TWI224969B - Use of anti-gp-39 antibodies for treatment and/or reversal of lupus and lupus associated kidney disease - Google Patents

Abstract

A method of treating lupus using anti-gp39 antibodies or fragments is provided. Such treatment has been shown to reverse disease, and in particular lupus-associated kidney disease, the major killer of lupus subjects.

Description

1224969 A7 _B7 V. Description of the invention (1) Related applications The present invention relates to an antagonist-receptor, which is also referred to in the literature as CD40CR, gp39 'or a CD D 1 which is called a CD 4 O B cell antigen. 54, and soluble ligands of this receptor, including fusion molecules containing at least a portion of the CD40 protein. This is based at least in part on the discovery that a soluble CD40 / immunoglobulin fusion protein can inhibit the activation of helper τ-cell-mediated cells by binding to a novel 39 kD protein on the helper τ_ cell membrane. The present invention proposes a substantially pure CD40CR receptor; a CD 40 CR soluble ligand, including an anti-gp 39 antibody and fragments thereof, and a fusion molecule containing at least a portion of a CD 40 protein; and control and cell activation The method of action is particularly useful for treating allergic or autoimmune diseases. More specifically, the present invention relates to the use of anti-gp 39 antibodies to treat systemic erythematosus (SLE) or drug-induced lupus. Background of the Invention Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Research by Mitchison, Benacerraf, and Raff first suggested that physical interactions between Th and B-cells are necessary to develop a humoral immune response. Later research proved that τ h can form a physical conjugate with antigens and presenting B cells that are compatible with major histocompatibility complexes (M HC) (Vitetta et al. 5 Immunol. Rev., 99 : 1 93 -23 9 (1 987)) 'and in these conjugates B cells are responsive to Th (Barrett et al., J. Immunol., 143: 1 745 · 1 754 (1 989)) . The discovery that Th-derived cardiac lymphoid hormones can show strong growth and differentiation on B cells can be inferred that 'soluble factors can be released in close proximity through the activation of activated B cells through the interaction of activated eight mediators. However, molecularly-selected lymphocytes -4- this paper rule money 1224969 B7 V. Description of the invention (2) No matter whether alone or in combination, none of them can show the ability to spit the entrance of B-cell circulation. The plasma membrane fraction is different from soluble factors, which are derived from activated T h defamable B-cell circulation entrance (Η 〇dgkineta 1 ·, J · Immunol., 14 5: 2025-2034 (1 990); Noelle et al J. Immunol., 146: 1118-1124 (1991)). Studies from purified plasma membrane fractions of activated Th suggest that proteins expressed on activated Th membranes are responsible for the initial humoral immunity (Noelle et al., J. Immunol., 146: 1 1 1 8- 1 1 24 (1 99 1); Bartlett et al., J. Immunol., 1 4 5: 3 956-3 962 (1 990)) o Purified plasma membrane (pmACT) from activated Th It has been used to examine the nature of this effect (Hodgkin et al., J. Immunol., 1 45: 2025-2034 (1 990); Noelle et al., J. Immunol., 146: 1118-1124 ( 1991). PMACT is derived from activated Th, not dormant Th (PMREST), and exhibits activity that induces beta-cell circulation entrances in an antigen-unspecific, π-unrestricted manner. In addition, it has been shown that PMACT is induced by PMACT The expressed activity requires 4-6 hours of activation, re-rNA synthesis' and is essentially a protein. (Bartlett et al, J lmrnun () 1, 145: 3956-3962 (1990)). Key points of invention Intellectual property of the Ministry of Economic Affairs The invention is printed by the Bureau's Consumer Cooperative, and it is related to antagonists and receptors, called CD4CR, is the receptor of CD4B cell antigen, and the receptor Soluble ligands, including fusion molecules containing at least part of the CD40 protein. At least part of it is based on the discovery that soluble CD40 / immunoglobulin fusion proteins can inhibit helper cell mediation by binding to novel 39kD receptor proteins Β fine 1224969

V. Description of the invention (3) Cell activation (called "CD40 antagonist-receptor" CD4CR "), and the discovery of a single antibody, called MR1, directly targeting this 39kD receptor, can inhibit helper T-cell mediation B-cell activation. The present invention proposes that the CD4 OCR soluble ligand of the CD4 OCR receptor is substantially pure, including antibodies, and fusion molecules containing at least a portion of the CD40 protein; and a method for controlling the activation of B cells. In a particular embodiment of the invention, the activation of B cells in an individual can be inhibited by contacting the individual's helper T cells with a therapeutically effective dose of a soluble ligand or CD40CR. This inhibitory effect of B-cell activation is particularly useful for treating allergic or autoimmune diseases. More specifically, the present invention proposes a method for treating lupus in an individual in need thereof, such as currently having systemic lupus erythematosus, or drug-scaring lupus, or even the advanced stage of the disease process (where kidney is often observed Damage), this method is to administer a therapeutically effective dose of an anti-gp39 antibody, such as an anti-human gp39 antibody or a fragment thereof, which is disclosed in the common US special case No. 08 / 475,847, published in June 1999 7th. One advantage of the present invention is that it can intervene in non-antigen-specific immune responses. Current therapies for allergies include desensitization of specific antigens and require each patient to be tested to identify antigens related to sensitivity. It's practical 'to conduct a detailed analysis of a patient's response to each and every possible allergen is virtually impossible. Furthermore, in most autoimmune conditions, the causative antigen is usually unknown or even unrelated to the disease process. The present invention is related to antigen-non-specific CD40 / CD40CR interactions, so it is not necessary to identify antigens associated with allergies or autoimmunity. Therefore, the present invention is suitable for the treatment of -6 · This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 public love) -J--------------- ^ — (Please First note on the back of ktr t write this page)-Printed by the Consumers' Cooperative of the Intellectual Property Bureau of the Ministry of Economy 9 A7-• ---------— R7 — V. Description of the invention (4) Allergies or It is particularly beneficial in autoimmune conditions, where the immunogen is unknown, or has multiple components (for example, in pollen fever, pukamidamide can induce lupus or systemic lupus erythematosus (SLE). It also Can be used in the acute treatment of immune activation, for example, it can be used for preventive treatment. Ig Immunoglobulin mab Monoclonal antibody PMact Prepared from the plasma membrane of dormant helper T cells Prepared from the plasma membrane of dormant helper T cells PAGE Polyacrylamide gel electrophoresis rIL4 Recombinant interleukin 4 rIL5 Recombinant interleukin 5 SN Supernatant Th Helper T-cell Thi refers to 1.6, aI-Ad-restricted rabbit immunoglobulin-specific pure line (please first Read the precautions on the back v. --- Write this page) Order _ · BRIEF DESCRIPTION OF THE FIGURES 1 ′ Strain antibody and CD40-Ig on PMACT-induced B-cell RNA synthesis. Figure A: Dormant B-cells co-cultured with pmtest4pMACT (from ThI), 25 μg / ml anti-CD4, anti-LFA] or Anti-JCAM-;!, Or their respective combinations (25 μg / ml each) were added to the holes containing pm ACT 'and then [3 Η] -uracil nucleus: y: incorporated into the detection of b cells rn A Synthesis. B-Cell RN A synthesis was performed by the culture system at 4 2-48 hours. The result presented is the arithmetic mean of the secondary culture 値 + / _ s · d, and it is 5 times. China National Standard (CNS) A4 Specification (210 X 297 mm) ί Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1224969 A7 — _ B7 V. Description of the Invention (5) Representatives of Examination B. Picture: Dormant B cells were co-cultured with ThACT (·, ^^ or Yashikou) PMACT. To the culture containing Thl PMact (Spring, ▲) was added incremental CD4 0-IG (A) or control protein CD7E -Ig (+). For cultures containing Th2 PMACT (□), incrementally increasing CD40-Ig was added, and B-cell rnA synthesis was evaluated 42-48 hours after culture. The results presented are the arithmetic mean 値 + / _ s.d · for three cultures, and are representative of three such experiments 图 Figure C: Dormant cells are co-cultured with LPS (50 μg / ml) or PMact. CD40-Ig (25 µg / ml; shaded) or CD7E-Ig (25 µg / ml; solid) was added to the culture. Add A diagram to determine RNA synthesis. The result presented is the arithmetic mean 値 + Λ s.d · of three cultures, and is representative of 3 experiments. Figure 2. CD40-Ig inhibits B-cell differentiation and increases cytokinesis. Figure A. Dormant B-cells were co-cultured with PMACT, rIL4 (10 ng / ml) and rIL5 (5 ng / ml). Add CD40-Ig or CD7E-Ig (25 μg / ml) at the beginning of the culture, or 1-2 or 3 days after the culture. On the 6th day of culture, the SNs in individual wells were recovered, and IgM (_) and IgGi (Y) were quantified using an anti-isotype-specific ELISA, as described (Noelle et al ·, J · Immunol ·, 146: 1 1 1 8-1 1 24 (1 99 1)) 0 In the presence of PMact, IL4 and IL5 (without CD40-Ig added), the concentrations of IgM and IgG1 were 4.6 μg / ml and 126 nanograms / ml, respectively. In the absence of IL4 and IL5, IgM and IgGi were not detected. The results are representative of three such experiments. This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) (Ktt-Notes on the back of this page) Packing-Line-Printed by the Intellectual Property Bureau of the Ministry of Economic Affairs and Consumer Cooperatives 1224969 A7 B7 Economy Printed by the Consumer Cooperative of the Ministry of Intellectual Property Bureau. 5. Description of the invention (6) Figure B: T h 1 dormant or activated with anti-CD 3 for 16 hours, irradiated and dormant with B-cells (4x10 4 / well) Culture in the presence (10 ng / mL). 0 and 25 micrograms / ml of bamboo fungus CD40-Ig (^) or CD7E-Ig (·) were added to the culture. At 66-72 hours after incubation, the holes were supplemented with 1 · 0 u C i [3 Η] -chest, and then recovered. The dashed line indicates the response of B-cells to resting T h. The result is the arithmetic mean 値 + /-s · d · of three cultivations, and it is representative of 2 experiments. Figure 3. CD40-Ig can detect molecules that appear on activated but not dormant TB. Dormant and activated Th are recovered and incubated with the fusion protein at 4 ° C for 20 minutes. Conjugate goat anti-hIgG (25 μg / ml). Analyze at least 5000 cells / sample to determine the percentage of positive cells and μFI. The results are representative of 6 such experiments. The outline shows that c D 4 0 -1 g is bound to 0. Figure 4, CD 4 0 -1 g can immunoprecipitate 39 kD protein from activated τ h 1 lysate. Thl was dormant or activated for 16 hours with insoluble anti-CD3. [35S] -labeled proteins from dormant or activated Th are immunoprecipitated with purified antibodies or fusion proteins (1-10 μg). The gel profile is representative of 3 experiments. Figure 5. Monoclonal antibody (mab) specific to the induced 39 kd Th membrane protein can inhibit PMACT's defamatory effect on B-cell RNA synthesis. Dormant B-cells and PMACT were co-cultured with anti-sedimentation / 0, anti-CD3, CD40-Ig, or MR1 each at 10 micrograms / liter. Determine the synthesis of RNA as shown in Figure 1. The result shown is the arithmetic mean 値 + / · s.d. Of three cultures, and is representative of three such experiments. (Please read the notes on the back of lr first to write this page) Binding-, Thread · This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) 1224969 A7 _ B7 V. Description of the invention (7) 6 'MR1 and CD40-Ig were confirmed to be activated. The same molecule. Panel A: Activated Th in MR1 or control group. Fluorescent marker. To assess whether CD40-Ig and MR1 compete for binding to activated Th, varying concentrations of MR1 or control hamster ig (anti-tcr) were added together with anti-CD40 (20 μg / ml). After incubation at 4 ° C for 20 minutes, the samples were washed and co-incubated with FITC-conjugated mab anti-human IgG. The results are representative of three such experiments. Panel B: Proteins from [3 5 S] -methionine-labeled activated Th were immunoprecipitated with MR1 (10 μg / sample) or CD4-0-Ig (10 μg / sample) and PAGE And fluorescence tomography analysis, the results are representative of two such experiments. Figure 7. Binding of CD 4 0 -1 g to human cell lines. Various human τ-cell lines and biotin-labeled CD40-Ig were exposed and evaluated by flow cell count. Figure 8, A: CD40 cDNA nucleotide sequence from Stamenkovic et al., EMB O J., 8: 1 403 _ 1 4 1 0 (1 989). The transmembrane area is underlined. Panel B: An illustration of the plastids that can be used to represent CD40-Ig. The amino acid sequence at the fusion position is shown below the CD40 diagram. Figure 9. Potentials of k- (ss) DNA antibodies produced by mouse populations from different treated NZB / NZW Fi. Open circles (—0 —) represent 4 to 10 months old mice treated with MR-1; open triangles) represent mice that did not respond to the development of proteinuria and received MR-1. Solid squares represent the development of proteinuria. Is indeed a response to the acceptance of MR-1 -10- ------------- S—— (Please read the precautions on the back to write this page) Order-·-丨 Line-Ministry of Economic Affairs Printed by the Intellectual Property Bureau's Consumer Co-operative Society on a moderate rule of paper 97 cm 2 x 10 2 / (\ standard A4 S) N (C quasi-standard home country 1224969 A7-B7 V. Description of the invention (8) Rat, and Filled circles (-··-) represent untreated mice. The number 値 is the average of the three titers of each group 値 +/- SEM. Figure 10, survival of different groups of treated NZB / NZW F1 mice. Open circles (—〇—) represents 4 to 10 months old mice treated with MR-1. Open triangles represent mice that did not respond to the development of proteinuria and received MR-1. Solid squares ---) represent developmental proteinuria Responding rats receiving MR-1 'and penetrating circles (-·-) represent untreated rats. DETAILED DESCRIPTION OF THE INVENTION The present invention proposes a substantially purified CD 4 CR receptor; soluble ligands of CD 4 CR, including anti-gp 39 antibodies and fragments thereof, and fusion molecules containing CD 4 0; and Method for controlling B cell activation by using soluble ligands. For the purpose of revealing and not limiting, the detailed description of the present invention is divided into sections: (i) a ligand capable of binding to CD40CR; (ii) a method for identifying CD40CR; (iii) a purified CD40CR Preparation; (iv) the use of ligands that can be combined with CD40CR; and (v) the use of CD40CR, printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs ---------------- ^ --- (Please write this page on the back of kt first note) --line- (iii) The preparation of purified CD40CR, especially for the treatment of lupus in the front, such as systemic lupus erythematosus, or drug-induced lupus. The present invention proposes soluble ligands for CD40CR, including (1) fusion molecules containing at least one portion of CD40 protein, and (ii) antibodies or antibody fragments that specifically bind to CD40CR, or gp-39, or antigens such as CD154 known. -11- This paper size is in accordance with the Chinese National Standard (CNS) A4 (210 X 297 mm). 5. Description of the invention (9) The so-called "soluble", so L tj used in this case knows that the ligand of the invention is not long-lasting: Binding to the plasma membrane of the cell, however, the soluble ligands of the present invention can be immobilized on a solid phase carrier of the cell, including lipid, protein or carbohydrate molecules 'beads' vesicles, magnetic particles, 蝓 ^? 、, 减 维 寺Or, it can be closed in implants or vesicles. The ability of this ligand to bind to CD 4 0 c R, can be, π ^ mouth) This knife can be bound to the same protein, such as CD40-Ig (? (Infra) or MR1 (infra), or another antibody that binds to amidine, as disclosed in the commonly-owned US Special No. 2008 / 475,847. The ligands of the invention may be included with a suitable carrier In a pharmaceutical composition. The present invention proposes a soluble fusion molecule that is a ligand of CD40CR. This fusion molecule contains at least a portion of the CD40 protein and adheres to a second molecule. The portion of CD40 preferably lacks a transmembrane region. CDs that can be used according to the invention The 40 protein portion is defined as any portion that can bind to (:) 〇4 () (:: 11, as shown to bind to the same protein as MR1 or CD40-Ig. A second molecule that can be used Including peptides and proteins, lipids and carbohydrates, and in a preferred embodiment of the present invention, it may be an immunoglobulin molecule, or a portion thereof (such as Fv, Fab, F (ab,) 2, or Fab1 fragment) or CD8, or another adhesion molecule, such as B7. The second molecule can be derived from non-human or human sources, or can be chimeric. The second molecule can also be an enzyme, a toxin 'growth factor', a lymphoid hormone, and antiproliferative Agents, alkylating agents, anti-metabolites, antibiotics' Changchun bioassay, complexes with platinum and chromium, radioisotopes, or fluorescent compounds. The fusion molecules of the present invention can be produced by chemical synthesis, or preferably using heavy

This paper rule is based on the Standard of Family Closed (CNS) A4 (210 X 297 printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1224969 A7 _ B7 V. Description of the invention (10) Group DNA technology. For example, encode at least one The CD40 protein nucleic acid sequence can be combined to encode a second molecule nucleic acid sequence and expressed in a suitable expression vector and then in a prokaryotic or preferably a prion nuclear expression system, such as yeast, baculovirus, or mammalian expression Including the introduction of foreign genetic animals. In addition, at least part of the CD40 protein can be expressed using electrophoresis technology, or affinity chromatography can be performed using ligands that bind to CD40 or a second molecule. The ligands that can bind to CD40 include the following, such as · · Anti-CD40 antibodies, such as G28-5, are produced by fusion tumors stored in ATCC with the number HB91 10, and CD40CR are described below. If the second molecule is an immunoglobulin or an immunoglobulin fragment, it can be used to contain Anti-immunoglobulin antibody affinity column; if the second molecule contains an Fc fragment, a protein A column can be used. According to a preferred embodiment of the present invention, the nucleic acid sequence can be used in the CD40 portion. This nucleic acid sequence can be produced by hydrolysing a plastid containing cDN A encoding a CD40 antigen, such as Stamenkovic et al., EMB OJ, 8: 1403-1410 (1 9 8 9), using ps 11 (P) and S au 3 A (S 3) restriction enzymes. The resulting P / S 3 fragment was then sub-bred to the used p And BamHI (B) are hydrolyzed by the same plastid to generate a truncated CD40 gene (see Figure 8). In particular, but not limited to, in the specific example of the present invention, it is used to generate a CD40 containing at least a portion And the expression vector of the ligand of the immunoglobulin sequence may preferably contain a virus-derived replication source, a bacterial replication source, a bacterial selective flag, a nuclear promoter and an enhancer sequence, and an immunoglobulin-fixing region. DNA sequences are separated by restricted positions, so that they can be substituted. 13- This paper size applies Chinese National Standard (CNS) A4 (210 X 297 mm). --- (Please read the winter precautions on the back of tr first I write this page) 1224969 A7 A --------- B7_ V. Description of the invention (11) -Part of the DNA sequence of CD40 is U, followed by the polyadenylation signal sequence (see Figure 8 · b). — — — — — — — — — — — — ^ -11 (please read 5 * on the back first) I note H write this page) In a specific embodiment of the present invention, the CD ^ gene can be subclassed to the immunoglobulin fusion plastid, such as Aruff〇et aL, CeH. 6 1.1 3 03- As described in 1 3 1 3 (1 990), it was hydrolyzed with MluI & B to form a plastid pCD40-Ig, which encodes a fusion molecule (: £) 40-1§ (see Figure 8). The CD40-Ig fusion protein is then produced by transducing pCD40_Ig plastids into eOs cells, forming a transitional expression system. The generated c D 4 〇_ 丨 g can be collected from the cos cell supernatant and the protein described in Aruff〇et al ·, Cell, 1 6 1: 1 3 03-1 3 1 3 (1 990) Purification by eight-column chromatography. Thread. The soluble ligand of the present invention preferably contains an antibody molecule, a monoclonal antibody molecule 'or fragments of these antibody molecules, which contain antigen-combination sites, which can cater to, preferably human gp39. This ligand may further contain a second molecule, which may be a protein, lipid, carbohydrate, enzyme 'toxin, growth factor, lymphoid hormone, antiproliferative agent, alkylating agent, antimetabolite, antibiotic, vinca alkaloid, platinum Complexes of complexes, radioisotopes, or glorious compounds can also be linked to antibody molecules or fragments. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. When the ligand is a monoclonal antibody, or a fragment thereof, the monoclonal antibody can be made to antagonize CD 4 0 CR (gp 3 9) by any technique. Cell lines can be used for the production of antibody molecules. For example, fusion tumor technology first developed by Kohler and Milstein, 1975, Nature, 256: 495-497), and other technologies that are more commonly used, such as the human B-cell fusion tumor technology (Kozbar et al., 19) 83, Immunology Today, 4:72)) and EBV · fusion tumor technology 'to generate human monoclonal antibodies (c0ie et -14- This paper is in accordance with China National Standard (CNS) A4 (210 X 297 mm) 1224969 A7 one

V. Description of the Invention (I2) a1 ·, 1 98 5, Monoclonal Antibodies and Cancer

Therapy, Alan R. Liss, Inc., pp 77-96) and so on are all within the scope of the present invention !! ^ i — (ktr back I note first write this page). Humanized and chimeric anti-gp3 9 antibodies are generally preferred because they are less likely to cause a defamatory immunogen response (HAM A response). Antibody fragments containing molecular isotypes can be produced by known techniques. For example, this fragment may contain the following, but is not limited to this: F (ab, h fragment, which can be produced by pepsin treatment of antibody molecules; Fab 'fragment, which can be produced by reducing the p (ab,) two-piece bi-stone tile bridge ; F (ab1) 2 fragment, which can be produced by pepsin treatment of antibody molecules; and 2Fab or Fab fragment, which can be produced by pepsin treatment of antibody molecules and reduction of disulfide bridges by reducing agents. As shown above, the present invention also proposes the Chimeric or human antibodies made by known techniques, as shown in Morrison et al, Proc Natl. Aead

Sci · U.S_A_ 81: 6 8 5 1 -6 8 5 5 (1 984) or European Patent No. 085 3 05604,2, publication number: No 0 1 73494, Morrison et al published in March 1986 3 5th. -· Line. Immunogens for antibody production produced by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economics can be from any source containing CD 40 CR. For example, activated Th, such as activated human Th cells can act as an immunogen. In addition, the substantially purified C D 4 0 C R is prepared as shown in 5 · 3 and can be used. If activated T h is used as the immunogen, antisera can be tested to antagonize the reactivity of activated (non-dormant) Th cells. Meanwhile, the immunogen may include recombinant gp39 or a fragment thereof. In this regard, D N A 's encoding murine and human g P 3 9 have been selected and expressed by recombinant methods. These proteins provide a potential immunogen that can produce anti-gp39 antibodies. 0 -15- This paper is in accordance with China National Standard (CNS) A4 (21〇χ 297 mm) 1224969 A7-B7 A7 V. Description of the invention In a preferred embodiment of the present invention, the soluble ligand is an anti-human gp39 monoclonal antibody 'and preferably a humanized or chimeric anti-human gp39 antibody. The following method can be used to generate! ^ 1 monoclonal antibody, which can specifically bind to §1) 39 'and can be used to generate other antibodies relative to CD4 OCR. The hamsters were immunized intraperitoneally with 5-106 activated Thi cells (D1.6) for 6 weeks at weekly intervals. When the serum titer of antagonizing murine Th 1 was greater than about 1: 10,000, cell fusion was performed with polyethylene glycol, and hamster spleen cells and N SI were used for immunization. Flowing cell counts were screened on dormant and activated Th, containing Sn in fused tumor holes that were growing. A special fusion tumor was further tested, which produced Thimab with selective confirmation of activation, and was subsequently cloned to derive MR1. MR 1 was produced in ascites and purified by ion exchange HPLC. In addition, and better, antibodies against human gp 39 can be prepared according to U.S. Patent No. 08 / 475,847, which case is incorporated herein by reference. The present invention also proposes a ligand containing a monoclonal antibody, and a fragment thereof, which can competitively inhibit the binding of MR1 to its target antigen or CD40-Ig to its receptor. CD4 0CR is characterized by (i) its ability to bind to CD40, fusion molecules containing at least a portion of CD40, and antibodies such as MR1; (ii) its functional properties that can stimulate B-cell circulation entrance, proliferation, and differentiation, And (丨 丨 丨) its cell distribution. CD40CR is characterized by its ability to bind to ligands such as CD40, fusion molecules containing CD40, and antibodies that antagonize CD40CR. As detailed below, a number of techniques can be used to identify CD 40 C R. For example, CD40-Ig and MR1 have been shown to confirm the same 39 kD molecule. It was found — 1 — — — — — — — — — — 11 (please read first — note on the back t write this page) Order: • Line • Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs -16-

1224969 A7---- B7 V. Explanation of the invention (14) Both CD40-Ig and MR 1 can immunoprecipitate 39kD protein from radiolabeled Th lysates (Figure 5b). Furthermore, 39kD protein was immunoprecipitated with CD40-Ig to remove the antigen that can be confirmed by MR 1 from Th lysate. CD4 OCR can also be identified for its ability to stimulate B · cell circulation entrance, proliferation, and differentiation. For example, it was found that the plasma membrane (PM) from activated (PMact) non-resting (PMrest) Th cells can induce B-cell RNA synthesis (Figure 1a); this defensive effect can be used as an indicator of B cell activation, and Not affected by antibodies such as anti-l FA-1, anti-CD4, anti-ICAM-1. However, CD40-Ig or MR1 can inhibit the activation of B cells induced by PMACT, as shown in Figure 1b and Figure 6. 0 B cell activation can also be detected using the following techniques, such as [3h] -uracil Incorporating RNA (such as B-cell differentiation, increased RNA synthesis), or MΗ] -thoracic inclusion, can detect DNA synthesis related to cell proliferation. For optimal detection of CD40CR in B cell proliferation, melanin-4 (IL-4) can be added to the culture medium at a concentration of about 10 ng / ml. In addition, B cell activation can be detected by the function of immunoglobulin secretion. For example, CD40CR, in a substantially purified form, or presented in pm, or another form, can be added to dormant B with IL-4 (10 ng / ml) and lL-5 (5 ng / ml). Cell. After 3 days of culture, additional amounts of medium can be added. On the 6th day of culture, supernatants from individual culture media can be recovered and IgM and Igl can be quantified as described in Noelle et al., J. Immunol., 146: 1 1 18-1 124 (1991). C D 40 C R can also be identified by its cell distribution. For example, it can be observed that • 17- This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) i !! ^ i — (Please read the 0 Cautions on the back first to write this page) • Line · Ministry of Economy Printed by the Intellectual Property Bureau Staff Consumer Cooperative 1224969 V. Description of Invention (15) CD40-Ig can be combined with activated, non-dormant Thl, as assessed by flow cell counts (Figure 3). Furthermore, CD4G_Ig can bind to bkat cells, HSB2 cells, and activated τ-cells (from human peripheral blood), but does not seem to bind significantly to CEM cells, HPBALL cells, or murine thyroid tumor cells. For example, without limitation, the presence of CD40CR on a specific cell type (π test cell ") can be evaluated as a flow cell count as follows. Test cells can be tested in parallel with dormant (negative control) and activated (positive control) Th cells. All cells were co-incubated with a ligand (such as CD40-Ig or MR1) for 20 minutes at a concentration of about 1 x 105 cells per 50 microliters, plus a fitc_conjugated anti-ligand antibody. Propidium iodide can be added to all samples to a final concentration of 2 μg / ml. Flow cell count analysis is then performed, such as on bd FACSCAN. Positive and negative scatter of cells and red electronegativity (for propidium iodide exclusion) confirm the logarithmic greenness of viable cells. In the present invention, the CD40CR is purified on a qualitative basis. This CD40CR can be prepared from cells exposed with CD40CR, such as activated helper τ cells, Jurkat and HS B 2 cells, using the following method. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs — — — — — — — — — — — — I — · III. Suitable cells, such as purified Th 丨 cells, use

Discontinuous gradient sedimentation of sucrose as described by Noelle et al., J. Immunol. 1 46: 1 1 1 8- 1 1 24 (1 99 1). CD4 0Cr can be separated by using a mild detergent to dissociate the crude membrane extract, and then subjected to size exclusion chromatography, followed by affinity chromatography, using a suitable ligand (such as MR1 or CD40- Ig), immunoprecipitation (such as the use of CD4 (MgsMR1) and / 18) This paper size applies Chinese National Standard (CNS) A4 specifications (210 X 297 public love) 1224969 A7 _ _ B7 V. Description of the invention (π) or gel Electrophoresis. The resulting protein is expected to have a molecular weight of about 39 kD. The present invention proposes a soluble CD4OCR (ie, cell-free) that can be included in a medical composition together with a suitable carrier. CD40CR is further proposed herein It is linked to a second molecule, which can be a peptide, protein, lipid, carbohydrate, enzyme, toxin, growth factor, lymphoid hormone, antiproliferative agent, alkylating agent, antimetabolite, antibiotic, vinca alkaloid, palladium Composite compounds, radioisotopes, or fluorescent compounds. The present invention further proposes a substantially purified CD 4 0 CR, which can be prepared using chemical synthesis or recombinant DNA technology. For example, the genes of CD4 OCR The c DNA prepared from activated helper T cells was embedded; the L g 11 0 expression line was isolated, and then screened by MR1 or CD40-Ig binding to identify CD 4 0 CR · expression pure lines. In addition, prepared from The cDNA of activated helper τ cells can be transfected into COS cells, and the supernatant can be screened with MR1 or CD40-Ig to identify the producer of CD40CR. The CD40CR gene is then used to express CD using known expression lines in the art 40 CR. The present invention proposes a method for controlling B-cell activation, in which a ligand capable of binding to CD4 OCR is used. In particular, it proposes to inhibit the friend of B-cell activation. This method includes combining B-cell and T The mixture of h cells is exposed to an effective concentration of a ligand capable of binding to CD 40 CR. The available ligands are described in paragraph 5.1 above. The method of the present invention can be carried out in a test tube or in a liquid. Effective concentration Refers to the concentration of ligands that can inhibit B-cell activation, as detected by any technique known in the art (including those shown in 5.2), which inhibits at least about 30%, and preferably about 75%. According to the present invention, Good, specific and non-limiting example, c D 4 0 -1 g -1 9- This paper size is in accordance with Chinese National Standard (CNS) A4 specification (210 X 297 issued) -------------- I --- (please read ^ Notes on the back side first to write this) Page) Order: • Line-Printed by Employee Consumer Cooperatives, Intellectual Property Bureau, Ministry of Economy 1224969

V. Description of the invention (π) — — — — — — — — — — — I I (please note on the back of this page-notes on this page) may not be used as a ligand, and the effective concentration can be at least about 10 micrograms / ml. In another specific and non-limiting specific example, the monoclonal antibody MR1 may be used, wherein the effective concentration may be at least about 10 µg / ml. If the method is performed in vivo, the effective concentration of the ligand may refer to the plasma concentration or local concentration of the ligand. For example, it is hoped that B-cell activation can be inhibited in localized areas to limit the overall effect on the immune system. In a specific embodiment, the present invention proposes a method for treating an individual suffering from dysfunction associated with B_cell activation, which method comprises administering to the individual a therapeutically effective dose of a ligand that can bind to CD40CR. The individual may be non-human, or preferably human. B-cell activation related disorders include, for example, allergic reactions (including allergies); autoimmune conditions include drug-induced lupus, systemic lupus erythematosus, adult rheumatoid arthritis, juvenile rheumatoid arthritis, hard Dermatosis, Sjogren's syndrome; and viral diseases involving B cells, including eb infections, and retroviral infections, including human immunodeficiency virus infections. --Line--Because B cell activation has been suggested to be related to the induction of human immunodeficiency virus replication from the incubation period, it is hoped that the ligands of the present invention can be administered to HI V positive individuals who have not yet developed AIDS or ARC. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, as discussed above, and described in the examples, special applications of anti-gp 3 9 antibodies or fragments thereof include treatment of lupus induced by drugs, or systemic lupus erythematosus . It has been found that, as described in experiments in basic data and examples, anti-gp39 antibody therapy can reduce the production of autoantibodies and kidney disease, resulting in prolonged survival in NZB / NZW, which is an acceptable animal for human SLE mode. Furthermore, it has been further shown in mice that it can be used for the development of 2-3 + proteinuria (live-20- this paper size applies Chinese National Standard (CNS) A4 specifications (210 X 297 mm) 1224969 A7-_ B7 Ministry of Economy Printed by the Intellectual Property Bureau's Consumer Cooperatives V. Description of the Invention (18) Lupus Disease Status) where the administration of anti-gp 3 9 antibody can indeed reverse the disease (the survival is prolonged after the antibody is injected and no proteinuria can be injected) know). Therefore, treatment with anti-gp39 antibodies has shown to reverse the course of lupus after developing nephropathy in an animal model that is acceptable for human lupus. This confirms the ability of anti-gp 3 9 antibodies to be used in human therapeutic applications in the treatment of lupus and other autoimmune diseases. In particular, it confirms that the antibody can be used to treat individuals with an actively ongoing disease, even at a more advanced stage of the epidemic. This therapy is effective and potentially even reverses the course of the disease, such as the kidney damage often caused in patients with lupus. The ligand can be administered in a suitable pharmaceutical carrier using methods well known in the art, including intravenous, intraperitoneal, subcutaneous, intrathecal, intra-articular or intramuscular injection, and oral, intranasal, intraocular and It is administered transanally and may be contained in microsphere 'liposomes and / or sustained release implants. A therapeutically effective dose of a ligand is defined as a dose that can significantly reduce the clinical effect of B-cell activation or poor T-cell activation, and can depend on the ligand used and the condition being treated. If CD40-ig is used, the therapeutic concentration can be about 0 μg / ml systemically (plasma concentration) or locally. If MR1 or another anti-gp39 antibody is used, such as a humanized or chimeric anti-human gp 39 antibody or a fragment thereof, the therapeutic concentration may be about 10 micrograms / ml systemically (plasma concentration) or locally. In a further specific example, the above method can utilize a ligand containing a toxin or an antimetabolite, so that T h cells can be killed or damaged, and B cell activation can be increased due to the destruction of T h cells. . The ligands of the present invention can also be used to label activated T cells. This technology can be used -21-(Please read the back page first) ij ♦ -Γ _ This paper size applies to China National Standard (CNS) A4 specifications (210 χ 297 mm) 1224969 A7 one

Description of the Invention (I9 Printed on the diagnosis of T cell disorders by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. At this point, it contains enzymes such as zygote 4, radioisotope, fluorescent compounds or other detectable marker ligands In the test tube, J is exposed to T cells in vivo, and the binding amount is quantified. The ligand of the present invention can also be used to deliver substance T-cells. The present invention proposes a method for controlling B cell activation by CD40CR or CD40CR-containing molecular B-cell activation method. This method includes exposing B cells to a CD4 0CR that is effective. This method can be carried out in vivo or in a test tube. Effective means that it can be used to defy B. Receptor concentration for cell activation, > Any technology known in the breadth field technology detection, at least about 30%, in specific and non-limiting, specific examples of the present invention, the CD40CR concentration may be about 1% local or systemic 〇 微 克克。 In a specific embodiment, the present invention proposes a method for treating an individual suffering from an immunodeficiency disorder associated with humoral immunity, which method comprises administering to the individual A dose of CD4CR. The individual can be non-human 'or preferably a human. Immunodeficiency disorders associated with weakened humoral immunity include, for example, acquired immunodeficiency caused by chemotherapy or radiation therapy, as well as those involving Disorders of humoral immunity. ^ CD4 0CR can be administered in a suitable pharmaceutical carrier by any method known in the art, including intravenous, intraperitoneal, subcutaneous, slightly intra-articular, or intramuscular. Injected, orally, intranasally, intraocularly, and transanally, and can be included in microspheres, liposomes, and / or sustained-release implants, such as growth factors, to activate them using the special features described above. In other words, it proposes to promote ---- I--i_ ^ i — please ^^ the back ^ notes on this page) Ί-Τ line-this paper rule fresh-22- 1224969 A7-___ B7 V. Description of the invention (20) The therapeutic dose of CD40CR can be defined as an amount that can increase the production of immunoglobulin by at least about 30%. In a further specific example, CD40CR can be conjugated to a toxin and administered to an individual under conditions that can differentially destroy B-cells that exhibit CD40. Examples of this condition include patients undergoing organ transplantation or suffering from multiple myeloma or another B-cell disease, or autoimmune disease. C D 4 0 C R can also be used to label B cells expressing c D 40. This technique can be used to diagnose B-cell disorders. Here, the receptors linked to enzymes, radioisotopes, fluorescent compounds or other detectable markers can be exposed to B cells in a liquid or test tube, and the amount of binding can be quantified. CD40CR can also be used to deliver molecules where it is linked to B-cells. Example 1 A novel receptor on activated helper T cells, c D 4 0 CR, can bind to CD 4 0 and can transduce signals for homologous activation materials and methods for B cells. Animals use DBA / 2J Mice (Jackson Laboratories, Bar Harbor, ME) prepared stuffed cells to support the growth of Th pure lines, and could be used to prepare dormant B-cells. The Consumer Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs printed D1.6, an I-Ad-restricted rabbit Ig-specific Thl pure line / kurt-Jones et al., J. Exp. Med., 1 6 6: 1 774 -1 787 (1 987)) from Worcester Massachusetts University, Dr. David Porker. D1.6 is referred to herein as Thl.

Th 1 (8x1 06 / holes) coated with 40 μg / 4 μl of PBS / holes -23- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) 1224969 A7 One B7 Five 2. Description of the invention (21) The clustered holes (6 holes, Corning, NY) were incubated with anti-CD3 for 16 hours, as described by Noelle et al (J. Immunoi, 146: 1118-! Ιι !! ^ · Ί (please note on the back of B3't 0 before I write this page) 1 124 (1 99 1) ”Plasma prepared by discontinuous glucose gradient sedimentation method, as described in Noelle et al. (Above) The dormant splenic B cells were prepared by sedimentation on a discontinuous Perc 11 gradient, as described by Defranco et al., (J. Exp. Med. 1 5 5: 1 523 (1 982)). Isolation From 70-75% (density of 1.087-1.097) cells at the Percoll interface typically have> 95% mlg +, with uniform and low-level almost forward light scanning, and do not respond to Con A. The following Mabs were purified from mouse ascites with ion exchange and PLC, which were irradiated and bone marrow recombination: anti-CD3] 45-2C.ll (Leo et al., Proc. Natl. Acad. Sci. USA, 84: 1 3 74- 1 3 78 (1 987); -Han, / ?: H57-597; anti-CD4; GK1.5 (Wilde et al., J. Immunol., 1 3 1: 2 1 7 8-2 1 8 3 (1 9 8 3); anti- 1 CAM: YN 1 / line · 1.7.4 (Prieto et al ·, Eur. J. Immunol ·, 19: 1551-1 557 (1 989)): Anti-LFA-lrFD 441.8 (Sarmiento et al ·, Immunol Rev 6 8: 1 3 5 (1 9 8 2)); and anti-rabbit / hamster K-chain: RG-7 (Springer, Hybrid, 1: 257-273 (1 982)). Employees of Intellectual Property Bureau of the Ministry of Economic Affairs employee consumption The CD40 fusion protein printed by the cooperative is prepared by using the restriction enzymes Pst im and S au.3 A (S3) to hydrolyze a plastid containing cDN A encoding CD40 antigen (Stamenkovic and Seed ·, EMBO J ·, 8: 1 403-1 4 1 0 (1 989)). This P / S 3 fragment was then subcultured to the same plastids that hydrolyzed. In this way, CD 4 0 △ can be prepared, and its coding is from the upstream of the film-passing area. • 24- This paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm) ο 6 9 24 12) A7 B7 V. Description of the invention ( 22) The truncated CD40 protein. The DNA fragment encoding CD40 △ was then subcultured to immunoglobulin fusion plastids (Aruffo et al., Cell, 61: 1 3 03-1 3 1 3 (1 990)) and hydrolyzed with Mlul and B. The production of the CD40-Ig fusion protein was transduced in cos cells and purified on a protein A column as described in Aruffo et al (Cell, 6 1: 1 3 03-1 3 1 3 (1990)). Interleukin 4 (IL4): Recombinant mouse IL4 was generously provided by Drs. C. Maliszewski and K. Grabstein, Immunex Corporation, Seattle, WA. Interleukin 5 (IL5): Recombinant mouse IL5 is from R & D Research, Sarrento, CA 0 3 x 1 0 4 Dormant B-cells are cultured in 50 μl of cRPMI in A / 2 microtiter wells ( Costar, Cambridge, MA). In these holes, add 0.5 micrograms of Thl or 1 \ 2 membrane protein. From 42-48 hours, the holes are supplemented with 2.5yCi ~-Urine taste dying ... England Nuclear, Boston, MA), recovered, and then the radioactivity is determined by the liquid flash spectrophotometry. Results are expressed as cpm / culture +/- s.d. Dormant B-cells are cultured as described above. 0.5 μg of Thl membrane protein, IL4 (10 ng / ml) and IL5 (5 ng / ml) were added to the culture wells. On day 3 of the culture, an additional 50 μl of cRPMI was added. On the sixth day of culture, SNs from individual wells were recovered, and IgM and IgGi were quantified, as described by Noelle et al. (J. Immunol., 146: 1118-1124 (1 9 9 1). 4x1 04 dormant Beta · 50 μl of cell culture in A / 2 microtiter pores -25- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) Please read first and read on the back---Precautions page Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economics, printed 1224969 A7 V. Description of the invention (23) In cRPMI (Costar, Cambridge, MA). Add 1 X 1 0 4 dormant or activated, irradiated ( 50,000 Ray) Ding Bu 丨 and IL4 (10 nanograms / liter). On the third day of culture, the holes were supplemented with 3H-thymidine of iu, as described above (NoeUe et al., (1991) j Immunol. 146: 1118-1124). Hamsters were immunized intraperitoneally with 5-10 \ 106 activated butane 111 (〇1.6) at a weekly interval for a total of 6 weeks. When antagonizing the serum effect of murine Th 1 Valence is greater than 1: 1 0, 0 0, 0, PEGylated hamster spleen cells and N s 丨 for cell fusion with polyethylene glycol. Self-contained gradual fusion SN was screened in the hole of the cell, and the flow cell count was performed on the dormant and activated Th 1. One of the special fusion tumors produced a mab that can selectively activate activated T h. Further testing and subsequent selection Colony to derive MR1. MR1 was generated in ascites and purified by ion-exchange HPLC. Dormant and activated Th (16 hours with anti-CD3) were recovered by line, and lxl05 cells / 50 microliters with fusion protein at 40%. Incubate for 20 minutes at C, followed by FITC_conjugated goat anti-human (h) Ig (J (25 μg / ml;

Southern Biotechnology, Birmingham, AL). Propionium iodide was added to all samples printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs at a final concentration of 2 μg / ml. Flow cell fluorescence analysis was performed on BOFSCSCAN. The positive and lateral scatter of the cells and the red electronegativity (used to break down the propionate exclusion effect) confirm the logarithmic green fluorescence of viable cells. To determine the percentage of positive cells and MFI, at least 5,000 viable cells must be analyzed. MR1, a mouse anti-rat / hamster k-key mab, was stained with FITC. Thl is dormant or activated with insoluble anti-Cd3 for 16 hours. From Hibernate-26- This paper size applies the Chinese National Standard (CNS) A4 specification (21〇χ 297 mm) 1224969 A7 B7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the invention (24 and activated 1 The protein (20/106 / ml) was labeled with 1111 (: [358] -methionine / cysteine for 1 hour, at this time it was washed twice with rpmi / 10% FCs. The cell mass is then lysed with an extraction buffer solution, as described above (NoeUe et al., (1 986) J. Immunol. 1 3 7: 1 7 1 8-1 726). The purified antibody or fusion protein (1 -10 μg) was added to 500 microliters of lysate (5 X 106 cell equivalents) at 4 ° C for 16 hours. At this time, the lysate was transferred to 50 microliters of tightly packed lysate. Protein A-Sepharose in test tube. Agglomerated protein A-Sepharose was resuspended, the tube was incubated at 4X: for 1 hour, and dropped to stir. The sample was washed with a high urgency washing buffer solution. Protein A was formed -Sepharose was resuspended in 30 μl of SDS sample buffer solution and performed on a 10% polyamidamine gel. After the gel was run, the gel was Fix and then perform fluorescence analysis. In order to define the cell surface molecules that can mediate PMACT-B cell cycle entrance defensive effect, add mabs of Th membrane protein to PMACT and B-cell culture. PMACT deflects B cell RNA synthesis compared 8 times more PMREST (Figure 1a). Adding anti-LF Α · 1, anti-CD4, anti-IC AM_1 alone, or in combination, cannot defame MPAC1 ^ iB cell RNA synthesis. In human systems, Anti-CD 4 0 mab has been shown to mediate B-cell proliferation (clark and Lane, (1991) Ann. Rev. Immunol, 9: 97-127), meaning that CD40 is an important promoter molecule for B-cells. In order to determine whether CD40 is involved in PMACT.B cell RNA synthesis, soluble extracellular region of human CD40 and human IgG, Fc part of soluble fusion protein (CD40-Ig) are added to PMACT and B cell culture. Can be prepared PMACT derived from Thl and Th2 and used to stimulate RNA synthesis of B cells-27- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) (Please read the precautions on the back to write this page )-Installation • Thread. 1224969 A7 -B7 V. Description of the Invention (25). Was added CD40-Ig, may result in inhibition of B-cell RNA synthesis by an amount of the agent, which is turned up by the abusive Thl and Th2 of PMACT from about 5 [mu] g / [mu] l CD40-Ig. The CD7E-Ig fusion protein (Damle and Aruffo, (1991) Proc. Natl. Acad. USA 88: 6403-6407) has no effect even when used up to 25 μg / ml. To examine whether CD4 0-Ig can inhibit the activation of B cells by T-independent activators, B-cells were cultured in the presence of LPs and CD40-Ig. On day 2, the synthesis of RN A was evaluated (Figure 1c). C D 4 0 · I g has no effect on L P S inhibiting B cell activation, but it can inhibit B cell response to P M A c τ. In the presence of PMACT, IL4 and IL-5, B cells can differentiate into multiple strains to produce Ig (Hodgkin et al., (1 990), J. Immunol. 1 45: 2025-2034; Noelle et al. 5 (1991) J. Immunol. 146: 1118-1124). To evaluate the prerequisites for CD40 communication during this process, CD40-Ig was added at the beginning of the culture or the next day. Adding CD40-Ig (Figure 2a) at the beginning of the culture can inhibit more than 95% of various IgM and IgG! Production, compared with the control level in the absence of it. In contrast, the addition of CD40-Ig on days 1 and 2 of the culture had little, if any, inhibitory effect on the production of IgM and IgG1. These data show that the communication via CD40 after 24 hours is no longer necessary for B-cell differentiation to Ig secretion. Data to date indicate that CD40 is involved in the activation of PMAC1 ^ iB cells. Studies have been performed to determine that CD 40 is also involved in the activation of B cells by intact, viable, activated Th. Th 1 was activated with insoluble anti-CD 3 for 16 hours, recovered and irradiated. The irradiated Th1 was co-cultured with B-cells in the presence of IL4, and the proliferation of B-cells was determined on the 3rd day of the culture. To reach -28- This paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) -------------- Ge --- (Please write on the back 6 notes (This page) Order: • Line. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economy 1224969 A7

V. Explanation of the invention (26) To T h 1 on the proliferation of B cells, an external source of IL 4 is necessary because Thl does not produce IL 4 (Noelle et al., (1 989) J. Immunol, 1 43: 1 807- 1 8 1 4). CD40-Ig inhibits irradiated

Th defies B-cell proliferation in a dose-dependent manner, similar to that observed in pm act (Figure 2b). The negative control group, c D 7 E -1 g, had no detectable effect. To examine whether activated T h 1 could show binding to c D 4 0, dormant and activated (16 hours) T h 1 was stained with CD 4 0 -1 g or CD 7 E -1 g. Take FIT C -Anti-H ig G. The combination of CD 4 0 -1 g was evaluated by flow cell count (Figure 3). T h 1 (16 hours with anti-C D 3 activation) instead of dormant T h 1 was 56% positive with CD 4 0 -1 g staining, but not c D 7 E · I g in the control group. In order to identify the CD4 0-Ig binding protein, the Thl protein was labeled with [35s] -sulfanine / cysteine biosynthesis, and the protein was immunoprecipitated with CD40-Ig or CD7E-Ig. The immunoprecipitated proteins were analyzed by S DS-PA G E fluorescence analysis (Figure 4). One main band has a 39 kD expressed molecular weight, and one low molecular weight band, B2-microglobulin. In the absence of mab, the main band has not been seen. The 39kd band can also be immunoprecipitated from activated Th, which is marked with on the carrier, confirming that the 39kD protein is a membrane protein. The Mabs printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs and specific antigens that selectively express on activated and dormant Th have been developed to identify Th molecules with negative 1 ^ effector phase activity. One of these mabs, MR1, confirms an antigen that can be selectively expressed on activated Th1. To see if MR1 and CD40-Ig could identify the same molecule, flow cell counting and blocking studies were performed. CD40-Ig and MR1 stained 56% and 61% of activated, non-dormant Th, respectively (Figure 5a). MR 1, but not -29- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) 1224969 A7

V. Description of the invention (27) Another hamster anti-T cell mab, anti-sink //? T C R, can block the staining of activated Th1 by CD4 0-Ig in a dose-dependent manner. These data show that CD4 0-Ig and MR1 can identify overlapping or identical epitopes on the 39 kD Th protein. In order to further prove that CD40-Ig and MR1 can confirm the same molecule, the antigen that can bind to MR1 can be identified by immunoprecipitating a protein from a radiolabeled Th lysate. Both CD40-Ig and MR1 can immunoprecipitate 39kD protein (Figure 1, 5b). Finally, 39 kD protein was immunoprecipitated with CD40-Ig to remove the antigen identified by MR1 in the radiolabeled lysates of activated Th, thus supporting the argument that the MR1 antigen and the CD40-binding protein are the same. Functional studies were performed with MR1 to highlight whether this mab can neutralize the activity exhibited by pmact. PMact & B-cells are cultured alone or in the presence of hamster mabs or CD40-Ig. There are two types of hamster mabs, anti-slave / 々TCR and α-CD3, which cannot inhibit the activation of pmact-dormantized B cells. In contrast, MR 1 or CD 4 0 -1 g can inhibit the activation of B cells (Figure 6). The data show that blocking the major Th surface molecules (LFA-1, CD4, ICAM-1, CD3, Han, 0TCR) with mabs fails to impede the ability of activated Th to circulate to the entrance of B cells. In contrast, c D 4 0 -1 g or ma b, which is specific for CD D 0 binding protein, can block τ h-dependent B cell activation in a dose-dependent manner. Moreover, the 39kD protein of the CD40 binding protein is the same, and it can be selectively expressed on membranes of Th that are activated rather than dormant. Both CD4 0-Ig and mab specific for 39 kD CD40 binding protein can block PMACT activation on B cells. Although there are many membrane proteins involved in Th · dependent B-cell messaging, this -30 · This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) --------- --II ^ --- Please ttt-Notes on the back of this page) Order-line · Printed by the Employees ’Cooperatives of the Intellectual Property Bureau of the Ministry of Economy 1224969 A7 B7 V. Description of Invention (28) Employees’ Cooperatives of the Intellectual Property Bureau of the Ministry of Economy The evidence presented in the printing caused the contribution of certain molecules to be eliminated (LFA-1, CD4, CD3 'Shen' 々TCR, ICAm-1) and suggested that CD40 is a B-cell receptor that is homologous to Dh. The data show that CD4o-Ig and mab specific for CD4o binding protein can inhibit τ h-dependent B-cell activation. The CD4 0 ligand is a 39kEM々 protein that can be expressed on activated but not dormant Th. Biochemical studies have shown that the 39kD protein is a single-stranded molecule that is not affected by reducing agents by movement on the electropherogram. According to the functional studies presented in this study, both activated Thl and Th2 exhibit 39kD CD40 binding proteins. This is consistent with a functional study showing that both Thl and Th2 can produce 3% cell cycle entrances. In an attempt to further identify the proteins, cDNAs (Cd53, CD27, and CD69) encoding CD proteins with a MW range of 39 kD were transiently transfected into cos cells, and the cells were tested for CD 4 0 -1 g binding ability. None of the transfected COS cells showed a protein capable of binding to CD40-Ig. It is therefore suspected that the 39 kD protein is not one of these CD proteins. The biochemical basis for signal transduction between Th and B cells has been avoided. The identification of cd4O as a T cell signal transduction molecule helps to focus attention on specific biochemical pathways known to couple to CD40 molecules. CD40 has four cysteine-rich features through its extracellular area, and it is known that it is a member of the nerve growth factor receptor (NGFR) family. Mab has been shown via CD40 communication (Uckun et al., J. Biol. chem. 2 6 6: 1 7478- 1 7485 (1 9 9 1)) involving the activation of tyrosine kinase, resulting in inositol triphosphate production The system increases the activation of 1 and at least 4 different serine / threonine kinases. Based on the information obtained from other members of the NG receptor family, it is expected that in the activated paper size • 31-This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 public love) Note

Page I Order 1224969 A7 A7-___ Β7 _____ V. Description of Invention (29)

The interaction between Th and B can cause many of the same biochemical processes. In the immunofluorescence binding study, the CD40Ig fusion protein was conjugated to biotin using biotin-B imine (Sigma). Two-step staining was used for flow cell count analysis. Phycoerythrin (PE) -bonded avidin (Becton-Dickinson) was used in conjunction with Coulter Epics C instrument. Individual results of screening multiple T cell lines are shown below. Xian found that jurkat and HSB2 cell lines specifically bind to the CD40 Ig fusion protein, while other T cell lines including CEM, HPBALL, and murine thyroid tumors do not (Figure 7). Example 2 Use of anti-gp 39 antibodies to treat or prevent lupus systemic lupus erythematosus (SLE) is a disease characterized by the production of a variety of pathogenic autoantibodies (Boumpas, Ann Int Med. 1995). Somatic antibodies cause damage, either directly after confirmation on normal cells, or indirectly through the formation of immune complexes, which are stored in normal tissue engineering and can activate the complement cascade. As for the normal antibody response, it has been clear in the studies of SLE and lupus-like disease in mouse strains that have been bred in the same line. according to. There is an example from the study of a typical mouse model of SL. The female offspring of NZB / NZ W mice suffer from diseases similar to human SL in many ways, including the production of autoantibodies and the development of immune complexes with glomerulonephritis. Treatment of these mice with depleted anti-CD4 antibodies prevented and indeed reversed nephritis. Unfortunately, from a therapeutic point of view, even in the case of human autoimmune diseases, even a short treatment attempt with anti-c D 4 antibodies can cause a critical subset of neutrons in this case. -32- Applicable to China National Standard (CNS) A4 specification (210 X 297 Public Love " 7 !! · ^ · — I (Please read the note on the back I write this page first)-> 0_ Printed by the Intellectual Property Bureau Staff Consumer Cooperatives System 1224969 A7------------ V. Description of the invention (30) Depletion is prolonged. Furthermore, anti-CD4 antibodies are used for small clinical mouth tests in diseases such as rheumatoid arthritis Its effectiveness is disappointing. Less specific immunosuppression is currently feasible, because it is known that the molecular-sense interaction of Xu Xi is involved in τ h cell assistance, allowing it to be targeted only at Actively involves Th cells that assist in the production of antibodies. An example is a secondary signal that is transmitted by a cross-action between CD2 8 / CTLA4 on Th cells and B 7 · 2 on B cells. Blocking antibodies can make τ cells inadequate in test tubes. Recently, rat CTLA_4 extracellular A fusion protein consisting of a domain connected to a murine Ig Cg2a chain has been shown to block the production of autoantibodies and prolong lifespan. When administered to NZB / NZW mice, even if they have a disease at the end stage, it is speculated Treated mice will competitively inhibit the combination of B 7.2 and endogenous CTL A-4. Experimental results Printed by our research by the Consumers' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs on NZB / NZW mice SLE 4 to 10 months old Anti-CD40L antibody administration. Anti-CD40L antibody treatment can significantly reduce the production of anti-DNA autoantibodies and kidney disease, and prolong the survival of these mice without inducing anti-antibody reactions, or Disruption of humoral immune system performance in treated mice. These data are shown in Figure 9. n-months old (anti-anti-D40k body-treated mice, non-untreated or ΗI g treated mice, 6 0 ° / 〇 is alive (Figure 9). Renal histological examination of long-term survivors after anti-c D 4 0 L antibody treatment showed no pathological changes in the mouth and no significant immune deposition in the glomeruli. The data obtained also suggest that anti-CD4 0L antibody therapy can Two issues related to the use of anti-lymphocyte-surface molecule antibodies in taking immunotherapy Anti-33- This paper size applies to the Chinese National Standard (CNS) A4 specification (21 × 297 mm) V. Description of the invention (31 ) The development of printed-antibody response and the extension of immune suppression after treatment in the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. In our research, mice that survived 11 days to respond to anti-CD40L antibody treatment did not develop The anti-antibody response is almost unchanged in animals receiving antibodies derived from another species. We speculate that this is due to the production of anti-c D 40 L carcasses to prevent their own antibodies. In the study of normal mice treated with the same anti-c D 4 OL antibody and MR-1, no anti-MRj response was seen, and it was concluded that the response observed in NZB / NZW mice could be representative of autologous Immune mice have a special overreaction. In any case, this observation has important implications for the therapeutic application of anti-human CD 4 0 L antibodies, as this serious complication of antibody therapy can be prevented by anti-CD 4 0 L antibody therapy, especially with the use of The `` humanized '' version of the antibody. In our research, the mice that did develop anti-anti-CD40 antibody responses did not produce or survive anti-DNA antibodies better than control mice, thus inferring that for whatever reason The antibodies produced by the collection were not prevented by anti-CD 4 0 L antibodies. The development of anti-anti-CD40L antibodies resulted in rapid clearance of the anti-CD40L antibodies administered next, and therefore lost therapeutic efficacy. In support of this argument, 'Mice treated with non-specific hamster I g G can detect the deposition of hamster antibodies in the glomeruli of a subset of mice treated with non-reactive anti-CD 4 0 L antibodies, And not in anti-CD40L antibody-responders. This observation is consistent with the anti-anti-CD40L antibody immune complexes that can be formed and precipitated in the kidney by continuous administration of anti-CD40L antibodies, which may be worsened rather than preventive, and that of glomerulonephritis. After removal, once the anti-CD 4 0 L antibody treatment is stopped, the ability to show anti-κ l 抗体 antibody response will be restored after up to 4 months, of which the antibody in the body fluid is free -34- 1224969 A7--B7 five 2. Description of the invention (32) The immune suppressive effect of the epidemic is reversible, and therefore anti-CD 4 0 L antibody therapy is suitable for human therapeutic applications. In these experiments, NZB / NZW F 1 mice were treated intraperitoneally with 200 micrograms of MR-1 twice a week, starting at 4-10 months of age. At the same time, the treatment strategy can also be used to achieve the treatment, increasing the dose to 250 mg MR-1, intraperitoneal injection three times a week, starting from 4 to 10 months old. This dose has been used in the collagen-blinded mode of rheumatoid arthritis and caused a 100/0 survival in the treated group of mice (Durie, Science, 1993). Using this strategy, NZB / NZW mice currently have a survival rate of 100% at 11 months (Figure 10), while both control mice died at 10 months. Treatment of established lupus disease Although the anti-gp3 9 antibody in Sle prevention in NZB / NXW F1 mice is interesting, it does not imply that this antibody can be used therapeutically in active disease states. In order to determine whether the treatment with antibodies can reverse the already confirmed disease, a group of mice is allowed to develop 2-3 + proteinuria (equivalent to 100 mg / day to 500 mg / day), which is determined by the urine metering method. At this time, the rats arbitrarily discontinued treatment or received 100 micrograms / day of MR-1. All 10 untreated rats died at 10 months of age. In contrast, 5 of the 10 MR-1 treated mice survived 11 months of age. Of these, 3 were just healthy and free of proteinuria. Interestingly, one of the mice did have high titer anti-DN A antibodies (Table 1). Two of the five survivors appeared to be sick and had high titer anti-DN A antibodies and 4+ proteinuria. Therefore, it was found that treatment with MR-1 after development of kidney disease can reverse the disease in some mice. Findings in our responding mice, that is, treatment with MR-1 -35- This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) !! Loading · — — (Please first kt #- Note on the back t page) > D _-· Line-Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 1224969 V. Invention Description (33 The Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs printed a reversible proteinuria instead of anti-antibodies -The production of DNA antibodies, which may further generate another mechanism for the action of MR-1, does not worsen the production of T-dependent antibodies. It also shows that NZB / NZW mice are treated with tumor necrosis factor (TNF-slave) 'Can significantly delay the development of nephritis (Jacob, Science, 1 988; Gordon, 189 89). Interestingly, the severe nephritis observed in NZB / NZW F1 mice can be partially attributed Because the dominant gene from the parent of NZ W 'which points to the H-2 complex. NZW mice have a reduced level of TNF-from the' It conforms to the polymorphism of the TNF-from the gene, which is located in the H-2 complex Within. NZB / NZW F1 mice also had significant levels of TNF- 戌. Furthermore, NZ was treated with anti-IL-10 antibodies B / NZW F1 mice can substantially delay the onset of nephritis and prolong survival. The beneficial effects seen in the treated mice appear to be TNF induced by IL-10-mediated by positive levels of regulation because of anti- Co-treatment with TNF-α antibodies can abolish the anti-ιιιιι response. In a preliminary experiment, we also administered "in the 4 mice of each group before and after the ruler-" and checked the level of TNF-n by ELISA. The levels are shown in Table 2. In terms of analysis of proteinuria and production of anti-DNA antibodies, only mice that responded to μ R -1 treatment could demonstrate that TNF- Shen was 2. 16 picograms / ml before treatment. Increased to 3 5 · 01 picograms / ml after treatment. These data can be deduced that at least part of the effect of μ r -1 is mediated by the increase of NZB / NZW F1 old-fashion TNF- 戌. Furthermore, these results confirm The anti-gp 3 9 antibody and its active fragments are useful in the treatment of human lupus, that is, systemic lupus erythematosus or drug-induced wolf cancer (Q). In particular, Yunsheng, J therapeutic intervention, and this type of lupus A common feature is nephritis. This antibody should be -36- National Standard (CNS) A4 MAGO (210 X 297 public love) (Please read the precautions on the back I. I this page) • Line · 1224969 A7 _B7__ V. Description of the invention (34) And may even reverse the course of the disease. Table 1 Age (months) with proteinuria after treatment printed by the Consumers' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs MR1 Treatment Period (yards) Post-treatment · Egg urinary relief • (week )). Re-treatment with MR1: Action period (weeks) 7 13 + 4 N / AN / A-9 6 0 '% 0 None-6 6 0 12 None i-5 6 0 11 • Yes 6 (0) 0 8 1 death-6 18 death-* ,, shop 6 6 '0 9 has 3 (+4) + 4 6 1 6 death-7 5 death---6 2 death-37- -------- ------ I --- (Please read the back ^ Notes and write this page first) 1 ^ 1 • Line · This paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm)

Claims (1)

1224969 Patent Application No. 088105322 Meeting of Chinese Patent Application (August 1993) g Scope of patent application 1. A pharmaceutical composition for treating lupus in an individual that causes a substantial increase in tumor necrosis factor-2 (TNF-a), and comprises a therapeutically effective dose of an antibody that can specifically bind to Tumor 39. 2. The pharmaceutical composition according to item 丨 of the application, wherein the lupus is whole body lupus erythematosus. 3. The pharmaceutical composition according to item 丨 of the application, wherein the lupus is a lupus induced by a drug. 4. The pharmaceutical composition according to item 丨 of the patent application scope, wherein the antibody is a humanized anti-human gp39 antibody. 5. The pharmaceutical composition according to item 1 of the application, wherein the antibody is an embedded anti-human gp 39 antibody. 6. The pharmaceutical composition according to item 丨 of the scope of patent application, which is administered systemically. 7. The pharmaceutical composition according to item 6 of the scope of patent application, which is used to treat kidney diseases related to lupus. 8. The pharmaceutical composition according to item 7 of the scope of patent application, which reduces and / or reverses the kidney disease by reducing proteinuria. 9. The pharmaceutical composition according to item 1 of the scope of patent application, which further contains tumor necrosis factor. 10. The pharmaceutical composition according to item 1 of the scope of patent application, which contains an anti-gp39 antibody in the range from 10 μg / ¾ to 1000 μg / ml. 57885-930810 This paper size applies to China Solid Standard (CNS) A4 (210X 297 mm)