KR20010072564A - USE OF ANTI-gp39 ANTIBODIES FOR TREATMENT AND/OR REVERSAL OF LUPUS AND ASSOCIATED KIDNEY DISEASE - Google Patents

Abstract

The present invention provides a method of treating lupus using anti gp39 antibodies or fragments. This treatment has been shown to reverse the disease, particularly lupus-associated kidney disease, which is the leading cause of death in lupus subjects.

Description

USE OF ANTI-gp39 ANTIBODIES FOR TREATMENT AND / OR REVERSAL OF LUPUS AND ASSOCIATED KIDNEY DISEASE}

First, studies of Mitchison, Benasecerraf and Raff suggested that physical interactions between T _h and B cells are essential for the development of humoral immune responses. Subsequent studies form a physical conjugate with the Class II major histocompatibility complex (MHC) of conformity, where T _h is an antigen presenting B cell. Vitetta et al., Immunol. Rev. , 99: 193-239 (1987)], B cells respond to T _h in the conjugate (Barrett et al., J. Immunol ., 143: 1745-1754 (1989)) It became. From the discovery that T _h induced lymphokines grow significantly on B cells to achieve a distinct effect, mediating activation of B cells with which soluble factor (s) released in close proximity by activated T _h interacts. Proposed. However, none of the molecularly cloned lymphokines clearly showed the ability to induce B cell cycle entry in combination alone. Unlike soluble factors, plasma membrane fractions from activated T _h induced B cell cycle entry. Hodgkin et al., J. Immunol ., 145: 2025-2034 (1990); Noelle et al., J. Immunol ., 146: 1118-1124 (1991). In using purified plasma membrane fractions from activated T _h of the study expressed in the film of the activated T _h protein, it has been proposed sikindago initiating humoral immunity [see: Noelle et al, J. Immunol, 146:.. 1118 -1124 (1991); Bartlett et al., J. Immunol ., 145: 3956-3962 (1990).

Purified plasma membranes (PM ^ACT ) from activated T _h have been used to characterize this effector function. Hodgkin et al., J. Immunol ., 145: 2025-2034 (1990); Noelle et al., J. Immunol ., 146: 1118-1124 (1991). PM ^ACT from activated T _h , but not resting T _h (PM ^REST ), was antigen specific and showed activity that induces B cell cycle entry in a class II unrestricted manner. In addition, the activity exhibited by PM ^ACT has been shown to require 4-6 hours of activation, de novo RNA synthesis, and native proteins. Bartlett et al., J. Immunol ., 145: 3956- 3962 (1990).

The present invention relates to a soluble ligand for the receptor comprising a counter receptor, which has been optionally mentioned in the literature as CD154 recently for CD40CR, gp39 or CD40 B cell antigens, and fusion molecules comprising at least a portion of the CD40 protein. will be. The present invention is based, at least in part, on the discovery that soluble CD40 / immunoglobulin fusion proteins can inhibit helper T cell mediated B cell activation by binding to a novel 39kD protein receptor on the helper T cell membrane. The present invention provides a substantially purified CD40CR receptor; Soluble ligands of CD40CR, including fusion molecules comprising an anti gp39 antibody and fragments thereof as well as at least a portion of the CD40 protein; And methods for controlling B cell activation that can be particularly useful in the treatment of allergic or autoimmune diseases. More specifically, the present invention relates to the use of anti-gp39 antibodies used to treat systemic lupus erythematosus (SLE) or drug-induced lupus.

Influence of monoclonal antibody and CD40-Ig on induction of B cell RNA synthesis by PM ^ACT .

Panel A. Resting B cells were cultured using Pmtest or PM ^ACT from T _h 1. 25 μg / ml of anti CD4, anti LFA-1 or anti ICAM-1 or a combination of each (25 μl / ml each) was added to the wells containing PM ^ACT and B cell RNA synthesis was [ ³ H ]-Was measured by incorporation of uridine. B cell RNA synthesis was performed for 42-48 hours after incubation. The results presented are for the arithmetic mean (+/- standard deviation) of the cultures from three experiments and represent five such experiments.

Panel B. Resting B cells were incubated with PM ^ACT from T _h 1 (•, ▲) or T _h 2 (□). T _h 1 PM ^ACT (•, ▲) containing the culture solution was increased in amount of CD40-Ig (▲) or control protein CD7E-Ig (Ø) was added. The amount of CD40-Ig was added to T _h 2 PM ^ACT (□) containing the culture solution. B cell RNA synthesis was assessed for 42-48 hours after incubation. The results presented are for the arithmetic mean (+/− standard deviation) of the cultures from three experiments and represent three experiments.

Panel C. Resting B cells were incubated with LPS (50 μg / ml) or PM ^ACT . CD40-Ig (25 μg / ml; rhombus) or CD7E-Ig (25 μg / ml; solid) was added to the culture. RNA synthesis was determined as described in panel A. The results presented are the arithmetic mean of the cultures from the three experiments (+/- standard deviation) and represent three such experiments.

CD40-Ig inhibited B cell differentiation and proliferation.

Panel A. Resting B cells were incubated with PM ^ACT , rIL4 (10 ng / ml) and rIL5 (5 ng / ml). At the start of the culture, or on day 1, 2 or 3 after the start of the culture, CD40-Ig or CD7E-Ig (25 μg / ml) was added. On day 6 of culture, supernatants (SN) were taken from individual wells and anti-isotypes as described in Noelle et al., J. Immunol ., 146: 1118-1124 (1991). isotype) specific ELISA was used to quantify IgM (■) and IgG ₁ (●). In the presence of PM ^ACT , IL4 and IL5 (in the absence of added CD40-Ig), the concentrations of IgM and IgG ₁ were 4.6 μg / ml and 126 ng / ml, respectively. In the absence of IL4 and IL5, no IgM or IgG ₁ was detected. The results are representative of three of these experiments.

Panel B. Th1 was left at rest for 16 hours or activated with anti-CD3, irradiated and incubated with resting B cells (4 × 10 ⁴ / culture) in the presence of IL4 (10 ng / ml). 0 to 25 μg / ml of CD40-Ig (▲) or CD7E-Ig (●) were added to the culture. After 66-72 hours of incubation, the wells were vibrated and taken up with 1.0 μCi of [ ³ H] -thymidine. The dashed line represents the response of B cells to Th at rest. The results presented are the arithmetic mean (+/- standard deviation) of the cultures from three experiments, representing two such experiments.

3. CD40-Ig detected molecules expressed on Th in the active state but not in the resting phase. The resting Th was taken and incubated with the fusion protein and then FITC conjugated goat anti hIgG (25 μg / ml) at 4 ° C. for 20 minutes. Percentage of cells and MFI was determined by analysis of at least 5000 cells / sample. CD40-Ig binding is shown in the form filled in the profile.

4. CD40-Ig immunoprecipitated 39 kD protein from the lysate of activated Th1. Th1 was activated for 16 hours at rest or undissolved anti-CD3. [ ³⁵ S] -labeled proteins from resting or activated Th were immunoprecipitated with purified antibodies or fusion proteins (1-10 μ). Gel profiles represent three of these experiments.

5. Monoclonal antibody specific for induced 39Kd Th membrane protein (mab) inhibited the induction of B cell RNA synthesis by PM ^ACT . Resting B cells and PM ^ACT were incubated with 10 μg / ml of each anti-α / β, anti-CD3, CD40-Ig or MR1. The results presented are the arithmetic mean of the cultures from the three experiments and represent the three experiments.

Figure 6. MR1 and CD40-Ig recognized the same molecule expressed on activated Th.

Panel A: Activated Th was fluorescently stained with MR1 or regulatory Ig. To assess whether CD40-Ig and MR1 compete with binding to activated Th, graded concentrations of MR1 or control hamster Ig (anti-α / β TCR) are added with anti-CD40 (20 μg / ml) It was. After incubation at 4 ° C. for 20 minutes, the samples were washed and incubated with FITC conjugated monoclonal antibody (mab) anti human IgG ₁ .

Panel B: Proteins from [ ³⁵ S] -methionine labeled activated Th were immunoprecipitated with MR1 (10 μg / sample) or CD40-Ig (10 μg / sample) and analyzed by PAGE and X-ray fluorescence. The results presented represent two such experiments.

Binding of CD40-Ig to human cell lines. Various human T-cell lines were exposed to biotin-labeled CD40-Ig and binding was assessed by flow cytometry.

Figure 8.

Panel A: Nucleotide sequence of CD40 cDNA from Stamenkovic et al., EMBO J. , 8: 1403-1410 (1989). The transmembrane region is highlighted.

Panel B: Schematic diagram of the plasmid that can be used to express CD40-Ig. The amino acid sequence at the fusion site of CD40 is shown below the schematic portion of CD40.

Figure 9.

Titer of anti- (ss) DNA antibody produced by different populations of treated NZB / NZW F ₁ mice. Open circles (--- ○ ---) indicate mice treated from 4 months to 10 months of age with MR-1; Open triangles (--- Δ ---) represent mice that received MR-1 after unresponsive proteinuria development; Closed squares (---> ---) represent mice that received MR-1 after responding proteinuria development; Closed circles (--- ● ---) represent untreated mice. Values are the average of three titers from each group (+/- SEM).

10.

Survival in different colonies of treated NZB / NZW F1 mice. Open circles (--- ○ ---) indicate mice treated from 4 months to 10 months of age using MR-1; Open triangles (--- Δ ---) represent mice that received MR-1 after unresponsive proteinuria development; Closed squares (---> ---) represent mice that received MR-1 after the onset of responded proteinuria; Closed circles (--- ● ---) represent untreated mice.

Detailed description of the invention

The present invention provides a substantially purified CD40CR receptor; Soluble ligands of CD40CR, including fusion molecules comprising CD40, as well as anti-gp39 antibodies and fragments thereof; And methods for controlling B cell activity using soluble ligands.

To describe more clearly and concisely without limiting the invention, the detailed description of the invention is subdivided as follows:

(i) a ligand that binds to CD40CR;

(ii) the method used to characterize the CD40CR;

(iii) preparation of purified CD40CR;

(iv) use of a ligand that binds to CD40CR; And

(v) Use of CD40CR.

(iii) purification of purified CD40CR; (Especially progressive lupus, such as systemic lupus erythematosus or drug-induced lupus).

The present invention includes (i) a fusion molecule comprising at least a portion of a CD40 protein and (ii) an antibody or antibody fragment that specifically binds CD40CR, or gp-39, or CD154, such antibodies are known. , Providing a soluble ligand of CD40CR.

As used herein, the term "soluble" indicates that the ligand of the present invention does not permanently bind to the cellular plasma membrane. However, the soluble ligands of the present invention may be attached to or included in implants or vesicles, including lipids, proteins, or noncellular solid supports including carbohydrate molecules, beads, vesicles, magnetic particles, fibers, and the like.

The ability of this ligand to bind to CD40CR is dependent on proteins such as CD40-Ig (below) or MR1 (below), or another antibody that binds to CD40CR as described in commonly assigned US patent application Ser. No. 08 / 475,847. Can be verified by demonstrating binding to.

The ligands of the present invention may also be included in pharmaceutical compositions with suitable carriers.

The present invention provides soluble fusion molecules that are ligands of CD40CR. Such a fusion molecule comprises at least a portion of a CO40 protein attached to a second molecule. It is preferred that some of the CD40 lack the CD40 transmembrane domain. A portion of the CD40 protein that may be used in accordance with the present invention is defined as any portion capable of binding to CD40CR, for example, such portion may be shown to bind to a protein such as MR1 or CD40-Ig.

Second molecules that may be used include peptides and proteins, lipids and carbohydrates, and in preferred embodiments of the invention immunoglobulin molecules, or portions thereof (Fv, Fab, F (ab ') ₂ , or Fab' fragments) or Another attachment molecule such as CD8, or B7. The second molecule may be derived from a non-human or human source or may be chimeric. The second molecule may also be an enzyme, toxin, growth factor, lymphokine, antiproliferative, alkylating agent, antimetabolic, antibiotic, vinca alkaloid, platinum coordination complex, radioisotope or fluorescent compound.

Fusion molecules of the invention can be produced by chemical synthesis, or preferably by recombinant DNA techniques.

For example, a nucleic acid sequence encoding at least a portion of a CD40 molecule can be combined with a nucleic acid sequence encoding a second molecule in a suitable expression vector, followed by a prokaryote or preferably eukaryotic expression system, eg, It is expressed in mammalian expression systems including enzymes, baculoviruses, or transgenic animals.

In addition, at least a portion of the CD40 protein can be expressed using electrophoretic methods or affinity chromatography using ligands that bind to CD40 or the second molecule. Ligands that bind CD40 include anti-CD40 antibodies, such as G28-5, produced by hybridomas deposited with the American Type Culture Collection under accession number HB9110 and CD40CR described in more detail below. However, the present invention is not limited thereto. If the second molecule is an immunoglobulin or an immunoglobulin fragment, an affinity column comprising an anti-immunoglobulin antibody can be used, and if the second molecule comprises an F _C fragment, a Protein A column can be used.

According to a preferred embodiment of the invention, a portion of the CD40 can be generated using a nucleic acid sequence encoding a CD40 protein truncated upstream from the transmembrane domain. This nucleic acid sequence contains cDNA encoding the CD40 antigen using PstI (P) and Sau 3A (S3) as described in Stamenkovic et al., EMBO J. , 8: 1403-1410 (1989). The plasmid can be prepared by restriction enzyme treatment. The resulting P / S3 fragment can be subcloned into the same plasmid digested with P and Bam HI (B).

In particular, in a non-limiting embodiment of the invention, the expression vector used to generate a ligand containing at least a portion of CD40 as well as an immunoglobulin sequence is preferably a viral origin, a replication origin, a bacterial origin, Eukaryotic promoter and amplifier sequences isolated from a selection marker, a DNA sequence encoding at least a portion of CD40, followed by a DNA sequence encoding an immunoglobulin constant region by restriction enzyme sites that allow subcloning of the polyadenylation signal sequence. May be included (see Figure 8.b.).

In a specific embodiment of the invention, the truncated CD40 gene is immunoglobulin as described in Aruffo et al., Cell , 61: 1303-1313 (1990) using Mlu I and B restriction enzyme treatment. Subcloning into the fusion plasmid may form plasmid pCD40-Ig encoding the fusion molecule CD40-Ig (see FIG. 8). CD40-Ig fusion proteins can then be generated by transfecting the PCD40-Ig plasmid with COS cells to form a short term expression system. The resulting CD40-I can be harvested from COS cell supernatants and purified by Protein A column chromatography as described in Aruffo et al., Cell , 61: 1303-1313 (1990).

Soluble ligands of the present invention will preferably comprise fragments of such antibody molecules that comprise an antibody molecule, a monoclonal antibody molecule or an antigen binding site that binds to CD40CR (gp39, preferably human gp39). Such ligands may be proteins, lipids, carbohydrates, enzymes, toxins, growth factors, lymphokines, antiproliferatives, alkylating agents, antimetabolic agents, antibiotics, vinca alkaloids, platinum coordination complexes, radioisotopes or fluorescent compounds It may further comprise a molecule and may be linked to an antibody molecule or fragment.

If the ligand is a monoclonal antibody or fragment thereof, the monoclonal antibody can be prepared for CD40CR (gp39) using the methods provided for the production of antibody molecules by continuous cell lines in culture. For example, the hybridoma method first developed by Kohler and Milstein (1975, Nature , 256: 495-497), as well as the human B cell hybridoma method (Kozbar et al. More recently available methods, such as 1983, Immunology Today , 4:72) and the EBV-hybridoma method for generating human monoclonal antibodies (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp 77-96) and the like are within the scope of the present invention. Humanized and chimeric anti-gp39 antibodies are generally preferred because these antibodies are less prone to inducing an immunogenic response (HAMA response) upon administration.

Antibody fragments containing the idiotype of the molecule can be produced by known techniques. For example, such fragments include F (ab ') ₂ fragments that can be produced by treating an antibody molecule with pepsin; Fab 'fragments that can be produced by reducing the disulfide bridges of F (ab') ₂ fragments; F (ab ') ₂ fragments that can be produced by treating an antibody molecule with papain; Antibody molecules include, but are not limited to, 2Fab or Fab fragments that can be produced by treating the antibody molecule with a reducing agent that reduces papain and disulfide bridges.

As described above, the present invention is also disclosed in Morrison et al . ( Proc. Natl. Acad. Sci . USA, 81: 6851-6855 (1984)) or European Patent Application No. 85305604.2, published March 5, 1996. Chimeric or human antibodies produced by such known techniques as described in published publication number 0173494 to Morrison et al.

Immunogens for making antibodies may be any source containing CD40CR. For example, activated T _h , eg activated human T _h cells, can be used as the immunogen. In addition, substantially purified CD40CRs prepared as mentioned in section 5.3 below may be used. If activated T _h is used as an immunogen, antisera may be tested for responsiveness to activated T _h cells rather than resting T _h cells.

Immunogens can also include recombinant gp39 or fragments thereof. In this regard, DNA encoding murine and human gp39 was cloned and expressed by recombinant methods. Such proteins provide an effective immunogen for preparing anti gp39 antibodies.

In a preferred embodiment of the invention, the soluble ligand is an antihuman gp39 monoclonal antibody, more preferably a humanized or chimeric antihuman gp39 antibody. The following method was used to prepare MR1 monoclonal antibodies, which specifically bind to murine gp39 and can be used to generate another antibody directed towards CD40CR.

Hamsters were immunized with intraperitoneal administration with 5-10 ⁶ activated T _h 1 (D1.6) at weekly intervals for 6 weeks. If serum titers to murine T _h were greater than about 1: 10,000, cell fusion was performed with polyethylene glycol using immune hamster splenocytes and NSI. SN from wells containing growing hybridomas were screened by resting cytometry and flow cytometry for activated T _h 1. One particular hybridoma that produces a monoclonal antibody (mab) that selectively recognizes activated Th was further tested and subcloned to induce MR1. A plurality of MR1s were generated and purified by ion HPLC.

Also, more preferably, antibodies against human gp39 can be prepared according to US Patent Application No. 08 / 475,847, which is incorporated herein by reference.

The invention also provides ligands comprising monoclonal antibodies and fragments thereof which can competitively inhibit binding of MR1 to a target antigen or to CD40-Ig to its receptor.

CD40CR comprises: i) binding capacity with at least a portion of CD40 and CD40, a CD40 fusion molecule comprising an antibody such as MR1; ii) functional properties capable of stimulating B cell cycle entry, proliferation and differentiation, and iii) their cell distribution.

CD40CR may be characterized by the ability to bind ligands, such as CD30 fusion molecules, including CD40, and by antibodies directed toward CD40CR.

As discussed in detail below, several techniques were used to characterize the CD40CR. For example, CD40-Ig and MR1 were found to recognize the same 39 kD molecule. Both CD40-Ig and MR1 were found to immunoprecipitate 39 kD protein from radiolabeled Th lysates (FIG. 5B). In addition, immunoprecipitation of the 39 kD protein with CD40-Ig removed the antigens recognized by MR1 from Th lysates.

CD40CR may also be characterized by its ability to stimulate B-cell cycle entry, proliferation and differentiation.

For example, plasma membranes (PM) from activated (PM ^ACT ) rather than dormant (PM ^REST ) have been found to induce B-cell RNA synthesis (FIG. 1A); This induction indicating B-cell activation is not affected by antibodies such as anti-LFA-1, anti-CD4, anti-ICAM-1. However, it has been found that CD40-Ig or MR1 can inhibit PM ^ACT induced B-cell activation as shown in FIGS. 1B and 6.

Induction of B-cell activation is [ ³ H] -uridine incorporation into RNA (RNA synthesis increases as B-cells differentiate) or [ ³ H] -thymidine incorporation measuring DNA synthesis associated with cell proliferation It can be measured by a technique such as For optimal determination of the CD40CR effect on B-cell proliferation, interleukin-4 (IL-4) can be added to the medium at a concentration of about 10 ng / ml.

In addition, B-cell activation can be measured as a function of immunoglobulin secretion. For example, CD40CR present in substantially purified form, PM or other forms, can be added to resting B-cells with IL-4 (10 ng / ml) and IL-5 (5 ng / ml). After 3 days of culture, additional volume of medium may be added. On day 6 of culture, supernatant (SN) from each culture was collected and Noelle et al. Immunol., 146: 1118-1124 (1991), can be quantified for IgM and Ig ₁ .

CD40CR may also be characterized by its cell distribution. For example, CD40-Ig was assessed by flow cytometry and did not bind to resting T _h 1 but was observed to bind to activated T _h 1 (FIG. 3). In addition, CD40-Ig was observed to bind to Jurkat cells, HSB2 cells and activated T-cells from human peripheral blood, but significantly binds to CEM cells, HPBALL cells or murine thyoma cells. It was not considered to be.

By way of example, and not by way of limitation, the presence of CD40CR on a particular cell type (“test cell”) can be assessed by flow cytometry as follows. Test cells can be tested simultaneously with resting (negative control) and activated (positive control) Th cells. All cells can be incubated with ligands (eg, CD40-Ig or MR1) for 20 minutes at 4 ° C. at a concentration of about 1 × 10 ⁵ cells / 50 μl, followed by incubation with FITC conjugated anti-ligand antibodies. have. Propidium iodide can be added to all samples at a final concentration of 2 μg / ml. Flow cytometry analysis can then be performed, for example on BD FACSCAN. After positive gating of cells by forward versus side scatter and red nagativity (for exclusion of propidium iodide), log green fluorescence of viable cells can be confirmed.

The present invention provides a substantially purified CD40CR. Such CD40CRs can be prepared from CD40CR containing cells, such as activated helper T-cells, Jurkat, and HSB2 cells by the following method.

Plasma membranes are described in Noel et al. Immunol., 146: 1118-1124 (1991), can be prepared by discontinuous sucrose gradient sedimentation from appropriate cells, such as activated T _h 1 cells. The CD40CR was then dissolved in a crude membrane extract with a mild detergent, followed by size exclusion chromatography, followed by affinity chromatography using a suitable ligand (e.g. MR1 or CD40-Ig) bound to a solid support. It can be isolated by performing chromatography, immunoprecipitation (eg, by CD40-Ig or MR1), and / or gel electrophoresis.

The resulting protein can be expected to have a molecular weight of about 39 kD.

The present invention provides soluble CD40CRs (ie, acellular) which may be included in pharmaceutical compositions with suitable carriers. The invention also relates to peptides, proteins, lipids, carbohydrates, enzymes, toxins, growth factors, lymphokines, proliferation inhibitors, alkylating agents, antimetabolic agents, antibiotics, vinca alkaloids, platinum coordinated complexes, radioisotopes or Provided is a CD40CR bound to a second molecule, which may be a fluorescent compound.

The present invention also provides a substantially purified CD40CR prepared by chemical synthetic or recombinant DNA techniques. For example, genes for CD40CR can be isolated by inserting cDNA prepared from activated helper T-cells into the λgt10 expression system and screening for MR1 or CD40-Ig binding to identify CD40CR expression clones. In addition, cDNA prepared from activated helper T-cells can be transfected into COS cells, and their supernatants can be screened with MR1 or CD40-Ig to identify CD40CR products. The gene for CD40CR can then be used to express CD40CR using expression systems known in the art.

The present invention provides a method of regulating B-cell activation using a ligand that binds to CD40CR. In particular, the present invention provides a method of inhibiting B-cell activation, including exposing a mixture of B-cells and Th cells to a ligand that binds to an effective concentration of CD40CR. Ligands that can be used are described in Section 5.1 above. The methods of the invention can be carried out in vitro or in vivo. By effective concentration is meant the concentration of a ligand that, by any technique known in the art (including those described in section 5.2 above), inhibits B-cell activation by at least about 30%, preferably about 75%. . According to a preferred, specific, non-limiting embodiment of the invention, CD40-Ig can be used as a ligand, in which case the effective concentration can be at least about 10 μg / ml. In another specific and non-limiting embodiment of the invention, monoclonal antibody MR1 can be used, in which case the effective concentration can be at least about 10 μg / ml. When the method of the present invention is carried out in vivo, the effective concentration of the ligand may refer to the plasma concentration or the local concentration of the ligand. For example, to limit the effect on the entire immune system, it may be desirable to inhibit B-cell activation in the local region.

In certain embodiments, the invention provides a method of treating a patient with a disease associated with B-cell activation, comprising administering to the patient a ligand that binds to a therapeutic amount of CD40CR. The patient may be an animal other than human, or preferably human.

Diseases associated with B-cell activation include allergies (including hypersensitivity); Autoimmune diseases including drug-induced lupus, systemic lupus erythematosus, adult rheumatoid arthritis, juvenile rheumatoid arthritis, scleroderma, Sjogren's syndrome and the like; And retroviral infections including, but not limited to, Epstein-Barr infection, and infection with human immunodeficiency virus.

Since B-cell activation has been proposed to be involved in the induction of human immunodeficiency virus replication from incubation periods, it may be desirable to administer the ligands of the present invention to HIV positive patients who have not yet exhibited symptoms of AIDS or ARC.

As discussed in the description above and in the Examples below, particularly preferred applications of anti-gp39 antibodies or fragments thereof include their use for the treatment of drug-induced lupus or systemic lupus erythematosus. As evidenced by the underlying data and experiments described in the Examples below, anti-gp39 antibody therapy mitigates autoantibody production and kidney disease and exhibits prolonged survival in NZB / NZW, an accepted animal model for human SLE. Was found.

In addition, it was further found that the administration of anti-gp39 actually reversed the disease in mice treated to exhibit 2-3 + proteinuria (active lupus disease state) (with prolonged survival after antibody administration and the absence of proteinuria). Proven). Thus, treatment with anti-gp39 antibodies has been shown to reverse lupus disease progression after kidney disease has appeared in an accepted animal model of human lupus. This demonstrates the potential of anti-gp39 antibodies for human treatment in the treatment of lupus and other autoimmune diseases. In particular, this demonstrates that such antibodies can be used to treat patients with active progressive disease even in the worsening stage of disease. Such treatment will effectively treat, possibly even reverse, a disease process, for example kidney damage that often causes the patient to lupus.

Ligands can be administered in a suitable pharmaceutical carrier by methods known in the art, including intravenous, intraperitoneal, subcutaneous, intravenous, intraarticular or intramuscular injection, and oral, intranasal, intraocular and rectal administration, and micro It may be constructed in the form of spheres, liposomes, and / or sustained release implants.

The therapeutic amount of ligand is defined as an amount that significantly reduces the deleterious clinical effects of B-cell activation or T-cell activation and can vary among the ligands used and the diseases treated. When using CD40-Ig, the therapeutic concentration may be about 10 μg / ml systemically (plasma concentration) or locally. When using MRI or other anti gp39 antibodies such as humanized or chimeric anti human gp39 antibodies or fragments thereof, the therapeutic concentration may be systemic (plasma concentration) or topically about 10 μg / ml.

In another embodiment of the present invention, the method may utilize a ligand comprising a toxin or an anti metabolite such that Th cells are killed or injured resulting in increased B cell activation as a result of Th cell destruction.

Ligands of the invention can also be used to label activated T cells, and such techniques can be useful for the diagnosis of T cell disease. Because of this, ligands comprising enzymes, radioisotopes, fluorescent compounds or other detectable labels can be exposed to T cells in vitro or in vivo to quantify the amount of binding.

Ligands of the invention can also be used to deliver substances, such as growth factors, to activated T-cells.

The present invention provides a method for regulating B cell activation using a molecule comprising CD40CR or a CD40CR prepared as described above. In particular, the present invention provides a method for promoting B cell activation comprising exposing B cells to an effective concentration of CD40CR. The method can be carried out in vivo or in vitro. Effective concentration refers to the concentration of receptors that induce B-cell activation of at least about 30%, as measured by techniques known in the art. In a detailed and non-limiting embodiment of the invention, the concentration of CD40CR can be about 10 μg / ml systemically or systemically.

In certain embodiments, the present invention provides a method of treating a subject having an immunodeficiency disease associated with humoral immunoreduction comprising administering to the subject a therapeutic amount of CD40CR. The subject is not human or is preferably human.

Immunodeficiency diseases associated with humoral immunodeficiency include inherited immunodeficiency syndrome induced by chemotherapy or radiation therapy, as well as genetic diseases that involve humoral immunity.

CD40CR may be administered in a suitable pharmaceutical carrier by methods known in the art, including intravenous, intraperitoneal, subcutaneous, intravenous, intraarticular or intramuscular injection, and oral, intranasal, intraocular and rectal administration, and micro In the form of spheres, liposomes, and / or sustained release implants.

The therapeutic amount of CD40CR for CD40 is defined as the amount that increases immunoglobulin production by at least about 30%.

In another embodiment, the CD40CR may be bound to a toxin and then administered to the subject under conditions favorable for destroying B cells expressing CD40. Examples of such conditions include when the patient receives an organ transplant or suffers from multiple myeloma or other B cell cancer, or an autoimmune disease.

CD40CR can also be used to label B cells expressing CD40, and such techniques may be useful for the diagnosis of B cell disease. Because of this, receptors bound to enzymes, radioisotopes, fluorescent compounds or other detectable labels can be exposed to B cells in vitro or in vivo to quantify the amount of binding.

CD40CR can also be used to deliver molecules that bind to B cells.

Summary of the Invention

The present invention relates to soluble ligands for such receptors comprising a counter receptor termed CD40CR and fusion molecules comprising at least a portion of a CD40 protein against a CD40 B cell antigen. The present invention, at least in part, inhibits helper T cell mediated B cell activation by binding a soluble CD40 / immunoglobulin fusion protein to a novel 39kD protein receptor (named "CD40CR" for the CD40 counter receptor) on the helper T cell membrane. It is based on the discovery that it can inhibit and the monoclonal antibody designated MR1, which is directed towards this 39kD receptor, can inhibit helper T cell mediated B cell activation.

The present invention relates to a CD40CR receptor for a soluble ligand of CD40CR comprising a fusion molecule comprising substantially an antibody and at least a portion of a CD40 protein; And a method of controlling B cell activation.

In certain embodiments of the invention, B cell activation in a subject can be inhibited by contacting the subject's helper T cells with a therapeutically effective amount of soluble ligand or CD40CR. Inhibition of such B cell activation may be particularly useful in the treatment of allergies or autoimmune diseases.

More specifically, the present invention is therapeutic for patients in need of such treatment, such as patients with systemic lupus erythematosus or drug-induced lupus, even in patients with worsening progression of these diseases (often with kidney damage observed). A method of treating lupus by administering an effective amount of an anti gp30 antibody, such as the anti human gp39 antibody or fragment thereof described in commonly assigned US patent application Ser. No. 08 / 475,847, filed June 7, 1995. To provide.

One advantage of the present invention is the ability to mediate immune responses that are not specific for the antigen. Several current therapies for allergies include desensitization to specific antigens and require testing each patient to identify antigens associated with sensitivity. As a practical matter, a thorough analysis of the patient's response to each and every potential allergen is virtually impossible. In addition, in most autoimmune diseases, the causative antigen is generally unknown or even unrelated to the progression of the disease. The present invention regarding antigen nonspecific CD40 / CD40CR interactions eliminates the need to characterize antigens associated with allergy or autoimmunity. Therefore, the present invention is particularly useful for the treatment of allergic or autoimmune diseases with multiple components, for which no immunogen is known or for example in hay fever, procaineamide-induced lupus or systemic lupus erythematosus (SLE). Can be. It is also useful for acute treatment of immune activation, for example for the treatment of hypersensitivity.

Abbreviation

Ig: immunoglobulins

mab: monoclonal antibody

PM ^ACT : Plasma membrane prepared from resting helper T cells

PM ^REST : plasma membrane prepared from resting helper T cells

PAGE: Polyacrylamide Gel Electrophoresis

rIL4: recombinant interleukin 4

rIL5: recombinant interleukin 5

SN: supernatant

T _h : helper T cells

T _h 1: D 1.6, IA ^d -limited, rabbit immunoglobulin specific clone.

Example 1

CD40CR, a novel receptor on activated helper T cells, binds to CD40

Converts Signals for Homologous Activation of B Cells

Materials and methods

animal

Female DBA / 2J mice (Jackson Laboratories, Bar Harbor, ME) were used for the preparation of packed cells to support the proliferation of Th clones and for the preparation of resting B cells.

D1.6, IA ^d restricted rabbit Ig-specific T _h 1 clone (Kurt-Jones et al., J. Exp. Med. , 166: 1774-1787 (1987)) by David Parker (University) of Massachusetts at Worcester. D1.6 is referred to herein as T _h 1.

As described by Noel et al . ( J. Immunol ., 146: 1118-1124 (1991)), T _h 1 was 16 in cluster wells (6 wells, Corning, NY) coated with 40 μg / 4 ml of PBS / well. Incubated with anti CD3 for 8 hours (8 × 10 ⁶ / well).

As described in Noelle et al. ( Id. ), Plasma membranes were prepared by discontinuous sucrose gradient settling.

As described in Defrano et al. ( J. Exp. Med. , 155: 1523 (1982)), resting spleen B cells were prepared by sedimentation upon discontinuous Percoll gradients. Cells isolated from the 70-75% (density 1.087-1.097) percol interface were generally greater than 95% mIg ⁺ , had a constant, low degree of proximal forward light scattering and were not responsive to Con A.

The following monoclonal antibodies (mabs) were purified by ion exchange HPLC from irradiated mouse ascites and reconstituted bone marrow: anti CD3: 145-2C11 (Leo et al., Proc. Natl. Acad. Sci. USA, 84 : 1374-1378 (1987); anti α, β: H57-597; anti CD4: GK1.5 (Wilde et al., J. Immunol., 131: 2178-2183 (1983); anti ICAM: YN1 / 1.7. 4 (Prieto et al., Eur. J. Immunol., 19: 1551-1557 (1989)); anti LFA-1: FD441.8 (Sarmiento et al., Immunol. Rev., 68: 135 (1982)) And anti rat / hamster K species: RG-7 (Springer, Hybrid. , 1: 257-273 (1982)).

CD40 fusion proteins were subjected to restriction enzyme treatment with the restriction enzymes Pst I (P) and Sau 3A (S3) by plasmids containing cDNA encoding CD40 antigen (Stamenkovic and Seed, EMBO J. 8: 1403-1410 (1989)). Prepared. This P / S3 fragment was subcloned into the same plasmid treated with P and Bam H1 (B). This allowed the production of CD40Δ encoding the CD40 protein cleavage upstream from the transmembrane domain. DNA fragments encoding CD40Δ were then subcloned into an immunoglobulin fusion plasmid (Aruffo et al., Cell, 61: 1302-1313 (1990)) using MluI and B restriction enzyme treatment. As described by Aruffo et al ( Cell, 61: 1302-1313 (1990)), CD40-Ig fusion proteins were prepared by short-term transfection in COS cells and purified on Protein A columns.

Interleukin 4 (IL4): Seeds recombinant mouse IL4. C. Maliszewski and K. Provided by Dr. K. Grabstein (Immunex Corporation, Seattle, WA).

Interleukin 5 (IL5): Recombinant mouse IL5 was purchased from R & D Research, Sarrento, CA.

3 × 10 ⁴ resting B cells were incubated in 50 μl cRPMI in A / 2 microtiter wells (Costar, Cambridge, MA). To this well, 0.5 μg of T _h 1 or T _h 2 membrane protein was added. 42-48 h, wells were pulsed with 2.5 μCi of ³ H-uridine (New England Nuclear, Boston, Mass.), Harvested and measured by liquid scintillation spectroscopy. The results are expressed in cpm / culture +/- sd.

Resting B cells were incubated as described above. To the culture wells, 0.5 μg T _h 1 membrane protein, IL4 (10 ng / ml) and IL5 (5 ng / ml) were added. On day 3 of culture, an additional 50 μl of cRPMI was added. On day 6 of culture, SN was taken from each well and quantified for IgM and IgG ₁ as described in Noelle et al ( J. Immunol. , 146: 1118-1124 (1991)).

4 × 10 ⁴ resting B cells were cultured in 50 μl cRPMI in A / 2 microtiter wells (Costar, Cambridge, Mass.). To this well, 1 × 10 ⁴ resting or activated, irradiated (500 rads) T _h 1 and IL4 (10 ng / ml) were added. On day 3 of culture, the wells were pulsed with ¹ μCi of ³ H-uridine, as described by Noelle et al ( J. Immunol ., 146: 1118-1124 (1991)).

Hamsters were immunized intraperitoneally with 5-10 × 10 ⁶ activated T _h 1 (D1.6) weekly for 6 weeks. The murine serum titers to T _h 1 1: a is greater than 10,000, and using immune hamster cell fusion ratio and NS1 cells was performed by polyethylene glycol. SN from wells containing growth hybridomas were screened by flow cytometry at rest and activation T _h 1. One particular hybridoma producing mab that selectively recognizes activation T _h was further tested and subcloned to induce MR1. MR1 was prepared in plural and purified by ion exchange HPLC.

Resting and activating T _h (16 _h with anti CD3) were recovered and incubated with fusion protein at 1 × 10 ⁵ cells / 50 μl at 4 ° C. for 20 min, followed by FITC-conjugated goat antihuman (h) IgG (25). Μg / ml; Southern Biotechnology, Birmingham, AL). Propidium iodide was added to all samples at a final concentration of 2 μg / ml. Flow cytometry was performed on BD FACSCAN. The cells were positively gated by fore-and-lateral scatter and then log green fluorescence of viable cells was confirmed by red negative (for propidium iodide exclusion). At least 5,000 viable cells were analyzed for determination of percent positive cells and MFI. Staining with MR1 used FITC-conjugated RG7, mouse anti rat / hamster κ chain mab.

T _h 1 was left at rest or activated with insoluble anti CD3 for 16 h. Proteins from resting and activated T _h (20 × 10 ⁶ / μl) were labeled with 1 mCi of [ ³⁵ S] -methionine / cysteine for 1 hour, then washed twice in RPMI / 10% FCS and cell pellets were extracted in extraction buffer. Noelle et al., (1986) J. Immunol . 137 : 1718-1726. Purified antibody or fusion protein (1-10 μg) was added to 500 μl of lysate (5 × 10 ⁶ cell equivalents) at 4 ° C. for 16 hours. The lysates were then transferred to a tube containing 50 μl of packed Protein A-Sepharose. The pelleted protein A-Sepharose was resuspended and the tube was incubated with stirring at 4 ° C. for 1 hour. The sample was then washed three times with high stringency wash buffer. Pelletized Protein A-Sepharose was resuspended in 30 μl SDS sample buffer and eluted on a 10% polyacrylamide gel. Then, the gel was fixed and fluorescence was performed.

To define cell surface molecules that mediate the induction of B cell cycle entry by PM ^ACT , mabs for T _h membrane proteins were added to the cultures of PM ^ACT and B cells. PM ^ACT induced B cell RNA synthesis eight times more than that observed in PM ^REST (FIG. 1A). The addition of anti LFA-1, anti CD4, anti ICAM-1 alone or a combination thereof did not inhibit the induction of B cell RNA synthesis by PM ^ACT .

In human systems, anti-CD40 mab has been shown to induce B cell proliferation [Clark and Lane, (1991) Ann. Rev. Immunol . 9 : 97-127], which associates CD40 as an important inducing molecule for B cells. To determine if CD40 is involved in the induction of B cell RNA synthesis by PM ^ACT , the soluble fusion protein (CD40-Ig) of the extracellular region of human CD40 and the F _c region of human IgG ₁ was converted to PM ^ACT and B cells. Added to the culture. PM ^ACT from T _h 1 and T _h 2 were prepared and used to stimulate B cell RNA synthesis. The addition of CD40-Ig to the culture resulting in dose dependent inhibition of B cell RNA synthesis induced by PM ^ACT from T _h 1 and T _h 2 was about 5 μg / ml of CD40-Ig. CD7E-Ig fusion protein [Damle and Aruffo, (1991) Proc. Natl. Acad. Sci. USA 88 : 6403-6407] had no effect even when used at 25 μg / ml.

To investigate whether CD40-Ig inhibits the activation of B cells by T-independent activators, B cells were cultured in the presence of LPS and CD40-Ig. After 2 days, RNA synthesis was assessed (FIG. 1C). CD40-Ig was ineffective at inhibiting B cell activation by LPS, but inhibited B cell response to PM ^ACT .

In the presence of PM ^ACT , IL4 and IL5, B cells differentiated polyclonal to produce Ig [Hodgkin et al., (1990) J. Immunol. 145 : 2025-2034; Noelle et al., (1991) J. Immunol. 146 : 1118-1124. To assess the need for CD40 signaling in this process, CD40-Ig was added at the beginning of the culture or after the culture. The addition of CD40-Ig at the start of culture (FIG. 2A) inhibited at least 95% of polyclonal IgM and IgG ₁ production compared to the control level in its absence. On the other hand, addition of CD40-Ig after 1 day and 2 days of culture hardly inhibited IgM and IgG ₁ production. These data indicated that signaling by CD40 after 24 hours is no longer essential for differentiation of B cells into Ig secretion.

The data so far indicate that CD40 is involved in the activation of B cells by PM ^ACT . To confirm that CD40 is complete and also involved in the activation of B cells by activated viable T _h , studies were conducted. T _h 1 was activated and recovered for 16 hours with insoluble anti-CD3 and irradiated. Irradiated T _h 1 was incubated with B cells in the presence of IL4 and B cell proliferation was measured 3 days after culture. Were a foreign source of IL4 need to achieve B-cell proliferation by T _h 1, because it does not produce a _{T h 1 IL4 [Noelle et al} ., (1989) J. Immunol. 143 : 1807-1814. CD40-Ig inhibited the induction of B cell proliferation by irradiated T _h 1 in a dose dependent manner, similar to that observed for PM ^ACT (FIG. 2B). Negative control, CD7E-Ig had no significant effect.

Activated T _h 1 is to investigate whether the expression of the binding protein for CD40, resting and activated (16 hours) in which a dyed T _h 1 with CD40-Ig or CD7E-Ig were stained with the following, FITC- anti HigG. Binding of CD40-Ig was assessed by flow cytometry (FIG. 3). T _h 1 activated for 16 h with anti-CD3 stained 56% positive with CD40-Ig, but not with control CD7E-Ig, and resting T _h 1 did not stain with CD40-Ig. To confirm that the CD40-Ig binding protein, T _h 1 protein of ^[35 S] - a biosynthetic typically labeled methionine / cysteine and then were immunoprecipitated proteins with CD40-Ig or CD7E-Ig. Immunoprecipitated proteins were analyzed by SDS-PAGE and fluorescence (Figure 4). Prominent and low molecular weight band, β2 microglobulin with an apparent molecular weight of 39 kD. In the absence of mab, no significant band was seen. The 39 kD band was also immunoprecipitated from ¹²⁵ T labeled activating T _h to confirm that the 39 kD protein is a membrane protein.

Mabs specific for antigens selectively expressed at activation versus resting T _h were developed to identify T _h molecules that are responsible for activity on T _h effector. One such mab, MR1, recognized an antigen selectively expressed on activating T _h 1. To investigate whether MR1 and CD40-Ig recognize the same molecule, flow cytometry and blocking studies were performed. CD40-Ig and MR1 stained activating T _h about 56% and 61%, respectively, but did not stain resting T _h (FIG. 5A). MR1 is a dose-dependent manner, but prevent the staining of activated T _h 1 by CD40-Ig, another hamster anti-T cell mab, anti-α / β TCR did not. These data suggested that CD40-Ig and MR1 recognize partially identical or identical epitopes on the 39 kD T _h protein. To further demonstrate that CD40-Ig and MR1 recognize the same molecule, MR1 binding antigens were identified by immunoprecipitation of proteins from radiolabeled T _h lysates. CD40-Ig and MR1 both immunoprecipitated the 39kD protein (FIG. 1. 5b). Finally, immunoprecipitation of 39kD protein by CD40-Ig eliminated the antigens recognized by MR1 from the radiolabeled lysate of activated T _h , which supports the idea that the MR1 antigen and CD40 binding protein are identical.

Functional studies of MR1 were performed to confirm that this mab neutralizes the activity expressed by PM ^ACT . PM ^ACT and B cells were cultured alone or in the presence of hamster mab or CD40-Ig. Both hamster mabs, anti α / β TCR and α-CD3 inhibited the activation of resting B cells by PM ^ACT . In contrast, MR1 or CD40-Ig inhibited B cell activation (FIG. 6).

The data show that blocking of marked T _h surface molecules (LFA-1, CD4, ICAM-1, CD3, α, β TCR) by mab did not interfere with the ability of activated T _h to induce B cell cycle entry. . In contrast, CD40-Ig or mab specific for CD40 binding protein inhibited T _h dependent B cell activation in a dose dependent manner. In addition, the CD40 binding protein was identified as a 39kD protein that is selectively expressed in the membrane of activated T _h but not expressed in resting T _h . Both CD40-Ig and mab specific for the 39kD CD40 binding protein inhibited B cell activation by PM ^ACT .

Although many membrane proteins have been associated with Th-dependent B-cell signaling, the evidence presented herein has lost the contribution of several molecules (LFA-1, CD4, CD3, alpha, beta, TCR, ICAm-1). And CD40 as a B-cell receptor for cognate signaling by Th. The data suggested that specific CD4Ig and mabs for CD40 binding protein inhibit Th-dependent B-cell activity.

The ligand for CD40 is 39Kd protein expressed on activated Th, not on resting period. Biochemical studies have suggested that the 39Kd protein is a short chain protein since it is not affected by the reducing agent in electrophoretic migration. Based on the functional studies presented herein, both activated Th1 and Th2 expressed 39kD CD40 binding protein. This is consistent with studies showing that both Th1 and Th2 induce B-cell cycle entries. To further characterize the 39 kD protein, cDNA encoding CD protein in the 39 kD range of MW (cD53, CD27, and CD69) was short-term transfected into COS cells and the cells were tested for CD40-Ig binding. None of the transfected COS cells expressed the protein bound to CD40-Ig. Thus, it was assumed that the 39 kD protein was not these CD proteins.

The biochemical basis for signal transduction between Th and B-cells is not clear. Identification of CD40 as a signaling molecule for T cell help has focused attention on specific biochemical pathways known to bind CD40 molecules. CD40 is a member of the neuronal growth factor (NGFR) family due to the presence of four cysteine enriched motifs in its extracellular domain. Signaling via CD40 by maps to include increased activity of inositol triphosphate and the activity of tyrosine kinases leading to the activation of four or more distinct serine / threonine kinases is described in UcKun et al., J. Biol. Chem. 266: 17478-17485 (1991). Based on information obtained from signaling through other members of the NGF receptor family, it was expected that the interaction between activated Th and B cells would result in many of the same biochemical processes.

For immunofluorescent binding studies, CD40 Ig fusion proteins were conjugated using biotinsuccinimide (sigma). Flow cytometry analysis was then performed in two steps of staining using phycoerythrin (PE) -streptavidin (Becton-Dickinson). Representative results of screening for multiple T-cell lines are shown below. The Jurkat and HSB2 cell lines were found to bind specifically, whereas CEM, HPBALL, and murine thyoma did not bind to the CD40 Ig fusion protein (FIG. 7).

Example 2

Use of anti-gp39 antibodies for the treatment and prevention of lupus

Systemic lupus erythematosus (SLE) is a disease characterized by the production of many toxic autoantibodies (Boumpas, Ann Int Med, 1995). These autoantibodies cause damage either directly through recognition of epitopes on normal cells or indirectly through the formation of immune complexes that accumulate in normal tissue or activate complementary cascades.

Studies of SLE and lupus disease in allogeneic lineages of mice, along with normal antibody responses, have clarified that lupus B cell autoimmune production depends on coordination from CD4 + T helper cells (Th). One embodiment was performed as a study of female offspring of NZB / NZW mice, a conventional SLE murine model infected with human SLE-like diseases in many respects, including the production of autoantibodies and the development of immune complex glomerulonephritis. Treatment for these mice depleting anti-CD4 antibodies prevented and substantially reversed nephritis. Unfortunately from a therapeutic point of view, simple therapeutic trials of anti-CD4 antibodies in human autoimmune disease have led to continued depletion of these crucial lymphocyte subsets in some cases. In addition, in diseases such as rheumatoid arthritis, clinical trials of a small number of anti-CD4 antibodies have disappointed in their effectiveness.

More specific immunosuppression has been made available due to the identification of several molecular interactions included in Th cell help, making it possible to target only Th cells actively involved in the aid of antibody preparation. One example is a second signal delivered by the interaction between B7.1 / B7.2 on B cells and CD28 / CTLA4 on Th cells. Blocking of antibodies against one or more participating molecules can provide in vitro hypoesponsiveness of T cells. Recently, fusion proteins consisting of extracellular domains of murine CTLA-4 linked to murine Ig Cg2a chains have been proposed to block antibody production and prolong survival when applied to NZB / NZW mice, even mice in advanced stages of exacerbation. This was presumably due to the competitive inhibition of binding of B7.2 and endogenous CTLA-4 in the treated mice.

Experiment result

We studied the effect of anti-CD40L antibody administration from SLE murine 4-10 months old in NZB / NZW mice. Anti-CD40L antibody treatment reduced anti-DNA autoantibody production and renal disease, and prolonged survival among these mice without causing a pronounced mismatch of the hormonal immune system in the generated or treated anti-antibody response. . These data are shown in FIG. At 11 months, 60% of the anti-CD40L antibody-treated mice survived, but none of the untreated or HIG treated mice survived (FIG. 9). Among the long-lived ones, histological examination of the kidneys showed negligible toxicity and no significant immune accumulation in the glomeruli.

The data obtained above also suggest that anti-CD40L antibody therapy may address two major concerns related to the use of anti-lymphocyte cell surface molecular antibodies for immunotherapy: development of anti-antibody responses and immunosuppressive treatment Lasting effect. Here, mice that reacted with anti-CD40L antibody treatment during 11 months of survival did not undergo anti-antibody responses, an almost inevitable phenomenon in animals that received antibodies derived from other species. We speculated that this was due to the prevention by the anti-CD40L antibody against the antibody preparation itself. In the study of normal mice treated with the same anti-CD40L antibody, MR-1, the absence of an anti-MR-1 response may indicate certain responsiveness that we observed in NZB / NZW mice in which the immune response was limited to autoimmune mice. Presented that. In some cases, these observations may be useful in the therapeutic application of anti-human CD40L antibodies, since the significant complexity of such antibody treatments can be avoided in combination with anti-CD40L antibody treatment, especially by using “humanized” forms of antibodies. Has an important relationship. In our study, mice undergoing anti-anti-CD40L antibody reactions were not as good as their controls in their anti-DNA antibody production or survival, which for any reason produced antibodies by a subset of the mice. It is shown that it was not inhibited by anti-CD40L. The development of anti-anti-CD40L antibodies subsequently led to the rapid removal of the administered anti-CD40L antibodies, resulting in a loss of therapeutic effect. In support of this finding, in mice treated with nonspecific hamster IgG, accumulation of hamster antibodies was identified in nephritis in a subset of mice treated with anti-CD40L antibody, but not in anti-CD40L antibody reactants. Did. This observation was consistent with anti-anti-CD40L antibody immune complexes formed and accumulated in consecutively administered kidneys of anti-CD40L and exacerbated rather than prevented glomerulonephritis.

Demonstration that the ability to undertake anti-KLH antibody responses is reversed at least four months later with the discontinuation of anti-CD40L antibody treatment, in which the immunosuppressive effects of antibodies on the humoral immune system are reversible, Thus, it has been confirmed that anti-CD40L antibody treatment may be suitable for therapeutic applications in humans.

In this example, NZB / NZW F1 mice were treated twice weekly with mice at 4-10 months of age with MR-1 200 ug. In addition, treatment was in accordance with the treatment regimen and the dose of MR-1 was increased to 250 mg three times a week for April to October mice. This dose was used in the collagen-induced model of rheumatoid arthritis and the treated mice survived 100% (Durie, Scirnce, 1993). Using this rule, NZB / NZW mice survived 100% in November, while the two mouse controls died by October (Figure 10).

Treatment of Chronic Lupus Disease

Although prevention of SLE is a concern in NZB / NXW F ₁ mice with anti-gp39 antibodies, this antibody is not a precursor to the utility of therapeutically inhibiting the active disease state. To determine if the chronicized disease can be reversed by antibody treatment, one group of mice developed 2-3 + proteinuria (equivalent to 100 mg / day to 500 mg / day) and was determined by urinary palpation. At this stage, mice were randomly divided and either continued treatment or injected with 100 μg MR-1 per day. All 10 untreated mice died at 10 months of age. In contrast, 5 of 10 mice treated with MR-1 were alive at 11 months of age. Three of these mice were healthy and free of proteinuria. Interestingly, one of these mice nevertheless had high titers of anti-DNA antibodies (see Table 1). Two of the five surviving animals became ill and exhibited high titers of anti DNA antibody and 4+ proteinuria.

Thus, it has been found that treatment with MR-1 after the development of kidney disease reverses the disease in some mice. Findings in mice responding that treatment with MR-1 reversed proteinuria but not anti-DNA antibody production raise the possibility of another mechanism for MR-1 effects in addition to the inhibition of T-dependent antibody production. .

It has also been shown that treatment of NZB / NZW mice with tumor necrosis factor (TNF-α) significantly delays the development of nephritis (Jacob, Science , 1988; Gordon, 1989]. Interestingly, the severe form of nephritis observed in NZB / NZW F ₁ mice is due in part to the dominant gene (s) from the NZW parent that is mapped to the H-2 complex. NZW mice had reduced levels of TNF-α correlated with the polymorphism of the TNF-α gene located in the H-2 complex. NZB / NZW F ₁ mice also significantly reduced TNF-α levels. In addition, treatment of NZB / NZW F ₁ mice with anti-IL-10 antibodies substantially delayed the onset of nephritis and prolonged survival. This beneficial effect is likely mediated by IL-10-induced upregulation of TNF-α in mice treated as a simultaneous treatment with anti-TNF-α antibodies that inhibited the anti IL-10 response.

In preliminary experiments, we also examined the levels of TNF-α by ELISA for mice from each of the four treatment groups before and after MR-1 administration. These levels are presented in Table 2. Only mice responding to MR-1 treatment with an increase in proteinuria and anti DNA antibody production showed an increase in TNF-α from 2.16 pg / ml before treatment to 35.01 pg / ml after treatment. These data suggest that MR-1 effects may be mediated, at least in part, by an increase in TNF-α in NZB / NZW F ₁ mice.

These results also demonstrate the utility of anti-gp39 antibodies and active fragments thereof for treating human lupus, ie systemic lupus erythematosus or drug-induced lupus. In particular, these antibodies can be used for the therapeutic adjustment of active and ongoing lupus, often characterized by nephritis. These antibodies even inhibited the progression of the disease and potentially reversed it.

Table 1

TABLE 2

mouse* Age of sample taken (months) TNF-α (pg / ml) # No treatment 2 5.5 9 3.1 MR-1 survival 2 2.2 9 35.0 MR-1 death 2 0.0 8 4.3

One mouse from each treatment group.

# Value representing the paired mean from the test using TNF-α ELISA kit from Genzyme, Cambridge, MA.

Various publications cited herein are incorporated by reference throughout this specification.

Claims (10)

A method of treating lupus in a subject in need of such treatment, comprising administering a therapeutically effective amount of an anti-gp39 antibody and fragment thereof that specifically binds gp39. The method of claim 1 wherein said lupus is systemic lupus erythematosus. The method of claim 1, wherein said lupus is drug induced lupus. The method of claim 1, wherein the antibody is an anti human gp39 antibody adapted to the human body. The method of claim 1, wherein the antibody is a chimeric anti human gp39 antibody. The method of claim 1, wherein said antibody is administered systemically. 7. The method of claim 6, wherein said antibody is administered to a lupus subject with lupus related kidney disease. 8. The method of claim 7, wherein said administration reduces and / or reverses renal disease as evidenced by reduced proteinuria after anti gp39 treatment. The method of claim 1, wherein the anti gp39 antibody is administered with a tumor necrosis factor. The method of claim 1, wherein the dosage of anti gp39 is in the range of 10 μg / ml to 1000 μg / ml.