WO1996025433A1 - Facteurs de differentiation et de proliferation des lymphocytes b et leurs procedes de preparation et d'utilisation - Google Patents

Facteurs de differentiation et de proliferation des lymphocytes b et leurs procedes de preparation et d'utilisation Download PDF

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WO1996025433A1
WO1996025433A1 PCT/US1996/002160 US9602160W WO9625433A1 WO 1996025433 A1 WO1996025433 A1 WO 1996025433A1 US 9602160 W US9602160 W US 9602160W WO 9625433 A1 WO9625433 A1 WO 9625433A1
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cells
bcdf
cell
mab
secretion
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Lloyd Mayer
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Mount Sinai School Of Medicine Of The City University Of New York
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5412—IL-6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation

Definitions

  • PHS Public Health Service
  • This invention relates to novel protein factors which cause proliferation or differentiation of B cells.
  • the invention also relates to methods for obtaining and purifying the factors and to B cells activated by the factors.
  • This invention further relates to monoclonal antibodies which inhibit the functional activity of these novel protein factors.
  • Lymphocytes are the primary cells involved in generating an immune response. There are two principal classes of lymphocytes, B cells and T cells. Mature B cells produce and secrete specific antibodies against antigens. T cells consist of several subpopulations which regulate the immune response in mammals by direct cell-cell interactions and by producing and releasing in the blood a variety of cytokines, protein factors which modulate immune responses.
  • lymphocytes differentiate from precursor stem cells which originate in the bone marrow.
  • B cells undergo the differentiation process in the bone marrow, and T cells differentiate in the thymus.
  • Both B and T cells have several stages of development. Resting, both immature and mature B cells express a B cell surface form of immunoglobulin ("Ig").
  • Mature cells respond to B cell differentiation factors to become plasma cells and secrete antibodies.
  • Ig immunoglobulin
  • T cells There are several stages in the maturation of T cells which occur in different regions of the thymus. In the thymus, differentiating T cells are known as thymocytes and express unique stage-specific cell surface protein "markers" .
  • T cells leave the thymus as long-lived small lymphocytes which circulate in the blood stream, lymphatic system and intercellular spaces. These mature T cells can be distinguished from the most immature thymocytes by the presence of the cell surface T cell receptor (TCR) for antigen.
  • T cells expressing TCR have immunological specificity; they can recognize and react with antigens such as those present on cell surfaces in cell mediated immune responses (e.g., graft rejection) .
  • T cells only recognize an antigen which is "presented” by a major histocompatibility complex (MHC) protein present on the surface of an antigen presenting cell (APC) and which binds specifically to the TCR.
  • MHC major histocompatibility complex
  • T cells may be stimulated to divide and proliferate so that many more T cells with the same antigenic specificity are produced.
  • the TCR not only recognizes a foreign antigen but signals the T cell to divide and proliferate.
  • activated T cells may destroy a cell expressing the specific antigen, a situation frequently found in virally infected cells which often have virus specific proteins on their surface. T cell activation enables the T cells to perform different tasks in the immune response. T cells can also be activated by binding of certain antibodies to the cell surface protein CD3 which is complexed to the TCR.
  • T cells Several subclasses of mature T cells have been recognized based on the different tasks they perform.
  • Helper T cells (T Tha) are required for promoting or enhancing B cell antibody production.
  • Cytotoxic/effector or killer cells (T k ) directly kill their target cells by cell lysis.
  • Target cells are usually virally infected, malignant or otherwise altered cells recognized by T k by virtue of the presence of specific antigenic markers.
  • Suppressor cells suppress or down-regulate immunological reactions.
  • T h cells express the cell surface CD4 protein whereas most T k and T, cells express the cell surface CD8 protein, Swain, "Evidence for Two Distinct Classes of Murine B Cell Growth Factors with Activities in Different Functional Assays", J. Exp. Med. , 158:822 (1983) .
  • B cells within lymph nodes and in the circulation are memory B cells having been primed earlier by the interaction with T cells, APCs and antigen.
  • T independent antigens There are three mechanisms by which B cells can be induced to undergo terminal maturation: 1) by antigen alone (T independent antigens) ; 2) by direct T cell: B cell linking in the context of antigen (cognate interaction) ; and 3) by factor mediated, noncognate, interactions.
  • T independent antigens There are several antigens (T independent antigens) that can activate and induce terminal maturation of B cells in the absence of T cells. These antigens are commonly bacterial or polysaccharide antigens with repeating antigenic units.
  • These antigens can "cross link” the B cell surface Ig molecules, thus inducing membrane depolarization and B cell activation and proliferation.
  • These antigens such as lipopolysaccharide, pneumococcal polysaccharides or Staphylococcus aureus organisms such as Staphylococcus aureus Cowan I (SAC) are potent B cell mitogens.
  • T dependent antigens are usually proteins which require activated T cells to induce B cell differentiation and proliferation. These cognate interactions involve close proximity to or linking of B and T cells. Additionally, some of the effects may be due to locally secreted factors.
  • B cells are generally activated in vivo by antigen or in vitro by molecules that can cross link surface Ig (anti-IgM, SAC), to induce an activated state which render the cells responsive to growth and differentiation stimuli.
  • BCGF B cell growth factors
  • BCDF B cell differentiation factors
  • Proliferation- independent BCDFs can also act on resting B cells inducing B cells to mature without undergoing division. These factors contribute to the nonspecific amplification of a very specific immune response. For example, the nonspecific amplification results in the proliferation of cells previously activated by antigen. An individual revaccinated or boosted with tetanus toxoid, for instance, exhibits a concomitant increase in antibody titer to other antigens such as measles and diphtheria. Other T cell derived factors have been found to induce or enhance B cell growth or differentiation. The distinct stages of normal murine and human B cell maturation are highly regulated.
  • This regulation appears, in large part, to be related to cytokines, predominantly T cell derived, which induce B cells to enter into the cell cycle and thus divide, proliferate and terminally differentiate.
  • cytokines predominantly T cell derived
  • IL-4 Interleukin-4
  • Yokota et al. "Isolation and Characterization of A Human Interleukin cDNA Clone, Homologous to Mouse B-cell Stimulatory Factor 1, that Expresses B cell and T cell Stimulating Activities", Proc. Natl. Acad. Sci. USA, 83:5894 (1986) .
  • B cell proliferation can be initiated by IL-2, IL-4 or IL-5. Defrance et al. , "B Cell Growth
  • IL-2 has effects on B cells and monocytes as well as activated T cells, and non-lymphoid cells can be regulated by IL-4, ⁇ -IFN and IL-6) . It is this last observation that has raised questions as to the specific physiological role(s) of each of these cytokines.
  • IL-2 is a potent synergistic signal for differentiation once a primary differentiation stimulus has been provided, yet alone it has little if any effect on B cells.
  • 7-IFN in mouse, but not man, is a potent B cell differentiation factor, but also requires other regulatory signals, such as BCDF, IL-4, or IL-5 to exert its effects.
  • Muraguchi et al. "The Essential Role of B Cell Stimulatory Factor 2 (BSF-l/IL-6) for the Terminal Differentiation of B Cells", J. Exp. Med., 167:332 (1988), identified a B cell differentiation factor derived from a Human T cell lymphotrophic virus (HTLV) I infected T cell line, and subsequently from an atrial myxoma cell line termed BSF-2, which induced weak but consistent increases in Ig secretion from the B cell line CESS.
  • HTLV Human T cell lymphotrophic virus
  • TRF T Cell-replacing Factor
  • BSF-2 Interleukin
  • IFN/J 2 hepatocyte stimulatory factor
  • plasmacytoma/hybridoma growth factor The major source of these cytokines was from fibroblasts and monocytes, and not T cells, although Kishimoto has recently demonstrated the delayed (4, 5 days) expression of IL-6 mRNA in activated T cells.
  • IL-6 has BCDF activity, it is unclear whether it was the major activity of this molecule because IL-6 is produced after the majority of T cell proliferation occurs (72 hours) .
  • IL-6 has been tested in several systems and, in addition to the activities described above, has been reported to perpetuate mitogen-activated T cell proliferation and augment proliferation of certain EBV transformed B cell lines. Only its anti-viral activity is in question.
  • the heterogeneity of activities for IL-6 may relate to the individual cell source (fibroblasts, monocytes, B cells, T cells, atrial myxoma cells) . but more recent evidence suggests that differential glycosylation, phosphorylation, and sulfation results in the multiple species (molecular weight) and functional heterogeneity.
  • BSF-2 Human Interleukin
  • monocytes Baczin, et al., J. Immunol., 139:780 (1987)
  • fibroblasts Van Damme et al. , "Identification Of The Human 26-kD Protein, Interferon Beta 2 (IFN-Beta 2) , As A B Cell Hybridoma/Plasmacytoma Growth Factor Induced By Interleukin 1 And Tumor Necrosis Factor", J. Exp. Med. 165:914 (1987)).
  • IFN-Beta 2 Interferon Beta 2
  • 26-kDa protein (Van Damme, et al., "Identification Of The Human 26-kD Protein, Interferon Beta 2 (IFN-Beta 2), As A B Cell Hybridoma/Plasmacytoma Growth Factor Induced By Interleukin 1 And Tumor Necrosis Factor", J. Exp.
  • HPGF hepatocyte-stimulating factor
  • HPSF hepatocyte-stimulating factor
  • HPGF primary structure has been partially sequenced and the cDNA of IFN-/J 2 and 26-kDa protein have been cloned.
  • Hirano et al. "Complementary DNA For A Novel Human Interleukin (BSF-2) That Induces B Lymphocytes To Produce Immunoglobulin", Nature, 324:73 (1986) .
  • BSF-2 Novel Human Interleukin
  • HPGF shows i munologic cross-specificity with IFN-0 2 . May, et al., "Synthesis And Secretion Of Multiple Forms of ⁇ 2-Interferon/B Cell Differentiation Factor 2/Hepatocyte Stimulatory Factor By Human Fibroblasts And Monocytes", J. Biol.
  • IL-6 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of natural IL-6 shows a multiple banding pattern ranging from about 20 to 35 kDa depending on the stimuli and the cell source. Under non-reducing conditions IL-6 can have a molecular weight (MW) as high as 65 to 70 kDa. Heterogeneity, under reducing conditions, is due to glycosylation, phosphorylation, and possibly sulfation. May, et al. , "Phosphorylation Of Secreted Forms Of Human Beta 2-Interferon/Hepatocyte Stimulating
  • BCGF B cell growth factor
  • BCDF B cell differentiation factor
  • mAb monoclonal antibodies
  • the 446-BCDF and 446-BCPF proteins are separable from other, previously identified cytokines and interferons.
  • This invention therefore provides 446-BCDF which has been characterized as a protein of about 30,000 daltons molecular weight, having a pi of about 6. Further, 446-BCDF is fully active in the presence of anti-IL-6 antibodies of sufficient quantity to completely neutralize the activity of IL-6.
  • the 446-BCPF provided by this invention has been characterized as a protein which elutes at a Na + concentration of lOOmM from an anion exchange column and induces resting B cell proliferation.
  • This invention also relates to a monoclonal antibody that specifically inhibits the functional activity of 446-BCDF.
  • Two monoclonal antibodies, 929 and 204 have been found to be specific for 446-BCDF in that they inhibit the B cell differentiation activity of 446-BCDF but fail to inhibit B cell differentiation in response to other cytokines such as IL-2 and IL-6 or mitogens such as pokeweed mitogen.
  • This invention provides a method of inducing B cells to differentiate comprising the steps of contacting activated B cells with an effective amount of 446-BCDF, allowing the B cells to incubate for a suitable length of time and determining B cell differentiation.
  • a suitable assay for B cell differentiation is the determination of antibody secretion.
  • Suitable B cells include but are not limited to B cells previously activated, for example, but not by limitation, by transformation with EBV or by exposure to fixed Staphylococcus aureus Cowan I strain organisms.
  • This invention also provides a method for inducing B cell proliferation comprising the steps of contacting a non-activated B cell sample with the 446-BCPF in a suitable concentration for a suitable length of time and adding a detectable marker, for instance [ 3 H] thymidine. After a suitable incubation time, the cells are harvested and assayed for the presence of the detectable marker.
  • This invention still further provides a method for obtaining BCDF and BCPF comprising the steps of exposing monocyte-depleted T cell cultures to purified anti-CD3 mAb, where a preferred mAb is 446, for a suitable length of time, then collecting the supernatant from the T cells. The supernatant may be used either directly as crude preparations of BCDF and BCPF or subjected to further purification steps prior to use.
  • This invention also provides a method for purifying 446-BCDF comprising the steps of providing a solid support, binding a monoclonal antibody selected from the group consisting of mAb 929, mAb 204 and combinations thereof to the solid support, contacting a sample comprising 446-BCDF thereby forming a 446-BCDF monoclonal antibody complex, and isolating purified 446-BCDF from the complex.
  • This invention further provides a method for detecting the presence of 446-BCDF in a sample comprising the steps of contacting a sample with a monoclonal antibody selected from the group consisting of mAb 929, mAb 204 and combinations thereof and assaying the B cell differentiation activity of the sample.
  • this invention provides a method for identifying the B cell receptors to which the novel BCDF and BCPF bind.
  • the method comprises obtaining mAb to B cells by any suitable means, and assaying the mAb for their ability to block the activities of either BCPF or BCDF.
  • Such antibodies are neutralizing and may be found to bind to the receptor.
  • the receptors can be isolated, purified and characterized.
  • labelled purified BCDF may be used to identify the B cell receptors.
  • Figs, la and lb are graphs showing T cell proliferation activity and induction of Ig secretion in normal B cells by anti-CD3 mAb preparations.
  • Anti-CD3 mAb preparations 454 (filled-in square) , 147 (open triangle) and 446 (open square) were incubated with normal T cells.
  • Figure 2 A graph showing anti-CD3 stimulated normal T cell supernatant induction of Ig secretion in normal B cells. Filled-in squares represent the presence of SAC and filled-in triangles represent the absence of SAC.
  • Figure 3 A graph showing induction of proliferation of normal B cells by anti-CD3-stimulated T cell supernatant. B cells were incubated in the presence (filled squares) or absence (filled diamonds) of SAC.
  • FIG. 4 A graph showing induction of Ig secretion in EBV transformed B cells by anti-CD3-stimulated T cell supernatant. Supernatants from mAb 454-activated T cells were incubated at the indicated concentrations for 3 days with 3xl0 3 cells/well AVB-21 (a) , AP3 (b) or EBE (c) EBV- transformed B cell lines.
  • Figure 5 A bar graph showing induction of (a) T cell proliferation and (b) normal B cell Ig secretion by B cells incubated with T cell supernatant by insolubilized and soluble anti-CD3 preparations.
  • Figure 7 A graph summarizing the results of gel filtration chromatography on a Superose 12 sizing column illustrating the molecular weight range of 446- BCDF of anti-CD3-activated T cell supernatant.
  • Figure 7. A graph summarizing the results of Anion-exchange chromatography of BCDF and BCPF active region from Superose 12 column chromatography illustrating fraction having BCPF activity are discernable as single peak while fractions having BCDF activity are discernable as two separate peaks.
  • Figure 8 A graph summarizing BCDF activity obtained by chromatofocused mAb 446-stimulated normal T cell supernatant in the absence of IL-6 activity. Fractions were incubated in the presence (open squares and shaded area) or absence (solid squares) of rabbit anti-IL-6 antiserum.
  • Figure 9 A graph showing the effect of anti gp39 mAb 5C8 on Ig secretion induced by 446-BCDF.
  • Figure 10 A graph showing the synergistic effect of 446-BCDF and anti-CD40 on Ig secretion.
  • FIGs. 11(a) and 11(b) are graphs showing the effect of mAb 929 and mAb MPC11 (isotype control mAb) , respectively, on B cell differentiation.
  • Figure 12. A graph showing the lack of inhibition of mAb on Ig secretion induced by IL-2 and SAC stimulated human peripheral blood B cells.
  • Figure 13 A graph showing the lack of inhibition of mAb 929 on Ig secretion in IL-6 activated CESS cells.
  • Figure 14 A graph showing the lack of inhibition by mAb 929 on Ig secretion induced by pokeweed mitogen stimulated human lymphocytes.
  • Figure 15 A graph summarizing the activity of 446-B cell differentiation factor after application to a mAb 929 affinity column and a control (MPC11) affinity column (unbound material) .
  • FIGs. 16(a) and 16(b) are graphs showing BCDF activity of two different BCDF preparations respectively, which have been eluted from a mAb 929 affinity column.
  • Figs. 17(a) and 17(b) show SDS-PAGE gels of 446-BCDF and 35 S labelled 446-BCDF purified by affinity chromatography.
  • Figure 18 A graph showing the effect of 446-BCDF on Ig secretion by tonsil cells in (a) presence or (b) absence of SAC.
  • Figure 19 A graph showing the polyclonal response of 446-BCDF tonsil cells in the (a) presence or (b) absence of SAC.
  • B cell differentiation factor derived from anti-CD3-stimulated T cells distinct from previously described cytokines, including IL-6, IL-2, IL-4, and IFN-7.
  • the novel method of anti-CD3 stimulation of normal peripheral blood T cells for the production of cytokines is more likely to mimic natural antigen stimulation in a polyclonal fashion and increase the number of ly phokines generated. This method also avoids introducing contaminating mitogens which could be difficult to completely remove.
  • mAb n 446 induces T cell production of a B cell differentiation factor termed "446-BCDF" and a B cell proliferation factor termed "446-BCPF”.
  • the 446-BCDF isolated after anti-CD3 stimulation appears to be secreted by T cells without the requirement for or contamination by monocytes which are a major source of IL-6.
  • Bazin, et al. "Role Of The Macrophage-Derived Hybridoma Growth Factor in the in vitro and in vivo Proliferation Of Newly Formed B Cell Hybridomas", J. Immunol., 139:780 (1987).
  • 446-BCDF is a protein of about 30 thousand daltons, molecular weight having a pi of about 6 and factually and biochemically distinct from IL-6.
  • 446- BCPF is a protein which elutes at a Na + concentration of lOOmM NaCl from an anion exchange column and is distinct from previously described B cell growth factors.
  • Activation of the T cells to produce these novel factors can be done by any known activating factor.
  • the preferred method is to activate using anti-CD-3 monoclonal antibodies.
  • the best results are effected using mAb 446. Therefore, a preferred embodiment is the use of mAb 446 to activate the T cells.
  • 446-BCDF shows both functional and physical analogy with IL-6 however, it is a distinctly different protein with enhanced function.
  • 446-BCDF induces Ig secretion in SAC activated normal B cells (Fig. 2, Fig. 4b) and in EBV-transformed B cell lines (Fig. 3) .
  • Molecular mass determination by gel filtration shows a broad activity peak with an average size at about 30 kDa (Fig. 6) .
  • SDS-PAGE followed by Western blot analysis shows 446-BCDF contains contaminating IL-6, however, the 446-BCDF activity is not inhibited by neutralization of IL-6 with polyclonal anti-IL-6 antiserum (Fig. 7) .
  • anti-CD3-stimulation of normal T cells induces a number of lymphokines including IL-6, a novel BCDF whose activity is not neutralized by anti-IL-6 antiserum and a novel BCPF.
  • lymphokines including IL-6, a novel BCDF whose activity is not neutralized by anti-IL-6 antiserum and a novel BCPF.
  • T cell cultures were progressively depleted of monocytes by plastic adherence and carbonyl iron ingestion. Such an approach results in the inability of T cells to respond to either antigen or mitogen. Mayer et al., "Evidence For A Defect in "Switch" T Cells In patients With Immunodeficiency And Hyperimmunoglobulinemia", N. Engl. J. Med., 314:409 (1986) .
  • 446-BCDF SAC- and/or plastic immobilized mAb 446 anti-CD3 induced both T cell proliferation and 446-BCDF secretion at a comparable level regardless of the number of contaminating monocytes (Table III) , therefore 446-BCDF is a T cell product and not a monokine.
  • 446-BCDF we have detected a factor(s) in the 446 stimulated T cell supernatants capable of inducing resting but not activated B cells to proliferate (Fig. 2), 446-BCPF.
  • This 446-BCPF is clearly separable from 446-BCDF by charge and MW characteristics. Additionally this proliferation factor exhibits a higher MW range than that described for either low (Mehta et al.
  • mAb 446 recognizes a unique CD3 epitope in that it cross-reacts with a cytoplasmic determinant within keratinocytes and certain epithelial cell lines.
  • mAb 446 recognizes a determinant solely on the 26-kDa subunit. Perturbation by mAb 446 of this unique CD3 epitope on the T cell surface may trigger intracellular biochemical pathways different from those resulting from interactions of surface CD3 with mAb directed at other epitopes. Thus, cross-linking another component of the CD3 molecule may transduce a signal resulting in IL-2 secretion whereas cross-linking another component of the CD3 complex may provide an alternate signal for BCDF secretion.
  • T h cells T h l and T h 2
  • secreted lymphokines IL-2 and IFN- ⁇ vs IL-4 and IL-5, respectively
  • Mosmann et al. "Two Types Of Murine Helper T Cell Clones: I. Definition According To Profiles Of Lymphokine Activities And Secreted Proteins", J. Immunol., 136:2348 (1986).
  • mAb 446 appears to induce IL-4, IL-6, BCDF, and IFN- ⁇ activity but not IL-2.
  • the receptor for 446-BCDF may play a role in therapeutics.
  • antibodies which recognize and bind to the receptor could down-regulate the polyclonal activation of B cells due to over production of BCDF as in the case of autoimmune disorders.
  • the present invention also provides for antibodies that inhibit the biological activity of the BCDF of the invention.
  • mAb 929 and mAb 204 described below, inhibit the B cell differentiating activity of 446-BCDF; such mAb may be used in methods of purifying 446-BCDF and may be used to detect the presence of 446-BCDF.
  • Such mAb may, as an example, be used to detect the presence of 446-BCDF in a blood sample taken from a patient, in which the presence of 446-BCDF, in amounts greater relative to the amount present in a normal subject, may correlate with a state of immune system activation such as caused by an infection.
  • aureus Cowan I strain were cultured in the presence of mAb 446 (anti-CD3) stimulated T cell supernatant whereas no significant increase in Ig secretion was noted with either mAb 454- or 147-induced T cell supernatant despite equivalent T cell proliferative responses to these antibodies.
  • IL-2 secretion was detectable in T cell supernatants from T cells stimulated with either mAb 454 or 147 but not 446.
  • Factors promoting B cell proliferation were detected in all antibody-stimulated T cell supernatants but, contrary to BCDF, appear to act only on non-activated B cells.
  • 446-BCDF has an apparent isoelectric point [pi] of about 6, in contrast to the reported pi of 5 for IL-6 and was more acidic than the documented basic pi of IFN-y. Lastly, peaks with BCDF activity were not active in assays for either IL-2 or IL-4. In addition, a rabbit anti-IL-6 heteroantiserum failed to inhibit the pi 6 BCDF (446-BCDF), suggesting that anti-CD3 induced BCDF is distinct from IL-6. Thus, anti-CD3 activated T cells generate both BCPF activity and BCDF as separate molecular entities distinct from IFN-7, IL-2, IL-4, and conventional IL-6.
  • PBMC Peripheral blood mononuclear cells
  • PBS phosphate buffered saline
  • T and non-T cells were further separated by rosetting with neuraminidase-treated sheep erythrocytes, following the method of Mayer et al. , "Human T Cell Hybridomas Secreting Factors For IgA-Specific Help, Polyclonal B Cell Activation and B Cell Proliferation", J. Exp.
  • Resultant T cells were 1% CD14 by FACS analysis.
  • Cells were cultured in RPMI 1640 (GIBCO, Grand Island, NY) , 2mM glutamine (GIBCO) , 100 U/ml penicillin/streptomycin (GIBCO) , and 10% heat inactivated FCS (Hazelton Research Products Inc., Lenexa, KS) henceforth CM.
  • RPMI 1640 GibbCO, Grand Island, NY
  • 2mM glutamine GIBCO
  • 100 U/ml penicillin/streptomycin GRB
  • 10% heat inactivated FCS Hazelton Research Products Inc., Lenexa, KS
  • peripheral blood B cells were immortalized. Briefly, isolated B cells (10 6 /ml) were incubated with an optimal concentration of supernatant containing Epstein Barr
  • EBV Epstein Barr Virus Nuclear Antigen
  • the human lymphoblastoid line CESS was obtained from Dr. Peter Ralph (Cetus Corporation, Emeryville, CA) as the cell line previously demonstrated to respond to IL-6. Hirano et al., "Purification To Homogeneity And Characterization Of Human B-Cell Differentiation Factor (BCDF or BSFp-2), Proc. Natl. Acad. Sci. USA, 82:5490 (1985).
  • BCDF or BSFp-2 BSFp-2
  • Monocyte-depleted T cell preparations were cultured in CM in T25 flasks (Falcon Labware, Lincoln Park, NJ) at 2 x 10 6 cells/ml in the presence of soluble or insolubilized (see below) purified anti-CD3 mAb 446, 147, or 454, as described by Stohl et al., "Induction of T Cell-Dependent B Cell Differentiation By Anti-CD3 Monoclonal Antibodies", J. Immunol., 138:1667 (1987), for 48 hours at 37°C.
  • Insolubilized anti-CD3 mAb was prepared by adsorbing purified anti-CD3 mAb (50 ⁇ g/ml) onto formalin fixed SAC (0.1%) according to the manufacturer's instructions (Pansorbin, Calbiochem, La Jolla, CA) for 2 hours at 4°C. After the incubation, unbound anti-CD3 was removed by washing twice in PBS. The insolubilized anti-CD3 mAb was then used at a final concentration of 0.001% (SAC) with normal monocyte depleted T cells. Control cultures containing untreated SAC alone with T cells were performed in the early experiments without induction of lymphokine secretion over base line. Thus, T cells were stimulated by anti-CD3 monoclonal antibodies.
  • B Cell Growth and Differentiation Assays for B cell proliferation as well as B cell differentiation were performed as previously described by Mayer et al. , "Human T Cell Hybridomas Secreting Factors For IgA-Specific Help, Polyclonal B Cell Activation and B Cell Proliferation", J. Exp. Med., 156:1860 (1982). Briefly, for B cell proliferation, 10 5 monocyte-depleted (by plastic adherence) non-T cells were cultured in triplicate 0.1 ml microwell cultures (Flow Laboratories Inc., McLean, VA) in the presence or absence of test T cell supernatants or column fractions at varying concentrations.
  • Cells were cultured for 96 hours and 1 ⁇ Ci [ 3 H]TdR/well (1CN Radiochemicals, Irvine, CA) was added during the last 18 hours of culture. Cells were harvested onto glass fiber filter mats (Skatron, Sterling, VA) and counts/min determined by liquid scintillation counting. In some cases, SAC (0.001%) was added to cultures along with the test supernatants at the onset of culture.
  • B cells were cultured as above in triplicate 0.1 ml cultures in CM for 144 h. Cell free supernatants were obtained after centrifugation and assayed for Ig secretion by ELISA.
  • IgG, IgA, and IgM secretion were determined in the initial enzyme-linked immunosorbant assay (ELISA), as previously described by Mayer et al. , "Evidence For A Defect In "Switch” T Cells In Patients With Immunodeficiency and Hyperimmunoglobulinemia", N. Engl. J. Med., 314:409 (1986). Thus, B cell proliferation and differentiation assays were performed.
  • Example 5 Assay for IL-2.
  • IL-6 and IFN-7 IL-2 was measured by the ability of T cell supernatant (TCS) to stimulate and maintain the growth of the murine cytotoxic T cell line HT2 or CTLL, as described by Gillis et al. , "T Cell Growth Factor: Parameters Of Production And A Quantitative Microassay For Activity", J. Immunol., 120:2027 (1978).
  • Units of IL-2 activity were determined by probit analysis, as described by Gillis et al., “T Cell Growth Factor: Parameters Of Production And A Quantitative Microassay For Activity", J. Immunol., 120:2027 (1978).
  • IL-4 secretion was determined by the ability of
  • TCS to induce CD23 expression on tonsil and peripheral blood B lymphocytes, according to the method of DeFrance et al., "Human Recombinant IL-4 Induces Fc Receptors (CD23) On Normal Human B Lymphocytes", J. Exp. Med., 165:1459 (1987). Briefly, test supernatants or column fractions were co-cultured with B cells for 18 hours in CM at 37°C. Expression of CD23 was determined by indirect immunofluorescence, according to Mayer et al., "Polyclonal Immunoglobulin Secretion In Patients With Common Variable Immunodeficiency Using Monoclonal B Cell Differentiation Factors", J. Clin.
  • IL-4 but not IL-2 or IFN-7 has been shown to be a potent inducer of CD23 on B cells, DeFrance, et al. (1987) (supra) .
  • IL-6 activity was measured using the B9 hybridoma cell line as described in Matsuda, T., "Establishment of an interleukin 6 (IL-6)/B cell stimulatory factor 2- dependent cell line and preparation of anti-IL-6 monoclonal antibodies", Eur. J. Immunol., 18:251 (1988) .
  • IFN-7 secretion was measured using a standard WISH cell bioassay, and assessing the ability of test supernatants to block VSV infection of WISH cells as previously described by Matricardi et al. , "Interferon Production In Primary Immunodeficiencies", J. Clin. Immunol., 4:388 (1984) .
  • Example 6 Chromatographic Separations 446-BCDF-containing supernatant was fractionated by passage through or elution from various Pharmacia fast protein liquid chromatography (FPLC) columns. Briefly, separations were generally carried out at 4°C with a flow rate of 0.4 ml/min and 0.5 ml column fractions. Molecular sizing was carried out by
  • Pharmacia Superose 12 (HR 10/30) column according to the manufacturer's instructions (Pharmacia, New Brunswick, NJ) using 0.2 ml. concentrated sample volumes and PBS as buffer for a total of 25 ml. Crude sample was concentrated by addition of solid ultrapure ammonium sulfate (Schwarz/Mann Biotech, Cleveland, OH) to saturation on ice, adjusted with NaOH to keep the pH neutral. The suspension was incubated for 2 hours at 4°C, centrifuged at 15,000 rpm for 10 min in a Sorval Centrifuge with an SS-34 rotor and resuspended in PBS. Anion-exchange chromatography was performed with a Pharmacia Mono Q (HR 5/5) column.
  • the sample was dialyzed in 20mM triethanolamine-HCl (TEA) , pH 7.4 before addition to the column. Elution was carried out with a linear gradient of 0 to 0.5 M NaCl/20mM TEA, pH 7.4, in a total volume of 20 ml. Residual protein was washed off the column with 2 ml of 2M NaCl/TEA, pH 7.4, followed by 8 ml of 0.5 M NaCl/TEA, pH 7.4. Then 0.5 ml of the high salt wash was desalted by a Pharmacia Fast Desalting column resulting in an approximate 10-fold dilution of sample.
  • TEA triethanolamine-HCl
  • Chromatofocusing was carried out with Pharmacia PBE 84 resin in an 8 ml (HR 10/10) column. Each sample was dialyzed exhaustively against 0.025 M histidine, pH 6.5, before addition to the column. This column was eluted with Pharmacia Polybuffer 74-HC1 (1/8 dilution), pH 4.0, with a total of 90 ml, 2 ml/fraction. Residual protein was washed off the column with 2 ml of Polybuffer 74-HC1 (1/8 dilution 2M NaCl), pH 4.0, followed by 18 ml of Polybuffer 74-HC1 (1/8 dilution), pH 4.0. The pH of all chromatofocusing fractions was determined prior to desalting as described above.
  • mAb anti-CD3 antibodies 147 (IgGl), 446 (IgGl), and 454 (IgG2a) each induced similar proliferative responses at 48 and 72 h. However, supernatants from these cultures displayed differences in lymphokine activity. IL-2 activity was substantial in culture supernatants of T cells stimulated by mAb 454 (Table I) although lower than that observed in phytohemagglutinin (PHA) -stimulated supernatants. mAb 147-stimulated TCS contained lesser amounts of IL-2 activity but, with the exception of one donor (no.
  • IL-2 activity was detectable in each experiment. In contrast, IL-2 activity was never detected in TCS from T cells stimulated with mAb 446. In other experiments using purified anti-CD3 mAb, culture supernatants of PBMC stimulated with mAb 446 at 10 to 1000 ng/ml never had detectable IL-2 activity whereas IL-2 activity was readily detectable in parallel supernatants of mAb 454 stimulated PBMC. No supernatants from mAb 446-stimulated cultures harvested from 3-48 hours contained detectable IL-2 activity.
  • Table I shows the results from five donors. Samples of PBMC were stimulated with anti-CD3 mAb, PHA, or medium alone for 24 h. The culture supernatants were harvested and assayed for IL-2 activity. Table II shows the induction of CD23 on tonsillar B cells by anti-CD3 stimulated TCS. In the target sample a total of 5x10 s isolated tonsillar B cells were cultured in CM in the presence or absence of 10% anti-CD3 stimulated TCS for 18 hours at 37°C. Cells were harvested and stained for the presence of CD23 by indirect immunofluorescence as described in example 5.
  • Figures la and lb show the T cell proliferation activity and induction of Ig secretion in normal B cells by anti-CD3 mAb preparations as described in previous examples.
  • TRF T Cell Replacing Factor
  • Figure 3 shows the induction of proliferation of normal B cells by anti-CD3-stimulated TCS.
  • B cells were incubated in the presence (black squares) or absence (black triangles) of SAC.
  • Proliferation was measured by incubation of supernatant with 1 x 10 5 normal B cells/well for two days, [ 3 H] TdR was added and the cells were harvested for scintillation counting on day three.
  • Stimulation index was calculated by determining the fractional increase of [ 3 H]TdR incorporation of the samples at the indicated supernatant concentration over that of medium control.
  • FIG. 4 shows induction of Ig secretion in EBV transformed cells by anti-CD3-stimulated TCS.
  • Supernatants from mAb 446 activated T cells were incubated at the indicated concentrations for three days with 3 x 10 5 cells/well AVB-21 (a) , AP3 (b) , or EBE (c) EBV transformed cell lines. On day three the cell lines were assayed for Ig secretion by ELISA.
  • 446-BCDF was combined with 446-induced TCS and added to cultures of three EBV-transformed B cell lines generated in this laboratory.
  • mAb 446-induced BCDF was capable of sustaining both IgM and IgG responses consistent with a single factor acting in an isotype non-restricted fashion (Figs. 3 and 4).
  • Freshly isolated tonsillar B cells secreted IgM and IgG in response to mAb 446-BCDF whereas peripheral blood B cells secreted predominantly IgG.
  • mAb-446 was immobilized onto SAC and used to stimulate T cells.
  • the response of T cells to soluble anti-CD3 mAb has been shown to be monocyte and FcR-dependent. Van Wauwe et al., "Mitogenic Actions Of Orthoclone 0KT3 On Human Peripheral Blood Lymphocytes: Effects Of Monocytes And Serum Components", Int. J.
  • FIG. 5 shows the induction of T cell proliferation and normal B cell Ig secretion by insolubilized and soluble anti-CD3 preparations.
  • TCS were prepared by incubation of normal T cells with mAb 446 preparations: (a) insolubilized 446 mAb (shadowed area) and SAC alone (solid area) ; (b) soluble mAb 446 (shadowed area) and no addition (solid area) .
  • normal T cells were stimulated with mAb 446 preparations for two days at 2 x 10 6 cells/ml, incubated with [ 3 H]TdR and harvested for scintillation counting on day three.
  • normal B cells were incubated with supernatant from mAb 446 activated T cells for 6 days in the presence of SAC and assayed for Ig secretion by ELISA.
  • insolubilized 446 was twofold more effective in inducing T cell proliferation than soluble anti-CD3 mAb. This increase in proliferation was correlated with an increase in BCDF activity (Fig. 5b) .
  • SAC-stimulated T cells did not secrete BCDF above baseline suggesting that CD3 crosslinking was necessary for the activation and lymphokine secretion and that protein A leaching off the SAC was not responsible for the demonstrated effects.
  • an i-CD3 mAb were bound to plastic and used to stimulate T cells the results obtained were identical to those described with SAC crosslinking, supporting the concept that T cells alone, and not SAC and T cells, are responsible for the BCDF activity.
  • T cells are the source of 446-BCDF obtained from the cultures. This was more rigorously demonstrated in cultures where monocytes had been depleted by plastic adherence and carbonyl iron ingestion and magnetic removal. Despite the sequential decrease in monocytes in these cultures, 446-BCDF secretion remained constant and T cell proliferation was unchanged (Table III) . These data support the concept that mAb 446-induced BCDF and IL-6 are distinct molecular entities.
  • Table III shows 446-BCDF activity with progressive monocyte depletion.
  • T cells were isolated as described in example 1 by SRBC rosetting and density gradient centrifugation as described above. After isolation, T cells were further purified (monocyte depletion) by sequential plastic adherence (1 hour in a T75 flask in CM at 37°C) and subject to carbonyl iron ingestion with removal of iron laden cells with a magnet. Isolated and monocyte depleted T cells were then analyzed for the presence of monocytes (CD14) by indirect immunofluorescence. T cells were then stimulated with the SAC immobilized mAB 446 for 72 hours in triplicate microwell cultures.
  • FIG. 6 shows gel filtration chromatography of anti-CD3 activated TCS.
  • mAb 446-activated TCS was concentrated by ammonium sulfate precipitation, dialyzed against PBS and applied to a Pharmacia
  • the eluted BCDF activity had a MW of approximately 30,000 Da. which is within the range of MW for the family of IL-6 proteins. May et al. , "Synthesis And Secretion Of Multiple Forms of "b2-Interferon/B Cell Differentiation Factor 2/Hepatocyte Stimulating Factor” By Human Fibroblasts And Monocytes", J. Biol. Chem., 263:7760 (1988) . B cell proliferation was noted only in “resting" B cells (Fig. 2) with activity observed at higher (although partly overlapping) MW. BCDF containing fractions, nos.
  • Figure 7 shows the results of anion-exchange chromatography of the BCDF active region from Superose 12 column chromatography.
  • Pooled fractions 26-31 from Superose 12 column chromatography ( Figure 6) were dialyzed against 20mM triethanolamine-HCl, pH 7.4, applied to a Pharmacia Mono Q fast protein liquid chromatography column and eluted with a linear 0 to 0.5 M NaCl gradient.
  • Proliferation (dotted square) was measured by incubation of fractions at 10% with 1 x 10 s normal B cells/well for two days, [ 3 H]TdR was added and the cells were harvested for scintillation counting on day three.
  • Ig secretion black diamond was measured by incubation of the fractions at 10% with 1 x 10 3 normal B cells/well co-cultured with SAC for 6 days and supernatant assayed by ELISA.
  • the proliferation factor activity came off as a single peak inducing an 8 fold increase in [ 3 H]TdR incorporation, whereas the BCDF activity was observed to reside in two discrete areas of activity, flanking B cell proliferation activity (two- and fivefold increases in Ig secretion, respectively) .
  • 446-BCDF and 446-BCPF activities were discernible as two distinct molecular entities.
  • FIG 8 shows BCDF activity by chromatofocused mAb 446-stimulated normal TCS in the absence of IL-6 activity.
  • Insolubilized mAb 446-stimulated TCS was concentrated by saturated ammonium sulfate, dialyzed in 25mM histidine, pH 6.5, and applied to a Pharmacia PBE 96 resin containing FPLC column. The resin was eluted with Polybuffer 74, pH 4.0 and fractions were desalted by a Pharmacia Fast Desalting column according to the manufacturer's instructions.
  • T cells in the presence or absence of monocytes can secrete a BCDF distinct from IL-6 which polyclonally induces normal activated B cells to undergo terminal maturation to Ig-secreting cells.
  • CD40 and its ligand gp39 have been identified and cloned (Noelle et al. , "CD40 and its ligand, an essential ligand-receptor pair for thymus- dependent B cell activation", Immunol. Today, 13:431 (1992)) . It has also been found that the interaction of CD40 on the surface of B cells and its ligand on the surface of activated T cells triggers B cell proliferation and Ig secretion (Armitage et al. , "Human B cell proliferation and Ig secretion induced by recombinant CD40 ligand are modulated by soluble cytokines", J. Immunol., 150:3671 (1993); Splawski et al.
  • mAb 5C8 in the range of .01 to 10 ⁇ g/ml were adding to corresponding wells containing 10% 446- BCDF. After incubating the mixture at 37°C for 2 hours SAC-activated PB B cells (10 5 PB B cells, 0.001% v/v SAC) were added. Ig secretion was measured by ELISA after seven days. As shown in Fig. 9, 446-BCDF induced a 7-fold increase in Ig secretion by B cells. Moreover, mAb 5C8 did not significantly block 446-BCDF activity.
  • mAb 5C8 completely inhibits anti-CD3 stimulated T cell induction of B cell differentiation in a coculture system (Y.D. Kuang, personal communication) . This indicates that soluble CD40L is not present in 446-BCDF preparations and does not contribute to 446-BCDF mediated B cell differentiation.
  • the IL-6 responsive CESS cell line does not respond to 446-BCDF, while lines AV21 and AP3 differentiate only in the presence of 446-BCDF and not IL-6. This difference can be exploited by the assumption that each cell bears a receptor for the specific BCDF to which it responds. Since Kishimoto et al., "IL-6 receptor and mechanism of signal transduction", Int. J. Immunopharmacology, 14:431 (1992) have recently cloned the IL-6R we can compare and contrast the 446-BCDF receptor, obtained by mAb detection or crosslinking, with that of IL-6. For example, a crosslinking agent may be added to a suspension of purified 446-BCDF and B cells.
  • An mAb specific for 446-BCDF such as mAb 929 or 204 may then be used to isolate the 446-BCDF/BCDF receptor complex from the mixture as described herein below in Example 19.
  • the BCDF receptor may then be separated from the complex by hydrolyzing or destroying the crosslink.
  • PB T Peripheral blood T cells (2-4xl0 6 /ml) were stimulated with purified anti-CD3 mAb (1 ⁇ g/ml) in phenol red free RPMI 1640 (Gibco, Grand Island, NY) , 1% bovine serum albumin (BSA) (Sigma, St. Louis, MO) , 1% penicillin/streptomycin (Gibco) , and 2mM glutamine (Gibco) for 48 hours.
  • T cell supernatants (TCS) were concentrated 20-50 fold by Amicon filtration (MW cutoff 10,000-Danvers, MA), and passed over an anti-IL6 affinity column (Amgen, Thousand Oaks, CA) at least three times to remove residual IL-6.
  • IL-6 activity was assessed by the absence of proliferation in a B9 cell line bioassay.
  • IL-6 depleted, concentrated TCS was dialyzed against lOmM Na 2 P0 4 (Sigma) and passed over a DEAE Sepharose column (Whatman) .
  • Semi-purified 446-BCDF was eluted in the 50mM salt fraction as described in Sherris, D., et al. , "Characterization of lymphokines mediating B cell growth and differentiation from monoclonal anti-CD3 antibody stimulated T cells", J. Immunol. 142:2343 (1989) .
  • the resultant enriched preparation was devoid of IL-2, IL-4, 7-IFN, and IL-6 activity measured by bioassay or ELISA.
  • IL-2, IL-4 and IL-6 activity was measured as in Example 5.
  • 7-IFN activity was measured by ELISA (Endogen, Boston, MA) .
  • This preparation was used in B cell differentiation assays described below, for immunization in the generation of anti-BCDF mAbs described below, or for further purification by affinity chromatography as described herein below.
  • hybridoma clones were selected in hypoxanthine/ aminopterin/thymidine (HAT) medium.
  • HAT hypoxanthine/ aminopterin/thymidine
  • Three levels of screening of growth positive wells were performed.
  • First concentrated crude mAb 446-stimulated TCS was allowed to air dry on ELISA plates (Nunc Immulon II) . After washing in PBS/Tween 20 (0.1%/sodium azide (0.02%), the plates were blocked with PBS/1% BSA for l hour at 22°C followed by incubation with test hybridoma supernatants (dilution # in PBS/Tween wash buffer) for 4 hours.
  • alkaline phosphate conjugated goat anti-mouse Ig (polyspecific, heavy and light chain - 10 ⁇ g/ml in PBS/Tween buffer - Tago, Burlingame, CA) was added for an additional 1 hour, and finally, substrate 104 (50 ⁇ g/ml - Sigma) was added. The color change was recorded by a MicroElida reader (Physica, NY) . Clones demonstrating positive binding in the ELISA assay were then tested for their ability to inhibit Ig secretion by normal B cells induced by partially purified 446-BCDF. Hybridoma supernatants (20% vol/vol) were added to 1x10 s SAC (Pansorbin, Calbiochem, Irvine, CA - 0.001% v/v) activated B cells (in 80 ⁇ l
  • CM in triplicate microwell cultures for 7 days in the presence or absence of partially purified 446-BCDF at a concentration previously shown to be optimal (10% v/v) for the induction of Ig secretion
  • Sherris, D., et al. "Characterization of lymphokines mediating B cell growth and differentiation from monoclonal anti-CD3 antibody stimulated T cells", J. Immunol. 142:2343 (1989) .
  • Total Ig secretion was measured by ELISA as previously described in Sherris et al., J. Immunol. 142:2343 (1989) .
  • Inhibition of induced Ig secretion was assessed by comparison of mAb treated cultures with irrelevant isotype control treated cultures.
  • Clones exhibiting >30% inhibition of BCDF induced Ig secretion were expanded, subcloned by limiting dilution and re- analyzed using varying concentrations of hybridoma supernatant in a BCDF assay. Inhibitory clones were further analyzed for their effects on B cell viability as assessed by Trypan Blue exclusion, their inability to stain isolated T cells, B cells or monocytes using staining procedures previously described for hybridoma supernatants, Gottling, A., et al., "A membrane protein selectively expressed on normal ly phoid cells cross reacting with epithelial stem cells", J. Amer. Acad. Derm.
  • the remaining two, 929 (IgG 2b ) and 204 (IgG 2 ,) demonstrated inhibition of 446-BCDF activity without evidence of toxicity or staining of PBMC.
  • IL-2. IL-6, PWM. and 446-BCDF bv mAb 929 lxlO 5 B cells were co-cultured with either SAC (0.001% v/v) in the presence of IL-2 (Boehringer Mannheim, Indianapolis, IN) or lxlO 3 T cells plus pokeweed mitogen (PWM) (Gibco) in triplicate 100 ⁇ l microwell cultures for 7 days in the presence or absence of Protein G (Pharmacia, Piscataway, NJ) purified mAb 929 or mAb 204 described herein below as follows. Ig secretion was assessed by ELISA as described in Sherris, D., et al. , "Characterization of lymphokines mediating B cell growth and differentiation from monoclonal anti-CD3 antibody stimulated T cells", J. Immunol. 142:2343 (1989).
  • Human peripheral blood B cells (1 x 10 5 ) were co- cultured with SAC (0.001%) and 446-BCDF (25% v/v).
  • mAb 929 and isotype control mAb MPCll were added at the onset of culture. Seven days later, the supernatants were assayed for Ig secretion by ELISA. As shown in Fig. 11(a), the addition of purified mAb 929 to SAC plus 446-BCDF stimulated normal B cells was capable of inhibiting terminal differentiation at concentrations as low as 0.6 ⁇ g/ml. A concentration of 0.6 ⁇ g/ml resulted in 30% inhibition, while 16 ⁇ g/ml resulted in 73% inhibition. In contrast as shown in Fig. 11(b), the addition of an isotype matched IgG 2b control, MPCll had no significant inhibitory effect.
  • mAb 929 The inhibition by mAb 929 appears to be specific to 446-BCDF in that mAb 929 had no effect on SAC plus IL-2 induced Ig secretion.
  • Human peripheral blood B cells (1 x 10 5 ) were also cultured with IL-2 (10 U/ml) and SAC (0.001%).
  • mAb 929 did not inhibit IL-2 induced Ig secretion. Furthermore it was found that mAb 929 had no effect on the IL-2 induced proliferation of CTLL cells. Thus it is clear that mAb 929 and mAb 204 specifically inhibit the ability of 446-BCDF to promote B cell differentiation.
  • Human peripheral blood B cells (1 x 10 3 ) were also cultured for seven days with 1 x 10 s T cells plus pokeweed mitogen (PWM) (1 ⁇ g/ml) .
  • PWM pokeweed mitogen
  • Anti-BCDF mAb 929 or isotype control MPCll (10 ⁇ g/ml) were added at onset of culture. The supernatants were assayed for Ig secretion by ELISA. The results are shown in Fig. 14. As was the case with respect to IL-2, Ig secretion by B and T cells plus PWM was equally unaffected by mAb 929. This provides strong evidence that mAb 929 was not mediating its inhibitory effect by nonspecific suppression or toxicity. Lastly, CESS cells were cultured with IL-6
  • IL-6 is a weak stimulus for Ig secretion by human peripheral blood B cells
  • rIL-6 40 U/ml was added to slg + CESS cells as described in Raynal, MC, et al. , "Interleukin 6 induces secretion of IgG ! by coordinated transcriptional activation and differential mRNA accumulation. Proc Natl Acad Sci, 86:8024 (1989) (10 4 /well in triplicate microwell cultures) . Cell free supernatants were analyzed for inhibition of IL-6 induced IgG- secretion by ELISA. As can be seen in Fig.
  • mAb 929 had no effect on IL-6 induced Ig secretion by CESS cells, nor was an effect observed on the IL-6 induced proliferation of B9 cells. Thus in three distinct B cell differentiation systems, mAb 929 demonstrated no inhibitory effect indicating that this mAb is 446-BCDF specific in that it specifically inhibits the ability of 446-BCDF to promote B cell differentiation.
  • Example 18 Preparation of a mAb 929 Affinity Column Purified mAb 929 or an IgG 2b isotype control were bound to CnBr activated Sepharose 4B beads available from Pharmacia. Protein binding was determined by measuring absorbence at 280A (Aj g0 ) of the residual antibody solution. Each preparation of beads represented >90% protein binding. An 8 ml column was packed in a syringe and washed extensively with .1 M glycine HC1 at pH 2.8 available from Sigma and phosphate buffered saline (PBS) prior to use.
  • PBS phosphat
  • Example 19 Absorption of 446-BCDF by mAb 929 Affinity Column Concentrated TCS (0.5 ml) containing 446-BCDF prepared according to Example 15 was passed over either an mAb 929 Sepharose 4B or isotype control MPCll (IgG 2b ) Sepharose 4B column as described in Example 18. The concentrated BCDF was passed over the column at least 3 times and the flow through was collected. Fractions of 0.3 ml were collected and tested for their ability to stimulate Ig secretion by SAC activated peripheral blood B cells. As seen in Fig. 15, BCDF activity in the flow through was significantly decreased. A maximum of 17% of total activity was observed after passage over the 929 column when compared to the isotype control column.
  • PBS phosphate buffered saline
  • a second fainter band was occasionally seen at 29 kDa.
  • This band could represent an alternative form of 446-BCDF resulting from differences in glycosylation, sulfation, etc. or some associated molecule. Elution of protein from the gel to test for bioactivity and determination of the properties of the 44 kDa and 29 kDa band could not be performed since the bands were only seen in TCA precipitates, a procedure which would denature the protein and destroy bioactivity. No bands were seen in the flow through or eluate from the isotype matched mAb control column. Thus from 400-800 ml of TCS, only a small band was detected by silver staining. Therefore, as is true for many previously described cytokines, 446-BCDF appears to be an extremely potent factor exhibiting significant activity at extremely low protein concentrations.
  • 446-BCDF enhances Ig secretion by SAC-activated PB B cells.
  • 10% ⁇ ⁇ / ⁇ ) of either isotype control TCS or 446-BCDF in the presence of SAC (0.001% ⁇ / ⁇ ) were used to stimulate PB B cells.
  • the supernatants were harvested and the total Ig and Ig isotype secretion was measured by ELISA.
  • the results expressed as the mean of triplicate cultures are shown in Table IV.
  • the stimulation index reported is the ratio of the amount of Ig induced by 446-BCDF over the amount of Ig induced by control TCS.
  • the Ig secretion induced by control TCS was not significantly higher than baseline Ig.
  • 446-BCDF induced a 7- to 123-fold increase in Ig secretion by SAC-activated PB B cells compared to isotype control stimulated TCS. Moreover, 446-BCDF induced a 6- to 141-fold increase in IgG secretion, a 22- to 362-fold increase in IgM secretion, 5- to 98-fold increase in IgA secretion, but no significant IgE secretion.
  • the IgE level of both control TCS and BCDF groups were lower than 0.5 ⁇ g/ml.
  • the ability of 446-BCDF to induce Ig secretion in stimulated and unstimulated human tonsil cells was determined as follows. Purified tonsil B cells (10 s cells/well) were cultured in triplicate with 446- BCDF (10% ⁇ / ⁇ ) in the presence or absence of 0.001% ⁇ / ⁇ SAC. After seven days, Ig secretion was measured by ELISA. As shown in Fig. 18 human tonsil cells respond to 446-BCDF both in the presence (Fig. 18A) and absence (Fig. 18B) of SAC. This may be due to the fact that tonsil cells are generally considered to be preactivated.
  • Ig isotypes were analyzed. As shown in Fig. 19 as with PB B cells, Ig secretion by 446 -BCDF tonsil cells was also polyclonal both in the presence or absence of SAC.
  • IgG ( ⁇ g/ml) IgM ( ⁇ g/ml) IgA ( ⁇ g/ml)
  • S.I. is stimulation index.
  • 446-BCDF induced an 8- to 44-fold increase in IgG secretion, 3- to 67-fold increase in IgM secretion, and a 6- to 27-fold increase in IgA secretion.

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Abstract

On obtient des nouveaux facteurs de différentiation et de croissance des lymphocytes B à partir de lymphocytes T activés par l'anticorps monoclonal dirigé contre CD3. Des anticorps monoclonaux spécifiques dirigés contre lesdits facteurs, utilisés pour la détection et la purification par chromatographie d'affinité sont également décrits.
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WO2021026312A1 (fr) * 2017-02-17 2021-02-11 Todaro George J Utilisation de polypeptides tgf-alpha ou d'anticorps anti-tgf-alpha pour le traitement de maladies et de troubles

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999007870A1 (fr) * 1997-08-11 1999-02-18 Chiron Corporation Methodes de modification genetique de lymphocytes t
US6114113A (en) * 1997-08-11 2000-09-05 Chiron Corporation High efficiency genetic modification method
WO2021026312A1 (fr) * 2017-02-17 2021-02-11 Todaro George J Utilisation de polypeptides tgf-alpha ou d'anticorps anti-tgf-alpha pour le traitement de maladies et de troubles
US11279753B2 (en) 2017-02-17 2022-03-22 George J. Todaro Use of TGF-alpha polypeptide or anti-TGF-alpha antibodies for the treatment of diseases and disorders

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