RELATED APPLICATION

[0001] The present disclosure claims priority to U.S. Provisional Application No.

62/781668, filed December 19, 2018, which is incorporated by reference herein in its entirety.

FIELD

[0002] The present disclosure is directed to anti-tumor necrosis factor (TNF)-alpha antibodies and antigen-binding fragments thereof, and their uses for treating pathological disorder mediated by TNF-alpha, e.g., autoimmune diseases or inflammatory disorders or conditions, e.g., inflammatory arthritis (e.g., rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis) .

BACKGROUND

[0003] Biological molecules are increasingly becoming part of the portfolio of biotechnology and pharmaceutical companies. This is based on their ability to be highly specific relative to small molecules. However, biological therapeutics run the risk of being recognized as foreign by a host immune system. Indeed, monoclonal antibodies have become a very important class of biological drugs, but there is an ongoing concern with the use of antibodies due to their potential immunogenicity. Some patients receiving these drugs produce anti-drug antibodies (ADAs) which are associated with various potential clinical consequences.

[0004] Five anti-tumor necrosis factor (TNF)-alpha biologic agents, including adalimumab, are currently approved by both the FDA and EMA for the treatment of rheumatoid arthritis (van Schouwenburg et al. (2013) Nat Rev Rheumatol. 9(3): 164-72). In some patients, the antibody adalimumab triggers an immune response that results in the formation of ADAs. Formation of ADAs is one of the causes of clinical non-response to treatment with adalimumab, and has been described in patients with rheumatoid arthritis (van de Putte et al. (2004) Ann. Rheum. Dis. 63:508-516). Hence, there is a need for modified TNF-alpha antibodies and antigen-binding fragments thereof with reduced immunogenicity relative to wild-type adalimumab.

[0005] Many factors influence the immunogenicity of anti-TNF -alpha biologic agents

- the drug' s characteristics and biophysical properties, the patient' s immune system activity, dose, duration, and administration route. Insight into how TNF-alpha therapeutic antibodies evoke an immune response is expected to lead to the development of strategies to minimize the adverse events of adalimumab.

SUMMARY OF THE INVENTION

[0006] The characteristics of patient-derived monoclonal antibodies for binding to adalimumab is described in van Schouwenburg et al. (2014) Jml. Biol. Chem. 289(50): 34482- 88. In particular, van Schouwenburg et al. (2014) analyzed the variable region sequences of sixteen B-cell clones, and used these sequences to generate a panel of eleven ADAs. These adalimumab ADAs were recombinantly expressed, and their affinities and specificities to adalimumab and adalimumab single-point mutants were investigated van Schouwenburg et al. (2014) show that, in ADA-positive patients, all ADAs were directed against a small region on adalimumab - a few hot spots in the framework regions and positions in the light chain and heavy chain CDRs. Nevertheless, van Schouwenburg et al. (2014) note that it may prove impossible to eliminate one or more of these immunogenic determinants without impairing TNF -alpha binding or introducing new immunogenic epitopes on adalimumab (van Schouwenburg et al. (2014), p. 34486).

[0007] We have now determined that it is possible to modify the variable regions of adalimumab to reduce immunogenicity, while still retaining TNF-alpha binding activity. In particular, spotted positions and regions were "back-mutated," i.e., one or more amino acid residues were mutated to the germline sequence of the corresponding framework groups.

These residues were identified by comparing the antibody framework sequences to the germline sequences from which the antibody was derived. Positions 36 and 37 in the adalimumab heavy chain variable region (VH) (numbering according to IMGT) and positions 67, 68 and 69 in the adalimumab light chain variable region (VL) were considered as main binding positions for adalimumab ADAs. However, site-directed mutagenesis of other positions in adalimumab, e.g., in VH 165, S I 14 and VL G28, V99 also have good effects on reducing adalimumab binding to ADAs.

[0008] Accordingly, the present disclosure is directed to anti-tumor necrosis factor

(TNF)-alpha antibodies and antigen-binding fragments thereof that are based on the binding regions of adalimumab (the active ingredient in HUMIRA). These variant antibodies and antigen-binding fragments thereof have mutations in the heavy chain variable region (VH), the light chain variable region (VL), or both the VH and VL relative to wild-type adalimumab. These mutations result in antibodies and antigen-binding fragments with reduced

immunogenicity and other desirable properties relative to wild-type adalimumab.

[0009] Disclosed herein are antibodies to human tumor necrosis factor (TNF)-alpha or antigen-binding fragments thereof, comprising: a) a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO:26; and b) a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 11, wherein at least one of the Xaa amino acids in SEQ ID NO:26 differs from the corresponding amino acid in the same position of an amino acid sequence as set forth in SEQ ID NO:4.

[0010] Additionally disclosed herein are antibodies to human TNF-alpha or antigen binding fragments thereof, comprising: a) a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO:4; and b) a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO:28, wherein at least one of the Xaa amino acids in SEQ ID NO:28 differs from the corresponding amino acid in the same position of an amino acid sequence as set forth in SEQ ID NO: 11.

[0011] Additionally disclosed herein are antibodies to human TNF-alpha or antigen binding fragments thereof, comprising: a) a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO:26; and b) a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO:28, wherein at least one of the Xaa amino acids in SEQ ID NO:26 differs from the corresponding amino acid in the same position of an amino acid sequence as set forth in SEQ ID NO:4 and at least one of the Xaa amino acids in SEQ ID NO:28 differs from the corresponding amino acid in the same position of an amino acid sequence as set forth in SEQ ID NO: 11.

[0012] Additionally disclosed herein are antibodies to human TNF-alpha or antigen binding fragments thereof, comprising a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO:4 and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 11, wherein the VH is mutated at one or more of positions 31, 32, 58 and 107 of SEQ ID NO:4.

[0013] Additionally disclosed herein are antibodies to human TNF-alpha or antigen binding fragments thereof, comprising a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO:4 and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 11, wherein the VL is mutated at one or more of positions 28, 53, 54, 55, 56, and 83 of SEQ ID NO: 11. [0014] Additionally disclosed herein are antibodies to human TNF-alpha, comprising a heavy chain variable region (VH), comprising an amino acid sequence as set forth in SEQ ID NO:4, and a light chain variable region (VL), comprising an amino acid sequence as set forth in SEQ ID NO: 11, wherein: a) the VH is mutated at one or more of positions 31, 32, 58 and 107 of SEQ ID NO:4; and b) the VL is mutated at one or more of positions 28, 53, 54, 55, 56, and 83 of SEQ ID NO: 11.

[0015] Additionally disclosed herein are antibodies to human TNF-alpha, comprising a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 6 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 13, wherein the heavy chain is mutated at one or more of positions 31, 32, 58 and 107 of SEQ ID NO:6

[0016] Additionally disclosed herein are antibodies to human TNF-alpha, comprising a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 6 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 13, wherein the light chain is mutated at one or more of positions 28, 53, 54, 55, 56 and 83 of SEQ ID NO: 13.

[0017] Additionally disclosed herein are antibodies to human TNF-alpha, comprising a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 6 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 13, wherein: a) the heavy chain is mutated at one or more of positions 31, 32, 58 and 107 of SEQ ID NO:6; and b) the light chain is mutated at one or more of positions 28, 53, 54, 55, 56 and 83 of SEQ ID NO: 13.

[0018] Additionally disclosed herein are antibodies to human TNF-alpha or antigen binding fragments thereof, comprising a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO:4 and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 11, wherein the VH is mutated at one or more of IMGT positions 36, 37, 65 and 114.

[0019] Additionally disclosed herein are antibodies to human TNF-alpha or antigen binding fragments thereof, comprising a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO:4 and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 11, wherein the VL is mutated at one or more of IMGT positions 28, 66, 67, 68, 69, and 99.

[0020] Additionally disclosed herein are antibodies to human TNF-alpha or antigen binding fragments thereof, comprising a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO:4 and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 11, wherein: a) the VH is mutated at one or more of IMGT positions 36, 37, 65 and 114; and b) the VL is mutated at one or more of IMGT positions 28, 66, 67, 68, 69 and 99.

[0021] Additionally disclosed herein are antibodies to human TNF-alpha, comprising a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 6 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 13, wherein the heavy chain is mutated at one or more of IMGT positions 36, 37, 65 and 114.

[0022] Additionally disclosed herein are antibodies to human TNF-alpha, comprising a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 6 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 13, wherein the light chain is mutated at one or more of IMGT positions 28, 66, 67, 68, 69 and 99.

[0023] Additionally disclosed herein are antibodies to human TNF-alpha, comprising a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 6 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 13, wherein: a) the heavy chain is mutated at one or more of IMGT positions 36, 37, 65 and 114; and b) the light chain is mutated at one or more of IMGT positions 28, 66, 67, 68, 69 and 99.

[0024] In preferred embodiments, the antibodies to human TNF-alpha or antigen binding fragments thereof comprise a heavy chain variable region (VH) and a light chain variable region (VL) having one of the mutations set forth in Table 1. In preferred embodiments, the antibodies to human TNF-alpha or antigen-binding fragments thereof comprise a heavy chain variable region (VH) and a light chain variable region (VL) set forth in a row of Table 2.

[0025] Additionally disclosed herein are antibodies to human TNF-alpha or antigen binding fragments thereof, comprising: a) a heavy chain variable region (VH) comprising, in sequence, a complementarity determining region (CDR) having an amino acid sequence as set forth in SEQ ID NO: 15; a CDR having an amino acid sequence as set forth in SEQ ID NO:2; and a CDR having an amino acid sequence as set forth in SEQ ID NO:3; and b) a light chain variable region (VL) comprising, in sequence, a CDR having an amino acid sequence as set forth in SEQ ID NO:8; a CDR having an amino acid sequence as set forth in SEQ ID NO:9; and a CDR having an amino acid sequence as set forth in SEQ ID NO: 10.

[0026] Additionally disclosed herein are pharmaceutical compositions, comprising an antibody or antigen-binding fragment thereof according to the disclosure. [0027] Additionally disclosed herein are uses of the disclosed antibodies or antigen binding fragments thereof in the manufacture of a medicament for use in the treatment of a pathological disorder mediated by TNF -alpha.

[0028] Additionally disclosed herein are uses of the disclosed pharmaceutical compositions in the manufacture of a medicament for use in the treatment of a pathological disorder mediated by TNF -alpha.

[0029] Additionally disclosed herein are methods of treating a pathological disorder mediated by TNF -alpha, said method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the disclosure to a subject in need thereof.

[0030] Additionally disclosed herein are methods of treating a pathological disorder mediated by TNF -alpha, said method comprising administering a pharmaceutical composition according to the disclosure to a subject in need thereof.

[0031] Additionally disclosed herein are isolated nucleic acid molecules encoding the disclosed antibodies and antigen-binding fragments thereof, cloning and expression vectors comprising one or more of the aforesaid nucleic acid molecules, host cell comprising one or more of the aforesaid cloning or expression vectors, and processes for the production of an antibody or antigen-binding fragment thereof according to the disclosure.

[0032] Additionally disclosed herein are diagnostic reagents comprising an antibody or antigen-binding fragment thereof according to the disclosure and a diagnostic label.

[0033] Additionally disclosed herein are kits comprising an antibody or antigen binding fragment thereof according to the disclosure or a pharmaceutical composition according to the disclosure.

[0034] Additional embodiments of the invention are provided in the following sections.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 shows the relative binding of adalimumab antibody variants 4 and 40 in comparison to adalimumab. Adalimumab is normalized and set to 100% relative binding. Variant 4 (4%) and 40 (8%) have significantly lower binding to ADAs.

[0036] Figure 2 shows increased hydrodynamic radius of adalimumab and adalimumab antibody variants upon thermal stress at a concentration of 10 mg/ml. Adalimumab (1) shows increased hydrodynamic radius starting at 50°C, whereas variants (4) and (40) show no significant change in hydrodynamic radius.

[0037] Figure 3 shows the expression yields of adalimumab and adalimumab antibody variants 4 and 40 before and after size exclusion chromatography. Variant 4 and 40 show higher expression in HEK293T cells after size exclusion chromatography.

[0038] Figure 4A-C shows temperature dependent hydrodynamic radius profiles of antibodies at concentrations of 40, 70 and 100 mg/ml. The profile of adalimumab is in

Figure 4A, the profile of variant 4 is in Figure 4B, and the profile of variant 40 is in Figure 4C.

DETAILED DESCRIPTION

General Matters

[0039] In order that the present invention may be more readily understood, certain terms are defined throughout the detailed description. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains.

[0040] Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings:

[0041] Tumor necrosis factor-alpha (TNF-alpha), also known as cachectin, is a well- known cytokine that plays a role in antitumor activity, immune modulation, inflammation, anorexia, cachexia, septic shock, viral replication, and hematopoiesis. TNF-alpha is expressed by a variety of cells. As used herein,“TNF-alpha” refers to human TNF-alpha. Further information on human TNF-alpha, including structure and sequence information, may be found at UniProtKB - P01375 and in various references, such Pennica et al. (1984) Nature 312:724-29, Shirai et a. (1985) Nature 313:803-06; Wang et a. (1985) Science 228: 149-54.

[0042] An amino acid given as“X” or“Xaa” can be any of the substituted amino acids in the respective position given in column 3 of Table 1, as well as the wild-type amino acid in the respective position given in column 2 of Table 1.

[0043] The term“antibody” as used herein refers to a polypeptide of the

immunoglobulin family that is capable of binding a corresponding antigen non-covalently, reversibly, and in a specific manner. For example, a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (U) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system ( e.g ., effector cells) and the first component (Clq) of the classical complement system.

[0044] The term“antibody” includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, and anti- idiotypic (anti -Id) antibodies (including, e.g., anti -Id antibodies to antibodies of the present disclosure). The antibodies can be of any isotype/class (e.g., IgG, IgE, IgM, IgD, IgA and IgY), or subclass (e.g., IgGi, IgG2, IgG3, IgG4, IgAi and IgAi).

[0045] “Complementarity-determining domains” or“complementarity-determining regions” (“CDRs”) interchangeably refer to the hypervariable regions ofVL and VH . The CDRs are the target protein-binding site of the antibody chains that harbors specificity for such target protein. There are three CDRs (CDRl-3, numbered sequentially from the N- terminus) in each human VL or VH , constituting in total about 15-20% of the variable domains. CDRs can be referred to by their region and order. For example,“VHCDR1” or “HCDRl” both refer to the first CDR of the heavy chain variable region. The CDRs are structurally complementary to the epitope of the target protein and are thus directly responsible for the binding specificity. The remaining stretches of the VL or VH , the so- called framework regions, exhibit less variation in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. W.H. Freeman & Co., New York, 2000).

[0046] The positions of the CDRs and framework regions can be determined using various well-known definitions in the art, e.g., Rabat, Chothia, IMGT, AbM, and combined definitions (see, e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al. , Nature, 342:877-883 (1989); Chothia et al, J. Mol. Biol., 227:799-817 (1992); Lefranc, M.P., Nucleic Acids Res., 29:207-209 (2001); Al-Lazikani et al. , J.Mol.Biol., 273:927-748 (1997)). Definitions of antigen combining sites are also described in the following: Ruiz et al., Nucleic Acids Res., 28:219- 221 (2000); MacCallum et al, J. Mol. Biol., 262:732-745 (1996); and Martin et al , Proc.

Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al , Methods Enzymol., 203: 121-153 (1991); and Rees et al, In Sternberg M.J.E. (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). In a combined Rabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to the amino acid residues that are part of a Rabat CDR, a Chothia CDR, or both. For instance, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in a VH, e.g., a mammalian VH , e.g., a human VH; and amino acid residues 24-34 (LCDR1), SO SO (LCDR2), and 89-97 (LCDR3) in a VL, e.g., a mammalian VL, e.g., a human VL. Under IMGT the CDR amino acid residues in the VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3) (numbering according to “Rabat”). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT/DomainGap Align.

[0047] Both the light and heavy chains are divided into regions of structural and functional homology. The terms“constant” and“variable” are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VL ) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CHI, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention, the numbering of the constant region domains increases as they become more distal from the antigen-binding site or amino- terminus of the antibody. The N-terminus is a variable region and at the C-terminus is a constant region; the CH3 and CL domains actually comprise the carboxy-terminal domains of the heavy and light chain, respectively.

[0048] The term“antigen-binding fragment,” as used herein, refers to one or more portions of an antibody that retain the ability to specifically interact with (e.g. , by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen. Examples of binding fragments include, but are not limited to, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), F(ab)2 fragment, Fab fragment, F(ab’)2, fragment F(ab') fragments, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of the VH and CHI domains (and optionally a portion of the hinge); an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., Nature 341:544-546, 1989), which consists of a VH domain; and an isolated complementarity determining region (CDR), or other epitope-binding fragments of an antibody.

[0049] Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (“scFv”); see, e.g., Bird et al., Science 242:423-426, 1988; and Huston et al, Proc. Natl. Acad. Sci. 85:5879-5883, 1988). Such single chain antibodies are also intended to be encompassed within the term“antigen binding fragment.” Often there is a peptide linker between the VH and VL domains. In preferred embodiments, scFvs of the disclosure have the general structures: MU-VL-linker- VH-COOH or NH2-VH-linker-VL-COOH. These antigen-binding fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0050] Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen-binding fragments can be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies).

[0051] Antigen-binding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen-binding regions (Zapata et al. , Protein Eng. 8: 1057-1062, 1995; and U.S. Pat. No. 5,641,870).

[0052] The term "monoclonal antibody" or "Mab" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies of the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. Such monoclonal antibody may be produced by a single clone of B cells or hybridoma. Monoclonal antibodies may also be recombinant, i.e., produced by protein engineering. Monoclonal antibodies may also be isolated from phage antibody libraries. In addition, in contrast with preparations of polyclonal antibodies, which typically include various antibodies directed against various determinants, or epitopes, each monoclonal antibody is directed against a single epitope of the antigen. Methods for generation of monoclonal antibodies using phage display technology are known in the art (Proetzel, G., Ebersbach, H. (Eds.) Antibody Methods and Protocols. Humana Press ISBN 978-1-61779-930-3; 2012). In preferred embodiments, the anti -TNF -alpha antibody is a monoclonal antibody.

[0053] The term“human antibody,” as used herein, includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al, J. Mol. Biol. 296:57-86, 2000). In a preferred embodiment, the anti-TNF- alpha antibody is a human antibody.

[0054] The human antibodies of the present disclosure can include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site- specific mutagenesis in vitro or by somatic mutation in vivo, or a conservative substitution to promote stability or manufacturing).

[0055] A "humanized" antibody is an antibody that retains the reactivity of a non human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts (i.e., the constant region as well as the framework portions of the variable region). See, e.g., Morrison et al. 1984, Proc. Natl. Acad. Sci. USA, 81:6851- 6855; Morrison and Oi, 1988, Adv. Immunol., 44:65-92; Verhoeyen et al. 1988, Science, 239: 1534-1536; Padlan 1991, Molec. Immun., 28:489-498; and Padlan 1994, Molec. Immun., 31: 169-217. Other examples of human engineering technology include, but are not limited to Xoma technology disclosed in US 5,766,886. In some embodiments, the anti -TNF -alpha antibody is a humanized or chimeric antibody.

[0056] Chimeric or humanized antibodies of the present disclosure can be prepared based on the sequence of a murine monoclonal antibody prepared as described above. DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, the murine variable regions can be linked to human constant regions using methods known in the art (see e.g., U.S. Patent No. 4,816,567 to Cabilly et ah). To create a humanized antibody, the murine CDR regions can be inserted into a human framework using methods known in the art. See e.g., U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.

[0057] The term“recognize” as used herein refers to an antibody or antigen-binding fragment thereof that finds and interacts (e.g., binds) with its epitope, whether that epitope is linear or conformational. The term“epitope” refers to a site on an antigen to which an antibody or antigen-binding fragment of the disclosure specifically binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3,

4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation.

Methods of determining spatial conformation of epitopes include techniques in the art, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)), or electron microscopy. A“paratope” is the part of the antibody which recognizes the epitope of the antigen.

[0058] The phrase“specifically binds” or“selectively binds,” when used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody, antibody fragment, or antibody-derived binding agent, refers to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biologies, e.g. , in a biological sample, e.g. , a blood, serum, plasma or tissue sample. For example, specific binding can be determined by a competition assay, e.g., a competitive ELISA. In some competition assays, specific binding is indicated if the binding of a labeled target to a probe is competitively inhibited by excess unlabeled target. Thus, under certain designated immunoassay conditions, the antibodies or binding agents with a particular binding specificity bind to a particular antigen at least two times the background and do not substantially bind in a significant amount to other antigens present in the sample. In one aspect, under designated immunoassay conditions, the antibody or binding agent with a particular binding specificity binds to a particular antigen at least ten (10) times the background and does not substantially bind in a significant amount to other antigens present in the sample. Specific binding to an antibody or binding agent under such conditions may require the antibody or agent to have been selected for its specificity for a particular protein. As desired or appropriate, this selection may be achieved by subtracting out antibodies that cross-react with molecules from other species (e.g., mouse or rat) or other subtypes.

Alternatively, in some aspects, antibodies or antibody fragments are selected that cross-react with certain desired molecules.

[0059] In some embodiments, specific binding of an antibody or antigen-binding fragment of the disclosure means binding with an equilibrium constant (KA) of at least 10 ² M ³ , at least 5xl0 ² M ^_1 , at least 10 ³ M ^_1 , at least 5xl0 ³ M ^_1 , at least 10 ⁴ M 'at least 5xl0 ⁴ M ^_1 , at least 10 ⁵ M ^_1 , at least 5xl0 ⁵ M ^_1 , at least 10 ⁶ M ^_1 , at least 5xl0 ⁶ M ^_1 , at least lCfiM ¹ , at least 5xl0 ⁷ M ^_1 , at least 10 ⁸ M ^_1 , at least 5xl0 ⁸ M ^_1 , at least 10 ⁹ M ^_1 , at least 5xl0 ⁹ M ^_1 , at least 10 ¹⁰ M ³ , at least 5c10 ¹⁰ M ^_1 , at least KWh/! ¹ , at least SXKWM ¹ , at least 10 ¹² M ^_1 , at least 5xl0 ¹² M ^_1 , at least 10 ¹³ M ^_1 , at least 5xl0 ¹³ M ¹ , at least 10 ¹⁴ M ^_1 , at least 5xl0 ¹⁴ M ^_1 , at least 10 ¹⁵ M ^_1 , or at least 5xl0 ¹⁵ M ^_1 .

[0060] In some embodiments, specific binding of an antibody or antigen-binding fragment of the disclosure means a dissociation rate constant (KD) of less than 5xlO ² M, less than 10 ² M, less than 5xl0 ³ M, less than 10 ³ M, less than 5xlO ⁴ M, less than 10 ⁴ M, less than 5xlO ⁵ M, less than 10 ⁵ M, less than 5xlO ⁶ M, less than 10 ⁶ M, less than 5xlO ⁷ M, less than 10 ⁷ M, less than 5xlO ⁸ M, less than 10 ⁸ M, less than 5xlO ⁹ M, less than 10 ⁹ M, less than 5x10 ¹⁰ M, less than 10 ¹⁰ M, less than 5xl0 ⁿ M, less than 10 ⁿ M, less than 5xlO ¹² M, less than 10 ¹² M, less than 5xl0 ¹³ M, less than 10 ¹³ M, less than 5xlO ¹⁴ M, less than 10 ¹⁴ M, less than 5xlO ¹⁵ M, or less than 10 ¹⁵ M or lower, and binds to the target antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., HSA).

[0061] The term“affinity” as used herein refers to the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody“arm” interacts through weak non-covalent forces with antigen at numerous sites; the more interactions, the stronger the affinity. As used herein, the term "high affinity" for an IgG antibody or fragment thereof (e.g., a Fab fragment) refers to an antibody having a KD of 10 ⁸ M or less, 10 ⁹ M or less, or 10 ¹⁰ M, or 10 ¹¹ M or less, or 10 ¹² M or less, or 10 ¹³ M or less for a target antigen. However, high affinity binding can vary for other antibody isotypes. For example, high affinity binding for an IgM isotype refers to an antibody having a KD of 10 ⁷ M or less, or 10 ⁸ M or less. One suitable assay for measuring affinity, e.g., KD involves the use of the BIACORE technology (e.g. by using the BIACORE 3000 instrument (BIACORE, Uppsala, Sweden)), which can measure the extent of interactions using surface plasmon resonance technology.

[0062] As used herein, the term "avidity" refers to an informative measure of the overall stability or strength of the antibody-antigen complex. It is controlled by three major factors: antibody epitope affinity; the valence of both the antigen and antibody; and the structural arrangement of the interacting parts. Ultimately these factors define the specificity of the antibody, that is, the likelihood that the particular antibody is binding to a precise antigen epitope.

[0063] The term“isolated antibody” refers to an antibody that is substantially free of other antibodies having different antigenic specificities. An isolated antibody that specifically binds to one antigen may, however, have cross-reactivity to other antigens. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

[0064] The term“corresponding human germline sequence” refers to the nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other all other known or inferred variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. The corresponding human germline sequence can also refer to the human variable region amino acid sequence or subsequence with the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other evaluated variable region amino acid sequences. The corresponding human germline sequence can be framework regions only, complementarity determining regions only, framework and complementary determining regions, a variable segment (as defined above), or other combinations of sequences or subsequences that comprise a variable region. Sequence identity can be determined using the methods described herein, for example, aligning two sequences using BUAST, AUIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91% 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference variable region nucleic acid or amino acid sequence.

[0065] A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase EUISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective binding reaction will produce a signal at least twice over the background signal and, more typically, at least 10 to 100 times over the background.

[0066] The term“equilibrium dissociation constant (KD, M)” refers to the dissociation rate constant (kd, time ¹ ) divided by the association rate constant (k _a , time ¹ , M ¹ ). Equilibrium dissociation constants can be measured using any known method in the art. The antibodies and fragments of the present disclosure generally will have an equilibrium dissociation constant of less than about 10 ⁶ or 10 ⁷ or 10 ⁸ M, for example, less than about 10 ⁹ M or 10 ¹⁰ M, in some aspects, less than about 10 ¹¹ M, 10 ¹² M or 10 ¹³ M.

[0067] The term "bioavailability" refers to the systemic availability (i.e., blood/plasma levels) of a given amount of drug administered to a patient. Bioavailability is an absolute term that indicates measurement of both the time (rate) and total amount (extent) of drug that reaches the general circulation from an administered dosage form.

[0068] A "modification" or“mutation” of an amino acid residue/position, as used herein, refers to a change of a primary amino acid sequence as compared to a starting amino acid sequence (e.g., a wild-type sequence), wherein the change results from a sequence alteration involving said amino acid residue/positions. For example, typical modifications include substitution of the residue (or at said position) with another amino acid (e.g., a conservative or non-conservative substitution), insertion of one or more amino acids adjacent to said residue/position, and deletion of said residue/position. An "amino acid mutation," or variation thereof, refers to the replacement of an existing amino acid residue in a

predetermined (starting) amino acid sequence with a different amino acid residue. Generally and preferably, the modification results in alteration in at least one physicobiochemical activity of the variant polypeptide compared to a polypeptide comprising the starting (or "wild type") amino acid sequence. For example, in the case of an antibody, a physicobiochemical activity that is altered can be binding affinity, binding capability and/or binding effect upon a target molecule.

[0069] The term“comprising” encompasses“including” as well as“consisting,” e.g., a composition“comprising” X may consist exclusively of X or may include something additional, e.g., X + Y. [0070] Unless otherwise specifically stated or clear from context, as used herein, the term“about” in relation to a numerical value is understood as being within the normal tolerance in the art, e.g., within two standard deviations of the mean. Thus,“about” can be within +/-10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.05%, or 0.01% of the stated value, preferably +/-10% of the stated value. When used in front of a numerical range or list of numbers, the term“about” applies to each number in the series, e.g., the phrase“about 1-5” should be interpreted as“about 1 - about 5”, or, e.g., the phrase“about 1, 2, 3, 4” should be interpreted as“about 1, about 2, about 3, about 4, etc.”

[0071] The phrase“diagnostic label” refers to labels for use in diagnosing a subject with a particular disease, monitoring patient treatment, monitoring and/or assessing the level of TNF-alpha in a particular sample, or for use in a research setting. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction. A diagnostic label (e.g., a chemiluminescent tag) may be combined with a disclosed anti -TNF-alpha antibody or antigen-binding fragment thereof to produce a diagnostic reagent.

[0072] As used herein,“selecting” and“selected” in reference to a patient is used to mean that a particular patient is specifically chosen from a larger group of patients due to the particular patient having a predetermined criterion. Similarly,“selectively treating a patient” refers to providing treatment to a patient that is specifically chosen from a larger group of patients due to the particular patient having a predetermined criteria. Similarly,“selectively administering” refers to administering a drug to a patient that is specifically chosen from a larger group of patients due to the particular patient having a predetermined criterion.

[0073] The word“substantially” does not exclude“completely,” e.g., a composition which is“substantially free” from Y may be completely free from Y. Where necessary, the word“substantially” may be omitted from the definition of the disclosure.

[0074] As used herein, the phrase“consisting essentially of’ refers to the genera or species of active pharmaceutical agents included in a method or composition, as well as any excipients inactive for the intended purpose of the methods or compositions. In some aspects, the phrase“consisting essentially of’ expressly excludes the inclusion of one or more additional active agents other than an anti -TNF-alpha antibody or fragment of the present disclosure. In some aspects, the phrase“consisting essentially of’ expressly excludes the inclusion of one or more additional active agents other than an anti -TNF -alpha antibody or fragment of the present disclosure and a second co-administered agent.

[0075] The term“amino acid” refers to naturally occurring, synthetic, and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, g-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.

Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0076] The term“conservatively modified variant” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are“silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0077] For polypeptide sequences,“conservatively modified variants” include individual substitutions, deletions or additions to a polypeptide sequence which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such

conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles. The following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (L), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)). In some aspects, the term "conservative sequence modifications" are used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.

[0078] The term“optimized” as used herein refers to a nucleotide sequence that has been altered to encode an amino acid sequence using codons that are preferred in the production cell or organism, generally a eukaryotic cell, for example, a yeast cell, a Pichia cell, a fungal cell, a Trichoderma cell, a Chinese Hamster Ovary cell (CHO) or a human cell. The optimized nucleotide sequence is engineered to retain completely or as much as possible the amino acid sequence originally encoded by the starting nucleotide sequence, which is also known as the“parental” sequence.

[0079] The terms“percent identical” or“percent identity,” in the context of two or more nucleic acids or polypeptide sequences, refers to the extent to which two or more sequences or subsequences that are the same. Two sequences are“identical” if they have the same sequence of amino acids or nucleotides over the region being compared. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 30 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0080] Lor sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0081] A“comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c (1970), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g. , Brent et al, Current Protocols in Molecular Biology, 2003).

[0082] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BEAST and BEAST 2.0 algorithms, which are described in Altschul et al, Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al , J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as a basis for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative -scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0083] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873- 5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0084] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, (Comput. Appl. Biosci. 4: 11-17, 1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch, (J. Mol. Biol. 48:444-453, 1970), algorithm which has been incorporated into the GAP program in the GCG software package (available from University of South Florida), using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10,

8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0085] Other than percentage of sequence identity noted above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0086] The term“nucleic acid” is used herein interchangeably with the term

“polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Examples of nucleic acids that are part of the disclosure include cDNA, genomic DNA, recombinant DNA, and RNA (e.g., mRNA). The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0087] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, as detailed below, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J. Biol. Chem. 260:2605-2608; and Rossolini et al, (1994) Mol. Cell. Probes 8:91-98).

[0088] The term“operably linked” in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some

transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

[0089] The terms“polypeptide” and“protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0090] The term“subject” includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, and reptiles. Except when noted, the terms “patient” or“subject” are used herein interchangeably.

[0091] As used herein, phrases such as "a patient in need of treatment" or "a subject in need of treatment" includes subjects, such as mammalian subjects, that would benefit from administration of an antibody or composition of the present disclosure used, e.g., for detection, for a diagnostic procedure and/or for treatment.

[0092] “IC50” (half-maximal inhibitory concentration) refers to the concentration of a particular antibody or fragment thereof which inhibits a signal halfway (50%) between the baseline control and the maximum possible signal.

[0093] “EC50” (half-maximal effective concentration) refers to the concentration of a particular antibody or fragment thereof which induces a response halfway (50%) between the baseline control and the maximum possible effect after a specific exposure or treatment time. For example, the EC50 is the concentration of antibody at which virus infection is reduced by 50%.

[0094] “EC90” refers to the concentration of a particular antibody or fragment thereof which induces a response corresponding to 90% of the maximum possible effect after a specific exposure or treatment time. For example, the EC90 is the concentration of an antibody or fragment thereof at which virus infection is reduced by 90%.

[0095] The term "treatment" or“treat” is herein defined as the application or administration of a TNF -alpha antibody or antigen-binding fragment according to the disclosure, or a pharmaceutical composition comprising said anti-TNF -alpha antibody, to a subject or to an isolated tissue or cell line from a subject, where the subject has a particular disease (e.g., arthritis), a symptom associated with the disease, or a predisposition towards development of the disease (if applicable), where the purpose is to cure (if applicable), delay the onset of, reduce the severity of, alleviate, ameliorate one or more symptoms of the disease, improve the disease, reduce or improve any associated symptoms of the disease or the predisposition toward the development of the disease. The term“treatment” or“treat” includes treating a patient suspected to have the disease as well as patients who are ill or who have been diagnosed as suffering from the disease or medical condition, and includes suppression of clinical relapse. The phrase“reducing the likelihood” refers to delaying the onset or development or progression of a disease, infection or disorder.

[0096] The term“therapeutically acceptable amount” or“therapeutically effective amount” or“therapeutically effective dose” interchangeably refer to an amount sufficient to effect the desired result (e.g., a reduction in disease activity, inhibition of disease progression, etc.). In some aspects, a therapeutically acceptable amount does not induce or cause undesirable side effects. A therapeutically acceptable amount can be determined by first administering a low dose, and then incrementally increasing that dose until the desired effect is achieved. A“prophylactically effective dosage,” and a“therapeutically effective dosage,” of the molecules of the present disclosure can prevent the onset of, or result in a decrease in severity of, respectively, disease symptoms, including symptoms associated with TNF-alpha activity.

[0097] The term“co-administer” refers to the simultaneous presence of two active agents in the blood of an individual. Active agents (e.g., additional therapeutic agents) that are co-administered with the disclosed antibodies and antigen-binding fragments can be concurrently or sequentially delivered.

Recombinant anti-TNF-alpha antibodies

[0098] The present disclosure provides antibodies and antibody fragments (e.g., antigen-binding fragments), that bind to TNF-alpha and reduce TNF-alpha activity, signal, etc. Furthermore, the present disclosure provides antibodies that have desirable

pharmacokinetic characteristics and other desirable attributes, and thus can be used for treating a pathological disorder mediated by TNF-alpha, e.g., an autoimmune disease or an inflammatory disorder. The present disclosure further provides pharmaceutical compositions comprising the antibodies and/or fragments thereof and methods of making and using such pharmaceutical compositions for the prevention and treatment of a pathological disorder mediated by TNF-alpha, e.g., an autoimmune disease or an inflammatory disorder.

[0099] The present disclosure provides for antibodies or antigen-binding fragments thereof that specifically bind to a TNF-alpha. Antibodies or antigen-binding fragments thereof of the present disclosure include, but are not limited to, the human monoclonal antibodies or antigen-binding fragments thereof, isolated as described in the Examples below. [00100] The active ingredient of HUMIRA, adalimumab, is a well-known TNF-alpha antibody. The heavy chain CDRs of adalimumab (HCDR1, HCDR2, HCDR3) according to the IMGT definition are set forth in the amino acid sequences of SEQ ID NOs: 1-3, respectively. The light chain CDRs of adalimumab (LCDR1, LCDR2, LCDR3) according to the IMGT definition are set forth in the amino acid sequences of SEQ ID NOs:8-10, respectively. The adalimumab VH is set forth in the amino acid sequence of SEQ ID NO:4, which is encoded by the nucleic acid sequence of SEQ ID NO:5. The adalimumab VL is set forth in the amino acid sequence of SEQ ID NO: 11, which is encoded by the nucleic acid sequence of SEQ ID NO: 12. The adalimumab heavy chain is set forth in the amino acid sequence of SEQ ID NO:6, which is encoded by the nucleic acid sequence of SEQ ID NO:7. The adalimumab light chain is set forth in the amino acid sequence of SEQ ID NO: 13, which is encoded by the nucleic acid sequence of SEQ ID NO: 14.

[00101] Administration of adalimumab to patients has been shown to result in ADAs, which inhibit binding of adaliumuab to TNF-alpha. To address this issue, we identified key residues for TNF-alpha binding in adalimumab, along with key residues that lead to the unwanted immunogenicity. Employing a germeline -guided approach, we generated single mutations and multiple mutations in the VH and VL of adalimumab, being careful to avoid mutating residues involved in binding to TNF-alpha.

[00102] It has been now been determined that IMGT residues D36, Y37, 165 and SI 14 of the adalimumab VH and heavy chain (positions 31, 32, 58, and 107 of SEQ ID NO:4 and SEQ ID NO:6) are amenable to de-immunizing substitution. Furthermore, IMGT residues G28, T66, L67, Q68, S69 and V99 of the adalimumab VL and light chain (positions 28, 53, 54, 55, 56, and 83 of SEQ ID NO: l 1 and SEQ ID NO: 13) are amenable to de-immunizing substitutions. Table 1, below, provides these preferred mutations for variant adalimumab- based anti-TNF antibodies and antigen-binding fragments according to the disclosure.

Table 1: Preferred mutations in variant adalimumab-based anti-TNF antibodies and antigen binding fragments according to the disclosure. Positions of mutations are based on the location in SEQ ID NOs:4/6 and SEQ ID NOs: 11/13 (column 1) or IMGT numbering (column 2). Preferred amino acid mutations (substitutions) at each position are given in column 3.

[00103] Based on the above, the amino acid sequence set forth in SEQ ID NO:26 generically represents the VH of the TNF-alpha antibodies and antigen-binding fragments of the invention, while amino acid sequence set forth in SEQ ID NO:28 generically represents the VL of the TNF-alpha antibodies and antigen-binding fragments of the invention.

Similarly, the amino acid sequence set forth in SEQ ID NO:27 generically represents the heavy chain of the TNF-alpha antibodies and antigen-binding fragments of the invention, while amino acid sequence set forth in SEQ ID NO:29 generically represents the light chain of the TNF-alpha antibodies and antigen-binding fragments of the invention.

[00104] In some embodiments, TNF-alpha antibodies and antigen-binding fragments of the disclosure comprise a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO:26; and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 11, wherein at least one of the Xaa amino acids in SEQ ID NO:26 differs from the corresponding amino acid in the same position of an amino acid sequence as set forth in SEQ ID NO:4. In some embodiments, TNF-alpha antibodies and antigen-binding fragments of the disclosure comprise a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO:4; and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO:28, wherein at least one of the Xaa amino acids in SEQ ID NO:28 differs from the corresponding amino acid in the same position of an amino acid sequence as set forth in SEQ ID NO: 11. In some embodiments, TNF-alpha antibodies and antigen-binding fragments of the disclosure comprise a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO:26; and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO:28, wherein at least one of the Xaa amino acids in SEQ ID NO:26 differs from the corresponding amino acid in the same position of an amino acid sequence as set forth in SEQ ID NO:4 and at least one of the Xaa amino acids in SEQ ID NO:28 differs from the corresponding amino acid in the same position of an amino acid sequence as set forth in SEQ ID NO: 11.

[00105] In some embodiments, TNF-alpha antibodies of the disclosure comprise a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:27; and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 13, wherein at least one of the Xaa amino acids in SEQ ID NO:27 differs from the corresponding amino acid in the same position of an amino acid sequence as set forth in SEQ ID NO:6. In some embodiments, TNF-alpha antibodies of the disclosure comprise a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:6; and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:29, wherein at least one of the Xaa amino acids in SEQ ID NO:29 differs from the corresponding amino acid in the same position of an amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, TNF-alpha antibodies of the disclosure comprise a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:27; and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:29, wherein at least one of the Xaa amino acids in SEQ ID NO:27 differs from the corresponding amino acid in the same position of an amino acid sequence as set forth in SEQ ID NO:6 and at least one of the Xaa amino acids in SEQ ID NO:29 differs from the corresponding amino acid in the same position of an amino acid sequence as set forth in SEQ ID NO: 13.

[00106] In some embodiments, one, two, three or four of the Xaa amino acids in SEQ

ID NO:26 differs from the corresponding amino acid in the same position of the amino acid sequence as set forth in SEQ ID NO:4.

[00107] In some embodiments, one, two, three or four of the Xaa amino acids in SEQ

ID NO:28 differs from the corresponding amino acid in the same position of the amino acid sequence as set forth in SEQ ID NO: 11.

[00108] In some embodiments, one, two, three or four of the Xaa amino acids in SEQ

ID NO:26 differs from the corresponding amino acid in the same position of the amino acid sequence as set forth in SEQ ID NO:4, and one, two, three or four of the Xaa amino acids in SEQ ID NO:28 differs from the corresponding amino acid in the same position of the amino acid sequence as set forth in SEQ ID NO: 11. [00109] In some embodiments, one, two, three or four of the Xaa amino acids in SEQ

ID NO:27 differs from the corresponding amino acid in the same position of the amino acid sequence as set forth in SEQ ID NO:6.

[00110] In some embodiments, one, two, three or four of the Xaa amino acids in SEQ

ID NO:29 differs from the corresponding amino acid in the same position of the amino acid sequence as set forth in SEQ ID NO: 13.

[00111] In some embodiments, one, two, three or four of the Xaa amino acids in SEQ

ID NO:27 differs from the corresponding amino acid in the same position of the amino acid sequence as set forth in SEQ ID NO: 6 and one, two, three or four of the Xaa amino acids in SEQ ID NO:29 differs from the corresponding amino acid in the same position of the amino acid sequence as set forth in SEQ ID NO: 13.

[00112] The present disclosure in certain aspects provides antibodies or antigen binding fragments thereof that specifically bind to TNF -alpha, said antibodies or antigen - binding fragments comprising a VH domain comprising an amino acid sequence of SEQ ID NO:4 having at least one of the VH mutations of Table 1 (D36S, D36N, Y37S, Y37F, I65T,

S 114A). IMGT positions 36, 37, 65, and 114 of the VH correspond to positions 31, 32, 58 and 107, respectively, of SEQ ID NO:4 and SEQ ID NO:6. In some embodiments, the antibodies or antigen-binding fragments thereof have 1, 2, 3 or 4 of the VH mutations of Table 1.

[00113] The present disclosure in certain aspects provides antibodies or antigen binding fragments thereof that specifically bind to TNF -alpha, said antibodies or antigen - binding fragments thereof comprising a VL domain comprising an amino acid sequence of SEQ ID NO: 11 having at least one of the VL mutations of Table 1 (G28S, L67R, Q68E, S69T, T66S, V99F, T66N, V99I). IMGT positions 28, 66, 67, 68, 69, and 99 of the VL correspond to positions 28, 53, 54, 55, 56 and 83, respectively, of SEQ ID NO: 11 and SEQ ID NO: 13. In some embodiments, the antibodies or antigen-binding fragments thereof have 1, 2, 3 or 4 of the VL mutations of Table 1.

[00114] In some embodiments, the antibodies or antigen-binding fragments thereof have 1, 2, 3 or 4 the VH mutations of Table 1 and no VL mutation. In some embodiments, the antibodies or antigen-binding fragments thereof have 1, 2, 3 or 4 of the VL mutations of Table 1 and no VH mutation. In some embodiments, the antibodies or antigen-binding fragments thereof have 1, 2, 3 or 4 of the VH mutations of Table 1 and 1, 2, 3 or 4 the VL mutations of Table 1. [00115] Accordingly, disclosed herein are antibodies to human tumor necrosis factor

(TNF)-alpha or antigen-binding fragments thereof, comprising a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO:4 and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 11, wherein the VH is mutated at one or more of positions 31, 32, 58 and 107 of SEQ ID NO:4. In some embodiments, the VH and VL mutations are selected from mutations set forth in Table 1. In some embodiments, the VH and VL regions are set forth in a row of Table 2.

[00116] Additionally disclosed herein are antibodies to human tumor necrosis factor

(TNF)-alpha or antigen-binding fragments thereof, comprising a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO:4 and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 11, wherein the VL is mutated at one or more of positions 28, 53, 54, 55, 56 and 83 of SEQ ID NO: 11. In some embodiments, the VH and VL mutations are selected from mutations set forth in Table 1. In some embodiments, the VH and VL regions are set forth in a row of Table 2.

[00117] Additionally disclosed herein are antibodies to human tumor necrosis factor

(TNF)-alpha or antigen-binding fragments thereof, comprising a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO:4 and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 11, wherein: the VH is mutated at one or more of positions 31, 32, 58 and 107 of SEQ ID NO:4; and the VL is mutated at one or more of positions 28, 53, 54, 55, 56 and 83 of SEQ ID NO: 11. In some embodiments, the VH and VL mutations are selected from mutations set forth in Table 1. In some embodiments, the VH and VL regions are set forth in a row of Table 2.

[00118] Additionally disclosed herein are antibodies to human tumor necrosis factor

(TNF)-alpha or antigen-binding fragments thereof, comprising an amino acid sequence as set forth in SEQ ID NO:4 and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 11, wherein the VH is mutated at one or more of IMGT positions 36, 37, 65 and 114. In some embodiments, the VH and VL mutations are selected from mutations set forth in Table 1. In some embodiments, the VH and VL regions are set forth in a row of Table 2.

[00119] Additionally disclosed herein are antibodies to human tumor necrosis factor

(TNF)-alpha or antigen-binding fragments thereof, comprising an amino acid sequence as set forth in SEQ ID NO:4 and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 11, wherein the VL is mutated at one or more of IMGT positions 28, 66, 67, 68, 69 and 99. In some embodiments, the VH and VL mutations are selected from mutations set forth in Table 1. In some embodiments, the VH and VL regions are set forth in a row of Table 2.

[00120] Additionally disclosed herein are antibodies to human tumor necrosis factor

(TNF)-alpha or antigen-binding fragments thereof, comprising a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO:4 and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 11, wherein the VH is mutated at one or more of IMGT positions 36, 37, 65 and 114; and the VL is mutated at one or more of IMGT positions 28, 66, 67, 68, 69 and 99. In some embodiments, the VH and VL mutations are selected from mutations set forth in Table 1. In some embodiments, the VH and VL regions are set forth in a row of Table 2.

[00121] Disclosed herein are also antibodies to human tumor necrosis factor (TNF)- alpha, comprising a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO:6 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 13, wherein the heavy chain is mutated at one or more of positions 31, 32, 58 and 107 of SEQ ID NO:6. In some embodiments, the heavy chain and light chain mutations are selected from mutations set forth in Table 1. In some embodiments, the heavy chain comprises a VH region and the light chain comprises a VL region, and the VH and VL regions are set forth in a row of Table 2.

[00122] Additionally disclosed herein are antibodies to human tumor necrosis factor

(TNF)-alpha, comprising a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO:6 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 13, wherein the light chain is mutated at one or more of positions 28, 53, 54, 55, 56 and 83 of SEQ ID NO: 13. In some embodiments, the heavy chain and light chain mutations are selected from mutations set forth in Table 1. In some embodiments, the heavy chain comprises a VH region and the light chain comprises a VL region, and the VH and VL regions are set forth in a row of Table 2.

[00123] Additionally disclosed herein are antibodies to human tumor necrosis factor

(TNF)-alpha, comprising a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO:6 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 13, wherein: the heavy chain is mutated at one or more of positions 31, 32, 58 and 107 of SEQ ID NO:6; and the light chain is mutated at one or more of positions 28, 53, 54, 55, 56 and 83 of SEQ ID NO: 13. In some embodiments, the heavy chain and light chain mutations are selected from mutations set forth in Table 1. In some embodiments, the heavy chain comprises a VH region and the light chain comprises a VL region, and the VH and VL regions are set forth in a row of Table 2.

[00124] Additionally disclosed herein are antibodies to human tumor necrosis factor

(TNF)-alpha, comprising a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO:6 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 13, wherein the heavy chain is mutated at one or more of IMGT positions 36, 37, 65 and 114. In some embodiments, the heavy chain and light chain mutations are selected from mutations set forth in Table 1. In some embodiments, the heavy chain comprises a VH region and the light chain comprises a VL region, and the VH and VL regions are set forth in a row of Table 2.

[00125] Additionally disclosed herein are antibodies to human tumor necrosis factor

(TNF)-alpha, comprising a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO:6 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 13, wherein the light chain is mutated at one or more of IMGT positions 28, 66, 67, 68,

69 and 99. In some embodiments, the heavy chain and light chain mutations are selected from mutations set forth in Table 1. In some embodiments, the heavy chain comprises a VH region and the light chain comprises a VL region, and the VH and VL regions are set forth in a row of Table 2.

[00126] Additionally disclosed herein are antibodies to human tumor necrosis factor

(TNF)-alpha, comprising a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO:6 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 13, wherein: the heavy chain is mutated at one or more of IMGT positions 36, 37, 65 and 114; and the light chain is mutated at one or more of IMGT positions 28, 66, 67, 68, 69, and/or 99. In some embodiments, the heavy chain and light chain mutations are selected from mutations set forth in Table 1. In some embodiments, the heavy chain comprises a VH region and the light chain comprises a VL region, and the VH and VL regions are set forth in a row of Table 2.

[00127] Disclosed herein are antibodies to human tumor necrosis factor (TNF)-alpha and antigen-binding fragments thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL) set forth in a row of Table 2.

[00128] Disclosed herein are antibodies to human tumor necrosis factor (TNF)-alpha, comprising a heavy chain and a light chain, said heavy chain comprising a heavy chain variable region (VH), said light chain comprising a light chain variable region (VL), wherein said VH and VL are set forth in a row of Table 2.

[00129] In preferred embodiments, the TNF-alpha antibody or antigen-binding fragment thereof has a VH Y37 (IMGT) mutation. In preferred embodiments, the TNF-alpha antibody or antigen-binding fragment thereof comprises a VL of SEQ ID NO: 11 and a VH of SEQ ID NO:4 comprising a VH Y37 (IMGT) mutation.

[00130] In preferred embodiments, the TNF-alpha antibody or antigen-binding fragment thereof has a VL S69 (IMGT) mutation. In preferred embodiments, the TNF-alpha antibody or antigen-binding fragment thereof comprises a VL of SEQ ID NO: 11 comprising a VL S69 (IMGT) mutation and a VH of SEQ ID NO:4.

[00131] In preferred embodiments, the TNF-alpha antibody or antigen-binding fragment thereof has a VH Y37S (IMGT). In preferred embodiments, the TNF-alpha antibody or antigen-binding fragment thereof comprises a VL of SEQ ID NO: 11 and a VH of SEQ ID NO:4 comprising a VH Y37S (IMGT) mutation.

[00132] In preferred embodiments, the TNF-alpha antibody or antigen-binding fragment thereof has a VL S69T (IMGT) mutation. In preferred embodiments, the TNF-alpha antibody or antigen-binding fragment thereof comprises a VL of SEQ ID NO: 11 comprising a VL S69T (IMGT) mutation and a VH of SEQ ID NO:4.

[00133] In preferred embodiments, the TNF-alpha antibody or antigen-binding fragment thereof has a VH Y37 mutation, and a VL S69 (IMGT) mutation. In preferred embodiments, the TNF-alpha antibody or antigen-binding fragment thereof comprises a VL of SEQ ID NO: l 1 comprising a VL S69 (IMGT) mutation and a VH of SEQ ID NO:4 comprising a VH Y37 (IMGT) mutation.

[00134] In preferred embodiments, the TNF-alpha antibody or antigen-binding fragment thereof has a VH Y37S mutation and a VL S69T (IMGT) mutation. In preferred embodiments, the TNF-alpha antibody or antigen-binding fragment thereof comprises a VL of SEQ ID NOT 1 comprising a VL S69T (IMGT) mutation and a VH of SEQ ID NO:4 comprising a VH Y37S (IMGT) mutation.

[00135] In a preferred embodiment, the TNF-alpha antibody or antigen-binding fragment thereof has a VH Y37S (IMGT) mutation and no VL mutation relative to wild-type adalimumab. This antibody or antigen-binding fragment is referred to as“Variant 4” or“v4”. The heavy chain CDRs of TNF-alpha antibody or antigen-binding fragment v4 (HCDR1, HCDR2, HCDR3) according to the IMGT definition are set forth in the amino acid sequences of SEQ ID NOs: 15, 2 and 3, respectively. The light chain CDRs of the TNF-alpha antibody or antigen-binding fragment v4 (LCDR1, HCDR2, HCDR3) according to the IMGT definition are set forth in the amino acid sequences of SEQ ID NOs: 8- 10, respectively. It is contemplated that the CDRs of TNF-alpha antibody or antigen-binding fragment v4 may be identified using other well-known definitions, e.g., Rabat, Chothia, combined Rabat and Chothia numbering scheme.

[00136] The TNF-alpha antibody or antigen-binding fragment v4 VH is set forth in the amino acid sequence of SEQ ID NO: 16, which is encoded by the nucleic acid sequence of SEQ ID NO: 17. The TNF-alpha antibody or antigen-binding fragment v4 VL is set forth in the amino acid sequence of SEQ ID NO: 11, which is encoded by the nucleic acid sequence of SEQ ID NO: 12. The TNF-alpha antibody v4 heavy chain is set forth in the amino acid sequence of SEQ ID NO: 18, which is encoded by the nucleic acid sequence of SEQ ID NO: 19. The TNF-alpha antibody v4 light chain is set forth in the amino acid sequence of SEQ ID NO: 13, which is encoded by the nucleic acid sequence of SEQ ID NO: 14.

[00137] In another preferred embodiment, the TNF-alpha antibody or antigen-binding fragment thereof has a VH Y37S (IMGT) mutation and a VL S69T (IMGT) mutation. This antibody or antigen-binding fragment is referred to as“Variant 40” or“v40”. The heavy chain CDRs of TNF-alpha antibody or antigen-binding fragment v40 (HCDR1, HCDR2, HCDR3) according to the IMGT definition are set forth in the amino acid sequences of SEQ ID NOs: 15, 2 and 3, respectively. The light chain CDRs of TNF-alpha antibody or antigen binding fragment v40 (LCDR1, HCDR2, HCDR3) according to the IMGT definition are set forth in the amino acid sequences of SEQ ID NOs:8-10, respectively. It is contemplated that the CDRs of TNF-alpha antibody or antigen-binding fragment v40 (preferably TNF-alpha antibody v4) may be identified using other well-known definitions, e.g., Rabat, Chothia, combined Rabat and Chothia numbering scheme.

[00138] The TNF-alpha antibody or antigen-binding fragment v40 VH is set forth in the amino acid sequence of SEQ ID NO: 16, which is encoded by the nucleic acid sequence of SEQ ID NO: 17. The TNF-alpha antibody or antigen-binding fragment v40 VL is set forth in the amino acid sequence of SEQ ID NO:20, which is encoded by the nucleic acid sequence of SEQ ID NO:21. The TNF-alpha antibody v4 heavy chain is set forth in the amino acid sequence of SEQ ID NO: 18, which is encoded by the nucleic acid sequence of SEQ ID NO: 19. The TNF-alpha antibody v4 light chain is set forth in the amino acid sequence of SEQ ID NO:22, which is encoded by the nucleic acid sequence of SEQ ID NO:23. [00139] Disclosed herein is an antibody to human tumor necrosis factor (TNF)-alpha or an antigen-binding fragment thereof, comprising: a heavy chain variable region (VH) comprising, in sequence, a complementarity determining region (CDR) having an amino acid sequence as set forth in SEQ ID NO: 15; a CDR having an amino acid sequence as set forth in SEQ ID NO:2; and a CDR having an amino acid sequence as set forth in SEQ ID NO:3; and a light chain variable region (VL) comprising, in sequence, a CDR having an amino acid sequence as set forth in SEQ ID NO: 8; a CDR having an amino acid sequence as set forth in SEQ ID NO:9; and a CDR having an amino acid sequence as set forth in SEQ ID NO: 10.

[00140] In some embodiments, the antibody to human tumor necrosis factor (TNF)- alpha or an antigen-binding fragment thereof, comprising: a heavy chain variable region (VH) comprising the three CDRs of the amino acid sequence as set forth in SEQ ID NO: 16, and a light chain variable region (VL) comprising the three CDRs of the amino acid sequence as set forth in SEQ ID NO: 11, wherein the CDRs are defined according to IMGT, Rabat, Chothia or a combined Rabat and Chothia numbering scheme. In one embodiment, the CDRs are defined according to IMGT. In one embodiment, the CDRs are defined according to Rabat.

In one embodiment, the CDRs are defined according to Chothia. In one embodiment, the CDRs are defined according to a combined Rabat and Chothia numbering scheme.

[00141] In some embodiments, the VH comprises an amino acid sequence as set forth in

SEQ ID NO: 16 and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 11.

[00142] In some embodiments, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 18 and said light chain comprises an amino acid sequence as set forth in SEQ ID NO: 13.

[00143] In some embodiments, the antibody to human tumor necrosis factor (TNF)- alpha or an antigen-binding fragment thereof, comprising: a heavy chain variable region (VH) comprising the three CDRs of the amino acid sequence as set forth in SEQ ID NO: 16, and a light chain variable region (VL) comprising the three CDRs of the amino acid sequence as set forth in SEQ ID NO:20, wherein the CDRs are defined according to IMGT, Rabat, Chothia or a combined Rabat and Chothia numbering scheme. In one embodiment, the CDRs are defined according to IMGT. In one embodiment, the CDRs are defined according to Rabat.

In one embodiment, the CDRs are defined according to Chothia. In one embodiment, the CDRs are defined according to a combined Rabat and Chothia numbering scheme.

[00144] In some embodiments, the VH comprises an amino acid sequence as set forth in

SEQ ID NO: 16 and the VL comprises an amino acid sequence as set forth in SEQ ID NO:20. [00145] In some embodiments, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 18 and said light chain comprises an amino acid sequence as set forth in SEQ ID NO:22.

[00146] In some embodiments, the antibody or antigen-binding fragment thereof is an antibody selected from a chimeric antibody, a humanized antibody, and a human antibody. The TNF-alpha antibody is preferably a monoclonal TNF -alpha antibody.

[00147] In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody.

[00148] The TNF-alpha antigen-binding fragment is preferably selected from a Fab, a

F(ab)2, and an scFv.

[00149] The VH and VL regions of preferred TNF-alpha antibodies and antigen-binding fragments according to disclosure are shown in Table 2.

fragments according to the disclosure. Each row of the table refers to a particular variant (e.g., Variant 1, Variant 2, etc.). Column 2 provides the mutation in the VH of a given variant relative to the wild-type adalimumab VH (SEQ ID NO: 4) and as an IMGT position. Column 3 provides the mutation in the VL of the variant relative to the wild-type adalimumab VL (SEQ ID NO: 11) and as an IMGT position. As an example, Variant 35 represents an antibody or antigen-binding fragment having a VH as set forth in SEQ ID NO:4 with a Y37F mutation (position 32 of SEQ ID NO:4) and a VL as set forth in SEQ ID NO: 11 with an L67R mutation (position 54 of SEQ ID NO: 11). *VKl-33 FR3 refers to a light chain having the following mutations in SEQ ID NO: 11 : T66N, Q68E, V99I (IMGT). ** VK1-13 FR3 refers to a light chain having the following mutations in SEQ ID NO: l 1 : T66S, Q68E, V99F (IMGT).

[00150] Table 3 sets forth amino acid sequences of the CDRs, VH, VL, full length heavy chain, and full length light chain of adalimumab and the v4 and v40 TNF -alpha antibodies and antigen-binding fragments, along with nucleic acid sequences that encode the VH, VL, full length heavy chain, and full length light chain of these molecules. Notably, these nucleic acid sequences can be optimized for expression in mammalian cells.

Table 3: Representative sequences, including generic sequences, v40 and v4 sequences, and sequences for wild-type adalimumab. An amino acid given as“X” can be any of the amino acids in the respective position given in column 3 of Table 1, as well as the wild-type amino acid of adalimumab in the respective position given in column 2 of Table 1.

[00151] Other antibodies of the present disclosure include those where additional amino acids (and their corresponding encoding nucleic acids) in the VH, VL, heavy chain or light chain framework regions have been mutated; yet have at least 60, 70, 80, 90 or 95 percent identity to the general TNF -alpha antibody and antigen-binding fragment amino acid sequences of SEQ ID NOs:26-29 described in Table 3.

[00152] Other antibodies or antigen-binding fragments thereof of the present disclosure include those in which additional amino acids (and their corresponding encoding nucleic acids) in the CDRs have been mutated, yet have at least 60, 70, 80, 90 or 95 percent identity in the CDR regions with the CDR regions of the general TNF-alpha antibody and antigen binding fragment amino acid sequences of SEQ ID NOs:26-29 described in Table 3. In some aspects, it includes mutant amino acid sequences wherein no more than 1, 2, 3, 4 or 5 additional amino acids have been mutated in the CDR regions when compared with the CDR regions of the general TNF-alpha antibody and antigen-binding fragment amino acid sequences of SEQ ID NOs:26-29 described in Table 3.

Further Alteration of the Framework and Fc Regions

[00153] The present disclosure provides specific anti-TNF-alpha antibodies and antigen-binding fragments thereof. These antibodies and fragments may comprise further modifications to framework residues within VH and/or VL, e.g. to improve the properties of the antibody. Such framework modifications are made to decrease the immunogenicity of the antibody.

[00154] For example, one approach is to "back-mutate" one or more additional framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline

configuration, the somatic mutations can be "back-mutated" to the germline sequence by, for example, site-directed mutagenesis. Such "back-mutated" antibodies are also intended to be encompassed.

[00155] Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T- cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent

Publication No. 2003/0153043 by Carr et al.

[00156] In addition, or alternative to modifications made within the framework or CDR regions, antibodies can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody can be chemically modified (e.g. , one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these aspects is described in further detail below.

[00157] In one aspect, the hinge region of CHI is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[00158] In another aspect, the Fc hinge region of an antibody or fragment is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.

[00159] In yet other aspects, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in, e.g., U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[00160] In another aspect, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in, e.g., U.S. Patent Nos. 6,194,551 by Idusogie et al.

[00161] In another aspect, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described in, e.g., the PCT Publication WO 94/29351 by Bodmer et al. In a specific aspect, one or more amino acids of an antibody or antigen-binding fragment thereof of the present disclosure are replaced by one or more allotypic amino acid residues, for the IgGi subclass and the kappa isotype. Allotypic amino acid residues also include, but are not limited to, the constant region of the heavy chain of the IgGi, IgG2, and IgG3 subclasses as well as the constant region of the light chain of the kappa isotype as described by Jefferis et al., MAbs. 1:332-338 (2009).

[00162] In yet another aspect, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids. This approach is described in, e.g., the PCT Publication WO 00/42072 by Presta. Moreover, the binding sites on human IgGl for FcyRl, FcyRII, F cy R II I and FcRn have been mapped and variants with improved binding have been described (see Shields et al., J. Biol. Chem.

276:6591-6604, 2001).

[00163] As used herein, the term“ADCC” or“antibody dependent cell cytotoxicity” activity refers to cell depleting activity. ADCC activity can be measured by standard ADCC assay, well known to a person skilled in the art.

[00164] In still another aspect, the glycosylation of an antibody or fragment is modified. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for "antigen.” Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in, e.g., U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[00165] Additionally, or alternatively, an antibody or fragment can be made that has an altered type of glycosylation, such as a hypofiicosylated antibody having reduced amounts of fiicosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies to thereby produce an antibody with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fiicosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofiicosylation of antibodies expressed in that host cell (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases ( e.g ., beta (1,4)-N acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17: 176-180, 1999).

[00166] In another aspect, the antibody or fragment is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, to increase the biological half-life, the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos.

5,869,046 and 6,121,022 by Presta et al.

[00167] In order to minimize the ADCC activity of an antibody, specific mutations in the Fc region result in“Fc silent” antibodies that have minimal interaction with effector cells. In general, the "IgG Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc region and variant Fc regions. The human IgG heavy chain Fc region is generally defined as comprising the amino acid residue from position C226 or from P230 to the carboxyl-terminus of the IgG antibody. The numbering of residues in the Fc region is that of the EU index of Kabat. The C- terminal lysine (residue K447) of the Fc region may be removed, for example, during production or purification of the antibody.

[00168] Silenced effector functions can be obtained by mutation in the Fc region of the antibodies and have been described in the art: LALA and N297A (Strohl, W., 2009, Curr. Opin. Biotechnol. vol. 20(6):685-691); and D265A (Baudino et al., 2008, J. Immunol. 181: 6664- 69) see also Heusser et al., WO2012065950. Examples of silent Fc lgGl antibodies are the LALA mutant comprising L234A and L235A mutation in the lgGl Fc amino acid sequence. Another example of a silent lgGi antibody is the DAPA (D265A, P329A) mutation (US 6,737,056). Another silent lgGl antibody comprises the N297A mutation, which results in aglycosylated/non-glycosylated antibodies.

[00169] Fc silent antibodies result in no or low ADCC activity, meaning that an Fc silent antibody exhibits an ADCC activity that is below 50% specific cell lysis (low ADCC activity), or that is below 1% specific cell lysis (no ADCC activity). [00170] Preferred modifications to the Fc include the“LS” mutation (M428L, N434S,

(EU numbering)) and the“YTE” mutation (M252Y, S254T, T256E (EU Numbering)) for half-life extension, and the“DAPA” mutation (D265A,P329A (EU Numbering)) for effector silencing.

Production of Antibodies and Fragments

[00171] Anti-TNF-alpha antibodies and antigen-binding fragments thereof can be produced by any means known in the art, including but not limited to, recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, whereas full- length monoclonal antibodies can be obtained by, e.g., hybridoma or recombinant production. Recombinant expression can be from any appropriate host cells known in the art, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[00172] Disclosed herein are isolated nucleic acid molecules, or a set of nucleic acid molecules, encoding an antibody or antigen-binding fragment as described herein. In some embodiments, the isolated nucleic acid molecule is complementary DNA (cDNA) or messenger RNA (mRNA).

[00173] The disclosure further provides polynucleotides encoding the antibodies described herein, e.g., polynucleotides encoding heavy or light chain variable regions or segments comprising the complementarity determining regions as described herein.

[00174] The VH and VL regions of the v4 antibody and antigen-binding fragment are encoded by the nucleic acid sequences of SEQ ID NOs: 17 and 12 of Table 3. The VH and VL regions of v40 antibody and antigen-binding fragment are set forth in the nucleic acid sequences of SEQ ID NOs: 17 and 21 of Table 3. The heavy and light chain regions of the v4 antibody and antigen-binding fragment are encoded by the nucleic acid sequences of SEQ ID NOs: 19 and 14 of Table 3. The heavy and light chain regions of v40 antibody and antigen binding fragment are encoded by the nucleic acid sequences of SEQ ID NOs: 19 and 23 of Table 3.

[00175] In some aspects, the polynucleotide encoding the VH has at least 85%, 89%,

90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO: 17 (Table 3). In some aspects, the

polynucleotide encoding the VL has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO: 12 or 21 (Table 3). [00176] In some aspects, the polynucleotide encoding the heavy chain has at least 85%,

89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO: 19 (Table 3). In some aspects, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NOs: 14 or 23 (Table 3).

[00177] Other preferred nucleic acids are those encoding the heavy chain variable region, light chain variable region, heavy chain and light chain amino acid sequences set forth in SEQ ID NOs: 26-29, wherein at least one of the Xaa of any of the aforementioned encoded sequences is mutated with respect to wild-type adalimumab.

[00178] In some aspects, the polynucleotide encodes a heavy chain variable region

(VH) comprising an amino acid sequence as set forth in SEQ ID NO:26, wherein at least one of the Xaa amino acids in SEQ ID NO:26 differs from the corresponding amino acid in the same position of an amino acid sequence as set forth in SEQ ID NO:4.

[00179] In some aspects, the polynucleotide encodes a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO:28, wherein at least one of the Xaa amino acids in SEQ ID NO:28 differs from the corresponding amino acid in the same position of an amino acid sequence as set forth in SEQ ID NO: 11.

[00180] In some aspects, the polynucleotide encodes a heavy chain that comprises an amino acid sequence as set forth in SEQ ID NO:27; wherein at least one of the Xaa amino acids in SEQ ID NO:27 differs from the corresponding amino acid in the same position of an amino acid sequence as set forth in SEQ ID NO:6.

[00181] In some aspects, the polynucleotide encodes a light chain that comprises an amino acid sequence as set forth in SEQ ID NO:29, wherein at least one of the Xaa amino acids in SEQ ID NO:29 differs from the corresponding amino acid in the same position of an amino acid sequence as set forth in SEQ ID NO: 13.

[00182] In some aspects, the polynucleotide encodes a polypeptide wherein one, two, three or four of the Xaa amino acids in SEQ ID NO:26 differs from the corresponding amino acid in the same position of the amino acid sequence as set forth in SEQ ID NO:4.

[00183] In some aspects, the polynucleotide encodes a polypeptide wherein one, two, three or four of the Xaa amino acids in SEQ ID NO:28 differs from the corresponding amino acid in the same position of the amino acid sequence as set forth in SEQ ID NO: 11. [00184] In some aspects, the polynucleotide encodes a polypeptide wherein one, two, three or four of the Xaa amino acids in SEQ ID NO:27 differs from the corresponding amino acid in the same position of the amino acid sequence as set forth in SEQ ID NO:6.

[00185] In some aspects, the polynucleotide encodes a polypeptide wherein one, two, three or four of the Xaa amino acids in SEQ ID NO:29 differs from the corresponding amino acid in the same position of the amino acid sequence as set forth in SEQ ID NO: 13.

[00186] In some aspects, the polynucleotide encodes a heavy chain variable region

(VH) comprising an amino acid sequence as set forth in SEQ ID NO:4, wherein the VH is mutated at one or more of positions 31, 32, 58 and 107 of SEQ ID NO:4.

[00187] In some aspects, the polynucleotide encodes a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 11, wherein the VL is mutated at one or more of positions 28, 53, 54, 55, 56, and 83 of SEQ ID NO: 11.

[00188] In some aspects, the polynucleotide encodes a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO:6, wherein the heavy chain is mutated at one or more of positions 31, 32, 58 and 107 of SEQ ID NO:6

[00189] In some aspects, the polynucleotide encodes a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 13, wherein the light chain is mutated at one or more of positions 28, 53, 54, 55, 56, and 83 of SEQ ID NO: 13.

[00190] In some aspects, the polynucleotide encodes a heavy chain variable region

(VH) comprising an amino acid sequence as set forth in SEQ ID NO:4, wherein the VH is mutated at one or more of IMGT positions 36, 37, 65 and 107.

[00191] In some aspects, the polynucleotide encodes a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 11, wherein the VL is mutated at one or more of IMGT positions 28, 66, 67, 68, 69, and 99.

[00192] In some aspects, the polynucleotide encodes a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO:6, wherein the heavy chain is mutated at one or more of IMGT positions 36, 37, 65 and 114.

[00193] In some aspects, the polynucleotide encodes a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 13, wherein the light chain is mutated at one or more of IMGT positions 28, 66, 67, 68, 69, and 99.

[00194] In some aspects, the polynucleotide encodes an antibody or antigen-binding fragment of the disclosure, comprising a VH having at least one of the mutations set forth in Table 1. [00195] In some aspects, the polynucleotide encodes an antibody or antigen-binding fragment of the disclosure, comprising a VL having at least one of the mutations set forth in Table 1.

[00196] In some aspects, the polynucleotide encodes an antibody or antigen-binding fragment of the disclosure, comprising a VL having at least one of the mutations set forth in Table 1 and a VH having at least one of the mutations set forth in Table 1.

[00197] In some aspects, the polynucleotide encodes an antibody or antigen-binding fragment of the disclosure, comprising a heavy chain having at least one of the mutations set forth in Table 1.

[00198] In some aspects, the polynucleotide encodes an antibody or antigen-binding fragment of the disclosure, comprising a light chain having at least one of the mutations set forth in Table 1.

[00199] In some aspects, the polynucleotide encodes an antibody or antigen-binding fragment of the disclosure, comprising a heavy chain having at least one of the mutations set forth in Table 1 and a light chain having at least one of the mutations set forth in Table 1.

[00200] In some aspects, the polynucleotide encodes an antibody or antigen-binding fragment of the disclosure, comprising a VH, wherein said VH region is set forth in a row of Table 2.

[00201] In some aspects, the polynucleotide encodes an antibody or antigen-binding fragment of the disclosure, comprising a VL, wherein said VL region is set forth in a row of Table 2.

[00202] In some aspects, the polynucleotide encodes an antibody or antigen-binding fragment of the disclosure, comprising a VH, wherein said VH region is set forth in a row of Table 2, and a VL, wherein said VL region is set forth in a row of Table 2.

[00203] The polynucleotides of the present disclosure can encode only the variable region sequence of an anti-TNF-alpha antibody or fragment. They can also encode both a variable region and a constant region of the antibody. Some of the polynucleotide sequences encode a polypeptide that comprises variable regions of both the heavy chain and the light chain of an exemplified anti-TNF-alpha antibody or fragment. Some other polynucleotides encode two polypeptide segments that respectively are substantially identical to the variable regions of the heavy chain and the light chain of one of the antibodies.

[00204] The polynucleotide sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence encoding an anti-TNF-alpha antibody or fragment. Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al. , Meth.

Enzymol. 68:90, 1979; the phosphodiester method of Brown et al, Meth. Enzymol. 68: 109, 1979; the diethylphosphoramidite method of Beaucage et al, Tetra. Lett., 22: 1859, 1981; and the solid support method of U.S. Patent No. 4,458,066. Introducing mutations to a polynucleotide sequence by PCR can be performed as described in, e.g., PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al, Nucleic Acids Res. 19:967, 1991; and Eckert et al, PCR Methods and Applications 1: 17, 1991.

[00205] Also provided in the present disclosure are expression vectors and host cells for producing the anti -TNF -alpha antibodies and fragments described above. Disclosed herein are cloning and expression vectors comprising one or more nucleic acid molecules, or set of nucleic acid molecules, encoding the anti -TNF -alpha antibodies and fragments described above, wherein the vector is suitable for the recombinant production of the antibody or antigen binding fragment thereof.

[00206] Various expression vectors can be employed to express the polynucleotides encoding the disclosed anti -TNF -alpha antibodies and fragments. Both viral-based and nonviral expression vectors can be used to produce the antibodies in a mammalian host cell. Nonviral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al, Nat Gen. 15:345, 1997). For example, nonviral vectors useful for expression of polynucleotides and polypeptides of the anti-TNF-alpha antibodies and fragments in mammalian (e.g., human) cells include pThioHis A, B & C, pcDNA3.1/His, pEBVHis A, B & C (Invitrogen, San Diego, CA), MPSV vectors, and numerous other vectors known in the art for expressing other proteins. Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al, supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al, Cell 68: 143, 1992.

[00207] The choice of expression vector depends on the intended host cells in which the vector is to be expressed. Typically, the expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) that are operably linked to the polynucleotides encoding an anti-TNF-alpha antibody or fragment thereof. In some aspects, an inducible promoter is employed to prevent expression of inserted sequences except under inducing conditions. Inducible promoters include, e.g., arabinose, lacZ, metallothionein promoter or a heat shock promoter. Cultures of transformed organisms can be expanded under non inducing conditions without biasing the population for coding sequences whose expression products are better tolerated by the host cells. In addition to promoters, other regulatory elements may also be required or desired for efficient expression of an anti-TNF-alpha antibody or fragment thereof. These elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences. In addition, the efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (see, e.g. , Scharf el al, Results Probl. Cell Differ. 20: 125, 1994; and Bittner el al, Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer may be used to increase expression in mammalian host cells.

[00208] The expression vectors may also provide a secretion signal sequence position to form a fusion protein with polypeptides encoded by inserted anti-TNF-alpha antibody or fragment sequences. More often, the inserted anti-TNF-alpha antibody or fragment sequences are linked to a signal sequences before inclusion in the vector. Vectors to be used to receive sequences encoding anti-TNF-alpha antibody or fragment VH and VL sometimes also encode constant regions or parts thereof. Such vectors allow expression of the variable regions as fusion proteins with the constant regions thereby leading to production of intact antibodies or fragments thereof. Typically, such constant regions are human.

[00209] Disclosed herein are host cells comprising one or more cloning or expression vectors as described herein. The host cells for harboring and expressing the anti-TNF-alpha antibody or fragment chains can be either prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which typically contain expression control sequences compatible with the host cell (e.g. , an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation. Other microbes, such as yeast, can also be employed to express anti-TNF-alpha antibodies or fragments thereof. Insect cells in combination with baculovirus vectors can also be used.

[00210] In other aspects, mammalian host cells are used to express and produce the anti-TNF-alpha antibodies or fragments of the present disclosure. For example, they can be either a hybridoma cell line expressing endogenous immunoglobulin genes (e.g., the myeloma hybridoma clones) or a mammalian cell line harboring an exogenous expression vector.

These include any normal mortal or normal or abnormal immortal animal or human cell. For example, a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including the CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B-cells and hybridomas. The use of mammalian tissue cell culture to express polypeptides is discussed generally in, e.g., Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y., 1987. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al, Immunol. Rev. 89:49-68, 1986), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific, and/or modulatable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline- inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[00211] Methods for introducing expression vectors containing the polynucleotide sequences of interest vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts (see generally Sambrook et al, supra). Other methods include, e.g., electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycatiomnucleic acid conjugates, naked DNA, artificial virions, fusion to the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), agent- enhanced uptake of DNA, and ex vivo transduction. For long-term, high-yield production of recombinant proteins, stable expression will often be desired. For example, cell lines which stably express anti-TNF-alpha antibody or fragment chains, can be prepared using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth of cells which successfully express the introduced sequences in selective media. Resistant, stably transfected cells can be proliferated using tissue culture techniques appropriate to the cell type.

[00212] In some embodiments, the anti-TNF-alpha antibody fragment is comprised of a single polypeptide chain that is encoded by a single nucleic acid, which may be inserted into a single cloning or expression vectors. In other embodiments, the anti-TNF-alpha antibody or fragment thereof is comprised of two polypeptide chains encoded by more than one nucleic acid, which is referred to herein as“a set of nucleic acid molecules”. In some embodiments, the nucleic acid encoding the first chain is inserted into a first cloning or expression vector and the nucleic acid encoding the second chain is inserted into a second cloning or expression vector. In this situation, the anti-TNF-alpha antibody or fragment thereof is expressed via a set of cloning or expression vectors. Alternatively, both nucleic acids may be inserted into a single cloning or expression vector.

[00213] Disclosed herein is a process for the production of the antibody and antigen binding fragments as described herein, comprising culturing a host cell as described herein under conditions sufficient to express said antibody or antigen-binding fragment thereof, and thereafter purifying and recovering said antibody or antigen-binding fragment thereof from the host cell culture as one polynucleotide chain.

Generation of transfectomas producing monoclonal antibodies

[00214] Antibodies of the disclosure can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (e.g., Morrison, S. 1985, Science 229: 1202).

[00215] For example, to express the antibodies, or antibody fragments thereof, DNAs encoding partial or full-length light and heavy chains can be obtained by standard molecular biology or biochemistry techniques (e.g., DNA chemical synthesis, PCR amplification or cDNA cloning using a hybridoma that expresses the antibody of interest) and the DNAs can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term "operatively linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). The light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide, also called leader sequence, which facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[00216] In addition to the antibody chain genes, the recombinant expression vectors of the disclosure carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel 1990, Gene Expression Technology. Methods in

Enzymology 185, Academic Press, San Diego, CA). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and polyoma. Alternatively, nonviral regulatory sequences may be used, such as the ubiquitin promoter or P-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe,

Y. et al. 1988, Mol. Cell. Biol. 8:466-472).

[00217] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the disclosure may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfir- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

[00218] For expression of the light and heavy chains, standard techniques were applied to transfect a host cell with the expression vector(s) encoding the heavy and light chains. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. It is theoretically possible to express the antibodies of the disclosure in either prokaryotic or eukaryotic host cells. Expression of antibodies in eukaryotic cells, for example mammalian host cells, yeast or filamentous fungi, is discussed because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.

[00219] In one specific embodiment, a cloning or expression vector according to the disclosure comprises at least one of the nucleic acid coding sequences of the disclosure, operatively linked to suitable promoter sequences. [00220] In one specific embodiment, a cloning or expression vector according to the disclosure comprises a nucleic acid encoding an antibody or antigen-binding fragment of the disclosure having a VL as set forth in Table 2.

[00221] In one specific embodiment, a cloning or expression vector according to the disclosure comprises a nucleic acid encoding an antibody or antigen-binding fragment of the disclosure having a VH as set forth in Table 2

[00222] In one specific embodiment, a cloning or expression vector according to the disclosure comprises a nucleic acid(s) encoding an antibody or antigen-binding fragment of the disclosure having a VH and VL as set forth in a row of Table 2.

[00223] In one specific embodiment, a cloning or expression vector according to the disclosure comprises at least one of the nucleic acid sequences from Table 3, operatively linked to suitable promoter sequences.

[00224] In one specific embodiment, a cloning or expression vector according to the disclosure comprises at least one of SEQ ID NOs: 12, 14, 17, 19, 21, 23 operatively linked to suitable promoter sequences.

[00225] In one specific embodiment, a cloning or expression vector according to the disclosure comprises at least one nucleic acid encoding at least one of SEQ ID NO:26-29, operatively linked to suitable promoter sequences, wherein Xaa in any of the aforementioned encoded sequences is mutated with respect to wild-type adalimumab.

[00226] Mammalian host cells for expressing the recombinant antibodies of the disclosure include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described Urlaub and Chasin 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220 used with a DH FR selectable marker, e.g., as described in R.J. Kaufman and P.A. Sharp 1982, Mol. Biol.

159:601-621), CHOK1 dhfr+ cell lines, NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma cells, another expression system is the GS gene expression system shown in PCT Publications WO 87/04462, WO 89/01036 and EP 0 338 841. In one embodiment, mammalian host cells for expressing the recombinant antibodies of the disclosure include mammalian cell lines deficient for FUT8 gene expression, for example as described in US Patent No. 6,946,292. [00227] When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods (See for example Abhinav et al. 2007, Journal of Chromatography 848:28-37).

[00228] In one specific embodiment, the host cell of the disclosure is a host cell transfected with an expression vector(s) having nucleic acid(s) encoding VH and/or VL amino acid sequences of the v4 or v40 antibody or antigen-binding fragment thereof from Table 3, suitable for the expression of any of the variants therein, operatively linked to suitable promoter sequences. In one specific embodiment, the host cell of the disclosure is a host cell transfected with an expression vector(s) having nucleic acid(s) encoding heavy and light chain amino acid sequences of the v4 or v40 antibody or antigen-binding fragment thereof from Table 3, suitable for the expression of any of the variants therein, operatively linked to suitable promoter sequences. In one specific embodiment, the host cell of the disclosure is a host cell transfected with an expression vector(s) having nucleic acid(s) selected from SEQ ID NOs: 12, 14, 17, 19, 21 and 23, suitable for the expression of the v4 and v40 antibodies and fragments, operatively linked to suitable promoter sequences.

[00229] In one embodiment, the host cell of the disclosure is a host cell transfected with an expression vector(s) having nucleic acid(s) encoding an antibody or antigen-binding fragment of the disclosure.

[00230] In one specific embodiment, the host cell of the disclosure is a host cell transfected with an expression vector(s) having nucleic acid(s) encoding an antibody or antigen-binding fragment of the disclosure having a VH as set forth in Table 2, suitable for the expression of the antibody or antigen-binding fragment, operatively linked to suitable promoter sequences.

[00231] In one specific embodiment, the host cell of the disclosure is a host cell transfected with an expression vector(s) having nucleic acid(s) encoding an antibody or antigen-binding fragment of the disclosure having a VL as set forth in Table 2, suitable for the expression of the antibody or antigen-binding fragment, operatively linked to suitable promoter sequences. [00232] In one specific embodiment, the host cell of the disclosure is a host cell transfected with an expression vector(s) having nucleic acid(s) encoding an antibody or antigen-binding fragment of the disclosure having a VH and VL as set forth in a row of Table 2, suitable for the expression of the antibody or antigen-binding fragment, operatively linked to suitable promoter sequences.

[00233] In one specific embodiment, the host cell of the disclosure is a host cell transfected with at least one expression vector having at least one nucleic acid from Table 3, suitable for the expression of an antibody or antigen-binding fragment of the disclosure, operatively linked to suitable promoter sequences.

[00234] These host cells may then be further cultured under suitable conditions for the expression and production of an antibody or antigen-binding fragment of the disclosure.

Immunoconiugates

[00235] In another aspect, the present disclosure features an anti-TNF-alpha antibody of the disclosure, or a fragment thereof, conjugated to an active or therapeutic moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin. Such conjugates are referred to herein as "immunoconjugates".

[00236] Immunoconjugates that include one or more cytotoxins are referred to as

"immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Examples include taxon, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, t. colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 - dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine), ablating agents (e.g., mechlorethamine, thioepa chloraxnbucil, meiphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti -mitotic agents (e.g., vincristine and vinblastine). [00237] Cytoxins can be conjugated to antibodies of the disclosure using linker technology available in the art. Examples of linker types that have been used to conjugate a cytotoxin to an antibody include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptide-containing linkers. A linker can be chosen that is, for example, susceptible to cleavage by low pH within the lysosomal compartment or susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).

[00238] For further discussion of types of cytotoxins, linkers and methods for conjugating therapeutic agents to antibodies, see also Saito, G. et al. 2003, Adv. Drug Deliv. Rev. 55: 199-215; Trail, P.A. et al. 2003, Cancer Immunol. Immunother. 52:328-337; Payne, G. 2003, Cancer Cell 3:207-212; Allen, T.M. 2002, Nat. Rev. Cancer 2:750-763; Pastan, I. and Kreitman, R. J. 2002, Curr. Opin. Investig. Drugs 3: 1089-1091; Senter, P.D. and

Springer, C.J. 2001, Adv. Drug Deliv. Rev. 53:247-264.

[00239] Antibodies of the present disclosure also can be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals, also referred to as

radioimmunoconjugates. Examples of radioactive isotopes that can be conjugated to antibodies for use diagnostically or therapeutically include, but are not limited to, iodine ¹³¹ , indium ¹¹¹ , yttrium ⁹⁰ , and lutetium ¹⁷⁷ . Method for preparing radioimmunconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including ZEVALIN (DEC Pharmaceuticals) and BEXXAR (Corixa Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the disclosure.

[00240] The antibody conjugates of the disclosure can be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon-g; or, biological response modifiers such as, for example, lymphokines, interleukin- 1 ("IL-1"), interleukin-2 ("IL-2"), interleukin- 6 ("IL6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors. [00241] Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Amon et al.1985, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56; Hellstrom et at. 1987, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 ; Thorpe 1985, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506; Thorpe et al. 1982, Immunol. Rev., 62: 119-58.

Kits

[00242] The disclosure also encompasses kits for treating a patient having a pathological disorder mediated by TNF -alpha, e.g., an autoimmune disease or an

inflammatory disorder or condition. Such kits comprise a therapeutically effective amount of a TNF-alpha antibody or antigen-binding fragment thereof according to the disclosure, e.g., v4 or v40 (ideally as part of a pharmaceutical composition). Additionally, such kits may comprise means for administering the TNF-alpha antibody or antigen-binding fragment (e.g., an autoinjector, a syringe and vial, a prefdled syringe, a prefilled pen) and instructions for use. These kits may contain additional therapeutic agents (described infra) for treating having a pathological disorder mediated by TNF-alpha, e.g., an autoimmune disease or an

inflammatory disorder or condition. Such kits may also comprise instructions for

administration of the TNF-alpha antibody or antigen-binding fragment thereof to treat the patient. Such instructions may provide the dose, route of administration, regimen, and total treatment duration for use with the enclosed TNF-alpha antibody or antigen-binding fragment, e.g., v4 or v40.

[00243] The phrase“means for administering” is used to indicate any available implement for systemically administering a drug to a patient, including, but not limited to, a pre-filled syringe, a vial and syringe, an injection pen, an autoinjector, an IV drip and bag, a pump, etc. With such items, a patient may self-administer the drug (i.e., administer the drug without the assistance of a physician) or a medical practitioner may administer the drug.

[00244] Disclosed herein are kits comprising an antibody or antigen-binding fragment or a pharmaceutical composition as described herein, wherein the kit additionally comprises instructions for use and means for administering said antibody or antigen-binding fragment thereof or said pharmaceutical composition to a subject in need thereof.

Therapeutic and Diagnostic Uses

[00245] The antibodies or proteins of the present disclosure have in vitro and in vivo diagnostic and therapeutic utilities. For example, these molecules can be administered to cells in culture, e.g. in vitro or in vivo, or in a subject, e.g., in vivo, to treat, prevent or diagnose a variety of disorders.

[00246] Disclosed herein are diagnostic reagents comprising an antibody or antigen binding fragment as described herein and a diagnostic label.

[00247] In one aspect, the antibodies or antigen-binding fragments thereof, are useful for detecting the presence of TNF-alpha in a biological sample. The term "detecting" as used herein encompasses quantitative or qualitative detection. In certain aspects, a biological sample comprises a cell or tissue. In certain aspects, such tissues include normal and/or cancerous tissues that express TNF-alpha at higher levels relative to other tissues.

[00248] In one aspect, the present disclosure provides a method of detecting the presence of TNF-alpha in a biological sample. In certain aspects, the method comprises contacting the biological sample with an anti -TNF-alpha antibody or fragment under conditions permissive for binding of the antibody to the antigen, and detecting whether a complex is formed between the antibody and the antigen. The biological sample can include, without limitation, urine or blood samples.

[00249] Also included is a method of diagnosing a disorder associated with expression of TNF-alpha. In certain aspects, the method comprises contacting a test cell with an anti- TNF -alpha antibody or fragment; determining the level of expression (either quantitatively or qualitatively) of TNF-alpha in the test cell by detecting binding of the antibody to TNF-alpha; and comparing the level of expression of TNF-alpha in the test cell with the level of expression of TNF-alpha in a control cell (e.g. , a normal cell of the same tissue origin as the test cell or a non-virus infected cell), wherein a higher level of presence of TNF-alpha in the test cell as compared to the control cell indicates the presence of a disorder associated with TNF-alpha. In certain aspects, the test cell is obtained from an individual suspected of having a pathological disorder mediated by TNF-alpha. [00250] In certain aspects, a method of diagnosis or detection, such as those described above, comprises detecting binding of a TNF-alpha antibody or fragment to a cell, e.g., using a "FACS" assay.

[00251] Certain other methods can be used to detect binding of a TNF-alpha antibody or fragments. Such methods include, but are not limited to, antigen-binding assays that are well known in the art, such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays, and immunohistochemistry (IHC).

[00252] In certain aspects, the TNF-alpha antibody or fragment is labeled. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g. , through an enzymatic reaction or molecular interaction.

[00253] In certain aspects, the TNF-alpha antibody or fragment is immobilized on an insoluble matrix. Immobilization entails separating the anti-TNF-alpha antibody or fragment from any proteins that remain free in solution. This conventionally is accomplished by either insolubilizing the TNF-alpha antibody or fragment before the assay procedure, as by adsorption to a water-insoluble matrix or surface (Bennich et al, U.S. Patent No. 3,720,760), or by covalent coupling (for example, using glutaraldehyde cross-linking), or by

insolubilizing the TNF-alpha antibody or fragment after formation of a complex between the TNF-alpha antibody or fragment and TNF-alpha, e.g. , by immunoprecipitation.

[00254] Any of the above aspects of diagnosis or detection can be carried out using an

TNF-alpha antibody or fragment of the present disclosure in place of or in addition to another TNF-alpha antibody or fragment (e.g., adalimumab).

[00255] The antibodies and antibody fragments (e.g. , antigen-binding fragments) of the present disclosure are useful in treating, reducing the likelihood of or ameliorating a pathological disorder mediated by TNF-alpha.

[00256] Additionally disclosed herein is the use of an antibody or antigen-binding fragment thereof or a pharmaceutical composition as described herein for use in the treatment of a pathological disorder mediated by TNF-alpha. In some embodiments, the antibody or antigen-binding fragment thereof or pharmaceutical composition as described herein is for use in combination with one or more additional therapeutic agents. [00257] Additionally disclosed herein is the use of an antibody or antigen-binding fragment thereof or a pharmaceutical composition as described herein in the manufacture of a medicament for use in the treatment of a pathological disorder mediated by TNF-alpha. In some embodiments, the medicament having the antibody or antigen-binding fragment thereof or pharmaceutical composition as described herein is for use in combination with one or more additional therapeutic agents.

[00258] Additionally disclosed herein is a method of treating a pathological disorder mediated by TNF-alpha, said method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof or a pharmaceutical composition as described herein to a subject in need thereof. In some embodiments, the antibody or antigen binding fragment thereof or pharmaceutical composition as described herein is administered to the subject with one or more additional therapeutic agents.

[00259] The phrase“pathological disorder mediated by TNF-alpha” encompasses all diseases and medical conditions in which TNF-alpha, whether directly or indirectly, in the disease or medical condition, including the causation, development, progress, persistence or pathology of the disease or condition. Accordingly these terms include conditions associated with or characterized by aberrant TNF-alpha levels and/or diseases or conditions that can be treated by reducing or suppressing TNF-alpha induced activity in target cells or tissues, e.g., the production of IL-6 or IL-1. These include autoimmune diseases and inflammatory disorders or conditions, such as arthritis, rheumatoid arthritis, or psoriasis. These further include allergies and allergic conditions, hypersensitivity reactions, chronic obstructive pulmonary disease, cystic fibrosis and organ or tissue transplant rejection.

[00260] For example, the antibodies or fragments of the disclosure may be used for the treatment of recipients of heart, lung, combined heart lung, liver, kidney, pancreatic, skin or comeal transplants, including allograft rejection or xenograft rejection, and for the prevention of graft versus host disease, such as following bone marrow transplant, and organ transplant associated arteriosclerosis.

[00261] The antibodies or fragments of the disclosure, whilst not being limited to, are useful for the treatment, prevention, or amelioration of autoimmune disease and/or inflammatory conditions and disorders, in particular inflammatory conditions with an etiology including an autoimmune component such as arthritis (for example rheumatoid arthritis, arthritis chronica progrediente and arthritis deformans) and rheumatic diseases, including inflammatory conditions and rheumatic diseases involving bone loss, inflammatory pain, spondyloarhropathies, including ankylosing spondylitis, Reiter syndrome, reactive arthritis, psoriatic arthritis, juvenile idiopathic arthritis and enterophathis arthritis, enthesitis, hypersensitivity (including both airways hypersensitivity and dermal hypersensitivity) and allergies.

[00262] The antibodies or fragments of the disclosure may also be useful for the treatment, prevention, or amelioration of arthritis, preferably rheumatoid arthritis, arthritis chronica progrediente, reactive arthritis, psoriatic arthritis, enterophathic arthritis and arthritis deformans, rheumatic diseases, spondyloarthropathies, ankylosing spondylitis, juvenile psoriasis, juvenile idiopathic arthritis, Reiter syndrome, hypersensitivity (including both airways hypersensitivity and dermal hypersensitivity), allergies, lupus (systemic lupus erythematosus, SLE, or lupus nephritis), inflammatory muscle disorders, polychondritis, sclerodoma, Wegener granulomatosis, dermatomyositis, idiopathic sprue, Steven-Johnson syndrome, chronic active hepatitis, myasthenia gravis, psoriasis, idiopathic sprue, autoimmune inflammatory bowel disease, ulcerative colitis, Crohn's disease, Irritable Bowel Syndrome, endocrine ophthalmopathy, Grave’s disease, sarcoidosis, fascular sarcoidosis, multiple sclerosis, primary biliary cirrhosis, juvenile diabetes (diabetes mellitus type I), autoimmune haematological disorders, hemolytic anaemia, aplastic anaemia, pure red cell anaemia, idiopathic thrombocytopenia, uveitis (anterior and posterior), keratoconjunctivitis sicca, vernal keratoconjunctivitis, fibrosis, interstitial lung fibrosis, idiopathic interstitial pneumonia, glomerulonephritis (with and without nephrotic syndrome), idiopathic nephrotic syndrome or minimal change nephropathy, tumors, inflammatory disease of skin, cornea inflammation, myositis, loosening of bone implants, metabolic disorders, atherosclerosis, diabetes, and dislipidemia, bone loss, osteoarthritis, osteoporosis, obstructive or inflammatory airways diseases, asthma, bronchitis, pneumoconiosis, pulmonary emphysema, acute and hyperacute inflammatory reactions, acute infections, septic shock, endotoxic shock, adult respiratory distress syndrome, meningitis, pneumonia, severe bums, cachexia wasting syndrome, stroke, Bechet' s disease, herpetic stromal keratitis, Hidradentis suppurativa, sepsis, neuromyelitis optica, cutaneous infection, cachexia, chronic obstructive pulmonary disease, heart failure, Kawasaki disease, Hashimoto's thyroiditis, ischemia, infarction, anal fistula, ichthyosis, acne, vitiligo, alopecia areata, atopic dermatitis, vasculitis, lichen pilaris, lichen striatus, generlized pustular psoriasis, tendinopathy, small and medium vessel primary vasculitis, bronchiolitis, large vessel vasculitides including giant cell arteritis, polymyalgia rheumatica, periprosthetic osteolysis, glomerulonephritis (with and without nephrotic syndrome, e.g. including idiopathic nephrotic syndrome or minimal change nephropathy), multiple myeloma other types of tumors, cancer, seborrhea, thyoma, primary biliary cirrhosis (PBC), scleroderma, autoimmune thyroid disease, polymyositis, Sjogren's syndrome, pernicious anemia, idiopathic thrombopenic purpura (ITP), loosening of bone implants, metabolic disorders, (such as obesity, atherosclerosis and other cardiovascular diseases including dilated cardiomyopathy, myocarditis, diabetes mellitus type II, and dyslipidemia), Addison's disease, systemic sclerosis, autoinflammatory fever syndromes, haematological disorders (including e.g. hemolytic anaemia, aplastic anaemia, autoimmune hemolytic anemia (AIHA), pure red cell anaemia and idiopathic thrombocytopenia), dermatitis herpetiformis, pemphigus erythematosus, pyoderma gangrenosum, ichthyoses, pytiriasis rubra pilaris, rosacea (e.g, papulopustular rosacea), lichen planopilaris, allergic contact dermatitis, viral infection and complications of viral infection (e.g., human papilloma virus (HPV)), morphoea, dry eye disease, inflammatory muscle diseases, autoimmune peripheral neurophaties, atherothrombosis, autoinflammatory fever syndromes, bullous diseases of the skin and mucous membranes, diseases of bone metabolism including osteoarthritis, osteoporosis and other inflammatory arthritis, and bone loss in general, including age-related bone loss, and in particular periodontal disease, chronic candidiasis and other chronic fungal diseases.

[00263] Anatomically, uveitis can be anterior, intermediate, posterior, or pan-uveitis. It can be chronic or acute. The etiology of uveitis can be autoimmune or non-infectious, infectious, associated with systemic disease, or a white-dot syndrome.

[00264] In one aspect, the disclosure provides for a method of treating, reducing the likelihood of or ameliorating a a pathological disorder mediated by TNF -alpha, comprising administering the antibodies, antibody fragments (e.g., antigen-binding fragments) disclosed herein, to a patient. In certain aspects, the pathological disorder mediated by TNF -alpha is an autoimmune disease or an inflammatory disorder or condition. The following list of conditions comprises particularly preferred targets for treatment with antibodies or an antigen binding portion thereof according to the disclosure: psoriatic arthritis, ankylosing spondylitis, axial spondyloarthris, and rheumatoid arthritis.

Combination Therapy [00265] In certain instances, the antibody or antibody fragment (e.g. , antigen-binding fragment), of the present disclosure is combined with one or more additional therapeutic agents, such as other anti-viral agents, anti-allergic agents, anti-nausea agents (or anti emetics), pain relievers, cytoprotective agents, immunosuppressants and combinations thereof.

[00266] The term“pharmaceutical combination” as used herein refers to either a fixed combination in one dosage unit form, or non-fixed combination or a kit of parts for the combined administration where two or more therapeutic agents may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g. synergistic effect.

[00267] An antibody or fragment of the disclosure may be used in combination with other agents and therapies that have use in treating various diseases, disorders and conditions (herein“additional therapeutic agent”).

[00268] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a therapeutic condition or infection described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration encompasses co-administration in multiple, or in separate containers (e.g., capsules, powders, and liquids) for each active ingredient.

Powders and/or liquids may be reconstituted or diluted to a desired dose prior to

administration. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein.

[00269] The combination therapy can provide "synergy" and prove "synergistic", i.e., the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the individual components separately. A synergistic effect can be attained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect can be attained when the individual components are administered or delivered sequentially, e.g., by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.

[00270] In one aspect, the present disclosure provides a method of treating a disease or disorder as disclosed herein by administering to a subject in need thereof an anti -TNF -alpha antibody or fragment in combination with an additional therapeutic agent, e.g., for the treatment or prevention of diseases mentioned above.

[00271] For example, the antibodies of the disclosure may be used in combination with a disease modifying anti-rheumatic drug (DMARD), e.g., Gold salts, sulphasalazine, antimalarias, methotrexate, D-penicillamine, azathioprine, mycophenolic acid, tacrolimus, sirolimus, minocycline, leflunomide, glucocorticoids; a calcineurin inhibitor, e.g. cyclosporin A or FK 506; a modulator of lymphocyte recirculation, e.g. FTY720 and FTY720 analogs; a mTOR inhibitor, e.g. rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, CCI779, ABT578, AP23573 or TAFA-93; an ascomycin having immuno-suppressive properties, e.g. ABT-281, ASM981, etc.; corticosteroids; cyclophosphamide; azathioprine; leflunomide; mizoribine; myco-pheno-late mofetil; 15-deoxyspergualine or an immunosuppressive homologue, analogue or derivative thereof; immunosuppressive monoclonal antibodies, e.g., monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40. CD45, CD58, CD80, CD86 or their ligands; other immunomodulatory compounds, e.g. a recombinant binding molecule having at least a portion of the extracellular domain of CTLA4 or a mutant thereof, e.g. an at least extracellular portion of CTLA4 or a mutant thereof joined to a non-CTLA4 protein sequence, e.g. CTLA4Ig (for ex. designated ATCC 68629) or a mutant thereof, e.g. LEA29Y ; adhesion molecule inhibitors, e.g. LFA-1 antagonists, ICAM-1 or -3 antagonists, VCAM-4 antagonists or VLA-4 antagonists; or a chemotherapeutic agent, e.g. paclitaxel, gemcitabine, cisplatinum, doxorubicin or 5- fluorouracil; other anti-TNF agents, e.g., monoclonal antibodies to TNF, e.g. infliximab, adalimumab, CDP870, or receptor constructs to TNF-RI or TNF-RII, e.g. Etanercept, PEG- TNF-RI; blockers of proinflammatory cytokines, IL-1 blockers, e.g. Anakinra or IL-1 trap, canakinumab, IL-13 blockers, IL-4 blockers, IL-6 blockers, IL-17 blockers (such as secukinumab, broadalumab, ixekizumab); chemokines blockers, e.g., inhibitors or activators of proteases, e.g., metalloproteases, anti-IL-15 antibodies, anti-IL-6 antibodies, anti-IL-4 antibodies, anti-IL-13 antibodies, anti-CD20 antibodies, NSAIDs, such as aspirin or an anti- infectious agent (list not limited to the agent mentioned). [00272] In accordance with the foregoing, the present disclosure provides in a yet further aspect, a method as defined above comprising co-administration, e.g., concomitantly or in sequence, of a therapeutically effective amount of an anti-TNF-alpha antibody or antigen-binding portion thereof as disclosed herein, and at least one additional therapeutic agent, said additional therapeutic agent being a immuno-suppressive, immunomodulatory, anti-inflammatory, chemotherapeutic or anti-infectious drug, e.g., as indicated above. The additional therapeutic agent may be provided with the anti-TNF-alpha antibody or antigen binding portion thereof as part of a kit.

[00273] Where the antibodies or antigen-binding portion thereof as disclosed herein are administered in conjunction with an immuno-suppressive, immunomodulatory, anti inflammatory, chemotherapeutic, or anti-infectious therapy, dosages of the co-administered combination compound will vary depending on the type of co-drug employed, e.g., whether it is a DMARD, anti-IL-17 antibody, IL-1 blocker or others, on the specific drug employed, on the condition being treated, and so forth.

Pharmaceutical Compositions

[00274] Disclosed herein are pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof as described herein, in combination with one or more pharmaceutically acceptable excipient, diluent or carrier.

[00275] Disclosed herein are pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof as described herein, in combination with one or more additional therapeutic agent.

[00276] To prepare pharmaceutical or sterile compositions including anti-TNF-alpha antibodies or fragments thereof, the antibodies of fragments of the present disclosure are mixed with a pharmaceutically acceptable carrier or excipient. The compositions can additionally contain one or more other therapeutic agents that are suitable for reducing the activity of TNF -alpha in a sample or patient.

[00277] The term“pharmaceutical composition” refers to a mixture of at least one active ingredient (e.g., an antibody or fragment of the disclosure) and at least one pharmaceutically-acceptable excipient, diluent or carrier.

[00278] A“medicament” refers to a substance used for medical treatment.

[00279] The phrase "pharmaceutically acceptable" means approved by a regulatory agency of a federal or a state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans. [00280] Pharmaceutically acceptable carriers includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). In one embodiment, the carrier should be suitable for subcutaneous route. Depending on the route of administration, the active compound, i.e., antibody, immunoconjugate, or bispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[00281] Formulations of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g. , lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, e.g., Hardman et al , Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y., 2001; Gennaro, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y., 2000; Avis, et al. (eds.), Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY, 1993; Lieberman, et al. (eds.),

Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY, 1990; Lieberman, et al. (eds.) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY, 1990; Weiner and Kotkoskie, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y., 2000).

[00282] The anti -TNF -alpha antibody of fragment may be produced as a lyophilisate in a vial. The lyophilisate can be reconstituted with water or a pharmaceutical carrier suitable for injection. For subsequent intravenous administration, the obtained solution will usually be further diluted into a carrier solution.

[00283] Selecting an administration regimen for a therapeutic depends on several factors, including the severity of the infection, the level of symptoms, and the accessibility of the target cells in the biological matrix. In certain aspects, an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available (see, e.g.,

Wawrzynczak, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK, 1996; Kresina

(ed.), Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y.,

1991; Bach (ed.), Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY., 1993; Baert et al. , New Engl. J. Med. 348:601-608, 2003; Milgrom et al, New Engl. J. Med. 341 : 1966-1973, 1999; Slamon et al , New Engl. J. Med. 344:783-792, 2001; Beniaminovitz et al, New Engl. J. Med. 342:613-619, 2000; Ghosh et al, New Engl. J. Med. 348:24-32, 2003; Lipsky et al , New Engl. J. Med. 343: 1594-1602, 2000).

[00284] Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., infusion reactions.

[00285] Actual dosage levels of the active ingredients in the pharmaceutical compositions with the anti-TNF -alpha antibodies and fragments can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the antibodies, the route of administration, the time of administration, the half-life of the antibody in the patient, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors known in the medical arts.

[00286] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. [00287] Compositions comprising antibodies or fragments thereof can be provided by continuous infusion, or by doses at intervals of, e.g., one day, one week, or 1-7 times per week. Doses can be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, or by inhalation. A specific dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects.

[00288] For administration of the antibody or protein, the dosage ranges from about

0.0001 to 150 mg/kg, such as 5, 15, and 50 mg/kg subcutaneous administration, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once per month, once every 3 months or once every three to 6 months. Dosage regimens for an anti -TNF -alpha antibody or fragment of the disclosure include 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg, 10 mg/kg, 20 mg/kg or 30 mg/kg by intravenous administration, with the antibody being given using one of the following dosing schedules: every four weeks for six dosages, then every three months; every three weeks; 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks. Preferred doses (e.g., adult doses) are 40 mg, 80 mg and 160 mg, preferably given subcutaneously.

[00289] Doses of the antibodies then can be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months.

[00290] An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method, route and dose of administration and the severity of side effects (see, e.g., Maynard et al, A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla., 1996; Dent, Good Laboratory and Good Clinical Practice, Urch Pubk, London, UK, 2001).

[00291] The route of administration may be by, e.g. , topical or cutaneous application, injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional, or by sustained release systems or an implant (see, e.g., Sidman et al., Biopolymers 22:547-556, 1983; Langer et al, J. Biomed. Mater. Res. 15: 167-277, 1981; Langer, Chem. Tech. 12:98-105, 1982; Epstein et al, Proc. Natl. Acad.

Sci. USA 82:3688-3692, 1985; Hwang et al, Proc. Natl. Acad. Sci. USA 77:4030-4034, 1980; U.S. Pat. Nos. 6,350,466 and 6,316,024). Where necessary, the composition may also include a solubilizing agent or a local anesthetic such as lidocaine to ease pain at the site of the injection, or both. In addition, pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g. , U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference their entirety.

[00292] A composition of the present disclosure can also be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Selected routes of administration for the antibodies include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. Parenteral administration can represent modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion. Alternatively, a composition of the present disclosure can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. In one aspect, the antibodies of the present disclosure are administered by infusion. In another aspect, the antibodies are administered subcutaneously.

[00293] If the antibodies of the present disclosure are administered in a controlled release or sustained release system, a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, CRC Crit. RefBiomed. Eng. 14:20, 1987; Buchwald et al , Surgery 88:507, 1980; Saudek et al, N. Engl. J. Med. 321 :574, 1989). Polymeric materials can be used to achieve controlled or sustained release of the therapies of the antibodies (see e.g. , Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York, 1984; Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61, 1983; see also Levy et al , Science 228: 190, 1985; During et al, Ann. Neurol. 25:351, 1989; Howard et al, J. Neurosurg. 7 1 : 105, 1989; U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly(2 -hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG),

polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,

polyethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and poly orthoesters. In one aspect, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. A controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical

Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984).

[00294] Controlled release systems are discussed in the review by Langer, Science

249: 1527-1533, 1990). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more antibodies of the present disclosure. See, e.g., U.S. Pat. No. 4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698, Ning et ciL, Radiotherapy & Oncology 39: 179-189, 1996; Song et ciL, PDA Journal of Pharmaceutical Science & Technology 50:372-397, 1995; Cleek et al, Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, 1997; and Lam et al, Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, 1997, each of which is incorporated herein by reference in their entirety.

[00295] Various means for administering therapeutic compositions are known in the art. For example, in one embodiment, a therapeutic composition of the disclosure can be administered with a needleless hypodermic injection device, such as the devices shown in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556. Examples of well-known implants and modules useful in the present disclosure include: U.S. Patent No. 4,487,603, which shows an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which shows a therapeutic device for administering medicants through the skin; U.S. Patent No. 4,447,233, which shows a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which shows a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which shows an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which shows an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art. In preferred embodiments, the means for administering the anti-TNF-alpha antibodies and fragments is selected from a syringe, an autoinjector, an injection pen, a vial and syringe, an infusion pump, a patch, or an infusion bag and needle.

[00296] If the antibodies of the disclosure are administered topically, they can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g. , Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity, in some instances, greater than water are typically employed. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g. , preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, in some instances, in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well-known in the art.

[00297] If the compositions comprising the antibodies are administered intranasally, it can be formulated in an aerosol form, spray, mist or in the form of drops. In particular, prophylactic or therapeutic agents for use according to the present disclosure can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane,

trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[00298] Methods for co-administration or treatment with an additional therapeutic agent, e.g , an immunosuppressant, a cytokine, steroid, chemotherapeutic agent, antibiotic, etc., are known in the art (see, e.g., Hardman el al, (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.). An effective amount of therapeutic may decrease the symptoms by at least 10%; by at least 20%; at least about 30%; at least 40%, or at least 50%.

[00299] Additional therapies (e.g. , prophylactic or therapeutic agents), which can be administered in combination with the anti -TNF -alpha antibodies may be administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart from the anti-TNF -alpha antibodies and fragments of the present disclosure. The two or more therapies may be administered within one same patient visit.

[00300] In certain aspects, anti -TNF -alpha antibodies and fragments can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the anti-TNF-alpha antibodies and fragments cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties, which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., Ranade, (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g. , U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa e/ a/. , (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (Bloeman et al , (1995) FEBS Lett. 357: 140; Owais et al , (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al, (1995) Am. J. Physiol. 1233: 134); p 120 (Schreier et al, (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346: 123; J. J. Killion; L J. Fidler (1994) Immunomethods 4:273.

[00301] The present disclosure provides protocols for the administration of pharmaceutical composition comprising anti-TNF-alpha antibodies and fragments alone or in combination with other therapies to a subject in need thereof. The combination therapies (e.g., prophylactic or therapeutic agents) can be administered concomitantly or sequentially to a subject. The therapy (e.g. , prophylactic or therapeutic agents) of the combination therapies can also be cyclically administered. Cycling therapy involves the administration of a first therapy (e.g. , a first prophylactic or therapeutic agent) for a period of time, followed by the administration of a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time and repeating this sequential administration, i.e., the cycle, in order to reduce the development of resistance to one of the therapies (e.g., agents) to avoid or reduce the side effects of one of the therapies (e.g. , agents), and/or to improve, the efficacy of the therapies.

[00302] The therapies (e.g. , prophylactic or therapeutic agents) of the combination therapies of the disclosure can be administered to a subject concurrently. The term

"concurrently" is not limited to the administration of therapies (e.g., prophylactic or therapeutic agents) at exactly the same time, but rather it is meant that a pharmaceutical composition comprising antibodies or fragments thereof are administered to a subject in a sequence and within a time interval such that the antibodies can act together with the other therapy(ies) to provide an increased benefit than if they were administered otherwise. For example, each therapy may be administered to a subject at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect. Each therapy can be administered to a subject separately, in any appropriate form and by any suitable route. In various aspects, the therapies (e.g., prophylactic or therapeutic agents) are administered to a subject less than 15 minutes, less than 30 minutes, less than 1 hour apart, at about 1 hour apart, at about 1 hour to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, 24 hours apart, 48 hours apart, 72 hours apart, or 1 week apart. In other aspects, two or more therapies (e.g., prophylactic or therapeutic agents) are administered to a within the same patient visit.

[00303] The prophylactic or therapeutic agents of the combination therapies can be administered to a subject in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents of the combination therapies can be administered concurrently to a subject in separate pharmaceutical compositions. The prophylactic or therapeutic agents may be administered to a subject by the same or different routes of administration.

[00304] The details of one or more embodiments of the disclosure are set forth in the accompanying description above. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications cited in this specification are incorporated by reference as applicable, unless otherwise indicated. The following Examples are presented in order to more fully illustrate the preferred embodiments of the disclosure. These examples should in no way be construed as limiting the scope of the disclosed subject matter, which is defined by the appended claims.

EXAMPLES

Example 1: Generation of anti-TNF-alpha antibodies and production of anti- adalimumab Fabs; Expression and Purification

[00305] Methods for generation of monoclonal antibodies using phage display technology are known in the art (Proetzel, G., Ebersbach, H. (Eds.) Antibody Methods and Protocols. Humana Press ISBN 978-1-61779-930-3; 2012). In brief, the anti-TNF-alpha antibodies described herein were generated as follows.

[00306] Heavy chain and light chain DNAs for various adalimumab antibody variants and Fab ADAs (see van Schouwenburg et al. (2014) Jml. Biol. Chem. 289(50): 34482-88) were synthesized at GeneArt (Regensburg, Germany) and cloned into mammalian expression vectors using restriction enzyme-ligation based cloning techniques. The resulting plasmids were co transfected into HEK293T cells. For transient expression of antibodies and Fabs, equal quantities of vector for each engineered chain were co-transfected into suspension-adapted HEK293T cells using Polyethylenimine (PEI; Cat# 24765 Polysciences, Inc.). Typically, 100 ml of cells in suspension at a density of 1-2 Mio cells per ml was transfected with DNA containing 50 pg of expression vector encoding the heavy chain and 50 pg expression vectors encoding the light chain. The recombinant expression vectors were then introduced into the host cells and the construct produced by further culturing of the cells for a period of 7 days to allow for secretion into the culture medium (HEK, serum-fee medium) supplemented with 0.1% pluronic acid, 4mM glutamine, and 0.25 pg/ml antibiotic.

[00307] The adalimumab antibody variants and Fabs produced by the expressed constructs were then purified from cell-free supernatant using immunoaffmity chromatography. Filtered conditioned media was mixed with 300 pi Protein A resin (CaptivA PriMab™, Repligen), equilibrated with PBS buffer pH 7.4. The resin was washed three times with 15 column volumes of PBS pH 7.4 before the antibody or Fab respectively, was eluted with 10 column volumes protein A elution buffer (50mM citrate, 90mM NaCl, pH 2.5).

Example 2: Surface Plasmon Resonance (SPR) binding analysis of adalimumab antibody variants to TNF-alpha

[00308] A direct binding assay was performed to characterize the binding of the engineered antibody variants against TNF-alpha.

[00309] Kinetic binding affinity constants (KD) were measured on protein-A captured protein using recombinant human antigens as analyte. Measurements were conducted on a BIAcore ^® T200 (GE Healthcare, Glattbrugg, Switzerland) at room temperature. For affinity measurements, the proteins were diluted in lOmM NaP, 150mM NaCl, 0.05% Tween 20, pH 5.8 and immobilized on the flow cells of a CM5 research grade sensor chip (GE Healthcare, ref BR- 1000- 14) using standard procedure according to the manufacturer’s recommendation (GE Healthcare). To serve as reference, one flow cell was blank immobilized. Binding data were acquired by subsequent injection of analyte dilutions series on the reference and measuring flow cell. Zero concentration samples (running buffer only) were included to allow double referencing during data evaluation. For data evaluation, doubled referenced sensograms were used and dissociation constants (KD) analyzed. Several adalimumab antibody variants bind to TNF-alpha in the same affinity range as wild-type (WT) adalimumab (results shown in Table

4).

Table 4 - Dissociation constants (K _D ) of adalimumab and antibody variants binding to TNF-alpha

*VKl-33 FR3 refers to a light chain having the following mutations: T66N, Q68E, V99I (IMGT). ** VK1-13 FR3 refers to a light chain having the following mutations in: T66S, Q68E, V99F (IMGT).

Example 3: Relative binding of adalimumab and adalimumab antibody variants to anti drug antibody Fabs (AD As)

[00310] Binding analysis of adalimumab and adalimumab antibody variants with ADA

Fabs was performed by SPR measurement using the Proteon XPR36 system (Bio-Rad).

[00311] Adalimumab and adalimumab antibody variants were immobilized on a Proteon

GLC chip (Bio-Rad ref. 1765011) by amine coupling. Kinetic data were acquired by injection of ADA Fabs diluted in lOmM NaP, 150mM NaCl, 0.05% Tween 20, pH 5.8. To serve as reference, one flow cell was blank immobilized. Maximum response (Rmax) rates of adalimumab antibody variants were extracted, normalized, and compared to maximum response rates observed for adalimumab. Relative binding levels of adalimumab and adalimumab variants are shown in Table 5. Figure 1 shows relative binding of variants 4 and 40 in comparison to adalimumab.

Table 5 - Relative binding levels of adalimumab and antibody variants to Fab ADAs

Example 4: Dynamic light scattering (DLS) of adalimumab and antibody variants

[00312] The aggregation propensity of adalimumab and adalimumab antibody variants was measured by dynamic light scattering (DLS, Wyatt). Dynamic light scattering was applied to measure the translational diffusion coefficients of adalimumab variants in solution by quantifying dynamic fluctuations in scattered light. Adalimumab variant size distributions without fractionation, providing polydispersity estimates as well as hydrodynamic radii were measured at a concentration of lOmg/ml. Hydrodynamic radii of adalimumab variants upon thermal stress were determined with a DynaPro™ plate reader (Wyatt Technology Europe GmbH, Dembach, Germany) combined with the software DYNAMICS (version 7.1.0.25, Wyatt). 50 pL of the undiluted and filtered (0.22 pm PVDF-Filter (Millex® Syringe-driven Filter Unit, Millipore, Billerica, USA)) protein solution was measured in a 384-well plate (384 round well plate, Polystyrol, Thermo Scientific, Fangenselbold, Germany). Higher molecular weight (MW) aggregates of the thermally stressed adalimumab sample could be identified, but not from adalimumab variants. Adalimumab shows increased hydrodynamic radius starting at 50°C, whereas antibody variants 4 and 40 retain the same radii until 60°C (Figure 2).

[00313] Based on the low number of mutations, same binding affinity to TNF-alpha

(compared to adalimumab wild-type), and low relative binding to ADAs, Variant 4 and 40 were considered for further studies, i.e., expression studies and biophysical characterization,

e.g., thermal stressed concentration dependent dynamic light scattering and differential scanning calorimetry.

Example 5 - Expression studies and additional DLS experiments with adalimumab, and antibody variants 4 and 40

[00314] For expression studies, adalimumab and antibody variants 4 and 40 were expressed in HEK293T cells (transient transfection, see Example 1). As shown in Figure 3, Adalimumab expresses up to 87 mg/L in HEK cells; antibody variant 4 shows slightly better expression (95mg/L), and antibody variant 40 has significantly better expression yield (132 mg/L). After polishing via size exclusion chromatography, yields of antibody variants 4 (78 mg/L) and variant 40 (112 mg/L) remain higher than adalimumab yield (65 mg/L).

[00315] For further analysis of adalimumab and antibody variants 4 and 40, the concentration of protein in samples was increased up to 100 mg/ml and characterized using DLS under thermal stress in order to measure increases in hydrodynamic radius, an indicator of aggregation formation. Adalimumab at concentrations of 70 mg/ml shows an increase of hydrodynamic radius, indicating aggregation formation above 50°C, whereas variant 4 shows slight formation of aggregation only at 100 mg/ml concentration above 60°C and variant 40 retain the hydrodynamic radius at the highest measured concentration (100 mg/ml) and temperature (60°C). Results are shown in Figure 4A-C.

Example 6: Differential scanning calorimetry (DSC) of adalimumab and antibody variants 4 and 40

[00316] The thermal stability of adalimumab and engineered variants 4 and 40 was compared using calorimetric measurements. Calorimetric measurements were carried out on a differential scanning micro calorimeter (Nano DSC, TA instruments). The cell volume was 0.5ml and the heating rate was l°C/min. All proteins were used at a concentration of lmg/ml in PBS (pH 7.4). The molar heat capacity of each protein was estimated by comparison with duplicate samples containing identical buffer from which the protein had been omitted. The partial molar heat capacities and melting curves were analyzed using standard procedure. Thermograms were baseline corrected and concentration normalized. Adalimumab, variant 4 and variant 40 show similar melting points (Tm) of Fabs, as shown in Table 6.

Table 6: Melting points of wild-type adalimumab and antibody variants 4 and 40.

Example 7: Formulation

[00317] The anti-TNF -alpha antibodies described herein are monoclonal antibodies that can be provided in a ready-to-use liquid formulation or as a lyophilisate. For subsequent intravenous administration, a lyophilisate will usually be further diluted into a carrier solution to prepare a liquic antibody solution for infusion. Important stability-indicating analytical methods to select the most stable formulation include, amongst others, size -exclusion chromatography to determine aggregation levels, subvisible particulate matter testing, and potency testing.

[00318] It is understood that the examples and aspects described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

[00319] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety (or as context dictates), to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

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145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175 Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190

Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255 lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr 290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly 305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie

325 330 335

Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu 370 375 380

Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 7

<211> 1353

<212> DNA

<213> artificial sequence

<220>

<223> DNA Heavy chain adaliumumab

<400> 7

gaagtgcagc tggtggaatc tggcggagga ctggttcagc ctggcagaag cctgagactg 60 tcttgtgccg ccagcggctt caccttcgat gattatgcca tgcactgggt ccgacaggcc 120 cctggaaaag gacttgaatg ggtgtccgcc atcacctgga acagcggcca catcgattac 180 gccgatagcg tggaaggccg gttcaccatc agcagagaca acgccaagaa cagcctgtac 240 ctccagatga actccctgag agccgaggat accgccgtgt actattgcgc caaggtgtcc 300 tacctgagca ccgcctcttc tctggattac tggggacagg gcaccctggt cacagtgagc 360 tcagctagca ccaagggccc cagcgtgttc cccctggcgc ccagcagcaa gagcaccagc 420 ggcggcacag ccgccctggg ctgcctggtg aaggactact tccccgagcc agtgaccgtg 480 tcctggaaca gcggagccct gacctccggc gtgcacacct tccccgccgt gctgcagagc 540 agcggcctgt acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag 600 acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gagagtggag 660 cccaagagct gcgacaagac ccacacctgc cccccctgtc ctgcccctga actgctgggc 720 ggaccctccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat cagccggacc 780 cccgaagtga cctgcgtggt ggtggacgtg tcccacgagg accctgaagt gaagttcaat 840 tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcccagaga ggaacagtac 900 aacagcacct accgggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 960 aaagagtaca agtgcaaagt ctccaacaag gccctgcctg cccccatcga gaaaaccatc 1020 agcaaggcca agggccagcc ccgcgagccc caggtgtaca cactgccccc cagccgggac 1080 gagctgacca agaaccaggt gtccctgacc tgcctggtca agggcttcta ccccagcgat 1140 atcgccgtgg aatgggagag caacggccag cccgagaaca actacaagac caccccccct 1200 gtgctggaca gcgacggctc attcttcctg tacagcaagc tgaccgtgga caagtcccgg 1260 tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320 acccagaagt ccctgagcct gagccccggc aaa 1353

<210> 8

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1 adalimumab and Variant 4 and variant 40

<400> 8

Gin Gly lie Arg Asn Tyr

1 5

<210> 9

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> LCDR2 adalimumab and Variant 4 and variant 40

<400> 9

A1a A1a Ser

1

<210> 10

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3 adaliumumab and Variant 4 and variant 40

<400> 10

Gin Arg Tyr Asn Arg Ala Pro Tyr Thr

1 5

<210> 11

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> VL adalimumab and Variant 4

<400> 11

Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15 Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Gly lie Arg Asn Tyr

20 25 30

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie

35 40 45

Tyr Ala Ala Ser Thr Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gin Arg Tyr Asn Arg Ala Pro Tyr

85 90 95

Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys

100 105

<210> 12

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> DNA VL adalimumab and Variant 4

<400> 12

gatatccaga tgacacagag ccctagcagc ctgtctgcct ctgtgggcga tagagtgacc 60 atcacatgtc gggccagcca gggcatcaga aactacctgg cctggtatca gcagaagccc 120 ggaaaggccc ctaagctgct gatctatgcc gcctctacac tccagagcgg cgtgccaagc 180 agattttctg gcagcggctc tggcaccgac ttcaccctga ccatttctag cctccagcca 240 gaggacgtgg ccacctacta ctgccagaga tacaacagag ccccttacac cttcggccag 300 ggcaccaagg ttgagatcaa g 321

<210> 13

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> light chain adalimumab and Variant 4

<400> 13

Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15 Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Gly lie Arg Asn Tyr 20 25 30

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie

35 40 45

Tyr Ala Ala Ser Thr Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gin Arg Tyr Asn Arg Ala Pro Tyr

85 90 95

Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140

Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160

Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 14

<211> 642

<212> DNA

<213> artificial sequence

<220>

<223> DNA light chain adalimumab and Variant 4

<400> 14 gatatccaga tgacacagag ccctagcagc ctgtctgcct ctgtgggcga tagagtgacc 60 atcacatgtc gggccagcca gggcatcaga aactacctgg cctggtatca gcagaagccc 120 ggaaaggccc ctaagctgct gatctatgcc gcctctacac tccagagcgg cgtgccaagc 180 agattttctg gcagcggctc tggcaccgac ttcaccctga ccatttctag cctccagcca 240 gaggacgtgg ccacctacta ctgccagaga tacaacagag ccccttacac cttcggccag 300 ggcaccaagg ttgagatcaa gcgtacggtg gcagctccgt ctgttttcat ctttccacct 360 agcgacgagc aactcaaaag tggtacagca tccgtggttt gtctgctgaa caatttttac 420 cccagggagg ctaaggtcca gtggaaagtc gataacgctc ttcagtctgg caacagtcag 480 gagagcgtca cagagcagga ctctaaggat agcacttata gtctgtcctc cacgctgaca 540 ctgtctaaag cggattatga gaagcacaag gtttacgcct gtgaggtaac gcaccaagga 600 ctctcctccc cagttaccaa atctttcaac agaggagaat gt 642

<210> 15

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1 Variant 4 and Variant 40

<400> 15

Gly Phe Thr Phe Asp Asp Ser Ala

1 5

<210> 16

<211> 121

<212> PRT

<213> artificial sequence

<220>

<223> VH variant 4 and Variant 40

<400> 16

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Ser

20 25 30

Ala Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala lie Thr Trp Asn Ser Gly His lie Asp Tyr Ala Asp Ser Val

50 55 60 Glu Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly

100 105 110

Gin Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 17

<211> 363

<212> DNA

<213> artificial sequence

<220>

<223> DNA VH Variant 4 and Variant 40

<400> 17

gaagtgcagc tggtggaatc tggcggagga ctggttcagc ctggcagaag cctgagactg 60 tcttgtgccg ccagcggctt caccttcgat gattctgcca tgcactgggt ccgacaggcc 120 cctggaaaag gacttgaatg ggtgtccgcc atcacctgga acagcggcca catcgattac 180 gccgatagcg tggaaggccg gttcaccatc agcagagaca acgccaagaa cagcctgtac 240 ctccagatga actccctgag agccgaggat accgccgtgt actattgcgc caaggtgtcc 300 tacctgagca ccgcctcttc tctggattac tggggacagg gcaccctggt cacagtgagc 360 tea 363

<210> 18

<211> 451

<212> PRT

<213> artificial sequence

<220>

<223> Heavy chain Variant 4 and variant 40

<400> 18

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Ser

20 25 30

Ala Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Ala lie Thr Trp Asn Ser Gly His lie Asp Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly

100 105 110

Gin Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255 lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie

325 330 335

Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 19

<211> 1353

<212> DNA

<213> artificial sequence

<220>

<223> DNA Heavy chain variant 4 and variant 40

<400> 19

gaagtgcagc tggtggaatc tggcggagga ctggttcagc ctggcagaag cctgagactg 60 tcttgtgccg ccagcggctt caccttcgat gattctgcca tgcactgggt ccgacaggcc 120 cctggaaaag gacttgaatg ggtgtccgcc atcacctgga acagcggcca catcgattac 180 gccgatagcg tggaaggccg gttcaccatc agcagagaca acgccaagaa cagcctgtac 240 ctccagatga actccctgag agccgaggat accgccgtgt actattgcgc caaggtgtcc 300 tacctgagca ccgcctcttc tctggattac tggggacagg gcaccctggt cacagtgagc 360 tcagctagca ccaagggccc cagcgtgttc cccctggcgc ccagcagcaa gagcaccagc 420 ggcggcacag ccgccctggg ctgcctggtg aaggactact tccccgagcc agtgaccgtg 480 tcctggaaca gcggagccct gacctccggc gtgcacacct tccccgccgt gctgcagagc 540 agcggcctgt acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag 600 acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gagagtggag 660 cccaagagct gcgacaagac ccacacctgc cccccctgtc ctgcccctga actgctgggc 720 ggaccctccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat cagccggacc 780 cccgaagtga cctgcgtggt ggtggacgtg tcccacgagg accctgaagt gaagttcaat 840 tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcccagaga ggaacagtac 900 aacagcacct accgggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 960 aaagagtaca agtgcaaagt ctccaacaag gccctgcctg cccccatcga gaaaaccatc 1020 agcaaggcca agggccagcc ccgcgagccc caggtgtaca cactgccccc cagccgggac 1080 gagctgacca agaaccaggt gtccctgacc tgcctggtca agggcttcta ccccagcgat 1140 atcgccgtgg aatgggagag caacggccag cccgagaaca actacaagac caccccccct 1200 gtgctggaca gcgacggctc attcttcctg tacagcaagc tgaccgtgga caagtcccgg 1260 tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320 acccagaagt ccctgagcct gagccccggc aaa 1353

<210> 20

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> VL Variant 40

<400> 20

Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Gly lie Arg Asn Tyr

20 25 30

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie

35 40 45 Tyr Ala Ala Ser Thr Leu Gin Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gin Arg Tyr Asn Arg Ala Pro Tyr

85 90 95

Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys

100 105

<210> 21

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> DNA VL Variant 40

<400> 21

gatatccaga tgacacagag ccctagcagc ctgtctgcct ctgtgggcga tagagtgacc 60 atcacatgtc gggccagcca gggcatcaga aactacctgg cctggtatca gcagaagccc 120 ggaaaggccc ctaagctgct gatctatgcc gcctctacac tccagacagg cgtgccaagc 180 agattttctg gcagcggctc tggcaccgac ttcaccctga ccatttctag cctccagcca 240 gaggacgtgg ccacctacta ctgccagaga tacaacagag ccccttacac cttcggccag 300 ggcaccaagg ttgagatcaa g 321

<210> 22

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> Light chain variant 40

<400> 22

Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Gly lie Arg Asn Tyr

20 25 30

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie

35 40 45 Tyr Ala Ala Ser Thr Leu Gin Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gin Arg Tyr Asn Arg Ala Pro Tyr

85 90 95

Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin

145 150 155 160

Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 23

<211> 642

<212> DNA

<213> artificial sequence

<220>

<223> DNA Light chain variant 40

<400> 23

gatatccaga tgacacagag ccctagcagc ctgtctgcct ctgtgggcga tagagtgacc 60 atcacatgtc gggccagcca gggcatcaga aactacctgg cctggtatca gcagaagccc 120 ggaaaggccc ctaagctgct gatctatgcc gcctctacac tccagacagg cgtgccaagc 180 agattttctg gcagcggctc tggcaccgac ttcaccctga ccatttctag cctccagcca 240 gaggacgtgg ccacctacta ctgccagaga tacaacagag ccccttacac cttcggccag 300 ggcaccaagg ttgagatcaa gcgtacggtg gcagctccgt ctgttttcat ctttccacct 360 agcgacgagc aactcaaaag tggtacagca tccgtggttt gtctgctgaa caatttttac 420 cccagggagg ctaaggtcca gtggaaagtc gataacgctc ttcagtctgg caacagtcag 480 gagagcgtca cagagcagga ctctaaggat agcacttata gtctgtcctc cacgctgaca 540 ctgtctaaag cggattatga gaagcacaag gtttacgcct gtgaggtaac gcaccaagga 600 ctctcctccc cagttaccaa atctttcaac agaggagaat gt 642

<210> 24

<211> 36

<212> PRT

<213> artificial sequence

<220>

<223> VK1-13 FR3

<400> 24

Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro Glu Asp Phe Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 25

<211> 36

<212> PRT

<213> artificial sequence

<220>

<223> VK1-33 FR3

<400> 25

Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Gin Pro Glu Asp lie Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 26 <211> 121

<212> PRT

<213> artificial sequence

<220>

<223> Generic Sequence Variant VH

<220>

<221> 31_Xaa

<222> (31) .. (31)

<223> X may be D (Asp) , S (Ser) , or N (Asn)

<220>

<221> 32_Xaa

<222> (32) .. (32)

<223> X may be Y (Tyr) , S (Ser), or F (Phe)

<220>

<221> 58 Xaa

<222> (5Ϊ) .. (58)

<223> X may be I (lie) or T (Thr)

<220>

<221> 107_Xaa

<222> (107) .. (107)

<223> X may be S (Ser) or A (Ala)

<400> 26

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Arg 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Xaa Xaa

20 25 30

Ala Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala lie Thr Trp Asn Ser Gly His Xaa Asp Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Xaa Leu Asp Tyr Trp Gly

100 105 110

Gin Gly Thr Leu Val Thr Val Ser Ser

115 120 <210> 27

<211> 451

<212> PRT

<213> artificial sequence

<220>

<223> Generic Variant Heavy Chain

<220>

<221> Xaa_31

<222> (31) .. (31)

<223> X may be D (Asp) , S (Ser) , or N (Asn)

<220>

<221> Xaa_32

<222> (32) .. (32)

<223> X may be Y (Tyr) , S (Ser), or F (Phe)

<220>

<221> Xaa 5

<222> (58T.

<223> X may (lie) or T (Thr)

<220>

<221> Xaa_l 07

<222> (107) .. (107)

<223> X may be S (Ser) or A (Ala)

<400> 27

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Arg 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Xaa Xaa

20 25 30

Ala Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala lie Thr Trp Asn Ser Gly His Xaa Asp Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Xaa Leu Asp Tyr Trp Gly

100 105 110

Gin Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255 lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr 290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly 305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie

325 330 335

Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser

355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu 370 375 380

Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 28

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> Generic Variant VL

<220>

<221> Xaa 28

<222> (287. · (28)

<223> X may be G (Gly) or S (Ser)

<220>

<221> Xaa 53

<222> (537- · (53)

<223> X may be T (Thr) , S (Ser) or N (Asn)

<220>

<221> Xaa 54

<222> (547- · (54)

<223> X may be L (Leu) or R (Arg)

<220>

<221> Xaa 55

<222> (557. · (55)

<223> X may be Q (Gin) or E (Glu)

<220>

<221> Xaa 56

<222> (567- · (56)

<223> X may be S (Ser) or T (Thr)

<220> <221> Xaa_83

<222> (83) .. (83)

<223> X may be V (Val) , F (Phe) or I (lie)

<400> 28

Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Xaa lie Arg Asn Tyr

20 25 30

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie

35 40 45

Tyr Ala Ala Ser Xaa Xaa Xaa Xaa Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 75 80

Glu Asp Xaa Ala Thr Tyr Tyr Cys Gin Arg Tyr Asn Arg Ala Pro Tyr

85 90 95

Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg

100 105

<210> 29

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> Generic Variant Light Chain

<220>

<221> Xaa_28

<222> (28) .. (28)

<223> X may be G (Gly) or S (Ser)

<220>

<221> Xaa 53

<222> (537.. (53)

<223> X may be T (Thr) , S (Ser) , or N (Asn)

<220>

<221> Xaa 54

<222> (547. · (54)

<223> X may be L (Leu) or R (Arg)

<220>

<221> Xaa 55

<222> (557. · (55) <223> X may be Q (Gin) or E (Glu)

<220>

<221> Xaa 56

<222> (567. · (56)

<223> X may be S (Ser) or T (Thr)

<220>

<221> Xaa_83

<222> (83) .. (83)

<223> X may be V (Val) , F (Phe) or I (lie)

<400> 29

Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Xaa lie Arg Asn Tyr

20 25 30

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie

35 40 45

Tyr Ala Ala Ser Xaa Xaa Xaa Xaa Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 75 80

Glu Asp Xaa Ala Thr Tyr Tyr Cys Gin Arg Tyr Asn Arg Ala Pro Tyr

85 90 95

Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140

Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160

Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205

Phe Asn Arg Gly Glu Cys

210