WO2001002013A1 - Predicting immunotherapy effectiveness through delayed type hypersensitivity - Google Patents

Abstract

The present invention in the field of immunotherapy, provides a method for predicting the likelihood of tumor shrinkage in response to administration of immunotherapeutic agents to patients having tumors or cancer, especially lymphoma or myeloma. The present invention further provides methods for optimizing a beneficial outcome of immunotherapy for treating cancer, immune system diseases and infections diseases in an anergic patient by boosting the immune responsiveness of the patient before beginning the immunotherapy, and methods for determining the optimum amount and schedule of an immunotherapeutic agent to administer to treat cancer and immune diseases.

Description

PREDICTING IMMUNOTHERAPY EFFECTIVENESS THROUGH DELAYED

TYPE HYPERSENSITIVITY

This application claims priority to US Provisional 60/142,058 which is ₅ incorporated herein in its entirety.

TECHNICAL FIELD

The present invention is in the field of immunotherapy, specifically, in the area of predicting the likelihood of tumor

10 shrinkage in response to administration of immunotherapeutic agents to patients having tumors or cancer. The present invention further provides methods for optimizing a beneficial outcome of immunotherapy for treating cancer, immune system diseases and infectious diseases in an anergic patient by

15 boosting the immune responsiveness of the patient before beginning the immunotherapy, and methods for determining the optimum amount and schedule of an immunotherapeutic agent to administer to treat cancer and immune diseases.

20 BACKGROUND

The first attempts to treat cancer with immunotherapy were made approximately 100 years ago on the assumption that tumor cells are recognized as 'foreign' by the immune system. Later on, a whole series of experimental animal tumor models were developed.

25 They included the use of syngeneic tumors, spontaneously arising tumors and human tumor xenografts in immunodeficient mice. The experimental data contributed to our understanding of the interaction between immunocompetent cells and their products on the one hand and tumor cells on the other. On this basis,

30 various immunotherapeutic protocols have been devised which included the use of 'nonspecific' components such as bacterial adjuvants, cytokines, NK cells and macrophages and attempts were made to raise specific T and B cell responses against tumor cells. Many human tumor-associated antigens have been

35 characterized, and various ways of increasing the immunogenicity of human tumor cells have been described. Moreover, more insight has been achieved in defining 'high risk' populations on the basis of genetic background, the role of environmental factors and the characterization of 'precancerous ' cells. Some cancer vaccines have been used in clinical trials, which have resulted in partially beneficial therapeutic effects but have not provided a full solution for a rational use of immunotherapy against human neoplasia.

Immune-based therapies have also been used to treat immune diseases, including autoimmune diseases and infectious diseases, especially hepatitis B and C.

Numerous investigators have shown a positive correlation between defective cell-mediated immunity, as indicated by anergy to multiple skin test antigens, and disseminated cancer. Johnson , W. C. et al . Role of delayed hypersensi tivi ty m predicting postoperative morbidi ty and mortali ty. Amer. J. Surgeryl 37 : 536- 541 (1979) ; Lamb, D. , Pilney, F. Kelly, W. D. and Good, R . A. A compara tive study of the incidence of anergy m pa tien ts wi th carcinoma , leukemia , Hodgkm ' s disease and other Iymphomas . J. Immunology 89 : 555-558 (1962) ; Eilber, F. R . and Morton , D. L . Impaired immunologic reactivi ty and recurrence following cancer surgery. Cancer25 : 362- 367 (1970) ; Hersh , E. M. et al . Chemotherapy , immunocompetence , immunosuppression and prognosis m acute leukemia . N. Engl . J.Med. 285 : 1211 -1216 (1971 ) . However this old body of knowledge has not resulted in a test that can be used in an individual patient to define appropriate therapy. This invention includes use of the method to both optimize and mdivisualize therapy.

In the above studies the occurrence of anergy often correlated with advanced stage disease because as patients become more ravaged by the disease, their immune systems begin to fail. In these studies, anergy was general. The demonstration of anergy can be a negative prognostic factor in certain malignant diseases since diminished cutaneous reactivity has been associated with poor prognosis. From the old literature it is known that, in general, patients capable of displaying normal delayed hypersensitivity skin test reactions to standard skin test antigens may have a slower progression of disease, whereas those who are anergic or who exhibit significantly impaired reactivity, have an increased rate of progression of disease. We have discovered that in cases a positive response to DTH testing (including those responses previously thought to represent "anergy") can, when combined with the appropriate therapy herein described, lead to a regression (as opposed to simply a slower progression of the disease. We have discovered that this relationship is so close that in individual patients, including those in good clinical condition, DTH responsiveness to can be used to determine appropriate therapy for an individual m order to have the best chance of actual amelioration of the disease.

There is a great need for methods to predict the success of therapy for any given disease, and to determine the optimum amount and administration schedule of immunotherapeutic agents to achieve a positive outcome (as opposed to a merely less negative outcome) and disease response. Despite peripherally relevant literature going back twenty years, as described above, a method robust enough to guide therapy in any population of patients has not been found. Further, methods are needed to effectively treat the anergic patient. The present invention is directed to providing such methods, as well as others.

OBJECTS AND SUMMARY OF THE INVENTION

It is an object of the invention to provide a method for predicting effectiveness of treating an immune disease or disorder or cancer, or treatment of tumors in an animal with an immunotherapeutic agent. This is done by: administering a multiantigen test for delayed type hypersensitivity (DTH) prior to treatment with the immunotherapeutic agent and then scoring DTH responsiveness as a function of at least one of induration and redness of an area surrounding a site of antigen exposure. A patient is classified as anergic if the animal displays no DTH response to any antigen, and as responsive if the animal displays a DTH positive response to any antigen in the DTH test.

DTH responsive patients are predicted to have a good response to immune therapy. For cancer this response is specifically tumor shrinkage. Anergic patients will likely not have tumor shrinkage or a positive response to immunotherapy.

It is a further object to provide a method for optimizing a beneficial outcome of immunotherapy for treating a disease in an anergic animal. To accomplish this, the animal is given a DTH test and responsiveness is measured for each antigen. If the patient is anergic, he is treated with a particular amount of a particular combination of one or more known immunostimulatory agents for a particular duration of time, and DTH testing is repeated one or more times to determine whether the patient has developed DTH responsiveness during the course of treatment. If the animal remains anergic after the initial treatment, the drug is administered at a different concentration for a duration of time. DTH testing is repeated. If the patient has developed a DTH response to at least one antigen, immunotherapy is begun. If he or she remains anergic, then different amounts of the immunostimulants are administered yet again, or a different combination of immunostimulants is administered and DTH testing is repeated until a positive DTH response is obtained, at which point the patient is ready for immunotherapy of his or her disease.

It is a further object to provide a method for determining the optimum amount of an immunotherapeutic agent to administer to an animal to treat a disease by screening patients having the disease for DTH activity and forming from among those anergic patients, two or more groups of initially anergic animals, each group having two or more members. To each respective group s administered respective different amounts of immunotherapeutic agent for a duration of time. DTH testing is repeated one or more times during the course of drug administration and the results are used to determine the number of anergic and DTH responsive animals in each group. The optimum amount of immunotherapeutic agent to administer to treat the disease in a general population of animals, is the amount that generated the strongest DTH response in one or more of the groups.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

An immunostimulant as defined herein is any compound or combination that acts to stimulate the immune system including chemicals, proteins, hormones, cytokines, chemokmes, vitamins, nutraceuticals, small thiol containing molecules, disulfide containing molecules, carboxy amide derivatives, antigens, adjuvants, minerals especially Zn, Cr and Se, interleukins, interferons including interferons alpha, beta, gamma and tau and mutants thereof, and growth factors.

Immune therapy as defined herein is synonymous with immunotherapy as used herein, and means any therapeutic regimen for treating a disease, that acts at least in part through manipulating the immune system of the diseased person. Such manipulation can include stimulating various immune system parameters and inhibiting other parameters that may be undesirably overactive.

Delayed type hypersensitivity (DTH) as used herein means a reaction exemplified by the tuberculin reaction which (as opposed to immediate hypersensitivity) takes 12 to 48 hours to develop. DTH is triggered by intradermal injection of antigen (skin test) in a sensitized individual and results in the formation of erythema and induration at the injection site characterized histologically by perivasular infiltration of monoccytes and lymphocytes. The term includes all aspects of cell-mediated immunity including contact dermatitis, granulomatosus reactions and allograft reactions.

Induration as used herein means swelling and/or increased firmness perceptible to the eye or finger.

The results of delayed type hypersensitivity (DTH) are used clinically to determine if a person has mounted an immune response to many antigens. The most frequent application is measurement of DTH m response to tuberculosis called a PPD or tine test. It is also used to determine if a patient, such as a cancer patient, has had a failure of the immune system and has become anergic. It was discovered that the initial level of DTH responsiveness in a cancer patient after a number of previous therapies, but before treatment with an immunotherapeutic, is useful for predicting a tumor shrinkage response upon treatment with an immunotherapeutic agent, as opposed to standard cytotoxic chemotherapy in general. It was found that anergic patients do not respond to immunotherapy with tumor shrinkage, while patients having measurable DTH activity to at least one common recall antigen before therapy, respond to immunotherapy with tumor shrinkage. According to the present invention, a patient is tested for delayed type hypersensitivity (DTH) for one or more ubiquitous antigens prior to treatment with the immunotherapeutic agent by measuring the DTH response to each antigen. Patients are classified as anergic if the patient displays no DTH response to any antigen in the DTH test, or as responsive if the patient has a DTH positive responsive to at least one antigen tested. It has been discovered that a poor outcome for immunotherapeutic treatment of the disease is predicted if the patient is anergic in prestudy DTH testing. By contrast, a good outcome such as the shrinkage of the benign tumor in a precancerous patient or a malignant tumor in a cancer patient is predicted if the patient is DTH positive, .e., DTH responsive. DTH responsiveness, as shown by redness or induration is required for at least one antigen, response to additional antigens and larger responses may be further predictive of ability to respond to the immunotherapeutic.

It was further discovered that initial DTH responsiveness in a patient can be used to identify patients who will respond to immunotherapy (these patients are DTH responsive in pretreatment testing) , as opposed to other types of drug therapy that do not work specifically through the immune system, and separate them from patients who are less likely to respond to immunotherapy (these patients are anergic) . Thus, the present invention is not limited to identifying only cancer patients who will respond favorably to immunotherapy, but to identifying patients having a disease for which effective immunotherapeutic agents are known, especially immune system diseases and disorders, and infectious diseases as enumerated herein.

The most common means of determining whether a patient has a DTH response to an antigen is measuring induration of at least 2 mm at the site of antigen injection under the skin. However, according to the present invention, a person of skill in the art can set his or her criteria own for DTH responsiveness based on observation of the patients having the same disease, or other criteria that are known or that may in the future be established by persons of skill in the art. A further aspect of this invention is that simple redness n response to an antigen skin test, which is not considered positive DTH by the standard definition, is sufficient to identify DTH responsive patients who will respond to certain immunostimulatory drugs with disease shrinkage. Thus another measurement of DTH responsiveness in this invention may include redness of the area surrounding the site of antigen exposure. In a preferred embodiment, a person is anergic if no response to any antigen tested has either induration or redness greater than or equal to 2 mm in diameter. In another preferred embodiment, a person is anergic if no response to any antigen tested has either an induration or redness greater than or equal to 5 mm in diameter. In yet another embodiment, a person is anergic if no response to any antigen tested has either an induration or redness greater than or equal to 1 mm in diameter

The patients being tested for immunotherapy are preferably cancer patients, precancer patients with non-malignant tumors or cells, or patients having an immune system disease, including infectious diseases. In an especially preferred embodiment, the patients have either B cell lymphoma, multiple myeloma, leukemia, breast, prostate, or urogenital cancer. However, the invention is not limited to treatment of these diseases, but can be used to predict the outcome of immunotherapy for treating any disease that responds favorably to immunotherapy. The present invention is further directed to a method for optimizing a beneficial outcome of immunotherapy for treating a disease in an anergic patient with an immunotherapeutic agent, by boosting the immune responsiveness of the patient before beginning the immunotherapy. To accomplish this, a patient demonstrated by DTH testing to be anergic, is given a particular amount of a particular combination of one or more drugs known to stimulate the immune system (immunostimulatory drugs) for a particular duration of time. At various intervals while receiving drug, the patient is tested for DTH responsiveness. If the initially anergic patient develops DTH responsiveness to one or more of the antigens tested, then the patient shows an increase in immune responsiveness that is sufficient to predict a good outcome of immunotherapeutic treatment for his or her disease. If the patient remains anergic after receiving the particular amount of immunostimulatory drug, then the amount is changed, usually increased, to a new amount, or the one or more immunostimulants are changed. The new amount of drug, or the new immunostimulant (s) combination as the case may be, is then administered for a duration of time, and the patient is retested for DTH responsiveness at various intervals. The amount of immunostimulant and the type of immunostimulants are varied until the patient develops a positive DTH response to one or more antigens. Once the patient exhibits a positive DTH response, he or she goes from being a poor candidate for immunotherapy of his or her disease to being a good candidate with a favorable prognosis.

A patient's response to immunostimulants is usually very individual, therefore, a compound that stimulates the immune system of patient A may not elicit a response in patient B. Also, a compound that elicits a DTH response in patient A reflecting a boost of cellular immunity or T cell activity, may elicit an increase in antibodies in patient B and have no effect on DTH activity. Therefore, various immunostimulants and combinations thereof may have to be administered sequentially to a patient to elicit the desired change from an anergic state to a DTH responsive state. Patients having the same disease or similar diseases may respond similarly to a given immunostimulant or drug combination. A pattern may emerge among patients having a given set of related diseases, for converting anergic patients to DTH responsive patients by administering immunostimulants . The present invention is also directed to a method for determining the optimum amount of an immunotherapeutic agent for treating a disease in a general population patients in need of such treatment. This is accomplished by forming two or more groups wherein all the members are anergic, then administering to respective groups of initially anergic patients, respective different amounts of immunotherapeutic agent for a duration of time. Each patient is periodically tested for DTH responsiveness one or more times during the period of receiving drug. In a preferred embodiment, a DTH test is also administered at or near the end of the period of administering drug. The results of each DTH test for each patient in each group are analyzed to determine the percentage of initially anergic patients in each group who become DTH responsive. The optimum amount of immunotherapeutic agent to administer to treat the disease in a general population of patients in need of such treatment is the amount that generates the strongest DTH response in one or more of the groups.

There are various ways to determine the group having the strongest DTH response. A person of skill in the art using the present methods can define "the strongest DTH response" in various ways. In a preferred embodiment the strongest DTH response is defined as the response that occurs in the group containing the highest percentage of initially anergic patients that develop a positive DTH response after treatment. In another preferred embodiment it is defined as the response that occurs in the group, the members of which collectively have the highest number of positive DTH responses to individual antigens, defined as the sum of the total number of DTH positive responses to individual antigens for each patient in the group, for all the DTH tests conducted; or a group, the members of which collectively have the highest sum of (1) the number of DTH responsive patients, and (2) the number of positive DTH responses to individual antigens which is defined as the sum of the total number of DTH positive responses to individual antigens for each patient in the group, for all the DTH tests conducted. However, the invention is not limited to these enumerated ways to determine the strongest DTH response. A person of skill in the art can define anergic and a positive DTH responsive based on various criteria and using various thresholds for responsiveness.

In the specific examples set forth below, patients having B cell lymphoma or multiple myeloma were treated with the anticancer drug beta-alethine (BETATHINE™, Beta LT™) known to affect the immune system. In addition to being an immunotherapeutic agent for cancer, beta-alethine is also a general immune stimulant. Predrug DTH tests administered to all patients revealed that many of the patients were anergic, i.e., showed no DTH response to any antigen, prior to therapy. A positive DTH response was defined as either induration or redness at the site of antigen injection that was greater than or equal to 2 mm in diameter.

An anergic patient exhibits no induration or redness at the site of antigen injection exceeding the selected threshold of 2 mm in diameter. For certain purposes, a positive DTH response can be defined as either induration or redness at the site of antigen injection that was greater than or equal to 5 mm in diameter.

The DTH test given to each patient was a multiantigen test for seven (7) common recall antigens: to seven common recall antigens: Tetanus Toxin, Diptheria toxoid, Streptococcus, Tuberculin, Glycerin Control, Candida, Trichophyton and Proteus. However, the invention is not limited to these antigens or to any particular antigens; nor is it limited to any particular number of antigens. Any antigen can be used. In a preferred embodiment the antigens are chosen from common environmental recall antigens like the seven listed above plus mumps antigen and flu peptides . In another preferred embodiment tumor antigens may be used, including antigens obtained from the patient's own tumor. In another preferred embodiment between one and ten antigens are tested; in a more preferred embodiment five antigens are tested; m the most preferred embodiment seven antigens are tested. DTH testing may be accomplished by injecting the one or more antigens manually, or by using known multiantigen test kits for cellular hypersensitivity such as MULTITEST CMI® made by Connaught, a Pasteur Merieux Company. _Other DTH tests known in the art may also be used, such as in_dividually prepared antigens that may be injected mtradermally. Other methods may be used to determine immune responsiveness of a patient in need of immunotherapy for treating a disease

DTH tests were readministered after the onset of beta-alethine therapy on days 31 and 73 of the 85 day study. It was discovered that patients testing DTH positive in prestudy DTH skin testing had a positive response to immunotherapy with beta- alethine, while anergic patients had a poor outcome. This positive response included actual tumor shrinkage. The power of this invention is that it documents the ability to improve the patients condition not jus the ability to predict how rapidly a patient will deteriorate.

Eight (8) lymphoma patients were studied, of which 4 were initially anergic patients and 4 initially DTH responsive patients. Of the four anergic patients, three developed increases in tumor size, and one remained stable. By contrast, none of the four responsive lymphoma patients had more than a 10% increase in tumor size, and three of the 4 responsive lymphoma patients displayed a decrease in tumor size. TABLE 1

Mean tumor size of the anergic group (sum of bi-dimensional products) was 48 ± 45 (SD) . It was unexpected that anergic patients did not have larger tumors than the responsive patients in prestudy testing. the responsive group, ,mean tumor size

(sum of bi-dimensional products) was 78 ± 25 (SD) in this group.

Thus, by comparing pre-study DTH testing and pre-study tumor size with DTH tests repeated during immunotherapy and end of study tumor size, it was discovered that patients who are initially anergic prior to immunotherapy do not show tumor shrinkage, even though they may respond with stable disease. Patients who are initially DTH responsive before immunotherapy are predicted to respond to immunotherapy with tumor shrinkage of at least 10%. In the case of lymphoma this tumor shrinkage significant and reproducible.

The effect of beta-alethine, an immunostimulatory compound, on six myeloma patients was also examined. Four of the six myeloma patients were initially anergic . Of these four, three developed a DTH positive immune response to one or more antigens during the trial on day 29, or near the end of the trial on day 73. Little change was seen in the DTH response of the initially responsive patients. TABLE 2

DTH Response of Initially Anergic Patients Only

These results show that some anergic cancer patients are capable of developing DTH positive responses to one or more antigens if treated with immunostimulants.

As mentioned earlier, the present invention applies to patients having cancer, and immune system diseases or disorders, including infectious diseases by bacteria, viruses, parasites and fungi. Further, the present invention is not limited to any particular immunostimulants for increasing immune responsiveness as measured by DTH activity prior to immunotherapy, nor is it limited to any particular immunotherapy or immunotherapeutic agent. The present invention includes any immunostimulants and any immunotherapeutic agents. The choice of immunostimulant and immunotherapeutic agent will vary depending on the patient's individual response and the disease being treated. In a preferred embodiment compounds of Formula I, which include beta- alethine, carbobenzoxy beta-alanyl taurine and beta-alanyl taurine, are preferred immunostimulants and immunotherapeutic agents. Compounds of Formula I have been shown to be effective as immunostimulants (US Patent No. ) and as immunotherapeutic agents for treating cancer, immune diseases, and infectious diseases including viral, bacterial, parasitic, and fungal infections. Importantly, compounds of Formula I, specifically beta-alethine and carbobenzoxy beta-alanyl taurine have been sown to stimulate delayed type hypersensitivity (DTH) in vivo. Compounds of the formula (I) have the following structure:

R¹ I N-L^x-CO-NH-L²-A (I) I

R²

wherein:

A is a group of the formula -P0₃H, -S0₃H, -OPO-(OH)₂, - 0SO₂OH, or -SH, or pharmaceutically acceptable salt thereof or physiologically hydrolyzable derivative thereof, or disulfide thereof when A is -SH. Suitable salts include sodium, potassium, calcium and zinc. Suitable hydrolyzable derivatizing groups include esters, such as substituted or unsubstituted lower alkyl (e.g. Ci to C₄) or arylalkyl (e.g. benzyl) esters; Ri is H, a linear or branched lower alkyl, for example, a C_! to C₆ alkyl, arylalkyl, for example, wherein the alkyl moiety is Ci to C alkyl and the aryl moiety is a substituted (e.g. lower alkyl or halogen) or unsubstituted phenyl group, or alkenyl (for example, C₂-C₆ alkenyl);

R₂ is H, a linear or branched lower alkyl, for example, a Ci to C₆ alkyl, an alkenyl, for example, a C₂-C₆ alkenyl, an arylalkyl, for example, wherein the alkyl moiety is a Ci to C₄ alkyl and the aryl moiety is a substituted (e.g. lower alkyl or halogen) or unsubstituted phenyl group; or an acyl, for example, acetyl, benzoyl, arylsulfonyl (for example, when the aryl moiety is phenyl); a carbonate ester such as alkoxycarbonyl (e.g., C₁-C7 alkoxy carbonyl) (for example, -OCOC (CH₃) ₃) ; allyloxy carbonyl (e.g. -OCOCH₂CH=CH₂ ) ; cycloalkoxycarbonyl (e.g. when the ring is C₃-C₈ (C₅-C₆ being preferred) and when the alkoxy moiety is Cι-C_B) (for example -OCOCH₂C₅H₉) ; or an unsubstituted arylalkoxycarbonyl (for example -OCOCH₂C₆H₅) or a substituted arylalkoxycarbonyl wherein the substituent is, for example, a halogen, a nitro group, an amino group or a methoxyl group; alternatively, R¹ and R² taken together form, with the nitrogen to which they are attached, a 5 to 7 membered ring (for example, R¹ and R² taken together can be -(CH₂)₄-, -(CH₂)₅ or - (CH₂)₆-); and

L¹ and L² are hydrocarbon linking groups, for example, a linear or branched chain alkyl of the formula -(C_nH_2n)- wherein n is, for example, 1 to 8 in the case of L¹ and 2 to 8 in the case of L² except when A is -P0₃H or -S0₃H in which case n can be 1-8; a cycloalkyl of 3 to 8 carbon atoms, preferably 5 or 6 carbon atoms; or an interphenylene (e. g. or ) .

Advantageously, L¹ and L² are -(C_nH_2n)- wherein n is 1 to 8 in the case of L¹ or 2 to 8 in the case of L² except when A is -P0₃H or -S0₃H in which case n can be 1-8 (examples of branched chain alkyls include -CH₂CHR-, -CH₂CHRCH₂-, -CHRCH₂CH₂-, and -CH₂CH₂CHR- wherein R is an alkyl group and wherein the total number of carbon atoms, including R, does not exceed 8) .

A particular group of compounds of the invention is of the formula (I) wherein A, R¹, R², L^x and L² are as defined above in said first embodiment with the proviso that when A is -S0₃H or pharmaceutically acceptable salt thereof or physiologically hydrolyzable derivative thereof, one of R¹ and R² is H, and L¹ and L² are (CH₂)₂, then the other of R¹ and R² is not H.

Another particular group of compounds of the invention is of the formula (I) wherein A, R¹, R², L¹ and L², are as defined above in the first embodiment with the proviso that when A is -S0₃H or pharmaceutically acceptable salt thereof or physiologically hydrolyzable derivative thereof, one of R¹ and R² is H, and L¹ and L² are (CH₂)₂, then the other of R¹ and R² is not C₆H₅CH₂OCO- . A further particular group of compounds of the invention is of the formula (I) wherein A is a group of the formula -P0₃H or -OPO(OH)₂, more particularly -P0₃H, or a pharmaceutically acceptable salt thereof or a physiologically hydrolyzable derivative thereof, and wherein R¹, R², L¹, and L² are as defined above in the first embodiment.

Another particular group of compounds of the invention is of the formula (I) wherein A is a group of the formula -S0₃H or -OS0₂OH, more particularly -OS0₂OH, or pharmaceutically acceptable salt thereof, or physiologically hydrolyzable derivative thereof, and wherein R¹, R², L¹ and L² are as defined above in the first embodiment. The provisos above can apply to this group of compounds as well.

A further particular group of compounds of the invention is of the formula (I) wherein at least one of R¹ and R² is an alkyl, advantageously a lower alkyl (e.g. Ci to C₆) , and wherein A, L', L² and the other of R¹ and R² are as defined above in the first embodimen .

Another particular group of compounds of the invention s of the formula (I) wherein R¹ is an alkyl and R² is acyl and wherein A, L¹ and L² are as defined above in the first embodiment.

A further particular group of compounds of the invention is of the formula (I) wherein L¹ is -(CH₂)- and wherein A, R¹, R', and L⁷ are as defined above n the first embodiment.

Yet another particular group of compounds of the invention is of the formula (I) wherein R¹ and R² are taken together and form, with the nitrogen to which they are attached, a 5 to 7 membered ring, and wherein A, L¹ and L² are as defined above in the first embodiment. The compounds of Formula I can be prepared using the methods provided in USP 6,007,819, USP 4,102,948 and USP 4,218,404, as appropriate. These patents are incorporated herein by reference in their entirety.

UTILITY The present invention relates to methods of using the results of DTH tests to one or more antigens administered before, during, and or at the end of drug administration, to predict outcome of immunotherapy for a disease, or to optimize a beneficial outcome of immunotherapy for treating a disease in an anergic patient with an immunotherapeutic agent, by boosting the immune responsiveness of the patient before beginning the immunotherapy. The invention also permits one to determine the optimum amount of an immunotherapeutic agent to administer for treating a disease in a patient based on the results of DTH testing. The present methods is not limited to use in humans, but are useful in any mammal.

The present methods is not limited to the immunotherapeutic treatment of any certain disease or group of diseases, but is generally useful for immunotherapy of any diseases. The invention covers situations where the immunotherapeutic treatment includes administration of the immunotherapeutic agent alone or as a composition that includes more than one active agent such as cancer chemotherapeutic agents, hormones, vitamins, cytokines, enzyme regulators, regulatory macromolecules, regulatory thiols or other small molecules.

The present methods are especially useful where the disease being treated with immunotherapy is cancer, including preneoplastic and neoplastic conditions, including both soft (e.g. hematolymphoid) and solid tumors (e.g. carcinomas and sarcomas) . More specifically, breast cancer, prostate cancer, glioblastomas, melanomas, myelomas, lymphomas, leukemias, lung cancer or tumors, skin cancer or tumors, bladder cancer or tumors, kidney cancer or tumors, brain cancer or tumors, ovarian cancer or tumors, head and neck cancer or tumors, pancreatic cancer or tumors, uterine cancer or tumors, bone cancer or tumors, colorectal cancer or tumors, cervical cancer and neuroectodermal cancer or tumors, and premalignant conditions, including, without limitation, monoclonal gammapothies, dysplasia, including, without limitation, cervical and oral dysplasia . The present methods are especially useful where the disease being treated with immunotherapy is an autoimmune diseases which may be loosely grouped into those primarily restricted to specific organs or tissues and those that affect the entire body. Examples of organ-specific disorders (with the organ affected) include multiple sclerosis (myelin coating on nerve processes), type I diabetes mellitus (pancreas), Hashimotos thyroiditis (thyroid gland) , pernicious anemia (stomach) , Addison's disease (adrenal glands), myasthenia gravis (acetylcholme receptors at neuromuscular junction) , rheumatoid arthritis (joint lining) , uveitis (eye) , psoriasis (skin) , Guillain-Barre Syndrome (nerve cells) and Grave's disease (thyroid) . Systemic autoimmune diseases include systemic lupus erythematosus, dermatomyositis, and psoriasis.

Other examples of hypersensitivity disorders treated with immunotherapy include asthma, eczema, atopical dermatitis, contact dermatitis, other eczematous dermatitides, seborrheic dermatitis, rhinitis, Lichen planus, Pemplugus, bullous Pemphigoid, Epidermolysis bullosa, uritcaπs, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Alopecia areata, atherosclerosis, primary biliary cirrhosis and nephrotic syndrome. Related diseases include intestinal inflammations, such as Coeliac disease, proctitis, eosinophilia gastroenteritis, mastocytosis, inflammatory bowel disease,

Chrohn ' s disease and ulcerative colitis, as well as food-related allergies .

Other immune system diseases and disorders include inflammatory, proliferative and hyperproliferative diseases, as well as cutaneous manifestations of lmmunologically mediated diseases. In particular, conditions relating to immune system hypersensitivity. There are four types of immune system hypersensitivity . Type I, or lmmediate/anaphylactic hypersensitivity, is due to mast cell degranulation in response to an allergen (e.g., pollen), and includes asthma, allergic rhinitis (hay fever) , urticaria (hives) , anaphylactic shock, and other illnesses of an allergic nature. Type II, or autoimmune hypersensitivity, is due to antibodies that are directed against perceived "antigens" on the body's own cells. Type III hypersensitivity is due to the formation of antigen/antibody immune complexes which lodge in various tissues and activate further immune responses, and is responsible for conditions such as serum sickness, allergic alveolitis, and the large swellings that sometimes form after booster vaccinations. Type IV hypersensitivity is due to the release of lymphokmes from sensitized T-cells, which results in an inflammatory reaction. Examples include contact dermatitis, the rash of measles, and "allergic" reactions to certain drugs.

The present methods are especially useful where the disease being treated with immunotherapy is an infectious such as a viral, bacterial, parasitic or fungal infection. Examples of such infectious diseases include: hepatitis B and C, pneumonia, meningitis, Tuberculosis, HIV, Pyogenic cocci (staphylococci, pharyngitis, tonsillitis, sinusitis, streptococci, pneumococci, meningococci, gonococci) , enteric bacilli (Escherichia coll, Klebsiella, Salmonella shigella) , cholera, pseudomonas (Pseudomonas aerugmosa, Pseudomonas mallei), bacteroides, mycobacteria (tuberculosis), spirochetes (Treponema pallidum (syphilis)), clostridia, Diphtheria hemophilus and Bordetella bacilli, Granuloma inguinale, brucella, tularemia, anthrax, plague, mycoplasma, listeriosis; rickettsial disorders: typhus group, Rocky Mountain spotted fever, Lyme disease, scrub typhus, Q fever; chlamydial disorders: trachoma and inclusion conjunctivitis, lymphogranuloma venereum, and psittacosis; viral diseases: cutaneous viral infections (chickenpox, herpes zoster, measles) , respiratory viral infections, viral diseases of the central nervous system, viral diseases of the liver, viral diseases of the salivary glands, and infectious mononucleosis; fungal diseases: Candida albicans, mucor, histoplasmosis, aspergillosis, blastomycosis, coccidioidomycosis, actinomycosis and nocardiosis; and protozoal (parasitic) diseases: pneumocystosis, amebiasis, malaria, toxoplasmosis, leishmaniasis, trypanosomiasis, and giardiasis; helminths diseases (worms): trichinosis, strongyloidiasis, enterobius vermicularis, filariasis, hookworm disease, ascaπasis, flukes, cestodes, tapeworms, and tπchuriasis; and other diseases: sarcoidosis, cat-scratch disease, legionnaires' disease .

The present methods can be used to predict and optimize the effectivenss of immunotherapy of drugs that affect the immune system, and that are administered either alone or in combination with other drugs no matter how they are administered, including topically, orally, rectally, mtravaginally intravenously, intraperitoneally, subcutaneously, intramuscularly or mtranasally, as appropriate for the effect sought. The compounds can also be administered transdermally using, for example, transdermal patches or transmucosally via sprays or other application.

The methods of the present invention are suitable for monitoring immunotherapy in humans and in the treatment of any animal having an immune system including veterinary treatment of similar conditions affecting warm-blooded patients, such as dogs, cats, horses and cattle.

Various aspects of the present invention are described in greater detail in the non-limiting Examples that follow.

Certain of the synthetic procedures described below correspond to those described by Knight et al, Cancer Research 54:5623

(1994) or in USP 4,218,404, or represent modif cations thereof. In addition, the disclosures of WO 92/00955 and PCT/US91/04725 are relevant here, including the portions therein that relate to syntheses, therapeutic regimens and cell culture treatment protocols, those regimens and protocols being applicable to the compounds of the present invention.

Example I

Immunotherapy of B cell Lymphoma wi th Beta -alethine and DTH Testing to Moni tor Pa tien ts

It has been discovered that periodically administering a multiple antigen a delayed type hypersensitivity (DTH) skin test before treatment and at various times during and after immunotherapy generates information that (1) can be used to predict whether a given patient will respond to immunotherapy of a disease, especially cancer, immune system diseases and infectious diseases, and (2) permits optimizing a beneficial outcome of immunotherapy for treating a disease in an anergic patient with an immunotherapeutic agent, by boosting the immune responsiveness of the patient before beginning the immunotherapy, and (3) permits one to determine the optimum amount of an immunotherapeutic agent for treating a disease in a general population patients in need of such treatment.

Human patients having B cell lymphoma were treated with the immune stimulant Beta-alethine (BETATHINE™/ Beta LT™) (hereinafter "BT") at very low doses of 2 micrograms BETATHINE™ per person. Beta-alethine was administered by subcutaneous injection every 14 days (q2w) . Drug was administered on days 1, 15, 29, 43, 57 and 71. Phase I/II Study to Assess the Safety and Efficacy of Low Doses of BETATHINE™ in Patients with Advanced B-Cell Lymphoma, Protocol No. DTI-98-02, IND control number 059191, These protocols are incorporated herein by reference in their entirety.

Trial Designs :

DTI-98-02: This is an open label, non-randomized Phase I/II study to assess the effects of a low dose (2 ug per patient) of Beta LT™ (beta-alethine, or BETATHINE™) in patients with low grade B-cell lymphoma. Study drug is administered by subcutaneous injection of 0.1 mLs of a 20 ug/mL solution once every two weeks for a total of six doses. Objective tumor response was assessed at days 43 and 85, and compared to baseline. DTH testing for both was done pre-study and was repeated at day 31 and day 73 after the onset of beta-alethine therapy.

Delayed Type Hypersensi tivi ty

To determine whether Beta-alethine, which is both an immunostimulant and an immunotherapeutic agent, was stimulating the immune system, the patients were given a multiple-antigen skin test to determine their DTH response prior to treatment. This DTH test was administered again at about four (4) weeks (day 31) after onset of drug therapy and at about ten (10) weeks (day 73) after onset of drug therapy.

The MULTITEST CMI® kit from Pasteur Merieux Connaught Canada was purchased and used to assess DTH responses to seven common recall antigens: Tetanus Toxoid, Diptheria toxoid, Streptococcus, Tuberculin OLD, Glycerin Control, Candida, Trichophyton and Proteus. Other antigens can also be used. Other methods or kits for testing DTH responsiveness in an animal in vivo may be used. The package insert describing the MULTITEST CMI® kit in detail was followed and is incorporated herein by reference in its entirety.

The delayed cutaneous responses associated with the ubiquitous antigens in the MULTITEST CMI® battery appear to be typical cellular hypersensitivity reactions. A relatively small amount of soluble antigen is introduced into the epidermis and superficial dermal tissue by puncture. Circulating T-cells (lymphocytes), sensitized to the antigen from prior contact, react with the antigens in the skin and induce a specific immune response which includes mitosis (blastogenesis) and the release of many soluble mediators (lymphokines) . Some lymphokines initiate inflammation (vasculitis and edema) that is manifest after several hours. If periodic testing is done more frequently than every two months, then the test sites should be rotated so that retesting is not conducted at the same site sooner than two months.

The test sites were read at 48 hours. The time for maximal reactivity to the various antigens may vary in different people. A positive reaction from any of the seven delayed hypersensitivity skin test antigens is typically induration of 2 mm or greater with The MULTITEST CMI® kit. For our studies, we measured induration and/or redness, and used a redness measurement of 2 mm or greater as indicating a DTH response to an antigen. The size of the typical DTH response will vary depending on how the antigen is injected and on how much antigen is injected and on how antigenic the antigen is. There are many different ways to administer antigen. Other methods are known to those skilled in the art. The results of the DTH tests are shown in tables below for each patient by patient accession numbers. The table indicates induration/redness scores. An antigen was scored as causing a positive DTH response if it caused redness of at least 2 mm around the antigen site if it caused induration of at least 2 mm around the antigen site. For example, the score on day 0 for patient 208 is 1 / 2 which indicates that 1 of the 7 antigens tested caused induration greater than or equal to 2mm, and 2 antigens of the 7 antigens tested caused redness greater than or equal to 2mm. Patient 207 is also DTH responsive with a score of 0 / 3, because the patient had at least one antigen causing redness of 2 mm or more.

The results in table 3 show that 4 patients (#202, 205, 208 and 207) had DTH responses on day 0, and the other 4 patients were anergic. By day 29, two of the initially anergic patients (#203, 204) developed a DTH positive response to at least one antigen. Patient #203 remained DTH responsive at day 73, patient #206 developed a DTH response for the first time, and patient #204 returned to anergic status.

The criteria for a DTH positive response can be varied as is shown by Table 4 where a positive response was scored as either induration or redness of at least 5 mm. Thus, a person of ordinary skill in the art can determine the criteria for a DTH positive response.

TABLE 3 DTH RESPONSES MEETING THE 2MM THRESHOLD IN LYMPHOMA

PATIENTS

204 nd / 0 nd / 4 nd / 0 206 nd / 0 nd / 0 0/1

202 nd / 1 nd / 1 nd / 1 205 nd / 2 0 Off study 208 1 / 2 2 / 5 2 / 2 207 0 / 3 0 / 1 1 / 1 DTH RESPONSIVENESS IS COUNTED IF THE PATIENT DISPLAYS REDNESS OF 2 MM OR GREATER DIAMETER AT THE ANTIGEN INJECTION SITE. INDURATION/REDNESS IS RECORDED; ND MEANS NOT DONE; THE NUMBER INDICATES THE NUMBER OF DIFFERENT ANTIGENS THAT CAUSED REDNESS OF 2MM OR GREATER.

TABLE 4 DTH RESPONSES MEETING THE 5 MM THRESHOLD

202! nd / 1 nd / 1 ¹ nd / 1

2051 nd / 0 0 Off study

208 0 / 2 0 / 5 1 / 2

207j 0 / 2 0 / 1 1 / 1

1

DTH RESPONSIVENESS IS COUNTED IF THE PATIENT DISPLAYS REDNESS OF 5 MM OR GREATER DIAMETER AT THE ANTIGEN INJECTION SITE. INDURATION/REDNESS IS RECORDED; ND MEANS NOT DONE; THE NUMBER INDICATES THE NUMBER OF DIFFERENT ANTIGENS THAT CAUSED REDNESS OF 5 MM OR GREATER.

The specific details of each lymphoma patient's response to those antigens that elicited a positive DTH response are listed below. Also indicated are those patients who were initially anergic, and remained anergic throughout the study.

TABLE 5

DTH RESPONSES FOR Lymphoma [All measurements are in centimeters . ]

Tumor measurements

The tumor size for each patient was measured at various points during the study as indicated in Table 6 which shows all tumor values as totals representing the sum of the result of multiplying the largest measured diameters on CAT scans for each lesion. None of the initially DTH responsive patients (i.e., patients who were DTH positive at pre-study testing) showed progressive disease during the study, which means that none had an increase of tumor size of more than 10%. Indeed, three out of the four initially responsive patients showed a downward trend in tumor size (16% decrease for #208, 25% for #202, and 32% for #205) . The 25% decrease at day 85 for patient #208 became a 53% decrease at 12 months. Thus DTH responsiveness in prestudy testing is an indication of good prognosis for immunotherapy.

In contrast, no long lasting improvement in the disease in all diseased areas was obtained in any of the initially anergic patients. Three of the four patients that were initially anergic (DTH negative), had tumor increases. One patient (#201) had a +155.66 percent increase in tumor volume which indicates progressive disease according to the protocol. Patient #201 was initially anergic and remained anergic despite treatment with BT, showed progression of the disease. Only one initially anergic patient (# 206) showed any decrease in tumor size. This Patient worsened during the study and eventually developed leukemia. A one-tailed Fisher's Exact test on the differences between three patients with DTH response and decreasing cancer and the three patients without DTH and with increasing cancer yielded a significant difference (p=.05). Thus, anergic status in prestudy DTH testing is predictive of a poor prognosis for immunotherapy .

Table 6. Lymphoma Tumor Measurements

Unmonitored data provided by clinical site

Values below the total size are the percent change (- or +) from day 0 (pre-study) to the day in that column. A tumor was considered stable if the % increase or % decrease in tumor size is less than 10%.

**For initially anergic patient #206, tumor size decreased during therapy but the disease worsened as the person developed leukemia and white blood cell counts became unacceptably high. 9.5 units WBC day 0; 28.4 at day 43; and 30.9 at day 85.

Example 2 Myeloma

DTH multiantigen skin testing was also done on myeloma patients before, and during a study wherein beta-alethine was administered as a chemotherapeutic agents in a Phase I/II Study to Assess the Safety and Efficacy of Low Doses of BETATHINE™ in Patients with Advanced Myeloma, Protocol No. DTI-98-01, IND control number 059148, incorporated by reference herein.

DTI-98-01: This is an open label, non-randomized Phase I/II study to assess the effects of a low dose (2 ug per patient) of Beta LT™ (beta-alethine, or BETATHINE™) in patients with myeloma. Study drug is administered by subcutaneous injection of 0.1 mLs of a 20 ug/mL solution once every two weeks for a total of six doses. Safety and immunologic tests are performed on the days of drug administration and three other days. Primary endpoints are: Objective tumor response measured as changes in M proteins (complete response, partial response, stable, or progression, assessed on days 29, 57 and 85) . DTH tests were given on days 0, 31 and 73 if the study. DTH positive responses were assessed and scored as described in Example 1.

Four of the six myeloma patients were initially anergic. Two of the six myeloma patients studied were initially DTH responsive, therefore no valid comparisons between the responsive and anergic myeloma groups could be made. However, the data shows that, like in lymphoma patients, many of the myeloma patients who were initially anergic responded to beta-alethine treatment by developing DTH responsiveness. TABLE 7

Redness 2 mm or higher indicates a positive DTH response.

induration / redness; nd=not done.

TABLE 8

Redness 5 mm or higher indicates a positive DTH response myeloma DO D31 D73

101 nd/0 nd /l nd / 2

102 nd/0 nd /l nd /0

103 0/0 0/0 0 / 1

105 0/0 0/0 Off study

106 0/0 0 / 1 0 / 2

104 nd /0 0 / 2 0 / 3 redness 5 mm or higher; induration / redness; nd=not done.

Table 9 below shows the unmonitored findings for the six myeloma patients. Three of the five initially anergic myeloma patients later showed an increased immune response, as measured by a response to at least one test antigen.

TABLE 9 MYELOMA DTH RESPONSES FOR INDIVIDUAL ANTIGENS

All DTH axb measurements are in centimeters. A positive DTH response was counted for an antigen if it caused redness or inundation of at least 2mm (0.2cm). 0 = no reaction; nd = not done

The data shows that using a 2 mm diameter redness as a threshold for scoring a DTH response, two of the four initially anergic myeloma patients developed DTH responsiveness during treatment with beta-alethine. In lymphoma, three out of four initially anergic patients developed DTH responses, supporting the conclusion that anergic patients can become responsive to DTH, indicating an increase in cellular immunity and T cell activity, with treatment with immunostimulants. The DTH responsive patients are then candidates for a good prognosis with immunotherapy.

TABLE 10

DTH Response of Initially Anergic Patients Only

These results show that some anergic cancer patients are capable of developing DTH positive responses to one or more antigens if treated with immunostimulants .

Most patients remained stable and some had slightly decreasing M-protem levels during the 85-day study. One patient progressed in the first 85 days.

One skilled in the art will appreciate from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. A method for predicting effectiveness of treating an immune disease or disorder in an animal with an immunotherapeutic agent, comprising the steps of: a) administering a test for delayed type hypersensitivity (DTH) for one or more antigens to the animal prior to treatment with the immunotherapeutic agent; b) measuring a DTH response to each antigen as a function of at least one of induration and redness of an area surrounding a site of antigen exposure; c) classifying the animal as anergic, if the animal displays no DTH response to any antigen in the DTH test and as responsive, if the animal displays a DTH positive response to any antigen in the DTH test; d) predicting a good outcome for immunotherapeutic treatment of the disease if the patient is responsive, and a poor outcome if the patient is anergic in prestudy DTH testing.

2. A method for predicting predicting likelihood of tumor shrinkage in an animal having cancer in response to immunotherapy with an immunotherapeutic agent, comprising the steps of: a) administering a test for delayed type hypersensitivity (DTH) for one or more antigens to the animal prior to treatment with the immunotherapeutic agent; b) measuring a DTH response to each antigen as a function of at least one of induration and redness of an area surrounding a site of antigen exposure; c) classifying the animal as anergic, if the animal displays no DTH response to any antigen in the DTH test, and as responsive if the animal displays a DTH positive response to any antigen in the DTH test; and d) predicting tumor shrinkage in response to immunotherapy with an immunotherapeutic agent if the patient is responsive, and predicting no tumor shrinkage if the patient is anergic in prestudy DTH testing.

3. A method for optimizing a beneficial outcome of immunotherapy for treating a disease in an anergic animal with an immunotherapeutic agent, comprising the steps of: a) administering a test for delayed type hypersensitivity (DTH) for one or more antigens to the animal, b} measuring the DTH response to each antigen as a function of at least one of an induration and a redness of an area surrounding a site of antigen exposure, c) if the animal displays a positive DTH response to one or more of the antigens, then cl) classifying the animal as responsive, and c2) initiating immunotherapy by administering the immunotherapeutic agent, d) if the animal displays no DTH response to any antigen in the DTH test, then dl) classifying the animal as anergic, d2) administering a particular amount of a particular combination of one or more known immunostimulatory agents for a particular duration of time; and d3} repeating steps a) through c) one or more times within the particular duration of time.

4. The method of claim 3, further comprising the step of: e) if the animal remains anergic after step d) , then el) setting the particular amount of the first combination equal to a different amount, and e2) repeating steps a) through d) .

5. The method of claim 4, wherein step d) further comprises setting the particular duration of time equal to a different duration of time.

6. The method of claim 5, further comprising the step of: f) if the animal remains anergic after step e) , then fl) changing the particular combination of one or more known immunostimulatory agents to consist of a different combination of one or more known immunostimulatory agents, and f) repeating steps a) through d) .

7. A method for determining the optimum amount of an immunotherapeutic agent to administer to an animal to treat a disease, comprising the steps of: a) screening a group of animals in need of such treatment by administering a test for delayed type hypersensitivity (DTH) for one or more antigens to the animal, b) measuring the DTH response of each animal in the group to each antigen as a function of at least one of an induration and a redness of an area surrounding a site of antigen exposure, bl) if the animal displays a positive DTH response to one or more of the antigens, then classifying the animal as responsive, b2) if the animal displays no DTH response to any antigen in the DTH test, then classifying the animal as anergic, c) forming two or more groups of initially anergic animals, each group having two or more members, d) administering to respective groups of initially anergic animals, respective different amounts of immunotherapeutic agent for a duration of time, e) repeating steps a)-b) for every animal one or more times during the course of administration the immunotherapeutic agent in step d) , f) analyzing the results of each DTH test for each animal to determine the number of anergic and DTH responsive animals in each group, and g) selecting as the optimum amount of immunotherapeutic agent to administer to treat the disease in a general population of animals, the amount that generated the strongest DTH response in one or more of the groups.

8. The method of 7, wherein the group having the strongest DTH response is the group containing the highest number of DTH responsive animals.

9. The method of 7, wherein the group having the strongest DTH response is the group, the members of which collectively have the highest number of positive DTH responses to individual antigens, defined as the sum of the total number of DTH positive responses to individual antigens for each animal in the group, for all the DTH tests conducted.

10. The method of 7, wherein the group having the strongest DTH response is the group, the members of which collectively have the highest sum of (1) number of DTH responsive animals, and (2) the number of positive DTH responses to individual antigens defined as the sum of the total number of DTH positive responses to individual antigens for each animal in the group, for all the DTH tests conducted.

10. The method as in one of claims 1-3 and 7 wherein the DTH test is a multiple antigen skin test.

11. The method as in one of claims 1-3 and 7 wherein the one or more antigens are selected from the group comprising Tetanus Toxoid, Diptheπa toxoid, Streptococcus, Tuberculin OLD, Glycerin Control, Candida, Trichophyton and Proteus .

12. The method of claim 3, wherein the amount of immunotherapeutic agent adjusted by varying the frequency of administration.

13. The method of claim 3, wherein the amount of immunotherapeutic agent is adjusted by increasing the concentration of agent administered in each administration.

14. The method as in one of claims 1-3, wherein the immunotherapeutic agent is a combination of two or more drugs that affect the immune system.

15. The method as in one of claims 3 and 7, wherein the disease is cancer.

16. The method as in one of claims 3 and 7, wherein the disease is an immune disease.

17. The method as in one of claims 1 and 18, wherein the immune disease is an autoimmune disease.

18. The method as in one of claims 1 and 18, wherein the immune disease is an infectious disease.

19. The method as in one of claims 1-3 and 7, wherein the immunotherapeutic agent is beta-alethine.

20. The method as in one of claims 1-3 and 7, wherein a positive DTH response is indicated by induration at the site of antigen injection that is greater than or equal to 1 mm in diameter.