TWI478720B - Compositions and methods for eliciting an immune response to escape mutants of targeted therapies to cancer - Google Patents

Description

Composition and method for inducing immune response to escaped mutants of cancer-targeted therapies

The present invention relates generally to compositions and methods for eliciting an immune response to a cancer-associated mutant polypeptide or to a mutant polypeptide exhibiting a cancer-associated mutant polypeptide, wherein the mutant polypeptide is known to be responsive to a target therapeutic or prophylactic agent The occurrence of a specific mutation or its appearance has occurred. The compositions and methods disclosed herein are useful for inducing an immune response, particularly a cell-mediated immune response, to a mutant polypeptide, including deletions and/or blockade of cells expressing a mutant polypeptide, in conjunction with administration of the target agent. The compositions and methods are useful for prophylactic or therapeutic use.

Recent discoveries in cancer biology have provided an opportunity to design target specific anticancer agents and promote drug development. These findings have made it possible to design molecules with high selectivity for specific targets in cancer cells. Segota & Bukowski, Cleveland Clinic J. Med., 71(7): 551-560 (2004). For example, the successful treatment of chronic myelogenous leukemia (CML) with the Bcr-Abl tyrosine kinase inhibitor imatinib (Gleevec) has led to great expectations for this standard approach. Capdeville et al, Nature Reviews, 1: 493-502 (2002). Targeted cancer therapies raise a key issue associated with it: the formation of targets ultimately leads to escape mutations in drug resistance. For example, it has been reported that mutations have been found in patients who are initially susceptible to Gleevec treatment but are therefore mutated to develop resistance to further Gleevec treatment. Gorre et al, science, 293: 876-880 (2001); Shah et al, Cancer Cell, 2: 117-125 (2002); Branford et al, Blood, 99(9): 3742-3745 (2002); Deininger Et al, Blood, 105(7): 2640-263 (2005). Similarly, the effects of therapeutic agents such as gefitinib (Iressa) or erlotinib (Tarceva), which are specifically targeted to the epidermal growth factor receptor (EGFR), have been reported to be present. Mutations in EGFR are found in patients with resistant non-small cell lung cancer (NSCLC). Kobayashi et al, N. Engl . J. Med, 352(8): 786-792 (2005). Therefore, the efficacy of these cancer drugs is significantly limited by the escape mutations.

Therefore, it is desirable to minimize the production of mutants that occur with the administration of therapeutic and/or prophylactic agents.

Methods for inducing an immune response are disclosed, for example, in Thyphronitis et al, Anticancer Research, Vol. 24: 2443-2454 (2004) and Plate et al, Journal of Cell Biology, Vol. 94: 1069 (2005). Yeast systems are disclosed, for example, in U.S. Patent No. 5,830,463, Stubbs et al, Nature Med 5:625-629 (2001); Lu et al, Cancer Research 64:5084-5088 (2004) and Franzusoff et al., Expert Opin. Bio. Ther. Vol. 5: 565-575 (2005).

All references, including patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.

Provided herein are compositions comprising a yeast vehicle and one or more mutant polypeptides comprising a mutated fragment or mimetope or a nucleic acid encoding a mutated polypeptide comprising a mutated fragment or an epitope mimetic, wherein The mutant polypeptide occurs in response to administration of at least one specific mutation of the subject therapeutic agent and/or prophylactic agent or has occurred in the process.

In one aspect, the invention provides a composition comprising one or more of the following: i) a yeast comprising a nucleic acid encoding at least one cancer-associated mutant polypeptide comprising a mutated fragment or an epitope mimetic Vehicle; ii) a yeast vehicle comprising at least one cancer-associated mutant polypeptide comprising a mutated fragment or epitope mimetic; iii) at least one cancer-associated mutant polypeptide comprising a mutated fragment or epitope a mimetic-binding yeast vehicle; iv) a yeast vehicle comprising a nucleic acid encoding at least one cancer-associated mutant polypeptide, comprising a mutated fragment or an epitope mimetic, in a cell-loaded dendritic cell; or v) a yeast vector for intracellular loading of dendritic cells and at least one cancer-associated mutant polypeptide comprising a mutated fragment or epitope mimetic, wherein the mutant polypeptide is known to be responsive to administration of a target cancer therapeutic agent and/or At least one specific mutation of the prophylactic agent occurs or has occurred by this process.

In some aspects, the cancer-associated mutant polypeptide contains at least one escape mutant. In other aspects, the escape mutation was T315I. In other aspects, the cancer-associated mutant polypeptide contains at least two escape mutations. In other aspects, the cancer-associated mutant polypeptide contains at least three escape mutants. In other aspects, the escape mutations were T315I, E255K, and M351T. In its ground state, the two escape mutants are selected from the group consisting of T315I, E255K and M351T. In other aspects, the subject therapeutic and/or prophylactic agent is Gleevec.

In other aspects, the yeast vehicle is a complete yeast, yeast protoplast spheroid, yeast cytoplast, yeast residue or subcellular yeast membrane extract or portion thereof. In other aspects , the yeast vehicle is selected from the group consisting of Saccharomyces , Schizosaccharomyces , Kluveromyces , Hansenula , Candida ( Candida ) and yeast of the genus Pichia are obtained.

In some aspects, in any of the above compositions, the yeast vehicle is obtained from Saccharomyces cerevisiae . In other aspects, in any of the above compositions, the mutant polypeptide, which comprises a mutated fragment or an epitope mimetic, is encoded by an oncogene, is a tumor associated antigen or is expressed by a cancer cell.

In other aspects, in any of the above compositions, the mutant polypeptide, comprising the mutated fragment or epitope mimetic, comprises at least one specific mutation that has occurred in response to administration of an agent selected from the group consisting of: Imatinib, gefitinib, erlotinib, kinase inhibitor, SB 203580, tyrosine kinase inhibitor or a combination thereof.

The invention also provides any of the above compositions additionally comprising a pharmaceutically acceptable excipient. In other aspects, any of the above compositions additionally comprise at least one adjuvant.

In other aspects, the invention provides any of the above compositions for use in ameliorating a cancer condition in a mammal comprising administering to the mammal an effective amount of the composition, wherein the disease is associated with a cancer that has responded to a target A therapeutic polypeptide and/or a prophylactic agent is associated with a mutant polypeptide.

In other aspects, the invention provides any of the above compositions for reducing resistance to administration of an agent having a cancer risk or suffering from cancer, comprising administering to the mammal an effective amount of the composition Wherein the cancer is associated with a mutant polypeptide that has been responsive to administration of a therapeutic agent and/or prophylactic agent.

The invention further provides a method for eliciting an immune response in a mammal to a cancer-associated mutant polypeptide, comprising a mutated fragment or a cancer-associated epitope mimetic, comprising administering to the mammal an effective amount of the above Any composition.

In one aspect, the invention provides a cancer-associated mutant polypeptide, a fragment comprising the same, or a cancer, in a mammal, by administering an effective amount of the above composition and the subject therapeutic and/or prophylactic agent A method by which an epitope mimic induces an immune response.

The invention further provides a method of reducing the resistance to administration of a medicament for a mammal having a cancer risk or suffering from cancer by administering to the mammal an effective amount of a composition of any of the foregoing compositions, wherein the cancer system It is associated with a mutant polypeptide that has been responsive to the administration of a therapeutic and/or prophylactic agent.

In one aspect, the invention provides a method for reducing resistance to a drug administered to a mammal having a cancer risk or suffering from cancer, comprising administering to the mammal an effective amount of the above composition and subject treatment And/or preventive agents.

The invention also provides the use of any of the foregoing compositions for the preparation of a medicament.

The invention also provides the use of any of the foregoing compositions for the preparation of a medicament for the use of a cancer-associated mutant polypeptide, a fragment comprising the same, or a cancer-associated epitope mimetic in a mammal Inducing an immune response.

The invention also provides the use of any of the foregoing compositions for the preparation of a medicament for binding to a cancer-related mutant polypeptide, comprising a mutant fragment, in a mammal, in combination with the subject therapeutic and/or prophylactic agent Or an epitope-like mimetic associated with cancer induces an immune response.

The invention also provides the use of any of the foregoing compositions for the preparation of a medicament for the treatment of cancer, wherein the cancer is a mutant polypeptide that has been responsive to administration of a therapeutic agent and/or prophylactic agent Related.

The invention also provides the use of any of the foregoing compositions for the preparation of a medicament for ameliorating the symptoms of a disease, wherein the disorder is associated with a mutation that has occurred in response to administration of the therapeutic agent and/or prophylactic agent Peptide related.

The invention also provides the use of any of the foregoing compositions for the manufacture of a medicament for reducing resistance to administration of a medicament for a mammal having a cancer risk or suffering from cancer, wherein the cancer system has responded It is associated with a mutant polypeptide that is present when the subject therapeutic agent and/or prophylactic agent is administered.

In other aspects, the invention provides a kit for eliciting an immune response to a cancer-associated mutant polypeptide, a fragment comprising the mutation, or an epitope mimic associated with cancer, in a mammal, wherein the mutant polypeptide is known Occurrence occurs in response to administration of at least one specific mutation of the subject therapeutic agent and/or prophylactic agent or has occurred thereby and wherein the kit comprises any of the foregoing compositions.

In other aspects, the invention provides a kit for eliciting an immune response to a cancer-associated mutant polypeptide, a fragment comprising the mutation, or an epitope mimic associated with cancer, in a mammal, wherein the mutant polypeptide is known Occurring in response to administration of at least one specific mutation of the subject therapeutic agent and/or prophylactic agent or which has occurred therefrom and wherein the kit comprises a yeast vehicle and at least one mutant polypeptide comprising a fragment or table of the mutation Bit mimic. In some aspects, the kit also includes the subject therapeutic and/or prophylactic agent. In other aspects, the kit additionally includes instructional materials regarding the use of the kit.

The invention also provides a method for the preparation of a yeast vehicle comprising a nucleic acid encoding a cancer-associated mutant polypeptide comprising a mutated fragment or a cancer-associated epitope mimetic, wherein the mutant polypeptide is known to be responsive to administration At least one specific mutation of the subject therapeutic agent and/or prophylactic agent occurs or has occurred thereby, and wherein the nucleic acid is transfected into the yeast vehicle by a technique selected from the group consisting of: diffusion, Active transport, ultrasonic treatment, electroporation, microinjection, lipofection, adsorption and protoplast fusion.

The present invention provides methods for preparing a yeast vehicle comprising a cancer-associated mutant polypeptide, a fragment comprising the mutation, or an epitope mimetic associated with cancer, wherein the mutant polypeptide is known to be responsive to administration of a target therapeutic agent and / or at least one specific mutation of the prophylactic agent or it has occurred by this process, and wherein the mutant polypeptide, which comprises a mutated fragment or an epitope mimetic, is placed by a technique selected from the group consisting of the following techniques In or associated with yeast vehicle: diffusion, active transport, liposome fusion, ultrasonic treatment, electroporation, phagocytosis, lipofection, freeze/thaw cycles, chemical cross-linking, bio-linking or mixing. In one aspect, the yeast vehicle is intracellularly loaded into dendritic cells or macrophages. In another aspect, the yeast vehicle is intracellularly loaded into dendritic cells.

The invention also provides a method of treating a mammal by administering an effective amount of any of the foregoing compositions to a mammal having chronic myelogenous leukemia, wherein the number of leukemia cells in the mammal is reduced.

The invention also provides a method of delaying the progression of chronic myelogenous leukemia in a mammal comprising administering to the mammal an effective amount of any one of the foregoing compositions, wherein the number of leukemia cells developed in the mammal is reduced.

There is a need for a prophylactic and therapeutic agent that specifically targets a unique pathway of tumor cells, but has the disadvantage that the tumor cells undergo mutations that allow them to escape the agent and/or become resistant. Provided herein are compositions and methods for the appearance of a mutant polypeptide that is known to respond to a target agent or that has responded to a target agent, or to an immune response to a cell that exhibits a mutant polypeptide. In some examples, the immune response is a cellular immune response; in some instances, the immune response is a humoral response; and in other examples, the immune response is a cellular and humoral response. In some instances, the cellular immune response is intended to clear cells, such as cancer cells or cells that support tumor progression, which have escaped control and contain mutations in the polypeptide of the targeted drug, but cell clearance is not required. In some instances, cellular and/or humoral immune responses will block cell proliferation or viral replication.

Accordingly, provided herein are methods for eliciting an immune response to a mutant polypeptide, or a nucleic acid comprising a mutant polypeptide, and/or a cell exhibiting the mutant polypeptide, the mutant polypeptide being known to be responsive to a specific mutation in a therapeutic or prophylactic agent And appear or have appeared through this process. In some instances, the immune response is a cellular immune response. In some instances, the immune response is a humoral immune response. In other examples, the immune response includes cellular and humoral immune responses. In some examples, the mutant polypeptide is encoded by an oncogene and/or expressed by a cancer cell. In some examples, the mutant polypeptide is associated with or manifested by a cancer cell. In some examples, the agent is targeted to cancer cells. In some examples, the agent is a small molecule or antibody.

Provided herein are methods for eliciting an immune response to a mutant polypeptide comprising a nucleic acid encoding a mutant polypeptide and/or a cell exhibiting the mutant polypeptide, wherein the mutant polypeptide is known to respond to a specific mutation in the administration of a therapeutic and/or prophylactic agent And the appearance or the appearance thereof, the method comprising administering an effective amount of the composition and the agent, wherein the composition comprises: a. a cell, a vector or a virus comprising a nucleic acid encoding the mutant polypeptide; b. a cell, vector or virus that binds; c. a mutant polypeptide, or a peptide that induces an immune response to a mutant polypeptide (epitope mimetic); or d. a nucleic acid encoding a mutant polypeptide, or a nucleic acid capable of binding to the nucleic acid, such as siRNA or Antisense RNA; wherein an effective amount of the composition is administered with the agent.

In some examples, the composition comprises one or more of: i) a yeast vehicle comprising a nucleic acid encoding at least one mutant polypeptide comprising a mutated fragment or an epitope mimetic; ii) comprising at least one a mutant polypeptide, a yeast vehicle comprising the mutated fragment or epitope mimetic; iii) a yeast vehicle that binds to at least one mutant polypeptide, which comprises a mutated fragment or an epitope mimetic; iv) intracellular loading of dendrites a yeast vehicle comprising a nucleic acid encoding at least one mutant polypeptide comprising a mutated fragment or an epitope mimetic; or v) a yeast vehicle loaded into dendritic cells and at least one mutant polypeptide, comprising A fragment or epitope mimetic of a mutation, wherein the mutant polypeptide has occurred or has occurred in response to administration of at least one specific mutation of the therapeutic agent and/or prophylactic agent.

In some examples, the composition is capable of eliciting a cellular immune response. In some examples, the composition is capable of inducing a humoral response. In other examples, the cellular immune response additionally comprises a humoral immune response.

In some examples of the method, the composition comprises an adjuvant. In other examples, the composition additionally comprises an agonist or ligand to the Toll-like receptor. In other examples, the composition comprises a yeast vehicle. In other examples, the composition comprises a CpG sequence. In other examples, the cell is a dendritic cell.

Provided herein are methods for ameliorating the symptoms of a disease in a mammal comprising administering to the mammal an effective amount of a composition together with a therapeutic and/or prophylactic agent, wherein a specific mutation is known to be responsive to administration of the agent Or a mutant polypeptide has been produced by this process, wherein the composition comprises: a. a cell, vector or virus comprising a nucleic acid encoding a mutant polypeptide; b. a cell, vector or virus that binds to the mutant polypeptide; c. a mutant polypeptide, or a peptide which induces an immune response by a mutant polypeptide (epitope mimetic); or d. a nucleic acid encoding a mutant polypeptide, or a nucleic acid capable of binding to the nucleic acid, such as siRNA or antisense RNA; wherein an effective amount of the composition is administered together with the agent Give.

In some examples, the composition comprises one or more of: i) a yeast vehicle comprising a nucleic acid encoding at least one mutant polypeptide comprising a mutated fragment or an epitope mimetic; ii) comprising at least one a mutant polypeptide, a yeast vehicle comprising the mutated fragment or epitope mimetic; iii) a yeast vehicle that binds to at least one mutant polypeptide, which comprises a mutated fragment or an epitope mimetic; iv) intracellular loading of dendrites a yeast vehicle comprising a nucleic acid encoding at least one mutant polypeptide comprising a mutated fragment or an epitope mimetic; or v) a yeast vehicle loaded into dendritic cells and at least one mutant polypeptide, comprising A fragment or epitope mimetic of a mutation, wherein the mutant polypeptide has occurred or has occurred in response to administration of at least one specific mutation of the therapeutic agent and/or prophylactic agent.

In some examples, the composition is capable of eliciting a cellular immune response. In some examples, the composition induces a humoral immune response. In other examples, the cellular immune response additionally comprises a humoral immune response.

In some examples of the method, the composition comprises an adjuvant. In other examples, the composition additionally comprises an agonist or ligand to the Toll-like receptor. In other examples, the composition comprises a yeast vehicle. In other examples, the composition comprises a CpG sequence. In other examples, the cell is a dendritic cell.

In some instances, the mammal is at risk for the disease and is administered prophylactically; in other instances, the mammal is afflicted with a disease and is administered therapeutically. In some examples, the disease is cancer.

Also provided herein are cells, vectors, viruses, compositions (such as immunogenic compositions) and kits comprising a mutant polypeptide or a nucleic acid encoding a mutant polypeptide, the mutant polypeptide being known to be responsive to the specificity of the therapeutic and/or prophylactic agent Sexual mutations occur or have emerged from this process. In some examples, the composition is capable of eliciting an immune response when administered to a mammal. In some examples, the composition is capable of eliciting a cellular immune response upon administration to a mammal. In some examples, the composition is capable of eliciting a humoral immune response when administered to a mammal. In other examples, the composition is capable of inducing cellular and humoral immune responses when administered to a mammal.

Provided herein are yeast vehicles (including yeast vectors), yeast-based compositions, and methods for mutating a polypeptide or a nucleic acid comprising a nucleic acid encoding a mutant polypeptide and/or a cell expressing a mutant polypeptide, the mutant polypeptide being known to be responsive to Specific mutations in therapeutic agents and/or prophylactic agents occur or have occurred in this process. In some examples, the yeast vehicle comprises a nucleic acid encoding a mutant polypeptide. In some examples, the yeast vehicle comprises a nucleic acid that exhibits a mutant polypeptide. In other examples, the yeast vehicle binds to the mutant polypeptide. In some examples, the yeast vehicle is loaded into a carrier cell, such as a dendritic cell or a macrophage.

Also provided herein are methods of making such yeast vehicles and yeast-based compositions comprising the same.

common technology

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained in detail in, for example, Methods of Enzymology, Vol. 194, Guthrie et al., Cold Spring Harbor Laboratory Press (1990); Biology and activities of yeasts, Skinner et al., Academic Press (1980); Methods in yeast genetics: a laboratory course manual, Rose et al, Cold Spring Harbor Laboratory Press (1990); The Yeast Saccharomyces: Cell Cycle and Cell Biology, Pringle et al., Cold Spring Harbor Laboratory Press (1997); The Yeast Saccharomyces : Gene Expression, Jones et al., Cold Spring Harbor Laboratory Press (1993); The Yeast Saccharomyces: Genome Dynamics, Protein Synthesis, and Energetics, Broach et al., Cold Spring Harbor Laboratory Press (1992); Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual, 3rd edition (Sambrook and Russell, 2001), (collectively referred to herein as "Sambrook"); Current Protocols in Molecular Biology (FMAusubel) Et al., 1987, including additions to 2001); PCR: The Polymerase Chain Reaction, (Mullis et al., ed., 1994); Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York; Harlow and Lane (1999) Using Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press , Cold Spring Harbor, NY (collectively referred to herein as "Harlow and Lane"), edited by Beaucage et al., Current Protocols in Nucleic Acid Chemistry John Wiley & Sons, Inc., New York, 2000) and Vaccines, S. Plotkin And W. Orenstein, eds., 3rd edition (1999).

definition

As used herein, the sing "

"Cancer" as used herein includes, but is not limited to, melanoma, squamous cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer. , skin cancer, brain cancer, hemangioma, angiosarcoma, mast cell tumor, primary liver cancer, lung cancer, pancreatic cancer, gastrointestinal cancer, renal cell carcinoma, hematopoietic tumor formation and metastatic cancer.

As used herein, a therapeutic and/or prophylactic "agent" or "agent" of a cell means that the agent is directed against a molecule that is associated with or has a function in causing cell transformation or supporting tumor progression in the context of cancer. Examples of therapeutic and/or prophylactic agents designed to target cells, such as cancer cells, are disclosed herein and are known in the art.

A "mutant polypeptide" as used herein encompasses a full-length polypeptide encoded within a genome and fragments thereof, as long as the fragment comprises a mutation that is known to be responsive to a specific mutation of an agent, such as a prophylactic and/or therapeutic agent. Appears or has appeared in this process. Such mutant polypeptides that occur in response to specific mutations in an agent (such as a prophylactic and/or therapeutic agent) that targets a cell, such as a cancer cell, are commonly referred to in the art as "escape mutants." Mutations can be found in any region of the full length polypeptide and can include amino acid substitutions, insertions or deletions, or combinations thereof, or non-contiguous sequence fusions (such as can be seen in a translocation event). In some instances, it is known that a mutant polypeptide that occurs in response to a specific mutation in an agent or that has occurred in the process is itself immunogenic, ie, without the need for an adjuvant or other carrier or vehicle (such as a yeast vehicle) ) combined, but not necessarily. In other instances, mutant polypeptides that occur in response to an agent or that have occurred in the process are known to be immunogenic in combination with an adjuvant that promotes their antigenicity.

An "adjuvant" as used herein includes, for example, a cytokine response that induces innate immunity and is reported to be an agonist ligand, CpG, of a Toll-like receptor (TLR) associated with antigen-presenting cell (APC) maturation and activation. Nucleotide sequence, single or double stranded RNA (TLR7 agonist), lipid fraction (such as lipopolysaccharide (LPS)), mannan and dextran, yeast component (reported via TLRs 2, 4 And 6 interact to function) yeast vehicle (such as the yeast vehicle described herein).

The administration of a mutant polypeptide or a nucleic acid encoding a mutant polypeptide (which may be made by any of the methods disclosed herein or known in the art) with an agent is not intended to mean that the mutant polypeptide is administered simultaneously with the agent, but this manner It is covered by the methods disclosed herein. The mutant polypeptide can be administered prior to, concurrently with, or after administration of the agent or in a combination of the above. The mutated polypeptide or nucleic acid encoding the mutated polypeptide can be administered hours, days or months after the agent. In some examples, a mutated polypeptide or nucleic acid encoding a mutated polypeptide (which can be made by any of the methods disclosed herein or known in the art) is administered after administration of the agent, and especially if the agent is known to be directly or Indirect interference with proliferation of any cell type can be additionally administered after administration of the agent.

As used herein, "epitope mimetic" refers to a polypeptide that is capable of mimicking the ability of a mutant polypeptide to elicit an immune response to a mutant polypeptide or a cell that exhibits a mutant polypeptide, and in some instances, a cellular and/or humoral immune response. An "epitope mimetic" as used herein includes, but is not limited to, mimicking one of a mutant polypeptide protein that is known to occur in response to a specific mutation in a therapeutic or prophylactic agent or has occurred in the process or a peptide of a plurality of epitopes; a non-protein immunogenic portion of the mutant polypeptide (eg, a carbohydrate structure); and a synthetic or natural organic molecule (including nucleic acid) having a structure similar to at least one epitope of the mutant polypeptide. Such epitope mimetics can be designed using computer generated structures of mutated polypeptides. Epitope mimetics can also induce immune responses, such as cells, by generating random samples of molecules, such as oligonucleotides, peptides, or other organic molecules, and by methods and assays described herein and known in the art. The ability of the immune response to screen these samples is obtained.

"Improving the symptoms of a disease or infection" as used herein includes alleviating, stabilizing, reversing, slowing or delaying any symptoms and/or progression of a disease state, which may be measured by clinical and/or subclinical criteria.

In some examples, an "effective amount" of a mutant polypeptide (or an epitope mimetic thereof) or a nucleic acid encoding a mutant polypeptide refers to an amount that is capable of eliciting an immune response when administered to a mammal. In some instances, the immune response is a cellular immune response. In other examples, the immune response is a humoral response. In some instances, the immune response is a cellular and humoral response.

"Mammal", "mammal" or "mammalian host" as used herein includes human and non-human primates, such as chimpanzees and other baboon and monkey species; livestock, such as cattle, sheep, pigs, hills and Horses; domesticated mammals such as dogs and cats; laboratory animals, including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds, such as chickens, turkeys and other pheasant birds , ducks, geese and their analogues. This term does not indicate a specific age. Therefore, it is intended to cover adult, juvenile and newborn individuals as well as prenatal mammals.

"Pharmaceutical" means an epitope (linear, conformational or both) that contains one or more immune systems that stimulate the host, such as the mammalian immune system, to produce antigen-specific humoral and/or cellular antigen-specific responses or The molecule of the immunogenic determinant. The agent may itself be an "immunogen" or be combined with an agent that promotes its antigenicity to become an "immunogen." The term "antigen" includes complete proteins, truncated proteins, protein fragments, and peptides. Antigens can be naturally produced or genetically engineered variants of proteins. The term "antigen" includes subunit antigens (i.e., antigens that are separated and separated from the complete organism to which the antigen is naturally associated). Antibodies, such as anti-idiotypic antibodies or fragments thereof, and synthetic peptide epitope mimetics (i.e., synthetic peptides that mimic antigens or antigenic determinants) are also encompassed by the definition of antigens used herein. In some instances, the antigen encompasses a mutant polypeptide that is known to have occurred in response to a specific mutation in a prophylactic and/or therapeutic agent or that has occurred in the process or may be self-reported in response to a prophylactic and/or therapeutic agent Mutant mutations occur or have been obtained from mutant polypeptides that occur in this process, and can be naturally produced or synthesized. The antigen can be as small as a single epitope, or slightly larger and can include multiple epitopes. Thus, the size of the antigen can be as small as about 5-12 amino acids (eg peptides) and up to full length proteins, including multimers and fusion proteins, chimeric proteins, whole cells, complete microorganisms or parts thereof (eg whole cells) Lysate or extract of microorganisms). It will be appreciated that in some instances (i.e., when the antigen is expressed by a vector (such as a yeast vector) or a virus from a recombinant nucleic acid molecule), the antigen includes, but is not limited to, a protein, a fragment thereof, a fusion protein, a chimeric protein, a multimer. Instead of whole cells or microorganisms.

An "epitope" as used herein is defined herein as a single antigenic site within a given antigen sufficient to elicit an immune response, which may be a cellular and/or humoral immune response. Those skilled in the art will recognize that the size and composition of T cell epitopes differ from B cell epitopes, and that epitopes presented via the class I major histocompatibility complex (MHC) pathway differ from those presented via the class II MHC pathway. The epitope. In general, a B cell epitope will comprise at least about 5 amino acids but as little as 3-4 amino acids. A T cell epitope (such as a cytotoxic T lymphocyte (CTL) epitope) will comprise at least about 7-9 amino acids, and the helper T cell epitope comprises at least about 12-20 amino acids. Typically, the epitope will comprise between about 7 and 15 amino acids, such as 8, 9, 10, 12 or 15 amino acids.

An immunological reaction to a mutant polypeptide (or epitope mimetic) or a nucleic acid encoding the polypeptide (or a nucleic acid capable of binding to a mutant polypeptide, such as siRNA or antisense RNA) or a composition comprising the polypeptide or nucleic acid, as used herein "Or" immune response" includes the formation of a cellular immune response that recognizes the polypeptide in a mammal. In some instances, the immune response is a humoral immune response. In some examples, the cellular immune response additionally includes a humoral immune response. The immune response can be specific to the mutant polypeptide, but this is not required. The immune response induced by administration of the mutant polypeptide or the nucleic acid encoding the polypeptide may be any aspect of the immune response (eg, cellular response, humoral response, cytokine production) compared to an immune response in which the polypeptide or nucleic acid is not administered. Detection increases. The immune response can be a specific reaction of the mutant polypeptide, but this need not be the case. Compositions which are capable of inducing an immune response in association with a mutant polypeptide (or an epitope mimetic thereof) or a nucleic acid encoding a mutant polypeptide are encompassed within the scope of the invention.

As used herein, "humoral immune response" refers to an immune response mediated by an antibody molecule or immunoglobulin. The antibody molecules of the present invention include IgG species (as well as IgG1, IgG2a, and IgG2b subtypes), IgM, IgA, IgD, and IgE. The antibody functionally includes an antibody to the primary immune response and a memory antibody response or a serum neutralizing antibody. For infectious diseases, the antibodies of the invention may, but need not, neutralize or reduce the infectivity of a virus encoding a mutant polypeptide, and/or mediate antibody-complement or antibody-dependent cellular cytotoxicity (ADCC) against a mutant polypeptide.

A "cellular immune response" as used herein is an immune response mediated by T lymphocytes and/or other white blood cells, including but not limited to natural killer (ND) cells and macrophages. The T lymphocytes of the present invention include T cells or gamma/delta receptors exhibiting a subunit of the α/β T cell receptor exhibiting T cells and may be effector or suppressor T cells.

As used herein, "T lymphocytes" or "T cells" are non-antibody-producing lymphocytes that form part of the cell-mediated antibody arm of the immune system. The T cell line is produced by immature lymphocytes that migrate from the bone marrow to the thymus and undergo a maturation process under the guidance of thymus hormones in the thymus. Mature T cells are immunologically active based on their ability to recognize and bind to specific antigens. Activation of immunocompetent T cells is triggered when the antigen binds to a surface receptor of lymphocytes. Known to produce a T cell response, the antigen must be synthesized intracellularly or introduced into the cell, subsequently processed into a small peptide by the proteasome complex and translocated to the endoplasmic reticulum/Golgi complex secretion pathway for eventual association with the primary class I tissue. Compatible complex (MHC) protein binding. Alternatively, the peptide antigen can be introduced extracellularly from a peptide that has been bound to the MHC-I or MHC-II receptor. Cellular immunity functionally includes antigen-specific cytotoxic T lymphocytes (CTLs).

As used herein, "antigen-specific killer T cell", "CTL" or "cytotoxic T cell" as used herein refers to a peptide antigen or human leukocyte antigen (HLA) that is present in association with an MHC protein (when A cell that has specificity when the protein is indicated in humans. The CTL of the present invention includes an activated CTL which is triggered by a specific antigen in the case of MHC; and a memory CTL or a recall CTL which refers to a T cell which is reactivated by re-exposure to the antigen; and a cross-reactivity or Cross-differentiation of CTL. The CTL of the present invention includes CD4+ and CD8+ T cells. The activated antigen-specific CTL of the present invention promotes cell destruction and/or lysis of a subject infected with a CTL-specific pathogen via secretory chemokines and cytokines (including but not limited to macrophage inflammatory protein 1α (MlP-1α) ), MlP-1β and RANTES) block the entry of pathogens; and promote the secretion of soluble factors that inhibit infection. Cellular immunity of the invention also refers to antigen-specific responses produced by helper T cell subsets of T cells. Helper T cells are used to assist in stimulating the function of non-specific effector cells against cells that exhibit peptides that bind to MHC molecules on the surface and to concentrate their activity. Cellular immune response also refers to the production of cytokines, cellular chemokines, and other such molecules, which are produced by activated T cells and/or other white blood cells, including those derived from CD4 and CD8 T cells and NK cells. A composition that induces a cellular immune response can be used to sensitize a mammal by presenting a peptide that binds to an MHC molecule on the cell surface. The cell-mediated immune response is localized to or near the cell on which the antigen is present. In addition, antigen-specific T lymphocytes can be produced to achieve long-term protection by the immunized host.

The ability of a particular polypeptide or antigen to stimulate a cell-mediated immunological response can be determined by a variety of assays known in the art, such as by lymphocyte proliferation (lymphocyte activation) assays, CTL killing, or by sensitization by assays. Antigen-specific T lymphocytes in the examiner. Such assays are well known in the art. See, for example, Erickson et al, J. Immunol. (1993) 151: 4189-4199; Doe et al, Eur. J. Immunol. (1994) 24: 2369-2376. Other methods of measuring cell-mediated immune responses include measuring intracellular cytokine secretion by T cell populations or by measuring epitope-specific T cells (eg, by tetramer technology) (McMichael, AJ, and O) 'Callaghan, CA, J. Exp. Med. 187 (9) 1367-1371, 1998; Mcheyzer-Williams, MG, et al, Immunol. Rev. 150: 5-21, 1996; Lalvani, A., et al, J. Exp. Med. 186: 859-865, 1997.).

An "immunological response" or "immune response" as used herein encompasses an immune response that stimulates CTL production and/or aids in T cell production or activation and/or antibody-mediated immune responses. "T lymphocytes" or "T cells" are non-antibody-producing lymphocytes that form part of the cell-mediated antibody arm of the immune system. The T cell line is produced by immature lymphocytes that migrate from the bone marrow to the thymus and undergo a maturation process under the guidance of thymus hormones in the thymus. Here, the rapid division of mature lymphocytes increases to a very large number. Mature T cells are immunologically active based on their ability to recognize and bind to specific antigens. Activation of immunocompetent T cells is triggered when the antigen binds to a surface receptor of lymphocytes in the presence of MHC/HLA receptors and co-receptors.

An "immunogenic composition" as used herein, includes a mutant polypeptide (or an epitope mimetic thereof) or encoding that is known to occur in response to a specific mutation in a therapeutic agent and/or prophylactic agent or that has occurred in the process. A composition of a nucleic acid that mutates a polypeptide, and may or may not comprise an adjuvant that promotes antigenicity of the mutant polypeptide, wherein administration of the composition to a mammal results in the formation of a cellular immune response, a humoral immune response, or a cellular and humoral immune response. Immunogenic compositions include compositions that are capable of eliciting a protective cellular immune response, but are not required to do so.

"Prophylactic composition" as used herein refers to a form of administration of an immunogenic state (na Ve) "or a mammalian individual or host that has not previously been exposed to a pathogen antigen or that has not produced an effective immune response to the pathogen to prevent diseases such as cancer and infection or reinfection (however, it is not necessary for the invention to completely prevent infection or reinfection) The prophylactic composition of the present invention does not necessarily produce sterilizing immunity in the administration host or individual.

A "therapeutic composition" as used herein refers to a composition that is administered to an individual or host that has cancer or has been infected with a virus (in the condition of an infectious disease) and, in some instances, has progressed to a certain disease state.

As used herein, "immune", "immunogenic" or "immunized" refers to the process of administering an effective amount of an immunogenic composition to a living mammalian subject or host to induce an immune response against the composition. In some examples, the immune response comprises a cellular immune response, such as a cytotoxic T cell response. In some examples, the immune response includes a humoral response, such as antibody production. In some instances, the immune response includes cellular and humoral responses.

Carrier and composition

Provided herein are vectors, such as bacterial, viral, insect, and yeast vectors, comprising a nucleic acid encoding a mutant polypeptide that is known to be responsive to an agent or that has undergone a specific mutation. Provided herein are compositions comprising the vectors and viruses that bind to the mutant polypeptide. Such vectors and viruses include, but are not limited to, bacterial vectors, naked plastid DNA, yeast vectors and vehicles, rAAV, adenovirus, canarypox virus, vaccinia virus, vaccinia virus, modified Ankara vaccinia (modified vaccinia Ankara) (MVA), alphaviruses, baculovirus, Venezuela equine encephalitis virus and baculovirus, which can be considered routine by those skilled in the art. Method made. A variety of viral based systems have been developed for gene transfer into mammalian cells. For example, the life cycle and genetics of AAV are discussed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992). Further disclosures regarding AAV systems are provided, for example, in RO Snyder et al, Human Gene Therapy 8:1891-1900 (1997); D Duan et al, J. Virol 8568-8577 (1998); Duan D et al, J. Virol 73:161-169 (1999); N Vincent-Lacaze et al, J. Virol 73: 1949-1955 (1999); C McKeon et al, NIDDK Workshop on AAV Vectors: Gene transfer into quiescent cells, Human Gene Therapy 7 :1615-1619 (1996); H Nakai et al, J. Virol 75: 6969-6976 (2001) and BC Schnepp et al, J. Virol. 11: 3495-350 (2003). The AAV system is described, for example, in U.S. Patent No. 5,786,211, U.S. Patent No. 5,871,982, and U.S. Patent No. 6,258,595. A variety of adenoviral vectors and expression systems have been described. See, for example, Haj-Ahmad and Graham, J. Virol 57:267-274 (1986); Bett et al, J. Virol 67:5911-5921 (1993); Mittereder et al, Human Gene Therapy 5:717-729 (1994) ); Seth et al, J. Virol. 68: 933-940 (1994); Barr et al, Gene Therapy 1: 51-58 (1994); Berkner, KL BioTechniques 6: 616-629 (1988) and Rich et al. Human Gene Therapy 4: 461-476 (1993). The poxvirus vaccine is described, for example, in Small, Jr., PA et al. (U.S. Patent No. 5,676,950, issued October 14, 1997). Other viral vectors include viral vectors derived from the vaccinia virus family, including vaccinia virus and fowlpox virus. Baculoviruses are described, for example, in U.S. Patent No. 5,731,182.

Provided herein are methods for eliciting an immune response, or in some instances, a cellular immune response, to a mutant polypeptide or a nucleic acid encoding the polypeptide. In some instances, the immune response is a humoral response, and in other examples, the immune response includes cellular and humoral immune responses. Illustrative examples of mutant polypeptides encoded by oncogenes and viruses that are known to occur in response to specific mutations in therapeutic and/or prophylactic agents are disclosed herein and other examples are known in the art. Responsive to the presence of a mutant polypeptide in the (etc.) agent with, for example, reduced sensitivity to the agent and/or resistance to the agent and/or increased clinical and/or subclinical symptoms of the disease (such as in HIV) In the case where there is a decrease in CD4 count). Mutant polypeptides (such as cellular immunity) that are known to occur in response to specific mutations in a cell or virus that will be administered, administered, or being administered to a mammal, or that have occurred in the process Benefits of the reaction may include prolonging the duration of efficacy of the agent, minimizing or reversing resistance to the agent, delaying or minimizing the occurrence of specific mutant polypeptides, and eliminating specific mutant polypeptides (but removal is not necessary so Have certain benefits) and minimize disease symptoms, reduce or reverse and / or slow disease progression.

Provided herein are vectors, for example, including but not limited to, bacterial, viral, insect, and yeast vectors comprising a mutant polypeptide that encodes a mutation that is known to occur in response to a therapeutic agent and/or a prophylactic agent or that has occurred in the process Nucleic acid, and a composition comprising the carrier. In some examples, the vector is a yeast vector. In other examples, the vector comprises a nucleic acid capable of binding to the mutant polypeptide, such as siRNA or antisense RNA, thereby inhibiting the performance of the mutant polypeptide. Provided herein are compositions comprising a carrier, for example, a bacterial, viral, insect, and yeast carrier, which are present or have occurred in response to a specific mutation in response to a therapeutic and/or prophylactic agent. The mutant polypeptide, which comprises a fragment of the mutation, and an epitope mimetic thereof. In some examples, the vector is a yeast vector or vehicle. In other examples, the composition comprising the mutated polypeptide further comprises an adjuvant. Examples of adjuvants are described herein and are known in the art. Provided herein are cells (including, but not limited to, dendritic cells) comprising a nucleic acid encoding a mutant polypeptide as described herein and/or vectors and/or viruses that exhibit a mutant polypeptide. In some examples, the dendritic cells comprise a mutant polypeptide as described herein. Also provided herein are compositions comprising a nucleic acid encoding a mutant polypeptide. Also provided herein are methods for making such cells, vectors, viruses, and compositions.

The mutated polypeptide or a nucleic acid comprising the mutated fragment or the nucleic acid encoding the polypeptide or fragment and/or the composition or vector comprising the same is administered together with a prophylactic and/or therapeutic agent and can be administered to a mammal having a risk of cancer or infection Or a mammal suffering from cancer or infection. The mutant polypeptide or a fragment thereof comprising the mutation or the nucleic acid encoding the polypeptide or fragment and/or a composition or vector comprising the same can be administered after the agent, whether or not the mutant polypeptide has been responsive to the agent and whether or not it has been detected To the resistance to the agent. Detection of a mutant polypeptide in response to the presence of the agent can be determined by methods known in the art. If the mutant polypeptide or its encoding nucleic acid and/or a composition or vector comprising the same are administered to a mammal together with an agent and it has been determined that the mutant polypeptide has been mutated in response to the specific mutation of the agent, the methods described herein are not necessarily eliminated. Mutant polypeptides are used to benefit mammals from administration.

Thus, a mutant polypeptide, which comprises a mutated fragment or epitope mimetic or a nucleic acid encoding the same, and/or a composition or vector comprising the same, can be administered in combination with an agent, in a method described herein, including A method for inducing an immune response (such as a cellular immune response, a humoral immune response, or both), a method for improving a cancer symptom in a mammal, a method for reducing resistance to an agent, and a method for increasing the efficacy of the agent, A method for increasing sensitivity to a pharmaceutical agent, a method for delaying or minimizing the occurrence of a mutant polypeptide, and a method for reducing clinical and/or subclinical symptoms of cancer. In some instances, the mutant polypeptide, or a nucleic acid encoding the same, and/or a vector thereof, is exemplified as compared to administering the agent alone (ie, without administering the mutant polypeptide or its encoding nucleic acid and/or a composition or vector comprising the same) Administration of the compositions or carriers together with the agent delays or minimizes the occurrence of the mutated polypeptide and/or increases the efficacy of the agent and/or reduces the clinical and/or subclinical symptoms of the cancer to a greater extent.

Yeast based composition and method

Provided herein are yeast vectors, yeast vehicles, and yeast-based compositions comprising a mutant polypeptide that is known to occur in response to a specific mutation of the agent or that has occurred in the process. The terms "yeast vector" and "yeast vehicle" as used herein are used interchangeably and include, but are not limited to, complete yeast, yeast protoplast spheroids, yeast cytoplasts, yeast residues, and subcellular yeast membrane extracts or Part of it. In some examples, yeast cells are prepared using yeast cells or yeast protoplast spheroids, which in some instances comprise a nucleic acid molecule encoding a mutant polypeptide such that the polypeptide is expressed by yeast cells or yeast protoplast spheroids. In some examples, the yeast vehicle can be obtained from a non-pathogenic yeast. In other examples, the yeast vehicle can be obtained from a yeast selected from the group consisting of: Mycobacterium, Schizosaccharomyces, Kluyveromyces, Hansenula, Candida, and Pichia pastoris. Genus. In some examples, the genus Bacillus is Saccharomyces cerevisiae.

In general, the yeast vehicle can be combined with the mutant polypeptide by any of the techniques described herein. In some examples, the yeast vehicle is loaded intracellularly with a mutant polypeptide. In other examples, the mutant polypeptide is covalently or non-covalently linked to the yeast vehicle. In other examples, the yeast vehicle is combined with the mutant polypeptide by mixing. In other examples, the mutant polypeptide is recombinantly expressed from a yeast vehicle or from a yeast cell or yeast protoplast spheroid from which the yeast vehicle is derived.

Accordingly, provided herein are yeast vehicles that encompass any yeast cell (eg, whole cell or intact cell) or a derivative thereof, which can be used in combination with the mutant polypeptide in the composition or as an adjuvant. Thus the yeast vehicle can include, but is not limited to, a surviving intact yeast organism (ie, a yeast cell having all components including the cell wall), a killed (dead) complete yeast organism, or a derivative thereof, including: yeast Protoplast spheroids (ie, yeast cells without cell walls), yeast cytoplasmic bodies (ie, yeast cells without cell walls and nucleus), yeast residues (ie, yeast cells without cell walls, nuclei, and cytoplasm) or subcellular yeast membranes Extract or part thereof (previously also referred to as subcellular yeast particles).

Yeast protoplast spheroids are typically produced by enzymatic digestion of yeast cell walls. This method is described, for example, in Franzusoff et al, Meth. Enzymol. 194: 662-674, (1991). Yeast cytoplasts are typically produced by enucleating yeast cells. This method is described, for example, in Coon, Natl. Cancer Inst. Monogr. 48:45-55 (1978). Yeast residues are typically produced by resealing permeabilized or lysed cells and may, but are not required to, contain at least some of the cellular organs of the cells. This method is described, for example, in Franzusoff et al, J. Biol. Chem. 258: 3608-3614 (1983) and Bussey et al, Biochim. Biophys. Acta 553: 185-196 (1979). The subcellular yeast membrane extract or a portion thereof refers to a yeast membrane free of natural nuclear or cytoplasm. The granules can be of any size, ranging from natural yeast film size to size within the size range of the microparticles prepared by ultrasonic treatment or subsequent re-sealing by other film disrupting methods known to those skilled in the art. Methods for making subcellular yeast membrane extracts are described, for example, in Franzusoff et al, Meth. Enzymol. 194:662-674 (1991). When a portion of the yeast membrane extract containing the yeast membrane fraction is also used, and when the antigen is expressed by yeast recombinantly prior to preparation of the yeast membrane extract, the antigen of interest is part of the extract. Yeast can also be electroporated or otherwise loaded with a target antigen (such as a peptide).

The yeast vehicle of the present invention can be prepared using any yeast strain. Yeast is a single-celled microorganism belonging to one of the following three classes: Ascomycetes, Basidiomycetes, and Fungi Imperfecti. Although pathogenic yeast strains or their non-pathogenic mutants can be used, in some instances, non-pathogenic yeast strains are used. Yeast strains for use in the compositions and methods disclosed herein include Brassica, Candida (which may be pathogenic), Cryptococcus , Hansenula, Kluyveromyces , Pichia, Rhodotorula , Schizosaccharomyces and Yarrowia . In one example, the yeast strains include Brassica, Candida, Hansenula, Pichia, and Schizosaccharomyces. In some examples, the yeast strain is a genus of the genus Bacillus. Yeast strain species include Saccharomyces cerevisiae, Saccharomyces carlsbergensis , Candida albicans , Candida kejyr , Candida tropicalis , Cryptococcus laurentii , Cryptococcus neoformans , Hansenula anomala , Hansenula polymorpha , Kluyveromyces fragilis , Kluyveromyces lactis , Marks Kluyveromyces marxianus var . lactis, Pichia pastoris , Rhodotorula rubra , Schizosaccharomyces pombe , and Yarrowia lipolytica . It should be understood that many such species include a variety of subspecies, bacterial types, subtypes, etc., which are intended to be included in the aforementioned species. In some examples, the yeast species include Saccharomyces cerevisiae, Candida albicans, Hansenula polymorpha, methanol yeast, and Schizosaccharomyces pombe. Saccharomyces cerevisiae is used in some instances due to its relatively easy handling and "generally considered safe" or "GRAS" for use as a food additive (GRAS, FDA proposed Rule 62 FR 18938, April 17, 1997). In some instances, a yeast strain capable of replicating a plastid to a particularly high number of replicas, such as a CIR strain of Saccharomyces cerevisiae, is used. Other suitable strains are known in the art.

In some embodiments, the yeast vehicle of the present invention is capable of fusing with a yeast vehicle and a cell type to which the mutant polypeptide is to be delivered, such as dendritic cells or macrophages, thereby effecting the yeast vehicle (and in many instances) The medium is an antigen) which is particularly efficiently delivered to the cell type. As used herein, yeast vehicle and target cell type fusion means that the yeast cell membrane or its particles are capable of fusing with a membrane of a target cell type (eg, dendritic cells or macrophages), resulting in the formation of fused cells. As used herein, a fused cell line is a protoplast polynucleate produced by cell fusion. However, it should be noted that although in some cases the target or fusion moiety may be required to be incorporated into the yeast vehicle, this need not be the case. Yeast vehicles have also been shown to be readily absorbed by dendritic cells (as well as other cells such as macrophages).

The yeast vehicle can be formulated into a yeast-based composition, including compositions intended to be administered directly to an individual having a risk of cancer or infection, or prior to administration, using a variety of techniques known to those skilled in the art. Loaded in a carrier such as dendritic cells in vitro.

Provided herein are yeast vehicles comprising at least one mutant polypeptide to be administered to a mammal and compositions comprising the same. Also provided herein are yeast vehicles comprising two or three mutant polypeptides to be administered to an animal, and compositions comprising the same. This may include yeast-based vaccines containing a mutant polypeptide having one, two or three or more of the escape mutants of a known or contemplated target or prophylactic agent. In one aspect, the yeast vehicle contains an escape mutation. In other aspects, the yeast vehicle contains two escape mutations. In other aspects, the yeast vehicle contains three escape mutants.

In some examples, the composition comprises one or more of: i) a yeast vehicle comprising a nucleic acid encoding at least one mutant polypeptide comprising a mutated fragment or an epitope mimetic; ii) comprising at least one a mutant polypeptide, a yeast vehicle comprising the mutated fragment or epitope mimetic; iii) a yeast vehicle that binds to at least one mutant polypeptide, which comprises a mutated fragment or an epitope mimetic; iv) intracellular loading of dendrites a yeast vehicle comprising a nucleic acid encoding at least one mutant polypeptide comprising a mutated fragment or an epitope mimetic; or v) a yeast vehicle loaded into dendritic cells and at least one mutant polypeptide, comprising A fragment or epitope mimetic of a mutation, wherein the mutant polypeptide has occurred or has occurred in response to administration of at least one specific mutation of the therapeutic agent and/or prophylactic agent.

Such compositions may include one, two, several, several or a plurality of mutant polypeptides comprising one or more immunogenic domains of one or more mutant polypeptides, as desired. As used herein, a polypeptide includes an "antigen." As used herein, an antigen includes any protein moiety (peptide, protein fragment, full length protein) in which the protein is naturally produced or synthetically derived, a cellular component (whole cell, cell lysate or disrupted cell), organism (complete organism) , a lysate or a disrupted cell), a carbohydrate, a lipid or other molecule or a portion thereof, wherein the antigen induces an antigen-specific immune response (humoral and/or cellular immune response). Yeast exhibits the particle characteristics of many immunostimulatory complexes with the additional advantage that it naturally possesses adjuvant-like properties and can be readily engineered to express mutant polypeptides (including antigens). Lu et al, Cancer Research 64, 5084-5088 (2004) demonstrated that yeast-based immunotherapy can induce a cell-mediated immune response to tumors that exhibit a Ras oncoprotein with a single amino acid mutation. The results demonstrate that yeast vehicle and yeast-based systems enable immunotherapy to target polypeptides with a single amino acid mutation. Accordingly, provided herein are yeast vehicle and yeast-based compositions comprising a mutant polypeptide that is known to be responsive to an agent or that has occurred in the process, and methods of using the same to induce an immune response to a mutant polypeptide. In some examples, the immune response is a cellular immune response. In some instances, the immune response is a humoral response. In other examples, the immune response is a cellular and humoral immune response. In some other examples, the yeast vehicle is genetically engineered to selectively deliver antigen to a desired cell type. Also provided herein are yeast vehicles comprising a yeast strain capable of producing a heterologous precursor protein having a dibasic amino acid treatment site. The yeast strain is capable of properly processing the precursor protein into at least one cleavage product protein.

In some examples, the mutant polypeptide is encoded by an oncogene, such as Ras. In some examples, the mutant polypeptide is a tumor associated antigen or a protein expressed by a cancer cell.

Preparation of carrier

Provided herein are compositions comprising a vector (such as a yeast vehicle) that binds to a mutant polypeptide. The binding comprises expressing the polypeptide by a carrier, such as a recombinant yeast, introducing the mutant polypeptide into a vector, ligating the polypeptide to a carrier entity, and mixing the vector with a mutant polypeptide, such as in a buffer or other solution for formulation. This method is familiar to those of ordinary skill in the art.

For illustrative purposes, the yeast carrier is described below. In some examples, a yeast cell used to prepare a yeast vehicle is transformed with a heterologous nucleic acid molecule encoding a mutant polypeptide to express the polypeptide from a yeast cell. The yeast is also referred to herein as recombinant yeast or recombinant yeast vehicle. The yeast cells can then be loaded into dendritic cells in intact cell form, the yeast cells can be killed, or derivatized, for example, by formation of yeast protoplast spheroids, cytoplasts, residues or subcellular particles, any of the above treatments The derivative is then loaded into dendritic cells. Yeast protoplast spheroids can also be directly transfected with recombinant nucleic acid molecules (e.g., protoplast spheroids from complete yeast and then transfected) to produce recombinant protoplast spheroids that exhibit antigen.

According to the invention, the isolated nucleic acid molecule or nucleic acid sequence is a nucleic acid molecule or sequence that has been removed from at least one component that is naturally associated therewith. Thus, "isolated" does not necessarily reflect the extent to which the nucleic acid molecule has been purified. Isolated nucleic acid molecules suitable for use in transfection vectors, such as yeast vehicles, include DNA, RNA or derivatives of DNA or RNA. The isolated nucleic acid molecule can be double-stranded or single-stranded. An isolated nucleic acid molecule suitable for use in the present invention includes a nucleic acid molecule encoding a protein or a fragment thereof, so long as the fragment contains at least one epitope suitable for use in the composition of the present invention.

Nucleic acid molecules can be transformed into a vector, such as a yeast vehicle, by any method known in the art, including, but not limited to, diffusion, active transport, liposome fusion, electroporation, water bath sonication And genetic engineering design.

A nucleic acid molecule that is transformed into a yeast vehicle can include a nucleic acid sequence encoding one or more mutant polypeptides. The nucleic acid molecule can comprise a partial or complete coding region, a regulatory region, or a combination thereof. One of the advantages of yeast strains is their ability to carry a variety of nucleic acid molecules and the ability to produce a variety of heterologous proteins. In some examples, the number of antigens produced by the yeast vehicle is any number of antigens that can be suitably produced by the yeast, and is generally in the range of at least one to at least about 5 or more. In one example, the yeast vehicle produces from about 2 to about 5 antigens.

The mutant polypeptide encoded by the nucleic acid molecule in the yeast vehicle can be a full length protein, or a functionally equivalent protein in which the amino acid has been deleted (eg, a truncated variant of the protein), inserted, inverted, substituted, and/or derivatized (eg, acetamidine). Glycosylation, glycosylation, phosphorylation, by glycerophospholipid inositol (GPI) anchoring of the stator, such that the modified protein has a biological function substantially similar to that of the native protein (or, if desired, its function compared to the native protein) Enhanced or inhibited). Modifications can be made by techniques known in the art, including, but not limited to, direct modification of the protein or modification of the nucleic acid sequence encoding the protein using, for example, typical or recombinant DNA techniques to effect random or target mutations.

Techniques known to those skilled in the art can be used to achieve the performance of the mutant polypeptide in a vector. Briefly, a nucleic acid molecule encoding at least one polypeptide to be mutated is inserted into a expression vector in such a manner that the nucleic acid molecule is operably linked to a transcriptional control sequence to enable constitutive of the nucleic acid molecule when transformed into a host yeast cell. Or adjust performance. A nucleic acid molecule encoding one or more mutant polypeptides can be operably linked to one or more transcription control sequences on one or more expression vectors.

In the recombinant molecule of the invention, the nucleic acid molecule is operably linked to a expression vector comprising a regulatory sequence, such as a transcriptional control sequence, a translation control sequence, a replication initiation site, and the like, which are compatible and controllable to the vector. A regulatory sequence in which a nucleic acid molecule behaves. In particular, a recombinant molecule of the invention comprises a nucleic acid molecule operably linked to one or more transcription control sequences. The phrase "operably linked" refers to a nucleic acid molecule that is linked to a transcriptional control sequence in a manner such that the molecule is expressed when transfected (ie, transformed, transduced, or transfected) into a host cell.

Transcriptional control sequences that control the amount of protein produced include sequences that control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are sequences that control the initiation of transcription, such as promoters and upstream activating sequences. A variety of upstream activating sequences (UAS) (also known as enhancers) are known and can be used in vectors.

Transfection of the nucleic acid molecule into the vector can be achieved by any method by which the nucleic acid molecule is administered to the cell, and such methods include, but are not limited to, diffusion, active transport, water bath ultrasonic treatment, electroporation, microscopy Injection, lipofection, adsorption and protoplast fusion. The transfected nucleic acid molecule can be integrated into the chromosome or maintained on an extrachromosomal vector using techniques known to those skilled in the art. In the case of yeast, the yeast cytoplast, yeast residue and subcellular yeast membrane extract or a part thereof can also be recombinantly produced by transfecting the intact yeast microorganism or the yeast protoplast spheroid with the desired nucleic acid molecule, thereby The antigen is produced therein, and the microorganism or protoplast spheroid is then further manipulated using techniques known to those skilled in the art to produce a cytoplast, residue or subcellular yeast membrane extract or portion thereof containing the desired antigen. Efficient conditions for the manufacture of recombinant vectors and vectors for expressing mutant polypeptides include effective media in which the vector can be cultured. Effective culture is generally an aqueous medium containing assimilable sources of carbohydrates, nitrogen and phosphorus, as well as suitable salts, minerals, metals and other nutrients such as vitamins and growth factors. The medium may comprise a composite nutrient or may be a defined inorganic medium. The vectors of the present invention can be cultured in a variety of containers including, but not limited to, bioreactors, Erlenmeyer flasks, test tubes, microplates, and Petri plates. The culture line is carried out at a temperature, pH and oxygen content suitable for the yeast strain. Such culture conditions are well within the knowledge of the general practitioner (see, for example, Guthrie et al. (eds.), 1991, Methods in Enzymology, Vol. 194, Academic Press, San Diego).

In one embodiment of the invention, a vector (such as a yeast vehicle) can be loaded intracellularly with a mutant polypeptide as an alternative to displaying the mutant polypeptide in a vector. Subsequently, the vector containing the mutant polypeptide in the current cell can be administered to a patient or loaded into a carrier such as a dendritic cell (described below). With regard to yeast vehicles, the mutant polypeptide can be directly inserted into the yeast vehicle of the present invention by techniques known to those skilled in the art, such as diffusion, active transport, liposome fusion, electroporation, phagocytosis, freezing. / Thaw cycle and water bath ultrasonic processing.

Yeast vehicles that can be directly loaded with mutant polypeptides include intact yeast as well as protoplast spheroids, residues or cytoplasts that can be loaded with antigen after production but prior to loading into dendritic cells. Alternatively, the intact yeast can be loaded with an antigen and then a protoplast spheroid, residue, cytoplast or subcellular particle can be prepared therefrom. Any number of antigens can be loaded into the yeast vehicle from at least 1, 2, 3, 4 or any integer up to hundreds or thousands of antigens, such as by loading a microorganism, for example by loading a mammal Tumor cells or parts thereof are provided.

In another example, the mutated antigen is physically linked to a carrier, such as a yeast vehicle. The physical linkage of the mutated polypeptide to the vector can be achieved by any suitable method in the art, including covalent and non-covalent binding methods including, but not limited to, chemically crosslinking the mutant polypeptide to the outer surface of the support, Alternatively, the mutant polypeptide can be biologically linked to the outer surface of the carrier, such as by using an antibody or other binding partner. Chemical crosslinking can be achieved, for example, by the following methods, including glutaraldehyde linkage, photoaffinity labeling, treatment with carbodiimide, treatment with chemicals capable of linking disulfide bonds, and use in the art Other cross-linking chemicals are treated as standard. Alternatively, in the case of yeast, the chemical can be contacted with a yeast vehicle that alters the charge of the lipid bilayer of the yeast membrane or the composition of the cell wall such that the surface of the yeast is more fused to an antigen having a particular charge characteristic or Combine. Targeting agents, such as antibodies, binding peptides, soluble receptors, and other ligands, can also be incorporated into a mutant antigen in the form of a fusion protein or otherwise allowed to bind to an antigen to bind the antigen to the carrier.

In another example, the vector and the mutant polypeptide are bound to each other by a more passive, non-specific or non-covalent binding mechanism, such as by gently mixing the vector with the antigen in a buffer or other suitable formulation.

In some embodiments of the invention, both the vector and the mutated antigen are intracellularly loaded into a cargo, such as a dendritic cell or macrophage, to form an immunogenic composition. Dendritic cells can be any dendritic cell known in the art. Dendritic cells are cells of monocytes and lymphocyte lines and are known to be the most potent antigen presenting cells (APCs) and stimulate antigen-specific T cell responses. Mature dendritic cells are generally recognized as having the following cell expression marker phenotypes: MAC3 ^- , CD80 ⁺ , CD86 ⁺ , CD401 ° w , CD54 ⁺ , MHC class I and MHC class II, and are capable of FITC-dextran absorption. In some examples, the dendritic cell line used in the compositions of the invention is isolated from the patient (i.e., autologous cells) to be administered the composition. Dendritic cells can be isolated from bone marrow or peripheral blood. Such cells can be produced, for example, from peripheral blood mononuclear cells by culturing in the presence of, for example, granulocyte macrophage colony stimulating factor, IL-4 and TNF-α. Other methods for isolating and producing dendritic cells are known in the art. (See, for example, Wilson et al, 1999, Immunol 162: 3070-8; Romani et al, 1994, J. Exp Med 180: 83-93; Caux et al., 1996, J. Exp Med 184: 695-706 and Kiertscher et al. , 1996, J. Leukoc . Biol 59: 208-18).

In order for dendritic cells to efficiently present antigen to native T cells, immature dendritic cells must be activated to mature, as defined by upregulation of MHC and costimulatory molecules. Yeast via Toll-like receptors (TLRs) (see, for example, Takeda K. and Akira S., 2005, International Immunology , p. 17: p. 1-14), mannan, dextran, and C-type lectin receptors Dendritic cells provide potent activation stimuli leading to costimulatory immune receptors, upregulation of MHC molecules, and secretion of immunoregulatory cytokines. In addition, when the yeast is preloaded with antigen prior to loading into the dendritic cells, the yeast provides antigen to the dendritic cells in a highly concentrated encapsulation in a highly affinity internalization, thereby effectively increasing the amount of antigen available for treatment. Those skilled in the art will appreciate that other vectors can be used to load dendritic cells.

The various forms that can be used to achieve the loading of the two components are discussed in more detail below. As used herein, the term "loading" and its derivatives refer to the insertion, introduction, or entry of components (such as dendritic cells) into a component (yeast vehicle and/or antigen). Intracellular loading of a component refers to the intracellular compartment in which a component is inserted or introduced into a cell (eg, through the plasma membrane and at least into the cytoplasm, phagosome, lysosome, or part of the intracellular space of the cell). Loading a component into a cell involves any environment in which the component is forced into the cell (eg, by electroporation) or placed in the component via a process (eg, phagocytosis) that is substantially likely to enter the cell (eg, A method of contacting a cell or in the vicinity of a cell. Loading techniques include, but are not limited to, diffusion, active transport, liposome fusion, electroporation, phagocytosis, and water bath sonication. In some instances, a passive mechanism for loading dendritic cells with yeast vehicle and/or antigen is used, including passive effects of dendritic cells on yeast vehicle and/or antigen.

In the case of yeast, the yeast vehicle can be loaded into the dendritic cells at about the same time or simultaneously with the mutant polypeptide, but one component can also be loaded into the cells and then loaded into another component over a period of time. In some examples, the yeast vehicle and the mutant polypeptide are bound to each other and subsequently loaded into dendritic cells. For example, a recombinant yeast vehicle or yeast vehicle that exhibits a mutant polypeptide can be loaded into dendritic cells with any other complex or mixture of mutant polypeptides. Dendritic cells can additionally be loaded with a free mutant polypeptide, i.e., a polypeptide that does not directly bind to the yeast vehicle when introduced (loaded) into dendritic cells. The addition of the free polypeptide to the yeast vehicle-antigen complex additionally enhances the immune response against the polypeptide. The free polypeptide loaded into the dendritic cells need not be the same as the polypeptide expressed by the yeast vehicle, the polypeptide loaded into the yeast vehicle, or otherwise bound to the yeast vehicle. In this way, an immune response against the target cell or virus can be enhanced.

In some examples, a composition comprising a mutant polypeptide or nucleic acid encoding the same includes one or more adjuvants (including adjuvants described herein) and/or a carrier, but is not required to do so. Adjuvants are generally substances which generally enhance the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, TLR agonists, CpG sequences as described herein (see, for example, Krieg et al. WO 96/02555), single-stranded RNA, double-stranded RNA, Freund's adjuvant, Other bacterial cell wall components (including LPS, flagellin), aluminum-based salts, calcium-based salts, cerium oxide, polynucleotides, toxoids, serum proteins, viral capsid proteins, other bacterial derivative preparations, gamma interferon, embedded Segment copolymer adjuvant (such as Hunter's Titermax adjuvant (CytRx.TM., Inc. Norcross, Ga.), Ribi adjuvant (available from ImmunoChem Research, Inc., Hamilton, Mont.)) and saponin and Derivatives (such as Quil A (available from Superfos Biosector A/S, Denmark)).

Carriers are generally compounds which increase the half-life of the therapeutic composition in the animal being treated. Suitable carriers include, but are not limited to, polymeric controlled release formulations, biodegradable implants, liposomes, oils, esters, and glycols.

The immunogenic compositions of the invention may also comprise one or more pharmaceutically acceptable excipients. As used herein, "pharmaceutically acceptable excipient" refers to any substance suitable for delivery of a composition useful in the methods of the invention to a suitable in vivo or ex vivo site. In some embodiments, a pharmaceutically acceptable excipient is capable of maintaining a vector (or a dendritic cell comprising a vector) in a form such that the vector (with mutation) after the vector or cell has reached the target cell, tissue or site in the body Peptide binding) or dendritic cells (loaded with vectors and mutant antigens) are capable of eliciting an immune response (including cellular immune responses, humoral immune responses, or both) at the site of the target (which should be noted throughout the body). Suitable excipients of the invention include excipients or formulations (also referred to herein as non-standard carriers) that are transported but are not intended to specifically target a composition or vaccine. Examples of pharmaceutically acceptable excipients include, but are not limited to, water, physiological saline, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solution, Hank's solution (Hank's solution), other aqueous physiological balance solutions, oils, esters and glycols. The aqueous carrier can contain, for example, suitable adjunctive substances required to enhance the physiological conditions of the recipient by enhancing chemical stability and isotonicity. Suitable auxiliary materials include, for example, sodium acetate, sodium chloride, calcium lactate, potassium chloride, calcium chloride, and other materials used to prepare phosphate buffers, Tris buffers, and bicarbonate buffers. The auxiliary substances may also include preservatives such as thimerosal, m-cresol or o-cresol, formalin and benzyl alcohol.

cancer

As used herein, cancer includes any type of tumor or neoplasia including, but not limited to, colorectal cancer, melanoma, squamous cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, Testicular cancer, prostate cancer, ovarian cancer, bladder cancer, skin cancer, brain cancer, hemangioma, angiosarcoma, mast cell tumor, primary liver cancer, lung cancer, pancreatic cancer, gastrointestinal cancer, renal cell carcinoma, hematopoietic tumor formation and Metastasis. For example, chronic myelogenous leukemia is a type of cancer that is expected to be treated and prevented by the present invention.

Examples of specific cancer antigens include, but are not limited to, MAGE (including but not limited to MAGE3, MAGEA6, MAGEA10), NY-ESO-1, gp100, tyrosinase, EGFR, PSA, PSMA, VEG-F, PDGFR, KIT, PMSA, CEA, HER2/neu, Muc-1, hTERT, MARTI, TRP-1, TRP-2, Bcr-Abl and p53 (TP53), p73, Ras, PTENSrc, p38, BRAF, APC (adenomatous) Colon polyps), myc, VHL (von Hippel Lindau protein), Rb-1 (retinal blastoma), Rb-2, BRCA1, BRCA2, AR (androgen receptor), Smad4, Mutant carcinogenic forms of MDR1 and FLT3.

In some examples, a cancer antigen is suitable for or obtainable from a molecule (such as a protein, peptide, glycoprotein or carbohydrate) that is targeted by a therapeutic and/or prophylactic agent. Molecular targets for therapeutic and/or prophylactic cancer agents are known in the art and include, but are not limited to, cell surface receptors (such as receptor tyrosine phosphatase, receptor serine/threonine) Kinases and tyrosine kinases, intracellular signal transduction molecules (such as intracellular tyrosine kinases or other second signal transduction molecules) and transcription factors, cell cycle regulators, proteasome components, and angiogenesis Protein and protein associated with apoptosis control.

Therapeutic and/or prophylactic agents that have been observed to cause cancer have resulted in the emergence of escape mutants, i.e., mutant polypeptides. For example, mutations have been found in Bcr-Abl, which have been reported to render individuals resistant to previous treatment with the Bcr-Abl tyrosine kinase inhibitor, imatinib (Gleevec), resistant to the treatment. Gorre et al, Science, 293:876-880 (2001); Shah et al, Cancer Cell, 2:117-125 (2002); Branford et al, Blood, 99(9): 3742-3745 (2002); Deininger Et al, Blood, 105(7): 2640-263 (2005). Similarly, mutations in EGFR have also been found in patients with non-small cell lung cancer (NSCLC), making them resistant to treatment with gefitinib or erlotinib (Tarceva). Kobayashi et al, N. Engl. J. Med., 352(8): 786-792 (2005). Therefore, the efficacy of such anticancer agents is significantly limited by the presence of mutant polypeptides.

Accordingly, provided herein are mutant polypeptides (or epitope mimetics thereof) that are encoded by an oncogene and/or expressed by a cancer cell, or that are known to have a specificity for the administration of a therapeutic agent and/or a prophylactic agent. An immunogenic composition of a nucleic acid of a mutated mutant polypeptide, and a method for eliciting an immune response to the mutant polypeptide or a cell expressing the mutant polypeptide. In some examples, the immune response is a cellular immune response. In some instances, the immune response is a humoral immune response. In other examples, the immune response includes cellular and humoral immune responses.

A polypeptide mutant of a cancer antigen can be pre-existing in a mammal (i.e., present at the time of diagnosis) and selectively occurs due to administration of a therapeutic agent and/or prevention. Alternatively, the polypeptide mutant can occur due to the pressure exerted by the agent. The mutation can be located at any amino acid position of the cancer antigen. Although the mutation of the polypeptide is described in the context of a single mutation, it is understood that the mutant polypeptide may comprise more than one (such as 2, 3, 4, 5 or more) amino acid mutations. For example, a cancer antigen can contain one or more mutations at different amino acid positions. In one aspect, the mutation is an E255K escape mutant. In another aspect, the mutation is a T315I mutation. In another aspect, the mutation is a M351T mutation. In other aspects, the mutation was a combination of all three E255K, T315I and M351T. In other aspects, the mutation is a combination of two of the three mutations disclosed above (eg, E255K/T315I or T315I/M351T or E255K/M351T). The cancer antigen may further contain other mutations, such as mutations associated with transformation events. Illustrative examples of mutant polypeptides of cancer antigens are disclosed in Table I.

In some examples, the mutant polypeptide can be a fusion polypeptide comprising a plurality of immunogenic domains from a mutant polypeptide of one or more cancer antigens. For example, it is known that there are several different mutations (eg, E255K, T315I, and M351T) in the Bcr-Abl protein that occurs after administration of Gleevec. The mutated polypeptide may comprise one or more Bcr-Abl mutations at the same position and/or at different positions and/or a combination of mutations at more than one position.

In some examples, the polypeptide mutant comprises a mutation in Bcr-Abl. Bcr-Abl is a reactive tyrosine kinase produced by DNA translocation between chromosomes 9 and 22. It is reported that Bcr-Abl is the cause of chronic myelogenous leukemia (CML) and its constitutive kinase activity is the key to its ability to transform hematopoietic cells in vivo. Imatinib (Gleevec, 2-phenylaminopyrimidine) is a tyrosine kinase inhibitor that is a therapeutic agent for CML. Multiple mutations in Bcr-Abl that render proteins resistant to drug therapy (eg, Gleevec treatment) have been identified in vivo and in vitro. Deininger et al, Blood, 105(7): 2640-2653 (2005); Azam et al, Cell, 112:831-43 (2003). Table I provides escape mutants of Bcr-Abl (and other cancer antigens) recognized in Gleevec-resistant mammals (mutations 1-28) and other mutations recognized by in vitro methods (mutations 29-132) An illustrative list of numbers). These mutations are located in different domains of Bcr-Abl, including but not limited to kinase domains (such as P-loop, A-loop, T315, C-helix, SH3 contact or SH2 contact), end-capped structures Domain, SH3 domain, SH2 domain and other connection zones. In one embodiment, the mutant polypeptide comprises E255K, T315I, and M351T. In other embodiments, the mutant polypeptide comprises T315I. In other embodiments, the mutant polypeptide comprises E255K. In other embodiments, the mutant polypeptide comprises M351T. In other embodiments, the mutant polypeptide comprises a combination of two of the following mutant polypeptides: E255K, T315I, and M351T (eg, E255K/T315I or T315I/M351T or E255K/M351T).

In some examples, the mutant polypeptide comprises a mutation in EGFR. EGFR is a receptor tyrosine kinase that plays a key role in the initiation of cell division in normal cells and cancer cells. In a variety of cancers, including non-small cell lung cancer (NSCLC) and glioblastoma (brain cancer), EGFR overexpression or mutations are reported, which are believed to be associated with tumor formation and growth. Two oral aniline quinazoline EGFR tyrosine kinase inhibitors, gefitinib (Iressa) and erlotinib (Tarceva), have been approved for the treatment of NSCLC in the United States. The escape mutant T790M was found in EGFR and was reported to render mammalian subjects resistant to Iressa or Tarceva treatment.

Accordingly, provided herein are compositions comprising a mutant polypeptide of EGFR (or an epitope mimetic thereof) or a nucleic acid encoding EGFR and methods for use in inducing an immune response. In some examples, the immune response is a cellular immune response. In some instances, the immune response is a humoral immune response. In other examples, the immune response includes cellular and humoral immune responses. In some examples, the mutant polypeptide comprises a mutation in the EGFR kinase domain. In some examples, the mutant polypeptide comprises a T790M mutation with respect to a wild-type EGFR polypeptide.

In some examples, the mutant polypeptide comprises a mutation in a platelet-derived growth factor receptor (PDGFR). PDGFR is a receptor tyrosine kinase. Activation of PDGFR has been reported to be critical for the progression of various types of cancer, such as glioblastoma, hyperplastic cutaneous fibrosarcoma, and CML. A single escape mutation (T674I) has been identified in patients with Gleevec-treated eosinophilia syndrome. See, for example, Cools et al, New Engl. J. Med., 348: 1201-1214 (2003). Accordingly, provided herein are compositions comprising a PDGFR mutant polypeptide (or an epitope mimetic thereof) or a nucleic acid encoding a PDGFR and methods for inducing an immune response. In some examples, the immune response is a cellular immune response. In some instances, the immune response is a humoral immune response. In other examples, the immune response includes cellular and humoral immune responses. In some examples, the mutant polypeptide comprises a mutation in the PDGFR kinase domain. In some examples, the mutant polypeptide comprises a T764I mutation with respect to a wild-type PDGFR polypeptide.

In some embodiments, the mutant polypeptide comprises a mutation in KIT. KIT is a stem cell factor (SCF) tyrosine kinase receptor. Activation of mutations in the kinase domain by KIT has been reported to be associated with gastrointestinal matrix tumors (GIST) and other types of tumors. The escape mutation (T670I) was found in KIT and was reported to render patients resistant to Gleevec treatment. See, for example, Tamborini et al, Gastroenterology, 127:294-299 (2004). Accordingly, provided herein are compositions comprising a mutant polypeptide of KIT (or an epitope mimetic thereof) or a nucleic acid encoding KIT and methods for use in inducing an immune response. In some examples, the immune response is a cellular immune response. In some instances, the immune response is a humoral immune response. In other examples, the immune response is a cellular and humoral immune response. In some examples, the mutant polypeptide comprises a mutation in the KIT kinase domain. In some examples, the mutant polypeptide comprises a T670I mutation with respect to a wild-type KIT polypeptide.

In some examples, the mutant polypeptide comprises a mutation in FLT3 that occurs in response to administration of the subject agent, comprising a population selected from the group consisting of A627T, N676D, N676S, F691L, F691I, G697R, and G697S relative to the wild-type FLT-3 polypeptide. Amino acid mutation. See, for example, Cools et al, Cancer Res., 64: 6385-6389 (2004). In some examples, the mutant polypeptide comprises a mutation in p38, including a mutation at a position relative to the wild type protein T106. In other examples, the mutant polypeptide comprises a mutation in Src, including a mutation at a position relative to the amino acid T341 amino acid. In some examples, the mutant polypeptide comprises a mutation in the FGFR, including an amino acid mutation at a position relative to the wild type protein V561.

Accordingly, provided herein are compositions comprising the mutant polypeptide (or an epitope mimetic thereof) or a nucleic acid encoding a mutant polypeptide, and methods of inducing an immune response. In some examples, the immune response is a cellular immune response. In some instances, the immune response is a humoral immune response. In other examples, the immune response includes cellular and humoral immune responses. In some instances, it is known that a mutant polypeptide that occurs in response to a specific mutation of an agent or that has occurred in the process itself is immunogenic, that is, does not require binding to an adjuvant, but is not required to do so. In other instances, mutant polypeptides that occur in response to an agent or that have emerged in the process are known to be immunogenic in combination with an adjuvant that promotes their antigenicity, such as a Toll-like receptor ligand or agonist. Or a CpG nucleotide sequence, or a vector or vehicle, such as a yeast vehicle.

Accordingly, the compositions described herein are useful for eliciting an immune response to a mutant polypeptide in a mammal comprising administering to the mammal an effective amount of the composition together with the subject therapeutic and/or prophylactic agent. In some examples, the composition comprises one or more of: i) a yeast vehicle comprising a nucleic acid encoding at least one mutant polypeptide comprising a mutated fragment or an epitope mimetic; ii) comprising at least one a mutant polypeptide, a yeast vehicle comprising the mutated fragment or epitope mimetic; iii) a yeast vehicle that binds to at least one mutant polypeptide, which comprises a mutated fragment or an epitope mimetic; iv) intracellular loading of dendrites a yeast vehicle comprising a nucleic acid encoding at least one mutant polypeptide comprising a mutated fragment or an epitope mimetic; or v) a yeast vehicle loaded into dendritic cells and at least one mutant polypeptide, comprising A fragment or epitope mimetic of a mutation, wherein the mutant polypeptide has occurred or has occurred in response to administration of at least one specific mutation of the therapeutic agent and/or prophylactic agent.

In addition, such compositions are useful in the manufacture or manufacture of a medicament for inducing an immune response to a mutant polypeptide in a mammal in combination with the subject therapeutic and/or prophylactic agent. In some examples, the mutant polypeptide is an oncogene, a tumor associated antigen, or a polypeptide expressed by a cancer cell. In some examples, the cancer cell line is selected from the group consisting of colorectal cancer, melanoma, squamous cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, testicular cancer, prostate Cancer, ovarian cancer, bladder cancer, skin cancer, brain cancer, hemangioma, angiosarcoma, mast cell tumor, primary liver cancer, lung cancer, pancreatic cancer, gastrointestinal cancer, renal cell carcinoma, hematopoietic tumor formation, and metastatic cancer.

In some examples, the immune response is a cellular immune response. In other examples, the immune response is a humoral immune response. In other examples, the immune response includes cellular and humoral immune responses.

Furthermore, the compositions described herein are for use in the treatment of a disease in a mammal comprising administering to the mammal an effective amount of a composition, wherein the disease is associated with a therapeutic and/or prophylactic agent known to be responsive to administration of the subject. At least one specific mutation occurs or has been associated with a mutant polypeptide that occurs in the process. In some examples, the compositions are used with the subject therapeutic and/or prophylactic agents. Additionally, the compositions can be used in the manufacture or manufacture of a medicament for the treatment of a disease in a mammal in combination with a subject therapeutic and/or prophylactic agent. In some examples, the mutant polypeptide is an oncogene, a tumor associated antigen, or a polypeptide expressed by a cancer cell. In some examples, the cancer cell line is selected from the group consisting of colorectal cancer, melanoma, squamous cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, testicular cancer, prostate Cancer, ovarian cancer, bladder cancer, skin cancer, brain cancer, hemangioma, angiosarcoma, mast cell tumor, primary liver cancer, lung cancer, pancreatic cancer, gastrointestinal cancer, renal cell carcinoma, hematopoietic tumor formation, and metastatic cancer. In some examples, the disease is cancer.

Methods for identifying novel mutant polypeptides in cancer antigens that arise from administration of a medicament are known in the art. Escape mutations identified by in vivo methods have been shown to be highly correlated with escape mutations produced in vivo. See, for example, Azam et al, Cell, 112:831-843 (2003); Cools et al, Cancer Research, 64:6385-6389 (2004); Blenkoke et al, Chem. Biol ., 11:691-701 (2004) . For example, Azam et al. provide a screening method for identifying resistant mutant polypeptides that target a targeted anticancer agent, which is generally applicable to any agent-mutant polypeptide pair (Azam et al, Biol. Proced. Online, 5 (1) ): 204-210 (2003)). Briefly, a cDNA encoding a target mutant polypeptide is cloned into a selection vector and subjected to subsequent mutations to produce a library of target cancer polypeptide mutations. The library is then introduced into cells that are sensitive to drug treatment. A pure line that is resistant to treatment with a drug is then selected in the presence of a therapeutic agent, which is isolated and sequenced to reveal putative mutations. To verify the resistance phenotype of each candidate mutation, mutations were also regenerated in the native cDNA by localization mutations. The mutated cDNA was introduced into drug-sensitive cells to confirm its drug resistance phenotype. Drug resistance can additionally be confirmed by cell proliferation assays. The structural results of the mutations can also be analyzed by mapping to a protein crystal structure model.

Provided herein are compositions comprising a vector that binds to a mutated polypeptide, including compositions that are administered directly to a patient or are first loaded with a carrier such as a dendritic cell using a variety of techniques known to those skilled in the art. For example, the carrier can be dried by lyophilization or by exposure to liquid nitrogen or dry ice. Compositions comprising yeast vehicles can also be prepared by compacting the yeast into a cake or lozenge, such as for use in yeast used in baking or brewing operations. In addition, the carrier may be combined with a pharmaceutically acceptable excipient, such as an isotonic buffer tolerated by the host cell, prior to loading into dendritic cells or other types of administration. Examples of such excipients include water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Non-aqueous vehicles such as fixed oils, sesame oil, ethyl oleate and triglycerides can also be used. Other suitable formulations include suspensions containing a viscosity enhancing agent such as polyethylene glycol (PEG), sodium carboxymethylcellulose, sorbitol, glycerol or dextran. Excipients may also contain minor amounts of additives such as substances that enhance isotonicity and chemical stability. Examples of the buffer include phosphate buffer, bicarbonate buffer, and Tris buffer, and examples of the preservative include thimerosal, m-cresol or o-cresol, formalin, and benzyl alcohol. The standard formulation can be a liquid injectable or a solid that can be absorbed into a suitable liquid to form an injectable suspension or solution. Thus, in a non-liquid formulation, the excipient may comprise, for example, dextrose, human serum albumin and/or a preservative, to which sterile water or physiological saline may be added prior to administration.

Provided herein are methods comprising administering to a mammal having a cancer or infection risk or suffering from cancer or infection, a composition comprising a vector that binds to a mutant antigen, such as an immunogenic composition. Such methods are generally useful for inducing an immune response, in some instances a cellular immune response in a mammal. It is believed that such methods can be used to induce a cellular immune response to a mutant polypeptide that has emerged in response to the agent or that would respond to the agent, thereby minimizing or reversing resistance to the agent, and/or extending the agent. The efficacy, and / or to minimize, reduce or reverse certain symptoms of the disease or infection.

Accordingly, provided herein is a method for minimizing resistance to a prophylactic and/or therapeutic agent in a mammal comprising administering to the mammal an effective amount comprising a binding to a mutant polypeptide that has been responsive to the agent. A composition of a carrier, such as a yeast vehicle. Also provided herein are methods for reducing the resistance to administration of a medicament having a risk of disease or infection or a mammal suffering from a disease or infection (prophylactic and/or therapeutic administration) comprising administering to the mammal an effective amount And a composition, wherein the composition comprises: a. a cell, vector or virus comprising a nucleic acid encoding a mutant polypeptide; b. a cell, vector or virus that binds to the mutant polypeptide; c. a mutant polypeptide, or induced to the mutant polypeptide An immunoreactive peptide (epitope mimetic); or d. a nucleic acid encoding a mutant polypeptide, or a nucleic acid that binds to the nucleic acid, such as siRNA or antisense RNA; wherein an effective amount of the composition is administered with the agent. In some examples, the composition comprises one or more of: i) a yeast vehicle comprising a nucleic acid encoding at least one mutant polypeptide comprising a mutated fragment or an epitope mimetic; ii) comprising at least one a mutant polypeptide, a yeast vehicle comprising the mutated fragment or epitope mimetic; iii) a yeast vehicle that binds to at least one mutant polypeptide, which comprises a mutated fragment or an epitope mimetic; iv) intracellular loading of dendrites a yeast vehicle comprising a nucleic acid encoding at least one mutant polypeptide comprising a mutated fragment or an epitope mimetic; or v) a yeast vehicle loaded into dendritic cells and at least one mutant polypeptide, comprising A fragment or epitope mimetic of a mutation, wherein the mutant polypeptide has occurred or has occurred in response to administration of at least one specific mutation of the therapeutic agent and/or prophylactic agent.

In some examples, the composition is capable of eliciting a cellular immune response. In other examples, the immune response is a humoral immune response. In other examples, the immune response includes cellular and humoral immune responses.

In some examples of such methods, the composition comprises an adjuvant. In other examples, the composition additionally comprises an agonist or ligand to the Toll-like receptor. In other examples, the composition comprises a yeast vehicle. In other examples, the composition comprises a CpG sequence. In other examples, the cell is a dendritic cell. In some examples, the mammal is a human.

Also provided herein are vectors (including, for example, yeast vehicles), viruses, and compositions (such as comprising a yeast vehicle) for use in a method of inducing a mutant polypeptide-specific immune response in a mammal that has been administered, will be administered, or is being administered a medicament. Yeast-based compositions), including immunogenic compositions. In some instances, the mammal is at risk for the disease, and the prophylactic administration of the mutated polypeptide, the fragment comprising the mutated fragment or the epitope mimetic, and/or the combination comprising the vector is administered prophylactically, simultaneously and/or after the agent. Things. In other examples, the mammal is afflicted with a disease, and the conjugated polypeptide-binding vector and/or composition comprising the vector is therapeutically administered prior to, concurrently with, and/or after the agent. For example, such yeast vehicles that are administered in combination with a mutant polypeptide can be used to enhance the sensitivity of the mammal to the therapeutic and/or prophylactic agent, and/or to enhance the therapeutic efficacy of the agent, and/or to extend the The effective life cycle of the drug. In some examples, a mutant polypeptide has been identified prior to administration of a vector or composition as described herein, while in other instances it is predicted that a mutant polypeptide will be present in response to the agent.

A composition comprising a vector (such as a yeast vehicle) comprising a mutated polypeptide, a fragment comprising the mutated or an epitope mimetic thereof, and a therapeutic agent or prophylactic agent, which may be administered simultaneously or sequentially, before or after administration of the agent, may be administered simultaneously or sequentially. Agent. Simultaneous administration is contemplated as being in one composition or in the form of individual compositions. In some examples, the agent is in a different formulation than the mutant polypeptide or its encoding nucleic acid system and is administered simultaneously or separately. It will be appreciated that in some instances, the agent and the mutant polypeptide or nucleic acid encoding the same are administered sequentially, for which it can be administered daily, weekly or monthly according to the appropriate judgment of the mammalian physician. The term "simultaneous administration" as used herein means the administration of a composition comprising a yeast vehicle and a therapeutic agent on the same day. The composition or therapeutic agent comprising the mutated polypeptide can be administered first. When administered simultaneously, the composition comprising the yeast vehicle and the therapeutic agent may be contained in the same dose (ie, a unit dose comprising the composition comprising the yeast vehicle and the therapeutic agent) or in discrete doses (eg, comprising a yeast vehicle) The composition is contained in one dosage form and the therapeutic agent is contained in another dosage form).

In some instances, a composition comprising a mutated polypeptide is administered as a "subsequent treatment", that is, after treatment with the agent has been initiated or after an increase in disease symptoms is observed. However, it is also possible to administer a composition comprising a carrier, such as a yeast vehicle, prior to initiation of treatment with a therapeutic or prophylactic agent.

The methods described herein can also comprise administering a vector and a mutant polypeptide to a mammal, wherein the vector and the polypeptide are not complexed to each other, i.e., the polypeptide is not recombinantly expressed by the vector, is loaded into the vector, or is linked to the vector. The vector and the mutant polypeptide may be admixed to the formulation or administered separately prior to administration to the subject. The administration process can be carried out ex vivo, for example by introduction of dendritic cells loaded with yeast vehicle; or ex vivo. Ex vivo administration refers to a partial regulatory step in vitro in a mammal, such as administering a composition of the invention to a population of cells (dendritic cells) removed from a mammal under conditions such that the vector and the mutant polypeptide are loaded into the cell and The cells return to the mammal. The composition comprising the carrier can then be returned to the mammal or administered to the mammal by any suitable mode of administration.

Compositions (including compositions comprising dendritic cells loaded with a carrier and a mutated polypeptide) can be administered, for example, systemically or mucosally. Routes of administration are well known to those skilled in the art, depending on the type of condition, the mutant polypeptide used, and/or the target cell population or tissue. Administration methods include, but are not limited to, intravenous administration, intraperitoneal administration, intramuscular administration, intranodal administration, intracoronary administration, intraarterial administration (eg, administration into the carotid artery), subcutaneous administration, transdermal administration, intratracheal administration. Administration, subcutaneous administration, intra-articular administration, intraventricular administration, inhalation (eg aerosol), intracranial, intraspinal, intraocular, intraocular, intranasal, oral, pulmonary administration, catheter infusion, and direct injection into tissues . Routes of administration include: intravenous, intraperitoneal, subcutaneous, intracortical, nodular, intramuscular, transdermal, inhalation, intranasal, oral, intraocular, intra-articular, intracranial, and spinal. Parenteral delivery includes intracortical, intramuscular, intraperitoneal, intrapleural, intrapulmonary, intravenous, subcutaneous, atrial catheter and venous catheter routes. Ear delivery can include ear drops, intranasal delivery can include nasal drops or intranasal injections, and intraocular delivery can include eye drops. Aerosol (absorption) delivery can also be carried out using standard methodologies in the art (see, for example, Stribling et al, Proc. Natl. Acad. Sci. USA 189: 11277-11281 (1992)). For example, in one example, a composition comprising a yeast vehicle can be formulated into a composition suitable for spray delivery using a suitable inhalation device or nebulizer. Oral delivery can include solids and liquids that can be used orally, and can be used to produce mucosal immunity, and compositions comprising yeast vehicles can be readily prepared for use, for example, in lozenges or capsules or formulated into foods and beverages. The product is delivered orally. Other routes of administration that regulate mucosal immunity can also be used to treat cancer or infectious diseases. Such routes include bronchial, intracortical, intramuscular, intranasal, other inhaler, rectal, subcutaneous, topical, transdermal, vaginal and urethral routes. The route of delivery can be any route of delivery of a composition comprising a yeast vehicle to the respiratory system including, but not limited to, inhalation, intranasal, intratracheal, and the like.

The effective administration of a cell, a vector (such as a yeast vehicle) or a virus, or a composition comprising a cell, vector, virus or yeast vehicle, as described herein, to a mammal having a disease risk or suffering from a disease does not require protection of the mammal. Free of disease. Effective dosage parameters can be determined using standard methods in the art, which are suitable for minimizing or minimizing disease symptoms or minimizing disease progression. Such methods include, for example, determining survival, side effects (i.e., toxicity), and progression or regression of the disease.

For the use of a yeast vehicle, a suitable single dose size is a dose that is capable of eliciting an immune response in a mammal when administered one or more times over a suitable period of time, the immune response being a cellular immune response in some instances, and It can be an antigen-specific immune response. As is known in the art, the dosage of the composition required to elicit an immune response will depend on a variety of factors. Those skilled in the art can readily determine the appropriate single dose size for administration based on the mammalian body size and route of administration.

A suitable single dose of a composition comprising a carrier that binds to a mutated polypeptide is an amount effective to provide an effective amount of an immune response to a given cell type, tissue or body area of the patient upon administration of one or more doses over a suitable period of time and/or Or the dose of the mutant polypeptide. In the case of yeast, a single dose of the yeast vehicle of the invention is from about 0.004 YU (4 x 10 ³ cells) to about 100 YU (1 x 10 ⁹ cells) per dose (i.e., per organism). , such as 0.1 YU (1 × 10 ⁶ cells) to about 100 YU (1 × 10 ⁹ cells), including any intermediate dose, in increments of 0.1 × 10 ⁶ cells (ie 1.1 × 10 ⁶ , 1.2 × 10 ⁶ , 1.3 × 10 ⁶ etc.). In one embodiment, a 2 YU (2 × 10 ⁶ yeast). This dosage range is effective for any organism of any size, including mice, monkeys, humans, and the like. When the composition is administered by loading the yeast vehicle and the mutant antigen into dendritic cells, the single dose of the composition described herein is from about 0.5 x ¹⁰⁶ to about 40 x 10 ⁶ per mammal per administration. Dendritic cells. In other examples, a single dose per body about about 1 × 10 ⁶ to 20 × 10 ⁶ dendritic cells per mammal other examples are about 1 × 10 ⁶ to about 10 × 10 ⁶ dendritic cells . An "enhancing" dose of a composition comprising a yeast vehicle as described herein may be administered in the event of a reduction in the immune response against the mutated antigen, or an enhanced dose may be administered as needed to provide an immune response or inducement against a particular mutant polypeptide. The memory response of a particular mutant polypeptide. The booster dose can be administered from about 1 week to several years after the initial administration. In one example, the administration process is a combination of administration of 1 x 10 ⁵ to about 1 x 10 ⁹ yeast cell equivalents per week for 3 months, followed by once a week for 1 month (5 doses) followed by monthly Do it once.

In some examples, described herein as the yeast vehicle comprising the composition comprising the dendritic cells per patient per single dose of about 0.5 × 10 ⁶ to about 40 × 10 ⁶ dendritic cells, while in another example of per patient per single dose of about 1 × 10 ⁶ to about 10 × 10 ⁶ dendritic cells. These doses are given to typical humans or other primates. Dosages suitable for use in other animals can be determined by those skilled in the art. For example, for mice, a suitable dose is from 1 x ¹⁰⁶ to about 3 x 10 ⁶ per single dose per mouse. Other dosages can be determined by those skilled in the art and are well within the capabilities of those skilled in the art. Effective administration of compositions containing mammalian each mammalian Per single dose of about 0.5 × 10 ⁶ to about 40 × 10 ⁶ dendritic cells.

It will be apparent to those skilled in the art that the number of agents administered to a mammal will depend on the nature of the yeast vehicle and the mammal's response to the administration. Accordingly, the number of suitable agents within the scope of the present invention includes any number desired for the desired purpose. For example, the administration can be repeated to increase the number of T cells available to attack the target cells. As will be apparent to those skilled in the art, the dosage and frequency of administration can be adjusted during the administration of the drug.

Those skilled in the art can also monitor the effects of administration by using animal models such as leukemia mouse models. Such mouse models are readily known to those skilled in the art. One method of monitoring the effects of administration is to monitor the survival rate of mice (inoculated and not vaccinated) after challenge or enhancement. Another method of monitoring the effects of administration is to measure the percentage of leukemia cells over time. There are some mouse models by which leukemia cells are labeled with a marker such as green fluorescent protein (GFP). GFP positive cells are indicative of leukemia cells and can be monitored in various intervals of the body, such as bone marrow.

Set

Provided herein are kits for performing any of the methods described herein. The kit of the invention may comprise at least one yeast vehicle and at least one cancer-associated mutant polypeptide that is known to have occurred or has occurred in response to a specific mutation in a therapeutic and/or prophylactic agent. The kit may additionally comprise a therapeutic and/or prophylactic agent. In some examples, the agent is targeted to cancer cells. The kit may additionally include instructions for performing the methods described herein.

A kit comprising a single component typically has a component enclosed in a container, such as a vial, ampoules or other suitable storage container. Likewise, a kit comprising more than one component can also have reagents in the container (either alone or in a mixture).

The description relating to the use of the kit for carrying out the invention generally describes how to use the contents of the kit to carry out the method of the invention. The description provided by the kit of the present invention is generally written on a label or package insert (such as a sheet included in a kit), but machine readable instructions (such as instructions on a magnetic or optical storage disc) ) also applies.

The invention includes a kit for immunizing a human patient against a poxvirus infection, the kit comprising an immunogenic amount of a soluble truncated poxvirus envelope protein encoded by an isolated nucleic acid encoding a vaccinia virus soluble truncated envelope protein.

Although the foregoing invention has been described in some detail by way of illustration Therefore, the description and examples should not be construed as limiting the scope of the invention.

Instance Example 1 Construction of a yeast vehicle containing escape mutants

The purpose of this example is to describe a yeast construct containing an escape mutant species known to be produced by Gleevec.

A yeast-based carrier of about 400 amino acids representing p210 Bcr-Abl was produced. These vectors are under the control of the constitutive TEF1 promoter (transcription elongation factor 1). The yeast containing the following construct was cultured, collected, washed with PBS, fired at 56 ° C for 1 hour, washed with PBS and then resuspended in PBS for administration to mice.

The following sequences were used to generate a variety of yeast constructs: Construct 1 VK13-BA5M-VAX E1.E2.E3

This construct contains three escape mutations: E255K, T315I and M351T. This sequence was altered at V227I and N336S, which corresponds to a change from a human residue to a residue consistent with the mouse residue. The MADEAP sequence has been added to the Bcr-Abl sequence at the start to achieve stability. Bold text shows mutations for the escape mutant (E255K, T315I and M351T). The last 6 residues are hexahistidine labels. The 328 amino acid lines between the MADEAP sequence and the hexahistidine tag are from the human Abl kinase domain. The last amino acid is the stop codon ( ^* ).

Structure 2 VK14-VAX only E2

This construct contains only one escape mutant: T315I. This sequence was altered at V227I and N336S, which corresponds to a change from a human residue to a residue consistent with the mouse residue. The MADEAP sequence has been added to the Bcr-Abl sequence at the start to achieve stability. The bold text shows the mutation corresponding to the escape mutant (T315I). The last 6 residues are hexahistidine labels. The sequence between the MADEAP sequence and the hexahistidine tag is from the human Abl kinase domain. The last amino acid is the stop codon ( ^* ).

Structure 3 VK15-JCN-BAM-VAX b3a2

This construct system has a wild type Bcr-Abl of mouse residues at the junction of Bcr and Abl. This sequence was changed at V227I and N336S and at two changes to the upstream residue of the human Bcr-Abl junction replaced by the corresponding mouse amino acid, which corresponds to residues from human residues to residues consistent with mouse residues. Changes (these changes are indicated by double underscores above). The MADEAP sequence has been added to the Bcr-Abl sequence at the start to achieve stability. The last 6 residues are hexahistidine labels. The sequence between the MADEAP sequence and the hexahistidine label was derived from human Bcr-Abl kinase containing 97 residues upstream of the Bcr-Abl junction. The last amino acid is the stop codon ( ^* ).

Structure 4 fully human only E2

This construct contains a fully human Bcr-Abl residue with a escape mutant (T315I). The MADEAP sequence has been added to the Bcr-Abl sequence at the start to achieve stability. The bold text is a mutation corresponding to the escape mutant (T315I). The last 6 residues are hexahistidine labels. The sequence of 328 amino acids between the MADEAP sequence and the hexahistidine tag is from the human Abl kinase domain. The last amino acid is the stop codon ( ^* ).

Construct 5 contains a complete human Bcr-Abl wild type sequence

This construct is a wild-type Bcr-Abl sequence containing a junction of Bcr and Abl. The MADEAP sequence has been added to the Bcr-Abl sequence at the start to achieve stability. The last 6 residues are hexahistidine labels. The sequence between the MADEAP sequence and the hexahistidine label was derived from the human Bcr-Abl sequence containing 97 residues upstream of the Bcr-Abl junction. The last amino acid is the stop codon ( ^* ).

Structure 6 fully human E1.E2.E3

This construct contains three escape mutations: E255K, T315I and M351T. The MADEAP sequence has been added to the Bcr-Abl sequence at the start to achieve stability. Bold text shows mutations for the escape mutant (E255K, T315I and M351T). The last 6 residues are hexahistidine labels. The 328 amino acid lines between the MADEAP sequence and the hexahistidine tag are from the human Abl kinase domain. The last amino acid is the stop codon ( ^* ).

Example 2 Leukemia Mouse Model

A leukemia mouse model was used to inject mice with retroviruses containing various forms of Bcr-Abl. These leukemia cells can also be labeled with GFP markers for monitoring purposes.

Mice were immunized 3 times with 2 YU yeast (20 million yeast) at two week intervals. 500,000 leukemia cells were challenged on the 7th day after the last inoculation.

The mouse study was divided into two parts: two groups of mice were present in Experiment 1: (1) The first group of mice were not immunized but challenged with wild-type CML (but Bcr-Abl had no escape mutation) (5 per group) only). (2) The second group of mice were immunized with a yeast-based vaccine containing three escape mutations found by Gleevec treatment (Bcr-Abl has three TAME epitopes - E255K, T315I, M351T) and then with wild-type CML (but Bcr) -Abl has no escape mutation) attack (5 in each group).

The results were as follows: Identification of the Carbamine curves of these groups - all mice were sacrificed on the same day (Day 10). However, there were fewer leukemia cells (based on percentage) in the blood and spleen of the inoculated mice (both on day 10). See Figure 1. The number of T cells and B cells in the spleen of the inoculated mice was also increased, thereby demonstrating the vaccine effect.

Three groups of mice were present in the Part 2 mouse study: (1) The first group was unimmunized but challenged with T315I CML (Bcr-Abl with one TAME mutation - T315I) (5 per group).

(2) The second group of mice were immunized with a yeast-based vaccine containing three escape mutants (Bcr-Abl has three TAME epitopes - E255K, T315I, M351T) and then T315I CML (Bcr-Abl has one TAME mutation - T315I) Attack (3 in each group).

(3) The third group of mice was immunized with a yeast-based vaccine with one escape mutation (Bcr-Abl has one TAME epitope-T315I) and then challenged with T315ICML (Bcr-Abl has one TAME mutation-T315I) 4 in each group).

The results are as follows: The Cabermeer curve shows a clear survival advantage for the vaccinated mice. In the control group (Group 1), all mice died (5/5 deaths). In contrast, mice in the vaccinated group survived. By the 13th day, none of the 7 mice in the vaccinated group died. See Figure 2.

When the percentage of leukemia cells was measured in the second part of the experiment, the percentage of leukemia cells in the blood of the inoculated mice was statistically significantly reduced compared to the uninoculated mice. These results are shown in Figure 3.

Figure 1 depicts the results of an experiment measuring the percentage of leukemia cells inoculated and unvaccinated mice. Briefly, mice were divided into two groups. The first group of mice were uninoculated and challenged with leukemia cells of wild-type Bcr-Abl (no escape mutant). The percentage of leukemia cells in these mice is shown on the left side of Figure 1. The second group of mice was immunized with a yeast construct containing three escape mutants (E255K, T315I and M351T) and then challenged with leukemia cells of wild-type Bcr-Abl (no escape mutant). The percentage of leukemia cells in these mice is shown on the right side of Figure 1.

Figure 2 shows the Kaplan Meier survival curve of three groups of mice. The first group of 5 mice were unimmunized and challenged with leukemia cells with Bcr-Abl with T315I escape mutant. The dotted line shows its survival curve. A second group of 3 mice was immunized with a yeast-based vaccine containing Bcr-Abl with three escape mutations (E255K, T315I and M351T) and then challenged with wild-type Bcr-Abl with the T315I mutation. A third group of 4 mice were immunized with a yeast-based vaccine containing Bcr-Abl with one escape mutant (T315I) and then challenged with wild-type Bcr-Abl with the T315I mutation. Survival of vaccinated mice (7 in total) is shown in solid lines in Figure 2.

Figure 3 depicts the results of the same experiment as Figure 2, whereby the percentage of leukemia cells was analyzed. The results of the unvaccinated mice are shown on the left side of Figure 3 and the results of the inoculated mice are shown on the right side.

<110> American Business Global Immunity <120> Compositions and methods for immune response induced by cancer-type therapy for escape mutants <130> 595432000142 <140> 096109502 <141> 2007-03-20 <160> 6 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 340 <212> PRT <213> Human Abl kinase <220><221> VARIANT <222> (1)...(6) <223> Protein stability label<220><221>VARIANT<222> (335)...(340) <223> Hexameric histidine label <220><221> VARIANT <222> 198,258,294 <223> Mutation corresponding to the escape mutant (E225K , T315I and M351T)<220><221> VARIANT <222> 168,279 <223> Change to mouse human residue (V227I, N336S) <400> 1 <210> 2 <211> 340 <212> PRT <213> Human Abl kinase <220><221> VARIANT <222> (1)...(6) <223> Protein stability label <220><221> VARIANT <222> (335)...(340) <223> Hexameric histidine label <220><221> VARIANT <222> 153,279 <223> Changed to mouse human residue (V212I, N336S) <220><221> VARIANT <222> 258 <223> Corresponding to the mutation of the escape mutant T315I <400> 2 <210> 3 <211> 437 <212> PRT <213> Wild-type human Bcr-Abl with mouse residues <220><221> VARIANT <222> (1)...(6) <223> Protein Stability Label <220><221> VARIANT <222> (432)...(437) <223> Hexameric histidine label <220><221> VARIANT <222> 18,33,265,376 <223> in Bcr- Change the human residue at the Abl junction to mouse <400> 3 <210> 4 <211> 340 <212> PRT <213> Human Abl Ester <220><221> VARIANT <222> (1)...(6) <223> Protein Stability Label <220><221> VARIANT <222> (335)...(340) <223> Hexameric histidine label <220><221> VARIANT <222> 258 <223> Mutation corresponding to the escape mutant T315I <400> 4 <210> 5 <211> 437 <212> PRT <213> Human Abl Kinase <220><221> VARIANT <222> (1)...(6) <223> Protein Stability Label <220><221> VARIANT <222> (431)...(437) <223> Hexameric histidine label <400> 5 <210> 6 <211> 340 <212> PRT <213> Human Abl kinase <220><221> VARIANT <222> (1)...(6) <223> Protein stability label <220><221> VARIANT <222> (335)...(340) <223> Hexameric histidine label <220><221> VARIANT <222> 198,258,294 <223> Mutations corresponding to escape mutants (E225K, T315I and M351T) <400> 6

Claims (21)

A composition comprising: i) a yeast vehicle and a nucleic acid molecule encoding a polypeptide comprising a human Bcr-Abl junction sequence; or ii) a yeast vehicle and a polypeptide comprising a human Bcr-Abl junction sequence, wherein The human Bcr-Abl junction sequence is selected from the group consisting of SEQ ID NO: 5 or SEQ ID NO: 3. The composition of claim 1, wherein the human Bcr-Abl junction sequence is SEQ ID NO: 5. The composition of claim 1, wherein the human Bcr-Abl junction sequence is SEQ ID NO: 3. A composition comprising: i) a yeast vehicle and a nucleic acid molecule encoding a mutant polypeptide selected from the group consisting of SEQ ID NO: 1, 2, 4 or 6; or ii) a yeast vehicle and selected from the group consisting of SEQ ID NO: Mutant polypeptide of 1, 2, 4 or 6. The composition of claim 4, wherein the mutant polypeptide is selected from the group consisting of SEQ ID NO: 4 or SEQ ID NO: 6. The composition of claim 1 or 4, wherein the yeast vehicle is a complete yeast, a yeast protoplast spheroid, a yeast cytoplast, a yeast residue or a subcellular yeast membrane extract or a fraction thereof. The composition of claim 1 or 4, wherein the yeast vehicle is complete yeast. The composition of claim 1 or 4, wherein the polypeptide is from the yeast vehicle Reorganized performance. The composition of claim 1 or 4, wherein the yeast vehicle is selected from the group consisting of Saccharomyces , Schizosaccharomyces , Kluveromyces , Hansenula , It is obtained from yeast of the group of Candida and Pichia . The composition of claim 1 or 4, wherein the yeast vehicle is obtained from Saccharomyces cerevisiae . The composition of claim 1 or 4, which further comprises at least one adjuvant. The composition of claim 1 or 4 further comprising a pharmaceutically acceptable excipient. The composition of claim 1 or 4 for use in ameliorating the symptoms of a cancer which exhibits Bcr-Abl in a mammal, comprising administering to the mammal an effective amount of the composition, wherein the cancer is associated with a The mutant Bcr-Abl polypeptide that is present in response to the targeted cancer therapeutic and/or prophylactic agent is associated. A composition according to claim 1 or 4 for use in reducing resistance to an agent administered to a mammal having a cancer risk or suffering from cancer, wherein the cancer exhibits Bcr-Abl, which comprises administering to the mammal An effective amount of the composition, wherein the cancer is associated with a mutant Bcr-Abl polypeptide that occurs in response to administration of the indicated therapeutic and/or prophylactic agent. Use of a composition according to claim 1 or 4 for the preparation of a medicament for eliciting an immune response to a cancer-associated mutant Bcr-Abl polypeptide. Use of a composition as claimed in claim 1 or 4 for preparation A drug that induces an immune response to a cancer-associated mutant Bcr-Abl polypeptide in combination with a subject therapeutic and/or prophylactic agent. Use of a composition according to claim 1 or 4 for the preparation of a medicament for the treatment of a cancer which exhibits Bcr-Abl, wherein the cancer occurs in response to administration of a therapeutic agent and/or prophylactic agent The mutant Bcr-Abl polypeptide is associated. Use of a composition according to claim 1 or 4 for the manufacture of a medicament for reducing the resistance to administration of a medicament for cancer or a mammal suffering from cancer, wherein the cancer exhibits Bcr-Abl, And wherein the cancer is associated with a mutant Bcr-Abl polypeptide that occurs in response to administration of the indicated therapeutic and/or prophylactic agent. A kit for inducing an immune response in a mammal to a mutant Bcr-Abl polypeptide associated with a cancer that exhibits Bcr-Abl, wherein the mutant polypeptide is known to react to the administered therapeutic and/or prophylactic agent At least one specific mutation occurs or has occurred, and wherein the kit comprises the composition of claim 1 or 4. The kit of claim 19, further comprising the subject therapeutic and/or prophylactic agent. The set of claim 19 further includes instructions for using the set.