmaceutical composition for the treatment of hereditary angioedema in a human subject in need thereof, comprising administering to the liver or muscle of said subject a therapeutically effective amount of a recombinant nucleotide expression vector containing a transgene encoding the antibody lanadelumab or an antigen binding fragment thereof operably linked to one or more regulatory sequences that control expression of the transgene in human liver cells or human muscle cells, and flanked by AAV ITR sequences so that a depot is formed that releases the HuPTM form of said mAb or antigen binding fragment thereof; wherein the transgene contains a nucleotide sequence encoding a heavy chain containing a heavy chain CDR i) GFTFSHYIMM (amino acids 26-35 of SEQ ID NO: 47); ii) GIYSSGGITVYADSVKG (amino acids 50-66 of SEQ ID NO: 47); and iii) YRRIGVPRRDEFDI (amino acids 98-111 of SEQ ID NO: 47); wherein the transgene contains a nucleotide sequence encoding a light chain containing a light chain CDR i) SASVGDRVTITCRASQSISSWLA (amino acids 12-34 of SEQ ID NO: 48); ii) KASTLES (amino acids 50-56 of SEQ ID NO: 48); and iii) QQYNTYWT (amino acids 89-96 of SEQ ID NO: 48); wherein the nucleotide sequence encoding the heavy chain and the nucleotide sequence encoding the light chain are separated by a nucleotide sequence encoding a cleavable linker or IRES; wherein said antibody lanadelumab is expressed in said subject. 11. Use according to claim 10, wherein the antigen binding fragment is a Fab, F(ab')2 or a full length antibody. 12. Use according to claim 11, wherein the antibody lanadelumab or an antigen binding fragment thereof comprises a heavy chain having the amino acid sequence of SEQ ID NO: 47 and a light chain having the amino acid sequence of SEQ ID NO: 48. 13. Use according to claim 12, wherein the transgene contains the nucleotide sequence SEQ ID NO: 147 encoding the heavy chain and the nucleotide sequence SEQ ID NO: 148 encoding the light chain. 14. Use according to any one of claims 10-13, wherein the cleavable linker contains a nucleotide poly- - 146 047128 sequence SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217 or SEQ ID NO: 218. 15. Use according to any one of claims 10 to 14, wherein the recombinant nucleotide expression vector is packaged into AAV8 or AAV9. 16. A method for producing recombinant AAVs, comprising (a) culturing a host cell containing (i) an artificial genome containing a cis-expression cassette flanked by an AAV ITR, wherein the cis-expression cassette contains a transgene encoding lanadelumab or an antigen-binding fragment operably linked with expression control elements that will control the expression of the transgene in human cells; (ii) a trans-expression cassette lacking the AAV ITRs, wherein the trans-expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that direct expression of the AAV rep and capsid proteins in a host cell in culture and provide proteins rep and cap in trans; (iii) sufficient accessory functions of the adenovirus for replication and packaging of the artificial genome by AAV capsid proteins; and (b) recovering recombinant AAV encapsulating the artificial genome from cell culture; wherein the transgene contains a nucleotide sequence encoding a heavy chain containing a heavy chain CDR i) GFTFSHYIMM (amino acids 26-35 of SEQ ID NO: 47); ii) GIYSSGGITVYADSVKG (amino acids 50-66 of SEQ ID NO: 47); and iii) YRRIGVPRRDEFDI (amino acids 98-111 of SEQ ID NO: 47); wherein the transgene contains a nucleotide sequence encoding a light chain containing a light chain CDR i) SASVGDRVTITCRASQSISSWLA (amino acids 12-34 of SEQ ID NO: 48); ii) KASTLES (amino acids 50-56 of SEQ ID NO: 48); and iii) QQYNTYWT (amino acids 89-96 of SEQ ID NO: 48); and wherein the nucleotide sequence encoding the heavy chain and the nucleotide sequence encoding the light chain are separated by a nucleotide sequence encoding a cleavable linker or IRES.