EP0625909A1 - Autoantibody assay and usage in the control of human disease - Google Patents

Abstract

The invention relates to methods for titrating autoantibodies and to their therapeutic use, in particular antithymocyte autoantibodies (AAAT). This invention describes methods of autoantibody titration and the use of autoantibodies to antithymocytes (AAAT), from an amount of autoantibodies relative to a target population of lymphocytes, to predict the body's resistance / susceptibility to disease. and tumors, as well as suggestion protocols for the treatment of various diseases, malignancies and autoimmune reactions. The main characteristic of the invention is that, according to the inventor's thesis, by modifying the quantity of autoantibodies (in the sera) which regulates the target cell population, diseases and / or malignancies can be suppressed or otherwise treated .

Description

AUTOANTIBODY ASSAY AND USAGE IN THE CONTROL OF

HUMAN DISEASE

BACKGROUND OF THE INVENTION 1. Field of the Invention

This invention relates generally to the field of biological cellular antigen assay and, specifically, to establishing a method of assay for autoantibodies in relation to regulation of the immune system. This includes, and herein teaches, a novel method for relating the assay to predict body resistivity (or susceptibility) to autoimmune diseases and tumors, thereafter suggesting a protocol for treatment of the aforesaid diseases by increase/reduction of the autoantibody titer.

2. Discussion of Relevant Publications

All species tested (rat, mouse, man etc.) have a demonstrable antibody (ATAA) in their serum which is capable of reacting with and sometimes killing { in vi tro) a selected population of their own thymocytes (autologous) or thymocytes of the same species (allogeneic) . This antibody will also react in certain species with bromelain- treated red blood cells and possibly dendritic epithelial cells, CD5 receptor and a presently unknown receptor specific for ATAA. During the last decade or two, various investigators have detailed a wide variety of autoantibodies [specific antibodies which react with almost any tissue or organ in the body, i.e. brain, skin, various serum proteins (including antibody molecules), all blood cells (including lymphocytes), intestinal tissues, heart cells, etc.]. It has never been clear whether these antibodies cause resultant disease or are the result of the disease, but in some cases they are interpreted as being causal.

One such autoantibody is one directed against antigens on thymocytes (ATAA) . There are many different types of thymocytes which originate in the thymus and are the precursors of T-lymphocytes which are found in blood and various lymphoid and non-lymphoid organs of the body. T- lymphocytes are concerned with cellular immunity, as differentiated from B-lymphocytes which produce antibodies and are responsible for the humoral immune response. The T-lymphocytes recognize antigens by recognition sites and accessory molecules which are of various types and which define each type of T-lymphocyte. The recognition sites are associated with the major histocompatibility complex. Thus, T-lymphocytes are of many different types, each identifiable by distinct antigenic differences and many of which have distinct functions. The development (ontogeny) of the various T-lymphocytes occur within the thymus gland and are present in various percentages, at various times, within the thymus. (See: Molecular and Cellular Events of T-Cell Development; B.J. Fowlkes and Drew M. Pardoll; Adv. In Immunology; Vol. 44, pp. 201-217; 1989).

Aside from classification of T-lymphocytes by their recognition sites and accessory molecules, there are several different functional subsets of T-lymphocytes: helper cells; suppressor cells; killer cells; etc. therefore, it is no surprise that investigators have been unable to understand the nature or the functional significance of the thymocyte autoantibody. It has been said that the serum levels of ATAA do not show etiological significance to disease (Eisenberg et al . , J . Immunology 722, p. 2272, 1979).

Measurements of ATAA are made in vi tro in which antibodies in serum are mixed with an animal ' s thymocytes and a reaction is observed which can lead to the binding of and/or death of a certain number of the cells. It is illogical to think that such antibodies and target cells could coexist in vi vo. Thus, it is not surprising that a disease causal effect has not been postulated. There has recently been described, in certain strains of mice (motheaten and NZB), a rapidly fatal condition characterized by a wide variety of autoimmune diseases and an extraordinarily high level of ATAA. It is of special interest that some of these animals have naturally occurring anti-tumor activity.

In the same vein as the above, Perper et al . disclose an IgG thymolytic autoantibody in rats which has specificity for a sub-population of T-cells (R.J. Perper, A.L. Oronsky and Maria Sanda; Immunology, paper 312; 1976). These researchers present an interesting disclosure wherein a cytotoxic anti-thymocyte IgG autoantibody is found present in Lewis rats which, in the presence of autologous complement, destroys (in vi tro) 12-28% of isologous or aubologous thymocytes, a small number of lymph node cells and splenocytes, but not bone marrow or circulating lymphocytes. The labile cells in the thymus represent a finite subpopulation which is autologous anti-thymocyte antibody sensitive and steroid resistant. The presence of the autoantibody is randomly distributed in outbred animals, whereas inbred Lewis rats, a strain in which the induction of some autoimmune reactions is under genetic control, the antibody is almost always present. In this strain, the susceptible T-cells and the quantity of circulating autoantibody is significantly depressed during the productive phase of the T-cell mediated disease (adjuvant polyarthritis) and returns to normal after the disease becomes stabilized. There is a direct relationship seen between the amount of susceptible cells in the thymus and the amount of antibody in circulation, which suggests that the antibody could serve as a marker for a specific subpopulation of thymocytes which may have a regulatory influence on T-cell reactivity. But Perper et al . stop at this point and, the aforesaid disclosure notwithstanding, recent patent and professional literature searches have failed to reveal a teaching of what I now term ATAA causality.

The concomitant presence of both ATAA and autoimmune diseases in the same animal continues to frustrate other investigators because none have yet been able to establish a causal role for ATAA, simply because they are making measurements of ATAA in the presence of disease. Interestingly enough, U.S. Patent No. 4,937,071, teaching a METHOD FOR AUGMENTING IMMUNE RESPONSE, discloses a method for enhancing the ability for humoral immune response in a mammal which comprises exposure of lymphocytes histocompatible with the lymphocytes of the mammal to the presence of delta-immunoglobulin at a concentration higher than that at which the lymphocytes would have been exposed while in the lymph or blood stream of the mammal and, thereafter introducing these lymphocytes to the blood stream of lymph of the mammal. This is clearly a method of augmentation, and no one suggests the use of a naturally occurring (or monoclonal replicate of an) autoantibody to regulate the immune response mechanism. This fact exists in spite of the proliferation of a great deal of related work such as the preparation of monoclonal antibody for inhibiting adhesion-dependent leukocyte functions, the use of monoclonal antibody for diagnostic procedures used to differentiate between normal cells and tumor cells, diagnostic and therapeutic uses of a monoclonal antibody against the antigen found on essentially all normal T-cells and cutaneous T-lymphoma cells, the identification of monoclonal anti-Sm antibodies and the disclosure of various compounds that are said to modulate immune response ( ibid. ) . Further to this abundance of antibody literature, Harley, in U.S. Patent No. 4,784,942, teaches the production of monoclonal antibodies, produced by a continuous hybridoma cell line, in methods for detecting the presence of selected autoimmune RNA proteins and antibodies against such proteins in biological samples, and which may be incorporated into diagnostic test kits for the similar purpose. The monoclonal antibodies used against autoimmune RNA proteins include proteins such as La/ssb, Ro/ssa, aNP and Sm. These monoclonal antibodies may be applied in methods for screening subjects for systemic lupus erythematosus, subacute cutaneous erythematosus, neonatal lupus, Sjδgren's syndrome, complete congenital heart block, and other disorders which involve the presence of antibodies against autoimmune RNA proteins. In spite of painstaking searches in the literature of the U.S. Patent Office and the professional journals, I have found no reference, of any kind, to the inculcation of a predictive value for imputing the susceptibility for autoimmune diseases .

The above mentioned shortcomings which appertain within the study of autoantibodies and their relationship to carcinomas and autoimmune diseases have been satisfactorily answered by my research and are revealed in the hereinafter disclosed assay and treatment methods.

SUMMARY I have found quite surprisingly that the target cell of ATAA regulates the development of the disease and the level of ATAA serves as a marker and may control the number of these (target) cells. I use the amount of ATAA to predict susceptibility/resistivity to disease and to regulate the quantity of these target cells so as to be able to manipulate body susceptibility/resistivity and the cause of both autoimmune diseases and malignancies. The quantitation of ATAA in circulation using the appropriate target cell (a subpopulation of thymocytes, the antigen of which might be present on various other cell types and detailed, but not limited to those mentioned earlier) predicts susceptibility to the development of allergic hypersensitivity reactions and autoimmune disease or resistance to tumor development. Furthermore, by either raising or lowering the level of ATAA, or the target (susceptible) cell, modification may be had of the course of either tumor development or autoimmune disease/allergic hypersensitivity diseases.

BRIEF DESCRIPTION OF THE DRAWINGS Of the drawings:

Figure 1 is a graphical representation showing the effect of pre-existing autoantibody titer on the severity of adjuvant disease (re: swelling) in Lewis rats;

Figure 2 is a graphical representation contrasting the level of pre-existing anti-thymocyte autoantibody (ATAA) with the change in body weight (day 8- 18) after the induction of EAE; and

Figure 3 is a graphical display which illustrates that, when the target cell of ATAA is eliminated, there exists an enhanced immune reactivity of the surviving cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The target cell of ATAA is able to suppress the cellular immune response (a suppressor cell in, and derived from, the thymus). High levels of ATAA reduce the number of suppressor cells and allow the development of autoimmune diseases. Alternatively, low levels of ATAA result in high levels of suppressor cells which prevent cellular immunity from rejecting malignancies. Thus, an inverse relationship between vulnerability to malignancy and autoimmunity exists. This relationship is regulated by the presence of ATAA. The reason that serum levels of ATAA do not correlate with the progression of disease is because, when an autoimmune disease appears, there may occur a production and release from the thymus of suppressor cells in an attempt to abort disease development. These cells consume circulating ATAA which results in a drop in its level in circulation. Thus, the (conventional) measurement of ATAA during disease will produce anomalous data that are completely misleading.

The induction of adjuvant arthritis in rats has been shown in multiple published studies to be a model of animal and human autoimmune ' disease, as has the induction of allergic encephalomyelitis (EAE) . In the arthritis model, the amount of ATAA (as measured by the ability of serum to kill a subpopulation of thymocytes —see description of assay hereinafter) predicts the severity of the arthritic condition (Figure 1). When EAE was induced in rats, the pre-existing quantity of ATAA predicted the development of disease as quantitated by the change in weight during the disease process (Figure 2). Therefore, when an individual has a high amount of antibody, he/she is more susceptible to the development of autoimmune disease induction. A reasonable assumption is that the antibody destroys a population of thymocytes which repress the development of the disease; and when a stimulus is applied, disease development runs unchecked. This is best exemplified by using another model system in which a suspension of foreign cells is injected into a rat. These cells are so constituted that they can react against the recipient, but the recipient is unable to react against the cells (graft versus host-recipient reaction). The reaction is quantitated by measuring the size of the lymph nodes draining the injection site. When cells are treated with ATAA, there is a greater than expected reaction (Figure 3) which indicates ATAA destroyed a cell which is capable of suppressing the reaction, thus resulting in a greater than expected (predictable) activity. Thus, Figure 3 shows the popliteal lymph node Graft versus Host (GVH) of Lewis rat thymocytes preincubated with isologous serum (naturally occurring ATAA) . The same volumes of cells at varying concentrations were injected in the sub-plantar location of F.Hybrids (L/BN) . Thymocytes were preincubated with either complement sufficient isologous serum (120 CH₅₀ units) or the same serum heat inactivated (0 CH₅₀ units). The control experiment in which 5.0 x 10⁷ F.Hybrid cells were preincubated with complement sufficient is shown as a single point (L/BN^~*L/BN) .

The reason that other investigators have missed the prognostic significance of ATAA and its relationship to disease is because they measure the levels of antibody while the disease is present . However, it is revealed through my studies, that after the induction of an autoimmune disease in rats (adjuvant arthritis), the susceptible cells as well as the amount of ATAA decrease markedly. Thus, any measurement of these parameters while (an active) disease exists would preclude any conclusion concerning etiological significance. This observation further helps to explain the presence of ATAA, in circulation, when the target cell is within the same body. Since there has been reported to exist a functional barrier between cells within thymus and antibodies in circulation, it is reasonable that ATAA exists in circulation coincident with target cells with the thymus. When, however, cells are released (due to induction of disease or due to genetic programming) the reaction between ATAA and target cells results in a permanent decrease in numbers of susceptible cells and a temporary decrease in ATAA. Once the susceptible cells are eliminated, the amount of antibody increases, since it is still being produced. Now, if cells were slowly released, they would be destroyed; but, ATAA would remain high, as we have previously shown that ATAA exists in excess in circulation.

Although I inculcate a theoretical role for the regulation of the immune response by ATAA, there is also the empirical datum that the production and presence of ATAA is controlled or linked to the same immunoregulatory gene (Ir) as that which causes the disease itself; i.e., one rat gets disease whereas another does not because of its genetic makeup and the expression of this gene is accompanied by the expression of the gene which results in ATAA production. In either case, however, I have discovered that the measurement of ATAA production and/or the target cell antigen(s) of ATAA has diagnostic, prognostic and/or therapeutic value.

I apply the same type of assay system as has been used in rats to a human situation. In this case, I use various human sera and fetal human thymocytes as target cells. Using this assay, I measured, in several human samples, the presence of ATAA which reacts with (kills) a small percentage of human fetal thymocytes. There was variability in the number of target cells killed between various human samples, including one in which an aberrant immune responsiveness existed historically. As mentioned earlier in the SUMMARY, the quantitation of ATAA in circulation while using the appropriate target cell, predicts susceptibility to the development of allergic hypersensitivity reactions and autoimmune disease (high level) or resistance to tumor development (low level). By either raising or lowering the level of ATAA or the susceptible cell, modification of a course of either tumor development or autoimmune disease (including allergic hypersensitivity diseases) is attained. There are other uses for an assay using ATAA and relevant target cell or target antigen. This is detailed in the following table:

Table A UTILITY OF ASSAY (Regulatory Relationship of Antibody to Target Cell Antigen) The assay permits:

1. The prediction of vulnerability for development of autoimmune diseases so that patients can be tested at regular intervals for the appearance of autoantibodies of various types. Treatment may be started early. These data may be used for genetic counseling.

2. The prediction for drug idiosyncratic reactions since many are autoimmune in origin.

3. The prediction for susceptibility to reactions to immunizations or any other allergic hyper¬ sensitivity reaction,

4. Use as a marker for following the efficacy of various treatments of an autoimmune disease.

5. A reference as necessary to monitor efforts to alter levels of ATAA and susceptible cells as outlined further in Table B.

6. The prediction vulnerability for development of tumors so that patients receive enhanced medical surveillance for early tumor detection, and thus, alerts a patient to avoid high risk activities. It is also important for genetic counseling concerning tumor development.

7. The monitoring of course and treatment of patients with tumors.

8. The use for development and assay of new therapeutic agents for treatment of tumor and autoimmune diseases. (drug screening tool).

9. The use to design new therapeutic agents which can modify ATAA or its susceptible cell.

10. Deletion of self reactive cells and/or suppressor cells.

11. Retardation of transplantation rejection.

12. The enrichment of suppressor cell population in bone marrow graphs in order to prevent graph versus host reactions.

Table B outlines methods for altering the in vivo levels of ATAA or the susceptible cell. This is simply an outline without excluding other possibilities currently known or which may become available, and the reader should be cautioned not to attribute arbitrary bounds to these methods.

Table B

METHODS OF ALTERING LEVELS OF ATAA AND SUSCEPTIBLE CELLS AND UTILITY FOR SAME

I • Raising Levels of ATAA

A. Passively transferring serum from donor with high levels of ATAA.

B. Production and transfer of monoclonal ATAA. C. Use of immuno stimulants. Utility

1. Enhance tumor rejection.

2. Prevention of tumor development.

II. Lowering Levels of ATAA

A. Passing patient's serum (extra-corporeal) over antigen containing immuno-absorbents .

B. In vi vo administration of purified target cell antigen.

C. Administration of pharmacological agents which are capable of lowering existing antibody levels (as yet to be described) .

Utility

1. Prevention and/or therapy of autoimmune disease.

2. Prevention of hypersensitivity reactions.

III . Increasing Number of Susceptible Cells

A. Lower levels of ATAA (see above) .

B. Adoptive transfer from fetal or adult source.

C. In vi tro: delete other ly phoid cell types resulting in functional increase in susceptible cell.

Utility

1. Same as lowering ATAA — in vi vo .

2 . In vi tro: allow for adoptive transfer of enriched cell population, especially in regard to G.V.H. reaction. IV. Decreasing Number of Susceptible Cells

A. Increased levels of ATAA (see above).

B. Extra-corporeal passage of patient's leukocytes over fixed monoclonal ATAA.

C. Use of heterologous antibody directed at cellular antigens.

D. Use of pharmacological or biological entities directed at biochemical or antigenic constituents of susceptible cell.

E. Treat cell suspension in vi tro with ATAA to reduce the number of susceptible cells.

Utility

1. Enhance tumor rejection.

2. Prevention of tumor development.

The following is a description of the Assay which was used in all experiments detailed in this application.

DESCRIPTION OF ASSAY

Essentially, serum from an individual animal is mixed with a pool of living thymocytes from about five to about ten donors. When using inbred animals (Lewis rats), this is basically the same as using their own cells since these animals are genetically alike. When using outbred animals (Wistar or Sprague Dawley rats) the pool of thymocytes is derived from about five to about ten individual donors all presumably with different histocompatibility antigens. The latter was preferred since it mimics the human situation where the amount of anti-thymocyte antibody (ATAA) will differ in each individual and it is tested against a common thymocyte antigen present in all individuals within the species. The assay involves mixing a vital dye (trypan blue) with the live thymocytes. The live cells will exclude the dye and will remain unstained (clear) when viewed under a microscope. The dead cells will allow the passage of dye internally so that they stain and appear blue. This assay, therefore, reflects the ability of the added serum to kill the target cells. There are innumerable other methods to measure the reaction of antibody in the serum (anti-thymocyte antibody) with the relevant target cells, i.e. (1) labeling the target cells with radioactive molecules and observing the release of radioactivity as a function of cell death; (2) measuring binding of antibody with cells such as using an ELISA assay; (3) measuring binding of antibody with target cells using the consumption of secondary serum molecules (complement), and so forth. The end result is the measurement of the reaction of antibody in serum with target thymocytes.

Thymus glands were obtained from either Lewis, Wistar or Sprague Dawley rats anaesthetized with ether. The cells were teased into cold Hanks balanced salt solution, washed twice in the same solution and resuspended at concentration of 5x10° cells per ml. in RPMI 1640 media. After warming at 37°C, 100 microliters of cell suspension was placed in each well of 300 microliter plates to which was added 50 microliters of serum at a 1:3 dilution at 37°C. All samples were tested in quadruplicate. Plates were incubated in a stirred water bath at 37°C for 30 minutes. Fifty microliters of cell suspension were mixed with 50 microliters of 0.2% trypan blue and the number of dead and live cells were counted in a hemocytometer plate. One hundred cells from each sample were separately counted.

EAE was induced by described methods [J. Neurosci. Res. 24 -. 222-230 (1989)] and the end point of disease development as a function of weight gain is described in detail elsewhere [J. Pharmacal. Exp. Therap., 242: 614-620 (1987)]. Adjuvant arthritis was induced as described elsewhere in the art [Proc. Soc. Exp. Biol., 737: 506 (1971)]. The aforementioned methods only are herein incorporated by these references.

It should be noted that the assay system used, although accurate, is simplistic and economically feasible. Increased sensitivity is also achieved by employing radioactive labeled cells or various types of binding measurement techniques. The most important assay development technique is to identify the target cell type, and then purify the relevant antigenic determinant. Once this is accomplished, the target cell (or antigen) suspension will be so enriched that, instead of identifying a small number of relevant cells, most or all will be reactive with the ATAA-containing serum. Thus, a more accurate quantitation of the amount of ATAA is made. In addition, when the relevant antigen is purified, the probability is higher that it will be found in more easily accessible cells or tissues, rather than having to use thymus cells. It is possible to quantitate the number of susceptible cells with which a person is constituted; in fact, once the antigen is properly identified it is possible to synthesize it. Thus, pure ATAA as a monoclonal antibody, and antigen, as a defined entity, determine the classification of individuals as to their immunological control mechanisms and therapy will involve the alteration of ATAA and other methods as outlined in Tables A and B.

Although much remains that can be done, it is felt that this disclosure shall serve as a benchmark for future efforts which employ its concepts and such is readily commended to the field consistent with the hereinafter appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for evaluating the functional viability of an individual's immune system comprising: determining an amount of an autoantibody and its regulatory relationship to an amount of its target lymphocyte cell subpopulation; and comparing said amounts to a standard wherein said comparing correlates a high autoantibody amount with a high risk factor for autoimmune disease, vaccine reactions or hypersensitivity reactions and a low risk factor or vulnerability to malignancies, while a low autoantibody amount indicates the probability of high target cell subpopulation, high vulnerability to malignancy and a low cellular immunity.

2. A method for preventing tumor development and/or enhancing tumor rejection by a body comprising increasing autoantibody amount relative to a target cell subpopulation, the target cell being that associated with progression of said development and/or rejection, the increasing accomplished by any of the following:

(a) passively transferring serum from a donor with high levels of said autoantibody to a patient donee;

(b) producing and transferring to a donee monoclonal autoantibody; and

(c) using in the donee immuno stimulants.

3. A method for preventing and/or treating autoimmune disease, vaccine reaction and hypersensitivity reaction comprising decreasing autoantibody amount relative to a target cell subpopulation in a patient/donee, said subpopulation being that of a cell causally related to said disease or said reactions, said autoantibody decreasing effected by one or more of the following:

(a) passing a patient's serum, i.e., extra- corporeal, over antigen containing immuno-absorbents;

(b) administering in vi vo a purified target cell antigen; and

(c) administering to said patient pharmacological agents which have a function of lowering existing antibody levels,

4. The method of Claim 2 wherein said autoantibody is an ATAA.

5. The method of Claim 3 wherein said autoantibody is an ATAA.

6. The method of Claim 2 wherein said increasing is accomplished by decreasing subpopulation of cells which are ATAA susceptible cells and said decreasing is accomplished by one or more of the following:

(a) increasing levels of ATAA;

(b) extra-corporeal passing of a patient's leukocytes over fixed monoclonal ATAA; (c) using heterologous antibody by directing same at cellular antigens;

(d) using pharmacological or biological entities which are directed at biochemical or antigenic constituents of said susceptible cell; and

(e) treating a cell suspension in vi tro with ATAA thereby reducing the number of susceptible cells.

7. The method of Claim 3 wherein said decreasing is accomplished by increasing a subpopulation of cells which are ATAA susceptible cells and wherein said increasing of the susceptible cells is accomplished by one or more of the following:

(a) lowering levels of ATAA;

(b) adoptively transferring said susceptible cells from a fetal or adult source; and

(c) deleting, in vi tro, other lymphoid cell types thereby causing a functional increase in said susceptible cell subpopulation.

8. A quantitation method for determining the amount of anti T-cell autoantibody, ATAA, relative to a subpopulation of target T-cells in an individual's serum comprising:

(a) obtaining a quantum of target T-cell antigen from an ATAA-recognized antigen pool;

(b) mixing with said quantum of target T-cell antigen a predetermined quantity of the individual's serum; and (c) determining by conventional quantification methods said amount of anti T-cell autoantibody and the number of target T-cells.

9. The method of Claim 8 further comprising predicting said individual's susceptibility/resistivity to disease or malignancy as such are related to amount of the target T- cells which may be helper, killer, suppressor or other cells involved with immunity in the individual.