ES2369265T3 - A BAFF SOLUBLE SPECIFIC ANTIBODY FOR USE IN CANCER TREATMENT. - Google Patents

Abstract

An antibody or a specific active fragment for soluble BAFF for use in the treatment of cancer.

Description

A specific antibody for soluble BAFF for use in the treatment of cancer

Field of the Invention

The invention relates to an antibody specific for soluble BAFF or an active fragment of that antibody for use in the treatment of cancer. The use of a BAFF ligand, a B cell activation factor belonging to the family of tumor necrosis and its blocking agents to stimulate or inhibit the expression of B cells and immunoglobulins, is described. This protein and its receptor may have anticancer and / or immunoregulatory applications as well as uses for the treatment of immunosuppressive disorders such as HIV. Specifically, the ligand and its blocking agents may play a role in the development of hypertension and its related disorders. In addition, cells transfected with genes for this ligand can be used in gene therapy to treat tumors, autoimmune diseases or inherited genetic disorders that involve B cells. Blocking agents, such as recombinant variants or antibodies specific for the ligand or its receptor, can have immunoregulatory applications as well. The use of BAFF as a B-cell stimulator for immuno suppressed diseases is considered, including, for example, uses for patients suffering from organ transplantation (for example, bone marrow transplantation) as well as the recovery of cancer treatments to stimulate the production of B cells. The use of BAFF as an adjuvant and / or costimulator to raise and / or restore B cell levels to approximate them to normal levels is also considered.

Foundation of the invention

Cytokines related to tumor necrosis factor (TNF) are mediators of host defense and immunoregulation. Members of this family exist in membrane-anchored forms, which act locally through cell-to-cell contact, or as secreted proteins capable of diffusing to more distant targets. A parallel family of receptors indicates the presence of these molecules that lead to the initiation of cell death or cell proliferation and differentiation in the target tissue. At present, the TNF family of ligands and receptors has at least 11 recognized ligand-receptor pairs, including: TNF: TNF-R; LT-α: TNF-R; LT-α / β: LT-β-R; FasL: Fas; CD40L: CD40; CD30L: CD30; CD27L: CD27; OX40L: OX40 and 4-1BBL: 4-1BB. The DNA sequences encoding these ligands have only about 25% to about 30% identity even in the most related cases, although the amino acid ratio is about 50%.

The defining characteristic of this family of cytokine receptors is found in the extracellular domain rich in cysteine initially revealed by the cloning of two distinct TNF receptors. This family of genes encodes characteristic glycoproteins of Type I transmembrane proteins with an extracellular ligand binding domain, a region that encompasses a single membrane and a cytoplasmic region involved in the activation of cellular functions. The cysteine-rich ligand binding region shows a central domain closely linked to disulfide, which, depending on the particular family member, is repeated multiple times. Most receivers have four domains, although there may be as few as three, or as many as six.

Proteins in the TNF family of ligands are characterized by a short N-terminal stretch of normally short hydrophilic amino acids, which often contain several arginine or lysine residues thought to serve as stop transfer sequences. It immediately follows a transmembrane region and an extracellular region of variable length, which separates the C-terminal receptor binding domain from the membrane. This region is sometimes called "stem". The C-terminal binding region comprises most of the protein, and often, but not always, contains glycosylation sites. These genes lack the characteristic classic signal sequences of Type I membrane proteins, Type II membrane proteins with the terminus C resting outside the cell, and a short N-terminal domain that resides in the cytoplasm. In some cases, for example, TNF and LT-α breakage in the stem region may occur soon during protein processing and the ligand is then mainly in secreted form. Most ligands, however, exist in the form of a membrane, mediating signalized localization.

The structure of these ligands has been well defined by crystallographic analyzes of TNF, LT-α, and CD-40. TNF and PPP lymphotoxin (LT-α) are structured in a double-folded sandwich-β antiparallels with the topology " jelly roll " or Greek key. The rms deviation between the Cα and β residues is 0.61 C, suggesting a high degree of similarity in their molecular topography. A structural feature that emerges from molecular studies of CD40L, TNF and LT-α is the propensity to meet in oligomeric complexes. Intrinsic to the oligomeric structure is the formation of the receptor binding point at the junction between the nearby subunits creating a multivalent bond. It has been observed that the quaternary structures of TNF, CD40L and LT-α exist as trimers by the analysis of their crystalline structures. Many of the amino acids conserved between the different ligands are in stretches of the scaffold of the β sheet. It is likely that the basic sandwich structure is conserved in all these molecules, since portions of these scaffolding sequences are preserved through the different family members. The quaternary structure can also be maintained since the conformation of the subunit probably remains similar.

Members of the TNF family can best be described as master switches in the immune system that control cell survival and differentiation. Only TNF and LTα are currently recognized as secreted cytokines that contrast with the other predominantly anchored members in the TNF family membrane. Although a membrane form of TNF has been well characterized and is likely to have unique biological roles, secreted TNF functions as a general alarm that signals cells more distant from the triggering event site. Thus the secretion of TNF can amplify an event that leads to well-described changes in the vasculature lining and in the inflammatory state of the cells. In contrast, membrane-bound family members send signals although TFN type receptors only to cells in direct contact. For example, T cells provide "help" CD40 mediated only to B cells brought into direct contact through cognated TCR interactions. Similar cell-cell contact limitations on the possibility of inducing cell death apply to the well studied Fas system.

It seems that TNF ligands can be segregated into three groups based on their ability to induce cell death. First, TNF, Fas and TRAIL ligand can effectively induce cell death in many lines and most likely their receptors have good canonical death domains. Presumably the ligand to DR-3 (TRAMP / WSL-1) would also be in this category. Below are those ligands that trigger a weaker death signal limited to some cell types and TWEAK, ligand CD30 and LTalb2 are examples of this class. How this group can trigger cell death in the absence of a canonical death domain is an interesting issue and suggests that there is a separate weaker death signaling mechanism. Finally, there are those members who cannot provide a death signal effectively. Probably all groups can have antiproliferative effects on some types of cells consistent with inducing cell differentiation, for example CD40. Funakoshi et al. (1994).

The TNF family has grown dramatically in recent years to cover at least 11 different signaling pathways that involve immune system regulation. The extended expression models of TWEAK and TRAIL indicate that there is an even more functional variety to discover in this family. This aspect has been especially highlighted recently in the discovery of two receptors that affect the ability of rous sarcoma and herpes simplex viruses to replicate, as well as historical observations that TNF has antiviral activity and syphilis viruses code for decode TNF receptors. Brojatsch et al. (nineteen ninety six); Montgomery et al. (nineteen ninety six); Smith et al. (1994), 76 Cell 959-962; Vassalli et al. (1992), 10 Immunol. 411-452.

TNF is a mediator of septic shock and cachexia, and is involved in the regulation of hematopoietic cell development. It seems to play an important role as a mediator of inflammation and defense against bacterial, viral and parasitic infections as well as having antitumor activity. TNF is also involved in different autoimmune diseases. TNF can be produced by several types of cells, including macrophages, fibroblasts, T cells and natural killer cells. TNF binds to two different receptors, each acting through specific intracellular signaling molecules, thus giving rise to different effects of TNF. TNF may exist as a form of membrane binding or as a soluble secreted cytokine.

LT-α shares many activities with TNF, that is, binding to TNF receptors, but unlike TNF, it appears to be secreted primarily by activated T cells and some lymphoblastoid-β tumors. The heteromeric complex of LT-α and LT-β is a membrane bound complex that binds to the LT-β receptor. The LT system (LTs and LT-R) seems to be involved in the development of peripheral lymphoid organs since the genetic disruption of LT-β leads to disorganization of T and B cells in the spleen and an absence of lymph nodes. The LT-β system is also involved in the cell death of some adenocarcinoma cell lines.

Fas-L, another member of the TNF family, predominantly expresses itself on activated T cells. It induces the death of cells that support its receptor, including tumor cells and HIV-infected cells, by a mechanism known as programmed cell death or apoptosis. In addition, deficiencies in Fas or Fas-L can lead to lymphoproliferative disorders, which confirm the role of the Fas system in the regulation of immune responses. The Fas system is also involved in the destruction of T cells in patients with HIV. TRAIL, another member of this family, also seems to be involved in the death of a wide variety of transformed cell lines of diverse origin.

CD40-L, another member of the TNF family, expresses itself on T cells and induces the regulation of B cells that support CD40. In addition, alterations in the CD40-L genes give rise to the disease known as X-linked hyper-IgM syndrome. The CD40 system is also involved in different autoimmune diseases and CD40-L is known to have antiviral properties. Although the CD40 system is involved in the release of apoptotic B cells, it induces apoptosis in nonimmune cells. Many additional lymphocyte members of the TNF family are also involved in costimulation.

Generally, members of the TNF family have fundamental regulatory roles in the control of the immune system and in the activation of acute host defense systems. Given the current progress in manipulating members of the TNF family for therapeutic benefit, it is likely that members of this family can provide unique means to control the disease. Some of the ligands in this family can directly induce apoptotic death of many transformed cells, for example, LT, TNF, Fas and TRAIL ligand. Nagata (1997) 88 Cell 355-365. Fas and possibly TNF and CD30 receptor activation can induce cell death in non-transformed lymphocytes that can play an immunoregulatory function. Amakawa et al. (1996) 84 Cell 551-562; Nagata (1997) 88 Cell 355-365; Sytwu et al. (nineteen ninety six); Zheng et al. (1995) 377 Nature 348-351. In general, death is triggered following the aggregation of death domains that reside on the cytoplasmic side of TNF receptors. The death domain implements the assembly of several signal transduction components that lead to the activation of the caspasse cascade. Nagata (1997) 88 Cell 355-365. Some receptors lack canonical death domains, for example, LTb and CD30 receptor (Browning et al. (1996); Lee et al. (1996)), can still induce cell death although more weakly. It is likely that these receptors function primarily to induce cell differentiation and death is an aberrant consequence in some transformed cell lines, although this picture is unclear, since studies on the CD30 null mouse suggest a role of death in negative selection in the scam. Amakawa et al. (1996) 84 Cell 551-562. Conversely, signaling through other pathways such as CD40 is required to maintain cell survival. Thus, there is a need to identify and characterize additional molecules that are members of the TNF family thus providing additional means of controlling diseases and manipulating the immune system.

EP 0 869 180 describes TL5 polypeptides and polynucleotides and methods for producing said polypeptides, as well as various medical uses thereof.

Here we characterize the functional properties of a new ligand of the TNF cytokine family. The new ligand, called BAFF (B cell activating factor belonging to the TNF family); It appears to be expressed by T cells and dendritic cells for the purpose of costimulation of B cell and may, therefore, play an important role in the control of cell B function. In addition, we have generated transgenic mice that overexpress BAFF under control. of a specific liver promoter. These mice have excessive numbers of mature B cells, spontaneous germ center reactions, secrete autoantibodies, and have high numbers of plasma cells in secondary lymphoid organs and Ig deposition in the kidney.

Summary of the invention

The invention relates to an antibody specific for soluble BAFF or an active fragment of that antibody for use in the treatment of cancer.

The use of BAFF ligands, blocking agents and antibodies for the ligand to stimulate or inhibit the growth of B cells and immunoglobulin secretion is also described. Uses are described for therapeutic applications in numerous diseases and disorders, as described in detail below, as well as for obtaining information about, and manipulating the immune system and its processes. In addition, a method for stimulating or inhibiting the growth of B cells and the secretion of immunoglobulins is described. The molecules associated with BAFF, as described in this invention, may also have utility in the treatment of autoimmune diseases, disorders related to proliferation and maturation of B cells, regulation of the BAFF ligand and inflammation. The regulation or prevention of hypertension and disorders related to hypertension of renal and cardiovascular tissue is described.

Features and advantages of the invention will be established in the description that follows, and in part they will be apparent from the description, or may be learned by practice of the invention. The objects and other advantages of the invention will be realized and obtained by the methods particularly indicated in the written description and its claims, as well as in the attached drawings.

A method for effecting the growth of B cells and the secretion of immunoglobulins by the administration of various BAFF ligands and related molecules is described.

Stimulation of the growth of B cells by the use of BAFF ligands or active fragments of the polypeptide is also described. The polypeptide can be used alone or with a CD40 ligand or an anti-murine antibody.

Uses of growth stimulation and maturation of B cells induced by dendritic cells by using BAFF ligands or active BAFF fragments are described. Again the polypeptide can be used alone or with CD40 ligand or anti-µ antibodies.

BAFF blocking agents and the BAFF receptor have been used to inhibit B cell growth and immunoglobulin secretion. These agents can be inoperable, recombinant BAFF, BAFF specific antibodies, BAFF receptor specific antibodies or a BAFF anti-ligand molecule.

BAFF uses, BAFF-related molecules and BAFF blocking agents for treating hypertension, hypertension-related disorders, immune disorders, autoimmune diseases, inflammation and B-cell lymphoproliferative disorders are described.

Uses of BAFF and BAFF-related molecules are described as agonists or antagonists to effect immune responses by effecting the growth and / or maturation of B cells and immunoglobulin secretion.

Soluble constructs comprising BAFF that can be used to directly trigger pharmacological events mediated by BAFF are described. Such events may have useful therapeutic benefits in the treatment of cancer, tumors, or in the manipulation of the immune system to treat immune diseases.

Additionally, the claimed invention relates to antibodies directed against soluble BAFF, which can be used for the treatment of cancers.

Gene therapy methods using BAFF genes are also described.

The pharmaceutical preparations of the invention may optionally include supports, auxiliaries, fillers or other pharmaceutical compositions, and may be administered in any of the numerous forms or routes known in the art.

It should be understood that the foregoing general description and the following detailed description are by way of example and explanation, and attempt to provide additional explanation of the claimed invention.

The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in, and constitute a part of this specification, illustrate various embodiments of the invention, and together with the description serve to explain the principles of the invention.

Description of the drawings

Figure 1 (A) represents the predicted amino acid sequence of human and mouse BAFF. The predicted transmembrane domain (TMD), dashed line), potential glycosylation sites linked to N (stars) and the natural processing site of human BAFF (arrow) are indicated. The double line above hBAFF indicates the sequence obtained by Edman degradation of the processed form of BAFF. (B) represents a comparison of the extracellular protein sequence of BAFF and some members of the TNF ligand family. Homologous and identical wastes are represented in black and shaded boxes, respectively. (C) represents dendrogram of TNF family ligands. Figure 2 is a schematic characterization of recombinant BAFF (A) Schematic representation of recombinant BAFF constructs. Soluble recombinant BAFFs beginning at Leu83 and Gin136 are expressed fused to an N-terminal Flag tag and a 6 amino acid linker. The long form is interrupted between Arg133 and Ala134 (arrow) in 293 T cells, to produce a processed form of BAFF. Asn124 and Asn242 belong to N-glycosylation consensus sites. The N-linked glycan present in Asn124 is shown as Y. TMD: transmembrane domain. (B) Treatment with recombinant BAFF N-glycanase F (PNgasaF) peptide. Concentrated supernatants containing APRIL and Flag-labeled BAFFs were deglycosylated and analyzed by Western blotting using polyclonal anti-BAFF or anti-Flag M2 antibodies, as indicated. All bands, except processed BAFF also reacted with anti-Flag M2 (data not shown). (C) Full length BAFF is processed to a soluble form. 293T cells were transfected transiently with full-length BAFF. Transfected cells and their concentrated supernatants were analyzed by Western blotting using polyclonal antiBAFF antibodies. Supernatants corresponding to 10 times the amount of cells were loaded onto the gel. (D) BAFF size exclusion chromatography on Superdex-200. Supernatants containing soluble / short BAFF were fractionated on a Superdex 200 column and the eluted fractions were analyzed by Western blotting using anti-Flag M2 antibody. The migration positions of the molecular mass markers (in kDa) are indicated on the left hand side for SDS-PAGE and in the upper part of the figure by size exclusion chromatography.

Figure 3 depicts expression of BAFF (A) Northern blots (2 µg poly A + RNA per lane) of several human tissues were tested with BAFF antisense mRNA. (B) BAFF reverse transcriptase amplification. IL-2 receptor alpha chain and purified blood T-cell RNA actin at several moments of PHA activation, negative E-Rosetting blood cells (B cells and monocytes), in vitro derived immature dendritic cells, 293 cells, and cells 293 sterile transfected with full-length BAFF (293-BAFF). Control amplifications were performed in the absence of added cDNA. The IL-2 receptor alpha chain was amplified as a T cell activation marker.

Figure 4 depicts BAFF binding to mature B cells. (A) binding of soluble BAFF to BJAB and Jurkat cell lines, and to purified CD19 + cells from umbilical cord blood. The cells were stained with the indicated amount (in ng / 50 µl) of Flag-BAFF and analyzed by flow cytometry. (B) Soluble BAFF binding to PBLs. PBLs were stained with anti-CD8-FITC or with anti-CD19-FITC (horizontal axis) and with Flag-BAFF plus M2-biotin and avidin-P (vertical axis). Flag-BAFF was omitted in controls.

Figure 5 represents that BAFF co-stimulates B cell proliferation. (A) Superficial expression of BAFF in stably transfected 293 cells. Wild type (wt) 293 and 293-BAFF cells were stained with antiBAFF mAb 43.9 and analyzed by flow cytometry. (B) Costimulation of PBLs by 293-BAFF cells. PBLs (105 / well) were incubated with 293 cells fixed to 15,000 glutaraldehyde (293 wt 293-BAFF) in the presence or absence of anti-B cell receptor antibody (anti-µ). 293 cells fixed alone incorporated 100 cpm. (C) Dose-dependent costimulation of PBL proliferation by soluble BAFF in the presence of anti-µ.

Proliferation was determined after 72 hours of incubation by incorporation of [3 H] -thymidine. Controls include cells treated with BAFF alone, with heat-denatured BAFF or with an antibody equated to an irrelevant isotype instead of anti-µ. (D) Comparison of the costimulatory effects of sCD40L and sBAFF on the proliferation of PBL. The experiment was performed as described in panel C. (E) BAFF co-stimulates the secretion of Ig from pre-activated human B cells. The purified CD19 + B cells were activated by coculturing with EL4T cells and activated T cell supernatants for 5-6 days, then they were re-isolated and cultured for another 7 days in the presence of only (-) medium or containing T cell supernatants 5% activated (T-SUP) or a mixture of cytokines (IL-2, IL-4, IL-10). The columns represent means of Ig concentrations for cultures with or without 1 µg / ml of BAFF. Mean values ± SD in terms of "times increase" were 1.23 ± 0.11 for medium only, 2.06 ± 0.18 with supernatant T cells (4 experiments) and 1.45 ± 0.06 with IL-2, IL-4 and IL-10 (2 experiments ). These were performed with peripheral blood (3 experiments) or umbilical cord blood B cells (one experiment; 2.3-fold increase with supernatant T cells, 1.5-fold increase with IL-2, IL-4 and IL- 10).

(F) The dose-response curve for the effect of BAFF in cultures with T-cell supernatants, as shown in panel D. Mean value ± SD of 3 experiments.

Figure 6 illustrates that BAFF acts as a cofactor for the proliferation of B cells. The proliferation of human PBL was measured alone (500 cpm), with the presence of BAFF ligand alone, with the presence of goat antimurin (mu) alone, and with BAFF and anti-mu ligand. The combination of anti-mu and BAFF significantly increased the proliferation of PBL when the concentration of BAFF increased, suggesting cofactor characteristics of BAFF.

Figure 7 represents increased B cell number in BAFF Tg mice

(TO)

Increased lymphocyte counts in BAFF Tg mice. The graph compares 12 control litters (left panel) with 12 Tg BAFF mice (right panel). Lymphocyte counts are shown with circles and granulocytes (including neutrophils, eosinophils, basophils) with diamonds.

(B)

Increased proportion of B cells in PBL of Tg BAFF mice. PBL was stained with anti-B220.FITC and anti-CD4-PE for FACS analysis and activated on living cells using the front lateral dispersion. The percentages of CD40 and B220 positive cells are indicated. A control mouse (left) and two Tg BAFF mice (right) are shown and the results are representative of 7 animals analyzed in each group.

(C)

FACS analysis of the ratio of B cells to T cells in PBL. The difference between control animals and Tg BAFF mice in (A) and (C) was statistically significant (P <0.001).

(D)

Increased expression of MHC class II on PBL B cells of BAFF Tg mice.

(AND)

 Increased Bcl-2 expression in PBL B cells of BAFF Tg mice.

MHC class II expression was analyzed by FACS.

Bcl-2 expression was measured by intracytoplasmic staining and the cells were analyzed by FACS.

In (D) and (E) living cells were activated on the front lateral dispersion. Four control litter partners / white bars are shown) and four Tg BAFF mice and are representative of at least 12 animals analyzed for each group. MFI: average fluorescence intensity. The difference between control animals and Tg BAFF mice was statistically significant (P <0.005).

(F)

Increased expression of effector T cells in BAFF Tg mice. PBL were stained with anti-CD4-Cychrome, anti-CD44-FITC and anti-L selectin-PE. CD4 + activated cells are shown. Percentages of CD44hi / Lselectinalo cells are indicated. A control mouse (left) and two Tg BAFF mice (right) are shown and the results were representative of 8 animals analyzed in each group.

Figure 8 shows B cell compartments augmented in the spleen but not in the bone marrow of Tg BAFF mice.

(TO)

FACS staining for mature B cells using anti-Ig-FITC and anti-B220-PE, in spleen (top panel), bone marrow (middle panel) and MLN (bottom panel). The percentages of mature B B220 + / IgM cells are indicated.

(B)

 FACS staining is indicated for preB cells (B220 + / CD43-) and proB cells (B220 + / CD43 +) in the bone marrow using anti-CD43-FITC, and anti-B220-Cychrome and anti-IgM-PE simultaneously. Activated cells are shown

about the negative IgM population. Percentages of preB cells (B220 + / CD43-) and proB cells (B220 + / CD43 +) are indicated.

For all figures (a and B) a control mouse (left) and two Tg BAFF mice (right) are shown and the results are representative of 7 animals analyzed for each group.

Figure 9 shows increased levels of Ig, RF and CIC in Tg BAFF mice.

(A) SDS-PAGE of two control sera (-) and 4 sera of BAFF (+) Tg mice side by side with the indicated amount of purified mouse IgG for reference. The intensity of the albumin band is similar in all streets indicating that the material loaded on the gel is equivalent for each sample.

ELISA based analysis of Ig (B), RF (C) and CIC (D) of total mice in the sera of 19 control littermates (white bars) and 21 Tg BAFF mice (black bars). In the absence of an appropriate RF control, the title (base log 2) for RF is defined as the dilution of the sera that gives an O.D: 3 times higher than that of the fund. The amount of CIC is defined as the amount of PAP required to generate an O.D. equivalent to that obtained with the serum tested. The difference between control animals and Tg BAFF mice was statistically significant (P <0.001 in (B) and (C), P <0.003 in (D)).

Figure 10 illustrates the presence of antissDNA and anti-dsDNA autoantibodies in some BAFF Tg mice.

(TO)

ELISA analysis of antissDNA antibodies in 19 control littermates (gray bars) and 21 Tg BAFFF mice (black bars).

(B)

ELISA analysis of anti-ssDNA autoantibodies in 5 control litter mates and the 5 animals showing levels of anti-ssDNA autoantibodies of (A).

(C)

Kidney paraffin sections of a control mouse (left) and a Tg BAFF mouse (right), stained with goat mouse anti-mouse Ig-HRP. Ig deposition is shown by a brown stain. These tables are representative of 6 Tg BAFF mice analyzed.

Figure 11 shows enlarged Peyer plates in Tg BAFF mice.

Photograph of Peyer's plaques (indicated by an arrow) on the small intestine of a control mouse (left) and a Tg BAFF mouse (right). This table is representative of at least 12 mice killed for each group. 5X magnification.

Figure 12 shows interrupted T and B cell organization, intense germ center reactions, decreased number of dendritic cells and increased number of plasma cells in the spleen of BAFF Tg mice.

A control mouse is shown in A, C, E and G and a BAFF Tg in B, D, F and H. The B cells are blue and the T cells are brown (A and B). The germ centers are shown with an arrow (C and D). Only some germ centers are seen in control mice (C). CD11c positive dendritic cells are brown and appear in the T cell zone, linking channels and the marginal zone (E). Very few are present in TFF BAFF (F) mice. Syndecan-1-positive plasma cells were only detectable in the red pulp of Tg BAFF (H) mice but not in control mice (G).

These tables are representative of at least 12 Tg BAFF mice analyzed and 12 control mice. The increase is 100X for all frames except C and D which is 50X. B = cell follicle B, T: PALS, WP: white pulp, RP: red pulp.

Figure 13 shows interrupted T and B cell organization, intense germ center reactions and large numbers of plasma cells in MLN and Tg BAFF mice.

The control mouse is shown in A, C, E and G and the Tg BAFF mouse is shown in B, D, F H. Immunohistochemistry was carried out as described in Figure 6. Staining of T and B cells It is shown in A and B, the germinal centers in C and D, the dendritic cells in E and F and the plasma cells in G and H. GC: germinal center. 100X magnification.

Detailed description of the invention

Detailed reference will now be made to the preferred embodiments of this invention. The use of BAFF and BAFF related molecules to effect the growth and maturation of B cells and immunoglobulin secretion is described. The use of BAFF and BAFF related molecules to effect immune system responses is described, when needed due to immune-related disorders. Additionally, this invention encompasses cancer treatment using a specific antibody for soluble BAFF.

The BAFF ligand and its homologs produced by hosts transformed with the sequences described herein, as well as native BAFF purified by the processes known in the art, or produced from known amino acid sequences, are useful in a variety of methods for anticancer applications, antitumor and immunoregulatory. They are also useful in therapy and methods aimed at other diseases,

Another aspect relates to the use of the polypeptide encoded by the isolated nucleic acid encoding the BAFF ligand in "antisense" therapy. As used here, "antisense" therapy refers to the administration or generation in situ of oligonucleotides or their derivatives that hybridize specifically under cellular conditions with the mRNA and / or cellular DNA encoding the ligand of interest, as well as inhibiting the expression of the encoded protein, that is, inhibiting the transcription and / or translation. The binding can be by complementarity of conventional base pairs, or, for example, in the case of DNA duplex binding, through specific interactions in the main groove of the double helix. In general, "antisense" therapy It refers to a range of techniques generally employed in the art, and includes any therapy that relies on specific binding to oligonucleotide sequences.

An antisense construct can be supplied, for example, as an expression plasmid, which, when transcribed in the cell, produces RNA that is complementary to at least a portion of the cellular mRNA encoding the Kay-ligand. Alternatively, the antisense construct can be an oligonucleotide probe that is generated ex vivo. Those oligonucleotide probes are preferably modified oligonucleotides that are resistant to endogenous nucleases, and are therefore stable in vivo. Example nucleic acid molecules for use as antisense oligonucleotides are phosphoramidates, phosphothioates and analogous methylphosphonates of DNA (See, for example, U.S. Patent 5,176,996; U.S. Patent 5,264,564; and U.S. Patent 5,256,775). Additionally, general approaches to construct oligomers useful in antisense therapy have been reviewed, for example, by Van Der Krol et al., (1988) Biotechniques 6: 958-976; and Stein et al. (1988) Cancer Res 48: 2659-2668.

C. BINDING BAFF

The BAFF ligand, as reported above, is a member of the TNF family and has been described in PCT application number PCT / US98 / 19037 (WO99 / 12964). The protein, its fragments or their counterparts can have wide therapeutic and diagnostic applications.

The BAFF ligand is present mainly in the spleen and in peripheral blood lymphocytes, markedly indicating a regulatory role in the immune system. Comparison of the claimed BAFF ligand sequences with other members of the human TNF family reveals considerable structural similarity. All proteins share several regions of sequence conservation in the extracellular domain.

Although the precise three-dimensional structure of the claimed ligand is not known, it is predicted that, as a member of the TNF family, it may share certain structural characteristics with other members of the family.

The new polypeptides specifically interact with a receptor that has not yet been identified. However, the peptides and methods described herein allow the identification of receptors that interact specifically with the BAFF ligand or fragments thereof.

Methods for using BAFF ligand derived peptides that have the ability to bind to their receptors are described. BAFF ligand fragments can be produced in several ways, for example, recombinantly, by PCR, proteolytic digestion or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by separating one or more nucleotides from one end or both ends of a nucleic acid encoding the polypeptide. Expression of mutagenized DNA produces polypeptide fragments.

Polypeptide fragments can also be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase t-boc or f-moc chemistry. For example, peptides and DNA sequences can be arbitrarily divided into fragments of desired length without overlapping the fragment, or divided into overlapping fragments of a desired length. Methods like these are described in detail below.

Generation of soluble forms of the BAFF ligand

Soluble forms of the BAFF ligand can be effectively labeled and can be administered as a drug that now mimics the natural shape of the membrane. It is possible that BAFF ligands are naturally secreted as soluble cytokines, however, if not, genes can be re-managed to force secretion. To create a secreted soluble form of BAFF ligand, the Nterminus transmembrane regions, and some portion of the stem region, would be removed at the DNA level, and replaced with a type I leader or alternatively type II leader sequence that will allow sufficient proteolytic cleavage in The chosen expression system. One skilled in the art could vary the amount of the stem region retained in the secretion expression construct to optimize receptor binding properties and secretion efficiency. For example, constructs containing all possible stem lengths, that is, N-terminal truncations, could be prepared so that proteins starting at amino acids 81 to 139 would result. The optimum length stem sequence would result of this type. of analysis.

E. Generation of antibodies reactive with the BAFF ligand

The invention also includes antibodies specifically reactive with the claimed BAFF ligand or its receptors. Antiprotein / antiseptic antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. By Harlow and Lane (Cold spring Harbor Press: 1988)). A mammal such as a mouse, a hamster or a rabbit can be immunized with an immunogenic form of the peptide. Techniques for conferring immunogenicity on a protein or peptide include conjugation to supports, or other techniques well known in the art.

An immunogenic portion of BAFF ligand or its receptors can be administered in the presence of an auxiliary. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as an antigen to assess antibody levels.

In a preferred embodiment, the subject antibodies are immunospecific for antigenic determinants of the BAFF ligand or its receptors, (e.g., antigenic determinants of a polypeptide of SEQ.ID.NO.:2, sequence described in PCT application number PCT / US98 / 19037 (WO99 / 12964) and is incorporated herein in its entirety, or a closely related human or non-human mammalian homologue (eg, 70, 80 or 90 percent homologous, more preferably 95% homologous). In yet a further embodiment of the present invention, the BAFF anti-ligand or BAFF ligand receptor antibodies have substantially no cross-reaction (ie, specifically react) with a protein that is, for example, less than 80% homologous. SEQ.ID.NO .: 2 or 6, said sequence described in PCT application number PCT / US98 / 19037 (WO99 / 12964) and incorporated herein in its entirety; preferably less than 90% homologous to SEQ.ID.NO.:2, sequence described in PCT application number PCT / US98 / 19037 (WO99 / 12964) and incorporated herein in its entirety; and most preferably less than 95% homologous to SEQ.ID.NO.:2, sequence described in PCT application number PCT / US98 / 19037 (WO99 / 12964) and incorporated herein in its entirety. By "substantially without cross-reaction", it is understood that the antibody has a binding affinity for a non-homologous protein that is less than 10%, more preferably less than 5%, and even more preferably less than 1%, of the affinity of binding for a protein of SEQ.ID.NO.:2, sequence described in PCT application number PCT / US98 / 19037 (WO99 / 12964) and incorporated herein in its entirety.

The term "antibody" as used herein is intended to include fragments thereof that are specifically reactive with the BAFF ligand or its receptors. The antibodies can be fragmented using conventional techniques and the fragments explored for their usefulness in the same manner as described above for complete antibodies. For example, F (ab ') 2 fragments can be generated by treating antibodies with pepsin. The resulting F (ab ') 2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. The antibodies of this invention are intended to include biospecific and chimeric molecules that have BAFF anti-ligand or BAFF anti-ligand receptor activity. Thus, monoclonal and polyclonal (Ab) antibodies directed against BAFF ligand, tumor ligand and its receptors, and antibody fragments such as Fab 'and F (ab') 2, can be used to block the action of the ligand and its respective receptor.

Various forms of antibodies can also be obtained using recombinant DNA techniques. Winter and Milstein (1991) Nature 349: 293-299. For example, chimeric antibodies can be constructed in which the antigen binding domain of an animal antibody binds to a human constant domain (eg, Cabilly et al., U.S. Patent 4,816,567) Chimeric antibodies can reduce Immunogenic responses elicited by animal antibodies when used in human clinical treatments.

In addition, "humanized antibodies" can be synthesized recombinants that recognize the BAFF ligand or its receptors. Humanized antibodies are chimeras that comprise mostly human IgG sequences in which the regions responsible for specific antigen binding have been inserted. The animals are immunized with the desired antigen, the corresponding antibodies are isolated, and the portion of the sequences of the variable region responsible for specific antigen binding is removed. The antigen-binding regions derived from animals are then cloned into the appropriate position of human antibody genes in which the antigen-binding regions have been deleted. Humanized antibodies minimize the use of heterologous sequences (i.e., inter species) in human antibodies, and thus are less likely to elicit immune responses in the treated subject.

The construction of different classes of recombinant antibodies can also be carried out by making chimeric or humanized antibodies comprising variable domains and human constant domains (CH1, CH2, CH3) isolated from different classes of immunoglobulins. For example, antibodies with increased antigen binding site valences can be recombinantly produced by cloning the antigen binding site into vectors that support the human chain constant regions. Arulanandam et al. (1993). J.Exp. Med., 177: 1349-1450.

In addition, recombinant DNA techniques can be used to alter the binding affinities of recombinant antibodies with their antigens by altering amino acid residues in the vicinity of the antigen binding sites. The antigen binding affinity of a humanized antibody can be increased by molecular modeling-based mutagenesis. Queen et al., (1989) Proc. Natl Acad. Sci. 86: 10029-33.

F. Analog generation: production of DNA sequences and altered peptides.

BAFF ligand analogs may differ from the BAFF ligand naturally present in the amino acid sequence,

or in modes that do not imply sequence, or in both. Non-sequence modifications include chemical derivatization in vivo or in vitro of the BAFF ligand. Non-sequence modifications include, but are not limited to, changes in acetylation, methylation, phosphorylation, carboxylation or glycosylation.

Preferred analogs include biologically active fragments of the BAFF ligand, whose sequences differ from the sequence given in SEQ.ID.NO.:2, sequence described in PCT application number PCT / US98 / 19037 (WO99 / 12964) and incorporated herein in its entirety, by one or more conservative amino acid substitutions, or by one or more non-conservative amino acid substitutions, deletions or insertions that do not abolish the activity of the BAFF ligand. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, for example, substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and, phenylalanine, tyrosine.

G. Materials and methods of the invention

The monoclonal anti-Flag M2 antibody, biotinylated anti-Flag M2 antibody and the agarose-coupled M2 anti-Flag antibody were obtained from Sigma. Reagents for cell culture were obtained from Life Sciences (Basel, Switzerland) and Biowhittaker (Walkersville, MD). Soluble human APRIL labeled Flag (residues K110-L250) was produced in 293 cells as described (10, 11) anti CD19, anti CD8 and anti CD4 labeled FITC antibodies were obtained from Pharmingen (San Diego, CA). Goat F (ab ') 2 specific for the Fc5μ fragment of human IgM was obtained from Jackson ImmunoResearch (West Grove, PA). Secondary antibodies were obtained from Pharmingen or Jackson ImmunoResearch and were used at the recommended dilutions.

Human embryonic kidney 293 T cells (12) and fibroblast cell lines (Table 1) were maintained in DMEM containing 10% heat-inactivated fetal bovine serum (FSC). Human embryonic kidney 293 cells were maintained in DMEM F12 nutrient mixture (1: 1) supplemented with 2% FCS. T cell lines, B cell lines and macrophage cell lines (Table 1) were developed in RPMI supplemented with 10% FCS. Molt-4 cells were cultured in an Iscove medium supplemented with 10% FCS. The epithelial cell lines developed in alpha-MEM medium containing 10% FSC, 0.5 mM non-essential amino acids, 10mM Na-Hepes and 1 mM sodium pyruvate. HUVECS were maintained in M199 medium supplemented with 20% FSC, 100 μg / ml of epithelial cell growth factor (Collaborative Research, Inotech, Dottikon, Switzerland) and 100 μg / ml of heparin sodium salt (Sigma). All media contained the antibiotics penicillin and streptomycin. Peripheral blood leukocytes were isolated from heparinized blood from healthy adult volunteers by Ficoll-Paque gradient centrifugation (Pharmacia, Uppsala, Sweden) and culture in RPMI, 10% FSC.

T cells were obtained from non-adherent PBLs by rosette formation with red blood cells from sheep treated with neuraminidase and separated from cells without rosettes (mostly B cells and monocytes) by Ficoll-Paque gradient centrifugation. The purified T cells were activated for 24 hours with phytohemagglutinin (Sigma) (1 µg / ml), washed and cultured in RPMI, 10% FSC, 20 U / ml IL-2. CD14 + monocytes were purified by magnetic cell sorting using anti-CD14 antibodies, goat anti-mouse coated micro accounts and a MINImacsTM device (Milteyi Biotech), and cultured in the presence of GM-CSF (800 U / ml), Leucomax® , Essex CEIME, Lucerne, Switzerland) and IL-4 (320 ng / ml, Lucerne Chem, Lucerne, Switzerland) for 5 days later with GM-CSF, IL-4 and TNF (200 U / ml, Bender, Vienna, Austria ) for an additional 3 days to obtain a CD83 +, population similar to dendritic cells. Human B cells of purity> 97% were isolated from peripheral blood or umbilical cord blood using anti-CD19 magnetic beads (M450, Dynal, Oslo Norway) as described (13).

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Northern Blot Analysis

Northern Blot analysis was carried out using Northern Blots I and II of Human Multiple Tissue (Clontech No. 7760 and No. 7759-1). The membranes were incubated in hybridization solution (50% formamide, 2.5 x Denhardt's, 0.2% SDS, 10 mM EDTA, 2xSSC, 50 mM NaH2PO4, pH 6.5, 200 μg / ml salmon sperm DNA sonicated) for 2 hours at 60 ° C. The antisense RNA probe containing nucleotides corresponding to amino acids 136-285 of hBAFF was denatured by heat and added at 2x106 cpm / ml in a recent hybridization solution. The membrane was hybridized 16 hours at 62 ° C, washed once in 2xSSC, 0.05% SDS (30 minutes at 25 ° C), once in 0.1xSSC, or, 1% SDS (20 minutes at 65 ° C) and exposed to -70 ° C to X-ray films.

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BAFF cDNA characterization

A partial sequence of human BAFF cDNA was contained in several EST clones (BanK Gen T87299 and AA166695 entry numbers) derived from fetal liver and spleen and ovarian cancer libraries. The 5 'portion of the cDNA was obtained by 5'RACE-PCR (Marathon-Ready cDNA, CLonetech, Palo Alto, CA) amplification with oligonucleotides AP1 and JT1013 (5'-ACTGTTTCTTCTGGACCCTGAACGGC-3') using the provided library cDNA from a reserve of human leukocytes as a model, recommended by the manufacturer. The resulting PCR product was cloned into blunt PCR-0 (Invitrogen NV Leek, Holand) and subcloned as an EcoRI / PstI fragment into PAC vector pT7T3 (Pharmacia) containing EST clone T87299. The full length hBAFF cDNA was therefore obtained by combining 5 'and 3' fragments using the internal PstI site of BAFF. The sequence has been assigned the GenBank entry number AF116456.

A partial 617 bp sequence of murine BAFF was contained in two overlapping EST clones (AA422749 and AA254047). A PCR fragment spanning nucleotides 158 to 391 of this sequence was used as a probe to explore the mouse spleen cDNA library (Stratagene, La Jolla, CA).

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Expression of recombinant BAFF

Full-length hBAFF was amplified using JT1069 (5'GACAAGCTTGCCACCATGGATGACTCCACA-3 ') and JT637 (5'-ACTAGTCACAGCAGTTTCAATGC-3'). The PCR product was cloned into blunt PCR-O and resubcloned as a HindIII / EcoRI fragment into a mammalian expression vector PCR-3. A short version of soluble BAFF (amino acids Q136-L285) was amplified using oligos JT636 (5 'CTGCAGGGTCCAGAAGAAACAG-3') and JT637. A long version of soluble BAFF (aaL83-L285) was obtained from full-length BAFF using internal PstI site. Soluble baffs were re-cloned as PSTI / EcoRI fragments behind the hemagglutinin signal peptide and the Flag sequence of a modified PCR-3 vector, and as PstI / SpeI fragments in a framework modified pQE16 bacterial expression vector with a N-terminal Flag sequence (14). The constructs were sequenced in both strands. The establishment of 293 cell lines expressing the soluble short form of full-length BAFF, and the expression and purification of recombinant soluble BAFF from bacteria and mammalian 293 cells was carried out as described (14,15).

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Reverse transcriptase PCR

Total RNA extracted from T cells, B cells, immature dendritic cells derived in vitro, 293 wt and 293-BAFF cells (full length) was reverse transcribed using the Ready to Go (Pharmacia) system according to the manufacturers instructions. BAFF and β-actin cDNAs were detected by PCR amplification with Taq DNA polymerase (1 minute steps at 94 ° C, 55 ° C and 72 ° C for 30 cycles) using specific oligonucleotides: for BAFF, JT1322 5'-GGAGAAGGCAACTCCAGTCAGAAC-3 'and JT1323 5'CAATTCATCCCCAAAGACATGGAC-3 '; for

an IL-2, 5'CTTCTCCTTCACCTGGAAACTGACTG-3 'alpha receptor chain; GCTGGAAGGTGGACAGCGA-3 '.

JT1368 for 5'-TCGGAACACAACGAAACAAGTC-3 'β-actin, 5'-GGCATCGTGATGGACTCCG-3' Y JT1369 and 5 '

- Gel permeation chromatography

293T cells were transiently transfected with the short form of soluble BAFF and developed in serum-free Optimem medium for 7 days. The conditioned supernatants were concentrated 20 times, mixed with internal catalase and ovalbumin standards, and loaded onto a Superdex-200 HR10 / 30 column. The proteins were eluted in PBS at 0.5 ml / minute and the fractions (0.25 ml) were precipitated with trichloroacetic acid and analyzed by Western blotting using anti-Flag M2 antibody. The column was calibrated with standard proteins: ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 Dka), chymotrypsinogen at (25 kDa) and ribonuclease A (13.7 kDa).

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PNGase F treatment

The samples were heated in 20 µl of 0.5% SDS, 1% 2-mercaptoethanol for 3 minutes at 95 ° C, then cooled and supplemented with 10% Nonidet P-40 (2 µl), 0 sodium phosphate, 5 M, pH 7.5 (2 µl) and Nglycanase Peptide F (125 units / µl, or without enzyme in controls). Samples were incubated for 3 hours at 37 ° C before analysis by Western blotting.

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EDMAN sequencing.

293 T cells were transiently transfected with the long form of soluble BAFF and developed in serum-free Optimem medium for 7 days. Conditioned supernatants were concentrated 20 times, fractionated by SDS-PAGE and stained on polyvinylidene difluoride membrane (BioRad Labs, Hercules, CA) as previously described (16) and then sequenced using a gas phase sequencer (ABI 120A, Perkin Elmer, Foster City, CA)) coupled to an analyzer (ABI 120A, Perkin Elmer) equipped with a C18 phenylthiohydantoin column 2.1x250 mm. The data was analyzed using ABI 610 software (Perkin Elmer).

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Antibodies

Polyclonal antibodies were generated by immunizing rabbits (Eurogentec, Seraing, Belgium) with recombinant soluble BAFF. Spleen from rats immunized with the same antigen were fused to x63Ag8.653 mouse myeloma cells, and the hybridoma was screened for BAFF-specific IgGs. One of these monoclonal antibodies, 43.9, is an IgG2A that specifically recognizes hBAFF.

The cells were stained in 50 µl of FACS buffer (PBS, 10% FCS, 0.02% NaN3) with 50 ng (or the indicated amount) of short soluble hBAFF labeled Flag for 20 minutes at 4 ° C, followed by antiFlag M2 ( 1 µg) and secondary antibody. Anti-BAFF mAB 43.9 was used at 40 µg / ml. For two-color FACS analysis, peripheral blood lymphocytes were stained with soluble BAFF labeled Flag / long (2 µg / ml), followed by biotinylated streptavidin anti-Flag M2 (1/400) and labeled PE, followed by anti CD4, anti CD8 or anti CD19 labeled FITC.

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PBL Proliferation Assay

Peripheral blood leukocytes were incubated in 96-well plates (105 cells / well in 100 µl of RPMI supplemented with 10% FCS) for 72 hours in the presence or absence of 2 µg / ml of goat anti-human chain antibody µ ( Sigma) or control F (ab ') 2 and with the indicated concentration of native or boiled soluble BAFF / long. Cells were pulsed for an additional 6 hours with [3 H] thymidine (1 µCi / well) and collected. The incorporation of [3 H] thymidine was monitored by liquid scintillation counting. In some experiments, the recombinant soluble BAFF was replaced by 293 cells stably transfected with full-length BAFF (or 293 wt as a control) that was fixed for 5 minutes at 25 ° C in 1% paraformaldehyde. The test was performed as described (17). In additional experiments, CD19 + cells were isolated to form PBL with magnetic beads and the remaining CD10 cells were irradiated (3,000 rads) before reconstitution with CD19 + cells. The proliferation assay with sBAFF was then performed as described above.

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B cell activation assay

Purified B cells were activated in the EL-4 culture system as described (13). Briefly, 104 B cells mixed with 5x104 irradiated murine EL-4 thymoma cells (clone B5) were cultured for 5-6 days in 200 µl of a medium containing 5% v / v of human T cell culture supernatants (106 / ml) that have been activated for 48 hours with PHA (1 µg / ml) and PMA (1 ng / ml). B cells were re-isolated with antiCD 19 beads and cultured for another 7 days (5x104 cells in 200 µl, duplicate or triplicate culture in flat-bottom 96-well plates) in a medium alone or in medium supplemented with 5% cell supernatants T, or with 50 ng / ml of IL-4 and IL-10 (Peprotech, London, United Kingdom), in the presence or absence of sBAFF. The anti-Flag M2 antibody was added at a concentration of 2 µg / ml and had no effect by itself. IgM, IgG and IgA in culture supernatants were quantified by ELISA assays as described (13).

Human BAFF was identified by sequence homology as a possible new member of the TNF ligand family although we scan the public databases using an improved profile search (18). A cDNA encoding the complete 285 amino acid protein (aa) was obtained by combining EST clones (covering the 3 'region) with a fragment (5' region) amplified by PCR. The absence of a signal peptide suggested that BAFF was a type II membrane protein that is typical of members of the TNF ligand family. The protein has a predicted cytoplasmic domain of 46 aa, a hydrophobic transmembrane region, and an extracellular domain of 218 aa that contains two potential N-glycosylation sites (Fig. 1A). The BAFF extracellular domain sequence shows higher homology with APRIL (33% amino acid identities, 48% homology), while the identity with other family members such as TNF, FasL, LTα, TRAIL or RANHL is below of 20% (Fig 1B, C). The mouse BAFF CDNA clone isolated from a spleen library encoded a slightly longer protein (309 aa) due to an insertion between the transmembrane region and the first of several β strands that constitute the receptor binding domain in all members TNF ligands (19). This ectodomain rich in β strand is almost identical in human and mouse BAFF (86% identity, 93% homology) suggesting that the BAFF gene has been conserved greatly during evolution (Fig. 1A).

Although members of the TNF family are synthesized as membrane-inserted ligands, rupture in the stem region between transmembrane and the receptor binding domain is frequently observed. For example, TNF or Fasl are easily broken from the cell surface by metalloproteinases (20, 21). Although producing several forms of recombinant BAFF in 293 T cells, we observe that a 32 kDA recombinant soluble form of BAFF (aa 83285, sBAFF / long), which contains the entire stem region and an N-terminal Flag tag in addition to the binding domain at the receptor, it was extensively processed to a smaller 18 kDa fragment (Fig. 2A, B). The rupture occurred in the stem region since the fragment was detectable only with antibodies raised against the interaction domain of the complete BAFF receptor but not with anti-Flag antibodies (data not shown). It was also revealed that only N124 (located on the stem) but not N242 (located at the entrance of the f-β sheet) was glycosylated, since the molecular mass of the unprocessed sBAFF / length was reduced from 32 kDa to 30 kDa per separation of the N-linked carbohydrates with PNgasa F while the cleaved form 18 kDa was insensitive to this treatment.

Peptide sequence analysis of the 18kDa fragment certainly showed that the break occurred between R133 and A134 (Fig. 1A). R133 is at the end of the polybasic region that is conserved between human (R-N-K-R) and mouse (R-N-R-R). To check whether the rupture was not simply an expression artifice of soluble unnatural forms of BAFF, full-length BAFF bound to the transmembrane was expressed in 293T cells (Fig. 2C). The complete 32 kDa BAFF and some higher molecular mass species (probably corresponding to undissociated dimers and trimers) were easily detected in cell extracts, but more than 95% of the BAFF recovered from the supernatant corresponded to the processed 18kDa form, indicating that BAFF was also processed when it was synthesized as a membrane bound ligand.

A soluble BAFF was engineered (Q136-L285, sBAFF / short) whose sequence began 2aa downstream of the processing site (Fig. 1B). As predicted, the N-terminus-bound Flag tag of this recombinant molecule was not separated (data not shown), which allowed its purification by an anti-Flag affinity column. To verify its correct folding, the purified BAFF / short was analyzed by gel filtration where the protein was eluted at an apparent molecular mass of 55 kDa (Fig. 2D). The BAFF / short is correctly assembled in a homotimer (3x20 kDa) in accordance with the quaternary structure of other members of the TNF family (19). Finally, unprocessed sBAFF / long was quickly expressed in bacteria, indicating that the fact of the break was specific for eukaryotic cells.

Northern blotting analysis of BAFF revealed that the 2.5kb BAFF mRNA was abundant in the spleen and PBLs (Fig. 3A). The thymus, heart, placenta, small intestine and lung showed weak expression. This restricted distribution suggested that the cells present in lymphoid tissues were the main source of BAFF. By PCR analysis, we found that BAFF mRNA was present in T cells and in dendritic cells derived from peripheral blood monocytes but not in B cells (Fig. 3B). Even simple, unstimulated T cells seemed to express some BAFF mRNA.

A tagged sequence site (STS, SHGC-36171) was found in the database that included the human BAFF sequence. This site presents a human chromosome 13 map in 9cM interval between markers D13S286 and D13S1315. In the cytogenetic map, this interval corresponds to 13q32-34. Of the members of the family of known TNF ligands, only RANKL (Trance) has been located on this chromosome (22) although completely distant from BAFF (13q14). In order for the ligand to exert maximum biological effects, the BAFF receptor (BAFF-R) is likely to be expressed in the same cells or in nearby cells present in lymphoid tissues. Using recombinant sBAFFs as a tool to specifically determine BAFF-R expression by FACS, we certainly found high levels of receptor expression in several B cell lines such as Burkitt Raji and BJAB lymphomas (Fig. 4A, Table 1). In contrast, T cell lines, fibroblastic, epithelial and endothelial origin were all negative. A very weak staining is observed with the THP-1 monocyte line which, however, could be due to binding to the Fc receptor. Thus, BAFF-R expression appears to be restricted to B cell lines. The two mouse B cell lines tested were negative using human BAFF as a probe, although weak binding is observed on mouse splenocytes (data not shown). The presence of BAFF-R on B cells was corroborated by analysis of umbilical cord lymphocytes and peripheral blood. While CD8 + and CD4 + T cells lacked BAFF-R (Fig. 4B and data not shown), abundant staining is observed on CD19 + B cells (Fig. 4A and 4B), which indicates that BAFF-R is expressed in all blood B cells, including naive and memory cells.

Since BAFF binds to blood-derived B cells, experiments were performed to determine if the ligand could deliver stimulatory or growth inhibitory signals. Peripheral blood lymphocytes (PBL) were stimulated with anti-IgM antibodies (µ) together with stably fixed 293 cells expressing superficial BAFF (Fig. 5A). [3 H] thymidine incorporation levels induced by anti-µ alone were not altered by the presence of control cells but were increased twice in the presence of cells transfected with BAFF (Fig. 5B). A dose-dependent proliferation of PBL was also obtained when BAFF transfected cells were replaced by purified BAFFs (Fig. 5C), indicating that BAFF does not require membrane binding to exert its activity. In this experimental test, sCD40L-induced proliferation required concentrations that exceed 1 µg / ml but was less dependent on the presence of anti-µ than that mediated by BAFF (Fig. 5D). When purified CD19 + B cells were co-cultured with autologous CD19-irradiated PBL, proliferation costimulation by BAFF was not affected, demonstrating that the taking of [3 H] thymidine was mainly due to proliferation of B cells and not to indirect stimulation of other cell type (data not shown). The B cell proliferation observed in response to BAFF was completely dependent on the presence of anti-µ antibodies, indicating that BAFF functioned as a costimulator for B cell proliferation.

To investigate a possible effect of BAFF on the secretion of immunoglobulin, purified umbilical cord or peripheral B cells, they were pre-activated by coculture with EL-4T cells in the presence of a mixture of cytokines from PHAA / PMA stimulated T cell supernatants ( 2. 3). These B cells were re-isolated to 98% purity and produced a two-fold increase in Ig secretion during a secondary culture in the presence of BAFF and activated T-cell cytokines compared to cytokines alone. A very modest effect occurred in the absence of exogenous cytokines, and an intermediate effect was observed in the presence of recombinant cytokines IL-2, IL-4 and IL-10 (Fig. 5E, F):

The biochemical analysis of BAFF is also consistent with the typical homotrimeric structure of the TNF family members. Among this family of ligands, BAFF exhibits the highest level of sequence similarity with APRIL that we have recently characterized as a ligand that stimulates the growth of several tumor cells (11). Unlike TNF and LT, which are two family members with equally high homology (33% identity) and whose genes are linked on chromosome 6, APRIL and BAFF are not grouped on the same chromosome. APRIL is located on the same chromosome 17 (J.L.B. unpublished data) while BAFF maps on the distal arm of human chromosome 13 (13q34). At this point abnormalities in Burkitt lymphomas were characterized as the second most frequent defect (24) in addition to the translocation that includes the myc gene at the Ig site (25). Considering the high levels of BAFF-R expression on all lymphoma cell lines analyzed (see Table 1), the intriguing possibility that some Burkitt lymphomas may have deregulated Baff expression is suggested, thus stimulating growth in an autocrine manner.

The role of antigen-specific B lymphocytes during the different stages of the immune response depends greatly on signals and contacts of auxiliary T cells and cells presenting antigens such as dendritic cells (20). B lymphocytes first receive these signals early during the immune response when they interact with T cells on the edge of B-cell follicles in lymphoid tissues, leading to their proliferation and differentiation in low affinity antibody-forming cells (18). At the same time, some antigen-specific B cells also migrate to the B cell follicle and contribute to the formation of germ centers, another B cell proliferation site, but also to maturation and affinity generation of memory B cells and memory cells. high affinity plasma (19).

Signals triggered by another member of the TNF CD40L superfamily have been shown to be critical for the function of B-lymphocytes at multiple stages of the T-cell-dependent immune response. However, several studies clearly showed that the CD40L / CD40 interaction does not justify all It helps B-cell contact-dependent T cells. Certainly, CD40L-deficient T cells isolated from mice or from knockout patients with X-linked hyper IgM syndrome have been found to successfully induce B cell proliferation and their differentiation into plasma cells. Studies using blocking antibodies against CD40L showed that a subset of surface IgD positive B cells isolated from human tonsils proliferate and differentiate in response to activated T cells in a CD40 independent manner. Other members of the TNF family such as membrane bound TNF and CD30L have also been implicated in a stimulation of B cells independent of surface Ig and CD40. Similar to our results with BAFF, it has been seen that CD40-deficient B cells can be stimulated to proliferate and differentiate in plasma cells by auxiliary T cells for as long as surface Ig receptors are triggered at the same time. BAFF, as well as CD30L and CD40L, are expressed by T cells but their originality lies in their expression by dendritic cells as well as the highly specific location of their receptor on B cells that is in contrast to CD40 CD30 and the TNF receptor, whose expression It has been described on many different cells. This observation suggests independent and specific BAFF-induced functions on B cells.

In support of a role for BAFF in the growth of B-cell induced by dendritic cells and by T-cell and in potential maturation, we find that BAFF co-stimulates the proliferation of blood-derived B cells concomitantly with cross-linking of B-cell receptors, and so, regardless of CD40 signaling. In addition, using differentiated CD19 positive B cells in vitro in a GC-like preplasma / B cell (14), we observed a costimulatory effect of BAFF on Ig secretion by these cells in the presence of activated T cell supernatant or a mixture of IL-2, IL-4 and IL-10. Interestingly, the costimulatory effect was stronger in the presence of the activated T cell supernatant compared to the cytokine mixture, suggesting additional soluble factors secreted by activated T cells involved in producing antibodies that can synergize with the same additional BAFF or BAFF. It is, therefore, possible that BAFF actively contributes to the differentiation of these GC-like B cells in plasma.

It is clear that BAFF can signal in naive B cells and in B cells delivered to GC in vitro. If this observation will be transferred or not during a normal immune response it will have to be confirmed by appropriate experiments in vivo.

The biological responses induced in B cells by BAFF are different from CD40L, since the proliferation triggered by CD40L was less dependent on an anti-µ co-stimulus (17) (and Fig. 5D). In addition, CD40L can counteract apoptotic signals in B cells after B cell receptor hooking (29), while BAFF was not able to rescue the R-cell line of R-apoptosis mediated by anti-µ, despite the fact that Ramos cells express BAFF-R (Table 1; FM and JLB, unpublished observations). It is likely, therefore, that CD40L and BAFF fulfill different functions. In this regard, it is notable that BAFF does not interact with any of the 16 recombinant TNF family receptors tested, including CD40 (P.S. and J: T., Unpublished observations).

B cell growth was effectively stimulated with recombinant soluble BAFF that lacked transmembrane domain. This activity is in contrast to several members of the TNF family that are active only as membrane bound ligands such as TRAIL, FasL and CD40L. Soluble forms of these ligands have poor biological activity that can be increased by crosslinking, therefore mimicking the membrane bound ligand. (15). In contrast, crosslinking of sBAFF labeled Flag with anti-Flag antibodies or the use of membrane-bound BAFF expressed on the surface of epithelial cells did not increase the mitogenic activity of BAFF, suggesting that it can act systematically as a secreted cytokine, as it does TNF This is in accordance with the observation that a polybasic sequence present in the stem of BAFF acted as a substrate for a protease. Similar polybasic sequences are also present in corresponding locations in APRIL and TWEAK and in both cases there is evidence of proteolytic processing (30) (N.H. and J.T., unpublished observation). Although the protease responsible for the breakdown has yet to be determined, it is unlikely that it is the metalloproteinase responsible for the release of membrane bound TNF since its sequence preferences differ completely (21). The multibasic motifs in BAFF (R-N-K-R), APRIL (R-K-R-R) and Tweak (R-P-R-R) are reminiscent of the minimum rupture signal for furin (R-X-K / R-R), the prototype of a family of proprotein convertase (31).

The practice of this invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, microbiology, recombinant DNA, protein chemistry, and immunology, which are within the skill of the art. These techniques are described in the bibliography. See, for example, Molecular Cloning: A Laboratory manual, 2nd edition. (Sambrook, Fritsch and Maniatis, eds.), Cold Spring Harbor Laboratory Press, 1989; DNA Cloning, Volumes I and II (D.N. Glover, ed.), 1985; Oligonucleotide Synthesis, (M. J. Gait, ed.), 1984; Culture of Animal Cells (R. I. Freshney, ed.). Alan R. Liss, Inc., 1987; Immobilized Cells and Enzymes, IRL Press, 1986; A Practical Guide to Molecular Cloning (B. Perbal), 1984; Methods in Enzymology, Volumes 154 and 155 (Wu et al., Eds.), Academic Press, New York; Gene Transfer Vectors for Mammalian Cells (J.H. Miller and M.P. Calos, eds.), 1987, Cold Sprin Harbor Laboratory; Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds.), Academic Press, London, 1987; Handbook of Experiment Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds.), 1986; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, 1986.

The following examples are provided to illustrate this invention, and should not be considered as limiting thereof.

Examples

The following experimental procedures were used in Examples 1-6.

DNA construct for the generation of Tg mice with murine BAFF

Both human and murine cDNA sequences have been previously described (Schneider et al., 1999). A PCR fragment encoding full-length murine BAFF was generated by RT-PCR. The first cDNA strand was synthesized from mouse lung polyA + (Clontech, Palo Alto, CA) using oligo dT according to the manufacturer's protocol (GibcoBRL, Grand Island, NY). The PCR reaction contained 1 x Pfu buffer (Stratagene, La Jolla, CA) 0.2mM dNtps, 10% DMSO, 12.5 pM primers, 5 units of Pfu enzyme (Stratagene) and the following primers with Not1 5 restriction sites '-TAAGAATGCGGCCGCGGAATGGAATGGATGAGTCTGCAAA-3' and 5'-TAAGAATGCGGCCGCGGGATCACGCACTCCAGCAA-3 '. The tempering was amplified for 30 cycles at 94 ° C for 1 minute, 54 ° C for 2 minutes and 72 ° C for 3 minutes followed by a 10 minute extension at 72 ° C. This sequence corresponds to nucleotides 214 to 1171 of the GenBank file AFI 19383. The PCR fragment was digested with Not1 and then cloned into a pCEP4 vector (Invitrogen, Carlsbad, CA). The fragment containing murine BAFF was separated with Xba1 in order to include the SV40 poly A addition site sequence. This fragment was cloned into a pUC-based vector where the promoter sequence was added. The promoter, a 1 Kb blunt Bg12Not1 fragment containing the human ApoE enhancer and the AAT promoter (alpha antitrypsin) was purified from plasmid clone 540B (a gift from Dr. Catherine Parker Ponder, Washington University, St Louis, MO). An EcoRV / Bg12 fragment was purified from the final vector and used for the generation of transgenic mice. Injected offspring of F1 (BDF1) female C57Bl / 6J x male DBA / 2J mice were backcrossed on C57BL / 6 mice. Microinjection techniques and generation of transgenic mice have been previously described (Mcknights et al., 1983).

Analytical methods:

Serum samples were subjected to reduced SDS-PAGE analysis using a 12.5% linear gel. Total mouse liver RNA was prepared and processed for Northern Blot analysis using Promega isolation equipment (Madison, WI) according to the manufacturer's guidelines. BAFF transgene-specific MRNA was detected using a probe that encompasses the SV40 poly A tail of the transgenic construct and was obtained by digesting the pCEP4 vector modified with Xba1 and BamH1. The probe recognizes a 1.8-2 Kd band corresponding to mRNA from the BAFF transgene. The PCR analysis of tail DNA of BAFF Tg mice was performed using 12.5 pM of the following 5'-GCAGTTTCACAGCGATGTCCT-3 'primers [SEQ. ID. NO .: 21] and 5'-GTCTCCGTTGCGTGAAATCTG-3 '[SEQ. ID. NO .: 22] in a reaction containing 1X Taq polymerase buffer (Stratagene), 0.2 nM dNTPs, 10% DMSO and 5 units of Taq polymerase (Stratagene). A 719 bp of the transgene was amplified for 35 cycles at 94 ° C for 30 seconds, 54 ° C for 1 minute and 72 ° C for 1.5 minutes, followed by a 10 minute extension at 72 ° C.

The presence of proteins in mouse urine was measured using Multistix 10 SG reagent bands for urine analysis (Bayer Corporation Division, Elkhart, IN).

Cell-dyn and cytofluorometric analysis (FACS).

Differential WBC counts of anticoagulated whole blood with recent EDTA were performed with an Abbott Cell Dyne 3500 (Chicago, IL). For FACS analysis, Fluorescein-labeled rat anti-mouse antibodies (FITC), Cychrome, and Phycoerthrin- (PE): anti-B220, anti-CD4, anti-Cd8, anti-CD43, anti-IgM, anti-CD25, anti -CD24, anti-CD38, anti-CD21, anti-CD44, anti-L-selectin and a series of anti-Bcl-2 hamster Ig / control hamster were obtained from Pharmingen (San Diego, CA). The production of recombinant E. coli as well as BAFF labeled mouse and human Flag derived from mammalian cells were previously described (Schenider et al., 1999). All antibodies were used conforming to the manufacturer's specifications. PBL was purified from mouse blood as follows: mouse blood was collected in microtubes containing EDTA and diluted 1/2 with PBS. Five hundred µl of diluted blood was applied on top of 1 ml of Ficoll (Celardane, Hornby, Notary, Canada) in a 4 ml glass tube, the gradient was carried out at 200 rpm for 30 minutes at room temperature and The lymphocyte-containing interface was collected and washed twice in PBS before staining with FACS. The spleen, bone marrow and mesenteric lymph nodes were ground in a single cell suspension in RPM medium (Life Technologies, Inc, Grand Island, NY), and washed in FACS buffer (PBS supplemented with 2% fetal calf serum (JRH Biosciences, Lenexa, KS.) The cells were first suspended in BAFF buffer supplemented with the following blocking reagents: 10 µg / ml of human Ig (Sandoz, Basel, Switzerland) and 10 µg / ml of Fc blocking antibody anti-mouse (Pharmingen) and incubated 30 minutes on ice before staining with fluorochrome-labeled antibodies All antibodies were diluted in FACS buffer with the blocking reagent mentioned above.The samples were analyzed using a FACScan cytofluorometer (Becton Dickinson).

Detection of total mouse Ig and rheumatoid factors in mouse serum by ELISA assays.

ELISA plates (Corning glass, Corning, NY) were coated overnight at 4 ° C with a 10 µg / ml solution of total goat anti-mouse Ig (Southern Biotechnology Associates, Inc. Birminghan, AL), in baking soda buffer 50 mM sodium pH 9.6. The plates were washed 3 times with PBS / 0.1% Tween and blocked overnight with 1% gelatin in PBS. One hundred µl / well of serial serum dilutions or standard dilutions were added to the plates for 30 minutes at 37 ° C. After one last wash, 3 times with PBS70, 1% Tween, the enzymatic reaction was developed using a 10 µg / ml solution of p-nitrophenylphosphate (Boehringer Mannheim, Indianapolis, IN) in 10% diethanolamine. The reaction was stopped by adding 100 µl of 3N NaOH / well. The optical density was measured at 405 nm using a spectrophotometer from Molecular Devices (Sunnyvale, CA). Standard curves were obtained using purified mouse Ig obtained from Southern Biotechnology Associates. In the case of detection of rheumatoid factors (RF), the plates were coated with normal goat Ig (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) instead of goat anti-mouse Ig and mouse Ig detection It was carried out as described before. The detection of mouse isotypes in the RF assay was done using IgA, IgM, IgG2A, IgG2b and IgG3 anti goat mouse labeled AP, as well as IgA, IgM, IgG2A, IgG2b and purified mouse IgG3 for standard curves (Southern Biotechnology Associates Inc.). All statistical comparisons were carried out by analysis of variance.

Detection of immune complexes in circulation and precipitation of cryoglobulins in mouse sera.

The assay was performed as previously described (June et al., 1979, Singh Tingle, 1982) with the following modifications: ELISA plates (Corning glass) were coated overnight at 4 ° C with 5 µg / ml human C1q (Quidel , San Diego, CA) in 50 mM sodium bicarbonate buffer pH 9.6. The plates were washed three times with PBS / 0.1% Tween. Fifty µl / well of 0.3 M EDTA was added to the plates plus 50 µl / well of serial serum dilutions or solutions of known concentrations of a standard immune complex (anti-mouse peroxidase peroxidase) (PAP) from DAKO (Carpentry, AC). The plates were incubated 30 minutes at 37 ° C. The plates were washed 3 times with PBS / 0.1% Tween. Mouse Ig in the immune complexes were detected using 100 µl / well of a 1 µg / ml solution of an AP labeled goat anti mouse mouse (Southern Biotechnology Associates, Inc.) as described above for ELISA assays. Cryoglobulins were detected by incubating overnight at 4 ° C mouse serum diluted 1/15 times in water and the precipitates were scored visually.

Single strand (SS) and anti-double strand (ds) DNA assays.

Anti-ssDNA was carried out using Immuno-NUNC Plate MaxiSorp plates (NUNC A / S, Denmark). Plates were coated overnight at 4 ° C first with 100 µg / ml methylated BSA (Carbochem Corp., La Jolla, CA), then with 50 µg / ml grade I calf thymus DNA (Sigma, St. Louis, MO). The calf thymus DNA was cut by sonication and then digested with S1 nuclease before use. For the anti-ssDNA assay, the DNA was boiled for 10 minutes and cooled on ice before use. After blocking, serial dilutions of the serum samples were added and incubated at room temperature for 2 hours. Autoantibodies were detected with goat anti-mouse IgG-AP (Sigma) and developed as described above for ELISA assays. Standard curves were obtained using known amounts of anti-DNA mAb 205, which is specific for ss-and dsDNA (Datta et al., 1987).

Immunochemistry

Spleen and lymph nodes were frozen in O.C.T. (Miles, Elkahrt, IN) and were mounted to section them with cryostat. Sections 7-10 µm thick were dried and fixed in acetone. All incubations of Ab (10 µg / ml) were done for 1 hour at room temperature in a humidified box after dilution in Tris-Temponated A saline solution (TBS-A, 0.05M Tris, 0.15M NaCl, 0 , 05% Tween-20 (v / v), 0.25% BSA), washed in TBS-B (0.05M Tris, 0.15M NaCl, 0.05% Tween-20) and fixed 1 minute in methanol before starting the enzymatic reaction. Horseradish peroxidase (HRP) and alkaline phosphatase (AP) activities were developed using the diaminobenzidine tablet substrate (DAB) equipment and the 5-bromo-4-chloro-3-indolyl phosphate / blue nitrotetrazolium (BCIP / NBT, Pierce, Rockford, IL), respectively. Dyed tissue sections were finally fixed 5 minutes in methanol and stained with Giemsa (Fluka, Buchs, Switzerland). Rat anti B220 with biotin labeled antibodies, anti-syndecan-1 as well as anti-CD4, anti-CD8α and anti-CD8β from unlabeled rat were obtained from Pharmingen. Peanut agglutinin labeled with biotin was obtained from Vector laboratories (Burlingame, CA). Anti-mouse rat labeled Ig (HRP) and streptavidin (HRP) were obtained from Jackson ImmunoResearch Laboratories, Inc and streptavidin labeled AP from Southern Biotechnology Associates, Inc. In the case of kidney tissue immunochemistry to detect Ig deposition, Paraffin section, wax-free and blocked using diluted Vector horse serum (Burlingame, CA), was used, followed by staining with HRP goat anti-mouse Ig from Jackson Immunoresearch. Detection was carried out as described above.

Example 1

Transgenic founding mice with BAFF (BAFF Tg) have an abnormal phenotype

Full-length murine BAFF was expressed in transgenic mice using the specific alpha-1 antitrypsin promoter with the APO E enhancer. The full length version was chosen in the hope that BAFF would be,

Either excised and would act systematically, or it would be retained in a form of membrane binding such that specific local liver abnormalities would be observed possibly providing functional indications. We obtained 13 positive founding mice for the BAFF transgene (Table 2). Four of these mice died at an early age. Routine pathology was carried out on 811 and 816 mice (Table 2). There was no obvious infection in these mice, however, cardiovascular and renal abnormalities were evident and similar to those described for severe hypertension (Fu, 1995) (Table 2). Sections of kidney tissue stained (H&E) with hematoxylin and eosin, showed that morphology of glomeruli in that mouse was abnormal, while the rest of the kidney tissue appeared normal (data not shown). Many transgenic founding mice with BAFF had proteinuria (Table 2). Immunochemistry on frozen tissue sections of the 816 mouse revealed abnormal and extensive B-cell staining, and reduced T-cell staining, and this observation was confirmed in the progeny (see below, Figure 12).

Using the two-color FACS analysis, the ratio in 0% of B220 positive B cells over% of CD4 positive T cells was calculated. This ratio was two to seven times higher in Tg BAFF founding mice when compared to control negative BDF1 mice (Table 2), suggesting an increase in B cell population in TFF BAFF mice. We selected nine of these founding mice to generate our different transgenic mouse lines as highlighted in Table 2. None of the remaining Tg BAFF founding mice or the derived progeny showed no sign of ill health months after the early death of the founders. 696, 700, 811 and 816, suggesting that these 4 mice could have expressed higher levels of BAFF that caused their death. Overexpression of BAFF in the liver of transgenic mice was confirmed by Northern Blot analysis (data not shown). In all histologically examined TFF BAFF mice, the livers showed no abnormalities indicating that local BAFF overexpression did not induce any immunological or pathological event. An ELISA assay for murine BAFF is not available; however, we showed that 2% serum from BAFF Tg mice, but not from control mice, blocked the binding of soluble mouse labeled Flag BAFF cells derived from mammalian cells to BJAB cells. In addition, 5% serum from BAFF Tg mice but not from control mice increased human B cell proliferation from PBL in the presence of anti-µ (data not shown). These data suggest that substantial amounts of soluble BAFF are present in the blood of Tg BAFF.

Example 2

Peripheral lymphocytosis in Tg mice with BAFF is due to high B cell numbers

It was found that the population of transgenic mice has more lymphocytes in the blood when compared to control negative littermates, reaching values as high as 13,000 lymphocytes / µ of blood (Figure 7A). In contrast, the number of granulocytes per µ of blood in TFF BAFF mice and control mice remained within normal limits (Figure 7A). As the FACS analysis, using anti-CD40 and anti-B220 antibodies, from peripheral blood cells (PBL) of 18 Tg BAFF mice supplied by six different founder mice, showed increased B / T ratios (Figure 7B and 7C), the elevated Lymphocyte levels resulted from a subset of expanded B cells. Similarly, using this method, the calculation of absolute numbers of T cells circulating with CD4 revealed a 50% reduction of this subset of T cells in BAFF Tg mice compared to control mice, and the same observation was made to the subset of CD8 T cells (data not shown). All PBL B cells of BAFF Tg mice have increased MHC class II and Bcl-2 expression compared to B cells of control mice (Figure 7D and 7E, respectively, indicating some level of B cell activation in PBL of Tg mice BAFF: T cells in the blood of BAFF Tg mice did not express early activation CD69 or CD25 markers; however, 40 to 56% of CD4 or CD8 cells were effector T cells activated with a CD44hi, L-selectin phenotype versus only 8% to 12% in control littermates (Figure 7F) Thus Tg BAFF mice clearly show signs of B cell lymphocytosis and global B cell activation along with T cell alterations.

Example 3

Expanded B cell compartments are composed of mature cells

To see if BAFF overexpression in transgenic mice was affecting the B cell compartment centrally in the bone marrow and peripherally in secondary lymphoid organs, we examined FACS spleen nodes, bone marrow nodes, and mesenteric lymph of a total of seven mice Tg BAFF and seven companions of control litter derived from four founding mice. The mature B cell compartment was analyzed by staining with anti-B220 and anti-IgM antibodies. Two representative BAFF Tg mice and a representative control litter partner are shown in Figure 8. The mature B cell compartment (Ig +, B220 +) was increased in spleen and mesenteric lymph nodes (Figure 8A, top and bottom panels , respectively). Analysis of B220 + / IgM + B cells (Figure 7A, middle panel) or proB cell compartments (CD43 + / B220 +) and preB cell (CD43- / B220 +) in the bone marrow showed that Tg BAFF mice and littermates Control were similar. These data indicated that BAFF overexpression is affecting the proliferation of mature B cells in the periphery but not of progenitor B cells in the bone marrow. FACS analysis of the subpopulations of B cells in the spleen revealed an increased proportion of marginal zone B cells (MZ) in Tg BAFF mice compared to control mice (Table 3). The follicular B cell population remains proportional in BAFF Tg mice and in control mice while the fraction of newly formed B cells is slightly decreased in BAFF Tg mice (Table 3). This result was also confirmed in splenic B220 + B cells using anti-CD38 antibodies versus anti-CD24 antibodies and anti-IgM antibodies versus anti-IgD antibodies and analyzing the population of MZ B cells in CD38 hi / CD24 + and IgMhi / IgD10 with respect to MZ B cell population, respectively, as previously described (Oliver et al., 1997) (data not shown). Immunohistochemical analysis using an anti-mouse IgM antibody revealed the expansion of the bright IgM MZ B cell area in the spleen of BAFF Tg mice compared to control mice (data not shown). All B220 + splenic B cells of Tg BAFF also express higher levels of MHC class II (Table 3) and Bcl-2 (data not shown) compared to splenic B cells of control mice, which indicate that splenic B cells as well as PBL B cells are in an activated state.

Example 4

Tg mice with BAFF have high levels of total immunoglobulins, rheumatoid factors and immune complexes in circulation in their serum

The increased B cell compartment in TFF BAFF mice suggested that the level of total Ig in the blood of these animals could also be increased. SDS-PAGE, serum analysis of Tg BAFF mice and control littermates showed that IgG bands of heavy and light chains were at least 10 times more intense in 3 of the 4 Tg BAFF mice compared to control sera (Figure 9A ). Similarly, an ELISA determination in the sera of BAFF Tg mice shows significantly higher levels of total GI than those of control mice (Figure 9B).

Despite the high levels seen by SDS-PAGE, excessively high levels of Ig seen by ELISA determination in some mice, for example, 697-5, 816-8-3 and 823-20, led us to suspect the presence of rheumatoid factors (RF) in the sera, or antibodies directed against antigenic determinants on the Fc fragment of IgG (Jefferis, 1995). These antibodies could bind to the goat anti-mouse Ig used to coat the ELISA plates and give erroneously high values. ELISA plates were coated with normal irrelevant goat Ig and Ig binding of Tg BAFFF to normal goat Ig. Figure 9C shows that the sera of most of the BAFF Tg mice contained Ig that reacts with normal goat Ig, while only two of the 19 control mice showed reactivity in the same assay. These RF were mainly of the IgM, IgA and IgG2A isotypes (data not shown).

The presence of RF can be associated with the presence of high levels of immune complexes (CLC) and cryoglobulin in the blood (Jefferis, 1995). To verify whether or not TFF BAFF mice have abnormal CIC serum levels, a Clq-based binding assay was used to detect CIC in Tg BAFF mice analyzed above. Only 5 Tg BAFF showed significantly high levels of CIC compared to control mice, however, these mice corresponded to animals that have the highest levels of total rheumatoid factor and Ig (Figure 9D). We also observed precipitate formation when the sera of Tg BAFF mice were diluted in water but not in the control sera indicating the presence of cryoglobulin in these mice (data not shown). Thus, in addition to B-cell hyperplasia, TFF BAFF mice have hyperglobulinemia associated with RF and CIC.

Example 5

Some TFF BAFF mice have high levels of double-stranded (ds) and single-stranded anti-DNA (ss) autoantibodies.

Initially, we observed kidney abnormalities reminiscent of a lupus-like disease in two of four founding mice (Table II). The presence of anti-DNA autoantibodies has also been described in SLE or mouse (SNF1) patients (SWR x NZB) similar to SLE (Datta et al., 1987). Anti-ssDNA autoantibody levels were detected in BAFF Tg mice that had previously been shown to have the highest level of total serum Ig (Figure 10 A). We analyzed the serum of two Tg BAFF negative mice for antibodies against ssDNA (697-5 and 816-1-1) and three transgenic mice that secrete anti-ssDNA antibodies (820-14, 816-8-3 and 820-7 ) for the presence of anti-dsDNA antibodies, in parallel with five control litter partners. Tg BAFF mice also secreted anti-dsDNA, however, secretion levels do not always correlate with those of anti-ssDNA antibodies, since Tg BAFF 697-5 mouse serum that did not contain detectable levels of anti-ssDNA antibodies , was clearly positive regarding the presence of anti-dsDNA (Figure 10B). Therefore, BAFF Tg mice that show the most severe hyperglobulinemia secrete pathological levels of anti-DNA autoantibodies. Additionally, and also reminiscent of a problem similar to lupus in these mice we detected immunoglobulin deposition in the kidney of six Tg BAFF analyzed (Figure 10 C), three of these mice did not secrete detectable levels of anti-DNA antibodies (data not shown) .

Example 6

Tg mice with BAFF have enlarged B-cell follicles, numerous germinal centers, reduced dendritic cell numbers and increased plasma cell numbers in both spleen and mesenteric lymph nodes (MLN)

Tg BAFF mice had large spleens, MLN (data not shown) and Peyer pieces (Figure 11). Immunohistochemistry showed the presence of enlarged B-cell follicles and reduced peripheral arteriolar lymphoid sheets (PALS or T-cell area) in TFF BAFF mice (Figure 12B). Interestingly, few germ centers were observed in non-immunized control littermates (and this is typical of this colony in general) and those present were small (Figure 12 C), while Tg BAFF mice possessed numerous germinal centers in the absence. of immunization (Figure 12D). Staining with anti-CD11c for dendritic cells in the T cell zone and in the marginal zone of control mice (Figure 12E) was considerably reduced in Tg BAFF mice (Figure 12F). Syndecan-1-positive plasma cells were almost undetectable in the spleen of control littermates (Figure 12G), although the red pulp of Tg BAFF mice was strongly positive for Syndecan-1 (Figure 12H). Very similar observations were made for the MLN (Figure 13). In MLN of BAFF Tg mice the B cell areas were greatly expanded (Figure 13B) in contrast to the normal node where B cell follicles were easily recognizable at the periphery of the node under the capsule with a typical paracortical T cell zone ( Figure 13A). The marrow of Tg BAFF mice was filled with syndecan-1 positive cells that are presumably plasma cells (Figure 13 H). In conclusion, the analysis of secondary lymphoid organs in Tg BAFF mice was consistent with the expanded B-cell phenotype that shows multiple cellular abnormalities and intense immune activity.

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Table I: MARCH expression in different cell lines

Cell type

Cell lines Binding of Specific details

MARCH

Similar to

HT-29 - colon adenocarcinoma

epithelial

A375 MCF-7 - / + - melanoma adenocarcinoma of the breast

MB260

- melanoma

Cos

+ monkey kidney cells

Fiber blasts

WI-38 H3-68 H3-27 --- lung foreskin foreskin

HUVEC

- umbilical vein

THP-1

- / + monocytes

Lines of

Molt-4 - lymphoblastic leukemia

T cells

Hut-78 - cutaneous lymphoma

Jurkat

- lymphoblastic leukemia

B cell lines

BJAB Namalawa Daudi Bouquets Raji JIYOYE SKW-64 RPMI 1788 IM-9 NC-37

+++ ++ + / ++ +++ + ++ +++ +++ +++

Burkitt lymphoma Burkitt lymphoma Burkitt lymphoma EBNA + VCA + Burkitt lymphoma EBV Burkitt lymphoma Burkitt EBV + lymphoma secreting peripheral blood IgM, IgM lymphoblast secretion, Ig lymphoblast secretion EBV +

WEHI-231 cell lines -A20 mouse B cell lymphoma -B cell lymphoma

The surface expression of MARCH was determined by FACS using MARCH labeled Flag as described in Material and Methods Table II of Tg founding mice BAFF Number of Proteinuriaa B / Tb mice

female 690 male 696 female 697 male 700 700 male 802 female 804 female 807 male 810 male / e 811 male 813 female / f 816 male 820 male 823 BDF1 Female control BDF1 Male control

ND 2 ND ND +++ ND ND ND ++ 4.6 +++ 5.4 ND 4 +++ 7.8 ND ND + 5.4 ++ ND ++ 3 ++ 2.9 +/- 1 , 5 +/- 2.5

a Proteinuria is measured using colored medical strips immersed in mouse urine and is defined as follows: -no proteinuria, +/- traces, + (30mg / dl), ++ (100mg / dl), +++ (300 mg / dl), ++++ (> 2000mg / ml.bB / T is the ratio of% B cells to% T cells in PBL determined by FACS analysis, using anti-B220 antibodies labeled PE and anti-CD4 labeled FITC. c Early death There is no transmission of transgenes in the progeny Cardiovascular and renal abnormalities observed during autopsy sacrificed due to the presence of blood in the urine Abnormalities in the kidney, heart and arteries evident after analysis of sections stained with H&E of all tissues The increase in the population of splenic B cells was determined by imunohistochemistry on frozen spleen sections using biotin-labeled anti-mouse B220 and anti-mouse CD4, followed by alkaline phosphatase-labeled streptavidin and peroxide-labeled anti-rat Ig of horseradish.

Underlined founding mice used for ND playback: not done

Table 3: Increased MHC class II expression on B cells and widened ratio of MZ B cells in the spleen of BAFF Tg mice.

MHC class II expression levels on B220 + B cells (MFI)

% of follicular B cells (B220 + / IgMlo / CD21int) % of MZ B cells (B220 + / IgMlo / CD21int) % of newly formed B cells (B220 + / IgMlo / CD21int)

Control mouse 816-1-10 802-21 823-1 Mouse 802-6 820-7 816-1-1

1170 1029 1240 1707 1900 2088 45 48 39 49 39 40 6 10.5 9 18 23 23 12 9 6.5 5.9 6.3 5.8

Splenocytes were analyzed by FACS and activated on the B220 + population. A representative experiment is shown. MFI: medium fluorescence intensity.

Claims (4)

1. An antibody or a specific active fragment for soluble BAFF for use in the treatment of cancer.

2. The antibody according to claim 1, wherein the antibody is a monoclonal antibody.

3. The antibody according to claims 1 or 2, wherein the active antibody fragment is selected from a Fab 'fragment and an F (ab') 2 fragment.

The antibody according to any one of claims 1 to 3, wherein the antibody is a chimeric antibody.

5. The antibody according to any one of claims 1 to 4, wherein the antibody is a humanized antibody fifteen 6. The antibody according to any one of claims 1 to 5 for use in the treatment of cancer, wherein the cancer is Burkitt lymphoma.