CA2000308A1 - Therapeutic and diagnostic methods using soluble t cell surface molecules - Google Patents

Abstract

ABSTRACT 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. Specific embodiments involve the diagnosis and monitoring of therapy using absolute values of such soluble molecules. Further embodiments involve detecting a change in the levels of such soluble molecules, in the diagnosis and therapy of diseases and disorders. In specific embodiments, detection of increases in both soluble IL2R and creatinine in the body fluid of a transplant patient can be used to differentially diagnose renal allograft rejection from infection. The invention is also directed to methods for measurement of soluble CD4 antigens, which measurements can be used, in a specific embodiment, to diagnose a state of immune activation, to diagnose rheumatoid arthritis, or to stage adult T cell leukemia in a patient. In another embodiment, soluble CD4 levels can be used to monitor the efficacy of disease treatments, a specific embodiment including monitoring therapy of HIV-infected patients. In another aspect, the invention relates to the detection, staging, or monitoring of therapy of diseases and disorders by measuring a plurality of soluble T cell markers.

Description

~s ~ o THERAPEUTIC AND DIAGNOSTIC METHODS
USING SOLUBLE_T CELL SURFA OE _MOLECULES

l. INTRODUCTION
The present inv~ntion is directed to the measurement of soluble T cell surface molecules, such as soluble T cell 5 growth factor receptors and T cell differentiation an~igens or fragment thereo~, and the application of such measurements in the diagnosis and theraE)y o~ diseases and dissrders. The ~easurement of ~uch molecules, and preferably a plurality of ~uch moleculec;, ca~ be us~d in 10 monitoring th~ ~ffect of a therapeutic treatment, detecting and/or staging disease or in ~i~ferential diagnosis of a physiological condition~

2. BACKGROUND OF THE INVENTION

2.l. T CELL GROWTH FACTORS AND RECEPTORS
T cells secrete a variety of polypeptide~ aff~cting i~munoregulation o~ hematopoiQtic cells and are themselves subject to r~gulation by hormone peptides interacting with 20 s~ecific receptors on th~ir c~ ur~ac~. Interleukin 2 (IL-2), originally ter~d T c~ll growth factor, i3 synthe~ized and secrQted by antig~n- or lectin-activated T
ly~phocyte~ in th~ ~r~3ence of ~crophage-æerived int~rl~ukin-~ and ~ust intaract with 9p~Ci~iC high-affinity 25 me~brane r~c~ptor~ to exert it3 bioloqical e~ects (Smith, X.A., l980, I~unol. Re~. 51:337-357; L~onard, W.J., et al., 1983, Proc. Natl. Acad. Sci. U~SoA~ 80 6957-69~ The int~rlQukin 2 r~c~ptor (IL2R, Tac antig~n) is not present on th~ ~urfaca Or r~sting T or ~ lymphocy~e.R. Upon ac~ivation 3D by SpeCi~iG ~ntig~n~ or mitog~ns, T cell proli~eration is mediated by an autocrin~ m~chani3~ whQreby activated cells secr~te I~-2 a~d al~o expr~ c~ll sUrXar~ r~ceptor~ ~or IL-2 (IL2R) (LQonard, W.J., ~t al., 1982, Natur~ 300:267:
Meuer~ S.C., et æl., 1~84, Proc. Natl. Acad. Sci. U.S.A.

81:1509). In addition to T cells, B cells tMingari, M.C., et al., 1984, Nature 312:641-3; Pike, B.L., et al., 1984, Proc. Natl. ~cad. Scio U.S.A. 81:7317-21; Saiki, O., et al., 1988, J. Immunol. 140:853-B), NK cells (Ortaldo, 3.R., et al., 1984, J. Immunol. 133:779-83; Kehrl, J.H., et al., 5 19~8, J. Clin. Invest. 81:200-5) and possibly monocytes (Herrmann, F., et al., 1985, J. Immunol. 162~1111-6; Holter, W., et al., 1986, J. Immunol. 136:2171-75) express a membrane-bound IL2R.
Current evidence suggests that both chains of the IL-2 1a heterodimer receptor expressed on the surface of activated T
cells, are encoded by a single gene on human chromosome 10 (Leonard, W.J., et al., 1984, Nature 311:626:31). The high affinity IL2R that functions to signal T cell cycle progression is composed of two distinct polypeptide chains, 1S each of which contains an IL-2 binding site (Teshigawara, K., et al., 1987, J. Exp. Med. 165:223). The larger IL-2 binding protein (75 kD molecular weight) is designated as the beta chain, whereas the smaller protein (55 kD molecular weight) is termed the alpha chain (Smith, K.A., 1988, Adv.
20 Immunol. 42:165-~8). The alpha chain was the first IL-2 binding protein to become recognized as an "activation antigenn on the sur~ace of activated T cells (hence the name anti Tac for ~T activated~) ~Uchiyama, T., ek al., 1981, J.
Immunol. 126:1393-7).
Interaction o~ IL-2 with its cell surface receptor result~ 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~. Mea~urement of IL2R provides information on the state of immune activation

3~ of the lymphoid population. This has been accomplished by measuring IL2R on cell ur~aces using flow cytometry or fluorescence microscopyc Using monoclonal antibodies which : define the IL-2 receptor, altered IL-2 receptor e~pression has been reported in a number o~ i~mune abnormalities (Greene and Leonard, supra; Depper, J.M., et al., lg84, J.
Immunol. 133:1691-1695). 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.
160:1450-1466), hairy cell leukemia (Waldmann et al., supra;
5 Korsmeyer, S.J., et al., 1983, Proc. Natl. Acad. Sci. U.S.A.
80:4522-4526), and human T cell leukemia virus (HTLV)-I
associated adult T cell leukemia (Depper, J.M., et al., 1984, J. Immunol. 133:1691-1695). The function of cellular IL2R in lymphoid malignancies has not been fully elucidated.
10 Several cases of common, pre-B or T cell acute lymphoblastic leukemia (ALL) have been induced to express IL2R after in vitro activation ~Touw, I., et al., 1985, Blood 66:556-561;
Touw, I., et al., lg86, Blood 68:1088-1094; Matsuoka, M., et al., 1986, Leuk. Res. 10:597-603) and, in some cases, 15 interleukin 2 stimulated subsequent colony formation o~
neoplastic progenitor cells ln vitro (Touw, 1985, supra;
Touw, 1986, supra).
Leukemia cells from some patients with T c211 chronic l~mphocytic leukemia were shown to have the receptors and a 20 good proliferativ~ response to exogenous interleukin 2 (Uchiyama, T., et al., 1985, J. Clin. Invest. ?6:446-453;
Tsudo, M., 1986, ~lood 67:316~321). However, HTL~-l associated adult T cell leukemia constitutively expressed high level~ o~ cell surface IL2R but had no or very poor 25 pxoliferative responses to interleukin 2 (Uchiyama, 1985, supra; Arya, S.R., et al., 1984, Science 223:1086-10~7).
Ebert et al. (1985, Clin. Immunol. Immunopathol. 37:283-297) have reported that T cells fro~ patients with AIDS virus lack the ability to express IL2R on thei~ surface even when 30 the cell is activated.
Utilizing immunohistochemical staining, Kurnick reported high number of IL2R and HLA-DR positive cells in lung tumor infiltrating l~mphocytes (Kurnick, J.T., et al., 1986, ClinO Immunol. Immunopath. 38:367-380).

2.2. T CELL SUR~ACE MOLECULES
Clusters of differentiation (CD) have been established which define human leukocyte differentiation antigens (Bernard and Boumsell, 1984, Hum. Immunol. 11:1-10), by the comparison of reactivities of monoclonal antibodies directed 5 against the differentiation antigens. The T cell surface antigens, their classification into epitope-defined subgroups, and their distributions on T cells have been studied by use of monoclonal antibodies directed against human T cells (Clark et al., 1983, Immunogenetics 18:599-10 615; Hansen et al., 1984, in Leucocyte Typing, Bernard, A.,et al., eds., Springer-Verlag, New York, ppO 195-212). Some of the T cell clusters of differentiation and other T cell surface molecules are listed in Table 1.

~S

-TABLE I
T CELL SURFACE MARKERS
T Cell ~olecular Detection Surface Weight Monoclonal.
5 Marker (kd) Expression Antibody Re~erence T Cell 90 All T Cells T40/25 Brenner, M~Bo ~
Antigen et al.,1984, J.
Receptor Exp. Med. 160:
541-55~
CD8 30/43 Suppressor/ OKT8 Reinherz, E~L., et cytotoxic Leu 2 al., 1979, PNAS USA
(subset of T 76:4061-4065;
cells) Ledbetter, J.A., ~t al., 1981 Monoclonal Anti-bodies and T Cell Hybridoma Elsevier/
North Holland, N.Y., pp. 16-22.
T6 4g Thymocytes ~ OKT6 Reinherz, 1979, Langerhans NAI/34 supra.
Cells Leukemia Cells 20 CD4 62 Helper/Inducer OK4 Kung, P.C., et al., Cells (subset L~u 3a 1979, Science 206:
of T cells~ 347-349 CD3 19 Pan T Cell OKT3 Kung, id.
TAC 50 IL 2 Receptor Anti- U hiyawa, T., et (Activated T TAC al., 1981, 3.
Cells~ Immunol.
126(4~:1393 1397 T9 94 ~ransferrin OKT9 Reinherz, E.L., Rsceptor et al., 1980, PNAS
(Activated T USA 77:1588-1592 Cells) CD2 50 All T Cells OKTll Verbi, W., et al., Leu 5 1982, Eur. J.
: Immunol. 12:81-86 VLA-l 130/165 Late Activated VLA-l Helmer, ~.E., et /210 T Cell~ al., 19~4, J.
Immunol. 132:3011-.

These 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. Several of the cell s~rface molecules have been studied in great detail and have been found to be important 5 in initiating and regulating immune funct:ions, and ar~
critical to communication processes between immun~ cells. T
cell antigen receptor, a surface molecule which comprises a disulfide-linker dimer of approximately 90 kilodaltons ~kd~, recognizes specific antigens and is responsible for 10 initiating a complex series of biochemical events which constitute the T cell activation process ~Meuer, S.C., et al., 1984, Ann. Rev. Immunol. 2:23-50; Acuto, O., et al., 1983, Cell 34:717-726). The CD3 structure is a three-chain complex associated with the T cell receptor (Kannellopoulos, 15 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.
157:705). Lymphokine receptors, ~ interleukin 2 (IL-2) receptor and interleukin 1 (IL-1) receptor, are essential 20 for the activation and proliferation of T cells (5mith, K.A.~ 1984, Ann. Rev. Immunol. 2:319-333; Dower, S.K., et al., 1985~ J. Exp. Med. 162:501-515). CD8 is a T cell specific surface glycoprotein exprassed on the surface of approximately 30% of T lymphocytes associated with 25 suppres~ion and cytotoxic functions and the ability to recognize antigen in the context of class I MHC antigens tSwain, S.L., 1983, Immunol. Rev. 74:129-42)~ CD4 (OKT4 antigen) is a 55 kd glycoprotein e~pressed on the surface of approximately 60% of all T lymphocytes and is associated 30 with helper function (Reinherz et al., 1979, Proc. Natl.
Acad. Sci. U.S.A. 76:4061-4065) and ~he ability to recognize antigens in the context of type II MHC antigens (Swain, 3~

supra; M~uer, S.C., et al., 1982, Proc. Natl. Acad. Sci.
U~S.A. 79:4395-99). 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). These various cell surface 5 markers have enormous clinical application potentials for the identification of lymphocyte populations and their functional status (Kren~ky, A.M. and Clayberger, C., 1985, Transplant. 39(4)o339-348; Kung, P.C., et al., 1984, Monoclonal Antibodies in Clinical Investigations, Clinical 10 Biochemistry-Contemporary Theories and Techniques, vol. 3, Academic Press, pp. 89-115; Kung, P.C., et al., 1983, Int.
J. Dermatol. 22(2):67-73).
Existing clinical methods of T cell typing involve the use of monoclonal antibodies which define T cell surface 15 markers to detect the presence of speci~ic cell surface markers on the T cell surface. Measuring the total numbers of T cells by surface markerR has been useful for the characterization and classification of lymphoid malignancies (Greaves, M., et al., 1981, Int. J. Immunopharmac.
20 3(3):283-300~. Changes in the relative percentage of T
helper and T suppressor/cytotoxic cells were found to be associated with immune events in renal transplantation due to viral infection (Colvin, R.B, et al., 1981, Proc. 8th Int. Congr. Nephrol., Athens, pp. 990-996~, autoimmune 25 disease~ (Veys, E.M., et al., 19~I, Int. J. Immunopharmac.
3(3):313-319), and AIDS (Gupta, S., 1986, Clin. Immunol.
Immunopathol. 38:93-100; Ebert, E.C., et al., 1985, Clin.
Im~unol. Immunopathol. 37:283-297).
The expr~ssion of T cell surface markers has also been 30 used ~or the assessment of the immune status of patients.
It has been sstablished that by measuring the relative number of distinct, functional T cell subsets, and/or the relative number of activated T c211s in peripheral blood or tissues, an assessment of the immunological condition of a patient is possible~ The activation antigens ~ . IL-2 receptor) appear to be involved in T cell growth and differentiation processes.
Antibodies to CD4 have been widely described (Kung, P.C., et al., 1979, Science 206:347~349) and are 5 colrunercially availableO A series of such antibodies reacting with non-competing epitopes on the CD4 molecule have been described. Such a set has been termed OK~r4, OKT4A, OKT4B, OXT4C, OKr4D, OKT4E, and O:E~T4F ~Rao, P.E., et al ., 1983 , Cell. Iml[unol . 80: 310) .
Antibodies directed against the CD4 or CD8 antigens have been shown to block cell function. Antibodies against CD4 block most helper T function~, mixed lymphocyte reaction~ and induction of T helper activity (Biddison, W.E., et al., 1984, J. Exp. Med. 159:783). Antibodie~
15 against CD8 block the cytotoxic activity o~ CD8 positive cytotoxic T lymphocytes ~Swain, S.L., 1981, Proc. Natl.
Acad. Sci. U.S.A. 78:7101~71û5). Antibodies against CD4 have al~o been described that are capable of activating CD4-positive T cells. CD4 is internaliz2d upon treatment of 20 the cells with phorbol esters: and resul~ing phosphorylation (Hoxie, J.A., et al., 19~6, J. Immunol. 137:1194-1201).
$he 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 (~
25 sheets held together by disulfide bridges) homology at the V
~variable)-like domain of C~4 to the V region of immlmoglobulin (Maddon, P.J., et al., 1985, Cell 42:93-104).
This V-like regiorl of the molecule is ~ollowed by a stretch of 263 amino acids with no known homology to other 30 molecules, followed by a tran~me~brane domain and highly charged cytoplasmic tail, containing serines which are phosphorylated upon activation ~Littman, D.R., et al., 1984, Nature 325:453-55).

Homology between CD4 and CD8 is quite low. CD8 exists on the cell surface as dimeric or multimeric structures composed of a 33 kD monomer (Snow, P.~., et al.l 1983, J.
Biol. Chem. 258:14675 14681).

Z.3. SOLUBT,E IM~INE CELL SURFACE MOLECULES
Several immune cell surface markers have been detected in the serum. The molecules of the hum,an major histocompatibility complex (HLA moleculles) are sets of cell surface glycoproteins involved in immune recognition. These 10 macromolPcular antigens have also been found to be present in body fluids such as serum (Pellegrino, M.A., et al., 1984, Meth. Enzymol. 108:614-62~). The serum levels of Class I HLA-A and HLA-B have been show~ to be present in sufficient quantity to perform HLA-typing in sera (Russo, 15 C., et al., lg83, Transplant. Proc. 15(1):66-68; Pelleyrino, M.A., et al., 1981, Transplant. Proc. 13t4):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.
66(2):279-283; Russo, C., et al., 1983, Transplant. Proc.
20 15(1):57-59). The serum HL~-DR (Ia) has been shown to be markedly depressed in tumor patients.
A soluble form of IL2R has been detected (Rubin et al., 1985, J. Im~unol. 135:3172-3177; Rubin et al., 1985, Fed. Proc. 4~:946; U.S. Patent No. 4,707,443 by Nelson et 25 al.) that i~ released by activated normal peripheral bloo~
mononuclear cells and synthesized in large amounts in vitro by ~TLV-I-infected leukemic cell line~. A sandwich enzyme .
im~unoassay wa~ used to guantitate th~ soluble IL2R.
Little i~ known about the functional significance of 30 soluble I~2R. Since soluble IL2R is capable of binding interleukin 2 ~Rubin, L.A., et al., 19~5, J. Immunol.
135:3172-3177), it may have an immunoregulatory role by competing with cellular IL2R for the ligan~ and thus down-regulating the immune response. In this regaxd, the solubl~

IL2R has been suggested to be a ~blocking ~actor~ producedby the malignant cells to inhibit the host's immune response to the tumor (id~).
Subsequent studies have disclosed comparable levels of soluble IL2R in cord blood and peripheral blood from normal 5 adults (Nelson, D.L., et al., 1986, Pediatr. Res. 20:136~
139). Increased serum levels of IL2R have been found in patients with certain B or T cell malignancies (Nelson, D.C., 1986, Fed. Proc. 45:377; Saadeh, C'., et al., 1986, Fed. Proc. 45:378 MacKeen, L., et al., 1986, Fed. Proc.
1~ 45.454; Reuben, J.M., et al., 1986, Blood 68(5), Supp.
1:213a). 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).
Several other cell surface markers which are pr:imarily present on T cells have also been îound in soluble form.
CD2, a T cell surface molecule present in all normal T cells ~nd a receptor for sheep red blood cells, has been detected at highar levels in the sera of certain cancer patients than 20 those found in normal control patients (Falcao, R.P., et al ., 1984, Clin. Lab. Immunol . 13 :141-143; Oh. S.-K., et al., 1985, Sca~d. J. Immunol. ~2:51-60). CD8 (Leu 2, OKT8), a surface marker fourld on the surface of suppressor/cytotoxic T cells and which may be involved in 25 cellular recognition, has also been report~d at highly el~vated l~vels in the serum of patients with ~ cell leukemia (Fujimoto, J. r et al., 1983, J. Exp. Med. 159:752-766). Leu-1, anot:her T cell surface molecule, was measured in seru~ following anti-Leu-l monoclonal ~ntibody treatment 30 (~iller, R.A., et al., 1982, New Engl. JO Med. 306:5ï7-520).
Oh et al. (1985, supra~ reported that less than half o~ the patients with malignancies in their study presented elevated levels of soluble O~rll receptor in their s~rum.
3~

However, not all T cell surface molecules are released into the serum (Fujimoto, J., et al., 1983, J. Exp. Med.
159:752-766). Leu l antigen was not detectable in the serum o~ normal or leukemic patients who have not received antibody therapy. Leu 3 antigens were ,also not detectabl~
5 in soluble form in ~ cell culture supernatants (id.).
Fujimoto et al. (1983, J. Exp. Med. 159:752-766) were unable to find evidence of released CD4 using a sandwich enzyme immunoassay based on Leu3b and Leu3a (Bec on-Dickinson). This assay worked well with detergent lysates 10 of cells but did not detect released CD4 in culture supernatants o~ CD4+ and CD8+ leukemic T cells which could be shown to release CD8, or in culture supernatants of CD4 T cells.
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~ concern~ 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 di~ferentiation antigens in the ~iagnosis and 25 therapy of various diseases and disorders.

3. SUMMARY OF ~HE INVENTION
~ he pre~ent invenkion is directed to the measurement o~ soluble ~ cell growth factor receptor~, soluble T cell 39 di~ferentiation antigens, or related solubl~ molecules or fragMents thereof, and the use of such measurements in the diagno~is and therapy o~ disease and disorders. The measurament of such molecule~ can be valuable in monitoring the e~fect of a therapeutic trea~ment on a subject, detecting and/or staging a disease in a subject, and in differen~ial diagnosis of a physiological condi~ion in a subject. These measurements can also aid in predicting therapeutic outcome and in evaluating and monitoring the immune status o~ patients.
In a specific embodiment, a rise in both solublP 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 in~ection in a transplant patient. In another particular embodiment, 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 th~ measurement of serum IL2R levels to stage nonwlymphatic malignancies.
1~ 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 samp~e from a patient can be used to diagnose a state of immune activation. Such an increase in soluble CD4 antigen levels in synovial ~luid can be used to diagnose rheumatoid arthritis. Soluble CD4 measurements can also be used to stage adult T cell leukemia, or determine the phenotype of a cell in culture. Soluble CD~ measurements can also be used to monitor AIDS patients undergoing therapy.
a~ The invention also relates to the measurement of a plurality of soluble T cell surface markers, for the detecting, staging, or monitoring of treatmsnt o~ a disease or disorder. In particulax embodiments, the measurement of a plurality of soluble T cell surface markers and their 3~ change relatlve tQ on another can be superior to the -13~

measurement of any soluble T cell surface marker alone, for the detecting, stag.ing, or monitoring of treatment of a disease or disorder.
In particular embodiments, measurements of the soluble T cell surface molecules can be accomplished by sandwich 5 enzyme immunoassays. Kits for such mea~surements are also provided.
3.1. DEFINITIONS
As used h~rein, the following abb3reviations will have the meanings indicated:
10 Staging a disease = assessing the degree of severity according to standard classifications AC = adenocarcinoma CsA = cyclosporin A
~5 HC = hairy cell leukemia HTLV III/LAV/HIV = Human T Cell Leukemia Virus Type I/Lymphadenopathy Associated Virus/Human Immunodeficiency Virus IL-1 = interleukin-1 IL-2 - interleukin-2 IL2R = interleukin-2 receptor mAb = monoclonal antibod~
PBMC = peripheral blood mononuclear 2~ cell PHA = phytohemagglutinin SCLC = s~uamous cell lung carcinoma Spontaneous rele se = release by normal or pathologic physiological processes of the cell AZT - azido-deoxythymidine RF = rheumatoid ~actor 3~

;` ,

4. DESCRIPTION OF THE FIGURES
Figure 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 Seotion 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 Prom a number of disease groups.
The assay used was as described in Section 6.202, infra.
SF: synovial fluid; EBV/mono: Epstein Barr : Virus/mononucleosis.
: Figure 3. Longitudinal studies of soluble CD~ levels - in sera of patients with Kaposi's sarcoma (KS) or with -AIDS-related complex (~RC). Closed diamonds: soluble CD8 1~ levels (U/ml): Open diamonds: HIV p24 levels (pg/ml) x 10.
Figure 4. Serial serum IL2R (units/ml) and creatinine (Cr) (mg/dl) levels in a renal allogra~t recipient who developed a rejection episode that responded to OKT3 antibody therapy (arrow marks day of biopsy diagnosis). The 2D rise in serum IL2R accompanied and preceded the rise in serum creatinin2.
Figure 5. Dot plots of the change in serum IL2R (A~, uri~e IL2R (B), or seru~ creatinine (Cr) (C~ concentrations by clinical status: stable ~S~, rejection (R), cyclosporine 26 toxicity (C), and infection (I~. Samples of rejection are from 2 days befora the first rise in Cr to the time o~
institution of antirejection therapy.
Fiyure 60 A plot of the sensitivity and specificity o~ the serum IL2R a~say and serum creatinine (Cr) for the ~9 diagnosis of rejection. The data from Figure 5(a,c) are given in the form of receiver operating characteristic tROC) curves. In this display, the threshold for a positiv~ test iB 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.

5. DE~AILED DESCRIPTION OF THE INVENTION
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.
As used herein, the term ~soluble~ shall mean those mslecules that are ~spontaneously released~; i.e., released by normal or pathologic physiological processes of the cell.
Such molecules are to be distinguished from nsolubilizedN
cel} surface forms of the molecules, whose solubilization is 15 brought about by ln v ro manipulation such as cell lysis by detergent. The soluble T cell markers ~antigens and receptors) of the invention are molecules which carry antig~nic determinants of their cell-surface counterparts.
Proteinaceous molecules, or fragments thereo~, derived 20 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 ~luid and not associated with the surface o~ a ~ cell are soluble T cell surface molecules of the 25 invention. These moleculeR can be either glycosylated or nonglycosylated and may be soluble by themselves or considered soluble by virtue of their association with other soluble molecuIes.
The measurement of the soluble molecules of the 30 invention can be valuable in monitoring the e~fect of a therapeutic treatment on a subject, detecting and/or staging a dis~asQ in a subject, and in dif~erential diagnosis of a physiological condition in a subject. These measurements can also aid in predicting therapeutic outcome and in evaluating and monitoring the immune s~atu~ of patients.
More ~han one type o~ 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.

5.l. MONITORING TXE EFFECT OF A THERAPEUTIC TREATMENT
The present invention provides a method for monitoring the affect of a therapeutic treatment on a subject who has undergon~ the therapeutic treatment. This method comprises 10 measuring at suitable time intervals the amount of a soluble molecule or soluble fragment thereo~ which comprises, or is immunologically related to, a T cell growth ~actor r~ceptor or T cell differ~ntiation antigen. Any change or absence o~
change in the amount of the soluble molecule can be 1~ identified and correlated with the effect of the therapeutic treatment on the subject. In a specific embodiment of the invention, soluble molecules immunologically related to the CD4 antigen can be measuxed in the serum of patients by a sandwich enzyme immunoassay in ~rder to evaluate the 20 therapeutic efficacy of, for example, administration of immunomodulators such as alpha-interferon, Cyclosporin A, and monoclonal antibody OKT3. In another embodiment of the invention, the levels of soluble CD4 molecules can be measured in the s~rum o~ ~IDS patients in order to evaluate 25 the therapeutic e~ficacy of tr~atments such as admin~stration of AZT, interferon or CD4 itself.
The therapeutic treatmants which may be evaluated according to the present invention include but are not li~ited to radiotherapy, drug administration, vaccine 3D administratioA immunosuppressive or immunoenhancive regimens, etc. The im~uno uppressant regimens include, but are not li~ited to administration o~ drugs such as Cyclosporin A, chlorambucil, cyclophosphamide, or azathioprine, and anti-T cell antibody such as anti-T3 3~

~17-monoclonal antibody and anti-thymocyte globulin, etc. The immunoenhancive regimens include, but are not limited to administra~ion of interleukln~1, interleukin-2, and other T
cell growth factors.

5 5.2. DETECTING ~ND~OR STAGING A DISEASE IN A SUBJECT
In another embodiment of the present invention, 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 i5 compared to a baseline level. This baseline level can be the amount which is established to be normally present in the body fluid of subje~ts with various degrees of the disease or disorder.
15 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 tha subject during a specific stage of the diseaso or disorder, is indicative o:E 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 disease. Diseas¢s 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, inra.

TABLE II

DISEASES AND DISORDERS WHICH MAY BE DETECTED ~ND/OR
STAGED IN A SUBJECT ACCORDING TO THE PRESENT I~VENTION
_ _ I. Inf~ctious Diseases Induced by virus:
Herpesvirus Cytomegalovirus Epstein-Barr Virus HTLV-I
HTLV-III/LAV/HIY (AIDS) II. Cancer T cell leukemia 1~ ~TLV-I-associated adult T cell leukemia T cell lymphoma ~urkitt's lymphoma Hairy cell leukemia Sezary syndro~e 2~ Hodgkin's disease Chronic lymphocytic leukemia ~ Non-~odgkin's lymphoma : ~ B-c~ll acute lymphoblastic leukemia : Solid tumor~
.
III. Autoimmune Diseases Rheumatoid arthritis Diabetes Multiple sclerosi~
::
Systemic lupu~ erythematosis ~: : IV. organ Allogra~t Rejection . . _ `: :

:: :

In specific embodiments of this aspect of the invention, measurements of plasma or serum levels of the soluble molecules can b~ used in the detection of dis~ase, or to determine disease stage and assign risk.
Rheumatoid arthritis can be monitored by measuring 5 soluble CD4 lev~ls in a patient. In a pref~rred embodiment, eleva~ed levels of soluble CD~ in synovial fluid relative to seru~ is a diagnostic indication o~ rheumatoid arthritis.
In anotAer embodiment of the invention, detection of an increase in soluble CD4 antigen in the body fluid of a 1~ patient can be used to diagnose a state of immune activation. 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, 1~ the detection of soluble CD4 in cell culture supernatants can be relied on as an indication of the CD4~ phenotype o~
the lymphocyte~ present.

5.3. DIFFERENTIAL DIAGNOSIS_OF A PHYSIOLOGICAL CONDITION
In another embodiment of the invention, the measure-ment of soluble T cell growth factor receptors, T cell surface antigens, or i~munologically related molecules can be u~ed to differentially diagnose in a subject a particular physiological condition as distinct from among two or more 2~ physiological conditions. To this end, the measured amount of the soluble mol~cule is compared with the amount of the soluble molecule normally pres~nt in a body fluid of a subject with one of the suspected phy iological conditions.
A measured amount of the soluble molecule similar to to the 30 amount normally present in a sub-~ect with one of the physiological conditions, and not normally present in a subject with one or more o the other phy~iological conditions, is indicative of the phy~iological condition of the subject.
3~ .

In a specific embodiment of this aspect of the invention, measurement of soluble molecules can be used in the differential diagnosis o~ renal allograft rejection, especially in distinguishing Cyclosporin A nephrotoxicity or infection. Similar differ ntial diagnosis of allograft rejection using the methods o~ the invention can be applied to other organ allogra~ts, including but not limited to liver, heart, and pancreas.
In a preferred ~mbodiment of the invention, the measurement of changes in the levels of soluble molecules, rather than measurement of the absolute levels of the soluble markers, can be used to differentially diagnose renal allograft rejection.

5.4. SOLUBLE T CELL GROWTH FACTOR
RECEPTORS, T CELL DIFFERENTIATION
ANTIG~NS, AND RELATED ~OLECULES
Any T cell surface molecule or immunologically related molecule which is present in soluble form in the body fluid at le~els which correlate with a dis~ase condition or disorder, or a stage thereof, may be u ed in the practice of the pxesent invention. T cell sur~ace markers which may potentially b~ used include but are not limited to those listed in Table Il supra.
In specific embodiments, the soluble ~orm of the CD4, CD8, and IL2R molecules may be measured.

5.4.l. XITS AND ASSAYS FOR ~EASUREMENT
Any procedures known in the art for the measurement of soluble molecules can b~ used in the practice of the instant : 30 invention. Such procedures include but are not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays, ~LISA (enzyme linked immunosorbent assay), ~sandwich~ immunoassays, precipitin reactions, gel di~usion precipitin reactions, immunodif~usion as~ays, agglutination assays, complement-fixa~ion assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and immunoelectro-phoresis assays, to name but a few.
In a preferred embodiment, a sandwich enzyme immunoassay can be used. One description of such an embodiment follows: A monoclonal antibody (capture antibody, mAb 1) directed against the soluble antigen is adsorbed onto a solid suhstratum. The soluble antiqen present in the sample binds to the antibody, and unreacted sample components are removed by washin~. An en~yme-conjugated monoclonal antibody (detection antibody, m~b 2)directed against a second epitope of the antigen hinds to the antigen captured by mAb 1 and completes the sandwich.
After removal of unbound mAb 2 by washing, a substrate solution is added to the wells. A colored product is formed 1~ in proportion to the amount of antigen present in the sample. The reaction is terminated by addition o~ stop solution and absorbance is measured spectrophotometrically.
A standard curve is prepared from known concentratlons of the soluble antigen, from which unknown sample values can be 20 determined. In particular embodiments, such an assay may be used to determine soluble IL2R levels or soluble T cell antigen levels. In a preferred embodiment for the measurement of IL2R levels, anti-IL2R mAbs 2R12 and 7G7 can be used as the capture and detection antibodies, respectiv~ly, in a sandwich immunoassay (such as the C~LLFREE0 IL2R Test Kit assay, T Cel~ Sciences, Cambridge, M~). In a preferred embodiment for the measurement of soluble CD8 antigen levels, anti-CD~ mAbs 4C9 and 5F4 can be used as the capture and detection antibodies, respectively, 3D in a sandwich enzy~e immunoassay (such as the CELLFREE~ T8 Test Kit assay, T Cell Sciences, Ca~bridge, M~). In a preferred embodiment for the measurement soluble CD4 antigen 3~

levels, anti;~CD4 mAbs 8F4 and R2B7 can be used ~s the capture and detection reagents, respectively, in a sandwich enz~me immunoassay (see Sections 5.4.3 and 6, infra).
Kits for carrying out the assays of, and for use in the practice of, th~ pxesent invention are also within the scope of the invention. For instance, such a kit can comprise a pair o~ antibodies to the same T cell marker (receptor or antigen), which antibodies; do not compete for the same binding site on the marker. In anoth~r embodiment, a kit can comprise more than on~ pair of such antibodies, 10 each pair directed against a different T c~ll marker, thus useful for the detection or measurement of a plurality of T
cell markers.

5.4.2. FORMULATION OF AN IMMUNOASSAY FOR

SOINBLE FORMS OF T CELL SURFACE
MARKERS OVER SOLUBILIZED FORMS
The p~esent invention also provide~ a way of deriving immunoassay systems which preferentially detect/quantitate physiologically released (soluble) forms of cell surface 20 markers over solubilized (e.g., detergent-treated) cell surface markers. Such a method involves the use of recombinant forms of the specific cell surface marker to be as~ayed, which have been genetically engineered to be physiologically soluble ~ ., by deletion of DNA sequences 2~ 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 epit~pes are .ikely also to be absent from the physiologically released form of the marker.
30 Thus, the recombinant molecule can be used to screen anti-ceIl surface marker antibodie~ for deter~ination of the appropriate antibodies to be used for pre~erential detection of the physiologically released ~or~ o~ the surfac~ marker.
Pairs of antibodies can be screened for optimization of a 3~

sandwich ELISA for detection of soluble cell-surface marker.
This aspect of the invention is illustrated by way of example in Section 6, infra, wher~ a soluble CD4 as~ay is devised that preferentially detects soluble CD4 relative to solubilized CD4.
Antibodies can be produced ~or 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.
Various procedures known in the art may be u~ed ~or the production of polyclonal antibodies to epitopes of a given T cell ~urface molecul~. For the production of antibody, various host animals can be immunized by injection with a T cell surface molecule, a recombinant version thereof, synthetic protein, or fragment thereof, including 1~ but not limited to rabbits, mice, rats, etc. In a preferred embodiment, the immunogen is a truncated recombinant soluble form of the T cell surface molecule. Various adjuvants may be used to increase the immunological response, depending on the host species, and i~cluding 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 h~man adjuvants such as BCG (bacille Calmette-Guerin) 2~ and corynebacterium parvum.
A monoclonal antibody to an epitope o~ the T cell surfac~ molecule can be pr~pared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not 30 limited to the hybridoma technique originally described by Kohler and Mil~tein (1975, Nature 256 495-497~, and the more rec~nt human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72) and EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
In one ~mbodiment, the monoclonal antibodies may be human monoclonal antihodies or chimeric human-mouse (or 5 other species) monoclonal antibodies. Human monoclonal antibodies may be made by any of numerous techniques known in the art (~ , Teng et al., 19~3, Proc. Natl. Acad. Sci.
U.S.A. 80:7308 7312; Kozbor et al,, 1983, Immunology Today 4:72-79; Olsson et al., 1982, Meth. Enzy~ol. 92:3-16).
10 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.
Acad. Sci. U.S.A. 81:6851; Takeda et al., 1985, Nature 314:452).
1~ A molecular clone of an antibody to an epitope of a T
cell surface molecule can be prepared by known technique~.
Recombinant DNA methodology (see e.g~, Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) may be used 20 to construct nucleic acid sequences which encode a monoclonal antibody molecule, or antigen binding rsgion thereof.
Antibody molecules may be purified by known techniques, e.y., im~unoabsorption or immunoaffinity 2~ chromatography, chromatographic methods such a~ HPLC (high per~ormance liguid chromatography), or a combination thereof, etc.
Antibod~ ~rag~ent~ which contain the idiotype of the ~olecule can be generated by known techniques. For example, 30 such fragment~ include but are not limited to: the F(ab')2 ~ra~ment which can be produced by pepsin digestion of the antibcdy 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 b2 generated by treating the antibody molecule with papain and a reducing agent.
Once specific antibodies are demonstrated to be suitable for use in the pre~erential del:ection of soluble T
5 cell-surface molecules, other such suitable antibodies may be selected by virtue of their having the same epitope specificity as the ormer antibodies. Such similar epitope specificity can be ascertained, for sxample, by obserYing the ability o a second antibody to inhibit binding of a first antibody to its antigen.

5.4.3. SOLUBLE CD4 AND ASSAY FOR ITS DETECTION
The inventinn is also directed to assays for mea urement of soluble (released) CM, which assays 1~ preferentially measure soluble CD4 over the solubilized membrane form o~ CD4. Examples o~ such assays are detailed infra in Section 6.
In a preferred embodiment for the detection or measurement of soluble CD4 antigen levels, a~ti-CD4 mAbs 8F4 20 and R2B7 can be used as the capture and detection raagents, respectively, in a sandwich immunoassay.
We also demonstrate in~ra that the physiologically released form of CD4 is physically dif~erent from that of the solubilized cell surface form, and that assays which 26 guantitata the solubilized cell surface form do not necessarily quantitake the released form.
Soluble CD4 has been ~pecifically quanti~ated, according to ths present invention, and has been shown to be a reliabla indicator o~ variou~ pathologic conditions (see 30 Section 6, infra). Thus, detection and/or measurement of soluble CD4 can be used to diagno~e, 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 ~or the detecting or staging of disease, or the monitoring of treatm~nt by 5 measuring a plurality (at least two) T cell surface markers (receptors or dif~erentiation antigens). For example, 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. Such lO diseases or disorders include those discussed supra in Sections 5.1 through 5.3 (e.g., see Table II). Soluhle marker levels can represent a measure of immune system function, paralleling disease course or treatment ef~icacy.
In a preferred embodiment, the prognostic indicator is the e6 observed change in different marker levels relative to one another, rather than the ab~olute levels of the soluble markers present at any one time. Since soluble CD4, soluble CD8 and soluble IL2R level~ are measures of the immune sy~tem itself, they should provi~e a much improved measure 2~ f the relative health of the immune system during various stages of disea6es or disorders.
In a preferred embodiment, measurements of a plurality of soluble T cell surface markers are used to detect~ stage, or ~onitor treat~ent of diseases and disorders caused by HIV
2~ (the c~usative agent of AIDS) infection.
Since the discovery of AIDS and the observation that the AIDS virus, HIV, binds to the T4, ox CD4, receptor, there have b~en several proposals for the treatment of AIDS
patient~ or for the development of vaccine~ ~or populations 3D f people at risk. These include the treatment of AIDS
patients with drugs such as AZ~ (azido-deoxythymid}ne), ~ or interferon~, and with soluble CD4, or it~ fragments and derivatives, and the production of potential AID5 vaccines, such as gpl20 peptides. What is very much needed is a procedure that can be used to monitor the efficacy of these .:

treatments or vaccines. To date, the levels of the HIV
antigen p24 have not proved sensitive enough. With the observation herein described that soluble CD4 in particular, but soluble CD8 and soluble IL2R receptors as well, can be identified and detected in HIV-infected patients with 5 dif~erent mani~estations of disease, it becomes possible to develop a sensitive immunoassay to monitor AIDS therapies and vaccines. The CELLFREE~ Test Kit (T C~ll Sciences, Cambridge, MA) assays can be use~ul ~or this purpose. Due to the intimate involv~ment of CD4 in the etiology o~ AIDS, 1~ it is expected that spontaneously released soluble CD~
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, a~ soluble CD4 is produced when CD~+ cells become activated, (see 16 Section 6.2, _ue~) as occurs during HIY infection.
Furthermore, measurements of other soluble T cell markers, such as soluble IL2R and soluble CD8, that also indicate the state o~ immune function should be valuable.
The best index for monitoring AIDS treatment or 2D disea~e progression can be a profile of soluble T cell ~
markers, such as ~oluble CD4, CD8 and I~2R, rather than any individual marker alone ~see Sections 6.3 and Section 7)0 Such a profile can be obtained by determining the soluble receptor levels o~ a panel of soluble receptors in 2~ longitudinal samples of sera from patients undergoing treatment.
In a preferred aspect, the approach that can be taken is to determine the l~vels of solubla CD4 (and soluble CD8 and soluble IL2R) levels in longitudinal time studies and to 30 compar~ these values with a baseline level. The baseline level can be either th~ level of the soluble marker present in normal t disease free individuals: and/or the levels t -28~

present prior to treatment, or during remission of disease, or during periods o~ stability. These level~ can then be correlated with the disease course or treatment outcome.

6. SOLUBL2 CD4 ANTI6EN
In the examples detail~d herein, ia sandwich immunoassay is described for the pre~erlsntial detection o~
the soluble form of CD4 antigen relative to the cell-surface form of CD4 antigen.

60l~ MATERIALS AND METHODS

6.l.1. ANTIBODIES
Antibody Leu3a, biotin or FITC labeled, was purchased from Becton Dickinson, Mountain View, California.
$6 Antibodies ORT4 and 0KT4A were obtained from Ortho Diagnostics, Raritan, New Jersey. Antibody IOT4 was obtained from Immunotech, Cedex, France and fur~h~r purified by ammonium sulfate precipitation. Antibodies B67.2 and B660l were from G. Trincherie, Wistar Institute. Ant~hody 2D 3G2 was from Sanchex ~adrid, Madrid, Spain. Antibody R2B7 was obtained fro~ a fu~ion of rat spleen cells (carried out : according to standard procedures), from an animal immuniz~d with whol~ human peripheral blood lymphocytes, with mouse SP2/0 myeloma cell~. Clone R2B7 was selected from this 2~ fusion based on its ability to stain populations of peripheral blood lymphocytes identical to these identified by OKT4.
Antibodies were purified eith~r by ammonîum sulfate pr~cipita~ion or by protein A sepharose using the Bior~d 30 MPAS buf~er ~ystem ¢BioRad Corporation, Richmond, CaIi~ornia). ~orseradish peroxidase (HRP) conjugates were prepared e sentially according to the method of Wilson and ~29-Nakan~ (lg78, ln Immunofluorescence ancl Related Techniques, Knapp, W., et al., eds. Elsevier, p. 215) using a molar HRP
to antibody ratio of four.
Antibodies generated from a fusion of mice immunized with intact T cells (Jurkat) were screened for their ability 5 to substitute for I.eu3A in the assay as follows: Plates were coated with R2B7 as described, blocked and incubated with recombinant ~oluble CD4 for 2 hour-s at 37C. Plates were washed and 50 ~1 of each hybridoma supernatant a 1-10 ~g~ml were added followed by 50 ~1 of biotinyl Leu3A.
10 Followin~ a 2 hour incubation, plate~ were washed and 100 ~1 of streptavidin per~xidase (0.5 ~g/ml) was added for 30 minutes. Plates were washed and developed as described below.

1~ 6.1.2. IMMUNOASSAY PROTOCOLS
6.1.2.1. INITIAL.ASSAY
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 2~ coate~ overnight at 4~C with a solution o~ murine monoclonal anti-human CD4 antibody in PBS, pH 7.4. Any remaining protein-binding site on the microtiter wells were then blocked for two hours at 37C with 300 ~1 per well o~ a solution of ~SA (1%) (Rirkegard and Perry Laboratories, 2b Maryland) an~ Tween 20 tO.05%) (2y~ed Laboratories, South San Francisco, California~ in phosphate bu fered saline (PBS), pH 7.4. The wells wera thsn washed three times with 350 ~1 per well of PBS (pH 7.4) with 0.05% Tween 20.
Following the fi~al wash step, the wash solution was 30 aspirated from the well~ and 50 ~1 of a sample diluent consisting o~ 0.15 M NaCl, 25 m~ Tris (pH 7.4) supplemented with bovine proteins was added to each well. Fifty ~1 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 ~or fifteen seconds.
The plate was then sealed and incubated at 37C 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 ~1 of PBS/Tween 20 as above. One hundred ~1 of horseradish peroxidase (H~P) conjugated murine monoclonal anti~human CD4 antibody was added to each well of the micxotiter plate, and the plate was again incubated at 37C
for 2 hours, as above. At the end of this incubation, the wells were once again washed three times with PBS/Tween 20 ~ as above. One hundxed ~1 o~ o-phenylenediamine (0.2%) dissolved in 0.1 M sodiu~ citrate buffer, pH 5.5, was then added to each well of the plate and incubated at 24C + 2C
for 30 minutes. At the end of this final incubation, 50 ~1 of 2 N H2S04 was added to each well to stop the reaction and 1~ the absorbance of each well was read at 490 nm.
For a~says involving biotinylated antibodies, biotin conjugates were substituted for the HRP antibody conjugate.
After the 2 hour incubation, wells were washed and 100 ~1 of streptavidin horseradish peroxidase (Zymed Laboratories) at 0.5 ~g/ml in 1% bovine serum albumin in tris buffered ~aline wa~ added. Following a 30 minute incubation at 37~, wells were washed and color developed as described above.
Where indicated, assays were also performed as a single step as~ay i~ which conjugated antibody was added at 2~ the same time as the sample and incubated for 4 hours at room temperature o~ a rotating sha~er platform, after which washing and color development w~re performed as described.

6.1.2.2. OPTIMI~ED ASSAY
3~ The co~figuration of the initial assay was modified by optimizing each o~ the assay reagents. This rssulted in an improved sensitivity for the overall assay where much lower ~6 levels of soluble CD4 cculd be reliably and reproducibly detected. The optimized assay configuration is given in Table III.

.

TABLE III
COMPARISON OF INITIAL &_OPTIMIZED ASSAY CONFIGURATIONS

INITIAL ASSAY OPTIMIZED ASSAY
Block1ng Buffer-1% BSA + 0.05% Tween 20 0.5% Casein, 0. 008% NP-40, 0.005% EDTA
Sample Diluent*
1~
0.15 M NaC1, 25mM Tris, 0.25% NP-40, supplemented supplemented with bovine with bovine proteins in PBS
proteins Conjugate Diluent*
25% FCS in Tris buffered 15% FCS, 0.15~ NP-40 2D saline + O.25% NP-40 * Aggregated IgG was added to ~oth the sample diluent and the conjugate diluent to remove any ef~ect of rheumatoid factors in various samples.
.
Occasionally, it was ob~erved that the presence of rheumatoid factor (RF) in ~ome of the samples led to erroneous determinations of soluble CD4 that appeared as false positive~. To remove this effect, aggregated IgG was add~d to the sa~ple and conjugate diluent buffers. The aggregated IgG was prepared by hea~ing a 100 ~g/ml solution of IgG in 100 m~ sodium phosphate buf~er, 0.9% NaCl, pH 5.56 at 56-60C for 50 minutes, follow2d by neutralization with dibasic sodium ph~sphate, 0.9% NaCl, pH 8 to giva a final pH
of 7.~.

~:

, 6.1~3. CELL PR0CEDURES
For stimulation experiments, peripheral blood mononucl~ar cells were prepared using Ficoll Hypaque gradients. Cells were put into culture along with phytohemagglutinin (P~A~ ~0.5 ~g/ml) or phorbol myristate acetate (1 ng/ml) and ionophore A2317 (0.1 ng/ml) or 0~T3 (anti-T3 monoclonal antibody) (2 ~g/ml)" Samples were taken daily. Fox long term cultures of cells from rheumatoid arthritis or lung cancer patients, cells were maintained on IL-2. Cells were removed from culture supernatants by centrifugation followed by filtration through 0~22 ~m ~ilters and stored frozen until analysis.
Cell surface phenotyping was parformed using a Cytofluorograph II (Ortho Diagnostic System, Westwood, Massachusetts) and FITC labeled OKT4 or OKT8 (Ortho 16 Diagnostic Systems, Raritan, New Jersey).
Recombinant souble CD4 was obtained from ce:ll culture supernatant of a chinase hamster ovary (CH0) cell line transfected with CD4 truncated ak the transmembrane exon (Fisher, R.A., et al., 1988, Nature 331:76-78).
2~
6.2. RESULTS
6.2.1. USING INITIAL ASSAY PROTOCOL
Table IV shows the initial results of screening serum samples for released CD4 using OgT4 or 0KT4A as capture 2~ reag~nt, and Leu3a a~ a detection r~agent.

-33~

TABLE IV

Capture Antibody:
OX~'4 0XT4A
Sample HPB T Cell Lysate 5 x 106 cells/ml 0.895 0.13 2.5 x 106 cells/ml 0.769 0.079 1.25 x 106 cells/ml 0.549 0.046 T Cell LeuXemia***
Patient Serum Sample 1184 0.000 0.000 Sample 1174 0.000 0.020 Sample 1195 0.004 0.040 Sample 1147 0.004 0.000 _ _ _ Values shown are OD , using the indicated capture antibody and bioti~ted Leu3a as detection antibody.
The indicated numbers of HP~ (human leukemia T cell line) : ~ells were lysed in 1 ml detergent buffsr.
Serum from patients with acute HTLV I associated T cell 2~ leukemia :
, While this a~say could detec~ solubilized CD4 in cell :lysates, no detectable soluble CD4 was observed in the sera of patients~with~HT1V I associated T cell leukemia, which is a disease~characterized by an intense activated population of T cells.
: Subsequent e~forts were focused on determining whether antibodies could be selected which ~ight preferentially 36 recognize a released (soluble) for~ of the CD4. Recombinant :::

' CD4, with the tran~membrane and cytoplasmic regions deleted at the gene level, was used as a model antigen. Antibodies were coated ~nto microtiter wells overnight and blocked as described. Samples containing eith0r buffer, detergent solubilized CD~ Prom the 3urkat T cell line a~ two di~ferent 5 dilutions, or recombinant CD4 were added, followed by a second incuba~ion with HRP-conjugated antibodieq (or biotinylated Leu3a followed by straptavidin HRR). Each antibody was evaluated on both a capture and detection mode with all other antibodie~ on each o~ the samples. The results are shown in Table. V and VI.

TABLE V

Ca~ture Ant ibody 3G2 B66 . 1 R2B7 - B67 . 2 oKr4 ~etection Antibo~r 0. 204 0 . ~27 0 . 154 0. 242 >2 . 00/l . 9 B66.1 0.053 0.064 1.420 0.053 >2.00/1.28 R2~7 0.230 >2.0/0.93 0.217 1.843 >2.09/>2 ~67.~ 0.027 00030 0.110 0.008 1.50 OKT4 0.037 0.0~4 0.040 0.000 0.008 Leu3A 0.196 0.206 >2.00/1.94 0.279 >2.00/>2 C~ll ly~at~l~ contained 5 x l0~ c~ l- Valu~ shown are OD 9 . Po~ tho~ antibody pairs wh~r~ absorbance6wa~ >2 . o ~o~ ~ x l0 c~lls/ml ly~ate, the valuQ for l x l0 c~lls/~l i~ ~hown in sa3a~a box prec:~ded by a sla~h.

TABLE VI

RECOMBINANT SOLUBLE CD4 DETECTIONt Cap~ure Antibody 3G2 B66.l R2B7 B67.2 OKT4 Detection 10 Antibod~_ 3G2 0.002 0.003 0.074 0.000 0.230 B66.l 0.002 0.012 0.170 0.004 0.0l0 R2B7 0.000 0.036 0.059 0.013 0.300 15 B67.2 0.000 0.000 0.008 0.000 0.009 OKT4 0.00l 0.000 0.0l0 0.002 0.000 Leu3A 0.008 0.000 1.765 0.000 0.231 t Values shown are OD4go~
: mAb B53.l was also used, but showed no positive results ~: when used as either capture or detect~ion reagent.
~ , 25 The da~a presented in Table~3 Y and VI reveals a wide range o~ assay efficacies for the detection o~ solubilized cell-surfac~ CD4 antigen (in celI lysa~e) or recombinant soluble :CD4:antigen.~ Optimal~comblnations~ for detection of reco:mbinant or lysate C~4 antigçn are shown in Table VII.

: ~ :

;
:: ~

, TABLE VII

OPTIMAL PAIRS OF ANTIBODIES FOR DETECTION
OF DETERGENT SOLUBILIZED OR R.F,COMBINANT CD4 Cap ure Antibody D~tection Antibody R2B7 Leu3A
R2B7 B66.1 B66.1 R2B7 B67.2 R2B7 OKT4 B66.1 OKT4 B67.2 1~ OKT4 Leu3A

_ _ _ _ _ Intexestingly, only the combination of R2B7 as a capture 2D antibody with Leu3A as a detection antibody gave signal with tha recombinant CD4 substantially equivalenk to that ssen in lysate, suggesting this pair might preferentially recognize soluble CD4.
Antibodies were generated fxom a mouse immunized with 2~ whol~ T cells and screened for heir ability to replace Leu3a in an a~say. 500 hybridoma clones were screened and three clones m~eting the above criteria were identified.
one of the~e clones, termed 8F4, showed the ability to block binding o~ ~ITC~ ~abeled Leu3A to CD4 positive T cell 30 surfaces.
An1:ibodies 8F4 and R2B7 were evaluated ~ith regard to optimal con~iguration in the assay. Tabla YIII shows that 8F4 u~ed as captur~ an~ibody with R2B7 used as detection antibody produced ~ ~ignificantly great:er 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.

lS

' :

,; ~
.

~: :

TABLE VIII
CD4 DET~CTION

Absorbance at 490 nm ..
8F4 Capture and R2B7 Capture and R2B7 Dstection 8F4 Detection -SAMPLE:
HPB Cell Lysate (cells/ml lysate) 2 x 1Q6 1.683 1.178 ; 1~1 x 106 1.005 0.619 5 x 105 0~583 ~.370 2.5 x 105 0.2~4 0.172 1.25 x 105 0.135 0.090 6.25 x 104 0.067 0.045 0 0.014 0.013 Recombinant Soluble CD4 Dilutions*
~"",___ __ _ .n 1:2 >2.0 1.411 1:4 >2.0 0.g9 20 1:8 >2.0 0.666 1:16 1.165 0.309 32 0.718 0.15 1:64 0.382 0.074 `::
,~ 2~ T Cell Cul~ure Superna~ants ~: ST16 T c~ll line : 0.187 0.135 5B4 T4 clone 0.152 0.104 6D11 T4 clone 0.171 0.120 5C8 T4 clone 0.331 0.246 TIL SC4 T8 clone 0.037 : 0.019 *
/ Ra~ios represent the dilutions o~ cell culture supernatant : ~ o~ transfected ~O cells expressi~g the recombinant ~: soluble ~D4 antigen.
~: Sam~le designation .

~ : :

The antibody used by Doumerc et al. (19~6, 6th Intl.
Congr~ss of Immunology, Toronto, Ontario, Canada, July 6-11, 1986, Abstr. 5.54.6, p. 708), IOT4, wa~ evaluated ~or its ability to measure the same form of CD4 antigen as that detected in the ~ssay using 2F4 with R2B7. Table IX shows the results when IOT4 antibody was used as both capture and detection reagent as was done by Doumerc et al.
.. _ . ...... ....... _ _ .
TABLE IX
~0 *

_ Detection_Antibody Capture Antibody 8F4 IOT4 R2B7 8F4 +NP40 .037 0.051 0.545 1~ -NP40 .013 0.035 0.423 IOT4 +NP40 .624 0.035 0.381 -NP40 .063 0.017 0.040 R2B7 ~NP40 ND 0.040 0.400 -NP40 ND 0.049 0.32Q

RECOMBINANT SOLUBLE CD4 DET~CTION
.
_ Detection Antibody Captur~
Antibody 8F4 IOT4 R~7 : 8F4 +NP40 .003 .142 1.341 -NP40 .005 .155 1.421 IOT4 +NP40 .004 ~010 0.010 -NP40 .006 .014 0.006 R2B~ +NP40 ND .133 1.18 -NP4 0 ND .13 4 1.18 3D Value~ sho~n ar~ OD . Where ind~cated, 0.25% NP40 wa~
pres~nt in both th~4~ple dilu~nt and con~uqat~ diluent of th~ ~andwi~h i~unoa~say. Reco~binant CD4 as~y~ W~rQ
carri~d out u~ing a ~taAdard dilution o~ c~ll cultur~
supexnatant Or transfact~d CHO c~ . ND: Not d~t~rmined -:`

.

Antibodies were evaluated for CD4 detection using di~ferent 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 conjugatle diluents (Ta~le IX).
IOT4 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 themselves and IOT4. When used as a capture reagent, IOT4 detected only C~4 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 t0 (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 itsel~ was capable of a strong signal for both recombinant and cell lysate samples. Inclusion o~ NP4 0 failed to disrupt this signal.
In cantrast, 8F4 did not show such behavior, reacting only weakly with both cell lysate and reco~binant material when used in both parts of the ~andwich. IOT4 also failed to give a signi~icant signal when paired with B66.1 and B67.2 for both cell lysate and recombinant samples.
Table X shows the results o~ scr~ening culture supernatants from T cell lines or clones derlved fro~
patients with rheumatoid arthritis or lung cancer.

3@~

. . _ . . _ .
TABLE X

Sample Soluble * Soluble *
Designation Phenotype CD8 (U/ml) CD4 (U/ml) 1 . CD4+ clone 112 249 2 CD4~ clone 102 241 3 CD4 clone 111 277 4 CD8 lone 2,388 CD4 clone 4,000 gQ
6 CD4 , CD8 clone 63 70 TIl 5 CD4 , CD8+ mixed line 568 61 TIl 6 CD4+, CD8+ mixed line 4,000 23B
TIl 6 2 CD4~, CD8+ mixed line 4,000 12Q
TIl 7 PBT CD4+, CD8~ mixed line 51 TIl 4 B cell line 5B4 CD4+ clone 120 20 6G7 CD4 clone 240 SAl CD4 clone 223 5C8 CD4+ clone 115 6G7 CD4+ clone 193 6D11 CD4+ clone 116 26 5B4 CD4~ olone 150 6D2 ~D4~ clone 53 176 5C4 CD4+ clone 117 STl 2Hl CD4 clone 52 178 STl lC10 CD4+ C10~2 160 3D S~l 13Gll CD8* ~lone 3,217 : ST2 13C6 CD4+ clone 132 ST2 13C6 2 CD4+ clone 53 5T2 llC12 C~8+ clonQ 1,0~6 ST2 13~10 CD4~ clone 369 94 .

ST2 CM CD4 , CD8 mixed line 221 ST2 13Hl CD8 clone 3,428 ST2 14A5 CD4 clone 72 142 ST2 13A5 CD4 clone 71 161 ST 13 PB CD4+, C~8+ mixed line 4,000 50 ST 16 CD4 , CD8 mixed line 901 247 ST5 PBT CD4 , CD8 mixed line 240 ST5 CD4 , CD8 mixed line TIl4 PBT CD4 , CD8mixed line 4,000 71 TIl4 lOF8 CD4 , CD8 mixed line 1,217 70 STl2 CD4 , CD8mixed line 4,000 S~ll CD4 , CD~mixed line 4,000 139 _ Blank values indicate undetectable levels. CD4 units were defined in terms o~ the amount ~f absorbance of CD4 1~ antigen found in a lysate of 10 Jurkat T cells in 1% NP40 buffer, as measured using 8F4 as capture and R2B7 as detection reagents. CD8 units were ~ased on a reference preparation of culture supernatant from Jurkat T cells used to standardize the CELLF~EE~ T8 (T Cell Sciences, Cambridge, MA) assay.

. ~ Cell phenotype was determined by flow cytometry. 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 2~ sandwich i~munoassay kit ~CELLFREE~ T8 Test Kit, T Cell Science~, Inc., Cambridge, ~A) was us~d to measure soluble CD8. The CD8 antigen detected by this assay has been ~ characterized previously as a 52-55 kD dimer composed of : mono~er polypeptide~, each with a molecular weight of 30 approximately 27 kD. As shown in Table X, 21 o~ 21 CD4+
cIones showed soluble CD4 in the supernatant. Zero o~ four CD~+ clones showed soluble CD4 in the supernatant. The cell `: ~
~ .

lines showed varying mixes of soluble CD4 and soluble CD8.
No correlation was observed between cell number and level o~
soluble CD4.
Table XI shows the rate of release of CD4 into the 5 media after in vitro stimulation of peripheral blood mononuclear cells.

TABLE XI

SOLUBLE CD4 LEVELS AFTER IN VITRO C L STIMUL~TION

;Type o~ Cell Days in Soluble Experiment # Stimulation* Culture CD4 (U/ml~
. _ __ !
1~ 1 PHA 1 4.4 2 6.~
3 15.0 17.4 . 20 1 O~T3 1 1.8 2 4.4 3 7.6 : 4 11.5 1 NONE 1 1.1 2 1.1 3 4.8 ~ 4 <~ 0 3D 2 PHA 1 7.6 :. ~ 3 24.3 4 43.9 41.6 3~

2 Phorbol myristate,l 10.3 acetate plus 3 12.9 ionophore A2317 4 12.7 - _ _ 5 7.8 Carried out as described in Section 23.1.3, supra.
CD4 units were as defined for Table XXIV.

In experiment #1, PHA showed significantly greater effect than stimulation with OKT3. Similarly, in experiment #2, PHA was significantly more effective than phorbol esters and ionophores at inducing CD4 release. No significant release occurred when cells were put into culture without mitogen, suggesting release is an active process and not merely due to cell death.
1~ Table XII shows levels o~ soluble CD4 detected in sera of individuals with HTLV-l associated adult T cell leukemia.

` . :

~: ' ' :

~ ,X
, ' . .
TABLE XII

Sample Soluble CD4*
Desiqnation Disea~e (units/~l) 1165 Acute Adult T Cell leuke!mia 3.51 1166 Acute Adult T Cell leukemia 37.56 1167 Acute Adult T Cell leukemia 7.90 1168 Acute Adult T Cell leukemia 5.61 1~ 1169 Acute Adult T CeIl leukemia 1.41 1170 Acute Adult T Cell leukemia 1.07 1171 Acute Adult T Cell leukemia 0.08 1172 Acute Adult T Cell leukemia 0.52 1173 Acute Adult T Cell leu~emia 9.~1 1174 Acute Adult T Cell leukemia 9.54 1175 Chronic Adult T Cell leukemia 8.92 1~ 1176 Chromic Adult r Cell leukemia 0.66 1177 Chronic Adult T Cell leukemia 2.54 1178 Chronic Adult T Cell leukemia 0.23 1179 Chronic Adult T Cell leukemia 0.31 1180 Chronic Adult T Cell leukemia 0.23 1181 Chronic Adult T Cell leukemia 1.83 1182 Chronic Adult T Cell leukemia 3.36 2~ 1183 Chronic Adult T Cell leukemia 2.21 ; 1184 Chronic Adult T Cell leukemia 3.78 1185 Smoldering Adult ~ Cell leukemia 0.87 1186 Smoldering Adult T Cell leukemia 0.59 1187 Smoldering Adult T Cell leukemia 0.24 1188 Smoldering Adult T Cell leukemia 0.31 1189 Smoldering Adult T Cell leukemia 1.34 : 2~ 1190 Smold~ring Adult T Cell leukemia 1.01 1191 Smoldering Adult T Cell leukemia 1.61 1132 Smoldering Adulk T Cell leukemia 0.91 Detected using R2B7 as capture reagent and biotinylated Leu3~ as de~ection reagent. ~D4 units were as defined for 3~ Tab~e :X.
.

Individuals with the most active stages of the disease had the highest levels of soluble CD4 in their sera.
Figure 1 shows the levels of CD4 in sera of normal individuals and in patients from a nun~er of disease groups.
Levels of CD4 in synovial fluid of rhe.umatoid 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 levals in longitudinal samples from patients on IL-2 therapy.

.

tS

. 2 :: :

~: :
;~

TABLE XIII
SOLUBLE CD4 LEVELS IN PATIENTS UNDEXGOING IL-2 ~IERAPY
.
Soluble Patient Date Soluble Soluble IL2R
Desi~tion (moO/day) CD8 (U/ml) CD4 (U/ml) (U/ml) A 5/04 492 19 >1600 A 5/06 531 28 >1600 A 5/10 529 27 >1600 A 5/11 490 22 >1600 10 B 1/2~ 325 10 385 B 2/04 595 19 >1600 B 2/10 1221 12 >1600 B 2/12 914 17 >1600 B 2/20 452 16 >1600 C 1/05 319 21 ~64 C 1/15 1232 26 >1600 1~ C 1/21 890 26 >1600 D 3/04 271 7 >1600 D 3/09 519 16 >1600 D 3/10 484 10 >1600 E 3/23 621 14 >1600 F 3/29 615 102 >1600 20 F . 4/05 898 140 >1600 Patient 1 333 9 494 Patient 2 222 22 >1600 Patient 3 274 12 683 Patient 4 149 11 : 650 ~Patient`5 1492 49 >1600 ::Patient 6 1008 74 >1600 2~ Pa~ient 7 608 8 >1600 Patient 8 2094 27 >1600 Patient g 779 16 >1600 Patient 10 1400 31 >1600 ;Normal 1** 476 9 466 Normal 2** 311 6 540 :Normal 3** 178 3 396 Norm~1 4** 5 39 Normal 5** 3 Normal 6** 4 ~:Normal 7** 6 :Normal 8** 6 Normal 9** 8 :Normal 10** 3 ' ~::

.: :
, :

* Soluble CD8 was measured using the CELLFREE~ T8 Test Xit (T Cell Sciences, Cambridge, MA). Soluble CD4 was detected using 8F4 capture, R2B7 detection. CD4 units were as defined for Tabla XXIV. Soluble IL2R was measured using the CELLFREE~ IL2R Test Kit (T Cell Sciences, Cambridge, MA).
**Healthy blood donors (not undergoing IL-2 therapy) The data of Table XIII shows that detectable levels of soluble CD4 are present in sera of patients being treated 1~ 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 CD~, along with ~fi soluble IL2R, in renal transplantation patients.

' 3~i TABLE XIV

Sample Patient Date Solubl~. So~uble ~Designation (mo./day) CD4* (U/ml) _2R (U/ml) : p 4/07 ~ .4 455 : 4/09 7.6 493 ; 10 4/14 20.4 4090 4/16 9.3 3865 5/02 6.8 1200 5/30 37.4 565 H 3/14 16.4 811 3/21 11,7 4~1 4/09 12.4 3~7 16 5/14 21.6 6g2 6/11 36.9 907 L 6/15 91.8 1965 6/16 29.2 2705 6/23 38.1 3990 6/30 48. ~ 7400

7/07 35.2 6300 G 4/30 26.1 1288 5/05 7.6 700 5/07 2~ .2 ~45 : 5/09 32.6 3625 : 5/12 17.1 3635 5/14 18.8 3035 26 ` 5/1~ ~1.2 3040 5/21 20.0 4080 6/04 46.5 2475 6/25 19.4 1995 S 6/13 6.6 1090 6/16 13.7 680 . 6/18 13.1 930 S/20 11.1 1705 6/23 8.3 27~8 6/25 19.5 5515 6/27 11.2 3460 6/30 9.9 2205 : 7/07 12 ~ 8 1825 : 3~ t Detectëd using 8F4 cap~ure, R2B7 d tection Analyzed uslng CELLFREE0 IL2R Test Kit -- ~ ~

,:

Elevated levels of CD4 did not show a correlation with IL2R
but did, like IL2R, show increases during rejection episodes.

6.2.2. RESULTS USING OPTIMIZED ASSAY PROTOCOL
Once the soluble CD4 assay was optimized and any effects of rheumatoid ~actor eliminati3d, it was possible to detect much lower levels of soluble CD4. For normal healthy individuals, the range of soluble CD4 was 8 U/~l to 36 U/ml with a mean of 17.2 U/ml. This was determined ~rom the assay of 189 normal samples. A high number of replicates was also run to achieve confidence at the low end o~ the range.
Using t~is improved assay, a series of renal transplant patient~ was analyzed, and the data is presented in Table XV.

TABLE XV

RENAL TRANSPLANT PATIENTS

~ P~TIENT Diagnosisl SolublP CD4 Soluble IL-2R Solubla CD8 :~ (U/ml)2 ~U/ml)3 (U/ml)4 2~
: Cl Cs~ 69 200 181 CsA 30 340 448 Bl Rejection15 240 47 Zl Stable 19 380 3}0 Stab}e 19 390 374 Stable 15 300 321 ~: 39 Rl Rejection24 420 481 Rej~ction33 820 120 Ml CsA 122 770 534 CsA 29 330 570 ~C

CsA 26 80 526 L1 Rejection 43 1040 ND
Rejection 30 1120 ND
S1 Rejection 25 6gO ND
P1 Rejection 44 2180 1894 Rejection 25 370 390 ~; S2 CsA 117 510 267 CsA 22 390 67 Al Stable 96 400 731 Stable 27 320 390 Stable 23 40 409 L2 Rejection 23 1240 228 Rejection too high 1000 1559 ~ejection 24 1050 452 1 Diagnosis was either cyclosporin A toxicity (CsA), stable :transplant, or rejection; multiple patient serum samples were taken at di~ferent timesO
lS 2 Soluble CD4 was measured using the improved assay (Section 6.1.2.2, supra).
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 (T Cell Scienceq, Cambridge, MA).
ND: Not Determined.

:The above data indicate~ that it was possible to detect soluble CD4 levels~in renal transplant patients in the pha~es o~ rejection, toXicity and stability. It was also possible to detect elevated levels o~ other solubla T
cell sur~ace markers, su::h as soluble CD8 and soluble IL2R.
This data show~ that soluble receptcr~ are present during : the course of transplant episodes. It is expected that a :~30: longitudinal study of each o~ these patients will provide data that will indicate how the levels of each oluble marker change with toxicity, rejection or stability episode~. Thus, a change in the o~sexved level for any particular marker, such as an increase or decrease or no 3C change, may be o~ more valu~ than the absolute level of a :: :
.

.
. ' . ' ~

.
. . ~ ..
. .
., ' marker present at any one point in time, for the diagnosis or monitoring of tr~atment in disease (see Section 8, infra). For comparison, the change in the obsarved level of a marker must be compared to a baseline level which could either be the level seen in normal inclividuals with no disease, the pre-transplant level in the renal patient, the value present in a stable situation or during remission of symptoms, etc.
In a preferred embodiment, the cliagnosis 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. Thus, for example, a better prognostic indicator can be a ris~ in one 1S marker relative to a simultaneous fall in another marker.
The resulting profile of soluble T cell marker expression should be an exquiæite indicator of minute changes in the immune system as its function is modi~ied by therapeutic treatments or disease progression (see Section 7, infra).
Table XVI gives the value~ of soluble CD4 levels seen during preliminary studies on patients with Acquired Immune ~eficiency Syndrome (AIDS) and other stages of HIV-induced disease including Kaposi's Sarcoma (KS), AIDS related complex (ARC) or as~mptomatic seropositive (ASYM).
~u TABLE XVI
LEVELS OF SOLUBLE RECEPTORS IN HIV-INFECTED PATIENTS

PATIENT Diagnosis Soluble CD4~ Soluble IL-2R* Soluble CD8 ( U/ml ) ( U/ml ) ( U/ml )

8 AIDS 1 1760 1007

9 AIDS 20 1964 626 1~ 11 AIDS 1 1529 343 A~C 71 1645 2646 ~ 19 ARC 177 150~ 1246 ARC -0 g~ 572 : 22 AR~ 16 1742 1279 23 ASY~ 15 740 2700 ~; 24 ASYM 2~ 1403 15 3 29 ASY~ 14 1308 1702 3} ASYM 17 1012 1386 32 AS~ 13 1541 5127 . : 33 A9YM: 42 1752 4022 ~ 3D 34 ASYM 10 :504 1598 : * Assays as indica~ed for Table XV.
i:
, ~
3~ :
.
.~

.
: ' ' From the above data, it is clear that although the values of soluble CD4 are low compared to the other soluble marker levels, they are easily detectable in sera from patients having di~ferent stages of HIV-induced disease. As discussed supra, improved prognostic indices based upon levels or changes in levels of these soluble markers are expected with longitudinal studies involving a panel of soluble receptor markers. Such studies should reveal a profile of soluble receptors that can be used to determine the stage of progression towards AIDS in patients or the response to treatment of such patients. ~ longitudinal study of the patients in Table XVI and others can be conducted to determine the soluble receptor profiles of the patients and to correlate these profiles with the efEicacy 1~ f their ongoing azido-deoxythymidine treatment tses Section 8, infra).
Using the improved assay format, paired samples o~
synovial fluid and sQrum were analyzed for severa} patients with rheumatoid arthritis. This data is presented in Table XVII.

TABLE XVII

2~ AND SERU~ SAMPLES FRO~ INDIVIDUAL PATIENTS
PATIENT Samplel Soluble CD42 Soluble C~4 tU/ml) + HA~U/ml) + IgG

1 serum 1613 3D fluid 4236 2 serum 1512 ~luid 6457 3 serum 1514 fluid 4439 3~' serum 21 16 fluid 103 96 serum 23 19 f luid 6 6 59 6 serum 13 14 ~luid 41 37 7 serum 22 22 fluid 78 84 8 serum 15 13 fluid 80 73 . g serum 15 13 ~luid 87 90 lo serum 19 15 ~luid 12g 125 Samples were obtained ~rom the serum or synovial fluid of each patient.

~A = heat aggregated IgG added to remove any false 1~ positive problems associated with occassional high RF
(rheumatoid factor) containing samples.
3 IgG = unaggregated IgG control to detect sampl~s that may have had RF problems.

The above data indicates that with the increased sensitivity of the e~ployed assay, it was possible to detect soluble CD4 levels in both serum and synovial fluid samples of rheumatoid arthritis patients. The levels observed in ~ serum samp}es wer~ within~ he normal range, however.
:~ ~ Furthermore, th~ levels of soluble CD4 were elevated in synovial:~luid sampleæ reLative to serum samples form 10 of

10 patients analyzed. This suggests a localized production and release of CD4 antigen.
:: : Lev~ls o~ soluble CD~ in sera of normal lndividuals and patient~ from a number of disease groups were m~asured, ; with the results ~hown in Figure 2. Eleva~ed levels of soluhle CD4 were observed in renal transplant patients, synovial:~luid of rheumatoid arthritis patient~, in some patients with EBV infections/ and in patients with various ~`~; ; 3~ leukemias. ~ :

';

~::

6.3. DISCUSSION
Assays have been described herein that allow the m~asurement of CD4 in a detergent solubilized membrane form, in a recombinant soluble form (genetically engineered to exclude the transmembrane region), and in a spontaneously released form from activated T cells. Nine different anti-CD4 antibodiest in a total of 63 different configurations (one configuration consisting of a single capture and a single detection antibody3 wer~ screened for suitability for deterting CD4 in cell lysates. Nine such suitable configurations were identifiedO Of these, only five configurations showed ~ignificant react:ivity with soluble recombinant CD4. Three of these five configurations involved R2B7 as one of the antibodies. R2B7 when paired 1~ with %F4 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 solubilized lysate CD4 is seen using 8F4 capture with R2B7 detection. This is rouqhly twice the ratio seen in the reverse confiyuration. It is possible that binding of one or more of the antibodies induces con~ormational changes in the molecule. This is supported by the observation that when IOT4 iR used as a capture reagent, only solubilized lysate CD4 may be detected (using 8F4 or R2B7 detection), 2~ whereas when 8F4 or R2~7 are used as capture in combination with IOT4 as detection, soluble CD4 is preferentially detected over solubilized lysate CD4. Recombinant CD4 serves as a good model for released CD4 since those antibody combinations which work be~t with recombinant soluble CD4 also work best with th~ spontaneously soluble form of the molecule. Thus, selection of the antibodies and their configuration in the sandwich immunoassay is crucial to successful detection of released CD4.

Doumerc et al. (1986, 6th Intl. Congress of Immunology, Toronto, Ontario, Canada, July 6-11, 1986, Abstr. 5.54.6, p. 70g) havP described an enzyme immunoassay based on the use of IOT4 as a capture and deteetion reagent to detect CD4 in serum. In our hands, this con~iguration works only to measure the membrane for~ of the molecule and fails to ad~quately measure the soluble recombinant or soluble spontaneously released form of the molecule described here. Doumerc et al. (id.) disclose increased serum CD4 during transient episodes of lymphocyte destruction. It is possible that the foxm o~ the molecule measured by Doumerc et al. represents a form still asæociated with pieces of membrane, not a truly soluble moiety. Such a membrane form will tend to aggregate or 1~ associate into micelles or vesicles, thus rendering it capable of detection in a sandwich immunoassay using the same antibody as both capture and detection reagent. Since soluble CD4 is not known to exist in multimeric structures, in the absence o~ repeating epitopes, the same antibody cannot be used as both capture and detection reagent for 2~
assay of ssluble CD4. Doumerc et al. ~urther suggest that the CD4 they measure correlates with total CD4 cell pool.
We suggest that CD4 release, like CD8, is rather a function of activation of cells. This is supported by the kinetics of released CD4 observed during in vitro stimulation. Thus, the molecule described by Doumerc is signi~icantly dif~erent from the molecule described herein.
CD4 release may be a function o~ the type and pathway of activa~ion. Phytohemagglutinin and T3 s~imula~ion both result~d in a release o~ small amounts of CD4. Stimulation with phorbo~ esters, known to cause phosphorylation and internalization o~ ~D4, or with ionophores, resulted in significantly less rel~ased CD4 than did PHA stimulation, despite intense cellular ac~ivation. The kinetics o~ CD4 3~ release were also significantly different between c811s -5~-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.
Cloned IL-2 dependent CD4 T cells or T cell lines containing CD4~ cells all show~d de ectable soluble CD4 in their culture supernatants. CD8+ c~lls showed only soluble CD8. Thus, th~ released molecules are an accurate reflection o~ the cell sur~ace phenotype of the cells. No correlation was observe~ between levels o~ soluble CD4 and 1~ the number of cells.
Low levels of CD4 were seen in sera from normal individuals. Elevated soluble CD4 was observed in certain individuals with EBV infection, lung cancer and with T cell leukemias, and correlated overall with the stage of disease l~ (Fig. 1,2). Elevations in CD4 antigen levels were also observed in di.corders due to HIV infection, and in some synovial fluid, but not sera, from patients with rheumatoid arthritis. Elevations were observed in certain patients on IL-2 therapy where there is activation of CD4~ as well as ao CD8 cells and natural killer cells. Finally, elevations were observed in sera of some patients undergoing renal allograft rejection or cyclosporin A toxicity. Soluble CD4 levels may thus be of value in the diagno~is and monitoring of a pathologic event.
2~ The relationship between spontaneously released CD4 and membrane CD4 can be determined f~om patterns of antibody reaativity. If the spontaneouæly released material were identical to the cell-sur~ace polypeptida, it should behave in the assays, which have detergent incorporated into them, like solubilized CD4 in cell lysate. If they are more analogou to the recombinant truncat~d version of CD4 they should behave like it. Th~ latter is the case; that is, those antibody pairs which afford suitable de~ection 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 op~imally with the released material. It is clear from antibody reactivity patterns that the released ~orm of the CD4 antigen differs significantly from the membrane form.
A key element in deriving a successful sandwicA
immunoassay for the detection of soluble CD4 was the strategy employed in antibody selection. A readily available model source of soluble CD4, recombinant truncated CD4, was used in a screening procedure to select antibodies with a preferential ability to identify the released form of the molecule. Using this criteria, a pair of antibodies was selected that could detect spontaneously released CD4 in sera and in culture supernatants. This strategy may be 1~ widely applicable to the detection and discovery of other released molecules.

7. EVALUATION OF THE ROLE OF THE SOLUBLE CDB RECEPTOR

IMMUNODEFICIENCY VIRUS INFECTION
2D We evaluated the levels of soluble CD8 and IL~R in patients with HIV infection. The levels of CD8 and IL2R
were compared with each other and with levels of plasma p24 antigen, CD4 cells, CDR cells, and CD4/CD8 ratios.

2~ 7.1. M~THODS
7.1.1. SAMPLE SELECTION
Stored serum from patients with ~IV infection was examined using either the CELLFREE~ IL-2R Test Kit or OE LLFREE~ T8 Test Kit tT Cell Sciences, Cambridge, MA). 63 3~ patients with HIV infection and 7 normal controls were studied. Patients were divided into 4 groups depending upon the mani~estations of HIV infection; these groups were: 21 assy~ptomatic seropositive ~ASSY~), 19 AIDS related complex (ARC), 13 Kaposi's sarcoma (XS) and 10 AIDS wi~h ' 311i opportunistic infection. Stored sera from 15 patients followed longitudinally over a period of 2 years were also examined.

7.1.2. HIV p24 ANTIGEN ASSAY
This assay utilized a sandwich ELISA microplate ~ormat. Highly specific rabbit polyclonal antibodies to HIV
p24 core antigen were immobilized on microtiter plat~ wells and used to capture ~IV p24 core antigen present in 450 ~1 f plasma. The captured HIV p24 core antigen was complexed with biotinylated polyclonal antibodies to HIV p24 core antigen and probed with a streptavidin-horseradish peroxidase conjugate. This complex was visualized by incubation with orthophenyldiamine-HCl producing a color 1S intensity directly proportional to the amount of HIV p24 core antigen captured. The results were also quantitated spectrophotometriGally and compared against the absorbance of an HIV p24 core antigen standard curve.

~0 7.1.3~ CD4/CD8 RATIO
The CD4 and CD8 ratios were determined by standard flow cytometry.
~.
7.2. RESULT5 Soluble CD8, soluble IL2R, p24 antigen, CD4/CD8 ratio, 26 +
CD4 cells and CD8' cells were measured in sampl~s from patients with AIDS, ARC, KS, ASSYM or normals, as shown in Table XVIII.

W N
+l +I fl +
) ~ O
. .. .
C~ O O1`O
o'P 11 a~
t~ l +l +l +l +l .~ ~r~ I` ~ a~
O t O CO ,~
~ ~ Q~ o o o o ~
x ~ ~ 8 ~
o o o o a a) a o ~
~:~ O ~ a~ :
+l +l +l +l ~
- a)'Q ~ ~r a~ I` I`' 1: ~1 S Qi ~

H
: Q ~ +l +l +l +l +~
; O ~ o ~ o R E3 I~ ~ O ~ ~ ~ +~ ~
~: :,: _I co Ln o ~ ~
Q) , ~ r ~ ~
Q 1~ *l +l +l +l ~ +l ~ ~
o t~ t o ~ 0 ,~ Il') u~ a) Q.
U~ a~
u~ v a ~ : ~
Z ~ , Using chi-square analysis, comparing each group with normal controls, the majority of patients with AIDS, ARC, ASSYM and KS showed levels of both IL2R and soluble CD8 which were great~r than the upper 95% value of normal ~p<0.00001).
IL2R was better than CD4/CD8, %CD4, and p24 for discriminating ASSYM from AIDS (p<0.0001), ASSYM from ARC
(p<0.002) and ARC from AIDS (p<0.0001). Of interest is the observed difference in soluble CD8 betw~een groups with ASSYM, KS and AIDS. This suggests that early on in the course of 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 (~alker, C.M. et al., 1986, 1~ Science, 234, 1563-1566). Soluble CD~ levels may be an accurate measure of the immune system's attempts to suppress HIV infection. In addition, by using a combination of both soluble IL2R and soluble CD8 levals in each patient group, it was possible to distinguish between normal and assymptomatic patients.
In addition to determining the value of each soluble marker level, a comparison was also made between different soluble marker values to determine how they correlated with one another~ The data prese~ted in Table XIX presents the correlation observed between several marker combinations.
The samples analyzed for this table do not represent longitudinal samples obtained from individual patients, but samples from the population of people belonging to the different HIV-infected groups.

3~ ' -~3-TABLE XIX
CORRELATIONS BETWEEN THE BEHAVXOR OF DIFFERENT
PARAMETERS IN AIDS PATIENTS*
S
ASSYM ARC KS .AIDS Combined HIV
Correlation Positive . . _ _ . . . _ sCD~ v sIL-2~ + +
sCD8 v CD4/CD8 - +
sCD8 v % CD4 sCD8 v % CD8 ~ +
p24 v ~L2R +
sIL2R v CD4/CD8 sIL2R v % CD4 - -sIL2R v ~ CD8 +
CD4/CD8 v % CD4 + ~ + +
1~ CD4/CD4 v % CD8 CD4 v ~ CD8 * Pearson Corxelation i expressed as ~ (positive : correlation between the two parameters) or - (negative correlation between the two parameters): blank values ~ indicate that the correlation had a probability :: : index > 0.05; s - soluble ; ~:

2~ It is clear that the occurrence of soluble markers is not independent of one another or of other markers of the immune system. Thus, the combined behavior of thes~
receptors should be even more valuable than the observance of any~single r~ceptor.
~ In addition:to the patient sa~ples analyzed abova, : : three patients with ARC and three with KS were followed longitudinally as shown in Figure 3. The results of this study indicated that soluble CD8 levels appeared to parallel ~64-the change in p24 core antigen levels. Since the p24 core antigen levels have not proved to ba sensitive enough tests of the progres ion of AIDS, we propose that th~ solubl2 CD8 levels, which r~flect the statu~ of the immun~ system itself, can be a much better indicator.

8. A CRITICAL ANALYSIS OF T~E DIAÇNOSTIC UTILITIES
OF I~MUNOASSAYS FOR SERUM AND URINE SOLUBLE INTER-A~ described herein, a study was conducted to evaluate 10 the diagnostic u~ility of assays for serum and urine soluble IL2R in renal allograft recipients. Serial serum and urine samples obtained pro~pectiv~ly were tested for soluble IL2R
level~ by ~andwich enzy~ immunoa~ay test, and correlations were sought with serum creatinine and episodes of rejection, 1~ cyclosporin A lC~) toxicity, and in~ection Our results demonstrated that a rise in serum IL2R between samples taken within a week predicted tha onset o~ rejcction bette:r than absolut~ serum IL2R level3 or urine values. For the diagno~is of acute re~ection, a ri~e in serum IL2R
2n (sensitivity 73%, spQcificity 87%) wa~ comparable in overall test p~rformanc~ with a ris~ in ~rum creatinine ~sen~itivity 70%, ~p~cificity ~4S). Overall, th~ two tests h~d equivalent recQiv~r op~rating characteristic curves.
Because the ~tiology o~ ~al~ positives in creatinine and 2B IL2R a 5~y~ di~red (pri~arily cyclo~porin~ toxicity and : inf~ction, ra~p~tively), th~ predictiv~ ~alu~ o~ the combin~d t~3t~ wa~ sup~rior to eith~r alone.

8.1~ ~ATERIALS AND ME~HODS

8.1.1. PATIENTS
The s~udy popula~ion consi~e~ o~ 33 adult~ who rec~ivad renal all ~ ra~t~ at th~ Ma~sachu~tt~ Gen~ral ~ospit21. Maint~n~nc~ im~unosuppres~ion con ist~d of -~5-cyclosporin A (CsA) and prednisone (Colvin, RoB~ ~ et al., 1987, Clin. Immunol. Immunopathol. 43:273-276). Episodes of rejection were treated with increased steroids, anti-T3 monoclonal antibody OKT3, or ATG (~nti-thymocyte globulin) (Delmonico, F.L., et al., 1987, Am. Surg. 236:649-654).
Rejection was diagnosed by a progressive rise in serum creatinine that respond~d to increased immunosuppressionO
Other causes of renal failure were excluded. Twenty-one episodes of rejection in 15 patients were monitored during the six month study period. Biopsies were obtained in ten patients and showed acute cellular rejection in all instances (Colvin, R.B., et al., 1987, Clin. Immunol.
Immunopathol. 43:273-276). CsA toxicity was diagnosed by a rise in creatinine that responded to decreased CsA dose.
S Nine toxicity episodes occurred in eight patients. Three viral episodes occurred in three patients: a severe cytomegaloviru~ infection, lymphoproliferation associated with Epstein Barr viru~, and a transient gastroenteritis accompanied by fever and lymphocytosis.
2~
8.1.2. IL2R ASSAY
Serum (N=481) and urine (N=274~ samples were obtained whenever possible prior to transplant and serially 1-2 times per week during hvspitalizatio~ and at most clinic visits for the first 3~6 months after transplantation. Samples were coded and stored frozen until assay. Soluble immunoreactive IL2R lev~ls were assayed by a ~andwich enzyme immunoassay test kit according to the specifications of the manufacturer (CELLFRE~, T Cell Sciences, Inc.) (Colvin, 3D 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
After decoding and classificatiQn of the clinical s~atus, the results of the serum and urine IL2R as~ay~ and the serum creatinine assays were compared according to standard statistical ~echniques, using a spread sheet data base. Samples taken within tb~ first four days after transplantation and during treatment for rej~ction and for two days afterward were excluded ~rom analysis. Various ~easurements (absolute and change bet:ween 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 Prsss, Cleveland, O~, pp. 1-65).
1~
8.2. RESULTS
The aggr~gate data ~or serum and urine IL2R levels are summarizcd in Table XXXI. The conaentration of ~erum IL2R
in 24 pretran~plant patients was elevated compared with normal controls, but ~ell after transplanta` ion in 20 of these patian~s. ~retreatment IL2R l~vels from patients who had no re~oction epi~ode~ did not di~r ~rom thos~ with sub~equ¢nt rQ~ec~lon ~pi~ode~. In ~ o~ th~ 5 patien~s with delay~d ons~t vf ~u~ct$on (craatinin~ not falllng b~low 4 by a~ day 7~, th~ s~ru~ IL2R l~vels r~ in~d ele~at~d }onger (transiently ~alling on day~ 1-2 in ~wo patients). Patients with no ~pi~od~s o~ r~ction during th~ ~onltoring period had mod~rat~ly ~l~Yated 9~rum I~R level3 (980 ~ 692, I75 sampl~s, 18 pa~i~nt~ ~ ~ .001) co~pared ~ith nor~als.
Sa~pl~ tha~ w~r~ tak~n during ~tabl~ p~riods from th~
patient~ that had an ~pi~od~ o~ r0~ction w~r~ si~ilar and have b~n i~clud~d in the ~stable~ category in Tabl~ XXXI.

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-Z days before the first rise in serum creatinine.
Four patients had a rise in serum IL2~ greater than the 90th percentile of stable patients. Overall, the mean serum IL~R was elevated 1-2 days prior to th~ rise in creatinine (P < .03). Fourteen episocles of rejection in g patients were sampled on the first day of creatinine elevation; 9 of 14 samples (64%) were eleva~ed above the 90th percentile of valu~s during periods of stability. The IL2R levels remained elevated after the creatinine rise and into the treatment period, finally declining during or lS 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 ro~e during episodes of infection (CMV, EBV, gastroenteritis) (Table XXXI). In contrast, serum IL2R levels were not significantly rais~d in patients with cyclosporine A toxicity.
The urine IL2R concentration rose in the immediate post-transplant pexiod and remained higher in periods of stability than in pratransplant levels (Table XXXI).
During episodes o~ rejection, urine IL2R rose in a pattern that was not diætinguishable from the serum values, except that somewhat greater sample-to-sample variation wa~ noted.
Urine IL2R al o ~ollowed the pattern of serum IL2R during episodes of infection and cyclosporine A toxici y 3~ .

..

o o oo U~
v v v v ,~ 3 ~ ~ 0 ~ o ~ a~
~ o ~ cn ~ o~ r~o ~ u~
~ ~ ~ 0 ~ 1 + 1 0 ,,~,_1 ,_1 .~ ,r, O ~ 0 * ~ V O
Z Z~ x,q 4 ~ Dl O o o c: ,~ 3 h v v v vv :~o ~0 m O O N ~o '1:1 u~ O o cn ~ ~oo ~
~ W. w 1:1 ~r ~ ~1 ,~ N O t' ~ ~ t` C~ ~ V E~ ,"
20E~ o ~ N ~ ~ ~ ~ ~ h ~: H ~

~ ~ .. C A ~ :

25~ p ~
~ . ~ h c~
.rl h~
30~ O ~ ~ a~D
_~ .rl I ~-1-3 G) ~ ~ ~ ~ O
~ ~ ~ q O ~ O-rl ~
1 X K g a) ~ n 0 L~ ~ 0 ~ O q) ~o o ~o ~ ~ o ~ -~ ~ ~: ~3 E~ ~1 U ~ h-~-~ ~ ~: I O :~
q~ U ~ V S'~ ~ o ~ ~ ~ c ~ ~ z ~ h ~ ) Ic U~ P4 3 U~ H S.l lc *
`: : :
:

,' ' ., ~" ~ .
' .
.
.

The absolute concentratlon of serum IL2R had a sensitivity of 46~3% and a specificity of 87.1% for the diagno~is 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 IL,2R had comparable sensitivity and specificity (44.0% and 8~.4%, respectively). A single serum creatinine level was no better, using the 9Qth percentile as a threshold for a 0 positive test (sensitivity 39.0%, specificity 88.9%~.

TABLE XXXII

COMPARISON OF ASSAYS FOR THE DIA~NOSIS
OF RENAL ALLOGRAFT REJECTION
. . _ Assay _ N Sensitivlty (%) specificitY ~L

Serum IL2R 328 46.3 87.1 Creatinine 328 39.0 88.9 ao Q I~2R 225 73.3 86.7 ~ Creatinine 225 70.3 84~0 Urine IL2R 156 44.0 86.4 2~
Q IL2R 111 52.4 87.8 Positive defined as >90th percentile of values from sta~le patients. Samples taken during the first 4 days after transplantation were excluded from this analysis.
Rejection samples were taken from 2 days befor~ the first ; 3D rise in creatinine until just before anti-rejection therapy was begun. N, number of samples. ~, ris~ in ** serial values taken within 1 week.
Number of samples ~C

In clinical practice, the absolute level of serum creatinine is less useful for the diagnosis of rejection than a rise in the level in serial samples. Based on this analogy, we determined whether the change in IL2R
concentration in serial samples ~aken within a week (delta IL2R) might be mora sensitive than the absolute level~ 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 1~ sensitivity (73.3~ than absolute serum IL2R levels ~or the diagnosis o~ 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).
1~ 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 testæ also had a comparable sensitivity specificity relationship overall, independent of 2D the threshold selected for definition o~ a positive test, as shown in the ROC curves (Fig. 6). However, because the sources of false positives in creatinine and IL2R assays di~er (primarily cyclosporine A toxicity and infection, respectively), ~he predictive value of the combined ~.ests 2~ was superior to either test alone (Table XXXIII).

3~

TABLE XXXIII

PREDICTIVE VALUE OF SERIAL SERUM IL2R ~ND CREATININE ASSAYS

Serum Values _ % o~ Samples with Each Diagnosis CsA
Cr ~ IL2R **Rejec- Toxi->O.1 m~/dl >139 U/ml N Stable _tion city In~ection 1~ Single Test:
- 16987.2 7.1 2.6 5.1 + 5633.944.6 14.3 5.4 - 17385.3 6.1 6.7 1.8 ~ 5230.851.9 1.9 15.4 Combined Tests:
- - 14392.3 3.5 2.8 1.4 + + 26 7.780.8 3.8 7.7 + - 3056.716.7 23.3 3.3 2D _ + 2653.823.1 0.0 23.

oYerall 22573.3 16.4 5. 4.9 . .
Rise in values 5 days to 6 months after transplantation between seria} samp}es taken within 1 week. Rejection samples were taken ~rom 2 days before the rise in creatinine until the institution of anti rejection therapyO ~Positive values were defined as >9Oth percentile of stable values.
**
~ N, Number o~ samples , '~

8.3. DISCUSSION
The develop~ent o~ a reliable, noninvasive, economical diagnostic test for immunologic rejection is an important goal. The renal biopsy is the reference standard, but is not without risk and expense. ~ine neledle aspirates have a lower risk, but have not ~een widely a~pplied because they require spacialized expertise for per~ormance and interpretation. Many less invasive blood tests have been proposed, only to be abandoned due to a high cost/banefit O ratio or lack of clinical predictive value beyond that of serum creatinine.
We have found that the specifisity and sensitivity o~
soluble serum IL2R assays are similar to those o serum creatinine for the diagnosis of rejection. Among the 1~ various IL2R measurements, the serial change in serum IL2R
was the most sensitive and specific. The absolute level of serum IL2R varied more from patient to patient and was elevated in chronic renal failure (Colvin, R.B., et al., 1987, Clin. Immunol. Immunopathol. 43:273-276). The urinary concentration of IL2R showed a similar pattern, but had greater sample to sample variation, probably due in part to differencas in urine output and urinary degradation of the immunoreactive determinants. Standardization with urine cxeatinine concentration and inhibition of protease activity can be used to improve the quality of the urinary assays.
A revealinq analysis of a diagnostic test is its R~C
curve, which formalizes the relationship between sensitivity and specificity, and i5 an intrinsic property of the test, independent of disease prevalence or the definition of a 3~ positive result (Fink, D.J~ and Galen, R.S., l9a2, in Computer Aids to Clinical ~ecisions, Vol. 2, William , B.T., ed., CRC Press, Cleveland, OH, pp. 1-65). ROC curves repor~ed in these studies demonstrate that the overall test performance o~ serum IL2R was indistinguishable from serum :' : ' .

creatinine for the diagnosis of rejection. However, each test had different sources of ~false positives" in the allograft recipients. CsA toxicity raised the cr atinine levels more than the soluble IL2R levels, and viral infection was associated primarily wit:h a marked rise in soluble IL2R. Thus, the combination of tests for serum IL2R
and creatinine had greater predictive value than either test alone for distinguishing acute reje~tion ~rom infection or CsA toxicity. The strength o~ the combination test is evident in Table XXXIII. If a rise in serum IL2R
accompanies a rise in creatinine, the odds ara 20:1 in favor of rejection over CsA toxicity. In contrast, if no IL2R
rise accompanies the rise in creatinine, the odds favor CsA
over rejection by about 3:2 (the majority of patients will 1~ be stable).
In these studies and others (Colvin, R.B., et al., 1987, Clin. Immunol. Immunopathol. 43:273-~76; Solc, V. and Krause, J.R., 1987, Diag. Clin. Immunol. 5:171-174), infection was accompanied by levels of serum I~2R (up to 33,825 units/ml during a cytomegalovirus infection in this series) even higher than those observed during allograft rejection, suggesting a guantitative or qualitative difference in T c~ll activation. Accordingly, extremely high levels o~ IL2R favor the diagnosis of infection over rejection. Finally, transient eleva~ions in serum IL2R are sometimes observed in clinically stable transplant recipients. These have been categorized here as ~false positives~ but may be caused by subclinical episod~s of re~ection or infection.
: 33 IL2R assays can also be valuable in the monitoring of immunologic activity in recipients of other organ allografts, such as the h~art, liver and pancreas, in which the early diagnosis of rejection is particularly difficult.

-7'1-9. DEPOSIT OF HYBRIDOMAS
The following hy~ridoma cell lines, producing the indicated monoclonal antibody, have been deposited with the American Type Culture Collection, Rockville, Maryland, and have been assigned the listed accession numbers:

Accession Hybridoma Monoclonal AntibodyNumber Cell line AM92/2R12 AM92/2Rl2 (anti-IL2R] HB 9341 Cell line 7G7 7G7 (anti-IL2R) HB 8784 Cell line 4C9 4C9 (anti-CD8) HB g340 Cell line 5F4/7Bl2 5F4/7Bl2 (anti-CD8) HB 9342 Cell line 8F4 8F4 (anti-CD4) HB 9843 Cell line R2B7 R2B7 (anti-CD4) HB 9842 16 The present invention is not to be limited in scope by the cell lines deposited since the deposited embodiments are intended as single illustrations oP one aspect of the invention and any cell lines which are functionally : equivalent are within the scope of thiq invention. Indeed) various modifications o~ the invention in addition to those shown and described herein will become apparent to those skilled in the art fr~m the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

:

.,~

Claims (62)

WHAT IS CLAIMED:

1. A method for detecting or measuring the amount in a sample of a soluble molecule carrying antigenic determinants of the CD4 T cell surface antigen comprising:
(a) contacting the sample with a first anti-CD4 monoclonal antibody and with a second anti-CD4 monoclonal antibody that does not compete for the same binding site on the CD4 antigen as the first anti-CD4 antibody under conditions which allow immunospecific binding; and (b) detecting whether immunospecific binding occurs of a component in the sample with both first and second anti-CD4 antibodies, in which immunospecific binding of a component of the sample with both first and second anti-CD4 antibodies indicates the presence of the soluble molecule, in which the sample contains only such soluble molecules as are spontaneously released.

2. The method according to claim 1 in which the second antibody is labeled so that it is capable of producing a detectable signal.

3. The method according to claim 1 in which the first antibody is immobilized.

4. The method according to claim 1 in which the first antibody is immobilized and the second antibody is labeled so that immunospecific binding is indicated by the detection of immobilized label.

5. The method according to claim 1, 2 or 4 in which the first antibody comprises monoclonal antibody 8F4 as produced by the hybridoma deposited with the ATCC and assigned accession number HB 9843.

6. The method according to claim 1, 2 or 4 in which the second antibody comprises monoclonal antibody R2B7 as produced by the hybridoma deposited with the ATCC and assigned accession number HB 9842.

7. The method according to claim 1, 2 or 4 in which the first antibody comprises monoclonal antibody 8F4 as produced by the hybridoma deposited with the ATCC and assigned accession number HB 9843, and the second antibody comprises monoclonal antibody R2B7 as produced by the hybridoma deposited with the ATCC and assigned accession number HB 9842.

8. The method according to claim 1 in which the first antibody has the same epitope specificity as that of monoclonal antibody 8F4 as produced by the hybridoma deposited with the ATCC and assigned accession number HB 9843.

9. The method according to claim 1 in which the second antibody has the same epitope specificity as that of monoclonal antibody R2B7 as produced by the hybridoma deposited with the ATCC and assigned accession number HB 9842.

10. The method according to claim 1 in which the first antibody has the same epitope specificity as that of monoclonal antibody 8F4 as produced by the hybridoma deposited with the ATCC and assigned accession number HB 9843, and the second antibody has the same epitope specificity as that of monoclonal antibody R2B7 as produced by the hybridoma deposited with the ATCC and assigned accession number HB 9842.

11. The method according to claim 1 or 4 in which the sample is serum.

12. A kit for measuring the level of a cell free soluble molecule carrying antigenic determinants of the CD4 T cell surface antigen comprising:
(a) a first anti-CD4 monoclonal antibody; and (b) a second anti-CD4 monoclonal antibody that does not compete for the same binding site on the CD4 antigen as the first anti-CD4 antibody.

13. The kit of claim 12 in which the second anti-CD4 antibody is labeled so that it is capable of producing detectable signal.

14. The kit of claim 13 in which the label is an enzyme.

15. The kit of claim 14 in which the enzyme is horseradish peroxidase.

16. The kit of claim 12 in which the first antibody comprises monoclonal antibody 8F4 as produced by the hybridoma deposited with the ATCC and assigned accession number HB 9843.

17. The kit of claim 12 in which the second antibody comprises monoclonal antibody R2B7 as produced by the hybridoma deposited with the ATCC and assigned accession number HB 9842.

18. The kit of claim 12 in which the first antibody comprises monoclonal antibody 8F4 as produced by the hybridoma deposited with the ATCC and assigned accession number HB 9843, and the second antibody comprises monoclonal antibody R2B7 as produced by the hybridoma deposited with the ATCC and assigned accession number HB 9842.

19. The kit of claim 17 in which monoclonal antibody R2B7 is labeled.

20. The kit of claim 18 in which monoclonal antibody R2B7 is labeled.

21. The kit of claim 20 in which the label is an enzyme.

22. The kit of claim 21 in which the enzyme is horseradish peroxidase.

23. The kit of claim 12 in which the first antibody has the same epitope specificity as that of monoclonal antibody 8F4 as produced by the hybridoma deposited with the ATCC and assigned accession number HB 9843.

24. The kit of claim 12 in which the second antibody has the same epitope specificity as that of monoclonal antibody R2B7 as produced by the hybridoma deposited with the ATCC and assigned accession number HB 9842.

250 The kit of claim 12 in which the first antibody has the same epitope specificity as that of monoclonal antibody 8F4 as produced by the hybridoma deposited with the ATCC and assigned accession number HB 9843, and the second antibody has the same epitope specificity as that of monoclonal antibody R2B7 as produced by the hybridoma deposited with the ATCC and assigned accession number HB
9842.

26. A method for diagnosing a state of immune activation in a subject comprising detecting or measuring an increase, relative to levels in a subject in an unactivated state, in the amount in a sample from the subject of a spontaneously released, soluble molecule carrying antigenic determinants of the CD4 T cell surface antigen.

27. A method for diagnosing a state of immune activation in a subject comprising detecting or measuring an increase, relative to levels in a subject in an unactivated state, in the amount in a sample from the subject of a soluble molecule carrying antigenic determinants of the CD4 T cell surface antigen, according to the method of claim 1.

28. A method for diagnosing a state of immune activation in a subject comprising detecting or measuring an increase, relative to levels in a subject in an unactivated state, in the amount in a sample from the subject of a soluble molecule carrying antigenic determinants of the CD4 T cell surface antigen, according to the method of claim 7.

29. A method for determining the phenotype of a T
cell comprising incubating the cell in vitro, and detecting or measuring the amount, in a sample of the cell culture fluid, of a spontaneously released, soluble molecule carrying antigenic determinants of the CD4 T cell surface antigen.

30. A method for staging adult T cell leukemia in a patient comprising measuring the amount of a spontaneously released, soluble molecule carrying antigenic determinants of the CD4 T cell surface antigen in a sample obtained from the patient, in which increases in amounts relative to healthy individuals or to the patient at an earlier time indicates more advanced stages of disease.

31. The method according to claim 30 in which the sample is serum.

32. A method for diagnosing rheumatoid arthritis in a patient comprising detecting or measuring an increase in the amount, relative to healthy individuals or to the patient at an earlier time, of a spontaneously released, soluble molecule carrying antigenic determinants of the CD4 T cell surface antigen in synovial fluid obtained from the patient.

33. A method for diagnosing rheumatoid arthritis in a patient comprising detecting an increase in the amount in synovial fluid relative to the amount in serum of a spontaneously released, soluble molecule carrying antigenic determinants of the CD4 T cell surface antigen,

34. A method for monitoring the treatment of a patient with a viral infection comprising measuring the level in a body fluid of the patient of a spontaneously released, soluble molecule carrying antigenic determinants of the CD4 T cell surface antigen.

35. The method according to claim 34 in which the viral infection is Human Immunodeficiency Virus infection.

36. A monoclonal antibody which preferentially binds to a spontaneously released, soluble molecule carrying antigenic determinants of the CD4 T cell surface antigen, relative to the CD4 T cell surface antigen.

37. A rat monoclonal antibody reactive with (a) a spontaneously released, soluble molecule carrying antigenic determinants of the CD4 T cell surface antigen, and (b) the CD4 T cell surface antigen.

38. Monoclonal antibody 8F4, as produced by the hybridoma deposited with the ATCC and assigned accession number HB 9843.

39. Monoclonal antibody R2B7, as produced by the hybridoma deposited with the ATCC and assigned accession number HB 9842.

40. The Fv, Fab, Fab', or F(ab')2 fragment of the monoclonal antibody of claim 36, 37, 38 or 39.

41. An antibody comprising the Fv, Fab, Fab', or F(ab')2 fragment of the monoclonal antibody of claim 36, 37, 38 or 39.

42. A method for differentially diagnosing allograft rejection from infection in a transplant patient comprising:
(a) measuring the amount of a spontaneously released, soluble molecule carrying antigenic determinants of the interleukin-2 receptor in a sample obtained from the transplant patient;
and (b) measuring the amount of creatinine in a body fluid obtained from the patient, in which increases in both the amount of the molecule and in the amount of creatinine, relative to healthy subjects without a transplant or not undergoing rejection or to the patient at an earlier time, indicates rejection or the allograft.

43. The method according to claim 42 in which the sample comprises serum, and the body fluid comprises serum.

44. The method according to claim 42 in which the sample comprises urine, and the body fluid comprises urine.

45. The method according to claim 42 in which the soluble molecule carrying antigenic determinants of the interleukin-2 receptor is detected by:
(a) contacting the sample with monoclonal antibody 2R12, as produced by the hybridoma deposited with the ATCC and assigned accession number HB 9341, and with monoclonal antibody 7G7, as produced by the hybridoma deposited with the ATCC and assigned accession number HB 8784, under conditions which allow immunospecific binding; and (b) detecting whether immunospecific binding occurs of a component in the sample with both monoclonal antibodies 2R12 and 7G7, in which such immunospecific binding indicates the presence of the soluble molecule in the sample.

46. The method according to claim 42 in which the allograft comprises a renal transplant.

47. The method according to claim 42 in which the allograft comprises a cardiac transplant.

48. A method for detecting, monitoring the treatment of, or staging a disease or disorder in a patient comprising measuring the levels in a body fluid of the patient of a plurality of spontaneously released, soluble molecules, which molecules are selected from the group consisting of molecules carrying antigenic determinants of the interleukin-2 receptor, molecules carrying antigenic determinants of the CD4 antigen, and molecules carrying antigenic determinants of the CD8 antigen.

49. The method according to claim 48 which further comprises measuring the changes in the levels of the molecules relative to the levels in healthy individuals or in the patient at an earlier time.

50. The method according to claim 48 in which the disease or disorder comprises transplant rejection.

51. The method according to claim 49 in which the disease or disorder comprises transplant rejection.

52. The method according to claim 50 in which the transplant is a renal transplant.

53. The method according to claim 52 in which renal transplant rejection is differentially diagnosed from cyolosporin A toxicity.

54. The method according to claim 48 in which the disease or disorder is caused by viral infection.

55. The method according to claim 49 in which the disease or disorder is caused by viral infection.

56. The method according to claim 54 or 55 in which the viral infection is Human Immunodeficiency Virus infection.

57. The method according to claim 48 in which the disease or disorder comprises rheumatoid arthritis.

58. The method according to claim 49 in which the disease or disorder comprises rheumatoid arthritis.

59. A kit for detecting, monitoring the treatment of, or staging a disease or disorder in a patient comprising:
(a) a pair of anti-interleukin-2 receptor monoclonal antibodies, which antibodies do not compete for the same binding site on the interleukin-2 receptor; and (b) a pair of anti-CD4 monoclonal antibodies, which antibodies do not compete for the same binding site on the CD4 antigen.

60. A kit for detecting, monitoring the treatment of, or staging a disease or disorder in a patient comprising:. :
(a) a pair of anti-interleukin-2 receptor monoclonal antibodies, which antibodies do not compete for the same binding site on the interleukin-2 receptor; and (b) a pair of anti-CD8 monoclonal antibodies, which antibodies do not compete for the same binding site on the CD8 antigen.

61. A kit for detecting, monitoring the treatment of, or staging a disease or disorder in a patient comprising:
(a) a pair of anti-CD8 monoclonal antibodies, which antibodies do not compete for the same binding site on the CD8 antigen; and b) a pair of anti-CD4 monoclonal antibodies, which antibodies do not compete for the same binding site on the CD4 antigen.

62. The kit of claim 59 which further comprises a pair of anti-CD8 monoclonal antibodies, which antibodies do not compete for the same binding site on the CD8 antigen.