JP2005514029A - Epitope synchronization in antigen-presenting cells - Google Patents

Abstract

  Disclosed herein are vaccines and methods for eliciting an immune response against cancer cells and cells infected by intracellular parasites. Disclosed are vaccines having housekeeping epitopes. Housekeeping epitopes are formed by housekeeping proteasomes in surrounding cells, but not by professional antigen presenting cells. Thus, vaccines containing housekeeping epitopes derived from antigens associated with peripheral target cells can induce an immune response against the target cells. Also disclosed is a method of treatment comprising administering a vaccine having a housekeeping epitope.

Description

[Background of the invention]
Field of the Invention The invention disclosed herein induces antigen-presenting cells to present specific epitopes to specific target cells, thereby promoting an effective cytotoxic T cell response to the target cells The present invention relates to methods and compositions for doing so.

  The present invention further relates to the identification of target cell epitopes and epitope clusters, as well as epitope encoding vectors, which can be used to purify immunologically active pharmaceutical compositions. When administered, these compositions can stimulate the subject's immune system and increase the immune response to target cells presenting the target antigen. Thus, the present invention is useful in the treatment and prevention of neoplastic and viral diseases.

[Description of related technology]
Neoplasia and immune system The neoplastic pathology, commonly known as cancer, is generally thought to be generally attributed to single cells that grow out of control. An uncontrolled growth state is typically due to a multi-step process in which a series of cell lines do not function, resulting in the origin of neoplastic cells. The resulting neoplastic cells themselves can rapidly proliferate and form one or more tumors, ultimately causing the death of the host.

  Because the precursors of neoplastic cells share the host's genetic material, neoplastic cells are largely immune from the host's immune system. During immune surveillance, the process by which the host immune system monitors and localizes foreign substances, neoplastic cells will emerge as “self” cells to the host immune surveillance mechanism.

Viruses and the immune system For cancer cells, viral infection clearly involves the expression of non-self antigens. As a result, many viral infections are successfully addressed by the immune system with minimal clinical sequelae. In addition, it was possible to develop effective vaccines for many of the infections that cause severe disease. Various vaccine approaches have been successfully used to combat various diseases. These approaches include subunit vaccines composed of individual proteins produced by recombinant DNA technology. Despite these advantages, the selection and effective administration of minimal epitopes for use as a viral vaccine remains problematic.

  In addition to the difficulties involved in epitope selection, there are viral problems that have evolved the ability to escape the host immune system. Many viruses, particularly those that establish persistent infections (eg, herpes and poxvirus family members) produce immunomodulatory molecules that allow the virus to escape the host immune system. The effect of these immunomodulatory molecules on antigen presentation can be overcome by targeting selected epitopes for administration as immunogenic compositions. In order to better understand the interaction of neoplastic cells and virus-infected cells with the host immune system, a description of immune components is given below.

The immune system functions to distinguish molecules that are endogenous to the organism (“self” molecules) from substances that are exogenous or foreign to the organism (“non-self” molecules). The immune system has an adaptive response to two types of foreign bodies: a humoral response and a cellular response based on the components that mediate the response. While the humoral response is mediated by antibodies, the cellular response involves cells that are classified as lymphocytes. Recent anticancer and antiviral strategies have focused on mobilizing the host immune system as a means of anticancer or antiviral therapy or therapy.

  The immune system functions in three phases: the cognitive phase, the activation phase and the effector phase to protect the host from foreign substances. In the cognitive phase, the immune system recognizes and signals the presence of foreign antigens or invaders in the body. The foreign antigen can be a cell surface marker derived from, for example, neoplastic cells or viral proteins. Once the system senses an invader, antigen-specific cells of the immune system proliferate and differentiate in response to signals elicited by the invader. The final stage is an effector stage in which effector cells of the immune system respond to and inactivate detected invaders.

  A series of effector cells carry out an immune response against the invader. One type of effector cell, B cells, produces antibodies that target foreign antigens encountered by the host. In combination with the complement system, antibodies direct the destruction of cells or organisms carrying the target antigen. Another type of effector cell is the natural killer cell (NK cell), a type of lymphocyte that has the ability to spontaneously recognize and destroy various virus-infected cells as well as malignant cell types. The method used by NK cells to recognize target cells is poorly understood.

  Another type of effector cell, T cells, has members that fall into three subcategories, each playing a different role in the immune response. Helper T cells secrete cytokines that stimulate the growth of other cells necessary to enhance an effective immune response, while suppressor T cells down regulate the immune response. A third category of T cells, cytotoxic T cells (CTL), can directly lyse target cells presenting foreign antigens on their surface.

Major histocompatibility complex and T cell target recognition T cells are antigen-specific immune cells that function in response to specific antigen signals. B lymphocytes and the antibodies they produce are also antigen-specific objects. However, unlike B lymphocytes, T cells do not respond to free or soluble forms of antigen. In order for T cells to respond to an antigen, it is necessary that the antigen be bound to a presentation complex known as the major histocompatibility complex (MHC).

MHC complex proteins provide a means for T cells to distinguish native or “self” cells from foreign cells. There are two types of MHC: class I MHC and class II MHC. T helper cells (CD4 ⁺ ) interact predominantly with class II MHC proteins, whereas cytolytic T cells (CD8 ⁺ ) interact predominantly with class I MHC proteins. Both MHC complexes are transmembrane proteins that have most of their structure on the outer surface of the cell. Furthermore, both classes of MHC have grooves on their outer part where the peptides bind. In this groove, small fragments of native or foreign proteins are bound and presented to the extracellular environment.

Cells called antigen-presenting cells (APCs) present antigens to T cells using MHC complexes. In order for a T cell to recognize an antigen, it must be presented on the MHC complex for recognition. This requirement is called MHC restriction, which is the mechanism by which T cells distinguish “self” cells from “non-self” cells. If the antigen is not presented by a recognizable MHC complex, the T cell does not recognize or act on the antigen signal. T cells specific for peptides bound to recognizable MHC complexes bind to these MHC-peptide complexes and progress to the next stage of the immune response.

  As mentioned above, neoplastic cells are largely spared by the immune system. A great deal of effort is now expended in attempting to utilize the host immune system to help fight the presence of neoplastic cells in the host. One such research area involves the production of anti-cancer vaccines.

Anti-cancer vaccines Among the various means available to oncologists in the fight against cancer are the patient's immune system. Research has been done in various attempts to fight the immune system against cancer or neoplastic diseases. Unfortunately, the results to date are largely disappointing. One area of particular interest includes the production and use of anti-cancer vaccines.

  In order to generate a vaccine or other immunogenic composition, it is necessary to introduce an antigen or epitope into the subject that can enhance the immune response. Although neoplastic cells are derived from normal cells and are therefore substantially consistent with normal cells in terms of gene level, many neoplastic cells are known to present tumor associated antigen (TuAA). Theoretically, these antigens can be used by the subject's immune system to recognize these antigens and attack neoplastic cells. Unfortunately, neoplastic cells appear to be overlooked by the host immune system.

  A number of different strategies have been developed in an attempt to generate vaccines with activity against neoplastic cells. These strategies include the use of tumor associated antigens as immunogens. For example, US Pat. No. 5,993,828 discloses urine tumor associated antigen on the cell surface and at least one tumor associated selected from the group consisting of GM-2, GD-2, fetal antigen and melanoma associated antigen. Described is a method for producing an immune response against a specific subunit of a urine tumor-associated antigen by administering to a subject an effective dose of a composition comprising inactivated tumor cells having the antigen. This patent therefore describes the use of whole inactivated tumor cells as an immunogen in an anti-cancer vaccine.

Another strategy using an anti-cancer vaccine involves administering a composition containing an isolated tumor antigen. In one approach, MAGE-A1 antigenic peptide was used as an immunogen (Chaux, P., et al., “Identification of Five MAGA-A1 Epitopes Recognized by
Cytolytic T Lymphocytes Obtained by In Vitro Stimulation with Dendritic Cells Transduced with MAGE-A1 ”, J. Immunol., 163 (5): 2928-2936 (1999)). There have been several therapeutic trials with MAGE-A1 peptides for vaccines, but the effectiveness of the vaccine treatment has been limited. Some results of these trials are shown in Vose, JM, `` Tumor Antigens Recognized by T Lymphocytes '' 10 ^th European Cancer Conference, Day 2, Sept.
14, discussed in 1999.

In another example of a tumor-associated antigen used as a vaccine, Scheinberg et al. Described 12 chronic myeloid leukemia (CML) patients who had already received interferon (IFN) or hydroxyurea and helper with adjuvant QS-21. Treated with 5 injections of class I related bcr-abl peptide along with the peptide. Scheinberg, DA, et al. “BCR-ABL Breakpoint Derived Oncogene Fusion Peptide Vaccines Generate Specific Immune Responses in Patients with Chronic Myelogenous Leukemia (CML)” [Abstract 1665], American Society of Clinical Oncology 35 ^th Annual Meeting, Atlanta (1999) . Proliferative and delayed type hypersensitivity (DHT) T cell responses showing T helper activity were elicited, but no cytolytic killer cell activity was observed in fresh blood samples.

Further examples of attempts to identify TAAs for use as vaccines can be found in recent studies such as Cebon et al. And Scheibenbogen et al. Cedon et al. Immunized patients with metastatic melanoma using the MART-1 _26-35 peptide administered intradermally with IL-12 in increasing doses given either subcutaneously or intravenously. Of the first 15 patients, 1 complete response, 1 partial response, and 1 mixed response were observed. Immunoassays for T cell generation included DTH, which was examined in patients with or without IL-12. A positive CTR assay was examined in patients with evidence of clinical benefit, but not in patients with tumor regression. Cebon, et al., “Phase I Studies of Immunization with Melan-A and IL-12 in HLA A2 + Positive Patients with Stage III and IV Malignant Melanoma”, [Abstract 1671], American Society of Clinical Oncology 35 ^th Annual Meeting, Atlanta (1999).

Scheibenbogen et al. Immunized 16 patients with metastatic black and 18 patients with 2 adjuvant patients with 4 HLA class I restricted tyrosinase peptides. Scheibenbogen, et al., `` Vaccination with Tyrosinase peptides and GM-CSF in Metastatic Melanoma:
a Phase II Trial ", [Abstract 1680], American Society of Clinical Oncology 35 ^th
Annual Meeting, Atlanta (1999). Increased CTR activity was observed in 4/15 patients, two adjuvant patients and two patients with tumor regression. Patients with progressive disease, like trials by Cebon et al., Did not show booster immunity. Despite the various efforts expended to date to produce effective anticancer vaccines, such compositions have not yet been developed.

  Vaccine strategies to protect against viral diseases have had many successes. Perhaps the most notable of these is the progress made against smallpox disease, which has been eradicated. The success of polio vaccines is similar.

  Virus vaccines are classified into three categories: smallpox vaccines, Sabin poliovirus vaccines and live attenuated virus vaccines such as measles, mumps and rubella mixed vaccines, soak polio virus vaccines, hepatitis A virus vaccines and typical influenza virus vaccines. Or killed or inactivated viral vaccines, as well as subunit vaccines such as hepatitis B. Due to the lack of a complete viral genome, subunit vaccines offer greater safety than whole virus-based vaccines.

  A successful subunit vaccine paradigm is a recombinant hepatitis B vaccine based on viral envelope proteins. Despite considerable academic interest in pushing the subunit concept beyond a single protein for individual epitopes, the effort has not yet yielded significant results. Viral vaccine research has also focused on eliciting antibody responses, but cellular responses are also performed. However, many subunit formulations are particularly poor at generating CTL responses.

[Summary of Invention]
The present invention relates to methods and compositions for inducing antigen-presenting cells to provide specific epitopes for specific target cells, thereby promoting a long-term directed cytotoxic T cell response to the target cells. About.

Vaccines comprising a housekeeping epitope In one aspect of the invention, a vaccine comprising a housekeeping epitope derived from an antigen associated with a target cell is provided. Suitably, the target cell may be a neoplastic cell. Neoplastic cells include leukemia, carcinoma, lymphoma, astrocytoma, sarcoma, glioma, retinoblastoma, melanoma, Wilmsoma, bladder cancer, breast cancer, colon cancer, hepatocellular carcinoma, pancreatic cancer, prostate cancer Any transformed cell associated with a solid tumor or lymphoma, such as lung cancer, liver cancer, stomach cancer, cervical cancer, testicular cancer, renal cell cancer, and brain cancer. Alternatively, target cells can be infected by intracellular parasites. For example, the intracellular parasites include adenovirus, cytomegalovirus, Epstein-Barr virus, herpes simplex virus, human herpes virus 6, varicella-zoster virus, hepatitis virus, papilloma virus, parvovirus, polyoma virus, measles virus. Or a virus such as rubella virus, human immunodeficiency virus (HIV), or human T cell leukemia virus. Intracellular parasites may be bacteria, protozoa, fungi, or prions. More specifically, intracellular parasites include Chlamydia, Listeria, Salmonella, Legionella, Brucella, Coccella, Rickettsia, Mycobacteria, Leishmania, Trypanosoma, Toxoplasma, and Plasmod It can be of the genus Ummus.

  The housekeeping epitope can be derived from an antigen associated with the target cell. The antigens are MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, CEA, RAGE, NY -ESO, SCP-1, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Bar Viral antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72 -4, CAM 17.1, NuMa, K-ras, β-cate , CDK4, Mum-1, p16, TAGE, PSAM, PSCA, CT7, telomerase, 43-9F, 5T4, 791 Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA125, CA 15-3 (CA 27. 29 \ BCAA), CA 195, CA242, CA-50, CAM43, CD68 \ KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18 NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein / cyclophilin C-related protein, TAAL6, TAG72, TLP, TPS, GA733-2 \ KSA and the like. Optionally, the antigen is In another aspect of the possible. The present invention in pulse-associated antigen, the antigen can be a parasite-associated antigen.

  In another aspect of the invention, the housekeeping epitope may comprise or encode a polypeptide from about 6 to about 23 amino acids in length. Preferably, the polypeptide is 9 or 10 amino acids long. The peptide may be a synthetic polypeptide. Suitably, the vaccine further comprises a buffer, a detergent, a surfactant, an antioxidant, or a reducing agent. In yet another aspect of the vaccine, the housekeeping epitope comprises a nucleic acid. In a preferred embodiment, the housekeeping epitope is specific for at least one allele of MHC. The allele can encode type A1, A2, A3, A11, A24, A26, A29, B7, B8, B14, B27, B35, B44, B62, B60, or B51.

  In yet another aspect of the invention, the vaccine may include an immune epitope. Optionally, the immune epitope is derived from a second antigen associated with the target cell. The first antigen and the second antigen may be the same or different. Preferably, the housekeeping epitope is specific for the first allele of MHC and the immune epitope is specific for the second allele of MHC. The first allele and the second allele may be the same or different.

  In yet another aspect of the invention, the vaccine comprises an epitope cluster (see below) comprising immune epitopes. The epitope cluster can be derived from a second antigen associated with the target cell. The first antigen and the second antigen may be the same or different. Preferably, the epitope cluster comprises or encodes a polypeptide having a length of at least 10 amino acids and less than about 60 amino acids. Preferably, the length of the polypeptide of the epitope cluster is less than about 80%, 50% or 20% of the length of the second antigen.

  In another aspect of the invention, the vaccine further comprises a second housekeeping epitope derived from a second antigen associated with a second target cell. Optionally, the first antigen and the second antigen can be the same. Alternatively, the first antigen and the second antigen are different. Similarly, the first target cell and the second target cell may be the same or different.

  The vaccine of the present invention preferably comprises a nucleic acid construct encoding a housekeeping epitope derived from an antigen associated with the target cell. Preferably, the target cell is a neoplastic cell. The neoplastic cells are leukemia, carcinoma, lymphoma, astrocytoma, sarcoma, glioma, retinoblastoma, melanoma, Wilmsoma, bladder cancer, breast cancer, colon cancer, hepatocellular carcinoma, pancreatic cancer, prostate It can be any transformed cell associated with a solid tumor or lymphoma such as cancer, lung cancer, liver cancer, gastric cancer, cervical cancer, testicular cancer, renal cell cancer, and brain cancer. In contrast, the target cells can be infected by intracellular parasites. The intracellular parasite may be a virus. In particular, the above viruses are adenovirus, cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1, herpes simplex virus 2, human herpes virus 6, varicella-zoster virus, hepatitis B virus, hepatitis D virus, papilloma virus. , Parvovirus B19, polyomavirus BK, polyomavirus JC, hepatitis C virus, measles virus, rubella virus, human immunodeficiency virus (HIV), human T cell leukemia virus I, or human T cell leukemia virus II There may be. Optionally, the intracellular parasite is a bacterium, protozoan, fungus, or prion. More specifically, the intracellular parasites include Chlamydia, Listeria, Salmonella, Legionella, Brucella, Coccella, Rickettsia, Mycobacteria, Leishmania, Trypanosoma, Toxoplasma, and Pluso It can be of the genus Modium.

  The antigens of the vaccine comprising the nucleic acid construct are MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE- 2, CEA, RAGE, NY-ESO, SCP-1, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm- 23H1, PSA, TAG-72-4, CAM 17.1, It may be NuMa, K-ras, β-catenin, CDK4, Mum-1, and p16. Alternatively, the antigen can be a virus or an antigen associated with a viral infection. In yet another embodiment, the antigen is an antigen associated with a non-viral intracellular parasite.

The housekeeping epitope preferably encodes a polypeptide from about 6 to about 23 amino acids in length. More preferably, the housekeeping epitope encodes a 9 to 10 amino acid long polypeptide. Preferably, the housekeeping epitope is specific for at least one allele of MHC. The alleles are type A1, A
2, A3, A11, A24, A26, A29, B7, B14, B18, B27, B35, B44, B62, B60, or B51 can be encoded.

  In another aspect of the invention, the vaccine further comprises an immune epitope. The immune epitope may be derived from a second antigen associated with the target cell. The first antigen and the second antigen may be the same or different. Preferably, the housekeeping epitope is specific for a first allele of MHC and the immune epitope is specific for a second allele of MHC. The first allele and the second allele may be the same or different.

  In yet another aspect of the invention, the vaccine having the nucleic acid construct further comprises an epitope cluster. The epitope cluster includes immune epitopes. Preferably, the epitope cluster is derived from a second antigen associated with the target cell. The first antigen and the second antigen may be the same or different.

  Suitably, the Eptoop cluster comprises or encodes a polypeptide having a length of at least 10 amino acids and less than about 60 amino acids. In preferred embodiments, the epitope cluster comprises or encodes a polypeptide having a length that is less than about 80% of the length of the second antigen. In another preferred embodiment, the length of the polypeptide is less than about 50% of the length of the second antigen. In particularly preferred embodiments, the length of the polypeptide is less than about 20% of the length of the second antigen.

  In yet another aspect of the invention, the vaccine comprising a nucleic acid construct further comprises a second housekeeping epitope, wherein the second housekeeping epitope is derived from a second antigen associated with a second target cell. To do. The first antigen and the second antigen can be the same or different. Preferably, the first target cell and the second target cell are different.

Nucleic acid construct The present invention provides a nucleic acid construct comprising a first coding region, wherein the first coding region comprises at least a first sequence encoding a first polypeptide, wherein the first polypeptide comprises A first housekeeping epitope derived from a first antigen associated with the first target cell. The first coding region can further comprise a second sequence encoding at least a second polypeptide, wherein the second polypeptide is derived from a second antigen associated with a second target cell. Including a second epitope. The first polypeptide and the second polypeptide can be contiguous or non-contiguous. The second epitope can be a housekeeping epitope or an immune epitope. The first antigen and the second antigen can be the same or different. Similarly, the first and second target cells can be the same or different.

The target cells are, for example, leukemia, carcinoma, lymphoma, astrocytoma, sarcoma, glioma, retinoblastoma, melanoma, Wilmsoma, bladder cancer, breast cancer, colon cancer, hepatocellular carcinoma, pancreatic cancer, prostate It can be a neoplastic cell such as cancer, lung cancer, liver cancer, gastric cancer, cervical cancer, testicular cancer, renal cell cancer, or brain cancer. Examples of the first antigen include MART-1 / MelanA, gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15. NY-ESO, the product of a member of the SSX gene family, CT-7, and the product of a member of the SCP gene family. Examples of the target cells include adenovirus, cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1 and 2, human herpes virus 6, varicella-zoster virus, hepatitis B virus, hepatitis D virus, papilloma virus, parvovirus. Viruses such as B19, polyomavirus BK, polyomavirus JC, hepatitis C virus, measles virus, rubella virus, human immunodeficiency virus (HIV), human T cell leukemia virus I, or human T cell leukemia virus II Can be infected. The target cells can also be infected by bacteria, protists, fungi, prions, or any other intracellular parasite, examples of which include Chlamydia, Listeria, Salmonella, Legionella, Brucella, They are the genus Koxiera, Rickettsia, Mycobacteria, Leishmania, Trypanosoma, Toxoplasma, and Plasmodium.

  The construct typically includes a first promoter sequence operably linked to the first coding region. The promoter can be, for example, cytomegalovirus (CMV), SV40, and retroviral long terminal repeat (LTR). The promoter can be a bidirectional promoter and / or the second promoter sequence can be operably linked to a second coding region. The nucleic acid construct can further include a poly A sequence operably linked to the first coding region, the second coding region, or both. The nucleic acid construct also includes an internal ribosome entry site (IRES) sequence, ubiquitin sequence, autocatalytic peptide sequence, enhancer, nuclear translocation sequence, immunostimulatory sequence, and expression cassette for cytokines, selectable markers, reporter molecules, etc. Can do. The first polypeptide is about 7 to about 15 amino acids long, preferably 9 or 10 amino acids long. The second polypeptide can be 9 or 10 amino acids in length, or it can be an epitope cluster from about 10 to about 75 amino acids in length. The first epitope and the second epitope can bind the same or different alleles of MHC.

  Other embodiments of the invention include vaccines comprising any of the above-described nucleic acid construct embodiments, methods of treating animals by administering such vaccines, and methods of producing the vaccines.

Identification of Epitope Clusters Other embodiments of the invention disclosed herein are epitopes used to generate a pharmaceutical composition capable of eliciting an immune response from a subject administered the composition. It relates to the identification of cluster regions. One embodiment of the disclosed invention relates to an epitope cluster that is derived from an antigen associated with the target, said cluster known or predicted for the groove to which the MHC receptor peptide binds. Which comprises or encodes at least two sequences having an affinity, wherein the cluster is a fragment of an antigen.

  In one aspect of the invention, the target is a neoplastic cell. Alternatively, the target may be a cell that is infected by an intracellular parasite. The intracellular parasite can be a virus, bacterium or protozoan. Optionally, the target is a pathogenic agent. The pathogenic agent may include a virus, bacterium, fungus, protozoan, prion, toxin, or venom.

  In another aspect of the invention, the MHC receptor may be a class I HLA receptor. Similarly, the MHC receptor may be a class II HLA receptor.

  In yet another aspect of the invention, the cluster comprises or encodes a polypeptide having a length, the length being at least 10 amino acids. Suitably, the length of the polypeptide may be less than about 75 amino acids.

In yet another embodiment of the invention, an antigen having a length is provided, wherein the cluster is composed of or encodes a polypeptide having a length, and the length of the polypeptide. Is less than about 80% of the length of the antigen. Preferably, the length of the polypeptide is less than about 50% of the length of the antigen. More preferably, the length of the polypeptide is less than about 20% of the length of the antigen.

（式中、Ｘはタンパク質配列中の天然に存在する任意のアミノ酸であり、
ＸａおよびＸ（｜ｂ｜−１）はそれぞれ長さ「ａ」および「｜ｂ｜−１」のこのようなアミノ酸の紐であり、
ａはＰ２_１およびＰ２_Ｎ間のアミノ酸の数を示し、そして（｜ｂ｜−１）はＰ２_ＮおよびＰ_１間のアミノ酸の数を表し、
Ｐ２_１は第１のエピトープの第１の主要アンカーおよび第２の残基であり、
Ｐ２_Ｎは第Ｎ番目のエピトープの第１の主要アンカーおよび第２の残基であり、
Ｐ _１は第１のエピトープの最後の主要アンカーおよびＣ末端残基であり、そして
Ｐ _Ｎは第Ｎ番目のエピトープの最後の主要アンカーおよびＣ末端残基であり、
２ Ｎ Ｎｃ、Ｎはクラスターの第Ｎ番目のエピトープを示し、Ｎｃはクラスター中のエピトープの総数を示し、
ａ_Ｎおよびｂ_Ｎは第１と第Ｎ番目のエピトープ間の位置関係を限定する）
を有し得る。さらに（Ｎｃ／Ｌｃ）＞（Ｎｐ／Ｌｐ）であり得り、クラスターおよび抗原はそれぞれある長さを有し、式中、Ｌｃはクラスターの長さであり、Ｌｐは抗原の長さであり、そしてＮｐは抗原中のエピトープの総数である。
さらにまた実施形態は、上記および本明細書に記載したような構造式に従ってエピトープクラスターを含む単離されたポリペプチドであって、アミノ酸配列が例えば抗原のアミノ酸配列の約８０％以下からなるポリペプチドに関する。実施形態は、単離されたポリペプチドを含むワクチンまたは免疫治療薬製品にも関する。別の態様では、単離されたポリペプチドは、例えば単離されたポリヌクレオチドによりコードされ得る。さらに別の態様では、ワクチンまたは免疫治療薬製品は、ポリヌクレオチドを含み得る。ポリヌクレオチドは、例えばＤＮＡであり得る。ポリヌクレオチドは、例えばＲＮＡであり得る。 Another embodiment of the disclosed invention is a method of identifying an epitope cluster comprising providing a sequence of antigens associated with a target cell, wherein an MHC receptor peptide binds to identify a putative MHC epitope Scoring candidate peptides within the sequence based on known or predicted affinity for the groove, and a region within the antigen, comprising at least two putative MHC epitopes, as a whole It relates to a method comprising the step of identifying a region comprising a putative MHC epitope density higher than the putative MHC epitope density in said antigen.
Another embodiment relates to an epitope cluster. The cluster can be derived from antigens associated with the target. The cluster may comprise or encode at least two sequences with known or predicted affinity for the groove to which the MHC receptor peptide binds. A cluster can be, for example, a fragment of an antigen. The cluster has the following structural formula:

Where X is any naturally occurring amino acid in the protein sequence;
Xa and X (| b | -1) are such amino acid strings of length "a" and "| b | -1", respectively;
a represents the number of amino acids between P2 ₁ and P2 _N , and (| b | -1) represents the number of amino acids between P2 _N and P ₁ ;
P2 ₁ is a first primary anchor and the second residue of the first epitope,
P2 _N is the first primary anchor and the second residue of the N-th epitope,
P ₁ is the last primary anchor and C-terminal residues of the first epitope, and P _N is the last primary anchor and C-terminal residues of the N-th epitope,
2 N Nc, N represents the Nth epitope of the cluster, Nc represents the total number of epitopes in the cluster,
a _N and b _N limits the positional relationship between the first and N-th epitope)
Can have. Furthermore, (Nc / Lc)> (Np / Lp), where the cluster and the antigen each have a certain length, where Lc is the length of the cluster and Lp is the length of the antigen; Np is the total number of epitopes in the antigen.
Furthermore, an embodiment is an isolated polypeptide comprising an epitope cluster according to a structural formula as described above and herein, wherein the amino acid sequence comprises, for example, about 80% or less of the amino acid sequence of the antigen About. Embodiments also relate to a vaccine or immunotherapeutic product comprising the isolated polypeptide. In another aspect, an isolated polypeptide can be encoded by, for example, an isolated polynucleotide. In yet another aspect, the vaccine or immunotherapeutic product may comprise a polynucleotide. The polynucleotide can be, for example, DNA. The polynucleotide can be, for example, RNA.

Method of treatment A method of treating an animal by administering a vaccine comprising a housekeeping epitope to the animal, wherein the housekeeping epitope is derived from a first antigen associated with the first target cell, according to the invention. Intended. Preferably, the administering step includes transdermal, intranodal, peri-nodal, oral, intravenous, intradermal, intramuscular, intraperitoneal, or transmucosal delivery modes.

  The animal treatment method may further comprise an assay step to determine characteristics indicative of the target cell status. Preferably, the assay step further includes a first assay step and a second assay step, wherein the first assay step is prior to the administration step, and the second assay step is the step of the administration step. Followed later. Preferably, the characteristics determined in the first assay step are compared with the characteristics determined in the second assay step to obtain a result. The result can be a reduction in the number of target cells, a reduction in the mass or size of the tumor containing the target cells, or a reduction in the number or concentration of intracellular parasites that infect the target cells.

  Preferably, the target cell is a neoplastic cell. The neoplastic cells are leukemia, carcinoma, lymphoma, astrocytoma, sarcoma, glioma, retinoblastoma, melanoma, Wilmsoma, bladder cancer, breast cancer, colon cancer, hepatocellular carcinoma, pancreatic cancer, prostate It can be any transformed cell associated with a solid tumor or lymphoma such as cancer, lung cancer, liver cancer, stomach cancer, cervical cancer, testicular cancer, renal cell cancer, and brain cancer. Alternatively, the target cell is infected by intracellular parasites. The intracellular parasite may be a virus. The viruses are adenovirus, cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1, herpes simplex virus 2, human herpes virus 6, varicella-zoster virus, hepatitis B virus, hepatitis D virus, papilloma virus, parvovirus Can be virus B19, polyoma virus BK, polyoma virus JC, hepatitis C virus, measles virus, rubella virus, human immunodeficiency virus (HIV), human T cell leukemia virus I, or human T cell leukemia virus II . The intracellular parasite may be a bacterium, protozoan, fungus, or prion. Preferably, the intracellular parasite is Chlamydia, Listeria, Salmonella, Legionella, Brucella, Coccella, Rickettsia, Mycobacteria, Leishmania, Trypanosoma, Toxoplasma, and Plasmodium It is a genus.

  In another aspect of the present invention, the antigen is MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE. -2, CEA, RAGE, NY-ESO, SCP-1, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK , MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm -23H1, PSA, TAG-72-4, CAM 17.1, NuMa , K-ras, β-catenin, CDK4, Mum-1, and p16. Alternatively, the antigen is associated with a virus or viral infection. In yet another aspect, the antigen is an antigen associated with a non-viral intracellular parasite.

  The housekeeping epitope may comprise or encode a polypeptide about 6 to about 23 amino acids in length. Preferably, the polypeptide is 9 or 10 amino acids long. The polypeptide may be synthetic. The vaccine may further include a buffer, a detergent, a surfactant, an antioxidant, or a reducing agent. The housekeeping epitope may preferably comprise a nucleic acid. Preferably, the housekeeping epitope is specific for at least one allele of MHC. The allele can encode type A1, A2, A3, A11, A24, A26, A29, B7, B8, B14, B18, B27, B35, B44, B62, B60, or B51.

In yet another aspect of the invention, the animal treatment method further comprises an immune epitope. The immune epitope may be derived from a second antigen associated with the target cell. Optionally, the first antigen and the second antigen are the same. The housekeeping epitope can be specific for the first allele of MHC and the immune epitope can be specific for the second allele of MHC. The first allele and the second allele may be the same or different.

  Suitably, the vaccine comprises an epitope cluster comprising the immune epitope. The epitope cluster may be derived from a second antigen associated with the target cell. Optionally, the first antigen and the second antigen are the same. The epitope cluster may comprise or encode a polypeptide having a length of at least 10 amino acids and less than about 60 amino acids.

  Preferably, the epitope cluster comprises or encodes a polypeptide having a length that is less than about 80% of the length of the second antigen. The length of the polypeptide can be less than about 50% of the length of the second antigen. In yet another aspect, the length of the polypeptide can be less than about 20% of the length of the second antigen.

  The animal treatment method further comprises a second housekeeping epitope, wherein the second housekeeping epitope is derived from a second antigen associated with a second target cell. The first antigen and the second antigen may be the same or different. Similarly, the first target cell and the second target cell may be the same or different.

  Also contemplated by the present invention is a method of treating an animal comprising administering to the animal a vaccine comprising a nucleic acid construct. The nucleic acid construct preferably encodes a housekeeping epitope. The housekeeping epitope may be derived from a first antigen associated with a first target cell.

  In another aspect of the invention, a method for producing a vaccine is provided. The method comprises the step of selecting a housekeeping epitope by identifying an epitope that is or can be produced from a particular antigen source by a houseskiing proteasome, wherein the housekeeping epitope is a first From a first antigen associated with the target cell, producing a vaccine comprising said housekeeping epitope, and preparing a vaccine composition comprising or encoding the selected housekeeping epitope.

  The above vaccine produced according to the method described above is also provided by the present invention. The vaccine can be administered to treat animals. Accordingly, methods for treating animals with the above vaccines are contemplated as well.

Housekeeping and Discovery of Other Epitopes Other embodiments of the invention disclosed herein are epitopes useful for generating vaccines capable of eliciting an immune response from a subject to which the composition has been administered. In particular, it relates to the identification of the most useful epitopes in the vaccine embodiments of the invention. One embodiment of the present invention is a method of finding an epitope comprising selecting an epitope from a population of peptide fragments of an antigen associated with a target cell, the fragment comprising a major histocompatibility complex class I receptor peptide Has a known or predicted affinity for the groove to which is bound, and the selected epitope relates to a method corresponding to the proteasome cleavage product of the target cell.

Another embodiment of the present invention is a method of discovering epitopes comprising: a sequence derived from a target cell that encodes or comprises a protein expressed in the target cell Identifying a peptide fragment population of the protein, the member of which has a known or predicted affinity for the groove to which a major histocompatibility complex class I receptor peptide binds And a step of selecting the epitope derived from the peptide fragment population, the epitope corresponding to a proteasome product active in the target cell.

  One aspect of this embodiment relates to epitopes discovered by the methods described above. Another aspect of this embodiment relates to a vaccine comprising the discovered epitope. Yet another aspect of the present invention relates to a method of treating an animal comprising administering the above vaccine to the animal.

  One embodiment of the disclosed invention is a method of discovering epitopes, comprising: providing a tumor cell, and a sequence, wherein the sequence comprises or encodes an antigen associated with the tumor cell Identifying a peptide fragment population predicted to have affinity for a groove to which a major histocompatibility complex class I receptor peptide binds, the peptide fragment population of the antigen, the peptide fragment population of the antigen, The present invention relates to a method comprising the step of selecting an epitope derived from a population, which is determined by an in vitro analysis to be a proteasome cleavage reaction product of a proteasome active in the neoplastic cell.

  One aspect of this embodiment relates to epitopes discovered by the methods described above. Another aspect of this embodiment relates to a vaccine comprising the discovered epitope. Yet another aspect of the present invention relates to a method of treating an animal comprising administering the above vaccine to the animal.

  Another embodiment of the disclosed invention is a method of epitope discovery comprising the step of selecting an epitope from a population of peptide fragments of an antigen associated with a target in a host, wherein the fragment is a major histocompatibility of the host Having a known or predicted affinity for the groove to which the sex complex class I or II receptor peptide binds, and the selected epitope is a product of proteolytic cleavage of the antigen in the host cell. It relates to the corresponding method.

One aspect of this embodiment relates to epitopes discovered by the methods described above. Another aspect of this embodiment relates to a vaccine comprising the discovered epitope. Yet another aspect of the invention relates to a method for treating an animal comprising administering the above-described vaccine to the animal.
Another embodiment relates to an isolated T cell that expresses a T cell receptor specific for an MHC-peptide complex comprising a first housekeeping epitope. The housekeeping epitope can be derived, for example, from a first antigen associated with the first target cell. A T cell clone may comprise, for example, a T cell. Furthermore, a polyclonal population of T cells can include, for example, T cells. T cells can be produced, for example, by in vitro immunization. T cells can be isolated from, for example, immunized animals.
Another embodiment relates to a method of manufacturing an adoptive immunotherapeutic. The method can include, for example, using a T cell as described herein in combination with a pharmaceutically acceptable adjuvant, carrier, diluent or excipient. T cells are initially obtained, for example, from a donor. Furthermore, the donor can be the intended recipient of an immunotherapeutic agent, for example. The donor can be immunologically untreated with respect to the first antigen. The donor may have been previously exposed to the first antigen, for example. The donor can be vaccinated with, for example, a housekeeping epitope prior to donation.
The method for producing an adoptive immunotherapeutic agent can further include a step of culturing T cells in vitro. T cells can be stimulated to proliferate, for example, by exposure to MHC-peptide complexes. T cells can be stimulated to proliferate, for example, by exposure to cytokines and the like. The culture may further comprise pAPC, adjuvant, combinations thereof and the like. The pAPC can be, for example, a dendritic cell. The adjuvant can be, for example, GM-CSF, G-CSF, IL-2, IL-12, BCG, tetanus toxoid, osteopontin / ETA-1, CD40 ligand and CTLA-4 blocker.
Embodiments of the invention also relate to the use of T cells as described herein in the manufacture of a medicament for use in adoptive immunotherapy. Other embodiments relate to a method of treating a disease comprising administering T cells as described herein to a recipient. Another embodiment is directed to a method of treating a disease comprising administering to a recipient an immunotherapeutic agent produced according to the methods described herein.

Detailed Description of Preferred Embodiments
Embodiments of the present invention provide epitopes, vaccines and therapeutic methods for inducing an effective immune response against target cells. The main basis of the present invention is a new and unexpected discovery that a large number of target cells display different epitopes than those displayed by professional antigen presenting cells (pAPC). Because of this difference, pAPC induces T cells against epitopes that are not present on target cells, and therefore T cells cannot recognize target cells. The methods and agents of the present invention can cause pAPCs to display the same epitope present on a target cell, resulting in a T cell that can correctly recognize and destroy the target cell. Strategies for commercializing vaccines according to this aspect of the invention are disclosed in US patent application Ser. No. 09 / 999,186 (title: METHODS OF COMMERCIALIZING AN ANTIGEN) (filed Nov. 7, 2001).

  The embodiments of the invention disclosed herein further provide methods for identifying epitopes of target antigens that can be used to generate immunologically effective vaccines. Such vaccines can stimulate the immune system to recognize and destroy target cells displaying the selected epitope. Embodiments of the present invention are particularly useful for the treatment and prevention of cancer and the treatment and prevention of cellular infection by intracellular parasites and for the treatment or prevention of symptoms associated with other pathogens, toxins and allergens.

  Certain types of targets are particularly vulnerable to the immune system. Among these are many types of cancer and cells infected by intracellular parasites such as viruses, bacteria and protozoa. A great deal of research has been done to identify antigens and epitopes useful for generating an effective immune response against such targets, but with little success. The present disclosure provides the basis for efficiently discovering new generations of effective epitopes that are effective against such vulnerable targets.

  The invention disclosed herein makes it possible to select epitope sequences with true biological relevance. For epitopes that have biological significance, ie function in stimulating an immune response, they must have an affinity to bind the major histocompatibility complex (MHC) receptor peptide groove. There are various means known in the art to predict whether an oligopeptide sequence has MHC binding affinity. However, most of the sequences predicted to have MHC binding affinity are not biologically relevant because they are not actually presented on the surface of the target cell or pAPC.

  The disclosed method of the invention allows vaccine designers to ignore peptides that are not useful because they cannot be presented by target cells, despite the expected high binding affinity for MHC. Thus, the methods and disclosures disclosed herein provide a major advance in vaccine design, one of which combines the power of antigen sequence analysis with basic immunological facts. The methods taught herein allow for easy and effective selection of meaningful epitopes for the production of MHC class I or class II vaccines using any polypeptide sequence that corresponds to the desired target. .

Further embodiments of the invention disclosed herein provide epitope cluster regions (ECR) for use in vaccines and in vaccine design and epitope discovery. In particular, embodiments of the invention relate to the identification of epitope clusters for use in generating immunologically active compositions directed against a target cell population and for use in the discovery of individual housekeeping epitopes and immune epitopes. In many cases, multiple putative class IMHC epitopes may be present in a single target associated antigen (TAA). Such putative epitopes are often found in clusters (ECRs), which are MHC epitopes that are relatively densely distributed within certain regions in the amino acid sequence of the parent TAA. Because these ECRs include a number of putative epitopes with biological activities that may be useful in inducing an immune response, they can be used in vitro or to identify epitopes that are particularly useful for vaccine design. Excellent material for in vivo analysis. And since the epitope clusters themselves are processed inside the cell to generate active MHC epitopes, the clusters can be used directly in the vaccine, and one or more putative epitopes in the cluster are active MHC epitopes. Actually processed in the epitope.

  The use of ECR in vaccines provides an important technological advance in the production of recombinant vaccines, and further provides a significant advance in safety over existing nucleic acid vaccines that encode the entire protein sequence. Recombinant vaccines generally rely on expensive and technically difficult production of total proteins in microbial fermenters. ECR offers the option to use chemically synthesized polypeptides, greatly simplifying development and manufacturing, and eliminating various safety issues. Similarly, the ability to use a nucleic acid sequence encoding ECR, which is typically a relatively short region of the entire sequence, is more expensive, time consuming, and perhaps difficult molecular biology for using the entire gene sequence Rather than an approach, it allows the use of synthetic oligonucleotide chemistry techniques in the development and manipulation of nucleic acid based vaccines.

  This can greatly improve the safety of nucleic acid vaccines because ECR is encoded by a relatively short nucleic acid sequence compared to that encoding the entire protein for which ECR is found. An important problem in the field of nucleic acid vaccines is the fact that the degree of sequence homology between the vaccine and the sequence in the animal to which the vaccine is administered determines the probability of integration of the vaccine sequence into the genome of the animal. The basic safety issue of nucleic acid vaccines is their ability to integrate into genomic sequences, which can result in deregulation of gene expression and tumor transformation. The Food and Drug Administration recommends that nucleic acids and recombinant vaccines should contain as little sequence homology as possible with human sequences. In the case of vaccines that deliver tumor-associated antigens, it is inevitable that the vaccine contains nucleic acid sequences that are homologous to those that encode proteins expressed in the patient's tumor cells. However, it is highly desirable to limit the extent of these sequences to the extent that it is minimally essential to promote the expression of epitopes to induce a therapeutic immune response. Thus, the use of ECR offers the dual advantage of incorporating multiple epitopes with possible therapeutic value while providing the minimal region of homology.

Aspects of the invention provide nucleic acid constructs that encode housekeeping epitopes. Housekeeping epitopes include peptide fragments produced by peripheral cell active proteasomes, as described in more detail below. The basis for the present invention is that any antigen associated with a target cell is differentially processed into two distinct sets of epitopes for presentation by the body's class I major histocompatibility complex (MHC) molecule. It is a discovery that you get. “Immune epitopes” are presented by pAPC and are generally also presented in surrounding cells that have been acutely infected or under active immunological attack by interferon (IFN) secreting cells. In contrast, “housekeeping epitopes” are presented by all other surrounding cells, such as generally tumor (cancerous) cells and chronically infected cells. Despite the presence of a functioning immune system in the host, this mismatch, or asynchrony, in the displayed epitope is responsible for the persistence and progression of cancer and chronic infection. Therefore, in order to elicit an effective cytolytic T lymphocyte (CTL) -mediated immune response, it is essential to generate a synchronized epitope presentation between pAPC and target cells.

  Tuning can be most reliably accomplished by providing a housekeeping epitope for pAPC. Often, a more robust response can be achieved by providing more than one epitope. Furthermore, once an effective immune response against the target cell is established, IFN secretion can lead to expression of the immune proteasome, thereby switching the epitope presentation to an immune epitope. For this reason, it may also be beneficial to include immune epitopes in addition to housekeeping epitopes in vaccines developed in accordance with the above disclosure. Providing immune epitopes in the form of ECR may have additional utility. Embodiments of the invention provide expression vectors that encode housekeeping epitopes and / or immune epitopes in various combinations. Preferred expression constructs encode at least one epitope that can stimulate a cellular immune response directed against the target cell. In one embodiment of the invention, the target cell is a neoplastic cell. In another embodiment, the target cell is any intracellularly infected host cell. Intracellular infection agents include persistent viruses and any other infectious organisms that have an intracellular stage of infection.

  The nucleic acid constructs of some embodiments are directed to strengthen the subject's immune system and to sensitize it to the presence of neoplastic cells in the host. In other embodiments, the nucleic acid construct promotes persistent viral infection as well as eradication of cells infected with intracellular parasites.

Definitions Unless otherwise apparent from the usage of the terms herein, the terms listed below shall generally have the designated meaning for the purpose of this description.

  Professional antigen presenting cell (pAPC)-A cell that carries a T cell costimulatory molecule and can induce a T cell response. Well characterized pAPC are dendritic cells, B cells and macrophages.

  Peripheral cells-cells that are not pAPC.

  Housekeeping proteasome—A proteasome that is normally active in surrounding cells and is generally not present or strongly active in pAPC.

  Immune proteasomes—proteasomes that are normally active in pAPC; immune proteasomes are also active in several surrounding cells in infected tissues.

  Epitope—A molecule or substance that can stimulate an immune response. In preferred embodiments, epitopes according to this definition include, but are not limited to, polypeptides and nucleic acids encoding the polypeptides, in which case the polypeptides may stimulate an immune response. In other preferred embodiments, epitopes according to this definition include peptides presented on the surface of cells that are non-covalently associated with a pocket of class I MHC so that they can interact with T cell receptors. However, it is not limited to this.

  MHC epitope—a polypeptide having a known or predicted affinity for a mammalian class I major histocompatibility complex (MHC) molecule.

  HLA epitope—a polypeptide having a known or predicted affinity for a human class I major histocompatibility complex (MHC) molecule.

  Housekeeping epitopes-In a preferred embodiment, a housekeeping epitope is defined as a polypeptide fragment that is an MHC epitope and is displayed on cells in which the housekeeping proteasome is predominantly active. In another preferred embodiment, a housekeeping epitope is defined as a polypeptide containing a housekeeping epitope according to the above definition, ie adjacent to one to several additional amino acids. In another preferred embodiment, a housekeeping epitope is defined as a nucleic acid encoding a housekeeping epitope according to any of the above definitions.

  Immune epitopes-In a preferred embodiment, immune epitopes are defined as polypeptide fragments that are MHC epitopes and are displayed on cells in which the immune proteasome is predominantly active. In another preferred embodiment, an immune epitope is defined as a polypeptide containing an immune epitope according to the above definition, ie adjacent to one to several additional amino acids. In another preferred embodiment, the immune epitope is defined as a polypeptide comprising an epitope cluster sequence and has at least two polypeptide sequences with known or predicted affinities for class I MHC. In yet another preferred embodiment, an immune epitope is defined as a nucleic acid encoding an immune epitope according to any of the above definitions.

  Target cells—cells targeted by the vaccines and methods of the invention. Examples of target cells according to this definition include, but are not limited to, neoplastic cells and cells carrying intracellular parasites such as viruses, bacteria or protozoa.

  Target associated antigen (TAA)-a protein or polypeptide present in a target cell.

  Tumor associated antigen (TuAA)-TAA where the target cell is a neoplastic cell.

  It should be noted that the following discussion describes the inventors' understanding of the practice of the present invention. However, this discussion is not intended to limit the patent to any particular theory of implementation that is not described in the claims.

Different proteasomes produce different epitopes Epitopes presented by class I MHC on the surface of pAPC or surrounding cells are produced by digestion of proteins within those cells by the proteasome. It has been reported that the proteasome of pAPC is not identical to the proteasome of the surrounding cells, but the significance of this difference has not been recognized to date. The present invention is the fact that when pAPC and surrounding cells process a given TAA, proteasomes active in pAPC produce epitope fragments that are different from those produced by proteasomes active in surrounding cells. Based on. For convenience of reference and as described above, proteasomes that are predominantly active in pAPC are referred to herein as “immune proteasomes”, whereas proteasomes that are normally active in surrounding cells are present. In the specification, it is called “housekeeping proteasome”.

The significance of differential processing of pAPC and surrounding cells cannot be exaggerated. This differential processing provides a unified explanation as to why certain target cells are resistant to recognition and attack by the immune system. pAPC can pick up TAA released from target cells and present it on their surface, but pAPC consequently stimulates the production of CTLs, resulting in "immune epitopes" (due to processing of TAA by the immune proteasome In contrast, the target cell displays a “housekeeping epitope” (a separate fragment of TAA produced by the housekeeping proteasome). Thus, the CTL response under physiological conditions is misdirected from the epitope on the target cell.

  Since CTL responses are induced by pAPC by definition, they target immune epitopes rather than housekeeping epitopes, so they do not recognize target cells and thus remain in the body. This basic “epitope compartmentalization” of the cellular immune response is the reason why some neoplastic cells can survive and form tumors. It is also why some viruses and intracellular parasites can chronically infect cells without being eradicated by the immune system. As for infectious agents, they usually cause the expression of the immune proteasome in the cells they infect. This results in the production of epitopes on the cell surface that are identical to those presented by pAPC to the immune system. Infection thus results in “epitope synchronization” between the immune system and the infected cell, subsequent destruction of the infected cell, as well as clearance of the infectious agent from the body. In the case of some infectious agents, particularly those that can establish chronic infections, they have evolved a means to prevent the expression of the immune proteasome in the cells they infect. In these cells the proteasome is maintained in a housekeeping manner, thereby preventing epitope tuning and attack by CTL. There is substantial evidence that this is a common mechanism used by virtually all chronic infectious agents.

  One way to overcome this failure to recognize and eradicate certain target cells in the part of the CTL is to provide vaccines and therapeutic methods that can “synchronize” epitope presentation. Epitope synchronization in this situation is made so that pAPC presents a housekeeping epitope, resulting in a CTL that recognizes the housekeeping epitope displayed on the target cell, thereby attacking and eliminating the target cell Means that.

  Thus, embodiments of the present invention are useful for treating neoplastic diseases such as solid tumors and lymphomas. Additional embodiments of the present invention have application in treating persistent viral diseases as well as parasitic infections where the infectious agent has an intracellular stage of infection. Appropriate administration of housekeeping epitopes corresponding to such target cells can activate specific cytotoxic T cell responses against the target cells.

Role of Epitope Differences in Cancer In some embodiments, the present invention is directed to treating neoplastic diseases. Cancer is caused by progressive unregulated growth of single abnormal cell progeny. The term “cancer” as used herein encompasses neoplastic diseases, neoplastic cells, tumors, tumor cells, malignant diseases and any transformed cells such as solid tumors and disseminated neoplastic diseases. . Historically, cancer cells have generally been thought to escape detection and destruction by the immune system because they contain the same genetic material as other non-cancerous cells of the body. The genetic identity or similarity between cancer cells in the body and healthy cells probably creates difficulties in distinguishing cancer cells from normal cells, so the immune system will be evidenced by the survival of cancer cells in the body In addition, the effective immune response cannot be enhanced.

  In contrast, various tumor-associated antigens (TuAA) have been described that can elicit and actually elicit an immune response. Numerous studies have described intratumoral infiltrating lymphocytes (TILs) that can kill target cells presenting various TuAA-derived peptides in vitro. However, as described in more detail below, TIL is unable to control cancer because of differences in epitopes produced and presented by cells that induce CTL activity, pAPC, and the desired target cells, ie, tumor cells. caused by. To understand the difference, it is necessary to understand the function and dynamics of the proteasome.

All cells contain a proteasome to degrade proteins. These proteasomes, which make up about 1% of the total protein content of the cell, function to regulate the protein half-life in the cell. In the proteolytic process, the proteasome generates a vast majority of peptide fragments involved in class I antigen presentation, and the proteasome cleavage pattern affects the availability of antigenic epitopes for presentation on class I molecules (FIG. 1). MHC epitopes are therefore produced by cellular proteasome activity. However, the proteolytic activity in pAPC is significantly different compared to surrounding cells. pAPC constitutively contains subunits that are typically only expressed in surrounding cells as part of the cellular immune response during infection or after exposure to various cytokines, particularly interferon (IFN). Contains the proteasome to be incorporated. As noted above, the different proteasome activities of pAPC and surrounding cells are referred to herein as the immune proteasome and housekeeping proteasome, respectively.

  Immune proteasomes and housekeeping proteasomes have the ability to cleave proteins at similar but distinct locations. The immune proteasome incorporates several subunits that distinguish it from its housekeeping equivalent. These immune subunits include LMP2, LMP7 and MECL1, which replace the catalytic subunit of the housekeeping proteasome, and PA28α and PA28β that provide regulatory functions (FIG. 2). Collectively, the incorporation of these subunits results in activity from the immune proteasome that differs qualitatively and quantitatively from the activity of the housekeeping proteasome. There is evidence that there are differences between housekeeping proteasomes and immune proteasomes with respect to the MHC epitopes they produce, but to date, these differences have been theoretically explained in quantitative terms. Others have suggested that the ultimate effect mediated by the immune proteasome is to promote the production of more peptides than different ones.

  Qualitative differences in antigen processing between immune and housekeeping proteasomes have significant implications for vaccine design. IFN-γ is produced by T lymphocytes, in which case it is involved in promoting the induction of a cellular immune response and, as described above, induces expression of the immune proteasome. In particular, IFN is also produced by virtually any other cell under certain conditions, ie when the cell becomes infected by a pathogen. In effect, viral infection typically results in IFN production by infected cells, which in turn induces the cell to be converted from a housekeeping proteasome shape to an immune proteasome shape. One explanation for this phenomenon is that infection and subsequent IFN upregulation function to align the pAPCs involved in stimulating the immune response against the virus with the infected cells with respect to the displayed antigen repertoire. This results in the processing of both the endogenous “self” protein normally expressed by the cell, as well as the protein for the infectious agent (“non-self”) in the same manner as antigen processing occurs in pAPC. Conversion of the proteasome of infected cells from a housekeeping form to an immune form results in “epitope synchronization” between infected cells and pAPC (FIG. 3).

  TuAA-specific MHC class I restricted CTLs are important components of the immune response against cancer. TuAAs are useful targets for tumor-specific T cell responses to the extent that they are not displayed on the surface of normal cells or are overexpressed by tumor cells or otherwise strongly characterized by tumor cells. is there. Numerous TuAAs are known and readily available to those skilled in the literature or commercially.

Indeed, some tumors have been found to lack IFN-γ induction of immune proteasomes. In these situations, CTL appear to target immune epitopes from TuAA processed by pAPC. Despite the large number of CTLs in these patients that are specifically activated against these immune epitopes, CTLs cannot find epitopes on cancer cells. The disease progresses, and accumulating CTL that eventually cannot locate the target becomes dysfunctional (Lee, et al. Nature
Medicine (1999) 5 [6]: 677-685). By providing a housekeeping epitope to pAPC, epitope presentation by pAPC can be synchronized with epitope presentation by the tumor and activate a CTL population that recognizes the housekeeping epitope present on the tumor.

  Thus, the discovery that immune proteasomes in pAPC produce qualitatively different epitopes than those produced by housekeeping proteasomes in surrounding cells provides an explanation for why TIL does not eradicate tumor cells. The above processing mechanism explains how T lymphocytes arrive in the tumor mass, but is relatively ineffective against the tumor cells themselves. Differential antigen processing between the immune proteasome of pAPC and the housekeeping proteasome of tumor cells may explain the high frequency observation of T lymphocytes specific for TuAA in patients with advanced cancer (Lee , et al. Nature Medicine (1999) 5 [6]: 677-685) (FIG. 4).

  Because of differences in proteasome activity, peripheral target cells, such as tumor cells, and some cells infected with viruses or other intracellular parasites, all of which express the housekeeping proteasome, are necessarily T cells However, it displays an epitope signal different from the epitope signal conditioned by pAPC for recognition. In light of this discovery, a powerful immunoregulatory role for the proteasome emerges. This discovery provides clues to the manipulation of the immune system, particularly pAPC, to induce beneficial and lethal cell-mediated attacks of target cells.

  Differential antigen processing explains why CTL specific for TuAA are often found during TIL without eradication of the disease. A T lymphocyte response is prepared against TuAA processed by pAPC. CTL found between TILs exclusively target class ITuAA that was present on pAPC but not on tumor cells (FIG. 4).

  The behavior of tumor cells in the body, ie migration, antigen shedding, induction of inflammatory response, etc., produces a strong immune response. Unfortunately, the natural mechanism of differential antigen processing between tumor cells and pAPC results in tumor epitope isolation. That is, the tumor has a different epitope feature than that of pAPC that processes TuAA, and thus the tumor epitope is “isolated” from the epitope from which CTL is derived by pAPC for recognition. The ability to predict and counteract this epitope isolation effect is critical to the development of new generations of therapeutic cancer vaccines. Overcoming epitope isolation results in epitope synchronization.

The role of epitope differences in infection by viruses and other intracellular parasites A wide variety of mechanisms are used by persistent pathogens to establish chronic infections in host organisms. A common attribute is a reduction or alteration of antigen expression. In some embodiments, the present invention is directed to the treatment and prevention of intracellular infections by various pathogens. Examples of such pathogens include, but are not limited to, any virus, bacterium, protozoan, prion, or other organism that has an intracellular stage of infection in the host.

  Viral antigen presentation by pAPC begins with digestion of the viral antigen into peptides by the proteasome. After the proteasome digests the protein into peptides, some of the peptides are loaded onto the class I complex in the endoplasmic reticulum and transported to the cell surface. On the cell surface, class I-peptide complexes are recognized by T cell receptors on the surface of CTL and the infected cells are killed.

  Herpesviruses and retroviruses are immune from detection and subsequent eradication by the host immune system through restricted viral gene expression. Other mechanisms by which certain viruses can evade the immune system have been proposed, including "immunologically exempted" sites of viral infection and antigenic mutations in key viral peptides . While these models may explain the persistence of certain viruses, the concept of epitope tuning or conversely epitope partitioning provides a solution. That is, the concept is that any virus or other cell that slips through the immune system by blocking immune proteasome expression in the host cell or otherwise preventing effective epitope synchronization between the infected cell and pAPC. It provides the basis for the vaccine to direct an effective cellular immune response against endoparasites (FIG. 5).

  Since infection of any cell with a pathogen usually causes the infected cell to produce IFN, the proteasome in the infected tissue typically switches from a housekeeping form to an immune form. Infection thus has the effect of strengthening infected cells with respect to that of pAPCs involved in stimulating immune responses against viruses or other intracellular pathogens and the antigen repertoire it displays on its surface. When virus-infected or parasitically infected cells are induced to express immune proteasomes rather than housekeeping proteasomes, the result is “epitope synchronization” between infected cells and pAPC, and subsequent eradication of infected cells by CTL. It becomes.

  However, certain viruses and other intracellular parasites can escape T cell recognition by down-regulating the expression of host molecules required for efficient T cell recognition of infected cells. There is evidence to suggest that many chronic viral infections interfere with the IFN cascade (see Table 1). Thus, due to the role of housekeeping proteasomes, immune proteasomes and epitope partitioning in a number of chronic infections, some embodiments of the present invention may include any relevant intracellular parasites such as viruses, bacteria and protozoa It is also applicable to vaccine design against (but not limited to). All intracellular parasites are targets for such vaccine design. Examples of these are viruses such as adenovirus, cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1, herpes simplex virus 2, human herpes virus 6, varicella-zoster virus, hepatitis B virus, hepatitis D. Virus, papillomavirus, parvovirus B19, polyomavirus BK, polyomavirus JC, hepatitis C virus, measles virus, rubella virus, human immunodeficiency virus (HIV), human T cell leukemia virus I and human T cell leukemia virus II; bacteria such as Chlamydia, Listeria, Salmonella, Legionella, Brucella, Coccella, Rickettsia, Mycobacterium; and protozoa such as Leishmania, trypanosomes, Toxoplasma and Plasmodium Genus including but not limited to.

Vaccines and Methods to Achieve Epitope Synchronization As discussed herein, effective cellular immunity is based on synchronized epitope presentation between pAPC and infected peripheral cells. In the absence of epitope synchronization, target cells are not recognized by T cells, even when those T cells are directed against TAA. Cancer cells and cells carrying viable intracellular parasites escape the cellular immune response because they avoid epitope synchronization. “Natural” epitope synchronization involves the activation of immune proteasomes in infected cells such that infected cells display immune epitopes and are therefore recognized by T cells induced by pAPC. Furthermore, cells infected by cancer as well as surviving intracellular parasites do not have active immune proteasomes and are therefore not recognized by normal arrays of induced T cells.

The vaccines and methods of the preferred embodiments of the present invention thus show essentially “reverse” epitope synchronization and allow pAPCs to display housekeeping epitopes, addressing the situation where target cells do not display immune epitopes (FIG. 6). And FIG. 7). Certain embodiments also provide a second wave of epitope tuning by inducing pAPC to display both housekeeping epitopes and immune epitopes corresponding to the selected target cells. Thus, in these dual epitope embodiments, if the target cell is effectively attacked by T cells that recognize the housekeeping epitope, switching by the target cell to immune proteasome processing does not result in a loss of immune recognition. This is due to the presence of immune epitopes in the vaccine that act to induce a population of T cells that recognize the immune epitope.

  Preferred embodiments of the invention are directed to vaccines and methods for presenting housekeeping epitopes corresponding to epitopes displayed on specific target cells to pAPCs or populations of pAPCs. In one embodiment, the housekeeping epitope is a TuAA epitope that is processed by a particular tumor type of housekeeping proteasome. In another embodiment, the housekeeping epitope is a virus-related epitope that is processed by the housekeeping proteasome of cells infected with the virus. This facilitates a specific T cell response against the target cell. Co-expression of multiple epitopes corresponding to different induction states (pre- and post-attack) by pAPC results in an effective CTL response against target cells since they display either housekeeping epitopes or immune epitopes (Figure 8).

  By having both housekeeping epitopes and immune epitopes presented on pAPC, this embodiment can optimize the cytotoxic T cell response to target cells. Using dual epitope expression, pAPC continues to maintain a CTL response to immunotype epitopes when tumor cells are switched from housekeeping proteasomes to immune proteasomes, eg, by induction with IFN that can be produced by tumor-infiltrating CTLs. obtain.

  In a preferred embodiment, immunization of a patient is by a vaccine that includes a housekeeping epitope. A number of preferred TAAs are exclusively associated with target cells, particularly in the case of infected cells. In another embodiment, a number of preferred TAAs are the result of deregulated gene expression in transformed cells, but can also be found in testis, ovary, and fetal tissues. In another embodiment, useful TAAs are expressed at higher levels in target cells than in other cells. In yet other embodiments, TAAs are not differentially expressed in target cells compared to other cells, but they are involved in certain functions of the cell and target cells are distant from most other surrounding cells. It is still useful for sorting. In such embodiments, healthy cells that also display TAA can be incidentally attacked by an induced T cell response, but such incidental damage is much better than the condition caused by the target cell. Conceivable.

  When neoplastic cells are the target, preferred antigens include TuAA. Examples of protein antigens suitable for use include differentiation antigens such as MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor specific multiple direct antigens such as MAGE- 1, MAGE-3, BAGE, GAGE-1, GAGE-2, CEA, RAGE, NY-ESO, SCP-1, Hom / Mel-40 and PRAME. Similarly, TuAAs include overexpressed oncogenes and mutant tumor suppressor genes such as p53, H-Ras and HER-2 / neu. In addition, unique TuAAs resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, and viral antigens such as Epstein-Barr virus antigen EBNA, and human papillomavirus (HPV) antigen E6 And E7. Other useful protein antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CAM 17. 1, NuMa, K-ras, β-catenin, CDK4, Mum-1 and p16, but are not limited thereto. These and other TuAAs and pathogen-related antigens are known and available in the literature or commercially to those skilled in the art.

  In a further embodiment, TAA is a virus specific antigen (see Table 2). In yet another embodiment of the invention, TAA is an antigen specific for non-viral intracellular parasites. Examples of parasite-specific antigens include nucleotides, proteins or other gene products associated with intracellular parasites. Suitable nucleotides or proteins can be found in the NCBI taxonomy database located at http://www.ncbi.nlm.nih.gov/Taxonomy/tax.html/. A more detailed description of gene products for parasites and other pathogens is provided on this website.

  The compounds and methods described herein are effective in any situation where the target cell displays a housekeeping epitope. Effective epitope discovery methods for use with the vaccines and therapeutic embodiments of the present invention are disclosed herein.

Proteolytic processing of antigens Epitopes displayed by MHC on target cells or on pAPC are cleavage products of large protein antigen precursors. For MHC I epitopes, protein antigens are digested by proteasomes that reside in the cell (see FIG. 1). Intracellular proteasomal digestion typically produces peptide fragments approximately 3 to 23 amino acids long. Additional proteolytic activity in the cell or in the extracellular environment can further trim and process these fragments. Processing of the MHC II epitope occurs via intracellular proteases from the lysosomal / endosomal compartment.

Perhaps most products of protein processing by proteasomes or other protease activities have little or no affinity for the binding groove of a particular MHC receptor peptide. However, processing products with such affinity would be presented at some level of abundance by MHC on the cell surface. Conversely, if a given oligopeptide sequence does not fully emerge from the antigen processing activity of the cell, it cannot be presented on the cell surface, regardless of the predicted affinity of the sequence for MHC.

  Vaccine designs that focus entirely on MHC affinity are fundamentally flawed. The mere fact that peptides have MHC binding affinity does not guarantee that such peptides are directed to functional immunogens. In order to provide an epitope that can elicit an effective immune response against TAA, the peptide must have MHC binding affinity and must be the product of a cellular peptide production system. The disclosed method of the invention utilizes both MHC binding affinity analysis and antigen processing analysis protocols to identify novel such epitopes.

Correlation of predicted or known MHC binding with proteolytic processing of antigens To identify epitopes that are believed to be effective as immunogenic compounds, prediction of MHC binding alone is the It is generally disadvantageous because fragments are not actually produced in the cell. Embodiments of the present invention combine analysis of MHC binding and analysis of proteolytic processing to identify epitopes that have both essential properties of useful epitopes: MHC affinity and precise proteolytic processing. Peptides with both of these properties are strong candidates for vaccines and immunotherapy. Peptides lacking any of these properties will not have any significant opportunity to function as an effective epitope.

  Embodiments of the invention can identify TAA-derived epitopes for use in vaccines. Target antigen is infected with neoplastic cells, cells infected with viruses or other intracellular parasites, or other pathogenic agents such as bacteria, fungi, protozoa, viruses, prions, toxins, toxins, allergens, etc. Obtained from cultured cells. In short, embodiments of the present method can be applied to virtually any protein sequence and can identify epitopes within it that can be produced by proteolysis and that can bind to MHC. Thus, the present invention is not limited to any particular target or medical condition, but instead encompasses the discovery of biologically relevant MHC epitopes from any useful source.

  In a preferred embodiment, TAA is characteristic of neoplastic cells and is therefore defined as a tumor associated antigen (Tu-AA). Preferred TuAAs include MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; generally excess Expressed embryonic antigens; overexpressed oncogenes and mutant tumor suppressor genes such as p53, Ras, HER-2 / neu; unique neoplastic antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4- RET, IGH-IGK, MYL-RAR1, and differentiation antigens such as viral antigens such as Epstein-Barr virus (EBVA) and human papillomavirus (HPV) antigens E6 and E7. Other antigens of interest include prostate specific antigen (PSA), prostate stem cell antigen (PSCA), MAAT-1, GP-100, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, p185erbB -2, p180erbB-3, c-met, nm-23H1, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, -Catenin, CDK4, Mum-1, p15 And p16. Other target antigens are also contemplated.

Various methods are available for identifying TuAA and are well known to those skilled in the art. Examples of these techniques include differential hybridization, such as the use of microarrays; subtractive hybridization cloning; differential display at the mRNA or protein speech level; EST sequencing; and SAGE (sequence analysis of gene expression) . These nucleic acid techniques are reviewed by Carulli, JP et al J. Cellular Biochem Suppl. 30/31: 286-296, 1998. The differential display of proteins includes, for example, comparison of two-dimensional polyacrylamide gel electrophoresis of cell lysates from tumors and normal tissues, the location of unique or overexpressed protein spots in the tumor, recovery of proteins from the gel, As well as protein identification using classical biochemical or mass spectroscopic sequencing techniques. Additional techniques for TuAA identification are described in Tureci, O., Sahin, U., and Pfreundschuh, M., “Serological analysis of human tumor antigens: molecular definition and implications”, Molecular Medicine Today, 3: 342, 1997. This is the SEREX technique discussed in. The use of these and other methods provides those skilled in the art with the techniques necessary to identify useful antigens for generating housekeeping and immune class I epitopes and class II epitopes for vaccines. However, in the practice of the invention, it is not necessary to identify a new TuAA or TAA. Rather, embodiments of the present invention make it possible to identify useful epitopes from any related protein sequence, whether the sequence is already known or novel.

Analysis of TAA fragments for MHC binding To identify biologically relevant epitopes, fragments within TAA that have known or predicted affinities for MHC are identified. The amino acid sequence of TAA can be analyzed by a number of different techniques to identify peptide fragments with known or predicted affinity for the groove to which the MHC peptide binds. In one embodiment of the invention, TAA fragments are analyzed for their predicted ability to bind to the groove to which the MHC peptide binds using a computer algorithm. Each allele of MHC identifies a specific epitope binding domain. Thus, for any given MHC allele, a candidate peptide can be screened for predicted affinity for it. Examples of suitable computer algorithms for this purpose include Hans-Georg Rammensee, Jutta Bachmann, Niels Emmerich, Stefan Stevanovic: SYFPEITHI: An Internet Database for MHC Ligands and Peptide Motifs (http: // access via: syfpeithi.bmi -heidelberg.com/scripts/MHCServer.dll/home.htm) found on the World Wide Web page. The results obtained from this method are discussed in Rammensee, et al., “MHC Ligands and Peptide Motifs,” Landes Bioscience Austin, TX, 224-227, 1997. Another such http: // site is “bimas.dcrt.nih.gov/molbio/hla_bind”, which also contains the appropriate algorithm. This website method is described by Parker, et al., “Scheme for ranking potential
HLA-A2 binding peptides based on independent binding of individual peptide side-chains, ”J. Immunol. 152: 163-175.

  The use of the NIH (Parker) algorithm in the method of the present invention selects peptides using a number of possible retention times to indicate the binding sequence. In one embodiment, peptides with infinite retention times are selected. In another embodiment, peptides having a retention time of 25 minutes or longer are selected to indicate the binding sequence. In yet another embodiment, a retention time of 15 minutes or longer is selected to indicate the binding sequence. In yet another embodiment, a retention time of 10 minutes or longer is selected to indicate the binding sequence. Retention times of 9, 8, 7, 6, 5, 4, 3, 2, and 1 minute are also considered.

  As an alternative to prediction algorithms, a number of standard in vitro receptor binding affinity assays are available to identify peptides with affinity for a particular allele of MHC. Thus, by the method of this aspect of the invention, the initial population of peptide fragments may be narrowed to include only peptides with actual or predicted affinity for selected alleles of MHC.

  Initially, peptide candidates for this analysis can include each possible sequence of about 6-24 contiguous amino acids from the entire protein sequence of TAA. In a preferred embodiment, the sequence can be about 7-20 amino acids long. In a further preferred embodiment, the sequence can be about 8-15 amino acids long. For sequence analysis to identify fragments with predicted affinity for MHC 1, the most preferred embodiment analyzes all possible sequences of 9 or 10 contiguous amino acid fragments of TAA. Analysis of fragment MHC affinity can be performed in vitro or by computer analysis of fragments.

  Selected common alleles of MHC 1 and their approximate frequencies are reported in Tables 3-5 below.

Tables 3, 4 and 5 show HLA Gene and Haplotype Frequencies in the North American
Population: The National Marrow Donor Program Donor Registry, Mori, M. et al. Determining Whether a Fragment with MHC Affinity is a Useful Epitope.

  As noted above, a preliminary step in the disclosed method is to select a sub-population of peptides with actual or predicted MHC affinity from among the original population of peptide fragments. Selected fragments are further analyzed to determine that they can be produced by cells under in vivo conditions that can result in binding of the peptide to selected MHC alleles. All peptides that meet both the MHC affinity and the exact proteolytic processing criteria are called “discovered epitopes”. Various methods are utilized to determine whether peptide fragments can be produced by proteolytic processing in vivo. These methods include elution of peptides from solubilized MHC and intact cells, computer sequence analysis of proteolytic cleavage motifs, and in vitro analysis of actual peptide fragments produced by cellular proteolytic mechanisms.

  In a preferred embodiment, a series of synthetic peptides can be generated that contain individual or clustered candidate peptide sequences in the center. Such peptides typically range from about 10 to about 75 amino acids in length. In a preferred embodiment, the synthetic peptide is about 20-60 amino acids in length. In a more preferred embodiment, the cluster is about 30-40 amino acids long. Using standard peptide synthesis chemistry, such as t-Boc protection chemistry, Fmoc protection chemistry, etc., one of ordinary skill in the art can generate a population of candidate peptides for subsequent screening.

Alternatively, peptide fragments containing candidate peptides can be generated in vitro by protease digestion or chemical cleavage of TAA or fragments thereof. Protease digestion to prepare such fragments of TAA can use a wide variety of known proteases, including proteasome protease, trypsin, -chymotrypsin, bromelain, clostripain, elastase, endoproteinase , Exoproteinase, proteinase K, ficin, papain, pepsin, plasmin, thermolysin, thrombin, trypsin, cathepsin and the like, but are not limited thereto. Chemical methods can also be used to generate peptide candidates. Suitable chemicals or chemical reactions for cleaving peptide bonds include mild acid cleavage, cyanogen bromide, hydroxylamine, iodosobenzoic acid, 2-nitro-5-thiocyanobenzoate and the like. In one embodiment, non-fragmented TAA can be used, but the use of a particularly large initial sequence can complicate the analysis.

  Regardless of how the fragment containing the candidate peptide is made, it is important to determine which epitopes are produced by cellular machinery. In one embodiment of the invention, proteasome digestion is used to estimate cellular epitope production. In this embodiment, the immune and housekeeping proteasomes are purified for in vitro use in order to assess the antigenic repertoire that is naturally produced from the two types of proteasomes.

  In general, proteasomes are prepared by affinity purification from cell extracts. In preferred embodiments, cell lysates are prepared using standard techniques. Lysate is removed by ultracentrifugation if red blood cells are not the original source material. The prepared cell lysate is then purified from other cell components using any one of a number of purification techniques, including various forms of chromatography.

  In one embodiment, affinity chromatography is used to purify the proteasome. The cell lysate is applied to an affinity column containing a monoclonal antibody (mAb) against one of the proteasome subunits. The column is then washed to purify the bound proteasome from other cellular material. After washing, the bound proteasome is then eluted from the column. The eluate is characterized with respect to protein content and proteolytic activity on a standard substrate.

  Cleavage analysis using both housekeeping proteasomes and immune proteasomes generates class I epitopes from various TAAs. The epitope presented by pAPC corresponds to the cleavage product of the immune proteasome, whereas the epitope presented by the tumor and by many cells chronically infected with intracellular parasites corresponds to the cleavage product of the housekeeping proteasome To do. Once digestion is performed, the specific molecular species produced are identified. In a preferred embodiment, this is accomplished by mass spectrometry. This allows for rapid identification of natural peptide fragments produced by either of the two types of proteasomes. In another embodiment, cleavage of the target antigen or fragment thereof by immunoproteasomes and housekeeping proteasomes or by endosome / lysosomal proteases (see below) is predicted by computer modeling based on a cleavage motif of related proteolytic activity. .

Class I MHC initially folds in the endoplasmic reticulum, so it is initially loaded with proteasome-derived peptides, while the class II MHC binding groove is blocked by the so-called invariant chain (Ii) in this compartment. . Peptide loading for Class II MHC occurs primarily in the endosomal compartment, utilizing peptides produced by endosomal and lysosomal proteases. Thus, if in vitro identification of MHC class II epitopes is desired, the preparation of proteases from endosomal and / or lysosomal fractions can be an alternative to proteasomes. Various methods for accomplishing this surrogate are described in the literature. For example, Kido & Ohshita, Anal. Biochem., 230: 41-7 (1995); Yamada, et al., J. Biochem. (Tokyo), 95: 1155-60 (1984); Kawashim
a, et al., Kidney Int., 54: 275-8 (1998); Nakabayshi & Ikezawa, Biochem. Int. 16: 1119-25 (1998); Kanaseki & Ohkuma, J. Biochem. (Tokyo), 110: 541-7 (1991); Wattiaux,
et al., J. Cell Biol., 78: 349-68 (1978); Lisman, et al., Biochem. J. 178: 79-87 (1979); Dean, B., Arch. Biochem. Biophys., 227: 154-63 (1983); Overdijk, et al., Adv. Exp. Med. Biol., 101: 601-10 (1978); Stromhaug, et al., Biochem. J., Biochem. J., 335 : 217-24 (1998); Escola, et al., J. Biol. Chem. 271: 27360-5 (1996); Hammond, et al., Am. J. Physiol., 267: F516-27 (1994) Williams & Smith, Arch. Biochem. Biophys. 305: 298-306 (1993); Marsh, M., Methods Cell Biol., 31: 319-34 (1989); and Schmid & and Mellman, Prog. Clin. Biol Res., 270: 35-49 (1988) all disclose methods for preparing suitable proteolytic preparations.

  In another embodiment, digestion to determine which epitopes are produced by cellular machinery occurs in cells that express TAA or fragments thereof. For class I epitopes, the type of proteasome expressed by the cell is preferably determined, for example, by Western blotting. MHC epitopes produced are from solubilized and purified MHC as described in Falk, K. et al. Nature 351: 290, 1991, or described in US Pat. No. 5,989,565. Can be eluted directly from such intact cells. The eluted fragments are then identified by mass spectrometry.

Analysis of target protein fragments The molecular species detected by mass spectrometry is compared to the candidate peptides predicted above. For the case of class I epitopes, peptides that are as long or longer than the candidate peptide and share the C-terminus are desirable. N-terminal trimming up to at least 25 amino acids can occur separately for the proteasome (Craiu, A. et al. Proc.
Natl. Acad. Sci. USA 94: 10850-55, 1997). Class II MHC is very tolerant with respect to the length of the peptide to which it binds, so the absence of cleavage at the center of the epitope is the primary criterion rather than the generation of appropriate ends.

  The selected digestion product is then synthesized and used as a standard in analytical methods such as HPLC for aliquots of the digest. This can provide further testing for the identity of the digested product and determine its yield. In rare cases, more than one possible product may have similar sufficient mass and chemical characteristics that cannot be reliably separated by these methods. In such cases, HPLC peaks can be collected and subjected to direct sequencing to confirm identity.

Analysis of peptides for MHC binding Epitopes are synthesized and tested for their ability to bind MHC receptors. For example, in one preferred assay, cells displaying MHC I receptors can be used to measure the binding affinity of candidate peptides labeled with a radionuclide. Another preferred approach uses a cell culture-based assay to measure the ability of a peptide to bind to the MHC I receptor. In this assay, cells lacking a transporter (TAP) associated with antigen processing are used to determine whether the candidate peptide has the ability to bind to the MHC I receptor. TAP ^- cells do not always properly fold class I MHC proteins and thus have a phenotype in which surface expression of MHC I is reduced or not expressed. When cells are flooded with exogenous peptides that can bind to the MHC I groove, receptor expression is restored. This can be monitored by several means, for example by RIA, FACS, etc. Using TAP ^- cells, one skilled in the art can screen a number of possible candidate peptides for receptor binding without performing a detailed binding affinity analysis.

  The analytical methods of the various embodiments of the present invention are useful for examining candidate peptides produced by various methods. For example, the described analysis can be used to evaluate a large number of candidate peptides generated by in vitro methods or by computer analysis to identify candidate sequences having MHC receptor binding properties. Preferred candidate peptides in this embodiment of the invention are those already known to be products of proteolytic production by housekeeping proteasomes and / or immune proteasomes. Both in vivo and in vitro cleavage products shown or predicted to bind to MHC are properly indicated as “discovered epitopes”. Epitope clusters for use with the present invention are disclosed herein.

ECR is processed into an MHC binding epitope in pAPC The immune system constantly examines the body for the presence of foreign antigens, partly due to the activity of pAPC. A pAPC endocytosis substance found in the extracellular environment processes the substance into a shorter oligopeptide of about 3 to 23 amino acids in length from the polypeptide form and converts some of the resulting peptides to the MHC complex of pAPC. Display for T cells. For example, tumor cells upon lysis release their cell contents, such as various proteins, to the extracellular environment. These released proteins are endocytosed by pAPC and processed into separate peptides, which are then displayed on the surface of pAPC via MHC. By this mechanism, it is not the entire target protein that is presented on the surface of pAPC, but rather only one or more separate fragments of that protein that are presented as MHC binding epitopes. If the displayed epitope is recognized by a T cell, that T cell is activated and an immune response results.

  Similarly, a scavenger receptor on pAPC can incorporate a naked or a recombinant organism containing a target nucleic acid sequence. Incorporation of the nucleic acid sequence into pAPC then results in expression of the encoded product. As described above, if the ECR can be processed into one or more useful epitopes, these products can be presented as MHC epitopes for recognition by T cells.

  MHC binding epitopes are often heterogeneously distributed throughout the protein sequence in the cluster. Embodiments of the invention are directed to identifying an epitope cluster region (ECR) in a specific region of the target protein. Candidate ECRs are thought to be natural substrates for various proteolytic enzymes and are thought to be processed into one or more epitopes for MHC display on the surface of pAPC. In contrast to more classical vaccines that deliver whole proteins or biological agents, ECR can be administered as a vaccine, where at least one epitope is present on MHC without the use of full-length sequences. Result in a high probability presented.

Use of ECR in the identification of distinct MHC binding epitopes Identification of putative MHC epitopes for use in vaccines is often available predictive algorithms that analyze protein or gene sequences to predict the binding affinity of peptide fragments for MHC Includes the use of These algorithms grade putative epitopes by predicted affinity or other properties associated with MHC binding. Examples of algorithms for this type of analysis include the Rammensee and NIH (Parker) algorithms. However, it has proved to be a difficult and laborious process to identify naturally occurring epitopes on the cell surface from among the predicted epitopes predicted using these algorithms. The use of ECR in the epitope identification process can greatly simplify the task of identifying distinct MHC binding epitopes.

In a preferred embodiment, ECR polypeptides are synthesized on an automated peptide synthesizer, and these ECRs are then subjected to in vitro digestion using proteolytic enzymes that are involved in protein processing for epitope presentation. Mass spectrometry and / or analytical HPLC are then used to identify the digested products, and an in vitro MHC binding test is used to assess the ability of these products to actually bind to MHC. If the epitopes contained in the ECR are shown to bind MHC, they can be incorporated into vaccines and used as diagnostic agents, as separate epitopes or in the context of ECR.

  The use of ECR in this preferred embodiment (because of its relatively short sequence can be generated by chemical synthesis) is a significant improvement over what would otherwise require the use of whole protein. . This is because the entire protein must be produced using recombinant expression vector systems and / or complex purification techniques. Simplification using chemically synthesized ECR allows for analysis and identification of a large number of epitopes, while greatly reducing the time and expense of the process when compared to other commonly used methods. The use of limited ECR also greatly simplifies mass spectroscopic analysis of the digest, since the product of ECR digestion is a small fraction of the digest of the total protein.

In another embodiment, a nucleic acid sequence encoding ECR is used to express a polypeptide in a cell or cell line to determine if the epitope is presented on the surface. Various means can be used to detect surface epitopes. Preferred embodiments include cell lysis and affinity purification of MHC, followed by peptide elution and analysis from MHC, or elution of epitopes from intact cells (Falk, K., respectively).
et al. Nature 351: 290, 1991 and US Pat. No. 5,989,565). A sensitive method for analyzing peptides eluted in this way from MHC uses capillary or nanocapillary HPLC ESI mass spectrometry and on-line sequencing.

Target associated antigens containing ECR TAAs from which ECR may be limited include those from TuAA, such as oncofetal, cancer-testis, deregulated genes, fusion genes from errant translocations, differentiation antigens, embryos Sex antigens, cell cycle proteins, mutant tumor suppressor genes, and overexpressed gene products such as oncogenes. Furthermore, ECR can be obtained from viral gene products, particularly those associated with viruses that cause chronic disease or are oncogenic, such as herpes virus, human papilloma virus, human immunodeficiency virus and human T cell leukemia virus. ECR can also be obtained from parasites such as trypanosoma, leishmania and other intracellular or parasite gene products.

Some of these TuAAs include α-fetoprotein, carcinoembryonic antigen (CEA), esophageal cancer-derived NY-ESO-1 and SSX genes, SCP-1, PRAME, MART-1 / MelanA (MART-1) , Gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-2, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed oncogenes and mutant tumors Suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR1 and viral antigens, EBNA1 , EBNA2, HPV-E6, -E7; prostate specific antigen (PSA), prostate stem cells Hara (PSCA), MAAT-1, GP-100, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, p185erbB-2, p185erbB-3, c-met, nm-23H1, TAG-72 , CA 19-9, CA72-4, CAM 17.1, NuMa, K-ras, -Catenin, CDK4, Mum-1, p15 and p16 Numerous other TAAs are also present in both pathogens and tumors Intended for. For TuAA, various methods are available and known in the art to identify genes and gene products that are differentially expressed in neoplastic cells relative to normal cells. Examples of these techniques include differential hybridization, such as the use of microarrays; subtractive hybridization cloning; differential display at the mRNA or protein expression level; EST sequencing; and SAGE (sequence analysis of gene expression) . These nucleic acid techniques are described in Carulli, JP et al J.
Cellular Biochem Suppl. 30/31: 286-296, 1998. Differential display of proteins, for example, comparison of two-dimensional polyacrylamide gel electrophoresis of cell lysates from tumors and normal tissues, location of unique or overexpressed protein spots in tumors, recovery of proteins from gels, And identification of proteins using classical biochemical or mass spectroscopic sequencing techniques. Additional techniques for TuAA identification are described in Tureci, O., Sahin, U., and Pfreundschuh, M., “Serological analysis of human tumor antigens: molecular definition and implications”, Molecular Medicine Today, 3: 342, 1997. This is the Serex technique discussed in (1).

  The use of these and other methods is necessary for identifying genes and gene products contained within target cells that can be used as possible candidate proteins for generating the disclosed epitopes of the present invention. Are provided to those skilled in the art. However, it is not necessary to identify a new TuAA or TAA in the practice of the invention. Rather, embodiments of the present invention make it possible to identify ECRs from any related protein sequence, whether the sequence is already known or novel.

Protein Sequence Analysis to Identify Epitope Clusters In a preferred embodiment of the invention, the identification of ECR involves two main processes: (1) identifying good putative epitopes, (2) these Limit the boundaries of any cluster where the putative epitopes are located. There are various preferred embodiments for each of these two processes, and the selected embodiment for the first process can be freely combined with the selected embodiment for the second process. The methods and embodiments disclosed herein for each of these processes are merely exemplary and are not intended to limit the scope of the invention in any way. Those skilled in the art will appreciate that special tools that can be applied to the analysis of a particular TAA and that such analysis can be performed in a number of ways in accordance with the present invention.

  Preferred embodiments for identifying good putative epitopes include grading putative epitopes to predict the binding affinity of peptide fragments to MHC, or according to predicted affinity or other characteristics associated with MHC binding. The use of any available prediction algorithm that analyzes the sequence of a protein or gene to apply is included. As mentioned above, examples of available algorithms for this type of analysis include the Rammensee and NIH (Parker) algorithms. Similarly, good putative epitopes can be identified by direct or indirect assays of MHC binding. In order to select a “good putative epitope” it is necessary to set a breakpoint with respect to the score reported by the prediction software or with respect to the assayed binding affinity. In some embodiments, such a division is absolute. For example, the split may be based on the measured or predicted half-life of dissociation between the epitope and the selected MHC allele. In such a case, the splitting embodiment is, for example, longer than 0.5 minutes, in a preferred embodiment longer than 2.5 minutes, in a more preferred embodiment longer than 5 minutes, and in a highly stringent embodiment 10 minutes. Or any dissociation half-life longer than 20 minutes or 25 minutes. In these embodiments, a good putative epitope is one that is predicted or identified to have good MHC binding properties, limited to be on the desired side of the designated split point. Similarly, splitting can be based on measured or predicted binding affinity between the epitope and the selected MHC allele. Furthermore, the absolute split can simply be a selected number of putative epitopes.

  In other embodiments, the division is relative. For example, a selected percentage of the total number of putative epitopes can be used to establish a partition to limit candidate sequences as good putative epitopes. Further features for grading epitopes are also obtained from measured or estimated MHC binding. The property used for such determination can be anything related to or indicative of binding. In preferred embodiments, the identification of good putative epitopes can combine a number of methods of grading candidate sequences. In such embodiments, good epitopes typically represent good epitope consensus based on different methods and parameters, or are particularly highly graded by at least one method.

  If several good putative epitopes are identified, their position relative to each other can be analyzed to determine the optimal cluster for use in a vaccine or vaccine design. This analysis is based on the density of selected epitopes characteristic within the sequence of TAA. The region that has the highest density characteristics or has a density that exceeds a selected cut is called ECR. Various embodiments of the present invention use different features for density analysis. For example, one preferred feature is simply the presence of any good putative epitope (as limited by any suitable method). In this embodiment, all putative epitopes above the split are treated equally in the density analysis, and the best cluster is the one with the highest density of good putative epitopes per amino acid residue. In another embodiment, preferred features are based on the parameter (s) conventionally used to score or grade the putative epitope. In this embodiment, a putative epitope that has twice the score of another putative epitope in the density analysis compared to other putative epitopes is doubly metered in the density analysis. Still other embodiments are scaled down, but consider score or grade by giving some putative epitopes a heavier weight than others in density analysis, for example using the logarithm or square root of the score.

  Depending on the length of the TAA being analyzed, the number of possible candidate epitopes, the number of good putative epitopes, the variability in scoring good putative epitopes, and other factors that will become apparent in any given analysis Various embodiments of the invention can be used alone or in combination to identify the ECR that is most useful for a given application. Iterative or parallel analysis using multiple approaches can be beneficial in many cases. ECR is a tool for increasing the efficiency of identification of true MHC epitopes and for efficient “packaging” of MHC epitopes into vaccines. Accordingly, any of the embodiments described herein, or other embodiments apparent to those of skill in the art based on this disclosure, can be obtained by using ECR instead of using full TAA for vaccines and vaccine design. Useful for enhancing the efficiency of attempts.

  Since many or most TAAs have regions with low density of predicted MHC epitopes, the use of ECR is a beneficial way to avoid the inefficiencies of including regions of low epitope density in vaccines and epitope identification protocols I will provide a. Thus, a useful ECR may be limited to any portion of TAA that is not total TAA, in which case that portion is more densely estimated than total TAA, or in particular any region of TAA that has a lower density of putative epitopes. Has an epitope. Thus, in this aspect of the invention, the ECR can be any fragment of TAA having a high epitope density. In some embodiments, the ECR may include a region up to about 80% of the length of the TAA. In preferred embodiments, the ECR may comprise a region up to about 50% of the length of the TAA. In a further preferred embodiment, the ECR may comprise a region up to about 30% of the length of the TAA. In the most preferred embodiment, the ECR may comprise a region that is 5-15% of the length of the TAA.

In another aspect of the invention, the ECR may be limited with respect to its absolute length. Thus, according to this definition, the smallest cluster for a 9-mer epitope contains 10 amino acid residues and has 2 overlapping 9-mers with 8 amino acids in common. In a preferred embodiment, the cluster is about 15-75 amino acids long. In a more preferred embodiment, the cluster is about 20-60 amino acids long. In the most preferred embodiment, the cluster is about 30-40 amino acids in length.

  In practice, as described above, ECR identification can use a simple density function, eg, the number of epitopes divided by the number of amino acids across those epitopes. Although it is not necessary that the epitopes overlap, the values for a single epitope are not significant. If only a single value is used for the percent split and no absolute split in epitope prediction is used, a single threshold can be set at this stage to limit the clusters. However, the use of absolute splitting and execution of the first process with different splitting percentages can result in variations in the overall density of candidate epitopes. Such variations may require further explanation or manipulation. For example, the overlap of two epitopes is more significant when only three candidate epitopes are considered than when 30 candidates are considered for any particular length protein. In order to take this feature into account, the weight given to a particular cluster can be further divided by the fraction of possible peptides actually under consideration to increase the significance of the calculation. This approximates the result to the average density of predicted epitopes in the parent protein.

  Similarly, some embodiments are based on scoring good putative epitopes with an average number of peptides considered per amino acid in the protein. The resulting ratio represents a factor in which the density of predicted epitopes in the putative cluster differs from the average density in the protein. Thus, ECR is limited in one embodiment as any region that contains two or more predicted epitopes with this ratio greater than 2, ie, any region that has twice the average density of epitopes. In other embodiments, the region is limited to ECR ratios greater than 1.5, 3, 4 or 5 or more.

  Consideration of the average number of peptides per amino acid in the target protein to calculate the presence of ECR highlights high density population ECR regardless of individual component scores / affinities. This is most appropriate for the use of score-based partitioning. However, ECRs that have only a few high-grade candidates can have several densely packed but low-graded candidates, especially if a small percentage of the total number of candidate peptides is selected as a good putative epitope. It can be more biologically significant than the cluster it has. Thus, in some embodiments it is appropriate to consider the score of individual peptides. This is most easily accomplished by replacing the sum of the peptide scores in the putative cluster instead of the number of peptides in the putative cluster in the above calculations.

  This total scoring method is more sensitive to sparsely clustered clusters containing high scoring epitopes. Scores rather than the score itself because the wide range of scores generated by the BIMAS-NIH / Parker algorithm (ie, the half-life of dissociation) can result in a single high scoring peptide that reduces the involvement of other possible epitopes Is preferably used for this approach.

（式中、Ｘはタンパク質配列中の天然に存在する任意のアミノ酸であり、式中のＸの各出現は、式中の任意の他のＸと異なるかまたは同一であるアミノ酸を示し得り、
ａはＰ２_１およびＰ２_２間のアミノ酸の数を示し、
ｂはＰ２_２およびＰ _１の相対位置を表し、
ＸａおよびＸ（｜ｂ｜−１）はそれぞれ長さ「ａ」および「｜ｂ｜−１」のこのようなアミノ酸の紐であり、
｜ｂ｜はｂの絶対値であり、
Ｐ２_１は第１のエピトープの第１の主要アンカーおよび第２の残基であり、
Ｐ２_２は第２のエピトープの第１の主要アンカーおよび第２の残基であり、
Ｐ_１は第１のエピトープの最後の主要アンカーおよびＣ末端残基であり、そして
Ｐ_２は第２のエピトープの最後の主要アンカーおよびＣ末端残基である）。
アンカー残基の同一点は、クラスターが関連するＭＨＣ型の結合モチーフによって、Ｘに関する可能性の特定の部分集合である。種々のＭＨＣ型に関する結合モチーフは当該技術分野において既知であり、いくつかの例が以下で考察される。特に、種々の種からの多数のＭＨＣ分子に対する主要および補助アンカーならびにその他の好ましい残基は、上記の参考文献により援用される「ＭＨＣリガンドおよびペプチドモチーフ（MHC Ligands and Peptide Motifs）」に報告されている。これらのデータは、ＳＹＦＰＥＩＴＨＩにより用いられる予測アルゴリズムの基礎を成し、クラスＩ ＨＬＡに関するそれらのデータが抽出されており、表６に示されている。ＢＩＭＡＳ−ＮＩＨ／Ｐａｒｋｅｒアルゴリズムにより用いられ、アンカー、好ましいおよび好ましくない残基も明示するクラスＩ ＨＬＡ係数表が、表７−１〜表７−４１に示されている。これらの表は、当業者にアクセス可能である有用な情報の種類の例証的な実例として提供される。表はこのような情報の完全
リストとして示されているわけではない。
９アミノ酸という最も一般的な長さを有するエピトープに関しては、以下に例示されるように「ａ」は０から７まで、そして「ｂ」は６から−１まで変わり得るが、但しａ＋ｂ＝６である。

Ｐ _１および第２のエピトープの第１の位置は、ｂ＝−１である場合、同一残基であり、ｂの負の記号は、式のこの実施形態において、Ｐ２_２が左でなくてＰ _１の右に置かれることを示す。Ｐ _１およびＰ２_２（第２のエピトープの第２の位置）は、ｂ＝０である場合に同一残基であり、それらの「間の」長さ｜ｂ｜−１は、−１の式値（formal value）を呈する。
８または１０アミノ酸長であるエピトープも一般に見出される。したがって他の実施形態では、構造式は、０ ａ Ｌｅ−２およびＬｅ−３ ｂ −１（ａ＋ｂ＝Ｌｅ−３であり、ここで、Ｌｅはエピトープの長さである）と設定することにより、他の長さのエピトープに関して一般化され得る。その場合クラスターの長さＬｃは、４＋２ａ＋ｂである。
したがってほとんどの実施形態において、「ａ」および「ｂ」は特定範囲で任意の値をとり得る。しかしながらＰ_１およびＰ２_２が一致する場合に関しては、例えば上記の９量体に関してはａ＝６、ｂ＝０であり、アンカー残基の特定化は排除構造式をもたらし得る。Ｐ２がＬまたはＭであり、ＰがＶまたはＬである単純ＨＬＡ−Ａ^＊０２０１結合モチーフ定義を用いて、ａ＝６、ｂ＝０は、Ｐ_１およびＰ２_２がともにＬである場合にのみ含入構造式を説明することが分かる。ａ＝６、ｂ＝０がまさに強制されるわけでないが、排除構造式を説明するような、Ｐ２およびＰに関する好ましい残基が互いに素な集合である種々のモチーフが存在する。
上記の構造式は、Ｐ２の位置に主要アンカーが常に存在するかのように上記の構造式は書かれているが、しかしこれは事実とは異なる。いくつかのモチーフ（例えばＨＬＡ−Ａ１）では、Ｐ２の位置の代わりにＰ３に主要アンカー残基が存在する。Ｐ３_１およびＰ３_２に関して上記の構造を書き直し、ａおよびｂに関する値の容認範囲を調整するには十分簡単であるが、しかしこれは必要でない。エピトープクラスターの特徴の理解によって、クラスＩＩ エピトープクラスターに適用可能であるよう式を調整することは同様に簡単である。したがってＰ３の位置に主要アンカー残基を有するモチーフを適応させるよう式をより簡単に修正するために、Ｐ２は、好ましいＰ３の残基（のアミノ側）に隣接するＸと定義され得るが、これはＨＬＡ−Ａ１の例ではＤおよびＥである。同様にＨＬＡ−Ｂ８に関する結合モチーフは、Ｐのほかに、Ｐ３およびＰ５の両方で主要アンカーを有し、それらの位置ではＫまたはＲが選択される。さらにまた、Ｐ２を配列Ｘ−Ｋ／Ｒ−Ｘ−Ｋ／Ｒにおける第１の残基と限定することにより、上記の鋳型が依然として用いられ得る。したがって実践者の選択および目的によって、第２のアンカーを含めた、そして結局はマトリックス定義を含めたより複雑なモチーフ定義が適用され得る。

「ａ」および「ｂ」に関して上記された値の範囲は、エピトープは少なくとも１つのアミノ酸だけ重複することを示す。場合によっては、エピトープは隣接するかまたは１〜数個のアミノ酸のギャップにより分離される情況を考えるのが適切であり得る。このクラスのクラスターの構造は、このような実施形態では、それぞれ接合面に関しては１、２、３・・・だけ、そして１、２・・・個のアミノ酸のギャップだけ、「ａ」の最大値を増大し、そして「ｂ」の最小値を低減することにより説明される。同様に、他の方向の「ａ」および「ｂ」の値の変更は、より大きい最小重複を保証する。
上に示した構造は、２つのエピトープからなるクラスターを限定しただけである。より多数のエピトープのクラスターを限定するための構造式の一般化は、いくつかの修正を要する。それにもかかわらず、アンカー残基、エピトープ長、ならびに「ａ」および「ｂ」の値に及ぼす限界変動の作用に関する種々の考察は、このより一般的な構造に等しく当てはまる。クラスター中により多くのエピトープを有する可能性は考え得る方法の数を増大するが、この場合、異なる個々のエピトープからの相互排除Ｐ２およびＰ アンカー残基の重複は、強制または排除構造を限定する。
上に示した構造式はクラスター中のエピトープの任意の隣接対に当てはまり得るため、
任意のサイズのクラスター中のエピトープの各連続対に定義を反復的に当てはめるために、指標模式図が考案され得る。あるいは、以下のように、エピトープクラスターの各連続成員と組合せて第１のエピトープに反復的に適用され得るよう、式が適合され得る：

（式中、２ Ｎ Ｎｃ；
Ｎはクラスターの第Ｎ番目のエピトープを示し、
Ｎｃはクラスター中のエピトープの総数を示し、
ａ_Ｎおよびｂ_Ｎは第１と第Ｎ番目のエピトープ間の位置関係を限定し、
上のａおよびｂと同様に、

および

上記からの結果、

上記は、特定ＭＨＣ分子に対する既知のまたは予測親和性を共有する単一長のエピトープを含むエピトープクラスターを説明する一般構造式である。これは、近接クラスターと呼ばれ得る。上に開示されたエピトープクラスターの一般定義は、クラスター内のエピトープの密度が全タンパク質中のエピトープの平均密度より大きいことを必要とする。したがってこの密度要件は、（Ｎｃ／Ｌｃ）＞（Ｎｐ／Ｌｐ）として表されるが、この場合、Ｎｐはタンパク質中のエピトープの総数であり、Ｌｐはタンパク質の長さである。 Various other calculations may be devised under certain conditions or other conditions. In general, the epitope density function is proportional to the number of predicted epitopes in a putative cluster, their score, their grade, etc., and inversely proportional to the number of amino acids or fractions of the proteins contained in that putative cluster, Built. Alternatively, the function can be evaluated with respect to a window of a selected number of consecutive amino acids. In either case, the function is evaluated for all putative epitopes of all proteins. If the ratio of values for the putative cluster (or window) and total protein is greater than, for example, 1.5, 2, 3, 4, 5 or more, the ECR is limited.
Epitope cluster structural formula An epitope cluster is a segment of a protein, such as a string of amino acids linked by peptide bonds. Within the protein in which it is a segment, its end is a half peptide bond. As an isolated macromolecule, it generally has terminal amino and carboxylic acid groups of other polypeptides, but any blocking group or other modification made at the end does not change the characteristic structure of the epitope cluster. . Any cluster has an amino acid sequence, but is not directly limited by that sequence. Rather, the cluster is limited by the alignment of epitopes associated with a particular MHC molecule within the protein sequence.
One example of epitope clustering within a protein (FIG. 9) is a position plot of a predicted HLA-A ^* 0201 epitope in tyrosinase. Specific sequence information has been generalized to symbols to illustrate the density and location of epitopes in this protein or any segment thereof, which indicates where the clusters exist and where they do not exist. Such plots are obtained from knowledge about protein sequences and predictive analysis (see Example 24: Tables 21-24 and FIG. 18), but are also obtained empirically. For example, by creating an ordered set of 9-mer fragments of tyrosinase and testing each fragment for HLA-A ^* 0201 binding, a plot very similar to FIG. 9 is obtained. Thus, according to this example, clusters can be identified without any reference to the basic sequence. Knowledge of the sequence facilitates and increases the usefulness of the cluster, but it is not their direct determinant.
Thus, by considering the sequence motifs present in the individual constituent epitopes of the cluster, a general structural formula describing the epitope cluster can be written.
The simplest cluster consists of two overlapping epitopes and can be represented by the following formula:

Wherein X is any naturally occurring amino acid in the protein sequence, and each occurrence of X in the formula may indicate an amino acid that is different from or identical to any other X in the formula;
a represents the number of amino acids between P2 ₁ and P2 _2,
b represents the relative position of P2 ₂ and _{P 1,}
Xa and X (| b | -1) are such amino acid strings of length "a" and "| b | -1", respectively;
| B | is the absolute value of b,
P2 ₁ is a first primary anchor and the second residue of the first epitope,
P2 ₂ is a first primary anchor and a second residue of a second epitope,
P ₁ is the last primary anchor and C-terminal residues of the first epitope, and P ₂ is the last primary anchor and C-terminal residues of the second epitope).
The identity of the anchor residues is a specific subset of possibilities for X, depending on the MHC-type binding motif with which the cluster is associated. Binding motifs for various MHC types are known in the art and some examples are discussed below. In particular, the main and auxiliary anchors and other preferred residues for a large number of MHC molecules from various species are reported in “MHC Ligands and Peptide Motifs”, which is incorporated by reference above. Yes. These data form the basis of the prediction algorithm used by SYFPEITHI and have been extracted for class I HLA and are shown in Table 6. Class I HLA coefficient tables used by the BIMAS-NIH / Parker algorithm and also specifying anchors, preferred and undesired residues are shown in Tables 7-1 to 7-41. These tables are provided as illustrative examples of useful information types that are accessible to those skilled in the art. The table is not shown as a complete list of such information.
For the epitope with the most common length of 9 amino acids, “a” can vary from 0 to 7 and “b” can vary from 6 to −1, as illustrated below, provided that a + b = 6 is there.

The first position of P ₁ and the second epitope is the same residue when b = −1, and the negative sign of b is P _{2 2} in this embodiment of the formula and P 22 is not left. Indicates that it is placed to the right of ₁ . P ₁ and P2 ₂ (second position of the second epitope) are the same residues when b = 0, and their “between” lengths | b | −1 is the formula of −1 Presents a formal value.
Epitopes that are 8 or 10 amino acids long are also commonly found. Thus, in other embodiments, by setting the structural formula as 0 a Le-2 and Le-3 b -1 (a + b = Le-3, where Le is the length of the epitope) It can be generalized for epitopes of other lengths. In that case, the length Lc of the cluster is 4 + 2a + b.
Thus, in most embodiments, “a” and “b” can take any value within a specified range. However, for the case where P ₁ and P2 ₂ match, eg a = 6, b = 0 for the above-mentioned 9-mer, the specification of the anchor residue may result in an excluded structural formula. P2 is L or M, using simple ^HLA-A * 0201 binding motif defined P is V or L, a = 6, b = 0 only if _{P 1} and P2 ₂ are both L It can be seen that the containing structural formula is explained. Although a = 6, b = 0 is not exactly enforced, there are a variety of motifs where the preferred residues for P2 and P are disjoint sets to account for the excluded structural formula.
The above structure is written as if the main anchor is always present at the position P2, but this is not the case. In some motifs (eg HLA-A1), there is a major anchor residue at P3 instead of at position P2. It is easy enough to rewrite the above structure for P3 ₁ and P3 ₂ and adjust the acceptable range of values for a and b, but this is not necessary. By understanding the characteristics of the epitope cluster, it is equally simple to adjust the formula to be applicable to a class II epitope cluster. Thus, in order to more easily modify the formula to accommodate a motif with a major anchor residue at position P3, P2 can be defined as X adjacent to the preferred P3 residue (on the amino side), Are D and E in the example of HLA-A1. Similarly, in addition to P, the binding motif for HLA-B8 has major anchors at both P3 and P5, and K or R is selected at those positions. Furthermore, by limiting P2 to the first residue in the sequence XK / RXK / R, the above template can still be used. Thus, depending on the practitioner's choice and purpose, more complex motif definitions can be applied, including the second anchor and eventually including the matrix definition.

The range of values described above for “a” and “b” indicates that the epitopes overlap by at least one amino acid. In some cases, it may be appropriate to consider situations where epitopes are adjacent or separated by a gap of one to several amino acids. The structure of this class of clusters is such that, in such an embodiment, the maximum of “a” is only 1, 2, 3,..., And 1, 2,. And decreasing the minimum value of “b”. Similarly, changing the values of “a” and “b” in the other directions guarantees a larger minimum overlap.
The structure shown above only limited a cluster of two epitopes. Generalization of the structural formula to limit a larger number of epitope clusters requires some modification. Nevertheless, various considerations regarding the effect of marginal variation on anchor residues, epitope length, and the values of “a” and “b” apply equally to this more general structure. The possibility of having more epitopes in the cluster increases the number of possible ways, but in this case the overlap of mutually excluded P2 and P anchor residues from different individual epitopes limits the forced or excluded structure.
Since the structural formula shown above can apply to any adjacent pair of epitopes in the cluster,
In order to apply the definition iteratively to each successive pair of epitopes in a cluster of any size, an indicator schematic can be devised. Alternatively, the formula can be adapted so that it can be applied iteratively to the first epitope in combination with each successive member of the epitope cluster as follows:

(Wherein 2 N Nc;
N represents the Nth epitope of the cluster,
Nc indicates the total number of epitopes in the cluster;
a _N and b _N define the positional relationship between the first and Nth epitopes;
Like a and b above,

and

The result from above,

The above is a general structural formula that describes an epitope cluster containing single-length epitopes that share a known or predicted affinity for a particular MHC molecule. This can be referred to as a proximity cluster. The general definition of an epitope cluster disclosed above requires that the density of epitopes within the cluster is greater than the average density of epitopes in the total protein. This density requirement is therefore expressed as (Nc / Lc)> (Np / Lp), where Np is the total number of epitopes in the protein and Lp is the length of the protein.

Analysis of target gene production for MHC binding Once a TAA has been identified, protein sequences can be used to identify putative epitopes with known or predicted affinity for the groove to which MHC binds. Peptide fragment testing can be performed in vitro, or the use of the sequence can be computationally analyzed to determine MHC receptor binding of the peptide fragment. In one embodiment of the invention, peptide fragments based on the amino acid sequence of the target protein are analyzed for their predictive ability to bind to the groove to which the MHC peptide binds. Examples of suitable computer algorithms for this purpose include the Rammensee / SYFPEITHI and NIH (Parker) sites referenced in the epitope discovery discussion above.

  As an alternative to prediction algorithms, a number of standard in vitro receptor binding affinity assays are available to identify peptides with affinity for a particular allele of MHC. Thus, by the method of this aspect of the invention, the initial population of peptide fragments can be narrowed to include only putative epitopes that have actual or predicted affinity for selected alleles of MHC. Selected common alleles of MHC and their approximate frequencies are reported in Tables 3-5 above.

  It has been observed that predicted epitopes are often clustered in one or more specific regions within the amino acid sequence of TAA. Such ECR identification provides a simple and practical solution to the problem of designing effective vaccines to stimulate cellular immunity. For vaccines where an immune epitope is desirable, ECR is directly useful as a vaccine. This is because the immune proteasome of pAPC correctly processes the cluster to release one or more containing MHC-binding peptides, and in the same way, cells with immune proteasomes are peptides that are completely TAA-derived Is processed and presented. Clusters are also useful as starting materials for the identification of housekeeping epitopes produced by housekeeping proteasomes active in surrounding cells.

Additional Considerations for Vaccine Design There are many alleles of MHC I in the human population. Thus, in a preferred embodiment, the vaccine design may take into account the patient's MHC I genotype in order to deliver an epitope with appropriate binding affinity for the particular patient's MHC allele (s). Since patients can be homozygous or heterozygous for related loci, in some embodiments of the invention the best epitope for a single MHC I allele is preferred, whereas in other embodiments Epitopes corresponding to different MHC alleles can be preferred. A partial list of the major class I MHC types, each generally encoded by multiple alleles, as well as their approximate frequency, is reported in Table 8.

  In yet another embodiment of the invention, the pAPC is equipped with housekeeping epitopes and epitope clusters. An epitope cluster is a peptide or nucleic acid sequence that contains or encodes at least two sequences with known or predicted affinity for MHC I. The housekeeping epitope is preferably provided to pAPC as a precursor that is manipulated in a fully processed state or in such a way that it can be processed in pAPC to be an effective housekeeping epitope, , Immune epitopes can be processed from large precursors by pAPC. This is because the immune proteasome is constitutively active in pAPC and is well-qualified to process an appropriate precursor of probably any length into an “exact” immune epitope.

Possible epitopes are generally not always found in clusters in separate segments of TAA containing multiple epitopes for the purpose of providing immune epitopes. If simply a polypeptide containing a cluster of possible epitopes, or a pAPC having a nucleic acid encoding the cluster, the recombinant organism expressing the cluster can cause the pAPC to produce at least one suitable immune epitope. Since epitope clusters generally contain possible epitopes for more than one class I MHC allele, in many embodiments, a single cluster is used to provide useful immune epitopes for more than one class I MHC allele. Can be produced.

  In a preferred embodiment, the patient is vaccinated with a vaccine comprising a housekeeping epitope from a selected TAA. The housekeeping epitope can be a polypeptide or a nucleic acid encoding the polypeptide, or the recombinant organism is engineered to express a distinct epitope. In addition to this “minimal” vaccine (whether a polypeptide, nucleic acid, or recombinant organism) that contains a housekeeping epitope, embodiments of the invention may include one or more other housekeeping epitopes, Alternatively, it includes a vaccine additionally having one or more immune epitopes, or any combination thereof. Such epitopes can be derived from the same TAA or they can be derived from different TAAs.

Preferred embodiments of the invention include methods of administering a vaccine comprising a housekeeping epitope to induce a therapeutic immune response. The vaccine is administered to the patient in a manner consistent with standard vaccine delivery protocols well known in the art. TAA epitope administration methods include, but are not limited to, transdermal, intranodal, peri-nodal, oral, intravenous, intradermal, intramuscular, intraperitoneal and mucosal administration. A particularly useful method of vaccine delivery to elicit a CTL response is PCT WO 99/01283 ("A METHOD OF INDUCING A CTL
RESPONSE ”(submitted on July 10, 1998).

  Vaccines for inducing specific T cell responses against target cells are also encompassed in preferred embodiments of the invention, since epitope synchronization systems have utility in inducing cell-mediated immune responses. The vaccine contains the housekeeping epitope at a concentration effective to cause the pAPC or population of pAPCs to display the housekeeping epitope. Advantageously, the vaccine may comprise a plurality of housekeeping epitopes, or one or more housekeeping epitopes, optionally in combination with one or more immune epitopes. The vaccine formulation contains peptides and / or nucleic acids at a concentration sufficient to cause pAPC to present the epitope. The formulation preferably contains the epitope at a total concentration of about 1 μg to 1 mg / 100 μl of vaccine formulation. Conventional dosages and administration for peptide vaccines and / or nucleic acid vaccines are used with the present invention, and such administration regimes are known in the art. In one embodiment, a single dose for an adult is beneficially from about 1 to about 5000 μl of such composition and is administered once or multiple times, eg, 1 week, 2 weeks, 1 month Or more, it may be 2, 3, 4 or more administrations. In a particularly preferred embodiment, such a composition is administered continuously for several days at a rate of at least 1 μl / hr by use of an insulin pump, directly to the lymph nodes. Such administration can be repeated periodically to maintain a CTL response, as more fully described in PCT WO 99/01283.

  The compositions and methods of the present invention disclosed herein are further intended to incorporate an adjuvant into the formulation to enhance vaccine performance. In particular, the addition of an adjuvant to the formulation is intended to enhance the delivery or uptake of the epitope by pAPC. Adjuvants contemplated by the present invention are known to those skilled in the art and include, for example, GMCSF, GCSF, IL-2, IL-12, BCG, tetanus toxin, osteopontin / ETA-1, CD40 ligand and CTLA-4 Examples include a blocking agent.

In further embodiments, housekeeping epitope-reactive T cells can be administered to a patient as adoptive immunotherapy. Such T cells are most easily obtained by in vitro immunization using cells from naïve donors, although the use of patients as donors may also be feasible. Techniques for in vitro immunization are known in the art (eg, Stauss et al., Pro. Natl. Acad. Sci. USA 89: 7871-7875, 1992; Kawakami et al., J. Immunol. 154: 3961-3968, 1995; Salgaller et al., Cancer Res. 55: 4972-4979, 1995; Tsai et al., J. Immunol. 158: 1796-1802, 1997; and Chung et al., J. Immunother 22: 279-287, 1999; and Bathe OF, J. Immunol. 167: 4511-4517, 2001). Also contemplated is the use of an immunized donor or the patient itself as an initial source of T cells (eg, Oelke, M. et al. Clin. Cancer Res. 6: 1997-2005, 2000; Gervois, N. et al. Clin. Cancer Res. 6: 1459-1467, 2000; Valmori, D. et al. Cancer Res. 59: 2167-3173, 1999; Tsai, V. et al. Crit. Rev. Immunol. 18: 65-75, 1998; Matsunaga, K. et al., Jpn. J.
Cancer Res. 90: 1007-1015, 1999; van Elsas, A. et al. Eur. J. Immunol. 26: 1683-1689, 1996; Alters, SE et al. Adv. Exp. Med. Biol. 417: 519 -524, 1997; and Dunbar, PR et al. J. Immunol. 62: 6959-6962, 1999; and Becker, C. et al., Nat. Med. 7: 1159-1162, 2001). Once generated, a sufficient number of such T cells can be obtained by expansion in vitro by stimulation with the vaccines and / or cytokines of the present invention (eg, Kurokawa, T. et al., Int. J Cancer 91: 749-746, 2001). These T cells may constitute a clone or polyclonal population that recognizes one or more epitopes. Typically transferred into mice on the order of 10 ⁵ to 10 ⁸ cells and into humans on the order of 10 ⁸ to 10 ¹¹ cells (eg Drobyski, WR et al. Blood 97: 2506-2513, 2001; Seeley BM et al. Otolaryngol. Head Neck Surg. 124: 436-441, 2001; Kanwar, JR et al. Cancer Res. 61: 1948-1956, 2001; Plautz, GE et al. Clin. Cancer Res. 6: 2209 -2218, 2000; Plautz, GE et al., J. Neurosurg. 89: 42-51, 1998; and Plautz, GE et al., Urology 54: 617-623, 1999). Clones and otherwise enriched populations generally require the transfer of a small number of cells. The recognized epitope can be a housekeeping epitope or a combination of housekeeping and immune epitopes. It is also contemplated that genetic engineering can be used to express the cloned TCR in a cell line suitable for use in adoptive immunotherapy. Examples of sources from which useful TCRs can be cloned include the T cells described above, as well as HLA-transgenic mice immunized with the vaccines of the present invention. Additional modifications will be apparent to those skilled in the art.
Adoptive immunotherapy refers to a therapeutic approach, eg, to treat cancer or infectious disease, but in some cases specific immunity to tumor cells and / or antigenic components, or tumor regression or infectious disease Immune cells are administered to the host for the purpose of directly or indirectly mediating the treatment of (see US Pat. Nos. 5,830,464, 5,985,270, 6,156,302). . Methods for adoptive immunotherapy of cancer and infectious diseases enhance host immunocompetence and immune effector cell activity.
Examples of cancers responsive to adoptive immunotherapy include human sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymph Endothelial sarcoma, synovial tumor, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell cancer, adenocarcinoma, sweat gland Cancer, sebaceous gland cancer, papillary cancer, papillary adenocarcinoma, cystadenocarcinoma, medullary cancer, bronchogenic cancer, renal cell cancer, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, fetal cancer, Wilms tumor, Cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ventricular ependymoma, pineal tumor, hemangioblastoma Acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemia, eg acute lymphocytic Hematology and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia) ); And polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease and the like.
Adoptive immunotherapy includes passive immunotherapy, which directly or indirectly mediates antitumor effects and has an established tumor-immunoreactivity that is not necessarily dependent on the intact host immune system. Delivery of functional reagents (eg, effector cells or antibodies). Embodiments of the invention related to the use of effector cells, such as T cells (eg, CTLs with specificity for an epitope). Preferably the epitope may comprise an immune epitope, more preferably a housekeeping epitope.
The use of autologous T lymphocytes in adoptive immunotherapy avoids the question of who should be selected as the donor of immune cells for adoptive transfer. In some cases, because of the risk that inactivated antigenic tumors may have grown in individuals who were immunized donors, to obtain immune cells, even normal individuals or even cancer patients Cannot immunize. Another problem that is avoided by the use of autologous immune cells is graft-versus-host disease that can be fatal if not successfully treated. The use of autologous T lymphocytes in adoptive immunotherapy can circumvent the question of who is chosen as the donor of immune cells for adoptive transfer.
T cells used for adoptive immunotherapy specifically kill tumor cells and have the ability to proliferate and persist after transfer, thus completely eliminating all residual or newly generated tumor cells Can do. T cells can be activated ex vivo polyclonally and then reinjected into the patient, in which case they respond directly to the tumor or to the tumor antigen presented by APC in the patient To do. A preferred method of obtaining a suitable number of T cells for adoptive immunotherapy is to isolate and clone out antigen-specific T cells in vitro.
Culture conditions for expanding single antigen-specific T cells into billions in total while retaining antigen recognition in vivo are known in the art. In vitro culture conditions usually utilize intermittent stimulation with antigen, often in the presence of cytokines (eg, IL-2) and non-dividing supporting cells. To generate a sufficient number of cells for immunotherapy, immunoreactive polypeptides can be used to rapidly develop antigen-specific T cell cultures. In particular, using various standard techniques known in the art, antigen-presenting cells (eg, dendritic cells, macrophages, mononuclear cells, fibroblasts or B cells) are pulse labeled with immunoreactive polypeptides. Alternatively, polynucleotide sequence (s) can be introduced into antigen presenting cells. For example, APC can be transfected or transduced with a polynucleotide sequence, in which case the sequence contains a promoter region suitable for increased expression and as part of a recombinant virus or other expression system. Can be expressed. A number of viral vectors (eg, poxvirus, vaccinia virus and adenovirus) can be used to transduce antigen presenting cells. Furthermore, antigen presenting cells are transfected with the polynucleotide sequence by various means (eg, gene gun technology, lipid mediated delivery, electroporation, osmotic shock and particle delivery mechanisms) and determined by one skilled in the art. As such, it can produce efficient and acceptable expression levels.
The effect of adoptive immunotherapy on the onset and progression of the disease can be determined by any method known to those skilled in the art, for example: a) delayed hypersensitivity as an assessment of cellular immunity, b) cytotoxicity T in vitro Cellular activity, c) levels of tumor-specific antigens (eg, carcinoembryonic antigen (CEA)), d) changes in tumor morphology using techniques such as computed tomography (CT) scans, e) high risk Can be monitored by measuring, but not limited to, changes in the level of estimated biomarkers of risk for a particular cancer in an individual, and f) changes in tumor morphology using sonograms.

In some embodiments of the invention, the vaccine may include a recombinant organism, such as a virus, bacterium, or parasite, that has been genetically engineered to express the epitope in the host. For example, Listeria monocytogenes, a gram-positive facultative intracellular bacterium, is a powerful vector for targeting TuAA to the immune system. In a preferred embodiment, the vector can be engineered to express a housekeeping epitope and induce a therapeutic response. The normal route of infection of this organism is the intestine, but can also be delivered orally. In another embodiment, an adenoviral (Ad) vector encoding a housekeeping epitope for TuAA can be used to induce an antiviral or antitumor response. Bone marrow derived dendritic cells can be transduced with a viral construct and then injected, or the virus can be delivered directly to the animal by subcutaneous injection to induce a strong T cell response. Another embodiment uses a recombinant vaccine virus engineered to encode an amino acid sequence corresponding to a housekeeping epitope for TAA. Vaccine viruses carrying constructs with appropriate nucleotide substitutions in the form of minigene constructs can direct the expression of housekeeping epitopes, resulting in a therapeutic T cell response to the epitope.

  Particularly useful nucleic acid constructs useful as vaccines according to the present invention are disclosed herein.

Epitope-encoding vector construct The present invention provides a nucleic acid construct for use as a therapeutic vaccine. The construct includes a coding region having a sequence encoding a polypeptide. The polypeptide is an epitope of TAA. In one embodiment, the target cell is a neoplastic cell and the polypeptide is a TuAA epitope or precursor of an epitope. In another embodiment, the target cell is any cell infected with an intracellular parasite. The term “parasite” as used herein includes any organism or infectious agent, such as a virus having an intracellular stage of infection within a host. Examples of these include viruses such as adenovirus, cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1, herpes simplex virus 2, human herpes virus 6, varicella-zoster virus, hepatitis B virus, hepatitis D Virus, papillomavirus, parvovirus B19, polyomavirus BK, polyomavirus JC, hepatitis C virus, measles virus, rubella virus, human immunodeficiency virus (HIV), human T cell leukemia virus I, and human T cell leukemia Virus II; bacteria such as Chlamydia, Listeria, Salmonella, Legionella, Brucella, Coccella, Rickettsia, Mycobacterium; and protozoa such as Leishmania, trypanosomes, Toxoplasma and Plasmodium Beam genus including but not limited to.

  The polypeptide (s) encoded by the nucleic acid construct can include a housekeeping epitope of TAA. In a preferred embodiment, the nucleic acid construct encodes multiple housekeeping epitopes. If the construct encodes such multiples, the multiple epitopes may all correspond to different segments of a single TAA, or they may correspond to different TAAs. In a preferred embodiment, the nucleic acid construct contains a housekeeping epitope and an immune epitope. In another preferred embodiment, the nucleic acid construct contains a housekeeping epitope and an epitope cluster region.

  In a preferred embodiment where the vaccine construct encodes both a housekeeping epitope and an immune epitope, the vaccine may stimulate a cellular immune response against target cells presenting either epitope. That is, the immune response can recognize the housekeeping epitope initially displayed by the target cell, and then also the immune epitope presented by the target cell after induction by IFN.

  Beneficially, the nucleic acid construct further comprises a third or fourth sequence, or more sequences, such sequences encoding a third or fourth epitope, or more additional epitopes, respectively. Such epitopes can be housekeeping epitopes or immune epitopes derived from a single TAA or from two or more different TAAs and in any combination. The constructs can be designed to encode epitopes corresponding to any other proteasome activity that plays a role in antigen processing in any target cell or pAPC.

The encoded MHC epitope is preferably about 7-15 amino acids long, more preferably 9 or 10 amino acids long. In general, the preferred peptide size for MHC I binding is 9 amino acids, but shorter and longer peptides may also bind MHC I in some cases. Similarly, many peptides that are much longer than 9 amino acids can be trimmed by exopeptidases or other proteases residing in cells to produce fragments that bind MHC I very efficiently. The size of the peptide containing the immune epitope sequence is not critical as long as the sequence contains the epitope. This is because the immune proteasome resident in pAPC, in combination with trimming exopeptidase and other proteases, properly retains full-length TAA and produces immune epitopes in its normal function. Thus, a nucleic acid sequence encoding an immune epitope may actually encode a much larger precursor, such as complete TAA. Such constructs preferably also encode a housekeeping epitope.

  Examples of TuAA and TAA suitable for use in the present invention include MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor specific multi-lineage antigens such as MAGE-1 , MAGE-3, BAGE, GAGE-1, GAGE-2, CEA, RAGE, NY-ESO, SCP-1, Hom / Mel-40 and PRAME, but are not limited thereto. Similarly, TuAAs include overexpressed oncogenes and mutant tumor suppressor genes such as p53, H-Ras, and HER-2 / neu. In addition, unique TuAAs resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigen E6 and E7 are mentioned. Other useful protein antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72. -4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, and p16. These and other TuAAs and pathogen-associated antigens are known and available to those of skill in the art, either in the literature or commercially.

  In a further embodiment, TAA is a virus specific antigen (see Table 2 above). In yet another embodiment of the invention, TAA is an antigen specific for non-viral intracellular parasites. Examples of parasite-specific antigens include nucleotides, proteins or other gene products associated with intracellular parasites. Suitable nucleotides or proteins can be found in the NCBI taxonomy database located at http://www.ncbi.nlm.nih.gov/Taxonomy/tax.html/. A more detailed description of gene products for parasites and other pathogens is provided on this website.

  Particularly preferred peptides are about 7-15 amino acids in length. An extensive list of peptides with MHC binding motifs is presented in Han-Georg Rammensee, Jutta Bachmann, and Stefan Stevanovic, “MHC Ligands and Peptide Motifs,” Springer-Verlag, Germany, (1997) Landes Bioscience, Austin, Texas ing.

  The epitope encoded by the construct has affinity for one or more MHC I alleles. In some embodiments where the patient is heterozygous for MHC I, the construct may encode epitopes corresponding to different MHC I alleles.

Preferred nucleic acid constructs include at least one promoter sequence operably linked to the 5 ′ end of the coding region of the construct. It will be appreciated by those skilled in the art that any promoter that is active in mammalian cells can be used. Preferred promoter sequences include CMV promoter, SV40 promoter and retroviral LTR promoter sequence, and may also include EF-1A, UbC, β-actin promoter. In some embodiments, the construct can include two or more promoters operably linked to the 5 ′ end of different polypeptide coding sequences. Similarly, constructs can use enhancers, nuclear import sequences, immunostimulatory sequences, and expression cassettes for cytokines, selectable markers, reporter molecules, and the like. In addition, immunostimulatory or other regulatory sequences can be attached to the vector via stably hybridized PNA peptide nucleic acids. In a preferred embodiment, the nucleic acid construct of the invention also includes a poly-A sequence operably linked to the 3 ′ end of the coding region. A nucleic acid construct comprising a nuclear import sequence and an immunostimulatory sequence is illustrated in FIG. 10A.

  In certain embodiments, the nucleic acid construct encodes an mRNA that is translated as a single polypeptide and then cleaved. In one such embodiment, the polypeptide consists of a linear array of epitopes, where the primary (N-terminal) sequence is one or more immune epitopes or epitope clusters and the secondary (C-terminal) The sequence is a housekeeping epitope, so the exact C-terminus of the housekeeping epitope is specified by a terminal codon, and all other HLA epitope ends are determined by proteasome processing and exopeptidase trimming. Strategies for the construction of vectors that can rely on such immunoproteasome processing and trimming for the release of housekeeping useful as vaccines according to the present invention are described in US Provisional Patent Application No. 60 / 336,968 (title) : EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS AND METHODS FOR THEIR DESIGN (filed on November 7, 2001).

  In another preferred embodiment, the nucleic acid construct encodes an amino acid sequence in which an immune epitope or epitope cluster is linked to a ubiquitin sequence. The ubiquitin sequence is similarly linked to a housekeeping epitope. The presence of ubiquitin between epitopes facilitates efficient delivery of immune epitopes to the proteasome for epitope processing. The ubiquitin sequence (with or without an N-terminal spacer to ensure the integrity of the preceding peptide) is between the first and second sequences, or between any other epitope coding sequences, Located within the frame. The so generated sequence 1-ubiquitin-sequence 2 polypeptide is rapidly (co-translationally) cleaved at the ubiquitin-sequence 2 junction by a ubiquitin-specific processing protease to yield sequence 1-ubiquitin and sequence 2. Occurs (see FIG. 11).

  Physiologically, ubiquitin primarily serves as a signal that targets proteins for degradation by the proteasome. It has only three conservative amino acid substitutions between yeast and human, among most conserved proteins in eukaryotes. Although the exact sequence of ubiquitin may vary somewhat, the sequence of a preferred embodiment is represented by SEQ ID NO: 5. Ubiquitin has two important features: 1) a C-terminal Gly residue involved in conjugation of ubiquitin to the Lys side chain of the protein substrate, and 2) a Lys residue at position 48 for the formation of multiple ubiquitin chains. It is a 76 amino acid long polypeptide.

  Ubiquitin genes are unique in the sense that all of them are synthesized as fusions with other polypeptides, such as other ubiquitins. In yeast beer yeast, four ubiquitin genes have been identified. The first three (UBI 1-3) are fused to ribosomal proteins and the fourth gene (UBI4) is synthesized as a fusion of five identical repeats of ubiquitin sequences. Thus, functional free ubiquitin is naturally produced after co-translational proteolytic processing by a universally expressed ubiquitin-specific hydrolase. Such natural mechanisms are developed by creating a C-terminal fusion between a single ubiquitin moiety and any desired polypeptide.

  Ubiquitin can exist in two conformations. The first is described above and consists of a linear fusion of a single ubiquitin and any desired polypeptide, in which the C-terminal Gly of ubiquitin is a polypeptide selected via a peptide bond. To the N-terminal amino acid. The second involves conjugation of a ubiquitin moiety and a protein substrate by Gly-Lys bond formation. In this case, the COOH group of ubiquitin Gly is linked to the ε (epsilon) side chain of the solvent-exposed Lys of the substrate (or another ubiquitin moiety). The ubiquitin signal for substrate degradation is related to the second conformation. Thus, in the sequence 1-ubiquitin-sequence 2 construct described above, sequence 2 is typically not targeted to the proteasome. Thus, the sequence 2 portion is preferably used for fully processed epitopes or those requiring only N-terminal trimming, typically housekeeping epitopes. The ubiquitin moiety that remains attached to Sequence 1 in the above construct is polyubiquitinated with Lys48, thereby targeting the fragment to the proteasome for processing, and the epitope contained in Sequence 1 Causes liberation. Note that when more than two sequences are linked together in a linear array by ubiquitin moieties, only the last sequence generally behaves in the manner of sequence 2, and the processing of all upstream sequences is similar to that of sequence 1. Should. To the extent that the constructs described herein are expressed in pPAC where the immune proteasome is predominantly active, the correct expression of housekeeping epitopes by these constructs is the sequence 2 position, or the epitope is in pAPC. Benefit from housekeeping epitopes present at corresponding positions that do not require proteasome processing at

In yet another embodiment, the nucleic acid construct of the present invention may comprise a self-proteolytic peptide coding sequence. Such sequences are located between the primary and secondary sequences, or between any other epitope coding sequences. An example of such a self-proteolytic sequence is intein. Also included are picornaviruses such as pliovirus, and other enteroviruses, rhinoviruses, cardioviruses and aphtoviruses 3C ^pro and 2A ^pro proteases, and equivalent coronavirus proteases. These proteases catalyze the post-translational cleavage of large precursor polyproteins produced by this family of viruses.

  In one embodiment, an autocatalytic protein sequence is inserted between two or more epitopes. In further embodiments, the sequence is inserted after two or more epitopes, but cleavage signals are found between the epitopes so that they are cleaved in two or more fully functional epitopes. The type of protease is not critical, it is only important that an appropriate cleavage signal is available for proper processing of the epitope.

Because the cleavage site and the sequence of the autocatalytic protein are known (reviewed recently by Seipelt, J. et al., Virus Research 62: 159-168, 1999), they produce polyproteins or biproteins Can be easily used for the construction of the vector to be In short, 3C ^pro predominantly recognizes the QG site as a cleavage signal, but other closely adjacent positions are important. Some 3C ^pros of these viruses adhere less closely to this general pattern, providing a higher degree of design flexibility. The restrictions imposed by these requirements are more formal than is practical, especially when the protease is placed between the expressed epitopes. In this alignment, upstream immune epitopes can be released by proteasome processing even if the viral protease is unable to cleave its N-terminus. The critical residues for cleavage at the C-terminus are internal to the 3C ^pro itself, generally leaving just 1 to 4 residues, if any, at the N-terminus of the downstream housekeeping epitope Is removed by exopeptidase trimming. 2A ^pro is often used in a similar manner and the cleavage site is preferably GP, but is understood to be somewhat more variable between these viruses. Its expression can result in a block of host cell protein synthesis with rapidity and integrity depending on the virus strain from which it is derived.

  Strictly speaking, the 2A proteins from cardiovirus and aft virus (ie hand-foot-and-mouth disease virus (FMDV)) are not proteases, but rather do not cause peptide termination at their C-terminus without causing termination of translation. Prevent (Ryan, MD, et al., Bioorganic Chemistry 27: 55-79, 1999). Thus, locating these 2A proteins between epitopes can cause cleavage within a single reading frame. The 2A protein from FMDV is very small, only 18 amino acids, making it particularly suitable for multi-epitope expression. A plasmid using the 2A protein is illustrated in FIG.

  In certain other embodiments, the nucleic acid construct encodes an mRNA that is translated as two or more polypeptides. In one such embodiment, the transcript may contain one or more internal ribosome entry site (IRES) sequences located between the primary and secondary sequences, or between any other epitope coding sequences. . The IRES sequence is used naturally by picornaviruses to direct internal cap-independent translation of mRNA. Such IRES sequences also allow separate translation of two or more consecutive open reading frames from the same messenger RNA. Although the IRES sequence of the various constructs can vary, a preferred embodiment IRES sequence is presented in SEQ ID NO: 6. The C-terminus of each epitope expressed is defined by a stop codon. Thus, the order of the sequence encoding the housekeeping epitope and the sequence encoding the immune epitope is not a problem and this provides flexibility in plasmid construction. Optionally, the sequence encoding the housekeeping epitope can precede the IRES sequence and the sequence encoding the immune epitope can be linked to the other end of the IRES sequence. Such vectors can usefully encode two or more housekeeping epitopes. They may further allow combinations of the various single polypeptide constructs described above to productively express multiple epitopes (see FIGS. 9A and 9B).

  In certain other embodiments, the nucleic acid construct encodes two or more mRNA transcripts. Each of these transcripts can encode a single epitope or any of the dual or multiple epitope transcripts described in the above embodiments. Two or more transcripts are the result of using multiple promoters. One skilled in the art will recognize that the use of more than one copy of a single promoter can result in instability of the growing plasmid. Thus, it is generally preferred to use two (or more) different promoters.

  Two or more transcripts can also be the result of the use of a bidirectional promoter. Bidirectional promoters can be found in a wide variety of organisms. Examples of such promoters include PDGF-A from humans, pcbAB and pcbC from Penicillium chrysogenum, neurophilic JC viruses, and BRCA1 from mice, dogs and humans. Thorough research on bi-directional promoters has started relatively recently, but there is a growing amount of information about sequences, regulation and other functions. For example, the human transcobalamin II promoter requires a 69 base pair (bp) fragment containing a GC box and an E box for full transcriptional activity. The dipeptidyl peptidase IV promoter has been shown to stimulate transcription from both sides with similar efficiency. Rat mitochondrial chaperonins 60 and 10 are head-to-head linked and share a bidirectional promoter. Thus, various working bidirectional promoters have been identified, sequenced, and cloned in a way that they can be used in nucleic acid constructs to express two genes.

  Thus, in a preferred embodiment, the nucleic acid construct contains a bidirectional promoter, as described above, linked to a nucleic acid sequence encoding, for example, a housekeeping epitope or precursor thereof. In a particularly preferred embodiment, the nucleic acid construct contains a bidirectional promoter linked to a nucleic acid sequence encoding a plurality of housekeeping epitopes. In another embodiment, the nucleic acid construct comprises a bidirectional promoter linked to nucleic acid sequences encoding housekeeping epitopes and immune epitopes, or to an epitope cluster region. Furthermore, bi-directional promoters can be regulated positively or negatively.

  If the nucleic acid construct contains more than one epitope, a bidirectional promoter can express multiple epitopes in comparable amounts, or some can be expressed at a higher level than others. Alternatively, certain epitopes are inducible and others are constitutive. In this way, temporal regulation of epitope expression can be achieved, where some epitopes are expressed early in the treatment and others are expressed late.

Analysis of Epitope Expression Several methods described in the literature can be used to determine whether an epitope has been presented on pAPC. An indirect but powerful method is the use of class I tetramer analysis to determine T cell frequency in animals before and after administration of housekeeping epitopes. Clonal expansion of T cells in response to an epitope occurs selectively only when the epitope is presented to T cells by pAPC. Thus, measurement of specific T cell frequencies for housekeeping epitopes before and after administration of the epitope to the animal is one means of determining whether the epitope is present on pAPC. An increase in the frequency of T cells specific for the epitope after administration indicates that the epitope was present on pAPC. Other methods of determining T cell frequency such as limited dilution analysis or ELISPOT can be used in principle in the same way as assessing housekeeping epitope presentation by pAPC. Similarly, any method of determining T cell frequency in an animal can be used.

  A direct method for determining housekeeping epitope presentation on pAPC involves purification of pAPC from the animal after epitope administration. After vaccination of animals with housekeeping epitopes, pAPC can be transformed into PBMCs, spleen cells by using monoclonal antibodies against specific markers present on pAPC, as well as monoclonal antibodies immobilized on magnetic beads using affinity purification. Or it can be recovered from lymph node cells. The optimal time for such recovery varies and depends on the animal to be vaccinated, the nature of the vaccine, and other factors such as dose, administration site, pharmacokinetics, and the like. Crude blood or spleen cell preparations can be enriched for pAPC using these techniques. Enriched pAPC can then be generated and used in proliferation assays against T cell clones that are specific for the housekeeping epitope. pAPC is co-incubated with T cell clones, and T cells are monitored for proliferative activity, for example, by measuring the uptake of radiolabeled thymidine by T cells. Proliferation indicates that T cells specific for the housekeeping epitope are stimulated by that epitope on pAPC.

  The following examples are intended for purposes of illustration and are not intended to limit the scope of the invention in any way.

[Example]
Characterization of proteolysis of HLA epitopes as housekeeping epitopes or immune epitopes Using the techniques described below, synthetic peptides of 13 or more amino acids containing candidate HLA epitopes are prepared intensively. Proteasomes are prepared from cells that express each type of proteasome, eg, red blood cells and large cells for housekeeping proteasomes and immune proteasomes, respectively. The peptide is digested with a proteasome preparation and the resulting fragment is identified by mass spectrometry. An HLA epitope is a housekeeping epitope if it is produced in significant yield in a preparation containing the housekeeping proteasome, one of which fragments is the common C-terminus with the HLA epitope. Similarly, if one of the fragments is C-terminal in common with the HLA epitope and is produced in significant yield by the immune proteasome and not in significant yield by the housekeeping proteasome, the HLA epitope is It is.

A. Peptide synthesis A synthetic or recombinant polypeptide is constructed comprising an HLA epitope and at least two residues proximal to its termini. These residues added to the end of a particular HLA epitope ensure that the proteasome complex encounters a processing environment similar to the environment in which it is found in the cell, and thus the proteasome normalizes its proteolytic function. Increase the likelihood of implementation. Additional residues commonly found near the end of the HLA epitope can be added if necessary to help increase the solubility of the peptide.

  Some HLA epitopes exhibit difficulty in lysis due to their high hydrophobicity. Certain peptides can be very difficult to purify because they do not dissolve in normal chromatographic eluates. Alternatively, they do not dissolve in the digestion buffer and can be very difficult to use once purified. This problem can be avoided by carefully selecting part of the sequence surrounding the HLA epitope for inclusion in a particular peptide construct or by extending the sequence as described in the preceding paragraph. Can do. If there are no proximal residues at the end of the HLA epitope that can help increase solubility, a short hydrophobic sequence can be added instead (eg, -EAEAE). This adds at least 3-5 residues ahead of the end of the HLA epitope to maintain the natural truncation site for the proteasome.

  In a preferred embodiment, the peptides are synthesized on an Applied Biosystems 433A peptide synthesizer using standard Fmoc solid phase synthesis methodology. The synthesizer is equipped with a conductivity feedback monitoring system that can increase the reaction time for sequences containing a series of residues that are difficult to deprotect and / or difficult to bind. After synthesis, the peptide is excised from the support with trifluoroacetic acid in the presence of a suitable scavenger, precipitated with ether and lyophilized.

  The crude peptide is then purified on the same preparative diphenyl HPLC column after first developing a gradient using an analytical diphenyl HPLC system. The major HPLC fraction from the first preparation injection of the peptide is analyzed by electrospray mass spectrometry to identify the target compound. Corresponding peaks from subsequent injections are collected, pooled, lyophilized, and samples are taken to verify retention time and chromatographic purity by analytical HPLC. These purified peptides are then prepared for digestion with a proteasome preparation.

B. Proteasome Assay The immune proteasome or housekeeping proteasome complex is isolated as detailed in Example 2 below.

The purified peptide is then dissolved in an appropriate buffer to a concentration of about 1 mM and about 2 volumes of the proteasome preparation is added. Replicate digests: Prepare one for mass spectrometry and one for HPLC analysis and prepare additional digests with a positive control peptide to verify the qualified functionality of the proteasome preparation used . The following peptides are suitable for use as control peptides for immune proteasome assays: MLLAVLYCLLWSFQTS (SEQ ID NO: 7), HSYTTAEEAAGITILT VI LGVL (SEQ ID NO: 8), EAASSSSTLVEVTLGE V PAAESPD (SEQ ID NO: 9), EFLWGPRAL V ETSYVK V LHHMVKI (SEQ ID NO: 10), APEEKIWEELSV L EVFEGR (SEQ ID NO: 11), and ELMEVDPIGHL Y IFAT (SEQ ID NO: 12). Underlined residues indicate proteolytic cleavage sites. The peptide FLWGPRALVETSYVK (SEQ ID NO: 13) is suitable as a control peptide for housekeeping proteasome assays. These are allowed to incubate in parallel for a period of time at 37 ° C., followed by termination of digestion by the addition of diluted trifluoroacetic acid and freezing of the samples on dry ice. One replicate and positive control is sent out for analysis using Lasermat 2000 (Finnigan Mat, LTD, UK). Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry etc. is reserved for HPLC.

C. MALDI-TOF mass spectrometry of digests Analysis of digests is performed using either “Peptide” software (Lighthouse Data) or software available from ThermoBioanalysis Ltd., UK. This software can generate the sequence and molecular weight of all possible fragments that meet both the requirement to have the exact C-terminus of any predicted epitope and the requirement to contain more than the full length of that epitope.

For example, a peptide encompassing an HLA epitope has the following sequence:
AAMLL AVLYCLLSEI AAAEEE (SEQ ID NO: 14)
(Where the underlined sequences are HLA epitopes), the program will identify all of the following sequences as potentially useful and assign each molecular weight.
AAMLL AVLYCLLSEI (SEQ ID NO: 15)
AMLL AVLYCLLSEI (SEQ ID NO: 16)
MLL AVLYCLLSEI (SEQ ID NO: 14)
LL AVLYCLLSEI (SEQ ID NO: 18)
L AVLYCLLSEI (SEQ ID NO: 19)
AVLYCLLSEI (SEQ ID NO: 20)

  If the MALDI-TOF results indicate that one or more of their molecular weights are represented in the digestion mixture, the corresponding peptide is synthesized, purified, identified by mass spectrometry, followed by analytical HPLC. Do and establish both standard retention time and approximate mass to peak area ratio. The stock digest is then diluted with an appropriate solvent and injected using the same HPLC method. If the digest gives a peak in good yield with the same retention time as the standard, it is almost certain that it is due to the presence of that sequence in the digest. If there is ambiguity due to possible generation of other fragments that give the same or similar mass analysis results, suspicious components can be collected and reserved for C-terminal sequencing to confirm identity.

Purification of Proteasome Complex A. Blood cell derived proteasome complex Concentrated red blood cell bags were obtained from a local blood bank (HemaCare, VanNuys, CA). The contents of each bag were poured into 200 ml centrifuge tubes and washed 3 times with PBS by centrifugation at 2000 RPM for 10 minutes at room temperature in a Megafuge 2.0 (Heraeus, Southplainfield, NJ) swing bucket rotor. After the final wash, the sample was subdivided into several centrifuge tubes after pouring one container to minimize variability between tubes. Cells at 2000 RPM
And centrifuged again for 10 minutes. The remaining PBS was aspirated. The pellet was stored at -70 ° C until used.

B. Tumor cell-derived proteasome complex Large cells, Burkitt lymphoma cell lines were obtained from ATCC (American Type Culture Collection, Manassas, Va.). Cells were grown using standard cell culture methods and stimulated with INF-γ (100-500 U / ml) (Pharmingen, San Diego, Calif.). The expression of immunoproteasome subunits was individually confirmed by immunohistochemistry in culture and SDS-PAGE in cell lysate samples. Cells were collected by a centrifuge, washed with PBS and stored at -70 ° C until use.

C. Further Processing of Protease Complexes Blood cell or lymphoma cell pellets (frozen) were thawed in a 30 ° C. bath and ddH ₂ O was added to each tube. The cell suspension was homogenized with a 40 ml Dounce homogenizer. In addition, for tumor cells, the cell homogenate was centrifuged at 2000 rpm to remove cell necrotic tissue debris. The supernatant was centrifuged at 10,000 rpm for 10 minutes at 4 ° C, and further centrifuged at 50,000 rpm for 30 minutes at 4 ° C in a T-1270 rotor (Sorval, Newtown, CT).

The homogenate was passed through filter paper to remove necrotic tissue pieces and subsequently pooled together. A 68% sucrose solution was added to the pooled homogenate sample. The antibody-sepharose preparation was incubated with this homogenate for 3 hours at room temperature on a rotator. The suspension was centrifuged, washed 3 times with TBS, and thoroughly washed 6-8 times with a vacuum funnel. The proteasome was eluted in TBS (pH 7.6), and the optical density of the eluate was measured. The proteasome preparation was dialyzed overnight at 4 ° C. against 20 mM Tris (pH 7.6) using cellulose membrane MWCO1000. The next day, the proteasome preparation was concentrated by ultrafiltration on a Millipore ULTRAFREE-15 centrifuge (Millipore, Danbury, Conn.). Proteasomes at a concentration of 4 mg / ml were collected and stored at −20 ° C. until use. Proteasomes were tested for activity and specificity by digestion of fluorescent substrates or control peptides that yielded known fragments. The following peptides are suitable for use as control peptides for immune proteasome assays: MLLAVLYCLLWSFQTS (SEQ ID NO: 21), HSYTTAEEAAGITILT VI LGVL (SEQ ID NO: 22), EAASSSSTLVEVTLGE V PAAESPD (SEQ ID NO: 23), EFLWGPRAL V ETSYVK V LHHMVKI (SEQ ID NO: 24), APEEKIWEELSV L EVFEGR (SEQ ID NO: 25), and ELMEVDPIGHL Y IFAT (SEQ ID NO: 26). Underlined residues indicate proteolytic cleavage sites. The peptide FLWGPRALVETSYVK (SEQ ID NO: 27) is suitable as a control peptide for housekeeping proteasome assays.

D. Quantification and activity analysis of proteasome preparations The above-described proteasome preparations were quantified using an enzyme linked immunosorbent assay (ELISA). ELISA techniques are known in the art, generally Ausubel, et al., "Short Protocols in Molecular Biology", 3 ^rd Ed., Are discussed in Unit 12.2 (1997). Hybridoma cells (MCP-21) producing a monoclonal anti-human proteasome antibody were obtained from European Collecction pf Cell Culture ((ECACC), UK) and maintained using standard cell culture techniques and equipment. Hybridoma supplement (Gibco BRL, Rockville, MD) was added to the antibody producing cells. When a cell density of 500,000 cells / ml was reached in a volume of about 2-3 liters, the cells were removed by centrifugation and the supernatant was collected. MAb secretion in the medium was monitored periodically by optical density (OD) using a Lambda 20 spectrophotometer (Perkin Elmer, Norwalk, CT).

  The supernatant was passed over a protein G sepharose column (Amersham / Pharmacia Biotech Piscataway, NJ). The column was washed with PBS, and the antibody was eluted with 0.1 M glycine buffer (pH 2.2). The optical density of the eluate fraction was measured at 280 nm and the positive fraction was collected. The antibody was dialyzed against 2 L of PBS for 2 days at 4 ° C. and stored until use.

  The antibody was conjugated to CNBr activated Sepharose 4B (Amersham Pharmacia Biotech Piscataway, NJ). The antibody-sepharose complex is washed 5-7 times alternately in 0.1 M sodium acetate saline (pH 4) and 0.1 M sodium borate saline (pH 8), and finally tris buffered saline. Suspended in (TBS) (pH 8). The preparation was stored at 4 ° C. until use.

Generation of peptides that bind to the groove of predicted MHC I peptides using algorithmic modeling Algorithms for candidate MHC I binding peptide populations generated from amino acid sequences of human carcinoembryonic antigen precursor (CEA) (GENBANK Accession P06731) It was created using The specific algorithm is available at << http://134.2.96.221/scripts/hlaserver.dll/EpPredict.htm >> as described above. Once this algorithm was accessed, the amino acid sequence for CEA was provided. Next, parameters for a given epitope length (decamer) and a specific MHC allele (H2-Db) were selected. After this, the data was submitted for algorithmic analysis. The resulting data is shown in Table 9.

  The above table optionally truncates a score of less than 15. This algorithm can produce a score of less than 15.

Peptide was synthesized using a 433A ABI synthesizer, digesting peptide precursors with immune and housekeeping proteasomes to determine the fragments produced by proteolytic digestion. Using Fastmoc chemistry, peptides were produced in an amount of 0.25 mmol. The peptides were tested for solubility and once dissolved, a 2 mM solution was prepared that was divided into approximately 25-30 μL aliquots and stored at −20 ° C. until further use. Periodic digestion reactions, typically consisting of 2 μl of peptide and 4 μl of proteasome, were performed with t = 0 as a control and incubation of the peptide with water instead of the proteasome as a further control. The reaction was carried out at 37 ° C. and was terminated by adding 10% TFA (trifluoroacetic acid) on dry ice. The frozen samples were then analyzed by MALDI-TOF mass spectrometry (MS) as described in Example 5 below.

An optional desalting step can be performed on the digest prior to MS analysis using the ZIP-TIP method (Millipore, Boston, Mass.). ZIP TIP is a specially designed pipette tip containing a spherical silica resin bed. The sample is bound to a chip pre-equilibrated with 0.1% TFA and then eluted with 50% acetonitrile 0.1% TFA elution buffer.

Identification and quantification of relevant proteolytic fragments by HPLC and mass spectrometry. Identification of sequences of therapeutic interest The amino acid sequence of a given protein is entered into a computer and an algorithm such as Rammensee et al. Is used to generate a 9 or 10 amino acid long sequence predicted to bind to a particular HLA receptor. The algorithm also ranks these predicted epitopes according to how well they match the binding motif.

  Next, a synthetic peptide containing the identified sequence of possible epitopes is constructed to encompass the epitope candidate sequence and at least 3 to 5 residues proximal to its ends. Residues added to the ends of specific epitope candidates ensure that the proteasome complex encounters a processing environment similar to that found in the cell, and thus the proteasome performs its proteolytic function normally Increase the possibility. Additional residues commonly found near the ends of epitope candidates can be added if necessary to help increase the solubility of the peptide.

  Peptides are synthesized on an Applied Biosystems 433A peptide synthesizer (Applied Biosystems, Norwalk, CT) using standard Fmoc solid phase synthesis methodology. The synthesizer is equipped with a conductivity feedback monitoring system that can increase the reaction time for sequences containing a series of residues that are difficult to deprotect and bind. After synthesis, the peptide is excised from the support with trifluoroacetic acid in the presence of a suitable scavenger, precipitated with ether and lyophilized.

  The crude peptide is then dissolved at 0.5 mg / ml in a suitable solvent. Subsequently, 5 microliters (5 μl) of this solution is analyzed by Shimadzu analytical reverse phase HPLC system (Shimadzu Scientific Instruments, Columbia, MD) using a 0.1% TFA water-acetonitrile gradient. Typically, a C-18 silica column (Machery-Nagel # 720051.40, (Machery-Nagel GmbH, Germany)) is used for hydrophilic peptides and a phenyl silica column (Vydac # 219TP5415 (The Separations Group, Inc., Hesperia, CA)) is used for hydrophobic peptides. The gradient used varies from 0-40% acetonitrile for hydrophilic peptides to 30-70% acetonitrile for hydrophobic peptides. The peptide is then purified on a Varian Prostar HPLC system (Varian, Inc., Palo Alto, Calif.) Using a similar gradient and semi-preparative format of the columns described above (Machery Nagel # 715802, and Vydac 219TP510). The main HPLC fraction from the first preparative injection of peptide is analyzed using a MALDI-TOF mass spectrometer to identify the desired component. Corresponding peaks from subsequent injections are collected, pooled, lyophilized, and samples are taken to verify retention time and chromatographic purity by analytical HPLC using the system described above. These purified peptides are then prepared for digestion with a proteasome preparation.

B. Proteasome Assay Isolation of immune proteasomes or housekeeping proteasome complexes by the Levy method described above (Morel, S., et al., Immunity 12: 107-117 (2000), references cited herein) To do. The purified peptide is dissolved in a suitable buffer to a concentration of about 1-2 mM and about 2 volumes of proteasome preparation is added. The chosen buffer must solvate the peptide without interfering with the digestion process. In order to verify the qualified functionality of the proteasome preparation used, further digests are prepared using the positive control peptide described above. These are incubated for up to 120 minutes at 37 ° C., followed by termination of digestion by the addition of diluted trifluoroacetic acid and the samples analyzed immediately by mass spectrometry or frozen on dry ice until analysis . The digestion reaction can also be stopped by placing the sample on ice for immediate analysis by mass spectrometry.

C. MALDI-TOF mass spectrometry of digests About 0.5 μl of each digest was directly mixed with an equal volume of matrix solution (10 mg / ml dihydroxybenzoic acid, pH 2-3 in 70% EtOH) on a sample slide and about 40 μl. Air dried at ° C. The samples were then analyzed on a Lasermat® MALDI-TOF mass spectrometer (Thermo Bioanalysis, Santa Fe NM) calibrated with the appropriate molecular weight standards.

  Computer programs developed for proteasome assays ("Peptide" software (Lighthouse Data), or "Dynamo" (Thermo Bioanalysis Ltd., UK) are required to have the exact C-terminus of any predicted epitope, and Generate the sequence and molecular weight of all possible fragments that fulfill both the requirement of containing more than the full length of the epitope.

  If the MALDI-TOF results indicate that a specific molecular weight is expressed in the digestion mixture, the corresponding peptide is synthesized and purified, identified by MALDI-TOF, followed by reverse phase analytical HPLC, Both retention time and approximate mass to peak area ratio were established. These approaches are very similar to those described above. The replicate proteasome digest is then diluted with a suitable solvent and analyzed using the same analytical HPLC method. If the digest gives a peak in good yield with the same retention time as the standard, it is almost certain that this is due to the presence of that sequence in the digest. If there is ambiguity due to possible generation of other fragments that give the same or similar mass analysis results, suspicious components can be collected and reserved for sequencing to confirm identity. Analytical HPLC also importantly provides relatively accurate quantification of the peptide product in the digest and determines whether a given peptide is a minor product or a major product of the digest This indicates whether the epitope is efficiently produced by the proteasome. Using the method described above, housekeeping epitopes were identified. FIG. 13 shows the results of a flow cytometry assay to verify HLA binding by these epitopes. This assay is described in Example 6.

Determination of MHC binding ability of selected peptides The binding of candidate peptides to HLA-A2.1 was assayed according to the method of Stauss et al. (Proc Natl Acad Sci USA 89 (17): 7871-5 (1992)). T2 cells expressing empty or unstable MHC molecules on the surface were washed twice and suspended at 5 × 10 ⁶ cells / ml in serum-free complete Iscove modified Dulbecco medium (IMDM). β ₂ microglobulin (Sigma, St. Louis, MO) was added at 5 μg / ml to distribute the cells at 5 × 10 ⁵ cells / well in a 96 well U-bottom plate. Peptides were added at 100, 10, 1 and 0.1 μg / ml. The plate was gently shaken for 2 minutes and incubated for 4 hours at 37 ° C. in a 5% CO ₂ incubator. Unbound peptide was removed by washing twice with IMDM and a saturating amount of monoclonal antibody W6 / 32 (Sigma) was added. After 30 minutes incubation at 4 ° C., cells in PBS (staining buffer) supplemented with 1% heat inactivated FCS, 0.1% (w: v) sodium azide, pH 7.4-7.6 Were washed, incubated with fluorescein isothiocyanate (FITC) -conjugated goat F (ab ′) anti-mouse IgG (Sigma) for 30 minutes at 4 ° C. and washed 4 times as before. Cells were resuspended in staining buffer and fixed by adding 1/4 volume of 2% paraformaldehyde. Analysis of surface HLA-A2.1 molecules stabilized by peptide bonds was performed by flow cytometry using a FACScan (Becton Dickinson, San Jose, Calif.).

  The experimental results are shown in FIG. Using the method described above, candidate tyrosinase housekeeping epitopes (tyrosinase 207-216, FLPWHRLFLL, SEQ ID NO: 85) identified by proteasome digestion are compared to the known A2.1 binder FLPSDYFPSV (SEQ ID NO: 86) (positive control). It was found to bind to HLA-A2.1 to a similar extent. The HLA-B44 binding peptide AEMGKYSFY (SEQ ID NO: 87) was used as a negative control. The fluorescence obtained from the negative control was similar to the signal obtained when the peptide was not used in the assay. Positive and negative control peptides were selected from Table 18.3.1 in Current Protocols in Immunology P18.3.2, John Wiley and Sons, New York, 1988.

Elution of HLA epitopes from tumors, tissue samples, immortalized cell lines, or tumor cell lines Rather than in vitro proteolysis to generate HLA epitopes, they can be used in tumors, tissue samples, tumor cell lines Or other immortalized cell lines can be identified after elution from HLA. A variety of such methods can be used, but one of the most powerful methods for identifying epitopes from the cell surface is by capillary HPLC or nanocapillary HPLC ESI mass spectrometry and online sequencing, as described in the published literature. Include. Elution techniques for solubilized HLA and intact cells are also described in Falk, K. et al. Nature 351: 290, 1991 and US Pat. No. 5,989,565, respectively. However, it is necessary to identify the type of proteasome expressed in cells that have undergone peptide elution and analysis in order to determine whether the identified epitope is the housekeeping epitope necessary to produce an effective vaccine The sex is not described in the literature. In order to definitively identify HLA epitopes as either housekeeping epitopes or immune epitopes, it is generally necessary to know which proteasome the cell source expresses. Proteasome expression can preferably be assessed by Western blotting as detailed below, and it can be assessed by RT-PCR, immunohistochemistry, or in situ hybridization.

IFN induction test Another assay to distinguish between housekeeping and immune epitopes is to test the ability of anti-peptide CTL to kill cells expressing the TAA. IFN can be used to induce expression of the immune proteasome (assuming it is not already constitutively expressed) and to compare CTL recognition of induced and non-induced cells. As mentioned above, the proteasome type should be confirmed, for example, by Western blotting. If IFN-induced cells die preferentially, the peptide constitutes an immune epitope. If non-induced cells die preferentially, the peptide constitutes a housekeeping epitope. Several epitopes can be produced by both proteasomes with different efficiencies, in which case cytolytic activity is observed for both populations. Such epitopes are classified as housekeeping epitopes because they are present on peripheral target cells.

Use of human peripheral blood mononuclear cells (PBMC) or tumor wet lymphocytes (TIL) to identify housekeeping epitopes Using TIL isolated from patient biopsies or PBMC from donor or patient blood Thus, housekeeping epitopes can be identified using methods generally described in the published literature. It is confirmed that the target cells used to test for active killing by PBMC or TIL to identify housekeeping epitopes express only housekeeping proteasomes and not significant levels of immune proteasomes . PBMC from donor blood is stimulated in vitro using a panel of peptide antigens with predicted affinity for class I HLA alleles expressed on the blood cells used. Each PBMC sample is stimulated with a specific class I peptide antigen for one week, preferably in combination with a cytokine such as IL-2 or IL-12 to increase T cell activity. This stimulation is repeated at least three times to induce T cell clonal expansion specific for the peptide. A standard chromium release assay is performed using target cells known to express the protein containing the epitope, exclusively the housekeeping proteasome. Evidence of target cell death as measured by chromium release indicates that the peptide used to stimulate PBMC exists as a housekeeping epitope on the surface of the target cell. Tumors that express this protein are therefore candidate targets for vaccines containing the epitope.

Identification of Housekeeping Proteasomes and / or Immunoproteasomes by Western Blotting Both of the following protocols begin with a membrane in which proteins extracted from a given cell are transcribed after electrophoretic separation.

A. Dye production protocol The membrane is washed for 5 minutes in 20 ml PBS-T (phosphate buffered saline, pH 7.4 + 0.1% Tween 20) at room temperature on an orbital shaker (RT / shaker).
PBS (Sigma, Cat.No.P-3813)
(Capacities can vary in different ways depending on the container type).
2. Incubate the membrane for 5 minutes in 20 ml PBS-T, 3% H ₂ O ₂ : 30% H ₂ O ₂ 2 ml + PBS-T 18 ml on RT / shaker.
3. Wash the membrane with PBS-T for 3 x 5 minutes on an RT / shaker.
4. Block overnight in 20 ml PBS-T / 5% non-fat dry milk: PBS-T 20 ml + 1 g milk on a shaker at 4.4 ° C.
5. Rinse the membrane with PBS-T.
6). Primary antibody (Affinity Research) in blocking buffer for 2 hours on RT / shaker
Products Ltd, United Kingdom) Incubate membrane in 5 ml:
α-LMP2 antiserum (mouse) (Cat. No. PW8205) 1: 5000
α-LMP2 antiserum (human) (Cat. No. PW8345) 1: 10000
α-LMP7 antiserum (Cat. No. PW8200) 1: 20000
α-20S proteasome α subunit monoclonal antibody
(Cat. No. PW8105) 1: 1000
These conditions are for the antibody only. The conditions for any antibody must be determined empirically.
7). The membrane is washed as in step 3.
8). Incubate the membrane in 5 ml of secondary antibody (Vector Laboratories, Inc., Burlingame, Calif.) In blocking buffer for 30 min on an RT / shaker:
GARB (goat anti-rabbit) (for antiserum) (Vector Labs Cat. No. BA-1000)
1: 2000
Horse anti-mouse (for monoclonal antibodies) (Vector Labs Cat. No. BA-2000)
1: 1000
9. The membrane is washed as in step 3.
10. Incubate the membrane for 30 minutes in 5 ml of ABC (Vector Laboratories, Cat. No. PK-6100) in PBS-T:
The ABC is made at least 30 minutes before use as follows.
A = 5 μl / 1 ml = 25 μl / 5 ml
B = 5 μl / 1 ml = 25 μl / 5 ml
5 μl A + 5 μl B>Mixing> Stand at 4 ° C.> Add PBS-T990 μl Dilute ABS in PBS-T just before use.
11. The membrane is washed as in step 3.
12 detection:
1) Transfer 5 ml of 0.2M PB to the first 15 ml tube.
0.4M phosphate buffer:
Monobasic sodium phosphate 90.4ml (1M)
Dibasic sodium phosphate 619.2 ml (0.5 M)
pH 7.4 or less QS 1L or less 2) Transfer 2.8 ml of 0.2M PB to the second 15 ml tube.
3) Transfer 2 ml of 1% glucose to the third 15 ml tube.
4) Weigh 6 mg of ANS (nickel ammonium sulfate), transfer it to the first 15 ml tube and vortex.
5) Add 110 l of glucose oxidase (Sigma, Cat. No. G-6891) to the Eppendorf tube.
6) Add DAB substrate (diaminobenzidine HCl, KPL, Maryland Cat. No. 71-00-46) 1101 to another Eppendorf tube.
7) Mix 5ml of PB + 2ml of glucose in food + GO110l
+ DAB110l
+ 0.2M PB 2.8ml
13. Apply the detection mixture onto the membrane and set up a timer. Record the length of incubation in the chromogen.
14 When the band is fully visible, wash the membrane three times with 0.2M PB.
15. Shake overnight in PBS at RT.

B. Chemiluminescence protocol Rinse the membrane twice in TBS-T (Tris-buffered saline, pH 7.6 + 0.1% Tween 20).
Tris buffered saline: Tris base 2.42 g (20 mM)
Sodium chloride 8g (137mM)
1M hydrochloric acid 3.8ml
Block overnight in 20 ml blocking buffer (TBS-T / 5% nonfat dry milk) at 2.4 ° C./shaker:
TBS-T20ml + milk 1g
The capacity depends on the container type.
3. Rinse the membrane twice with TBS-T.
6). Primary antibody in blocking buffer (Affinity Research for 2 hours on RT / shaker)
Products Ltd, United Kingdom) Incubate membrane in 5 ml:
α-LMP2 antiserum (mouse) (Cat. No. PW8205) 1: 5000
α-LMP2 antiserum (human) (Cat. No. PW8345) 1: 10000
α-LMP7 antiserum (Cat. No. PW8200) 1: 20000
α-20S proteasome α subunit monoclonal antibody
(Cat. No. PW8105) 1: 1000
5. Wash the membrane in 20 ml TBS-T on RT / shaker:
Briefly, after rinsing the membrane using two changes of TBS-T, it is washed once with freshly changed wash buffer for 15 minutes at room temperature and twice for 5 minutes.
6). Incubate membrane in 5 ml 1: 1000 dilution of HRP-labeled (horseradish peroxidase-labeled) secondary antibody (Amersham; Cat # NIF 824 or NIF 825) in blocking buffer for 1 hour on RT / shaker To do.
7). The membrane is washed as in step 5.
8). Equal volumes of detection solution 1 (Amersham; Cat # RPN2109) and detection solution 2 (Amersham; Cat # RPN2109) (1 ml + 1 ml) are mixed.
9. Drain excess buffer from the washed membrane and place it on a piece of Saran wrap with the protein facing up. Detection reagent is added to cover the membrane.
10. Incubate at room temperature for 1 minute without agitation.
11. Excess detection reagent is drained and the membrane is transferred to the Kodak Digital Science Image Station 440CF with the protein on the bottom. Develop and quantify signal according to manufacturer's instructions.

The presence of housekeeping specific subunits (in either protocol) is assessed directly using:
α-β1 (Y) subunit monoclonal antibody (Cat. No. PW8140)
1: 1000
α-β2 (Z) subunit monoclonal antibody (Cat. No. PW8145)
1: 1000
(Affinity Research Products Ltd, United Kingdom)

Preparation of Housekeeping Epitope Peptide Vaccine Sequences identified as housekeeping epitopes are synthesized using a commercially available peptide synthesizer. Certain peptides can be formulated in various ways, alone or in adjuvants such as CFA, IFA or melacin, or IL-2, IL-12 to achieve the effect of stimulating T cells against epitopes in animals. Alternatively, it is administered in combination with a cytokine such as GM-CSF. Peptides can also be formulated with controlled release substances such as PLGA microspheres or other biodegradable substances to modify the pharmacokinetics of the peptides or improve immunogenicity. Peptides are also formulated for oral delivery with substances that promote priming of the immune response by ingestion into GALT (gastrointestinal tract-related lymphoid tissue). Peptides are also attached to fine gold particles so that they can be delivered using a “gene gun”.

A. Synthesis of GMP grade peptides Peptides are synthesized using FMOC or tBOC solid phase synthesis methodology. After synthesis, peptides are excised from their supports with trifluoroacetic acid or hydrogen fluoride, respectively, in the presence of a suitable protective scavenger. After removing the acid by evaporation, the peptide is extracted with ether, scavengers and crude are removed, and the precipitated peptide is lyophilized. The purity of the crude peptide is measured by HPLC, sequence analysis, amino acid analysis, counter ion content analysis and other suitable means. If the crude peptides are sufficiently pure (greater than about 90% purity), they can be used as is. If purification is required to meet drug substance specifications, use one or a combination of the following: reprecipitation, reverse phase, ion exchange, size exclusion or hydrophobic interaction chromatography, or alternating current distribution The peptide is purified.

B. Drug Product Formulation GMP grade peptides are formulated in parenterally acceptable aqueous, organic, or aqueous-organic buffers or solvent systems where they are physically and chemically stable; It also remains biologically powerful. In general, a buffer, or a combination of buffers, or a combination of a buffer and an organic solvent is appropriate. The pH range is typically 6-9. Organic modifiers or other excipients can be added to help the solubility and stability of the peptide. These include detergents, lipids, cosolvents, antioxidants, chelating agents, and reducing agents. In the case of lyophilized products, sucrose or mannitol or other lyophilization aids can be added. Peptide solutions are sterilized by membrane filtration into their final container closure system and lyophilized for dissolution in the clinic or stored until use.

Delivery of Housekeeping Epitope Peptide Vaccine A. Intranodular delivery Formulations containing peptides in aqueous buffer solutions with antibacterial, antioxidant, and immunomodulatory cytokines are inguinal lymphatics using a mini-pumping system (MiniMed; Northridge, CA) developed for insulin delivery Nodes were injected continuously over several days. This injection cycle was chosen to mimic the kinetics of antigen presentation of natural infection. Further embodiments to this form of vaccine delivery useful according to the present invention are described in PCT publication WO 99/01283, US patent application 09 / 380,534 (title: A METHOD OF INDUCING A CTL RESPONSE) (September 1, 1999). Application) and US patent application Ser. No. 09 / 776,232 (title: A METHOD OF INDUCING A CTL RESPONSE) (filed Feb. 2, 2001).

B. Controlled release Controlled PLGA microspheres are used to deliver peptide formulations, modify peptide pharmacokinetics, and improve immunogenicity. This formulation is injected or taken orally.

C. Gene gun delivery A peptide formulation is prepared in which the peptide is adhered to gold microparticles. The particles are delivered with a gene gun and accelerated at high speed to penetrate the skin and deliver the pAPC-containing particles to the skin tissue.

D. Aerosol delivery Peptide formulations are inhaled as an aerosol for ingestion into appropriate vascular or lymphoid tissues in the lung.

Nucleic Acid Vaccine Preparation The carrier plasmid vector pVAX1 (Invitrogen, Carlsbad, Calif.) Containing the kanamycin resistance gene and the CMV promoter was modified to contain two sequences containing the desired epitope. In addition, it contains an IRES sequence located between two epitopes and allows their co-expression using one promoter. The appropriate E. coli strain was then transfected with the plasmid and plated out on selective media. Several colonies were grown in suspension culture and positive clones were identified by restriction mapping. The positive clones were then grown and sorted into storage vials and stored at -70 ° C.

Next, a plasmid miniprep (QIAprep Spin Mini-prep: Qiagen, Valencia, Calif.) Is made from a sample of these cells and the construct has the desired sequence using automated fluorescent dideoxy sequence analysis. It was confirmed. Additional nucleic acid vaccine vectors and formulations are described in previous sections herein and in Examples 18-20 below. As with vaccine manufacture, certain modifications of the plasmid backbone useful in conjunction with large-scale production of plasmids are described in US patent application Ser. No. 09 / 715,835 (title: AVOIDANCE OF UNDESIRABLE REPLICATION INTERMEDIATES IN PLASMID PROPAGATION ) (Filed on Nov. 16, 2000).

Delivery of nucleic acid vaccine
A small pumping system such as a MiniMed insulin pump is used to inject the nucleic acid vaccine into the lymph nodes. To mimic the antigen presentation dynamics of natural infection, the nucleic acid construct formulated in a buffered aqueous solution containing antibacterial agents, antioxidants, and immunomodulatory cytokines is delivered over several days in the infusion cycle. Further embodiments to this form of vaccine delivery useful according to the present invention are disclosed in PCT publication WO 99/01283 and US patent application 09 / 776,232.

  Optionally, controlled release materials such as PLGA microspheres or other biodegradable materials are used to deliver the nucleic acid construct. These substances are injected or taken up orally. Nucleic acid vaccines prime the immune response by ingestion into GALT tissue, given using oral delivery. Alternatively, the nucleic acid vaccine is delivered using a gene gun, where the nucleic acid vaccine is attached to fine gold particles. The nucleic acid construct can also be inhaled as an aerosol for ingestion into appropriate blood vessels or lymphoid tissues in the lung.

Assay for presentation of housekeeping epitopes on pAPC Tetramer Analysis Class I tetramer analysis is used to determine T cell frequency in animals before and after administration of housekeeping epitopes. T cell clonal expansion in response to the epitope indicates that the epitope is presented to the T cell by pAPC. The specific T cell frequency is measured against housekeeping epitopes before and after administration of the epitope to the animal to determine whether the epitope is present on pAPC. An increase in T cell frequency specific for the epitope after administration means that the epitope was presented on pAPC.

B. Proliferation assay Approximately 24 hours after vaccination of animals with housekeeping epitopes, pAPCs are collected from PBMCs, glandular cells or lymph node cells using monoclonal antibodies directed against specific markers present on pAPCs and magnetic beads for affinity purification. Fix it. Crude blood or splenocyte preparations are enriched for pAPC using this technique. The enriched pAPC is then used in a proliferation assay against the generated T cell clones specific for a given housekeeping epitope. T cells are monitored for proliferative activity by co-incubating pAPC with T cell clones and measuring the incorporation of radiolabeled thymidine by T cells. Proliferation indicates that T cells specific for housekeeping epitopes are stimulated by that epitope on pAPC.

Assay for effectiveness of housekeeping epitopes as anti-cancer treatments Surrogate indicators or survival are used to determine the effectiveness of epitope-tuned vaccines in cancer treatment.

A. T cell frequency analysis A useful surrogate indicator is the determination of T cell frequency for the housekeeping epitope used in immunization. Patients who show an increase in T cell frequency against a particular TuAA epitope used in tumor immunotherapy are compared to patients who have been immunized with the same epitope but do not show an increase in T cell frequency against the epitope. And has significantly better survival. Tetramer analysis, ELISPOT analysis, or limiting dilution analysis is used to assess T cell frequency against housekeeping epitopes before and after immunization with epitopes, indicating the anticancer efficacy of housekeeping epitopes in vaccines.

B. Tumor burden / survival analysis Animals with existing tumors are evaluated for tumor burden before and after immunization with housekeeping epitopes. Partial or complete tumor regression represents an effective therapeutic intervention and correlates with improved survival. In the laboratory environment, several animals are inoculated with tumors in parallel. Next, some of the animals are immunized with a housekeeping epitope vaccine. The survival of animals immunized with housekeeping epitopes is compared to the survival of animals given a control epitope or placebo to determine vaccine efficacy.

C. Chromium release assay Animals genetically modified to express human class IMHC are immunized with housekeeping epitopes. T cells from these animals are used in standard chromium release assays using human tumor targets or targets engineered to express the same class IMHC. Target T cell killing indicates that stimulation of T cells in the patient is effective in killing tumors that express similar TuAAs.

Identification of Unique Housekeeping Epitopes Epitopes useful in the vaccines and methods of the present invention can be readily identified as disclosed herein. For example, three unique housekeeping epitopes that are not produced by pAPC were identified as follows.

A. Isolation and purification The immune proteasome or housekeeping proteasome complex is isolated. The purified peptide is dissolved in a suitable buffer to a concentration of about 1-2 mM and added to about 2 volumes of the proteasome preparation. The chosen buffer must solvate the peptide without interfering with the digestion process. In order to verify the qualified functionality of the proteasome preparation used, further digests are prepared using the positive control peptide described above. These are incubated for up to 120 minutes at 37 ° C., followed by termination of digestion by addition of diluted trifluoroacetic acid and analysis of the samples immediately by mass spectrometry or freezing on dry ice until analysis . The digestion reaction can also be stopped by placing the sample on ice for immediate analysis by mass spectrometry.

B. MALDI-TOF mass spectrometry of digests About 0.5 μl of each digest was directly mixed with an equal volume of matrix solution (10 mg / ml dihydroxybenzoic acid in 70% EtOH, pH 2-3) on a sample slide and about 40 μl. Air dried at ° C. Samples were then analyzed on a Lasermat® MALDI-TOF mass spectrometer calibrated with the appropriate molecular weight standards.

  The computer program developed for the proteasome assay is the sequence and molecular weight of all possible fragments that fulfill both the requirement to have the exact C-terminus of any predicted epitope and the requirement to contain more than the full length of that epitope. Is generated.

If the MALDI-TOF results indicate that a specific molecular weight is expressed in the digestion mixture, the corresponding peptide is synthesized and purified, identified by MALDI-TOF, followed by reverse phase analytical HPLC, Both retention time and approximate mass to peak area ratio were established. These approaches are very similar to those described above. The replicate proteasome digest is then diluted with an appropriate solvent and analyzed using the same analytical HPLC method. If the digest gives a peak in good yield with the same retention time as the standard, it is almost certain that it is due to the presence of that sequence in the digest. If there is ambiguity due to possible generation of other fragments that give the same or similar mass analysis results, suspicious components can be collected and reserved for sequencing to confirm identity. Using the method described above, housekeeping epitopes were identified.

C. Further unique housekeeping epitopes The binding of candidate peptides to HLA-A2.1 was assayed according to the method of Stauss et al. (Proc Natl Acad Sci USA 89 (17): 7871-5 (1992)). T2 cells expressing empty or unstable MHC molecules on the surface were washed twice and suspended at 5 × 10 ⁶ cells / ml in serum-free complete Iscove modified Dulbecco medium (IMDM). β ₂ microglobulin (Sigma, St. Louis, MO) was added at 5 μg / ml to distribute the cells at 5 × 10 ⁵ cells / well in a 96 well U-bottom plate. Peptides were added at 100, 10, 1 and 0.1 μg / ml. The plate was gently shaken for 2 minutes and then incubated for 4 hours at 37 ° C. in a 5% CO ₂ incubator. Unbound peptide was removed by washing twice with IMDM and a saturating amount of monoclonal antibody W6 / 32 (Sigma) was added. After 30 minutes incubation at 4 ° C., cells in PBS (staining buffer) supplemented with 1% heat inactivated FCS, 0.1% (w: v) sodium azide, pH 7.4-7.6 Were washed, incubated with fluorescein isothiocyanate (FITC) -conjugated goat F (ab ′) anti-mouse IgG (Sigma) for 30 minutes at 4 ° C. and washed 4 times as before. Cells were resuspended in staining buffer and fixed by adding 1/4 volume of 2% paraformaldehyde. Analysis of surface HLA-A2.1 molecules stabilized by peptide bonds was performed by flow cytometry using a FACScan (Becton Dickinson, San Jose, Calif.).

Using the method described above, candidate tyrosinase housekeeping epitopes (tyrosinase 207-216, FLPWHRLFLL, SEQ ID NO: 85) identified by proteasome digestion are compared to the known A2.1 binder FLPSDYFPSV (SEQ ID NO: 86) (positive control). It was found to bind to HLA-A2.1 to a similar extent. The HLA-B44 binding peptide AEMGKYSFY (SEQ ID NO: 87) was used as a negative control. The fluorescence obtained from the negative control was similar to the signal obtained when the peptide was not used in the assay. Positive and negative control peptides were selected from Table 18.3.1 in Current Protocols in Immunology P18.3.2, John Wiley and Sons, New York, 1998.
A number of additional housekeeping epitopes characterized in accordance with the present invention include two US provisional patent applications (Title: EPITOPE SEQUENCES) Nos. 60/282, 211 (filed Apr. 6, 2001) and No. / (2001). Filed on Nov. 7).

Construction of pVAX-EP1-IRES-EP2 Overview:
The starting plasmid for this construct is pVAX1 purchased from Invitrogen (Carlsbad, CA). Epitopes EP1 and EP2 were synthesized by GIBCO BRL (Rockville, MD). The IRES was excised from pIRES purchased from Clontech (Palo Alto, Calif.). See FIG. 10B.
Method:
1. Digest pIRES with EcoRI and NotI. The digested fragments are separated on an agarose gel and the IRES fragment is purified by gel purification.
2. Digest pVAX1 with EcoRI and NotI. The pVAX1 fragment is gel purified.
3. Set up a ligation containing the purified pVAX1 and IRES fragments.
4). Transform competent DH5α with the ligation mixture.
5. Remove 4 colonies and make a miniprep.
6). Perform restriction enzyme digestion analysis of miniprep DNA. One recombinant colony with an IRES insert was used for further insertion of EP1 and EP2. This intermediate construct was called pVAX-IRES.
7). EP1 and EP2 are synthesized.
8). EP1 is subcloned into pVAX-IRES between the AflII and EcoRI sites to create pVAX-EP1-IRES.
9. Between SalI and NotI, EP2 is subcloned into pVAX-EP1-IRES to produce the final construct PVAX-EP1-IRES-EP2.
10. To confirm the sequence, the EP1-IRES-EP2 insert is sequenced.

Construction of pVAX-EP1-IRES-EP2-ISS-NIS Overview:
The starting plasmid for this construct is pVAX-EP1-IRES-EP2 (Example 18). The ISS (immunoregulatory sequence) (SEQ ID NO: 89) introduced into this construct is AACGTT and the NIS used (present for nuclear translocation sequence) (SEQ ID NO: 88) is a SV40 72 bp repeat sequence. . ISS-NIS was synthesized by Gibco BRL. See FIG. 910A.
Method:
1. Digest pVAX-EP1-IRES-EP2 with NruI. The linearized plasmid is gel purified.
2. Synthesize ISS-NIS.
3. Set up a ligation reaction containing purified linearized pVAX-EP1-IRES-EP2 and synthesized ISS-NIS.
4). Transform competent DH5α with the ligation product.
5). Remove the colony and make a miniprep.
6). Perform a miniprep restriction enzyme digestion.
7). The plasmid with the insert is sequenced.

Construction of pVAX-EP-2-UB-EP1 Summary: The starting plasmid for this construct is pVAX1 (Invitrogen). EP2 and EP1 were synthesized by GIBCO BRL. Wild-type ubiquitin cDNA encoding 76 amino acids in the construct was cloned from yeast. Please refer to FIG.
Method:
1. RT-PCR is performed using yeast mRNA. Primers were designed to amplify the complete coding sequence of yeast ubiquitin.
2. Analyze the RT-PCR product using an agarose gel. A band with the expected size is gel purified.
3. The purified DNA band is subcloned into pZERO1 at the EcoRV site. The resulting clone was called pZERO-UB.
4). Several clones of pZERO-UB are sequenced. The ubiquitin sequence is confirmed before further manipulation.
5). EP1 and EP2 are synthesized.
6). EP2, ubiquitin and EP1 are ligated and the insert is cloned into pVAX1 between BamHI and EcoRI and it is under the control of the CMV promoter.
7). The sequence of the insert EP2-UB-EP1 is confirmed by sequencing.

IRES (SEQ ID NO: 6)
Reference: Clontech PT3266-5

Ubiquitin (SEQ ID NO: 5)
Atgcagattttcgtcaagactttgaccggtaaaaccataacattggaagttgaatcttccgataccatcgacaacgttaagtcgaaaattcaagacaaggaaggtatccctccagatcaacaaagattgatctttgccggtaagcagctagaagacggtagaacgctgtctgattacaacattcagaaggagtccaccttacatcttgtgctaaggctaagaggtggc reference: Ozkaynak, E., Finley, D., Solomon, MJ and Varshavsky, A., The yeast ubiquitin genes:
a family of natural gene fusions. EMBO J. 6 (5), 1429-1439 (1987).

NIS (SEQ ID NO: 88) Tggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacc

Reference: Dean DA, Dean BS, Muller S, Smith LC, Sequence requirements for plasmid nuclear import. Exp. Cell Res. 253 (2): 713-22 (1999).

ISS (SEQ ID NO: 89)
aacgtt

See: Sato Y, Roman M, Tighe H, Lee D, Corr M, Nguyen M, Silverman GJ, Lotz M, Carson DA and Raz E, Immunostimulatory DNA sequences necessary for effective intradermal gene immunization. Science, 273: 352-354 ( 1996).

  Examples 21-24 are all related to the prediction of the 9-mer epitope presented by HLA-A2.1, but the approach is equally applicable to any HLA type, or epitope length, MHC binding assays are available.

ECR in Melan-A / MART-1 (SEQ ID NO: 1)
This melanoma tumor associated antigen (TAA) is 118 amino acids long. Of the 110 possible 9-mers, 16 are given a score of 16 by the SYFPEITHI / Rammensee algorithm (see Table 10). These represent an average epitope density on 14.5% of the possible peptides and 0.136 proteins per amino acid. These 12 overlaps covering amino acids 22-49 of SEQ ID NO: 1 give an epitope density for a cluster of 0.428, giving the above-mentioned ratio of 3.15. Another two predicted epitopes overlap with amino acids 56-69 of SEQ ID NO: 1, giving an epitope density for a cluster of 0.143, with a ratio of just 1.05, clearly different from the average. See FIG.

  By limiting the analysis to a 9-mer predicted to have a dissociation half-life of 5 minutes according to the BIMAS-NIH / Parker algorithm, there are only 5 (see Table 11). The average density of epitopes in the protein is here only 0.042 per amino acid. The overlapping peptides of the protein cover amino acids 31-48 of SEQ ID NO: 1 and the other two cover 56-69 of SEQ ID NO: 1 as described above, giving ratios of 3.93 and 3.40, respectively ( (See Table 12).

ECR in SSX-2 / HOM-MEL-40 (SEQ ID NO: 2)
This melanoma tumor associated antigen (TAA) is 188 amino acids long. Of the 180 possible 9-mers, 11 are given a score of 16 by the SYFPEITHI / Rammensee algorithm (see 11 Table 13). These represent an average epitope density on the protein of 6.1% of possible peptides and 0.059 per amino acid. Three of these overlaps covering amino acids 99-114 of SEQ ID NO: 2 result in an epitope density for a cluster of 0.188, giving the above ratio of 3.18. There are also overlapping pairs of predicted epitopes at amino acids 16-28, 57-67, and 167-183 of SEQ ID NO: 2, giving ratios of 2.63, 3.11, and 2.01, respectively. There are additional predicted epitope clusters covering amino acids 5 to 28 of SEQ ID NO: 2. Evaluating regions 5-28 of SEQ ID NO: 2 containing 3 epitopes provides an epitope density of 0.125 and a ratio of 2.14 (see Table 14).

By limiting the analysis to a 9-mer predicted to have a dissociation half-life of 5 minutes according to the BIMAS-NIH / Parker algorithm, there are only 6 (see Table 15). The average density of epitopes in the protein is here only 0.032 per amino acid. Only a single pair overlaps 167-180 of SEQ ID NO: 2, giving a ratio of 4.48. However, the top peptide is close to another single predicted epitope when the region is evaluated to have a ratio of amino acids 41-65 of SEQ ID NO: 2 of 2.51, with a substantial difference from the mean. Represent (
(See FIG. 16 and Table 16).

ECR in NY-ESO (SEQ ID NO: 3)
This melanoma tumor associated antigen (TAA) is 180 amino acids long. Of the 172 possible 9-mers, 25 are given a score of 16 by the SYFPEITHI / Rammensee algorithm (see Table 17). Similar to Melan-A above, these represent 14.5% of the possible peptides and an average epitope density on the protein of 0.136 per amino acid. However, the distribution is quite different. Almost half of the proteins are empty with exactly one predicted epitope in the first 78 amino acids. Unlike Melan-A, where very close clusters of highly overlapping peptides existed, in NY-ESO, the overlap is smaller and extends over most of the rest of the protein. One group of 19 overlapping peptides covers amino acids 108-174 of SEQ ID NO: 3, resulting in a ratio of 2.04. Another 5 predicted epitopes cover 79-104 of SEQ ID NO: 3, providing a ratio of just 1.38 (see Table 18).

  Instead, if you choose an approach that considers only the predicted epitope, in this case only the top 5% of the 9 peptides, then test whether a good cluster is weakened by a peptide that is predicted to be less likely to bind to MHC be able to. When these predicted epitopes are just considered, it can be seen that regions 108-140 of SEQ ID NO: 3 contain 6 overlapping peptides with a ratio of 3.64. There are also two adjacent peptides in regions 148-167 of SEQ ID NO: 3 with a ratio of 2.00. Thus, the large clusters 108-174 of SEQ ID NO: 3 can be subdivided into two small clusters that cover many of the same sequences (see Table 18).

Consider 14 peptides by limiting the analysis to a 9-mer predicted to have a dissociation half-life of 5 minutes according to the BIMAS-NIH / Parker algorithm (see Table 19). The average density of epitopes in the protein is here 0.078 per amino acid. A single group of 10 overlapping peptides is observed to cover amino acids 144-171 of SEQ ID NO: 3 in a ratio of 4.59. All 14 peptides fit within the region 86-171 of SEQ ID NO: 3, an average density of epitopes in the protein of 2.09 times. If such a large cluster is larger than what we consider ideal, it further provides a significant advantage by operating with the whole protein (see FIG. 17 and Table 20).

ECR in tyrosinase (SEQ ID NO: 4)
This melanoma tumor associated antigen (TAA) is 529 amino acids long. Of the 521 possible 9-mers, 52 are given a score of 16 by the SYFPEITHI / Rammensee algorithm (see Table 21). Similar to Melan-A above, these represent 10% of the possible peptides and an average epitope density on the protein of 0.098 per amino acid. There are 5 groups of overlapping peptides, each containing 2-13 predicted epitopes, each having a ratio in the range of 2.03-4.41. There are seven additional groups of overlapping peptides each containing 2-4 predicted epitopes, each with a ratio in the range of 1.20-1.85. Seventeen peptides in regions 444-506 of SEQ ID NO: 4, including the 13 overlapping peptides, constitute a cluster having a ratio of 2.20 (see Table 22).

  Consider 28 peptides by limiting the analysis to a 9-mer predicted to have a dissociation half-life of 5 minutes according to the BIMAS-NIH / Parker algorithm (see Table 23). The average density of epitopes in the protein under these conditions is 0.053 per amino acid. At this density, any overlap represents the average density of epitopes over twice. There are 5 groups of overlapping peptides, each containing 2-7 predicted epitopes, each with a ratio in the range 2.22-4.9 (see Table 24). Only three of these clusters are common to the two algorithms. Some (but not all) of these clusters can be expanded by evaluating regions containing them and adjacent predicted epitopes (see FIG. 18).

Pulse labeling of dendritic cells (DC) with 40 μg / ml tyrosinase _207-215 epitope at a cell concentration of 1-2 × 10 ⁶ / ml for 4 hours at 20 ° C. in the presence of 3 μg / ml β2- _{microbrovulin} And irradiation (4200 rad) before use. CD8 + T cells purified using immunomagnetic beads (Dynal) are used as responders. 0.25 ml of cytokine producing DC (1 × 10 ⁵ cells / ml) mixed with 0.25 ml of CD8 + T cells (20 × 10 ⁵ cells / ml) in each well in the presence of 10 ng / ml IL-7 CTL primary immunoculture is established in 48-well tissue culture plates (Costar). Twenty-four hours after the start of culture, 10 ng / ml recombinant IL-10 (Endogen, Cambridge, MA) is added to each well. Re-stimulate CTL cultures (each individual well) every 7 days (up to 6 cycles) using irradiated (3300 rad) autologous mononuclear cells pulsed with tyrosinase _207-215 . After each restimulation cycle, recombinant IL-10 (10 ng / ml) and recombinant IL-2 (10 IU / ml) are added on days 1 and 2, respectively. Complete medium consists of RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 5% AB male human serum, L-glutamine, sodium pyruvate and non-essential amino acids (all from Life Technologies) at the recommended concentrations. Be replaced by tyrosinase _207-215 tyrosinase _207-216 may use this same protocol.
US Provisional Patent Application No. 60 / 274,063 (Title: ANTI-NEOVASCULATURE VACCINES FOR
CANCER) (filed March 7, 2001) is also useful consistent with the subject matter disclosed herein.

FIG. 2 represents a schematic diagram of a portion of cells involved in protein processing and epitope presentation by the proteasome. Comparison of housekeeping proteasome and immune proteasome. FIG. 4 represents a schematic diagram of epitope synchronization between infected cells and pAPC. The presentation of different epitopes by pAPC and tumor cells is shown. The presentation of different epitopes by pAPC and infected cells is shown. FIG. 6 represents tumor cell presentation of both housekeeping and immune epitopes due to induction by IFN-γ. FIG. 5 shows the attack of virus-infected cells by T cells induced to recognize housekeeping epitopes. Shows a double attack against both housekeeping and immune epitopes. Position plot of predicted HLA-A ^* 0201 epitope in tyrosinase Position plot of predicted HLA-A ^* 0201 epitope in tyrosinase Represents a component of the plasmid pVAX-EPI-IRES-EP2-ISS-NIS. Represents the components of plasmid pVAX-EP1-IRES-EP2. Represents the components of plasmid pVAX-EP2-UB-EP1. Represents the components of plasmid pVAX-EP2-2A-EPI. Figure 3 represents the results of a flow cytometry assay that verifies HLA binding by a Melan-A epitope. Figure 3 represents the results of a flow cytometry assay that verifies HLA binding by tyrosinase peptides 207-216. 1 represents the sequence of Melan-A (SEQ ID NO: 1) showing clustering of class I HLA epitopes. Figure 3 represents the sequence of SSX-2 (SEQ ID NO: 2) showing clustering of class I HLA epitopes. FIG. 5 represents the sequence of NY-ESO (SEQ ID NO: 3) showing clustering of class I HLA epitopes. The top of the sequence line represents the sequence of tyrosinase (SEQ ID NO: 4) showing clustering of class I HLA epitopes represented by the BIMAS-NIH / Parker algorithm and at the bottom by the SYFPEITHI / Rammensee algorithm.

[Sequence Listing]

Claims (28)

  An isolated T cell expressing a T cell receptor specific for an MHC-peptide complex comprising a first housekeeping epitope, wherein the housekeeping epitope is associated with a first target cell-associated first cell. Isolated T cells derived from an antigen.   A T cell clone comprising the T cell according to claim 1.   A polyclonal population of T cells comprising the T cells of claim 1.   The T cell according to claim 1, which is produced by in vitro immunization.   2. The T cell of claim 1 isolated from an immunized animal. A method of manufacturing an adoptive immunotherapy drug,
6. A method comprising combining the T cells of any one of claims 1-5 with a pharmaceutically acceptable adjuvant, carrier, diluent or excipient.   7. The method of claim 6, wherein the T cell is initially obtained from a donor.   8. The method of claim 7, wherein the donor is an intended recipient of an immunotherapeutic agent.   8. The method of claim 7, wherein the donor is immunologically untreated with respect to the first antigen.   8. The method of claim 7, wherein the donor has been previously exposed to the first antigen.   8. The method of claim 7, wherein the donor is vaccinated with the housekeeping epitope prior to donation.   The method according to claim 6, further comprising culturing the T cell in vitro.   13. The method of claim 12, wherein the T cells are stimulated to proliferate upon exposure to the MHC-peptide complex.   13. The method of claim 12, wherein the T cells are stimulated to proliferate upon exposure to cytokines.   13. The method of claim 12, wherein the culture further comprises pAPC, an adjuvant or a combination thereof.   16. The method of claim 15, wherein the pAPC is a dendritic cell.   16. The adjuvant according to claim 15, wherein the adjuvant is selected from the group consisting of GM-CSF, G-CSF, IL-2, IL-12, BCG, tetanus toxoid, osteopontin / ETA-1, CD40 ligand and CTLA-4 blocker. The method described.   Use of the T cell according to any one of claims 1 to 5 in the manufacture of a medicament for use in adoptive immunotherapy.   A method for treating a disease, comprising administering the T cell according to any one of claims 1 to 5 to a recipient.   A method for treating a disease, comprising administering the immunotherapeutic agent according to claim 6 to a recipient. An epitope cluster wherein the cluster is derived from an antigen associated with a target and comprises or encodes at least two sequences having known or predicted affinity for the groove to which the MHC receptor peptide binds, the cluster comprising an antigen And the cluster has the structural formula:

Where X is any naturally occurring amino acid in the protein sequence;
Xa and X (| b | -1) are such amino acid strings of length "a" and "| b | -1", respectively;
a represents the number of amino acids between P2 ₁ and P2 _N , and (| b | -1) represents the number of amino acids between P2 _N and P ₁ ;
P2 ₁ is a first primary anchor and the second residue of the first epitope,
P2 _N is the first primary anchor and the second residue of the N-th epitope,
P ₁ is the last major anchor and C-terminal residue of the first epitope, and _PN is the last major anchor and C-terminal residue of the Nth epitope;
2 N Nc, N represents the Nth epitope of the cluster, Nc represents the total number of epitopes in the cluster,
a _N and b _N define the positional relationship between the first and Nth epitopes)
Epitope cluster having   (Nc / Lc)> (Np / Lp), the cluster and the antigen each have a certain length, where Lc is the length of the cluster and Lp is the length of the antigen And Np is the total number of epitopes in the antigen.   24. An isolated polypeptide comprising the epitope cluster of claim 21, wherein the amino acid sequence consists of about 80% or less of the amino acid sequence of the antigen.   24. A vaccine or immunotherapeutic product comprising the polypeptide of claim 23.   24. An isolated polynucleotide encoding the polypeptide of claim 23.   26. A vaccine or immunotherapeutic product comprising the polynucleotide of claim 25.   26. The polynucleotide of claim 25, which is DNA.   26. The polynucleotide of claim 25, which is RNA.

Images