MXPA00007534A - Antibodies against human cd40 - Google Patents

Abstract

The Applicants have discovered novel chimeric and humanized anti-human CD40 antibodies which block the interaction between gp39 and CD40. The anti-CD40 antibodies of the present invention are effective in modulating humoral immune responses against T cell-dependent antigens, collagen induced arthritis, and skin transplantation, and are also useful for their anti-inflammatory properties.

Description

ANTIBODIES AGAINST CD40 HUMAN BACKGROUND OF THE INVENTION Immuno / inflammatory responses are mediated by complex series of interaction. A receptor / ligand pair that showed to be important in that process is CD40 / gp39. The gp39 / CD40 interaction mediates a significant number of signaling events between activated T cells and other effective immune system cells that lead to the amplification of an immune / inflammatory response. Responses to signaling through CD40 include help from T cell to B cells in the humoral immune response, the induction of cytokines by monocytes, and the expression of adhesion molecules by endothelial cells.

CD40 is a type of cell surface receptor I and a member of the tumor necrosis factor receptor (TNRF) supergene family. Although originally identified as a B cell antigen, CD40 is now believed to be expressed by all cells presenting antigen (APC), including dendritic cells, keratinocytes, and monocytes. CD40 is also expressed by cell types that can act as APC under certain conditions, such as vascular endothelial cells, or cells involved in direct interactions with T cells or T cell precursors REF .: 121688 as epithelial cells. Very recently, it has also been reported that CD40 can be expressed by fibroblasts, eosinophils, and activated T cells. The expression CD40 has also been seen in cancer cells. Evidence for this is primarily derived from the identification of some carcinoma and melanoma derived from cell lines which are CD40 + T cells (Clark and Ledbetter, Proc.Nat.Acid.Sci. (19d6) 83: 4494-98; Scheriever et al. ., J. Exp. Med. (1989) 169: 2043-56; Caux et al., J. Exp. Med. (1994) 180: 1263-72; Alderson et al., J. Exp. Med. (1993). ) 178: 669-74; Young et al., Int. J. Cancer (1989) 43: 786-94;; Paulie et al., Cancer I munol. Immunother .. (1985) 20: 23-28 Denfeld et al. , Eur. Im unol. (1996) 26: 2329-34 Gaspari et al., Eur. J. Immunol, (1996) 26: 1371-77 Peguet-Navarro et al., J. I Munol. (1997) 158: 144-52 Hollenbaugh et al., J. Exp. Med. (1995) 182: 33-40; Galy and Spits J. Im unol. (1992) 149: 775-82. Gp 39 is also known as CD40L, TRAP, T-BAM, and now has the official designation of the Leukocyte Workshop of CD154. In in vi tro tests, gp39 appears in T cells approximately 2-4 hours following T cell activation and peak levels in 6-8 hours. The protein level then declines rapidly and is not detectable 24 hours after stimulation. The expression gp39 has also been detected in eosinophilic cells and mast cells (Noelle et al., Proc. Nati, Acad. Sci. (1992) 89: 6550-54; Hollenbaugh et al., EMBO J. (1992) 11: 4313 -21; Spriggs et al., J. Exp. Med. (1992) 176: 1543-50; Graf et al., Eur. J. Immunol. (1992) 22: 3191-94; et al., Covey et al. ., Mol. Immunol. (1994) 31: 471-84; Castle et al., J. Immunol. (1993) 151: 1777-88; Roy et al., J. Immunol. (1993) 151: 2497-2510; Gauchat et al., Nature (1993) 365: 340-43; Gauchat et al., Eur. J. Immunol. (1995) 25: 863-65; Koshy et al., J. Clin. Inv. (1996) 98: 826-37; Desai-Mehta et al., J. Clin. Inv. (1996) 97: 2063-73.

CD40 is a potent signaling receptor, which provides a mechanism for activated T cells to regulate a wide range of immune and inflammatory responses. In vitro and in vivo studies with recombinant forms of the gp39 ligand and anti-CD40 mAbs have shown that signaling through this receptor that leads to a cellular response in all CD40 + cells, and that the product does not only vary by type of cell but is also modulated by concurrent signaling events through other receivers. In B cells, for example, CD40 signaling in conjunction with signaling by the IL-4 receptor leads to B cell proliferation and the production of IgE isotype antibodies (Gordon et al., Eur. J. Immunol. 1987) 17: 1535-38; Rousset et al., J. Exp. Med. (1991) 173: 705-710; Jabara et al., J. Exp. Med. (1990) 172: 1861-64; Gasean et al. al., J. I munol. (1991) 147: 8-13). Gp39 mediating CD40 signaling can play a very important role in cellular immunity through the induction of CD80 and CD86, the important T-cell co-stimulatory molecules which bind CD28 and CTLA4 (Goldstein et al., Mol. Immunol (1996) 33: 541-52).

The CD40 / gp39 receptor / ligand system is one of many systems which are involved in the productive interaction between activated T cells and other cells of the immune system. However, a number of findings suggest that this interaction is unique and central to the regulation of the humoral immune response in humans. In particular, defects in gp39 expression or structure have been shown to be the cause of human immunodeficiency known as hyperlinked X-linked IgM (X-HIM) syndrome. This immunodeficiency is characterized by the inability of affected individuals to produce other antibodies than those of the IgM isotype, indicating that the productive interaction between gp39 and CD40 is required by an effective humoral immune response (Alien et al., Science (1993) 259: 990-93; Aruffo et al., Cell (1993) 72: 291-300; Di Santo et al-, Nature (1993) 361: 541-43; Fuleihan, et al. al., Proc. Nati, Acad. Sci. (1993) 90 (6): 2170-73; Korthauer et al., Nature (1993) 361: 539-541; Notarangelo et al., Immunodef. Rev. (1992) 3: 101-22). In the same way, recent data indicate that no X-linked HIM syndrome in humans is caused by defects in the CD40 molecule. Using knock technology that knocks out the gene, mice lacking CD40 or gp39 have been generated.

These mice exhibit a phenotype which has the same characteristics as HIM syndrome suggesting that mice may be an appropriate model in which to test the effects of in vivo treatment with both anti-CD40 or anti-gp39 mAbs that blocks the interaction between CD40 and gp39 (Kawabe et al., Im unit (1994) 1: 167-78; Xu et al., Im unity (1994) 1: 423-431; Renshaw et al., J. Exp. Med. (1994) 180 : 1889-1900; Castigli et al., Proc. Nati, Acad. Sci. USA (1994) 91: 12135-39).

The effects of in vivo inhibition of the CD40 / gp39 interaction have been extensively studied in normal mice and mouse models of disease using a hamster anti-mouse mAb gp39 (MRI). The immunosuppressive capacity of the antibodies is reflected in their ability to completely inhibit the humoral immune response for T-cell dependent antigens (Foy, et al., J. Exp. Med. (1993) 178: 1567-75). Some mouse models of immune diseases have also been shown to be inhibited by treatment with the antibody, including those mediated by cellular immune responses. Disease models that were shown to be inhibited by treatment with anti-gp39 include collagen-induced arthritis, experimental allergic encephalomelitis, lupus nephritis, transplant rejection disease and graft vs. host (Durie et al., Science (1993) 261: 1328-30; Berry, et al., unpublished; Gerritse et al., Proc. Nati. Acad. Sci. USA (1995) 93: 2499-504; Mohán et al., J. Immunol. (1995) 154: 1470-1480; Larsen et al., Transplantation (1996) 61: 4-9; Hancock et al., Proc. Nati Acad. Sci.USA (1996) 93: 13967-72; Parker et al., Proc. Nati Acad. Sci.

USA (1995) 92: 9560-64; Durie, et al., J. Clin. Invest. (1994) 94: 1333-38, Wallace, et al., Unpublished). The role of CD40 / gp39 in the amplification of a cellular immune response can be direct, through the stimulation of a subset of activated T cells that are capable of expressing CD40, or indirectly, through the induction of cytokines and the expression of important co-stimulatory cell surface molecules such as CDdO and CD86, which bind the CD28 and CTLA-4 T cell receptors. The anti-inflammatory effects of the inhibitor have been demonstrated by studies in a mouse model of lung damage by oxygen induced. The effects on inflammation in vivo are suggested by the results showing that the stimulation of CD40 on vascular endothelial cells and monocytes which result in the expression of cell adhesion molecules, nitric oxide (NO), metalloproteinase and proinflammatory matrix cytokines (Kiener et al., J. Immunol. (1995) 155: 3952-60; Hollenbaugh et al., J. Exp. Med. (1995) 182: 33-40).

Biological studies with anti-human gp39 mAbs in monkeys have shown that these biologicals that inhibit the interaction between gp39 and CD40 in vivo are effective immunosuppressive agents in primates. Anti-gp39 mAbs have been shown to be effective for the inhibition of antibody responses to T cell-dependent antigens, and to protect allografts from rejection, similar results have been observed in rodents.

Collectively previous studies have shown that agents which break the interaction between gp39 and CD40 could be potent immunosuppressants and anti-inflammatory agents. Therefore, there is a need in the art for an effective method of blocking the interaction of CD40 / gp39 to provide an immunosuppressive or anti-inflammatory effect. One purpose of the present invention is to provide an antibody, which blocks the interaction between gp39 and CD40.

Another objective of the present invention is to provide an effective chimeric antibody to block the interaction between gp39 and CD40.

Another objective of the present invention is to provide an effective humanized antibody to block the interaction between CD40 and gp39.

A further object of the present invention is a method of modulating an immune response by administering an antibody, chimeric antibody, or humanized antibody of the present invention. The method can be useful in treating any number of autoimmune diseases, such as skin or other organ transplantation.

BRIEF DESCRIPTION OF THE INVENTION The present invention comprises a novel antibody, more preferably a chimerized anti-human monoclonal CD40 antibody (mAb), which blocks the interaction between gp39 and CD40. In one embodiment of the present invention, a particularly preferred chimerized CD40 anti-human mAb is referred to as "chi220". Chi220 is a chimeric antibody that comprises murine variable and human kappa and constant regions of gamma 1. Chi220, like its parent mouse mAb, binds CD40 and, as a result, effectively blocks humoral immune responses from T cell-dependent antigens and a dependent on the dose.

Also within the scope of the present invention were the humanized anti-CD40 antibodies which block the interaction between gp39 and CD40. In one embodiment of the present invention, a humanized antibody is referred to as F4; in another embodiment, the humanized antibody is referred to as L3.17. Preferred humanized antibodies of the present invention comprise varying variable and heavy human variable regions with murine CDRs grafted thereinto.

The anti-CD40 antibodies of the present invention, preferably the chimeric and humanized antibodies set forth herein, are effective in modulating humoral immune responses against T cell-dependent antigens, collagen-induced arthritis, and transplant rejection. The anti-CD40 antibodies of the present invention, preferably the chimeric and humanized antibodies set forth herein, are also useful for their anti-inflammatory properties (which are similar to those observed with anti-gp39).

The antibodies of the present invention, particularly the chimeric anti-CD40 antibody, chi 220 and humanized anti-CD40, F4 and L3.17 antibodies, have extensive therapeutic applications, including autoimmune diseases, inflammatory diseases and transplants. Because the expression of CD40 observed on malignant cells of some histological types, the potential of oncological applications of anti-CD40 antibodies, particularly the chimeric and humanized antibodies of the present invention, are evident.

The following abbreviations with used through the present application and are known to those skilled in the art: APC (cells presenting antigen); CDR (complementary-determinant region); CHO (Chinese hamster ovary); CIA (collagen-induced arthritis); Cmax (maximum serum concentration); COS (african green monkey fibroblast cell line); DMARD (modified anti-rheumatic disease medications); ELISA (enzyme-linked immunosorbent assay); EPT (Endpoint Titration); EU (endotoxin units); Fab (fragment of antigen binding); FITC (fluoroisothiocinate); HU (humanized); hl06-2 (anti-gp39 humanized mAb); HAMA (human-anti-mouse antibodies); im (intramuscular); KHL (bocacalle limpet hemocyanin) mAb (monoclonal antibody); MTX (methotrexate); OVA (ovalbumin); PBS (phosphate buffer saline); PCR (polymerase chain reaction); PE (phycoerythrin); se (subcutaneous); SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis); SEC (size exclusion chromatography); SRBC (red blood cells of sheep); STR (tank reactor with agitation); TNF (tumor necrosis factor); VL (antibody light chain variable region); VH (antibody heavy chain variable region).

A nucleic acid. encoding a preferred light chain of the chimeric antibody of the present invention (chimeric antibody 2220) has been deposited with the American Type Culture Collection (ATCC) and was given the ATCC Accession Number. A nucleic acid encoding a preferred heavy chain of a chimeric antibody of the present invention (2,220) has been deposited with the American Type Culture Collection (ATCC) and was given the ATCC Accession Number.

A nucleic acid encoding a preferred light chain of the humanized antibody of the present invention (humanized antibody F4) has been deposited with the American Type Culture Collection (ATCC) and given the ATCC Accession Number. A nucleic acid encoding an additional preferred light chain of the humanized antibody of the present invention (humanized antibody L3.17) has been deposited with the American Type Culture Collection (ATCC) and given the ATCC Accession Number. A nucleic acid encoding a preferred heavy chain of the humanized antibody of the present invention (F4 and L3.17) has been deposited with the American Type Culture Collection (ATCC) and given the ATCC Accession Number.

The deposits referred to herein will be maintained under the terms of the Budapest Treaty in the International Recognition of the Deposit of Microorganisms for purposes of Patent Procedures. These deposits are provided merely as convenience to those qualified in the art and are not in admission that a deposit is required under 35 U.S.C. § 112. The polynucleotide sequence (s) contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded therein, are incorporated herein by reference and are controlled in the case of any conflict with any description of sequences in this writing. A license may be required to make, use or sell the deposited materials, and such a license is not granted here.

All references cited in this application, whether s? Pra or infra are hereby incorporated by reference in their entirety.

Brief Description of the Drawings Figure 1 shows the inhibition - of sgp39 linked to Raji cells by anti-human CD40 mAbs.

Figure 2 is a schematic description of a primate study protocol. The days of treatment are indicated with diamonds. Immunization with SRBC and KLH are indicated with rectangles and triangles, respectively. The animals treated with 2.36 were not studied after Phase I and the animals treated with 1,106 were not studied after Phase II.

Figure 3 shows the response of anti-SRBC antibody in primates. Figure 3a shows the results of the analysis for anti-SRBC IgM antibodies. Figure 3b shows the results of the analysis for anti-SRBC IgG antibodies.

Figure 4a shows the variable region sequence of the chi220 light chain in bold (SEQ ID NO: 1), Figure 4b shows the variable region sequence of the chi220 heavy chain in bold (SEQ ID NO: 2). ). The sequences underlined in Figure 4a and 4b are the signal sequences inserted from the human antibody with the closest homology which have been used as a humanization standard.

Figure 5 shows the results of the in vitro tests testing the chimeric and humanized antibody of the present invention. Figure 5a shows the binding of chi220 and h220v3 to hCD40-mG2b in an ELISA-based assay. Figure 5b shows the inhibition of sgp39 mediated co-stimulation of human B cells with anti-human CD40 mAbs.

Figure 6 shows the anti-SRBC IgM antibody response. Figure 6a shows the results of monkeys receiving 10, 40 or 100 mg / kg chi220. Figure 6b shows the results of the monkeys that received 0.1 or 1 mg / kg chi220.

Figure 7 shows the anti-SRBC IgG antibody response. Figure 7a shows the results of monkeys receiving 10, 40 or 100 mg / kg chi220. Figure 7b shows the results of monkeys receiving 0.1 or 1 mg / kg chi220.

Figure 8 shows the anti-OVA antibody response in primates. Figure 8a shows the results of the analysis for anti-OVA IgM antibodies. Figure 8b shows the results of the anti-OVA IgG antibody analysis.

Figure 9 shows the anti-KLH antibody response in primates. Figure 9a shows the results of the analysis for anti-KLH IgM antibodies. Figure 9b shows the results of the anti-KLH IgG antibody analysis.

Figure 10 shows a comparison of the ability of the antibody 7E1-G1 and 7E1-G2b to suppress the IgG antibody response to SRBC.

Figure 11 shows the response dose of inhibition of antibody response to SRBC with 7E1-G2b.

Figure 12 shows the maps of the expression vector for the heavy chain region and the light chain region of a chimeric antibody of the present invention.

Figure 13 provides a nucleic acid sequence for an expression vector capable of expressing a heavy chain of a chimeric antibody of the present invention. The initial ATG (nucleotides 1000-1002), encoding the initial Met of the inserted human antibody signal sequence, is in bold. Nucleotides 1057 through 1422 (SEQ ID NO: 5), underlined, provide a preferred nucleic acid sequence encoding a variable heavy chain of an antibody of the present invention.

Figure 14 provides a nucleic acid sequence for an expression vector capable of expressing a chimeric antibody light chain of the present invention. The initial ATG (nucleotides 1005-1007), encoding the initial Met of the inserted human antibody signal sequence, is in bold. Nucleotides 1065 through 1388 (SC ID NO: 6), underlined, provide a preferred nucleic acid sequence encoding a variable light chain of an antibody of the present invention.

Figure 15 shows an alignment of murine anti-CD40 variable regions and human pattern sequences. The amino acid sequences of murine anti-CDH40 H and L chain variable regions were used to identify homologous human germ line sequences. The numbering of residues and the definition of CDRs (underlined) were based on Kabat et al. (Kabat, EA et al., (1991) Sequences of proteins of immunological interest (5th Ed.) Whashington DC: United States Department of Health and Human Services; Kabat, EA, et al., (1977) J. Biol. Chem. 252: 66o9-6616). The differences in sequence are indicated by vertical lines and structure positions characterized in the combinatorial expression library are marked with an asterisk.

Figure 16 shows the titration results of humanized anti-CD40 variants on immobilized antigen. The chimeric anti-CD40 Fab variants expressed in bacteria and the variants selected from each of the libraries were characterized. Fab chimeric (full circles), Hu I-19C11 (open circles), Hu II-CW43 (open squares), Hu III-2B8 (filled triangles), and an irrelevant (filled squares) were released from the periplasmic space of 15 ml of Bacterial cultures and serial dilutions were incubated with immobilized CD40-Ig antigen in microtiter plates. The bound antibody was quantified as described below.

Figure 17 demonstrates how the affinity of the antibody correlates with the inhibition of soluble gp-39 bound to CD40-Ig. The ligand for the CD40 receptor, gp39, was captured in a microtiter plate. Subsequently, variable quantities of purified chimeric Fabs (full circles), Hu II-CW43 (open squares), Hu III-2B8 (filled triangles), Hu II / III-2B12 (open circles), and an irrelevant (filled squares) were co-incubated with 2 μg / ml CD40-Ig on microtiter plate. The binding of CD40-Ig to gp39 was quantified as described below.

Figure 18 shows the quantification of residues of the murine structure in active variants. The variable regions of most of the active anti-CD40 variants of the structure optimization library Hu I (A) and of the structure optimization library / HCDR3, Hu II (B) were sequenced to identify the amino acids in the positions of the structure library. Each unique variant was categorized based on the total number of murine residues retained in library positions of structure 8. Thirty-four clones from the Hu I library and fourteen clones from the HU II library were sequenced, leading to the identification of the unique variants 24 and 10, respectively. The solid line indicates the expected sequence distribution of an equal number of randomly selected variants.

Detailed description of the invention The present inventors have developed chimeric and humanized anti-human CD40 antibodies with immunosuppressive properties. Such anti-human CD40 antibodies have obvious applications as a therapeutic. The present inventors have also developed a close anti-mouse mAb pair CD40 (close pair for anti-human mAb CD40) which is useful for studying the effects of anti-CD40 mAb therapy on a number of mouse models of immune and inflammatory disease . The development of anti-CD40 antibodies is complicated by the fact that CD40 is a potent signaling molecule. Antibodies that bind to this antigen can be categorized based on the ability to stimulate CD40 signaling as well as the ability to block the CD40 / gp39 interaction.

Applicants for anti-human mAb CD40, which blocks the CD40 / gp39 interaction, was selected from an extensive panel of anti-CD40 mAbs. The antibody, labeled 2220, was chimerized and humanized. The "chimeric" antibodies comprise a light chain and a heavy chain: the light chain is comprised in a variable region of light chain and a constant region of light chain; the heavy chain is comprised in a heavy chain variable region and a heavy chain constant region. Chimeric antibodies comprise variable regions of a species and constant regions of other species (e.g., mouse variable regions linked with constant human regions). (See, e.g., U.S. Patents 4,816,397 and 4,816, 567). Each light chain variable region (VL) and each heavy chain variable region (VH) consists of "structure" regions interrupted by three hypervariable regions called "determinant complemental regions" or "CDRs." "Humanized" antibodies comprise antibodies with regions of human structure combined with immunoglobulin CDRs from a donor mouse or a rat. (See, e.g. U.S. Patent 5,530,101). Included within the scope of the present invention are humanized antibodies which comprise CDRs derived from the murine variable chains published herein.

Most direct approaches to humanize an antibody consist of grafting the CDRs of the mAb donor onto a human structure (Jones, P.T., et al., (1986) Nature 321: 522-525). However, certain residue supports of CDR structures, and murine CDRs grafted onto contact antigen in human structure standards can decrease the binding activity of the resulting humanized mAb (Foote, J., et al., (1992) J Mol. Biol. 224: 487-499). The evaluation of the potential contribution of structure-specific residues to antibody affinity has two problems. First, for a particular mAb it is difficult to predict which structure residues play a critical role in maintaining affinity and specificity. Second, for structure positions that differ between the parent mAb and the human pattern, it is difficult to predict whether the amino acid derived from the parent murine or human pattern will yield a more active mAb. Consequently, antibody humanization methods that rely exclusively on structural predictions are not always successful.

The prior art contains a description of a general antibody engineering strategy that points out the difficulty of maintaining the antibody binding activity followed by humanization (Rosok, M., et al., (1996) J. Biol. . Chem. 271: 22611-22618). The potentially important structure residues that differ between the parent mAb and the human standard are characterized in a single step by synthesizing and expressing a combinatorial antibody library containing all possible combinations of amino acids of the parent and the human standard at the positions of structure in question. The variants showing the structure of the optimal structure are identified by protection and subsequently, the structure (s) of the optimal structure are determined by DNA sequencing. Typically, multiple sequencing of active clones reveals critical structure positions that require the expression of a particular amino acid. Conversely, the expression of a murine or human amino acid in the structure position of the library at an equivalent frequency in the active clones is consistent with a less important function that particular structure position. In this way, a humanized version of the antibody that preserves the binding activity of the parent mAb is rapidly identified based on functional linkage.

The processes of antibody humanization and affinity maturation are often performed in discrete stages (Rosok (1996), supra; Yelton, D.E. et al., (1995) J.

Immunol. 155: 1994-2004; Wu, H., et al., (1998) Proc.

Nati Acad. Sci. USA 95: 6037-6042; Baca, M., et al., (1997) J. Biol. . Chem. 272: 10678-10684; Marks, J.D., et al., (1992) J. Biol. Chem. 267: 16007-16010). Using a modified strategy described below, multiple humanized versions of murine mAb 2.220 showing equivalent or better affinities than chimeric Fab were generated.

The antibody of the chimeric anti-CD40 application of the present invention referred to herein as "chi220". Applicants for the near pair anti-mouse mAb CD40 referred to herein as "7EI". Applicants for humanized anti-CD40 antibodies of the present invention are referred to herein as "F4" and "L3.17".

Two different isotype variants of 7E1 were generated. These two variants of 7E1 are useful in examining the role of the Fc portion of the molecule in. mAb anti-CD40 therapy in preclinical models of immuno and inflammatory diseases. The generation of anti-mouse mAb CD40, the criteria used to select one which makes the properties of chi220 mate, the generation of isotype variants of mAb and their activity in vivo in mouse models of immune diseases are also presented here. Studies with both chi220 and murine father mñb 2,220 in monkeys, as well as studies with 7E1 in mice, showed that these anti-CD40 mAbs are potent immunosuppressive agents, and will be discussed in more detail below. The studies described here were carried out using standard technology known to those skilled in the art.

In summary, the antibodies of the application have been shown to suppress the humoral immune response in monkeys. In the same way, two isotype variants of a close partner of anti-mouse mAb CD40, 7E1, showed immunosuppressive activity in a number of preclinical models of human disease. Taken together, these findings indicate that chi220, F4 and L3.17 are useful for clinical application in the treatment of autoimmune diseases and transplants.

The following examples are for illustrative purposes and do not limit the scope of the invention sought, which is defined only by the claims.

Example 1 Selection of Antibody Murine Antibody CD40 A. Isolation and characterization of live Jn A panel of monoclonal antibodies was generated against human CD40 using standard hybridoma technology with human CD40 fusion protein as the immunogen. The antibodies were protected by binding to CD40 using both a CD40 + cell line and fusion proteins. Bonding tests of gp39 for CD40 and functional tests of stimulation through CD40 were used to characterize cloned antibodies. Selected antibodies were then characterized by cross-activity with primate cells to test the availability of antibodies for use in primate preclinical models. 1. Immunization and Fusion. Two fusions were carried out to generate hybridomas producing anti-human CD40 mAbs. Immunizations to generate immune lymphocytes were carried out in 6-8 week old female BALB / c mice using as the immunogen a recombinant fusion protein consisting of the extracellular domain of human CD40 fused to the main one, the CH2 and CH3 domains of a Murine IgG2b antibody (hCD40-mG2b).

For 40-1 fusion, the mouse was initially immunized subcutaneously at sites 3-4 with an emulsion (total of 200 ul) of 30 ug hCD40-mG2b in complete Freund's adjuvant. The animal was similarly boosted on day 21 with hCD40-mG2b in incomplete Freund's adjuvant and then given a final pre-fusion immunization on day 37 by intervenous injection of 30ug of hCD40-mG2b in PBS. Immunizations for 40-2 fusion were similarly carried out except that the adjuster Ribi (R-730) was replaced by Freund's adjutor. Potentiating immunizations were on days 21 and 42 with final pre-fusion potential on day 58.

The three days following the final potentiation injections, vessel leukocytes and lymph nodes were harvested and fused at a ratio of 3: 1 with mouse myeloma cells X63-Ag8.653 using standard methods (Kearney et al., J. Immunol. (1979) 123: 1548-50; Lane, J. Immunol. (1985) 81: 223-28). Cell suspensions from each fusion were seeded in ten boxes of void cell cultures 96 at a culture density of approximately 170,000 total cells (prefusion) per cast. 2. Protection and Cloning Two test formats were used to identify mAbs with specificity for native human CD40. The cell culture supernatants from all the casts were initially protected by their ability to bind to CD40 positive, human B cell line transformed EBV (1A2-2C) in a base ELISA format. Each supernatant was then tested in an ELISA-based format for reactivity with a recombinant, purified fusion protein consisting of the extracellular domain of human CD40 fused to the main one, the CH2 and CH3 domains of a human IgGl antibody, hCD40-Ig, and a similarly constructed irrelevant human Ig fusion protein, Leu8-hlg "(Hollenbaugh, et al., EMBO J. (1992) 11: 4313-4321) Reactivity with the former and not with the latter last fusion protein, coupled with the cell-binding data, he established the presence of the native CD40-specific antibody in approximately 200 master castings.

A key functional property for the desired anti-CD40 mAb was the ability to completely block the interaction of CD40 and its ligand, gp39. Thus, as the next step in antibody selection, all CD40-specific masked supernatants were tested for their ability to inhibit the binding of the soluble, recombinant murine human-CD gp39 fusion protein, sgp39, to immobilized immobilized IgG-40. in an ELISA base format. (Those that completely inhibited this interaction were subsequently titled in the same format to establish which fills contained the highest titers of inhibitory antibody.) From this analysis, 10 of the strongest empty master inhibitors were selected for cloning.

The cloning of the appropriate antibody secreting cells was carried out in a two-step process. The cells of each master dump were first "miniclonated" at a seeding density of 10 cells / void after which the highest titrated, the CD40-specific "miniclone" void was formally cloned by a limiting dilution method. 3. Posterior Characterization Six test formats were used for subsequent antibody characterization. These were sgp39 inhibition bound to human B cells, inhibition of B cell proliferation induced by spg39 plus anti-IgM, "inhibition of the synthesis of the antibody in vi tro by B cells induced by activated T cells, direct co-stimulation of B cells with anti-IgM, direct co-stimulation of B cells with anti-IgM in the presence of chain antibody light cross-linked anti-kappa, and direct co-stimulation of B cells with anti-IgM in the presence of a second anti-CD40 mAb, G28-5. This mAb was known to possess strong co-stimulant activity and incompletely block CD40 / gp39 interaction has been included for comparison purposes in many of the tests.

This analysis led to the selection of four mAbs: 1.66 (IgG2b), 2.36 (IgG2a), 2.174 (IgGl) and 2.220 (IgG2a). The tests were run to characterize the mAbs. In one experiment, Raji cells human B cell line were incubated with 2 or 20 μg / ml of several anti-CD40 mAbs followed by a second incubation in undiluted COS cell supernatant containing mCD8-gp39 fusion proteins (sgp39). Sgp39 bound was detected by subsequent incubation of cells with FITC labeled mAb anti-mCD8 and analysis of cells in a FACScan. The percent inhibition was calculated by dividing the mean fluorescence of the samples incubated with the antibody by the mean fluorescence of the samples without antibody in the first incubation (Figure 1).

As shown in Figure 1, each of the four mAbs was able to completely inhibit the binding of the sgp39 fusion protein to a human B cell line expressing high levels of CD40, although in the case of 2174, a relatively high concentration of antibodies was required for complete blockade. Similar data were obtained using human amygdala B cells. These data were in parallel for two functional tests. First, it was shown that each mAb was able to completely block the costutration mediated by sgp39 of human amygdala B cells. Second, each significantly inhibited the production of IgG and IgM in a T-cell-dependent B cell antibody synthesis assay.

Three out of four antibodies showed limited ability to co-stimulate B cell proliferation in the presence of anti-IgM. MAb 2.220 was more consistent in its ability to induce weak co-stimulatory activity. With the addition of an anti-kappa light chain antibody, used to crosslink anti-CD40 mAbs, 2.36 gained significant co-stimulatory activity, while the activity of three other antibodies was not affected. The co-stimulatory ability of G28-5 was shown to be differentially modulated when paired in combination with each of the four new anti-CD40 mAbs. MAbs 1.66 and especially 2,174 improved co-stimulation G28-5, while 2,220 and 2.36 suppressed it.

The following selection based on evaluations in in vitro human systems, the four anti-CD40 mAbs were subsequently examined for suitability for in vivo evaluation in non-human primate studies. Two key points of analysis were the relative potency of each to bind primate B cells and in vitro suppression, B-cell antibody synthesis dependent on T cells. All four mAbs were found to cross-react with B cells of cynomolgus macaque (Macaca fascicularis). Links 2.36 and 2.220 with higher avidity than 2.174 and 1.66. The apparent lower linkage of mAbs 2174 and 1.66 was not due to their particular isotypes, as another isotype pair mAbs anti-CD40 demonstrated binding levels comparable to 2.36 and 2.220. { e.g., G28-5 and 2.118). These results were in contrast to those observed with human B cells where each of the mAbs demonstrated comparable binding. The ability of the four mAbs to suppress antibody synthesis for monkey B cells was found parallel to the ability to bind.

B. In vivo characterization.

Two studies were carried out in non-human primates using the murine anti-human CD40 mAbs to assess the adequacy of anti-CD40 as an immunosuppressive agent and to select the appropriate antibody for further development. First, the in vivo clearance and acute toxicity of the four selected anti-CD40 mAbs were compared. These results were used to select two antibodies, 2.36 and 2.220, which were then tested in a second study designed to evaluate efficacy in the inhibition of antibody response to T-dependent antigen and acute toxicity.

Primate Efficacy Study with 2.36 and 2.220 Based on previous findings, mAbs 2.36 and 2.220 were evaluated for their ability to suppress T-dependent antibody response following intravenous administration to cynomolgus monkeys. This study was divided into three phases (Figure 2). In Phase I, four consistent groups of one or two female and male cynomolgus monkeys each were immunized intravenously one day with red blood sheep cells (SRBCs), and then treated with 20 mg / kg of mAbs 2.36. 2,220, 106 (Murine IgGl anti-human gp39, positive control), or L6 (IgG2a murine anti-human tumor antigen, negative control) on days 1.3, and 5. Titrations of IgM and IgG for in SRBC immunogen, serum levels of test and control articles, the presence of anti-test and control article antibodies, serum immunoglobulin levels, peripheral blood leukocyte counts, and frequencies of several subpopulations of peripheral blood lymphocytes were determined. In phase II, after control and test items were cleared, the animals were immunized with SRBCs and a second antigen, limpet hemocyanin (KLH), to evaluate the induction of immunological tolerance and the reversibility of the immunosuppression observed. In phase III, selected animals were re-immunized to determine whether they initially suppressed the response of the recovered anti-SRBC antibody by following an additional challenge with SRBCs and to evaluate the secondary antibody response to KLH.

An experiment was performed to show that mAb 2220 significantly suppressed the primary antibody response to SRBCs (Figure 3). The monkeys were treated with 20 mg / kg both of mAb 1.106, L6, 2.36, or 2.220 in Phase I on Days 1, 3, and 5. The monkeys were immunized with SRBC on Day 1 of Phase I, II, and III. Figure 3a shows the results of the serum samples that were analyzed for anti-SRBC IgM antibodies; the Figure 3b shows the results of the serum samples that were analyzed for anti-SRBC IgG antibodies. The data are expressed as the geometric mean of the anti-SRBC titration for each group (n = 3 or 4).

The peak primary response was inhibited 85% and 98% for IgM and IgG, respectively. Following the clearance of mAb 2220 in serum for lower detectable levels, the peak secondary response for SRBCs was still inhibited 79% and 56% for IgM and IgG, respectively, compared to the negative control response in Phase I. This was in contrast to the positive control, mAb 1.106, with which a strong secondary antibody response to SRBCs was observed. The tertiary response to SRBCs was not inhibited, indicating that mAb 2220 induced prolonged immunosuppression, but not immunological tolerance. All animals immunized with KLH had a primary and secondary anti-KLH response, suggesting that immunosuppression was reversible. The animals treated with 2.36 were not included in phase II because no significant inhibition was seen in phase I of the study.

Mean peak serum concentrations, presented immediately after the last dose, were 744 and 405 μg / ml for mAbs 2220 and 2.36, respectively. While mAb 2.36 cleared serum for lower detectable levels by day 15, mAb 2220 did not clear until day 29. Both mAbs 2.36 and 2220 were immunogenic.

There were no clinical observations related to the medication, changes in body weight or food intake, or alterations in hematology or serum Ig levels in any animal. The related findings observed with the medication were only transient decreases of 70% and 43% in the percentages of peripheral B cells with mAbs 2.36 and 2.220, respectively. Recovery of B cells to normal levels occurred within 2-3 weeks post-treatment. In summary, mAb 2.220 significantly suppressed the antibody response to SRBCs and 2.36 did not. Based on these findings, mAb 2.220 was selected for further development.

Example 2 Generation of Chimeric Antibody chi220 To direct immunogenicity of recombinant forms of murine anti-human mAb 2220 in which the variable regions are fused to human constant regions were generated and compared for in vitro efficacy. The two approaches used were chimeric antibody generation, containing the unaltered murine variable regions, and humanized forms in which murine hypervariable regions (CDRs) were grafted onto sequences of human structure within the variable regions. Chimeric antibodies retain the binding properties of the parent antibody antigen, but may be more likely to be immunogenic. Humanized antibodies are less likely to be immunogenic, but mutations introduced in humanization may affect the antigen binding.

A. Construction and Characterization Tn Vitro of Humanized and Chimeric Antibodies.

The VL and VH regions of anti-CD40 mAb 2.220 were obtained by PCR. cDNA was generated from RNA isolated from the hybridoma expressing mAb 2.220 using a specific IgG1 or a Cβ-specific anti-sense primer to obtain the VH and VL regions, respectively. A poly-G termination was added to these unique strands cDNAs. The variable regions were then amplified by PCR using as a sense primer an oligonucleotide containing a poly-C sequence, in addition to the poly-G terminus, and a nested set of antisense primers. The PCR product obtained was then inserted into a bacterial vector using restriction sites included in the primers. The multiple clones were then sequenced by dideoxynucleotide sequencing. Two independent experiments were carried out, starting at an RNA state and the sequences obtained were the same.

To generate a chimeric form of the antibody, the variable regions were amplified by PCR using primers that introduced a sequence encoding the human antibody signal sequence found to closely match the 2,220 sequence, as shown in Figure 4. The underlined portions of the variable sequence of the light chain (Figure 4a) and the variable sequence of the heavy chain (Figure 4b) designated the signal sequences inserted from the human antibody with the homologous closest to murine 2220. The PCR products were inserted into a vector containing the sequences encoding the constant regions of human kappa or of λ to generate light or heavy chain, respectively. The vectors also contained genes resistant to the appropriate drug for the generation and amplification of stable lines expressing the protein. The protein for the initial characterization was produced by transient expression of COS cells followed by protein A purification.

As an example, the chimeric antibody producing cell line was generated by co-transfecting CHO DG44 cells with separate expression vectors for the light and heavy chains of the chimeric antibody, and the high copy number electroporation method was used to promote co-integration (See, U.S. Patent 4,956,288). The heavy and light chains of chi220 were cloned in the expression vectors pD17 and pD16, respectively. Both vectors are derived from the pcDNA3 plasmid In Vitrogen, and have the following characteristics (Figure 12): (1) neomycin-resistant gene from pcDNA 3 was replaced with the murine dihydrofolate reductase gene. { DHFR) under control of the SV40 promoter minus enhancer (also referred to as the "weakened DHFR", note that only the promoter was weakened, not the DHFR enzyme - the less enhancer promoter still contains the SV40 origin of replication, such that these vectors they can be used in transient COS transfectations); (2) the gene of interest is expressed from the CMV promoter, and the polyadenylation signals are from the bovine growth hormone gene; (3) the expression box for the gene of interest is flanked by transcription termination sequences (i.e., 5 'for the promoter and 3' for the poly A site); (4) the vectors contain two different restriction site poly-links, one 3 'for the promoter for cloning the gene of interest, and one 5' for the promoter for the linearization of the vector prior to transfection; and (5) the gene resistant to ampicillin and ColEl origin for propagation of the plasmid in E. coli.

The light and heavy chain genes used were genomic constructions, with the following modifications: (1) the sequences encoding the heavy chain signal peptide, variable region and the CH1 domain were contiguous (i.e., did not contain introns); (2) sequences coding for light chain and variable region signal peptide were contiguous.

Other expression vectors known to those skilled in the art, and capable of expressing a chimeric antibody of the present invention, are considered by the present invention. A nucleic acid sequence useful in the expression vector capable of expressing a heavy chain of a chimeric antibody of the present invention is shown in Figure 13; A nucleic acid sequence useful in an expression vector capable of expressing a chain of a chimeric antibody of the present invention is shown in Figure 14.

The complete amino acid sequence of the light and heavy chains of the chimeric antibody ("chi220"), includes the variable and constant regions, as follows (The amino acids in bold indicate variable variable and light weight): Heavy Chain Sequence (SEQ ID NO: 3) QIQ VQSGPE LKKPGETVRT. SCKASGYAFT TTGMQWVQEM PG GLKWIG 50 INTHSGVPKY VEDFKGRFAF SLETSANTAY LQISNLNED TATYFCVRSG 100 NGNYDIAYFA AND GQGTXVTV SAASTKGPSV FPIAPSSKST SGGTAALGC 150 VKDYFPEPVT V3WNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPS? SLGT 200 QTYICNVNHK P? NTKVDKKV? PKSCDKTET CPPCPAP? LL GGPSVFLFPP 250 KP DT MI5R TPEVTCWVD VSH? DP? NWYVDGVEVH NAKTKPREEQ 300 YNSTYRWSV VKF LTVLHQDWLN GK? KALPAPIEKT ISKAKGQPRE 350 PQVYTLPPSR YKCKVSN D? LTKNQVSL DIAVE TCLVKGFYPS MSNF QPENNYKTT? 400 PVLD? DGSFF LYSKLTVDKS R QQGNVFSC SVMHEALHNK YTQKS 3 S? 450 GK 452 Light Chain Sequence (SEQ ID NO: 4) DrvXTQSPAT LSVTPGDRVS IiSCRASQSIS DY H YQQKS HESPRLLSKY 50 ASHSISGSPS RFSGSGSGSD TLSINSVEP EDVGIYYCQH GHSFPWT GG 100 GnOiEIKRTV AAPSVFIFPP S? EQLKSGTA SWCLLNNFY PR? AKVQW V 150 DNALQSGNSQ? SVTEQDSKD STYSLSSTLT LSKADYE H VYACEVTHQO 200? SPVTKSFN RGEC 214 Some humanized forms of 220 were generated. This process involves the identification of murine and germline sequences with the closest homologies for the VH and VL domains. The murine germ line sequences were used to identify probable locations of somatic mutations that have arisen during the affinity maturation process. The human sequences were then used as a standard and regions of the known sequence or suspected to be important for binding specificity are replaced in human sequences by both VH and VL. The structures of the sequences were then modeled using as a standard the protein with the closest homology for which a crystal structure has been solved. The plasmids encoding the humanized forms were generated using PCR-directed mutagenesis and used to generate the antibody by transient expression of COS cells. In vitro tests were carried out with the chimeric and humanized antibodies of the present invention, and the results are described in Figure 5. Figure 5a shows the results of a binding test that tests the binding of chi229 and h220v3 for hCD40- mG2b in a basic ELISA test. The Immulon-2 cast plates were coated with hCD40-mG2b at a concentration of 10 ng / ml in PBS for 2 hr. The emptying plates were blocked with Specimen Diluent (Genetic Systems), and the antibodies were added at the indicated concentrations. Following one hour of incubation, the emptying plates were washed, and the presence of antibodies detected using IgG peroxidase-conjugated goat anti-human antibody. H220v3 is a humanized form of mAb 2.220. The values are the average of the duplicate dumps and the error bars represent the standard deviation.

Figure 5b shows the results of an assay that tests the inhibition of sgp39-mediated co-stimulation of human B cells with anti-human CD40 mAbs. Resting human tonsil cells (50, 000 / emptying plate) were incubated with sgp39 fusion protein, immunochamas coated with 20 μg / ml anti-human rabbit IgM with and the indicated concentrations of anti-CD40 mAbs or control in 96 emptying plates. 72 hours after the initiation of cultures, all the emptying boxes were pulsed with [H] thymidine 1 uCi / emptying plate and the cells cultured for an additional 18 hours. The cells were then harvested and incorporated [3 H] thymidine as measured in a flash counter.

Based on the results of the in vi tro tests (Figures 5a and 5b, which show both chimeric and humanized antibodies effectively bound CD40 and inhibited B cell stimulation) the chimeric antibody was selected for further study.

Example 3 Effectiveness of chi220 A. • Chimeric chi220: Study of the Efficacy of the Single Dose in Non-Human Primates.

Chi 220 was evaluated in cynomolgus monkeys for its ability to suppress primary and secondary humoral immune responses for T cell-dependent antigens. In one study, groups of four monkeys were immunized with sheep erythrocytes (SRBCs) and given a second immunization of Ovalbumin (OVA) immediately prior to receiving a single intravenous ball dose of either chi220 to 10, 40, or 100 mg / kg or phosphate buffered saline (PBS) as control. Substantial suppression of the primary humoral immune response against SRBCs was observed at all three dose levels, demonstrating efficacy of chi220 in primates. A transient exhaustion dependent on peripheral blood B-cell dose was observed in all monkeys treated with chi220, with time to recover it is also dose dependent. At two higher doses, mild transient decreases in the mean absolute numbers of the peripheral blood T-cell group were observed. Minimal transient decreases in serum IgM levels were observed, no changes related to the drug were observed in serum IgG or IgA levels.

To evaluate the induction of immunological tolerance and reversibility of immunosuppressive activity, all monkeys were immunized with OVA, SRBCs, and a neopannin, limpet hemocyanin (KLH) on day 149, when serum levels of chi220 in the Group of 100 mg / kg were the lowest levels believing themselves to be immuno.supressive (~ 10 μg / ml) and the B numbers of peripheral blood had returned to predose levels. The anti-SRBC response at the lowest dose levels was generally comparable to the response of the primary anti-SRBC antibody in control monkeys. However, the antibody response to SRBCs was still partially or substantially suppressed in monkeys treated at the two highest dose levels.

To further explore the dependence of the dose of immunosuppression and B cell depletion, a second study was carried out in which additional monkeys (four / group) were immunized with SRBCs, and then given a dose of chi220 at 0.1 or 1.0 mg / kg or PBS. Suboptimal immunosuppression of the antibody response to SRBCs was observed for both dose levels. Moderate B-cell depletion of peripheral blood was evident in monkeys receiving 1.0 mg / kg chi 220 by Day 8 reverting by Day 29. At 0.1 mg / kg, a decrease in the mean number and percentage of B cells of Peripheral blood was observed, but the values were not outside the normal historical ranges for peripheral blood B cells. Minimal transient decreases in peripheral blood T cell numbers and mild decreases in ex vivo T cell proliferation were observed in monkeys receiving 1 mg / kg chi220. Finally, there was no evidence of complete activation or changes related to the drug in serum levels of IF-6 or TNFa. Ex vivo T cell activation, complete activation, and serum cytosine levels were not evaluated in monkeys treated with 10, 40, or 100 mg / kg chi220.

In both studies, the serum samples were examined, following the administration of chi220 for circulation levels of the test article, and to evaluate the formation of antibody against the tested article. The pharmacokinetic analysis indicated that the mean peak serum concentration (Cmax) of chi220 did not increase in a manner proportional to the increase in dose, and that the half-life of chi220 became prolonged when the dose administered was increased. Chi220 was found to be immunogenic when administered at 0.1, 1.0 or 10 mg / kg. At circulating concentrations above 10 μg / ml, it appears that chi220 can suppress the antibody response directed against the. 1. Experimental Protocol. In the initial study mentioned above, cynomolgus monkeys were assigned to four groups consisting of two males and two females each. All monkeys were immunized 28 days prior to chi220 or the administration of control articles with OVA (5 mg / kg, im and 10 mg / kg, cs). On Day 1, all monkeys were immunized with SRBCs (1.7 ml / kg of a 10% suspension, iv) and given a second OVA immunization (5 mg / kg, im and 10 mg / kg, cs) immediately prior to receiving a single intravenous ball dose of either chi220 to 10, 40, or 100 mg / kg or sterile PBS as a control. On Day 149, after the chi220 serum levels had putatively fallen below the immunosuppressive levels (~ 10 μg / ml) and the levels of peripheral blood B cells had returned to the predose levels in all groups, the monkeys were immunized with OVA, SRBCs, and KLH (10 mg / animal, im) The purpose of the KLH immunization was to show that the monkeys were able to achieve an immune response to neoantigen after being treated with chi220.

To demonstrate a better dose response with respect to immunosuppression and peripheral blood B-cell depletion, additional monkeys in a second study (two / sex / group) were immunized with SRBCs, and then given a single dose of either chi220 at 10, 40, or 100 mg / kg or PBS as a control on Day 1. Haematological parameters and subpopulations of peripheral blood lymphocytes were monitored at selected time points during both studies. The chemical parameters of the serum were monitored in monkeys that received dose levels of 10, 40, or 100 mg / kg of chi220, but were not monitored at the dose levels of 0.1 and 1.0 mg / kg because no findings related to the medication at the highest doses. In addition, serum levels of IgM, IgG, IgA, and chi220 were determined. To assess efficacy, the formation of specific IgM and IgG antibodies against the SRBC and OVA immunogens was determined in the appropriate serum samples obtained just before the immunogenic administration and weekly thereafter. The specific formation of IgM and IgG antibody against the test article for monkeys receiving chi220 was determined prior to administration of the test article on Day 1, and weekly thereafter. Geometric mean titers were used when the antibody responses between the groups were compared. In addition, total hemolytic complement activity (CH50) and C4d fragment levels were measured, and levels of TNF-or and IL-6 were determined in monkeys receiving 0.1 or 1 mg / kg chi220 at time points. selected following the administration of chi220. Activation of peripheral blood T cell ex vivo was also evaluated following stimulation with concanavilin A in monkeys receiving 0.1 and 1 mg / kg of chi 220 on Days 17 and 31 to evaluate the effects of chi220 on T cell sensitivity to mitogen. Finally, all monkeys were observed daily for clinical signs of toxicity, body weights were recorded weekly, and feed intake was monitored daily.

The monkeys were immunized with SRBC prior to receiving the vehicle or 10, 40, or 100 mg / kg of chi220 (Figure 6a) or 0.1 or 1 mg / kg of chi220 (Figure 6b) on Day 1. The serum samples were analyzed for anti-SRBC IgM antibodies by ELISA. The data expressed as geometric mean endpoint titer of anti-SRBC antibody (EPT) for each group (n = 2 [100 mg / kg group beyond Day 15] or 4), where EPT is equivalent to the reciprocal of the larger dilution of serum with a larger absorbance than twice the background of medium plaque.

Results a. Response of Anti-SRBC Antibody When administered to monkeys 10, 40, or 100 mg / kg, chi220 was substantially effective in suppressing the. Primary antibody response against SRBCs. On the peak day of the response of the control anti-SRBC IgM primary antibody (Day 8), the response of the primary mean anti-SRBC IgM antibody was suppressed approximately 92-94% in the monkeys treated with 10, 40, or 100 mg / kg of chi220, compared to the control (Figure 6a). The response of the anti-SRBC antibody IgM group mean did not become positive until Day 85 at dose levels 10, 40, or 100 mg / kg. On the peak day the response of the primary anti-SRBC IgG antibody to the control (Day 15), the measurement of the primary anti-SRBC IgG antibody response was suppressed at 98%, 99% and 85% in monkeys receiving 10, 40 , or 100 mg / kg chi220, respectively, compared to the controls Figure 7a). The anti-SRBC antibody titers of the highest general predose in the 100 mg / kg group can be taken into account for the apparent lack of dose-dependent immunosuppression. In summary, monkeys treated with 10 or 100 mg / kg of chi200 did not raise a primary anti-SRBC IgG antibody response until Day 85. However, two of the monkeys treated with 40 mg / kg of chi220 had a delay of Primary anti-SRBC IgG antibody response to SRBCs (comparable to control response in magnitude), which becomes positive for Day 36 and peak on Day 51.

On day 149, after serum chi220 levels fell more below putatively immunosuppressive levels (~ 10 μg / ml) and peripheral blood B-cell levels had a return to predose levels at all levels. groups, the monkeys were immunized a second time with SRBCs. As expected, the control monkeys increased a strong secondary anti-SRBC IgG antibody response to SRBCs. Monkeys treated with 10 mg / kg chi220 raised the primary anti-SRBC IgG antibody responses to SRBCs that were generally comparable to the primary antibody response in the control monkeys. However, the antibody response to SRBCs was still partially suppressed at the dose level of 40 mg / kg and substantially suppressed at the dose level of 100 mg / kg. Although two of the monkeys treated with 40 mg / kg of chi220 that had been previously treated raised the weak primary antibody responses to SRBCs they developed anti-SRBC IgM and IgG antibody titers characteristic of a secondary antibody response, the anti-antibody responses -SRBC in two of the other monkeys in that group and the remainder of the monkeys treated with 100 mg / kg of chi220 was approximately 90% suppressed compared to the primary anti-SRBC antibody response of the control monkeys.

Suboptimal immunosuppression of antibody response to SRBCs was observed followed by administration of 0.1 mg / kg or 1.0 mg / kg of chi220. { Figures 6b and 7b). While all monkeys treated with chi220 raised the IgM antibody response positive for the SRBC antigen, the total mean peak response of anti-SRBC IgM antibody was suppressed approximately 56% in monkeys treated with 1 mg / kg chi220 compared with the average peak control response. No suppression of the anti-SRBC IgM antibody response was observed in monkeys treated with 0.1 mg / kg chi220. The average response of anti-SRBC IgM antibody reached the peak on Day 15 in control monkeys, and on Day 8 in monkeys receiving 0.1 and 1.0 mg / kg of chi220. In total, the peak mean response of anti-SRBC IgM antibody was suppressed 56% and 42% in monkeys treated with 0.1 and 1.0 mg / kg chi220, respectively. The mean response of anti-SRBC IgG antibody reached the peak on Day 15 in control monkeys and in monkeys treated with 1 mg / kg chi220, on Day 8 in monkeys receiving 0.1 mg / kg chi220.

Answer Anti-OVA Antibody The monkeys were administered with intravenous doses of 10, 40, or 100 mg / kg of chi220 on Day 1. In addition all monkeys were immunized with OVA on Days 28, 1 and 149. The serum samples were analyzed for antibodies IgM (Figure 8a) or IgG (Figure 8b) anti-OVA.

The data were expressed as geometric mean of the anti-OVA endpoint titer (EPT) for each group (n = 2 [100 mg / kg group beyond Day 15] or 4), where EPTs are equivalent to the reciprocal of the dilution larger serum with an absorbance of more than twice the background of the middle plate.

The specific formation of the IgM and IgG antibody against OVA was monitored weekly during the study in monkeys receiving 10, 40, or 100 mg / kg of chi220. Primary and secondary anti-Ova antibody responses were highly variable and generally weak in all monkeys (Figure 8). Monkeys programmed to receive chi220 on Day 1 had higher anti-OVA antibody titers than monkeys in the control group.

On Day 149, the monkeys were given a third OVA immunization. All monkeys raised OVA positive OVA antibody responses to OVA within 7 days of challenge. Control monkeys and monkeys treated with 10 mg / kg of chi220 had characteristic titers of tertiary antibody response, while monkeys treated with either 40 or 100 mg / kg of chi220 developed antibody titers that were more characteristic of a response tertiary c. Answer Anti-KLH Antibody Monkeys were administered with intravenous doses of 10, 40, or 100 mg / kg of chi220 on Day 1. In addition, all monkeys were immunized with KLH on Day 149. The serum samples were analyzed for IgM antibodies (Figure 9a) or IgG (Figure 9b) anti-KLH. The data were expressed as the geometric mean of the anti-KLH endpoint titer (EPT) for each group (n = 2 [100 mg / kg group beyond Day 15] or 4), where EPTs are equivalent to the reciprocal of the dilution larger serum with an absorbance of more than twice the background of the middle plate.

On Day 149, after serum chi220 levels fell further below immunosuppressive levels putatively (~ 10 μg / ml) and peripheral blood B-cell levels had a return to predose levels in all patients. groups, the monkeys were immunized with KLH (10 mg / animal, im). All the monkeys raised the IgM and IgG antibody responses to KLH, demonstrating that the ability to respond to a new antigen was not compromised (Figure 9). d. Serum Levels of the Test Item and Antibody Response and Anti-Test Article The serum samples were examined following the administration of chi220 to determine the circulation levels of the test article and to evaluate the formation of antibody against the test article . The mean peak serum concentration (Cmax) of chi220 occurred three minutes following the administration of the doses of 10 or 40 mg / kg and at six hours followed by the administration of the 100 mg / kg dose. The Cmax values of chi220 were 329, 2429, and 2343 μg / ml in monkeys treated with 10, 40 or 100 mg / kg chi220, respectively. There was, however, considerable variation in the Cmax of the individual monkeys in the groups of 40 and 100 mg / kg. The mean serum half-life of chi220 was estimated to be approximately 114, 173 and 315 hours in monkeys treated with 10, 40 or 100 mg / kg chi2_20, respectively.

Mean Cmax values were presented at three minutes after the administration of chi220, were 1.77 and 33 μg / ml for doses of 0.1 and 1 mg / kg, respectively. No genders were related to differences in chi220 levels that were observed with each dose level. The values of the mean AUCinf were 15.5 and 847 ug.h / ml, for doses of 0.1 and 1 mg / kg, respectively. Taken together, studies suggest that the half-life of chi220 becomes prolonged when the dose administered increases. Furthermore, it appears that Cmax of chi220 increases the dose increase disproportionately.

Although the IgM anti-test article response was minimal or absent in monkeys receiving 10, 40, or 100 mg / kg chi229, a significant anti-test IgM response was observed in monkeys receiving 10 mg / kg of chi220. The anti-test IgA response medium in monkeys receiving 10 mg / kg of chi220 became positive on Day 29, approximately 1 week after the average serum concentration of the chi220 group had fallen below 10 μg / ml , and reached the peak on Day 36 and 43 to a geometric mean title of 12,627. The appearance of the anti-IgG antibody test article in monkeys that were treated with 10 mg / kg of chi220 also coincided with the first increase detected in B-cell numbers followed by depletion. For the last day measured (Day 149), monkeys receiving 40 or 100 mg / kg of chi220 had not yet raised a positive antibody response against chi220, although the mean chi220 serum levels of the group were lower than 10 μg / ml for Day 57 (40 mg / kg of the group) or Day 92 (100 mg / kg of the group).

Chi220 was immunogenic when administered at 0.1 or 1 mg / kg. Three or four monkeys that received either 0.1 or 1 mg / kg of chi220 had weak IgM anti-positive test antibody responses by Day 15 during the study. Three or four monkeys treated with 1 mg / kg of chi220 had significant IgA anti-test item IgA antibody responses Day 22, having the peak to the titration of the geometric mean end point of 16,618. Overall, the response of the IgM antibody to the test article of the geometric mean was not positive in the monkeys receiving 0.1 mg / kg of chi220, and only a monkey that received 0.1 mg / kg of chi220 had a weakly positive response from the chi220. IgM antibody anti-test article, reaching the final titration peak of 2430 on Day 22. Collectively, these data suggest that chi220 is able to immunosuppress the antibody response itself at serum levels higher than approximately 10 μg / ml.

Example 4 Generation of Humanized Anti-CD40 Antibodies F4 and L3.17 A variety of methods known in the art have been used for the humanization of mAbs. The structure-based enfogues have proven useful but the complexity that arises from the large number of structure residues potentially involved in the link activity decreases the success rate. Rather than predict the optimal structure based on modeling, the approach of the antibody library described below allows the identification of conformations of the active structure based on numerous combinations of protection. Mutagenesis approaches coupled with selection methods allow the analysis of many variants and mimic the maturation process in vivo (reviewed in Mark, JD, et al., (1992) J. Biol. Chem. 267: 16007-16010 ). Codon-based mutagenesis allows the construction of libraries that characterize the contribution of specific residues and, thus, is more efficient than random mutagenesis approaches. For example, error-prone PCR can not be used to synthesize the libraries of combinatorial structures described below. Moreover, random mutagenesis creates larger and more diverse libraries and unfortunately, most mutations do not improve the binding activity of mAb. As a result, larger numbers of clones must be protected to identify active variants.

A strategy called "guided selection" has been used to isolate human mAbs from a phage library published in a two-step process that uses a rodent mAb as a standard (Jespers, L. S., et al., (1994) Biol. / technology 12: 899-903). Recently, a guided selection variation using published phage technologies was described in which a Fd quimperic fragment was used to select an L chain from a library containing human L chains with murine grafted CDR3 (Rader, C, et al., (1998) Proc. Nati Acad. Sci. USA 95: 8910-8915).

Subsequently, the most active L chain was used to select an H chain from the human H chain library containing the murine HCDR3. The mAbs isolated by these approaches are entirely human (Jespers, supra) or primarily human (Rader, supra), but the diversity of the long antibody introduced at each stage of the process necessitates the use of affinity enrichment methods.

The following materials and methods were used to generate the anti-CD40, F4 and L3.17 antibodies of the present invention. 1. Construction of chimeric anti-CD40.

Based on the sequence of murine anti-CD mAb 2,220 overlaying oligonucleotides encoding VH and VL (69-75 bases in length) were synthesized and purified. The variable domains of H and L were synthesized separately by combining 25 pmol of each of the overlapping oligonucleotides with Pfu DNA polymerase.

(Stratagene) in a 50 μl PCR reaction consisting of cycles of: denaturation at 94 ° C for 20 sec, annealing at 50 ° C for 30 sec, heating at 72 ° C for one minute, and holding at 72 ° C for 30 sec. Subsequently, the annealing temperature was increased to 55 ° C for 25 cycles. A reverse primer and a biotinylated forward primer were used for subsequent amplification of 1 μl of the fusion product in a 100 μl PCR reaction using the same program. The products were purified by agarose gel electrophoresis, electroeluted, and phosphorylated by T4 polynucleotide kinase (Boehringer Mannheim) and then incubated with streptavidin magnetic beds (Boehringer Mannheim) in 5mM Tris-Cl pH 7.5, 0.5mM EDTA, 1M NaCl , and 0.05% Tween 20 for 15 min at 25 ° C. The beds were washed and not biotinylated, less DNA strand was eluted by incubation with 0.15 M NaOH at 25 ° C for 10 min. Anti-CD40 chimeric Fab was synthesized in a modified vector M13IX104 (Kristensson, K., et al., (1995) Vaccines 95, pp. 39-43, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) called M13IX104CS, by mutagenesis Hybridization (Rosok, MJ, et al., (1996) J. Biol. Chem. 271: 22611-22618; Kunkel, T.A. (1985) Proc. Nati Acad. Sci * USA 82: 488-492) using VH and VL oligonucleotide in three-fold molar excess of the uridinylated vector standard. The vector M13IX104 was modified by relocation of cysteine residues in the final regions of the kappa and constant of? L with serine. The reaction was electroporated into DH10B cells and titrated on a Blue XL-1 flax. 2. Construction of Combinatorial System and System Libraries / CDR3.

The library of the combinatorial structure (Hu I) was synthesized by the same method used to construct chimeric anti-CD40, with modifications. The overlapping oligonucleotides encoding the regions regions of the structure of the variable domains of H and L of the human and murine anti-CD40 CDRs as defined by Kabat et al. (Kabat, EA, et al., (1991) Sequences of proteins of immunological interest (5th Ed.), Washington DC: United States Department of Health and Human Services; Kabat, EA, et al., (1977) J. Biol. Chem. 252: 6609-6616) were synthesized. The degenerate oligonucleotides that encode both the murine and human amino acids at the position of structure seven of VH and one of VK were synthesized (Figure 15, residues marked with an asterisk).

The libraries of the structure / HCDR3 (Hu II) and structure / HCDR3 / LCDR3 (Hu III) were synthesized by the same method as the library of the combinatorial structure, with modifications. The CDR residues selected by mutagenesis were: Ser95-Tyr102 in HCDR3 and Gln89-Thr97 in LCDR3 (Figure 15, underlined). Oligonucleotides encoding HCDR3 and LCDR3 were designed to mutate a single residue of CDR and were synthesized by introducing NN (G / T) at each position as described in the art (Glaser, SM, et al., (1992) J Immunol. 149: 3903-3913). The overlapping oligonucleotides encoding the library structure and not the library of murine CDRs were combined with 25 pmol of the oligonucleotides encoding mutated HCDR3 or with 25 pmol each of the oligonucleotides encoding mutated HCDR3 and LCDR3. 3. Protection of Expression Libraries in Phages The Hu II and HU III libraries were initially protected by a modified plaque removal approach known in the art, called capture survey (Watkins, JD, et al- (1998) Anal. Biochem. 256: 169-177) . Briefly, nytocellulose filters (82-mm) were coated with goat anti-human kapa, blocked with 1% BSA, and applied to an agar plate containing bacterial flax infected with phages. In the initial protection, the phages were seeded at 105 phages / 100-mm plate. After capture of variants of anti-CD40 Fabs expressed on phages, the filters were incubated 3 h at 25 ° C with 5 ng / ml of CD40-Ig in PBS containing 1% BSA. The filters were rinsed four times with PBS containing 0.1% Tween 20 and incubated with goat anti-mouse IgG2b conjugated alkaline phosphatase (Southern Biotechnology) diluted 3000-fold in PBS containing 1% BSA for 1 h at 25 ° C. The filters were washed four times with PBS containing 0.1% Tween 20 and developed as described (Watkins (1998), supra). To isolate individual clones, the positive plates of the initial protection were collected, plated at a lower density (<103 phages / 100 mm plate), and protected by the same approach.

The combinatorial library Hu 1 was first protected by an ELISA that allows the rapid evaluation of relative affinities of the variants (Watkins, JD, et al., (1997) Anal. Biochem. 253: 37-45), In addition, the ELISA was used to characterize clones identified by survey protection by capture. Briefly, microtiter plates were coated with 5 μg / ml goat anti-human kapa (Southern Biotechnology) and blocked with 3% BSA in PBS. Then, 50 μl of Fab culture supernatant of Escherichia coli or cell lysate was incubated with the plate for 1 h at 25 ° C, the plate was washed three times with PBS containing 0.1% of Tween 20 and 0.1 μg / ml of anti-mouse IgG2b-conjugated goat alkaline phosphatase diluted 3000-fold in PBS containing 1% BSA was added per lh at 25 ° C. The plate was washed three times with PBS containing 0.1% Tween 20 and was developed as described in the art (Watkins (1997), supra). 4. DNA sequencing Single filament DNA was isolated and the variable region genes of the H and L chain of the humanized antibodies were sequenced by the fluorescent dideoxynucleotide termination method (Perkin-Elmer, Foster City, CA).

The nucleic acid sequence (SEQ ID NO: 7) and amino acid sequence (SEQ ID NO: 8) of the variable light chain of the humanized antibody F4 is as follows: GAA ATT GTG TTG AC CAG TCT CCA GCC ACC CTG TCT TTG TCT 42 I V T T Q s P A T i. S L s 14 CCA GGG GAA AGA GCC ACC CTC tcc TGC AGG GCC AGT CAG AGT B4 p G E R A T L s C R A S Q s 28 ATT AGC GAT TAC TTA CAT TGG TAC CA CAG AAA CCT GGC CAG 126 I S D and L H W Y Q Q K P G Q 42 GCT CCC AGG CTC CTC ATC TAT TAC GCA CC CAC TCC ATC TCT 168 GG CCA GCC AGG '' ") AGT GGC AGT GGG TCT GGG ACA GAC 210 p A R S G S c G D 70 1T ACT CTC ACC ATC. . AGC AGC CTA GAG CCT GAA TTT GCA 252 T S S L E P E D A 84 TAT TAC CAG CAT GGC CAC CT CCT TGG ACC TC 294 V Y C Q H G E S F P F 98 GGA ACC AAG GTG GAA ATT AAA 321 G G G K V E T K 107 The nucleic acid sequence (SEQ ID NO: 9) and amino acid sequence (SEQ ID NO: 10) of the variable heavy chain of the humanized antibody F4 and L3.17 is as follows: CAG GTG CAG CTG GTG CAA GGG TCT GAG TTG AAG AAG CCT 42 Q V Q V Q S G S K K P 14 GCC TCA GTG AAG GTT TCC TGC AAG GCT CT GGA TAC GCC 84 G A S V K V £ C K A S G V A 28 TTC ACT ACC ACT or ATG CAG TGG GTG CGA CAG GCC .GGA 126 F T T T G M Q W V R Q A p G 42 CA GAG TGG ATG GGA TGG ATC AAC ACC CAC AGC GGG 168 Q G L W W G G I N T H S G 56 GTC CCA AAG TAT GTC GAG GAC TTC AAA GGA CGG - -. - GTC TTC 210 V p K V V E D F K G R F V 70 CC TTG GAC ACC TCT GTC AGC ACG GCA TAT CTG CAG ATC AGC 252 S L D T s V S T A V L Q I S 84 AGC CTA AAG GCT GAG GAC ACT GCC GTG TAT TAC TGT b.u AGA 294 S K A E D T A V Y V C A R 98 GGC AAT GGG AAC TAT GAC CTG GCA TAC AAG TAT TGG 336 s G N G N V D L A Y F K V W 112 GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA 366 G Q G T L V T V S S 122 The nucleic acid sequence (SEQ ID NO: 11) and amino acid sequence (SEQ ID NO: 12) of the variable light chain of the humanized antibody F4 and L3.17 is as follows: GAA ATT GTG TTG ACA CAG TCT CCA GCC ACC CTG TCT TTG TCT 42 EIVTQSPATLSLS 14 CCA GGG GAA AGA GCC ACC CTC TCC TGC AGG GCC AGT CAG AGT 84? C? R A T L S C R A S S S 28 a-CT AGC CAT TA TO CA TGG TAC CA CAG AAA CCT GGC CAG 126 S D Y K Y Q Q K P G Q 2 GCT CCC AGG CTC CTC ATC TAT TAC GCA TCC CAC TCC ATC TCT 168 P R I Y Y A S E S? S 56 GGC ATC CCA GCC AGG TTC AGT GGC AGT GGG TCT GGG AC GAC 210 G I P A F S G S G S G T D 70 TTC ACT CTC ACC ACT AGC AGC CTA GAG CCT GAA GAT TTT GCA 252 F T T I S S L E P E D F 84 GTT TAT TAC TGT CAG CAT GGC CAC TCT TAT CCT TGG ACC TTC 294 V Y Y C C H G H S Y P W T F 98 GGA GGG GGG ACC AAG GTG GAA ATT AAA 321 G G G T K V E I K 107 5. Expression and Purification of Fab Certain Fabs were cloned into an expression vector under the control of the BAD promoter regulated by arabinose. In addition, a histidine-six label was fused to the carboxyl terminus of the H chain to allow purification with nickel chelation resins. The purified Fab was quantified as described (Watkins (1997), supra). 6. Characterization Tests Immulon II microtiter plates were coated with 0.1 μg / ml CD40-Ig in PBS for 16 h at 4 ° C and blocked with 3% BSA in PBS. The plates were washed three times in PBS containing 0.1% Tween 20 and the Fab released from the periplasmic space was serially diluted three times in PBS containing 1% BSA and incubated with the plate 2 h at 25 ° C. Subsequently, the plate was washed four times with PBS containing 0.1% Tween 20 and the antibody binding was detected by incubation with alkaline phosphatase conjugated goat kappa anti-human diluted 2000-fold in PBS containing 1% BSA was added by lh to 25 ° C. The plate was washed four times with PBS containing 0.1% Tween 20 and was developed colorimetrically (Watkins (1997), supra).

To test the variants for inhibition of ligand binding, Immulon II microtiter plates were coated with 2 μg / ml CD8 anti-urine to capture the sgp39 fusion protein which expresses the CD8 domain. Plates were rinsed once with PBS containing 0.05% Tween 20, and blocked with 3% BSA in PBS. The plate was washed once with PBS containing 0.05% Tween and was incubated with cell culture medium containing saturated levels of sgp39 for 2 h at 25 ° C. The unbound sgp39 was aspirated and the plate was washed twice with PBS containing 0.05% Tween 20. Next, 25 μl of 4 μg / ml of CD40-human Ig in PBS. The plates were incubated 2 h at 25 ° C and were washed three times with PBS containing 0.05% Tween 20. CD40-Ig bound was detected following an incubation of 1 h at 25 ° C with horseradish peroxidase-conjugated specific F (ab ') 2 anti-human goat IgG Fcy (Jackson) diluted 10,000-fold in PBS. The plate was washed four times with PBS containing 0.05% Tween 20 and the binding was quantified colorimetrically by incubation with 1 mg / ml o-phenylenediamine dihydrochloride and 0.003% hydrogen peroxide in 50 mM citric acid, 100 mM Na2HP0, pH 5. The reaction was terminated by the addition of H2SO4 to a final concentration of 0.36 M and the absorbance at 490 nm was determined. 7. BIAcore Analysis The kinetic constants for the interaction between CD40 and the anti-CD40 variants were determined by surface plasma resonance (BIAcore). The CD40 fusion protein was immobilized to a (1-ethyl-3- [3-dimethylaminopropyl] -carbodiimido hydrochloride) and an activated sensor microprocessor of N-hydrosuccinamide CM5 by injection of 8 μl of 10 μg / ml CD40-Ig in 10 mM sodium acetate, pH 4. CD40-Ig was immobilized at a low density (-150 RU) to prevent Fab re-binding during the dissociation phase. To obtain the association rate constants (kon), the binding rate of six different Fab concentrations varied between 25-600 nM in PBS was determined at a flow rate of 20 μl / min. The dissociation rate constants (k0 ± f) were the average of six measurements obtained by analysis of the dissociation phase. The sensorgrams were analyzed with program 3.0 of BIAevaluation. Kd was calculated from Kd = koe / kon. The residual Fab was removed after each measurement by prolonged dissociation.

The results of the kinetic analysis for humanized antibodies F4 and L3.17 compared to a chimeric Fab are shown in Table 1 below: Table 1 Results of Humanization As discussed above, the sequences of the variable region structure of murine anti-CD40 mAb were used to identify the majority of the sequences of the human germline homologs. The residues of the structure of the H chain were 74% identical to the human germ line VH7 (7-4.1) and the JH4 sequences while the L chain was 74% identical to the human germline VKIII (L6) and the JK sequences . Alignment of the variable sequences of the H and L chains is shown in Figure 15. The CDR residues, as defined by Kabat et al. (Kabat, E. A. et al., (1991). Sequences of proteins of immunological interest (5th Ed.) Whashington DC: United States Department of Health and Human Services; Kabat, E.A., et al., (1977) J. Biol. Chem. 252: 6609-6616) are underlined and were excluded from the homology analysis. Residues of the structure that differed between murine mAb and human standards were evaluated individually.

Based on sequence and structural analysis, the CDRs antibody with the exception of HCDR3 shows a limited number of main chain conformations called canonical structures (Chothia, C, et al., (1987) J. Mol. Biol. 196: 901 -917; (Chothia, C, et al., (1987) Nature 342: 877-883) Furthermore, certain critical residues have been identified (Chothia (1987), supra; Chothia (1989) supra). The residues of the canonical structure of murine anti-CD40 were identified accordingly, and it was determined that the amino acids in critical canonical positions in the structures of the H and L chain of the human standards were identical to the corresponding murine residues.

Murine amino acids on the surface not normally found in human antibodies are likely to contribute to the immunogenicity of humanized mAb (Padlan, E.A. (1991) Mol.Immunol.28: 489-498). Therefore, structure residues that differ between murine anti-CD40 and human standards were analyzed and based on solvent exposure were predicted to be hidden or localized on the surface of the antibody (Padlan (1991), supra) . The distances from the center of the residues of the structure exposed to solvent to CDRs are not expected to contribute significantly to the antigen binding and thus, with the exception of two H chain residues all were changed to the corresponding human amino acid to decrease the potential immunogenicity . Chain residues H 28 and 46 were predicted to be exposed to solvent. However, H28 is located within the HCDR1 region as defined by Chothia et al., Supra, and potentially interacts with the antigen. In addition, lysine in H46 in murine mAb is somewhat unusual and significantly different from glutamic acid in the human standard. Therefore, the murine and human residues in H28 and H26 were expressed in the combinatorial library (Figure 15, asterisks).

Residues of structure that differ from the remnant, all predicted to be the majority hidden within the antibody, were evaluated for: (1) proximity to CDRs; (2) potential to contact the opposite domain in the VK-VH interface; (3) relationship of amino acids that differ; and (4) predicted importance in modulating CDR activity as defined by Studinicka et al. (Studinicka, G.M., et al., (1994) Protein Eng. 7: 805-814). Most of the structure differences of the L chain in hidden residues were amino acids related to positions considered to be not likely to be directly involved in the conformation of the CDR. However, L49 is located adjacent to LCDR2, potentially contacts the VH domain, is not related to human waste, and may be involved in determining the conformation of LCDR2. For these reasons, murine and human amino acids in L49 were both expressed in the combinatorial structure library (Figure 15, asterisks).

The analysis of the H chain sequence of murine and the human standard was more complex. The H9 residue is a proline in murine mAb while the human standard contains a non-related serine residue. The H9 position can also play a role in modulating the conformation of the CDR and in this way, it was selected as a site of the combinatorial library (Figure 15, asterisks). The remnant hidden structure residues that differed between murine anti-CD40 and the H chain pattern were at the positions of structure 38, 39, 48, and 91. Murine MAb anti-CD40 contained glutamine and glutamic acid in H38 and H39, respectively, while the human standard contained arginine and glutamine. The residue H38 is in proximity to HCDR1, glutamine - > changes arginine is not conserved, an expression of glutamine in this site in murine Abs is somewhat unusual. Similarly, glutamic acid - glutamine is not a conservative difference for hidden amino acids, H39 is a residual of contact V? potential, and glutamic acid is somewhat unusual in murine mAbs. Residue H48 is in close proximity to HCDR2 and H91 was predicted to be a high-risk site (Studnicka (1994), supra; Harris, L. et al., (1995) Prot Sci. 4: 306-310) potentially contacting the domain V ?. In this way, both murine and human residues were expressed in H38, 39, 48, and 91 (Figure 15, asterisks).

In summary, the structure library consisted of murine CDRs grafted into human patterns. In addition, a structure residue in the L chain and seven structure residues in the H chain were considered potentially important for maintaining mAb activity. All these sites were characterized by synthesizing a combinatorial library that expressed all possible combinations of murine and human amino acids found in these residues. The total diversity of this library, called Hu I, was 28 or 256 variants (Table 2 below).

Table 2: Summary of anti-CD40 antibody libraries expressed in phages. Library Positions in Size * Protected * the Hu library I Structure 256 2.4 x 103 Hu II Structure, 1.1 x 105 2.0 x 106 HCDR3 Hu III Structure, 3.1 x 107 5.5 x 105 HCDR3, LCDR3 * Number of unique clones based on the DNA sequence. Thirty-two codons are used to encode all 20 amino acids at each CDR position. * The Hu I library was protected by ELISA using antibodies expressed in cultures of small-scale bacteria (Watkins (1997), supra). The Hu II and Hu III libraries were seeded on agar plates / Blue XL-1 of 105 per 10-mm box and were protected by capture-by-catch (Watkins (1998), supra).

The Hu I library was expressed in small-scale bacterial cultures (<1 ml), uniform amounts of Fab released from the periplasmic space were captured in a microtiter plate, and the binding activity of the antibodies was directly compared by ELISA (Watkins (1997), supra). Although variants that bind the target antigen with comparable, or better than, chimeric Fab affinities were identified, most of the protected Hu I clones were less active than chimeric anti-CD40 Fabs. Approximately 6% of the randomly selected variants showed binding activities comparable to chimeric Fab (data not shown). The identification of Hu I variants with activity comparable to chimeric CD40 is consistent with the interpretation that most of the critical structure residues were included in the combinatorial library.

The clones were subsequently characterized by titration on the immobilized antigen, to confirm the identification of the multiple variants with affinity. For example, clone 1911 binds the CD40 receptor with higher affinity than the chimeric Fab, as demonstrated by the change in the titration profile (Figure 16, open circles vs. full circles). DNA sequencing of the 34 most active clones led to the identification of 24 unique structure combinations, each containing murine 2-6 structure residues.

LCDR3 and HCDR3 contact the antigen directly, interact with the other CDRs, and also affect the affinity and specificity of the antibodies significantly (Wilson, IA et al., (1993) Curr. Opin. Struc. Biol. 3: 113-118; Padlan, EA, (1994) Mol Immunol., 31: 169-217). In addition, the conformations of LCDR3 and HCDR3 are determined in part by certain residues. To identify the most active antibody, codon-based mutagenesis (Glaser, SM, et al, (1992), J. Immunol., 149: 3903-3913) was used to synthesize oligonucleotides that introduce mutations in each position in HCDR3, one at a time. , resulting in the expression of all 20 amino acids in each CDR residue. Each oligonucleotide encoded no more than one unique amino acid alteration. The fusion of oligonucleotides encoding the HCDR3 library was mixed with the superposition of oligonucleotides encoding the combinatorial structure and other CDRs to generate a structure library / HGDR3. The diversity of this library, called Hu II, was 1.1 x 105 (Table 2, above). A library for LCDR3 was synthesized in a similar way. The oligonucleotides encoding LCDR3, HCDR3, and the combinatorial structure were used to create a structure library / HCDR3 / LCDR3, called Hu III. The largest number of structure / CDR3 combinations resulted in a library with complexity of 3.1 x 107 (Table 2, above).

Combining mutations in LCDR3 and / or HCDR3 with the structure library increased the potential diversity of humanized anti-CD40 variants from 256 to more than 107. To protect these large libraries more efficiently a modified plate-lift test, called capture-by-capture, it was used (Watkins (1998), supra). In summary, phage-infected bacteria were seeded on solid agar plates and subsequently, they were superimposed with nitrocellulose filters that had been covered with specific Fab reagent. Following the close capture of uniform amounts of Fab expressed in phages, the filters were tested with 5 ng / ml of CD40-Ig fusion protein. Because the filters were tested with antigen at a concentration substantially below Kd of the Fab, only variants that showed improved affinity were detected. The multiple clones that showed the highest affinities were identified following the protection of > 106 variants of Hu II and of > 105 variants of the Hu III library using 82 mm filters containing ~ 105 variants per filter (Table 2).

Due to the high density of phages in the filters, the positive plates were collected, planted at a lower density, and protected again. Subsequently, the variants that produced the most intense colorimetric signal in the capture-by-capture test were subsequently characterized by ELISA. As expected, most of the clones identified by capture-by-capture protection linked CD40 better than chimeric Fab. Titration of the immobilized CD40-Ig variants identified multiple clones that showed greater affinities than chimeric and humanized Fab (Figure 16, compare open squares and triangles filled with circles).

The structure / CDR mutations that confer improved affinity were identified by DNA sequencing. Single variable region sequences were identified in the Hu II 10/13 variants and the Hu III 3/4 variants. Both Hu II and Hu III variants contained murine structure residues 1-5 and CDR3 0-2 mutations, as summarized in the Table below. Table 3. Simultaneous optimization of the structure and CDR residues that identify the variants with the highest affinity.

* Number of murine structure residues that differ from most of the homologous human germline sequence based on the CDRs definition of Kabat et al., Supra. The number of murine structure residues that differ from the human pattern is indicated in parentheses. All these structure differences between the murine mAb and the humanized versions are located in the H (H) chain at the indicated positions using a numbering system of Kabat et al.

The affinities of chimeric Fab expressed in bacteria and the variants selected from each of the libraries were more fully characterized using surface resonance measurements of plasma to determine the rates of association and dissociation of purified Fab with immobilized CD40-Ig. The immobilized chimeric anti-CD40 had a constant dissociation O = 3.14 nM and, consistent with the protection results, many of the variants showed higher affinities. Two of the best clones, F4 and L3.17, had Kd of 0.24 nM and 0.10 nM, respectively (Table 1) . The improved affinities of the anti-CD40 variants were predominantly the result of lower dissociation rates as the rates of association were very similar for all variants (variations from 0.9 to 3.2 x 106 M "1s ~ 1).

Finally, variants that showed improved affinity were tested for their ability to block the binding of the gp39 ligand to the CD40 receptor. All variants inhibited the binding of the soluble CD40-Ig fusion protein to immobilize the gp39 antigen in a dose-dependent manner that correlated with the affinity of the Fabs (Figure 17). For example, the most potent ligand binding inhibitor binding the CD40-Ig fusion protein was the 2B8 variant, which was also the variant with the highest affinity for CD40 (Figure 17). The 2B8 variant showed ~ 17 times more - high affinity for CD40 than the one that had chimeric Fab and inhibited ligand binding ~ 7 times more effectively.

Use 5 Mouse Model System The applicants also developed and tested in vivo a rat anti-murine CD40 mAb designated 7E1-G2b and its predecessor, 7E1-G1. The generation of this antibody was performed to explore the potential of anti - CD40 therapy in models of. Murine of autoimmune, inflammatory and transplant disease. The primary objective of the mouse model system was to generate an anti-murine counterpart that completely mimicked 2.220 and potent blocker of the gp39 / CD40 interaction while possessing weak co-stimulatory activity, and testing it in vivo in standard experimental disease models.

A. Isolation and Characterization of Anti-Mutin Monoclonal Antibodies CD40 7E1 and 7E1-G2b 1. Immunization, Fusion and Characterization A recombinant murine CD40 immunoglobulin fusion protein consisting of the extracellular region of mouse CD40 fused to the major, the CH2 and CH3 domains of a mouse IgG2 antibody (mCD40-mIg) was used to immunize a Lewis rat female of 8 weeks via inoculation in the foot plant. Three days after the last immunization, leukocytes drained from the lymph nodes were fused mouse myeloma cells X63-Ag8.653 to create heterohybridomas from mouse x rat. Casings containing antibody specific for CD40 native mouse were identified by reactivity with the original immunogen mCD40-mIg by ELISA, and by reactivity with a CD40 positive B cell lymphoma cell line (WEHI-231, ATCC CRL-1702). The supernatants were then tested for the ability to inhibit the binding of mCD40-mIg to solubilize, recombinant mp8-murine gp39 fusion protein, mgp39, the murine equivalent of sgp39. Approximately twelve of the most potent inhibitory master castings were cloned by the limiting dilution method.

Following cloning, functional tests were performed with culture supernatants and purified antibody to more precisely test the ability of anti-CD40 mAbs to inhibit the interaction of murine gp39 with CD40 and to determine its stimulatory properties. The inhibitory properties were measured by the ability to inhibit the binding of mgp39 to WEHI-231 using standard procedures known in the art. The stimulatory properties were measured by the adjusted induction, homotypic adhesion of WEHI-231 cells and proliferation of spleen B cells in the presence of the antibody and anti-IgM using procedures known in the art. Of these results, three mAbs (5A3, 7E1-G1 and 8E1) were determined to be the most similar to anti-human mAb 2.220 CD40 with respect to gp39 / CD40 block and level of co-stimulatory activity. 2. Selection of 7E1 as the Anti-Murine CD40 mAb guide. In vivo studies in mice were performed to identify which of the anti-CD40 blocking / non-stimulatory mAbs most potently suppressed antibody responses specific for T-dependent antigen. The suppression of the IgG antibody response to SRBCs of mice with anti-murine CD40 mAb was studied. Groups of five BALB / c mice were immunized IV with 1 x 108 SRBCs and concurrently treated ip with 1 mg of anti-murine mAbs CD40, 5A3, 7E1-G1 and 8E1. As controls, groups of similarly immunized mice were treated with MR1 (hamster anti-murine gp39, positive control, 250 ug), 6E9 (rat anti-human gp39, negative control, Img) or PBS. Mice were evaluated by anti-SRBC IgG titers by ELISA at 7, 14, 21 and 35 days. The results indicated that when administered as a single dose of antibody at the time of antigen challenge with SRBCs, mAb 7E1-G1 showed to be more effective suppressor of the anti-SRBC IgG response compared to 5A3 and 8E1, and was therefore selected as the direction of anti-CD40 mAb for murine studies. 3. Variant of Change of Isotype of mAb 7E1-G1 7E1-G1 does not possess causative functional characteristics comparable to those of the chimeric 2220 anti-human mAb CD40 (i.e., rat IgG1 is not as efficient as human IgG1 in complement fixation and Fc receptor interaction) and the specific antibody suppression profile in vivo for 7E1 was not as complete as that seen with 2,220 mAb in primates. In this way, an antibody having 7E1 specificity but with a rat isotype more similar to human IgG in its causative capacities was seen. For this purpose, a natural isotype change variant of 7E1, from an IgG to an IgG2b, was generated by the sib selection technique (Hale et al., J. Immunol. (1987) 103 (1): 59-67). Briefly, an anti-CD40 mAb of an OgG2b isotype was identified by ELISA between supernatants from flush plates 96 that had been seeded at 1000 cells / flush plate with the original 7E1 hybridoma. Subsequent rounds of sowing and identification of positive IgG2b casts at planting densities of 200 and then 20 cells / emptying plate followed by two rounds of cloning by limiting dilution led to isolation of a clonal IgG2b variant of 7E1, 7E1-G2b . 7E1-G2b is a legitimate change variant of IgG1 as demonstrated by three groups of data. First, N-terminal sequencing of the heavy chain showed that both versions were identical for the first 35 amino acid residues. Second, PCR analysis using specific primers for the variable heavy chain CDRs of 7E1-G1 yielded - an appropriate size band of cDNA obtained from either 7E1-G1 or 7E1-G2b, and not two other unrelated antibodies. Finally, the evaluation of the binding activity of purified batches of the two versions for mCD40-hIg immobilized in an ELISA using an anti-kappa tracer reagent yielded essentially identical titration curves.

B. In vivo studies In vivo comparison of 7E1-G1 or 7E1-G2b in Antibody Response Model 7E1-G1 was compared to 7E1-G2b for efficiency in vivo using SRBC's as T cell-dependent antigen. Groups of three to five animals immunized iv with SRBC and concurrently treated ip with the antibody 7E1-G1 or 7E1-G2b, at 1, 0.25, or 0.1 mg of the compound on day 0 as indicated in Figure 10. MAb anti-murine gp39 MR1 served as a positive control for immunosuppressive effect. MAb 6E9 and PBS served as an irrelevant mAb and no mAb controls, respectively. The mice were evaluated by anti-SRBC titers by ELISA on days 7, 14 and 21. The titration represents the calculated dilution of serum to yield a value of OD = 0.3 in the ELISA. As shown in Figure 10, 7E1-G2b suppressed the IgG response to SRBCs at doses where 7E1-G1 did not suppress it. 2. Response of the 7E1-G2b Dose in the Mouse Model T-dependent Antigen 7E1-G2b was examined in a T-cell dependent primary immune response model using SRBC as antigen. 7E1-G2b was tested at several doses to determine the lowest effective dose. BALB / c mice (n = 5) were injected IV with 1 x 108 SRBCs and treated with a single injection of 7E1-G2b at indicated doses or MRI (anti-murine gp39) or PBS administered at the same time as the antigen in the day 0. Shown in Figure 11 is the anti-SRBC IgG response on days 7, 16, and 28. The values reported are the ELISA absorbance values at the 1/50 serum dilution. The error bars indicate standard deviation.

As shown in Figure 11, a single treatment with 7E1-G2b at 25 μg / mouse (1.25 mg / kg) suppressed the IgG immune response by 87% at Day 16 and complete suppression was obtained at doses of 50 or 100 μg at Day 16. On day 28, 50 μg / mouse suppressed the IgG response by 89%, and 100 μg / mouse suppressed it completely. Note that MRI was used as a positive control for immunosuppression at a suboptimal dose of 100 μg / mouse. 3. 7E1-G2b in Collagen-Induced Arthritis Prevention Mouse Model (CIA) A standard experimental murine model for rumatoid arthritis, the model, of collagen-induced arthritis (CIA), was used to determine the effect of 7E1-G2b in the prevention of arthritis. DBA / 1J male mice (6-8 weeks) were injected with 200 μg of type II chicken collagen (CII) in complete Freund adjuster intradermally on day 0. Treatment with 7E1-G2b at 250 μg / dose was administered IP every four days starting on day 1. The control group was treated with PBS in the same dosing schedule. All mice were promoted with CII with incomplete Freund adjuster on day 21. The mice were observed daily for inflammation in the claw and subjectively recorded on a scale of 0-3 with 3 equal to the maximum inflammation of the erythema. The claws were measured with calibrator daily. The reported clinical record was derived from the sum of the records of each claw at the time of slaughter and dividing by the total number of animals in each group. The values reported are the range of the median of the groups.

The development of arthritis, and hence joint inflammation in mice, was completely inhibited by 7E1-G2b therapy as shown in Table 4 below. Mice treated with 7E1-G2b were completely free of the disease for 90 days.

Table 4 Treatment of Collagen-Induced Arthritis Group Median Median Incidence (Range) (Range) Tx of (Range) Outcome Arthritis Measure Clinical Claw Day start 7E1-G1 0/5 0 0 0.075 7E1-G2b 0/5 0 0 0.075 Control 4/4 30 (27- 3.5 (3-4] 0.114 (0.110- PBS 32) 0.117) As demonstrated above, the antibodies of the present invention are potent immunomodulators, with therapeutic uses against a variety of diseases.

The present invention comprises chimeric and humanized antibodies as described above with additional conservative amino acid substitutions which have no effect substantially on the CD40 bond. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions with the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine.

In one aspect, the present invention is directed to produce the chimeric and humanized antibodies as described above by expressing recombinant DNA segments encoding the light variable chain and the variable murine (or portions thereof) chain attached to DNA segments that encode human constant regions. Exemplary DNA sequences designed in accordance with the code of the present invention for polypeptide chains comprising all or a portion of the variable region of the light chain as shown in SEQ ID NO: 1 or its deposited ATCC clone, and / or all or a portion of the variable region of the heavy chain as shown in SEQ ID NO: 2 or its deposited ATCC clone.

Also included in the present invention are the light and heavy chain variable regions and their functional or active parts thereof. The immunologically functional or competent form of the protein or part thereof is also referred to herein as a "light / heavy chain variable region or a biologically active portion thereof". In the present case, the biologically active portion thereof comprises a portion of said light or heavy chain, which when incorporated into the antibody, still allows the antibody to bind to human CD40.

Specifically comprised within the present invention are the nucleic acid sequences encoding the variable heavy chain and the variable light chain of an antibody of the present invention. For example, nucleotides 1057 through 1422 (SEQ ID NO: 5) of Figure 13 provides a preferred nucleic acid sequence encoding a heavy chain of an antibody of the present invention; nucleotides 1065 through 1388 (SEQ ID NO: 6) of Figure 14 provides a preferred nucleic acid sequence encoding a light chain of an antibody of the present invention. SEQ ID NO: 7 and SEQ ID NO: 11 show the preferred nucleic acid sequences encoding the variable light chains of the humanized antibodies of the present invention; SEQ ID NO: 9 shows the preferred nucleic acid sequence encoding the variable heavy chain of a humanized antibody of the present invention. The plasmids comprising the polynucleotides shown in SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 have been deposited with the ATCC.

Chimeric and / or humanized antibodies that bind to human CD40 and that comprise polypeptides that are substantially homologous to, or exhibit substantial sequence identity for, the light and heavy variable chain sequences published herein are also contemplated by the present invention. For example, chimeric antibodies comprising a light chain region exhibiting at least about 85% sequence identity, more preferably at least about 90% sequence identity, and even more preferably at least about 95% identity of sequence, and most preferred at least about 98% sequence identity with the light chain region as shown in SEQ ID NO: 4 are included within the scope of the present invention. Most particularly, the chimeric antibodies comprise a variable light chain region exhibiting at least about 85% sequence identity, more preferably at least about 90% sequence identity, and even more preferably at least about 95% of sequence identity, and most preferred at least about 98% sequence identity with the variable light chain region as shown in SEQ ID NO: 1 are also included within the scope of the present invention. Also within the scope of the present invention are humanized antibodies comprising a light chain region exhibiting at least about 65% sequence identity, more preferably at least about 90% sequence identity, and even more preferably at least about 95% sequence identity, and most preferred at least about 98% sequence identity with the light chain region as shown in SEQ ID NO: d and / or SEQ ID NO: 12 are also included within the scope of the present invention.

Additionally, chimeric antibodies comprising a heavy chain region exhibiting at least about 85% sequence identity, more preferably at least about 90% sequence identity, and even more preferably at least about 95% sequence identity sequence identity, and most preferred at least about 98% sequence identity with the heavy chain region as shown in SEQ ID NO: 3 are included within the scope of the present invention. Most particularly, the chimeric antibodies comprise a variable heavy chain region exhibiting at least about 85% sequence identity, more preferably at least about 90% sequence identity, and even more preferably at least about 95% sequence identity, and most preferred at least about 98% sequence identity with the variable heavy chain region as shown in SEQ ID NO: 2 are also included within the scope of the present invention. Additionally, humanized antibodies comprising a heavy chain region exhibiting at least about 85% sequence identity, more preferably at least about 90% sequence identity, and even more preferably at least about 95% sequence identity sequence identity, and most preferred at least about 98% sequence identity with the heavy chain region as shown in SEQ ID NO: 10 are also included within the scope of the present invention.

The DNA segments typically further comprise a control DNA sequence expression operably linked to the chimeric or humanized antibody encoding sequences, including the naturally associated regions or the heterologous promoter regions. Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts can also be used. Once the vector has been incorporated into an appropriate host, the host is maintained under conditions suitable for high level expression of nucleotide sequences and, if desired, the collection or purification of the variable light chain, heavy chain, dimers. of light / heavy chain or intact antibody, binding fragments or other form of immunoglobulin may follow. (See, Beychok, S., "Cells of Immunoglobulin Synthesis," Academic Press, N.Y. (1979)). Single chain antibodies can also be produced by joining nucleic acid sequences encoding the VH and VL regions published herein with DNA encoding a polypeptide linkage.

Prokaryotic hosts, such as E. coli, and other microbes, such as yeast, can be used to express an antibody of the present invention. In addition to the microorganisms, mammalian tissue cell cultures can also be used to express and produce the antibodies of the present invention. Eukaryotic cells may be preferred, because a number of suitable host cell lines capable of secreting immunod, intact lobulins have been developed in the art, and include CHO cell lines, several COS cell lines, HeLa cells, lines of myeloma cells, and hybridomas. Expression vectors for these cells can include expression control sequences, such as a promoter or enhancer, and necessarily processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional termination, all known in the art.

Vectors containing the DNA segments of interest (eg, light and / or heavy chains encoding sequences and expression control sequences) can be transferred into host cells by methods well known in the art, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cellular hosts. (See, e.g., Maniatis, et al., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Press (1982)).

Once expressed, the whole antibodies, their dimers, light and heavy chains, or other forms of immunoglobulin of the present invention, can be purified according to procedures standard in the art, including precipitation with ammonium sulfate, affinity columns, chromatography. of column, gel electrophoresis and the like. Pure immunoglobulins of substantially at least 90 to 95% homogeneity are preferred, and 98 to 99% or more of homogeneity are most preferred, for pharmaceutical uses.

The antibodies of the present invention will typically find use in treating mediated antibody and / or mid-T cell disorders. Convenient disease states typical for treatment include grafts versus host disease and transplant rejection, and autoimmune diseases such as Type 1 diabetes, psorasis. , multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, and severe miestenia.

The antibodies and pharmaceutical compounds of the present invention are particularly useful for parenteral administration, i.e., subcutaneously, intramuscularly or intravenously. Pharmaceutical compounds for parenteral administration will commonly comprise a solution of the antibody dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, all well known in the art, e.g., water, buffer water, saline, glycine and the like. These solutions are sterile and generally free of particular matter. These pharmaceutical compounds can be sterilized by well-known sterilization techniques. The compounds may contain pharmaceutically acceptable excipients required to approximate physiological conditions such as adjusted pH and buffering agents, toxicity adjusting agents and the like, eg, sodium acetate, sodium chloride, potassium chloride, calcium chloride, lactate of sodium, human albumin, etc.

The compounds containing antibodies of the present invention can be administered for prophylactic and / or therapeutic treatments. In the therapeutic application, the compounds are administered to the patient who already suffers from a disease, in an amount sufficient to cure or at least partially stop the disease and its complications. An adequate amount to accomplish this is defined as a "therapeutically effective dose". The effective amounts for this use will depend on the severity of the disease state and the general state of the patient's immune system, and can be determined by any skilled in the art.

In prophylactic applications, the compounds containing antibodies of the present invention are administered to the patient who is not yet in a disease state to improve the patient's resistance (suppressive immune response). Such an amount is defined to be a "prophylactically effective dose". In this use, the precise amount again depends on the health status of the patient and the overall level of immunity. A preferred prophylactic use is for prevention of transplant rejection, e.g. rejection of kidney transplant.

Although the present invention has been described in some detail as a form of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Having described the invention as above, the content of the following is claimed as property

Claims (30)

Claims

1. A light chain variable region characterized in that it comprises all or a biologically active portion of an amino acid sequence as shown in SEQ ID NO: 1 (Figure 4a).

2. A heavy chain variable region characterized in that it comprises all or a biologically active portion of an amino acid sequence as shown in SEQ ID NO: 2 (Figure 4b).

3. A chimeric antibody which binds to human CD40 characterized in that it comprises a light chain of a heavy chain, the light chain comprises the variable region of the heavy chain of claim 1.

4. The chimeric antibody which binds to human CD40 characterized in that it comprises a light chain of a heavy chain, the heavy chain comprises the variable region of the heavy chain of claim 2.

5. The chimeric antibody of claim 3 characterized in that the heavy chain comprises the variable region of the heavy chain of claim 2.

6. A chimeric antibody which binds human CD40, comprising a light chain and a heavy chain, characterized in that the light chain comprises all or a biologically active portion of an amino acid sequence as shown in SEQ ID NO: 4 and the heavy chain comprises all or a biologically active portion of an amino acid sequence as shown in SEQ ID NO: 3.

7. A nucleic acid molecule characterized in that it comprises a nucleotide sequence encoding the variable region of the light chain of claim 1.

8. A nucleic acid molecule characterized in that it comprises a nucleotide sequence encoding the variable region of the heavy chain of claim 2.

9. An expression vector characterized in that it comprises a nucleic acid sequence of claim 7.

10. An expression vector characterized in that it comprises a nucleic acid sequence of claim 8.

11. A humanized antibody characterized in that it comprises a portion of the variable region of the light chain of claim 1.

12. A humanized antibody characterized in that it comprises a portion of the variable region of the heavy chain of claim 2.

13. A pharmaceutical compound characterized in that it comprises the chimeric antibody of claim 5.

14. A pharmaceutical compound characterized in that it comprises the chimeric antibody of claim 6.

15. A chimeric antibody which binds to human CD40 characterized in that it comprises a light chain variable region and a heavy chain variable region, characterized in that. the variable region of the light chain comprises an amino acid sequence having at least 90% of the sequence identity for the variable region of the light chain of claim 1.

16. A chimeric antibody which binds to human CD40 characterized in that. comprises a light chain variable region and a heavy chain variable region, characterized in that the variable region of the heavy chain comprises an amino acid sequence having at least 90% sequence identity for the variable region of the heavy chain of the claim 2.

17. A method of treating a patient suffering from a mediated disorder of T cells, characterized in that the method comprises administering to the patient a therapeutically effective dose of a pharmaceutical compound of claim 14.

18. The nucleic acid molecule of claim 7 characterized in that it comprises the nucleotide sequence as shown in SEQ ID NO: 6.

19. The nucleic acid molecule of claim 8 characterized in that it comprises the nucleotide sequence as shown in SEQ ID NO: 5.

20. The chimeric antibody of claim 6 characterized by comprising a light chain amino acid sequence as shown in SEQ ID NO: 4 and a heavy chain amino acid sequence as shown in SEQ ID NO: 3.

21. The humanized antibody of claim 11 characterized in that it comprises a light chain variable region as shown in SEQ ID NO. 8

22. The humanized antibody of claim 11 characterized in that it comprises a heavy chain variable region as shown in SEQ ID NO. 10

23. The humanized antibody of claim 12 characterized in that it comprises a light chain variable region as shown in SEQ ID NO. 8

24. The humanized antibody of claim 12 characterized in that it comprises a heavy chain variable region as shown in SEQ ID NO. 10

25. The chimeric antibody of claim 11 characterized in that it comprises a light chain variable region as shown in SEQ ID NO: 8 and a heavy chain variable region as shown in SEQ ID NO: 10.

26. The humanized antibody of claim 11 characterized in that it comprises a light chain variable region as shown in SEQ ID NO. 12

27. The humanized antibody of claim 26 characterized in that it comprises a heavy chain variable region as it is. shows in SEC ID NO. 10

28. The humanized antibody of claim 11 characterized in that it comprises a light chain variable region as shown in SEQ ID NO: 12 and a heavy chain variable region as shown in SEQ ID NO: 10.

29. A pharmaceutical compound characterized in that it comprises a humanized antibody of claim 25.

30. A pharmaceutical compound characterized in that it comprises a humanized antibody of claim 28.