ES2353222T3 - ANTI-BODIES ANTI-CD154 (BINDING CD40) AGLICOSILADOS AND USES OF THE SAME. - Google Patents

Abstract

Aglycosylated anti-CD154 antibody antibody or antibody, characterized by a modification in the N-linked site conserved of the CH2 domains of the Fc part of said antibody.

Description

Technical Field of the Invention [0001] The present invention relates to aglycosylated anti-CD154 antibodies or antibody derivatives thereof, which block the interaction of CD154 and CD40 molecules. Methods for producing aglycosylated anti-CD154 antibodies and antibody derivatives are described herein. The antibodies and antibody derivatives of the present invention are useful in the treatment and prevention of diseases that involve undesirable immune responses, and that are mediated by CD154-CD40 interactions.

Background of the invention [0002] The generation of humoral and cell-mediated immunity is orchestrated by the interaction of activated auxiliary T cells with antigen presenting cells ("APC") and effector T cells. The activation of the auxiliary T cells is not only dependent on the interaction of the receptor of the antigen-specific T cells ("TCR") with its cognate peptide ligand-MHC, but also requires coordinated binding and activation by numerous molecules of cell adhesion and costimulators [Salazar-Fontana, 2001]. [0003] A critical costimulatory molecule is CD154 (also known as ligand of CD40, CD40L, gp39, T-BAM, T-cell Activating Molecule, TRAP), a Type II transmembrane protein that is expressed temporarily and dependent on surface activation of CD4 + T cells. CD154 is also expressed, after activation, in a subset of CD8 + T cells, basophils, mast cells, eosinophils, natural cytolytic cells, B cells, macrophages, dendritic cells and platelets. [0004] The CD154 counterreceptor, CD40, is a Type I membrane protein that is expressed constitutively and generally on the surface of many cell types, including APCs [Foy, 1996]. [0005] The signaling through CD40 by CD154 initiates a cascade of events that produce the activation of the CD40 carrier cells and the optimal activation of the CD4 + T cells. More specifically, the cognate interaction between CD154 and CD4 promotes the differentiation of B cells into antibody secreting cells and memory B cells [Burkly, 2001]. Additionally, the interaction of CD154CD40 promotes cell-mediated immunity through the activation of macrophages and dendritic cells and the generation of natural cytolytic cells and cytotoxic T lymphocytes [Burkly, 2001].

[0006] The critical role of CD154 in regulating the function of the humoral and cell-mediated immune response has caused great interest in the use of inhibitors of this pathway for therapeutic immunomodulation [US Patent 5,474,771]. As such, it has been shown that anti-CD154 antibodies are beneficial in a wide variety of immune response models with other therapeutic proteins or gene therapy, allergens, autoimmunity and transplantation [US Patent 5,474,771; Burkly, 2001]. [0007] The CD40-CD154 interaction has been shown to be important in several experimentally induced autoimmune diseases, such as collagen-induced arthritis, experimental allergic encephalomyelitis ("EAE"), oophoritis, colitis, and drug-induced lupus nephritis. Specifically, it has been shown that disease induction in all these models can be blocked with CD154 antagonists at the time of antigen administration [Burkly, 2001]. [0008] Blocking the disease using anti-CD154 antagonists has also been observed in animal models of spontaneous autoimmune disease, including insulin-dependent diabetes and lupus nephritis, as well as in graft versus host disease, transplantation, pulmonary fibrosis. , and models of atherosclerosis disease [Burkly, 2001]. [0009] While it has been proven that glycosylated anti-CD154 antibodies are useful for the prevention and treatment of various diseases related to the immune response, in some subjects, the therapies that use them are sometimes complicated by thromboembolic activity [ Biogen Press Release, 2001; IDEC Press Release, 2001]. While the mechanism of this side effect is unknown, it may involve colligation by the anti-CD154 antibody, or its aggregates, of FcγRIIa and CD154 on platelets, which lead to inappropriate platelet activation. The binding of other Fcγ receptors and complement could also enhance this effect. Consequently, anti-CD154 antibody forms that do not bind effector receptors may be safer and / or more effective for therapeutic use. [0010] The mechanism by which anti-CD154 antibodies inhibit immune function may be more complex than simple binding to CD154 to block interactions with CD40 and, in effect, may include contributions by effector pathways. For example, antibody-antigen binding can induce suppression of activated T cells through the Fc domain of binding to Fcγ receptors or complement components. Alternatively, the binding of the antibody to CD154 can be increased by the formation of a framework of the cell surface of the antibody on the Fcγ receptor carrying cells. In addition, antibody access to its site of action can be promoted by Fcγ receptor binding interactions. [0011] In glycosylated antibodies, which include anti-CD154 antibodies, glycans bound to the N-linked site conserved in the CH2 domains of the Fc dimer are included among the CH2 domains, with the sugar residues that make contact with specific amino acid residues in the opposite CH2 domain [Jeffries, 1998]. In vitro studies with several glycosylated antibodies have shown that the removal of CH2 glycans alters the structure of Fc so that the binding of the antibody to the Fc receptors and the C1Q complement protein is greatly reduced [Nose, 1983; Leatherbarrow, 1985; Tao, 1989; Lund, 1990; Dorai, 1991; Hand, 1992; Leader, 1991; Pound, 1993; Boyd, 1995]. In vivo studies have confirmed the reduction of the effector function of aglycosylated antibodies. For example, an anti-CD8 aglycosylated antibody is unable to reduce CD8 carrier cells in mice [Isaacs, 1992] and an anti-CD3 aglycosylated antibody does not induce cytokine release syndrome in mice or humans [Boyd, 1995; Friend, 1999]. [0012] While the removal of glycans from the CH2 domain appears to have a significant effect on effector function, other functional and physical properties of the antibody remain unchanged. Specifically, it has been shown that the elimination of glycans had little to no effect on serum half-life and antigen binding [Nose, 1983; Tao, 1989; Dorai, 1991; Hand, 1992; Hobbs, 1992].

Summary of the invention [0013] In accordance with the present invention, the effector function of Fc involved in the mechanism of action of anti-CD154 antibodies is diluted by the use of an anti-CD154 antibody in which the effector function of Fc it has been reduced by a modification of the N-linked site conserved of the CH2 domains of the Fc dimer, which produces the "aglycosylated" anti-CD154 antibodies. Examples of such modifications include the mutation of the N-linked site conserved in the CH2 domains of the Fc dimer, the removal of glycans from the N-linked site of the CH2 domains and the impediment of glycosylation. To find out if the mechanism of inhibition with the anti-CD154 antibody depends on its Fc effector interactions, the anti-CD154 antibody and its aglycosylated counterpart were compared with respect to their ability to inhibit various diseases by blocking the CD154- interaction. CD40 The results reported herein demonstrate that the aglycosylated forms of a CD154 antibody are equally protective as the glycosylated forms of the anti-CD154 antibody. [0014] Because the aglycosylated anti-CD154 antibodies of the present invention are characterized by decreased effector function, these antibodies are particularly suitable for use in subjects where the potential for undesirable thromboembolic activity exists. Additionally, reducing the Fc effector function of aglycosylated anti-CD154 antibodies can reduce or eliminate other potential side effects of anti-CD154 antibody therapies, such as suppression of activated T cells and other populations of induced cells to express CD154 or Fc-dependent activation of monocytes / macrophages. [0015] Specifically, the present invention provides aglycosylated anti-CD154 antibodies that recognize CD154. More particularly, the present invention provides an aglycosylated, humanized anti-CD154 antibody - namely "aglycosylated hu5c8", and a murine aglycosylated anti-CD154 antibody - namely "muMR1-aglycosylated". [0016] In one embodiment of the present invention, the aglycosylated hu5c8 antibody is produced from the NS0 aglycosylated hu5c8 cell line, which was deposited in the American Type Culture Collection ("ATCC"), 10801 University Blvd., Manassas, Virginia on January 14, 2003 (Access No. PTA-4931), and the aglycosylated MR1 antibody is produced from the allycosylated murine MR1 cell line that was deposited with the ATCC on January 14, 2003 (Access No. PTA -4934). [0017] In one embodiment of the present invention, aglycosylated anti-CD154 antibodies are capable of inhibiting the interaction between CD154 and CD40. [0018] In another embodiment of the present invention, aglycosylated anti-CD154 antibodies can be associated with CD154 so as to block, directly or indirectly, the activation of CD40 carrier cells. [0019] Also described herein is a method of inhibiting an immune response in a subject, comprising administering to an individual an aglycosylated anti-CD154 antibody or an antibody derivative thereof, wherein the antibody or antibody derivative is administered in an amount effective to inhibit the activation of immune cells in the subject. [0020] Also described herein is a method of treating or preventing, in a subject, a condition or disease dependent on the immune response, which comprises administering to the subject an aglycosylated anti-CD154 antibody or one of its antibody derivatives, the antibody or antibody derivative that is administered in an amount effective to inhibit the activation of immune cells in the subject and thereby treat or prevent the condition or disease dependent on the immune response.

Brief description of the drawings [0021] FIG. 1 illustrates that the aglycosylated hu5c8 monoclonal antibody ("mAb") and glycosylated hu5c8 mAb bind to human CD154 with the same relative affinity. Binding of the biotinylated hu5c8 mAb to the cell surface CD154 competed with titers of the glycosylated hu5c8 mAb or unlabeled aglycosylated hu5c8 mAb. The mean fluorescence intensity of the biotinylated antibody detected with streptavidin-PE was plotted versus the concentration of the unlabeled antibody. The four parameter curve settings are illustrated collectively. [0022] FIG. 2 illustrates that mAb hu5c8 aglycosylated has better FcR binding capabilities. The ability of an anti-CD154 antibody, namely aglycosylated hu5c8 mAb, to form a bridge between huCD154 and FcγRI + cells (a) or between huCD154 + CHO cells and FcγRIII + cells (b) was evaluated. Fluorescence labeled FcγR + cells were added to microtiter plates containing glycosylated hu5c8 mAb or aglycosylated hu5c8 mAb that are bound to CD154. Bound FcγR + cells were detected by measuring the relative fluorescent units ("UFR") in each well using excitation / emission spectra, 485/530 nm. [0023] FIG. 3 illustrates that glycosylated hu5c8 mAb and aglycosylated hu5c8 mAb have the same serum half-life in cynomolgus monkeys. The concentrations of glycosylated hu5c8 mAb and aglycosylated hu5c8 mAb were measured in the serum of cynomolgus monkeys after administration of a single intravenous dose of 20 mg / kg. The mean serum concentrations for each treatment are represented ± standard deviation ("SD"). [0024] FIG. 4 illustrates that glycosylated hu5c8 mAb and aglycosylated hu5c8 mAb inhibit a primary immune response to tetanus toxoid ("TT"). The results are plotted for cynomolgus monkeys in the groups treated with mAb and control with saline solution in the study of glycosylated hu5c8 mAb (closed symbols) and in the aglycosylated hu5c8 mAb (open symbols). [0025] FIG. 5 illustrates that the aglycosylated hu5c8 mAb inhibits an immune response due to TT. Total primary antibody responses (closed bars) and secondary (open bars) (EAUC) antibody to TT for individual cynomolgus monkeys are plotted. Group 1A animals received saline solution before primary and secondary TT stimuli. Group 1B animals received saline solution before the primary TT stimulus and aglycosylated hu5c8 mAb before the secondary TT stimulus.

[0026] FIG. 6 illustrates the pharmacokinetics of the glycosylated murine chimeric MR1 antibody ("muMR1") and aglycosylated muMR1 in BALB / c mice. The results are plotted for the glycosylated muMR1 antibody (rhombus symbol) and aglycosylated muMR1 antibody (square symbol). [0027] FIG. 7 (A & B) illustrates that the aglycosylated anti-CD154 antibody decreases the autoantibody response to single chain (A) and double chain (B) DNA in SNF1 mice. The results for muMR1 antibody (rhombus symbol) and aglycosylated muMR1 antibody (square symbol) are plotted. The mulgG2a control antibody is shown as a triangle symbol. [0028] FIG. 8 illustrates that the aglycosylated anti-CD154 antibody decreases the development of glomerular nephritis in SNF1 mice. The histological scores of the compound are illustrated for mice treated with mulgG2a (controls), muMR1 and agMosylated muMR1. [0023] FIG. 9 illustrates that aglycosylated anti-CD154 antibodies delay the onset of glomerular nephritis in SNF1 mice. The results for muMR1 antibody (rhombus symbol) and aglylated muMR1 antibody (square symbol) are shown. The mulgG2a control antibody is shown as a triangle symbol. [0030] FIG. 10 illustrates that aglycosylated anti-CD154 antibodies delay the onset of increased blood urea nitrogen ("BUN") levels in SNF1 mice. The results for muMR1 antibody (rhombus symbol) and aglylated muMR1 antibody (square symbol) are shown. The mulgG2a control antibody is shown as a triangle symbol. [0031] FIG. 11 illustrates that aglycosylated anti-CD154 antibodies prevent an increase in serum creatinine in SNF1 mice. The results for muMR1 antibody (rhombus symbol) and aglylated muMR1 antibody (square symbol) are shown. The mulgG2a control antibody is shown as a triangle symbol. [0032] FIG. 12 illustrates that mice treated with the muMR1 antibody did not develop symptoms of experimental autoimmune encephalomyelitis ("EAE"), compared to mice treated with the P1.17 isotype control antibody. The results for muMR1 antibody (open circles) and control antibody P1.17 (closed circles) are illustrated. [0033] FIG. 13 illustrates that in mice treated with the aglycosylated muMR1 antibody, the aglycosylated muMR1 antibody was as effective in inhibiting the clinical signs of EAE as the muMR1 antibody. The results are illustrated indicating disability score (mean + standard error of the mean - "SEM") and% of the initial weight (mean + SEM) related to the days after the induction of the disease. P1.17 is an Ig control. [0034] FIG. 14 represents fasting blood glucose levels ("FBG") in rhesus monkeys after allogeneic islet transplantation. Acute rejection was defined as FBG> 100 mg / dl. Animals treated with aglycosylated hu5c8 MAb (broken lines) and glycosylated hu5c8 mAb (full lines) are illustrated.

Detailed description of the invention [0035] In order for the present invention to be fully understood, the following detailed description is set forth. Unless otherwise defined, all technical and scientific terms used herein have the same meaning commonly understood by those skilled in the art to which the present invention belongs. Examples of methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in the practice of the present invention and will be apparent to those skilled in the art. [0036] Throughout this application, several publications and references are mentioned in parentheses. The descriptions of these publications and references, in their entirety, are incorporated herein by reference in this application to more fully describe the state of the art to which the present invention belongs. The bibliographic citation for these references can be found in the text or listed by number after the Experimental Details section. In case of conflict, the present specification will govern. The materials, methods and examples are illustrative only and are not intended to be limiting. [0037] Standard reference works that set forth the general principles of recombinant DNA technology known to those skilled in the art include Ausubel et al., Current Protocols In Molecular Biology, John Wiley & Sons, New York (1998 and Supplements to 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Plainview, New York (1989); Kaufman et al., Eds., Handbook Of Molecular And Cellular Methods In Biology And Medicine, CRC Press, Boca Raton (1995); McPherson, Ed., Directed Mutagenesis: A Practical Approach, IRL Press, Oxford (1991). [0038] Standard reference works that set forth the general principles of immunology known to those skilled in the art include: Harlow and Lane, Antibodies: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1999); and Roitt et al., Immunology, 3d Ed., Mosby-Year Book Europe Limited, London (1993). Standard reference works that expose the general principles of physiology and medical pharmacology known to those skilled in the art include: Fauci et al., Eds., Harrison's Principles Of Internal Medicine, 14th Ed., McGraw-Hill Companies, Inc. ( 1998). [0039] The present description contemplates the use of antiglycosylated antibodies or derivatives thereof, to inhibit immune responses and treat diseases and conditions induced by immune responses - for example: autoimmune disease, allergy, transplant rejection, inflammation, graft versus host disease, fibrosis, and atherosclerosis. [0040] More specifically, aglycosylated anti-CD154 antibodies used for treatment, in particular for human treatment, include human antibodies, humanized antibodies, chimeric antibodies, polyclonal antibodies and multimeric antibodies. ANTIBODIES [0041] An antibody is an approximately 150 kD PM glycoprotein, which is produced by the humoral branch of the vertebrate immune system in response to the presence of foreign molecules in the body. An antibody or antibody derivative is capable of recognizing and binding to its specific antigen in vitro or in vivo, and can initiate any of the subsequent actions associated with antibody binding, including, for example, direct cytotoxicity, complement-dependent cytotoxicity ( "CDC"), antibody dependent cytotoxicity ("ADCC"), and antibody production. [0042] After binding to the antigen, the antibodies activate one or more of the effector systems of the immune system that contribute to the neutralization, destruction and elimination of the infecting microorganism or other entity that contains the antigen, for example, cancer cell. [0043] While natural antibodies are derived from a single species, genetically engineered antibodies and antibody fragments can be derived from more than one species of animal, for example, chimeric antibodies. To date, mouse (murine) / human and murine / non-human primate chimeric antibodies have been generated, although other combinations of species are possible. [0044] In one embodiment, the aglycosylated anti-CD154 antibodies of the present invention are chimeric antibodies. Normally, the chimeric antibodies include the heavy and / or light chain variable regions, which include residues of the complementarity determining region ("CDR") and structure, of one species, (usually mouse) fused with the constant regions of another species (normally human). These mouse / human chimeric antibodies contain approximately 75% human and 25% mouse amino acid sequences, respectively. Human sequences represent the counting regions of the antibody, while mouse sequences represent the variable regions (and thus contain the antigen binding sites) of the antibody. [0045] The rationale for using such chimeras is to retain the specificity of the mouse antibody antigen but reduce the immunogenicity of the mouse antibody (a mouse antibody can cause an immune response against it in different species than the mouse) and consequently be able to use the chimera in human therapies. [0046] In another specific embodiment, the aglycosylated anti-CD154 antibodies of the present invention include chimeric antibodies comprising structural regions of one antibody and CDR regions of another antibody. [0047] In a more specific embodiment, the aglycosylated anti-CD154 antibodies of the present invention include chimeric antibodies comprising CDR regions of different human antibodies. [0048] In another specific embodiment, the aglycosylated anti-CD154 antibodies of the present invention include chimeric antibodies comprising the CDR regions of at least two different human antibodies. [0049] The methods of obtaining all the chimeric antibodies described above are well known to those skilled in the art [US Patent 5,807,715; Morrison, 1984; Sharon, 1984; Takeda, 1985]. [0050] In another embodiment of the present invention, aglycosylated anti-CD154 antibodies also include primatized, humanized and fully human antibodies. Primatized and humanized antibodies normally include heavy and / or light chain CDR of a murine antibody grafted onto a structural region V of the non-human or human primate antibody, usually also comprising a human constant region [Riechmann, 1988; Co, 1991; U.S. Patents 6,054,297; 5,821,337; 5,770,196; 5,766,886; 5,821,123; 5,869,619; 6,180,377; 6,013,256; 5,693,761; and 6,180,370].

1. Humanized antibodies [0051] A humanized antibody is an antibody produced by recombinant DNA technology, in which some or all of the amino acids of a light or heavy chain of human immunoglobulin that are not necessary for antigen binding (for example, the constant regions and the structural regions of the variable domains) are used to replace with the corresponding amino acids of the light or heavy chain of the cognate non-human antibody. By way of example, a humanized version of a murine antibody for a given antigen has in its heavy and light chains (1) constant regions of a human antibody; (2) structural regions of the variable domains of a human antibody; and (3) CDR of the murine antibody. When necessary, one or more residues of the human structural regions can be exchanged for residues in the corresponding positions of the murine antibody so as to preserve the binding affinity of the humanized antibody to the antigen. This change is sometimes called "retromutation." Generally, humanized antibodies are less likely to induce an immune response in humans compared to chimeric human antibodies because the former contain considerably less non-human components. The methods for obtaining humanized antibodies are well known to those skilled in the art of antibodies [European Patent 239400; Jones, 1986; Riechmann, 1988; Verhoeyen, 1988; Queen, 1989; Orlandi, 1989; US patent 6,180,370]. [0052] In one embodiment of the present invention, humanized antibodies are generated by the transplantation of murine (or other non-human) CDRs into a human antibody. More specifically, this is obtained as follows: (1) cDNAs encoding the heavy and light chain variable domains are isolated from a hybridoma; (2) the DNA sequences of the variable domains, which include the CDRs, are determined by sequencing; (3) the DNAs encoding the CDRs are transferred to the corresponding regions of a heavy or light chain variable domain coding sequence of the human antibody by site-directed mutagenesis; and (4) the segments of the human constant region gene of a desired isotype (for example, 1 for CH and k for CL) are added. Finally, the humanized heavy and light chain genes are coexpressed in mammalian host cells (eg, CHO or NS0 cells) to produce soluble humanized antibody. [0053] Sometimes, the direct transfer of the CDRs to the human structure leads to the loss of antigen binding affinity of the resulting antibody. This is because in some cognitive antibodies, certain amino acids in the structural regions interact with the CDRs and consequently influence the affinity of binding to the total antigen of the antibody. In such cases, it may be critical to introduce "retromutations" into the structural regions of the acceptor antibody in order to retain the antigen binding activity of the cognate antibody. The general methods of obtaining retromutations are well known to those skilled in the art [Queen, 1989; Co, 1991; PCT patent application WO 90/07861; Tempest, 1991].

2. Human Antibodies [0054] In one embodiment of the present invention, antibodies and antibody derivatives are fully human aglycosylated anti-CD154 antibodies. [0055] In a more particular embodiment of the present invention, fully human antibodies are prepared using in vitro activated human splenocytes, [Boerner, 1991] or phage deployed antibody libraries [US Patent 6,300,064]. [0056] In a more particular embodiment of the present invention, fully human antibodies are prepared by cloning the repertoire [Persson, 1991; Huang and Stollar, 1991]. In addition, US Patent 5,798,230 describes the preparation of human monoclonal antibodies of human B cells, in which the B cells producing the human antibody are immortalized by infection with an Epstein-Barr virus or a derivative thereof, which expresses the nuclear antigen of the Epstein-Barr 2 virus ("EBNA2"), a protein required for immortalization. The EBNA2 function is subsequently deactivated, resulting in an increase in antibody production. [0057] Other methods for producing fully human antibodies involve the use of non-human animals that have inactivated endogenous IG loci and are transgenic for the heavy chain and light chain genes of the rearranged human antibody. Such transgenic animals can be immunized with activated T cells or D1.1 protein [US Patent 5,474,771; U.S. Patent 6,331,433; US Patent 6,455,044] and hybridomas can be generated from the B cells derived therefrom. The details of these methods are described in the art. See, for example, the various publications / patents of GenPharm / Medarex (Palo Alto, CA) for transgenic mice containing minilocus of human Ig, including US Patent 5,789,650; the various publications / patents of Abgenix (Fremont, CA) with respect to XENOMOUSE® mice, which include US patents 6,075,181, 6,150,584 and 6,162,963; Green, 1997; Mendez, 1997; and the various publications / patents of Kirin (Japan) regarding "transomic" mice, which include European Patent 843961 and Tomizuka, 1997.

GENERATION OF DEGLYCOSILATED ANTIBODIES [0058] There are currently two ways to reduce the effector function of a mAb while retaining the other valuable attributes of the Fc part of it. One way to modify an antibody is to mutate amino acids on the surface of the mAb that are involved in effector binding interactions [European Patent 239400; Jefferies, 1998]. While it is likely that some combination of the mutations will result in adequate reduction of effector function, surface mutant antibodies that have been tested so far appear to retain residual activity. Another problem with this approach is that changes in amino acids on the surface of the mAb can cause immunogenicity. [0059] The present invention relates to aglycosylated anti-CD154 antibodies or antibody derivatives with diminished effector function, characterized by a modification in the N-linked site conserved in the CH2 domains of the Fc part of said antibody. [0060] In one embodiment of the present invention, the modification comprises a mutation at the glycosylation site of the heavy chain to prevent glycosylation at the site. Accordingly, in a preferred embodiment of the present invention, aglycosylated anti-CD154 antibodies or antibody derivatives are prepared by mutation of the heavy chain glycosylation site, i.e., the N298Q mutation (N297 using EU numbering from Kabat) and express in an appropriate host cell. For example, this mutation can be obtained following the manufacturer's recommended protocol for the Amersham-Pharmacia Biotech single site mutagenesis kit (Piscataway, NJ, USA). The mutated antibody can be stably expressed in a host cell (eg, NS0 or CHO cell) and subsequently purified. As an example, purification can be carried out using protein A chromatography and gel filtration. It will be apparent to those skilled in the art that additional methods of expression and purification can also be used. [0061] In another embodiment of the present invention, aglycosylated anti-CD154 antibodies or antibody derivatives have decreased effector function, in which the modification at the N-linked site conserved of the CH2 domains of the Fc part of said antibody or antibody derivative comprises the removal of glycans from the CH2 domain, that is, deglycosylation. These aglycosylated anti-CD154 antibodies can be generated by conventional methods and subsequently enzymatically deglycosylated. Methods for enzymatic deglycosylation of antibodies are well known to those skilled in the art [Williams, 1973; Winkelhake & Nicolson, 1976]. [0062] In another embodiment of the present invention, deglycosylation can be obtained using the tunicamycin glycosylation inhibitor [Nose & Wigzell, 1983]. That is, the modification is the impediment of glycosylation at the N-linked site conserved in the CH2 domains of the Fc part of said antibody. [0063] In other embodiments of the present invention, recombinant CD154 polypeptides (or cell or cell membranes containing said polypeptides) can be used as an antigen to generate an anti-CD154 antibody or antibody derivatives, which can subsequently be deglycosylated. . The antigen can be mixed with an adjuvant or ligated to a hapten to increase antibody production. [0064] Whether the modifications of the aglycosylated antibodies or derivatives of the antibody of the present invention are produced by the site-directed mutation or by enzymatic deglycosylation methods described above, the basis for antibody generation is well known to those skilled in the art. in the technique For example, protocols for immunizing non-human mammals are well established in the art [Harlow, 1998; Coligan, 2001; Zola, 2000]. [0065] After immunization, the antibodies or antibody derivatives of the present invention can be produced using any conventional technique. See, for example, Howard, 2000; Harlow, 1998; Davis, 1995; Delves, 1997; Kenney, 1997. [0066] In some embodiments of the present invention, the host cells may be, for example, (1) bacterial cells, such as, E. coli, Caulobacter crescentus, Streptomyces species, and Salmonella typhimurium; (2) yeast cells, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Pichia methanolica; (3) insect cell lines, such as those of Spodoptera frugiperda for example, Sf9 and Sf21 cell lines, and expresSF ™ cells (Protein Sciences Corp., Meriden, CT, USA) - Drosófila S2 and Trichoplusia cells in High Five® Cells (Invitrogen, Carlsbad, CA, USA); or (4) mammalian cells. Typical mammalian cells include COS1 and COS7 cells, Chinese hamster ovary (CHO) cells, NS0 myeloma cells, NIH 3T3 cells, 293 cells, HEPG2 cells, HeLa cells, L cells, HeLa, MDCK, HEK293, WI38 , murine ES cell lines (eg, from strains 129 / SV, C57 / BL6, DBA-1, 129 / SVJ), K562, Jurkat cells, and BW5147. Other mammalian cell lines are well known and readily available in the American Type Culture Collection ("ATCC") (Manassas, VA, USA) and the National Institute of General Medical Sciences (NIGMS) Human Genetic Cell Repository at the Coriell Cell Repositories ( Camden, NJ, USA). These cell types are representative only and do not mean an exhaustive list. [0067] In another embodiment of the present invention, aglycosylated anti-CD154 antibodies or antibody derivatives are prepared by cell free translation. [0068] In another embodiment of the present invention, aglycosylated anti-CD154 antibodies or antibody derivatives are produced in bioreactors containing cells expressing the antibody, in order to facilitate large-scale production. [0069] In another embodiment of the present invention, aglycosylated anti-CD154 antibodies or antibody derivatives are produced in transgenic mammals (eg, goats, cows or sheep) that express the antibody in milk, in order to facilitate production in large-scale aglycosylated anti-CD154 antibodies [US Patent 5,827,690; Pollock, 1999]. [0070] As indicated above, aglycosylated anti-CD154 antibodies or antibody derivatives of the present invention can be produced in prokaryotic and eukaryotic cells. The invention accordingly also provides cells that express the antibodies of the present invention, which include hybridoma cells, B cells, plasma cells, as well as recombinantly modified host cells to express the antibodies of the present invention. [0071] Among other considerations, some of which were described above, a host cell strain can be chosen for its ability to process the expressed CD154 protein as desired. In addition to aglycosylation, such post-translation modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, phosphorylation, lipidation and acylation, and it is an aspect of the present invention to provide aglycosylated anti-CD154 antibodies or antibody derivatives with a

or more of these post-translation modifications. ANTIBODY MODIFICATIONS [0072] When administered, antibodies are often rapidly removed from the circulation and can consequently induce a relatively short pharmacological activity. Consequently, frequent injections of relatively large doses of antibodies may be necessary to support the therapeutic efficacy of the antibody treatment. [0073] In one embodiment of the present invention, aglycosylated anti-CD154 antibodies or antibody derivatives can be modified (ie, binding to other moieties) to increase the integrity and longevity of the antibody in vivo. For example, aglycosylated anti-CD154 antibodies or antibody derivatives of the present invention can be modified to include a moiety that can increase stabilization, thereby prolonging the serum half-life of the antibody. [0074] In some embodiments of the present invention the aglycosylated anti-CD154 antibodies are modified by the covalent bonding of water-soluble polymers, such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone

- all of which are known to exhibit substantially longer half-lives in blood after intravenous injection than the corresponding unmodified proteins [Abuchowski, 1981; Anderson, 1992; Newmark, 1982; Katre, 1987]. [0075] Antibody modifications can also increase the solubility of the protein in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the protein, and greatly reduce the immunogenicity and antigenicity of the protein. As a result, the desired in vivo biological activity can be obtained by the administration of such polymeric protein adducts less frequently or in smaller doses than with the unmodified protein. [0076] In some embodiments of the present invention, aglycosylated anti-CD154 antibodies are modified by labeling with a detectable label, for example, a radioactive isotope, enzyme, dye or biotin. [0077] In some embodiments of the present invention, aglycosylated anti-CD154 antibodies or antibody derivatives are modified by conjugating with a therapeutic agent, for example, a radioisotope or radionuclide (eg, 111In or 90Y), toxin residue ( for example, tetanus toxoid or ricin), toxoid or chemotherapeutic agent [US Patent 6,307,026]. [0078] In some embodiments of the present invention, aglycosylated anti-CD154 antibodies or antibody derivatives are modified by conjugating with an image display agent. Image display agents may include, for example, a labeling group (eg, biotin, fluorescent moieties, radioactive moieties, histidine label or other peptide tags) for simple isolation or detection. ANTIBODY DERIVATIVES [0079] The present invention also relates to derivatives of the aglycosylated anti-CD154 antibody. All the methods and reagents described above with respect to aglycosylated anti-CD154 antibodies can be used to produce derivatives of the aglycosylated anti-CD154 antibody of the present invention. [0080] In some embodiments of the present invention, derivatives of the aglycosylated anti-CD154 antibody include heteromeric antibody complexes and antibody fusions, such as bispecific antibodies, hemidimeric antibodies, multivalent antibodies (ie, tetravalent antibodies) and chain antibodies simple. A hemidimeric antibody is made up of an Fc part and a Fab part. A single chain antibody is made up of variable regions linked by protein spacers in a single protein chain. [0081] In some embodiments of the present invention, derivatives of the aglycosylated anti-CD154 antibodies of the present invention may also include proteins containing one or more light chains and / or heavy immunoglobulin chains, such as monomers and homo-o hetero-multimers (for example, dimers or trimers) of these chains, where these chains are optionally sulfur-linked or otherwise crosslinked. These antibody derivatives may be able to bind to one or more antigens. [0082] According to an alternative embodiment, the present invention includes aglycosylated antigen binding fragments of whole antibodies, such as Fab, Fab ’, F (ab’) 2 and F (v) fragments. In a further embodiment, the present invention includes antigen binding fragments of whole antibodies, such as Fab, Fab ’, F (ab’) 2 and F (v) antibody fragments. CELL LINES [0083] The present invention also provides cell lines that produce the aglycosylated anti-CD154 antibodies described herein. One such cell line, which produces the aglycosylated hu5c8 antibody, was deposited on January 14, 2003 at the ATCC, 10801n University Blvd., Manassas, Virginia, 20110-2209, USA, under the conditions of the Budapest Treaty for Recognition International Deposit of Microorganisms for the Purpose of Patent Procedure and was assigned ATCC Access No. PTA-4931. A second cell line, which produces the murine chimeric aglycosylated mu5c8 antibody, was deposited on January 14, 2003 at the ATCC, 10801 University Blvd., Manassas, Virginia, 20110-2209, USA, under the conditions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms with the Purpose of Patent Procedure and was assigned ATCC Access No. PTA-4934. THERAPEUTIC METHODS [0084] In one embodiment of the present invention, an aglycosylated anti-CD154 antibody, or an antibody derivative thereof, or a pharmaceutical composition comprising the antibody or antibody derivative, is capable of inhibiting an immune response in a subject. The antibody, antibody derivative or pharmaceutical composition is administered to the subject in an effective inhibitory amount. [0085] An "effective inhibitory amount" of an antibody, antibody derivative or pharmaceutical composition is any amount that is effective to inhibit the CD154-CD40 interaction in the subject to which it is administered. The methods of determining an "inhibitory amount" are well known to those skilled in the art and depend on factors that include, but are not limited to the type of subject involved, the size of the subject and the therapeutic agent administered. [0086] In a specific embodiment of the present invention, the aglycosylated antiCD154 antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative is capable of binding to the CD154 protein molecule. [0087] In a specific embodiment of the present invention, the aglycosylated antiCD154 antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative is capable of binding to the CD154 protein to which the hu5c8 specifically binds aglycosylated produced by the cell line that has the ATCC Access No. PTA-4931. [0088] In a specific embodiment of the present invention, the aglycosylated antiCD154 antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative is capable of binding to the epitope of CD154 to which the aglycosylated hu5c8 produced by the cell line having the ATCC Access No. PTA-4931, and in which the aglycosylated anti-CD154 antibody or antibody derivative is characterized by a mutation of N298Q (N297 using Kabat EU numbering). [0089] In a specific embodiment of the present invention, the aglycosylated antiCD154 antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative does not bind to an effector receptor. In a more specific embodiment of the present invention, the aglycosylated anti-CD154 antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative is capable of binding to the CD154 protein to which the produced aglycosylated hu5c8 specifically binds by the cell line having ATCC Access No. PTA-4931, and in which the anti-CD154 aglycosylated antibody or derivative of antibody or pharmaceutical composition does not bind to an effector receptor. [0090] In a specific embodiment of the present invention, the aglycosylated anti-CD154 antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative does not cause thrombosis. In a more specific embodiment of the present invention, the aglycosylated anti-CD154 antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative is capable of binding to the CD154 protein to which the produced aglycosylated hu5c8 specifically binds by the cell line that has the ATCC Access No. PTA-4931, and in which the aglycosylated anti-CD154 antibody or derivative of antibody or pharmaceutical composition does not cause thrombosis. [0091] In another specific embodiment of the present invention, the aglycosylated antiCD154 antibody, antibody derived or pharmaceutical composition comprising the antibody or antibody derivative is capable of inhibiting the immune response by inhibiting the CD154-CD40 interaction. [0092] In one embodiment of the present invention, the aglycosylated anti-CD154 antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative is capable of inhibiting inflammation. For the purposes of the present invention, inflammatory responses are characterized by redness, edema, heat and pain, as a consequence of capillary dilation with edema and migration of phagocytic leukocytes. Some examples of inflammatory responses include: arthritis, contact dermatitis, hyper-IgE syndrome, inflammatory bowel disease, allergic asthma, and idiopathic inflammatory disease [Gallin, 1989]. Some examples of arthritis include: rheumatoid arthritis, non-rheumatoid inflammatory arthritis, arthritis associated with Lyme disease and inflammatory osteoarthritis. Some examples of idiopathic inflammatory disease include: psoriasis and systemic lupus. [0093] In one embodiment of the present invention, the aglycosylated anti-CD154 antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative is capable of inhibiting rejection by the subject of a transplanted organ. [0094] In a more specific embodiment of the present invention, the aglycosylated antiCD154 antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative is capable of inhibiting the rejection by the subject of a heart, kidney, liver transplant , skin, cells of the pancreatic islet or bone marrow. [0095] In one embodiment of the present invention, the aglycosylated anti-CD154 antibody, antibody derived or pharmaceutical composition comprising the antibody or antibody derivative is capable of inhibiting graft versus host disease in a subject. [0096] In one embodiment of the present invention, the aglycosylated anti-CD154 antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative is capable of inhibiting allergic responses in a subject - for example, hay fever or an allergy to penicillin or other drugs. [0097] In one embodiment of the present invention, the aglycosylated anti-CD154 antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative is capable of inhibiting the autoimmune response in a subject suffering from autoimmune disease. Examples of autoimmune disease include, but are not limited to: rheumatoid arthritis, myasthenia gravis, systemic lupus erythematosus, Graves disease, idiopathic thrombocytopenic purpura, hemolytic anemia, diabetes mellitus, inflammatory bowel disease, Crohn's disease, multiple sclerosis, psoriasis, and diseases drug-induced autoimmune, for example, drug-induced lupus. [0098] In one embodiment of the present invention, the aglycosylated antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative is capable of inhibiting an autoimmune response in a subject suffering from an autoimmune response deriving from a disease. infectious [0099] In one embodiment of the present invention, the aglycosylated antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative is capable of inhibiting an autoimmune response in a subject suffering from an autoimmune response derived from the syndrome of Reiter, spondyloarthritis, Lyme disease, HIV infections, syphilis or tuberculosis. [0100] In one embodiment of the present invention, the aglycosylated antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative is capable of inhibiting fibrosis in a subject. [0101] Some examples of fibrosis include: pulmonary fibrosis or fibrotic disease. Some examples of pulmonary fibrosis include: pulmonary fibrosis resulting from adult respiratory distress syndrome, drug-induced pulmonary fibrosis, idiopathic pulmonary fibrosis, or hypersensitivity pneumonitis. Some examples of fibrotic diseases include: hepatitis C; hepatitis B; cirrhosis; cirrhosis of the liver as a result of a toxic attack; cirrhosis of the liver as a result of drugs; liver cirrhosis due to a viral infection; and cirrhosis of the liver due to an autoimmune disease. [0102] In one embodiment of the present invention, the aglycosylated antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative is capable of inhibiting gastrointestinal disease. Some examples of gastrointestinal disease include: esophageal dysmotility, inflammatory bowel disease and scleroderma. [0103] In one embodiment of the present invention, the aglycosylated antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative is capable of inhibiting vascular disease. Some examples of vascular disease include: atherosclerosis or reperfusion injury. [0104] In one embodiment of the present invention, the aglycosylated antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative is capable of inhibiting the proliferation of T-cell tumor cells in a subject suffering from a cancer of T cells, for example, a leukemia

or T-cell lymphoma. Such aglycosylated anti-CD154 antibody or antibody derivative or pharmaceutical composition can be administered to the subject in an amount effective to inhibit the proliferation of T-cell tumor cells in this subject. [0105] In one embodiment of the present invention, the aglycosylated antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative is capable of inhibiting viral infection of a subject's T cells by HTLV I virus. Such aglycosylated anti-CD154 antibody, antibody derivative or pharmaceutical composition can be administered to the subject in an amount effective to inhibit viral infection. [0106] In one embodiment of the present invention, the aglycosylated antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative is capable of detecting by tumor cells or neoplastic cells in a subject that expresses a protein that is specifically recognized by the aglycosylated hu5c8 produced by the cell line that has ATCC Access No. PTA-4931. A method of detecting tumor cells or neoplastic cells in a subject by image comprises the steps of: administering to the subject an effective amount of aglycosylated anti-CD154 antibody, derived from antibody, or the pharmaceutical composition under conditions that allow the formation of a complex between the antibody or antibody derivative and a protein on the surface of tumor cells or neoplastic cells; and to detect by image any antibody / protein complex or antibody derivative / complex formed, in this way any of the tumor cells or neoplastic cells in the subject are detected by image. [0107] In one embodiment of the present invention, the aglycosylated antibody, antibody derivative or pharmaceutical composition comprising the antibody or antibody derivative is capable of detecting the presence of tumor cells or neoplastic cells in a subject expressing a protein that is specifically recognized by the aglycosylated hu5c8 produced by the cell line that has ATCC Access No. PTA-4931. One of these methods for detecting the presence of tumor cells or neoplastic cells in a subject comprises the steps of: administering to the subject an effective amount of aglycosylated antibody, derived from antibody, or the pharmaceutical composition under conditions that allow the formation of a complex between the antibody or antibody derivative and a protein; remove any unbound image display agent from the subject; and detecting the presence of any antibody / protein complex or antibody derivative / complex formed, the presence of such a complex indicates the presence of tumor cells or neoplastic cells in the subject. PHARMACEUTICAL COMPOSITIONS [0108] The present invention provides pharmaceutical compositions comprising an aglycosylated anti-CD154 antibody or antibody derivative, which is described herein. [0109] In one embodiment of the present invention, the pharmaceutical composition comprises one or more aglycosylated anti-CD154 antibodies or antibody derivatives. [0110] In another embodiment of the present invention, the pharmaceutical compositions may further comprise a pharmaceutically acceptable carrier, an adjuvant, an administration vehicle, a buffer or a stabilizer. [0111] In a more particular embodiment of the present invention, the pharmaceutically acceptable carrier is phosphate buffered saline, physiological saline, water, citrate / sucrose / Tween formulations and emulsions - for example, oil / water emulsions. [0112] In one case, the pharmaceutical composition can be administered in a microencapsulation device to reduce or prevent the host's immune response against the protein. The antibody or antibody derivative can also be administered microencapsulated in a membrane, such as a liposome. [0113] In one embodiment of the present invention, the pharmaceutical composition may be in the form of a sterile injectable preparation, for example, a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to techniques known in the art using suitable dispersing agents, humectants and suspending agents. [0114] In one case, the pharmaceutical composition can be administered orally, topically or intravenously. [0115] In a more specific embodiment of the present invention, for oral administration, the pharmaceutical composition is formulated in a suitable capsule, tablet, suspension or aqueous solution. The solid formulations of the compositions for oral administration may contain suitable carriers or excipients, such as corn starch, gelatin, lactose, acacia, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, calcium carbonate, sodium chloride

or alginic acid. Disintegrants that can be used include, without limitation, microcrystalline cellulose, corn starch, sodium starch glycolate and alginic acid. Tablet binders that can be used include acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine (Povidone ™), hydroxypropyl methylcellulose, sucrose, starch, and ethyl cellulose. Lubricants that can be used include magnesium stearates, stearic acid, silicone fluid, talc, waxes, oils and colloidal silica. [0116] In a more specific embodiment of the present invention, for topical applications, pharmaceutical compositions may be formulated in a suitable ointment. Some examples of formulations of a composition for topical use include: drops, tinctures, lotions, creams, solutions, and ointments containing the active ingredient and various carriers and carriers. [0117] In one embodiment of the present invention, a topical semi-solid ointment formulation typically comprises a concentration of the active ingredient from about 1 to 20%, for example, 5 to 10%, in a carrier, such as a cream base. Pharmaceutical [0118] In one embodiment of the present invention, pharmaceutical compositions for inhalation and transdermal compositions can also be easily prepared. [0119] In one embodiment of the present invention, liquid formulations of a pharmaceutical composition for oral administration prepared in water or other aqueous vehicles may contain various suspending agents such as methylcellulose, alginates, tragacanth, pectin, kelgina, carrageenan, acacia , polyvinylpyrrolidone, and polyvinyl alcohol. Liquid formulations of the pharmaceutical compositions of the present invention may also include

solutions, emulsions, syrups and elixirs containing, together with the active compounds, wetting agents, sweeteners, and coloring and flavoring agents. Various liquid and powder formulations of the pharmaceutical compositions can be prepared by conventional methods for inhalation in the lungs of the treated mammal. [0120] In one embodiment of the present invention, the liquid formulations of a pharmaceutical composition for injection comprise various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols - that is , glycerol, propylene glycol, liquid polyethylene glycol, and the like. In some embodiments, the composition includes a citrate / sucrose / tween carrier. For intravenous injections, water soluble versions of the compositions can be administered by the drip method, by which a pharmaceutical formulation containing the antifungal agent and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients. A suitable insoluble form of the composition can be prepared and administered as a suspension in an aqueous base

or a pharmaceutically acceptable oily base, such as an ester of a long chain fatty acid - for example, ethyl oleate. [0121] In one embodiment of the present invention, the pharmaceutical composition comprises about 0.1 to 90% by weight (such as 1 to 20% or 1 to 10%) of an aglycosylated anti-CD154 antibody or antibody derivative of this, in a pharmaceutically acceptable carrier. [0122] In one embodiment of the present invention, the optimal percentage of the antibody

or antibody derivative in each pharmaceutical composition varies according to the formulation itself and the desired therapeutic effect in the specific pathologies and correlated therapeutic regimens. The pharmaceutical formulation is well established in the art [Gennaro, 2000; Ansel, 1999; Kibbe, 2000]. Conventional methods, known to those skilled in the art of medicine, can be used to administer the pharmaceutical composition to the subject. [0123] In some embodiments of the present invention, the pharmaceutical composition further comprises an immunosuppressive or immunomodulatory compound. For example, such an immunosuppressive or immunomodulatory compound may be one of the following: an agent that interrupts costimulatory signaling of T cells by means of CD28; an agent that interrupts calcineurin signaling, a corticosteroid, a

antiproliferative agent, and an antibody that specifically binds to a protein expressed on the surface of immune cells, including but not limited to CD45, CD2, IL2R, CD4, CD8 and RANK FcR, B7, CTLA4, TNF, LTβ, and VLA-4. [0124] In some embodiments of the present invention, the immunosuppressive or immunomodulatory compound is tacrolimus, sirolimus, mycophenolate mofetil, mizorubin, deoxyspergualin, brequinar sodium, leflunomide, rapamycin

or azaspirano. [0125] In other embodiments of the present invention, antibodies, antibody derivatives or pharmaceutical compositions comprising them may be included in a container, container or dispenser alone or as part of a kit with labels and instructions for administration. ADMINISTRATION AND ROUTES OF APPLICATION [0126] Aglycosylated anti-CD154 antibodies or antibody derivatives thereof, and the pharmaceutical compositions of the present invention may be administered to a subject in any manner that is medically acceptable. For the purposes of the present invention, "administration" means any of the standard methods of administering an antibody, antibody derivative or pharmaceutical composition known to those skilled in the art, and should not be limited to the examples provided herein. [0127] In some cases, aglycosylated anti-CD154 antibodies, antibody derivatives and pharmaceutical compositions can be administered to a subject by injection intravenously, subcutaneously, intraperitoneally, intramuscularly, intramedullary, intraventricular, intraepidural, intraarterial, intravascular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intraspinal, intratumoral, intracranial, enteral, intrapulmonary, transmucosal, intrauterine, sublingual, or local at sites of inflammation or tumor growth by standard methods. [0128] In some cases, aglycosylated anti-CD154 antibodies, antibody derivatives and pharmaceutical compositions can be administered to a subject by routes including oral, nasal, ophthalmic, rectal, or topical. [0129] In a more specific case, aglycosylated anti-CD154 antibodies, antibody derivatives and pharmaceutical compositions of the present invention can be administered to a subject orally in the form of capsules, tablets, suspensions or aqueous solutions. [0130] In a more specific case, aglycosylated anti-CD154 antibodies, antibody derivatives and pharmaceutical compositions can be administered to

a subject topically by the application of a cream, ointment or the like. [0131] In other cases, aglycosylated anti-CD154 antibodies, antibody derivatives and pharmaceutical compositions of the present invention can also be administered by inhalation through the use of a nebulizer, a dry powder inhaler or a metered dose inhaler. [0132] In other cases, aglycosylated anti-CD154 antibodies, antibody derivatives and pharmaceutical compositions can be administered to a subject by sustained release administration, such as depot injections of erodible implants directly applied during surgery. or by the implantation of an infusion pump or a biocompatible sustained release implant in the subject. [0133] In a more specific case, aglycosylated anti-CD154 antibodies, antibody derivatives and pharmaceutical compositions of the present invention can be administered to a subject by routes of administration of an injectable reservoir, such as by the use of materials. and biodegradable or injectable methods of 1, 3 or 6 months deposit. [0134] In a more specific case, aglycosylated anti-CD154 antibodies, antibody derivatives and pharmaceutical compositions of the present invention can be administered to a subject by application on the subject's skin of a transdermal patch containing the antibody. , the antibody derivative or the pharmaceutical composition, and leaves the patch in contact with the subject's skin, generally for 1 to 5 hours per patch. [0135] In other cases, aglycosylated anti-CD154 antibodies, antibody derivatives and pharmaceutical compositions can be administered to a subject at any dose by body weight and any dosage frequency that is medically acceptable. The acceptable dose includes a range between about 0.01 and 200 mg / kg of the subject's body weight. [0136] In other cases, aglycosylated anti-CD154 antibodies, antibody derivatives and pharmaceutical compositions of the present invention can be administered to a subject repeatedly at intervals ranging from one every day to one each month. [0137] In one case, aglycosylated anti-CD154 antibodies, antibody derivatives and pharmaceutical compositions can be administered in multiple doses per day, if desired, to obtain the total desired daily dose. The effectiveness of the treatment method can be assessed by the subject's control for known signs or symptoms of a disorder. [0138] In all cases, the dose and dose rate of aglycosylated anti-CD154 antibodies, antibody derivatives thereof and pharmaceutical compositions of the present invention effective to produce the desired effects will depend on a variety of factors, such as the nature of the disease to be treated, the size of the subject, the purpose of the treatment, the specific pharmaceutical composition used and the criteria of the treating physician. [0139] Aglycosylated anti-CD154 antibodies, antibody derivatives thereof and the pharmaceutical compositions of the present invention can be administered as a single dose for certain indications, such as preventing the immune response to an antigen to which a subject is exposed. for a short time, such as an exogenous antigen administered in a single day of treatment. Examples of such therapy may include co-administration of the antibody or antibody derivative of the invention together with a therapeutic agent, for example an antigenic pharmaceutical agent, an allergen or a blood product or a gene therapy vector. In indications where the antigen is chronically present, such as in the control of the immune reaction in the transplanted tissue or to chronically administer antigenic pharmaceutical agents, the antibodies, the antibody derivatives or the pharmaceutical compositions of the invention are administered at intervals during the medically indicated time, which varies from days or weeks to the subject's life. [0140] In one case, the subject that can be treated by the methods described above is an animal. Preferably, the animal is a mammal. Examples of mammals that can be treated include, but are not limited to, humans, nonhuman primates, rodents (including rats, mice, hamsters and guinea pigs) cow, horse, sheep, goat, pig, dog and cat. Preferably, the mammal is a human being. [0141] The present invention can be better understood on the basis of the following Examples. However, one skilled in the art will readily appreciate that the specific methods and results described are only illustrative of the invention that is more fully described in the Embodiments of the Invention that follow hereafter.

Experimental details EXAMPLES [0142] The following examples illustrate the products of the present invention and their uses. Appropriate modifications and adaptations of the conditions and parameters described normally found in the molecular biology technique that are apparent to those skilled in the art are within the spirit and scope of the present invention.

Example 1: Generation and evaluation of the aglycosylated hu5c8 antibody

Expression and characterization of the aglycosylated hu5c8 mAb

[0143] In order to reduce the effector function of the hu5c8 mAb, an aglycosylated form was created by changing the Asn-linked site of the CH2 domain of the heavy chain by a Gln residue. [0144] A competitive binding assay demonstrated that the ability of the aglycosylated hu5c8 mAb to bind to CD154 of the cell surface was not altered, compared to the glycosylated hu5c8 mAb (FIG. 1). [0145] The reduction of effector function was measured in vitro using a bridge formation assay format. The relative binding of the aglycosylated hu5c8 mAb to FcγRl decreased twenty-fivefold, compared to the glycosylated hu5c8 mAb (FIG. 2A). No residual binding of the aglycosylated hu5c8 mAb to FcγRIII at concentrations of up to 5 mg / ml could be demonstrated, while normal glycosylated hu5c8 mAb gave an EC50 of 50 ng / ml in the same assay format (FIG. 2B).

Pharmacokinetics of mAb hu5c8 aglycosylated in cynomolgus monkeys

[0146] The serum concentration-time profiles after a single dose of 20 mg / kg of mAb hu5c8 and mAb hu5c8 aglycosylated from two independent studies were subjected to pharmacokinetic analysis using a two-compartment model with a constant velocity of First order removal (WinNolin Professional Software v3.1, Pharsight Corp., Cary, NC). FIG. 3 contains the pharmacokinetic profiles and Table 1 contains the average pharmacokinetic parameters for mAb hu5c8 and mAb hu5c8 aglycosylated. The clearance and volume of distribution for mAb hu5c8 was slightly higher than for mAb hu5c8 aglycosylated.

Table 1

Pharmacokinetic parameters after a single intravenous dose of 20 mg / kg of hu5c8 or Hu5c8 aglycosylated to CynomolgusA monkeys

CmxB CIC VssD t1 / 2E antibody (μg / ml) (mlh / kg) (ml / kg) (d)

hu5c8 515 (± 16) 4.61 (± 0.70) 71 (± 10) 11.5 (± 2.5) agli. hu5c8 869 (± 360) 3.10 (± 1.10) 47 (± 11) 11.8 (± 3.0)

Adults reported as arithmetic mean ± standard deviation, n = 3 for the hu5c8 treatment group, n = 4 for the aglycosylated hu5c8 treatment group. Maximum serum concentration, systemic clearance, Volume of steady-state distribution, E half-life of terminal phase in serum.

METHODS -Example 1 [0147] 1. Generation of antibodies. The selection, cloning and humanization of the hu5c8 mAb has been previously described. See Lederman, 1992 and Karpusas, 2001,

5 respectively. The hybridoma of mAb hu5c8 is available in ATCC (HB10916). Mutation of the glycosylation site of the N298Q heavy chain (N297 using EU numbering) in the glycosylated hu5c8 mAb was performed by single site removal mutagenesis using an Amersham-Pharmacia Biotech kit (Piscataway, NJ, USA) following the recommended protocol by the manufacturer. The resulting aglycosylated hu5c8

10 was stably expressed in NSO myeloma cells and purified by Protein A chromatography and gel filtration. The aglycosylated hu5c8 antibody producing cell line is available from ATCC (PTA-4931). SDS-PAGE and analytical gel filtration chromatography showed that the protein formed the expected disulfide bound tetramer.

15 [0148] 2. CD154 binding assay. A competitive binding assay based on FACS was performed on huCD154 + D1.1 cells (gift from Dr. Leonard Chess, Columbia University, also available at ATCC (CRL-10915)). The binding of 0.1 mg / ml of biotinylated hu5c8 mAb to CD154 of the cell surface competed with titres of hu5c8 mAb and aglycosylated hu5c8 mAb. The biotinylated hu5c8 mAb

20 cell-bound was detected with streptavidin-phycoerythrin (PE) (BD-PharMingen San Diego, CA, USA). Relative binding affinities were inferred from the IC50 values of the four parameter curve settings. [0149] 3. CD154-FcγR bridge formation tests. FcγR binding affinities were measured using assays based on the ability of the antibody to form

25 a "bridge" between the antigen and an FcγR carrier cell (see below). The FcγRl bridge formation assay (CD64) was performed by coating Maxisorb 96-well ELISA plates (Nalge-Nunc Rochester, NY, USA) overnight at 4 ° C with 1 mg / ml recombinant soluble human CD154 (Biogen, Karpusas, 1995) in PBS and subsequently blocked with 1% BSA in PBS. The degrees

30 of hu5c8 mAb (glycosylated or aglycosylated) were subsequently bound to CD154 for 30 minutes at 37 ° C, the plates were washed, and the binding of U937 fluorescently labeled cells (CD64 +) was measured. U937 cells were cultured in RPMI medium with 10% FBS, 10 mM HEPES, L-glutamine, and penicillin / streptomycin, division 1: 2, and activated for one day before testing with 1000 units / ml of IFNγ for increase the expression of FcγRI. [0150] FcγRIII bridge formation assays (CD16) were performed using a monolayer of Chinese hamster ovary (CHO) cells expressing CD154 (Biogen) cultured in 96-well tissue culture plates (Corning Life Sciences Acton , MA, USA), with the measurement of mAb-dependent binding of fluorescently labeled Jurkat cells transfected with CD16 (gift from Dana Farber Institute, Boston, MA, USA). CHO-CD154 + cells were seeded in 96-well plates at a rate of 1x105 cells / ml and grown to confluence in an α-MEM medium with 10% dialyzed FBS, 100 nM methotrexate, Lglutamine, and penicillin / streptomycin (all reagents from Gibco-BRL Rockville, MD, USA). Jurkat CD16 + cells, which grow in RPMI with 10% FBS, 400 mg / ml geneticin, 10 mM HEPES, sodium pyruvate, L-glutamine, and penicillin / streptomycin (all Gibco-BRL reagents), are They divided 1: 2 a day before the test. [0151] In the assays for FcγRI and FcγRIII receptors, Fc receptor carrying cells were labeled with 2 ', 7'-bis- (2-carboxy-ethyl) -5- (and-6) carboxyfluorescein (BCECF) acetoxymethyl ester -AM) (Molecular Probes Eugene, OR, USA) for 20 minutes at 37 ° C. After washing to remove excess BCECF-AM, 1x105 of the labeled cells were incubated in the assay for 30 minutes at 37 ° C. Unbound FcγR + cells were washed several times and the plates were read on a Cytofluor 2350 fluorescent microplate reader (Millipore Corporation Bedford, MA, USA) with an excitation wavelength of 485 nm and an emission wavelength 530 nm

Example 2: Aglycosylated hu5c8 antibody inhibits primary and secondary humoral responses

Inhibition of a primary humoral response to tetanus toxoid (TT) antigen in cynomolgus monkeys

[0152] The ability of a single dose of 20 mg / kg of each of aglycosylated hu5c8 mAb and glycosylated hu5c8 mAb, prepared according to Example 1, to inhibit a primary antibody response to TT in separate studies was evaluated. Administration of aglycosylated hu5c8 mAb or glycosylated hu5c8 mAb resulted in 70% and 77% reductions, respectively, of the total primary immune response (EAUC), compared to saline treated groups. FIG. 4 shows the titers of TT antibody up to day 42 in graphic form, which demonstrates that aglycosylated hu5c8 mAb inhibits a primary humoral response to a degree comparable to glycosylated hu5c8 mAb, despite its decreased FcγR binding capabilities. [0153] The immunogenicity of humanized mAbs is another measure of their efficacy in this non-human primate model. Three out of four animals treated with a single dose of 20 mg / kg of aglycosylated hu5c8 mAb developed a low titer of hu5c8 antibodies around day 82, immediately after the drug was removed from the serum, in a manner compatible with inhibition of response. humoral while the aglycosylated mAb was present (data not shown). [0154] As an additional measure of primary response inhibition, inguinal lymph node biopsies showed that the presence of germ centers ("GC") in animals treated with aglycosylated hu5c8 mAb decreased greatly, compared to controls. In the treated animals, the GC were rare and small, occupying less than 20% of the cortex. Control animals presented multiple GC with moderate to reagent secondary follicles. The moderate to severe degree of lymph node hypoplasia observed was consistent with that previously observed with glycosylated hu5c8 mAb (data not shown). [0155] No gross changes in hematological parameters were observed after administration of a single dose of 20 mg / kg of glycosylated or aglycosylated hu5c8 mAb in cynomolgus monkeys and there was no significant change in the total amount of lymphocytes. On the other hand, the ratio of CD4 / CD8 T cells remained constant, indicating that there was no extended reduction of CD4 + T cells (data not shown).

[0156] Inhibition of a humoral response due to tetanus toxoid (TT) antigen in cynomolgus monkeys

[0157] The ability of a single dose of 20 mg / kg of aglycosylated hu5c8 mAb to inhibit an immune response was evaluated as a result of giving a second TT stimulus to the eight saline control animals that had a normal primary response in the phase previously described from the study of mAb hu5c8 aglycosylated. Prior to the second stimulus with TT, four of these animals received 20 mg / kg of aglycosylated hu5c8 mAb (Group 1B) and four received saline (Group 1A). [0158] Secondary immune responses are characterized by faster onset and

they are of greater magnitude than the primary immune responses. The magnitude of the secondary response was calculated with respect to the primary response for individual animals (secondary EAUC / primary EAUC) (Table 2). FIG. 5 shows the individual primary and secondary total immune responses. It should be noted that there was considerable variability in the degree of immune response among individual animals. On average, a secondary TT stimulus in saline controls (Group 1A) produced a total antibody response that was 6.5 times higher than its primary response to this antigen, while animals that received aglycosylated hu5c8 mAb ( Group 1B) produced an antibody response

10 total secondary that was, on average, only 2.0 times higher than its primary response. Consequently, administration of the aglycosylated hu5c8 mAb resulted in a 70% reduction in the magnitude of the secondary antibody response, compared to controls with saline solution. Table 2

Total primary and secondary responses (EAUC) to tetanus toxoid in cynomolgus monkeys

Treatment group

Group 1A (saline-saline) A Group 1B (saline -hu5c8 aglycosylated) B

Animal #

1 TO 1 1A-2 1A-3 1A4 1B-1 1B-2 1B-3 1B-4

Primary EAUC (x 105)

2.1 1.1 2.0 0.3 3.9 0.9 1.6 2.6

Secondary EAUC (x 105)

8.7 5.2 9.6 6.0 8.3 1.5 3.3 5.5

Magnitude

4.2 4.8 4.8 18.8 2.1 1.6 2.1 2.2

Average magnitude of the group

6.5 2.0

A Animals of group 1A received saline before stimulation with primary and secondary TT. Group 1B animals received saline before stimulation with primary TT and aglycosylated hu5c8 before stimulation with secondary TT. B The magnitude of the secondary response is represented by the ratio of secondary EAUC / primary EAUC.

METHODS - Example 2 [0159] 1. Humoral immune response to TT. Two independent studies were carried out on cynomolgus monkeys, using the same monkeys as in Example 1. Blood samples were collected from each study and the whole blood for immunotyping was maintained at room temperature and the analysis was performed on the day at That was extracted. [0160] This study of aglycosylated hu5c8 mAb included two treatment groups. Group 1, which consists of four males and four females, received saline solution on day 1 and served as untreated controls. Group 2, consisting of two males and two females, received a single dose of 20 mg / kg of intravenous aglycosylated hu5c8 mAb on day 1 (described above). Four hours after treatment, all animals received an intramuscular dose (IM) of 5 Lf (flocculant doses of lime) of the adsorbed TT. Pre-and post-treatment blood was collected on day 1 and on selected days up to 190 days after dosing. Lymph node biopsy samples were taken on day 15. [0161] The study of glycosylated hu5c8 mAb was composed of five treatment groups, each with three females. On Day 1, Group 1 received saline solution (untreated controls) and Groups 2 to 5 received a single dose of 0.2, 1.5 or 20 mg / kg of glycosylated hu5c8 mAb respectively, available from the ATCC ( CRL10915). Four hours after treatment, all animals received a single IM injection of 5 Lf of adsorbed TT. Blood was collected from all pre-and post-treatment groups on day 1 and on selected days up to 42 days after dosing. To allow comparability of these two independent studies, the selected serum samples were analyzed in parallel with the anti-TTA ELISA. [0162] On day 230 after the primary stimulus with TT in the aglycosylated study described above, the control Group 1 was divided into two groups. Group 1A served as untreated controls, while Group 1B was treated with aglycosylated hu5c8 mAb to assess its ability to inhibit a secondary immune response. The animals were treated as follows: Group 1B received a single dose of 20 mg / kg of intravenous aglycosylated mAb hu5c8 on day 1. Group 1A received an equivalent intravenous dose volume of phosphate buffered saline in on day 1. Four hours after treatment, all animals received an IM dose of 5 Lf of adsorbed TT. Pre-and post-treatment blood was collected on day 1 and on selected days up to 85 days after dosing. [0163] 2. Evaluation of immune responses. Immune responses were evaluated using non-compartmental analysis. The calculated immune parameters included the maximum titer value (Emáx), the time to reach this maximum value (tmax), and the total antibody response to the antigen administered from the moment of antigen administration until the last time point of sampling (EAUC (0final)). Pharmacokinetic analysis was performed using a two-compartment model with a first-order elimination rate constant (WinNolin Professional Software v3.1, Pharsight Corp., Cary, NC, USA). The pharmacokinetic parameters determined included the maximum serum concentration (Cmax), the systemic clearance rate (C1), the volume of steady state distribution (Vss) and the half-life of the terminal phase (t1 / 2) of the antibody. Statistical analyzes were performed, including mean, standard deviation and geometric mean using the Microsoft Excel version 5.0 computer program (Microsoft Corp., Redmond, WA, USA). [0164] 3. Immunophenotyping of cynomolgus monkeys. Lymphocyte immunophenotyping was performed using a two-color whole blood staining protocol followed by FACS analysis. Briefly, 100 µl of whole blood treated with EDTA was incubated for 20 min at room temperature with one of the following combinations of labeled mAbs: clone CD20-FITC 2H7 (BD-Pharmingen San Diego CA, USA) and CD2-PE clone RPA-2.10 (BD-Pharmingen), CD3FITC clone SP-34 (BD-Pharmingen) and CD4-PE clone OKT4 (Ortho Diagnostic Systems Raritan, NJ, USA), or CD3-FITC and CD8-PE clone DK-25 (Dako Corporation Carpinteria, CA, USA). The erythrocytes were lysed with 2 ml of 1X FACS lysis solution (Becton-Dickinson Franklin Lakes, NJ, USA). The lymphocytes were fixed with 1% paraformaldehyde and analyzed with a FACScan equipped with a Cellquest computer program (Becton-Dickinson). The number of total lymphocytes was determined by the sum of all CD2 and CD20 positive cells. B cells were identified as CD20 positive cells. Subsets of T cells were identified as double positive for CD3 and CD4 or CD3 and CD8. Total lymphocytes, B cells, CD4 + T cells, and CD8 + T cells were represented as the percentage of positive cells within the lymphocyte analysis window. The ratio of CD4 / CD8 for each data set was calculated. [0165] 4. ELISA for the pharmacokinetics of mAb hu5c8. ELISA plates (Nalge-Nunc Rochester, NY, USA) were coated with 5 μg / ml of soluble recombinant human CD154 (Biogen, see also Karpusas 1995) in PBS overnight at 4 ° C and blocked with 2% of ass serum (Jackson ImmunoResearch Laboratories West Grove, PA, USA - Catalog # 017-000-121). Serial dilutions of serum and a standard curve of hu5c8 mAb from 8-500 ng / ml were captured during one hour incubation at room temperature. The bound hu5c8 mAb was detected using anti-human horseradish peroxidase (HRP) IgG (Jackson ImmunoResearch Laboratories West Grove, PA, USA) followed by development with a 3.3 ', 5.5'- substrate kit tetramethylbenzidine ("TMB") (Pierce Biotechnology Rockland, IL, USA). The plates were read at 450 nm using a Spectromax plate reader (Molecular Devices Sunnyvale, CA, USA). A Softmax Pro Software (Molecular Devices) was used to update the diluted serum to the linear part of a four parameter curve fit of the standards. [0166] 5. ELISA to determine the responses of the anti-hu5c8 antibody. ELISA plates (Corning-Costar) were coated with 1 µg / ml of aglycosylated hu5c8 mAb (described above) in a pH 9.6 bicarbonate buffer overnight at 4 ° C and blocked with 1% BSA. Serial dilutions of cynomolgus monkey serum were incubated for 1.5 hours at room temperature. The bound anti-hu5c8 mAb was detected using 100 ng / ml of biotinylated hu5c8 mAb followed by streptavidin-HRP (Pierce Biotechnology) and development with a TMB substrate kit (Pierce Biotechnology). The plates were read at 450 nm using a Spectromax plate reader (Molecular Devices). The antibody titer was defined as the reciprocal of the highest dilution that produced> 0.100 units of D.O with respect to the pre-bleed values. [0167] 6. ELISA for the anti-TT response. ELISA plates (Corning-Costar) were coated with 5 µg / ml of TT (Massachusetts Public Health Biological Laboratories Boston, MA, USA) in a pH 9.6 bicarbonate buffer overnight at 4 ° C. Cynomolgus serum was added in serial dilutions to the blocked plate for 2 hours at room temperature. Anti-TT antibodies were detected using a rabbit antimono IgG-HRP (Cappel-Organon Teknika Durham, NC, USA) followed by development with a TMB substrate kit (Pierce Biotechnology). The plates were read at 450 nm using a Spectromax plate reader (Molecular Devices). The Softmax Pro (Molecular Devices) computer program was used to create a four-parameter curve fit for each serum sample of the serial dilution. The antibody titer was defined as the reciprocal of the highest dilution that produced 0.100 units of D.O with respect to the pre-bleed values. [0168] 7. Hematology. Anticoagulated blood samples were collected with potassium EDTA and analyzed monthly to determine the following hematological parameters: total leukocyte count, erythrocyte count, hemoglobin concentration, hematocrit value, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration , platelet count and blood smear evaluation (which includes differential counts). [0169] 8. Lymph node biopsy. An inguinal lymph node was collected from all animals on day 15 of the aglycosylated hu5c8 mAb, study of the primary TT response. Lymph node tissue was cut, included in paraffin, sectioned and mounted on glass slides. The slides were stained with hematoxylin and eosin. The slides were visually analyzed to determine the presence of the germ centers and quantitatively evaluated on the basis of the frequency and size of germ centers.

Example 3: The aglycosylated muMR1 antibody inhibits lupus nephritis [0170] Systemic lupus erythematosus ("SLE") is an autoimmune disease that arises spontaneously, with a predominance in women, which is characterized by the production of a variety of anti- autoantibodies. pathogenic nuclear. In lupus nephritis, kidney damage is mediated by cellular and humoral immune mechanisms, which include the formation of immune complexes that deposit in the renal globules and activate the complement cascade, resulting in glomerulonephritis. It has been previously established that the production of anti-nuclear autoantibodies in human and mouse SLE is driven by cognated interactions between selected populations of Th cells and autoimmune B cells. [Kalled et al., 1998]. [0171] Previous studies have shown that for mice (SWR x NZB) F1 (SNF1) with established lupus nephritis, long-term treatment with the antiCD154 mAb, hamster MR1 (haMR1), beginning at 5.5 months of Age prolonged survival and decreased the incidence of severe nephritis. [0172] In this example, the efficacy of two murine chimeric MR1 mAbs in this model is described. The murine chimeric MR1 ("muMRl") consists of the original hamster heavy and light chain variable domains fused to the heavy and light chain constant domains of murine IgG2a. An aglycosylated version of the muMRl was created by the mutation of the N-linked glycosylation site in the CH2 domain of the IgG2a Fc. Using these two antibodies, the role of glycosylation of Fc on the potency of anti-CD154 mAbs in lupus nephritis was evaluated. [0173] The results of this example demonstrate that both chimeric mAbs, muMR1 and aglycosylated muMR1, retained the ability to inhibit autoantibody responses. Specifically, similar to the wild type, the MR1 of the parent hamster, both murinized antibodies retained the ability to decrease renal inflammation, fibrosis, sclerosis and vasculitis in mice treated with them, compared to mice treated with a mAb control of murine IgG2a.

Pharmacokinetics of muMR1 and muMR1 aglycosylated

[0174] An analysis of the pharmacokinetics of muMR1 and agMylated muMR1 antibodies revealed that both antibodies exhibited the same serum kinetic profile of BALB / c mice (FIG. 6). Specifically, it is estimated that the half-life of these chimeric molecules in normal mice is ~ 9 days, similar to hamster MR1 (data not shown).

Autoantibody response analysts

[0175] Treatment with muMR1 or agglicosylated muMR1 greatly decreased the autoantibody response to dsDNA and ssDNA compared to control animals (FIG. 7 A and B).

Histological analysis

[0176] An analysis of kidney histology showed that three of the five isotype control animals had severe end stage renal disease characterized by the numerous features described in the scoring scheme. The animals in the treated groups clearly had less severe disease with few exceptions. The differences between animals treated with the wild-type (n = 11) and aglycosylated (n = 12) forms of muMR1 were minimal although the composite disease scores for the wild-type forms were slightly lower. FIG. 8 shows the histology scores composed of the group for the kidneys. Of the animals treated with aglycosylated muMR1, most had moderate early changes in the glomeruli with little or no significant tubulo-interstitial change. All animals treated with muMR1 (n = 11) had moderate early changes and no significant tubulo-interstitial change. From these results, it is evident that both muMR1 and aglycosylated muMR1 are effective for the prevention of the histological changes characteristic of lupus nephritis. [0177] The degree of lymphoid expansion in the areas of B and T cells was evaluated by histology for each spleen of treated animals. The identification of the secondary follicles was difficult due to the artifacts associated with the preparation of the frozen section. The correlation between the extent of splenic lymphoid area expansion and renal disease scores was not evident. However, the degree of expansion of the periarteriolar lymphocytic sheath (PALS) appeared to be clearly greater in isotype control and animals treated with aglycosylated muMR1, compared to animals treated with muMR1 (data not shown).

Renal Function Analysts

[0178] To assess renal function, proteinuria (PU) levels were measured, as well as serum creatinine and blood urea nitrogen (BUN) measurements of each animal. Both chimeric muMR1 mAbs delayed the onset of severe nephritis in SNF1 animals, measured by protein content in the urine. When compared to control animals, mice treated with anti-CD154 had lower PU values at the time points between 6 and 9 months (FIG. 9). However, at -10 months, all groups had PU values indicating renal failure (FIG. 9). These results suggest that anti-CD154 treatment delays the onset of proteinuria. [0179] FIG. 11 shows the serum creatinine levels of SNF1 mice, and FIG. 10 shows the levels of BUN in the serum of SNF1 mice. The animals in the control group had serum creatinine and elevated BUN levels in the range of 7.25-10.5 months. In contrast, mice treated with glycosylated muMR1 remained stable throughout the study, without substantial rise or decrease in serum creatinine or BUN. Animals treated with aglycosylated MuMR1 maintained normal serum creatinine levels until -9 months of age, when a slight increase was observed. Similarly, BUN levels in this group rose to 11 months of age.

Survival assessment

[0180] Treatment with anti-CD154 mAb prolonged the survival of SNF1 mice. At 11 months of age, more than 90% of the treated mice were alive compared to 56% of the controls. At 14 months, all control mice died, while 86% and 75% of the mice with muMR1 and aglycosylated muMR1 were still alive, respectively. (data not revealed). [0181] Together, these experiments demonstrate that long-term treatment of nephritic SNF1 mice with muMR1 or aglycosylated muMR1 prolonged survival, decreased autoantibody production and delayed the development of renal pathology. Slightly different results obtained with glycosylated and aglycosylated muMR1 indicate a minor role of Fc glycosylation on the efficacy of anti-CD154 mAbs in lupus. Consequently, aglycosylated anti-CD154 mAb represents an effective treatment for lupus nephritis.

METHODS -Example 3

[0182] 1. Mice. BALB / c, SWR and NZB mice were purchased from Jackson Laboratory (Bar Harbor, ME). The hybrids (SWR x NZB) F1 (SNF1) were raised in the Biogen bioterium under conventional barrier conditions. Female SNF1 mice were used for all lupus studies. BALB / c mice were used for pharmacokinetic studies ("PK"). [0183] 2. Antibodies. The MR1 hybridoma (ATCC # CRL-2580), which produces Armenian Hamster anti-mouse CD154 mAb, was purchased from the American Type Culture Collection (Rockville, MD). [0184] 3. Treatment protocol. All injections were administered intraperitoneally. The lupus study consisted of a control group of SNF1 mice that received PI.17 muIgG2a and treated SNF1 groups that received one of the chimeric antiCD154 mAbs. A single dose of 500 μg of mAb was administered once a week during the first six weeks, followed by a single injection of 500 μg once per month until the death of the animal or completion of the study. Studies began when the animals were -5.5 months old and serum samples were collected once a month. For the PK study, BALB / c mice received a single dose of 100 μg of muMR1 or aglycosylated muMR1 and blood samples were collected after 4 hours and on days 1, 2, 4, 7, 9, 11 and 14. [0185] 4. ELISA tests. For the detection of MR1 mAbs in serum, NUNC Maxisorp plates were coated with 15 µg / ml of soluble recombinant murine CD154 overnight at 4 ° C. The next day the plates were blocked, diluted sera were added, followed by anti-mouse IgG2a conjugated to horseradish peroxidase (Southern Biotech) and development with the TMB substrate. The reaction was stopped with 2 N sulfuric acid and the plates were read at 450 nm in a Spectramax plate reader (Molecular Devices, CA). Anti-single-stranded DNA (ADNss) and double-stranded ("ADNds") ELISAs were performed using NUNC MaxiSorp plates. The plates were coated overnight at 4 ° C with 10 μg / ml of methylated BSA (Calbiochem Corp. La Jolla, CA) and subsequently with 5 μg / ml of grade I thymus DNA (SIGMA, St. Louis, MO) for two hours at 25 ° C. The ram thymus DNA was broken down by sonication and subsequently digested with SI nuclease before use.For the anti-ASDNss assay, the DNA was boiled for 10 min and cooled on ice before use.After blocking, serial dilutions of serum samples were added and incubated at room temperature for 2 hours Autoantibodies were detected with goat anti-mouse IgG-Alkaline Phosphatase (SIGMA, ST. LOUIS, MO) and developed with p-nitrophenyl phosphate (SIGMA, ST. LOUIS, MO) in 1 M diethanolamine buffer Plates were read at 405 nm, and standard curves were obtained using known amounts of mAb 205 or mAb 5c6 anti-DNA, which are specific for ADNss and ADNds. Anti-DNA titers were defined as the reciprocal dilution to 0.1 OD units with respect to the background. [0186] 5. Histology. Cryosections and tissues included in paraffin, fixed in kidney and spleen formalin, were stained with hematoxylin-eosin ("H&E") to determine inflammatory infiltration. The kidneys were also stained with Masson Trichrome for fibrosis and Schiff's periodic acid staining ("PAS") for thickening of the basement membrane. Dyed tissue sections were classified by a veterinary pathologist. The total histopathological score score of lupus nephritis was based on glomerular, interstitial and tubular changes. Grades 0 to 4+ are based on the percentage of compromise of the structure examined (ie glomeruli, vessels, etc.) and are as follows: 0, without significant injuries; 1+, 1% 30% of affected architecture; 2+, 30% -60% of affected architecture; 3+,> 60% architecture affected to some degree; 4+,> 60% of architecture severely affected. [0187] 6. Urine and serum analysis. The urine of each mouse was monitored weekly with Albustix (Bayer Corp., Tarrytown, NY) to measure proteinuria ("PU"). The proteinuria level was rated as follows: 0.5+, 15 to 30 mg / dl; 1+, 30 mg / dl; 2+, 100 mg / dl; 3+, 300 mg / dl; 4+,> 2000 mg / dl. Creatinine and blood urea nitrogen (BUN) in serum were measured on a COBAS chemistry analyzer (Roche) intermittently during the study to provide measures of renal function in addition to PU.

Example 4: The aglycosylated muMR1 antibody inhibits experimental autoimmune encephalomyelitis (EAE) [0188] It has been shown that blocking the CD40 ligand at the time of immunization suppresses the development of EAE [Samoilova, 1997]. In this example, muMR1 and aglycosylated muMR1 antibodies were analyzed for their ability to inhibit EAE. This example also assessed whether the inhibition of EAE by the anti-CD154 mAb was associated with an active suppression mechanism and to what extent it was mediated by a mechanism that is dependent on antibody-dependent Fc-dependent interactions. [0189] The results of this example demonstrate that the aglycosylated muMR1 mAb is an EAE inhibitor as effective as the wild-type glycosylated hamster MR1 mAb for blocking the development of clinical disease. More specifically, all MR1 mAbs completely inhibited the development of the disease, evaluated by the average maximum clinical score and the average severity of the disease, with the exception of a mouse from the group treated with hamster MR1. These results suggest that the underlying protection mechanism against EAE by anti-CD154 mAbs does not appear to involve induction of regulatory T. They also show that the Fc-dependent effector functions of the mAb do not play an important role with respect to clinical efficacy but appear to contribute to the mechanism of inhibition underlying the autoimmune establishment of EAE.

Inhibition of clinical EAE with glycosylated muMR1

[0190] Figure 12 demonstrates that mice treated with muMR1 did not develop disease symptoms after primary peptide immunization, compared to mice treated with the P1.17 isotype control. Animals treated with muMR1 also did not develop clinical symptoms during the 80-day follow-up period (data not shown). When mice were reinmunized with PLP139-151 emulsified in complete Freund's adjuvant, there was an increase in severity of clinical symptoms in animals treated with P1.17. Mice that had been treated on days 0, 2 and 4 with muMR1 developed EAE after re-stimulation, with a severity that amounted to the first phase of EAE in animals treated with P1.17. These results demonstrate that treatment with anti-CD154 mAb did not produce active suppression. It was also studied if the transfer of 20 x 106 spleen cells, collected from mice 1 or 3 weeks after EAE induction and treatment with muMR1, could return to recipient mice, without previous treatment, resistant to subsequent EAE induction active. This was not the case (data not shown).

Inhibition of clinical EAE with aglycosylated muMR1

[0191] Figure 13 and Table 3 demonstrate that the aglycosylated muMR1 and muMR1 mAbs were effective in inhibiting the clinical signs of EAE during the entire follow-up period, when administered as 3 doses of 200 μg, compared with Ig control P1.17. In this aspect, the muMR1 and agMosylated muMR1 antibodies were equally effective as the hamster MR1 (data not shown). The administration of lower doses of these antibodies revealed no major differences between the antibodies, with respect to their ability to inhibit EAE.

Inhibition of CNS inflammatory infiltrates [0192] To assess whether antibodies differed in inhibition of inflammatory infiltrates within the central nervous system ("CNS"), separate groups of 4 to 5 mice, treated with different amounts of antibodies (muMR1 and aglycosylated muMR1, or 3 doses of 200 μg and control IgG P1.17), were sacrificed on day 16, at the peak of disease activity in mice treated with the isotype control antibody.

5 Table 3: Glycosylation is not required for the inhibitory effect of anti-CD154

Antibody

Dose (μg) N Incidence Maximum average score ± SD1 Average cumulative score ± SD1

P1.17

3 x 200 14 13 2.4 ± 0.8 32.1 ± 32.5

muMR1

3 x 200 13 0 0 ± 0§ 0 ± 0 #

muMR1

3 x 75 6 0 0 ± 0§ 0.8 ± 1.2 # #

muMR1

3 x 25 6 3 1.4 ± 1.6§§ 39.3 ± 53.1

MR1 Aglycosylated

3 x 200 14 0 0 ± 0§ 0 ± 0 #

MR1 Aglycosylated

3 x 75 6 one 0.6 ± 1. 4§ 19.7 ± 47.9

MR1 Aglycosylated

3 x 25 6 2 0.8 ± 1.3§ 9 ± 17.4 *

1. The development of the disease is presented in Figure 1. For each individual mouse the maximum disability score and the cumulative score during the entire monitoring period were evaluated separately before calculating the group mean. § p <0.0005, compared to mice treated with P1.17; §§ p <0.05, compared to mice treated with P1.17 # p = 0.002, compared to mice treated with P1.17; # # p <0.02, compared to mice treated with P1.17 * p = 0.05, compared to 25 μg of muMR1

[0193] As shown in Table 4, the antibodies showed no difference with respect to their ability to suppress the development of inflammatory infiltrates within the CNS during the early stage of disease development. Because

10 It was doubtful if the mouse can develop subclinical activity in the absence of EAE signs, CNS tissues of 6 to 14 mice per group were analyzed at the end point of this study (day 58). [0194] In contrast to mice treated with 200 µg of muMR1, both animals treated with P1.17 and mice treated with 200 µg of agliMR1 showed evidence of moderate inflammatory infiltrates (Table 5) that were predominantly located in the cerebellum. However, treatment with smaller amounts of antibodies revealed that agliMR1 was similar although somewhat more protective.

5 than its glycosylated form. These results demonstrate that both antibodies also inhibit the development of clinical EAE.

METHODS -Example 4 [0195] 1. Induction of EAE. Female SJL mice (10-12 weeks, Harlan) were immunized subcutaneously with 50 µg of PLP139-151 emulsified in adjuvant

10 of complete Freund (Difco, Detroit, MI). Three days later, the mice were injected with 109 heat-inactivated B. pertussis microorganisms (RIVM, Bilthoven, The Netherlands) intravenously. The development of EAE was monitored by the daily assessment of body weight and a disability score. This score varies from 0: no symptoms, 0.5: partial loss of tail tone, 1: complete loss of

15 tail tone, 2: limb weakness, 2.5:

Table 4: Effect of anti-CD154 on CNS infiltration (day 16)

Antibody

Dose (µg) Number of animals with infiltrates

None

Sporadic Moderate Severe

P1.17

3 x 200 0 3 one one

muMR1

3 x 200 3 one 0 0

muMR1

3 x 75 3 2 0 0

muMR1

3 x 25 one one 3 0

MR1 Aglycosylated

3 x 200 5 0 0 0

MR1 Aglycosylated

3 x 75 4 one 0 0

MR1 Aglycosylated

3 x 25 2 one 2 0

Table 5: Effect of anti-CD154 on CNS infiltration (day 58)

Antibody

Dose (µg) Number of animals with infiltrates

None

Sporadic Moderate Severe

P1.17 Isotype

200 3 8 3 0

muMR

200 12 one 0 0

muMR

75 3 3 0 0

muMR

25 0 5 one 0

MR1 Aglycosylated

200 8 5 one 0

MR1 Aglycosylated

75 4 2 0 0

MR1 Aglycosylated

25 4 2 0 0

partial paresis, 3: complete paralysis of hind limbs, 3,5: complete paralysis of the diaphragm and hind limbs, incontinence, 4: dying, at 5: death due to EAE.

5 [0196] 2. Generation of antibodies. The variable domains of the heavy and light chains of the MR1 CD154 hamster anti-mouse mAb were cloned by RT-PCR of the total hybridoma RNA. Expression vectors for the hamster / mouse chimeric mAb were constructed by genetic manipulation of murine IgG2a cDNAs or

10 constant region of the murine kappa chain (derived from the full-length cDNA clones of the heavy and light chains of the anti-human CD154 mAb - that is, glycosylated hu5c8) in the variable domains of the heavy and light chain, respectively , using standard recombinant DNA techniques. It was shown that the chimeric MR1 mAb transiently expressed, designated muMR1, recapitulates

15 the CD154 binding properties of the hamster mAb by flow cytometry and immunoprecipitation. [0197] The chimeric aglycosylated MR1, called agliMR1, was constructed by heavy chain site-directed mutagenesis to change the asparagine residue at the N-linked glycosylation site of Fc (N297 in the Kabat RU nomenclature)

20 for a glutamine residue. Stable expression vectors containing tandem transcription cassettes directed by CMV-IE promoter for light and heavy immunoglobulin chains and a glutamine synthetase gene were constructed as a selectable marker for IgG2a muMR1 and aglimuMR1, mAb kappa. Expression vectors were transfected into NS0 cells and stable clones were isolated by selection in glutamine free medium. [0198] MuMR1 and agliMR1 were purified by affinity of the cell supernatants of the Bioreactor in Protein A Sepharose followed by size exclusion chromatography in Sephacril 300 to remove aggregates. Chromatography resins were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). The mAbs were shown to be> 95% pure by SDS-PAGE and the endotoxin analysis guaranteed the safety of these reagents for use in vivo. It was found that MuMR1 and agliMR1 have the same relative affinity for cell surface muCD154 in in vitro assays and the same pharmacokinetic half-life in vivo in BALB / c mice (data not shown). The murine IgG2a isotype control mAb, P1.17 (ATCC # TIB-10), was purified by Protein A from ascites in Protos Immunoresearch (Burlingame, CA) under Biogen's contract. [0199] 3. Administration of anti-CD154 antibodies. On days 0, 2 and 4, each mouse received 200 μl of PBS i.p., which also contained the following: Group 1 = PBS (control); Group 2 = 200 μg of muMR1; Group 3 -200 μg of hamster Ig (Ig control); Group 4 = 200 μg of murine MR1; Group 5 = 200 μg of control of IgG2a P1.17; Group 6 = 200 μg of aglycosylated muMR1. [0200] In some experiments, mice were re-immunized on day 80 with 50 µg of PLP139-151 emulsified with the complete Freund's adjuvant. [0201] 4. Evaluation of the disease. Mice were weighed daily and monitored by clinical activity for a period of 56 days, according to the following scoring system: 0 = no symptoms; 0.5 = loss of tail tone 1 = complete loss of tail tone; 2 = weakness of the member; 2.5 = partial paresis; 3 = complete paralysis of the hind limb; 3.5 = complete paralysis of the diaphragm and hind limbs, incontinence; 4 = dying; and 5 = death due to EAE. [0202] 5. Histology. The brain tissue and spinal cord of each individual mouse were fixed in 10% formalin and included in paraffin. From each individual mouse, three to six sections of the spinal cord (4 μm) and six brain sections (comprising cerebellum, brain, brainstem and subarachnoid space) analyzed by 100 μm were analyzed with respect to the extent of inflammatory infiltrates after of staining with hematoxylin. [0203] Each individual section was classified according to the following scale: 0 = no infiltrates; 1 = sporadic, mild perivascular infiltration (less than two inflammatory lesions per section); 2 = = multifocal perivascular infiltration; 4 = infiltration

Severe perivascular multifocal accompanied by propagation in the parenchyma. Based on the average of all sections, the mice were categorized as without infiltration, with sporadic, moderate or severe infiltration. [0204] 6. Statistical analysis. The results were analyzed by one-way ANOVA, followed by retrospective analysis using the LSD test. P values <0.05 are They considered significant.

Example 5: Aglycosylated hu5c8 antibody inhibits islet cell transplantation [0205] Previous studies have shown that rhesus monkeys, treated with a 20 mg / kg dose induction / maintenance regime of glycosylated hu5c8 maintained renal allograft function and none experienced an episode of rejection during the six-month dosing period, while untreated monkeys who received renal allografts quickly rejected their transplants within eight days [Kirk, 1999]. In a related study by the Kirk research group, it was shown that aglycosylated hu5c8 was not effective for the treatment of transplant rejection. [0206] In striking contrast to the Kirk findings, the results of the present invention demonstrate that an aglycosylated form of the hu5c8 mAb may indeed be effective for the treatment of transplant rejection in other contexts.

Islet cell allograft transplantation in Rhesus monkeys

[0207] It has previously been shown that glycosylated hu5c8 allows the implantation of the islet and maintains the survival of the allograft [Kenyon, 1999]. [0208] Four rhesus monkeys treated with a 20 mg / kg dose induction / maintenance regimen achieved insulin independence for> 213,> 255,> 269, and> 341 post-transplant days. The results of this study showed that untreated control monkeys that received islet allografts exhibited acute rejection on day 8, which was evidenced by persistent hyperglycemia and lack of production of c-peptide (a product of endogenous insulin production) in the day 11-14 (data not shown). [0209] In contrast to untreated control animals, FIG. 14 shows the successful treatment of one of the two rhesus monkeys treated with a dose induction / maintenance regime of aglycosylated hu5c8 (described above). Although both monkeys experienced hyperglycemia and initial rejection on day 7, when the monkeys were treated with insulin and continued treatment with aglycosylated hu5c8, one of the two monkeys exhibited partial allograft function measured by the presence of c-peptide on day 28 (open rhombus), and remained until day 45. [0210] These results suggest that aglycosylated anti-CD154 mAbs are useful in treatment regimens in a transplant context. However, because the immune response during transplantation is a strong response, that is, it involves humoral, cellular and inflammatory immune responses, it is possible that aglycosylated anti-CD154 antibodies may be more effective when administered in combination with an immunosuppressive compound. or immunomodulator. For example, an agent that interrupts costimulatory signaling of T cells by means of CD28; an agent that disrupts calcineurin signaling, a corticosteroid, an antiproliferative agent, or other antibody that specifically binds to a protein expressed on the surface of immune cells, which includes but is not limited to CD45, CD2, IL2R, CD4, CD8 and RANK FcR, B7, CTLA4, TNF, LTβ and VLA-4.

METHODS - Example 5 [0211] 1. Islet cell allograft transplantation. These studies were performed as previously described [Kenyon, 1999]. In short, pairs of donor-receptor recipients of rhesus monkeys were chosen based on the reactivity of the positive mixed lymphocyte culture. The recipients underwent pancreatectomy and intraportal allogeneic islet transplantation on day 0. [0212] 2. Antibody treatment. Dosing was performed using an induction consisting of 20 mg / kg on Days -1, 0, 3, 10 and 18 and maintenance consisted of a dose of 20 mg / kg per month from day 28. [0213] 3. Evaluation of graft function. Islet grafting function was monitored daily by fasting and postprandial blood glucose levels. Islet failure (absence of primary function or acute rejection) was defined as an absence of fasting and stimulated c-peptide production (an endogenous insulin product). The absence of primary function was defined as the failure of the transplanted tissue to function in the immediate post-transplant period and was characterized by persistent hyperglycemia and unstable glycemic control. Episodes of acute rejection were defined as fasting glucose> 100 mg / dl and postprandial glycemia> 150-175 mg / dl. The overall graft function was assessed by controlling the amount of exogenous insulin required to maintain normal blood glucose levels.

Example 6: Use of the aglycosylated hu5c8 antibody (anti-CD154) to prevent anti-CD154-mediated T-cell alloreactivity [0214] Monoclonal antibodies that target CD154 of the T cells have been shown to partially prevent in vitro reactivity thus as in vivo. However, it is not evident whether the suppressive activity of the anti-CD154 mAb depends on the blockage of stimulatory CD40-CD154 interactions or, alternatively, on the direct administration of the inhibitory signals through CD154. [0215] To assess the effects of stimulatory CD40-CD154 interactions as opposed to the stimulation of CD154 (ie, direct administration of inhibitory signals through CD154) on the in vitro reactivity of human T cells, PBMCs Unfractionated, or purified CD4 + T cells, were cocultured with poorly paired stimulatory cells with HLA, allogeneic (CD40 positive cells) in the presence of a humanized anti-CD154 mAb (hu5c8, Biogen Inc., MA, USA), soluble or immobilized in The culture wells. Most of the blocking experiments were performed with a genetically engineered variant of the soluble anti-CD154 (aglycosylated hu5c8) that has reduced Fc effector function and consequently may have a reduced ability to crosslink CD154. The aglycosylated hu5c8 used in this example is the same as the aglycosylated hu5c8 mAb described throughout the present specification. See, for example, Example 1, ¶ 15 and ¶ 83, supra. Soluble but uncoated anti-CD154 antibodies inhibited the proliferation of allogeneic T cells in cultures of primary mixed lymphocytes ("MLC ') (inhibition of 40 ± 23% versus 3 ± 18%, n = 4). Similarly, proliferation of alloantigen-specific T lymphocytes in secondary MLC (n = 5 experiments) was suppressed by activation of blockade with CD154 (with soluble antibodies), while increased by stimulation with CD154 (coated antibodies). with the coated anti-CD154 antibodies, the generation of alloantigen-specific CTL effectors was strongly increased, while CTLs were not affected by the CD154 block. [0216] To analyze whether the stimulatory activity of anti-CD154 required costimulatory interactions B7- CD28, MLC was performed in the presence of CTLA4Ig, a molecule that prevents the binding of B7 molecules to CD28. Activation in the presence of CTLA4-Ig suppressed proliferation n of alloantigen-specific T lymphocytes in primary and secondary MLC, respectively, by 75 ± 14% (n = 3) and 64 ± 28% (n = 6) and inhibited the generation of CTL in 48 ± 23% (n = 2 ). Signaling of the coated anti-CD154 antibodies significantly increased the independent proliferation of residual CD28 of the alloantigen-specific T cells both primary (in 58 ± 0.6%, n = 2) and secondary (in 61 ± 49%, n = 6), although it did not completely nullify the inhibition induced by CTLA4-Ig. However, the inhibitory effect of CTLA4-Ig on the generation of CTL was nullified by stimulation with CD154 (by means of coated anti-CD154 antibodies). The expression of CD25, HLA-DR and CD95 on alloreactive T cells was strongly increased by stimulation of CD154 (n = 2), compared to control cultures with or without CTLA4-Ig. [0217] Our data show that anti-CD154 antibodies can increase, rather than block, the alorreactivity of T cells, when they are immobilized on the culture plate. Blocking of CD154 by soluble glycosylated hu5c8 amAb could not increase the activation of T cells in vitro and therefore may be advantageous in vivo and, based on these findings, may be effective, in experimental transplant models, for reduce T-cell responses to alloantigens in vivo, either alone or in combination with molecules that block other costimulatory pathways.

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Claims (56)

one.

 Aglycosylated anti-CD154 antibody antibody or antibody, characterized by a modification in the conserved N-linked site of the CH2 domains of the Fc part of said antibody.

2.

Aglycosylated anti-CD154 antibody antibody or antibody according to claim 1, wherein the modification comprises a mutation at the glycosylation site of the heavy chain, wherein the mutation prevents glycosylation at the site.

3.

Aglycosylated anti-CD154 antibody antibody or antibody according to claim 2, wherein the modification comprises a mutation of N298Q (N297 using Kabat EU numbering).

Four.

Aglycosylated anti-CD154 antibody antibody or antibody according to claim 1, wherein the modification comprises the removal of glycans from the CH2 domain.

5.

Aglycosylated anti-CD154 antibody antibody or antibody according to claim 1, wherein the modification comprises preventing glycosylation in the CH2 domain.

6.

 Aglycosylated anti-CD154 antibody antibody or derivative according to any one of claims 1 to 5, wherein said antibody or aglycosylated anti-CD154 antibody derivative does not bind to an effector receptor.

7.

 Aglycosylated anti-CD154 antibody antibody or antibody according to any one of claims 1 to 5, wherein said antibody or aglycosylated anti-CD154 antibody derivative does not cause thrombosis.

8.

 Aglycosylated anti-CD154 antibody antibody or antibody according to any one of claims 1 to 5, wherein said antibody is selected from the group consisting of: a monoclonal antibody, a polyclonal antibody, a murine antibody, a chimeric antibody, a primatized antibody, a humanized antibody, and a fully human antibody.

9.

Aglycosylated anti-CD154 antibody antibody or antibody according to any one of claims 1 to 5, wherein said antibody is selected from the group consisting of: a multimeric antibody, a heterodimeric antibody, a hemidimeric antibody, a tetravalent antibody, and an antibody bispecific.

10.

 Antibody or derivative of aglycosylated anti-CD154 antibody according to any of claims 1 to 5, wherein said antibody is aglycosylated hu5c8 produced by the cell line having ATCC Access No. PTA-4931.

eleven.

 Aglycosylated anti-CD154 antibody antibody or antibody according to any one of claims 1 to 5, wherein said antibody or antibody derivative is labeled with a detectable label.

12.

 Aglycosylated anti-CD154 antibody antibody or antibody according to claim 11, wherein the detectable label is a radioactive isotope, enzyme, dye or biotin.

13.

 Aglycosylated anti-CD154 antibody antibody or antibody according to any one of claims 1 to 5, wherein said antibody or antibody derivative is conjugated to a therapeutic agent.

14.

 Aglycosylated anti-CD154 antibody antibody or antibody according to claim 13, wherein said therapeutic agent is a radioisotope, radionuclide, toxin, toxoid or chemotherapeutic agent.

fifteen.

 Aglycosylated anti-CD154 antibody antibody or antibody according to any one of claims 1 to 5, wherein said antibody or antibody derivative is conjugated to an image display agent.

16.

 Aglycosylated anti-CD154 antibody antibody or antibody according to claim 15, wherein the image display agent is a labeling group.

17.

 An aglycosylated anti-CD154 antibody antibody or antibody according to claim 15, wherein the image display agent is a biotin, a fluorescent group, a radioactive group, a histidine tag, or a peptide tag.

18.

 Pharmaceutical composition comprising the antibody or aglycosylated anti-CD154 antibody derivative according to any one of claims 1 to 5.

19.

 Pharmaceutical composition according to claim 18, further comprising a pharmaceutically acceptable carrier.

twenty.

 Pharmaceutical composition according to claim 18, further comprising an immunosuppressive or immunomodulatory compound.

twenty-one.

 Cell line producing the antibody or aglycosylated anti-CD154 antibody according to any one of claims 1 to 5.

22

 Cell line according to claim 21, wherein the cell line produces the aglycosylated hu5c8 (Access ATCC No. PTA-4931).

2. 3.

Use of an aglycosylated anti-CD154 antibody or an aglycosylated antiCD154 antibody derivative according to any one of claims 1 to 5, or a pharmaceutical composition comprising said antibody or antibody derivative, for the

manufacture of a medicament to inhibit an immune response, an inflammatory response, transplant rejection, graft versus host disease, an allergic response, an autoimmune response, fibrosis, viral T-cell infection by the HTLV I virus, gastrointestinal disease, vascular disease, or proliferation of T-cell tumor cells, in a subject.

24.

 Use according to claim 23, wherein the aglycosylated anti-CD154 antibody, antibody derivative or pharmaceutical composition inhibits the binding of CD154 to CD40.

25.

 Use according to claim 23, wherein the aglycosylated anti-CD154 antibody, antibody derivative or pharmaceutical composition is capable of specifically binding to a protein that is specifically recognized by aglycosylated hu5c8, which is produced by the cell line having ATCC Access No. PTA4931.

26.

 Use according to claim 23, wherein the aglycosylated anti-CD154 antibody, antibody derivative or pharmaceutical composition specifically binds to the epitope to which the aglycosylated hu5c8 (ATCC Access No. PTA-4931) specifically binds.

27.

 Use according to claim 23, wherein the inflammatory response is selected from the group consisting of: arthritis, contact dermatitis, hyper-IgE syndrome, inflammatory bowel disease, allergic asthma and idiopathic inflammatory disease.

28.

 Use according to claim 27, wherein the arthritis is selected from the group consisting of: rheumatoid arthritis, non-rheumatoid inflammatory arthritis, arthritis associated with Lyme disease and inflammatory osteoarthritis.

29.

 Use according to claim 27, wherein the idiopathic inflammatory disease is selected from the group consisting of: psoriasis and systemic lupus.

30

 Use according to claim 23, wherein the transplant rejection involves transplantation of heart, kidney, liver, skin, pancreatic islet cells or bone marrow.

31.

 Use according to claim 23, wherein the allergic response is selected from the group consisting of: hay fever or an allergy to penicillin or other drugs.

32

 Use according to claim 23, wherein the autoimmune response derives from an infectious disease.

33.

 Use according to claim 23, wherein the autoimmune response derives from Reiter's syndrome, spondyloarthritis, Lyme disease, HIV infections, syphilis or tuberculosis.

3. 4.

 Use according to claim 23, wherein the autoimmune response is selected from the group consisting of: rheumatoid arthritis, myasthenia gravis, lupus erythematosus

systemic, Graves' disease, idiopathic thrombocytopenic purpura, hemolytic anemia, diabetes mellitus, inflammatory bowel disease, Crohn's disease, multiple sclerosis, psoriasis, drug-induced autoimmune diseases, and drug-induced lupus.

35

 Use according to claim 23, wherein the fibrosis is selected from the group consisting of: pulmonary fibrosis and fibrotic disease.

36.

 Use according to claim 35, wherein the pulmonary fibrosis is selected from the group consisting of: pulmonary fibrosis resulting from adult respiratory distress syndrome, drug-induced pulmonary fibrosis, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis.

37.

 Use according to claim 35, wherein the fibrotic disease is selected from the group consisting of: Hepatitis C; Hepatitis B; cirrhosis; liver cirrhosis due to a toxic attack; liver cirrhosis as a result of drugs, liver cirrhosis as a result of a viral infection and liver cirrhosis as a result of an autoimmune disease.

38.

 Use according to claim 23, wherein the gastrointestinal disease is selected from the group consisting of: esophageal dysmotility, inflammatory bowel disease and scleroderma.

39.

 Use according to claim 23, wherein the vascular disease is selected from the group consisting of: atherosclerosis or reperfusion injury.

40

 Use according to any one of claims 23 to 39, wherein the aglycosylated antiCD154 antibody or derivative of the aglycosylated anti-CD154 antibody is selected from the group consisting of: a monoclonal antibody, a polyclonal antibody, a murine antibody, a chimeric antibody, a primatized antibody, a humanized antibody, and a fully human antibody.

41.

 Use according to any one of claims 23 to 39, wherein the aglycosylated antiCD154 antibody or derivative of the aglycosylated anti-CD154 antibody is selected from the group consisting of: a multimeric antibody, a heterodimeric antibody, a hemidimeric antibody, a tetravalent antibody, and a bispecific antibody.

42

 Use according to any one of claims 23 to 39, wherein the aglycosylated antiCD154 antibody or aglycosylated anti-CD154 antibody or pharmaceutical composition comprising said antibody or antibody derivative is administered to said subject by intravenous, subcutaneous, intraperitoneal, intramuscular injection , intramedullary, intraventricular, intraepidural, intraarterial, intravascular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intraspinal, intratumoral,

intracranial; by an enteric, intrapulmonary, transmucosal, intrauterine, or sublingual route of administration; or locally at sites of inflammation or tumor growth.

43

 Use according to any one of claims 23 to 39, wherein the aglycosylated antiCD154 antibody, derived from aglycosylated anti-CD154 antibody or pharmaceutical composition comprising said antibody or antibody derivative is administered to said subject by a route selected from the group consisting of : oral, nasal, ophthalmic, rectal and topical routes.

44.

 Use according to claim 43, wherein the aglycosylated anti-CD154 antibody, aglycosylated anti-CD154 antibody derivative or pharmaceutical composition comprising said antibody or antibody derivative is administered to said subject orally in the form of capsules, tablets, suspensions or aqueous solutions.

Four. Five.

 Use according to claim 43, wherein the aglycosylated anti-CD154 antibody, derived from aglycosylated anti-CD154 antibody or pharmaceutical composition comprising said antibody or antibody derivative is administered to said subject topically by the application of a cream, ointment or similar.

46.

 Use according to claim 43, wherein the aglycosylated anti-CD154 antibody, aglycosylated anti-CD154 antibody derivative or pharmaceutical composition comprising said antibody or antibody derivative is administered to said subject by inhalation through the use of a nebulizer, an inhaler of dry powder or a metered dose inhaler.

47

 Use according to any one of claims 23 to 39, wherein the aglycosylated antiCD154 antibody, derived from aglycosylated anti-CD154 antibody or pharmaceutical composition comprising said antibody or antibody derivative is administered to said subject by sustained release administration.

48.

 Use according to any one of claims 23 to 39, wherein the aglycosylated antiCD154 antibody, derived from aglycosylated anti-CD154 antibody or pharmaceutical composition comprising said antibody or antibody derivative is administered to said subject together with a therapeutic agent.

49.

 Use according to any of claims 23 to 39, wherein the aglycosylated antiCD154 antibody, derived from aglycosylated anti-CD154 antibody or pharmaceutical composition comprising said antibody or antibody derivative is administered to said subject in multiple doses per day.

fifty.

 Use according to any one of claims 23 to 39, wherein the aglycosylated antiCD154 antibody, derived from aglycosylated anti-CD154 antibody or composition

Pharmaceutical comprising said antibody or antibody derivative is administered to said subject repeatedly at intervals ranging from every day to every two months.

51.

Use according to any of claims 23 to 39, wherein the aglycosylated antiCD154 antibody, derived from aglycosylated anti-CD154 antibody or pharmaceutical composition comprising said antibody or antibody derivative is administered to said subject at intervals for as long as it is medically indicated. , which varies from days or weeks to the entire life of the subject.

52

 Use according to any of claims 23 to 39, wherein the aglycosylated antiCD154 antibody, derived from aglycosylated anti-CD154 antibody or pharmaceutical composition comprising the antibody or antibody derivative is administered to the subject with an immunomodulatory or immunosuppressive compound.

53.

 Use according to claim 52, wherein the immunomodulatory or immunosuppressive compound is selected from the group consisting of:

(to)

 an agent that interrupts costimulatory signaling of T cells by means of CD28;

(b)

 an agent that interrupts calcineurin signaling,

(C)

 a corticosteroid,

(d)

 an antiproliferative agent; Y

(and)

an antibody that specifically binds to a protein expressed on the surface of immune cells, including but not limited to CD45, CD2, IL2R, CD4, CD8 and RANK FcR, B7, CTLA4, TNF, LTP, and VLA-4.

54

 Use according to claim 52, wherein the immunosuppressant or immunomodulatory compound is selected from the group consisting of: tacrolimus, sirolimus, mycophenolate mofetil, mizoribine, deoxyspergualin, brequinar sodium, leflunomide, rapamycin and azaspiran.

55.

 Use of the aglycosylated anti-CD154 antibody or antibody derivative according to any one of claims 1 to 5, for the manufacture of a composition for use in a method of imaging tumor cells or neoplastic cells in a subject expressing a protein that is recognized specifically by the aglycosylated hu5c8 produced by the cell line that has ATCC Access No. PTA-4931, said method comprising the steps of:

(to)

 administering to the subject an effective amount of the composition under conditions that allow the formation of a complex between the antibody or the antibody derivative and a protein on the surface of the tumor cells or neoplastic cells; Y

(b)

 visualize by image any antibody / protein complex or antibody derivative / complex formed, thereby visualizing any of the tumor cells or neoplastic cells in the subject.

56. Use of the antibody or derivative of aglycosylated anti-CD154 antibody according to Any one of claims 1 to 5, for the manufacture of a composition for use in a method for detecting the presence of tumor cells or neoplastic cells in a subject expressing a protein that is specifically recognized by the aglycosylated hu5c8 produced by the cell line which has ATCC Access No. PTA-4931, said method comprising the steps of: 10 (a) administering to the subject an effective amount of the composition under conditions that allow the formation of a complex between the antibody or the antibody derivative and the protein;

(b)

 remove any unbound image agent from the subject; Y

(C)

 detect the presence of any antibody / protein complex or derivative of

The antibody / complex formed, indicating the presence of such a complex the presence of tumor cells or neoplastic cells in the subject.