/N VITRO CULTURE SYSTEM TO EVALUATE SYNERGY IN TARGETING IMMUNE SUPPRESSIVE PATHWAYS CONCURRENT TO IMMUNOTHERAPY

Reference to Related Applications

This application claims the benefit of the filing date of the U.S. provisional application No. 60/897,155, filed on January 23, 2007, the entire content of the application is incorporated herein by reference.

The invention described herein also expressly incorporates the entire content of U.S.S.N. 10/831 ,886, filed on April 26, 2004 by Schultes et al, titled "Combination therapy for treating disease" (now published as US-2005-0063976-A1 on March 24, 2005). U.S.S.N. 10/831,886 is a continuation-in-part of U.S. application Ser. No. 09/871 ,339, filed on May 31, 2001, which is a continuation of U.S. application Ser. No. 08/913,290, filed on March 20, 1998, now U.S. Pat. No. 6,241,985, which is a U.S. national stage application filed under 35 U.S.C. 371 based on PCT application number PCT/IB96/00461, on May 15, 1996; and is a continuation-in-part of PCT application number PCT/IB02/05794, filed on Oct. 28, 2002, which claims priority to U.S. provisional application No. 60/339,240, filed on Oct. 26, 2001, each of the referred-to applications is hereby incorporated in its entirety by reference. PCT applications PCT/IB96/00461 and PCT/IB02/05794 were filed and published in English.

Background of the Invention

Despite the progress that modern medicine has made in treating cancer, cancer recurrence remains a concern. For a majority of cancers, typical treatment includes surgery followed by high doses of chemotherapy. A majority of these patients relapse and do not respond to other chemotherapeutic treatments. These patients then avail themselves to experimental or salvage treatments.

Current experimental regimens focus on mixing chemotherapies in an attempt to overcome resistance issues. Most of these treatments result in serious blood toxicities such as neutropenia, and thrombocytopenia. Other serious and frustrating symptoms to the patient include hair loss and nausea.

Although many have turned to the use of chemotherapy in conjunction with antibody

treatments, many of these have also presented similar toxicities to the chemotherapy.

Another problem is that tumor-induced immune suppression is an obstacle to cancer immunotherapy, and it involves many different pathways exploited by the tumor.

Thus, there remains a need to identify new treatments that not only treat the initial symptoms of a disease, but also alleviate and/or prevent recurrence of those symptoms.

Summary of the Invention

The invention relates to immunology. More particularly, the invention relates to the use of agents targeting / antagonizing immune suppressive pathways concurrent to immunotherapy, or concurrent to immunotherapy in combination with chemotherapy. The agents block one or more dominant suppressive pathways to improve outcomes of tumor immunotherapi es .

One aspect of the invention provides a method for treating cancer, comprising concurrently administering a binding agent, such as a monoclonal antibody (e.g., a xenotypic monoclonal antibody), a chemotherapeutic drug, and an agent that antagonizes one or more immune suppressive pathways to a patient suffering from cancer.

Another aspect of the invention provides a method for treating cancer, comprising concurrently administering a binding agent, such as a monoclonal antibody (e.g., a xenotypic monoclonal antibody), and an agent that antagonizes one or more immune suppressive pathways to a patient suffering from cancer.

In certain embodiments, the binding agent is a monoclonal antibody, such as a xenotypic monoclonal antibody. In certain embodiments, the xenotypic monoclonal antibody is murine. Exemplary xenotypic monoclonal antibody is AIt-I , Alt-2, Alt-3, Alt-4, Alt-5, or Alt-6.

In certain embodiments, the patient is a human.

In certain embodiments, the agent comprises a cytokine.

In certain embodiments, the agent comprises IL-15, TNF-alpha, FLT-3L, IL- 12, IL- 21 , IL-23, or combination thereof.

In certain embodiments, the agent comprises an antibody.

In certain embodiments, the antibody blocks an immune regulatory mechanism (e.g., blocks one or more inhibitory receptors), or inhibits, inactivates, or removes T _{1 eg} .

In certain embodiments, the antibody is effective at increasing T-cell reactivity particularly to the tumor.

In certain embodiments, the antibody comprises an anti-CD25 antibody, an anti-GITR antibody, an anti-B7-Hl antibody, an anti-TGF-β antibody, or a combination thereof.

In certain embodiments, the agent comprises an inhibitor of IDO or an Arginase inhibitor.

In certain embodiments, the agent comprises NOHA or 1 -MT.

In certain embodiments, the agent comprises a cytokine, an antibody, an inhibitor of IDO or an Arginase inhibitor, or a combination thereof.

In certain embodiments, the agent comprises one or more of IL- 15, TNF-alpha, FLT- 3L, IL-12, IL-21, IL-23, an anti-CD25 antibody, an anti-GITR antibody, an anti-B7-Hl antibody, an anti-TGF-β antibody, NOHA or 1-MT, or a combination thereof; wherein the chemotherapeutic drug is Gemcitabine or Topotecan; and wherein the xenotypic monoclonal antibody is an anti-CA 125 antibody (such as OVAREX ^® MAb or MAb B43.13) or an anti- MUC-I antibody (such as BREV AREX ^® MAb or MAb-AR20.5).

In certain embodiments, the chemotherapeutic drug is administered within a week before the monoclonal antibody. In certain embodiments, the chemotherapeutic drug is administered within a week after the monoclonal antibody. In certain embodiments, the chemotherapeutic drug is administered simultaneously (e.g., concurrently or at about the same time or within the same day) with the monoclonal antibody.

In certain embodiments, the monoclonal antibody is administered in a dose of less than or equal to 10 mg, 5 mg, 2 mg, 1 mg, 0.5 mg.

In certain embodiments, the monoclonal antibody is administered in a dose of about 0.1 μg to 2 mg per kg of body weight, or about about 0.2 μg to 1 mg per kg of body weight, or about 0.5 μg to 500 μg per kg of body weight, or about 1 μg to 250 μg per kg of body

weight, or about 2 μg to 100 μg per kg of body weight, or about 5 μg to 50 μg per kg of body weight, etc.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being highly toxic to the patient.

In certain embodiments, the method further comprises surgical removal of the cancer. In certain embodiments, the cancer is surgically removed concurrent with the administration of the chemotherapeutic drug and the monoclonal antibody in a dose (1) equal to or less than 10 mg, 5 mg, 2 mg, 1 mg, 0.5 mg; or (2) about 0.1 μg to 2 mg per kg of body weight, or about about 0.2 μg to 1 mg per kg of body weight, or about 0.5 μg to 500 μg per kg of body weight, or about 1 μg to 250 μg per kg of body weight, or about 2 μg to 100 μg per kg of body weight, or about 5 μg to 50 μg per kg of body weight, etc.

In certain embodiments, the monoclonal Ab is Alt- 1 , Alt-2, Alt-3, Alt-4, Alt-5, or AIt- 6.

In certain embodiments, administration of the monoclonal antibody comprises a 20 minute intravenous infusion.

In certain embodiments, the chemotherapeutic drug is administered within seven days prior to the administration of the monoclonal antibody.

In certain embodiments, the chemotherapeutic drug is administered within seven days following the administration of the monoclonal antibody.

In certain embodiments, the chemotherapeutic drug is administered every four weeks for six cycles.

In certain embodiments, the method further comprises the step of administering the monoclonal antibody every twelve weeks for up to two years.

In certain embodiments, the monoclonal antibody and chemotherapeutic drug are administered at weeks 1, 4, and 8, followed by further administration of the chemotherapeutic drug alone at weeks 12 and 16, followed by concurrent administration of the

chemotherapeutic drug and xenotypic monoclonal antibody at week 20.

In certain embodiments, the concurrent administration of the monoclonal antibody and the chemotherapeutic drug occurs at week 1, followed by administration of the chemotherapeutic drug at week 4, wherein the concurrent administration is repeated for six cycles and followed by administration of the monoclonal antibody every twelve weeks for up to two years.

In certain embodiments, the monoclonal antibody is administered at weeks 1, 3, 5, 7 and 9, followed by concurrent administration of the chemotherapeutic drug and the monoclonal antibody in a dose less than or equal to 2 mg at week 12. The dosage level may be varied asindicated herein above, or be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being highly toxic to the patient.

In certain embodiments, the concurrent administration of the chemotherapeutic drug and monoclonal antibody is repeated every four weeks for up to 6 cycles.

In certain embodiments, the method further comprises administering the monoclonal antibody every twelve weeks for up to two years.

In certain embodiments, the method comprises administering the monoclonal antibody and the chemotherapeutic agents according to the schemes shown in Figure 13 or described in Example IV.

In certain embodiments, the method comprises administering the chemotherapeutic drug at Weeks 0, 4, 8, 12, 16, and 20 (Cycles 1-6, respectively), and administering the monoclonal antibody either concurrently with, or about one week after administering the chemotherapeutic agent (e.g., carboplatin/paclitaxel for ovarian cancer), at Day 1 of Week 0 (Cycle 1), Week 8 (cycle 3), and Week 16 (cycle 5), (about 4 weeks between cycles), followed by administering the monoclonal antibody every twelve weeks for up to two years or until disease progression.

The method of claim 1, 2, or 20, wherein the concurrent administration of the monoclonal antibody and the chemotherapeutic drug occurs at Day 1 , followed by administration of the chemotherapeutic drug at Weeks 4, 8, 12, 16, and 20, and wherein the

concurrent administration of the monoclonal antibody is repeated at Weeks 8 and 16 followed by administration of the monoclonal antibody every twelve weeks for up to two years.

Another aspect of the invention provides a method for inducing a host immune response in a patient against a multi-epitopic in vivo tumor antigen, which antigen does not elicit an effective host immune response, comprising concurrently administering to the patient: (1) an agent that antagonizes one or more immune suppressive pathways to a patient suffering from cancer; (2) a chemotherapeutic drug; and, (3) a composition comprising a binding agent that specifically binds to a first epitope on the antigen and allowing the binding agent to form a binding agent/antigen pair, wherein a host immune response is elicited against a second epitope on the antigen.

In certain embodiments, the binding agent is a murine monoclonal antibody.

In certain embodiments, the murine MAb is AIt-I, Alt-2, Alt-3, Alt-4, Alt-5, or Alt-6.

In certain embodiments, the patient is human.

In certain embodiments, the chemotherapeutic drug is administered within a week before the binding agent. In certain embodiments, the chemotherapeutic drug is administered within a week after the binding agent. In certain embodiments, the chemotherapeutic drug is administered simultaneously {e.g., concurrently or at about the same time or within the same day) with the binding agent.

In certain embodiments, the binding agent is administered in a dose (1) equal to or less than 10 mg, 5 mg, 2 mg, 1 mg, 0.5 mg; or (2) about 0.1 μg to 2 mg per kg of body weight, or about about 0.2 μg to 1 mg per kg of body weight, or about 0.5 μg to 500 μg per kg of body weight, or about 1 μg to 250 μg per kg of body weight, or about 2 μg to 100 μg per kg of body weight, or about 5 μg to 50 μg per kg of body weight, etc.

In certain embodiments, the method further comprises surgical removal of the cancer.

Another aspect of the invention provides a method for treating cancer, comprising concurrent administration to a patient suffering from cancer a chemotherapeutic drug, a binding agent, an antigen, and an agent that antagonizes one or more immune suppressive pathways.

In certain embodiments, the binding agent is a murine monoclonal antibody, such as AIt-I, Alt-2, Alt-3, Alt-4, Alt-5, or Alt-6.

In certain embodiments, the patient is human.

In certain embodiments, the chemotherapeutic is administered within a week before the murine monoclonal antibody. In certain embodiments, the chemotherapeutic is administered within a week after the murine monoclonal antibody. In certain embodiments, the chemotherapeutic is administered simultaneously (e.g., concurrently or at about the same time or within the same day) with the murine monoclonal antibody.

In certain embodiments, the murine monoclonal antibody is administered in a dose (1) equal to or less than 10 mg, 5 mg, 2 mg, 1 mg, 0.5 mg; or (2) about 0.1 μg to 2 mg per kg of body weight, or about about 0.2 μg to 1 mg per kg of body weight, or about 0.5 μg to 500 μg per kg of body weight, or about 1 μg to 250 μg per kg of body weight, or about 2 μg to 100 μg per kg of body weight, or about 5 μg to 50 μg per kg of body weight, etc.

Another aspect of the invention provides a method for inducing a host immune response in a patient against a multi-epitopic in vivo tumor antigen, which antigen does not elicit an effective host immune response, comprising concurrently administering to the patient: (1) an agent that antagonizes one or more immune suppressive pathways; (2) a chemotherapeutic drug; (3) a composition comprising a binding agent present in an amount of from 0.1 μg to 2 mg per kg of body weight of the host, and wherein the binding agent specifically binds to an epitope on the antigen and an effective host immune response is elicited against a second epitope on the antigen.

Another aspect of the invention provides a method for treating cancer, comprising administering a xenotypic antibody, a chemotherapeutic drug, and an agent that antagonizes one or more immune suppressive pathways to a patient suffering from cancer.

Another aspect of the invention provides a therapeutic composition comprising: (a) at least one monoclonal antibody, each specific for an antigen associated with a disease; and, (b) at least one antagonist of an inhibitory component of the immune system, such as an agent that antagonizes one or more immune suppressive pathways, reduces immune suppression, or inhibits or removes T _reg .

In certain embodiments, the therapeutic composition further comprises a chemotherapeutic drug.

In certain embodiments, the antagonist is an antibody. In certain embodiments, the antagonist is a cytokine.

In certain embodiments, the antagonist is selected from the group consisting of: IL- 15, TNF-alpha, FLT-3L, IL- 12, IL-21 , IL-23, an anti-CD25 antibody, an anti-GITR antibody, an anti-B7-Hl antibody, an anti-TGF-β antibody, NOHA or 1-MT, and a combination thereof.

In certain embodiments, at least one of said monoclonal antibody is specific for a cancer antigen (e.g., specifically binding to an epitope on a cancer antigen and an effective host immune response is elicited against a second epitope on the antigen).

In certain embodiments, the antigens are associated with the same disease.

In certain embodiments, at least one of said monoclonal antibody is an anti-CA 125 antibody, an anti-MUC-1 antibody, an anti-PSA antibody, an anti-CA 19.9 antibody, or a combination thereof.

In certain embodiments, at least one of said monoclonal antibody is selected from the group consisting of: AIt-I, Alt-2, Alt-3, Alt-4, Alt-5, and Alt-6.

In certain embodiments, the antigens are cancer antigens.

Another aspect of the invention provides a kit, comprising: (a) at least one monoclonal antibody each specific for an antigen associated with a disease; and, (b) at least one antagonist of an inhibitory component of the immune system, such as an agent that antagonizes one or more immune suppressive pathways, reduces immune suppression, or inhibits or removes T _reg .

In certain embodiments, the kit further comprises a chemotherapeutic drug.

It is contemplated that any one embodiment of the invention can be combined with any other embodiments where appropriate, unless explicitly indicated to the contrary.

Brief Description of the Drawings

Figure 1 is a diagram showing a non-limiting embodiment of the invention. Patients can continue chemotherapy up to 6 cycles and OVAREX ^® MAb up to 2 years. Endpoints: Time to progression, QOL, Safety, Survival.

Figure 2 is a graph showing the difference in the numbers between Ab2 responders (open squares, effective immune response) and Ab2 non-responders (black squares, ineffective immune response) over time in ovarian cancer patients.

Figure 3 is a schematic representation of an exemplary experimental set up for the experiment described in Example II.

Figures 4A-4D show the cytokine results described in Example II.

Figures 5A-5D show the antibody results described in Example II.

Figures 6A-6D show the IDO/Arginase inhibitor results described in Example II.

Figure 7 shows the result of a successful combinations therapy in Example II.

Figure 8 shows that IC could induce CD4 ⁺ T helper cells, and CD8 ⁺ IFN-γ producing CTL cells, whereas PSA alone or PSA in complex with a non-specific antibody mainly stimulated CD4 ⁺ T cells, using human immune cells.

Figure 9A shows that immune complexes of PSA and MAb-AR47.47 induce CTL specific for PSA in PSA-transgenic mice. Figure 9B shows that immune complexes of PSA and MAb-AR47.47 also induce ThI and Th2 helper responses to PSA (based on cytokine ELISA assay and ICC assay) in PSA-transgenic mice.

Figures 1OA and 1OB show that cross-linking MAb-AR47.47 increased humoral but not cellular responses.

Figures 1 IA &1 IB show that increasing the immunogenicity of MAb-AR47.47 enhanced T helper but not CTL responses. Figures 1 1 C & 1 1 D show that increasing the immunogenicity of the anti-PSA antibody in form of a goat antibody in mice enhanced T helper 2 but not T helper 1 or CTL responses.

Figures 12A and 12B show that MAb-AR47.47 is able to control tumor growth, and

that MAb-AR47.47 in combination with cyclophosphamide is able to reject tumors more so than AR47.47 or cyclophosphamide alone.

Figure 13 shows the study design for OVA-Gy-18 described in Example IV. Figure 14 shows certain results of the experiment described in Example IV.

Specifically, assessment of baseline plasma cytokines for VEGF, IFN-γ, TNF-α, IL- 2R, IL-IO and IL- 15 was carried out using SEARCHLLIGHT™ technology conducted by Pierce Biotechnology ThermoFisher Scientific. SIM=simultaneous infusion arm; OWD=one week delayed arm.

In ELISPOT analysis of the immune response to CA 125, autologous dendritic cells (DCs) were pulsed with CA 125 4 hours prior to maturation. Pulsed and non-pulsed DC were incubated with PBMCs from each time point in triplicate on anti-IFN-γ coated ELISPOT plates, which were stained after 18 hrs. The pre-immunization and maximum response measured post immunization are displayed. The ELISPOT assay was conducted at SBI.

Absolute lymphocytes were measured at the clinical laboratories. T regulatory cells and TCR zeta T cells in peripheral blood were characterized by flow cytometry using phycoerythrin-labeled anti-human CD25 and phycoerythrin-cyanine 5-labeled CD4, both purchased from Beckman Coulter (Fullerton, CA) and fluorescein isothiocyanate-labeled FoxP3 (eBiosciences, San Diego, CA). TCR zeta chain was characterized using a PE-anti- TCR zeta chain antibody from Beckman Coulter. The flow cytometry analysis was conducted at Strategic Business Initiatives (SBI), Pittsburgh, PA. C=chemotherapy cycle.

Figure 15 shows certain results of the experiment described in Example IV.

Specifically, clinical responses in the form of PFS outcomes are shown in panels A - C. (A) Kaplan-Meier graph of PFS, ITT Population. Twelve month PFS estimate from Kaplan-Meier analysis. Kaplan-Meier graph of (B) PFS and (C) overall survival dichotomized by treatment emergent CA 125 specific T-cell response (responder or non- responder).

Safety data are shown in the two tables.

Detailed Description of the Invention

The invention relates to immunology. More particularly the invention relates to the use of immunotherapy in combination with chemotherapy.

The present invention partly stems from the discovery that a combination of immunotherapy with traditional chemotherapy and/or radiotherapy alleviates and/or prevents the recurrence of cancer, or improves the treatment of cancer. The presence of a host anti- xenotypic antibody response in a patient will stimulate an immune response, and the administration of an agent that antagonizes one or more immune suppressive pathways promotes host immune response. The inventors have exploited this discovery to develop therapeutics containing binding agents and agents that antagonize one or more immune suppressive pathways useful in immunotherapy, and chemotherapeutic or radiotherapeutic drugs, as well as methods for using these therapeutics. The patents and publications cited herein reflect the level of skill in this field and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference.

Accordingly in one embodiment, the invention provides a method for treating cancer, comprising concurrently administering a binding agent, such as a monoclonal antibody (e.g., a xenotypic monoclonal antibody), a chemotherapeutic drug, and an agent that antagonizes one or more immune suppressive pathways to a patient suffering from cancer.

In a related embodiment, the invention relates to a method for treating cancer, comprising concurrently administering a binding agent, such as a monoclonal antibody (e.g., a xenotypic monoclonal antibody), and an agent that antagonizes one or more immune suppressive pathways to a patient suffering from cancer.

There are many agents that antagonize one or more immune suppressive pathways in cancer patients, such as those described herein below.

In certain embodiments, such agent may comprise a cytokine. For example, the cytokine can be IL-15, TNF-alpha, FLT-3L, IL-12, IL-21 , IL-23, or combination thereof.

In certain embodiments, the agent comprises an antibody. For example, the antibody blocks an immune regulatory mechanism, e.g., blocks one or more inhibitory receptors, or

inhibits, inactivates, or removes T _reg (regulatory T cells).

In certain embodiments, the antibody is effective at increasing T-cell reactivity particularly to the tumor. Exemplary such antibodies include an anti-CD25 antibody, an anti- GITR antibody, an anti-B7-Hl antibody, an anti-TGF-β antibody, or a combination thereof.

In certain embodiments, the agent comprises an inhibitor of IDO or an Arginase inhibitor. For example, the agent may comprise NOHA or 1-MT.

In certain embodiments, the agent comprises a cytokine, an antibody, an inhibitor of IDO or an Arginase inhibitor, or a combination thereof.

In certain preferred embodiments, the agent comprises one or more of IL- 15, TNF- alpha, FLT-3L, IL-12, IL-21, IL-23, an anti-CD25 antibody, an anti-GITR antibody, an anti- B7-H1 antibody, an anti-TGF-β antibody, NOHA or 1-MT, or a combination thereof; wherein the chemotherapeutic drug is gemcitabine or topotecan; and wherein the xenotypic monoclonal antibody is an anti-CA 125 antibody (such as OVAREX ^® MAb or MAb B43.13) or an anti-MUC-1 antibody (such as BREV AREX ^® MAb or MAb-AR20.5).

Numerous binding agents are within the scope of the invention.

For example, in some embodiments, the binding agent is an antibody, such as a monoclonal antibody. The monoclonal antibody may be a xenotypic monoclonal antibody, such as a murine monoclonal antibody when, for example, the patient is a human.

In some embodiments, the binding by the (xenotypic) monoclonal antibody of a first epitope exposes a second distinct epitope on the antigen or enables an immune response to such second distinct epitope.

In some embodiments of the invention, the (xenotypic) monoclonal antibody, when bound to the antigen, forms an immunogenic complex. Exemplary (xenotypic) monoclonal antibodies ("MAb"), preferably include IgGl antibodies; chimeric monoclonal antibodies ("C-MAb"); humanized antibodies; genetically engineered monoclonal antibodies ("G- MAb"); fragments of monoclonal antibodies (including but not limited to "F(ab) ₂ ", "F(ab)" and "Dab"); and single chains representing the reactive portion of monoclonal antibodies ("SC-MAb"). The binding agent may be labeled or unlabeled.

Where the patient is human, preferred xenotypic monoclonal antibodies include, without limitation, murine monoclonal antibodies. Particularly preferred murine monoclonal antibodies (preferably murine IgGl monoclonal antibodies) include those with the same binding specificity, or those binding to the same epitope as that bound by: AIt-I (murine IgGl , specifically binds to MUC-I ; ATCC No. PTA-975; American Type Culture Collection, Manassas, Va.), Alt-2 (OVAREX ^® MAb B43.13, murine IgGl, specifically binds to CA 125; ATCC No. PTA-1883), Alt3 (murine IgG3, specifically binds to CA 19.9; ATCC No. PTA- 2691), Alt-4 (murine IgM, specifically binds to CA 19.9; ATCC No. PTA-2692), Alt-5 (murine IgGl, specifically binds to CA 19.9; ATCC No. PTA-2690); or Alt-6 (murine IgGl, specifically binds to prostate specific antigen (PSA); ATCC No. HBl 2526).

In certain embodiments of the invention, the chemotherapeutic drug used is commercially available. Merely to illustrate, the chemotherapeutic can be an inhibitor of chromatin function, a topoisomerase inhibitor, a microtubule inhibiting drug, a DNA damaging agent, an antimetabolite (such as folate antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), a DNA synthesis inhibitor, a DNA interactive agent (such as an intercalating agent), and/or a DNA repair inhibitor. Some non limiting examples include carboplatin, cisplatin, docetaxel, paclitaxel, doxorubicin, HCl liposome injection, topotecan, hydrochloride, gemcitabine, cyclophosphamide, and etoposide or any combination thereof.

In certain embodiments, preferred chemotherapeutic drug is gemcitabine, topotecan and cyclophosphamide.

Chemotherapeutic agents may be categorized by their mechanism of action into, for example, the following groups: anti-metabolites/anti-cancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan,

camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, Cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP 16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L- asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); antiproliferative/antimitot- ic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab, rituximab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-1 1) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers, toxins such as Cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, or diphtheria toxin, and

caspase activators; and chromatin disruptors. Preferred dosages of the chemotherapeutic agents are consistent with currently prescribed dosages.

Various administration schemes may be used for the agents, antibodies, and/or chemotherapeutic drugs of the subject invention.

In certain preferred embodiments, the chemotherapeutic drug is administered within a week before or after the murine monoclonal antibody (as binding agent).

The methods according to the invention are useful for providing a therapeutic benefit to patients suffering from cancer. As used herein, the term "cancer" is used to mean a condition in which a cell in a patient's body undergoes abnormal, uncontrolled proliferation. The abnormal cell may proliferate to form a solid tumor, or may proliferate to form a multitude of cells (e.g., leukemia). Note that because cancer is the abnormal, uncontrolled proliferation of a patient's cell, the term does not encompass the normal proliferation of a cell, such as a stem cell other than a cancer stem cell or a spermatocyte.

By "treating a patient suffering from cancer" is meant that the patient's symptoms are alleviated following treatment according to the invention. In one non-limiting example, a patient suffering from a highly metastatic cancer (e.g., breast cancer) is treated where additional metastasis either do not occur, or are reduced in number as compared to a patient who does not receive treatment. In another non-limiting example, a patient is treated where the patient's solid cancer either becomes reduced in size or does not increase in size as compared to a patient who does not receive treatment. In yet another non-limiting example, the number of cancer cells (e.g., leukemia cells) in a treated patient either does not increase or is reduced as compared to the number of cancer cells in a patient who does not receive treatment. In preferred embodiments the patient is human.

It will be appreciated that a "patient suffering from cancer" of the invention may express the mutant protein and not yet be symptomatic for the disease. For example, where the cancer is colon cancer (which is associated with the mutant K-ras protein), a patient with a mutant K-ras protein in some cells of the colon is a patient according to the invention even though that patient may not yet be symptomatic for colon cancer. "Associated with a mutant protein" means signs or symptoms of illness in a majority of patients are present when the mutant protein is present in the patient's body, but in which signs or symptoms of illness are

absent when the mutant protein is absent from the patient's body. "Signs or symptoms of illness" are clinically recognized manifestations or indications of disease.

In one embodiment of the present invention, the patient in need of treatment is suffering from cancer of the prostate, ovaries, breast, stomach, lung, colon, and skin. In a preferred embodiment, the patient in need of treatment is a human.

Preferably, the therapeutic compositions of the invention further comprise a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant a carrier that is physiologically acceptable to the administered patient. One exemplary pharmaceutically acceptable carrier is physiological saline. Other pharmaceutically- acceptable carriers and their formulations are well-known and generally described in, for example, Remington's Pharmaceutical Sciences (18th Ed., ed. A. Gennaro, Mack Publishing Co., Easton, Pa., 1990).

"Administering" as used herein means providing the composition to the patient in a manner that results in the composition being inside the patient's body. Such an administration can be by any route including, without limitation, parenteral, sub-cutaneous, intradermal, intravenous, intra-arterial, intraperitoneal, and intramuscular.

Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the subject pharmaceutical compositions, are formulated into pharmaceutically acceptable dosage forms such as described below or by other conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being highly toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and

prior medical history of the patient being treated, and like factors well known in the medical arts.

In certain embodiments, the method further comprises surgery, administration of a chemotherapeutic drug, and administration of a (xenotypic) monoclonal antibody in a dose equal to or less than 2 mg given by intravenous infusion over 20 minutes during weeks 1, 3, 5, 9, then every 8 weeks, followed by administration of a chemotherapeutic drug within 5 days of the administration of the binding agent.

In certain non-limiting embodiments of the invention, the xenotypic antibody, e.g., Alt-2, is administered as a 2 mg dose dissolved in 50 mL saline and infused slowly preferably over approximately 20 minutes. If an allergic or other reaction occurs that may limit the completion of the dose, then a lower dose may be employed at that time or with subsequent treatments, so that the expected dose range would be 1 -2 mg per treatment. Premedication with oral or intravenous dyphenhydramine (25 to 50 mg) is usually administered to lessen the risk of allergic reaction to the protein. The schedule used for combined Alt-2 and chemotherapy for example, comprises administering Alt-2 at the dose above at weeks 1, 3, 5, 7, 9 with chemotherapy administered with Alt-2 on weeks 12 through 26. Administration of Alt-2 may be started after recovery from any required surgery that is done prior to the chemotherapy and then continued up to, and during, the chemotherapy treatment period. The chemotherapy can be given in 3-4 week cycles or other schedules according to the treating physician and common clinical practice. Chemotherapy may continue for up to six cycles followed by the xenotypic antibody administration every twelve weeks for up to two years.

In another aspect, the method comprises surgery, followed within seven days by administration of a (xenotypic) monoclonal antibody in a dose equal to or less than 2 mg given by intravenous infusion over 20 minutes during weeks 1 , 3, 5, 9, then every 8 weeks with concurrent administration of a chemotherapeutic drug at week 3 and thereafter.

In certain embodiments, the murine antibody is administered at week 1 after completing standard surgery but has not yet begun chemotherapy. The murine antibody is administered in a dose equal to or less than 2 mg via a 20 minute intravenous infusion followed by a second treatment and concurrent administration of a chemotherapeutic drug on weeks 6 and beyond. "Concurrent Administration" means administration within a relatively

short time period from each other. Preferably such time period is less than 2 weeks, more preferably less than 7 days, most preferably less than 1 day and could even be administered simultaneously.

In certain embodiments, the method comprises administering the chemotherapeutic drug at Weeks 0, 4, 8, 12, 16, and 20 (Cycles 1-6, respectively), and administering the monoclonal antibody either concurrently with, or about one week after administering the chemotherapeutic agent (e.g., carboplatin/paclitaxel for ovarian cancer), at Day 1 of Week 0 (Cycle 1), Week 8 (cycle 3), and Week 16 (cycle 5), (about 4 weeks between cycles), followed by administering the monoclonal antibody every twelve weeks for up to two years or until progression.

The expected progression-free survival times may be measured in months to years, depending on prognostic factors including the number of relapses, stage of disease, and other factors. Overall survival is also measured in months to years. In the case of ovarian cancer, the addition of the xenotypic monoclonal antibody, Alt-2 is expected to increase the time to recurrence or progression, and may also prolong the survival time. Any improvement of 2 months or longer is usually considered to be clinically meaningful.

In another aspect, the invention provides a method for inducing a host immune response in a patient against a multi-epitopic in vivo tumor antigen present in the host's serum, which antigen does not elicit a host immune response, comprising administering to the patient an agent that antagonizes one or more immune suppressive pathways, a chemotherapeutic drug, and a composition comprising a binding agent that specifically binds to a first epitope on the antigen and allowing the binding agent to form a binding agent/antigen pair, wherein a host immune response is elicited against a second epitope on the antigen.

Exemplary multi-epitopic antigens are described in and herein incorporated by reference in Nicodemus C. F. et al, Expert Rev. Vaccines 1(1): 34-48 (2002); Qi et al, Hybridoma and Hybridomics 20: 313-323 (2001); and Berlyn et al., Clin. Immunol. 101 : 276-283, (2001).

A "binding agent", as used herein, refers to one member of a binding pair, including an immunologic pair, e.g., a binding moiety that is capable of binding to an antigen,

preferably a single epitope expressed on the antigen, such as a predetermined tumor antigen. In some embodiments of the invention, the binding of a first single epitope exposes a second distinct epitope on the antigen. In one embodiment of the invention, the binding agent, when bound to the antigen, forms an immunogenic complex. Exemplary binding agents include, but are not limited to: antibodies, monoclonal antibodies ("MAb"), preferably IgGl antibodies; chimeric monoclonal antibodies ("C-MAb"); humanized antibodies; genetically engineered monoclonal antibodies ("G-MAb"); fragments of monoclonal antibodies (including but not limited to "F(Ab). sub.2", "F(Ab)" and "Dab"); single chains representing the reactive portion of monoclonal antibodies ("SC-MAb"); fusion proteins of antibody fragments and cytokines, antigen-binding peptides; tumor-binding peptides; a protein, including receptor proteins; peptide; polypeptide; glycoprotein; lipoprotein, or the like, e.g., growth factors; lymphokines and cytokines; enzymes, immune modulators; hormones, for example, somatostatin; any of the above joined to a molecule that mediates an effector function; and mimics or fragments of any of the above. The binding agent may be labeled or unlabeled.

Preferred binding agents of the invention are monoclonal antibodies. Where the patient is human, these xenotypic monoclonal antibodies include, without limitation, murine monoclonal antibodies. Particularly preferred murine monoclonal antibodies include those with the same binding specificity, or those binding to the same epitope as that bounds by: AIt-I (murine IgGl, specifically binds to MUC-I; ATCC No. PTA-975; American Type Culture Collection, Manassas, Va.), Alt-2 (OVAREX ^® Mab-B43.13, murine IgGl, specifically binds to CA 125; ATCC No. PTA-1883), Alt3 (murine IgG3, specifically binds to CA 19.9; ATCC No. PTA-2691), Alt-4 (murine IgM, specifically binds to CA 19.9; ATCC No. PTA-2692), Alt-5 (murine IgGl , specifically binds to CA 19.9; ATCC No. PTA-2690); and Alt-6 (murine IgGl, specifically binds to prostate specific antigen (PSA); ATCC No. HB-12526).

A "multi-epitopic in vivo tumor antigen" is an antigen that presents multiple epitopes on its surface. Some non-limiting examples of such antigens include CA 125, MUC-I, PSA, CA 19.9, and TAG-72. Although they are called antigens, these molecules, while aberrant, are frequently tolerated in cancer patients and as such their presence does not by itself evoke a remedial immune reaction.

"Inducing a host immune response" means that the patient experiences alleviation or reduction of signs or symptoms of illness, and specifically includes, without limitation, prolongation of survival. In certain preferred embodiments of the methods according to the invention, a CD8 ⁺ IFN-γ producing T cell is activated to induce a cytotoxic T lymphocyte (CTL) immune response in the patient administered the murine monoclonal antibody. In certain embodiments of the methods according to the invention, a CD4 ⁺ IFN-γ producing T cell is activated to induce a helper T cell immune response in the patient administered with the composition. These activated CD4 ⁺ IFN-γ producing T cells (i.e., helper T cells) provide necessary immunological help (e.g. by release of cytokines) to induce and maintain CTL. In certain embodiments of the methods according to the invention, a CD4 ⁺ IL-4 or IL-5 producing T cell is activated to induce a helper T cell immune response in the patient administered with the composition. Activated CD4 ⁺ IL-4 or IL-5 producing T cells (i.e., helper T cells) provide necessary immunological help (e.g. by release of cytokines) to induce and maintain a humoral immune response mediated by B cells. Thus, in certain embodiments of the methods according to the invention, a humoral response to the antigen is activated in the patient administered with the composition.

Activation of a CD8 ⁺ and/or CD4 ⁺ IFN-γ or IL-4, producing T cells means causing T cells that have the ability to produce IFN-γ or IL-4 to actually produce IFN-γ or IL-4, or to increase their production of IFN-γ or IL-4.

"Induction of CTL" means causing potentially cytotoxic T lymphocytes to exhibit antigen specific cytotoxicity. "Antigen specific cytotoxicity" means cytotoxicity against a cell presenting an epitope that is associated with the antigen associated with the cancer that is greater than an antigen that is not associated with the cancer. "Cytotoxicity" refers to the ability of the cytotoxic T lymphocyte to kill the target cell. Preferably, such antigen-specific cytotoxicity is at least 3-fold, more preferably 10-fold greater, more preferably more than 100-fold greater than cytotoxicity against a cell not presenting the antigen not associated with the cancer.

In a related aspect of the invention, the method comprises concurrent administration of a chemotherapeutic drug, a binding agent, an antigen, and an agent that antagonizes one or more immune suppressive pathways.

In a further aspect, the invention provides a method for inducing a host immune response in a patient against a multi-epitopic in vivo tumor antigen, which antigen does not elicit an effective host immune response, comprising concurrently administering to the patient an agent that antagonizes one or more immune suppressive pathways, a chemotherapeutic drug, and a composition comprising a binding agent present in an amount of from 0.1 μg to 2 mg per kg of body weight of the host, and wherein the binding agent specifically binds to an epitope on the antigen and an effective host immune response is elicited against a second epitope on the antigen.

Examples

The following example is intended to further illustrate certain particularly preferred embodiments of the invention and is not intended to limit the scope of the invention.

Example I Clinical and Immunologic Outcomes of Patients with Recurrent Epithelial Ovarian Cancer (EOC) Treated with B43.13 and Chemotherapy (Ct)- Interim Immunology and Clinical Results from Study OVA-Gy-12

Patients with recurrence after platinum therapy and a first surgery and were enrolled if they were candidates for secondary surgery and continued chemotherapy. Alt-2 was administered by 20-minute infusion in weeks 1, 3, 5, and 9 prior to initiation of chemotherapy, and then an option to continue every 8 weeks x 2 doses concurrent with chemotherapy on weeks 12 and 26. Humoral immune responses, including HAMA, Ab2 and anti-CA 125 antibody, were assessed at baseline and serially. Using a gamma-interferon ELISPOT assay, T cell responses were evaluated for activation by Alt-2, CA 125, or autologous tumor.

20 patients were enrolled; median follow-up was 6 months ranging up to 2 years. AIt- 2 was well tolerated and did not produce drug-related serious adverse reactions. In 15 of 19 (79%) evaluable patients, robust treatment-emergent humoral responses were observed to the constant (HAMA) and variable region of the antibody (Ab2). Overall, 11 of 18 (61 %) evaluable patients demonstrated functionally active T cells, stimulated by CA 125 or by autologous tumor. T cell responses to Alt-2 were demonstrated in 7 of 18 (39%) patients. T

cell responses were MHC class I and II restricted, indicating the activation of CTL (cytotoxic T lymphocytes) and T helper cells. Immune responses were commonly induced by wk 12 after 4 doses, and were generally maintained in patients continuing combined treatment with Alt-2 and chemotherapy. About 75% were still alive and median survival had not been reached at 120 weeks. Some data from the experiments described herein are listed below and in FIGs. 1 and 2.

The table below illustrates the results of three clinical studies where Alt-2 is administered concurrently with a chemotherapeutic drug.

CHEMOTHERAPY/IMMUNOTHERAPY COMBINATIONS OVAREX ^® MAb + Salvage Chemotherapy

• Clinical experience in 3 studies

• More data emerging from Study 12 (20 patients treated)

• Encouraging clinical and immune responses especially CR & PRs with carboplatin, Doxil and other agents in salvage setting

The table below illustrates the different disease characteristics of Ab2 responders vs. Ab2 non-responders.

Evidence for Drug Effect in Ab2 Responders: Disease characteristics of Ab 2 responders vs non-responders

*No statistical difference compared to placebo or to patients (pts) with Ab2 > 100.

The data demonstrates that Alt-2 is well tolerated and induces multiple antigen- specific immune responses, even when combined with chemotherapy. In advanced EOC, these data are among the first to demonstrate induction of tumor-specific T cells.

Example II In vitro System for Ovarian Cancer

Applicants have developed an in vitro system for ovarian cancer using human dendritic cells (DC), T cells, and HLA-A2 matched live NIH:OVCAR-3 tumor cells to assess the presence of tumor-induced regulatory pathways and to study agents that impact such mechanisms. The system generates regulatory T cells (T _reg ), non-responsive T cells, DC with an immature phenotype, and high levels of TGF-β. The co-cultures were treated with antibodies to neutralize soluble factors such as TGF-β or VEGF, antibodies to block immune suppressive cell surface molecules like GITR and B7-H1, cytokines that promote T cell activation (IL-2, IL-7, IL-12, IL-15, IL-21, IL-23) or DC recruitment or activation (GM-CSF, TNF-α, Flt-3L), antibodies to reduce T _reg (rat and human anti-CD25), or inhibitors of indolamine-2,3-dioxygenase (IDO) or arginase (1-MT, NOHA, L-NMMA) for 7 days in the presence or absence of the immune activating antibodies, oregovomab (OVAREX MAb- B43.13, anti-CA 125) or BREV AREX ^® MAb-AR20.5 (anti-MUC-1).

DC maturation could be improved by addition of anti-CD25 antibodies and TNF-α. A reduction in the number of T _ieg (CD4 ⁺ CD25brightFoxP3 ⁺ ) in the co-culture was achieved most potently with anti-CD25 antibodies, but also with anti-TGF-β, anti-GITR, anti-B7-Hl, as well as Flt-3L, IL-12, IL-15, IL-15+IL-21, IL-7, TNF-α, and inhibitors of IDO and arginase. These agents also enhanced non-specific T cell proliferation; however, tumor- specific T cell responses were only restored with anti-TGF-β, anti-CD25, anti-B7-Hl, anti- GITR, Flt-3L, IL-15, 1-MT and L-NMMA.

Synergy in inducing tumor-specific T cell responses was observed when MAb-B43.13 or MAb-AR20.5 was combined with anti-TGF-β, low-dose anti-CD25, anti-B7-Hl , anti- GITR, IL-12, IL-15, and inhibitors of IDO and arginase. Combinations of several immune-

modulating agents are expected to exhibit synergistic effects. Dendritic Cells (DCs), T Cells and Tumor Cells:

In these experiments, DC were generated from monocytes for 7 days in GM-CSF and IL-4 in 12-well plates (5x10 ⁵ cells/well). Immature DC were washed, and NIH:OVCAR-3 tumor cells (5x10 ⁴ cells/well) were added on Day 7. The cultures were treated with cytokines, antibodies to regulatory molecules, inhibitors to IDO and arginase, or multiple combinations of effective biologies with each other or with chemotherapeutics, with or without addition of MAb-B43.13, MAb-AR20.5 or control IgGl (5 μg/mL) for 1 h. T cells isolated from the same donor as the DC were added (2x10 ⁶ cells/well) and the cultures incubated for 7 days. Controls included cultures without drug or tumor cells, cultures with antibodies alone, and tumor cells grown separated from DC and T cells by a semi-permeable membrane.

Functional and Phenotypic Assays:

T cells were harvested and stained for CD4-FITC, CD25-PE/Cy5 (CyC) and intracellular FoxP3-PE (eBiosciences) and tested for T cell proliferation against media (background), anti-CD3/CD28 beads, or apoptotic tumor cells (BrdU ELISA, EMD- Calbiochem). DC were harvested and stained with anti-CD40-FITC, CD86-PE and CD83- CyC. Supernatants were tested for TGF-b by ELISA. The averages of 3-5 experiments are shown. The experimental lay-out is described in Figure 3 and the results described herein are shown in Figures 4-7.

Cytokines:

Cytokines with reported effects in cancer immunotherapy were tested at different concentrations in the co-culture system. Cytokines effective at increasing T cell reactivity particularly to the tumor were tested further in combination with anti-CA 125 and anti-MUC- 1 antibodies at optimum concentrations.

IL-15, TNF-α, FLT-3L, IL-12, IL-21 and IL-23 were able to enhance non-specific and tumor-specific T cell proliferation in a concentration dependent manner.

Those cytokines were tested in combination with tumor specific antibodies (MAb- B43.13 and MAb-AR20.5) and an isotype control (MOPC-21). The specific antibodies

showed some enhancement of tumor specific T cell response alone and showed particularly strong responses in combination with IL- 15, TNF-α, Flt-3L and IL-23).

IL- 15, IL-23 and to a lesser degree TNF-α reduced T _ieg induction.

IL- 12 was the only cytokine that affected DC maturation (upregulated CD40 and CD86), but it didn't translate into enhanced T cell activation.

IL-15, TNF-α, FLT-3L, IL- 12, IL-21, and IL-23 were able to enhance T cell proliferation to non-specific stimuli (anti-CD3/CD28) as well as to the co-cultured tumor cells. The effect was enhanced in combination with MAb-B43.13 and MAb-AR20.5 for IL- 15, TNF-α, FLT-3L and IL-23.

Results described herein are shown in Figures 4A-4D. Antibodies:

Antibodies that block known regulatory mechanisms or remove T _ieg were tested at different concentrations in the in vitro system. Antibodies effective at increasing T cell reactivity particularly to the tumor were tested further in combination with anti-CA 125 and anti-MUC-1 antibodies under optimized conditions.

Antibodies that block TGF-β, GITR or B7-H1 or that remove/ inactivate T _reg (anti- CD25) were able to enhance non-specific and tumor-specific T cell proliferation in a concentration dependent manner.

The effect of anti-CD25 antibodies (at low cone.) and anti-GITR or anti-B7-Hl (at blocking cone.) was further enhanced in the presence of rumor-specific antibodies.

Both anti-CD25 antibodies were very potent in removing T, _eg .

The monoclonal and polyclonal anti-GITR antibodies enhanced DC maturation (upregulated CD40, CD86 and CD83; further increased when combined with the tumor- specific antibodies), whereas the humanized anti-CD25 antibody (ZENAPAX ) down- regulated CD83 on co-cultured DC.

Antibodies to TGF-β, CD25, GITR and B7-H1 could enhance T cell stimulation to the co-cultured tumor. Anti-CD25, -GITR and -B7-H1 also showed additive effects in combination with the studied anti-CA 125 and anti-MUC-1 antibodies.

Results described herein are shown in Figures 5A-5D. IDO/Arginase Inhibitors:

Inhibitors for IDO and arginase were tested in the co-culture system alone and in combination at several concentrations. Effective doses were tested further in combination with anti-CA 125 and anti-MUC-1 antibodies.

Inhibitors of IDO and arginase showed a small but statistically significant effect in enhancing T cell responses in the co-culture system.

When combined with the antibodies against CA 125 and MUC-I , only 1 -MT showed additive enhancement of tumor-specific T cell responses.

None of the inhibitors revealed a significant effect in reducing the percentage of FoxP3 ⁺ CD4 ⁺ CD25 ^bl T, _eg cells. The addition of MAb-B43.13 or MAb-AR20.5 did not significantly alter the T _reg profile.

The arginase inhibitor NOHA enhanced CD40 expression on co-cultured DC, IDO inhibitor 1-MT in combination with MAb-B43.13 and MAb-AR20.5 increased CD40 and CD83.

Inhibitors for IDO and arginase had only marginal effects in this system. Results described herein are shown in Figures 6A-6D. Multiple Combinations:

The most successful agents were combined with each other for up to triple combinations. The effects of the most potent cytokines or IDO/arginase inhibitors could not be further enhanced when combined with other cytokines, inhibitors or antibodies. However, combinations of multiple antibodies or combination of antibodies with chemotherapeutics, particularly gemcitabine and topotecan, could further enhance tumor-specific T cell responses. Most potent effects were seen when antibodies against CD25 were combined with gemcitabine or anti-CD25, -GITR or -B7H1 were combined with topotecan. The results are depicted in Figure 7.

Conclusions:

The data presented herein demonstrates that co-cultures of iDCs, live tumor cells and

T cells showed induction of T _reg and inhibition of T cell responses and DC maturation, compared to T cells cultured without tumor cells. Tumor cells separated by a semipermeable membrane induced less T _reg and did not inhibit T cell proliferation.

Some of the immune suppressive pathways could be reversed by adding certain cytokines (IL- 15, TNF-α, FLT-3L, IL-23), or antibodies that block inhibitory receptors or T _reg (anti-CD25, anti-GITR, anti-B7-Hl).

These data suggest that immunotherapy with agents such as OVAREX ^® MAb or BREVAREX ^® MAb could be enhanced in patients with tumor burden and multiple immune regulatory pathways when combined with those cytokines or antibodies.

One promising strategy according to data generated in this in vitro ovarian cancer system would be to combine OVAREX ^® MAb or BREVAREX ^® MAb with chemotherapy (particularly gemcitabine or topotecan) and anti-CD25 antibodies (which are commercially available). Schedules can be further optimized in vivo, in view of specific patient parameters.

Certain exemplary (non-limiting) experimental details are provided below for illustrative purpose only.

Antibodies: MAb-B43.13 and MAb-AR20.5 are murine monoclonal IgGl antibodies to CA 125 and MUC-I, respectively (AltaRex Medical Corp.). MOPC21 (Biolegend) was used as isotype control antibody. Anti-TGF-β, anti-B7-Hl and anti-GITR antibodies were purchased from R&D Systems. Rat anti-CD25 was from Abeam and humanized anti-CD25 (ZENAP AX ^® ) was supplied by Roche.

Cytokines: All cytokines were acquired from Biosource with the exception of Flt-3L, IL- 12 and IL-23 (R&D Systems).

Cell Lines: NIH:OVCAR-3 ovarian tumor cells were purchased from ATCC.

Human PBL: Leukaphoresis samples from healthy normal donors were obtained from SeraCare Life Science Inc. (Oceanside, CA). Peripheral Blood Leukocytes (PBL) were purified on Ficoll (Histopaque 1.077, Sigma).

Cell Preparations: Human DC were prepared from PBL by negative selection with anti-CD3, CD 16 and CD 19, followed by anti-mouse-Ig-magnetic beads (Dynal, Monocyte Isolation Kit). Cells were cultured in GM-CSF and IL-4 (1000 U/ml each, BioSource) for 4-

6 days. Human T cells were isolated using Dynal's Negative T Cell Isolation kit.

Chemotherapeutic Drugs: Paclitaxel and topotecan were purchased from LKT labs; gemcitabine was obtained from Lilly.

Co-culture Experiments: Drugs were used at IC90 concentrations in co-culture experiments with NIH:OVCAR-3 tumor cells, immature DC and T cells from HLA-A*0201 positive donors as outlined below. T cells were harvested and stained for presence of CD4 ⁺ CD25 ^bπght FoxP3 ⁺ T cells (regulatory phenotype) and analyzed for T cell proliferation to non-specific (anti-CD3/CD28 beads, Dynal) and specific (apoptotic NIH:OVCAR-3 tumor cells) stimuli. Dendritic cells were analyzed for maturation markers (CD40, CD83, CD86; BioLegend) by flow cytometry. Supernatants were quantitated for TGF-β by ELISA (BD BioSciences). Figure 3 is a schematic representation of an exemplary experimental set up.

Example IH Immunization with anti-PSA Antibody in Complex with PSA overcomes T and B Cell Tolerance to PSA in PSA-transgenic Mice

PSA is shed into the blood, and is accessible for immune complex formation, but it is not expressed on the cell-surface. However, PSA-derived peptides are expressed on MHC class I and II for T cell recognition. In prostate cancer treatment, CTL and T helper 1 responses are considered to be most important, while antibodies to PSA are not expected to have direct effects.

The murine MAb-AR47.47 (IgGIk) recognizes all circulating forms of PSA, and targets the epitope EPEEFLT (SEQ ID NO: 1). While not wishing to be bound by any particular theory, MAb-AR47.47 is thought to bind PSA and forms an immune complex (IC). The IC is then taken up by antigen processing cells (APCs), such as DCs, via Fc Receptor and MMR. In vitro studies showed that human and murine dendritic cells process PSA more efficiently in immune complex (IC) form with MAb-AR47.47. IC could induce CD4 ⁺ T helper cells, and CD8 ⁺ IFN-γ producing CTL cells, whereas PSA alone or PSA in complex with a non-specific antibody mainly stimulated CD4 ⁺ T cells (see Figure 8).

We have further investigated the activation of PSA-specific immune responses with IC in human PSA-transgenic mice, which are tolerant to human PSA (as a self-antigen) on the CD4 and CD8 T cell level. The mice were generated by, and provided as a gift from Dr.

Frelinger (U. of Rochester). Essentially, expression of the full-length human PSA is driven by the murine PSA promoter. The expression is restricted to prostate. Both B and T cell compartments are tolerant to human PSA. Several PSA-transfected tumor cells are thus available as excellent transplanted tumor models useful for studying anti-tumor effect of vaccines as well as combination therapies because they are not being spontaneously rejected. These tumor models feature secreted PSA.

In an exemplary experiment, nine PSA-transgenic mice (3 groups with 3 mice/group) were immunized s.c. at weeks 0, 3, 6 and 9 with PSA (10 μg/mouse), MAb-AR47.47 alone (50 μg/mouse), or IC consisting of MAb-AR47.47 and PSA (10 μg of PSA + 50 μg of MAb), respectively, at various concentrations. PBS was used as control. B cell and cytokine responses were analyzed at baseline, weeks 4, 7 and 10. T cell responses were analyzed at week 10. That is, test bleeds for antibody and cytokine responses were taken after 2, 3 and 4 immunizations. One week after the last immunization, mice were sacrificed and spleen cells isolated for analysis of T cell responses (CTL and ICC).

To investigate the need for foreign antibody in inducing immune responses, a polyclonal rabbit and goat anti-PSA antibody or MAb-AR47.47-ovalbumin was tested in parallel in additional experiments.

Mice were immunized 3-times s.c. with PSA (2, 10 and 50 μg/mouse), MAb-AR47.47 (50 μg/mouse), PSA + MAb-AR47.47 (2, 10, and 50 + 50 μg/mouse), PSA + MAb-AR47.47 + rabbit anti-mouse IgG (2, 10, and 50 + 50 + 50 μg/mouse), or PBS. Test bleeds for antibody responses were taken after 2 and 3 immunizations. One week after the last immunization, mice were sacrificed and spleen cells isolated for analysis of T cell responses by IFN-γ ICC.

Serum samples were analyzed for antibodies to PSA.

Further, mice were immunized 4-times s.c. with PSA + MAb-AR47.47 (10 and 50 μg/mouse), PSA + MAb-AR47.47-OVA (10 and 50 μg/mouse), or PBS. One week after the last immunization, mice were sacrificed and spleen cells isolated for analysis of T cell responses by T cell proliferation and CTL assays.

In another study, mice were immunized 4-times s.c. with PSA (0.4, 2, and 10

μg/mouse), MAb-AR47.47 (10 μg/mouse), PSA + MAb-AR47.47 (0.4, 2, and 10 μg/mouse + 10 μg/mouse), goat anti-PSA (10 μg/mouse), PSA + goat-anti-PSA (0.4, 2, and 10 μg/mouse + 10 μg/mouse), PSA-KLH (10 μg/mouse in RIBI adjuvant) or PBS. Test bleeds for antibody responses were taken after 3 and 4 immunizations. One week after the last immunization, mice were sacrificed and spleen cells isolated for analysis of T cell responses by IFN-γ and IL-10 ICC. Serum samples were analyzed for antibodies to PSA.

The results show that immune complexes of PSA and MAb-AR47.47 induce CTL specific for PSA (Figure 9A). Immune complexes of PSA and MAb-AR47.47 also induce ThI and Th2 helper responses to PSA (based on cytokine ELISA assay and ICC assay). See Figure 9B. T helper (p<0.05) and particularly CTL (p<0.001) responses were superior in mice immunized with IC than in mice immunized with PSA alone. Interestingly, IC with PSA and the polyclonal rabbit or goat antibody as well as with MAb-AR47.47-ovalbumin induced enhanced T helper cell responses to PSA, but resulted in weaker CTL induction relative to IC with MAb-AR47.47. See Figures 10 and 1 1.

Interestingly, cross-linking MAb-AR47.47 increased humoral but not cellular responses (see Figures 1OA and 10B). Specifically, T cells specific for PSA were highest in the PSA-AR47.47 group. PSA concentration of about 2-10 μg/mL were found to be effective in complex (equimolar concentration or slight Ab excess). Cross-linking Ab by using rabbit anti-mouse IgG made the IC less effective than the non-cross-linked Ab-PSA complexes. In contrast, increased antibody responses were observed with PSA-AR47.47 IC and IC cross- linked with anti-mouse IgG compared to PSA alone. Cross-linking also enabled antibody responses at lower immunogen concentrations.

Furthermore, increasing the immunogenicity of MAb-AR47.47 enhanced T helper but not CTL responses (Figures 1 IA & 1 IB). It was found that the s.c. route of administration is more potent than the i.v. route for induction of CTL. In addition, conjugation of MAb- AR47.47 to OVA enhances T cell proliferation but not CTL induction.

In addition, increasing the immunogenicity of the anti-PSA antibody by using goat anti-PSA in comparison to MAb-AR47.47 enhanced T helper 2 but not T helper 1 or CTL responses (Figures 1 I C-I ID). One week after the fourth immunization, IFN-γ T cell responses were highest in the groups immunized with PSA+MAb-AR47.47, while IL-10 T

cell responses were strongest in groups immunized with PSA+goat anti-PSA.

Although immune complexes with murine MAb-AR47.47 and PSA are able to induce ThI and CTL responses, IC with goat anti-PSA Ab and PSA are more potent in inducing Th2 responses. The strongest T cell responses were achieved with equimolar ratios of Ab to Ag or slight Ab excess.

These experiments demonstrate that, although mice are largely non-responsive to immunizations with PSA or PSA-KLH; particularly on the T cell level, IC (such as AR47.47- PSA IC) can reverse unresponsiveness to PSA and prime antibodies, T helper cells and CTL against this self tumor antigen in vivo. T helper 1 and CTL responses (MAb-facilitated class I processing) could be induced by immunization with IC but not with PSA or Ab alone. Attempts to make the antibody more foreign in the mice (conjugation to KLH, cross-linking with rabbit Ab, goat anti-PSA, etc.) resulted in better T helper 2 responses, but not T helper 1 or CTL responses.

Further experiments were conducted to compare the effect of anti-PSA antibodies (such as AR47.47) with PSA alone in treating a mouse model of prostate cancer (Figures 12A & 12B). In this experiment, mice were transplanted at Day 0 with 2 x 10 ⁵ CT26.PSA tumor cells (which expresses PSA) at the dorsal flank. When tumors reached about 4-6 mm in diameter (about Day 10), mice were immunized weekly five times with: PSA (2 μg/mouse), AR47.47 (10 μg/mouse), PSA+AR47.47 (2+10 μg/mouse) or PBS (s.c.) control. Tumors were measured every other day and volumes calculated. Mice were sacrificed when tumors reached about 1.5 cm ³ .

Mice treated with AR47.47 had much slower tumor growth, such that in this treatment group, the first mouse did not die until after Day 30. About 50% of the mice survived past Day 45. In contrast, the first mouse died around Day 25 in the "PSA alone" treatment group, merely 1 or 2 days after the first mouse death in the control (PBS) group. In addition, about 50% of the mice in the PSA treatment group survived past Day 31, an entire 2 weeks less than that of the AR47.47 group. The survival data in the AR47.47 treatment group is statistically significant over the PBS control (p = 0.0019).

Another experiment was conducted to compare the effect of anti-PSA antibodies (such as AR47.47) to PSA in treating a mouse model of prostate cancer in combination with

cyclophosphamide. In this experiment, mice were transplanted at Day 0 with 2 x 10 ⁵ CT26.PSA tumor cells (which expresses PSA) at the dorsal flank. When tumors reached about 4-6 mm in diameter (about Day 10), mice were treated i.p. with the chemotherapeutic drug cyclophosphamide or "Cy" (100 mg/kg), followed by immunization 2 days later with: PSA (2 μg/mouse), AR47.47 (10 μg/mouse), or PBS (s.c.) control. Cy treatment was repeated once and immunizations 4 times on a weekly schedule. Tumors were measured every other day and volumes calculated.

Mice treated with AR47.47 in combination with Cy rejected the transplanted tumors in 7 of 8 animals (about 88% remission rate), while PBS control with Cy is only about 50% successful.

In both experiments, the mice used are PSA-transgenic mice developed by J. Frelinger (see Wei et al., Tissue-specific expression of the human prostate-specific antigen gene in transgenic mice: implications for tolerance and immunotherapy. Proc Nat'l Acad Sci U.S.A. 94: 6369-6374, 1997). These BALB/c mice express human PSA under the probasin promotor in the mouse prostate. Since the human PSA protein is expressed from a transgene in a normal mouse tissue, it is treated as a "self-antigen" by the transgenic mice. As a result, the mice are tolerant to human PSA on the CD4 and CD8 T cell level. Yet the survival experiments and combination therapy experiments described above clearly demonstrate that the anti-human PSA antibody can break the immune tolerance in the transgenic mice, and effectively prolong the survival of cancer-bearing mice in the survival experiment, or even completely reject the tumor in the majority of the cancer-bearing mice in the combination therapy.

These experiments demonstrate that AR47.47 can (1) significantly reduce tumor growth in mice transplanted with PSA-expressing prostatic tumor cells; (2) significantly prolong survival of mice transplanted with PSA-expressing prostatic tumor cells; and (3) more effectively reject tumor in combination therapy in mice transplanted with PSA- expressing prostatic tumor cells.

These experiments also provide direct evidence that self-tolerance to tumor antigen can be overcome, and demonstrate that the certain anti-PSA antibodies can be used to treat prostate cancer in an in vivo mouse model having PSA-expressing prostate cancer cells.

Specifically, MAb-AR47.47 alone (in the presence of circulating PSA) and in immune complex form can control tumor growth. MAb-AR47.47 in combination with cyclophosphamide was able to reject tumors in 88% of the mice, compared to 50% of the mice treated with Cy alone or 25% with AR47.47 alone.

Certain materials and methods used in this exemplary example described herein are provided below for illustrative purpose only.

Antibodies: MAb-AR47.47 (murine IgGl ; AltaRex Medical Corp.) recognizes free and complexed PSA at the linear epitope EPEEFLT (SEQ ID NO: xxx, see Berlyn et al., Generation of CD4(+) and CD8(+) T lymphocyte responses by dendritic cells armed with PSA/anti-PSA (antigen/antibody) complexes. Clin Immunol 101 : 276-283, 2001), which is involved in the induction of cellular immune response. The antibody is a mouse IgG lκ, and has an affinity of approximately 3 x 10 ⁹ M ¹ to all soluble forms of PSA.

Antigens: PSA, purified from seminal fluid was purchased from Scripps or Maine Biotechnology Associates.

Cell lines: Human PSA and control vector (neo) transfected BALB/c tumor cell lines P815 and CT26 were kindly provided by J.F. Frelinger (U. of Rochester, Rochester, NY).

PSA-transgenic mice: The mice (BALB/c) were developed by J.F. Frelinger (Wei et al., Tissue-specific expression of the human prostate-specific antigen gene in transgenic mice: implications for tolerance and immunotherapy. Proc Natl Acad Sci USA. 94: 6369- 6374, 1997), which express human PSA under the probasin promotor in the mouse prostate. The mice are tolerant to hu PSA on the CD4 and CD8 T cell level.

Immunizations: Mice were immunized with PSA, MAb-AR47.47 or IC consisting of MAb-AR47.47 and PSA as well as appropriate controls. Immunizations were given s.c. or i.v. every 2 weeks for a total of 4-5 doses. Mice were monitored for antibodies to PSA and cytokines (IL-4, IFN-γ) by ELISA, and for T cell responses by IFN-γ ICC, IL-10 ICC, T cell proliferation assay, or CTL assay.

Assessment of chemotherapeutics: CT26.PSA tumor cells were transplanted s.c. into the rear flank of the mice at 2x10 ⁵ cells/mouse. When tumors reached approximately 5 mm in diameter, mice were treated with docetaxel, irinotecan, topotecan, or cyclophosphamide (Cy).

Only Cy showed an anti-tumor effect in this model.

Studies with Cyclophosphamide: CT26.PSA tumor cells were transplanted s.c. into the rear flank of the mice at 2x10 cells/mouse. When tumors reached approximately 5 mm in diameter, mice were treated with cyclophosphamide (100 mg/kg) 2 days prior to vaccination with AR47.47 (50 μg/mouse), PSA (10 μg/mouse), AR47.47+PSA or PBS control. Tumors were measured every other day and volumes calculated based on the formula: Volume = (width/2) x π x length.

Example IV Combination Chemo-Immunotherapy in Ovarian Cancer

Ovarian cancer is the most lethal gynecological cancer in the US. Modest increases in median survival have occurred in the past 20 years, but patients with advanced disease still have disappointing long term survival. Thus, there is a medical need to explore alternative treatment approaches such as combination therapy.

One antibody in clinical development, Alt-2 is a CA 125-specific MAb that stimulates immune responses to CA 125. A phase II front-line clinical trial was conducted to examine the feasibility and immunogenicity of chemo-immunotherapy under two different schedules of chemo-immunotherapy (C-IT) following debulking laparotomy.

Qualifying patients (see Patient Demographics in the table below) were randomized to concurrent chemotherapy (carboplatin/paclitaxel) with MAb administered at cycles 1, 3, and 5 (Arm A) or 1 week following cycles 1 , 3, and 5 (Arm B) of C. Both arms received oregovomab every 12 weeks until progression; response to C-IT was assessed at 36 weeks. The study design is shown in Figure 13.

Forty patients were randomized, 18 to Arm A and 22 to Arm B. Complete clinical response to surgery and C-IT was achieved in 15 patients (83%) in Arm A and 18 patients (82%) in Arm B. The activity of C-IT was assessed by serial measurements of CA 125, as well as clinical parameters for progression-free survival and survival. Immunological endpoints included development of humoral responses to the MAb (HAMA, Ab2) and cellular immune responses to CA 125 (IFN-γ ELISPOT). Patients were assessed for baseline and on-study immune status including quantitation of tumor infiltrating lymphocytes (TIL), phenotype of PBMC including TCR-ζ chain expression and plasma cytokines levels.

After 4 infusions with oregovomab, robust humoral responses were measured in more than 75% of patients (88% and 68% for HAMA, and 94% and 68% for Ab2 in Arm A and B,

respectively). The kinetics of the humoral response was more rapid in Arm A with 31% of patients developing a robust humoral response vs. 0% of patients in Arm B after one infusion with Alt-2. CA 125-specific T cell responses were observed in 44% vs. 21% of patients in Arm A vs. B. The percentage of FoxP3 ⁺ /CD4 ⁺ /CD25 ⁺ T _reg in the TIL and PBMC was consistent between the two arms. TCR-ζ chain expression, a marker of functionality of T cells, was decreased in these patients; however, there was a treatment-emergent increase in TCR-ζ chain expression on CD4 ⁺ cells in both treatment arms (54% and 53% Arms A and B respectively). Patients in both treatment arms had elevated VEGF, IL-10, IL-2R, and TNF-α at baseline. The immune response to Alt-2 was not abrogated by concurrent administration of chemotherapy or by potential immunosuppression associated with tumor burden present during treatment in the front-line setting. In fact, surgery and front-line C appeared to have an immune-adjuvant effect on both humoral and cellular responses. The results of the experiment are represented in Figures 14 & 15.

The results show that Chemo-immunotherapy with Alt-2 in conjunction with frontline carboplatin-paclitaxel is feasible. Schedule of dosing the combination is important to achieve the optimal result. In this set of experiments, baseline parameter of immune regulatory state did not appear to predict immune response or pattern of clinical responses. Applicants also note that accelerated immune response in Arm A was concurrent to a measured reduction in lymphocytes relative to Arm B. Clinical outcomes, and cellular and humoral immune response outcomes were consistent in favoring simultaneous infusion of Alt-2 and chemotherapy.

Chemo-immunotherapy had a similar safety profile to chemotherapy alone. A randomized front-line efficacy study of Alt-2-carboplatin-paclitaxel chemo-immunotherapy administered on the same day is justified to further explore use of Alt-2 beyond maintenance mono-immunotherapy.

The example demonstrates that combination therapy with chemotherapy and immunotherapy is feasible, and, in combination with any agents that antagonize one or more immune suppressive pathways, the therapy can be used to efficiently treat a patient suffering from cancer.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

All of the above-cited references and publications are hereby incorporated by reference in their entireties.