Human antibodies capable to bond human tnf alpha

The subject of the invention is an isolated human antibody or antigen binding portion thereof, recombinant human antibody or antigen binding portion thereof, pharmaceutical compositions, isolated nucleic acids, recombinant expression vector, host cell, a method of synthesizing a human antibody that binds human TNFα, a method of inhibiting human activity In vitro TNFα, an antibody or antigen binding portion thereof, uses of the antibody or antigen binding portion thereof.

Tumor necrosis factor a (TNFα) is a cytokine produced by many cell types, including monocytes and macrophages, which was initially identified based on its ability to cause necrosis of certain mouse tumors (see e.g. Old (1985) Science 230: 630- 632). It was then shown that the factor called cachexia associated with cachexia is the same molecule as TNFα. TNFa is associated with shock induction (see, e.g., Beutler and Cerami (1988) Annu. Rev. Biochem. 57: 505-518; Beutler and Cerami (1989) Annu. Rev. Immunol. 7: 625-655). In addition, TNFα is associated with the pathophysiology of various other human diseases and disorders, including sepsis, infections, autoimmune diseases, transplant rejection and graft versus host reactions (see, e.g., Moeller et al. (1990) Cytokine 2: 162-169; U.S. Patent No. 5,231,024, Moeller et al .; European Patent Application No. 260610B1, Moeller et al., Vasilli (1992) Annu. Rev. Immunol. 10: 411-452; Tracey and Cerami (1994) Annu. Rev. Med. 45: 491-503).

Because of the harmful role of human TNF? (HTNF?) In various human disorders, therapeutic strategies have been developed to inhibit or counteract hNNF? Activity. In particular, antibodies have been sought that bind and neutralize hTNFa as a means to inhibit the action of hTNFa. Some of the earlier antibodies were mouse monoclonal antibodies (mAb) secreted by hybridomas made from lymphocytes of mice immunized with hTNFa (see e.g. Hahn et al. (1985) Proc. Natl. Acad. Sei. USA 82: 3814-3818; Liang et al. (1986) Biochem. Biophys. Res. Commun. 137: 847-854; Hirai et al. (1987) J. Immunol. Meth. 96: 57-62; Fendly et al. (1987) Hybridoma 6: 359-370; Moeller et al. (1990) Cytokine 2: 162-169; U.S. Patent No. 5,231,024 to Moeller; European Patent Application No. 186833B1, Wallach; Published European Patent Application No. 218868A1, Old et al .; European Patent Publication No. 260610B1, Moeller et al.). While these antibodies usually showed high affinity for hTNFa (e.g. Kd <10 '⁹ M)

188 192 and were able to neutralize hTNFa activity, their in vivo use may be limited by problems associated with the administration of murine antibodies to humans, such as short serum half-life, inability to induce some human effector functions and induction of an adverse immune response in humans against murine antibodies (human antibody response to mouse antibodies; HAMA). To overcome the problems associated with the use of fully murine antibodies in humans, murine anti-hTNFa antibodies have been genetically engineered to make them more similar to human ones. For example, chimeric antibodies have been produced in which the variable regions of the antibody chains are of murine origin and the constant regions of the antibody chains are of human origin (Knight et al. (1993) Mol. Immunol. 30: 1443-1453; PCT publication WO 92/16553, Daddon et al.). In addition, humanized antibodies have also been produced in which the hypervariable domains of antibody variable regions are of mouse origin, while the remainder of the variable regions and constant regions of the antibody are of human origin (PCT publication WO 92/11383, Adair et al.). However, because such chimeric and humanized antibodies still retain some of the mouse sequences, they can still elicit an unwanted immune response, the response of human antibodies against chimeric antibodies (HACA), especially when administered for a long time, e.g. for chronic indications such as rheumatoid arthritis ( see, e.g., Elliot et al. (1994) Lancet 344: 1125-1127; Elliot et al. (1994) Lancet 344: 1105-1110).

The hTNFa inhibitory agent that is beneficial to murine mAbs or derivatives thereof (e.g., chimeric or humanized antibodies) should be entirely a human anti-hTNFa antibody, since such an agent should not induce HAMA, even when administered for a long time. Human monoclonal antibodies to hTNFa were generated using human hybridoma techniques (Boyle et al. (1993) Cell. Immunol. 152: 556-568; Boyle et al. (1993) Cell. Immunol. 152: 569-581; published European Patent Application No. 614984A2, Boyle et al.). However, it has been reported that these hybridoma derived monoclonal autoantibodies have hTNFa affinity too low to be calculated by conventional methods, were unable to bind soluble hTNFa and unable to neutralize hTNFa-induced cytotoxicity (see Boyle et al., Supra). In addition, the success of the human hybridoma technique depends on the natural presence in human peripheral blood of lymphocytes that produce autoantibodies against hTNFa. Some studies have detected serous autoantibodies against hTNFa in human subjects (Fomsgaard et al. (1989) Scand. J. Immunol. 30: 219-223; Bendtzen et al. (1990) Prog. Leucocyte Biol. 106: 447-452) , while others do not (Leusch et al. (1991) J. Immunol. Meth. 139: 145-147).

An alternative to naturally occurring human anti-hTNFa antibodies should be a recombinant anti-hTNFa antibody. Recombinant human antibodies that bind to hTNFa with relatively low affinity (i.e., Kd ~ 10 'have been described⁷ M) and high disconnection speed (i.e. K_ABOUTff ~ 10 '² s') (Griffith et al., (1993) EMBO J. 12: 725-734). However, due to their relatively fast dissociation kinetics, these antibodies may not be useful for therapeutic applications. In addition, it has been reported that recombinant human anti-hTNFa antibodies do not neutralize hTNFa activity, but rather increase the binding of hTNFa to the cell surface and increase the internalization of hTNFa (Lidbury et al. (1994) Biotechnol. Ther. 5: 27-45; PCT Publication No. WO 92/03145, Aston et al.).

Therefore, human antibodies, such as recombinant human antibodies, that bind soluble hTNFa with high affinity and slow dissociation kinetics are still needed, and which have the ability to neutralize hTNFa activity, including hTNFa-induced cytotoxicity (in vitro and in vivo) and activation of hTNFa-induced cells .

Summary of the Invention

The invention provides an isolated human antibody or antigen binding portion thereof that has the following characteristics:

a) dissociates from the Judean TNF with speed steel K_aboutfr equal to 1 χ 1 0 '³s' ¹ or mmej, as determined by surface plasmon resonance;

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b) has a light chain CDR3 domain comprising the amino acid sequence of Sequence Identifier. No. 3 or modified based on SEQ ID. # 3 by single alanine replacement at positions 1,4, 5, 7 or 8, or by one to five conservative amino acid replacements at positions 1, 3, 4, 6, 7, 8 and / or 9;

c) has a heavy chain CDR3 domain comprising the amino acid sequence of Sequence Identifier. No. 4 or modified based on SEQ ID. No. 4 by single alanine replacement at positions 2, 3, 4, 5, 6, 8, 9, 10 or 11, or by one to five conservative amino acid replacements at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and / or 12.

In a preferred embodiment of the invention, the human antibody or antigen binding portion thereof dissociates from human TNFα with a Kd constant of 1 x 10 '⁸ M or less and with a Koff speed constant of 1 x 10 '³s⁴ or less, determined by surface plasmon resonance and neutralizes the cytotoxicity of human TNFα in a standard in vitro L929 assay with an IC50 of 1 x 10 '⁷ M or less, more preferably an IC50 of 1 x 10 '⁸ M or less, more preferably an IC50 of 1 x 10 '⁹ M or less, even more preferably an IC50 of 5 x 10 '¹⁰ M or less.

In one embodiment, the invention provides an isolated human antibody or antigen binding portion thereof, which is a recombinant antibody or antigen binding portion thereof.

In a preferred embodiment, such an antibody inhibits human TNFα expression of ELAM-1 on human umbilical vein endothelial cells.

In another embodiment of the invention, the isolated human antibody or antigen binding portion thereof dissociates from human TNFα at a rate constant K ", f equal to 5 x W ^ s⁴ or less, more preferably with a Kof rate constant of 1 x 10⁴s ⁴ or less.

The invention includes an isolated human antibody or antigen binding portion thereof comprising a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of Sequence Identifier. No. 3 or modified based on SEQ ID. No. 3 by single replacement with alanine at positions 1, 4, 5, 7 or 8 and a heavy chain variable region (HCVR) with a CDR3 domain comprising the amino acid sequence SEQ ID NO: No. 4 or modified based on SEQ ID. No. 4 by a single replacement with alanine in positions 2, 3, 4, 5, 6, 8, 9, 10 or 11.

Preferably, the LCVR further has a CDR2 domain comprising the amino acid sequence with Sequence Identifier. No. 5 and HCVR also has a CDR2 domain comprising the Sequence ID amino acid sequence. No. 6, more preferably LCVR also has a CDR1 domain comprising the amino acid sequence SEQ ID '. No. 7 and HCVR also has a CDR1 domain comprising the amino acid sequence SEQ ID NO: No. 8

In another embodiment of the invention, the isolated human antibody or antigen binding portion thereof has a light chain variable region (LCVR) having a CDR3 domain comprising an amino acid sequence selected from the group consisting of: Seq Identifier '. No. 3, Sequential Identifier '. No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26 or a heavy chain variable region (HCVR) having a CDR3 domain comprising an amino acid sequence selected from the group consisting of: No. 4, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33 and SEQ ID No. 34.

Another aspect of the invention provides an isolated human antibody or antigen-binding portion thereof that has a light chain variable region (LCVR) comprising the amino acid sequence of Sequence Identifier. # 1 and heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: No. 2

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In a preferred embodiment of this aspect of the invention, the isolated human antibody or antigen binding portion thereof has an IgG1 heavy chain constant region, preferably IgG4.

In another preferred embodiment, the isolated human antibody is a Fab fragment, more preferably is a single chain Fv fragment.

In a further embodiment, the invention provides a recombinant human antibody or antigen binding portion thereof that neutralizes human TNFα activity but does not neutralize human TNFp activity, and possesses the identifying characteristics of the antibody of the invention. Preferably, such an antibody also neutralizes the chimpanzee TNFa activity and at least one additional primate TNFa selected from the group consisting of orangutan TNFa, marmoset TNFa, cynomolgus TNFa and rhesus TNFa, more preferably also neutralizes the dog's TNFa activity, more preferably also neutralizes the pig's TNFa activity.

Another aspect of the invention provides a pharmaceutical composition, comprising the antibody or antigen binding portion thereof of the invention and a pharmaceutically acceptable carrier, preferably the composition comprises at least one additional therapeutic agent for treating a disease in which TNFα activity is harmful. In a preferred embodiment of the composition, the additional therapeutic agents are selected from the group consisting of nonsteroidal anti-inflammatory agents, anti-inflammatory cytokine inhibitory agents, CDP-571 / BAY-10-3356, cA2, 75 kdTNFR-IgG, 55 kdTNFR-IgG, IDEC-CE9.1 / SB210396, DAB486-IL-2, DAB 389-IL-2, Anti-Tac, IL-4, IL-10, IL-4 agonists, IL-10 agonists, IL-1RA, TNF-bp / s-TNFR, S284, R973401, MK-966, Iloprost, methotrexate, thalidomide, thallidomide-related agents, leflunonide, tranexamic acid, T-614, prostaglandin El, Tenidap, Naproxen, Meloxicam, Piroxicam, Diclofenac, Indomethacin, Sulfasalazine, Azathioprine, ICE inhibitors, zap-70 inhibitors, drug inhibitors, VEGF inhibitors, VEGF-R inhibitors, corticosteroids convertase, anti-IL-12 antibodies, interleukin 11 inhibitors, interleukin 13, interleukin-17, gold, penicillamine, chloroquine, hydroxychloroquine, chlorambucil, cyclophosphamide, cyclosporin, anti-thymocyte globulin, anti-CD4, CD5-toxin antibodies, orally administered peptides, collagen, disodium lobenzarite, regulatory factors for HP228 and HP466 cytokines, antisense ICAM-1 phosphorothioate oligodeoxynucleotides, soluble receptor 1 complement, prednisone, glycosin, minocycline polysulfine , anti-IL2R antibodies, marine lipids, plant lipids, auranofin, phenylbutazone, meclofenamic acid, flufenamic acid, intravenous immune globulins, zileuton, mycophenolic acid, tacrolimus, sirolimus, amiprilos, cladribine, azarbin, budenside, epidermal growth factor, aminosalicylates, 6-mercaptopurine, metronidazole, lipoxygenase inhibitors, mesalamine, balsamides -1, monoclonal antibodies against IL-β, monoclonal antibodies against IL-6, growth factors, elastase inhibitors, pyridinyl-imidazole compounds, prednisolone glucuronide-conjugated prodrugs, dexamethasone or budesonide, dextran-conjugated prednisolone prodrugs, slow-release mesalazine, platelet activating factor (PAF) antagonists Platelet Activating Factor), ciprofloxacin, lingocaine, predisolone, methylpredizone, cyclophosphamide, 4-aminopyridine, tizanidine, interferon pla, interferon pib, copolymer 1, hyperbaric oxygen, intravenous immunoglobulin, clbribine, hypertonophilic, hemoglobin antagonist, salt continuous cytokines such as TNFα, IL-βΙ, IL-6 and / or IL-8, SK&F 107647, tetravalent guanylhydrazone CNI-1493, tissue factor pathway inhibitor, PHP, iron chelating agents and chelates, including diethylenetriaminepentaacetic acid-iron (III), lysophylline, PGG-glucan, lipid-reconstituted A-1 apolipoprotein, chiral hydroxamic acids, endotoxin antibodies, E5531, rBPI₂and, synthetic anti-endotoxin peptides, replacement therapy surfactants and anti-IL-8 antibodies.

In a further embodiment of the invention it provides an isolated nucleic acid encoding the light chain of an antibody of the invention, whose CDR3 domain comprises the sequence

188 192 amino acid with SEQ ID No. 3 or modified based on SEQ ID. No. 3 by single alanine replacement at positions 1, 4, 5, 7 or 8, or by one to five conservative amino acid replacements at positions 1, 3, 4, 6, 7, 8 and / or 9, preferably a light chain variable region ( LCVR) of the antibody, preferably the CDR2 domain of the LCVR antibody comprises the amino acid sequence of Sequence Identifier. No. 5, more preferably with SEQ ID. No. 7 In a preferred embodiment, the CDR3 domain comprises the amino acid sequence of Sequence Identifier. No. 4 or modified based on SEQ ID. No. 4 by single alanine replacement at positions 2, 3, 4, 5, 6, 8, 9, 10 or 11, or by one to five conservative amino acid replacements at positions 2, 3,4, 5, 6, 8, 9, 10, 11 and / or 12.

In another preferred embodiment, the isolated nucleic acid of the invention encodes an antibody heavy chain variable region (HCVR), preferably the antibody HCVR CDR2 domain comprises an amino acid sequence having a SEQ ID. No. 6, more preferably with SEQ ID No. 8

In another embodiment of the invention, an isolated nucleic acid encoding the light or heavy chain of an antibody of the invention, wherein the CDR3 domain comprises an amino acid sequence selected from the group consisting of:

a) light chain with SEQ ID No. 3, SEQ ID No. 11-26;

b) a heavy chain with SEQ ID. No. 4, SEQ ID No. 27-34.

The invention also provides an isolated nucleic acid encoding an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 1. No. 1. Preferably, the isolated nucleic acid of the invention encodes the antibody light chain variable region and antibody light chain constant region, preferably the isolated nucleic acid of the invention is a recombinant expression vector. The invention further provides an isolated nucleic acid encoding an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1. # 2. In a preferred embodiment, it encodes the heavy chain variable region of the antibody and heavy chain constant region of the antibody, preferably the heavy chain constant region of the antibody is an IgG1 constant region, more preferably the heavy chain constant region of the antibody is an IgG4 constant region. In a preferred embodiment, the isolated nucleic acid of the invention is a recombinant expression vector.

In yet another embodiment, the invention provides a recombinant expression vector encoding:

a) an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and

b) an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: No. 2

The invention also provides a host cell into which the recombinant expression vector of the invention has been introduced.

Another embodiment of the invention provides a method of synthesizing a human antibody that binds human TNFα, which comprises culturing the host cell of the invention in culture medium until the cell synthesizes the human antibody that binds human TNFα.

Another object of the invention is also a method of inhibiting human TNFα activity in vitro, which comprises contacting human TNFα with an antibody or antigen binding portion thereof of the invention, as a result of which TNFα activity is inhibited.

A further aspect of the invention provides an antibody or antigen binding portion thereof of the invention for use in inhibiting human TNFα activity in a human suffering from a disease in which TNFα activity is harmful.

In a preferred embodiment of this aspect of the invention, the disease is sepsis, preferably the antibody is administered to a human along with a cytokine, interleukin-6 (IL-6) or administered to a human having a serum or plasma IL-6 concentration above 500 pg / ml.

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In another preferred embodiment, the disease is an autoimmune disease, preferably the autoimmune disease is selected from the group consisting of rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, gouty arthritis, allergies, multiple sclerosis, autoimmune diabetes, autoimmune uveitis and nephrotic syndrome.

In further preferred embodiments, the antibody or antigen-binding portion thereof of the invention for use in inhibiting human TNFα activity in a human suffering from a disease in which TNFα activity is harmful is used when the disease is an infectious disease, transplant rejection or graft versus host disease, cancer , lung disease, intestinal disease, heart disease, the disease is selected from the group consisting of inflammatory bone disease, bone resorptive diseases, alcoholic hepatitis, viral hepatitis, hepatitis fulminans, coagulation disorders, burns, post-reperfusion injury, keloid formation, scar tissue formation, fever, periodontal disease, obesity and radiation toxicity.

A further aspect of the invention relates to the use of an antibody or antigen binding portion thereof of the invention for the manufacture of a medicament for the treatment of a disease in which TNFα activity is harmful when the disease is sepsis, preferably the antibody is administered to a human along with a cytokine, interleukin-6 (IL-6) or administered a person with a serum or plasma IL-6 concentration above 500 pg / ml.

A further preferred embodiment of the invention relates to the use of an antibody or antigen binding portion thereof of the invention for the manufacture of a medicament for the treatment of a disease in which TNFα activity is harmful when the disease is rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, gouty arthritis, allergy , multiple sclerosis, diabetic autoaggression, autoimmune uveitis and nephrotic syndrome.

In further preferred embodiments, the antibody or antigen-binding portion thereof of the invention for use in the manufacture of a medicament for the treatment of a disease in which TNFα activity is detrimental is used when graft rejection or graft versus host reaction is neoplastic disease, the disease is selected from the group consisting of inflammatory bone disease, bone resorptive disease, alcoholic hepatitis, viral hepatitis, hepatitis fulminans, coagulation disorders, burns, post-reperfusion injury, keloid formation, scar tissue formation, fever, periodontal disease, obesity, and radiation toxicity.

Another aspect of the invention provides the antibody or antigen binding portion thereof of the invention for use in therapy. '

In yet another embodiment, the invention provides an antibody or antigen binding portion thereof of the invention in combination with at least one additional therapeutic agent for use in treating a disease in which TNFα activity is detrimental. In a preferred embodiment, the additional therapeutic agent is selected from the group consisting of nonsteroidal anti-inflammatory agents, anti-inflammatory cytokine inhibitory agents, CDP-571 / BAY-10-3356, cA2, 75 kdTNFR-IgG, 55 kdTNFR-IgG, IDEC-CE9.1 / SB210396, DAB486-IL-2, DAB 389-IL-2, Anti-Tac, IL-4, IL-10, IL-4 agonists, IL-10 agonists, IL-1RA, TNF-bp / s-TNFR, S284 , R973401, mK-966, Iloprost, methotrexate, thalidomide, related agents for thalidomide, leflunomide, tranexamic acid, T-614, prostaglandins El, Tenidap, Naproxen, Meloxicam, Piroxicam, Diclofenac, Indomethacin, Sulfasalazine, Azathioprine, ICE inhibitors, zap-70 inhibitors, drug inhibitors, VEGF inhibitors, VEGF-R inhibitors, corticosteroids, anti-TNF-IL-inhibitors , interleukin 11, interleukin 13, interleukin-17, gold, penicillamine, chloroquine, hydroxychloroquine, chlorambucil, cyclophosphamide, cyclosporin, anti-thymocyte globulin, anti-CD4 antibodies, CD5-toxins, orally administered peptides, collagen, disodium lobenzarite salt, regulatory agents for HP228 and HP466 cytokines, ICS-1 antisense phosphotothioate oligodeoxynucleotides, soluble receptor 1 complement, prednisone, orgotein, glycosamine 188 192 glycan, sea salt, glycosamine 182 192 , plant lipids, fenclobutazene auranofin, nucleofunamic acid, flufenamic acid, intrinsic resistance globulins, zileuton, mckefunelovic acid, tacrolimus, sirolimus, amiprilos, claciribine, azarbin, budensozide, umaline growth factor, aminosalicylates, 6-mercaptepurinc, metrenidazel, lipexynase, antagonists, balsalphase, lipase antagonists, anti-IL-1 β moneclene, menoclene anti-IL-6, growth factors, elastase inhibitors, pyyUyleleimUazel compounds, prunnizing prodrugs conjugated with glucuronide, dexamutazene or budesonide, prednisellon prodrugs conjugated with dextran, slow-releasing mesalazine, platelet activating factor (PAF) antagonists Platelet Activating Factor), cipreflexacin, lingocaine, predisolone, mutylpreUizelene, ccklofeefamiUu, 4-aminopyridine, tizaniUcnc, interferon [la, interferon βΐb, copolymer 1, hyperbaric oxygen, intraplyclinic salt antagonists of cytokines such as TNFα, IL-βΙ, IL-6 and / or IL-8, SK&F 107647, tetravalent guanylohu Urazene CNI-1493, tissue factor pathway inhibitor, PHP, iron chelating agents and chelates, including Uiethylinotriaminopuntaectic-iron (III) complex, lysefilinc, PGG-glucan, lipid-reconstituted A-1 apelipoprotein, chiral hcdrekeamic acids, anti-endotoxin antibodies, E5531, rBPI21, synthetic endotoxins and anti-IL-8 antibodies.

Short description of the figures

Figures 1A and 1B show the amino acid sequences of the light chain variable region D2E7 (D2E7 VL; also shown in SEQ ID NO: 1), mutants obtained by VL D2E7 alanine scanning (LD2E7 * .A1, LD2E7 * .A3, LD2E7 * .A4, LD2E7 * .A5, LD2E7 * .A7 and LD2E7 * .A8) light chain variable region of the D2E7, 2SD4 related antibody (VL 2SD4; also shown on SEQ ID No. No. 9) and other related heavy chain variable regions (EP B12, VL10E4, VL100A9, VL100D12, VL10F4, LOE5, VLLOF9, VLLOFIO, VLLOG7, VLLOG9, YLLOHIO, VL1B7, VL1C1, VL1C7, VL0.1F4 LOE7.A and LOE7.T). Figure 1A shows the FR1, CDR1, FR2 and cDr2 domains. Figure 1B shows the FR3, CDR3 and FR4 domains. The light chain domains CDR1 ("CDR L1"), CDR2 ("CDR L2") and CDR3 ("CDR L3") are boxed.

Figures 2A and 2b show the amino acid sequences of the heavy chain variable region D2E7 (D2E7 VH; also shown on SEQ ID. No. 2), mutants obtained by alanine scanning method D2E7 VH (HD2E7 * .A1, HD2E7 * .A2, HD2E7 * .A3, HD2E7 * .A4, HD2E7 * .A5, HD2E7 * .A6, HD2E7 * .A7, HD2E7 *. A8, HD2E7 * .A9), the heavy chain variable region of the D2E7-related antibody, 2SD4 (VH 2SD4; also shown in SEQ ID NO: 10) and other heavy chain variable regions related to D2E7 (VH1B11, VH1D8, VH1A11, VH1B12, VH1B12 D2, VH1E4, VH1F6, VH1G1, 3C-H2, VH1D2.N and VH1-D2.Y). Figure 2 shows the FR3, CDR3 and FR4 domains. The heavy chain domains CDR1 ("CDR H1"), CDR2 ("CDR H2") and CDR3 ("CDR H3") are boxed.

Figure 3 is a graph showing inhibition of TNFα-induced L929 cells by human anti-hTNFa antibodies, D2E7, compared to the mouse anti-hTNFa antibody, MAK 195.

Figure 4 is a graph showing inhibition of rhTNFa binding to hTNFa receptors on U-937 cells by human anti-hTNFa antibodies, D2E7, compared to the mouse anti-hTNFa antibody, MAK 195.

Figure 5 is a graph showing inhibition of expression of ELAM-1 on TNFα-induced HUVEC cells by human anti-hTNFa antibodies, D2E7, compared to the mouse anti-hTNFα antibody, MAK 195.

Figure 6 is a bar graph showing protection against TNFα induced lethality in D-galactosamine sensitized mice after administration of the human anti-hTNF α antibody, D2E7 (black bars), compared to the anti-hTNFa mouse antibody, MAK 195 (hatched bars).

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Figure 7 shows the nucleotide sequence of the light chain variable region D2E7, with the predicted amino acid sequence below the nucleotide sequence. The CDR L1, CDR L2 and CDR L3 regions are underlined.

Figure 8 shows the nucleotide sequence of the D2E7 heavy chain variable region, with the predicted amino acid sequence below the nucleotide sequence. The CDR HI, CDR H2 and CDR H3 regions are underlined.

Figure 9 is a graph showing the effect of D2E7 antibody treatment on the average joint size of Tgl97 transgenic mice as a polyarthritis model.

Detailed description of the invention

The invention provides isolated human antibodies or antigen-binding fragments thereof that bind to human TNFα with high affinity, slow detachment (dissociation) rate and high neutralization capacity. Various aspects of the invention relate to antibodies and antibody fragments and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for the production of such antibodies and fragments. Methods of using the antibodies of the invention to detect human TNFα or to inhibit human TNFα activity, both in vitro and in vivo, are also encompassed by the invention.

In order to better explain the invention, certain terms will first be defined.

The term "human TNFa" (abbreviated as hTNFa or simply hTNF) as used herein refers to a human cytokine that occurs as a 17 kDa ed / Ielnic / a form and a 26 kDa membrane associated form whose biologically active form consists of a non-covalently trimer associated 17 kDa units. The structure of hTNFa is further described in, for example, Pennica et al. (1984) Nature 312: 724-729; Davies et al. (1987) Biochemistry 26: 1322-1326; and Jones et al. (1989) Nature 338: 225-228. In killing, the term TNFa includes recombinant human TNFa (rhTNFa), which can be produced by standard recombinant expression methods or purchased commercially (R&D Systems, Cat. No. 210-TA, Minneapolis, MN).

The term "antibody" as used herein is intended to include immunoglobulin molecules composed of four polypeptide chains, two heavy (H) and two light (L) chains joined together by disispercell bonds. Each heavy chain includes the heavy chain variable region (abbreviated HCVR) or VH) and the heavy chain constant region. The heavy chain constant region includes three domains, CHI, CH2 and CH3. Each light chain includes a light chain variable region (abbreviated LCVR or VL) and a light chain constant region. The light chain constant region comprises one CL domain. The VH and VL regions can be further subdivided into hypervariable regions, referred to as complementarity determining regions (CDRs), separated by more conserved regions, referred to as framework regions (FRs). Each VL and VH consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, Fr2, CDR2, FR3, CDR3, FR4.

The term "antigen-binding portion" of an antibody (or simply "the portion of an antibody") as used herein refers to one or more antibody fragments that retain the ability to specifically bind to an antigen (eg, hTNFa). It has been shown that the antigen-binding function of an antibody can be performed by full-length antibody fragments. Examples of binding fragments encompassed by the term "antigen binding portion" of an antibody include:

(i) Fab fragment, a monovalent fragment comprising the VL, VH, CL and CHI domains;

(ii) the F (ab ') 2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region;

(iii) the Fd fragment comprising the VH and CHI domains;

(iv) an Fv fragment comprising the VL and VH domains of one arm of the antibody, (v) a dAb fragment (Ward et al. (1989) Nature 341: 544-546) which includes the VH domain; and (vi) an isolated complementarity determining fragment (CDR).

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In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombinant methods with a synthetic linker that allows them to be produced as a single protein chain in which a pair of VL and VH regions form a monovalent molecule (known also as single chain Fv (scFv); see, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also encompassed by the term "antigen binding portion" of the antibody. Other forms of single chain antibodies, such as dual specificity antibodies are also included. Such antibodies are bivalent dual specificity antibodies in which the VH and VL domains are expressed as a single polypeptide chain, but using a linker that is short enough to prevent pairs between domains of the same chain, forcing them to pair with complementary other chain domains and the formation of two antigen binding sites (see, Holliger et al (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak et al. (1994) Structure 2: 1121-1123).

Still further, the antibody or antigen-binding portion thereof may be part of a larger immunoadhesive molecule produced by covalent or non-covalent association of the antibody or portion of the antibody with one or more proteins or peptides. Examples of such immunoadhesive molecules include the use of the streptavidin core region to produce the tFameric scFv molecule (Kipriyanov et al. (1995) Human Antibodiesand Hybridomas 6: 93-101) and the use of a cysteine ​​residue, a tag peptide and a C-terminal polyhistidine tag to produce bivalent and biotinated scFv molecules (Kipriyanov et al., (1994) Mol. Immunol. 31: 1047-1058). Antibody portions such as Fab or F (ab ') 2 fragments can be made from whole antibodies using conventional techniques such as papain or pepsin digestion of whole antibodies, respectively. In addition, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques as described herein.

The term "human antibody" as used herein includes antibodies with constant and variable regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo), for example in CDRs, especially in CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of a mammal of another species, such as a mouse, have been grafted into human framework sequences.

The term "recombinant human antibody" as used herein is intended to include all human antibodies that are produced, expressed, generated or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (described later in Chapter II, below ), antibodies isolated from a recombinant combinatorial human antibody library (described later in Chapter III, below), antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (see, e.g., Taylor et al., (1992) Nuci. Acids Res. 30: 6287-6295) or antibodies produced, expressed, generated or isolated by any other method that includes assembly of human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In some embodiments, however, such recombinant human antibodies undergo mutagenesis in vitro (or, when a transgenic animal is used for human Ig sequences, in vivo somatic mutagenesis), hence the amino acid sequences of the recombinant antibody VH and VL regions are sequences that are derived or being related to human VH and VL germline sequences may not occur naturally in the in vivo human germline repertoire.

188 192 "Isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having other antigenic specificities (eg, isolated hTNFa binding antibody is substantially free of antibodies that specifically bind non-hTNFa antigens). However, an isolated antibody that specifically binds hTNFa may exhibit cross-activity with other antigens, such as other species TNFa molecules (as discussed in detail below). In addition, the isolated antibody may be substantially free of other cell components and / or chemical compounds.

"Neutralizing antibody" as used herein (or "antibody neutralizing hTNFa activity") is intended to refer to an antibody whose binding to hTNFa causes inhibition of hTNFa biological activity. This inhibition of hTNFa biological activity can be determined by measuring one or more indicators of hTNFa biological activity, such as hTNFa-induced cytotoxicity (in vitro and in vivo), hTNFa-induced cell activation, and binding of hTNFa to the hTNFa receptor. These indicators of biological activity can be determined by one or more known standard in vitro or in vivo assays (see example 4). Preferably, the ability of the antibody to neutralize hTNFa activity is assessed by inhibiting hTNFa-induced cytotoxicity of L929 cells. As an additional or alternative parameter for hTNFa activity, the ability of the antibody to inhibit hTNFa-induced ELAM-1 expression on HUVEC can be assessed as a measure of hTNFa cellular stimulation.

The term "surface plasmon resonance method" as used herein refers to an optical phenomenon that enables the analysis of specific biological interactions in real time by detecting changes in the protein concentration in the biological sensor matrix, for example using a BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway , NJ). For further description, see example 1 and Jonsson et al. (1993) Ann. Biol. Clin. 51: 1926; Jonsson et al. (1991) Biotechniques 11: 620-627; Johnsson et al. (1995) J. Mol. Recognit. 8: 125-131; and Johnsson et al. (1991) Anal. Biochem. 198: 268-277.

The term "Kefr" as used herein is intended to refer to constant disconnection rate for dissociation of the antibody from the antibody / antigen complex.

The term "Kd" as used herein refers to the intent of dissociation constant of a particular antibody-antigen interaction.

The term "nucleic acid molecule" as used herein includes DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.

The term "isolated nucleic acid molecule" as used herein refers to nucleic acids encoding antibodies or antibody portions (e.g. VH, VL, CDR3) that bind to hTNFa, refers to a nucleic acid molecule in which the nucleotide sequences encoding the antibody or part of the antibody are free of other nucleotide sequences encoding the antibodies or parts of the antibody that binds antigens other than hTNFa, which sequences can be acid flanked in nature nucleic acid in human genomic DNA. Thus, for example, the isolated nucleic acid of the invention encoding the VH region of an anti-hTNFa antibody does not contain other sequences encoding other VH regions that bind non-hTNFa antigens.

The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been attached. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which an additional DNA segment can be incorporated. Another type of vector is a viral vector in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they were introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may integrate into the host cell genome when introduced into the host cell and thus replicate with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they have been operably linked. Such vectors are referred to as "recombinant expression vectors" (or after

188 192 simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably because the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (e.g. non-replicable retroviruses, adenoviruses and adeno-associated viruses) that perform equivalent functions.

The term "recombinant host cell" (or simply "host cell") refers to the cell into which the recombinant expression vector has been introduced. It should be understood that such terms are intended not only for a particular cell, but also for its progeny. Because some modifications may occur in subsequent generations due to mutations or environmental influences, such offspring may not actually be identical to the parent cell, but are still within the scope of the term "host cell".

Various aspects of the invention will be described in detail in the following subsections.

I. Human antibodies that bind human TNFα

The invention provides isolated human antibodies or antigen-binding portions thereof that bind human TNFα with high affinity, slow detachment rates and high neutralizing capacity. Preferably, the human antibodies of the invention are recombinant, neutralizing human anti-hTNFa antibodies. The most preferred recombinant neutralizing antibody of the invention is referred to as D2E7 and has the VL and VH sequences shown in Figures 1A and 1B and Figures 2A and 2B, respectively (the amino acid sequence of the VL region D2E7 is also shown on SEQ ID NO: 1; amino acid sequence of the VH region D2E7 is shown on SEQ ID No. 2). The binding properties of D2E7, compared to mouse MAK 195 anti-hTNFa mAb, which shows high affinity and slow dissociation kinetics and other human anti-hTNFa antibody, related to the D2E7 sequence, 2SD4 is summarized below:

<td>Antibody</td><td>Koff s'</td><td>Kon M''s · '</td><td>Kd M.</td><td>stoichiometry</td>

<td>D2E7 IgG1</td><td>8.81 x 10 '⁵</td><td>1.91 x 10'5</td><td>6.09 x 10 '¹⁰</td><td> 1,2</td>

<td>2SD4 IgG4</td><td>8.40 x 10 '³</td><td>4.20 x 10'5</td><td>2.00 x 10 '⁸</td><td> 0,8</td>

<td>MAK 197 F (ab ') 2</td><td>8.70 x 10 '⁵</td><td>1.90 x 10</td><td>4.60 x 10'O</td><td> 1,4</td>

Antibody D2E7 and related antibodies also show strong ability to neutralize hTNFa activity, as assessed in several in vitro and in vivo tests (see example 4). For example, these antibodies neutralize hTNFa-induced cytotoxicity of L929 cells with an IC50 in the range of about 10 '⁷ M to about 1Ó '¹⁰ M. D2E7, expressed as a full-length IgG1 antibody, neutralizes hTNFa-induced cytotoxicity of L929 cells with an IC50 of about 1.25 x 10 * 0 M. In addition, the ability to neutralize D2E7 is preserved when the antibody is expressed as a Fab, F fragment ( ab ') 2 or scFv. D2E7 also inhibits hTNF? Induced cell activation, as measured by hTNF? Induced ELAM-1 expression on HUVEC (IC₅o = about 1.85 x 10 '* 0 M) and binding of hTNFa to the hTNFa receptor on U-937 cells (IC50 = about 1.56 x 10'OM). In relation to the latter, D2E7 inhibits binding of hTNFa to the p55 and p75 receptor. In addition, the antibody inhibits hTNF? Induced lethal effect in vivo in mice (ED50 = 1-2.5 μg / mouse).

With respect to D2E7 binding specificity, this antibody binds to human TNFα in various forms, including soluble hTNFa, transmembrane hTNFa and hTNFa associated with the cell receptor. D2E7 does not specifically bind to other cytokines such as lymphotoxin (TNFfi) IL-1a, IL-1-β, IL-2, IL-4, IL-6, IL-8, IFNy and TGFβ. However, D2E7 has cross-activity with other forms of tumor necrosis factor. For example, the antibody neutralizes the activity of at least five TNFa of other primates (rapeseed, orangutan, marmoset, cynomolgus and rhesus) with values

188 192

IC50 approximately equivalent as in the case of neutralization of hTNFa (see example 4, subsection E). D2E7 also neutralizes the activity of mouse TNFα, albeit about 1000 - weaker than human TNFα (see example 4, subsection E). D2E7 also binds to dog and pig TNFa.

In one aspect, the invention provides D2E7 antibodies and antibody portions, D2E7 related antibodies and antibody portions, and other human antibodies and antibody portions having D2E7 equivalent properties, such as high binding affinity for hTNFa with slow dissection kinetics and high neutralization capacity. In one embodiment, the invention provides an isolated human antibody or antigen binding portion thereof that dissociates from human TNFa with a Kz of 1 x 10 '⁸ M or less and a Kof speed constant equal to 1 x 10 '³ s'¹ or less, both values ​​determined by surface plasmon resonance and neutralizes hTNFa-induced cytotoxicity against L929 cells in a standard in vitro assay with an IC50 of 1 x 10-⁷ M or less. Even more preferably, the isolated human antibody or antigen binding portion thereof dissociates from human TNFα with Kof · equal to 5 x 10⁴ s- or smaller, or even more preferably with Kof equal to 1 x 10-s⁴ or less. Even more preferably, the isolated human antibody or antigen-binding portion thereof neutralizes the cytotoxicity of human TNFα in a standard in vitro test with L929 with IC50 = 1 x 10 * 8 M gjbo less, even more preferably with IC50 = 1 x 10 '⁹ M or less, and even more preferably an IC50-5 x 10 ^ M or less. In a most preferred embodiment, the antibody is an isolated human recombinant antibody or antigen binding portion thereof. In another most preferred embodiment, the antibody also neutralizes TNFα-induced cell activation, as assessed using a standard in vitro test for hTNFa-induced ELAM-1 expression on human umbilical cord vein cells (HUVEC).

Surface plasmon resonance analysis for Kd and Kof determination can be performed as described in Example 1. A standard in vitro assay with L929 cells for IC50 values ​​is described in Example 4, subsection A. Standard in vitro assay for hTNFa induced ELAM-1 expression on human umbilical cord vein cells (HUVEC) are described in Example 4, subsection C. Examples of recombinant human antibodies that meet the aforementioned kinetic and neutralization criteria include antibodies having the following [VH / LH] pairs, the sequences of which are shown in FIG. 1A, 1B, 2A and 2B (see also example 2, 3 and 4, kinetics and neutralization analysis): [D2E7 VH / D2E7 VL]; [HD2E7 * .A1 / DaE7 VL], [HD2E7 * .A2 / D2E7 VL], [HD2E7 * .An / DaE7 VL], [HD2E7 * .A4 / D2E7 VL], [HDaE7 * .A5 / D2E7 VL], [HDaE7 * .A6 / DaE7 VL], [HD2E7 * -. A7 / D2E7 VL], [HDaE7 * .A8 / DaE7 VL], [HD2E7 * .A9 / DaE7 VL], [D2E7 VH / LD2E7 * .A1] , [D2E7 VH / LD2E7 * .A4], [D2E7 VH / LD2E7 * .A5], [D2E7 VH / LD2E7 * .A7], [D2E7 VH / LDaE7 * .A8], [HD2E7 * .A9 / LD2E7 *. A1], [VH1-D2 / LOE7], [VH1-D2.N / LOE7.T], [VH1-Da.Y / LOE7.A], [VH1-D2.N / LOE7.A], ^ 1 ^ 2 ^ B12] and [3C-H2 / LOE7].

The light and heavy chain CDR3 domains are known to play an important role in the specificity / affinity of binding the antibody to the antigen. In another aspect, the invention provides human antibodies exhibiting slow kinetics of enjoyment from the combination with hTNFa and whose light and heavy chain CDR3 domains are structurally identical or related to D2E7. As shown in Example 3, position 9 of CDR3 VL D2E7 can be taken by Ala or Thr without significantly affecting Koff. Accordingly, the compatibility motif for CDR3 VL D2E7 includes the amino acid sequence: QRYNRPY- (T / A) (SEQ ID NO: 3). In addition, position 12 CDR3 VH D2E7 can be occupied by Tyr or Asn without significantly affecting Kof. Accordingly, the compliance motif for CDR3 VH D2E7 includes the amino acid sequence: VSYLSTASSLD- (Y / N) (SEQ ID NO: 4). In addition, as shown in Example 2, the CDR3 domain of the D2E7 light and heavy chains may be substituted by single alanine residues (at positions 1, 4, 5, 7 or 8 in CDR3 VL or at positions 2, 3, 4, 5, 6, 8 , 9, 10 or 11 in CDR3 VH) without significant effect on Kof. Still further, one of ordinary skill in the art will recognize that, given the possibility of substitution of CDR3 VL and VH D2E7 with alanine, it is possible to replace the amino acids in CDR3 while maintaining a low antibody detachment constant, in particular for canned amino acid substitutions. "Replacement with a conservative amino acid" as used herein means that one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined, including basic side chains (e.g. lysine, arginine, histidine), acid side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), branched side chains in the beta position (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Preferably, no more than 1 to 5 substitutions are made with conservative amino acids in the VL and / or VH D2E7 CDR3 domains. Even more preferably, no more than 1 to 3 substitutions are made with conservative amino acids in the CDR3 VL and / or VH D2E7 domains. In addition, conservative amino acid substitutions should not be made at critical sites for hTNFa binding. As shown in example 3, positions 2 and 5 in CDR3 VL d2E7 and positions 1 and 7 of CDR3 VH D2E7 appear to be critical positions for interaction with hTNFa, hence the substitution with conservative amino acids at these positions is preferably not performed (although alanine substitution in position 5 CDR3 VL D2E7 as described above).

In another embodiment, the invention provides an isolated human antibody or antigen binding portion thereof with the following properties:

a) dissociates from human TNFa with a Koff rate constant of 1 x 10 '³s1 or less as determined by surface plasmon resonance;

b) has a light chain CDR3 domain comprising the amino acid sequence of Sequence Identifier. No. 3 or modified based on SEQ ID. No. 3 by replacing with alanine at positions 1, 4, 5, 7 or 8 by one to five substitutions with conservative amino acids at positions 1, 3, 4, 6, 7, 8 and / or 9;

c) has a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO. No. 4 or modified based on SEQ ID. No. 4 by replacing with alanine in positions 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservative amino acids in positions 2, 3,4, 5, 6, 8,9, 10, 11 and / or 12.

More preferably, the antibody or antigen binding portion thereof dissociates from human TNFα with a Kof equal to 5 x 10 6 * s'1 or less. Even more preferably, the antibody or antigen binding portion thereof dissociates from human TNFα with a Kof-equal to 1 x 10-4 s- or less.

In yet another embodiment, the invention provides an isolated human antibody, or antigen binding portion thereof, with a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence SEQ ID '. No. 3 or modified based on SEQ ID. No. 3 by alanine at positions 1, 4, 5, 7 or 8 and with the heavy chain variable region (HCVR) having the CDR3 domain comprising the amino acid sequence SEQ ID NO: No. 4 or modified based on SEQ ID. No. 4 by alanine replacement at positions 2, 3, 4, 5, 6, 8, 9, 10 or 11. Preferably, the LCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: No. 5 (i.e. CDR2 VL D2E7), and HCVR still has a CDR2 domain comprising the amino acid sequence of Sequence Identifier. No. 6 (i.e. CDR2 VH D2E7). Even more preferably, the LCVR still has a CDR1 domain comprising the amino acid sequence with Sequence Identifier. No. 7 (i.e. CDR1 (VL D2E7)), and HCVR still has a CDR1 domain comprising the amino acid sequence with SEQ ID. No. 8 (i.e. CDRJ VH D2E7). The VL framework regions are preferably derived from the human V1 I germline family, more preferably the V20 human germline A20 gene, and most preferably the V2 D2E7 framework sequences shown in Figures 1A and iB. The Vk framework regions are preferably derived from the human germline Vh3, more preferably the human germline VH gene DP-31, and most preferably the VH D2E7 framework shown in Figures 2A and 2B.

In yet another embodiment, the invention provides an isolated human antibody or light chain-binding portion thereof (LCVR) comprising the amino acid sequence of the SEQ ID NO. No. 1 (i.e. VL D2E7) and region

188 192 heavy chain variable (HCVR) comprising the amino acid sequence of SEQ ID NO: No. 2 (i.e. VH D2E7). In some embodiments, the antibody comprises a heavy chain constant region such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. Preferably, the heavy chain constant region is an IgG1 or IgG4 heavy chain constant region. In addition, the antibody may include a light chain constant region, a kappa or lambda light chain constant region. Preferably, the antibody comprises a kappa light chain constant region. Alternatively, a portion of the antibody may be, for example, a Fab fragment or a single Fv unit.

The most preferred recombinant antibody of the invention, designated D2E7, has a light chain CDR3 domain comprising the amino acid sequence of the Sequence Identifier. No. 3 and the heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: No. 4. Preferably, the D2E7 antibody has a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO. # 1 and heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO. No. 2

In yet other embodiments, the invention provides an isolated human antibody or antigen-binding portion thereof, having D2E7-related CDR3 VL and VH domains, for example antibodies or antigen-binding fragments thereof with a light chain variable region (LCVR) having CDR3 domains comprising an amino acid sequence selected from the group consisting of Sequential ID No. 3, SEQ ID No. 11, Sequential Identifier No. 12, Sequential Identifier No. 13, Sequential Identifier No. 14, Sequential Identifier No. 15, SEQ ID No. 16, Sequential Identifier No. 17, SEQ ID No. 18, Sequential Identifier No. 19, Sequential Identifier No. 20, SEQ ID No. 21, Sequential Identifier No. 22, SEQ ID No. 23, SEQ ID: No. 24, SEQ ID No. 25 and SEQ ID NO: 26 or with a heavy chain variable region (HCVR) having CDR3 domains comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID: No. 31, SEQ ID. No. 32, Sequential Identifier No. 33, SEQ ID No. 34 and SEQ ID No. 35

In yet another embodiment, the invention provides a recombinant human antibody or antigen binding portion thereof that neutralizes activity of human TNFα but not human TNF11. Preferably, the antibody or antigen-binding portion thereof also neutralizes the chimpanzee TNFa activity and at least one additional primate TNFa selected from the group consisting of orangutan TNFa, marmoset TNFa, rinimolgus TNFa and rhesus TNFa. Preferably, the antibody or antigen-binding portion thereof neutralizes human, chimpanzee and / or an additional primate TNFα in a standard in vitro assay with L929 cells with an IC50 value of 1 x 10 '⁸ M or smaller, more preferably equal to 1 x 10 '⁹ M or less, and even more preferably equal to 5 x 10 '¹⁰ M or less. In one embodiment, the antibody also neutralizes the dog's TNFα activity, preferably in a standard in vitro assay with L929 cells with an IC50 value of 1 x 10'7 M or less, more preferably of 1 x 10 '⁸ M or less, and even more preferably equal to 5 x Kr M or less. In another embodiment, the antibody also neutralizes porcine TNFα activity, preferably in a standard in vitro assay with L929 cells with an IC50 value of 1x10⁵ M or smaller, more preferably equal to 1 x 10 '⁶ M or less, and even more preferably equal to 5 x 10 '⁷ M or less. In yet another embodiment, the antibody also neutralizes mouse TNFα activity, preferably in a standard in vitro assay with L929 cells with an IC50 value of 1 x 10 '⁴ M or less more preferably equal to 1 x 10 '⁵M or smaller and even more preferably equal to 5 x 10'6 M or smaller.

The antibody or antigen binding portion thereof can be derivatized or combined with another functional molecule (e.g., another peptide or protein). Antibodies or antigen binding portions thereof of the invention include durcanized and otherwise altered forms of human anti-hTNFa antibodies, including immunity molecules. For example, the antibody or antigen-binding portion thereof of the invention may be operably linked (chemical binding, gene fusion, myofusion linkage, or

188 192 yet differently) with one or more particles, such as another antibody (e.g. dual-specific antibody), a detectable agent, a cytotoxic agent, a pharmaceutical agent and / or a protein or peptide that can mediate the binding of the antibody or antibody portion to another molecule (such as streptavidin core region or polyhistidine tag).

One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or different types, e.g. to produce dual specificity antibodies). Suitable crosslinkers include heterobifunctional linkers having two different reacting groups separated by a suitable spacer (e.g. m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g. disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, IL.

Useful detection agents that can be derivatized with antibodies or parts thereof include fluorescent compounds. Exemplary fluorescent detection agents include fluorescein, fluorescent isothiocyanine, rhodamine, 5-dimethylamino-1-naphthalenesulfonyl chloride, phycoerythrin, etc. The antibody can also be derivatized with a detectable enzyme such as alkaline phosphatase, horseradish peroxidase, glucose oxidase etc. When the antibody is derivatized with a detectable enzyme, it is detected using appropriate reagents that the enzyme uses to produce a detectable reaction product. For example, when there is a detectable enzyme - horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine leads to the formation of a colored reaction product that is detectable. The antibody can also be derivatized with biotin and detected by indirect measurement of avidin or streptavidin binding.

II. Antibody expression

An antibody or portion thereof of the invention may be produced by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. To express the antibody recombinantly, the host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the light and heavy chains of the antibody, such that the light and heavy chains are expressed in the host cell and preferably secreted into the medium in which the host cell is cultured and from which antibodies can be recovered. Standard recombinant DNA methodology can be used to obtain antibody light and heavy chain genes, incorporate these genes into expression vectors, and introduce vectors into host cells as described in Sambrook et al. (Ed.) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989), Ausubel et al. (ed.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and Boss et al., U.S. Patent No. 4,816,397.

To express d2E7 and D2E7 related antibody, DNA fragments can be obtained by amplification and modification of the germline heavy and light chain sequences using a polymerase chain reaction (PCR). DNA germline sequences for human light and heavy chain variable region genes are known (see e.g. human germline sequence database "Vbase"; see also, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, fifth edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson et al. (1992) J. Mol. Biol. 227: 776-798; Cox et al., (1994) "A Directory of Human Germ-line Vk Segments Reveals a Strong Bias in their Usage" Eur. J. Immunol. 24: 827-836; whose contents are hereby incorporated by reference). To obtain a DNA fragment encoding the D2E7 heavy chain variable region or D2E7 related antibody, one member of the Vh3 human germline VH3 family is amplified by standard PCR. More preferably, the VH DP-31 germline sequence is amplified. To obtain the Dna fragment encoding the D2E7 light chain variable region or D2E7 related antibody, one of the members of the human germline VrI family of VL genes is amplified by standard PCR. More preferably, the a20 VL germline sequence is amplified. PCR primers useful for use in ampli28

188 The germline sequence of the VH DP-31 and VL A20 germline sequences can be designed based on the nucleotide sequences disclosed in the references cited above using standard methods.

Once the germline VL and VH fragments are obtained, these sequences can be mutated in such a way as to encode the D2E7 or related D2E7 amino acid sequences disclosed herein. The amino acid sequences encoded by the germline VH and VL DNA sequences are compared with the D2E7 VH and VL or D2E7 related amino acid sequences to identify amino acid residues that differ from D2E7 or D2E7 related germline sequences. Then, the corresponding nucleotides in the germline DNA are mutated in such a way that the mutated germline sequences encode the D2E7 or D2E7 related amino acid sequence using the genetic code to determine what nucleotide changes should be made. Mutagenesis of germline sequences is carried out by standard methods such as PCR mutagenesis (in which mutated nucleotides are incorporated into PCR primers in such a way that the PCR product contains mutations) or site-directed mutagenesis.

In addition, it should be noted that if the "germline" sequences obtained by PCR encode amino acids different in the framework region than in the original germline configuration (i.e. differences in the amplified sequence relative to actual germline sequences, for example due to somatic mutation) it may be desirable to change these amino acid differences back to the germline sequence (ie, "backmutation" of framework residues to the germline configuration).

When DNA fragments encoding D2E7 VH and VL segments or D2E7 related (obtained by amplification and mutagenesis of germline VL and VH genes as described above) are obtained, these fragments can be further manipulated with standard recombinant DNA techniques, for example to transform variable region genes into full length antibody chain genes, Fab fragment genes, or scFv gene. In these manipulations, the DNA fragments encoding VL and VH are operably linked to another DNA fragment encoding another protein, such as an antibody constant region or flexible linker. The term "operably linked" as used herein means that the two DNA fragments are joined in such a way that the amino acid sequences encoded by the two DNA fragments remain in one reading frame.

Isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH encoding DNA to another DNA molecule encoding the heavy chain constant regions (CHI, CH2 and CH3). The heavy chain constant region gene sequences are known (see, for example, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, fifth edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments covering these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgG1 or IgG4 constant region. For the Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

Isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a Fab heavy chain gene) by operably linking the VL-encoding DNA to another DNA molecule encoding the light chain constant CL. Light chain constant region gene sequences are known (see, for example, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, fifth edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments covering these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region, but is most preferably a kappa constant region.

To generate the scFv gene, the DNA fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser) 3 in such a way that the VH and VL sequences can be expressed as a continuous single chain protein, VL and VH regions connected by a flexible connector (see Bird

188 192 et al. (1988) Science 242: 423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al., Nature (1990) 348: 552-554).

To express the antibodies or parts of the antibodies of the invention, the DNA encoding the full length light and heavy chains obtained as described above is introduced into expression vectors in such a way that the genes are operably linked to transcription and translation control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into the vector in such a way that the transcription and translation control sequences in the vector serve ⁱⁿ their function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected in such a way that they are compatible with the host cell used. The antibody light chain gene can be inserted into a separate vector or, more often, both genes are inserted into the same expression vector. Antibody genes are introduced into the expression vector by standard methods (e.g. ligation of complementary restriction sites in the antibody gene fragment and vector, or ligated with blunt ends (no restriction sites present). Before introducing the light or heavy D2E7 or related D2E7 sequences, the expression vector may already carry the antibody constant region sequences. For example, one approach to converting D2E7 or D2E7 related sequences into full-length antibody genes involves inserting them into expression vectors already encoding light chain and heavy chain constant regions, respectively, in such a way that the VH segment is operably linked to the segment (segments) CH in the vector, while the VL segment is operably linked to the CL segment in the vector. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector in such a way that the signal peptide is linked in one frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polidenylation signals) that control the transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Meth. in Enzymol. 185, Academic Press, San Diego, CA (1990). The skilled artisan will recognize that the design of the expression vector, including the choice of regulatory sequences, may depend on factors such as the choice of transformed host cell, the level of desired protein expression, etc. Preferred regulatory sequences for mammalian host cells include viral elements that direct the expression of high levels of protein in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (such as the CMV enhancer / promoter), simian virus (SV40) (such as the SV40 promoter / enhancer), adenovirus (e.g., major adenovirus late promoter (AdMLP)) and polyoma. Other descriptions of viral regulatory elements are found, for example, in US Patent Nos. 5,168,062, Stinski; No. 4,510,245 to Bell et al .; No. 4,968,615, Schaffner et al.

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate vector replication in a host cell (e.g., origin of replication), and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., US Patent Nos. 4,399,216, 4,634,665 and 5,179,017, Axel et al.). For example, typically a selectable marker gene provides resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in co-

188 192 dhfr 'cells with selection / amplification using methotrexate) and the neo gene (for selection using G418).

For expression of light and heavy chains, the expression vector (s) encoding the light and heavy chains are transfected into the host cell by standard methods. The various forms of "trafficking" include a great variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, e.g. transfection by electroporation, calcium phosphate precipitation, dextranDEAE, etc. Although it is possible to theoretically express the antibodies of the invention in other prokaryotic or eukaryotic host cells, the expression of antibodies in eukaryotic cells, and preferably in mammalian host cells is most preferred because such cells, compared to prokaryotic cells, are more likely to produce and secrete properly folded and immunologically active antibodies. Prokaryotic expression of antibody genes has been described as ineffective for the production of significant amounts of active antibody (Boss and Wood (1985) Immunology Today 6: 12-13).

Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese hamster ovary (CHO) cells (including CHO dhfr 'cells, described by Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, used with a label enabling DHFR selection, e.g. as described in Kaufman and Sharp (1982) Mol. Biol. 159: 601-621), NSO myeloma cells, COS cells and SP2. When the recombinant expression vectors encoding the antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient for expression of the antibody by the host cells, or even more preferably, secreting the antibodies to the culture medium in which the host cell is grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

Host cells can be used to generate parts of whole antibodies, such as Fab fragments or scFv molecules. It should be understood that variations of the above procedure are within the scope of the invention. For example, it may be desirable to transfect a DNA host cell encoding a heavy chain or light chain (or both) of the antibodies of the invention. Recombinant DNA technology can also be used to remove some or all of the DNA encoding one or both light and heavy chains, which is necessary for binding to hTNFa. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention. In addition, bispecific antibodies can be prepared in which one light and heavy chain are the antibodies of the invention and the other light and heavy chain are specific for the antigen other than hTNFa by cross-linking the antibody of the invention with the other antibody by standard chemical cross-linking methods.

In a preferred system of recombinant expression of the antibody or antigen-binding portion thereof of the invention, the recombinant expression vector encoding both antibody chains, light and heavy, is introduced into CHO dhfr 'cells by calcium phosphate transfection. In the recombinant expression vector, the light and heavy chain genes of the antibody are operably linked to each other with enhancer / promoter regulatory elements (e.g. derived from SV40, CMV, adenovirus etc., such as the CMV enhancer regulatory element / AdMLP promoter or SV40 enhancer regulatory element / AdMLP promoter), to drive high levels of gene transcription. The recombinant expression vector also carries the DHFR gene, which allows the selection of CHO cells that have been transfected with the vector using methotrexate amplification selection. Selected host cell transformants are cultured to allow them to express the light and heavy chains of the antibody and all antibody is recovered from the culture medium. Standard molecular biology techniques are used to generate recombinant expression vectors, transfect the host cells, select transformants, grow host cells and recover the antibody from the culture medium.

188 192

In view of the above, another aspect of the invention provides a nucleic acid, vector and host cell composition that can be used to recombinantly express antibodies and parts of the antibodies of the invention. The nuclide sequence coding for the light chain variable region D2E7 is shown in Figure 7 and SEQ ID. No. 36. The CDR1 LCVR domain comprises nucleotides 70-102, the CDR2 domain comprises nucleetcdc 148-168, and the CDR3 domain comprises mucluetides 265-291. The nucleotide sequence coding for the heavy chain variable region is shown in Figure 8 and SEQ ID. No. 37. The CDR1 HcVR domain comprises nucleotides 91-105, the CDR2 domain comprises nucluetcdy 148-198, and the CDR3 domain includes 295-330 nucleotides. One of ordinary skill in the art will recognize that nucleotide sequences encoding D2E7 related antibodies or parts thereof (e.g. a CDR domain (such as a CDR3 domain) can be obtained from the nuclide sequences encoding LCVR and HCvR D2E7 using the genetic code and standard molecular biology techniques.

In one embodiment, the invention provides an isolated nucleic acid encoding a light chain CDR3 domain comprising the amino acid sequence of SEQ ID. No. 3 (i.e. CDR3 VL D2E7) or modified based on SEQ ID. No. 3 by replacing with alanine at positions 1, 4, 5, 7, or 8 by one to five substitutions with conservative amino acids at positions 1, 3, 4, 6, 7, 8 and / or 9. This nucleic acid may encode only the CDR3 region or more preferably the entire antibody light chain variable region (LCVR). For example, the nucleic acid may encode an LCVR with a CDR2 domain comprising the amino acid sequence on Sequence Identifier: No. 5 (i.e. CDR2 VL D2E7) and a CDR1 domain comprising the amino acid sequence of the Sequence Identifier. No. 7 (i.e. CDR1 VL D2E7).

In another embodiment, the invention provides an isolated nucleic acid encoding a heavy chain CDR3 domain comprising the amino acid sequence of Sequence Identifier. No. 4 either modified based on SEQ ID No. 4 by replacement with alanine at positions 2, 3, 4, 5, 6, 8, 9, 10 or 11, or by one to five substitutions with conservative amino acids at positions 2, 3, 4, 5 , 6, 8, 9, 10, 11 and / or 12. This nucleic acid may encode only the CDR3 region or more preferably the entire heavy chain variable region of the antibody (HCVR). For example, the nucleic acid may encode HCVR with a CDR2 domain comprising the amino acid sequence on the Sequence Identifier. No. 6 (i.e. CDR2 VH D2E7) and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: No. 8 (i.e. CDR1 VH D2E7).

In yet another embodiment, the invention provides nucleic acids encoding a CDR3 related domain ^from D2E7, e.g., comprising an aminoquasese sequence selected from the group consisting of SEQ ID. No. 2, Sequential Identifier No. 3, SEQ ID No. 11, Sequential Identifier No. 12, Sequential Identifier No. 13, Sequential Identifier No. 14, Sequential Identifier No. 15, SEQ ID No. 16, Sequential Identifier No. 17, SEQ ID No. 18, Sequential Identifier '. No. 19, Sequential Identifier No. 20, SEQ ID No. 21, Sequential Identifier No. 22, SEQ ID: No. 23, SEQ ID No. 24, Sequential Identifier: No. 25, SEQ ID No. 26, SEQ ID No. 27, Identifier SeRw. No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, Sequential Identifier No. 32, Sequential Identifier No. 33, Sequential Identifier: No. 34 and Sequential Identifier. No. 35

In yet another embodiment, the invention provides an isolated nucleic acid encoding an antibody light chain variable region comprising the amino acid sequence of Sequence Identifier :. No. 1 (i.e. LCVR D2E7). Preferably, the nucleic acid comprises a nucleotide sequence with SEQ ID NO: No. 36, however, one of ordinary skill in the art will recognize that due to the degeneracy of the genetic code, other muketyUewe sequences may encode the amino acid sequence of SEQ ID NO. No. 1. The nucleic acid may encode only LCVR or may encode the heavy chain constant region of antibodies operably linked to LCVR. In one embodiment, the nucleic acid is in a recombinant expression vector.

In yet another embodiment, the invention provides an isolated nucleic acid encoding an antibody heavy chain variable region comprising the amino acid sequence of Sequence Identifier :. No. 2 (i.e. HCYR D2E7). Preferably, this nucleic acid

188 192 comprises the nucleotide sequence of SEQ ID NO: No. 37, however, one of ordinary skill in the art will recognize that due to the degeneracy of the genetic code, other nucleotide sequences may encode the amino acid sequence of SEQ ID NO. No. 2. Nucleic acid may encode only HCVR or may encode the heavy chain constant region of antibodies operably linked to HCVR. For example, the nucleic acid may comprise an IgG1 or IgG4 constant region. In one embodiment, the nucleic acid is contained in a recombinant expression vector.

The invention also provides recombinant expression vectors encoding both the heavy and light chains of the antibody. For example, in one embodiment, the invention provides a recombinant expression vector encoding:

a) an antibody light chain with a variable region comprising the amino acid sequence ⁰ SEQ ID. No. 1 (i.e. LCVR D2E7); and

b) a heavy chain antibody with a variable region comprising the amino acid sequence of Sequence Identifier. No. 2 (i.e. HCVR D2E7).

The invention also provides host cells into which one or more recombinant expression vectors of the invention have been introduced. Preferably, the host cell is a mammalian host cell, even more preferably the host cell is a CHO cell, NSO cell or a cOs cell.

Still further, the invention provides a method of synthesizing a recombinant human antibody of the invention by culturing the host cells of the invention in a suitable medium until the recombinant human antibody of the invention has been synthesized. The method may further comprise isolating the recombinant human antibody from the culture medium.

III. Seeection of &lt;3&gt; &lt;3&gt; anti-antibody antibodies

The recombinant human antibodies of the invention in addition to the D2E7 and D2E7 related antibodies disclosed herein can be isolated by screening recombinant combinatorial antibody libraries, preferably scFv phage libraries, generated using human VL and VH cDNAs produced from human lymphocyte mRNAs. Methods for producing and screening such libraries are known. In addition to commercially available kits for the production of phage libraries (e.g. Recombinant Phage Antibody System, Pharmacia, cat. No. 27-9400-01; and SurfZAP ™ set, Stratagene, cat. no. 240612) examples of methods and reagents particularly useful for use in the preparation and screening of antibody expression libraries can be found, for example, in Ladner et al., US Patent No. 5,223,409; Kang et al., PCT publication WO 92/18619; Dower et al., PCT publication WO 91/17271; Winter et al., PCT publication WO 92/20791; Markland et al., PCT publication WO 92/15679; Breitling et al., PCT publication WO 93/01288; McCafferty publication PCT WO 92/01047; Garrard et al., PCT publication WO 92/09690; Fuchs et al. (1991) Bio / Technology 9: 1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3: 81-85; Huse et al. (1989) Science 246: 1275-1281; McCafferty et al. (1990) 348: 552-554; Griffith et al. (1993) EMBO J. 12: 725-735; Hawkins et al. (1992) J. Mol. Biol. 226: 889-896; Clackson et al. (1991) Nature 352: 624-628; Gram et al. (1992) PNAS 89: 3576-3580; Garrard et al. (1991) Bio / Technology 9: 1373-1377; Hoogenboom et al., (1991) Nuci. Acids Res. 19: 4133-4137; Barbas et al. (1991) PNAS 88: 7978-7982.

In the most preferred embodiment, to isolate human antibodies with high affinity and low detachment rate constant from hTNFa, a murine anti-hTNFa antibody showing high affinity and low detachment rate constant from TNFa (e.g. MAK 195, hybridoma with deposit access number ECAcC 87 050801) was used to select human light and heavy chain sequences having similar binding activity to hTNFa using the "epitope imprint" method or targeted selection as described in Hoogenboom et al., PCT publication WO 93/06213. The antibody libraries used in the method are preferably scFv libraries generated and screened as described in McCafferty et al., PCT publication WO 92/01047; McCafferty et al. (1990) Nature 348: 552-554; and Griffith et al. (1993) EMBO J. 12: 725-734. The scFv antibody libraries are preferably screened using recombinant hTNFa as the antigen.

When the VL and VH segments are preselected, "mixing and matching" experiments are performed in which different pairs of initially selected VL and VH segments are screened for hTNFa binding to select preferred combinations of VL / VH pairs. In addition, to further improve the affinity and / or low rate of detachment during hTNFa binding, the VL and VH segments of the preferred VL / VH pair can be randomly mutated, preferably in the VH and / or VL CDR3 region, in a process analogous to an in vivo somatic mutation process responsible for the affinity maturation of antibodies during a natural immune response. This in vitro affinity maturation can be achieved by amplifying VH and VL regions using PCR primers complementary to VH CDR3 or VL CDR3, which were "sharpened" by a random mixture of four nucleotide bases at certain positions in such a way that the resulting PCR products encode VH and V1 segments in which random mutations were introduced into the VH and / or VL CDR3 regions. These randomly mutated VH and VL segments can be screened again for binding to hTNFa and sequences that exhibit high binding affinity and low hTNFa detachment rate constant can be selected.

The amino acid sequences of the selected light and heavy chains of the antibody can be compared with the germline sequences of the light and heavy chains. In cases where specific framework residues of the selected VL and / or VH chains differ from the framework configuration (e.g. as a result of the somatic mutation of the immunoglobulin genes used to generate the phage library) it may be desirable to "reverse flow" the altered framework residues of the selected antibodies into the germinal configuration (ie, change the amino acid sequence of the framework region of the selected antibody in such a way that they are the same as the amino acid sequences of the germline region framework). Such "back imitation" (or "germline") framework residues can be achieved by standard molecular biology methods for introducing specific mutations (e.g., site-directed mutagenesis; PCR mutagenesis, etc.).

After screening and isolating the anti-hTNFa antibodies of the invention from a phage library of recombinant immunoglobulins, the nucleic acid encoding the selected antibody can be recovered from the expression package (e.g., from the phage genome) and cloned into other expression vectors by standard recombinant DNA techniques. If desired, the nucleic acid can be further manipulated to create other forms of the antibodies of the invention (e.g. attach a nucleic acid encoding additional immunoglobulin domains (such as additional constant regions). To express recombinant human antibodies isolated by screening the combinatorial library, the DNA encoding the antibody is cloned into a recombinant expression vector and introduced into a mammalian host cell as described in detail in chapter II above.

IV. Pharmaceutical compositions and administration

Antibodies and antibody portions of the invention can be incorporated into pharmaceutical compositions useful for administration to patients. Typically, the pharmaceutical composition comprises an antibody or part of an antibody of the invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., which are physiologically acceptable. Examples of physiologically acceptable carriers include one or more of: water, saline, phosphate buffered saline, dextrose, glycerin, ethanol etc., as well as combinations thereof. In many cases, it is preferred to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition. Pharmaceutically acceptable carriers can further comprise smaller amounts of additives such as wetting or emulsifying agents, preservatives or buffers that increase the shelf life or efficacy of the antibody or antibody portion.

The compositions of the invention may exist in many forms. These include, for example, liquid, semi-solid and solid dosage forms, such as solutions (e.g., solutions

188 192 for injection or transfusion), dispersions or suspensions, tablets, pills, powders, liposomes or suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of solutions for injection or transfusion, such as compositions similar to those used for passive immunization of humans with other antibodies. Parenteral (e.g. intravenous, subcutaneous, intraperitoneal, intramuscular) administration is the preferred method of administration. In a preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.

Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions can be prepared as solutions, microemulsions, dispersions, liposomes or other structures useful for achieving high drug concentration. Sterile injectable solutions can be prepared by incorporating the active ingredient (i.e. antibodies or antibody portions) in the desired amount to a suitable solvent with one or more combinations of the ingredients listed above, if necessary, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle which contains the basic dispersion medium and the other ingredients required as mentioned above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred production method is drying under vacuum and freeze drying, which allows obtaining the active ingredient powder with additional desired component from a previously sterile filtered solution. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the desired particle size in the case of dispersions and the use of surfactants. Prolonged absorption of the injectable compositions can be induced by incorporating an absorption delaying agent, for example, salts of monostearate and gelatin into the composition.

Antibodies and antibody portions of the invention can be administered by a variety of known methods, although for many therapeutic applications the preferred route / method of administration is intravenous injection or transfusion. As noted by a specialist, the route and / or method of administration will vary depending on the desired result. In some embodiments, the active ingredient may be prepared with a carrier that prevents the rapid release of the compound, such as a controlled release form, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and poly lactic acid can be used. Many methods of making such forms have been patented and known, see, e.g., Sustained and Controlled Release Drug Delivery Systems, Robinson (ed.), Marcel Dekker, Inc., New York, 1978.

In some embodiments, the antibody or portion of the antibody can be administered orally, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) can also be enclosed in a hard or soft gelatin capsule, compressed into a tablet, or incorporated directly into the subject's diet. For oral administration, the compounds may be incorporated into the excipient and used in the form of edible tablets, cheek tablets, troches, capsules, elixirs, suspensions, syrups, cachets, etc. In order to administer the compound of the invention by other means than parenteral administration, it may be desirable to coat the compound or co-administration with an inactivation agent.

Supplementary active ingredients can be incorporated into the compositions. In specific embodiments, the antibody or antibody portion of the invention combines with agents that are useful for treating disorders in which hTNFa activity is detrimental. For example, an anti-hTNFa antibody or portion of an antibody of the invention may be combined and / or administered simultaneously with one or more additional antibodies that bind to other targets (e.g. antibodies that bind other cytokines or bind cell surface molecules), one or more cytokines, soluble TNFα receptors (see e.g. PCT publication WO 94/06476) and / or one or more chemical agents that inhibit the production or activity of hTNFa (such as cyclohexanoylene derivatives described in PCT publication WO 93/19751). In addition, one or more antibodies

188 192 of the invention can be used in combination with two or more of the above therapeutic agents. Such combination therapies may advantageously utilize lower doses of administered therapeutic agents, avoiding possible toxicity or complications associated with various monoterspism.

Non-limiting examples of therapeutic agents for rheumatoid arthritis with which the antibody or portion of an antibody of the invention may be combined include: nonsteroidal anti-inflammatory agents (NSAIDs); anti-inflammatory cytokine inhibitory agents (CSAID); CDP-571 / BAY-10-335ó (humanized anti-hTNFa antibody; Celltech / Bayer); cA2 (chimeric anti-HTNFa antibody; Centocor); 75 kdTNFR-IgG (75 kDa and IgG TNF receptor fusion protein; Immunex; see Arthritis &amp; Rheumatism (1994) 37: S295 ·; J. Invest. Med. (1996) 44: 235A); 55 kdTNFR-IgG (55 kDa and IgG TNF receptor fusion protein; Hoffmann-La Roche); IDEC-CE9.1 / SB 210396 (anti-CD4 non-deleting antibody; IDEC / SmithKline; see e.g. Arthritis &amp; Rheumatism (1995) 38: S 185); DAB 486-IL-2 and / or DAB 389-IL-2 (IL-2 fusion protein; Seragen; see e.g. Arthritis &amp; Rheumatism (1993) 36: 1223); .At-Tsa (humanized ppz / eCIecCaSo IL-2Ra subunit; Protem Desiig Labs / Roche); IL-4 (anti-inflammatory cytokine; DNAX / Schering); IL-10 (SCH 52000; recombinant IL-10, anti-inflammatory cytokine; DNAX / Schering); IL-4 agonists; IL-10 and / or IL-4 (see sgonistic / ne antibodies); IL-1RA (IL-1 receptor agonist; Synergen / Amgen); TNFbp / s-TNFR (soluble TNF binding protein; see, e.g., Arthritis &amp; Rheumatism (1996) 39: 9 (supplement), S284; Amer. J. Physiol. - Heart and Circulatory Physiology (1995) 268: 37-42); R973401 (type IV phosphodiester / y inhibitor; see, e.g., Arthritis &amp; Rheumatism (1996) 39: (supplement) S282); MK-966 (COX-2 inhibitor; see e.g. Arthritis &amp; Rheumatism (1996) 39: (supplement), S81); Iloprost (see Arthritis &amp; Rheumatism (1996) 39: (supplement), S82); methotrexate (Arthritis &amp; Rheumatism (1996) 39: (supplement), S82; thalidomide (Arthritis &amp; Rheumatism (1996) 39: (supplement), S282) and thalidomide related drugs (e.g. Celgene); leflunomide (anti-inflammatory and cytokine inhibitor; see e.g. Arthritis &amp; Rheumatism (1996) 39: (supplement), S131; Inflammation Res. (1996) 45: 103-107); tranexamic acid (an inhibitor of plasminogen activation; see e.g. Arthritis &amp; Rheumatism (1996) 39: (supplement), S282); prostaglandin E1 (see e.g. Arthritis &amp; Rheumatism (1996) 39: (supplement), S282); Tenidap (a nonsteroidal anti-inflammatory drug; see e.g. Arthritis &amp; Rheumatism (1996) 39: (supplement), S280); naproxen (a nonsteroidal anti-inflammatory drug; see, e.g., Neuro Report (1996) 7: 1209-1213); meloxicam (a nonsteroidal anti-inflammatory drug); Ibuprofen (a nonsteroidal anti-inflammatory drug); piroksicam (a nonsteroidal anti-inflammatory drug); diclofenac (a nonsteroidal anti-inflammatory drug); indomethacin (a nonsteroidal anti-inflammatory drug); sulfasazine (see, e.g., Arthritis &amp; Rheumatism (1996) 39: (supplement), S281); azathioprine (see e.g. Arthritis &amp; Rheumatism (1996) 39: (supplement), S281); ICE inhibitor (IL-1P converting enzyme inhibitor); zap-70 and / or drug inhibitor (zap-70 or 1ck tyrosine kinase inhibitor); vEGf inhibitor and / or VEGF-R inhibitor (vascular epithelial growth factor inhibitors or vascular epithelial growth factor receptor; angiogenesis inhibitors); corticosteroid anti-inflammatory drugs (e.g. SB203580); TNF-converting enzyme inhibitors; anti-IL-12 antibodies, interleukin 11 (see e.g. Arthritis &amp; Rheumatism (1996) 39: (supplement), S296); interleukin-13 (see, e.g., Arthritis &amp; Rheumatism (1996) 39: (supplement), S308); interleukin-17 inhibitors (see e.g. Arthritis &amp; Rheumatism (1996) 39: (supplement), p. 120); gold, penicillamine, chloroquine, hydroxychloroquine, chlorambucil, cyclophosphamide, cyclosporin, total irradiation of lymphoid organs, anti-thymocyte globulin, anti-CD4 antibodies, CD5-toxin, oral peptides and collagen, lobenzarite disodium salt; Cytokine Regulatory Factors (CRA) HP228 and HP466 (Houghten PHsrmaceutlcsls, Inc.); anti-ICS-1 antisense phosphothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble receptor 1 complement (TP10; T Cell Sciences, Inc.); prednisone, orgotein, pollsiarc / sn glycosminoglycan, minocycline, anti-IL-2R antibodies; plant and marine lipids (fish fat and plant seed acids; see, e.g., DeLuca et al (1995) Rheum. Dis. Clin. North Am. 21: 759-777); aursnofins, phenylbuts / on, meclofenamic acid, flufenamic acid, intravenous immunity globulins36

188 192 we, zileuton, mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); amiprilose (therafectin); cladribine (2-chlorodeoxyadenosine) and azaribine.

Non-limiting examples of therapeutic agents for inflammatory bowel disease with which an antibody or portion of an antibody of the invention may be combined include: budensozide, epidermal growth factor, corticosteroids, cyclosporin, sulfasalazine, aminosalicylates, 6-mercaptopurine, azathioprine, metronidazole, lipoxygenase inhibitors, mesalamine, olsalazine, balsalazide, antioxidants, thromboxane inhibitors, IL-1 antagonists anti-IL-6 monoclonal antibodies, growth factors, elastase inhibitors, pyridinyl-imidazole compounds; CDP-571 / BAY-10-3356 (humanized anti-hTNFa antibody; Celltech / Bayer); cA2 (chimeric anti-hTNFa antibody; Centocor); 75 kdTNFR-IgG (75 kDa and IgG TNF receptor fusion protein; Immunex; see Arthritis &amp; Rheumatism (1994) 37: S295; J. Invest. Med. (1996) 44: 235A); 55 kdTNFR-IgG (55 kDa and IgG TnF receptor fusion protein; Hoffmaim-La Roche); IDEC-CE9.1 / SB 210396 (anti-CD4 non-deleting antibody; IDEC / SmithKline; see e.g. Arthritis &amp; Rheumatism (1995) 38: 3185); DAB 486-IL-2 and / or DAB 389-IL-2 (IL-2 fusion protein; Seragen; see e.g. Arthritis &amp; Rheumatism (1993) 36: 1223); Anti-Tac (humanized anti-IL-2Ra antibody; Protein Design Labs / Roche); IL-4 (anti-inflammatory cytokine; DNAX / Schering); IL-10 (SCH 52000; recombinant IL-10, anti-inflammatory cytokine; DNAX / Schering); IL-4 agonists; IL-10 and / or IL-4 (see agonist antibodies); interleukin-11, prodrugs of prednisolone, dexamethasone or budesonide conjugated to glucuronide or dextran, anti-sense phosphotothioate oligodeoxynucleotides against ICAM-1 (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement 1 receptor (TP10; T Cell Sciences, Inc.); slow release mesalazine, methotrexate, platelet activating factor (PAF) antagonist; ciprofloxacin and lignocaine.

Non-limiting examples of multiple sclerosis therapeutic agents with which an antibody or portion of an antibody of the invention may be combined include: corticosteroids, prednisolone, methylprednisolone, azathioprine, cyclophosphamide, cyclosporine, methotrexate, 4-aminopyridine, tizanidine, interferon-pia (Avonex ™, Biogen), interferon-pib (Betaseron ™, ChirońBerlex), Copolymer 1 Cop-1 Teva Pharmaceutical Industries, Inc.), hyperbaric oxygen, intravenous immunoglobulins, CDP-571 / BAY-10-3356 (humanized anti-hTNEa antibody; Celltech / Bayer); cA2 (chimeric anti-hTNFa antibody; Centocor); 75 kdTNFR-IgG (75 kDa and IgG TNF receptor fusion protein; Immunex; see Arthritis &amp; Rheumatism (1994) 37: 3295; J. Invest. Med. (1996) 44: 235A); 55 kdTNFR-IgG (55 kDa and IgG TNF receptor fusion protein; Hoffmann-La Roche); IL-10, IL-4 and IL-10 and / or IL-4 agonists (e.g. agonist antibodies).

Non-limiting examples of sepsis therapeutic agents with which an antibody or portion of an antibody of the invention may be combined include: hypertonic saline solutions, antibiotics, intravenous gamma globulins, continuous hemofiltration, carbapenems (e.g. meropenem), cytokine antagonists such as TNFα, IL-1β, IL-6 / or IL-8, CDP -571 / BAY-10-3356 (humanized anti-hTNFa antibody; Celltech / Bayer); cA2 (chimeric anti-hTNFa antibody; Centocor); 75 kdTNFR-IgG (TNF receptor thyroid protein 75 kDa and IgG; Immunex; see, Arthritis &amp; Rheumatism (1994) 37 -S295; J. Invest. Med. (1996) 44: 235A); 55 kdTNFR-IgG (55 kDa and IgG tNf receptor fusion protein; Hoffmann-La Roche), Cytokine Regulatory Agents (CRA) HP228 and HP466 (Houghten Pharmaceuticals, Inc.), SHF 107647 (low molecular weight peptide; SmithKline Beecham), tetravalent guanylhydrazone CNI-1493 (Picower Institute), tissue factor pathway inhibitor (TFP1, Chiron), pLXSHD + E6 / E7 (chemically modified hemoglobin, APEX Bioscience), iron chelating agents and chelates, including diethylenetetraiminopenta complex iron (III) (DTPA-iron (III); Molichem Medicines); lysofillin (synthetic low molecular weight methylxanthine; Cell Therapeutics, Inc.), PGGglucan (water-soluble β1,3-glucan, Alpha-Beta Technology), lipid-reconstituted A-1 apolipoprotein, chiral hydroxamic acids (synthetic anti-bacterial agents that inhibit lipid A biosynthesis), anti-endotoxin antibodies; E5531 (synthetic

188 192 lipid A antagonist, Eisai America, Inc.), rBPI21 (recombinant N-terminal fragment of the human bacturicidal / permeable protein) and synthetic anti-endotoxic peptides (SAEP, BiosYnth Research Laboratories).

Non-limiting examples of therapeutic agents for adult respiratory distress syndrome (ARDS) with which an antibody or portion of an antibody of the invention may be combined include: anti-IL-8 antibodies, surfactant replacement therapy, CDP-571 / BAY-10-3356 (humanized antibody against hTNFa; Celltuch / Bacer); cA2 (chimeric anti-hTNFa antibody; Centocor); 75 kdTNFR-IgG (75 kDa and IgG TNF receptor fusion protein; ImmuneK; see Arthritis &amp; Rheumatism (1994) 37: S295 "; J. Invest. Muu. (1996) 44: 235A); 55 kdTNFR-IgG (55 kDa and IgG TNF receptor fusion protein; Hoffmann-La Roche).

The use of antibodies or parts of antibodies of the invention in combination with other therapeutic agents is discussed more extensively in Section IV.

The pharmaceutical composition of the invention may comprise a "therapeutically effective amount" or "prophylactically effective amount" of an antibody or portion of an antibody of the invention. The term "therapeutically effective amount" refers to an effective amount in the necessary doses and for an appropriate period of time to achieve the desired therapeutic effect. The therapeutically effective amount of the antibody or antibody portion may vary depending on factors such as disease state, age, sex and body weight of the subject, and the ability of the antibody or antibody portion to elicit the desired response in the subject. A therapeutically effective amount is also the amount at which the toxic or harmful effects do not outweigh the therapeutically beneficial effects. The term "prophylactically effective amount" refers to an effective amount in the necessary doses and for an appropriate time to achieve the desired prophylactic effect. Usually, because the prophylactic dose is applied to the subject before or at the early stages of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

Dosage regimens can be tuned to provide the optimal desired response (e.g., therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses over time, or the dose may be proportionally reduced or increased as indicated by the need for a therapeutic situation. It is particularly advantageous to formulate the parenteral composition in the form of dosage units to facilitate administration and uniformity of dosage. The unit dosage form as used herein refers to separate units physically useful as single doses for the treated mammalian subject, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the necessary pharmaceutical carrier. Details of the unit dosage form according to the invention are required and directly dependent on the (U) unique characteristics of the active ingredient and the particular therapeutic or prophylactic effect to be achieved; and (b) domain-related restrictions, such as the active ingredient for treating sensitivity in individuals.

An exemplary and non-limiting range of the therapeutically or prophylactically effective amount of the antibody or antibody portion of the invention is 0.1-20 mg / kg, preferably 1-10 mg / kg. It should be noted that the dose values ​​may vary depending on the type and severity of the condition to be alleviated. It should also be understood that the hives of any particular individual should have a specific dosage regimen tailored to the individual needs and professional judgment of the person administering or supervising the administration of the composition and the range of doses given as exemplary and not intended to limit the scope or practice of the claimed composition.

IV. The use of antibodies according to the invention

Based on their ability to bind to hTNFa, anti-hTNFa antibodies or parts thereof of the invention can be used to detect hTNFa (e.g., in a biological sample, such as serum or plasma), using conventional immunoassays such as the enzyme-linked immunoassay (ELISA) , rαdieimmunolegiczmc test

188 192 (RIA) or tissue immunohistochemistry. The invention provides a method for detecting hTNFa in a biological sample comprising contacting the biological sample with an antibody or antibody portion of the invention and detecting the antibody (or antibody portion) associated with hTNFa or unbound antibody (or antibody portion) and thereby detecting hTNFa in the sample. The antibody is directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibody. O-rings detectable substances include various enzymes, prosthetic groups, fluorescent, luminescent and radioactive substances. Examples of useful enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylchlinesterase; examples of useful prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of useful fluorescent substances include umbelliferone, fluzrcecein, fluorescein isothiocyanate, rhodamine, dichlorotriazinamine fluorescein, danzyl chloride and phycoerythrin; examples of luminescent substances include luminol; and examples of useful radioactive substances include ^ and, ^η,Ι, ³5S 1³H.

Alternatively, for labeling hTNFa antibodies can be tested in body fluids by a competitive immunoassay using a rhTNFa standard labeled with a detectable substance and an unlabeled anti-rhTNFa antibody. In this assay, a biological sample, a labeled rhTNFa standard and an anti-rhTNFa antibody are combined and the amount of labeled rhTNFa standard bound to the unlabeled antibody is gelled. The amount of hTNFa is inversely proportional to the amount of labeled rhTNFa standard bound to the anti-rhTNFa antibody.

The D2E7 antibody of the invention can also be used to detect TNFa of non-human species, in particular primate TNFa (e.g. chimpanzee, orangutan, mannosets, cynomolgus and rhesus), pigs and mice, because D2E7 can bind to any of these TNFa (discussed further) in example 4, subsection E).

Antibodies and antibody portions of the invention are capable of neutralizing hTNFa activity in vitro and in vivo (see Example 4). In addition, at least some of the antibodies of the invention, such as D2E7, can neutralize TNFα activity in other species. Antibodies and antibody portions of the invention can be used to inhibit TNFα activity, e.g. in cell culture containing hTNFa, in human or other mammalian individuals having TNFa with which the antibody of the invention cross-reacts (e.g., chimpanzee, orangutan, marmosets, cynomzlgnsα, rhesus, pig and mouse). In one embodiment, the invention provides a method of inhibiting TNFα activity by contacting TNFα with an antibody or antibody portion of the invention in such a way that TNFα activity is inhibited. Preferably, TNFα is human TNFα. For example, to a cell culture containing, or suspected of containing, hTNFa, an antibody or portion of an antibody of the invention may be added to the culture medium to inhibit hTNFa activity in the culture.

In another embodiment, the invention provides a method of inhibiting TNFα activity in an individual suffering from a disorder in which TNFα activity is harmful. TNFa is associated with the pathophysiology of many diseases (see, e.g., Moeller et al., (1990) Cytokine 2: 162-169; US Patent No. 5,231,024, Moeller et al .; European Patent Publication No. 260,610 BI, Moeller et al. ). The invention provides methods of TNFα activity in a subject suffering from such a disorder that comprises administering to the subject an antibody or portion of an antibody of the invention in such a way that the subject's TNFα activity is inhibited. Preferably, TNFα is human TNFα and the subject is human. Alternatively, the subject may be a TNFα expressing mammal with which the antibody of the invention cross-reacts. Still further, the subject may be a mammal into which hTNFa has been introduced (e.g., by administration of TNFα or expression of the TNFα transgene). The antibody of the invention may be administered to a human subject for therapeutic purposes (more widely discussed below). In addition, the antibody of the invention may be administered to a non-human mammal expressing TNFα with which the antibody of the invention cross-reacts (e.g. Primates, pigs or mice) for veterinary purposes or as an animal model. With respect to the latter, such animal models may be useful for testing the therapeutic efficacy of the antibodies of the invention (e.g., testing dosing and timing of administration).

As used herein, "a disorder in which TNFα activity is harmful" includes diseases and other disorders in which the presence of TNFa has been demonstrated in the body of a subject suffering from the disorder or suspected that TNFa is responsible for the pathophysiology of the disease, or is a factor causing disease worsening. A disease in which TNF? Activity is harmful is one in which inhibition of TNF? Activity should alleviate the symptoms and / or progression of the disease. Such disorders can be determined, for example, by an increase in TNFα concentration in the body fluid of the subject suffering from the disorder (e.g., an increase in the concentration of TNFa in the serum, plasma, joint fluid, etc.) of the subject, which can be determined, e.g. . There are numerous examples of disorders in which TNFα activity is harmful. The use of antibodies or parts of antibodies to treat specific disorders is discussed further below.

A. Sepsis

Tumor necrosis factor has an established role in the pathophysiology of sepsis, with biological effects including hypotension, myocardial suppression, vascular leak syndrome, organ necrosis, stimulation of the release of secondary toxic mediators and activation of the coagulation cascade (see e.g. Moeller et al. (1990) Cytokine 2 : 162-169; US Patent No. 5,231,024, Moeller et al .; European Patent Publication No. 260,610 BI, Moeller et al .; Tracey and Cerami (1994) Annu. Rev. Med. 45: 491-503; Russel and Thompson (1993) Curr. Opin. Biotech. 4: 714-721). Antibodies or parts thereof of the invention may be used to treat sepsis in any of its clinical forms, including septic shock, endotoxic shock, Gram-negative sepsis and toxic shock syndrome.

In addition, to treat sepsis, the anti-hTNFa antibody or portion of an antibody of the invention can be co-administered with one or more additional therapeutic agents that are intended to further alleviate sepsis, such as an interleukin-1 inhibitor (such as described in PCT Publication WO 92/16221 and WO 92/17583), cytokine, interleukin-6 (see e.g. PCT publication WO 93/11793) or platelet activating factor antagonist (see e.g. European Patent Publication No. EP 374 510). Other combination therapies for the treatment of sepsis are discussed in Section III.

In addition, in a most preferred embodiment, the anti-TNFα antibody or a portion of the antibody of the invention is administered to a human subject in a subset of patients with sepsis having plasma IL-6 or serum concentrations above 500 pg / ml, preferably 1000 pg / ml at the time of treatment (see publication PCT No. WO 95/20978, Daum et al.).

B. Autoimmune diseases

Tumor necrosis factor is associated with the pathophysiology of various autoimmune diseases. For example, TNFα binds to the activation of inflammation in tissues and causing joint damage in rheumatoid arthritis (see, e.g., Moeller et al. (1990) Cytokine 2: 162-169; US Patent No. 5,231,024, Moeller et al .; European Patent Publication No. 260 610 BI, Moeller et al .; Tracey and Cerami, supra; Arend and Dayer (1995) Arth, Rheum. 38: 151-160; Fava et al. (1993) Clin. Exp. Immunol. 94: 261-266). TNFa is also associated with the promotion of islet cell death and induction of insulin resistance in diabetes (see, e.g., Tracey and Cerami, PCT Publication WO 94/08609). TNFa is also associated with oligodendrocyte cytotoxicity and inflammatory plaques in multiple sclerosis (see, e.g., Tracey and Cerami, supra). Chimeric and humanized murine anti-hTNFa antibodies undergo clinical trials in the treatment of rheumatoid arthritis (see, e.g., Elliot et al., (1994) Lancet 344: 1125-1127; Elliot et al. (1994) Lancet 344: 1105-1110; Rankin et al. (1995) Br. J. Rheum. 34: 334-342).

Human antibodies or parts of the antibodies of the invention may be used to treat autoimmune diseases, in particular those associated with inflammation, including rheumatoid arthritis, rheumatoid condylitis, inflammation

188 192 osteoarticular and gout arthritis, allergies, multiple sclerosis, autoimmune diabetes, autoimmune uveitis and nephrotic syndrome. Typically, the antibody or antibody portion is administered systemically, although in some diseases it may be beneficial to administer the antibody or antibody portion to the site of inflammation (e.g. topical administration to the joint in rheumatoid arthritis or local administration in ulcers in the course of diabetes, alone or in combination with a cyclohexanoylidene derivative as described in pCt publication WO 93/19751). The antibody or antibody portion of the invention may also be administered with one or more additional agents useful in the treatment of autoimmune diseases, as discussed more extensively in Section III.

C. Infectious diseases

Tumor necrosis factor is associated with the biological effects observed in various infectious diseases. For example, TNFa is associated with causing inflammation of the brain, capillary thrombosis and malaria infarction. It also participates in causing encephalitis, including breaking the blood-brain barrier, triggering septic shock syndrome and causing venous infarction in meningitis. It is also associated with cachexia, stimulation of virus proliferation and induction of central nervous system damage in acquired immune deficiency syndrome (AIDS). Antibodies and antibody portions of the invention can be used to treat a variety of infectious diseases, including bacterial meningitis (see e.g. published European patent application EP 585 705), brain malaria, AIDS and AIDS related syndrome (ARC) (see published European patent application EP 230 574), as well as secondary to transplant cytomegalovirus infection (see e.g. Fietze et al. (1994 ) Transplantation 58: 675-680). The antibodies or portions of the antibody of the invention may therefore be used to alleviate the symptoms associated with infectious diseases, including fever, muscle pain caused by infection (such as influenza), and secondary cachexia to infection (e.g., secondary to AIDS or ARC).

D. Transplantation

Tumor necrosis factor is considered to be a key mediator of allograft rejection and graft versus host (GvHD) and a mediator of adverse effects seen when rat OKT3 antibody directed against the T lymphocyte receptor CD3 complex is administered to inhibit renal transplant rejection (see Eason et al. ., (1995) Transplantation 59: 300-305; Suthanthiran and Storm (1994) New Engl. J. Med. 331: 365-375). Antibodies and antibody portions of the invention can be used to inhibit transplant rejection, including allogeneic and xenogeneic transplant rejection, and GvHD inhibition. Although the antibody or antibody portion can be used alone, it is more preferable to use it in combination with one or more other factors that inhibit the immune response against allogeneic transplantation or inhibit GvHD. For example, in one embodiment, the antibody or antibody portion of the invention is used in combination with OKT3 to inhibit OKT3-induced reactions. In another embodiment, the antibody or antibody portion of the invention is used in combination with one or more antibodies directed against other targets associated with regulation of the immune response, such as the CD25 (IL-2 receptor-α) surface molecule, CDIa (LFA-1), CD54 (ICAM-1), CD4, CD45, CD28 / CTLA4, CD80 (B7-1) and / or (CD86 (B7-2). In yet another embodiment, the antibody or antibody portion of the invention is used in combination with one or more general immunosuppressive agents, such as cyclosporin A or FK506.

E. Cancer

Tumor necrosis factor is associated with cachexia, tumor growth, metastatic potential and cytotoxicity in cancer. Antibodies or antibody portions of the invention can be used to treat cancer, inhibiting tumor growth

188 192 or metastasis and / or amelioration of secondary cachexia to cancer. The antibody or antibody portion can be administered generally or topically to the tumor.

F. Lung diseases

Tumor necrosis factor is associated with the pathophysiology of adult respiratory distress syndrome (ARDS), including stimulation of leukocyte-endothelial activation, targeting of cytotoxicity to pneumocytes and induction of vascular leak syndrome. The antibodies and antibody portions of the invention can be used to treat a variety of lung diseases, including adult type respiratory distress syndrome (see e.g. PCT publication WO 91/04054), "concussive lung", chronic inflammatory lung disease, lung sarcoidosis, pulmonary fibrosis and silicosis. The antibody or antibody portion of the invention may be administered systemically or locally to the lung, for example in the form of an aerosol. The antibody or antibody portion of the invention may also be administered with one or more therapeutic agents useful in the treatment of lung diseases, as discussed more extensively in Section III.

G. Intestinal diseases

Tumor necrosis factor is associated with the pathophysiology of inflammatory bowel disease (see, e.g., Tracy et al. (1986) Science 234: 470-474; Sun et al. (1988) J. Clin. Invest. 81: 1328-1331; MacDonald et al. (1990) Clin. Exp. Immunol., 81: 301-305). Chimeric mouse anti-hTNFa antibodies are undergoing clinical trials in Crohn's disease (van Dullemen et al. (1995) Gastroenterology 109: 129-135). Human antibodies or parts of the antibodies of the invention can also be used to treat intestinal diseases such as idiopathic inflammatory bowel disease, which includes two syndromes: Crohn's disease and ulcerative colitis. The antibody or antibody portion of the invention may also be administered with one or more additional therapeutic agents useful in the treatment of intestinal diseases, as discussed more extensively in Section III.

H. Heart disease

Antibodies and antibody portions of the invention can also be used to treat a variety of heart conditions, including cardiac ischemia (see e.g. published European Patent Application No. EP 453 898) and heart failure (myocardial weakness) (see e.g. PCT Publication WO 94/20139 ).

And other

Antibodies and antibody portions of the invention can also be used to treat a variety of disorders in which TNFα is harmful. Examples of other diseases and disorders in which TNFα activity is associated with pathophysiology and which can be treated using the antibodies or portions of the antibody of the invention include: inflammatory bone diseases and bone resorptive diseases (see, e.g., Bertolini et al., (1986) Nature 319: 516 -518; Konig et al. (1988) J. Bone Miner. Res. 3: 621-627; Lemer and Ohlin (1993) J. Bone Miner. Res. 8: 147-155; Shankar and Stern (1993) Bone 14: 871-876), hepatitis including alcoholic hepatitis (see e.g. McClain and Cohen (1989) Hepatology 9: 349-351; Felver et al. (1990) Alcohol. Clin Exp. Res. 14: 255-259; and Hansen et al. (1994) Hepatology 20: 461-474), viral hepatitis (Sheron et al. (1991) J. Hepatol. 12: 241-245; and Hussain et al., (1994) J. Clin Paahol. 44: 1111-1115) o ^^ from hepatitis fulminans, inclination of fever (paatr e.g. van der Poll et al., (1990) N. Engl. J. Med. 322: 1622-1627; and van der Poll (1991) Prog. Clin. Biol. Res. 367: 55-60), burns (see e.g. Giroir et al. (1994) Am. J. Physiol. 267: HI 18-124; and Liu et al. (1994) Bums 20: 40-44), aero-fusion injury (see, e.g., Scales et al., (1994) Am. J. Physiol. 267: G1122-1127; Serrick et al. (1994) Transplantation 58: 1158-1162; and Yao et al. (1995) Resuscitation 29: 157-168), keloid formation (see e.g. McCauley et al. (1992) J. Clin. Immunol. 12: 300-308), scar tissue formation, fever, periodontal disease, obesity and radiation toxicity.

The invention is further illustrated by the following examples, which should not be considered as limiting. The contents of all references, patents and published applications cited in the description are incorporated as references.

188 192

Example 1. Kinetic analysis of human antibody binding to hTNFa

The binding interaction between the ligand (biotinated rukembimewamcm human TNFa (rhTNFa) immobilized in the biological sensor matrix) and analytical reagent (antibodies in solution) was measured in real time by surface plasmon resonance (SPR) using a BIAcore system (Pharmacia Biosumser, Piscataway, NJ) . The system uses SPR optical properties to detect changes in protein concentration in the dextran matrix of a biological sensor. Proteins are covalently bound to the dextran matrix at a known concentration. Antibodies are injected through the dextran matrix, and specific binding between the injected antibodies and the immobilized ligand causes an increase in protein concentration in the matrix and a change in the SPR signal. Here, changes in the SPR signal are recorded as resonance units (RUs) and displayed with respect to time on the y axis of euneorogrαmu.

In the culu to facilitate the immobilization of biomethic rhTNFa on the matrix of the biological sensor, streptavidin covalently binds to free amino groups of the dextran matrix by activating the carboxyl groups on the matrix using 100 mM N-Hydrexysucccnimid (NHS) and 400 mM N-etc 3-N 'hydrochloride salt -UUTUTYLAMINEPROPYLO) -Carbodiimide (EDC). Then, streptawiUin is injected through the activated matrix. 35 pl streptavidin (25 pg / ml) diluted with sodium acetate, pH 4.5, the activated biological sensor is injected and free amino groups are bound on the protein directly bound to the activated carboxyl groups. Unreacted matrix DCS esters are inactivated by IM injection of ethanolamine. Streptavidne-coupled biosensor systems are also commercially available (Pharmacia BR-1000-16, Pharmacia Biose ^ Piscataway, NY).

Bietamic rhTNFa was prepared by dissolving 5.0 mg of biotin (D-Biethylcyl-ε-aminocapromewe and N-hydrocecuccinimideUu ester; Boehringer Mannheim cat. No. 1008 960) in 500 (il dimethyl sulfoxide forming a solution of 10 mg / ml. 10 pl biotin per ml added to rhTNFa (at a concentration of 2.65 mg / ml) resulting in a molar ratio of biotin to rhTNFa of 2: 1. The reaction was mixed gently and incubated for two hours at room temperature in the dark. PD-10 column, Suphadex G25M (Pharmacia Cat. No. 17-0851-01) was equilibrated with 20 ml cold PBS and loaded with 2 ml rhTNFα-biotcmc per column. The column was eluted with 10 x 1 ml cold PBS. Fractions were collected and OD280 read (1.0 OD = 1.25 mg / ml). The appropriate fractions were pooled and stored at -80 ° C until use. RhTNFa Biething is also commercially available (R&amp;D Systems Cat. No. FTAOO, Minneapolis, MN).

Bietcmewanc rhTNFa, which is to be immobilized on the matrix by streptavidmina, was diluted in PBS working buffer (Gibco catalog No. 14190-144, Gibco BRL, Grand Island, NY) with the addition of 0.05% (BIAcoru) surfactant P20 (Pharmacia BR-1000- 54, Pharmacia Biosensor, Piscataway, NJ). To determine the binding capacity of immobilized rhTNFa by rhTNFa-specific antibodies, the binding assay was carried out as follows: biotinne or rhTNFa aliquots (25 nM; 10µl aliquots) injected with streptavidin-coupled dextran matrix at a rate of 5 µl / min. PBS alone was passed through each flow chamber before and immediately after protein injection. The signal difference between basal and about 30 seconds after the end of the injection of bietcnewαnuge rhTNFa was taken as the binding value (about 500 RU). The binding of rhTNFa specific antibody to immobilized rhTNFa was measured directly. Antibodies (20 pg / ml) were diluted in PBS working buffer and 25 µl aliquots were injected through immobilized protein matrices at 5 µl / min. After injection of protein and immediately thereafter, PBS alone was passed through each flow chamber. The signal difference between basal and approximately 30 seconds after the end of the injection of bietcnewαnege rhTNFa was taken as the binding value for a particular sample. Biosensor templates were regenerated using 100 mM HCl before injecting the next sample. In the culu determination of the detachment constant (Kof) the attachment constant (Kon), association constant (Ka) and deduction constant (Ku) BIAcor kinetics testing software (version 2.1) was used.

Representative results of binding of D2E7 (full length IgG4) to bietcmowαmcm rhTNFa, compared to MAK 195 mAb (fragment F (ab ') 2) are shown in Table 1 below'

188 192

Table 1

Binding of IgG4 D2E7 or MAK 195 to biotinylated rhTNF a

<td>Antibody</td><td>[Ab], nM</td><td>rhTNF a, compound RUs</td><td>Ab, related RUs</td><td>rhTNF a / Ab</td><td>Kof, sec ', (Avg.)</td>

<td>D2E7</td><td> 267</td><td> 373</td><td> 1215</td><td> 1,14</td><td>8.45 x 10-5</td>

<td></td><td> 133</td><td> 420</td><td> 1569</td><td> 1,30</td><td>5.42 x 10'5</td>

<td></td><td> 67</td><td> 434</td><td> 1633</td><td> 1,31</td><td>4.75 x 10 '⁵</td>

<td></td><td> 33</td><td> 450</td><td> 1532</td><td> 1,19</td><td>4.46x 10-5</td>

<td></td><td> 17</td><td> 460</td><td> 1296</td><td> 0,98</td><td>3.47 x 10-5</td>

<td></td><td> 8</td><td> 486</td><td> 936</td><td> 0,67</td><td>2.63 x 10 '⁵</td>

<td></td><td> 4</td><td> 489</td><td> 536</td><td> 0,38</td><td>2.17 x 10-5</td>

<td></td><td> 2</td><td> 470</td><td> 244</td><td> 0,18</td><td>3.68 x 10 '5</td>

<td></td><td></td><td></td><td></td><td></td><td>(4.38 x O '⁵)</td>

<td>MAK 195</td><td> 400</td><td> 375</td><td> 881</td><td> 1,20</td><td>5.38 x 10 '⁵</td>

<td></td><td> 200</td><td> 400</td><td> 1080</td><td> 1,38</td><td>4.54 x 10'5</td>

<td></td><td> 100</td><td> 419</td><td> 1141</td><td> 1,39</td><td>3.54 x 10 * 5</td>

<td></td><td> 50</td><td> 427</td><td> 1106</td><td> 1,32</td><td>3.67 x 10</td>

<td></td><td> 25</td><td> 446</td><td> 957</td><td> 1,09</td><td>4.41 x 10 '⁵</td>

<td></td><td> 13</td><td> 464</td><td> 708</td><td> 0,78</td><td>3.66 x 10'5</td>

<td></td><td> 6</td><td> 474</td><td> 433</td><td> 0,47</td><td>7.37 x 10'5</td>

<td></td><td> 3</td><td> 451</td><td> 231</td><td> 0,26</td><td>6.95 x 10'5</td>

<td></td><td></td><td></td><td></td><td></td><td>(4.94 x 10'5)</td>

In a second series of experiments, the kinetics of molecular interactions between the full-length IgG1 form D2E7 and biotinylated rhTNF were quantitatively analyzed using BIAcore technology as described above. The results of kinetic constants are summarized below in Tables 2, 3 and 4.

Table 2

Interaction dissociation constants between D2E7 and biotinylated rhTNF a

<td>Experience</td><td>Kd (s'j</td>

<td> 1</td><td>9.58 x 10 '^s</td>

<td> 2</td><td>9.26 x 10-</td>

<td> 3</td><td>7.60 x 10'5</td>

<td>Average</td><td>8.81 ± 1.06 x 10'5</td>

188 192

Table 3

Association interaction constants between D2E7 and biotinylated rhTNFa

<td>Experience</td><td>KAM- · s')</td>

<td> 1</td><td>1.33 x 10</td>

<td> 2</td><td>1.05 x 10-5</td>

<td> 3</td><td>3.36 x 10</td>

<td>Average</td><td>1.91 ± 1.26 x 10-5</td>

Table 4

Kinetics and affinity constants between D2E7 and biotinylated rhTNF a

<td>Experience</td><td>KAM- • s-)</td><td>Kz (s')</td><td>Kd (M)</td>

<td> 1</td><td>1.33 x 105</td><td>9.5 8 x 10-5</td><td>7.20 x 10-0</td>

<td> 2</td><td>1.05 x 105</td><td>9.26 x 10-5</td><td>8.82 x 10-0</td>

<td> 3</td><td>3.3 6 x 105</td><td>7.60 x 10</td><td>2.26 x 10-0</td>

<td>Average</td><td>1.91 ± 1.26 x 10-5</td><td>8.81 ± 1.06 x 10-5</td><td>6.09 ± 3.42 x 10-0</td>

Association and dissociation constants were calculated by BIAcore analysis of the regions of association and dissociation of sensorograms. Conventional kinetics of chemical reactions have been adopted for the interaction between D2E7 and the biotinylated rhTNFa molecule; zero order dissociation kinetics and first order association kinetics. For analysis, the selection of molecular models for kinetic analysis only considered the interaction of one arm of the bivalent D2E7 antibody and one unit of trimeric biotinylated rhTNFa. Three independent experiments were performed and the results were analyzed separately. The mean dissociation constant (kd) of the interaction between D2E7 and biotinated rhTNFa was 8.81 ± 1.06 x 10'5 s'¹, while the mean of the association constant k_;and was 1.91 ± 1.26 x 10 ^ 1 The intrinsic dissociation constant (Kd) was calculated from the formula Kd = kj / ka. Thus, the mean Kz of D2E7 to rhTNFa antibody obtained from these parameters was 6.09 ± 3.42 x 10 ^ M. Slight differences in kinetic values ​​for the IgG1 D2E7 form (shown in Tables 2, 3 and 4) and the IgG4 D2E7 form (shown in Table 1 and in Examples 2 and 3) are not the reason for the significant differences caused by the presence of IgG1 or IgG4 constant regions, but rather they can be attributed to a more accurate measurement of the concentration of antibody used to analyze the IgG1 kinetics. The kinetic values ​​for the D2E7 IgG1 form shown here seem to most closely correspond to the actual kinetic parameters of the D2E7 antibody.

Example 2. Mutagenesis by alanine scanning of CDR3 D2E7 domains

A standard method introduced a number of single mutations into alanine along the CDR3 domain of the VL D2E7 and VH D2E7 regions. Light chain mutations are shown in Figure IB (LD2E7 * .A1, LD2E7 * .A3, LD2E7 * .A4, LD2E7 * .A5, LD2E7 * .A7 and LD2E7 * .A8 with mutations to alanine in positions 1, 3, respectively , 4, 5, 7 or 8 CDR3 VL D2E7 domains). Mutations in the heavy chain are shown in Figure 2b (Hd2E7 * .A1, HD2E7 * .A2, HDaE7^φ.An, HD2E7 * .A4, HD2E7 * .A5, HD2E7 * .A6, HD2E7 * .A7, HD2E7 * .A8, HD2E7 * .A9 with mutation to alanine in positions 2, 3, 4, 5, 6, respectively, 8, 9, 10 or 11 CDR3 VH D2E7 domains). The kinetics of rhTNFa interaction with the wild type VL and VH D2E7 antibody was compared to the antibody consisting of:

1) wild-type VL D2E7 in combination with alanine substituted VH D2E7;

2) wild type VH D2E7 paired with alanine substituted VL D2E7; or

188 192

3) VL D2E7 substituted with alanine in combination with VH D2E7 substituted with alanine.

All antibodies were tested as full length IgG4 molecules.

The kinetics of antibody interaction with rhTNFa was determined by surface plasma resonance, as described in Example 1. The Koff values ​​for various VL / VH pairs are summarized in Table 5 below.

Table 5

Binding of mutants formed by scanning with alanine D2E7 to biotinylated rhTNFa

<td>VH</td><td>VL</td><td>koff</td>

<td>D2E7 VH</td><td>D2E7 VL</td><td>9.65 x 10-4</td>

<td>* HD2E7 .A1</td><td>D2E7 VL</td><td>1.40 x 10-4</td>

<td>* HD2E7 .A2</td><td>D2E7 VL</td><td>4.60 x 10⁴</td>

<td>* HD2E7 .A3</td><td>D2E7 VL</td><td>8.15 x 10</td>

<td>* HD2E7 .A4</td><td>D2E7 VL</td><td>1.80 x 10-4</td>

<td>* HD2E7 .A5</td><td>D2E7 VL</td><td>2.35 x 10-4</td>

<td>* HD2E7 .A6</td><td>D2E7YL</td><td>2.90 x 10-4</td>

<td>* HD2E7 .A7</td><td>D2E7 VL</td><td>1.00 x 10'4</td>

<td>* HD2E7 .A8</td><td>D2E7 VL</td><td>3.10 x 10-4</td>

<td>* HD2E7 .A9</td><td>D2E7 VL</td><td>8.1 x 10-4</td>

<td>D2E7 VH</td><td>* LD2E7 .A1</td><td>6.6 x 10-⁵</td>

<td>D2E7 VH</td><td>* LD2E7 .A3</td><td> —</td>

<td>D2E7 VH</td><td>* LD2E7 .A4</td><td>1.75 x 10-4</td>

<td>D2E7 VH</td><td>LD2E7 * A5</td><td>1.80 x 10-4</td>

<td>D2E7 VH</td><td>* LD2E7, A7</td><td>1.40 x 10-4</td>

<td>D2E7 VH</td><td>* LD2E7 .A8</td><td>3.65 x 10</td>

<td>* HD2E7 .A9</td><td>* LD2E7 .A1</td><td>1.05 x 10-4</td>

These results show that most of the CDR3 domain positions of the V2 and VH D2E7 regions can be replaced by a single alanine residue. Replacement with single alanineⁱⁿ positions 1, 4, 5 or 7 of the CDR3 VL D2E7, or positions 2, 5, 6, 8, 9 or 10 of the CDR3 VH D2E7 do not significantly affect the rate of rhTNF? binding disconnection compared to the parent antibody D2E7. Replacement with alanine in position 8 of CDR3 vL D2E7 or in position 3 of CDR3 VH D2E7 gives a 4-fold higher Koff value, and replacement with alanine in position 4 or 11 of CDR3 VH D2E7 gives an 8-fold higher Koff value, indicating that these positions are more critical for binding to hTNFa. However, single alanine substitution at positions 1,4, 5, 7 or 8 of the CDR3 VL D2E7, or positions 2, 3, 4, 5, 6, 8, 9, 10 or 11 of the CDR3 VH D2E7 constantly causes rhTNFa binding at the rate of binding disconnection A kof of 1 x 10-3 s or less.

Example 3. Analysis of binding of D2E7 related antibodies

A series of antibodies related to the sequence with D2E7 were analyzed for their binding to rhTNFa compared to D2E7 by surface plasma resonance as described in Example 1. The amino acid sequences of the VL regions tested are shown in Figures 1A and IB. The amino acid sequences of the HL regions tested are shown in Figures 2A and 2B. The Koff values ​​for the different VH / VL pairs (in the marked format, as full-length IgG1 or IgG4 antibody, or as scFv) are summarized in Table 6.

188 192

Table 6

Binding of D2E7 related antibodies to biotinylated rhTNFa

<td>VH</td><td>VL</td><td>Format</td><td>Kfsec ')</td>

<td>D2E7 VH</td><td>D2E7 VL</td><td>IgG1 / IgG4</td><td>9.65 x 10 '⁵</td>

<td>Kappa-D2</td><td>LOE7</td><td>lgGl / IgG4</td><td>7.70 x 10'5</td>

<td>VH1-D2</td><td>LOE7</td><td>scFv</td><td>4.60 x 10</td>

<td>VH1-D2.N</td><td>LOE7.T</td><td>IgG4</td><td>2.10 x 10-5</td>

<td>VH1-D2.Y</td><td>LOE7.A</td><td>IgG4</td><td>2.70 x 10-5</td>

<td>VH1-D2.N</td><td>LOE7.A</td><td>IgG4</td><td>3.20 x 10-5</td>

<td>VH1-D2</td><td>EPB12</td><td>scFv</td><td>8.0 x 10-</td>

<td>VH1-D2</td><td>2SD4 VL</td><td>scFv</td><td>1.94 x 10</td>

<td>3C-H2</td><td>LOE7</td><td>scFv</td><td>1.50 x 10'-</td>

<td>2SD4 VH</td><td>LOE7</td><td>scFv</td><td>6.07 x 10-3</td>

<td>2SD4 VH</td><td>2SD4 VL</td><td>scFv</td><td>1.37 x 10 '²</td>

<td>VH1A11</td><td>2SD4 VL</td><td>scFv</td><td>· 1,34x10²</td>

<td>VH1B12</td><td>2SD4 VL</td><td>scFv</td><td>1.01 x 10</td>

<td>VH1B11</td><td>2SD4 VL</td><td>scFv</td><td>9.80 x 10-3</td>

<td>VH1E4</td><td>2SD4 VL</td><td>scFv</td><td>1.59 x 10-</td>

<td>VH1F6</td><td>2SD4 VL</td><td>scFv</td><td>2.29 x 10-2</td>

<td>VH1D8</td><td>2SD4 VL</td><td>scFv</td><td>9.50 x 10 '³</td>

<td>VH1G1</td><td>2SD4 VL</td><td>scFv</td><td>2.14 x 10-2</td>

<td>2SD4 VH</td><td>EPB12</td><td>scFv</td><td>6.70 x 10-3</td>

<td>2SD4 VH</td><td>VL10E4</td><td>scFv</td><td>9.60 x 10 -</td>

<td>2SD4 VH</td><td>VL100A9</td><td>scFv</td><td>1.33 x 10</td>

<td>2SD4 VH</td><td>VL100D2</td><td>scFv</td><td>1.41 x 10-</td>

<td>2SD4 VH</td><td>VL10F4</td><td>scFv</td><td>1.11 x 10-2</td>

<td>2SD4VH</td><td>VLLOE5</td><td>scFv</td><td>1.16 x 10-</td>

<td>2SD4VH</td><td>VLLOF9</td><td>scFv</td><td>6.09 x 10-2</td>

<td>2SD4VH</td><td>VLLOF10</td><td>scFv</td><td>1.34 x 10-2</td>

<td>2SD4VH</td><td>VLLOG7</td><td>scFv</td><td>1.56 x 10-</td>

<td>2SD4 VH</td><td>VLLOG9</td><td>scFv</td><td>1.46 x 10-</td>

<td>2SD4 VH</td><td>VLLOH1</td><td>scFv</td><td>1.17 x O-²</td>

<td>2SD4 VH</td><td>VLLOH10</td><td>scFv</td><td>1.12 x 10-</td>

<td>2SD4 VH</td><td>VL1B7</td><td>scFv</td><td>1.30 x 10</td>

<td>2SD4 VH</td><td>VL1C1</td><td>scFv</td><td>1.36 x 10-2</td>

<td>2SD4 VH</td><td>VL1C7</td><td>scFv</td><td>2.00 x 10-2</td>

<td>2SD4 VH</td><td>VL0.1F4</td><td>scFv</td><td>1.76 x 10-2</td>

<td>2SD4 VH</td><td>VL0.1H8</td><td>scFv</td><td>1.14 x 10-</td>

188 192

Slow disconnection rate (i.e. K_ABOUTf '&lt;1 x 10' s'¹) for full length antibodies (i.e. IgG format) having a VL selected from the group consisting of D2E7, LOE7, LOE7.T and LOE7.A which have threonine or alanine at position 9 indicates that position 9 of CDR3 VL D2E7 can be occupied by one of these two residues with no significant effect on Kff. The CDR3 VL D2E7 compliance motif includes QRYNRPY- (T / A) (SEQ ID NO: 3). In addition, a slow disconnection rate (i.e., Kof &lt;1 x 10 's'¹) for antibodies having VH selected from the group consisting of D2E7, VH1-D2.N and VH1-D2.Y, which have tyrosine or asparagine at position 12, indicates that position 12 of CDR3 VH D2E7 can be occupied by these two residues without affecting K_aboutf. Accordingly, the CDR3 VH D2E7 compliance motif includes the amino acid sequence: VSYLSTASSLD- (Y / N) (SEQ ID NO: 4).

The results shown in Table 6 show that in scFv format antibodies containing CDR3 of the VL or VH 2SD4 region show faster Kof (i.e. Kof&gt; 1 x 10 '³ s'¹) compared with antibodies containing the VL or Vh d2e7 region CDR3. In CDR3, VL 2SD4 differs from D2E7 at positions 2, 5 and 9. However, as discussed above, positions 9 can be occupied by Ala (as in 2SD4) or Thr (as in D2E7) without significantly affecting Kof. Thus, by comparing 2SD4 and D2E7, positions 2 and 5 of the CDR3 VL D2E7, both arginines, can be identified as crucial for antibody binding to hTNFa. These residues may be directly involved as contact residues at the antibody binding site or may be critical in maintaining the scaffold architecture of the antibody molecule in this region. Regarding the nature of position 2, the replacement of Arg (in LOE7, which has the same CDR3 as D2E7) by Lys (in EP B12) accelerates Kof twice. Regarding the nature of position 5, the replacement of Arg (in D2E7) by Ala (in LD2E7 * .A5), as described in Example 2, also accelerates Kof twice. In addition, without Arg in positions 2 and 5 (in 2SD4) the disconnection speed is five times faster. However, it should be noted that although position 5 is important for improving binding to hTNFa, the change in this position can be abolished by changes in other positions as seen in VLL0E4, VLL0H1 or VL0.1H8.

In CDR3 VH, 2SD4 differs from D2E7 in positions 1, 7 and 12. As discussed above, however, position 12 can be occupied by Asn (as in 2SD4) or Tyr (as in D2E7) without significantly affecting Kof. Thus, by comparing 2SD4 and D2E7, positions 1 and 7 of the CDR3 VH D2E7 can be identified as key for binding to hTNF? As discussed above, these residues may be directly involved as contact residues at the antibody binding site or may be critical to maintaining the scaffold architecture of the antibody molecule in this region. Both positions are important for binding to hTNFa because when using CDR3 VH 2C-H2 (which has valine converted to alanine in position 1 relative to CDR3 VH D2E7) scFv shows 3 times faster detachment than from CDR3 Vh D2E7, but this rate is still 4 times lower than when using CDR3 VH 2SD4 (which has changes in positions 1 and 7 relative to CDR3 VH D2E7).

Example 4. Functional D2E7 activity

To test the functional activity of D2E7, the antibody was used in several tests that measure the ability of the antibody to inhibit hTNFa activity in vitro and in vivo.

A. Neutralization of TNFα-induced cytotoxicity to L929 cells

Human recombinant TNFα (rhTNFa) causes cellular cytotoxicity of murine L929 cells after incubation for 18-24 hours. Human anti-hTNFa antibodies were tested in the L929 assay by co-incubating the antibodies with hTNF α and cells as follows. The contents of a 96-well microtiter plate containing 100 μ.1 anti-hTNFa antibodies were serially diluted 1/3 using RPMI medium containing 10% fetal bovine serum (FBS). 50 ul rhTNF was added to a final concentration of 500 pg / ml in each well. The plates were then incubated for 30 minutes at room temperature. Then 50 μΐ fiUrobroblasts L929 susceptible to hTNFa were added at a final density of 5 x 10⁴ cells per well, including 1 μg / ml actinomycin-D. Controls included medium with cells and rhTNFa, with cells. These controls and a TNFa standard curve, ranging from 2 ng / ml to 8.2 pg / ml, were used

188 192 to determine the quality of the test and provide a neutralization window. Plates were incubated overnight (18-24 hours) at 37 ° C and 5% CO2

100 pl medium was taken from each well and 50 μΐ 5 mg / ml 3, (4,4-dimethylthiazol-2-yl) -2,) diphenyl-trazrazole bromide (MTT, commercially available from Sigma Chem ^ al Co ., St. Louis, MO) in PBS. Plates were again incubated for 4 hours at 37 ° C. Then 50 µl of 20% sodium dodecyl sulfate (SDS) was added to each well and the plates were incubated overnight at 37 ° C. Optical density was measured at 570 / / 630 n wavelength, curves plotted for each sample and IC 50 determined by standard methods.

Representative results for human antibodies comprising different VL and VH pairs, compared to MAK 195, are shown in Figure 3 and Table 7 below.

Table 7

Neutralization of TNF α-induced L929 cytotoxicity

<td>VH</td><td>VL</td><td>Structure</td><td>IC50, M</td>

<td>D2E7</td><td>D2E7</td><td>scFv</td><td>1.1 x10-0</td>

<td>D2E7</td><td>D2E7</td><td>IgG4</td><td>4.7 x 10-11</td>

<td>2SD4</td><td>2SD4</td><td>scFv / IgG1 / IgG4</td><td>3.0 x10⁷</td>

<td>2SD4</td><td>LOE7</td><td>scFv</td><td>4.3 x 10-</td>

<td>VH1-D2</td><td>2SD4</td><td>scFv</td><td>1.0 x 10-8</td>

<td>VH1-D2</td><td>LOE7</td><td>scFv / IgG1 / IgG4</td><td>3.4 x 10-0</td>

<td>VH1.D2.Y</td><td>LOE7.T</td><td>IgG4</td><td>8.1 x 10-1</td>

<td>VH1-D2.N</td><td>LOE7.T</td><td>IgG4</td><td>1.3 x 10-0</td>

<td>VH1-D2.Y</td><td>LOE7 A</td><td>IgG4</td><td>2.8 x 10-1 '</td>

<td>VH1-D2.N</td><td>LOE7.A</td><td>IgG4</td><td>6.2 x 10-1</td>

<td>MAK 195</td><td>MAK 195</td><td>scFv</td><td>1.9 x 10'8</td>

<td>MAK 195</td><td>MAK 195</td><td>^ flab</td><td>6.2 x 101 '</td>

The results in Figure 3 and Table 7 show that human D2E7 anti-hTNFa antibody and various D2E7 related antibodies neutralize TNFa-induced L929 cytotoxicity with a force approximately equivalent to that of mouse anti-hTNF a MAK 195 MAb.

In another series of experiments, the ability of the IgG1 D2E7 form to neutralize TNFα induced L929 cytotoxicity was tested as described above. The results from three independent experiments and their average are given in Table 8.

Table 8

Neutralization of TNF-induced and L929 cytotoxicity by IgG1 D2E7

<td>Experience</td><td>IC »[M]</td>

<td> 1</td><td>1.26 x 10-Ό</td>

<td> 2</td><td>1.33 x 10-0</td>

<td> 3</td><td>1.15 x 10-0</td>

<td>Average</td><td>1.25 ± 0.01 x 10-0</td>

188 192

This series of experiments confirmed that D2E7 in the form of full-length IgG1 neutralizes TNF-induced L929 cytotoxicity with an IC50 mean of 1.25 ± 0.01 χ 10 '' M.

B. Inhibition of TNF? Binding to TNF? Receptors on U-937 cells

The ability of human anti-hTNFa antibodies to inhibit the binding of hTNFa to cell surface hTNFa receptors was tested using cell line U-937 (ATCC CRL 1593), a human histiocyte line expressing hTNFa receptors. U-937 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (Hyclone A-1111, Hyclone Laboratories, Logan, Utah), L-glutamine (4 nM), HEPES buffer solution (10 mM), penicillin (100 pg / ml) and streptomycin (100 pg / ml). To test full-length IgG antibody activity, U-937 cells were pre-incubated with PBS supplemented with 1 mg / ml human IgG (Sigma 1-4506, Sigma Chemical Co., St. Louis, MO) for 45 minutes on ice, followed by cells were washed three times with binding buffer. For binding assay, U-937 cells (5x 10⁶ cells / ml) were incubated in binding buffer (PBS with 0.2% bovine serum albumin) in 96-well microtiter plates (Costar 3799, Costar Corp., Cambridge, MA) together with labeled rhTNFa ¹²⁵I (3 χ 10 '¹⁰ M; 25 pCi / ml; NEN Research Products, Wilmington, DE) with or without anti-rhTNFa antibodies, in a final volume of 0.2 ml. Plates were incubated on ice for 1.5 hours. Then 75 pl of each sample was transferred to 1.0 ml tubes (Sarstedt 72.700, Sarstedt Corp., Princeton, NJ) containing dibutylphthalate (Sigma D-2270, Sigma Chemical Co., St. Louis, MO) and dininbutylphalan (ICN 210733, ICN , Irvine, CA). The tubes contained 300 [mu] l mixtures of dibutylphthalate and dininbutylphthalate in a 2: 1 volume ratio. Free (i.e. unbound) labeled rhTNFa¹²⁵And removed by centrifugation for 5 minutes. Then, the bottom of each tube containing cell pellet was cut off using tube scissors (BelArt 210180001, Bel-Art Products, Pequannock, NJ). The cell pellet contained '25i-labeled rhTNFa bound to the TNFa p60 and p80 receptors, while the aqueous phase above the oil mixture contained an excess of free-labeled rhTNFa^r“⁵I. All cell pellets were collected in counting tubes (Falcon 2052, Becton Dickinson Labware, Lincoln Park, NJ) and counted in a scintillation counter.

Representative results are shown in Figure 4. The IC50 value for inhibition of hTNFa binding to hTNFa receptors on U-937 cells by D2E7 is about 3 x W '¹'M. The results show that human anti-rhTNFa D2E7 antibody inhibits the binding of rhTNFa to hTNFa receptors on U-937 cells at a concentration approximately equal to that of the murine anti-rhTNF mAb MAK 195.

In another series of experiments, the ability of the D2E7 IgG form to inhibit the binding of rhTNFa to hTNFa receptors on U-937 cells was tested as described above. The results of three independent experiments and their average are summarized in Table 9.

Table 9

Inhibition of TNF binding to the receptor on U-937 cells by IgG1 D2E7

<td>Experience</td><td>IC50 [M]</td>

<td> 1</td><td>1.70 x 10'O</td>

<td> 2</td><td>1.49 χ 10'0</td>

<td> 3</td><td>1.50 χ 10'Ό</td>

<td>Average</td><td>1.56 ± 0.12 χ Ό '¹’</td>

This series of experiments confirmed that D2E7 in the form of full-length IgG1 inhibits the binding of TNF to the receptor on U-937 cells with an IC50 mean of 1.56 ± 0.12 χ 10 *¹'M.

To test the inhibitory force of D2E7 binding⁵I-rhTNFa with individual p55 and p75 receptors, solid-radioimmunoassay was performed. To measure D2E7 IC50 values ​​for separate TNF receptors, different antibody concentrations were incubated with the concentration¹²⁵I-rhTNFa equal to 3 χ 10 '¹⁰. The mixture was tested in separate plates containing p55 or p75 receptors in a dose-dependent manner. The results are summarized in Table 10.

188 192

Table 10

Inhibition of TNF binding to p55 and p75 TNFR receptors by IgG1 D2E7

<td></td><td colspan="2">IC50 [M]</td>

<td>Reagent</td><td>p55 TNFR</td><td>p75 TNFR</td>

<td>D2E7</td><td>1.47 x 10'9</td><td>1.26 x 10'9</td>

<td>rhTNF</td><td>2.31 x 10-9</td><td>2.70 x 10-9</td>

Inhibition of the binding of? 11-rhTNFa to TNF p55 and p75 receptors on U-937 cells from D2E7 followed the usual sigmoidal curve, indicating IC50 values ​​similar for each receptor. In a radieimmum mum-based cm (RIA) test with recombinant TNF receptors, IC50 values ​​for inhibition of the binding of '^ I-rhTNFa to p55 and p75 receptors by D2E7 were calculated as 1.47 x 10', respectively⁹and 1.25 x 10'9 M. The decrease in IC50 values ​​in the solid-state assay was probably due to higher receptor density in the RIA format, since unlabelled rhTNFa also inhibited with similar IC50 values. IC50 values ​​for inhibition of the binding of ^ I-rhTNFa to p55 and p75 receptors not labeled with rhTNFa were 2.31 x 109 and 2.70 x 10-M, respectively.

C. Inhibition of ELAM-1 expression on HUVEC

Human umbilical vein endothelial cells (HUVEC) can be induced to express the endothelial cell leukocyte adhesion molecule 1 (ELAM-1) on their surface by rhTNFa treatment, which can be detected by reacting HUVEC treated with rhTNFa treated with mouse anti-human ELAM-1 antibody. The ability of human anti-rhTNFa antibodies to inhibit TNFα-induced ELAM-1 expression on HUVEC was tested as follows: HUVEC (ATCC CRL 1730) was plated in 96 well plates (5 x 10 'cells / ml) and incubated overnight at 37 ° C. The next day, serial dilutions of human anti-hTNFa antibody (1:10) were made in microtiter plates, ranging from 20-100 pg / ml antibody. The rhTNFa stock solution was made at a concentration of 4.5 ng / ml, aliquots of rhTNFa were added to each well containing the antibody and the contents mixed well. Controls contained medium alone, medium with anti-hTNFa antibody, and rhTNFa medium. HUVEC plates were removed from overnight incubation at 37 ° C and medium was gently removed from each well. 200 µl of the antibody and rhTNFa mixture were transferred to each well of the hUvEC plates. HUVEC plates were further incubated at 37 ° C for 4 hours. Then, the stock solution of the murine anti-ELAM-1 antibodies was diluted 1.1000 in RPMI. The medium in each well of the HUVEC plates was gently removed and 50 µl / well of anti-ELAM-1 antibody solution added and the HUVEC plates were incubated for 60 minutes at room temperature. A solution of anti-mouse IgG labeled with I I was prepared in RPMI (approximately 50,000 cpm at 50 μΐ). The medium in each well was harvested, the wells were rinsed twice with RPMI and 50 µl of anti-mouse Ig-labeled I Ig antibody was added to each well. Plates were incubated for one hour at room temperature and then each well was washed with RPMI. 180 µl of 5% SDS solution was added to each well to break down the cells. Cell lysate from each well was transferred to the tube and counted in a scintillation counter.

Representative results are shown in Figure 5. The IC50 value of hTNFa induced HAM inhibition of EL2-1 expression on HUVEC D2E7 was about 6 x 10ⁿ M. These results show that human anti-hTNFa antibody inhibits hTNFa-induced ELAM-1 expression on HUVEC at concentrations approximately equivalent to mouse anti-HTNFa MAK 195 mAb.

In another series of experiments, the ability of the IgG1 D2E7 form to inhibit TNFα induced ELAM-1 expression on HUVEC was tested as described above. The results of three independent experiments and their average are summarized in Table 11.

188 192

Table 11

Inhibition of TNF-induced ELAM-1 expression by IgG1 D2E7

<td>Experience</td><td>IC50 [M]</td>

<td> 1</td><td>1.95 x 10-0</td>

<td> 2</td><td>169x 10-0</td>

<td> 3</td><td>1.90 x 10-Ό</td>

<td>Average</td><td>1.85 ± 0.14 x W¹⁰</td>

This series of experiments confirmed that D2E7 in the form of full-length IgG1 inhibits TNFα-induced ELAM-1 naHUVEC expression with an IC50 mean of 1.85 ± 0.14 x 10 ^ M.

The potency of D2E7 IgG1 neutralization was also tested for rhTNFa-induced expression of two other adhesion molecules, ICAM- and VCAM-1. Since the rhTNFa titration curve for ICAM-1 expression at 16 hours was very similar to the ELAM-1 expression curve, the same concentration of rhTNFa was used in antibody neutralization experiments. HUVEC was incubated with rhTNFa in the presence of various concentrations of D2E7 in a CO2 incubator at 37 ° C for 16 hours, and ICAM-1 expression was measured with mouse anti-ICAM-1 antibody and sheep anti-mouse antibody, labeled ^Π5Ι. Two independent experiments were performed and IC50 values ​​calculated. Unrelated IgG1 antibody did not inhibit ICAM-1 expression.

The experimental procedure for testing VCAM-1 expression was identical to the ELAM-1 expression testing procedure except that the anti-VCAM-1 mAb was used instead of the anti-ELAM-1 mAb. Three independent experiments were performed and IC50 values ​​calculated. Unrelated human IgG1 antibody did not inhibit VCAM-1 expression. The results are summarized in Table 12.

Table 12

Inhibition of ICAM-1 and VCAM-1 expression by IgG1 D2E7

<td colspan="2">ICAM-1 inhibition</td><td colspan="2">IC₅0 [M]</td>

<td>Experience</td><td>IC50 [M]</td><td>Experience</td><td>IC₅₀ [M]</td>

<td> 1</td><td>1.84 x 10- °</td><td> 1</td><td>1.03 x 10-0</td>

<td> 2</td><td>2.49 x 10-0</td><td> 2</td><td>9.26 x 10-0</td>

<td></td><td></td><td> 3</td><td>1.06 x 10</td>

<td>Average</td><td>2.1 7 ± 0.46 x 10-0</td><td>Average</td><td>1.01 ± 0.01 x 10-0</td>

These experiments show that treatment of primary human umbilical vein endothelial cells with rhTNFa leads to optimal expression of ELAM-1 and VCAM-1 adhesion molecules after 4 hours, and maximum overregulation of ICAM-1 expression after 16 hours. D2E7 was able to inhibit the expression of three adhesion molecules in a dose dependent manner. The IC50 values ​​for ELAM-1, ICAM-1 and VCAM-1 inhibition were 1.85 x 10-0, 2.17 x 10-0 and 1.01 x 10-0 M., respectively. These values ​​are very similar, indicating similar dose requirements for the activation signal from rhTNFa to induce ELAM-1, ICAM-1 and VCAM-1 expression. Interestingly, D2E7 was equally effective in the longer ICAM-1 expression inhibition test. The ICAM-1 inhibition test required 16 hours of incubation of rhTNFa with D2E7 with HUVEC compared with 4 hours for the ELAM-1 and VCAM-1 inhibition test. Because D2E7 has a slow rate of rhTNFa detachment, it is convincing that during 16-hour incubation there was no significant competition for the TNF receptor on HUYEC.

188 192

D. RhTNF neutralization in vivo

Three different systems in vivo were used to demonstrate the effectiveness of D2E7 in inhibiting hTNFa activity in vivo.

I. Inhibition of leeahi \ RNF-induced effkku in e ^ -gatehSoozLmiee mice

Injection of recombinant human TNFa (rhTNFa) into D-galactosamine sensitized mice causes death within 24 hours. TNFα neutralizing agents have been shown to prevent mortality in this system. To test the ability of human anti-hTNFa antibodies to neutralize hTNFa in vivo in this model, C57B1 / 6 mice were injected with different concentrations of D2E7-IgG1 or control protein, intraperitoneally (ip) in PBS. Mice were exposed 30 minutes later to 1 μg rhTNFa and 20 mg D-galactosamine in PBS ip and observed for 24 hours. It has been pre-determined that these amounts of rhTNFa and D-ghlhktozhmiec induce approximately 80-90% mortality in mice.

Representative results presented as a bar graph of% survival versus antibody concentration are shown in Figure 6. Black bars represent D2E7 and dashed bars represent MAK 195. Injection of 2.5-25 pg of D2E7 antibody on the mouse protected the animals against TNFα induced lethal effect. The ED50 value was about 1-2.5 μg / mcsz. The positive control antibody, MAK 195, had similar protective capacity. Injection of D2E7 in the absence of rhTNFa had no harmful effects on mice. Injection of non-specific human IgG1 antibody did not provide protection against TNFα.

In the second experiment, 49 mice were divided into 7 equal groups. Each group received different doses of d2E7 30 minutes prior to receiving LD &lt;1&gt; of rhTNFα / D-gh-z o.o. mixture (1.0 μg rhTNFa and 20 mg D-gala ^ for mouse). Control group 7 received normal human IgG1 kappa antibody at a dose of 25 μg / mouse. Mice were tested 24 hours later. The survival rates for each group are summarized in Table 13 below.

Table 13

Survival within 24 hours after treatment with D2E7

<td>Group</td><td>Survival rate (live / total)</td><td>Survivability (%)</td>

<td>1 (without antibody)</td><td> 0/7</td><td> 0</td>

<td>2 (1 pg)</td><td> 1/7</td><td> 14</td>

<td>3 (2.6 pg)</td><td> 5/7</td><td> 71</td>

<td>4 (5.2 pg)</td><td> 6/7</td><td> 86</td>

<td>5 (26 pg)</td><td> 6/7</td><td> 86</td>

<td>6 (26 pg; without rhTNF)</td><td> 7/7</td><td> 100</td>

<td>7 (25 pg Hu IgG1)</td><td> 1/7</td><td> 14</td>

II. Inhibition of fever in TNF-induced rabbits

The effectiveness of D2E7 in inhibiting rhTNFa-induced fever was studied in rabbits. Groups of three female NZW rabbits, weighing about 2.5 kg, were injected intravenously with D2E7, rhTNFa and DrE7 and rhTNFa immune complexes. Rectal temperature was measured with thermistor probes on a Kaye recorder every minute for about 4 hours. Recombinant human TNF in saline was injected at a dose of 5 μg / kg, causing a temperature increase greater than 0.4 ° C approximately 45 minutes after injection. The antibody preparation alone, in the saline solution at a dose of 138 μg / kg, did not induce a temperature increase for 140 minutes after injection. In further experiments, D2E7 or control reagent (human IgG1 or vehicle) was injected into rabbits iv 15 minutes after injection of rhTNFa in saline at 5 μg / gk iv. Representative results of several experiments are summarized in Table 14.

188 192

Table 14

Inhibition of rhTNFa induced fever in rabbits by D2E7

<td></td><td colspan="2">Temperature rise *, ° C</td><td></td><td>Stack. Moth.</td><td>Temp. Peak.</td>

<td>D2E7 dose (pg / kg)</td><td>rhTNF</td><td>rhTNF + D2E7</td><td>% inhibitions. **</td><td>D2E7: rhTNF</td><td>minutes after rhTNF</td>

<td> 14</td><td> 0,53</td><td> 0,25</td><td> 53</td><td> 1</td><td> 60</td>

<td> 24</td><td> 0,43</td><td> 0,13</td><td> 70</td><td> 1,6</td><td> 40</td>

<td> 48</td><td> 0,53</td><td> 0,03</td><td> 94</td><td> 3,3</td><td> 50</td>

<td> 137</td><td> 0,53</td><td> 0,00</td><td> 100</td><td> 9,5</td><td> 60</td>

<td> 792</td><td> 0,80</td><td> 0,00</td><td> 100</td><td> 55</td><td> 60</td>

* = peak temperature ** =% inhibition = (1- {temperature increase with rhTNF a + D2E7 / temperature increase with rhTNF a} alone) x 100

Intravenous D2E7 treatment at a dose of 14 gg / kg partially inhibited the pyrogenic reaction compared to salt-treated rabbits. D2E7 administered at a dose of 137 gg / kg completely inhibited the pyrogenic reaction to rhTNFa in the same experiment. In the second experiment, D2E7 administered at a dose of 24 gg / kg also partially inhibited the pyrogenic reaction compared to rabbits treated with salt alone. The molar ratio of D2E7 to rhTNFa in this experiment was 1/6: 1. In the third experiment, D2E7 injected iv at a dose of 48 gg / kg (molar ratio D2E7 to rhTNFa = 3.3: 1) completely inhibited the pyrogenic reaction compared to rabbits treated with control human IgG1 in a salt solution at a dose of 30 μg / kg. In the final experiment, rabbits treated with D2E7 (792 gg / kg) in a very high molar ratio to rhTNFa (55: 1) did not develop any increase in temperature within 4 hours of observation. Treatment of rabbits with immune complexes made from a mixture of D2E7 and rhTNFa by incubation at 37 ° C for an hour in a 55: 1 molar ratio without subsequent administration of rhTNF and also did not induce any temperature increase.

III. Prevention of polyarthritis in Tg197 transgenic mice

The effect of D2E7 on disease development was studied in a model of arthritis in transgenic mice. Transgenic mice (Tg 197) that express wild-type human TNF (modified in the 3 'region outside the coding sequence) were generated and these mice developed chronic polyarthritis with 100% with an incidence at 4-7 weeks (see EMBO J ( 1991) 10: 4025-4031, further description of the model Tg197).

Transgenic animals were identified by PCR at the age of 3 days. Transgenic animal litters were divided into six groups. Transgenic mice were verified by slot-blot analysis at 15 days of age. Treatment protocols for the six groups were as follows: group 1 = no treatment; group 2 = salt (carrier), group 3 = D2E7 1.5 Higkg, group 4 = D2E7 15 gg / kg, group 5 = D2E7 30 gg / kg and group 6 = IgG1 isotype control 30 gg / kg. The study also included a non-transgenic litter as a control ((group 7 = non-transgenic, no treatment). Each group received three ip injections per week for specific treatment. Injections were carried out for 10 weeks. Every week, macroscopic changes in joint morphology were recorded in each animal. At 10 weeks of age, all mice were killed and mouse tissue was removed for formalin, and tissue microscopic examination was performed.

At the beginning of each week, all mice were individually weighed. At the same time, joints (in mm) were measured as a measure of the severity of the disease. Joint size was assessed as the average of three measurements on the right front elbow using a micrometer. Arthritis was assessed weekly as follows: 0 = no arthritis (normal appearance and mobility); + = mild arthritis (joint sprain); ++ = moderation54

188 192 mucosal arthritis (edema, joint deformity) and +++ = severe arthritis (ankylosis detected during flexion and severe impairment of mobility).

Histopathological evaluation based on staining of joint sections with hematoxylin / eosin was performed as follows: 0 = no detectable disease; 1 = synovial proliferation; 2 = significant synovial thickening and 3 = cartilage destruction and bone erosion.

The effect of D2E7 treatment on the average joint size of Tg197 transgenic mice is shown in Figure 9. The histopathological and arthritis assessments of Tg 197 transgenic mice at 11 weeks of age are summarized in Table 15 below.

Table 15

Effect of D2E7 on histological assessment and arthritis in Tg197 mice

<td>Group</td><td>Treatment</td><td>Histopathology assessment</td><td>Arteretta rating.</td>

<td> 1</td><td>no</td><td> 3 (7/70)</td><td> +++ (7/7)</td>

<td> 2</td><td>salt</td><td> 3 (8/8)</td><td> +++ (8/8)</td>

<td> 6</td><td>IgG1 control</td><td> 3 (9/9)</td><td> +++ (7/9)</td>

<td> 3</td><td>D2E7 1.5 pg / g</td><td> 0 (6/8)</td><td> 0 (8/8)</td>

<td> 4</td><td>D2E7 15 pg / g</td><td> 0 (7/8)</td><td> 0 (8/8)</td>

<td> 5</td><td>D2E7 30 pg / g</td><td> 0 (8/8)</td><td> 0 (8/8)</td>

This experiment showed that the D2E7 antibody has a definitely beneficial effect on transgenic mice expressing wild-type human TNF (Tg 197) without visible arthritis during the study.

E. Neutralization of TNF and other species by D2E7

The binding specificity of D2E7 was tested by measuring its ability to neutralize tumor necrosis factor of various primates and mice using the L929 cytotoxicity test (as described in Example 4, subsection A, above). The results are summarized in Table 16 below.

Table 16

The ability of D2E7 to neutralize TNF of different species in the L929 test

<td>TNF *</td><td>Source</td><td>IC50 for neutralization by D2E7 [M] **</td>

<td>man</td><td>recombinant</td><td>7.8 x 10'11</td>

<td>chimpanzee</td><td>PBMC stimulated LPS</td><td>5.5 x 10-11</td>

<td>orangutan</td><td>recombinant</td><td>6.0 x 10 ·</td>

<td>Marmoset</td><td>PBMC stimulated LPS</td><td>4.0 x 10'O</td>

<td>cynomolgus</td><td>PBMC stimulated LPS</td><td>8.0 x 10'11</td>

<td>rhesus</td><td>PBMC stimulated LPS</td><td>3.0 x 10'11</td>

<td>dog</td><td>WBC stimulated LPS</td><td>2.2 x 10'O</td>

<td>Pig</td><td>recombinant</td><td>1.0 x 10'7</td>

<td>mouse</td><td>recombinant</td><td>&gt; 1.0 x 10'7</td>

188 192

The results in Table 16 show that D2E7 is able to neutralize TNFa activity of five primate species in a manner equivalent to human TNFa and also can neutralize canine TNFa activity (about 10 times less than human TNFa) and porcine and mouse TNFa (about 1000-times less than human TNFa. In addition, the binding of D2E7 to rhTNFa in solution was not inhibited by other cytokines such as lymphotoxin (TNF β), IL-1a, IL-1 β, IL-2, IL-4, IL-6, IL-8, IFNy and TGFp, indicating that D2E7 is specific for the TNFα ligand.

F. No cytokine release by whole human blood incubated with D2E7

In this example, the ability of D2E7 to induce cytokine secretion or exfoliation of surface molecules by cells of normal human blood was examined. D2E7 was incubated with diluted whole blood from three different normal donors at various concentrations for 24 hours. At the same time, a positive control was performed with LPS at a concentration that, as previously determined, stimulates immunocompetent blood cells to secrete cytokines. Supernatants were collected and tested in an ELISA panel for soluble cytokines, receptors and adhesion molecules: IL-1x, IL-β, IL-1 receptor antagonist, IL-6, IL-8, TNFα, soluble TNFα receptor I and TNFα soluble receptor II , soluble ICAM-1 and soluble E-selectin. No significant amounts of cytokines or purified surface molecules were detected by incubation with D2E7 at concentrations up to 343 pg / ml. Control cultures without antibody addition also did not produce detectable amounts of cytokines, whereas LPS-controlled cultures gave elevated values ​​in the higher ranges of picograms and lower nanograms. These results indicate that D2E7 does not induce cytokine secretion and purification of blood cell surface proteins above the values ​​normally found in px vivo cultures.

Part of the disclosure is a sequence list, the contents of which are summarized in the following table.

<td>Id. Seq. No.</td><td>Antibody chain</td><td>Region</td><td>Sequence Type</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td> 1</td><td>D2E7</td><td>VL</td><td>amino acid</td>

<td> 2</td><td>D2E7</td><td>VH</td><td>amino acid</td>

<td> 3</td><td>D2E7</td><td>VL CDR3</td><td>amino acid</td>

<td> 4</td><td>D2E7</td><td>VH CDR3</td><td>amino acid</td>

<td> 5</td><td>D2E7</td><td>VLCDR2</td><td>amino acid</td>

<td> 6</td><td>D2E7</td><td>VHCDR2</td><td>amino acid</td>

<td> 7</td><td>D2E7</td><td>VLCDR1</td><td>amino acid</td>

<td> 8</td><td>D2E7</td><td>VH CDR1</td><td>amino acid</td>

<td> 9</td><td>2SD4</td><td>VL</td><td>amino acid</td>

<td> 10</td><td>2SD4</td><td>VH</td><td>amino acid</td>

<td> 11</td><td>2SD4</td><td>VL CDR3</td><td>amino acid</td>

<td> 12</td><td>EPB12</td><td>VL CDR3</td><td>amino acid</td>

<td> 13</td><td>VL10E4</td><td>VLCDR3</td><td>amino acid</td>

<td> 14</td><td>VL100A9</td><td>VLCDR3</td><td>amino acid</td>

<td> 15</td><td>VLL100D2</td><td>VL CDR3</td><td>amino acid</td>

<td> 16</td><td>VLLOF4</td><td>VL CDR3</td><td>amino acid</td>

<td> 17</td><td>LOE5</td><td>VL CDR3</td><td>amino acid</td>

<td> 18</td><td>VLLOG7</td><td>VL CDR3</td><td>amino acid</td>

<td> 19</td><td>VLLOG9</td><td>VL CDR3</td><td>amino acid</td>

188 192 table continued

<td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td> 20</td><td>VLLOHI</td><td>VL CDR3</td><td>amino acid</td>

<td> 21</td><td>VLLOH10</td><td>VL CDR3</td><td>amino acid</td>

<td> 22</td><td>VL1B7</td><td>VL CDR3</td><td>amino acid</td>

<td> 23</td><td>VL1C1</td><td>VL CDR3</td><td>amino acid</td>

<td> 24</td><td>VLO. 1F4</td><td>VL CDR3</td><td>amino acid</td>

<td> 25</td><td>VLO. 1H8</td><td>VL CDR3</td><td>amino acid</td>

<td> 26</td><td>LOE7. AND</td><td>VL CDR3</td><td>amino acid</td>

<td> 27</td><td>2SD4</td><td>VH CDR3</td><td>amino acid</td>

<td> 28</td><td>VH1B11</td><td>VH CDR3</td><td>amino acid</td>

<td> 29</td><td>VH1D8</td><td>VH CDR3</td><td>amino acid</td>

<td> 30</td><td>VH1A11</td><td>VH CDR3</td><td>amino acid</td>

<td> 31</td><td>VH1B12</td><td>VH CDR3</td><td>amino acid</td>

<td> 32</td><td>VH1E4</td><td>VH CDR3</td><td>amino acid</td>

<td> 33</td><td>VH1F6</td><td>VH CDR3</td><td>amino acid</td>

<td> 34</td><td>3C-H2</td><td>VH CDR3</td><td>amino acid</td>

<td> 35</td><td>VH1-m. N</td><td>VH CDR3</td><td>amino acid</td>

<td> 36</td><td>D2E7</td><td>VL</td><td>nucleic acid</td>

<td> 37</td><td>D2E7</td><td>VH</td><td>nucleic acid</td>

equivalents

Those skilled in the art will recognize or be able to determine, using no more than a few routine experiments, many substitutes for specific embodiments of the invention. Such substitutes are intended to be subject to the following reservations.

188 192

SEQUENCE LIST (1) GENERAL INFORMATION:

(i) NOTIFIER:

(A) NAME: BASF Aktiengesellschaft (B) STREET: Carl-Bosch Str. 38 (C) CITY: 67056 Ludwigshafen (D) STATE: Rheinland-Pfalz (E) COUNTRY: Germany (ii) TITLE OF THE INVENTION: TNFa binding antibodies (iii ) NUMBER OF SEQUENCES: 37 (iv) ADDRESS FOR CORRESPONDENCE:

(A) ADDRESS: LAHIVE &amp; COCKFIELD (B) STREET: 60 State Street, suite 510 (C) CITY: Boston (D) STATE: Massachusetts (E) COUNTRY: USA (F) CODE: 02109-1875 (v) POSSIBLE TO READ BY THE COMPUTER:

(A) MEDIA TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OS: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Release # 1.0, Version # 1.25 (vi) DETAILS OF CURRENT APPLICATION:

(A) APPLICATION NUMBER:

(B) DATE OF NOTIFICATION:

(C) CLASSIFICATION:

(vii) DETAILS OF A PREVIOUS NOTIFICATION:

(A) APPLICATION NUMBER: US 08 / 599,226 (B) DATE OF APPLICATION: 09-FEB-1996 (C) CLASSIFICATION:

(vii) DATA ON PREVIOUS NOTIFICATION:

(A) APPLICATION NUMBER: US 60/031, 476 (B) DATE OF APPLICATION: 25-NOV-1996 (C) CLASSIFICATION:

(viii) INFORMATION ON THE NAME:

(A) NAME: DeConti, Giulio A., Jr.

(B) REGISTRATION NUMBER: 31,503 (C) CASE NUMBER: BBI-043CPPC (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617)227-7400 (B) TELEFAX: (617)227-5941 (2) INFORMATION FOR SEQ ID NO. NO. 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 107 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear

188 192 (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) OI? I ^ £ 5 SEQUENCES: IDENTIFICATOR SEQ. NO. 1:

<td>Asp 1</td><td colspan="3">Ila GGn Met</td><td>Thr 5</td><td>Gln</td><td>Cheese</td><td>Pro</td><td>Cheese</td><td>Cheese 1S</td><td colspan="2">Leu Cheese</td><td colspan="2">Ala Ser</td><td colspan="2">Val Gly 10</td>

<td>Asp</td><td>Aa.</td><td>val</td><td>T1a</td><td>How much:</td><td>RSR</td><td>ces</td><td>ar</td><td>A la</td><td>Oops!</td><td>glr</td><td>G ly</td><td>H p</td><td>TSG</td><td>own</td><td>at p</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Leu</td><td>ala</td><td>Trp</td><td>Tyr</td><td>Gln</td><td>RSN</td><td>lys</td><td>Pro</td><td>G ly</td><td>lys</td><td>APR</td><td>sro</td><td>lys</td><td>Leu</td><td>Seu</td><td>ai p</td>

<td></td><td></td><td> 30</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Tyr</td><td>Ali.</td><td>ala</td><td>ana</td><td>Thr</td><td>RSU</td><td>Gln</td><td>Cheese</td><td>Gly</td><td>val</td><td>PSE</td><td>Cheese</td><td>Ay p</td><td>pte</td><td>Cheese</td><td>ro p</td>

<td></td><td> 00</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Cheese</td><td>GGA</td><td>SSr</td><td>Gly</td><td>Thr</td><td>Asp</td><td>phe</td><td>Th.r</td><td>Leu</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Gln</td><td>Pro</td>

<td> 60</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Glu</td><td>alp</td><td>val</td><td>ala</td><td>Thr</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gln</td><td>Arg</td><td>Tyr</td><td>apt</td><td>Arg</td><td>ala</td><td>Pro</td><td>Tyr</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 90</td><td></td>

<td>Thr</td><td>PPh</td><td>Gly</td><td>Gin</td><td>Gly</td><td>TSR</td><td>lys</td><td>val</td><td>Glu</td><td>ISS</td><td>lti</td><td></td><td></td><td></td><td></td><td></td>

100 105 (2) INFORMATION FOR THE SECOND ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 121 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 2:

<td>Glu 1</td><td>Thallium</td><td>Gln</td><td>Leu</td><td>val 5</td><td>Glu</td><td>ses</td><td>Gly</td><td>Gly</td><td>Gly 1S</td><td>Leu</td><td>val</td><td>Gin</td><td>Pro</td><td>Gly 15</td><td>Arg</td>

<td>Cheese</td><td>Leu</td><td>This.</td><td>Leu twenty</td><td>Cheese</td><td>Cys</td><td>ala</td><td>AAL a</td><td>Cheese 25</td><td>Gly</td><td>plie</td><td>Thr</td><td>phe</td><td>Asp thirty</td><td>Asp</td><td>Tyr</td>

<td>ala</td><td>Underworld</td><td>His thirty</td><td>Trp</td><td>val</td><td>Arg</td><td>Gln</td><td>ala 4S</td><td>Pro</td><td>Gly</td><td>lys</td><td>Gly</td><td>Leu 45</td><td>Glu</td><td>τη?</td><td>val</td>

<td>Cheese</td><td>ala 00</td><td>How much</td><td>background</td><td>TRT</td><td>RSN</td><td>ter 5S</td><td>Gly</td><td>T Ss</td><td>How much</td><td>Asp</td><td>T yr 60</td><td>AS p</td><td>Aia</td><td>Cheese</td><td>SpS</td>

<td>Glu 60</td><td>GGA</td><td>Cord</td><td>PPe</td><td>Thr</td><td>How much 70</td><td>Cheese</td><td>Arg</td><td>Asp</td><td>own</td><td>ala 75</td><td>lys</td><td>own</td><td>Cheese</td><td>Leu</td><td>Tyr 80</td>

<td>Leu</td><td>Gln</td><td>mee</td><td>own</td><td>Cheese 80</td><td>Leu</td><td>ATg</td><td>ala</td><td>Glu</td><td>ASp £ 30</td><td>Thr</td><td>ala</td><td>val</td><td>Tyr</td><td>Tyr 90</td><td>Cym</td>

188 192

Ala Lys Val Ser Tyr Leu Cheese Thr Ala Ser Ser Leu Asp Tyr Trp Gly 100 105 HO

Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 (2) INFORMATION FOR THE SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (ix) FEATURE

(A) NAME / KEY: Modified place (B) LOCATION: 9 (D) OTHER INFORMATION: / note = Xaa to Thr or Ala (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Gln Arg Tyr Asn Arg Ala Pro Tyr Xaa 1 5 (2) INFORMATION FOR THE SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (ix) FEATURE

(A) NAME / KEY: Modified place (B) LOCATION: 12 (D) OTHER INFORMATION: / note = "Xaa is Tyr or Asn (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Val Ser Tyr Leu Cheese Thr Ala Cheese Leu Asp Xaa 15 10 (2) INFORMATION FOR THE SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide

188 192 (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Ala Ala Ser Thr Leu Gln Ser 1 5 (2) INFORMATION FOR THE SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 6:

Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu 15 10 11

Gly (2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 7:

Arg Ala Cheese Gln Gly Ile Arg Asn Tyr Leu Ala 15 10 (2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 8:

Asp Tyr Ala Met His 1 5

188 192 (2) INFORMATION FOR THE SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 107 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal

<td>(Xi)</td><td>DESCRIPTION OF THE SEQUENCE:</td><td>SEQ ID</td><td>NO: 9:</td>

<td>Asp</td><td>Ile Gln Met Thr</td><td>Gln Cheese Pro Cheese Ser</td><td>Leu Cheese Ala Cheese Ile Gly</td>

<td> 1</td><td> 5</td><td> 10</td><td> 15</td>

<td>Asp</td><td>Arg Val Thr Ile</td><td>Thr Cys Arg Ala Ser</td><td>Gln Gly Ile Arg Asn Tyr</td>

<td></td><td> 20</td><td> 25</td><td> 30</td>

<td>Leu</td><td>Ala Trp Tyr Gln</td><td>Gln Lys Pro Gly Lys</td><td>Ala Pro Lys Leu Leu Ile</td>

<td></td><td> 35</td><td> 40</td><td> 45</td>

<td>Tyr</td><td>Ala Ala Ser Thr</td><td>Leu Gln Ser Gly Val</td><td>Pro Ser Arg Phe Ser Gly</td>

<td></td><td> 50</td><td> 55</td><td> 60</td>

<td>Cheese</td><td>Gly Ser Gly Thr</td><td>Asp Phe Thr Leu Thr</td><td>How much Cheese Leu Gln Cheese Pro</td>

<td> 65</td><td></td><td> 70</td><td> 75 80</td>

<td>Glu</td><td>Asp Val Ala Thr</td><td>Tyr Tyr Cys Gln Lys</td><td>Tyr Asn Cheese Ala Pro Tyr</td>

<td></td><td> 85</td><td> 90</td><td> 95</td>

<td>ala</td><td>Phe Gly Gln Gly</td><td>Thr Lys Val Glu Ile</td><td>lys</td>

100 105 (2) INFORMATION FOR THE SEQ ID NO: 10:

<td>(and)</td><td colspan="3">SEQUENCE CHARACTERISTICS: (A) LENGTH: 121 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>(Ii)</td><td colspan="2">PARTICLE TYPE: peptide</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>(V)</td><td colspan="3">FRAGMENT TYPE: internal</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>(Xi)</td><td>DESCRIPTION OF THE SEQUENCE:</td><td colspan="3">SEQ ID</td><td>NO:</td><td> 10:</td><td></td><td></td><td></td><td></td>

<td>Gln 1</td><td>Val Gln Leu Val 5</td><td>Glu Cheese Gly</td><td>Gly</td><td>Gly 10</td><td>Leu</td><td>val</td><td>Gln</td><td>Pro</td><td>Gly 15</td><td>Arg</td>

<td>Cheese</td><td>Leu Arg Leu Cheese 20</td><td>Ala Ala's</td><td>Cheese 25</td><td>Gly</td><td>phe</td><td>Thr</td><td>phe</td><td>Asp thirty</td><td>Asp</td><td>Tyr</td>

<td>ala</td><td>Met His Trp Val 35</td><td>Arg Gln Ala 40</td><td>Pro</td><td>Gly</td><td>lys</td><td>Gly</td><td>Leu 45</td><td>Asp</td><td>Trp</td><td>val</td>

<td>Cheese</td><td>Ala Ile Thr Trp 50</td><td>Asn Ser Gly 55</td><td>His</td><td>How much</td><td>Asp</td><td>Tyr 60</td><td>ala</td><td>Asp</td><td>Cheese</td><td>val</td>

188 192

<td>Glu</td><td>Gly</td><td>tog</td><td>phe</td><td>ala</td><td>val</td><td>Cheese</td><td>Arg</td><td>Asp</td><td>A3n</td><td>ala</td><td>Lyr</td><td>own</td><td>ala</td><td>Leu</td><td>Tyr</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Leu</td><td>Gln</td><td>Underworld</td><td>own</td><td>Cheese</td><td>Leu</td><td>Arg</td><td>Pro</td><td>Glu</td><td>Asp</td><td>Thr</td><td>ala</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td>9r</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Thr</td><td>lys</td><td>ALA</td><td>Cheese</td><td>Tyr</td><td>Leu</td><td>srr</td><td>The</td><td>rer</td><td>er</td><td>Cheese</td><td>5EU</td><td>Asp</td><td>own</td><td>Trp</td><td>G 5y</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Gln</td><td>Gly</td><td>Thr</td><td>Leu</td><td>val</td><td>Thr</td><td>val</td><td>Cheese</td><td>Cheese</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

115 120 (2) INFORMATION FOR THE SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 11:

Gln Lys Tyr Asn Cheese Ala Pro Tyr Ala 1 5 (2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 12:

Gln Lys Tyr Asn Arg Ala Pro Tyr Ala 1 5 (2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal

188 192 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Gln Lys Tyr Gln Arg Ala Pro Tyr Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 14:

Gln Lys Tyr Cheese Ala Pro Tyr Cheese Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 15:

Gln Lys Tyr Asn Cheese Ala Pro Tyr Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 16:

Gln Lys Tyr Asn Arg Ala Pro Tyr Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

188 192 (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 17:

Gln Lys Tyr Asn Cheese Ala Pro Tyr Tyr 1 5 (2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 18:

Gln Lys Tyr Asn Cheese Ala Pro Tyr Asn 1 5 (2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 19:

Gln Lys Tyr Thr Ser Ala Pro Tyr Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 20:

188 192

Gln Lys Tyr Asn Arg Ala Pro Tyr Asn 1 5 (2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 21:

Gln Lys Tyr Asn Cheese Ala Ala Tyr Cheese 1 5 (2) INFORMATION FOR THE SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 22:

Gln Gln Tyr Asn Cheese Ala Pro Asp Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 23:

Gln Lys Tyr Asn Ser Asp Pro Tyr Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

188 192 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 24:

Gln Lys Tyr Ile Ser Ala Pro Tyr Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 25:

Gln Lys Tyr Asn Arg Pro Pro Tyr Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 26:

Gln Arg Tyr Asn Arg Ala Pro Tyr Ala 1 5 (2) INFORMATION FOR THE SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal &lt;xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 27:

188 192

Ala Ser Tyr Leu Cheese Thr Cheese Ser Cheese Leu Asp Asn 15 10 (2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 28:

Ala Ser Tyr Leu Cheese Thr Cheese Ser Cheese Leu Asp Lys 15 10 (2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 29:

Ala Ser Tyr Leu Cheese Thr Cheese Ser Cheese Leu Asp Tyr 15 10 (2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 30:

Ala Ser Tyr Leu Cheese Thr Cheese Cheese Leu Asp Asp 15 10 (2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids (B) TYPE: amino acid

188 192 (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 31:

Ala Cheese Tyr Leu Cheese Thr Cheese Phe Cheese Leu Asp Tyr 15 10 (2) INFORMATION FOR THE SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 32:

Ala Ser Tyr Leu Cheese Thr Cheese Ser Cheese Leu His Tyr 15 10 (2) INFORMATION FOR THE SEQ ID NO: 33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids (B, TYPE: amino acid (D, TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) RAGMENT TYPE : internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

Ala Ser Phe Leu Cheese Thr Cheese Ser Cheese Leu Glu Tyr 15 10 (2) INFORMATION FOR THE SEQ ID NO: 34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 34:

Ala Ser Tyr Leu Cheese Thr Ala Ser Ser Leu Glu Tyr 15 10

188 192 (2) INFORMATION FOR THE SEQ ID NO: 35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) PARTICLE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 35:

Val Ser Tyr Leu Cheese Thr Ala Cheese Leu Asp Asn 15 10 (2) INFORMATION FOR SEQ ID NO: 36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 321 base pairs (B) TYPE: nucleic acid (C) THREADS: double (D) TOPOLOGY: linear (ii) PARTICLE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO. NO: 36:

GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGGAGGGGA CAGAGGT ^ CC

ATCACTTGTC GAACACATCA GGGAAGACGA AATTACTTAG CAATGAGCTA CAAAACACCA

AAAGGGAAAC ATAGGATACT GCTCTCTACT GCATCCACTT TGAAGCGAGG CAATAAATGC

AAATTAAATG GACGTAAATC TGGAACAAAG TTCACTCTCA CAACTAGGAG CACGCAGAAC

AAGGCTATTG CAGCTTATGC CTGTAGCAGG TATATiCCGTG CACAAAGGGC CTTTGAACAC

AAACCCAGGA TGGAAATAAA A (2) INFORMATION FOR THE SEQ ID. NO 37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 363 base pairs (B) TYPE: nucleic acid (C) THREADS: double (D) TOPOLOGY: linear (ii) PARTICLE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

AAAGTACAAC TAATGGAGTA TAAAAAAAGA TTTTTACAGC CCAGAAGAAT CCAGAGAGAG

TCCTATAAGA CCTCTGGATT AACATTTAAT GATTATGCCA ATGAGAGAAA CCGGACAGAA

120

180

240

300

321

120

188 192

<td>CCAGGGAAGG</td><td>GCCTGGAATG</td><td>GGTCTCAGCT</td><td>ATCACTTGGA</td><td>ATAGTGGTCA</td><td>CATAGACTAT</td><td> 180</td>

<td>GCGGACTCTG</td><td>TGGAGGGCCG</td><td>ATTCACCACC</td><td>TCCAGAGACA</td><td>ACGCCCAAGGAA</td><td>CTCCCTGTAT</td><td> 240</td>

<td>CTGCAAATGA</td><td>ACAGTCTGAG</td><td>AGCTGAGGAT</td><td>ACGGCCGTAT</td><td>ATTACTGTGC</td><td>CGAAAGTCTCG</td><td> 300</td>

<td>TACCTTAGCA</td><td>CCGCGTCCTC</td><td>CCTCTACTAT</td><td>TGGGGCCCAG</td><td>GTACCCTGGT</td><td>CACCGTCTCG</td><td> 360</td>

<td>AGT</td><td></td><td></td><td></td><td></td><td></td><td> 363</td>

188 192

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<td>ABOUT</td><td>FROM</td><td> &gt;</td><td>FROM</td><td>from</td><td>FROM</td><td> &gt;</td><td> &gt;</td><td> &gt;</td><td>m</td><td> 1</td><td> 1</td><td>ω</td><td>r *</td><td>About&gt;</td><td>r *</td><td>ABOUT*</td><td>r-</td><td>about</td><td>ο-</td><td>r *</td><td>about*</td>

<td>WHAT</td><td> &gt;</td><td></td><td> &gt;</td><td> &gt;</td><td> &gt;</td><td></td><td></td><td></td><td></td><td> ^4</td><td>• H</td><td>OJ</td><td>ω</td><td>ω</td><td>at</td><td>ω</td><td>in</td><td>ω</td><td>AT]</td><td>ω</td><td>ω</td>

<td>OJ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>FROM</td><td>FROM</td><td>ABOUT</td><td>OJ</td><td>OJ</td><td>ol</td><td>OJ</td><td>OJ</td><td>OJ</td><td>OJ</td><td>OJ</td><td>ol</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> &gt;</td><td> &gt;</td><td></td><td>about</td><td>at</td><td>Q</td><td>about</td><td>about</td><td>Q</td><td>about</td><td>about</td><td>ABOUT</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>from</td><td>from</td><td>FROM</td><td>from</td><td>X</td><td>FROM</td><td>X</td><td>from</td><td>FROM</td>

188 192 sts

L&gt;

in ω

&gt;

t&gt;

j

ΕΟ σ

about

CZ) ........

IN.........

&gt;.........

E -.........

&gt;.........

J .........

E -.........

ABOUT.........

ABOUT.........

ABOUT.........

2.........

Fig-2B

<img file="PL188192B1_D0005.tif" />

H &lt;&lt;&lt;&lt;&lt;&lt;&lt;&lt;&lt; &lt;

U ........... U&gt; · ........... ► »► ► ...........&gt; &lt;

&gt;.......&lt; · · · &gt;

&lt;........... &lt;

E- · ........... EQ ...........

ω ........... ω cu ........... «χ

K ........... Oi

J ........... J

IN ........... CZ)

2 ........... Z

from ........... from θ '........... θ'

J ........... J&gt; ...........&gt;

J ........... J &lt;........... CZ)

Z ........... Z

X ........... «&lt;........... &lt;

from ........... from

Ω ........... o

X ........... ai

IN ........... CZ)&gt;

* C

Cl.

K e -.........

ω .........

«.........

<td>• r</td><td>f-i</td><td>HIM</td><td>Γ "&lt;</td><td>CN</td><td>CN</td><td>• τΤ</td><td></td><td>τΉ</td><td>CN</td><td>from</td><td></td><td colspan="2"> —4</td><td>CN</td><td>m</td><td> *3*</td><td>in</td><td>vo</td><td>r ·</td><td>What</td><td>er »</td>

<td> &gt;</td><td></td><td>Q</td><td></td><td>and K ™</td><td>about</td><td>ω</td><td>Cl.</td><td>about</td><td>X</td><td> •</td><td></td><td>X</td><td></td><td> &lt;</td><td> &lt;</td><td> &lt;</td><td> &lt;</td><td></td><td>rt</td><td> &lt;</td><td>and</td>

<td></td><td>cq</td><td> ♦-(</td><td> &lt;</td><td>m</td><td></td><td>rH</td><td></td><td></td><td> 1</td><td>CN</td><td>CN</td><td> &gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> «—&lt;</td><td>X</td><td>~ r &lt;</td><td> »—4</td><td> ♦-&lt;</td><td>X</td><td>X</td><td>X</td><td>at</td><td>Q</td><td>about</td><td></td><td> *</td><td> *</td><td> *</td><td> ♦</td><td> «</td><td> ♦</td><td> *</td><td> *</td><td> ♦</td>

<td>Q</td><td>X</td><td> &gt;</td><td>X</td><td></td><td>X</td><td> &gt;</td><td> &gt;</td><td> &gt;</td><td>m</td><td> 1</td><td> 1</td><td>r-</td><td>r *</td><td></td><td>r-</td><td>r *</td><td>Γ *</td><td>r *</td><td>r ~</td><td>Γ *</td><td>r *</td>

<td>CZL</td><td> &gt;</td><td></td><td> &gt;</td><td>and&gt;</td><td> &gt;</td><td></td><td></td><td></td><td></td><td>rH</td><td></td><td>ω</td><td>ω</td><td>ω</td><td>cd</td><td>cd</td><td>cd</td><td>ω</td><td>cd</td><td>cd</td><td>cd</td>

<td>CN</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>X</td><td>X</td><td>CN</td><td>CN</td><td>CN</td><td>CN</td><td>CN</td><td>CN</td><td>CN</td><td>CN</td><td>CN</td><td>CN</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> &gt;</td><td> &gt;</td><td>about</td><td>α</td><td>α</td><td>Q</td><td>Q</td><td>α</td><td>about</td><td>about</td><td>about</td><td>Q</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td><td>X</td>

188 192

FROM 57Ο / 63Ό nm

FIG. 3

<img file="PL188192B1_D0006.tif" />

Antibody concentration, M

D2E7-IgG4

MAK195-IgG1

2SD4-IgG1 MAK195 F (ab ') 2

188 192% inhibition

FIG. 4

<img file="PL188192B1_D0007.tif" />

D2E7-IgG4

MAK195-IgG4

2SD4-IgG4 MAK195 F (ab ') 2

188 192% inhibition

FIG. 5

<img file="PL188192B1_D0008.tif" />

Antibody concentration, M

D2E7-IgG4

MAK195-IgG1

2SD4-! GG1 MAK195 F (ab ') 2

188 192

Φ

Φ k_

<img file="PL188192B1_D0009.tif" />

σ »θ 3 σ» ιω

Cd σ&gt;!

&lt;ΙΩ ¹Continued

ΙΩ

Continued

ΙΩ σ * □

ΙΩ

Cd cn ω

οο

ο.

Cd &lt;- ο

X ο

σ «

HLD

cd

Ο

<img file="PL188192B1_D0010.tif" />

fO

CD about

X st

about

J?

ΙΩ

Φ □

188 192

D2E7 VL

GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA diqmtqspsslsasv

CDR LI

GGG GAC AGA GTC ACC ATC ACT TGT CGG GCA AGT CAG GGC ATC AGA ^GDRVT ITCRASQGIR

AAT TAC TTA GCC TGG TAT CAG CAA AAA CCA GGG AAA GCC CCT AAG

NYLA WYQQKPGKAPK

LR CDR

CTC CTG ATC TAT GCT GCA TCC ACT TTG CAA TCA GGG GTC CCA TCT

LLIY ST STLQSGVPS

CGG TTC AGT GGC AGT GGA TCT GGG ACA GAT TTC ACT CTC ACC ATC RFSG SGSGTDFTLTI

AGC AGC CTA CAG CCT GAA GAT GTT GCA ACT TAT TAC TGT CAA AGG SSLQ PEDVATYYC _Q_R_

LR CDR

TAT AAC CGT GCA CCG TAT ACT TTT GGC CAG GGG ACC AAG GTG GAA YNRA p YT FGQGTKVE

ATC AAA IK

FIGURE 7

188 192

D2E7 VH

<td colspan="4">GAG GTG CAG CTG</td><td>GTG</td><td>GAG</td><td colspan="2">TCT GGG</td><td colspan="2">GGA GGC</td><td>TTG</td><td colspan="2">GTA CAG</td><td>CCC</td><td>GGC</td>

<td>E</td><td>V</td><td>Q</td><td>L</td><td>V</td><td>E</td><td>S</td><td>G</td><td>G</td><td>G</td><td>L</td><td>V</td><td>Q</td><td>P</td><td>G</td>

<td colspan="2">AGG TCC</td><td rowspan="2">CTG L</td><td rowspan="2">AGA R</td><td rowspan="2">CTC L</td><td rowspan="2">TCC S</td><td colspan="2">TGT GCG</td><td rowspan="2">GCC AND</td><td rowspan="2">TCT S</td><td colspan="5">GGA TTC ACC TTT GAT</td>

<td>R</td><td>S</td><td>C</td><td>AND</td><td>G</td><td>F</td><td>T</td><td>F</td><td>D</td>

<td>GAT</td><td colspan="3">CDR Hl TAT GCC ATG</td><td>CAC</td><td>TGG</td><td>GTC</td><td>CGG</td><td>CAA</td><td>GCT</td><td>CCA</td><td>GGG</td><td>AAG</td><td>GGC</td><td>CTG</td>

<td></td><td>Y</td><td>AND</td><td>M</td><td>H</td><td>IN</td><td>V</td><td>R</td><td> 0</td><td>AND</td><td>P</td><td>G</td><td>K</td><td>G</td><td>L</td>

CDR H2

<td colspan="3">GAA TGG GTC</td><td>TCA</td><td colspan="2">GCT ATC</td><td>ACT</td><td colspan="4">TGG AAT AGT GGT</td><td>CAC</td><td>ATA</td><td colspan="2">GAC TAT</td>

<td>E</td><td>IN</td><td>V</td><td>S</td><td></td><td>AND</td><td>T</td><td>IN</td><td></td><td> 3</td><td>G</td><td>H</td><td>AND</td><td>P</td><td>Y</td>

<td>GCG</td><td>GAC</td><td>TCT</td><td>GTG</td><td>GAG</td><td>GGC</td><td>CGA</td><td>TTC</td><td>ACC</td><td>ATC</td><td>TCC</td><td>AGA</td><td>GAC</td><td>AAC</td><td>GCC</td>

<td>AND</td><td>D</td><td>S</td><td>-V.</td><td>E</td><td></td><td>R</td><td>F</td><td>T</td><td>AND</td><td>s</td><td>R</td><td>D</td><td>N</td><td>AND</td>

<td colspan="2">AAG AAC</td><td rowspan="2">TCC S</td><td rowspan="2">CTG L</td><td rowspan="2">TAT Y</td><td rowspan="2">CTG L</td><td colspan="8">CAA ATG AAC AGT CTG AGA GCT GAG</td><td rowspan="2">GAT D</td>

<td>K</td><td>N</td><td>Q</td><td>M</td><td>N</td><td>S</td><td>L</td><td>R</td><td>AND</td><td>E</td>

<td>ACG</td><td>GCC</td><td>GTA</td><td>TAT</td><td>TAC</td><td>TGT</td><td>GCG</td><td>AAA</td><td>GTC</td><td>TCG</td><td>TAC</td><td>CDR CTT</td><td>H3 AGC</td><td>ACC</td><td>GCG</td>

<td>T</td><td>AND</td><td>V</td><td>Y</td><td>Y</td><td>C</td><td>AND</td><td>K</td><td>V</td><td> 5</td><td>, -Y-</td><td>L</td><td>S</td><td>T</td><td>AND</td>

<td colspan="2">TCC TCC</td><td colspan="2">CTT GAC</td><td>TAT</td><td>TGG</td><td colspan="2">GGC CAA</td><td>GGT</td><td>ACC</td><td>CTG</td><td>GTC</td><td>ACC</td><td>GTC</td><td>TCG</td>

<td></td><td>--S</td><td>L</td><td>D</td><td>Y</td><td>IN</td><td>G</td><td>Q</td><td>G</td><td>T</td><td>L</td><td>V</td><td>T</td><td>V</td><td>S</td>

AGT

S

FIGURE 8

188 192

5η

FIG. 9

<img file="PL188192B1_D0011.tif" />

"Ij-_r

8 10

Mouse age (weeks)

<img file="PL188192B1_D0012.tif" />

group 7: non-transgenic group 1: without treatment group 2: saline group 6: isotype control 30 Ig / g antibody group 3: 1.5 Ig antibody / g group 4: 15 Ig antibody / g group 5: 30 Ig antibody / g

188 192

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