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WO1999027963A1 - Procedes de fabrication d'anticorps en rapport avec la rupture de la tolerance peripherique dans les lymphocytes b - Google Patents

Procedes de fabrication d'anticorps en rapport avec la rupture de la tolerance peripherique dans les lymphocytes b Download PDF

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Publication number
WO1999027963A1
WO1999027963A1 PCT/US1998/025253 US9825253W WO9927963A1 WO 1999027963 A1 WO1999027963 A1 WO 1999027963A1 US 9825253 W US9825253 W US 9825253W WO 9927963 A1 WO9927963 A1 WO 9927963A1
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Prior art keywords
antibody
mice
cells
animal
antibodies
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Thomas F. Tedder
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Duke University
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Duke University
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Priority to US09/555,349 priority Critical patent/US6921846B1/en
Priority to AU16087/99A priority patent/AU1608799A/en
Publication of WO1999027963A1 publication Critical patent/WO1999027963A1/fr
Anticipated expiration legal-status Critical
Priority to US11/188,577 priority patent/US20060021069A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the subject invention relates generally to a method for the production of monoclonal antibodies. More particularly, the subject invention utilizes an animal having antibody-forming cells, such as B lymphocytes, with disrupted peripheral tolerance. Preferably, the animal comprises a transgenic animal.
  • the invention also provides a method for the use of such monoclonal antibodies, and polyclonal antibodies derived from an animal having antibody-forming cells, such as B lymphocytes, with disrupted peripheral tolerance, for in vitro and in vivo clinical diagnostics and therapeutics.
  • C complement usually followed by a number from 1 to 9 when referencing the factors of the complement system in the immune system
  • PrPc a normal prion protein epitope PrPSc - a disease related prion protein epitope sHEL - soluble hen egg lysozyme
  • the hybridoma secreted a single type of immunoglobulin; moreover, like the myeloma cells, the hybridoma had the potential for indefinite cell division.
  • the combination of these two features offered distinct advantages over conventional antisera.
  • Antisera derived from vaccinated animals are variable mixtures of polyclonal antibodies which never can be reproduced identically.
  • Monoclonal antibodies are highly specific immunoglobulins of a single type.
  • the single type of immunoglobulins secreted by a hybridoma is specific to one and only one antigenic determinant, or epitope, on the antigen, a complex molecule having a multiplicity of antigenic determinants.
  • an antigenic determinant may be one of the many peptide sequences (generally 6-7 amino acids in length; Atassi, M. Z. (1980) Molec. Cell. Biochem. 32:21-43) within the entire protein molecule.
  • monoclonal antibodies raised against a single antigen may be distinct from each other depending on the determinant that induced their formation. For any given hybridoma, however, all of the antibodies it produces are identical. Furthermore, the hybridoma cell line is easily propagated in vitro or in vivo, and. yields monoclonal antibodies in extremely high concentration.
  • a monoclonal antibody can be utilized as a probe to detect its antigen.
  • monoclonal antibodies have been used in in vitro diagnostics, for example, radioimmunoassays and enzyme-linked immunoassays (ELISA), and in in vivo diagnostics, e.g. in vivo imaging with a radio-labeled monoclonal antibody.
  • ELISA enzyme-linked immunoassays
  • a monoclonal antibody can be utilized as a vehicle for drug delivery to such antibodies' antigen.
  • This procedure is carried out by screening the hybridomas that are formed to determine which hybridomas, if any, produce a monoclonal antibody that is capable of binding to the target antigen.
  • This screening procedure can be very tedious in that numerous, for example, perhaps several thousand, monoclonal antibodies may have to be screened before a hybridoma that produces an antibody that is capable of binding the target antigen is identified. Accordingly, there is a need for a method for the production of monoclonal antibodies that increases the likelihood that the hybridoma will produce an antibody to the target antigen.
  • the immune systems of conventional animals used in the production of monoclonal antibodies cannot recognize epitopes that are highly conserved among vertebrate, and particularly mammalian species, as "non- self because of "self tolerance.
  • a method for the production of monoclonal antibodies to an antigen comprising:
  • the subject invention also provides methods for utilizing a monoclonal antibody or a polyclonal antibody derived from an animal having antibody-forming cells with disrupted peripheral tolerance.
  • the present invention provides a process of detecting an antigen, wherein the process comprises immunoreacting the antigen with an antibody prepared according to the process described above to form an antibody-polypeptide conjugate, and detecting the conjugate.
  • the present invention contemplates a diagnostic assay kit for detecting the presence of an antigen in a biological sample, where the kit comprises a first container containing a first antibody capable of immunoreacting with antigen, with the first antibody present in an amount sufficient to perform at least one assay, and wherein the antibody is produced by the process described above.
  • an assay kit of the invention further comprises a second container containing a second antibody that immunoreacts with the first antibody, wherein the second antibody is produced by the processes described above.
  • the antibodies used in an assay kit of the present invention are monoclonal antibodies.
  • the first antibody is affixed to a solid support.
  • the first and second antibodies comprise an indicator.
  • the indicator is a radioactive label or an enzyme.
  • the present invention contemplates a diagnostic assay kit for detecting the presence, in a biological sample, of an antibody immunoreactive with an antigen, the kit comprising a first container containing the antigen that immunoreacts with the antibody, with the antigen present in an amount sufficient to perform at least one assay, and wherein the antibody is produced by the processes described above.
  • the present invention contemplates a method of producing a non-human animal with an immune system having cells with a predetermined characteristic.
  • the method comprises the steps of:
  • step (b) obtaining another animal having immune system cells with either the same or a different characteristic from the animal of step (a);
  • step (c) breeding the animal of step (a) with the animal of step (b) to produce an animal with an immune system having cells with a predetermined characteristic.
  • Figure 1 depicts anti-HEL lgM a antibody levels in lg HEL and sHEL/lg HEL mice that overexpress CD19.
  • Each value indicates serum levels of HEL-specific lgM a from individual 2-month-old (2 mo) or 5- to 10-month old (>5 mo) mice measured by ELISA.
  • Horizontal bars indicating mean anti-HEL antibody concentrations for each group are provided for reference.
  • the dashed horizontal line (arrowhead) delimits the 95% confidence interval for the log normal distribution of anti-HEL antibody levels observed in unimmunized 2-month-old sHEL/lg HE mice as described in Materials and Methods.
  • Figure 2 depicts humoral immune responses of (A) sHEL/lg HE and (B) lg HE mice that overexpress CD19 in response to immunization with HEL.
  • Two-month-old mice were injected i.p. with HEL or PBS mixed with CFA on days 0 and 21 (arrows), and were bled at the indicated times.
  • Levels of serum anti-HEL lgM a antibodies for individual mice were determined by ELISA.
  • Mean antibody levels are shown as solid (hCD19 +/+ ) or dashed (hCD19 " ⁇ ) lines. The dashed horizontal lines (arrowhead) delimit the 95% confidence interval for the log normal distribution of anti-HEL antibody levels observed in unimmunized sHEL/lg HEL mice.
  • Figure 3 depicts signal transduction through surface IgM and CD19 in B cells from (A)sHEL/lg HEL /hCD19 +/+ or (B) lg HEL /hCD19 + + mice.
  • Relative [Ca + levels were assessed by flow cytometry after gating on the B220 + population of indo-1 loaded splenocytes. Baseline fluorescence ratios were collected for 1 min before HEL and/or specific monoclonal antibodies were added (arrows) at final concentrations of: HEL, 100 ng/ml; anti-mouse CD19, 40 ⁇ g/ml; anti-human CD19, 40 ⁇ g/ml.
  • Figure 4 depicts affinity measurements of anti-NP antibodies from hybridomas of CD19TG and CD19KO mice compared with affinities of antibodies generated wild-type C57BL/6 mice.
  • Representative anti-NP antibodies were purified and their affinities for NP (Ka) measured by fluorescence quenching (bars).
  • the days following NP immunization that the antibodies were isolated from mice is indicated.
  • the thick vertical line indicates the lower limit of detection in the fluorescence quinch assay and the thin vertical line indicates the average Ka for anti-NP antibodies generated by germinal center B cells.
  • Figure 5 depicts reactivity of anti-NP antibodies with self antigens.
  • Figure 5A depicts hybridoma supernatant fluid was assessed for reactivity with ssDNA by ELISA.
  • P r mice was used as a positive control.
  • Values represent mean OD values ( ⁇ SD) from triplicate wells. Similar results were obtained in three independent experiments.
  • Figure 5B depicts reactivity of purified TG7-83 antibody with ssDNA compared with two established anti-ssDNA antibodies of the lgG1 isotype (452s.69 and 165s.3g). Reactivity was significantly higher ( p ⁇ 0.05, * p ⁇ 0.01 ) than the negative control antibodies (B1-8 or TG18-161 ).
  • transgenic mouse models for autoreactive B cells provide a mechanism for determining the role of CD19 signaling in regulating peripheral tolerance in autoimmunity.
  • the CD19 cell surface molecule regulates signal transduction events critical for B lymphocyte development and humoral immunity. Increasing the density of CD19 expression renders B lymphocytes hyper-responsive to transmembrane signals, and transgenic mice that over-express CD19 have increased levels of autoantibodies.
  • CD19 The role of CD19 in tolerance regulation and auto-antibody generation was therefore examined by crossing mice that overexpress a human CD19 transgene with transgenic mice expressing a model autoantigen (soluble hen egg lysozyme, sHEL) and high-affinity HEL-specific lgM a and lgD a (lg HE antigen receptors).
  • a model autoantigen soluble hen egg lysozyme, sHEL
  • HEL-specific lgM a and lgD a lg HE antigen receptors
  • B cells in sHEL/lg HEL double-transgenic mice are functionally anergic and do not produce autoantibodies.
  • overexpression of CD19 in sHEL/lg HE double-transgenic mice resulted in a breakdown of peripheral tolerance and the production of anti-HEL antibodies at levels similar to those observed in lg HEL mice lacking the sHEL autoantigen. Therefore, altered signaling thresholds due to CD19 overexpression resulted in the breakdown of peripheral tolerance.
  • CD 19 overexpression shifts the balance between tolerance and immunity to autoimmunity by augmenting antigen receptor signaling.
  • the immune system of the CD19 over-expressing transgenic mouse and particularly the B lymphocytes of the mouse's immune system, recognize the highly conservative mammalian epitope as a particle foreign to the mouse's system ("non-self), which would instigate an immune response.
  • This immune response is developed to produce monoclonal antibodies as described more fully herein.
  • the demonstrated ability to break down peripheral tolerance as described herein and the breeding experiments described herein provide a method for manipulating the immune system of an animal such that an animal having an altered immune system with desired characteristics can be produced, as more fully described in Example 3.
  • immune system includes all the cells, tissues, systems, structures and processes, including non-specific and specific categories, that provide a defense against "non-self molecules, including potential pathogens, in an animal.
  • the non-specific immune system includes phagocytositic cells such as neutrophils, monocytes, tissue macrophages, Kupffer cells, alveolar macrophages and microglia.
  • the specific immune system refers to the cells and other structures that impart specific immunity within a host. Included among these cells are the lymphocytes, particularly the
  • B cell lymphocytes and the T cell lymphocytes include natural killer (NK) cells.
  • NK natural killer
  • antibody-producing cells like B lymphocytes, and the antibodies produced by the antibody-producing cells are also included within the term "immune system".
  • peripheral lymphoid tissues refer to the lymph node-, spleen-, or gut- associated lymphoid tissues wherein cells, such as B lymphocytes, of the immune system are developed.
  • peripheral in the context of the term “peripheral tolerance” indicates a tolerance, or failure to recognize an antigen, by a cell of the immune system, such as a B lymphocyte, in the peripheral lymphoid tissues wherein such cells usually react with antigens.
  • damaged peripheral tolerance means any manipulation or alteration of the peripheral tolerance of the antibody-producing cells of the immune system.
  • damaged peripheral tolerance is meant to refer to the break down of peripheral tolerance, which facilities monoclonal antibody production in accordance with the methods of the present invention.
  • the term "anergy” means a condition in which the immune system of an animal fails to respond to the injection of an antigen.
  • peripheral anergy means a condition in which the peripheral immune system of an animal fails to respond to the injection of an antigen.
  • autoantibody means an antibody formed against an epitope native to the animal.
  • antibody-producing cell refers to any antibody-producing cell within the immune system. Preferably, it is meant to refer to B lymphocytes.
  • complement is meant to refer to the non-specific defense system that is activated by the bonding of antibodies to antigens and by this means is directed against specific invaders that have been identified by antibodies. Eleven complement proteins have been characterized in the field and are generally referred to by those having ordinary skill in the art as C1-C9. The complement proteins act generally along a cascade wherein they contribute to (1 ) recognition (C1 ); (2) activation (C4, C2, and C3, in that order); and (3) attack (C5-C9).
  • complement proteins attach to the cell membrane and destroy the victim cell in a process known as complement fixation.
  • the complement system is well known in the art and is more fully described in Fox, Human Physiology, William C. Brown Pub., DuBuque, Iowa (1987).
  • human immunity is meant to refer to the form of acquired immunity in which antibody molecules are secreted in response to antigenic stimulation.
  • cell-mediated immunity is meantto refertothe immunological defense provided by T cell lymphocytes, which come into close proximity to their victim cells.
  • B cell lymphocytes or “B lymphocytes” are meant to refer to a type of lymphocyte that can be transformed by antigens into plasma cells that secrete antibodies, and are thus responsible for humoral immunity.
  • T cell lymphocytes is meant to refer to a type of lymphocyte that provides cell-mediated immunity, in contrast to B lymphocytes that provide humoral immunity to the secretion of antibodies.
  • cytotoxic There are three sub- populations of T cells: cytotoxic, helper, and suppressor.
  • overexpress overexpressing and “overexpressed” refer to any level of expression of a gene or protein, whether the gene be a transgene or a normal gene, that exceeds normal or expected levels of expression by any amount.
  • transgenic mice which overexpress CD19 is described as a preferred embodiment of the instant invention.
  • Such transgenic mice have been developed in the field according to published techniques (see, for example, Engel et al. (1995) Immunity 3:39-50 and Zhou (1994) Mol. Cell. Biol. 14:3884-3894). Thus, these mice are conveniently available as starting materials.
  • any animal can be utilized in the subject invention, including mouse, pig, rat, rabbit, guinea pig, goat, sheep, primate, and poultry.
  • CD 19 overexpressing transgenic mice are preferred because the break down in peripheral tolerance of antibody-producing cells found in these mice
  • other animals having a manipulated or altered characteristics in the cells of their immune system are contemplated to be within the scope of this invention.
  • Moderate levels of disrupted peripheral tolerance have been described in the art with respect to the manipulation of CD45 and with respect to the manipulation of LPR mice. But, there has been no disclosure of the production of monoclonal antibodies until the instant disclosure.
  • Example 3 Production of Monoclonal Antibodies
  • the animal having antibody-forming cells with disrupted peripheral tolerance is utilized for the production of monoclonal antibodies.
  • the system can be utilized to produce a monoclonal antibody to any antigen that the animal not having antibody-forming cells with disrupted peripheral tolerance could produce.
  • An exemplary list of antigens appears in U.S. Pat. No.3,935,074, the contents of which are herein incorporated by reference.
  • the animal having antibody-forming cells such as B lymphocytes
  • disrupted peripheral tolerance provides a much enhanced immune response to the antigen.
  • the system having antibody-forming cells with disrupted peripheral tolerance system is particularly useful for generating a highly specific antibody for those antigens with numerous epitopes.
  • the system having antibody-forming cells with disrupted peripheral tolerance is used to generate monoclonal antibodies to epitopes that are highly conserved among vertebrate and particularly, mammalian species.
  • Animals not having antibody-forming cells with disrupted peripheral tolerance do not typically respond to such highly conserved epitopes because of self- tolerance.
  • the immune systems of conventional animals cannot recognize highly conserved epitopes as "non-self and therefore cannot produce antibodies against such an epitope.
  • the immune systems of the animals of the instant invention can recognize highly conserved epitopes as "non-self because of the antibody-forming cells with disrupted peripheral tolerance.
  • the animals of the instant invention can be immunized by standard techniques.
  • the animal be immunized at least two times with at least about three weeks between each immunization, followed by a prefusion booster.
  • Somatic cells of the animal having the potential for producing antibody and, in particular B lymphocytes are suitable for fusion with a B-cell myeloma line.
  • Somatic cells can be derived from the lymph nodes, spleens and peripheral blood of primed animals, and the lymphatic cells of choice depend to a large extent on their empirical usefulness in the particular fusion system. However, somatic cells derived from the spleen are generally preferred.
  • animals having antibody-forming cells with disrupted peripheral tolerance can be used as a source of antibody-producing lymphocytes.
  • mice are the preferred animals for use in making monoclonal antibodies because of the availability of excellent cell lines to use as fusion partners.
  • the use of antibody-producing cells from other animals is also possible.
  • the choice of a particular animal depends on the choice of antigen, for it is important that the animal have a B-lymphocyte in its repertoire of B-lymphocytes that can produce an antibody to such antigen.
  • myeloma cell lines have been developed from lymphocyte tumors for use in hybridoma-producing fusion procedures (G. Kohler and C. Milstein (1976) Eur. J. Immunol. 6:511-519; M. Schulman et al. (1978) Nature 276:269-270).
  • the cell lines have been developed for at least three reasons. The first reason is to facilitate the selection of fused myeloma cells. Usually, this is accomplished by using myelomas with enzyme deficiencies that render them incapable of growing in certain selective media that support the growth of hybridomas. The second reason arises from the inherent ability of lymphocyte tumor cells to produce their own antibodies.
  • the purpose of using monoclonal techniques is to obtain immortal fused hybrid cell lines that produce the desired single specific antibody genetically directed by the somatic cell component of the hybridoma.
  • myeloma cell lines incapable of producing light or heavy immunoglobulin chains or those deficient in antibody secretion mechanisms are used.
  • a third reason for selection of these cell lines is their suitability and efficiency for fusion.
  • myeloma cell lines can be used for the production of fused cell hybrids, including NS-1 , X63-Ag8, NIS-Ag4/1 , MPC11-45.6TG1.7, X63-Ag8.653, Sp2/O-Agf14, FO, and S194/5XXO.Bu.1., all derived from mice, and 210 -.RCY3.Agl.+B 2.3 +L derived from rats.
  • Fusion Methods for generating hybrids of antibody-producing spleen or lymph node cells and immortalizing cells generally comprise mixing somatic cells with immortalizing cells in a proportion which can vary from about 20:1 to about 1 :1 in the presence of an agent or agents (chemical, viral or electrical) that promote the fusion of cell membranes. It is often preferred that the same species of animal serve as the source of the somatic and immortalizing cells used in the fusion procedure. Fusion methods have been described by Kohler and Milstein (1975), Nature 256:495-497; (1976), Eur. J. Immunol. 6:511-519; by Gefter et al. (1977), Somatic Cell Genet.
  • the fused cells are cultured in selective media, for instance HAT medium, which contains hypoxanthine, aminopterin and thymidine.
  • HAT medium permits the proliferation of hybrid cells and prevents growth of unfused myeloma cells which normally would continue to divide indefinitely.
  • Aminopterin blocks de novo purine and pyrimidine synthesis by inhibiting the production of tetrahydrofolate.
  • the addition of thymidine bypasses the block in pyrimidine synthesis, while hypoxanthine is included in the media so that inhibited cells can synthesize purine using the nucleotide salvage pathway.
  • the myeloma cells employed are mutants lacking hypoxanthine phosphoribosyl transferase (HPRT) and thus cannot utilize the salvage pathway.
  • HPRT hypoxanthine phosphoribosyl transferase
  • the B lymphocyte supplies genetic information for production of this enzyme. Since B lymphocytes themselves have a limited life span in culture (approximately two weeks), the only cells which can proliferate in HAT media are hybrids formed from myeloma and spleen cells.
  • the mixture of fused myeloma and B-lymphocytes is diluted in HAT medium and cultured in multiple wells of microtiter plates. In two to three weeks, when hybrid clones become visible microscopically, the supernatant fluid of the individual wells containing hybrid clones is assayed for specific antibody production.
  • the assay must be sensitive, simple and rapid. Assay techniques include radioimmunoassays, enzyme immunoassays, cytotoxicity assays, and plaque assays.
  • each cell line can be propagated in either of two standard ways.
  • a sample of the hybridoma can be injected into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
  • the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can be tapped to provide monoclonal antibodies in high concentration.
  • the individual cell lines can be propagated in vitro in laboratory culture vessels.
  • the culture medium containing high concentrations of a single specific monoclonal antibody, can be harvested by decantation, filtration or centrifugation.
  • the monoclonal antibodies made by the method of the subject invention can be utilized in any technique known or to be developed in the future that utilizes a monoclonal antibody.
  • a major use of monoclonal antibodies is in an immunoassay, which is the measurement of the antigen-antibody interaction.
  • immunoassays are generally heterogeneous or homogeneous.
  • the immunological reaction usually involves the specific antibody, a labeled analyte, and the sample of interest.
  • the signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte.
  • Both the immunological reaction and detection of the extent thereof are carried out in a homogeneous solution.
  • Immunochemical labels which may be employed include free radicals, fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth.
  • the major advantage of a homogeneous immunoassay is that the specific antibody need not be separated from the labeled analyte.
  • the reagents are usually the specimen, the specific antibody, and means for producing a detectable signal.
  • the specimen is generally placed on a support, such as a plate or a slide, and contacted with the antibody in a liquid phase.
  • the support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal.
  • the signal is related to the presence of the analyte in the specimen.
  • Means for producing a detectable signal include the use of radioactive labels, fluorescers, enzymes, and so forth.
  • Exemplary of heterogeneous immunoassays are the radioimmunossay, immunofluoroescence methods, enzyme-linked immunoassays, and the like.
  • the monoclonal antibodies can be labeled with radioactive compounds, for instance, radioactive iodine, and administered to a patient intravenously.
  • the antibody can also be labeled with a magnetic probe.
  • NMR can then be utilized to pinpoint the antigen. After localization of the antibodies at the antigen, the antigen can be detected by emission tomographical and radionuclear scanning techniques, thereby pinpointing the location of the antigen.
  • the purified monoclonal antibody is suspended in an appropriate carrier, e.g., saline, with or without human albumin, at an appropriate dosage and is administered intravenously, e.g., by continuous intravenous infusion over several hours, as in Miller et al., In Hybridomas in Cancer Diagnosis and Therapy (1982), incorporated herein by reference.
  • an appropriate carrier e.g., saline
  • human albumin e.g., aline
  • intravenously e.g., by continuous intravenous infusion over several hours, as in Miller et al., In Hybridomas in Cancer Diagnosis and Therapy (1982), incorporated herein by reference.
  • the monoclonal antibodies of subject invention can be used therapeutically. Antibodies with the proper biological properties are useful directly as therapeutic agents. Alternatively, the antibodies can be bound to a toxin to form an immunotoxin or to a radioactive material or drug to form a radiopharmaceutical or pharmaceutical. Methods for producing immunotoxins and radiopharmaceuticals of antibodies are well-known (see, for example, Cancer Treatment Reports (1984) 68:317-328).
  • polyclonal antibodies derived from an animal having antibody-producing cells with disrupted peripheral tolerance also can be utilized in immunoassays and provide an improved result as compared to polyclonal antibodies derived from a conventional animal.
  • Polyclonal antibodies derived from an animal having antibody-producing cells with disrupted peripheral tolerance can be made by utilizing such an animal, as described hereinabove, and immunization techniques, as described hereinabove, followed by separating the polyclonal antibodies from the animal by conventional techniques, e.g. by separating the serum from the animal.
  • the present invention provides pharmaceutical compositions comprising a monoclonal antibody produced by a process of the present invention and a physiologically acceptable carrier.
  • a composition has a variety of uses, including, for example but not limited to, use as a delivery agent for a cytotoxic substance as described herein.
  • composition of the present invention is typically administered parenterally in dosage unit formulations containing standard, well-known nontoxic physiologically acceptable carriers, adjuvants, and vehicles as desired.
  • parenteral as used herein includes intravenous, intra-muscular, intraarterial injection, or infusion techniques.
  • Injectable preparations for example sterile injectable aqueous or oleaginous suspensions, are formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1 ,3-butanediol.
  • Suitable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or di-glycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • Preferred carriers include neutral saline solutions buffered with phosphate, lactate, Tris, and the like. Assay Kits
  • the present invention contemplates diagnostic assay kits for detecting the presence of an antigen in biological samples, where the kits comprise a first container containing a first antibody capable of immunoreacting with the antigen with the first antibody present in an amount sufficient to perform at least one assay, the antibody obtained from an animal having antibody-producing cells with disrupted peripheral tolerance.
  • the assay kits of the invention further comprise a second container containing a second antibody that immunoreacts with the first antibody, the second antibody obtained from an animal having antibody-producing cells with disrupted peripheral tolerance.
  • the antibodies used in the assay kits of the present invention are monoclonal antibodies.
  • the first antibody is affixed to a solid support. More preferably still, the first and second antibodies comprise an indicator, and, preferably, the indicator is a radioactive label or an enzyme.
  • B lymphocyte tolerance to self antigens is achieved by the negative selection and elimination of immature B cells that express high-affinity IgM receptors for autoantigens.
  • Negative selection is antigen receptor-dependent but also relies on established triggering thresholds for intracellular signals (Goodnow, C. C. (1996) Proc. Natl. Acad. Sci.
  • Intracellular signaling thresholds are likely to also play a major role in the regulation and maintenance of peripheral tolerance.
  • the CD19 cell surface molecule regulates intracellular signaling thresholds critical for B cell development and humoral immunity. Tedder et al. (1997) Immunity 6: 07 A 18; Fearon et al. (1996) Science 272:50-54; Carter et al. (1992) Science 256:105-107; Dempsey et al. (1996) Science 271 :348-350; Engel et al. (1995) Immunity 3:39-50; Rickert et al. (1995) Nature 376:352-355.
  • B lymphocytes from mice that overexpress CD19 are hyper-responsive to antigen receptor crosslinking, which results in serum immunoglobulin (Ig) levels that are increased by about 40% and humoral responses that are augmented several fold.
  • Ig serum immunoglobulin
  • Transgenic mouse models for autoreactive B cells provide a mechanism for determining the role of CD19 signaling in regulating peripheral tolerance and autoimmunity.
  • B cells from transgenic mice expressing a model autoantigen (soluble hen egg lysozyme, sHEL) and high-affinity HEL-specific lgM a and lgD a (lg HEL ) antigen receptors enter the peripheral pool but are anergic to antigen receptor ligation and produce little, if any, spontaneous HEL-specific antibody.
  • sHEL/lg HEL double-transgenic mice were crossed with hCD19 transgenic mice to determine whether tolerance would be maintained in sHEL/lg HE /hCD19 transgenic mice or autoantibodies would be generated.
  • CD19 overexpression in sHEL/lg HEL double-transgenic mice resulted in the production of anti-HEL antibodies at levels similar to those observed in lg HEL mice lacking this model self antigen. Therefore, lowered signaling thresholds due to CD 19 overexpression resulted in the breakdown of peripheral tolerance in sHEL/lg HEL double-transgenic mice.
  • mice hCD19 transgenic mice (hi 9-1 line, C57BL/6) were produced as described in Engel et al. (1995) Immunity 3:39-50 and in Zhou et al. (1994) Mol. Cell. Biol. 14:3884-3894). In the h19-1 line of mice, 9-14 copies of the hCD19 transgene are integrated into a single (or closely linked) site(s). These h19-1 mice used in this study were backcrossed onto a wild-type C57BL/6 background for 8 to 10 generations without a diminution of hCD19 expression and all mice express similar levels of cell-surface hCD19.
  • mice expressing sHEL (ML5 line) and lg HEL (MD4 line) were as described (Goodnow et al. (1988) Nature 334:676-682; Hartley et al. (1991 ) Nature 353:765-769).
  • sHEL/lg HEL /hCD19 triple-transgenic mice were generated by appropriate backcrosses of sHEL/lg HEL double-transgenic mice with hCD19 + + mice. Transgene expression was assessed as described in (Engel et al. (1995) Immunity 3:39-50; Zhou et al. (1994) Mol. Cell. Biol. 14:3884-3894; Goodnow et al.
  • mice Two-month-old mice were immunized i.p. with 100 ⁇ g of HEL in complete Freund's adjuvant (CFA, Sigma Chemical Co.) or PBS in CFA at day 0 and were boosted at day 21. Animals were bled just before the first immunization and 7, 14, and 28 days later.
  • CFA complete Freund's adjuvant
  • HEL-specific IgM allotype a (lgM a ) antibody Serum levels of HEL-specific IgM allotype a (lgM a ) antibody were measured by ELISA on HEL-coated plates as described (Goodnow et al. (1989) Nature 342:385-391 ). Absolute antibody concentrations were determined relative to a standard curve of HEL-specific lgM a monoclonal antibody (E1 clone) generated from an lgHEL transgenic mouse immunized with HEL. The ELISA sensitivity limit was about 20 ng/ml of anti-HEL lgM a antibody.
  • Antibodies used in this study included: FITC-conjugated and biotin-coupled goat anti-mouse IgM isotype-specific antibodies (Southern Biotechnology Associates, Inc., Birmingham, AL); anti-B220 (CD45RA, RA3-6B2, provided by R. L.
  • Phycoerythrin-conjugated goat anti-rat IgG antibodies (Caltag, Burlingame, CA) were used to visualize anti-CD5 monoclonal antibody staining.
  • Cells reacting with biotin-coupled HEL were stained with phycoerythrin-conjugated streptavidin.
  • Isolated lymphocytes were analyzed on a FACScan® flow cytometer (Becton-Dickinson, San Jose, CA) as described (Sato et al. (1996) J. Immunol. 156:4371-4378.)
  • Serum anti-HEL lgM a autoantibody levels in lg HEL transgenic, sHEL/lg HEL double-transgenic, and sHEL/lg HEL /hCD19 triple-transgenic mice were determined to assess the status of B cell tolerance in each set of mice. Serum antibody levels for each individual mouse are shown in figure 1 and mean autoantibody levels for each set of mice are provided to simplify discussion of the results. Two-month-old sHEL/lg HEL double transgenic mice produced very low or undetectable levels of anti-HEL lgM a antibodies (mean levels 31 ng/ml) when compared with lg HE transgenic mice (mean 16,700 ng/ml, Fig. 1 ).
  • mice 45% (14 of 33) of sHEL/lg HE hCD19 +/" mice had anti-HEL lgM a autoantibody levels (mean 2,430 ng/ml) that were significantly greater than those found in sHEL/lg HEL mice (P ⁇ 0.001 , Fig. 1 ).
  • Anti-HEL lgM a antibody levels were also elevated in 38% (14 of 36) of sHEL/lg HEL /hCD19 +/+ mice (mean 10,500 ng/ml, P ⁇ O.01 , Fig. 1 ).
  • FIG. 2A A measurable antibody response was only detected in sHEL/lg HE mice after secondary immunization.
  • Fig. 2A A striking result was that the inflammation induced by CFA alone induced sHEL/lg HE 7hCD19 + + mice to produce anti-HEL antibodies in response to endogenous sHEL autoantigen (Fig. 2A).
  • the mean secondary antibody response was 4 thousandfold higher than in sHEL/lg HEL mice (P ⁇ 0.05).
  • the anti-HEL antibody levels induced in some sHEL/lg HEL /hCD19 + + mice were equivalent to those of lg HEL mice (Fig. 2B).
  • B cell development in lg HE and sHEL/lg HEL mice that overexpress CD19 was analyzed using flow cytometry. The results are discussed herein below. Representative two-color immunofluorescence staining of B cells from A) bone marrow, B) blood, C) spleens, and D) peritoneum of littermate pairs was performed. B lymphocytes were revealed by B220 or IgM expression. Quadrants delineated by squares indicated the pre-B cell (B220'°lgM " ), immature B cell (B220'°lgM + ) and mature B cell (B220 hi lgM + ) compartments, with numbers representing the percentage of cells within quadrants.
  • the gates that defined mature B lymphocytes for sHEL/lg HE mice were different from the gate used for lgHEL mice since surface IgM levels are downregulated in sHEL/lg HEL mice. Spleen cells were also stained for B220 or IgM and counterstained for sHEL binding or l-A expression.
  • Additional gates were used to determine the frequency of the CD5 + B220 + population and CD5 " B220 + population of cells for Table 1.
  • Populations of cells lacking surface antigen expression were determined using unreactive monoclonal antibodies as controls. All samples were stained in parallel and analyzed sequentially by flow cytometry with identical instrument settings. Relative fluorescence intensity was shown on a four decade log scale, with 50% log density contour levels. Horizontal dashed lines in some histograms were provided for reference. Similar results were obtained with at least five sets of mice. Equivalent results were obtained by using anti-lgM a antibody instead of anti-lgM antibody.
  • Peripheral B cell numbers were significantly reduced in both lg HEL mice and sHEL/lg HE mice overexpressing CD19 (Table 1 ). Overexpression of CD19 reduced circulating B cell numbers by 87% in lg HEL mice and 78% in sHEL/lg HEL mice. CD19 overexpression reduced spleen B cell numbers by 42% in lg HEL mice and 48% in sHEL/lg HE mice. Conventional B cells within the peritoneum were also reduced by > 90% in lg HE 7hCD19 + + and sHEL/lg HE 7hCD19 + + mice.
  • B cells from sHEL/lg HE /hCD19 +/+ mice still bound sHEL in vitro in proportion to their lgM a density.
  • B cells from lg HEL /hCD19 +/+ transgenic mice had an intermediate lgM'°l-A hi phenotype even in the absence of sHEL (Table 1 ).
  • B cells from mice that overexpressed CD19 also expressed significantly elevated levels of cell surface CD86 (B7-2). Therefore, CD19 overexpression appeared to augment the phenotypic outcome of signaling through the B cell antigen receptor in the absence or presence of autoantigen.
  • B cells from sHEL/lg HEL /hCD19 + + mice still exhibited a phenotype that is characteristic of anergic B cells.
  • Peripheral tolerance in sHEL/lg HEL mice results in the failure of anergic B cells to mobilize intracellular Ca ++ in response to HEL-mediated antigen receptor crosslinking in vitro.
  • B cells from sHEL/lg HEL /hCD19 +/+ mice were equivalent to anergic B cells from sHEL/lg HE mice in their failure to mobilize Ca ++ in response to HEL (Fig. 3A).
  • one pathway to autoantibody production in sHEL/lg HEL mice may be via concomitant CD19 overexpression, chronic antigen receptor ligation, and the influence of inflammatory mediators triggering the simultaneous breakdown of tolerance and autoantibody production in anergic lg HEL B cells.
  • inflammatory mediators such as those generated by CFA administration may induce the expansion or differentiation of antigen-stimulated
  • the latter possibility is supported by the finding that B cells from mice that overexpressed CD 19 maintained a phenotype characteristic of anergic B cells and failed to generate Ca ++ responses following antigen receptor ligation (Fig. 3).
  • the spontaneous development of autoantibodies in sHEL/lg HEL /hCD19 mice may also require a breakdown in T-cell tolerance, since HEL-specific helper T cells are anergic due to chronic sHEL exposure. Adelstein et al. (1991 ) Science 251 :1223-1225.
  • Applicant also has generated and analyzed 7 independent lines of hCD19 transgenic mice. Zhou et al. (1994) Mol. Cell. Biol. 14:3884-3894. In all cases, B cells from each mouse line demonstrate identical functional abnormalities. In lines subjected to analysis, hyper-responsive B cells and enhanced autoantibody production was observed. The magnitude of these abnormalities correlates directly and linearly with the level of hCD19 overexpression. Sato etal. (1997) J. Immunol. 158:4662-4669. In addition, the abnormalities observed in hCD19 transgenic mice are reciprocal of what applicant has observed in CD19-deficient mice (Engel et al. (1995) Immunity 3:39-50; Sato et al. (1995) Proc.
  • CD19 is a signaling component of a multimeric complex that includes
  • CD21 the receptor for the C3d fragment of complement that covalently associates with antigens during complement activation.
  • C3d binding to CD21 can thereby act as a ligand for the CD19 complex that links complement activation with B-cell function.
  • CD21 -deficient mice manifest developmental and functional defects similar to those of CD19-deficient mice (Engel et al. (1995) Immunity 3:39-50; Rickert et al. (1995) Nature 376:352-355; Ahearn et al. (1996) Immunity 4:251-262; Molina et al. (1996) Proc. Natl. Acad. Sci. USA 93:3357-3361 ) overexpression of CD19 in vivo may mimic C3d ligation of CD21 by augmented signaling through the CD19 complex. Tedder et al. (1997) Immunity 6:107-118.
  • C3 cleavage products binding to the CD19 complex may provide a molecular mechanism for bypassing peripheral B-cell anergy in vivo.
  • the inappropriate or prolonged generation of C3d during inflammatory or infectious episodes in vivo may increase the responsiveness of autoreactive B cells to weak self antigens through augmented CD19 function, resulting in a breakdown of tolerance and the clonal amplification of autoantibody-producing B cells.
  • CD19 function may be a molecular mechanism linking inflammation with the development of autoimmune disease.
  • Tissue Phenotype Frequency (%) and Number (# x 10 ⁇ 6 ) of B cells* bone marrow %lgMB220'° 14 ⁇ 2 14 ⁇ 2 13 ⁇ 2 13 ⁇ 2
  • * Cumulative mean ( ⁇ SEM) frequencies of different cell populations from at least five two-month-old mice of ea genotype. Flow cytometry gates were used to determine the frequency of each cell type within the lymphocyte populatio
  • B-cell numbers for blood indicate the number of cells/ml. B-cell numbers from spleen and peritoneum were determined bas on the total number of lymphocytes recovered.
  • mice have been generated that are hyporesponsive to transmembrane signals, while other mice have been generated that are hyper-responsive to transmembrane signals. This has considerable ramifications for the abilities of these mice to recognize and generate humoral antibody responses against a variety of antigens, particularly self antigens. Moreover, this provides a powerful new approach to developing a new class of mAbs that react with molecules which are highly conserved during recent mammalian evolution.
  • PrPsc Unconventional agents termed prion proteins
  • CJD Creutzfeldt-Jakob disease
  • PrPsc antigenic sites are species-directed, involve non- self sites and are common to both the normal host precursor (PrPc) and the disease form (PrPsc).
  • mice are immunized intra-pertioneally (ip) with the test proteins and both primary and secondary humoral immune responses are assessed by ELISA. Multiple tolerance-deficient mice strains are assessed and are compared with wild-type mice of the same genetic background.
  • MHC major histocompatability complex
  • mice have a C57BI/6 background. These mice are bred into a BALB/c background for at least five generations. Humoral immune responses in mice is assessed following initial crosses to assess whether this has an advantageous influence on humoral immune responses. Following these crosses, these mice are used for the generation of mAbs.
  • mice generated above and immunized with optimal antigens are used for the generation of mAbs using standard approaches, such as those described above.
  • PrPO/0 tolerance-deficient mice Generate mAbs with PrPO/0 tolerance-deficient mice. Mice rendered deficient in PrPc by homologous recombination gene targeting are obtained. PrPO/0 mice generate sera capable of specifically precipitating in vitro synthesized human prion proteins, while wild-type mice do not generate humoral immune responses. Drasemann et al. (1996) J. Immunol. Methods 199:109-118). This provides considerable advantages for generating mAbs reactive with this self-protein should responses with tolerance-deficient mice not be optimal. Furthermore, this may allow the generation of mAbs reactive with novel epitopes that may not be optimally identified in tolerance-deficient mice. PrPO/0 tolerance-deficient mice generate vigorous polyclonal immune responses after immunization with human prion gene sequences. These mice will be used as spleen donors for mAb production.
  • high affinity monoclonal antibodies are prepared by the methods described in this Example. Monoclonal antibodies having high affinity for an antigen are desirable in that high affinity provides for biological activity in vivo. Some monoclonal antibodies produced by conventional methods often react with antigens in a biological sample, such as blood, but the affinity of such reactions is low.
  • affinity and “high affinity” have well-recognized meanings in the art. Particularly, the term “affinity” refers to the goodness of the fit of an antigenic determinant to a single antigen-binding site, and it is independent of the number of sites.
  • high affinity refers to a particular good fit of an antigenic determinant to a single antigen-binding site. Typically, the affinity of an antibody and an antigenic determinant is judged by the use of an affinity constant K.
  • Antigens (Ags) and antibodies (Abs) interact according to the reversible equilibrium equation:
  • K [AgAb]/[Ag][Ab], wherein the units for K are liters per mole.
  • a high affinity antigen-antibody interaction is therefore typically described as having an affinity constant K of greater that 1 * 10 5 liters per mole. See Alberts et al. Molecular Biology of the Cell, Garland Publishing, Inc. (New York and London. 1983), p. 970. Such high affinity monoclonal antibodies have application, for example, in a pharmaceutical setting.
  • an antigen such as a cancer antigen
  • an antigen can prevent the antigen from carrying out its biological activity in the cell with which it is associated, thereby killing the cell or slowing its growth.
  • monoclonal antibodies have application as drug carriers, for delivery of, for example, a cytotoxic agent to the cancer cell.
  • Example 1 describes breeding of transgenic mice, including a transgenic mouse which over-expresses CD19, wherein progeny of such mice possess antibody-forming cells having disrupted peripheral tolerance. Given the disclosure of Example 1 , then, a practitioner having ordinary skill in the art can breed and/or prepare lines transgenic mice for cross-breeding, wherein each line of mice have immune systems with certain characteristics, to produce progeny animals having combinations of the characteristics. Such animals would be very useful, inter alia, in the preparation of monoclonal antibodies as described herein or in the preparation of vaccines. This Example describes the preparation of such an animal.
  • An example of a desirable characteristic is hyper-sensitivity to antigens so that antibodies, and particularly monoclonal antibodies, can be made against epitopes that are highly conservative among mammalian or other vertebrate species. Indeed, an animal having a hyper-sensitive immune system due to a breakdown in peripheral tolerance is described in Example 1.
  • transgenic animal which includes a transgene, wherein expression of the transgene in the animal imparts a predetermined or desired characteristic to a cell or a type of cells within the animal's immune system.
  • transgenic mice which over-express CD19 are used to provide disruption of peripheral tolerance in B lymphocytes in mice.
  • the preferred next step in the method is to prepare a second line of transgenic animals which include a transgene, wherein expression of the transgene in the animal imparts a predetermined or desired characteristic to a cell or a type of cells within the animal's immune system.
  • the predetermined characteristic of the cell or cells within the immune system of the second strain of transgenic animals can be the same or different from the predetermined characteristic of the cell or cells within the immune system of the first strain of animals.
  • the second and subsequent lines of animals included sHEL (ML5 line) and lg HEL (ML5 line) mice, as well as double transgenic sHEL/lg HEL mice.
  • the resulting progeny are then screened to determine that transgenes are inherited and are subsequently expressed. Screening protocols include the well known techniques of PCR amplification, northern blot analysis and southern blot analysis using nucleic acid probes or segments from the transgene initially used to prepare the transgenic animal. Additional protocols are described in Engel et al. (1995) Immunity 3:39-50 and in Zhou (1994) Mol. Cell. Biol. 14:3884-3894.
  • Immune cells within the progeny are then screened to determine if the predetermined characteristics have been imparted to the cells. Such screening methods are provided in Example 1 and include isotype-specific ELISA assays and immunofluorescence assays. Finally, progeny having immune cell or cells, such as antibody-producing cells like B lymphocytes, are bred according to well-known techniques to propagate a line of mice which have within their immune systems cells which demonstrate the predetermined or desired characteristics.
  • SHP1 may play a key role in setting thresholds for negative selection in the bone marrow, while CD19 regulates peripheral tolerance.
  • tolerance may be finely tuned, with CD19 and SHP1 altering signaling strengths to differing extents.
  • animals can be produced according to the methods of this Example wherein the animals have cells in their immune systems that display characteristics associated with altered SHP1 and CD19 expression.
  • CD19 forms a complex with CD21 (also known in the art as complement receptor Type II [CR2]), CD81 and Leu-13. Tedder et al. (1994) Immunology Today 15:437-442. Additionally, the structure and in vitro function of the CD19-CD21 complex has been characterized in the art. Tedder et al. (1994), Immunol. Today 15:437-442. Summarily, CD19 is a member of the immunoglobulin superfamily with a cytoplasmic region of approximately 240 amino acids.
  • CD19 The amino acid sequences of the cytoplasmic of human CD19 (hCD19), mouse CD19 (mCD19), and guinea pig CD19 are highly homologous, which is consistent with a critical role for this region in CD19 function.
  • CD19 physically associates with CD21 on the surface of human B cells.
  • CD21 contains an extracellular domain of 15 or 16 repeating structural elements called short consensus repeats (SCRs), a membrane spanning region, and a 34-amino acid cytoplasmic domain.
  • SCRs short consensus repeats
  • Human and mouse forms have been identified.
  • the human form, hCD21 can physically associate with a structurally similar complement receptor, CD35 (CR1 ) and generate a receptor complex that does not contain CD19.
  • the human CD35 is expressed by B cells, erythrocytes, neutrophils, monocytes, and some T cells. Thus, interactions of these molecules can be manipulated to prepare a non-human animal having an immune system with predetermined or desired characteristics associated with such a manipulation.
  • CD19 also associates directly with CD81 , a member of the tetrospans family of proteins that includes CD9, CD37, CD53, CD63 and CD82. Bradberry et al. (1992), J. Immunol. 149:2841-2850 and Levy et al. (1991 ) J. Biol. Chem. 266:14597-14602. CD81 is over-expressed by most B lineage cells and by a wide variety of cell types including most lymphocytes, natural killer cells, thymocytes, eosinophils, neuroblastomas, melanomas, and fibroblasts.
  • interactions of CD19 and CD81 within the complex can be manipulated to prepare a non-human animal having an immune system with predetermined or desired characteristics associated with such a manipulation.
  • Table 2 presents phenotypic characteristics of mice with genetically altered response regulators of B lymphocyte signal transduction. The table presents both negative and positive effects, each such effect being a potential predetermined characteristic for an animal prepared according to the methods of this Example.
  • a novel C3 mRNA transcript has been identified that encodes a truncated C3 protein.
  • Cell lines transfected with the related cDNA secrete a co-stimulatory factor that augments the proliferation of B cells in assays with macrophage- depleted mouse splenic B cells.
  • Such a cDNA thus provides a candidate for use in the production of a transgenic animal according to the methods of this Example.
  • CD20 and CD35, or CD21/35 play a role in immune response, and are thus candidates for manipulation in an animal according to the method of this Example.
  • mice with a mCD21/35 MAb blocks both T-cell dependent and independent immune responses in the generation of immunological memory. See, for example, Gustavsson et al. (1995) J. Immunol. 154:6524-6528. Chimeric mice with normal levels of CD21/35 on their follicuiar dendritic cells, but not on their B cells, have defects in humoral responses to antigens similar to those of CD21/35 deficient mice. Croix et al. (1996) J. Exp. Med. 183:1857-1864.
  • CD5 B cells and CD5 + B-1 cells given their documented role in pathogenic autoantibody responses, such as the pathogenic autoantibody responses of systemic lupus erythematosus (SLE).
  • SLE is characterized by production of antibodies to DNA within the body in association with systemic inflammation. Shirai et al. (1991 ) Clin. Immunol. Immunopathol. 59:173-186. See also Murakami and Honjo (1995), Immunol. Today 16:534-539, discussing that high affinity pathogenic IgG antibodies are generally produced by CD5 " B cells.
  • multiple response regulators govern signaling thresholds in the cells of an animal's immune system, particularly B cells.
  • Response regulators with positive or negative effects influence signaling through the B cell antigen-receptor complex.
  • the resulting signaling thresholds regulate negative selection in the bone marrow, the magnitude of antibody response in the periphery, autoimmunity, and peripheral tolerance. Therefore, preparing an animal having cells in its immune system with predetermined characteristics derived from manipulation of these response regulators is highly useful in the characterization of immune response, development of monoclonal antibodies, and vaccines, and in the treatment of autoimmune disorders.
  • transgenic any animal can be utilized in the methods.of this Example, including mouse, pig, rat, rabbit, guinea pig, goat, sheep, primate, and poultry, with a mouse being preferred.
  • transgenic it is meant any animal having a genome altered by the hand of man in any manner.
  • transgenic includes the insertion of desired transgene, any of the well-known “knock-in” approaches, and any of the well-known “knock-out” approaches.
  • the preparation of lines of animals having immune system cells demonstrating a predetermined or desired characteristic for use in subsequent breeding is not limited to transgenic protocols. Any suitable protocol that generates an alteration in the cells of the animal's is contemplated to be within the scope of the method of this Example. Such protocols include, among others, exposure to mutagens, as described in Cyster et al. (1995) Immunity 2: 13-24.
  • C57BL/6 mice generate T cell-dependent humoral responses to the (4- hydroxy-3-nitrophenyl)acetyl (NP) hapten that are dominated by canonical antibodies composed of a single VH gene, V186.2, and ⁇ 1 light chain. Selection for this receptor is thought to be driven by its frequency and affinity.
  • lowering the activation threshold of B lymphocytes by overexpression of a single cell-surface molecule, CD19 resulted in anti-NP antibodies comprising an unprecedented diverse repertoire of VH and V ⁇ _ rearrangements with no or few mutations.
  • antigen-receptor selection is regulated by endogenous B lymphocyte signaling thresholds and not antigen receptor affinity.
  • mice Despite the enormous diversity of antibodies, inbred strains of mice often respond to haptens and simple antigens by producing remarkably homogenous antibodies (Blier and Bothwell, 1988).
  • One of the best examples is the response of C57BL/6 (lgh b ) mice to the (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten (Imanishi and Makela, 1975).
  • V186.2-to-DFL16.1 heavy chain rearrangement paired with the ⁇ 1 light chain is referred to as the canonical anti-NP B cell antigen receptor (Reth et al., 1978; Reth et al., 1979).
  • the homogeneity of the anti-NP response in lgh b mice is mirrored in the response of BALB/c mice to phosphorylcholine (Crews et al., 1981 ); antibodies produced against p- azophenylarsonate in strain A mice (Pawlak et al., 1973); the 2- phenyloxazolone response in BALB/c and DBA/2 mice (Makela et al., 1978); and the response of BALB/c mice to poly(Glu 60 -Ala 30 -Tyr 10 ) (Theze and Somme, 1979). The cause of low genetic variance in these antibody responses remains obscure.
  • Transmembrane signals generated through the B cell antigen receptor complex regulate B cell responses to antigen binding and may thereby also regulate repertoire selection. Other cell surface molecules can also modifying B cell responses to antigen. Transmembrane signals generated through the B cell antigen receptor complex and other surface receptors are critically regulated by CD19 expression (reviewed in Fearon and Locksley, 1996; Tedder et al., 1997). CD19 functions as a general regulator of B cell proliferation, differentiation, clonal expansion in the peripheral B cell pool, and of peripheral tolerance, as described above.
  • CD19-deficient mice are hypo-responsive to transmembrane signals while B lymphocytes from transgenic mice that overexpress CD19 (CD19-TG) are hyper-responsive (Engel et al., 1995; Sato et al., 1995; Zhou et al., 1994).
  • CD19-deficient and CD19- overexpressing mice serve as model systems where CD19 is a general response regulator of cell-surface receptor signaling and cellular signal transduction thresholds.
  • CD19-TG, CD19KO, and wildtype C57BL/6 mice were immunized with NP coupled to the T-cell-dependent antigen, chicken gamma globulin (CGG).
  • CGG chicken gamma globulin
  • Mice that overexpressed CD19 generated anti-NP humoral immune responses that were quantitatively similar to those of wildtype C57BL/6 mice, while CD19KO mice generated only modest responses.
  • the antibody response generated by mice that overexpressed CD19 were qualitatively distinct from those of C57BL/6 controls.
  • VH gene segment and gene family use were unprecedently diverse in CD19TG mice and none of the anti-NP antibodies produced carried ⁇ 1 light chains.
  • CD19TG and wildtype C57BL/6 mice were immunized with NP-i ⁇ -CGG to assess their humoral immune responses to NP.
  • Serum anti-NP antibody concentrations at the time of immunization (day 0) and on subsequent days (4, 8, 10, 16, and 58) were determined by ELISA using NP25-BSA. Values represented either mean antibody levels ( ⁇ SEM) from 4-5 individual mice per time-point following immunization relative to isotype-matched anti-NP monoclonal antibodies used to generate standard curves, or represented antibody concentrations relative to control serum from a C57BL/6 mouse immunized with NP-i ⁇ -CGG.
  • CD19TG mice generated serum IgM responses quantitatively similar to those of wildtype mice, despite an overall (-80%) reduction in peripheral B cell numbers (Engel et al., 1995).
  • IgGI responses of CD19TG mice were -10-fold lower than in wildtype mice.
  • lgG2a, lgG2b, and lgG3 responses in CD19TG mice were also below those of wildtype mice. All antibody responses were of the lgh b allotype. Remarkably, ⁇ 1 antibody responses were poor in CD19TG mice, while ⁇ + antibody responses were similar in CD19TG and wildtype mice.
  • the primary NP-specific antibody response in CD19TG mice is dominated by IgM and IgGI , with the vast majority of antibodies bearing light chains other than ⁇ 1.
  • the relative affinity/avidity of the antibody response in CD19TG mice was assessed by comparing antisera binding to highly- (NP25-BSA) or sparsely- (NP5-BSA) substituted NP-bovine serum albumin (BSA) substrates over a wide range of antibody concentrations as described previously (Herzenberg et al., 1980). Values represented mean ( ⁇ SEM) ratios of anti-NPs versus anti-NP25 antibody concentrations from 4-5 individual mice per time- point. In cases where anti-NPs antibody levels were not detectable, values of 0 were used for generating means.
  • CD19TG and wildtype mice Differences between CD19TG and wildtype mice were significant, p ⁇ 0.05, at about 3 days, 10 days and 14 days after immunization for IgM; at about 11 days, 18 days and 58 days for ⁇ 1 ; and at about 58 days for K.
  • mice overexpressing CD19 generate high affinity IgGI antibody that does not bear ⁇ 1 light chain.
  • the relative frequency of anti-NP antibody-producing B cells in the spleen and bone marrow of CD19TG mice was assessed using ELISpot assays. Determined values represented mean AFC numbers ( ⁇ SEM) from 4- 14 individual mice per time-point following immunization on day 0. Differences between CD19TG and wildtype mice were significant, p ⁇ 0.05, at about days 8 and 11 for IgM in spleen, and at about day 58 for IgGI in bone marrow.
  • NP-specific IgM antibody producing cells were 2- to 7-fold higher among CD19TG mouse splenocytes than among wildtype splenocytes following immunization.
  • the frequency of IgGI anti-NP antibody-forming cells among CD19TG splenocytes were not significantly different from wildtype controls.
  • CD19TG mice also had consistently higher frequencies of IgM-secreting cells and lower frequencies of IgG-secreting cells among bone marrow-derived anti- NP antibody-forming cells when compared with wildtype controls.
  • CD19KO mice were immunized with NP-is-CGG to compare their humoral immune responses with wildtype littermates.
  • Mean serum anti-NP antibody concentrations ( ⁇ SEM) from 4-5 individual mice per time-point following immunization (day 0) were determined by ELISA using NP25-BSA as described above.
  • the average relative affinity of serum anti-NP antibodies was estimated by determining the mean concentration of NPs-binding and NP25- binding antibodies at each time-point by ELISA as described above.
  • CD19KO and wildtype Differences between CD19KO and wildtype were significant, p ⁇ 0.05, at about 3 days and 18 days after immunization for IgM; at about 18 days and 58 days for IgGI ; at about 58 days for ⁇ 1 ; and at about 18 and 58 days for K.
  • the frequency of spleen and bone marrow cells secreting anti-NP25- binding antibody was determined by ELISpot assays as described above. Values represent mean antibody-forming cell (AFC) numbers ( ⁇ SEM) from 4-5 individual mice per time-point following immunization on day 0. Differences between CD19KO and wildtype mice were significant, p ⁇ 0.05, at about days 10 and 18 for IgGI in spleen, and at about days 10, 16 and 58 for IgGI in bone marrow.
  • Serum IgM and IgGI antibody responses to NP were 10-fold and >100- fold lower in CD19KO mice than in wildtype mice. ⁇ 1- and ⁇ -bearing antibody responses to NP were also suppressed in CD19KO mice. Affinity maturation was also delayed and reduced in CD19KO mice when compared with wildtype mice.
  • the relative frequency of B cells producing IgGI anti-NP antibodies in the spleens and bone marrow of CD19KO mice was markedly lower than in wildtype mice at each time point following immunization. Therefore, antibody responses to NP in CD19KO mice were markedly diminished beyond what would be expected with the overall (40-60%) reduction in peripheral B cell numbers in these mice.
  • Germinal center B cell responses in CD19TG and CD19KO mice were assessed after immunization with NP-i ⁇ -CGG. Histologic sections of spleen were stained with the GL7 monoclonal antibody and/or peanut agglutinin (PNA) to identify germinal center B cells (Laszlo et al., 1993; Rose et al., 1980). Antibody specific for mouse K light chain was used to visualize the B cell zones (follicles) within the splenic white pulp. Overall, B cell follicles were smaller in CD19TG mice than in wildtype mice both before and after NP immunization. Correspondingly, T cell zones occupied a larger portion of CD19TG mouse spleens.
  • PNA peanut agglutinin
  • the overall frequency of follicles was also significantly (p ⁇ 0.01 ) lower in CD19TG mice than in wildtype mice, reflecting reduced B cell numbers in these mice.
  • the frequency of PNA + germinal centers within follicles of CD19TG mice increased in response to immunization, although the germinal centers were usually smaller in CD19TG mice than in wildtype controls.
  • the frequency of germinal centers per follicle was also significantly reduced in CD19TG mice. Nonetheless, the percentage of splenic B cells induced to express the GL7 antigen was similar in CD19TG and wildtype mice as assessed by flow cytometry, although GL7 expression kinetics were delayed in CD19TG mice.
  • CD19TG mice displayed different phenotypic properties than observed in wildtype mice by the dissociation of the PNA and GL7 markers.
  • germinal centers in CD19TG mice did not contain ⁇ 1 + B cells after immunization with NP-CGG.
  • the repertoire of anti-NP antibodies elicited in CD19TG mice was examined by generating hybridomas from splenocytes of individual CD19TG mice immunized with NPi ⁇ -CGG.
  • the hybridomas were labeled TG2, TG3, TG7, or TG18 depending on the day of splenocyte isolation post-immunization (Table 3). Splenocytes from mice boosted with NP-i ⁇ -CGG on day 15 were used to generate TG18 hybridomas.
  • the TG2, TG3, TG7 and TG18 fusions generated 164, 36, 210, and 275 hybridomas total with 3, 1 , 31 , and 10 monoclonal hybridoma lines isolated that secreted antibodies reactive with NP- BSA but not BSA in ELISA assays. Surprisingly, none of these hybridomas secreted ⁇ 1 -bearing antibodies (Table 3).
  • the heavy chain genes of the TG2, TG3 and TG7 hybridomas were sequenced by PCR amplification of cDNA made from hybridoma RNA. Eight of 31 antibodies (26%) were encoded by VDJ rearrangements containing VH gene segments common in the anti-NP B cells of C57BL/6 mice, V186.2, V23, and C1 H4 (Table 3). Three of these antibodies, TG7-14, -17, and -99, may have arisen from a common progenitor since each had identical VDJ sequences. These were the only antibodies to display canonical VDJ sequences for anti-NP antibodies with the preferred YYGS motif in CDR3 (Table 3).
  • CD19TG mice are capable of generating antibody heavy chains typical of those obtained in lgh b mice to NP, although these represented a minority of the antibodies and none of these heavy chains paired with ⁇ 1.
  • VH genes not normally found in NP-specific B cells from C57BL/6 mice (Table 3).
  • Sixteen of these antibodies used VH segments encoded by known members of the J558 family; 86.22 (1 hybridoma), G4D11 (1 hybridoma), V130 (5 hybridomas, 2 were related), 671.5 (8 related hybridomas), and C1A4 (1 hybridoma).
  • the majority of VH regions did not contain somatic mutations.
  • VH8 One anti-NP antibody, TG7-83, used a previously unidentified VH segment similar to the 5D3 gene (Kaartinen et al., 1988) of the J558 family although six nucleotide differences at the 5' end were homologous with the 186.2 VH gene sequence.
  • This VH sequence is similar to that of the dC5 antibody from a C57BL/6 mouse (GenBank accession number AF045488) and the germiine VHII gene, H30, isolated from BALB/c mice (Schiff etal., 1985) and is therefore likely to represent a heretofore unidentified VH gene segment in C57BL/6 mice.
  • TG7 hybridomas utilized VH gene segments from the 7183, Q52 and IX gene families (Table 3).
  • the TG7-3 antibody was encoded by a novel VH7183 family member most similar to the VH61 -1 P gene of BALB/c mice (Chukwuocha et al., 1994).
  • a C57BL/6 VH gene that only differs from TG7-3 at four positions was identified, although these differences are unlikely to represent somatic diversification since these residues are found in other VH7183 family members of C57BL/6 mice.
  • the TG7-50, -108, and -110 hybridomas generated unrelated antibody products using VH segments almost identical to the OX-2 gene segment, a VH Q52 family member of BALB/c mice (Lawler et al., 1987).
  • the TG7-125 hybridoma utilized a VH region identical to the BALB/c germiine OX-1 VH gene, another member of the Q52 family.
  • the TG7-118 hybridoma utilized a VH region most homologous with the VGAM3-8 VH gene of C57BL/6 mice, an IX gene family member (Winter et al., 1985).
  • the TG7-188 VH sequence was also 98% homologous with VH regions of two hybridomas, 5G6 and 264, from C57BL/6 mice (GenBank accession number AF045504, Nottenburg et al., 1987).
  • the TG7-188 VH segment may therefore represent a new member of the IX gene family in C57BL/6 mice.
  • the AGTC changes at the 5' end may represent somatic diversification since we were unable to detect similar sequences in the C57BL/6 genome.
  • Considerable diversity in DH and JH use by all of the hybridomas was apparent (Table 3), but relatively few antibodies contained the CDR3 YYGS motif typical of anti-NP antibodies in C57BL/6 mice. All JH sequences were of the b allotype.
  • TG 18-43 carried a member of the J558 family, V23, bearing 2 point mutations.
  • the TG18-161 hybridoma VH gene segment matched the V23 sequence except for 4 nucleotide differences; 3 were clustered at codons 9,10 and 11 which were identical to the V186.2 gene.
  • the TG18-161 heavy chain gene rearrangement may be derived from a hybrid of two well characterized VH gene segments, V23 and V186.2, or from a previously unknown J558 family member.
  • hybridomas utilized a VH gene segment that differed from those utilized by the unrelated TG7-50, -108 and -110 hybridomas at three positions that are potential sites of hypermutation.
  • DH and JH gene utilization was also diverse among the TG18 hybridoma set (Table 3), although none of the antibodies encoded the YYGS motif.
  • the repertoire of the anti-NP antibody response of CD19TG mice substantially diverges from the response of wildtype lgh b mice with only 20% (2/10) of the TG18 antibodies encoded by members of the J558 VH gene family and none carried the ⁇ 1 light chain.
  • CD19KO mice were immunized with NP-is-CGG on day 0 , boosted on day 7, with hybridomas generated on day 10.
  • the KO10 fusions generated 615 hybridomas total.
  • Only six clonal hybridomas were isolated ( ⁇ 1 %) that secreted ⁇ , K antibodies with low affinities/avidities for NP25-BSA, but not BSA, in ELISA screens (Table 3).
  • Four antibodies were encoded by non-canonical, germiine VH genes of the J558 family (Table 3).
  • the KO10-613 antibody was encoded by a newly described member of the J558 family, L350- 7 (Kasturi et al., 1994). Two identical antibodies were encoded by a new member of the IX VH gene family. Thus, there was little affinity maturation or selection for canonical sequences in CD19-deficient mice. Affinity Analysis of Anti-NP Antibodies
  • Antibodies representing most NP-specific hybridomas from primary and secondary responses were purified and used for NP affinity determinations by fluorescence quenching (Azuma et al., 1987; Eisen and McGuigan 1971 ; Jones et al., 1986).
  • This assay measures antibody binding to NP-caproate, a monovalent derivative of the immunizing hapten. Under the conditions utilized the assay was sensitive to 7.0 X 10 3 M _1 , below which NP-specific binding was not detected.
  • the K a s ranged between a low of 7.2 X 10 3 M-1 for TG7-125 and a high of 1.6 X 105 M-1 for TG7-180, with an average affinity of 7.2 X 104 M" 1 (Fig. 4).
  • the KO10-613 antibody had a Ka of 1.3 X 10 4 M" 1 .
  • all three TG18 antibodies had relatively high affinities, 1.9 to 2.9 X 106 M" 1 .
  • the K a s of the NP-specific hybridomas antibodies were also compared with K a s of antibodies generated by transfectomas producing canonical anti-NP antibodies and representative antibodies utilized by B cells isolated from NP- specific foci or germinal centers according to art-recognized techniques.
  • the NP-specific antibodies generated in CD19TG mice were of lower affinity than canonical anti-NP antibodies represented by the B1-8 ⁇ 1 control antibody (Fig. 4).
  • the TG18 antibodies were uniformly of higher affinity than canonical antibodies or antibodies isolated from germinal centers.
  • the CD19TG mouse generates noncanonical anti-NP antibodies of higher affinity than canonical antibodies generated in wild-type C57BL/6 mice.
  • TG7-83 antibody for ssDNA was comparable with the binding of two well-characterized, isotype- matched anti-ssDNA autoantibodies (Krishnan etal., 1995; Tillman etal. , 1992) over a range of antibody concentrations (Fig. 5B).
  • the KO10-613, TG3-471 , TG7-75, and TG7- 68 antibodies also reacted with self protein antigens.
  • hCD19 transgenic mice CD19TG, h19-1 line, C57BL/6
  • B lymphocytes of the h19-1 line of CD19TG mice express 3-fold higher levels of total cell surface CD19 (Sato et al., 1996; Sato et al., 1997) and have 9-14 copies of the hCD19 transgene integrated into a single (or closely linked) genomic site(s) on chromosome 7.
  • mice used in this study were backcrossed with C57BL/6 mice (Jackson laboratory, Bar Harbor, ME) for 8 to 10 generations without a diminution of hCD19 expression and all mice expressed similar levels of cell-surface hCD19.
  • CD19KO mice were backcrossed with C57BL/6 mice for 8 to 10 generations.
  • Flow cytometric analysis demonstrated that B lymphocytes from all mice expressed the IgM ⁇ but not lgM a allotype, and the mice only produce antibodies of the b allotype. All mice were 2-3 months of age at the time of use and were housed under identical conditions in a specific pathogen free barrier-facility. All studies and procedures were approved by the Animal Care and Use Committee of Duke University. Antigens and Immunizations
  • Serum IgM, IgGI , lgG2a, lgG2b, lgG3, IgA, K light chain, and A1 light chain antibodies specific for NP were quantified by ELISA.
  • Wells of 96-well flat bottom plates (Costar, Cambridge, MA) were coated with either 5 ⁇ g/ml NP5- BSA or NP25-BSA in 0.1 M borate-buffered saline (pH 8.4) at 4°C overnight before the wells were blocked with phosphate-buffered saline (pH 7.4) containing 2% gelatin and 1 % BSA.
  • Serially-diluted mouse sera were then added to each well at room temperature for 1.5 hours.
  • alkaline phosphatase (ALP)-labeled goat antibody specific for mouse IgM, IgGI , lgG2a, lgG2b, lgG3, IgA, or K light chain was added and incubated at room temperature for 1.5 hours.
  • A1 light chain-bearing antibody binding was assessed using biotinylated Ls136 (anti-A1 ) monoclonal antibody (Reth et al., 1978) and ALP- conjugated streptavidin (Southern Biotechnology Associates).
  • ALP activity was visualized using p-nitrophenyl phosphate substrate (Southern Biotechnology Associates) and optical densities were determined at 405 nm.
  • the concentrations of IgM, IgGI , A1 , or K anti-NP antibodies were estimated by comparisons to standard curves generated using serially diluted control monoclonal antibodies on each plate.
  • the standard for IgGI and A1 anti-NP antibodies was H33L ⁇ 1 , as is known in the art.
  • the standard for IgM was B1-8, an IgM anti-NP monoclonal antibody (Reth et al., 1978).
  • the K antibody standard was TG 18-43 (Table 3).
  • the standard for lgG2a, lgG2b, igG3, IgA anti-NP antibodies was serially diluted serum from a C57BL/6 mouse obtained 10 days following immunization with NP-is-CGG. Enzyme-Linked Immunospot Assays
  • NP-specific antibody-forming cells AFC
  • NP25-BSA conjugates The frequency of NP-specific antibody-forming cells (AFC) from single- cell splenocyte and bone marrow suspensions were estimated by enzyme- linked immunospot (ELISpot) assays using NP5-BSA and NP25-BSA conjugates as has been described in the art.
  • ELISpot enzyme- linked immunospot
  • Single cell suspensions of mouse splenocytes were incubated with anti- FcgRI/RI I monoclonal antibody (clone 2.4G2, PharMingen, San Diego, CA) for 10 min on ice to block Fey receptor function.
  • anti- FcgRI/RI I monoclonal antibody clone 2.4G2, PharMingen, San Diego, CA
  • splenocytes were subsequently incubated with FITC-labeled GL7 monoclonal antibody (PharMingen), phycoerythrin- conjugated anti-B220 monoclonal antibody (RA3-6B2, Caltag, South San Francisco, CA), and 7-aminoactinomycin D (Molecular Probes Inc., Eugene, OR) for 30 min on ice before washing.
  • the cells were subsequently analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). The percentage of GL7 + , B220 + cells was calculated from live lymphocytes selected by forward-side scatter patterns and exclusion of 7-aminoactinomycin D.
  • splenocytes were incubated with FITC-labeled anti- B220 antibody (RA3-6B2) and biotinylated Ls136 antibody, washed, and incubated with phycoerythrin-conjugated streptavidin. After washing, the frequency of A1 + cells among viable B220 + lymphocytes was determined by flow cytometry. Immunohistochemistrv
  • HRP-conjugated PNA and biotinylated GL7 antibody were also stained with HRP-conjugated PNA and biotinylated GL7 antibody, followed by streptavidin/alkaline phosphatase, or stained with HRP-conjugated PNA and ALP-labeled anti- ⁇ light chain antibodies.
  • HRP and ALP activities were visualized using 3- aminoethyl carbasole (Sigma Chemical Co.) and naphthol AS-MX phosphate/fast blue BB (Sigma Chemical Co.), respectively (Jacob et al., 1991 ).
  • TdT Terminal deoxynucleotidyl transferase
  • TUNEL Terminal deoxynucleotidyl transferase
  • MEBSTAIN Apoptosis kit Immunotech, Westbrook, ME
  • splenocytes from one ortwo immunized mice were fused with nonsecreting P3X63-Ag8.653 myeloma cells as described (Kearney et al., 1979) and subdivided into ten 96 well tissue culture plates.
  • One CD19TG mouse was boosted on day 15 with the same antigen and its splenocytes were fused with the myeloma cell line on day 18.
  • Two CD19KO mice were boosted on day 7 with 100 ⁇ g of NP-CGG conjugate and their splenocytes were fused with myeloma cells on day 10.
  • hybridomas generated from these fusions were named based on the splenocyte source and the fusion day following immunization: for example, K010 hybridomas were generated from splenocytes of CD19KO mice 10 days after the first immunization.
  • Monoclonal hybridomas secreting anti-NP antibodies were identified by ELISA. Culture supernatant fluid from each hybridoma was added to NP45- BSA-coated 96-well flat bottom ELISA plates. After washing, ALP-labeled goat anti-mouse IgM+lgG+lgA antibodies were added to each well and ALP activity was visualized using p-nitrophenyl phosphate substrate. Hybridomas generating BSA-reactive antibodies were identified by ELISA using BSA-coated plates and were eliminated.
  • NP-reactive antibodies were determined by ELISA with NP45-BSA coated plates and by using mouse monoclonal antibody isotyping kits (Amersham Life Sciences, Arlington Heights, IL).
  • Hybridomas secreting A light chain antibodies were identified by ELISA using plates coated with goat anti-mouse whole Ig antibodies and ALP- labeled goat anti-mouse A light chain antibodies (Southern Biotechnology Associates) as the developing reagent.
  • Hybridomas secreting A light chain antibodies were identified by immunohistochemical staining using cytospin preparations of each hybridoma. Hybridomas were centrifuged onto glass slides, dried for 2 hours, then fixed with acetone at 4°C for 10 min. The slides were stained with biotinylated Ls136 antibody, followed by incubation with HRP-conjugated streptavidin which was visualized as above.
  • NP-specific hybridomas were grown in miniPerm bioreactors (Heraeus, South Plainfield, NJ). Their culture supernatant fluid was concentrated and the antibody product was purified over protein G-Sepharose (Pierce, Rockford, IL) or mannose-binding columns (Pierce, Rockford, IL) . Antibody protein concentrations and purity were determined by light absorption and by antibody isotype-specific sandwich ELISA. VH and Light Chain Gene Utilization
  • Cytoplasmic RNA was extracted from 0.1 - 1 X 10 6 hybridoma cells using the RNeasy Mini Kit (Qiagen Chatsworth, CA). First strand cDNA was synthesized from cytoplasmic RNA using oligo-dT primers (dT-i ⁇ ) and a Superscript Kit (Gibco BRL, Gaitherburg, MD). One ⁇ l of cDNA solution was used as template for PCR amplification of VH genes.
  • PCR reactions were carried out in a 100- ⁇ l volume of a reaction mixture composed of 10 mM Tris- HCl (pH 8.3), 50 mM KCi, 1.5 mM MgCI 2 , 200 ⁇ M dNTP (Perkin Elmer, Foster City, CA), 50 pmol of each primer, and 5 U of Taq DNA polymerase (ISC Bioexpress, Kaysville, UT). Amplification was for 30 cycles (94°C for 1 min, 58 °C for 1 min, 72 °C for 1 min; Thermocycler, Perkin Elmer).
  • V186.2-related VH genes were amplified using a sense primer complementary to the 5' region of the V186.2 gene (primer V186.2; 5' TCTAG AATTC AGGTC CAACT GCAGC AGCC 3" - SEQ ID NO: 1 ) and antisense primers complementary to the C ⁇ coding region (primer C ⁇ -in; 5' GAGGG GGAAG ACATT TGGGA AGGAC TG 3' - SEQ ID NO:2) or the Cy region (primer C ⁇ 1 ; 5' GAGTT CCAGG TCACT GTCAC TGGC 3' - SEQ ID NO:3).
  • V H genes not amplified using the V186.2 primer were amplified using a promiscuous 5' VH primer (MSVHE; 5' GGGAA TTCGA GGTGC AGCTG CAGGA GTCTGG 3' - SEQ ID NO:4) as previously described (Kantor et al., 1996).
  • a light chain cDNA was amplified using a V ⁇ primer (5' AACTG CAGGC TGTTG TGACT CAGGA ATC - SEQ ID NO:5) and a CA primer (CGGGA TCCGC TCTTC AGAGG AAGGT GGAAA CA - SEQ ID NO:6).
  • Amplified PCR products were purified from agarose gels using the QIAquick gel purification kit (Qiagen) and were sequenced directly in both directions using an ABI 377 PRISM DNA sequencer after amplification using the Perkin Elmer Dye Terminator Sequencing system with AmpliTaq DNA polymerase and the same primers for initial PCR amplification. Sequences were compared with known VH sequences using the BLAST search program provided by the National Center for Biotechnology Information. VH genes belonging to the J558 family were analyzed as described (Bothwell et al., 1981 ; Gu et al., 1991 ).
  • K a s of purified anti-NP antibodies was determined by fluorescence quenching as described (Azuma et al., 1987; Eisen and McGuigan, 1971 ; Jones et al., 1986). Briefly, K a s for NP and NIP haptens were measured by fluorescence quenching in a Shimadzu RF-3501 fiuorospectrophotometer (Shimadzu Scientific Instruments, Columbia, MD). Excitation and emission wavelengths were 280 and 340 nm, respectively: temperature (25°C) and pH (7.4) were held constant.
  • Calf thymus DNA (Sigma Chemical Co.) was purified by repeated phenol/chloroform extraction followed by ethanol precipitation. The DNA suspension was boiled for 10 min before immersion in an ice bath to generate ssDNA. ELISA assays were carried out in 96 well Immulon II microtiter plates (Dynatech Laboratories, Chantilly, VA) that were coated overnight at 4°C with ssDNA (5 mg/ml in 0.1 M Na citrate buffer containing 0.15 M NaCI, pH 8.0). Plates were washed three times with PBS (pH 7.3).
  • Supernatant fluid from individual hybridomas was diluted (generally 1 :2) in PBS containing 1% BSA (Sigma) and 0.05% Tween-20 and added to triplicate wells of the antigen- coated ELISA plates.
  • Sera from individual MRL autoimmune mice were used as positive controls which were diluted (1 :100) in PBS containing 1 % BSA (Sigma Chemical Co.) and 0.05% Tween-20.
  • Peroxidase-conjugated goat anti- mouse Ig antibody diluted in PBS containing 1% BSA and 0.05% Tween-20 was added to the wells for 1 h before washing three times with PBS.
  • B cells were purified from single cell splenocyte suspensions by removing T cells with anti-Thyl .2 antibody-coated magnetic beads (Dynal, Inc., Lake Success, NY). B cell suspensions were analyzed by flow cytometry following isolation to assess purity. B cell preparations from CD19KO and C57BL/6 mice were >95% B220 + while preparations from CD19TG mice were >75% B220 + . B cells were seeded in 24-well flat bottom plates (Costar) at 1 x 10 6 cells per well with various concentration of F(ab')2 fragments of goat anti- mouse IgM antibodies (Cappel, Durham, NC) and cultured for 16 hrs or 48 hrs in a CO2 incubator.
  • % apoptosis [(% TUNEL+ cells / (% TUNEL+ cells + % live cells)] X 100.
  • Cultured cells were also washed with PBS containing 0.2% BSA followed by PBS containing 1 % glucose and fixed with ice-cold 70% ethanol overnight. The fixed cells were stained with 0.05 mg/ml propidium iodide (PI; Sigma chemical Co.) solution containing 100 U/ml RNase A (Sigma Chemical Co.). Stained cells were analyzed by flow cytometry and cells with hypodiploid nuclei were considered apoptotic. Data analysis All data are shown as mean values ⁇ SEM unless indicated otherwise.
  • IgM antibody (B1-8, 10 ⁇ g/ml) generated mean OD values of 1.18 while an IgGI antibody (H33L ⁇ l, 1.0 ⁇ g/ml) generated OD values of 1.76. All OD values were significantly greater (p ⁇ 0.05) than those obtained with control culture media (IgM ELISA, 0.072 ⁇ 0.001 ; IgG ELISA 0.077 ⁇ 0.001 ) or supernatant fluid from isotope-matched negative control hybridomas. These results are representative of those obtained in at least three experiments.
  • b Parenthesis indicate that the V H genes used are similar to those cited, but are likely to be distinct genes.
  • c ND not determined because the homologous gene has not been identified in C57BL/6 mice, the size of the D region was too small, or there were ambiguities in the sequence of TG7-138.
  • ADDRESSEE JEFFREY L. WILSON
  • B STREET: SUITE 1400, UNIVERSITY TOWER, 3100 TOWER
  • ATTORNEY/AGENT INFORMATION (A) NAME: JEFFREY L. WILSON (B) REGISTRATION NUMBER: 36,058

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Abstract

La présente invention se rapporte à un procédé de fabrication d'anticorps monoclonaux. Dans ce procédé, on utilise un animal immunisé possédant des cellules fabriquant des anticorps à tolérance périphérique rompue. L'invention concerne également un procédé d'utilisation de ces anticorps monoclonaux et des anticorps polyclonaux dérivés de l'animal immunisé possédant des cellules fabriquant des anticorps à tolérance périphérique rompue, l'invention pouvant servir au diagnostic et à la thérapie cliniques in vitro et in vivo.
PCT/US1998/025253 1997-11-28 1998-11-25 Procedes de fabrication d'anticorps en rapport avec la rupture de la tolerance peripherique dans les lymphocytes b Ceased WO1999027963A1 (fr)

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US7300911B2 (en) 2000-03-04 2007-11-27 Henkel Kommanditgesellschaft Auf Aktien Method of preparing multiphase laundry detergent and cleaning product shaped bodies having noncompressed parts

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