A vector containing a sequence encoding an anti-TNF antibody and an inflammation-inducible promoter

Abstract

The present invention provides a vector comprising a nucleotide sequence encoding an anti-TNF antibody or a fragment thereof, wherein the nucleotide sequence encoding the anti-TNF antibody or a fragment thereof is operably linked to an inflammation-inducible promoter.

Description

Technical Field

[0001] The present invention relates to a vector for preventing or treating inflammatory eye diseases.

Background Art

[0002] Chronic inflammation in the eye can lead to cumulative damage that ultimately causes significant vision loss. Chronic non-infectious uveitis is a vision-threatening intraocular inflammation that accounts for 10% of blindness in the working-age population and has a disproportionately large economic burden (see, for example, Joltikov, K.A. and Lobo-Chan, A.M., 2021. Frontiers in Medicine, 8:695904). Uveitis can include intraocular inflammation that affects the uvea and adjacent structures, such as the cornea, vitreous humor, retina, and optic nerve. Most commonly, uveitis is idiopathic, but it can be associated with infections, malignancies, or underlying inflammatory conditions, such as spondyloarthritis, sarcoidosis, juvenile idiopathic arthritis (JIA), inflammatory bowel disease, rheumatoid arthritis, tubulointerstitial nephritis, and other autoinflammatory diseases (see, for example, Rosenbaum, J.T. et al., 2019. Seminars in Arthritis and Rheumatism, 49(3), pages 438-445).

[0003] Currently, the first-line treatment for non-infectious uveitis is corticosteroids, which can be administered locally, periocularly, intravitreally, or systemically. However, there are problems associated with this treatment option. Systemic administration of corticosteroids has several well-known side effects that can lead to adverse events, and while local administration of corticosteroids can reduce the required concentration, repeated injections are required because the intravitreal drug concentration decreases over time. Due to the recurrence of the condition, patients may need to maintain continuous systemic corticosteroid treatment, which can cause a number of adverse effects (see, for example, Valenzuela, R.A. et al., 2020. Frontiers in Pharmacology, 11:655). Local treatment options, including steroid-based intravitreal implants and intravitreal injections, have not significantly improved the clinical situation. These are effective in reducing the recurrence of milder diseases but are associated with serious adverse events such as cataracts and glaucoma.

[0004] Immunosuppressive therapy (IMT) is an alternative to corticosteroid therapy and includes antimetabolites, calcineurin inhibitors, and alkylating agents. When conventional corticosteroids and IMT are ineffective, biological agents and biologic drugs, such as TNF inhibitors, IL-1 blockers, and anti-CD20, can be used. However, these drugs are associated with adverse events. For example, the adverse effects associated with TNF inhibitors include the development of autoimmune diseases, an increased risk of infections, reactions at the injection site, an increased risk of malignancies, and the exacerbation of demyelinating disorders (see, for example, Valenzuela, R.A. et al., 2020. Frontiers in Pharmacology, 11:655). SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0005] Therefore, a new approach for treating or preventing inflammatory eye diseases, such as uveitis, is needed. MEANS FOR SOLVING THE PROBLEMS

[0006] The inventors have developed a gene therapy for treating or preventing an inflammatory eye disease, such as uveitis, in which an anti-inflammatory TNF inhibitor is delivered to the eye.

[0007] The inventors have surprisingly demonstrated that a vector encoding a TNF inhibitor under the control of an inflammation-inducible promoter can enable inflammation-inducible expression of the TNF inhibitor in the eye. When the expression of the TNF inhibitor is associated with an inflammation-inducible promoter, gene therapy can thus provide a dosage level that is adaptable and responsive for preventing or treating an inflammatory eye disease. Such gene therapy can prevent recurrence of inflammation and / or maintain inflammation at a subclinical level, thereby preventing cumulative damage while reducing the occurrence of adverse events.

[0008] In one aspect, the present invention provides a vector comprising a nucleotide sequence encoding a TNF inhibitor.

[0009] In a preferred embodiment, the TNF inhibitor is an anti-TNF antibody or a fragment thereof. Any suitable anti-TNF antibody or fragment thereof can be used. In some embodiments, the TNF inhibitor is any one of adalimumab or a fragment thereof, infliximab or a fragment thereof, golimumab or a fragment thereof, or certolizumab or a fragment thereof. In some embodiments, the TNF inhibitor is adalimumab or a fragment thereof or infliximab or a fragment thereof.

[0010] In some embodiments, the TNF inhibitor is an anti-TNF antibody fragment. Any suitable anti-TNF antibody fragment can be used. In some embodiments, the anti-TNF antibody fragment is an antigen-binding fragment (Fab), a fragment antibody (F(ab')2), a single-chain antibody (scFv), or a single-domain antibody (sdAb). In some embodiments, the anti-TNF antibody fragment is an antigen-binding fragment (Fab).

[0011] In some embodiments, the TNF inhibitor is adalimumab or a fragment thereof. In some embodiments, the TNF inhibitor is an antigen-binding fragment (Fab) of adalimumab. In some embodiments, the TNF inhibitor is an anti-TNF antibody or a fragment thereof comprising one or more CDR regions selected from SEQ ID NOs: 1-6 or derivatives thereof containing one amino acid substitution. In some embodiments, the TNF inhibitor is an anti-TNF antibody or a fragment thereof comprising CDR regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each comprising SEQ ID NOs: 1, 2, 3, 4, 5, and 6 or derivatives thereof containing one amino acid substitution, or consisting of the foregoing. In some embodiments, the TNF inhibitor is an anti-TNF antibody or a fragment thereof comprising a heavy chain comprising or consisting of a sequence having at least 70% identity to SEQ ID NO: 7 and / or a light chain comprising or consisting of a sequence having at least 70% identity to SEQ ID NO: 8.

[0012] In some embodiments, the heavy chain is encoded by a nucleotide sequence having at least 70% identity to SEQ ID NO: 47, and / or the light chain is encoded by a nucleotide sequence having at least 70% identity to SEQ ID NO: 48. The nucleotide sequence encoding the heavy chain and the nucleotide sequence encoding the light chain may be connected via a linker sequence. Preferably, the linker sequence encodes a 2A self-cleaving peptide and / or an enzymatically cleavable peptide motif. In some embodiments, the linker sequence encodes a 2A self-cleaving peptide having at least 70% sequence identity to any of SEQ ID NOs: 55-58. The nucleotide sequence encoding the heavy chain and / or the nucleotide sequence encoding the light chain may each be operably linked to a signal sequence. In some embodiments, the signal sequence encodes a signal peptide selected from any of the human growth hormone (HGH) signal peptide, interleukin-2 (IL-2) signal peptide, CD5 signal peptide, immunoglobulin kappa light chain signal peptide, trypsinogen signal peptide, serum albumin signal peptide, and prolactin signal peptide.

[0013] In some embodiments, the nucleotide sequence encoding the TNF inhibitor comprises or consists of a heavy chain having a sequence with at least 70% identity to SEQ ID NO: 7, optionally a 2A self-cleaving peptide having at least 70% sequence identity to any of SEQ ID NOs: 55-58, and a light chain having a sequence with at least 70% identity to SEQ ID NO: 8, or encodes an anti-TNF antibody or fragment comprising or consisting of the foregoing. In some embodiments, the nucleotide sequence encoding the TNF inhibitor encodes an anti-TNF antibody or fragment comprising or consisting of an amino acid sequence having at least 70% identity to SEQ ID NO: 65.

[0014] In some embodiments, the nucleotide sequence encoding the TNF inhibitor comprises or consists of a nucleotide sequence having at least 70% identity to SEQ ID NO: 47, a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 59 or 60, and a nucleotide sequence having at least 70% identity to SEQ ID NO: 48. In some embodiments, the nucleotide sequence encoding the TNF inhibitor comprises or consists of a nucleotide sequence having at least 70% identity to SEQ ID NO: 66.

[0015] In a preferred embodiment, the nucleotide sequence encoding the TNF inhibitor is operably linked to an inflammation-inducible promoter. Any suitable inflammation-inducible promoter can be used. Preferably, the inflammation-inducible promoter comprises one or more inflammation-inducible transcription factor binding motifs selected from an AP-1 transcription factor binding motif; an NF-κB transcription factor binding motif; an IRF transcription factor binding motif; a STAT transcription factor binding motif; and an NFAT transcription factor binding motif, or any combination thereof.

[0016] In some embodiments, the inflammation-inducing promoter comprises one or more AP-1 binding motifs and / or one or more NF-κB binding motifs. In some embodiments, the inflammation-inducing promoter comprises two or more AP-1 binding motifs and / or two or more NF-κB binding motifs, three or more AP-1 binding motifs and / or three or more NF-κB binding motifs, four or more AP-1 binding motifs and / or four or more NF-κB binding motifs, or five or more AP-1 binding motifs and / or five or more NF-κB binding motifs. In some embodiments, the inflammation-inducing promoter comprises at least one AP-1 binding motif coupled to at least one NF-κB binding motif. In some embodiments, the inflammation-inducing promoter comprises five AP-1 binding motifs coupled to five NF-κB binding motifs. Preferably, the AP-1 binding motif comprises or consists of SEQ ID NO: 70, or comprises or consists of any of SEQ ID NOs: 71-73 or derivatives thereof comprising one nucleotide substitution. Preferably, the NF-κB binding motif comprises or consists of SEQ ID NO: 74, or comprises or consists of SEQ ID NO: 75 or derivatives thereof comprising two or fewer nucleotide substitutions. In some embodiments, the inflammation-inducing promoter comprises or consists of a nucleotide sequence having at least 70% identity to SEQ ID NO: 76.

[0017] In some embodiments, the vector comprises a nucleotide sequence having at least 70% identity to SEQ ID NO: 77.

[0018] The vector may contain any other suitable vector element. The nucleotide sequence encoding the TNF inhibitor may be operably linked to a polyadenylation sequence. Preferably, the polyadenylation sequence is selected from any of the bovine growth hormone (bGH) polyadenylation sequence, the SV40 polyadenylation sequence, and the rabbit beta-globin polyadenylation sequence. In some embodiments, the polyadenylation sequence comprises or consists of a nucleotide sequence having at least 70% identity with SEQ ID NO: 78. The nucleotide sequence encoding the TNF inhibitor may be operably linked to a woodchuck hepatitis post-transcriptional regulatory element (WPRE). In some embodiments, the WPRE comprises or consists of a nucleotide sequence having at least 70% identity with SEQ ID NO: 79. The nucleotide sequence encoding the TNF inhibitor may be operably linked to an intron. Preferably, the intron is selected from the beta-globin intron or the SV40 intron. In some embodiments, the intron comprises or consists of a nucleotide sequence having at least 70% identity with SEQ ID NO: 80.

[0019] In a preferred embodiment, the vector is a viral vector. Any suitable viral vector can be used. Preferably, the viral vector is any of a parvovirus vector, preferably an adeno-associated virus (AAV) vector, an adenovirus vector, a herpes simplex virus vector, an anellovirus vector, a retrovirus vector, or a lentivirus vector.

[0020] In a preferred embodiment, the vector is an adeno-associated virus (AAV) vector. In a preferred embodiment, the vector is an AAV vector particle. The AAV vector particle can be pseudotyped to confer tropism for eye tissue. Preferably, the AAV vector particle contains an AAV2 capsid protein or an AAV2 capsid variant protein, and optionally the AAV2 capsid variant is selected from any of AAV2.tYF, AAV2.7m8, R100, AAV2.GL, and AAV2.NN. The vector may contain one or more inverted terminal repeats (ITRs).

[0021] In some embodiments, the vector comprises or consists of a nucleotide sequence having at least 70% identity with SEQ ID NO: 91.

[0022] In one aspect, the present invention provides a vector comprising or consisting of a nucleotide sequence having at least 70% identity with SEQ ID NO: 91. The vector may be a viral vector. The vector may be an AAV vector.

[0023] In one aspect, the present invention provides a cell comprising the vector of the present invention. The cell may be an isolated cell.

[0024] In one aspect, the present invention provides a kit for producing the vector of the present invention.

[0025] In one aspect, the present invention provides a pharmaceutical composition comprising the vector of the present invention or the cell of the present invention. The vector or cell may be combined with a pharmaceutically acceptable carrier, diluent or excipient.

[0026] In one aspect, the present invention provides a vector, a cell and / or a pharmaceutical composition according to the present invention for use as a medicament.

[0027] In one aspect, the present invention provides the use of a vector, a cell or a pharmaceutical composition according to the present invention for the manufacture of a medicament.

[0028] In one aspect, the present invention provides a method comprising administering to a subject in need thereof a vector, a cell or a pharmaceutical composition according to the present invention.

[0029] In one aspect, the present invention provides a vector for use in the prevention or treatment of an inflammatory eye disease, wherein the vector comprises a nucleotide sequence encoding a TNF inhibitor, and the nucleotide sequence encoding the TNF inhibitor is operably linked to an inflammation-inducible promoter.

[0030] In one aspect, the present invention provides the use of a vector in the manufacture of a medicament for preventing or treating an inflammatory eye disease, wherein the vector comprises a nucleotide sequence encoding a TNF inhibitor, and the nucleotide sequence encoding the TNF inhibitor is operably linked to an inflammation-inducing promoter.

[0031] In one aspect, the present invention provides a method for preventing or treating an inflammatory eye disease, the method comprising administering a vector to a subject in need thereof, wherein the vector comprises a nucleotide sequence encoding a TNF inhibitor, and the nucleotide sequence encoding the TNF inhibitor is operably linked to an inflammation-inducing promoter.

[0032] In one aspect, the present invention provides the vector according to the present invention or the pharmaceutical composition according to the present invention for use in the prevention or treatment of an inflammatory eye disease.

[0033] In one aspect, the present invention provides the use of the vector according to the present invention or the pharmaceutical composition according to the present invention in the manufacture of a medicament for preventing or treating an inflammatory eye disease.

[0034] In one aspect, the present invention provides a method for preventing or treating an inflammatory eye disease, comprising administering the vector according to the present invention or the pharmaceutical composition according to the present invention to a subject in need thereof.

[0035] The inflammatory eye disease can be any inflammatory eye disease. Preferably, the inflammatory eye disease is uveitis. The vector or pharmaceutical composition can be administered in response to a recurrence of the inflammatory eye disease, particularly when the inflammatory eye disease is uveitis.

[0036] The vector or pharmaceutical composition can be administered by any suitable route. Preferably, the vector or pharmaceutical composition is administered intravitreally. In some embodiments, the vector or pharmaceutical composition is administered by intravitreal, subretinal, direct retinal, subconjunctival, sub-Tenon's capsule or suprachoroidal injection. In some embodiments, the vector or pharmaceutical composition is administered by intravitreal injection.

[0037] The vector or pharmaceutical composition can be administered in any suitable regimen. Preferably, the vector or pharmaceutical composition is administered as a single dose. Preferably, the vector is administered at a dose of at least about 1E10 vg / mL, at least about 1E11 vg / mL, at least about 1E12 vg / mL or at least about 5E12 vg / mL. Preferably, the vector is administered at a dose of at least about 1E9 vg / eye, at least about 1E10 vg / eye or at least about 1E11 vg / eye. Preferably, the vector is administered at a dose of about 1E9 vg / eye to about 5E12 vg / eye.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0039] Here, various preferred features and embodiments of the present invention will be described by way of non-limiting examples.

[0040] It should be noted that as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0041] As used herein, the terms "comprising", "comprises", and "consisting of" are synonymous with "including", "includes", "containing", or "contains", are inclusive or open-ended, and do not exclude additional unrecited members, elements, or steps. The terms "comprising", "comprises", and "consisting of" also include the term "consisting of".

[0042] Numeric ranges include the numbers defining the range. Unless otherwise indicated, each nucleic acid sequence is written left to right in the 5' to 3' direction, and each amino acid sequence is written left to right in the amino to carboxy direction.

[0043] The publications discussed in this specification are provided solely for their disclosure prior to the filing date of the present application. This specification should not be construed as an admission that such publications constitute prior art against the claims appended hereto. All publications mentioned in this specification are incorporated herein by reference.

[0044] The present disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Those skilled in the art will understand that all features of the invention disclosed herein can be combined without departing from the scope of the disclosed invention.

[0045] TNF inhibitor The vector of the present invention comprises a nucleotide sequence encoding a TNF inhibitor. The inventors have surprisingly shown that such a vector can be used for preventing or treating inflammatory eye diseases.

[0046] As used herein, a "TNF inhibitor" can be any protein that suppresses the inflammatory response to TNF. Tumor necrosis factor (TNF), also known as cachexin or cachectin, is sometimes also known as tumor necrosis factor alpha (TNF-α). TNF is synthesized as a transmembrane protein (mTNF) and cleaved into soluble TNF (sTNF). There are two types of TNF, TNF-alpha and TNF-beta, which are very closely related. The activity of both TNFs is mediated through binding to the TNF receptors TNFR1 and TNFR2. Binding of TNF can activate several signaling pathways, including activation of transcription factors, proteases, and protein kinases. This signaling can lead to activation of target cells that results in inflammation and immune responses through the release of several cytokines and initiation of the apoptosis pathway (see Gerriets, V. et al., 2021. "Tumor necrosis factor inhibitors" in StatPearls).

[0047] Exemplary TNF inhibitors include adalimumab, infliximab, golimumab, certolizumab pegol, etanercept, XPro1595, XENP345, R1antTNF, Atrosab and Atrosimab (see, for example, Lis, K., Kuzawinska, O. and Balkowiec-Iskra, E., 2014. AMS, 10(6), page 1175; and Fischer, R. et al., 2020. Frontiers in cell and developmental biology, 8, 401). Preferably, the TNF inhibitor can inhibit TNF activity by directly binding to TNF. For example, the TNF inhibitor may be an anti-TNF antibody or a fragment thereof (e.g., adalimumab, infliximab, golimumab, certolizumab) or may include the TNF binding domain of the TNFR receptor (e.g., etanercept). Alternatively, the TNF inhibitor can inhibit TNF activity by binding to the TNF receptor. For example, the TNF inhibitor may be a TNF mutein (e.g., XPro1595, XENP345, R1antTNF) or an anti-TNFR antibody or a fragment thereof (e.g., Atrosab and Atrosimab).

[0048] Anti-TNF antibody or a fragment thereof In a preferred embodiment, the TNF inhibitor is an anti-TNF antibody or a fragment thereof.

[0049] Antibodies are glycoproteins belonging to the immunoglobulin superfamily. Antibodies are typically made from a basic structural unit each having two heavy chains and two light chains. Antibodies can recognize antigens via the fragment antigen binding (Fab) variable region. The fragment crystallizable region (Fc region) is the tail region of the antibody that can enable the antibody to activate the immune system. The hinge region is a stretch of the heavy chain that links the Fab and Fc regions.

[0050] "Heavy chain variable region" or "VH" refers to a fragment of the heavy chain of an antibody that contains three CDRs intervening between adjacent stretches known as framework regions that form a scaffold to support the CDRs. "Light chain variable region" or "VL" refers to a fragment of the light chain of an antibody that contains three CDRs intervening between framework regions.

[0051] With respect to an antibody or an antigen-binding fragment thereof, "complementary determining region" or "CDR" refers to highly variable loops in the variable region of the heavy or light chain of the antibody. The CDRs interact with the conformation of the antigen and can mainly determine the binding to the antigen. The heavy chain variable region and the light chain variable region each contain three CDRs (numbered from the amino terminus to the carboxy terminus, heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3).

[0052] The CDRs of the variable regions of the heavy and light chains of an antibody can be predicted from the sequences of the variable regions of the heavy and light chains of the antibody using predictive software available in the art, for example, using the Abysis algorithm or the IMGT / V-QUEST software (see, for example, Lefranc et al., 2009 NAR 37:D1006-D1012 and Lefranc 2003, Leukemia 17: 260-266). The CDR regions identified by any algorithm are considered to be equally suitable for use in the present invention. The length of the CDRs can vary depending on the antibody for which the CDRs are predicted and between the heavy and light chains. Thus, the three heavy chain CDRs of an intact antibody may be of different lengths (or may be of the same length), and the three light chain CDRs of an intact antibody may be of different lengths (or may be of the same length). For example, the CDRs may range in length from 2 or 3 amino acids to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In particular, the CDRs may be 3 to 14 amino acids in length, for example at least 3 amino acids and less than 15 amino acids.

[0053] Suitable anti-TNF antibodies are known in the art. Furthermore, anti-TNF antibodies and their fragments and / or derivatives can be prepared using methods known to those skilled in the art. Such methods include phage display, methods for generating human or humanized antibodies, or methods using transgenic animals or plants engineered to produce human antibodies. Phage display libraries of partial or fully synthetic antibodies are available, and antibodies or fragments thereof that can bind to a target molecule can be screened. Phage display libraries of human antibodies are also available. Once identified, the amino acid sequence or polynucleotide sequence encoding the antibody (or its fragment and / or derivative) can be isolated and / or determined. The sequence of the antibody can be used to design its appropriate fragments and / or derivatives.

[0054] In a preferred embodiment, the anti-TNF antibody or its fragment is an anti-TNF antibody fragment. An "anti-TNF antibody fragment" can be a fragment of an anti-TNF antibody or a genetically engineered product of one or more fragments of an anti-TNF antibody, and the fragment is involved in binding to TNF. Examples include antigen-binding fragment (Fab), fragment antibody (F(ab')2), variable region (Fv), single-chain antibody (scFv), single-domain antibody (sdAb), and camel antibody (VHH). The use of anti-TNF antibody fragments can be advantageous as it may reduce inflammation associated with the Fc region.

[0055] In some embodiments, the anti-TNF antibody fragment is an antigen-binding fragment (Fab), fragment antibody (F(ab')2), single-chain antibody (scFv), or single-domain antibody (sdAb).

[0056] In a preferred embodiment, the anti-TNF antibody fragment is an antigen-binding fragment (Fab). An "antigen-binding fragment" (Fab) refers to the region on an antibody that binds to an antigen. It is composed of one constant region and one variable region of each of the heavy and light chains.

[0057] In other embodiments, the anti-TNF antibody fragment is a fragment antibody (F(ab')2). The "fragment antibody" (F(ab')2) refers to the region on the antibody that remains after digestion of the Fc region while leaving a part of the hinge region intact.

[0058] In other embodiments, the anti-TNF antibody fragment is a single-chain antibody (scFv). The "single-chain antibody" (scFv) refers to an engineered antibody consisting of a light-chain variable region and a heavy-chain variable region that are connected to each other directly or via a peptide linker sequence. The peptide linker sequence is usually about 10 to 25 amino acids in length and is rich in glycine for flexibility and serine or threonine for solubility. The peptide linker sequence can connect the N-terminus of the heavy-chain variable region to the C-terminus of the light-chain variable region or vice versa.

[0059] In other embodiments, the anti-TNF antibody fragment is a single-domain antibody (sdAb). The "single-domain antibody" (sdAb), also known as a nanobody, refers to an antibody fragment consisting of a single monomeric variable antibody domain. Thus, the sdAb can be a heavy-chain variable region (VH) or a light-chain variable region (VL). Examples of single-domain antibodies include, but are not limited to, VHH fragments and VNAR fragments. Single-domain antibodies can also be generated by splitting the dimeric variable domain from a common IgG molecule into monomers.

[0060] The TNF inhibitor may comprise at least one CDR (e.g., HCDR3) that can be predicted from an anti-TNF antibody (or a variant of such a predicted CDR, e.g., a variant having 1, 2, or 3 amino acid substitutions). It will be understood that a molecule containing 3 or fewer CDR regions (e.g., a single CDR or even a part thereof) may be able to retain the antigen-binding activity of the antibody from which the CDR is derived. Molecules containing two CDR regions are described in the art as being able to bind to a target antigen, for example, in the form of a minibody (see, e.g., Vaughan and Sollazzo, 2001, Combinational Chemistry & High Throughput Screening, 4, 417-430). Molecules containing a single CDR have been described as being able to exhibit strong binding activity to a target (see, e.g., Nicaise et al., 2004, Protein Science, 13: 1882-91).

[0061] The TNF inhibitor may comprise one or more variable heavy chain CDRs, e.g., 1, 2, or 3 variable heavy chain CDRs. Alternatively or additionally, the TNF inhibitor may comprise one or more variable light chain CDRs, e.g., 1, 2, or 3 variable light chain CDRs. The TNF inhibitor may comprise 3 heavy chain CDRs and / or 3 light chain CDRs (more specifically, a variable heavy chain region containing 3 CDRs and / or a variable light chain region containing 3 CDRs), and at least one CDR, preferably all CDRs, may be derived from an anti-TNF antibody or may be selected from one of the CDR sequences provided below.

[0062] The TNF inhibitor may include any combination of variable heavy chain CDRs and variable light chain CDRs, such as one variable heavy chain CDR and one variable light chain CDR, two variable heavy chain CDRs and one variable light chain CDR, two variable heavy chain CDRs and two variable light chain CDRs, three variable heavy chain CDRs and one or two variable light chain CDRs, one variable heavy chain CDR and two or three variable light chain CDRs, or three variable heavy chain CDRs and three variable light chain CDRs. Preferably, the TNF inhibitor includes three variable heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and / or three variable light chain CDRs (LCDR1, LCDR2, and LCDR3).

[0063] One or more CDRs present within the TNF inhibitor do not all have to be from the same antibody as long as the domain has the above binding activity. Thus, one CDR may be predicted from the heavy or light chain of an anti-TNF antibody, while another CDR present may be predicted from a different anti-TNF antibody. In this example, it may be suitable for CDR3 to be predicted from an anti-TNF antibody. However, especially when there is more than one CDR in the TNF inhibitor, it is preferred that the CDRs be predicted from anti-TNF antibodies. Combinations of CDRs may be used from different antibodies, especially antibodies that bind to the same desired region or epitope.

[0064] In a preferred embodiment, the TNF inhibitor includes three CDRs predicted from the variable heavy chain sequence of an anti-TNF antibody and / or three CDRs predicted from the variable light chain sequence of an anti-TNF antibody.

[0065] The present invention includes "variants" of the CDR regions described below. As used herein, the term "variant" is defined in the following "Variants, Derivatives and Fragments" section. It will be understood that one or more amino acid substitutions may be made in the CDR while retaining antigen-binding ability. For example, a CDR variant may include three or fewer amino acid substitutions, such as three amino acid substitutions, two amino acid substitutions, or one amino acid substitution. In particular, in some embodiments, the CDR variant includes one amino acid substitution and retains antigen-binding ability. The variant may be a variant having at least 80% or 90% identity with the CDR.

[0066] Examples of antibodies and their fragments and / or derivatives that can be used in the present invention are further described below. The TNF inhibitor may comprise or consist of an amino acid sequence comprising the CDRs described herein, and substitutions, variations, modifications, replacements, deletions and / or additions of one or more amino acid residues may occur in the framework region. The derivatives described herein may retain TNF-binding ability. The derivative may enable binding to TNF at a level of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the level of the corresponding reference amino acid sequence.

[0067] TNF binding affinity can be determined by the equilibrium binding constant (KD), which can be determined by any suitable assay, such as surface plasmon resonance (see, for example, Shealy, D.J. et al., 2010. mAbs, 2(4), pp. 428-439). Preferably, the anti-TNF antibody or fragment thereof can bind to soluble TNF with a binding affinity of about 1000 pM or less, about 900 pM or less, about 800 pM or less, about 700 pM or less, about 600 pM or less, about 500 pM or less, about 400 pM or less, about 300 pM or less, about 200 pM or less, or about 100 pM or less, as determined by, for example, surface plasmon resonance. Preferably, the anti-TNF antibody or fragment thereof can bind to soluble TNF with a binding affinity of about 0.1 pM to about 1000 pM, about 1 pM to about 1000 pM, about 10 pM to about 1000 pM, 0.1 pM to about 100 pM, about 1 pM to about 100 pM, or about 10 pM to about 100 pM, as determined by, for example, surface plasmon resonance. Preferably, the anti-TNF antibody or fragment thereof can bind to transmembrane TNF with a binding affinity of about 10000 pM or less, about 9000 pM or less, about 8000 pM or less, about 7000 pM or less, about 6000 pM or less, about 5000 pM or less, or about 4000 pM or less, as determined by, for example, surface plasmon resonance.

[0068] In some embodiments, the TNF inhibitor is selected from any of adalimumab or a fragment and / or derivative thereof; infliximab or a fragment and / or derivative thereof; golimumab or a fragment and / or derivative thereof; and certolizumab or a fragment and / or derivative thereof.

[0069] In some embodiments, the TNF inhibitor is selected from adalimumab or a fragment and / or derivative thereof; and infliximab or a fragment and / or derivative thereof.

[0070] In some embodiments, the TNF inhibitor is selected from any of an adalimumab fragment or a derivative thereof; an infliximab fragment or a derivative thereof; a golimumab fragment or a derivative thereof; and a certolizumab fragment or a derivative thereof.

[0071] In some embodiments, the TNF inhibitor is selected from either an adalimumab fragment or its derivative; and an infliximab fragment or its derivative.

[0072] Adalimumab In a preferred embodiment, the TNF inhibitor is adalimumab or a fragment and / or derivative thereof. In some embodiments, the TNF inhibitor is adalimumab Fab, adalimumab F(ab')2, adalimumab scFv, adalimumab sdAb or a derivative thereof. In some embodiments, the TNF inhibitor is adalimumab Fab or a derivative thereof.

[0073] Adalimumab (Humira®) is a recombinant fully human IgG1 monoclonal antibody, which specifically binds to TNF and thereby neutralizes the activity of the cytokine. Those skilled in the art will be able to generate adalimumab derivatives using knowledge of conservative mutations and / or the TNF inhibitory mechanism of adalimumab (see, for example, Hu, S. et al., 2013. Journal of Biological Chemistry, 288(38), pp. 27059 - 27067). For example, the following adalimumab variants have been shown to have KD values equivalent to wild - type adalimumab: L178K, L178N, Q160N, L116N, T118N, A122N, Q179N, L183N and T199N (see, for example, Reslan, M. et al., 2020. International Journal of Biological Macromolecules, 158, pp. 189 - 196).

[0074] In some embodiments, the TNF inhibitor is a fragment of adalimumab or its derivative. In some embodiments, the TNF inhibitor is adalimumab Fab, adalimumab F(ab')2, adalimumab scFv or adalimumab sdAb or a derivative thereof. In some embodiments, the TNF inhibitor is adalimumab Fab or a derivative thereof.

[0075] Suitable adalimumab Fab derivatives include those described in Yoshikawa, M. et al., 2022. The Journal of Biochemistry, mvac040; and Nakamura, H. et al., 2020. Biological and Pharmaceutical Bulletin, 43(3), pages 418 - 423. Preferably, the adalimumab Fab derivative can be selected from one or more of H:K137C - L:I117C, H:K137C - L:F209C, H:S138C - L:F116C, H:S140C - L:S114C, and H:V177C - L:Q160C.

[0076] In some embodiments, the TNF inhibitor is an anti - TNF antibody (such as Fab, F(ab')2, scFv, or sdAb) or is derived therefrom, and the antibody contains one or more CDR regions selected from SEQ ID NOs: 1 - 6 or variants thereof. In other words, in some embodiments, the TNF inhibitor contains one or more CDR regions selected from SEQ ID NOs: 1 - 6 or variants thereof.

[0077] In some embodiments, the TNF inhibitor is (i) HCDR1 having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, HCDR2 having the amino acid sequence of SEQ ID NO: 2 or a variant thereof, and / or HCDR3 having the amino acid sequence of SEQ ID NO: 3 or a variant thereof; and / or (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 4 or a variant thereof, LCDR2 having the amino acid sequence of SEQ ID NO: 5 or a variant thereof, and / or LCDR3 having the amino acid sequence of SEQ ID NO: 6 or a variant thereof and includes.

[0078] In some embodiments, the TNF inhibitor comprises an HCDR2 having the amino acid sequence of SEQ ID NO: 2 or a variant thereof and / or an LCDR2 having the amino acid sequence of SEQ ID NO: 5 or a variant thereof. In adalimumab, L2 and H2 of the CDRs contribute most to the interaction with the antigen (see Hu, S. et al., 2013. Journal of Biological Chemistry, 288(38), pp. 27059-27067).

[0079] In some embodiments, the TNF inhibitor is (i) an HCDR1 having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, an HCDR2 having the amino acid sequence of SEQ ID NO: 2 or a variant thereof, and an HCDR3 having the amino acid sequence of SEQ ID NO: 3 or a variant thereof; and / or (ii) an LCDR1 having the amino acid sequence of SEQ ID NO: 4 or a variant thereof, an LCDR2 having the amino acid sequence of SEQ ID NO: 5 or a variant thereof, and an LCDR3 having the amino acid sequence of SEQ ID NO: 6 or a variant thereof comprises.

[0080] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain, and the heavy chain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with SEQ ID NO: 7.

[0081] In some embodiments, the TNF inhibitor comprises or consists of a light chain, and the light chain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with SEQ ID NO: 8.

[0082] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 7, and the light chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 8.

[0083] In some embodiments, the TNF inhibitor is (i) a heavy chain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 7, the amino acid sequence comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, an HCDR2 having the amino acid sequence of SEQ ID NO: 2 or a variant thereof, and an HCDR3 having the amino acid sequence of SEQ ID NO: 3 or a variant thereof; and / or (ii) a light chain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 8, the amino acid sequence comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 4 or a variant thereof, an LCDR2 having the amino acid sequence of SEQ ID NO: 5 or a variant thereof, and an LCDR3 having the amino acid sequence of SEQ ID NO: 6 or a variant thereof comprising or consisting of the same.

[0084] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain, the heavy chain comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 9.

[0085] In some embodiments, the TNF inhibitor comprises or consists of a light chain, the light chain comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 11.

[0086] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain and a light chain, the heavy chain comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 9, and the light chain comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 11.

[0087] In some embodiments, the TNF inhibitor is (i) a heavy chain comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 9, the amino acid sequence comprising HCDR1 having the amino acid sequence of SEQ ID NO: 1, HCDR2 having the amino acid sequence of SEQ ID NO: 2, and HCDR3 having the amino acid sequence of SEQ ID NO: 3; and / or (ii) a light chain comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 11, wherein the amino acid sequence comprises LCDR1 having the amino acid sequence of SEQ ID NO: 4, LCDR2 having the amino acid sequence of SEQ ID NO: 5 and LCDR3 having the amino acid sequence of SEQ ID NO: 6 comprising or consisting of them.

[0088] [Table 1]

[0089] Infliximab In some embodiments, the TNF inhibitor is infliximab or a fragment and / or derivative thereof. In some embodiments, the TNF inhibitor is infliximab Fab, infliximab F(ab')2, infliximab scFv, infliximab sdAb or a derivative thereof. In some embodiments, the TNF inhibitor is infliximab Fab or a derivative thereof.

[0090] Infliximab (Remicade®) is a chimeric monoclonal antibody against human TNF. It binds to both the soluble and transmembrane forms of TNF at picomolar concentrations. Those skilled in the art will be able to generate infliximab derivatives using knowledge of conservative mutations and / or the TNF inhibitory mechanism of infliximab (see, for example, Liang, S. et al., 2013. Journal of Biological Chemistry, 288(19), pp. 13799-13807).

[0091] In some embodiments, the TNF inhibitor is a fragment or derivative of infliximab. In some embodiments, the TNF inhibitor is infliximab Fab, infliximab F(ab')2, infliximab scFv or infliximab sdAb or a derivative thereof. In some embodiments, the TNF inhibitor is infliximab Fab or a derivative thereof.

[0092] In some embodiments, the TNF inhibitor is or is derived from an anti-TNF antibody (e.g., a Fab, F(ab')2, scFv or sdAb), and the antibody comprises one or more CDR regions selected from SEQ ID NOs: 12-17 or variants thereof. In other words, in some embodiments, the TNF inhibitor comprises one or more CDR regions selected from SEQ ID NOs: 12-17 or variants thereof.

[0093] In some embodiments, the TNF inhibitor is (i) an HCDR1 having the amino acid sequence of SEQ ID NO: 12 or a variant thereof, an HCDR2 having the amino acid sequence of SEQ ID NO: 13 or a variant thereof and / or an HCDR3 having the amino acid sequence of SEQ ID NO: 14 or a variant thereof; and / or (ii) an LCDR1 having the amino acid sequence of SEQ ID NO: 15 or a variant thereof, an LCDR2 having the amino acid sequence of SEQ ID NO: 16 or a variant thereof and / or an LCDR3 having the amino acid sequence of SEQ ID NO: 17 or a variant thereof comprises.

[0094] In some embodiments, the TNF inhibitor is (i) an HCDR1 having the amino acid sequence of SEQ ID NO: 12 or a variant thereof, an HCDR2 having the amino acid sequence of SEQ ID NO: 13 or a variant thereof and an HCDR3 having the amino acid sequence of SEQ ID NO: 14 or a variant thereof; and / or (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 15 or a variant thereof, LCDR2 having the amino acid sequence of SEQ ID NO: 16 or a variant thereof, and LCDR3 having the amino acid sequence of SEQ ID NO: 17 or a variant thereof comprises.

[0095] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain, and the heavy chain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with SEQ ID NO: 18.

[0096] In some embodiments, the TNF inhibitor comprises or consists of a light chain, and the light chain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with SEQ ID NO: 19.

[0097] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain and a light chain, the heavy chain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with SEQ ID NO: 18, and the light chain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with SEQ ID NO: 19.

[0098] In some embodiments, the TNF inhibitor is (i) A heavy chain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 18, wherein the amino acid sequence comprises an HCDR1 having the amino acid sequence of SEQ ID NO: 12 or a variant thereof, an HCDR2 having the amino acid sequence of SEQ ID NO: 13 or a variant thereof, and an HCDR3 having the amino acid sequence of SEQ ID NO: 14 or a variant thereof; and / or (ii) A light chain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 19, wherein the amino acid sequence comprises an LCDR1 having the amino acid sequence of SEQ ID NO: 15 or a variant thereof, an LCDR2 having the amino acid sequence of SEQ ID NO: 16 or a variant thereof, and an LCDR3 having the amino acid sequence of SEQ ID NO: 17 or a variant thereof comprising or consisting of these.

[0099] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain, and the heavy chain comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with SEQ ID NO: 20 or 21.

[0100] In some embodiments, the TNF inhibitor comprises or consists of a light chain, and the light chain comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with SEQ ID NO: 22.

[0101] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain and a light chain, the heavy chain comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 20, and the light chain comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 22.

[0102] In some embodiments, the TNF inhibitor is (i) a heavy chain comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 20, the amino acid sequence comprising HCDR1 having the amino acid sequence of SEQ ID NO: 12, HCDR2 having the amino acid sequence of SEQ ID NO: 13, and HCDR3 having the amino acid sequence of SEQ ID NO: 14; and / or (ii) a light chain comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 22, the amino acid sequence comprising LCDR1 having the amino acid sequence of SEQ ID NO: 15, LCDR2 having the amino acid sequence of SEQ ID NO: 16, and LCDR3 having the amino acid sequence of SEQ ID NO: 17 and comprises or consists of them.

[0103]

Table 2

[0104] Golimumab In some embodiments, the TNF inhibitor is golimumab or a fragment and / or derivative thereof. In some embodiments, the TNF inhibitor is golimumab Fab, golimumab F(ab')2, golimumab scFv, golimumab sdAb, or a derivative thereof. In some embodiments, the TNF inhibitor is golimumab Fab or a derivative thereof.

[0105] Golimumab (Simponi®) is a human IgG1 TNF antagonist monoclonal antibody. Those skilled in the art will be able to generate golimumab derivatives using knowledge of conservative mutations and / or the TNF inhibitory mechanism of golimumab (see, e.g., Shealy, D.J. et al., 2010. Mabs, 2(4), pages 428-439).

[0106] In some embodiments, the TNF inhibitor is a fragment of golimumab or a derivative thereof. In some embodiments, the TNF inhibitor is golimumab Fab, golimumab F(ab')2, golimumab scFv, or golimumab sdAb, or a derivative thereof. In some embodiments, the TNF inhibitor is golimumab Fab or a derivative thereof.

[0107] In some embodiments, the TNF inhibitor is an anti-TNF antibody (e.g., Fab, F(ab')2, scFv, or sdAb) or is derived therefrom, and the antibody comprises one or more CDR regions selected from SEQ ID NOs: 23-28 or variants thereof. In other words, in some embodiments, the TNF inhibitor comprises one or more CDR regions selected from SEQ ID NOs: 23-28 or variants thereof.

[0108] In some embodiments, the TNF inhibitor is (i) HCDR1 having the amino acid sequence of SEQ ID NO: 23 or a variant thereof, HCDR2 having the amino acid sequence of SEQ ID NO: 24 or a variant thereof, and / or HCDR3 having the amino acid sequence of SEQ ID NO: 25 or a variant thereof; and / or (ii) an LCDR1 having the amino acid sequence of SEQ ID NO: 26 or a variant thereof, an LCDR2 having the amino acid sequence of SEQ ID NO: 27 or a variant thereof, and / or an LCDR3 having the amino acid sequence of SEQ ID NO: 28 or a variant thereof comprises.

[0109] In some embodiments, the TNF inhibitor is (i) an HCDR1 having the amino acid sequence of SEQ ID NO: 23 or a variant thereof, an HCDR2 having the amino acid sequence of SEQ ID NO: 24 or a variant thereof, and an HCDR3 having the amino acid sequence of SEQ ID NO: 25 or a variant thereof; and / or (ii) an LCDR1 having the amino acid sequence of SEQ ID NO: 26 or a variant thereof, an LCDR2 having the amino acid sequence of SEQ ID NO: 27 or a variant thereof, and an LCDR3 having the amino acid sequence of SEQ ID NO: 28 or a variant thereof comprises.

[0110] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain, and the heavy chain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with SEQ ID NO: 29.

[0111] In some embodiments, the TNF inhibitor comprises or consists of a light chain, and the light chain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with SEQ ID NO: 30.

[0112] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 29, and the light chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 30.

[0113] In some embodiments, the TNF inhibitor is (i) a heavy chain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 29, the amino acid sequence comprising HCDR1 having the amino acid sequence of SEQ ID NO: 23 or a variant thereof, HCDR2 having the amino acid sequence of SEQ ID NO: 24 or a variant thereof, and HCDR3 having the amino acid sequence of SEQ ID NO: 25 or a variant thereof; and / or (ii) a light chain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 30, the amino acid sequence comprising LCDR1 having the amino acid sequence of SEQ ID NO: 26 or a variant thereof, LCDR2 having the amino acid sequence of SEQ ID NO: 27 or a variant thereof, and LCDR3 having the amino acid sequence of SEQ ID NO: 28 or a variant thereof and comprises or consists of them.

[0114] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain, the heavy chain comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 31.

[0115] In some embodiments, the TNF inhibitor comprises or consists of a light chain, the light chain comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 33.

[0116] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain and a light chain, the heavy chain comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 31, and the light chain comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 33.

[0117] In some embodiments, the TNF inhibitor is (i) a heavy chain comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 31, the amino acid sequence comprising HCDR1 having the amino acid sequence of SEQ ID NO: 23, HCDR2 having the amino acid sequence of SEQ ID NO: 24, and HCDR3 having the amino acid sequence of SEQ ID NO: 25; and / or (ii) A light chain comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 33, wherein the amino acid sequence comprises LCDR1 having the amino acid sequence of SEQ ID NO: 26, LCDR2 having the amino acid sequence of SEQ ID NO: 27, and LCDR3 having the amino acid sequence of SEQ ID NO: 28 Comprising or consisting of them.

[0118]

Table 3

[0119] Certolizumab In some embodiments, the TNF inhibitor is certolizumab or a fragment and / or derivative thereof. In some embodiments, the TNF inhibitor is certolizumab Fab, certolizumab F(ab')2, certolizumab scFv, certolizumab sdAb or a derivative thereof. In some embodiments, the TNF inhibitor is certolizumab Fab or a derivative thereof.

[0120] Certolizumab is a humanized antigen-binding fragment (Fab') of a monoclonal antibody, which is usually administered in a form conjugated to polyethylene glycol (Cimzia®). Those skilled in the art could generate certolizumab derivatives using knowledge of conservative mutations and / or the TNF inhibitory mechanism of certolizumab (see, for example, Lee, J.U. et al., 2017. International journal of molecular sciences, 18(1), page 228).

[0121] In some embodiments, the TNF inhibitor is a fragment of certolizumab or a derivative thereof. In some embodiments, the TNF inhibitor is certolizumab Fab, certolizumab F(ab')2, certolizumab scFv or certolizumab sdAb or a derivative thereof. In some embodiments, the TNF inhibitor is certolizumab Fab or a derivative thereof.

[0122] In some embodiments, the TNF inhibitor is or is derived from an anti-TNF antibody (e.g., a Fab, F(ab')2, scFv, or sdAb), and the antibody comprises one or more CDR regions selected from SEQ ID NOs: 34-39 or variants thereof. In other words, in some embodiments, the TNF inhibitor comprises one or more CDR regions selected from SEQ ID NOs: 34-39 or variants thereof.

[0123] In some embodiments, the TNF inhibitor is (i) an HCDR1 having the amino acid sequence of SEQ ID NO: 34 or a variant thereof, an HCDR2 having the amino acid sequence of SEQ ID NO: 35 or a variant thereof, and / or an HCDR3 having the amino acid sequence of SEQ ID NO: 36 or a variant thereof; and / or (ii) an LCDR1 having the amino acid sequence of SEQ ID NO: 37 or a variant thereof, an LCDR2 having the amino acid sequence of SEQ ID NO: 38 or a variant thereof, and / or an LCDR3 having the amino acid sequence of SEQ ID NO: 39 or a variant thereof and comprises.

[0124] In some embodiments, the TNF inhibitor is (i) an HCDR1 having the amino acid sequence of SEQ ID NO: 34 or a variant thereof, an HCDR2 having the amino acid sequence of SEQ ID NO: 35 or a variant thereof, and an HCDR3 having the amino acid sequence of SEQ ID NO: 36 or a variant thereof; and / or (ii) an LCDR1 having the amino acid sequence of SEQ ID NO: 37 or a variant thereof, an LCDR2 having the amino acid sequence of SEQ ID NO: 38 or a variant thereof, and an LCDR3 having the amino acid sequence of SEQ ID NO: 39 or a variant thereof and comprises.

[0125] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain, the heavy chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 40.

[0126] In some embodiments, the TNF inhibitor comprises or consists of a light chain, the light chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 41.

[0127] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 40, and the light chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 41.

[0128] In some embodiments, the TNF inhibitor is (i) a heavy chain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 40, the amino acid sequence comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 34 or a variant thereof, an HCDR2 having the amino acid sequence of SEQ ID NO: 35 or a variant thereof, and an HCDR3 having the amino acid sequence of SEQ ID NO: 36 or a variant thereof; and / or (ii) a light chain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 41, wherein the amino acid sequence comprises an LCDR1 having the amino acid sequence of SEQ ID NO: 37 or a variant thereof, an LCDR2 having the amino acid sequence of SEQ ID NO: 38 or a variant thereof, and an LCDR3 having the amino acid sequence of SEQ ID NO: 39 or a variant thereof comprising or consisting of them.

[0129] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain, the heavy chain comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity to SEQ ID NO: 42 or 43.

[0130] In some embodiments, the TNF inhibitor comprises or consists of a light chain, the light chain comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity to SEQ ID NO: 44.

[0131] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain and a light chain, the heavy chain comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity to SEQ ID NO: 42, and the light chain comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity to SEQ ID NO: 44.

[0132] In some embodiments, the TNF inhibitor is (i) a heavy chain comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 42, the amino acid sequence comprising HCDR1 having the amino acid sequence of SEQ ID NO: 34, HCDR2 having the amino acid sequence of SEQ ID NO: 35, and HCDR3 having the amino acid sequence of SEQ ID NO: 36; and / or (ii) a light chain comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 44, the amino acid sequence comprising LCDR1 having the amino acid sequence of SEQ ID NO: 34, LCDR2 having the amino acid sequence of SEQ ID NO: 35, and LCDR3 having the amino acid sequence of SEQ ID NO: 36 comprising or consisting of them.

[0133] [Table 4]

[0134] TNFR domain In some embodiments, the TNF inhibitor comprises the TNF binding domain of a TNF receptor (TNFR).

[0135] TNF signals through two receptors (TNFR1 and TNFR2) that share a similar structural arrangement with an N-terminal extracellular domain (ECD) composed of four cysteine-rich domains (CRDs), an α-helix transmembrane domain, and a cytoplasmic domain. The two receptors are most different in their cytoplasmic domains, and TNFR1 has a death domain that TNFR2 does not have (see, for example, Bodmer, J.L. et al., Trends in biochemical sciences, 27(1), pages 19-26).

[0136] In some embodiments, the TNF inhibitor comprises a soluble form of a TNF receptor (TNFR). In some embodiments, the TNF inhibitor comprises a soluble form of TNFR1 or TNFR2. In some embodiments, the TNF inhibitor comprises or consists of a soluble form of TNFR2.

[0137] The TNF binding domain and / or the soluble form of the TNF receptor can be fused to any suitable domain. Preferably, the TNF binding domain and / or the soluble form of the TNF receptor can be coupled to an Fc domain (e.g., the Fc portion of human IgG1).

[0138] Etanercept In some embodiments, the TNF inhibitor is etanercept or a derivative thereof.

[0139] Etanercept (Enbrel®) is a fusion protein consisting of the TNFR2 domain coupled to the Fc portion of human IgG1. Those skilled in the art could generate etanercept derivatives using knowledge of conservative mutations and / or the TNF inhibitory mechanism of etanercept (see, for example, Lamanna, W.C. et al., 2017. Scientific reports, 7(1), pp. 1-8).

[0140] In some embodiments, the TNF inhibitor comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 45. In some embodiments, the TNF inhibitor comprises or consists of the amino acid sequence of SEQ ID NO: 45.

[0141] TIFF2025522813000005.tif38150 Example of the etanercept sequence (SEQ ID NO: 45)

[0142] Other TNF inhibitors In some embodiments, the TNF inhibitor is a fusion protein consisting of the extracellular domain of TNFR1 coupled to the Fc portion of human IgG1.

[0143] In some embodiments, the TNF inhibitor comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with SEQ ID NO: 93. In some embodiments, the TNF inhibitor comprises or consists of the amino acid sequence of SEQ ID NO: 93.

[0144] TIFF2025522813000006.tif38150 Example of fusion protein sequence (SEQ ID NO: 93)

[0145] Signal peptide The TNF inhibitor can be operably linked to one or more signal peptides.

[0146] "Signal peptide" can refer to a short peptide that induces the insertion of a protein into the endoplasmic reticulum membrane. Signal peptides are typically N-terminal extensions of newly synthesized secretory and membrane proteins, are 16 - 30 amino acid residues in length, and consist of a hydrophilic and usually positively charged N-terminal region, a central hydrophobic domain, and a C-terminal region with a cleavage site for signal peptidase. In addition to these common features, signal peptides do not share sequence similarity and some are over 50 amino acid residues in length (see, for example, Kapp, K. et al., 2009. Protein transport into the endoplasmic reticulum, pages 1 - 16).

[0147] The TNF inhibitor can be functionally linked to any suitable signal peptide. The SPdb Signal Peptide Database is a repository of experimentally determined and computationally predicted signal peptides (see, for example, Choo, K.H. et al., 2005. BMC bioinformatics, 6(1), pp. 1 - 8). Suitable signal peptides include human growth hormone (HGH) signal peptide, interleukin - 2 (IL - 2) signal peptide, CD5 signal peptide, immunoglobulin kappa light chain signal peptide, trypsinogen signal peptide, serum albumin signal peptide, or prolactin signal peptide.

[0148] In some embodiments, the TNF inhibitor is functionally linked to one or more signal peptides selected from any of the human growth hormone (HGH) signal peptide, interleukin - 2 (IL - 2) signal peptide, CD5 signal peptide, immunoglobulin kappa light chain signal peptide, trypsinogen signal peptide, serum albumin signal peptide, and prolactin signal peptide. In some embodiments, the TNF inhibitor is functionally linked to one or more human growth hormone (HGH) signal peptides.

[0149] In some embodiments, the TNF inhibitor is functionally linked to one or more signal peptides comprising or consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with SEQ ID NO: 46. In some embodiments, the TNF inhibitor is functionally linked to one or more signal peptides comprising or consisting of the amino acid sequence of SEQ ID NO: 46.

[0150] TIFF2025522813000007.tif7145Example of HGH signal peptide (SEQ ID NO: 46)

[0151] Example of nucleotide sequence The TNF inhibitor can be encoded by any suitable nucleotide sequence.

[0152] In some embodiments, the nucleotide sequence is codon-optimized, for example, codon-optimized for expression in humans. Different cells have different usage of specific codons. This codon bias corresponds to the bias in the relative abundance of specific tRNAs in the cell type. By changing the codons in the sequence to match the relative abundance of the corresponding tRNA, expression can be increased. Similarly, expression can be decreased by intentionally selecting codons that are known to be rare for the corresponding tRNA in a particular cell type. Thus, an additional degree of translational control becomes available. Codon usage tables are known in the art for various organisms as well as for mammalian cells (e.g., human).

[0153] In a preferred embodiment, the TNF inhibitor is an anti-TNF antigen-binding fragment (Fab). Preferably, the nucleotide sequence encoding the anti-TNF Fab can include, from 5' to 3': a first signal sequence; nucleotides encoding a heavy chain; a linker sequence; a second signal sequence; and nucleotides encoding a light chain. Preferably, the nucleotide sequence encoding the anti-TNF Fab can include, from 5' to 3': a first signal sequence; nucleotides encoding a light chain; a linker sequence; a second signal sequence; and nucleotides encoding a heavy chain.

[0154] Heavy and light chains The nucleotides encoding the heavy chain and the nucleotides encoding the light chain can encode any combination of heavy and light chains described herein. For example, the nucleotide sequence encoding the anti-TNF Fab can include, from 5' to 3': (a) a first signal sequence; nucleotides encoding an adalimumab heavy chain or a derivative thereof; a linker sequence; a second signal sequence; and nucleotides encoding an adalimumab light chain or a derivative thereof; (b) The first signal sequence; nucleotides encoding infliximab heavy chain or its derivative; linker sequence; the second signal sequence; and nucleotides encoding infliximab light chain or its derivative; (c) The first signal sequence; nucleotides encoding golimumab heavy chain or its derivative; linker sequence; the second signal sequence; and nucleotides encoding golimumab light chain or its derivative; (d) The first signal sequence; nucleotides encoding certolizumab heavy chain or its derivative; linker sequence; the second signal sequence; and nucleotides encoding certolizumab light chain or its derivative; (e) The first signal sequence; nucleotides encoding adalimumab light chain or its derivative; linker sequence; the second signal sequence; and nucleotides encoding adalimumab heavy chain or its derivative; (f) The first signal sequence; nucleotides encoding infliximab light chain or its derivative; linker sequence; the second signal sequence; and nucleotides encoding infliximab heavy chain or its derivative; (g) The first signal sequence; nucleotides encoding golimumab light chain or its derivative; linker sequence; the second signal sequence; and nucleotides encoding golimumab heavy chain or its derivative; or (h) The first signal sequence; nucleotides encoding certolizumab light chain or its derivative; linker sequence; the second signal sequence; and nucleotides encoding certolizumab heavy chain or its derivative may include.

[0155] In some embodiments, the nucleotide sequence encoding the anti-TNF Fab may comprise, from 5' to 3': a first signal sequence; nucleotides encoding the adalimumab heavy chain or a derivative thereof; a linker sequence; a second signal sequence; and nucleotides encoding the adalimumab light chain or a derivative thereof. In some embodiments, the nucleotide sequence encoding the anti-TNF Fab may comprise, from 5' to 3': a first signal sequence; nucleotides encoding the adalimumab light chain or a derivative thereof; a linker sequence; a second signal sequence; and nucleotides encoding the adalimumab heavy chain or a derivative thereof.

[0156] In some embodiments, the nucleotide sequence encoding the adalimumab heavy chain comprises or consists of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with SEQ ID NO: 47. In some embodiments, the nucleotide sequence encoding the adalimumab heavy chain comprises or consists of SEQ ID NO: 47.

[0157] TIFF2025522813000008.tif50150Example of the nucleotide sequence encoding the adalimumab heavy chain (SEQ ID NO: 47)

[0158] In some embodiments, the nucleotide sequence encoding the adalimumab light chain comprises or consists of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with SEQ ID NO: 48. In some embodiments, the nucleotide sequence encoding the adalimumab light chain comprises or consists of SEQ ID NO: 48.

[0159] TIFF2025522813000009.tif50150Example of the nucleotide sequence encoding the adalimumab light chain (SEQ ID NO: 48)

[0160] In some embodiments, the nucleotide sequence encoding the anti-TNF Fab may include, from 5' to 3': a first signal sequence; nucleotides encoding infliximab heavy chain or a derivative thereof; a linker sequence; a second signal sequence; and nucleotides encoding infliximab light chain or a derivative thereof. In some embodiments, the nucleotide sequence encoding the anti-TNF Fab may include, from 5' to 3': a first signal sequence; nucleotides encoding infliximab light chain or a derivative thereof; a linker sequence; a second signal sequence; and nucleotides encoding infliximab heavy chain or a derivative thereof.

[0161] In some embodiments, the nucleotide sequence encoding the infliximab heavy chain includes or consists of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 49. In some embodiments, the nucleotide sequence encoding the infliximab light chain includes or consists of SEQ ID NO: 49.

[0162] TIFF2025522813000010.tif50149 Example of nucleotide sequence encoding infliximab heavy chain (SEQ ID NO: 49)

[0163] In some embodiments, the nucleotide sequence encoding the infliximab heavy chain includes or consists of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 50. In some embodiments, the nucleotide sequence encoding the infliximab light chain includes or consists of SEQ ID NO: 50.

[0164] TIFF2025522813000011.tif50149 Example of nucleotide sequence encoding infliximab light chain (SEQ ID NO: 50)

[0165] Signal sequence The first signal sequence and the second signal sequence may encode the same signal peptide or different signal peptides. In some embodiments, the first signal sequence and the second signal sequence encode the same signal peptide. The first signal sequence and the second signal sequence may be any signal sequence described herein.

[0166] In some embodiments, the first signal sequence and / or the second signal sequence encodes any one of a human growth hormone (HGH) signal peptide, an interleukin-2 (IL-2) signal peptide, a CD5 signal peptide, an immunoglobulin kappa light chain signal peptide, a trypsinogen signal peptide, a serum albumin signal peptide, and a prolactin signal peptide.

[0167] In some embodiments, the first signal sequence and / or the second signal sequence encodes a human growth hormone (HGH) signal peptide. In some embodiments, the first signal sequence and the second signal sequence encode a human growth hormone (HGH) signal peptide.

[0168] In some embodiments, the first signal sequence and / or the second signal sequence comprises or consists of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with any one of SEQ ID NOs: 51-54. In some embodiments, the first signal sequence and / or the second signal sequence comprises or consists of a nucleotide sequence of any one of SEQ ID NOs: 51-54.

[0169] TIFF2025522813000012.tif11150Example of HGH signal sequence (SEQ ID NO: 51) TIFF2025522813000013.tif10148Example of HGH signal sequence (SEQ ID NO: 52) TIFF2025522813000014.tif10148Example of HGH signal sequence (SEQ ID NO: 53) TIFF2025522813000015.tif10148Example of HGH signal sequence (SEQ ID NO: 54)

[0170] Linker sequence The linker sequence may include one or more cleavage sites. As used herein, "cleavage site" may include a nucleotide sequence encoding a specific peptide sequence at which a site-specific protease can cleave or excise a peptide (also known as an enzymatically cleavable peptide motif) and a nucleotide sequence encoding a self-cleaving peptide.

[0171] In preferred embodiments, the linker sequence encodes a cleavage site. In some embodiments, the linker sequence encodes a self-cleaving peptide and / or an enzymatically cleavable peptide motif.

[0172] In some embodiments, the linker sequence encodes a 2A self-cleaving peptide. 2A self-cleaving peptides are a class of peptides 18 - 22 aa in length that can induce ribosome skipping during protein translation in cells. Suitable 2A self-cleaving peptides include T2A, P2A, E2A, and F2A or derivatives thereof.

[0173] In some embodiments, the linker sequence encodes a 2A self-cleaving peptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with any of SEQ ID NOs: 55-58. In some embodiments, the linker sequence encodes a 2A self-cleaving peptide comprising or consisting of any of SEQ ID NOs: 55-58.

[0174] In some embodiments, the linker sequence encodes a 2A self-cleaving peptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with SEQ ID NO: 55. In some embodiments, the linker sequence encodes a 2A self-cleaving peptide comprising or consisting of SEQ ID NO: 55.

[0175] TIFF2025522813000016.tif7131P2A peptide sequence example (SEQ ID NO: 55) TIFF2025522813000017.tif7131T2A peptide sequence example (SEQ ID NO: 56) TIFF2025522813000018.tif7131E2A peptide sequence example (SEQ ID NO: 57) TIFF2025522813000019.tif7137F2A peptide sequence example (SEQ ID NO: 58)

[0176] In some embodiments, the linker sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with any of SEQ ID NOs: 59-60. In some embodiments, the linker sequence comprises any of SEQ ID NOs: 59-60.

[0177] TIFF2025522813000020.tif Example of 7139P2A nucleotide sequence (SEQ ID NO: 59) TIFF2025522813000021.tif Example of 7139P2A nucleotide sequence (SEQ ID NO: 60)

[0178] The linker sequence can include any other suitable nucleotide sequence, for example, a nucleotide sequence that aids in the expression of anti-TNF Fab. Preferably, the linker sequence can include a Furin site and / or a fusion protein linker sequence.

[0179] In some embodiments, the linker sequence includes a Furin site. Furin is a protease enzyme that can cleave at the conserved polybasic RNRR site. Preferably, the Furin site encodes RKRR. In some embodiments, the linker sequence includes a fusion protein linker sequence. The fusion protein linker sequence can join two protein domains together. Preferably, the fusion protein linker sequence encodes SGSG.

[0180] In some embodiments, the linker sequence encodes, from 5' to 3': a Furin site, a fusion protein linker sequence, and a 2A self-cleaving peptide.

[0181] In some embodiments, the linker sequence encodes an amino acid having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity to SEQ ID NO: 61. In some embodiments, the linker sequence encodes an amino acid comprising or consisting of SEQ ID NO: 61.

[0182] TIFF2025522813000022.tif Example of linker peptide sequence (SEQ ID NO: 61)

[0183] In some embodiments, the linker sequence comprises or consists of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with any of SEQ ID NOs: 62-63. In some embodiments, the linker sequence comprises or consists of any of SEQ ID NOs: 62-63.

[0184] TIFF2025522813000023.tif10149 Example of linker sequence (SEQ ID NO: 62) TIFF2025522813000024.tif10149 Example of linker sequence (SEQ ID NO: 63)

[0185] Other sequences The nucleotide sequence encoding the anti-TNF Fab may include any other suitable sequence. For example, the anti-TNF Fab may include an HA tag for detection. Preferably, the HA tag may comprise or consist of SEQ ID NO: 64.

[0186] TIFF2025522813000025.tif7138 Example of HA tag (SEQ ID NO: 64)

[0187] Examples of anti-TNF Fab sequences In some embodiments, the nucleotide sequence encoding the anti-TNF Fab encodes an amino acid sequence that, from 5' to 3', comprises or consists of an amino acid sequence having at least 70% identity with SEQ ID NO: 46, an amino acid sequence having at least 70% identity with SEQ ID NO: 7, an amino acid sequence having at least 70% sequence identity with any of SEQ ID NOs: 55-58, an amino acid sequence having at least 70% identity with SEQ ID NO: 46, and an amino acid sequence having at least 70% identity with SEQ ID NO: 8.

[0188] In some embodiments, the nucleotide sequence encoding the anti-TNF Fab encodes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with SEQ ID NO: 65. In some embodiments, the nucleotide sequence encoding the anti-TNF Fab encodes the amino acid sequence of SEQ ID NO: 65.

[0189] TIFF2025522813000026.tif42149 Example of adalimumab Fab amino acid sequence (SEQ ID NO: 65)

[0190] In some embodiments, the nucleotide sequence encoding the anti-TNF Fab comprises or consists of a nucleotide sequence having at least 70% identity with any one of SEQ ID NOs: 51-54 from 5' to 3', a nucleotide sequence having at least 70% identity with SEQ ID NO: 47, a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 59 or 60, a nucleotide sequence having at least 70% identity with any one of SEQ ID NOs: 51-54 and a nucleotide sequence having at least 70% identity with SEQ ID NO: 48.

[0191] In some embodiments, the nucleotide sequence encoding the anti-TNF Fab comprises or consists of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with SEQ ID NO: 66. In some embodiments, the nucleotide sequence encoding the anti-TNF Fab comprises or consists of SEQ ID NO: 66.

[0192] TIFF2025522813000027.tif112150 Example of adalimumab Fab nucleotide sequence (SEQ ID NO: 66)

[0193] In some embodiments, the nucleotide sequence encoding the anti-TNF Fab encodes an amino acid sequence that, from 5' to 3', has at least 70% identity with the amino acid sequence of SEQ ID NO: 46, at least 70% identity with the amino acid sequence of SEQ ID NO: 18, at least 70% sequence identity with any one of SEQ ID NOs: 55-58, at least 70% identity with the amino acid sequence of SEQ ID NO: 46, and at least 70% identity with the amino acid sequence of SEQ ID NO: 19, or consists of or comprises such amino acid sequences.

[0194] In some embodiments, the nucleotide sequence encoding the anti-TNF Fab encodes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with SEQ ID NO: 67. In some embodiments, the nucleotide sequence encoding the anti-TNF Fab encodes the amino acid sequence of SEQ ID NO: 67.

[0195] TIFF2025522813000028.tif42150 Example of infliximab Fab amino acid sequence (SEQ ID NO: 67)

[0196] In some embodiments, the nucleotide sequence encoding the anti-TNF Fab, from 5' to 3', comprises or consists of a nucleotide sequence having at least 70% identity with any one of SEQ ID NOs: 51-54, a nucleotide sequence having at least 70% identity with SEQ ID NO: 49, a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 59 or 60, a nucleotide sequence having at least 70% identity with any one of SEQ ID NOs: 51-54, and a nucleotide sequence having at least 70% identity with SEQ ID NO: 50.

[0197] In some embodiments, the nucleotide sequence encoding the anti-TNF Fab comprises or consists of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 68. In some embodiments, the nucleotide sequence encoding the anti-TNF Fab comprises or consists of SEQ ID NO: 68.

[0198] TIFF2025522813000029.tif77149TIFF2025522813000030.tifExample of infliximab Fab nucleotide sequence (SEQ ID NO: 68)

[0199] Promoter The vectors of the invention may include a promoter, preferably an inflammation-inducible promoter. Preferably, the promoter can be operably linked to a nucleotide sequence encoding a TNF inhibitor (e.g., an anti-TNF antibody fragment). The term "operably linked" may mean that the recited components are in a relationship that enables them to function in their intended manner.

[0200] A "promoter" may refer to a region of DNA that causes the initiation of transcription of a gene. A promoter is typically located near the transcription start site of a gene, upstream of the DNA (towards the 5' region of the sense strand). Any suitable promoter may be used, and the selection can be readily made by those skilled in the art.

[0201] Promoters typically include "core" and "proximal" regions. The "core promoter region" can include promoter elements such as the transcription start site, RNA polymerase binding site, and general transcription factor binding sites (e.g., TATA box, B recognition element). The "proximal promoter region" can include, for example, primary regulatory elements and specific transcription factor binding sites required to promote efficient and controllable transcription. The sizes and components of both the core promoter region and the proximal promoter region typically vary in a gene-specific manner.

[0202] In some embodiments, the promoter is an eye tissue-specific promoter. As used herein, an "eye tissue-specific promoter" is a promoter that preferentially promotes the expression of a gene in eye cells (e.g., photoreceptors, RPE cells, retinal ganglion cells, etc.).

[0203] In other embodiments, the promoter is a constitutive promoter. As used herein, a "constitutive promoter" is a promoter that is always active. Exemplary constitutive promoters include the chicken beta-actin (CBA) promoter or variants or fragments thereof. The promoter may comprise or consist of the CAG promoter or variants or fragments thereof.

[0204] Inflammation-inducible promoter The vector of the present invention preferably includes an inflammation-inducible promoter. Preferably, a nucleotide sequence encoding a TNF inhibitor (e.g., an anti-TNF antibody fragment) is operably linked to the inflammation-inducible promoter. The promoter can promote the expression of the TNF inhibitor in response to inflammation.

[0205] The use of an inflammation-inducible promoter is advantageous because it can express a TNF inhibitor when inflammation is occurring at a subclinical level, thereby preventing damage caused by a flare-up before symptoms occur.

[0206] As used herein, an "inflammation-inducible promoter" may refer to a promoter that preferentially promotes the expression of a transgene operably linked in response to inflammation. Inflammation can be characterized at the tissue level by redness, swelling, heat, pain, and / or loss of tissue function (see, for example, Chen, L. et al., 2018. Oncotarget, 9(6), page 7204). Inflammation can be characterized at the cellular level by an increase in the levels of inflammatory cytokines, activated immune cells, or acute-phase proteins. Inflammation may be considered acute, i.e., an immediate response to a harmful stimulus, or chronic, i.e., long-term inflammation.

[0207] Preferably, an inflammation-inducible promoter can promote higher expression of a transgene operably linked in response to a higher level of inflammation. Higher expression can be measured, for example, by measuring the expression of a transgene operably linked to the promoter, such as green fluorescent protein (GFP), and the expression of the transgene correlates with the ability of the promoter to promote gene expression. The level of inflammation can be determined by appropriate methods known in the art (see, for example, Menzel et al., 2021, Antioxidants, 10(3), page 414). For example, measurement of the level of an inflammation marker can be performed from an appropriate medium, such as a body fluid. Suitable body fluids may include blood, urine, or vitreous humor.

[0208] Suitably, the inflammation-inducible promoter can promote higher expression of a functionally linked transgene in eye cells (such as photoreceptors, RPE cells, retinal ganglion cells, etc.) in response to a higher level of inflammation in the eye cells. The level of inflammation in the eye can be determined by appropriate non-invasive methods known in the art. For example, by grading eye inflammation (see, e.g., McNeil, R., 2016. Eye news, 22(5), pages 1-4-19) or by laser flare photometry (see, e.g., Tugal-Tutkun, I. and Herbort, C.P., 2010. International ophthalmology, 30(5), pages 453-464).

[0209] The inflammation-inducible promoter may (or may be derived from) a promoter associated with a gene whose expression increases in response to inflammation. Suitably, the inflammation-inducible promoter may (or may be derived from) a promoter associated with a gene whose expression increases in eye cells in response to inflammation (such as uveitis). For example, genes whose expression increases in uveitis include CCL16, IL15, CCL7, CYSLTR1, IL17, IL4, CCL8, IL15RA, CCR1, CDC25A, LAT, MEF2B, CSF3, MAP2K7, IL20, TYK2, MAF, COL1A1, IKBKG, MGC27165, IL10, CIITA, NFATC2IP, MEF2D, GSK3A, TH1L, IL3, IL10RB, SMAD9, C19orf10, IL13RA2, TGFB2, LEP, HAVCR2, SOCS5, IRF1, TXLNA, RFX2, FADD, TGIF, CCR4, RFX1, MIF, IL10RA, CXCL5, LTA, IGFBP3, NFKBIB, CXCL13, IKBKE, MEF2A, LAG3, ICAM1, AMHR2, CCL15 and PDGFB (see, e.g., Li, Z. et al., 2008. The Journal of Immunology, 181(7), pages 5147-5157). Methods for identifying promoters associated with genes will be known to those skilled in the art.

[0210] In some embodiments, the inflammation-inducing promoter comprises an IFN-beta minimal promoter or a fragment or derivative thereof. In some embodiments, the inflammation-inducing promoter comprises a nucleotide sequence that is at least 70% identical to SEQ ID NO: 69 or a fragment thereof. In some embodiments, the inflammation-inducing promoter comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 69 or a fragment thereof. In some embodiments, the inflammation-inducing promoter comprises SEQ ID NO: 69.

[0211] TIFF2025522813000031.tif15149Example of IFN-beta minimal promoter (SEQ ID NO: 69)

[0212] The inflammation-inducing promoter may comprise one or more inflammation-related transcription factor binding motifs. The "inflammation-related transcription factor binding motif" or "inflammation-related transcription factor binding site" may refer to a nucleotide sequence to which an inflammation-related transcription factor binds. Exemplary inflammation-related transcription factors include, but are not limited to, AP-1, NF-κB, IRF, STAT, and NFAT (see, for example, Smale ST. Cell. 2010 Mar 19;140(6):833-44., Platanitis E, Decker T. Front Immunol. 2018 Nov 13;9:2542., Pessler F, Dai L, Cron RQ, Schumacher HR. Autoimmun Rev. 2006 Feb;5(2):106-10). Exemplary inflammation-related transcription factor binding motifs may include, but are not limited to, an AP-1 binding motif, an NF-κB binding motif (κB site), an interferon-stimulated response element (ISRE), a gamma-interferon activation sequence (GAS), and an NFAT binding motif.

[0213] In some embodiments, the inflammation-inducing promoter comprises at least one inflammation-related transcription factor binding motif selected from: an AP-1 transcription factor binding motif; an NF-κB transcription factor binding motif; an IRF transcription factor binding motif; a STAT transcription factor binding motif; and an NFAT transcription factor binding motif; or any combination thereof.

[0214] In some embodiments, the inflammation-inducing promoter comprises two or more inflammation-related transcription factor binding motifs. In some embodiments, the inflammation-inducing promoter comprises three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more inflammation-related transcription factor binding motifs.

[0215] AP-1 binding motif In some embodiments, the inflammation-inducing promoter comprises one or more AP-1 binding motifs.

[0216] Activator protein 1 (AP-1) is a transcription factor that regulates gene expression in response to various stimuli, including cytokines, growth factors, stress, and bacterial and viral infections. The "AP-1 binding motif", also known as the "AP-1 transcription factor binding motif" or "AP-1 promoter site", is a DNA sequence to which the AP-1 transcription factor can bind (see, for example, Kim, H. et al., 1997. Biochemical Journal, 324(2), pp. 547-553).

[0217] In some embodiments, the inflammation-inducing promoter comprises two or more AP-1 binding motifs. In some embodiments, the inflammation-inducing promoter comprises three or more AP-1 binding motifs. In some embodiments, the inflammation-inducing promoter comprises four or more AP-1 binding motifs. In some embodiments, the inflammation-inducing promoter comprises five or more AP-1 binding motifs. In some embodiments, the inflammation-inducing promoter comprises five AP-1 binding motifs.

[0218] Exemplary AP-1 binding motifs are shown as SEQ ID NO: 70 (where "s" is g or c and "m" is a or c), SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 73. Any other variant or derivative to which AP-1 binds may be used in the present invention.

[0219] In some embodiments, the inflammation-inducible promoter comprises the nucleotide sequence of SEQ ID NO: 70.

[0220] TIFF2025522813000032.tif6136Example of an AP-1 binding consensus motif (SEQ ID NO: 70)

[0221] In some embodiments, the inflammation-inducible promoter comprises SEQ ID NO: 71 or a derivative thereof having one or two nucleotide substitutions.

[0222] TIFF2025522813000033.tif6136Example of an AP-1 binding motif 1 (SEQ ID NO: 71)

[0223] In some embodiments, the inflammation-inducible promoter comprises SEQ ID NO: 72 or a derivative thereof having one or two nucleotide substitutions.

[0224] TIFF2025522813000034.tif6136Example of an AP-1 binding motif 2 (SEQ ID NO: 72)

[0225] In some embodiments, the inflammation-inducible promoter comprises SEQ ID NO: 73 or a derivative thereof having one or two nucleotide substitutions.

[0226] TIFF2025522813000035.tif6136Example of an AP-1 binding motif 3 (SEQ ID NO: 73)

[0227] NF-kB binding motif In some embodiments, the inflammation-inducible promoter comprises one or more NF-kB binding motifs.

[0228] The nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) is a protein complex that controls DNA transcription in response to stimuli such as stress, cytokines, free radicals, heavy metals, ultraviolet irradiation, oxidized LDL, and antigens of bacteria or viruses. The "NF-κB binding motif", also known as the "NF-κB transcription factor binding motif" or "NF-κB promoter site", is a DNA sequence to which the NF-κB transcription factor can bind (see, for example, Natoli, G., 2006. FEBS letters, 580(12), pages 2843-2849).

[0229] In some embodiments, the inflammation-inducible promoter comprises two or more NF-κB binding motifs. In some embodiments, the inflammation-inducible promoter comprises three or more NF-κB binding motifs. In some embodiments, the inflammation-inducible promoter comprises four or more NF-κB binding motifs. In some embodiments, the inflammation-inducible promoter comprises five or more NF-κB binding motifs. In some embodiments, the inflammation-inducible promoter comprises five NF-κB binding motifs.

[0230] Exemplary NF-κB binding motifs are shown as SEQ ID NO: 74 (where "r" is a purine, "y" is a pyrimidine, and "n" is any nucleotide) and SEQ ID NO: 75. Any other variant or derivative to which NF-κB binds may be used in the present invention.

[0231] In some embodiments, the inflammation-inducible promoter comprises SEQ ID NO: 74 of the nucleotide sequence.

[0232] TIFF2025522813000036.tif6136Example of an NF-κB binding consensus motif (SEQ ID NO: 74)

[0233] In some embodiments, the inflammation-inducible promoter comprises SEQ ID NO: 75 of the nucleotide sequence or a derivative thereof having one or two nucleotide substitutions.

[0234] TIFF2025522813000037.tif6136Example of NF-κB binding motif (SEQ ID NO: 75)

[0235] Other inflammation-related transcription factor binding motifs In some embodiments, the inflammation-inducible promoter comprises one or more, two or more, three or more, four or more, or five or more interferon-stimulated response elements (ISREs). An ISRE (also known as an IRF transcription factor binding motif) is a DNA sequence to which an IRF transcription factor can bind. All members of the IRF family have a DNA binding domain at the N-terminus that recognizes an ISRE, which can be characterized by the consensus sequence AANNGAAA (see, for example, Yanai, H. et al., 2012. Oncoimmunology, 1(8), pages 1376-1386).

[0236] In some embodiments, the inflammation-inducible promoter comprises one or more, two or more, three or more, four or more, or five or more gamma-interferon activation sequences (GASs). A GAS (also known as a "STAT transcription factor binding motif") is a DNA sequence to which a STAT transcription factor can bind. Some members of the STAT protein family, particularly STAT1, STAT2, STAT3, STAT4, and STAT6, act as transcription factors that modulate the inflammatory response. STAT transcription factors bind to similar sequences and can have a palindromic core motif TTCN 2-4 GAA (see, for example, Ehret, G.B. et al., 2001. Journal of Biological Chemistry, 276(9), pages 6675-6688).

[0237] In some embodiments, the inflammation-inducible promoter comprises one or more, two or more, three or more, four or more, or five or more NFAT binding motifs. An NFAT transcription factor binding motif is a DNA sequence to which an NFAT transcription factor can bind. The NFAT family acts synergistically with AP-1 proteins on DNA elements containing adjacent NFAT and AP-1 binding sites to regulate the expression of inducible genes. The NFAT binding motif can be characterized by the consensus sequence TGGAAA (see, for example, Macian, F. et al., 2001. Oncogene, 20(19), pages 2476-2489).

[0238] Examples of inflammation-inducible promoters In some embodiments, the inflammation-inducible promoter comprises a combination of two or more different inflammation-related transcription factor binding motifs. For example, the inflammation-inducible promoter may comprise any combination of one or more AP-1 binding motifs, one or more NF-κB binding motifs, one or more gamma-interferon activation sequences (GAS), one or more interferon-stimulated response elements (ISRE), and one or more NFAT binding motifs.

[0239] In some embodiments, the inflammation-inducible promoter comprises one or more AP-1 binding motifs and / or one or more NF-κB binding motifs. In some embodiments, the inflammation-inducible promoter comprises two or more AP-1 binding motifs and / or two or more NF-κB binding motifs. In some embodiments, the inflammation-inducible promoter comprises three or more AP-1 binding motifs and / or three or more NF-κB binding motifs. In some embodiments, the inflammation-inducible promoter comprises four or more AP-1 binding motifs and / or four or more NF-κB binding motifs. In some embodiments, the inflammation-inducible promoter comprises five or more AP-1 binding motifs and / or five or more NF-κB binding motifs.

[0240] Inflammatory-related transcription factor binding motifs can be coupled. The term "coupled" may mean that the inflammatory-related transcription factor binding motifs are in a relationship that enables them to function in the intended manner (e.g., bind to inflammatory-related transcription factors). Preferably, the inflammatory-related transcription factor binding motifs may be linked by short nucleotide sequences or directly linked, or any combination thereof. Preferably, the inflammatory-related transcription factor binding motifs are linked by a linker sequence of 1 to about 20 nucleotides or 1 to about 10 nucleotides. Preferably, the inflammatory-related transcription factor binding motifs are directly linked (i.e., there is no linker sequence in between).

[0241] Preferably, the inflammation-inducible promoter may include one or more inflammatory-related transcription factor binding sites and a minimal promoter (e.g., the IFN-beta minimal promoter). Preferably, the inflammation-inducible promoter may include a proximal promoter region containing one or more inflammatory-related transcription factor binding sites. Preferably, the inflammation-inducible promoter may include a proximal promoter region containing one or more inflammatory-related transcription factor binding sites and a core promoter region derived from a gene that is selectively expressed in response to inflammation.

[0242] In some embodiments, the inflammation-inducible promoter comprises or consists of a nucleotide sequence that is at least 70% identical to SEQ ID NO: 76 or a fragment thereof. Preferably, the inflammation-inducible promoter comprises or consists of a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 76 or a fragment thereof. In some embodiments, the inflammation-inducible promoter comprises or consists of the sequence of SEQ ID NO: 76 or a fragment thereof.

[0243] TIFF2025522813000038.tif24149 Example of an inflammation-inducible promoter (SEQ ID NO: 76)

[0244] In some embodiments, the nucleotide sequence encoding a TNF inhibitor operably linked to an inflammation-inducing promoter comprises or consists of a nucleotide sequence that is at least 70% identical to SEQ ID NO: 77 or a fragment thereof. Preferably, the nucleotide sequence encoding a TNF inhibitor operably linked to an inflammation-inducing promoter comprises or consists of a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 77 or a fragment thereof. In some embodiments, the nucleotide sequence encoding a TNF inhibitor operably linked to an inflammation-inducing promoter has a nucleotide sequence that comprises or consists of the sequence of SEQ ID NO: 77 or a fragment thereof.

[0245] TIFF2025522813000039.tif109149TIFF2025522813000040.tif42149Examples of inflammation-inducing promoter and adalimumab Fab nucleotide sequence (SEQ ID NO: 77)

[0246] Other regulatory elements The vectors of the present invention may include one or more additional regulatory elements that can act before or after transcription. Preferably, the nucleotide sequence encoding a TNF inhibitor is operably linked to one or more additional regulatory elements that can act before or after transcription.

[0247] As used herein, "regulatory element" may refer to any nucleotide sequence that promotes the expression of a polypeptide, for example, that acts to increase the expression of a transcript or to enhance the stability of mRNA. Suitable regulatory elements include, for example, enhancer elements, post-transcriptional regulatory elements, introns, polyadenylation sites and Kozak sequences.

[0248] Enhancer The vector of the present invention may contain an enhancer. Preferably, the nucleotide sequence encoding the TNF inhibitor is operably linked to the enhancer. The enhancer can promote the expression of the TNF inhibitor in eye cells (e.g., retinal ganglion cells, RPE cells, photoreceptors, glial cells).

[0249] The term "enhancer" or "enhancer element" may refer to a region of DNA to which a protein (activator) can bind to increase the likelihood of transcription of a particular gene occurring. Enhancers are cis-acting. They can be located up to 1 Mbp (1,000,000 bp) away from the gene, either upstream or downstream from the start site.

[0250] The vector of the present invention may contain an eye tissue-specific enhancer. Preferably, the enhancer can be operably linked to the nucleotide sequence encoding the TNF inhibitor.

[0251] As used herein, a "tissue-specific enhancer" is an enhancer that preferentially promotes the expression of a gene in a particular cell or tissue. Preferably, the tissue-specific enhancer can promote higher expression of a gene in a particular cell type compared to other cell types. Higher expression can be measured, for example, by measuring the expression of a transgene operably linked to the enhancer, such as green fluorescent protein (GFP), and the expression of the transgene correlates with the ability of the enhancer to promote gene expression. Suitable tissue-specific enhancers will be known to those skilled in the art. The enhancer may be a retina-specific enhancer, preferably a retinal ganglion-specific enhancer. Preferably, the enhancer may be (or be derived from) an enhancer associated with a gene that is selectively expressed in human retinal cells. Methods for identifying enhancer regions associated with a gene will be known to those skilled in the art.

[0252] Polyadenylation sequence The vector of the present invention may contain a polyadenylation sequence. Preferably, the nucleotide sequence encoding the TNF inhibitor is operably linked to a polyadenylation sequence. The polyadenylation sequence may be inserted after the nucleotide sequence to improve the expression of the transgene.

[0253] The polyadenylation sequence typically includes a polyadenylation signal, a polyadenylation site, and a downstream element: the polyadenylation signal includes a sequence motif recognized by the RNA cleavage complex; the polyadenylation site is the cleavage site where the polyA tail is added to the mRNA; the downstream element is a GT-rich region, which is usually immediately downstream of the polyadenylation site and is important for efficient processing.

[0254] Suitable polyadenylation sequences will be known to those skilled in the art (see, for example, Schambach, A. et al., 2007. Molecular Therapy, 15(6), pages 1167-1173; and Choi, J.H. et al., 2014. Molecular brain, 7(1), pages 1-10). Examples of polyadenylation sequences include the bovine growth hormone (bGH) polyadenylation sequence, the SV40 polyadenylation sequence, and the rabbit beta-globin polyadenylation sequence.

[0255] In some embodiments, the polyadenylation sequence comprises or consists of a nucleotide sequence that is at least 70% identical to SEQ ID NO: 78 or a fragment thereof. Preferably, the polyadenylation sequence comprises or consists of a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 78 or a fragment thereof.

[0256] In some embodiments, the polyadenylation sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 78 or a fragment thereof.

[0257] TIFF2025522813000041.tif Example of 20148bGH polyadenylation sequence (SEQ ID NO: 78)

[0258] Post-transcriptional regulatory element The vector of the present invention may contain a post-transcriptional regulatory element. Preferably, the nucleotide sequence encoding the TNF inhibitor is operably linked to the post-transcriptional regulatory element.

[0259] The vector of the present invention may contain a woodchuck hepatitis post-transcriptional regulatory element (WPRE). Preferably, the nucleotide sequence encoding the TNF inhibitor is operably linked to the WPRE.

[0260] Suitable WPRE sequences will be known to those skilled in the art (see, for example, Zufferey, R. et al., 1999. Journal of virology, 73(4), pages 2886-2892; and Zanta-Boussif, M.A. et al., 2009. Gene therapy, 16(5), pages 605-619). Preferably, the WPRE is a wild-type WPRE or a mutant WPRE. For example, the WPRE may be mutated to suppress the translation of the woodchuck hepatitis virus X protein (WHX) by, for example, mutating the translation initiation codon of the WHX ORF.

[0261] In some embodiments, the WPRE comprises or consists of a nucleotide sequence that is at least 70% identical to SEQ ID NO: 79 or a fragment thereof. Preferably, the WPRE comprises or consists of a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 79 or a fragment thereof.

[0262] In some embodiments, the WPRE comprises or consists of the nucleotide sequence of SEQ ID NO: 79 or a fragment thereof.

[0263] TIFF2025522813000042.tif42149 Example of WPRE array (SEQ ID NO: 79)

[0264] Intron The vector of the present invention may contain an intron. Preferably, the nucleotide sequence encoding the TNF inhibitor is operably linked to the intron. The intron can be inserted between the promoter and the nucleotide sequence encoding the TNF inhibitor in order to increase expression.

[0265] Suitable introns are known to those skilled in the art (see, for example, Powell, S.K. et al., 2015. Discovery medicine, 19(102), page 49), and may include the MVM intron, the F.IX shortened intron 1, the chimeric β-globin / immunoglobulin heavy chain intron, the chimeric adenovirus / immunoglobulin intron, and the SV40 intron.

[0266] In some embodiments, the intron is the SV40 intron.

[0267] In some embodiments, the intron comprises or consists of a nucleotide sequence that is at least 70% identical to SEQ ID NO: 80 or a fragment thereof. Preferably, the intron comprises or consists of a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 80 or a fragment thereof.

[0268] In some embodiments, the intron comprises or consists of the nucleotide sequence of SEQ ID NO: 80 or a fragment thereof.

[0269] TIFF2025522813000043.tif15149 Example of SV40 intron (SEQ ID NO: 80)

[0270] Kozak sequence The vector of the present invention may contain a Kozak sequence. Preferably, the nucleotide sequence encoding the TNF inhibitor is operably linked to the Kozak sequence. The Kozak sequence may be inserted before the start codon to improve the initiation of translation.

[0271] Suitable Kozak sequences will be known to those skilled in the art (see, for example, Kozak, M., 1987. Nucleic acids research, 15(20), pages 8125-8148). The consensus Kozak sequence in vertebrates may have the sequence of SEQ ID NO: 95 or SEQ ID NO: 96.

[0272] Preferably, the Kozak sequence may comprise or consist of the nucleotide sequence of SEQ ID NO: 95 or 96 or a variant thereof having five or fewer deletions, substitutions or insertions. Preferably, the variant may have four or fewer, three or fewer, two or fewer or one deletion, substitution or insertion. Preferably, the variant may have three or fewer, two or fewer or one deletion and / or three or fewer, two or fewer or one substitution. Preferably, the variant may have three or fewer, two or fewer or one deletion and / or three or fewer, two or fewer or one substitution. Preferably, the variant may have one deletion and / or one substitution. Preferably, the variant may have one deletion and one substitution.

[0273] TIFF2025522813000044.tif6142 Example of consensus Kozak sequence 1 (SEQ ID NO: 95) TIFF2025522813000045.tif6142 Example of consensus Kozak sequence 2 (SEQ ID NO: 96)

[0274] Inflammatory inhibitory oligonucleotide The vector of the present invention may contain a sequence encoding an inflammatory inhibitory oligonucleotide. Preferably, the nucleotide sequence encoding the TNF inhibitor is operably linked to the sequence encoding the inflammatory inhibitory oligonucleotide.

[0275] Insertion of a sequence encoding an anti-inflammatory oligonucleotide into a vector can suppress innate and T cell responses and enhanced gene expression by "shielding" the vector so that it does not induce unwanted immune responses. Suitable sequences will be known to those skilled in the art (see, for example, Chan, Y.K. et al., 2021. Science translational medicine, 13(580), p.eabd3438).

[0276] The sequence encoding the anti-inflammatory oligonucleotide can antagonize TLR9 activation. In some embodiments, the anti-inflammatory oligonucleotide may be a TLR9 inhibitory oligonucleotide. In some embodiments, the sequence encoding the anti-inflammatory oligonucleotide comprises one or more TLR9i sequences, for example, one or more, two or more, three or more TLR9i sequences. Suitable TLR9i sequences are known in the art, and a suitable TLR9i sequence is shown in SEQ ID NO: 81. In some embodiments, the TLR9i sequence comprises or consists of a nucleotide sequence that is at least 92%, at least 96% or 100% identical to SEQ ID NO: 81. The TLR9i sequences may be separated by any suitable linker (e.g., AAAAA linker).

[0277] TIFF2025522813000046.tif7142Example of TLR9i sequence (SEQ ID NO: 81)

[0278] In some embodiments, the sequence encoding the anti-inflammatory oligonucleotide comprises or consists of a nucleotide sequence that is at least 70% identical to SEQ ID NO: 82 or a fragment thereof. Preferably, the sequence encoding the anti-inflammatory oligonucleotide comprises or consists of a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 82 or a fragment thereof.

[0279] In some embodiments, the sequence encoding the anti-inflammatory oligonucleotide comprises or consists of the nucleotide sequence of SEQ ID NO: 82 or a fragment thereof.

[0280] TIFF2025522813000047.tif16148io2 sequence example (SEQ ID NO: 82)

[0281] Vector The vectors of the present invention may be capable of transducing eye cells (e.g., retinal ganglion cells, RPE cells, photoreceptors, glial cells). In some embodiments, the vectors of the present invention can specifically transduce eye cells.

[0282] In some embodiments, the vectors of the present invention can transduce retinal cells. In some embodiments, the vectors of the present invention can specifically transduce retinal cells. The retina is a multilayered membrane that lines the posterior chamber of the eye and senses the image of the visual world that is transmitted to the brain via the optic nerve. From the inside to the outside of the eye, the retina includes a layer of neurosensory retina and retinal pigment epithelium, and there is a choroid outside the retinal pigment epithelium.

[0283] In some embodiments, the vectors of the present invention can transduce retinal ganglion cells. In some embodiments, the vectors of the present invention can specifically transduce retinal ganglion cells. Retinal ganglion cells are a type of nerve cell located near the inner surface of the retina of the eye.

[0284] Vectors suitable for transducing eye cells include viral vectors such as parvovirus vectors (e.g., AAV vectors), lentiviral vectors, adenoviral vectors, and also non-viral delivery systems (see, e.g., Rodrigues, G.A. et al. 2019. Pharmaceutical research, 36(2), 1-20).

[0285] The vector of the present invention may be a viral vector. The viral vector of the present invention is preferably an adeno-associated virus (AAV), although it is contemplated that other viral vectors may be used. In some embodiments, the viral vector is any of a parvovirus vector, an adenovirus vector, a herpes simplex virus vector, an anellovirus vector, a retrovirus vector or a lentivirus vector.

[0286] The vector of the present invention may be in the form of viral vector particles. In some embodiments, the viral vector is any of a parvovirus vector particle, an adenovirus vector particle, a herpes simplex virus vector particle, an anellovirus vector particle, a retrovirus vector particle or a lentivirus vector particle. Preferably, the viral vector of the present invention is in the form of AAV vector particles.

[0287] Methods for preparing and modifying viral vectors and viral vector particles, such as those derived from AAV, are known in the art. Suitable methods are described in Ayuso, E. et al., 2010. Current gene therapy, 10(6), pages 423-436, Merten, O.W. et al., 2016. Molecular Therapy-Methods & Clinical Development, 3, 16017; and Nadeau, I. and Kamen, A., 2003. Biotechnology advances, 20(7-8), pages 475-489.

[0288] Parvovirus vector The vector of the present invention may be a parvovirus vector. The vector of the present invention may be in the form of parvovirus vector particles.

[0289] Parvoviruses and in particular adeno-associated viruses (AAV) provide a highly versatile platform for the rational design of human gene therapy vectors. Typically, all parvoviruses are composed of small non-enveloped capsids containing single-stranded DNA genomes. Preferably, the parvovirus vector is derived from the Parvovirinae subfamily, including Dependoparvovirus, Protoparvovirus, and Bocaparvovirus. The vectors of the present invention may be parvovirus-based hybrid gene therapy vectors (see, for example, Fakhiri, J. and Grimm, D., 2021. Molecular Therapy, 29(12), pp. 3359-3382).

[0290] In some embodiments, the vector of the present invention is a dependoparvovirus vector. In some embodiments, the vector of the present invention is in the form of a dependoparvovirus vector. Some dependoparvoviruses are also known as adeno-associated viruses because they cannot replicate productively in host cells unless the cells are co-infected with a helper virus, such as an adenovirus.

[0291] In preferred embodiments, the vector of the present invention is an adeno-associated virus (AAV) vector. In preferred embodiments, the vector of the present invention is in the form of AAV vector particles.

[0292] AAV genome An AAV vector or AAV vector particle may contain an AAV genome or a fragment or derivative thereof. The AAV genome is a polynucleotide sequence that may encode functions required for the production of AAV particles. These functions include functions that act in the replication and packaging cycle of AAV in host cells, including capsid formation of the AAV genome into AAV particles. Naturally occurring AAV is replication-deficient and also depends on the supply of helper functions in trans for completion of the replication and packaging cycle. Thus, the AAV genome of the AAV vectors of the present invention is typically replication-deficient.

[0293] The AAV genome may be in either a single-stranded form, either plus or minus strand, or alternatively in a double-stranded form. Use of the double-stranded form allows avoidance of the DNA replication step in target cells, and thus can accelerate the expression of the transgene.

[0294] Naturally occurring AAV can be classified according to various biological systems. The AAV genome may be from any natural origin serotype, isolate or clade of AAV.

[0295] AAV can be referred to in terms of its serotype. A serotype corresponds to a variant subspecies of AAV, and the variant subspecies has characteristic reactivity that can be used to distinguish it from other variant subspecies due to the profile of expression of its capsid surface antigen. Typically, AAV vector particles having a particular AAV serotype do not efficiently cross-react with neutralizing antibodies specific for any other AAV serotype. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11. In some embodiments, the AAV vectors of the present invention may be of the AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype, or variants thereof. In some embodiments, the AAV vectors of the present invention are of the AAV2 serotype or variants thereof.

[0296] The AAV genome may similarly contain packaging genes, such as the rep and / or cap genes that encode the packaging function for AAV particles. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52, and Rep40 or variants thereof. The cap gene encodes one or more capsid proteins, such as VP1, VP2, and VP3 or variants thereof. These proteins constitute the capsid of the AAV particle, which determines the AAV serotype.

[0297] The AAV genome may be the entire genome of naturally occurring AAV. For example, AAV vectors or vector particles may be prepared by using a vector containing the entire AAV genome.

[0298] Preferably, the AAV genome is derivatized for the purpose of administration to a patient. Such derivatization is standard in the art, and the present invention encompasses the use of any known derivative of the AAV genome and derivatives that can be prepared by applying techniques known in the art. The AAV genome may be a derivative of any naturally occurring AAV. Preferably, the AAV genome is a derivative of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. Preferably, the AAV genome is a derivative of AAV2.

[0299] Derivatives of the AAV genome include any truncated or modified AAV genome that enables the expression of the transgene from the AAV vector of the present invention in vivo. Typically, the AAV genome can be significantly truncated while containing a minimal viral sequence but retaining the above functions. This is preferred for safety reasons as it reduces the risk of recombination with the wild-type virus of the vector and further avoids the induction of a cellular immune response due to the presence of viral gene proteins in target cells.

[0300] Typically, the derivative will comprise at least one inverted terminal repeat (ITR), preferably two or more ITRs, such as two ITRs or more. One or more of the ITRs may be derived from AAV genomes having different serotypes, or may be chimeric or mutant ITRs. Suitable mutant ITRs are those having a deletion of the trs (terminal resolution site). This deletion enables the continuous replication of the genome, thereby creating a single-stranded genome containing both the coding sequence and the complementary sequence, i.e., a self-complementary AAV genome. This allows for the avoidance of DNA replication in target cells, and thus the expression of the transgene can be accelerated.

[0301] The AAV genome may comprise one or more ITR sequences or variants thereof from any natural serotype, isolate or clade of AAV. The AAV genome may comprise at least one, such as two, ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or variants thereof.

[0302] Preferably, the AAV genome may comprise at least one, such as two, AAV2 ITRs or variants thereof. In some embodiments, the AAV genome comprises the AAV2 5' ITR and / or the AAV2 3' ITR.

[0303] In some embodiments, the AAV genome comprises a 5' ITR having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 83. In some embodiments, the AAV genome comprises a 5' ITR comprising or consisting of SEQ ID NO: 83.

[0304] TIFF2025522813000048.tif15148Example of AAV2 5’ ITR (SEQ ID NO: 83)

[0305] In some embodiments, the AAV genome comprises a 3' ITR having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 84. In some embodiments, the AAV genome comprises a 3' ITR comprising or consisting of SEQ ID NO: 84.

[0306] TIFF2025522813000049.tif15148AAV2 3’ITR example (SEQ ID NO: 84)

[0307] One or more ITRs may flank the nucleotide sequence encoding the TNF inhibitor at either end. Inclusion of one or more ITRs is preferred, for example, to assist in concatemer formation of the AAV vector in the nucleus of the host cell after conversion of single-stranded vector DNA to double-stranded DNA by the action of the host cell's DNA polymerase. Formation of such episomal concatemers protects the AAV vector during the lifespan of the host cell, thereby enabling long-term expression of the transgene in vivo.

[0308] Preferably, the AAV genome may comprise one or more AAV2 ITR sequences flanking the nucleotide sequence encoding the TNF inhibitor. Preferably, the AAV genome may comprise two AAV2 ITR sequences flanking either side of the nucleotide sequence encoding the TNF inhibitor.

[0309] Suitably, only the ITR elements will be sequences retained from the native AAV genome in the derivative. The derivative will preferably not contain the rep and / or cap genes of the native genome and any other sequences of the native genome. This is preferred both for the reasons above and to reduce the likelihood of the vector integrating into the host cell genome. Furthermore, by reducing the size of the AAV genome, flexibility can be increased in incorporating not only the transgene but also other sequence elements (e.g., regulatory elements) into the vector.

[0310] Thus, in the derivatives of the invention, the following parts can be removed: one inverted terminal repeat (ITR) sequence, the replication (rep) and capsid (cap) genes. However, the derivative may further contain one or more rep and / or cap genes or other viral sequences of the AAV genome. Naturally occurring AAV integrates at a specific site on human chromosome 19 with high frequency and shows a negligible frequency of random integration. Thus, retention of the integration ability in AAV vectors can be tolerated in a therapeutic setting.

[0311] The invention further includes providing the sequences of the AAV genome in an order and arrangement different from that of the native AAV genome sequence. The invention also includes substitution of one or more AAV sequences or genes with a chimeric gene composed of a sequence from another virus or sequences from two or more viruses. Such chimeric genes may be composed of sequences from two or more related viral proteins of different viral species.

[0312] AAV capsid protein The AAV vector particles may have a capsid formed by a capsid protein. The serotype can facilitate the transduction of eye cells (e.g., retinal ganglion cells, RPE cells, photoreceptors (visual cells), glial cells), for example, the specific transduction of eye cells. The AAV vector particles may be eye tissue-specific vector particles. The AAV vector particles may have a capsid formed by an eye tissue-specific capsid. The AAV vector particles may contain an eye tissue-specific capsid protein.

[0313] In some embodiments, the AAV vector particles are retinal-specific vector particles. In some embodiments, the AAV vector particles are capsidated by a retinal-specific capsid. In some embodiments, the AAV vector particles contain a retinal-specific capsid protein.

[0314] In some embodiments, the AAV vector particles are retinal ganglion-specific vector particles. In some embodiments, the AAV vector particles are capsidated by a retinal ganglion-specific capsid. In some embodiments, the AAV vector particles contain a retinal ganglion-specific capsid protein.

[0315] Preferably, the AAV vector particles may be in a transcapsidated form in which an AAV genome or derivative having ITRs of one serotype is packaged in a capsid of a different serotype. The AAV vector particles also include a mosaic form in which a mixture of unmodified capsid proteins from two or more different serotypes constitutes the viral capsid. The AAV vector particles also include a chemically modified form having a ligand adsorbed on the surface of its capsid. For example, such ligands can include antibodies for targeting specific cell surface receptors.

[0316] When the derivative contains a capsid protein, i.e., VP1, VP2, and / or VP3, the derivative may be a chimeric, shuffled, or capsid-modified derivative of one or more naturally occurring AAVs. In particular, the present invention encompasses the provision of capsid protein sequences from different serotypes, clades, clones, or isolates of AAV into the same vector (i.e., pseudotyped vectors). The AAV vector may be in the form of pseudotyped AAV vector particles.

[0317] Chimeric, shuffled, or capsid-modified derivatives will typically be selected to provide one or more desired functionalities to the AAV vector. Thus, these derivatives may exhibit an increase in the efficiency of gene delivery, a decrease in immunogenicity (humoral or cellular), a change in tropism range, and / or an improvement in the targeting of retinal cells as compared to an AAV vector containing a naturally occurring AAV genome. The increase in the efficiency of gene delivery can be achieved by an improvement in receptor or co-receptor binding at the cell surface, an improvement in internalization, an improvement in transport into the cell and nucleus, an improvement in uncoating of viral particles, and an improvement in the conversion of the single-stranded genome into the double-stranded form. The increase in efficiency may also be related to a change in tropism range or targeting of retinal cells such that the vector dose is not diluted by administration to tissues that do not require it.

[0318] Chimeric capsid proteins include those made by recombination between the capsid-encoding sequences of two or more naturally occurring AAV serotypes. This may be carried out, for example, by a markerless rescue method in which a non-infectious capsid sequence of one serotype is co-transfected with a capsid sequence of a different serotype and the capsid sequence having the desired properties is selected using directed selection. The capsid sequences of the different serotypes can be altered by homologous recombination intracellularly to produce a novel chimeric capsid protein.

[0319] For example, a directed evolution approach has been utilized to create novel AAV vectors that more effectively cross biological barriers and target specific cell types. For example, the identification of the AAV.7m8 variant enables efficient gene delivery to the entire retina of both mice and primates after intravitreal injection. Similarly, SH10, an AAV6 variant, has been shown to increase tropism for glial cells after intravitreal delivery and rescue retinal function in a rat model of RP (e.g., Rodrigues, G.A., et al. Pharmaceutical research, 36(2), pp.1-20).

[0320] Chimeric capsid proteins also include those created by manipulation of the capsid protein sequence to transfer a specific capsid protein domain, surface loop, or specific amino acid residues between two or more capsid proteins, such as between two or more capsid proteins of different serotypes.

[0321] Shuffled or chimeric capsid proteins may also be created by DNA shuffling or by error-prone PCR. Hybrid AAV capsid genes can be created by randomly fragmenting the sequences of related AAV genes, such as AAV genes encoding capsid proteins of multiple different serotypes, and then reconstructing the fragments in a self-priming polymerase reaction that can also cause crossovers between regions with sequence homology. By screening a library of hybrid AAV genes created by shuffling the capsid genes of several serotypes in this way, viral clones with the desired functionality can be identified. Similarly, a diverse library of variants that can later be selected for desired properties may be created by randomly mutating the AAV capsid gene using error-prone PCR.

[0322] The sequence of the capsid gene may also be genetically modified to introduce specific deletions, substitutions or insertions relative to the natural wild-type sequence. In particular, the capsid gene may be modified by insertion of the sequence of an unrelated protein or peptide within the open reading frame of the capsid coding sequence, or at the N-terminus and / or C-terminus of the capsid coding sequence. The unrelated protein or peptide may advantageously function as a ligand for a specific cell type, thereby providing improved binding to target cells or improving the specificity of targeting of the vector to a specific cell population. The unrelated protein may also be one that aids in the purification of the virus particles as part of the production process, i.e., an epitope or affinity tag. The site of insertion will typically be selected so as not to interfere with other functions of the virus particle, such as internalization and transport of the virus particle.

[0323] For example, AAV variants are generated by site-directed mutagenesis of surface-exposed tyrosine residues, thereby preventing phosphorylation of the capsid followed by ubiquitination and proteasome-mediated degradation. AAV2, AAV8 and AAV9 with these mutations have been shown to have improved transduction efficiency both in vitro and in vivo (see, for example, Petrs-Silva, H., et al. Molecular therapy, 17(3), pp.463-471).

[0324] The capsid protein may be an artificial capsid protein. As used herein, the term "artificial capsid" means that the capsid particle contains an amino acid sequence that does not occur naturally, or contains an amino acid sequence that has been engineered (e.g., modified) from a naturally occurring capsid amino acid sequence. In other words, the artificial capsid protein contains mutations or variations in the amino acid sequence when the artificial capsid amino acid sequence is aligned with the parental capsid amino acid sequence, as compared to the sequence of the parental capsid from which the artificial capsid amino acid sequence is derived.

[0325] The capsid protein may contain mutations or modifications that improve the ability to transduce eye cells compared to the wild-type capsid protein or compared to unmodified or wild-type virus particles. The improvement in the ability to transduce eye cells may be measured, for example, by measuring the expression of a transgene, such as GFP, carried by the AAV vector particles, and the expression of the transgene in the eye cells correlates with the ability of the AAV vector particles to transduce the eye cells.

[0326] Preferably, the AAV vector particles of the present invention are AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 vector particles, or variants thereof. AAV vector particles having these serotypes can transduce eye cells.

[0327] The AAV vector particles of the present invention may contain an AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 capsid protein, or a variant thereof. Preferably, the AAV vector particles may contain the AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 capsid proteins VP1, VP2, and VP3, or variants thereof.

[0328] In one embodiment, the AAV vector particles include one or more AAV2 ITR sequences adjacent to a nucleotide sequence encoding a TNF inhibitor, and an AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 capsid protein or a variant thereof. In one embodiment, the AAV vector particles include an AAV2 genome and an AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 capsid protein or a variant thereof.

[0329] AAV2 vectors and variants thereof In some embodiments, the AAV vector particles are AAV2 vector particles or variants thereof. In some embodiments, the AAV vector particles include the AAV2 capsid protein or variants thereof. Preferably, the AAV vector particles may include the AAV2 capsid proteins VP1, VP2, and VP3 or variants thereof.

[0330] AAV2 variants include AAV2.tYF, AAV2.7m8, R100, AAV2.GL, AAV2.NN, LSV1, R195-003, and dyno-86m. In some embodiments, the AAV vector particles are AAV2 vector particles, AAV2.tYF vector particles, AAV2.7m8 vector particles, R100 vector particles, AAV2.GL vector particles, AAV2.NN vector particles, LSV1 vector particles, R195-003 vector particles, or dyno-86m vector particles. In some embodiments, the AAV vector particles include the AAV2 capsid protein, AAV2.tYF capsid protein, AAV2.7m8 capsid protein, R100 capsid protein, AAV2.GL capsid protein, AAV2.NN capsid protein, LSV1 capsid protein, R195-003 capsid protein, or dyno-86m capsid protein. Preferably, the AAV vector particles may include the AAV2 capsid proteins VP1, VP2, and VP3, the AAV2.tYF capsid proteins VP1, VP2, and VP3, the AAV2.7m8 capsid proteins VP1, VP2, and VP3, the R100 capsid proteins VP1, VP2, and VP3, the AAV2.GL capsid proteins VP1, VP2, and VP3, the AAV2.NN capsid proteins VP1, VP2, and VP3, the LSV1 capsid proteins VP1, VP2, and VP3, the R195-003 capsid proteins VP1, VP2, and VP3, or the dyno-86m capsid proteins VP1, VP2, and VP3.

[0331] In some embodiments, the AAV vector particles are AAV2 vector particles, AAV2.tYF vector particles, AAV2.7m8 vector particles, R100 vector particles, AAV2.GL vector particles, or AAV2.NN vector particles. In some embodiments, the AAV vector particles comprise an AAV2 capsid protein, an AAV2.tYF capsid protein, an AAV2.7m8 capsid protein, an R100 capsid protein, an AAV2.GL capsid protein, or an AAV2.NN capsid protein. Preferably, the AAV vector particles can comprise the AAV2 capsid proteins VP1, VP2, and VP3, the AAV2.tYF capsid proteins VP1, VP2, and VP3, the AAV2.7m8 capsid proteins VP1, VP2, and VP3, the R100 capsid proteins VP1, VP2, and VP3, the AAV2.GL capsid proteins VP1, VP2, and VP3, or the AAV2.NN capsid proteins VP1, VP2, and VP3.

[0332] In some embodiments, the AAV vector particles are AAV2 vector particles. In some embodiments, the AAV vector particles comprise an AAV2 capsid protein. In some embodiments, the AAV vector particles comprise the AAV2 capsid proteins VP1, VP2, and VP3.

[0333] Preferably, the AAV2 VP1 capsid protein can comprise or consist of the amino acid sequence of SEQ ID NO: 85, or a variant that is at least 90% identical to SEQ ID NO: 85. Preferably, the variant can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 85. Preferably, the AAV2 VP2 and VP3 capsid proteins can be N-terminal truncations of SEQ ID NO: 85, or N-terminal truncations of a variant that is at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 85.

[0334] TIFF2025522813000050.tif55150Example of AAV2 VP1 capsid protein (SEQ ID NO: 85)

[0335] In some embodiments, the AAV vector particles are AAV2.tYF vector particles. In some embodiments, the AAV vector particles comprise the AAV2.tYF capsid protein. In some embodiments, the AAV vector particles comprise the AAV2.tYF capsid proteins VP1, VP2, and VP3. The single substitution of phenylalanine (F) for tyrosine (Y) increased the potency of AAV2 after intravitreal injection (see, e.g., Petrs-Silva, H., et al. Molecular therapy, 17(3), pp.463-471).

[0336] Preferably, the AAV2.tYF VP1 capsid protein can comprise or consist of the amino acid sequence of SEQ ID NO: 86, or a variant that is at least 90% identical to SEQ ID NO: 86. Preferably, the variant can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 86. Preferably, the AAV2.tYF VP2 and VP3 capsid proteins can be N-terminal truncations of SEQ ID NO: 86, or N-terminal truncations of variants that are at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 86.

[0337] TIFF2025522813000051.tif55150Example of AAV2.tYF VP1 capsid protein (SEQ ID NO: 86)

[0338] In some embodiments, the AAV vector particles are AAV2.7m8 vector particles. In some embodiments, the AAV vector particles comprise the AAV2.7m8 capsid protein. In some embodiments, the AAV vector particles comprise the AAV2.7m8 capsid proteins VP1, VP2, and VP3. AAV2.7m8 is a engineered capsid with 10 amino acids inserted into the adeno-associated virus (AAV) surface variable region VIII (VR-VIII), and as a result, has the ability to efficiently transduce retinal cells after intravitreal administration (see, e.g., Bennett, A., et al., 2020. Journal of structural biology, 209(2), p.107433).

[0339] Preferably, the AAV2.7m8 VP1 capsid protein can comprise or consist of the amino acid sequence of SEQ ID NO: 87, or a variant that is at least 90% identical to SEQ ID NO: 87. Preferably, the variant can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 87. Preferably, the AAV2.7m8 VP2 and VP3 capsid proteins can be N-terminal truncations of SEQ ID NO: 87, or N-terminal truncations of variants that are at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 87.

[0340] TIFF2025522813000052.tif59149Example of AAV2.7m8 VP1 capsid protein (SEQ ID NO: 87)

[0341] In some embodiments, the AAV vector particles are R100 vector particles. In some embodiments, the AAV vector particles comprise the R100 capsid protein. In some embodiments, the AAV vector particles comprise the R100 capsid proteins VP1, VP2, and VP3. R100 has shown superior transduction of human retinal cells compared to wild-type AAV (see, e.g., Kotterman, M., et al., 2021. bioRxiv 2021.06.24.449775).

[0342] Preferably, the R100 VP1 capsid protein can comprise or consist of the amino acid sequence of SEQ ID NO: 88, or a variant that is at least 90% identical to SEQ ID NO: 88. Preferably, the variant can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 88. Preferably, the R100 and VP3 capsid proteins can be N-terminal truncations of SEQ ID NO: 88, or N-terminal truncations of a variant that is at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 88.

[0343] TIFF2025522813000053.tif59149Example of R100 VP1 capsid protein (SEQ ID NO: 88)

[0344] In some embodiments, the AAV vector particles are AAV2.GL vector particles. In some embodiments, the AAV vector particles comprise an AAV2.GL capsid protein. In some embodiments, the AAV vector particles comprise AAV2.GL capsid proteins VP1, VP2, and VP3. In some embodiments, the AAV vector particles are AAV2.NN vector particles. In some embodiments, the AAV vector particles comprise an AAV2.NN capsid protein. In some embodiments, the AAV vector particles comprise AAV2.NN capsid proteins VP1, VP2, and VP3. AAV2.GL and AAV2.NN mediate extensive and high-level retinal transduction after intravitreal injection in mice, dogs, and non-human primates (see, e.g., Pavlou, M., et al. EMBO molecular medicine, 13(4), p.e13392).

[0345] Preferably, the AAV2.GL VP1 capsid protein can comprise or consist of the amino acid sequence of SEQ ID NO: 89, or a variant that is at least 90% identical to SEQ ID NO: 89. Preferably, the variant can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 89. Preferably, the AAV2.GL VP2 and VP3 capsid proteins can be N-terminal truncations of SEQ ID NO: 89, or N-terminal truncations of variants that are at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 89.

[0346] TIFF2025522813000054.tif59149Example of AAV2.GL VP1 capsid protein (SEQ ID NO: 89)

[0347] Suitably, the AAV2.NN VP1 capsid protein can comprise or consist of the amino acid sequence of SEQ ID NO: 90, or a variant that is at least 90% identical to SEQ ID NO: 90. Suitably, the variant can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 90. Suitably, the AAV2.NN VP2 and VP3 capsid proteins can be N-terminal truncations of SEQ ID NO: 90, or N-terminal truncations of a variant that is at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 90.

[0348] TIFF2025522813000055.tif59149Example of AAV2.NN VP1 capsid protein (SEQ ID NO: 90)

[0349] In some embodiments, the AAV vector particle is an LSV1 vector particle. In some embodiments, the AAV vector particle comprises the LSV1 capsid protein. In some embodiments, the AAV vector particle comprises the LSV1 capsid proteins VP1, VP2, and VP3. Loop-swap variant 1 (LSV1) transduces the retina and retinal pigment epithelium (RPE) from the vitreous, which is based on a substitution from amino acids 571 - 579 of AAV2.5T (see, e.g., Baker, C.K. et al., 2022, Molecular Therapy, 30(4), p.575).

[0350] Preferably, the LSV1 VP1 capsid protein can comprise or consist of the amino acid sequence of SEQ ID NO: 94, or a variant that is at least 90% identical to SEQ ID NO: 94. Preferably, the variant can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 94. Preferably, the LSV1 VP2 and VP3 capsid proteins can be N-terminal truncations of SEQ ID NO: 94, or N-terminal truncations of variants that are at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 94.

[0351] TIFF2025522813000056.tif24148TIFF2025522813000057.tif32149Example of LSV1 VP1 capsid protein (SEQ ID NO: 94)

[0352] In some embodiments, the AAV vector particles are R195-003 vector particles. In some embodiments, the AAV vector particles comprise the R195-003 capsid protein. In some embodiments, the AAV vector particles comprise the R195-003 capsid proteins VP1, VP2, and VP3 (see, e.g., Human Gene Therapy Methods 2022; 33 (23-24): A27-A28).

[0353] In some embodiments, the AAV vector particles are dyno-86m vector particles. In some embodiments, the AAV vector particles comprise the dyno-86m capsid protein. In some embodiments, the AAV vector particles comprise the dyno-86m capsid proteins VP1, VP2, and VP3 (see, e.g., Molecular Therapy 2023; 31(4), S1, p.1284).

[0354] In some embodiments, the AAV vector particles comprise one or more AAV2 ITR sequences adjacent to a nucleotide sequence encoding a TNF inhibitor, and an AAV2 capsid protein or a variant thereof. In some embodiments, the AAV vector particles comprise an AAV2 genome and an AAV2 capsid protein or a variant thereof.

[0355] Other parvovirus vectors In some embodiments, the vector of the present invention is a protoparvovirus vector. In some embodiments, the vector of the present invention is in the form of a protoparvovirus vector. Protoparvoviruses have been extensively studied and are used as vectors such as minute virus of mice (MVM), rat parvovirus H1, and LuIII virus. Recently discovered human variants include bufavirus (BuV), tusavirus (TuV), and cutter virus (CuV) (see, for example, Fakhiri, J. and Grimm, D., 2021. Molecular Therapy, 29(12), pp.3359-3382).

[0356] In some embodiments, the vector of the present invention is a bocaparvovirus vector. In some embodiments, the vector of the present invention is in the form of a bocaparvovirus vector. The use of human bocavirus 1 (HBoV1) as a parvovirus vector for gene delivery has been described (see, for example, Shao, L., et al., 2021. Frontiers in Microbiology, 12, p.1463).

[0357] Other viral vectors Retrovirus and lentivirus vectors The vector of the present invention may be a retrovirus vector or a lentivirus vector. The vector of the present invention may be a retrovirus vector particle or a lentivirus vector particle.

[0358] Retroviral vectors may be derived from or derivable from any suitable retrovirus. A number of different retroviruses have been identified. Examples include murine leukemia virus (MLV), human T-cell leukemia virus (HTLV), murine mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29), and avian erythroblastosis virus (AEV).

[0359] Retroviruses can be broadly classified into two categories, "simple" and "complex". Retroviruses can be further classified into seven groups. Five of these groups are retroviruses with oncogenic potential. The remaining two groups are lentiviruses and spumaviruses.

[0360] The basic structure of retroviral and lentiviral genomes share many common features, such as 5' LTR and 3' LTR. Between or within these, there are packaging signals to enable packaging of the genome, primer binding sites, integration sites to enable integration into the host cell genome, and the gag, pol, and env genes that encode packaging components (which are polypeptides necessary for the construction of viral particles). Lentiviruses have additional features, such as the rev gene and RRE sequence in HIV, which enable efficient transport of the integrated proviral RNA transcript from the nucleus of the infected target cell to the cytoplasm.

[0361] In proviruses, these genes are flanked at both ends by regions called long terminal repeats (LTR). The LTR is involved in proviral integration and transcription. The LTR also functions as an enhancer-promoter sequence and can control the expression of viral genes.

[0362] LTRs are the same sequences that can themselves be divided into three elements: U3, R, and U5. U3 is derived from a sequence specific to the 3' end of the RNA. R is derived from a sequence that repeats at both ends of the RNA. U5 is derived from a sequence specific to the 5' end of the RNA. The sizes of these three elements can vary quite a bit between different retroviruses.

[0363] In defective retroviral vector genomes, gag, pol, and env may be absent or non-functional.

[0364] In a typical retroviral vector, at least a portion of one or more protein-coding regions essential for replication may be removed from the virus, rendering the viral vector replication-defective. Also, to create a vector that has the ability to transduce a target host cell with a portion of the viral genome and / or integrate its genome into the host genome, the regulatory control regions and reporter portions within the vector genome may be replaced with a library encoding candidate modulating moieties that are operably linked.

[0365] Lentiviral vectors are part of a larger group of retroviral vectors. Briefly, lentiviruses can be classified into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, the human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV). Examples of non-primate lentiviruses include the prototype "slow virus" visna-maedi virus (VMV), as well as the related caprine arthritis encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

[0366] The lentivirus family differs from retroviruses in that lentiviruses have the ability to infect both dividing and non-dividing cells. In contrast, other retroviruses, such as MLV, cannot infect non-dividing cells or cells with slow division, such as cells that make up muscle, brain, lung, and liver tissues.

[0367] As used herein, a lentiviral vector is a vector that contains at least one component part derivable from a lentivirus. Preferably, the component part is involved in the biological mechanism by which the vector infects cells and expresses or replicates genes.

[0368] The lentiviral vector may be a "primate" vector. The lentiviral vector may also be a "non-primate" vector (i.e., it may be derived from a virus that does not naturally infect primates, particularly humans). Examples of non-primate lentiviruses may be any member of the lentivirus family that does not naturally infect primates.

[0369] Examples of lentivirus-based vectors include HIV-1 and HIV-2-based vectors, which are described below.

[0370] HIV-1 vectors contain cis-acting elements also found in simple retroviruses. Sequences spanning the gag open reading frame have been shown to be important for HIV-1 packaging. Thus, HIV-1 vectors often contain related portions of gag with mutated translation initiation codons. Furthermore, most HIV-1 vectors also contain a portion of the env gene that includes the RRE. Rev binds to the RRE, thereby allowing the transport of full-length or singly spliced mRNA from the nucleus to the cytoplasm. In the absence of Rev and / or the RRE, full-length HIV-1 RNA accumulates in the nucleus. Alternatively, the requirement for Rev and the RRE can be relaxed by using a constitutive transport element obtained from certain simple retroviruses, such as Mason-Pfizer monkey virus. Efficient transcription from the HIV-1 LTR promoter requires the viral protein Tat.

[0371] Most HIV-2-based vectors are structurally very similar to HIV-1 vectors. Similar to HIV-1-based vectors, HIV-2 vectors also require the RRE for efficient transport of full-length or singly spliced viral RNA.

[0372] Preferably, the viral vectors used in the present invention have a minimal viral genome. By "minimal viral genome" it should be understood that the viral vector has been engineered to remove non-essential elements and retain essential elements in order to confer the functionality necessary for the viral vector to infect, transduce, and deliver a nucleotide sequence of interest to a target host cell. Further details of this method can be found in WO 1998 / 017815.

[0373] Preferably, the plasmid vector used to produce the viral genome in the host cell / packaging cell will have sufficient lentiviral genetic information to allow packaging of the RNA genome in the presence of packaging components into virus particles that have the ability to infect target cells but do not have the ability to replicate independently to produce infectious virus particles in the final target cell. Preferably, the vector is deficient in a functional gag-pol and / or env gene and / or other genes essential for replication.

[0374] However, the plasmid vector used to produce the viral genome in the host cell / packaging cell will also include transcriptional regulatory control sequences operably linked in a functional form to the lentiviral genome to induce transcription of the genome in the host cell / packaging cell. These regulatory sequences may be native sequences associated with the viral sequences to be transcribed (i.e., the 5' U3 region), or they may be heterologous promoters, such as another viral promoter (e.g., the CMV promoter).

[0375] The vector may be a self-inactivating (SIN) vector lacking viral enhancer and promoter sequences. SIN vectors can be generated and transduced into non-dividing cells in vivo with an efficacy similar to that of wild-type vectors. Transcriptional inactivation of the long terminal repeat (LTR) in the SIN provirus should prevent mobilization by replication-competent viruses. This should also allow regulated expression of genes from internal promoters by eliminating any cis-acting effects of the LTR.

[0376] The vector may be integration-defective. An integration-defective lentiviral vector (IDLV) can be produced, for example, by packaging the vector using a catalytically inactive integrase (e.g., HIV integrase having a D64V mutation in the catalytic site), or by modifying or deleting the essential att sequences from the LTR of the vector, or by a combination of the foregoing.

[0377] Adenovirus vector The vector of the present invention may be an adenovirus vector. The vector of the present invention may be an adenovirus vector particle.

[0378] Adenovirus is a double-stranded linear DNA virus that does not pass through an RNA intermediate. There are more than 50 different human adenovirus serotypes, which are classified into six subgroups based on genetic sequence homology. The natural targets of adenovirus are the respiratory and gastrointestinal epithelia, which generally cause only mild symptoms. Serotypes 2 and 5 (having 95% sequence homology) are most commonly used in adenovirus vector systems, and they are usually associated with upper respiratory tract infections in the young.

[0379] Adenovirus is used as a vector for gene therapy and for the expression of heterologous genes. The large (36 kb) genome can accommodate up to 8 kb of foreign insert DNA and, by being efficiently replicated in a complementing cell line, can produce a very high titer of up to 10 12 which can result in a very high titer. Thus, adenovirus is one of the best systems for studying gene expression in primary non-replicating cells.

[0380] Expression of viral or foreign genes from the adenovirus genome does not require replicating cells. Adenovirus vectors enter cells by receptor - dependent endocytosis. Once inside the cell, adenovirus vectors rarely integrate into the host chromosome. Instead, they function episomally (independently of the host genome) as linear genomes in the host nucleus. Thus, the use of recombinant adenoviruses reduces the problems associated with random integration into the host genome.

[0381] Herpes simplex virus vector The vector of the present invention may be a herpes simplex virus vector. The vector of the present invention may be a herpes simplex virus vector particle.

[0382] Herpes simplex virus (HSV) is a neurotropic DNA virus with advantageous properties as a gene delivery vector. HSV is highly infectious, so HSV vectors are efficient vehicles for delivering foreign genetic material into cells. Viral replication can be easily blocked by null mutations in immediate - early genes that can be complemented in trans in vitro, which allows for the easy production of high - titer, pure preparations of non - pathogenic vectors. Its genome is large (152 Kb), and many of the viral genes are unnecessary for replication in vitro, so they can be replaced with large or multiple transgenes. Latent infection by wild - type virus results in the persistence of episomal virus in the sensory neuron nuclei during the lifetime of the host. The vector is non - pathogenic and cannot reactivate and persist long - term. By utilizing latency - active promoter complexes in vector design, stable transgene expression over a long period in the nervous system can be obtained. Due to the broad expression pattern of cell receptors recognized by the virus, HSV vectors transduce a wide variety of tissues. Understanding the processes involved in cell entry has enabled the targeting of the tropism of HSV vectors.

[0383] Vaccinia virus vector The vector of the present invention may be a vaccinia virus vector. The vector of the present invention may be a vaccinia virus vector particle.

[0384] Vaccinia virus is a large enveloped virus having a linear double-stranded DNA genome of approximately 190 kb. Vaccinia virus can accommodate up to approximately 25 kb of foreign DNA, which makes the virus useful for the delivery of large genes. A number of attenuated vaccinia virus strains suitable for gene therapy applications, such as the MVA and NYVAC strains, are known in the art.

[0385] Anellovirus vector The vector of the present invention may be an anellovirus vector. The vector of the present invention may be an anellovirus vector particle.

[0386] Anellovirus is a small single-stranded circular DNA virus. Anelloviruses are highly diverse and have not been associated with disease to date. Anelloviruses have the potential to infect the entire human population, and there is no evidence of disease association or viral clearance from infected individuals (see, for example, Venkataraman, T., et al., 2022. bioRxiv 2022.03.28.486145).

[0387] Non-viral delivery systems In some embodiments, the vector is a non-viral vector. Suitable non-viral delivery systems will be known to those skilled in the art (for example, Zulliger, R., et al., 2015. Journal of Controlled Release, 219, pp.471-487; and Oliveira, A.V., et al., 2017. Materials Science and Engineering: C, 77, pp.1275-1289).

[0388] In some embodiments, the vector is a plasmid. In some embodiments, the plasmid is modified to facilitate uptake into cells and / or the nucleus. In some embodiments, the plasmid is included within non-viral particles, such as lipoplex particles or polyplex particles.

[0389] Non-viral delivery systems include, but are not limited to, transfection methods. Here, transfection includes the process of delivering a gene to a target cell using a non-viral vector. Exemplary transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compact DNA-mediated transfection, liposomes, immunoliposomes, lipofectins, cationic agent-mediated transfection, cationic facial amphiphiles (CFAs), and combinations thereof.

[0390] Exemplary vectors The vectors of the present invention may include, from 5' to 3': a promoter (e.g., an inflammation-inducible promoter) and a nucleotide sequence encoding a TNF inhibitor.

[0391] The vectors of the present invention may further include any other element described herein or any other suitable element, such as one or more spacer sequences. The spacer sequence(s) may include, for example, at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotide bases. The spacer sequence may include restriction sites to allow for the insertion of one or more additional elements.

[0392] In a preferred embodiment, the vector of the present invention is an AAV vector. In some embodiments, the AAV genome comprises, from 5' to 3': a 5' ITR; an inflammation-inducible promoter; a nucleotide sequence encoding a TNF inhibitor; and a 3' ITR. In some embodiments, the AAV genome comprises, from 5' to 3': a 5' ITR; an inflammation-inducible promoter; a nucleotide sequence encoding a TNF inhibitor; a polyadenylation sequence; and a 3' ITR. In some embodiments, the AAV genome comprises, from 5' to 3': a 5' ITR; an inflammation-inducible promoter; a nucleotide sequence encoding a TNF inhibitor; a WPRE; a polyadenylation sequence; and a 3' ITR. In some embodiments, the AAV genome comprises, from 5' to 3': a 5' ITR; an inflammation-inducible promoter; an intron; a nucleotide sequence encoding a TNF inhibitor; a WPRE; a polyadenylation sequence; and a 3' ITR. In some embodiments, the AAV genome comprises, from 5' to 3': a 5' ITR; an inflammation-inducible promoter; an intron; a nucleotide sequence encoding a TNF inhibitor; a WPRE; a polyadenylation sequence; a sequence encoding an inflammation-suppressing oligonucleotide; and a 3' ITR.

[0393] In one aspect, the present invention provides a vector comprising or consisting of a nucleotide sequence that is at least 70% identical to SEQ ID NO: 91 or a fragment thereof. Preferably, the vector comprises or consists of a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 91 or a fragment thereof. In some embodiments, the vector comprises or consists of the nucleotide sequence of SEQ ID NO: 91 or a fragment thereof.

[0394] In some embodiments, the AAV genome comprises or consists of a nucleotide sequence that is at least 70% identical to SEQ ID NO: 91 or a fragment thereof. Preferably, the AAV genome comprises or consists of a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 91 or a fragment thereof. In some embodiments, the AAV genome comprises or consists of the nucleotide sequence of SEQ ID NO: 91 or a fragment thereof.

[0395] TIFF2025522813000058.tif231150TIFF2025522813000059.tif20148 Example of an AAV vector encoding adalimumab Fab (SEQ ID NO: 91)

[0396] In one aspect, the present invention provides a vector comprising or consisting of a nucleotide sequence that is at least 70% identical to SEQ ID NO: 92 or a fragment thereof. Preferably, the vector comprises or consists of a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 92 or a fragment thereof. In some embodiments, the vector comprises or consists of the nucleotide sequence of SEQ ID NO: 92 or a fragment thereof.

[0397] In some embodiments, the AAV genome comprises or consists of a nucleotide sequence that is at least 70% identical to SEQ ID NO: 92 or a fragment thereof. Preferably, the AAV genome comprises or consists of a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 92 or a fragment thereof. In some embodiments, the vector comprises or consists of the nucleotide sequence of SEQ ID NO: 92 or a fragment thereof.

[0398] TIFF2025522813000060.tif130149TIFF2025522813000061.tif121149 Example of an AAV vector encoding infliximab Fab (SEQ ID NO: 92)

[0399] Variants, derivatives, analogs and fragments In addition to the specific polypeptides and polynucleotides described herein, the present invention also encompasses variants, derivatives and fragments thereof.

[0400] In the context of the present invention, a "variant" of any given sequence is a sequence in which the residues of a particular sequence (whether amino acid residues or nucleic acid residues) are modified in such a way that the polypeptide or polynucleotide retains at least one or all of its endogenous functions. Variant sequences can be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue present in a naturally occurring polypeptide or polynucleotide.

[0401] As used herein with respect to a protein or polypeptide of the present invention, the term "derivative" includes any substitution, variation, modification, replacement, deletion and / or addition of one (or more) amino acid residues from or to its sequence, provided that the resulting protein or polypeptide retains at least one or all of its endogenous functions.

[0402] Typically, amino acid substitutions may be made, for example, from 1, 2 or 3 to 10 or 20 substitutions, provided that the modified sequence retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogs.

[0403] The polypeptides used in the present invention may also have deletions, insertions or substitutions of amino acid residues that result in silent changes and yield functionally equivalent polypeptides. Conservative amino acid substitutions may be made based on similarity in properties such as polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathicity of the residues, as long as the intrinsic function is maintained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids having uncharged polar head groups with similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.

[0404] Conservative substitutions may be made, for example, according to the following table. Amino acids listed in the same column of the second row and the same row of the third row may be substituted for each other:

[0405] [Table 5]

[0406] The effects of additions, deletions, substitutions, alterations, replacements and / or variations can be predicted using any suitable prediction tool, such as SIFT (Vaser, R., et al., 2016. Nature protocols, 11(1), pp.1-9), PolyPhen-2 (Adzhubei, I., et al., 2013. Current protocols in human genetics, 76(1), pp.7-20), CADD (Rentzsch, P., et al., 2021. Genome medicine, 13(1), pp.1-12), REVEL (Ioannidis, N.M., et al., 2016. The American Journal of Human Genetics, 99(4), pp.877-885), MetaLR (Dong, C., et al., 2015. Human molecular genetics, 24(8), pp.2125-2137), and / or MutationAssessor (R Reva, B., et al., 2011. Nucleic acids research, 39(17), pp.e118-e118), or based on clinical data, such as by ClinVar (Landrum, M.J., et al., 2016. Nucleic acids research, 44(D1), pp.D862-D868). Suitable additions, deletions, substitutions, alterations, replacements and / or variations are considered to be tolerated, benign, and / or likely to be benign.

[0407] Typically, a variant may have a certain identity with the wild-type amino acid sequence or the wild-type nucleotide sequence.

[0408] In this context, a variant sequence is considered to include an amino acid sequence that can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical to the target sequence, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical. A variant can also be considered from the perspective of similarity (i.e., amino acid residues having similar chemical properties / functions), but in the context of the present invention, it is preferably expressed from the perspective of sequence identity.

[0409] In this context, a variant sequence is considered to include a nucleotide sequence that can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical to the target sequence, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical. A variant can also be considered from the perspective of similarity, but in the context of the present invention, it is preferably expressed from the perspective of sequence identity.

[0410] Preferably, when referring to a sequence showing a percentage of identity to any one of the sequence numbers detailed herein, it refers to the sequence showing the described percentage of identity over the full length of the recited sequence number.

[0411] Sequence identity comparisons can be performed visually or, more commonly, by employing readily available sequence comparison programs. These commercially available computer programs can calculate the percentage of identity between two or more sequences.

[0412] The percent identity may be calculated over a continuous array, i.e., after aligning one array with the other, each amino acid or nucleotide in one array is directly compared one residue at a time with the corresponding amino acid or nucleotide in the other array. This is referred to as "gapless" alignment. Typically, such gapless alignment is performed only on a relatively small number of residues.

[0413] This is a very simple and consistent method, but this method does not take into account, for example, that in otherwise identical sequence pairs, one insertion or deletion in an amino acid or nucleotide sequence can squeeze subsequent residues or codons out of alignment, and thus may result in a significant decrease in the percent identity when performing a global alignment. As a result, most sequence comparison methods are designed to generate an optimal alignment that takes into account possible insertions and deletions without overly penalizing the overall identity score. This is achieved by inserting "gaps" into the sequence alignment for the purpose of trying to maximize local identity.

[0414] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment such that, for the same number of identical amino acids or nucleotides, an alignment that contains the fewest gaps reflecting a higher relatedness between the two compared sequences will obtain a higher score than one that contains a large number of gaps. Typically, an "affine gap cost" is used that sets a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. A high gap penalty results in an optimized alignment that contains fewer gaps. Most alignment programs allow the gap penalty to be changed. However, when using such software for sequence comparison, it is preferable to use the default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for the gap and -4 for each extension.

[0415] Calculation of the percent identity thus first requires the creation of an optimal alignment that takes into account a gap penalty. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (see, e.g., Devereux, J., et al., 1984. Nucleic acids research, 12(1), pp.387-395). Examples of other software capable of performing sequence comparisons include, but are not limited to, the BLAST package (see, e.g., Altschul, S.F., et al., 1990. Journal of molecular biology, 215(3), pp.403-410), BLAST 2 (see, e.g., Tatusova, T.A. and Madden, T.L., 1999. FEMS microbiology letters, 174(2), pp.247-250), FASTA (see, e.g., Pearson, W.R. and Lipman, D.J., 1988. PNAS, 85(8), pp.2444-2448), EMBOSS Needle (Madeira, F., et al., 2019. Nucleic acids research, 47(W1), pp.W636-W641), and the comparison tools of the GENEWORKS suite.

[0416] The final percent identity can be measured, but the alignment process itself is typically not based on all-or-nothing pairwise comparisons. Instead, a scaled similarity score matrix that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance is usually used. One example of such a commonly used matrix is the BLOSUM62 matrix.

[0417] If optimal alignment is generated by software, the percent sequence identity can be calculated. The software typically performs this as part of a sequence comparison and creates a numerical result. The percent sequence identity may be calculated as the number of identical residues as a proportion of the total residues in the recited sequence number.

[0418] A "fragment" is also a variant, and this term typically refers, functionally or, for example, in an assay, to a selected region of a polypeptide or polynucleotide of interest. A "fragment" thus refers to an amino acid or nucleic acid sequence that is part of a full-length polypeptide or polynucleotide.

[0419] Such variants, derivatives, and fragments may be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. When making an insert, synthetic DNA encoding the insert may be made along with 5' and 3' flanking regions corresponding to naturally occurring sequences on either side of the insertion site. The flanking regions will contain convenient restriction sites corresponding to sites in the sequences such that the naturally occurring sequences are cleaved by appropriate enzyme(s) and the synthetic DNA is ligated to the cleavage site. The DNA is then expressed according to the invention to produce the encoded protein. These methods are merely illustrative of the many standard techniques known in the art for the manipulation of DNA sequences, and other known techniques may also be used.

[0420] Vectors, Kits, and Systems In one aspect, when the vector is a viral vector, the invention provides a vector encoding the viral genome of the invention. The vector may be a transfer vector, as described herein. For example, the vector may be a plasmid, and / or the viral genome may be operably linked to a promoter (e.g., a viral promoter such as the CMV promoter).

[0421] In one aspect, the present invention provides a kit or system for producing a vector (e.g., a viral vector) of the present invention. The kit or system may be a viral packaging kit or system, or alternatively a viral production kit or system. As used herein, a "viral packaging kit or system" may include one or more components for packaging a viral vector of the present invention, and optionally instructions. As used herein, a "viral production kit or system" may include one or more components for producing a viral vector of the present invention, and optionally instructions.

[0422] The kit or system may include a transfer vector encoding the viral genome of the present invention, and optionally one or more helper vectors. The kit or system may further include a host cell (e.g., a packaging cell or a producer cell) and / or other reagents (e.g., a transfection reagent, a culture medium, etc.). The kit or system may further include any other suitable components, and optionally instructions for packaging and / or producing a viral vector of the present invention.

[0423] Cell In one aspect, the present invention provides a cell comprising a vector (e.g., a viral vector) of the present invention. The cell may be an isolated cell. Preferably, the cell is a mammalian cell, e.g., a human cell. The cell may be an isolated human cell.

[0424] Preferably, the cell may be a producer cell. The term "producer cell" includes a cell that produces the viral particles after transient transfection, stable transfection or vector transduction of all the elements necessary to produce the viral particles, or any cell engineered to stably contain the elements necessary to produce the viral particles. In some embodiments, the producer cell is an AAV producer cell. Suitable producer cells are known to those skilled in the art (see, for example, Martin, J., et al. 2013. Human gene therapy methods, 24(4), pp.253-269), and include HEK293, COS-1, COS-7, CV-1, HeLa, CHO, and A549 cell lines. In some embodiments, the producer cell is a HEK293 cell, or a derivative thereof (e.g., HEK293T cells).

[0425] Preferably, the cell may be a packaging cell. The term "packaging cell" includes a cell that contains some or all of the elements necessary to package a recombinant viral genome. Typically, such a packaging cell contains one or more vectors capable of expressing the viral structural proteins (e.g., the AAV rep and cap genes), and / or one or more genes encoding the viral structural proteins are integrated into the genome of the packaging cell. Cells that contain only some of the elements required for the production of enveloped viral particles are useful as intermediate reactants in the generation of a viral particle producer cell line through subsequent steps of transient transfection, transduction or stable integration of each of the additional required elements. These intermediate reactants are included within the term "packaging cell". In some embodiments, the packaging cell is an AAV packaging cell. Suitable packaging cells will be known to those skilled in the art (see, for example, Martin, J., et al. 2013. Human gene therapy methods, 24(4), pp.253-269).

[0426] Pharmaceutical composition In one aspect, the present invention provides a pharmaceutical composition comprising the vector or cell of the present invention. In a preferred embodiment, the pharmaceutical composition comprises the vector of the present invention in the form of viral vector particles.

[0427] A pharmaceutical composition is a composition comprising or consisting of a therapeutically effective amount of a pharmaceutically active substance, i.e., a vector. The pharmaceutical composition preferably comprises a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).

[0428] "Pharmaceutically acceptable" includes that the preparation is sterile and free of pyrogens. The carrier, diluent, and / or excipient must be "acceptable" in the sense that it is compatible with the vector and not harmful to its recipient. Typically, the carrier, diluent, and excipient will be sterile and pyrogen-free saline or infusion media, but other acceptable carriers, diluents, and excipients may be used.

[0429] Acceptable carriers, diluents, and excipients for therapeutic use are well known in the pharmaceutical art. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may contain, as (or in addition to) the carrier, excipient or diluent, any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilizing agent(s).

[0430] Examples of pharmaceutically acceptable carriers include, for example, water, saline solutions, alcohol, silicone, wax, petrolatum, vegetable oils, polyethylene glycol, propylene glycol, liposomes, saccharides, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, essential oils, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

[0431] The vector, cell or pharmaceutical composition according to the present invention may be administered in a manner suitable for the treatment and / or prevention of the diseases described herein. Suitable administration routes are known to those skilled in the art. The dosage and frequency of administration will be determined by those skilled in the art according to factors such as, for example, the health status of the subject and the type and severity of the disease of the subject. The pharmaceutical composition may be formulated as appropriate.

[0432] The vector, cell or pharmaceutical composition according to the present invention can be administered locally to the eye. Preferably, the vector, cell or pharmaceutical composition according to the present invention is administered by subretinal injection, direct retinal injection, subconjunctival injection, sub-Tenon's injection, periocular injection, suprachoroidal injection, or intravitreal injection. The pharmaceutical composition of the present invention can be formulated as appropriate.

[0433] In some embodiments, the vector, cell, or pharmaceutical composition of the present invention is administered intravitreally. The term "intravitreal" may refer to the interior of the eyeball, and thus intravitreal administration may be related to administration into the interior of the subject's eyeball. In some embodiments, the vector, cell, or pharmaceutical composition is administered to the subject's eye by subretinal injection, direct retinal injection, suprachoroidal injection, or intravitreal injection. Those skilled in the art are familiar with and will be able to fully perform individual subretinal injection, direct retinal injection, suprachoroidal injection, or intravitreal injection (see, for example, Hartman, R.R. and Kompella, U.B., 2018. Journal of Ocular Pharmacology and Therapeutics, 34(1-2), pp.141-153).

[0434] In some embodiments, the vector, cell, or pharmaceutical composition is administered to the subject's eye by subretinal, suprachoroidal, or intravitreal injection. In a preferred embodiment, the vector, cell, or pharmaceutical composition of the present invention is administered by intravitreal injection.

[0435] The pharmaceutical composition may contain the vector or cell of the present invention in an infusion medium, such as a sterile isotonic solution. The pharmaceutical composition may be enclosed in an ampoule made of glass or plastic, a disposable syringe or a vial for multiple administrations.

[0436] The vector, cell or pharmaceutical composition may be administered as a single or multiple doses. Preferably, the vector, cell or pharmaceutical composition may be administered as a single, one-time-only dose. The pharmaceutical composition can be formulated as appropriate.

[0437] The vector, cell or pharmaceutical composition may be administered at various doses (e.g., measured in viral genomes (vg) / mL). In any case, a physician can determine the actual dosage that would be most appropriate for any given individual subject, and the dosage may vary, for example, depending on the age, weight and response of that particular subject.

[0438] Preferably, the vector of the present invention is at least about 10 10 vg / mL, at least about 10 11 vg / mL, at least about 10 12 vg / mL, or at least about 5x10 12 vg / mL. Preferably, the vector of the present invention is administered at a dose of about 10 13 vg / mL or less, about 10 12 vg / mL or less, or about 10 11 vg / mL or less. Preferably, the vector of the present invention is administered at a dose of about 10 10 ~ about 10 13 vg / mL, or about 10 11 ~ about 10 13 vg / L. Preferably, the vector of the present invention is administered at a dose of about 10 10 ~ about 10 12 vg / mL. Preferably, the vector of the present invention is administered at a dose of about 10 11 ~ about 10 13 vg / mL. Preferably, the vector of the present invention is administered at a dose of about 10 12 ~ about 10 13 vg / mL. Preferably, the vector of the present invention is administered at a dose of about 10 12~about 5 x 10 12 It is administered at a dose of vg / mL. The pharmaceutical composition can be formulated accordingly.

[0439] Preferably, the vector of the present invention is at least about 10 9 vg / eye, at least about 2 x 10 9 vg / eye, at least about 5 x 10 9 vg / eye, at least about 10 10 vg / eye, at least about 2 x 10 10 vg / eye, at least about 5 x 10 10 vg / eye, or at least about 10 11 vg / eye. Preferably, the vector of the present invention is administered at a dose of about 10 13 vg / eye or less or about 5 x 10 12 vg / eye or less. Preferably, the vector of the present invention is administered at a dose of about 10 9 vg / eye to about 5 x 10 12 vg / eye, about 10 10 vg / eye to about 5 x 10 12 vg / eye, about 10 10 vg / eye to about 10 12 vg / eye, or about 10 10 vg / eye to about 5 x 10 11 vg / eye.

[0440] Preferably, the vector of the present invention can be administered in combination with one or more other therapeutic agents. The one or more other therapeutic agents can be administered separately, simultaneously, or sequentially. The pharmaceutical composition may further contain one or more other therapeutic agents. For example, the vector of the present invention can be administered in combination with one or more immunosuppressive agents (e.g., antimetabolites, calcineurin inhibitors, and alkylating agents).

[0441] The vector of the present invention can reduce the need for immunosuppressive therapy. In some embodiments, the vector of the present invention is administered in the absence of immunosuppressive therapy (i.e., the subject does not receive immunosuppressive therapy).

[0442] The present invention further includes a kit comprising the vector, cell and / or pharmaceutical composition of the present invention. Preferably, the kit is for use in the methods as described herein and is used as described herein, for example, in the treatment methods described herein. Preferably, the kit includes instructions for use of the components of the kit.

[0443] Method for treating and / or preventing a disease In one aspect, the present invention provides the vector, cell and / or pharmaceutical composition according to the present invention for use as a medicament.

[0444] In one aspect, the present invention provides the use of the vector, cell or pharmaceutical composition according to the present invention in the manufacture of a medicament.

[0445] In one aspect, the present invention provides a method of administering a therapeutically effective amount of the vector, cell or pharmaceutical composition according to the present invention to a subject in need thereof.

[0446] The vector, cell or pharmaceutical composition can be administered to any subject in need thereof. The subject can be a mammal (e.g., human). The vector, cell or pharmaceutical composition according to the present invention can be administered to a subject having or at risk of having an inflammatory eye disease.

[0447] Inflammatory eye disease The vector, cell or pharmaceutical composition according to the present invention can be used to prevent and / or treat an inflammatory eye disease.

[0448] In one aspect, the present invention provides the vector, cell or pharmaceutical composition according to the present invention for use in the prevention and / or treatment of an inflammatory eye disease.

[0449] In one aspect, the present invention provides the use of the vector, cell or pharmaceutical composition according to the present invention in the manufacture of a medicament for preventing or treating an inflammatory eye disease.

[0450] In one aspect, the present invention provides a method for preventing or treating an inflammatory eye disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a vector, cell or pharmaceutical composition according to the present invention.

[0451] As used herein, "inflammatory eye disease" refers to any disorder associated with inflammation of the eye, including uveitis, scleritis, keratitis, conjunctivitis, iritis, retinochoroiditis, choroiditis, retinitis and retinochoroiditis.

[0452] After administration of the vector, cell and / or pharmaceutical composition according to the present invention, one or more symptoms of the inflammatory eye disease can be prevented and / or treated in the subject. Any suitable method for determining the severity of the inflammatory eye disease may be used (see, for example, McNeil, R., 2016. Eye news, 22(5), pages 1-4).

[0453] The vector, cell and / or pharmaceutical composition according to the present invention can prevent and / or reduce intraocular inflammation. Any suitable method may be used to determine intraocular inflammation. A suitable method for quantifying intraocular inflammation includes laser flare photometry (see, for example, Tugal-Tutkun, I. and Herbort, C.P., 2010. International ophthalmology, 30(5), pages 453-464).

[0454] The vector, cell and / or pharmaceutical composition according to the present invention can reduce the recurrence rate of the inflammatory eye disease. Any suitable method may be used to determine the recurrence rate. For example, recurrence (or relapse) of uveitis is typically defined as grading of 2+ or more vitreous haze using anterior chamber cells and / or the SUN grading system (see, for example, McNeil, R., 2016. Eye news, 22(5), pages 1-4).

[0455] The vector, cell and / or pharmaceutical composition according to the present invention can prevent and / or reduce vision loss. Inflammatory eye diseases, such as uveitis, are a major cause of visual morbidity (see, for example, Durrani, O.M. et al., 2004. British Journal of Ophthalmology, 88(9), pages 1159 - 1162). The vector, cell and / or pharmaceutical composition according to the present invention can maintain or improve vision.

[0456] Uveitis In a preferred embodiment, the inflammatory eye disease is uveitis.

[0457] In one aspect, the present invention provides a vector, cell or pharmaceutical composition according to the present invention for use in the prevention and / or treatment of uveitis.

[0458] In one aspect, the present invention provides the use of a vector, cell or pharmaceutical composition according to the present invention in the manufacture of a medicament for preventing or treating uveitis.

[0459] In one aspect, the present invention provides a method for preventing or treating uveitis, the method comprising administering a therapeutically effective amount of a vector, cell or pharmaceutical composition according to the present invention to a subject in need thereof.

[0460] Uveitis refers to a group of intraocular inflammatory diseases of the uvea (i.e., the iris, ciliary body, and choroid) and adjacent structures including the cornea, vitreous humor, retina, and optic nerve. Uveitis can be classified based on the major anatomical site of inflammation (i.e., anterior, intermediate, and posterior) and / or etiological origin including infectious, non-infectious, or masquerade (see, for example, Rosenbaum, J.T. et al., 2019. Seminars in Arthritis and Rheumatism, 49(3), 438-445; and Standardization of Uveitis Nomenclature (SUN) Working Group, 2005. American journal of ophthalmology, 140(3), 509-516).

[0461] Uveitis may be anterior uveitis, intermediate uveitis, posterior uveitis, or panuveitis. Uveitis may be infectious, non-infectious, or masquerade. In a preferred embodiment, uveitis is non-infectious uveitis.

[0462] In some embodiments, uveitis is selected from one or more of sympathetic ophthalmia, birdshot chorioretinopathy, sarcoid uveitis, intermediate uveitis, Vogt Koyanaga Harada syndrome, JIA-related uveitis, idiopathic retinal vasculitis, HLA-B27-related non-anterior uveitis.

[0463] In some embodiments, uveitis is sympathetic ophthalmia. Sympathetic ophthalmia is a rare bilateral granulomatous uveitis that occurs after surgical or accidental trauma to one eye (see, for example, Damico, F.M. et al., 2005. Seminars in ophthalmology, 20(3), 191-197).

[0464] In some embodiments, the uveitis is punctate choroiditis. Punctate choroiditis is a rare chronic bilateral posterior uveitis (see, e.g., Levinson, R.D. et al., 2006. American journal of ophthalmology, 141(1), pp. 185-187).

[0465] In some embodiments, the uveitis is sarcoid uveitis. Sarcoid uveitis may also be known as sarcoid-related uveitis. Sarcoidosis is a disease that causes non-caseating granulomatous inflammation in one or more organs. The most common ocular symptoms are uveitis, dry eye, and conjunctival nodules (see, e.g., Jamilloux, Y. et al., 2014. Autoimmunity reviews, 13(8), pp. 840-849).

[0466] In some embodiments, the uveitis is intermediate uveitis. Intermediate uveitis may refer to inflammation in the anterior vitreous, ciliary body, and peripheral retina (see, e.g., Babu, B.M. and Rathinam, S.R., 2010. Indian journal of ophthalmology, 58(1), p. 21).

[0467] In some embodiments, the uveitis is Vogt-Koyanaga-Harada syndrome. Vogt-Koyanagi-Harada syndrome is a bilateral chronic diffuse granulomatous panuveitis that frequently presents with neurological, auditory, and cutaneous symptoms (see, e.g., Fang, W. and Yang, P., 2008. Current eye research, 33(7), pp. 517-523).

[0468] In some embodiments, the uveitis is JIA-related uveitis. JIA is the most common rheumatic disease in childhood, and JIA-related uveitis has its most common extra-articular manifestation (see, for example, Clarke, S.L. et al., 2016. Pediatric Rheumatology, 14(1), pp. 1-11).

[0469] In some embodiments, the uveitis is idiopathic retinal vasculitis. Retinal vasculitis is an inflammatory eye condition that threatens vision and involves the retinal blood vessels. Based on etiology, retinal vasculitis can be classified as either idiopathic or secondary to infection, neoplasm, or a systemic inflammatory disease (see, for example, Talat, L. et al., 2014. Journal of ophthalmology, p. 197675).

[0470] In some embodiments, the uveitis is HLA-B27-associated non-anterior uveitis. Human leukocyte antigen (HLA)-B27-associated uveitis is the most commonly diagnosed cause of acute anterior uveitis (see, for example, Loh, A.R. and Acharya, N.R., 2010. American journal of ophthalmology, 150(4), pp. 534-542).

[0471] [Examples] Here, the present invention will be further illustrated by examples, which are intended to be helpful to those skilled in the art in practicing the present invention and are not intended to limit the scope of the present invention in any way.

[0472] [Example 1] Local administration of an anti-TNF antibody fragment suppresses experimental autoimmune uveoretinitis (EAU) in vivo B10.RIII mice were immunized against experimental autoimmune uveoretinitis (EAU), and after day 10, the eyes were monitored using topical endoscopic fundus imaging (TEFI) to select experimental mice showing clinically obvious disease. On day 10, 15 μg of infliximab or vehicle control (EAU) was injected into the mouse group via the intravitreal route. The eyes were enucleated (day 14), and retinal infiltrates were characterized. The efficacy of intravitreal infliximab-derived Fab molecules in suppressing inflammation and retinal infiltration in the B10.RIII EAU model was demonstrated by representative fundus images (Figure 1A), clinical disease scores (Figure 1B), and flow cytometry analysis of total CD45+ cell counts from a single eye on day 14 (Figure 1C). **P < 0.005; data are shown as mean + / - SEM and are representative of two independent experiments.

[0473] [Example 2] Therapeutic vector design Figure 2 is a schematic diagram showing the vector constructs of CMV.infliximab Fab (constitutive CMV promoter) and AP1-NFkB.infliximab Fab (inflammatory-inducible promoter composed of five repeated AP1 and NFkB binding sites). The heavy and light chains of infliximab Fab are separated by a self-cleaving 2A peptide to generate two separate peptides, which then form Fab in situ. After cell transduction, AAV persists in the nucleus as episomal DNA.

[0474] [Example 3] Evaluation of constitutive transgene expression in vitro and in vivo To evaluate constitutive infliximab Fab transgene expression, HEK-293T (a standard cell line for AAV development) or ARPE-19 (an ocular cell line) cells were transduced with AAV2.CMV.infliximab or AAV.CMV.NULL vector [MOI 1E5 vg / cell], and the culture supernatants were assayed using a clinical IFX ELISA kit (Figure 3A). Detectable expression of infliximab Fab from both cell types was evident by 72 hours (∼30 ng / ml). ****P<0.0001. Data are shown as mean + / − SEM.

[0475] To evaluate transgene expression in vivo, AAV2.CMV.infliximab or AAV.CMV.NULL was administered by intravitreal (IVT) injection at 5E12 vg / ml into the eyes of healthy B10.RIII mice. Mice were sacrificed 4 weeks after AAV, eyes were dissected, and supernatants (retina and vitreous) were assayed using a clinical IFX ELISA kit (Figure 3B). Detectable expression of infliximab Fab (∼1.5 ng / ml) in vivo was observed in eyes that received the 5E12 vg / ml dose. *P<0.05. Data are shown as mean + / − SEM, and each data point represents a single eye.

[0476] These data demonstrate that AAV can mediate constitutive expression of anti-TNF Fab.

[0477] [Example 4] Evaluation of Inducible Transgene Expression in Clinical Models In Vitro and In Vivo To demonstrate the inducibility of the inflammatory response-responsive promoter design, HEK-293T cells transduced with AAV2.AP1-NFkB.EGFP (reporter vector) or AAV2.AP1-NFkB.infliximab (therapeutic vector) were stimulated with recIL-1β (2 ng / mL). Activation resulted in visible GFP expression within 24 hours, with intensity increasing up to 72 hours, while no GFP signal was observed with AAV2.AP1-NFkB.NULL (control vector) (Figure 4A). Higher GFP signals were observed with the constitutive AAV.CMV.EGFP vector. Stimulation rapidly induced the expression of infliximab Fab (8 hours), with the accumulation reaching approximately 20 ng / ml at 72 hours (Figure 4B). Images were taken with EVOS FL at 10x magnification. ****P<0.0001; data are shown as mean + / − SEM.

[0478] To demonstrate the in vivo inducibility of the inflammatory response-responsive promoter design, AAV2.AP1-NFkB.EGFP (reporter) or AAV.AP1-NFkB.NULL (control) were administered to C57BL / 6J mice by intravitreal (IVT) injection at a dose of 5E12 vg / ml. Four weeks after AAV injection, the mice were immunized to induce experimental autoimmune uveoretinitis (EAU) and imaged to monitor the onset of ocular inflammation. On day 14 of EAU, representative fundus and OCT images demonstrated clear clinical signs of the disease (perivascular sheathing and vitreous infiltration) and induction of GFP expression (Figure 5A). Neither clinical signs of the disease nor expression of the GFP transgene were observed in mice receiving AAV only (without EAU).

[0479] To demonstrate the in vivo inducibility of the therapeutic transgene, groups of mice were injected in the contralateral eyes with AAV2.AP1-NFkB.infliximab or AAV2.AP1-NFkB.NULL, and EAU was induced 4 weeks after AAV. At 3 weeks after EAU, the mice were sacrificed, the eyes were dissected, and the supernatants (retina and vitreous) were assayed using a clinical IFX ELISA kit (Figure 5B). Detectable expression of infliximab Fab was observed only in the eyes with EAU that received the therapeutic vector and not in the controls. *P<0.05. Data are shown as mean + / − SEM, and each data point represents a single eye.

[0480] These data demonstrate the inducibility of an inflammatory-responsive promoter design that expresses either a reporter (eGFP) or a therapeutic (anti-TNF Fab) transgene in vitro and in vivo.

[0481] [Example 5] Evaluation of the efficacy of a constitutive therapeutic transgene in vivo To demonstrate the efficacy of the constitutive therapeutic transgene, groups of mice were injected in the contralateral eyes with AAV7m8.CMV.infliximab or AAV7m8.CMV.NULL, followed by intravitreal administration of recombinant human TNF (rec_hTNF) 4 weeks after AAV. At 18 hours (the peak of the inflammatory response to rec_hTNF), representative fundus and OCT images demonstrate increased inflammation (vitreous infiltration) in the controls (NULL) compared to the eyes with infliximab (Figure 6A). No clinical signs of disease were observed in mice given AAV only. At 18 hours, the mice were sacrificed, and eyes were prepared for flow cytometry analysis to determine the absolute number of Ly6C+ monocytes (the dominant infiltration in this model) from a single eye. A significant decrease in the number of monocytes was observed in eyes that received the therapeutic vector and not in the controls (Figure 6B), and this effect was further emphasized in a paired (contralateral eye) analysis (Figure 6C). **Wilcoxon signed-rank test; *P<0.05 Wilcoxon matched-pair analysis.

[0482] These data demonstrate the efficacy of the design of a constitutive promoter encoding a therapeutic (infliximab Fab) transgene that suppresses human TNF-mediated inflammation in vivo.

[0483] [Example 6] Constitutive expression of other anti-TNF biologics in vitro and in vivo To evaluate the constitutive expression of additional anti-TNF biologics, HEK-293T cells were transfected with the huTNFRI-huIgG plasmid and the culture supernatant was assayed using an anti-human TNF antibody ELISA kit (Figure 7A). Detectable expression of huTNFRI-huIgG was evident by 48 hours. To evaluate AAV-mediated transgene expression, HEK-293T cells were transduced with AAV7m8.CMV.huTNFRI-huIgG or AAV7m8.CMV.NULL vector [MOI 1E5 vg / cell] and the culture supernatant was assayed using an anti-human TNF antibody ELISA kit (Figure 7B). Detectable expression of huTNFRI-IgG was evident by 72 hours (approx. 30 ng / ml).

[0484] To evaluate the constitutive transgene expression of huTNFRI-huIgG in vivo, AAV7m8.CMV.huTNFRI-huIgG or AAV7m8.CMV.NULL was administered by intravitreal (IVT) injection into the eyes of healthy C57BL / 6J mice at a range of doses [2E8 or 2E9 vg / eye]. Mice were sacrificed 4 weeks after AAV and the eyes were dissected and the supernatant (retina and vitreous) was assayed using an anti-human TNF antibody ELISA kit (Figure 7C). Detectable expression of huTNFRI-huIgG in vivo was observed in eyes receiving doses of 2E8 and 2E9 vg / eye at approximately 4 ng / ml and 9 ng / ml, respectively.

[0485] These data provide additional evidence that gene therapy vectors can mediate the constitutive expression of multiple anti-TNF biologics relevant to the clinic.

[0486] [Example 7] Biological Activity of a Constitutively Expressed Anti-TNF Transgene In Vitro To determine whether the transgene products were bioactive and functional, the inventors evaluated AAV plasmids encoding two modified constructs engineered to express adalimumab Fab, huTNFRI-huIgG, and the extracellular domain of huTNFRI fused to murine Fc (huTNFRI-msIgG) and the murine version (msTNFRI-msIgG). The inventors also evaluated the biological activity of infliximab Fab using the AAV2.CMV.infliximab viral vector.

[0487] First, HEK-BLUE TNF reporter cells were transfected with AAV.CMV.huTNFRI-huIgG, AAV.CMV.huTNFRI-msIgG, AAV.CMV.msTNFRI-msIgG, AAV.CMV.ADALIMUMAB plasmid, or AAV2.CMV.infliximab for 48 hours. The cells were further stimulated with recombinant human TNF or murine TNF (0.5 ng / ml) for 24 hours, and NFkB activation was evaluated. All TNFRI antibody-like plasmid constructs were bioactive, inhibiting huTNF-mediated activation and msTNF-mediated activation in the reporter cell line compared to NULL or recTNF alone (Figure 8A). For monoclonal Fab-based anti-TNF biologics (both human-specific), plasmid expression of adalimumab Fab (Figure 8B) or AAV-mediated infliximab Fab expression (Figure 8C) both inhibited activation by huTNF.

[0488] These data demonstrate that vectorized anti-TNF biologics, including huTNFRI-huIgG and adalimumab, are bioactive and neutralize TNF-mediated signaling and NFkB activation in vitro.

[0489] [Example 8] Evaluation of Expression and Biological Activity of an Inducible Anti-TNF Transgene in Clinical Disease Models In Vitro and In Vivo To further demonstrate the inducibility of anti-TNF biologics via an inflammatory response-responsive promoter, HEK-293T cells were transduced with AAV7m8.AP1-NFkB.huTNFRI-huIgG or AAV7m8.AP1-NFkB.NULL vector [MOI 1E5 vg / cell] and stimulated with IL-1b (2 ng / ml). The inventors selected this construct for further characterization because huTNFRI-huIgG can potently neutralize both human and mouse TNF, facilitating in vivo efficacy testing in mice. Stimulation resulted in robust induction of huTNFRI-huIgG expression by 24 hours, with the accumulated amount reaching approximately 25 ng / ml at 48 hours (Figure 9A).

[0490] To determine the bioactivity of the induced huTNFRI-huIgG protein, conditioned media (cell supernatant recovered from the expression assay at 48 hours) was "spiked" with recombinant human or mouse TNF (final concentration 10 ng / ml) and incubated with HEK-BLUE reporter cells for 24 hours. NFkB activation was robustly induced in reporter cells in response to both stimulation with huTNF or mTNF alone and conditioned media from AAV7m8.AP1-NFkB.NULL. Supplementation with conditioned media from cells transduced with AAV7m8.AP1-NFkB.huTNFRI-huIgG and stimulated with IL-1b completely suppressed NFkB activation in reporter cells, indicating potent bioactivity of the induced transgene (Figure 9B).

[0491] To demonstrate the in vivo inducibility of the therapeutic transgene, mice were injected with AAV7m8.AP1-NFkB.huTNFRI-huIgG, and EAU was induced 4 weeks after AAV. On day 19 after EAU, mice were sacrificed when mild to moderate clinical signs of inflammation (not yet peak disease) were observed, the eyes were dissected, and the supernatant (retina and vitreous) was assayed using anti-human TNF antibody ELISA. The inventors found that clinical disease drove detectable expression of huTNFRI-huIgG in the eyes of EAU that received the therapeutic vector, but not in the control (AAV only) (Figure 10A).

[0492] Using the recombinant human TNF model, the inventors also evaluated the inducibility of the huTNFRI-huIgG transgene in response to acute inflammatory stimuli. Mice were injected with AAV7m8.AP1-NFkB.huTNFRI-huIgG and AAV7m8.AP1-NFkB.NULL (contralateral eye control) at 2E9 vg / eye. Four weeks after AAV, the mice received bilateral administration of recombinant human TNF (rec_hTNF), were sacrificed 18 hours later, the eyes were dissected, and the eye supernatants (retina and vitreous) were assayed for huTNFRI-huIgG expression. Acute activation induces a significant increase in the expression of huTNFRI-huIgG compared to the control (Figure 10B).

[0493] These data demonstrate the inducibility and sensitivity of an inflammatory-responsive promoter design for expressing a bioactive therapeutic (anti-TNF biologic) transgene in vitro and in vivo using two different disease models, one with chronic low-grade inflammation and the other with acute ocular inflammation.

[0494] [Example 9] Evaluation of the Efficacy of an Inducible Therapeutic Transgene In Vivo To demonstrate the efficacy of the inducible treatment-introduced gene, B10.RIII mice were injected with AAV7m8.AP1-NFkB.huTNFRI-huIgG or AAV7m8.CMV.NULL[2E9 vg / eye] into the contralateral eye, and then immunized against EAU 4 weeks after AAV. On day 11, representative fundus and OCT images demonstrated increased inflammation (perivascular cuffing and vitreous infiltration) in the eyes that received the control NULL vector (Figure 11A). The contralateral eyes of the same 4 animals that received the inducible treatment vector showed substantially reduced clinical inflammation in both perivascular cuffing and vitreous infiltration. At this time point, the mice were sacrificed, and eyes were prepared for flow cytometry analysis to determine the absolute numbers of CD45+ (total leukocytes), CD3+ (lymphocytes), CD4+ (Th T cells), and CD11b+ (macrophages and monocytes) populations from the enucleated eyes (Figure 11B). The inventors observed a tendency for reduced immune cell infiltration by AAV7m8.AP1-NFkB.huTNFRI-huIgG compared to AAV7m8.CMV.NULL.

[0495] These data demonstrate the efficacy of inducible anti-TNF in locally suppressing ocular inflammation in a mouse model of human uveitis in vivo.

[0496] [Example 10] Method Viral vector: Plasmid design, cloning, and sequencing were performed in-house. Production, QC, and quantification of ultra-high purity AAV2 preparations were performed by Vector Builder. Production, QC, and quantification of ultra-high purity AAV7m8 preparations were performed by Vector BioLabs.

[0497] [Table 6]

[0498] Cell lines: HEK 293T or ARPE-19 cells were cultured using standard complete medium. Cell plasmid transfection was performed using Lipofectamine at a concentration of 0.25 μg / ml. Cell AAV transduction was performed at a certain range of multiplicity of infection (MOI; 1E4 - 1E5 vg / cell).

[0499] Anti-TNF constructs: Infliximab Fab was expressed by encoding the heavy and light chains of infliximab Fab separated by a self-cleaving 2A peptide to generate two separate peptides, which then formed Fab in situ. Adalimumab Fab was also expressed, encoding the heavy and light chains of adalimumab Fab. For both Fabs, a human growth hormone (HGH) signal peptide was added to the N-terminus of both the heavy and light chains to enable secretion.

[0500] For antibody-like molecules, huTNFRI-huIgG was expressed by encoding the extracellular domain of human tumor necrosis factor receptor type 1 (TNFRI, also known as the p55 receptor, 211 amino acid residues at the N-terminus including the native signal peptide), followed by encoding the Fc region of human IgG1. Similarly, huTNFRI-msIgG was expressed, but the Fc region of human IgG1 was replaced with the Fc region of mouse IgG1. Similarly, msTNFRI-msIgG was expressed, and furthermore, the extracellular domain of huTNFRI (211 amino acid residues at the N-terminus including the native signal peptide) was replaced with the extracellular domain of msTNFRI (212 amino acid residues at the N-terminus including the native signal peptide). These soluble fusion proteins bind to and neutralize TNF.

[0501] Characterization of infliximab Fab and huTNFRI-huIgG: The levels of secreted Fab or IgG (cell culture or ex vivo retinal supernatant) were assayed using a clinical IFX ELISA kit (R-Biopharm) or an anti-human TNF antibody ELISA kit (LS Bio; LS-F55832). The bioactivity of infliximab Fab or huTNFRI-huIgG (i.e., the ability to neutralize human / mouse TNF) was evaluated using TNF-α reporter HEK293 cells (Invivogen) that enable monitoring of NF-κB pathway activation.

[0502] Therapeutic intervention (AAV or biologic agent); Intravitreal injections were performed using a 33G Hamilton syringe under ketamine-based recovery anesthesia with a surgical microscope to inject a maximum volume of 2 μl.

[0503] Uveitis model: Experimental autoimmune uveoretinitis (EAU) is an established preclinical model of human non-infectious uveitis (Khalili, H. et al., Sci Rep, 2016. 6: p. 36905). It was induced using a standard immunization protocol with retinal peptide (RBP-3) via an appropriate systemic route (e.g., subcutaneous or intraperitoneal injection) and additional adjuvants (CFA and pertussis toxin). EAU susceptibility depends on the mouse strain, with different RBP-3 epitopes inducing acute and severe inflammation (B10.RIII) or a persistent disease with reduced disease severity (C57BL / 6J). The B10.RIII strain was used in the initial therapeutic AAV2.infliximab vector trial, and both strains were used in subsequent therapeutic AAV7m8.huTNFRI-huIgG vector trials.

[0504] Recombinant human TNF model: To evaluate the efficacy of therapeutic anti-TNF vectors against human proteins, acute inflammation can be induced by intravitreal injection of recombinant human TNF (20 ng / eye). Susceptibility does not depend on the mouse strain, and similar inflammatory kinetics are observed in B10.RIII or C57BL / 6J.

[0505] Clinical evaluation: With the Micron IV system, in vivo eye evaluations (fundus, fluorescence, OCT, and ERG) of disease severity can be repeatedly performed. Ex vivo, a single retina is processed for routine 15-color flow cytometry (FACS) to determine and quantify the immunophenotype of CD45+ infiltrating cell populations.

[0506] The practice of the present invention, unless otherwise indicated, uses conventional techniques of chemistry, biochemistry, molecular biology, microbiology, and immunology, which are within the capabilities of those skilled in the art. Such techniques are described in the literature. For example, see Skoog, D.A. et al., (2013) Fundamentals of Analytical Chemistry, 9th Edition, Cengage learning; Walker J.M. (2009) The Protein Protocols Handbook, 3rd Edition, Springer Nature; Green, M.R. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al., (2003) Current Protocols in Molecular Biology, John Wiley & Sons; Hill, A.J. (2013) DNA Sequencing Protocols, Humana Press; Nielsen, B.S. and Jones, J. (2021) In Situ Hybridization Protocols, Springer US; Herdewijn, P. (2010) Oligonucleotide Synthesis: Methods and Applications, Humana Press; and Luo, Y. (2019) CRISPR Gene Editing: Methods and Protocols, Springer New York. Each of these general texts is incorporated herein by reference.

Claims

1. A vector comprising a nucleotide sequence encoding an anti-TNFα antibody or a fragment thereof, wherein the nucleotide sequence encoding the anti-TNFα antibody or a fragment thereof is functionally linked to an inducible inflammation promoter.

2. The anti-TNFα antibody or a fragment thereof is an antibody fragment, preferably the antibody fragment is an antigen-binding fragment (Fab), a fragment antibody (F(ab') 2 The vector according to claim 1, which is a single-chain antibody (scFv) or a single-domain antibody (sdAb).

3. The nucleotide sequence encoding the anti-TNFα antibody fragment is functionally linked to the inducible inflammation promoter, and in some cases the inducible inflammation promoter, (a) comprising one or more inflammation-inducing transcription factor-binding motifs selected from AP-1 transcription factor-binding motifs; NF-κB transcription factor-binding motifs; IRF transcription factor-binding motifs; STAT transcription factor-binding motifs; and NFAT transcription factor-binding motifs or any combination thereof; (b) comprising one or more AP-1 binding motifs and / or one or more NF-κB binding motifs; (c) comprising two or more AP-1 binding motifs and / or two or more NF-κB binding motifs, three or more AP-1 binding motifs and / or three or more NF-κB binding motifs, four or more AP-1 binding motifs and / or four or more NF-κB binding motifs, or five or more AP-1 binding motifs and / or five or more NF-κB binding motifs; (d) comprising at least one AP-1 binding motif coupled to at least one NF-κB binding motif; (e) comprising five AP-1 binding motifs coupled to five NF-κB binding motifs; and / or (f) A nucleotide sequence having at least 70% identity with SEQ ID NO: 76, or comprising such a sequence, The AP-1 binding motif contains or consists of SEQ ID NO: 70, or the AP-1 binding motif contains or consists of a derivative thereof containing any of SEQ ID NOs: 71-73 or one nucleotide substitution, or The NF-κB binding motif contains or consists of SEQ ID NO: 74, or the NF-κB binding motif contains or consists of SEQ ID NO: 75 or a derivative thereof containing two or fewer nucleotide substitutions. The vector according to claim 1.

4. Anti-TNFα antibodies or fragments thereof Adalimumab or its fragments, infliximab or its fragments, golimumab or its fragments, or certolizumab or its fragments, Adalimumab or fragments thereof, or infliximab or fragments thereof, Adalimumab or fragment thereof Antigen-binding fragment (Fab), and / or Adalimumab antigen-binding fragment (Fab) The vector according to claim 1.

5. Anti-TNFα antibodies or fragments thereof (a) comprising one or more CDR regions selected from SEQ ID NOs: 1-6 or their derivatives containing one amino acid substitution, (b) Each comprising SEQ ID NOs: 1, 2, 3, 4, 5, and 6 or derivatives thereof comprising one amino acid substitution, or comprising CDR regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or (c) A heavy chain comprising or having a sequence having at least 70% identity with SEQ ID NO: 7 and / or a light chain comprising a sequence having at least 70% identity with SEQ ID NO: 8, and optionally, The heavy chain is encoded by a nucleotide sequence having at least 70% identity with SEQ ID NO: 47, and / or the light chain is encoded by a nucleotide sequence having at least 70% identity with SEQ ID NO:

48. The nucleotide sequences encoding the heavy chain and the nucleotide sequences encoding the light chain are linked via a linker sequence. The linker sequence encodes a 2A self-cleaving peptide and / or an enzymatically cleavable peptide motif, preferably the linker sequence encodes a 2A self-cleaving peptide having at least 70% sequence identity with any of SEQ ID NOs. 55-58, and / or A nucleotide sequence encoding a heavy chain and / or a nucleotide sequence encoding a light chain are functionally linked to a signal sequence, and the signal sequence may encode a signal peptide selected from among human growth hormone (HGH) signal peptide, interleukin-2 (IL-2) signal peptide, CD5 signal peptide, immunoglobulin kappa light chain signal peptide, trypsinogen signal peptide, serum albumin signal peptide, and prolactin signal peptide. The vector according to claim 1.

6. A nucleotide sequence encoding an anti-TNFα antibody or a fragment thereof, A heavy chain comprising or consisting of a sequence having at least 70% identity with SEQ ID NO: 7, a 2A self-cleaving peptide having at least 70% sequence identity with any of SEQ ID NOs: 55-58, and a light chain comprising or consisting of a sequence having at least 70% identity with SEQ ID NO:

8. A nucleotide sequence having at least 70% identity with SEQ ID NO: 47, a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 59 or 60, and a nucleotide sequence having at least 70% identity with SEQ ID NO: 48, or comprising such a sequence and / or A nucleotide sequence having at least 70% identity with sequence number 66, or consisting thereof. The vector according to claim 1.

7. A nucleotide sequence encoding an anti-TNFα antibody or a fragment thereof, (a) Functionally linked to a polyadenylated sequence, wherein the polyadenylated sequence is selected from a bovine growth hormone (bGH) polyadenylated sequence, an SV40 polyadenylated sequence, and a rabbit beta-globin polyadenylated sequence, and wherein the polyadenylated sequence contains or consists of a nucleotide sequence having at least 70% identity with SEQ ID NO:

78. (b) A nucleotide sequence functionally linked to a woodchuck hepatitis post-transcriptional regulatory element (WPRE), wherein the WPRE has at least 70% identity with SEQ ID NO: 79, and / or (c) Functionally linked to an intron, the intron being selected from a beta-globin intron or an SV40 intron, and the intron containing or consisting of a nucleotide sequence having at least 70% identity with SEQ ID NO: 80 The vector according to claim 1.

8. The vector is (a) A nucleotide sequence having at least 70% identity with SEQ ID NO: 77, (b) Selected from the group consisting of viral vectors, parvovirus vectors, adenovirus vectors, herpes simplex virus vectors, anerovirus vectors, retrovirus vectors or lentivirus vectors, and adeno-associated virus (AAV) vectors. (c) AAV vector particles, which are optionally pseudotyped to confer ocular tissue tropism, and / or AAV vector particles comprising an AAV2 capsid protein or an AAV2 capsid variant protein, which optionally comprises an AAV2 capsid variant selected from AAV2.tYF, AAV2.7m8, R100, AAV2.GL and AAV2.NN. (d) containing one or more terminal inverted repeat sequences (ITRs), and / or (e) A nucleotide sequence having at least 70% identity with SEQ ID NO: 91, The vector according to claim 1.

9. A vector containing a nucleotide sequence having at least 70% identity with sequence number 91.

10. A kit for manufacturing the vector according to any one of claims 1 to 9.

11. A pharmaceutical composition or isolated cells comprising the vector according to any one of claims 1 to 9, in combination with a pharmaceutically acceptable carrier, diluent, or excipient.

12. A composition comprising the vector according to any one of claims 1 to 9 for use as a pharmaceutical.

13. Use of the vector according to any one of claims 1 to 9 for the manufacture of a pharmaceutical product.

14. A composition comprising a vector for use in the prevention or treatment of inflammatory eye diseases, wherein the vector comprises a nucleotide sequence encoding a TNF inhibitor, and the nucleotide sequence encoding the TNF inhibitor is functionally linked to an inducible inflammation promoter.

15. Use of a vector in the manufacture of a pharmaceutical product for the prevention or treatment of inflammatory eye disease, wherein the vector comprises a nucleotide sequence encoding a TNF inhibitor, and the nucleotide sequence encoding the TNF inhibitor is functionally linked to an inducible inflammation promoter.

16. A composition comprising the vector according to any one of claims 1 to 9 for use in the prevention or treatment of inflammatory eye diseases, optionally, Uveitis is an inflammatory eye disease. The vector is administered into the eye. The vector is administered by intravitreous, subretinal, direct retinal, subconjunctival, sub-Tenon's capsule, or suprachoroidal injection. The vector is administered as a single dose. The vector is administered in doses of at least approximately 1E10 vg / mL, at least approximately 1E11 vg / mL, at least approximately 1E12 vg / mL, or at least approximately 5E12 vg / mL. The vector is administered at a dose of at least about 1E9vg / eye, at least about 1E10vg / eye, or at least about 1E11vg / eye, preferably at a dose of about 1E9vg / eye to about 5E12vg / eye, and / or The vector is administered in response to a recurrence of an inflammatory eye disease, preferably the inflammatory eye disease being uveitis. composition.

17. A pharmaceutical composition according to claim 11 for use in the prevention or treatment of inflammatory eye disease, wherein, Uveitis is an inflammatory eye disease. The pharmaceutical composition is administered into the eye. The pharmaceutical composition is administered by intravitreous, subretinal, direct retinal, subconjunctival, sub-Tenon's capsule, or suprachoroidal injection. The pharmaceutical composition is administered as a single dose. The pharmaceutical composition is administered in doses of at least about 1E10 vg / mL, at least about 1E11 vg / mL, at least about 1E12 vg / mL, or at least about 5E12 vg / mL. The pharmaceutical composition is administered in doses of at least about 1E9vg / eye, at least about 1E10vg / eye, or at least about 1E11vg / eye, preferably in doses of about 1E9vg / eye to about 5E12vg / eye, and / or The pharmaceutical composition is administered in response to a recurrence of an inflammatory eye disease, preferably the inflammatory eye disease being uveitis. Pharmaceutical composition.

18. Use of the vector according to any one of claims 1 to 9 for the manufacture of a medicament for the prevention or treatment of inflammatory eye disease, wherein, Uveitis is an inflammatory eye disease. The vector is administered into the eye. The vector is administered by intravitreous, subretinal, direct retinal, subconjunctival, sub-Tenon's capsule, or suprachoroidal injection. The vector is administered as a single dose. The vector is administered in doses of at least approximately 1E10 vg / mL, at least approximately 1E11 vg / mL, at least approximately 1E12 vg / mL, or at least approximately 5E12 vg / mL. The vector is administered at a dose of at least about 1E9vg / eye, at least about 1E10vg / eye, or at least about 1E11vg / eye, preferably at a dose of about 1E9vg / eye to about 5E12vg / eye, and / or The vector is administered in response to a recurrence of an inflammatory eye disease, preferably the inflammatory eye disease being uveitis. use.

19. Use of the pharmaceutical composition according to claim 11 for the manufacture of a medicament for the prevention or treatment of inflammatory eye disease, wherein, Uveitis is an inflammatory eye disease. The pharmaceutical composition is administered into the eye. The pharmaceutical composition is administered by intravitreous, subretinal, direct retinal, subconjunctival, sub-Tenon's capsule, or suprachoroidal injection. The pharmaceutical composition is administered as a single dose. The pharmaceutical composition is administered in doses of at least about 1E10 vg / mL, at least about 1E11 vg / mL, at least about 1E12 vg / mL, or at least about 5E12 vg / mL. The pharmaceutical composition is administered in doses of at least about 1E9vg / eye, at least about 1E10vg / eye, or at least about 1E11vg / eye, preferably in doses of about 1E9vg / eye to about 5E12vg / eye, and / or The pharmaceutical composition is administered in response to a recurrence of an inflammatory eye disease, preferably the inflammatory eye disease being uveitis. use.

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