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WO1990004180A1 - Procedes therapeutiques et diagnostiques utilisant des molecules de surface de cellules t - Google Patents

Procedes therapeutiques et diagnostiques utilisant des molecules de surface de cellules t Download PDF

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Publication number
WO1990004180A1
WO1990004180A1 PCT/US1989/004413 US8904413W WO9004180A1 WO 1990004180 A1 WO1990004180 A1 WO 1990004180A1 US 8904413 W US8904413 W US 8904413W WO 9004180 A1 WO9004180 A1 WO 9004180A1
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soluble
antibody
monoclonal antibody
cell
patient
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Patrick C. Kung
Stephen H. Ip
Michael C. Brown
Linda A. Mackeen
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T Cell Sciences Inc
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T Cell Sciences Inc
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Priority to KR1019900701202A priority Critical patent/KR900702370A/ko
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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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2812—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2815—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD8
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56977—HLA or MHC typing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • G01N2800/245—Transplantation related diseases, e.g. graft versus host disease

Definitions

  • the present invention is directed to the measurement of soluble T cell surface molecules, such as soluble T cell growth factor receptors and T cell differentiation antigens or fragments thereof, and the application of such
  • a plurality of such molecules can be used in monitoring the effect of a therapeutic treatment, detecting and/or staging disease or in differential diagnosis of a physiological condition.
  • T CELL GROWTH FACTORS AND RECEPTORS T cells secrete a variety of polypeptides affecting immunoregulation of hematopoietic cells and are themselves subject to regulation by hormone peptides interacting with specific receptors on their cell surface.
  • InterleuJcin 2 (IL-2) originally termed T cell growth factor, is
  • interleuJcin-1 and must interact with specific high-affinity membrane receptors to exert its biological effects (Smith, K.A., 1980, Immunol. Rev. 51:337-357; Leonard, W.J., et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:6957-6961).
  • the interleuJcin 2 receptor (IL2R, Tac antigen) is not present on the surface of resting T or B lymphocytes.
  • T cell proliferation mediated by an autocrine mechanism whereby activated cells secrete IL-2 and also express cell surface receptors for
  • IL-2 (IL2R) (Leonard, W.J., et al., 1982, Nature 300:267;
  • B cells In addition to T cells, B cells (Mingari, M.C., et al., 1984, Nature 312:641-3; Pike, B.L., et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:7917-21; Saiki, O., et al., 1988, J. Immunol. 140:853-8), NK cells (Ortaldo, J.R., et al., 1984, J. Immunol. 133:779-83; Kehrl, J.H., et al., 1988, J. Clin. Invest. 81:200-5) and possibly monocytes
  • IL-2 binding site Teshigawara, K., et al., 1987, J. Exp. Med. 165:223
  • the larger IL-2 binding protein 75 kD molecular weight
  • the smaller protein 55 kD molecular weight
  • the alpha chain was the first IL-2 binding protein to become recognized as an "activation antigen" on the surface of activated T cells (hence the name anti-Tac for "T activated") (Uchiyama, T., et al., 1981, J. Immunol. 126:1393-7).
  • IL-2 Interaction of IL-2 with its cell surface receptor results in a continuous T cell proliferation (Greene, W.C. and Leonard, W.J., 1986, Ann. Rev. Immunol. 4:69-95; Smith, K.A., 1984, Ann, Rev. Immunol. 2:319-333).
  • Measurement of IL2R provides information on the state of immune activation of the lymphoid population. This has been accomplished by measuring IL2R on cell surfaces using flow cytometry or fluorescence microscopy. Using monoclonal antibodies which define the IL-2 receptor, altered IL-2 receptor expression has been reported in a number of immune abnormalities (Greene and Leonard, supra; Depper, J.M., et al., 1984, J. Immunol.
  • Membrane IL2R has been found on certain B- or T-cell malignancies including Burkitt's lymphoma (Waldmann, T.A., et al., 1984, J. Exp. Med.
  • ALL T cell acute lymphoblastic leukemia
  • Leukemia cells from some patients with T cell chronic lymphocytic leukemia were shown to have the receptors and a good proliferative response to exogenous interleukin 2
  • CD Clusters of differentiation
  • T cell clusters of differentiation and other T cell surface molecules are listed in Table I.
  • T cell surface markers serve as markers of the cell lineage, the identity of the functional T cell subset to which the T cell belongs, and the activation state of the T cell.
  • T cell antigen receptor a surface molecule which comprises a disulfide-linker dimer of approximately 90 kilodaltons (kd)
  • kd kilodaltons
  • the CD3 structure is a three-chain complex associated with the T cell receptor (Kannellopoulos, J.M., et al., 1983, EMBO J. 2:1807; Borst, J., et al., 1983, Eur. J. Immunol 13:576; Van Den Elsen, P, et al., 1984, Nature 312:413; Meuer, S.C., et al., 1983, J. Exp. Med.
  • Lymphokine receptors e.g. interleukin 2 (IL-2) receptor and interleukin 1 (IL-1) receptor
  • IL-2 interleukin 2
  • IL-1 interleukin 1
  • CD8 is a T cell specific surface glycoprotein expressed on the surface of approximately 30% of T lymphocytes associated with
  • CD4 (OKT4 antigen) is a 55 kd glycoprotein expressed on the surface of approximately 60% of all T lymphocytes and is associated with helper function (Reinherz et al., 1979, Proc. Natl.
  • CD4 has also been identified as the receptor for the HTLV-III virus associated with acquired immune deficiency syndrome (AIDS) (McDougal, J.S., et al., 1986, Science 231:382-385).
  • AIDS acquired immune deficiency syndrome
  • T cell typing involves the use of monoclonal antibodies which define T cell surface markers to detect the presence of specific cell surface markers on the T cell surface. Measuring the total numbers of T cells by surface markers has been useful for the characterization and classification of lymphoid malignancies (Greaves, M., et al., 1981, Int. J. Immiinopharmac.
  • T cell surface markers has also been used for the assessment of the immune status of patients.
  • the activation antigens e.g. IL-2 receptor
  • Antibodies to CD4 have been widely described (Kung, P.C., et al., 1979, Science 206:347-349) and are
  • OKT4 Such a set has been termed OKT4,
  • OKT4A, OKT4B, OKT4C, OKT4D, OKT4E, and OKT4F (Rao, P.E., et al., 1983, Cell. Immunol. 80:310).
  • Antibodies directed against the CD4 or CD8 antigens have been shown to block cell function.
  • Antibodies against CD4 block most helper T functions, mixed lymphocyte
  • CD4 is internalized upon treatment of the cells with phorbol esters and resulting phosphorylation (Hoxie, J.A., et al., 1986, J. Immunol. 137:1194-1201).
  • CD4 The cloning of the gene encoding CD4 reveals that it, like CD8, is a member of the immunoglobulin supergene family, containing both amino acid (32%) and structural ( ⁇ sheets held together by disulfide bridges) homology at the V (variable)-like domain of CD4 to the V region of
  • CD8 exists on the cell surface as dimeric or multimeric structures composed of a 33 kD monomer (Snow, P.M., et al., 1983, J. Biol. Chem. 258:14675-14681). 2.3. SOLUBLE IMMUNE CELL SURFACE MOLECULES
  • HLA molecules are sets of cell surface glycoproteins involved in immune recognition. These macromolecular antigens have also been found to be present in body fluids such as serum (Pellegrino, M.A., et al., 1984, Meth. Enzymol. 108:614-624).
  • the serum levels of Class I HLA-A and HLA-B have been shown to be present in sufficient quantity to perform HLA-typing in sera (Russo, C. , et al., 1983, Transplant. Proc. 15(l):66-68; Pellegrino, M.A., et al., 1981, Transplant. Proc. 13 (4) : 1935-1938).
  • the presence of Class II HLA-DR in serum has also been detected (Sandrin, M.S., et al., 1981, J. Natl. Cancer Inst.
  • IL2R A soluble form of IL2R has been detected (Rubin et al., 1985, J. Immunol. 135:3172-3177; Rubin et al., 1985, Fed. Proc. 44:946; U.S. Patent No. 4,707,443 by Nelson et al.) that is released by activated normal peripheral blood mononuclear cells and synthesized in large amounts in vitro by HTLV-I-infected leukemic cell lines. A sandwich enzyme immunoassay was used to quantitate the soluble IL2R.
  • soluble IL2R is capable of binding interleukin 2 (Rubin, L.A., et al., 1985, J. Immunol.
  • the soluble IL2R has been suggested to be a "blocking factor" produced by the malignant cells to inhibit the host's immune response to the tumor (id.).
  • Elevated levels of soluble IL2R have also been reported present in the serum of aged subjects (Saadeh, C., et al., 1986, Fed. Proc. 45:378), and in patients with AIDS (Saadeh, supra).
  • CD2 a T cell surface molecule present in all normal T cells and a receptor for sheep red blood cells, has been detected at higher levels in the sera of certain cancer patients than those found in normal control patients (Falcao, R.P., et al., 1984, Clin. Lab. Immunol. 13:141-143; Oh. S.-K., et al., 1985, Scand. J. Immunol. 22:51-60).
  • CD8 Leu 2, OKT8
  • Leu-1 another T cell surface molecule, was measured in serum following anti-Leu-1 monoclonal antibody treatment (Miller, R.A., et al., 1982, New Engl. J. Med. 306:517-520).
  • Oh et al. (1985, supra) reported that less than half of the patients with malignancies in their study presented elevated levels of soluble OKT11 receptor in their serum. However, not all T cell surface molecules are released into the serum (Fujimoto, J., et al., 1983, J. Exp. Med. 159:752-766).
  • Leu 1 antigen was not detectable in the serum of normal or leukemic patients who have not received
  • Leu 3 antigens were also not detectable in soluble form in T cell culture supernatants (id.).
  • PCT Publication No. WO 87/03600 published June 18, 1987, entitled "Assay Systems for Detecting Cell Free T Cell Antigen Receptor Related Molecules and Clinical Utilities of the Assays" concerns methods for diagnosing diseases and for monitoring diseased conditions by measuring the amount of soluble T cell antigen receptor in a subject's body fluid.
  • PCT publication No. WO 87/05912, published October 8, 1987, entitled “Therapeutic and Diagnostic Methods Using Soluble T Cell Surface Molecules” relates to the measurement of certain soluble T cell growth factor receptors and soluble T cell differentiation antigens in the diagnosis and therapy of various diseases and disorders.
  • the present invention is directed to the measurement of soluble T cell growth factor receptors, soluble T cell differentiation antigens, or related soluble molecules or fragments thereof, and the use of such measurements in the diagnosis and therapy of diseases and disorders.
  • the measurement of such molecules can be valuable in monitoring the effect of a therapeutic treatment on a subject. detecting and/or staging a disease in a subject, and in differential diagnosis of a physiological condition in a subject. These measurements can also aid in predicting therapeutic outcome and in evaluating and monitoring the immune status of patients.
  • a rise in both soluble IL2R and creatinine in a body fluid of a patient can be used to predict allograft rejection or to differentially diagnose renal allograft rejection from infection in a transplant patient.
  • a change in serum IL2R concentrations in serial samples can be more sensitive than the absolute level of serum IL2R for the diagnosis of rejection.
  • the invention is also directed to the measurement of serum IL2R levels to stage non-lymphatic malignancies.
  • the invention also relates to immunoassays which preferentially detect soluble CD4 over the cell-surface CD4.
  • An increase in soluble CD4 antigen levels in a sample from a patient can be used to diagnose a state of immune activation.
  • Such an increase in soluble CD4 antigen levels in synovial fluid can be used to diagnose rheumatoid
  • Soluble CD4 measurements can also be used to stage adult T cell leukemia, or determine the phenotype of a cell in culture. Soluble CD4 measurements can also be used to monitor AIDS patients undergoing therapy.
  • the invention also relates to the measurement of a plurality of soluble T cell surface markers, for the
  • the measurement of a plurality of soluble T cell surface markers and their change relative to one another can be superior to the measurement of any soluble T cell surface marker alone, for the detecting, staging, or monitoring of treatment of a disease or disorder.
  • measurements of the soluble T cell surface molecules can be accomplished by sandwich enzyme immunoassays. Kits for such measurements are also provided.
  • Staging a disease assessing the degree of severity according to standard
  • AC adenocarcinoma
  • HC hairy cell leukemia
  • HTLV III/LAV/HIV Human T Cell Leukemia Virus
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • PBMC peripheral blood mononuclear cell
  • PHA phytohemagglutinin
  • SCLC squamous cell lung carcinoma
  • Spontaneous release release by normal or pathologic physiological processes of the cell
  • AZT azido-deoxythymidine
  • RF rheumatoid factor 4 .
  • FIG. 1 Levels of soluble CD4 in sera of normal individuals and patients from a number of disease groups.
  • the assay used was as described in Section 6.2.1, infra.
  • CD4 antigen was detected using mAb 8F4 as capture reagent and mAb R2B7 as detection reagent in a sandwich immunoassay.
  • the limit of sensitivity for the assay was 20 units.
  • Figure 2 Levels of soluble CD4 in sera of normal individuals and patients from a number of disease groups. The assay used was as described in Section 6.2.2, infra. SF: synovial fluid; EBV/mono: Epstein Barr
  • AIDS-related complex ARC
  • Closed diamonds soluble CD8 levels (U/ml); Open diamonds: HIV p24 levels (pg/ml) x 10.
  • Figure 5 Dot plots of the change in serum IL2R (A), urine IL2R (B), or serum creatinine (Cr) (C) concentrations by clinical status: stable (S), rejection (R), cyclosporine toxicity (C), and infection (I). Samples of rejection are from 2 days before the first rise in Cr to the time of institution of antirejection therapy.
  • Figure 6 A plot of the sensitivity and specificity of the serum IL2R assay and serum creatinine (Cr) for the diagnosis of rejection.
  • the data from Figure 5(a,c) are given in the form of receiver operating characteristic (ROC) curves.
  • ROC receiver operating characteristic
  • the threshold for a positive test is varied over a broad range (here from the 70th to the 99th percentile of stable patients). The further the curves are from the diagonal line (which represents chance alone), the better the discrimination of the test.
  • the curves for IL2R and Cr are equivalent.
  • the present invention is directed to the measurement of soluble T cell growth factor receptors, soluble T cell differentiation antigens, or related soluble molecules or fragments thereof, and the use of such measurements in the diagnosis and therapy of diseases and disorders.
  • soluble shall mean those molecules that are “spontaneously released”; i.e., released by normal or pathologic physiological processes of the cell. Such molecules are to be distinguished from “solubilized” cell surface forms of the molecules, whose solubilization is brought about by in vitro manipulation such as cell lysis by detergent.
  • the soluble T cell markers (antigens and others).
  • receptors of the invention are molecules which carry antigenic determinants of their cell-surface counterparts.
  • Proteinaceous molecules, or fragments thereof, derived from the surface of T cells, and proteinaceous molecules which have immunologically similar counterparts present on the surface of T cells or activated T cells, which are present in a body fluid and not associated with the surface of a T cell are soluble T cell surface molecules of the invention. These molecules can be either glycosylated or nonglycosylated and may be soluble by themselves or
  • the measurement of the soluble molecules of the invention can be valuable in monitoring the effect of a therapeutic treatment on a subject, detecting and/or staging a disease in a subject, and in differential diagnosis of a physiological condition in a subject. These measurements can also aid in predicting therapeutic outcome and in evaluating and monitoring the immune status of patients.
  • More than one type of soluble molecule can be measured.
  • the soluble molecules can be measured in any body fluid of the subject including but not limited to serum, plasma, urine, and saliva.
  • the present invention provides a method for monitoring the effect of a therapeutic treatment on a subject who has undergone the therapeutic treatment.
  • This method comprises measuring at suitable time intervals the amount of a soluble molecule or soluble fragment thereof which comprises, or is immunologically related to, a T cell growth factor receptor or T cell differentiation antigen. Any change or absence of change in the amount of the soluble molecule can be
  • soluble molecules immunologically related to the CD4 antigen can be measured in the serum of patients by a sandwich enzyme immunoassay in order to evaluate the
  • the levels of soluble CD4 molecules can be measured in the serum of AIDS patients in order to evaluate the therapeutic efficacy of treatments such as
  • the therapeutic treatments which may be evaluated according to the present invention include but are not limited to radiotherapy, drug administration, vaccine administration immunosuppressive or immunoenhancive
  • the immunosuppressant regimens include, but are not limited to administration of drugs such as
  • Cyclosporin A chlorambucil, cyclophosphamide, or
  • the immunoenhancive regimens include, but are not limited to administration of interleukin-1, interleukin-2, and other T cell growth factors.
  • measurement of a soluble molecule which comprises, or is immunologically related to, a T cell growth factor receptor or T cell differentiation antigen can be used to detect and/or stage a disease or disorder in a subject.
  • the measured amount of the soluble molecule is compared to a baseline level.
  • This baseline level can be the amount which is established to be normally present in the body fluid of subjects with various degrees of the disease or disorder.
  • An amount present in the body fluid of the subject which is similar to a standard amount, established to be normally present in the body fluid of the subject during a specific stage of the disease or disorder, is indicative of the stage of the disease in the subject.
  • the baseline level could also be the level present in the subject prior to the onset of disease or the amount present during remission of
  • Diseases or disorders which may be detected and/or staged in a subject according to the present invention include but are not limited to those listed in Table II, infra.
  • measurements of plasma or serum levels of the soluble molecules can be used in the detection of disease, or to determine disease stage and assign risk.
  • Rheumatoid arthritis can be monitored by measuring soluble CD4 levels in a patient.
  • elevated levels of soluble CD4 in synovial fluid relative to serum is a diagnostic indication of rheumatoid arthritis.
  • detection of an increase in soluble CD4 antigen in the body fluid of a patient can be used to diagnose a state of immune
  • Soluble CD4 measurements can also be used to detect and/or stage adult T cell leukemia. Elevation of CD4 antigen levels in the synovial fluid of a patient can indicate rheumatoid arthritis. In yet another embodiment, the detection of soluble CD4 in cell culture supernatants can be relied on as an indication of the CD4 phenotype of the lymphocytes present.
  • the measurement of soluble T cell growth factor receptors, T cell surface antigens, or immunologically related molecules can be used to differentially diagnose in a subject a particular physiological condition as distinct from among two or more physiological conditions.
  • the measured amount of the soluble molecule is compared with the amount of the soluble molecule normally present in a body fluid of a subject with one of the suspected physiological conditions.
  • a measured amount of the soluble molecule similar to to the amount normally present in a subject with one of the physiological conditions, and not normally present in a subject with one or more of the other physiological
  • measurement of soluble molecules can be used in the differential diagnosis of renal allograft rejection, especially in distinguishing Cyclosporin A nephrotoxicity or infection. Similar differential diagnosis of allograft rejection using the methods of the invention can be applied to other organ allografts, including but not limited to liver, heart, and pancreas.
  • the measurement of changes in the levels of soluble molecules can be used to differentially diagnose renal allograft rejection.
  • T cell surface molecule or immunologically related molecule which is present in soluble form in the body fluid at levels which correlate with a disease condition or disorder, or a stage thereof, may be used in the practice of the present invention.
  • T cell surface markers which may potentially be used include but are not limited to those listed in Table I, supra.
  • CD4, CD8, and IL2R molecules may be measured.
  • immunodiffusion assays agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and immunoelectrophoresis assays, to name but a few.
  • a sandwich enzyme In a preferred embodiment, a sandwich enzyme
  • mAb 1 directed against the soluble antigen is adsorbed onto a solid substratum.
  • the soluble antigen present in the sample binds to the antibody, and unreacted sample components are removed by washing.
  • An enzymeconjugated monoclonal antibody (detection antibody, mAb 2) directed against a second epitope of the antigen binds to the antigen captured by mAb 1 and completes the sandwich.
  • a substrate solution is added to the wells.
  • a colored product is formed in proportion to the amount of antigen present in the sample.
  • the reaction is terminated by addition of stop solution and absorbance is measured spectrophotometrically.
  • a standard curve is prepared from known concentrations of the soluble antigen, from which unknown sample values can be determined.
  • such an assay may be used to determine soluble IL2R levels or soluble T cell antigen levels.
  • anti-IL2R mAbs 2R12 and 7G7 can be used as the capture and detection antibodies
  • anti-CD8 mAbs 4C9 and 5F4 can be used as the capture and detection antibodies, respectively, in a sandwich enzyme immunoassay (such as the CELLFREE ® T8 Test Kit assay, T Cell Sciences, Cambridge, MA).
  • anti-CD4 mAbs 8F4 and R2B7 can be used as the capture and detection reagents, respectively, in a sandwich enzyme immunoassay (see Sections 5.4.3 and 6, infra).
  • Kits for carrying out the assays of, and for use in the practice of, the present invention are also within the scope of the invention.
  • a kit can comprise a pair of antibodies to the same T cell marker (receptor or antigen), which antibodies do not compete for the same binding site on the marker.
  • a kit can comprise more than one pair of such antibodies, each pair directed against a different T cell marker, thus useful for the detection or measurement of a plurality of T cell markers.
  • the present invention also provides a way of deriving immunoassay systems which preferentially detect/quantitate physiologically released (soluble) forms of cell surface markers over solubilized (e.g., detergent-treated) cell surface markers.
  • solubilized e.g., detergent-treated
  • the recombinant forms of the specific cell surface marker to be assayed which have been genetically engineered to be physiologically soluble (i.e., by deletion of DNA sequences encoding the transmembrane region).
  • Such recombinant forms are likely to lack epitopes found on the transmembrane region, which epitopes are thus specific to the solubilized cell surface marker and which epitopes are likely also to be absent from the physiologically released form of the marker.
  • the recombinant molecule can be used to screen anti- cell surface marker antibodies for determination of the appropriate antibodies to be used for preferential detection of the physiologically released form of the surface marker. Pairs of antibodies can be screened for optimization of a sandwich ELISA for detection of soluble cell-surface marker. This aspect of the invention is illustrated by way of example in Section 6, infra, where a soluble CD4 assay is devised that preferentially detects soluble CD4 relative to solubilized CD4.
  • Antibodies can be produced for testing for suitability for use in the detection of soluble forms of T cell surface markers. Such antibodies can be polyclonal or monoclonal. Monoclonal antibodies are preferred for use.
  • T cell surface molecule Various procedures known in the art may be used for the production of polyclonal antibodies to epitopes of a given T cell surface molecule.
  • various host animals can be immunized by injection with a T cell surface molecule, a recombinant version thereof, synthetic protein, or fragment thereof, including but not limited to rabbits, mice, rats, etc.
  • the immunogen is a truncated recombinant soluble form of the T cell surface molecule.
  • adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • a monoclonal antibody to an epitope of the T cell surface molecule can be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497), and the more recent human B cell hybridoma technique (Kozbor et al.. 1983, Immunology Today 4:72) and EBV-hybridoma technique
  • the monoclonal antibodies may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies.
  • Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci.
  • chimeric antibody molecules may be prepared containing a mouse (or rat, or other species) antigen-binding domain with human constant regions (Morrison et al., 1984, Proc. Natl.
  • a molecular clone of an antibody to an epitope of a T cell surface molecule can be prepared by known techniques.
  • nucleic acid sequences which encode a
  • Antibody molecules may be purified by known
  • Antibody fragments which contain the idiotype of the molecule can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • the invention is also directed to assays for
  • anti-CD4 mAbs 8F4 and R2B7 can be used as the capture and detection reagents, respectively, in a sandwich immunoassay.
  • Soluble CD4 has been specifically quantitated, according to the present invention, and has been shown to be a reliable indicator of various pathologic conditions (see section 6, infra). Thus, detection and/or measurement of soluble CD4 can be used to diagnose, to monitor, and/or to stage various diseases and disorders involving the immune system. 5.4.4. DETECTING OR STAGING OF DISEASE OR MONITORING OF RESPONSE TO TREATMENT IN PATIENTS BY MEASUREMENT OF A PLURALITY OF SOLUBLE T CELL SURFACE MARKERS
  • the present invention also provides for the detecting or staging of disease, or the monitoring of treatment by measuring a plurality (at least two) T cell surface markers (receptors or differentiation antigens).
  • a plurality of soluble T cell markers selected from among soluble IL2R, CD4, and CD8 can be measured to diagnose, stage, or monitor treatment of diseases or disorders.
  • diseases or disorders include those discussed supra in
  • Soluble marker levels can represent a measure of immune system function, paralleling disease course or treatment efficacy.
  • the prognostic indicator is the observed change in different marker levels relative to one another, rather than the absolute levels of the soluble markers present at any one time. Since soluble CD4, soluble CD8 and soluble IL2R levels are measures of the immune system itself, they should provide a much improved measure of the relative health of the immune system during various stages of diseases or disorders.
  • measurements of a plurality of soluble T cell surface markers are used to detect, stage, or monitor treatment of diseases and disorders caused by HIV (the causative agent of AIDS) infection.
  • soluble CD4 in particular, but soluble CD8 and soluble IL2R receptors as well, can be identified and detected in HIV-infected patients with different manifestations of disease, it becomes possible to develop a sensitive immunoassay to monitor AIDS therapies and vaccines.
  • the CELLFREE® Test Kit T Cell Sciences, Cambridge, MA assays can be useful for this purpose. Due to the intimate involvement of CD4 in the etiology of AIDS, it is expected that spontaneously released soluble CD4 levels should be extremely sensitive markers of the state of immune function during various stages of HIV infection and therapeutic treatments. This is especially true, as soluble CD4 is produced when CD4 cells become activated, (see
  • soluble T cell markers such as soluble IL2R and soluble CD8, that also indicate the state of immune function should be valuable.
  • the best index for monitoring AIDS treatment or disease progression can be a profile of soluble T cell markers, such as soluble CD4, CD8 and IL2R, rather than any individual marker alone (see Sections 6.3 and Section 7).
  • soluble T cell markers such as soluble CD4, CD8 and IL2R
  • Such a profile can be obtained by determining the soluble receptor levels of a panel of soluble receptors in
  • the approach that can be taken is to determine the levels of soluble CD4 (and soluble CD8 and soluble IL2R) levels in longitudinal time studies and to compare these values with a baseline level.
  • the baseline level can be either the level of the soluble marker present in normal, disease free individuals; and/or the levels present prior to treatment, or during remission of disease, or during periods of stability. These levels can then be correlated with the disease course or treatment outcome.
  • Antibody Leu3a biotin or FITC labeled, was purchased from Becton Dickinson, Mountain View, California.
  • Antibodies B67.2 and B66.1 were from G. Trincherie, Wistar Institute.
  • Antibody 3G2 was from Sanchex Madrid, Madrid, Spain.
  • Antibody R2B7 was obtained from a fusion of rat spleen cells (carried out according to standard procedures), from an animal immunized with whole human peripheral blood lymphocytes, with mouse SP2/0 myeloma cells. Clone R2B7 was selected from this fusion based on its ability to stain populations of
  • peripheral blood lymphocytes identical to these identified by OKT4.
  • Antibodies were purified either by ammonium sulfate precipitation or by protein A sepharose using the Biorad MPAS buffer system (BioRad Corporation, Richmond,
  • HRP horseradish peroxidase
  • Antibodies generated from a fusion of mice immunized with intact T cells were screened for their ability to substitute for Leu3A in the assay as follows: Plates were coated with R2B7 as described, blocked and incubated with recombinant soluble CD4 for 2 hours at 37oC. Plates were washed and 50 ⁇ l of each hybridoma supernatant at 1-10 ⁇ g/ml were added followed by 50 ⁇ l of biotinyl Leu3A.
  • the enzyme immunoassay for the CD4 antigen was based on the sandwich immunoassay technique. Briefly, each well of a microtiter plate (Nunc, certified high binding) was coated overnight at 4oC with a solution of murine monoclonal anti-human CD4 antibody in PBS, pH 7.4. Any remaining protein-binding sites on the microtiter wells were then blocked for two hours at 37oC with 300 ⁇ l per well of a solution of BSA (1%) (Kirkegard and Perry Laboratories, Maryland) and Tween 20 (0.05%) (Zymed Laboratories, South San Francisco, California) in phosphate buffered saline (PBS), pH 7.4. The wells were then washed three times with 350 ⁇ l per well of PBS (pH 7.4) with 0.05% Tween 20.
  • the wash solution was aspirated from the wells and 50 ⁇ l of a sample diluent consisting of 0.15 M NaCl, 25 mM Tris (pH 7.4) supplemented with bovine proteins was added to each well. Fifty ⁇ l of standard or sample were added to the appropriate wells in duplicate. The solution in the wells was mixed thoroughly by gently tapping the side of the plate for fifteen seconds. The plate was then sealed and incubated at 37 o C for 2 hours . At the end of this incubation period, the solution was aspirated from the plate and each well was washed three times with 350 ⁇ l of PBS/Tween 20 as above.
  • HRP horseradish peroxidase
  • biotin conjugates were substituted for the HRP antibody conjugate. After the 2 hour incubation, wells were washed and 100 ⁇ l of streptavidin horseradish peroxidase (Zymed Laboratories) at 0.5 ⁇ g/ml in 1% bovine serum albumin in tris buffered saline was added. Following a 30 minute incubation at 37oC, wells were washed and color developed as described above.
  • assays were also performed as a single step assay in which conjugated antibody was added at the same time as the sample and incubated for 4 hours at room temperature on a rotating shaker platform, after which washing and color development were performed as described.
  • the configuration of the initial assay was modified by optimizing each of the assay reagents. This resulted in an improved sensitivity for the overall assay where much lower levels of soluble CD4 could be reliably and reproducibly detected.
  • the optimized assay configuration is given in Table III.
  • aggregated IgG was added to the sample and conjugate diluent buffers.
  • the aggregated IgG was prepared by heating a 100 ⁇ g/ml solution of IgG in 100 mM sodium phosphate buffer, 0.9% NaCl, pH 5.56 at 56-60oC for 50 minutes, followed by neutralization with dibasic sodium phosphate, 0.9% NaCl, pH 8 to give a final pH of 7.4. 6.1.3. CELL PROCEDURES
  • mononuclear cells were prepared using Ficoll Hypaque gradients. Cells were put into culture along with
  • PHA phytohemagglutinin
  • phorbol myristate acetate 1 ng/ml
  • ionophore A2317 0.1 ng/ml
  • OKT3 anti-T3 monoclonal antibody
  • Cytofluorograph II (Ortho Diagnostic System, Westwood, Massachusetts) and FITC labeled 0KT4 or OKT8 (Ortho
  • Recombinant souble CD4 was obtained from cell culture supernatant of a Chinese hamster ovary (CHO) cell line transfected with CD4 truncated at the transmembrane exon (Fisher, R.A., et al., 1988, Nature 331:76-78).
  • Antibodies were generated from a mouse immunized with whole T cells and screened for their ability to replace Leu3a in an assay. 500 hybridoma clones were screened and three clones meeting the above criteria were identified. One of these clones, termed 8F4, showed the ability to block binding of FITC labeled Leu3A to CD4 positive T cell
  • Antibodies 8F4 and R2B7 were evaluated with regard to optimal configuration in the assay.
  • Table VIII shows that 8F4 used as capture antibody with R2B7 used as detection antibody produced a significantly greater ratio of signal observed using recombinant soluble CD4 to signal observed using detergent solubilized membrane CD4, compared to the ratio of signal observed with R2B7 as a capture antibody and 8F4 as detection.
  • Antibodies were evaluated for CD4 detection using different combinations of capture and detection reagents in assay matrices. Assays were carried out using 25% fetal calf serum in tris buffered saline, with and without Nonidet P40 (NP40) in both sample and conjugate diluents (Table IX). I0T4 reacted with detergent solubilized CD4 but failed to react with the recombinant soluble CD4. Also shown in Table IX are antibodies 8F4 and R2B7 in combination with
  • IOT4 When used as a capture reagent, IOT4 detected only CD4 in cell lysates. Interestingly, however, when IOT4 was used as a detection reagent with 8F4 or R2B7 used as capture reagent, a much stronger signal is seen for the recombinant CD4 antigen than is seen with solubilized CD4 in cell lysates. It should be noted that this signal (for recombinant CD4) is significantly less than the signal obtained when R2B7 is paired with 8F4 as either detection or capture reagent. R2B7 when paired with itself was capable of a strong signal for both recombinant and cell lysate samples. Inclusion of NP40 failed to disrupt this signal.
  • Table X shows the results of screening culture
  • Soluble CD4 was determined using 100 ⁇ l cell culture supernatant in a single-step assay using R2B7 as the antibody immobilized on the solid phase with biotinyl Leu3a and streptavidin peroxidase used for detection.
  • a commercially available sandwich immunoassay kit (CELLFREE ® T8 Test Kit, T Cell Sciences, Inc., Cambridge, MA) was used to measure soluble CD8.
  • the CD8 antigen detected by this assay has been characterized previously as a 52-55 kD dimer composed of monomer polypeptides, each with a molecular weight of approximately 27 kD.
  • Table XI shows the rate of release of CD4 into the media after in vitro stimulation of peripheral blood
  • Table XII shows levels of soluble CD4 detected in sera of individuals with HTLV-1 associated adult T cell leukemia.
  • Figure 1 shows the levels of CD4 in sera of normal individuals and in patients from a number of disease groups. Levels of CD4 in synovial fluid of rheumatoid arthritis patients and in sera of lung cancer patients were elevated as compared to the levels in sera from normal individuals.
  • Table XIII shows CD4 levels in longitudinal samples from patients on IL-2 therapy.
  • Soluble CD8 was measured using the CELLFREE* T8 Test Kit (T Cell Sciences, Cambridge, MA). Soluble CD4 was measured using the CELLFREE* T8 Test Kit (T Cell Sciences, Cambridge, MA). Soluble CD4 was measured using the CELLFREE* T8 Test Kit (T Cell Sciences, Cambridge, MA). Soluble CD4 was measured using the CELLFREE* T8 Test Kit (T Cell Sciences, Cambridge, MA). Soluble CD4 was measured using the CELLFREE* T8 Test Kit (T Cell Sciences, Cambridge, MA). Soluble CD4 was
  • Table XIII shows that detectable levels of soluble CD4 are present in sera of patients being treated with IL-2.
  • One of the events observed in IL-2 therapy is an increase in circulating activated CD4 positive lymphocytes. Soluble CD4 levels in these patients fluctuate throughout the course of therapy and may have prognostic value.
  • Table XIV shows levels of soluble CD4, along with soluble IL2R, in renal transplantation patients.
  • Diagnosis was either cyclosporin A toxicity (CsA), stable transplant, or rejection; multiple patient serum samples were taken at different times.
  • CsA cyclosporin A toxicity
  • Soluble IL2R was measured using the CELLFREE ® IL-2R Test kit (T Cell Sciences, Cambridge, MA).
  • Soluble T8 was measured using the CELLFREE ® T8 Test kit
  • a change in the observed level for any particular marker such as an increase or decrease or no change, may be of more value than the absolute level of a marker present at any one point in time, for the diagnosis or monitoring of treatment in disease (see Section 8,
  • the change in the observed level of a marker must be compared to a baseline level which could either be the level seen in normal individuals with no disease, the pre-transplant level in the renal patient, the value present in a stable situation or during remission of symptoms, etc.
  • the diagnosis of disease or monitoring of treatments of patients with renal transplants or other diseases and states of immune activation will be through an analysis of a panel of soluble T cell markers, rather than from only one individual marker.
  • a better prognostic indicator can be a rise in one marker relative to a simultaneous fall in another marker.
  • the resulting profile of soluble T cell marker expression should be an extraordinarily variable changes in the immune system as its function is modified by therapeutic treatments or disease progression (see Section 7, infra).
  • Table XVI gives the values of soluble CD4 levels seen during preliminary studies on patients with Acquired Immune Deficiency Syndrome (AIDS) and other stages of HIV-induced disease including Kaposi's Sarcoma (KS), AIDS related complex (ARC) or asymptomatic seropositive (ASYM).
  • AIDS Acquired Immune Deficiency Syndrome
  • KS Kaposi's Sarcoma
  • ARC AIDS related complex
  • ASYM asymptomatic seropositive
  • HA heat aggregated IgG added to remove any false positive problems associated with occassional high RF (rheumatoid factor) containing samples.
  • IgG unaggregated IgG control to detect samples that may have had RF problems.
  • R2B7 as one of the antibodies.
  • R2B7 when paired with 8F4 showed greatest sensitivity for detecting soluble CD4 both from recombinant and natural sources.
  • the greatest ratio of signal from a soluble recombinant CD4 to signal from sdlubilized lysate CD4 is seen using 8F4 capture with R2B7 detection. This is roughly twice the ratio seen in the reverse configuration. It is possible that binding of one or more of the antibodies induces conformational changes in the molecule.
  • Abstr. 5.54.6, p. 708 have described an enzyme immunoassay based on the use of IOT4 as a capture and detection reagent to detect CD4 in serum. In our hands, this configuration works only to measure the membrane form of the molecule and fails to adequately measure the soluble recombinant or soluble spontaneously released form of the molecule
  • CD4 release may be a function of the type and pathway of activation. Phytohemagglutinin and T3 stimulation both resulted in a release of small amounts of CD4. Stimulation with phorbol esters, known to cause phosphorylation and internalization of CD4, or with ionophores, resulted in significantly less released CD4 than did PHA stimulation, despite intense cellular activation. The kinetics of CD4 release were also significantly different between cells stimulated by phorbol esters and those stimulated by PHA. Release can also not be attributed to simple membrane turnover. No CD4 is released by resting cells in vitro.
  • CD4 T cells or T cell lines containing CD4 cells all showed detectable soluble CD4 in their culture supernatants.
  • CD8 cells showed only soluble CD8.
  • spontaneously released CD4 and membrane CD4 can be determined from patterns of antibody reactivity. If the spontaneously released material were identical to the cell-surface polypeptide, it should behave in the assays, which have detergent incorporated into them, like solubilized CD4 in cell lysate. If they are more analogous to the recombinant truncated version of CD4 they should behave like it. The latter is the case; that is, those antibody pairs which afford suitable detection of solubilized lysate CD4, but not recombinant soluble CD4, yielded poor detection of the soluble CD4 from T cell culture supernatant, whereas those antibody pairs showing optimal reactivity with recombinant soluble CD4 also reacted optimally with the released material. It is clear from antibody reactivity patterns that the released form of the CD4 antigen differs significantly from the membrane form.
  • This assay utilized a sandwich ELISA microplate format. Highly specific rabbit polyclonal antibodies to HIV p24 core antigen were immobilized on microtiter plate wells and used to capture HIV p24 core antigen present in 450 ⁇ l of plasma. The captured HIV p24 core antigen was complexed with biotinylated polyclonal antibodies to HIV p24 core antigen and probed with a streptavidin-horseradish
  • the CD4 and CD8 ratios were determined by standard flow cytometry.
  • CD4 + cells and CD8 + cells were measured in samples from patients with AIDS, ARC, KS, ASSYM or normals, as shown in
  • ASSYM and KS showed levels of both IL2R and soluble CD8 which were greater than the upper 95% value of normal (p ⁇ 0.00001).
  • IL2R was better than CD4/CD8, %CD4, and p24 for
  • HIV infection elevated soluble CD8 levels may reflect host immune response to HIV. It has been demonstrated that CD8 positive cells are able to control HIV infection in vitro by suppressing viral replication (Walker, C.M. et al., 1986,
  • Soluble CD8 levels may be an accurate measure of the immune system's attempts to suppress
  • the study population consisted of 33 adults who received renal allografts at the Massachusetts General
  • CsA cyclosporin A
  • prednisone Cold-B., et al., 1987, Clin. Immunol. Immunopathol. 43:273-276.
  • Episodes of rejection were treated with increased steroids, anti-T3 monoclonal antibody OKT3, or ATG (anti-thymocyte globulin) (Delmonico, F.L., et al., 1987, Am. Surg. 206:649-654).
  • CsA toxicity was diagnosed by a rise in creatinine that responded to decreased CsA dose.
  • cytomegalovirus infection lymphoproliteration associated with Epstein Barr virus, and a transient gastroenteritis accompanied by fever and lymphocytosis.
  • immunoreactive IL2R levels were assayed by a sandwich enzyme immunoassay test kit according to the specifications of the manufacturer (CELLFREE ® , T Cell Sciences, Inc.) (Colvin, R.B., et al., 1987, Clin. Immunol. Immunopathol. 43:273- 276). Assays were performed in batches calibrated to units based on a standard supernatant from phytohemagglutinin activated lymphocytes. 8.1.3. DATA ANALYSIS
  • the results of the serum and urine IL2R assays and the serum creatinine assays were compared according to standard statistical techniques, using a spread sheet data base. Samples taken within the first four days after transplantation and during treatment for rejection and for two days afterward were excluded from analysis. Various measurements (absolute and change between serial samples) were compared for their clinical utility, as judged by sensitivity, specificity, predictive value, and Receiver Operating Characteristic (ROC) curves (Fink, D.J. and Galen, R.S., 1982, in Computer Aids to Clinical Decisions, Vol. 2, Williams, B.T., ed., CRC Press, Cleveland, OH, pp. 1-65).
  • ROC Receiver Operating Characteristic
  • the aggregate data for serum and urine IL2R levels are summarized in Table XXXI.
  • the concentration of serum IL2R in 24 pretransplant patients was elevated compared with normal controls, but fell after transplantation in 20 of these patients.
  • Pretreatment IL2R levels from patients who had no rejection episodes did not differ from those with subsequent rejection episodes.
  • the serum IL2R levels remained elevated longer
  • Serum IL2R usually rose during episodes of rejection and fell after successful antirejection immunosuppression was instituted.
  • Figure 4 illustrates a representative patient. Seven patients with rejection episodes were tested 1-2 days before the first rise in serum creatinine. Four patients had a rise in serum IL2R greater than the 90th percentile of stable patients. Overall, the mean serum IL2R was elevated 1-2 days prior to the rise in creatinine (P ⁇ .03). Fourteen episodes of rejection in 9 patients were sampled on the first day of creatinine elevation; 9 of 14 samples (64%) were elevated above the 90th percentile of values during periods of stability.
  • the IL2R levels remained elevated after the creatinine rise and into the treatment period, finally declining during or after antirejection therapy to the "stable" levels noted above. If only biopsy-confirmed rejection episodes are analyzed, the values are not significantly different (Table XXXI). Serum IL2R also rose during episodes of infection (CMV, EBV, gastroenteritis) (Table XXXI). In contrast, serum IL2R levels were not significantly raised in 8 patients with cyclosporine A toxicity.
  • Urine IL2R During episodes of rejection, urine IL2R rose in a pattern that was not distinguishable from the serum values, except that somewhat greater sample-to-sample variation was noted. Urine IL2R also followed the pattern of serum IL2R during episodes of infection and cyclosporine A toxicity.
  • the absolute concentration of serum IL2R had a sensitivity of 46.3% and a specificity of 87.1% for the diagnosis of rejection, using the 90th percentile of stable patients as the threshold (Table XXXII). Even lower sensitivity occurred with a higher threshold (17% using the 95th percentile). Urine IL2R had comparable sensitivity and specificity (44.0% and 86.4%,
  • delta IL2R concentration in serial samples taken within a week
  • the individual data on serum and urine IL2R and serum creatinine are given in a dot plot (Fig. 5), and are summarized in Table XXXII.
  • the rise in serum IL2R had a much greater sensitivity (73.3%) than absolute serum IL2R levels for the diagnosis of rejection with no loss of specificity (86.7%).
  • the delta urine IL2R measurement was not appreciably better than the absolute urine level and less sensitive (52.4%) than the delta serum IL2R (Table XXXII).
  • a rise in serum IL2R was comparable in sensitivity and specificity to a rise in serum creatinine using the 90th percentile of stable patients to define a positive test (Table XXXII). The two tests also had a comparable
  • IL2R assays can also be valuable in the monitoring of immunologic activity in recipients of other organ

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Abstract

L'invention concerne la mesure de récepteurs solubles du facteur de croissance de cellules T, d'antigènes de différentiation solubles des cellules T, ou des molécules ou fragments de molécules apparentés, et l'utilisation de ces mesures pour le diganostic et la thérapie de maladies et de troubles pathologiques. Des modes de réalisation spécifiques impliquent le diagnostic et le contrôle d'une thérapie au moyen des valeurs absolues de ces molécules solubles. D'autres modes de réalisation impliquent la détection de modifications du niveau de ces mooécules solubles, le diagnostic et la thérapie de maladies et de troubles pathologiques. Dans des modes spécifiques de réalisation, la détection d'une augmentation de la quantité de IL2R et de créatinine solubles dans le fluide corporel d'un patient ayant subi une transplantation sert à diagnostiquer de manière différenciée un rejet d'une allogreffe rénale ou une injection. Des procédés servent à mesurer les antigènes CD4 solubles, ces mesures pouvant être utilisées, dans un mode spécifique de réalisation, afin de diagnostiquer un état d'activation immunitaire, de diagnostiquer le rhumatisme articulaire, ou de déterminer la période d'évolution d'une leucémie des cellules T adultes chez un patient. Dans un autre mode de réalisation, on peut utiliser les niveaux de CD4 soluble afin de contrôler l'efficacité de traitement de maladies, un mode de réalisation spécifique servant à contrôler la thérapie de patients infectés par le VIH. Dans un autre aspect, l'invention concerne la détection, la détection de la période d'évolution ou le contrôle de la thérapie de maladies et de troubles pathologiques au moyen de mesures d'une pluralité d'indicateurs solubles des cellules T.
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* Cited by examiner, † Cited by third party
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EP0556272A4 (fr) * 1986-03-31 1994-04-27 T Cell Diagnostics, Inc.
WO1996025670A1 (fr) * 1995-02-17 1996-08-22 E.I. Du Pont De Nemours And Company Dosage immunologique de numeration cellulaire
EP0749476A4 (fr) * 1993-12-21 1998-10-14 Harvard College Mediateurs du rejet chronique d'allogreffes
WO1999015553A3 (fr) * 1997-09-23 1999-05-20 Bundesrepublik Deutschland Let Polypeptide costimulant de lymphocytes t, anticorps monoclonaux, leur production et leur utilisation
EP0945727B1 (fr) * 1998-03-24 2003-06-11 Jcr Pharmaceuticals Co., Ltd. Méthode de diagnostic pour détecter l'infection avec VIH
US7125551B2 (en) 1997-09-23 2006-10-24 Bundersrepublik Deutschalnd Methods for treatment of asthmatic disorders using a monoclonal antibody to 8F4 polypeptide
US8318905B2 (en) 2004-04-23 2012-11-27 Richard Kroczek Antibodies for depletion of ICOS-positive cells in vivo
WO2014140197A1 (fr) * 2013-03-15 2014-09-18 Westfaelische Wilhelms-Universitaet Muenster Détection du rejet aigu d'une allogreffe rénale
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US6077948A (en) * 1993-12-21 2000-06-20 President And Fellows Of Harvard College Mediators of chronic allograft rejection (AIF-1) and DNA encoding them
EP0749476A4 (fr) * 1993-12-21 1998-10-14 Harvard College Mediateurs du rejet chronique d'allogreffes
WO1996025670A1 (fr) * 1995-02-17 1996-08-22 E.I. Du Pont De Nemours And Company Dosage immunologique de numeration cellulaire
US5658745A (en) * 1995-02-17 1997-08-19 E. I. Du Pont De Nemours And Company Cell enumeration immunoassay
US7125551B2 (en) 1997-09-23 2006-10-24 Bundersrepublik Deutschalnd Methods for treatment of asthmatic disorders using a monoclonal antibody to 8F4 polypeptide
WO1999015553A3 (fr) * 1997-09-23 1999-05-20 Bundesrepublik Deutschland Let Polypeptide costimulant de lymphocytes t, anticorps monoclonaux, leur production et leur utilisation
US7132099B2 (en) 1997-09-23 2006-11-07 Bundersrepublik Deutschland Methods of modulating T lymphocyte costimulation with an antibody to an 8F4 polypeptide
US7259247B1 (en) 1997-09-23 2007-08-21 Bundersrespublik Deutschaland Letztvertreten Durch Den Direktor Des Robert-Koch-Institutes Anti-human T-cell costimulating polypeptide monoclonal antibodies
US7722872B2 (en) 1997-09-23 2010-05-25 Bundersrepublik Deutschland Letztvertreten Durch Den Direktor Des Robert-Koch-Institutes Treatment of cancer with antibodies to costimulating polypeptide of T cells
EP0945727B1 (fr) * 1998-03-24 2003-06-11 Jcr Pharmaceuticals Co., Ltd. Méthode de diagnostic pour détecter l'infection avec VIH
US8318905B2 (en) 2004-04-23 2012-11-27 Richard Kroczek Antibodies for depletion of ICOS-positive cells in vivo
US8916155B2 (en) 2004-04-23 2014-12-23 Bundesrepublik Deutschland letztvertreten durch das Robert-Koch-Institut vertreten durch seinen Präsidenten Method for the treatment of T cell mediated conditions by depletion of ICOS-positive cells in vivo
EP2788507A4 (fr) * 2011-12-09 2015-07-08 Sequenta Inc Procédé de mesure de l'activation immunitaire
WO2014140197A1 (fr) * 2013-03-15 2014-09-18 Westfaelische Wilhelms-Universitaet Muenster Détection du rejet aigu d'une allogreffe rénale

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