WO2008125903A2 - Method of inhibiting an undesired immune response - Google Patents

Abstract

The invention provides a method of suppressing an immune response to a therapeutic agent, the method comprising administering to a subject in need thereof a combination of a purified first therapeutic agent and a second therapeutic agent, wherein the combined dose of the first and second therapeutic agents suppresses an immune response to the first therapeutic agent in the subject. The invention also provides a method of suppressing an immune response to auto- antigens, the method comprising administering to a subject in need thereof a combination of an auto antigen or a mixture of auto-antigens and a second therapeutic agent, wherein the combined dose of the first and second therapeutic agents suppresses an immune response to auto-antigens in the subject.

Description

Method of Inhibiting an Undesired Immune Response

Related Applications

This application claims the benefit of U.S. Provisional Application No. 60/874,687, filed December 12, 2006 the contents of which is incorporated herein by reference in its entirety.

Field of the Invention

The invention generally relates to methods and compositions for suppressing immune responses.

Background of the Invention

There are a multitude of biologically-derived therapeutic proteinaceous agents used in current medicine. However, many of these agents elicit an immune response by the treated patient, causing generation of neutralizing antibodies against the proteinaceous drug and/or hypersensitivity reactions, which can limit the effectiveness of the proteinaceous agents. Similarly, many chronic inflammatory disorders are believed to be driven by auto-antigens. Unfortunately, successful treatments selectively targeting these antigens are not available.

Summary of the Invention The present invention is based, in part, upon the discovery of compositions and methods for reducing undesired immune response to proteinaceous therapeutic agents and to autoimmune antigens.

In one aspect, the invention provides a method of suppressing an immune response to a therapeutic agent. The method includes administering to a subject in need thereof a combination of a purified first therapeutic agent and a second therapeutic agent. The combined dose of the first and second therapeutic agents suppresses an immune response to the first therapeutic agent in the subject.

The method optionally includes assessing the immune response to the first therapeutic agent following administering of the combination. In some embodiments, the method includes repeating administration of sub-therapeutic dose of the first therapeutic agent and/or the second therapeutic agent to the subject. For example, administration of the second therapeutic agent can be initiated within 12 months prior to administration of the first therapeutic agent, and continued thereafter. The first therapeutic agent and the second therapeutic agent can be administered simultaneously or separately to the subject.

The combination of a subtherapeutic dose of the first therapeutic agent can be administered to the subject prior to or after administering a therapeutic dose of the first agent. In some embodiments, the combination is administered to the subject when the immune response to a therapeutic dose of the first therapeutic agent in the subject inhibits the effectiveness of the first therapeutic agent in the subject or elicits side effects secondary to the first agent in the subject.

In some embodiments, the second therapeutic agent is administered in an amount sufficient to delete memory immune cells specific for first therapeutic agent, and/or render them tolerant towards the first therapeutic agent.

In some embodiments, the second therapeutic agent is administered in an amount sufficient to reduce a hypersensitive immune response to the first therapeutic agent in the subject. In some embodiments, the second therapeutic agent is administered in an amount sufficient to reduce rapid clearance or inactivation of the first therapeutic agent, by circulating antibodies specific for the first therapeutic agent.

In some embodiments, the second therapeutic agent and/or a sub-therapeutic dose of the first therapeutic agent is administered continuously for at least one hour. For example, in some embodiments the second therapeutic agent is administered continuously for at least 2, 6, 12, 24, 28, 96 hours, or one week.

The first therapeutic agent and/or second therapeutic agent is preferably formulated for administration to a human subject.

The first therapeutic agent and second therapeutic agent can be administered in any route that elicits the intended effect. Thus, in various embodiments, the first therapeutic agent and/or the second therapeutic agent are administered in an oral, nasal, topical, parenteral, subcutaneous, intramuscular, intravenous, rectal, buccal, or transdermal route. The first therapeutic agent and second therapeutic agent can be administered in the same route or in a different route.

In various embodiments the first therapeutic agent is, e.g, a polypeptide, protein, glycoprotein, lipoprotein, glycopeptides, lipopeptide, or an antisense oligonucleotide.

When the first therapeutic agent is a polypeptide or a protein, the polypeptide or protein can be, e.g., a hormone, cytokine, naturally occurring anti-cytokine (IL-IRA), a clotting factor, endothelial cell growth factor, or coagulation factor. Examples of suitable polypeptides or proteins that can be used as a first therapeutic agent include, e.g., insulin, human growth hormone, EPO, G-CSF, CG-CSF, GM-CSF, α-interferon, β-interferon, IL-1 1, IL- 12 , factor VIII, factor VII, or factor IX.

A polypeptide or protein first therapeutic agent can also be an antibody or a fragment of an antibody, e.g., an antigen-binding fragment of an antibody. The antibody can be, e.g., a polyclonal antibody or a monoclonal antibody. The monoclonal antibody can be, e.g., a chimeric antibody, including a humanized monoclonal antibody. In various embodiments, the antibody used as a first therapeutic agent is, e.g., an anti-cytokine antibody, anti-cytokine receptor antibody, anti-chemokine antibody, or an anti-chemokine receptor antibody.

Specific examples of antibodies that can be used as a first therapeutic agent include, e.g, an anti-TNFα antibody, an anti-EGF receptor antibody, an anti-IL-2 receptor antibody, an anti- HER2 antibody, anti-VEGF antibody, anti-CD20 antibody, anti-IL-12 antibody, anti-IL-23, anti- INF-gamma, anti-CTLA-4, anti-VLAl, anti-VLA-4, anti alpha4beta7 integrin, anti IL- 12 P40, anti CD 25, anti IL-6 receptor, anti IL-4, or anti IL-5 antibody.

In other embodiments, a polypeptide or protein first therapeutic agent is a polypeptide or protein that includes an extracellular portion of a cytokine receptor. In some embodiments, the extracellular portion of the cytokine receptor is provided as a fusion protein. An example of a suitable cytokine receptor is a cytokine receptor is a receptor for TNFα.

In some embodiments, the first therapeutic agent is a fragment of a polypeptide. For example the fragment can include a segment of a naturally-occurring human or non-human protein and which binds to a human major histocompatibility complex (MHC) class I or II allotype.

If desired, the first therapeutic agent is administered with an adjuvant. The first therapeutic agent may be linked (e.g., via a covalent or non-covalent linkage) to the adjuvant. Alternatively, the first therapeutic agent can be unlinked to the adjuvant. When unlinked, the adjuvant may be administered simultaneously with the first therapeutic agent or administered at a time distinct from when the first therapeutic agent is administered to the subject.

In general, any amount of a first therapeutic agent can be used in conjunction with a second therapeutic agent provided it is administered in a subtherapeutic amount to the subject. By "subtherapeutic" amount is meant that the first therapeutic agent is administered to a subject in an amount that does not elicit a therapeutic response or does not suppress an immune response of the subject when administered in the absence of the second therapeutic agent.

The amount of a subtherapeutic dose of the first therapeutic agent will be determined by the particular first therapeutic agent used, the subject. In some embodiments the subtherapeutic dose of the first therapeutic agent will correspond to about lpg to 50mg/kg. In some embodiments, the dose of the first therapeutic agent is 100 pg/kg to about 1 mg/kg body weight of the subject, e.g., about 1 ng/kg to about 500 μg/kg body weight, 10 ng/kg to about 50 μg/kg body weight, about 100 ng/kg to about 5 μg/kg body weight, about 500 ng/kg to about 1 μg/kg body weight of the subject, or about 1 ng/kg to about 500 μg/kg body weight of the subject.

In some embodiments, the second therapeutic agent is a cell cycle inhibitor, e.g, an inhibitor of purine or pyrimidines nucleotide biosynthesis, or a folate antagonist. Examples of suitable second therapeutic agent include, e.g., methotrexate, 6-mercaptopurine, azathiopurine, thioguanosine, mycophenolic acid, rapamycin, cytosine arabinoside,5-fluorouracil (5-FU), a 5- FU prodrug (e.g. ftorafur, 5'-deoxyfluorouridine, carmofur), fluorouridine, T- deoxyfluorouridine, a prodrug derivative of fluorouridine or 2'-deoxyfluorouridine, fluorocytosine, 5'fluorouracil, fluorodeoxyuridine, arabinosyl cytosine, a prodrug of arabinosyl cytosine, cyclocytidine, 5-aza-2'-deoxycytidine, arabinosyl 5-azacytosine, N-phosphonoacetyl-L- aspartic acid (PALA), pyrazofurin, 6-azauridine, azaribine, 6-azacytidine, trifluoro-methyl-2'- deoxyuridine, thymidine, 3-deazauridine, a vinca alkaloid, a nitrogen mustard alykylating agent (including cyclophosphamide), daunorubicin, doxorubicin, a podophyllotoxin, cisplatin, or taxol.

If desired, the method can further include administering a third therapeutic agent. The third therapeutic agent can be, e.g, an agent that inhibits B cell growth, an agent that inhibits T cell growth, an IL-2 inhibitor, an antihistamine, or a corticosteroid.

The methods and composition described herein can be used on any suitable subject. By "subject" is meant a mammal, including, e.g, a human or a non-human mammal such as a primate, dog, cat, pig, cow, sheep, goat, horse, rat, or mouse.

In another aspect, the invention provides a method of suppressing an autoimmune response in a subject. The method includes administering to a subject in need thereof a combination of a sub-therapeutic dose of an autoantigen or a fragment of the autoantigen and a second therapeutic agent, wherein the combined dose of the first and second therapeutic agents suppresses an autoimmune response in the subject.

In some embodiments, the autoimmune response is associated with multiple sclerosis, asthma, diabetes, rheumatoid arthritis, systemic lupus erythematosus, polymyositis, dermatomyositis, autoimmune hepatitis, auto-immune thyroiditis, celiac disease, inflammatory bowel disease, psoriasis, Lichen planus, Pemphigus, auto-immune thrombocytopenia, sepsis, rheumatic disease, Sjogrens syndrome, or a transplantation-related immune response. The transplantation-related immune response can be, e.g., graft- versus-host-disease or posttransfusion thrombocytopenia.

When the autoimmune response is associated with multiple sclerosis, a suitable autoantigen for use as a first therapeutic agent is, e.g., myelin basic protein (MBP) or myelin oligodendrocyte glycoprotein (MOG), or a fragment or derivative of either of the these proteins.

When the autoimmune response is associated with rheumatoid arthritis, a suitable autoantigen for use as a first therapeutic agent is collagen type II, human chondrocyte glycoprotein 39 and proteoglycans, citrullinated filaggrin, glucose-6-phosphate isomerase, p205, or a heat shock protein present in elevated levels in patients with rheumatoid arthritis (such as HSC 70 kDa and HSP 90 kDa).

If desired the method optionally includes assessing the immune response to the first therapeutic agent following administering of the combination.

In some embodiments, the method includes repeating administration of sub-therapeutic dose of the first therapeutic agent and/or the second therapeutic agent to the subject. For example, administration of the second therapeutic agent can be initiated within 12 months prior to administration of the first therapeutic agent, and continued thereafter.

The first therapeutic agent and the second therapeutic agent can be administered simultaneously or separately to the subject.

The combination of a subtherapeutic dose of the first therapeutic agent can be administered to the subject prior to or after administering a therapeutic dose of the first agent.

In some embodiments, the combination is administered to the subject when the immune response to a therapeutic dose of the first therapeutic agent in the subject inhibits the effectiveness of the first therapeutic agent in the subject or elicits side effects secondary to the first agent in the subject. In some embodiments, the second therapeutic agent is administered in an amount sufficient to delete memory immune cells specific for first therapeutic agent, and/or render them tolerant towards the first therapeutic agent.

In some embodiments, the second therapeutic agent is administered in an amount sufficient to reduce a hypersensitive immune response to the first therapeutic agent in the subject.

In some embodiments, the second therapeutic agent is administered in an amount sufficient to reduce rapid clearance or inactivation of the first therapeutic agent, by circulating antibodies specific for the first therapeutic agent. In some embodiments, the second therapeutic agent and/or a sub-therapeutic dose of the first therapeutic agent is administered continuously for at least one hour. For example, in some embodiments the second therapeutic agent is administered continuously for at least 2, 6, 12, 24, 28, 96 hours, or one week. The first therapeutic agent and/or second therapeutic agent is preferably formulated for administration to a human subject.

The first therapeutic agent and second therapeutic agent can be administered in any route that elicits the intended effect. Thus, in various embodiments, the first therapeutic agent and/or the second therapeutic agent are administered in an oral, nasal, topical, parenteral, subcutaneous, intramuscular, intravenous, rectal, buccal, or transdermal route. The first therapeutic agent and second therapeutic agent can be administered in the same route or in a different route.

In various embodiments, the first therapeutic agent is, e.g, a polypeptide, protein, glycoprotein, lipoprotein, glycopeptides, lipopeptide, or an antisense oligonucleotide.

In some embodiments, the first therapeutic agent is a fragment of a polypeptide. For example the fragment can include a segment of a naturally-occurring human or non-human protein and which binds to a human major histocompatibility complex (MHC) class I or II allotype.

If desired, the first therapeutic agent is administered with an adjuvant. The first therapeutic agent may be linked (e.g., via a covalent or non-covalent linkage) to the adjuvant. Alternatively, the first therapeutic agent can be unlinked to the adjuvant. When unlinked, the adjuvant may be administered simultaneously with the first therapeutic agent or administered at a time distinct from when the first therapeutic agent is administered to the subject.

In general, any amount of a first therapeutic agent can be used in conjunction with a second therapeutic agent provided it is administered in a subtherapeutic amount to the subject. By "subtherapeutic" amount is meant that the first therapeutic agent is administered to a subject in an amount that does not elicit a therapeutic response or does not suppress an immune response of the subject when administered in the absence of the second therapeutic agent.

The amount of a subtherapeutic dose of the first therapeutic agent will typically be determined by the particular first therapeutic agent and/or second therapeutic agent used. In some embodiments, the subtherapeutic dose of the first therapeutic agent corresponds to about lpg to 50mg/kg. For example, in various embodiments the dose of the first therapeutic agent is 100 pg/kg to about 1 mg/kg body weight of the subject, e.g., about 1 ng/kg to about 500 μg/kg body weight, 10 ng/kg to about 50 μg/kg body weight, about 100 ng/kg to about 5 μg/kg body weight, about 500 ng/kg to about 1 μg/kg body weight of the subject, or about 1 ng/kg to about 500 μg/kg body weight of the subject.

In some embodiments, the second therapeutic agent is a cell cycle inhibitor, e.g, an inhibitor of purine or pyrimidines nucleotide biosynthesis, or a folate antagonist. Examples of suitable second therapeutic agent include, e.g., methotrexate, 6-mercaptopurine, azathiopurine, thioguanosine, mycophenolic acid, rapamycin, cytosine arabinoside,5-fluorouracil (5-FU), a 5- FU prodrug (e.g. ftorafur, 5'-deoxyfluorouridine, carmofur), fluorouridine, T- deoxyfluorouridine, a prodrug derivative of fluorouridine or 2'-deoxyfluorouridine, fluorocytosine, 5'fluorouracil, fluorodeoxyuridine, arabinosyl cytosine, a prodrug of arabinosyl cytosine, cyclocytidine, 5-aza-2'-deoxycytidine, arabinosyl 5-azacytosine, N-phosphonoacetyl-L- aspartic acid (PALA), pyrazofurin, 6-azauridine, azaribine, 6-azacytidine, trifiuoro-methyl-2'- deoxyuridine, thymidine, 3-deazauridine, a vinca alkaloid, a nitrogen mustard alykylating agent (including cyclophosphamide), daunorubicin, doxorubicin, a podophyllotoxin, cisplatin, or taxol. If desired the method can further include administering a third therapeutic agent. The third therapeutic agent can be, e.g, an agent that inhibits B cell growth, an agent that inhibits T cell growth, an IL-2 inhibitor, an antihistamine, or a corticosteroid.

The methods and composition described herein can be used on any suitable subject. By "subject" is meant a mammal, including, e.g, a human or a non-human mammal such as a primate, dog, cat, pig, cow, sheep, goat, horse, rat, or mouse

In a further aspect, the invention provides a method of increasing the effectiveness of a therapeutic agent in a subject. The method includes administering a sub-therapeutic dose of a combination of a first therapeutic agent described herein along with a second therapeutic agent described herein, wherein the combined dose of the first and second therapeutic agents suppresses an immune response to the first therapeutic agent in the subject and thereby increasing the effectiveness of the first therapeutic agent in the subject.

In another aspect, the invention provides a pharmaceutical composition comprising a first therapeutic agent described herein and a sub-therapeutic dose of a second therapeutic agent described herein or fragment of the second therapeutic agent described herein, along with a pharmaceutically acceptable carrier. In some embodiment, the combined dose of the first and second therapeutic agents in the pharmaceutical composition suppresses an immune response in a subject. In some embodiments, the first therapeutic agent or second therapeutic agent or fragment thereof, is formulated for delivery to a human subject. The first therapeutic agent and/or second therapeutic agent in the pharmaceutical composition is preferably formulated for administration to a human subject.

Also within the invention is a kit comprising a sub-therapeutic dose of a first therapeutic agent described herein and a second therapeutic agent or fragment of the second therapeutic agent described herein. In some embodiments, the first therapeutic agent and second therapeutic agent are formulated so that the combined dose of the first and second therapeutic agents suppresses an immune response in a subject. The kit may optionally include one or more pharmaceutically acceptable carriers.

In a further aspect, the invention provides a method of identifying a treatment regimen for suppressing an immune response. The method includes administering to a test subject a candidate sub-therapeutic dose of a first therapeutic agent as described herein and a candidate dose of a second therapeutic agent described herein, detecting the immune response to the candidate dose in the subject, and comparing the immune response to the candidate dose to the immune response to the first therapeutic ageing in a control subject not administered the candidate dose of the first therapeutic agent and second therapeutic agent. A decreased immune response to the candidate dose in the test subject relative to the control subject indicates the candidate dose is a therapeutic regimen for suppressing an immune response.

In yet another aspect the invention provides methods of detecting a humoral immune (e.g. antibody) response to a therapeutic antibody in a subject by contacting a subject sample know to or suspected of containing an antibody specific for the therapeutic antibody with a Fab₂ fragment of said therapeutic antibody and detecting the antibody- Fab₂ fragment complex. The presence of the antibody- Fab₂ fragment complex indicates the presence a humoral immune response to the therapeutic antibody in the subject. Whereas the absence of the antibody- Fab₂ fragment complex indicates the absence a humoral immune response to the therapeutic antibody in the subject. The subject sample and the Fab₂ fragment of said therapeutic antibody are contacted under conditions permitting the antibody to specifically bind the Fab₂ fragment to form an antibody- Fab₂ fragment complex. The therapeutic antibody is for example, an anti-TNF antibody such as is Infliximab. The antibody- Fab₂ fragment complex is detected by method known in the art such as a Western Blot. Optionally, the Fab₂ fragment is immobilized on a solid phase such as nitrocellulose.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description and claims.

Brief Description of the Drawings

Figure IA. is a schematic illustration showing the results of FACS analysis demonstrating proliferation of stimulated T-cells is abrogated by thiopurines. Proliferation of CFSE-labeled CD4+ T-cells at day 5 after stimulation with plate bound anti-CD3 (OKT3) and soluble anti-CD28, in the presence or absence of medium alone, 6-MP (5μM), 6-TG (5 μM) or AZA (5μM). Figure IB is a histrogram showing mean proliferation ±SEM of PB CD4+ T-cells triggered by the protocol of Figure IA. Results shown are a summary of 21 different experiments performed.

Figure 1C is a schematic illustration showing the results of FACS analysis demonstrating proliferation of CFSE-labled CD8+ T-cells at day 5 after stimulation with plate bound anti-CD3 (OKT3) and soluble anti-CD28, in the presence or absence of 6-MP (5μM), 6- TG (5 μM) or Azathoiprine (5μM).

Figure 2 A is a histogram showing Thiopurines induce apoptosis of stimulated T-cells no earlier than day 5 post-stimulation, and do not affect resting T-cells. Mean percentage±SEM of anti-CD3/CD28 stimulated CD4+ cells undergoing early apoptosis (Annexin+PI-) and late apoptosis/necrosis (Annexin+PI+) after 5 days co-incubation with the above noted concentrations of thiopurines, or cyclohexamide. * denotes P<0.05 for the comparison with stimulated cells cultured alone (untreated cells), n=18.

Figure 2B is a schematic illustration showing the results of FACS analysis demonstrating the percentage of anti-CD3/CD28 stimulated CD4+ cells undergoing apoptosis (Annexin V+ cells), with respect to dividing cells (CFSElow, left upper quadrants) and non- dividing cells (CFSEhigh, right upper quadrants), after 5 days co-incubation with the designated agents. Shown is a representative example out of 5 experiments performed.

Figure 2C is a histogram showing mean percentage±SEM of non-stimulated CD4+ cells undergoing early apoptosis (Annexin V+PI-) and late apoptosis (Annexin V+PI+) after 5 days of culture with the designated agents, or in media alone (untreated). * denotes P<0.05 for the comparison with cells cultured in media alone (untreated cells).

Figure 3A is a illustration of an Eexperimental design for examining the effect of successive antigen encounters and prolonged administration of 6-MP on the antigen-specific memory pool. Two different experiments were performed with 6 mice in each group.

Figure 3B is a histogram showing the mean percentage±SEM of dividing CD4+ splenocytes in response to the designated antigens or medium alone, for 6-MP treated versus non-treated mice, sacrificed after 4 weeks of the immunization protocol (see Example 1).

Figure 3C is a histogram showing mean percentage ±SEM of dividing CD4+ splenocytes in response to the designated antigens or medium alone, for 6-MP treated versus non-treated mice, sacrificed after 20 weeks of the immunization protocol, or versus non-immunized mice ('non-immunized').

Figure 3D is a histogram showing mean spleen weight and splenocyte numbers in mice sacrificed after 20 weeks of the immunization protocol, for the 6-MP treated versus non-treated group.

Figure 4 is a photograph of a western blotting of serums of patient serums treated with Infliximab (patients IS1,IS2,IS3,BS4) vs. Infliximab-naive patients (NC) , with Fc or Fab2 fragments of Infliximab. Shown is a representative example out of 10 Infliximab treated and 4 Infliximab-naϊve patients studied.

Detailed Description of the Invention

The invention provides a method of suppressing an immune response to a therapeutic agent, the method comprising administering to a subject in need thereof a combination of a purified first therapeutic agent and a second therapeutic agent, wherein the combined dose of the first and second therapeutic agents suppresses an immune response to the first therapeutic agent in the subject.

A "purified first therapeutic agent", as used herein, refers to a therapeutic agent that has been separated from other cellular components, including proteins, lipids, and nucleic acids with which it is naturally associated. The first therapeutic agent can constitute at least 10, 20, 50 70, 80 or 95% by dry weight of the purified preparation.

The first therapeutic agent is preferably provided in a sub-therapeutic dose. By "subtherapeutic" amount is meant that the first therapeutic agent is administered to a subject in an amount that does not elicit a therapeutic response or does not suppress an immune response to the subject when administered in the absence of the second therapeutic agent. In some embodiments, periodic immunization of a subject is performed with minute quantities of a first therapeutic agent (e.g., a therapeutic protein or a break-down immunogenic product of a therapeutic protein), while concurrently, or nearly concurrently, administering a second therapeutic agent that selectively blocks actively cycling cells. While not wishing to be bound by theory, it is believed that the result of the combined administration is a gradual reduction of the T-cell and B-cell memory pool specific for the first therapeutic agent, which results in the diminution of an immune response directed by the subject against the first therapeutic agent.

As is explained in more detail below, the first and second therapeutic agent can be administered via the same or different routes (e.g oral or subcutaneous delivery), and/or coupled to adjuvants. The antigens may be broken down to immunogenic peptides prior to administration. Recurrent immunization, if coupled with administering one of the existing drugs that arrest cell divisions (e.g azathioprine, 6-mercaptopurine, or methotrexate), is predicted to result in deletion of the specific memory cells targeting the first therapeutic agent. Deletion of these cells, in turn, can lead to amelioration and elimination of the hypersensitivity response towards these agents, and also to reduce their rapid clearance brought upon by circulating antibodies.

The method can also be adapted for treating auto-immune diseases, which result from inappropriate aggressive response of the immune system against auto-antigens of the patient. In this aspect of the invention, a sub-therapeutic dose of a first therapeutic antigen corresponding to an auto-antigen associated with the autoimmune response is administered to a subject along with the second therapeutic agent. Repeated immunization with the putative antigens will result in the dwindling of the auto-aggressive memory T-cell pool, thereby tolerizing the immune system to the culprit auto-antigens and leading to resolution of the inflammatory process. The first and second therapeutic agents may be administered along with agents aimed to reduce side effects such as antihistaminic corticosteroids, or an additional agent or agents that arrest cells from growth, yet allow them to mount an immune response. Such additional agents may include anti cytokine therapies (such as anti-IL-2), or agents that target specific cells that participate in the immune response of the targeted cell (i.e. T and B cells for example). The compositions and methods may also be used to target less specific cells of the immune system, such as NK cells or cells from a monocytic lineage, which are also arrested from growth and division, yet retain their functional capacity. These can be used in situations where such cells are considered to have a primary pathologic role. First Therapeutic Agent

In general, the first therapeutic agent is any therapeutic agent that, when administered in a therapeutic dose to a subject, elicits an immune response that either inhibits the effectiveness of the therapeutic agent or otherwise causes an adverse immune reaction in the subject. The first therapeutic agent is typically a polypeptide or protein, an antigenic fragment of the polypeptide or protein, glycoprotein, lipo-protein, glycoptpeide, lipopeptide, or polysaccharide. For example, the fragment can be the result of enzymatic cleavage or a metabolic breakdown product. The protein can be a modified.

In other embodiments, the first therapeutic agent is an oligonucleotide or polynucleotide, or derivative of an oligonucleotide or polynucleotide. For example, the first therapeutic agent can be, e.g., an antisense nucleic acid, an siRNA, or a ribozyme that selectively binds to a nucleic acid. Examples of suitable oligonucleotide agent is an anti-sense oligonucleotide of intercellular adhesion molecule 1 (ICAM-I) (see, e.g., US Patent No. 6,096,722).

In general, any therapeutic polypeptide or protein can be used. Examples include, e.g., a hormone, cytokine, a naturally occurring or non-naturally occurring anti-cytokine, a clotting factor, endothelial cell growth factor, or coagulation factor. Examples of specific polypeptides include, e.g., insulin, human growth hormone, EPO, G-CSF, CG-CSF, GM-CSF, α-interferon, β- interferon, IL-1 1, IL- 12, factor VIII, factor VII, factor IX, and IL-IRA.

In some embodiments, the first therapeutic agent polypeptide includes an extracellular portion of a cytokine receptor. For example, the extracellular portion of the cytokine receptor can be provided as a fusion protein. The fusion protein may optionally include a full-length immunoglobulin polypeptide, or less than full-length immunoglobulin polypeptide, e.g., a heavy chain, light chain, Fab, Fab₂, Fv, or Fc.

In some embodiments, the first therapeutic agent is a fragment that elicits a B cell or T cell-mediated immune response. For example, a polypeptide fragment can be a segment of a naturally-occurring human or non-human protein, or with a sequence corresponding to a sequence of a segment of a naturally-occurring human or non-human protein, and which binds to a human major histocompatibility complex (MHC) class I or II allotype. In other embodiments, the polypeptide or polypeptide fragment has a sequence that is not found in a naturally occurring peptide or polypeptide.

A further class of first therapeutic agent is an antibody or a fragment of an antibody. "Antibody" refers to a polypeptide ligand substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen). The antibody can be provided as, e.g., an intact immunoglobulin or as fragment, e.g., a fragment produced by digestion with various peptidases. This includes, e.g., Fab¹ and F(ab)'₂ Fv (defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains); and (5) Single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. The term "antibody" also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. It includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single chain antibodies. "Fc" portion of an antibody refers to that portion of an immunoglobulin heavy chain that comprises one or more heavy chain constant region domains, CHl, CH2 and CH3, but does not include the heavy chain variable region.

Antibodies or antibody fragments that can used as first therapeutic agents include those specific for polypeptides such as insulin, human growth hormone, EPO, G-CSF, CG-CSF, GM- CSF, α-interferon, β-interferon, IL- 1 1 , IL- 12, factor VIII, factor VII, factor IX, and IL- 1 RA.

Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).

The humanized form of a non-human (e.g., murine) antibody can be, e.g., a chimeric molecule of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323- 329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

General examples of antibodies suitable as first therapeutic agents include an anti- cytokine antibody, anti-cytokine receptor antibody, anti-chemokine antibody, or anti-chemokine receptor antibody. More specific examples of antibodies as first therapeutic agents include an anti-TNFα antibody, an anti-EGF receptor antibody, an anti-IL-2 receptor antibody, an anti- HER2 antibody, anti-VEGF antibody, anti-CD20 antibody, anti-IL-12 antibody, anti-IL-23, anti- INF-gamma, anti-CTLA-4, anti-VLAl, anti-VLA-4, and an anti alpha4beta7 integrin.

Second Therapeutic Agent

In general, a suitable second therapeutic agent is any compound that disrupts B cell or T cell replication or function such that the B cell or T cell can not replicate so as to bring about an immune response to the first therapeutic agent. A suitable second therapeutic agent can be a cell cycle inhibitor, an antineoplastic agent and/or various other therapeutic nucleoside analogs that act by affecting nucleoside or nucleotide biosynthesis, utilization, or metabolism.

For example, the second agent can be, e.g, methotrexate, 6-mercaptopurine, azathioprine, thioguanosine, mycophenolic acid, rapamycin, cytosine arabinoside, 5-fluorouracil (5-FU), a 5- FU prodrug (e.g. ftorafur, 5'-deoxyfluorouridine, carmofur), fluorouridine, 2'- deoxyfluorouridine, a prodrug derivative of fluorouridine or 2'-deoxyfluorouridine, fluorocytosine, 5'fluorouracil, fluorodeoxyuridine, arabinosyl cytosine, a prodrug of arabinosyl cytosine, cyclocytidine, 5-aza-2'-deoxycytidine, arabinosyl 5-azacytosine, N-phosphonoacetyl-L- aspartic acid (PALA), pyrazofurin, 6-azauridine, azaribine, 6-azacytidine, trifluoro-methyl-2'- deoxyuridine, thymidine, 3-deazauridine, a vinca alkaloid, a nitrogen mustard alykylating agent (including cyclophosphamide), daunorubicin, doxorubicin, a podophyllotoxin, cisplatin, taxol, or mycophenolate, or derivatives of mycophenolate.

Additional Therapeutic Agents

If desired a third and/or additional agents can be administered with the first and second therapeutic agent. Suitable agents can include, e.g. an agent that inhibits B cell and/or T cell growth, an antihistamine, an IL-I inhibitor, and a corticosteroid. Specific examples of additional agents include, e.g. prednisolone, methyl-prednisolone, cortisone, dexamethasone, cyclosporine, FK 506, and diphenhydramine. Selecting a Combination of a First Therapeutic Agent and a Second Therapeutic Agent

A therapeutic regimen using a combination of a first therapeutic agent and a second therapeutic agent can be identified by administering to a subject a candidate sub-therapeutic dose of a first therapeutic agent and a candidate dose of a second therapeutic agent. The immune response to the candidate dose in the subject is detected and compared to the immune response by the subject or a similarly situated subject that is not administered the candidate dose of the first therapeutic agent and/or the second therapeutic agent. A decreased immune response to the candidate dose in the test subject relative to the control subject indicates the candidate dose is a therapeutic regimen for suppressing an immune response. The dose of the first agent, and/or the dose of the second agent can be modified in subsequent administrations to the subject to achieve the desired suppression of the immune response.

The exact dose of the first and second therapeutic agents is chosen by the individual physician in view of the patient to be treated. In general, dosage and administration are adjusted to provide an effective amount of the first and second therapeutic agents sufficient to inhibit an immune response to the first therapeutic agent. The effective amount of first and second therapeutic agents may vary depending on such factors as the desired biological endpoint, the drug to be delivered, the target tissue, the route of administration, etc. For example, the effective amount of first and second therapeutic agents might be the amount that results in a decrease in the titer of antibodies directed against first therapeutic agent as measured by ELISA, or a decrease in the cellular response to first therapeutic agent as measured by IFN-gamma production by ELISA, or by thymidine and/or CFSE based proliferation assays.

Additional factors which may be taken into account include the severity of the disease state; age, weight and gender of the patient being treated; diet, time and frequency of administration; drug combinations; reaction sensitivities; and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular composition.

For first and second therapeutic agents, the appropriate dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

Therapeutic efficacy and toxicity of first and second therapeutic agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose is therapeutically effective in 50% of the population) and LD50 (the dose is lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use.

In various embodiments, the first therapeutic agent is provided at a dose of 100 pg to 500 mg/kg, e.g., 500 pg to 250 mg; 1 ng to 100 mg/ per body weight, 10 ng to 50 mg, 100 ng to 10 mg, 250 ng to 5 mg, 500 ng to 1 mg. In various embodiments, the second therapeutic agent is provided at a dose of 0.5-3 mg/kg for azathioprine, 0.25-2 mg/kg for 6-MP, and 5-25 mg/week for methotrexate.

Pharmaceutical compositions for administering first and second therapeutic agents

The first and second therapeutic agents are typically provided, either separately or together, in a pharmaceutically acceptable carrier suitable for administering the pharmaceutical composition to a subject. In some embodiments, the first and/or second therapeutic agents are provided in reagents of a grade suitable for administration to a human patient. The carriers may be chosen based on the route of administration as described below, the location of the target issue, the drug being delivered, the time course of delivery of the drug, etc.

Preferably, a combination of a first therapeutic agent and second therapeutic agent for the methods and compositions of the invention is non-toxic when administered to the subject. The term "nontoxic" is used in a relative sense and is intended to designate any substance that has been approved by the United States Food and Drug Administration ("FDA") for administration to humans or, in keeping with established regulatory criteria and practice, is susceptible to approval by the FDA for administration to humans.

The term "pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Remington 's Pharmaceutical Sciences Ed. by Gennaro, Mack Publishing, Easton, Pa., 1995 discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as TWEEN™ 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

The first and second therapeutic agents of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "dosage unit form" as used herein refers to a physically discrete unit of first and second therapeutic agents appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.

If several different therapeutic modalities are to be administered simultaneously then they may be combined into a single pharmaceutical composition, which is described in more detail below. Alternatively, they may be prepared as separate compositions that are then mixed or simply administered one after the other. If several different first and second therapeutic agents (e.g., with different first and second therapeutic agents drugs) are to be administered at different times then they are preferably prepared as separate compositions. The first and second therapeutic agents can be added to one or more of these pharmaceutical compositions or prepared as separate compositions. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, U. S. P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as a solvent or suspending medium. For this purpose any bland fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. In on embodiment, the first and/or second therapeutic agent is suspended in carrier fluid comprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) TWEEN™ 80. The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the first and/or second therapeutic agent with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the first and/or second therapeutic agent.

Dosage forms for topical or transdermal administration of a pharmaceutical composition include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The therapeutic agent or agents is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulations, ear drops and eye drops are also contemplated as being within the scope of this invention. The ointments, pastes, creams and gels may contain, in addition to the first and/or second therapeutic agent or agents, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the first and/or second therapeutic agent in a proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the first and/or second therapeutic agent in a polymer matrix or gel. Powders and sprays can also contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these drugs. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.

When administered orally, the first and/or second therapeutic agent or agents are optionally encapsulated. A variety of suitable encapsulation systems are known in the art ("Microcapsules and Nanoparticles in Medicine and Pharmacy," Edited by Doubrow, M., CRC Press, Boca Raton, 1992; Mathiowitz and Langer J. Control. Release 5: 13, 1987; Mathiowitz et al., Reactive Polymers 6:275, 1987; Mathiowitz et al., J. Appl. Polymer ScL 35:755, 1988; Langer Ace. Chem. Res. 33:94,2000; Langer J. Control. Release 62:7, 1999; Uhrich et al., Chem. Rev. 99:3181, 1999; Zhou et al., J. Control. Release 75:27, 2001; and Hanes et al., Pharm. Biotechnol. 6:389, 1995). For example, the first and/or second therapeutic agent or agents are encapsulated within biodegradable polymeric microspheres or liposomes. Examples of natural and synthetic polymers useful in the preparation of biodegradable microspheres include carbohydrates such as alginate, cellulose, polyhydroxyalkanoates, polyamides, polyphosphazenes, polypropylfumarates, polyethers, polyacetals, polycyanoacrylates, biodegradable polyurethanes, polycarbonates, polyanhydrides, polyhydroxyacids, poly(ortho esters) and other biodegradable polyesters. Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides. Pharmaceutical compositions for oral administration can be liquid or solid. Liquid dosage forms suitable for oral administration of the first and/or second therapeutic agent or agents include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to an encapsulated or unencapsulated the first and/or second therapeutic agent or agents, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents. As used herein, the term "adjuvant" refers to any compound which is a nonspecific modulator of the immune response. In certain preferred embodiments, the adjuvant stimulates the immune response. Any adjuvant may be used in accordance with the present invention. A large number of adjuvant compounds are known in the art (Allison, Dev. Biol. Stand. 92:3, 1998; Unkeless et al., Annu. Rev. Immunol. 6:251, 1998; and Phillips et al., Vaccine 10: 151, 1992).

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the encapsulated or unencapsulated the first and/or second therapeutic agent is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents. Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art.

By "pharmaceutically acceptable carrier" is meant a carrier that is physiologically acceptable to an administered animal while retaining the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences, (18^th edition),. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa.

The combination of the first therapeutic agent and second therapeutic agent may be administered in either a local or systemic manner or in a depot or sustained release fashion. The two agents may be delivered in an oral, transdermal or intranasal formulation. The first therapeutic agent and second therapeutic agent are preferably administered in a manner that provides the desired effect from the first and second agents in the combination. Optionally, the first and second agents are admixed into a single formulation before they are introduced into a subject. The combination may be conveniently sub-divided in unit doses containing appropriate quantities of the first and second agents. The unit dosage form may be, for example, a capsule or tablet itself or it can be an appropriate number of such compositions in package form. The quantity of the active ingredients in the unit dosage forms may be varied or adjusted according to the particular need of the condition being treated. Alternatively, the first therapeutic agent and second therapeutic agent are not mixed until after they are introduced into the subject. Thus, the term "combination" encompasses embodiments where first therapeutic agent and second therapeutic agent are provided in separate formulations and are administered sequentially. For example, the first therapeutic agent and second therapeutic agent may be administered to the subject separately within 180 days, 90 days, 60 days, 30 days, 14 days, 7 days, 2 days, 1 day, 18 hours, 12 hours, one hour, a half hour, 15 minutes, or less of each other. Each agent may be provided in multiple, single capsules or tablets that are administered separately to the subject. Alternatively, the first therapeutic agent and second therapeutic agent are separated from each other in a pharmaceutical composition such that they are not mixed until after the pharmaceutical composition has been introduced into the subject. The mixing may occur just prior to administration to the subject or well in advance of administering the combination to the subject. If desired, the first therapeutic agent and second therapeutic agent may be administered to the subject in association with other therapeutic modalities, e.g., drug, surgical, or other interventional treatment regimens. Accordingly, the combination described herein may be administered simultaneously or within 180 days, 90 days, 60 days, 30 days, 14 days, 7 days, 5 days, 3 days, one day, 12 hours, 6 hours, 3 hours, or one hour of additional therapeutic modalities. Where the combination includes a non-drug treatment, the non-drug treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination and the other therapeutic modalities is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non- drug treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks. The first therapeutic agent and/or second therapeutic agent can be administered via oral, nasal, topical (e.g., as a powder, creams, ointments, or drops), parenteral, subcutaneous, intramuscular, intravenous, rectal, buccal, by inhalation (as by sprays) and transdermal administration.

The first therapeutic agent and/or second therapeutic agent can be packaged as a kit that includes single or multiple doses of one or more of the agents, each packaged or formulated individually, or single or multiple doses of two or more agents packaged or formulated in combination. Thus, one or more agents can be present in first container, and the kit can optionally include one or more agents in a second container. The container or containers are placed within a package, and the package can optionally include administration or dosage instructions. A kit can include additional components such as syringes or other means for administering the agents as well as diluents or other means for formulation.

Methods of Suppressing Autoimmune Responses

The method can also be adapted to suppress an unwanted autoimmune response in a subject. A combination of a sub-therapeutic dose of an autoantigen, or a fragment of the autoantigen, or a tissue-derived mixture of antigens (when the putative antigen is unknown) , and a second therapeutic agent is administered to a subject at risk for or suffering from an autoimmune disorder. The combined dose of the first and second therapeutic agents preferably suppresses an autoimmune response in the subject. The autoimmune response can be, e.g., an autoimmune response associated with multiple sclerosis, asthma, diabetes, rheumatoid arthritis, systemic lupus erythematosus, polymyositis, dermatomyositis, autoimmune hepatitis, auto-immune thyroiditis, celiac disease, inflammatory bowel disease, psoriasis, Lichen planus, Pemphigus, auto-immune thrombocytopenia, sepsis, rheumatic disease, Sjogrens syndrome, a transplantation-related immune response (such as graft- versus-host-disease, post- transfusion thrombocytopenia). Additional diseases or conditions include, e.g., inflammatory bowel diseases such as Crohn's disease and ulcerative colitis in which the tissue is damaged by the activity of the immune system, including damaged caused to the resident flora. The first therapeutic antigen is chosen so that it corresponds to an autoantigen associated with, or causing, the disease. For example, for multiple sclerosis the first therapeutic agent can be myelin basic protein (MBP) or myelin oligodendrocyte glycoprotein (MOG). Other suitable autoantigens for the condition rheumatoid arthritis include, e.g., collagen type II, human chondrocyte glycoprotein 39 and proteoglycans, citrullinated filaggrin, glucose-6-phosphate isomerase, p205, and heat shock proteins (including HSC 70 kDa and HSP 90 kDa).

In some embodiments, the method can be adapted for organ or tissue-specific inhibition of an immune response to an antigen or immunogen. A suitable organ or tissue can include, e.g., intestinal mucosa or other mucosal surfaces.

Method of Increasing the Effectiveness of a Therapeutic Agent

The methods and compositions described herein can also be used to increase the effectiveness of a therapeutic agent in a subject. A sub-therapeutic dose of a combination of a first therapeutic agent is administered along with a second therapeutic agent so that the combined dose of the first and second therapeutic agents suppresses an immune response to the first therapeutic agent in the subject.

Methods of Detecting a Humoral Immune Response

Also included in the invention is an assay to detect the presence of a humoral (e.g., antibody) response in a subject to a therapeutic antibody. The therapeutic antibody include an anti-cytokine antibody, anti-cytokine receptor antibody, anti-chemokine antibody, or anti- chemokine receptor antibody. More specific examples of therapeutic antibodies include an anti- TNFα antibody, an anti-EGF receptor antibody, an anti-IL-2 receptor antibody, an anti-HER2 antibody, anti-VEGF antibody, anti-CD20 antibody, anti-IL-12 antibody, anti-IL-23, anti-INF- gamma, anti-CTLA-4, anti-VLAl, anti-VLA-4, and an anti alpha4beta7 integrin. A humoral response to a therapeutic antibody is detected by contacting, e.g. exposing) a subject sample with a fragment of the therapeutic antibody. The fragment is a Fab, Fab₂, Fv, or Fc fragment or a subfragment thereof. The sample is a subject derived sample known to or suspected of containing an antibody to a therapeutic antibody. For example, the sample is blood, serum, plasma, urine or saliva. The subject has or is receiving the therapeutic antibody. The subject sample and the fragment of the therapeutic antibody are contacted under conditions permitting an antibody- therapeutic antibody fragment complex. Complex formation is detected if present. The presence of an antibody- therapeutic antibody fragment complex indicated the presence of a humoral immune response to the therapeutic antibody in the subject. In contrast, the absence of a antibody- therapeutic antibody fragment complex indicates the absence of a humoral immune response to the therapeutic antibody in the subject.

The step of detecting the reaction product (i.e., antibody- therapeutic antibody fragment complex) may be carried out with any suitable immunoassay. Immunoassays carried out in accordance with the present invention may be homogeneous assays or heterogeneous assays. The signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte. Both the immunological reaction and detection of the extent thereof are carried out in a homogeneous solution. Immunochemical labels which may be employed include free radicals, radioisotopes, fluorescent dyes, enzymes, bacteriophages, or coenzymes. , In a heterogeneous assay approach, the reagents are usually the sample, the therapeutic antibody fragment, and means for producing a detectable signal. Samples as described above may be used. The therapeutic antibody fragment is generally immobilized on a support, such as a bead, plate or slide, and contacted with the specimen suspected of containing the anti- therapeutic antibody in a liquid phase. The support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal. The signal is related to the presence of the anti- therapeutic antibody in the sample. Means for producing a detectable signal include the use of radioactive labels, fluorescent labels, or enzyme labels. For example, if the antigen, e.g., anti- therapeutic antibody to be detected contains a second binding site, an antibody which binds to that site can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step. The presence of the detectable group on the solid support indicates the presence of the antigen, e.g., anti- therapeutic antibody in the test sample. Examples of suitable immunoassays are radioimmunoassays, immunofluorescence methods, or enzyme-linked immunoassays. Those skilled in the art will be familiar with numerous specific immunoassay formats and variations thereof which may be useful for carrying out the method disclosed herein. See generally E. Maggio, Enzyme-Immunoassay, (1980) (CRC Press, Inc., Boca Raton, FIa.); see also U.S. Pat. No. 4,727,022 to Skold et al. titled "Methods for Modulating Ligand-Receptor Interactions and their Application," U.S. Pat. No. 4,659,678 to Forrest et al. titled "Immunoassay of Antigens," U.S. Pat. No. 4,376,110 to David et al., titled "Immunometric Assays Using Monoclonal Antibodies," U.S. Pat. No. 4,275,149 to Litman et al., titled "Macromolecular Environment Control in Specific Receptor Assays," U.S. Pat. No. 4,233,402 to Maggio et al., titled "Reagents and Method Employing Channeling," and U.S. Pat. No. 4,230,767 to Boguslaski et al., titled "Heterogenous Specific Binding Assay Employing a Coenzyme as Label."

Therapeutic antibody fragments are conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation. Therapeutic antibody fragments as described herein may likewise be conjugated to detectable groups such as radiolabels (e.g., 35 S, 125 I, 131 I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with known techniques.

Diagnostic kits for carrying out the methods described herein are produced in a number of ways. In one embodiment, the diagnostic kit comprises (a) an therapeutic antibody fragments (e.g., Infliximab Fab₂ fragment) conjugated to a solid support and (b) a second antibody of the invention conjugated to a detectable group. The reagents may also include ancillary agents such as buffering agents and protein stabilizing agents, e.g., polysaccharides and the like. The diagnostic kit may further include, where necessary, other members of the signal-producing system of which system the detectable group is a member (e.g., enzyme substrates), agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like. The test kit may be packaged in any suitable manner, typically with all elements in a single container along with a sheet of printed instructions for carrying out the test.

The invention will be illustrated in the following non-limiting examples.

Example 1.: General Methods

Human subjects: The study was approved by the Institutional Ethics Committee at the Chaim Sheba Medical Center. Peripheral blood samples were obtained from healthy blood donors and from patients with Crohn's disease attending the Gastroenterology Department at the Chaim Sheba Medical Center.

Reagents. Interleukin (IL) -2 was purchased from PeproTech (NJ, USA). Azathioprine,

Cyclohexamide and Prednisolone (Sigma- Aldrich, St. Louis,USA) were solublized in DMSO. 6- Mercaptopurine (6-MP) and 6-Thioguanine (6-TG) (Sigma), and dissolved in NaOH. Thiopurines were then diluted in RPMI- 1640 (GIBCO, Invitrogen, CA, USA) to stock concentrations (ImM), and cyclohexamide and prednisolone were kept in DMSO stock concentration of 2mg/ml or 3mg/ml, respectively. Drugs were then added to cultures at the designated final concentrations. Collagen IV and Fibronectin were from Sigma-Aldrich.

MAb and FACS analysis.

The various fluorescent-conjugated anti CD3, anti CD4, anti CD8, anti CD14, anti VLA- 4, anti CD25, anti CD69, anti-IFN-γ, anti-TNF-α and anti-IL-2, and the corresponding fluorochrome-conjugated mouse IgG isotype controls were all purchased from PharMingen (San Diego, California, USA). Anti CD56 was from Immunotech (Marseille, France). Stained cells were analyzed on a Becton Dickinson FACScaliber cytofluorograph (NJ, USA) after gating on viable lymphocyte populations in forward / side scatter dot plots.

Separation of lymphocyte subsets and monocytes

PBMC were isolated from heparinized blood samples by density centrifugation on a Ficoll-Hypaque gradient (Sigma). CD4+, CD8+ and CD 14+ cell populations were isolated using positive immunomagnetic selection on separation columns as specified by the manufacturer (BD Biosciences Pharmingen, CA, USA). The positive fraction routinely contained >90% of the desired cell subset as analyzed by FACS immediately after separation.

Isolation of Lamina propria lymphocytes (LPL)

LPL were obtained from intestinal biopsies of patients undergoing colonoscopy in Sheba Medical Center for Crohn's disease or for unrelated reasons (e.g. cancer screening, irritable bowel syndrome). LPL for experiments performed at Mount Sinai Medical Center were obtained from patients undergoing surgical resection for refractory Crohn's disease, cancer, or other inflammatory disease (e.g. diverticulitis). Briefly, samples were washed twice with 10ml PBS+3mM EDTA+antibiotics (lOOmg/ml Streptomycin and lOOu/ml Penicillin), and then twice with 10ml Hanks' salt solution+lmM EDTA+ antibiotics. Samples were then passed 4 times through an 18G needle and incubated twice at 37c for 30 minutes in RPMI+FCS 20%+300mg/ml collagenase A (Sigma Aldrich) +10mg/ml DNase (Sigma) with antibiotics and 25mg/ml

Amphothericin with lOmmol/1 HEPES buffer. Cells from the supernatant of the digested samples were then collected, filtered and washed. Cells were subsequently subjected to centrifugation through a Percoll gradient (Sigma), and the lymphocytes at the 40/80% interface were collected. The cells obtained were routinely composed of >70% CD3+ T-cells as shown by FACS analysis. In vitro T cell and monocyte activation and cell cultures

Isolated CD4+ or CD8+ T-cells were resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 μg/ml) (Life Technologies, Grand Island, N. Y.)- Cells were placed in 96 well flat bottom tissue culture plates (Corning Inc. Corning, NY) at 3x10⁵ cells/well containing 200Dl of medium. In stimulation experiments, wells were pre-coated with anti-CD3 (OKT3) at 2μg/ml overnight, and 1 μg/ml Anti-CD28 (BD Pharmingen) was added to the cell suspension upon plating. Interleukin (IL)-2 (40 IU/ml) was added on day 3. LPLs were similarly cultured after triggering by 1 μg/ml pre-plated anti-CD2 (BD Pharmingen) with soluble anti-CD28. Isolated CD 14+ cells were resuspended in RPMI 1640 medium supplemented with 10% FBS, 2 mM L- glutamine, penicillin (100 U/ml) and streptomycin (100 μg/ml) (Life Technologies, Grand Island, N.Y.). Cells were plated in 96 well flat bottom tissue culture plates (Corning Inc. Corning, NY), stimulated with Lipopolysaccharide (LPS, Sigma-Aldrich) at 1 μg g/ml and cultured for the designated time-points before harvesting.

Analysis of cell apoptosis by annexin V staining.

Apoptotic cells were detected by staining with anti-annexin V and propidium iodide using the Annexin-V apoptosis detection kit-I (IQ-Products, Groningen, Netherlands). Cells were washed twice in PBS and the pellet was resuspended in annexin V binding buffer (PharMingen) at a concentration of 10⁶ cells /ml. Annexin V FITC and propidium iodide were added (5 μl of each per 10⁵ cells). Samples were gently mixed and incubated for 20 minutes on ice ih the dark before FACS analysis. In some experiments, when apoptosis was determined along with CFSE content, cells were stained with Annexin-V PE and AAD according to the manufacturer's instructions (AnnexinV/AAD kit, BD Pharmingen). Analyzing T cell proliferation using the CFSE dilution assay

The FACS based analysis of T proliferation by serial halving of the fluorescence intensity of the vital dye, CFSE, has been described previously [16]. Briefly, T-cells (10 ⁷ /ml) were suspended in 1 ml PBS and then incubated with 2μM CFSE (Molecular Probes Inc., Eugene, Oregon, USA) for 15 minutes at 37°C with constant shaking. Subsequently, the cells were washed twice with large volumes of RPMI medium. The desired subsets of cells were purified and triggered with polyclonal OKT3 stimulation. The cells were then cultured for the indicated periods, harvested and analyzed for CFSE content and various surface markers. Mice immunization experiments

Balb/C female mice were obtained from Harlan (Rehovot, Israel) and acclimated for one week prior to experimentation. Mice were kept at the animal facility of the Sheba Medical Center. All experiments were approved by the Institutional Ethics Committee at the Sheba Medical Center. Mice were co-immunized subcutaneously withlOOμg HEL emulsified in IFA (both from Sigma), and with lOOμg Ovalbumin (Sigma) with IFA. All mice were immunized twice over 4 weeks with both antigens concurrently, and were then rested for 4 weeks, until week 8. Thereafter, mice were divided into two groups, one treated with daily i.p. 6-MP at 3mg/kg, and one with vehicle alone. Both groups continued to receive bi-weekly i.p immunizations with HEL antigen, while Ovalbumin was no longer administered. Mice were sacrificed after 4 weeks and after 20 weeks of this protocol. The spleens were aseptically extracted, weighed, and splenocytes were isolated and counted. Subsequently, splenocytes were labeled with CFSE as above, and resuspended in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 μg/ml). Cells were placed in 96 well flat bottom tissue culture plates at 5X10⁵ cells/well containing 200μl of medium. Splenocytes were stimulated by HEL (70μM), Ovalbumin (20 μM), medium alone, or PHA (lOμg/ml) as a positive control. IL-2 (40 IU/ml) was added on day 3. Cells were harvested on day 7, and cellular proliferation was determined by FACS analysis of the CFSE content of CD4+ gated cells. Since some dye is lost from the parental generation and some T cells can slowly proliferate in response to soluble factors in the medium (bystander effect), we considered for data analysis antigen-responsive cells to be only those CD4+ T cells that have undergone more than two cellular divisions [16].

Digestion of Infliximab and Western Blotting

The infliximab was dissolved in ddH2O and the salts were washed by dialysis in 2OmM sodium phosphate + 1OmM EDTA pH=7.0. Hydrolysis was done using Immobilized Papain (commercial kit of Pierce), followed by purification by protein A (part of a commercial kit Immunopure - Fab preparation kit of Pierce). The Fab fractions were collected from the first elution fraction and the FC fractions were collected after adding the elution buffer. We used denaturing gel electrophoresis to insure the specificity of Fab fractions by size in the eluents expected to contain the Fab region. Thereafter, the fractions were washed by dialysis with PBS. Fractions expected to contain the Fc region were purified on DEAE-FF columns (HiTrap, Amersham). Infliximab was further hydrolyzed by Papain from a different source (Sigma) and purified using Protein G columns. The above protocols were repeated and followed by ultrafiltration using different cutoffs. The protein fractions obtained were washed by dialysis with PBS and freezes. Specificity of Fc fragment western blotting was verified by gel electrophoresis and size separation of the products.

Statistical analysis. P values were calculated by the paired Student t-test, Wilcoxon rank sum test or the Mann-Whitney test as appropriate, using the MedCalc software (Mariakerke, Belgium). Values <0.05 were considered significant.

Example 2. 6-MP and 6-TG abrogate the proliferation of lymphocytes after stimulation with either anti CD3/CD28 or TSST-I

Although previous studies have demonstrated the anti-proliferative effect of Thiopurines on stimulated T-cells, the precise quantization of this effect in terms of cellular expansion was difficult, because most studies employed DNA incorporation assays [18,19]. Therefore, this set of experiments were designed to gauge the inhibition of T-cell proliferation by these drugs, at the single cell level, using Thiopurine concentrations that have been shown to correspond with intra- leukocyte 6-TG levels during active treatment [6, 20]. Thus, PB CD4+ T-cells from IBD patients and healthy controls were isolated, loaded with CFSE, and triggered by plate-bounded anti-CD3 (OKT3) and soluble anti-CD28 in the absence or the presence of the indicated concentrations of AZA, 6-MP or 6-TG. Cellular divisions were determined on day 5 by FACS measurements of CFSE dilution. As depicted in Figure IA for a specific donor, it was found that addition of AZA, 6-TG and 6-MP at pharmacologically relevant concentrations, significantly abrogated the proliferation of CD4+ T-cells in response to T-cell receptor (TCR) ligation. Across the multiple experiments performed, 6-TG, 6-MP and AZA reduced the proliferation of CD4+ cells by more than 80% (n=21, 51±3.2%, 8±1.9%, 8.6±2% 12±3.8% mean proliferation±SEM, for OKT3 alone, 6-TG, 6-MP or AZA, respectively, P<0.01 for all comparisons, Wilcoxon rank sum test, and see Figure IB). Of note, no statistically significant difference was found between the magnitude of proliferation inhibition by the different drugs in the healthy controls (n=13) versus the Crohn's patients tested (n=8, P=NS for all comparisons, Mann-Whitney test).

A similar inhibition of proliferation after anti CD3/CD28 stimulation was observed for isolated CD8 cells (Figure 1C), and abrogation of CD4+ cellular division was also found after stimulation with the super-antigen TSST-I (data not shown). Example 3. Thiopurines do not induce apoptosis of resting T-cells, and a modest apoptosis of activated T-cell is induced only at day 5 following stimulation

It was recently suggested that in addition to cell cycle arrest, exposure of CD4+ T-cells to 6-MP and 6-TG in vitro may result in significant apoptosis of activated cells [6]. Because it is conceptually hard to reconcile this reported pro-apoptotic effect with the absence of early response to therapy, the apoptotic effect of the Thiopurines on activated T-cells was examiner.

CD4+ cells were isolated from IBD patients and healthy controls as above. Cells were incubated in medium alone as negative control, or stimulated by plated anti-CD3 and CD28, with or without the different Thiopurines at the designated concentrations or with lOμg/ml cyclohexamide as a positive control. Apoptosis on day 5 of CD4+ T-cells was determined by Annexin V/PI staining. As shown on Figures 2A and 2B, it was found that the co-culture of TCR-ligated CD4+ cells with 6-TG, 6-MP or AZA resulted in a modest but statistically significant increase in the percentage of early apoptotic (Annexin+/PI-) cells by day 5 (4.1±2.9%, vs. 7.1±3.3%, 6.8±3.2%, 8.2±4.2%, for the percentage of apoptotic cells after stimulation alone, 6-TG, 6-MP or AZA, respectively, n=18, PO.05 for all comparisons,

Wilcoxon rank sum test). A similar effect was observed with respect to the percentage of the late apoptotic-necrotic (Annexin+/PI+) cells at day 5 [Figure 2a]. No significant difference was found in the mean percentage of apoptotic cells in patients with Crohn's disease (n=7) compared with healthy donors (n=l 1, data not shown). ' Although these results were in agreement with previous observations [6], it was of interest to elucidate the seeming contradiction between the Thiopurines¹ ability to inhibit T-cell proliferation and to induce apoptosis, and their inability to rapidly control the immune-driven inflammatory process in human disorders. Therefore, the kinetics of the pro-apoptoic effect of these drugs on activated lymphocytes, and the fate of these cells during the first days after stimulation was examined

To further elucidate this association between cellular division and induction of apoptosis, the apoptotic fate of individual cells in relation to their proliferative status was determined. CD4+ cells were isolated, labeled with CFSE, and triggered with plate-bound anti-CD3 and soluble CD28, in the absence or the presence of the designated drugs. Apoptosis of CFSE- labeled CD4+ cells was determined on day 5 by AnnexinV PE/ AAD staining. Using this experimental approach, it was found that in the presence of Thiopurines, apoptosis was preferentially induced in CFSE-low cells, i.e. cells that were actively dividing [Figure 2b, left upper quadrants]. In contrast, in the untreated culture only a fraction of the actively dividing cells underwent cell death [Figure 2b, left dot plot - left upper quadrant]. Additionally, whereas apoptosis induction by Thiopurines was tightly coupled to cellular division, the pro-apoptotic effect of cyclohexamide was independent of cellular division [Figure 2b].

Because these data suggested that the modest pro-apoptotic effect of 6-MP and 6-TG was associated with post-stimulation cellular events, it was of interest to examine their effect on unstimulated T-cells. Therefore, apoptosis induction by Thiopurines in CD4+ cells cultured without exogenous stimuli was investigated. It was found, that even at day 5 none of the Thiopurines exerted any apoptotic effect on CD4+ cells cultured in media alone, in contrast to its effect on stimulated cells [Figure 2c]. Taken together, these results suggest that Thiopurines do not induce apoptosis of resting

T-cells, and that apoptosis of activated cells is coupled to cell-cycle.

Example 4. Prolonged administration of 6-MP in vivo, causes shrinkage of the specific CD4+ memory pool to repeatedly encountered antigens, but not to previously encountered antigens. The in vitro findings indicated that whereas Thiopurines cause cell cycle arrest and a modest increase in activation-dependent apoptosis (at day 5), they do not significantly inhibit the acquisition of immediate effector functions by stimulated immunocytes. These findings may provide an explanation for the lack of early anti-inflammatory effect of these drugs, by suggesting that repeated bouts of T-cell activation can result in tissue damage mediated by cycle- arrested yet functional immunocytes. However, it also raises a question as to the mechanism responsible for the eventual late anti-inflammatory effect of Thiopurines. Thus, it was of interest to study this seeming paradox in mice in the context of repeated antigen reencounter, in vivo, over the time-course of treatment with 6-MP. In order to focus on the specific effects of 6-MP on the memory T cell clones re-encountering antigen versus its effects on other memory T cells, a a novel experimental design was employed, as schematically shown in Figure 3A. Specifically,

Balb/C mice were co-immunized twice with HEL and Ovalbumin over a 4 week period, and then rested for additional 4 weeks to allow for the development of a resting antigen-specific memory T-cell population. Mice were then divided into two groups, one treated with daily i.p. 6-MP and the other with vehicle alone. Both groups continued to be challenged by HEL antigen SQ every other week, whereas Ovalbumin was no longer administered. Mice were sacrificed either after 4 weeks or after 20 weeks of this protocol. The magnitude of the splenic CD4+ memory response was determined ex vivo by CFSE-based measurement of the proliferation of CD4+ T splenocytes in response to either antigen. It was found that short-term administration of 6-MP for 4 weeks, had no effect on the memory response to either antigen [Figure 3b]. In contrast, the prolonged administration of 6-MP for 20 weeks, markedly abrogated the memory T cell response to the re- encountered HEL antigen, compared to mice treated with vehicle alone while re-exposed to this antigen [7.6±6.1% responding cells Vs. 23.8±3.2%, P=0.01, Figure 3c]. Importantly, in these same mice, the prolonged administration of 6-MP did not affect the magnitude of the memory T cell response to the previously-primed OVA antigen that was not re-introduced during 6-MP treatment [22±3.2% Vs. 29.9±6.8%, P>0.1 , Figure 3c]. Furthermore, the mean spleen weight of the mice treated with 6-MP for 20 weeks was significantly lower compared to mice treated with vehicle alone. Accordingly, the average total number of splenocytes was proportionally reduced [Figure 3d].

Thus, these in vivo results suggest that repeated antigen encounters over a prolonged course of time are a pre-requisite for specific memory deletion induced in the context of long- term Thiopurine treatment.

Example 5: Identification of the Immunogenic Fraction of Infliximab Infliximab is a chimeric monoclonal anti-TNF alpha antibody which is used or treatment of patients with diverse inflammatory disorders. Its use is limited by the development of anti- Infliximab neutralizing antibodies (ATI) in the recipient patient after several courses of treatment. To identify the immunogenic portion of Infliximab, the Infliximab immunoglobulin was digested into a FAb2 fragment and a Fc fragment. A western blot assay to detect the presence of antibodies in the serum against each of these fragments was performed. It was found that that serum from patients not exposed to Infliximab did not react with any of the fragments, attesting to the specificity of the assay [Figure 4]. In contrast, most serums obtained from patients treated with Infliximab and clinically suspected to have developed ATI, have reacted against the Fab2 fragment but not against the Fc one. These data indicate that the Fab2 is the immunogenic fragment of Infliximab. These findings may pave the way for using only the Fab fragment in combination with cell-cycle drugs such as thiopurines, in order to delete specific memory clones and reduce the production of ATI by patients receiving Infliximab. Moreover, these findings suggest a new assay to assess the development of ATI, which may be more sensitive than the currently available assay.

Other Embodiments

Also within the invention is a method of inhibiting an immune response using a composition that includes a therapeutic dose of both the first agent and a second agent. In this aspect of the invention, either the first therapeutic agent or second therapeutic agent, or both, are preferably administered in different route than typically administered for the first and/or second therapeutic agent.

Claims

What is claimed is:

1. A method of suppressing an immune response to a therapeutic agent, said method comprising administering to a subject in need thereof a combination of a purified first therapeutic agent and a second therapeutic agent, wherein the combined dose of the first and second therapeutic agents suppresses an immune response to said first therapeutic agent in said subject.

2. The method of claim 1, further comprising assessing the immune response to said first therapeutic agent following administering of said combination.

3. The method of claim 1, further comprising repeating administration of sub-therapeutic dose of said first therapeutic agent to said subject.

4. The method of claim 1, further comprising repeating administration of said second therapeutic agent to said subject.

5. The method of claim 4, wherein administration of said second therapeutic agent is continuous for at least one hour.

6. The method of claim 4, wherein administration of said second therapeutic agent is continuous for at least 2, 6, 12, 24, 28, 96 hours, or one week.

7. The method of claim 1, wherein said combination is administered prior to administering a therapeutic dose of said first therapeutic agent to said patient.

8. The method of claim 1, wherein said combination is administered to said subject when the immune response to a therapeutic dose of said first therapeutic agent in said subject inhibits the effectiveness of said first therapeutic agent in said subject or elicits side effects secondary to the first agent in said subject.

9. The method of claim 1 , wherein said second therapeutic agent is administered in an amount sufficient to delete memory immune cells specific for first therapeutic agent, and/or render them tolerant towards said first therapeutic agent

10. The method of claim 1, wherein said second therapeutic agent is administered in an amount sufficient to reduce a hypersensitive immune response to said first therapeutic agent in said subject.

1 1. The method of claim 1, wherein said second therapeutic agent is administered in an amount sufficient to reduce rapid clearance or inactivation of said first therapeutic agent, by circulating antibodies specific for said first therapeutic agent.

12. The method of claim 1, wherein said first therapeutic agent is formulated for administration to a human subject.

13. The method of claim 1, wherein said first therapeutic agent and said second therapeutic agent are administered simultaneously.

14. The method of claim 1, wherein administration of said second therapeutic agent is initiated within 12 months prior to administration of said first therapeutic agent, and continued thereafter.

15. The method of claim 1, wherein said first therapeutic agent is administered in a route selected from the group consisting of oral, nasal, topical, parenteral, subcutaneous, intramuscular, intravenous, rectal, buccal, and transdermal administration.

16. The method of claim 1, wherein said second therapeutic agent is administered in a route selected from the group consisting of oral, nasal, topical, parenteral, subcutaneous, intramuscular, intravenous, rectal and transdermal administration.

17. The method of claim 1 , wherein said first therapeutic agent is a polypeptide or protein, glycoprotein, lipoprotein, glycopeptides, lipopeptide, or an antisense oligonucleotide.

18. The method of claim 17, wherein said polypeptide is a hormone, cytokine, naturally occurring anti-cytokine (IL-IRA), a clotting factor, endothelial cell growth factor, or coagulation factor.

19. The method of claim 18, wherein said polypeptide is insulin, human growth hormone, EPO, G-CSF, CG-CSF, GM-CSF, α-interferon, β-interferon, IL-1 1, IL- 12 , factor VIII, factor VII, or factor IX.

20. The method of claim 17, wherein said polypeptide is an antibody or a fragment of an antibody.

21. The method of claim 20, wherein said antibody is a monoclonal antibody.

22. The method of claim 21, wherein said monoclonal antibody is a chimeric antibody.

23. The method of claim 20, wherein said antibody is an anti-cytokine antibody, anti- cytokine receptor antibody, anti-chemokine antibody, or an anti-chemokine receptor antibody.

24. The method of claim 20, wherein said antibody is an anti-TNFα antibody, an anti-

EGF receptor antibody, an anti-IL-2 receptor antibody, an anti-HER2 antibody, anti-VEGF antibody, anti-CD20 antibody, anti-IL-12 antibody, anti-IL-23, anti-INF-gamma, anti-CTLA-4, anti-VLAl, anti-VLA-4, anti alpha4beta7 integrin, anti IL-12 P40, anti CD 25, anti IL-6 receptor, anti IL-4, or anti IL-5 antibody.

25. The method of claim 17, wherein said polypeptide comprises an extracellular portion of a cytokine receptor.

26. The method of claim 25, wherein said extracellular portion of said cytokine receptor is provided as a fusion protein.

27. The method of claim 25, wherein said cytokine receptor is a receptor for TNFα.

28. The method of claim 1 , wherein said first therapeutic agent is a fragment of a polypeptide.

_; 29. The method of claim 28, wherein said fragment is a segment of a naturally-occurring human or non-human protein and which binds to a human major histocompatibility complex (MHC) class I or II allotype.

30. The method of claim 1, wherein said first therapeutic agent is administered with an adjuvant.

31. The method of claim 30, wherein said first therapeutic agent is linked to said adjuvant.

32. The method of claim 1, wherein said subtherapeutic dose of said first therapeutic agent corresponds to about 1 pg/kg to about 50 mg/kg body weight of said subject.

33. The method of claim 1, wherein said subtherapeutic dose of said first therapeutic agent corresponds to about 1 ng/kg to about 500 μg/kg body weight of said subject.

34. The method of claim 1, wherein said subtherapeutic dose of said first therapeutic agent corresponds to about 1 ng/kg to about 50 μg/kg body weight of said subject.

35. The method of claim 1, wherein said subtherapeutic dose of said first therapeutic agent corresponds to about 10 ng/kg to about 5 μg/kg body weight of said subject.

36. The method of claim 1, wherein said subtherapeutic dose of said first therapeutic agent corresponds to about 500 ng/kg to about 1 μg/kg body weight of said subject.

37. The method of claim 1, wherein said subtherapeutic dose of said first therapeutic agent corresponds to about 1 ng/kg to about 500 μg/kg body weight of said subject.

38. The method of claim 1, wherein said subject is human.

39. The method of claim 1, wherein said second therapeutic agent is a cell cycle inhibitor.

40. The method of claim 39, wherein said cell cycle inhibitor is a folate antagonist

41. The method of claim 40, wherein said folate antagonist is methotrexate.

42. The method of claim 39, wherein said cell cycle inhibitor is 5-fluorouraccil.

43. The method of claim 39, wherein said cell cycle inhibitor inhibits purine biosynthesis.

44. The method of claim 1, wherein said second therapeutic agent is methotrexate, 6- mercaptopurine, azathiopurine, thioguanosine, mycophenolic acid, rapamycin, cytosine arabinoside, 5-fluorouracil (5-FU), a 5-FU prodrug, ftorafur, 5'-deoxyfluorouridine, carmofur, fluorouridine, 2'-deoxyfluorouridine, a prodrug derivative of fluorouridine or T- deoxyfluorouridine, fluorocytosine, 5'fluorouracil, fluorodeoxyuridine, arabinosyl cytosine, a prodrug of arabinosyl cytosine, cyclocytidine, 5-aza-2'-deoxycytidine, arabinosyl 5-azacytosine, N-phosphonoacetyl-L-aspartic acid (PALA), pyrazofurin, 6-azauridine, azaribine, 6-azacytidine, trifluoro-methyl-2'-deoxyuridine, thymidine, 3-deazauridine, a vinca alkaloid, a nitrogen mustard alykylating agent, cyclophosphamide, daunorubicin, doxorubicin, a podophyllotoxin, cisplatin, or taxol.

45. The method of claim 1, further comprising administering a third therapeutic agent.

46. The method of claim 45, wherein said third therapeutic agent inhibits B cell growth.

47. The method of claim 45, wherein said third therapeutic agent inhibits T cell growth.

48. The method of claim 45, wherein said third therapeutic agent is an IL-2 inhibitor.

49. The method of claim 45, wherein said third therapeutic agent is an antihistamine.

50. The method of claim 45, wherein said third therapeutic agent is a corticosteroid.

51. A method of suppressing an autoimmune response in a subject, said method comprising administering to a subject in need thereof a combination of a sub-therapeutic dose of an autoantigen or a fragment of said autoantigen, or a mixture of auto-antingens, and a second therapeutic agent, wherein the combined dose of the first and second therapeutic agents suppresses an autoimmune response in said subject.

52. The method of claim 41, wherein said autoimmune response is associated with multiple sclerosis, asthma, diabetes, rheumatoid arthritis, systemic lupus erythematosus, polymyositis, dermatomyositis, autoimmune hepatitis, auto-immune thyroiditis, celiac disease, inflammatory bowel disease, psoriasis, Lichen planus, Pemphigus, auto-immune thrombocytopenia, sepsis, rheumatic disease, Sjogrens syndrome, or a transplantation-related immune response.

53. The method of claim 52, wherein the transplantation-related immune response is graft- versus-host-disease or post-transfusion thrombocytopenia.

54. The method of claim 41, wherein said autoimmune response is associated with multiple sclerosis.

55. The method of claim 42, wherein said autoantigen is myelin basic protein (MBP) or myelin oligodendrocyte glycoprotein (MOG).

56. The method of claim 41, wherein said autoimmune response is associated with rheumatoid arthritis.

57. The method of claim 56, wherein said autoimmune antigen is collagen type II, human chondrocyte glycoprotein 39 and proteoglycans, citrullinated filaggrin, glucose-6- phosphate isomerase, p205, or a heat shock protein present in elevated levels in patients with rheumatoid arthritis.

58. A method of increasing the effectiveness of a therapeutic agent in a subject, the method comprising administering a sub-therapeutic dose of a combination of a first therapeutic agent along with a second therapeutic agent, wherein the combined dose of the first and second therapeutic agents suppresses an immune response to said first therapeutic agent in said subject., thereby increasing the effectiveness of said first therapeutic agent in said subject.

59. A pharmaceutical composition comprising a first therapeutic agent; a sub-therapeutic dose of a second therapeutic agent or fragment of said second therapeutic agent, and a pharmaceutically acceptable carrier; wherein the combined dose of the first and second therapeutic agents suppresses an immune response in a subject.

60. The pharmaceutical composition of claim 59, wherein said first therapeutic agent or second therapeutic agent or fragment thereof, is formulated for delivery to a human subject.

61. A kit comprising a sub-therapeutic dose of a first therapeutic agent; and a second therapeutic agent or fragment of said second therapeutic agent, wherein the combined dose of the first and second therapeutic agents suppresses an immune response in a subject.

62. The kit of claim 61, further comprising a pharmaceutically acceptable carrier;

63. A method of identifying a treatment regimen for suppressing an immune response, said method comprising administering to a test subject a candidate sub-therapeutic dose of a first therapeutic agent and a candidate dose of a second therapeutic agent; and detecting the immune response to said candidate dose in said subject, and comparing the immune response to said candidate dose to the immune response to said first therapeutic ageing in a control subject not administered said candidate dose of said first therapeutic agent and second therapeutic agent, wherein a decreased immune response to said candidate dose in said test subject relative to said control subject indicates said candidate dose is a therapeutic regimen for suppressing an immune response.

64. A method of detecting a humoral immune response to an therapeutic antibody in a subject comprising contacting a subject sample know to or suspected of containing an antibody specific for said therapeutic antibody with a fragment of said therapeutic antibody under conditions permitting said antibody to specifically bind said fragment to form an antibody- fragment complex; and detecting the antibody- fragment complex, wherein the presence of the antibody- fragment complex indicates the presence a humoral immune response to said therapeutic antibody in said subject .

65. The method of claim 64, wherein said fragment is a Fab fragment, a Fab₂ fragment, a Fv fragment, a Fc fragment or fragment thereof.

66. The method of claim 64, wherein said therapeutic antibody is an anti-TNF antibody.

67. The method of claim 65, wherein said anti-TNF antibody is Infliximab.

68. The method of claim 64, wherein said fragment is immobilized on a solid phase.