CN116410331A - Chimeric antigen receptors targeting CS1, bispecific chimeric antigen receptors targeting BCMA/CS1 and applications thereof - Google Patents

Abstract

The present invention provides a chimeric antigen receptor targeting CS1, which comprises an extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain; wherein: the extracellular antigen recognition domain comprises an anti-CS1 scFv antibody, the amino acid sequences of the VH complementarity determining regions CDR1, CDR2, and CDR3 of the scFv antibody include the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 respectively, and the scFv antibody The amino acid sequences of the VL complementarity determining regions CDR1, CDR2, and CDR3 respectively include the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

Description

Chimeric antigen receptor targeting CS1, bispecific chimeric antigen targeting BCMA/CS1 Receptors and their applications

technical field

The present application relates to the field of biomedicine, in particular to a chimeric antigen receptor targeting CS1, a bispecific chimeric antigen receptor targeting BCMA/CS1 and applications thereof.

Background technique

Multiple myeloma, defined as the malignant proliferation of plasma cells in the bone marrow, is the second most common hematological malignancy, accounting for 1% of all cancers. Studies have shown that multiple myeloma has a high incidence in the elderly over 60 years old and the incidence has increased steadily in recent years. For most patients, multiple myeloma is incurable and will eventually develop into relapsed/refractory multiple myeloma. The survival period of patients with relapsed/refractory multiple myeloma who are ineffective with existing multiple myeloma treatments (such as immunomodulators, proteasome inhibitors, antibody drugs) is only about 13 months.

CS1, also known as SLAMF7, CRACC or CD319, is a glycoprotein expressed on the surface of myeloma cells, which is a strong marker between normal plasma cells and malignant plasma cells in multiple myeloma. Chu et al. developed two second-generation CARs for the treatment of multiple myeloma, one target antigen is BCMA, and the other target antigen is CS1; in vitro experiments, although two CAR-Ts with different targets showed Similar killing activity, but in vivo experiments in mice, CS1 CAR-T cells have stronger anti-tumor activity.

A chimeric antigen receptor (Chimeric Antigen Receptor, CAR) is the core component of a CAR cell therapy drug, which may include a targeting moiety (for example, a part that binds a tumor-associated antigen (Tumor-Associated Antigen, TAA)), a hinge region, a spanning Membrane domains and intracellular domains. CAR-T cell immunotherapy is considered to be one of the most promising means to overcome tumors. CAR-T cells use genetic modification to enable T cells to express CAR proteins. This CAR protein has the ability to recognize the intact protein on the membrane surface without relying on antigen presentation, thereby causing the activation and functional effects of T cells.

In 2021, Bristol-Myers Squibb and Bluebird Bio jointly announced that the U.S. Food and Drug Administration (FDA) has approved its BCMA-targeted CAR-T cell therapy (bb2121) for use after 4 lines of treatment (including immunomodulators, proteases, etc.) Adult patients with relapsed or refractory multiple myeloma treated with body inhibitors and antibody drugs), but no CS1-targeting CAR-T cell therapy has been marketed. It is of practical significance to develop better cell therapy methods targeting CS1.

Contents of the invention

The present application provides a chimeric antigen receptor targeting CS1, a bispecific chimeric antigen receptor targeting BCMA/CS1 and applications thereof. The inventors used multiple CS1-targeting scFvs to construct chimeric antigen receptor expression vectors and prepare CS1-targeting CAR-T cells, and also verified that CS1 CAR-T cells have good tumor-suppressing functions at the cellular level and determined that Optimal chimeric antigen receptor targeting CS1.

A chimeric antigen receptor targeting CS1, comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain; wherein: the extracellular antigen recognition domain comprises an anti-CS1 scFv antibody, The amino acid sequences of the VH complementarity determining regions CDR1, CDR2, and CDR3 of the scFv antibody include the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the VL complementarity determining regions of the scFv antibody The amino acid sequences of regions CDR1, CDR2, and CDR3 include the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

In certain embodiments, the VH sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO: 7, and the VL sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO: 8. amino acid sequence.

In certain embodiments of the above-mentioned chimeric antigen receptor, the scFv antibody is a humanized antibody.

In certain embodiments, the VH sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO: 9, and the VL sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO: 10. amino acid sequence.

In certain embodiments of the above-mentioned chimeric antigen receptor, the scFv antibody is a rabbit-derived antibody.

In certain embodiments of the above-mentioned chimeric antigen receptor, there is a connecting region between VH and VL in the scFv antibody, and the connecting region is selected from one or more of the following: SEQ ID NOs: 59-61.

In certain embodiments of the above-mentioned chimeric antigen receptor, the sequence of the scFv antibody is shown in SEQ ID NO:11 or SEQ ID NO:12.

In certain embodiments of the above-mentioned chimeric antigen receptor, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α.

In some embodiments, the above-mentioned chimeric antigen receptor, the transmembrane region is derived from one of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70 or Various.

In some embodiments of the above-mentioned chimeric antigen receptor, wherein the intracellular domain includes an intracellular signal transduction region; optionally, it also includes a co-stimulatory signal transduction region.

In some embodiments, the above-mentioned chimeric antigen receptor, wherein the intracellular signaling region is derived from CD3δ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, Syk one or more of.

In certain embodiments of the above-mentioned chimeric antigen receptor, wherein the co-stimulatory signaling region is derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1 One, two or more of , ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, MyD88.

In some embodiments, the above chimeric antigen receptor further comprises a leader peptide located at the N-terminal of the amino acid sequence of the chimeric antigen receptor; optionally, the leader peptide is derived from CD8α.

In some embodiments of the above chimeric antigen receptor, the extracellular antigen recognition domain further comprises scFv antibodies against one of the following targets: CD138, NKG2D, CD38, BCMA, CD19, CD70, CD44v6, Lewis Y.

In certain embodiments, the above-mentioned chimeric antigen receptors, the extracellular antigen recognition domain comprises anti-BCMA scFv VL, anti-CS1 scFv VL, anti-CS1 scFv VH and anti-BCMA scFv VH, the anti- The amino acid sequences of the scFv VH complementarity-determining regions CDR1, CDR2, and CDR3 of CS1 include the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 respectively, and the scFv VL complementarity-determining region of the anti-CS1 The amino acid sequences of CDR1, CDR2, and CDR3 respectively include the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the amino acid sequences of the scFv VH complementarity determining regions CDR1, CDR2, and CDR3 of the anti-BCMA The sequences include the amino acid sequences shown in SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53 respectively, and the amino acid sequences of the anti-BCMA scFv VL complementarity determining regions CDR1, CDR2, and CDR3 include SEQ ID NO: 54. The amino acid sequence shown in SEQ ID NO:55, SEQ ID NO:56.

In certain embodiments, the VH sequence of the anti-CS1 scFv antibody includes the amino acid sequence shown in SEQ ID NO: 7, and the VL sequence of the anti-CS1 scFv antibody includes SEQ ID NO : Amino acid sequence shown in 8.

In certain embodiments, the VH sequence of the anti-BCMA scFv antibody includes the amino acid sequence shown in SEQ ID NO: 57, and the VL sequence of the anti-BCMA scFv antibody includes SEQ ID The amino acid sequence shown in NO:58.

In certain embodiments of the above-mentioned chimeric antigen receptor, the extracellular antigen recognition domain includes the amino acid sequence shown in SEQ ID NO:50.

The present application also provides an isolated nucleic acid molecule comprising the nucleotide sequence encoding the above-mentioned chimeric antigen receptor.

The present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule.

In some embodiments, the above-mentioned vector is an expression vector; in some embodiments, the vector is a viral vector; in some embodiments, it is a lentiviral vector.

The present application also provides an engineered immune effector cell, which comprises the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned carrier.

In some embodiments, the immune effector cells are selected from T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, and differentiated pluripotent stem cells. One or more of T cells, NK cells differentiated from pluripotent stem cells, and embryonic stem cells.

In some embodiments, the above-mentioned immune effector cells are T lymphocytes; optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.

The present application also provides a pharmaceutical composition, which includes the above-mentioned engineered immune effector cells and pharmaceutically acceptable auxiliary materials.

In some embodiments of the above pharmaceutical composition, the pharmaceutically acceptable excipients include protective agents.

In some embodiments of the above pharmaceutical composition, the pharmaceutically acceptable adjuvant includes cell cryopreservation solution.

In some embodiments, the above-mentioned pharmaceutical composition is an intravenous injection.

The present application also provides the use of the above-mentioned chimeric antigen receptor, nucleic acid molecule, carrier or immune effector cell in the preparation of medicines for treating diseases or conditions related to the expression of CS1.

In some embodiments, the above-mentioned use, the disease or disease associated with the expression of CS1 is cancer; optionally, the cancer is multiple myeloma; further optionally, the cancer is refractory or recurrent multiple myeloma.

The above uses In certain embodiments, the disease or disorder associated with the expression of CS1 may be an autoimmune disease.

In some embodiments of the above use, the autoimmune disease may be selected from the following: systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis or autoimmune hemolytic anemia.

The above use In some embodiments, the drug is an intravenous injection.

The present application also provides a method for treating diseases or disorders related to the expression of CS1, comprising the following steps: administering an effective amount of the above-mentioned immune effector cells or the pharmaceutical composition to a disease or disorder associated with the expression of CS1. subjects in need.

In some embodiments of the above method, the disease or disorder associated with the expression of CS1 is cancer; optionally, the cancer is multiple myeloma; further optionally, the cancer is refractory or recurrent multiple myeloma.

The above methods In certain embodiments, the disease or condition associated with the expression of CS1 may be an autoimmune disease.

In some embodiments of the above method, the autoimmune disease may be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, or autoimmune hemolytic anemia.

In some embodiments of the above methods, the administration is by intravenous injection.

In some embodiments of the above method, the administering method is to administer an effective amount of the immune effector cells or the pharmaceutical composition to the subject in a single injection.

In some embodiments of the above method, the effective amount of immune effector cells or the pharmaceutical composition is 1×10 ⁵ to 1×10 ⁷ cells/kg.

The present application also provides the above-mentioned immune effector cells or the above-mentioned pharmaceutical composition for treating diseases or diseases related to the expression of CS1.

In some embodiments, the above-mentioned immune effector cells or the above-mentioned pharmaceutical composition, the disease or disease related to the expression of CS1 is cancer; optionally, the cancer is multiple myeloma; further optionally, the cancer is Refractory or relapsed multiple myeloma.

In certain embodiments of the above-mentioned immune effector cells or the above-mentioned pharmaceutical composition, the disease or disorder associated with the expression of CS1 may be an autoimmune disease.

The above-mentioned immune effector cells or the above-mentioned pharmaceutical composition In some embodiments, the autoimmune disease can be selected from the following: systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis or autoimmune Immune hemolytic anemia.

Description of drawings

Figure 1 shows a schematic diagram of the CS1 CAR structure in Example 1 of the present application.

Figure 2 shows the staining of UTD cells (T cells not transduced with CAR), CS1 CAR-T cells 21G-27G, and positive control CS1 CAR-T cells LUC90V2 with PE fluorescence-labeled CS1 antigen in Example 2 of the present application. The results of detecting the percentage of CAR molecules expressed on the surface.

Figure 3 shows the results of staining UTD cells, CS1CAR-T cells 21G-27G, and positive control CAR-T cells 02G with fluorescently-labeled anti-human IgG antibodies in Example 2 of the present application to detect the percentage of CAR molecules expressed on their surfaces .

Figure 4 shows the release of IL-2 detected by CS1 CAR-T cells activated by CS1 positive target cells in Example 3 of the present application. For K562 cells and K562-CS1 cells, from left to right are UTD, 21G cells, The release of IL-2 from 22G cells, 23G cells, 24G cells, 25G cells and 26G cells.

Figure 5 shows the killing effect of CS1 CAR-T cells on CS1 positive target cells in Example 3 of the present application. For K562 cells and K562-CS1 cells, from left to right are the killing effects of UTD, 21G cells, 22G cells, 23G cells, 24G cells, 25G cells, 26G cells and positive control LUC90V2 cells on target cells.

Figure 6A shows the continuous proliferation of CD3+ cells after multiple rounds of antigen stimulation in Example 4 of the present application to activate CS1 CAR-T cells; Figure 6B shows the activation of CS1 CAR-T cells after multiple rounds of antigen stimulation in Example 4 of the present application , Continuous proliferation of CAR+ cells.

Fig. 7 shows a schematic diagram of the construction of the BCMA-CS1 bispecific CAR in Example 5 of the present application.

Figure 8 shows the results of flow cytometry for the detection of the positive rate of BCMA-CS1 bispecific CAR-T cells in each group in Example 6 of the present application.

Figure 9A, Figure 9B, and Figure 9C show the effects of BCMA-CS1 bispecific CAR-T cells, negative control cells, and positive control cells in Example 7 of the present application on double-negative target cells K562 and exogenous BCMA-positive cells K562- BCMA, exogenous CS1 positive cell K562-CS1 cell killing experiment results.

Detailed ways

The implementation of the invention of the present application will be described in the following specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

The application is further described as follows: In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation steps used herein are all terms and routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

In this application, the term "Chimeric Antigen Receptor" (Chimeric Antigen Receptor, CAR) is the core component of CAR cell therapy drugs, which may include extracellular antigen recognition domains (for example, binding tumor-associated antigen (Tumor-Associated Antigen , part of TAA), hinge region, transmembrane region and intracellular domain. CAR-T (Chimeric Antigen Receptor T) cell immunotherapy is considered to be one of the most promising means to overcome tumors. CAR-T cells use genetic modification to enable T cells to express CAR proteins. This CAR protein has the ability to recognize the intact protein on the membrane surface without relying on antigen presentation, thereby causing the activation and functional effects of T cells.

In the present application, the term "extracellular antigen recognition domain" refers to the antigen recognition domain (AntigenRecognition Domain, ARD). The ability of CAR cell therapy products (such as CAR-T cells) to specifically recognize and/or bind to target antigens expressed by tumor cells depends on the extracellular antigen recognition domain. Chain variable region (Single Chain Variable Fragment, abbreviated as scFv), or derived from receptor-ligand interaction, TCR mimics, variable lymphocyte receptors (Variable Lymphocyte Receptors, VLR). So far, the most common source is the scFv segment of the antibody. The scFv includes the variable region of the heavy chain and the variable region of the light chain of the antibody, and the two are connected by a peptide chain, such as: a linking sequence consisting of 18 amino acids GSTSGSGKPGSGEGSTKG .

In this application, the term "specific recognition and/or binding" refers to the recognition and/or binding between CAR and a specific target, with greater affinity, avidity, easier, and/or bind the target for a greater duration.

In the present application, the term "humanized antibody" is also referred to as a humanized engineered antibody, which is obtained by combining the complementarity-determining regions (CDRs) of non-human mammalian antibodies, such as mouse antibodies, rat antibodies, and rabbit antibodies. ) are transplanted into the CDRs of human antibodies for preparation, and conventional recombinant DNA techniques for preparing humanized antibodies are known (eg WO96/02576). For example, when the CDRs are obtained from a rabbit antibody, primers can be synthesized (corresponding primers can be obtained by referring to the method described in WO98/13388) and used to link the CDRs of the rabbit antibody to the framework regions (FRs) of the human antibody. As for human antibody FRs linked to CDRs, those are selected such that the CDR regions form good antigen-binding sites.

In this application, the term "hinge region" refers to the link between the extracellular antigen recognition domain and the transmembrane domain. This region allows CAR to recognize antigen by giving the antigen recognition domain a certain range of motion. Currently used hinge regions are mainly derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α. In addition, the typical hinge region also contains residues that are involved in CAR dimerization and contribute to enhanced antigen sensitivity.

In this application, "transmembrane region" refers to the transmembrane domain that connects the intracellular and extracellular components of the CAR structure. Different transmembrane domains can affect the expression and stability of CAR to a certain extent, but they are not directly involved in signal transmission, and the downstream signal transmission can be improved through interaction. The transmembrane region can be derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70.

Within this application, the term "intracellular domain" includes intracellular signaling regions and may also include co-stimulatory signaling regions.

In the present application, the term "intracellular signaling region" refers to the activation of at least one normal effector function of an immune effector cell responsible for the expression of CAR. The intracellular signaling region can be derived from one or more of CD3δ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk.

In this application, the term "co-stimulatory signaling domain" exists because, in addition to the stimulation of antigen-specific signals, many immune effector cells also require co-stimulation to promote cell proliferation, differentiation and survival, and activation of cell activation. Effector function. In some embodiments, the CAR may also include one or more co-stimulatory signaling regions, wherein the co-stimulatory signaling regions may be derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4- One, two or more of 1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, MyD88.

In the present application, the term "isolated" generally means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of. The term "isolated" does not exclude artificial or synthetic substances obtained from the natural state by artificial means, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

In this application, the term "guide peptide" refers to a short peptide before the extracellular antigen recognition domain (such as scFv sequence), which functions to guide the export of recombinant protein synthesized in the cell to the outside of the cell. Commonly used guide peptides are human CD8α signal peptide, or human GM-CSF receptor α signal peptide.

In this application, one of the key factors determining the efficacy of CAR-immune cell therapy is the selection of tumor target antigens. In this application, the term "CS1" is also called SLAMF7 (signaling-lymphocyte-activating molecule F7) or CD319, which is a glycoprotein on the cell surface, expressed in plasma cells, NK cells, CD8+ T cells, activated B Cells and dendritic cells and other normal tissues, but not expressed in hematopoietic stem cells and non-hematopoietic organs, can participate in the mutual adhesion between myeloma cells and bone marrow stromal cells. The term "BCMA" refers to B cell maturation antigen, a member of the tumor necrosis factor receptor superfamily. Human BCMA is expressed almost exclusively in plasma cells and multiple myeloma cells. BCMA may be a suitable tumor antigen target for immunotherapeutics against multiple myeloma. However, due to the heterogeneity of specific antigens on the surface of multiple myeloma cells, the selection of its antigen target is not necessarily single. By selecting appropriate targets, the anti-tumor activity of CAR-T cells can be optimized.

In this application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, which may be isolated from its natural environment or a synthetic analogue .

In this application, when referring to CAR gene transduction/transfection and target gene expression, gene transduction/transfection methods mainly include viral and non-viral methods. Such as: through γ-retroviral vectors, lentiviral vectors, adeno-associated viral vectors, plasmid DNA-dependent vectors, transposon-dependent gene transfer, and mRNA-mediated gene transduction.

The term "vector" generally refers to a nucleic acid delivery tool into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. The vector can transform, transduce or transfect the host cell, so that the genetic material elements carried by it can be expressed in the host cell. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); phage such as lambda phage or M13 phage and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillary polyoma vacuoles Viruses (such as SV40). A vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication. Vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes or protein coats, but not only. The term "transposon" refers to a discontinuous DNA segment that has the ability to migrate between chromosomal sites and carry genetic information, such as: Sleeping Beauty SB system and PB system derived from Lepidoptera insects. In some embodiments, electroporation can also be used to transduce mRNA into T cells.

In this application, the term "immune effector cell" generally refers to a cell that participates in an immune response, eg, promotes an immune effector response. Immune effector cells may be selected from the group consisting of: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T lymphocytes differentiated from pluripotent stem cells, One or more of NK cells and embryonic stem cells.

In this application, the term "pharmaceutical composition" generally refers to a pharmaceutical composition suitable for administration to patients, which may contain the immune effector cells described in this application, and may also contain one or more pharmaceutically acceptable excipients, such as : one or more of carrier, protective agent, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, preservative. In some embodiments, the pharmaceutically acceptable excipients include protective agents, such as cell cryopreservation solution. In some embodiments, the pharmaceutical composition of the present application is a cell suspension or frozen cells thereof.

In this application, the term "subject" generally refers to human or non-human animals, including but not limited to mice, rats, cats, dogs, rabbits, horses, pigs, cows, sheep or monkeys.

In this application, the term "comprising" generally means including specifically specified features, but not excluding other elements.

In the present application, the term "about" usually refers to the fluctuation range acceptable to those skilled in the art above or below the specified value, such as: within the range of ±0.5%-10%, for example, 0.5% above or below the specified value %, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% range.

Chimeric antigen receptors, nucleic acids, vectors, immune effector cells, pharmaceutical compositions

In one aspect, the present application provides a chimeric antigen receptor targeting CS1, which comprises an extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain; wherein: the extracellular antigen recognition domain comprises Anti-CS1 scFv antibody, the amino acid sequences of the VH complementarity determining regions CDR1, CDR2, and CDR3 of the scFv antibody include the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 respectively, and the scFv The amino acid sequences of the VL complementarity determining regions CDR1, CDR2, and CDR3 of the antibody include the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

In some embodiments, the VH sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO:7, and the VL sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO:8.

In some embodiments, the scFv antibody is a humanized antibody.

In some embodiments, the VH sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO:9, and the VL sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO:10.

In some embodiments, the scFv antibody is a rabbit antibody.

In some embodiments, there is a connecting region between VH and VL in the scFv antibody, and the connecting region is selected from one or more of the following: SEQ ID NOs: 59-61.

In some embodiments, the sequence of the scFv antibody is shown in SEQ ID NO:11 or SEQ ID NO:12.

In some embodiments, the present application also includes substitution, deletion, addition and/or insertion of one or more amino acids in the amino acid sequence of any one of the above chimeric antigen receptors, and it has the equivalent of any one of the above Chimeric Antigen Receptor Activity. Those skilled in the art know that during the process of humanization, amino acids in the scFv FR region can be substituted so that the CDR region of the engineered antibody can retain a suitable antigen-binding site. In the case of the above CDRs, the humanized transformation of the FR region in the scFv is different from the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10. And those skilled in the art also know that in order to make the CDR region of the modified antibody retain a suitable antigen-binding site during the process of humanization, if necessary, one, two, three or none of the CDRs More than 10% of the amino acid sequence may be substituted, deleted, added and/or inserted, and these are also included in this application.

In some embodiments, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α; optionally, the amino acid sequence of the hinge region is derived from CD8α; further Optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO:13.

In some embodiments, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70; alternatively, The amino acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:14.

In some embodiments, wherein the intracellular domain comprises an intracellular signaling region; optionally, also includes a co-stimulatory signaling region; further optionally, wherein the intracellular signaling region is derived from CD3δ, CD3γ , one or more of CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, Syk; still further optionally, the intracellular signaling region is derived from CD3δ, For example: the amino acid sequence of the intracellular signaling region includes the amino acid sequence shown in SEQ ID NO:15.

In some embodiments, wherein the co-stimulatory signaling region is derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, One, two, or more than three of NKG2D, DAP10, B7-H3, and MyD88; optionally, the costimulatory signal transduction region is derived from CD28 or 4-1BB; further optionally, the costimulatory signal transduction region The amino acid sequence of comprises the amino acid sequence shown in SEQ ID NO:16.

In some embodiments, the chimeric antigen receptor further comprises a guide peptide located at the N-terminal of the chimeric antigen receptor amino acid sequence; optionally, wherein the guide peptide is derived from CD8α; further optionally, The amino acid sequence of the leader peptide comprises the amino acid sequence shown in SEQ ID NO:17.

In some embodiments, the chimeric antigen receptor of the present invention comprises the amino acid sequence shown in SEQ ID NO:32.

In some embodiments, the extracellular antigen recognition domain further comprises scFv antibodies against any of the following targets: CD138, NKG2D, CD38, BCMA, CD19, CD70, CD44v6, Lewis Y.

In some embodiments, the extracellular antigen recognition domain sequentially comprises anti-BCMA scFv VL, anti-CS1 scFv VL, anti-CS1 scFv VH and anti-BCMA scFv VH, and the anti-CS1 scFv VH complementarity determining region The amino acid sequences of CDR1, CDR2, and CDR3 respectively include the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the amino acids of the scFv VL complementarity determining regions CDR1, CDR2, and CDR3 of the anti-CS1 The sequences respectively include the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the amino acid sequences of the anti-BCMA scFv VH complementarity determining regions CDR1, CDR2, and CDR3 include SEQ ID NO: 51. The amino acid sequences shown in SEQ ID NO:52 and SEQ ID NO:53, the amino acid sequences of the anti-BCMA scFv VL complementary determining regions CDR1, CDR2, and CDR3 respectively include SEQ ID NO:54, SEQ ID NO:55, Amino acid sequence shown in SEQ ID NO:56.

In some embodiments, the VH sequence of the anti-CS1 scFv antibody includes the amino acid sequence shown in SEQ ID NO:7, and the VL sequence of the anti-CS1 scFv antibody includes the amino acid sequence shown in SEQ ID NO:8 sequence.

In some embodiments, the VH sequence of the anti-BCMA scFv antibody includes the amino acid sequence shown in SEQ ID NO:57, and the VL sequence of the anti-BCMA scFv antibody includes the amino acid sequence shown in SEQ ID NO:58 sequence.

In some embodiments, the extracellular antigen recognition domain includes the amino acid sequence shown in SEQ ID NO:50.

On the other hand, the present application also provides an isolated nucleic acid molecule comprising the nucleotide sequence encoding the above-mentioned chimeric antigen receptor.

On the other hand, the present application provides an isolated nucleic acid molecule encoding a chimeric antigen receptor, which is selected from one of the following nucleic acid molecules: comprising the nucleotide sequence shown in SEQ ID NO: 18, or with SEQ ID NO Nucleic acid molecule that is similar to the nucleotide sequence shown in :18 and encodes the same chimeric antigen receptor nucleotide sequence.

In some embodiments, the similarity to the nucleotide sequence shown in SEQ ID NO: 18 refers to at least 70%, 75%, 80%, 85% of the nucleotide sequence shown in SEQ ID NO: 18 , 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

In some embodiments, the similarity to the nucleotide sequence shown in SEQ ID NO: 18 refers to the wobble (degeneracy) of the third base of the nucleic acid codon and the sequence shown in SEQ ID NO: 18 Nucleotide sequences are different, but they encode the same chimeric antigen receptor nucleic acid molecule. Optionally, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:39.

On the other hand, the present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule. Vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); phages such as lambda phage or M13 phage and animal viruses, etc. . Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillary polyoma vacuoles Viruses (such as SV40).

In some embodiments, the vector is an expression vector; optionally, the vector is a viral vector; further optionally, a lentiviral vector.

On the other hand, the present application also provides an engineered immune effector cell, which comprises the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned carrier.

In some embodiments, the immune effector cells are selected from T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T cells differentiated from pluripotent stem cells, pluripotent One or more of NK cells and embryonic stem cells differentiated from stem cells.

In some embodiments, the immune effector cells are T lymphocytes; optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.

In some embodiments, the surface of the immune effector cells may express the chimeric antigen receptors described herein.

On the other hand, the present application also provides a pharmaceutical composition, which includes the above-mentioned engineered immune effector cells and pharmaceutically acceptable auxiliary materials. The pharmaceutically acceptable auxiliary materials include: one or more of carriers, protective agents, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives.

In some embodiments, the pharmaceutically acceptable excipients include protective agents, such as cell cryopreservation solution.

In some embodiments, the pharmaceutical composition is a cell suspension or frozen cells thereof.

In some embodiments, the pharmaceutical composition is an intravenous injection.

Preparation method and use

On the other hand, the present application also provides a method for preparing immune effector cells, which includes the following steps: transducing the vector described in the present application into the immune effector cells.

In some embodiments, the immune effector cells are selected from T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T cells differentiated from pluripotent stem cells, pluripotent One or more of NK cells and embryonic stem cells differentiated from stem cells.

In some embodiments, the immune effector cells are T lymphocytes; optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.

On the other hand, the present application also provides the use of the chimeric antigen receptor described in the present application, the nucleic acid molecule, the carrier and/or the immune effector cell for the preparation of a drug, wherein the drug For use in the treatment of a disease or condition associated with the expression of CS1.

On the other hand, the present application also provides a method for treating a disease or disorder associated with the expression of CS1, the method comprising administering an effective dose of the present invention to a subject in need of treating a disease or disorder associated with the expression of CS1. The chimeric antigen receptor, the nucleic acid molecule, the carrier and/or the immune effector cell.

In some embodiments, the administration can be performed by different means, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. For example, the mode of administration may be administered to a subject by intravenous injection. In some embodiments, the effective dose of immune effector cells or the pharmaceutical composition can be administered to the subject once, or dividedly administered to the subject within a certain period of time, such as once a week, once every two weeks, Once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months.

In some embodiments, for different indications, the dosage may be different; for patients with different disease severity, the dosage may also be different. The administered dose may range from 1×10 ⁵ CAR-positive T cells/kg to 1×10 ⁷ CAR-positive T cells/kg, for example, 1×10 ⁵ CAR-positive T cells/kg to 1×10 ⁶ CAR-positive T cells/kg, 1×10 ⁶ CAR-positive T cells/kg to 1×10 ⁷ CAR-positive T cells/kg.

In some embodiments, the subjects can include humans and non-human animals. For example, the subject may include, but is not limited to, mice, rats, cats, dogs, horses, pigs, cows, sheep, rabbits or monkeys.

On the other hand, the present application also provides the chimeric antigen receptor, the nucleic acid molecule, the vector and/or the immune effector cell, which can be used to treat diseases related to the expression of CS1 or illness.

In some embodiments, the disease or condition associated with the expression of CS1 may include non-solid tumors, and optionally, the non-solid tumors are hematological tumors.

In some embodiments, the disease or disorder associated with the expression of CS1 may include multiple myeloma.

In some embodiments, the multiple myeloma is relapsed or refractory multiple myeloma.

Without intending to be limited by any theory, the following examples are only for explaining the chimeric antigen receptor, immune effector cell, preparation method and application of the present application, and are not intended to limit the scope of the present invention. The Examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, to insert genes encoding proteins into such vectors and plasmids, or to introduce plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

Example 1. Acquisition of CS1 CAR-T cells

We have 7 CS1-specific humanized scFv sequences (the amino acid sequences of these scFv are respectively as SEQ ID NO: 19, SEQ ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 , SEQ ID NO: 23, and SEQ ID NO: 24, the amino acid sequences of these 7 scFvs are respectively referred to as abbreviations by numbering 21G, 22G, 23G, 24G, 25G, 26G, and 27G in this application. Encoding the above-mentioned 21G～27G scFv The nucleotide sequences are respectively shown in SEQ ID NO:25, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 ). Since the functional verification of scFv at the protein level cannot reflect its function at the cellular level, we chose to screen candidate CS1 scFv sequences on the second-generation CAR structure. The schematic diagram of the CAR structure is shown in Figure 1. We use the CD8α guide chain as the signal peptide (its amino acid sequence is shown in SEQ ID NO:17), use CS1 scFv as the extracellular tumor antigen recognition region, and use CD8α for the hinge region and transmembrane region structure (its amino acid sequence is shown in SEQ ID NO:13 and SEQ ID NO:14), 4-1BB is an intracellular co-stimulatory signal (its amino acid sequence is shown in SEQ ID NO:16), and CD3δ is T Cell activation signal (its amino acid sequence is shown in SEQ ID NO: 15).

1. Construction of lentiviral vector

Artificially synthesize CAR structure fragments comprising 21G-27G scFv in the present application (the amino acid sequences of these CAR structures are respectively as SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID As shown in NO:35, SEQ ID NO:36, and SEQ ID NO:37, the nucleotide sequences encoding the above-mentioned CAR structure are respectively as SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO : 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44), and respectively constructed to the transformed empty lentiviral vector (manufacturer: SBI company, article number: CD500-CD800, such as WO2021/ 121227 The conventional resistance transformation described in Example 1) obtained the CAR expression vector, and then the CAR expression vector and three packaging plasmids were transfected into 293T cells, and a functional lentiviral vector was obtained after collection and purification. The three packaging plasmids are pMD2.0G (purchased from Biovector, product number Biovector012259), pMDLg-/pRRE (purchased from Biovector, product number Biovector012251), and pRSV-Rev (purchased from Biovector, product number Biovector012253).

2. Prepare 7 kinds of CS1 CAR-T cells by lentiviral transduction

The transduction experiment was carried out according to conventional methods known to those skilled in the art, and the transduction steps are briefly described as follows:

1) Sorting T cells

Peripheral blood mononuclear cells (PBMC) were isolated from human single blood cells, and then T cells were sorted from PBMC cells.

2) Activate T cells

Separated T cells with complete lymphocyte culture medium (X-VIVO15 medium+5%FBS+300IU/ml IL-2 or X-VIVO15 medium+5%FBS+5ng/ml IL-15+10ng/ml IL -7) Resuspend to make the final concentration (1～2)×10 ⁶ cells/ml, and add 5～10 μl of CD3/CD28 magnetic beads to stimulate, mix well and place in an incubator for culture, the culture condition is 37 ℃ + 5% CO2, culture time at least 24 hours.

3) Lentiviral transduction of T cells

Take out the activated T cells, add polybrene (polybrene) with a final concentration of 8 μg/ml, mix well, and slowly add lentiviral vector according to MOI=2, put it in a centrifuge after mixing, and centrifuge at 1500rpm 1.5 hours. Then place it in an incubator for cultivation, the cultivation condition is 37° C.+5% CO2, and the cultivation time is at least 24 hours.

4) Expansion and culture of T cells after transduction

The transduced cells were taken out, and the cell density was monitored to maintain the cells at (0.5-1)×10 ⁶ cells/ml for use in subsequent examples.

The T cells obtained after infecting T cells with lentiviruses containing 21G-27G scFv were named CS1CAR-T cells 21G-27G, respectively. Next, we screened seven CAR structures at the cellular level to finally determine an optimal candidate scFv sequence.

Example 2. Detection of CAR molecules expressed on the surface of CS1 CAR-T cells 21G-27G

The CAR protein molecules expressed on the surface of 21G-27G of the seven CS1 CAR-T cells obtained in Example 1 were detected. We used PE fluorescence-labeled CS1 antigen (manufacturer: ACRO Biosystems, product number: SL7-HP2H3) to stain 7 kinds of CS1 CAR-T cells, UTD cells (T cells not transduced with CAR), LUC90V2 CAR-T cells (LUC90V2 is positive Control CS1 CAR sequence, the CAR sequence is shown in SEQ ID NO: 63, the scFv in the CAR sequence is a mouse sequence, the method for obtaining the CAR-T cells is also the method in Example 1), by flow cytometry CAR positive ratio detection and analysis were carried out after surgery. The results are shown in Figure 2: the CAR positive ratios of CS1 CAR-T cells 22G, 26G, 21G, and 23G were high, reaching 96.70%, 82.61%, 77.61%, and 68.99%, respectively, but the CS1 CAR-T cells 24G, 25G, and 27G CAR protein expression was barely detected. In addition, since the CS1 antigen was used for staining in the experiment, the above results also suggested that the ability of these CAR proteins to specifically recognize the CS1 antigen was different.

In the above experiment of staining CS1 CAR-T cells with PE fluorescently labeled CS1 antigen, almost no CAR protein expression was detected in CS1 CAR-T cells 24G, 25G, and 27G. Whether CS1 CAR is not expressed or CS1 CAR is expressed but cannot recognize CS1 antigen, we further use fluorescently labeled anti-human IgG antibody (manufacturer: invitrogen, product number: A11013) to detect 7 kinds of CS1 CAR-T cells, UTD cells, positive Control BCMA CAR-T cell 02G staining (because scFv 21G-27G are all humanized antibodies, which contain human FR regions, and human T cells basically do not express IgG on the surface, so anti-human IgG antibody was used for staining It can be clarified whether CS1 CAR-T cells 24G, 25G, and 27G do not express CS1 CAR on the surface or they cannot recognize CS1 antigen), and the CAR positive ratio is detected and analyzed by flow cytometry. In addition, since the scFv in the LUC90V2 CAR sequence is a mouse sequence and does not contain a human FR region, BCMA CAR-T cell 02G containing another humanized scFv is used here as a positive control in this method (the sequence of the BCMA02G CAR is shown in SEQ ID NO:62). The results are shown in Figure 3: In this method, the CAR-positive ratios of CS1 CAR-T cells 22G, 26G, 21G, and 23G were still high, reaching 85.14%, 79.76%, 79.95%, and 79.55%, respectively; CS1CAR-T cells Both 24G and 25G can clearly detect CAR-positive cells, indicating that the CAR protein on the surface of CS1 CAR-T cells 24G and 25G can be expressed, but it may not be able to recognize the CS1 antigen, while the CS1 CAR-T cell 27G still does not detect CAR protein expression .

Example 3, CS1 CAR-T cells were subjected to cytokine release experiments and cell killing experiments

1. Cytokine release experiment: The CS1 CAR-T cells 21G-26G obtained in Example 1 (because there is no CAR protein expression on the surface of CS1CAR-T cells 27G, so this cell will not be involved in the follow-up experiment) and the target cells according to the After CAR+ cells and target cells were co-cultured in X-VIVO15 medium for 24 hours under the condition of an effect-to-target ratio of 1:1, the concentration of IL-2 in the cell supernatant was detected by ELISA method (manufacturer: Dakewe, article number: 1110202), where : K562 is a CS1-negative target cell, K562-CS1 is a positive target cell expressing CS1 exogenously, and the UTD group is used as a negative control. The results of cytokine release experiments are shown in Figure 4: the IL-2 release levels of CS1 CAR-T cells 21G, 22G, and 26G were significantly higher than those of CS1 CAR-T cells 23G, 24G, 25G, and UTD groups.

2. Cell killing experiment: CS1 CAR-T cells 21G-26G obtained in Example 1 and target cells were co-cultured in X-VIVO15 medium for 48 hours according to the condition of CAR+ cells and target cell effect-to-target ratio of 1:1. The survival ratio of target cells was detected by detecting the luciferase activity stably expressed in the target cells (the detection reagent was purchased from Promega, product number: E2520), and the results are shown in Figure 5: CS1 CAR-T cells 21G, 22G, 23G, 26G and positive The control CS1 CAR-T cells LUC90V2 all had better killing effect on K562-CS1 positive target cells expressing CS1 exogenously.

Example 4, Sustained Proliferation of CS1 CAR-T Cells

Antigen stimulation can activate CAR-T cells to proliferate CAR-T cells, and the continuous activation of T cells will lead to cell exhaustion, and the proliferation ability and effector function of exhausted T cells will decrease. We detected CS1 CAR-T cells 21G The proliferation of CD3+ cells (i.e., the proliferation of T cells, CD3 is a marker to distinguish whether they are T cells) and the proliferation of CAR+ cells after multiple rounds of antigen stimulation experiments of ~26G, to verify the continuous proliferation of CS1 CAR-T cells .

Before antigen stimulation, the CAR-positive ratio of CS1 CAR-T cells in each group was adjusted to a level consistent with that of the CAR-T cell group with the lowest CAR-positive ratio using T cells that were not transduced with CAR. In multiple rounds of antigen stimulation experiments, CS1 CAR-T cells in each group were co-cultured with CS1 endogenous positive target cells MM.1S (human multiple myeloma cells) in a 24-well plate at an effect-to-target ratio of 1:2. , 2ml X-VIVO15 medium per well, repeat 3 wells for each group of cells. After 3 to 4 days, 500 μl of cells were stained with fluorescently labeled CD3 antibody (manufacturer: BioLegend, product number: 317318) and CS1 antigen (same as Example 2) for CD3 and CAR, and detected and analyzed by flow cytometry, showing CD3 positive cells The proportion, number and fluorescence intensity of CAR-positive cells in the sample can also be calculated according to the conversion of the volume multiple, and the number of CAR-positive cells in the CD3-positive cells in the sample can be calculated, and then the CAR-T cells are taken out from each group according to the calculation results according to the effect-to-target ratio: 1: 2 Add MM.1S cells for a new round of stimulation, and repeat until CAR-T cell proliferation stagnates, ending antigen stimulation.

The results of sustained proliferation are shown in Figure 6A and Figure 6B: only CS1 CAR-T cells 22G and 26G can maintain the expansion ability for 23 days under the condition of repeated antigen stimulation, among which CD3+ cells and CAR+ cells in CS1 CAR-T cells 22G Cell proliferation was the best and was significantly better than that of CD3+ cells and CAR+ cells in 26G.

In summary, in vitro pharmacodynamic tests show that the expression of 22G CAR on the surface of T cells is superior to other candidates; it is superior to other candidate CAR-T in terms of cytokine release, in vitro cell killing, and sustained proliferation of CAR-T. . CDR1, CDR2, and CDR3 of VH in 22G scFv are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and CDR1, CDR2, and CDR3 of VL in 22G scFv are shown in SEQ ID NO:4, Shown in SEQ ID NO:5 and SEQ ID NO:6.

Example 5, Preparation of BCMA-CS1 bispecific CAR-T cells

We chose to screen candidate BCMA-CS1 bispecific scFv sequences on the second-generation CAR construct. Selected 02G scFv for the BCMA target (the CDR1, CDR2, and CDR3 of its VH are shown in SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53 respectively, and the CDR1, CDR2, and CDR3 of its VL are shown in SEQ ID NO:53, respectively. ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, the amino acid sequence of scFv VH is shown in SEQ ID NO: 57, the amino acid sequence of scFv VL is shown in SEQ ID NO: 58), for CS1 After the 22G scFv target is selected, the construction of BCMA-CS1 bispecific CAR begins. Considering the changes in the sequence of target BCMA scFv and CS1 scFv, the sequence of VH and VL in each target scFv, the type of linker, and the different structures of tandem and loop, the structural design of BCMA-CS1 bispecific CAR has There are many options, including four tandem structures and two loop structures as shown in Figure 7, and are built into the second-generation CAR structure. We use CD8α guide chain as signal peptide, BCMA-CS1 bispecific scFv as extracellular tumor antigen recognition region, hinge region and transmembrane region as CD8α structure, 4-1BB as intracellular co-stimulatory signal, CD3δ as T Cell activation signal. In addition to replacing the CAR sequence, the method for preparing CS1 CAR-T cells in Example 1 was still used to prepare BCMA-CS1 bispecific CAR-T cells 41A, 41B, 42A, 42B, 43A, 43B (the sequence of the corresponding scFv Respectively such as: SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50), the amino acid sequence of the module in the second generation CAR structure and Example 1 is the same.

Example 6. Positive rate detection of BCMA-CS1 bispecific CAR-T cells

BCMA-CS1 bispecific CAR-T cells 41A, 41B, 42A, 42B, 43A, and 43B were tested for CAR positive rate, among which: UTD cells were used as negative controls, and BCMA CAR-T cells 02G (replacing the CAR sequence, still using Prepared by the method for preparing CS1 CAR-T cells in Example 1) and CS1 CAR-T cells 22G (prepared in Example 1) were used as positive controls, and FITC fluorescently labeled BCMA antigen (manufacturer: ACRO Biosystems, article number: BCA-HF254) and PE fluorescently labeled CS1 antigen (same as Example 2) were stained, and the results are shown in Figure 8: CAR-T cells with four tandem structures (41A, 41B, 42A, 42B) were almost not detected When it comes to CAR-positive cells, both 43A and 43B can clearly detect CAR-positive cells, but most of the positive cells of 43A only recognize BCMA antigens, while the positive cells of 43B can recognize both BCMA and CS1 antigens, indicating that different structural designs of scFv will Affects its own antigen-binding ability.

Example 7, BCMA-CS1 CAR-T cell killing experiment

In Example 6, since only BCMA-CS1 bispecific CAR-T cells 43A and 43B clearly detected CAR-positive cells, in the cell killing experiment, 43A, 43B and BCMA CAR-T cells 02G, CS1 CAR-T cells The CAR positive rate of 22G was adjusted to be consistent. Since the positive rate of CAR in other groups was very low, the positive rate of CAR in all groups was not uniformly adjusted to the lowest level. In the end, there were no adjusted cells in each group (41A, 41B, 42A, 42B) Only the same number of T cells can be added in the experiment, but the same number of CAR+T cells cannot be satisfied. In the cell killing experiment, the obtained BCMA-CS1 bispecific CAR-T cells 41A, 41B, 42A, 42B, 43A, 43B and target cells were placed in X-VIVO15 medium according to the conditions of different effect-to-target ratios between CAR+ cells and target cells. After co-cultivation for 48 hours, the survival rate of target cells was detected by detecting the stably expressed luciferase activity in the target cells (same as in Example 3). and the killing ability of target cells expressing exogenous CS1, and UTD cells were used as negative controls, and BCMACAR-T cells 02G and CS1 CAR-T cells 22G were used as positive controls. The results of the killing test are shown in Figure 9A, Figure 9B and Figure 9C: It can be seen from the results that BCMA-CS1 dual-target CAR-T cells 43A and 43B have a good ability to kill K562-BCMA cells, while K562 - In the killing experiment of CS1 cells, only 43B has obvious killing ability. Among the six structures screened, only 43B has complete dual-target killing activity of BCMA and CS1, and the other groups only have different degrees of ability to kill BCMA-positive target cells.

sequence description

SEQ ID NO: 1: 22G scFv VH CDR1;

SEQ ID NO: 2: 22G scFv VH CDR2;

SEQ ID NO: 3: 22G scFv VH CDR3;

SEQ ID NO: 4: 22G scFv VL CDR1;

SEQ ID NO: 5: 22G scFv VL CDR2;

SEQ ID NO: 6: 22G scFv VL CDR3;

SEQ ID NO: 7: amino acid sequence of humanized 22G scFv VH;

SEQ ID NO:8: amino acid sequence of humanized 22G scFv VL;

SEQ ID NO: 9: amino acid sequence of rabbit source 22G scFv VH;

SEQ ID NO: 10: amino acid sequence of rabbit-derived 22G scFv VL;

SEQ ID NO: 11: amino acid sequence of humanized 22G scFv;

SEQ ID NO:12: amino acid sequence of 22G scFv from rabbit;

SEQ ID NO: 13: the amino acid sequence of the hinge region;

SEQ ID NO: 14: amino acid sequence of the transmembrane region;

SEQ ID NO: 15: amino acid sequence of intracellular signaling region;

SEQ ID NO: 16: amino acid sequence of co-stimulatory signaling region;

SEQ ID NO:17: the amino acid sequence of the leader peptide (ie signal peptide);

SEQ ID NO: 18: nucleotide sequence encoding 22G scFv amino acid sequence;

SEQ ID NO: 19: 21G scFv amino acid sequence;

SEQ ID NO:20: 23G scFv amino acid sequence;

SEQ ID NO:21: 24G scFv amino acid sequence;

SEQ ID NO:22: 25G scFv amino acid sequence;

SEQ ID NO:23: 26G scFv amino acid sequence;

SEQ ID NO:24: 27G scFv amino acid sequence;

SEQ ID NO:25: nucleotide sequence encoding 21G scFv amino acid sequence;

SEQ ID NO:26: nucleotide sequence encoding 23G scFv amino acid sequence;

SEQ ID NO:27: nucleotide sequence encoding 24G scFv amino acid sequence;

SEQ ID NO:28: nucleotide sequence encoding 25G scFv amino acid sequence;

SEQ ID NO:29: nucleotide sequence encoding 26G scFv amino acid sequence;

SEQ ID NO: 30: nucleotide sequence encoding 27G scFv amino acid sequence;

SEQ ID NO:31: 21G CAR amino acid sequence;

SEQ ID NO:32: 22G CAR amino acid sequence;

SEQ ID NO:33: 23G CAR amino acid sequence;

SEQ ID NO:34: 24G CAR amino acid sequence;

SEQ ID NO:35: 25G CAR amino acid sequence;

SEQ ID NO:36: 26G CAR amino acid sequence;

SEQ ID NO:37: 27G CAR amino acid sequence;

SEQ ID NO:38: nucleotide sequence encoding 21G CAR amino acid sequence;

SEQ ID NO:39: nucleotide sequence encoding 22G CAR amino acid sequence;

SEQ ID NO:40: nucleotide sequence encoding 23G CAR amino acid sequence;

SEQ ID NO:41: nucleotide sequence encoding 24G CAR amino acid sequence;

SEQ ID NO:42: nucleotide sequence encoding 25G CAR amino acid sequence;

SEQ ID NO:43: nucleotide sequence encoding 26G CAR amino acid sequence;

SEQ ID NO:44: nucleotide sequence encoding 27G CAR amino acid sequence;

SEQ ID NO:45: bispecific scFv 41A amino acid sequence;

SEQ ID NO:46: bispecific scFv 41B amino acid sequence;

SEQ ID NO:47: bispecific scFv 42A amino acid sequence;

SEQ ID NO:48: bispecific scFv 42B amino acid sequence;

SEQ ID NO:49: bispecific scFv 43A amino acid sequence;

SEQ ID NO:50: bispecific scFv 43B amino acid sequence;

SEQ ID NO:51:02G scFv VH CDR1;

SEQ ID NO:52:02G scFv VH CDR2;

SEQ ID NO:53:02G scFv VH CDR3;

SEQ ID NO:54:02G scFv VL CDR1;

SEQ ID NO:55:02G scFv VL CDR2;

SEQ ID NO:56:02G scFv VL CDR3;

SEQ ID NO:57: VH amino acid sequence of humanized 02G scFv antibody;

SEQ ID NO:58: VL amino acid sequence of humanized 02G scFv antibody;

SEQ ID NOs:59-61: the amino acid sequence of the linking region connecting VH and VL;

SEQ ID NO:62: BCMA 02G CAR;

SEQ ID NO:63: LUC90V2 positive control CS1 CAR sequence.

sequence listing

<110> Heyuan Biotechnology (Tianjin) Co., Ltd.

<120> Chimeric antigen receptors targeting CS1, bispecific chimeric antigen receptors targeting BCMA/CS1 and their applications

<130> CP1211280/CB

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Gly Phe Ser Leu Ser Asn Tyr Gly

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<212> PRT

<213> Synthetic Sequence

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Ile Gly Thr Ile Gly Ala Thr

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<212> PRT

<213> Synthetic Sequence

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Ala Arg Gly Ile Tyr Gly Asp Ile Tyr Val Tyr Ala Phe Asp Ile

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<212> PRT

<213> Synthetic Sequence

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Gln Ser Val Arg Asp Asn Gly Asp

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<213> Synthetic Sequence

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Asp Val Ser

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Ala Gly Gly Tyr Ile Ala Gly Ser Asp Arg Trp Val

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Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu Ser Asn Tyr

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Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

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Gly Phe Ile Gly Thr Ile Gly Ala Thr Leu Tyr Ala Asn Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala

85 90 95

Arg Gly Ile Tyr Gly Asp Ile Tyr Val Tyr Ala Phe Asp Ile Trp Gly

100 105 110

Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

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Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

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Asp Arg Val Ile Ile Asn Cys Gln Ala Ser Gln Ser Val Arg Asp Asn

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Gly Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

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Leu Ile Tyr Asp Val Ser Ala Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Ser Leu

65 70 75 80

Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ile Ala

85 90 95

Gly Ser Asp Arg Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu

100 105 110

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Gln Ser Val Glu Glu Ser Arg Gly Gly Leu Ile Lys Pro Thr Asp Thr

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Val Ser Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Tyr Ile Gly

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Phe Ile Gly Thr Ile Gly Ala Thr Leu Tyr Ala Asn Trp Ala Lys Ser

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Arg Ser Thr Ile Thr Arg Asn Thr Asn Leu Asn Thr Val Thr Leu Lys

65 70 75 80

Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Arg

85 90 95

Gly Ile Tyr Gly Asp Ile Tyr Val Tyr Ala Phe Asp Ile Trp Gly Pro

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 10

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Ala Ala Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Ala Val Gly

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Gly Thr Val Thr Ile Ser Cys Gln Ala Ser Gln Ser Val Arg Asp Asn

20 25 30

Gly Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu

35 40 45

Leu Ile Tyr Asp Val Ser Ala Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Lys Gly Arg Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu

65 70 75 80

Glu Cys Asp Asp Ala Ala Thr Tyr Ser Cys Ala Gly Gly Tyr Ile Ala

85 90 95

Gly Ser Asp Arg Trp Val Phe Gly Gly Gly Thr Glu Val Val Val Lys

100 105 110

<210> 11

<211> 248

<212> PRT

<213> Synthetic Sequence

<400> 11

Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ile Ile Asn Cys Gln Ala Ser Gln Ser Val Arg Asp Asn

20 25 30

Gly Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Asp Val Ser Ala Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Ser Leu

65 70 75 80

Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ile Ala

85 90 95

Gly Ser Asp Arg Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

115 120 125

Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

130 135 140

Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu Ser Asn Tyr Gly

145 150 155 160

Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly

165 170 175

Phe Ile Gly Thr Ile Gly Ala Thr Leu Tyr Ala Asn Trp Ala Lys Gly

180 185 190

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln

195 200 205

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg

210 215 220

Gly Ile Tyr Gly Asp Ile Tyr Val Tyr Ala Phe Asp Ile Trp Gly Pro

225 230 235 240

Gly Thr Leu Val Thr Val Ser Ser

245

<210> 12

<211> 375

<212> PRT

<213> Synthetic Sequence

<400> 12

Ala Ala Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Ala Val Gly

1 5 10 15

Gly Thr Val Thr Ile Ser Cys Gln Ala Ser Gln Ser Val Arg Asp Asn

20 25 30

Gly Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu

35 40 45

Leu Ile Tyr Asp Val Ser Ala Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Lys Gly Arg Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu

65 70 75 80

Glu Cys Asp Asp Ala Ala Thr Tyr Ser Cys Ala Gly Gly Tyr Ile Ala

85 90 95

Gly Ser Asp Arg Trp Val Phe Gly Gly Gly Thr Glu Val Val Val Lys

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

115 120 125

Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly Asp

130 135 140

Arg Val Ile Ile Asn Cys Gln Ala Ser Gln Ser Val Arg Asp Asn Gly

145 150 155 160

Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

165 170 175

Ile Tyr Asp Val Ser Ala Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

180 185 190

Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln

195 200 205

Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ile Ala Gly

210 215 220

Ser Asp Arg Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

245 250 255

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

260 265 270

Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu Ser Asn Tyr Gly Val

275 280 285

Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Phe

290 295 300

Ile Gly Thr Ile Gly Ala Thr Leu Tyr Ala Asn Trp Ala Lys Gly Arg

305 310 315 320

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met

325 330 335

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg Gly

340 345 350

Ile Tyr Gly Asp Ile Tyr Val Tyr Ala Phe Asp Ile Trp Gly Pro Gly

355 360 365

Thr Leu Val Thr Val Ser Ser

370 375

<210> 13

<211> 45

<212> PRT

<213> Synthetic Sequence

<400> 13

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 14

<211> 24

<212> PRT

<213> Synthetic Sequence

<400> 14

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 15

<211> 112

<212> PRT

<213> Synthetic Sequence

<400> 15

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 16

<211> 42

<212> PRT

<213> Synthetic Sequence

<400> 16

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 17

<211> 21

<212> PRT

<213> Synthetic Sequence

<400> 17

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 18

<211> 744

<212>DNA

<213> Synthetic Sequence

<400> 18

gagatcgtga tgacccagtc cccaagtaca ctgagcgcct ccgtgggcga ccgcgtgatc 60

ataaactgtc aggccagtca gagtgttcgt gataatggcg acttagcctg gtatcaacag 120

aagcccggta aggcccccaa actgctcatt tacgatgtat ccgctctggc atccggggtg 180

ccaagccgct tctccggggag tgggtctggc gccgagttca ccctgaccat atcttccctg 240

cagcccgacg acttcgcaac gtactattgc gcaggcggtt acatcgctgg ttctgatagg 300

tgggttttcg ggcagggcac gaagttgacc gtgctgggcg gagggggctc aggaggtggc 360

ggtagcggag gaggcggctc agaagtgcag ctggtggagt ccggcggtgg actggtgcaa 420

ccgggaggct cactcagatt gtcatgcacc gcctctggct ttagtctcag taactatgga 480

gtgagttggg tgaggcaggc acccggcaag ggcctggaat ggatcggctt cattggtacc 540

attggtgcca cattgtacgc gaactgggcg aagggcaggt ttaccatcag ccgcgacaac 600

agcaagaata ccgtttacct gcagatgaat agcctgaggg ccgaagacac ggcggtctat 660

ttctgtgcac gggggatata tggtgatatt tatgtttatg cctttgacat ctggggccct 720

gggaccctcg taactgtgag cagc 744

<210> 19

<211> 249

<212> PRT

<213> Synthetic Sequence

<400> 19

Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ile Ile Asn Cys Gln Ala Ser Gln Ser Ile Ser Ser Ser Tyr

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Cys Thr Tyr Gly Thr Phe His

85 90 95

Ser Ser Gly Tyr Gly Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

115 120 125

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

130 135 140

Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu Ser Ser Tyr Gly Met

145 150 155 160

Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Ser

165 170 175

Ile Asn Thr Asp Gly Ser Thr Tyr Tyr Ala Thr Trp Ala Lys Gly Arg

180 185 190

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly

210 215 220

Tyr Pro Gly Tyr Ile Thr Asp Ser Tyr Tyr Tyr Phe Asn Ile Trp Gly

225 230 235 240

Pro Gly Thr Leu Val Thr Val Ser Ser

245

<210> 20

<211> 246

<212> PRT

<213> Synthetic Sequence

<400> 20

Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ile Ile Asn Cys Gln Ala Ser Glu Asn Ile Tyr Ser Ser

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Ser Tyr Tyr Asp Thr Gly Arg

85 90 95

Ala Ser Phe Ala Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly Gly

100 105 110

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln

115 120 125

Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

130 135 140

Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu Ser Ala Tyr Ala Met Gly

145 150 155 160

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Ile Ile

165 170 175

Ser Asp Ser Ala Ser Thr Phe Tyr Ala Thr Trp Ala Lys Gly Arg Phe

180 185 190

Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn

195 200 205

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg Ala Tyr

210 215 220

Tyr Val Val Asp Asn Asp Ser Pro Phe Asn Met Trp Gly Pro Gly Thr

225 230 235 240

Leu Val Thr Val Ser Ser

245

<210> 21

<211> 239

<212> PRT

<213> Synthetic Sequence

<400> 21

Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ile Ile Asn Cys Gln Ala Ser Gln Asn Val Tyr Asp Asn

20 25 30

Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Asp Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Ser Leu

65 70 75 80

Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Thr Tyr Asp Cys

85 90 95

Ile Ser Asp Cys Asp Thr Phe Gly Gln Gly Thr Lys Leu Thr Val Leu

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

115 120 125

Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

130 135 140

Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu Ser Thr Tyr Ala

145 150 155 160

Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly

165 170 175

Phe Ile Gly Ser Gly Gly Gly Ala Tyr Tyr Ala Ser Trp Ala Lys Gly

180 185 190

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln

195 200 205

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg

210 215 220

Leu Val Ala Phe Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

225 230 235

<210> 22

<211> 242

<212> PRT

<213> Synthetic Sequence

<400> 22

Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ile Ile Asn Cys Gln Ser Ser Gln Ser Val Val Asn Asn

20 25 30

Asn Glu Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Glu Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Ser Leu

65 70 75 80

Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gly Thr Tyr Tyr Ser

85 90 95

Ser Gly Phe Tyr Val Thr Phe Gly Gln Gly Thr Lys Leu Thr Val Leu

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

115 120 125

Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

130 135 140

Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Ile Asn Asn Val Asp

145 150 155 160

Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly

165 170 175

Val Ile Ser Ser Gly Ala Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

180 185 190

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln

195 200 205

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg

210 215 220

Gly Gly Val Leu Phe Asp Pro Trp Gly Pro Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser

<210> 23

<211> 242

<212> PRT

<213> Synthetic Sequence

<400> 23

Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ile Ile Asn Cys Gln Ala Ser Glu Arg Ile Asp Tyr Ser

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Ser Tyr Tyr Asp Ile Ser Ser

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val

115 120 125

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

130 135 140

Cys Thr Ala Ser Gly Phe Pro Leu Ser Val Tyr Tyr Met Ser Trp Val

145 150 155 160

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Ile Phe Tyr Asp

165 170 175

Gly Thr Thr Tyr Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser

180 185 190

Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg

195 200 205

Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg Val Met Tyr Val Ala

210 215 220

Asp Tyr Gly Ala Leu Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser

<210> 24

<211> 239

<212> PRT

<213> Synthetic Sequence

<400> 24

Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ile Ile Asn Cys Gln Ala Ser Gln Ser Val Tyr Lys Ser

20 25 30

Asn Tyr Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

35 40 45

Leu Ile Gly Ser Ala Ser Gln Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Ser Leu

65 70 75 80

Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Ile Tyr Asp Cys

85 90 95

Ser Ser Ala Asp Cys Asn Ala Phe Gly Gln Gly Thr Lys Leu Thr Val

100 105 110

Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

130 135 140

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu Ser Ser Ser Tyr

145 150 155 160

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

165 170 175

Gly Ala Ile Gly Thr Asn His Asn Thr Tyr Tyr Pro Ser Trp Ala Lys

180 185 190

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

195 200 205

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Thr

210 215 220

Thr Gly Asp Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

225 230 235

<210> 25

<211> 747

<212>DNA

<213> Synthetic Sequence

<400> 25

gagatcgtga tgacccagtc cccaagtaca ctgagcgcct ccgtgggcga ccgcgtgatc 60

ataaactgtc aggccagtca gagcattagt agttacttat cctggtatca acagaagccc 120

ggtaaggccc ccaaactgct catttacagg gcatccactc tggcatccgg ggtgccaagc 180

cgcttctccg ggagtgggtc tggcgccgag ttcaccctga ccatatcttc cctgcagccc 240

gacgacttcg caacgtacta ttgccaatgc acttatggta cttttcatag tagtggttat 300

ggtttcgggc agggcacgaa gttgaccgtg ctgggcggag ggggctcagg aggtggcggt 360

agcgggaggag gcggctcaga agtgcagctg gtggagtccg gcggtggact ggtgcaaccg 420

ggaggctcac tcagattgtc atgcaccgcc tctggcttta gtctcagtag ctatggaatg 480

atctgggtga ggcaggcacc cggcaagggc ctggaatgga tcggctccat caatactgat 540

ggtagcacat actacgcaac ctgggcgaag ggcaggtta ccatcagccg cgacaacagc 600

aagaataccg tttacctgca gatgaatagc ctgagggccg aagacacggc ggtctattac 660

tgtgcacggg gttatcctgg ttatattact gtagttaact actactttaa catctggggc 720

cctgggaccc tcgtaactgt gagcagc 747

<210> 26

<211> 738

<212> DNA

<213> Synthetic Sequence

<400> 26

gagatcgtga tgacccagtc cccaagtaca ctgagcgcct ccgtgggcga ccgcgtgatc 60

ataaactgtc aggccagtga gaacatttac agctctttag cctggtatca acagaagccc 120

ggtaaggccc ccaaactgct catttacgct gcatccaagc tggcatccgg ggtgccaagc 180

cgcttctccg ggagtgggtc tggcgccgag ttcaccctga ccatatcttc cctgcagccc 240

gacgacttcg caacgtacta ttgccaaagt tattatgata ctggtcgtgc tagttttgct 300

ttcgggcagg gcacgaagtt gaccgtgctg ggcggagggg gctcaggagg tggcggtagc 360

ggaggaggcg gctcagaagt gcagctggtg gagtccggcg gtggactggt gcaaccggga 420

ggctcactca gattgtcatg caccgcctct ggctttagtc tcagtgccta tgcaatgggc 480

tgggtgaggc aggcacccgg caagggcctg gaatggatcg gcatcattag tgatagtgct 540

agcacatttt acgcgacctg ggcgaagggc aggtttacca tcagccgcga caacagcaag 600

aataccgttt acctgcagat gaatagcctg agggccgaag aacacggcggt ctatttctgt 660

gcacggggcct attatgttgt cgataatgat tcccctttta atatgtgggg ccctgggacc 720

ctcgtaactg tgagcagc 738

<210> 27

<211> 717

<212>DNA

<213> Synthetic Sequence

<400> 27

gagatcgtga tgacccagtc cccaagtaca ctgagcgcct ccgtgggcga ccgcgtgatc 60

ataaactgtc aggccagtca gaatgtttat gataacaacc gcttatcctg gtatcaacag 120

aagcccggta aggcccccaa actgctcatt tacgatgcat ccaaactggc atccggggtg 180

ccaagccgct tctccggggag tgggtctggc gccgagttca ccctgaccat atcttccctg 240

cagcccgacg acttcgcaac gtactattgc ctaggcactt atgattgtat tagtgattgt 300

gatactttcg ggcagggcac gaagttgacc gtgctgggcg gagggggctc aggaggtggc 360

ggtagcggag gaggcggctc agaagtgcag ctggtggagt ccggcggtgg actggtgcaa 420

ccgggaggct cactcagatt gtcatgcacc gcctctggct ttagtctcag tacctatgca 480

ataaactggg tgaggcaggc acccggcaag ggcctggaat ggatcggctt cattggtagt 540

ggtggtggcg catactacgc gagctgggcg aagggcaggt ttaccatcag ccgcgacaac 600

agcaagaata ccgtttacct gcagatgaat agcctgaggg ccgaagacac ggcggtctat 660

ttctgtgcac ggttggttgc cttctggggc cctgggaccc tcgtaactgt gagcagc 717

<210> 28

<211> 726

<212> DNA

<213> Synthetic Sequence

<400> 28

gagatcgtga tgacccagtc cccaagtaca ctgagcgcct ccgtgggcga ccgcgtgatc 60

ataaactgtc agtccagtca gagtgttgtt aataacaacg aattatcctg gtatcaacag 120

aagcccggta aggcccccaa actgctcatt tacgaggcat ccaaactggc atccggggtg 180

ccaagccgct tctccggggag tgggtctggc gccgagttca ccctgaccat atcttccctg 240

cagcccgacg acttcgcaac gtactattgc caaggcactt attatagtag tggtttttac 300

gttactttcg ggcagggcac gaagttgacc gtgctgggcg gagggggctc aggaggtggc 360

ggtagcggag gaggcggctc agaagtgcag ctggtggagt ccggcggtgg actggtgcaa 420

ccgggaggct cactcagatt gtcatgcacc gcctctggct ttaccatcaa taacgtcgac 480

atgagctggg tgaggcaggc acccggcaag ggcctggaat ggatcggcgt cattagtagt 540

ggtgctaaca catactacgc gagctgggcg aagggcaggt ttaccatcag ccgcgacaac 600

agcaagaata ccgtttacct gcagatgaat agcctgaggg ccgaagacac ggcggtctat 660

ttctgtgcac ggggcggcgt tctttttgat ccctggggcc ctgggaccct cgtaactgtg 720

agcagc 726

<210> 29

<211> 726

<212>DNA

<213> Synthetic Sequence

<400> 29

gagatcgtga tgacccagtc cccaagtaca ctgagcgcct ccgtgggcga ccgcgtgatc 60

ataaactgtc aggccagtga gaggattgat tatagtttgg cctggtatca acagaagccc 120

ggtaaggccc ccaaactgct catttacgct gcatccaaac tggcatccgg ggtgccaagc 180

cgcttctccg ggagtgggtc tggcgccgag ttcaccctga ccatatcttc cctgcagccc 240

gacgacttcg caacgtacta ttgccaaagc tattatgata ttagtagtta tactttcggg 300

cagggcacga agttgaccgt gctgggcgga gggggctcag gaggtggcgg tagcggagga 360

ggcggctcag aagtgcagct ggtggagtcc ggcggtggac tggtgcaacc gggaggctca 420

ctcagattgt catgcaccgc ctctggcttt ccccttagtg tctactacat gagctgggtg 480

aggcaggcac ccggcaaggg cctggaatgg atcggcatct tttatgatgg taccacatac 540

tacgcgagct gggcgaaggg caggtttacc atcagccgcg acaacagcaa gaataccgtt 600

tacctgcaga tgaatagcct gagggccgaa gacacggcgg tctatttctg tgcacgggtt 660

atgtatgttg ctgactatgg tgccctgaac atctggggcc ctgggaccct cgtaactgtg 720

agcagc 726

<210> 30

<211> 717

<212>DNA

<213> Synthetic Sequence

<400> 30

gagatcgtga tgacccagtc cccaagtaca ctgagcgcct ccgtgggcga ccgcgtgatc 60

ataaactgtc aggccagtca gagtgtttat aagagcaatt acttaggctg gtatcaacag 120

aagcccggta aggcccccaa actgctcatt ggttctgcat cccaactggc atccggggtg 180

ccaagccgct tctccggggag tgggtctggc gccgagttca ccctgaccat atcttccctg 240

cagcccgacg acttcgcaac gtactattgc ctaggcattt atgattgtag tagtgctgat 300

tgtaatgctt tcgggcaggg cacgaagttg accgtgctgg gcggaggggg ctcaggaggt 360

ggcggtagcg gaggaggcgg ctcagaagtg cagctggtgg agtccggcgg tggactggtg 420

caaccgggag gctcactcag attgtcatgc accgcctctg gctttagtct cagtagctat 480

gcaataaact gggtgaggca ggcacccggc aagggcctgg aatggatcgg cgccattggt 540

actaatcata acacataacta cccgagctgg gcgaagggca ggtttaccat cagccgcgac 600

aacagcaaga ataccgttta cctgcagatg aatagcctga gggccgaaga cacggcggtc 660

tatttctgta cgactggtga catctggggc cctgggaccc tcgtaactgt gagcagc 717

<210> 31

<211> 493

<212> PRT

<213> Synthetic Sequence

<400> 31

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu

20 25 30

Ser Ala Ser Val Gly Asp Arg Val Ile Ile Asn Cys Gln Ala Ser Gln

35 40 45

Ser Ile Ser Ser Ser Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala

50 55 60

Pro Lys Leu Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Cys Thr

100 105 110

Tyr Gly Thr Phe His Ser Ser Ser Gly Tyr Gly Phe Gly Gln Gly Thr Lys

115 120 125

Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

145 150 155 160

Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu

165 170 175

Ser Ser Tyr Gly Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

180 185 190

Glu Trp Ile Gly Ser Ile Asn Thr Asp Gly Ser Thr Tyr Tyr Ala Thr

195 200 205

Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

210 215 220

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

225 230 235 240

Tyr Cys Ala Arg Gly Tyr Pro Gly Tyr Ile Thr Asp Ser Tyr Tyr Tyr

245 250 255

Phe Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Thr Thr

260 265 270

Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln

275 280 285

Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala

290 295 300

Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala

305 310 315 320

Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr

325 330 335

Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

340 345 350

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

355 360 365

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys

370 375 380

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

385 390 395 400

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

405 410 415

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

420 425 430

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

435 440 445

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

450 455 460

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

465 470 475 480

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 32

<211> 492

<212> PRT

<213> Synthetic Sequence

<400> 32

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu

20 25 30

Ser Ala Ser Val Gly Asp Arg Val Ile Ile Asn Cys Gln Ala Ser Gln

35 40 45

Ser Val Arg Asp Asn Gly Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly

50 55 60

Lys Ala Pro Lys Leu Leu Ile Tyr Asp Val Ser Ala Leu Ala Ser Gly

65 70 75 80

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu

85 90 95

Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala

100 105 110

Gly Gly Tyr Ile Ala Gly Ser Asp Arg Trp Val Phe Gly Gln Gly Thr

115 120 125

Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

145 150 155 160

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser

165 170 175

Leu Ser Asn Tyr Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Phe Ile Gly Thr Ile Gly Ala Thr Leu Tyr Ala

195 200 205

Asn Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

210 215 220

Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

225 230 235 240

Tyr Phe Cys Ala Arg Gly Ile Tyr Gly Asp Ile Tyr Val Tyr Ala Phe

245 250 255

Asp Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Thr

260 265 270

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

275 280 285

Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val

290 295 300

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

305 310 315 320

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

325 330 335

Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

340 345 350

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

355 360 365

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

370 375 380

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

385 390 395 400

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

405 410 415

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

420 425 430

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

435 440 445

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

450 455 460

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

465 470 475 480

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 33

<211> 490

<212> PRT

<213> Synthetic Sequence

<400> 33

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu

20 25 30

Ser Ala Ser Val Gly Asp Arg Val Ile Ile Asn Cys Gln Ala Ser Glu

35 40 45

Asn Ile Tyr Ser Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala

50 55 60

Pro Lys Leu Leu Ile Tyr Ala Ala Ser Lys Leu Ala Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Ser Tyr

100 105 110

Tyr Asp Thr Gly Arg Ala Ser Phe Ala Phe Gly Gln Gly Thr Lys Leu

115 120 125

Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

145 150 155 160

Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu Ser

165 170 175

Ala Tyr Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

180 185 190

Trp Ile Gly Ile Ile Ser Asp Ser Ala Ser Thr Phe Tyr Ala Thr Trp

195 200 205

Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val

210 215 220

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe

225 230 235 240

Cys Ala Arg Ala Tyr Tyr Val Val Asp Asp Asp Ser Pro Phe Asn Met

245 250 255

Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala

260 265 270

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

275 280 285

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

290 295 300

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

305 310 315 320

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

325 330 335

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

340 345 350

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

355 360 365

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

370 375 380

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

385 390 395 400

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

405 410 415

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

420 425 430

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

435 440 445

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Arg Gly Lys Gly His

450 455 460

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

465 470 475 480

Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 34

<211> 483

<212> PRT

<213> Synthetic Sequence

<400> 34

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu

20 25 30

Ser Ala Ser Val Gly Asp Arg Val Ile Ile Asn Cys Gln Ala Ser Gln

35 40 45

Asn Val Tyr Asp Asn Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly

50 55 60

Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Lys Leu Ala Ser Gly

65 70 75 80

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu

85 90 95

Thr Ile Ser Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Leu

100 105 110

Gly Thr Tyr Asp Cys Ile Ser Asp Cys Asp Thr Phe Gly Gln Gly Thr

115 120 125

Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

145 150 155 160

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser

165 170 175

Leu Ser Thr Tyr Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Phe Ile Gly Ser Gly Gly Gly Ala Tyr Tyr Ala

195 200 205

Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

210 215 220

Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

225 230 235 240

Tyr Phe Cys Ala Arg Leu Val Ala Phe Trp Gly Pro Gly Thr Leu Val

245 250 255

Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala

260 265 270

Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg

275 280 285

Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys

290 295 300

Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu

305 310 315 320

Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu

325 330 335

Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

340 345 350

Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Glu Gly Gly

355 360 365

Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

370 375 380

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

385 390 395 400

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

405 410 415

Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

420 425 430

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

435 440 445

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

450 455 460

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

465 470 475 480

Pro Pro Arg

<210> 35

<211> 486

<212> PRT

<213> Synthetic Sequence

<400> 35

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu

20 25 30

Ser Ala Ser Val Gly Asp Arg Val Ile Ile Asn Cys Gln Ser Ser Gln

35 40 45

Ser Val Val Asn Asn Asn Asn Glu Leu Ser Trp Tyr Gln Gln Lys Pro Gly

50 55 60

Lys Ala Pro Lys Leu Leu Ile Tyr Glu Ala Ser Lys Leu Ala Ser Gly

65 70 75 80

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu

85 90 95

Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln

100 105 110

Gly Thr Tyr Tyr Ser Ser Gly Phe Tyr Val Thr Phe Gly Gln Gly Thr

115 120 125

Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

145 150 155 160

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr

165 170 175

Ile Asn Asn Val Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Val Ile Ser Ser Gly Ala Asn Thr Tyr Tyr Ala

195 200 205

Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

210 215 220

Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

225 230 235 240

Tyr Phe Cys Ala Arg Gly Gly Val Leu Phe Asp Pro Trp Gly Pro Gly

245 250 255

Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 36

<211> 486

<212> PRT

<213> Synthetic Sequence

<400> 36

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu

20 25 30

Ser Ala Ser Val Gly Asp Arg Val Ile Ile Asn Cys Gln Ala Ser Glu

35 40 45

Arg Ile Asp Tyr Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala

50 55 60

Pro Lys Leu Leu Ile Tyr Ala Ala Ser Lys Leu Ala Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Ser Tyr

100 105 110

Tyr Asp Ile Ser Ser Tyr Thr Phe Gly Gln Gly Thr Lys Leu Thr Val

115 120 125

Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

145 150 155 160

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Pro Leu Ser Val Tyr

165 170 175

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

180 185 190

Gly Ile Phe Tyr Asp Gly Thr Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

195 200 205

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln

210 215 220

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg

225 230 235 240

Val Met Tyr Val Ala Asp Tyr Gly Ala Leu Asn Ile Trp Gly Pro Gly

245 250 255

Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 37

<211> 483

<212> PRT

<213> Synthetic Sequence

<400> 37

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu

20 25 30

Ser Ala Ser Val Gly Asp Arg Val Ile Ile Asn Cys Gln Ala Ser Gln

35 40 45

Ser Val Tyr Lys Ser Asn Tyr Leu Gly Trp Tyr Gln Gln Lys Pro Gly

50 55 60

Lys Ala Pro Lys Leu Leu Ile Gly Ser Ala Ser Gln Leu Ala Ser Gly

65 70 75 80

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu

85 90 95

Thr Ile Ser Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Leu

100 105 110

Gly Ile Tyr Asp Cys Ser Ser Ala Asp Cys Asn Ala Phe Gly Gln Gly

115 120 125

Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

145 150 155 160

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe

165 170 175

Ser Leu Ser Ser Ser Tyr Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys

180 185 190

Gly Leu Glu Trp Ile Gly Ala Ile Gly Thr Asn His Asn Thr Tyr Tyr

195 200 205

Pro Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

210 215 220

Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

225 230 235 240

Val Tyr Phe Cys Thr Thr Gly Asp Ile Trp Gly Pro Gly Thr Leu Val

245 250 255

Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala

260 265 270

Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg

275 280 285

Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys

290 295 300

Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu

305 310 315 320

Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu

325 330 335

Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

340 345 350

Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Glu Gly Gly

355 360 365

Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

370 375 380

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

385 390 395 400

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

405 410 415

Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

420 425 430

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

435 440 445

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

450 455 460

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

465 470 475 480

Pro Pro Arg

<210> 38

<211> 1482

<212>DNA

<213> Synthetic Sequence

<400> 38

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggagatcg tgatgaccca gtccccaagt acactgagcg cctccgtggg cgaccgcgtg 120

atcataaact gtcaggccag tcagagcatt agtagttact tatcctggta tcaacagaag 180

cccggtaagg cccccaaact gctcatttac agggcatcca ctctggcatc cggggtgcca 240

agccgcttct ccgggagtgg gtctggcgcc gagttcaccc tgaccatatc ttccctgcag 300

cccgacgact tcgcaacgta ctattgccaa tgcacttatg gtacttttca tagtagtggt 360

tatggtttcg ggcagggcac gaagttgacc gtgctgggcg gagggggctc aggaggtggc 420

ggtagcggag gaggcggctc agaagtgcag ctggtggagt ccggcggtgg actggtgcaa 480

ccgggaggct cactcagatt gtcatgcacc gcctctggct ttagtctcag tagctatgga 540

atgatctggg tgaggcaggc acccggcaag ggcctggaat ggatcggctc catcaatact 600

gatggtagca catactacgc aacctgggcg aagggcaggt ttaccatcag ccgcgacaac 660

agcaagaata ccgtttacct gcagatgaat agcctgaggg ccgaagacac ggcggtctat 720

tactgtgcac ggggttatcc tggttatatt actgatagtt actactactt taacatctgg 780

ggccctggga ccctcgtaac tgtgagcagc accacgacgc cagcgccgcg accaccaaca 840

ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg 900

gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgatatcta catctgggcg 960

cccttggccg ggacttgtgg ggtccttctc ctgtcactgg ttatcaccct ttactgcaaa 1020

cggggcagaa agaaactcct gtatatattc aaacaaccat ttatgagacc agtacaaact 1080

actcaagagg aagatggctg tagctgccga tttccagaag aagaagaagg aggatgtgaa 1140

ctgagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 1200

ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 1260

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1320

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1380

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1440

acctacgacg cccttcacat gcaggccctg ccccctcgct aa 1482

<210> 39

<211> 1479

<212>DNA

<213> Synthetic Sequence

<400> 39

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggagatcg tgatgaccca gtccccaagt acactgagcg cctccgtggg cgaccgcgtg 120

atcataaact gtcaggccag tcagagtgtt cgtgataatg gcgacttagc ctggtatcaa 180

cagaagcccg gtaaggcccc caaactgctc atttacgatg tatccgctct ggcatccggg 240

gtgccaagcc gcttctccgg gagtgggtct ggcgccgagt tcaccctgac catatcttcc 300

ctgcagcccg acgacttcgc aacgtactat tgcgcaggcg gttacatcgc tggttctgat 360

aggtgggttt tcgggcaggg cacgaagttg accgtgctgg gcggaggggg ctcaggaggt 420

ggcggtagcg gaggaggcgg ctcagaagtg cagctggtgg agtccggcgg tggactggtg 480

caaccgggag gctcactcag attgtcatgc accgcctctg gctttagtct cagtaactat 540

ggagtgagtt gggtgaggca ggcacccggc aagggcctgg aatggatcgg cttcattggt 600

accattggtg ccacattgta cgcgaactgg gcgaagggca ggtttaccat cagccgcgac 660

aacagcaaga ataccgttta cctgcagatg aatagcctga gggccgaaga cacggcggtc 720

tatttctgtg cacgggggat atatggtgat atttatgttt atgcctttga catctggggc 780

cctgggaccc tcgtaactgt gagcagcacc acgacgccag cgccgcgacc accaacaccg 840

gcgcccacca tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg 900

gggggcgcag tgcacacgag ggggctggac ttcgcctgtg atatctacat ctgggcgccc 960

ttggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaaacgg 1020

ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact 1080

caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 1140

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 1200

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 1260

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 1320

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 1380

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1440

tacgacgccc ttcacatgca ggccctgccc cctcgctaa 1479

<210> 40

<211> 1473

<212>DNA

<213> Synthetic Sequence

<400> 40

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggagatcg tgatgaccca gtccccaagt acactgagcg cctccgtggg cgaccgcgtg 120

atcataaact gtcaggccag tgagaacatt tacagctctt tagcctggta tcaacagaag 180

cccggtaagg cccccaaact gctcatttac gctgcatcca agctggcatc cggggtgcca 240

agccgcttct ccgggagtgg gtctggcgcc gagttcaccc tgaccatatc ttccctgcag 300

cccgacgact tcgcaacgta ctattgccaa agttattatg atactggtcg tgctagtttt 360

gctttcgggc agggcacgaa gttgaccgtg ctgggcggag ggggctcagg aggtggcggt 420

agcggaggag gcggctcaga agtgcagctg gtggagtccg gcggtggact ggtgcaaccg 480

ggaggctcac tcagattgtc atgcaccgcc tctggcttta gtctcagtgc ctatgcaatg 540

ggctgggtga ggcaggcacc cggcaagggc ctggaatgga tcggcatcat tagtgatagt 600

gctagcacat tttacgcgac ctgggcgaag ggcaggttta ccatcagccg cgacaacagc 660

aagaataccg tttacctgca gatgaatagc ctgagggccg aagacacggc ggtctatttc 720

tgtgcacggg cctattatgt tgtcgataat gattcccctt ttaatatgtg gggccctggg 780

accctcgtaa ctgtgagcag caccacgacg ccagcgccgc gaccaccaac accggcgccc 840

accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900

gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc 960

gggacttgtg gggtcccttct cctgtcactg gttatcaccc tttactgcaa acggggcaga 1020

aagaaactcc tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag 1080

gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg 1140

aagttcagca ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac 1200

gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 1260

cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg 1320

cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1380

ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1440

gcccttcaca tgcaggccct gccccctcgc taa 1473

<210> 41

<211> 1452

<212>DNA

<213> Synthetic Sequence

<400> 41

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggagatcg tgatgaccca gtccccaagt acactgagcg cctccgtggg cgaccgcgtg 120

atcataaact gtcaggccag tcagaatgtt tatgataaca accgcttatc ctggtatcaa 180

cagaagcccg gtaaggcccc caaactgctc atttacgatg catccaaact ggcatccggg 240

gtgccaagcc gcttctccgg gagtgggtct ggcgccgagt tcaccctgac catatcttcc 300

ctgcagcccg acgacttcgc aacgtactat tgcctaggca cttatgattg tattagtgat 360

tgtgatactt tcgggcaggg cacgaagttg accgtgctgg gcggaggggg ctcaggaggt 420

ggcggtagcg gaggaggcgg ctcagaagtg cagctggtgg agtccggcgg tggactggtg 480

caaccgggag gctcactcag attgtcatgc accgcctctg gctttagtct cagtacctat 540

gcaataaact gggtgaggca ggcacccggc aagggcctgg aatggatcgg cttcattggt 600

agtggtggtg gcgcatacta cgcgagctgg gcgaagggca ggtttaccat cagccgcgac 660

aacagcaaga ataccgttta cctgcagatg aatagcctga gggccgaaga cacggcggtc 720

tatttctgtg cacggttggt tgccttctgg ggccctggga ccctcgtaac tgtgagcagc 780

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 840

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 900

gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 960

ctgtcactgg ttatcaccct ttactgcaaa cggggcagaa agaaactcct gtatatattc 1020

aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga 1080

tttccagaag aagaagaagg aggatgtgaa ctgagagtga agttcagcag gagcgcagac 1140

gcccccgcgt accagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 1200

gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 1260

agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 1320

gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaaggggca cgatggcctt 1380

taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 1440

ccccctcgct aa 1452

<210> 42

<211> 1461

<212>DNA

<213> Synthetic Sequence

<400> 42

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggagatcg tgatgaccca gtccccaagt acactgagcg cctccgtggg cgaccgcgtg 120

atcataaact gtcagtccag tcagagtgtt gttaataaca acgaattatc ctggtatcaa 180

cagaagcccg gtaaggcccc caaactgctc atttacgagg catccaaact ggcatccggg 240

gtgccaagcc gcttctccgg gagtgggtct ggcgccgagt tcaccctgac catatcttcc 300

ctgcagcccg acgacttcgc aacgtactat tgccaaggca cttattatag tagtggtttt 360

tacgttactt tcgggcaggg cacgaagttg accgtgctgg gcggaggggg ctcaggaggt 420

ggcggtagcg gaggaggcgg ctcagaagtg cagctggtgg agtccggcgg tggactggtg 480

caaccgggag gctcactcag attgtcatgc accgcctctg gctttaccat caataacgtc 540

gacatgagct gggtgaggca ggcacccggc aagggcctgg aatggatcgg cgtcattagt 600

agtggtgcta acacataacta cgcgagctgg gcgaagggca ggtttaccat cagccgcgac 660

aacagcaaga ataccgttta cctgcagatg aatagcctga gggccgaaga cacggcggtc 720

tatttctgtg cacggggcgg cgttcttttt gatccctggg gccctgggac cctcgtaact 780

gtgagcagca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900

aggggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1020

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1080

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccgggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcta a 1461

<210> 43

<211> 1461

<212>DNA

<213> Synthetic Sequence

<400> 43

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggagatcg tgatgaccca gtccccaagt acactgagcg cctccgtggg cgaccgcgtg 120

atcataaact gtcaggccag tgagaggatt gattatagtt tggcctggta tcaacagaag 180

cccggtaagg cccccaaact gctcatttac gctgcatcca aactggcatc cggggtgcca 240

agccgcttct ccgggagtgg gtctggcgcc gagttcaccc tgaccatatc ttccctgcag 300

cccgacgact tcgcaacgta ctattgccaa agctattatg atattagtag ttatactttc 360

gggcagggca cgaagttgac cgtgctgggc ggagggggct caggaggtgg cggtagcgga 420

ggaggcggct cagaagtgca gctggtggag tccggcggtg gactggtgca accgggaggc 480

tcactcagat tgtcatgcac cgcctctggc tttcccctta gtgtctacta catgagctgg 540

gtgaggcagg cacccggcaa gggcctggaa tggatcggca tcttttatga tggtaccaca 600

tactacgcga gctgggcgaa gggcaggttt accatcagcc gcgacaacag caagaatacc 660

gtttacctgc agatgaatag cctgagggcc gaagacacgg cggtctattt ctgtgcacgg 720

gttatgtatg ttgctgacta tggtgccctg aacatctggg gccctgggac cctcgtaact 780

gtgagcagca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900

aggggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1020

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1080

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccgggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcta a 1461

<210> 44

<211> 1452

<212>DNA

<213> Synthetic Sequence

<400> 44

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggagatcg tgatgaccca gtccccaagt acactgagcg cctccgtggg cgaccgcgtg 120

atcataaact gtcaggccag tcagagtgtt tataagagca attacktagg ctggtatcaa 180

cagaagcccg gtaaggcccc caaactgctc attggttctg catcccaact ggcatccggg 240

gtgccaagcc gcttctccgg gagtgggtct ggcgccgagt tcaccctgac catatcttcc 300

ctgcagcccg acgacttcgc aacgtactat tgcctaggca tttatgattg tagtagtgct 360

gattgtaatg ctttcgggca gggcacgaag ttgaccgtgc tgggcggagg gggctcagga 420

ggtggcggta gcggaggagg cggctcagaa gtgcagctgg tggagtccgg cggtggactg 480

gtgcaaccgg gaggctcact cagattgtca tgcaccgcct ctggctttag tctcagtagc 540

tatgcaataa actgggtgag gcaggcaccc ggcaagggcc tggaatggat cggcgccatt 600

ggtactaatc ataacacata ctacccgagc tgggcgaagg gcaggtttac catcagccgc 660

gacaacagca agaataccgt ttacctgcag atgaatagcc tgagggccga agaacacggcg 720

gtctatttct gtacgactgg tgacatctgg ggccctggga ccctcgtaac tgtgagcagc 780

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 840

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 900

gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 960

ctgtcactgg ttatcaccct ttactgcaaa cggggcagaa agaaactcct gtatatattc 1020

aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga 1080

tttccagaag aagaagaagg aggatgtgaa ctgagagtga agttcagcag gagcgcagac 1140

gcccccgcgt accagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 1200

gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 1260

agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 1320

gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaaggggca cgatggcctt 1380

taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 1440

ccccctcgct aa 1452

<210> 45

<211> 512

<212> PRT

<213> Synthetic Sequence

<400> 45

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu Ser Asn Tyr

20 25 30

Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Phe Ile Gly Thr Ile Gly Ala Thr Leu Tyr Ala Asn Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala

85 90 95

Arg Gly Ile Tyr Gly Asp Ile Tyr Val Tyr Ala Phe Asp Ile Trp Gly

100 105 110

Pro Gly Thr Leu Val Thr Val Ser Ser Gly Ser Thr Ser Gly Gly Gly

115 120 125

Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu Ile Val Met Thr

130 135 140

Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly Asp Arg Val Ile Ile

145 150 155 160

Asn Cys Gln Ala Ser Gln Ser Val Arg Asp Asn Gly Asp Leu Ala Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Val

180 185 190

Ser Ala Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe

210 215 220

Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ile Ala Gly Ser Asp Arg Trp

225 230 235 240

Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser

245 250 255

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

260 265 270

Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly Asp

275 280 285

Arg Val Ile Ile Asn Cys Gln Ser Ser Pro Ser Val Tyr Asn Asn Tyr

290 295 300

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

305 310 315 320

Tyr Glu Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

325 330 335

Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

340 345 350

Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Thr Tyr Val Ser Gly Asp

355 360 365

Arg Arg Ala Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly Gly Gly

370 375 380

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

385 390 395 400

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

405 410 415

Ser Cys Thr Ala Ser Gly Phe Ser Leu Ser Thr Tyr His Met Thr Trp

420 425 430

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Ser

435 440 445

Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr

450 455 460

Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser

465 470 475 480

Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg Asp Leu Asp

485 490 495

Tyr Val Ile Asp Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

500 505 510

<210> 46

<211> 507

<212> PRT

<213> Synthetic Sequence

<400> 46

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu Ser Asn Tyr

20 25 30

Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Phe Ile Gly Thr Ile Gly Ala Thr Leu Tyr Ala Asn Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala

85 90 95

Arg Gly Ile Tyr Gly Asp Ile Tyr Val Tyr Ala Phe Asp Ile Trp Gly

100 105 110

Pro Gly Thr Leu Val Thr Val Ser Ser Gly Ser Thr Ser Gly Gly Gly

115 120 125

Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu Ile Val Met Thr

130 135 140

Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly Asp Arg Val Ile Ile

145 150 155 160

Asn Cys Gln Ala Ser Gln Ser Val Arg Asp Asn Gly Asp Leu Ala Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Val

180 185 190

Ser Ala Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe

210 215 220

Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ile Ala Gly Ser Asp Arg Trp

225 230 235 240

Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Glu Ala Ala Ala Lys

245 250 255

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ile Val Met Thr Gln

260 265 270

Ser Pro Ser Thr Leu Ser Ala Ser Val Gly Asp Arg Val Ile Ile Asn

275 280 285

Cys Gln Ser Ser Pro Ser Val Tyr Asn Asn Tyr Leu Ser Trp Tyr Gln

290 295 300

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Glu Thr Ser Ser Thr

305 310 315 320

Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ala

325 330 335

Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr

340 345 350

Tyr Tyr Cys Ala Gly Thr Tyr Val Ser Gly Asp Arg Arg Ala Phe Gly

355 360 365

Gln Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly

370 375 380

Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

385 390 395 400

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser

405 410 415

Gly Phe Ser Leu Ser Thr Tyr His Met Thr Trp Val Arg Gln Ala Pro

420 425 430

Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Ser Ser Ser Gly Ser Thr

435 440 445

Tyr Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

450 455 460

Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

465 470 475 480

Thr Ala Val Tyr Phe Cys Ala Arg Asp Leu Asp Tyr Val Ile Asp Leu

485 490 495

Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

500 505

<210> 47

<211> 512

<212> PRT

<213> Synthetic Sequence

<400> 47

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

His Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala

85 90 95

Arg Asp Leu Asp Tyr Val Ile Asp Leu Trp Gly Pro Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala

130 135 140

Ser Val Gly Asp Arg Val Ile Ile Asn Cys Gln Ser Ser Pro Ser Val

145 150 155 160

Tyr Asn Asn Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

165 170 175

Lys Leu Leu Ile Tyr Glu Thr Ser Thr Leu Ala Ser Gly Val Pro Ser

180 185 190

Arg Phe Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser

195 200 205

Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Thr Tyr

210 215 220

Val Ser Gly Asp Arg Arg Ala Phe Gly Gln Gly Thr Lys Leu Thr Val

225 230 235 240

Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

245 250 255

Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu

260 265 270

Ser Ala Ser Val Gly Asp Arg Val Ile Ile Asn Cys Gln Ala Ser Gln

275 280 285

Ser Val Arg Asp Asn Gly Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly

290 295 300

Lys Ala Pro Lys Leu Leu Ile Tyr Asp Val Ser Ala Leu Ala Ser Gly

305 310 315 320

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu

325 330 335

Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala

340 345 350

Gly Gly Tyr Ile Ala Gly Ser Asp Arg Trp Val Phe Gly Gln Gly Thr

355 360 365

Lys Leu Thr Val Leu Gly Ser Thr Ser Gly Gly Gly Ser Gly Gly Gly

370 375 380

Ser Gly Gly Gly Gly Ser Ser Ser Glu Val Gln Leu Val Glu Ser Gly Gly

385 390 395 400

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser

405 410 415

Gly Phe Ser Leu Ser Asn Tyr Gly Val Ser Trp Val Arg Gln Ala Pro

420 425 430

Gly Lys Gly Leu Glu Trp Ile Gly Phe Ile Gly Thr Ile Gly Ala Thr

435 440 445

Leu Tyr Ala Asn Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

450 455 460

Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

465 470 475 480

Thr Ala Val Tyr Phe Cys Ala Arg Gly Ile Tyr Gly Asp Ile Tyr Val

485 490 495

Tyr Ala Phe Asp Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

500 505 510

<210> 48

<211> 507

<212> PRT

<213> Synthetic Sequence

<400> 48

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

His Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala

85 90 95

Arg Asp Leu Asp Tyr Val Ile Asp Leu Trp Gly Pro Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala

130 135 140

Ser Val Gly Asp Arg Val Ile Ile Asn Cys Gln Ser Ser Pro Ser Val

145 150 155 160

Tyr Asn Asn Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

165 170 175

Lys Leu Leu Ile Tyr Glu Thr Ser Thr Leu Ala Ser Gly Val Pro Ser

180 185 190

Arg Phe Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser

195 200 205

Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Thr Tyr

210 215 220

Val Ser Gly Asp Arg Arg Ala Phe Gly Gln Gly Thr Lys Leu Thr Val

225 230 235 240

Leu Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

245 250 255

Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

260 265 270

Asp Arg Val Ile Ile Asn Cys Gln Ala Ser Gln Ser Val Arg Asp Asn

275 280 285

Gly Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

290 295 300

Leu Ile Tyr Asp Val Ser Ala Leu Ala Ser Gly Val Pro Ser Arg Phe

305 310 315 320

Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Ser Leu

325 330 335

Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ile Ala

340 345 350

Gly Ser Asp Arg Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu

355 360 365

Gly Ser Thr Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly

370 375 380

Ser Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

385 390 395 400

Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu Ser

405 410 415

Asn Tyr Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

420 425 430

Trp Ile Gly Phe Ile Gly Thr Ile Gly Ala Thr Leu Tyr Ala Asn Trp

435 440 445

Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val

450 455 460

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe

465 470 475 480

Cys Ala Arg Gly Ile Tyr Gly Asp Ile Tyr Val Tyr Ala Phe Asp Ile

485 490 495

Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

500 505

<210> 49

<211> 487

<212> PRT

<213> Synthetic Sequence

<400> 49

Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ile Ile Asn Cys Gln Ser Ser Pro Ser Val Tyr Asn Asn

20 25 30

Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Glu Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Thr Tyr Val Ser Gly

85 90 95

Asp Arg Arg Ala Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly Gly

100 105 110

Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

115 120 125

Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu

130 135 140

Ser Asn Tyr Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

145 150 155 160

Glu Trp Ile Gly Phe Ile Gly Thr Ile Gly Ala Thr Leu Tyr Ala Asn

165 170 175

Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

180 185 190

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

195 200 205

Phe Cys Ala Arg Gly Ile Tyr Gly Asp Ile Tyr Val Tyr Ala Phe Asp

210 215 220

Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Ser Thr Ser

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu Ile

245 250 255

Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly Asp Arg

260 265 270

Val Ile Ile Asn Cys Gln Ala Ser Gln Ser Val Arg Asp Asn Gly Asp

275 280 285

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

290 295 300

Tyr Asp Val Ser Ala Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

305 310 315 320

Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

325 330 335

Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ile Ala Gly Ser

340 345 350

Asp Arg Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly Gly

355 360 365

Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

370 375 380

Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu

385 390 395 400

Ser Thr Tyr His Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

405 410 415

Glu Trp Ile Gly Val Ile Ser Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser

420 425 430

Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

435 440 445

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

450 455 460

Phe Cys Ala Arg Asp Leu Asp Tyr Val Ile Asp Leu Trp Gly Pro Gly

465 470 475 480

Thr Leu Val Thr Val Ser Ser

485

<210> 50

<211> 487

<212> PRT

<213> Synthetic Sequence

<400> 50

Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ile Ile Asn Cys Gln Ser Ser Pro Ser Val Tyr Asn Asn

20 25 30

Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Glu Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Thr Tyr Val Ser Gly

85 90 95

Asp Arg Arg Ala Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly Gly

100 105 110

Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala

115 120 125

Ser Val Gly Asp Arg Val Ile Ile Asn Cys Gln Ala Ser Gln Ser Val

130 135 140

Arg Asp Asn Gly Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala

145 150 155 160

Pro Lys Leu Leu Ile Tyr Asp Val Ser Ala Leu Ala Ser Gly Val Pro

165 170 175

Ser Arg Phe Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile

180 185 190

Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly

195 200 205

Tyr Ile Ala Gly Ser Asp Arg Trp Val Phe Gly Gln Gly Thr Lys Leu

210 215 220

Thr Val Leu Gly Ser Thr Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly

225 230 235 240

Gly Gly Gly Ser Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Gly Leu

245 250 255

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe

260 265 270

Ser Leu Ser Asn Tyr Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys

275 280 285

Gly Leu Glu Trp Ile Gly Phe Ile Gly Thr Ile Gly Ala Thr Leu Tyr

290 295 300

Ala Asn Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

305 310 315 320

Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

325 330 335

Val Tyr Phe Cys Ala Arg Gly Ile Tyr Gly Asp Ile Tyr Val Tyr Ala

340 345 350

Phe Asp Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Gly

355 360 365

Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

370 375 380

Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu

385 390 395 400

Ser Thr Tyr His Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

405 410 415

Glu Trp Ile Gly Val Ile Ser Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser

420 425 430

Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

435 440 445

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

450 455 460

Phe Cys Ala Arg Asp Leu Asp Tyr Val Ile Asp Leu Trp Gly Pro Gly

465 470 475 480

Thr Leu Val Thr Val Ser Ser

485

<210> 51

<211> 8

<212> PRT

<213> Synthetic Sequence

<400> 51

Gly Phe Ser Leu Ser Thr Tyr His

1 5

<210> 52

<211> 7

<212> PRT

<213> Synthetic Sequence

<400> 52

Ile Ser Ser Ser Gly Ser Thr

1 5

<210> 53

<211> 10

<212> PRT

<213> Synthetic Sequence

<400> 53

Ala Arg Asp Leu Asp Tyr Val Ile Asp Leu

1 5 10

<210> 54

<211> 7

<212> PRT

<213> Synthetic Sequence

<400> 54

Pro Ser Val Tyr Asn Asn Tyr

1 5

<210> 55

<211> 3

<212> PRT

<213> Synthetic Sequence

<400> 55

Glu Thr Ser

1

<210> 56

<211> 11

<212> PRT

<213> Synthetic Sequence

<400> 56

Ala Gly Thr Tyr Val Ser Gly Asp Arg Arg Ala

1 5 10

<210> 57

<211> 116

<212> PRT

<213> Synthetic Sequence

<400> 57

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

His Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala

85 90 95

Arg Asp Leu Asp Tyr Val Ile Asp Leu Trp Gly Pro Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 58

<211> 110

<212> PRT

<213> Synthetic Sequence

<400> 58

Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ile Ile Asn Cys Gln Ser Ser Pro Ser Val Tyr Asn Asn

20 25 30

Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Glu Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Thr Tyr Val Ser Gly

85 90 95

Asp Arg Arg Ala Phe Gly Gln Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 59

<211> 15

<212> PRT

<213> Synthetic Sequence

<400> 59

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 60

<211> 18

<212> PRT

<213> Synthetic Sequence

<400> 60

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

1 5 10 15

Lys Gly

<210> 61

<211> 18

<212> PRT

<213> Synthetic Sequence

<400> 61

Gly Ser Thr Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly

1 5 10 15

Ser Ser

<210> 62

<211> 485

<212> PRT

<213> Synthetic Sequence

<400> 62

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu

20 25 30

Ser Ala Ser Val Gly Asp Arg Val Ile Ile Asn Cys Gln Ser Ser Pro

35 40 45

Ser Val Tyr Asn Asn Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Glu Thr Ser Thr Leu Ala Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly

100 105 110

Thr Tyr Val Ser Gly Asp Arg Arg Ala Phe Gly Gln Gly Thr Lys Leu

115 120 125

Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

145 150 155 160

Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu Ser

165 170 175

Thr Tyr His Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

180 185 190

Trp Ile Gly Val Ile Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp

195 200 205

Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val

210 215 220

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe

225 230 235 240

Cys Ala Arg Asp Leu Asp Tyr Val Ile Asp Leu Trp Gly Pro Gly Thr

245 250 255

Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

260 265 270

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

275 280 285

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

290 295 300

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

305 310 315 320

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys

325 330 335

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

340 345 350

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Glu

355 360 365

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

370 375 380

Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

385 390 395 400

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

405 410 415

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

420 425 430

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

435 440 445

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

450 455 460

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

465 470 475 480

Ala Leu Pro Pro Arg

485

<210> 63

<211> 486

<212> PRT

<213> Synthetic Sequence

<400> 63

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu

20 25 30

Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr

35 40 45

Ser Phe Thr Thr Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln

50 55 60

Gly Leu Glu Trp Ile Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg

65 70 75 80

Leu Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser

85 90 95

Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser

100 105 110

Ala Val Tyr Tyr Cys Ala Arg Ser Thr Met Ile Ala Thr Arg Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met

145 150 155 160

Thr Gln Ser Gln Lys Ser Met Ser Thr Ser Val Gly Asp Arg Val Ser

165 170 175

Ile Thr Cys Lys Ala Ser Gln Asp Val Ile Thr Gly Val Ala Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Ser Ala Ser

195 200 205

Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly

210 215 220

Thr Asp Phe Thr Phe Thr Ile Ser Asn Val Gln Ala Glu Asp Leu Ala

225 230 235 240

Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu Thr Phe Gly Ala

245 250 255

Gly Thr Lys Leu Glu Leu Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

Claims (21)

1. A CS 1-targeted chimeric antigen receptor comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain; wherein the extracellular antigen recognition domain comprises an anti-CS 1 scFv antibody, the amino acid sequences of the VH complementarity determining regions CDR1, CDR2 and CDR3 of the scFv antibody comprise the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 respectively, and the amino acid sequences of the VL complementarity determining regions CDR1, CDR2 and CDR3 of the scFv antibody comprise the amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively.

2. The chimeric antigen receptor according to claim 1, wherein the scFv antibody is a humanized antibody, optionally the VH sequence of the scFv antibody comprises the amino acid sequence shown in SEQ ID No. 7 and the VL sequence comprises the amino acid sequence shown in SEQ ID No. 8;

or, the scFv antibody is a rabbit antibody, optionally, the VH sequence of the scFv antibody comprises an amino acid sequence shown as SEQ ID NO. 9, and the VL sequence of the scFv antibody comprises an amino acid sequence shown as SEQ ID NO. 10.

3. The chimeric antigen receptor of claim 2, wherein the scFv antibody has a linking region between VH and VL selected from one or more of the following: SEQ ID NOs 59-61.

4. The chimeric antigen receptor according to claim 1, wherein the scFv antibody has the sequence shown in SEQ ID No. 11 or SEQ ID No. 12.

5. The chimeric antigen receptor of any one of claims 1-4, wherein the hinge region is derived from one or more of IgG1, igG4, CD7, CD28, CD84, CD8 a; optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown as SEQ ID NO. 13;

and/or the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8 a, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, fc 70; optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown as SEQ ID NO. 14;

And/or, the intracellular domain comprises an intracellular signaling region; optionally, a costimulatory signaling region is also included.

6. The chimeric antigen receptor of claim 5, wherein the intracellular signaling region is derived from one or more of CD3 delta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, fcrgamma, fcrbeta, CD66d, DAP10, DAP12, syk; alternatively, the intracellular signaling region is derived from cd3δ; further alternatively, the amino acid sequence of the intracellular signaling region comprises the amino acid sequence shown as SEQ ID NO. 15;

and/or the costimulatory signaling region is derived from one, two or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, myD 88; optionally, the costimulatory signaling region is derived from CD28 or 4-1BB; further alternatively, the amino acid sequence of the costimulatory signaling region comprises the amino acid sequence depicted as SEQ ID NO. 16.

7. The chimeric antigen receptor according to any one of claims 1-4, further comprising a leader peptide at the N-terminus of its amino acid sequence; optionally, wherein the guide peptide is derived from CD8 a; further alternatively, the amino acid sequence of the leader peptide comprises the amino acid sequence shown as SEQ ID NO. 17.

8. The chimeric antigen receptor according to any one of claims 1-4, comprising the amino acid sequence shown in SEQ ID NO. 32.

9. The chimeric antigen receptor according to any one of claims 1-4, wherein the extracellular antigen-recognition domain further comprises an scFv antibody against one of the following targets: CD138, NKG2D, CD, BCMA, CD19, CD70, CD44v6, lewis Y.

10. The chimeric antigen receptor according to claim 9, wherein the extracellular antigen recognition domain comprises in sequence an anti-BCMA scFv VL, an anti-CS 1 scFv VH, and an anti-BCMA scFv VH, the amino acid sequences of the anti-CS 1 scFv VH complementarity determining regions CDR1, CDR2, CDR3 comprise the amino acid sequences of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, the amino acid sequences of the anti-CS 1 scFv VL complementarity determining regions CDR1, CDR2, CDR3 comprise the amino acid sequences of SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, the amino acid sequences of the anti-BCMA scFv VH complementarity determining regions CDR1, CDR2, CDR3 comprise the amino acid sequences of SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, and the amino acid sequences of the anti-BCMA scFv VL complementarity determining regions CDR1, CDR2, CDR3 comprise the amino acid sequences of SEQ ID No. 54, SEQ ID No. 55, SEQ ID No. 56, SEQ ID No. 3, respectively.

11. The chimeric antigen receptor according to claim 10, wherein the VH sequence of the anti-CS 1 scFv antibody comprises the amino acid sequence shown in SEQ ID No. 7 and the VL sequence of the anti-CS 1 scFv antibody comprises the amino acid sequence shown in SEQ ID No. 8; the VH sequence of the anti-BCMA scFv antibody comprises an amino acid sequence shown as SEQ ID NO:57, and the VL sequence of the anti-BCMA scFv antibody comprises an amino acid sequence shown as SEQ ID NO: 58.

12. The chimeric antigen receptor according to claim 10 or 11, wherein the extracellular antigen-recognition domain comprises an amino acid sequence as set forth in SEQ ID No. 50.

13. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor of any one of claims 1-12; alternatively, the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO. 18, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 18 and encoding the same chimeric antigen receptor; further alternatively, the nucleic acid molecule comprises the nucleotide sequence shown as SEQ ID NO. 39.

14. A vector comprising the nucleic acid molecule of claim 13; optionally, the vector is an expression vector; further alternatively, the vector is a viral vector; still further alternatively, the vector is a lentiviral vector.

15. An engineered immune effector cell comprising the chimeric antigen receptor of any one of claims 1-12, the isolated nucleic acid molecule of claim 13, or the vector of claim 14.

16. The immune effector cell of claim 15, which is selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T cells into which pluripotent stem cells differentiate, NK cells into which pluripotent stem cells differentiate, embryonic stem cells; alternatively, the immune effector cell is a T lymphocyte; further alternatively, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.

17. A pharmaceutical composition comprising the engineered immune effector cell of claim 15 or 16 and a pharmaceutically acceptable adjuvant; optionally, the pharmaceutically acceptable excipients include a protective agent; further alternatively, the protective agent comprises a cell cryopreservation solution.

18. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition is an intravenous injection.

19. Use of the chimeric antigen receptor of any one of claims 1-12, the isolated nucleic acid molecule of claim 13, the vector of claim 14, or the engineered immune effector cell of claim 15 or 16 in the manufacture of a medicament for treating a disease or disorder associated with expression of CS 1.

20. The use of claim 19, wherein the disease or disorder associated with expression of CS1 is cancer; optionally, the cancer is multiple myeloma; further alternatively, the multiple myeloma is refractory or recurrent multiple myeloma.

21. The use of claim 19, wherein the disease or disorder associated with expression of CS1 is an autoimmune disease; alternatively, the autoimmune disease may be selected from the group consisting of: systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis or autoimmune hemolytic anemia.

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