Antibody that binds to TNFRSF25

Abstract

The present invention provides an antibody and an antibody fragment that bind to human TNFRSF25. The antibody may be a monoclonal antibody and / or a biparatope antibody and / or a single-chain variable fragment (scFv) antibody. A method for treating or preventing a disease or disorder associated with inflammation and / or autoimmunity is provided, comprising the step of administering an effective amount of a human TNFRSF25-binding antibody to a patient in need thereof.

Description

Technology Field

[0001] Reference to related applications

[0002] The present application claims the benefit of priority of U.S. Provisional Application No. 63 / 624,600 filed January 24, 2024; U.S. Provisional Application No. 63 / 570,593 filed March 27, 2024; U.S. Provisional Application No. 63 / 677,519 filed July 31, 2024; U.S. Provisional Application No. 63 / 700,056 filed September 27, 2024; U.S. Provisional Application No. 63 / 706,966 filed October 14, 2024; and U.S. Provisional Application No. 63 / 720,903 filed November 15, 2024, the full contents of each of which are incorporated herein by reference.

[0003] Reference to sequence list

[0004] The present application comprises a sequence listing XML that was submitted electronically and whose entirety is incorporated by reference. The sequence listing XML created on January 24, 2025, is named STKLP0006WO.xml and has a size of 150,605 bytes. Background Technology

[0005] 1. Field

[0006] The present invention generally relates to the fields of medicine and immunology. More specifically, it relates to an antibody that binds to TNFRSF25 and a method of using the same.

[0007] 2. Description of Related Technology

[0008] Tumor necrosis factor receptor super-family 25 (TNFRSF25) is a member of the tumor necrosis factor receptor super-family (TNFRSF). TNFRSF25 is also known as death receptor 3 (DR3), lymphocyte-associated receptor of death (LARD), APO-3, TRAMP, and WSL-1. TNFRSF25 is expressed on T cells and some other cells, including endothelial cells, epithelial cells, osteoblasts, B cells, natural killer T (NKT) cells, and type 2 innate lymphocytes (ILC2).

[0009] TNFRSF25 activation is mediated by tumor necrosis factor (TNF)-like cytokine 1A (TL1A; also known as TNF superfamily 15; TNFSF15), which is expressed by lymphocytes, plasma cells, dendritic cells, macrophages, monocytes, and synovial fibroblasts as well as endothelial cells. TL1A is also expressed in the kidneys, lungs, prostate, and thymus. By binding to TNFRSF25, TL1A exerts multiple effects on adaptive immune cells, which influence the activation, proliferation, and differentiation of different immune cells such as helper T cells, regulatory T cells, and B cells, as well as the production of cytokines such as interferon-γ (IFN-γ), interleukin (IL)-2, IL-13, tumor necrosis factor alpha (TNF-α), and granulocyte macrophage-colony-stimulating factor (GM-CSF). TL1A and TNFRSF25 are also involved in promoting apoptosis and necroptotic cell death.

[0010] Binding of TL1A to TNFRSF25 has been associated with several inflammatory diseases such as inflammatory bowel disease, ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriasis, atherosclerosis, asthma, multiple sclerosis, primary biliary cirrhosis, systemic lupus erythematosus, and ankylosing spondylitis. Soluble TL1A can be detected in the serum and body fluids of patients with T-cell-mediated inflammatory autoimmune diseases such as rheumatoid arthritis, psoriasis, and ankylosing spondylitis.

[0011] Blocking TL1A from binding to TNFRSF25 and / or blocking the activation of TNFRSF25 can reduce inflammatory responses. However, soluble TL1A is not constitutively expressed and has a short serum half-life; TL1A expression is strictly regulated by TLR stimulation. As such, anti-TL1A antibodies must act within a short window to avoid pathway activation. On the other hand, TNFRSF25 is constitutively expressed. However, previously known anti-TNFRSF25 antibodies have been reported to possess agonist activity, which can promote harmful inflammatory responses such as T cell proliferation and cytokine production by T cells.

[0012] Thus, a TNFRSF25-binding antibody is required that blocks TL1A from binding to TNFRSF25 and / or blocks the activation of TNFRSF25 without residual agonist action of TNFRSF25.

[0013] The present invention provides monoclonal antibodies and antibody fragments that bind to TNFRSF25, wherein the antibody or antibody fragment is (a) SEQ NO 50 and SEQ NO 51; (b) SEQ NO 52 and SEQ NO 53; (c) SEQ NO 54 and SEQ NO 55; (d) SEQ NO 94 and SEQ NO 95; (e) SEQ NO 96 and SEQ NO 97; (f) SEQ NO 98 and SEQ NO 99; (g) SEQ NO 100 and SEQ NO 97; (h) SEQ NO 94 and SEQ NO 97; (i) SEQ NO 94 and SEQ NO 99; (j) SEQ NO 96 and SEQ NO 95; (k) SEQ NO 96 and SEQ NO 99; (l) SEQ NO 98 and SEQ NO 95; (m) SEQ NO 98 and SEQ NO 97; (n) SEQ NO 100 and SEQ NO 95; or (o) clone-paired heavy and light chain CDR sequences derived from clone-paired heavy and light chain variable sequences of SEQ ID NO. 100 and SEQ ID NO. 99. For example, clone-paired heavy and light chain CDR sequences include (a) SEQ ID NOs. 7 to 12; (b) SEQ ID NOs. 13 to 18; (c) SEQ ID NOs. 19 to 24; (d) SEQ ID NOs. 30 to 35; (e) SEQ ID NOs. 36 to 41; (f) SEQ ID NOs. 42 to 47; (g) SEQ ID NOs. 7, 8, 77 and 10 to 12; (h) SEQ ID NOs. 7, 8, 78, 79, 11 and 80; (i) SEQ ID NOs. 81, 8, 79, 83, 11 and 84; (j) SEQ ID NOs 7 to 9, 86, 11 and 87; (k) SEQ ID NOs 7, 8, 77, 79, 11 and 80; (l) SEQ ID NOs 7, 8, 77, 83, 11 and 84; (m) SEQ ID NOs 7, 8, 77, 86, 11 and 87; (n) SEQ ID NOs 7, 8, 78 and 10 to 12; (o) SEQ ID NOs 7, 8, 78, 83, 11 and 84; (p) SEQ ID NOs 7, 8, 78, 86, 11 and 87; (q) SEQ ID NOs 81, 8, 82 and 10 to 12;(r) Sequence Nos. 81, 8, 82, 79, 11 and 80; (s) Sequence Nos. 81, 8, 82, 86, 11 and 87; (t) Sequence Nos. 7, 8, 85 and 10 to 12; (u) Sequence Nos. 7, 8, 85, 79, 11 and 80; (v) Sequence Nos. 7, 8, 85, 83, 11 and 84; (w) Sequence Nos. 30, 31, 88, 33, 34 and 35; (x) Sequence Nos. 30, 31, 89, 90, 34 and 80; (y) Sequence Nos. 30, 31, 91, 90, 34 and 80; (z) Sequence Nos. 30 to 32, 90, 34 and 80; (a') Sequence Nos. 30, 31, 88, 90, 34 and 80; (b') Sequence Nos. 30, 31, 88, 90, 34 and 80; (c') Sequence Nos. 30, 31, 88, 90, 34 and 80; (d') Sequence Nos. 30, 31, 89 and 33 to 35; (e') Sequence Nos. 30, 31, 89, 90, 34 and 80; (f') Sequence Nos. 30, 31, 89, 90, 34 and 80; (g') Sequence Nos. 30, 31, 91 and 33 to 35; (h') Sequence Nos. 30, 31, 91, 90, 34 and 80; (i') SEQ ID NOs 30, 31, 91, 90, 34, and 80; (j') SEQ ID NOs 30 to 32, 90, 34, and 80; (k') SEQ ID NOs 30 to 32, 90, 34, and 80; (l') SEQ ID NOs 138 to 143; or (m') SEQ ID NOs 144 to 147, 29, and 148. The CDR sequences of the monoclonal antibody and antibody fragment may be (a) SEQ ID NOs 50 and 51; (b) SEQ ID NOs 52 and 53; (c) SEQ ID NOs 54 and 55; (d) SEQ ID NOs 94 and 95; (e) SEQ ID NOs 96 and 97; (f) SEQ ID NOs 98 and 99; (g) SEQ ID NOs 100 and 97; (h) Sequence No. 94 and Sequence No. 97; (i) Sequence No. 94 and Sequence No. 99; (j) Sequence No. 96 and Sequence No. 95;(k) SEQ NO 96 and SEQ NO 99; (l) SEQ NO 98 and SEQ NO 95; (m) SEQ NO 98 and SEQ NO 97; (n) SEQ NO 100 and SEQ NO 95; (o) SEQ NO 100 and SEQ NO 99; or (p) SEQ NO 92 and SEQ NO 93 may comprise 1, 2, or 3 substitutions, deletions, and / or insertions for the CDR sequence derived from the clonal-paired heavy and light chain variable sequences of SEQ NO 92 and SEQ NO 93. The monoclonal antibodies and antibody fragments include (a) SEQ NO 50 and SEQ NO 51; (b) SEQ NO 52 and SEQ NO 53; (c) SEQ NO 54 and SEQ NO 55; (d) SEQ NO 94 and SEQ NO 95; (e) SEQ NO 96 and SEQ NO 97; (f) SEQ NO 98 and SEQ NO 99; (g) SEQ ID NO 100 and SEQ ID NO 97; (h) SEQ ID NO 94 and SEQ ID NO 97; (i) SEQ ID NO 94 and SEQ ID NO 99; (j) SEQ ID NO 96 and SEQ ID NO 95; (k) SEQ ID NO 96 and SEQ ID NO 99; (l) SEQ ID NO 98 and SEQ ID NO 95; (m) SEQ ID NO 98 and SEQ ID NO 97; (n) SEQ ID NO 100 and SEQ ID NO 95; (o) SEQ ID NO 100 and SEQ ID NO 99; or (p) may include clone-paired heavy and light chain variable sequences having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity independently of the sequences of SEQ ID NO 92 and SEQ ID NO 93. Monoclonal antibodies and antibody fragments are (a) SEQ NO 59 and SEQ NO 60; (b) SEQ NO 61 and SEQ NO 62; (c) SEQ NO 63 and SEQ NO 64; or (d) SEQ NO 127 and SEQ NO 128; (e) SEQ NO 129 and SEQ NO 130; (f) SEQ NO 131 and SEQ NO 132; (g) SEQ NO 133 and SEQ NO 130; (h) SEQ NO 127 and SEQ NO 130; (i) SEQ NO 127 and SEQ NO 132;(j) SEQ ID NO 129 and SEQ ID NO 128; (k) SEQ ID NO 129 and SEQ ID NO 132; (l) SEQ ID NO 131 and SEQ ID NO 128; (m) SEQ ID NO 131 and SEQ ID NO 130; (n) SEQ ID NO 133 and SEQ ID NO 128; (o) SEQ ID NO 133 and SEQ ID NO 132; (p) SEQ ID NO 151 and SEQ ID NO 152; or (q) may include heavy and light chain sequences forming clone pairs having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity independently with respect to the sequences of SEQ ID NO 137 and SEQ ID NO 128.;

[0014] The present invention provides a monoclonal antibody and an antibody fragment that may comprise clone-paired heavy and light chain variable sequences having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity independently with respect to the sequences of SEQ ID NO. 48 and SEQ ID NO. 49. The monoclonal antibody and antibody fragment may comprise (a) SEQ ID NO. 56 and SEQ ID NO. 57; (b) SEQ ID NO. 58 and SEQ ID NO. 57; (c) SEQ ID NO. 134 and SEQ ID NO. 57; (d) SEQ ID NO. 150 and SEQ ID NO. 57; (e) SEQ ID NO. 153 and SEQ ID NO. 57; (f) SEQ ID NO. 151 and SEQ ID NO. 152; Or (g) may include clone-paired heavy and light chain sequences having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity independently of the sequences of SEQ ID NO. 135 and SEQ ID NO. 57.

[0015] The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, an F(ab')2 fragment, or an Fv fragment. The antibody may be a chimeric antibody, a biparatopic antibody, or a bispecific antibody. The antibody and antibody fragment may be an IgG antibody or a recombinant IgG antibody or antibody fragment.

[0016] The antibody fragments are (a) SEQ NO 50 and SEQ NO 51; (b) SEQ NO 52 and SEQ NO 53; (c) SEQ NO 54 and SEQ NO 55; (d) SEQ NO 94 and SEQ NO 95; (e) SEQ NO 96 and SEQ NO 97; (f) SEQ NO 98 and SEQ NO 99; (g) SEQ NO 100 and SEQ NO 97; (h) SEQ NO 94 and SEQ NO 97; (i) SEQ NO 94 and SEQ NO 99; (j) SEQ NO 96 and SEQ NO 95; (k) SEQ NO 96 and SEQ NO 99; (l) SEQ NO 98 and SEQ NO 95; (m) SEQ NO 98 and SEQ NO 97; (n) SEQ NO 100 and SEQ NO 95; (o) SEQ NO 100 and SEQ NO 99; Or (p) a recombinant scFv (single-strand fragment variable) antibody comprising a clonal-paired heavy chain and light chain variable sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity independently with respect to the sequences of SEQ ID NO. 92 and SEQ ID NO. 93. The heavy chain and light chain variable sequences may be linked by a flexible linker, for example, a Gly-Ser linker. The flexible linker may include or be composed of the sequence GGRGSGGGGSGSGGS (SEQ ID NO. 101) or GGGGSGGGGSGGGGS (SEQ ID NO. 102). The antibody fragment may be a recombinant scFv (single-strand fragment variable) antibody comprising or composed of a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with respect to any one of sequence numbers 103 to 126.

[0017] When antibodies or antibody fragments are measured by surface plasmon resonance or bio-layer interferometry, K of 50 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, 0.75 nM or less, 0.5 nM or less, 0.1 nM or less, 75 pM or less, 50 pM or less, 25 pM or less, 10 pM or less, 7.5 pM or less, or 5 pM or less D It can be combined with TNFRSF25.

[0018] The present invention provides a dual paratope antibody comprising a first binding part that binds to a first epitope of human TNFRSF25 and a second binding part that binds to a second epitope of human TNFRSF25, wherein the first binding part comprises a clone-paired heavy chain and light chain CDR sequence derived from the clone-paired heavy chain and light chain variable sequences of SEQ ID NO. 48 and SEQ ID NO. 49, and the second binding part comprises (a) SEQ ID NO. 50 and SEQ ID NO. 51; (b) SEQ ID NO. 52 and SEQ ID NO. 53; or (c) a clone-paired heavy chain and light chain CDR sequence derived from the clone-paired heavy chain and light chain variable sequences of SEQ ID NO. 54 and SEQ ID NO. 55. The first binding portion may include (a) clone-paired heavy and light chain CDR sequences of SEQ ID NOs 1 to 6 or (b) SEQ ID NOs 25 to 29 and 6, and the second binding portion may include (a) SEQ ID NOs 7 to 12; (b) SEQ ID NOs 13 to 18; (c) SEQ ID NOs 19 to 24; (d) SEQ ID NOs 30 to 35; (e) SEQ ID NOs 36 to 41; or (f) clone-paired heavy and light chain CDR sequences of SEQ ID NOs 42 to 47. The first binding portion may include a clone-paired heavy chain and light chain variable sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity independently with respect to the sequences of SEQ ID NO. 48 and SEQ ID NO. 49, and the second binding portion may include a clone-paired heavy chain and light chain variable sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity independently with respect to (a) SEQ ID NO. 50 and SEQ ID NO. 51; (b) SEQ ID NO. 52 and SEQ ID NO. 53; or (c) SEQ ID NO. 54 and SEQ ID NO. 55, heavy chain and light chain variable sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity independently with respect to the sequences of SEQ ID NO. 50 and SEQ ID NO. 55.The first binding portion may comprise heavy and light chain sequences forming clone-pairs having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity independently with respect to the sequences of SEQ ID NO. 136 and SEQ ID NO. 57, and the second binding portion may comprise (a) SEQ ID NO. 59 and SEQ ID NO. 60; (b) SEQ ID NO. 61 and SEQ ID NO. 62; (c) SEQ ID NO. 63 and SEQ ID NO. 64; (d) SEQ ID NO. 66 and SEQ ID NO. 60; (e) SEQ ID NO. 67 and SEQ ID NO. 62; Or (f) may include heavy chain and light chain sequences forming clone pairs having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity independently of the sequences of SEQ ID NO. 68 and SEQ ID NO. 64.

[0019] The present invention provides an isolated nucleic acid molecule encoding a heavy chain variable region, a light chain variable region, a heavy chain, or a light chain of any one of the antibodies provided herein. The present invention provides an isolated nucleic acid molecule encoding scFv provided herein. The present invention also provides an expression vector comprising said nucleic acid.

[0020] The present invention provides a hybridoma or engineered cell comprising a nucleic acid molecule encoding a heavy chain variable region, a light chain variable region, or a heavy chain or a light chain of any one of the antibodies provided herein. The present invention provides a method for producing any one of the antibodies provided herein, the method comprising the steps of culturing the hybridoma or engineered cell under conditions allowing the expression of the antibody or antibody fragment or biparatop antibody, and optionally isolating the antibody or antibody fragment or biparatop antibody from the culture.

[0021] The present invention provides a pharmaceutical formulation comprising any one of the antibodies provided to the present invention.

[0022] The present invention provides a method for treating a patient having a disease or disorder associated with inflammation and / or autoimmunity, the method comprising the step of administering to the patient a pharmaceutical formulation comprising any one of the antibodies provided in the present invention. The disease or disorder may be ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriasis, atherosclerosis, asthma, multiple sclerosis, primary biliary cirrhosis, systemic lupus erythematosus, or ankylosing spondylitis. Administration may preserve epithelial barrier integrity. Administration may protect against intestinal injury, such as TL1A-mediated intestinal injury, for example.

[0023] Any of the antibodies, antibody fragments, or biparatope antibodies provided herein may be used to treat or prevent diseases or disorders related to inflammation and / or autoimmunity in patients.

[0024] The present invention provides the use of antibodies or antibody fragments or dual-paratop antibodies or pharmaceutical formulations as provided herein in the manufacture of agents for treating or preventing diseases or disorders related to inflammation and / or autoimmunity.

[0025] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, the detailed description and specific embodiments are to be understood as being provided only as examples, although they represent preferred embodiments of the present invention, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. Brief explanation of the drawing

[0026] The following drawings constitute part of this specification and are incorporated to further demonstrate specific aspects of the invention. The invention may be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. Fig. 1. MSD plate analysis of antibody binding to recombinant biotin-conjugated human TNFRSF25 protein. Recombinant biotin-conjugated human TNFRSF25 protein was immobilized on streptavidin-coated MSD plates and allowed to bind to anti-TNFRSF25 mAbs in solution. After a washing step, the bound antibodies were detected using sulfo-tagged anti-human Fc antibodies. FIGS. 2a to 2b. Recombinant TNFRSF25 binding and TL1A blockade by NH24 scFv variants. (Fig. 2a) TNFRSF25 was pre-coated onto an MSD assay plate, and the scFv supernatant was added at a 2-fold serial dilution, after which the myc tag present on the C-terminus of the scFv was detected. (Fig. 2b) The scFv supernatant was serially diluted and added to an MSD plate pre-coated with TNFRSF25. Next, TL1A was added to the scFv-TNFRSF25 complex, and residual TL1A was detected. The less residual TL1A, the higher the blockade activity of the given scFv. Some background blockade by the negative control resulted from a matrix effect due to the unpurified nature of the scFv supernatant and is therefore considered a background signal. Figs. 3a to 3c. Evaluation of anti-TNFRSF25 antibodies containing various IgG1 Fc-silencing mutations. (Fig. 3a) CD16-mediated NFAT activation. O33-scFv-derived anti-TNFRSF25 antibodies were first incubated with CHOK1 cells expressing TNFRSF25 for 1 hour. Antibody-opsonized CHOK1 cells were further incubated with Jurkat cells expressing cell surface Fc receptor CD16A and NFAT-luciferase reporter genes in a 1:4 ratio. Luciferase activity resulting from CD16-mediated NFAT activation was measured using a luminometer after 6 hours. (Fig. 3b) Binding of anti-TNFRSF25 to FcγR1 containing Fc-silencing mutations as determined by BLI. The sensorgram shows the BLI trace of the analytes (at 300 nM) as indicated in the legend. (Fig. 3c) SL-061 (and SL-034) do not possess detectable FcγR1 (CD64)-mediated DR3 receptor activation or residual agonistic function. HEK293 cells (parental or CD64-expressing) were seeded into separate 96-well plates and incubated for 5 hours to allow cell attachment. Subsequently, anti-TNFRSF25 antibody [32 pM] and an equal number of DR3 + Jurkat-NFkB-luciferase reporter cells into individual HEK293 cells (maternal and CD64 + It was added to a plate containing ) and incubated for 5 hours. BioGlo luciferase substrate (Promega) was added at a 1:1 ratio and incubated at room temperature for 10 minutes. HEK293 parent and CD64 + The plate containing the cells was subsequently read on a luminescence plate reader. Data analysis was performed for each antibody using CD64 +The effects of CD64-mediated crosslinking and activation were measured by subtracting the parent plate value from the plate. Fig. 4. Binding affinity of anti-TNFRSF25 antibody to TNFRSF25. BLI analysis was performed using an Octet Red 96e instrument. Biotinylated recombinant human TNFRSF25-His tag (Acro #TN5-H52H3) was loaded as a ligand onto a streptavidin biosensor. During the binding step, the biosensor was incubated with various concentrations of anti-TNFRSF25 antibody for 600 seconds, followed by a 600-second dissociation step. Figs. 5a to 5c. Binding affinity of SL-061 (L234A / L235A + G237A) to TNFRSF25 by BLI / Octet and SPR. (Fig. 5a) BLI analysis was performed using an Octet Red 96e instrument. Biotinylated recombinant human TNFRSF25-His tag (Acro #TN5-H52H3) was loaded onto the streptavidin biosensor as a ligand. During the binding step, the biosensor was incubated with SL-061 at the indicated concentration for 600 seconds, followed by a dissociation step of 600 seconds. Complete kinetic parameters could not be determined by BLI. (Fig. 5b) SPR was performed on a Biacore 8K instrument. Biotinylated recombinant human TNFRSF25-His tag (Acro #TN5-H52H3) was loaded at 50 nM onto the streptavidin sensor chip SA (Cytiva), and then SL-061 at the indicated concentrations was flowed for a 2-minute binding step followed by a 30-minute dissociation step. (Fig. 5c) Binding specificity of SL-033 to recombinant receptors. TNFRSF family proteins TNFR1 or TNFR2, as well as the decoy receptor DcR3, were immobilized on MSD plates and allowed to bind to various concentrations of SL-033 (or positive controls). After the washing step, SL-033 was detected using a sulfo-tagged anti-human Fc antibody (or an anti-HIS sulfo-tagged antibody suitable for the positive control). SL-033 did not exhibit detectable binding to the tested receptors. Fig. 5d. FcRn binding kinetics by anti-TNFRSF25 antibody. BLI analysis was performed using an Octet Red 96e instrument. Biotinylated recombinant human (h) FcRn-His tag protein (Acro#FCM-H82W7) was loaded onto the streptavidin biosensor as a ligand. During the binding step, the biosensor was incubated with various concentrations of anti-DR3 candidate antibodies for 600 seconds, followed by a 600-second dissociation step. Both SL061_YTE and SL061_LS showed approximately a 10-fold improvement in binding affinity to hFcRn at pH 6.0 (acidic) compared to pH 7.4 (neutral), which is mainly attributed to their slower dissociation rates at pH 6.0. Fig. 6. TL1A blocking activity of anti-TNFRSF25 antibody. Human TNFRSF25 was pre-coated onto MSD assay plates and incubated with various concentrations of anti-TNFRSF25 antibody. The plates were washed and incubated with a fixed concentration of TL1A-rabbit(r) Fc protein (0.4 μg / ml). For the analysis of the anti-TL1A benchmark antibody control, anti-TL1A antibodies (labeled with α-TL1A1 and α-TL1A2) were first incubated with a fixed amount (0.4 μg / ml) of TL1A-rFc in a separate 96-well plate before being added to the TNFRSF25-coated MSD plates. The TNFRSF25:TL1A protein complex was detected using a sulfo-tagged anti-rabbit secondary antibody. Determine the TL1A blocking % (top right panel) by normalizing the raw MSD signal against the anti-DR3 no (0 nM) well or the anti-TNFRSF log 10 Plotted as a function of (nM) concentration (bottom panel). Fig. 7a. CHOK1-TNFRSF25 by flow cytometry +Natural TNFRSF25 binding by anti-TNFRSF25 antibodies on cells. Natural TNFRSF25 binding was evaluated using CHOK1 cell lines engineered to overexpress human TNFRSF25 protein via stable transfection with a mammalian expression vector (pcDNA) encoding the human TNFRSF25 cDNA sequence (NP_683866). CHOK1-TNFRSF25 + Cells were incubated with various concentrations of anti-TNFRSF25 antibody at 4°C for 1 hour. Cells were washed and further incubated with APC-conjugated anti-human Fc antibody to detect antibody-drug bound cells by flow cytometry. Fig. 7b. SL-061 is specific for binding to natural TNFRSF25. Several TNFRSF receptors overexpressed in engineered mammalian cells were incubated with various concentrations of SL-061. Cells were washed and further incubated with an APC-conjugated anti-human Fc antibody to detect antibody-drug conjugated cells by flow cytometry. Fig. 7c. SL-061 does not induce internalization of the TNFRSF25 receptor in engineered mammalian cells. Engineered CHOK1 cells overexpressing TNFRSF25 (cDNA sequence NP_683866) were incubated with various concentrations of SL-061 at 4°C or 37°C for 30 minutes. Cells were washed and further incubated with an APC-conjugated anti-human Fc antibody to detect antibody-drug conjugated cells by flow cytometry. Fig. 7d. SL-061 shows binding to natural cynomolgus TNFRSF25. The cynomolgus TNFRSF25 sequence (REFSEQ accession number: XM_015443331.1) was cloned into a pCDNA vector for cell-surface expression as a dichistrogenic gene for co-expressing cleavable cytoplasmic GFP. The vector was transiently transfected into HEK293 cells. 24 hours after transfection, cells were incubated with various concentrations of SL-061 or 4C12 (positive control). Next, cells were washed and further incubated with an APC-conjugated anti-human Fc antibody to detect antibody-drug conjugated cells by flow cytometry. Fig. 8a. The anti-TNFRSF25 antibody competitively blocks TL1A binding to natural TNFRSF25 on CHOK1 cells. To evaluate the ability of the TNFRSF25 antagonist antibody to bind to TNFRSF25 and block TL1A binding, CHOK1-TNFRSF25 + Cells were first incubated with various concentrations of anti-TNFRSF25 antibody at 4°C for 30 minutes. Anti-TL1A benchmark antibodies (α-TL1A1 and α-TL1A2) were used as comparative controls in this analysis. A fixed concentration of biotinylated human TL1A-Fc protein (156 nM) was subsequently added to the cells, and they were incubated for an additional 1 hour at 4°C. After washing to remove excess antibody and TL1A, cells were further incubated with PE-conjugated streptavidin for the detection of cells bound to TL1A by flow cytometry. The average fluorescence intensity of TL1A (PE) is shown. Fig. 8b. The anti-TNFRSF25 antibody SL-061 efficiently blocked TL1A-induced IFNγ secretion in both soluble and membrane-bound forms from human PBMCs. Human PBMCs were stimulated with anti-CD3 / CD28 and treated with soluble TL1A (100 ng / mL), or 1 x 10⁶ HEK293-TL1A or HEK293 parent cells, each not expressing TL1A on the surface. 4 Co-cultured with dogs. After 72 hours, the supernatant was harvested and the levels of IFNγ were analyzed using MSD. The dotted line represents the levels of IFNγ produced by PBMCs in the absence of TL1A. An anti-TL1A benchmark antibody (α-TL1A1) was used as a comparative control in this analysis. The bottom graph indicates that IFNγ secretion was indeed induced by membrane-expressed TL1A, as HEK293 parent cells did not induce IFNγ secretion even in the absence of the SL-061 antibody. Fig. 9. Binding of SL-033 to TNFRSF25 on lymphocytes in human PBMCs. Human PBMCs were left unstimulated or stimulated for 24 hours with IL12+IL18 or anti-CD3 / CD28 (used at sub-optimal doses) to evaluate the binding of anti-TNFRSF25 (SL-033) to lymphocyte subsets. Cells were harvested and incubated with a fixed concentration of SL-033 (5 μg / mL), then incubated with APC-anti-human Fc (detection of SL-033) and lineage-specific antibodies against T and NK cells, and analyzed by flow cytometry. Histograms of SL-033 intensity are shown for each cell type. Fig. 10. Dose-dependent binding of SL-061 to TNFRSF25 on lymphocytes in human PBMCs. Human PBMCs were stimulated with either IL12+IL18 or anti-CD3 / CD28 for 24 hours to evaluate the binding of anti-TNFRSF25 (SL-061) to lymphocyte subsets. Cells were harvested and incubated with various concentrations of SL-061, then incubated with APC-anti-human Fc (detection of SL-061) and lineage-specific antibodies against T and NK cells, and analyzed by flow cytometry. The mean fluorescence intensity (MFI) of SL-061 binding is shown for each cell type. Fig. 11. Anti-TNFRSF25 antibody efficiently blocks TL1A-induced IFNγ secretion from human PBMCs (IL12+IL18 stimulation). TNFRSF25 expression was upregulated in human PBMCs by stimulating them with IL-12 and IL-18 for 24 hours. After 24 hours, cells were treated with various concentrations of anti-TNFRSF25 antibody in the presence or absence of TL1A (100 ng / mL). After 48 hours, the supernatant was harvested and the levels of IFNγ were analyzed using MSD. The dotted line represents the levels of IFNγ produced by PBMCs in the absence of TL1A. Fig. 12. Enhanced blockade of IFNγ secretion by SL-033 with Fc-silencing mutation (IL12 / IL18 stimulation). Human PBMCs were stimulated with IL-12 and IL-18 for 24 hours to upregulate TNFRSF25 expression. After 24 hours, cells were treated with various concentrations of anti-TNFRSF25 in the presence or absence of TL1A (100 ng / mL). After 48 hours, the supernatant was harvested and IFNγ levels were analyzed using MSD. IFNγ concentrations were normalized to levels produced by untreated PBMCs stimulated with IL12 / 18+TL1A. Fig. 13. Similar IFNγ blockade by SL-033 (L234A / L235A) and SL-061 (L234A / L235A + G237A) under CD3 / CD28 stimulation conditions without evidence of residual agonist action. Human PBMCs were stimulated with CD3 / CD28 complex + / - TL1A (100 ng / mL) and treated with various concentrations of anti-TNFRSF25 antibody. After 72 hours, the supernatant was harvested and IFNγ levels were analyzed using MSD. The dotted line represents the level of IFNγ produced by CD3 / CD28-stimulated cells in the absence of TL1A. Fig. 14. SL-033 (L234A / L235A) blocks the secretion of TL1A-induced TNFα, IL-6, and GM-CSF without evidence of residual agonist action. Human PBMCs were stimulated with CD3 / CD28 complex + / - TL1A (100 ng / mL) and treated with various concentrations of anti-TNFRSF25 antibody. After 72 hours, the supernatant was harvested and cytokine levels were analyzed using MSD. The dotted line represents the cytokine levels produced by CD3 / CD28-stimulated cells in the absence of TL1A. Fig. 15. SL-061 (L234A / L235A + G237A) blocks TL1A-induced IFNγ secretion by CD3 / CD28-stimulated lymphocytes (3 healthy donors). Human PBMCs from 3 individual healthy donors were stimulated with CD3 / CD28 complex + / - TL1A (100 ng / mL) and treated with various concentrations of anti-TNFRSF25 antibody. After 72 hours, the supernatant was harvested and IFNγ levels were analyzed using MSD. The dotted line represents the level of IFNγ produced by CD3 / CD28-stimulated cells in the absence of TL1A. Fig. 16. SL-061 (L234A / L235A + G237A) blocks TL1A-induced IFNγ secretion from IBD patient PBMCs without evidence of residual agonist action. Human PBMCs derived from donors with ulcerative colitis (UC) or Crohn's disease (CD) were stimulated with CD3 / CD28 complex + / - TL1A (100 ng / mL) and treated with various concentrations of anti-TNFRSF25 antibody. After 72 hours, the supernatant was harvested and IFNγ levels were analyzed using MSD. The dotted line represents the level of IFNγ produced by CD3 / CD28-stimulated cells in the absence of TL1A. Fig. 17a. Treatment with SL-061 did not result in toxicity to the intestinal epithelial barrier. The toxicity of the SL-061 antibody was evaluated under inflammatory conditions using a RepliGut® intestinal culture system. Human intestinal stem cells were seeded into a transwell system featuring a semipermeable membrane coated with a biomimetic scaffold composed of collagen hydrogel, which promotes the differentiation of stem cells into a monolayer of intestinal epithelial cells that closely mimics the in vivo intestinal epithelium. Cells were cultured according to the RepliGut® protocol to allow for the formation of a fully differentiated, polarized epithelial monolayer. The resulting epithelial monolayer contained a population of polarized and differentiated cells, representing a physiologically relevant model system for toxicity evaluation. Once the monolayer was established (days 6 to 7), the cultured cells were exposed to a fixed concentration of 5 μg / mL of SL-061 in the presence of various concentrations of the pro-inflammatory cytokines TNFα and IFNγ. These cytokines were added to mimic the inflammatory environment, and their concentrations were varied to evaluate dose-dependent effects. Barrier function was evaluated by measuring transepithelial electrical resistance (TEER) daily throughout the experiment. Fig. 17b. SL-061 inhibited TL1A-mediated intestinal epithelial barrier disruption and the production of inflammatory cytokines by PBMCs. The effects of SL-061 on intestinal barrier function were evaluated using a co-culture system of pre-activated peripheral blood mononuclear cells (PBMCs) and the RepliGut® intestinal epithelial model. PBMCs derived from healthy donors were pre-activated with the anti-CD3 / CD28 complex and recombinant TL1A to stimulate the production of inflammatory cytokines. After 24 hours, pre-activated PBMCs were added to the basal compartment of the RepliGut® intestinal culture system, while SL-061 (5 μg / mL) was applied to both the apical and basal compartments. Intestinal barrier integrity was assessed daily by TEER measurements. TEER values ​​were normalized to the maximum value in each well to account for inter-well variability. Figs. 18a to 18c. Receptor occupancy and immunophenotype analysis in non-human primates. Fig. 18a. SL-061 receptor occupancy (indirect): Blocking of anti-TNFRSF25 (clone 4C12) binding to Treg and proliferating CD4+ T cells (Ki-67+). Fig. 18b. SL-061 treatment did not result in an increase in the frequency of Treg or proliferating CD4+ T cells. Fig. 18c. SL-061 treatment did not result in an expansion of T cells. FIGS. 19a to 19b. Receptor occupancy. NHPs were administered SL-061 at 1, 10, or 100 mg / kg or as a vehicle control on days 1, 15, and 29. (Fig. 19a) The percentage of DR3-positive Tregs was measured by flow cytometry using the anti-DR3 antibody (clone 4C12). The dashed line indicates the background staining level determined using the isotype control antibody. (Fig. 19b) Receptor occupancy was calculated by normalizing the DR3+ percentage against the vehicle control (set to 100%) and isotype control staining (set to 0%). Data are presented as mean ± SD. The indicated time points are before administration on day 1 and 2 hours after the initial administration, and days 7, 15, 22, 43, and 57. The time point on day 57 includes only recovered animals in the vehicle and 100 mg / kg groups. FIGS. 20a to 20c. Absolute cell counts. NHPs were administered SL-061 at 1, 10, or 100 mg / kg or as a vehicle control on days 1, 15, and 29. (Fig. 20a) The absolute number of CD4+ T cells, (Fig. 20b) CD8+ T cells, and (Fig. 20c) Tregs was determined by flow cytometry using count beads. Data are presented as mean ± SD. Time points indicated are before administration on day 1 and 2 hours after the initial administration, and days 7, 15, 22, 43, and 57. The time point on day 57 includes only recovered animals in the vehicle and 100 mg / kg groups. FIGS. 21a to 21c. T-cell activation and proliferation. SL-061 was administered to NHPs at 1, 10, or 100 mg / kg or as a vehicle control on days 1, 15, and 29. (Fig. 21a) The percentage of CD25+ cells among CD4+ T cells (left) and CD8+ T cells (right) was measured to assess the T-cell activation status. (Fig. 21b) T-cell proliferation was assessed by measuring the percentage of Ki67+ cells among CD4+ T cells (left) and CD8+ T cells (right). (Fig. 21c) The frequency of Treg cells within CD4+ T cells (left) and the percentage of Ki67+ Treg cells (right) were determined by flow cytometry. Data are presented as mean ± SD. The indicated time points are before administration on day 1 and 2 hours after the initial administration, and on days 7, 15, 22, 43, and 57. The 57-day mark includes only recovered animals in the vehicle and 100 mg / kg groups. Specific details for implementing the invention

[0027] The present invention provides monoclonal antibodies and biparatop antibodies having clonal-paired heavy and light chain variable regions as exemplified in Table 3, as well as clonal-paired heavy and light chain variable regions as exemplified in Table 3, which bind to human TNFRSF25. Additionally, clonal-paired heavy and light chains for the disclosed antibodies are exemplified in Table 4. An exemplary biparatop antibody is exemplified in Table 5. An exemplary scFv construct is exemplified in Table 6. These antibodies may be produced using the methods described herein. These antibodies block TL1A binding to cell-surface TNFRSF25; block TL1A-induced secretion of TNF-alpha, IL-6, GM-CSF, and IFN-gamma; do not agonize cell-surface TNFRSF25; and / or do not induce internalization of cell-surface TNFRSF25.

[0028] [Table 1]

[0029]

[0030]

[0031]

[0032] [Table 2]

[0033]

[0034]

[0035]

[0036] [Table 3]

[0037]

[0038]

[0039]

[0040] [Table 4]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048] [Table 5]

[0049]

[0050]

[0051]

[0052] [Table 6]

[0053]

[0054]

[0055]

[0056] I. Definition

[0057] As used herein, “one(a)” or “one(an)” may mean one or more. As used herein in the claim(s), when used with the word “comprising,” the word “one” or “one” may mean one or more than one.

[0058] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer only to the alternative or the alternatives are mutually exclusive; however, the present disclosure supports definitions referring only to the alternative and referring to and “and / or.” As used herein, “another” may mean at least a second or more.

[0059] Throughout this application, the term “approximately” is used to indicate that the value includes inherent error variation for the device, inherent variation of the method used to determine the value, variation existing among subjects of study, or a value within 10% of the specified value.

[0060] As used herein, “essentially absent” in relation to a specific component is used herein to mean that none of the specific components have been intentionally incorporated into the composition and / or are present only as contaminants or trace amounts. Accordingly, the total amount of the specific component resulting from any unintended contamination of the composition is well less than 0.05%, preferably less than 0.01%. Most preferably, the composition is one in which no amount of the specific component can be detected by standard analytical methods.

[0061] “Nucleic acid,” “nucleic acid sequence,” “oligonucleotide,” “polynucleotide,” or other grammatical equivalents as used herein refer to at least two nucleotides joined together by covalent bonds, namely deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides are polymers of any length and include, for example, 20, 50, 100, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. Polynucleotides described herein generally include phosphodiester bonds, but in some cases, include at least one different linkage, for example, a phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkage, and nucleic acid analogs that may have a peptide nucleic acid backbone and linkage. Mixtures of naturally occurring polynucleotides and analogs may be prepared; alternatively, mixtures of different polynucleotide analogs and mixtures of naturally occurring polynucleotides and analogs may be prepared. The following are non-limiting examples of polynucleotides: genes or gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, cRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. Where present, modifications to the nucleotide structure may be imparted before or after the assembly of the polymer. The sequence of the nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with labeling components. This term also includes both double- and single-stranded molecules.Unless otherwise specified or required, the term polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form. A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is an alphabetic representation of a polynucleotide molecule. Unless otherwise specified, a specific polynucleotide sequence also implicitly encompasses not only the explicitly stated sequence but also its conservedly modified variants (e.g., degenerate codon substitutions) and complementary sequences. Specifically, degenerate codon substitutions can be achieved by producing a sequence in which the third position of one or more selected codons (or all) is substituted with a mixed-base and / or deoxyinosine residue.

[0062] As used herein, the terms “peptide,” “polypeptide,” and “protein” refer to polymers of amino acid residues. These terms also apply to amino acid polymers in which one or more amino acid residues are synthetic chemical mimics of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymers. In this case, the term “polypeptide” encompasses antibodies or fragments thereof.

[0063] Terms used herein in the fields of recombinant nucleic acid technology, immunology, antibody engineering, and other molecular and cellular biology will be generally understood by those skilled in the art.

[0064] II. Antibodies and antibody modification

[0065] The antibodies of the present invention have various applications, including as immunotherapy in patients in need. As such, in some cases, the antibodies of the present invention may be used to treat inflammatory diseases and autoimmune diseases. The antibodies may be mutated or modified as further discussed below. Methods for preparing and characterizing antibodies are well known in the art (see, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent 4,196,265).

[0066] “Antibody” is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv, Fd, Fd', single-stranded antibody (ScFv), diabodies, linear antibodies), mutants thereof, naturally occurring variants thereof, fusion proteins comprising antibody portions having antigen recognition sites of required specificity, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule comprising antigen recognition sites of required specificity.

[0067] “Isolated antibodies” are antibodies separated from and / or recovered from components of their natural environment. Contaminating components of their natural environment are substances that may interfere with the diagnostic or therapeutic use of the antibodies and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain examples, the antibodies are purified as follows: (1) exceeding 95% by weight of the antibody as determined by the Lowry method, and most particularly exceeding 99% by weight; (2) sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by the use of a spinning cup sequencer; or (3) homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. Since the isolated antibodies will not contain at least one component of the antibody’s natural environment, in situ ( in situ It includes antibodies. However, typically, the isolated antibodies will be prepared by at least one purification step.

[0068] The basic 4-chain antibody unit is a heterotemeric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). As used herein, the term “heavy chain” refers to a larger immunoglobulin subunit that binds to the immunoglobulin light chain through its amino-terminal region. The heavy chain has a variable region (V H ) and invariant region(C H Includes ). The invariant region is C H 1, hinge, C H 2, and C H It additionally includes 3 domains. In the case of IgE, IgM, and IgY, the heavy chain is C HIt contains four domains but does not have a hinge domain. Those skilled in the art will understand that the heavy chain is classified into gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and that there are some subclasses among them (e.g., γ1-γ4, α1-α2). The properties of this chain determine the “class” of the antibody as IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc., are well characterized and are known to confer functional specialization.

[0069] As used herein, the term “light chain” refers to a smaller immunoglobulin subunit that binds to the amino-terminal region of the heavy chain. Like the heavy chain, the light chain has a variable region (V L ) and invariant region(C L It includes ). The light chain is its invariant domain (C L Based on the amino acid sequence of the light chain, it is classified as either kappa or lambda (κ, λ). These pairs can combine with any pair of heavy chains to form an immunoglobulin molecule. Additionally, the meaning of light chain encompasses light chains having a lambda variable region (V-lambda) connected to a kappa constant region (C-kappa) or a kappa variable region (V-kappa) connected to a lambda constant region (C-lambda).

[0070] For example, IgM antibodies consist of five basic heterotemeric units along with an additional polypeptide called the J-chain and thus contain 10 antigen-binding sites, whereas secreted IgA antibodies can polymerize to form polyvalent aggregates containing two to five basic 4-chain units along with the J-chain. In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each L-chain is connected to the H-chain by a single covalent disulfide bond, whereas two H-chains are connected to each other by one or more disulfide bonds depending on the H-chain isotype. Each H and L-chain also has regularly spaced intrachain disulfide bridges. Each H-chain has a variable region (V) at the N-terminus. H It has ), followed by three invariant domains (C) for each of the alpha and gamma chains. H ) and 4 C for mu and isotypes H Each L-chain has a domain. Each L-chain has a variable region (V-) at the N-terminus. L It has ), followed by an invariant domain (C) at the other end thereof L has ). V L is V H Aligned with and C L The first invariant domain of the heavy chain (C H It aligns with 1). Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable regions. V H and V L The pairing of these forms a single antigen-binding site together. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr, and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, p. 71, and Chapter 6.

[0071] The “variable region” of an antibody refers, alone or in combination, to the variable region of the antibody light chain or the variable region of the antibody heavy chain. The term “variable” refers to the fact that specific segments of the variable region differ widely in sequence among antibodies. Light chain (V L ) and heavy chain (V H The variable regions of both parts mediate antigen binding and define the specificity of a specific antibody for a specific antigen. However, variability is not uniformly distributed across the entire variable region. Instead, the variable region consists of relatively invariant stretches called framework regions (FRs) separated by shorter regions of extreme variability, called complementarity determining regions (CDRs) or hypervariability regions. The variable regions of natural heavy and light chains each contain four FRs, generally adopt a beta-sheet configuration, and are connected by three CDRs; these form loops to link the beta-sheet structures, and in some cases, form part of them. CDRs complement the shape of the antigen and determine the affinity and specificity of the antibody for the antigen. V L and V H Both have six CDRs. The CDRs of each chain are held close together by FRs and, together with CDRs from other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0072] As used herein, the term “hypervariable region” refers to the amino acid residue of the antibody responsible for antigen binding. The hypervariable region is generally an amino acid residue from the “complementary determination region” or “CDR” (e.g., V when numbered according to the Kabat numbering system).L At approximately residuals 24-34 (L1), 50-56 (L2), and 89-97 (L3), and V H At approximately 31–35 (H1), 50–65 (H2), and 95–102 (H3); Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and / or residues from “hypervariable loops” (e.g., V when numbered according to the Chothia numbering system). L In residuals 24-34(L1), 50-56(L2) and 89-97(L3), and V H In 26-32(H1), 52-56(H2), and 95-101(H3); Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); and / or residues from the “supervariable loop” / CDR (e.g., V when numbered according to the IMGT numbering system) L In residuals 27-38(L1), 56-65(L2) and 105-120(L3), and V H Includes 27-38(H1), 56-65(H2), and 105-120(H3); Lefranc, MP et al. Nucl. Acids Res. 27:209-212 (1999), Ruiz, M. et al. Nucl. Acids Res. 28:219-221 (2000)). Optionally, antibodies are V when numbered according to AHo. L From the next points 28, 36(L1), 63, 74-75(L2) and 123(L3), and V subH has symmetric insertions in one or more of 28, 36 (H1), 63, 74-75 (H2) and 123 (H3); Honneger, A. and Plunkthun, AJ Mol. Biol. 309:657-670 (2001)). As used herein, CDR may refer to a CDR defined by any of these numbering approaches, by a combination of approaches, or by other preferred approaches. Additionally, new definitions of highly conserved cores, boundaries, and hypervariable regions may be used.

[0073] The “constant region” of an antibody refers, alone or in combination, to the constant region of the antibody light chain or the constant region of the antibody heavy chain. Light chain (C L ) and heavy chain (C H 1, C H 2 or C H 3, or in the case of IgM and IgE, C H The constant region of 4) confers important biological properties such as secretion, transplacental mobility, Fc receptor binding, and complement binding. By convention, the numbering of constant region domains increases as they move further away from the antigen binding site or the amino-terminus of the antibody. Although the constant region is not directly involved in binding the antibody to the antigen, it exhibits various effector functions, such as the antibody's participation in antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), and antibody-dependent complement deposition (ADCD).

[0074] A heavy chain variable region sequence, e.g., V of any one of SEQ ID NOs 48, 50, 52 and 54 H The sequence, or any variant thereof, may be covalently linked to various heavy chain constant region sequences known in the art. Similarly, V of any one of light chain variable region sequences, e.g., SEQ ID NOs 49, 51, 53, and 55L , or any variant thereof is considered to be covalently linked to various light chain invariant region sequences known in the art.

[0075] For example, the antibody or antibody fragment may have a heavy chain constant region selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; in particular, it may have a heavy chain constant region selected from, for example, the (e.g., human) heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4. The antibody or antibody fragment may have a light chain constant region selected from, for example, the (e.g., human) light chain constant regions of kappa or lambda. The constant regions may be mutated and altered, for example, to modify the properties of the antibody or antibody fragment (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, and / or complement function). The antibody or antibody fragment may not recruit effector cells or fix complement. The antibody or antibody fragment may have a reduced or absent ability to bind to the Fc receptor. For example, this is an isotype or subtype, fragment, or other mutant that does not support binding to the Fc receptor, and, for example, this has a mutated or deleted Fc receptor binding region.

[0076] The constant region of the heavy chain of the antibody or antibody fragment may be a human IgG1 isotype having the following amino acid sequence:

[0077]

[0078] The human IgG1 constant region can be modified at amino acid Asn297 (boxed in SEQ ID NO. 69 of the preceding paragraph) to prevent glycosylation of the antibody, e.g., Asn297Ala (N297A). The constant region of the antibody can be modified at amino acid Leu235 (boxed in SEQ ID NO. 69 of the preceding paragraph) to alter Fc receptor interactions, e.g., Leu235Glu (L235E) or Leu235Ala (L235A). The constant region of the antibody can be modified at amino acid Leu234 (boxed in SEQ ID NO. 69 of the preceding paragraph) to alter Fc receptor interactions, e.g., Leu234Ala (L234A). The constant region of the antibody may be altered at both amino acids 234 and 235, e.g., Leu234Ala and Leu235Ala (L234A / L235A). The constant region of the antibody may be modified at amino acid Gly237 (boxed at SEQ ID No. 69 in the preceding paragraph) to alter Fc receptor interactions, e.g., Gly237Ala (G237A). The constant region of the antibody may be altered at amino acids 234, 234, and 237, e.g., Leu234Ala, Leu235Ala, and Gly237Ala (L234A / L235A / G237A). All residue numbers are EU numbering (Kabat, EA, et al. This is based on (1991) Sequences Of Proteins Of Immunological Interest, 5th edition, US Department of Health and Human Services, NIH Publication No. 91-3242).

[0079] The constant region of the antibody's heavy chain may be a human IgG1 isotype having an amino acid sequence:

[0080]

[0081] The human IgG1 constant region can be modified at amino acid Asn297 (boxed in SEQ ID NO. 70 of the preceding paragraph) to prevent glycosylation of the antibody, e.g., Asn297Ala (N297A). The constant region of the antibody can be modified at amino acid Leu235 (boxed in SEQ ID NO. 70 of the preceding paragraph) to alter Fc receptor interactions, e.g., Leu235Glu (L235E) or Leu235Ala (L235A). The constant region of the antibody can be modified at amino acid Leu234 (boxed in SEQ ID NO. 70 of the preceding paragraph) to alter Fc receptor interactions, e.g., Leu234Ala (L234A). The constant region of the antibody may be altered at both amino acids 234 and 235, e.g., Leu234Ala and Leu235Ala (L234A / L235A). The constant region of the antibody may be modified at amino acid Gly237 (boxed at SEQ ID No. 70 in the preceding paragraph) to alter Fc receptor interactions, e.g., Gly237Ala (G237A). The constant region of the antibody may be altered at amino acids 234, 234, and 237, e.g., Leu234Ala, Leu235Ala, and Gly237Ala (L234A / L235A / G237A). All residue numbers are EU numbering (Kabat, EA, et al., This is based on the above literature (supra).

[0082] Human IgG1 constant region C H 3. The domain may be modified to include either a “knob” mutation or a “hole” mutation for heterodimerization with the 2nd invariant region (residual numbers are EU numbering (Kabat, EA, et al.,According to the aforementioned literature). A specific knob mutation is one or more amino acid substitutions to increase the contact surface between two domains by introducing one or more amino acids that provide additional protuberances to the beta-strand structure, and is, for example, one or more knob mutations selected from the group consisting of T366Y, T366W, T394W, and F405A (numbered according to Kabat's EU index). The knob mutation specifically provides a matching (homogeneous) surface to bind to another antibody domain modified to contain one or more hole mutations. The domain having the knob mutation may further contain the substitution N208D / Q295E / N384D / Q418E / N421D.

[0083] A specific hole mutation is one or more amino acid substitutions to increase the contact surface between two domains by introducing one or more amino acids that provide an additional cave of the beta-strand structure, and is one or more hole mutations selected from the group consisting of, for example, T366S, L368A, Y407T, and Y407V (numbered according to Kabat's EU index). The hole mutation specifically provides a matching (cognate) surface to bind to another antibody domain modified to include one or more knob mutations.

[0084] The matching knob-into-hole mutation is, for example, T366Y on one CH3 domain and the matching Y407′ on the second CH3 domain of the CH3 domain pair, referred herein as T366Y / Y407′. Exemplary matching mutations are T366Y / Y407′T; F405A / T394′W; T366Y:F405A / T394′W:Y407′T; T366W / Y407′A; and S354C:T366W / Y349′C:T366′S:L368′A:Y407′V.

[0085] The constant region of the antibody's heavy chain may be a human IgG1 isotype, for example, an allotype of the human IgG1 isotype, for example, an IgG1 G1m3 allotype. An exemplary human IgG1 allotype is Magdelaine-Beuzelin et al. (2009) Pharmacogenet. Genomics 19(5):383-7.

[0086] The constant region of the antibody's heavy chain may be a human IgG2 isotype having the following amino acid sequence:

[0087]

[0088] The human IgG2 constant region can be modified at amino acid Asn297 (boxed at sequence number 71 in the previous paragraph) to prevent glycosylation of the antibody, for example, as Asn297Ala (N297A), where the residue number is EU numbering (Kabat, EA, et al., This is based on the above literature.

[0089] The constant region of the antibody's heavy chain may be a human IgG3 isotype having the following amino acid sequence:

[0090]

[0091] The human IgG3 constant region can be modified at amino acid Asn297 (boxed in SEQ ID No. 72 of the previous paragraph) to prevent glycosylation of the antibody, e.g., Asn297Ala (N297A). The human IgG3 constant region can be modified at amino acid Arg435 (boxed in SEQ ID No. 72 of the previous paragraph) to extend the half-life, e.g., Arg435H (R435H). All residue numbers are EU numbering (Kabat, EA, et al., This is based on the above literature.

[0092] The constant region of the antibody's heavy chain may be a human IgG4 isotype having the following amino acid sequence:

[0093]

[0094] The human IgG4 constant region can be modified within the hinge region to prevent or reduce strand exchange, for example, the human IgG4 constant region is modified at Ser228 (boxed in SEQ ID NO. 73 of the preceding paragraph), e.g., Ser228Pro (S228P). The human IgG4 constant region can be modified at amino acid Leu235 (boxed in SEQ ID NO. 73 of the preceding paragraph) to alter Fc receptor interactions, e.g., Leu235Glu (L235E). The human IgG4 constant region can be modified at both Ser228 and Leu335, e.g., Ser228Pro and Leu235Glu (S228P / L235E). The human IgG4 constant region can be modified at amino acid Asn297 (boxed in SEQ ID No. 73 of the previous paragraph) to prevent glycosylation of the antibody, for example, as Asn297Ala (N297A). All residue numbers are EU numbering (Kabat, EA, et al., This is based on the above literature.

[0095] The human IgG constant region can be modified to enhance FcRn binding. Examples of Fc mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, and Thr256Glu (M252Y, S254T, and T256E, respectively) (Dall'Acqua et al. (2006) J. Biol. Chem. 281(33): 23514-23524), or Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al. (2010) Nature Biotech. 28(2): 157-159). All residue numbers are in EU numbering (Kabat, EA, et al., This is based on the above literature.

[0096] The constant region of the antibody's light chain may be a human kappa constant region, for example, a human kappa constant region having the following amino acid sequence:

[0097] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 74).

[0098] The constant region of the antibody's light chain may be a human kappa constant region, for example, a human kappa constant region having the following amino acid sequence:

[0099] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 75).

[0100] The constant region of the antibody's light chain may be a human lambda constant region, for example, a human lambda constant region having the following amino acid sequence:

[0101] GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC (SEQ ID NO: 76).

[0102] An antibody may be an antibody fragment. An “antibody fragment” comprises only a portion of an intact antibody and generally includes the antigen-binding site of the intact antibody, thereby retaining the ability to bind to an antigen. Examples of antibody fragments covered by this definition include the following: (i) V L , C L , V H and C H1 Fab fragment having a domain; (ii) C H1 Fab' fragment, which is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the domain; (iii) VH and C H Fd fragment having 1 domain; (iv) V H and C H 1 Domain and C H 1. Fd' fragment having one or more cysteine ​​residues at the C-terminus of the domain; (v) V of the monoclonal antibody L and V H Fv fragment having a domain; (vi) V H (vii) a dAb fragment consisting of a domain; (viii) an isolated CDR region; (viii) a divalent fragment F(ab')2 fragment comprising two Fab' fragments connected by a disulfide bridge at a hinge region; (ix) a single-stranded antibody molecule (e.g., single-stranded Fv; scFv); (x) a light chain variable domain (V) in the same polypeptide chain L Heavy chain variable domain (V) connected to ) H “Diabadi” having two antigen-binding sites including ); (xi) a pair of tandem Fd segments (V) forming a pair of antigen-binding regions together with a complementary light chain polypeptide. H -C H 1-V H -C H “Linear antibody” including 1)

[0103] Antibodies can be chimeric antibodies. “Chimeric antibodies” refer to antibodies in which one part of the amino acid sequence of each heavy chain and light chain is homologous to a corresponding sequence within an antibody of a specific species or belonging to a specific class, while the remaining segment of the chain is homologous to a corresponding sequence within another.

[0104] A. monoclonal antibodies

[0105] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific because they are directed toward a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations containing different antibodies directed toward different determinants (epitopes), each monoclonal antibody is directed toward a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized without contamination by other antibodies. The modifier “monoclonal” is not interpreted to require the production of the antibody by any specific method. For example, monoclonal antibodies useful for the present disclosure may be produced by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or by using recombinant DNA methods in bacterial, eukaryotic, or plant cells after single-cell sorting of antigen-specific B cells, antigen-specific plasmablasts responding to infection or immunization, or after capture of linked heavy and light chains from single cells within a bulk sorted antigen-specific collection (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies may also be isolated from a phage antibody library using techniques described, for example, Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0106] Methods for producing various types of monoclonal antibodies, including chimeric and fully human, are well known and highly predictable in the art. For example, the following U.S. patents and patent applications provide a feasible description of such methods: U.S. Patent Application Publications Nos. 2004 / 0126828 and 2002 / 0172677; and U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,196,265; 4,275,149; 4,277,437; 4,366,241; 4,469,797; 4,472,509; 4,606,855; 4,703,003; 4,742,159; 4,767,720; 4,816,567; 4,867,973; 4,938,948; 4,946,778; 5,021,236; 5,164,296; 5,196,066; 5,223,409; 5,403,484; 5,420,253; 5,565,332; 5,571,698; 5,627,052; 5,656,434; 5,770,376; 5,789,208; 5,821,337; 5,844,091; 5,858,657; 5,861,155; 5,871,907; 5,969,108; 6,054,297; 6,165,464; 6,365,157; ​​6,406,867; 6,709,659; 6,709,873; 6,753,407; 6,814,965; 6,849,259; 6,861,572; 6,875,434; and 6,891,024, each of which is incorporated herein by reference.

[0107] B. Single-chain antibodies

[0108] A single-chain variable fragment (scFv) is a fusion of the variable regions of the heavy and light chains of an immunoglobulin linked with a short linker. This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker peptide. This modification generally leaves the specificity unchanged. scFvs can be generated directly from subcloned heavy and light chains derived from hybridomas or B cells. Single-chain variable fragments lack the constant Fc region found in complete antibody molecules and, consequently, the common binding site (e.g., proteins A / G) used for antibody purification. These fragments can often be purified / immobilized using protein L because protein L interacts with the variable region of the kappa light chain.

[0109] Flexible linkers are generally composed of helix- and turn-promoting amino acid residues such as alanine, serine, and glycine. However, other residues can also function. For example, the linker is V H It may have a proline residue at the second residue after the C-terminus and may have arginine and proline abundant at other positions. As another example, the linker may have the sequence GGRGSGGGGSGSGGS (Sequence No. 101) or GGGGSGGGGSGGGGS (Sequence No. 102).

[0110] Single-stranded antibodies can also be produced by combining the receptor light and heavy chains using non-peptide linkers or chemical units. Typically, the light and heavy chains are produced in separate cells, purified, and then linked together in an appropriate manner (i.e., the N-terminus of the heavy chain is attached to the C-terminus of the light chain via an appropriate chemical bridge).

[0111] Crosslinking reagents are used to form molecular bridges that link the functional groups of two different molecules, for example, stabilizers and coagulants. However, it is considered that heteromeric complexes composed of dimers or polymers of the same analogue or different analogues may be formed. To link two different compounds in a stepwise manner, hetero-difunctional crosslinking agents may be used to eliminate the formation of unwanted homopolymers.

[0112] An exemplary hetero-difunctional crosslinker comprises two reactive groups: one reacts with a primary amine group (e.g., N-hydroxysuccinimide) and the other reacts with a thiol group (e.g., pyridyl disulfide, maleimide, halogen, etc.). Through the primary amine reactive group, the crosslinker can react with lysine residue(s) of one protein (e.g., selected antibody or fragment), and through the thiol reactive group, the crosslinker already bound to the first protein reacts with a cysteine ​​residue (free sulfhydryl group) of another protein (e.g., selected agent).

[0113] It is desirable to use a crosslinking agent that has reasonable stability in blood. Numerous types of disulfide-containing linkers are known that can be successfully used to conjugate targeting and therapeutic / preventive agents. Linkers containing sterically disrupted disulfide bonds can be proven to confer greater stability in vivo, thereby preventing the release of targeting peptides before reaching the site of action. Therefore, these linkers constitute a group of linking agents.

[0114] For example, SMPT is a difunctional crosslinker containing a disulfide bond that is “sterically hindered” by an adjacent benzene ring and a methyl group. The steric hindrance of the disulfide bond is believed to function to protect the bond from attack by thioleate anions, such as glutathione, which may be present in tissues and blood, thereby helping to prevent the dissociation of the conjugate before delivery of the attached agent to the target site. Like many other known crosslinkers, SMPT crosslinkers confer the ability to crosslink functional groups such as the SH of cysteine ​​or primary amines (e.g., the epsilon-amino group of lysine). Another possible type of crosslinker includes heterodifunctional photoreactive phenylazides containing cleavable disulfide bonds, such as sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiophorphionate. The N-hydroxy-succinimidyl group reacts with primary amino groups, and phenylazide reacts non-selectively with any amino acid residue (during photolysis).

[0115] In addition to hindered crosslinkers, unhindered linkers may also be used accordingly. Other useful crosslinkers that are not considered to contain or produce protected disulfides include SATA, SPDP, and 2-iminothiolane. The use of these crosslinkers is well understood in the art. Flexible linkers may also be used.

[0116] U.S. Patent 4,680,338 describes a difunctional linker useful for producing conjugates of ligands and amine-containing polymers and / or proteins, particularly a difunctional linker useful for forming antibody conjugates with chelators, drugs, enzymes, detectable labels, etc. U.S. Patents 5,141,648 and 5,563,250 disclose cleavable conjugates comprising unstable bonds that can be cleaved under various mild conditions. These linkers are particularly useful in that the agent of interest can be directly bound to the linker, and cleavage results in the release of the active agent. Specific uses include adding free amino groups or free sulfhydryl groups to proteins such as antibodies, or to drugs.

[0117] U.S. Patent 5,856,456 provides a peptide linker for use in linking polypeptide components to produce fusion proteins, e.g., single-strand antibodies. The linker has a length of up to about 50 amino acids and includes at least one occurrence of a proline-followed charged amino acid (preferably arginine or lysine), and is characterized by greater stability and reduced aggregation. U.S. Patent 5,880,270 discloses an aminooxy-containing linker useful in various immunodiagnostic and isolation technologies.

[0118] C. Bispecific and multispecific antibodies

[0119] Antibodies may be bispecific or multispecific. A “bispecific antibody” is an antibody having binding specificity for at least two different epitopes. Such an antibody combines a first binding site for a first epitope with a second binding site for a second epitope. The first epitope and the second epitope may be two different epitopes of a single antigen. Such an antibody may be called a biparatope. Alternatively, the first epitope and the second epitope may be on two different antigens. Bispecific antibodies may be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0120] Methods for manufacturing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-to-light chain pairs, where the two chains have different specificities. Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, typically performed by an affinity chromatography step, is somewhat cumbersome, and the yield of the product is low.

[0121] According to another approach, an antibody variable region having the desired binding specificity (antibody-antigen combination site) is fused to the immunoglobulin constant domain sequence. Preferably, the fusion is hinged, C H2 and C H3 It is a fusion with an Ig heavy chain constant domain comprising at least a portion of the region. A first heavy chain constant region (C) comprising a site necessary for light chain binding. H1It is preferable that ) be present in at least one of the fusions. The immunoglobulin heavy chain fusion and, if desired, the DNA encoding the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into suitable host cells. This provides greater flexibility in adjusting the mutual ratios of the three polypeptide fragments when the unequal ratio of the three polypeptide chains used in the composition provides the optimal yield of the desired bispecific antibody. However, if expression of at least two polypeptide chains in equal proportions results in a high yield or the ratio does not significantly affect the yield of the desired chain combination, it is possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector.

[0122] A bispecific antibody may consist of a hybrid immunoglobulin heavy chain having first binding specificity to one arm, and a hybrid immunoglobulin heavy-to-light chain pair (providing second binding specificity) to the other arm. This asymmetric structure facilitates the separation of the desired bispecific compound from the unwanted immunoglobulin chain combination, as it provides a method of easy separation where the immunoglobulin light chain is present in only half of the bispecific molecule. This approach is disclosed in WO 94 / 04690. For further details on generating bispecific antibodies, for example, Suresh et al. See , Methods in Enzymology, 121:210 (1986).

[0123] According to another approach described in U.S. Patent No. 5,731,168, the interface between a pair of antibody molecules can be manipulated to maximize the percentage of heteromers recovered from a recombinant cell culture. A preferred interface is C H3It includes at least a portion of the domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). A compensatory “cavity” of the same or similar size to the large side chain(s) is formed on the interface of the second antibody molecule by replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism to increase the yield of the heterodimer compared to other unwanted end-products, such as the homodimer.

[0124] Bispecific antibodies include cross-linked or “heteroconjugated” antibodies. For example, one of the antibodies in the heteroconjugation may be coupled to avidin, and the other may be coupled to biotin. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980). Heteroconjugated antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980 along with a number of cross-linking techniques.

[0125] Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be prepared using chemical linkages. Brennan et al., Science, 229: 81 (1985) describes a procedure in which an intact antibody is proteolytically cleaved to produce an F(ab')2 fragment. This fragment is reduced in the presence of sodium arsenite, a dithiol complexing agent, to stabilize adjacent dithiols and prevent the formation of intermolecular disulfides. The resulting Fab' fragment is subsequently converted into a thionitrobenzoate (TNB) derivative. One of the Fab'-TNB derivatives is then reconverted to Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody. The produced bispecific antibody can be used as a preparation for the selective immobilization of an enzyme.

[0126] E. coli ( E. coli Techniques exist to facilitate the direct recovery of Fab'-SH fragments from ), which can be chemically coupled to form bispecific antibodies. Shalaby et al. J. Exp. Med., 175: 217-225 (1992) describes the production of a bispecific antibody F(ab')₂ molecule. Each Fab' fragment was secreted separately from E. coli and formed a bispecific antibody through targeted chemical coupling in vitro. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as induce lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0127] Various techniques for directly preparing and isolating bispecific antibody fragments from recombinant cell cultures are also described (Merchant et al. , Nat. Biotechnol . 16, 677-681 (1998)). For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., J. Immunol., 148(5):1547-1553, 1992). Leucine zipper peptides from Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homomers were reduced at the hinge region to form monomers, and then reoxidized to form antibody heteromers. This method can also be utilized for the production of antibody homomers. Hollinger et al. The “diabadi” technique described by , Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) provided an alternative mechanism for manufacturing bispecific antibody fragments. The fragment is V by a linker that is too short to allow pairing between two domains on the same chain. L V connected to H It includes. Therefore, one fragment's V H and V L The domain is the complementary V of another fragment L and V H It is forced to pair with the domain, thereby forming two antigen-binding sites. Another strategy for preparing bispecific antibody fragments by using single-stranded Fv (sFv) dimers has also been reported. Gruber et al. See , J. Immunol., 152:5368 (1994).

[0128] Bispecific or multispecific antibodies may be formed as DOCK-AND-LOCK™ (DNL™) complexes (see, e.g., U.S. Patent Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143 and 7,666,400). Generally, this technique utilizes specific, high-affinity binding interactions occurring between the dimerization and docking domain (DDD) sequence of the regulatory (R) subunit of cAMP-dependent protein kinase (PKA) and the anchor domain (AD) sequence derived from any of the various AKAP proteins (Baillie et al. , FEBS Letters. 2005; 579:3264; Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004; 5: 959). DDD and AD peptides can be attached to any protein, peptide, or other molecule. Because the DDD sequence spontaneously dimerizes and binds to the AD sequence, this technique allows for the formation of a complex between any selected molecule that can be attached to the DDD or AD sequence.

[0129] Antibodies having a valence of more than two are considered. For example, triple-specific antibodies can be prepared (Tutt et al. , J. Immunol. 147:60, 1991; Xu et al. , Science Antibodies may also contain sequences or moietyes that allow for the dimerization or multimerization of the receptor. These sequences include those derived from IgA that allow for the formation of a multimer along with a J-chain. Another multimerization domain is the Gal4 dimerization domain.

[0130] Polyvalent antibodies can be internalized (and / or catabolized) more rapidly than divalent antibodies by cells expressing the antigen to which the antibody binds. The antibody of the present disclosure may be a polyvalent antibody having three or more antigen-binding sites (e.g., a tetravalent antibody), which can be readily produced by the recombination expression of a nucleic acid encoding the polypeptide chain of the antibody. The polyvalent antibody may comprise a dimerization domain and three or more antigen-binding sites. A preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen-binding sites that are amino-termined to the Fc region. The polyvalent antibody may comprise (or consist of) three to about eight, e.g., four antigen-binding sites. The polyvalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable regions. For example, polypeptide chain(s) are VD1-(X1).sub.n-VD2-(X2) n It may include -Fc, where VD1 is a first variable region and VD2 is a second variable region, Fc is one polypeptide chain of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, polypeptide chain(s) are V H -C H 1-Gayosung Linker-V H -C H 1-Fc region chain; or V H -C H 1-V H -C HIt may include a 1-Fc region chain. The polyvalent antibody of the present invention may further include at least two (and preferably four) light chain variable region polypeptides. The polyvalent antibody of the present invention may include, for example, about two to about eight light chain variable region polypeptides. The light chain variable region polypeptides considered herein include a light chain variable region and optionally C L Includes additional domains.

[0131] Charge modification is particularly useful in the context of multispecific antibodies, where amino acid substitution in the Fab molecule affects V in one of their binding arms (or one or more in the case of molecules containing more than two antigen-binding Fab molecules). H / V L This results in a reduction of mispairing of mismatched heavy and light chains (Bence-Jones-type byproducts) that may occur in the production of Fab-based dual- / multispecific antigen-binding molecules having exchanges (see also PCT publication number WO 2015 / 150447, in particular the examples contained therein, the whole of which is incorporated herein by reference).

[0132] D. antibody conjugate

[0133] The antibody of the present disclosure may be linked to at least one formulation to form an antibody conjugate. The conjugate may be, for example, an antibody conjugated to another proteinaceous, carbohydrate, lipid, or mixed moiety molecule(s). Such antibody conjugates include, but are not limited to, modifications comprising linking the antibody to one or more polymers. For example, the antibody may be linked to one or more water-soluble polymers. Linking to water-soluble polymers reduces the likelihood of the antibody precipitating in an aqueous environment, such as a physiological environment. Those skilled in the art may select a suitable water-soluble polymer based on considerations including, but not limited to, whether the polymer / antibody conjugate will be used for the treatment of a patient and, if so, the pharmacological profile of the antibody (e.g., half-life, dosage, activity, antigenicity, and / or other factors).

[0134] To increase the efficacy of antibody molecules as diagnostic agents or therapeutic agents, it is common practice to link, covalently bond, or complex at least one desired molecule or moiety. Such molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules include molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules attached to antibodies include toxins, anti-tumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or polynucleotides. In contrast, reporter molecules are defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, enzymes (e.g., catalyzing colorimetric or fluorometric reactions), substrates, and solid matrices such as biotin. The antibody may contain one, two, or more of any of these labels.

[0135] Antibody conjugates are also used as diagnostic agents. Antibody diagnostics are generally classified into two classes: those intended for use in in vitro diagnostics, such as in various immunoassays, and those intended for use in in vivo diagnostic protocols, generally known as “antibody-directed imaging.” Many suitable imaging agents are known in the art, and methods for attaching them to antibodies are also known (see, for example, U.S. Patents 5,021,236, 4,938,948 and 4,472,509). The imaging moiety used may be a paramagnetic ion, a radioisotope, a fluorescent dye, an NMR-detectable material, and an X-ray imaging agent.

[0136] Paramagnetic ions considered for use as conjugates include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and / or erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include, but are not limited to, lanthanum (III), gold (III), lead (II), and bismuth (III).

[0137] The radioisotope being considered for use as a conjugate is astatin 211 , 14 carbon, 51 chrome, 36 chlorine, 57 cobalt, 58 Cobalt, copper 67 , 152 Eu, Gallium 67 , 3 Hydrogen, iodine 123 , iodine 125 , iodine 131 , indium 111 , 59 steel, 32 Phosphorus, rhenium 186 , rhenium 188 , 75 Selenium, 35 Sulfur, Technetium 99m and / or yttrium 90 Includes 125 I is often desirable. Technetium 99m and / or indium 111These are also often preferred due to their suitability for low-energy and long-range detection. The radiolabeled monoclonal antibodies of the present disclosure can be produced according to methods well known in the art. For example, the monoclonal antibodies may be iodinated by contact with chemical oxidizers such as sodium and / or potassium iodide and sodium hypochlorite, or enzymatic oxidizers such as lactoperoxidase. The monoclonal antibodies according to the present disclosure may be produced by a ligand exchange process, for example, by reducing pertechnate with a tin solution, chelating the reduced technetium on a Sephadex column, and applying the antibody to this column. 99m It can be labeled as such. Alternatively, direct labeling techniques may be used, for example, by incubating the antibody with a reducing agent such as pertechnetate, SNCl2, a buffer solution such as sodium-potassium phthalate solution. The intermediate functional group often used to bind radioisotopes existing as metal ions to antibodies is diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0138] Fluorescent labels considered for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and / or Texas Red.

[0139] Additional types of antibodies considered in this disclosure are intended primarily for in vitro use, wherein the antibody is linked to an enzyme (enzyme tag) that produces a colored product upon contact with a secondary binding ligand and / or a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxide, or glucose oxidase. Preferred secondary binding ligands are biotin and avidin and streptavidin compounds.

[0140] Several methods for attaching or conjugating antibodies to their conjugate moiety are known in the art. Some attachment methods involve the use of metal chelate complexes using organic chelating agents, such as, for example, diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3α-6α-diphenylglycuryl-3 attached to the antibody (U.S. Patents 4,472,509 and 4,938,948). Monoclonal antibodies may also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodates. Conjugates with fluorescein markers are prepared in the presence of such coupling agents or by reaction with isothiocyanates. In U.S. Patent 4,938,948, imaging of a breast tumor is achieved using a monoclonal antibody, and a detectable imaging moiety is bound to the antibody using a linker such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.

[0141] Another known method for site-specifically attaching molecules to antibodies involves the reaction of antibodies with hapten-based affinity labels. Essentially, hapten-based affinity labels react with amino acids within the antigen-binding site, thereby disrupting the site and blocking the specific antigen response. However, this may not be advantageous as it results in the loss of antigen binding by the antibody conjugate.

[0142] Molecules containing an azido group can also be used to form covalent bonds to proteins through reactive nitrene intermediates generated by low-intensity ultraviolet light. In particular, 2- and 8-azido analogs of purine nucleotides have been used as site-directed optical probes to identify nucleotide-binding proteins in zygous cell extracts. 2- and 8-azido nucleotides have also been used to map the nucleotide-binding domains of purified proteins and can be used as antibody binding agents.

[0143] Derivatization of immunoglobulins by selectively introducing sulfhydryl groups into the Fc region of immunoglobulins using reaction conditions that do not alter the antibody binding site is also considered. Antibody conjugates produced according to this methodology are disclosed to exhibit improved lifetime, specificity, and sensitivity (U.S. Patent 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, in which the reporter or effector molecule is conjugated to a carbohydrate residue within the Fc region, is also disclosed in the literature. This approach is reported to produce antibodies that are diagnostically and therapeutically promising and are currently undergoing clinical evaluation.

[0144] E. Production and purification of antibodies

[0145] The method for generating monoclonal antibodies generally starts along the same lines as the method for manufacturing polyclonal antibodies. The first step for both of these methods is the immunization of an appropriate host. As is well known in the art, a given composition for immunization can vary in its immunogenicity. Therefore, it is often necessary to enhance the host immune system, which can be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins, such as ovalbumin, mouse serum albumin, or rabbit serum albumin, may also be used as carriers. Means of conjugating polypeptides to carrier proteins are well known in the art and include glutaraldehyde, m-maleimidovencoyl-N-hydroxysuccinimide ester, carbodiimide, and bis-biazotized benzidine. Also, as is well known in the art, the immunogenicity of a specific immunogenic composition can be enhanced by the use of a non-specific stimulant of the immune response known as an adjuvant. An exemplary and preferred adjuvant in animals is a complete Freund adjuvant (killed Mycobacterium tuberculosis ( Mycobacterium tuberculosisIt includes non-specific stimulants of immune responses including ), incomplete Freund adjuvants and aluminum hydroxide adjuvants, and in humans, a combination of alum, CpG, MFP59, and immunostimulatory molecules (such as “adjuvant systems”). Additional experimental forms of inoculation for inducing antigen-specific B cells are possible, which include a gene-coding antigen delivered as a DNA or RNA gene in a nanoparticle vaccine or physical delivery system (e.g., on lipid nanoparticles or gold biolistic beads) and delivered via a needle, gene gun, or transdermal electroporation device. The antigen gene may also be carried as encoded by a replicable or defective viral vector such as an adenovirus, adeno-associated virus, poxvirus, herpesvirus, or alphavirus replicon, or alternatively by a virus-like particle.

[0146] Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells typically involve mixing somatic cells with myeloma cells in a 2:1 ratio in the presence of agents or agents (chemical or electrical) that promote cell membrane fusion, although the ratio may vary from about 20:1 to about 1:1. In some cases, as an initial step, transformation of human B cells with Epstein-Barr virus (EBV) increases the size of the B cells, thereby enhancing fusion with relatively large myeloma cells. The efficiency of EBV transformation is enhanced by using CpG and Chk2 inhibitor drugs in the transformation medium. Alternatively, human B cells can be activated by co-culture with a transfected cell line expressing the CD40 ligand (CD154) in a medium containing additional soluble factors such as IL-21 and human B cell activating factor (BAFF), a type II member of the TNF superfamily. Fusion methods using Sendai virus or polyethylene glycol (PEG) are also known. The use of electrically induced fusion methods is also appropriate. The fusion procedure typically involves about 1 x 10⁻³ -6 Up to 1 x 10 -8Although viable hybrids are produced at a low frequency, fusion efficiency close to 1 in 200 can be achieved through optimized procedures. However, the relatively low efficiency of fusion does not cause problems, as viable, fused hybrids are distinguished from the parent's unfused cells (especially unfused myeloma cells that would normally divide indefinitely) by culturing them in a selective medium. Selective media generally contain agents that block the novel synthesis of nucleotides in tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azacerin. Aminopterin and methotrexate block the novel synthesis of both purines and pyrimidines, whereas azacerin blocks purine synthesis only. When aminopterin or methotrexate is used, the medium is supplemented with hypoxanthine and thymidine as sources of nucleotides (HAT medium). When azacerin is used, the medium is supplemented with hypoxanthine. If the B cell source is an EBV-transformed human B cell line, Ouabain is added to remove the EBV-transformed cell line that is not fused to the myeloma.

[0147] The preferred selection medium is HAT or HAT containing auabain. Only cells capable of operating the nucleotide salvage pathway can survive in HAT medium. Myeloma cells cannot survive because they are defective in key enzymes of the salvage pathway, such as hypoxanthine phosphoribosyl transferase (HPRT). B cells can operate this pathway, but they have a limited lifespan in culture and generally die within about two weeks. Therefore, the only cells capable of surviving in the selection medium are hybrids formed from myeloma and B cells. When the source of B cells used for fusion is a cell line of EBV-transformed B cells as in the present invention, auabain can also be used for drug selection of the hybrid, because EBV-transformed B cells are susceptible to drug killing, whereas the myeloma partner used is selected to be resistant to auabain.

[0148] Culture provides a population of hybridomas from which specific hybridomas are selected. Typically, hybridoma selection is performed by culturing cells in monoclonal dilutions on microtiter plates and then testing individual clonal supernatants for desired reactivity (after about 2 to 3 weeks). The analysis must be sensitive, simple, and rapid, such as radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, and point immunocouplement assay. The selected hybridomas are subsequently serially diluted or sorted by flow cytometry and cloned into individual antibody-producing cell lines; these clones can then be proliferated indefinitely to provide monoclonal antibodies. Cell lines can be utilized for monoclonal antibody production in two basic ways. Samples of the hybridoma can be injected into animals (e.g., mice) (often into the peritoneal cavity). Optionally, the animals are primed with hydrocarbons, particularly oils such as pristane (tetramethylpentadecane), prior to injection. When human hybridomas are used in this manner, it is optimal to inject them into immunodeficient mice, such as SCID mice, to prevent tumor rejection. The injected animals develop tumors that secrete specific monoclonal antibodies produced by the fused cell hybrids. Animal body fluids, such as serum or ascites fluid, can then be collected to provide high concentrations of monoclonal antibodies. Individual cell lines can also be cultured in vitro, where monoclonal antibodies are naturally secreted into the culture medium, from which they can be readily obtained at high concentrations. Alternatively, human hybridoma cell lines can be used in vitro to produce immunoglobulins from the cell supernatant. Cell lines can be adapted for growth in serum-free media to optimize the ability to recover high-purity human monoclonal immunoglobulins.

[0149] After culturing the hybridoma, the cells are lysed, and total RNA can be extracted. Random hexamers can be used with RT to generate cDNA copies of the RNA, and then PCR can be performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. The PCR product is cloned into the pGEM-T Easy vector and then sequenced by automated DNA sequencing using standard vector primers. Binding and neutralization analyses can be performed using antibodies collected from the hybridoma supernatant and purified by FPLC using a Protein G column.

[0150] Recombinant full-length IgG antibodies can be produced by subcloning heavy and light chain Fv DNA from a cloning vector into an IgG plasmid vector and transfecting 293 (e.g., Freestyle) cells or CHO cells, and the antibodies can be collected and purified from the supernatant of 293 or CHO cells. Other suitable host cell systems include bacteria such as E. coli, insect cells (S2, Sf9, Sf29, High Five), plant cells (e.g., tobacco with or without manipulation for human-like glycans), birds, or various non-human transgenic contexts such as mice, rats, goats, or cattle.

[0151] For both the purpose of subsequent antibody purification and host immunization, the expression of the nucleic acid encoding the antibody is also considered. The antibody-coding sequence can be RNA such as natural RNA or modified RNA. Modified RNA refers to specific chemical modifications that confer increased stability and low immunogenicity to the mRNA, thereby facilitating the expression of therapeutically important proteins. For example, N1-methyl-pseudouridine (N1mΨ) is superior to various other nucleoside modifications and combinations thereof in terms of translational capacity. In addition to disabling the immuno- / eIF2α phosphorylation-dependent inhibition of translation, the introduced N1mΨ nucleotides dramatically alter the kinetics of the translation process by increasing ribosomal arrest and density on the mRNA. The increased ribosomal loading of the modified mRNA makes them more receptive to initiation by favoring either the recycling of ribosomes or the recruitment of new ribosomes on the same mRNA. Such modifications can be used to enhance antibody expression in vivo after inoculation with RNA. RNA can be delivered as naked RNA or in delivery vehicles such as lipid nanoparticles, whether natural or modified.

[0152] Alternatively, DNA encoding antibodies can be used for the same purpose. The DNA is contained in an expression cassette that includes a promoter active in the host cell in which it is designed. The expression cassette is advantageously contained in a replicable vector, such as a conventional plasmid or minivector. Vectors are considered to include viral vectors such as poxviruses, adenoviruses, herpesviruses, adeno-associated viruses, and lentiviruses. Replicons encoding antibody genes, such as alphavirus replicons based on VEE viruses or Sindbis viruses, are also considered. Delivery of these vectors can be performed via needle, intramuscular, subcutaneous, or intradermal routes, or by transdermal electroporation, if in vivo expression is desired.

[0153] Alternatively, molecular cloning approaches can be used to generate monoclonal antibodies. Single B cells labeled with the antigen of interest can be physically sorted using paramagnetic bead selection or flow cytometry sorting, after which RNA can be isolated from the single cells and antibody genes can be amplified by RT-PCR. Alternatively, the antigen-specific bulk sorted cell population can be separated into microvesicles, and the matched heavy and light chain variable genes from the single cells can be recovered using the physical linkage of heavy and light chain amplicons or common barcoding of the heavy and light chain genes from the vesicles. The matched heavy and light chain genes from single cells can also be obtained from a population of antigen-specific B cells by treating the cells with cell-penetrating nanoparticles containing RT-PCR primers and barcodes to label transcripts with one barcode per cell. Antibody variable genes can also be isolated by RNA extraction from hybridoma cell lines, and antibody genes can be obtained by RT-PCR and cloned into immunoglobulin expression vectors. Alternatively, a combination immunoglobulin phagemid library is prepared from RNA isolated from cell lines, and phagemids expressing appropriate antibodies are selected by panning using viral antigens. Compared to conventional hybridoma techniques, the advantage of this approach is approximately 10 in a single round. 4 The fact is that many antibodies can be produced and screened, and that new specificity is generated by the combination of H and L chains, further increasing the possibility of finding a suitable antibody.

[0154] Other U.S. patents, each incorporated herein by reference, that teach the production of antibodies useful in the present disclosure include U.S. Patent 5,565,332, which describes the production of chimeric antibodies using a combination approach; U.S. Patent 4,816,567, which describes recombinant immunoglobulin preparations; and U.S. Patent 4,867,973, which describes antibody-therapeutic conjugates.

[0155] Monoclonal antibodies produced by any means may be purified, if desired, by filtration, centrifugation, and various chromatographic methods such as FPLC or affinity chromatography. Fragments of the monoclonal antibodies of the present disclosure may be obtained from monoclonal antibodies purified by methods including digestion using enzymes such as pepsin or papain, and / or cleavage of disulfide bonds by chemical reduction. Alternatively, the monoclonal antibody fragments covered by the present disclosure may be synthesized using an automated peptide synthesizer.

[0156] The antibodies of the present disclosure may be purified. The term “purified” as used herein is intended to refer to a composition that can be isolated from other components, wherein the protein is purified to any degree relative to its naturally obtainable state. Thus, purified protein also refers to a protein that is free from the environment in which it may naturally occur. Where the term “substantially purified” is used, this designation will refer to a composition in which a protein or peptide forms the major component of the composition, for example, a composition comprising about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the protein in the composition.

[0157] Protein purification techniques are well known to those skilled in the art. At one level, these techniques involve crude fractionation of the cellular environment into polypeptide and non-polypeptide fractions. After separating polypeptides from other proteins, polypeptides of interest may be further purified using chromatography and electrophoresis techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for the preparation of pure peptides are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; and isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies, etc., or centrifugation following heat denaturation; gel filtration, reverse phase, hydroxyapatite, and affinity chromatography; and combinations of these techniques and other techniques.

[0158] In purifying the antibody of the present disclosure, it may be preferable to express the polypeptide in a prokaryotic or eukaryotic expression system and to extract the protein using denaturation conditions. The polypeptide may be purified from other cellular components using an affinity column that binds to the tagged portion of the polypeptide. As is generally known in the art, it is believed that the order of various purification steps may be changed, or certain steps may be omitted, and that this may still result in a suitable method for producing substantially purified proteins or peptides.

[0159] Typically, complete antibodies are fractionated using a preparation (i.e., protein A) that binds to the Fc portion of the antibody. Alternatively, antigens can be used to simultaneously purify and select appropriate antibodies. These methods often utilize a selection preparation bound to a support such as a column, filter, or bead. The antibody is bound to the support, contaminants are removed (e.g., by washing), and the antibody is released by applying conditions (salt, heat, etc.).

[0160] Various methods for quantifying the degree of purification of a protein or peptide will be known to those skilled in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction or evaluating the amount of polypeptide in the fraction by SDS / PAGE analysis. Another method for evaluating the purity of a fraction is to calculate the specific activity of the fraction, compare it to the specific activity of the initial extract, and thereby calculate the degree of purity. The actual unit used to express the amount of activity will depend on the specific analytical technique selected to track purification, as well as whether the expressed protein or peptide exhibits detectable activity.

[0161] It is known that polypeptide migration can sometimes vary significantly depending on different SDS / PAGE conditions. Therefore, it will be understood that the apparent molecular weight of purified or partially purified expression products may vary under different electrophoretic conditions.

[0162] F. Antibody modification

[0163] The sequence of the antibody may be modified for various reasons, such as improved expression, improved cross-reactivity, or reduced off-target binding. The modified antibody may be prepared by any technique known to those skilled in the art, including expression through standard molecular biological techniques or chemical synthesis of the polypeptide.

[0164] For example, one may wish to perform modifications, such as introducing conservative changes into antibody molecules. In performing such changes, the hydrophobicity index of amino acids may be considered. The importance of the hydrophobic amino acid index in conferring interactive biological functions to proteins is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydrophobicity of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interactions between the protein and other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0165] Substitution of similar amino acids can be effectively achieved based on hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local mean hydrophilicity of a protein, governed by the hydrophilicity of adjacent amino acids, correlates with the biological properties of the protein. As described in detail in U.S. Patent 4,554,101, the following hydrophilic values ​​are assigned to amino acid residues: Basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); Acidic amino acids: aspartate (+3.0 ± 1), glutamate (+3.0 ± 1), asparagine (+0.2), and glutamine (+0.2); Hydrophilic, nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4); Sulfur-containing amino acids: cysteine ​​(-1.0) and methionine (-1.3); Hydrophobic, non-aromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5 ± 1), alanine (-0.5), and glycine (0); hydrophobic, aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5), and tyrosine (-2.3).

[0166] Amino acids can be substituted with other amino acids having similar hydrophilicity, and biologically or immunologically modified proteins can be produced. In such modifications, substitution with amino acids having a hydrophilicity value within ± 2 is preferred, within ± 1 is particularly preferred, and within ± 0.5 is much more particularly preferred.

[0167] Amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions considering the various characteristics mentioned above are well known to those skilled in the art and include arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0168] The present disclosure also considers isotype modifications. Different functionalities can be achieved by modifying the Fc region to have different isotypes. For example, changing to IgG1 can increase antibody-dependent cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve valence.

[0169] The Fc region of an antibody having a modified effector function can be designed, for example, by modifying C1q binding and / or FcγR binding and thereby altering CDC activity and / or ADCC activity. “Effector function” is responsible for activating or reducing biological activity (e.g., in a target). Examples of effector functions include, but are not limited to, C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptors; BCR). Such effector functions may require the Fc region to be combined with a binding domain (e.g., antibody variable domain) and can be evaluated using various assays (e.g., Fc binding assay, ADCC assay, CDC assay, etc.).

[0170] For example, a variant Fc region of an antibody having improved C1q binding and improved FcγRIII binding (e.g., having both improved ADCC activity and improved CDC activity) can be generated. Alternatively, if it is desired that the effector function be reduced or eliminated, a variant Fc region having reduced CDC activity and / or reduced ADCC activity can be engineered. In other embodiments, only one of these activities may be increased, and optionally, the other activity may also be reduced (e.g., generating an Fc region variant having improved ADCC activity but reduced CDC activity, and vice versa).

[0171] The isolated monoclonal antibody, or its antigen-binding fragment, may comprise a substantially homogeneous glycan without sialic acid, galactose, or fucose. The aforementioned substantially homogeneous glycan may be covalently attached to a heavy chain constant region.

[0172] Monoclonal antibodies may possess novel Fc glycosylation patterns. Glycosylation of the Fc region is typically either N-linking or O-linking. N-linking refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. O-linking glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. The recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain peptide sequence are asparagine-X-serine and asparagine-X-threonine, where X is any amino acid excluding proline. Therefore, the presence of either of these peptide sequences in the polypeptide creates a potential glycosylation site.

[0173] The glycosylation pattern may be modified, for example, by deleting one or more glycosylation sites found in the polypeptide and / or by adding one or more glycosylation sites not present in the polypeptide. The addition of a glycosylation site to the Fc region of the antibody (in the case of an N-linked glycosylation site) is conveniently achieved by modifying the amino acid sequence to include one or more of the tripeptide sequences described above. An exemplary glycosylation variant has an amino acid substitution of the heavy chain residue Asn 297. The modification may also be made by adding one or more serine or threonine residues to the sequence of the original polypeptide (in the case of an O-linked glycosylation site), or by substitution. Additionally, a change of Asn 297 to Ala may remove one of the glycosylation sites.

[0174] Isolated monoclonal antibodies, or their antigen-binding fragments, may be present in a substantially homogeneous composition represented by GNGN or G1 / G2 glycoforms, which exhibit increased binding affinity for Fc gamma RI and Fc gamma RIII compared to the same antibodies that lack a substantially homogeneous GNGN glycoform and possess glycoforms containing G0, G1F, G2F, GNF, GNGNF, or GNGNFX. Fc glycosylation plays a significant role in the antiviral and anticancer properties of therapeutic mAbs. The removal of core fucose dramatically improves the ADCC activity of mAbs mediated by natural killer (NK) cells, but appears to have the opposite effect on the ADCC activity of polymorphonuclear (PMN) cells.

[0175] Isolated monoclonal antibodies, or their antigen-binding fragments, can be expressed in cells expressing beta(1,4)-N-acetylglucosaminyltransferase III (GnT III), thereby causing GnT III to add GlcNAc to the antibody. Methods for producing antibodies in this manner are disclosed in WO / 9954342 and WO / 03011878. Cell lines can be modified using genome editing technologies, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), to enhance, reduce, or eliminate specific post-translational modifications, such as glycosylation. For example, CRISPR technology can be used to remove genes encoding glycosylation enzymes in 293 or CHO cells used to express monoclonal antibodies.

[0176] Antibody variable gene sequences obtained from human B cells can be manipulated to enhance their manufacturability and safety. Potential protein sequence fragility can be identified by searching for sequence motifs associated with regions including the following:

[0177] 1) Unpaired Cys residue,

[0178] 2) N-linked glycosylation,

[0179] 3) Asn deamidation,

[0180] 4) Asp isomerization,

[0181] 5) SYE cutting,

[0182] 6) Met oxidation,

[0183] 7) Trp oxidation,

[0184] 8) N-terminal glutamate,

[0185] 9) Integrin bond,

[0186] 10) CD11c / CD18 combined, or

[0187] 11) Fragmentation

[0188] These motifs can be removed by modifying synthetic genes containing cDNA that encode antibodies.

[0189] Antibodies can be engineered to improve solubility. For example, some hydrophilic residues, such as aspartic acid, glutamic acid, and serine, contribute significantly more favorably to protein solubility than other hydrophilic residues, such as asparagine, glutamine, threonine, lysine, and arginine.

[0190] Deep sequencing of the human B cell repertoire from blood donors has been performed on an extensive scale. Sequence information on a significant portion of the human antibody repertoire facilitates the statistical evaluation of antibody sequence characteristics common to healthy humans. Based on knowledge of antibody sequence characteristics within the Human Recombinant Antibody Variable Gene Reference Database, the degree of position-specific “human similarity” (HL) of antibody sequences can be estimated. HL has been shown to be useful in the development of antibodies for clinical use, such as therapeutic antibodies or antibodies as vaccines. The goal is to increase the human similarity of antibodies to reduce potential side effects and anti-antibody immune responses that could significantly diminish the efficacy of antibody drugs or induce serious health effects. Antibody characteristics of the combined antibody repertoire from three healthy human blood donors, totaling approximately 400 million sequences, could be evaluated, and a novel “Relative Human Similarity” (rHL) score was generated focusing on the hypervariable regions of antibodies. The rHL score enables easy distinction between human (positive score) and non-human sequences (negative score). Antibodies can be engineered to remove residues that are uncommon in the human repertoire.

[0191] G. Characterization of antibodies

[0192] Antibodies according to the present disclosure may first be defined by their binding specificity. A person skilled in the art may determine whether such antibodies fall within the scope of the present claims by evaluating the binding specificity / affinity of a given antibody using techniques well known to a person skilled in the art. For example, the epitope to which a given antibody binds may consist of a single adjacent sequence of three or more amino acids (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) located within the antigen molecule (e.g., a linear epitope within a domain). Alternatively, the epitope may consist of a plurality of non-adjacent amino acids (or amino acid sequences) located within the antigen molecule (e.g., a stereochemical epitope).

[0193] Various techniques known to those skilled in the art may be used to determine whether an antibody interacts with “one or more amino acids” within a polypeptide or protein. Exemplary techniques include conventional cross-blocking assays, such as those described in *Antibodies, Harlow and Lane* (Cold Spring Harbor Press, Cold Spring Harbor, NY). Cross-blocking can be measured in various binding assays, such as ELISA, biolayer interferometry, or surface plasmon resonance. Other methods include alanine scanning mutation assays, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63), peptide cleavage assays, single-particle reconstruction, high-resolution electron microscopy techniques using cryoEM or tomography, crystallographic studies, and NMR analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of the antigen may be used (Tomer (2000) Prot. Sci. 9: 487-496). Another method that can be used to identify amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange, detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves deuterium-labeling the protein of interest and then binding an antibody to the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water, and exchangeable protons within the amino acids protected by the antibody complex undergo deuterium-to-hydrogen back-exchange at a slower rate than exchangeable protons within amino acids that are not part of the interface. Consequently, amino acids forming part of the protein / antibody interface can retain deuterium and thus exhibit a relatively higher mass compared to amino acids not included in the interface. After the antibody dissociates, the target protein undergoes protease cleavage and mass spectrometric analysis, thereby revealing deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts.For example, see Ehring (1999) Analytical Biochemistry 267: 252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A.

[0194] The term “epitope” refers to a region on an antigen to which B cells and / or T cells respond. B-cell epitopes can be formed from both adjacent amino acids and non-adjacent amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed from adjacent amino acids are generally retained when exposed to a denaturing solvent, whereas epitopes formed by tertiary folding are generally lost when treated with a denaturing solvent. Epitopes typically comprise at least three, and more typically at least five or eight to ten amino acids, in a unique spatial stereostructure.

[0195] Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP), is a method for classifying a large number of monoclonal antibodies directed toward the same antigen based on the similarity of the binding profiles of each antibody to a chemically or enzymatically modified antigen surface (see US 2004 / 0101920, the whole of which is specifically incorporated herein by reference). Each category may reflect a unique epitope that is clearly different from or partially overlaps with the epitope expressed by another category. This technique allows for the rapid filtering of genetically identical antibodies, enabling characterization to focus on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce monoclonal antibodies with desired characteristics. MAP can be used to classify the antibodies of the present disclosure into groups of antibodies that bind to different epitopes.

[0196] The present disclosure comprises an antibody capable of binding to the same epitope, or a portion of the same epitope. Whether an antibody binds to the same epitope as a reference antibody, or competes for binding, can be easily determined using conventional methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference antibody, the reference antibody is allowed to bind to a target molecule under saturation conditions. Next, the ability of the test antibody to bind to the target molecule is evaluated. If the test antibody is able to bind to the target molecule after saturation binding with the reference antibody, it can be concluded that the test antibody binds to an epitope different from that of the reference antibody. On the other hand, if the test antibody is unable to bind to the target molecule after saturation binding with the reference antibody, the test antibody may bind to the same epitope as the epitope bound by the reference antibody.

[0197] To determine whether the test antibody competes for binding with the initiating antibody, the binding methodology described above is performed in two directions: in the first direction, the binding of the test antibody to TNFRSF25 is evaluated after the initiating antibody is allowed to bind to TNFRSF25 under saturation conditions. In the second direction, the binding of the initiating antibody to TNFRSF25 is evaluated after the test antibody is allowed to bind to TNFRSF25 under saturation conditions. If, in both directions, only the first (saturated) antibody is able to bind to TNFRSF25, it is concluded that the test antibody and the initiating antibody compete for binding to TNFRSF25. As understood by those skilled in the art, the test antibody competing for binding with the initiating antibody may not necessarily bind to the same epitope as the initiating antibody, but may sterically block the binding of the initiating antibody by binding to an overlapping or adjacent epitope.

[0198] Two antibodies bind to the same or overlapping epitopes when each competitively inhibits (blocks) the binding of the other antibody to the antigen. That is, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits the binding of the other antibody by at least 50%, but preferably 75%, 90%, or even 99%, as measured in competitive binding assays (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, if virtually all amino acid mutations within the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody, the two antibodies have the same epitope. If some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody, the two antibodies have overlapping epitopes.

[0199] Additional conventional experiments (e.g., peptide mutation and binding assays) may subsequently be performed to determine whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody or whether steric blockage (or another phenomenon) is the cause of the observed lack of binding. These types of experiments may be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody-binding assay available in the art.

[0200] In another aspect, antibodies can be defined by their variable sequences, which include an additional “framework” region. This is presented in Table 3, which shows the entire variable region. Additionally, antibody sequences can be optionally varied from these sequences using methods discussed in more detail below. For example, the nucleic acid sequence may be (a) a variable region separated from the constant domains of the light and heavy chains, (b) the nucleic acid may differ from those presented above without affecting the residues encoded thereby, (c) the nucleic acid may differ from those presented above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology, (d) the nucleic acid may differ from those presented above by its ability to hybridize under high rigor conditions exemplified by low salt and / or high temperature conditions, e.g., conditions provided by about 0.02 M to about 0.15 M NaCl at a temperature of about 50°C to about 70°C, and (e) amino acids by a given percentage, e.g., 80%, 85%, 90%, 91%, (f) It may differ from what is presented above by 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology, or it may differ from what is presented above by allowing conservative substitutions. Each of the foregoing applies to the amino acid sequences in Table 2.

[0201] When comparing polynucleotide and polypeptide sequences, two sequences are said to be “identical” if the sequences of nucleotides or amino acids in the two sequences are identical when aligned for maximum correspondence as described below. Comparison between two sequences is typically performed by comparing the sequences across a comparison window to identify and compare local regions of sequence similarity. A “comparison window” as used herein refers to at least about 20 adjacent positions, usually 30 to about 75, or 40 to about 50 segments, where the sequences can be compared with reference sequences at the same number of adjacent positions after the two sequences are optimally aligned.

[0202] Optimal alignment of sequences for comparison can be performed using the Megalign program within the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.) using basic parameters. Alternatively, optimal alignment of sequences for comparison can be performed by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the similarity method search of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA within the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

[0203] One specific example of an algorithm suitable for determining sequence identity percentage and sequence similarity is Altschul, respectively. et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul. et al. (1990) J. Mol. Biol. 215:403-410 describes the BLAST and BLAST 2.0 algorithms. BLAST and BLAST 2.0 can be used to determine the percentage of sequence identity for the polynucleotides and polypeptides of the present disclosure, for example, together with the parameters described herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The rearranged nature of antibody sequences and the variable length of each gene require multiple rounds of BLAST search for a single antibody sequence. Furthermore, manual assembly of different genes is difficult and error-prone. The sequencing tool IgBLAST (world-wide-web at ncbi.nlm.nih.gov / igblast / ) identifies matching for germline V, D, and J genes, details of rearranged junctions, and the distinction between Ig V domain framework regions and complementarity determination regions. IgBLAST can analyze nucleotide or protein sequences, process sequences in batches, and allow simultaneous searching of germline gene databases and other sequence databases to minimize the possibility of missing the best possible matching germline V gene.

[0204] “Grayline nucleic acid residues” refer to nucleic acid residues that occur naturally in germline genes that encode constant or variable regions. “Grayline genes” are DNA found in germ cells (i.e., cells destined to become eggs or sperm). “Grayline mutations” refer to heritable changes in specific DNA that occur in germ cells or zygotes at the single-cell stage, and when passed to offspring, these mutations are integrated into all cells of the body. Germline mutations are contrasted with somatic mutations, which are acquired in a single body cell. In some cases, nucleotides within germline DNA sequences that encode variable regions are mutated (i.e., somatic mutations) and replaced by different nucleotides.

[0205] In one approach, the “percentage of sequence identity” is determined by comparing two optimally aligned sequences across a comparison window of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence within the comparison window may contain 20 percent or less, typically 5 to 15 percent, or 10 to 12 percent of additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to calculate the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to calculate the percentage of sequence identity.

[0206] Another way of defining antibodies is as any of the antibodies provided herein and “derivatives” of their antigen-binding fragments. Derivative antibodies or antibody fragments may be modified by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formulation, and metabolic synthesis of tunicamycin. In one embodiment, the antibody derivative will possess a function similar to or identical to that of the parent antibody. In another embodiment, the antibody derivative will exhibit altered activity compared to the parent antibody. For example, the derivative antibody (or its fragment) may bind more tightly to its epitope or be more resistant to proteolysis than the parent antibody.

[0207] The term “derivative” refers to an antibody or its antigen-binding fragment that binds immunospecifically to an antigen but contains one, two, three, four, five, or more amino acid substitutions, additions, deletions, or modifications compared to the “maternal” (or wild-type) molecule. Such amino acid substitutions or additions may introduce naturally occurring (i.e., DNA-coded) or non-naturally occurring amino acid residues. The term “derivative” refers to a modified C to form, for example, an antibody having a variant Fc region exhibiting enhanced or impaired effector or binding properties. H 1, hinge, C H 2, C H 3 or C HIt encompasses variants having four regions. The term “derivative” additionally encompasses non-amino acid modifications, e.g., amino acids that can be glycosylated (e.g., having content of modified mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolneuraminic acid, etc.), acetylation, pegylation, phosphorylation, amidation, derivatization by known protective / blocking groups, proteolytic cleavage, amino acids that can be linked to cellular ligands or other proteins, etc. In some embodiments, modified carbohydrate modification modulates one or more of antibody solubilization, facilitation of intracellular transport and secretion of antibodies, promotion of antibody assembly, stereochemical integrity, and antibody-mediated effector function. In certain embodiments, modified carbohydrate modification enhances antibody-mediated effector function compared to antibodies lacking carbohydrate modification. Carbohydrate modifications that result in altered antibody-mediated effector function are well known in the industry.

[0208] Derivative antibodies or antibody fragments may be produced in a sequence or glycosylated state engineered to confer a desired level of activity in antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), or antibody-dependent complement deposition (ADCD) functions, as measured by bead-based or cell-based assays or in vivo studies in animal models.

[0209] Derivative antibodies or antibody fragments may be modified by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formulation, and metabolic synthesis of tunicamycin. In one embodiment, the antibody derivative will possess a function similar to or identical to that of the parent antibody. In another embodiment, the antibody derivative will exhibit altered activity compared to the parent antibody. For example, the derivative antibody (or its fragment) may bind more tightly to its epitope or be more resistant to proteolysis than the parent antibody.

[0210] The biophysical properties of antibodies can be determined. Elevated temperatures can be used to unfold antibodies to determine relative stability using the average apparent melting temperature. Differential Scanning Calorimetry (DSC) determines the heat capacity of molecules as a function of temperature, C p It measures (per degree, the heat required to warm it). DSC can be used to study the thermal stability of antibodies. DSC data for mAbs sometimes resolves the unfolding of individual domains within the mAb structure, showing up to three peaks (Fab, C) in the thermogram. H 2 and C H It is particularly interesting because it generates (from the unfolding of the 3 domains). Typically, the unfolding of the Fab domain produces the strongest peak. The DSC profile and relative stability of the Fc portion show characteristic differences for human IgG1, IgG2, IgG3, and IgG4 subclasses (Garber and Demarest, Biochem. Biophys. Res. Commun.355, 751-757, 2007). Additionally, the average apparent melting temperature can be determined using circular dichroism (CD) performed with a CD spectrometer. Far-UV CD spectra will be measured for the antibody in the range of 200 to 260 nm at 0.5 nm increments. The final spectrum can be determined as the average of 20 cumulative measurements. The residual ellipsometry value can be calculated after background subtraction. Thermal spreading of the antibody (0.1 mg / mL) can be monitored at 235 nm at 25 to 95°C and a heating rate of 1°C / min. Dynamic light scattering (DLS) can be used to evaluate aggregation tendencies. DLS is used to characterize the size of various particles, including proteins. If the system is not dispersed in terms of size, the average effective diameter of the particles can be determined. This measurement depends on the size of the particle core, the size of the surface structure, and the particle concentration. Since DLS essentially measures variations in scattered light intensity caused by particles, the diffusion coefficient of the particles can be determined. DLS software on commercial DLA instruments displays particle populations at different diameters. Stability studies can be conveniently performed using DLS. DLS measurements of a sample can indicate whether particles aggregate over time or with changes in temperature by determining whether the hydrodynamic radius of the particles increases. If particles aggregate, larger populations of particles with larger radii can be observed. Temperature-dependent stability can be analyzed by controlling the temperature in situ. Capillary electrophoresis (ECE) technology includes a proven methodology for determining antibody stability characteristics. The iCE approach can be used to decompose antibody protein charge variants resulting from deamidation, C-terminal lysine, sialylation, oxidation, glycosylation, and any other changes to the protein that may lead to changes in the protein's pI.Each expressed antibody protein can be evaluated by high-throughput, free-solution isoelectric point focusing (IEF) on a capillary column (cIEF) using the Protein Simple Maurice instrument. Whole-column UV absorption detection can be performed every 30 seconds for real-time monitoring of molecules focused at the isoelectric point (pI). This approach combines the high resolution of traditional gel IEF with the advantages of quantification and automation found in column-based separations, while eliminating the need for a mobilization step. This technique yields a reproducible and quantitative analysis of identity, purity, and heterogeneity profiles for the expressed antibodies. The results identify charge heterogeneity and molecular size measurements on the antibodies using both absorbance and natural fluorescence detection modes with detection sensitivities up to 0.7 μg / mL.

[0211] The intrinsic solubility score of the antibody sequence can be determined. The intrinsic solubility score is CamSol Intrinsic (Sormanni et al. , J Mol Biol It can be calculated using (427, 478-490, 2015). The amino acid sequences for residues 95–102 (Kabat numbering) in HCDR3 of each antibody fragment, such as scFv, can be evaluated via online programs to calculate solubility scores. Additionally, solubility can be determined using laboratory techniques. Various techniques exist, including adding lyophilized protein to the solution until it is saturated and reaches the solubility limit, or concentrating by ultrafiltration in a microconcentrator with a suitable molecular weight cut-off. The most direct method is the induction of amorphous precipitation, which measures protein solubility using methods involving protein precipitation with ammonium sulfate (Trevino et al. , J Mol Biol(. , 366: 449-460, 2007). Ammonium sulfate precipitation provides rapid and accurate information regarding relative solubility values. Ammonium sulfate precipitation produces a precipitated solution with well-defined aqueous and solid phases and requires a relatively small amount of protein. Solubility measurements performed using the induction of amorphous precipitation by ammonium sulfate can also be easily performed at different pH values. Protein solubility is highly dependent on pH, and pH is considered the most important exogenous factor affecting solubility.

[0212] III. Pharmaceutical Dosage Forms

[0213] The present disclosure provides a pharmaceutical composition comprising an antibody that binds to human TNFRSF25. Such composition comprises an amount of antibody or a fragment thereof that is effective prophylactically or therapeutically and a pharmaceutically acceptable carrier.

[0214] The phrase “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce adverse effects, allergies, or other undesirable reactions when appropriately administered to animals such as humans. The preparation of pharmaceutical compositions comprising antibodies or additional active ingredients will be known to those skilled in the art in light of this disclosure. Additionally, it will be understood that for administration to animals (e.g., humans), the formulation must meet sterility, pyrogenity, general safety, and purity standards as required by the FDA Office of Biological Standards.

[0215] As used herein, “pharmaceuticalally acceptable carriers” include, as known to those skilled in the art, any and all aqueous solvents (e.g., water, alcohol / aqueous solutions, saline solutions, sodium chloride, parenteral vehicles such as Ringer’s dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coating agents, surfactants, antioxidants, preservatives (e.g., antimicrobial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption retardants, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluids and nutritional supplements, similar substances and combinations thereof. Water is a specific carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. The pH and precise concentrations of various components within the pharmaceutical composition are adjusted according to well-known parameters.

[0216] The active ingredient may be formulated for parenteral administration and may be formulated for injection via, for example, intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. Typically, such compositions may be prepared as either a liquid solution or a suspension; a solid form suitable for use in preparing a solution or suspension when adding liquid prior to injection may also be prepared; and the formulation may also be emulsified.

[0217] Protein compositions may be formulated in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), which are formed with inorganic acids such as, for example, hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0218] The composition may also include a small amount of a wetting agent or emulsifier, or a pH buffer, if desired. Such compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical formulations are described in Remington's Pharmaceutical Sciences. Such compositions will contain a prophylactically or therapeutically effective amount of antibody or a fragment thereof, preferably in a purified form, along with a suitable amount of carrier, to provide a form for appropriate administration to a patient.

[0219] Passive delivery of antibodies will generally involve the use of intravenous or intramuscular injection. The antibody may be in the form of a monoclonal antibody. The antibody will be formulated in a carrier suitable for injection, that is, a sterile carrier that can be used with a syringe.

[0220] The terms “unit dose” or “dosage” refer to physically distinct units suitable for use in a subject, each unit comprising a predetermined quantity of the therapeutic composition calculated to produce the desired response discussed above in relation to its administration, i.e., the appropriate route and therapeutic regimen. The amount to be administered depends on the desired effect depending on both the number of treatments and the unit dose. The actual dose of the composition of the present embodiment administered to a patient or subject may be determined by physical and physiological factors such as body weight, the subject’s age, health and sex, the type of disease being treated, the extent of disease penetration, previous or concurrent therapeutic interventions, the patient’s idiosyncratic constitution, the route of administration, and the efficacy, safety, and toxicity of the specific therapeutic substance. For example, the dose may also include about 1 μg / kg / body weight per administration to about 1000 mg / kg / body weight (such ranges include intermediate doses) or greater, and any range derivable therefrom. In non-limiting examples of ranges derivable from the figures listed herein, ranges such as about 5 μg / kg / body weight to about 100 mg / kg / body weight, about 5 μg / kg / body weight to about 500 mg / kg / body weight may be administered. In any case, the practitioner in charge of administration will determine the concentration of the active ingredient(s) in the composition and the appropriate dose(s) for the individual subject.

[0221] IV. Treatment Methods

[0222] The antibodies or antibody compositions provided herein may be used to treat, improve, or prevent the diseases, disorders, or symptoms described herein. The antibodies or antibody compositions provided herein may be used to treat, improve, or prevent diseases, disorders, or symptoms associated with inflammation and / or autoimmunity. In certain embodiments, the compositions and methods provided herein involve administering the antibodies or antibody compositions provided herein in combination with a second or additional therapy.

[0223] The antibodies or antibody compositions provided herein are for inflammatory bowel disease (e.g., Crohn's disease and / or ulcerative colitis), allergy, acute or chronic reactive airway disease, allergic rhinitis, allergic dermatitis, atopic diseases, atopic asthma, atopic dermatitis, bronchial asthma, eosinophil invasive asthma, chronic obstructive pulmonary diseases, arthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, primary biliary cirrhosis, systemic lupus erythematosus, It can be used to treat inflammatory diseases such as psoriasis, atherosclerosis, osteoporosis, and / or multiple sclerosis.

[0224] The antibodies or antibody compositions provided herein are for multiple sclerosis, rheumatoid arthritis, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura (ITP), autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, neuritis, uveitis ophthalmia, and multiple Polyendocrinopathies, purpura (e.g., Henloch-Schoenlein purpura), Reiter's disease, Stiff-Man Syndrome, autoimmune pulmonary inflammation, autism, Guillain-Barre Syndrome, insulin-dependent diabetes mellitus, and autoimmune inflammatory eye disorders, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis),Systemic lupus erhythematosus, Goodpasture's syndrome, pemphigus, receptor autoimmunities (e.g., Graves' Disease, Myasthenia Gravis, and insulin resistance), autoimmune thrombocytopenic purpura, rheumatoid arthritis, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis / dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, chronic renal failure, Glomerulonephritis, nephrosis, IgA nephropathy, bullous pemphigoid, Sjogren's syndrome, adrenergic drug resistance (asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria,It can be used to treat autoimmune diseases such as atopic dermatitis, inflammatory myopathies, and other inflammatory, granulomatous, degenerative, and atrophic diseases.

[0225] In some aspects, the present invention provides a method for treating inflammatory bowel disease (IBD) in a subject by administering the anti-TNFRSF25 antibody provided herein to the subject. The IBD may be Crohn's disease (CD) or ulcerative colitis (UC). The IBD may be a severe form of IBD characterized by a subclinical phenotype. The IBD may be a moderate to severe form of IBD. The IBD may be a moderate form of IBD.

[0226] The subclinical phenotypes of IBD include, but are not limited to, non-stenotic, stenotic, penetrating, isolated internal penetrating, disease, and perianal disease (pCD). Stenosis is progressive narrowing of the intestine. Internal penetrating disease creates an abnormal passage (fistula) between the intestine and other structures. pCD is a form of Crohn's disease that causes inflammation around the anus.

[0227] IBD can be refractory. As used herein, the term “refractory” refers to a condition where standard treatment fails to induce remission of the disease. Non-limiting examples of standard treatments include glucocorticosteroids, anti-TNF therapy, anti-α4-β7 therapy (vedolizumab), anti-IL12p40 therapy (ustekinumab), thalidomide, and cytoxin.

[0228] “Therapeutics” and “treating” refer to administering or applying a therapeutic agent to a subject or performing a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit for a disease or health-related condition. For example, therapy may include the administration of a pharmaceutically effective amount of at least one antibody targeting TNFRSF25, either alone or in combination with other therapies.

[0229] The term “subject” as used herein refers to any individual or patient on whom the subject method of the present invention is performed. Generally, the subject is a human being, but as understood by those skilled in the art, the subject may be an animal. Thus, other animals, including mammals such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, farm animals (including cattle, horses, goats, sheep, pigs, etc.), and primates (including monkeys, chimpanzees, orangutans, and gorillas), are included within the definition of the subject.

[0230] The terms “therapeutic benefit” or “therapeutically effective,” as used throughout this application, refer to any that promote or improve the well-being of the subject in connection with medical treatment of such conditions. This includes, but is not limited to, a reduction in the frequency or severity of signs or symptoms of the disease. For example, treatment of Crohn's disease may entail, for example, a reduction in the Crohn's Disease Activity Index (CDAI) score. Treatment of ulcerative colitis may refer to an improvement in the Mayo score, the Mayo bowel frequency subscore and / or the Mayo endoscopic subscore and / or histological-endoscopic mucosal improvement and / or endoscopic normalization and / or the ulcerative colitis disease activity index and / or ulcerative colitis endoscopic severity index.

[0231] The term “Crohn’s Disease Activity Index” or “CDAI” encompasses research tools used to quantify the symptoms of patients with Crohn’s disease. The CDAI is generally used to define the response or remission of CD. The CDAI consists of eight factors, each adjusted by a weighting factor and then summed. The components and weighting factors of the CDAI are as follows:

[0232]

[0233] 1 point is added for each set of complications: presence of arthralgia or apparent arthritis; inflammation of the iris or uveitis; presence of erythema nodosum, pyoderma gangrene, or aphthous ulcers; anal fissure, fistula, or abscess; other fistulas; and / or fever during the previous week.

[0234] Remission of Crohn's disease is typically defined as a drop in the CDAI to less than 150 points. Severe disease is typically defined as a value greater than 450 points. In a specific aspect, a response to a specific drug in patients with Crohn's disease is defined as a drop in the CDAI to greater than 70 points.

[0235] As used herein, the “Mayo score” refers to an index system for evaluating the severity of ulcerative colitis. Schoeder et al. See , N Engl J Med 1987; 317:1625-9. The Mayo score ranges from 0 to 12, with subscores from 0 to 3, where higher scores indicate more severe disease. In some aspects, subscores may be graded for bowel frequency, rectal bleeding, mucosal appearance on endoscopy, and physician's overall assessment (PGA).

[0236] As used herein, the “Endoscopic Severity Index of Ulcerative Colitis” or “UCEIS” refers to an index for evaluating endoscopic disease activity. The index evaluates three criteria, including vascular patterns, hemorrhage, and erosion and ulcers. Travis et al. See , Gut., 61(4):535-42 (2012). Higher scores reflect increased disease severity.

[0237] As used herein, the “Ulcerative Colitis Disease Activity Index” or “UCDAI” refers to an index system for evaluating the symptomatic severity or response of patients with ulcerative colitis. The index evaluates four variables, including bowel frequency, severity of bleeding, colonic mucosal appearance, and a physician’s overall assessment of disease activity. Sutherland et al. See , Gastroenterology. 1987; 92:1894-8. Each variable is scored from 0 to 3, with a total index score ranging from 0 to 12; 0-2: remission; 3-6: mild; 7-10: moderate; >10: severe ulcerative colitis.

[0238] In addition to being used as monotherapy, the antibodies of the present invention may also find use in combination therapy. Effective combination therapy may be achieved with a single composition or pharmacological formulation comprising both agents, or with two separate compositions or formulations administered simultaneously, wherein one composition comprises at least one antibody of the present invention and the other comprises a second agent(s). Alternatively, antibody therapy may precede or follow treatment with other agents by an interval ranging from minutes to months.

[0239] Various combinations may be used, such as when the antibody of the present invention is “A” and “B” represents the second agent, and non-limiting examples thereof are described below:

[0240]

[0241] In various aspects, the anti-TNFRSF25 antibody is administered to the subject in a series of treatments. The anti-TNFRSF25 antibody and the second treatment may be administered in any order or simultaneously. The anti-TNFRSF25 antibody may be administered to a patient who has previously received treatment with the second treatment. The anti-TNFRSF25 antibody and the second treatment may be administered substantially simultaneously or concurrently. For example, the subject may receive the anti-TNFRSF25 antibody during a course of treatment using the second treatment. The anti-TNFRSF25 antibody may be administered within one year of treatment using the second treatment. The anti-TNFRSF25 antibody may be administered within 10, 8, 6, 4, or 2 months of any treatment using the second treatment. The anti-TNFRSF25 antibody may be administered within 4, 3, 2, or 1 week of any treatment using the second treatment. The anti-TNFRSF25 antibody may be administered within 5, 4, 3, 2, or 1 day of any treatment using the second treatment. The two treatments may be administered to the subject within hours or minutes (i.e., simultaneously).

[0242] Other treatments include 1) anti-inflammatory drugs (e.g., sulfasalazine, azulpidine, 5-aminosalicylates, mesalamine, Asacol, Lyalda, Lowasa, Canasa, valsalazide, collazal, and aminosalicylates including but not limited to olsalazine and Defentum); 2) corticosteroids (e.g., prednisone and hydrocortisone); 3) Immunosystem suppressants (e.g., azathioprine, azasan, Imuran, mercaptopurine, purinetole, purisan, cyclosporine, Zengraf, Neoral and Sandimune, infliximab, Remicade, adalimumab, Humira, golimumab and Simponi, tumor necrosis factor (TNF)-alpha inhibitors (e.g., infliximab), methotrexate, rhumatrex, natalizumab, tysabri, vedolizumab, Entyvio, ustekinumab and Stelara); 4) Antibiotics (e.g., metronidazole, flazyl, ciprofloxacin, cipro); 5) Antidiarrheals (e.g., fiber supplements—metamucil or citrucel) or loperamide; 6) Analgesics (e.g., Tylenol, ibuprofen, naproxen sodium and diclofenac sodium); and 7) surgery (e.g., removal of the colon, partial gastrointestinal resection, colectomy, rectocolectomy and / or stenosisplasty), including but not limited to these. In some embodiments, this treatment may be administered in combination with an anti-TNFRSF25 antibody. Treatment with the antibody may occur before, simultaneously with, or after the administration of another treatment. Concurrent administration may include co-administration using a single pharmaceutical formulation or separate formulations, or sequential administration in any order, but generally within a time period in which all active agents can simultaneously exert their biological activity. Any administration schedule for this treatment may also be used as determined by a skilled practitioner.

[0243] In some embodiments, the second treatment comprises another antibody. Thus, the treatment may involve, but is not limited to, the co-administration of the antibody provided herein with another antibody against an additional antigen, such as tumor necrosis factor (TNF)-alpha. Co-administration may include co-administration using a single pharmaceutical formulation or separate formulations, or sequential administration in any order, but generally within a time period in which all active agents can simultaneously exert their biological activity.

[0244] V. Detection Method

[0245] This disclosure also relates to an immunodetection method for detecting human TNFRSF25. A wide variety of analytical formats are considered for detecting protein products, to name a few, including immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiography, fluorescence immunoassay, chemiluminescence assay, bioluminescence assay, dot blotting, FACS analysis, and Western blotting. Steps for various useful immunodetection methods are described in the scientific literature. Generally, an immunolinking method involves obtaining a sample and, in some cases, contacting the sample with an antibody specific to the protein to be detected under conditions effective for allowing the formation of an immunocomplex. Generally, the detection of immunocomplex formation is well known in the art and can be achieved through the application of numerous approaches. These methods are generally based on the detection of labels or markers, such as any of radioactive, fluorescent, biological, and enzymatic tags. Of course, as is known in the art, additional benefits can be found through the use of secondary binding ligands, such as second antibodies and / or biotin / avidin ligand binding arrays.

[0246] The antibody itself used for detection may be linked to a detectable label, thereby allowing the amount of primary immune complex in the composition to be determined simply by detecting this label. Alternatively, the first antibody bound to the primary immune complex may be detected by a second binding ligand having a binding affinity for the antibody. In this case, the second binding ligand may be linked to a detectable label. The second binding ligand itself is often an antibody and may therefore be referred to as a “secondary” antibody. The primary immune complex is contacted with the labeled secondary binding ligand or antibody for a sufficient amount of time under conditions effective for allowing the formation of the secondary immune complex. The secondary immune complex is then washed, generally to remove any non-specifically bound labeled secondary antibody or ligand, and the remaining label within the secondary immune complex is subsequently detected.

[0247] As used herein, the term “sample” refers to any sample suitable for the detection method provided by the present invention. The sample may be any sample containing a substance suitable for detection or isolation. Sources of the sample include blood, pleural fluid, peritoneal fluid, urine, saliva, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, and bronchial lavage fluid. In one aspect, the sample is a blood sample containing, for example, whole blood or any fraction or component thereof. Blood samples suitable for use with the present invention may be extracted from any known source containing blood cells or components thereof, such as veins, arteries, peripheral tissues, umbilical cords, etc. For example, the sample may be obtained and processed using well-known and conventional clinical methods (e.g., procedures for collecting and processing whole blood). In one aspect, an exemplary sample may be peripheral blood collected from a subject having cancer. In some aspects, the biological sample comprises a plurality of cells. In certain aspects, the biological sample comprises fresh or frozen tissue. In certain aspects, biological samples include formalin-fixed or paraffin-embedded tissues. In some aspects, biological samples are tissue biopsies, fine-needle aspirates, blood, serum, plasma, cerebrospinal fluid, urine, feces, saliva, circulating tumor cells, exosomes, or bodily fluid secretions such as aspirates and sweat. In some aspects, biological samples include cell-free DNA.

[0248] VI. Kit

[0249] The present invention also provides a kit comprising a therapeutic agent and / or other therapeutic agents and a delivery agent. In some embodiments, a kit for preparing and / or administering the therapy of the embodiment is provided. The kit may comprise one or more sealed vials containing any of the pharmaceutical compositions of the embodiment. The kit may comprise, for example, at least one anti-TNFRSF25 antibody as well as reagents for preparing, formulating and / or administering the components of the embodiment or performing one or more steps of the method of the present invention. In some embodiments, the kit may also comprise a suitable container, which is a container that does not react with the components of the kit, such as an Eppendorf tube, an assay plate, a syringe, a vial, or a tube. The container may be made of a sterile material such as plastic or glass.

[0250] Additionally, an immunodetection kit for use with the immunodetection method described above is provided. Since an antibody can be used to detect TNFRSF25, the antibody may be included in the kit. Accordingly, the immunodetection kit will comprise a first antibody that binds to TNFRSF25 and optionally an immunodetection reagent within a suitable container means.

[0251] The kit may further include instructions outlining the procedural steps of the method presented herein and will follow substantially the same procedures as those described herein or known to those skilled in the art. The instruction information may be in a computer-readable medium containing machine-readable instructions that, when executed using a computer, give rise to an indication of a real or virtual procedure delivering a pharmaceutically effective amount of the therapeutic agent.

[0252] Ⅶ. Examples

[0253] The following examples are included to demonstrate preferred embodiments of the present invention. Those skilled in the art should recognize that the technology disclosed in the following examples represents technology discovered by the inventors to function well in the practice of the present invention and may therefore be considered to constitute a preferred mode for practicing the invention. However, those skilled in the art should recognize that, in light of this disclosure, many modifications may be made to the specific embodiments disclosed and similar or similar results may still be obtained without departing from the spirit and scope of the present invention.

[0254] Example 1

[0255] Monoclonal and biparatop antibodies specifically targeting human TNFRSF25 are provided herein. Monoclonal antibodies were produced in engineered CHOK1 cell lines by transfecting cells with a vector genetically encoding heavy and light chain antibody sequences and then performing single-step affinity-capture chromatography using FcXL resin. The final monoclonal antibody formulations were buffered with PBS, pH 7.4. The production of biparatop antibodies followed a two-step process. In the first step of the process, so-called Knob-half (or Hall-half) antibodies were produced by transfecting CHOK1 cell lines engineered with a vector genetically encoding the Knob (or Hall) heavy chain and corresponding light chain sequences. CHOK1 cell harvests from the Knob-half antibodies and Hall-half antibodies were subsequently purified by protein A column-based chromatography. In the second step of the process, the two half-antibodies were reassembled into the desired biparatope form by quenching the reaction with a buffer exchange to PBS, pH 7.4, following a mild reduction step. To detect antibody binding to human TNFRSF25, recombinant biotin-conjugated human TNFRSF25 protein was immobilized on streptavidin-coated MSD (Meso Scale Discovery) plates and allowed to bind to anti-TNFRSF25 mAbs in solution. After a washing step, the bound antibodies were detected using sulfo-tagged anti-human Fc antibodies. The results of these MSD analyses are shown in arbitrary units (au) on the y-axis of Figure 1. To determine the binding affinity of antibodies to recombinant human TNFRSF25 protein, recombinant biotin-conjugated human TNFRSF25 protein was immobilized on streptavidin-coated BLI sensors and allowed to bind to anti-TNFRSF25 mAbs in solution. Binding affinity (K D) was calculated using the binding rate (Ka) and dissociation rate (Kd) of mAb binding to the target using a 1:1 Langmuir binding model in ForteBio data analysis software. The results of this BLI / octet analysis are shown in Table 7.

[0256] [Table 7]

[0257]

[0258] Affinity-matured NH24 variants were expressed in their scFv form in the peripheral cytoplasm of E. coli, and expression was induced overnight. The supernatant from the overnight E. coli culture was collected as unpurified scFv and used to test the ability to block binding to recombinant human TNFRSF25 and the binding of recombinant human TL1A to TNFRSF25. Compared to the original NH24 clone, all four variants tested exhibited significantly higher levels of binding to TNFRS25 (Fig. 2a). Significantly higher blocking by the affinity-matured scFv was observed compared to the original NH24 clone (Fig. 2b).

[0259] The effects of various IgG1 Fc-silencing mutations were evaluated with anti-TNFRSF25 antibodies. To this end, anti-TNFRSF25 antibodies containing various Fc-silencing mutations within the IgG1-Fc domain (L234A / L235A [clone 033], L234A / L245A + N297A [clone 034], L234A / L235A + G237A [clone 061]) were generated. To evaluate the Fc-mediated antibody-dependent cytotoxicity (ADCC) activity by anti-TNFRSF25 variants, the antibodies were first incubated with CHOK1 cells expressing TNFRSF25 for 1 hour. Antibody-opsonized CHOK1 cells were further incubated with Jurkat cells expressing the cell surface Fc receptor FcγRIIIa (CD16A) and NFAT-luciferase reporter gene in a 1:4 ratio. Luciferase activity resulting from CD16-mediated NFAT activation was measured using a luminescence analyzer after 6 hours. Fc-mediated ADCC activity was significantly reduced or silenced in the anti-TNFRSF25 variants compared to an mAb [clone 062] containing the wild-type sequence of the IgG1-Fc domain (Fig. 3a). Additionally, to minimize / eliminate the risk of anti-TNFRSF25 cross-linking that could occur via Fcγ receptor binding following DR3 receptor binding on the cell, the lack of FcγR1 (CD64) binding in the anti-TNFSF25 antibody containing the Fc-silencing mutation was confirmed by BLI (Fig. 3b). Figure 3c demonstrates that the Fc-competent (IgG1 WT) anti-TNFRSF25 antibody (SL-062) can activate the NFkB pathway through the simultaneous binding of DR3 (on Jurkat reporter cells) and FcγR1 (CD64) receptors (on engineered HEK293 cells).SL-033 (Fig. 3b), which retains residual FcγR1 binding, was able to activate the NFkB pathway, albeit to a lower degree than SL-062, whereas the complete Fc-silencing anti-TNFRSF25 antibodies (SL-034 and SL-061) did not activate the DR3-driven NFkB pathway.

[0260] To detect the binding of specific whole antibodies to human TNFRSF25, a biotinylated recombinant human TNFRSF25-His tag (Acro #TN5-H52H3) was loaded as a ligand onto a streptavidin biosensor. During the binding step, the biosensor was incubated with various concentrations of anti-TNFRSF25 antibodies for 600 seconds, followed by a dissociation step of 600 seconds. The TNFRSF25 binding affinities of whole antibodies SL-033 [clone 033], NH24, and NH46 (each having L234A / L235A G1 constant region substitutions) are provided in Figure 4 and Table 8.

[0261] [Table 8]

[0262]

[0263] To detect the binding of the L234A / L235A + G237A [Clone 061] antibody to human TNFRSF25, a biotinylated recombinant human TNFRSF25-His tag (Acro #TN5-H52H3) was loaded as a ligand onto a streptavidin biosensor. During the binding step, the biosensor was incubated with the L234A / L235A + G237A [Clone 061] antibody at the concentrations indicated in Fig. 5a for 600 seconds, followed by a dissociation step of 600 seconds. Complete kinetic parameters could not be determined by BLI. SPR was performed by loading biotinylated recombinant human TNFRSF25-His tag (Acro #TN5-H52H3) at 50 nM onto the streptavidin sensor chip SA (Cytiva), followed by flowing L234A / L235A + G237A [Clone 061] antibodies at the concentrations indicated in Fig. 5b for a 2-minute binding step and a subsequent 30-minute dissociation step. d 1.36 pM(k as analyzed by Biacore Insight Evaluation software for 1:1 bond stoichiometry on = 2.85E6 1 / Ms and k off It was estimated that = 3.89E-6 1 / s). As shown in Fig. 5c, no binding to DcR3, TNFR1, or TNFR2 was observed for SL-033 by MSD. SL-061 also does not bind to DcR3, TNFR1, or TNFR2.

[0264] To detect the binding of specific whole antibodies to the neonatal Fc receptor (FcRn) under neutral and acidic pH conditions, biotinylated recombinant human FcRn-His tag protein (Acro#FCM-H82W7) was loaded onto a streptavidin biosensor as a ligand. During the binding step, the biosensor was incubated with various concentrations of anti-TNFRSF25 antibody for 600 seconds, followed by a dissociation step of 600 seconds. The FcRn binding characteristics of whole antibodies SL-061, SL-061_YTE, SL-061_LS, and SL-062 under neutral and acidic pH (pH 6.0) conditions are presented in Figure 5d and Table 9. Both SL061_YTE and SL061_LS showed a binding affinity for FcRn that was about 10-fold higher at pH 6.0 (acidic) compared to pH 7.4 (neutral), which is mainly due to their slower dissociation rate at pH 6.0.

[0265] [Table 9]

[0266]

[0267] To determine the TL1A blocking activity of specific whole antibodies against human TNFRSF25, recombinant biotin-conjugated human TNFRSF25 protein was immobilized on streptavidin-coated MSD (Meso Scale Discovery) plates and bound to anti-TNFRSF25 mAbs in solution. After a washing step, the plates were incubated with a fixed concentration of TL1A-rabbit(r) Fc protein (0.4 μg / ml). For the analysis of the anti-TL1A benchmark antibody control, anti-TL1A was first incubated with a fixed amount (0.4 μg / ml) of TL1A-rFc in a separate 96-well plate before being added to the TNFRSF25-coated MSD plates. The DR3:TL1A protein complex was detected using a sulfo-tagged anti-rabbit secondary antibody. The TL1A blocking percentage was determined by normalizing the raw MSD signal to wells without anti-TNFRSF25 (0 nM). SL-033 was found to be superior to anti-TL1A in blocking TL1A binding to DR3 (Fig. 6 and Table 10). SL-061 showed an IC of 0.056 nM via MSD. 50 It blocked the binding of recombinant TL1A to TNFRSF25. In addition, SL-061_YTE and SL-061_LS variants blocked the binding of TL1A to recombinant TNFRSF25 with efficacy similar to SL-061.

[0268] [Table 10]

[0269]

[0270] Natural TNFRSF25 binding was evaluated using a CHOK1 cell line engineered to overexpress the human TNFRSF25 protein via stable transfection with a mammalian expression vector (pcDNA) encoding the human TNFRSF25 cDNA sequence (NP_683866). CHOK1-TNFRSF25 +Cells were incubated with various concentrations of anti-TNFRSF25 antibodies at 4°C for 1 hour. Cells were washed and further incubated with APC-conjugated anti-human Fc antibodies to detect antibody-drug bound cells by flow cytometry (Fig. 7a). SL-033 was found to bind more tightly to natural TNFRSF25 than NH46 and NH24 (Fig. 7a and Table 11).

[0271] [Table 11]

[0272]

[0273] To demonstrate that SL-061 is specific for binding to natural TNFRSF25, several TNFRSF receptors overexpressed in engineered mammalian cells were incubated with various concentrations of SL-061. The cells were washed and further incubated with an APC-conjugated anti-human Fc antibody to detect antibody-drug bound cells by flow cytometry. SL-061 did not exhibit detectable binding to the other TNFRSF receptors tested and was specific only to TNFRSF25 (Fig. 7b).

[0274] To demonstrate that SL-061 does not induce the internalization of TNFRSF25, engineered CHOK1 cells overexpressing TNFRSF25 (cDNA sequence NP_683866) were incubated with various concentrations of SL-061 at 4°C or 37°C for 30 minutes. The cells were washed and further incubated with an APC-conjugated anti-human Fc antibody to detect antibody-drug bound cells by flow cytometry. TNFRSF25 binding by SL-061 was not altered at 4°C or 37°C (Fig. 7c), suggesting that there was no internalization of the TNFRSF25 receptor at 37°C.

[0275] To demonstrate that SL-061 binds to natural cyanomolgus TNFRSF25, the cyanomolgus TNFRSF25 sequence (REFSEQ accession number: XM_015443331.1) was cloned into a pCDNA vector for cell-surface expression as a dichistrogenic gene for co-expressing cleavable cytoplasmic GFP. The vector was transiently transfected into HEK293 cells. 24 hours after transfection, cells were incubated with various concentrations of SL-061 or 4C12 (positive control). Next, cells were washed and further incubated with an APC-conjugated anti-human Fc antibody to detect antibody-drug conjugated cells by flow cytometry (Fig. 7d).

[0276] To evaluate the ability of the TNFRSF25 antagonist antibody to bind to TNFRSF25 and block TL1A binding, CHOK1-TNFRSF25 + Cells were first incubated with various concentrations of anti-TNFRSF25 antibodies at 4°C for 30 minutes. A fixed concentration of biotinylated human TL1A-Fc protein (156 nM) was then added to the cells, and they were incubated for an additional 1 hour at 4°C. After washing to remove excess antibody and TL1A, cells were further incubated with PE-conjugated streptavidin to provide for the detection of cells bound to TL1A by flow cytometry. The average fluorescence intensity of TL1A (PE) is shown (Fig. 8a). SL-033 and SL-061 competitively blocked TL1A binding to natural TNFRSF25 on CHOK1 cells and were superior to anti-TL1A antibodies in blocking TL1A binding to TNFRSF25 on the cells. SL-061 has an IC50 approximately 13-fold lower than the benchmark anti-TL1A antibody in blocking TL1A binding to TNFRSF25 on cells. 50It was found to have . In addition, SL-061_YTE and SL-061_LS variants block TL1A binding to natural TNFRSF25 with efficacy similar to SL-061.

[0277] The anti-TNFRSF25 antibody SL-061 was tested for its ability to efficiently block soluble and membrane-bound TL1A-induced IFNγ secretion from human PBMCs following anti-CD3 / CD28 stimulation, compared to a benchmark anti-TL1A antibody. Human PBMCs were stimulated with anti-CD3 / CD28 and co-cultured with either HEK293-TL1A cells or HEK293 parent cells that do not express TL1A on the surface, or treated with soluble TL1A (100 ng / mL). After 72 hours, the supernatant was harvested and IFNγ levels were analyzed using MSD (Fig. 8b). The bottom graph of Fig. 8b shows that IFNγ secretion was indeed induced by membrane-expressed TL1A, as HEK293 parent cells did not induce IFNγ secretion even in the absence of the SL-061 antibody.

[0278] Human PBMCs were stimulated with either IL12+IL18 or anti-CD3 / CD28 for 24 hours to evaluate the binding of anti-TNFRSF25 to lymphocyte subsets. Cells were harvested and incubated with SL-033 or SL-061 (5 μg / mL), then incubated with APC-anti-human Fc (for detection of SL-033 or SL-061) and lineage-specific antibodies against T and NK cells, and analyzed by flow cytometry. Histograms of SL-033 intensity (Fig. 9) and mean fluorescence intensity (MFI) of SL-061 binding (Fig. 10) for each cell type are shown.

[0279] SL-033 CD4 under stimulated and non-stimulated conditions + and CD8 +It bound similarly to T cells, which is consistent with the constitutive expression of TNFRSF25 on T cells. NK cells minimally express TNFRSF25 in the resting state, but binding of SL-033 increased in NK cells upon stimulation with IL12 and IL18 (Fig. 9).

[0280] The ability of anti-TNFRSF25 antibodies to efficiently block TL1A-induced IFNγ secretion in human PBMCs following IL-12+IL-18 stimulation was tested. TNFRSF25 expression was upregulated by stimulating human PBMCs with IL-12 and IL-18 for 24 hours. After 24 hours, cells were treated with various concentrations of anti-TNFRSF25 antibodies in the presence or absence of TL1A (100 ng / mL). After 48 hours, the supernatant was harvested and IFNγ levels were analyzed using MSD. SL-033 blocked TL1A-induced IFNγ secretion more efficiently than NH24-35 (Fig. 11). Additionally, enhanced blockade of IFNγ secretion was observed for SL-033 with an Fc-silencing mutation (Fig. 12).

[0281] The anti-TNFRSF25 antibody was tested for its ability to efficiently block TL1A-induced IFNγ secretion from human PBMCs after stimulation with the CD3 / CD28 complex for 72 hours in the presence or absence of TL1A (100 ng / mL). After 72 hours, the supernatant was harvested and the levels of IFNγ were analyzed by MSD. SL-033 (L234A / L235A) and SL-061 (L234A / L235A + G237A) comparablely blocked IFNγ secretion (Fig. 13). In the same analysis, the supernatant was harvested after 72 hours for MSD analysis of additional cytokines. SL-033 (L234A / L235A) blocked the secretion of TL1A-induced TNFα, IL-6, and GM-CSF without evidence of residual agonist action (Fig. 14).

[0282] Human PBMCs from three healthy donors were stimulated with an anti-CD3 / CD28 complex in the presence or absence of TL1A (100 ng / mL) and treated with various concentrations of anti-TNFRSF25 antibody. After 72 hours, the supernatant was harvested and IFNγ levels were analyzed using MSD. SL-061 (L234A / L235A + G237A) blocked TL1A-induced IFNγ secretion from CD3 / CD28-stimulated lymphocytes (Fig. 15).

[0283] Human PBMCs derived from donors with ulcerative colitis (UC) or Crohn's disease (CD) were stimulated with the CD3 / CD28 complex in the presence or absence of TL1A (100 ng / mL) and treated with various concentrations of anti-TNFRSF25 antibody. After 72 hours, the supernatant was harvested and cytokine levels were analyzed using MSD. SL-061 (L234A / L235A + G237A) blocked TL1A-induced IFNγ secretion from PBMCs derived from IBD patients (Fig. 16).

[0284] The anti-TNFRSF25 antibody was tested for toxicity against the intestinal epithelial barrier in an in vitro inflammation model. A RepliGut® intestinal culture system was used to evaluate the toxicity of the anti-DR3 antibody under inflammatory conditions. Human intestinal stem cells were seeded into a Transwell system featuring a semipermeable membrane coated with a biomimetic scaffold composed of collagen hydrogel, which promotes the differentiation of stem cells into a monolayer of intestinal epithelial cells that closely mimics the in vivo intestinal epithelium. Cells were cultured according to the RepliGut® protocol to allow for the formation of a fully differentiated, polarized epithelial monolayer. The resulting epithelial monolayer contained a population of polarized and differentiated cells, representing a physiologically relevant model system for toxicity evaluation. Once the monolayer was established (days 6 to 7), the cultured cells were exposed to a fixed concentration of 5 μg / mL of SL-061 in the presence of varying concentrations of the pro-inflammatory cytokines TNFα and IFNγ. These cytokines were added to mimic the inflammatory environment, and their concentrations were varied to evaluate dose-dependent effects. Barrier function was assessed by measuring transepithelial electrical resistance (TEER) daily throughout the experiment. TEER measurements provide a quantitative assessment of the integrity of tight junctions between epithelial cells and serve as an indicator of the overall barrier function of the intestinal monolayer. The combination of TNFα and IFNγ induced epithelial barrier disruption, as indicated by a decrease in TEER 48 hours after treatment (Fig. 17a). Furthermore, the addition of SL-061 did not exacerbate the epithelial barrier disruption induced by TNFα and IFNγ (Fig. 17a).

[0285] The effects of SL-061 on intestinal barrier function were also evaluated using a co-culture system of pre-activated peripheral blood mononuclear cells (PBMCs) and the RepliGut® intestinal epithelial model. PBMCs derived from healthy donors were pre-activated with the anti-CD3 / CD28 complex and recombinant TL1A to stimulate the production of inflammatory cytokines. After 24 hours, pre-activated PBMCs were added to the basal compartment of the RepliGut® intestinal culture system, while SL-061 (5 μg / mL) was applied to both the apical and basal compartments. Intestinal barrier integrity was assessed by daily TEER measurements. TEER values ​​were normalized to the maximum value in each well to account for inter-well variability. The addition of TL1A to pre-activated PBMCs resulted in a significant decrease in TEER after 5 days of treatment, indicating a loss of epithelial barrier function (Fig. 17b, left panel). In contrast, the addition of SL-61 did not result in a decrease in TEER at the same time point, confirming the protective effect of anti-TNFRSF25 against TL1A-mediated intestinal injury (Fig. 17b, left panel). The presence of inflammatory cytokines was assessed by MSD in the culture supernatant 6 days after treatment. The addition of TL1A resulted in an increase in TNFα concentration, which was attenuated by SL-061 treatment in the co-culture system (Fig. 17b, right panel).

[0286] To evaluate the safety and pharmacokinetic profile of the mAb, receptor occupancy (RO) and immunophenotype analysis of SL-061 were evaluated in a non-human primate (NHP) toxicology study over a 4-week period. Cynomolgus macaques were administered three intravenous doses of 1, 10, or 100 mg / kg of SL-061 every two weeks (i.e., on days 1, 15, and 29). Flow cytometry was performed on whole blood samples collected at various time points before and after treatment. Plasma was collected at different time points to evaluate pharmacokinetic parameters.

[0287] For analysis, red blood cells were lysed, and samples were stained with fluorescently labeled antibodies targeting cell surface markers (CD3, CD4, CD8, CD25, CD127, TNRFSF25 [clone 4C12]) and intracellular markers (FoxP3, Ki-67). T-cell subsets were identified using the following marker combinations:

[0288] CD4+ T cells: CD3 + CD4 + CD8 - lymphocytes

[0289] CD8+ T cells: CD3 + CD4 - CD8 + lymphocytes

[0290] Regulatory T cells (Treg): CD3 + CD4 + CD8 - CD25 + FoxP3 + lymphocytes

[0291] Proliferative CD4+ T cells: CD3 + CD4 + CD8 - Ki67 + lymphocytes

[0292] RO is a Treg and proliferative CD4 expressing the highest levels of TNFRSF25 + It was evaluated by measuring the SL-061-mediated inhibition of anti-TNFRSF25 (clone 4C12) binding to T cells. TNFRSF25 +The percentage of cells was determined against isotype antibody control staining. Complete RO was observed across all dose levels and was maintained for at least 2 weeks after administration (Fig. 18a) and was maintained at the 4-week mark after the final administration (Fig. 19a). The absence of dose-dependent inhibition of anti-TNFRSF25 (clone 4C12) binding suggests that complete RO was achieved at ≤1 mg / kg (Figs. 18a and 19a). These data suggest sustained target binding at the 4-week mark after the final administration (Fig. 19b).

[0293] Treg and proliferative CD4 + T cells (Figs. 18b, 20a, and 20b), proliferative CD8 + No change was observed in the proportion of T cells (Fig. 20c) or the absolute number of T cell subsets (Fig. 18c), indicating a lack of agonist activity after SL-061 binding on T cells. Additionally, compared to the vehicle control, CD4 + T cells and CD8 + CD25 among T cells + The frequency of cells was not significantly altered by SL-061 treatment (Fig. 21a). Similarly, Ki67 + The percentage of cells remained largely unchanged after SL-061 administration across all dose groups (Fig. 21b). Notably, despite the high receptor occupancy of SL-061 on Treg, CD4 + Proportion of Tregs or proliferative Ki67 within a T cell population + There was no observable change in the percentage of Treg (Fig. 21c). Overall, despite target binding as evidenced by receptor occupancy data, SL-061 treatment did not result in substantial changes in T-cell number, activation status, or proliferation at any dose level tested.

[0294] Toxokinetic analysis of SL-061 demonstrated that the antibody was cleared over time without any observable clinical or toxicological effects on the subjects. Collected in vivo and recovery study data showed no abnormalities in leukocyte, erythrocyte, platelet, or neutrophil counts after injection of SL-061. Toxicological evaluations showed no identifiable toxic effects when treating non-human primates, even at the highest dose level of 100 mg / kg.

[0295] Example 2

[0296] All Fc silencing antibodies tested at our institution were successfully produced in small quantities (<5 mg) via protein A capture chromatography after transient production runs in CHOK1-derived cells. However, in larger-scale production, the SL-034 (L234A / L235A + N297A) antibody precipitated after affinity chromatography capture, whereas the SL-061 (L234A / L235A + G237A) antibody did not precipitate.

[0297] * * *

[0298] All methods disclosed and claimed herein can be manufactured and practiced without excessive experimentation in light of this disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods and steps or the order of steps described herein without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain chemically and physiologically related agents may replace the agents described herein, and that the same or similar results may be achieved. All such similar substitutions and variations apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

Claims

Claim 1 A polymeric protein comprising a first polypeptide having the amino acid sequence of SEQ ID NO. 134 and a second polypeptide having the amino acid sequence of SEQ ID NO.

57. Claim 2 As a monoclonal antibody or antibody fragment binding to TNFRSF25, the antibody or antibody fragment comprises (a) SEQ ID NO. 50 and SEQ ID NO. 51; (b) SEQ ID NO. 52 and SEQ ID NO. 53; (c) SEQ ID NO. 54 and SEQ ID NO. 55; (d) SEQ ID NO. 94 and SEQ ID NO. 95; (e) SEQ ID NO. 96 and SEQ ID NO. 97; (f) SEQ ID NO. 98 and SEQ ID NO. 99; (g) SEQ ID NO. 100 and SEQ ID NO. 97; (h) SEQ ID NO. 94 and SEQ ID NO. 97; (i) SEQ ID NO. 94 and SEQ ID NO. 99; (j) SEQ ID NO. 96 and SEQ ID NO. 95; (k) SEQ ID NO. 96 and SEQ ID NO. 99; (l) SEQ ID NO. 98 and SEQ ID NO. 95; (m) SEQ ID NO. 98 and SEQ ID NO. 97; (n) SEQ ID NO. 100 and SEQ ID NO. 95; (o) SEQ ID NO 100 and SEQ ID NO 99; or (p) a monoclonal antibody or antibody fragment comprising a clone-paired heavy chain and light chain CDR sequence derived from the clone-paired heavy chain and light chain variable sequences of SEQ ID NO 92 and SEQ ID NO 93. Claim 3 In paragraph 2, the antibody or antibody fragment is (a) SEQ ID NOs 7 to 12; (b) SEQ ID NOs 13 to 18; (c) SEQ ID NOs 19 to 24; (d) SEQ ID NOs 30 to 35; (e) SEQ ID NOs 36 to 41; (f) SEQ ID NOs 42 to 47; (g) SEQ ID NOs 7, 8, 77 and 10 to 12; (h) SEQ ID NOs 7, 8, 78, 79, 11 and 80; (i) SEQ ID NOs 81, 8, 79, 83, 11 and 84; (j) SEQ ID NOs 7 to 9, 86, 11 and 87; (k) SEQ ID NOs 7, 8, 77, 79, 11 and 80; (l) SEQ ID NOs 7, 8, 77, 83, 11 and 84; (m) SEQ ID NOs 7, 8, 77, 86, 11 and 87; (n) SEQ ID NOs 7, 8, 78 and 10 to 12; (o) SEQ ID NOs 7, 8, 78, 83, 11 and 84; (p) SEQ ID NOs 7, 8, 78, 86, 11 and 87; (q) SEQ ID NOs 81, 8, 82 and 10 to 12; (r) SEQ ID NOs 81, 8, 82, 79, 11 and 80; (s) SEQ ID NOs 81, 8, 82, 86, 11 and 87; (t) SEQ ID NOs 7, 8, 85 and 10 to 12; (u) SEQ ID NOs 7, 8, 85, 79, 11 and 80; (v) Sequence Nos. 7, 8, 85, 83, 11 and 84; (w) Sequence Nos. 30, 31, 88, 33, 34 and 35; (x) Sequence Nos. 30, 31, 89, 90, 34 and 80; (y) Sequence Nos. 30, 31, 91, 90, 34 and 80; (z) Sequence Nos. 30 to 32, 90, 34 and 80; (a') Sequence Nos. 30, 31, 88, 90, 34 and 80; (b') Sequence Nos. 30, 31, 88, 90, 34 and 80; (c') Sequence Nos. 30, 31, 88, 90, 34 and 80; (d') Sequence Nos. 30, 31, 89 and 33 to 35; (e') Sequence Nos. 30, 31, 89, 90, 34 and 80; (f') Sequence Nos. 30, 31, 89, 90, 34 and 80;(g') SEQ ID NOs 30, 31, 91 and 33 to 35; (h') SEQ ID NOs 30, 31, 91, 90, 34 and 80; (i') SEQ ID NOs 30, 31, 91, 90, 34 and 80; (j') SEQ ID NOs 30 to 32, 90, 34 and 80; (k') SEQ ID NOs 30 to 32, 90, 34 and 80; (l') SEQ ID NOs 138 to 143; or (m') a clone-paired heavy chain and light chain CDR sequence comprising SEQ ID NOs 144 to 147, 29 and 148, a monoclonal antibody or antibody fragment comprising Claim 4 In claim 2 or 3, the antibody or antibody fragment is (a) SEQ ID NO 50 and SEQ ID NO 51; (b) SEQ ID NO 52 and SEQ ID NO 53; (c) SEQ ID NO 54 and SEQ ID NO 55; (d) SEQ ID NO 94 and SEQ ID NO 95; (e) SEQ ID NO 96 and SEQ ID NO 97; (f) SEQ ID NO 98 and SEQ ID NO 99; (g) SEQ ID NO 100 and SEQ ID NO 97; (h) SEQ ID NO 94 and SEQ ID NO 97; (i) SEQ ID NO 94 and SEQ ID NO 99; (j) SEQ ID NO 96 and SEQ ID NO 95; (k) SEQ ID NO 96 and SEQ ID NO 99; (l) SEQ ID NO 98 and SEQ ID NO 95; (m) SEQ ID NO 98 and SEQ ID NO 97; (n) SEQ ID NO 100 and SEQ ID NO 95; (o) SEQ ID NO 100 and SEQ ID NO 99; or (p) a monoclonal antibody or antibody fragment comprising a clone-paired heavy chain and light chain variable sequence having at least 70%, 80%, or 90% identity independently with respect to the sequences of SEQ ID NO. 92 and SEQ ID NO.

93. Claim 5 In any one of paragraphs 2 to 4, the antibody or antibody fragment is (a) SEQ ID NO 50 and SEQ ID NO 51; (b) SEQ ID NO 52 and SEQ ID NO 53; (c) SEQ ID NO 54 and SEQ ID NO 55; (d) SEQ ID NO 94 and SEQ ID NO 95; (e) SEQ ID NO 96 and SEQ ID NO 97; (f) SEQ ID NO 98 and SEQ ID NO 99; (g) SEQ ID NO 100 and SEQ ID NO 97; (h) SEQ ID NO 94 and SEQ ID NO 97; (i) SEQ ID NO 94 and SEQ ID NO 99; (j) SEQ ID NO 96 and SEQ ID NO 95; (k) SEQ ID NO 96 and SEQ ID NO 99; (l) SEQ ID NO 98 and SEQ ID NO 95; (m) SEQ ID NO 98 and SEQ ID NO 97; (n) SEQ ID NO 100 and SEQ ID NO 95; (o) SEQ ID NO 100 and SEQ ID NO 99; or (p) a monoclonal antibody or antibody fragment comprising a clone-paired heavy chain and light chain variable sequence having at least 95% identity with respect to the sequences of SEQ ID NO. 92 and SEQ ID NO. 93, respectively. Claim 6 In any one of claims 2 to 5, the antibody or antibody fragment is (a) SEQ ID NO 50 and SEQ ID NO 51; (b) SEQ ID NO 52 and SEQ ID NO 53; (c) SEQ ID NO 54 and SEQ ID NO 55; (d) SEQ ID NO 94 and SEQ ID NO 95; (e) SEQ ID NO 96 and SEQ ID NO 97; (f) SEQ ID NO 98 and SEQ ID NO 99; (g) SEQ ID NO 100 and SEQ ID NO 97; (h) SEQ ID NO 94 and SEQ ID NO 97; (i) SEQ ID NO 94 and SEQ ID NO 99; (j) SEQ ID NO 96 and SEQ ID NO 95; (k) SEQ ID NO 96 and SEQ ID NO 99; (l) SEQ ID NO 98 and SEQ ID NO 95; (m) SEQ ID NO 98 and SEQ ID NO 97; (n) SEQ ID NO 100 and SEQ ID NO 95; (o) SEQ ID NO 100 and SEQ ID NO 99; or (p) a monoclonal antibody or antibody fragment comprising a clonal-paired heavy chain and light chain variable sequence of SEQ ID NO. 92 and SEQ ID NO.

93. Claim 7 In any one of claims 2 to 6, the antibody or antibody fragment is (a) SEQ ID NO. 59 and SEQ ID NO. 60; (b) SEQ ID NO. 61 and SEQ ID NO. 62; (c) SEQ ID NO. 63 and SEQ ID NO. 64; or (d) SEQ ID NO. 127 and SEQ ID NO. 128; (e) SEQ ID NO. 129 and SEQ ID NO. 130; (f) SEQ ID NO. 131 and SEQ ID NO. 132; (g) SEQ ID NO. 133 and SEQ ID NO. 130; (h) SEQ ID NO. 127 and SEQ ID NO. 130; (i) SEQ ID NO. 127 and SEQ ID NO. 132; (j) SEQ ID NO. 129 and SEQ ID NO. 128; (k) SEQ ID NO. 129 and SEQ ID NO. 132; (l) SEQ ID NO. 131 and SEQ ID NO. 128; (m) SEQ ID NO. 131 and SEQ ID NO. 130; (n) SEQ ID NO 133 and SEQ ID NO 128; (o) SEQ ID NO 133 and SEQ ID NO 132; (p) SEQ ID NO 151 and SEQ ID NO 152; or (q) a monoclonal antibody or antibody fragment comprising clone-paired heavy and light chain sequences having at least 70%, 80%, or 90% identity independently with respect to the sequences of SEQ ID NO 137 and SEQ ID NO 128. Claim 8 In any one of claims 2 to 7, the antibody or antibody fragment is (a) SEQ ID NO. 59 and SEQ ID NO. 60; (b) SEQ ID NO. 61 and SEQ ID NO. 62; (c) SEQ ID NO. 63 and SEQ ID NO. 64; or (d) SEQ ID NO. 127 and SEQ ID NO. 128; (e) SEQ ID NO. 129 and SEQ ID NO. 130; (f) SEQ ID NO. 131 and SEQ ID NO. 132; (g) SEQ ID NO. 133 and SEQ ID NO. 130; (h) SEQ ID NO. 127 and SEQ ID NO. 130; (i) SEQ ID NO. 127 and SEQ ID NO. 132; (j) SEQ ID NO. 129 and SEQ ID NO. 128; (k) SEQ ID NO. 129 and SEQ ID NO. 132; (l) SEQ ID NO. 131 and SEQ ID NO. 128; (m) SEQ ID NO. 131 and SEQ ID NO. 130; A monoclonal antibody or antibody fragment comprising a clone-paired heavy chain and light chain sequence having at least 95% identity with respect to the sequences of (n) SEQ ID NO. 133 and SEQ ID NO. 128; (o) SEQ ID NO. 133 and SEQ ID NO. 132; or (p) SEQ ID NO. 151 and SEQ ID NO. 152; or (q) SEQ ID NO. 137 and SEQ ID NO.

128. Claim 9 In any one of claims 2 to 8, the antibody or antibody fragment is (a) SEQ ID NO. 59 and SEQ ID NO. 60; (b) SEQ ID NO. 61 and SEQ ID NO. 62; (c) SEQ ID NO. 63 and SEQ ID NO. 64; or (d) SEQ ID NO. 127 and SEQ ID NO. 128; (e) SEQ ID NO. 129 and SEQ ID NO. 130; (f) SEQ ID NO. 131 and SEQ ID NO. 132; (g) SEQ ID NO. 133 and SEQ ID NO. 130; (h) SEQ ID NO. 127 and SEQ ID NO. 130; (i) SEQ ID NO. 127 and SEQ ID NO. 132; (j) SEQ ID NO. 129 and SEQ ID NO. 128; (k) SEQ ID NO. 129 and SEQ ID NO. 132; (l) SEQ ID NO. 131 and SEQ ID NO. 128; (m) SEQ ID NO. 131 and SEQ ID NO. 130; A monoclonal antibody or antibody fragment comprising (n) SEQ ID NO 133 and SEQ ID NO 128; (o) SEQ ID NO 133 and SEQ ID NO 132; or (p) SEQ ID NO 151 and SEQ ID NO 152; or (q) SEQ ID NO 137 and SEQ ID NO 128, comprising a clone-paired heavy chain and light chain sequence. Claim 10 A monoclonal antibody or antibody fragment that binds to TNFRSF25, wherein the antibody or antibody fragment comprises (a) SEQ ID NO 134 and SEQ ID NO 57; (b) SEQ ID NO 56 and SEQ ID NO 57; (c) SEQ ID NO 58 and SEQ ID NO 57; (d) SEQ ID NO 150 and SEQ ID NO 57; (e) SEQ ID NO 153 and SEQ ID NO 57; (f) SEQ ID NO 151 and SEQ ID NO 152; or (g) a clone-paired heavy chain and light chain sequence of SEQ ID NO 135 and SEQ ID NO 57. Claim 11 In any one of paragraphs 2 to 10, the antibody fragment is a monoclonal antibody or antibody fragment that is a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, an F(ab')₂ fragment, or an Fv fragment. Claim 12 In any one of claims 2 to 11, the antibody fragment is (a) SEQ ID NO 50 and SEQ ID NO 51; (b) SEQ ID NO 52 and SEQ ID NO 53; (c) SEQ ID NO 54 and SEQ ID NO 55; (d) SEQ ID NO 94 and SEQ ID NO 95; (e) SEQ ID NO 96 and SEQ ID NO 97; (f) SEQ ID NO 98 and SEQ ID NO 99; (g) SEQ ID NO 100 and SEQ ID NO 97; (h) SEQ ID NO 94 and SEQ ID NO 97; (i) SEQ ID NO 94 and SEQ ID NO 99; (j) SEQ ID NO 96 and SEQ ID NO 95; (k) SEQ ID NO 96 and SEQ ID NO 99; (l) SEQ ID NO 98 and SEQ ID NO 95; (m) SEQ ID NO 98 and SEQ ID NO 97; (n) SEQ ID NO 100 and SEQ ID NO 95; (o) SEQ ID NO 100 and SEQ ID NO 99; or (p) a monoclonal antibody or antibody fragment comprising a recombinant scFv (single-strand fragment variable) antibody comprising a clonal-paired heavy chain and light chain variable sequence having at least 70%, 80%, or 90% identity independently with respect to the sequences of SEQ ID NO. 92 and SEQ ID NO.

93. Claim 13 In any one of claims 2 to 12, the antibody fragment is (a) SEQ ID NO 50 and SEQ ID NO 51; (b) SEQ ID NO 52 and SEQ ID NO 53; (c) SEQ ID NO 54 and SEQ ID NO 55; (d) SEQ ID NO 94 and SEQ ID NO 95; (e) SEQ ID NO 96 and SEQ ID NO 97; (f) SEQ ID NO 98 and SEQ ID NO 99; (g) SEQ ID NO 100 and SEQ ID NO 97; (h) SEQ ID NO 94 and SEQ ID NO 97; (i) SEQ ID NO 94 and SEQ ID NO 99; (j) SEQ ID NO 96 and SEQ ID NO 95; (k) SEQ ID NO 96 and SEQ ID NO 99; (l) SEQ ID NO 98 and SEQ ID NO 95; (m) SEQ ID NO 98 and SEQ ID NO 97; (n) SEQ ID NO 100 and SEQ ID NO 95; (o) SEQ ID NO 100 and SEQ ID NO 99; or (p) a monoclonal antibody or antibody fragment comprising a recombinant scFv (single-strand fragment variable) antibody comprising a clonal-paired heavy chain and light chain variable sequence having at least 95% identity with respect to the sequences of SEQ ID NO. 92 and SEQ ID NO. 93, respectively. Claim 14 In any one of claims 2 to 13, the antibody fragment is (a) SEQ ID NO 50 and SEQ ID NO 51; (b) SEQ ID NO 52 and SEQ ID NO 53; (c) SEQ ID NO 54 and SEQ ID NO 55; (d) SEQ ID NO 94 and SEQ ID NO 95; (e) SEQ ID NO 96 and SEQ ID NO 97; (f) SEQ ID NO 98 and SEQ ID NO 99; (g) SEQ ID NO 100 and SEQ ID NO 97; (h) SEQ ID NO 94 and SEQ ID NO 97; (i) SEQ ID NO 94 and SEQ ID NO 99; (j) SEQ ID NO 96 and SEQ ID NO 95; (k) SEQ ID NO 96 and SEQ ID NO 99; (l) SEQ ID NO 98 and SEQ ID NO 95; (m) SEQ ID NO 98 and SEQ ID NO 97; (n) SEQ ID NO 100 and SEQ ID NO 95; (o) SEQ ID NO 100 and SEQ ID NO 99; or (p) a monoclonal antibody or antibody fragment comprising a recombinant scFv (single-strand fragment variable) antibody comprising a clone-paired heavy chain and light chain variable sequence of SEQ ID NO. 92 and SEQ ID NO.

93. Claim 15 A monoclonal antibody or antibody fragment according to any one of claims 2 to 14, wherein the antibody fragment is a recombinant scFv (single-chain fragment variable) antibody, wherein the heavy chain and light chain variable sequences are connected by a flexible linker. Claim 16 In paragraph 15, the flexible linker is a monoclonal antibody or antibody fragment that is a Gly-Ser linker. Claim 17 In claim 16, the flexible linker comprises or consists of the sequence GGRGSGGGGSGSGGS (SEQ No. 101) or GGGGSGGGGSGGGGS (SEQ No. 102), a monoclonal antibody or antibody fragment. Claim 18 A monoclonal antibody or antibody fragment, wherein the antibody fragment comprises or consists of a sequence having at least 70%, 80%, or 90% identity with respect to any one of SEQ ID NOs 103 to 126, and is a recombinant scFv (single-strand fragment variable) antibody. Claim 19 A monoclonal antibody or antibody fragment, wherein the antibody fragment comprises or is composed of a sequence having at least 95% identity with respect to any one of SEQ ID NOs 103 to 126, and is a recombinant scFv (single-strand fragment variable) antibody. Claim 20 A monoclonal antibody or antibody fragment, wherein the antibody fragment is a recombinant scFv (single-strand fragment variable) antibody comprising or composed of any one of the sequences of SEQ ID NOs 103 to 126. Claim 21 In any one of paragraphs 2 through 10, the antibody is a monoclonal antibody or antibody fragment that is a chimeric antibody, a biparatopic antibody, or a bispecific antibody. Claim 22 In any one of paragraphs 2 through 10, the antibody is a monoclonal antibody or antibody fragment, which is an IgG antibody or a recombinant IgG antibody or an antibody fragment. Claim 23 A biparatope antibody comprising a first binding portion that binds to a first epitope of human TNFRSF25 and a second binding portion that binds to a second epitope of human TNFRSF25, wherein the first binding portion comprises a clone-paired heavy chain and light chain CDR sequence derived from the clone-paired heavy chain and light chain variable sequences of SEQ ID NO. 48 and SEQ ID NO. 49, and the second binding portion comprises (a) SEQ ID NO. 50 and SEQ ID NO. 51; (b) SEQ ID NO. 52 and SEQ ID NO. 53; or (c) a clone-paired heavy chain and light chain CDR sequence derived from the clone-paired heavy chain and light chain variable sequences of SEQ ID NO. 54 and SEQ ID NO.

55. Claim 24 A double paratop antibody according to claim 23, wherein the first binding portion comprises (a) clone-paired heavy and light chain CDR sequences of SEQ ID NOs 1 to 6 or (b) SEQ ID NOs 25 to 29 and 6, and the second binding portion comprises (a) SEQ ID NOs 7 to 12; (b) SEQ ID NOs 13 to 18; (c) SEQ ID NOs 19 to 24; (d) SEQ ID NOs 30 to 35; (e) SEQ ID NOs 36 to 41; or (f) clone-paired heavy and light chain CDR sequences of SEQ ID NOs 42 to 47. Claim 25 A double paratop antibody according to claim 23 or 24, wherein the first binding portion comprises clone-paired heavy and light chain variable sequences having at least 70%, 80%, or 90% identity independently with respect to the sequences of SEQ ID NO. 48 and SEQ ID NO. 49, and the second binding portion comprises clone-paired heavy and light chain variable sequences having at least 70%, 80%, or 90% identity independently with respect to the sequences of SEQ ID NO. 50 and SEQ ID NO. 51; (b) SEQ ID NO. 52 and SEQ ID NO. 53; or (c) clone-paired heavy and light chain variable sequences having at least 70%, 80%, or 90% identity independently with respect to the sequences of SEQ ID NO. 54 and SEQ ID NO.

55. Claim 26 A double paratop antibody according to any one of claims 23 to 25, wherein the first binding portion comprises clone-paired heavy and light chain variable sequences having at least 95% identity with respect to the sequences of SEQ ID NO. 48 and SEQ ID NO. 49, and the second binding portion comprises clone-paired heavy and light chain variable sequences having at least 95% identity with respect to the sequences of SEQ ID NO. 50 and SEQ ID NO. 51; (b) SEQ ID NO. 52 and SEQ ID NO. 53; or (c) clone-paired heavy and light chain variable sequences having at least 95% identity with respect to the sequences of SEQ ID NO. 54 and SEQ ID NO.

55. Claim 27 A double-paratop antibody according to any one of claims 23 to 26, wherein the first binding portion comprises a clone-paired heavy and light chain variable sequence of SEQ ID NO 48 and SEQ ID NO 49, and the second binding portion comprises (a) SEQ ID NO 50 and SEQ ID NO 51; (b) SEQ ID NO 52 and SEQ ID NO 53; or (c) a clone-paired heavy and light chain variable sequence of SEQ ID NO 54 and SEQ ID NO 55. Claim 28 In any one of claims 23 to 27, the first coupling part comprises (a) SEQ ID NO. 56 and SEQ ID NO. 57; (b) SEQ ID NO. 58 and SEQ ID NO. 57; (c) SEQ ID NO. 65 and SEQ ID NO. 57; or (d) heavy chain and light chain sequences forming clone-pairs having at least 70%, 80%, or 90% identity independently with respect to the sequences of SEQ ID NO. 136 and SEQ ID NO. 57, and the second coupling part comprises (a) SEQ ID NO. 59 and SEQ ID NO. 60; (b) SEQ ID NO. 61 and SEQ ID NO. 62; (c) SEQ ID NO. 63 and SEQ ID NO. 64; (d) SEQ ID NO. 66 and SEQ ID NO. 60; (e) SEQ ID NO. 67 and SEQ ID NO. 62; or (f) a double paratop antibody comprising a clonal-paired heavy chain and light chain sequence having at least 70%, 80% or 90% identity independently with respect to the sequences of SEQ ID NO. 68 and SEQ ID NO.

64. Claim 29 A double paratop antibody according to any one of claims 23 to 28, wherein the first binding portion comprises (a) SEQ ID NO 56 and SEQ ID NO 57; (b) SEQ ID NO 58 and SEQ ID NO 57; (c) SEQ ID NO 65 and SEQ ID NO 57; or (d) clone-paired heavy and light chain sequences having at least 95% identity with respect to the sequences of SEQ ID NO 136 and SEQ ID NO 57, and the second binding portion comprises (a) SEQ ID NO 59 and SEQ ID NO 60; (b) SEQ ID NO 61 and SEQ ID NO 62; (c) SEQ ID NO 63 and SEQ ID NO 64; (d) SEQ ID NO 66 and SEQ ID NO 60; (e) SEQ ID NO 67 and SEQ ID NO 62; or (f) clone-paired heavy and light chain sequences having at least 95% identity with respect to the sequences of SEQ ID NO 68 and SEQ ID NO 64. Claim 30 A double paratop antibody according to any one of claims 23 to 29, wherein the first binding portion comprises (a) SEQ ID NO 56 and SEQ ID NO 57; (b) SEQ ID NO 58 and SEQ ID NO 57; (c) SEQ ID NO 65 and SEQ ID NO 57; or (d) clone-paired heavy and light chain sequences of SEQ ID NO 136 and SEQ ID NO 57, and the second binding portion comprises (a) SEQ ID NO 59 and SEQ ID NO 60; (b) SEQ ID NO 61 and SEQ ID NO 62; (c) SEQ ID NO 63 and SEQ ID NO 64; (d) SEQ ID NO 66 and SEQ ID NO 60; (e) SEQ ID NO 67 and SEQ ID NO 62; or (f) clone-paired heavy and light chain sequences of SEQ ID NO 68 and SEQ ID NO 64. Claim 31 An isolated nucleic acid encoding the first and / or second polypeptide of the polymeric protein of claim 1, the variable region of the antibody heavy chain and / or light chain of the antibody or antibody fragment of any one of claims 2 to 22, the antibody heavy chain and / or light chain region of the antibody or antibody fragment of any one of claims 2 to 22, the scFv of any one of claims 11 to 20, or the first or second binding portion of the biparatopic antibody of any one of claims 23 to 30. Claim 32 An expression vector containing the nucleic acid of claim 31. Claim 33 A hybridoma or engineered cell comprising a nucleic acid encoding an antibody or antibody fragment of any one of paragraphs 2 to 22 or a biparatopic antibody of any one of paragraphs 23 to 30. Claim 34 A hybridoma or engineered cell containing the nucleic acid of paragraph 31. Claim 35 A method for producing a monoclonal antibody or antibody fragment of any one of claims 2 to 22 or a biparatop antibody of any one of claims 23 to 30, the method comprising the step of culturing a hybridoma or engineered cell of claim 33 or 34 under conditions allowing the expression of the antibody or antibody fragment or biparatop antibody, and optionally isolating the antibody or antibody fragment or biparatop antibody from the culture. Claim 36 A pharmaceutical formulation comprising the polymeric protein of claim 1, one or more antibodies or antibody fragments of any one of claims 2 to 22, or a biparatopic antibody of any one of claims 23 to 30. Claim 37 A method for treating a patient having a disease or disorder related to inflammation and / or autoimmunity, the method comprising the step of administering the pharmaceutical formulation of claim 36 to the patient. Claim 38 In paragraph 37, the disease or disorder is ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriasis, atherosclerosis, asthma, multiple sclerosis, primary biliary cirrhosis, systemic lupus erythematosus, or ankylosing spondylitis. Claim 39 In paragraph 37 or 38, the administration is a method that preserves the integrity of the epithelial barrier. Claim 40 A polymeric protein of claim 1, an antibody or antibody fragment of any one of claims 2 to 22, a biparatope antibody of any one of claims 23 to 30, or a pharmaceutical formulation of claim 36 for use in treating or preventing diseases or disorders related to inflammation and / or autoimmunity in patients. Claim 41 The use of a polymeric protein of claim 1, an antibody or antibody fragment of any one of claims 2 to 22, or a biparatopic antibody of any one of claims 23 to 30 in the manufacture of a drug for treating or preventing diseases or disorders related to inflammation and / or autoimmunity.

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